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| + | == Abstract == |
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| − | ''' The Effects of GlyNAC on the Regeneration Rate and Movement of Dugesia tigrina'''</div>
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| − | ==Abstract== | + | == Document == |
| − | | + | <pdf>Media:Review_337222142936-6874-document.pdf</pdf> |
| − | This study investigates the effects of GlyNAC—a combination of glycine and N-acetylcysteine—on tissue regeneration in Dugesia tigrina (planarians). Aging is associated with physiological decline, including reduced tissue repair and increased oxidative stress. GlyNAC has emerged as a promising anti-aging supplement due to its ability to elevate intracellular glutathione levels, a critical antioxidant that mitigates oxidative damage and supports cellular health. Planarians are used as a model organism in this study due to their robust regenerative capabilities, presence of a central nervous system, and endogenous glutathione production, which parallel key human physiological processes. The experimental design includes one control group fed standard ground beef and three treatment groups fed ground beef supplemented with varying concentrations of GlyNAC. Regenerative capacity will be assessed by measuring the length of planarian regrowth post-amputation. Additionally, motility will be evaluated by recording planarian movement over a 30-minute interval using grid-lined Petri dishes. This study aims to determine whether GlyNAC supplementation enhances tissue regeneration and movement in planarians. Future research should expand the sample size and explore molecular assays to validate findings at a biochemical level.
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| − | ==Introduction==
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| − | ''GlyNAC''
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| − | In recent years, interest in anti-aging research has grown significantly due to one’s fear of aging and the many symptoms that come with it, such as muscle fatigue, inflammation, a decrease in tissue regeneration, and oxidative damage[1]. Oxidative stress occurs when there's an imbalance between the production of reactive oxygen species (ROS) and the body's ability to detoxify them [2]. Excessive oxidative stress leads to aging and cell death, while low oxidative stresssupports cell regeneration [3].
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| − | There is limited research on why older people face these symptoms and the lack of effective treatments to treat these symptoms. Despite this, many try to preserve their health and prevent these symptoms from happening through various means such as dieting, supplements, exercise, and antioxidants. Previous experiments have shown that both glycine and N-acetylcysteine, which are antioxidants that can protect against oxidative stress [4]. These components are precursors to glutathione(GSH), an important antioxidant that protects against oxidative stress, increased energy, cell proliferation, etc. Therefore, GlyNAC is expected to boost glutathione production by combining Glycine and N-acetylcysteine [1].
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| − | As one ages, there is a deficiency of GSH due to the lack of glycine and cysteine needed to synthesize the GSH. With this deficiency of GSH, fewer antioxidant defenses are protecting against oxidative stress that can damage cells that promote a healthy body. Therefore, GlyNAC demonstrates that it has the potential and ability to benefit those who are aging.
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| − | ''Planarian''
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| − | Planaria, specifically Dugesia tigrina, are flatworms known for their remarkable tissue regeneration abilities and nervous system. This renders them an ideal model for studying tissue regeneration and neurobiological processes, which helps provide insights into human tissue regeneration and the nervous system since both planaria and humans use stem cells for this process and have similar nervous systems. Planaria are famously used to research tissue regeneration, toxicology, and other pharmacology studies. Planarians can regenerate their tissue by using stem cells that are unspecialized cells that can develop into various types of cells, such as skin and nerve cells [5]. Additionally, planaria have glutathione in their redox system and glutathione is an essential component in balancing the redox system [6]. With this planaria can create glutathione in balancing their redox system.
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| − | ==Materials and Method:==
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| − | ''Preparation of GlyNAC treatment''
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| − | Glycine and N-acetylcysteine were purchased on Amazon (Nutricost). Glycine and N-acetylcysteine were mixed with a 1:1 ratio to make GlyNAC. The GlyNAC was mixed into the ground beef with their desired mass concentrations 0%, 1%, 5%, and 10% (Table 1.)
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| − | '''Table 1. The concentration of GlyNAC in Ground Beef.'''
