Edwards, Hunter et al. (2021) International Worm Meeting "Effect of individual members of gut microbiome on C. elegans stress resistance, lifespan and healthspan"

Vidal, Daniela, Edwards, Hunter, Samuel, Buck, Vanpalli, Siva, Anupom, Taslim, Zhang, Fan

[International Worm Meeting, 2021]

Dysregulation of the human gut microbiome has been linked to the development of many human diseases, including inflammatory disorders such as irritable bowel syndrome and ulcerative colitis. Moreover, studies have shown that the microorganisms colonizing the GI tract interact extensively with host signaling pathways, including the highly conserved ageing-related Insulin/IGF signaling pathway. The mechanisms by which the gut microbiota influences host gene expression and physiology remains unclear. With a rapid lifespan of less than one month, C. elegans have been used to study the relationship between host phenotypes and gene expression for decades. C. elegans are typically grown on a non-native singular food source, E. coli OP50, selected for its accessibility and ease of growth in the laboratory environment. The versatility of the soil dwelling nematode offers a unique platform to study complex microbial communities in a well-defined gnotobiotic environment, however very little is known about the effects of non-E.coli bacteria on C. elegans health and survival. Here we conduct comprehensive screen of select individual bacteria isolated from natural C. elegans microbiome. Sterile C. elegans can be housed in pillared microfluidic chambers where bacterial membership can be precisely controlled and readily delivered to control quality and quantity of the bacteria. Using this platform, we show that individual members of the natural microbiome colonize the C. elegans gut and exert variable effects on host physiology including delayed development and growth, stress resistance and survival. Specifically, we find that two bacterial isolates, which dominate the guts of C. elegans, Ochrobactrum BH3 and Myroides BIGb0244, extend lifespan and healthspan in our semi-liquid microfluidic environment. Our results lay the foundation for future, high-throughput screens of larger communities and panels of microbes, such as the BIGbiome and CeMbio model microbiomes. This robust system will allow for simultaneous and comprehensive assessment of the effects of both individual isolates and multi-member communities on host gene expression and aging related phenotypes.

Weiner A (2003) Mol Cell "Soaking up RNAi."

Weiner A

[Mol Cell, 2003]

RNA interference (RNAi) describes the ability of double-stranded RNA (dsRNA) to inhibit homologous gene expression at the RNA or DNA level. In a recent paper, Feinberg and Hunter report that a single transmembrane dsRNA transport protein may enable RNAi to spread systemically from one cell to another.

Michael C Fitzgerald et al. (2004) East Coast Worm Meeting "Specificity of the C. elegans Putative Transmembrane Channel SID-1"

Craig P Hunter, Michael C Fitzgerald

[East Coast Worm Meeting, 2004]

SID-1 was identified in a genetic screen for mutants capable of cell autonomous RNAi but deficient for systemic RNAi (Winston et al., 2002). The systemic RNAi defect is likely due to the inability of cells lacking SID-1 to import double-stranded RNA (dsRNA) from neighboring cells. The nature of SID-1 activity was elucidated using a heterologous system whereby either wild-type SID-1 or, as a negative control, a missense mutant form of SID-1 was transiently expressed in Drosophila S2 cells (Feinberg & Hunter 2003). These investigations showed that SID-1 enables efficient RNAi by adding dsRNA to the media of transfected S2 cells (soaking RNAi) and uptake of labeled dsRNA into cells. Furthermore, long dsRNA was shown to be more effective than short dsRNA for SID-1 mediated soaking RNAi in S2 cells and for systemic RNAi in C. elegans . We are investigating the length dependence of silencing as well as substrate specificity of the SID-1 channel. We have shown that SID-1 is extremely efficient, enabling soaking RNAi with less than one molecule of dsRNA per transfected cell and will present evidence that transport is extremely rapid. We will also report the results of ongoing analyses of length-dependent transport and channel selectivity, which may have implications for the use of SID-1 as a tool for molecular biology. W. M. Winston, C. Molodowitch, C. P. Hunter, Science 295 , 2456-59 (2002). Systemic RNAi in C. elegans requires the putative transmembrane protein SID-1. E. H. Feinberg, C. P. Hunter, Science 301 , 1545-7 (2003). Transport of dsRNA into cells by the transmembrane protein SID-1.

Julia M Claggett et al. (2004) East Coast Worm Meeting "An Investigation into the potential regulatory relationships between components of a network specified by pal-1."

