Pepper, Judy S. et al. (2011) International Worm Meeting "Mutants that fail to respond to exogenous serotonin identify new serotonin signaling genes."

Koelle, Michael R., Pepper, Judy S.

[International Worm Meeting, 2011]

Signaling by the neurotransmitter serotonin is thought to be reduced in the brain during depression, but the mechanisms that alter serotonin signaling remain unclear. We are using a genetic approach in C. elegans to delineate the molecules that mediate serotonin signaling to shed light on this issue. Several C. elegans behaviors are regulated by serotonin signaling, including locomotion, egg-laying, and pharyngeal pumping. Serotonin released when food-deprived animals encounter food slows locomotion, and high concentrations of exogenous serotonin paralyzes animals, presumably by exaggerating this response. To identify additional signaling molecules that mediate the effects of serotonin, we carried out a screen for mutants resistant to the paralytic effects of exogenous serotonin. Individual F1 progeny of mutagenized animals were cultured for one generation in 96-well microtiter dishes and 30 mM serotonin was added. This concentration paralyzes nearly all wild-type animals within minutes, and we looked for wells in which ~25% of the F2 progeny (mutant homozygotes) continued to thrash. We recovered 13 serotonin resistant mutants from a screen of 12,000 mutagenized haploid genomes. Using single nucleotide polymorphism (SNP) mapping and sequencing we identified the mutations causing serotonin resistance in the five strongest mutants. Four mutations were in genes previously known to be required for serotonin response. One mutation was in the gene encoding G protein coupled receptor SER-4 and two were in the goa-1 G protein gene, suggesting serotonin acts through SER-4 to activate GOA-1 signaling and slow locomotion. An additional mutation was in the serotonin-gated chloride channel MOD-1, suggesting serotonin has an additional role in slowing locomotion through this receptor. We found that the fifth strong serotonin resistance mutation, vs119, changes a glycine to a glutamate in the enzymatic core of the catalytic subunit of a conserved protein complex whose only known biochemical function is to post-translationally modify other proteins on lysine residues by acetylating them. Two additional alleles for this lysine acetylase gene as well as a mutation that disrupts another subunit of the complex all cause strong resistance to exogenous serotonin. We hypothesize that vs119 may function in serotonin signaling by modifying one or more of the other proteins required for serotonin response: the SER-4 or MOD-1 serotonin receptors, or the G protein, GOA-1. We previously found that the GOA-1 protein runs a series of species of different charge on an isoelectric focusing gel, and we are currently testing the hypothesis that this is due to variable modifications of GOA-1 by the acetylase.

Judy S Pepper et al. (2004) East Coast Worm Meeting "A Screen for Mutants Resistant to Serotonin: a Search for Genes Involved in Neurotransmitter Signaling and Centrosome Movement"

Judy S Pepper, Michael R Koelle

[East Coast Worm Meeting, 2004]

Serotonin is a neurotransmitter that influences human behaviors and psychiatric disorders, including depression. Several C. elegans behaviors are also mediated through serotonin signaling, including locomotion, egg-laying, and pharyngeal pumping. Serotonin signals in adult neurons through the G a o protein, GOA-1, to control these behaviors (1). G a o also regulates an essential early embryonic process, centrosome movement, during embryonic cell division (2). Our lab recently characterized maternal-effect lethal mutations in a gene, rgs-7 , that regulates G a o for its embryonic function (3). We hypothesize that there may be other G a o signaling components used in both adult neurotransmitter signaling and embryonic cell divisions. We are carrying out a two-phase screen that will allow us to identify such signaling components. Our primary screen is to isolate mutants resistant to the paralytic effects of exogenous serotonin, which should identify adult animals defective for G a o signaling. The screen is carried out in a "clonal" fashion, such that maternal-effect lethal mutations can be identified and recovered. Our secondary screen will identify mutations from the primary screen that also cause lethality due to defects in embryonic cell divisions. Previous screens would have missed G a o signaling genes that function in both embryos and adult neurons because non-clonal screens require both viability and fertility. Our primary screen used the following approach: the F1 progeny of ethylmethane sulfonate mutagenized animals were cloned into liquid culture medium in 96-well microtiter dishes and allowed to grow for one generation, after which 30mM serotonin was added to each culture. 30mM serotonin induces paralysis of wild-type animals within minutes, while mutants for the G a o gene goa-1 or the serotonin-gated ion channel gene mod-1 continue to thrash under such treatment (4). We looked for new mutants that showed similar serotonin resistance. Wells containing ~25% resistant (F2 homozygous mutant) animals were identified, and we recovered the heterozygous mutant siblings found in the same wells to recover any maternal-effect lethal mutations We have completed a primary screen of 10,000 mutagenized haploid genomes and have recovered 13 isolates. Each mutation isolated will be outcrossed, genetically mapped, and analyzed in our secondary screen for defects in embryonic cell divisions. 1. Segalat, L., Elkes, D.A. and Kaplan, J.M. (1995). Science 267: 1648-1651. 2. Gotta M., and Ahringer, J. (2001). Nature Cell Biol. 3: 297-300. 3. Hess, H.A. and Koelle, M.R., (2004) submitted 4. Ranganathan, R, Canon S.C., and Horvitz, H.R. (2000). Nature 408: 470-475.

