SQ Volume 19 (2021-2022) by Saltman Quarterly

Undergraduate Research Journal Volume 19 2021–22 sqonline.ucsd.edu

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.

FRONT COVER A mining bee, Andrena spp., on a blade of grass. Photo by Bridget Spencer.

INSIDE COVER A white-fronted bee-eater, Merops bullockoides, sitting on a branch at the San Diego Zoo. Photo by Andrea Farrell.

BACK COVER Ring-billed gull, Larus delawarensis, at La Jolla Shores Beach. Photo by Adamari Martinez.

GENEROUSLY UNDERWRITTEN BY

THE SALTMAN FAMILY AND SUPPORTED BY

LETTER FROM THE EDITORS

Dear Reader,

photo by BRIDGET SPENCER

Within the folds of this magazine lies the diligent collaboration of countless minds and a legacy that has been proudly celebrated for nearly two decades. Over the last three years of those two decades, we have had the pleasure to witness the resilience, grit, and co-operation that has allowed SQ to persist through these exceptionally dynamic years. Finding ourselves transitioning between different modalities of life over the course of this year has been a reminder of the dynamic nature of science itself. Whether it be public health policy changes backed by research, the continued surveillance of evolving variants of a virus, or the addition of experiments to a research project upon interpreting new data, it is fair to say that science thrives in the malleable state of flux that enables the collaborative art of discovery. Although the rejection of previous beliefs can be uncomfortable, refining hypotheses is essential for progress. In the midst of this pandemic, we found ourselves in need of honest, precise communication about COVID-19 vaccine development and public health research. Science communicators, then, must capture snapshots of not only the final product of research, but also the ever-evolving and interdependent nature of science that the scientific method

demands. In these periods of transit and turmoil, our collective humanity and willingness to collaborate have been the constants that ensure the delivery of in-depth science stories. From explaining the mechanisms behind bioluminescence to covering the invisible universe of marine microbes, this issue of SQ hopes to capture the expansive variety of research from the vast ocean to organisms invisible to the naked eye. From covering stories about marine compounds as a promising treatment for glioblastoma to the use of autopsies in search of a cure for HIV infection, this issue of SQ celebrates research that illuminates hope for better health outcomes from unexpected sources. Ultimately, Saltman Quarterly is a collection of the many bridges that connect the brilliant minds of researchers, writers, illustrators, designers, editors, reviewers, and every other valuable member of our community, coming together to play our small part to bridge the gap between science and humanity. With that, we present you with this token of our gratitude for the collaboration behind this publication. We sincerely hope you enjoy Volume 19 of Saltman Quarterly.

Warmly,

Anjali Iyangar & Nicole Adamson Editors-in-Chief, Saltman Quarterly 2021-22

TABLE OF

CONTENTS

FEATURES

An Invisible Universe by Annika So One "Last Gift" by Katelyn Nguyen Marine Microbes: The Eureka of Current Cancer Research by Emily White Plant Warfare by Kevin Martinez Shining Blue Stars in the Ocean by Angela Wang

S A LT M A N D E D I CAT I O N

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 not only to the student body at UC San Diego, but also to surrounding communities.

RESEARCH, BREVIAS, AND REVIEWS

SENIOR HONORS THESIS

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

The Path of Most Resistance: Molecule Equisetin Found in Marin Sponges Is a Possible Breakthrough Against Multi Drug-Resistant Bacteria by Akshay S. Bharadwaj Incorporation of Non-Canonical Amino Acids In Vivo Via The Quadruplet Codon System by Nandika Mishra Meta-analysis of the Pacific Oyster Microbiome: Characterizing Microbiome Environment Association and Core Tissue Microbiomes by Yash Garodia The Effect of the Amino-Terminal Fragment (ATF) on the Activity and Inhibition of Urokinase-Type Plasminogen Activator (uPA) by Harriet J. Song Identifying Drivers of Neuropathic Pain in Arthritis by Asim Mohiuddin

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STAFF

Meet the members of the 2021-2022 Saltman Quarterly staff who worked throughout the year to bring you the issue, as well as our online content, quarterly newsletters, and community outreach initiatives.

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DR. PAUL SALTMAN He viewed the relationship between t e a c h e r s a n d st u d e n t s a s sy m b o l i c , w i t h e a c h p a r t y "g i v i n g a n d sharing" to help the other reach their full human potential.

he early stages of the COVID-19 pandemic were filled with hope that the development of a vaccine would bring an end to the global crisis that has affected billions of people worldwide. In time, the pandemic has brought various health-related scientific and technological advances. However, despite the universal pain felt by most and the tremendous global effort to develop vaccines to assist in preventing the spread of the illness, many people continue to deny the ongoing risk of the pandemic or question the efficacy and safety of the vaccines that have been developed. The recent events that have unfolded are evidence of the multidisciplinary nature of the development and implementation of the vaccine; they require us to closely examine the relationship between science and society to not only provide an explanation for the persistence of the pandemic, but to bring the pandemic to an end. The social component of scientific and technological development requires effective education and communication efforts to deliver honest, thoughtful, and intelligible messages regarding the role of science in our communities, all of which are qualities embodied by Dr. Paul Saltman’s education philosophy and our work here at Saltman Quarterly. Dr. Paul Saltman earned a B.S. in Chemistry in 1949 followed by a Ph.D. in Biochemistry in 1953 from the California Institute of Technology. He completed his postdoctoral studies at the College de France in Paris, followed by professorship at the University of Copenhagen and Murdoch University in Australia. He then joined the teaching faculty at the department of Biochemistry and Molecular Biology at the University of Southern California’s Keck School of Medicine. Dr. Saltman joined UC San Diego as the provost of Revelle College in 1967, marking the beginning of the next 32 years of his life he dedicated to UC San Diego. At the time, UC San Diego was still a young university which, like Dr. Saltman, had a proven dedication to pushing the boundaries of the frontiers of science and education. Dr. Saltman later went on sqonline.ucsd.edu

to serve as the Vice Chancellor of Academic Affairs from 1972 until 1980, after which he ultimately returned to directly dedicate his full time to his passions, research and education, as a professor at UC San Diego. Dr. Saltman had earned the utmost respect from peers, students, faculty, and administrators alike. He charmed masses of students with his charisma and quick wit, both within and outside the classroom. For Dr. Saltman, teaching was a force through which he nurtured and cared. He viewed the relationship between teachers and students as symbiotic, with each party “giving and sharing” to help the other reach their full human potential. He believed that educators have a responsibility to be knowledgeable in their area of study and its relation to other disciplines, to appreciate the eclecticism of academica, present with humility and the absence of hypocrisy, and to encourage creativity and expression. Dr. Saltman placed high value in understanding the epistemology of academic science, thus he took great efforts to contextualize science and technology through an interdisciplinary approach to help the general public develop a more holistic view of the fields and their developments. As a professor at UC San Diego, he created a course series for non-stem majors to learn about the applications of science within a liberal arts context. Titled The Frontiers of Science, the series was developed to inspire students to analyze the intersection of current scientific and technological findings within the context of their personal, social, and cultural values.

Tr u e t o h i s values, Saltman Quarterly st r i v e s t o communicate cutting edge biological science and technology in an accessible manner . . .

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Additionally, as an educator Dr. Saltman was concerned with establishing quality scientific education for students and the general public outside the university. While serving on the National Science Foundation Committee for Science Communication, he established the Program for Teacher Enhancement in Science and Technology to help educate elementary and secondary school teachers on the current and recent frontiers of science. In 1999, Dr. Saltman was posthumously awarded Honorary Alumnus of the Year by the UC San Diego Alumni Association. Later that year, UC San Diego established the Paul D. Saltman Endowed Chair in Science Education to honor his legacy in science education within and beyond the UC San Diego community. In the lab, Dr. Saltman’s expertise was in nutritional science; he focused his investigations on the importance of trace metals, such as iron, copper, zinc, and manganese, in our diets. His efforts to translate academic nutritional findings from the lab to popular science for the general public to better their health and diets extended his influence as an educator well beyond the pages of academic journals. Dr. Saltman not only served on the editorial boards for various scientific journals, but actively engaged in an interdisciplinary approach to science communication. He wrote numerous articles published in popular science magazines and journals and authored The University of California San Diego Nutrition Book to help readers “make the best personal decisions about [their] diet[s].” His efforts to strive for accurate scientific communication and education is also notable in his dedication to debunking dietary myths in popular science. For example, with his scientific expertise he publicly argued that traditionally “unhealthy” foods such as twinkies, pizza, and hamburgers all offered health benefits when consumed in dietary balance with other nutritional necessities. In addition, Dr. Saltman featured in many interviews and talk shows, did a television series called “Patterns of Life” for National Education Television, and another for PBS. His dedication to science education led him to make significant contributions beyond the academic

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community by ensuring that the knowledge and scientific jargon was clear and accessible to the general public. In honor of Dr. Saltman’s dedication to accessible science knowledge and writing, Saltman Quarterly was established in 2004. True to his values, Saltman Quarterly strives to communicate cutting edge biological science and technology in an accessible manner while simultaneously supporting students’ creativity and expression within biology. Our popular science publications Under the Scope and SQ Online feature clear, nuanced, and thought provoking discussions about the interdisciplinary nature of recent developments in biology and science, as well as their positionalities within a sociocultural context. The Saltman Quarterly flagship publication, which you are reading right now, highlights student and faculty research at UC San Diego to encourage students to interact with and learn about creative and cutting edge biology research on campus. The community outreach and media teams help extend Dr. Saltman’s vision beyond the UC San Diego community. As a hub for discussion on the wondrous biological phenomena around us today today, Saltman Quarterly strives to learn from the community just as much, if not more than we share. Amidst the COVID-19 pandemic, it is crucial that we seek such discourse in equilibrium between teaching and learning to continue to adapt to a new normal.

WRITTEN BY MEGHA SRIVATSA Megha is a Molecular and Cell Biology student in Warren College. They are minoring in Psychology and Health Care—Social Issues. They will be graduating in 2024.

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FEATURES 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.

Vintage Wine flower, Echinacea purpurea, at the High Line park in New York City. Photo by Zina Patel sqonline.ucsd.edu

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illustrated by ANGEL WILLIAMSON

MILLIONS OF MICROBES ON A SINGLE SHELL by written SO ANNIKA

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features As new sequencing technologies emerge, scientists can characterize previously unknown microbes living on the shells of inside marine invertebrate hosts. UC San Diego Roy lab investigates how these microbial universes fluctuate with our ever-changing climate and what that means for their hosts and for us.

hile sunbathing or surfing at La Jolla’s beaches, you may find a diverse array of marine invertebrates, ranging from barnacles to periwinkles to mussels. You might have even gone tide pooling to see what marine life you can find. However, few give thought to the invisible, yet key players in the intertidal ecosystem—symbiotic microbes. Microbes associated with marine invertebrates, seemingly insignificant pieces of the marine ecological puzzle, still remain poorly studied due to limitations in technology. However, microbe-host interactions in vertebrate systems are increasingly well-characterized: in particular, we know that the human microbiome plays an essential role in our digestion, immunity, and stress mechanisms.1 However, the mechanisms by which the microbiome of invertebrates, such as mussels and sea snails, affects their host is not yet clear.2 Using what we know about microbe-vertebrate interactions, researchers can infer that microbes greatly influence the physiology and metabolism of marine invertebrates as well. In particular, it is important to explore how these microbial-invertebrate interactions are affected by environmental factors such as climate change.2 If environmental changes cause microbial systems to divert from their normal distributions, what would happen to their invertebrate hosts? All current models of ecosystem responses to anthropogenic, or human-caused, climate change only factor in plant and animal interactions, excluding microbial interactions. To create a more accurate model, we need to evaluate the ecosystem holistically by including how bacteria respond to climate change and other anthropogenic stressors. Otherwise, there is potential for errors in the predictions of cli-

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mate change effects on invertebrate hosts. This limitation of current models could lead to many consequences beyond the biology itself—for example, communities that depend on these fisheries for their livelihood could be negatively affected.