L Ryan Baugh, Julia M Claggett, Craig P Hunter

[East Coast Worm Meeting, 2004]

PAL-1 protein, contributed both maternally and zygotically, is necessary and sufficient to specify and maintain the C blastomere lineage in the C. elegans embryo (Hunter and Kenyon, 1996). A number of this master regulator's targets were identified by microarrays comparing the transcript abundance in wild-type and mutant embryos either lacking or containing extra C blastomeres. Furthermore, we collected these embryos at defined time points, thus additionally providing temporal information. Target genes could then be separated by their transcriptional initiation into four consecutive temporal phases defined by a singular cell cycle beginning with the 2C-cell stage (Baugh et al, 2003). Using reporter YFP constructs for thirteen of the targets and a volume-rendering program, the 3D spatial expression pattern of each target gene was established. On the basis of this spatial information and knowledge of the temporal phase to which each target belongs, we have proposed a set of regulatory relationships between the components. We are currently testing these hypotheses by disrupting potential (capital O, grave accent)upstream(capital O, acute accent) regulators via RNAi and/or mutation and either observing the effect on individual (capital O, grave accent)downstream(capital O, acute accent) reporters or analyzing the effect on transcript abundance using QPCR. We hope that such measurements will give us insight into how the genes within the pal-1 network regulate each other in order to establish and maintain the various cell fates within the C blastomere lineage. Hunter, C.P. and Kenyon, C. (1996). Spatial and temporal controls target pal-1 blastomere-specification activity to a single blastomere lineage in C. elegans embryos. Cell 87, 217-26. Baugh, L.R., Hill, A.A., Slonim, D.K., Brown, E.L. and Hunter, C.P. (2003). Composition and dynamics of the Caenorhabditis elegans early embryonic transcriptome. Development 130, 889-900.

Rahman, Mizanur et al. (2021) International Worm Meeting "A microfluidics-based chemical screening platform for lifespan and healthspan extension in Caenorhabditis elegans"

Gupta, Siddhartha, Rahman, Mizanur, Szewczyk, Nathaniel, Vanapalli, Siva, Anupom, Taslim, Driscoll, Monica, Edwards, Hunter

[International Worm Meeting, 2021]

C. elegans is a premier model organism used to identify pharmacological interventions that enhance lifespan and healthspan. Generally, animals are cultured on agar surface where drugs are either dissolved in agar or spread over solidified agar. The effects of the drug on lifespan are measured by the change of median or mean lifespan. The agar-based screening method has some severe limitations, including (i) limited throughput, (ii) progeny blocking methods can complicate outcomes, (iii) drug molecules may not be accessible to the animal due to transport limitation, (iv) bacteria can metabolize drug molecules, (vi) animals may avoid the bacterial lawn and therefore minimize drug exposure, and (vii) animals may be lost from the assay due to desiccation or burrowing. We present a microfluidic technology named an "Infinity screening system," where animals are cultured in a confined, micro-structured liquid environment with an integrated fluid processing and imaging hardware. Food and drugs are prepared in a batch (no FuDR) for the entire lifespan experiment for precise control over the consistency of daily dose of food and drug quality. Images of animals moving in the chip pillar environment are acquired each day and processed for live/dead count and locomotion-based cohorts with a software built in-house. This study used seven well-studied anti-aging drugs to benchmark the infinity platform for the drug effectiveness and consumption for whole-life analysis. We used three concentrations for each drug, concentration identified in plate-based assays, 1/10th, and 1/100th of the plate-based dosage. The entire experiments require approximately 3 man-hr/day and 30 days to complete one biological replicates. We found that infinity chip requires approximately 7.5 -750 times less drugs to achieve similar effects to that of agar plates. In this study, we found Thioflavin T as toxic in the microfluidic environment at the highest concentration. The actual dependency of drug type on the effective concentration is still unclear. In general, we observed significant increase in mean lifespan without appreciable changes in maximum lifespan at an effective concentration. Moreover, alpha-Ketoglutarate was able to rescue plate like lifespan enhancement in infinity chip contrasting the lifespan outcome from Lifespan Machine. Although, all the drugs extend lifespan, only a few of them helped maintain a larger fraction of animal highly active in the older age.

Tawe WN et al. (1997) Worm Breeder's Gazette "Paraquat mediates differential gene expression in C. elegans"

Henkle-Duehrsen K, Tawe WN, Eschbach M-L, Walter RD

[Worm Breeder's Gazette, 1997]

