Evaluating the Effect of Ashwagandha on Lipid Accumulation in N2 Caenorhabditis elegans

S.K. Rowe, H.M. Van Auken & A.M. Brody

Department of Science, Principles of Experimental Design in Biotechnology, Rock Canyon High School, Highlands Ranch, Colorado

April 29, 2021

Abstract

The medicinal herb ashwagandha has been used for centuries to treat various health conditions and regulate weight. Having a high body mass index (BMI) predisposes individuals to a number of deathly conditions; therefore, discovering ways to lower the population of overweight individuals to limit adverse health outcomes is increasingly important. This study examined the effect of ashwagandha on lipid accumulation in N2 Caenorhabditis elegans (C. elegans). It was hypothesized that ashwagandha would decrease lipid accumulation within C. elegans due to its biologically active components that have caused weight loss individually. Research supporting this hypothesis could lead to further studies in ashwagandha’s application toward human weight regulation. C. elegans exposed to a 100 μg/mL solution of ashwagandha root extract on OP50 Escherichia coli (E. coli) seeded plates were compared to a no-treatment negative control and a DMSO-only control. Age-synchronized C. elegans were incubated at 21°C for 72 hours. Nile Red (NR) staining was used to illuminate lipid content within the C. elegans, and data was quantified through ImageJ. Results indicate an average fluorescence of 26.407 for the E. coli group, 24.529 for the DMSO group, and 26.387 for the ashwagandha group. Two-sample t-tests indicated statistical insignificance for each comparison with p-values 0.989 for the E. coli/ashwagandha comparison and 0.175 for the E. coli/DMSO comparison. It can be concluded that ashwagandha has no effect on lipid accumulation within N2 C. elegans. Future experiments that combine ashwagandha with other supplements may draw different conclusions.

Keywords: Ashwagandha, N2 Caenorhadbditis elegans (C. elegans), Nile Red Stain, Obesity, Lipid Accumulation.

Introduction

In recent years, obesity has become a significant, worldwide public health issue. This chronic disorder currently affects about 13% of the world’s population and about 42% of United States citizens, with projections that nearly 50% of U.S. citizens will be obese by 2030 (Graph 1) (Centers for Disease Control and Prevention, 2021). This condition is defined as a body mass index (BMI) of 30 or greater in adults; in children, a BMI of above the 95th percentile for age is considered obese. Obesity is associated with serious health risks, including type II diabetes, cardiovascular disease, nonalcoholic fatty liver disease, and certain forms of cancer (Ogden et al., 2007). Obesity and obesity-related conditions are also among the leading causes of preventable, premature death in the U.S. (Centers for Disease Control and Prevention, 2021). Due to this epidemic, research on obesity prevention and mitigation has increased significantly in recent years. One pathway that scientists are currently investigating is supplement-based obesity mitigation; specifically, their ability to regulate metabolism and lipid accumulation. One such supplement, ashwagandha extract, has shown promise in obesity reduction and may play a role in lipid metabolism (Choudhary et al., 2016).

Ashwagandha

Withania somnifera, commonly known as ashwagandha, is a member of the Solanaceae family of flowering plants and is most commonly administered as a stress relieving supplement (Pic. 1). The herb has been traced back to use in India in 6000 BCE with the traditional medical system known as Ayurvedic medicine. In particular, ashwagandha has been used for thousands of years for Rasayana, a term within Ayurvedic medicine which refers to herbal substances promoting a youthful mental and physical state. Ashwagandha has been identified as one of the most valuable out of the many Ayurvedic Rasayana herbs, believed to aid in immune system support and inflammation reduction, as well as mitigating side effects associated with insomnia, aging, arthritis, and anxiety (Singh et al., 2011). Additionally, several scientific studies have demonstrated a correlation to lower blood pressure, ulcer prevention, lower cholesterol, blood-sugar stabilization, and an increased red blood cell count (Umadevi et al., 2012).

