PCR Spring 2021 by The Pingry School

PC R. Spring 2021.

Research Week. Special Edition.

Contents. AP Biology Consumption Rate of Bran and Polystyrene on Mealworms 9 Diet’s Effect on the Cognition of Concussed Fruit Flies 10 Different Nutrients and Their Effects on Saccharomyces cerevisiae 10 Effect of Over the Counter Drugs on the Cognitive Function of Fruit Flies After Trauma 11 Effect of Saline Water on Nutrient Uptake Efficiency of Duckweed 11 Effect of UVC Light On Saccharomyces Cerevisiae Growth 12 Eutrophication Via Fertilizer in Lemna minor 12 The Effect of Diet on Tenebrio Molitor Survivorship and Fertilizer Potential 13 The Effect of Nutrient Limitation on the Reproductive Rate of Yeast 14 The Effect of pH on the Growth of Lemna Minor 14 The Effects of Ultraviolet Radiation on Cell Death in Saccharomyce Cerevisiae 15 The Effects of Vinegar on Duckweed Growth 16 Removal of Ammonia (NH4OH) in Water by Lemna minor 16 2

Methods in Molecular Biology Research Summary of Research Class 17 Plastic Degrading Enzymes 18 Bioinformatic Characterization of 3DS8 19-20

SMART Team Fatty Acid Regulation of Toxicity in Vibrio Cholerae: A Structural Analysis of Unbound, Ligand Bound, and DNA Bound ToxT 21-23 Understanding the Functions and Variants of the SARS-CoV-2 Virus 23-24

HIRT Empathizing With “The Uncanny” in Children’s Storytelling and Bargaining 25-27 Afrofuturism Research Project 27

Contents. IRT Artificial Collagen for Use as a Biomaterial 29 Drover: Drone-Rover Communication for Pathfinding 30 Effect of Dietary Restrictions on the Cognitive Ability of Drosophila melanogaster 31 Metagenomic Analysis of a Compost Microbiome 32 Plastic Degrading Enzymes 33 Protein Purification of Shigella dysenteriae Transcription Factor VirF 34 Quantifying the Effect of Structure on Neural Network Training 35 Quantifying the Phototactic Memory of Chlamydomonas Reinhardtii 36 The Effect of KIF11 Activity on YAP Localization 37 The Effect of Memory Enhancing Supplements on Amyloid-ß 42 Expression in a Drosophila melanogaster Model for Alzheimer’s Disease 38

Club Highlights Anatomy Club 39 FYI Sci 39-40 Pingry Community Research 41 Girl Code 41 Robotics 42

Editor’s Note. Welcome to the Research Week edition of the Pingry Community Research

(PCR) Journal. We are excited to showcase Pingry’s top scientific talent, both in terms of research skills and knowledge of scientific concepts and discoveries. The PCR journal provides students the opportunity to publish novel research. Through a written medium, students demonstrate their in-depth understanding of complex, collegiate-level scientific topics, and their applications in research at Pingry. This special edition of PCR serves as a written complement to the in person poster presentations occurring throughout research week. Readers can preview abstracts, figures, and summaries reflecting the research conducted in advanced courses and extracurriculars, such as AP Biology, Independent Research Teams (IRT), and the Methods in Molecular Biology Research Class, among others. Through the PCR journal, we hope to spark intellectual curiosity and promote scientific inquiry amongst the next generation of Pingry researchers. Dive into the wonders of Pingry Research through this special edition of PCR: Pingry’s foremost journal of scientific research. Kristin Osika (V), Editor-in-Chief Caitlin Schwarz (V), Head Copy Editor Christine Guo (V), Head Layout Editor 6

Editorial Staff. Editor-in-Chief: Kristin Osika (V), current Aneesh Karuppur (VI), former Head Copy Editor: Caitlin Schwarz (V) Copy Editors: Ryan Arrazcaeta (V) Brian Li (V) Aanya Patel (V) Ashleigh Provoost (V) Sam Wexler (V) Max Watzky (IV)

Head Layout Editor: Christine Guo (V) Layout Editors: Mirika Jambudi (IV) Sarah Gu (III) Art Editor: Kelly Cao (III) Faculty Advisor: Mr. D. Maxwell

AP Biology. Consumption Rate of Bran and Polystyrene on Mealworms by Kenan Mushayandebvu (V) and Ulysses Smith (V) The twenty-first century is a period marked by innovation of a caliber never before seen. Unfortunately, such magnificent strides in human achievement come at the expense of the environment. Time and time again, the atmosphere, forests, and oceans are tossed aside and treated as collateral damage in modern society. The rise in waste products raises a particularly difficult problem: where should all of the trash go? Recycling is often cited as the solution to combat pollution, yet it is not efficient enough to compensate for the amount of waste produced. Furthermore, recycling is a very difficult task.

(mealworm’s normal diet). We will compare the consumption rate of the aforementioned substances over the course of several months. In our experiment, we filled 9 plastic containers with 10 mealworms and their respective diets. Then we covered the containers with plastic lids and poked holes in them for air. We put 25 grams of bran in 3 of the containers, 1 gram of polystyrene foam in 3 containers, and 1 gram of polystyrene film in the last 3 containers. Additionally, 10 grams of apples were put in each container for hydration. Every week the amount of food left in the containers was measured to determine the quantity of food that the mealworms consumed. The results lead us to conclude that that the mealworms consumed more bran than both polystyrene foam and polystyrene film as well as similar amounts of both types of polystyrene.

Such is the case with a substance like polystyrene (styrofoam), which is difficult to get rid of because it cannot be burned or reused. If polystyrene is burned, it produces lethal carbon monoxide gas, meaning that nearly all styrofoam is dumped into landfills. A potential solution to this problem is biodegradation of styrofoam via mealworms, which are able to safely eat styrofoam without major side effects. Accordingly, mealworms are being looked at as a potential way to recycle styrofoam into biomaterial. We seek to determine if the mealworms are efficient enough at digesting the polystyrene to keep up with the growing amount of waste produced by humans. Previous research concluded that mealworms are able to survive on styrofoam without any severe side effects. With this knowledge, we hypothesize that the mealworms would be able to consume styrofoam and polystyrene film at a near-identical rate at which they consume bran

Diet’s Effect on the Cognition of Concussed Fruit Flies by Sabrina Schneider (V), Aanya Patel (V), and Chris Halada (VI) Concussions affect about 2 million people globally each year. Our study aims to provide insight on specific nutrients capacity to treat fly brains. In early trials we analyzed post-mortem brain tissue of flies struck by the HIT device to determine that the device is an effective means to concuss fruit flies. A secondary study proved that beta-hydrobuatante can increase the cognition of concussed fruit flies through a quantitative analysis the variance between the aggression of the two groups varied. For a future trial, we used the conclusions drawn from these two studies to quantitatively test, though a t-maze, whether beta-hydrobuatante increases the cognition of flies concussed using the HIT device. Our goal is to determine if a ketone body diet, in comparison to their normal diet, would cause fruit flies to have improved cognitive function after being concussed.

Different Nutrients and Their Effects on Saccharomyces Cerevisiae by Anna Riley (V) My study aims to further research into a more efficient means of fermentation to create better biofuels that benefit our planet. Saccharomyces cerevisiae undergoes alcoholic fermentation as a direct result of anaerobic respiration. When presented with monosaccharides, S. cerevisiae ferments and emits carbon dioxide. S. cerevisiae was treated with five separate substrates to determine the optimal nutrient source for fermentation. The amount of carbon dioxide was measured in centimeters. In an effort to limit confounding variables, all experiments were completed inside six test tubes per substrate, each time using the same measurements. The solids were measured in grams, while H2O was measured

in milliliters. My data lead me to conclude that sugar as a substrate generates the largest amount of foam, therefore making it the most successful nutrient for the fermentation of S. cerevisiae.

Effect of Over the Counter Drugs on the Cognitive Function of Fruit Flies After Trauma by Olivia Hauck (V), Rohan Prabhu (V), and Maureen Schwarz (V) Our experiment is designed to determine which over-the-counter (OTC) drugs, if any, have an impact on fruit fly cognition following a traumatic episode. We used a trauma infliction device and T-maze (with fly repellant on one end and food on the other) to determine the flies’ ability to recognize their food, which allowed us to determine the change in their cognitive abilities after sustaining a TBI. Subsequently, we treated the flies with Ibuprofen (Advil), Aspirin, Excedrin (a mixture of Aspirin, Acetaminophen, and Caffeine),

and Diphenhydramine hydrochloride (Benadryl). Based on prior research and drug property similarities, we hypothesize that Excedrin, Aspirin, and Advil would have a negative effect on fruit flies cognitive abilities, while Benadryl would have a positive effect on brain cognition. Our data suggest that Advil and Aspirin did, in fact, negatively impact fruit fly cognition following a traumatic episode. Through our project, we hope to distinguish which medications are most effective at improving cognitive function after TBI in flies.

