Applications & Publications

A defining member of the new cysteine-cradle family is an aECM protein signalling skin damage in C. elegans

Apical extracellular matrices (aECMs) act as crucial barriers, and communicate with the epidermis to trigger protective responses following injury or infection. In Caenorhabditis elegans, the skin aECM, the cuticle, is produced by the epidermis and is decorated with periodic circumferential furrows. We previously showed that mutants lacking cuticle furrows exhibit persistent immune activation (PIA), providing a valuable model to study the link between cuticle damage and immune response. In a genetic suppressor screen, we identified spia-1 as a key gene downstream of furrow collagens and upstream of immune signalling. spia-1 expression oscillates during larval development, peaking between each moult together with patterning cuticular components. It encodes a secreted protein that localises to furrows. SPIA-1 shares a novel cysteine-cradle domain with other aECM proteins. SPIA-1 mediates immune activation in response to furrow loss and is proposed to act as an extracellular signal activator of cuticle damage. This research provides a molecular insight into intricate interplay between cuticle integrity and epidermal immune activation in C. elegans.

Induction of proteostasis pathways in a C. elegans model of exercise

Exercise is one of the most potent interventions known that is able to prevent and even treat dozens of age-related dysfunctions and diseases however much remains unknown about how its benefits are derived. Because exercise exerts such wide-ranging effects, and because a decline in protein homeostasis (proteostasis) with age has been connected to numerous age-related diseases, we hypothesized that exercise could increase the activity of proteostasis pathways like the proteasome and autophagy and that this could ameliorate age-related declines in function. We investigated the effects of exercise has on proteostasis in Caenorhabditis elegans. We utilized a involuntary movement exercise paradigm to investigate acute exercise and the effects of multiple days of exercise on aging and proteostasis, including the autophagy-lysosome system, the proteasome, and neurotoxic peptides. We found that exercise is able to increase proteasomal activity and autophagic flux in vivo, improved resistance to toxic peptides, and increased lifespan. One of the primary rationales for studying the mechanisms of exercise is to uncover potential mediators that can be repurposed to deliver the benefits of exercise to those unable to engage in physical activity. We hypothesized that exercise-elevated metabolite α-ketoglutarate, already demonstrated to improve age-related declines in flies and mice, would mimic the effects of exercise on proteostasis. Treatment with α-ketoglutarate showed resulted in complex proteostatic outcomes, indicative of the challenges in recapitulating a multi-functional domain phenomenon like exercise.

Utilization of Artificial Intelligence Coupled with a High-Throughput, High-Content Platform in the Exploration of Neurodevelopmental Toxicity of Individual and Combined PFAS

Per- and polyfluoroalkyl substances (PFAS) are synthetic chemicals used in various products, such as firefighting foams and non-stick cookware, due to their resistance to heat and degradation. However, these same properties make them persistent in the environment and human body, raising public health concerns. This study selected eleven PFAS commonly found in drinking water and exposed Caenorhabditis elegans to concentrations ranging from 0.1 to 200 µM to assess neurodevelopmental toxicity using a high-throughput, high-content screening (HTS) platform coupled with artificial intelligence for image analysis. Our findings showed that PFAS such as 6:2 FTS, HFPO-DA, PFBA, PFBS, PFHxA, and PFOS inhibited dopaminergic neuron activity, with fluorescence intensity reductions observed across concentrations from 0.1 to 100 µM. PFOS and PFBS also disrupted synaptic transmission, causing reduced motility and increased paralysis in aldicarb-induced assays, with the most pronounced effects at higher concentrations. These impairments in both neuron activity and synaptic function led to behavioral deficits. Notably, PFOS was one of the most toxic PFAS, affecting multiple neurodevelopmental endpoints. These results emphasize the developmental risks of PFAS exposure, highlighting the impact of both individual compounds and mixtures on neurodevelopment. This knowledge is essential for assessing PFAS-related health risks and informing mitigation strategies.

Naturally occurring variation in a cytochrome P450 modifies thiabendazole responses independently of beta-tubulin

Widespread anthelmintic resistance has complicated the management of parasitic nematodes. Resistance to the benzimidazole (BZ) drug class is nearly ubiquitous in many species and is associated with mutations in beta-tubulin genes. However, mutations in beta-tubulin alone do not fully explain all BZ resistance. We performed a genome-wide association study using a genetically diverse panel of Caenorhabditis elegans strains to identify loci that contribute to resistance to the BZ drug thiabendazole (TBZ). We identified a quantitative trait locus (QTL) on chromosome V independent of all beta-tubulin genes and overlapping with two promising candidate genes, the cytochrome P450 gene cyp-35D1 and the nuclear hormone receptor nhr-176. Both genes were previously demonstrated to play a role in TBZ metabolism. NHR-176 binds TBZ and induces the expression of CYP-35D1, which metabolizes TBZ. We generated single gene deletions of cyp-35D1 and nhr-176 and found that both genes play a role in TBZ response. A predicted high-impact lysine-to-glutamate substitution at position 267 (K267E) in CYP-35D1 was identified in a sensitive strain, and reciprocal allele replacement strains in different genetic backgrounds were used to show that the lysine allele conferred increased TBZ resistance. Using competitive fitness assays, we found that neither allele was deleterious, but the lysine allele was selected in the presence of TBZ. Additionally, we found that the lysine allele significantly increased the rate of TBZ metabolism compared to the glutamate allele. Moreover, yeast expression assays showed that the lysine version of CYP-35D1 had twice the enzymatic activity of the glutamate allele. To connect our results to parasitic nematodes, we analyzed four Haemonchus contortus cytochrome P450 orthologs but did not find variation at the 267 position in fenbendazole-resistant populations. Overall, we confirmed that variation in this cytochrome P450 gene is the first locus independent of beta-tubulin to play a role in BZ resistance.

A microbial natural product fractionation library screen with HRMS/MS dereplication identifies new lipopeptaibiotics against Candida auris

The rise of drug-resistant fungal pathogens, including Candida auris, highlights the urgent need for novel antifungal therapies. We developed a cost-effective platform combining microbial extract prefractionation with rapid MS/MS-bioinformatics-based dereplication to efficiently prioritize new antifungal scaffolds. Screening C. auris and C. albicans revealed novel lipopeptaibiotics, coniotins, from Coniochaeta hoffmannii WAC11161, which were undetectable in crude extracts. Coniotins exhibited potent activity against critical fungal pathogens on the WHO Fungal Priority Pathogens List, including C. albicans, C. neoformans, multidrug-resistant C. auris, and Aspergillus fumigatus, with high selectivity and low resistance potential. Coniotin A targets β-glucan, compromising fungal cell wall integrity, remodelling, and sensitizing C. auris to caspofungin. Identification of a PKS-NRPS biosynthetic gene cluster further enables the discovery of related clusters encoding potential novel lipopeptaibiotics. This study demonstrates the power of natural product prefractionation in uncovering bioactive scaffolds and introduces coniotins as promising candidates for combating multidrug-resistant fungal pathogens.

High-throughput tracking enables systematic phenotyping and drug repurposing in C. elegans disease models

There are thousands of Mendelian diseases with more being discovered weekly and the majority have no approved treatments. To address this need, we require scalable approaches that are relatively inexpensive compared to traditional drug development. In the absence of a validated drug target, phenotypic screening in model organisms provides a route for identifying candidate treatments. Success requires a screenable phenotype. However, the right phenotype and assay may not be obvious for pleiotropic neuromuscular disorders. Here, we show that high-throughput imaging and quantitative phenotyping can be conducted systematically on a panel of C. elegans disease model strains. We used CRISPR genome-editing to create 25 worm models of human Mendelian diseases and phenotyped them using a single standardised assay. All but two strains were significantly different from wild-type controls in at least one feature. The observed phenotypes were diverse, but mutations of genes predicted to have related functions led to similar behavioural differences in worms. As a proof-of-concept, we performed a drug repurposing screen of an FDA-approved compound library, and identified two compounds that rescued the behavioural phenotype of a model of UNC80 deficiency. Our results show that a single assay to measure multiple phenotypes can be applied systematically to diverse Mendelian disease models. The relatively short time and low cost associated with creating and phenotyping multiple strains suggest that high-throughput worm tracking could provide a scalable approach to drug repurposing commensurate with the number of Mendelian diseases.

Yin Yang 1 and guanine quadruplexes protect dopaminergic neurons from cellular stress via transmissive dormancy

Neurons deploy diverse adaptive strategies to ensure survival and neurotransmission amid cellular stress. When these adaptive pathways are overwhelmed, functional impairment or neurodegeneration follows. Here we show that stressed neurons actively induce a state of transmissive dormancy as a protective measure. Extending observations of neurotrauma in C. elegans and mice, human dopaminergic neurons capable of surviving severe cellular challenges both decrease spontaneous activity and modulate dopamine homeostasis through the transcriptional regulator Yin Yang 1 (YY1). To bolster stress resilience and mitigate dopamine toxicity, YY1 increases expression of the vesicular monoamine transporter 2, vMAT2, while coordinately inhibiting dopamine synthesis through stabilization of a guanine quadruplex in intron 10 of tyrosine hydroxylase, TH. This dopaminergic stress response has the potential to cause circuit inactivation, yet safeguards neurons by minimizing the toxic accumulation of cytosolic dopamine and inducing a state of neuronal dormancy. In essence, neurons appear to actively prioritize viability over functionality.

The replicative helicase CMG is required for the divergence of cell fates during asymmetric cell division in vivo

We report that the eukaryotic replicative helicase CMG (Cdc45-MCM-GINS) is required for differential gene expression in cells produced by asymmetric cell divisions in C. elegans. We found that the C. elegans CMG component, PSF-2 GINS2, is necessary for transcriptional upregulation of the pro-apoptotic gene egl-1 BH3-only that occurs in cells programmed to die after they are produced through asymmetric cell divisions. We propose that CMG’s histone chaperone activity causes epigenetic changes at the egl-1 locus during replication in mother cells, and that these changes are required for egl-1 upregulation in cells programmed to die. We find that PSF-2 is also required for the divergence of other cell fates during C. elegans development, suggesting that this function is not unique to egl-1 expression. Our work uncovers an unexpected role of CMG in cell fate decisions and an intrinsic mechanism for gene expression plasticity in the context of asymmetric cell division.

Exposure to benzyl butyl phthalate (BBP) leads to increased double-strand break formation and germline dysfunction in Caenorhabditis elegans

Benzyl butyl phthalate (BBP), a plasticizer found in a wide range of consumer products including vinyl flooring, carpet backing, food packaging, personal care products, and children’s toys, is an endocrine-disrupting chemical linked to impaired reproduction and development in humans. Despite evidence that BBP exposure perturbs the integrity of male and female gametes, its direct effect on early meiotic events is understudied. Here, using the nematode Caenorhabditis elegans, we show that BBP exposure elicits a non-monotonic dose response on the rate of X-chromosome nondisjunction measured using a high-throughput screening platform. From among the range of doses tested (1, 10, 100 and 500 μM BBP), we found that 10 μM BBP elicited the strongest effect on the germline, resulting in increased germ cell apoptosis and chromosome organization defects. Mass spectrometry analysis shows that C. elegans efficiently metabolizes BBP into its primary metabolites, monobutyl phthalate (MBP) and monobenzyl phthalate (MBzP), and that the levels of BBP, MBP, and MBzP detected in the worm are within the range detected in human biological samples. Exposure to 10 μM BBP leads to germlines with enlarged mitotic nuclei, altered meiotic progression, activation of a p53/CEP-1-dependent DNA damage checkpoint, increased double-strand break levels throughout the germline, chromosome morphology defects in oocytes at diakinesis, and increased oxidative stress. RNA sequencing analysis indicates that BBP exposure results in the altered expression of genes involved in xenobiotic metabolic processes, extracellular matrix organization, oocyte morphogenesis, meiotic cell cycle, and oxidoreduction. Taken together, we propose that C. elegans exposure to BBP leads to increased oxidative stress and double-strand break formation, thereby compromising germline genomic integrity and chromosome segregation.

Assessment of the effects of cannabidiol and a CBD-rich hemp extract in Caenorhabditis elegans

Consumer use of cannabidiol (CBD) is growing, but there are still data gaps regarding its possible adverse effects on reproduction and development. Multiple pathways and signaling cascades involved in organismal development and neuronal function, including endocannabinoid synthesis and signaling systems, are well conserved across phyla, suggesting that Caenorhabditis elegans can model the in vivo effects of exogenous cannabinoids. The effects in C. elegans on oxidative stress response (OxStrR), developmental timing, juvenile and adult spontaneous locomotor activity, reproductive output, and organismal CBD concentrations were assessed after exposure to purified CBD or a hemp extract suspended in 0.5% sesame oil emulsions. In C. elegans, this emulsion vehicle is equivalent to a high-fat diet (HFD). As in mammals, HFD was associated with oxidative-stress-related gene expression in C. elegans adults. CBD reduced HFD-induced OxStrR in transgenic adults and counteracted the hypoactivity observed in HFD-exposed wild-type adults. In C. elegans exposed to CBD from the onset of feeding, delays in later milestone acquisition were irreversible, while later juvenile locomotor activity effects were reversible after the removal of CBD exposure. CBD-induced reductions in mean juvenile population body size were cumulative when chronic exposures were initiated at parental reproductive maturity. Purified CBD was slightly more toxic than matched concentrations of CBD in hemp extract for all tested endpoints, and both were more toxic to juveniles than to adults. Dosimetry indicated that all adverse effect levels observed in C. elegans far exceeded recommended CBD dosages for humans.

Effect of altered production and storage of dopamine on development and behavior in C. elegans

The nematode, Caenorhabditis elegans (C. elegans), is an advantageous model for studying developmental toxicology due to its well-defined developmental stages and homology to humans. It has been established that across species, dopaminergic neurons are highly vulnerable to neurotoxicant exposure, resulting in developmental neuronal dysfunction and age-induced degeneration. C. elegans, with genetic perturbations in dopamine system proteins, can provide insight into the mechanisms of dopaminergic neurotoxicants. In this study, we present a comprehensive analysis on the effect of gene mutations in dopamine-related proteins on body size, development, and behavior in C. elegans.

Caenorhabditis elegans RIG-I-like receptor DRH-1 signals via CARDs to activate antiviral immunity in intestinal cells

Reproductive regulation of the mitochondrial stress response in Caenorhabditis elegans

Olfaction regulates peripheral mitophagy and mitochondrial function

The central nervous system coordinates peripheral cellular stress responses, including the unfolded protein response of the mitochondria (UPRMT); however, the contexts for which this regulatory capability evolved are unknown. UPRMT is up-regulated upon pathogenic infection and in metabolic flux, and the olfactory nervous system has been shown to regulate pathogen resistance and peripheral metabolic activity. Therefore, we asked whether the olfactory nervous system in Caenorhabditis elegans controls the UPRMT cell nonautonomously. We found that silencing a single inhibitory olfactory neuron pair, AWC, led to robust induction of UPRMT and reduction of oxidative phosphorylation dependent on serotonin signaling and parkin-mediated mitophagy. Further, AWC ablation confers resistance to the pathogenic bacteria Pseudomonas aeruginosa partially dependent on the UPRMT transcription factor atfs-1 and fully dependent on mitophagy machinery. These data illustrate a role for the olfactory nervous system in regulating whole-organism mitochondrial dynamics, perhaps in preparation for postprandial metabolic stress or pathogenic infection.

ADR-2 regulates fertility and oocyte fate in C. elegans

RNA binding proteins play essential roles in coordinating germline gene expression and development in all organisms. Here, we report that loss of ADR-2, a member of the Adenosine DeAminase acting on RNA (ADAR) family of RNA binding proteins and the sole adenosine-to-inosine RNA editing enzyme in C. elegans, can improve fertility in multiple genetic backgrounds. First, we show that loss of RNA editing by ADR-2 restores normal embryo production to subfertile animals that transgenically express a vitellogenin (yolk protein) fusion to green fluorescent protein. Using this phenotype, a high-throughput screen was designed to identify RNA binding proteins that when depleted yield synthetic phenotypes with loss of adr-2. The screen uncovered a genetic interaction between ADR-2 and SQD-1, a member of the heterogenous nuclear ribonucleoprotein (hnRNP) family of RNA binding proteins. Microscopy, reproductive assays, and high-throughput sequencing reveal that sqd-1 is essential for the onset of oogenesis and oogenic gene expression in young adult animals, and that loss of adr-2 can counteract the effects of loss of sqd-1 on gene expression and rescue the switch from spermatogenesis to oogenesis. Together, these data demonstrate that ADR-2 can contribute to the suppression of fertility and suggest novel roles for both RNA editing-dependent and independent mechanisms in regulating embryogenesis.

Cytosolic dopamine determines hypersensitivity to blunt force trauma

The selective vulnerability of dopaminergic neurons to trauma-induced neurodegeneration is conserved across species, from nematodes to humans. However, the molecular mechanisms underlying this hypersensitivity to blunt force trauma remain elusive. We find that extravesicular dopamine, a key driver of Parkinson’s disease, extends its toxic role to the acute challenges associated with injury. Ectopic dopamine synthesis in serotonergic neurons sensitizes this resilient neuronal subtype to trauma-induced degeneration. While dopaminergic neurons normally maintain dopamine in a functional and benign state, trauma-induced subcellular redox imbalances elicit dopamine-dependent cytotoxicity. Cytosolic dopamine accumulation, through perturbations to its synthesis, metabolism, or packaging, is necessary and sufficient to drive neurodegeneration upon injury and during aging. Additionally, degeneration is further exacerbated by rapid upregulation of the rate-limiting enzyme in dopamine synthesis, cat-2, via the FOS-1 transcription factor. Fundamentally, our study in C. elegans unravels the molecular intricacies rendering dopaminergic neurons uniquely prone to physical perturbation across evolutionary lines.

