Luke, Cliff J. et al. (2011) International Worm Meeting "Controlling protease-driven lysosomal dependent "necrotic" cell death and beyond."

Silverman, Gary A., Miedel, Mark M., Bhatia, Sangeeta R., Luke, Cliff J., Graf, Nathan J.

[International Worm Meeting, 2011]

Cell necrosis is a common pathologic feature of many disorders including ischemic injury, hemorrhagic shock and trauma. Necrotic cell death results from perturbations in the protease-inhibitor balance, which leads to excessive intracellular proteolysis, loss of membrane integrity and dissolution of sub-cellular architecture. The strong association between promiscuous intracellular proteolysis and cell death suggests that protease inhibitors serve as necrotic cell death regulators. Extensive searches within the largest family of protease inhibitors, the serpins (e.g., antithrombin and alpha-1-antitrypsin), failed to yield candidate regulatory genes. However, this result was not surprising as most serpins are secreted and unlikely to play a regulatory role intracellularly. We identified a subset of serpins that are abundantly expressed within the cytoplasm of metazoan epithelial cells. Since some of these intracellular serpins (serpinsIC) neutralize lysosomal cysteine and serine proteases, we hypothesized that they regulate intracellular proteolysis and enhance cell survival. This hypothesis was confirmed by showing that the C. elegans serpinIC, SRP-6, exhibits a pro-survival function by blocking necrotic cell death. The newly defined necrotic cell death pathway was dependent upon calpains and lysosomal cysteine proteases, two in vitro targets of SRP-6. SRP-6 provided protection by blocking both the induction of, and the lethal effects from, lysosomal injury. Human intracellular serpins also play a role in cytoprotection as SERPINB3 (a human cysteine protease inhibitory serpin) could function in place of SRP-6, as well as protecting mammalian cell lines from cell death. We now hypothesize that multiple noxious stimuli converge upon an evolutionarily conserved, protease-driven core stress-response pathway that, in the absence of serpinIC regulation, leads to necrotic cell death rather than cell survival. The role of protease inhibitors in the necrosis pathway and the potential for therapeutic intervention will be discussed.

Sternberg, Paul et al. (2015) International Worm Meeting "WormBase 2015."

Berriman, Matt, Howe, Kevin, Kersey, Paul, Stein, Lincoln, Harris, Todd, Sternberg, Paul, Schedl, Tim

[International Worm Meeting, 2015]

WormBase has existed for 15 years and has evolved in many ways. The new website is fully operational and has made the process of adding new data types, displays, and tools easier. Behind the scenes we are piloting an overhaul of the underlying database infrastructure to allow us to handle the ever increasing data, have the website perform faster, and allow more frequent updates of information. This is a critical time for the project, as we face considerable pressure from two directions. The first is that our funders really want us to do more with less. We are responding to this by leading the way in making curation (the process of extracting information from papers and data sets into computable form) more efficient using a new version of Textpresso (to be released later this calendar year); by discussing with other model organism information resources ways to work together to be more efficient and inter-connected; and by seeking additional sources of funding. The second, delightful, pressure is an increase in data and results generated by the C. elegans and nematode communities. While we are handling this increase by changes in our software for curation, the database infrastructure, and the website, we do need your help. Many of you have helped us over the last few years to identify data in your papers or by sending us data directly. We now need you to help with a few types of information by submitting the data via specially designed, user-friendly forms that ensure good quality and the use of standard terminology. In particular, we have a large backlog of uncurated information associating alleles with phenotypes. We pledge to make this process as painless as possible, and to improve WormBase's description of phenotypes with your feedback, starting at this meeting at the WormBase booth, workshops and posters. With your help, continual improvement of our efficiency, and additional sources of funding, we are optimistic that we can do much more with even somewhat less effort.Consortium: Paul Davis, Michael Paulini, Gary Williams, Bruce Bolt, Thomas Down, Jane Lomax, Todd Harris, Sibyl Gao, Scott Cain, Xiaodong Wang, Karen Yook, Juancarlos Chan, Wen Chen, Chris Grove, Mary Ann Tuli, Kimberly Van Auken, D. Wang, Ranjana Kishore, Raymond Lee, John DeModena, James Done, Yuling Li, H.-M. Mueller, Cecilia Nakamura, Daniela Raciti, Gary Schindelman.