Although very little research has scientifically validated the potential uses of ashwagandha, extensive studies have identified over 35 chemical constituents within ashwagandha, each with their own unique applications (Mishra et al., 2000). A number of biologically active compounds within ashwagandha have an effect on human organ system functioning. Of the many chemical constituents, withaferin-A, withanolide-D, withanone, withanosides, and sitoindosides have demonstrated the most significant stress relieving properties (Fig. 1) (Jain & Mathur, 2020). Scientists believe that these bioactive components in ashwagandha may also contribute to weight loss or reduce weight gain. This effect might result either through their own biochemical interactions, or through craving and overeating regulation from their stress-reduction properties (Choudhary et al., 2016). However, no definitive research has been conducted that supports the use of ashwagandha as a medical supplement targeted towards weight loss. Another study conducted to investigate the adaptogenic effects and medicinal uses of ashwagandha stated that the supplement may cause weight loss by the alternative mechanisms of regulating digestion, managing fluctuations in diet, and boosting metabolism. These other potential benefits of ashwagandha are hypothesized to result from nitrogenous organic compounds and bioactive amino acids within the plant (Umadevi et al., 2012). To begin the investigation into ashwagandha as a potential weight loss supplement, research into the effects in a model organism with a similar bodily system to humans is necessary.

Research Question/Hypothesis

Our research attempted to determine the effect of ashwagandha on lipid accumulation within N2 C. elegans. We hypothesized that ashwagandha would decrease fat storage within N2 C. elegans’ hypodermal and intestinal cells due to antioxidants within the supplement that decrease inflammation and speed up metabolism. The presence of Vitamin B6 and other antioxidants have been demonstrated to help the body metabolize and burn stored fat (Novin et al., 2019). A statistically significant reduction in lipids would demonstrate that ashwagandha causes weight reduction in C. elegans. Potential lipid reduction was quantified through Nile Red staining and the ImageJ processing program.

Caenorhabditis elegans

        Traditionally, rodents are used as model organisms in obesity research. The high maintenance costs of rodent studies, however, make C. elegans a more cost-effective and time-efficient model to study obesity. C. elegans are small nematodes that reach about 1mm in length upon adulthood. Wild-type C. elegans (N2) have a life span of roughly 21 days and a generation time of 3 days (Zheng & Greenway, 2011). Their quick growth allows for rapid turnover and results, making research involving C. elegans simpler, quicker, and cheaper. The similarity of C. elegans’ reproductive, nervous, and digestive systems to humans, combined with their ability to be easily genetically modified, make them an extremely valuable model for studying human functions and disease (Yokoyama, 2020). In this study, we will use wild type C. elegans at the L4 stage (Fig. 2). It is not necessary for the nematodes to have any genetic alterations—N2 C. elegans already contain lipid genes conserved in humans that are associated with obesity and other metabolic diseases (Escorcia et al., 2018). We are attempting to discover the effects of ashwagandha on the general population; therefore, wild type C. elegans will provide the most basic and effective model for our research. C. elegans store fat mainly within their hypodermal and intestinal cells, which can be directly visualized via the use of fixative or non fixative staining methods (Zhang et al., 2013).

Staining

Stains, such as Nile Red, Sudan black, and Oil Red, can quantify lipid storage by measuring the intensity of accumulated dye in fat cells through C. elegans’ transparent bodies (Pic. 2). Direct observation of fat storage controls for confounding variables, such as water intake and size-dependent metabolism, that are present in weight-based rodent studies. A previous study has concluded that the label-free coherent anti-Stokes Raman scattering (CARS) microscopy is the most reliable method to assay for fat stores; however, this technology is unavailable to us. As stated by the same article, the Nile Red fixative staining method is recommended for the greatest accuracy if CARS microscopy is not feasible (Yen et al., 2010). The Nile Red stain has also been deemed the most accurate stain for determining lipid concentration in C. elegans out of all the fixative staining methods (Zheng et al., 2016). The stain was visualized using the green fluorescent lens of the EVOS microscope.

