SQ Volume 20 (2022-2023)

Front Cover

Northern Elephant Seal (Mirounnga angustirostris) sleeping among other eleephant seals, San Simeon, Calfiornia.

Photo by Jordan Chalmers

Inside Cover

Canyon Tree Frog (Hyla arenicolor) at night from the Mary Vagle Nature Center in Florida, California.

Photo by Bridget Spencer

Back Cover

Bighorn Sheep (Ovis canadensis) at Mount Evans, Colorado.

by Andrea Farrell

GENEROUSLY UNDERWRITTEN BY THE SALTMAN FAMILY

AND SUPPORTED BY

The views expressed in this publication are solely those of Saltman Quarterly, its principal members, and the authors of the content of this publication. While the publisher of this publication is a registered student organization at UC San Diego, the content, opinions, statements, and views expressed in this or any other publication published and/ or distributed by Saltman Quarterly are not endorsed by and do not represent the views, opinions, policies, or positions of the ASUCSD, GSAUCSD, UC San Diego, the University of California and the Regents or their offices, employees, or agents. The publisher of this publication bears and assumes the full responsibility and liability for the content of this publication.

In an effort to engage the UC San Diego Community, Saltman Quarterly holds an annual photo contest. The winners of this contest have their images featured on the cover and interior pages of the journal.

Photo

LETTER from the EDITORS

Photo by Andrea Farrell

Dear Readers,

Where to start? We've jumped back into the full swing of things—rushing from Galbraith to our next class in Warren, attending professors' office hours in the depths of the H&SS building, and spending late nights on the 7th floor of Geisel. As a collective, we've acquired a heightened sense of awareness about our time commitments. Committees were hard-pressed to find a 30-minute slot that works with everyone's schedules. But through the process of re-orienting ourselves in the chaos of college life, Saltman Quarterly has been our steady home base.

Within and surrounding SQ exists a desire to return, to connect. After the nerves and shifty awkwardness of the first few meetings wore off, we were reminded of just how vibrant SQ can be. This year, our staff aimed to foster this community while expanding SQ into new creative realms of science communication. We launched our inaugural podcast episode, highlighting student and faculty voices on campus in a new medium. We created Research Features, which summarizes our primary scientific manuscripts into accessible language for a broader audience on our website. Our website also now proudly boasts SQ B-Sides, a portfolio for biology-related artistic works designed to inspire and encourage our members to share their creative side.

This year's edition of Saltman Quarterly would not exist without the spirit and vitality of the biology community at UC San Diego, which remains the driving force of our work. Our Features articles cover snippets of ongoing biological research in several little corners of campus, ranging from efflux transporter proteins in sea urchins to the prediction of inflammatory disease severity using the Boolean method. The primary research manuscripts and literature reviews on pages 26 through 45, written by UC San Diego undergraduates, as well as the Senior Honors The-

ses on page 46, are proof that UC San Diego is home to enthusiastic undergraduate student researchers who are highly involved in their projects, often conducting research independently.

It’s clear that all over campus, stories are waiting to be told. In the pages of this magazine, we've narrowed in on a select few, highlighting the creative and imaginative work that arises from unwavering passion for science—a passion that has carried on despite the chaos. As more stories unfold, we’re confident that Saltman Quarterly’s staff will continue to bring them to life in a way that invites new, curious minds into the ever-expanding world of biology.

Our time in this organization has given us a deep understanding of the necessity for engaging communication of scientific findings. Though many of us may not end up working in science journalism down the line, the value of storytelling in science is one we’ll carry with us into our respective careers. As you read, we hope you’ll come to share this value, too.

We’re excited to present you with the 20th volume of Saltman Quarterly.

Sincerely,

Lina Lew and Sharanya Sriram Editors-in-Chief, Saltman Quarterly 2022-23

TABLE of CONTENTS Saltman Dedication Features

Saltman Quarterly thanks the Saltman family for their generosity and support. Their contributions have allowed SQ to continue to spread Dr. Paul Saltman’s ideals of science, communication, and education.

The Guardian of the Genome byAnanya Prasad

The New Era of Biology: Differentiating Rare Inflammatory Diseases With the Boolean Method by Anna Grinberg

Nightclub Bouncer in a Sea Urchin: The Efflux Transporter Proteins Embedded in Cell Membranes by QianqianTao

How Your Hands Talk to Your Brain: On Movement & the Senses by Emma Chen

What to Expect When You're Expecting: The Female Pelvic Floor, Pregnancy & Aging byAmy Li

Research, Brevia and Reviews

05 26 60 03

The Double-Edged Sword: The Reciprocal Relationship Between Chronic Pain and Depression and Anxiety by Leslie Ngo

Homology Arms Influence Homology Directed CRISPR/Cas9 Genome Editing by Andres Maximus Canedo Sandoval

Aerial Insect Abundance Predicts Myotis Bat Habitat Use Along Streams by Ari Brisco Schofield,Tazlina Dentinger, Richard Klein, and Brandi Sanchez

Analyzing Effects of UV-B Radiation Exposure on Escherichia coli Survival in Sur face Water Bodies by Leanne Liaw

Senior Honors Theses Staff

Undergraduates in the Biology Honors program are required to complete a written thesis detailing their scientific research. The Senior Honors Theses section, which presents the abstracts of their individual theses, highlights the achievements of accomplished student researchers.

Meet the members of the 2022-2023 Saltman

Quarterly staff who worked throughout the year to bring you this issue, as well as our online content, quarterly insiders, and community outreach initiatives.

Through many professors cross our lives and influence our learning, career development, and personal growth, few have left as indelible a mark on the biology department at UC San Diego as Professor Paul Saltman. Even fewer can say their legacy is enshrined in a student-run publication and is inspired by their outlook on scientific communication. Twenty-four years after his passing, Dr. Saltman is still remembered today as a pioneering nutritional biochemist, dedicated teacher, and supportive mentor whose passion for science and commitment to sharing his expertise with others has shaped generations of students that carry on his legacy today.

Dr. Saltman earned his B.S. in Chemistry and his Ph.D. in Biochemistry from the California Institute of Technology in Pasadena, California. After completing schooling, he became a professor at the University of Copenhagen and at Murdoch University in Australia before moving back to Los Angeles to serve as a faculty member at the University of Southern California’s Keck School of Medicine. Fourteen years later, he received an offer in 1967 to join UC San Diego as the Provost of Revelle College, and later took on the role of Vice

Chancellor for Academic Affairs. He was also a keen athlete and a founding member of the San Onofre Surfing Association, a true Southern California native.

Dr. Saltman’s impactful research in nutrition focused on understanding how trace metals like iron, copper, zinc, and manganese interact with human biochemistry. With wide-reaching implications in preventing anemia and enhancing skeletal metabolism, his work improved dietary plans and informed nutritional supplements for human health.

Dr. Saltman also served as an advisor in the food industry for Procter and

DR. PAUL SALTMAN

“I don’t want kids to grow up to be Paul. I want kids to grow up to the limits of their own human potential.”

...he believed scientists have the responsibility to use their knowledge for the betterment of society and make a positive impact on the world around us...

Gamble. His brand of scientific curiosity, which melded sharp intellect and scientific rigor to debunk prevalent diet myths, made him a well-respected figure amongst his colleagues.

The legacy Paul Saltman leaves behind is not one simply based on research accomplishments, but also his notable dedication to mentorship for young scientists. He was known to be charismatic and accessible, taking time to get to know students after class. His students said no other professor took a personal interest in their wellbeing and career directions at the level of commitment he did. On mentorship, he said “I don’t want kids to grow up to be Paul. I want kids to grow up to the limits of their own human potential.” He understood the mark of a good mentor was allowing students to find and grow their own strengths with his guidance. Dr. Saltman inspired generations of biologists coming through UC San Diego, many of whom became successful scientists and leaders in their own right. The outpouring of kind letters he received from past students often credited him as a factor in their success.

In fact, Dr. Saltman was among the first recipients of the Career Teaching Award from UC San Diego in 1994. Throughout his career, he was given Teaching Awards from Revelle, Muir, Warren, and Marshall Colleges. After his passing, the title of Paul D. Saltman Chair in Science Education for a professor in the biology department was established in his honor. These accolades reflect his ability to communicate to students, and proof of the quality and depth of the education he provided.

Beyond the classroom, Dr. Saltman was deeply invested in communicating scientific findings to the greater public in an engaging and approachable manner. His efforts to increase public awareness of healthy diets involved filming TV segments, such as one titled “Patterns of Life” for National Educational Television, and a series for PBS. He also wrote a popular book, the University of California San Diego Nutrition Book, to introduce nutritional science concepts to the layperson. His main message was that of a balanced diet. Everything in moderation, including junk food, was key to

healthy development beyond what any fad diet could provide. In addition, he supported science education below the college level, serving on the National Science Foundation Committee on Science Education and teaching summer classes to enhance the scientific knowledge of high school teachers. Ultimately, he believed scientists have the responsibility to use their knowledge for the betterment of society and make a positive impact on the world around us, which is a sentiment Saltman Quarterly, as an organization, shares today. In turn, we believe research in academia cannot effect change until it leaves the academic bubble and is made accessible to the general public.

Saltman Quarterly was established in 2004 by undergraduates at UC San Diego as a continuation of Professor Paul Saltman’s legacy. To carry on the vision of its namesake, Saltman Quarterly strives to make biology research at UC San Diego accessible to its audience and inform readers about cutting-edge research. Beyond the flagship Saltman Quarterly publication you are reading now, which highlights faculty and student research, the student club has expanded to include publications such as Under the Scope, online articles, and blogs, which aim to make sense of the intersectional relationship between biology, culture, and society. Committees such as community outreach, media, and the podcast team take our messages beyond the UC San Diego community, just as Dr. Saltman intended with his public broadcasts. In commemoration of the 20th issue of Saltman Quarterly, here’s to all the mentors whose passion for their fields push us to the limits of our potential and inspire us to step up for the next generation.

Written by Annika So

Annika So is a 4th year Biochemistry/Chemistry major from John Muir College. She will be graduating in 2023.

FEATURES

Meerkat (Suricata suricatta) at the San Diego Zoo, California.

UC San Diego is at the forefront of scientific discovery and exploration as a hub of biological research. The Features section highlights some of the groundbreaking work accomplished by researchers affiliated with the UC San Diego campus.

Photo by Andrea Farrell

Nightclub Bouncer in a Sea Urchin:

The Efflux Transporter Proteins Embedded

in Cell Membranes

Written by Qianqian Tao

Illustrated by Zaid Dibis

The ATP-binding cassette (ABC) superfamily includes efflux transporter proteins that constitute the first line of defense against environmental toxicants in many organisms. The Hamdoun Lab at Scripps Institution of Oceanography investigates the activity of these transporters in sea urchins–an ideal animal model–to demystify why chemicals are processed differently in distinct cell types.

When you walk barefoot around the rocky intertidal zone, you might notice these otherworldly, spiny creatures burrowed in rock crevices. Just by looking at them, you would never believe how closely related they are to humans, but evidence shows that sea urchins could be a powerful genetic model for studying human diseases.

Sea urchins hold a closer position to humans in the phylogenetic tree than fruit flies and nematodes and share early developmental pathways with us, making them ideal for understanding the mechanisms of disease. Sea urchins and humans are deuterostomes, which means that our embryos develop an anus before a mouth, while other animals, such as fruit flies and nematodes, form their mouths first. They also share many genes with us. The first whole genome sequencing results of a non-chordate deuterostome were obtained from the purple sea urchin Strongylocentrotus purpuratus, revealing that sea urchins share 70% of their genes with humans. These shared genes include ones that regulate disease response and cause genetic disorders. For example, a gene that encodes for an efflux transporter protein from the ATP-binding cassette (ABC) superfamily can lead to blood disorders in humans when mutated.

ABC transporters constitute the first line of defense against toxicants in many organisms. They are embedded in cell membranes to prevent foreign molecules, known as xenobiotics, from entering and harming the cells. If the transporters let in a toxic molecule, they may expel this molecule after intracellular detoxifying enzymes modify it.1 Problems may arise when the transporters fail to recognize a chemical, or their efflux ability is hindered. Professor Amro Hamdoun, a marine biologist from Scripps Institution of Oceanography, believes that studying the activity of the ABC transporter superfamily can help demystify how chemical exposure may cause disease. Why does a toxic molecule penetrate and kill healthy cells, while cancer drugs have limited drug efficacy due to the failure to enter tumor cells? With 400 out of 23,300 genes in the genome encoding for defense systems against chemical stressors, sea

urchins became the lab’s primary research model for studying ABC transporters.

EXPRESSION PATTERNS OF ABC TRANSPORTERS IN EMBRYOS

Embryos are considered the most fragile stage of life due to a lack of a fully developed immune system. However, sea urchin embryos express a high level of ABC transporters, which implies a sophisticated defense system against xenobiotics. Expression levels aren’t uniform throughout the embryo, though.2 To understand how transporter activity levels differ in tissues of the embryo, Dr. Hamdoun’s Lab characterized the spatial and temporal expression of three essential subfamilies of ABC transporters in embryos of the purple sea urchin Strongylocentrotus purpuratus. The three subfamilies explored in this study include ABCB, ABCC and ABCG transporters, which differ in their number of transmembrane and nucleotide (i.e. ATP) binding domains. The ABCB transporters act on a chemical before it enters the cell. The ABCC transporters, on the other hand, can efflux both normal metabolites and xenobiotics modified by intracellular detoxification processes. The ABCG transporters, also known as the breast cancer resistance protein, are responsible for translocating nutrients from the mother to the baby during pregnancy and lactation, but could also inadvertently transport toxic chemicals to the baby.3

The lab used two technologies, in-situ hybridization and laser scanning confocal microscopy, to provide accurate spatial and temporal information about transporter expression levels in embryos.4 Researchers could get a large amount of test subjects by injecting KCl through the tissue around the mouth of a sea urchin, triggering it to release millions of reproductive cells. These egg or sperm cells were collected and fertilized externally to obtain a large amount of embryos. In-situ hybridization hybridized mRNA strands with corresponding colorimetric tags that are visible to naked eyes to stain the embryo. Areas in the embryo with high levels of stain revealed locations with elevated levels of transporter expression. Cyclins is a family of signal proteins from

1. Expression of ABCB transporter genes in sea urchin embryos

Staining indicates ABCB1 and ABCB4 expression throughout embryonic development. ABCB1 is ubiquitously expressed and becomes enriched in the gut while ABCB4 is exclusively expressed in the gut.

maternal eggs that maintain synchronous cell division during normal embryonic development. These proteins promoted synchronized development of embryos in the experiment, ensuring adequate test subjects at any time point. The laser scanning confocal microscopy tool took images of these stained embryos at high resolution. Researchers then analyzed these images captured at successive stages of development to determine temporal and spatial patterns of high levels of ABC transporters expression.

With the assistance of in-situ hybridization and confocal imaging, the lab became the first to characterize the expression patterns of ABC transporters in deuterostome embryos. They found that the activity of ABC transporters exhibited temporal and spatial specificity, making certain cell types more resistant to xenobiotics than others. This highly regulated transporter activity also allowed for the translocation and function of certain

signaling molecules to different parts of the embryo. One observation was that ABCC4 was highly expressed in the mesoderm, the middle layer of the embryo which later differentiates into various body tissues, and may be responsible for gut differentiation as it transports signaling molecules. Another pattern was an extensive expression of ABC transporters including ABCB1, ABCB4, and ABCG2 within the gut, ensuring a strong protection from xenobiotics when the embryo starts to feed. These sophisticated expression patterns imply that the activities of ABC transporters are highly controlled and involved in key embryogenesis processes. This study also provides insight into how certain toxic chemicals that cannot be recognized and effluxed by transporters will start to interfere with normal cell function during early embryonic development. By pinpointing which areas of the embryo have higher or lower transporter expression, we can extrapolate the effect that toxic molecules have on adult organs, as we know which regions of the embryo will develop into certain organs.4

DEVELOPING A KNOCKOUT LINE IN SEA URCHIN

Another direction the Hamdoun lab took to investigate the function of ABC transporters was to knock out the gene of a transporter and maintain a stable knockout line in sea urchin to determine how the loss of transporter functionality affected the uptake and efflux of a molecule. The transporter ABCB1a, also known as permeability glycoprotein (P-gp), derives its name from its ability to transport a wide range of molecules through cell membranes. This transporter acts like a bouncer in a nightclub that “kicks” undesirable guests out of the party. This family is also widely studied in therapeutic drug development for being the major obstacle for drug efficacy because the transporters continuously expel the drug taken in by patients.3

The significance of ABCB transporters led the lab to utilize CRIS-

PR-Cas9 to knock out the ABCB1a gene–a major contributor to an animal’s ability to efflux xenobiotics–in the painted sea urchin Lytechinus pictus. The lab used a micron needle to inject CRISPR-Cas9 into embryos of the F0 generation. Sea urchin embryos have a physical disruption repair mechanism that fixes the broken cell membrane and ensures normal embryonic development after injection. Sperm from edited adult males were fertilized with eggs from wild-type adult females to generate the heterozygous F1 generation. The F1 generation was in-crossed manually to make homozygous mutants in the F2 generation. The F2 generation grew to the juvenile stage, solidifying a stable knockout line with non-functional ABCB1a. The reduced activity of the ABCB1a gene in the F2 generation was confirmed by measuring the accumulation of a molecule that only fluoresces when it accumulates inside a cell. A higher level of fluorescence was detected in embryos where ABCB1 a was knocked out as more foreign molecules accumulated in cells. The methods used in this study to achieve a stable knockout line omitted repetitive procedures (e.g. mRNA injection) and can be easily reproduced in other labs, accelerating research on disease modeling in sea urchins. This stable line also reduces the variability introduced in collecting wild animals for research by providing a stable source of sea urchins with predictable genotypes. Sea urchin lines have the potential to be applied in the pharmaceutical industry to test therapeutic drugs, gene function and protein production.5

MAN-MADE CHEMICALS BECOME TRANSPORTER INHIBITORS

The sophisticated efflux transportation mechanisms in sea urchins have evolved to adapt to naturally occuring toxic molecules, which can change over time. However, the rate at which new man-made chemicals are introduced to the environment exceeds the limits of evolution. These chemicals might inhibit transporter proteins by occupying active site pockets, which prevents the transporters from exporting other molecules.6 Going back to the nightclub analogy, if there are too many bad individuals that meddle and confuse the bouncers, it will be challenging for the bouncers to ensure all the bad individuals are out. Thus, the efflux efficacy

Figure

Figure 2. Membrane topology models of ABC transporters.

Subfamilies of ABC transporters consist of different numbers of nucleotide binding domains (NBDs) and transmembrane domains (TMDs).

is greatly reduced. One transporter inhibitor is synthetic musk fragrance. This chemical can accumulate in human bodies through the use of perfume and leak into the ocean through sewage. Studies on mussels reveal that synthetic musk results in a long-term loss of efflux transporter activities. While the activities were prohibited, the animal could accumulate toxic molecules that should have been effluxed under normal transporter function.3 Inversely, transporter inhibitors can have positive implications in therapeutic design. Researchers are developing transporter inhibitors that can be administered alongside cancer drugs to prevent them from being exported by ABC transporters.

Sea urchins and other marine organisms are not the only ones that need to cope with toxic chemicals leaked into the environment. In our daily lives, we are also exposed to various xenobiotics that can accumulate in our bodies and even pass on to future generations, such as flame retardants, persistent pesticides, and molecules in non-stick surfaces. These chemicals can not be efficiently effluxed by ABC transporters; therefore, they accumulate in cells, disrupt normal cell function, and induce disease.

Dr. Hamdoun’s Lab intends to use sea urchin models to understand how ABC transporter activity influences the toxicity of relevant chemicals. Sea urchins are a strong animal model for exploring these human diseases because of their genetic similarities to humans, unique embryonic features, and sophisticated defense systems. The lab’s progress in characterizing the temporal and spatial expression of three ABC transporters in sea urchin embryos provides insight into how human embryos interact with xenobiotics from the womb. Additionally, the stable knockout line in sea urchins they generated promotes the extensive and

sustainable usage of the sea urchin in labs. There are still many questions that remain unsolved in this field: What does studying chemical defense systems in sea urchin embryos inform us about human embryonic development? Can the relationship between ABC transporters and small molecule inhibitors inform pharmaceutical development? More generally, what can we do to make sea urchins more readily available for experimental use? The Hamdoun lab continues to be intrigued by these curious sea creatures. Dr. Hamdoun anticipates a promising future in the field as creative undergraduate and graduate researchers in the lab work towards answering these questions.

REFERENCES

[1] Goldstone, J. V., et al. "The chemical defensome: environmental sensing and response genes in the Strongylocentrotus purpuratus genome." Developmental biology 300.1 (2006): 366-384.

[2] Hamdoun, A., & Epel, D. (2007). Embryo stability and vulnerability in an always changing world. Proceedings of the National Academy of Sciences, 104(6), 1745-1750.

[3] Epel, David, et al. "Efflux transport-

[4] Schrankel, Catherine S., and Amro Hamdoun. "Early patterning of ABCB, ABCC, and ABCG transporters establishes unique territories of small molecule transport in embryonic mesoderm and endoderm." Developmental biology 472 (2021): 115-124.

[5] Vyas, Himanshu, et al. "Generation of a homozygous mutant drug transporter (ABCB1) knockout line in the sea urchin Lytechinus pictus." Development 149.11 (2022): dev200644.

[6] Nicklisch, Sascha CT, et al. "Global marine pollutants inhibit P-glycoprotein: Environmental levels, inhibitory effects, and cocrystal structure." Science advances 2.4 (2016): e1600001.

Qianqian is a 4th year Marine Biology major from Roger Revelle College. ers: newly appreciated roles in protection against pollutants." (2008): 3914-3920.

Written by Qianqian Tao

The Guardian of the Genome

Written by Ananya Prasad Illustrated by Simran Patel

A deep dive into the workings of p53, a crucial regulatory protein, and its connection to tumor formation in pancreatic ductal adenocarcinoma and breast cancer.

Cancer is the poster child for cell proliferation, yet even after decades of research, we haven't com pletely understood its behavior in the human body. What factors in a cell’s surroundings transform it into a can cerous cell? Which molecules control a cell’s ability to multiply at a fatally high rate? Which genes are responsible for preventing a cell from going rogue? If we answer these questions, we might be able to prevent a cell from becoming cancerous. To start, one place to look are the mechanisms to maintain cell growth, which includes the protein p53, also known as “The Guardian of the Genome.”

In 1979, p53 was first discovered bound to an oncogenic protein in simian virus 40 (SV40) infected cells. This association led to the belief that p53 was an oncoprotein (a protein that can transform a normal cell into a tumor cell) due to its high levels of expression in cancer cells. A paradigm shift in the understanding of p53 function occurred in 1989 with use of the “two-hit” test, a method which hypothesizes that for genes where mutations cause cancer, an oncogene only has one mutated allele, while tumor suppressor genes have homozygous mutations. When performed on chromosome 17, the test revealed both copies of p53 were mutated in cancer cells, indicating it was a tumor suppressor. Today, p53 is known as an essential transcription factor that suppresses tumorigenesis by regulating cell division and death through its ability to recognize small amounts of DNA damage, arrest growth of genetically unstable cells, and kill cells with irreparable chromosomes. In fact, the gene that encodes for p53 is the most frequently mutated gene in human cancers, pointing to its role in preventing cancer formation.1

a dynamic tumor ecosystem composed of 2 main types of cells: around 10% of the tumor is made up of pathogenic pancreatic epithelial cells containing mutant p53 that cannot regulate DNA damage, while the other 90% is characterized by a dense fibrotic stroma known as desmoplasia that facilitates tumor growth and drug resistance by acting as a virtually impenetrable shelter for the malignant cells harbored within. Desmolpasia can be better understood as a type of fibrosis composed of extracellular matrix (ECM) proteins, immune cells, and pancreatic stellate cells.2,3

Dr. Geoffrey M. Wahl, a professor at the Salk Institute for Biological Studies, began research in this area out of curiosity as to how normal epithelial cells, with diploid chromosomes, transform into cancer cells with an abnormal number of chromosomes.

Dr. Geoffrey M. Wahl, a professor at the Salk Institute for Biological Studies, began research in this area out of curiosity as to how normal epithelial cells, with diploid chromosomes, transform into cancer cells with an abnormal number of chromosomes. This led to him studying p53 and how it regulated chromosomal stability. To further understand the implications p53 had for cancer, he turned to pancreatic ductal adenocarcinoma (PDAC) as a model. Like most cancers, PDAC contains

As a type of fibroblast, pancreatic stellate cells are normally quiescent, but in the presence of cancerous cells actively divide to form a desmoplasia. Since cancer-associated pancreatic stellate cells (caPSCs) are not part of the core tumor, it was unknown whether they contain the wild type or mutant copy of the p53 gene. To test this, Dr. Wahl’s Lab isolated fibroblasts from 8 different patients with PDAC and treated them with Nutlin-3a, a small molecule that allows the accumulation of p53. Nutlin-3a works as an Mdm2 inhibitor, competitively binding to this p53 regulator protein which ubiquitinates p53 to signal for its degradation. Mdm2 itself is a transcriptional target of the p53 protein, thereby creating a negative feedback loop that regulates both

p53 and Mdm2 levels. However, mutant p53 present in cancer cells are not transcriptionally active, preventing the regulatory negative feedback loop from functioning and leading to elevated levels of p53 in cancer.

