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[
Genome Biol,
2007]
ABSTRACT: BACKGROUND: Although Caenorhabditis elegans is an important model for the study of DNA damage- and repair-related processes such as aging, neurodegeneration and carcinogenesis, DNA repair is poorly characterized in this organism. We adapted a quantitative PCR assay to characterize repair of UVC radiation-induced DNA damage in C elegans, and then tested whether DNA repair rates were affected by age in adults. RESULTS: UVC radiation induced lesions in young adult C elegans with a slope of 0.4-0.5 lesions per 10kb DNA per 100 Joules/m2, in both nuclear and mitochondrial targets. L1 and dauer larvae were >5-fold more sensitive to lesion formation than young adults. Nuclear repair kinetics in a well-expressed nuclear gene were biphasic in non-gravid adult nematodes: a faster, first order (
t1/2 ~16 h) phase lasting ~24 h and resulting in removal of ~60% of the photoproducts was followed by a much slower phase. Repair in 10 nuclear DNA regions was 15% and 50% higher in more actively transcribed regions in young and aging adults, respectively. Finally, repair was reduced 30-50% in each of the 10 nuclear regions in older adults. However, this decrease in repair could not be explained by a reduction in expression of nucleotide excision repair genes, and we present a plausible mechanism, based on gene expression data, to explain this decrease. CONCLUSIONS: Repair of UVC-induced DNA damage in C elegans is similar kinetically and genetically to repair in humans. Furthermore, this important repair process slows significantly in aging C elegans, the first whole organism in which this question has been addressed.
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[
Aquat Toxicol,
2010]
Silver nanoparticles (AgNPs) are frequently used as antimicrobials. While the mechanism(s) by which AgNPs are toxic are unclear, their increasing use raises the concern that release into the environment could lead to environmental toxicity. We characterized the physicochemical behavior, uptake, toxicity (growth inhibition), and mechanism of toxicity of three AgNPs with different sizes and polyvinylpyrrolidone (PVP) or citrate coatings to the nematode Caenorhabditis elegans. We used wild-type (N2) C. elegans and strains expected to be sensitive to oxidative stress (
nth-1,
sod-2 and
mev-1), genotoxins (
xpa-1 and
nth-1), and metals (
mtl-2). Using traditional and novel analytical methods, we observed significant aggregation and extra-organismal dissolution of silver, organismal uptake and, in one case, transgenerational transfer of AgNPs. We also observed growth inhibition by all tested AgNPs at concentrations in the low mg/L levels. A metallothionein-deficient (
mtl-2) strain was the only mutant tested that exhibited consistently greater AgNP sensitivity than wild-type. Although all tested AgNPs were internalized (passed cell membranes) in C. elegans, at least part of the toxicity observed was mediated by ionic silver. Finally, we describe a modified growth assay that permits differentiation between direct growth-inhibitory effects and indirect inhibition mediated by toxicity to the food source.
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[
Autophagy,
2012]
Mitochondrial DNA (mtDNA) is different in many ways from nuclear DNA. A key difference is that certain types of DNA damage are not repaired in the mitochondrial genome. What, then, is the fate of such damage? What are the effects? Both questions are important from a health perspective because irreparable mtDNA damage is caused by many common environmental stressors including ultraviolet C radiation (UVC). We found that UVC-induced mtDNA damage is removed slowly in the nematode Caenorhabditis elegans via a mechanism dependent on mitochondrial fusion, fission, and autophagy. However, knockdown or knockout of genes involved in these processes-many of which have homologs involved in human mitochondrial diseases-had very different effects on the organismal response to UVC. Reduced mitochondrial fission and autophagy caused no or small effects, while reduced mitochondrial fusion had dramatic effects.
