Olmedo M et al. (2009) European Worm Neurobiology Meeting "The circadian clock in C. elegans"

Olmedo M, Merrow M, van der Pol M

[European Worm Neurobiology Meeting, 2009]

Circadian clocks are present in most organisms from cyanobacteria to humans. Circadian rhythms have a period of about 24 hours in constant conditions and are entrained to the 24-hours day by environmental signals (zeitgebers). In entrainment, clocks organize gene expression and metabolism to anticipate the daily changes of the environment. Several reports have shown evidence of a circadian clock in Caenorhabditis elegans, using either activity or resistance to osmotic shock. We find these nematode circadian phenotypes subtle so we are working to develop a more efficient system for circadian experimentation with these animals. A fundamental property of circadian clocks is their pervasive regulation of physiology. We thus adapted a chemotaxis assay based on olfaction to monitor changes in olfactory sensitivity over the day. Age-matched adult worms were raised in entraining temperature cycles. They were exposed to the repellant 1-octanol for 15-min, a sub-threshold condition. We find that 1-octanol is an effective repellant for C. elegans in presence of E. coli. Preliminary results show a rhythmic response pattern to 1-octanol over the day.

Olmedo M et al. (2015) Genetics "A High-Throughput Method for the Analysis of Larval Developmental Phenotypes in Caenorhabditis elegans."

Olmedo M, Artal-Sanz M, Merrow M, Geibel M

[Genetics, 2015]

Caenorhabditis elegans postembryonic development consists of four discrete larval stages separated by molts. Typically, the speed of progression through these larval stages is investigated by visual inspection of the molting process. Here, we describe an automated method to monitor the timing of these discrete phases of C. elegans maturation, from the first larval stage through adulthood, using bioluminescence. The method was validated with a lin-42 mutant strain that shows delayed development relative to wild-type animals and with a daf-2 mutant that shows an extended second larval stage. This new method is inherently high-throughput and will finally allow dissecting the molecular machinery governing the speed of the developmental clock, which has so far been hampered by the lack of a method suitable for genetic screens.

Olmedo M et al. (2017) Adv Genet "Sleeping Beauty? Developmental Timing, Sleep, and the Circadian Clock in Caenorhabditis elegans."

Merrow M, Geibel M, Olmedo M

[Adv Genet, 2017]

The genetics toolkit is pretty successful in drilling down into minutiae. The big challenge is to integrate the information from this specialty as well as those of biochemistry, physiology, behavior, and anatomy to explain how fundamental biological processes really work. Sleep, the circadian clock and development all qualify as overarching processes that encompass levels from molecule to behavior as part of their known mechanisms. They overlap each other, such that understanding the mechanisms of one can lead to insights into one of the others. In this essay, we consider how the experimental approaches and findings relating to Caenorhabditis elegans development and lethargus on one hand, and to the circadian clock and sleep in higher organisms on the other, could complement and enhance one another.

Olmedo M et al. (2012) Proc Natl Acad Sci U S A "Circadian regulation of olfaction and an evolutionarily conserved, nontranscriptional marker in Caenorhabditis elegans."

Edgar RS, Valekunja UK, O'Neill JS, Merrow M, Olmedo M, Reddy AB

[Proc Natl Acad Sci U S A, 2012]

Circadian clocks provide a temporal structure to processes from gene expression to behavior in organisms from all phyla. Most clocks are synchronized to the environment by alternations of light and dark. However, many organisms experience only muted daily environmental cycles due to their lightless spatial niches (e.g., caves or soil). This has led to speculation that they may dispense with the daily clock. However, recent reports contradict this notion, showing various behavioral and molecular rhythms in Caenorhabditis elegans and in blind cave fish. Based on the ecology of nematodes, we applied low-amplitude temperature cycles to synchronize populations of animals through development. This entrainment regime reveals rhythms on multiple levels: in olfactory cued behavior, in RNA and protein abundance, and in the oxidation state of a broadly conserved peroxiredoxin protein. Our work links the nematode clock with that of other clock model systems; it also emphasizes the importance of daily rhythms in sensory functions that are likely to impact on organism fitness and population structure.

Olmedo M et al. (2019) Aging Cell "Prolonged quiescence delays somatic stem cell-like divisions in Caenorhabditis elegans and is controlled by insulin signaling."

