Dana L. Miller et al. (2007) International Worm Meeting "Adaptation to hydrogen sulfide increases thermotolerance and lifespan."

Mark B. Roth, Dana L. Miller

[International Worm Meeting, 2007]

Hydrogen sulfide (H₂S), which is naturally produced in animal cells, has been shown to effect physiological changes that improve the capacity of mammals to survive environmental changes. We have investigated the physiological response of C. elegans to H₂S to begin to elucidate the molecular mechanisms of H₂S action. We show that nematodes exposed to H₂S are apparently healthy and do not exhibit phenotypes consistent with metabolic inhibition. However, we observed that animals exposed to H₂S had increased thermotolerance and lifespan and survived subsequent exposure to otherwise lethal concentrations of H₂S. Increased thermotolerance and lifespan is not observed in the sir-2.1(ok434) deletion mutant exposed to H₂S. However, mutants in the insulin signaling pathway (both daf-2 and daf-16), animals with mitochondrial dysfunction (isp-1 and clk-1) and a genetic model of caloric restriction (eat-2) all exhibit H₂S-induced increased thermotolerance. These data suggest that H₂S activates a pathway including SIR-2.1 that is separate from dietary restriction and insulin signaling that results in increased lifespan. Moreover, these studies suggest that SIR-2.1 activity may translate environmental change into physiological alterations that improve survival. It is interesting to consider the possibility that the mechanisms by which H₂S increases thermotolerance and lifespan in nematodes are conserved, and that studies using C. elegans may help explain beneficial effects observed in mammals exposed to H₂S.

Abergel, Z. et al. (2017) International Worm Meeting "Adaptation of the nematode C. elegans to hypoxia and reoxygenation stress reveals an unexpected function of the neuroglobin GLB-5 in innate immunity."

Zuckerman, B., Zelmanovich, V., Abergel, Z., Abergel, R., Gross, E., Smith, Y., Romero, L., Livshits, L.

[International Worm Meeting, 2017]

Deprivation of oxygen (hypoxia) followed by reoxygenation (H/R stress) is a major component in several pathological conditions such as vascular inflammation, myocardial ischemia, and stroke. However how animals adapt and recover from H/R stress remains an open question. Previous studies showed that the neuroglobin GLB-5(Haw) is essential for the fast recovery of the nematode Caenorhabditis elegans (C. elegans) from H/R stress. Here, we characterize the changes in neuronal gene expression during the adaptation of worms to hypoxia and recovery from H/R stress. Our analysis shows that innate immunity genes are differentially expressed during both adaptation to hypoxia and recovery from reoxygenation stress. Moreover, we reveal that the prolyl hydroxylase EGL-9, a known regulator of both adaptation to hypoxia and the innate immune response, inhibits the fast recovery from H/R stress through its activity in the O2-sensing neurons AQR, PQR, and URX. Finally, we show that GLB-5(Haw) acts in AQR, PQR, and URX to increase the tolerance of worms to bacterial pathogenesis. Together, our studies suggest that innate immunity and recovery from H/R stress are regulated by overlapping signaling pathways.

Hill, Robin L. (2011) International Worm Meeting "Characterizing wild nematode isolates in an undergraduate research lab."

Hill, Robin L.

[International Worm Meeting, 2011]

