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[
International Worm Meeting,
2021]
In the absence of food, C. elegans are able to arrest development in order to survive extended periods of starvation. This period of quiescence is reversible upon feeding, and the ability to start and stop development is dependent on the transcriptional effector of insulin-like signaling,
daf-16/FoxO.
daf-16 is responsible for inhibiting pathways that promote development and activating stress-response genes, and it is regulated by the insulin receptor
daf-2/InsR via the PI3K pathway. We are interested in exploring the gene regulatory mechanisms of
daf-2 that promote starvation resistance in
daf-16-dependent and independent manners. Gene expression data revealed that the histone variant
hil-1/H1-0 is activated by
daf-16 during starvation, and that its expression decreases upon feeding. Assays for survival, growth and reproduction following recovery from starvation using
hil-1 mutant alleles
tm1442 and
gk229 suggest that
hil-1 plays a critical role in C. elegans' ability to survive and recover from starvation. We hypothesize that HIL-1 alters chromatin structure, regulating expression of nutrient-responsive genes. In addition, RNA-seq-based epistasis analysis suggests a function of
daf-2 independent of
daf-16. Phenotypic assays of
daf-2 and
daf-16 single and double mutants revealed that simultaneous disruption of
daf-2 and
daf-16 leads to increased starvation resistance over the
daf-16 single mutant. Together these results suggest DAF-2 regulates starvation resistance through one or more effectors in addition to DAF-16. Identifying
daf-2 signaling effector mechanisms as well as the DAF-16-dependent function of
hil-1 in cellular quiescence will be valuable for understanding the genetic basis of physiological adaptation to nutrient availability with implications for aging and the pathological consequences of nutrient stress in humans.
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Baugh, L. Ryan, Miska, Eric, Willis, Alexandra, Reinke, Aaron, Burton, Nick, Price, Jon, Braukmann, Fabian, Stevens, Lewis, Fisher, Kinsey
[
International Worm Meeting,
2021]
Despite reports of parental exposure to stress promoting adaptations in progeny in diverse organisms, there remains considerable debate over the ecological significance and evolutionary conservation of these multigenerational effects. Here, we investigate four independent examples of intergenerational adaptations to stress in C. elegans - bacterial infection, microsporidia infection, osmotic stress and starvation - across four different Caenorhabditis species. We found that all four intergenerational adaptations to stress are conserved in at least one other species, that the responses and evolutionary conservation patterns are stress specific, and that intergenerational adaptive effects have deleterious trade-offs in mismatched environments. By profiling the intergenerational and transgenerational effects of different stresses on gene expression across species, we identified 3,174 genes that exhibited intergenerational changes in expression in multiple species in response to stress. Furthermore we found that an inversion in the expression of certain stress response genes required for intergenerational adaptations, from increased expression in the offspring of stressed parents to decreased expression in the offspring of stressed parents, correlates with an inversion of an adaptive response to infection in C. elegans and C. kamaaina to a deleterious intergenerational effect in C. briggsae. By contrast, we did not observe any conserved transgenerational changes in gene expression in response to stress, suggesting that the intergenerational effects of stress on offspring gene expression are not maintained transgenerationally. Our results demonstrate that intergenerational responses to stress play a substantial, evolutionarily conserved, and largely reversible role in regulating animal physiology.