Ashwagandha and C. elegans

The effects of administering ashwagandha to C. elegans have only been superficially investigated. One study examined its effect on the lifespan of C. elegans. The root extract, both crude and water soluble, was administered to N2 C. elegans as well as the acr-16 (ok789) strain. The study concluded that while the administration of ashwagandha had no effect on the lifespan of wild type C. elegans, the C. elegans could uptake the purified compounds mixture (PI-RE) of the root extract at concentrations of 5 ng/mL and 10 ng/mL with no ill effects (Fig. 3) (Kumar et al., 2013). Another study also administered ashwagandha as one of six Ayurvedic herbs to determine its effect on the BZ555 and NL5901 strains of C. elegans, which model Parkinson’s Disease (PD). A stock solution of 66 µg/mL of the ashwagandha was prepared using dimethyl sulfoxide (DMSO) and administered to the C. elegans through the NGM agar. This study reported no toxicity of ashwagandha on C. elegans and concluded that the extract of ashwagandha was the most effective of the six ayurvedic herbs tested in inhibiting the aggregation of the protein α-synuclein, which is linked to Parkinson’s disease (Anjaneyulu et al., 2020). These articles discuss the potential weight loss effects of ashwagandha on C. elegans, but neither article specifically states any data found regarding this hypothesis (Anjaneyulu et al., 2020) (Kumar et al., 2013).

Methods Overview

In order to test our hypothesis, we administered a 100 µg/mL ashwagandha solution through the media to N2 wild type C. elegans embryos that had been isolated via age synchronization. The C. elegans were incubated at 21°C for 72 hours before being washed from the plate and pelleted. The worm pellet remained at room temperature overnight before being stained with Nile Red stain, which binds specifically to intracellular lipid droplets to dye them using fluorescent microscopy (Rumin et al., 2015). The stained C. elegans were imaged under the green fluorescent lens of the EVOS microscope. Images were then uploaded to the ImageJ processing program in order to obtain a fluorescence score for each C. elegans. Mean fluorescent scores between the control and experimental groups were compared using two-sample t-tests to determine statistical significance.

Methods

This experiment measured the effects of ashwagandha on lipid cell accumulation in C. elegans. N2 C. elegans were cultured on 60 mm NGM-lite plates seeded with OP50 E. coli. Approximately 100 µL of 100 µg/mL ashwagandha 1% DMSO solution was added to the plates in the experimental group. One control group of N2 C. elegans was not exposed to treatments, and a second control group of N2 C. elegans was exposed to a 1% DMSO solution alone. C. elegans were stained with Nile Red stain, and the green channel of the EVOS fluorescence microscope was used to visualize lipid droplets in each nematode. ImageJ was used to quantify lipid droplets through fluorescent scores. Two-sample t-tests compared average fluorescence scores between each group to determine treatment efficacy.

Pre-Trials

Pre-trials were conducted to determine the concentration of experimental ashwagandha treatment, refine the lipid quantification assay, and solidify basic husbandry procedures. To determine the most viable concentration of ashwagandha to administer to the C. elegans, we tested a 80 µg/mL concentration extrapolated from the previously described study on the effects of ashwagandha on protein aggregation in the BZ555 and NL5901 strains of C. elegans (Anjaneyulu et al., 2020). Ashwagandha was dissolved in 1% DMSO to ensure solubility (Galvao et al., 2014). After testing this concentration and observing no qualitative change in lipid accumulation, we increased the ashwagandha concentration. Pre-trials concluded with a concentration of 100 µg/mL of ashwagandha diluted to 1% in DMSO LB broth/E. Coli media, the concentration with the highest likelihood to have an effect on C. elegans lipid accumulation without necrosis. With this concentration, the average mortality rate did not exceed 50% (e.g. LD50). It was also determined during pre-trials that the green fluorescent channel of the EVOS microscope was optimized for lipid quantification.

