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Driscoll M, Phillips PC, Coleman-Hulbert AL, Sedore CA, Melentijevic I, Falkowski R, Banse SA, Abbott M, Ange JS, Blue BW, Guo M, Jarrett CM, Johnson E, Lithgow GJ
[
J Biol Methods,
2020]
<i>Caenorhabditis elegans</i> (<i>C. elegans</i>) lifespan assays constitute a broadly used approach for investigating the fundamental biology of longevity. Traditional <i>C. elegans</i> lifespan assays require labor-intensive microscopic monitoring of individual animals to evaluate life/death over a period of weeks, making large-scale high throughput studies impractical. The lifespan machine developed by Stroustrup <i>et al</i>. (2013) adapted flatbed scanner technologies to contribute a major technical advance in the efficiency of <i>C. elegans</i> survival assays. Introducing a platform in which large portions of a lifespan assay are automated enabled longevity studies of a scope not possible with previous exclusively manual assays and facilitated novel discovery. Still, as initially described, constructing and operating scanner-based lifespan machines requires considerable effort and expertise. Here we report on design modifications that simplify construction, decrease cost, eliminate certain mechanical failures, and decrease assay workload requirements. The modifications we document should make the lifespan machine more accessible to interested laboratories.
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[
International Worm Meeting,
2019]
RNA interference is a powerful tool for the dissection of fundamental mechanisms in C. elegans. Past whole-genome RNAi screens in C. elegans have provided fascinating insights into nematode biology, however, manual screening protocols can be prohibitively resource- and time-intensive. We have developed a highly automated, high-throughput whole-genome RNAi screening platform that can be implemented at a fraction of the time and cost of manual screens. Our screening protocol employs robotics, coupled with a high content imaging system. Unlike other automated systems, our platform utilizes solid media instead of liquid media for worm growth, thus avoiding the stresses induced by a continuous swimming environment. Multiple fluorescent channels and pattern recognition algorithms enable the analysis of multiple parameters simultaneously. Our methods for animal growth and imaging enable screening of the entire genome in approximately one week, fast enough to make epistasis studies routine and feasible. We elected to run trials based on the expression levels of Pcysl-2GFP.
cysl-2 is involved in the hydrogen cyanide assimilation pathway. It is also directly upregulated by
hif-1 and is therefore a good surrogate for hypoxia experiments. The Pcysl-2GFP reporter had the advantage of an easy to measure phenotype with a well characterized pathway to confirm results. We completed trials on known regulators and 3X whole genome repeat trials for modulators of a Pcysl-2GFP reporter strain to identify genes that affect
cysl-2 expression. We then rescreened positive RNAi clones using a
hif-1 knockout strain to determine
hif-1 dependency. Our development work established proof-of-principle for primary and epistasis screens using this technology. We will show how measures of fertility levels, survivability and brood size scores can be readily executed with our platform. Our current experiments are focused on a genome wide-screens for modifiers of a higher-resolution and more challenging phenotype-the neuronal production of exophers, large membrane-enclosed extrusions that can mediate expulsion of protein aggregates and damaged organelles.1 This project is of potential high importance as the biology of aggregate extrusion is largely uncharacterized and the process of aggregate transfer is prevalent in human neurodegenerative disease. Of general technical interest, this screen features detection of sub-cellular size structures. We will also present data on screens for exopher modulators. 1. C. elegans Neurons Jettison Protein Aggregates and Mitochondria Under Neurotoxic Stress Ilija Melentijevic, et. Al. Nature. 2017 Feb 16; 542(7641): 367-371.
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St. Ange, J., Anusionwu, I., Parekh, M., Melentijevic, I., Guasp, R., Driscoll, M., Abbott, M., Szot, M., Wang, G.