Small Public Liberal Arts Colleges pose multiple challenges, particularly in teaching laboratory courses. Resources are limited and the majority of students have no lab experience. In addition, faculty struggle to maintain a full-time active research program due to heavy teaching loads. A significant percentage of students expressing an interest in the sciences also express a desire for graduate or professional studies. As many of these programs desire "hands-on" scientific experience, providing research opportunities could be vital to their future success. To address these issues, a full semester research-based project was developed around isolating and identifying wild nematode isolates. An overview of nematode phylogeny and population genetics studies is coupled with students working in small groups to develop a research project, isolate nematodes and characterize the isolates using morphological and molecular analysis. Results are presented in a format similar to graduate lab meetings. The molecular techniques utilized during the pilot project included species-specific PCR (glp-1) (1) as well as sequencing of the 18S ribosomal RNA gene (2). I adapted universal rice primers (URP) as a fingerprint assay (3). Morphological analysis included buccal (lips, buccal tube, pharynx) and tail (shape, papillae and spicule structure) morphology as well as mating system (gonochoristic vs. hermaphroditic). Strains from the CGC have been used for comparison (both morphological and molecular) to wild isolates. Mating crosses with C.elegans and C.briggsae males have been performed with hermaphroditic species. Males from gonochoristic strains can be employed for gonochoristic isolates. Hermaphroditic isolates were obtained from leaf litter in wooded settings, open grasslands and under a rotting pumpkin. Species-specific PCR as well as mating crosses reject identification as C.elegans or C.briggsae. Sequencing of the 18S RNA gene is ongoing. Gonorchorisitc isolates were obtained from both household and grass cutting compost. Morphological analysis based on tail morphology suggests the genus Rhabditis, subgenus Rhabditella and Cephalopoides. Student feedback was uniformly positive. Isolates were easily obtained and the self-directed nature of the projects deepened their understanding of lab methods and research design. The range of analysis methods allows the course to be adapted to resource availability, therefore providing a method of research experience while enhancing knowledge of the model species. (1) Barriere, A., and Felix M.A. (2005). Curr. Biol. 15,1176-184. (2) Floyd, R. et al. (2002). Mol Ecol. 11, 839-850. (3) Kang, H. W. et al. (2002). Mol. Cells. 2, 281-287.

Rasika Kalamegham et al. (2004) East Coast Worm Meeting "EGL-26 belongs to the NlpC/P60 superfamily of putative enzymes and is closely related to a mammalian acyl transferase"

Wendy Hanna-Rose, Rasika Kalamegham

[East Coast Worm Meeting, 2004]

egl-26 was identified in a genetic screen to uncover mutants with vulval morphology defects. In egl-26 mutants the morphology of a single vulval toroid (vulF) is abnormal and a proper connection to the uterus is not made leading to the egg-laying defect. EGL-26 is a member of the NlpC/P60 superfamily of enzymes, which is characterized by a Histidine containing domain and a Cysteine containing domain (H-box and NC domain, respectively). EGL-26 along with other eukaryotic proteins belongs to a distinct subclass of NlpC/P60-related putative enzymes. The mammalian proteins lecithin: retinol acyltransferase or LRAT and H-ras revertant 107 or H-Rev107 are the most closely related to EGL-26. Both LRAT and H-Rev107 contain putative transmembrane domains in addition to the H-box and NC domains. Although EGL-26 contains no putative transmembrane domains, it is localized at the apical membrane of cells where it is expressed. Proper localization of LRAT within the retinal pigment epithelium is essential for its function. Significantly, an S-F substitution at amino acid 275 of EGL-26 found in the egl-26 (n481) allele causes mislocalization of an EGL-26::GFP fusion leading to general cytoplasmic expression as opposed to normal apical membrane localization. The corresponding Serine residue is conserved in both LRAT and H-Rev107. We are attempting to analyze the relationship between the mammalian proteins and EGL-26 by attempting a rescue of egl-26 mutants by expression of either LRAT or H-Rev107 or both. We plan to test the importance of membrane localization by restoring membrane localization to EGL-26n481 via addition of alternative membrane localization signals.

Schwartz, Hillel et al. (2013) International Worm Meeting "Developing Heterorhabditis nematodes as an experimental system for the study of mutualistic symbiosis."

Schwartz, Hillel, Sternberg, Paul

[International Worm Meeting, 2013]