Experimental Design

The experiment consisted of three groups: a no-treatment control to provide a baseline for normal lipid reduction, a DMSO control to control for the potential effects of DMSO on lipid reduction, and the ashwagandha experimental group. The experimental group was administered a concentration of 100 µg/mL of ashwagandha in 1% DMSO, the DMSO control group received 1% DMSO concentration diluted in 5 mL of media, and the E. coli group received no treatment. We conducted three trials for each group, with C. elegans quantified as a single data point. Each trial plate consisted of 20 imaged C. elegans, totalling 60 data points per trial (Fig. 4).

Plate Preparation

100 µL of OP50 E. coli overnight cultures in lysogeny broth (LB) incubated overnight at 37o C and were used to seed all experimental plates. Four plates were prepared for each experimental group. The ashwagandha treatment solution was prepared by adding 0.002 g of ashwagandha powder to 200 µL of DMSO, and subsequently diluting that solution in 19.8 mL of culture. The DMSO-only control was prepared by adding 50 µL of DMSO to 4.95 mL of culture. Approximately 100 µL of each overnight culture was spread onto ⅔ of four unseeded plates per culture, totaling 12 experimental plates (Pic. 3). After 24 hours, these seeded plates were parafilmed and stored at 4°C to preserve viability (Stiernagle, 2006). Stock populations were maintained throughout the experiment on 60 mm stock plates of untreated NGM-Lite agar through the chunking method of C. elegans transfer.

Population Synchronization

        Population synchronization was necessary to ensure the age of the C. elegans remained constant for each trial of data collection. This process was done by creating a bleach solution consisting of 0.67g sodium hydroxide, 6.67 mL of household bleach (7.4% sodium hypochlorite), and 25 mL deionized (DI) water; we took a large drop of the solution and placed it along the rim of the plate. A bacterial loop was utilized to collect as many gravid adults and eggs as possible from our stock plates and place them in the bleach solution. Each plate was then examined to ensure successful synchronization (Pic. 4). Plates were incubated at 21℃ for 72 hours before our first data collection at the L4 stage.

Nile Red Stain Assay

The Nile Red (NR) staining method was used to stain and visualize the lipids within the C. elegans. The 5 mg/mL stock solution of Nile Red was created by adding 10 mg of NR powder to 20 mL of 100% acetone. NR is extremely light sensitive, so the stock solution was stored in a tightly sealed bottle in a dark room at room temperature. For each sample, 1,006 µL of fresh NR working solution was prepared, following a ratio of 6 µL of NR stock solution per 1 mL of 40% isopropanol.

To prepare for NR staining, the C. elegans were washed off of the plate with 1 mL of 1X PBS + 0.01% Triton X-100 (PBST) solution. About 500 mL of stock solution was made prior to all stain assays by adding 0.05 mL of Triton X-100 to 499.95 mL of 1X PBS. The washed worms were pelleted at 560 x g for 1 minute (Bikandi et al., 2004) before the supernatant was removed and 100 µL of 40% isopropanol was added to the worm pellet. The pellet then incubated at room temperature for 3 minutes, fixing the worms. After incubating, the worms were pelleted again and the supernatant was removed. The worms were left to sit overnight with the lids partially open to ensure full evaporation of leftover alcohol. These procedures were completed approximately 20 hours prior to staining and data collection.

To begin lipid staining, 150 µL of NR working solution was added to each sample, inverting the tube three times to fully mix the worms with NR solution. Each sample was then wrapped in foil and rocked on the rocking platform for 30 minutes at 100 rpm (Pic. 5). After incubation, the worms were pelleted at 100 x g for 1 minute and the supernatant was removed. Then, 1 mL of C. elegans wash was added to each sample and centrifuged for 1 minute at 100 x g. The remaining supernatant was removed, leaving only the worm pellet in each microcentrifuge tube.