Judy S Pepper et al. (2005) International Worm Meeting "A Screen for Mutants Resistant to Serotonin: a Search for Genes Involved in Neurotransmitter Signaling and Mitotic Spindle Positioning"

Michael R Koelle, Judy S Pepper

[International Worm Meeting, 2005]

Serotonin is a neurotransmitter that modulates human behaviors and improper levels are associated with psychiatric disorders, such as depression. Several C. elegans behaviors are also mediated through serotonin signaling, including locomotion, egg-laying, and pharyngeal pumping. Serotonin signals in adult neurons through the Go protein homolog, GOA-1, to control these behaviors (1). Go also regulates an essential early embryonic process, mitotic spindle positioning, during asymmetric cell divisions (2). Our lab recently characterized maternal-effect lethal mutations in a gene, rgs-7, that regulates Go for its embryonic function (3). We hypothesized that there are other Go signaling components used in both adult neurotransmitter signaling and embryonic cell divisions. We carried out a two-phase screen to identify such signaling components. Treatment with 30mM serotonin induces paralysis of wild-type animals within minutes. Our primary screen was for mutants resistant to this effect, and thus identified adult animals defective for Go -mediated serotonin signaling. The screen was carried out in a clonal fashion, such that maternal-effect lethal mutations could be identified and recovered. The secondary screen identified mutations from the primary screen that also caused lethality due to defects in embryonic cell divisions.We completed a primary screen of 12,000 mutagenized haploid genomes and recovered 13 serotonin-resistant mutants. We have identified four of the mutations; two are alleles of goa-1, one is an allele of the serotonin-gated ion channel MOD-1 (4), and one is an allele of the putative G protein coupled serotonin receptor SER-4. One mutation, vs119, causes strong serotonin resistance and appears to identify an additional signaling gene that we plan to clone. Two mutations, vs113 and vs114, cause temperature sensitive lethality when grown at 25C and preliminary results show defects in mitotic spindle positioning in early embryos. vs113 embryos are defective for rotation of the P0nucleo-centrosomal complex that occurs just prior to mitosis. vs114 embryos show a defect in the rocking movements of the mitotic spindle during anaphase of the P0 cell division the same defect is seen in embryos lacking Go. We are currently mapping these mutations.1. Segalat, L., Elkes, D.A. and Kaplan, J.M. (1995). Science 267: 1648-1651. 2. Gotta M., and Ahringer, J. (2001). Nature Cell Biol. 3: 297-300. 3. Hess, H.A., Rper, J.-C, Grill, S.W., and Koelle, M.R. (2004). Cell 119: 209-218. 4. Ranganathan, R., Canon, S.C., and Horvitz, H.R. (2000). Nature 408: 470-475.

Lee K et al. (2014) Appl Microbiol Biotechnol "Anti-biofilm, anti-hemolysis, and anti-virulence activities of black pepper, cananga, myrrh oils, and nerolidol against Staphylococcus aureus."

Cho MH, Kim SI, Lee J, Lee JH, Lee K

[Appl Microbiol Biotechnol, 2014]

The long-term usage of antibiotics has resulted in the evolution of multidrug-resistant bacteria. Unlike antibiotics, anti-virulence approaches target bacterial virulence without affecting cell viability, which may be less prone to develop drug resistance. Staphylococcus aureus is a major human pathogen that produces diverse virulence factors, such as -toxin, which is hemolytic. Also, biofilm formation of S. aureus is one of the mechanisms of its drug resistance. In this study, anti-biofilm screening of 83 essential oils showed that black pepper, cananga, and myrrh oils and their common constituent cis-nerolidol at 0.01 % markedly inhibited S. aureus biofilm formation. Furthermore, the three essential oils and cis-nerolidol at below 0.005 % almost abolished the hemolytic activity of S. aureus. Transcriptional analyses showed that black pepper oil down-regulated the expressions of the -toxin gene (hla), the nuclease genes, and the regulatory genes. In addition, black pepper, cananga, and myrrh oils and cis-nerolidol attenuated S. aureus virulence in the nematode Caenorhabditis elegans. This study is one of the most extensive on anti-virulence screening using diverse essential oils and provides comprehensive data on the subject. This finding implies other beneficial effects of essential oils and suggests that black pepper, cananga, and myrrh oils have potential use as anti-virulence strategies against persistent S. aureus infections.

