Pukkila-Worley, Read et al. (2011) International Worm Meeting "Immuno-stimulatory small molecules rescue Caenorhabditis elegans from infection with antibiotic-resistant Gram-positive and Gram-negative bacteria."

Feinbaum, Rhonda, Conery, Annie L., Kirienko, Natalia V., Larkins-Ford, Jonah, Chen, Kevin, Pukkila-Worley, Read, Mylonakis, Eleftherios, Ausubel, Frederick M.

[International Worm Meeting, 2011]

Antibiotic-resistant bacteria pose a major threat to public health. As a means to identify novel antimicrobial therapies, we previously screened 33,931 compounds for those that cured Caenorhbaditis elegans of infection with the Gram-positive bacterial pathogen Enterococcus faecalis. We identified 33 small molecules that rescued infected nematodes at a concentration more than ten-fold lower than required to inhibit bacterial growth in vitro. We therefore postulated that a subset of these compounds cured infection by directly stimulating host immunity. To test this hypothesis, we used C. elegans transcriptional reporters to provide a visual readout of immune gene activation and found that five of the 33 compounds strongly activated at least one immune reporter. We predicted that immuno-stimulatory compounds would cure nematodes infected with diverse and antimicrobial-resistant pathogens. Indeed, we found that two compounds (referred to here as #1 and #2) were able to rescue worms infected with the clinically troublesome pathogens, Pseudomonas aeruginosa (a Gram-negative bacteria) and Vancomycin-Resistant E. faecalis (VRE). Using genome-wide, transcription-profiling analyses, we found that compound #1 induced the expression of genes in a pattern that overlapped significantly with those upregulated after exposure to P. aeruginosa. Interestingly, compound #1-mediated activation of immune response genes and resistance to P. aeruginosa infection was entirely dependent on ATF-7, a conserved transcription factor orthologous to the mammalian ATF2/ATF7 and target of the p38 MAP-kinase PMK-1, a central regulator of immunity in the nematode. In a separate transcriptome analysis, we found that compound #2 induced a strong detoxification response that involved upregulation of defense response genes. In summary, we have identified two compounds that cure nematodes infected with E. faecalis, VRE and P. aeruginosa infection by directly stimulating host immunity. Taken together, these data suggest that pharmacologic manipulation of innate defense responses may be a strategy to combat infection with multi-drug resistant microbes.

Cheesman, Hilary et al. (2015) International Worm Meeting "Aberrant activation of p38 MAP kinase-dependent innate immune responses is toxic to C. elegans."

Feinbaum, Rhonda, Cheesman, Hilary, Dowen, Robert, Pukkila-Worley, Read

[International Worm Meeting, 2015]

Inappropriate activation of innate immune responses in intestinal epithelial cells underlies the pathophysiology of inflammatory disorders of the intestine. Here we examine the physiological effects of immune hyperactivation in the intestine of the model nematode C. elegans. We previously identified an immunostimulatory xenobiotic, called RPW-24, that protects C. elegans from bacterial infection by inducing immune effector expression via the conserved p38 MAP kinase pathway, but was toxic to nematodes developing in the absence of pathogen. To investigate a possible connection between the toxicity and immunostimulatory properties of this xenobiotic, we conducted a forward genetic screen for C. elegans mutants that are resistant to the toxic effects of the compound. We identified seven toxicity suppressors from a screen of approximately 100,000 mutagenized haploid genomes, and found that five of these strains contain loss-of-function mutations in each of the known components of the p38 MAP kinase cassette (tir-1, nsy-1, sek-1 and pmk-1). These data indicate that the toxicity of RPW-24 is directly related to its stimulatory effects on the p38 MAPK pathway. We therefore predicted that a genetic screen aimed at identifying endogenous activators of the p38 MAP kinase pathway would uncover mutants that phenocopy the addition of RPW-24 to wild-type animals. We screened approximately 170,000 haploid genomes for dominant mutations that activated the p38 MAPK-dependent pathogen response gene (F08G5.6::GFP) and identified a single allele, which had a gain-of-function (gof) mutation in nsy-1, the MAP kinase kinase kinase that acts upstream of p38 MAPK PMK-1. We found that the nsy-1(gof) allele had greater levels of phosphorylated p38 MAPK, caused hyperinduction of p38 MAPK PMK-1-dependent immune effectors, and was more resistant to killing by the bacterial pathogen Pseudomonas aeruginosa compared to wild-type controls. As was the case in wild-type worms that were exposed to RPW-24, a nsy-1(gof) mutation was toxic to developing animals, a phenotype that could be suppressed through RNAi-mediated knockdown of the nsy-1 gene. Thus, a gain-of-function mutation in nsy-1 and xenobiotic-induced hyperactivation of the p38 MAPK pathway each drive immune responses that are protective during infection, but are toxic to nematodes under normal growth conditions. These data suggest that there are physiological mechanisms that regulate the p38 MAPK pathway to ensure cellular homeostasis in C. elegans.