The C. elegans SET1 histone methyltransferase SET-2 is not required for transgenerational memory of silencing

Sex-specific developmental gene expression atlas unveils dimorphic gene networks in C. elegans

Parallel evolution of alternate morphotypes of Chryseobacterium gleum during experimental evolution with Caenorhabditis elegans

Microbial evolution within polymicrobial communities is a complex process. Here, we report within-species diversification within multispecies microbial communities during experimental evolution with the nematode Caenorhabditis elegans. We describe morphological diversity in the target species Chryseobacterium gleum, which developed a novel colony morphotype in a small number of replicate communities. Alternate morphotypes coexisted with original morphotypes in communities, as well as in single-species experiments using evolved isolates. We found that the original and alternate morphotypes differed in motility and in spatial expansion in the presence of C. elegans. This study provides insight into the emergence and maintenance of intraspecies diversity in the context of microbial communities.

In Vitro and In Vivo Insights into a Broccoli Byproduct as a Healthy Ingredient for the Management of Alzheimer's Disease and Aging through Redox Biology

Broccoli has gained popularity as a highly consumed vegetable due to its nutritional and health properties. This study aimed to evaluate the composition profile and the antioxidant capacity of a hydrophilic extract derived from broccoli byproducts, as well as its influence on redox biology, Alzheimer’s disease markers, and aging in the Caenorhabditis elegans model. The presence of glucosinolate was observed and antioxidant capacity was demonstrated both in vitro and in vivo. The in vitro acetylcholinesterase inhibitory capacity was quantified, and the treatment ameliorated the amyloid-β- and tau-induced proteotoxicity in transgenic strains via SOD-3 and SKN-1, respectively, and HSP-16.2 for both parameters. Furthermore, a preliminary study on aging indicated that the extract effectively reduced reactive oxygen species levels in aged worms and extended their lifespan. Utilizing broccoli byproducts for nutraceutical or functional foods could manage vegetable processing waste, enhancing productivity and sustainability while providing significant health benefits.

Bioactive Properties of Tagetes erecta Edible Flowers: Polyphenol and Antioxidant Characterization and Therapeutic Activity against Ovarian Tumoral Cells and Caenorhabditis elegans Tauopathy

Tagetes erecta is an edible flower deeply rooted in traditional Mexican culture. It holds a central role in the most popular and iconic Mexican celebration, “the Day of the Dead”. Furthermore, it is currently receiving interest as a potential therapeutic agent, motivated mainly by its polyphenol content. The present study aims to evaluate the biological activity of an extract synthesized from the petals of the edible flower T. erecta. This extract showed significant antioxidant scores measured by the most common in vitro methodologies (FRAP, ABTS, and DPPH), with values of 1475.3 μM trolox/g extr, 1950.3 μM trolox/g extr, and 977.7 μM trolox/g extr, respectively. In addition, up to 36 individual polyphenols were identified by chromatography. Regarding the biomedical aspects of the petal extract, it exhibited antitumoral activity against ovarian carcinoma cells evaluated by the MTS assay, revealing a lower value of IC50 compared to other flower extracts. For example, the extract from T. erecta reported an IC50 value half as low as an extract from Rosa × hybrida and six times lower than another extract from Tulbaghia violacea. This antitumoral effect of T. erecta arises from the induction of the apoptotic process; thus, incubating ovarian carcinoma cells with the petal extract increased the rate of apoptotic cells measured by flow cytometry. Moreover, the extract also demonstrated efficacy as a therapeutic agent against tauopathy, a feature of Alzheimer’s disease (AD) in the Caenorhabditis elegans experimental model. Treating worms with the experimental extract prevented disfunction in several motility parameters such as wavelength and swimming speed. Furthermore, the T. erecta petal extract prevented the release of Reactive Oxygen Species (ROS), which are associated with the progression of AD. Thus, treatment with the extract resulted in an approximate 20% reduction in ROS production. These findings suggest that these petals could serve as a suitable source of polyphenols for biomedical applications.

Lessons from assembling a microbial natural product and pre-fractionated extract library in an academic laboratory

Microbial natural products are specialized metabolites that are sources of many bioactive compounds including antibiotics, antifungals, antiparasitics, anticancer agents, and probes of biology. The assembly of libraries of producers of natural products has traditionally been the province of the pharmaceutical industry. This sector has gathered significant historical collections of bacteria and fungi to identify new drug leads with outstanding outcomes—upwards of 60% of drug scaffolds originate from such libraries. Despite this success, the repeated rediscovery of known compounds and the resultant diminishing chemical novelty contributed to a pivot from this source of bioactive compounds toward more tractable synthetic compounds in the drug industry. The advent of advanced mass spectrometry tools, along with rapid whole genome sequencing and in silico identification of biosynthetic gene clusters that encode the machinery necessary for the synthesis of specialized metabolites, offers the opportunity to revisit microbial natural product libraries with renewed vigor. Assembling a suitable library of microbes and extracts for screening requires the investment of resources and the development of methods that have customarily been the proprietary purview of large pharmaceutical companies. Here, we report a perspective on our efforts to assemble a library of natural product-producing microbes and the establishment of methods to extract and fractionate bioactive compounds using resources available to most academic labs. We validate the library and approach through a series of screens for antimicrobial and cytotoxic agents. This work serves as a blueprint for establishing libraries of microbial natural product producers and bioactive extract fractions suitable for screens of bioactive compounds.

Life-Cycle-Dependent Toxicities of Mono- and Bifunctional Alkylating Agents in the 3R-Compliant Model Organism C. elegans

Caenorhabditis elegans (C. elegans) is gaining recognition and importance as an organismic model for toxicity testing in line with the 3Rs principle (replace, reduce, refine). In this study, we explored the use of C. elegans to examine the toxicities of alkylating sulphur mustard analogues, specifically the monofunctional agent 2-chloroethyl-ethyl sulphide (CEES) and the bifunctional, crosslinking agent mechlorethamine (HN2). We exposed wild-type worms at different life cycle stages (from larvae L1 to adulthood day 10) to CEES or HN2 and scored their viability 24 h later. The susceptibility of C. elegans to CEES and HN2 paralleled that of human cells, with HN2 exhibiting higher toxicity than CEES, reflected in LC50 values in the high µM to low mM range. Importantly, the effects were dependent on the worms’ developmental stage as well as organismic age: the highest susceptibility was observed in L1, whereas the lowest was observed in L4 worms. In adult worms, susceptibility to alkylating agents increased with advanced age, especially to HN2. To examine reproductive effects, L4 worms were exposed to CEES and HN2, and both the offspring and the percentage of unhatched eggs were assessed. Moreover, germline apoptosis was assessed by using ced-1p::GFP (MD701) worms. In contrast to concentrations that elicited low toxicities to L4 worms, CEES and HN2 were highly toxic to germline cells, manifesting as increased germline apoptosis as well as reduced offspring number and percentage of eggs hatched. Again, HN2 exhibited stronger effects than CEES. Compound specificity was also evident in toxicities to dopaminergic neurons–HN2 exposure affected expression of dopamine transporter DAT-1 (strain BY200) at lower concentrations than CEES, suggesting a higher neurotoxic effect. Mechanistically, nicotinamide adenine dinucleotide (NAD+) has been linked to mustard agent toxicities. Therefore, the NAD+-dependent system was investigated in the response to CEES and HN2 treatment. Overall NAD+ levels in worm extracts were revealed to be largely resistant to mustard exposure except for high concentrations, which lowered the NAD+ levels in L4 worms 24 h post-treatment. Interestingly, however, mutant worms lacking components of NAD+-dependent pathways involved in genome maintenance, namely pme-2, parg-2, and sirt-2.1 showed a higher and compound-specific susceptibility, indicating an active role of NAD+ in genotoxic stress response. In conclusion, the present results demonstrate that C. elegans represents an attractive model to study the toxicology of alkylating agents, which supports its use in mechanistic as well as intervention studies with major strength in the possibility to analyze toxicities at different life cycle stages.

Proton Microbeam Targeted Irradiation of the Gonad Primordium Region Induces Developmental Alterations Associated with Heat Shock Responses and Cuticle Defense in Caenorhabditis elegans

We describe a methodology to manipulate Caenorhabditis elegans (C. elegans) and irradiate the stem progenitor gonad region using three MeV protons at a specific developmental stage (L1). The consequences of the targeted irradiation were first investigated by considering the organogenesis of the vulva and gonad, two well-defined and characterized developmental systems in C. elegans. In addition, we adapted high-throughput analysis protocols, using cell-sorting assays (COPAS) and whole transcriptome analysis, to the limited number of worms (>300) imposed by the selective irradiation approach. Here, the presented status report validated protocols to (i) deliver a controlled dose in specific regions of the worms; (ii) immobilize synchronized worm populations (>300); (iii) specifically target dedicated cells; (iv) study the radiation-induced developmental alterations and gene induction involved in cellular stress (heat shock protein) and cuticle injury responses that were found.

Effect of altered production and storage of dopamine on development and behavior in C. elegans

The nematode, Caenorhabditis elegans, is an advantageous model for studying developmental toxicology due to its homology to humans and well-defined developmental stages. Similarly to humans, C. elegans utilize dopamine as a neurotransmitter to regulate motor behavior. We have previously reported behavioral deficits in a genetic model of C. elegans (OK411) that lack the neurotransmitter transporter necessary for packaging dopamine into synaptic vesicles. Anecdotally, we observed these C. elegans appeared to have a smaller body size, which is supported by prior studies that observed a larger body size in C. elegans that lack the enzyme that catalyzes dopamine synthesis, suggesting a complex regulatory system in which dopamine mediates body size in C. elegans. However, the question of whether body size abnormalities apparent in C. elegans with disruptions to their dopamine system are developmental or purely based on body size remains unanswered. Here, we present data characterizing the effect of gene mutations in dopamine-related proteins on body size, development, and behavior. We analyzed C. elegans that lack the ability to sequester dopamine (OK411), that overproduce dopamine (UA57), and a novel strain (MBIA) generated through crossing OK411 and UA57, which lacks the ability to sequester dopamine into vesicles and additionally endogenously overproduces dopamine. This novel strain was generated to address the hypothesis that an endogenous increase in production of dopamine can rescue deficits caused by a lack of vesicular dopamine sequestration. Compared to wild type, OK411 have shorter body lengths and behavioral deficits in early life stages. In contrast, the MBIA strain have similar body lengths to wild-type by early adulthood and display similar behavior to wild-type by early adulthood. Our data suggests that endogenously increasing the production of dopamine is able to mitigate deficits in C. elegans lacking the ability to package dopamine into synaptic vesicles. These results provide evidence that the dopamine system impacts development, growth, and reproduction in C. elegans.

Glial-derived mitochondrial signals affect neuronal proteostasis and aging

Proteostasis is differentially modulated by inhibition of translation initiation or elongation

Recent work has revealed an increasingly important role for mRNA translation in maintaining proteostasis. Here, we use chemical inhibitors targeting discrete steps of translation to compare how lowering the concentration of all or only translation initiation-dependent proteins rescues Caenorhabditis elegans from proteotoxic stress. We systematically challenge proteostasis and show that pharmacologically inhibiting translation initiation or elongation elicits a distinct protective profile. Inhibiting elongation protects from heat and proteasome dysfunction independently from HSF-1 but does not protect from age-associated protein aggregation. Conversely, inhibition of initiation protects from heat and age-associated protein aggregation and increases lifespan, dependent on hsf-1, but does not protect from proteotoxicity caused by proteasome dysfunction. Surprisingly, we find that the ability of the translation initiation machinery to control the concentration of newly synthesized proteins depends on HSF-1. Inhibition of translation initiation in wild-type animals reduces the concentration of newly synthesized proteins but increases it in hsf-1 mutants. Our findings suggest that the HSF-1 pathway is not only a downstream target of translation but also directly cooperates with the translation initiation machinery to control the concentration of newly synthesized proteins to restore proteostasis.

azyx-1 is a new gene that overlaps with zyxin and affects its translation in C. elegans, impacting muscular integrity and locomotion

Development of a workflow for the selection, identification and optimization of lactic acid bacteria with high γ-aminobutyric acid production

Lactic acid bacteria produce γ-aminobutyric acid (GABA) as an acid stress response. GABA is a neurotransmitter that may improve sleep and resilience to mental stress. This study focused on the selection, identification and optimization of a bacterial strain with high GABA production, for development as a probiotic supplement. The scientific literature and an industry database were searched for probiotics and potential GABA producers. In silico screening was conducted to identify genes involved in GABA production. Subsequently, 17 candidates were screened for in vitro GABA production using thin layer chromatography, which identified three candidate probiotic strains Levilactobacillus brevis DSM 20054, Lactococcus lactis DS75843and Bifidobacterium adolescentis DSM 24849 as producing GABA. Two biosensors capable of detecting GABA were developed: 1. a transcription factor-based biosensor characterized by the interaction with the transcriptional regulator GabR was developed in Corynebacterium glutamicum; and 2. a growth factor-based biosensor was built in Escherichia coli, which used auxotrophic complementation by expressing 4-aminobutyrate transaminase (GABA-T) that transfers the GABA amino group to pyruvate, hereby forming alanine. Consequently, the feasibility of developing a workflow based on co-culture with producer strains and a biosensor was tested. The three GABA producers were identified and the biosensors were encapsulated in nanoliter reactors (NLRs) as alginate beads in defined gut-like conditions. The E. coli growth factor-based biosensor was able to detect changes in GABA concentrations in liquid culture and under gut-like conditions. L. brevis and L. lactis were successfully encapsulated in the NLRs and showed growth under miniaturized intestinal conditions.

Lipid homeostasis is essential for a maximal ER stress response

Changes in lipid metabolism are associated with aging and age-related diseases, including proteopathies. The endoplasmic reticulum (ER) is uniquely a major hub for protein and lipid synthesis, making its function essential for both protein and lipid homeostasis. However, it is less clear how lipid metabolism and protein quality may impact each other. Here, we identified let-767, a putative hydroxysteroid dehydrogenase in Caenorhabditis elegans, as an essential gene for both lipid and ER protein homeostasis. Knockdown of let-767 reduces lipid stores, alters ER morphology in a lipid-dependent manner, and blocks induction of the Unfolded Protein Response of the ER (UPRER). Interestingly, a global reduction in lipogenic pathways restores UPRER induction in animals with reduced let-767. Specifically, we find that supplementation of 3-oxoacyl, the predicted metabolite directly upstream of let-767, is sufficient to block induction of the UPRER. This study highlights a novel interaction through which changes in lipid metabolism can alter a cell's response to protein-induced stress.

Fast and easy method to culture and obtain large populations of male nematodes

Multiple pals gene modules control a balance between immunity and development in Caenorhabditis elegans

The immune system continually battles against pathogen-induced pressures, which often leads to the evolutionary expansion of immune gene families in a species-specific manner. For example, the pals gene family expanded to 39 members in the Caenorhabditis elegans genome, in comparison to a single mammalian pals ortholog. Our previous studies have revealed that two members of this family, pals-22 and pals-25, act as antagonistic paralogs to control the Intracellular Pathogen Response (IPR). The IPR is a protective transcriptional response, which is activated upon infection by two molecularly distinct natural intracellular pathogens of C. elegans–the Orsay virus and the fungus Nematocida parisii from the microsporidia phylum. In this study, we identify a previously uncharacterized member of the pals family, pals-17, as a newly described negative regulator of the IPR. pals-17 mutants show constitutive upregulation of IPR gene expression, increased immunity against intracellular pathogens, as well as impaired development and reproduction. We also find that two other previously uncharacterized pals genes, pals-20 and pals-16, are positive regulators of the IPR, acting downstream of pals-17. These positive regulators reverse the effects caused by the loss of pals-17 on IPR gene expression, immunity, and development. We show that the negative IPR regulator protein PALS-17 and the positive IPR regulator protein PALS-20 colocalize inside and at the apical side of intestinal epithelial cells, which are the sites of infection for IPR-inducing pathogens. In summary, our study demonstrates that several pals genes from the expanded pals gene family act as ON/OFF switch modules to regulate a balance between organismal development and immunity against natural intracellular pathogens in C. elegans.

Characterizing short germline-specific promoters with a range of expression levels in C. elegans

Endocytic coelomocytes are required for lifespan extension by axenic dietary restriction

A rather peculiar but very potent means of achieving longevity is through axenic dietary restriction (ADR), where animals feed on (semi-)defined culture medium in absence of any other lifeform. The little knowledge we already have on ADR is mainly derived from studies using the model organism Caenorhabditis elegans, where ADR more than doubles organismal lifespan. What is underlying this extreme longevity so far remains enigmatic, as ADR seems distinct from other forms of DR and bypasses well-known longevity factors. We here focus first on CUP-4, a protein present in the coelomocytes, which are endocytic cells with a presumed immune function. Our results show that loss of cup-4 or of the coelomocytes affects ADR-mediated longevity to a similar extent. As the coelomocytes have been suggested to have an immune function, we then investigated different central players of innate immune signalling, but could prove no causal links with axenic lifespan extension. We propose that future research focuses further on the role of the coelomocytes in endocytosis and recycling in the context of longevity.

