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Welcome to STEM Class

STEM is a class designed to help students with their year long STEM project and to help students learn about the whole process of designing and testing their project thoroughly. This is a very innovative class with lots of help from peers and the teacher. The many presentations and posters made help students learn how to formulate their ideas and describe hard topics to an audience.

A Study Utilizing a Sanfilippo Drosophila model to Investigate the Impact of Isoflavones on Motor Control

Sanfilippo Syndrome is a rare genetic condition that causes learning and motor skills loss, much like Alzheimer’s. Children experience a buildup of sugar in their bodies which destroys their cells. This buildup can be prevented with isoflavones, a chemical found in soybeans.

Abstract

GraphicAbstract

Sanfilippo Syndrome is a progressive genetic disorder classified as a lysosomal storage disorder. People with this condition have little control over their motor skills and children lose the ability to walk and feed themselves over time due to a buildup of glycosaminoglycans (GAG) in their bodies. A Drosophila melanogaster model that has an SGSH mutation, a mutation that exhibits Sanfilippo Syndrome in flies, can help identify which medications can treat those symptoms. The overall aim of this project hypothesized that the motor control of a person with this condition would improve after being treated with a dose of isoflavones such as genistein, daidzein, and glycitein. SGSH flies carried out two assays, a feeding test to measure their feeding abilities and a negative geotaxis test that measured their long-distance climbing abilities. The locomotion test was carried out in a tube of height 17.5 cm, and after a period of one minute, the height reached was recorded. A statistical analysis using a paired t-test was carried out (p-value being 0.05). The mutated flies treated with isoflavones had a statistically significant difference in their climbing ability compared to untreated mutated flies with an average height of 13.4 cm. This meant that medicated SGSH flies climbed to a height similar to the average height of regular wild-type flies which was around 12.7 cm. The difference that occurred with the use of isoflavones in flies means that in the near future, a child with Sanfilippo Syndrome can be prevented from losing their motor skills. Keywords: Sanfilippo Syndrome, isoflavones, Drosophila, motor control, climbing assay.

Project Proposal

To learn more about my project proposal, click here:STEM PAGE 2  

Literature Review

To learn more about my literature review, click here:STEM PAGE 2  

Research Question

Can various isoflavones that affect the decrease of GAG improve motor control? Particular types of soy extract may cause the quantity of GAG to decrease in Drosophila melanogaster because soy isoflavones, like genistein, have the ability to inhibit the production of tyrosine kinase, a protein that is needed for GAG synthesis.

Hypothesis

If a Sanfilippo Syndrome model is given isoflavones as a medication, then their motor skills should improve because isoflavones have the capability to decrease the synthesis glycosaminoglycans in the brain.

Background: Sanfilippo Syndrome

Sanfilippo Syndrome, also called MPS 3, is an autosomal recessive inherited lysosomal storage disorder that causes progressive neurodegeneration affecting the central nervous system. Children can suffer from sleep disturbance, hyper activeness, aggression problems, impulsiveness, and may present several signs of Autism as well (Fraser, 2005). At the end of their lives, children are unable to walk, talk, or feed themselves, eventually resulting in a vegetative state (Agrawal, 2012). Lysosomal storage disorders are caused by the mutation of lysosomal enzymes that are needed to break down glycosaminoglycans (GAGs), sugars composed of uronic acid and sulfated amino (Casale, 2021). There are four different types of MPS disorders, each being caused by a different missing enzyme; Type A is known as being the most common as well as the most severe caused by a missing enzyme called heparan sulfate sulfatase. Without the enzyme, the sugar proceeds to fill the lysosomal chamber, preventing the cell from carrying out the needed function and eventually killing the cell (Zelei, 2018). Children can be diagnosed with this syndrome by testing the amount of GAG present in urine and comparing it to normal levels of GAG. Despite diagnostic tests being available at hospitals, there are no established therapies, medicines, or cures for this disorder. Previous trials that have focused on finding therapies to cure this syndrome have been about enzyme replacement therapy as well as gene replacement therapy (Agrawal, 2012).

Background: Isoflavones

Substrate reduction therapy is focuses on fixing the unbalanced number of substrates, or GAG. This therapy blocks the body from producing more glycosaminoglycans, which prevents the amount of these substrates from overgrowing a given capacity (Arfi, 2010). Isoflavones, products found in soy products, are thought to make this possible. Genistein, a particular type of isoflavone, can cross the blood-brain barrier and can inhibit the tyrosine kinase activity of the epidermal growth factor (Akiyama, 1986). This EGF receptor is known as one of the proteins that allows GAG to accumulate, therefore, having a medication to stop that protein would result in the synthesis of GAG to stop (Ghosh, 2021).