In order to identify genes that are differentially expressed as a consequence of stress due to paraquat, we used the differential display technique (1) to compare mRNA expression patterns in Caenorhabditis elegans. A C. elegans mixed-stage worm population, as well as a homogeneous larval population were treated with 100 mM paraquat, parallel to controls. Over 50 mRNA species that are potentially up-regulated in response to paraquat were identified. Sixteen of these candidates were re-amplified, ligated into plasmid vectors and the nucleotide sequences were determined. The induction of four of these expressed sequence tags (ESTs) designated L1, M47, M96 and M132 were confirmed in two independent stress / differential display experiments, as well as by northern blot analysis with RNA from stressed and unstressed worms. The nucleotide sequences of the independently isolated L1 and M47 ESTs were found to be identical. Their corresponding mRNA level increased more than 40-fold in the larval stage, and to a lesser extent in the mixed-stage worm population, in response to paraquat. Induction levels of 3 - 5 fold were observed for the M132 and M96 ESTs, in both larval and mixed populations. All of the isolated ESTs showed homology to portions of the C. elegans cosmids. The L1/M47 EST is derived from a gene encoding one of the putative C. elegans glutathione S-transferases. The paraquat-inducible M132 EST was also identified, and encodes a putative C2H2 - type zinc finger protein, possessing an N-terminal leucine zipper. However, the M96 EST appears to be a novel gene whose product has not yet been identified. Searches of other eukaryotic nucleotide sequence databases did not reveal any significant homologies to known sequences from any organism. Since paraquat is known to generate superoxide radicals in vivo, and the cellular superoxide dismutase (SOD) enzyme complex is responsible for the quenching of the deleterious effects of these radicals, the response of the C. elegans superoxide dismutases (2,3,4) to paraquat was also investigated in this study. Northern blot experiments demonstrated that mRNA steady state levels of the C. elegans manganese type and the copper/zinc type superoxide dismutases increased two-fold in response to paraquat, in the larval population. In contrast, mixed-stage populations did not show any apparent increase in the levels of these SOD mRNAs in response to paraquat. References: 1. Liang, P., Pardee, A.B. (1992). Science 257: 967 - 971 2. Giglio, M-P., Hunter, T., Bannister, J.V., Bannister, W.H., Hunter, G.J. (1994). Biochem. Mol. Biol. Int. 33 (1): 37-40 3. Giglio, A. M., Hunter, T., Bannister, J.V., Bannister, W.H., Hunter, G.J. (1994) . Biochem. Mol. Biol. Int. 33 (1): 37-40 4. Larsen, P.L. (1993). Proc. Natl. Acad. Sci. USA, 90: 8905-8909

Rahman, Mizanur et al. (2017) International Worm Meeting "Microfluidics-based evaluation of neuromuscular healthspan in C.elegans."

Blawzdziewicz, Jerzy, Van-Bussel, Frank, Vanapalli, Siva, Szewczyk, Nathaniel, Edwards, Hunter, Rahman, Mizanur, Driscoll, Monica

[International Worm Meeting, 2017]

Aging studies in C.elegans based on functional metrics and molecular markers are essential to identify genetic and environmental determinants of healthspan. Although functional measures based on locomotion, pharyngeal pumping and oxidative stress have been used to interrogate healthspan of C.elegans, our understanding on maintenance and deterioration of the neuromuscular system with age is far from complete. Here, we propose muscle strength and stimulated reversals as two novel functional measures to report on the neuromuscular health of C.elegans. These measures are possible due to two technological advances made in our laboratory: NemaFlex - an image-based force-sensing micropillars that can quantify muscle strength and NemaLife - a microfluidics-based culturing apparatus that enables longitudinal aging experiments without the need for drug-induced blocking of progeny. We profiled strength across the lifespan of a wild-type population, and our results show that strength increases 5-fold from young adult to mid-life followed by a sharp decline - providing the first direct evidence of muscle strength loss due to age in C. elegans. With respect to stimulated reversals, we find that worms maintain a high reversal speed during their reproductive period and then declines at a rate that is nearly identical to the animal mortality rate. Interestingly, the decline in reversal speed precedes the onset of strength decline by about 3 days indicating that reversal speed is an early predictor of mortality. Moreover, since reversal speed is an indicator of motor activity, the early decline in reversal speed suggests that the presynaptic loss of function occurs early in life, consistent with prior electrophysiological studies. The stochastic nature of aging produces a wide distribution of lifespans in isogenic C. elegans populations. We observed that long lived worms generally maintain muscle strength long after median lifespan although their motor activity significantly declines. We observed four major classes of strength profiles among individual worms, each of which has a unique healthspan and motor activity pattern. For example, individuals starting with high reversal speed do not necessarily have long lifespan but all strong and long lived worms maintain very high motor activity during the late reproductive period. In summary, maintenance of strength together with motor activity is a possible route for increasing locomotory healthspan.

Michael A Beer (2005) International Worm Meeting "Systematic genome-wide identification of transcriptional cis-regulatory logic in C. elegans."