Since cancerous cells possess inactive mutant p53 (leading to lack of Mdm2), Nut lin-3a should have no effect on p53 levels. However, an increase in p53 expression was observed in all 8 samples, supporting the hypothesis that caPSCs contain functional wild type p53. The Nutlin-3a treatment also induced phe notypic changes consistent with cell quiescence (such as cytoplasmic lipid droplet accumulation and downregulation of smooth muscle alpha-actin) in the caPSCs. To further understand the genome-wide effects of p53, Dr. Wahl’s team analyzed the transcriptome of human caPSC lines cultured with Nutlin-3a and found that wild type p53 expression results in decreased expression of oncogenic genes, suggesting that p53 plays a role in caPSC inactivation.

With these promising findings in vitro, Dr. Wahl and his team wanted to see whether these results could be translated in animal models to decrease desmoplastic density. Thus, they decided to also test p53 activation in vivo on mice with PDAC. They injected these mice with a clinical version of Nutlin-3a, increasing p53 expression.

However, the change in the density of the desmoplasia was not enough to produce a significant difference in chemotherapeutic drug treatment response. This goes to show that even with a sound hypothesis, the translation from in vitro to in is not always seamless.4

As the guardian of the genome, mutated p53 can create tumor heterogeneity. It was known that particular oncogenes were responsible for reprogramming normal cells into undifferentiated pluripotent cells. Under normal conditions, it activates p53 so that it arrests the cell cycle and induces cell death. However, inactivating p53 causes cellular reprogramming to go ahead, where a mature, specialized cell reverts to an earlier stem-like state from which the cell can re-specialize. This causes intratumoral heterogeneity, where cells within a tumor can have distinct morphological and phenotypic profiles, such as those observed in PDAC.

Dr. Wahl and his team hypothe sized that given the high frequency of p53 mutations in cancers such as breast cancer, the oncogenes responsible for cellular repro gramming could be working while mutated p53 was unable to arrest this process. This made the cell more vulnera ble to reprogramming into a primitive, plastic state and marked the progression of a cancer. To test this hypothesis, one experiment compared the transcriptome (the set of all RNA transcripts in coding and non-coding regions of the DNA) of induced pluripotent cells (normal cells that had been induced to pluripotency by inactivating p53 and activating oncogenes) to an array of breast cancer cells. A high similarity was

found between the transcriptome gene signatures of the induced pluripotent cells and basal-like breast cancer cells, a type of aggressive breast cancer. Studying various mouse models has

Inactivating p53 causes cellular reprogramming to go ahead, where a mature, specialized cell reverts to an earlier stem-like state from which the cell can re-specialize.

shown that tumors mimicking basal-like breast cancer can be generated by inactivating or mutating a multitude of genes such as BRCA1 and PIK3ca in luminal breast cells. These genetic mutations become more possible when p53 is inactivated and the genome

Figure 1. Desmoplasia

Pathogenic pancreatic epithelial cells surrounded by a hard shell of cancer-associated pancreatic stellate cells that prevent drug penetration, i.e. desmoplasia.

The stem cells that the gene signatures of basal-like breast cancer cells matched were normal stem cells that develop into mammary gland cells. These are only abundant during the embryonic stage and should cease to exist after birth. So how did the basal-like breast cancer cells have a similar gene signature to that of pluripotent cells? To further investigate this similarity, Dr. Wahl isolated normal mammary gland stem cells and compared its transcriptome to the basal-like breast cancer cells. He discovered that the transcriptome of basal-like breast cancer cells was highly related to that of the embryonic stem cells. This led to the conclusion that basal-like breast cancer involved cellular reprogramming from a normal differentiated breast cell to a developmentally plastic stem-like cell. Similar instances of cell state reprogramming can be found in other cancers and diseases. In colon cancer, cells reprogram into stomach-like cells by going through a fetal intermediate state. In Barrett's Esophagus (damage to the lower portion of the esophagus), tissue irritation can result in reprogramming of adult cells into embryonic stem-like cells. In pancreatic ductal ligation, which causes

degradation of acinar cells (the functional unit of the exocrine pancreas), cells reprogram to an embryonic state to generate acinar cells and ductal cells (cells lining the pancreatic duct) to repair the damaged pancreas. The common theme in all these cases of cellular reprogramming is the inactivation of p53. Reducing the expression of p53 is not always an indication of cancer. Cells often need to repair injury, which requires a lower level of p53 expression. However, being in this state makes the cell highly vulnerable to accumulate mutations, increasing the risk of cancer.5

Studies in Dr. Wahl’s Lab began with finding answers to basic scientific questions, such as how cancer cells acquire and survive with chromosomally unstable genomes. Their pioneering research led to fundamental findings in the field of cancer biology, further cementing p53’s role as a master regulator of both the genome and cell state integrity and stability. Knowing how p53 mutations can propel tumor development has made p53 a major field in cancer research. Further exploring the mechanisms that mediate cell reprogramming and the factors that contribute to genome instability and tumor heterogeneity can be pivotal in finding therapies to remedy these occurrences. For example, investigating the extent of change in desmoplastic density due to p53 activation may yield more effective drug therapies for diseases like PDAC. Perhaps with further targeted research and innovation, we will be equipped with the tools to treat one of mankind’s most puzzling diseases.

REFERENCES

[1] Vogelstein, B., Sur, S. & Prives, C. (2010) p53: The Most Frequently Altered Gene in Human Cancers. Nature Education 3(9):6

[2] Sarantis, Panagiotis et al. “Pancreatic ductal adenocarcinoma: Treatment hurdles, tumor microenvironment and immunotherapy.” World journal of gastrointestinal oncology vol. 12,2 (2020): 173-181. doi:10.4251/ wjgo.v12.i2.173

[3] Pandol, Stephen et al. “Desmoplasia of pancreatic ductal adenocarcinoma.” Clinical gastroenterology and hepatology: the official clinical practice journal of the American Gastroenterological Association vol. 7,11 Suppl (2009): S44-7. doi:10.1016/j. cgh.2009.07.039

[4] Saison-Ridinger, Maya et al. “Reprogramming pancreatic stellate cells via p53 activation: A putative target for pancreatic cancer therapy.” PloS one vol. 12,12 e0189051. 6 Dec. 2017, doi:10.1371/journal. pone.0189051

[5] Wahl, Geoffrey M, and Benjamin T Spike. “Cell state plasticity, stem cells, EMT, and the generation of intra-tumoral het erogeneity.” NPJ breast cancer vol. 3 14. 19 Apr. 2017, doi:10.1038/ s41523-017-0012-z

Figure 2: Cell Reprogramming

Reversion of a specialized cell into a primitive, plastic stem-like state followed by re-specialization of that cell, i.e. cell reprogramming.

Written by Ananya Prasad

Ananya is a 3rd year Bioinformatics major from Seventh College.

What to Expect When You're Expecting:

The Female Pelvic Floor, Pregnancy, & Aging

Written by Amy Li

Illustrated by Hanh Nguyen

Photo by Harnoor Sidhu

Human female pelvic floor muscles have a seemingly contradictory potential for plasticity and susceptibility to degeneration. The Alperin Lab at UC San Diego aims to understand the mechanisms underlying these two properties in order to improve care for patients suffering from pelvic floor disorders.

In the final week before childbirth, the soon-to-be mother supports the weight equivalent of a small pumpkin inside their womb. During vaginal delivery, the strain of expelling that pumpkin-sized baby results in the pelvic floor muscles stretching up to 300% of their resting length, five times the threshold for injury in other skeletal muscles.1 Interestingly, the mother may never develop pelvic floor dysfunction, while another woman who has never had children might.

Female pelvic floor muscles (PFMs), perhaps most commonly known as the muscles activated in Kegel exercises, are a group of skeletal muscles that support the pelvic organs (i.e. the uterus, bladder, and rectum). Their ability to (usually) withstand the stresses of pregnancy and delivery with no apparent consequence points to a unique capacity for plasticity and regeneration, just as the appearance of PFM dysfunction in those with no history of vaginal delivery warns of a unique vulnerability to degeneration.

PFM dysfunction often develops into one or more medical conditions known as pelvic floor disorders (PFDs), which affect an estimated 25-50% of those with female PFMs over age 50 worldwide. Examples of PFDs include pelvic organ prolapse, in which the pelvic organs slip and bulge painfully into the vagina, and stress urinary incontinence, which is the involuntary leakage of urine.2 Despite their prevalence, there is a lack of preventative measures for PFDs and treatment options are often ineffective or limited: physical therapy, surgery, or botox injections (a treatment that offers pain relief without addressing any underlying causes.) A treatment that does address underlying causes is a tall order, given how understudied and poorly understood those causes are—all too common for women's health as a whole.

This state of affairs motivates the interdisciplinary research of physician-scientist Dr. Marianna Alperin, a female pelvic medicine and reconstructive surgery specialist who leads a research lab at UC San Diego. The Alperin lab seeks to understand the roles of two notable risk factors—vaginal childbirth and aging—in the pathophysiology of PFM dysfunction in order to find better clinical solutions. Over the course of their research, two significant biological constitu-

ents that have emerged in PFM regeneration and dysfunction are muscle stem cells and intramuscular extracellular matrix (ECM) collagen content.

REGENERATION: MUSCLE STEM

CELLS

Muscle stem cells (MuSCs) are a subtype of stem cells—undifferentiated cells with the ability to remain dormant until they are activated to either self-renew their population or differentiate into specialized cells. MuSCs specifically form new myofibers (muscle fibers) or fuse with existing ones to achieve muscle regeneration. As people age, the MuSC reservoir is depleted (with depletion in PFMs being two times greater than in other skeletal muscles) and the remaining MuSCs become less effective.3 Researchers believe there is potential here for stem cell therapy—in which stem cells are transplanted into a patient to repair or even regenerate damaged tissue—to treat PFDs brought on by aging. However, they first need to better understand female PFM MuSCs' properties.

A significant barrier to the study of female PFMs is their anatomic location deep inside the pelvis which makes the collection of tissue samples from living people—especially from pregnant individuals—technically difficult and ethically dubious. These constraints prompted the Alperin Lab to develop a novel method for isolating the PFM MuSCs in rats, an excellent model for human PFMs due to significant similarities in muscle architecture. Researchers identified the cell surface protein CD106, necessary for the maintenance of quiescent MuSCs and prevention of their premature entry into the cell cycle, as a unique marker for MuSCs. An antibody that binds specifically to CD106 was added to a mixture of rat PFM cells, which was then coupled with a fluorescent dye, allowing the now-fluorescent MuSCs to be sorted with a fluorescence-activated cell sorting machine. They confirmed the successful isolation of MuSCs using multiple criteria, including the presence of identifying transcription factors and the cells' ability to form myofibers in growth media. cost-effective method will enable the Alp erin Lab's upcoming studies on the effect of pregnancy on MuSCs and the differences between

PFM MuSCs and other skeletal MuSCs, without the need for samples from pregnant people.

STABILITY YET STIFFNESS: ECM COLLAGEN

One of the elements responsible for PFM plasticity and degeneration is intramuscular ECM collagen, an important structural protein found in connective tissues. Alperin Lab researchers demonstrated that collagen can promote PFM plasticity during pregnancy by increasing their collagen levels when under mechanical stress. In a study that involved inserting weighted beads into the uteri of rat models to simulate pregnancy without the confounding effect of its accompanying hormonal changes, the intramuscular collagen content in "pregnant" PFMs was compared to control PFMs and other skeletal muscles in the "pregnant" rat. They found that the pregnant PFMs had more collagen compared to both the control and other skeletal muscles, leading the researchers to conclude that PFMs have the unique ability to increase their intramuscular collagen content in response to pregnancy. They hypothesize that the collagen stabilizes sarcomeres (repeating contractile functional units in myofibers) which elongate in response to the growing fetus's increased mechanical load. This would explain how PFMs are able to withstand up to five times more stretching than other skeletal muscles.

However, in a second study done on human female cadavers, increased collagen caused stiffness in the PFMs (but, strangely enough, not in other skeletal muscles) of older individuals, reducing aged PFMs' capacity for supporting the pelvic organs' weight.4

Alperin Lab researchers conducted a follow-up study revealing that excessive collagen disrupted myofibrillar packing and caused fibrosis, where muscle scar tissue replaces functional myofibers in a process called degeneration.5 Stiffness and fibrotic degeneration lead to mechanical dysfunction and PFM weakness, offering a mechanistic explanation for why PFD risk increases with age and exposing the fine line between benefit and harm when it comes to elevated PFM collagen levels. Collagen stabilizes muscles in pregnancy, yet stiffens them in old age. This leads to the unanswered question across both studies: how much collagen is too much? It remains unclear at what point increased collagen shifts from providing necessary structural support to damaging PFM function.

Still, the Alperin Lab's discovery that aging PFMs undergo degeneration rather than solely a loss of muscle cross-sectional area, known as atrophy,5 is a significant one: it explains why current physical therapy treatments for PFD sufferers often fail. Exercising atrophying muscle can restore muscle mass, but exercising degenerating muscle only further injures myofibers and encourages replacement by fibrotic tissue.

PUTTING IT TOGETHER

The fibrosis arising from increased collagen content and MuSCs are not isolated features; understanding their interplay is key to understanding PFM dysfunction. This is where fibroadipogenic progenitor cells (FAPs) come in. FAPs have two major roles: 1) they differentiate into fat cells and fibroblasts (cells that secrete collagen), and 2) they contribute to MuSC activation via cytokine secretion, which then signals for immune response and inflammation following an injury. Typically when inju-

floor.

The bladder, uterus, and rectum (left to right) are supported by the PFMs, consisting of the coccygeus and levator ani muscles, with the levator ani made up of the iliococcygeus and pubovisceralis muscles.

ry occurs, the tissue's FAP population proliferates and supports regeneration by activating MuSCs and differentiating into fibroblasts, which lay down the collagen needed to support healing tissues. However, due to MuSC depletion with age, injured myofibers are no longer replaced by new myofibers, but instead by fibrotic and fatty tissue created by FAPs, making them also responsible for degeneration.5,6

Aging throws FAPs' roles in creating fibroblasts and recruiting MuSCs out of balance. The Alperin lab assessed the effect of aging on PFM injuries in rat models by using irradiation to induce cellular senescence, a state where cells can no longer undergo cell division, which contributes to aging. Researchers used embryonic myosin heavy chains as a marker for newly regenerated myofibers. The irradiated injured muscles had fewer fibers positive for embryonic myosin heavy chain compared to non-irradiated controls. Further examination revealed that the impaired regeneration of irradiated muscles was attributable to lower FAP proliferation and subsequently reduced MuSC recruitment.6 Regulation of FAP differentiation, then, is another potential therapeutic strategy for PFDs, particularly if used in coordination with stem cell therapy.

Beyond identifying potential therapeutic targets, the Alperin Lab is also developing new, concrete treatments. In collaboration with researchers from UC San Diego's Bioengineering department, the Alperin Lab has created a new biomaterials treatment for birth injury. Highlighted in a promising preclinical trial, skeletal muscle (SKM) hydrogel is a gelatin-like injection made from isolating pig muscle ECM from its inhabiting cells in a process called decellularization. SKM was shown to prompt upregulation of genes associated with MuSC pool expansion and reduce excessive collagen levels.7 SKM is also affordable, minimally invasive, and effective both post-delivery and four weeks later at the post-partum visit. These patient-oriented considerations were taken into account thanks to Dr. Alperin's perspective as a physician-scientist, ever conscious of how her research will translate into improving her

Figure 1: Anatomy of the human female pelvic

patients' lives.

MARGINALIZATION OF WOMEN'S HEALTH

The difficulty with centering research around patient experiences lies in attracting more physicians like Dr. Alperin into research; underfunding likely discourages potential physician-scientists from entering women's health research. In a study that examined NIH funding, 64% of female-dominant diseases were underfunded in 2019, compared to only 8% in male-dominant diseases. To make matters worse, the degree of underfunding was severe: female-dominant diseases received, on average, only 37% of the funding that the disease's economic burden warranted.8 These numbers speak to a systemic issue in medical research, where women's health has historically been and continues to be dismissed.

Though a common health issue, public awareness of PFDs is limited because of the stigma surrounding women's health. Americans are just beginning to move past the conditioned embarrassment surrounding menstruation, a non-pathological process, so one can imagine the shame that someone suffering from a PFD may feel about their symptoms. One way to destigmatize PFDs is for physicians to include preventative care into routine checkups, which can serve the dual purposes of normalizing discussions about PFDs and preventing PFDs. According to Dr.

Figure 2: FAPs' response to myofiber injury.

Normally (left), FAPs increase leads to MuSC activation, then tissue repair. In pathological states (right), such as in aged tissue, there are not enough MuSCs for the FAPs to activate, causing collagen and fat to take over the injured myofibers.

Alperin, although physical therapy is often ineffective after the onset of PFDs, exercising the PFMs (i.e. squeezing, or doing Kegel exercises) does work as a preventative measure. But many of the patients she's worked with are not performing their Kegels correctly; they're clenching their gluteal or inner thigh muscles, not the PFMs. OB/GYNs and even primary care providers can promote these exercises and verify that people with female PFMs are squeezing the right muscles during checkups.

PFDs have long been poorly understood and consequently written off as just an inevitable part of childbirth and aging—but they don't have to be. Thanks to the Alperin Lab's work, we are closer to understanding how the mother who vaginally delivered a pumpkin-sized child can never develop pelvic floor dysfunction—her PFMs adapted to avoid birth injury by increasing their intramuscular collagen content; and how another woman who has never had children might—her PFMs underwent fibrotic degeneration due to aging. There's still a lot we don't know: how does pregnancy affect PFM MuSCs? How much collagen is too much collagen? Luckily, progress in PFM research is rapid, since researchers can borrow existing tools and methods from more mature disciplines—a silver lining in a field where much is left unanswered.

REFERENCES

[1] Rieger MM, Wong M, Burnett LA, Sesillo FB, Baynes BB, Alperin M. 2022. Mechanisms governing protective pregnancy-induced adaptations of the pelvic floor muscles in the rat preclinical model. Am J Obstet Gynecol. 226(5):708.e1-708. e13. doi:10.1016/j.ajog.2021.11.1353.

[2] Alperin M, Abramowitch S, Alarab M, Bortolini

M, Brown B, Burnett LA, Connell KA, Damaser M, de Vita R, Gargett CE, and others. 2022. Foundational science and mechanistic insights for a shared disease model: an expert consensus. Int Urogynecol J. 33(6):1387-1392. doi:10.1007/ s00192-022-05253-y.

[3] Sesillo FB, Wong M, Cortez A, Alperin M. 2022. Isolation of muscle stem cells from rat skeletal muscles. Stem Cell Res. 43:101684. doi:10.1016/j.scr.2019.101684.

[4] Burnett LA, Cook M, Shah S, Wong M, Kado DM, Alperin M. 2020. Age-associated changes in the mechanical properties of human cadaveric pelvic floor muscles. J Biomech. 98:109436. doi:10.1016/j.jbiomech.2019.109436.

[5] Rieger M, Duran P, Cook M, Schenk S, Shah M, Jacobs M, Christman K, Kado DM, Alperin M. 2021. Quantifying the Effects of Aging on Morphological and Cellular Properties of Human Female Pelvic Floor Muscles. Ann Biomed Eng. 49(8):1836-1847. doi:10.1007/s10439-02102748-5.

[6] Sesillo FB, Rajesh V, Wong M, Duran P, Baynes B, Laurent LC, Christman KL, Sacco A, Alperin M. 2021. The role of muscle stem cells and fibro-adipogenic progenitors in female pelvic floor muscle regeneration following birth injury. bioRxiv. doi:https://doi.org/10.1101/2021.07.30.454534.

[7] Duran P, Sesillo FB, Burnett L, Menefee SA, Cook M, Zazueta-Damian G, Dzieciatkowska M, Do E, French S, Shah MM, and others. 2021. Pro-regenerative Extracellular Matrix Hydrogel Prevents and Mitigates Pathological Alterations of Pelvic Muscles Following Birth Injury. bioRxiv. doi:https://doi.org/10.1101/2021.05.28.446170.

[8] Mirin AA. 2021. Gender Disparity in the Funding of Diseases by the U.S. National Institutes of Health. Journal of Women's Health. 30(7):956963. doi:https://doi.org/10.1089/jwh.2020.8682.

Written by Amy Li

Amy is a 2nd year Neurobiology and Biochemistry double major from Thurgood Marshall College.

The New Era of Biology:

Differentiating Rare Inflammatory Diseases with the Boolean Method

Written by Anna Grinberg

Illustrated by Oliver Kelley

Computational analysis of gene sequences has been a valuable tool for identifying molecular pathways which contribute to the understanding of medical conditions. The Boolean Lab at UC San Diego analyzes the activity of gene expression to compare host-immune responses for two different diseases that may aid in tracking prognosis and drug development.

Alarge amount of genetic information is accessible to biologists who don't know how to wrangle the data, and computer scientists who can’t interpret the biology. A bridge between applying computational formulas to data sets and identifying correlations between various molecular mechanisms is the new era of biology. For example, transcriptomes which contain the portion of RNA code for protein expression may be biomarkers which correlate to a certain disease.1 A biomarker may be a set of genes that express proteins, known as a gene signature, which contribute to a subsequent medical condition.1 Identifying and quantifying biomarkers corresponding to specific diseases can aid in revealing distinct molecular pathways and inform therapy development. However, this process is easier said than done. In the clinic, small patient sample numbers, especially with rare diseases, makes identifying biomarkers hard. In the laboratory, petri dish-based experimental models may not reflect human physiology. However, with computational assistance, these barriers can be overcome. One application could be a formula that simplifies clinical transcriptomic data into a correlational relationship with disease type and severity. This can be analyzed to differentiate between two symptomatically similar medical conditions and serve as a step towards accurate diagnosis and tracing disease severity.

In the clinic, separate patients may present with indistinguishable symptoms, with diagnoses requiring very different treatments. In recent years, a condition linked to post-COVID-19 infection, known as multisystem inflammatory disease in children (MIS-C) caused an inflammatory response in children, including skin rashes and a heart defect known as myocardi-

al dysfunction. As MIS-C case counts accumulated, doctors found these symptoms hard to distinguish from another pediatric disease, Kawasaki disease (KD). This acute inflammatory disease was first described in Japan, 1967.2 Patients show symptoms of red eyes, hands, and feet. As an immune response to an unidentified antigen trigger, doctors observe high white blood cell count and long-lasting vascular damage which causes coronary artery aneurysms (inflamed arteries). Fortunately, MIS-C patients who present vascular damage do not experience permanent

Identifying and quantifying biomarkers corresponding to specific diseases can aid in revealing distinct molecular pathways and inform therapy development.

effects. In contrast, 25% of KD patients, if left with untreated coronary arterial walls, will develop life-threatening aneurysms.2 Although the early onset symptoms of these two diseases are similar, their heart conditions require distinct treatments to prevent exacerbation. An appealing way to distinguish KD from MIS-C and provide appropriate treatment is through computational differentiation of their mRNA expression signatures which actively transcribes protein products. Using mRNA sequencing and further analysis to find a disease’s unique transcriptome signature shows promise towards locating inflammatory pathways that correlate with disease and symptom severity and could possibly serve as targets for drug development.

Dr. Debashis Sahoo, who leads the Boolean Lab at UC San Diego, took on the challenge to simplify these large transcriptomic datasets into a biomarker indicative of viral infection in collaboration with Dr. Jane C. Burns and Dr. Pradipta Ghosh. RNA sequencing data has allowed researchers to identify protein products that are correlated to host immune responses of certain viral infections. However, a biomarker that pertains to viral infections amongst these host immune responses has not been identified. To do this, researchers trained an artificial intelligence model on mRNA transcriptomes of patients infected by COVID-19 and influenza. To identify genes upregulated in COVID-19 infections, publicly accessible mRNA sequences of virally infected and healthy cells were analyzed against a specific gene’s expression levels as a reference point. Known as the ‘seed’ gene, this gene is chosen as the baseline from which correlational relationships can be derived.3 The re-

In acute KD, ViP and sViP signatures could classify patients with large coronary arterial aneurysms from those with small ones. Additionally, the sViP signature could differentiate MIS-C patients with major or minor myocardial dysfunction.

ceptor angiotensin-converting enzyme 2 (ACE2), which enables COVID-19 entry, was chosen as the ‘seed’ gene since it is associated with COVID-19 infection, and high levels are indicative of a viral response.3 Boolean analysis, a way to mathematically detect the dependent relationship between two values, identified 166 genes which were upand downregulated in correlation to ACE2 mRNA expression levels.4 This group of genes were coined the viral pandemic (ViP)

signature, which could be used as a fundamental viral marker. This 166-gene pool was further enriched for genes involved with unique pathways and trained, by the AI model, on influenza patients annotated with disease severity. This model obtained a 20-gene subset—coined the severe viral pandemic (sViP) signature. These ViP signatures served as mechanistic biomarkers that pro vided the framework for uncover ing and differentiating diseases.

To test the accuracy of the ViP signature, the model was tested against patient data. Dr. Jane C. Burns, a practicing pediatrician and Kawasaki disease expert at the UC San Diego School of Medicine, has experience with both KD and MIS-C patients. In 2020, she and her team collected whole blood samples from children in the emergency room presenting with fevers or any inflammatory symptoms. The blood samples were then isolated for RNA and the transcriptome was sequenced via next-gen eration sequencing, a concurrent examination of numerous gene fragments to detect sequence variations. This dataset was then given to Dr. Sahoo, whose group analyzed the 166-gene ViP and 20-gene sViP signatures using data sets of MIS-C and KD patients. Surprisingly, both KD and MIS-C patients showed upregulation in the genes associated with the ViP signatures, sharing the same viral pandemic signature and indicating they were on the same continuum of host immune response. Curiously, no unique viral source has been recognized in KD, and compelling evidence exists pointing to KD as a disease not caused by a viral infection; however it shares an inflammation gene signature that is linked to serious viral infections.

Figure 1.