Effect of Saline Water on Nutrient Uptake Efficiency of Duckweed by Choe Chung (V), Christine Guo (V), and Alina Irvine (VI) Duckweed is a small flowering aquatic plant that floats on the surface of still-moving bodies. The plant grows efficiently in areas with nutrient-rich water containing phosphates and nitrates. It absorbs the majority of its nutrients through the frond; however, certain species of duckweed, such as Lemna Minor duckweed, use their roots for additional nutrient uptake. Recently, due to climate change, rising precipitation and temperature levels have increased saltwater intrusion into freshwater. Due to water pollution, many species and ecosystems have been endangered. However, indicator species, such as duckweed, can help prevent the eradication of freshwater sources by warning scientists of water pollution. Therefore our experiment examines duckweed’s sensitivity to saltwater and more specifically, how different concentrations of salt water affect the plant’s nutrient uptake efficiency.

To set up the experiment, we grew Lemna Minor duckweed in 32oz containers and dissolved varying concentrations of NaCl into each container. We then used nutrient tablets to measure the nutrient levels of each container every other day for 10-12 days.

Our results show that as the concentration of NaCl increased, the concentration of phosphate and nitrate in the water increased. Thus, NaCl inhibits duckweed’s ability to store nutrients.

Effect of UVC Light On Saccharomyces Cerevisiae Growth by Jill Dugan (VI), Emily Samaro (V), and Sydney Stovall (VI) Yeast are single-celled organisms that are easy to study and share a similar eukaryotic structure with human skin cells.eukaryotic cells.To simulate the damage to skin cells caused by the sun’s UV rays, we designed an experiment that measures the growth of yeast cells exposed to UVC light. UVC light has a high energy level and is the most damaging type of UV ray because its short wavelength allows the energy to penetrate deep into cell membranes, which damages DNA. To test the intensity of UVC light damage on cells, we measured the rate of growth for yeast cells that were exposed to UVC light for various lengths of time. When yeast ferments in water with sugar, carbon dioxide is produced, which

creates a layer of foam on top of the solution. We hypothesize that increased exposure to UVC light decreases the growth of yeast cells - and, therefore, the growth of foam. However, we have been unable to accept this hypothesis since our data has not been statistically significant. Rather, our data suggests that there is no correlation between UVC light exposure and the growth of yeast cells.

Eutrophication Via Fertilizer in Lemna Minor by Lily Arrom (VI) and Cal Mahoney (VI) Although fertilizer is helpful for plant growth, it is detrimental in excess. Overfertilization can lead to sudden plant growth with insufficient root systems, as well as a process known as eutrophication: an excess of nutrients on the water’s surface. Overly fertilized water can lead to an increase in the production of phytoplankton, algae, and other microorganisms. In large quantities, these organisms will consume oxygen molecules dissolved in the water, which are needed by plants and animals. Eventually, these organisms are oxygen deprived for too long, which causes them to die.

in pond water were done, and individual fronds of duckweed were grown in 2mL wells. Data was analyzed via image processing software to track the changes in frond surface area. Gaining a better understanding of duckweed’s optimal growing conditions can help to maximize its real-world uses, such as a biofuel and animal feed.

Our research sought to find the optimal level of fertilizer for Lemna minor (common duckweed), and at which concentration the fertilizer causes eutrophication. Serial dilutions of fertilizer

The Effect of Diet on Tenebrio Molitor Survivorship and Fertilizer Potential by Caitlin Schwarz (V) and Carolyn Coyne (VI) Tenebrio molitor, the larva state of mealworm beetles, is of particular interest to at-home research due to the fact that they are easy to handle and have a relatively short larvae stage (four to six weeks). Various studies have determined that tenebrio molitor have the ability to effectively metabolize non-biodegradable waste because of their gut bacteria. However, it was previously unclear whether mealworm survivorship is adversely impacted by a plastic-based diet. Four groups of mealworms fed polystyrene, cardboard, food scraps, or oats (standard diet) were confined to their respective habitats for fifteen days and observations on transformation and survivorship were made every three days.

Figure 1: Trial Average: Mass of Mealworms as a Function of Diet

Figure 2: Death, Transformation, and Beatle Counts at Day 15

Our trials lead to the conclusion that diets consisting of exclusively non-biodegradable plastic, food scraps, or cardboard do not have adverse effects on transformation and survivorship of tenebrio mulitor. Furthermore, via an additional experiment, we determined that fertilizing duckweed populations with frass from each respective mealworm group does not have an adverse effect on plant growth. Further trials are needed to prove if the frass fertilizer has a positive impact rather than merely a neutral one. Through our study, we hope to contribute to an important body of scientific knowledge by further proving how mealworms could help with humanity’s current waste crisis.

The Effect of Nutrient Limitation on the Reproductive Rate of Yeast by Bella Vieser (V) and Sammi Barr (V) Our research project tests the change in reproductive rates in yeast when the intake of glucose is limited. The similarity between yeast and animal cells will allow the conclusions drawn from our yeast study to provide an inference on effects in animal cells as well. Yeast is a single-cell eukaryotic organism that shares many common properties with human cells and therefore can be used in cellular studies. Yeast cells can sense the quality and quantity of nutrients they receive externally due to their nutritional signaling networks.

they have available and maintain homeostasis. We hypothesize that cells will adjust their reproductive rate to their nutrient availability. We hypothesize that the amount of colonies in dishes with limited glucose will stay constant throughout the 7 day cycle, and there will be colony growth in the control.

Figure 1: Example of Yeast Colony Growth and the Grid Counting Method

Nutrient studies were done to test how different levels of availability of key nutrients, such as sugars, amino acids, and nitrogen compounds, would affect the rate of cell growth. All of the studies we found concluded that yeast will adjust their cellular activities based on glucose supply (Kayikci 2015). Their growth rates slowed in order to conserve the energy

The Effect of pH on the Growth of Lemna Minor by Sanjana Biswas (VI) and Meghana Pentyala (V) Lemna Minor is a species of duckweed that was previously proven to exhibit cleaning properties in heavily polluted environments, filtering out pollutants, such as some forms of plastic and light aluminum. Still, there is a lack of research investigating the effects of L. Minor in water contaminated with ammonia (a common pollutant that alters surface water’s pH). The optimal pH for duckweed growth is often unequal to that of surface water and is dependent upon the species of duckweed. To investigate the effects that acidic and basic solutions have on L. Minor, the most common species of duckweed, we performed 3 trials. L. Minor

growth was measured by ImageJ volume calculating software and individual leaves were counted each day. Average survival time was also recorded to find the optimal growing environment, which was found to proliferate most successfully at pH 8. Our data suggest that L. Minor will grow and filter pollutants in ammonia-contaminated water.

The Effects of Ultraviolet Radiation on Cell Death in Saccharomyce Cerevisiae by Michelle Lee (V), Ansley Higgins (V), and Ryder Stine (VI) Saccharomyces cerevisiae is a species of yeast commonly known as brewer’s yeast or baker’s yeast. Yeast, single-celled organisms classified as fungi, are eukaryotic and have DNA in the cell’s nucleus. Yeast is commonly used as a model organism in research because human cells and yeast cells share many cellular processes. More specifically, yeast cell division, also known as budding, closely mirrors cellular division in human cells. In past research, scientists have concluded that Ultraviolet (UV) radiation harms and in some instances kills yeast cells; specifically, UV radiation causes large quantities of phosphate-containing compounds to be lost from yeast cells. It has been proven that a decrease in phosphate directly relates to a decreased reproduction rate of yeast (7). In some cases, high doses of UV light resulted in complete inhibition of proliferation (3). Additionally, time-course studies show that cells treated with relatively small doses of radiation had a slow rate of cell loss over a 24-hour period, while cells treated with large doses of radiation had a high rate of cell loss, reaching a maximum total loss within 4 hours (6). High doses of radiation increase the size of vacuoles in yeast cells; however, when the vacuoles eventually decrease, the cell collapses (5). To determine how UV radiation affects yeast cells, we studied the effect that different strengths of UV radiation had on cell death. In order to test this, the distance between the Petri dishes and the UV light is varied for each experimental group. There are three experimental groups and one control group, with a sample size of three. The control group has no exposure to light and will be kept in a dark environment. The three experimental groups are six inches away from the UV light, twelve inches away from the UV light, and twenty-four inches away from the UV light, re-

spectively. We hypothesis that increased strengths of UV exposure will result in increased cell death. Determining the effects of different strengths of UV radiation on cell death in yeast will lead to a better understanding of how UV radiation affects human cells and leads to cancer.