Biofilms: Microbial Cities

The unique composition of microbial species living in a region of an organism is known as a microbiome. In invertebrate organisms with a shell, one of the crucial microbiomes consists of a shell surface biofilm. Biofilms are a conglomeration of microbes bound by a slimy extracellular matrix that enables them to bind to foreign surfaces. When a bacterium adheres to a surface, it secretes a polymeric mucus that recruits other bacteria, protozoans, and even eukaryotes to adhere to the matrix and form a biofilm. Biofilm communities are crucial to the wellbeing of an ecosystem. Primarily, biofilms can protect microbes from environmental threats faced by free-living bacteria. Additionally, biofilms increase microbial access and absorption of nutrients. The invertebrate’s shell surface biofilm has a host-specific, unique microbial composition different from that in marine water and sand.This specificity of microbial composition is determined by various levels of host regulation, such as nitrogen excretion, oxygen metabolism, and symbiotic feedback of the microbiome feeding into its host.3 If some of these environmental conditions change, it is hypothesized that the microbes that depend on such aspects are also affected and subsequently react to the change. Dr. Kaustuv Roy’s lab in UC San Diego’s Division of Biological Sciences has investigated the biological and physical processes that generate large scale gradients in marine biodiversity for the past 30 years. Currently, the lab is investigating the impact of anthropogenic climate change on microbiome-invertebrate host systems using genomics, environmental data, and geographical mapping. Dr. Roy's work is focused on understanding how changes such as nutrient availability, temperature and pH affect the

distribution of the microbial communities growing on these marine invertebrates.

Latitude and Diversity

One way to probe these environmental factors is to investigate how microbe communities on marine hosts differ with geographical latitude. The latitudinal diversity gradient proposes that animal and plant species diversity is generally highest in the tropical areas and declines towards higher latitudes. To investigate whether the same is true for microbiome diversity, the Roy lab chose the invertebrate host model of Mytilus californianus, or the California mussel. This mussel has a large geographic distribution from Baja California to southern Alaska and is abundant in the intertidal zone, making it easy to find and sample. Additionally, marine bivalves such as mussels follow the common trend of higher diversity near the equator with a decline towards the poles. To identify whether the microbes on the host followed the same trend, the lab gathered biofilm samples by swabbing the gills and shells of the mussel at various latitudes ranging from Alaska to La Jolla.4 Generally, microbes in a large sample are identified by genomic sequencing. To do this, particular regions of the bacterial genome are compared across various species. Microbiologists commonly use the 16S ribosomal RNA (16S rRNA) gene sequence to identify bacterial species. As a component of ribosomal RNA, this gene is essential for microbial survival as it plays a crucial role in protein synthesis. For these reasons, the 16S rRNA gene is evolutionarily conserved throughout all prokaryotes. Specifically, it consists of highly conserved regions, shared across all bacteria, which are interspersed with variable regions that closely correlate with taxonomy grouping and can be used to identify particular species of microbes. Amplification and sequencing these variable regions can thus allow us to compare and identify the various microbes through sequence homology to databases of known species.5 SALTMAN QUARTERLY

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Figure 1.

The 16S ribosomal RNA sequences of microbes consist of conserved regions (grey) identical across all species interspersed with variable (colored) sequences that are used to identify species through bioinformatic analyses.

Though large-scale genetic sequencing is a powerful methodology to identify short sequences from a pooled sample, this technology has several drawbacks. Since databases of poorly studied and non-model organisms are incomplete, and only short segments are sequenced in 16S amplification, microbes can often only be identified to the phylum level. The Roy group gathered 103 California mussel shell and gill samples at various latitudes, generating more than 13,000 variable sequences. These sequences were analyzed against a database of the 16S rRNA sequences of known species, and via sequence homology comparison, the possible types of microbes that were present on the shell at various latitudes could be narrowed down and identified.4 The Roy group discovered that hostassociated microbes on mollusk shell surfaces do not follow the same latitude diversity gradient displayed by marine invertebrate hosts. In fact, the microbiome on the shell tends to display an inverse latitude diversity gradient, becoming more diverse in mid or high latitudes rather than further south. An increase in the species pool of free-floating bacteria in the ocean 8

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which correlated with an increase in latitude could explain the increased taxonomic diversity of the shell surface microbes. Additionally, other environmental factors such as temperature, ocean pH, and host characteristics—all of which change with latitude—could explain the microbial diversity trends. On the other hand, gill community diversity had no significant trend with latitude. These results suggest that environmental factors that change with latitude play a smaller role than local conditions, such as host physiology and diet, on the gill microbiome of the California mussel, while shell microbiome composition is regulated by a different set of variables.4

Time Will Tell

Additionally, there is a demonstrated need to understand microbial communities’ temporal responses to environmental changes in temperature and ocean pH, so we can predict how biological systems will react to future changes in each respective factor across the globe. Currently, it is known that some hosts have evolved a tight symbiotic relationship with their microbes. As the climate continues to

drastically change, this symbiosis creates possibility for co-extinction, where a host and its symbiont go extinct at the same time.2 As a result, in a process known as “dark extinction,” we might lose microbial diversity when a host goes extinct before being documented or studied. To address how microbial communities on hosts change as a result of local climate fluctuations created over large spans of time, the lab carried out a temporal study at the local La Jolla Shores beach. Researchers in Dr. Roy’s lab and at the Scripps Institute of Oceanography (SIO) analyzed the microbial diversity of samples of Donax gouldii, a small seawater clam, dating back to 2008. From the samples analyzed, it was concluded that microbial species diversity, quantified by the number of unique species present, was maintained following local environmental fluxes such as temperature changes and algal blooms. However, the identity of species constituting this constant level of biodiversity present on the hosts was completely different from year to year, with a constant turnover of microbes. Although the mechanism for how the clam keeps stable biodiversity is unclear, it is possible that the host metabolism, such as its immune system and space limitation, could be a factor.2

The Two Sides of Panama

Another experiment investigating the effect of the environment on microbe-host interactions examined microbial biodiversity in Panama, a prime location for marine ecologists because of its unique geography. In particular, the nutrient-rich Pacific Ocean and less nutrient-rich Caribbean are separated by the Isthmus of Panama, a land mass formed 3.5 million years ago between the two bodies of water. As a result, divergent evolution from the same parent species created invertebrate groups on either side of the isthmus that differed based on the oceanic environment they occupied. Samples from seven different marine snails were collected on opposite sides of the Isthmus of Panama, and the host-microbes were sequenced and identified. Surprisingly, microbial taxa associated with hosts do not show the expected pattern of divergence following the rise of the isthmus, unlike their hosts which sqonline.ucsd.edu

shared more recent common ancestors.6 Dr. Roy hypothesizes that because many marine host associated microbes tend to be geographically restricted, individual microbial species were not widely distributed across Panama even before the Isthmus formed. So when a land mass formed to isolate the Pacific and Atlantic oceans, the microbes associated with host populations on each side of the isthmus were already different. This study was a good example of how evolutionary divergence trends in eukaryotic hosts may not always align with those seen in their microbiomes.

More Questions than Answers

While the Roy lab has observed that various climate factors affect the microbiome, how specific factors impact microbe community composition remains to be explored. Through manipulation of temperature and pH in a tank, researchers can observe changes in invertebrate microbial composition in a controlled environment. Additionally, the Roy group may explore the impact of the structure, texture, and material of the shell on microbial biofilm formation. How do environmental

factors such as ocean acidification affect invertebrate shell surface biofilms? Working with other labs in the Jacobs School of Engineering and SIO, the group plans to investigate the effect of different shell materials and textures on biofilm formation. This research also has implications for marine fouling, where certain microbial colonization of boat and underwater pipe surfaces recruits organisms such as barnacles to attach themselves to these surfaces. To prevent biofouling, structures are often painted with anti-biofouling paint, commonly toxic to marine life. By studying the interaction between invertebrate larvae and microbes during biofilm formation, methods to prevent biofouling without marine toxins could be elucidated. The microbiome of an organism is a dynamic, ever-changing community, and we are just starting to explore how microbial communities associated with marine invertebrates function. Emerging technologies such as long-read sequencing platforms will soon allow scientists to sequence longer genomic segments to identify bacteria species with greater specificity, producing a finer insight into

how microbial communities change over time with respect to climate changes. As researchers grow more mindful of the importance of studying large timescale perturbations of climate, the Roy lab is archiving microbial samples in the university collection to provide resources for those who want to do a temporal study on mollusk microbiota a decade down the line. The microcosmos on the surface of marine invertebrates still has a wealth of information to divulge, and maybe it’ll be you who makes the next discovery.

References: 1. Carabotti, M.; Scirocco, A.; Maselli, M. A.; Severi, C. The Gut-Brain Axis: Interactions between Enteric Microbiota, Central and Enteric Nervous Systems. Ann Gastroenterol 2015, 28 (2), 203–209. 2. Neu, A. T.; Hughes, I. V.; Allen, E. E.; Roy, K. Decade‐scale Stability and Change in a Marine Bivalve Microbiome. Mol Ecol 2021, 30 (5), 1237–1250. 3. de Carvalho, C. C. C. R. Marine Biofilms: A Successful Microbial Strategy With Economic Implications. Front. Mar. Sci. 2018, 5, 126. 4. Neu, A. T.; Allen, E. E.; Roy, K. Do Host‐associated Microbes Show a Contrarian Latitudinal Diversity Gradient? Insights from Mytilus Californianus , an Intertidal Foundation Host. J Biogeogr 2021, 48 (11), 2839–2852. 5. Johnson, J. S.; Spakowicz, D. J.; Hong, B.Y.; Petersen, L. M.; Demkowicz, P.; Chen, L.; Leopold, S. R.; Hanson, B. M.; Agresta, H. O.; Gerstein, M.; Sodergren, E.; Weinstock, G. M. Evaluation of 16S RRNA Gene Sequencing for Species and Strain-Level Microbiome Analysis. Nat Commun 2019, 10 (1), 5029. 6. Neu, A. T.; Torchin, M. E.; Allen, E. E.; Roy, K. Microbiome Divergence of Marine Gastropod Species Separated by the Isthmus of Panama; preprint; bioRxiv.

Figure 2.

To examine the changes in shellfish microbiota composition over a decade, researchers swabbed the shell and gills of the Donax gouldii (small seawater clam). They observed fluctuations and turnover in species that occupied the host, although the overall diversity remained constant.

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ONE "LAST GIFT" Hidden HIV Reservoirs Uncovered

illustrated by

ABBY JONES 10

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written by

KATELYN NGUYEN sqonline.ucsd.edu

features

In 2017, Dr. David "Davey" Smith, an infectious disease physician at UC San Diego, and his research group launched the "Last Gift Study," an end-of-life study that recruits individuals living with both HIV and a terminal non-AIDS related illness to investigate and identify the hiding places of HIV before and after death.

ost people are familiar with the concept of hide-andseek, a popular children's game in which the “hiders” conceal themselves within a given environment and the “seeker” searches for these hidden players. Indeed, many of us have fond memories participating in this game in our youth. However, this game is not only played by schoolchildren in the comfort of their own neighborhood circles—there are examples of the “hideand-seek” tango in nature that determine the survival of both participants. With that in mind, consider this: what happens when the given environment happens to be our own bodies, and the “hiders” are deadly pathogens? 37.7 million individuals around the world are living with the Human Immunodeficiency Virus (HIV). Thanks to significant advances in medicine, a good number of these patients are able to live relatively normal lives. Nonetheless, scientists in the role of "seeker" continue striving to detect and identify where these potentially devastating "hider" virions are located.

