Skn-1 Dependence of Cocoa Powder Lifespan Extension on Caenorhabditis elegans Batul Dhariwala Mentor: Dr. Kirsten Crossgrove University of Wisconsin – Whitewater, Department of Biology Caenorhabditis elegans Introduction Organisms such as Caenorhabditis elegans are beneficial organisms to aid in lifespan studies. They reproduce quickly, have relatively short lifespans, are easy to maintain, and have similar molecular pathways to humans and other organisms.  Previous research has shown that cocoa powder extends the lifespan of C. elegans; to counteract the cocoa powder effects, we are using RNA interference to disable the skn-1 gene which is necessary for lifespan extension. We are using the gene, skn-1 which has been known to extend the lifespan of C. elegans due to its resistance to stress. The purpose of this study is to test whether the skn-1gene is necessary for lifespan extension via cocoa powder by activating the skn-1 stress response pathway. We will be looking at how long the organism is alive and how many days it is active. Literature Review Olsen, A., Vantipalli, M.C. and Lithgow, G.J. (2006), Using Caenorhabditis elegans as a Model for Aging and Age-Related Disease. Annals of the New York Academy of Sciences, 1067: 120-128. doi:10.1196/annals.1354.015 Mutations in the age-1 and daf-2 genes have shown to cause a increase in nematode lifespan and stress resistance. Curtis, R., O’Connor, G. and DiStefano, P.s. (2006), Aging Networks in Caenorhabditis elegans: AMP-Activated Protein Kinase (aak-2) Links Multiple Aging and Metabolism Pathways. Aging cell, 5: 119-126. doi:10.1111/j.1474-9726.2006.00205.x It was learned that genetic and metabolic control can be manipulated to extend lifespan and vitality of an organism; specifically by limiting caloric intake. Cabreiro, Fillipe et al. Metformin Retards Aging in C. elegans by Altering Microbial Folate and Methionine Metabolism. Cell, Volume 153, Issue 1,228-239. doi: 10.1016/j.cell.2013.02.035 Metformin at 25,50, and 100 mM increased C. elegans mean lifespan by 18%, 36%, and 3%. Collins, J.J., et al. The measurement and analysis of age-related changes in Caenorhabditis elegans (December 7,2007), WormBook, ed. The C. elegans Research Community, WormBook, doi/10.1895/wormbook.1.137.1,http://www.wormbook.org. 40% of known genes are known to be associated with human genes have clear orthologs in C. elegan genome. Age Related changes in tissue morphology and function are controlled by common mechanisms and/or casually linked in series. Martorell, P., Forment, J. V., de Llanos, R., Monton, F., Llopis, S., Gonzalez, N., … Ramon, D. (2011). Use of Saccharomyces cerevisiae and Caenorhabditis elegans as Model Organisms To Study the Effect of Cocoa Polyphenols in the Resistance to Oxidative Stress. Journal of Agricultural and Food Chemistry, 59(5), 2077–2085. Worms fed with cocoa powder were able to resist the oxidative stressor hydrogen peroxide. Cocoa powder analysed on a genetic level has many beneficial effects by using C. elegans mutant strains; polyphenol-enriched cocoa powder does not protect sir-2.1 and daf-16 mutants against oxidative stress. Figure 4: Overexpression of skin-1 extends the lifespan of c. elegans because they have more resistance to stress. Source: www.semnaticscholar.org Figure 3: SKN-1 C. elegan life cycle Source: www.semnaticscholar.org Research Question & Hypothesis Research Question: Is lifespan extension caused by cocoa powder skn-1 dependent? Hypothesis: We hypothesize cocoa powder extends C. elegans lifespan by activating skn-1. Figure 5: The view of C. elegans on a agar plate through a microscope Source: http://ericmiskalab.org/ SKN-1 SKiNhead is a protein that encodes bZip to Nrf (Nuclear factor-erythroid-related factor) transcription factors. SKN-1 works with the p38 MAPK pathway to administer oxidative stress response conjointly with DAF-16/FOXO signaling pathway that regulates the adult worms lifespan (wormbase). Anticipated Results We anticipate a difference in lifespan in the cocoa powder plates that contain skn-1 and dsRNAi. We are expecting either a neutral effect if the cocoa powder over powers the skn-1 gene; or a decreased lifespan because the RNAi has an increased effect in stopping the skn-1 gene from stress resistance. Methods The experimental plates that were made consisted of 10 regular RNAi, 10 regular control, 10 Cocoa RNAi, and 10 Cocoa Control plates were labeled using a number code to reduce bias. The strain fem-1 (BA-17) C. elegans were used because they do not lay any eggs. This is beneficial so the worms do not have to be transferred to fresh plates often. A synchronized egg population was obtained by egg prep. By adding a NaOH and bleach solution we were able to kill off any adult larvae and keep the eggs. A M9 (3 g KH2PO4, 6 g Na2HPO4, 5 g NaCl, add H2O to 1 liter, 1 M MgSO4) buffer was added to the solution to wash the eggs and remove excess NaOH and bleach. The larvae were then transferred to agar plates placed at 25°C to allow for reproduction After 3 days, the larvae were transferred onto the experimental plates. No more than 10-12 larvae a plate. The following day the organisms were counted. If the organisms did not move, they were scored dead. The worms were then transferred onto new experimental plates and accounted for each day until organisms were all dead and immobile when prodded. Figure 1: Because SKN-1 is stress resistant, it is able to ensure a longer life and in turn more growth for C. elegans Source:http://mcb.asm.org/content/33/17/3524/F8.expansion.html Future Plans With the data we are collecting, we will be computing the results into SPSS and creating a log rank function to make a survival curve. We will have a comparison of the different conditions; control dsRNAi, cocoa powder control dsRNAi, control plate skn-1 and dsRNAI, and cocoa powder skn-1 and dsRNAi. double stranded RNA interference dsRNAi is transferred to the worms via bacteria; their food source. dsRNA is expressed in the L4440 plasmid in the bacteria. RNAi is known to stop the expression of skn-1 in C. elegans, in turn shortening their lifespan. Figure 2: RNAi is known to interfere with skn-1, causing a loss in stress resistance.. Image Source: https://openi.nlm.nih.gov/imgs/512/355/17598/PMC17598_gb-2000-2-1-research0002-1.png Figure 6: Previously computed by Kristin Hauen. A survival curve is shown for each of the three lifespan experiments conducted (control, cocoa powder 2g/L, cocoa powder 4g/L). Acknowledgements University of Wisconsin-Whitewater,Biology Department Dr. Kirsten Crossgrove (Mentor) Whitney Supianoski (Director-McNair Scholars Program) Austin Chriske