Issuu

BROAD STREET SCIENTIFIC

The North Carolina School of Science and Mathematics – Durham Journal of Student STEM Research

Front Cover

Marrus orthocanna is a deep-sea siphonophore more alien than earthly. Orthocanna is composed of many individual organisms that work to function as one. These zooids, shimmering on orthocanna's blazing orange nectosome, showcase how evolution can sculpt forms as otherworldly as anything imagined beyond our planet.

Credit: Kevin Raskoff, California State University

Biology Section

The image shows a male siamese fighting fish (Betta splendens) with a striking crowntail fin type, featuring long, spiky, and dramatically fringed fins that resemble a crown. Its vibrant colors and elaborate fin structure highlight its aggressive yet captivating beauty.

Credit: Anandarajkumar, used under terms of the GNU Free Documentation License

Chemistry Section

The image shows Enypniastes eximia, also known as the headless chicken fish, a deep-sea sea cucumber with a translucent, gelatinous body. It has fin-like appendages that allow it to swim gracefully through the water column above the ocean floor.

Credit: Creative Commons License

Engineering Section

This image shows an unidentified coral sample from the Indian Ocean. It is currently in the colection of the Cantonal Museum of Geology in Lausanne, Switzerland.

Credit: (C) adafruit.com used with permission

Mathematics and Computer Science Section

The image shows a very large strawberry squid (Histioteuthis), notable for its reddish, speckled body that resembles a strawberry. One of its most striking features is its mismatched eyes, one large and one small, adapted for detecting light in the deep sea.

Credit: National Oceanic and Atmospheric Administration Fisheries

Physics Section

The image shows Pelagia noctiluca, a luminous jellyfish known for its glowing, pinkishpurple bell and long, trailing tentacles. Commonly called the mauve stinger, it’s often seen drifting through open oceans, emitting bioluminescent flashes when disturbed.

Credit: Matt Alaniz, under Unsplash License

SAJAN PATEL, 2026

BEN SHEEGOG, 2026 ONLINE

VINCENT SHEN, 2025

CHARLOTTE GOEBEL, 2025

Cloning the Anhydrobiosis Protein CAHS-89226 from Hypsibius exemplaris to Construct Desiccation-Tolerant Azotobacter vinelandii and Escherichia coli

ARETHA DATTA, 2025

24 Effects of Hypercholesterolemia and the Inhibition of AGE-RAGE Pathway on Exopher Formation in C. elegans

ANNELIESE HEYDER, 2025

31 Enhancing the Efficacy of Riparian Buffer Zones Using Native Grasses and Symbiotic Fungi in Relation to Overflowing Hog Waste in Rural North Carolina

SKYLER QU, 2025

44 Menthol Modified Carbon Dot Based Vesicle Nanoparticles with Enhanced Blood Barrier Penetration and Tumor Targeting Ability

VINCENT BARBORIAK, 2025

52 The Development, Synthesis, and Testing of Low-Spin Mn(I)-Centered Catalysts to Aid in Chemical Recycling of Plastic Waste

DYLAN DEES, 2025

59 Design, Synthesis, and Testing of Novel Inhibitor-Based Pharmacological Chaperones for Tay-Sachs Disease

LEAH NIKHIL, 2025

Engineering

68 Assessing the Mechanical Properties and Morphology of Biomimetic Nanofiber Hydrogel Composites for Articular Cartilage Scaffolds

ARIANNA LEE, 2025

Mathematics and Computer Science

77 Improving Robotic Arm Precision and Accuracy Using a Novel Quantum Kolmogorov- Arnold Network Algorithm

SPENCER HIRSCH, 2026 ONLINE

87 "Can You Even?" An Exploration of Polyominos

PEYTON JACKSON 2025, TATIANA MEDVED 2025, MATIAS RELYEA 2025

95 Unified Deep Learning and Machine Learning Ensembles for Robust Depression Detection

RICHARD SHAN, 2026

Physics

107 Analytically Modeling the Gravitational Radiation Generated from a Quasistar System

JEREMY ADAM, 2025

115 Investigating the Morphologies of Local-Scale Dust Storms in the Northern Hemisphere of Mars

JOHNATHAN STRICKLAND, 2025

LETTER from the CHANCELLOR

“The

good thing about science is that it’s true whether or not you believe in it”

Neil deGrasse Tyson

I am proud to introduce the fourteenth edition of the North Carolina School of Science and Mathematics’ (NCSSM) Durham campus scientific journal, Broad Street Scientific. Each year students at NCSSM-Durham conduct significant scientific research, and Broad Street Scientific is a student-led and student-produced showcase of some of the impressive research being done by students on our Durham campus.

Opened in 1980, NCSSM was the nation’s first public residential high school where students study a specialized curriculum emphasizing science and mathematics. Teaching students to do research and providing them with opportunities to conduct high-level research in biology, chemistry, physics, computational science, engineering and computer science, mathematics, humanities, and the social sciences is a critical component of NCSSM’s mission to educate academically talented students to become state, national, and global leaders in science, technology, engineering, and mathematics. Today, nearly 90% of NCSSM students conduct research during their two years at NCSSM. I am grateful for our talented NCSSM faculty and mentors at institutions across

our state who provide these incredible opportunities for students, on two campuses and in our online program, to learn research methods and conduct high quality research. I am proud that at NCSSM, we strongly support developing the next generation of scientists and researchers who will use science to help develop new technologies and solve our most challenging problems for many years to come. Amazing things happen when you bring talented student researchers together with incredible faculty and I am excited that this year’s publication of Broad Street Scientific provides another opportunity to share with the broader community the outstanding research being conducted by NCSSM students.

I would like to thank all of the students and faculty involved in producing Broad Street Scientific, particularly faculty sponsors Dr. Jonathan Bennett and Dr. Michael Falvo and senior editors Advika Arun, Ankit Biswas, Joshua Chilukuri, John Guo, Adrian Tejada, and Nikhil Vemuri. Explore and enjoy!

Todd Roberts

Dr.

Chancellor

WORDS from the EDITORS

Hello and welcome to the Broad Street Scientific, NCSSM Durham’s official journal of student research in science, technology, engineering, and mathematics! Now in its second decade of publication, the Broad Street Scientific continues to evolve and thrive as a journal. This past year we have expanded enrollment in our JTerm course offering, providing a structured introduction to scientific writing and research to a record number of NCSSM students.

These changes have allowed Broad Street Scientific to more fully meet its underlying mission: highlighting and celebrating the excellence and breadth of student research at NCSSM. This year’s edition of the Broad Street Scientific does just that. From explorations into the theoretical physics of quasistars to the development of novel methods for plastic recycling, the student research presented in this edition pushes the boundary of science and innovation.

This exploration of the seemingly familiar is celebrated in the journal’s theme this year: the deep sea. The deep sea is where life began, nurtured by nutrientrich hydrothermal vents thousands of feet below the ocean’s surface. From humble beginnings and simple chemical reactions, the organisms that call the deep sea home gave rise to a vast tree of life that expanded to the land, air, and sea. And yet, the deep sea remains one of humanity's most unexplored frontiers, more alien to us than the surface of Mars.

This past year has been one of great growth and improvement for the journal. It has expanded its efforts to educate on scientific writing and editing, welcoming more students than ever to its annual, 2-week JTerm class. As the journal continues to move through its second decade of publication, it has also remained truer than ever to its goal of celebrating the excellence and breadth of student research at NCSSM. These improvements in our 14th edition allow us to continue showcasing exceptional research, innovative ideas, and collaboration within the students of North Carolina School of Science and Mathematics.

We would like to thank the faculty, staff, and administration of NCSSM, particularly Chancellor Dr. Todd Roberts, Dean of Science Dr. Amy Sheck, and Director of Mentorship and Research for the Durham campus Dr. Sarah Shoemaker. They continue to support and nurture a stimulating academic environment that encourages motivated students to apply their interests towards solving real-world problems. For the next generation of young people who will no doubt change the world, NCSSM serves as a nurturing environment of passion and determination. We also extend special thanks to Dr. Jonathan Bennett and Dr. Michael Falvo for their invaluable support and guidance throughout the publication process. Lastly, we would like to acknowledge Dr. Daphne Klotsa for a fascinating conversation on active matter.