Kim, Seongseop et al. (2015) International Worm Meeting "An atlas of G protein-coupled neurotransmitter and neuropeptide receptor expression patterns in C. elegans."

Fernandez, Robert, Koelle, Michael R., Pepper, Judy, Kim, Seongseop

[International Worm Meeting, 2015]

Many neurotransmitters and neuropeptides signal by activating G protein-coupled receptors (GPCRs), but there is a limited understanding of the biological purpose of such signaling in large part because there are mutant phenotypes for only a tiny fraction of the GPCRs. The few C. elegans GPCRs that have been studied in detail are expressed in a very limited number of cells, and mutants show very specific defects in the behaviors controlled by those cells. Although it is now possible to get knockout mutations for any receptor, it has not been possible to identify any defects since we do not know what very specific defects to look for. If we knew the expression patterns for all the neural GPCRs, then we could analyze in detail knockouts of just the GPCRs expressed within the circuit that controls a behavior of interest for specific defects in that behavior. This strategy would provide a path to finally understand the functions of neurotransmitter/neuropeptide signaling through GPCRs.We are analyzing the cellular expression patterns of the ~100 neuropeptide receptor homologs and ~23 GPCRs for small molecule neurotransmitters that are found in C. elegans. We are using C-terminal translational gfp fusion transgenes generated by Sarov et al. (2012). These fosmid-based transgenes contain long stretches of 5' and 3' regulatory sequences and thus should show accurate and complete expression patterns. We are generating high-copy transgenes by microinjection to get bright GFP expression and also to generate possible over-expression phenotypes. We identify GFP-expressing cells by confocal microscopy, using several RFP transgenes to label specific cells as landmarks.Our pilot experiments have confirmed and added to previously known expression patterns of several neurotransmitter receptors. For example, our egl-6::gfp fosmid transgene showed expression in the HSN and GLR cells, as did a smaller transgene used by Ringstad and Horvitz (2008). However our fosmid transgene also showed additional expression in head, vulval, anal sphincter muscles, the gonadal sheath cells, and several additional neurons.Our receptor expression atlas will help identify neurotransmitter signaling events in all neural circuits, but we will first demonstrate its utility by focusing on the egg-laying circuit. This circuit consists of just four cell types, and we hope to identify all the neurotransmitter receptors that act in this circuit and to characterize their functions in generating the pattern of activity by which this circuit controls egg-laying behavior.

Dicklow MB et al. (1993) Journal of Chemical Ecology "A novel streptomyces species for controlling plant-parasitic nematodes"

Dicklow MB, Acosta N, Zuckerman BM

[Journal of Chemical Ecology, 1993]

A novel species of Steptromyces isolated from nematode suppressive soils in Costa Rica was evaluated for efficacy in controlling plant-parasitic nematodes. This isolate, designated CR-43, was shown to inhibit reproduction of Caenorhabditis elegans in a laboratory assay. Greenhouse trials utilizing three different methods of treatment with CR-43 gave significant reductions of tomato root galling due to Meloidogyne incognita. In a field experiment in Puerto Rico, [West Indies] in soil naturally infested with M. incognita, CR-43-treated pepper showed significant reductions in root galling and significant increases in yield as compared to untreated controls. In a second experiment in Puerto Rico, a significant reduction in tomato root galling and a slight reduction in root galling on pepper occurred. In this trial, yields on both tomato and pepper were higher in CR-43 treatments, but these differences were not statistically significant. In both experiments populations of Rotylenchulus reniformis were reduced by CR-43 treatment. In a field trial on strawberry in Massachusetts, CR-43-treated plants had lower numbers of Pratylenchus penetrans within roots and showed a significant decrease in black root rot disease. Studies on sterile filtrates from CR-43 cultures indicated that a major determinant of CR-43 antinematodal activity was mostly thermostable macromolecules of molecular weight higher than 6000. Culture filtrates of CR-43 exhibited antifungal activity in vitro.

Almotayri, A. et al. (2019) International Worm Meeting "Culinary spices reduce the body fat content of Caenorhabditis elegans."

Thomas, J., Almotayri, A., Jois, M., Munasinghe, M.

