Burns RH (1996) East Coast Worm Meeting "Sub-lethal chronic exposure to lead or cadmium affects fecundity and development of Caenorhabditis elegans"

Burns RH

[East Coast Worm Meeting, 1996]

The effects of chronic exposure to two heavy metals, lead and cadmium, were studied for the nematode, Caenorhabditis elegans. Ambient exposure to two concentrations of each metal was examined. Lead concentrations were 0.5 ug/l or 5 ug/l. Cadmium concentrations were 10 ug/l or 100 ug/l. For each metal, the later concentration is the highest for which no significant increase in lethality was noted. The lower concentration is varied from the higher by one order of magnitude. Number of progeny produced per hour of egg-laying was determined for nematodes raised on plates containing either lead or cadmium. Nematodes raised on lead showed no difference in average progeny produced per hour of egg-laying at either concentration when subjects were considered as a group. However, two populations of worms were observed. One group produced more progeny than controls apparently due to the dumping of eggs in large number. The other group produced zero or very few progeny. The number of nematodes producing no offspring within the time allotted for egg-laying was substantially greater than for controls. Chronic exposure to cadmium produced a significant decrease in progeny produced per hour of egg-laying at both concentrations. Developmental effects were also examined. For both metals, at both concentrations, the attainment of developmental milestones was delayed. At all stages of development, size was reduced, and normal adult length was never reached. Finally, we have quantified a phenomenon noted as an annoyance in earlier experiments. Subjects sometimes had to be dropped from experimental sets because they disappeared. This occurs with greater frequency than the occasional "A.W.O.L." seen under standard conditions which suggests navigational problems or, possibly, avoidance of the contaminated environment.

Burns RH et al. (1987) International C. elegans Meeting "effects of AChE inhibitors on movement rates of wild-type and ChAT-deficient C elegans."

Russell RL, Burns RH

[International C. elegans Meeting, 1987]

Singh, Akanksha (2021) International Worm Meeting "CRISPR- Nanobodies from C. elegans as an therapeutic approach for Erythroblastosis Fetalis"

Singh, Akanksha

[International Worm Meeting, 2021]

Erythroblastosis fetalis is a consequence of incompatibility amongst Rh-negative antigen from mother and Rh-positive antigen of foetus thus resulting in hemolysis. The hemolysis usually results in respiratory problems, neurological disorders or even heart failure. C.elegans is known to be homologous to some extent with human genome and produces monoclonal antibodies. The nanobodies from C. elegans are engineered with CRISPR Cas 9 technology where Cas 9 protein has been incorporated into nanobodies that will accelerate its efficiency. The engineered nanobodies will target genes like RHD and RhCE that are prominent in RBC membranous environment. The nanobodies with Cas-9 might block the interaction of Rh-negative antibodies with Rh-positive antibodies of foetus as by refusing the recruitment of Rh-negative antibodies in placenta. Antibodies from the C. elegans are extracted and engineered to produce nanobodies with encoded Cas 9 protein. The haemoglobin obtained from foetus are mixed with haemoglobin from mother with Rh-antigens in vitro. After a period of 24-48 hours, when engineered nanobodies are treated with blood by ELISA technique indicated reduced count of Rh-positive antibodies thus blocking RHD gene not allowing the interaction with Rh-negative antibodies. The aim of the study was to study molecular interaction occurring after exposure of nanobodies to Rh- antigens present in foetal blood. Keywords: Erythroblastosis fetalis, Rh-negative, hemolysis, C. elegans, CRISPR Cas 9, RHD , RHCE, haemoglobin, nanobodies.

Flavia I Pellerone et al. (2003) International Worm Meeting "Investigation of the functional role of Rh proteins in the nematode Caenorhabditis elegans."

Susan M Howitt, Flavia I Pellerone, Carolyn A Behm, Inge J Stewart

[International Worm Meeting, 2003]

Rhesus (Rh) factors have been extensively studied as blood group antigens. Recently it has become evident that Rh proteins share ~20% homology with methylamine permeases (mep) and ammonium transporters (amt) of yeast, bacteria and plants. Non-erythroid Rh proteins, expressed in kidney and liver, have also recently been identified in mammals. This implies that Rh proteins may function as the elusive ammonium transporter in animals. Putative Rh homologues also occur in lower organisms (green algae, slime mold) and invertebrates such as Caenorhabditis elegans and Drosophila melanogaster. Thus Rh proteins have functions not restricted to red blood cells. The model nematode C. elegans expresses four amt genes, plus two Rh genes (rhr-1 and rhr-2) that share significant identity with human RhAG genes. The considerable biological resources of C. elegans provide an excellent tool for studying the functions of these genes. To test whether the Rh proteins transport ammonium we have commenced complementation studies in a yeast strain lacking all three native ammonium transporters. In initial experiments, rhr-2 cDNA failed to complement but further work is under way. We will also undertake functional characterization of the Rh proteins by expressing them in Xenopus oocytes. We have used RNA interference to investigate the function of these proteins. Silencing expression of rhr-1 and rhr-2, individually or together, reduced mRNA expression by ~80% but induced no obvious phenotype in C. elegans. However, two dimensional gel electrophoresis of protein extracts from these worms has revealed differentially expressed proteins, which are undergoing further analysis.