[
International Worm Meeting,
2019]
RNA interference is a powerful tool for the dissection of fundamental mechanisms in C. elegans. Past whole-genome RNAi screens in C. elegans have provided fascinating insights into nematode biology, however, manual screening protocols can be prohibitively resource- and time-intensive. We have developed a highly automated, high-throughput whole-genome RNAi screening platform that can be implemented at a fraction of the time and cost of manual screens. Our screening protocol employs robotics, coupled with a high content imaging system. Unlike other automated systems, our platform utilizes solid media instead of liquid media for worm growth, thus avoiding the stresses induced by a continuous swimming environment. Multiple fluorescent channels and pattern recognition algorithms enable the analysis of multiple parameters simultaneously. Our methods for animal growth and imaging enable screening of the entire genome in approximately one week, fast enough to make epistasis studies routine and feasible. We elected to run trials based on the expression levels of Pcysl-2GFP.
cysl-2 is involved in the hydrogen cyanide assimilation pathway.
cysl-2 is a cysteine synthase that converts hydrogen cyanide to hydrogen sulfide. It is also directly upregulated by
hif-1 and is therefore a good surrogate for hypoxia experiments. The Pcysl-2GFP reporter had the advantage of an easy to measure phenotype with a well characterized pathway to confirm results. We completed trials on known regulators and 3X whole genome repeat trials for modulators of a Pcysl-2GFP reporter strain to identify genes that affect
cysl-2 expression. We then rescreened positive RNAi clones using a
hif-1 knockout strain to determine
hif-1 dependency. We will report on methods development and biological findings of these screens using our platform.
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[
International Worm Meeting,
2017]
RNA interference is a powerful tool for the exploration of gene function in C. elegans. Past whole genome RNAi screens in C. elegans have provided fascinating insights into nematode biology, however, current screening protocols can be prohibitively resource and time intensive. We are developing a highly automated, high-throughput whole-genome RNAi screening platform that can be implemented at a fraction of the time and cost of manual screens. Our screening protocol employs robotic dispensers and aspirators, coupled with a high content imaging system for animals grown and measured in a 96-well plate format. Multiple fluorescent channels and pattern recognition algorithms enable the analysis of multiple parameters simultaneously. We have developed methods for animal growth and imaging that should enable screening of the entire genome in approximately one week, fast enough to make epistatic studies feasible. Our initial experiments investigate genes that influence production of exophers, large extracellular vesicles that can mediate expulsion of protein aggregates and damaged organelles from neurons1. If conserved, exophers may play important roles in neurodegenerative diseases characterized by protein aggregation, including Parkinson's and Alzheimer's. In our model, we track an aggregating mCherry protein that is expelled in exophers. The mCherry protein exits touch neurons as exopher cargo, transits through the hypodermis, and can later be found concentrated in coelomocytes. When coelomocyte or hypodermal uptake is genetically disrupted, fluorescent exopher contents accumulate in the body in a phenotype we call "starry night." We will present preliminary screening data and images demonstrating the viability of the technology to screen suppressors and enhancers of the starry night phenotype. 1 Melentijevic, I. et al. C. elegans neurons jettison protein aggregates and mitochondria under neurotoxic stress. Nature 542, 367-371, doi:10.1038/nature21362 (2017).
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[
Biochemistry,
2012]
Decapping scavenger (DcpS) enzymes catalyze the cleavage of a residual cap structure following 3' 5' mRNA decay. Some previous studies suggested that both m(7)GpppG and m(7)GDP were substrates for DcpS hydrolysis. Herein, we show that mononucleoside diphosphates, m(7)GDP (7-methylguanosine diphosphate) and m(3)(2,2,7)GDP (2,2,7-trimethylguanosine diphosphate), resulting from mRNA decapping by the Dcp1/2 complex in the 5' 3' mRNA decay, are not degraded by recombinant DcpS proteins (human, nematode, and yeast). Furthermore, whereas mononucleoside diphosphates (m(7)GDP and m(3)(2,2,7)GDP) are not hydrolyzed by DcpS, mononucleoside triphosphates (m(7)GTP and m(3)(2,2,7)GTP) are, demonstrating the importance of a triphosphate chain for DcpS hydrolytic activity. m(7)GTP and m(3)(2,2,7)GTP are cleaved at a slower rate than their corresponding dinucleotides (m(7)GpppG and m(3)(2,2,7)GpppG, respectively), indicating an involvement of the second nucleoside for efficient DcpS-mediated digestion. Although DcpS enzymes cannot hydrolyze m(7)GDP, they have a high binding affinity for m(7)GDP and m(7)GDP potently inhibits DcpS hydrolysis of m(7)GpppG, suggesting that m(7)GDP may function as an efficient DcpS inhibitor. Our data have important implications for the regulatory role of m(7)GDP in mRNA metabolic pathways due to its possible interactions with different cap-binding proteins, such as DcpS or eIF4E.