Molecular basis of the TRAP complex function in ER protein biogenesis

The translocon-associated protein (TRAP) complex resides in the endoplasmic reticulum (ER) membrane and interacts with the Sec translocon and the ribosome to facilitate biogenesis of secretory and membrane proteins. TRAP plays a key role in the secretion of many hormones, including insulin. Here we reveal the molecular architecture of the mammalian TRAP complex and how it engages the translating ribosome associated with Sec61 translocon on the ER membrane. The TRAP complex is anchored to the ribosome via a long tether and its position is further stabilized by a finger-like loop. This positions a cradle-like lumenal domain of TRAP below the translocon for interactions with translocated nascent chains. Our structure-guided TRAP mutations in Caenorhabditis elegans lead to growth deficits associated with increased ER stress and defects in protein hormone secretion. These findings elucidate the molecular basis of the TRAP complex in the biogenesis and translocation of proteins at the ER.

Lipid droplets and peroxisomes are co-regulated to drive lifespan extension in response to mono-unsaturated fatty acids

Dietary mono-unsaturated fatty acids (MUFAs) are linked to longevity in several species. But the mechanisms by which MUFAs extend lifespan remain unclear. Here we show that an organelle network involving lipid droplets and peroxisomes is critical for MUFA-induced longevity in Caenorhabditis elegans. MUFAs upregulate the number of lipid droplets in fat storage tissues. Increased lipid droplet number is necessary for MUFA-induced longevity and predicts remaining lifespan. Lipidomics datasets reveal that MUFAs also modify the ratio of membrane lipids and ether lipids—a signature associated with decreased lipid oxidation. In agreement with this, MUFAs decrease lipid oxidation in middle-aged individuals. Intriguingly, MUFAs upregulate not only lipid droplet number but also peroxisome number. A targeted screen identifies genes involved in the co-regulation of lipid droplets and peroxisomes, and reveals that induction of both organelles is optimal for longevity. Our study uncovers an organelle network involved in lipid homeostasis and lifespan regulation, opening new avenues for interventions to delay aging.

Examining Sporadic Cancer Mutations Uncovers a Set of Genes Involved in Mitochondrial Maintenance

Reproductive-Toxicity-Related Endpoints in C. elegans Are Consistent with Reduced Concern for Dimethylarsinic Acid Exposure Relative to Inorganic Arsenic

Exposures to arsenic and mercury are known to pose significant threats to human health; however, the effects specific to organic vs. inorganic forms are not fully understood. Caenorhabditis elegans’ (C. elegans) transparent cuticle, along with the conservation of key genetic pathways regulating developmental and reproductive toxicology (DART)-related processes such as germ stem cell renewal and differentiation, meiosis, and embryonic tissue differentiation and growth, support this model’s potential to address the need for quicker and more dependable testing methods for DART hazard identification. Organic and inorganic forms of mercury and arsenic had different effects on reproductive-related endpoints in C. elegans, with methylmercury (meHgCl) having effects at lower concentrations than mercury chloride (HgCl2), and sodium arsenite (NaAsO2) having effects at lower concentrations than dimethylarsinic acid (DMA). Progeny to adult ratio changes and germline apoptosis were seen at concentrations that also affected gravid adult gross morphology. For both forms of arsenic tested, germline histone regulation was altered at concentrations below those that affected progeny/adult ratios, while concentrations for these two endpoints were similar for the mercury compounds. These C. elegans findings are consistent with corresponding mammalian data, where available, suggesting that small animal model test systems may help to fill critical data gaps by contributing to weight of evidence assessments.

Microfluidic-Assisted Caenorhabditis elegans Sorting: Current Status and Future Prospects

Caenorhabditis elegans (C. elegans) has been a popular model organism for several decades since its first discovery of the huge research potential for modeling human diseases and genetics. Sorting is an important means of providing stage- or age-synchronized worm populations for many worm-based bioassays. However, conventional manual techniques for C. elegans sorting are tedious and inefficient, and commercial complex object parametric analyzer and sorter is too expensive and bulky for most laboratories. Recently, the development of lab-on-a-chip (microfluidics) technology has greatly facilitated C. elegans studies where large numbers of synchronized worm populations are required and advances of new designs, mechanisms, and automation algorithms. Most previous reviews have focused on the development of microfluidic devices but lacked the summaries and discussion of the biological research demands of C. elegans, and are hard to read for worm researchers. We aim to comprehensively review the up-to-date microfluidic-assisted C. elegans sorting developments from several angles to suit different background researchers, i.e., biologists and engineers. First, we highlighted the microfluidic C. elegans sorting devices' advantages and limitations compared to the conventional commercialized worm sorting tools. Second, to benefit the engineers, we reviewed the current devices from the perspectives of active or passive sorting, sorting strategies, target populations, and sorting criteria. Third, to benefit the biologists, we reviewed the contributions of sorting to biological research. We expect, by providing this comprehensive review, that each researcher from this multidisciplinary community can effectively find the needed information and, in turn, facilitate future research.

Yolk-deprived Caenorhabditis elegans secure brood size at the expense of competitive fitness

Oviparous animals support reproduction via the incorporation of yolk as a nutrient source into the eggs. In Caenorhabditis elegans, however, yolk proteins seem dispensable for fecundity, despite constituting the vast majority of the embryonic protein pool and acting as carriers for nutrient-rich lipids. Here, we used yolk protein–deprived C. elegans mutants to gain insight into the traits that may yet be influenced by yolk rationing. We show that massive yolk provisioning confers a temporal advantage during embryogenesis, while also increasing early juvenile body size and promoting competitive fitness. Opposite to species that reduce egg production under yolk deprivation, our results indicate that C. elegans relies on yolk as a fail-safe to secure offspring survival, rather than to maintain offspring numbers.

Meisosomes, folded membrane microdomains between the apical extracellular matrix and epidermis

Apical extracellular matrices (aECMs) form a physical barrier to the environment. In Caenorhabditis elegans, the epidermal aECM, the cuticle, is composed mainly of different types of collagen, associated in circumferential ridges separated by furrows. Here, we show that in mutants lacking furrows, the normal intimate connection between the epidermis and the cuticle is lost, specifically at the lateral epidermis, where, in contrast to the dorsal and ventral epidermis, there are no hemidesmosomes. At the ultrastructural level, there is a profound alteration of structures that we term ‘meisosomes,’ in reference to eisosomes in yeast. We show that meisosomes are composed of stacked parallel folds of the epidermal plasma membrane, alternately filled with cuticle. We propose that just as hemidesmosomes connect the dorsal and ventral epidermis, above the muscles, to the cuticle, meisosomes connect the lateral epidermis to it. Moreover, furrow mutants present marked modifications of the biomechanical properties of their skin and exhibit a constitutive damage response in the epidermis. As meisosomes co-localise to macrodomains enriched in phosphatidylinositol (4,5) bisphosphate, they could conceivably act, like eisosomes, as signalling platforms, to relay tensile information from the aECM to the underlying epidermis, as part of an integrated stress response to damage.

Molecular evidence of widespread benzimidazole drug resistance in Ancylostoma caninum from domestic dogs throughout the USA and discovery of a novel ß-tubulin benzimidazole resistance mutation

Ancylostoma caninum is an important zoonotic gastrointestinal nematode of dogs worldwide and a close relative of human hookworms. We recently reported that racing greyhound dogs in the USA are infected with A. caninum that are commonly resistant to multiple anthelmintics. Benzimidazole resistance in A. caninum in greyhounds was associated with a high frequency of the canonical F167Y(TTC>TAC) isotype-1 β-tubulin mutation. In this work, we show that benzimidazole resistance is remarkably widespread in A. caninum from domestic dogs across the USA. First, we identified and showed the functional significance of a novel benzimidazole isotype-1 β-tubulin resistance mutation, Q134H(CAA>CAT). Several benzimidazole resistant A. caninum isolates from greyhounds with a low frequency of the F167Y(TTC>TAC) mutation had a high frequency of a Q134H(CAA>CAT) mutation not previously reported from any eukaryotic pathogen in the field. Structural modeling predicted that the Q134 residue is directly involved in benzimidazole drug binding and that the 134H substitution would significantly reduce binding affinity. Introduction of the Q134H substitution into the C. elegans β-tubulin gene ben-1, by CRISPR-Cas9 editing, conferred similar levels of resistance as a ben-1 null allele. Deep amplicon sequencing on A. caninum eggs from 685 hookworm positive pet dog fecal samples revealed that both mutations were widespread across the USA, with prevalences of 49.7% (overall mean frequency 54.0%) and 31.1% (overall mean frequency 16.4%) for F167Y(TTC>TAC) and Q134H(CAA>CAT), respectively. Canonical codon 198 and 200 benzimidazole resistance mutations were absent. The F167Y(TTC>TAC) mutation had a significantly higher prevalence and frequency in Western USA than in other regions, which we hypothesize is due to differences in refugia. This work has important implications for companion animal parasite control and the potential emergence of drug resistance in human hookworms.

In Vivo Anti-Alzheimer and Antioxidant Properties of Avocado (Persea americana Mill.) Honey from Southern Spain

There is growing evidence that Alzheimer’s disease (AD) can be prevented by reducing risk factors involved in its pathophysiology. Food-derived bioactive molecules can help in the prevention and reduction of the progression of AD. Honey, a good source of antioxidants and bioactive molecules, has been tied to many health benefits, including those from neurological origin. Monofloral avocado honey (AH) has recently been characterized but its biomedical properties are still unknown. The aim of this study is to further its characterization, focusing on the phenolic profile. Moreover, its antioxidant capacity was assayed both in vitro and in vivo. Finally, a deep analysis on the pathophysiological features of AD such as oxidative stress, amyloid-β aggregation, and protein-tau-induced neurotoxicity were evaluated by using the experimental model C. elegans. AH exerted a high antioxidant capacity in vitro and in vivo. No toxicity was found in C. elegans at the dosages used. AH prevented ROS accumulation under AAPH-induced oxidative stress. Additionally, AH exerted a great anti-amyloidogenic capacity, which is relevant from the point of view of AD prevention. AH exacerbated the locomotive impairment in a C. elegans model of tauopathy, although the real contribution of AH remains unclear. The mechanisms under the observed effects might be attributed to an upregulation of daf-16 as well as to a strong ROS scavenging activity. These results increase the interest to study the biomedical applications of AH; however, more research is needed to deepen the mechanisms under the observed effects.

A role for worm cutl-24 in background- and parent-of-origin-dependent ER stress resistance

Glia of C. elegans coordinate a protective organismal heat shock response independent of the neuronal thermosensory circuit

Aging organisms lose the ability to induce stress responses, becoming vulnerable to protein toxicity and tissue damage. Neurons can signal to peripheral tissues to induce protective organelle-specific stress responses. Recent work shows that glia can independently induce such responses. Here, we show that overexpression of heat shock factor 1 (hsf-1) in the four astrocyte-like cephalic sheath cells of Caenorhabditis elegans induces a non–cell-autonomous cytosolic unfolded protein response, also known as the heat shock response (HSR). These animals have increased lifespan and heat stress resistance and decreased protein aggregation. Glial HSR regulation is independent of canonical thermosensory circuitry and known neurotransmitters but requires the small clear vesicle release protein UNC-13. HSF-1 and the FOXO transcription factor DAF-16 are partially required in peripheral tissues for non–cell-autonomous HSR, longevity, and thermotolerance. Cephalic sheath glial hsf-1 overexpression also leads to pathogen resistance, suggesting a role for this signaling pathway in immune function.

ER-associated RNA silencing promotes ER quality control

The endoplasmic reticulum (ER) coordinates mRNA translation and processing of secreted and endomembrane proteins. ER-associated degradation (ERAD) prevents the accumulation of misfolded proteins in the ER, but the physiological regulation of this process remains poorly characterized. Here, in a genetic screen using an ERAD model substrate in Caenorhabditis elegans, we identified an anti-viral RNA interference pathway, referred to as ER-associated RNA silencing (ERAS), which acts together with ERAD to preserve ER homeostasis and function. Induced by ER stress, ERAS is mediated by the Argonaute protein RDE-1/AGO2, is conserved in mammals and promotes ER-associated RNA turnover. ERAS and ERAD are complementary, as simultaneous inactivation of both quality-control pathways leads to increased ER stress, reduced protein quality control and impaired intestinal integrity. Collectively, our findings indicate that ER homeostasis and organismal health are protected by synergistic functions of ERAS and ERAD.

Benzimidazoles cause lethality by inhibiting the function of Caenorhabditis elegans neuronal beta-tubulin

Dichloroacetate and thiamine improve survival and mitochondrial stress in a C. elegans model of dihydrolipoamide dehydrogenase deficiency

Dihydrolipoamide dehydrogenase (DLD) deficiency is a recessive mitochondrial disorder caused by depletion of DLD from α-ketoacid dehydrogenase complexes. Caenorhabditis elegans animal models of DLD deficiency generated by graded feeding of dld-1(RNAi) revealed that full or partial reduction of DLD-1 expression recapitulated increased pyruvate levels typical of pyruvate dehydrogenase complex deficiency and significantly altered animal survival and health, with reductions in brood size, adult length, and neuromuscular function. DLD-1 deficiency dramatically increased mitochondrial unfolded protein stress response induction and adaptive mitochondrial proliferation. While ATP levels were reduced, respiratory chain enzyme activities and in vivo mitochondrial membrane potential were not significantly altered. DLD-1 depletion directly correlated with the induction of mitochondrial stress and impairment of worm growth and neuromuscular function. The safety and efficacy of dichloroacetate, thiamine, riboflavin, 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR), l-carnitine, and lipoic acid supplemental therapies empirically used for human DLD disease were objectively evaluated by life span and mitochondrial stress response studies. Only dichloroacetate and thiamine showed individual and synergistic therapeutic benefits. Collectively, these C. elegans dld-1(RNAi) animal model studies demonstrate the translational relevance of preclinical modeling of disease mechanisms and therapeutic candidates. Results suggest that clinical trials are warranted to evaluate the safety and efficacy of dichloroacetate and thiamine in human DLD disease.

Loss of heat shock factor initiates intracellular lipid surveillance by actin destabilization

Cells sense stress and initiate response pathways to maintain lipid and protein homeostasis. However, the interplay between these adaptive mechanisms is unclear. Herein, we demonstrate how imbalances in cytosolic protein homeostasis affect intracellular lipid surveillance. Independent of its ancient thermo-protective properties, the heat shock factor, HSF-1, modulates lipid metabolism and age regulation through the metazoan-specific nuclear hormone receptor, NHR-49. Reduced hsf-1 expression destabilizes the Caenorhabditis elegans enteric actin network, subsequently disrupting Rab GTPase-mediated trafficking and cell-surface residency of nutrient transporters. The ensuing malabsorption limits lipid availability, thereby activating the intracellular lipid surveillance response through vesicular release and nuclear translocation of NHR-49 to both increase nutrient absorption and restore lipid homeostasis. Overall, cooperation between these regulators of cytosolic protein homeostasis and lipid surveillance ensures metabolic health and age progression through actin integrity, endocytic recycling, and lipid sensing.

A pals-25 gain-of-function allele triggers systemic resistance against natural pathogens of C. elegans

Regulation of immunity throughout an organism is critical for host defense. Previous studies in the nematode Caenorhabditis elegans have described an “ON/OFF” immune switch comprised of the antagonistic paralogs PALS-25 and PALS-22, which regulate resistance against intestinal and epidermal pathogens. Here, we identify and characterize a PALS-25 gain-of-function mutant protein with a premature stop (Q293*), which we find is freed from physical repression by its negative regulator, the PALS-22 protein. PALS-25(Q293*) activates two related gene expression programs, the Oomycete Recognition Response (ORR) against natural pathogens of the epidermis, and the Intracellular Pathogen Response (IPR) against natural intracellular pathogens of the intestine. A subset of ORR/IPR genes is upregulated in pals-25(Q293*) mutants, and they are resistant to oomycete infection in the epidermis, and microsporidia and virus infection in the intestine, but without compromising growth. Surprisingly, we find that activation of PALS-25 seems to primarily stimulate the downstream bZIP transcription factor ZIP-1 in the epidermis, with upregulation of gene expression in both the epidermis and in the intestine. Interestingly, we find that PALS-22/25-regulated epidermal-to-intestinal signaling promotes resistance to the N. parisii intestinal pathogen, demonstrating cross-tissue protective immune induction from one epithelial tissue to another in C. elegans.

Reduced bone morphogenic protein signaling along the gut-neuron axis by heat shock factor promotes longevity

Aging is a complex and highly regulated process of interwoven signaling mechanisms. As an ancient transcriptional regulator of thermal adaptation and protein homeostasis, the Heat Shock Factor, HSF-1, has evolved functions within the nervous system to control age progression; however, the molecular details and signaling dynamics by which HSF-1 modulates age across tissues remain unclear. Herein, we report a nonautonomous mode of age regulation by HSF-1 in the Caenorhabditis elegans nervous system that works through the bone morphogenic protein, BMP, signaling pathway to modulate membrane trafficking in peripheral tissues. In particular, HSF-1 represses the expression of the neuron-specific BMP ligand, DBL-1, and initiates a complementary negative feedback loop within the intestine. By reducing receipt of DBL-1 in the periphery, the SMAD transcriptional coactivator, SMA-3, represses the expression of critical membrane trafficking regulators including Rab GTPases involved in early (RAB-5), late (RAB-7), and recycling (RAB-11.1) endosomal dynamics and the BMP receptor binding protein, SMA-10. This reduces cell surface residency and steady-state levels of the type I BMP receptor, SMA-6, in the intestine and further dampens signal transmission to the periphery. Thus, the ability of HSF-1 to coordinate BMP signaling along the gut–brain axis is an important determinate in age progression.