Tanaka, R. et al. (2013) International Worm Meeting "Quiescence of entomo-phoretic nematode Caenorhabditis japonica."

Kanzaki, N., Hirooka, Y., Kikuchi, T., Tanaka, R.

[International Worm Meeting, 2013]

Entomo-phoretic nematodes are not parasitic to insects but use insects for their transportation. Caenorhabditis japonica, a bacteria-feeding nematode, has a species-specific phoresy with a shield bug, Parastrachia japonensis, and its lifecycle is synchronized to the bug's life. The active dauer larvae (DL) show intensive host-seeking, high sensitivity to oxidative stress and less than 15 days of longevity without attaching the bug. On the other hand, quiescent DL on the bug survive more than 11 months. Thus, the quiescence associated with the bug seems an essential factor for nematode survivability. In the present study, survivabilities of quiescent and active DL were compared under several different conditions to examine the involvement of the bugs in nematode's longevity. Then transcripts and proteins were analyzed to compare gene expression between quiescent and active DL. The quiescent DL on the bug and active DL were kept in a container with 85% (dehydrated condition) or 97% (lightly dehydrated condition which immobilize the DL) of relative humidity (RH). The most active DL died in one week because of desiccation (85% RH) or fungal infection (97% RH). On the other hands, quiescent DL on the bug showed significantly higher survival rate under the same conditions. Further, the survivability of surface-sterilized active DL kept in 97% RH was almost the same as quiescent DL. Therefore, the bugs are likely to work as the shelter from dehydration, and are also providing anti-microbe activity to DL. The expressed genes and proteins also differed between quiescent and active DL. Expression of genes and proteins involved in several stress resistance, metabolic regulation and cuticle formation were significantly higher in quiescent DL. On the other hand, expression of genes and proteins involved in several metabolic related (activity regulation) were significantly higher in active DL. Our results suggest C. japonica DL use their host bug not only for transportation, but also as shelter from environmental stresses and microbe infection. Further, the quiescent stage-specific gene expression allows the extraordinary longevity of this stage.

Nettell JJ et al. (1998) European Worm Meeting "EXPRESSION PATTERN OF C. ELEGANS RH (RHESUS) HOMOLOGUES -AN ATTEMPT TO ASSIGN FUNCTION TO HUMAN RH PROTEINS"

Nettell JJ, Tanner MJ, Kuwabara PE

[European Worm Meeting, 1998]

Rh antigens are highly immunogenic in man and are important in clinical haematology and blood transfusions. They form the major cause of haemolytic disease of the newborn in caucasian populations. The antigens are expressed on two 30kDa non-glycosylated membrane proteins (Rh30) and complex with a glycoprotein (Rhgp) of very close homology. The function of these erythroid specific proteins is unknown. In an attempt to elucidate the function of Rh we have been studying two recently discovered Rhgp homologues in the nematode C. elegans (CERH1/2). We have begun to localise the expression of both genes by constructing GFP-tagged promoter vectors containing a nuclear localisation signal (NLS). The gonad of N2 hermaphrodite C. elegans was injected and double rol-6/GFP transformed worms were visualised by laser confocal microscopy. GFP fluorescence, resulting from CERH promoter activity, indicated that expression predominates in seam cells and in some of the large polyploid nuclei of the intestine. The results also suggest CERH1 and CERH2 co-localise in these cell types. Further work should confirm this co-localisation and determine protein targeting by protein-GFP fusion constructs. Immunocytochemistry, using prepared anti-CERH peptide antibodies, will further investigate such expression patterns.

Vasquez-Rifo, Alejandro et al. (2015) International Worm Meeting "A Novel bacterial factor involved in the infection of Caenorhabditis elegans by Pseudomonas aeruginosa."

Vasquez-Rifo, Alejandro, Moore, Melissa, Ambros, Victor, Ricci, Emiliano

[International Worm Meeting, 2015]

Pseudomonas aeruginosa (P. aeruginosa) is widely recognized as a human pathogenic bacteria, responsible for life threatening infections in the context of cystic fibrosis, burns, and in immunocompromised patients. P. aeruginosa can also lethally infect the nematode Caenorhabditis elegans (C. elegans), using virulence processes and mechanisms that it also deploys against mammalian hosts. P. aeruginosa virulence factors include enzymes that promote tissue damage such as elastase, collagenase and phospholipase C, toxic bacterial metabolites (siderophores), and enzymes (such as endotoxin A) that inactive physiological processes of the host cells required for an adequate immune response, such as protein translation.We are investigating how P. aeruginosa infection of C.elegans impinges on distinct classes of worm RNAs. We observed that upon infection, worm RNA profiles are significant altered. In particular, this profiling evidenced a novel RNA nuclease activity. The activity does not require the action of the host pmk-1/p38 or apoptotic pathways, and,our analysis indicates that the RNA nuclease responsible for the observed activity is encoded by the bacteria and is likely actively secreted into the host cells. A bacterial mutant that does not induce the nuclease activity displays attenuated virulence against C. elegans suggesting a role for the nuclease as a virulence factor. We are currently characterizing this nuclease to define its precise roles during infection.