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| − | {| style="width: 100%;border-collapse: collapse;"
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| − | | style="border: 1pt solid black;"|Group
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| − | | style="border: 1pt solid black;"|Percentage of GlyNAC in Beef (g/g)
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| − | | style="border: 1pt solid black;"|Amount of GlyNAC used (g)
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| − | | style="border: 1pt solid black;"|Amount of Glycine (g)
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| − | | style="border: 1pt solid black;"|Amount of N-acetylcysteine(g)
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| − | | style="border: 1pt solid black;"|Amount of
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| − | Ground beef (g)
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| − | | style="border: 1pt solid black;"|Number of planaria
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| − | |-
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| − | | style="border: 1pt solid black;"|A
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| − | | style="border: 1pt solid black;"|0
| + | |
| − | | style="border: 1pt solid black;"|0
| + | |
| − | | style="border: 1pt solid black;"|0
| + | |
| − | | style="border: 1pt solid black;"|0
| + | |
| − | | style="border: 1pt solid black;"|20
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| − | | style="border: 1pt solid black;"|10
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| − | |-
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| − | | style="border: 1pt solid black;"|B
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| − | | style="border: 1pt solid black;"|1%
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| − | | style="border: 1pt solid black;"|0.2
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| − | | style="border: 1pt solid black;"|0.1
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| − | | style="border: 1pt solid black;"|0.1
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| − | | style="border: 1pt solid black;"|19.8
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| − | | style="border: 1pt solid black;"|10
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| − | |-
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| − | | style="border: 1pt solid black;"|C
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| − | | style="border: 1pt solid black;"|5%
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| − | | style="border: 1pt solid black;"|1.0
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| − | | style="border: 1pt solid black;"|0.5
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| − | | style="border: 1pt solid black;"|0.5
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| − | | style="border: 1pt solid black;"|19
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| − | | style="border: 1pt solid black;"|10
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| − | |-
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| − | | style="border: 1pt solid black;"|D
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| − | | style="border: 1pt solid black;"|10%
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| − | | style="border: 1pt solid black;"|2.0
| + | |
| − | | style="border: 1pt solid black;"|1.0
| + | |
| − | | style="border: 1pt solid black;"|1.0
| + | |
| − | | style="border: 1pt solid black;"|18
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| − | | style="border: 1pt solid black;"|10
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| − | |}
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| − | ''Preparation of Planarians''
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| − | 40 planarians have been ordered from the website, Carolina.com, and the catalog number is 132954. The planarians were kept in loosely sealed jars of Poland spring water. They were then separated into 4 groups of 10. Which were each used for different concentrations of GlyNAC. Afterwards, the planarian jars were stored in the cabinet with a gap. The planarians were fed ground beef for a week.
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| − | ''Treating Planarian''
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| − | Before the planarians are used, they must be nurtured. When working with them, safety equipment was worn and materials used in the experiment, as well as the workspace, was disinfected beforehand. The groups were treated with 20g of correspondent food according to Table 1. This was done a maximum of 3 times a week. After feeding, leftover beef was removed from the container using a pipette. Additionally, dirtied water was replaced with clean Poland spring water using a pipette, avoiding taking any planarians along with the dirtied water. The planarians were treated for 7 days before beginning the experiment.
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| − | ''Mobility Measurement''
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| − | Before adding the treatment, the behaviors of planarians were measured. To do this, safety gear was worn, and all materials, gear, and workspace was disinfected. Next, 30 mL of Poland spring water was poured into 4 petri dishes. Then, the planarians were put into the petri dishes based on their respective groups. Afterwards, graph paper (Figure 1) was placed below the petri dishes, and a camera was used to record the planarian groups for 30 minutes.
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| − | '''Figure 1. Experimental setup for behaviors and movement'''
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| − | <div class="center" style="width: auto; margin-left: auto; margin-right: auto;"> | + | |
| − | [[Image:Review_337222142936-image9.png|246px|alt='''Planarain Cutting'''|''Planarain Cutting'']] </div>
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| − | ''Planarain Cutting''
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| − | Before cutting the planarians, the scalpel was disinfected, which was done by wetting a paper towel with ethanol. Next, group A planarians were put onto ice for one minute and all the planarians were cut individually with the disinfected scalpel before the pharynx. The scalpel was disinfected every 3 cuts and repeated the process for groups B, C, and D. Then, a small jar was used and labeled as “AH” and another jar was labeled as “AT”. The tails of group A planarian were put into the “AT” jar and the heads of group A planarian were put into the “AH”. This process was repeated for the other groups but the letter of the labels was changed to represent the appropriate group. After that, the planarians were measured for the average mass of the planaria groups, AH, BH, CH, DH, AT, BT, CT, and DT to record the measurements.