Advika Arun, Ankit Biswas, Joshua Chilukuri, John Guo, Adrian Tejada, and Nikhil Vemuri

Editors-in-Chief

BROAD STREET SCIENTIFIC STAFF

Editors-in-Chief

Advika Arun, 2025

Adrian Tejada, 2025

Nikhil Vemuri, 2025

Publication Editors

John Guo, 2026

Sneha Khetan, 2026

Sindhu Paladugu, 2025

Zoey Zhang, 2026

Biology Editors

Jiah Lee, 2025

Adarsh Magesh, 2025

Grace Pan, 2026

Chemistry Editors

Pragathi Akula, 2026

Chloe Harnphanich, 2026

Vishnu Vanapalli, 2025

Engineering Editors

Sanika Agarkar, 2026

Sarah Zhou, 2026

Mathematics and Computer Science Editors

Physics Editors

Joshua Chilukuri, 2026

Tanuj Karthikeyan, 2026

Ankit Biswas, 2026

Jahnavi Bondada, 2026

Faculty Advisors

Dr. Jonathan Bennett

Dr. Michael Falvo

LIT FROM WITHIN

Nandhini Thangamani

Nandhini Thangamani was selected as the winner of the 2025 Broad Street Scientific Essay Contest. Her award included the opportunity to interview Dr. Daphne Klotsa, Associate Professor in Applied Physical Science at the University of North Carolina at Chapel Hill.

“We are stardust brought to life, then empowered by the universe to figure itself out…”

Pick a leaf off a tree and trace its edges; notice how the thin lines branch from the center stem. Look up at the tree, and study how the tree’s branches bloom from the trunk in the same way. Peripheral nerves spanning your body branch out from the spinal cord just like the tree. Your ear’s curve follows the same mathematical pattern as the swirls of a conch shell, the Fibonacci sequence hidden in plain sight. The whorls of your fingerprints are reminiscent of the grooves of a ram’s horns, a tug of war between phenotypes and genotypes. Eight possible patterns at the tips of your fingers: radial, ulnar, double, central pocket, arch, tented arch, whorl, and accidental. Combinations are practically limitless, infinite possibilities right at your fingertips, comparable to the number of stars in the sky.

The elements forged in the hearts of stars: nitrogen, carbon, hydrogen. Stars, composed of the hydrogen that runs through your veins.

"Are we made of stardust or are the stars made of us?"

We see the stars as beautiful extraterrestrial creations far away from our humble Earth, even though we might be made of the same elements. If stardust truly runs through the veins of the Earth and within every living being, shouldn’t we regard Earth with the same reverence as we do the stars?

To figure out whether we are made of stardust, we first have to find the chemical makeup for each star. We look at starlight through a prism and its absorption spectrum, showing a unique fingerprint for every element in the star. We thought we had an answer then, that their makeups would vary as widely as math can compute. But Cecilia Payne discovered that stars are mostly hydrogen, a little helium, and almost nothing else– “No, this can’t be right.”

“Are we different from everything that surrounds us?”

Looking through the center of nebulas, huge clouds of dust and gas, lit from within like a nightlight by nearby stars, we found the same hydrogen and helium. Stars are so similar to each other, but not to Earth, forming an extraterrestrial community we cannot dream of joining. So, we learn and we theorize and we research.

We look up at the stars for answers, but they just twinkle silently, shining with laughter.

"Maybe our answers will be closer than we thought."

"Maybe, we are the universe studying itself."

Biology echoes and ripples, repeating its own history. Thousands of years of evolution within the theropod lineage led to the sharp look in a rooster’s eyes as it watches the sunrise, the same as the Tyrannosaurus rex as it hunted down its prey.

Physics sings through your fingers as you hold your hand up to the sun, particles of light scattering as you glow orange from within. An entire world of subatomic particles, so small they go unobserved, yet they form our entire world and all of us in it. Electrons play, jumping from energy states like children on the playground, releasing photons with wavelengths of visible light, painting our world.

Mathematics translates these wavelength patterns into a way we understand. It's the language of the universe, taking those waves and describing them through Fourier transfers, as a sequence of sine and cosine functions. It makes the world tangible to our human minds. Disease spreads like wildfire through populations and differential equations trace its origin through finding the rate of change of the infected.

We have always sought to chart these patterns, the desire to understand the world around us transcending generations. Hipparchus, an ancient astronomer, craned his neck up at the sky and cataloged more than 850 stars by hand, painting constellations of unicorns and dragons. We seek meaning beyond what is confined by Earth when we create meaning with every bit of research we conduct.

Research and innovation is driven by our innate curiosity and sense of wonder. Every invention on Earth is made from love. A surgeon created medical latex gloves for his wife who would come home from her nursing shifts with irritated hands. To let his wife enjoy baked goods even though she was allergic to yeast, a chemist formulated baking soda. The original Band-aids were invented by a man who wanted to prevent his wife’s small cuts on her fingers while cooking.

The very first sign of civilization was a healed femur. A broken thigh bone several hundred years ago was a mark

of death, leaving one unable to run from predators or forage for food. Unless they were taken care of for several months, even though they could not contribute to the tribe. They were fed, dressed, protected, and warmed by love.

William Shatner ventured to space, yearning for meaning greater than what is weighed down by Earth’s gravity. He spent his life popularizing the idea that space is the final frontier on the show Star Trek. But when he actually reached this magical destination, floating through black infinity, he turned back to look at us. He saw the blue curve of the atmosphere, ridges of beige and green, white clouds swirling and shifting. So far from humanity, he realised everything worth marveling at is already on Earth.

The smile lines carved into your face like rivers meandering through the land and the delicate mole hidden under your collarbone are unique and special to only you. What you are made of is the same as everyone and everything around you, yet nothing can compare to what is in you. The secrets that you hide in your dreams, wishes blown away on dandelions, and pinky promises made in the corners of your hometown carve out your spot in the universe.

This coincidence that brought us here together– it’s not insignificant and shouldn’t be brushed over. Sit by the ocean and breathe, let the waves send wavelengths to your ears and hear the thrumming chorale of millions of drops of water. Do they sound like discordant crashes, or the steady beat of a strong heart?

Stare at the ocean during nighttime, dangling your legs above its surface and watching it come alive. A black void, mirroring the sky above. A sea full of stars blinking back at you.

We’ve mapped less of the seafloor than we have of the surface of Mars. A pool full of mysteries beneath our feet, yet few dare to venture into its depths.

Octopi potentially dream. If so, these invertebrates have developed a two-cycle sleep similar to vertebrates completely independently, with millions of years of evolution between them. During states of high rapid eye movement (REM) sleep, we dream the most. Yet no one knows why we dream. Whether it is our brain’s subconscious coming to the forefront, routine cleansing of the memory, or another theory, we constantly dream of new ideas for this mystery. Meanwhile, the little octopus slumbers, unbothered.

Dive deeper into the ocean and find almost alien life, not foreign to Earth but foreign to our preconceived notions of what life on Earth resembles. The first fish that crawled on land marked the fork of evolution; one side with land adaptations and the other with aquatic adaptations. With little to no light and a full range of movement unconstrained by gravity, ocean

life has evolved unpredictably. The Barreleye Fish with a transparent head so its huge green eyes, built like barrels, can look up, but also swivel to face forward. The Mystery Mollusc–initially confused to be a sea snail, hence the name–uses its hood in a similar fashion to a Venus Fly Trap plant to ensnare prey. More than 91% of ocean creatures have yet to be discovered, unlike anything we’ve thought possible on Earth. Perhaps the jaw-dropping life we’ve assumed only exists on a distant planet roams the very same Earth as us.

Our own Earth, unlike any other we know, ravaged by climate change and rising carbon emissions, still has so many wonders to discover. Looking around with the eyes of a scientist, itching to understand, can change your perspective of the world, and thus, eventually change the world itself. Geologists find pleasure in a pile of rocks and ornithologists spend hours bird-watching. Love and passion for the sciences drives us to the point of invention, continuously building upon generations of discoveries to add more research and benefit humanity. The stars live and die in brilliant supernovas, but their light travels on, illuminating the universe for years to come.