Yuen, Grace J. et al. (2011) International Worm Meeting "Enterococcus infection of Caenorhabditis elegans as a model of innate immunity."

Ausubel, Frederick M., Pukkila-Worley, Read, Yuen, Grace J.

[International Worm Meeting, 2011]

Enterococcus is a Gram-positive commensal that is found in the gastrointestinal and biliary tracts of all healthy humans. It is also an important opportunistic pathogen, causing nosocomial urinary tract and wound infections that are often complicated by antibiotic drug resistance. Most human enterococcal infections are caused by either E. faecalis or E. faecium. Our laboratory has modeled Enterococcus infection in C. elegans using both strains. We have previously shown that infection with either strain leads to gut distention, but only E. faecalis is able to establish a persistent infection and kill the nematode. We now provide evidence that at least two canonical C. elegans immune signaling pathways are important for survival during infection with both E. faecalis and E. faecium. While the lifespan of wild-type worms is unaffected by an E. faecium infection, mutations in the PMK-1 and FSHR-1 immune signaling pathways lead to an immunocompromised phenotype, where pmk-1 and fshr-1 mutants die rapidly upon E. faecium feeding. This new finding suggests that an active immune response is required to keep E. faecium infection "in check" in the worm intestine, and that E. faecium is indeed pathogenic to the nematode. We are now using microscopy and genetic studies to understand the biology of the Enterococcus infection in C. elegans and to identify novel Enterococcus-activated immune signaling pathways.

Ding, Wei et al. (2017) International Worm Meeting "Differential requirements for the methyl donor SAM in stress-induced gene expression."

Higgins, Daniel, Walker, Amy, Pukkila-Worley, Read, Ding, Wei

[International Worm Meeting, 2017]