Publically available transcriptome sequencing datasets enable scientists to computationally analyze gene expression in patients and gain understanding of a disease. This data is obtained from patients by collecting blood samples. These samples go into databases and are accessible to download for research purposes.

Beyond possible targets for drug treatments, the ViP signatures can po-

tentially predict the severity of disease and diagnosis. In acute KD, ViP and sViP signatures could classify patients with large coronary arterial aneurysms from those with small ones. Additionally, the sViP signature could differentiate MIS-C patients with major or minor myocardial dysfunction. Samples with higher levels of sViP signature expression revealed to be patients with major myocardial dysfunction, and a low level of sViP correlated to patients with

minor myocardial dysfunction.5 Blood transcriptomes of MIS-C patients were used to evaluate the protein products of marked genes that were tracked with clinical and laboratory outcomes such as the unique heart dysfunction of MIS-C patients.5 The results demonstrated that cytokine cellular communication and interferon upregulation was in higher quantities for MIS-C than KD. The greater magnitude of expression of these molecular factors involved in host-immune response in MIS-C determined that the severity and diagnosis of MIS-C may be quantitatively distinguished from KD. As computational methods help identify distinct gene expression biomarkers and highlight distinctive immune response features in rare diseases, it may aid in better understanding the nature of the disease.

KD and MIS-C, both inflammatory diseases, result in host-immune responses that stimulate immune cells, causing over-secretion of signaling proteins called cytokines.6 The extent of cytokine expression can differentiate inflammatory diseases. Researchers in the Boolean lab used KD and MIS-C blood samples and serum cytokine arrays to identify the cytokine-receptor pair, IL15 and IL15RA, as a prominent component of a pathway which induces the ViP signature. IL15 codes for a cytokine which binds to IL15RA receptors on immune cells to trigger an inflammation cascade. Though both KD and MIS-C shared an IL15 cytokine response, other responses were more unique to certain diseases.5 Upregulation of two other cytokines, TNFα and IL1β, was unique to MIS-C samples.5 The identification of TNFα and IL1β as upregulated cytokines in MIS-C may make them a target for anti-inflammatory drugs. Another distinctive quality of MIS-C based on analysis of serum cytokines showed that it had higher cytokine levels than KD, indicating a higher intensity immune response. Cytokine levels in MIS-C and KD as mechanistic biomarkers contribute to the broader understanding of MIS-C as a disease. This may help discern disease severity and proposes a target for therapeutics.

The Boolean lab has shown that an artificial intelligence approach to applying statistical concepts to biological data enables the identification of genetic biomarkers across a wide range of diseases. The strategy works in diverse contexts, such as mice, humans, or even pandemics, demonstrating how small sample sizes can be used to glean large insights through computer algorithms. This is especially important for rare diseases such as MIS-C and KD, where there isn't a large sample population. Biology contains complexities, uncertainties and exceptions, but Boolean logic can be used to control this noise.

REFERENCES

[1] Gönen M. 2009. Statistical aspects of gene signatures and molecular targets. Gastrointest Cancer Res.(2 Suppl):S19-21.

[2] Kushner, H. I.Turner, C. L., Bastian, J. F., Burns, J. C. 2004. The Narratives of Kawasaki Disease. Bulletin of the History of Medicine, vol. 78, no. 2, pp. 410–39.

[3] Sahoo D, Katkar G. D., Khandelwal S, Behroozikhah M, Claire A, Castillo V, et al. 2011. Al-guided discovery of the invariant host response to viral pandemics. eBiomMedicine, The LANCET vol. 68.

[4] Sahoo, D., Dill, D.L., Gentles, A.J., et al. 2008. Boolean implication networks derived from large scale, whole genome microarray datasets. Genome Biol 9, R157.

[5] Ghosh, P., Katkar, G.D, Shimizu, C. et al. 2022 An Artificial Intelligence-guided signature reveals the shared host immune response in MIS-C and Kawasaki disease. Nat Commun 13, 2687 (2022).

[6] Zhang JM, An J. 2007. Cytokines, inflammation, and pain. Int Anesthesiol Clin. Spring; 45(2):27-37.

Identifying unique markers of a disease helps improve our understanding of what mechanisms are involved with severity of the disease, which may aid in setting guidelines for treatment.

Written by Anna Grinberg

Anna is a 3rd year Human Biology major from Eleanor Roosevelt College.

How Your Hands Talk to Your Brain

Written by Emma Chen

Photo by Inaya Nicholls

On Movement & the Senses

Illustrated by Kristiana Wong

Prosthetic limbs and humanoid robots alike are plagued with issues involving poor coordination and unrefined movement. What sets the human capacity for dexterous movement apart from these robotics is our nervous system’s ability for feedback refinement. The Azim Lab at the Salk Institute studies senso rimotor pathways to better understand the relationship between the brain, body and movement.

Our hands partake in one of the most fundamental aspects of our lives: the exploration and manipulation of our surroundings. Consider your fingers, gripping a pencil as you scrawl out notes. Though a familiar movement, such an action requires dozens of muscles, coordinated with speed and precision. As we move, neural pathways throughout our body inform us of our present action and position, allowing us to coordinate subsequent movements. This mechanism is crucial to performing precise and skilled movements, such as handwriting. However, not all the information we receive from our sensory systems is equally relevant to the given moment. When someone pokes us in the arm, we perk up and become alert; but, when our own hands rest on our bodies, we heed little attention. The stimuli we receive need filtering so that they do not overwhelm our mental capacities.

Dr. Eiman Azim, an associate professor at the Salk Institute, is interested in how our nervous system controls dexterous movement, especially given challenges the brain faces like the large influx of sensory information we receive from our bodies. To explain the interaction between the brain and sensory inputs, Dr. Azim explores aspects of ‘gain control,’ where certain sensory signals are amplified by the brain to ‘increase the gain,’ while other less relevant feedback is dampened to prevent interference. For example, when writing, your brain can magnify the sensations of holding and controlling a pencil so your penmanship is smooth and legible, while simultaneously tuning down the sensations of the chair underneath you or the texture of the paper. Together, this allows you to remain focused on performing the movement at hand. To detangle the pathways involved, Dr. Azim and his lab use mouse models and molecular biology tools to better understand and functionally dissect the neurological components involved.

The basic anatomy of the sensory pathways from the hands to the brain is fairly well

understood. Neurons that innervate the hands send sensory information to the cuneate nucleus, a structure in the brainstem that specifically receives feedback from the upper body. Cuneate neurons then send their projections across the midline to the thalamus and on to the sensory cortex where the signals are integrated with other inputs.1 The neural mechanisms by which these sensory signals are regulat ed, however, remains unclear.

Dr. Azim and Dr. Jim Conner, a staff researcher lab, postulated that some level of signal modulation may be occurring at the cuneate nucleus. After mapping exactly how neurons that process touch information from the hand project to the brainstem cuneate nucleus in mice, Dr. Azim and Dr.

The stimuli we receive need filtering so that they do not overwhelm our mental capacities.

Conner began looking at other inputs onto the recipient cuneate cells that may regulate how efficiently this sensory signal is transferred to higher order brain centers. By using a modified rabies virus that travels backwards across synapses (the sites of signal transmission between neurons), they found that cuneate cells were being targeted by a cluster of inhibitory cells in the immediate vicinity of the cuneate.1 Dr. Azim and Dr. Conner then postulated that these local inhibitory cells could diminish the intensity of the incoming sensory information as it travels through the cuneate by reducing the efficiency of signal transmission from the sensory neurons to the cuneate cells.

By performing viral tracing in the mice, Dr. Azim and his

team identified the inhibitory neurons in the pathway as a potential candidate for modulating sensory gain. To affirm that these neurons actually inhibit sensory information from the hands, they went on to record neural activity from the cuneate nucleus while delivering stimuli to the mouse’s paw at regular intervals.1

The researchers sought to learn what would happen if the local inhibitory neurons became activated when a sensory signal was passing through the cuneate. To accomplish this, the investigators used a technique known as optogenetics, which involves inserting light-sensitive channels into the inhibitory neurons that would allow them to manipulate the neurons by simply shining light on them. When the researchers activated the now light-sensitive inhibitory neurons with light, the neurons in the cuneate nucleus demonstrated decreased sensitivity to tactile stimulation. Under normal conditions, a

The mice also struggled to exert the appropriate amount of grip strength, suggesting that the decreased sensitivity to touch in their palms affected their ability to perform the desired movement."

light touch to the hand surface evokes robust responses in neural activity in cuneate neurons. The decreased activity observed suggests that inhibitory neurons could impact the intensity of sensory feedback. Conversely, suppressing the activity of the inhibitory neurons using optogenetics caused cuneate cells to become hypersensitive and fire even when little or no sensory input was trans-

This finding reaffirmed the role of inhibitory neurons on the sensory information received: the lack of inhibition resulted in an excess response. Through these two experiments, the

researchers demonstrated that the inhibitory neurons had the potential to modulate the gain of sensory feedback ascending into the brain.

Figure 1. Mice Performing a Rope-Grasping Task.

In one condition, light was shone on their genetically modified inhibitory neurons to repress their activity, which evoked reactions in the mice including confusion, hunching over and ignoring the task at hand.

Next, the researchers asked what this modulatory influence has to do with the ability of an animal to perform a task involving dexterous movement with their hands. Mice were trained to perform a rope-grasping task that mimicked a climbing motion for a food reward. Using optogenetics to access the relevant circuitry once more, inhibitory neurons were either activated or inhibited to observe their effects on the movements of the mice. When the inhibitory neurons were activated, the animal made more mistakes and had difficulties coordinating its grasping movements. The mice also struggled to exert the appropriate amount of grip strength, suggesting that the decreased sensitivity to touch in their palms affected their ability to perform the desired movement. Next, the inhibitory neurons were repressed, causing the animals to demonstrate a range of reactions. In severe cases, the mouse ignored the rope task and hunched over, grasping at its own hand. Dr. Azim

speculates the animal was experiencing paresthesia, a sensation of tingling and prickling similar to pins-and-needles. In many cases, however, the mouse performed the task sufficiently well, suggesting that with reward motivation, the animal can adapt and perform the task as needed, despite excessive stimuli.1

Finally, the researchers designed an experiment to isolate the role of tactile feedback received by the hands when performing dexterous tasks. The experiment involved a ridged pedestal that the mouse was required to align to a fixed orientation from a random starting orientation, like spinning a lock into place. To determine the effects of inhibition on the performance of the mice, the researchers had the animals perform the task while activating their

inhibitory neurons. Though the mice were provided with a ridged pedestal, the animals took a longer amount of time to complete the task as the amount of sensory feedback they received was reduced. To expand on the experiment, researchers removed the ridges on top of the pedestal and presented a smooth pedestal to mice while leaving the inhibitory neurons unperturbed. With a smooth pedestal, the mice no longer had the ridges as sensory cues to help them assess whether the pedestal was in the correct orientation. Instead, the mice had to opt for a ‘turn-and-wait’ strategy in which the animals turned the pedestal in increments as they searched for the correct pedestal orientation that would result in reward. Similar to mice who had their inhibitory neurons activated, the mice with a smooth pedestal took a longer amount of time to complete the task.1 These experiments demonstrate that excessive inhibition in the cuneate removes the advantage provided by tactile information when performing dexterous movements and affirm the importance of precisely controlling appropriate levels of feedback modulation.

The research performed by Dr. Azim and his team pave a way for greater understanding of the relationship between the brain, body, and movement. These findings can also inform exciting advancements in more applied fields, such as that of prosthetics and robotics. Although a seemingly ordinary aspect of our lives, Dr. Azim believes movement to be the primary driving force for the way the nervous system has evolved: “without movement there is no food, there is no propagation of the species, there is nothing,” he says, “so the main reason the nervous system has evolved these basic structures and circuit motifs is to get the body to move effectively.” As we improve our knowledge of the rela tionship between sensory feedback and movement, perhaps we too could forge similar advancements in augmenting mobility with technology.

REFERENCES

[1] Conner, J.M.; Bohannon, A,; Igarashi, M.; Taniguchi, J.; Baltar, N.; Azim, E.; Modula tion of tactile feedback for the execution of dexterous movement. Science 2021. 374 (6565):316–323. doi: 10.1126/science.

Figure 2. A mouse attempting to spin a ridged pedestal to the correct orientation

Using light, the researchers activated its inhibitory neurons, increasing the level of signal inhibition taking place and increased the amount of time the mouse took to complete the task.

abh1123.

Written by Emma Chen

Emma is a 2nd year Neurobiology major from Roger Revelle College.

RESEARCH

Biology students at UC San Diego often choose to enrich their educational experience by joining labs and conducting their own research. This section showcases original research manuscripts and review papers produced and written entirely by undergraduate students.

American Flamingo (Phoenicopterus ruber) at the San Diego Zoo.

Photo by Andrea Farrell

Letter from the Research Editor

Dear Reader,

When I started engaging with immunology research as a first year undergraduate student at UC San Diego, I spent hours reading and rereading scientific literature, cross-referencing an ever-growing list of sources to piece together seemingly endless walls of text. I was tackling subject matter far too complex to understand with only general biology and chemistry courses under my belt.

That same year, the COVID-19 pandemic heightened general skepticism toward academia, exposing a rift between the scientific community and the broader public. Crucial public health findings regarding viral transmission, treatment, and vaccine development were disguised behind jargon, rendering these results indiscernible to most people. Inaccessible reports leave large margins of error for interpretation and disengage people with fewer opportunities to attain higher education, thereby enabling the widespread misinformation that has plagued recent public health efforts.

In my first year as Research Editor on Saltman Quarterly Vol. 19, I struggled to balance the accessibility that we strive for with the deserved integrity of our students' literature. This year, I returned to the position with a novel approach for deconstructing these educational barriers. Saltman Quarterly Vol. 20 presents the first edition of Research Features articles. These pieces have been carefully crafted to cover each publication accurately and accessibly, while introducing readers to the people behind the technical expertise.

As you make your way through the section, you will find each article is accompanied by a QR code. I invite you to scan each QR code to read the accompanying Research Feature for each publication.

Students at Saltman Quarterly are uniquely positioned to increase the accessibility of crucial ongoing research for the broader community. Research Features is my manifestation of this vision, and I hope that its addition will continue to deconstruct barriers to academic literature in future volumes.

In community,

Megha Srivatsa Research Editor

Saltman Quarterly 2022-23

The Double-Edged Sword: The Reciprocal Relationship Between Chronic Pain & Depressive and Anxiety Disorders

Leslie Ngo

UC San Diego, Class of 2023, Marshall College, B.S. Environmental Systems: Earth Sciences, Scripps Institution of Oceanography, Scripps Isotope Geochemistry Lab

ABSTRACT

Chronic pain is an extended physiological condition that is often accompanied by anxiety and depressive disorders. Understanding the structural, functional, and neurochemical mechanisms of nociceptive signaling provides insight into how alterations to these processes may lead to the advancement of pain from an acute to a chronic state. However, further research must be conducted to better understand the neurological interactions among sensory, emotional, and cognitive systems to better understand the development of comorbid anxiety and depressive disorders associated with chronic pain.This scientific review discusses overlapping structural and neurochemical mechanisms of chronic pain and emotional processing systems to evaluate the effectiveness of current interventions used to holistically treat patients diagnosed with chronic pain. An improved mechanistic understanding of the neural changes that occur in overlapping nociceptive and emotional processing pathways during the transition from acute to chronic pain will allow for the development of more targeted treatments unique to the needs of each patient.

INTRODUCTION

Chronic pain, a debilitating condition affecting more than 100 million American adults, consists of pain that persists despite prolonged treatment.1 Symptoms often limit a patient’s physical movement, thus reducing their ability to complete daily physical and social activities.2 The resulting immobility and social isolation can negatively impact mental health, as research has shown that those with chronic pain are more likely to experience anxiety and depression.2 In a study on 18,980 people with chronic pain in the United States, about 43.4% were diagnosed with depression. In contrast, depression affects 5% of the general population.2

Chronic pain results from a failure to resolve acute, short-lived pain. When pain is in an acute state, it is often provoked suddenly by an injury and does not last longer than six months.2 Acute pain is often resolved by treatment. However, when this pain prolongs for more than six months it is categorized as being in a chronic state. Chronic pain is experienced when the pain signals

remain active in the nervous system for an extended period of time, however, the cause of this extended neural activity is unclear.

The neural and physiological relationship between mental health and chronic pain is complex. Neuroimaging studies suggest a close relationship between brain regions associated with the emotional and sensory features of pain and brain structures affected by depression.1 These studies indicate patients

experiencing anxiety and depression may have a greater emotional reactivity to pain, rendering it more difficult for these patients to regulate pain through physiological treatments such as physical therapy, and mind and body techniques such as acupuncture.1 Studies investigating the relationship between physical health and mental health evidence that the two share a strong positive correlation, therefore harm to one can negatively impact the other.2 Consequently, although the positive correlation between chronic pain and mental health is well documented, the specific mechanisms involved in their relationship are unclear.2 As a result, patients experiencing both conditions are often underdiagnosed and therefore undertreated. This complex relationship can be explored by investigating the physiological mechanisms of chronic pain development. Understanding these overlapping mechanisms will increase the potential to develop effective treatments for patients experiencing anxiety and depressive disorders in conjunction with chronic pain.

Structural and Neurochemical Mechanisms of Chronic Pain

Recent studies have shown that changes in areas of the central nervous system that modulate pain are strongly associated with the development of chronic pain.3 The rostral ventral medulla (RVM), primary and secondary somatosensory cortices (S1 and S2), periaqueductal gray matter (PAG), thalamus, prefrontal cortex (PFC), anterior cortex (ACC), and amygdala (AMG) are all brain structures associated with the perception and transmittance of pain from the body to the brain.3 Changes in emotional and motivational states can also affect the intensity of pain as the nucleus accumbens (NAc), ACC, and amygdala are all brain structures that regulate emotional and motivational responses as part of the neural reward circuit and are also associated with chronic pain pathways (Fig. 1).3

Understanding how bodies process pain signals provides insight into how alterations to these processes may lead to the advancement of pain from an acute to chronic state. Pain signals in the spinal cord ascend from the spinal dorsal horn to the RVM and PAG of the brain stem.3 The RVM receives innervation from the PAG by RVM neurons. When the RVM receives the signal it inhibits inputs from the PAG, which inhibits incoming sensory activity. After

Figure 1. Overlapping neural pathways between pain and emotion processing.9

Brain structures that process nociceptive signals overlap with structures along the neural reward circuit. These overlapping pathways evidence a biological basis for the comorbid occurrence of chronic pain and anxiety and depressive disorders. Structures primarily involved in processing pain are indicated in white while structures primarily involved in processing emotion are indicated in blue.

passing through these regions, the signal is transmitted to the thalamus, which acts as a relay system for nociceptive signals, or pain signals received from the brain areas. Nociceptive signals are then passed on to the S1 and S2, which process the bodily sensations. Following, the signal triggers activation in the ACC and then the PFC, inducing an emotional response that is finally processed in the amygdala and NAc.3 When the ACC is activated, it processes the emotional context associated with the sensation, which is further processed in the PFC. Once the signals are processed by AMG and NAc, the sensations are dispersed along with the emotional context.

Various neurotransmitters also play an important role in processing pain by modulating synaptic transmission patterns.4 Glutamate is a neurotransmitter that mediates transmission and the storage of pain signals in the amygdala, using the N-methyl-D-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-r-isoxazolepropionic acid (AMPA), and kainite (KA) receptors.1 Both NMDA and AMPA receptors regulate the transmission of excitatory pain signals, while KA receptors regulate the intensity of these signals. An increase in glutamate activity leads to an increase in the binding of NMDA, AMPA, and KA receptors, resulting in persistent pain. Extended glutamate activity is often

caused by traumatic stress from an injury. As a result, extended signal transmission contributes to the advancement from acute to chronic pain. Another neurotransmitter involved in the development of chronic pain is called Gamma-Aminobutyric Acid (GABA).1 GABA modulates pain signals by regulating the activity of GABA receptors, GABAA, and GABAB, which are located in the dorsal horn of the cervical column of the spinal cord.1 GABA functions as an inhibitory neurotransmitter, as binding to GABAA and GABAB sends signals for reduced neuronal excitation. A lack of GABA reduced neural inhibition in various brain structures related to pain processing pathways, including ACC, PFC, amygdala, S1 and S2, thalamus, RVM, and PAG.4 Hyperactivity in these regions leads to higher pain perception, and therefore extended experiences of pain.1

Neural Development of Chronic Pain

Understanding the structural, functional, and neurochemical changes that occur within the brain during the development of chronic pain can expose novel avenues to treat chronic pain. Neurological studies show that activity in the amygdala increases when a patient experiences pain sensations, evidencing a biological correlation between emotional distress and pain perception. There-

fore, persistent pain sensations may lead to the development of a persistent state of emotional distress, which over time, may develop into emotional disorders such as depressive and anxiety disorders.1 In addition, when pain inputs are sent from the thalamus to the ACC, which processes and modulates pain, the ACC integrates input from various structures to project to the amygdala, generating a complete pain response.3

In addition, the hippocampus is another brain structure that is involved in the development of chronic pain as the hippocampus regulates the transmission of pain signals through various pathways. One example is the spinothalamic and parabrachial ascending pathway which carries pain signals from the skin to the thalamus. The nociceptive signal is processed in the thalamus and then transmitted to the primary sensory cortex for pain perception. In addition, the hippocampus also directs nociceptive inputs from the spinal cord to regulate the hypothalamic pituitary adrenal axis.1 The hypothalamic pituitary adrenal axis is a neural mechanism that mediates the neural impact of stressors. These neural effects are mediated by regulating autonomic nervous system processes such as metabolic and immunological responses.

Brain regions such as the RVM, PFC, NAc, amygdala, and thalamus have known affective aspects of pain and regulate associated emotions, however, the interstructural neural mechanisms of the signaling are unclear. In addition, many factors that play a role in the transition from acute to chronic pain and changes in neurological structures that mediate nociceptive signaling have yet to be studied to better understand the interactions among sensory, emotional, and cognitive aspects of chronic pain.

Treatment and Practice for Chronic Pain and Depression and Anxiety

Chronic pain and comorbid anxiety and depressive disorders can be treated using pharmacological interventions. However, current knowledge regarding overlapping neurological pathways remains limited. As a result, developing effective medication to manage chronic pain and its associated emotional disorders remains a challenge.2 NMDA receptor antagonists such as dextromethorphan and ketamine inhibit the binding of glutamate to NMDA receptors and therefore inhibit the transmission of nociceptive pain signals. Currently, pre-

scribing NMDA receptor antagonists in combination with antidepressants such as serotonin and norepinephrine reuptake inhibitors (SNRIs) are relatively effective in managing chronic pain and comorbid anxiety and depressive disorders.2 Serotonin and norepinephrine are neurotransmitters that are crucial regulators of chronic pain, depression, and anxiety. SNRIs prevent the neural reuptake of both neurotransmitters, inducing their signaling in the descending inhibitory pathways. SNRI induced signals project from the RVM and PAG to the locus coeruleus (LC) of the brain stem, and then to the dorsal horn of the spinal cord.3 As they reach the supraspinal regions, norepinephrine inhibits pain signals by binding to α2-adrenergic receptors while serotonin binds to 5-HT3 receptors. Binding these receptors hyperpolarizes GABAergic and glutamatergic neurons in the signal pathway, allowing neurotransmitters such as GABA and glutamate to further inhibit the pain signal. Just as serotonin and norepinephrine inhibit the transmission of pain signals, they also inhibit signals disruptive to the prefrontal cortex. Imaging studies indicate that depressive and anxiety disorders are associated with abnormal functionality in the prefrontal cortex. Therefore, prescribing antidepressant medications such as SNRIs not only reduces pain signals but can also combat emotional disorders that are associated with chronic pain.4 Another pharmacological intervention often prescribed to treat chronic pain are opioids. Opioids inhibit nociceptive signals by blocking calcium channels on nociceptive afferent nerves of the dorsal horn in the spinal cord, therefore inhibiting the release of excitatory neurotransmitters such as glutamate. However, there are several risks associated with prescribing opioids to patients with chronic pain. Such risks include infection, cardiovascular complications, and addiction. High rates of overdoses have been associated with opioid use, likely a result of high addiction rates. As a result, physicians are trying to rely less on strong pharmacological interventions and are exploring alternative forms of treating chronic pain and comorbid emotional disorders.

In addition to pharmacological interventions, psychological interventions such as cognitive behavioral therapy (CBT) and behavioral therapy have also shown promise in alleviating comorbid depressive and anxiety symptoms in patients with chronic pain. CBT focuses on developing positive pat-

terns of logic and reason in relation to chronic pain such as medication usage, mood, physical functioning, and quality of life. CBT emphasizes identifying and improving maladaptive emotions, thoughts, and behaviors to more sustainable and healthy habits.4 Treatments use strategies for relaxation, behavioral pacing, and cognitive restructuring for distorted thoughts.4 Research suggests that the emotional response most likely to develop in response to negative cognitions from chronic pain is fear and anxiety.4 Fear of physiological pain being triggered from physical activity leads to avoidance behaviors, a hallmark of anxiety disorders. Avoidance behaviors reduce patients' willingness to participate in activities that they perceive may potentially worsen their physical condition. Current practices in CBT focus on decreasing perceptions that physical activity will exacerbate chronic pain conditions.4 In conjunction, behavioral therapy focuses on identifying individuals behavior in response to some stimulus.4 Treatment focuses on exposing patients to physical activity to challenge the belief that physical activity will exacerbate chronic pain conditions to ultimately relieve pain-related fear and anxiety.4 To identify potential behavioral treatments for anxiety disorders, researchers studied exposure-based treatments for chronic pain patients. Patients engaged in low-impact activities, such as walking, to help them realize they are able to experience physical exercise without experiencing painful symptoms. The subjects regularly met with primary care physicians for 8 weeks to rate on a 5-point scale the level of pain anticipated before participating in some activity, and the level of pain experienced after engaging in the activity.4 Most of the subjects rated anticipated pain as a 4, but after engaging in the activity the reported the pain experienced, averaged between the 1-2 rating.4 These results suggest that patients with chronic pain often perceive physical activity as more harmful than in actuality. As a result, CBT and behavioral therapy offer a promising approach to encouraging chronic pain patients to engage in physical activity, improving their outlook on their illness, and engaging in mood-boosting physical activity.