Entomopathogenic nematodes of the genus Heterorhabditis are insect killers that live in mutually beneficial symbiosis with pathogenic Photorhabdus bacteria. Photorhabdus is rapidly lethal to insects and to other nematodes, including C. elegans, but is required for Heterorhabditis growth in culture and for the insect-killing that defines the entomopathogenic lifestyle. The symbiosis between Heterorhabditis and Photorhabdus offers the potential to study the molecular genetic basis of their cooperative relationship. We developing tools to make such studies more feasible: we have been studying multiple nematodes of the genus Heterorhabditis and developing tools for the molecular genetic analysis of Heterorhabditis bacteriophora. Many species of Heterorhabditis and variants of Photorhabdus have been isolated; some pairings show specificity in their ability to establish a symbiotic relationship. To better understand these interactions and other variations in the lifestyles of Heterorhabditis, we have sequenced H. indica, H. megidis, H. sonorensis, and H. zealandica; a H. bacteriophora genome sequence is available. A comparison of these closely related species may help us to identify mechanisms that regulate the response to bacterial interactions and to find variations that correlate with differences in lifestyle or bacterial compatibility. In addition to genomics, we are developing H. bacteriophora as a laboratory organism. H. bacteriophora grows well on plates, has been reported to be susceptible to RNAi and transgenesis, and can develop as a selfing hermaphrodite, and so should be a powerful system for the molecular genetic study of the aspects of biology to which it is uniquely well suited, most prominently symbiosis. This potential is severely diminished by inconvenient sex determination: the self-progeny of hermaphrodites are mostly females with some males; at low density, their progeny are almost exclusively females. We have screened for and isolated a constitutively hermaphroditic mutant for use in molecular genetic studies of symbiosis. This mutant also offers the opportunity to explore the basis of hermaphrodite sex determination in H. bacteriophora.

Schwartz H et al. (2010) Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI "Towards a SNP map for Heterorhabditis bacteriophora"

Sternberg P, Schwartz H

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

Heterorhabditis bacteriophora is a species of insect-parasitic nematode that lives in mutually beneficial symbiosis with pathogenic Photorhabdus luminescens bacteria. P. luminescens bacteria are lethal to insects and to other nematodes, including the soil nematode Caenorhabditis elegans, but are required for H. bacteriophora growth. The symbiosis between H. bacteriophora and P. luminescens therefore offers the potential to study the molecular genetic basis of their cooperative relationship. We are interested in developing tools to make such studies more feasible; in particular, we propose to generate tools for genetic mapping in H. bacteriophora. We will test independent isolates of H. bacteriophora to ensure that the isolates are cross-fertile. We will then examine the abilities of these isolates to grow on and to become infected with different wild-type and mutant Photorhabdus bacteria. From these tests we will select an isolate on which we will use next-generation high-throughput sequencing technology for the purpose of refining the existing draft genome sequence and for the creation of a SNP map. We anticipate that this SNP map will enable us and the wider insect-parasitic nematode community to identify induced mutations and natural variations affecting the interactions between H. bacteriophora nematodes and pathogenic Photorhabdus bacteria.

Chu, Yu-De et al. (2013) International Worm Meeting "An RNAi-based screen for the DExD/H-box RNA helicases involved in Caenorhabditis elegans microRNA function."

Chan, Shih-Peng, Chen, Shin-Kai, Chu, Yu-De, Chen, Guan-Rong, Huang, Tao

[International Worm Meeting, 2013]

MicroRNAs (miRNAs) are small non-coding regulatory RNAs that regulate gene expression at the post-transcriptional level and are involved in a broad spectrum of biological processes. Rearrangements of inter- or intra-molecular RNA structures and conformational changes of ribonucleoprotein complexes in the multiple processes of the miRNA pathway have led to the involvement of RNA helicase activities but little is known so far. In eukaryotes, RNA helicases generally belong to the superfamily 2 (SF2) in helicase classification, especially the DExD/H-box helicase family. The DExD/H-box proteins have been shown in association with many cellular processes involving RNA. To better understand the possible roles of DExD/H-box RNA helicases in miRNA function, we employed RNAi screen to identify genetic interaction between C. elegans DExD/H-box RNA helicases and the let-7 miRNA, which controls the timing of cell cycle exit and terminal differentiation. In addition to the RNA helicase p72, a component of Drosha Microprocessor complex, and CGH-1 that has been reported to facilitate the function of miRNA-induced silencing complex (miRISC), we found several DExD/H-box RNA helicases, which are involved in ribosomal RNA processing, pre-mRNA splicing and mRNA surveillance, may also take part in miRNA biogenesis and/or function. (Support: National Science Council, Taiwan. NSC 100-2311-B-002-006-MY3).