After each sample was stained, we prepared the slides for microscope imaging. About 5 µL of the worm pellet was placed on each microscope slide and covered with an airtight coverslip. Only one slide was prepared at one time, to ensure NR imaging remained constant across samples and to prevent the slide from drying out (Escorcia et al., 2018).

Data Collection & Analysis

The stained C. elegans were analyzed and imaged under the EVOS microscope (Pic. 6) and then uploaded for interpretation in the ImageJ processing program. In each group, 60 data points were analyzed. Each sample (microscope slide) was imaged separately under the EVOS microscope. The samples were imaged under the green fluorescence protein (GFP) channel at 10X magnification and 40% light exposure. For each sample, 20 random, separate worms were imaged, totalling 60 images after all three trials were performed. These images displayed the stained lipids within each nematode, which allowed for a visual representation of qualitative data (Pic. 7). To produce quantitative results, the images were uploaded to ImageJ, a downloadable Java-based image processing program developed by the National Institutes of Health (U.S. Department of Health and Human Services, n.d.). The Polygon selection tool was used to delineate each imaged nematode, and the ‘Measure’ function quantified the fluorescence intensity for the selected area. The process was repeated for 60 C. elegans per group per trial.

Constant Variables

The N2 strain of C. elegans was exclusively used and nematodes were consistently cultured on NGM-Lite 60 mm plates seeded with 100 µL of OP50 E. coli. Nematodes were kept at 21°C and assayed at the L4 stage after 72 hours of incubation. The treatment on the plates was kept constant as well: experimental plates were treated with 100 µg/mL of ashwagandha and the control plate with DMSO was treated with 1% DMSO. All procedures described above were held constant.

Results

In this experiment, we tested the effect of ashwagandha on lipid accumulation in N2 C. elegans. Three trials of N2 C. elegans exposed to an ashwagandha/DMSO solution were compared to three trials of N2 C. elegans with no exposure as well as three trials of N2 C. elegans with a 1% DMSO exposure. Each trial yielded 20 C. elegans, resulting in a total of 60 data points obtained from each group. The mean fluorescence scores, as determined through the ImageJ analysis program, were 26.407 for the E. coli group, 24.529 for the DMSO group, and 26.3867 for the ashwagandha group (Graph 2). Although consistent procedures were followed in all trials, a slight decrease in mean fluorescence scores was observed from trial 1 to trial 3 (Fig. 5). However, this change was not visible as the stain appeared the same color prior to being administered and during the staining and imaging process—the decrease was only noted at the final stage when analyzing through ImageJ and obtaining the fluorescence scores. A two-sample t-test was conducted between the DMSO group and E. coli group (test 1) as well as between the ashwagandha group and E. coli group (test 2). Both tests were conducted at the 0.05 significance level. The results indicated that the data was insignificant for both comparisons. A p-value of 0.175 was obtained for test 1, and test 2 resulted in a p-value of 0.989.

Discussion

It has become increasingly important to find solutions to the rising obesity rates in the United States and the world as a whole. Our research aimed to target this issue by learning how administering ashwagandha, an Ayurvedic supplement, to C. elegans would affect lipid accumulation within their bodies. We hypothesized that exposure to ashwagandha would decrease lipids in N2 C. elegans due to the bioactive components within ashwagandha, such as withaferin-A, withanolide-D, withanone, withansidoes, and sitoindosides, which have each contributed to weight loss individually. To examine the validity of our hypothesis, we exposed N2 C. elegans to powdered ashwagandha dissolved in DMSO and performed a stain assay to fluoresce the lipids in order to quantify the amount of lipids present in each C. elegans.

The mean fluorescence scores, as determined through the ImageJ analysis program, were 26.407 for the E. coli group, 24.529 for the DMSO group, and 26.3867 for the ashwagandha grouh. Although consistent procedures were followed in all trials, a slight decrease in mean fluorescence scores was observed from trial 1 to trial 3. However, this change was not visible as the stain appeared the same color prior.