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[
J Infect Dis,
2015]
BACKGROUND: Elimination of onchocerciasis and lymphatic filariasis is targeted for 2020. Given the coincident Loa loa infections in Central Africa and the potential for drug resistance development, the need for new microfilaricides and macrofilaricides has never been greater. With the genomes of L. loa, Onchocerca volvulus, Wuchereria bancrofti, and Brugia malayi available, new drug targets have been identified. METHODS: The effects of the tyrosine kinase inhibitors imatinib, nilotinib, and dasatinib on B. malayi adult males, adult females, L3 larvae, and microfilariae were assessed using a wide dose range (0-100 M) in vitro. RESULTS: For microfilariae, median inhibitory concentrations (IC50 values) on day 6 were 6.06 M for imatinib, 3.72 M for dasatinib, and 81.35 M for nilotinib; for L3 larvae, 11.27 M, 13.64 M, and 70.98 M, respectively; for adult males, 41.6 M, 3.87 M, and 68.22 M, respectively; and for adult females, 42.89 M, 9.8 M, and >100 M, respectively. Three-dimensional modeling suggests how these tyrosine kinase inhibitors bind and inhibit filarial protein activity. CONCLUSIONS: Given the safety of imatinib in humans, plans are underway for pilot clinical trials to assess its efficacy in patients with filarial infections.
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[
Worm Breeder's Gazette,
1976]
We have studied maternal effects in 23 zyg ts mutants to estimate the times of expression of genes whose products are required in embryogenesis. We have used the following three tests, called arbitrarily A, B, and C. A test: Heterozygous (m/+) L4's are shifted to 25 C and allowed to self-fertilize. If 100% of their eggs yield larvae (25% of which express the mutant phenotype as adults), then the mutant is scored as maternal (M). If 25% of the F1 eggs fail to hatch, then the mutant is scored as non-maternal (N). An M result indicates that expression of the + allele in the parent allows m/m zygotes to hatch and grow to adulthood. A result of N indicates the opposite: that the + allele must be expressed in the zygote for hatching to occur. Out of 23 zyg mutants tested, 3 were scored N and 20 were scored M in the A test. Therefore, for most of the genes defined by these mutants, expression in the parent is sufficient for zygote survival, even if the gene is not expressed in the zygote. B test: Homozygous (m/m) hermaphrodites reared at 25 C are mated with N2 (+/+) males. If eggs fail to hatch at 25 C, but mated hermaphrodites shifted to 16 C produce cross progeny to give proof of mating, then the mutant is scored M. If cross progeny appear in the 25 C mating, then the mutant is scored N. An M result indicates that expression of the + allele in the zygote is not sufficient to allow m/+ progeny of an m/m hermaphrodite to survive. Conversely an N result indicates either that zygotic expression of the + allele is sufficient for survival, or that a sperm function or factor needed for early embryogenesis can be supplied paternally (see C test below). Out of the 23 zyg mutants tested, 11 were scored M and 12 were scored N. The combined results of A and B tests and their simplest interpretation are as follows. Ten mutants are M,M; the genes defined by these mutants must be expressed in the hermaphrodite parent for the zygote to survive. Ten mutants are M,N; these genes can be expressed either in the parent or in the zygote. Two mutants are N,N; these genes must be expressed in the zygote. One mutant is N,M; this gene must be expressed both in the maternal parent and in the zygote. C test: Homozygous (m/m) hermaphrodites reared at 25 C are mated with heterozygous (m/+) males. If rescue by a +/+ male in the B test depends on the + allele, then only half the cross progeny zygotes of a C test mating (m/+ male x m/m hermaphrodite) should survive. However, if rescue depends on a function or cytoplasmic component from the male sperm, then all the cross progeny zygotes in a C test should survive. Of the 10 M,N mutants, 6 have been C tested; one exhibited paternal rescue independent of the + allele. The A and B tests also were carried out on 16 mutants that arrest before the L3 molt (acc mutants). In the A test on 2 of these mutants, all m/m progeny of m/+ parents grew to adulthood at 25 C. Therefore, parental contributions are sufficient to overcome a progeny mutational block as late as the L2 stage. All 16 acc mutants scored N in the B test.
