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[
International Worm Meeting,
2013]
Parasitic worms, called helminths, infect plants, animals, and humans worldwide leading to a decreased food supply, economic hardship, and significant levels of morbidity and mortality. Anthelmintics that combat these infections are represented by only five major classes of compounds. Misuse of these pharmaceuticals has contributed to widespread anthelmintic resistance in worms that infect livestock and emerging drug resistance in human-infecting helminths. The identification of new means to target helminths is imperative. The non-parasitic nematode Caenorhabditis elegans has long been a model system for helminths. Previously, our group screened a series of novel, synthetic compounds for anthelmintic activity in C. elegans. These compounds are derivatives of a natural product stilbene that have generated great interest due to their broad anti-microbial effects. Two microassays were used: the motility assay that screened for paralysis and the developmental assay that screened for developmental delays, developmental arrests, a decrease in fecundity, or death. Six compounds demonstrated significant anthelmintic activity and were prioritized for further study. This research project aims to determine if these six stilbenoid derivatives act via a novel mechanism. To this end, the compounds are being tested against mutant C. elegans that are resistant to existing anthelmintics using the two assays described above, along with an NGM plate-based assay. Work to date has primarily focused on the compound CL-5, which showed the strongest activity. Ivermectin is one of the most widely used anthelmintics today, and ivermectin resistance has been documented in helminth strains. Interestingly, CL-5 affects ivermectin-resistant C. elegans in a dose-dependent manner, similar to wildtype worms. Whereas ivermectin acts on ligand-gated chloride channels, the anthelmintic benomyl disturbs the microtubule cytoskeleton. Benomyl-resistant C. elegans are also sensitive to CL-5 and exhibit developmental defects similar to CL-5-treated N2 worms. We are currently testing mutant strains that are resistant to levamisole and emodepside. In addition, we are pursuing some other interesting effects of CL-5 treatment.
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[
International Worm Meeting,
2015]
Established and emerging drug resistance in infective organisms is a growing problem that threatens humans, livestock, and crops and causes morbidity, mortality, and economic woes. The need to identify pharmaceuticals with novel modes of action is imperative. To this end, an interdisciplinary team of scientists at the University of Wisconsin-La Crosse pursued the rich reservoir of metabolites produced in plants and fungi as a potential source of medicinal agents. A compound with strong antimicrobial activity was first isolated from Comptonia peregrina (sweet fern) and used as a template for the synthesis of hundreds of derivatives. This novel library of compounds has wide-ranging activities against different organisms, including the nematode Caenorhabditis elegans.Although non-parasitic, C. elegans has long been a model system for parasitic worms, called helminths. Anthelmintic drugs affect C. elegans and helminths similarly, and the mechanisms of action for commercially available anthelmintics have been elucidated in C. elegans. Previously, we reported the identification of multiple compounds from our library that affect worm motility and/or survival based on two microscale, liquid-based assays. Work to date has primarily focused on the compound CL-5, which showed the strongest activity. CL-5 is effective against mutant strains that are resistant to the major anthelmintics on the market, including ivermectin, benomyl, and levamisole. These data suggest that CL-5 acts via a different molecular mechanism. Recent experimentation has been driven by the fact that CL-5, along with other compounds in the library, is structurally related to resveratrol. Resveratrol alone does not affect the worms in our assay, nor does it attenuate or augment the effects of CL-5. Resveratrol is reported to lower oxidative stress in C. elegans, thereby increasing longevity (1). Preliminary experiments show that treatment with CL-5 leads to increased expression of
sod-3::GFP, an indirect indicator of oxidative stress. Current experiments aim to detect reactive oxygen species directly and these results will be reported.1. Chen, W. et al. Influence of resveratrol on oxidative stress resistance and life span in Caenorhabditis elegans. J Pharm Pharmacol. 65(5): 682-88 (2013).
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[
International Worm Meeting,
2021]
In C. elegans embryos, PLK-1 is pivotal for cell division and it orchestrates polarity establishment together with its binding partner, MEX-5. To achieve this, their localization/activity must be precisely regulated. MEX-5 enrichment at the anterior cytoplasm results from a change in its diffusivity following uneven phosphorylation along the embryo axis. We know PLK-1 relocalization to the anterior depends on MEX-5. However, the biological and physical mechanisms behind the dynamics of this protein are still poorly described. To address this, PLK-1 and MEX-5 gradient formation was measured in two CRISPR strains and significant discrepancies were revealed between the two proteins in terms of: 1) gradient steepness, as PLK-1 forms a less steep gradient compared to MEX-5; 2) dynamics, with PLK-1 gradient establishment delayed and slower; 3) diffusivity, as PLK-1 diffusion coefficient does not correspond to MEX-5's one from anterior to posterior. To shed light on PLK-1 dynamics, and how it is intertwined to MEX-5, we developed a novel Monte Carlo simulation framework able to recreate the protein motions in the C. elegans one-cell embryo. Thanks to our computational approach, we were able to postulate on the biological mechanisms behind MEX-5 and PLK-1 dynamics during the whole cell division, from early embryos to the steady-state before cytokinesis. The simulations succeed in reproducing PLK-1 gradient formation, in agreement with experimental measurements, if: 1) PLK-1 binds to phosphorylated MEX-5; 2) the binding is triggered after a defined time delay; 3) PLK-1 dynamically interacts with MEX-5, leading to a continuous replenishment of a pool of unbound PLK-1. The Monte Carlo framework we propose can eventually be applied to other polarity-related factors or mutants in which polarization is perturbed, to understand if it can be traced back to a failure in PLK-1 localization. Finally, conditions where gradient formation is altered, like after stress, can be simulated.
