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Bowlin, Peter, Li, Zihao, Fouad, Anthony, Du, Angelica, Kassouni, Alexander, Bhirgoo, Priya, Teng, Christopher, Fang-Yen, Christopher
[
International Worm Meeting,
2021]
The time and labor required for worm picking is a major bottleneck for many C. elegans experiments, especially those requiring a large number of strains. Many genetic screens and genetic manipulations would benefit greatly from an automated method for worm pushing. We developed a robot capable of transferring worms between agar plates using movements similar to those used for manual worm picking. The robot contains a motorized 3D stage that positions a wire loop pick mounted on a robotic arm to manipulate worms on an array of standard plates. Capacitive touch sensing is used to monitor contact between the pick and the agar substrate and provide feedback for the fine movements needed for picking. We constructed a dual-magnification fluorescence and bright field microscope capable of identifying developmental, morphological, or fluorescence based phenotypes of individual worms at high resolution while simultaneously imaging the entire plate at low resolution. We developed software to identify, classify, and track worms using a combination of machine vision methods, including motion detection, adaptive thresholding, and a convolutional neural network trained to recognize worms. In a test of its fluorescence-based sorting capabilities, the robot accurately identified, phenotyped, picked, and transferred worms to other plates at a rate of about 3 animals per minute. We are developing a high-level scripting language that will enable the robot to autonomously perform multi-step procedures, such as integrating extrachromosal arrays, performing genetic crosses, generating clonal populations of mutagenized worms, and other tasks. Automation of worm manipulation will both increase researchers' productivity and enable experiments that are impractical using standard methods.
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[
J Pharmacol Exp Ther,
2015]
NaCT (SLC13A5) is a Na(+)-coupled transporter for Krebs cycle intermediates and is expressed predominantly in the liver. Human NaCT is relatively specific for citrate compared with other Krebs cycle intermediates. The transport activity of human NaCT is stimulated by Li(+), whereas that of rat NaCT is inhibited by Li(+). We studied the influence of Li(+) on NaCTs cloned from eight different species. Li(+) stimulated the activity of only NaCTs from primates (human, chimpanzee, and monkey); by contrast, NaCTs from nonprimate species (mouse, rat, dog, and zebrafish) were inhibited by Li(+). Caenorhabditis elegans NaCT was not affected by Li(+). With human NaCT, the Li(+)-induced increase in transport activity was associated with the conversion of the transporter from a low-affinity/high-capacity type to a high-affinity/low-capacity type. H(+) was able to substitute for Li(+) in eliciting the stimulatory effect. The amino acid Phe500 in human NaCT was critical for Li(+)/H(+)-induced stimulation. Mutation of this amino acid to tryptophan (F500W) markedly increased the basal transport activity of human NaCT in the absence of Li(+), but the ability of Li(+) to stimulate the transporter was almost completely lost with this mutant. Substitution of Phe500 with tryptophan in human NaCT converted the transporter from a low-affinity/high-capacity type to a high-affinity/low-capacity type, an effect similar to that of Li(+) on the wild-type NaCT. These studies show that Li(+)-induced activation of NaCT is specific for the transporter in primates and that the region surrounding Phe500 in primate NaCTs is important for the Li(+) effect.
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[
International C. elegans Meeting,
1997]
Lithium (Li) has long been known to have teratogenic effects on the development of many organisms, including sea urchin, Xenopus and Dictyostelium. In Li-treated Xenopus embryos, ventral blastmeres are respecified to develop into dorsal structures, leading to dorsalized embryos lacking ventral mesodermal tissues. In Dictyostelium, Li alters the fate of prespore cells to become prestalk cells instead. Besides teratogenic effects, Li is also known to be a most effective treatment of manic-depressive illness.!@Although several models have been proposed to explain Li action, the molecular mechanism has remained unclear. Recently, GSK (glycogen synthase kinase)-3b was proposed to be a target of Li, suggesting that Li affects the wnt signaling pathway. To understand the mechanism of Li action, I first examined the effect of Li on C. elegans embryogenesis. I inoculated N2 animals at the late L4 stage onto NG plates containing 20 mM LiCl, incubated them at 20 oC. The number of eggs produced by treated animals was reduced to about half of the untreated control. Although cell division seemed to proceed, no embryos hatched on Li plates. Treated-embryos developed to produce gut granules, but did not execute normal morphogenesis at later embryonic stages. To identify genes involved in the action of Li, I have begun to screen for Li-resistant mutants that propagated on Li-containing medium. Several mutants were isolated, and one of them was mapped on the left side of
unc-42 on chromosome V. On Li plates, the hatching rate of mutant eggs cross-fertilized by wild-type males was essentially the same as that for self-fertilized mutant eggs. On the contrary, no wild-type eggs cross-fertilized by mutant males hatched on Li-containing plates. So, this mutation appeared to be maternal. Further genetic analyses of the mutants and the observation on the cellular phenotype of Li-treated embryos are underway.
