Jeong J et al. (2019) Nanotoxicology "Developing adverse outcome pathways on silver nanoparticle-induced reproductive toxicity via oxidative stress in the nematode Caenorhabditis elegans using a Bayesian network model."

Choi I, Song T, Choi J, Chatterjee N, Jeong J, Cha YK

[Nanotoxicology, 2019]

An adverse outcome pathway (AOP) is a framework that organizes the mechanistic or predictive relationships between molecular initiating events (MIEs), key events (KEs), and adverse outcomes (AOs). Previously, we intensively investigated the molecular mechanism that underlies toxicity caused by AgNPs in the nematode Caenorhabditis elegans. Using transcriptomics, functional genetics, and various molecular/biochemical tools, we identified oxidative stress as the major mechanism underlying toxicity and reproduction failure as the outcome. With this information, here we conducted a case study of building an AOP to link oxidative stress with reproductive toxicity. To validate this AOP, we filled the gaps by conducting further experiments on its elements, such as NADPH oxidase, ROS formation, PMK-1 P38 MAPK activation, HIF-1 activation, mitochondrial damage, DNA damage, and apoptosis. The establishment of a causal link between the MIE and AO is critical for the construction of an AOP. Therefore, causal relationships between each KE and AO were verified by using functional genetic mutants of each KE. By combining these experimental data with our previously published results, we established causal relationships between the MIE, KEs, and AO using a Bayesian network (BN) model, culminating in an AOP entitled 'NADPH oxidase and P38 MAPK activation leading to reproductive failure in C. elegans ( https://aopwiki.org/aops/207)' . Overall, our approach shows that an AOP can be developed using existing data and further experiments can be conducted to fill the gaps between the MIE, KEs, and the AO. This study also shows that BN modeling has the potential to identify causal relationships in an AOP.

Turaga PS et al. (2000) Infect Immun "Immunity to onchocerciasis: cells from putatively immune individuals produce enhanced levels of interleukin-5, gamma interferon, and granulocyte-macrophage colony-stimulating factor in response to Onchocerca volvulus larval and male worm antigens."

Lustigman S, Simonek SC, Bennett KE, Tierney TJ, McCarthy MC, Enyong PA, Turaga PS, Moukatte DW

[Infect Immun, 2000]

Antigen-specific interleukin-5 (IL-5), gamma interferon (IFN-gamma), and granulocyte-macrophage colony-stimulating factor (GM-CSF) responses in individuals living in an area of hyperendemicity for onchocerciasis in Cameroon were examined. The responses against antigens prepared from Onchocerca volvulus third-stage larvae (L3), molting L3 (mL3), and crude extract from adult males (M-OvAg) were compared to the responses against antigens from adult female worms and skin microfilariae. Cytokine responses for the putatively immune individuals (PI) and the infected individuals (INF) were compared. A differential cytokine profile of IL-5 (Th2 phenotype) and IFN-gamma (Th1 phenotype) was found in these individuals in response to the antigens. In both the PI and the INF, Th2 responses against all the antigens tested were dominant. However, in the PI group as a whole, there was an enhanced Th2 response against the larval antigens and the adult male and adult female antigens, and a Th1 response in a subgroup of the PI (27 to 54.5%) against L3, mL3, and M-OvAg antigens was present. While the PI produced significantly higher levels of GM-CSF against L3, mL3, and M-OvAg antigens than the INF, there was no difference in the GM-CSF responses of the groups against the other antigens. The present study indicated that, in comparison to the INF, the PI have distinct larva-specific and adult male-specific cytokine responses, thus supporting the premise that immunological studies of the PI would lead to the identification of immune mechanisms and the target genes that play a role in protective immunity.

Roussell DL et al. (1994) Parasitol Today "Germ-line determination in Caenorhabditis and Ascaris: will a helicase begin to unravel the mystery?"

Bennett KL, Roussell DL, Gruidl ME

[Parasitol Today, 1994]

How cell lineages are established during development in higher eukaryotes is being addressed by geneticists and by developmental and molecular biologists. In Drosophila melanogaster, a gene corresponding to a germ-line-specific RNA helicase, vasa, has been shown to be a component o f the posteriorly localized germ granules o f the developing embryo. A putative RNA helicase, glh-I r which appears germ-line specific in its expression, has recently been reported from the free-living nematode Caenorhabditis elegans. Parasitologists studying the nematode Ascaris lumbricoides var. suum have found it to be a useful complement to Caenorhabditis. Deborah Roussell, Michael Gruidl and Karen Bennett predict that Ascaris will be valuable in determining the role played by germ-line helicases in development.