The Effects of Vinegar on Duckweed Growth by Caeley Feeney (VI), Olivia Gallucci (V), and Gia Graziano (VI) Lemnoideae, an aquatic plant which lies on the surface of still or slow-moving bodies of water, acts as a purifier and supplement for certain animals. The change in growth of Lemnoideae in an acidic environment was tested through a series of three trials. The Lemnoideae growth was tested in four different environments: 75% vinegar-based solution, 50% vinegar-based solution, 25% vinegar-based solution, and a 0% vinegar-based solution. The results suggest that the quantity of Lemnoideae decreases quicker in an acidic environment than in its natural environment (water). These results confirm our hypothesis that the environment in which Lemnoideae inhabits has an effect on its growth and lifespan.

Removal of Ammonia (NH4OH) in Water by Lemna Minor by Jessica Lin (VI) and Sahdev Patel (VI) Ammonia, a chemical that has detrimental effects to human health, is considered a common waterborne contaminant. Commonly found in household cleaners and fertilizers, ammonia can drain into wastewater. High levels of ammonia are toxic for humans and especially aquatic life, as the ammonia runoff contaminates the natural water systems they inhabit. Because no recent studies have tested how well duckweed can absorb ammonia, we sought out to determine whether duckweed has the potential to absorb ammonia (specifically the NH4OH) from wastewater due to its prominence in water contamination. We tested the effi-

cacy of Lemna minor (a widely available duckweed species) in removing ammonia from lab-simulated sewage water by measuring the pH levels in the water, as ammonia’s strong basic properties ensure that there is a direct correlation between lower pH and lower ammonia concentration.

Methods in

Molecular Biology Research. Summary of Research Class

by Anushka Agrawal (VI), Ameera Ebrahim (VI), Massa Godbold (VI), Chris Halada (VI), Alina Irvine (VI), Nav Jha (V), Ariel Li (VI), Kayla Morriello (VI), Kristin Osika (V), Aanya Patel (V), Luca Pizzale (V), Francesca Rainuzzo (VI), Diana Severineanu (V), Carson Shilts (V), and Jason Xiong (VI)

This class, structured around the BASIL (Biochemistry Authentic Scientific Inquiry Lab) Curriculum, works to identify the unknown function of proteins whose structures were recently discovered. Our class is in the process of working to identify the functions of 3CBW, 4DIU, 3H04, 3N2C, 3L1W, 3B7F, 2QRU and 2O14 (Figure 1). We began our investigation by engineering E. coli bacteria to express these proteins for future lab work. Subsequently, we investigated the function of these proteins using computational tools, such as ProMol and BLAST platforms, which allow us to compare these proteins to proteins with known functions. Moving forward, we plan on purifying our proteins in order to test possible substrates, allowing us to determine the enzyme’s function.

Figure 1: The structures of the eight proteins under investigation and their PDB ID code. These proteins are being studied by Carson Shilts and Kristin Osika (2QRU), Francesca Rainuzzo and Kayla Morriello (3H04), Massa Godbold and Aanya Patel (3B7F), Jason Xiong and Chris Halada (4DIU), Anushka Agrawal and Diana Severineanu (3L1W), Ariel Li (3CBW), Alina Irvine and Nav Jha (3N2C), and Luca Pizzale and Ameera Ebrahim (2O14).

Plastic Degrading Enzymes by Sanjana Biswas (VI), Jemma Kushen (VI), and Dr. M. D’Ausilio The rapid and excessive production of plastic has major environmental impacts, adding to air, ocean, and terrestrial pollution. Though traditional recycling methods allow for the reuse of plastic, more than 91% of all plastics are not recycled. Additionally, the current chemical method of plastic recycling produces an inferior grade of plastic that cannot be reused to remake the same item. A growing body of research suggests that microbial plastic degradation methods may produce reusable plastic of equal quality to virgin plastic (plastic made from raw materials, not recycled). Localized strains of bacteria have been found to successfully break down plastic polymers into monomers. The Plastics Microbial Biodegradation Database (PMDB) identifies the sequence of three strains of Streptomyces bacteria from Australia and Russia hypothesized to degrade PET, the most prevalent form of plastic. Structure characterization is an important next step in understanding the kinematics of microbial plastic degradation and confirming the function of these enzymes. Our goal is to purify these putative PETases after expression in E. coli and send the protein samples to a crystallography lab to determine their structures, which can then be used for further bioinformatic analysis of enzyme activity. by Jerry O’Mara (VI) and Jordan Mandel (VI) Present estimates suggest that of the 359 million tons of plastics produced annually worldwide, 150–200 million tons accumulate in landfills or the natural environment. Polyethylene terephthalate, also known as PET, is the most abundant polyester plastic, with almost 70 million tons manufactured annually worldwide for textiles and packaging. Current recycling methods are incredibly costly, de-incentivizing large companies from recycling plastic. However, climate change and pollution have forced scientists to research and develop sustainable and green cutinase-catalyzed PET recycling methods. PET degradation enzymes can be used to hydrolyze polyethylene terephthalate (PET) to its monomeric units so that it can be recycled completely. During our research, we found a leaf and branch compost cutinase (LCC), also known as 4EB01. 4EB0 is a hydrolase that can be easily expressed in E. coli and is well suited for our research given its low activation temperature, relatively high PET hydrolysis activity, and overall thermostability. In our research, we have studied PET degrading enzymes to understand which enzymes would work best in an experiment carried out at Pingry. We have transformed and expressed the protein of interest in E. coli cells and wish to purify 4EB0 by attempting to follow an assay that, if done correctly, will depolymerize up to 90% of a plastic water bottle in ten hours1, breaking it down to its original monomers. These monomers are the building blocks that create PET products and will allow easy reuse by recombining the monomers into new products with no loss of function to the plastic. LCC and other cutinases can help eliminate the need to produce more PET and instead help recycle the existing supply of plastic sitting in landfills. Works Cited Silva, C., Da, S., Silva, N., Matamá, T., Araújo, R., Martins, M., Chen, S., Chen, J., Wu, J., Casal, M. and Cavaco‐Paulo, A. (2011), Engineered Thermobifida fusca cutinase with increased activity on polyester substrates. Biotechnology Journal, 6: 1230-1239. https://doi.org/10.1002/biot.201000391 PlasticsEurope. Plastics—the facts 2019. An analysis of European plastics production, demand and waste data. PlasticsEurope https://www.plasticseurope.org/application/files/1115/7236/4388/FINAL_web_version_Plastics_the_facts2019_14102019.pdf(2019).

Bioinformatic Characterization of 3DS8 by Sanjana Biswas (VI) and Jemma Kushen (VI) Introduction Following the development of crystallization techniques, the early 2000s saw an initiative to solve the structure and sequence of many proteins. This project resulted in a plethora of proteins with known structures and uncharacterized functions. Our goal is to classify the function of our protein of interest: 3DS8 (PDB ID). To determine the function of 3DS8, we conducted plasmid purification. 3DS8 was transformed into bacterial cells and purified through lysis. During this time, we began bioinformatic analysis of homologous proteins (ProMOL, Dali). After finding a predicted E.C., 3.1.1.3, further analysis (PyRx) was conducted to find ligands and develop an assay. Based on our research, we have determined the putative function of 3DS8, triacylglycerol lipase, and we plan to confirm this hypothesis through an assay.

aligned to query protein; nres is the total length of the protein. Higher z-scores indicate more significant results. Homolog Sequence Alignment

Figure 3: These potential homologs all aligned with the putative active site (Ser102, Asp188, His222) of 3DS8 Potential Ligands

Structural Homologs

Figure 1: ProMOL active site analysis results for query protein 3DS8: Levenshtein distance of 0 indicates all residues in query protein were found in match. RMSD indicates the average distance between alpha carbons of the query protein aligned with the match, centered on the catalytic residues. Figure 4: Prediction of substrate binding using PyRx and ProMOL. Known ligands of the putative EC (3.1.1.3) were chosen, and their binding affinity and binding location was determined through PyRx analysis. Polar interaction of substrate to active site was determined though ProMOL.

