Cyprocide selectively kills nematodes via cytochrome P450 bioactivation.

Left unchecked, plant-parasitic nematodes have the potential to devastate crops globally. Highly effective but non-selective nematicides are justifiably being phased-out, leaving farmers with limited options for managing nematode infestation. Here, we report our discovery of a 1,3,4-oxadiazole thioether scaffold called Cyprocide that selectively kills nematodes including diverse species of plant-parasitic nematodes. Cyprocide is bioactivated into a lethal reactive electrophilic metabolite by specific nematode cytochrome P450 enzymes. Cyprocide fails to kill organisms beyond nematodes, suggesting that the targeted lethality of this pro-nematicide derives from P450 substrate selectivity. Our findings demonstrate that Cyprocide is a selective nematicidal scaffold with broad-spectrum activity that holds the potential to help safeguard our global food supply.

PGP-14 establishes a polar lipid permeability barrier within the C. elegans pharyngeal cuticle.

The cuticles of ecdysozoan animals are barriers to material loss and xenobiotic insult. Key to this barrier is lipid content, the establishment of which is poorly understood. Here, we show that the p-glycoprotein PGP-14 functions coincidently with the sphingomyelin synthase SMS-5 to establish a polar lipid barrier within the pharyngeal cuticle of the nematode C. elegans. We show that PGP-14 and SMS-5 are coincidentally expressed in the epithelium that surrounds the anterior pharyngeal cuticle where PGP-14 localizes to the apical membrane. pgp-14 and sms-5 also peak in expression at the time of new cuticle synthesis. Loss of PGP-14 and SMS-5 dramatically reduces pharyngeal cuticle staining by Nile Red, a key marker of polar lipids, and coincidently alters the nematode's response to a wide-range of xenobiotics. We infer that PGP-14 exports polar lipids into the developing pharyngeal cuticle in an SMS-5-dependent manner to safeguard the nematode from environmental insult.

Selective control of parasitic nematodes using bioactivated nematicides.

Parasitic nematodes are a major threat to global food security, particularly as the world amasses 10 billion people amid limited arable land1-4. Most traditional nematicides have been banned owing to poor nematode selectivity, leaving farmers with inadequate means of pest control4-12. Here we use the model nematode Caenorhabditis elegans to identify a family of selective imidazothiazole nematicides, called selectivins, that undergo cytochrome-p450-mediated bioactivation in nematodes. At low parts-per-million concentrations, selectivins perform comparably well with commercial nematicides to control root infection by Meloidogyne incognita, a highly destructive plant-parasitic nematode. Tests against numerous phylogenetically diverse non-target systems demonstrate that selectivins are more nematode-selective than most marketed nematicides. Selectivins are first-in-class bioactivated nematode controls that provide efficacy and nematode selectivity.

Nemacol is a small molecule inhibitor of C. elegans vesicular acetylcholine transporter with anthelmintic potential.

Nematode parasites of humans and livestock pose a significant burden to human health, economic development, and food security. Anthelmintic drug resistance is widespread among parasites of livestock and many nematode parasites of humans lack effective treatments. Here, we present a nitrophenyl-piperazine scaffold that induces motor defects rapidly in the model nematode Caenorhabditis elegans. We call this scaffold Nemacol and show that it inhibits the vesicular acetylcholine transporter (VAChT), a target recognized by commercial animal and crop health groups as a viable anthelmintic target. We demonstrate that it is possible to create Nemacol analogs that maintain potent in vivo activity whilst lowering their affinity to the mammalian VAChT 10-fold. We also show that Nemacol enhances the ability of the anthelmintic Ivermectin to paralyze C. elegans and the ruminant nematode parasite Haemonchus contortus. Hence, Nemacol represents a promising new anthelmintic scaffold that acts through a validated anthelmintic target.

Development of a yeast whole-cell biocatalyst for MHET conversion into terephthalic acid and ethylene glycol.