KA Kuznicki et al. (1999) International C. elegans Meeting "Unwinding the P granules: evidence from RNAi and glh mutants"

KL Bennett, JW Kirchner, PA Smith, KA Kuznicki

[International C. elegans Meeting, 1999]

The Bennett laboratory studies the four g erm l ine RNA h elicases (GLHs) , components of the C. elegans P granules. The GLHs are members of the DEAD-box family of RNA helicases and contain retroviral-like CCHC zinc fingers and uncharged glycine-rich repeats (Roussell and Bennett 1993 PNAS 90 :9300-9304, Gruidl et al . 1996 PNAS 93 :13837-13842). GLH-3 and GLH-4, more recently discovered helicases, differ from GLH-1 and GLH-2; GLH-3 lacks the uncharged glycine-rich repeats of the others, while GLH-4 is the most unique. RNAi implies that each GLH has a different role. RNAi of glh-1 or glh-2 produces sterility in a small percentage of the F 1 generation at the restrictive temperature. By contrast, glh-3 RNAi causes smaller brood sizes in the injected worm at the restrictive temperature. Using a combination of the helicase regions of glh-1 , glh-2 , and glh-3 results in a more dramatic temperature sensitive phenotype. Approximately 75% of the F 1 generation is infertile. Other combinatorial glh RNAi injections will further determine if the glhs function synergistically. A glh-3 - strain with a 1.7 kb deletion was identified in our laboratory from a TMP/UV library we generated. Preliminary analysis of the glh-3 - strain indicates that it has a mild temperature sensitive fertility defect, with brood size approximately 60-70% of that seen with wild type worms at 25-26degC. This result is consistent with that seen in the glh-3 RNAi experiments. Ongoing experiments include glh-4 RNAi and screening for additional glh mutants.

MacDonald AJ et al. (2002) Infect Immun "Differential cytokine and antibody responses to adult and larval stages of Onchocerca volvulus consistent with the development of concomitant immunity."

MacDonald AJ, Bennett KE, Simonek SC, Enyong PA, Lustigman S, Harmon-Brown C, Tierney TJ, McCarthy MC, Turaga PS, Moukatte DW

[Infect Immun, 2002]

The possibility of concomitant immunity and its potential mechanisms in Onchocerca volvulus infection were examined by analyzing cytokine and antibody responses to infective larval (third-stage larvae [L3] and molting L3 [mL3]), adult female worm (F-OvAg), and skin microfilaria (Smf) antigens in infected individuals in a region of hyperendemicity in Cameroon as a function of age. Peripheral blood mononuclear cell interleukin 5 (IL-5) responses to F-OvAg and Smf declined significantly with age (equivalent to years of exposure to O. volvulus). In contrast, IL-5 secretion in response to L3 and mL3 remained elevated with increasing age. Gamma interferon responses to L3, mL3, and F-OvAg were low or suppressed and unrelated to age, except for responses to Smf in older subjects. IL-10 levels were uniformly elevated, regardless of age, in response to L3, mL3, and F-OvAg but not to Smf, for which levels declined with age. A total of 49 to 60% of subjects had granulocyte-macrophage colony-stimulating factor responses to all O. volvulus antigens unrelated to age. Analysis of levels of stage-specific immunoglobulin G3 (IgG3) and IgE revealed a striking, age-dependent dissociation between antibody responses to larval antigens (L3 and a recombinant L3-specific protein, O. volvulus ALT-1) which were significantly increased or maintained with age and antibody responses to F-OvAg, which decreased. Levels of IgG1 to L3 and F-OvAg were elevated regardless of age, and levels of IgG4 increased significantly with age, although not to O. volvulus ALT-1, which may have unique L3-specific epitopes. Immunofluorescence staining of whole larvae showed that total anti-L3 immunoglobulin levels also increased with the age of the serum donor. The separate and distinct cytokine and antibody responses to adult and infective larval stages of O. volvulus which are age related are consistent with the acquisition of concomitant immunity in infected individuals.