Sequence Homologs

Figure 2: DALI structure analysis results for query protein 3DS8: L-align is the length of sequence \

Conclusions and Further Research Due to 3DS8’s structural similarity to 1AZW and 3LIP (ProMOL), we believe that 3DS8 is a triacylglycerol lipase (3.1.1.3). It is predicted that RIL and OLA, ligands known to catalyze, will fit in 3DS8’s binding pocket (PyRx). To test potential fatty acid ligands, we will use Kwon and Rhee’s simple colorimetric method.

Protein Purification

Figure 5: Whole-cell lysate SDS-PAGE analysis of protein induction: BL21(DE3) cells transformed with pMCSG19. Lane C = uninduced control culture. Lane I = induced culture. Band shown is around the expected 73.3 kDa for the protein of interest.

Figure 6: Analysis of column chromatography purification of 3DS8. SDS-PAGE analysis of Ni-NTA column chromatography performed on BL21(DE3) cells transformed with pMCSG19. Arrow indicates expected size (73.3 kDa) of protein of interest. Lanes are in chronological order from lysis to elution, with protein applied to resin between the steps that produced “supernatant” and “supernatant flow-through”. The leftmost ladder is labelled with molecular weights in kDa.

SMART Team. Fatty Acid Regulation of Toxicity in Vibrio Cholerae: A Structural Analysis of Unbound, Ligand Bound, and DNA Bound ToxT by Cole Morriello (V), Kristin Osika (V), Ashleigh Provoost (V), Belinda Poh (V), Caitlin Schwarz (V), Maureen Schwarz (V), Anton Volchenkov (V), Allen Wu (V), Dr. F. J. Kull, and Dr. M. D’Ausilio Abstract Vibrio cholerae is an infectious bacterium that causes activation of transmembrane ion channels responsible for mediating the tonicity of cells in the small intestine. Activation of these ion channels results in osmotic movement into the small intestine, thus dehydrating cells and the infected individual. The transcription factor, ToxT, upregulates production of cholera toxin (CT) and the toxin coregulated pilus (TCP), key virulence factors in the progression of cholera. ToxT, a member of the AraC family of transcriptional regulators, has been crystallized in the presence of an unsaturated fatty acid (UFA) revealing a repressed form of the transcription factor in a conformation that prevents DNA binding. Two α-helices in the DNA binding domain (DBD), α6 and α9, must be parallel to bind consecutive major grooves in the DNA. In the UFA-bound structure, these helices are non-parallel, supporting the model that ligand binding to ToxT prevents DNA binding. ToxT has been shown biochemically to be homodimeric when bound to DNA; however, crystallization of the DNA bound form of ToxT has been challenging due to instability of the complex. Recently, a natural variant of ToxT with increased solubility has been crystallized in the presence and absence of UFA. Crystallization of the apo form of ToxT required a lysine to alanine substitution at position 231 in the ligand-binding pocket of the protein to prevent UFA binding. Although the structure of apo-ToxT is high-

ly similar to the UFA-bound structure, the apo version was shown to be more flexible than ligand-bound ToxT, supporting a model in which α6 and α9 in each member of dimeric ToxT are parallel and capable of binding to DNA. Using the structures described in this study, The Pingry School SMART (Students Modeling A Research Topic) Team used a 3D-printer from the Milwaukee School of Engineering (MSOE) to model the apo structure and the dimeric DNA-bound model of ToxT (Figures 1 and 2). These models support an in-depth analysis of ToxT structural conformations in support of the proposed mechanism of regulation. A detailed structural understanding of ToxT may support future development of vaccines and therapeutics for cholera and other pathogens whose toxicity is regulated by AraC family members. Background Cholera is caused by the bacterium Vibrio cholerae, which infects the lumen of the small intestine. In order for Vibrio cholerae to be infectious, it must produce two virulence factors, TCP and CT, that are both regulated by the transcription factor ToxT. ToxT is a regulator of the AraC family that promotes gene expression. Each year, there are around 1.3 to 4.0 million cases of cholera leading to around 21,000 to 143,000 deaths. Most cases are asymptomatic, but about 10% of the cases annually cause severe symptoms such as diarrhea and vomit-

Figure 1: ToxT dimer complex bound to DNA. The regulatory domain is highlighted in blue and alpha helix 3 is highlighted in green. Alpha helix 3 helps the subunit to dimerize. It is still unclear whether ToxT needs DNA to dimerize or if the dimerization of ToxT creates a favorable environment for dimerization. In white is the DNA binding domain and in purple are helices 6 and 9. The helices are the DNA binding helices, which will bind to the major grooves of DNA. ing, which subsequently lead to dehydration and death. Cholera is spread through drinking water, or though the ingestion of food that has been contaminated with cholera. As a result, cholera affects mostly places that do not have clean drinking water or poor sanitation. Apo and Ligand Bound Conformations of ToxT In ToxT’s apo conformation, alpha helices 6 and 9, highlighted in magenta, are able to dimerize to DNA due to their flexible conformation (Figure 2). Conversely, in the ligand bind confirmation ToxT adapts a more rigid structure and the helices form an antiparallel arrangement. Alpha helix 3, highlighted in green, is hypothesized to be partially responsible for the ability of ToxT to dimerize to DNA. The dimerization domain is highlighted in blue, interacting with the DNA binding domain shown in white.

Figure 2: Model of apo ToxT. It is hypothesized that in this state, ToxT assumes a more flexible conformation.

Significance Currently, the structure of ToxT bound to DNA is modeled based on synthesized data due to the fact that the structure is too unstable to crystalize in full. It is necessary to identify the definite structure and sequence of ToxT in order to further understand its mechanisms. This information would prove useful in designing specific inhibitors that would bind more preferentially to ToxT’s regulatory domain, even after it has entered the intestine; the exact structure is needed to create the most ideal inhibitor that would bind with the highest affinity to the regulatory domain of ToxT. Additionally, ToxT is an AraC family transcriptional regulator, so research regarding ToxT would promote a greater understanding of

AraC family proteins as a whole. These proteins all have similar mechanisms and respond to fatty acids in a similar way; once bound to a fatty acid, a conformational change inhibits their binding to DNA. This conformational change is associated with loss of flexibility in the alpha helices 6 and 9, which causes the helices to become antiparallel, therefore, binding with DNA is inhibited. If one molecule can inhibit all AraC family proteins, it will prove useful in the treatment of disease, especially in countries where infections caused by AraC family regulators are most prevalent. The structure of ToxT helps us to better understand its mechanisms, which ultimately will be highly useful in formulating vaccines and treatments for diseases caused by AraC family proteins.

Understanding the Functions and Variants of the SARS-CoV-2 Virus by Cayden Barrison (IV), Ainsli Shah (IV), Katie Lin (IV), Camille Collins (IV), Lauren Kim (IV), Alexander Henry (IV), Adrian Kurylko (IV), Spencer Jahng (IV), Charles Jiang (IV), and Dr. M. D’Ausilio Since it was first detected in Wuhan, China in December 2019, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has evolved into a global pandemic that has resulted in over 2.8 million deaths worldwide. SARS-CoV-2 reproduces via viral entry and subsequent hijacking of the host cells via the human angiotensin-converting enzyme 2 (ACE2) receptor to generate daughter virions. The primary protein involved in this process is the viral spike (S) protein, a 1273 amino acid long protein embedded in the lipid membrane of SARS-CoV-2, which is divided into two domains: the S1 subunit and the S2 subunit (Figure 1). The S1 subunit contains a receptor binding domain (RBD) which can recognize and bind to the ACE2 receptor, which is located among negatively charged ridges on the host cell’s surface. Once the virion (complete viral particle outside of a host cell) is bound to the host cell’s surface, the fusion peptide (located in the S2 subunit) fuses the host and viral membranes, allowing viral entry.