Swenson SI et al. (1995) International C. elegans Meeting "THE STEROID RECEPTOR HOMOLOGUE GENE, nhr-1, IN C. ELEGANS AND C. BRIGGSAE"

Purac A, Eagen RM, Honda BM, Swenson SI, Davison PJ

[International C. elegans Meeting, 1995]

We are studying the nhr-1 (nuclear hormone receptor) gene, one of a number of steroid receptor homologues identified in C. elegans. Sequencing of the cDNA has shown interesting features for this putative "orphan" receptor, including an unusual sequence in the P box of the DNA binding domain, which might indicate a very different DNA binding specificity and function, and a very long 3' untranslated region. The nhr-1 gene has been mapped to the right arm of the X chromosome, very close to sup-10. We are now characterizing the genomic organization of nhr-1 in C. elegans. We have also initiated work with C. briggsae in order to identify conserved, functionally important regions. The presence of an apparent homologue in C. briggsae was of special interest to us, given the unusual properties of nhr-1 (different P box, the long 3'UT region), and the apparent viability of homozygous deficiencies in this region around sup-10 (C. Cummins and P. Anderson). The C. elegans nhr-1 gene appears to contain 12 exons spread over 9.5 kb, including several relatively large introns, 1.3 to 2.3 kb in size. Work to date with genomic clones from C. briggsae shows conserved exons which share approximately 81-84% homology at the nucleotide level with the nhr-1 gene in C. elegans. This conservation between species, in the absence of a lethal phenotype for the homozygous deficiency, suggests that the nhr-1 gene may nevertheless have an evolutionarily relevant function. We wish to thank Ann Sluder, Gary Ruvkun, Claudia Cummins and Dave Baillie et al. for their assistance, and Alan Coulson et al. for help with mapping clones. Supported by a Research Reorientation Associateship (NSERC Canada) to SS, and an NSERC Operating grant to BMH.

John Willis et al. (2001) International C. elegans Meeting "PHYSIOLOGICAL ROLE OF K-CL COTRANSPORTERS IN C. ELEGANS"

John Willis, Edward Kipreos

[International C. elegans Meeting, 2001]

In vertebrate cells the K-Cl cotransporter has the capacity to reduce cellular concentration of K and Cl. This plays several documented roles: regulation of cytoplasmic chloride concentration, reduction of cell size in development, defense against cell swelling under hypoosmotic and isoosmotic conditions. The last, which includes the defense against swelling during moderate warming due to hyperactivity of the Na-K pump, has received the least attention but may be the most physiologically relevant. The gene sequences for two mammalian isoforms of this cotransporter were identified in 1996 and at that time two similar sequences were recognized in the C. elegans database (KO2A2.3, "KO2", on chromosome 2, and R13A1.2, "R13", on chromosome 4). Both C. elegans isoforms are expressed,as demonstrated by their presence in EST's and cDNA libraries. K02 has been shown to have K-Cl cotransport activity when transfected into mammalian cells. We have carried out dsRNAi blockages against both of these genes, singly and in combination, and found that about a third of the injected parents produced progeny that exhibit reduced survival at elevated temperature (33degC and 37degC) and increased sensitivity (seen as immobilization) to isoosmotic challenge induced by exposure to 100 or 200 mM ammonium chloride in M9 medium. A deletion mutant of K02 obtained from Gary Moulder at U. Oklahoma has not shown a phenotype by these tests and is now being used as a target for RNAi knockout of the R13 gene. GFP's expressed from upstream sequences of the two genes indicate that both isoforms are consistently expressed in pharyngeal muscle. The extent to which either is expressed elsewhere is still being determined. These studies may lead to establishing the role of K-Cl cotransporters in the whole organism and lay the foundation for a genetic analysis of their control

Monsalve, G.C. et al. (2009) International Worm Meeting "Timing the Molting Cycle."

Monsalve, G.C., Davie, J., Frand, A.R.