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[
Worm Breeder's Gazette,
1996]
Lithium (Li) has long been known to have teratogenic effects on the development of many organisms, including sea urchin, Xenopus and Dictyostelium. In Li-treated Xenopus embryos, ventral blastmeres are respecified to develop into dorsal structures, leading to dorsalized embryos lacking ventral mesodermal tissues. In Dictyostelium, Li alters the fate of prespore cells to become prestalk cells instead. Besides teratogenic effects, Li is also known to bea most effective treatment of manic-depressive illness. Although several models have been proposed to explain Li action, the molecular mechansm remains unclear. The most widely accepted model is the inositol depletion hypothesis, in which Li is thought to affect inositol phosphate turnover by inhibiting inositol monophosphatase, thus resulting in the depletion of endogenous inositol. To understand the mechanism of Li action, I first examined the effect of Li on C. elegans embryogenesis. I inoculated N2 animals at the late L4 stage onto NG plates containing 20 mM LiCl, incubated them at 20 C and observed the laid embryos. The number of eggs produced by treated animals was reduced to about half of the untreated control. Although cell division seemed to proceed, no embryos hatched on Li plates. Treated-embryos developed to produce gut granules, but did not execute normal morphogenesis at later embryonic stages. To identify genes involved in the action of Li, I have begun to screen for Li-resistant mutants, which propagated on Li-containing medium. So far, I obtained one mutant. The mutation was tentatively assigned to chromosome V. Preliminary genetic analysis showed that the mutation showed maternal effect. On Li plates, the hatching rate of mutant eggs cross-fertilized by wild-type males was essentially the same as that for self-fertilized mutant eggs. On the contrary, no wild-type eggs cross-fertilized by mutant males hatched on Li-containing plates. I am now trying to isolate other mutants and also to identify early defects of embryogenesis caused by lithium treatment. I would like to thank J. Miwa for encouragement and discussions.
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[
International Worm Meeting,
2021]
The platinum worm pick, a fixture in C. elegans laboratories for decades, has two drawbacks: (1) the high cost of platinum, a significant problem in many educational settings, and (2) the reliance on an open flame for sterilization, which presents safety hazards. To address the first drawback, we evaluated whether platinum could be replaced with an alternative metal. An ideal worm pick cools quickly after heating, withstands high temperature without degradation, can be flattened and shaped easily, and is inexpensive. With these criteria in mind, we compared 90% platinum, 10% iridium wire (PT9010) with 5 alternatives: stainless steel (SS), Nickel 200, two nickel chromium (Nichrome) alloys, and iron-chromium-aluminum (FeCrAl). To measure cooling rate we built a circuit to resistively heat wires (all 255 microm in diameter) to 800 C and measured the time it took them to cool to 25 C. We found that PT9010 and FeCrAl cooled more rapidly (6-7 s) than the other metals tested (8-9 s). To assay heat resistance we conducted a bending test after 3000 heating cycles of duration 4 s at 800 C. All materials except SS showed good heat resistance, withstanding >50 bends after 3000 heating cycles. SS exhibited poor heat resistance, breaking spontaneously after ~300 cycles. All materials could be easily flattened using standard tools. With regard to cost, all alternative materials were < 0.20 USD/m, as compared to 140 USD/m for PT9010. These results show that all metal alloys tested except for SS represent reasonable, economical alternatives for worm picks. The most promising is FeCrAl which cools as rapidly as platinum, exhibits good heat resistance, and is available at a fraction of the cost. Next, to explore an alternative to flame sterilization, we designed an electric worm pick consisting of a loop of PT9010 or FeCrAl wire attached to a handle containing a rechargeable battery and circuit board. Depressing a button causes current to flow through the loop, heating it to about 800 C within 2 s. A battery charge lasts for ~500 sterilizations. Worm researchers who tested the device reported that the wire loop could be used similar to a worm pick and that electric sterilization promoted faster work since no movements to a flame were necessary. Our device represents a convenient and safer alternative to flame-sterilized worm picks. We are using a similar loop-based worm picking technique in our automated worm picking system (see abstract by Zihao Li et al).
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[
J Appl Toxicol,
2015]
Lithium (Li) has been widely used to treat bipolar disorder, and industrial use of Li has been increasing; thus, environmental pollution and ecological impacts of Li have become a concern. This study was conducted to clarify the potential biological effects of LiCl and Li(2)CO(3) on a nematode, Caenorhabditis elegans as a model system for evaluating soil contaminated with Li. Exposure of C. elegans to LiCl and Li(2)CO(3) decreased growth/maturation and reproduction. The lowest observed effect concentrations for growth, maturation and reproduction were 1250, 313 and 10 000m, respectively, for LiCl and 750, 750 and 3000m, respectively, for Li(2)CO(3). We also investigated the physiological function of LiCl and LiCO(3) in C. elegans using DNA microarray analysis as an eco-toxicogenomic approach. Among approximately 300 unique genes, including metabolic genes, the exposure to 78m LiCl downregulated the expression of 36 cytochrome P450, 16 ABC transporter, 10 glutathione S-transferase, 16 lipid metabolism and two vitellogenin genes. On the other hand, exposure to 375m Li(2)CO(3) downregulated the expression of 11 cytochrome P450, 13 ABC transporter, 13 lipid metabolism and one vitellogenin genes. No gene was upregulated by LiCl or Li(2)CO(3). These results suggest that LiCl and Li(2)CO(3) potentially affect the biological and physiological function in C. elegans associated with alteration of the gene expression such as metabolic genes. Our data also provide experimental support for the utility of toxicogenomics by integrating gene expression profiling into a toxicological study of an environmentally important organism such as C. elegans.