Current HIV Treatment Options HIV poses a major threat to humanity, particularly to those in developing nations suffering high poverty rates, a lack of barrier contraceptives, and scarce medical interventions. There are three stages of infection: acute, chronic, and acquired immunodeficiency syndrome (AIDS). During the acute stage, where risk of transmission is highest, a large amount of HIV accumulates in the bloodstream. This causes occasional flu-like symptoms that align with the body’s natural response to infection by various pathogens. The chronic, or latent, stage is defined by little to no symptoms due to low levels of HIV replication. However, even if there is still a small amount of detectable virus in the blood, HIV is still infectious to other individuals, particularly during the exchange of bodily fluids such as blood or bodily secretions.

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AIDS, the final stage of HIV infection, is characterized by a severely damaged immune system that facilitates typically low-pathogenic microbes to take advantage of the host's impaired immunity, eventually causing additional infections. Fortunately, few individuals in the United States progress to this third and final stage due to the prevalence of antiretroviral therapy (ART), which has transformed HIV infection from a death sentence into a chronic condition. ART reduces the amount of HIV in the blood to undetectable levels, making it so that HIV+ individuals are no longer infectious to others. Though it is not a cure, ART does prevent HIV from replicating, locking the virus in its chronic stage. Despite the considerable success of ART, many challenges remain in developing additional therapeutics for HIV. Unlike acute infections such as influenza, HIV remains within people for their entire lives, even when not actively replicating. In particular, HIV searches for convenient pockets of the host’s body to hide in. One such location is brain tissue, presenting a dilemma to potential therapeutics because antiretroviral drugs cannot cross the bloodbrain barrier. Thus, for a cure for HIV to be developed, it will be imperative to eliminate these HIV reservoirs in patients before ART is ceased.1 However, since HIV is never truly removed from the hidden reservoirs in the body during ART, the virus rebounds rapidly if patients stop taking ART for even a short time, making it difficult for researchers to pin down where exactly HIV populates the body during the latent stage of infection. Thus, it is necessary to thoroughly analyze tissue samples from all over the bodies of HIV patients. This feat is only possible with full-body autopsies—an examinations of the body after a person has passed away.

The Last Gift Study

In response to this issue, Dr. David “Davey” Smith, an infectious disease physician and translational virologist at UC San Diego, embarked on a journey to find the hiding spots of dormant HIV in the body. This endeavor was inspired by one of Dr. Smith’s late patients who had regularly enrolled in various other HIV clinical trials until he became too ill to qualify for any more. Thus, the Last Gift Study was established as an ongoing collaboration of Dr. Smith, the National Institute of Allergy and Infectious Disease (NIAID), and the HIV Neurobehavioral Research Program in San Diego.2 The Last Gift Study consists of a clinical trial that enrolls and studies HIV+ patients without AIDS-related damage to

HIGHER CONCENTRATION

LOWER CONCENTRATION

Figure 1. Heat diagram of the body organs with the relative abundances of HIV virions. Red compartments (stomach, intestines, lymph nodes) indicate higher levels of HIV, while orange compartments (liver, brain, and kidney) have less viral load.

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1. HIV binds host receptors on T-cells or macrophages, fusing with the cellular membrane 2. HIV releases its dsRNA and protein contents into the cell cytoplasm 3. HIV dsRNA is reverse transcribed into dsDNA 4. HIV dsDNA enters the cells nucleus and integrates with host dsDNA 5. Recombinant host viral ds DNA replicates its genome and exits the nucleus 6. HIV self-assembles its virion from its replicated genome 7. The mature HIV virion buds off the cell membrane to infect other cells

Figure 1. Diagram of the HIV viral life cycle The cycle consists of HIV converting its double-stranded RNA (dsRNA) to double-stranded DNA (dsDNA) via reverse transcription, and integrating the dsDNA segment into the host genome.

their immune system, but with a separate terminal illness with a life prognosis of less than six months. The ultimate purpose of this study is to identify the hiding spots of HIV throughout the body to further prototype therapeutic interventions that may interrupt HIV migration from and around its hideouts in host reservoirs. While participating in this study, patients elect to provide tissue and blood samples from regions of the body that do not require invasive surgeries. These include gut and rectal biopsies—extractions of small tissue samples—as well as blood, genital secretions, and cerebral-spinal fluid samples. However, very little is known about HIV reservoirs in less accessible locations such as the brain. Therefore, patients in this trial also consent to a fullbody donation, and a rapid full-body autopsy is performed within six hours of the time of death to observe how HIV populates various tissues. As such, the samples collected before a patient’s death provide a baseline that can be compared against the post-death analysis of HIV replication. Most patients undergo ART during

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the study, though some have elected to cease treatment as they near the end of their prognosis. These incredibly personal decisions may provide researchers with additional insight on how HIV reservoirs change in those not undergoing ART. End-of-life studies have existed in cancer research for quite some time, but the Last Gift Study is the first of its kind for HIV-related research. One large benefit of these end-of-life studies is the perceived risk-benefit ratio—patients with terminal illnesses may be more willing to endure frequent tissue sample collections as a means of giving back to their communities, an enormously selfless decision. In addition, owing to their terminal conditions, such patients may be willing to test experimental therapies, which is not possible in those with HIV viral loads well-controlled by ART.

HIV Hiding Places Dr. Smith's team has determined valuable insights on HIV reservoirs which enable viral rebound after ART. Researchers mea-

sured the amount of HIV DNA specimens within the cells of a given blood sample and found that HIV was undetectable in the blood of patients taking ART.3 However, after death, HIV was detected in nearly all tissues. Because ART prevents HIV viral replication and ceases to function properly after a patient passes away, this result is not unexpected. More notably, researchers quantified higher HIV levels in the gut and lymph nodes than in kidney, brain, and liver tissues. This is expected, as macrophages and T-cells, or the immune cells which HIV infects, are more common in the gut and lymph nodes than in the brain. However, it is important to note that while HIV does not populate the brain to the same extent as it populates other tissues, the brain still remains a major hub for HIV latency. Microglial cells, the brain’s resident macrophages, act as a brain HIV reservoir.4 Since ART and other therapeutic drugs often have difficulty crossing the blood-brain barrier, understanding how HIV utilizes the brain

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as a safe hiding place is crucial to clear HIV from the body entirely. On another front, the Last Gift Study is careful to ensure that their research findings are not confounded by any comorbidities, or other medical conditions existing simultaneously within the same individual. For example, most HIV resides in the gut, but if an individual has colon or gastrointestinal cancer, investigations of the gut are better represented in other enrolled patients. Thus, this individual’s tissue may be more suited to study HIV in the brain. As a result, no one participant can contribute to the study of every tissue, but every patient can contribute something.

HIV Evolutionary Diversity The Last Gift study also contributes to explorations of HIV evolutionary diversity. During infection, HIV converts its RNA genome into DNA via reverse transcription and integrates these DNA segments into host genomes with the help of virus-encoded cleavage enzymes.5 The integrated HIV DNA segment then remains in host chromosomes for months to years, lying undetected by the host’s immune system. Simultaneously, the cell continues to produce HIV virions that replicate and infect various cells across the body. During each instance of integration, the host attempts to mount antiviral defenses by introducing mutations into HIV DNA segments during reverse transcription. As a result, a diverse HIV group of variants is created across all of the HIV-infected cells in the body.

The Future of the Last Gift Study Though the Last Gift Study is currently in its exploratory stage, Dr. Smith plans to implement various clinical trials that manipulate HIV to see if altering reservoirs across the body is possible. Eventually, Dr. Smith hopes to take the Last Gift Study further into testing possible therapeutics for HIV. In fact, CART-T cell therapy—a procedure in which immune cells are taken from patients, adapted to combat cancers, and subsequently reintroduced to the same patient—has been a main contender for HIV cure-related research. So far, the Last Gift Study has established that HIV replicates and hides throughout the body and at different levels. Genomic sequence analyses permit researchers to track HIV as it moves between tissues. Additionally, HIV viral rebound occurs when patients cease ART, sqonline.ucsd.edu

providing important insight as to how HIV might repopulate in the body. Though there is still much to be learned in the development of HIV cure-related therapies, with the advent of the Last Gift Study, humanity is many more steps ahead in this game of hide-and-seek. Perhaps one day, we can build a future where all of the hiding spots HIV takes advantage of are cleared from its threatening presence.

Refrences

WRITTEN BY KATELYN NGUYEN Katelyn is a Microbiology Major from Thurgood Marshall College. She will be graduating in 2022.

1. Richman DD, Margolis DM, Delaney M, Greene WC, Hazuda D, Pomerantz RJ. 2009. The Challenge of Finding a Cure for HIV Infection. Science. 323(5919):1304–1307. 2. Reardon S. 2018. Virus detectives test wholebody scans in search of HIV’s hiding places. Nature. 562(7728):472–473. 3. Chaillon A, Gianella S, Dellicour S, Rawlings SA, Schlub TE, Oliveira MFD, Ignacio C, Porrachia M, Vrancken B, Smith DM. 2020. HIV persists throughout deep tissues with repopulation from multiple anatomical sources. The Journal of Clinical Investigation. 130(4):1699– 1712. https://www.jci.org/articles/view/13481. 4. Wallet C, De Rovere M, Van Assche J, Daouad F, De Wit S, Gautier V, Mallon PWG, Marcello A, Van Lint C, Rohr O, et al. 2019. Microglial Cells: The Main HIV-1 Reservoir in the Brain. Frontiers in Cellular and Infection Microbiology. 5. Kirchhoff F. 2013. HIV Life Cycle: Overview. Encyclopedia of AIDS.:1–9.

Note From the Editors Dr. Smith is looking for student volunteers to assist with this project. Anyone interested in getting involved in the project should email Dr. Smith at d13smith@health.ucsd.edu.

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Marine Microbes illustrated by DIANA PRESAS-RAMOS

written by EMILY WHITE

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features A marine microbe Salinispora tropica produces the salinosporamide A proteasome inhibitor that exhibits potent anti-cancer activity targeted particularly against glioblastoma. The Moore Lab at UC San Diego currently researches salinosporamide A, opening up the potential of future proteasome inhibiting drugs.

eep in the Caribbean Sea, a small marine microbe known as Salinispora tropica coexists with its environment, relying on intracellular chemical communication to interact with neighboring microorganisms. Over centuries of evolution, this microbe has become reliant on certain specialized metabolites used for ecological interaction rather than organismal growth to potentially defend against microbial predators by inhibiting certain cellular components, as well as maintain homeostasis. Surprisingly, diseased cancer cells are reliant on similar cellular components to rapidly proliferate, and thus these microbial metabolites have the potential to inhibit these proliferative mechanisms. According to Dr. Bradley Moore of Scripps Institute of Oceanography and Skaggs School of Pharmacy, marine microbes serve as a promising source of biogenic molecules with varying biomedical applications, bolstered by the vast diversity and ingenious evolutionary mechanisms among the numerous marine microbiomes in the ocean. In particular, Dr. Moore has been exploring S. tropica's unique metabolic products to elucidate whether these compounds may serve as potential treatments against glioblastoma, a particularly dangerous form of brain cancer.

Current Glioblastoma Treaments Glioblastoma is the most common type of brain tumor, and accounts for almost half of all brain cancer cases. Glioblastoma forms in glial cells which are cells in the central nervous system (CNS) that support and protect neuronal structure, function, and wellbeing. Specifically, an extremely abundant form of glial cells known as astrocytes are essential in maintaining the proper function of neuronal synaptic connections, thus sustaining proper neuronal communication in the brain. Glioblastoma, in turn, forms from cancerous astrocyte cell groups and moves aggressively to infiltrate healthy astrocytes surrounding the tumors, disrupting the astrocytes' role in synaptic connection and impairing cognitive function. As in all forms of cancer, dysregulation of the normal cell cycle prevents the cycle

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from ending, causing excessive astrocytic growth in the form of glioblastoma.1 Anticancer drug therapies are promising options to treat various types of cancers, and effective chemotherapies remove tumors where surgical removal is not feasible. Glioblastoma cannot be removed surgically because the processes of cancerous astrocytes surround the endothelial cells of the blood brain barrier and branch out with their long tentacle-like extensions, making the glioblastoma located near the delicate vasculature difficult to remove. With a median patient survival time without treatment of six months from onset, and a survival rate of only 40% in the first year after diagnosis, alternative treatment options are clearly needed to address this illness. However, treating brain tumors involves a complicating factor: the bloodbrain barrier (BBB). Analogous to a security screener, the BBB is impermeable to large polar compounds, while selectively allowing lipid-based, water insoluble compounds to enter the brain. This feature protects the brain from harmful toxins potentially circulating in the bloodstream and vitally controls the compounds that can enter the brain from the bloodstream.2 However, the BBB’s selectivity prevents most anticancer drugs from entering the CNS due to their polarity and large size, which present a serious difficulty in treating glioblastoma with pharmaceuticals. With many current candidate anticancer drugs unable to pass through the barrier, there remains a need for drugs with disparate structures. Interestingly, the Moore Lab has found that such a biochemical solution may be within the deep-sea bacteria S. tropica.