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[
International Worm Meeting,
2019]
Mitochondrial biology has become an area of intense research owing to its unique role in several physiological processes and pathologies. The model organism Caenorhabditis elegans has been extensively used to study ageing and stress responses, which are often tightly associated with mitochondrial metabolism, highlighting the importance of improving our basic understanding about this organelle throughout worm lifespan. The Seahorse Extracellular Flux (XF) Analyzer measures oxygen consumption rates (OCR) of different biological samples in real-time, and has been a great asset to study mitochondrial function with relatively high throughput. The aim of this study was to characterize different mitochondrial parameters in all larval stages, as well as in young and older adult worms using the 24-well XF analyzer. Besides measuring whole worm basal OCR, with the use of different mitochondrial inhibitors we can also assess mitochondrial bioenergetics such as mitochondrial OCR, spare capacity, maximal OCR, ATP-linked OCR, and proton-leak, as well as non-mitochondrial OCR. In order to perform these measurements, we are first optimizing worm numbers and mitochondrial inhibitors concentrations for each life stage. During the optimization process we are also testing different worm media and possible drug interactions. Our preliminary results revealed that (i) the complex IV inhibitor sodium azide response seems to be affected if injected subsequently to the mitochondrial uncoupler FCCP and the ATPase inhibitor DCCD. We have also found that (ii) although L1 worms have very low OCR, their relative spare capacity can be even greater than in adult worms, which are known to have a higher metabolic rate. Moreover, (iii) we found significant differences between basal OCR in worms within a same larval stage. Late L3 worms had approximately a 2-fold increase in basal OCR when compared to early L3s that were only 3h younger. And finally, (iv) OCR measurements in adult worms appear to be much more variable than in larval stages, therefore slight changes in mitochondrial responses of adult worms might not be easily detectable. The careful characterization of the worm mitochondrial metabolism throughout different life stages using the XF analyzer should be a useful asset to the C. elegans research field, and so far has revealed that care must be taken with possible drug interactions, when choosing which life stage to analyze, and when treatments might affect the worm development.
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[
International Worm Meeting,
2019]
Growing evidence has shown that mitochondrial dysfunction not only compromises the energetic metabolism of cells, but also plays key roles in other physiological processes such as immunomodulation. We hypothesize that mitochondrial toxicity can be a common link between increased prevalence in immune-related disorders and toxic environmental exposures. To test this hypothesis, we are using the pesticide rotenone -a widely known complex I inhibitor- and the model organism Caenorhabditis elegans. Synchronized N2 eggs were exposed to rotenone (0-0.5 M) or 0.25% DMSO (control) in liquid with food (HB101) and harvested 52h later (L4 stage). After a further 48h depuration period, worm survival was followed in the presence of the pathogens Pseudomonas aeruginosa strain PA14, and Salmonella enterica serovar Typhimurium strain SL1344. Our first finding was that rotenone caused a dose-dependent decrease in worm size, which was associated to developmental delay. Worm vulval development was assessed to precisely determine the hours of developmental delay. Stage-synchronized worms exposed to 0.5 M rotenone had a longer median survival in SL1344 than control animals (~40%); but were more susceptible to PA14 (~15%). To validate whether these altered pathogen responses were due to rotenone-induced mitochondrial dysfunction, we analyzed different mitochondrial parameters. No significant differences were observed in preliminary measurements of worm basal oxygen consumption rate (OCR), spare capacity and ATP-linked OCR, or the ratio of mitochondrial to nuclear DNA copy number. This apparent lack of mitochondrial dysfunction after a developmental rotenone exposure may be due to a metabolic compensation in the worms, most likely through upregulation of complex II activity and the glyoxylate cycle, according to previous work. Thus, this appears to be a great model to study signaling between mitochondria and the immune system caused by metabolism shifts, without the detrimental effects of overt mitochondrial dysfunction. Now we are investigating the mechanisms by which mitochondrial signaling might be regulating the observed shifts in resistance to pathogens.