Us, made of stardust on a planet unlike any star. The odd one out in the known universe.

Alien.

Marvel at Earth’s wonders as if they were extraordinary. For the extraterrestrial is already here.

“…and we have only just begun.”

- Neil deGrasse Tyson

PHOTOGRAPHY:

Sajan Patel, Ben Sheegog, Vincent Shen, and Charlotte Goebel

Sajan Patel, Ben Sheegog, Vincent Shen, and Charlotte Goebel were selected as the winners of the 2025 Broad Street Scientific Photo Contest. Their awards included the opportunity to have their photographs featured in the 2025 volume of the Broad Street Scientific.

The Silent Guardians of the Patagonian Coast by Sajan Patel

Pictured are two Magellanic penguins standing in the fairly desolate landscape of Isla Martillo, located in the coastal Tierra del Fuego of Patagonia. Rugged terrain, sparse vegetation, and barren trees silhouette against a brooding sky. As a near-threatened species, their conservation must be prioritized to maintain Argentina’s natural biodiversity.

The Waking City by Ben Sheegog

This image captures a city waking beneath a blanket of fog as its skyline emerges in soft golden hues. The muted lights hint at lives awakening below, and the mountainous backdrop reminds us of where their roots are planted.

The wilderness of Alaska’s interior includes the New Hampshire-sized Denali National Park, which boasts North America's tallest peak. This photograph was captured from inside an airplane in late May, with the summer season seeing 16-19 hours of sunlight a day.

This blonde bear grazing in Glacier National Park, Montana, is an American black bear, disinguishable by its tall ears. It seems to smile in the fading summer light, enjoying the Park's serenity.

Interior Alaska by Vincent Shen

Glacier Brown Bear by Charlotte Goebel

CLONING THE ANHYDROBIOSIS PROTEIN CAHS-89226 FROM HYPSIBIUS EXEMPLARIS TO CONSTRUCT

DESICCATION-TOLERANT AZOTOBACTER VINELANDII AND ESCHERICHIA COLI

Aretha Datta

Abstract

Many rare diseases remain uncured, resulting in numerous global deaths annually. While cryopreservation—a way to pause aging through freezing—has been successful in animals, it has yet to be effectively applied to humans. Additionally, with the commercialization of space travel and NASA’s initiatives to colonize Mars and the Moon for resources, space travel and interplanetary colonization sectors have begun to grow, requiring enhanced spacecrafts for prolonged space travel. However, current space travel methods present significant challenges, including detrimental health effects and high resource costs, making long-term missions unfeasible. To address these challenges, mechanisms similar to anhydrobiosis, or desiccation, as observed in tardigrades like Hypsibius exemplaris, should be explored. This study aims to clone the CAHS-89226 gene—which is believed to play a role in desiccation tolerance in H. exemplaris and insert it into Azotobacter vinelandii, a bacteria known for using cysts as a method of tolerating desiccation, and Escherichia coli, a bacteria known for its relatively low desiccation tolerance. Although H. exemplaris is readily available in laboratory settings, its genome remains incompletely sequenced, making the confirmation of CAHS-89226 an important finding. The successful extraction of CAHS-89226 with sticky ends paves the way for future recombinant plasmid design. Importantly, this study reveals no significant loss in cell viability following electroporation and heat-shock transformations for A. vinelandii, but the opposite for E. coli. This research lays the groundwork for future investigations into the relationship of CAHS-89226 with other stress response genes. Ultimately, mechanisms of desiccation may provide a more economically viable and practical alternative to cryopreservation, potentially facilitating advancements in space travel and improving survival prospects for humanity.

1. Introduction

1.1 Space Travel

As space travel becomes increasingly commercialized and no longer solely government-controlled, the colonization of other planets emerges as a common question. Upcoming missions to Mars and the Moon are paving the way for the broader human expansion into space (Moon to Mars Architecture - NASA, 2024). In the future, such colonization may occur on distant planets suitable for human settlement. For example, NASA’s Kepler mission discovered a larger, older Earthlike planet called Kepler-452b about 1,400 light years away (Jenkins et al., 2015). With current spacecraft and aviation technology, the expected travel time to Kepler452b is 26 million years (Creighton, 2017). For this level of space travel, intergenerational travel would be necessary, posing multiple risks, including a large resource expense,

spaceflight-borne chronic and infectious illnesses, and negative psychological effects (Yin et al., 2023). Moreover, various risks are associated with spaceflight, such as circadian rhythm dysregulation (Yin et al., 2023), early onset cell senescence (Garbacki et al., 2023), muscle and bone degradation (Man et al., 2022), loss of vision or hearing (Buckey et al., 2018), reduced immune function (Lv et al., 2023), and mitochondrial dysfunction (Waisberg et al., 2024).

To mitigate these risks, scientists are exploring cryptobiotic capabilities—the ability to pause biological functions under extreme conditions—to preserve human health during transit. This approach would allow travelers to enter a dormant state, potentially pausing the aging process and reducing resource needs for essentials like fuel, food, and water. The reduced human activity aboard the vessel would also help minimize health risks during the journey.

1.2 Cryonics

Over 6,500 rare diseases currently lack a cure, and when more common diseases are considered, the number of untreatable conditions is significant (Rare Diseases, 2020). Such diseases, like rabies, are fatal, and often lead to excruciatingly painful deaths, highlighting the need for solutions beyond current medical technology. Cryonics, the practice of freezing recently deceased individuals in hopes of future revival, offers one potential pathway to overcome the limitations of today’s medicine. Despite successful cryopreservation in animals, humans have yet to be successfully cryopreserved (Ekpo et al., 2022). Since human cryopreservation remains unachievable, studying organisms like tardigrades, which can enter a state of cryptobiosis, could offer promising insights. Cryptobiotic capabilities in tardigrades might one day be adapted for human applications, potentially allowing the safe preservation of human tissues—or even whole bodies—until medical advancements are available.

1.3 Hypsibius exemplaris

Tardigrades are an extremotolerant species (Yoshida et al., 2021; Joseph, 2023), meaning they can survive the harshest environments in the world, including outer space (Møbjerg & Neves, 2021). Since tardigrades are limnoterrestrial, or only active when covered by a thin film of water, they are not well equipped to survive dry climates. Thus, when tardigrades are exposed to an arid environment, they enter cryptobiosis, more commonly known as a “tun state”, and do not exit it until they have enough water exposure (Møbjerg & Neves, 2021). In this state, they reduce body volume by 85-90% (Møbjerg & Neves, 2021), retaining only 2-3% of their extracellular and intra-cellular water content (Møbjerg & Neves, 2021). Tardigrades have five different forms of cryptobiosis they use to survive: anhydrobiosis (desiccation), osmobiosis (rise in external pressure), cryobiosis (freezing), anoxybiosis (lack of oxygen), and chemobiosis (exposure to environmental toxicants) (Møbjerg & Neves, 2021; Wełnicz et al., 2011; Roszkowska et al., 2021; Lim et al., 2024; Hibshman et al., 2020). In this state, they do not biologically age (Kasianchuk et al., 2023; Sieger et al., 2022), allowing them to be well-suited for long-term travel. The 1,300 different species of tardigrades on planet Earth have varying levels of anhydrobiosis capabilities (Tardigrade). Many of these anhydrobiotic capabilities are believed to be due to three main proteins: CAHS (Cytoplasm-Abundant Heat Soluble), SAHS (SecretoryAbundant Heat Soluble), and MAHS (MitochondrialAbundant Heat Soluble) (Kasianchuk et al., 2023; Boothby et al., 2017).

Hypsibius exemplaris is an herbivorous species of tardigrade commonly used for evolutionary biology and astrobiology research due to its anhydrobiotic capabilities (Poprawa et al., 2022; Gąsiorek et al., 2018). However, currently, only fractions of H. exemplaris’s genome have been sequenced and annotated. As Ramazzottius varieornatus was fully sequenced by Takekazu Kuneida, it was proposed as a suitable alternative. However, due to its unavailability in North Carolina and the challenges of rearing it, the next most accessible tardigrade, H. exemplaris, which is easy to rear and maintain, was selected instead (Hashimoto et al., 2016).