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[
Worm Breeder's Gazette,
1994]
cej-1 Encodes a Novel Protein with Poly-Threonine Motif M. L. A. Khanl, M. Tabish, T. Fukushigel1 S. Tsukita2, M. Itoh , Sh. Tsukita , and S. S. Siddiqui. (1): Lab. of Molecular Biology, Dept of Ecological Engg. Toyohashi Univ. Technology, Toyohashi 441, and (2). National Institute for Physiological Sciences, Okazaki 444, Japan.
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[
Mech Ageing Dev,
2009]
Energy production via oxidative phosphorylation generates a mitochondrial membrane potential (DeltaPsi(m)) across the inner membrane. In this work, we show that a lower DeltaPsi(m) is associated with increased lifespan in Caenorhabditis elegans. The long-lived mutants
daf-2(
e1370),
age-1(
hx546),
clk-1(
qm30),
isp-1(
qm150) and
eat-2(
ad465) all have a lower DeltaPsi(m) than wild type animals. The lower DeltaPsi(m) of
daf-2(
e1370) is
daf-16 dependent, indicating that the insulin-like signaling pathway not only regulates lifespan but also mitochondrial energetics. RNA interference (RNAi) against 17 genes shown to extend lifespan also decrease DeltaPsi(m). Furthermore, lifespan can be significantly extended with the uncoupler carbonylcyanide-3-chlorophenylhydrazone (CCCP), which dissipates DeltaPsi(m). We conclude that longevity pathways converge on the mitochondria and lead to a decreased DeltaPsi(m). Our results are consistent with the 'uncoupling to survive' hypothesis, which states that dissipation of the DeltaPsi(m) will extend lifespan.
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[
Arch Environ Contam Toxicol,
2005]
Fungi (Cunninghamella elegans ATCC 9245, Mucor ramannianus R-56, Aspergillus niger VKMF-1119, and Phanerochaete chrysosporium BKMF-1767) were tested to elucidate the biologic fate of the topical insect repellent N,N-diethyl-m-toluamide (DEET). The elution profile obtained from analysis by high-pressure liquid chromatography equipped with a reverse-phase C-18 column, showed that three peaks occurred after incubation of C. elegans, with which 1 mM DEET was combined as a final concentration. The peaks were not detected in the control experiments with either DEET alone or tested fungus alone. The metabolites produced by C. elegans exhibited a molecular mass of 207 with a fragment ion (m/z) at 135, a molecular mass of 179 with an m/z at 135, and a molecular mass of 163 with an m/z at 119, all of which correspond to N,N-diethyl-m-toluamide-N-oxide, N-ethyl-m-toluamide-N-oxide, and N-ethyl-m-toluamide, respectively. M. ramannianus R-56 also produced N, N-diethyl-m-toluamide-N-oxide and N-ethyl-m-toluamide but did not produce N-ethyl-m-toluamide-N-oxide. For the biologic toxicity test with DEET and its metabolites, the freshwater zooplankton Daphnia magna was used. The biologic sensitivity in decreasing order was DEET > N-ethyl-m-toluamide > N,N-diethyl-m-toluamide-N-oxide. Although DEET and its fungal metabolites showed relatively low mortality compared with other insecticides, the toxicity was increased at longer exposure periods. These are the first reports of the metabolism of DEET by fungi and of the biologic toxicity of DEET and its fungal metabolites to the freshwater zooplankton D. magna.