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[
International Worm Meeting,
2017]
C. elegans is widely used as a model system for monitoring stimulus-evoked Ca2+ transients in neurons. The ASH sensory neuron is the subject of several such studies, primarily due to its key importance as a polymodal nociceptor. However, despite the pivotal role of ASH in C. elegans, the overall biology and the characteristics of its Ca2+ transients (e.g., the "off" response), no mathematical model has been developed to describe the full mechanism of ASH Ca2+ dynamics. We propose a phenomenological computational model which captures the Ca2+ transients in the C. elegans ASH neuron upon its activation. The model is built on biophysical cascades that unfold as part of the neuron's Ca2+ signaling events and homeostatic mechanism (e.g., TRPV channels and voltage-gated channels activation, Ca2+ release from intracellular stores, IP3 dynamics, PMCA and SERCA pumps function). The state of the ion channels is described based on Hodgkin-Huxley equations and the remaining molecular states are based on kinetic equations with phenomenological adjustments. We fit the model using experimental data of osmotic stimulus-evoked ASH Ca2+ transients, detected with a FRET sensor (TN-XL), in young and aged worms, both untreated and exposed to oxidative stress. We use a multi-objective genetic algorithm to find the parameters for young untreated worms' data set. Parameters are estimated using a hybrid method that consists of a genetic algorithm and nonlinear least-squares. We use the same approach to fit the model for the other groups of experimental data. We validate the model using data from the literature, from ASH activation by stimuli of several strengths and durations. Finally, we demonstrate how our model can be used to predict the ASH Ca2+ response to stimulation pulses that are challenging to achieve experimentally (stimuli sequences of varying durations/lengths, or ramp stimuli). Our model includes for the first time the changes in ASH cytoplasmic Ca2+ flux observed both upon delivery and withdrawal of the stimulus (i.e., the "on" and "off" responses). This effort is the first to propose a quantitative dynamic model of the Ca2+ transients generating mechanism in a C. elegans neuron, based on essential biochemical pathways of the Ca2+ homeostasis machinery.
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[
International Worm Meeting,
2015]
Despite the extensive work performed on C. elegans sensing a variety of physical stimuli (electrical, thermal, hyper/hypo-osmotic), little is known about their sensing of magnetic field and the effect it has on their physiology or behavior. To elucidate this area, we study worm behavior when exposed to magnetic fields both internal to the organism and external. We used two electromagnets to expose worms to magnetic fields and tracked their locomotion pattern. To track the worms we used two tracking systems, a custom made and a commercially available (WormLab). We tested a number of configurations for the relative position of the magnets and the petri dish, the distance between worms and the magnets as well as on and off time intervals of applied magnetic field. Moreover, in a second set of experiments, worms were fed with superparamagnetic nanoparticles and magnetic microparticles, so as to investigate the effect of secondary magnetic field generated by the internalized particles. Particles of diameters 40nm, 70nm, 100nm and 1um were used. Secondary magnetic field generated by magnetized microparticles was simulated to estimate their magnetic field characteristics. The presence of microparticles in the gut, intestine and fat tissue was verified by fluorescent microscopy, confocal microscopy and SEM. Our results show that 1) iron core magnetic microparticles can be introduced by having the worms feed on a mixture of particles and E. coli OP50. Particles translocate in specific tissues, based on their size and they remain inside the worm's body for a time period reversely analogous to their size, 2) our tracking system offers results comparable with the commercially available system, yet we examined only velocity and distance travelled measurements thus far, 3) the magnetic field used does not seem to have any effect on untreated animals and nematodes fed with 40, 70 and 100nm diameter particles; however, worms containing particles of 1um diameter display changes in their locomotion behavior with their velocity decreased significantly. Therefore we conclude that the magnetic field with the intensity and specification we used does not seem to have an effect, for the time period applied, on the locomotion pattern of wild type animals. Moreover, it takes relatively large particles introduced in worms to observe a significant change to their locomotion parameters. Based on these results, we continue by applying stronger magnetic fields, further analyzing worm behavior to locate other possible changes, monitoring the effects of magnetic fields on specific neuron firing and using different types of particles so as to achieve different localization patterns which may affect differently the nervous system.
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[
International Worm Meeting,
2017]
During DNA replication Half of the genome is duplicated discontinuously on the lagging strand in the form of Okazaki Fragments (OFs). Despite many years of research, the precise location of replication origins, efficiency, and abundance in metazoa remains elusive. We have recently isolated and sequenced OFs from C. elegans embryos and developing larva, and have generated a first comprehensive genome wide map of DNA replication in multi cellular organism1. Aligning sequence reads of OFs to the C. elegans genome reveals the complementary enrichment of Okazaki fragments to either the Watson or Crick strands, which is the hallmark of a replication origin1,2. Importantly, we found that replication origins are associated with transcriptionally active regions of chromatin and replication start sites are strongly associated with histone modifications that define genetic enhancers such as H3K27 acetylation. By mapping DNA replication origins in various embryonic stages, we have demonstrated that origins are pre-defined prior to the onset of zygotic transcription. However, at the time of gastrulation, when zygotic transcription initiates, it does so in proximity of pre- defined replication origins. Replication origins and transcription are correlated until the last wave of embryonic cell division. When cell division ceases, this association breaks down and transcription shifts away from replication initiation sites and anti-correlates with replication origins in differentiated cells. Whilst majority of embryonic cells are terminally differentiated at the time of hatching, 53 somatic blast cells in newly hatched L1 larvae divide post embryonically to generate additional 403 cells in C. elegans hermaphrodites. The majority of the somatic blast cell lineage generate vulva, muscle, hypodermal, and neuronal cells. We have shown that the position and the efficiency of somatic replication origins are distinguishable from the embryonic origins yet remain associated with somatic H3K27 acetylation. Interestingly, genes located in proximity of somatic replication start sites are functionally related to vulva, muscle, hypodermal and neuronal cells. Our data strongly suggest that in rapidly dividing embryonic cells, replication is a fundamental regulator of gene activity, while during notably slower post embryonic cell division, transcriptional activity shapes the DNA replication profile. Currently we are investigating the causal link between transcription and somatic replication. 1.Spatiotemporal coupling and decoupling of gene transcription with DNA replication origins during embryogenesis in C. elegans. Ehsan Pourkarimi, James M Bellush, Iestyn Whitehouse. eLIFE, 2016. 2. Intrinsic coupling of lagging-strand synthesis to chromatin assembly. Duncan J. Smith & Iestyn Whitehouse. Nature, 2012