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[
J Vis Exp,
2017]
Next generation sequencing (NGS) technologies have revolutionized the nature of biological investigation. Of these, RNA Sequencing (RNA-Seq) has emerged as a powerful tool for gene-expression analysis and transcriptome mapping. However, handling RNA-Seq datasets requires sophisticated computational expertise and poses inherent challenges for biology researchers. This bottleneck has been mitigated by the open access Galaxy project that allows users without bioinformatics skills to analyze RNA-Seq data, and the Database for Annotation, Visualization, and Integrated Discovery (DAVID), a Gene Ontology (GO) term analysis suite that helps derive biological meaning from large data sets. However, for first-time users and bioinformatics' amateurs, self-learning and familiarization with these platforms can be time-consuming and daunting. We describe a straightforward workflow that will help C. elegans researchers to isolate worm RNA, conduct an RNA-Seq experiment and analyze the data using Galaxy and DAVID platforms. This protocol provides stepwise instructions for using the various Galaxy modules for accessing raw NGS data, quality-control checks, alignment, and differential gene expression analysis, guiding the user with parameters at every step to generate a gene list that can be screened for enrichment of gene classes or biological processes using DAVID. Overall, we anticipate that this article will provide information to C. elegans researchers undertaking RNA-Seq experiments for the first time as well as frequent users running a small number of samples.
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[
Development,
2021]
A dynamic pattern of histone methylation and demethylation controls gene expression during development, with some processes such as formation of the zygote involving large-scale reprogramming of methylation states. A new paper in Development investigates how inherited histone methylation regulates developmental timing and the germline/soma distinction in <i>Caenorhabditis elegans</i> To hear more about the story we caught up with first author and postdoctoral researcher Brandon Carpenter, and his supervisor David Katz, Associate Professor in the Department of Cell Biology at Emory University School of Medicine in Atlanta, Georgia.
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J Lipid Res,
2019]
Sterols represent one of the most ubiquitous and diverse classes of biological molecules derived from the common precursor mevalonic acid. While there are thematically similar modes by which various organisms synthesize sterols, there also are some unique twists in the pathways by which such organisms produce sterols as well as differences in the chemical nature of the dominant resident sterol present at steady-state in a given organism or cell type. In this issue of Journal of Lipid Research, David Nes and colleagues (Zhou et al. (1)) present a compelling and novel story, wherein they have elucidated a previously unknown alternative biosynthetic pathway utilized by nematodes (roundworms, of which Caenorhabditis elegans is an exemplar) to generate C4methyl sterols. This provides an evolutionary "missing link" along the continuum from prokaryotes to eukaryotes or eventually a singular divergent evolution of roundworms with regard to diversification of sterols and the metabolic routes accessible to them to achieve such diversity.
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[
Parasitol Today,
1992]
A major challenge to the development of vaccines against human lymphatic filariasis and onchocerciasis is to direct the immune response toward elimination of the early, prepathogenic larval stages and away from responses that mediate pathology. In this review, James Lok and David Abraham discuss the various animal models that have been used to investigate the pathways leading to immunity, immunological tolerance and chronic pathology in these diseases. Owing to the strict host specificities of the human-dwelling filariae, no single model serves to duplicate exactly all these aspects. Nevertheless, it has been possible to demonstrate a protective immune response invoked by and directed against incoming third-stage larvae of both lymphatic and skin-dwelling filariae. The fact that subsets of the sequelae of human filarial infection can be duplicated in animal systems should also aid in unravelling the mechanisms determining the course of infection and in ensuring that vaccine candidates do not produce an inappropriate immunopathological response. A proposed scheme for using animal models in screening candidates for a vaccine against Onchocerca volvulus is presented.
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Morshead, Mackenzie L, Guo, Max, Plummer, W Todd, Jones, E Grace, Garrett, Theo, Sedore, Christine A, Lithgow, Gordon, Phillips, Patrick C, Driscoll, Monica, Lucanic, Mark, Hall, David
[
MicroPubl Biol,
2020]
The Caenorhabditis Intervention Testing Program (CITP) is a multi-institutional, National Institute on Aging (NIA)-funded consortium. The goal of the program is to identify chemical compounds that extend lifespan robustly and reproducibly across genetically diverse Caenorhabditis strains (Lucanic et al. 2017). The CITP test compounds are selected if they are consistently highly ranked via computational prediction for lifespan or healthspan effects (Coleman-Hulbert et al. 2019), if they are predicted or known to interact with known lifespan-regulating pathways, or if they have previously been reported as extending lifespan or healthspan in laboratory animals. Obeticholic acid is an analog of the natural bile acid chenode oxycholic acid, which acts as an agonist of the farnesoid X receptor (FXR) (Neuschwander-Teri et al. 2015), a nuclear receptor (NR) closely involved with hepatic triglyceride homeostasis. Obeticholic acid is most commonly used to treat the autoimmune liver disease, primary biliary cholangitis. The most likely homolog of FXR in C. elegans is DAF-12, which can bind and be activated by human bile acids (Held et al. 2006; Zhi et al. 2011). DAF-12 modulation is of particular interest because it is closely linked to dauer formation, lifespan extension, and metabolism homeostasis (Antebi 2015).
