Schwartz M C et al. (2010) Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI "HMT-1 and Coelomocytes Detoxify Multiple Heavy Metals"

Schwartz M C, Sharma A K, Vatamaniuk O K, Benci J L, Fares H, Dang H, Selote D S, Chen A GY

[Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI, 2010]

Our past studies have established that pcs-1 and hmt-1, encoding phytochelatin (PC) synthase and a half molecule ATP binding cassette (ABC) transporter respectively, are acutely required for the detoxification of Cd2+, but despite what was thought previously, do not act in concert in heavy metal detoxification (Vatamaniuk et al 2001, 2005). Here, we report that in addition to Cd2+, pcs-1 and hmt-1 confer tolerance to As3+ and Cu2+. Consistent with the notion that pcs-1 and hmt-1 operate in distinct metal detoxification pathways, pcs-1;hmt-1 double mutants were more sensitive to these metals than pcs-1 or hmt-1 single mutants, and were expressed in distinct tissues: hmt-1 was expressed in intestinal cells and head and tail neurons, whereas pcs-1 was expressed in pharyngeal grinder, pharyngeal-intestinal valve, somatic and vulval muscles. Interestingly, pcs-1 and hmt-1 were co-expressed in coelomocytes. Since the biological role of coelomocytes in C. elegans is not known, the latter finding was intriguing and raised the possibility that coelomocytes are involved in heavy metal detoxification. We tested this hypothesis, by analyzing the heavy metal sensitivity of the coelomocyte-deficient mutant strain, NP717. In doing so we established that NP717 worms were sensitive to Cd2+, Cu2+ and As3+ but not to reactive oxygen species, H2O2. Since coelomocytes deficiency did not increase the sensitivity of worms to H2O2, we concluded that these cell types detoxify heavy metals, but not the oxidative stress, a consequence of many stresses as well as heavy metal toxicity. We also showed that although hmt-1 may function in coelomocytes, it acted mainly outside these cell types in metal detoxification. Since HMT-1 detoxified multiple heavy metals and was expressed in liver-like cells, the coelomocytes, as well as head neurons and intestinal cells, which are the cell types that are affected by heavy metal poisoning in humans and since coelomocytes are involved in metal detoxification, the HMT-1-dependent detoxification pathway and coelomocytes emerge as novel models for studies of heavy metal-promoted diseases.

Wypijewska A et al. (2012) Biochemistry "7-methylguanosine diphosphate (m(7)GDP) is not hydrolyzed but strongly bound by decapping scavenger (DcpS) enzymes and potently inhibits their activity."

Wypijewska A, Darzynkiewicz E, Davis RE, Jemielity J, Bojarska E, Lukaszewicz M, Stepinski J

[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.

O'Connell EM et al. (2015) J Infect Dis "Targeting Filarial Abl-like Kinases: Orally Available, Food and Drug Administration-Approved Tyrosine Kinase Inhibitors Are Microfilaricidal and Macrofilaricidal."

Nutman TB, O'Connell EM, Bennuru S, Steel C, Dolan MA

[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.

Yang B et al. (2020) Genes Dev "In vivo CRISPR screening for phenotypic targets of the mir-35-42 family in C. elegans."

Yang B, Schwartz M, McJunkin K

[Genes Dev, 2020]

Identifying miRNA target genes is difficult, and delineating which targets are the most biologically important is even more difficult. We devised a novel strategy to test the phenotypic impact of individual microRNA-target interactions by disrupting each predicted miRNA-binding site by CRISPR-Cas9 genome editing in <i>C. elegans</i> We developed a multiplexed negative selection screening approach in which edited loci are deep sequenced, and candidate sites are prioritized based on apparent selection pressure against mutations that disrupt miRNA binding. Importantly, our screen was conducted in vivo on mutant animals, allowing us to interrogate organism-level phenotypes. We used this approach to screen for phenotypic targets of the essential <i>mir-35-42</i> family. By generating 1130 novel 3'UTR alleles across all predicted targets, we identified <i>egl-1</i> as a phenotypic target whose derepression partially phenocopies the <i>mir-35-42</i> mutant phenotype by inducing embryonic lethality and low fecundity. These phenotypes can be rescued by compensatory CRISPR mutations that retarget <i>mir-35</i> to the mutant <i>egl-1</i> 3'UTR. This study demonstrates that the application of in vivo whole organismal CRISPR screening has great potential to accelerate the discovery of phenotypic negative regulatory elements in the noncoding genome.

Yang B et al. (2021) Genes Dev "Corrigendum: In vivo CRISPR screening for phenotypic targets of the mir-35-42 family in C. elegans."

McJunkin K, Schwartz M, Yang B

[Genes Dev, 2021]

Vuong, Thanh TK et al. (2013) International Worm Meeting "Mechanical forces in C. elegans embryo elongation."