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Baugh, Ryan, Meyer, Joel, Bijwadia, Shefali, Wang, Yang, Mello, Danielle, Chitrakar, Rojin, Bergemann, Christina, Caldwell, Alexis, Fisher, Kinsey
[
International Worm Meeting,
2021]
Mitochondria are central players in host immunometabolism as they function not only as metabolic hubs but also as signaling platforms regulating innate immunity. Environmental exposures to mitochondrial toxicants occur widely and are increasingly frequent. Many pesticides target mitochondrial function, including the well-characterized complex I inhibitor, rotenone (Rot). Exposures to these mitotoxicants can pose a serious threat to organismal health and the onset of diseases by disrupting immunometabolic pathways. Our hypothesis is that Rot can disrupt C. elegans immunometabolism and consequently alter pathogen survival. C. elegans eggs were exposed to Rot (0.5 microM) or vehicle (Ctrl - 0.125 uM DMSO) in liquid and harvested once they reached the L4 larval stage (which was ~24h later for the Rot treatment, as it caused growth delay). Inhibition of mitochondrial respiration by Rot was confirmed by measuring the worm oxygen consumption rate. To explore pathways that were modulated by Rot, we performed a transcriptomic analysis and found 179 differentially expressed genes. WormCat analysis revealed that the two major broad enriched categories were stress response -which was mostly represented by pathogen response and detoxification genes- and metabolism -which was mostly represented by lipid and mitochondrial metabolism genes. Next, Ctrl and Rot-exposed worms were depurated for 48h, and further exposed to Pseudomonas aeruginosa (PA14), and Salmonella enterica (SL1344). Rot-exposed worms were more resistant to SL1344 but more susceptible to PA14. The mitochondrial unfolded protein response (mitoUPR) is a well-known immunometabolic pathway in C. elegans which links mitochondria and immunity and provides resistance to pathogen infection. Rot activated the mitoUPR pathway, which was evidenced by increased
hsp-6:GFP expression. Activation was also observed after 24h of exposure to PA14 and SL1344, however, Ctrl PA14-infected worms displayed lower
hsp-6:GFP expression, and Rot rescued its expression only to the level of Ctrl OP50-raised worms. Thus, mitoUPR activation could be involved in the increased resistance to SL1344 but the level of activation in PA14 worms might not have been sufficient to promote resistance. By further exploring our transcriptomic dataset using WormExp and "module-weighted annotations" analysis tools, we identified genes that are known to confer resistance to PA14 that were downregulated by the Rot exposure, including HIF-dependent genes, which may underlie the increased susceptibility to PA14. Together, these results demonstrate that the mitotoxicant rotenone can modulate important pathways associated to the C. elegans immunometabolism and alter pathogen resistance.
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Antoshechkin, Igor, Baugh, Ryan, Fisher, Kinsey, Chen, Jingxian, Webster, Amy, Stevens, Lewis, Powell, Maya, Tanny, Robyn, Chitrakar, Rojin, Evans, Kathryn, Andersen, Erik
[
International Worm Meeting,
2021]
The genetic basis of natural variation in starvation resistance is not well understood though it is a fundamental, biomedically important trait. We developed a population selection and sequencing approach (MIP-seq) to measure starvation resistance for a large number of wild C. elegans strains in a single culture. We identified three quantitative trait loci (QTL) affecting starvation resistance. These QTL overlap with hyper-divergent regions and contain multiple members of several large gene families involved in environmental interactions. In particular, we identified 16 members of the insulin/EGF receptor-like domain (irld) family with variants within starvation resistance QTL. We generated and assayed loss-of-function mutations for four irld family members, all of which increased starvation resistance. We show that the transcription factor
daf-16/FoxO, a critical effector of insulin/insulin-like growth factor signaling (IIS) known to promote starvation resistance, is required for increased resistance of
irld-39;
irld-52 mutants, that these mutants affect DAF-16 target gene expression, and that the IIS receptor
daf-2/InsR is epistatic to these irld genes. We propose that IRLD proteins bind insulin-like peptides (ILPs) to modify signaling in the sensory nervous system thereby affecting organismal physiology. This work demonstrates the efficacy of using population sequencing to investigate natural variation of a complex trait, and it identifies irld genes that regulate IIS and starvation resistance. Furthermore, it shows that variation in a rapidly evolving large gene family modifies activity of a deeply conserved signaling pathway to affect a fitness-proximal trait.