The Unusual Structure of Salinosporamide A S. tropica belongs to the genus Salinispora discovered off the Bahamas by UC San Diego Professors William Fenical and Paul Jensen, who also pioneered the discovery of salinosporamide A compound. This microbe is part of a marine bacterium family called actinomycetes. These filament-shaped microbes contain specialized metabolites

that serve as appealing pharmaceutical candidates. These specialized metabolites are not essential for organismal growth and reproduction, and instead are critical to the bacterium's environmental response mechanisms, such as adaptation and defense systems.3 After genomic sequencing of S. tropica, various chemical laboratory techniques revealed unique biochemical structures that fascinate the Moore group; however, the specialized metabolite toxin known as salinosporamide A poses particular promise to anticancer drug therapy research. Salinosporamide A has potent proteasome inhibitory activity, causing a chain of chemical reactions that can ultimately result in potent anticancer activity and use as an anticancer drug. The proteasome is a large protein complex that regulates protein deterioration, with the ability to promote apoptosis, or programmed cell death. When cellular proteins misfold or become damaged, the enzyme ubiquitin ligase attaches a ubiquitin protein tag to the damaged protein, signaling for its degradation by the proteasome. Oftentimes, the damaged proteins are hydrolyzed into their amino acid building blocks which can be used to make new, healthy proteins.4 However, cancerous tumors often form large amounts of damaged proteins that are tagged and broken down in the proteasome, allowing them to be reused and contribute to effective cancerous growth. This results in greater proteasome activity in cancerous cells, and this greater activity is crucial to the cancer proliferative mechanism.5 When salinosporamide A binds to and inhibits proteasome 20S in astrocyte cells, protein recycling is blocked, allowing the cell to apoptose and thus preventing excessive cancerous growth with relatively minimal cytotoxicity in healthy cells. While this inhibitory regulation mechanism seems straightforward, the process is complex and relies on the unusual chemical structure of the salinosporamide A.

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Figure 1. The 20S Proteasome

The proteasome, shown above with 19S subunits above and below the 20S subunit, degrades damaged proteins tagged by ubiquitin so they can be recycled into amino acids and used for future protein synthesis.

There are several biochemical characteristics of salinosporamide A that enable its proteasome regulation effects. The presence of cyclohexenylalanine, a six-membered carbon ring with amine and carboxylic acid groups, enables salinosporamide A to react with the 20S proteasome by positioning it for proper binding. Another vital structural component of salinosporamide A is a beta lactone, or a square-shaped heteroring composed of three carbons and an oxygen attached to an additional carbonyl group, an oxygen double-bonded to a carbon. This beta lactone has similar features to beta lactam rings, which are found in the structures of many antibiotics. A nucleophilic threonine residue hydroxyl in the proteasome attacks the electron deficient beta lactone carbonyl group, which after a cyclic rearrangement, creates a permanent bond between the proteasome and the lactone. Finally, a chlorine atom on one of the salinosporamide A side chains displaces to allow the beta lactone ring, after being opened by the threonine residue nucleophilic attack, to cyclicize, thus giving salinosporamide A the structural space to bind to the proteasome irreversibly. This stable bonding enables the metabolite to hold a longer residence time on the proteasome than other proteasome inhibitors, a quality crucial to its application in a pharmaceutical setting.6 Unlike many other pharmaceutical molecules, salinosporamide A can permeate through the BBB by facilitated transport, or through se16

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lective channels spanning across the BBB. The Moore Lab determined that the structure of salinosporamide A mimics that of an amino acid due to its small, cyclic, and folded protein-like features. As such, it is classified as a nonproteinogenic amino acid since it contains an amine group and a carboxyl group connected to an organic carbon chain. This nonproteinogenic distinction comes from it being structurally distinct from the twenty-two amino acids created naturally from the genome. Amino acids can be transported across the BBB by facilitated transport, and thus salinosporamide A's structural similarity to endogenous amino acids permits it to disguise itself as an amino acid and enter the CNS. However, how does S. tropica create salinosporamide A without damaging its own proteasome? Normally, S. tropica produces both the proteasome inhibitor salinosporamide A and two copies of a proteasome during its natural metabolic processes. While this may seem counterintuitive, the bacterium evolved to introduce a mutation in the PSMB5 gene that allows one of the two co-synthesized proteasomes to retain functionality despite the existence of the toxin salinosporamide A. Interestingly, this fact may also shine light on ways cancer cells might resist treatment with salinosporamide A. As more drugs are created to treat various diseases, evolutionary pressure drives biological components targeted by these drugs to evolve mutations that resist pharmaceutical treatments, resulting in resistance to these drugs accumulating over time. Proteasome resistance appeared in patients taking Bortezomib, the

first proteasome inhibiting drug approved by the Food and Drug Administration for treatment of multiple myeloma. Although poorly understood, the resistance of this drug involves mutation in the gene PSMB5, coding for the subunit beta 5 of the 20S proteasome, which is partially responsible for breaking down proteins. Surprisingly, these mutations are similar to those in S. tropica, and occurs in the same location on the PSMB5 gene as the acquired resistance to Bortezomib in humans, suggesting a similar association between the drug resistance in humans and the evolutionary resistance in S. tropicas’ redundant proteasome.7 With further scientific exploration into how the secondary proteasome interacts with salinosporamide A and functions within the greater S. tropica proteasome complex, the mechanics of resistance to proteasome inhibiting drugs may become better understood in order to combat the growing health concern.

Synthesizing Salinosporamide The many unique characteristics of salinosporamide A make it a viable pharmaceutical inhibitor qualified for further research. The Moore Lab now organically synthesizes the molecule, as opposed to deriving it from the S. tropica organisms' natural metabolic processes. Now that mass production is viable, the compound has been clinically denoted as Marizomib, an oral anticancer drug currently in stage three clinical trials at UC San Diego for treatment against glioblastoma. The Moore Lab is currently exploring how the chemical structure is formed biologically, specifically the unique beta lactone ring. Kate Bauman, a graduate student heading the project on salinosporamide A in the Moore Lab, recently identified a novel enzyme that S. tropica utilizes to form the beta lactone, as well as a gamma lactam, or a pentagon ring structure. While necessary but not directly responsible for the inhibitory properties of the toxin mentioned earlier, the gamma lactam is particularly difficult to artificially synthesize, making the enzyme’s production of it especially notable. This enzyme is responsible for the final chemical assembly of salinospora-

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mide A, and the use of this enzyme in the lab may replace the need to synthesize the molecule through artificial organic methods. The structure of the beta lactone and gamma lactam require a complicated and expensive synthesis process that produces small yields of Marizomib, creating limited access for salinosporamide A production. Using the natural enzyme to form this compound will cut down the economic and environmental costs of its production, allowing higher yields and accessibility to the compound. Additionally, the ability to synthesize beta lactone and gamma lactam will enable the creation of a whole class of salinosporamides, each with their own slight variation in chemical structure. Specifically, a salinosporamide that is not naturally made could be tailored to inhibit the proteasomes of particular diseases with increased proteasome activity, for example non-glioblastoma cancers. While the impact of this enzyme discovery on Marizomib is yet to be revealed, there certainly is a multitude of potential applications in utilizing the drug to achieve proteasome inhibition. With this major breakthrough, Dr. Moore is hopeful that glioblastoma treatment could be just one application for salinosporamide molecules. The potential of proteasome inhibition extends to the multiple types of proteasomes other than the 20S protease involved in glioblastoma; other potential targets include immunoproteasomes, which are associated with neurological diseases such as Huntington’s Disease. Additionally, infectious diseases such as tuberculosis could be affect-

ed by proteasome misregulation. Clearly, further research is essential to revealing the numerous applications of proteasome inhibition in current medicine. The wide possibilities of proteasome inhibitor treatments provide a glance into the potential of natural product research. If just one toxin from a tiny microbe living at the bottom of the ocean can provide such promising treatment against brain cancer, imagine how many other organisms hold the secret to curing diseases within their unique molecular structure and fascinating biosignaling pathways. With time, and additional scientific innovation, exploration, and research, perhaps we will become accustomed to searching for solutions to humanity's most daunting health challenges in the hidden science of the natural world.

Resistance. Journal of Medicinal Chemistry. 55:10317-10327. 6. Feling RH, Buchanan GO, Mincer TJ, Kauffman CA, Jensen PR, Fenical W. 2003. Salinosporamide A: A Highly Cytotoxic Proteasome Inhibitor from a Novel Microbial Source, a Marine Bacterium of the New Genus Salinospora. 2003. Angewandte Chemie International Edition. 42(3):355-357. 7. Niewerth D, JAnsen G, Riethoff LFV, Meerloo JV, Kale AJ, Moore BS, Assaraf YG, Anderl JL, Zweegman S, Kaspers GJL, Cloos J. 2014. Antileukemic activity and mechanism of drug resistance to the marine Salinispora tropica proteasome inhibitor salinosporamide A (Marizomib). Molecular Pharmacology. 86(1):12-9.

References 1. Wirsching HG, Galanis E, Weller M. 2016. Glioblastoma. 134:381-97. 2. Mikitsh JL, Chacko AM. 2014. Pathways for Small Molecule Delivery to the Central Nervous System Across the Blood-Brain Barrier. Perspectives in Medicinal Chemistry. 6:11-24. 3. Jensen PR, Moore BS, Fenical W. 2015. The Marine Actinomycete Genus Salinispora: A Model Organism for Secondary Metabolite Discovery. Natural Product Report. 32(5):738-751. 4. Adams J. 2003. The proteasome: structure, function, and role in the cell. Cancer Treatment Reviews. 29(1):3-9. 5. Kale AJ, Moore BS. 2012. Molecular Mechanisms of Acquired Proteasome Inhibitor

WRITTEN BY EMILY WHITE Emily is a Molecular and Cell Biology/Public Health Major from Seventh College. She will be graduating in 2024.

Figure 2. Salinosporamide A Chemical Structure

The chemical structure of salinosporamide A, including a cyclohexenylalanine, beta lactone, and chlorine atom, allows it to act as a proteasome inhibitor with potent anti-cancer activity. sqonline.ucsd.edu

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PLANT WARFARE PERCEPTION & DEFENSE AGAINST INSECT ATTACKS written by KEVIN MARTINEZ

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illustrated by KRISTIANA WONG

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features Plants have specialized molecular mechanisms that allow them to recognize and respond to herbivory by releasing volatile plant hormones that attract predators and stunt the growth of the feeding insects. The discovery of this mechanism by Dr. Eric Schmelz has opened doors for the future of crop protection, given that the use of Bacillus thuringiensis transgenes is quickly becoming less effective.

nlike mammals, plants cannot run away when threatened by a feeding pest. Although they cannot move, plants interact more with their ecosystems than you might expect. While humans protect their livelihood with warfare, one way plants protect themselves from being eaten is by releasing volatile compounds into their surroundings to attract defenders, such as parasitoids and predatory insects, to attack the damaging herbivores. Without a nose to smell or eyes to see, plants need specialized perception systems to specifically recognize when an insect takes a bite. Over the course of human civilization, we have gone to great lengths to improve crop production; however, many modern year-round crops remain susceptible to pest attack. One method developed to protect plants from generalist pests is the introduction of bacterial Bacillus thuringiensis (Bt) transgenes that encode an insecticidal crystal (Cry) protein. There are many variations of Bt genes that encode diverse Cry toxins, each producing a variant toxin that targets different insects. Though this approach has successfully protected crops for some time, wide scale use of Bt genes created intense selective pressure that drives generalist insects to evolve adaptations to evade Cry toxin poisoning. As a result, our use of Cry toxins is at risk of becoming obsolete, rendering many crops vulnerable to insect attack. To combat pest evolution, researchers ventured to investigate whether naturally occurring plant defense mechanisms can be better leveraged to combat crop pests. A better understanding of dynamic plant defenses may lead to novel ways of effectively protecting crops in the future with diverse plant treatments.