Another promising treatment for chronic pain involves mindfulness-based therapies, which educate patients about their bodies to better understand and accept pain.4 Mindfulness involves consistently practicing activities such

as meditation, exercise, and journaling, as well as implementing emotional and behavioral coping skills that reduce unpleasant emotions and ease the cognitive and emotional toll that accompanies their experience with pain.5 Comparative studies have shown that GABA is released during exercise, inhibiting the progression of pain signals.4 In these studies, subjects with no significant physiological or psychiatric disorders regularly engaged in either yoga or hour-long walks. MRS data showed an increase in GABA levels, an increase in a positive mood, and a decrease in anxiety for the group regularly engaging in yoga. In contrast, the group that regularly engaged in walking experienced little to no change in GABA levels, mood, and anxiety. These results suggest that engaging in more intensive exercise has the ability to neurochemically encourage the inhibition of nociceptive transmission while improving the mood state of chronic pain patients.

The above results evidence a well-documented physiological association between physical and mental health, however, many patients diagnosed with chronic pain report that physicians are not proactive in patients discussing mental health during treatment.6 In a descriptive analysis of 11 patients diagnosed with chronic pain, 8 patients felt their physicians were providing insufficient treatment for comorbid emotional disorders, while the other 3 felt their physicians lacked compassion and were not understanding of their concerns.7 Rather than waiting for patients to report symptoms of developing mood disorders there is a need for physicians to be more proactive in initiating conversations about mental health with patients diagnosed with chronic pain.2 Feeling heard by physicians plays an important role in patients perceptions of their treatment. Because the relationship between mental health and chronic pain is individual and complex, treating chronic pain does not follow a “one size fits all” approach. It is important that treatment and care that are developed are patient-centered to ensure each patient’s unique needs are addressed.6

CONCLUSION

Chronic pain is a persistent medical diagnosis that can reduce the quality of life and increase risk of developing comorbid anxiety and depression. Additional research is required to better understand the mechanisms that un-

derlie the development of pain from an acute to a chronic state. Identifying key features of these conditions should allow for more targeted treatments to improve health outcomes and recovery rates. Understanding how bodies receive and process pain signals provides insight regarding the development and transmission of chronic pain. Treating chronic pain, depression, and anxiety requires an interdisciplinary integration of mental health treatments, physical therapies, and pharmacological intervention. In addition, it is crucial to conduct additional research to identify the biological mechanisms that facilitate the development of comorbid anxiety and depressive disorders from chronic pain in order to better target each condition for treatment. Improved mechanistic understanding of the development of chronic pain and the complex relationship between chronic pain and comorbid anxiety and depressive disorders will create opportunities to improve comprehensive treatments unique to the needs of each patient.

REFERENCES

[1] Kohrt, B. A., Griffith, J. L., & Patel, V. (2018). Chronic pain and mental health: integrated solutions for global problems. Pain, 159 Suppl 1(Suppl 1), S85–S90.

[2] Goesling, J., Lin, L. A., & Clauw, D. J. (2018). Psychiatry and Pain Management: at the Intersection of Chronic Pain and Mental Health. Current psychiatry reports, 20(2), 12.

[3] Obata H. (2017). Analgesic Mechanisms of Antidepressants for Neuropathic Pain. International journal of molecular sciences, 18(11), 2483.

[4] Vadivelu, N., Kai, A. M., Kodumudi, G., Babayan, K., Fontes, M., & Burg, M. M. (2017). Pain and Psychology-A Reciprocal Relationship. The Ochsner journal, 17(2), 173–180.

[5] Marks, D. M., Shah, M. J., Patkar, A. A., Masand, P. S., Park, G. Y., & Pae, C. U. (2009). Serotonin-norepinephrine reuptake inhibitors for pain control: premise and promise. Current neuropharmacology, 7(4), 331–336.

[6] Hooten W. M. (2016). Chronic Pain and Mental Health Disorders: Shared Neural Mechanisms, Epidemiology, and Treatment. Mayo Clinic proceedings, 91(7), 955–970.

[7] Yang S, Chang MC. Chronic Pain: Structural and Functional Changes in Brain Structures and Associated Negative Affective States. Int J Mol Sci. 2019 Jun 26;20(13):3130. doi: 10.3390/ijms20133130. PMID: 31248061; PMCID: PMC6650904.

Homology Arms Influence Homology Directed CRISPR/Cas9

Genome Editing

Andres Maximus Canedo Sandoval

UC

San Diego, Class of 2023, Revelle College, B.S. General Biology with Minor in Business, Dr. Lisa McDonnell, Department of Cell and Developmental Biology

ABSTRACT

Clustered Regularly Interspaced Short Palindromic Repeats, or CRISPR, is a groundbreaking tool that provides scientists with the ability to precisely edit genomes. Previous studies have found moderate success incorporating edits using homology arms on the HDR template that flank the target sequence downstream of the cut site. This study sought to evaluate the impact of different homology arm configurations on mutation success rate by constructing homology arms which flank either downstream, upstream, or both downstream and upstream regions of the cut site. Saccharomyces Cerevisiae were transformed with a pML104 plasmid designed with a gRNA and Cas9 to target the ADE2 gene. Our results suggest that edits occur with the highest efficiency when homology arms flank both upstream and downstream of the Cas9 cut site. These findings can be applied to optimize the structure of key components of CRISPR and improve efficiency of gene editing.

INTRODUCTION

CRISPR editing is a revolutionary genetic engineering system reshaping the scientific community for its versatility to edit the mammalian genome.1 Currently, researchers are harnessing CRISPR within the agricultural and medical fields with the goal of correcting human genetic disorders and expediting crop and livestock breeding. Recently, CRISPR was utilized to test gene drive on population fitness and led to the proposal that the introduction of genes which encode resistance to malaria could reduce rates of the disease within the human population.2 Meanwhile, in the wake of global warming and climate change, scientists have suggested that CRISPR can help to combat food insecurity, allowing society to withstand greater populations and various environmental changes.3 Within the medical field, CRISPR has been applied in animals to treat underlying genetic mechanisms of various life threatening diseases, and may soon be employed clinically to treat humans.4,5 As such, CRISPR holds the potential to improve the efficiency of medical treatment and nutritional resources.5

A multitude of CRISPR systems exist naturally within many bacteria and archaea and generally involve the as-

sembly of endonuclease proteins such as Cas9. These proteins are coupled with some form of transcribed guide RNA (gRNA) to target and cleave a specific location within the genome that is complementary to the gRNA transcript.6 Endogenous CRISPR systems often target invading DNA (e.g., viruses and plasmids). Utilizing the Cas9 and gRNA in other cell types can allow scientists to target a specific genomic region for cleavage. After cleavage, the cell must harness repair mechanisms to ameliorate the damage. The repair mechanism is the stage that allows for the potential insertion of a mutation.

The Type II CRISPR-Cas system is more commonly used for incorporating designed mutations via repair mechanisms, and is therefore the CRISPR system utilized within this study. CRISPR-Cas9 differs from other classifications of CRISPR as it relies on a singular large protein for its nuclease activity, known as the Cas9 nuclease, rather than multiple subunits of other CRISPR nucleases.7 For the purpose of this experiment, the gRNA was designed to be 20 nucleotides in length and complementary to a region of interest near a protospacer adjacent motif (PAM) site within the target genome. The PAM site is a nucleotide sequence recognized by the Cas9 protein. It is encoded by 5’-

NGG-3’, with ‘N’ being any nucleotide and ‘G’ representing guanine. This specific location marks the target cleavage site. As the gRNA is designed to be complementary to the region of interest, the sequence guides the Cas9 protein to the target location for target-specific cutting.8 Once the gRNA is transcribed and Cas9 is translated, they complex together into the Cas9-gRNA. The gRNA then binds to the complementary region of the DNA to create a double-stranded break (DSB) about 3 nucleotides upstream of the PAM site.9 DNA repair must then occur via non-homologous end joining (NHEJ) or homology-directed repair (HDR) in order to prevent genetic degradation.10

NHEJ is a non-template form of repair in which nucleotides are randomly ligated back together. NHEJ is most often associated with the insertion or deletion of nucleotides near the cut site, making this method of DNA repair more error-prone than HDR.9 The insertions and deletions caused by the random assembly of nucleotides leads to a frameshift within the ribosomal reading frame of a protein-coding gene. Normally, the reading frame encodes the correct sequence for a specific protein. However, during a frameshift, the amino acid sequence of the encoded protein is now altered, changing the gene product and sequence. Frameshift mutations often result in a loss-of-function.

On the other hand, HDR requires a homologous strand to serve as a template sequence for repair.9 Naturally occurring HDR takes place If a homologous chromosome is present to serve as a template for the broken fragment.11 Following the double stranded break, DNA resection occurs at the 5’ ends of the DNA. Nucleotides near the DSB are degraded by a resection protein com-

Resection and HDR Resection and HDR

Damaged Chromosomes

Figure 1. Resection is integral in Homology Directed Repair. Depiction of the 3’ single stranded overhangs created by resection. The resected areas are able to base pair to a homologous chromosome and complete Homology Directed Repair.

Double Stranded Break

Double Stranded Break

Resection

Resection

Overhang

Homology

Directed Repair

Homology Directed Repair

Homologous Chromosomes

plex and create single stranded overhangs on both strands of the broken chromosome, allowing access to additional nucleotides to increase the chances of the broken strand to base pair to the homologous strand (Fig. 1).12 Cellular repair mechanisms then utilize the complementary sequence of a homologous chromosome to repair the damaged chromosome. Since the strands are homologous, the regions of homology, or homology arms, help align the two strands together for proper repair. Homology arms refer to identical regions within a chromosome. These regions are crucial for HDR completion because base pairing must occur between the DNA from the cut chromosome and the repair template. The cell can then repair

the damaged chromosome by replicating the corresponding region of the DNA template. Using CRISPR systems, researchers are harnessing HDR to insert mutations into the genome by introducing designed template strands to function as homologous strands within the cells targeted by Cas9-gRNA to produce desired edits within a genome.

In a study from DiCarlo et al., CRISPR was used in yeast to introduce intentional mutations using HDR to test whether CRISPR works in a target-specific mutating manner. Results suggested that efficient HDR occurred even if the HDR template does not have homology arms on either side of the Cas9 cut site. Given that HDR may be used if base pairing can occur between single strands after DNA resection, researchers unexpectedly found HDR occurring without homology arms flanking the cut site.1 In turn, we wished to test the influence of homology arm configuration in a more targeted fashion.

Our study aimed to test the influence of homology arm configuration relative to the Cas9 cut site, and whether homology arms are needed to flank both upstream and downstream of the

PAM site to successfully introduce mutations encoded in the HDR template. We aimed to analyze the efficiency of incorporating mutations by using HDR templates that have homology arms either upstream or downstream of the DSB, or flanking the DSB in both the upstream and downstream regions. Saccharomyces Cerevisiae, commonly known as budding yeast, were used as the genetic model. The yeast strain used were haploid, rather than diploid, as the use of a designed homologous template strand to incorporate specific mutations is most efficient within haploid cells.

METHODS

To test the influence of homology arms on HDR editing, we targeted the ADE2 gene, a gene involved in purine biosynthesis, and that is a non-essential gene when yeast are grown on rich media.13 Functional ADE2 is associated with a white phenotype, however when ADE2 is dysfunctional, the colony displays a red phenotype, an identifier for ADE2 disruption.13

In this study, we utilized the pML104 plasmid as the expression vector for our Cas9 gene and gRNA.14 Our gRNA was designed to be 20 nucleotides in length and target a specific region upstream of the PAM sequence near the beginning of the open reading frame (ORF) of ADE2 (Fig. 2). Yeast cells carrying the vector were able to generate the Cas9 and gRNA components. In addition to

Figure 2. The guide RNA target sequence and homology directed repair templates used. The top row depicts the wildtype sequence, with the gRNA target region, PAM site, and Cas9 cut site indicated. Above the wildtype sequence are the locations of seven silent mutations encoded within the combination of HDR templates used. HDR B contains all 7 mutations, and homology arms flank the Cas9 cut site. HDR C contains mutations 4-7 downstream of the cut site, and is homologous to the target region largely downstream of the Cas9 cut site. HDR D contains mutations 1-3 upstream of the cut site, and mutation 4 just downstream of the cut site, and is homologous to the target region largely upstream of the Cas9 cut site. All templates contained mutation 4 which altered the PAM site. All HDR templates were the same length. Downstream regions refer to regions following the Cas9 cut site, and upstream regions being regions prior to the Cas9 cut site. In HDR C, there exist an additional ~20 nucleotides downstream of mutation 7 that are not depicted here. For HDR D, there exists an additional ~20 nucleotides upstream of mutation 1 that are not depicted here. HDR B has both the upstream and downstream homology arms that extend an additional ~20 nucleotides not depicted here.

the vector, yeast cells were transformed with various HDR templates: HDR B, C and D (Fig. 2). Specifically, HDR-B had homologous regions that flanked the Cas9 cut site. HDR-C was largely homologous to the target only downstream of the Cas9 cut site, and HDR-D was largely homologous to the target only upstream of the Cas9 cut site.

We transformed yeast with the pML-gRNA construct and HDR templates to induce a DSB in the ADE2 gene. Through sequencing of the ADE2 gene we were able to observe whether NHEJ occurred or if HDR mutations were successfully incorporated. White colonies were selected for sequencing as a white phenotype indicates either a wildtype ADE2 sequence or the incorporation of the HDR-encoded silent mutations. By comparing the rate of inclusion of the silent mutations via HDR B, C, and D, we could investigate if homology arms flanking (HDR B), homology arms mostly upstream (HDR D), or homology arms mostly downstream (HDR C) result in the greatest HDR efficiency.

RESULTS

To test the effect homology arms have on the incorporation of HDR-mutations via CRISPR editing, yeast were transformed with three different HDR templates with homology arms either flanking, upstream, or downstream from the Cas9 cut site. After sequencing the resulting white colonies, HDR templates with homology regions both upstream and downstream of the cut site (HDR B) were the only transformations that resulted in yeast colonies with successfully incorporated mutations in the ADE2 gene via HDR.

With a sample size of 100 sequences, it was found that when homology arms exist only downstream of the cut site (HDR C) or only upstream of the cut site (HDR D), HDR was unsuccessful as it was observed that 0% template mutations were incorporated into the genome. In contrast, when homology arms flanked both sides of the cut side (HDR B), we observed the incorporation of the designed silent mutations. Nearly 30% of white-colony sequences from HDR B transformations incorporated at least one silent mutation. Incorporation of all seven point mutations was observed to be less frequent, as 11% of the white-colony sequences from yeast transformed with HDR B incorporated all seven of the mutations.

Figure 3. Mutations were only introduced when the HDR template had homology arms that flanked the Cas9 cut site. The x-axis represents the five combinations of silent mutations and the wildtype nucleotides that we found among the 100 sequenced samples. CGCATCG represents an ADE2 sequence that contains all seven point mutations encoded in HDR-B, while TTGGCAA is all of the wildtype nucleotides for the locations of the seven point mutations targeted by HDR-B. Three other combinations of wildtype and mutated nucleotides were found within the sequenced samples: TGCATCG contains silent mutations 2 through 7 (see Fig. 1), and could only be possible with HDR-B. TTGACAA contains only mutation 4, which could be possible with HDR-B, C, or D. TTGATCG contains mutations 4 through 7, which could occur via HDR-B or HDR-C. The 100 sequences analyzed were from white yeast colonies after transformation with pML-gRNA and one of the HDR templates (and growing on selective media indicating uptake of the pML-gRNA plasmid). All of the sequences analyzed were free of other mutations (such as insertions or deletions). pML104 and pML104-gRNA were used as control groups as we expected them to return to wildtype.

DISCUSSION

Based on our results, it can be concluded that HDR incorporation is more likely to occur when homology arms flank the cut site in comparison to models where homology arms are found either only downstream or upstream of the cut site as HDR B was the only HDR template condition to incorporate any HDR mutation into the chromosome. This is in comparison to the 0% mutation efficiency when HDR templates are designed with only upstream or downstream homology arms.

These results are in contrast to DiCarlo et al. as the study reported that HDR was able to occur when homology arms only flanked downstream of the cut site.1 However, our results contradict that finding as the HDR templates we used to transform yeast successfully

incorporated mutations only when homology arms flanked both sides of the cut site. In HDR, the repair mechanism relies on the homologous regions in order to base pair the damaged and template sequence correctly. Correct base pairing between the template and damaged chromosome ensures the accuracy of repair, which is largely influenced by DNA resectioning. It may be possible that after resectioning, flanking homology arms increase the likelihood of base pairing to one of the single stranded resected fragments from the broken chromosome. The contradictions in findings between DiCarlo and our results may be due to differences in the lengths between the homology arms themselves. DiCarlo notes that the HDR templates utilized homology arms of ~90 nucleotides, while our study utilized homology arms of ~20 nucleotides on each end of our designed templates.

CGCATCG TGCATCG TTGACAA TTGATCG TTGGCAA

In addition, researchers have found that ADE2 gene HDR editing in yeast is more successful within double stranded HDR templates compared to single stranded HDR templates.21 This demonstrates proportionality between homology and successful occurrences of HDR. The homology arms of the double-stranded templates allow for more base-pairing when a DSB occurs, suggesting that the level of homology is correlated with higher rates of HDR occurrence. As a result, DiCarlo may have produced different results due to the differing lengths between the designed homology arms. It may be that if base pairing occurs within both strands of the broken DNA, HDR is more likely to occur. Consequently, flanking homology arms would result in a higher likelihood of successful HDR. The homologous regions for our templates with only upstream or downstream homology arms were unrecognizable by the host cell, and therefore rejected HDR as the repair mechanism so the mutations were not implemented. Additionally, results from the conducted study were also based upon the data of 100 sequences while Dicarlo et al. measured the rate of HDR, with a reported occurrence of 0.01%, based upon a phenotypic change in their yeast.1 It could be very likely that our sample size was too small to detect HDR occuring within the templates that did not have homology both up and downstream of the cut site (HDR C and D). Henceforth, we would need to sequence more colonies in order to truly determine the accuracy of DiCarlo’s findings, or modify the assay to one that relies on phenotypic change.

A study conducted by Zhang and colleagues measuring efficiency of knock-ins, or the addition of sequences to the target genome, by HDR found that homology arms with less than 300 base pairs are inefficient, and that homology arms should be around 600 base pairs for optimal efficiency.15 Findings from Zhang also suggest that homology arm length is proportional to increased HDR efficiency and the number of HDR mutations integrated into the genome. Accordingly, longer homology arms corresponded with the successful integration of implementation of larger edits.15 Since our HDR templates had homology arms of significantly shorter length, the homology arms were a factor regarding the low HDR mutation success rate as our results measured only 11% incorporation of all HDR point mutations. For future studies we should consider test-

ing the effect of longer homology arms on mutation success rate via HDR.

CONCLUSION

By identifying mechanisms which increase CRISPR editing efficiency, we grow the potential to develop more efficient strategies to optimize gene editing techniques. As we progress from experiment to practice, CRISPR editing has already been harnessed in multiple outlets across the globe by enabling agriculture industries to produce genetically modified produce. CRISPR editing has generated crops of optimal yield and resistant to viruses, fungi, and other stressors.16 By understanding the CRISPR-Cas9 editing in yeast, we can identify strategies to increase the editing success rate and efficiency to achieve desired outcomes in research, agriculture, and medicine.

ACKNOWLEDGEMENT

The work completed within this manuscript was part of an upper-division upper biology lab class (BIMM 101) under the supervision of Professor Lisa McDonnell - a class that aims to explore how CRISPR editing works.

CITATIONS

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[2] Hammond, Andrew, Paola Pollegioni, Tania Persampieri, Ace North, Roxana Minuz, Alessandro Trusso, Alessandro Bucci, et al. “Gene-Drive Suppression of Mosquito Populations in Large Cages as a Bridge between Lab and Field.” Nature Communications 12, no. 1 (July 28, 2021): 4589. https:// doi.org/10.1038/s41467-021-24790-6.

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[5] Barrangou, Rodolphe, and Jennifer A. Doudna. “Applications of CRISPR Technologies in Research and Beyond.” Nature Biotechnology 34, no. 9 (September 2016): 933–41. https://doi. org/10.1038/nbt.3659.

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[7] Briner, Alexandra E., and Rodolphe Barrangou. “Guide RNAs: A Glimpse at the Sequences That Drive CRISPR–Cas Systems.” Cold Spring Harbor Protocols 2016, no. 7 (July 1, 2016): pdb.top090902. https://doi.org/10.1101/pdb. top090902.

[8] Liao, Chunyu, and Chase L. Beisel. “The TracrRNA in CRISPR Biology and Technologies.” Annual Review of Genetics 55, no. 1 (2021): 161–81. https://doi.org/10.1146/annurev-genet-071719-022559.

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Aerial Insect Abundance

Predicts Myotis Bat Habitat Use Along Streams

Ari Brisco Schofield1*, Tazlina Dentinger2, Richard Klein3,

and Brandi Sanchez4

[1] UC San Diego, Eleanor Roosevelt College, Ecology, Behavior and Evolution, Class of 2024; [2] UC Davis, College of Engineering, Biological Systems Engineering, Class of 2025; [3] University of California, Berkeley, Conservation and Resource Studies, 2023; [4] UC San Diego, Revelle College, Ecology, Behavior and Evolution, Class of 2023; *Corresponding Author: Ari Brisco Schofield, abriscos@ucsd.edu

ABSTRACT

Bats have become increasingly threatened by anthropogenic disturbances in recent decades. Threats to bat populations are a cause for concern because bats provide crucial ecosystem services such as pest control and pollination. The ability to predict geographic localization of bat populations can improve bat species surveillance and lead to more informed management decisions to aid conservation. Understanding specific factors that affect bat habitat use, like prey abundance and physical site characteristics, can enable the construction of predictive occurrence models. To better understand the habitat use of Yuma myotis (Myotis yumanensis), we conducted a study in the riverine habitats located within LandelsHill Big Creek Reserve in Monterey County, CA, during the summer. Currently, it is unclear how to predict bat habitat use patterns and if management activities will adversely affect bat populations. We found that the number of Myotis bat calls is positively correlated with aerial insect abundance along streams, but is not correlated with stream width. Our results suggest that, during the summer, insect abundance is a stronger predictor of Yuma myotis habitat use than physical site-level characteristics, likely because bats track insect populations. Therefore, monitoring insect abundance is a promising step for developing future bat population predictive occurrence models. These models can serve as a basis to improve targeted monitoring of bat populations, which will be critical for future conservation efforts as bats continue to be threatened.

INTRODUCTION

Bats are important components of ecosystems as they provide irreplaceable support for many organisms, including humans. For example, they serve as crucial means of pest control by safeguarding agricultural crops and mitigating disease vectors such as mosquitos, bolstering public health overall.1,2 It is estimated that in the United States alone, bats provide a near equivalent of $22.9 billion in pest control per year through the consumption of insects.1 Yet in recent decades bat species around the world have been increasingly threatened by anthropogenic threats such as habitat loss and degradation, pesticides, water contamination, and fatal diseases (e.g. white-nose syndrome).3,4 In order to effectively monitor and conserve bat

species, wildlife managers must develop a greater understanding of where and when specific populations of bats are likely to occur.

An understanding of which factors influence the distribution of particular bat species can be used to create more accurate predictive models that enable wildlife managers to anticipate where populations are likely to be found.5 Monitoring bat populations can be difficult as they are small and nocturnal.3 As a result, there is currently very little data on the status of bat populations, which is necessary to make well-informed land management decisions to preserve these species. Predictive modeling allows wildlife managers to target areas for observing bats, resulting in improved population monitoring that can be used

to advise management activities.

However, constructing predictive occurrence models for widespread bat species is particularly challenging as current data show there is low variability in bat habitat use characteristics. Consequently, more data is needed to draw statistically significant conclusions regarding bat population statuses. One such widespread bat group are those within the genus Myotis (family Vespertilionidae), also known as mouse-eared bats.6,7 With at least 120 species, Myotis is the largest and most widely distributed genus globally within the order Chiroptera, found in a variety of habitats. As a result, not only is it difficult to analyze what factors change Myotis habitat use, but the genus is more likely to be impacted by anthropogenic threats because of their widespread distribution.8 Due to their abundance, Myotis spp. are also responsible for a large proportion of the ecosystem services bats provide, so their loss would be particularly harmful to ecosystems worldwide.

Compared to other bat genera, all Myotis are relatively small, and their diets are composed of a wide variety of insects, often dependent on regional variation in insect communities.6 Like other bats, all Myotis spp. use echolocation to navigate and detect objects in their surroundings, meaning that acoustic surveys can be done as a metric of habitat use.9 Within the genus Myotis there are several described foraging strategies, each of which is connected with the distribution, diet, and morphology of specific Myotis

One bat foraging strategy is trawling, the primary strategy used by the M. yumanensis. 6 Trawler bats fly a few centimeters over water and catch insects from the surface with their hind feet. In addition, Yuma myotises also catch aeri-

al insects close to both land and water depending on the availability of prey. The morphology of M. yumanesis allows for high maneuverability during flight, granting them the ability to fly in tight spaces. However, they are routinely found foraging in open, uncluttered habitats close to and above flowing bodies of freshwater. Since M. yumanesis are opportunistic feeders, they tend to have a highly variable diet; a majority of their diet consists of insects in the orders Lepidoptera, Diptera, Trichoptera, Ephemeroptera, and Coleoptera.10 Understanding whether foraging site characteristics and insect orders are or are not associated with foraging M. yumanesis presence would assist in predicting these bats’ location.