1.4 CAHS-89226

CAHS (Cytoplasm-Abundant Heat Soluble) is a TDP (tardigrade-intrinsically disordered protein) and is thought to be used in tardigrades to protect and preserve cellular components through desiccation (Barilla et al., 2024). CAHS is believed to produce a “bioglass” out of the cytoplasm, effectively suspending the cell in a certain position, thus stopping all metabolic processes, as no part of the cell moves or reacts (Barilla et al., 2024). In Hypsibius exemplaris, the CAHS-89226 gene is proposed to exist, though not confirmed, as the entire genome has not been sequenced (Hypsibius Exemplaris Genome Assembly nHd_3.1, n.d.). Additionally, the relationship between CAHS-D and trehalose, a sugar used for long-term cell desiccation (Tapia et al., 2015), is synergetic. Trehalose is not synergistic with numerous other desiccationresistant proteins, like Bovine Serum Albumin (Nguyen et al., 2022). This tells us that CAHS plays a role in desiccation tolerance, as trehalose alongside CAHS had a greater survival rate than either factor alone. Thus, many scientists believe that CAHS plays a role in the superior desiccation tolerance of tardigrades. Understanding the role of CAHS protein in anhydrobiosis can further our knowledge of desiccation tolerance in tardigrades and the possible uses of the protein in other organisms.

1.5 Azotobacter vinelandii

Azotobacter vinelandii, a common gram-negative, soildwelling, nitrogen-fixing bacterium, has been widely studied for its ability to produce nitrogen even in the presence of oxygen (Katsuyama, 2019). This is unique compared to most aerobic organisms that switch to using oxygen if any is available (Castillo et al., 2020). Additionally, it produces two biopolymers of interest: alginate and poly(3-hydroxybutyrate) (Castillo et al., 2020).

Generally best suited for more tropical environments, this bacterium uses Late-Embryo Abundant (LEA) proteins, which help form a hydrophobic layer and

facilitate the formation of cysts that protect the cells, allowing A. vinelandii to survive for up to ten years (Vela, 1974; Rodriguez-Salazar et al., 2017).

By inserting H. exemplaris’s CAHS-89226 gene into A. vinelandii, one can understand the role of this gene in association with LEA and potentially improve desiccation tolerance.

1.6 Escherichia coli

E. coli K-12 is a widely used laboratory strain of this gram-negative bacterium. While many strains of E. coli are harmless, some pathogenic variants can cause intestinal issues in mammals and other organisms. Additionally, E. coli shares a significant portion of its genome with Salmonella, having diverged from it approximately 100 million years ago (Noar & BrunoBárcena, 2018). Compared to other bacteria, E. coli is relatively susceptible to desiccation.

In this project, using E. coli as a control to study the effects of the CAHS-89226 gene will help assess the viability and functional impact of the CAHS-89226 gene, providing insights into its role in enhancing desiccation tolerance in H. exemplaris and possibly other organisms as well.

1.7 Hypothesis

Ho: The CAHS-89226 gene, in tandem with the LEA-1 gene, will not change desiccation tolerance in Azotobacter vinelandii or Escherichia coli, leading to no significant difference between growth and replication of A. vinelandii and E. coli pre- and post-desiccation.

H A: The CAHS-89226 gene, in tandem with the LEA1 gene, should lead to improved desiccation tolerance in Azotobacter vinelandii and Escherichia coli, leading to greater growth and replication of A. vinelandii and E. coli post-desiccation.

2. Materials and Methods

2.1 Tardigrade Cultures

Tardigrade cultures (Hypsibius exemplaris, strain Z151) were purchased from Carolina Biological and aerated two to four times a week using a pre-sterilized bulb pipette. Spring water was replaced once every month. Cultures arrived from Carolina Biological in containers that were 1 inch tall and 1.5 inches in diameter, containing around 1000 tardigrades each, and were kept in those containers for the entirety of the project. Cultures were placed under fluorescent light with the lid slightly opened to promote algal growth and provide food (chlorella, from Carolina Biological) for tardigrades. Food was added once a week.

2.2 Azotobacter vinelandii Cultures

Azotobacter vinelandii culture was purchased from Ward’s Scientific. The bacteria were then placed in 10 mL of Burk’s Medium (MgSO4 (0.2 g/L), K2HPO4 (0.8 g/L), KH2PO4 (0.2 g/L), CaSO4 (0.13 g/L), FeCl3 (0.00145 g/L), Na2MoO4 (0.000253 g/L), sucrose (20 g/L)), (to make it extra-rich, add 0.5 g/L yeast extract) and 10 replicates were created. Soil extract was made by putting air-dried garden soil (400 g) and tap water (1L) into the autoclave for 1 hour at 121 °C, followed by vortexing for two minutes and then straining through a cheesecloth into a sterile beaker. Two months later, the Azotobacter vinelandii was placed into the new Burk’s Medium. These refreshed cultures were then plated on Azotobacter Agar (K2HPO4 (1 g/L), MgSO4 (0.2 g/L), NaCl (0.2 g/L), FeSO4 (trace amounts), soil extract (5 g/L), mannitol (20 g/L), agar (15 g/L)) one month later. The cultures were left on a lab table near a light source for two days and grew to cover the entire plate. They were then wrapped in Parafilm and placed in a refrigerator for five weeks.

2.3 Escherichia coli Cultures

Escherichia coli K-12 was purchased from Carolina Biological and cultured in 10 mL of LB (Luria-Bertani) Broth (25 g/L LB Powder). These cultures were left in a shaking water bath at 37 °C overnight and then plated (100 μL per petri dish) on LB Agar (40 g/L of Miller’s LB Agar in 1L dH2O, autoclaved for 15 minutes). Plates were placed into the incubator at 37 °C overnight, then wrapped in Parafilm and placed in the refrigerator until future use.

2.4 DNA Extraction From Hypsibius exemplaris

Stock solutions of 10X buffer in dH2O (100 mM Tris (pH 8.2), 500 mM KCl, 15 mM MgCl2), detergent mix in dH2O (4.5% Tween-20, 4.5% Tris-X-100), and lysis buffer (100 μL/mL 10X buffer, 300 μL/mL detergent mix, 30 μL/ mL Proteinase K, 570 dH2O μL/mL dH2O) were created. For long-term storage, 10X buffer was stored at -20 °C, detergent mix was stored at room temperature away from direct sunlight, and the lysis buffer was stored at -26 °C (though it is recommended to store at -70 °C). Crushed glass was created by crushing microscope slides using a mortar and pestle until the glass shards were fine and white.

For DNA extraction, 80 μL of lysis buffer and crushed glass were added to a 1.5 mL microcentrifuge tube. About 30-40 adult tardigrades (~150 μL of concentrated tardigrade culture, more is better) were added to the cap of the microcentrifuge tube and then centrifuged, vortexed, and centrifuged again briefly (about 30 seconds

for each step). Tubes were then frozen for at least 15 minutes at -26 °Cs. Tubes were then thawed and 7.5 μL of lysate was transferred to a PCR tube. Tubes were heated for 60 minutes at 65 °C, then at 95 °C for 5 minutes, and finally centrifuged for 30 seconds.

2.5 PCR and Gel Electrophoresis

To extract CAHS-89226 from Hypsibius exemplaris, PCR and gel electrophoresis were used. First, to identify what genes existed in H. exemplaris, multiple primers –all within a range of 86.0-99.4% confidence of accuracy – were designed. Then, PCR and gel electrophoresis were run to identify which genes existed within the genome of the H. exemplaris used. After this, gels were viewed through an Accuris SmartDocTM box.

The CAHS-89226 gene showed a promising gel electrophoresis and was selected for insertion into Azotobacter vinelandii.

Primers were ordered from Eton Bioscience and came in a 10X concentration stock solution. Working solutions were created by performing a 1:10 dilution and were used for PCR. The PCR reaction setup included: 5 μL DNA template, 2 μL forward primer, 2 μL reverse primer, 2 μL dNTP mix ( NEB), 2 μL Standard Taq Buffer with MgCl2 (10X, NEB), 1.5 μL MgCl2 , 4.5 μL nuclease-free water, and 1 μL Taq DNA Polymerase. PCR tubes were then loaded into VeritiPro 96-Well ThermoCycler and the following temperatures and times were used: 95°C for 30 sec, 95°C for 30 sec, 58.5°C for 1 min, 68°C for 1 min (steps 2-4 were repeated 31 times), 68°C for 5 min, and 4°C thereafter.