Dr. Eric Schmelz, Professor of Cell and Developmental Biology at UC San Diego, in conjunction with Dr. Adam Steinbrenner and Dr. Alisa Huffaker, have recently identified a legume molecular recognition mechanism for insect herbivory.3 This molecular recognition mechanism is mediated by a particular plant receptor that binds to a specific plant protein fragment elicitor.3 Elicitors are compounds commonly found in caterpillar oral secretions deposited on the plant leaves during leaf digestion. When the plant receptor is activated by such an elicitor, this receptor stimulates the plant to synthesize and secrete many plant defense compounds. In bean plants, precisely defined oral secretion elicitors trigger plant volatile emissions.4 These emissions attract parasitoid wasps that use attacking caterpillars as hosts for their developing young. Therefore, through the oral secretion-stimulated production of volatile defense biochemicals, plants signal that their leaves are being chewed on and recruit natural enemy bodyguards that eliminate diverse groups of generalist pests. The ability to precisely identify elicitor molecules that activate plant signaling cascades was crucial to understanding

plant perception of insect herbivores. Dr. Schmelz’s contributions toward understanding the molecular cascade started in 2006 when he reported that cowpea plants recognize protein fragment elicitors derived from ATP synthase in fall armyworm oral secretions and initiate the rapid production of defense-related phytohormones, or plant hormones. The biochemical elicitors present in attacking herbivores, and perceived by plants, are the products of an ancient yet ongoing evolutionary arms race. The contents of insect oral secretions depend on their diet, yet bean plants have evolved adaptations to perceive highly conserved elicitor fragments from insects that vary very little across diverse plant groups.2 The cowpea plant’s response to herbivory and the content of herbivore secretions were characterized by capturing the released volatile biochemicals in a solvent, and upon heating and volatizing that

1. Caterpillar creates oral secretion during leaf digestion 4. Recrution of natural enemy bodyguards

2. Inceptin binds to plant receptor

Response to Attack In 1990, scientists noticed that both corn and bean plants release distinct volatile odors minutes to hours after an insect takes a bite from them. Subsequent studies revealed that nearly all plants respond to herbivory by synthesizing and emitting volatile biochemicals, yet how insect attack was perceived remained unclear.

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3. Synthesis of molecules, volatille emissions

Figure 1: Overview of plant perception of herbivory. This process includes recognition of inceptin in herbivore saliva via receptor binding on the plant surface, production of plant hormones and their release to attract predators.

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solvent, deciphering the identities of the trapped biochemicals within. Other separation assays of insect oral secretion samples were used to identify the elicitors capable of triggering plant volatile hormone production. The Schmelz lab isolated the protein fragment inceptin from insect oral secretions, a protein derived during the decomposition of plant chloroplastic ATP synthase protein subunits. Inceptin contains a highly conserved amino acid motif present in nearly all green plant tissues. Thus, when armyworms chew on such green leaves, trace amounts of inceptin from the gut of the herbivore are released onto the wound surface, which activates a receptor that subsequently stimulates the synthesis of phytohormones like ethylene, jasmonate and salicylic acid. Each class of phytohormones then activates transcription factors that initiate the transcription and translation of various enzymes. These enzymes not only produce volatile biochemicals that attract predators but also anti-nutrients that reduce caterpillar growth rates.

Adaptation Arms Race Over the years, several other elicitors were identified, which hints to a potentially vast diversity among plants’ pest-perception mechanisms. On one side of the battlefield, plants are evolving to recognize diverse compounds present in pest oral secretions, as evidenced by plant species recognizing molecules specific to arthropods. This recognition is derived from millions of years of plant-herbivore interactions driving the evolution of plant resistance. However, herbivores are similarly engaged in their side of the evolutionary arms race.4 Specifically, herbivore evolution is driven by the significant selective pressure to create slight chemical changes in the oral secretion composition, enabling the pest to evade plant perception. Tracking this prediction, Dr. Schmelz identified a truncated and inactive form of

1. Mutation induced 2. Response recorded

3. Loci identified 2__09070 2__22561

2_22560

219700 Vigun07g219600

Figure 2.

Plant response to inceptin-like protein coupled with Quantitative Trait Locus studies to identify possible genetic locations for the inceptin receptor. The region with the highest scores on the graph indicates a chromosomal location most likely to be associated with the receptor. 20

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inceptin that dominates in the oral secretions of the legume-specializing pest, the velvetbean caterpillar.1 The altered inceptin elicitor containing a single amino acid loss still binds to the receptor yet fails to activate plant defense responses. Similar to a camouflaged attacker who uses elements of their environment to hide in plain sight, velvetbean caterpillars hide from bean plants by evading oral secretion recognition with altered inceptin elicitors. Thus, while plants are evolving methods to identify diverse pest oral secretions, pests themselves are evolving methods to hide from plant defense systems. This finding defined the role of molecular elicitors in triggering plant volatile emissions as adaptations evolved in the specialist pest velvetbean caterpillar to escape plants’ inceptin perception mechanisms. However, the underlying receptors on the plants involved in eliciting the defense mechanism itself remained unclear.

The Mysterious Receptor By 2015, Dr. Schmelz and many other scientists were developing refined models of how herbivore-associated elicitors in oral secretions activate plant defense outputs. However, these models lacked the precise identity of the plant cell-surface receptor that binds inceptin. To identify this receptor, Dr. Schmelz and his team devised an assay to screen diverse bean germ cells for detectable phytohormone release in response to an inactive form of inceptin. This assay aimed to address whether wild plants are regaining ground in the evolutionary arms race against evolving pest oral secretions resistant to plant detection. The assay consisted of treating leaves with either water, inceptin, or inactive inceptin, and measuring defense responses to each. It was hypothesized that inactive inceptin would only elicit a plant defense response if the plant acquired mutations to allow it to detect this modified inceptin variant. Small yet reproducible plant responses were detected following application of the altered inceptin, suggesting that variation in the receptor gene could evoke variation in the plant elicitor's perception.

Answers in Legume Genomes Once the model of herbivore perception by plants was validated, the next step was to locate the specific genetic locations, or loci, on the bean chromosomes that might contain inceptin receptor (INR) candidates. Following the careful analysqonline.ucsd.edu

sis of 400 different bean plant lines and over 2000 biochemical samples, Dr. Schmelz and his group used both a Genome Wide Association Study (GWAS) and a separate Quantitative Trait Locus (QTL) mapping study to identify chromosome positions that were statistically associated with the rare positive plant elicitation responses to the inactive inceptin variant.2 QTL mapping is a statistical description of the association of a chromosomal region with the phenotypic trait data, while GWAS involves looking at several plants of the same species and deciding which traits may be associated by genetic variation. Both mapping approaches identified the same shared genetic position associated with antagonist responsiveness located inside the coding region of a receptor-like protein candidate, termed the inceptin receptor (INR). To understand whether the INR candidate interacts only with herbivory-associated molecules, an experiment was designed to compare INR’s mediated response to inceptin versus INR’s mediated response to a bacterial derived pathogen elicitor. Transgenic expression of INR in non-native tobacco plants demonstrated that plants transiently expressing INR proteins respond to inceptin, while bacterial elicitors did not activate any INR response, showing how such bacterial elicitors require different receptors. Therefore, newly imparted defense phytohormone release was only detected following inceptin treatment and predictable interactions with the INR. Having identified the INR, the Schmelz group next investigated whether the INR gene is unique to cowpea and bean plants, and whether there are variants of the receptor conferring the same defense response in other species.2 To determine this, they looked at shared genetic loci of different related legume plants with additional conserved marker genes nearby. It was found that only legumes with predicted INR genes responded to inceptin elicitation, while when expressed in tobacco, only candidate INR transgenes with more than 90% amino acid similarity produced defense responses. Closely related key crops such as soybean lack predicted functional copies of INR, and subsequently lack inceptin responses. Why soybean plants lost this perception mechanism remains a mystery. Given that INR expression equipped plants with the ability to recognize attack, Dr. Schmelz examined the ability of dissqonline.ucsd.edu

tantly-related plants to acquire inceptin perception. Stable transgenic tobacco plants expressing INR demonstrated that the INR transgene could be stably transferred to distantly related plants that lack inceptin responses and successfully boosts the production of antiherbivore defenses following INR activation. This discovery shows promise for enhancing natural crop protection mechanisms by staking the best of diverse plant immunity traits.

Plant-driven Defense in Agriculture

molecular patterns.” The Plant journal : for cell and molecular biology, 10.1111/tpj.15510. 26 Sep. 2021 3. Steinbrenner, Adam D., et al. "A receptor-like protein mediates plant immune responses to herbivore-associated molecular patterns." Proceedings of the National Academy of Sciences 117.49 (2020): 31510-31518. 4. Schmelz, Eric A. "Impacts of insect oral secretions on defoliation-induced plant defense." Current Opinion in Insect Science 9 (2015): 7-15.

Recent efforts in bean and corn plant models by Dr. Schmelz and Dr. Huffaker are pushing the boundaries of natural plant defense systems. Plants are incapable of fleeing from threats, yet their ability to call for help and bolster their defenses when they perceive danger extends their physical reach in a way that is not reproducible by humans. This defense behavior in plants has the potential to change the way we manage and optimize agricultural settings. The expression of diverse plant derived INR transgenes in naive crops enables us to boost natural protective responses of plants without further human inputs. The ability of a plant to perceive danger changes the interaction—an army that is not expecting attack is vulnerable. Receptors for common insect elicitors that activate plant defense responses provide a powerful radar, and are almost certain to be useful in bolstering crop protection in an environmentally sustainable fashion.

References 1. Eric A. Schmelz, Alisa Huffaker, Mark J. Carroll, Hans T. Alborn, Jared G. Ali, Peter E.A. Teal, An Amino Acid Substitution Inhibits Specialist Herbivore Production of an Antagonist Effector and Recovers Insect-Induced Plant Defenses, Plant Physiology, Volume 160, Issue 3, November 2012, Pages 1468–1478 2. Poretsky, Elly et al. “Comparative analyses of responses to exogenous and endogenous antiherbivore elicitors enable a forward genetics approach to identify maize gene candidates mediating sensitivity to herbivore-associated

WRITTEN BY KEVIN MARTINEZ Kevin is a Biochemistry and Cell Biology Major from Roger Revelle College. He will be graduating in 2022.

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SHINING BLUE STARS IN THE OCEAN THE SECRETS OF BIOLUMINESCENCE

written

by illustrated

YICHEN WANG

by ANGELA WANG

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features Dinoflagellates emit bioluminescent light to survive from predators through a complex biochemical cascade, currently being investigated by Dr. Michael Latz at UC San Diego.

hen was the last time you looked up at the night sky and were amazed by the spectacular scene of distant stars thousands of times larger than our sun? How about when you last looked down into the ocean to see an even more glamorous—albeit microscopic—shade of neon blue created by microorganisms hundreds of times smaller than a grain of rice? Bioluminescence, or light produced by living organisms, has been observed for eons and has fascinated scientists for many centuries. Organisms like jellyfish, shrimp, and zooplankton acquired this ability and started to use it to defend themselves against predators, attract mates, and hide themselves in the deep ocean. Notably, one of the most heavenly sights to see here in La Jolla, California has to be our neon blue tide. However, we must not forget to thank the group of organisms that emit this gorgeous light— dinoflagellates.