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| − | ''Tissue regeneration''
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| − | Every monday, wednesday, and friday, the planarians’ eyespot and length would be recorded and observed. The amputated planarian tail groups were observed under a microscope to see the time when the planarians eye spot appeared; this process was repeated for other groups. Then, the planarians’ lengths were measured in millimeters and the lengths were recorded down in an excel sheet of all planarians in group A and then the process was repeated for other groups. After the trials, the planarians were given to the supervisor to be autoclaved.
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| − | ''Data analysis''
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| − | The planarian motility was collected by observing the number of line tiles crossed by each planarian under a grid paper and was recorded down on an excel sheet before and after the treatment for 2 weeks. The means and Standard Deviation were done by excel standard functions. Then created into a line graph.
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| − | The planarian regeneration will be done by comparing how fast the eye spots grow between the tails groups. The length was measured between both the heads and tails. The means and Standard Deviation were done by excel standard functions. With this bar graphs were made to further understand the tissue regeneration of the planarian.
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| − | ''Results''
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| − | ''Results''
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| − | | style="border: 1pt solid black;vertical-align: top;"|'' [[Image:Review_337222142936-image6-c.png|600px]] ''
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| − | Figure 2. The Effect of GlyNAC on Planarians Movement-Trial 1. Each line represents the different concentrations of GlyNAC (A: 0% of GlyNAC, B: 1% of GlyNAC, C: 5% of GlyNAC, and D: 10% of GlyNAC ). There were 10 planarians in each group. The control group A had a decreased grid lines crossed. Group B initially increased but decreased after day 1. The higher concentrations, group C and D had an increasing amount of grid lines crossed. There were no significant differences.
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| − | | style="border: 1pt solid black;vertical-align: top;"|'' [[Image:Review_337222142936-image1-c.png|600px]] ''
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| − | Figure 3. The Effect of GlyNAC on Planarian’s Tail Regeneration-Trial 1. Each bar represents the planarian’s tail length difference from day 1 to day 11 in their respective treatment. There were 10 planarians in each group. The standard deviation was not significantly different. The trend of this graph shows that GlyNAC groups had delayed regeneration.
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| − | | style="border: 1pt solid black;vertical-align: top;"|'' [[Image:Review_337222142936-image7-c.png|600px]] ''
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| − | Figure 4. The Effect of GlyNAC on Planarian’s Head Regeneration-Trial 1. Each bar represents the planarian’s head length difference from day 1 to day 11 in their respective treatment. There were 10 planarians in each group. The standard deviation was not significantly different. The trend of this graph shows that the highest concentration of GlyNAC had a higher regeneration rate difference to the control group.
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| − | | style="border: 1pt solid black;vertical-align: top;"|'' [[Image:Review_337222142936-image3-c.png|600px]] ''
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| − | Figure 5. The Effects of GlyNAC on Planarian’s Eyespot Regeneration - Trial 1. Each line represents the different concentrations of GlyNAC. There were 10 planarians in each group. The data was not significantly different. The trend of this graph reveals that group C and B had a faster rate of regenerating eyespots in group A.
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| − | | style="border: 1pt solid black;vertical-align: top;"|'' [[Image:Review_337222142936-image2-c.png|600px]] ''
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| − | Figure 6. The Effect of GlyNAC on Planarians Movement-Trial 2. Each line represents the different concentrations of GlyNAC. There were 10 planarians in each group. The trend of this data indicates that group D had a higher rate of movement than all the groups. The remaining groups had minimal effects on movement. There were no significant differences.
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| − | Figure 7. The Effects of GlyNAC on Planarian’s Eyespot Regeneration - Trial 2. Each line represents the different concentrations of GlyNAC. There were 10 planarians in each group. The data was not significantly different. The trend of this graph reveals that group D has a higher rate of regeneration among all the groups.