After PCR, a gel digest (New England Biolabs #T1020S) was used to extract the DNA from the gel. The DNA was then double-digested using Sac-I-HF and Sal-I-HF (NEB) for 15 minutes at 37°C in S1X rCutSmart buffer (NEB).

2.6 Preparation of the Plasmid

Plasmids (nifF in pET21b), as shown in Figure 1, were purchased from AddGene and were placed in the refrigerator upon arrival. Plasmids in E. coli were then grown in a Luria-Bertani Broth (LB Broth) and 100 micrograms per milliliter mixture overnight in a shaking water bath at 37 °C.

Additionally, plasmids were also grown in a transformation medium (MgSO4 (1.9718 g/L), CaSO4 (0.0136 g/L), CH5COONH4 (1.1 g/L), glucose (10 g), KH2PO4 (0.25 g/L), K2HPO4 (0.55 g/L), 1.8% agar (18 g), autoclaved for 15 minutes) and 100 μg/mL ampicillin mixture overnight in a shaking water bath at 37 °C to test which medium yielded the best results. Previous work suggested the use of transformation media would increase plasmid overnight growth for Azotobacter vinelandii (Natzke et al., 2018). Those in the transformation medium did not grow, but those in LB Broth did.

Overnight cultures were mini-prepped using the NEB #T1010S kit and protocol. A sequential digest using Sac1-HF and Sal-1-HF enzymes from NEB was performed to prepare plasmids for uptake of the CAHS-89226 gene. The sequential digest was composed of 11 μL of nuclease-free water, 2 μL of 1x rCutSmart buffer, 1 μL of enzyme (SacI-HF or Sal-I-HF), and 5 μL of plasmid DNA. The digest

Figure 1. nifF in pET21b plasmid with cut sites, enzyme names, and tardigrade primer sequences. Image of plasmid taken from Addgene and cut sites added by author.

reaction mixture containing Sal-I-HF was incubated at 37 °C for 15 minutes and heat-inactivated at 65 °C before Sac-I-HF was added to the tube. Due to the compatible buffer between both enzymes, a PCR and DNA Cleanup (NEB #T1130) were not used between digestion reactions. Subsequently, a CIP (Calf Intestinal Alkaline Phosphatase) was performed with 6 μL of nuclease-free water, 3 μL of 1x rCutSmart buffer, and 1 μL of CIP enzyme (NEB). This enzyme reaction tube was then incubated at 37 °C for 10 minutes, and 80 °C for 2 minutes to heat-inactivate the enzyme.

Post-CIP, a gel was run to confirm the quality and concentration of the plasmid, and a PCR and DNA Cleanup (NEB #T1030S) was used to isolate the opened plasmid DNA from the reagents and enzymes and then was used to ligate.

2.7 Ligation

Six ligations were done: three different insert ratios of vector:insert (1:1, 1:3, and 1:6), a vector-only, and a plasmid-only. Plasmid-only was used to confirm the success of the electroporation protocol, vector-only was used to confirm the success of the CIP and sequential digest, and insert ratios were used as the desired endproduct of the transformations.

All ligation reaction mixtures included 1 μL vector (opened plasmid DNA), 1 μL T4 DNA Ligase (NEB), 2 μL T4 DNA Ligase Buffer (1x), and varying amounts of nuclease-free water, and insert based on the insert ratio.

Ligation reactions were incubated at room temperature for 10 minutes, inactivated at 65 °C for 10 minutes, and chilled on ice for 5 minutes. Ligations were then cleaned using a PCR and DNA Cleanup Kit from NEB (#T1030S) and stored at -20 °C until use.

2.8 Preparation of Electrocompetent Azotobacter vinelandii

To make Azotobacter vinelandii electrocompetent, cells were cultivated with vigorous shaking in a water bath at 30 °C for two hours. Cells were then harvested by centrifugation at 5000 rpm and 4 °C for 10 minutes. The supernatant was removed and cells were washed and resuspended with an original culture volume of icecold 10% glycerol. Cells were then centrifuged again for 10 minutes at 5000 rpm and 4 °C. This step was repeated three times using half and a quarter of the original volume, and finally 2-4 mL of the glycerol solution.

After harvesting, cells were placed into 0.8 μL microcentrifuge tubes as 40 μL aliquots and stored at -26 °C until electroporation.

2.9 Preparation of Electrocompetent Escherichia coli

To make electrocompetent Escherichia coli, 100 μL of overnight E. coli was inoculated in 10 mL of LB Broth and left to sit for 2-3 hours. Cells were then pelleted by centrifugation for 5 minutes at 6000 RPM and 4 °C. The supernatant was discarded and cells were resuspended in 10 mL of ice-cold 10% glycerol. Cells were centrifuged again for 5 minutes at 6000 rpm and 4 °C. This step was repeated four more times and then resuspended in 100 μL of ice-cold 10% glycerol.

After harvesting, cells were placed into 0.8 μL microcentrifuge tubes as 40 μL aliquots and stored at -26 °C until electroporation.

2.10 Electroporation

For electroporation, electrocompetent cells were thawed on ice. Each sample had a 1.5 mL microcentrifuge tube with SOC Medium (NEB) and an electroporation cuvette placed on ice. In the aliquoted cell tubes, 5 μL of ligated recombinant plasmid was added and incubated on ice for a minute. All the sample was then transferred to a cold electroporation cuvette and inserted into the shock pod of the Gene Pulser XCell. The 1 mm cuvette, E. coli pre-set protocol was used, and cells were pulsed once. Immediately after, 1 mL of cold Extra-Rich Burk’s Medium or SOC Medium (NEB) for A. vinelandii and E. coli, respectively, was added to resuspend cells. Then, the cell suspension was transferred to the 1.5 mL microcentrifuge tube and left to incubate overnight in a shaking water bath of 225 rpm at 37 °C.

The next morning, cells were placed onto Burk’s Medium Agar + 100 μg/mL ampicillin or LB Agar + 100 μg/mL ampicillin plates (for A. vinelandii and E. coli, respectively). Plates were incubated at 37 °C overnight and swabbed the following day to grow selectively in Burk’s Medium with 100 μg/mL ampicillin or LB Broth with 100 μg/mL ampicillin. Cultures were incubated in a shaking water bath at 37 °C overnight.

2.11 Preparation of Chemically Competent Escherichia coli

Lyophilized E. coli K-12 HB101 (Bio-Rad) was rehydrated with LB Broth and incubated overnight as instructed in the Bio-Rad pGLO lab. Cells were then placed in 250 μL of transformation solution (CaCl2), and vortexed. Cells were now ready to use.

2.12 Heat Shock

In the cells and transformation solution mixture, 10 μL of ligated plasmid was added and incubated on ice for 10 minutes, per the pGLO lab Quick Guide. Tubes were then heat-shocked at 42 °C for 50 seconds, followed by an ice bath for 2 minutes. Subsequently, 250 μL of LB Broth was added, and tubes were incubated at room temperature for 10 minutes. Bacteria were resuspended, and 100 μL of cell suspension was plated on selective agar plates.

Plates were incubated at 37 °C overnight and swabbed the following day to grow selectively in Burk’s Medium with 100 μg/mL ampicillin or LB Broth with 100 μg/mL ampicillin. Cultures were incubated in a shaking water bath at 37 °C overnight.

2.13 Miniprep and Gel Electrophoresis Confirmation

For confirmation, 2 mL of each selective liquid culture was miniprepped using the NEB #T1010 kit. Miniprep product was then double digested with Sac-I-HF and SalI-HF (NEB). The double digest reaction mixture contained 5 μL DNA, 2 μL 1X rCutSmart Buffer, 1 μL Sal-I-HF, 1 μL Sac-I-HF, and 11 μL nuclease-free water to make a 20 μL reaction tube. Tubes were placed in VeritiPro 96-Well ThermoCycler and allowed to incubate for 15 minutes at 37 °C. A CIP was performed to ensure no re-ligation between insert and plasmid, and then a 2.5% gel was run to confirm plasmid insertion and insert into A. vinelandii and E. coli.