Figure 1:The SARS-CoV-2 spike protein

The 2020-2021 Pingry SMART (Students Modeling A Research Topic) created structural models of the spike protein and researched previous studies that analyze several aspects of SARSCoV-2. The team focused on three specific topics: interactions between the spike protein and

Figure 2: The predominant mutation for the UK COVID Variant (N501Y). The SARS-CoV-2 spike protein is shown in blue, while the ACE2 receptor is in green.

the human ACE2 receptor, variants of the spike protein, and drug targets on the spike protein. The SARS-CoV-2 virus is closely genetically related to SARS-CoV, which emerged in 2002 as the first coronavirus to be a pandemic threat. Our research dove into important residues, such as Lys417, that are involved in binding to ACE2, while analyzing the few structural differences between SARS-CoV-2 and SARS-CoV that potentially effectuate the greater virulence of SARS-CoV-2. While focusing on variants of the spike protein, recent and prevailing strains of SARS-CoV-2, such as B.1.1.7, B.1.351, and P.1 were investigated. Our aim is to determine what distinguishes the spike proteins, ascertain how the spike proteins affect transmissibility and deadliness, as well as create models of the variant spike proteins. Ultimately, this information is helpful to better understand how these new strains have heightened binding affinity and increased infection rates as well as the fatality of the virus. Figure 3: a (left). Receptor binding motif of the SARS-CoV-2 spike protein (blue) and part of the ACE2 receptor (green). b, c (below). A closer look at the amino acid interactions between the aformentioned proteins.

a 24

HIRT.

Humanities Independent Research Teams

Empathizing With “The Uncanny” in Children’s Storytelling and Bargaining

by Carolyn Coyne (VI), Claire Keller (VI), Justin Li (VI), Sophie Pollard (VI), Sanjana Biswas (VI), Olivia Telamaque (V), and Andrew Wong (V) Background In previous years, the project’s data established a correlation between how children bargain with humans and how they represent humans in their stories according to the altruistic punishment framework. We want to see if this correlation endures when applied to non-human entities in the Uncanny Valley. Fundamentally, bargaining is a comparison between value propositions that determine what is fair, therefore, writing can be used as a mechanism for setting, testing, and evolving human altruism. If children bargain more favorably with animals, this would demonstrate the development of a moral imperative to advance the cause of animals. Method Fifty-eight third graders and fifty-five fifth graders were asked to write a story with specific instructions to include at least one non-human character, a story arc (beginning, middle, and end), and illustrations of their characters at the conclusion of their story. Assuming that a system based on altruistic punishment is prevalent in each story, the role of a non-human character within each story was measured on a scale of 0 to 2:

gaining with different players or opponents (whose pictures are shown on a computer). There are always ten tickets to be split. At the end of the game, the subject can go over to the ticket booth and cash in their tickets for stickers (the players would do the same). The more tickets the subject gets, the more stickers they get. The subject will play 50 “rounds” of the game, each with a new, unique player. There are ten human players, ten dog players, ten robot players, ten squirrel players, and ten humanoid robot/android players. First, the human players will split the tickets. The first split offered will be 5,5 (subject gets 5 tickets, human gets 5). The splits will then numerically proceed up to a 10,0 split and then down to a 0,10 split. If the subject says yes to a proposed split, then the deal happens as offered. However, if the subject says no, no one gets anything. Saying “no” goes against self-interest and has

0: the antagonist, or “bad guy.” 1: neutral, not the causal force behind the conflict 2: the protagonist, or “good guy.” The students were then asked to play an ultimatum game. The game was as follows: the game’s proctor will explain that the subject will be bar-

Figure 1: Our Modified Uncanny Valley Graph à la Masahiro Mori

Figure 2: Table of Average Treatment and Bargaining of all Entities been proven by earlier experiments to line up with the altruistic punishment framework; the subject is punishing someone for being unfair. After the ten initial human deals, the other four groups will try to bargain with the subject in the same way. Finally, the roles will be reversed. Now, the proctor states that the same rules apply, and the subject states what they would offer each type of player (human, dog, robot, squirrel, humanoid robot/android). All data points (accepts, rejects, and final splits) will be recorded, yielding 55 data points from each subject.

Figure 3: Average Treatment of Non-Human Entities To make this experience as life-like as possible, the proctor will explain to the subject that the tokens offered to the dog, or the other specified entity, will be cashed in by that entity at the end of the game for a prize of their choosing. Through the analogy of cashing in tokens at an arcade, the experience becomes accessible for the young subject, who may not understand the complexities of the game.

After the students completed their stories and the ultimatum games, we analyzed the results, and all 115 characters in the children’s stories were put into one of these three entity buckets that correspond to the simplified version of Masahiro Mori’s Uncanny Valley graph (Figure 1): Dog (most familiar to subject) Squirrel Humanoid Robot/Android (least familiar to subject) The entity that best represented the character was then examined in the bargaining data to find the subject’s split (the number of prizes s/he offered to that entity on a scale of zero to ten) with the corresponding entity. This method was used to determine the correlation between a subject’s portrayal of a character in a story and the same subject’s offer to that character in the ultimatum game. Some data points were pruned out of the dataset, either due to the student’s story not meeting the criteria we specified, or the student not understanding the ultimatum game. An average, separated by character entities, was taken of the treatment and bargain (data from third and fifth grade was pooled together). The raw averages are listed in Figure 2, while the treatment and bargaining averages are plotted in Figures 3 and 4, respectively. Analysis From our data, we observed a positive correlation between the familiarity of non-human characters on the uncanny valley (Figure 1) and average treatment of the entities. The less familiar a non-human character was, the less its average treatment and offered split was. This relationship between

familiarity, treatment in the story, and bargaining in non-human entities is in accordance with our Modified Uncanny Valley Graph in Figure 1. It is important to consider the bias of our sample. Further surveys of our data set indicated that a significant portion of the subjects had or had been exposed to dogs and other creatures. In effect, many of our students had some form of an emotional connection to dogs, and we speculate that all of the students in our data set were naturally predisposed to treat dogs as morally superior beings. Thus, we saw inflated numbers in treatment and bargaining for dogs. When it comes to the data regarding human characters, the data does not reflect what Figure 1 predicts. For average treatment, human characters,

Figure 4: Average Bargaining of Non-Human Entities

who had an average treatment of 1.11, were generally treated slightly worse than dogs (1.36), but better than squirrels (1.07). Surprisingly, humans received a score of 3.375 for average bargaining, the lowest average bargaining of any entity. Outside research points to a potential explanation for the difference in treatment of human characters and non-human characters. Wall Street Journal columnist and professor at the Santa Fe Institute Dr. Alison Gopnik claims that young children do not have the complex known as the uncanny valley, meaning that they are unbothered by animations or robots, which appear nearly human to adults. This points to the idea that children are more familiar with non-human entities, such as animals and robots, than adults. This would mean that younger children would be more empathetic towards animals and robots, offering more generous splits and awarding them protagonistic roles in their stories, compared to older 5th-grade children. Further experiments will be needed to investigate this correlation and determine at which general age children’s preferences change. Due to the limited scope of our data, a larger data set will also be needed. From our current data and future studies, we hope to better understand young children’s empathy and connection to the animal world and technology. In a rapidly changing technoscape, our findings will become more relevant.

Afrofuturism Research Project by Sydney Stovall (VI), Zara Jacob (VI), Herbert Toler (V), Josephine Alston (VI), Ms. E. Lear, and Ms. J. Watkins Currently, one of our largest projects is creating a survey titled “Decolonizing the English Curriculum.” The survey focuses on understanding necessary changes to the 9th and 10th grade English curriculums (if any).This project includes perspective questions that ask what students have taken away from their two years of non-elective English courses. Our motivation for this project has been the recent overhauls in the history curriculums for the 9th and 10th grades and our desire to hear students’ opinions on their experiences in English class. Moreover, we would like to know if they believe their voices are reflected in curricular texts.

IRT.

Independent Research Teams

Artificial Collagen for Use as a Biomaterial by Luc Francis (VI), Michelle Lee (V), James Houghton (V), Alexa Drovetsky (IV), and Dr. P. Haven

Collagen, the most abundant protein in the human body, plays important structural and functional roles in many physiological and pathogenic processes. At the molecular level, collagen is made of three polypeptide chains that form a secondary triple helical structure, which self-assembles in a lateral staggered association to create fibrils. The unique 67 nm gap-overlap repeat– known as D period– and behavior of the collagen molecule arise from the repeating Glycine-X-Y pattern in its primary structure. The ultimate goal of our project is to create a collagen mimetic peptide that can model the physical and chemical properties of natural collagen. FACT-152 (Fragment of A Collagen Trimer), our designed collagen peptide, is a 152 residue protein composed of three separate, highly stable regions selected from the α-I chain of type I human collagen. In the first stage of our research, we are working to create the FACT-152 gene. Our plasmid design includes the FACT-152 gene as well as various regions that aid stability, bacterial expression, and protein purification in vitro. Currently, collagen is most commonly purified from animals for use in the medical field. This process is expensive, tedious, and often results in high degrees of variation. Generating collagen mimetic peptides that successfully replicate human collagen could provide a safer, more cost-effective alternative. We plan to provide critical research that deepens our knowledge of collagen’s structure as well as determine the best method of creating viable artificial collagen that can be used as a biomaterial.