Transcriptional processes are tightly linked to metabolic products. For example, the 1-carbon (1CC) cycle produces SAM (s-adenosylmethionine), the methyl donor used for histone and nucleic acid modification. While 1CC disruption has been proposed to broadly affect epigenetic processes, it is surprising that shifts in SAM levels affect specific methylation events, such as activating H3K4 methylation, suggesting that changes in SAM metabolism may affect specific genes or biological processes. Because the mammalian H3K4 methyltransferase (HMT) family is complex, it has also been difficult to discern specific roles for individual HMTs. Using C. elegans, we found that reducing SAM caused defects in H3K4me3 accumulation on promoters of selected infection response genes during pathogen exposure, that induction of these genes failed and that these animals had reduced survival. Furthermore, set-16/MLL, appeared to have critical role in this process. This suggests SAM may be important for stress responsive transcription and that distinct H3K4 methyl transferases may have specific roles. To test this model, we treated C. elegans with three stresses, bacterial, detoxification and heat, then examined survival and genome wide mRNA expression patterns in animals with low SAM (sams-1(RNAi)), or after knockdown of H3K4 HMTs set-2/SET1 or set-16/MLL. We found that transcriptional responses to bacterial or detoxification stress were blunted in sams-1(RNAi) animals. set-2/SET1 or set-16/MLL RNAi had negligible effects on in control animals, however, elicited distinct effects in response to bacterial, detoxification stress. set-2/SET1 appears less broadly required during bacterial or detoxification stress. Many bacterial or detoxification stress genes were also strongly attenuated after set-16/MLL knockdown. Canonical heat shock gene expression, however, increased normally in response to heat after sams-1, set-2 and set-16 RNAi. Finally, our biological assays revealed two important effects. First, sams-1, set-2/SET1 and set-16/MLL RNAi animals all died rapidly from the pathogen, despite variations in transcriptional responses. Interestingly, other 1CC genes, such the SAM synthases sams-2 and sams-3, are normally induced in response to a pathogen, but fail in sams-1, set-2/SET1 and set-16/MLL RNAi animals, suggesting feedback loops maintaining 1CC function may be critical for pathogenic challenge. Finally, set-16/MLL was sensitive to all three stresses, whereas set-2/SET1 animals survived normally on the detoxification agent and after heat shock. Thus, set-16/MLL may have a more general role in stress responsive transcription. Our data suggests that depletion of SAM could reduce the ability of a tissue to respond to stress, thereby increasing potential for injury and exacerbating disease progression.

Giese SH et al. (2014) Bioinformatics "Specificity control for read alignments using an artificial reference genome-guided false discovery rate."

Giese SH, Zickmann F, Renard BY

[Bioinformatics, 2014]

MOTIVATION: Accurate estimation, comparison and evaluation of read mapping error rates is a crucial step in the processing of next-generation sequencing data, as further analysis steps and interpretation assume the correctness of the mapping results. Current approaches are either focused on sensitivity estimation and thereby disregard specificity or are based on read simulations. Although continuously improving, read simulations are still prone to introduce a bias into the mapping error quantitation and cannot capture all characteristics of an individual dataset. RESULTS: We introduce ARDEN (artificial reference driven estimation of false positives in next-generation sequencing data), a novel benchmark method that estimates error rates of read mappers based on real experimental reads, using an additionally generated artificial reference genome. It allows a dataset-specific computation of error rates and the construction of a receiver operating characteristic curve. Thereby, it can be used for optimization of parameters for read mappers, selection of read mappers for a specific problem or for filtering alignments based on quality estimation. The use of ARDEN is demonstrated in a general read mapper comparison, a parameter optimization for one read mapper and an application example in single-nucleotide polymorphism discovery with a significant reduction in the number of false positive identifications. AVAILABILITY: The ARDEN source code is freely available at http://sourceforge.net/projects/arden/.

Colonnello A et al. (2020) Neurotox Res "Correction to: Comparing the Neuroprotective Effects of Caffeic Acid in Rat Cortical Slices and Caenorhabditis elegans: Involvement of Nrf2 and SKN-1 Signaling Pathways."

Tunez I, Rubio-Lopez LC, Aguilera-Portillo G, Silva-Palacios A, Evaristo-Priego Y, Santamaria A, Galvan-Arzate S, Cortez-Nunez S, Aschner M, Rangel-Lopez E, Chen P, Colonnello A, Robles-Banuelos B, Garcia-Contreras R

[Neurotox Res, 2020]

One of the authors has incorrect family name spelling in the original article_. Michael Ashner should read as Michael Aschner.

Lee EB et al. (2019) Arch Pharm Res "Correction to: Lifespan-extending property of 6-shogaol from Zingiber officinale Roscoe in Caenorhabditis elegans."

Lee EB, Noh YJ, Kim SJ, Kim JH, Kim DK, Kim YJ, Hwang IH

[Arch Pharm Res, 2019]

The author would like to include conflict of interest statement of the online published article. The correct conflict of interest statement should read as: The authors declare no conflict of interest.

