71 Papers found (0.01 seconds)

Burns, Andrew et al. (2011) International Worm Meeting "An RNAi screen for defecation mutants."

Bennett, Jessica, Burns, Andrew, Porscher, Latarsha, Immerman, Lois, McCright, Samuel, Peters, Maureen, Miller, Matthew, Mullan, Hillary, Raible, Monica

[

International Worm Meeting,

2011]

Defecation in C. elegans is a highly regulated process that takes place approximately every 50 seconds in a feeding animal. The cycle is composed of three distinct muscle contractions: a posterior body contraction, an anterior body contraction, and an enteric muscle contraction. This one-minute biological rhythm is coordinated by a multi-tissue signaling system that utilizes several signaling molecules including calcium, protons, peptides, and classical neurotransmitters. The cycle's timekeeping mechanism is located in the intestine and requires calcium flux. Proton exchange at the intestinal membrane elicits contraction of the overlying body wall muscles. The subsequent contractions, anterior body and enteric muscle contractions, require the release of either peptidergic signals or a combination of peptidergic and GABAergic signals from neurons. Such signaling components have been identified primarily by forward genetic mutations that affect individual or multiple muscle contractions and/or cycle timing. Since these forward genetic screens have not been saturated, other critical components of the defecation cycle remain to be discovered. A reverse genetic RNA interference (RNAi) screen may permit the identification of additional defecation cycle components. RNAi is induced in eri-1(mg366) animals by feeding bacterial strains from the ORFeome RNAi library. Animals are treated and grown on NGM plates with a transcriptional inducer present. F1 progeny are visually scored using a standard dissecting microscope. The primary phenotype of interest is constipation as evidenced by a distended intestinal lumen. Other traits are also noted. The swollen intestinal lumen is indicative of constipation and defective defecation and is common to cycle timing and contraction mutants. Approximately 6,000 of the approximately 11,000 genes in this RNAi library have been scored to date. Thirty-three gene knockdowns have resulted in a constipated phenotype. Moreover, our study has verified genes known to affect cycle timing, posterior body contraction, and joint processes required for both anterior and enteric body contraction. This provides an independent confirmation of our screening procedure, as it has identified successfully known defecation cycle mutants. New defecation mutants have been discovered. The majority of our constipated phenotypes can be attributed to knockdowns of genes not previously shown to act in defecation. Further analysis of candidate defecation genes will provide a greater understanding of the cell and molecular pathways governing defecation.

Burns, Andrew et al. (2021) International Worm Meeting "Selective Control of Parasitic Nematodes with Bioactivated Nematicides"

[

International Worm Meeting,

2021]

Global food security is threatened as the world amasses 10 billion people amid limited arable land. While nematode pests are a major barrier to agricultural intensification, most traditional nematicides are now banned because of poor nematode-selectivity, leaving farmers with inadequate controls. Here, we describe a screen carried out in the model nematode Caenorhabditis elegans that enriches for selective nematicides by identifying molecules that are bioactivated by cytochrome P450s, which are phylogenetically diverse. We identify a family of structures, called nemactivins, that are robustly bioactivated to a toxic metabolite selectively in nematodes. At low parts-per-million concentrations, nemactivins perform comparably well with commercial nematicides at controlling infection by the world's most destructive plant-parasitic nematode Meloidogyne incognita. Hence, nemactivins are first-in-class bioactivated nematicides that provide much needed nematode-selectivity.

Burns, Andrew R. et al. (2015) International Worm Meeting "Caenorhabditis elegans is a useful model for anthelmintic discovery."

[

International Worm Meeting,

2015]

Parasitic nematodes infect one quarter of the world's population and impact all humans through widespread infection of crops and livestock. Resistance to current anthelmintics has prompted the search for new drugs. Traditional screens that rely on parasitic worms are costly and labor intensive, and target-based approaches have failed to yield novel anthelmintics. Here, we present our screen of 67,012 compounds to identify those that kill the non-parasitic nematode C. elegans. We rescreened our hits in two parasitic nematode species and two vertebrate models, and identified 30 structurally distinct anthelmintic lead molecules. Genetic screens of 19 million C. elegans mutants reveal those nematicides for which the generation of resistance is and is not likely. We discovered the target of one lead with nematode specificity and nanomolar potency as complex II of the electron transport chain. This work establishes C. elegans as an effective and cost-efficient model system for anthelmintic discovery.

Burns, Andrew R. et al. (2011) International Worm Meeting "Samamide is a new small-molecule probe of behavioural quiescence in C. elegans."

Hampson, David R., Roy, Peter J., Tharmalingalam, Suji, Burns, Andrew R., Ryu, William S., Cutler, Sean R.