Michael A Beer

[International Worm Meeting, 2005]

We have developed a systematic approach for inferring cis-regulatory logic from whole-genome microarray expression data.[1] This approach identifies local DNA sequence elements and the combinatorial and positional constraints that determine their context-dependent role in transcriptional regulation. We use a Bayesian probabilistic framework that relates general DNA sequence features to mRNA expression patterns. By breaking the expression data into training and test sets of genes, we are able to evaluate the predictive accuracy of our inferred Bayesian network. Applied to S. cerevisiae, our inferred combinatorial regulatory rules correctly predict expression patterns for most of the genes. Applied to microarray data from C. elegans[2], we identify novel regulatory elements and combinatorial rules that control the phased temporal expression of transcription factors, histones, and germline specific genes during embryonic and larval development. While many of the DNA elements we find in S. cerevisiae are known transcription factor binding sites, the vast majority of the DNA elements we find in C. elegans and the inferred regulatory rules are novel, and provide focused mechanistic hypotheses for experimental validation. Successful DNA element detection is a limiting factor in our ability to infer predictive combinatorial rules, and the larger regulatory regions in C. elegans make this more challenging than in yeast. Here we extend our previous algorithm to explicitly use conservation of regulatory regions in C. briggsae to focus the search for DNA elements. In addition, we expand the range of regulatory programs we identify by applying to more diverse microarray datasets.[3] 1. Beer MA and Tavazoie S. Cell 117, 185-198 (2004). 2. Baugh LR, Hill AA, Slonim DK, Brown EL, and Hunter, CP. Development 130, 889-900 (2003); Hill AA, Hunter CP, Tsung BT, Tucker-Kellogg G, and Brown EL. Science 290, 809812 (2000). 3. Baugh LR, Hill AA, Claggett JM, Hill-Harfe K, Wen JC, Slonim DK, Brown EL, and Hunter, CP. Development 132, 1843-1854 (2005); Murphy CT, McCarroll SA, Bargmann CI, Fraser A, Kamath RS, Ahringer J, Li H, and Kenyon C. Nature 424 277-283 (2003); Reinke V, Smith HE, Nance J, Wang J, Van Doren C, Begley R, Jones SJ, Davis EB, Scherer S, Ward S, and Kim SK. Mol Cell 6 605-616 (2000).

Joanne C Wen et al. (2003) International Worm Meeting "Functional Characterization of Early Embryonic PAL-1 Targets"

Craig P Hunter, Joanne C Wen, L Ryan Baugh

[International Worm Meeting, 2003]

The activation and maintenance of C lineage specification occurs through maternal and zygotic PAL-1 activity, respectively (Hunter & Kenyon, 1996; Edgar et al, 2001). A set of targets of this master regulatory transcription factor were identified by transcript profiling embryos with perturbed PAL-1 activity (see abstract by Baugh et al). To functionally characterize PAL-1 targets, we have used RNAi to assess the lethality and terminal phenotypes following loss of function. To identify interactions between targets, we are performing epistasis analysis both by scoring synthetic lethality and by examining the effect of RNAi against one target on the expression of reporters for other targets. Our hope is that such functional characterization of a key set of PAL-1 targets will generate the data necessary to begin modeling the PAL-1 regulatory network.

Michael C Fitzgerald et al. (2005) International Worm Meeting "SID-1 dependent intercellular RNA transport: substrates, kinetics, and mechanism"

Michael C Fitzgerald, Craig P Hunter, Joseph D Shih

[International Worm Meeting, 2005]

RNA interference (RNAi) is a phenomenon in which introduction of double stranded RNA (dsRNA) triggers gene-specific post-transcriptional gene silencing. There is tremendous potential for RNAi in therapeutic applications because of its promise to silence specific genes without adverse side affects. However efficient delivery of dsRNA into target cells and tissues is crucial for the success of such therapies. In C. elegans, silencing readily spreads between cells and tissues, a process termed systemic RNAi. However, the mechanisms behind systemic RNAi are not yet understood. To address this problem, the Hunter lab conducted a screen using a C. elegans strain that visually differentiates systemic silencing from cell autonomous RNAi. Five different systemic interference defective (sid) mutants were identified that define three different pathways for RNA transport: intercellular transport (sid-1 dependent), dsRNA uptake from the environment (sid-2 dependent), and signal export or transport from the intestine to other tissues (sid-3 dependent).Recent work by the Hunter lab with transiently transfected Drosophila S2 cells indicates that SID-1, the protein predicted to be the channel responsible for transport of the RNAi silencing signal between cells, is a nucleic acid transporter capable of mediating the entry of long double-stranded nucleic acids into cells2. This process occurs on the seconds timescale for dsRNA and does not require ATP in S2 cells. Additional studies have revealed that although silencing efficiency is strongly dependent on dsRNA length, 100bp and longer dsRNA are transported with similar kinetics. Poisoning assays using a missense mutant SID-1 reveal that functional SID-1 is most likely a homomultimer. Further studies seek to place SID-1 in a defined transporter class (ligand gated, co-transporter ion, etc.) for future patch clamp studies.