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[
J Neurosci Methods,
2012]
Caenorhabditis elegans is a popular model organism to study how neural circuits and genes regulate behavior. To reliably correlate circuit function with behavior, it is important to record neuronal activity in freely behaving worms. As neural circuits are composed of multiple neurons that cooperate to process information, it is highly desirable to simultaneously record the activity of multiple neurons in the circuitry. However, such a system has not been available in C. elegans. Here, we report the CARIBN II (Calcium Ratiometric Imaging of Behaving Nematodes version II) system. This system provides smoother data collection and more importantly permits simultaneous imaging of calcium transients from multiple neurons in freely behaving worms. Using this system, we imaged the activity of AVA and RIM, two key neurons in the locomotion circuitry that regulate backward movement (reversal) in locomotion behavior. We found that AVA activity increases while RIM activity decreases during the same reversal events in spontaneous locomotion, consistent with the recent report that the AVA and RIM are involved in promoting the initiation of reversals. The CARIBN II system provides a valuable tool for dissecting the neural basis of behavior in C. elegans.
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[
Nat Genet,
2005]
Cell-fate specification and cell-cell signaling have been well studied during vulva development in Caenorhabditis elegans and provide a paradigm in evolutionary developmental biology. Pristionchus pacificus has been developed as a 'satellite' organism with an integrated physical and genetic map that allows detailed comparisons to C. elegans. A common aspect of vulva formation in both species is the polarization of the P7.p lineage, which is responsible for vulval symmetry. In C. elegans, Wnt signaling is crucial for P7.p cell-fate patterning; nothing is known about vulval symmetry in P. pacificus. We isolated mutations that disrupt polarization of the P7.p lineage in P. pacificus and found that the corresponding gene encodes a Frizzled-like molecule. In addition, mutations in
Ppa-lin-17 (encoding Frizzled) and morpholino knock-down of
Ppa-lin-44 (encoding Wnt),
Ppa-egl-20 (encoding Wnt),
Ppa-mig-5 (encoding Dsh),
Ppa-apr-1 (encoding APC) and
Ppa-bar-1 (encoding beta-catenin) results in gonad-independent vulva differentiation, indicating that these genes have a role in a negative signaling process. In contrast, in C. elegans, Wnt signaling has a positive role in vulva induction, and mutations in
bar-1 result in a hypoinduced phenotype. Therefore, whereas the molecular mechanisms that generate vulval symmetry are conserved, the genetic control of vulva induction diversified during evolution.
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[
J Neurosci Methods,
2012]
Motor activity of Caenorhabditis elegans is widely used to study the mechanisms ranging from basic neuronal functions to human neurodegenerative diseases. It may also serve as a paradigm to screen for potential therapeutic reagents treating these diseases. Here, we developed an automated, 96-well plate and liquid phase based system that quantifies nematode motor activity in real time. Using this system, we identified an adult-onset, ageing-associated motor activity loss in a transgenic nematode line expressing human pathogenic G2019S mutant LRRK2 (leucine-rich repeat kinase 2), the leading genetic cause of Parkinson's disease characterized by dopaminergic neurodegeneration associated motor deficient mainly in elder citizens. Thus, our system may be used as a platform to screen for potential therapeutic drugs treating Parkinson's disease. It can also be used to monitor motor activity of nematodes in liquid phase at similar scenario.
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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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[
Worm Breeder's Gazette,
2002]
We describe the molecular characterization of laboratory strains of the nematode genus Pristionchus , which lays a foundation for microevolutionary analyses of vulva development. We isolated 13 laboratory strains of the Pristionchus genus that are derived from natural isolates from around the world. Mating experiments and ITS sequence analysis indicated that these 13 strains represent four different species; the gonochoristic species P. lheritieri and three hermaphroditic species, P. pacificus , P. maupasi and an as yet undescribed species Pristionchus sp., respectively. P. pacificus is represented by five different strains isolated from California, Washington, Hawaii, Ontario and Poland. Our working 'wild type' strain is the California strain. Since polymorphisms are becoming an important tool in modern day genomic analysis, which facilitate cloning of mutations, we decided to search for polymorphisms in the various Pristionchus pacificus strains. We performed amplified restriction fragment length polymorphism (AFLP) analyses of the different P. pacificus strains and found that the American strains are highly polymorphic (Srinivasan et al., 2001). We observed the largest genetic variation between the strain from California on the one hand and Washington and Hawaii on the other hand. In contrast, the developmentally distinct strain from Poland is identical to the Californian strain. Hence, we chose the Washington strain as our polymorphic strain for future experiments. These results provide us a framework for further studies on microevolution in developmental processes. Reference: Srinivasan, J., Pires-daSilva, A.; Gutierrez, A.; Jungblut, B.; Zheng, M.; Witte, H.; Schlak., I. & Ralf J. Sommer, Evolution & Development (2001) 3 : 229-240.
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[
Neuron,
2015]
Sensory-motor reflex circuits are the basic units from which animal nervous systems are constructed, yet little is known regarding how connections within these simple networks are established. In papers in Cell Reports and in this issue of Neuron, Zheng et al. (2015a, 2015b) demonstrate that coordinate activities of Hox genes in sensory neurons and interneurons govern connectivity within touch-reflex circuits in C. elegans.
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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.