Linkage mapping reveals loci that underlie differences in Caenorhabditis elegans growth

Growth rate and body size are complex traits that contribute to the fitness of organisms. The identification of loci that underlie differences in these traits provides insights into the genetic contributions to development. Leveraging Caenorhabditis elegans as a tractable metazoan model for quantitative genetics, we can identify genomic regions that underlie differences in growth. We measured postembryonic growth of the laboratory-adapted wild-type strain (N2) and a wild strain from Hawaii (CB4856) and found differences in body size. Using linkage mapping, we identified three distinct quantitative trait loci (QTL) on chromosomes IV, V, and X that are associated with variation in body growth. We further examined these growth-associated quantitative trait loci using chromosome substitution strains and near-isogenic lines and validated the chromosome X quantitative trait loci. In addition, we generated a list of candidate genes for the chromosome X quantitative trait loci. These genes could potentially contribute to differences in animal growth and should be evaluated in subsequent studies. Our work reveals the genetic architecture underlying animal growth variation and highlights the genetic complexity of growth in Caenorhabditis elegans natural populations.

Modular safe-harbor transgene insertion for targeted single-copy and extrachromosomal array integration in Caenorhabditis elegans

Efficient and reproducible transgenesis facilitates and accelerates research using genetic model organisms. Here, we describe a modular safe-harbor transgene insertion (MosTI) for use in Caenorhabditis elegans which improves targeted insertion of single-copy transgenes by homology directed repair and targeted integration of extrachromosomal arrays by nonhomologous end-joining. MosTI allows easy conversion between selection markers at insertion site and a collection of universal targeting vectors with commonly used promoters and fluorophores. Insertions are targeted at three permissive safe-harbor intergenic locations and transgenes are reproducibly expressed in somatic and germ cells. Chromosomal integration is mediated by CRISPR/Cas9, and positive selection is based on a set of split markers (unc-119, hygroR, and gfp) where only animals with chromosomal insertions are rescued, resistant to antibiotics, or fluorescent, respectively. Single-copy insertion is efficient using either constitutive or heat-shock inducible Cas9 expression (25–75%) and insertions can be generated from a multiplexed injection mix. Extrachromosomal array integration is also efficient (7–44%) at modular safe-harbor transgene insertion landing sites or at the endogenous unc-119 locus. We use short-read sequencing to estimate the plasmid copy numbers for 8 integrated arrays (6–37 copies) and long-read Nanopore sequencing to determine the structure and size (5.4 Mb) of 1 array. Using universal targeting vectors, standardized insertion strains, and optimized protocols, it is possible to construct complex transgenic strains which should facilitate the study of increasingly complex biological problems in C. elegans.

SKN-1 regulates stress resistance downstream of amino catabolism pathways

The deleterious potential to generate oxidative stress is a fundamental challenge to metabolism. The oxidative stress response transcription factor, SKN-1/NRF2, can sense and respond to changes in metabolic state, although the mechanism and consequences of this remain unknown. Here, we performed a genetic screen in C. elegans targeting amino acid catabolism and identified multiple metabolic pathways as regulators of SKN-1 activity. We found that knockdown of the conserved amidohydrolase T12A2.1/amdh-1 activates a unique subset of SKN-1 regulated genes. Interestingly, this transcriptional program is independent of canonical P38-MAPK signaling components but requires ELT-3, NHR-49 and MDT-15. This activation of SKN-1 is dependent on upstream histidine catabolism genes HALY-1 and Y51H4A.7/UROC-1 and may occur through accumulation of a catabolite, 4-imidazolone-5-propanoate. Activating SKN-1 results in increased oxidative stress resistance but decreased survival to heat stress. Together, our data suggest that SKN-1 acts downstream of key catabolic pathways to influence physiology and stress resistance.

The Smart Soil Organism Detector: An instrument and machine learning pipeline for soil species identification

An automated approach to quantify chemotaxis index in C. elegans

Chemotaxis assays are used extensively to study behavioral responses of Caenorhabditis nematodes to environmental cues. These assays result in a chemotaxis index (CI) that denotes the behavioral response of a population of nematodes to a particular compound and can range from 1 (maximum attraction) to -1 (maximum avoidance). Traditional chemotaxis assays have low throughput because researchers must manually setup experimental populations and score CIs. Here, we describe an automated methodology that increases throughput by using liquid-handling robots to setup experimental populations and a custom image analysis package, ct, to automate the scoring of CIs from plate images.

Intracellular lipid surveillance by small G protein geranylgeranylation

Assessment of the effects of organic vs. inorganic arsenic and mercury in Caenorhabditis elegans

Exposures to mercury and arsenic are known to pose significant threats to human health. Effects specific to organic vs. inorganic forms of these toxic elements are less understood however, especially for organic dimethylarsinic acid (DMA), which has recently been detected in pups of rodent dams orally exposed to inorganic sodium (meta)arsenite (NaAsO2). Caenorhabditis elegans is a small animal alternative toxicity model. To fill data gaps on the effects of DMA relative to NaAsO2, C. elegans were exposed to these two compounds alongside more thoroughly researched inorganic mercury chloride (HgCl2) and organic methylmercury chloride (meHgCl). For timing of developmental milestone acquisition in C. elegans, meHgCl was 2 to 4-fold more toxic than HgCl2, and NaAsO2 was 20-fold more toxic than DMA, ranking the four compounds meHgCl > HgCl2 > NaAsO2 » DMA for developmental toxicity. Methylmercury induced significant decreases in population locomotor activity levels in developing C. elegans. DMA was also associated with developmental hypoactivity, but at >100-fold higher concentrations than meHgCl. Transcriptional alterations in native genes were observed in wild type C. elegans adults exposed to concentrations equitoxic for developmental delay in juveniles. Both forms of arsenic induced genes involved in immune defense and oxidative stress response, while the two mercury species induced proportionally more genes involved in transcriptional regulation. A transgenic bioreporter for activation of conserved proteosome specific unfolded protein response was strongly activated by NaAsO2, but not DMA at tested concentrations. HgCl2 and meHgCl had opposite effects on a bioreporter for unfolded protein response in the endoplasmic reticulum. Presented experiments indicating low toxicity for DMA in C. elegans are consistent with human epidemiologic data correlating higher arsenic methylation capacity with resistance to arsenic toxicity. This work contributes to the understanding of the accuracy and fit-for-use categories for C. elegans toxicity screening and its usefulness to prioritize compounds of concern for further testing.

The gut efflux pump MRP-1 exports oxidized glutathione as a danger signal that stimulates behavioral immunity and aversive learning

Innate immune surveillance, which monitors the presence of potentially harmful microorganisms and the perturbations of host physiology that occur in response to infections, is critical to distinguish pathogens from beneficial microbes. Here, we show that multidrug resistance-associated protein-1 (MRP-1) functions in the basolateral membrane of intestinal cells to transport byproducts of cellular redox reactions to control both molecular and behavioral immunity in Caenorhabditis elegans. Pseudomonas aeruginosa infection disrupts glutathione homeostasis, leading to the excess production of the MRP-1 substrate, oxidized glutathione (GSSG). Extracellular GSSG triggers pathogen avoidance behavior and primes naïve C. elegans to induce aversive learning behavior via neural NMDA class glutamate receptor-1 (NMR-1). Our results indicate that MRP-1 transports GSSG, which acts as a danger signal capable of warning C. elegans of changes in intestinal homeostasis, thereby initiating a gut neural signal that elicits an appropriate host defense response.

Oral Elesclomol Treatment Alleviates Copper Deficiency in Animal Models

Copper (Cu) is an essential trace element for key biochemical reactions. Dietary or genetic copper deficiencies are associated with anemia, cardiomyopathy, and neurodegeneration. The essential requirement for copper in humans is illustrated by Menkes disease, a fatal neurodegenerative disorder of early childhood caused by mutations in the ATP7A copper transporter. Recent groundbreaking studies have demonstrated that a copper delivery small molecule compound, elesclomol (ES), is able to substantially ameliorate pathology and lethality in a mouse model of Menkes disease when injected as an ES-Cu2+ complex. It is well appreciated that drugs administered through oral means are more convenient with better efficacy than injection methods. Here we show, using genetic models of copper-deficient C. elegans and mice, that dietary ES supplementation fully rescues copper deficiency phenotypes. Worms lacking either the homolog of the CTR1 copper importer or the ATP7 copper exporter showed normal development when fed ES. Oral gavage with ES rescued intestine-specific Ctr1 knockout mice from early postnatal lethality without additional copper supplementation. Our findings reveal that ES facilitates copper delivery from dietary sources independent of the intestinal copper transporter CTR1 and provide insight into oral administration of ES as an optimal therapeutic for Menkes disease and possibly other disorders of copper insufficiency.

An Olive-Derived Extract 20% Rich in Hydroxytyrosol Prevents ß-Amyloid Aggregation and Oxidative Stress, Two Features of Alzheimer Disease, via SKN-1/NRF2 and HSP-16.2 in Caenorhabditis elegans

Olive milling produces olive oil and different by-products, all of them very rich in different bioactive compounds like the phenolic alcohol hydroxytyrosol. The aim of the present study was to investigate the effects of an olive fruit extract 20% rich in hydroxytyrosol on the molecular mechanisms associated with Alzheimer disease features like Aβ- and tau- induced toxicity, as well as on oxidative stress in Caenorhabditis elegans. Moreover, characterization of the extracts, regarding the profile and content of phenolics, as well as total antioxidant ability, was investigated. The study of lethality, growth, pharyngeal pumping, and longevity in vivo demonstrated the lack of toxicity of the extract. One hundred μg/mL of extract treatment revealed prevention of oxidative stress and a delay in Aβ-induced paralysis related with a lower presence of Aβ aggregates. Indeed, the extract showed the ability to avoid a certain degree of proteotoxicity associated with aggregation of the tau protein. According to RNAi tests, SKN-1/NRF2 transcription factor and the overexpression of HSP-16.2 were mechanistically associated in the observed effects.

Megapixel camera arrays enable high-resolution animal tracking in multiwell plates

Tracking small laboratory animals such as flies, fish, and worms is used for phenotyping in neuroscience, genetics, disease modelling, and drug discovery. An imaging system with sufficient throughput and spatiotemporal resolution would be capable of imaging a large number of animals, estimating their pose, and quantifying detailed behavioural differences at a scale where hundreds of treatments could be tested simultaneously. Here we report an array of six 12-megapixel cameras that record all the wells of a 96-well plate with sufficient resolution to estimate the pose of C. elegans worms and to extract high-dimensional phenotypic fingerprints. We use the system to study behavioural variability across wild isolates, the sensitisation of worms to repeated blue light stimulation, the phenotypes of worm disease models, and worms’ behavioural responses to drug treatment. Because the system is compatible with standard multiwell plates, it makes computational ethological approaches accessible in existing high-throughput pipelines.

Box C/D small nucleolar ribonucleoproteins regulate mitochondrial surveillance and innate immunity

Monitoring mitochondrial function is crucial for organismal survival. This task is performed by mitochondrial surveillance or quality control pathways, which are activated by signals originating from mitochondria and relayed to the nucleus (retrograde response) to start transcription of protective genes. In Caenorhabditis elegans, several systems are known to play this role, including the UPRmt, MAPKmt, and the ESRE pathways. These pathways are highly conserved and their loss compromises survival following mitochondrial stress. In this study, we found a novel interaction between the box C/D snoRNA core proteins (snoRNPs) and mitochondrial surveillance and innate immune pathways. We showed that box C/D, but not box H/ACA, snoRNPs are required for the full function of UPRmt and ESRE upon stress. The loss of box C/D snoRNPs reduced mitochondrial mass, mitochondrial membrane potential, and oxygen consumption rate, indicating overall degradation of mitochondrial function. Concomitantly, the loss of C/D snoRNPs increased immune response and reduced host intestinal colonization by infectious bacteria, improving host resistance to pathogenesis. Our data may indicate a model wherein box C/D snoRNP machinery regulates a "switch" of the cell's activity between mitochondrial surveillance and innate immune activation. Understanding this mechanism is likely to be important for understanding multifactorial processes, including responses to infection and aging.

The transcription factor HLH-26 controls probiotic-mediated protection against intestinal infection through up-regulation of the Wnt/BAR-1 pathway

Probiotics play a critical role in the control of host intestinal microbial balance, protecting the host from gastrointestinal pathogens, modulating the host immune response, and decreasing host susceptibility to infection. To understand the mechanism underlying the protective effect of probiotics against infections through immune regulation, we examined protection against Salmonella enterica infection following exposure to nonpathogenic Enterococcus faecium in the nematode Caenorhabditis elegans. We found that the transcription factor HLH-26, a REF-1 family member of basic helix-loop-helix transcription factors, was required in the intestine for E. faecium-mediated protection of C. elegans against a lethal S. enterica infection. In addition, we uncovered that defense response genes controlled by the canonical Wnt/BAR-1 pathway were activated upon exposure to E. faecium in an HLH-26-dependent manner. Our findings highlight a role for REF-1/HLH-26 in the control of the Wnt/BAR-1 pathway in probiotic-mediated protection against gut infection.

Robust regulatory architecture of pan-neuronal gene expression

Pan-neuronally expressed genes, such as genes involved in the synaptic vesicle cycle or in neuropeptide maturation, are critical for proper function of all neurons, but the transcriptional control mechanisms that direct such genes to all neurons of a nervous system remain poorly understood. We show here that six members of the CUT family of homeobox genes control pan-neuronal identity specification in Caenorhabditis elegans. Single CUT mutants show barely any effects on pan-neuronal gene expression or global nervous system function, but such effects become apparent and progressively worsen upon removal of additional CUT family members, indicating a critical role of gene dosage. Overexpression of each individual CUT gene rescued the phenotype of compound mutants, corroborating that gene dosage, rather than the activity of specific members of the gene family, is critical for CUT gene family function. Genome-wide binding profiles, as well as mutation of CUT homeodomain binding sites by CRISPR/Cas9 genome engineering show that CUT genes directly control the expression of pan-neuronal features. Moreover, CUT genes act in conjunction with neuron-type-specific transcription factors to control pan-neuronal gene expression. Our study, therefore, provides a previously missing key insight into how neuronal gene expression programs are specified and reveals a highly buffered and robust mechanism that controls the most critical functional features of all neuronal cell types.

A laboratory and simulation platform to integrate individual life history traits and population dynamics.

ATFS-1 plays no repressive role in the regulation of epidermal immune response

Fungal infection triggers the induction of antimicrobial peptide (AMP) genes in the epidermis (Pujol et al, 2008). We previously showed that this effect is suppressed by the mitochondrial unfolded protein response (UPRmt), which can be activated by knockdown of select genes including the mitochondrial metalloprotease spg-7 (Zugasti et al, 2016). Here, we confirm that RNAi against spg-7 triggers the UPRmt and blocks AMP induction during infection, whereas infection itself does not trigger the UPRmt. ATFS-1 is a key factor in the UPRmt, mediating much of the associated transcriptional response. We find that, surprisingly, ATFS-1 is not required for the suppression of AMP induction provoked by spg-7(RNAi). These data show that the mitochondrial dysfunction that blocks the immune response upon infection or wounding is independent of ATFS-1.

piRNAs initiate transcriptional silencing of spermatogenic genes during C. elegans germline development

Eukaryotic genomes harbor invading transposable elements that are silenced by PIWI-interacting RNAs (piRNAs) to maintain genome integrity in animal germ cells. However, whether piRNAs also regulate endogenous gene expression programs remains unclear. Here, we show that C. elegans piRNAs trigger the transcriptional silencing of hundreds of spermatogenic genes during spermatogenesis, promoting sperm differentiation and function. This silencing signal requires piRNA-dependent small RNA biogenesis and loading into downstream nuclear effectors, which correlates with the dynamic reorganization of two distinct perinuclear biomolecular condensates present in germ cells. In addition, the silencing capacity of piRNAs is temporally counteracted by the Argonaute CSR-1, which targets and licenses spermatogenic gene transcription. The spatial and temporal overlap between these opposing small RNA pathways contributes to setting up the timing of the spermatogenic differentiation program. Thus, our work identifies a prominent role for piRNAs as direct regulators of endogenous transcriptional programs during germline development and gamete differentiation.

The transcription factor ZIP-1 promotes resistance to intracellular infection in Caenorhabditis elegans

Defense against intracellular infection has been extensively studied in vertebrate hosts, but less is known about invertebrate hosts; specifically, the transcription factors that induce defense against intracellular intestinal infection in the model nematode Caenorhabditis elegans remain understudied. Two different types of intracellular pathogens that naturally infect the C. elegans intestine are the Orsay virus, which is an RNA virus, and microsporidia, which comprise a phylum of fungal pathogens. Despite their molecular differences, these pathogens induce a common host transcriptional response called the intracellular pathogen response (IPR). Here we show that zip-1 is an IPR regulator that functions downstream of all known IPR-activating and regulatory pathways. zip-1 encodes a putative bZIP transcription factor, and we show that zip-1 controls induction of a subset of genes upon IPR activation. ZIP-1 protein is expressed in the nuclei of intestinal cells, and is at least partially required in the intestine to upregulate IPR gene expression. Importantly, zip-1 promotes resistance to infection by the Orsay virus and by microsporidia in intestinal cells. Altogether, our results indicate that zip-1 represents a central hub for triggers of the IPR, and that this transcription factor has a protective function against intracellular pathogen infection in C. elegans.