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[
Mol Brain,
2021]
Aim: Experimental animals, such as non-human primates (NHPs), mice, Zebrafish, and Drosophila, are frequently employed as models to gain insights into human physiology and pathology. In developmental neuroscience and related research fields, information about the similarities of developmental gene expression patterns between animal models and humans is vital to choose what animal models to employ. Here, we aimed to statistically compare the similarities of developmental changes of gene expression patterns in the brains of humans with those of animal models frequently used in the neuroscience field.Methods: The developmental gene expression datasets that we analyzed consist of the fold-changes and P values of gene expression in the brains of animals of various ages compared with those of the youngest postnatal animals available in the dataset. By employing the running Fisher algorithm in a bioinformatics platform, BaseSpace, we assessed similarities between the developmental changes of gene expression patterns in the human (Homo sapiens) hippocampus with those in the dentate gyrus (DG) of the rhesus monkey (Macaca mulatta), the DG of the mouse (Mus musculus), the whole brain of Zebrafish (Danio rerio), and the whole brain of Drosophila (D. melanogaster).Results: Among all possible comparisons of different ages and animals in developmental changes in gene expression patterns within the datasets, those between rhesus monkeys and mice were highly similar to those of humans with significant overlap P-value as assessed by the running Fisher algorithm. There was the highest degree of gene expression similarity between 40-59-year-old humans and 6-12-year-old rhesus monkeys (overlap P-value = 2.1 10- 72). The gene expression similarity between 20-39-year-old humans and 29-day-old mice was also significant (overlap P = 1.1 10- 44). Moreover, there was a similarity in developmental changes of gene expression patterns between 1-2-year-old Zebrafish and 40-59-year-old humans (Overlap P-value = 1.4 10- 6). The overlap P-value of developmental gene expression patterns between Drosophila and humans failed to reach significance (30 days Drosophila and 6-11-year-old humans; overlap P-value = 0.0614).Conclusions: These results indicate that the developmental gene expression changes in the brains of the rhesus monkey, mouse, and Zebrafish recapitulate, to a certain degree, those in humans. Our findings support the idea that these animal models are a valid tool for investigating the development of the brain in neurophysiological and neuropsychiatric studies.
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[
CBE Life Sci Educ,
2008]
The skill set required of biomedical researchers continues to grow and evolve as biology matures as a natural science. Science necessitates creative yet critical thinking, persuasive communication skills, purposeful use of time, and adeptness at the laboratory bench. Teaching these skills can be effectively accomplished in an inquiry-based, active-learning environment at a primarily undergraduate institution. Cell Biology Techniques, an upper-level cell biology laboratory course at St. John Fisher College, features two independent projects that take advantage of the biology of the nematode Caenorhabditis elegans, a premier yet simple model organism. First, students perform a miniature epigenetic screen for novel phenotypes using RNA interference. The results of this screen combined with literature research direct students toward a singe gene that they attempt to subclone in the second project. The biology of the chosen gene/protein also becomes an individualized focal point with respect to the content of the laboratory. Progress toward course goals is evaluated using written, oral, and group-produced assignments, including a concept map. Pre- and postassessment indicates a significant increase in the understanding of broad concepts in cell biological research.