Alexandre Neves et al. (2006) Development & Evolution Meeting "A Notch-GATA Transcription Code for Endoderm-Specific Gene Activation in C.elegans"

James Priess, Alexandre Neves

[Development & Evolution Meeting, 2006]

Several Notch interactions occur in rapid succession during early C.elegans embryogenesis, each resulting in a distinct fate. We have previously shown that ref-1, a gene distantly related to Drosophila E(spl), is a direct Notch target in all these interactions. We show here that ref-1 expression is controlled by multiple, Notch-dependent enhancers that are specific for each interaction. We have identified a 150bp enhancer that is specific to Notch interactions in the endoderm, and found similar enhancers with the same activity in C.briggsae and C.remanei. The endoderm enhancer contains multiple, conserved binding sites for LAG-1/Su(H) and GATA transcription factors. We demonstrate that all LAG-1/Su(H) and GATA sites are required for full activity of this enhancer. We provide evidence that a GATA transcription factor called ELT-2 is a cooperative factor for Notch in the endoderm. Ectopic expression of ELT-2 in non-endodermal lineages caused activation of the endoderm enhancer only in Notch-signaled cells, suggesting that the presence of the cooperative factor dictates in which Notch interaction a Notch-dependent enhancer becomes responsive in vivo. Previous studies in Drosophila showed that synergy between Su(H) and the bHLH transcription factor Daughterless requires a specific configuration of Su(H) sites, known as a Su(H) paired site, SPS. The endoderm enhancer contains a putative SPS. However, we find that Notch-GATA synergy does not require a SPS, and instead requires a non-SPS configuration of oriented LAG-1/Su(H) sites. Thus, it appears that that each configuration couples Notch signaling with a specific class of transcription factor.

Alan Winter et al. (2002) European Worm Meeting "The unique forms of the prolyl 4-hydroxylase complex found in C. elegans are essential for development due to their cuticular collagen modifying activity"

Antony Page, Alan Winter

[European Worm Meeting, 2002]

Unique forms of the prolyl 4-hydroxylase (P4-H) complex modify the collagen rich cuticular ECM of C. elegans. ER localised P4-H enzyme complexes hydroxylate proline residues within the Gly-X-Y repeat regions of procollagen molecules. The nematode cuticle acts as an exoskeleton and is required for maintenance of worm body morphology. Mutations in collagens forming this cuticle, and the enzymes that process and modify procollagen, cause lethality and severe alterations to body shape as illustrated by the sqt-3 and bli-4 mutant phenotypes.

Choi, Jinhee et al. (2011) International Worm Meeting "Functional toxicogenomic analysis of multi-wall carbon nanotube in Caenohabditis elegans."

Choi, Jinhee, Bruno, Maribel, Ge, Yue, Roh, Ji-Yeon

[International Worm Meeting, 2011]

In the present study, toxicity of multi-wall carbon nanotube (MWCNT) was investigated in Caenohabditis elegans using microarray and mutant analyses. Whole genome microarray was conducted to screen the global changes in C. elegans transcription profiles 4 and 24 h after MWCNT exposure. Interactome analysis was subsequently conducted on differentially expressed genes using Ingenuity Pathways Analysis (IPA) software. After 4 h exposure to MWCNT, 846 genes were differentially expressed in C.elegans, whereas 24 h after exposure, 2247 genes were affected. Interactive gene networks corresponding to Eukaryotic Initiation Factors 4 (EIF4) pathways were highly overexpressed 4 h after MWCNT exposure, whereas NF-kB pathways were downregulated 24 h after exposure. Toxicity of MWCNT was also investigated on 27 potentially stress response C.elegans mutants using survival and reproduction as endpoints. Among the tested mutants, akt-1 (AKT signaling), nsy-1, sek-1 (p38 MAPK signaling) and cep-1 (p53) mutants showed more sensitive response to MWCNT exposure than wildtype did, in terms of reproduction potential. Gene expression analysis, subsequently conducted on selected genes in MAPK, AKT, Apoptosis signaling pathways, revealed that expression of nsy, mpk-2, sgk-1 and ape-1 genes was increased in C.elegans exposed to MWCNT. Overall results suggest that MWCNT possess considerable potential of causing toxicity in C.elegans, and stress signaling pathways seem to be involved in it. Acknowledgement: This work was supported by National Research Foundation of Korea (NRF) grant (2010-0016195).