Advances in our understanding of nematode ion channels as potential anthelmintic targets

Ion channels are specialized multimeric proteins that underlie cell excitability. These channels integrate with a variety of neuromuscular and biological functions. In nematodes, the physiological behaviors including locomotion, navigation, feeding and reproduction, are regulated by these protein entities. Majority of the antinematodal chemotherapeutics target the ion channels to disrupt essential biological functions. Here, we have summarized current advances in our understanding of nematode ion channel pharmacology. We review cys-loop ligand gated ion channels (LGICs), including nicotinic acetylcholine receptors (nAChRs), acetylcholine-chloride gated ion channels (ACCs), glutamate-gated chloride channels (GluCls), and GABA (γ-aminobutyric acid) receptors, and other ionotropic receptors (transient receptor potential (TRP) channels and potassium ion channels). We have provided an update on the pharmacological properties of these channels from various nematodes. This article catalogs the differences in ion channel composition and resulting pharmacology in the phylum Nematoda. This diversity in ion channel subunit repertoire and pharmacology emphasizes the importance of pursuing species-specific drug target research. In this review, we have provided an overview of recent advances in techniques and functional assays available for screening ion channel properties and their application.

Increased Pathogenicity of the Nematophagous Fungus Drechmeria coniospora Following Long-Term Laboratory Culture

Domestication provides a window into adaptive change. Over the course of 2 decades of laboratory culture, a strain of the nematode-specific fungus Drechmeria coniospora became more virulent during its infection of Caenorhabditis elegans. Through a close comparative examination of the genome sequences of the original strain and its more pathogenic derivative, we identified a small number of non-synonymous mutations in protein-coding genes. In one case, the mutation was predicted to affect a gene involved in hypoxia resistance and we provide direct corroborative evidence for such an effect. The mutated genes with functional annotation were all predicted to impact the general physiology of the fungus and this was reflected in an increased in vitro growth, even in the absence of C. elegans. While most cases involved single nucleotide substitutions predicted to lead to a loss of function, we also observed a predicted restoration of gene function through deletion of an extraneous tandem repeat. This latter change affected the regulatory subunit of a cAMP-dependent protein kinase. Remarkably, we also found a mutation in a gene for a second protein of the same, protein kinase A, pathway. Together, we predict that they result in a stronger repression of the pathway for given levels of ATP and adenylate cyclase activity. Finally, we also identified mutations in a few lineage-specific genes of unknown function that are candidates for factors that influence virulence in a more direct manner.

Cross-species screening platforms identify EPS-8 as a critical link for mitochondrial stress and actin stabilization

The dysfunction of mitochondria is associated with the physiological consequences of aging and many age-related diseases. Therefore, critical quality control mechanisms exist to protect mitochondrial functions, including the unfolded protein response of the mitochondria (UPRMT). However, it is still unclear how UPRMT is regulated in mammals with mechanistic discrepancies between previous studies. Here, we reasoned that a study of conserved mechanisms could provide a uniquely powerful way to reveal previously uncharacterized components of the mammalian UPRMT. We performed cross-species comparison of genetic requirements for survival under—and in response to—mitochondrial stress between karyotypically normal human stem cells and the nematode Caenorhabditis elegans. We identified a role for EPS-8/EPS8 (epidermal growth factor receptor pathway substrate 8), a signaling protein adaptor, in general mitochondrial homeostasis and UPRMT regulation through integrin-mediated remodeling of the actin cytoskeleton. This study also highlights the use of cross-species comparisons in genetic screens to interrogate cellular pathways.

DREAM represses distinct targets by cooperating with different THAP domain proteins

The DREAM (dimerization partner [DP], retinoblastoma [Rb]-like, E2F, and MuvB) complex controls cellular quiescence by repressing cell-cycle and other genes, but its mechanism of action is unclear. Here, we demonstrate that two C. elegans THAP domain proteins, LIN-15B and LIN-36, co-localize with DREAM and function by different mechanisms for repression of distinct sets of targets. LIN-36 represses classical cell-cycle targets by promoting DREAM binding and gene body enrichment of H2A.Z, and we find that DREAM subunit EFL-1/E2F is specific for LIN-36 targets. In contrast, LIN-15B represses germline-specific targets in the soma by facilitating H3K9me2 promoter marking. We further find that LIN-36 and LIN-15B differently regulate DREAM binding. In humans, THAP proteins have been implicated in cell-cycle regulation by poorly understood mechanisms. We propose that THAP domain proteins are key mediators of Rb/DREAM function.

Increased fidelity of protein synthesis extends lifespan.

Mitochondria-affecting small molecules ameliorate proteostasis defects associated with neurodegenerative diseases

Reduced peroxisomal import triggers peroxisomal retrograde signaling

Divergent Nodes of Non-autonomous UPR^ER Signaling through Serotonergic and Dopaminergic Neurons

Innate Immunity Promotes Sleep through Epidermal Antimicrobial Peptides

Three Rules Explain Transgenerational Small RNA Inheritance in C. elegans

Genetic Variation in Complex Traits in Transgenic a-Synuclein Strains of Caenorhabditis elegans

The neutrally charged diarylurea compound PQ401 kills antibiotic-resistant and antibiotic-tolerant Staphylococcus aureus

A cullin-RING ubiquitin ligase promotes thermotolerance as part of the intracellular pathogen response in Caenorhabditis elegans

Developmental ROS Individualizes Organismal Stress Resistance and Lifespan

Effect of soil microbial feeding on gut microbiome and cadmium toxicity in Caenorhabditis elegans

Fabrication of sharp silicon arrays to wound Caenorhabditis elegans

Early life exposure of a biocide, CMIT/MIT causes metabolic toxicity via the O-GlcNAc transferase pathway in the nematode C. elegans.

High-throughput COPAS assay for screening of developmental and reproductive toxicity of nanoparticles using the nematode Caenorhabditis elegans.

Evolution of sex ratio through gene loss

A Novel Gene Underlies Bleomycin-Response Variation in Caenorhabditis elegans.

High-throughput assessment of toxic effects of metal mixtures of cadmium(Cd), lead(Pb), and manganese(Mn) in nematode Caenorhabditis elegans.

MANF deletion abrogates early larval Caenorhabditis elegans stress response to tunicamycin and Pseudomonas aeruginosa.

Natural variation in C. elegans arsenic toxicity is explained by differences in branched chain amino acid metabolism

Interplay between mitochondria and diet mediates pathogen and stress resistance in Caenorhabditis elegans

Dual Role of Ribosome-Binding Domain of NAC as a Potent Suppressor of Protein Aggregation and Aging-Related Proteinopathies

Hazard potential of perovskite solar cell technology for potential implementation of “safe-by-design” approach

In Silico Molecular Docking and In Vivo Validation with Caenorhabditis elegans to Discover Molecular Initiating Events in Adverse Outcome Pathway Framework: Case Study on Endocrine-Disrupting Chemicals with Estrogen and Androgen Receptors

A tetracycline-dependent ribozyme switch allows conditional induction of gene expression in Caenorhabditis elegans

Translation attenuation by minocycline enhances longevity and proteostasis in old post-stress-responsive organisms

Ophiopogon japonicus herbal tea ameliorates oxidative stress and extends lifespan in caenorhabditis elegans

MRG-1/MRG15 is a barrier for germ cell to neuron reprogramming in Caenorhabditis elegans

Non-proteolytic activity of 19S proteasome subunit RPT-6 regulates GATA transcriptionduring response to infection

Food perception without ingestion leads to metabolic changes and irreversible developmental arrest in C. elegans

Evolutionary plasticity in the innate immune function of Akirin

Combined flow cytometry and high-throughput image analysis for the study of essential genes in Caenorhabditis elegans.

Modulatory upregulation of an insulin peptide gene by different pathogens in C. elegans.

A Damage Sensor Associated with the Cuticle Coordinates Three Core Environmental StressResponses in Caenorhabditis elegans.

Although extracellular matrices function as protective barriers to many types of environmental insult, their role in sensing stress and regulating adaptive gene induction responses has not been studied carefully...

Extracellular matrix barriers and inducible cytoprotective genes form successive lines of defense against chemical and microbial environmental stressors. The barrier in nematodes is a collagenous extracellular matrix called the cuticle. In Caenorhabditis elegans, disruption of some cuticle collagen genes activates osmolyte and antimicrobial response genes. Physical damage to the epidermis also activates antimicrobial responses. Here, we assayed the effect of knocking down genes required for cuticle and epidermal integrity on diverse cellular stress responses. We found that disruption of specific bands of collagen, called annular furrows, coactivates detoxification, hyperosmotic, and antimicrobial response genes, but not other stress responses. Disruption of other cuticle structures and epidermal integrity does not have the same effect. Several transcription factors act downstream of furrow loss. SKN-1/Nrf and ELT-3/GATA are required for detoxification, SKN-1/Nrf is partially required for the osmolyte response, and STA-2/Stat and ELT-3/GATA for antimicrobial gene expression. Our results are consistent with a cuticle-associated damage sensor that coordinates detoxification, hyperosmotic, and antimicrobial responses through overlapping, but distinct, downstream signaling.

Comprehensive list of SUMO targets in Caenorhabditis elegans and its implication for evolutionary conservation of SUMO signaling

An Antimicrobial Peptide and Its Neuronal Receptor Regulate Dendrite Degeneration in Aging and Infection.

JAK/STAT and TGF-ß activation as potential adverse outcome pathway of TiO2NPs phototoxicity in Caenorhabditis elegans

The combinatorial control of alternative splicing in C. elegans

Nuclear localized C9orf72 associated arginine containing dipeptides exhibit age-dependent toxicity in C. elegans

Neuronal inhibition of the autophagy nucleation complex extends life span in post-reproductive C. elegans

Autophagy is a ubiquitous catabolic process that causes cellular bulk degradation of cytoplasmic components and is generally associated with positive effects on health and longevity. Inactivation of autophagy has been linked with detrimental effects on cells and organisms. The antagonistic pleiotropy theory postulates that some fitness-promoting genes during youth are harmful during aging. On this basis, we examined genes mediating post-reproductive longevity using an RNAi screen. From this screen, we identified 30 novel regulators of post-reproductive longevity, including pha-4. Through downstream analysis of pha-4, we identified that the inactivation of genes governing the early stages of autophagy up until the stage of vesicle nucleation, such as bec-1, strongly extend both life span and health span. Furthermore, our data demonstrate that the improvements in health and longevity are mediated through the neurons, resulting in reduced neurodegeneration and sarcopenia. We propose that autophagy switches from advantageous to harmful in the context of an age-associated dysfunction.

Natural variation in a single amino acid substitution underlies physiological responses to topoisomerase II poisons.

Inter-organ signalling by HRG-7 promotes systemic haem homeostasis.

A systematic RNAi screen reveals a novel role of a spindle assembly checkpoint protein BuGZ in synaptic transmission in C. elegans

Larval crowding accelerates C. elegans development and reduces lifespan

Environmental conditions experienced during animal development are thought to have sustained impact on maturation and adult lifespan. Here we show that in the model organism C. elegans developmental rate and adult lifespan depend on larval population density, and that this effect is mediated by excreted small molecules. By using the time point of first egg laying as a marker for full maturity, we found that wildtype hermaphrodites raised under high density conditions developed significantly faster than animals raised in isolation. Population density-dependent acceleration of development (Pdda) was dramatically enhanced in fatty acid β-oxidation mutants that are defective in the biosynthesis of ascarosides, small-molecule signals that induce developmental diapause. In contrast, Pdda is abolished by synthetic ascarosides and steroidal ligands of the nuclear hormone receptor DAF-12. We show that neither ascarosides nor any known steroid hormones are required for Pdda and that another chemical signal mediates this phenotype, in part via the nuclear hormone receptor NHR-8. Our results demonstrate that C. elegans development is regulated by a push-pull mechanism, based on two antagonistic chemical signals: chemosensation of ascarosides slows down development, whereas population-density dependent accumulation of a different chemical signal accelerates development. We further show that the effects of high larval population density persist through adulthood, as C. elegans larvae raised at high densities exhibit significantly reduced adult lifespan and respond differently to exogenous chemical signals compared to larvae raised at low densities, independent of density during adulthood. Our results demonstrate how inter-organismal signaling during development regulates reproductive maturation and longevity.

Transaldolase inhibition impairs mitochondrial respiration and induces a starvation-like longevity response in Caenorhabditis elegans

Copper Oxide Nanoparticles Impact Several Toxicological Endpoints and Cause Neurodegeneration in Caenorhabditis elegans

The Intestinal Copper Exporter CUA-1 is required for Systemic Copper Homeostasis in Caenorhabditis elegans

Next-Generation Sequencing-Based Approaches for Mutation Mapping and Identification in Caenorhabditis elegans

Selection on a Subunit of the NURF Chromatin Remodeler Modifies Life History Traits in a Domesticated Strain of Caenorhabditis elegans.

An Abundant Class of Non-coding DNA Can Prevent Stochastic Gene Silencing in the C. elegans Germline.

Comparative Genomic Analysis of Drechmeria coniospora Reveals Core and Specific Genetic Requirements for Fungal Endoparasitism of Nematodes

A quantitative genome-wide RNAi screen in C. elegans for antifungal innate immunity genes

¹Centre d’Immunologie de Marseille-Luminy, Aix Marseille Université UM2,Inserm, U1104, CNRS UMR7280, 13288 Marseille, France. ²Institut de Mathématiques de Marseille, Aix Marseille Université, I2M Centrale Marseille,CNRS UMR 7373, 13453 Marseille, France. ³Present address: Section of Hematology/Oncology, Department of Pediatrics, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA. ⁴Present address: Institut de Biologie du Développement de Marseille, CNRS, UMR6216, Case 907, Marseille, France. ⁵Present address: Institut de Genomique Fonctionnelle, 141,rue de la Cardonille, 34094 Montpellier Cedex 05, France.

The MADD-3 LAMMER Kinase Interacts with a p38 MAP Kinase Pathway to Regulate the Display of the EVA-1 Guidance Receptor in Caenorhabditis elegans

¹ Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada, ² The Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada, ³ The Collaborative Programme in Developmental Biology, University of Toronto, Toronto, Ontario, Canada
 

Mitochondrial Stress Induces Chromatin Reorganization to Promote Longevity and UPRmt.

The C. elegans CCAAT-Enhancer-Binding Protein Gamma Is Required for Surveillance Immunity

¹ Division of Biological Sciences, Section of Cell and Developmental Biology, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA

Gait-specific adaptation of locomotor activity in response to dietary restriction in Caenorhabditis elegans.

¹Department of Molecular Prevention, Institute of Human Nutrition and Food Science, Christian-Albrechts-University of Kiel, Heinrich-Hecht-Platz 10, 24118 Kiel, Germany.

²Department of Molecular Prevention, Institute of Human Nutrition and Food Science, Christian-Albrechts-University of Kiel, Heinrich-Hecht-Platz 10, 24118 Kiel, Germany.

The principle of antagonism ensures protein targeting specificity at the endoplasmic reticulum

View Supplementary Material for this publication.

The ribonucleotidyl transferase USIP-1 acts with SART3 to promote U6 snRNA recycling

The DREAM complex promotes gene body H2A.Z for target repression

¹The Gurdon Institute, University of Cambridge, Cambridge CB2 1QN, United Kingdom

Genome-wide screen identifies a novel p97/CDC-48-dependent pathway regulating ER-stress-induced gene transcription

¹Team “Endoplasmic Reticulum stress and cancer”, INSERM, UMR1053, Bordeaux, France

²University of Bordeaux, Bordeaux, France eric.chevet@inserm.fr

Insect-Derived Cecropins Display Activity against Acinetobacter baumannii in a Whole-Animal High-Throughput Caenorhabditis elegans Model.

¹Division of Infectious Diseases, Rhode Island Hospital, Alpert Medical School of Brown University, Providence, Rhode Island, USA Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA.

DAF-16/FOXO and EGL-27/GATA promote developmental growth in response to persistent somatic DNA damage

¹Institute for Genome Stability in Ageing and Disease, Medical Faculty, University of Cologne, Joseph-Stelzmann-Str. 26 50931 Cologne, Germany

²Cologne Excellence Cluster for Cellular Stress Responses in Aging-Associated Diseases (CECAD) Research Center and Systems Biology of Ageing Cologne, University of Cologne, Joseph-Stelzmann-Str. 26 50931 Cologne, Germany

Bioactivity of nanosilver in Caenorhabditis elegans: Effects of size, coat, and shape

^aCenter for Food Safety and Applied Nutrition, FDA, Laurel, MD, United States

COPASutils: An R Package for Reading, Processing, and Visualizing Data from COPAS Large-Particle Flow Cytometers

Department of Molecular Biosciences, Northwestern University, Evanston, Illinois, United States of America

Activation of a G protein-coupled receptor by its endogenous ligand triggers the innate immune response of Caenorhabditis elegans.

¹1] Centre d'Immunologie de Marseille-Luminy, UM2 Aix-Marseille Université, Case 906, Marseille, France. [2] INSERM, U1104, 13288 Marseille, France. [3] CNRS, UMR7280, Marseille, France.

Backbone-independent nucleic acid binding by splicing factor SUP-12 reveals key aspects of molecular recognition

Institut Européen de Chimie et Biologie, IECB, Univ. Bordeaux, 2 rue Robert Escarpit, F-33607 Pessac, France

Inserm, U869, ARNA Laboratory, 146 rue Léo Saignat, F-33076 Bordeaux, France

ICeE: An Interface for C. elegans Experiments

A high-content assay for identifying small molecules that reprogram C. elegans germ cell fate.