Procedure: Negative Geotaxis Assay

infographic

The first technique used was a negative geotaxis assay which was performed to test the climbing abilities of SGSH mutated flies before and after medication. To perform a negative geotaxis assay all that was needed is a vial reaching a height of at least 17 cm. Flies were be placed inside the vial without anesthesia and left to assimilate for a couple of minutes. Flies were expected to pass the 17 cm boundary mark. To begin the test, the vial was tapped the against a hard surface a couple of times until all the flies fell to the bottom. Flies were timed for one minute and their heights were measured at the end of that time.

Procedure: Capillary Feeding

The second technique used was a Capillary Feeder Assay which was performed to test the ability of flies to feed themselves as well as the food intake of mutated vs nonmutated flies. A sucrose stock solution was prepared by adding 102.6 g sucrose to 100 mL of water. Five drops of food were added to the stock solution to see the sucrose solution clearly. To avoid a bias due to the dye color, two different colors were used, red and blue. To create a proper vial that could hold capillaries, four small round openings were drilled into the plastic cap of the vial and pipette tips of 2 - 20 µL volume were inserted into the holes to hold the capillaries in place. Flies were left to feed for 24 hours, and the amount of food gone was measured after that time.

feeding climbing

Example of climbing assay setup on left and example of feeding assay setup on right. Nichols, Charles D., et al. “Methods to Assay Drosophila Behavior.” Journal of Visualized Experiments : JoVE, no. 61, Mar. 2012, p. 3795. PubMed Central, https://doi.org/10.3791/3795.

Analysis

climbingraph

My negative geotaxis assay found that the non-medicated Sanfilippo Drosophila group had the lowest climbing heights with 5.8 and 9.9 cm. The medicated Sanfilippo Drosophila group experienced the highest climb heights with heights of 14.1 and 12.5 cm. Interestingly, the control flies, or wild-type flies, had similar but lower climb heights than the medicated mutated flies.

feedingraph

My capillary feeding assay found that the control groups of wild-type flies had the lowest feeding amounts with the amount of sucrose eaten being 4.2 cm. Mutated Sanfilippo flies experienced an increase in their feeding from 5.2 cm while un-medicated to 8.6 cm while medicated. One observation made was that flies seemed to favor blue capillaries over red ones.

Conclusion

There was a statistically significant increase in the motor function of mutated Drosophila melanogaster flies after being medicated with genistein for two days. Sanfilippo fly groups who were medicated saw in increase in their feeding and climbing abilities when compared to their unmedicated groups. However, the wildtype flies didn’t set the bar as high as they should’ve. Particularly in the feeding assay, wild type flies consumed drastically below the amount of food that un-medicated flies were able to consume. It is also important to note that in some cases, older flies who had genistein in their system began to experience trouble with climbing indicating that genistein had a limited effect lasting only a couple of days. Further research will be conducted to test out how daidzein and glycitein can affect motor control in a person affected by Sanfilippo Syndrome. These new isoflavones could show a better increase in motor function, resulting in higher climbing heights, than genistein.

Preliminary Poster

This is an outline of my poster with all of the needed sections written down. An edited or changed form will be presented at the STEM fair.