2.14 Desiccation and Survival Assay for Azotobacter vinelandii

Burk’s Buffer plates (MgSO4 (0.2 g/L), K2HPO4 (0.8 g/L), KH2PO4 (0.2 g/L), CaSO4 (0.13 g/L), FeCl3 (0.00145 g/L), Na2MoO4 (0.000253 g/L), N-Butanol (1-Butanol) (15 μL/L), agar (20 g/L)) were also created for the desiccation protocol. Azotobacter from the new batch was then plated, covered in aluminum foil, and then placed in a dark storage closet for five weeks. When retrieved, Burk’s Buffer plates showed signs of cyst formation. All plated samples were re-suspended in Burk’s Medium and allowed to grow for 2 weeks. An OD-620 was then performed to quantify growth post-desiccation. Post-electroporation and confirmation, new cultures of Azotobacter vinelandii were plated onto Burk’s Buffer plates, covered in aluminum foil, and placed in a dark storage closet for five weeks. When retrieved, Burk’s Buffer plates showed signs of cyst formation, like the control plates. All plated samples were re-suspended in Burk’s Medium and allowed to grow for two weeks. An OD-620 was then performed to quantify growth postdesiccation.

2.15 Desiccation and Survival Assay For Escherichia coli

Overnight cultures of E. coli in LB Broth (control) and LB + Amp Broth (electroporated E. coli) were created and placed in a shaking water bath at 37 °C. All cultures were then diluted to an OD-620 reading of 0.2. Diluted samples were centrifuged at 10,000g for 5 minutes. The supernatant was then discarded and replaced by 100 μL of sterile water. Cells were gently vortexed to resuspend and then placed in a desiccator with silica gel for 1 hour. E. coli was rehydrated using 900 μL LB Broth, and vortexed gently to resuspend the cells. The cell suspension was then incubated at 37 °C for one hour in a shaking water bath to allow for recovery. Finally, OD-620 values were measured to determine cell viability and significance of CAHS-89226.

For controls, diluted samples were centrifuged at 10,000g for 5 minutes, and the supernatant was discarded. Cells were then re-suspended in 900 μL LB Broth and incubated at 37 °C for one hour in a shaking water bath. After, OD-620 values were taken from each sample.

2.16 Data Analysis

Data were analyzed in RStudio using the car, ggplot2, and dplyr packages. For the A. vinelandii desiccation survival assay, a Shapiro-Wilk’s test was used to check for normality for each group, a Levene’s test for equal variances, and a Welch’s Two Sample T-Test to determine the significance between the desiccated, post-electroporation desiccated, and non-desiccated test groups. For the E. coli desiccation survival assay, an ANOVA was used to identify significant differences between the different groups, and then a Tukey’s posthoc was used to analyze statistical differences between individual groups. Following, graphs were created, with standard error bars and significant differences starred.

3. Results

3.1 PCR and Gel Extraction Results For Extraction of CAHS-89226 Gene

As shown in Figure 2, gel electrophoresis was performed on a 1% agarose gel at 100 V for 30 minutes to verify the presence and size of the CAHS-89226 gene. The gel was stained with Gel Loading Dye (NEB #B7025) for visualization, and a DNA ladder (NEB) served as a size reference alongside a negative control that lacked the gene. A distinct band appeared at approximately 250 bp in the CAHS-89226 lane, confirming the successful amplification and extraction of the gene, which was

absent in the control lane.

Figure 2. 1% gel electrophoresis of CAHS-89226 gene with sticky ends and negative control. Image taken by author.

3.2 Plasmid Extraction Gel

In Figure 3, gel electrophoresis was conducted on a 1% agarose gel at 100 V for 30 minutes to verify the presence of the nifF plasmid obtained from Addgene. The gel was stained with Gel Loading Dye (NEB #B7025) for visualization, with a DNA ladder (NEB) included as a size reference. A distinct band at approximately 6000 bp was observed in nearly all lanes, confirming the expected size of the plasmid. Samples consisted of equal volumes of miniprep and double digest products from overnight cultures of E. coli containing the plasmid in LB broth.

Figure 3. 1% gel electrophoresis of nifF plasmid in pET21b post-miniprep and double digestion. Image taken by author.

3.3 Escherichia coli Electroporation Results PostPlating

Figure 4 shows LB agar plates (bottom row) are a control to confirm cell viability post-electroporation, showing that cells survived and proliferated. The LB agar + ampicillin plates (top row) test for plasmid transformation; the presence of colonies indicates successful transformation through electroporation. However, as discussed in Section 3.5, the transformed colonies did not incorporate the insert gene.

Figure 4. Electroporated E. coli with a 1:6 plasmid:insert ratio plated on LB agar + 100 μg/mL ampicillin (top row) and LB agar (bottom row). Image taken by author.

3.4 Azotobacter vinelandii Electroporation

Results Post-Plating

Figure 5 shows the growth of A. vinelandii in every plate as expected in a successful transformation - the presence of the plasmid causes growth in all plates, as the plasmid contains the ampicillin resistance gene.

Figure 5. Electroporated plasmid without insert, 1:6 concentration, and 1:1 concentration in Azotobacter vinelandii plates of Burk’s medium agar and ampicillin stock solution of 300 μg/mL. Images taken by the author.

In Figure 6, the presence of A. vinelandii on all vectoronly plates at varying concentrations of ampicillin suggests the presence of a closed plasmid, suggesting that either the sequential digest or the CIP did not work.

Figure 6. Open vectors in Azotobacter vinelandii on plates of Burk’s medium agar and ampicillin stock solution of 100, 200, and 300 μg/mL. Images taken by author.

3.5

Escherichia

coli

Heat Shock Results Post-Plating

In Figure 7, the LB agar plates suggest cell viability during the heat-shock protocol, as all control groups displayed growth. However, no growth was observed on any LB agar + ampicillin plates, indicating that transformation was unsuccessful. The Control (no plasmid) plate on LB agar + ampicillin showed no growth, confirming ampicillin efficacy. The absence of colonies on the Vector Only and 1:6 plasmid:insert plates suggests two possibilities: either transformation or ligation was unsuccessful.

Figure 7. Heat-shocked E. coli plated on LB agar (column 1) and LB agar + 100 μg/mL ampicillin (columns 2-4). The top row follows an extended heatshock protocol, while the bottom uses the Bio-Rad Quick Guide protocol. All plates contain HB101 E. coli (K-12 strain) from the Bio-Rad pGLO kit. From left to right, the plates include Control (no plasmid) on LB agar, Control (no plasmid) on LB agar + ampicillin, Vector Only (no insert) on LB agar + ampicillin, and 1:6 plasmid:insert on LB agar + ampicillin. Image taken by author.

3.6 Plasmid Extraction Gel Post-Electroporation (For Transformation Confirmation)

Figure 8 shows a gel electrophoresis performed on a 2.5% agarose gel at 100 V for 30 minutes to confirm the presence of the nifF plasmid in electroporated A. vinelandii and E. coli. The gel was stained with Gel Loading Dye (NEB #B7025) for visualization, and a DNA ladder

(NEB) was included as a size reference. A distinct band appeared at approximately 6000 bp in almost all lanes, confirming the expected size of the plasmid. The samples were loaded as show in Table 1.

Figure 8. 2.5% gel electrophoresis of nifF plasmid grown overnight in varying concentrations of ampicillin and growth medium for Azotobacter vinelandii and Escherichia coli. Image taken by author.

Table 1. Growth conditions for A. vinelandii and E. coli cultures with varying plasmid ratios and DNA concentrations. Table created by the author.

The results confirm the successful transformation of the nifF plasmid into A. vinelandii and E. coli; however, they also indicate that the insert is not present in the plasmid.