Labouesse, Michel, Vuong, Thanh TK

[International Worm Meeting, 2013]

Embryo development requires precise coordination of mechanical forces and their failure can lead to diseases. During the morphogenesis of C. elegans embryo, the cooperation of epidermal acto-myosin network and muscle contractions is essential. The acto-myosin activity in the epidermis, which has been shown to be more important in lateral than in dorsal-ventral cells, squeeze the embryo and make it elongate [1]. Muscle contractions become active around 1.7-1.8 fold stage. They have been showed to induce a mechano-transduction pathway [2], which is important for elongation. However, it is unclear how the contractions along the anterior-posterior axis help to increase the length of the embryo. Our project aims to elucidate the mechanical role of muscle contractions and its coordination with acto-myosin forces. The experiments are designed following a working model where muscle contractions induce a change in the elasticity of the embryo. We are using a laser nano-dissection technique to investigate cortical tension and elasticity of epidermal cells before and after the onset of muscle contractions. In parallel, we are evaluating the relative changes of acto-myosin forces with a FRET sensor [3] inserted in HMP-1 - a component of the adherens junctions. I will present our observations and preliminary results of the epidermal cortex nano-dissection experiments and measures of acto-myosin forces exerted on adherens junctions. References 1.Gally C, Wissler F, Zahreddine H, Quintin S, Landmann F, Labouesse M. Myosin II regulation during C. elegans embryonic elongation: LET-502/ROCK, MRCK-1 and PAK-1, three kinases with different roles. Development. 2009 Sep;136(18):3109-19. Epub 2009 Aug 12. 2.Zhang H, Landmann F, Zahreddine H, Rodriguez D, Koch M, Labouesse M. A tension-induced mechanotransduction pathway promotes epithelial morphogenesis. Nature. 2011 Mar 3;471(7336):99-103. 3.Grashoff C, Hoffman B, Brenner M, Zhou R, Parsons M, Yang M, McLean M, Sligar S, Chen C, Ha T, Schwartz M. Measuring mechanical tension across vinculin reveals regulation of focal adhesion dynamics. Nature. 2010 July 8; 466(7303): 263-266.

Wood WB (1976) Worm Breeder's Gazette "maternal effects among ts zygote-defective (zyg) mutants."

Wood WB

[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.

Ali Khan L et al. (1994) Worm Breeder's Gazette "cej-1 Encodes a Novel Protein With Poly-Threonine Motif"

Siddiqui SS, Fukushige T, Ali Khan L, Tsukita Sh, Tabish M, Itoh M

[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.

Lemire BD et al. (2009) Mech Ageing Dev "C. elegans longevity pathways converge to decrease mitochondrial membrane potential."

Behrendt M, Decorby A, Lemire BD, Gaskova D

[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.

Seo J et al. (2005) Arch Environ Contam Toxicol "Biodegradation of the insecticide N,N-diethyl-m-toluamide by fungi: identification and toxicity of metabolites."

Kim SD, Cha CJ, Hur HG, Lee YG, Seo J, Ahn JH

[Arch Environ Contam Toxicol, 2005]

Fungi (Cunninghamella elegans ATCC 9245, Mucor ramannianus R-56, Aspergillus niger VKMF-1119, and Phanerochaete chrysosporium BKMF-1767) were tested to elucidate the biologic fate of the topical insect repellent N,N-diethyl-m-toluamide (DEET). The elution profile obtained from analysis by high-pressure liquid chromatography equipped with a reverse-phase C-18 column, showed that three peaks occurred after incubation of C. elegans, with which 1 mM DEET was combined as a final concentration. The peaks were not detected in the control experiments with either DEET alone or tested fungus alone. The metabolites produced by C. elegans exhibited a molecular mass of 207 with a fragment ion (m/z) at 135, a molecular mass of 179 with an m/z at 135, and a molecular mass of 163 with an m/z at 119, all of which correspond to N,N-diethyl-m-toluamide-N-oxide, N-ethyl-m-toluamide-N-oxide, and N-ethyl-m-toluamide, respectively. M. ramannianus R-56 also produced N, N-diethyl-m-toluamide-N-oxide and N-ethyl-m-toluamide but did not produce N-ethyl-m-toluamide-N-oxide. For the biologic toxicity test with DEET and its metabolites, the freshwater zooplankton Daphnia magna was used. The biologic sensitivity in decreasing order was DEET > N-ethyl-m-toluamide > N,N-diethyl-m-toluamide-N-oxide. Although DEET and its fungal metabolites showed relatively low mortality compared with other insecticides, the toxicity was increased at longer exposure periods. These are the first reports of the metabolism of DEET by fungi and of the biologic toxicity of DEET and its fungal metabolites to the freshwater zooplankton D. magna.