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[
International C. elegans Meeting,
1997]
Pairing and recombination between homologous chromosomes are essential for their faithful segregation during the first meiotic division. The mechanisms leading to homologous chromosome pairing and synapsis are still only poorly understood in any experimental organism. C. elegans provides a highly advantageous model system in which genetic and cytological approaches can be combined to study this process. We are developing tools and methods to enable us to examine chromosome pairing in the light microscope in order to address several specific questions: Do special sites on each chromosome (particularly the genetically-defined !pairing centers!) become associated earlier than others? Do chromosome rearrangements that act as crossover suppressors disrupt homologous pairing as dramatically as genetic evidence seems to suggest? Are gene products that are required for meiotic recombination associated with particular regions of the chromosomes? To answer these and other questions, we are using several approaches, including fluorescence in situ hybridization (FISH) methods, and labeling of chromosomes in living worms using the GFP-Lac repressor binding method developed by Aaron Straight, Andrew Murray, and Andy Belmont for other experimental systems. By applying high-resolution 3-dimensional microscopy, we hope to obtain a better understanding of rules governing the organization and interaction of chromosomes in the meiotic prophase nucleus.
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[
Development & Evolution Meeting,
2008]
An understanding of the forces acting on cells in the early embryo can provide important information for how cells interact to determine their shapes, movements and fates. These forces can be both physical and genetic and can be intracellular or extracellular. The early Caenorhabditis elegans embryo provides an excellent environment to explore the forces acting during embryogenesis and to develop techniques that can be applied later to more advanced biological events. We report the development of a 4D GGH (Glazier-Granier-Hogeweg) model to simulate the 4-cell stage of embryogenesis. GGH modeling uses a Metropolis Monte Carlo algorithm to describe the evolution of systems based on the idea that many systems transform to minimize their overall energy. By using a fixed-lattice environment governed by the Hamiltonian, an overall energy equation, GGH modeling can simplify the description of cell-based phenomena. The early stages of embryogenesis offer an environment where components of the Hamiltonian can be identified and tested. Our Hamiltonian includes cell-cell adhesion, cell-shell contact, centromere rotation and elongation, and constraints of surface area and volume. Our GGH model allows for the incorporation of several biological components that have not be explored in previous models of C. elegans embryogenesis. In addition, several aspects of our model broaden the possible applications of GGH modeling.
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[
International Worm Meeting,
2017]
Phenotypic plasticity allows species to respond to environmental changes. In the wild, populations of the nematode Caenorhabditis elegans develop and grow in nutrient-rich, ephemeral habitats. Movement between these ephemeral patches, and long term genotype survival, depends on the development of dauer larvae. Within these natural populations, survival is therefore dependent upon a critical development decision, as worms must commit to either a reproductive fate to increase local numbers, or a migratory fate to find new resources to exploit. Failure to disperse at the correct time will result in loss of the local population with the resources in the ephemeral habitat. Under laboratory conditions, the decision between dauer and non-dauer larval development is driven by ascaroside signalling - which acts as a proxy for population size - by food availability, and by temperature. However, phenotypic differences between genotypes in reaction to these factors is substantial. For example, ascaroside production profiles vary between genotypes as do the responses to specific ascarosides and to mixtures of ascarosides. There is also evidence that suggests that ascaroside signalling by worms may be manipulative. Here we present the results of Markov Chain Monte Carlo (MCMC) simulations of modelled C. elegans genotypes optimising the developmental switch under different environmental pressures. The simulation results are compared with a number of wild-type strains. The evolution of ascaroside signalling is examined with the additional effects of exposure to signal distortion and noise as may be present in heterogeneous wild habitats. The model is presented in the form of a generalizable method for studying developmental decisions and other phenotypically plastic traits under a variety of environmental conditions.