After comparing the E. coli control with the ashwagandha experimental group and the DMSO control with the experimental group, the results of this experiment did not display statistical significance. We do not have convincing evidence that the true mean fluorescence score for C. elegans exposed to ashwagandha is different from the true mean fluorescence score of C. elegans in either of the control groups. Therefore, we fail to reject the null hypothesis and conclude that ashwagandha has no effect on lipid accumulation in N2 C. elegans. Although further studies pertaining to our research question must be conducted in order to draw a reliable conclusion, from our research alone, we can infer that a 100 μg/mL concentration of ashwagandha can not be considered as a viable alternative fat-loss supplement for N2 C. elegans. The scope of our research did not allow us to conduct an additional assay to quantify how much of the ashwagandha was taken up by the C. elegans. Additionally, we were unable to test ashwagandha’s effects on a strain predisposed to obesity, such as the RB1600 tub-1 strain. A study found that mutations in the tub-1 strain promoted greater fat deposition than the wild type nematode strain (Mukhopadhyay et al., 2005). Our research revolved around finding a potential supplement to combat rising obesity rates in the United States; by using a wild-type strain, we focused more on addressing the general public rather than those already affected by obesity. Research involving ashwagandha and the tub-1 strain of C. elegans could better address obese subjects and conclude how the supplement affects organisms that are already affected by obesity. Research has recently concluded that the TUB homolog not only exists in humans, but also affects obesity (Carroll et al., 2004). A study involving mutations in the tubby gene of mice found high expression of TUB in the hypothalamus, especially in areas involved in body weight regulation, as well as a correlation between obesity and TUB expression in adipose tissue (Nies et al., 2017). This study indicates that the TUB gene is involved in regulating fat and food intake in humans. These findings show that the tub-1 strain, rather than a wild type strain, would be better suited to test the effects of ashwagandha on predisposed obesity and could conclude different findings.

When originally designing our research project, we found significant research on anti-stress properties of ashwagandha in humans. A study involving human subjects found that high-concentration full-spectrum ashwagandha root extract improved an individual’s resistance towards stress over a period of 60 days. The study concluded that these results may be due to the anti-stress activity of sitoindoside VII and sitoindoside VIII, both of which are found in the plant (Chandrasekhar et al., 2012). Stress (high cortisol levels) have been shown to cause weight gain; furthermore, patients with abdominal obesity are proven to have elevated cortisol levels (Hewagalamulage et al., 2016). With both of these findings in mind, we made the connection that ashwagandha could treat obesity through its anti-stress properties. In order to fully test these effects, further research involving the tub-1 strain (discussed above) alongside the wild-type strain of C. elegans could be conducted testing how cortisol levels change in C. elegans and how these changes affect lipid accumulation in the model organism. A study of these parameters would have to involve a consumption assay, a cortisol assay, and a stain assay, which would be out of the scope of research of our lab.

One potential source of error came from the Nile Red stain stock solution. Although the Nile Red stain was created and stored following exact procedures from a prior study, including keeping it covered in foil to avoid any light exposure, the stain may have still slightly degraded over time. This possible degradation could explain the slight decrease in mean fluorescence scores from trial 1 to trial 3. However, this decrease was not significant enough to alter the viability of the results. Finally, due to time constraints, we were unable to perform a consumption assay to measure the uptake of ashwagandha by the C. elegans. Therefore, our study is based on the assumption that the C. elegans were consuming the supplement and eating their E. coli media at a proportionate rate, and that discrepancies between amounts consumed did not have an effect on the results.

Because the results of our study were inconclusive, it would not make sense to move to a higher model organism. However, additional studies may be necessary to fully conclude that ashwagandha is ineffective in causing a decrease in lipids and ultimately in weight loss. Potential future studies could implement more trials with differing concentrations of ashwagandha or even be conducted on a different strain of C. elegans, such as tub-1. Future research could also focus on the anti-stress properties of ashwagandha and how these may contribute to weight gain/loss in humans. Tests on consumption, cortisol levels, and lipid accumulation could lead to conclusions on ashwagandha’s effects on stress and whether or not this contributes to overall weight gain or loss.