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| − | Figure 8. The Effect of GlyNAC on Planarian’s Tail Regeneration - Trial 2. Each line represents the growth of the planarian’s head length in their respective treatments. There were 10 planarians in each group. The error bars were not displayed for it not being significant. The trend shows that there is a positive effect of GlyNAC on planarian’s tail regeneration but there is no statistical significance.
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| − | Figure 9. The Effect of GlyNAC on Planarians Movement-Trial 2. Each line represents the relationship between the days and the number of gridlines crossed per planarian in their respective treatment. There were 10 planarians in each group. The error bars were not displayed for it not being significant. The trend shows that there is minimal positive effect of GlyNAC on planarian’s movement, but there is no statistical significance.
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| − | ''Discussion/Conclusion''
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| − | The data reveals that the GlyNAC had slightly increased the regeneration of eyespots and length. This supports the hypothesis that GlyNAC has a positive effect on the regeneration of a planarian. The implications is that GlyNAC will have a positive effect on tissue regeneration of humans. This is because the increase in tissue regeneration of planarians could give us a good understanding of how GlyNAC will affect the tissue regeneration of humans due to their similarity.
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| − | Several limitations within this experiment could have introduced potential sources of error. During the data collection, the researchers were manually counting the amount of lines crossed by the planarians; this can lead to human error and time consuming. This issue was solved by having the video recording be shortened. This allows for the researchers to be more time efficient. On the contrary, this did not solve the problem of human error. Another limitation is holidays, where data can not be collected. This can make comparing data from different trials hard. Trials were also not long enough to see the full extent of GlyNAC’s impact. Due to the time constraints this was not resolved.
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| − | ''Future Research''
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| − | GlyNAC supplementation demonstrates the greatest efficacy in older populations due to age-associated glutathione deficiencies. Future research should use a thermal camera or an artificial intelligence to track the movements of the planarian better, because when counting it manually it can be more inaccurate. Additionally, future research should focus on a better way of treating the planarians. Such as soaking the planarian in a GlyNAC solution. Lastly, future researchers should extend the time giving planarians GlyNAC and increase the sample size.
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| − | Acknowledgements
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| − | Dr. D. Marmor, Mrs. N. Barbar, Dr. L. Wang, Ms. Zhu, Mrs. Macleod, Dr. J. Cohen, Dr. S. Lin, Mr. Z. Liang, Ms. DePietro
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| − | References:
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| − | [1] Kumar, P., Liu, C., Hsu, J. W., Chacko, S., Minard, C., Jahoor, F., & Sekhar, R. V. (2021). Glycine and N‐acetylcysteine (GlyNAC) supplementation in older adults improves glutathione deficiency, oxidative stress, mitochondrial dysfunction, inflammation, insulin resistance, endothelial dysfunction, genotoxicity, muscle strength, and cognition: results of a pilot clinical trial. Clinical and translational medicine, 11(3), e372.
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| − | [2] Pizzino, G., Irrera, N., Cucinotta, M., Pallio, G., Mannino, F., Arcoraci, V., ... & Bitto, A. (2017). Oxidative stress: harms and benefits for human health. Oxidative medicine and cellular longevity, 2017.
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| − | [3] Lee, J., Cho, Y. S., Jung, H., & Choi, I. (2018). Pharmacological regulation of oxidative stress in stem cells. Oxidative Medicine and Cellular Longevity, 2018.
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| − | [4] Pawelec, Graham, David Goldeck, and Evelyna Derhovanessian. "Inflammation, aging, and chronic disease." Current opinion in immunology 29 (2014): 23-28.
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| − | [5] Reddien, P. W. (2018). The cellular and molecular basis for planarian regeneration. Cell, 175(2), 327-345.
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| − | [6] Bijnens, K., Jaenen, V., Wouters, A., Leynen, N., Pirotte, N., Artois, T., & Smeets, K. (2021). A spatiotemporal characterization of redox molecules in planarians, with a focus on the role of glutathione during regeneration. Biomolecules, 11(5), 714.
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