Jodie F Wren et al. (2005) International Worm Meeting "Environmental Genomics of Fluoranthene Exposure"

Stephen R Sturzenbaum, Jodie F Wren, A John Morgan, Peter K Hankard, Nick Redshaw, Suresh C Swain, Claus Svendsen, Martijs J Jonker, David J Spurgeon

[International Worm Meeting, 2005]

Benzo(j)Fluoranthene, is a 5-ring polycyclic aromatic hydrocarbon (PAH), known to occur as a product of plant biosynthesis, but also as a byproduct of incomplete combustion of organic material. Fluoranthene has been identified in cigarette smoke, char-broiled foods, drinking water, lake sediments and ambient air. It is thought to interact with DNA, cause heritable genetic disorders and has been associated with lung, intestinal and pharyngeal cancer. Information regarding the toxicity of fluoranthene in C.elegans still remains scarce, a shortfall we are aiming to redress. Using GE-31 (an embryonic lethal TS mutant) an elaborate toxicity test identified a dose dependant slippery pharynx phenotype and effects on specific demographic endpoints, such as brood size, volumetric growth and life span. Exploiting whole genome oligo array technology, we are currently exploring the stage-specific toxic effects of fluoranthene at a transcriptional level.

Duong, Trang (2017) International Worm Meeting "Investigating the biosynthesis and functions of newly identify isiRNA in C. elegans."

Duong, Trang

[International Worm Meeting, 2017]

Small non-coding RNAs of 20-30 nucleotides can target both chromatin and transcripts in a sequence-specific manner to regulate gene expression. The first small regulatory RNA was discovered in C. elegans in 1993(Lee, Feinbaum, & Ambros, 1993). Recent progress in high-throughput sequencing has uncovered an astounding landscape of small RNAs. Various small RNAs have been found and can be categorized into three main classes according to their mechanism of biogenesis and the Argonaute protein to which they bind: micro RNAs (miRNAs), Piwi-interacting RNAs (piRNAs), and endogenous small interfering RNAs (endo-siRNAs) (Czech and Hannon, 2011). Those small interference RNAs have diverse expression patterns and might regulate various developmental and physiology processes. In this study, we developed a pipeline to analyze siRNA sequence libraries. We used fastX-clipper, fastX-trimmer, then align to the WS253 reference genome using bowtie 2 program, and lastly a custom python script to keep only anti-sense reads. Our analysis across hundreds of libraries from dozen of different laboratories revealed that there is another class of siRNA that mapped to intronic regions of the gene (isiRNA). Though abundant, the function and biogenesis of these isiRNAs have yet to be described in C. elegans. We investigated the characteristics of introns that have isiRNA mapping to them, and we found that the median of these introns is 10-fold longer compared to the median of genomic introns. Through characterizing genes that have isiRNA mapping to them, we discovered that genes with alternative splicing have higher probability of having isiRNA mapping compared to genes without alternative splicing. By analyzing high-throughput sequences of co-immunoprecipation libraries we found that this isiRNA bound specifically to some endo-siRNA Argonautes but not to miRNA Argonautes. My current study is focusing on understanding the biogenesis and function of this newly identified isiRNA. Our study will broaden our understanding of the complex function and biogenesis of small non-coding RNA.

Jay M Maniar et al. (2005) International Worm Meeting "On the Endogenous Roles of EGO-1"

Andrew Z Fire, Jay M Maniar

[International Worm Meeting, 2005]