BACKGROUND: Over the 70 years since the introduction of plastic into everyday items, plastic waste has become an increasing problem. With over 360 million tonnes of plastics produced every year, solutions for plastic recycling and plastic waste reduction are sorely needed. Recently, multiple enzymes capable of degrading PET (polyethylene terephthalate) plastic have been identified and engineered. In particular, the enzymes PETase and MHETase from Ideonella sakaiensis depolymerize PET into the two building blocks used for its synthesis, ethylene glycol (EG) and terephthalic acid (TPA). Importantly, EG and TPA can be re-used for PET synthesis allowing complete and sustainable PET recycling. RESULTS: In this study we used Saccharomyces cerevisiae, a species utilized widely in bioindustrial fermentation processes, as a platform to develop a whole-cell catalyst expressing the MHETase enzyme, which converts monohydroxyethyl terephthalate (MHET) into TPA and EG. We assessed six expression architectures and identified those resulting in efficient MHETase expression on the yeast cell surface. We show that the MHETase whole-cell catalyst has activity comparable to recombinant MHETase purified from Escherichia coli. Finally, we demonstrate that surface displayed MHETase is active across a range of pHs, temperatures, and for at least 12 days at room temperature. CONCLUSIONS: We demonstrate the feasibility of using S. cerevisiae as a platform for the expression and surface display of PET degrading enzymes and predict that the whole-cell catalyst will be a viable alternative to protein purification-based approaches for plastic degradation.

A spatiotemporal reconstruction of the C. elegans pharyngeal cuticle reveals a structure rich in phase-separating proteins.

How the cuticles of the roughly 4.5 million species of ecdysozoan animals are constructed is not well understood. Here, we systematically mine gene expression datasets to uncover the spatiotemporal blueprint for how the chitin-based pharyngeal cuticle of the nematode Caenorhabditis elegans is built. We demonstrate that the blueprint correctly predicts expression patterns and functional relevance to cuticle development. We find that as larvae prepare to molt, catabolic enzymes are upregulated and the genes that encode chitin synthase, chitin cross-linkers, and homologs of amyloid regulators subsequently peak in expression. Forty-eight percent of the gene products secreted during the molt are predicted to be intrinsically disordered proteins (IDPs), many of which belong to four distinct families whose transcripts are expressed in overlapping waves. These include the IDPAs, IDPBs, and IDPCs, which are introduced for the first time here. All four families have sequence properties that drive phase separation and we demonstrate phase separation for one exemplar in vitro. This systematic analysis represents the first blueprint for cuticle construction and highlights the massive contribution that phase-separating materials make to the structure.

High-throughput small molecule screen identifies inhibitors of microsporidia invasion and proliferation in C. elegans.

Microsporidia are a diverse group of fungal-related obligate intracellular parasites that infect most animal phyla. Despite the emerging threat that microsporidia represent to humans and agricultural animals, few reliable treatment options exist. Here, we develop a high-throughput screening method for the identification of chemical inhibitors of microsporidia infection, using liquid cultures of Caenorhabditis elegans infected with the microsporidia species Nematocida parisii. We screen a collection of 2560 FDA-approved compounds and natural products, and identify 11 candidate microsporidia inhibitors. Five compounds prevent microsporidia infection by inhibiting spore firing, whereas one compound, dexrazoxane, slows infection progression. The compounds have in vitro activity against several other microsporidia species, including those known to infect humans. Together, our results highlight the effectiveness of C. elegans as a model host for drug discovery against intracellular pathogens, and provide a scalable high-throughput system for the identification and characterization of microsporidia inhibitors.

Egg-laying and locomotory screens with C. elegans yield a nematode-selective small molecule stimulator of neurotransmitter release.

Nematode parasites of humans, livestock and crops dramatically impact human health and welfare. Alarmingly, parasitic nematodes of animals have rapidly evolved resistance to anthelmintic drugs, and traditional nematicides that protect crops are facing increasing restrictions because of poor phylogenetic selectivity. Here, we exploit multiple motor outputs of the model nematode C. elegans towards nematicide discovery. This work yielded multiple compounds that selectively kill and/or immobilize diverse nematode parasites. We focus on one compound that induces violent convulsions and paralysis that we call nementin. We find that nementin stimulates neuronal dense core vesicle release, which in turn enhances cholinergic signaling. Consequently, nementin synergistically enhances the potency of widely-used non-selective acetylcholinesterase (AChE) inhibitors, but in a nematode-selective manner. Nementin therefore has the potential to reduce the environmental impact of toxic AChE inhibitors that are used to control nematode infections and infestations.

Temporal Regulation of Gene Expression in Post-Mitotic Cells is Revealed from a Synchronized Population of C. elegans Larvae.

