culturing-screening-plant-parasitic-nematode-ditylenchus

Culturing and Screening the Plant Parasitic Nematode <em>Ditylenchus dipsaci</em>

Plant-parasitic nematodes (PPNs) destroy over 12% of global food crops every year, which equates to roughly 157 billion dollars (USD) lost annually. With ...

University of Toronto

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  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  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  and  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.

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

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.

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

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  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 Ca release from wild type cells while promoting intracellular Ca release in  knockout cells. Lastly, we demonstrated that p38 inhibition blunts the aberrant temperature-dependent increase in resting Ca 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.

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

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.

Article	Year
Systematic pharmacological screens uncover novel pathways involved in cerebral cavernous malformations. EMBO molecular medicine\| PubMed ID: 30181117	2018
The marginal cells of the Caenorhabditis elegans pharynx scavenge cholesterol and other hydrophobic small molecules. Nature communications\| PubMed ID: 31477732	2019
Identification of drug modifiers for RYR1-related myopathy using a multi-species discovery pipeline. eLife\| PubMed ID: 32223895	2020
A survey of the kinome pharmacopeia reveals multiple scaffolds and targets for the development of novel anthelmintics. Scientific reports\| PubMed ID: 33911106	2021

Jessica Knox

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