3.7 Azotobacter vinelandii Desiccation And Survival Assay

Statistical analysis revealed significant differences in OD-620 values between the desiccated and nondesiccated groups (p = 3.7e-16) and between the desiccated post-electroporation and non-desiccated groups (p = 3.0e-14) in Figure 9. However, no significant difference was found between the desiccated and desiccated post-electroporation groups (p = 0.41), likely due to an incomplete double digest that prevented the plasmid from accepting the insert gene. Despite this, the data suggest that the desiccated post-electroporation groups exhibited slightly higher tolerance, indicating that A. vinelandii cell viability and cyst formation ability are not adversely affected by the electroporation process or the introduction of the nifF gene.

9. Graph of mean OD-620 values for desiccated (n=10), desiccated post-electroporation (n=5), and non-desiccated treatment groups (n=7). Graph created in RStudio by author.

3.8 Escherichia coli desiccation and survival assay

In Figure 10, the lack of significant differences between the desiccated electroporated cells and desiccated control groups can be attributed to an incomplete double digest, preventing the plasmid from accepting the insert gene. Gel electrophoresis results confirmed these findings, with a band at approximately 6000 bp for the plasmid, but none around 250 bp for the cut insert gene. Interestingly, unlike in A. vinelandii, desiccated electroporated cells had lower cell viability than the desiccated controls significantly. This suggests that E. coli cell viability and cyst formation ability remain unaffected by electroporation or the introduction of the nifF gene in the pET21b plasmid.

Figure

Figure 10. Graph of mean OD-620 values for desiccated (n=6), desiccated post-electroporation (n=6), non-desiccated (n=8) and non-desiccated post-electroporation (n=8) treatment groups.

4. Discussion

This study investigated the use of the CAHS-89226 gene in the desiccation tolerance of tardigrades. The protocol followed consisted of three main parts: gene extraction, recombinant plasmid creation with insert, and subsequent survival assays to test desiccation tolerance.

4.1 Successful Extraction of CAHS-89226 from Hypsibius exemplaris

Though H. exemplaris is widely used in laboratories, its entire genome has neither been sequenced nor annotated, leading to only a 93% confidence in the existence of CAHS-89226 within the genome (Hypsibius Exemplaris Genome Assembly nHd_3.1, n.d.). For this reason, extracting the gene proved difficult, as not all primers were consistently and accurately able to extract the gene. Thus, the successful extraction of CAHS-89226 opens the door to future uses of the gene in other projects.

4.2 Implications of the Survival Assays

In Azotobacter vinelandii, the significant differences between the desiccated and non-desiccated groups suggest that desiccation, while possible, greatly harms the A. vinelandii population, and not all survive. Additionally, the insignificant differences between the dessicated post-electroporation and desiccated groups suggest that the plasmid had no effect on desiccation tolerance on its own. Moreover, the insignificant difference between the desiccated post-electroporation and desiccated groups suggests the viability and non-impaired desiccation tolerance of A. vinelandii cells post-electroporation.

In Escherichia coli, the significant differences between the desiccated and non-desiccated groups suggest that desiccation, while possible, significantly harms

E. coli populations, with very select few surviving. The insignificant difference between the desiccated postelectroporation and desiccated groups suggests nonimpaired cell viability post-electroporation and also suggests that the plasmid did not accept the insert.

4.3

Limitations

4.3.1

Gene Interactions

Due to time constraints, this study examined CAHS89226 in isolation, though it’s known that tardigrade desiccation tolerance often relies on the collaborative effects of multiple genes, such as LEA-1 and SAHS. Future studies should investigate CAHS-89226 alongside other desiccation tolerance genes to understand their combined role in stress response and survival mechanisms.

4.3.2

Absence of Trehalose

Although trehalose is often co-expressed with CAHS proteins to enhance desiccation tolerance, it was not used in this protocol. Since trehalose may work synergistically with CAHS genes, its absence could have limited the observable effects of CAHS-89226 in this study. Due to the previously studied positive relationship between trehalose and CAHS, it can be assumed that desiccation tolerance would improve.

4.3.3 Host Limitations

E. coli and A. vinelandii may not be optimal models for studying tardigrade genes, as they lack cellular pathways and molecular structures native to eukaryotes like tardigrades. Future research could consider alternative hosts or in vitro systems better suited for examining the desiccation resilience expressed by tardigrade genes. Despite these limitations, the study provides a foundational understanding of CAHS-89226’s potential role in desiccation tolerance and highlights several pathways for further investigation.

5. Conclusion, Implications, and Future Work

5.1 Conclusion

This study aimed to advance the understanding of the role of CAHS-89226 in desiccation tolerance by isolating the gene from Hypsibius exemplaris and inserting it into Escherichia coli and Azotobacter vinelandii for desiccation survival assays. The successful extraction of the CAHS89226 gene brings the scientific community a step closer to understanding the use of the TDPs in cryptobiosis of tardigrades. Additionally, the extraction also confirms

the existence of the CAHS-89226 gene in H. exemplaris, as the organism has not been sequenced, leading to doubt in the existence of certain genes.

5.2

Implications and Future Work

This research lays essential groundwork for future studies on desiccation tolerance in H. exemplaris and uncovers broader potentials for cross-species application of stress tolerance genes. Additionally, it allows for future cloning studies of the CAHS-89226 gene, improving understanding of TDPs, as well as possibly improving the anhydrobiotic capabilities of other organisms.

Engineering desiccation-tolerant organisms could have far-reaching applications in the future. Since tardigrades, from which CAHS-89226 was derived, can pause aging in extreme conditions, this gene may similarly affect cellular aging in other species. Further studies should examine whether CAHS-89226 can modulate aging processes, potentially paving the way for its use in space travel, where paused aging could make long-term missions more feasible through the reduction of the need for resources and detrimental health effects from being active in space.

6. Acknowledgements

I would like to express my heartfelt gratitude to Dr. Heather Mallory for all her support. It has meant more to me than I can express. I am also deeply thankful to Dr. Kimberly Monahan for sharing her expertise in molecular genetics.

To the NCSSM RBio Class of ‘25, thank you for your encouragement, humor, and hope—you have all inspired me to push my limits and pursue a research career.

Finally, I would like to acknowledge the NCSSM Foundation, the Burroughs Wellcome Fund, and GlaxoSmithKline for their generous funding. This project would not have been possible without your support.

7. Works Cited

Barilla, M. A., Shuster, S. O., & Davis, C. (2024). Tardigrade CAHS hydrogels protect proteins through concentrationdependent mechanism. Biophysical Journal, 123(3), 75a–76a.

Boothby, T. C., Tapia, H., Brozena, A. H., Piszkiewicz, S., Smith, A. E., Giovannini, I., Rebecchi, L., Pielak, G. J., Koshland, D., & Goldstein, B. (2017). Tardigrades use intrinsically disordered proteins to survive desiccation. Molecular Cell, 65(6), 975-984.e5.

Buckey, J. C., Phillips, S. D., Anderson, A. P., Chepko, A. B., Archambault-Leger, V., Gui, J., & Fellows, A. M. (2018). Microgravity-induced ocular changes are related to body weight. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology, 315(3), R496.

Castillo, T., García, A., Padilla-Córdova, C., Díaz-Barrera, A., & Peña, C. (2020). Respiration in Azotobacter vinelandii and its relationship with the synthesis of biopolymers. Electronic Journal of Biotechnology, 48, 36–45.

Cryonics | Description, Process, Popularization, & Facts | Britannica

Ekpo, M. D., Boafo, G. F., Gambo, S. S., Hu, Y., Liu, X., Xie, J., & Tan, S. (2022). Cryopreservation of Animals and Cryonics: Current Technical Progress, Difficulties and Possible Research Directions. Frontiers in Veterinary Science, 9.

Escherichia Coli K 12—An overview | ScienceDirect Topics. (n.d.-a).

Garbacki, N., Willems, J., Neutelings, T., Lambert, C., Deroanne, C., Adrian, A., Franz, M., Maurer, M., De Gieter, P., Nusgens, B., & Colige, A. (2023). Microgravity triggers ferroptosis and accelerates senescence in the MG-63 cell model of osteoblastic cells. Npj Microgravity, 9(1), 1–16.

Gąsiorek, P., Stec, D., Morek, W., & Michalczyk, Ł. (2018). An integrative redescription of Hypsibius dujardini (Doyère, 1840), the nominal taxon for Hypsibioidea (Tardigrada: Eutardigrada). Zootaxa, 4415(1), 45–75.