RNA interference (RNAi) is a conserved mechanism in which double-stranded RNA (dsRNA) induces targeted degradation of homologous mRNA. Much work has focused on mechanistic analysis and technical applications of RNAi1. It has been shown in Caenorhabditis elegans that introduction of only a few molecules of dsRNA per cell is effective in silencing even a highly expressed gene. Genetic screens in C. elegans have been useful in identifying components required for RNAi and, hence the mechanism by which the pathway degrades target mRNA. Interestingly, several genes that have been shown to be essential for RNAi (e.g. rde-1 and rde-4) show no other obvious defects2. Meanwhile, a second group of genes are required for both RNAi and viability. The RNA-dependent RNA polymerase gene ego-1 is a member of this second class of genes3, 4. We hope that further analysis of this class of genes may provide insight into the role of RNA-directed RNA synthesis in normal development as well as in response to foreign nucleic acid. This observation leads to a simple, albeit overreaching question: what is the role of this second group of genes both inside and outside of our traditional view of RNAi? We have chosen to address this question using three approaches. First, by characterizing the spectrum of germline silencing processes that require (or do not require) EGO-1. Second, by characterizing the molecular and cellular defects in ego-1 mutant germlines and embryos. Finally, through characterization of the genetic and molecular requirements for lethality in the absence of EGO-1 function. 1Denli AM, Hannon GJ. 2003. RNAi: an ever-growing puzzle. TRENDS in Biochemical Sciences. 28:196:201. 2Tabara H, et al. 1999. The rde-1 gene, RNA interference, and transposon silencing in C. elegans. Cell. 99(2):123-32. 3Qiao L, et al. 1995. Enhancers of glp-1, a gene required for cell-signaling in Caenorhabditis elegans, define a set of genes required for germline development. Genetics. 141:551-69.4Smardon A, et al. 2000. EGO-1 is related to RNA-directed RNA polymerase and functions in germ-line development and RNA interference in C. elegans. Curr. Biology. 10:169-78.

Limke, Annette et al. (2021) International Worm Meeting "Food additives target the gut-neural axis: Impaired peptide trafficking and amyloid protein aggregation lead to premature aging phenotypes"

Berwanger, Lutz, Scharf, Andrea, Limke, Annette, von Mikecz, Anna

[International Worm Meeting, 2021]

Nanoparticles like nano silica (E551) are commonly used as food additives although bio-interactions with the gut are not well understood. Nutrients, like oligopeptides, together with nano silica are taken up via the same route via the pharynx to the intestinal tract. At the apical domain of intestinal cells, oligopeptides are transferred by the OPT-2/PEP-2 transporter to the cytoplasm and degraded downstream by hydrolases to provide amino acids for protein biosynthesis. For analyses of the peptide transport, the fluorophore-conjugated dipeptide B-Ala-Lys (AMCA) was used which normally distributes diffusely throughout the cytoplasm. Exposure with nano silica lead to an altered morphology of the intestine and disturbed peptide trafficking. Dipeptides accumulated in spherical subdomains that grow over time to a diameter of ≥6 microm. The peptide subdomains did not colocalize with known organelles such as gut granules and formed independently of the insulin/IGF-1 receptor (DAF-2) signaling pathway. Mutation of the daf-2 tyrosine kinase domain showed distinct patterns of peptide subdomains along the apical membrane of intestinal cells which indicated specific trafficking defects (Piechulek et al., 2019). We suggest that nano silica-induced peptide subdomains represent new compartments of dipeptide storage leading to the inhibition of hydrolysis and peptide metabolism which in turn results in a decline of translation. Aberrant segregation of dipeptides by phase separation leads to a petite phenotype resembling OPT-2/PEP-2 transporter deficient mutants. In addition, nano silica induce global amyloid protein aggregation that segregates components of the protein homeostasis to an insoluble aggregome. Candidate proteins include components of the translational machinery and ribosomal RNA processing (Scharf et al., 2016). Taken together, the results suggest that impairment of translation occurred by a lack of amino acid supply and amyloid segregation of proteins critically involved in protein synthesis. As translation was shown to be involved in the generation of both, premature aging and neurodegenerative diseases, further investigation of its role in the organ-cross talk between intestine and the neural system in xenobiotic-exposed C. elegans is required.

Katic, I. et al. (2019) International Worm Meeting "A reagent toolkit for tagging genes with compound tags."

Towbin, B., Katic, I., Gro beta hans, H.

[International Worm Meeting, 2019]