Shining to Live

Dinoflagellates are single-cell aquatic phytoplankton that usually emit light at night to avoid predators like copepods.1 Copepods are the most abundant zooplankton in the ocean, and therefore exert top-down control on the population of dinoflagellates. Top-down control refers to top consumers, or the predators in a food web, controlling the population distribution of lower trophic levels, or the prey. Just like cows on land, copepods have grazing behavior; however, instead of grass, they graze on phytoplankton like dinoflagellates. Thus far, there are three major hypotheses describing how dinoflagellates utilize bioluminescence to evade copepod predation. The first hypothesis is that dinoflagellates produce a flash of light to startle copepods. This light interrupts copepod grazing behavior, allowing dinoflagellates time to escape from their predator.2 In addition, high light intensities make copepods swim faster and straighter, poFigure 1. Purpose of bioluminescence

tentially forcing them to swim away from an area with a large density of dinoflagellates. It has been observed that copepod swimming behavior is indeed affected by natural and artificial dinoflagellate flashes, making this theory very persuasive.1 Interestingly, only marine and estuarine copepods exposed to dinoflagellates in their environment respond to light flashes with increased burst swimming—freshwater copepods do not respond to dinoflagellate flashes. This observation indicates that the marine copepods’ light-evasive burst swimming behavior evolved separately from their relatives in other envi-

ronments. The divergence could be due to the exclusive presence of bioluminescent dinoflagellates in marine environments and the fact that copepods’ visual predators attack them more often in the light than in the dark. Another popular theory on how bioluminescence enhances dinoflagellate evasion from copepod predation is that their blue flashes of light may attract copepods’ predators. It has been observed that lipid compounds released by copepods cause certain species of dinoflagellates to emit high-intensity light even at low colony densities.1 In this way, visual predators

Dinoflagellate

Hypothesis I Dinoflagellates produce flashes of light to startle copepods, which allows dinoflagellates time to escape. Copepod

Hypothesis II

Copepods' predator

Dinoflagellates produce flashes of light to attract copepods' predators.

Hypothesis III Dinoflagellates flashes of light serve as a warning signal that shows dinoflagellates are toxic to predators

Three theories on how dinoflagellates use bioluminescence to escape from copepods.

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UC San Diego's Scripps Institution of OceanogMechanical forces raphy, is interested in 1. Mechanical forces elucidating the mechaG-proteins activate G proteins. nism and scientific applications of dinoflagellate bioluminescence. 2. An increase in Specifically, he seeks to intracellular calcium. understand how exter3. Depolarizes the nal forces such as wamembrane of the ter flow are converted vacuole associated into cellular chemical with the scintillion. signals that ultimately result in the chemical Vacuole reaction between luciferin and luciferase, producing luminescence. Luciferin is a generic term for a Scintillon class of light-emitLuciferin Binding Protein ting compounds that exist in biolumines4. Protons flow into cent organisms. These the scintillion, lowercompounds typicaling the pH inside. ly undergo an enzyme-catalyzed reac5. Lucifren is retion with oxygen that leased from Lucifren produces an excited Binding Protien and state luciferin interLuciferase binds to Luciferase. Luciferin mediate that emits energy in the form of Figure 2. Mechanism of visible light when decaying back to the bioluminescence ground state. This light emitted during Signal transduction pathway of emitting light. the decay process creates the organism's bioluminescent emission. The enzyme that catalyzes the luciferin oxidation relike fish or squid might be attracted by the action is a protein called luciferase. In dibright light to feed on the copepods. As a noflagellates, both luciferin and luciferase result, not only do dinoflagellates remain are stored in vesicles called scintillons. In uneaten and their predators are removed, order to only emit light upon stimulation, but the copepods’ ability to habituate to luciferin is typically bound to the luciferin the light flash is also minimized, since binding protein, which protects luciferin the copepods who adapt and do not swim from oxidizing and thus keeps it inactiaway have a higher chance of being eaten vate inside the scintillons until the actiby their predators. vating stimulus is received. The third theory hypothesizes that The production and activation of bioluminescence serves as an aposematluciferin is also tightly regulated within ic—or warning—signal indicating that didinoflagellates. After luciferin is synthenoflagellates are toxic to predators. There sized, it is loaded into scintillons located are about twelve species of bioluminesnext to the vacuole, a large storage vesicle cent dinoflagellates that are known to be inside the cell that contains many protons. toxic to copepods when ingested. HowThe inside of the vacuole has a pH of less ever, though their colors might be a very than 6, while the pH of the scintillons is effective aposematic signal, it is not clear if more than 8.0, which creates the proton warning predators is the main purpose of gradient that is crucial for the activation bioluminescence, as copepods can often of bioluminescence. Interestingly, the distinguish between toxic and non-toxic membrane of the vacuole is electrically dinoflagellates using other visual means excitable, similar to a nerve cell. When or chemical sensing.3 the vacuole membrane is depolarized, a Excitement for Light proton-selective channel opens and conducts protons into scintillons, changing So how do dinoflagellates emit light? the conformation of the luciferase to exDr. Michael Latz, a marine biologist at 24

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pose its luciferin binding site and allowing the chemical reaction to proceed.4 Additionally, another ingenious structural strategy in dinoflagellates is that scintillons contain all the components necessary for emitting light and stabilizing it: luciferin, luciferase, and luciferin binding protein. The physical proximity among these three compounds make emitting light a rapid, readily accessible process. The mechanotransduction reaction cascade starts when dinoflagellates sense mechanical forces, such as the force produced by a predator grabbing them or shear stress caused by water flow, which activate GTP-binding proteins embedded in the plasma membrane.1 Then, transient receptor potential (TRP) channels on the cell membrane will open, allowing both a release of calcium ions from intracellular stores and an influx of calcium ions into the cell from the extracellular space. This increase in intracellular calcium depolarizes the membrane of the acidic vacuole associated with the scintillons. In turn, this depolarization of the vacuole membrane initiates a self-propagating action potential that travels along the vacuole membrane and opens voltage-gated proton channels in the vacuole membrane. Once these channels open, protons flow into the scintillons resulting in a rapid decrease of pH inside the vesicles that stimulates the release of luciferin from the luciferin binding protein and the activation of luciferase.1 Finally, now that luciferin can bind luciferase, luciferase facilitates the luciferin oxidation, which emits light. Through this complex mechanism, dinoflagellates are able to respond to external mechanical stimuli within 20 milliseconds—an immensely fast response time for a mechanosensing process in any organism. One of the key players in the transduction pathway, TRP channels, are essential in many sensory system responses in various organisms. Playing a key role in human temperature and pain sensing, TRP channels are also at the center of biological research in recent years. In fact, The Nobel Prize in Physiology or Medicine 2021 was awarded to scientists who first discovered TRP channels. By understanding their function in dinoflagellates, which might be the most ancient organism to have these channels, researchers may get a glimpse into the origins of TRP channel functions in eukaryotes. sqonline.ucsd.edu

Figure 3. Infinity Cube Photo of Dr. Latz in the Infinity Cube. exhibit at the Birch Aquarium.

ic expressions, which would help us to confirm the role of related proteins in the bioluminescent signaling pathway inside those shining blue stars of the ocean.

References

1. Jenny L, Michael IL. Bioluminescence in Eukaryotic Microbes. Encyclopedia of Microbiology. 4, 526-535 (2019). 2. E. J. Buskey. Swimming pattern as an indicator of the roles of copepod sensory systems in the recognition of food. Marine Biology. 79, 165-175 (1984).

Color of Science Displays of bioluminescence are known all over the world, from glowing caves in New Zealand to sparkling beaches in the Caribbean and Pacific Ocean. In addition to being visually appealing, light from marine animals has many more real-world applications, such as the use of biofluorescent organism-derived markers like asmCherry (red fluorescent proteins) and GFP (green fluorescent proteins) to stain cells. Unlike bioluminescence, where light is produced from a chemical reaction in an organism, biofluorescent organisms have proteins that absorb light and reemit it at a lower energy level. Dr. Roger Tsien, the late professor of Pharmacology, Chemistry and Biochemistry at UC San Diego and a 2008 Nobel Prize laureate in Chemistry, along with Dr. Shimomura and Dr. Chalfie, drastically increased GFP's fluorescent signal intensity and photostability.5 Since then, GFP has become a mature and popular tool for microscopy. Similar to the fact that GFP can be used for visualization, dinoflagellate bioluminescent proteins can be used to image flow fields including breaking waves, flow over seabeds, and boundary flows of moving animals and ships in the ocean.1 Since dinoflagellates can sense flow velocity change and respond to it almost instantaneously by emitting luminescence, they are very useful in visualizing moving animals. Additionally, researchers have used dinoflagellates to track dolphin movement and predict the motion of moving ships for various purposes. Another application of bioluminescence is to raise peoples' awareness of the connection between humanity and nature. In 2018, Dr. Latz collaborated with sqonline.ucsd.edu

3. Ali HA. et al. The Influence of the Toxin Producing Dinoflagellate, Alexandrium catenella (1119/27), on the Feeding and Survival of the Marine Copepod, Acartia tonsa. Harmful Algae. 98, (2020).

artist Iyvone Khoo to put together a bioluminescence exhibition called Infinity Cube at the Birch Aquarium at Scripps. Khoo was inspired to create the exhibit by a midnight walk at a beach in Mexico where she witnessed the co-shining of the Milky Way and the blue tides that pushed her to ponder about the microcosm and macrocosm, with humans right in the middle. Partnering with Dr. Latz, she composed her unique exhibit: a reflective cube with a media projection of dinoflagellate bioluminescence. Visitors were able to walk into the cube and immerse themselves into a marvelous ocean of blue lights to experience nature as their only illumination in the boundless darkness.6 Bioluminescence demonstrations not only engage the public but also provide an opportunity to learn about the wonders of bioluminescence and its role in nature.

4. Juan DR. et al. Identification of a vacuolar proton channel that triggers the bioluminescent flash in dinoflagellates. PLoS One. 12(2): e0171594 (2017). 5. Roger H, Andrew BC, Roger YT. Improved green fluorescence. Nature. 373, 663–664 (1995). 6. Michael IL. The Making of Infinity Cube, a Bioluminescence Art Exhibit. Bulletin. 28(4), 130-134 (2019).

Glance into Future The ability of dinoflagellates to bioluminesce is a true wonder; however, not all dinoflagellates possess this unique capacity. According to Dr. Latz, only about 18 out of 130 total dinoflagellate genera emit light, which raises the question of what makes the bioluminescent genera so special? To answer this question, the Latz group plans to search for the unique genes that code for proteins which enable bioluminescent species to shine. However, one challenge to answering this question is related to a peculiar property of dinoflagellates: their genome is hundreds of times larger than the human genome. Nevertheless, Dr. Latz is confident that with the incredibly fast advancements in molecular biology and genetics, soon it will not be a daydream to have the proper molecular tools to manipulate dinoflagellate genet-

WRITTEN BY ANGELA WANG Angela is a Neurobiology Major from Seventh College. She will be graduating in 2023.

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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.

Mosquito resting on a leaf after rainfall in the temperate rainforests of Olympic National Park, Washington. Photo by Dephny Duan.