We assayed lifespan in response to different concentrations of obeticholic acid exposure in three Caenorhabditis species using the flatbed scanner-based Automated Lifespan Machine (ALM) workflow previously published (Banse et al. 2019). To summarize, the worms were age synchronized by egg-lays on standard 60 mm diameter Nematode Growth Media (NGM) plates with lawns of Escherichia coli OP50-1, and transferred to compound-treated 38 mm NGM plates containing 51 M 5-Fluoro-2-deoxyuridine (FUdR) at a density of 50 worms per plate on day one of adulthood. For treatment plates, we used standardized protocols (Caenorhabditis Intervention Testing Program 2020); in short, obeticholic acid (Apexbio Technology) was dissolved in dimethyl sulfoxide (DMSO) and diluted appropriately such that addition of 7.5 l of stock solution and 125 l water for 35 mm diameter plates, and 17.5 l of stock solution and 232.5 l water for 50 mm diameter scanner plates would generate 50, 100, and 150 M final obeticholic acid concentrations. For control plates, DMSO was added instead of stock solution using the same method. The worms were maintained at 20C and transferred to new treatment plates again on day two of adulthood. One week after age-synchronization (day five of adulthood for C. elegans and C. briggsae, day four for C. tropicalis), the worms were transferred to compound-treated scanner plates and loaded onto the ALM. At this point, automated survival monitoring began, and the scanner data was collected and analyzed using Lifespan Machine software (https://github.com/nstroustrup/lifespan; Stroustrup et al.. 2013).
Our results indicate that obeticholic acid does not have a consistent beneficial effect on lifespan in any of the C. elegans or C. briggsae strains tested at the concentrations used. Although we did see some significant differences from the control for some of the concentrations in the C. tropicalis strains, overall the difference was not robust. We actually saw a significant decrease in lifespan in C. tropicalis JU1630, a weakly significant increase in C. tropicalis QG834 at some concentrations, and a relatively significant increase in C. tropicalis JU1373, but with only a 5.7-7.9% change in mean survival from the control (Fig. 1). In summary, our results do not indicate a robust effect of obeticholic acid on Caenorhabditis lifespan. This conclusion is based upon two biological replicates at each concentration performed in one lab, resulting in an average of 104 individuals measured per strain and concentration, and should be considered preliminary. The effect on lifespan in this study may pertain to a lack of physiological relevance of obeticholic acid to Caenorhabditis. Obeticholic acid was of interest to the CITP because of its effect on the mammalian NR FXR. Although DAF-12 has been identified as a potential Caenorhabditis homolog of FXR and other bile acids have been shown to bind with DAF-12 (Zhi et al. 2011), it is possible that obeticholic acid was not able to bind with high affinity to the receptor, therefore eliciting little to no effect on lifespan. Alternatively, obeticholic acid may be rapidly metabolized in Caenorhabditis.
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[
RNA Biol,
2019]
Worm biologists from the United States, Canada, and the United Kingdom gathered at the Colorado State University Todos Santos Center in Baja California Sur, Mexico, April 3-5, 2019 for the Todos Santos Small RNA Symposium. Meeting participants, many of whom were still recovering from the bomb cyclone that struck a large swath of North America just days earlier, were greeted by the warmth and sunshine that is nearly ubiquitous in the sleepy seaside town of Todos Santos. With only 24 speakers, the meeting had the sort of laid-back vibe you might expect amongst the palm trees and ocean breeze of the Pacific coast of Mexico. The meeting started with tracing the laboratory lineages of participants. Not surprisingly, the most common parental lineages represented at the meeting were Dr. Craig Mello, Dr. Gary Ruvkun, and Dr. Victor Ambros, whom, together with Dr. Andy Fire and Dr. David Baulcombe, pioneered the small RNA field. In sad irony, on the closing day of the meeting, participants were met with the news of Dr. Sydney Brenner's passing. By establishing the worm, <i>Caenorhabditis elegans</i>, as a model system Dr. Brenner paved the way for much of the research discussed here.