Jesus Rodriguez-Palero M, Merrow M, Fernandez-Yanez A, Olmedo M, Artal-Sanz M, Garcia-Sanchez S, Mata-Cabana A

[Aging Cell, 2019]

Cells can enter quiescence in adverse conditions and resume proliferation when the environment becomes favorable. Prolonged quiescence comes with a cost, reducing the subsequent speed and potential to return to proliferation. Here, we show that a similar process happens during Caenorhabditis elegans development, providing an in vivo model to study proliferative capacity after quiescence. Hatching under starvation provokes the arrest of blast cell divisions that normally take place during the first larval stage (L1). We have used a novel method to precisely quantify each stage of postembryonic development to analyze the consequences of prolonged L1 quiescence. We report that prolonged L1 quiescence delays the reactivation of blast cell divisions in C. elegans, leading to a delay in the initiation of postembryonic development. The transcription factor DAF-16/FOXO is necessary for rapid recovery after extended arrest, and this effect is independent from its role as a suppressor of cell proliferation. Instead, the activation of DAF-16 by decreased insulin signaling reduces the rate of L1 aging, increasing proliferative potential. We also show that yolk provisioning affects the proliferative potential after L1 arrest modulating the rate of L1 aging, providing a possible mechanistic link between insulin signaling and the maintenance of proliferative potential. Furthermore, variable yolk provisioning in embryos is one of the sources of interindividual variability in recovery after quiescence of genetically identical animals. Our results support the relevance of L1 arrest as an in vivo model to study stem cell-like aging and the mechanisms for maintenance of proliferation potential after quiescence.

Olmedo, M. et al. (2017) International Worm Meeting "Larval density during L1 arrest impacts recovery from starvation through changes in DAF-16 nuclear localization."

Olmedo, M., Artal-Sanz, M., Cerero, L., Mata-Cabana, A., Merrow, M., Fernandez-Yanez, A., Rodriguez-Palero, M. J.

[International Worm Meeting, 2017]

The developmental progression of Caenorhabditis elegans can be slowed down or interrupted when animals face unfavourable conditions like food scarcity. C. elegans larvae arrest as L1s when hatching in the absence of food. Arrested L1s can survive several weeks without food and present increased resistance to stress. Several genetic and environmental factors impact the survival of arrested L1s. These include the Insulin/insulin-like growth factor signalling (IIS) pathway and larval density during arrest 1,2. The effect of density is mediated by exposure of unknown compounds secreted after hatching 2. When food becomes available, larvae resume postembryonic development but animals that have been starved for long periods of time take longer to reach adulthood 3. This effect of starvation was attributed to a developmental delay after extended starvation. However, using a method we developed to measure developmental timing 4 we have been able to measure the duration of each stage of development after extended starvation. We have observed that developmental speed is resilient to time in starvation. The later entry into adulthood after extended arrest corresponds, instead, with a delay to resume development. That is, extended starvation increases recovery time. Using the same method, we observe that recovery time is affected by interventions that affect survival to starvation, like mutations in the IIS pathway and density of animals during arrest. In addition, we show that high density of animals contributes to the maintenance of DAF-16 nuclear localization during arrest, contributing to longer survival and fast recovery from arrest. Our results indicate that density and low insulin signalling impact DAF-16 function in a similar manner, possibly allowing integration of the two signals to control developmental arrest. 1. Baugh, L. R. To Grow or Not to Grow: Nutritional Control of Development During Caenorhabditis elegans L1 Arrest. Genetics 194, (2013). 2. Artyukhin, A. B., Schroeder, F. C. & Avery, L. Density dependence in Caenorhabditis larval starvation. (2013). 3. Lee, I., Hendrix, A., Kim, J., Yoshimoto, J. & You, Y.-J. Metabolic Rate Regulates L1 Longevity in C. elegans. (2012). 4. Olmedo, M., Geibel, M., Artal-Sanz, M. & Merrow, M. A High-Throughput Method for the Analysis of Larval Developmental Phenotypes in Caenorhabditis elegans. Genetics (2015).

Wypijewska A et al. (2012) Biochemistry "7-methylguanosine diphosphate (m(7)GDP) is not hydrolyzed but strongly bound by decapping scavenger (DcpS) enzymes and potently inhibits their activity."

Wypijewska A, Darzynkiewicz E, Davis RE, Jemielity J, Bojarska E, Lukaszewicz M, Stepinski J

[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.

O'Connell EM et al. (2015) J Infect Dis "Targeting Filarial Abl-like Kinases: Orally Available, Food and Drug Administration-Approved Tyrosine Kinase Inhibitors Are Microfilaricidal and Macrofilaricidal."

Nutman TB, O'Connell EM, Bennuru S, Steel C, Dolan MA

[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.

Wood WB (1976) Worm Breeder's Gazette "maternal effects among ts zygote-defective (zyg) mutants."

Wood WB

[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.

Ali Khan L et al. (1994) Worm Breeder's Gazette "cej-1 Encodes a Novel Protein With Poly-Threonine Motif"

Siddiqui SS, Fukushige T, Ali Khan L, Tsukita Sh, Tabish M, Itoh M

[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.