Figure 1: Hierarchical Organization of Collagen Structure A. Collagen Fibrils under an Electron Microscope B. Staggered and Intertwined arrangement of triple helices within a fibril C. Different stages of Fibrillogenesis from the Primary Structure (Bottom)

Drover: Drone-Rover Communication for Pathfinding by Noah Bergam (VI), Justin Li (VI), Micah Elwyn (VI), Sam Henriques (V), Diego Pasini (IV), Shaan Lehal (IV), and Dr. M.P. Jolly Due to their unique abilities and visual perspectives, integrating an unmanned aerial vehicle (UAV, or drone) and an unmanned ground vehicle (UGV, or rover) into a single autonomous system (a drover) is an enticing prospect, with applications currently being explored in wildfire tracking, garbage collection, and extraterrestrial reconnaissance. This project is concerned with building a drover cheaply and effectively, and enabling it to pathfind in unknown environments. The primary challenge in developing this pathfinding system involves the implementation of a multi-robot simultaneous localization and mapping (SLAM) protocol that can integrate the perspectives of the drone and the rover into a global map in real time. This project is currently developing a solution by implementing an Extended Kalman Filter SLAM scheme which can function on three sources of information: 3D images captured by a stereo vision setup on the rover, 2D images captured by a downward-facing camera on the drone, and the states of both the drone and rover according to GPS and other sensor readings. In addition to SLAM, the drover must be able to effectively maneuver through unknown environments. As such, this project proposes a cooperative pathfinding protocol called Longterm Direction, Short-term Correction (LDSC), where the drone draws and dictates long-term paths for the rover, and the rover identifies smaller objects in its path and dodges them in the short-term. The drone’s path-planning ability involves turning images into node maps and applying Djikstra’s Shortest Path algorithm from a starting point to an end point. In developing the drone end of the LDSC algorithm, it was found that weighing the nodes by color, rather than predetermining the path viability of each pixel on an absolute basis, was more effective for pre-planning paths on roads. Ultimately, the LDSC algorithm

will be able to generate these paths on continuous footage, translate them into motion instructions for the rover, and allow the rover to efficiently alter these instructions in the short term.

Effect of Dietary Restrictions on the Cognitive Ability of Drosophila melanogaster by Ameera Ebrahim (VI), Bella Vieser (V), Kimberly Wang (V), Mirika Jambudi (IV), and Dr. C. Kirkhart Previous research has proven that a calorierestricted diet increases the lifespan of Drosophila melanogaster. Our experiment aims to test the proportionality between the diet-induced lifespan elongation and the flies’ cognitive skills. Our experiment consists of two groups of flies: a control group fed molasses-based fly food (control) and an experimental group fed diet food (dilution of the regular food with an agar solution). After breeding flies on the two diets, we will test the memory of both groups using an odor T-maze. Last year, we ran a trial to test the lifespan of flies fed either the control diet or the calo-

rie-restricted diet. However, due to complications posed by the pandemic, we were unable to carry out our experiment in its entirety. This year, we repeated our lifespan experiment to verify last year’s results. Ultimately, the data supports our hypothesis that a calorie-restrictive diet correlates with an increase in the lifespan of drosophila melanogaster (Figure 1). Now that our initial hypothesis has been confirmed, we plan on continuing to experiment with the odor T-maze to determine how cognitive and memory abilities correlate with this proven extended lifespan.

Figure 1: The results are a composition of data from 9 different vials: 4 control vials and 5 calorie-restricted vials from the lifespan experiment. The total number of flies remaining in control and calorie-restricted vials were counted over time, and the data standardized to show the percentage of flies remaining in each group over a period of 100 days. Over time, the difference between the number of flies alive in the control and calorie-restricted vials increased. As the number of control flies approached zero, the calorie-restricted diet flies still had 1/3 of their total population alive. The data indicates that a calorie-restricted diet keeps the flies alive for longer than the control diet does.

Metagenomic Analysis of a Compost Microbiome by Ryan Arrazcaeta (V), Ainsley Ellison (V), Milenka Men (IV), Max Watzky (IV), and Dr. C. Sparrow

Metagenomics is the study of genetic material taken from a population of organisms. The composition of microorganism populations changes in response to factors in the environment such as temperature and pH level. By performing metagenomic analyses of genetic material from the Pingry composter, we hope to understand changes in the microbial community of the composter in response to these conditions. During the 2019-20 school year, the team harvested a small number of compost samples and successfully isolated DNA, submitted it for next-generation sequencing, and obtained interesting and useful data on the complex microbial community. This year, the team collected compost samples approximately once per month, both at the distal end (finished compost) as well as 10 feet from the distal end. To further characterize the composter environment, we used a HOBO data logger to mea-

sure the internal temperature of the composter. The data logger was put in the composter, and we let it naturally move through the composter as it is turned. The data logger takes the temperature every hour, and it takes about 4 months to go through the composter. Further characterization of the composter material includes measurement of pH on fresh samples. The remaining samples are dried in a desiccator for several days, and then DNA is extracted using the Qiagen power soil pro kit. Several purity tests are required for quality control including agarose gel electrophoresis and spectroscopy. This year’s DNA samples will be sent to GeneWiz for next-generation sequencing, which returns several different types of data. Our research will provide a natural history of the composter and will contribute practical information for the future operation of the composter.

Figure 2: The Pingry Composter

Figure 1: Relative abundance of bacteria families from our 2019 data set.

Plastic Degrading Enzymes by Helen Liu (VI), Tyler Headley (V), Lukas Strelecky (V), Maya Khan (IV), John Paul Salvatore (IV), and Dr. M. D’Ausilio The United States of America only recycles about 35% of its total waste, sending nearly 150 million tons of trash to landfills each year. When plastic is sent to landfills, it takes up to 450 years for it to fully biodegrade; often, the plastic ends up invading oceans or forests, where it poses a serious threat to native species. It is necessary to find an efficient way to recycle plastic in order to mitigate the damage done to our home planet. In our project, we work with a PET (polyethylene terephthalate, a commonly used plastic) depolymerase, often abbreviated as PETase. It has the ability to break PET polymers into smaller monomers, which can be reused to make new plastic. Since plastic in the environment is becoming an increasingly serious issue, the scientific com-

munity is seeking to study and improve PETase – namely, its efficiency and thermostability. We plan on contributing to the understanding of PETase by analyzing the effects that various mutations have on PETase. In collaboration with a molecular biology class at Deerfield Academy, we have obtained various PETase mutations. We will induce protein expression and use a plate-clearing assay to determine their efficiency. In future trials, we will create more mutations and develop an assay to measure thermostability. Overall, though our project is new, our hopes for it are high. We are continuing our mutation-based exploration of a protein in a pertinent field and are excited for all of the possibilities that it will bring.

Protein Purification of Shigella dysenteriae Transcription Factor VirF by Emma Huang (VI), Anushka Agrawal (VI), Arjun Sen (V), Brian Li (VI), Maria Loss (IV), and Dr. M. D’Ausilio Abstract Shigella is a pathogen that is a leading cause of bacterial foodborne illness worldwide. The virulence cascade of shigellosis requires one protein of the AraC superfamily transcription factors, VirF. Other members of this superfamily, such as ToxT, which is involved in the virulence cascade of Vibrio cholerae, show inhibition of DNA binding by a small fatty acid. We wish to determine whether or not VirF can be inhibited in a similar manner. Additionally, we are working to express, purify, and ultimately solve the crystal structures of VirF, which will help reveal the mechanism of transcriptional regulation in S. dysenteriae and could lead to development of novel treatments for Shigella related illnesses. Introduction The Shigella bacterium causes shigellosis, specifically mammalian gastroenteritis. There are an estimated 80-165 million cases of gastroenteritis globally each year due to the Shigella species– 600,000 of which result in death. Symptoms vary from intestinal inflammation and diarrhea to nausea and vomiting. Infection begins with the ingestion of contaminated food or water. Many of the genes required for intestinal penetration, invasion of host cells, intestinal disease, and diarrheal disease are carried out by the major regulatory protein VirF located on the PINV protein as part of the regulatory cascade. This member of the AraC protein family is a DNA-binding protein that activates toxicity factors genes: icsA and virB. These genes are then able to activate other downstream effectors involved invasion of the cell wall and host infection. Thus, inhibiting VirF would inhibit the whole virulence cascade of shigellosis.