¹Department of Pediatrics, University of Pittsburgh School of Medicine, Children's Hospital of Pittsburgh of UPMC, 4401 Penn Avenue, Pittsburgh, PA 15224, USA.

Genome evolution and plasticity of Serratia marcescens, an important multidrug resistant nosocomial pathogen

¹ Interdisciplinary Research Organization, University of Miyazaki, Miyazaki, Japan

²Department of Infectious Diseases, Faculty of Medicine, University of Miyazaki, Miyazaki, Japan

  ³Department of Genomics and Bioenvironmental Science, Frontier Science Research Center, University of Miyazaki, Miyazaki, Japan

A pair of RNA-binding proteins controls networks of splicing events contributing to specialization of neural celltypes.

·         ¹FAS Center for Systems Biology, Harvard University, 52 Oxford Street, Cambridge, MA 02138, USA.

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A genome-wide RNAi screen identifies potential drug targets in a C. elegans model of a1-antitrypsin deficiency.

·         ¹Department of Pediatrics, Cell Biology, University of Pittsburgh School of Medicine, Children's Hospital of Pittsburgh of UPMC and Magee-Womens Hospital Research Institute, 4401 Penn Avenue, Pittsburgh, PA 15224, USA.

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Control of Metazoan Heme Homeostasis by a Conserved Multidrug Resistance Protein.

·         ¹Department of Animal and Avian Sciences and Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742, USA.

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Progressive Degeneration of Dopaminergic Neurons through TRP Channel-Induced Cell Death.

¹Center for Organelle Research, University of Stavanger, 4036 Stavanger, Norway, Norwegian Center for Movement Disorders, Stavanger University Hospital, 4011 Stavanger, Norway

The C. elegans lifespan assay toolkit.

¹Department of Pediatrics, University of Pittsburgh School of Medicine, 7129 Rangos Research Centre, Children's Hospital of Pittsburgh, 4401 Penn Avenue, Pittsburgh, PA 15224, United States.
²Department of Pediatrics, University of Pittsburgh School of Medicine, 7129 Rangos Research Centre, Children's Hospital of Pittsburgh, 4401 Penn Avenue, Pittsburgh, PA 15224, United States. Electronic address: arjumand.ghazi@chp.edu.

Comparative toxicity of silver nanoparticles on oxidative stress and DNA damage in the nematode,Caenorhabditis elegans.

·         ¹School of Environmental Engineering, University of Seoul, Seoul 130-743, Republic of Korea; Graduate School of Energy and Environmental system Engineering, University of Seoul, Seoul 130-743, Republic of Korea.

·         ²Nicholas School of the Environment and Center for the Environmental Implications of Nanotechnology, Duke University, Durham, NC, USA.

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Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen

The wormsorter is an instrument analogous to a FACS machine that is used in studies of Caenorhabditis elegans, typically to sort worms based on expression of a fluorescent reporter. Here, we highlight an alternative usage of this instrument, for sorting worms according to their degree of colonization by a GFP-expressing pathogen. This new usage allowed us to address the relationship between colonization of the worm intestine and induction of immune responses. While C. elegans immune responses to different pathogens have been documented, it is still unknown what initiates them. The two main possibilities (which are not mutually exclusive) are recognition of pathogen-associated molecular patterns, and detection of damage caused by infection. To differentiate between the two possibilities, exposure to the pathogen must be dissociated from the damage it causes. The wormsorter enabled separation of worms that were extensively-colonized by the Gram-negative pathogen Pseudomonas aeruginosa, with the damage likely caused by pathogen load, from worms that were similarly exposed, but not, or marginally, colonized. These distinct populations were used to assess the relationship between pathogen load and the induction of transcriptional immune responses. The results suggest that the two are dissociated, supporting the possibility of pathogen recognition.

Defects in the C. elegans acyl-CoA Synthase, acs-3, and Nuclear Hormone Receptor, nhr-25, Cause Sensitivity to Distinct, but Overlapping Stresses.

Metazoan transcription factors control distinct networks of genes in specific tissues, yet understanding how these networks are integrated into physiology, development, and homeostasis remains challenging. Inactivation of the nuclear hormone receptor nhr-25 ameliorates developmental and metabolic phenotypes associated with loss of function of an acyl-CoA synthetase gene, acs-3. ACS-3 activity prevents aberrantly high NHR-25 activity. Here, we investigated this relationship further by examining gene expression patterns following acs-3 and nhr-25 inactivation. Unexpectedly, we found that the acs-3 mutation or nhr-25 RNAi resulted in similar transcriptomes with enrichment in innate immunity and stress response gene expression. Mutants of either gene exhibited distinct sensitivities to pathogens and environmental stresses. Only nhr-25 was required for wild-type levels of resistance to the bacterial pathogen P. aeruginosa and only acs-3 was required for wild-type levels of resistance to osmotic stress and the oxidative stress generator, juglone. Inactivation of either acs-3 or nhr-25 compromised lifespan and resistance to the fungal pathogen D. coniospora. Double mutants exhibited more severe defects in the lifespan and P. aeruginosa assays, but were similar to the single mutants in other assays. Finally, acs-3 mutants displayed defects in their epidermal surface barrier, potentially accounting for the observed sensitivities. Together, these data indicate that inactivation of either acs-3 or nhr-25 causes stress sensitivity and increased expression of innate immunity/stress genes, most likely by different mechanisms. Elevated expression of these immune/stress genes appears to abrogate the transcriptional signatures relevant to metabolism and development.

Silver Nanoparticle Behavior, Uptake, and Toxicity in Caenorhabditis elegans: Effects of Natural Organic Matter.

1) Nicholas School of the Environment and ‡Department of Civil and Environmental Engineering, Center for the Environmental Implications of Nanotechnology, Duke University , Durham, North Carolina 27708-0328, United States.

High-Throughput Screening for Novel Anti-Infectives Using a C. elegans Pathogenesis Model.

1) Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts; Department of Genetics, Harvard Medical School, Boston, Massachusetts.

The dsRBP and inactive editor ADR-1 utilizes dsRNA binding to regulate A-to-I RNA editing across the C. elegans transcriptome.

Inadequate adenosine-to-inosine editing of noncoding regions occurs in disease but is often uncorrelated with ADAR levels, underscoring the need to study deaminase-independent control of editing. C. elegans have two ADAR proteins, ADR-2 and the theoretically catalytically inactive ADR-1. Using high-throughput RNA sequencing of wild-type and adr mutant worms, we expand the repertoire of C. elegans edited transcripts over 5-fold and confirm that ADR-2 is the only active deaminase in vivo. Despite lacking deaminase function, ADR-1 affects editing of over 60 adenosines within the 3' UTRs of 16 different mRNAs. Furthermore, ADR-1 interacts directly with ADR-2 substrates, even in the absence of ADR-2, and mutations within its double-stranded RNA (dsRNA) binding domains abolish both binding and editing regulation. We conclude that ADR-1 acts as a major regulator of editing by binding ADR-2 substrates in vivo. These results raise the possibility that other dsRNA binding proteins, including the inactive human ADARs, regulate RNA editing through deaminase-independent mechanisms.

A comparative study into alterations of coenzyme Q redox status in ageing pigs, mice, and worms.

1) Division of Molecular Prevention, Institute of Human Nutrition and Food Science, Christian Albrechts University Kiel, Kiel, Germany.

A Variant in the Neuropeptide Receptor npr-1 is a Major Determinant of Caenorhabditis elegans Growth and Physiology

1) Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, New Jersey, United States of America 2) Department of Molecular Biology, Princeton University, Princeton, New Jersey, United States of America 3) Departments of Human Genetics and Biological Chemistry, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, United States of America 4) Howard Hughes Medical Institute, Chevy Chase, Maryland, United States of America

PRDE-1 is a nuclear factor essential for the biogenesis of Ruby motif-dependent piRNAs in C. elegans

1) Wellcome Trust Cancer Research UK Gurdon Institute, Department of Genetics, University of Cambridge, Cambridge CB2 1QN, United Kingdom; 2) MRC Clinical Sciences Centre, Imperial College Faculty of Medicine, London W12 0NN, United Kingdom

Evaluation of The Mitochondrial Redox Status in Caenorhabditis elegans: An Assay Based on The COPAS Biosort Flow Cytometer

¹Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China

²University of the Chinese Academy of Sciences, Beijing 100049, China

S-Adenosyl methionine synthetase 1 limits fat storage in Caenorhabditis elegans

Department of Molecular Prevention, Institute of Human Nutrition and Food Sciences, University of Kiel, Heinrich-Hecht-Platz 10, 24118 Kiel, Germany Department of Developmental Genetics, Institute of Genetics, TU Braunschweig, Spielmannstr. 7, 38106 Brunswick, Germany

Comparative RNAi Screens in C. elegans and C. briggsae Reveal the Impact of Developmental System Drift on Gene Function

1) The Donnelly Centre, University of Toronto, Toronto, Ontario, Canada 2) Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada 3) Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, United States of America 4) University of California Davis, United States of America

Fluphenazine Reduces Proteotoxicity in C. elegans and Mammalian Models of Alpha-1-Antitrypsin Deficiency

The classical form of α1-antitrypsin deficiency (ATD) is associated with hepatic fibrosis and hepatocellular carcinoma. It is caused by the proteotoxic effect of a mutant secretory protein that aberrantly accumulates in the endoplasmic reticulum of liver cells. Recently we developed a model of this deficiency in C. elegans and adapted it for high-content drug screening using an automated, image-based array scanning. Screening of the Library of Pharmacologically Active Compounds identified fluphenazine (Flu) among several other compounds as a drug which reduced intracellular accumulation of mutant α1-antitrypsin Z (ATZ). Because it is representative of the phenothiazine drug class that appears to have autophagy enhancer properties in addition to mood stabilizing activity, and can be relatively easily re-purposed, we further investigated its effects on mutant ATZ. The results indicate that Flu reverses the phenotypic effects of ATZ accumulation in the C. elegans model of ATD at doses which increase the number of autophagosomes in vivo. Furthermore, in nanomolar concentrations, Flu enhances the rate of intracellular degradation of ATZ and reduces the cellular ATZ load in mammalian cell line models. In the PiZ mouse model Flu reduces the accumulation of ATZ in the liver and mediates a decrease in hepatic fibrosis. These results show that Flu can reduce the proteotoxicity of ATZ accumulation in vivo and, because it has been used safely in humans, this drug can be moved rapidly into trials for liver disease due to ATD. The results also provide further validation for drug discovery using C. elegans models that can be adapted to high-content drug screening platforms and used together with mammalian cell line and animal models.

Forward and reverse mutagenesis in C. elegans.

1) Laboratory of Developmental Genetics, The Rockefeller University, New York NY 10065, USA

Sulfidation of silver nanoparticles: natural antidote to their toxicity.

RNA-binding protein GLD-1/quaking genetically interacts with the mir-35 and the let-7 miRNA pathways in Caenorhabditis elegans

1)Centre for Gene Regulation and Expression, University of Dundee, Dundee DD1 5EH, UK 2)Wellcome Trust Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge CB2 1QN, UK 3)Friedrich Miescher Institute for Biomedical Research, Basel 4002, Switzerland

Modular Control of Glutamatergic Neuronal Identity in C. elegans by Distinct Homeodomain Proteins.

Department of Biochemistry and Molecular Biophysics, HHMI, Columbia University Medical Center, New York, NY 10032, USA. Electronic address: es2754@columbia.edu.

Comparative toxicogenomic responses of mercuric and methyl-mercury.

* Corresponding author: Jonathan H Freedman freedma1@niehs.nih.gov Author Affiliations 1)Laboratory of Toxicology and Pharmacology, National Institute of Environmental Health Sciences, NIH, 111 T.W Alexander Drive, Research Triangle Park, P.O. Box 12233, 27709 Durham, NC, USA 2)SRA International, Inc., Durham, NC, USA 3)Microarray and Genome Informatics Group, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, Durham, NC, USA 4)Current address: Department of Biostatistical Sciences, Wake Forest University School of Medicine, Medical Center Boulevard, 27157 Winston-Salem, NC, USA

Comparison of the toxicity of fluoridation compounds in the nematode C. elegans.

Biomolecular Screening Branch, Division of the National Toxicology Program, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, USA.

A Pro-Cathepsin L Mutant Is a Luminal Substrate for Endoplasmic-Reticulum-Associated Degradation in C.elegans

Department of Pediatrics, Cell Biology and Physiology, University of Pittsburgh School of Medicine, Children’s Hospital of Pittsburgh of UPMC and Magee-Womens Hospital of UPMC, Pittsburgh, Pennsylvania, United States of America

EGL-13/SoxD specifies distinct O2 and CO2 sensory neuron fates in Caenorhabditis elegans.

Biotech Research and Innovation Centre, University of Copenhagen, Copenhagen, Denmark.

EGL-13/SoxD specifies distinct O2 and CO2 sensory neuron fates in Caenorhabditis elegans.

Active transcriptomic and proteomic reprogramming in the C. elegans nucleotide excision repair mutant xpa-1.

piRNAs can trigger a multigenerational epigenetic memory in the germline of C. elegans.

Wellcome Trust Cancer Research UK Gurdon Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, UK.

Toxicity ranking of heavy metals with screening method using adult Caenorhabditis elegans and propidium iodide replicates toxicity ranking in rat

United States Food and Drug Administration, Center for Food Safety and Applied Nutrition, Office of Applied Research and Safety Assessment, Division of Toxicology,8301 Muirkirk Road, Laurel, MD 20708, United States

* Corresponding author. Address: Mod. 1, Room 2002, 8301 Muirkirk Road,Laurel, MD 20708-2476, USA. Tel.: +1 301 210 8897.1) Address: Mod. 1, Room 2011, 8301 Muirkirk Road, Laurel, MD 20708-2476, USA. Tel.: +1 301 210 6375. 2) Address: Mod. 1, Room 2017, 8301 Muirkirk Road, Laurel, MD 20708-2476, USA. Tel.: +1 301 210 6329.

E-mail addresses: Piper.Hunt@fda.hhs.gov (P.R. Hunt), Nicholas.Olejnik@fda.hhs.gov (N. Olejnik), Sprando@fda.hhs.gov (R.L. Sprando).

Caenorhabditis elegans as a model in developmental toxicology.

Biomolecular Screening Branch, National Toxicology Program, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC, USA.

The Pseudokinase NIPI-4 Is a Novel Regulator of Antimicrobial Peptide Gene Expression

Daf-2 signaling modifies mutant SOD1 toxicity in C. elegans

The DAF-2 Insulin/IGF-1 signaling (IIS) pathway is a strong modifier of Caenorhabditis elegans longevity and healthspan. As aging is the greatest risk factor for developing neurodegenerative diseases such as Amyotrophic Lateral Sclerosis (ALS), we were interested in determining if DAF-2 signaling modifies disease pathology in mutant superoxide dismutase 1 (SOD1) expressing C. elegans. Worms with pan-neuronal G85R SOD1 expression demonstrate significantly impaired locomotion as compared to WT SOD1 expressing controls and they develop insoluble SOD1 aggregates. Reductions in DAF-2 signaling, either through a hypomorphic allele or neuronally targeted RNAi, decreases the abundance of aggregated SOD1 and results in improved locomotion in a DAF-16 dependant manner. These results suggest that manipulation of the DAF-2 Insulin/IGF-1 signaling pathway may have therapeutic potential for the treatment of ALS.

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

Topologically conserved residues direct heme transport in HRG-1-related proteins.

Department of Animal & Avian Sciences and Department of Cell Biology & Molecular Genetics, University of Maryland, College Park, Maryland 20742, USA.

A Genome-Wide Collection of Mos1 Transposon InsertionMutants for the C. elegans Research Community

 1)Centre de Génétique et de Physiologie Moléculaires et Cellulaires, CNRS UMR 5534, Campus de la Doua, Villeurbanne, France, 2) Université Claude Bernard Lyon 1,Villeurbanne, France, 3) School of Biochemistry, University of Bristol, Bristol, United Kingdom, 4) Centre d’Immunologie de Marseille-Luminy, Aix-Marseille University,Marseille, France, 5) INSERM, U1104, Marseille, France, 6) CNRS, UMR7280, Marseille, France, 7) Plateforme ‘‘Biologie de Caenorhabditis elegans’’, CNRS UMS3421, Campus dela Doua, Villeurbanne, France

* E-mail: ewbank@ciml.univ-mrs.fr

The Majority of Animal Genes Are Required for Wild-Type Fitness

1) The Donnelly Centre, University of Toronto, 160 College Street, Toronto, Ontario M5S 3E1, Canada 2) Department of Molecular Genetics, University of Toronto, 1 King’s College Circle, Toronto, Ontario M5S 1A8, Canada

*Correspondence: andyfraser.utoronto@gmail.com

HIF-1 Regulates Iron Homeostasis in Caenorhabditis elegans by Activation and Inhibition of Genes Involved in Iron Uptake and Storage

Steven Joshua Romney, Elizabeth A. Leibold

Department of Medicine, University of Utah, Salt Lake City, Utah, United States of America

Ben S. Newman

University of Washington, Seattle, Washington, United States of America

Colin Thacker

Department of Biology, University of Utah, Salt Lake City, Utah, United States of America

Elizabeth A. Leibold

Department of Oncological Sciences, University of Utah, Salt Lake City, Utah, United States of America

An intercellular heme-trafficking protein delivers maternal heme to the embryo during development in C. elegans.