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The Path of Most Resistance: Molecule Equisetin Found in Marine Sponges is a Possible Breakthrough Against Multi Drug-Resistant Bacteria

ntibiotics are crucial in protecting humans from a large range of bacterial diseases. Scientists have created several types of antibiotics that are now prescribed regularly to treat many infections. Unfortunately, the misapplication of antibiotics for viral infections and the unnecessary use of antibiotics as household cleaners have increased antibiotic resistance, in which bacteria evolve over generations, such that these treatments are no longer effective. In addition to killing harmful bacteria, the overuse and misuse of antibiotics eliminates beneficial bacteria, creating ideal conditions for malignant populations to grow and infect the host. A commonly-cited example of an antibiotic-resistant bacteria strain is methicillin-resistant Staphylococcus aureus (MRSA). Staphylococcus aureus has grown resistant to the antibiotic methicillin because of horizontal gene transfer, which lets bacteria distribute their genetic information among various species, augmenting the number of resistant genes a strain has.1,11 Consequently, MRSA develops antibiotic resistance and has the potential to invade immune systems and kill individuals. Over time, bacterial resistance has increased faster than the discovery and distribution of new antibiotics.1 This creates a pressing need for a new source of antibiotics that are effective against numerous bacteria strains. This review highlights a possible solution: equisetin. Found in the marine sponge species Fusarium equiseti, equisetin poses a breakthrough in the field of medicinal chemistry, with the ability to fight multi drug-resistant bacteria by inhibiting bacterial cell wall synthesis. One key chemical mechanism driving the effectiveness of antibiotics is their ability to block bacterial cell wall synthesis. Beta-lactam antibiotics are a family of antibiotics containing a beta-lactam ring, known as beta-lactamase. This four-membered cyclic amide ring7 attaches to the beta-lactam receptors found in bacterial cell walls and inhibits its function and repair mechanisms, resulting in the cells breaking apart.8 Marine fungi species are known to carry these lactamases. Equisetin, derived from one such species, contains this ring, contributing to its efficacy in overcoming antibiotic resistance. These findings suggest that more fungal species should be observed for the possibility of discovering more antibiotic species with capabilities similar to equisetin. Marine sponges are a well-known source of medicinal biosynthetic compounds.2 They contain numerous microorganisms—cyanobacteria, algae, and viruses—which biosynthesize compounds that can be used as immunosuppressants, neurodegenerative disease supplements, and antibiotics. Many antibiotics in the current pharmaceutical market are derived from marine organisms. For example, cephalosporins, used to treat skin infections, are derived from the fungi genus Acremonium.2 These marine organisms produce polypeptides that act as antibiotics by permeabilizing the cell membrane of bacteria, allowing toxic substances to enter and destroy bacterial particles.3 Another example is myticusin-1, derived from the mussel Mytilus coruscus, which has the potential to target bacterial species such as Bacillus subti-

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review Akshay S. Bharadwaj

B.S., Microbiology, Class of 2024, Warren College, University of California, San Diego. La Jolla, CA 92093, United States

lis, Staphylococcus aureus, Sarcina luteus, and Bacillus megaterium by blocking bacterial cell wall synthesis.13 This review primarily focuses on a research article in which scientists performed multiple experiments with equisetin to determine its efficacy. The first step for the scientists was identifying which fungal species contained equisetin. This was done using antiSMASH, a computer database containing software that recognizes secondary metabolites.4 Using antiSMASH, the scientists found a gene cluster in the marine fungi species F. equiseti that was 91-96% identical to the compound equisetin. After identifica-

Figure 1 (top). Staph bacteria are treated with various antibiotic substances at a dosage level of 10x the minimum inhibitory concentration (MIC). In the same amount of time as vancomycin, equisetin produced a lower viable bacterial number, indicating that it can kill off bacteria faster. Reprinted with permission.5 Figure 2 (bottom). With colistin on its own without equisetin, the fractional inhibitory concentration (FIC) is quite high. FIC is the MIC of the drug in combination divided by the MIC of drug acting alone.11 However, when combined with equisetin, colistin acted strongly synergistically, evidenced by the drop in FIC. This would suggest that equisetin works well in tandem with the antibiotic to eliminate Gram-negative bacteria as well. Reprinted with permission.5

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Figure 3 (top). This figure shows resistance development with equisetin. Both oxacillin and equisetin were injected at the same dosage into two staph bacteria cultures. The MIC required to eliminate bacteria inserted with oxacillin increased eight times more than the MIC for bacteria with equisetin. More importantly, the MIC for oxacillin continued to increase, while the MIC growth rate for equisetin flattened, suggesting that equisetin can more effectively impede antibiotic resistance. Reprinted with permission.5 Figure 4 (bottom). The x-axis depicts the time in hours that the various antibiotics circulated in the mice, and the y-axis depicts the survival rate of the mice. Mice injected with equisetin had a higher survival rate compared to those injected with vancomycin. Reprinted with permission.5

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tion in the fungi species, this molecule was isolated from deep-sea sediment and fermented in Petri dishes.5 The equisetin was then purified and separated from the sponge using an analytical chemistry technique called high-performance liquid chromatography (HPLC). The product of this method was a pale red powder: equisetin in a functional form for experimentation. Once the equisetin was procured, multiple bioassays were performed to examine the antibiotic abilities of equisetin against multi drug-resistant (MDR) bacteria.5 Experiments were conducted with gram-positive bacteria (bacteria containing thick cell walls), such as MRSA. In these experiments, equisetin proved more effective against multiple strains of these bacteria than the antibiotic vancomycin. This was shown with equisetin’s lower minimum inhibitory concentration (MIC), defined as “the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism after overnight incubation” (Figure 1).5 Equisetin on its own did not perform better than vancomycin on gram-negative bacteria, as both resulted in a MIC over 128 after being tested on strains such as Escherichia coli and Pseudomonas aeruginosa. However, further experimentation showed that equisetin acts well in synergy with certain antibiotics and helps eliminate such bacterial strains. A previous experiment measured the efficacy of seven gram-negative bacteria-targeting antibiotics. Among the seven, colistin had the best synergy with equisetin. When combined, the treatments performed very well against E. coli and S. aureus, requiring a lower dosage to achieve the efficacy of colistin alone.5 In fact, the equisetin-colistin combination is even powerful against a colistin-resistant strain of E. coli (Figure 2). Using multiple strains of E. coli with different concentrations of antibiotics revealed that a combination of equisetin and colistin inhibited 100% of the bacterial strains and killed 99% of the bacteria within 1 hour.9 With colistin and equisetin established as a powerful synergistic duo, the scientists performed studies on the biological mechanisms behind their efficacy. Using fluorescent dyes, they discovered that the equisetin-colistin combination breaks down both the outer membrane and plasma membrane of the bacteria, making it more susceptible to outsider attacks. In an experiment where staph bacteria were treated with low levels of equisetin over 100 days to develop resistance, the MIC required to kill the bacteria increased only by a factor of four and the bacterial growth rate flattened.5 The MIC for oxacillin, the standard antibiotic used for staph infections, increased by a factor of 64. A higher MIC indicates that more of the antibiotic is required to eliminate the bacterial strain. Thus, compared to oxacillin, equisetin is a far more effective repressor of resistance (Figure 3). Furthermore, the bacterial strains treated with equisetin were shown to develop collateral sensitivity; as the strain becomes resistant to one antibiotic, it is more susceptible to another antibiotic. Equisetin-treated bacterial strains became vulnerable to many different antibiotics such as daptomycin, gentamicin, and erythromycin, while developing resistance to equisetin.The MIC of equisetin required to eliminate antibiotic-resistant strains of certain bacteria such as S. aureus and VRE is two times less than what is required to eliminate the wild-type strain of the same bacteria. This suggests that a lower antibiotic dosage is required for bacteria strains treated with equisetin that are resistant to other antibiotics. Finally, equisetin was injected into animal models to analyze its in-vivo efficacy. In the Galleria mellonella larvae model, MRSA was injected first, followed by equisetin at a dose gradient ranging from 1 mg/kg to 10 mg/kg. The results showed that a standard dose of 10 mg/kg of equisetin can inhibit MRSA from infecting the larvae host and, as a result, decrease bacterial load (the quantity of sqonline.ucsd.edu

bacteria in an organism).5 In addition, the larvae models injected with equisetin had a higher survival rate than the ones injected with the antibiotic vancomycin. Similar results were produced in the rat models: the equisetin-injected mice experienced higher survival rates and these mice decreased bacterial loads in multiple organs (Figure 4). This suggests that equisetin has the potential to be a novel antibiotic for other animals and possibly even humans. Through experiments with bacterial cell cultures and in-vivo models, scientists have shown that equisetin can be effective in killing many strains of gram-positive and -negative bacteria while serving as an aid to other antibiotics like colistin. Scientists’ next steps could be to extend previous mice experiments into primate studies, eventually culminating in human trials. This would be useful in determining whether equisetum is effective in humans, while also revealing certain risks and side-effects that were not seen in the bacterial and mice models. As more fungal species such as F. equiseti continue to be found, strides will be made towards alleviating the medical problem of antibiotic resistance, providing necessary treatment for many patients globally.

ACKNOWLEDGMENTS This review was completed as part of a project titled “Chemistry in Context” in Dr. Stacey Brydges’ General Chemistry Class. I would sincerely like to thank Dr. Brydges for her assistance and guidance throughout this project. In addition, I would also like to express my gratitude for the researchers of the original paper that this review is based on.

REFERENCES 1. Jensen, S. O.; Lyon, B. R. Genetics of Antimicrobial Resistance in Staphylococcus Aureus. Future Microbiol. 2009, 4 (5), 565–582. 2. Häder, D.-P. Marine Sponges: Source of Novel Biotechnological Substances. In Natural Bioactive Compounds; Elsevier, 2021; pp 363–379. 3. Polypeptide Antibiotics: Bacitracin, Colistin, Polymyxin https://www.msdmanuals.com/professional/infectious diseases/bacteria-and-antibacterial drugs/polypeptide-antibiotics-bacitracin,-colistin,- polymyxin-b (accessed May 18, 2021). 4.antiSMASHbacterialversionhttps://antismash.secondarymetabolites. org/#!/about(accessed May 18, 2021). 5. Chen, S.; Liu, D.; Zhang, Q.; Guo, P.; Ding, S.; Shen, J.; Zhu, K.; Lin, W. A Marine Antibiotic Kills Multidrug-Resistant Bacteria without Detectable High-Level Resistance. ACS Infect. Dis. 2021, 7 (4), 884–893. 6. HPLC | High performance liquid chromatography https://www.youtube.com/watch?v=ZN7euA1fS4Y (accessed May 31, 2021). 7. Gao, M.; Glenn, A. E.; Blacutt, A. A.; Gold, S. E. Fungal Lactamases: Their Occurrence and Function. Front. Microbiol. 2017, 8, 1775. 8. Kohanski, M. A.; Dwyer, D. J.; Collins, J. J. How Antibiotics Kill Bacteria: From Targets to Networks. Nat. Rev. Microbiol. 2010, 8 (6), 423–435. 9. Zhang Q, Chen S, Liu X, Lin W, Zhu K. Equisetin Restores Colistin Sensitivity against Multi-Drug Resistant Gram-Negative Bacteria. Antibiotics (Basel). 2021;10(10):1263. Published 2021 Oct 18. doi:10.3390/ antibiotics10101263 10. Andrews JM. Determination of minimum inhibitory concentrations. J Antimicrob Chemother. 2001 Jul;48 Suppl 1:5-16. doi: 10.1093/jac/48. suppl_1.5. Erratum in: J Antimicrob Chemother 2002 Jun;49(6):1049. PMID: 11420333. 11. Sun, D., Jeannot, K., Xiao, Y., & Knapp, C. W. (2019, August 27). Editorial: Horizontal gene transfer mediated bacterial antibiotic resistance. Frontiers. Retrieved March 6, 2022, from https://www.frontiersin.org/ articles/10.3389/fmicb.2019.01933/full