Methods Our team will be working at the laboratory at The Pingry School under the guidance of Dr. M. D’Ausilio and Dr. C. Kirkhart. We received the VirF plasmid dried on filter paper from Dartmouth’s Kull Laboratory in January 2021. We rehydrated the plasmid in distilled water and proceeded to conduct bacterial transformation to replicate the plasmid. In the coming weeks, miniprep will be used to isolate the plasmid from the bacteria. Subsequently, PCR will be used to amplify DNA, and expression vector plasmid with an N-terminal his-tag will be used to express the VirF gene. Once successfully expressed, VirF will be transformed into bacterial cells to grow colonies on a Petri dish. Final analysis will be done by isolating the VirF protein using affinity chromatography. Future Steps Our goal is to isolate the VirF proteins, which will help us to understand the mechanism of transcriptional regulation in S. dysenteriae. Understanding how to inhibit these structures could lead to development of novel treatments for Shigella related illnesses. We were able to transform the VirF plasmid for replication, but our time in the lab has been limited due to scheduling complications from COVID-19. When we are back in the lab, our next steps will be to perform miniprep to isolate the plasmid from bacteria. In the interim, we are planning to develop a plasmid map that will help us better understand the DNA, which could show us other genes of interest, restriction enzyme sites, and the name and length of our plasmid.

Quantifying the Effect of Structure on Neural Network Training by Rhea Kapur (VI), Emmet Houghton (V), Katherine Xie (V), Olivia Taylor (IV), Evan Wen (IV), and Dr. M.P. Jolly With neural networks becoming increasingly common tools for many diverse applications of computer science (self-driving cars, language processing, image recognition, healthcare – the list goes on), it is of the utmost importance that they are as accurate and efficient as possible. It is believed that the structure of neural networks (number of nodes, layers, connections, etc.) has an impact on both of these variables, but neural network structure is understudied and the extent of this structural impact is unknown. The ShallowMind project seeks to answer this central question: How, and to what extent, does

the structure of a neural net affect how efficiently and effectively it trains? To examine this, we create our own mock datasets with varying boundary functions and levels of noise. We then generate thousands of neural networks with varying structures and test each one against a fixed dataset in a classification problem, collecting data at each epoch on the accuracy, overfitting index, weights, and other attributes of the network. Through subsequent data analysis, we identify and examine underperforming structures, the impact of increasing structural complexity on the efficiency of a network, and much more.

Figure 1: Examples of linear and parabolic datasets and varying noise generation.

Figure 2: Effect of structural complexity (nodes) on overfitting, as measured by the numerical difference between validation and training accuracy. In the parabolic dataset (with noise), after the total number of nodes in the structure exceeds 13, the overfitting index increases drastically; this indicates that these larger structures are unnecessarily complex and overanalyze the noise in the relatively simple parabolic dataset.

Figure 3: Structure Volatility vs Final Accuracy. This graph confirms our hypothesis that there is an inverse correlation between structure volatility and the accuracy of a structure. Structure volatility is a measure that we created to define how much the number of nodes in adjacent hidden layers varies.

Quantifying the Phototactic Memory of Chlamydomonas Reinhardtii by Julian Lee (VI), Christine Guo (V), Sarah Kloss (V), Max Liu (IV), Leo Xu (IV), and Dr. A. Samadani Phototaxis, the behavior describing the movement of organisms towards or away from light, allows some organisms to find ideal locations for absorbing light for photosynthesis, which plays a crucial role in cell survival. Chlamydomonas reinhardtii, a unicellular green algae that phototaxes towards green light, serves as a popular model organism for phototaxis. Chlamydomonas reinhardtii has been studied at the molecular level to examine organisms’ use of Rhodopsin proteins to cause phototaxis and at the population level to examine collective motility. We have designed an Figure 1: Sample of cells tracked by the fullyalgorithm to track cells in the second and third automated algorithm dimensions, which has allowed us to quantify the behavior of C. reinhardtii during directed motility to random motility. We quantify this behavior through the use of phototaxis indices, a value ranging from -1 to 1, that describes the correlation between the cell’s movement and the location of the light source. We aim to determine if C. reinhardtii has phototactic memory, which can be quantified as the time in which the algae continues moving in the direction of a light source after the light source has been turned off. The existence of a phototactic memory would prompt further research into the mechanism that allows for this Figure 2: Sample of cells tracked by the semimemory, as well as the potential impact of phoautomated algorithm totactic memory for functions beyond motility.

Figure 3: Experimental setup depicting cell placement under the microscope.

The Effect of KIF11 Activity on YAP Localization by Lily Arrom (VI), Luca Pizzale (V), Elodie Wardle (V), Matthew Oatman (IV), and Dr. C. Sparrow

Our project is focused on the relationship between two proteins: KIF11 and YAP. KIF11 is a kinesin, or the “tractor of the cell”, which is responsible for pulling apart chromosomes during mitosis and transporting other cellular components. YAP, on the other hand, is a transcription factor that activates cell growth and sometimes plays a role in cancer. Inactive YAP– located in the cytoplasm– has phosphate groups, but when the Hippo pathway dephosphorylates it, YAP moves into the nucleus and becomes active (Figure 1). We have been in touch with Dr. Gao and his lab at Rutgers University, where they hypothesize that KIF11 and YAP are linked together by their interactions with other proteins. Our goal is to test this hypothesis by culturing SW480 cells, manipulating their KIF11 activity, and performing western blots to observe changes in YAP activation. Finding a new way to control YAP could prove helpful in targeted cancer therapies. In the 2019-2020 school year, we inhibited KIF11 in SW480 cells by treating them with varying concentrations of a drug called Ispinesib (a small-molecule inhibitor). Subsequently, we separated these cells’ nuclear components from the cytosol via centrifugation to track YAP’s localization. Once we ran a western blot with these samples (Figure 2), we observed that as the concentration of Ispinesib increased, the amount of YAP in the nucleus decreased. Our results suggest that the inhibition of KIF11 prevents YAP from being activated, but more data is needed before drawing conclusions. Dr. Gao suggested that we repeat the experiment with two additional antibodies: one for histones (nuclear protein) to see if we succeeded in separating the nucleus from the cytosol and another for phosphorylated YAP to see if the Hippo pathway is still dephosphorylating YAP in its predicted location. We hope to complete these experiments with a fresh batch of SW480 cells once it is safe to work in the cell culture room.

Figure 1: YAP’s location in the cell changes with its activation

Figure 2: Inhibition of KIF11 decreases nuclear YAP. Samples taken in February 2020. The darker a band is, the more YAP is present in that location. Note how nuclear (active) YAP fades away as the Ispinesib concentration increases

The Effect of Memory Enhancing Supplements on Amyloid-ß 42 Expression in a Drosophila melanogaster Model for Alzheimer’s Disease

by Aneesh Karuppur (VI), Eva Schiller (VI), Shannen Gallagher (V), Rachel Zhang (IV), Morgan McDonald (IV), and Dr. C. Kirkhart Alzheimer’s Disease (AD), the most common cause of dementia internationally and the sixth leading cause of death in the United States, is an irreversible neurodegenerative disorder that causes loss of memory and cognitive skills. Currently, there is no known cause; however, previous research has associated AD with buildup of amyloid plaques in the brain, formed by a protein called amyloid beta 42 (Aβ42). We aim to determine whether commercially available memory-enhancing supplements lessen or exacerbate the neurodegenerative effects associated with AD by evaluating their impact on Aβ42 expression. Because past research has shown that selective expression of Aβ42 in the eye of Drosophila melanogaster, the fruit fly, creates a detectable rough eye phenotype (Fig. 1), we have chosen it as a suitable model for our project. This year, we established a control data set on our Drosophila melanogaster model of AD, and are currently testing the effects of Donepezil, an FDA-approved drug that specifically targets Aβ42. Last year, we determined that Ginkgo Biloba had no effect on Aβ42 production. In the future, we will continue to perform screens of multiple memory enhancing drugs and observe which have an effect on Aβ42 expression and subsequently AD. By the end of this school year, we will have data for Huperzine A specifically.