Gross, E. et al. (2013) International Worm Meeting "The neuroglobin GLB-5 regulates C. elegans responses to hypoxic exposure."

Soltesz, Z., Gross, E., Zelmanovich, V., de-Bono, M.

[International Worm Meeting, 2013]

Fine-tuned O2 metabolism is essential for most animals, requiring animals to monitor and adapt to changes in O2 levels. We previously identified a polymorphic neuroglobin, GLB-5, that acts in O2-sensing neurons and enables C. elegans to respond to small changes in O2 concentration (Persson et al, 2009). Here we show that GLB-5 is essential for fast behavioural recovery after exposure to hypoxia. Whereas glb-5(Haw) animals recovered from hypoxia within minutes, glb-5(Bri) recovered slowly, over four hours. By combining genetics, biochemistry, and Calcium imaging we provide evidence that glb-5 enables fast recovery of O2-sensing neurons after prolonged hypoxic exposure. We designed mutagenesis screens to explore how GLB-5 regulates recovery from hypoxia. One mutant we identified disrupts a conserved chaperone that regulates the activity of the O2 sensing neurons. The chaperone regulates the spatial organization of soluble guanylate cyclases in these neurons, altering the way they interact with the neuroglobin to control the O2 response properties of these neurons. Persson A, Gross E, Laurent P, Busch KE, Bretes H, de Bono M (2009) Natural variation in a neural globin tunes oxygen sensing in wild Caenorhabditis elegans. Nature 458: 1030-1033.

Richards JP et al. (1991) Worm Breeder's Gazette "The Cloning of a Putative C. elegans Cyclin B Gene Using the Polymerase Chain Reaction."

Bennett KL, Richards JP, Temenak JJ

[Worm Breeder's Gazette, 1991]

Cyclin proteins are intimately involved in cell cycle regulation. Two types of cyclin proteins have been identified, cyclin A and cyclin B. The cyclin A protein has been shown to be critical to embryonic development in Drosophila; homozygous mutant embryos lacking cyclin A die when zygotic transcription fails to provide cyclin A. The presence of cyclin B protein cannot rescue these mutants (C.F. Lehner and P.H. O'Farrell, Cell 1990. 61:535-547). No mutants in cyclin B have been reported in Drosophila. Cyclin B protein has been shown to be involved in meiotic maturation in clams (Westendorf et al., J. Cell Biol. 1989. 108:1431-1444). Perhaps surprisingly, in Drosophila the mRNA for cyclin B localizes by in situ hybridization to the Drosophila pole cells, the fly equivalents of the nematode P germline cell lineage (Whitfield et al., Nature 1989. 338:337340). The Bennett laboratory is interested in the nematode germline lineage and in identifying the components of the germline P granules ( Strome and Wood, Cell 1983. 35:15-25 and K. Bennett, WBG 9:1 55). We plan to determine if nematode cyclin B RNA is similarly localized to the nematode germline precursor cells, and would like to address what possible role the sequestration of cyclin B RNA plays in the determination of the germline. Using primers to conserved cyclin B regions, we have isolated, cloned and sequenced a PCR product from genomic Caenorhabditis DNA which is a putative cyclin B. As illustrated below, this fragment has 72% exact match at the amino acid level to the corresponding region of the clam or sea urchin cyclin B genes; this is exactly the same match ( 41/57 aa) that sea urchin and clam show to each other in this region. Southern blot analysis using this fragment as a probe with C. elegans DNA indicates a single copy gene. The PCR fragment recognizes a single 1.4 kb message on Northern analysis using polyA+ RNA from mixed stage worms; this size is large enough to accommodate the coding region of the cyclin B protein. We have recently screened R. Barstead's lambda ZAP mixed stage cDNA library. Thirty putative cyclin B cDNAs were detected on duplicate lifts when 120,000 phage were screened. [See Figure 1]

Spicher A et al. (1988) Worm Breeder's Gazette "homeo box containing genes in Ascaris lumbricoides and C elegans."