-
[
International Worm Meeting,
2019]
Arsenic is a well-established environmental toxicant that contributes to the pathogenesis of a number of diseases including cardiomyopathy, neuropathy, and cancers. Carcinogenic effects may be regulated via epigenetic mechanisms, suggesting that effects of arsenic exposure may persist through multiple generations. Arsenic inhibits a number of enzymes in energy production that results in metabolic shifts that support disease pathogenesis, making mitochondria an important target of arsenic toxicity to further study. Here, we use the model organism, C. elegans to study the effect of early-life arsenic exposure on mitochondrial function in the parental generation, and three subsequent generations of unexposed progeny. Age-synchronous worms were exposed to sodium arsenite at concentrations ranging from zero to 100% of the 48-hour LC10. In the exposed parental generation, there was a significant decrease in larval growth after 48 hours of exposure at all concentrations compared to controls. However, in each of the three subsequent generations, 48-hour larval growth was indistinguishable in the progeny of exposed worms compared to control progeny. No changes were observed in mitochondrial copy number or ATP content after exposure in the parental generation; oxygen consumption and reproduction analyses are ongoing. Thus, these levels of sodium arsenite exposure do not appear to have a dramatic effect on mitochondrial function in the parental generation. Further, in contrast to a recent publication on arsenite effects in worms, we have not yet detected transgenerational effects. Future studies include characterizing the epigenome in the parental generation and three subsequent generations to determine if epigenetic modifications are inherited even though we do not currently observe phenotypic inheritance of mitochondrial defects.
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[
Cell,
2009]
Meiotic chromosome pairs must receive at least one crossover to ensure proper segregation at the first meiotic division. Mets and Meyer (2009) now present compelling evidence that the establishment of higher-order chromosome structure by a condensin complex regulates crossover recombination by controlling the distribution and frequency of meiotic double-strand breaks.
-
[
Worm Breeder's Gazette,
1987]
Mutations in
dpy-27 and
dpy-28 affect the viability of XX but not XO animals. In addition, these mutations disrupt dosage compensation resulting in XX but not XO animals over-expressing their X-linked genes (as assayed by Northern analysis [Meyer and Casson, Cell 47:871 1986] and by morphogenetic assay [DeLong, et al. Genetics, in press]).
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[
Nat Methods,
2011]
Engineering precise genetic changes in a genome is powerful way to study gene function, and several recent papers describe new applications of gene-editing tools. Working with researchers at Sangamo BioSciences, Howard Hughes Medical Institute investigator Barbara Meyer and her colleagues at the University of California, Berkeley, described the first systems for making targeted genomic modifications in the roundworm Caenorhabditis elegans, a valuable model organism (Wood et al., 2011).
-
Gordon, K.L., Driscoll, M., Sherwood, D.R., Meyer, J.N., Laranjeiro, R., Hartman, J.H.
[
International Worm Meeting,
2017]
Health, disease, and aging are determined by genetic factors, environment, and lifestyle. In humans, environmental contributions to long-term health arise from ambient factors such as environmental chemical exposures and sun exposure, while lifestyle impacts health through mental health and stress, diet, drug usage, and exercise. In laboratory animals, the impact of environment and lifestyle are minimized through carefully controlled experimental conditions, and can therefore be modulated to study the effects of these factors. The effect of exercise on general health has been reported: positive impacts on cognitive function, maintenance of skeletal muscle, and protection from age-related diseases are increasingly recognized. However, molecular mechanisms underlying those protections are not well understood. Furthermore, it is unknown what impacts regular exercise training may have on other health-modifying factors such as toxic exposures from the environment. In this study, we used C. elegans to study the impact of regular exercise training on mitochondrial health and chemical toxicity. For exercise experiments, beginning at L4 stage, animals were transferred to unseeded agar plates without (control) or with liquid (causing worms to swim/thrash) for 90 minutes twice daily. This regimen was carried out for six days, and mitochondrial and toxicity outcomes were tested following exercise on adult day 6 and adult day 10. Preliminary results show that mitochondrial morphology is not significantly different between control and exercise groups at adult day 6 (p=0.64); however, on day 10, control animals have highly fragmented and disorganized mitochondria, while exercised animals exhibit significantly healthier mitochondrial morphology (p=0.0065). Furthermore, mitochondrial respiration significantly differed in spare capacity (p<0.001) on adult day 6, with exercised animals showing increased spare capacity compared to controls. Respiration experiments with day 10 adults are underway; total ATP, mitochondrial DNA copy number, and mitochondrial DNA lesions are also being investigated. Preliminary toxicity experiments showed that exercise-induced changes in mitochondrial health were accompanied by a 30-50% reduction in lethality induced by the mitochondrial toxicants arsenite and rotenone. Together, these data demonstrate that changes in physical activity result in altered mitochondrial health, which extends to protection against chemical toxicants known to damage mitochondria. Ongoing and future experiments will further explore the biochemical and metabolic changes underlying this phenomenon.