[International Worm Meeting, 2019]

Oxidative stress and inflammation are critical components in the development of obesity. Culinary herbs and spices, abundant in anti-oxidant and anti-inflammatory phytochemicals, may be useful in the prevention of obesity. In the present study, we report strong anti-obesity properties of culinary spices. Wild-type Bristol N2 strain (Caenorhabditis Genetics Centre) were grown on NGM plates seeded with E. coli (OP50). Worms were exposed to extract (1mg of dry powder per 1ml of NGM) of red chilli, black pepper, ginger, and turmeric. Fat content was determined on day 5 (adult stage) by staining with Oil-Red O. Compared to control group, chilli, ginger and pepper reduced the fat content of the worms by 92, 89 and 57% respectively. Turmeric had no effect on the fat content of C elegans. In conclusion, culinary herbs and spices may be useful in the prevention of obesity and C. elegans is a useful model for screening the anti-obesity properties of culinary spices.

Dalfo, Diana et al. (2009) International Worm Meeting "RNAi-based identification of genes involved in germline proliferation."

Dalfo, Diana, Albert Hubbard, E. Jane

[International Worm Meeting, 2009]

Precise control of cell proliferation is necessary to create organs of the correct size and shape. We are interested in understanding the molecular basis for the control cell proliferation in vivo, and we are using the C. elegans germ line as a model. In C. elegans, as in other organisms, germline proliferation is a key step in the development of a reproductively competent individual. Previously, we isolated mutants that disrupt the pattern of germline development and ultimately produce a large ectopic mass of proliferating germ cells (a germline tumor) in the proximal part of the gonad arm (Pepper et al., 2003a,b; Killian and Hubbard, 2004; Voutev et al., 2006). This phenotype, proximal proliferation (Pro), is observed with certain mutant alleles of glp-1 (Pepper et al., 2003). glp-1 encodes a receptor of the LIN-12/Notch family, and its activity promotes the proliferative or undifferentiated state and/or inhibits differentiation (meiosis) in the germ line (Austin and Kimble, 1987; Yochem and Greenwald, 1989). We are performing a genome-wide RNAi screen for enhancers of the Pro phenotype to look for factors that affect early germline proliferation and initial meiotic entry.

Nkambeu B et al. (2020) Neurochem Res "Capsaicin and Its Analogues Impede Nocifensive Response of Caenorhabditis elegans to Noxious Heat."

Beaudry F, Nkambeu B, Salem JB

[Neurochem Res, 2020]

Capsaicin is the most abundant pungent molecule identified in red chili peppers, and it is widely used for food flavoring, in pepper spray for self-defense devices and recently in ointments for the relief of neuropathic pain. Capsaicin and several other related vanilloid compounds are secondary plant metabolites. Capsaicin is a selective agonist of the transient receptor potential channel, vanilloid subfamily member 1 (TRPV1). After exposition to vanilloid solution, Caenorhabditis elegans wild type (N2) and mutants were placed on petri dishes divided in quadrants for heat stimulation. Thermal avoidance index was used to phenotype each tested C. elegans experimental groups. The data revealed for the first-time that capsaicin can impede nocifensive response of C. elegans to noxious heat (32-35 C) following a sustained exposition. The effect was reversed 6 h post capsaicin exposition. Additionally, we identified the capsaicin target, the C. elegans transient receptor potential channel OCR-2 and not OSM-9. Further experiments also undoubtedly revealed anti-nociceptive effect for capsaicin analogues, including olvanil, gingerol, shogaol and curcumin.

Hsu TY et al. (2018) Methods Mol Biol "The Use of Caenorhabditis elegans to Study Progranulin in the Regulation of Programmed Cell Death and Stress Response."

Kao AW, Hsu TY, Butler VJ

[Methods Mol Biol, 2018]

The nematode Caenorhabditis elegans (C. elegans) has proven to be a powerful model organism for the study of many biological processes, with major implications for human health and disease. As progranulin is a pleiotropic, secreted protein with both cell autonomous and non-autonomous roles, a multicellular organism such as C. elegans is ideal for the investigation of its normal function and pathological effects. The C. elegans genome contains a progranulin-like gene known as pgrn-1. The nematode pgrn-1 encodes a protein with three cysteine-rich granulin domains, compared to the seven and a half granulins in the human protein. We have shown that C. elegans mutants lacking pgrn-1 appear grossly normal, but exhibit accelerated apoptotic cell engulfment as well as a stress resistance phenotype (Kao et al., Proc Natl Acad Sci U S A 108:4441-4446, 2011; Judy et al., PLoS Genet 9:e1003714, 2013). In addition, the roles of individual granulins can also be dissected in C. elegans (Salazar et al., J Neurosci 35:9315-9328, 2015). Here, we describe methods for studying apoptosis and stress response in C. elegans.