Acknowledgements

We would like to thank the following people for supporting, authorizing, and funding our research project. Thank you to Dr. Frank Greenway—creator of the Harbor-UCLA obesity research clinic and current professor at Pennington Biomedical Research Center at Louisiana State University—for advising, providing expertise, and recommending possible supplements to look into during our initial planning process. Thank you to Dr. Jolene Zheng—assistant professor at Louisiana State University and researcher at Pennington Biomedical Research Center—for providing knowledge pertaining to the efficacy of using C. elegans as a model organism in obesity research. We would also like to thank Karilee Reed—a PhD student at Colorado State University—for offering feedback and suggestions throughout our research. Additionally, we offer thanks to Daniel Grammar and Sara Black (our Biotechnology Year Three mentors) for their assistance in designing our research project, assisting during pre-trials and experimental trials, editing our papers, and helping us prepare for presentations. Thank you Susanne Petri (our Experimental Design teacher) for her support and expertise throughout the research process. Furthermore, we thank Shawndra Fordham (our former Experimental Design teacher) for her guidance and brainstorming. Thank you to Kerry Hinton (Rock Canyon High School’s Chemical safety manager) for allowing us to use the precise analytical scale in her classroom and ensuring safe disposal of all chemicals. We would like to offer thanks to Bailey Thayer at Thermo Fisher for providing a discount on our Nile Red Stain. Lastly, we would like to thank Rock Canyon High School, Douglas County School District, and the Rock Canyon Science Department for providing us with lab space, funding, and all of the required materials for our research.

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About the Authors

Sadie Rowe is a senior at Rock Canyon High School. She has always had a passion for science and medicine, which has been fostered by the Biotechnology program and associated opportunities offered through RCHS. The Experimental Design class that facilitated this research has provided her with necessary skills to thrive in a future STEM related career. Notably, lab experience, time management, and technical writing skills have been an invaluable aspect of her learning, allowing Sadie to grow as a student and beyond. Additionally, having the opportunity to conduct such tangible research in a high school laboratory has allowed Sadie to expand her horizons and further inspired her to pursue a career within the field of biology. Next year, she will be attending the University of Wisconsin-Madison to study biomedical engineering where she looks forward to exploring the more technical aspects of science.

Hailey Van Auken is a senior at Rock Canyon High School who is extremely interested in and passionate about all things STEM. She applied for the Biotechnology Research Course after taking Intro to Biotechnology and AP Chemistry and loving both. As a student in this class, she learned how to properly research, design an experiment, and present in front of distinguished audience members. The hands-on laboratory experience that she performed this year has given her a strong foundation for laboratory skills and inspired her to pursue research in college. Additionally, Hailey contributed greatly to the mathematics component of the research this year, so she feels very prepared to take these skills to the collegiate level. She noted that her presentation skills have improved dramatically from the beginning of the year, and she is excited for more opportunities to speak in the future. After Hailey graduates, she will be attending California Polytechnic State University in San Luis Obispo, California. She will be studying Aerospace Engineering with a potential minor in business.

Ava Brody is a senior at Rock Canyon High School who applied for the Biotechnology Experimental Design course after greatly enjoying her experience in the initial Biotechnology class she took Junior year. While she is not certain if her upper level education or career path will involve biotechnology, the research development process has allowed her to advance her knowledge and critical thinking skills which will be invaluable to any of her future endeavors. In addition, Ava noted that through the creation of the research proposal, conducting the research, analyzing the data, and reflecting on the results she has developed her time-management, creativity, technical writing skills, and upper level thought processing. She very much appreciated having had the opportunity to conduct this research project with her peers in such an advanced laboratory at the high school level. While she is unsure of what her future may hold, Ava is looking forward to attending Emory University next year where she will be able to explore her variety of interests.