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[
Worm Breeder's Gazette,
1992]
After tabulating the results of the Worm Plate Survey. we have come up with some interesting results. Most notably. the high variability in prices that labs are paying for their plates, even for the exact same plates from the same supplier, and the fact that most plates are marked up considerably over the actual cost. The replies can be separated into 4 categories: Labs that get plates from Fisher ($29-$58). but wish they had non-vented plates Labs that get non-vented plates via Applied Scientific (~$38) Labs that get plates from Falcon (vented) or Nunc (non-vented) and pay much more Most labs' plates were "slipable" or "semi-stackable", but all labs wanted plates that stack well for easy manual pouring, seeding, carrying, and using. Everyone wanted plates with shallow lids such that the bottoms can be lifted out of the tops for inverted use. Some labs expressed an interest in plates slightly smaller than "60 mm". That number is in quotes because all of the companies' plates have bottoms smaller than 60 mm (e.g. Fisher -54 x 14 mm). We have negotiated with the plastic companies that really make the plates for Fisher, Applied Scientific, etc. (that actually just resell them to you). I have come to the conclusion that we can provide you with better worm plates, the same worm plates cheaper, or in most cases better worm plates cheaper. This is true for every lab. The bottom line is that we can get you top quality non-vented "60 mm" plates (like Applied Scientific's, except fully stackable) for about $29 per 500 case INCLUDING shipping depending on your usage and how many cases you can receive at one time. Several labs have found the non-vented plates last longer without drying out or getting contaminated, compared with normal vented plates, so you should save that way, too. We offer full service shipping (e.g. standing orders and same-day telephone orders, free. Similarly low prices are available on 100 mm and 150 mm plates that exceed industry standards for flatness (reducing media usage) and clarity. The 100 mm are about $27 per 500 case plus shipping; The 150 mm dishes (good for DNA & RNA preps and library platings, with more than 2.25x the surface area of 100 mm dishes) are made thicker and deeper than industry standards and are about $21.50 per 100 case plus shipping. The shipping charge is very low for labs, or groups of labs in one city, that can take delivery of many cases in a single shipment. You can even suggest that your stockroom order plates from us. Call us for an exact price quote depending on your usage and how many cases you can receive at one time. In any case, we'll work things out to save you money. In the future, we can offer inexpensive 35 mm dishes if the community at large can order about 2000 cases per year, so let me know about your needs for other sizes. The response was very mixed about pre-poured plates. We may set that up later, but for now we can help the most by saving you lots on empty petri dishes (and later, maybe media .supplies). We are happy to send out free samples so you can examine the dishes. If we haven't contacted you yet, just give us a call. Respondents: 38 (including 5 anonymous) "Winners": Horvitz = 550, Meyer = 400, Thomas = 400, Greenwald = 300 200-299 cases 8 labs 100-199 cases 7 labs 4-99 cases 19 labs Highest price per case: US = 118.75, Canada = $117 (non-vented) Lowest price per case: US = $29, Canada = $25 (vented) Farthest away response: Malta! No responses from MRC or anyone else in Europe or Asia. It is possible that we can save money and/or provide better plates for these labs, including, shipping, too. Let us know.
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Fisher GM, Preston S, Ansell BRE, Nowell C, Davis RA, Andrews KT, Auwerx J, Jabbar A, Gasser RB, Gijs MAM, Crawford S, Sternberg PW, Korhonen PK, Mouchiroud L, Cornaglia M, Hofmann A, Baell J, McGee SL, Chang BCH, Young ND
[
FASEB J,
2017]
As a result of limited classes of anthelmintics and an over-reliance on chemical control, there is a great need to discover new compounds to combat drug resistance in parasitic nematodes. Here, we show that deguelin, a plant-derived rotenoid, selectively and potently inhibits the motility and development of nematodes, which supports its potential as a lead candidate for drug development. Furthermore, we demonstrate that deguelin treatment significantly increases gene transcription that is associated with energy metabolism, particularly oxidative phosphorylation and mito-ribosomal protein production before inhibiting motility. Mitochondrial tracking confirmed enhanced oxidative phosphorylation. In accordance, real-time measurements of oxidative phosphorylation in response to deguelin treatment demonstrated an immediate decrease in oxygen consumption in both parasitic (Haemonchus contortus) and free-living (Caenorhabditis elegans) nematodes. Consequently, we hypothesize that deguelin is exerting its toxic effect on nematodes as a modulator of oxidative phosphorylation. This study highlights the dynamic biologic response of multicellular organisms to deguelin perturbation.-Preston, S., Korhonen, P. K., Mouchiroud, L., Cornaglia, M., McGee, S. L., Young, N. D., Davis, R. A., Crawford, S., Nowell, C., Ansell, B. R. E., Fisher, G. M., Andrews, K. T., Chang, B. C. H., Gijs, M. A. M., Sternberg, P. W., Auwerx, J., Baell, J., Hofmann, A., Jabbar, A., Gasser, R. B. Deguelin exerts potent nematocidal activity via the mitochondrial respiratory chain.