[International Worm Meeting, 2009]

Nematodes develop through periodic molts but molecular mechanisms that set the pace of the molting cycle are not well understood. Molting involves detachment of the old exoskeleton from the underlying hypodermis and synthesis of the new exoskeleton, an extracellular matrix composed largely of collagens. Epidermal stem cells divide in sync with the first three molts but terminally differentiate at the final molt. A period of behavioral quiescence (lethargus) accompanies remodeling of the exoskeleton. Animals subsequently use specialized movements to escape the old skeleton (ecdyse). Improper coordination of these cell biological and behavioral events can be fatal. We aim to identify genes and molecules that regulate the timing of the molting cycle. The best-characterized biological timers control circadian rhythms in the physiology and behavior of humans and other animals. In theory, worm counterparts of the core circadian clock proteins might also regulate the ultradian rhythm of the molting cycle. We are therefore investigating the role of clock gene homologs in the reiterative molecular and behavioral events characteristic of molting. As a complementary approach, we conducted a forward genetic screen for mutants that molt prematurely, using expression of the mlt-10p::gfp-pest reporter to indicate synthesis of a new cuticle. In a pilot screen of approximately 18,000 genomes, we indentified one candidate that expressed the mlt-10 reporter too early and then partly shed the L1 exoskeleton. This particular animal perished, possibly due to the exposure of an incomplete L2 skeleton to the environment. In ongoing work, a clonal screen will allow the recovery of lethal mutations from siblings of affected animals. To further investigate the physiologic regulation of the molting cycle, we have cloned and characterized new mutations that cause supernumerary molts after puberty. These alleles emerged from a genetic screen conducted by Alison Frand and Sascha Russel in the laboratory of Gary Ruvkun. By studying the regulation of the molting cycle, we expect to uncover novel but conserved mechanisms for the endocrine control of development and behavior.

Huang, Huiyan et al. (2021) International Worm Meeting "C. elegans modeling and human studies of a rare RAB5B patient variant reveal a novel role of RAB5B in regulated secretion of pulmonary surfactant"

Huang, Huiyan, Lindsey, Anika, Dawson, Zachary, Spielberg, David, Chen, Jian, Schedl, Tim, Mohammad, Ariz, Burrage, Lindsay, Kim, Hyori, Baldridge, Dustin, Pan, Jiehong, Rosenfeld, Jill, Scott, Daryl, Pak, Stephen, Hanchard, Neil, Murdock, David, Morrison, Stephanie, Dai, Hongzheng, Brody, Steven, Zhu, Alex, Tycksen, Eric, Silverman, Gary, Ramu, Avinash

[International Worm Meeting, 2021]

Many patients with severe, chronic diseases remain without a diagnosis despite extensive medical evaluation, including some cases with clinical exome sequencing. The goal of the NIH-funded Undiagnosed Diseases Network (UDN) is to provide a diagnosis for these challenging cases and to identify biological characteristics of newly discovered disease genes. The UDN uses a collaborative multidisciplinary approach that combines comprehensive medical workup, exome/genome sequencing, bioinformatic analysis, with functional studies in model organisms including zebrafish, Drosophila, and C. elegans. Through the UDN, we evaluated a child with interstitial lung disease suggestive of surfactant deficiency. Variants in known surfactant dysfunction disorder genes were not found in trio exome sequencing. Instead, a de novo heterozygous variant, p.Asp136His, in the Ras/Rab GTPases family nucleotide binding domain in RAB5B was identified. Functional studies were performed in the C. elegans Model Organism Screening Center at Washington University in St. Louis. We used CRISPR/Cas9 to knock the proband variant into the conserved position (Asp135) of the ortholog, rab-5. Analyses of organismal phenotypes such as locomotion and size demonstrated that rab-5[Asp135His] is damaging. We also show data indicating that rab-5[Asp135His] heterozygotes were defective in endocytosis and early endosome (EE) fusion. Dosage analysis by adding extra copies of wild type rab-5 transgene revealed that rab-5[D135H] has a strong dominant negative effect, requiring three wild type copies to suppress the variant's poisonous effect. Immunostaining studies of the proband's lung biopsy revealed that RAB5B and EE marker EEA1 were significantly reduced in type II pneumocytes, and mature SP-B and SP-C were significantly reduced, while ProSP-B and ProSP-C were normal. Furthermore, staining of normal lung showed co-localization of RAB5B and EEA1 with ProSP-B and ProSP-C. These findings indicate that dominant negative-acting RAB5B Asp136His and EE dysfunction cause a defect in processing/trafficking to produce mature SP-B and SP-C, inducing interstitial lung disease, and that RAB5B and EEs normally function in the regulated surfactant secretion pathway. Together, the data suggest a non-canonical function for RAB5B and identify RAB5B p.Asp136His as a genetic mechanism for surfactant dysfunction disorder.