Department of Animal & Avian Sciences and Department of Cell Biology & Molecular Genetics, University of Maryland, College Park, MD 20742, USA.

Fluorescence-based fixative and vital staining of lipid droplets in Caenorhabditis elegans eveal fat stores using microscopy and flow cytometry approaches

Institute of Human Nutrition and Food Science, Research Group Molecular Prevention, University of Kiel, Kiel, Germany.

The Caenorhabditis elegans mucin-like protein OSM-8 negatively regulates osmosensitive physiology via the transmembrane protein PTR-23.

The molecular mechanisms of animal cell osmoregulation are poorly understood. Genetic studies of osmoregulation in yeast have identified mucin-like proteins as critical regulators of osmosensitive signaling and gene expression. Whether mucins play similar roles in higher organisms is not known. Here, we show that mutations in the Caenorhabditis elegans mucin-like gene osm-8 specifically disrupt osmoregulatory physiological processes. In osm-8 mutants, normal physiological responses to hypertonic stress, such as the accumulation of organic osmolytes and activation of osmoresponsive gene expression, are constitutively activated. As a result, osm-8 mutants exhibit resistance to normally lethal levels of hypertonic stress and have an osmotic stress resistance (Osr) phenotype. To identify genes required for Osm-8 phenotypes, we performed a genome-wide RNAi osm-8 suppressor screen. After screening ~18,000 gene knockdowns, we identified 27 suppressors that specifically affect the constitutive osmosensitive gene expression and Osr phenotypes of osm-8 mutants. We found that one suppressor, the transmembrane protein PTR-23, is co-expressed with osm-8 in the hypodermis and strongly suppresses several Osm-8 phenotypes, including the transcriptional activation of many osmosensitive mRNAs, constitutive glycerol accumulation, and osmotic stress resistance. Our studies are the first to show that an extracellular mucin-like protein plays an important role in animal osmoregulation in a manner that requires the activity of a novel transmembrane protein. Given that mucins and transmembrane proteins play similar roles in yeast osmoregulation, our findings suggest a possible evolutionarily conserved role for the mucin-plasma membrane interface in eukaryotic osmoregulation.

Proteome changes of Caenorhabditis elegans upon a Staphylococcus aureus infection

Automated High-Content Live Animal Drug Screening Using C. elegans Expressing the Aggregation Prone Serpin a1-antitrypsin Z.

1) Department of Pediatrics, Cell Biology and Physiology, University of Pittsburgh School of Medicine, Children's Hospital of Pittsburgh of UPMC and Magee-Womens Hospital Research Institute, Pittsburgh, Pennsylvania, United States of America, 2) Department of Pharmacology and Chemical Biology, University of Pittsburgh Drug Discovery Institute, University of Pittsburgh, Pennsylvania, United States of America

Insulin-like signaling determines survival during stress via posttranscriptional mechanisms in C. elegans .

The Mental Health Research Institute, Parkville, Victoria, Australia. gmccoll@mhri.edu.au

An antibiotic selection marker for nematode transgenesis.

1) Genome Regulation and Evolution, INSERM U869, Institut Européen de Chimie et Biologie, Pessac, France. 2) Center for Cancer Systems Biology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA. 3) Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA. 4) Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA. 5) Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia, Canada.

Caenorhabditis elegans as a screening tool to find active compounds and targets in rare neuromuscular diseases.

CNRS UMR5534 – Centre de Génétique Moléculaire et Cellulaire, 16 rue Raphaël Dubois – bâtiment Mendel, Campus de La Doua – 69100 Villeurbanne - FRANCE

Optimizing analysis of high-throughput screens with the UBI Biosort

 1) Centre d'Immunologie de Marseille Luminy, Case 906, 13288 Marseille Cedex 9, France, 2) Modul-Bio, Parc Scientifique Luminy Biotech II, Case 935,13288 Marseille Cedex 9

A suite of MATLAB-based computational tools for automated analysis of COPAS Biosort data

Department of Physiology, University of Pennsylvania, Philadelphia, PA, USA

A differential proteomics study of Caenorhabditis elegans: infected with Aeromonas hydrophila.

Research Group of Functional Genomics and Proteomics, K.U. Leuven, Naamsestraat 59, 3000 Leuven, Belgium. annelies.bogaerts@bio.kuleuven.be

The fatty acid synthase fasn-1 acts upstream of WNK and Ste20/GCK-VI kinases to modulate anitmicrobial peptide expression in C. elegans epidermis

1) Centre d’Immunologie de Marseille-Luminy; Université de la Méditerranée; Marseille, France; 2) INSERM; U631; Marseille, France; 3) CNRS; UMR6102; Marseille, France; 4)Howard Hughes Medical Institute and Dept of MCDB; University of Colorado at Boulder; Colorado, USA

High-throughput screening and small animal models, where are we?

Current high-throughput screening methods for drug discovery rely on the existence of targets. Moreover, most of the hits generated during screenings turn out to be invalid after further testing in animal models. To by-pass these limitations, efforts are now being made to screen chemical libraries on whole animals. One of the most commonly used animal model in biology is the murine model Mus musculus. However, its cost limit its use in large-scale therapeutic screening. In contrast, the nematode Caenorhabditis elegans, the fruit fly Drosophila melanogaster, and the fish Danio rerio are gaining momentum as screening tools. These organisms combine genetic amenability, low cost and culture conditions that are compatible with large-scale screens. Their main advantage is to allow high-throughput screening in a whole-animal context. Moreover, their use is not dependent on the prior identification of a target and permits the selection of compounds with an improved safety profile. This review surveys the versatility of these animal models for drug discovery and discuss the options available at this day.

A Conserved PMK-1/p38 MAPK Is Required in Caenorhabditis elegans: Tissue-specific Immune Response to Yersinia pestis Infection*

LIN-28 and the poly(U) polymerase PUP-2 regulate let-7 microRNA processing in Caenorhabditis elegans .

Wellcome Trust Cancer Research UK Gurdon Institute, University of Cambridge, The Henry Wellcome Building of Cancer and Developmental Biology, Cambridge, UK.

Identification of Antifungal Compounds Active against Candida albicans Using an Improved High-Throughput Caenorhabditis elegans Assay

1 Division of Infectious Diseases, Massachusetts General Hospital, Boston, Massachusetts, United States of America, 2 Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, Massachusetts, United States of America,

Automated screening for mutants affecting dopaminergic-neuron specification in C. elegans

We describe an automated method to isolate mutant Caenorhabditis elegans that do not appropriately execute cellular differentiation programs. We used a fluorescence-activated sorting mechanism implemented in the COPAS Biosort machine to isolate mutants with subtle alterations in the cellular specificity of GFP expression. This methodology is considerably more efficient than comparable manual screens and enabled us to isolate mutants in which dopamine neurons do not differentiate appropriately.

High-Throughput Screen for Novel Antimicrobials using a Whole Animal Infection Model

†Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, ‡Department of Molecular Biology, §Center for Computational and Integrative Biology, and Center for Systems Biology, Massachusetts General Hospital,Boston, Massachusetts 02114, _Department of Biology, Northeastern University, Boston, Massachusetts 02115, and_Imaging Platform, Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142

Whole-Animal High-Throughput Screens: The C. elegans Model

Antifungal Innate Immunity in :C. elegans PKCdelta Links G Protein Signaling and a Conserved p38 MAPK Cascade.

A method to rank order water soluble compounds according to their toxicity using Caenorhabditis elegans: a Complex Object Parametric Analyzer and Sorter, and axenic liquid media.

Division of Toxicology, United States Food and Drug Administration, Center for Food Safety and Applied Nutrition, Office of Applied Research and Safety Assessment, Laural, MD 20708, United States. Robert.sprando@fda.hhs.gov

Neuroimmune regulation of antimicrobial peptide expression by a noncanonical TGF-beta signaling pathway in Caenorhabditis elegans epidermis.

Negative regulation of Caenorhabditis elegans epidermal damage responses by death-associated protein kinase.

A C. elegans -based, whole animal, in vivo screen for the identification of antifungal compounds

 Division of Infectious Diseases, Massachusetts General Hospital, Gray-Jackson 504, 55 Fruit Street, Boston, Massachusetts 02114, USA.

Anti-Fungal Innate Immunity in C. elegans Is Enhanced by Evolutionary Diversification of Antimicrobial Peptides

1 Centre d'Immunologie de Marseille-Luminy, Université de la Méditerranée, Case 906, Marseille, France   2 INSERM, U631, Marseille, France   3 CNRS, UMR6102, Marseille, France   4 Department of Animal Evolutionary Ecology, Zoological Institute, University of Tuebingen, Tuebingen, Germany

Distinct Innate Immune Responses to Infection and Wounding in the C. elegans Epidermis.

Centre d'Immunologie de Marseille-Luminy, Université de la Méditerranée, Case 906, 13288 Marseille cedex 9, France.

Discovery of a cholecystokinin-gastrin-like signaling system in nematodes.

Effects of Genetic Mutations and Chemical Exposures on Caenorhabditis elegans Feeding: Evaluation of a Novel, High-Throughput Screening Assay

1 Laboratory of Molecular Toxicology, National Toxicology Program, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, United States of America   2 Nicholas School of the Environment and Earth Sciences, Duke University, Durham, North Carolina, United States of America

Abstract
Government agencies have defined a need to reduce, refine or replace current mammalian-based bioassays with testing methods that use alternative species. Invertebrate species, such as Caenorhabditis elegans, provide an attractive option because of their short life cycles, inexpensive maintenance, and high degree of evolutionary conservation with higher eukaryotes. The C. elegans pharynx is a favorable model for studying neuromuscular function, and the effects of chemicals on neuromuscular activity, i.e., feeding. Current feeding methodologies, however, are labor intensive and only semi-quantitative.

Here a high-throughput assay is described that uses flow cytometry to measure C. elegans feeding by determining the size and intestinal fluorescence of hundreds of nematodes after exposure to fluorescent-labeled microspheres. . . .

A global analysis of genetic interactions in Caenorhabditis elegans

Addresses: *Department of Medical Genetics and Microbiology, The Terrence Donnelly Centre for Cellular and Biomolecular Research, 160 College St, University of Toronto, Toronto, ON, M5S 3E1, Canada. ^†Collaborative Program in Developmental Biology, University of Toronto, Toronto, ON, M5S 3E1, Canada. ^‡Department of Biomolecular Engineering, 1156 High Street, Mail Stop SOE2, University of California, Santa Cruz, CA 95064, USA.

Correspondence: Peter J Roy. Email: peter.roy@utoronto.ca; Joshua M Stuart. Email: jstuart@soe.ucsc.edu

16th International C. elegans Conference 2007 June 27 - 30

Click on the titles of the following posters to view an abstract:

Genome-scale analysis of in vivo spatiotemporal promoter activity in C. elegans.
Denis Dupuy, Nicolas Bertin, César Hidalgo, Kavitha Venkatesan, Domena Tu, David Lee, Nenad Svrzikapa, Anne-Ruxandra Carvunis, Rock Pulak, Ian Hope, John Reece-Hoyes, Rebecca Hunt-Newbury, Ryan Viveiros, William Mohler, Murat Tasan, Frederick Roth, Donald Moerman, Albert-László Barabási, David Baillie, Marc Vidal.

Genetic dissection of the transcriptional response to wounding and fungal infection.
Nathalie Pujol, C. Leopold Kurz, Sophie Cypowyj, Daniel Wong, Andrew Chisholm, Jonathan Ewbank.

A high throughput screen for novel anti-infective compounds.
Terence I. Moy, Annie L. Conery, Anne E. Carpenter, Anthony R. Ball, Kim Lewis, Frederick M. Ausubel.

Characterization of the role of peni( fr8) in innate immunity against fungal infection.
Kwang-Zin Lee, Nathalie Pujol, Andrew Chisholm, Jonathan Ewbank.

Automated drug screening and chemical genetics in C. elegans models of rare diseases.
Jean Giacomotto, Maité Carre-Pierrat, Laurent Segalat.

Isolation and characterization of C. elegans mutants deficient in PMK-1-dependent immunity.
Robert Shivers, Tristan Kooistra, Bethany Redding, Dennis H. Kim.

A Sodium: Neurotransmitter symporter Family protein required for the induction of antimicrobial peptides in C. elegans.
Katja Ziegler, Jonathan Ewbank, Nathalie Pujol.

High-throughput chemical screening using C. elegans growth and development.
Windy A. Boyd, Sandra J. McBride, Marjolein V. Smith, Grace E. Kissling, Julie R. Rice, Daniel W. Snyder, Christopher J. Portier, Jonathan H. Freedman.

Using a C. elegans feeding response assay in high-throughput chemical and genetic screening.
Windy A. Boyd, Sandra J. McBride, Jonathan H. Freedman.

Genetic dissection of Spinal Muscular Atrophy in Drosophila and C. elegans.
Jevede D. Harris, Tom Barsby, Amy K. Walker, Anne C. Hart. Gelsomino, Paolo Bazzicalupo.

Large scale genetic screens for cell fate mutants using worm sorter technology.
Vincent Bertrand, Nuria Flames, Maria Doitsidou, Richard Poole, Janine Recio, Oliver Hobert.

Semi-automated genetic screen for temperature sensitive mutations that abolish the early embryonic expression of the hox gene ceh-13.
Stephan Knierer, Adrian Streit.

The C. elegans dysferlin homolog fer-1 is expressed in muscle.
Todd Lamitina, Olga Lozynska, Tejvir Khurana.

Toward the development of a database for the storage and integration of COPAS™ BioSorter™ data.
Lisa R. Matthews, Philippe Vaglio, Jonathan Ewbank.

A French functional genomics platform.
Yohann Duverger, Sarah Scaglione, Daniel Wong, Jérôme Reboul, Jonathan Ewbank.

A semi-automated high-throughput approach to the generation of transposon insertion mutants.
Yohann Duverger, Jérôme Belougne, Sarah Scaglione, Dominique Brandli, Christophe Beclin, Jonathan Ewbank.

A green light to expression in time and space.

Genome-scale analysis of in vivo spatiotemporal promoter activity in Caenorhabditis elegans.

Differential regulation of gene expression is essential for cell fate specification in metazoans. Characterizing the transcriptional activity of gene promoters, in time and in space, is therefore a critical step toward understanding complex biological systems. Here we present an in vivo spatiotemporal analysis for approximately 900 predicted C. elegans promoters (approximately 5% of the predicted protein-coding genes), each driving the expression of green fluorescent protein (GFP). Using a flow-cytometer adapted for nematode profiling, we generated 'chronograms', two-dimensional representations of fluorescence intensity along the body axis and throughout development from early larvae to adults. Automated comparison and clustering of the obtained in vivo expression patterns show that genes coexpressed in space and time tend to belong to common functional categories. Moreover, integration of this data set with C. elegans protein-protein interactome data sets enables prediction of anatomical and temporal interaction territories between protein partners.

A semi-automated high-throughput approach to the generation of transposon insertion mutants in the nematode Caenorhabditis elegans

The generation of a large collection of defined transposon insertion mutants is of general interest to the Caenorhabditis elegans research community and has been supported by the European Union. We describe here a semi-automated high-throughput method for mutant production and screening, using the heterologous transposon Mos1 . The procedure allows routine culture of several thousand independent nematode strains in parallel for multiple generations before stereotyped molecular analyses. Using this method, we have already generated >17 500 individual strains carrying Mos1 insertions. It could be easily adapted to forward and reverse genetic screens and may influence researchers faced with making a choice of model organism.

Transgenic alternative-splicing reporters reveal tissue-specific expression profiles and regulation mechanisms in vivo

¹School of Biomedical Science and ²Medical Research Institute, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo 113-8510, Japan. ³Department of Physiology, Tokyo Women’s Medical University School of Medicine, Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan. ⁴CREST, JST, Hon-cho, Kawaguchi, Saitama 332-0012, Japan.

A small-molecule screen in C. elegans yields a new calcium channel antagonist.

Department of Medical Genetics and Microbiology, and The Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, 160 College Street, Toronto, Ontario M5S 3E1, Canada.

Genome-wide RNAi screening identifies protein damage as a regulator of osmoprotective gene expression.

Departments of Anesthesiology and Pharmacology, Vanderbilt University, T4208 Medical  Center North, 1161 21st Avenue South, Nashville, TN 37232.

The detection, stabilization, and repair of stress-induced damage are essential requirements for cellular life. All cells respond to osmotic stress-induced water loss with increased expression of genes that mediate accumulation of organic osmolytes, solutes that function as chemical chaperones and restore osmotic homeostasis. The signals and signaling mechanisms that regulate osmoprotective gene expression in animal cells are poorly understood. Here, we show that gpdh-1 and gpdh-2, genes that mediate the accumulation of the organic osmolyte glycerol, are essential for survival of the nematode Caenorhabditis elegans during osmotic stress. Expression of GFP driven by the gpdh-1 promoter (Pgpdh-1::GFP) is detected only during hypertonic stress but is not induced by other stressors. Using Pgpdh-1::GFP expression as a phenotype, we screened approximately 16,000 genes by RNAi feeding and identified 122 that cause constitutive activation of gpdh-1 expression and glycerol accumulation. Many of these genes function to regulate protein translation and cotranslational protein folding and to target and degrade denatured proteins, suggesting that the accumulation of misfolded proteins functions as a signal to activate osmoprotective gene expression and organic osmolyte accumulation in animal cells. Consistent with this hypothesis, 73% of these protein-homeostasis genes have been shown to slow age-dependent protein aggregation in C. elegans. Because diverse environmental stressors and numerous disease states result in protein misfolding, mechanisms must exist that discriminate between osmotically induced and other forms of stress-induced protein damage. Our findings provide a foundation for understanding how these damage-selectivity mechanisms function.