Unsupervised Uniform Manifold Approximation and Projection (UMAP) plots of single cell sequencing data from synchronized Caenorhabditis elegans larvae yield tissue-specific data clusters, some of which are plotted as elongated archipelagos. These archipelagos likely represent a single cell type. I show that the pharyngeal archipelagos express a myriad of asynchronous temporally regulated genes, which likely accounts for their elongated topology. With one archipelago, I show that there is a high correlation between a) the base pair distance between the binding sites of an archipelago-specific transcription factor (HLH-6) and the transcriptional start site of the targeted genes and b) the timing of peak gene expression of those genes that are expressed in an archipelago-specific manner. Despite the correlation being made with only four genes, it prompts the hypothesis that the physical distance between a transcription factor and the relevant transcription start site may be an important factor in determining the temporal onset of transcription and transcript abundance.

Culturing and Screening the Plant Parasitic Nematode Ditylenchus dipsaci.

Plant-parasitic nematodes (PPNs) destroy over 12% of global food crops every year, which equates to roughly 157 billion dollars (USD) lost annually. With a growing global population and limited arable land, controlling PPN infestation is critical for food production. Compounding the challenge of maximizing crop yields are the mounting restrictions on effective pesticides because of a lack of nematode selectivity. Hence, developing new and safe chemical nematicides is vital to food security. In this protocol, the culture and collection of the PPN species Ditylenchus dipsaci are demonstrated. D. dipsaci is both economically damaging and relatively resistant to most modern nematicides. The current work also explains how to use these nematodes in screens for novel small molecule nematicides and reports on data collection and analysis methodologies. The demonstrated pipeline affords a throughput of thousands of compounds per week and can be easily adapted for use with other PPN species such as Pratylenchus penetrans. The techniques described herein can be used to discover new nematicides, which may, in turn, be further developed into highly selective commercial products that safely combat PPNs to help feed an increasingly hungry world.

A survey of the kinome pharmacopeia reveals multiple scaffolds and targets for the development of novel anthelmintics.

Over one billion people are currently infected with a parasitic nematode. Symptoms can include anemia, malnutrition, developmental delay, and in severe cases, death. Resistance is emerging to the anthelmintics currently used to treat nematode infection, prompting the need to develop new anthelmintics. Towards this end, we identified a set of kinases that may be targeted in a nematode-selective manner. We first screened 2040 inhibitors of vertebrate kinases for those that impair the model nematode Caenorhabditis elegans. By determining whether the terminal phenotype induced by each kinase inhibitor matched that of the predicted target mutant in C. elegans, we identified 17 druggable nematode kinase targets. Of these, we found that nematode EGFR, MEK1, and PLK1 kinases have diverged from vertebrates within their drug-binding pocket. For each of these targets, we identified small molecule scaffolds that may be further modified to develop nematode-selective inhibitors. Nematode EGFR, MEK1, and PLK1 therefore represent key targets for the development of new anthelmintic medicines.

Author Correction: Caenorhabditis elegans is a useful model for anthelmintic discovery.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Identification of drug modifiers for RYR1-related myopathy using a multi-species discovery pipeline.

Ryanodine receptor type I-related myopathies (RYR1-RMs) are a common group of childhood muscle diseases associated with severe disabilities and early mortality for which there are no available treatments. The goal of this study is to identify new therapeutic targets for RYR1-RMs. To accomplish this, we developed a discovery pipeline using nematode, zebrafish, and mammalian cell models. We first performed large-scale drug screens in C. elegans which uncovered 74 hits. Targeted testing in zebrafish yielded positive results for two p38 inhibitors. Using mouse myotubes, we found that either pharmacological inhibition or siRNA silencing of p38 impaired caffeine-induced Ca2+ release from wild type cells while promoting intracellular Ca2+ release in Ryr1 knockout cells. Lastly, we demonstrated that p38 inhibition blunts the aberrant temperature-dependent increase in resting Ca2+ in myotubes from an RYR1-RM mouse model. This unique platform for RYR1-RM therapy development is potentially applicable to a broad range of neuromuscular disorders.