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[
J Biol Chem,
2008]
Lithium (Li+) has been used to treat mood affect disorders, including bipolar, for decades (1;2). This drug is neuroprotective and has several identified molecular targets. However, it has a narrow therapeutic range and the underlying mechanism(s) of its therapeutic action is not understood. Here we describe a pharmacogenetic study of Li+ in the nematode Caenorhabditis elegans. Exposure to Li+ at clinically relevant concentrations throughout adulthood increases survival during normal aging (up to 46% median increase). Longevity is extended via a novel mechanism with altered expression of genes encoding nucleosome-associated functions. Li+ treatment results in reduced expression of the worm ortholog of LSD-1 (T08D10.2), a histone demethylase; knockdown by RNA interference (RNAi) of T08D10.2 is sufficient to extend longevity (~25% median increase), suggesting Li+ regulates survival by modulating histone methylation and chromatin structure.
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[
International C. elegans Meeting,
2001]
Little is known about how ions permeate the C. elegans gut and cuticle. How various ions influence development and behavior is also not understood. One ion with considerable impact in human biology is Li + , which modulates bipolar disorder by an unknown mechanism. In particular, the targets of lithium that cause side effects remain to be identified. We are using C. elegans to genetically identify targets of lithium and to elaborate on mechanisms of transport, which may have an impact on human health. Li + has dosage-dependent effects on C. elegans embryos. When L4 animals mature on NGM plates with Li + added to the final concentrations of 10mM-20mM, they produce embryos that are unable to hatch. We demonstrated that this failure to hatch is due to defects in cytokinesis that result in multi-nucleated embryos and symmetrically partitioned cells. Li + also has a dosage-dependent effect on larval development. When adult hermaphrodites are permitted to lay embryos on 10mM-20mM Li + NGM plates, the resulting offspring experience a developmental delay proportional to the concentration of Li + . The offspring become progressively paralyzed as they reach adulthood. We demonstrated earlier that there is a delay in the entry into the S phase of the cell cycle larval stages. To learn more about the biology of Li + sensitivity, we screened for Li + resistant mutants. We developed three screens that took advantage of the embryonic arrest and the larval delay caused by Li + . We isolated one mutant
bz71 , in a screen of 44,000 haploid genomes, that is resistant to both the larval and embryonic blocks of 16mM Li + .
bz71 is dominant. Using classical mapping techniques, we positioned it on LGIII between
unc-32 and
dpy-18 . We are currently using SNP strategy to obtain a higher resolution map and we hope to report on the identification and cloning of this locus.
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[
Worm Breeder's Gazette,
1992]
Characterization of the axonal guidance and outgrowth gene
unc-33 W. Li, R. K. Herman and J. E. Shaw Department of Genetics and Cell biology, University of Minnesota, St Paul, MN 55108
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[
J Biol Chem,
2004]
Sodium-calcium exchangers have long been considered inert with respect to monovalent cations such as lithium, choline, and N-methyl-d-glucamine. A key question that has remained unsolved is how despite this, Li(+) catalyzes calcium exchange in mammalian tissues. Here we report that a Na(+)/Ca(2+) exchanger, NCLX cloned from human cells (known as FLJ22233), is distinct from both known forms of the exchanger, NCX and NCKX in structure and kinetics. Surprisingly, NCLX catalyzes active Li(+)/Ca(2+) exchange, thereby explaining the exchange of these ions in mammalian tissues. The NCLX protein, detected as both 70- and 55-KDa polypeptides, is highly expressed in rat pancreas, skeletal muscle, and stomach. We demonstrate, moreover, that NCLX is a K(+)-independent exchanger that catalyzes Ca(2+) flux at a rate comparable with NCX1 but without promoting Na(+)/Ba(2+) exchange. The activity of NCLX is strongly inhibited by zinc, although it does not transport this cation. NCLX activity is only partially inhibited by the NCX inhibitor, KB-R7943. Our results provide a cogent explanation for a fundamental question. How can Li(+) promote Ca(2+) exchange whereas the known exchangers are inert to Li(+) ions? Identification of this novel member of the Na(+)/Ca(2+) superfamily, with distinct characteristics, including the ability to transport Li(+), may provide an explanation for this phenomenon.