Background Information Investigating the Impact of Oxidative Stress on Tetrahymena thermophile Sirtuin, THD18 Emmanuel C. Dubuisson,New York City College of Technology Ralph Alcendor PhD, Department of Biological Sciences, New York City College of Technology Results Background Information Summary of Results INSTRUCTIONS: Insert text in text boxes as indicated on the template. You can modify color, background, font, font size, etc. by using PowerPoint features noted in the tabs. You can add borders around text boxes and you can add lines or other graphics where desired. To add more text boxes, you can copy an existing one and move it to the desired location. You can add any graphic by dragging it onto the slide or by copying/pasting it. Be sure that your graphic has high dots per inch. Images/graphics must be cited if not original work. Use Sans Serif fonts for Titles, labels and section headers. Use Serif fonts for text in text boxes. CONTENT: *Introduction, Background, and/or Abstract (a place to quickly summarize your topic and trigger your audience’s interest). Usually in narrative, paragraph format. *Materials and Methods or Process (a place to describe your process and what led to your results). Using bullet points can be a helpful way to present information. *Results (the place where the results of your experiment are explained). Using bullet points can be a helpful way to present information. *Conclusions or Discussion (the place where you explain why your results are conclusive and provide the reader with a short but solid justification of your hypothesis). Usually in narrative, paragraph format. *References or Literature Cited (This is where you make a list of the literature you have cited regarding this project. List the names of authors, publications and publishing dates.) To save space, consider noting “selected references” – those references essential to the project. Citation style must follow rules designated by discipline, e.g., MLA, APA, etc. *Acknowledgments (this is where you acknowledge grants and research programs. Also, use this section to thank the people who helped with your project. Can sometimes include your Contact Information) *Depending on your type of research and where you are in your project, you may have to use some variation of the above. Sirtuins are a family of enzymes that fulfill various important biological functions. Investigators have looked for the implication of Sirtuin genes in cell signaling mechanisms, in the formation and silencing of heterochromatin, in the regulation of ion channels, and in the modulation of the cellular redox reactions[2]. Different model organisms have been previously used to conduct these studies; among them, there are yeasts, nematodes, and fruit flies. Each one has made some valuable contribution into the vast body of knowledge related to this field. However, gaps in the understanding of Sirtuins functions still remain to be filled. In this optic, the purpose of this experiment is to uncover the impact of oxidative stress on Tetrahymena thermophila Sirtuin, THD18. This protist offers several advantages. In addition, of being widespread, it is easy to culture and to reproduce in the laboratory environment, inexpensive to maintain, and have a short generation time. From a phylogenetic perspective, mammalian Sirtuins share some homology with those found in Tetrahymena. Therefore, results of scientific investigations conducted on Tetrahymena Sirtuins can have direct applications to other fields such as human medicine. At a molecular level, oxidation reactions in living organisms are catalyzed by enzymes. In human beings for example, enzymes called SIRT3, and SIRT4, which are Sirtuins, participate in oxidative reactions. While SIRT3 protects against oxidative stress by acetylating and activating the Super Oxide Dismutase 2 (SOD2), which is an enzyme found in the mitochondria, SIRT4 on the other hand has the opposite effect, and tends to facilitate the oxidation of fatty acid in the liver and muscle cells[1]. Based on this fact, we hypothesized that the level of mRNA of T. thermophila sirtuin genes will increase in response to high levels of oxidative stress. To determine the impact of oxidative stress on Tetrahymena thermophila, the cells were cultured, exposed to hydrogen peroxide, and then evaluated for their metabolic activity. mRNA was extracted from the cells using the TRizol protocol. cDNA was synthesized, amplified, and analyzed with a gel electrophoresis. The results show Hydrogen peroxide induced oxidative stress in T. thermophila (figure1). The induction of oxidative stress was concentration dependent. Hydrogen peroxide induced cell death. 2 and 4 mM showed the highest levels of cell death (figure 2). Hydrogen peroxide lead to an increase in the mRNA expression levels of THH13 and THD-190. The highest level was in the presence of 2 mM of hydrogen peroxide. At 4 mM the mRNA levels decreased. Figure 1. DCF stained cells. Cells exposed to 1 – 4 mM of hydrogen peroxide had high levels of oxidative stress. Green fluorescence indicates presence of oxidative stress. Conclusion In conclusion, induction of oxidative stress by hydrogen peroxide lead to high levels of cell death. The expression of sirtuin THD13 and THD-190 increased in the presence of hydrogen peroxide induced oxidative stress. Consistent with the hypothesis, these results suggest sirruins may be involved in oxidative stress protect and or regulation. Materials and Methods Cell culture Cells were exposed to increasing concentration of hydrogen peroxide, for 24-48 hours at 30 °C with constant shaking. mRNA Isolation mRNA was extracted using 0.75 ml of TRIzol and 0.2 ml chloroform. The aqueous later was extracted and 500 µl of propanol was added and centrifuged. The pellet was washed with 1 ml of 75% ethanol and centrifuged for 5 minutes. The dried pellet was suspended in 100 µl of nuclease free water. Concentration of RNA was determined using a NanoDrop spectrophotometer. DCF Stain DCF ( 2',7'-dichlorofluorescein) was added to 200 µl of cells and incubated for 30 min., following which cells were viewed using a fluorescent microscope. Synthesis of cDNA This process was done using High Capacity cDNA Reverse Transcription kits. 10 µl of RNA was add to 10 µl of cDNA synthesizing mixture and incubated for 10 at 25 °C, 120 min at 56 ° C and 5 min at 85 ° C. PCR Reaction Reverse and forward primers, as well as dNTP and cDNA were combined for this reaction. Two µl of the primer were mixed with 20 µl of buffer (GoTAQ master mix). Samples were incubated at 95 ° C for 3 min., 95 ° C for 30 sec., 56 ° C for 30 sec and 72 ° C for 30 sec. The cycle was repeated 28 times. Final extension was set for 72 ° C for 10 min. Future Experiments Repeat the experiments Examine the effects of hydrogen peroxide on oxidative stress genes and genes involved in cell death Figure 2. Light microscope images of cells treated with hydrogen peroxide. 2 and 4 mM of hydrogen peroxide showed high number of cell death Acknowledgement We are very grateful to the STEM program at City Tech for giving students the possibility to get involved in scientific research at an early stage of their career. We also extent our gratitude to Professor Alcendor for his mentorship, and for accompanying us along our journey as future scientists References [1] Marcus, J. M., & Andrabi, S. A. (2018). SIRT3 Regulation Under Cellular Stress: Making Sense of the Ups and Downs. Frontiers in neuroscience, 12, 799. doi:10.3389/fnins.2018.00799 [2] (2014). The controversial world of sirtuins. Drug discovery today. Technologies, 12, e9-e17. Figure 3. mRNA expression of sirtuin genes. Both sirtuins mRNA increased in the presence of hydrogen peroxide. 18S is a control gene.