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[
International Worm Meeting,
2005]
It is possible to model the key features of a biological phenomenon using Simulations. Computer simulations may be used to check whether a particular theory of a physical mechanism is internally consistent, and whether the parameters they contain are sufficient to account for experimental observations. The problem we address is the asymmetric positioning of the mitotic spindle in the first cleavage in C.elegans, which results in daughter cells of unequal sizes. During the first cell division in wild type C.elegans embryos, the spindle is initially set up in the center of the cell. At anaphase, the posterior spindle pole is displaced horizontally towards the posterior of the egg while oscillating transversely. The anterior spindle pole is not displaced, but also oscillates transversely. As a result, the cell divides with the cleavage furrow displaced to the posterior, producing a large anterior AB cell and a smaller, posterior P1 cell. The spindle pole oscillations are thought to arise from the pulling of astral microtubules towards the cortex by a limited number of force generators (Grill et al., 2001, 2003). To understand the fine orchestration of the forces involved in positioning the spindle, we modeled the microtubules and motors in the C.elegans embryo, using Cytosim ( www.cytosim.org ). The program uses Brownian dynamics to model the microtubules, and Monte Carlo methods to simulate the stochastic binding and unbinding of molecular motors. Cytosim computes the mechanical and dynamic properties of microtubules, but not the tubulin molecules. For this reason, the computational load is sufficiently small, allowing the simulation of an entire cell in three dimensions, in a relatively short time. Two alternative models were built, which reproduce the trajectories of the spindle poles in the anaphase embryo. Experiments were then designed to distinguish between the models. The results were consistent with the latter model, from which we propose that pulling by molecular motors, pushing by microtubules on the cortex, and modulated dynamic instability, are necessary and sufficient to reproduce the spindle positioning process in the C.elegans embryo. By successively refining this model by experiments, increasingly accurate models will be constructed, aiding our understanding of this complex phenomenon.
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[
International Worm Meeting,
2009]
Ten nematode genomes have been sequenced, two completed and the other eight shotgun sequenced and assembled, representing four of the twelve nematode clades. We have used comparative sequence analysis to identify conserved non-coding sequences and their distribution among these nematodes. Comparative sequence analysis can identify functional sequence elements by seqeunce conservation. While analysis of a pair of genomes identifies many of these functional elements, additional conserved sequence elements are identified as additional genomes are analyzed and the species range of an element can be used to estimate its age. These conserved sequence elements are often regulatory elements although they are difficult to classify with in silico analysis. Analysis of the four sequenced Caenorhabditis species identifies extensive conserved non-coding sequence, approximately double the conserved sequence identified comparing a pair of species. P. pacificus and H. glycines were found to have few putative promoter sequence elements conserved with C. elegans, much less than expected based on gene conservation indicating that gene regulation in these species has diverged considerably. In contrast, promoter conservation with M. hapla is more extensive than expected. We have developed web-based software to allow researchers to explore and visualize sequence conservation in the analyzed nematode genomes
(http://daf.uky.edu/id_plot/). Gene regulatory boundaries in the nematode C. elegans were identified by examining evolutionary chromosomal recombination events. Conserved sequence elements were linked with particular genes identifying the natural boundaries between genes and the extent of worm promoters. We have used the set of identified C. elegans and C. briggsae conserved promoter elements to quantify and analyze promoter complexity. Monte Carlo sampling was used to identify GO and KEGG annotated gene groups that appear to have significantly low or high promoter complexity. Genes annotated as developmental genes and signaling genes especially G-protein coupled receptors and cell-cell signalling genes have high promoter complexity scores. Gene expression in the complete set of published C. elegans microarray experiments was analyzed and a strong positive correlation between gene expression variation and promoter complexity was discovered. Genes showing considerable regulation in microarray experiments tend to have complex promoters.
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[
C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting,
2008]
With multiple genomes related to each metazoan model organism being sequenced promoter analysis is becoming an especially useful tool in genomic analysis. Comparative sequence analysis can identify functional sequence elements for pairs of organisms that diverged far enough in the past to allow mutational drift of non-conserved sequences. While analysis of a pair of genomes identifies many of these functional elements adding additional genomes allows additional information to be elicited. Additional conserved sequence elements are identified as additional genomes are analyzed. These conserved sequence elements are often regulatory elements although they are difficult to classify with in silico analysis. For an individual gene, the set of associated conserved sequence elements and the organisms they are found in provides insight into the evolutionary history of the regulation of the gene. The eight complete and unfinished shotgun sequenced nematode genomes and the dozen informative insect genome sequences were used to analyze conserved non-coding sequences in these groups of organisms. Web-based software was developed to allow researchers to explore and visualize sequence conservation that expands upon previous work by analyzing conserved sequences in each pair of organisms rather than with respect to a single reference genome. We have used this analysis to identify gene regulatory boundaries in the nematode C. elegans. The genomes of C. elegans and other nematodes have diverged enough that syntenic regions are typically a few genes long. I was able to associate conserved sequence elements to particular genes identifying the natural boundaries between genes and the extent of worm promoters. We have used the set of identified C. elegans and C. briggsae conserved promoter elements construct a promoter complexity score. Promoter complexity identifies which genes have particularly interesting regulation, identifying gene groups with a strong promoter complexity signal and cases where a gene''s promoter complexity differs from the group''s promoter complexity. Monte Carlo random sampling was used to identify Gene Ontology and KEGG Pathway annotated gene groups that appear to have significantly low or high complexity. Genes annotated as developmental genes and signaling genes especially G-protein coupled receptors and cell-cell signaling genes have high promoter complexity scores. We also examined gene expression in the published C. elegans microarray experiments and found a strong positive correlation between gene expression variation and promoter complexity. Genes showing considerable regulation in microarray experiments tend to have complex promoters.