Hashimoto, T., Horikawa, D. D., Saito, Y., Kuwahara, H., Kozuka-Hata, H., Shin-I, T., Minakuchi, Y., Ohishi, K., Motoyama, A., Aizu, T., Enomoto, A., Kondo, K., Tanaka, S., Hara, Y., Koshikawa, S., Sagara, H., Miura, T., Yokobori, S., Miyagawa, K., Kunieda, T. (2016). Extremotolerant tardigrade genome and improved radiotolerance of human cultured cells by tardigrade-unique protein. Nature Communications, 7(1), 12808.

Hibshman, J. D., Clegg, J. S., & Goldstein, B. (2020). Mechanisms of Desiccation Tolerance: Themes and Variations in Brine Shrimp, Roundworms, and Tardigrades. Frontiers in Physiology, 11.

Hypsibius exemplaris genome assembly nHd_3.1. (n.d.). NCBI.

Jenkins, J. M., Twicken, J. D., Batalha, N. M., Caldwell, D. A., Cochran, W. D., Endl, M., Latham, D. W., Esquerdo, G. A., Seader, S., Bieryla, A., Petigura, E., Ciardi, D. R., Marcy, G. W., Isaacson, H., Huber, D., Rowe, J. F., Torres, G., Bryson, S. T., Buchhave, L., Borucki, W. J. (2015). Discovery and validation of Kepler-452b: a 1.6 R super earth exoplanet in the habitable zone of a G2 star. The Astronomical Journal, 150(2), 56.

Joseph, A. (2023). Chapter 5—Extremophiles— Organisms that survive and thrive in extreme environmental conditions. In A. Joseph (Ed.), Water Worlds in the Solar System (pp. 201–254). Elsevier.

Kasianchuk, N., Rzymski, P., & Kaczmarek, Ł. (2023). The biomedical potential of tardigrade proteins: A review. Biomedicine & Pharmacotherapy, 158, 114063.

Katsuyama, Y. (2019). Microbial Type III Polyketide Synthases. In Reference Module in Chemistry, Molecular Sciences and Chemical Engineering. Elsevier.

Lim, S., Reilly, C. B., Barghouti, Z., Marelli, B., Way, J. C., & Silver, P. A. (2024). Tardigrade secretory proteins protect biological structures from desiccation. Communications Biology, 7(1), 1–10.

Lv, H., Yang, H., Jiang, C., Shi, J., Chen, R., Huang, Q., & Shao, D. (2023). Microgravity and immune cells. Journal of the Royal Society Interface, 20(199).

Man, J., Graham, T., Squires-Donelly, G., & Laslett, A. L. (2022). The effects of microgravity on bone structure and function. Npj Microgravity, 8(1), 1–15.

Møbjerg, N., & Neves, R. C. (2021). New insights into survival strategies of tardigrades. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 254.

Moon to Mars Architecture—NASA (2024).

Natzke, J., Noar, J., & Bruno-Bárcena, J. M. (2018). Azotobacter vinelandii nitrogenase activity, hydrogen production, and response to oxygen exposure Applied and Environmental Microbiology, 84(16), e01208-18.

Nguyen, K., Kc, S., Gonzalez, T., Tapia, H., & Boothby, T. C. (2022a). Trehalose and tardigrade CAHS proteins work synergistically to promote desiccation tolerance. Communications Biology, 5(1), Article 1.

Noar, J. D., & Bruno-Bárcena, J. M. (2018a). Azotobacter vinelandii: The source of 100 years of discoveries and many more to come. Microbiology (Reading, England), 164(4), 421–436.

Poprawa, I., Bartylak, T., Kulpla, A., Erdmann, W., Roszkowska, M., Chajec, Ł., Kaczmarek, Ł., Karachitos, A., & Kmita, H. (2022). Verification of Hypsibius exemplaris (Eutardigrada; Hypsibiidae) application in anhydrobiosis research. PLoS ONE, 17(3), e0261485.

Rare Diseases. (2020, February 7). National Institutes of Health (NIH).

Rodriguez-Salazar, J., Moreno, S., & Espín, G. (2017a). LEA proteins are involved in cyst desiccation resistance and other abiotic stresses in Azotobacter vinelandii Cell Stress and Chaperones, 22(3), 397–408.

Roszkowska, M., Gołdyn, B., Wojciechowska, D., Kosicki, J. Z., Fiałkowska, E., Kmita, H., & Kaczmarek, Ł. (2021). Tolerance to anhydrobiotic conditions among two coexisting tardigrade species differing in life strategies. Zoological Studies, 60, e74.

Sieger, J., Brümmer, F., Ahn, H., Lee, G., Kim, S., & Schill, R. O. (2022). Reduced ageing in the frozen state in the tardigrade Milnesium inceptum (Eutardigrada: Apochela). Journal of Zoology, 318(4), 253–259.

Tapia, H., Young, L., Fox, D., Bertozzi, C. R., & Koshland, D. (2015). Increasing intracellular trehalose is sufficient to confer desiccation tolerance to Saccharomyces cerevisiae Proceedings of the National Academy of Sciences, 112(19), 6122–6127.

Tardigrade. (2020, March 13). National Geographic.

Vela, G. R. (1974). Survival of Azotobacter in Dry Soil. Applied Microbiology, 28(1), 77–79.

Waisberg, E., Ong, J., Masalkhi, M., Mao, X. W., Beheshti, A., & Lee, A. G. (2024). Mitochondrial dysfunction in Spaceflight Associated Neuro-Ocular Syndrome (SANS): A molecular hypothesis in pathogenesis. Eye, 38(8), 1409–1411.

Wełnicz, W., Grohme, M. A., Kaczmarek, Ł., Schill, R. O., & Frohme, M. (2011). Anhydrobiosis in tardigrades—The last decade. Journal of Insect Physiology, 57(5), 577–583.

Yin, Y., Liu, J., Fan, Q., Zhao, S., Wu, X., Wang, J., Liu, Y., Li, Y., & Lu, W. (2023). Long-term spaceflight composite stress induces depression and cognitive impairment in astronauts—Insights from neuroplasticity. Translational Psychiatry, 13(1), 1–7.

Yoshida, Y., Horikawa, D. D., Sakashita, T., Yokota, Y., Kobayashi, Y., Tomita, M., & Arakawa, K. (2021). RNA sequencing data for gamma radiation response in the extremotolerant tardigrade Ramazzottius varieornatus. Data in Brief, 36.

EFFECTS OF HYPERCHOLESTEROLEMIA AND THE INHIBITION OF THE AGE-RAGE PATHWAY ON EXOPHER FORMATION IN C. ELEGANS

Anneliese Heyder

Abstract

Aggregation of proteins and mitochondrial dysfunction have been linked to neurodegenerative diseases such as Alzheimer’s, Huntington’s, and Parkinson’s across species (Arnold et al., 2020). Exophers are large, membrane-bound vesicles that can sort out neurotoxins or protein aggregates and carry them out of the cell for degradation (Wang et al., 2023). Loss of proteostasis can be the effect of many different stressors; one link researchers have found is elevated cholesterol levels. Abnormally high cholesterol levels in the brain have been linked to multiple neurological disorders such as Parkinson’s, Huntington’s, amyotrophic lateral sclerosis (ALS), and atherosclerosis (Jin et al., 2019). The AGERAGE (Advanced Glycation End Products - Advanced Glycation End Products) signaling pathway leads to the expression of inflammatory cytokines and ROS (reactive oxidation species), which are associated with many neurological disorders (Reddy et al., 2023). In this experiment, the effect of different cholesterol levels on protein aggregation and exopher formation through the AGE-RAGE pathway in C. elegans was investigated. Worms were exposed to cholesterol for 48 hours, and a thrashing and exopher assay was performed. Worms were then exposed to Azeliragon, a RAGE inhibitor, for 24 hours and another thrashing and exopher assay was performed. The results support that cholesterol can induce neurodegeneration by decreasing dopamine levels and that neurotoxins produced by cholesterol cause exophergenesis to become activated and increase production, which is dependent on the AGE-RAGE pathway.