Lee H et al. (2023) BMC Genomics "Benchmarking datasets for assembly-based variant calling using high-fidelity long reads."

Kim J, Lee J, Lee H

[BMC Genomics, 2023]

BACKGROUND: Recent advances in long-read sequencing technologies have enabled accurate identification of all genetic variants in individuals or cells; this procedure is known as variant calling. However, benchmarking studies on variant calling using different long-read sequencing technologies are still lacking. RESULTS: We used two Caenorhabditis elegans strains to measure several variant calling metrics. These two strains shared true-positive genetic variants that were introduced during strain generation. In addition, both strains contained common and distinguishable variants induced by DNA damage, possibly leading to false-positive estimation. We obtained accurate and noisy long reads from both strains using high-fidelity (HiFi) and continuous long-read (CLR) sequencing platforms, and compared the variant calling performance of the two platforms. HiFi identified a 1.65-fold higher number of true-positive variants on average, with 60% fewer false-positive variants, than CLR did. We also compared read-based and assembly-based variant calling methods in combination with subsampling of various sequencing depths and demonstrated that variant calling after genome assembly was particularly effective for detection of large insertions, even with 10 × sequencing depth of accurate long-read sequencing data. CONCLUSIONS: By directly comparing the two long-read sequencing technologies, we demonstrated that variant calling after genome assembly with 10 × or more depth of accurate long-read sequencing data allowed reliable detection of true-positive variants. Considering the high cost of HiFi sequencing, we herein propose appropriate methodologies for performing cost-effective and high-quality variant calling: 10 × assembly-based variant calling. The results of the present study may facilitate the development of methods for identifying all genetic variants at the population level.

Lesack K et al. (2022) PLoS One "Different structural variant prediction tools yield considerably different results in Caenorhabditis elegans."

Lesack K, Andersen EC, Wasmuth JD, Mariene GM

[PLoS One, 2022]

The accurate characterization of structural variation is crucial for our understanding of how large chromosomal alterations affect phenotypic differences and contribute to genome evolution. Whole-genome sequencing is a popular approach for identifying structural variants, but the accuracy of popular tools remains unclear due to the limitations of existing benchmarks. Moreover, the performance of these tools for predicting variants in non-human genomes is less certain, as most tools were developed and benchmarked using data from the human genome. To evaluate the use of long-read data for the validation of short-read structural variant calls, the agreement between predictions from a short-read ensemble learning method and long-read tools were compared using real and simulated data from Caenorhabditis elegans. The results obtained from simulated data indicate that the best performing tool is contingent on the type and size of the variant, as well as the sequencing depth of coverage. These results also highlight the need for reference datasets generated from real data that can be used as 'ground truth' in benchmarks.

Milani, M et al. (2005) Scanning "Read-out of soft X-ray contact microscopy microradiographs by focused ion beam/scanning electron microscope."

Zrimec, A, Drobne, D, Tatti, F, Batani, D, Orsini, F, Poletti, G, Zullini, A, Milani, M

[Scanning, 2005]

A novel focused ion beam-based technique is presented for the read-out of microradiographs of Caenorhabditis elegans nematodes generated by soft x-ray contact microscopy (SXCM). In previous studies, the read-out was performed by atomic force microscopy (AFM), but in our work SXCM microradiographs were imaged by scanning ion microscopy (SIM) in a focused ion beam/scanning electron microscope (FIB/SEM). It allows an ad libitum selection of a sample region for gross morphologic to nanometric investigations, with a sequence of imaging and cutting. The FIB/SEM is less sensitive to height variation of the relief, and sectioning makes it possible to analyse the sample further. The SXCM can be coupled to SIM in a more efficient and faster way than to AFM. Scanning ion microscopy is the method of choice for the read-out of microradiographs of small multicellular organisms.