AF Dernburg et al. (1999) International C. elegans Meeting "Getting Intimate with the Right Partner"

AF Dernburg, AM Villeneuve

[International C. elegans Meeting, 1999]

A unique and ubiquitous feature of sexual reproduction is the pairing between homologous chromosomes that occurs during meiosis. This intimate association allows partner chromosomes to undergo genetic exchange, which is in turn essential for accurate chromosome segregation at the first meiotic division. Although this process has intrigued biologists for decades, many fundamental questions about its mechanism remain; e.g., how do chromosomes first encounter their homologs? Once physical contact is made, how is their homology recognized? Using FISH and high-resolution 3-D microscopy, we can visualize chromosome pairing at specific chromosomal loci within intact gonads. Since the entire temporal sequence of meiosis is present within each adult worm, this approach quickly revealed the stage at which meiotic pairing occurs. Combined with reverse genetics, these cytological methods have resolved another crucial question: Conflicting results from yeast and Drosophila had fueled a debate as to whether recombination is essential for meiotic chromosome pairing. By knocking out a gene, spo-11 , whose function is necessary to initiate recombination (in collaboration with Mike Dresser, Gary Moulder, and Bob Barstead) we abolished all meiotic exchange, but found that pairing and synapsis were unperturbed in this mutant. This study demonstrated pairing can occur in the absence of recombination in a metazoan organism, and that this paradigm is probably extremely widespread. We are now focusing on the role of "pairing centers" in meiotic chromosome pairing and synapsis. Cytological examination of worms lacking X -chromosome pairing centers has revealed that these distal regions are indeed essential for intimate association between the X s. We are testing whether synaptonemal complex components can associate with defective X chromosomes. Two additional approaches have enabled us to perturb normal homolog pairing: First, genetic screens for Him mutants have uncovered trans-acting factors involved in pairing (see MacQueen abstract). Another strategy is to study rearrangements, which require the pairing machinery to contend with chromosomes that do not share homology along their entire lengths. We are currently testing the long-standing assumption that balancer chromosomes suppress recombination by disrupting pairing between homologous sequences.

Gary Williams et al. (2006) European Worm Meeting "Curating Wormbase Gene Structures"

Tamberlyn Bieri, Darin Blasiar, John Spieth, Anthony Rogers, Gary Williams, Philip Ozersky, Paul Davis

[European Worm Meeting, 2006]

Gary Williams1, Paul Davis1, Anthony Rogers1, Philip Ozersky2, John Spieth2, Tamberlyn Bieri2, Darin Blasiar2. WormBase is an international consortium of biologists and computer scientists from Caltech (USA), Cold Spring Harbor Laboratory (USA), Wellcome Trust Sanger Institute (UK) and the Genome Sequencing Center at Washington University (USA).. WormBase is dedicated to providing the research community with accurate, current, accessible information concerning the genetics, genomics and biology of C. elegans, and some related nematodes. WormBase can be freely accessed at www.wormbase.org, and is also available for download at ftp://ftp.wormbase.org/pub/wormbase A new data release is produced every three weeks. Recently some new datasets have been added to the database to aid curation.The protein products from automated gene prediction in C. remanei have been mapped to the C. elegans genome; InterPro motifs are no longer taken from SwissProt annotations, but are directly predicted from the C. elegans proteins; contigs of ESTs from other nematode species produced by the NEMBASE and Nematode.net projects have been mapped to the genome and a new set of TEC-REDs have been mapped to the genome resulting in 3,325 new trans-spliced sites which mark the 5'' ends of genes or isoforms. Work is underway to classify and curate transposons and we intend to add Mass-Spec data from several sources to improve coding sequence validation.. Over the last year the curated coding sequences have increased from 19,854 to 20,060 and alternately spliced isoforms have increased from 2,772 to 2,883. The number of genes where every base of every exon is confirmed by EST evidence has increased from 6,427 to 6,584. In the C. elegans proteins there have been 630 modified entries, 440 deleted entries, 709 new entries and 64 reappeared entries.. WormBase is supported by a grant from the National Human Genome Research Institute at the US National Institute of Health #P41 HG02223 and the British Medical Research Council.