[

International Worm Meeting,

2011]

C. elegans displays at least three distinct behavioural quiescent states. First, a sleep-like state is induced by epidermal growth factor signalling before each larval molt. Second, satiation induces a quiescent state that is regulated by both TGF-b and insulin signalling. Finally, adult worms cycle through periods of quiescence after prolonged swimming. In all three cases, the C. elegans cGMP-dependent protein kinase (PKG) homolog egl-4 promotes quiescence. It has been shown that increased PKG activity in Drosophila promotes sleep, and PKG1 inhibition in mouse brain alters the sleep-wake distribution, suggesting phylogenetic conservation of factors that regulate sleep and perhaps behavioural quiescence in general. In a screen of 4,000 drug-like chemicals we identified a previously uncharacterized molecule that we hypothesize is promoting quiescence during swimming. We call this molecule 'samamide'. Reduced swimming activity is observed in adult worms treated acutely with samamide, suggesting that the chemical alters the physiological state of the animal. Chemical-genetic analyses suggest that samamide promotes quiescence by modulating metabotropic glutamate receptor activity. Samamide holds promise as a unique tool to better understand behavioural quiescence in animals.

Andrew Burns et al. (2006) Development & Evolution Meeting "Xenobiotic Uptake and Metabolism in C. elegans"

Peter Roy, Andrew Burns, Sean Cutler

[

Development & Evolution Meeting,

2006]

Parasitic nematode worms present a serious health concern to humans and other agriculturally important animals. There are many nematicides used to treat parasitic infection, but resistance to these compounds is a growing problem. Work from our lab has shown that drug uptake is necessary for observable bioactivity in C. elegans. Resistance may therefore be due to detoxifying proteins such as drug-metabolizing enzymes and xenobiotic efflux pumps that prevent drug uptake. The majority of these detoxifiers remain largely uncharacterized in C. elegans. For example, Cytochrome p450 (Cyp) enzymes are a major class of Phase I metabolic enzymes. There are approximately 80 putative Cyp genes in worms. Some of these genes have shown homology to mammalian drug-metabolizing Cyps, but all 80 remain largely uncharacterized. To assay drug uptake and metabolism in C. elegans we have developed a high-throughput high performance liquid chromatography (HT-HPLC) protocol. In brief, N2 worms are incubated in a variety of small lipophilic molecules, washed, lysed, and their contents are separated using HPLC. Drug uptake and metabolism can be directly visualized and quantified. The metabolites can be purified and analyzed by mass spectrometry to determine the modification that has been made. We are currently in the process of assaying ~2000 pharmacologically active small molecules. So far our work has shown that only a small fraction (~20%) of these small molecules are taken up to an observable extent by N2 worms, and of these about 10% are metabolized. The same HT-HPLC analysis will be performed on HA and 12 other wild-type isolates of C. elegans. Any differences between the isolates with respect to drug uptake and metabolism will be noted, and the genes involved will be mapped genetically and characterized. These analyses will lead to a better understanding of the basic mechanisms of xenobiotic detoxification in a whole animal model system. It is likely that this novel whole animal approach to studying drug uptake and metabolism may uncover new mechanisms of drug resistance and detoxification that would otherwise be missed in cell- or tissue-based assays. Differences between isolates will provide insight into C. elegans intra-species pharmacogenetic variation, and will highlight the ways in which segregated wild-type worm populations have developed diverse mechanisms of xenobiotic detoxification.

Andrew Burns et al. (2008) C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting "A Predictive Model for Small Molecule Accumulation and Bioactivity in C. elegans"

Mike TYERS, Iain Wallace, Jan Wildenhain, Corey Nislow, Guri Giaever, Peter Roy, Sean Cutler, Andrew Burns

[

C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting,

2008]

We are using C. elegans as a model system to discover new small molecule tools for biological analyses. One obstacle to finding such tools is the large number of xenobiotic defense factors encoded by the worm genome (Lindblom et al., 2006). For example, in a screen of 3,265 small molecules that are bioactive in other systems, we found only 64 (2%) that induce robust phenotypes in the worm (Kwok et al., 2006). We hypothesized that this hit rate is relatively low because most small molecules fail to accumulate in worms. To test this, we developed an HPLC-based assay to measure small molecule accumulation in worm tissue. We surveyed 1,018 compounds from the Spectrum library (Microsource Inc.) for accumulation in whole worms after 6 hours of incubation in 40uM of the small molecules. To ensure confidence in our assignments we established a detection limit of 18uM. Of the 361/1018 compounds that satisfied this criterion, only 25 (6.9%) accumulate in worms. Notably, 2 out of 25 accumulating molecules induce robust phenotype in the worm, compared to 0 out of 336 non-accumulating molecules. We also assayed 25 nematicides obtained from our other small molecule screens, and found that 17 (68%) accumulate. These results support our hypothesis that worms are generally resistant to small molecule accumulation, and show that accumulating small molecules are enriched for bioactivity in C. elegans. Next, the accumulation of an additional 77 compounds from our other screens was assayed, for a complete dataset of 463 small molecules. We used this dataset to build a predictive structure-based model of accumulation. We compiled 4,166 structural descriptors of the small molecules in our dataset, and built a model using a Bayesian Classifier machine learning method. Five-fold cross validation of the model estimates a prediction accuracy of 75.36 +/- 2.04%. We used the model to rank 9,742 compounds of a DIVERSet library (Chembridge Corp.), of which 48 induce robust phenotype in the worm. Encouragingly, 12 of the top-scoring 200 molecules induce phenotype, representing a 12.2-fold enrichment for bioactivity compared to the entire library (p10). None of the bottom-scoring 200 molecules induce phenotype. These data demonstrate that our model is effective at predicting small molecule accumulation and bioactivity in C. elegans. We hope to use this model to increase our efficiency at identifying new small molecule probes for biological analysis, and to aid the development of potential drug leads using C. elegans as a model.