Aisa Nakashima et al. (2008) European Worm Meeting "Two new germ cell immortality mutants"

Jan Laroque, Theresa M Zucchero, Julie Hall, Aisa Nakashima, Shawn Ahmed, Lauren Garwood

[European Worm Meeting, 2008]

Germ cells represent canonical stem cells that possess the remarkable. quality of being able to proliferate from one generation to the next,. indefinitely, free of replicative damage. mortal germline mutants initially. display normal levels of fertility, but become progressively sterile when. grown for multiple generations. Of 16 original mortal germline mutants (1),. most were temperature-sensitive and only became sterile when grown at 25oC.. Of the ts mutants, two were resistant to RNAi feeding constructs targeting. embryonic lethal genes at all temperatures: 1c and 10i. The 10i mutation. mapped to the right arm of chromosome IV, between unc-22 and dpy-4, and. fails to complement rsd-2 for its RNAi resistance phenotype. rsd-2 has been. reported to be is deficient for spreading of dsRNA-mediated RNAi from. somatic cells to germ cells (2), suggesting that this function may be. relevant to maintaining germ cells over multiple generations during growth. at high temperatures. The mechanism by which this occurs is being studied.. (1) Ahmed S, Hodgkin J (2000). MRT-2 checkpoint protein is required for. germline immortality and telomere replication in C. elegans. Nature.. (2) Tijsterman M, May RC, Simmer F, Okihara KL, Plasterk RH (2004). Genes. required for systemic RNA interference in Caenorhabditis elegans. Current. Biology.

K Brouwer et al. (2004) European Worm Meeting "MUTATOR GENES THAT PREVENT MICROSATELLITE INSTABILITY IN C. ELEGANS"

J Pothof, R Plasterk, M Tijsterman, K Brouwer

[European Worm Meeting, 2004]

Proper functioning of DNA repair and signaling pathways that maintain the stability of the genome is essential in the prevention of cancer. To identify genes contributing to genomic stability, a frameshift reporter was developed in our lab, which was subsequently used in a genome-wide RNAi screen. This led to the identification of 61 genes that prevent an increase in genomic instability (1). Thirteen genes were shown to be specifically involved in maintaining the stability of microsatellites (short sequence repeats). Among these genes are the four genes known to be involved in the mismatch repair pathway and that are linked to the human cancer syndrome HNPCC (hereditary non-polyposis colon cancer). For three of the yet uncharacterized genes (Y76A2B.5, K09B11.2 and rev-1) we have isolated null alleles. We are currently working on the characterization of these genes by performing epistasis experiments with the mismatch repair pathway and analyzing the mutator phenotypes of the mutants in further detail. 1 Pothof J, van Haaften G, Thijssen K, Kamath RS, Fraser AG, Ahringer J, Plasterk RH, Tijsterman M., Identification of genes that protect the C. elegans genome against mutations by genome-wide RNAi. Genes Dev. 2003 Feb 15;17(4):443-8.

Kirienko, Daniel et al. (2013) International Worm Meeting "A C. elegans-P. aeruginosa Liquid Assay Identifies Novel Small Molecules with Anti-Infective Properties."

Kirienko, Daniel, Kirienko, Natalia, Ausubel, Frederick

[International Worm Meeting, 2013]

Pseudomonas aeruginosa is a Gram-negative bacterium that causes serious, life-threatening infections for patients with burns, compromised immune systems, medical implants, or cystic fibrosis. Problematically, the bacterium is ubiquitous and exhibits, or readily acquires, resistance to many antimicrobials. These factors make it a serious medical concern, and development of novel anti-infectives is a major human health priority. C. elegans is a useful model host for P. aeruginosa infection. Many innate immune pathways are shared by C. elegans and humans, despite the relative simplicity of the innate immune system of the former. Moreover, many of the same Pseudomonas virulence mechanisms are used during infection of C. elegans and humans, making it an attractive option for studying host-pathogen interactions. We developed a liquid-based, C. elegans-P. aeruginosa pathogenesis assay to permit high-throughput screening of small molecules. Approximately 86,000 compounds were tested for the ability to ameliorate C. elegans killing. 202 hits were recovered, giving an overall hit rate of 0.23%. 93 hits were known antibiotics, 32 were molecules with known (but non-antibiotic) functions, and 70 were compounds with unknown functions. Using additional assays, we further investigated 55 of the 70 compounds with unknown functions. Interestingly, 21 of these compounds also rescued C. elegans from a lethal infection with Enterococcus faecalis, a Gram-positive pathogen. Furthermore, 32 of these compounds rescued at concentrations substantially lower than what was necessary to inhibit bacterial growth. The compounds appear to act via several separable mechanisms, as they exhibit differential abilities to rescue infections of P. aeruginosa on solid media, E. faecalis infections in liquid media, and P. aeruginosa-mediated pathogenesis in liquid. Several of these molecules are likely to promote immune stimulation, which may translate to mammalian pathogenesis.