PMID: 16880390 [PubMed - as supplied by publisher]

A stress-sensitive reporter predicts longevity in isogenic populations of Caenorhabditis elegans

1) Institute for Behavioral Genetics, University of Colorado at Boulder, Box 447, Boulder, Colorado 80309, USA. 2) Max Planck Institute for Demographic Research, Konrad-Zuse-Strasse 1, D-18057, Rostock, Germany. 3) Department of Integrative Biology, University of Colorado at Boulder, Box 447, Boulder, Colorado 80309, USA. 4) These authors contributed equally to this work. Published online: 24 July 2005.

A non-biased, in vivo genetic screen for genes that protect against necrosis. (Poster 758A)

Dept Mol Biol & Biochem, Rutgers Univ, Piscataway, NJ.

Multi-parameter axial profiling of transgenic C. elegans expressing fluorescent proteins from various cell-specific, tissue specific and developmentally regulated promoters. (Poster 1172A)

1) Union Biometrica, Holliston, MA; 2) Division of Biology, Kansas State University, Manhattan, Kansas.

A French functional genomics platform. (Poster 1294C)

1) Centre d’Immunologie de Marseille-Luminy, INSERM/CNRS/Université de la Méditerranée, Marseille, France; 2) INSERM UMR 599, Institut Paoli Calmette, Marseille, France.

The C. elegans Localizome Project: a beginning. (Plenary Session 212)

1) Center for Cancer Systems Biology (CCSB)/Dana-Farber Cancer Institute/Harvard Medical school, Boston, MA; 2) Union Biometrica, Holliston, MA; 3) University of Leeds, Leeds, UK; 4) Department Molecular Biology and Biochemistry, Simon Fraser University, Vancouver, BC; 5) Department of Zoology, The University of British Columbia, BC; 6) Dept. of Genetics and Developmental Biology University of Connecticut Health Center, Farmington, CT.

Identification of new innate immunity genes. (Parallel Session 294)

Centre d’Immunologie de Marseille-Luminy, INSERM/CNRS/Univ. de la Méditerranée, Marseille, France.

Stochastic Effects Make a Big Difference in How Long You Will Live (If You Are a Worm). (Plenary session 311)

Inst Behavioral Genetics, University of Colorado, Boulder, CO.

A novel method of quantifying C. elegans feeding. (Poster 408B)

Nicholas School of the Environment and Earth Sciences, Duke University, Durham, NC.

Development of medium-throughput toxicity screens using C. elegans. (Poster 409C)

Nicholas School of the Environment and Earth Sciences, Duke University, Durham, NC.

Studies of Interactions between PAR Proteins. (Poster 1105C)

1) Dept Molecular Biol & Genetics, Cornell Univ, Ithaca, NY; 2) Union Biometrica, Holliston, MA; 3) Department of Morphoregulaltion, Institute for Frontier Medical Sciences, Kyoto Univ, Kyoto, Japan.

Genetic analysis of osmotically regulated gene expression in C. elegans

Department of Anesthesiology, Vanderbilt University, 1161 21st Avenue South, MCN T4208, Nashville, TN, 37232.

XNP-1/ATR-X acts with RB, HP1 and the NuRD complex during larval development in C. elegans

^aINSERM U491, Faculte´ de Me´decine la Timone, 27, Bd Jean Moulin, 13385 Marseille Cedex 5, France ^bCentre d'Immunologie de Marseille Luminy, INSERM/CNRS/Universite´ de la Me´diterrane´e, Campus Luminy Case 906, 13288 Marseille Cedex 9, France.

Diversity and specificity in the interaction between Caenorhabditis elegans and the pathogen Serratia marcescens

Co-evolutionary arms races between parasites and hosts are considered to be of immense importance in the evolution of living organisms, potentially leading to highly dynamic life-history changes. The outcome of such arms races is in many cases thought to be determined by frequency dependent selection, which relies on genetic variation in host susceptibility and parasite virulence, and also genotype-specific interactions between host and parasite. Empirical evidence for these two prerequisites is scarce, however, especially for invertebrate hosts. We addressed this topic by analysing the interaction between natural isolates of the soil nematode Caenorhabditis elegans and the pathogenic soil bacterium Serratia marcescens.

High-throughput sublethal toxicity tests using the nematode Caenorhabditis elegans.

1) Duke University, Durham, NC, USA

Automated assays to study longevity in C. elegans

Maren Hertweck(1), and Ralf Baumeister, Bio3/Bioinformatics and Molecular Genetics,  Albert-Ludwigs University of Freiburg, Schänzlestr. 1, D-79104 Freiburg, Germany.

An Unlikely Star of Science: Jonathan Freedman Looks to Microscopic Roundworms to Document the Effects of Toxic Chemicals.

Long-term effects of sterol depletion in C. elegans : Sterol content of synchronized wild-type and mutant populations.

Three major long-term effects of sterol deprivation in Caenorhabditis elegans are described. 1) The life expectancy of sterol-deprived wild-type animals is decreased by more than 40%. Similar decreases are found in animals carrying mutations in the daf-9, daf-12, daf-16, and clk-1 genes, suggesting that previously described aging pathways involving these genes are not involved in the life-extending effects of sterols. 2) There is a premature loss of motility, measured by response to mild touch. 3) There is a rapid postreproductive onset of sarcopenia (muscle wasting) as measured by total body fluorescence in a myo3::GFP-expressing strain. We also report that five sterols (the desmethylsterols cholesterol, 7-dehydrocholesterol, and lathosterol and the 4α-methyl sterols lophenol and 4α-methyl-cholesta-Δ8(14)-en-3β-ol) are found in significant amounts at all stages of development and aging in cholesterol-fed animals. Supplying any one of these as the sole sterol confers similar protection from the long-term effects of sterol deprivation.

These findings suggest that sterols are required continuously throughout the animal's life.

Chapter 4: Practical applications of RNAi in C. elegans, "RNA interference: From Basic Biology to Drug Development,"

Discusses the use of the COPAS BIOSORT to score RNAi phenotypes complete with illustrations and pictures. SUMMER, 2004

TLR-independent control of innate immunity in Caenorhabditis elegans by the TIR domain adaptor protein TIR-1, an ortholog of human SARM

Both plants and animals respond to infection by synthesizing compounds that directly inhibit or kill invading pathogens. We report here the identification of infection-inducible antimicrobial peptides in Caenorhabditis elegans. Expression of two of these peptides, NLP-29 and NLP-31, was differentially regulated by fungal and bacterial infection and was controlled in part by tir-1, which encodes an ortholog of SARM, a Toll–interleukin 1 receptor (TIR) domain protein. Inactivation of tir-1 by RNA interference caused increased susceptibility to infection. We identify protein partners for TIR-1 and show that the small GTPase Rab1 and the f subunit of ATP synthase participate specifically in the control of antimicrobial peptide gene expression. As the activity of tir-1 was independent of the single nematode Toll-like receptor, TIR-1 may represent a component of a previously uncharacterized, but conserved, innate immune signaling pathway.

Model Organisms in Drug Discovery (2003): Chapter 3: Caenorhabditis elegans Functional Genomics in Drug Discovery: Expanding Paradigms.

Devgen NV, Technologiepark 9, B-9052 Ghent-Zwijnaarde, Belgium

Book Editor(s): Pamela M. Carroll, Kevin Fitzgerald Copyright © 2003 John Wiley & Sons, Ltd

Published Online: 05 Jan 2005
Print ISBN: 0470848936; Online ISBN: 0470014067

A worm's life [Med Sci Paris]

Centre d'Immunologie de Marseille Luminy, Cnrs UMR 6102, Inserm U. 136, Universite de la Mediterranee, Case 906, 13288 Marseille Cedex 09, France.

Use of the COPAS Biosort in a hi-throughput screen for genes that regulate lifespan.

Department of Biochemistry and Biophysics, University of California, San Francisco.

Imaging strategies and data types for genome-wide comparison and pattern-matching of GFP expression patterns: GLO-Worm, Program #41.

Genetics and Dev. Bio., UConn Health Center, Farmington USA.

Isolation of Long-Lived Individuals Within an Isogenic Population, Program # 145.

Institute for Behavioral Genetics, University of Colorado at Boulder, Boulder, CO USA.

Anti-fungal innate immune defence in C. elegans, Program #287.

1) CIML, Marseille, France; 2) IBMC, Strasbourg, France; 3) CBS, Montpellier, France; 4) NIG, Mishima, Japan.

GFP screens for regulators of motor neuron differentiation, Program #520A.

Neuroscience Program Cell and Developmental Biology, Vanderbilt University, Nashville, TN USA.

Applications of the COPAS (Complex Object Parametric Analysis and Sorting) Biosort to functional genomic studies, Program #1111A.

The Wellcome Trust Sanger Institute, Cambridge, UK.

Mos1 mutagenesis and the Union Biometrica worm sorter: complementary tools for a genetic screen for worms that resist bacterial infection, Program #314B.

CIML, Marseille, FRANCE.

A high throughput screening method to detect youthful nematodes, Program #356B.

Molecular Biology and Biochem, Rutgers University, Piscataway, NJ USA.

Measurement and analysis of stress induced responses in transgenic Caenorhabditis elegans, Program #389B.

Union Biometrica, Holliston, MA USA.

Developing an automating worm-based screen for bacterial virulence factors using the Union Biometrica sorter, Program #312C.

CIML, Marseille, FRANCE.

Genetic analysis of IFT and ARPKD: Isolating osm-5 suppressors, Program #510C.

1) Laboratory of Genetics, University of Wisconsin, Madison, WI USA; 2) Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT USA; 3) School of Pharmacy, University of Wisconsin, Madison, WI, USA.

An Automationed High-Throughput Assay for Survival of the Nematode Caenorhabditis elegans

Buck Institute, Novato, CA (USA). PII: S0891-5849(03)00328-9. SEPTEMBER, 2003

Caenorhabditis elegans: an emerging genetic model for the study of innate immunity

Invaluable insights into how animals, humans included, defend themselves against infection have been provided by more than a decade of genetic studies that have used fruitflies. In the past few years, attention has also turned to another simple animal model, the nematode worm Caenorhabditis elegans. What exactly have we learned from the work in Drosophila? And will research with C. elegans teach us anything new about our response to pathogen attack?

Measurement and analysis of stress induced responses in transgenic Caenorhabditis elegans, #L340.

Life Sciences, Union Biometrica, Inc., Holliston, MA.

GFP screens for regulators of motor neuron differentiation, #242.

Dept. of Cell and Developmental Biology, Vanderbilt University Med. Ctr., Nashville, TN

Check out my Profile! Isolation of chemotaxis defective mutants with altered str-1 expression levels using automated, high-sensitivity fluorescence Profiling, #12

1) Union Biometrica, Inc., 35 Medford St, Holliston, MA 01746, 2) Union Biometrica, GMBH, Cipalstraat 3, B-2440, Geel, Belgium

The application of scale space and the spatial color model in microscopy.

* Union Biometrica N.V., European Scientific Operations, Geel, Belgium. ** ISIS, Faculty of Science, University of Amsterdam, Amsterdam, the Netherlands. *** TU Eindhoven, Faculty Biomedical Technology, Biomedical Imageprocessing, Den Dolech, Eindhoven, the Netherlands

The Multi-Mode Mosaic framework for automated microscopy and analysis.

* Union Biometrica N.V., European Scientific Operations. ** ISIS, Faculty of Science, University of Amsterdam, Amsterdam, the Netherlands

Suppressors of gon-1, #626415

1) Department of Genetics, University of Wisconsin-Madison, Madison, WI; 2) Department of Biochemistry, University of Wisconsin-Madison, Madison, WI; 3) Howard Hughes Medical Institute, University of Wisconsin-Madison, 433 Babcock Drive, Madison, WI

Isolation of chemotaxis defective mutants with altered str-1 expression levels using automated, high-sensitivity fluorescence profiling, #496373.

Union Biometrica, Inc., Holliston, MA.

Application of linear scale space and the spatial color model in microscopy

* Union Biometrica N.V., European Scientific Operations, Cipalstraat 3, B-2440 Geel, Belgium, ** Intelligent Sensory Information Systems, Faculty of Science, UvA,  Amsterdam, The Netherlands, *** BioMedische Technologie, Technische Universiteit Eindhoven, Eindhoven, The Netherlands.

Post-embryonic Developmental Expression Chronograms: a new functional genomics data type generated using a nematode fluorescence sorting system, #178.

1) University of Connecticut Health Center, Farmington CT, 2) Union Biometrica, Holliston MA

Check out my Profile! Isolation of mutants with altered str-1 expression levels using automated, high-sensitivity fluorescence profiling, #112.

Union Biometrica, Inc., Holliston, MA

Multi-parameter & Dual Color Fluorescence Analysis and Flow Sorting of C. elegans, #199.

Union Biometrica, Inc., Holliston, MA

Enhanced Analytical Performance of the C. elegans Flow Sorter COPAS BIOSORT: Automated Re-analysis of Populations in Multi-well Plates and Reading of Axially Distributed Positional Fluorescent Signals, #32.

Union Biometrica Inc. & European Scientific Operations(*)

Enhanced One-step Nematode Recognition on micrographs of Living C. elegans Cultures in 384-well Plates using Linear Scale Space Mathematics.

*Union Biometrica, European Scientific Operations, Geel, Belgium; **Intelligent Sensory Information Systems, UvA, Amsterdam, the Netherlands.

Multi-parametric & dual color fluorescent analysis and flow sorting of C. elegans.

*Union Biometrica, European Scientific Operations, Geel, Belgium; **Union Biometrica, Holliston, MA, USA.

Fully Automated Instrumentation for Analysis in C elegans, #1089.

Union Biometrica, Inc., Holliston, MA.

Application of the COPAS (Complex Object Parametric Analysis and Sorting) dispenser to functional genomic studies, #1096.

The Sanger Centre, Wellcome Trust Genome Campus, Hinxton, UK

Automated Sorting of C. elegans Muv Mutants According to Pseudovulva Number, #1097.

Union Biometrica, Inc., Holliston, MA

Boy Is My Bursa Red: Automated Detection and Sorting of Fluorescent Stained C. elegans Males From a Mixed Population, #89.

Union Biometrica, Inc. (Holliston, MA).

Adapting a manual clonal screen to semi-automation, #858.

1) MCB, Harvard University, Cambridge, MA, 02138., 2) Union Biometrica, 19 Ward St., Holliston, MA 01746, 3) Axys, Nemapharm Group, South San Francisco, CA

Automated sorting and dispensing of C. elegans to wells of microtiter plates, #443.

1) Axys Pharmaceuticals, NemaPharm Group, South San Francisco, CA, 2) Union Biometrica, Inc., Holliston, MA.

Commentary: Classical genetics goes high-tech

In vivo quantitative high-throughput screening for drug discovery and comparative toxicology

Quantitative high-throughput screening (qHTS) pharmacologically evaluates chemical libraries for therapeutic uses, toxicological risk and, increasingly, for academic probe discovery. Phenotypic high-throughput screening assays interrogate molecular pathways, often relying on cell culture systems, historically less focused on multicellular organisms. Caenorhabditis elegans has served as a eukaryotic model organism for human biology by virtue of genetic conservation and experimental tractability. Here, a paradigm enabling C. elegans qHTS using 384-well microtiter plate laser-scanning cytometry is described, in which GFP-expressing organisms revealing phenotype-modifying structure–activity relationships guide subsequent life-stage and proteomic analyses, and Escherichia coli bacterial ghosts, a non-replicating nutrient source, allow compound exposures over two life cycles, mitigating bacterial overgrowth complications. We demonstrate the method with libraries of anti-infective agents, or substances of toxicological concern. Each was tested in seven-point titration to assess the feasibility of nematode-based in vivo qHTS, and examples of follow-up strategies were provided to study organism-based chemotype selectivity and subsequent network perturbations with a physiological impact. We anticipate that this qHTS approach will enable analysis of C. elegans orthologous phenotypes of human pathologies to facilitate drug library profiling for a range of therapeutic indications.

Methods to extract and study the biological effects of murine gut microbiota using Caenorhabditis elegans as a screening host

Gut microbiota has been established as a main regulator of health. However, how changes in gut microbiota are directly associated with physiological and cellular alterations has been difficult to tackle on a large-scale basis, notably because of the cost and labor-extensive resources required for rigorous experiments in mammals. In the present study, we used the nematode Caenorhabditis elegans as a model organism to elucidate microbiota-host interactions. We developed a method to extract gut microbiota (MCB) from murine feces, and tested its potential as food source for and its impact on C. elegans biology compared to the standard bacterial diet Escherichia coli OP50. Although less preferred than OP50, MCB was not avoided but had a lower energy density (triglycerides and glucose). Consistently, MCB-fed worms exhibited smaller body length and size, lower fertility, and lower fat content than OP50-fed worms, but had a longer mean lifespan, which resembles the effects of calorie restriction in mammals. However, these outcomes were altered when bacteria were inactivated, suggesting an important role of symbiosis of MCB beyond nutrient source. Taken together, our findings support the effectiveness of gut MCB processing to test its effects in C. elegans. More work comparing MCB of differently treated mice or humans is required to further validate relevance to mammals before large-scale screening assays.

Characterization of larval growth in C. elegans cuticle mutants