To study M. yumanesis habitat preference, we observed the effects of insect abundance and the availability of open flight paths on the summertime occurrence of M. yumanesis in riverine habitats. More specifically, we asked if the number of M. yumanesis varies with aerial insect availability and stream width, using the number of calls as an indicator of the abundance of M. yumanesis foraging at each site. We expected that higher aerial insect abundance, regardless of the orders of insects present, and more open flight paths surrounding water bodies would yield more bat calls indicating more bat habitat use in a particular area.

MATERIALS & METHODS

2.1

Natural History of Study System

This study was conducted at the Landels-Hill Big Creek Reserve, Big Sur, CA, USA from July 29 to August 3, 2022. This reserve is in the Santa Lucia Mountain range in Monterey County and ranges from 0 to 1,067 m in elevation.11 The coast receives an average of 62 cm of precipitation per year. Average temperatures from 0 to 30 m elevation in August range from 10.9 to 20.9ºC. Riverine habitats within the reserve are found along Big Creek and Devil’s Creek, which are both perennial streams that flow through the reserve. The plant communities vary across the streams, starting as coastal scrub and hardwood in downstream regions and transitioning to more mixed hardwood and redwood further upstream. In addition, the reserve is part of a pristine watershed, defined as functioning close to natural conditions.12 This makes the reserve a good baseline study system because

it is relatively unaffected by humans compared to degraded watersheds. The streams on the reserve provide foraging habitat for a variety of insectivorous bat species, including several Myotis species.

The Landels-Hill Big Creek Reserve is known to contain twelve species of bats across its various habitats.13 Myotis species known to live in the reserve are the Yuma myotis (M. yumanensis), California myotis (Myotis californicus), little brown myotis (Myotis lucifugus), longeared myotis (Myotis evotis), long-legged myotis (Myotis volans), and fringed myotis (Myotis thysanodes). Other bat species known to visit the riparian habitat include the canyon bat (Parastrellus hesperus) and Western red bat (Lasiurus blossevillii). M. yumanensiswas the focal species of our study because it is relatively common, especially within the riverine areas, which are also populated by a variety of aerial insects known to be prey for M. yumanensis. 14 Past studies have found insects from the orders Diptera, Ephemeroptera, Plecoptera, Trichoptera, Hemiptera, Hymenoptera, Lepidoptera, Neuroptera, and Coleoptera at the streams we studied.15

2.2 Research Methods

To examine how Myotis habitat use varies with habitat characteristics and insect populations, we observed

Figure 1. Map of site locations. All study site locations surveyed for bat call and flying insect abundance were located along streams in the Landels-Hill Big Creek Reserve in Monterey County, CA, USA. Sites 1 and 2 are both located on Lower Big Creek. Site 3 is located at the confluence of Upper Big Creek and Devil’s Creek. Site 4 is located on Upper Big Creek and Site 5 is located on Devil’s Creek. Sites varied in flight path openness based on stream width.

five different sites with variable characteristics (Fig. 1). Two sites were on Lower Big Creek: one near the Pacific Ocean and one further inland. The third site was at the confluence of Upper Big Creek and Devil’s Creek. The fourth and fifth sites were located on Upper Big Creek and Devil’s Creek respectively, be-

fore the creeks converge. At each site, we recorded stream width, bat calls, and aerial insects. We measured the stream width by averaging the width at two accessible points 10 m apart, one on either side of the site where we sampled insects.

We surveyed bat calls and aerial insects at one site each day from 8:00 P.M. to 10:00 P.M., when they begin their foraging. Bat echolocation calls were surveyed using the Echo Meter Touch 2 module with the Echo Meter Touch Bat Detector application version 2.8.14 (Wildlife Acoustics, Inc. 2022), which detected and recorded the calls as spectrograms. It detected bat calls with a trigger minimum frequency of 6 kHz, a trigger window (delay after end of detected call) of 1 second, and a maximum trigger length (recording length) of 30 seconds. Myotis calls were identified and separated from non-Myotis bat calls by the spectrograms and frequencies in Kaleidoscope Pro version 5.4.8 (Wildlife Acoustics, Inc. 2021) using the Humboldt State University Bat Lab’s 2018 table on bat echolocation call characteristics.16 The Echo Meter Touch Bat Detector application can also automatically identify bat species using the spectrograms, but there is some error involved in this identification due to noise interference or calls being too short or quiet. Given that multiple species within the Myotis genus produce calls with similar spectrograms, confidently identifying a specific species can be difficult even with software. The Echo Meter Touch 2 is capable of measuring the number of bat calls but cannot directly

Table 1. Site locations and variables. All sites surveyed for bat call and flying insect abundance were located along Big Creek and Devil’s Creek in the Landels-Hill Big Creek Reserve in Monterey County, California. The sites are numbered by how far inland they are located (Fig. 1). The number of Myotis calls and aerial insects were gathered over five nights in summer 2022. Bat calls were gathered with the Echo Meter Touch 2 module and Echo Meter Touch Bat application, then identified to the Myotis genus with Kaleidoscope Pro. Aerial insects were counted every 30 minutes using a light trap.

measure the abundance of individual bats as repeated calls could be from the same bats. It also did not account for the length of calls or periods when multiple bats were calling at the same time, which can occur in heavily populated sites. A larger number of calls in a time period, or call abundance, can indicate that an individual bat is spending more time in the area, or that there are more bats in the area. Either way, this indicates a high level of habitat use by bats, so we used Myotis call abundance as a metric for habitat use.

To examine aerial insect communities at each site, we used a white sheet with a 0.5 m by 0.5 m quadrat divided into 25 squares and set it up near the edge of the stream with an ultraviolet and a white flashlight pointed at it to attract insects, which is a standard method for collecting nocturnal aerial insects. In our pilot study, we allowed insects to accumulate on the sheet for 20 minutes, which resulted in enough insects to reasonably estimate the population and community composition of aerial insects in the area while still being possible to manually count. We then spent five minutes counting the insects in the quadrat before turning off the light and removing the insects from the sheet to avoid counting them in the next trial. Be-

Table 2. Absolute Abundances of Insect Orders. Results from insect surveys along five sites on Devil’s Creek and Big Creek at Landels-Hill Big Creek Reserve in Monterey County, CA, USA. Sites are ordered from closest to the Pacific Ocean to furthest inland (Fig. 1).

fore repeating the trial, we allowed the insects to disperse for five minutes, and conducted four trials each night for four nights. While counting the number of insects, we also classified them according to their respective order. In cases where there were too many similar-looking insects to count in five minutes, we counted the number of insects in two sets of

five squares, then multiplied by 2.5 to estimate the number in all 25 squares. Insects that we could not identify onsite were aspirated, retained in individual specimen jars, and identified under a microscope in conjunction with A Field Guide to Insects. 17

2.3 Statistical Analyses

All statistical analyses were performed using JMP Pro software version 16.0.0 (SAS Institute, Inc. 2021). Bat call abundance and insect abundance data were normalized using a natural log transformation. For each half-hour period from each site, we used a linear regression to analyze the effect of the abundance of all aerial insects on the number of Myotis calls in the same period, with time as a random effect (Fig. 2). We also used linear regressions to investigate whether lepidopteran and dipteran insect order abundance influence the number of Myotis calls, with time as a random effect (Fig. 3-4), and to analyze how stream width affects overall abundance of Myotis calls and aerial insects at each site (Fig. 4). We ran two one-way ANOVAs to examine the differences in overall number of Myotis calls and the number of aerial insects between sites.

RESULTS

We recorded a total of 1,639 bat calls over the course of our study. The Echo Meter Touch 2 Bat Detector application identified 812 of the recorded calls to the species level. Approximately 80% of the automatically-identified 812 calls belonged to M. yumanensis and 15% belonged to M. californicus. Two belonged to western small-footed myotises (Myotis ciliolabrum), and one be-

Figure 2. Myotis call abundance increases with aerial insect abundance. Five creek sites were surveyed along Big Creek and Devil’s Creek in LandelsHill Big Creek Reserve, Big Sur, CA, USA. Data on flying insect abundance and Myotis bat call abundance was recorded in half-hour chunks over 5 nights in summer 2022, with one sampling period per site. A linear regression was used to analyze the effect of the number of aerial insects on the number of Myotis calls, with time as a random effect. Myotis call abundance increased with the abundance of aerial insects collected (N = 20, R2 = 0.32, p = 0.0013).

longed to M. volans. To identify recorded calls that were not identified by the application, we manually compared the recorded spectrograms to known Myotis spectrograms. In total, we identified 1,620 Myotis spp. calls, though due to the similarity of calls within the Myotis genus, we were not able to identify all of the calls to the species level. Stream width varied from 3.5 m to 7.5 m (Table 1). We found 1,431 insects, with approximately 53% from order Lepidoptera, 36% from order Diptera, 4% from order Neuroptera, 3% from order Trichoptera, and the remaining 4% composed of small numbers from orders Homoptera, Coleoptera, Hymenoptera, Phasmatodea, Ephemeroptera, Hemiptera, and Plecoptera (Table 2).

We found that the number of Myotis calls increased with the number of observed aerial insects. Myotis calls in a half-hour period were most strongly correlated with the overall abundance of aerial insects found in the same period (N = 20, R2 = 0.32, p = 0.0013; Fig. 2). When analyzed in relation to specific orders, calls were correlated with absolute dipteran abundance (N = 20, R2 = 0.16, p = 0.0064; Fig. 3a) but only marginally and not significantly correlated with absolute lepidopteran abundance (N = 20, R2 = 0.14, p = 0.076; Fig. 3b). Specifically, there is a marginal difference between sites regarding the number of Myotis calls (N = 20, F = 2.76, p = 0.067; Fig. 4a) and number of aerial insects (N = 20, F = 2.49, p = 0.088; Fig. 4b) per site. Insects from the other orders we examined were far less abundant than lepidopterans and dipterans, so there was insufficient data to reliably

Figure 3. Myotis call abundance and specified insect orders. (a) Myotis call abundance increases with dipteran abundance. Five creek sites were surveyed along Big Creek and Devil’s Creek in Landels-Hill Big Creek Reserve, Big Sur, CA, USA. Data on dipteran abundance and Myotis call abundance was recorded in half-hour chunks over 5 nights in summer 2022, with one sampling period per site. A linear regression was run to test the effect of the number of dipterans on the number of Myotis calls, with time as a random effect. Myotis call abundance increased with the abundance of dipterans collected (N = 20, R2 = 0.16, p = 0.0064). (b) Myotis call abundance marginally increases with lepidopteran abundance. Five creek sites were surveyed along Big Creek and Devil’s Creek in Landels-Hill Big Creek Reserve, Big Sur, CA, USA. Data on lepidopteran abundance and Myotis call abundance was recorded in half-hour chunks over 5 nights in summer 2022, with one sampling period per site. A linear regression was run to test the effect of the number of lepidopterans on the number of Myotis calls, with time as a random effect. Myotis call abundance marginally increased with the abundance of lepidopterans collected (N = 20, R2 = 0.14, p = 0.076), but the effect was not statistically significant.

Figure 4. Myotis call and aerial insect abundances at each site. (a) Myotis call abundance at each site. Five creek sites were surveyed along Big Creek and Devil’s Creek in Landels-Hill Big Creek Reserve, Big Sur, CA, USA. Data on Myotis call abundance was recorded in half-hour chunks over 5 nights in summer 2022, with one sampling period per site. A one-way ANOVA was run to test the effect of site on the number of Myotis calls using the natural log of the number of calls. There is a marginal difference between sites for Myotis call abundance (N = 20, F = 2.76, p = 0.067) but not statistically significant. (b) Aerial insect abundance at each site. Five creek sites were surveyed along Big Creek and Devil’s Creek in Landels-Hill Big Creek Reserve, Big Sur, CA, USA. Data on aerial insect abundance was recorded in half-hour chunks over 5 nights in summer 2022, with one sampling period per site. A one-way ANOVA was run to test the effect of site on the number of aerial insects collected using the natural log of the number insects. There is a marginal difference between sites for aerial insect abundance (N = 20, F = 2.49, p = 0.088) but not statistically significant.

determine any correlation with Myotis calls. While it is not statistically significant, there is a marginal difference between sites regarding the number of Myotis calls (N = 20, F = 2.76, p = 0.067; Fig. 4a) and number of aerial insects (N = 20, F = 2.49, p = 0.088; Fig. 4b) per site. We did not find any relationship between stream width and the total abundance of aerial insects at each site (N = 5, R2 = 0.18, p = 0.57) or between stream width and the total abundance of Myotis calls (N = 5, R2 = 0.21, p = 0.44).

DISCUSSION

Overall, we found that Myotis call abundance increased with total aerial insect abundance, which was consistent with our hypothesis (Fig. 2). We found a marginal difference between sites for both Myotis call abundance and total aerial insect abundance (Fig. 4). However, in contrast to our predictions, we did not find a correlation between the size of the stream and Myotis call abundance.

Our results suggest that summertime Myotis habitat use may be driven more by availability of prey rather than by habitat physical characteristics. Stream width did not affect Myotis call abundance, despite previous studies indicating that they prefer open areas.10 This could be due to the stream width differences not being large enough be-

tween sites, or other site factors affecting bat abundance more than stream width (Table 1). Bats preferentially foraging in areas of larger prey abundance is supported by a previous study which found that bats were capable of tracking insect hatches over tens of kilometers.10 The marginal differences among sites in Myotis call and aerial insect abundance suggest that a site-level factor other than stream width could potentially affect where M. yumanensis and insects can be found. However, because we did not find statistical significance for call abundance and insect abundance overall across sites, more data would need to be collected to determine which site-level characteristics, if any, are important in determining bat habitat use. These characteristics could include nearby tree species, proximity to bat roosts, or stream flow rate, which could all impact either the insect community composition or the number of bats able to easily access the site.

Previous studies on bats suggest differences in abundance could also be due to variable environmental factors during the study, such as temperature, wind speed, and air pressure.18 For example, insect abundances for Site 3 were low in comparison to other sites (Table 2). This may have been the result of an abnormally cold, humid, or windy night, which were not controlled

for in the study. Gathering data at multiple sites on the same day, or several times over the course of a longer time scale would better indicate year-round habitat use. Recording more site-level variables, such as temperature, wind speed, and air pressure could also lead to a better understanding of the habitat characteristics that affect M. yumanensis habitat use.

Our results also showed that the relationship between insect Myotis bat calls and the abundance of insects varied based on the orders of the insects (Fig. 3). Specifically, there was a significant correlation between bat calls and dipteran abundance, but not between bat calls and lepidopteran abundance, despite a higher abundance of lepidopterans overall. This may indicate a dietary preference for dipterans over lepidopterans. However, this trend may have been created by our sampling methods, as our insect collection was based only on aerial insects that were attracted to white and/or UV light (Table 2). Previous studies have used emergence traps to track aquatic insects emerging from streams in addition to aerial insects.19 Expanding this study to include these methods may provide a more accurate representation of riparian insect communities.

While we found the presence of M. yumanensis to be most associated with dipterans in our study (Fig. 3a), it is also possible that there are shifts in dietary preferences throughout the year depending on seasonal availability of prey. Insect swarm composition varies over time due to differences in hatching times, resulting in temporal changes in the insects present in a given area over time.10 Furthermore, species composition and population abundances of bats are not static due to migration and hibernation.20 Since our observations were limited to the summer season and abundances of bats and insects can change seasonally, future studies should encompass larger time scales across multiple seasons. Additionally, expanding this study to include more sites across a larger area would more accurately capture overall foraging behavior. Individual bats are capable of traveling large distances, so variation between two sites that are relatively far apart could factor into bat abundance at those sites.14

The passive acoustic monitoring methods used in this study are a practical way to assess bat abundance, but more direct methods may be more accurate in determining abundance and identifying specific bat species.21 Studies that seek more direct methods could employ mist nets for capturing and identifying bats. They could also employ pellet analysis to provide a more accurate measure of dietary preferences. It should be noted that the Echo Meter Touch Bat Detector application’s automatic species identification software is known to have some error, such as misidentifying species. While it indicated that 80% of the automatically identified bat echolocation calls were from the species M. yumanensis, there were 825 calls out of 1,639 that it was not able to identify to a species level. Many of these (a total of 1,620) were manually identified. A more accurate species assessment would indicate whether our results may be applicable to Yuma myotis specifically or to Myotis spp. in general.

CONCLUSION

Since Myotis bat calls and insect abundance are correlated, insect populations may provide an indirect way to measure which areas bats are more likely to forage in. Knowing where populations of bats are likely to forage helps prioritizing areas for bat conservation, such as riverine areas with rich insect communities that provide plenty of prey

for foraging bats. The correlation between bats and their insect prey could also be used to target specific areas to directly monitor the bat population. Due to the difficulty in monitoring bat populations, developing more effective and efficient methods for conducting population surveys can assist in land management decision-making and conservation. This is increasingly relevant as anthropogenic activities and disease put growing stress on bat populations.

Data provided by this study and future studies could aid in creating improved bat population predictive occurrence models to be specifically based on insect abundance. This data could also help reduce the cost of more direct bat surveys by identifying likely sites of bat foraging. Doing so is necessary to monitor bat populations’ response to various threats. Bats are integral components of many ecosystems that provide humans with valuable ecosystem services such as pest management in agricultural areas and consumption of insects that are potential disease vectors. Despite their ecological value, there is limited research on bat populations, and thus, there continues to be a need for improved monitoring and assessment of bat populations.

ACKNOWLEDGEMENTS

This work was performed at the University of California Natural Reserve System Landels-Hill Big Creek Reserve.

Special acknowledgements to course professors Timothy Miller and Renske Kirchholtes, teaching assistant Amy Van Scoyoc, and course assistants Athena Lynch and Maria Carias.

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[15] Rundio, D.E., and Lindley, S.T.. Reciprocal fluxes of stream and riparian invertebrates in a coastal California basin with Mediterranean climate. Ecological Research 27, 539–550 (2012).

[16] Szewczak, J.. Echolocation Call Characteristics of California Bats. Humboldt State University Bat Lab (2018).

[17] Borror, D.J. and White, R.E.. A Field Guide to the Insects - America north of Mexico. Second edition. Boston, New York, Houghton Mifflin Company (1998).

[18] Gorman, K.M., Barr, E.L., Ries, L., Nocera, T., and Ford, W.M.. Bat activity patterns relative to temporal and weather effects in a temperate coastal environment. Global Ecology and Conservation 30, e01769 (2021).

[19] Rainey, W., Mary, P., and Sandra, C.. Temporal and spatial variation in aquatic insect emergence and bat activity in a restored floodplain wetland. CALFED, 54 (2006).

[20] Kuenzi, A.J., and Morrison, M.L.. Temporal Patterns of Bat Activity in Southern Arizona. The Journal of Wildlife Management 67, 52–64 (2003).

[21] Szewczak, J.M.. “Advanced analysis techniques for identifying bat species.” Brigham, M., Kalko, E., Jones, G., Parsons, S., and Limpens, H., editors. Bat echolocation research: tools, techniques and analysis. Bat Conservation International, 175 (2002).

Analyzing effects of UV-B radiation exposure on Escherichia coli survival in surface water bodies

Leanne Liaw

UC San Diego, Class of 2025, Marshall College, B.S. Neurobiology and Public Health, PI: Ms. Erin Meyers, International Baccalaureate Higher Level Biology Mentor

ABSTRACT

Microbial contamination of potable surface water leads to the deaths of approximately 1.7 million people annually on a global scale. Previous research has studied the impact of environmental conditions on the growth patterns of enteric bacteria in land-based microcosms. However, the myriad of abiotic and biotic factors that regulate the microcosm's environmental homeostasis made it challenging to distinguish how each independent factor influenced bacterial growth. Therefore, this study sought to isolate particular biotic factors to determine their influence on bacterial growth. To better understand the influence of UV-B radiation on enteric Escherichia coli (E. coli), samples were suspended in standardized aqueous solutions and subject to varying UV-B exposure conditions. Observed growth patterns show that E. coli growth negatively correlates with UV-B exposure. More broadly, these results suggest that global regions experiencing higher UV-B radiation will have decreased enteric bacterial survival in potable surface water bodies. Observing global UV-B radiation trends could contribute to the development of predictive microbial models to allow for the detection of physical regions that require additional resources for more intensive water treatment to mitigate waterborne enteric disease.

INTRODUCTION

The largest proportion of fatalities due to water consumption are those under five years of age.1 An additional 663 million individuals lack access to safe drinking water sources globally, leaving them vulnerable to a myriad of waterborne diseases that stem from ingesting contaminated water.2 Particularly, the invasion of E.coli in intestinal epithelial cells can cause diseases including mild diarrhea, gastroenteritis, and haemorrhagic colitis.3 Thus, the removal of biochemical contaminants from common drinking water collection sources is integral to ameliorating water safety.

A common route of enteric pathogen transmission is fecal contamination of drinking water. Diarrhoeagenic Escherichia coli (E. coli) is one of many prokaryotic microorganisms that proliferate within a variety of environments, ranging from mammalian intestines to the surfaces of water.4 These microorganisms can exchange various virulence or resistance mechanisms between each

other to support their survival. This is achieved by the exchange of mobile genetic elements, or genes that are transmitted between bacterial cells and can integrate into a new host genome.5

One form of mobile genetic element is a plasmid, or a DNA fragment that can either directly integrate into the chromosome or survive in the cytoplasm of bacteria to code for proteins.6 Plasmids containing antibiotic resistance genes can also transfer from one bacterium to another. While not all plasmids contain antibiotic resistance genes, a study on contaminated surface water sources in rural and urban Bangladesh noted a correlation between overabundance of human gut bacteria and plasmids carrying resistance genes.7 Thus, consuming stagnant surface water without sufficient sanitation treatment leaves individuals highly susceptible to diseases caused by opportunistic pathogens.

The integrity of DNA fragments like plasmids is influenced by multiple factors such as ultraviolet (UV) radiation.

The UV radiation range of the electromagnetic spectrum is subdivided into three categories: UV-A (315 - 400 nm), UV-B (290-315 nm), and UV-C (200290 nm).8 The majority of UV-C and a fraction of UV-B radiation are absorbed by the earth’s atmosphere due to their shorter wavelengths. As such, they are generally more difficult to detect on a ground level than the longer wavelengths of UV-A radiation.

Extended UV-B exposure is known to cause mutations in cellular genomes. One such example is pyrimidine dimerization. During pyrimidine dimerization, thymine (T) or cytosine (C) nitrogenous bases adjacent on a DNA strand will crosslink to form T-T, C-C, or even sometimes T-C dimers.8 Unless dimers are repaired, the abnormal base pairing disrupts the transcription process and ultimately hinders DNA replication.9 Under increased UV exposure, elevated rates of dimerization make it increasingly difficult for the enzymes to repair mutations. In fact, consistent UV radiation exposure within genes that encode enzymatic repair mechanisms themselves can produce nonfunctional products.10 Furthermore, sustained UV radiation may even induce phosphodiester bond breakage in DNA, which could lead to double stranded breaks and chromosomal breakage within the genome.8

Multiple studies have sought to understand how enteric bacteria, particularly pathogenic E. coli, proliferate when exposed to UV-B radiation in the context of land-based environments.7,11 However, these studies measured irradiation alongside influential biotic (e.g., amphibian or human presence) and abiotic (e.g., temperature and pH variations) components. Therefore, it is difficult to extrapolate how individual biotic and abiotic factors definitively affect bacterial growth. Therefore, it is hy-

Figure 1. Boxplot comparisons of turbidities. Comparisons indicate consistent decrease in turbidity as UV-B exposure increases. Standard deviations varied across conditions: standard deviation values increased alongside decreased UV-B exposure. The larger standard deviation of the condition without UV-B exposure suggests that change in turbidity was more highly variable. When comparing all boxplot trends, it appears that as UV-B exposure decreases, turbidity values increase by statistically significant margins. This trend suggests an exponential proliferation of E. coli colonies in aqueous environs when UV-B presence decreases. Uncertainties were calculated by subtracting the average NTU value of the dataset from the farthest measured value.

pothesized that the growth rate of E. coli colonies should consistently decrease when solely influenced by increasing exposure of UV-B irradiation. As irradiation intensifies, the DNA within these colonies will photodegrade and reduce cell proliferation. Gauging optimal light conditions for E. coli growth would aid in identifying which bodies of water are more susceptible to bacterial contamination and therefore require more thorough sanitation measures.

MATERIALS & METHODS

This study investigated the effect of UV-B radiation on Escherichia coli populations within an aqueous environment. Diluted nutrient broth was utilized to replicate bacterial growth conditions in an aqueous environment, while granulated sugar was added as a carbon source for ATP synthesis. E. coli colonies within aliquots of 20 mL of nutrient broth were exposed to the three UV-B exposure conditions.

Incubation of Bacteria

The K-12 strain of E. coli was used as a model system to test the effects of UV radiation. This strain is nonpathogenic and therefore provides the lowest level of risk of infection while still mimicking the rapid division rate of pathogenic E. coli. Additionally, this strain can be grown without the use of an incubator, closely resembling the pathogenic E. coli strains which grow in stagnant bodies of water.

