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.

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.

Lesanpezeshki, Leila et al. (2021) International Worm Meeting "Burrrowing chip: A microfluidic platform to visualize and quantitate the burrowing behavior of C. elegans"

Anupom, Taslim, Laranjeiro, Ricardo, Norouzi Darabad, Masoud, Lesanpezeshki, Leila, Driscoll, Monica, Blawzdziewicz, Jerzy, A. Vanapalli, Siva

[International Worm Meeting, 2021]

Caenorhabditis elegans has been widely used to study the genetics of behavior and neuromuscular function. Most of the behavioral studies in C. elegans are limited to two-dimensional (2D) assessment of animal locomotion on agar plates (crawling) or in liquid (swimming), and it was not until recently that C. elegans studies began to focus on 3D motion. 3D locomotion where animals burrow through a matrix is known to be physiologically distinct from 2D crawling and swimming (Bilbao et al. 2018) and 3D movement evokes different gene expression responses (Hewitt et al. 2020). We have previously deployed a Pluronic hydrogel environment to assess the burrowing performance of muscle mutants. The stiffness of the gel can be tuned to challenge the animals to burrow through a higher mechanical resistance environment to increase the assay sensitivity (Lesanpezeshki et al. 2019). However, due to the assay geometry being in a well-plate, the full burrowing trajectory was not observable, and the animals could be only visualized at the top surface, precluding evaluation of the burrowing behavior throughout the gel layers. To address the above issues, we have developed a microfluidic platform called a burrowing chip, that enables visualization and assessment of burrowing behavior in the same Pluronic hydrogel environment. The microfluidic format provides excellent control of the chemotactic gradient and animal/gel loading. The shallow depth of the channel enables use of consumer cameras for visualizing and recording burrowing behavior. We show that the device provides a 3D environment to assess the animal's burrowing performance and the animal distribution along the channel during chemotaxis. Next, we identify two burrowing classes of behaviorally slow and fast movement, based on the time it takes animals to reach the attractant. Slow burrowers weakly maintain their direction toward the attractant. We have discovered pause intervals as a novel phenotype for stimulated burrowing animals, as the burrowing animals exhibit long pauses. Finally, we show that the burrowing chip is capable of characterizing the muscle mutants based on their lower burrowing velocity and higher frequency of pauses compared to wild-type animals. We suggest that the burrowing chip can be used to provide insights into the genetic basis of burrowing behavior and to assess the neuromuscular health in C. elegans, paving the road to identify therapeutic targets for age-related diseases and decline.