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[
Biochem J,
1999]
The cAMP-dependent protein kinase (protein kinase A, PK-A) is multifunctional in nature, with key roles in the control of diverse aspects of eukaryotic cellular activity. In the case of the free-living nematode, Caenorhabditis elegans, a gene encoding the PK-A catalytic subunit has been identified and two isoforms of this subunit, arising from a C-terminal alternative-splicing event, have been characterized [Gross, Bagchi, Lu and Rubin (1990) J. Biol. Chem. 265, 6896-6907]. Here we report the occurrence of N-terminal alternative-splicing events that, in addition to generating a multiplicity of non-myristoylatable isoforms, also generate the myristoylated variant(s) of the catalytic subunit that we have recently characterized [Aspbury, Fisher, Rees and Clegg (1997) Biochem. Biophys. Res. Commun. 238, 523-527]. The gene spans more than 36 kb and is divided into a total of 13 exons. Each of the mature transcripts contains only 7 exons. In addition to the already characterized exon 1, the 5'-untranslated region and first intron actually contain 5 other exons, any one of which may be alternatively spliced on to exon 2 at the 5' end of the pre-mRNA. This N-terminal alternative splicing occurs in combination with either of the already characterized C-terminal alternative exons. Thus, C. elegans expresses at least 12 different isoforms of the catalytic subunit of PK-A. The significance of this unprecedented structural diversity in the family of PK-A catalytic subunits is discussed.
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[
International Worm Meeting,
2007]
Alpha-synuclein is the key molecule involved in synucleinopathies since multiplication of the wildtype alpha-synuclein locus is sufficient for progressive loss of dopamine (DA) neurons and the development of Parkinsons disease. We have constructed two isogenic strains of C. elegans that co-express GFP and alpha-synuclein under the
dat-1 promoter and have found that both lines exhibit DA neurodegeneration. Notably, the level of DA neurodegeneration corresponds to the level of alpha-synuclein expression as determined by semi-quantitative RT-PCR. Furthermore, both strains exhibit an age-related decline in DA neuron survival. For example, in worms expressing the higher level of alpha-synuclein, there is almost no degeneration when the animals are 3 days old, while 7 and 10 day-old animals have 25 and 36% degeneration, respectively. In mammalian systems, DA is implicated in the neurotoxicity of alpha-synuclein in DA neurons. We have discovered that the dorsal CEP neurons, which are post-synaptic to the ADE dopamine-producing neurons, degenerate significantly more often than the ventral CEP neurons, which do not synapse onto other dopamine producing neurons (p=0.02; Fisher Exact Test). DA neurodegeneration induced by alpha-synuclein overexpression was significantly rescued by overexpression of several genes implicated in Parkinsons disease (Cao et. al., J Neuroscience 25:3801; Cooper et al., Science 313:324; Hamamichi et al., 16th International Worm Meeting). Furthermore, several chemicals isolated from ongoing drug screening efforts significantly enhance DA neuron survival in our model. Alpha-synuclein-expressing worms were also tested for deficits in the DA-specific behavior of basal slowing in response to the presence of bacteria (Sawin et al., Neuron 26:619). We found that these worms displayed a locomotory defect for this phenotype that was often observed prior to the loss of DA neurons. Thus, it is possible that the behavioral deficit represents a more sensitive readout for DA neuron loss than visual inspection of the neurons. Furthermore, genetic and chemical enhancers of DA neuron survival often rescued this DA-specific behavioral phenotype.