Sze JY (2000) West Coast Worm Meeting "Transcriptional regulation of the tryptophan hydroxylase gene tph-1"

Sze JY

[West Coast Worm Meeting, 2000]

tph-1 encodes a tryptophan hydroxylase, the key enzyme for serotonin biosynthesis. When I was in Gary Ruvkun's lab, I collaborated with Martin Victor in Yang Shi's lab and isolated a tph-1 deletion mutation, tph-1(mg280). tph-1(mg280) shows no detectable serotonin based on anti-serotonin antibody staining and exhibits several behavioral and metabolic defects (Sze et al., 2000). It has been proposed that in mammals changes in the level of TPH mRNA correlate with long-term changes in the cellular serotonin level and that the regulation of TPH mRNA tends to be "inducer"- and region-specific (Semple-Rowland et al., 1996; Clark and Russo, 1997; Siuciak et al., 1998. Bethea, et al., 2000). Thus, the regulation of TPH expression may be a key step by which the nervous system adjusts its long-term synaptic serotonin levels. To identify genes regulating tph-1, I have started a genetic screen for mutations that alter tph-1::gfp expression in specific neurons. Thus far, mutations identified can be classified into three classes. (1) I have previously reported that mutations in the POU-homeodomain transcription factor UNC-86 abolish tph-1::gfp expression in NSM and HSN, but the expression in ADF is not affected. UNC-86 is expressed in HSN and NSM, but not in ADF. (2) I have identified 18 mutations where tph-1::gfp expression in ADF is reduced or eliminated. Laser ablation of ADF and ASI causes animals to form dauers (Bargmann and Horvitz, 1993; Schackwitz et al., 1996) and the tph-1(mg280) mutation downregulates daf-7::gfp expression and enhances daf-7(e1372) dauer formation at 15oC. To test the role of ADF-produced serotonin is important for a non-dauer growth, I am testing if these mutations enhance daf-7(e1372) dauer formation at 15oC. (3) I have identified one mutation that has tph-1::gfp expressed in an extra neuron. These mutants provide me as useful means to characterize the role of serotonin produced in specific neurons.

Rader J et al. (1997) International C. elegans Meeting "Ion Channels in HSN Pathfinding"

Rader J, Garriga G, Wightman BC

[International C. elegans Meeting, 1997]

Dominant, gain-of-function, mutations in egl-19 and unc-58 disrupt the outgrowth of the HSN axons. Recessive alleles of either gene do not cause pathfinding defects. Here, we report a genetic interaction between these two genes. egl-19 encodes an alpha-1 subunit of a voltage-gated Ca2+ channel (L. Lobel, et. al.WBG 13(1):46). Some time ago, Gary Ruvkun found that the paralysis of unc-58(dm) mutants could be rescued by growing the animals on endosulfan, a drug that blocks GABA-gated Cl- channels in insects. We found that endosulfan also rescued the axon outgrowth defect in unc-58(dm) mutants. To address the question of whether these dominant mutations might alter a common pathway, double mutants were constructed that carry the unc-58(e665dm) mutation and recessive mutations in either egl-19 or unc-36. unc-36 encodes a homolog of the alpha-2 subunit of voltage gated Ca2+ channels and has been shown to interact genetically with egl-19 (L. Lobel, et. al. WBG 13(2):71). Recessive mutations in both egl-19 and unc-36 suppressed the HSN pathfinding defects of unc-58(e665dm) mutants. The observation that these mutations interact genetically further supports the idea that mutations in ion channels can affect axonal pathfinding. However, the observation that a mutation in egl-19, which encodes the principle subunit of a Ca2+ channel, and mutations in unc-58 ,which might encode a Cl- channel, or act through such a channel, both cause a similar defect in ventrally directed growth of HSN suggests that this step in HSN outgrowth might be particularly vulnerable to generic changes in membrane potential and/or intracellular Ca2+ levels. The suppression of the HSN pathfinding defect displayed by unc-58 gain-of-function mutations by loss-of-function mutations in Ca2+ channel subunits suggests that unc-58 might cause its effect by influencing the intracellular Ca2+ levels.