A starter plate of agar media was used to ensure the viability of the bacterial strain. The selected strain of E. coli K-12 was streaked across the plate

and incubated at 37ºC to grow individual colonies. Prominent colonies were identified and selected to inoculate the nutrient broth in each tube. Differing initial amounts of E. coli would potentially exacerbate differences in final turbidity of the UV-B exposure condition. To prevent discrepancies, tubes were measured before and after E. coli input using a 0.01 g precision scale. The same E. coli deposit standardization process was repeated for remaining tubes.

Each of the three light conditions were prepared with 10 culture tubes containing 100 mL of diluted nutrient. Each set was allocated to a different exposure condition. The total homogenous growth medium was created via 0.3 mL of nutrient broth, 0.3 mL of distilled H2O, and 6.0 g of granulated sugar. Each test tube was firmly stoppered to induce samples to restrict growth to anaerobic respiration pathways and therefore control for growth rates that may result from variations in pathways for ATP synthesis. Each set contained one tube of diluted nutrient broth to serve as a negative control.

Exposure to UV-B Radiation

E. coli was analyzed under three UV-B exposure conditions: constant exposure, 12 hours of exposure, and no exposure. This experimental paradigm was chosen to stimulate how E. coli proliferates when exposed to UV-B radiation in a regular day-night cycle, as well as how its proliferation rate will differ when exposed to either extreme of constant or no UV-B exposure.

To prepare the constant exposure condition, the light bulb was placed

0.75 m above the samples to ensure samples received full coverage of the UV-B light source while minimizing the potential for thermal radiation to influence bacterial proliferation. Constant exposure was used in this condition to replicate E. coli growth in the midday sun while the 12 hour cycle of exposure and non-exposure light condition stimulated a normal day-night cycle. The third light condition involved placing samples inside a dark opaque container the entire duration of the experiment.

All tubes were finally placed into tube racks at their respective conditions and were left undisturbed for 72 hours at a constant controlled temperature of 23.5˚C and a 45% relative humidity. Since E. coli populations typically double approximately every 20 minutes when grown in nutrient broth, 72 hours were deemed a suitable time period for measurable growth.15

Measuring Bacterial Proliferation

The presence of particles in some solute can be measured by nephelometric turbidity units (NTU). In this experiment, higher turbidity values translate to higher levels of bacterial proliferation while lower turbidity values suggest lower levels of bacterial proliferation. Change in turbidity can also be noted qualitatively, as a higher the concentration of matriculates in the water will produce a more opaque solution, indicative of a higher turbidity value. Initial and final turbidites of the tubes were measured by placing each tube into a Vernier turbidity sensor and recording the NTU value.

RESULTS

Three trials were performed: constant UV-B exposure, 12 hours of UV-B exposure, and no UV-B exposure. Each condition had one negative control test tube containing no E. coli to ensure potential contamination within the nutrient broth did not impact bacterial growth in the experiment. After 72 hours, turbidites were measured in all tubes.

Samples under the condition not exposed to UV-B experienced the greatest population growth at an average difference of approximately 500 ± 200 NTU between initial and final observations. Samples which experienced constant UV-B exposure experienced the smallest change in growth, with an average of 20 ± 30 NTU. It may be worth noting that several individual samples actually had decreased NTU values than initially recorded, indicating a decrease in E. coli presence (Fig. 1).

The decrease in bacterial growth driven by increased levels of UV-B il-

lumination periods indicate that UV exposure decreased the amount of bacterial growth in the suspension solution. The inverse relationship between UV-B exposure and E. coli growth is further noted as the increased UV-B exposure translates to a higher turbidity value (Fig. 2).

Correlation between net NTU values is also supported through the R2 value. The resulting correlation coefficient is calculated to be ≃0.93, suggesting a strong correlation between decreasing UV-B exposure and increasing E. coli population growth. Overall, there is a statistically significant difference between the turbidites of each of the conditions, therefore it can be concluded that UV-B radiation is inversely proportional to bacterial proliferation in aqueous environments.

DISCUSSION

This study investigated the impact of UV-B exposure on E. coli colonies within an aqueous environment. Col-

onies were exposed to constant UV-B light, UV-B light exposure in 12 hour intervals, and no UV-B light to determine how surface water sources may act as a gastroenteric disease vector. It was determined that bodies of water exposed to constant UV-B light sustained the least bacterial growth, thus demonstrating that UV-B light restricted E. coli growth.

The inverse relationship between UV-B exposure and E. coli replication is postulated to be caused by UV-B’s light-induced mutations of DNA. Samples under constant UV-B exposure likely had more frequent opportunities for photo-mutagenesis to occur. Increases in electron excitation would have resulted in an increase in electron dimerization, and therefore required increased photolyase activity.

An older, more expansive experimental model supports the findings of this experiment by noting only residual microbial DNA remained after 58 days of exposure to moderately-high UV-B

Figure 2. Frequencies of changes in turbidites. The above histograms showcase the frequencies in turbidity changes across exposure conditions. While observing the individual-condition histograms, it can be seen that the data increasingly skewed to the left as UV-B exposure decreased. Constant UV-B exposure appears to be generally symmetrical with the highest frequency in the 15 - 25 NTU interval. The 12 hour exposure condition experienced a greater left-skew with its highest frequency at its 275 - 325 NTU interval, and the no UV-B exposure condition experienced the greatest left was skew with the majority of its values in the 550-650 NTU interval.

wavelengths.11 This model used both gram-positive and gram-negative bacteria (i.e., Staphylococcus epidermidis, Pseudomonas fluorescens, Corynebacterium xerosis) and prepared their growth medium by leaving Lithobates catesbeianus tadpoles in water-filled polypropylene tanks for 10 days to stimulate a naturally-occurring aquatic environment. It was determined that over time bacterial DNA degraded in an exponential manner. Given that data collection for the model of focus was only conducted before and after the 72 hour time period, increasing the incubation time period and monitoring turbidity in three day intervals could reveal particular growth patterns of the effect of UV-B light modulation on E. coli growth. Additionally, as nutrient broth is not a typically-occurring source in freshwater aquatic ecosystems, creating microcosms containing common freshwater organisms like crustaceans to mimic natural nutrient deposition could allow for an improved representation of the effect of UV-B exposure on often-used potable water sources. Furthermore, the inclusion of multiple fecal coliforms would allow for the determination of whether the E. coli’s behavior under UV-B exposure was experienced by E. coli alone or can be generalized to the entire bacterial subgroup.

Identifying whether UV-B resistance is possible in this bacterial subgroup is integral. Fecal coliforms are

commonly utilized to determine deterioration in water quality. However, external factors like pH or temperature overriding the impact of UV-B’s anti-microbial properties could force populations living in those areas to find additional means of decontaminating their potable water supply.13 Presence of UV-B resistant, gram-positive bacteria has been noted in a few high-altitude freshwater lakes in the Andean mountain region.14 By quantifying the colony-forming units (CFU) in water aliquots replicating increasingly concentrated amounts of Andes lake-water, it was determined that bacterial communities generally grew well in this environment when exposed to UV-B. Concerningly, there were even cases where UV-B stimulated bacterial growth.13 This bacterial growth could have been stimulated due to the higher turbidity of the Andean Lakes: though considered freshwater, these lakes have a slightly-higher salt concentration. As salts dissolve into the solvent-acting H2O, the increased presence of salt in the water bodies increases the presence of molecules in the solution, thus increasing the turbidity of the water.15 Freshwater bodies where potable water is typically attained have been noted to have lesser turbidity than their saltwater counterparts.14 It thus appears that while UV-B radiation has the potential to treat bacteria in common sources of freshwater, high turbidity is an important consideration given its ability to hinder UV-B penetration and kill bacteria.

Tables 1-3. ANOVA statistics for all UV-B conditions. Using a one-way ANOVA test, it was determined that the p-value for the dataset is lower than 0.05. Thus, the differences among the data for each condition is statistically significant and the null hypothesis is rejected.

As populations like the Andean mountain region utilize saltier bodies of water, it appears that additional measures must be determined to counteract the effect of turbidity from additional particles on UV-B radiation and resulting bacterial growth.

Additional fecal indicator bacteria (e.g., Pseudomonas aeruginosa and Staphylococcus aureus) were also inactivated to a significant degree over a 1.67 hour exposure period.16 Therefore, although high water turbidity initially decreases the potency of UV-B light’s microbial elimination, increased duration of exposure appears to eliminate the majority of more-resilient pathogens.

While public understanding of the necessity of sustainable access to safe drinking water has increased dramatically since 1995, according to the WHO’s 2015 assessment on the progress of sanitation and drinking water almost 2.4 billion people lack access to water sanitation facilities.2 Additionally, the majority of these regions are still developing and a significant portion of their populations live in rural areas and practice open defecation.2 Given the evident potency of UV-B radiation on decreasing E. coli presence, the development of microbial predictive occurrence models for waterborne pathogens like E. coli would be immensely beneficial to determining which regions experience less ground-absorption of UV-B and thus require more intensive water sanitation. In addition, UV-B’s efficacy in controlling E. coli presence could further be extended to several other enteric pathogens including Vibrio cholerae, Salmonella enterica, Burkholderia mallei, and especially Francisella tularensis, whose colonies can persist in water or mud for years and a time and are a significant source of morbidity on a global scale.17

CONCLUSION

This study indicates increased UV-B exposure decreases E. coli growth and survival within an aqueous environment. These results aid in understanding the importance of UV ra-

diation on microbial survival in potable surface water bodies. The data trends noted in this study could contribute to the formation of microbial predictive occurrence models to identify physical regions that require more intensive water sanitation to mitigate waterborne enteric disease. Further research on how other physicochemical factors behave in conjunction with UV radiation could be used to develop more effective techniques to diminish the presence of aqueous pathogens.

While contaminated water can be sterilized via pasteurization or chlorination, such strategies have limited availability in developing countries. At least 33% of the population in developing countries does not have access to safe drinking water. However, many developing nations are located in regions where sunlight and UV radiation is abundant. Preliminary UV-disinfecting bottles already appear to be in partial circulation in Togo, Colombia, Bolivia, Indonesia, and Thailand.18 Harnessing the bactericidal qualities of UV radiation by creating water containers that transmit UV-B wavelengths could ameliorate the quality of life in such regions by bolstering hygienic standards.

REFERENCES

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[2] World Health Organization. Progress on Sanitation and Drinking Water – 2015 update and MDG assessment. Geneva, Switzerland: World Health Organization; 2015

[3] Park J, Kim JS, Kim S, et al. A waterborne outbreak of multiple diarrhoeagenic Escherichia coli infections associated with drinking water at a school camp. International Journal of Infectious Diseases. 2018;66:45-50. doi:10.1016/j. ijid.2017.09.021

[4] Coleman BL, Louie M, Salvadori MI, et al. Contamination of Canadian private drinking water sources with antimicrobial resistant Escherichia coli. Water Research. 2013;47(9):3026-3036. doi:10.1016/j.watres.2013.03.008

[5] Frost LS, Leplae R, Summers AO, Toussaint A. Mobile genetic elements: the agents of open source evolution. Nature Reviews Microbiology. 2005;3(9):722-732. doi:10.1038/nrmicro1235

[6] Foxman B. Molecular Tools and Infectious Disease Epidemiology. Academic Press; 2010

[7] McInnes RS, uz-Zaman MH, Alam IT, et al. Metagenome-Wide Analysis of Rural and Urban Surface Waters and Sediments in Bangladesh Identifies Human Waste as a Driver of Antibiotic Resistance. Bulman Z, ed. American Society for Microbiology. 2021;6(4).

[8] Bolognia JL, Schaffer JV, Cerroni L. Dermatology. 4th ed. Elsevier; 2017:1536-1541.

[9] Case C, Funke B, Tortora G. Microbiology: An Introduction. 13th ed. (Beauparlant S, ed.). Pearson; 2018:120-225.

[10] Shapovalova OE, Levy D, Avnir D, Vinogradov VV. Protection of enzymes from photodegradation by entrapment within alumina. Colloids and Surfaces B, Biointerfaces. 2016;146:731-736. doi:https:// doi.org/10.1016/j.colsurfb.2016.07.020

[11] Strickler KM, Fremier AK, Goldberg CS. Quantifying effects of UV-B, temperature, and pH on eDNA degradation in aquatic microcosms. Biological Conservation. 2015;183(10):85-92. doi:10.1016/j.biocon.2014.11.038

[12] Favero MS, Carson LA, Bond WW, Petersen NJ. Pseudomonas aeruginosa: Growth in Distilled Water from Hospitals. Science. 1971;173(3999):836838. Accessed February 24, 2023. https://www. jstor.org/stable/1732137

[13] Pachepsky YA, Shelton DR. Escherichia Coliand Fecal Coliforms in Freshwater and Estuarine Sediments. Critical Reviews in Environmental Science and Technology. 2011;41(12):1067-1110. doi:https://doi.org/10.1080/10643380903392718

[14] R. Flores M, F. Ordoñez O, J. Maldonado M, E. Farías M. Isolation of UV-B Resistant Bacteria from Two High Altitude Andean Lakes (4,400 m) with Saline and Non Saline Conditions. The Journal of General and Applied Microbiology. 2009;55(6):447-458. doi:https://doi.org/10.2323/ jgam.55.447

[15] Onabule OA, Mitchell SB, Couceiro F. The Effects of Freshwater Flow and Salinity on Turbidity and Dissolved Oxygen in a Shallow Macrotidal Estuary: a Case Study of Portsmouth Harbour. Ocean & Coastal Management. 2020;191:105179. doi:https://doi.org/10.1016/j. ocecoaman.2020.105179

[16] Deller S, Mascher F, Platzer S, Reinthaler FF, Marth E. Effect of solar radiation on survival of indicator bacteria in bathing waters. Central European Journal of Public Health. 2006;14(3):133-137. doi:10.21101/cejph.a3380

[17] Magana-Arachchi DN, Wanigatunge RP. Ubiquitous Waterborne Pathogens. Waterborne Pathogens. 2020;1(1):15-42. doi:https://doi. org/10.1016/B978-0-12-818783-8.00002-5

[18] Fisher MB, Iriarte M, Nelson KL. Solar water disinfection (SODIS) of Escherichia coli, Enterococcus spp., and MS2 coliphage: Effects of additives and alternative container materials. Water Research. 2012;46(6):1745-1754. doi:10.1016/j. watres.2011.12.048

SENIOR HONORS THESES

UC San Diego's Senior Honors Thesis Program allows undergraduate biology majors to work one-on-one with faculty mentors to pursue independent lab research. These are the abstracts of all the exceptional research projects conducted by honors students this past year.

Salton Sea, California.

Photo by Bridget Spencer

SOPHIA BARBER

REVELLE COLLEGE NEUROBIOLOGY MAJOR COMPUTER SCIENCE MINOR

PI: Chengbiao Wu, Ph.D., UCSD School of Medicine, Department of Neurosciences

Huntington’s Disease and Alzheimer’s Disease

Impacts on MMP and Lysosomal Speed

Lysosomes and mitochondria serve critical roles in the cell as the major digestive and energy generating organelles. Lysosomal function has been heavily implicated in major metabolic regulation pathways and, outside of energy production, mitochondria demonstrate key roles in calcium storage and apoptosis decision making. Dysfunction of lysosomes and mitochondria has been linked to the pathogenesis of various neurodegenerative disorders, including Huntington’s disease and Alzheimer’s disease. However, previous models of Huntington’s disease and Alzheimer’s disease have demonstrated incongruent results in regards to the relative lysosome and mitochondrial expression in these conditions. Using primary neuron cultures from E18 mice we studied the distinct changes in lysosomal speed and mitochondrial membrane potential in the BACHD and PSAPP mice models used to demonstrate Huntington’s disease and Alzheimer’s disease respectively. Our results demonstrate increased mitochondrial membrane potential (MMP) in Alzheimer’s disease cortical neurons and decreased lysosomal speed in Huntington’s disease cortical neurons.

ZOE ADELSHEIM

PI: Michael J. McCarthy, M.D., Ph.D.,

UC San Diego Department of Psychiatry and Center for Circadian Biology

Contributions of Bipolar Disorder-Associated Risk Genes to Rhythmic Transcription Across the Genome

Bipolar disorder (BD) is a severe, mood disorder that has genetic risk factors and causes disrupted circadian rhythms, but how BD-associated risk genes regulate circadian rhythms remains undetermined. Presently, four BD-associated risk genes, ARNTL , ANK3, TCF4, and CACNA1C were knocked down using small interfering RNA in neuronal precursor cells derived from induced pluripotent stem cells from healthy donors. Rhythmic gene expression changes were measured using Per2-luc, a circadian reporter and whole-transcriptome RNA sequencing (RNAseq). Knockdown of BD-associated genes altered Per2-luc rhythm properties. RNAseq further revealed that BD-associated gene knockdowns altered rhythmic expression across the genome, including clock genes and other transcripts. Further analysis revealed altered rhythms in transcripts related to pathways previously implicated in BD. Collectively, this work highlights that rhythmic gene expression may be disrupted by variation in BD-associated risk loci. Further understanding of these mechanisms may allow us to better understand the relationship between BD and circadian rhythms.

ROOSEVELT COLLEGE NEUROBIOLOGY MAJOR HEALTH CARE SOCIAL ISSUES MINOR

JULIAN BAHRAMIPOUR

PI: Derek Welsbie M.D., Ph.D., Shiley Eye Institute, Department of Ophthalmology

In Vitro Screen for Dominant Negatives of Apoptotic Dual Leucine Zipper Kinase (DLK)

Glaucoma is one of the leading causes of irreversible blindness in the world. The hallmark of glaucomatous neurodegeneration is progressive retinal ganglion cell (RGC) apoptosis. RGC cell death is dependent on mitogen activated protein kinase (MAPK) family signaling, notably dual leucine zipper kinase (DLK). In candidate-based screens of DLK dominant negatives, kinase-dead mutants have been shown to ameliorate RGC degeneration. Here, we report a high-throughput, in vitro screen to identify novel dominant negative DLK cDNAs. Efficacious dominant negatives will be utilized to prevent DLK-mediated apoptosis signaling and to reveal novel residues critical to DLK activity.

REVELLE COLLEGE HUMAN BIOLOGY MAJOR GLOBAL HEALTH MINOR

GULSHANBIR BAIDWAN

PI: Tariq Rana, Ph.D., UC San Diego School of Medicine, Department of Pediatrics

Azidothymidine-induced Neurotoxicity in HIV-Associated Neurocognitive Disorders

Today, more than 38 million people are living with HIV/AIDS. While there is no cure, antiretroviral therapies (ART) do exist to control it. Azidothymidine (AZT), one of the oldest ART drugs, remains an important component of most ART “cocktails.” Despite reduction in viral loads, individuals undergoing ART often develop HIV associated neurocognitive disorders (HAND). The mechanism behind this remains less understood. Previously, the Rana lab established that cerebral organoids can serve as a model system for studying drug-induced neuroinflammation. To identify possible mechanisms of this neurocognitive damage, we treated cerebral organoids with AZT and studied the effects of treatment via single cell RNA sequencing. We analyzed 6,035 cells from five control organoids and 6,695 cells from five AZT-treated organoids and found that apoptotic and neuroinflammatory genes were upregulated in the neurons, astrocytes, and glial cells of AZT-treated organoids. Further studies will investigate AZT-induced neurodegeneration in neurons in vitro.

ROOSEVELT COLLEGE BIOLOGY WITH SPECIALIZATION IN BIOINFORMATICS MAJOR

HANNAH BUDROE

REVELLE COLLEGE GENERAL BIOLOGY & HISTORY MAJOR

PI: Moira Décima, M.S., Ph.D, Scripps

Institution of Oceanography, UCSD Depth-Resolved Patterns in Zooplankton Diversity in Subtropical and Subantarctic Waters: a Size-Structured Metabarcoding Approach

Zooplankton play critical ecological roles in trophic transfer and biogeochemical cycling, and their community composition is closely associated with the physical conditions of their environment. Zooplankton species can often be used as sentinels of oceanographic change, because they can be associated with very specific water masses. In this study, I use a DNA metabarcoding approach, based on 18s rRNA amplicon sequencing, to assess the similarities and variability of community composition in three size fractions (0.2-0.5, 0.5-1, 1-2mm) across depths and water types. The study region sees the mixing of Subantarctic (SA) and Subtropical (ST) waters with distinct physicochemical conditions. Preliminary analysis shows that there are distinct mesozooplankton communities between SA and ST waters. Community composition of the smaller (0.2-1mm) zooplankton is different between epipelagic (200m depth) and mesopelagic environments. This study forms a baseline survey of zooplankton diversity, enabling the detection of Southern Ocean ecosystem shifts in response to climate change.

MANAN CHOPRA

MARSHALL COLLEGE BIOLOGY WITH SPECIALIZATION IN BIOINFORMATICS MAJOR

PI:

Karl J.

Wahlin, Ph.D., UCSD Health, Department of Ophthalmology

Alternative Splicing and DNA Methylation Analysis of Age-Related Macular Degeneration (AMD) Human Retinal Tissue

Retinal degenerative diseases such as glaucoma, age-related macular degeneration (AMD), and retinitis pigmentosa affect 285 million people worldwide, and can all result in poor vision/vision loss that has varying etiologies and genetic factors involved. Most share a common neurodegenerative phenotype reflecting malfunction or total loss of retinal neurons. Oxford Nanopore Technologies long read sequencing has been increasingly used to investigate DNA methylation and gene expression patterns. These large reads that span hundreds of kilobases can capture entire transcripts in one read, enabling discovery of differentially expressed isoforms of genes of interest. I am taking advantage of this technology to investigate how AMD effects the methylation patterns of certain regions of the genome and the expression of certain genes/isoforms in contrast with healthy tissue. With the information, I hope to elucidate important genes in AMD processes for further research into the mechanisms and treatment of this disease.

ALLISON CHRISTIAN

REVELLE COLLEGE MOLECULAR & CELL BIOLOGY MAJOR

PI: Derek Welsbie, M.D., Ph.D., UCSD Health, Department of Ophthalmology Cloning of S. marcescens Endonuclease for AAV Purification

Adeno-associated virus (AAV) is the leading viral vector for gene therapies, primarily for its ability to efficiently deliver transgenes with relatively low toxicity and risk of host-genome integration. In the production of AAV, endonucleases are often required to digest producer cell genomic DNA. This reduces viral lysate viscosity and degrades contaminating nucleic acids—both of which decrease DNA fouling and maximize AAV column binding during chromatography purification. While benzonase is most commonly used for viral production, its prohibitive cost makes AAV purification broadly expensive and inaccessible. Here, we outline chromatography purification of the S. marcescens endonuclease as an efficacious alternative for producer cell genomic DNA digestion. This method lowers the overall cost and increases the yield of endonuclease, which can make way for higher throughput, larger scale AAV production imperative for the future of gene therapies.

MARTHA CHOW

MUIR COLLEGE MOLECULAR & CELL BIOLOGY MAJOR DATA SCIENCE MINOR

PI: Omar S. Akbari, Ph.D., UCSD Division of Biological Sciences, Department of Cell & Developmental Biology

Eliminating Malaria Vector Anopheles gambiae using the Precision-Guided Sterile Insect Technique (pgSIT)

Despite efforts to control mosquitoes using physical and chemical (eg. bednets and insecticides) methods, mosquito-borne diseases are on the rise. Malaria in particular is responsible for half a million deaths annually. Controlling Anopheles gambiae, the primary malaria vector in Africa, via the Precision-guided Sterile Insect Technique (pgSIT) would provide a sustainable, controllable, confinable, and scalable solution to control disease spread. The technology has been shown to suppress insect populations by releasing sterile ‘pgSIT males’ to mate with monandrous wild females. Using a binary CRISPR-Cas9 system, pgSIT knocks out genes essential for female development and male fertility. Here, we adapt pgSIT to A. gambiae, showing that our design facilitates genetic sexing and male sterilization. We also demonstrate that pgSIT males are fit; they mate with wild females competitively and survive as long as wild males. Our results indicate that pgSIT is a promising alternative to current control methods for A. gambiae

CHIHIN FENG

ROOSEVELT COLLEGE GENERAL BIOLOGY MAJOR

PI: Yishi Jin, Ph.D., School of Biological Sciences, Department of Neurobiology, Co-Mentor: Junxiang Zhou, Ph.D., School of Biological Sciences, Department of Neurobiology

PI: Jose

NHAN ALBERT DANG VU

Pruneda-Paz,

Ph.D., Department

of Cell and Developmental Biology, School of Biological Sciences, UCSD

Investigating the Role of cis-Regulatory Motifs on the Regulation of the Plant Circadian Clock Function

Almost every plant growth and developmental response is regulated by an endogenous biological clock function, which synchronizes daily rhythms in organismal physiology with daily oscillation of environmental cues. At the molecular level, the plant clock (studied in Arabidopsis thaliana), was shown to depend on the rhythmic expression of TOC1 gene, which is regulated by transcription factors that bind to a cis-regulatory motif in the TOC1 promoter called the Evening Element (EE). While the EE was shown to be critical for regulating the clock function, its function was never analyzed in the context of the endogenous TOC1 gene locus. To fill this gap in knowledge we generated Arabidopsis mutant lines carrying a genomic deletion of this EE, which will be used to determine its essentiality for the function of the plant clock.

ROOSEVELT COLLEGE HUMAN BIOLOGY & MARINE BIOLOGY MAJOR

YUFEI DENG

PI: Tariq Rana, Ph.D., UCSD School of Medicine, Department of Pediatrics Differential miRNAs Expressions in Anti-PD1 and Anti-CTLA4

Immunotherapy

Effects Of Microtubule Isotypes On Neuronal Morphology, Locomotion, And Growth Of

C. elegans

Most animal cells express multiple alpha and beta tubulin isoforms that heterodimerize to form microtubules which are critical cellular components required for maintaining cellular structure, transporting organelles and vesicles, and regulating signaling pathways. In C.elegans, BEN-1 beta-tubulin is specifically expressed in neurons while TBB-2 beta-tubulin is globally expressed. In my research, I investigate how combination of a BEN1 gain-of-function and a TBB-2 loss of function mutation affects neuronal morphology, locomotion, and growth of C. elegans. Through the CRISPR and Cre/loxP system, I created C. elegans strains in which I selectively removed TBB-2 in specific types of neurons and re-examined all the morphological and behavioral parameters. I find that the global microtubule disruptions cause severe defects in neuronal morphology, locomotion, and growth, while animals with the neuron-specific disruptions of TBB-2 are superficially wildtype. My result suggests that microtubule disruptions in neurons do not cause severe defects in locomotion and growth.