Figure 1: Drosophila melanogaster Rough Eye Phentoype

Club Highlights. by Lauren Kim (IV) and Maria Loss (IV)

Anatomy Club provides students with the opportunity to learn the anatomy of various organisms through hands-on dissections. This club is dedicated to fostering interest in and preparing students for potential careers in the biomedical field. The dissections are meant to enhance students’ curiosity and knowledge of this fundamental aspect of biology. Anatomy Club is advised by Ms. L. Torres (Pingry’s anatomy teacher)

and led by student leaders Maria Loss (IV) and Lauren Kim (IV). The dissections are guided by club leaders; therefore, club members are not required to have any past experience with anatomy or dissection procedures. The club is open to all high school students, regardless of grade level. This year, the club will be presenting an in-person dissection of a sheep lung, a fetal pig, and a snake during Pingry’s Research Week!

by Kristin Osika (V) , Jill Dugan (VI), Ainsley Ellison (V), Anika Govil (V), Emily Shen (V), and Sam Wexler (V)

FYI Sci is a student-run organization which aims to convey scientific research discoveries to the Pingry student body through the creation of blogs, presentations, videos, and more. Even in light of the COVID-19 pandemic and Pingry’s hybrid learning model, the 2020-2021 school year has been an incredible success for FYI Sci. Over the summer, Club President Kristin Osika (V) began organizing resources and creating forms, presentations, and emails, culminating in a cohesive plan for the year. In September, FYI

Sci accepted over twenty new members into our four departments: blogs, videos and slideshows, podcasts, and communications. Our members engaged in department-specific projects covering a broad variety of topics. More information can be found on our website: www.fyisci.pingry.org. This school year has been the most prolific to date for our blogs department, which is run by Ainsley Ellison (V). Members have written about topics ranging from “The Symbiotic Infection of Herpes” to “Discoveries in

ing events, and we recently added a Linktree to consolidate supplementary links and resources.

the SARS-CoV-2 Genome” throughout the year. Blogs can be complex and time-consuming projects, which makes the incredible success of the department all the more impressive! Under the leadership of Anika Govil (V), the videos and slideshows department was also incredibly productive. They released a video titled “The Effects of Sleep,” detailing the impact of sleep on the immune system. Members are working on a special video to promote research at Pingry in preparation for Pingry’s Research Week. In the podcasts department, led by Jill Dugan (VI), members write scripts, and record in groups for seven to ten minutes. Our newest podcast, “Gene Therapy,” and past podcast recordings can be found on our website and on Soundcloud.

This year, the communications department is run by Emily Shen (V); this department has amplified community outreach while maintaining a strong social media presence. The communications department has established “fact of the week” on the Upper School announcements document, and has created and distributed a bimonthly newsletter, in which recent projects from all departments are summarized and linked in a single publication for the Pingry community. At the heart of communications are Instagram posts, for which members find photos and write captions describing a topic of interest. This year, we posted about telomeres, chromotherapy, tectonic plates, and more. Instagram also serves as a platform for FYI Sci to promote other projects, including podcasts, blogs, and upcom-

In addition to social media, FYI Sci also maintains its own website, which is frequently updated by our Head Web Editor, Sam Wexler (V). Sam has posted new content, maintained website security, coded embedded forms, and ensured that our website looks wonderful. New to FYI Sci this year is a COVID-19 task force, which was created to tackle the ever-growing body of scientific knowledge surrounding coronavirus. With the support of the head of the podcast department, Jill Dugan (VI), the task force’s primary project was a podcast series; the first episode featured interviews with Dr. M. D’Ausilio and Dr. C. Kirkhart, Pingry biology faculty and research advisors. After winter break, the task force created and distributed a survey to assess Pingry student and faculty knowledge of COVID-19, compiling hundreds of responses in preparation for an interview with Dr. C. Sparrow, another Pingry research advisor who has over 25 years of background in the pharmaceutical industry. This interview will comprise the task force’s second podcast. Finally, FYI Sci will be presenting at Research Week! Our team will lead multiple experiments, engaging students in hands-on activities. During Research Week, we will also assuredly celebrate our members and their projects, as they are the core of FYI Sci. Building off of the progress made during this markedly strong year, FYI Sci hopes to continue to expand its reach for years to come, guided by our motto, “Bringing science to you!”

by Kristin Osika (V), Aneesh Karuppur (VI), Caitlin Schwarz (V), and Christine Guo (V) The Pingry Community Research (PCR) journal highlights student contributions to novel scientific research and promotes students’ interest in science, while emphasizing the importance of critical thinking and the diverse, interdisciplinary nature of scientific research fields. Though PCR only included AP Biology and IRT papers in prior years, our submission criteria have recently become more inclusive to reflect our contributors’ varied interests and their diversity in submission format; we now accept investigative articles, commentary, summer research, and more. This year, Pingry Community Research also produced this

special journal issue, highlighting the work of students who are presenting at Research Week! Pingry Community Research unveiled a new look with its Spring 2021 issue, publishing both in print and online. In the future, Pingry Community Research hopes to increase reader accessibility through the use of digital media. We recently began developing a website, which provides background information on Pingry Community Research and highlights current and past journal issues. In the future, we hope to increase our online presence further.

Girl Code by Rhea Kapur (VI) and Emma Huang (VI) Girl Code is a club focused on empowering women at Pingry to explore and delve into computer science. We have held a wide range of events in the past focused on everything from teaching programming languages to hosting female speakers with experience in CS-related nonprofit or industry work. However, our main highlight is always bringing our club members to the annual FemmeHacks, an all-female hackathon at the University of Pennsylvania! While FemmeHacks has traditionally been held in-person at the Pennovation Center, the hackathon was remote this year due to the pandemic. Girl Code had a great time participating virtually, with three teams submitting projects. Daniela Henriques, Ananya Sanyal, and Julia Eng created HabitTracker, an app for tracking daily tasks that feature inspirational messages upon completion. Divya Subramanian, Zala Bhan, Mirika Jambudi, and Francesca Zarbin created bliss, a self-care and mood-boosting mental health tracking app. And, Rhea Kapur, Emma Huang, Eva Schiller, and Olivia Taylor coded AllerScan, an iOS application

that uses vision and text recognition techniques from Google Cloud’s Firebase ML Toolkit as well as Google Cloud’s translation services to scan food labels (multiple languages supported) for a given user’s allergies. AllerScan won first place for the Best Use of Google Cloud prize, awarded by a team of Google engineers. Girl Code looks to close out the year with a techie movie night or similar bonding event and hopes to be back at FemmeHacks in person next year!

Robotics by Monica Chan (VI), Jemma Kushen (V), Alina Irvine (VI), Dwayne Bazil (IV), Olivia Taylor (IV), Diego Pasini (IV), Guanyun Liang (VI), Laura Liu (III), Alan Zhong (III), Sarah Gu (III), and Keira Chen (III) Do you like robots? Of course you do! Come see Pingry Robotics in action by interacting with our world-championship robots; they can do pull-ups, climb stairs, make half-court shots, raise discs ten feet in the air, and more. At our exhibit during Research Week, you will be able to drive some of our robots, see our robots that got to the world championships, and ask questions to the brilliant team behind them. The Pingry robotics team builds robots to complete specific tasks in time constrained competitions to win points. We compete in FTC (FIRST Tech Challenge) and FRC (FIRST Robotics Competition) and mentor the middle school team. In this year’s FTC competition, points are scored by launching frisbee-like rings into targets. This year’s small robot uses a set of wheels to roll rings scattered around the field into the robot. More wheels guide the rings up a steep ramp and into a temporary storage. The rings are then dropped onto a ramp where a servo pushes them into a flywheel that shoots rings at small targets with tremendous accuracy and speed. Our autonomous program moves the robot according to preprogrammed instructions that use sensor inputs to identify its location on the field and shoot accurately. Throughout the past few years we have been using our skills and experience from FTC and FRC in order to help those around us. Our school values of “intellectual engagement”, “discovery”, and “innovation” were key to our determination and dedication in spite of COVID-19.

Join PCR today. Develop your scientific literacy while learning about current research at Pingry. Email Kristin Osika (V): kosika2022@pingry.org Caitlin Schwarz (V): cschwarz2022@pingry.org Christine Guo (V): cguo2022@pingry.org Mr. D. Maxwell: dmaxwell@pingry.org

The Pingry School 131 Martinsville Road Basking Ridge, NJ 07920 908-647-5555