Andrew R. Burns et al. (2007) International Worm Meeting "A predictive model for drug accumulation in Caenorhabditis elegans."

Jan Wildenhain, Mike TYERS, Andrew R. Burns, Sean R. Cutler, Peter J. Roy

[

International Worm Meeting,

2007]

The C. elegans genome encodes an expansive array of potential xenobiotic defense factors that likely help the worm survive environmental toxins. As a consequence, the worm is relatively resistant to xenobiotic perturbation in a laboratory setting. For example, we surveyed over 14,000 small molecules for the induction of obvious phenotypes in the worm. The fraction of compounds with previously characterized bioactivity in other systems that induced phenotype in worms (3.2%, n=3280) is similar to that of 11,000 compounds without documented bioactivity (2.2%). We hypothesized that the low hit rate for the known bioactive compounds is because the worm is generally resistant to small molecule accumulation. To investigate xenobiotic retention in worms, we developed a high-throughput high performance liquid chromatography (HTP-HPLC) assay. We analyzed 1,110 molecules of the Spectrum library (Microsource Inc.) and found that only 177 (16%) are retained. Of these, 15.7% induce phenotype, representing a four-fold enrichment for molecules with bioactivity in worms (p<1.6E-13). These results support our hypothesis that C. elegans is generally resistant to small molecule accumulation. Next, we used our dataset of retained and non-retained molecules to build a model that predicts small molecule accumulation in the tissues of C. elegans. Such a model would allow us, and others, to screen small molecule libraries with greater efficiency, as robust retention is a key prerequisite for robust target inhibition. We compiled over 6,000 physicochemical descriptors of small molecules, and have identified over 100 that distinguish the 177 retained molecules from those not retained. Based on this analysis we built a Bayesian model of retention and used this model to rank 1258 molecules of the LOPAC library (Sigma). The top 50 molecules predicted to be retained are 4.4-fold enriched for molecules that induce phenotype in the worm (p<6.9E-4). We then analyzed the retention of the top 50 and bottom 50 ranked molecules, and found that 48% of the top-ranked molecules are retained in worm tissue. In contrast, only 8% of the bottom-ranked molecules are retained. This demonstrates that we have developed a good model to predict small molecule retention in C. elegans. Our model will hopefully facilitate the design of new nematicides to treat parasitic nematode infections, aid in the design of novel drug leads using C. elegans as a model for disease, and expand the use of small molecules as tools to probe worm biology.

Fire A et al. (1994) Worm Breeder's Gazette "Vectorology I: New lacZ Vectors (Building a Better Gene Trap)."

Fire A, Xu SQ

[

Worm Breeder's Gazette,

1994]

Vectorology I: New lacZ vectors ("building a better gene trap") Andrew Fire and SiQun Xu Carnegie Institution of Washington, Baltimore, Md 21210

Mehra A et al. (1994) Worm Breeder's Gazette "Direct Interaction Between FEM-3 and a Carboxy-Terminal Fragment of TRA-2A"

Spence AM, Heck L, Kuwabara PE, Mehra A

[

Worm Breeder's Gazette,

1994]

Direct Interaction between FEM-3 and a Carboxy-Terminal Fragment Or TRA-2A Arun Mehra, Linda Heck, Patricia Kuwabara*, and Andrew Spence Dept. of Medical Genetics, University of Toronto, 1 King's College Circle, Toronto, Canada, and *MRC Laboratory of Molecular Biology, Hills Rd., Cambridge, UK

Vairoletti, Franco et al. (2021) MicroPubl Biol

Vairoletti, Franco, Salinas, Gustavo, Mahler, Graciela, Saiz, Cecilia, Baron, Antonela

[

MicroPubl Biol,

2021]

Parasitic nematodes constitute a health problem for humans, livestock and crops, and cause huge economic losses to developing-country economies. Due to the spread of nematicide resistance, there is an urgent need for new drugs. C. elegans is now recognized as a cost-effective alternative for the screening of compound libraries with potential nematicidal activity, as parasitic organisms are hard to maintain under laboratory conditions. Here we describe an adaptation of a previously reported high throughput (HTP) infrared-based motility assay that leads to increased sensitivity. The modified assay uses L1 instead of L4 stage worms and matches the sensitivity reported by Burns et al. (2015) for the anthelmintic benzamides Wact11 and Wact11p. In addition, this method presents practical advantages over Burns et al. (2015) and other image-based protocols and provides a robust assay with a fast and simple readout ideal for HTP drug discovery.

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