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Lucanic, Mark, Hall, David, Osman, Hadley C, Sedore, Christine A, Foulger, Anna, Jackson, E Grace, Battistoni, Elena T, Lithgow, Gordon J, Guo, Max, Driscoll, Monica, Phillips, Patrick
[
MicroPubl Biol,
2021]
The Caenorhabditis Intervention Testing Program (CITP) is a multi-institutional, National Institute on Aging (NIA)-funded consortium with the goal of identifying compounds that will robustly extend lifespan with reproducible effects across genetically diverse Caenorhabditis species and strains (Lucanic et al., 2017). Compounds are prioritized for testing based on computational prediction for lifespan or healthspan effects (Coleman-Hulbert et al., 2019), predicted or known interactions with documented lifespan-regulating pathways, or previous reports for lifespan or healthspan extension in laboratory animals. In this case, diuron was of interest to CITP based on the report of a positive effect on the lifespan of C. elegans strain TJ1060 (
spe-9;
fer-15) (Lucanic et al., 2018; personal communication). Diuron (1-(3,4-dichlorophenyl)-3,3-dimethylurea) is an herbicide that inhibits photosynthesis by binding photosystem II (Haynes et al., 2000). Diuron has been previously tested in nematode species for toxicity (Neury-Ormanni et al., 2019) and reproductive effects (Mugova et al., 2018), and in both cases showed detrimental effects at high doses only.
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[
Protein J,
2018]
Phosphatases are well known to carry out important functions via counter activity of kinases and they serve as mechanism for dephosphorylating the monophosphate esters from the phosphorylated serine, threonine, tyrosine and histidine residues. The biological relevance of phosphatases could be explored further employing newer technologies and models. Caenorhabditis elegans is a powerful genetic model system that bears significant homology with humans, hence providing with a precious tool towards studying important signalling pathways. We carried out the present study to catalogue the C. elegans protein phosphatome, referred here as 'C.el phosphatome' and annotated the corresponding dataset. We further classified these phosphatases based on presence of catalytic conserved motif; GDxHG, GDxVDRG, GNHE, RxxD, DGxxG, DG, GxxDN for Ser/Thr phosphatases, HC(x)5 R for tyrosine phosphatases and DxDxT/V for aspartate based phosphatases. Bioinformatics tool DAVID was employed to decipher the biological relevance of phosphatases. Our findings show Ser/Thr phosphatases (114), Tyr phosphatases (121) and Asp phosphatases (0) in C. elegans genome based on the hallmark sequence identification. Amongst them, 34 and 57 Ser/Thr and Tyr phosphatases respectively contain the catalytic motif. This catalogue offers a precious tool for further studies towards understanding important biological processes and disease conditions.
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[
Nucleic Acids Res,
2010]
The recent publication of the Caenorhabditis elegans cisRED database has provided an extensive catalog of upstream elements that are conserved between nematode genomes. We have performed a secondary analysis to determine which subsequences of the cisRED motifs are found in multiple locations throughout the C. elegans genome. We used the word-counting motif discovery algorithm DME to form the motifs into groups based on sequence similarity. We then examined the genes associated with each motif group using DAVID and Ontologizer to determine which groups are associated with genes that also have significant functional associations in the Gene Ontology and other gene annotation sources. Of the 3265 motif groups formed, 612 (19%) had significant functional associations with respect to GO terms. Eight of the first 20 motif groups based on frequent dodecamers among the cisRED motif sequences were specifically associated with ribosomal protein genes; two of these were similar to mouse EBP-45, rat HNF3-family and Drosophila Zeste transcription factor binding sites. Additionally, seven motif groups were extensions of the canonical C. elegans trans-splice acceptor site. One motif group was tested for regulatory function in a series of green fluorescent protein expression experiments and was shown to be involved in pharyngeal expression.
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[
Mol Biol Cell,
2004]
Monitoring Editor: David Drubin Generating specific actin structures via controlled actin polymerization is a prerequisite for eukaryote development and reproduction. We here report on an essential C. elegans protein tetraThymosinbeta expressed in developing neurons and crucial during oocyte maturation in adults. TetraThymosinbeta has four repeats each related to the actin monomer sequestering protein thymosinbeta4 and assists in actin filament elongation. For homologues with similar multirepeat structure a profilin-like mechanism of ushering actin onto filament barbed ends, based on the formation of a 1:1 complex, is proposed to underlie this activity. We, however, demonstrate that tetraThymosinbeta binds multiple actin monomers via different repeats and in addition also interacts with filamentous actin. All repeats need to be functional for attaining wild-type activity in various in vitro assays. The activities on actin are thus a direct consequence of the repeated structure. In containing both G- and F-actin interaction sites, tetraThymosinbeta may be reminiscent of nonhomologous multimodular actin regulatory proteins implicated in actin filament dynamics. A mutation that suppresses expression of tetraThymosinbeta is homozygous lethal. Mutant organisms develop into adults but display a dumpy phenotype and fail to reproduce as their oocytes lack essential actin structures. This strongly suggests that the activity of tetraThymosinbeta is of crucial importance at specific developmental stages requiring actin polymerization.