Immunotherapy aims to boost host immune response against various diseases including cancers. Immune checkpoint inhibitors against PD1 and CTLA4 have achieved therapeutic effects to cancer, but the response rate remains low in colon cancer patients. To improve anti-PD1 immunotherapy, combinational therapy using both immunotherapy and other immune modulators should be developed. MicroRNAs (miRNAs) are non-coding small RNAs of 18-25 nucleotides. miRNAs play important roles in tumorigenesis and immune response, and therefore can be candidates as immune modulators. Colorectal cancer mouse models, MC38 and CT26, are used to discover and validate the functions of miRNAs. Analyzing differential small RNA expression between no treatment, monotherapy (anti-PD1 only), and combination therapy (anti-PD1 and anti-CTLA4) will tell the up-regulated or down-regulated miRNAs in responding tumors. Our findings will shed lights on the discovery of small molecule immunotherapy boosters and help the patients with partial, no-response or relapse.

SIXTH COLLEGE GENERAL BIOLOGY & COGNITIVE-BEHAVIORAL NEUROSCIENCE MAJOR

FARAH FAROUQ

PI: Stefan Leutgeb, Department of Neurobiology, School of Biological Sciences, University of California San Diego

Assessing a Potential Role of Sequential Coding for Working Memory

Working memory temporarily holds information during cognitive tasks and requires the medial prefrontal cortex (mPFC) and hippocampus. Hippocampal time cells are thought to support working memory tasks through sequence coding during delay phases, which has been hypothesized to require coordination by hippocampal theta oscillations. To test the role of theta for sequence coding, we performed a delayed alternation task that included running on a treadmill during the delay to control the persistence of theta. We then recorded from mPFC and along the septotemporal axis of the hippocampus, using a silicon and Neuropixels probe. We found that hippocampal time cells were only found during the first few seconds of the delay period, regardless of whether running was imposed or not. If we also find that prefrontal neuronal activity patterns occur independent of theta, we will determine that brain oscillations are less critical for sustaining working-memory related brain activity than previously proposed.

REVELLE COLLEGE GENERAL BIOLOGY MAJOR

MARSHALL COLLEGE MOLECULAR & CELL BIOLOGY MAJOR

LILI FOLLETT

PI: Andrew Muroyama, Ph.D,

UC San Diego, Division of Biological Sciences

Dissecting the Mechanistic Basis of Nuclear Migration during Stomatal Formation in Arabidopsis thaliana

Cellular pathways that control stem cell divisions are key to plant development. Oriented nuclear migrations before cell division allow the creation of daughter cells with differing sizes, fates, and placements. Plant development relies on asymmetric cell division to maintain stem cells, promote growth, and encourage cell diversity during leaf formation in Arabidopsis thaliana. While the nuclear behavior during asymmetric divisions has been characterized, associated regulators of the cytoskeleton and cell cycle remain unknown. We are testing whether Arp2/3-mediated actin polymerization is required for nuclear migration through time-lapse imaging of Arabidopsis seedlings treated with an Arp2/3 inhibitor (CK-666) and an associated control compound (CK-689). This work is being complemented by phenotypic analysis of stomatal formation in mutants in the protein components linking the cytoskeleton to the nuclear envelope. Determining the mechanism behind nuclear migration will give insight into how organelle segregation controls asymmetric cell divisions and stomatal development.

SIDDHARTH GAYWALA

PI: Richard Daneman, Ph.D., UCSD School of Medicine, Department of Pharmacology

How do Brain Endothelial Cells Modulate Neuroinflammation?

QINGQING GONG

ROOSEVELT COLLEGE MOLECULAR & CELL BIOLOGY MAJOR VISUAL ARTS (STUDIO) & COMPUTER SCIENCE MINOR

PI: Alon Goren, Ph.D., Department of Medicine, Division of Genetics

ROOSEVELT COLLEGE BIOINFORMATICS MAJOR COMPUTER SCIENCE MINOR

The unique properties of the brain vasculature, termed the blood-brain barrier (BBB), are essential to maintain brain health and become dysregulated in neuroinflammatory disease states, such as multiple sclerosis, epilepsy, and Alzheimer’s disease (AD). In preliminary studies, we identified Elovl7 to be downregulated at the protein and transcript level in post-mortem AD-patient vasculature. Elovl7, a long-chain fatty acid (LCFA) elongase, is highly and specifically expressed in brain endothelial cells (BECs), which harbor BBB properties. However, Elovl7’s role in AD pathology is unknown. As many LCFAs function as inflammatory modulators, we hypothesize that Elovl7 is involved in modulating neuroinflammation, and that its downregulation exacerbates neuroinflammation in disease. To test this, we generated a novel Elovl7-floxed mouse line, allowing us to specifically knockout Elovl7 in BECs with temporal control. This project will provide insight into the pathophysiology of AD and novel avenues by which the BBB protects the brain in health.

NATHAN GLONEK

WARREN COLLEGE MICROBIOLOGY MAJOR HISTORY MINOR

PI: Karsten Zengler, Ph.D., UCSD School of Medicine, Department of Pediatrics Understanding Microbiome Assembly Using Synthetically Reconstructed Communities (SynCom)

Synthetic bacterial communities (SynComs) serve as effective microbiological models to better understand the interactions between bacteria, their hosts, and other microbes in their environment. To make these SynComs relevant and representative, a variety of bacterial species must be included to mirror the function of the in vivo community. The Zengler Lab has developed a novel approach that investigates bacterial protein regulation to determine which members of a SynCom work together, and thus, have a similar influence on community assembly and function, i.e. they belong to the same bacterial guild. With this information, I will be utilizing a 16-species SynCom, representing a rhizosphere bacterial community to investigate how microbes exhibit resilience and respond to various perturbations such as antibiotic treatment. This will allow us to better understand how microbial communities assemble and interact, enabling us to leverage their diverse capabilities for industrial and therapeutic applications in the future.

Systematic Analysis of the Impact of Short Tandem Repeats on Gene Expression

Short tandem repeats (STRs) are genomic sequences with a repeat unit of 1-6bp, representing an intriguing type of genetic variation given their polymorphisms and high mutation rate. Our lab has previously shown that variations in the repeat number of numerous STRs is associated with the expression of nearby genes. To experimentally study this observation, we employ a massively parallel reporter assay to test a series of promoter proximal human STRs. Our results reveal that CG-rich STRs exhibit induction of reporter activity that is higher than AT-rich ones, which is solely an effect of the repeat unit patterns, with no consensus trend in terms of STR length. Among the STRs that exhibited reporter activity, the majority demonstrated a positive correlation between repeat number and reporter expression. We additionally performed the experiment in multiple cell lines, and observe that some of the STRs are impacting gene expression in a celltype specific manner.

WILLIAM HULSY

REVELLE COLLEGE BIOLOGY WITH SPECIALIZATION IN BIOINFORMATICS MAJOR

PI: Chi-Hua Chen, Ph.D., UCSD School of Medicine, Department of Radiology

Analysis of Expression Patterns of Genes In Different

Locations of the Brain

In every region of the brain, we are able to detect different levels of gene expression, and understanding how these differing results form from the same genetic code is vital towards understanding of neurodevelopment. We propose that it is possible to detect differing patterns of the gene expression when traveling through the brain’s regions in an anterior-posterior or dorsal-ventral direction. By generating neural maps of fifty-eight different genes found throughout these regions, we are able to generate spatial correspondence between gene expression. Current open-source programs, such as the spin test, assist us by generating permutations and helping us understand the relationship between cortical datasets. Another consideration is how regions are affected by sets of two factors: one tends to influence over frontal and temporal regions, and the other which tends to influence over occipital and parietal regions, along with regions such as the precuneus which is influenced by both.

MAX GRUBER

PI: Christina J. Sigurdson, DVM,

PhD, Departments of Pathology and Medicine, UC San Diego

Mechanisms of Prion Protein-Induced Neurodegeneration in the Central Nervous System of Drosophila melanogaster

Prion diseases are fatal neurodegenerative disorders characterized by neuronal loss and the accumulation of prion protein aggregates (PrPSc) in the brain. PrPC, the normal cellular prion protein, has been implicated in binding oligomers, including PrPSc, that form in patient brains, leading to neurotoxicity. The mechanism by which PrPC interacts with PrPSc to cause neuronal loss is unclear. The 92N-PrPC point mutation may help to elucidate the neurotoxic pathway, as it has been shown to result in spontaneous neuronal degeneration in mice. We generated transgenic 92N-PrPC and wild type PrP-expressing Drosophila. Western blot revealed that the 92N-PrPC is expressed in flies. Lifetime and climbing assays were used to test behavioral differences. These experiments show the generation of a mutant prion protein expressing Drosophila , and future experiments will focus on assessing the behavior changes, histopathologic lesions, and toxic signaling pathways towards a goal of understanding how mutant prion protein induces neuronal death.

REVELLE COLLEGE NEUROBIOLOGY MAJOR MUSIC MINOR

WAI LAM "ADELE" HONG

PI: Andrew Muroyama, Ph.D., School of Biological Sciences, Department of Cell and Developmental Biology

Exploring the Role of Organelle Segregation during Asymmetric Cell Divisions in Arabidopsis thaliana

Asymmetric cell divisions (ACD) promote cell diversity by maintaining progenitor cells while producing differentiated daughter cells. In many systems, organelle segregation is essential for proper ACD, but it remains mysterious in aerial tissue. Our research explores the dynamics of organelle segregation in stomatal lineage cells from two angles. 1) We will identify genes required for successful organelle segregation in Arabidopsis thaliana by first screening atpiezo1 CRISPR mutants for stomatal defects, as AtPIEZO1 is necessary for proper vacuole morphology. Quantification indicates that atpiezo1 mutants have an increased stomatal density, suggesting that proper vacuole morphology is required for stomatal development. 2) To quantify how ACDs are affected by perturbations of organelle segregation, movies of mitochondrial inheritance in basl, mutants with perturbed ACDs, are analyzed. Future studies will elucidate mechanisms of vacuolar and mitochondrial segregation. Our findings provide mechanistic insight into stomatal development, with implications for the development of other plant tissues.

LEYI HUANG

PI: Jin Zhang, Ph.D., UCSD, Department of Pharmacology, Departments of Chemistry & Biochemistry and Bioengineering New Fluorescent Biosensors of Protein Kinase A activity

cAMP-dependent protein kinase (PKA) pathway is known to integrate diverse extracellular inputs and regulate various physiology functions through phosphorylating downstream signaling molecules. The development of biosensors made of fluorescent proteins (FPs) enables researchers to directly visualize signaling events and investigate their spatiotemporal regulation in living cells. FP based PKA biosensors, known as A-Kinase Activity Reporter (AKAR), serve as surrogate substrate of PKA and report PKA activity via fluorescence changes. The projects I am involved in focus on developing new AKAR with enhanced capabilities. Using biochemical techniques combined with live-cell imaging and analytical tools, I constructed and tested several FP-based AKARs. A novel single-fluorophore AKAR has been developed based on the brightest GFP, mNeonGreen. In addition, optimization of specific components of the biosensor has led to improved FRET-AKARs with enhanced sensitivity and selectivity. Here, we report the design and development of new single-fluorophore and FRET-based AKARs which enable sensitive and robust detection of subtle changes of PKA activity in living cells.

REVELLE COLLEGE GENERAL BIOLOGY MAJOR MARSHALL COLLEGE MOLECULAR & CELL BIOLOGY MAJOR

ZICHEN (CARDIFF) JIANG

PI: Ludmil B. Alexandrov, Ph.D., Department of Cellular and Molecular Medicine and Department of Bioengineering and Moores Cancer Center

Enrichment of POLD1 Mutations in Microsatellite Unstable Colorectal Cancers

WARREN COLLEGE BIOLOGY WITH SPECIALIZATION IN BIOINFORMATICS MAJOR COGNITIVE SCIENCE MINOR

Colorectal cancer (CRC) is the fourth most common and second deadliest cancer in the United States. Molecularly, CRC has been previously separated into ultra-hypermutated, microsatellite unstable, and microsatellite stable subtypes. Ultra-hypermutated CRCs exhibit better overall survival and they have been linked to mutations in polymerase epsilon (POLE). Microsatellite unstable CRC harbor deficiency in DNA mismatch repair genes and generally have good response to immunotherapy. Here, we report that microsatellite unstable CRC have an enrichment of missense mutations in the exonuclease domain of polymerase delta (POLD1). Specifically, 3.95-fold enrichment (p-value: 0.04) was observed in 559 whole-exome sequenced CRC from The Cancer Genome Project. This finding was externally validated using 1,645 panel sequenced samples independently generated by Memorial Sloan Kettering Cancer Center. This study characterizes the relationship between POLD1 and microsatellite unstable CRC. As next steps, we plan to further confirm the finding in additional publicly available CRC cohorts and evaluate the overall survival of patients with and without mutation in POLD1.

ARCHISHMA KAVALIPATI

PI:

Gene Yeo,

Ph.D, MBA,

Department of Cellular and Molecular Medicine, UC San Diego Understanding Regulators and Targets of Small Nucleolar RNAs Using Enhanced CLIP Data

ELISE KIM

SIXTH COLLEGE MOLECULAR & CELL BIOLOGY MAJOR MUSIC MINOR

PI: Robert Rissman, Ph.D., UCSD School of Medicine, Department of Neurosciences

SIXTH COLLEGE BIOINFORMATICS MAJOR COMPUTER SCIENCE MINOR

Small nucleolar RNAs (snoRNAs) are a class of noncoding RNA known to guide post-transcriptional modifications on other RNAs by complexing with RNA-binding proteins (RBPs) to form small nucleolar ribonucleoproteins (snoRNPs). Dysregulation of snoRNAs is implicated in cancer and neurological diseases— for example, in amyotrophic lateral sclerosis (ALS), a mutation in FUS dysregulates a subset of snoRNAs responsible for ribosomal RNA modification, hampering translation efficiency. To systematically study snoRNA biogenesis and function, I aim to build a framework that utilizes >200 publicly available enhanced crosslinking and immunoprecipitation (eCLIP) datasets to probe RBP binding sites on snoRNAs and identify potential snoRNPs using clustering analysis. To understand the mechanisms by which a RBP mutation leads to the dysregulation of a subset of snoRNAs, I identify RBPs and protein-complexes with enriched binding sites on these dysregulated snoRNAs. With this computational framework, we are able to better understand the biogenesis and function of snoRNAs.

JOSEPH KENNEDY

ROOSEVELT COLLEGE NEUROBIOLOGY MAJOR

SPANISH LITERATURE MINOR

PI: Terry Jernigan, PhD., UCSD Distinguished Professor of Cognitive Science, Psychiatry, and Radiology; Director, Center for Human Development; Co-Director, ABCD Study Coordinating Center

Vertex-wise Associations Between Cortical Morphology and Behavioral Problems in Adolescents

The brain undergoes dynamic changes throughout adolescence, and non-invasive imaging techniques can help probe these developmental processes. Magnetic resonance imaging provides the opportunity to measure how brain development is related to observable behaviors. However, few studies have applied vertex-wise analyses to probe these relationships within high dimensional data. In this study, we applied vertex-wise linear mixed effects models to test the association between emotional and behavioral problems and cortical morphology, on a longitudinal scale, in the Adolescent Brain Cognitive Development Study (ABCD Study®). We found regional patterns in associations when examining associations at the vertex-wise level, indicating potential avenues for future research, particularly in structural MRI metrics (e.g., cortical surface area and thickness) while diffusion MRI measures (e.g., restricted spectrum imaging) lacked associations of similar strength. This study highlights the importance of applying whole-brain models to test the associations between brain structure and adolescent behaviors.

Identifying Blood-Based Biomarkers in Down Syndrome-Alzheimer’s Disease (DS-AD) Patients

Down syndrome (DS) is associated with a high prevalence of cognitive impairment and Alzheimer’s disease (AD) dementia in middle age and older adults (DS-AD). Oxidative stress and changes in inflammatory tone may cause an imbalance in extracellular vesicle sorting, formation, and cargoes, which is hypothesized to be causal in the neuropathology observed in DS-AD patients. Unfortunately, an unexplored area is the detection of early AD neuropathology prior to the development of dementia in DS-AD. Thus, there is a need for developing reliable blood-based AD biomarkers for the DS population. We have used bio-banked samples from a previously conducted study, “Vitamin E in Ageing Persons with Down Syndrome” (Sano et al.,2016), in order to observe concentrations of AD biomarkers. We found that anti-inflammatory treatment with Vitamin E had no effect on changing levels of amyloid-beta or phosphorylated tau or cognitive change. Analysis of participant subgroups is underway to determine whether the biology of specific individuals makes them more suitable to anti-inflammatory treatment. We are using proinflammatory panels and hope that the results from this current study will allow us to design a more precision-medicine based model for treatment of DS-AD.

YANLIN LIU

MARSHALL COLLEGE NEUROBIOLOGY & CLINICAL PSYCHOLOGY MAJOR

PI: Yimin Zou, Ph.D., UCSD School of Biological Sciences, Department of Neurobiology

The Distribution of Excitatory Synapses in the Hippocampus of Prickle Mutation Mice Across Developmental Stages

Prickle1 (PK1) and Prickle2 (PK2) are core components of planar cell polarity signaling pathway and their mutations are associated with autism in human. PCP signaling pathway plays important roles in synapse formation and maintenance. We examined the synaptic puncta changes in the hippocampus of Prickle point mutation mice. Both Prickle1 (PK1) R104Q and Prickle 2 (PK2) E8Q point mutation mice have been generated in the lab. The mice brains across three age groups – young, P14, and adult, two-month and six-month old – were sectioned, stained for excitatory synapse formation with presynaptic marker (Bassoon), postsynaptic markers (PSD95, SAP102). Fluorescence images were acquired using the confocal microscope and the quantification and co-localization of the synaptic proteins were analyzed with ImageJ. We found changes in the number of excitatory synapses in the hippocampus of PK1 R104Q and PK2 E8Q homozygous mice when compared to the heterozygous and wildtype mice at different age groups.

JUSTIN LAM

PI: David Cheresh, Ph.D., Distinguished Professor, Vice Chair of Pathology, Sanford Consortium of Regenerative Medicine, Moores Cancer Center Investigating Cellular Stress Responses Mediated by STAT3 in Pancreatic Cancer

Signal transducer and activator of transcription 3 (STAT3) is a transcription factor reported to be involved with tumor initiation and progression. Additionally, STAT3 activation is associated with adaptive responses to stress commonly associated with the tumor microenvironment such as oxidative stress, hypoxia, and nutrient deprivation. Similarly, cellular stress have been reported to stimulate tumor progression. To investigate STAT3’s role in response to cellular stress, we engineered STAT3 knock-out (KO) cells using the CRISPR Cas9 system. Here we report the establishment of STAT3-KO clones generated in a pancreatic cancer cell line model. Furthermore, STAT3 depletion and their functional inactivation of STAT3 signaling was validated through western blot analysis. Though STAT3 is widely accepted as an oncogene, STAT3 targets genes involved in its adaptive stress responses remain poorly understood. These STAT3-KO clones will be instrumental for future studies investigating STAT3’s role in cellular stress responses and the identification of its downstream effectors.

ABBY LEE

REVELLE COLLEGE HUMAN BIOLOGY MAJOR PSYCHOLOGY MINOR

PI: Deborah Yelon, Ph.D., UCSD School of Biological Sciences, Department of Cell and Developmental Biology

Role of Twist1 Transcription Factors in Pectoral Fin Development in Zebrafish

Twist1, a basic helix-loop-helix (bHLH) transcription factor, is essential for normal limb development. Twist1 null mice die at embryonic day 11.5, and display unclosed cranial neural tubes, defective head mesenchyme and stunted forelimb growth. However, to the best of our knowledge, the role of twist1 genes in zebrafish fin development has not yet been investigated. Moreover, the embryonic lethal phenotype of Twist1 null mice make extended investigation of Twist1’s role in limb development challenging. In this project, we investigate the roles of twist1a and twist1b in the initiation and maintenance of proximodistal fin outgrowth and anteroposterior fin patterning. Thus far, our data indicate a previously unappreciated role of twist1 genes in maintaining the apical ectodermal ridge of the developing fin and thereby coordinating the precise spatiotemporal processes of growth and patterning.

SIXTH COLLEGE HUMAN BIOLOGY MAJOR PSYCHOLOGY MINOR

JINA LEE

PI: Joseph Pogliano, Ph.D., School of Biological Sciences, Molecular Biology

Identifying and Characterizing the Core Genome of the Nucleus-forming Bacteriophage Family

The Pogliano lab has recently discovered a unique replication mechanism in bacteriophages in which the viral DNA is sequestered within a virally-encoded compartment during infection. This structure is analogous to the eukaryotic nucleus and protects the phage from many bacterial defense systems. However, the molecular machinery underlying this replication mechanism remains poorly characterized. Through comparative genomics, we identified 72 genes that make up the core genome of nucleus-forming phages, 21 of which are unique to these phages, and all but one have unknown functions. To identify the function of these proteins, we utilized a catalytically inactivated CRISPR-Cas13 (dCas13) to selectively knock down phage proteins. When knocking down a major nuclear component, we saw an arrest at the earliest stages of infection through fluorescence microscopy and plaque assays. In the future, we will use this tool to characterize the function of unknown and unique core genes of nucleus-based phage replication.

MUIR COLLEGE BIOLOGY WITH SPECIALIZATION IN BIOINFORMATICS MAJOR

CAROLINA LOPEZ

MARSHALL COLLEGE

HUMAN BIOLOGY MAJOR PSYCHOLOGY MINOR

MARSHALL COLLEGE MICROBIOLOGY MAJOR GLOBAL HEALTH MINOR

PI:

Amy L. Non, Ph.D., UC San Diego, Department of Anthropology, Institute of Genomic Medicine, and Human Milk Institute Identifying the Association Between Maternal Perceived Stress and Human Milk miRNA Expression

Early life is a sensitive period of development during which stressful experiences of a mother may have long-term epigenetic consequences for her baby. MicroRNAs (miRNAs), short non-coding RNA sequences, function as post-transcriptional regulators of gene expression and are highly abundant in human milk. They can be transmitted to a baby’s gut via exosomes, where they are absorbed into the blood stream potentially affecting infants’ growth and development. We will analyze exosomal miRNA content in 100 frozen milk samples that have been previously collected from mothers who donated milk to the UC San Diego Mommy’s Milk Research Biorepository, along with perceived stress and mood data. MiRNA will be extracted and sequenced from exosomes isolated by size exclusion chromatography. We hypothesize that maternal perceived stress, anxiety, and depression will negatively correlate with miRNA expression and are working to investigate this association.

DAVID MA

PI: Daniel Hollern, Ph.D., Salk Institute, Department of Immunobiology and Microbial Pathogenesis Generating Murine Models with Autoantibody Responses using Recombinant E. coli

Despite mechanisms such as central and peripheral tolerance, autoreactive B-cells are able to escape these safe measures into circulating lymphatics. Autoantibodies against our own proteins have major consequences to health as seen in illnesses such as autoimmune disease and cancer. In cancer, despite prognostic capability and association, little is known about the downstream consequences of autoantibodies. To elucidate these autoantibody consequences, this project intends to generate mice models that have an autoantibody responses. By injecting mice with self protein-expressing E. coli alongside adjuvants, we intend to generate autoreactive B cell responses. We hypothesize that these models will develop self-antigen specific B cells. When this work is complete, I will have established a method for generating models of self-antigen specific B cells. Moreover, when my project is complete we can use these models to study auto-reactive B cells role in cancer progression.

RAMINA MORTAZAVI

MARSHALL COLLEGE NEUROBIOLOGY MAJOR

PI: Shelley Halpain, Ph.D., Department of Neurobiology, & Sanford Consortium of Regenerative Medicine

Characterization of ER F-actin in the Somatodendritic Compartment of Hippocampal Neurons and its Role in Ischemic Stress Proteinopathy Cellular Model

Filamentous actin (F-actin) is a cytoskeletal protein concentrated in dendritic spines – the postsynaptic specializations at neuronal glutamate synapses. However, when undergoing ischemic stress driven by over-activation of NMDA receptors, F-actin disassembles in the dendritic spines and reassembles within the dendritic shaft, a pro-survival mechanism known as actinification. Actinification is dependent on the actin nucleating protein inverted formin-2 (INF2), which can be both cytosolic and endoplasmic reticulum (ER) -resident. Therefore, to properly assess whether ER F-actin starts at the ER in the shaft and whether ER F-actin has a role in the NMDA-induced assembly and organization of somatodendritic F-actin, fluorescent genetic tools were used to either monitor the early phase of actinification or to prevent assembly of F-actin near the ER. Our findings show that ER F-actin is essential for actinification, suggesting that one important role of this pro-survival response might be to protect the ER during ischemic stress.