Muscle cells have storage compartments stuffed full of calcium, which they release to trigger a contraction. This process depends on a channel-shaped protein called the ryanodine receptor, or RYR1 for short. When RYR1 is activated, it releases calcium from storage, which floods the muscle cell. Mutations in the gene that codes for RYR1 in humans cause a group of rare diseases called RYR1-related myopathies. The mutations change calcium release in muscle cells, which can make movement difficult, and make it hard for people to breathe. At the moment, RYR1 myopathies have no treatment. It is possible that repurposing existing drugs could benefit people with RYR1-related myopathies, but trialing treatments takes time. The fastest and cheapest way to test whether compounds might be effective is to try them on very simple animals, like nematode worms. But even though worms and humans share certain genes, treatments that work for worms do not always work for humans. Luckily, it is sometimes possible to test whether compounds might be effective by trying them out on complex mammals, like mice. Unfortunately, these experiments are slow and expensive. A compromise involves testing on animals such as zebrafish. So far, none of these methods has been successful in discovering treatments for RYR1-related myopathies. To maximize the strengths of each animal model, Volpatti et al. combined them, developing a fast and powerful way to test new drugs. The first step is an automated screening process that trials thousands of chemicals on nematode worms. This takes just two weeks. The second step is to group the best treatments according to their chemical similarities and test them again in zebrafish. This takes a month. The third and final stage is to test promising chemicals from the zebrafish in mouse muscle cells. Of the thousands of compounds tested here, one group of chemicals stood out ­ treatments that block the activity of a protein called p38. Volpatti et al. found that blocking the p38 protein, either with drugs or by inactivating the gene that codes for it, changed muscle calcium release. This suggests p38 blockers may have potential as a treatment for RYR1-related myopathies in mammals. Using three types of animal to test new drugs maximizes the benefits of each model. This type of pipeline could identify new treatments, not just for RYR1-related myopathies, but for other diseases that involve genes or proteins that are similar across species. For RYR1-related myopathies specifically, the next step is to test p38 blocking treatments in mice. This could reveal whether the treatments have the potential to improve symptoms.

The marginal cells of the Caenorhabditis elegans pharynx scavenge cholesterol and other hydrophobic small molecules.

The nematode Caenorhabditis elegans is a bacterivore filter feeder. Through the contraction of the worm's pharynx, a bacterial suspension is sucked into the pharynx's lumen. Excess liquid is then shunted out of the buccal cavity through ancillary channels made by surrounding marginal cells. We find that many worm-bioactive small molecules (a.k.a. wactives) accumulate inside of the marginal cells as crystals or globular spheres. Through screens for mutants that resist the lethality associated with one crystallizing wactive we identify a presumptive sphingomyelin-synthesis pathway that is necessary for crystal and sphere accumulation. We find that expression of sphingomyelin synthase 5 (SMS-5) in the marginal cells is not only sufficient for wactive accumulation but is also important for absorbing exogenous cholesterol, without which C. elegans cannot develop. We conclude that sphingomyelin-rich marginal cells act as a sink to scavenge important nutrients from filtered liquid that might otherwise be shunted back into the environment.

Systematic pharmacological screens uncover novel pathways involved in cerebral cavernous malformations.

Cerebral cavernous malformations (CCMs) are vascular lesions in the central nervous system causing strokes and seizures which currently can only be treated through neurosurgery. The disease arises through changes in the regulatory networks of endothelial cells that must be comprehensively understood to develop alternative, non-invasive pharmacological therapies. Here, we present the results of several unbiased small-molecule suppression screens in which we applied a total of 5,268 unique substances to CCM mutant worm, zebrafish, mouse, or human endothelial cells. We used a systems biology-based target prediction tool to integrate the results with the whole-transcriptome profile of zebrafish CCM2 mutants, revealing signaling pathways relevant to the disease and potential targets for small-molecule-based therapies. We found indirubin-3-monoxime to alleviate the lesion burden in murine preclinical models of CCM2 and CCM3 and suppress the loss-of-CCM phenotypes in human endothelial cells. Our multi-organism-based approach reveals new components of the CCM regulatory network and foreshadows novel small-molecule-based therapeutic applications for suppressing this devastating disease in patients.

The novel nematicide wact-86 interacts with aldicarb to kill nematodes.