Works Cited

  1. Agrawal, U., Meshram, A., Vagha, J., Swarnkar, K., & Palandurkar, K. (2012). Diagnosis of Sanfilippo Disease Correlating Clinical, Radiological and Biochemical Findings–A Case Report. Indian Journal of Clinical Biochemistry, 27(4), 417–421. https://doi.org/10.1007/s12291-012-0211-1
  2. Arfi, A., Richard, M., Gandolphe, C., & Scherman, D. (2010). Storage correction in cells of patients suffering from mucopolysaccharidoses types IIIA and VII after treatment with genistein and other isoflavones. Journal of Inherited Metabolic Disease, 33(1), 61–67. https://doi.org/10.1007/s10545-009-9029-2
  3. Akiyama, T., Ishida, J., & Nakagawa, S. (1986). Genistein, a Specific Inhibitor of Tyrosine-specific Protein Kinases. THE JOURNAL OF BIOLOGICAL CHEMISTRY, 262.
  4. Casale, Jarett, and Jonathan S. Crane (2021). Biochemistry, Glycosaminoglycans. StatPearls Publishing, PubMed. http://www.ncbi.nlm.nih.gov/books/NBK544295/.
  5. Fraser, J. (2005). Sleep disturbance in Sanfilippo syndrome: a parental questionnaire study. Archives of Disease in Childhood, 90(12), 1239–1242. https://doi.org/10.1136/adc.2004.065482
  6. Ghosh, A., Rust, S., Weisberg, D., Canal, M., Wijburg, F. A., Vaz, F. M., Tylee, K. L., Church, H. J., Hepburn, M., Bigger, B. W., & Jones, S. A. (2018). High dose genistein aglycone in Sanfilippo syndrome: Results of a randomised, double-blinded, placebo controlled clinical trial. Molecular Genetics and Metabolism, 123(2), S51–S52. https://doi.org/10.1016/j.ymgme.2017.12.121
  7. Grynkiewicz, G., Wegrzyn, G., Szechner, B., & Szeja, W. (2012). ISOFLAVONES FOR TREATING MUCOPOLYSACCHARIDOSES (20120190642). United States Patent Application. https://www.freepatentsonline.com/y2012/0190642.html
  8. Kloska, A., Jakóbkiewicz-Banecka, J., Narajczyk, M., Banecka-Majkutewicz, Z., & Węgrzyn, G. (2011). Effects of flavonoids on glycosaminoglycan synthesis: implications for substrate reduction therapy in Sanfilippo disease and other mucopolysaccharidoses. Metabolic Brain Disease, 26(1), 1–8. https://doi.org/10.1007/s11011-011-9233-2
  9. Madabattula, S. T., Strautman, J. C., Bysice, A. M., O’Sullivan, J. A., Androschuk, A., Rosenfelt, C., Doucet, K., Rouleau, G., & Bolduc, F. (2015). Quantitative Analysis of Climbing Defects in a Drosophila Model of Neurodegenerative Disorders. Journal of Visualized Experiments, 100. https://doi.org/10.3791/52741
  10. Morgan, Freya; Kavanagh, David; Swol, Elizabeth Van; Mokashi, Sneha S; Anholt, Robert; Mackay, Trudy (2020): A Drosophila model for Sanfilippo Syndrome. TAGC 2020. Poster. https://doi.org/10.6084/m9.figshare.12149238.v1
  11. Nichols, C. D., Becnel, J., & Pandey, U. B. (2012). Methods to Assay Drosophila Behavior. Journal of Visualized Experiments, 61. https://doi.org/10.3791/3795
  12. Parker, H., Ellison, S. M., Holley, R. J., O’Leary, C., Liao, A., Asadi, J., Glover, E., Ghosh, A., Jones, S., Wilkinson, F. L., Brough, D., Pinteaux, E., Boutin, H., & Bigger, B. W. (2020). Haematopoietic stem cell gene therapy with IL ‐1Ra rescues cognitive loss in mucopolysaccharidosis IIIA. EMBO Molecular Medicine, 12(3). https://doi.org/10.15252/emmm.201911185
  13. Piotrowska, E., Jakóbkiewicz-Banecka, J., & Węgrzyn, G. (2009). Different amounts of isoflavones in various commercially available soy extracts in the light of gene expression-targeted isoflavone therapy. Phytotherapy Research, 24(S1), S109–S113. https://doi.org/10.1002/ptr.2944
  14. Piotrowska, E., Jakóbkiewicz-Banecka, J., Tylki-Szymanska, A., Liberek, A., Maryniak, A., Malinowska, M., Czartoryska, B., Puk, E., Kloska, A., Liberek, T., Baranska, S., Wegrzyn, A., & Wegrzyn, G. (2008). Genistin-rich soy isoflavone extract in substrate reduction therapy for Sanfilippo syndrome: An open-label, pilot study in 10 pediatric patients. Current Therapeutic Research, 69(2), 166–179. https://doi.org/10.1016/j.curtheres.2008.04.002
  15. Rigon, L., de Filippis, C., Napoli, B., Tomanin, R., & Orso, G. (2021). Exploiting the Potential of Drosophila Models in Lysosomal Storage Disorders: Pathological Mechanisms and Drug Discovery. Biomedicines, 9(3), 268. https://doi.org/10.3390/biomedicines9030268
  16. Sun, M. Y., Ye, Y., Xiao, L., Rahman, K., Xia, W., & Zhang, H. (2016). Daidzein: A review of pharmacological effects. African Journal of Traditional, Complementary and Alternative Medicines, 13(3), 117. https://doi.org/10.4314/ajtcam.v13i3.15
  17. Zelei, T., Csetneki, K., Vokó, Z., & Siffel, C. (2018). Epidemiology of Sanfilippo syndrome: results of a systematic literature review. Orphanet Journal of Rare Diseases, 13(1). https://doi.org/10.1186/s13023-018-0796-4