ALEX MENG

REVELLE COLLEGE HUMAN BIOLOGY & GENERAL LINGUISTICS MAJOR

PI: John Ravits, M.D., UCSD School of Medicine, Department of Neurosciences

Investigating Sigma-1 Receptor Abnormalities in an ALS-related TDP-43 Proteinopathy Cellular Model

Abnormal TDP-43 protein expression, indicated by nuclear depletion and hyperphosphorylated cytoplasmic aggregate in motor neurons, is a hallmark pathology of amyotrophic lateral sclerosis (ALS). The upregulation of ER chaperone protein sigma-1 receptor (S1R) in neurons is known to alleviate the neurodegeneration in ALS models. Studies have shown that S1R interacts with nuclear envelope proteins and co-localizes with RanGAP, a nuclear pore protein, but the implications in ALS are not fully understood. In this project, I intend to study how TDP-43 pathology affects S1R’s localization and interaction at the nuclear envelope using a wild-type TDP-43 over-expression cell model. Preliminary data shows that increases in TDP-43 protein decrease S1R protein expression level. I will further study S1R cellular localization using nuclear-cytoplasmic fractionation and its interaction with RanGAP using immunofluorescence microscopy and molecular methods. Finally, I will conduct a neuropathological analysis of post-mortem ALS tissue to confirm and expand my findings.

MARCUS SAWAMURA

REVELLE COLLEGE GENERAL BIOLOGY MAJOR BUSINESS MINOR

PI: Derek Welsbie M.D., Ph.D., Shiley Eye Institute, Department of Ophthalmology

Optimization of AAV production for Gene therapy

The use of recombinant adeno associated virus (rAAV) as a gene therapy vector has been rapidly increasing because of the ability to deliver genes with low immunogenicity and the ability to infect a wide variety of target cell types. Many labs and companies are producing rAAV, but the process is very tedious and comes with an expensive price tag. Here we described a process that streamlines the production of rAAV production. Moreover, by testing various transfection protocols we describe an approach to reduce AAV production costs while maintaining high titer. Specifically, one of the commonly-used transfection reagents is modified polyethylenimine (PEI). It is used because of its adaptability, reproducibility and scalability, but it is also prohibitively expensive. Chemical synthesis of a deaminated PEI derivative, believed to be an acceptable alternative, was unexpectedly poor at transfection and led to poor virus production. As an alternative, we identified a method to reduce modified PEI amounts and improve viral production.

ASHNA NISAL

PI: Joseph Gleeson, M.D., UCSD School of Medicine, Department of Neurosciences

Neural Tube Defects and the

role

of Folic Acid and Somitogenesis

Neural tube defects (NTDs) are developmental malformations caused by incomplete closure of the neural tube (NT), precursor of brain and spinal cord during development. It’s a well-established fact that sufficient periconceptional levels of folic acid (FA) significantly reduce the risk of NTDs. Nevertheless, the underlying mechanism by which FA alleviates the risk of NTDs still remains unknown. Our preliminary data suggests that mice treated with low periconceptional FA had embryos with fewer somites than normal FA condition. Therefore, we are investigating how FA affects the rate of somite formation and how this in turn may affect NT closure. We differentiated mouse Epiblast Stem Cells (mEPiSC), expressing Luciferase under the promotor for somite-clock regulating gene Hes7, into Presomitic Mesoderm (PSM) that segments to form somites. My preliminary shows that inhibiting FA pathway with methotrexate during the PSM induction increases the period of Hes7 cycles, suggesting that FA may affect rate of somitogenesis by slowing the somite segmentation clock.

WARREN COLLEGE NEUROBIOLOGY MAJOR BUSINESS MINOR

DEEVYA RAMAN

PI: Emily Troemel, Ph.D., UCSD School of Biological Sciences, Department of Cell and Developmental Biology

Investigating C. elegans Intracellular Pathogen Response Genes for Roles in Resistance Against Infection

Activating an immune response is critical for defense against infection. The Intracellular Pathogen Response (IPR) is an immune response induced by two natural intracellular pathogens of the nematode Caenorhabditis elegans: the Orsay virus and the Microsporidia species Nematocida parisii. The C. elegans IPR consists of ~80 genes, and mutants with upregulation of all these genes have increased pathogen resistance, but which specific genes promote resistance is unclear. Here, we used genetic crosses and RNA interference to investigate the highly upregulated pals genes (of unknown function) for possible roles in resistance to the Orsay virus and N. parisii, quantifying pathogen load by fluorescence in situ hybridization. While we have not yet found evidence that upregulated pals genes promote immunity, we are currently optimizing the pathogen load assays. With these improved assays we will continue testing pals genes, as well as non-pals IPR genes to determine which promote immunity.

REVELLE COLLEGE HUMAN BIOLOGY MAJOR PSYCHOLOGY MINOR

SRIMAYE SAMUDRALA

PI: Cristian Achim, M.D, PhD., UCSD School of Medicine, Department of Psychiatry

Immunophilin and GR Association in HIV+ patients with a History of Depression

Immunophilins (IP) FKBP51 and FKBP52 are chaperone proteins abundant in the brain as they respond to signals surrounding the glial neurons mediated by the glucocorticoid receptor (GR). They are regulators in Major Depressive Disorder (MDD) within the hypothalamic-pituitary-adrenal (HPA) axis. This negative feedback loop is the central regulator of the stress response in the body. Heat shock protein 90 (Hsp90) and FKBP51 IP suppress control glucocorticoid receptors (GR trafficking to the nucleus and downstream gene expression) allowing for GR to enter the nucleus to upregulate the FKBP5 gene during the stress response, which is correlated with depression. We are examining FKBP52 FKBP51 and GR receptor correlation in the frontal cortex and basal ganglia by using immunohistochemistry stainings in autopsy brain tissues from HIV+ patient autopsies. All of these patients have a diagnosis of having with or without a history of depression, anywhere from major depressive disorder to episodal depression and have a wide age range. By examining immunophilin interactions with the HPA axis, it we plan can be used to better understand specific HIV+patients cases with depression mechanisms of disease in depression and other mood disorders and identify potential therapeutic targets.

REVELLE COLLEGE HUMAN BIOLOGY MAJOR

ROOSEVELT COLLEGE MOLECULAR & CELL BIOLOGY MAJOR

CELINA SHEN

PI: Christina Towers, Ph.D.,

The Salk Institute of of Biological Studies, Molecular and Cell Biology Laboratories

Mitochondrial-Derived Vesicles in Cancer Cells

Healthy mitochondria are essential to cancer cell survival through functions such as biomolecule synthesis and cell signaling. Damaged mitochondria are degraded in lysosomes via autophagy and the products may be recycled. Several cancer cell lines are dependent on autophagy for survival. However, some autophagy-dependent cells can develop resistance to autophagy inhibition and instead utilize mitochondrial-derived vesicles (MDVs). MDVs are a recently observed method of mitochondrial quality control that shuttle oxidized mitochondrial proteins to the lysosome independently of phagophores which characterize canonical autophagy. MDVs are significantly upregulated in cells resistant to autophagy-inhibition, demonstrating the dynamic nature of these structures and their strength in compensating for the loss of core metabolic pathways. Despite these important functions, much has yet to be elucidated about MDVs, including their mechanism of formation. In our group’s studies, we investigate potential MDV machinery to better characterize their formation and release. For example, DRP1 is a GTPase responsible for mitochondrial fission events during mitochondrial autophagy (mitophagy), but genetic deletion of DRP1 does not inhibit MDV formation. These studies additionally improve methods of studying MDVs by blocking other mitochondrial quality control processes to pinpoint MDV functions and machinery in isolation.

DAWEI TANG

REVELLE COLLEGE NEUROBIOLOGY MAJOR

PI: Edward Callaway, Ph.D, Salk Institute for Biological Studies

Exploring the Selectivity to Visual Texture Coarseness in Mouse Visual Cortex

We aim to understand how visual cortex extracts and encodes information collected in retina and sent to the brain. Amongst the stimulus features that mice recognize, visual textures are not well studied. In contrast, there have been extensive studies of representations of spatial frequency (SF) and spatial phase which together contribute to texture discrimination. How do mice pool first order SF and phase signals to discriminate coarseness of visual textures? We hypothesize neurons in the mouse visual cortex can evaluate the SF distribution of visual stimuli, enabling them to respond selectivity to the coarseness of visual texture. Using in vivo electrophysiology with silicone probes and Gabor generated texture border stimuli, I investigate whether neurons in mouse primary visual cortex can recognize texture borders defined by differences in the distributions of SF, in order to advance our understanding of how coarseness perception in mice arises from compressing SF and phase signals.

ELEANOR TERNER

PI: Michael Perry, PhD, Assistant Professor, Department of Cell & Developmental Biology Characterization of Sexual Dimorphism in Distantly Related Insect Visual Systems

MEGHA SRIVATSA

WARREN COLLEGE

MOLECULAR & CELL

BIOLOGY MAJOR HEALTH CARE SOCIAL ISSUES AND PSYCHOLOGY MINORS

PI: Christopher Benedict, Ph.D., La Jolla Institute for Immunology, Center for Autoimmunity and Inflammation and Center for Infectious Disease and Vaccine Research

Analyzing the Spatial Distribution and Genomic Expression of CD4+ and CD8+ T Cells Within and Surrounding Sites of Murine Cytomegalovirus Infection

ROOSEVELT COLLEGE GENERAL BIOLOGY MAJOR ENVIRONMENTAL SYSTEMS MINOR

Insects are strikingly diverse yet adept at recognizing conspecifics. To signal one another, some species use elaborate cues such as bright colors or aerial courtships. Their visual systems are often optimized to detect these cues and can exhibit dramatic sexually-specific differences. We used the insect eye and wing as models for understanding how the genome encodes different morphologies between sexes. The insect compound eye contains different kinds of photoreceptors that allow for color comparisons and motion vision. The Perry laboratory used single-cell sequencing to characterize a specialized eye region in male house flies, Musca domestica, known as the “Love Spot,” and found that it is regulated by the gene doublesex (dsx). We utilized immunohistochemistry and CRISPR/Cas9 tests of gene function in distantly related species such as butterflies and honeybees to determine whether similar mechanisms underlie the production of sexually-specific morphologies and cell types within and across species.

Cytomegalovirus (CMV) encodes one of the largest known DNA viral genomes of ~230kB. A large portion of this genome has evolved to evade host immune responses. To determine the immunological spatial and molecular distribution of CMV infection in vivo, female B6 mice were infected with mouse CMV (MCMV) encoding a mCherry reporter. FFPE slides of infected tissues were prepared and immunostained with CD4+, CD8+, mCherry, and nuclear fluorescent markers to visualize the spatial dispersion of T cells using confocal microscopy. Immunostaining of samples with mCherry signals was optimized for subsequent spatial transcriptomic sequencing. Bulk RNAseq directly within and surrounding MCMV infected cells will allow for comparative analysis and identification of immunological gene expression induced by infection of different cell types in different tissues. Profiling T cell gene expression will help elucidate their effector functions, as well as identifying defense mechanisms employed by MCMV to evade immune responses.

ERICA XIONG

SEVENTH

COLLEGE MOLECULAR & CELL BIOLOGY MAJOR

PI: Jens Lykke-Andersen, Ph.D., Professor, UCSD School of Biological Sciences

The Role of the Sm Complex in TOE1 Recognition of U1 snRNA

Small nuclear RNAs (snRNAs) are central components of spliceosomes. Besides canonical snRNA genes, many snRNA pseudogenes in the human genome can transcribe into variant snRNAs. Our lab identified TOE1, a deadenylase that specifically trims 3’ ends of RNA Polymerase II transcribed canonical snRNAs to the mature length but does not process tested variant snRNAs. Previous research in our lab has demonstrated that the Sm complex is critical for TOE1-mediated snRNA 3’ end processing but whether TOE1 directly recognizes the Sm complex remains unknown. I purified recombinant Flag-tagged TOE1 and used an antisense oligo to isolate regular U1 snRNPs (small nuclear ribonucleoproteins) or U1 snRNPs deficient in Sm-complex assembly from human cells. I then performed TOE1 in-vitro processing on these snRNPs and performed 3’ end RNA sequencing. The results revealed that TOE1 processed snRNPs deficient in Sm-complex assembly significantly less than regular snRNPs, indicating that the Sm complex plays a direct role in recruiting TOE1 to process regular snRNA 3’ ends to the mature length.

CAITLYN TRUONG

PI: Laura E. Crotty Alexander, M.D., Division of Pulmonary, Critical Care, Sleep Medicine and Physiology, Department of Medicine, UCSD

Elevated Levels of Inflammatory Cells in Sputum and Nasal Cytology of Nicotine and Tetrahydrocannabinol (THC) E-cigarette Vapers

Identifying and understanding the effects of nicotine e-cigarettes and tetrahydrocannabinol (THC) vapes becomes increasingly critical as young adults continue to perceive these vapes as less dangerous alternatives to combustible tobacco and marijuana. Further, with the e-cigarette or vaping device use associated lung injury (EVALI) epidemic that primarily occurs in THC e-cigarette users, there is high concern that vaped THC causes lung inflammation. Here we assessed differences in inflammatory immune cells in the airways of e-cigarette users, THC vapers, dual users, and non-vaping/non-smoking controls. Nasal cytology and induced sputum samples were collected from nicotine and THC e-cigarette users and controls who underwent informed consent. Overall, all inhalant users showed elevated inflammatory immune cell levels relative to controls. Based on the observed macrophage, neutrophil, lymphocyte, and eosinophil levels observed in our nasal and sputum samples, inhalant users are at risk for inflammatory lung diseases, including pneumonia, pneumonitis, and potential carcinoma.

MUIR COLLEGE BIOLOGY WITH SPECIALIZATION IN BIOINFORMATICS MAJOR COMPUTER SCIENCE MINOR

ANGELA WANG

PI: Joseph Gleeson, M.D., UCSD School of Medicine, Department of Neurosciences

Contribution of Inherited Variants to Neural Tube Defect Pathogenesis Based on Whole-Exome Sequencing Data

Neural tube defect (NTD) is a developmental neurological disorder affecting more than 300,000 newborns per year worldwide. One of the most severe types of NTD is meningomyelocele, where sections of the spinal cord protrude from the back, not protected by the spinal column. Despite the severity of meningomyelocele, its cause is largely unknown. Thus, in my project, I aim to investigate the contribution of inherited mutations to the pathogenesis of meningomyelocele. With a large cohort of meningomyelocele probands with both of their parents’ whole-exome sequencing data, I developed bioinformatics pipelines to filter for inherited variants and aim to investigate how different types of mutations affect meningomyelocele pathogenesis. By uncovering the genetic contributions to meningomyelocele, I hope to lay the foundation for future discoveries on its prevention and treatment.

SIXTH COLLEGE NEUROBIOLOGY AND DATA SCIENCE MAJOR

ANNA WILKE

PI: Cory Root, Ph.D., UCSD School of Biological Sciences, Department of Neurobiology Intercalated Cells of the Amygdala: Insights into Connectivity and Behavioral Implications

The amygdala, a brain structure critical to emotion and olfaction, contains numerous subnuclei. One inhibitory neuron group, the intercalated cells (ITCs), is particularly understudied. ITCs specifically express FoxP2, allowing cell type-specific targeting via Cre recombinase. Using FoxP2 transgenic mice, we have revealed the connectivity and function of the ITCs. First, ITC input-output mapping identified multiple novel reciprocal connections with cortical, subcortical, basal, amygdalar, hippocampal, and thalamic structures. Given these connections, we hypothesized that ITCs may mediate amygdalar valence control by broadly integrating and disseminating information to and from these areas. To investigate the role of ITCs in olfactory aversion responses, we utilized chemogenetics to selectively activate or inhibit ITCs in response to a variety of neutral, appetitive, and aversive olfactory stimuli. Our results suggest that ITCs modulate innate olfactory behaviors, which could have broader implications and provide therapeutic strategies for anxiety and trauma-related psychiatric disorders.

SIXTH COLLEGE NEUROBIOLOGY MAJOR

ROOSEVELT COLLEGE NEUROBIOLOGY AND COGNITIVE SCIENCE

MAJOR, SPECIALIZATION IN MACHINE LEARNING & NEURAL COMPUTATION

MARSHALL COLLEGE NEUROBIOLOGY MAJOR PSYCHOLOGY MINOR

JINGYUE XU

PI: Ralph J. Greenspan, Ph.D., Kavli Institute for Brain & Mind, UCSD Department of Neurobiology Inhibitory Connections Underlying Trace Memory Dynamics in Drosophila Ring Network

In trace conditioning, a form of associative learning, a temporal gap separates the conditioned stimulus (CS) from the unconditioned stimulus (US). Working memory (WM) involvement is therefore implicated over this trace interval. In theory, strong inhibitory-to-inhibitory (I-I) connections enable long neuronal timescales and facilitate working memory retention. Rewiring of these I-I synapses in turn disrupts WM task performance (Kim et al., 2020). Visual trace learning has been observed in Drosophila , which occurs in correlation with the mobilization of R2 and R4m ring neurons in the central complex (Grover et al., 2022). We hereby examine the Drosophila connectomics and showed that R2 and R4m ring neurons, along with other ring neuron classes, are interconnected and GABAergic (Turner-Evans et al., 2020). We set up genetic crosses to selectively perturb neuronal excitability or block inhibitory signaling among clusters of ring neurons. Trace learning is retained after increasing excitability of R3 neurons, while other effects remain to be futher investigated. These results will contribute to the understanding of the network mechanisms of higher-level visual working memory in Drosophila

RUBEN YBARRA

PI: Jody Corey-Bloom, M.D., Ph.D., UCSD Department of Neuroscience Investigating Plasma Levels of Astrocyte-Associated Markers, Glial Fibrillary Acidic Protein (GFAP) and Chitinase 3-like 1 (YKL-40), as Potential Biomarkers for Huntington’s Disease

Huntington’s disease (HD) is a genetic, neurodegenerative disorder, caused by a mutation in the Huntingtin gene. While the length of the genetic mutation has been associated with symptom onset and severity, variability still remains. Identifying biomarkers may enhance our ability to estimate disease onset, assess symptom severity, and track therapeutic responses. Much of HD biomarker research has focused on plasma neurofilament light (NfL), a neuronal marker of axonal degeneration, which has been significantly correlated with estimated years to disease onset, while glial markers have seldom been studied. We compared plasma levels of two astrocyte-associated markers, glial fibrillary acidic protein (GFAP) and chitinase 3-like 1 (YKL-40), with levels of NfL, as well as cognitive measures, to determine their potential association with HD symptom onset and severity. We conclude that both plasma GFAP and YKL-40 are significantly, albeit moderately, associated with symptom presentation and have potential as peripheral fluid biomarkers in HD.

ROOSEVELT COLLEGE NEUROBIOLOGY MAJOR HISTORY MINOR

SAMANTHA YIP

PI: Nicola Allen, Ph.D., Salk Institute, Molecular Neurobiology Laboratory Delivering an Astrocyte Secreted Factor to Target Synapse Loss in Alzheimer’s Disease

Alzheimer’s disease (AD), one of the most common forms of dementia, is characterized by memory and cognitive deficits. Synaptic dysfunction is observed in the early stages of AD and has been correlated with memory loss in patients. Previous research has shown that astrocytes release proteins which aid in synapse maturation and stability, characteristics of synapses that are reported to decrease in AD. Therefore, we hypothesize that overexpressing these proteins in the astrocytes of TauP301S AD mice may help prevent or rescue the synapse loss and consequent memory loss observed in AD. We delivered a candidate factor to AD and wildtype mice using a viral vector approach and assessed its effects on learning and memory with behavioral tests. Upon treatment, the mice showed improved performances in memory tasks. We are now addressing the factor’s effects on a cellular level with immunohistochemical analysis of glia reactivity and pathological Tau accumulation.

HAORAN ZHANG

PI: Qingfei Jiang, Ph.D., UCSD School of Medicine, Division of Regenerative Medicine

Profiling and Interrupting the aberrant RNA Editing Activity in T-cell Acute Lymphoblastic Leukemia

T-cell lymphoblastic leukemias (T-ALL) generate aggressive leukemias during the malignant transformation of T-cell progenitors. Leukemia-initiating cells (LICs) are enriched in relapsed or refractory T-ALL with enhanced survival and self-renewal capacities. Adenosine deaminase acting on RNA 1 (ADAR1) is an innate immune enzyme responsible for adenosine-to-inosine (A-to-I) hyper-editing in immunogenetic double-stranded RNA (dsRNA). 70% of T-ALL patients exhibited the high aberrant expression of ADAR1 p150 isoform, and inhibition of this overexpression can impair the LICs’ stemness. In this honor thesis research, I investigated the function and mechanism of the aberrant ADAR1 in promoting the stemness of LICs and RNA hyper-editing targets in T-ALL, mainly using the RNA editing analysis pipeline. In addition, potential RNA editing biomarkers were discovered. Based on these results, I developed an RT-qPCR platform to detect the RNA editing biomarkers in T-ALL accurately. This researchhas implications for developing novel therapies and biomarkers for relapsed T-ALL.

SIXTH COLLEGE BIOLOGY SPECIALIZATION IN BIOINFORMATICS AND BIOCHEMISTRY/CHEMISTRY

MAJOR

QILIN ZHAO

PI: Brenda Bloodgood, PhD., School of Biological Sciences, Department of Neurobiology; Eugene Yeo, PhD., MBA., UCSD School of Medicine, Department of Cellular and Molecular Medicine

Medium Spiny Neuron Excitability in an Organoid Model of Huntington’s Disease Plant Circadian Clock Function

Huntington’s disease (HD) is a severe neurodegenerative disease caused by an increased number of CAG replicates in the huntingtin gene which leads to cognitive decline, loss of motor functions, and other detrimental symptoms. Although HD onset mostly happens after adolescence, there are neuropathological derangements that precede the symptoms. Some of the earliest derangements include medium spiny neuron (MSN) hyperexcitability and degeneration in the striatum. In this project, we developed a striatal organoid model of HD from patient-derived iPSCs to study such MSN dysfunctions. Using slice electrophysiology, glutamate assay, viral labeling, and immunohistochemistry, we demonstrated that there are mature MSNs in the striatal organoids, and the extracellular glutamate concentration positively correlate with the number of CAG replicates in the cell line. Our work shows that the organoid model resembles physiological and molecular changes observed in human HD patients and pave a pathway for future studies of HD mechanisms and drug development.

SIXTH COLLEGE BIOINFORMATICS AND COGNITIVE SCIENCE MAJOR COMPUTER SCIENCE MINOR

HEAD ADVISORS

James Cooke, Ph.D.

Assistant Teaching Professor of Neurobiology

Adriana Elaine Scott, M.A.

Biology Student Engagement Coordinator

FACULTY ADVISORY BOARD

Lisa McDonnell, Ph.D.

Chih-Ying Su, Ph.D.

Elsa Cleland, Ph.D.

Douglass Forbes, Ph.D.

REVIEW BOARD

Anna Wilke

Anjali Hamada

Stephanie Collin

Anjali Hamada

Heidi Wong

Arasu Arunachalam

Helen Bell

Selina Luo

Shreyas Sankaranarayanan

Martin Korec

Leonardo Gonzalez

Anisah Moutra

Alistair Russell, Ph.D.

Ashley Juavinett, Ph.D.

Sara Jackrel, Ph.D.

Samia Rahman

Joshua Jin

Denise Arroyo

Sriram Selvakumaran

Sujana Sreenivasan

Leanne Liaw

Karthy Sajeev

SPECIAL THANKS TO PATERA DESIGN

Photo by Bridget Spencer

EDITORS-IN-CHIEF

Lina Lew & Sharanya Sriram

EDITOR AT LARGE

Nicole Adamson

EXECUTIVE EDITOR

Anjali Iyangar

HEAD PRODUCTION EDITORS

Tania Gallardo & Amber Hauw

SPECIAL SECTIONS DESIGN EDITOR

Irum Aamer

RESEARCH DESIGN EDITOR

Jacqueline Cheung

RESEARCH EDITOR

Megha Srivatsa

RESEARCH OUTREACH DIRECTOR

Syni Solanki

RESEARCH FEATURES WRITERS

Leslie Ngo

Andres Mazimus Candeo Sandoval

Ari Brisco Schofield

Leanne Liaw

FEATURES EDITOR

Annika So

FEATURES WRITERS

Ananya Prasad

Qianqian Tao

Emma Chen

Amy Li

Anna Grinberg

BS/MS LIAISON

Anna Hakimi

STAFF

HEAD TECHNICAL EDITOR

Nicolas Bello

TECHNICAL EDITORS

Ariya Uyeno

Daniel John

Sarah Malko

Irene Tsaur

Maya Wallace

Ishrak Ramzan

Gulshanbir Baidwan

Megan Hackbarth

PODCAST MANAGERS

Christina Do & Soleil Madonia

WEB DIRECTOR

Caitlyn Truong

WEB ASSISTANTS

Katie Lin

Bernice Hwang

Ritviksiddha Penchala

ONLINE EDITOR

Chiraag Kambalimath

ONLINE REPORTERS

Vidisha Marwaha

Mansi Joshi

April Lin

Sarah Mirsaidi Madjdabadi

Jhanvi Nataranjan

BLOG EDITOR

Roxana Shahmohammadi

BLOGGERS

Jihyun In

Malleka Suy

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Snehita Vallumchetla

REVIEW BOARD MANAGER

Ava Selami

COMMUNITY

OUTREACH CHAIR

Amoolya Chandrabhatta

COMMUNITY

OUTREACH COMMITTEE

Marcella Ku

Mery Kaltakdjian

Sahiti Yenumula

Hannah Budroe

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HEAD ILLUSTRATOR

Kristiana Wong

ILLUSTRATORS

Taryn Cornell

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Angel Williamson

Abby Jones

Zaid Dibis

Hanh Nguyen

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HEAD PHOTOGRAPHER

Bridget Spencer

PHOTOGRAPHERS

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MEDIA DIRECTORS

Diana Presas-Ramos & Sevim

Bianchi

MEDIA TEAM

Chen Xiang

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