Parasitic nematodes negatively impact human and animal health worldwide. The market withdrawal of nematicidal agents due to unfavourable toxicities has limited the available treatment options. In principle, co-administering nematicides at lower doses along with molecules that potentiate their activity could mitigate adverse toxicities without compromising efficacy. Here, we screened for new small molecules that interact with aldicarb, which is a highly effective treatment for plant-parasitic nematodes whose toxicity hampers its utility. From our collection of 638 worm-bioactive compounds, we identified 20 molecules that interact positively with aldicarb to either kill or arrest the growth of the model nematode Caenorhabditis elegans. We investigated the mechanism of interaction between aldicarb and one of these novel nematicides called wact-86. We found that the carboxylesterase enzyme GES-1 hydrolyzes wact-86, and that the interaction is manifested by aldicarb's inhibition of wact-86's metabolism by GES-1. This work demonstrates the utility of C. elegans as a platform to search for new molecules that can positively interact with industrial nematicides, and provides proof-of-concept for prospective discovery efforts.

Ancestral morphology of crown-group molluscs revealed by a new Ordovician stem aculiferan.

Exceptionally preserved fossils provide crucial insights into extinct body plans and organismal evolution. Molluscs, one of the most disparate animal phyla, radiated rapidly during the early Cambrian period (approximately 535-520 million years ago (Ma)). The problematic fossil taxa Halkieria and Orthrozanclus (grouped in Sachitida) have been assigned variously to stem-group annelids, brachiopods, stem-group molluscs or stem-group aculiferans (Polyplacophora and Aplacophora), but their affinities have remained controversial owing to a lack of preserved diagnostic characters. Here we describe a new early sachitid, Calvapilosa kroegeri gen. et sp. nov. from the Fezouata biota of Morocco (Early Ordovician epoch, around 478 Ma). The new taxon is characterized by the presence of a single large anterior shell plate and polystichous radula bearing a median tooth and several lateral and uncinal teeth in more than 125 rows. Its flattened body is covered by hollow spinose sclerites, and a smooth, ventral girdle flanks an extensive mantle cavity. Phylogenetic analyses resolve C. kroegeri as a stem-group aculiferan together with other single-plated forms such as Maikhanella (Siphogonuchites) and Orthrozanclus; Halkieria is recovered closer to the aculiferan crown. These genera document the stepwise evolution of the aculiferan body plan from forms with a single, almost conchiferan-like shell through two-plated taxa such as Halkieria, to the eight-plated crown-group aculiferans. C. kroegeri therefore provides key evidence concerning the long debate about the crown molluscan affinities of sachitids. This new discovery strongly suggests that the possession of only a single calcareous shell plate and the presence of unmineralised sclerites are plesiomorphic (an ancestral trait) for the molluscan crown.

Morphological Asymmetry of the Superior Cervical Facets from C3 through C7 due to Degeneration.

INTRODUCTION: Knowledge about facet morphology has already been discussed extensively in literature but is limited regarding asymmetry and its relation to facet degeneration. METHOD: Facet dimensions, surface area, curvature, and degeneration of the superior facets were measured in 85 dried human vertebrae from the anatomical collection of the Vrije Universiteit Brussel. The vertebrae were analysed using the Microscribe G2X digitizer (Immersion Co., San Jose, CA) and a grading system for the evaluation of cervical facet degeneration. Coordinates were processed mathematically to evaluate articular tropism. The statistical analysis includes the paired t-test and the Pearson correlation. RESULTS: On average, no systematic differences between the left and right facets were found concerning morphology and degeneration. However, there were significant differences regardless of the side-occurrence. There was a significant correlation between the dimensions of the total facet surface and the degree of degeneration but not for the recognizable joint surface. CONCLUSIONS: Facet tropism of the upper joint facets occurred often in the cervical spine but without side preference. A bigger difference in degeneration asymmetry was associated with a bigger difference in facet joint dimension asymmetry.

A validation study of a new instrument for low cost bite force measurement.

Quantitative assessment of force in masticatory muscles is not a routine clinical test, probably due to the lack of an "easy-to-use" device. Aim of this study is (1) to present a low cost bite force instrument located in a custom-made housing, designed to guarantee a comfortable and effective bite action, (2) to evaluate its mechanical characteristics, in order to implement it in clinical settings and in experimental setups. Linearity, repeatability and adaptation over time were assessed on a set of four different sensors in bare and housed condition. Application of the housing to the transducer may appreciably alter the transducer's response. Calibration of the housed transducer is thus necessary in order to correctly record real bite force. This solution may represent a low cost and reliable option for biting force measurement and objective assessment of individual force control in the scientific and clinical setting.