Precise editing using CRISPR/Cas9-induced DNA breaks and subsequent homology-directed repair (HDR) have become a routine part of C. elegans research. Emerging consensus in the field is that single-stranded oligonucleotides work as highest-efficiency HDR templates for creating small inserts or sequence changes, as well as for generating defined deletions. Several recent methods have achieved increased efficiency of precise insertions of larger fragments (Farboud et al., 2019; Paix et al., 2016; Dokshin, Ghanta et al., 2018), using double-stranded or partially single stranded templates and ribonucleoprotein complex delivery of CRISPR-Cas9. To support the increased use of longer endogenous tagging by CRISPR/Cas9 gene editing in C. elegans, we present a set of plasmids that can serve as templates for generating linear double- or partially single-stranded HDR templates that are selection marker independent. The plasmids each contain a fluorescent protein coding region, as well as affinity tags and/or the auxin-dependent degron domain (Zhang et al., 2015). We have used this kit routinely for >40 tagging experiments with a 98% success rate. The kit can easily be expanded to encompass additional fluorescent proteins or other simple tags. Farboud, B., A. F. Severson, and B. Meyer. 2019 Strategies for Efficient Genome Editing Using CRISPR-Cas9. Genetics 211: 431-457. https://doi.org/10.1534/genetics.118.301775 Paix, A., H. Schmidt, and G. Seydoux. 2016 Cas9-assisted recombineering in C. elegans: genome editing using in vivo assembly of linear DNAs. Nucleic Acid Res. doi: 10.1093/nar/gkw502 Dokshin, G. A., K. S. Ghanta, K. M. Piscopo, and C. C. Mello, 2018 Robust genome editing with short single-stranded and long, partially single-stranded DNA donors in Caenorhabditis elegans. Genetics 210: 781-787. https://doi.org/10.1534/ge- netics.118.301532 Zhang, L., J. D. Ward, Z. Cheng, and A. F. Dernburg, 2015. The auxin-inducible degradation (AID) system enables versatile conditional protein depletion in C. elegans. Development 142: 4374-4384. doi: 10.1242/dev.129635

Yu, Xinwei et al. (2021) International Worm Meeting "Fast deep learning correspondence for neuron tracking and identification in C. elegans using semi-synthetic training"

Sharma, Anuj K., Linderman, Scott W., Randi, Francesco, Yu, Xinwei, Leifer, Andrew M., Creamer, Matthew S.

[International Worm Meeting, 2021]

The nervous system of the nematode C.elegans is well characterized, such that each of the 302 neurons is named and has stereotyped locations across animals(Witvliet et al., 2020). The capability to find corresponding neurons across animals is essential to investigate neural coding and neural dynamics across animals. Despite the worm's overall stereotypy, the variability in neurons' spatial arrangement is sufficient to make predicting neural correspondence a challenge. We present an automated method to track and identify neurons in C. elegans, called "fast Deep Learning Correspondence" or fDLC, based on the transformer network architecture(Vaswani et al., 2017). The transformer has shown great success in natural language processing tasks by modeling the dependencies between words in a sentence. We reasoned this same architecture would be well-suited to extract spatial relationships between neurons in order to build a representation that facilitates finding correspondence to neurons in a template worm. The model is trained once on empirically derived semi-synthetic data and then predicts neural correspondence across held-out real animals via transfer learning. The same pre-trained model both tracks neurons across time and identifies corresponding neurons across individuals. Performance is evaluated against hand-annotated datasets, including NeuroPAL(Yemini et al., 2020). Using only position information, the method achieves 80.0% accuracy at tracking neurons within an individual and 65.8% accuracy at identifying neurons across individuals. Accuracy is even higher on a published dataset(Chaudhary et al., 2021). Accuracy reaches 76.5% when using color information from NeuroPAL. Unlike previous methods, fDLC does not require straightening or transforming the animal into a canonical coordinate system. The method is fast and predicts correspondence in 10 ms making it suitable for future real-time applications. Witvliet D, Mulcahy B, Mitchell JK, Meirovitch Y, Berger DR, Wu Y, et al.Connectomes across development reveal principles of brain maturation in C.elegans. bioRxiv. 2020;doi:10.1101/2020.04.30.066209. Vaswani A, Shazeer N, Parmar N, Uszkoreit J, Jones L, Gomez AN, et al.Attention is All You Need; 2017.Available from:https://arxiv.org/pdf/1706.03762.pdf. Yemini E, Lin A, Nejatbakhsh A, Varol E, Sun R, Mena GE, et al. NeuroPAL: A multicolor atlas for whole-brain neuronal identification in C. Elegans. Cell.2020;doi:10.1016/j.cell.2020.12.012. Chaudhary S, Lee SA, Li Y, Patel DS, Lu H. Graphical-model framework for automated annotation of cell identities in dense cellular images. eLife.2021;10:e60321. doi:10.7554/eLife.60321.