Schaller CWD, Mueller F, Wittmann C, Tobler H, Spicher A

[Worm Breeder's Gazette, 1988]

The study of homeo box containing genes in nematodes has been approached by using the homeo boxes of the Drosophila genes fushi tarazu (ftz) and Antennapedia (Antp) as hybridization probes. From a genomic library of A. lumbricoides, several positive clones were isolated and one of them was further investigated. Sequence analyses revealed the existence of a 180bp lone Ascaris homeo box (AHB-1), located within a short, 243bp lone open reading frame. The AHB-1 encodes an amino acid sequence which is 78% homologous to the Drosophila Antp homeo domain. By using the Ascaris sequence AHB-1 as a probe, we succeeded in isolating two different homeo box containing clones from a C. elegans genomic library, kindly provided by Drs. S. Ward and K. Bennett. The sequence of one of them (CELHO) was analyzed and was shown to encode a polypeptide which is 57% homologous to the Drosophila Antp homeo domain. Neither the Ascaris nor the C. elegans homeobox contains an intron. Furthermore, the C. elegans homeobox was used as probe for screening the Ahringerer cDNA library of eggs. Three cDNA clones were picked. We are currently investigating the pattern of expression of these genes during development. [See Figure 1]

Kreutzer MA et al. (1995) International C. elegans Meeting "CHARACTERIZATION OF THE CAENORHABDITIS ELEGANS CYCLIN GENES"

Richards JP, Bennett KL, Kreutzer MA

[International C. elegans Meeting, 1995]

The Bennett laboratory has identified genes that may be essential for nematode germline determination. Drosophila cyclin B RNA has been shown to localize to the Drosophila polar granules, germ granules that many be equivalent to the nematode P granules. Therefore, cyclins may play a role in the development of the germline. We have cloned cDNAs for the C. elegans cyclins A1, B and B3 (Kreutzer, et al. in press). Cyclins A1 and B are most closely related to either A- or B- type cyclins of other species while cyclin B3 is a distant ancestral cyclin gene. While cyclin A1 is a member of a large A-type multigene family that corresponds to a single transcript on northern blots, cyclin B3 appears to be a single gene recognizing a single transcript. In contrast, cyclin B recognizes three transcripts with one cyclin B transcript specific for the paternal germline. Our results suggest that the largest and smallest cyclin B transcripts use different polyadenylation sites. By northern analyses, the two other cyclin B as well as the cyclin A1 and cyclin B3 transcripts are most abundant in the maternal germline. In addition, two types of 3'UTR translational regulatory elements, ACEs and NREs, are found in the 3' untranslated region of each of these genes. We are currently using probes to the cyclin genes for in situ hybridization to wild type and germline defective adult worms and embryos to determine the localization of these RNAs during germline proliferation and embryogenesis.

Te-Wen Lo et al. (2007) International Worm Meeting "C. elegans FGF Receptor Signaling Can Occur via Direct Binding of the SEM-5/Grb2 Adaptor Protein."

Michael Stern, Te-Wen Lo

[International Worm Meeting, 2007]

The components of receptor tyrosine kinase(RTK)signaling complexes help to define the specificity of the effects of their activation. The C. elegans fibroblast growth factor receptor (FGFR), EGL-15, regulates a number of processes, including sex myoblast (SM) migration guidance and fluid homeostasis, both of which require a Grb2/Sos/Ras cassette of signaling components. Here we show that SEM-5/Grb2 can bind directly to EGL-15 to mediate SM chemoattraction. A yeast two-hybrid screen identified SEM-5 as able to interact with the carboxy-terminal domain (CTD) of EGL-15, a domain that is specifically required for SM chemoattraction. This interaction requires the SEM-5 SH2 binding motifs present in the CTD (Y1009 and Y1087), and these sites are required for the CTD role of EGL-15 in SM chemoattraction. Although SEM-5 is required for the fluid homeostasis function of EGL-15, these sites are not required for that function, indicating that SEM-5 can link to EGL-15 through an alternative mechanism. The multisubstrate adaptor protein FRS2 serves to link vertebrate FGFRs to GRB2. In C. elegans, an FRS2-like gene, rog-1, functions upstream of a Ras/MAPK pathway for oocyte maturation1 but is not required for EGL-15 function2 . Thus, unlike the vertebrate FGFRs, which require the multisubstrate adaptor FRS2 to recruit Grb2, EGL-15 can recruit SEM-5/Grb2 directly. 1Matusbara et al., 2007. Genes to Cells; 12(3): 407-420. 2see D. Bennett abstract.