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Incorporation of Non-Canonical Amino Acids in Vivo Via the Quadruplet Codon System Nandika Mishra Seventh College, Molecular and Cellular Biology, Class of 2025

he structure and function of proteins in the human body is largely limited by the number of amino acids that are naturally generated by the genetic code. Out of 64 codons, 61 of them code for only 20 amino acids, and the other three function as stop codons. This severely limits the proteins that our bodies can create, as well as their different functions. This is why finding ways to integrate artificial amino acids, or noncanonical amino acids (ncAAs), into the human body is so crucial, as they have the potential to enhance and expand protein structure and function. The quadruplet codon system8 is an innovative concept that aims to expand our original genetic system to consist of 256 codons instead of 64. This paper will discuss the development of the quadruplet codon system, alternative methods to the system, and the challenges that may come about in utilizing quadruplet codons, along with potential solutions. Although over 500 amino acids have been found in nature,1 only 20 are used to encode proteins within the human body. However, over the past 30 years, scientists have incorporated amino acids that do not naturally occur within the human body, known as non-canonical amino acids (ncAA), into human-made proteins. One of the earliest displays of the incorporation of ncAAs into proteins was via a suppressor tRNA with a stop codon as its corresponding codon. The tRNA codes for an ncAA, which is then incorporated into the enzyme beta-lactamase in place of the stop codon.2 At the time, this field and method were relatively novel and paved the way for similar experiments. A contemporary example of the use of ncAAs is in gene editing, a process where an organism is genetically modified to be dependent on an ncAA by introducing it into an essential gene. This also calls for an orthogonal translation system, a system containing the engineered enzyme needed to attach an ncAA to its corresponding tRNA. Modifying organisms such as viruses in this way means they cannot interact with regular cells. This doubles as an advantage for vaccination, because when using a genetically modified organism-based vaccine, horizontal gene transfer cannot occur between different systems. This means the virus can merely infect, not replicate. An immune response can then be successfully carried out.3

Another use of ncAAs includes changing the functions of certain proteins. One group took cinnamycin, a peptide antibiotic made by strains of the bacteria Streptomyces, and incorporated different ncAAs at various positions, testing out the effects of each of them. They found that with some of the new incorporations, the antibiotic activity of the now altered cinnamycin was increased4. Other uses of ncAAs include cancer therapy,5 post-translational modifications within proteins,6 and more. A more intricate application includes ncAA use in biosensors, specifically involving a phenomenon known as fluorescence resonance energy transfer (FRET) (Figure 1). In FRET, energy is transferred between two fluorophores—a donor molecule and an acceptor molecule, both typically fused to a binder protein. The closer these two molecules are to each other, the higher the FRET efficiency. Initially, researchers used a Snifit, which is an indicator consisting of a SNAP-tag (a self-labeling protein tag), a CLIP-tag (a fluorescent protein which can be tagged with a fluorophore), and a binding protein (BP). Afterwards, they replaced the CLIPtag with a fluorophore-tagged ncAA, which is much smaller than the CLIP-tag. This decreased the distance between the two fluorophores, which increased FRET efficiency. These new Snifits were labeled uSnifits, and the scientists found that the dynamic range of the sensors increased. They also found that the labeling process could be carried out in vivo when using an ncAA.7 In order to incorporate new ncAAs into humans, however, it is necessary to develop a way to expand our genetic code. Multiple methods exist for this purpose. However, one of the most promising methods to appear in recent years is known as the quadruplet codon system. This system introduces new ncAAs into the body by creating and utilizing codons consisting of four nucleotides rather than the traditional three. This novel approach increases the number of possible codons by four times the original amount, giving the body more diversity when creating proteins. The main problem with this approach is that the body’s natural machinery is engineered to process triplet codons, not quadruplets. When a ribosome reads a strand of RNA, the ribosome will often select a tRNA which corresponds to a triplet codon instead of a quadruplet codon, incorporating a canonical amino acid instead of an ncAA. To counter this, scientists have been working on engineering orthogonal (meaning that it cannot interact with the natural machinery of the cell) ribosomes that read for quadruplet codons.24 Along with the prospect of 256 codons as opposed to 64, the quadruplet codon system is shaping up to be one of the best candidates for the incorporation of ncAAs in vivo. Quadruplet codons have been the subject of many experi-

Figure 1. a) Conventional Snifit system using CLIP-tag. In this case, FRET is being exhibited by two fluorophores, represented by the green star on the CLIP-tag and the red star. b) uSnifit system, where the CLIP-tag has been replaced with a fluorophore-tagged ncAA, represented by the gray star. Since the distance between the two fluorophores has decreased, FRET efficiency has increased. Republished with permission.7

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review ments since the 1970s.9 One of the first influential studies in the field dates back to 1981, when a group of scientists experimented with the concept of frameshift suppression where frameshift mutations are overcome and a suppressor tRNA is able to read four nucleotides instead of three. The isolation of a novel suppressor, dubbed sufJ, revealed its ability to read exactly three different four-base codons upon recognition of the ACC triplet in mRNA: ACCA, ACCU and ACCC.10 Although frameshift suppression has been tested before,9, 11, 12 previous suppressors only worked for one or two codons of a single base, with minimal base changes. However, sufJ had low efficiency in decoding codons, around 1-2%. In 2000, one group of scientists managed to engineer a tRNA in E. coli such that its anticodon loop was expanded by one extra nucleotide—specifically, the tRNA Su6, which contains the anticodon for the stop codon UAG and is a mutation of the tRNA used for leucine. Altering this tRNA allowed it to decode the codon UAGA with an efficiency of 13-26%, which was ten times more efficient than frameshift suppression.13 Further experiments were performed in the E. coli strain MRA8, as it has a temperature-sensitive release factor 1 (the protein needed to terminate translation in E. coli), which reduces competition between termination of translation and decoding of quadruplet codons. Variants of Su6 were created to assess the decoding efficiency of one UAGA codon, two UAGA codons in tandem, and a UAGA and a UACA codon. The former two were found to have an efficiency of 40%, meaning that they were correctly decoded 40% of the time. The latter had an efficiency of 10%. The major problem lay with competition with canonical amino acids. Since this tRNA was a mutation of the tRNA assigned to leucine, that amino acid was found to be a product of translation around 50% of the time.13 The past few decades have brought about significant findings in overcoming competition from triplet codons, eventually continuing to the engineering of orthogonal tRNAs.13 In 2004, J. C. Anderson et. al. created an orthogonal tRNA and aminoacyl-tRNA synthetase (aaRS) pair that was able to incorporate an ncAA, L-homoglutamine, in response to the quadruplet codon AGGA, specifically in E. coli. It was challenging to engineer an aaRS that would be specific to the ncAA, and not to any endogenous amino acids. For this reason, E. coli tRNA could not be used, because the tRNA had to be orthogonal in the organism. The tRNA was based on a prokaryotic tRNA for lysine from the bacterium Pyrococcus horikoshii. The crystal structure of its aaRS was available for study, giving a base from which the artificial aaRS could be derived. Plus, this particular bacterium is tolerant to the introduction of new nucleotides into its tRNAs. The group performed further experiments in order to test the simultaneous incorporation of two ncAAs using a second orthogonal tRNA and aaRS pair. This pair was derived from the tRNA for tyrosine, from the organism Methanococcus jannaschii, for the artificial amino acid O-methyl-L-tyrosine. The two pairs were tested and found to be mutually orthogonal, meaning that one aaRS could not work with the tRNA from the other pair. Hence, these two were able to work together to concurrently incorporate two ncAAs into the same protein, representing a major breakthrough.14 Another group that worked on the simultaneous incorporation of two ncAAs was Hankore and colleagues.15 They decoded two quadruplet codons, UAGA and AGGA, then used them to simultaneously introduce two ncAAs into a single protein within E. coli. This group also derived their tRNA and aaRS pairs from organisms similar to those in previous studies—Methanocaldosqonline.ucsd.edu

coccus jannaschii and Methanosarcina barkeri. These pairs were mutually orthogonal to both the natural amino acids within E. coli as well as each other. However, they found that the original pair derived from M. jannaschii had to be further altered through directed evolution in order to achieve greater decoding efficiency. Eventually, this was observed in certain mutant versions of the original tRNA and aaRS pair. With both the derived tRNA and aaRS pairs, they were able to successfully perform site-specific simultaneous incorporation of two ncAAs in response to two quadruplet codons.15 In 2013, researchers conducted further experiments on an orthogonal tRNA and aaRS pair, creating mutated versions of each to see which one had the highest decoding efficiency for the quadruplet codon AGGA (and thus, the highest efficiency in incorporating ncAAs), within E. coli. This experiment was significant as they found that these mutated versions also operated well in mammalian cells. This meant that the tRNA and aaRS pair used in E. coli, a prokaryote, could also be used in mammalian eukaryotic cells.16 Although many of these experiments were often tested using only one or two quadruplet codons, it is likely that these approaches or similar ones can be used for many different quadruplet codon combinations. Research has shown that site-specific incorporations within proteins allow specific, targeted changes in structure or function. The simultaneous incorporation of ncAAs also shows two mutually orthogonal tRNA and aaRS pairs working together. The studies conducted thus far demonstrate the potential of the quadruplet codon system, not only for specific incorporation of ncAAs, but also for expanding the genetic code overall through the integration of new codons into certain organisms. While the quadruplet codon system is a promising method of expanding the genetic code, other aforementioned methods also exist for this purpose. The first method is called amber suppression. This method refers to using an orthogonal tRNA and aaRS pair in response to the amber stop codon, UAG, to insert an ncAA instead of terminating translation, which would extend the polypeptide. However, it also extends to the other two stop codons: the ochre codon (UAA) and the opal codon (UGA). In 2010, a group of scientists demonstrated in vitro incorporation of ncAAs using the three stop codons, as well as engineering tRNAs from ones existing in E. coli and yeast. Modifying the tRNA used for cysteine allowed them to insert a modified version of cysteine in response to these stop codons.17 Another group in 2010 converted the UAG codon from a stop codon to a sense codon in E. coli by removing its release factor to eliminate the possibility of translation termination. However, to avoid any harmful effects of codon alteration, the E. coli strain had to be genetically modified. It was found that the UAG codon still retained the function of stopping translation, as well as that of incorporating an ncAA, before the removal of the release factor. This problem falls under the “ambiguous intermediate” theory, where despite reassigning the genomic function of a codon, the codon still retains its previous function while shifting to the new one, prior to the elimination of the release factor. To prevent this from happening, they replaced a certain amount of UAG codons with UAA codons, to make sure that the UAG codon only had the function of incorporating an amino acid.18 A similar result was found by O’Donoghue and colleagues19 who used E. coli with the orthogonal tRNA and aaRS pair for the ncAA pyrrolysine, which works in response to UAG. They found that even though it is the only known pair to have naturally evolved to insert pyrrolysine SALTMAN QUARTERLY

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in response to a stop codon, it cannot completely overcome the competition between this reaction and the normal termination response.19 This method of stop codon suppression is advantageous because existing codons can additionally be used for their natural functions. However, amber, ochre, and opal suppression gives us a mere three codons to work with, whereas the quadruplet codon system provides the promise of many more opportunities for ncAA incorporation because of the variety of codons that can be created. Also, when taking into consideration the fact that one codon is still required to stop translation, only two stop codons are left to assign to ncAAs. Another disadvantage arises from competition among the release factors that terminate translation. In order to experiment with amber suppression, it is necessary to mutate the bacterial strains or alter the organisms such that their release factor is not present. However, it has been found that removal of release factors can lead to a decrease in the fitness of the cell,20 as ribosomes stall upon encountering the stop codons intended to be recognized by the release factors. Additionally, reassigning the function of amber codons by replacing some or all of them with UAA codons can lead to off-target mutations.8 The next method for expanding the genetic code comes from reassigning sense codons. In humans, 64 codons make up the genetic code, but three to four codons are generally assigned to the same amino acid. As a result, these codons only correspond to 20 amino acids. This is probably to account for possible mutations in our DNA. The codons assigned to one amino acid are very similar, so the chances that a nucleotide change will alter the amino acid are low. Reassigning sense codons involves employing infrequently utilized sense codons and having them code for an ncAA. For example, a group in 2014 reassigned the AGG codon in E. coli to code for ncAAs using the pyrrolysine tRNA and aaRS pair. This codon normally codes for arginine, so arginine would sometimes be inserted in response to AGG instead of an ncAA. The group was unable to resolve this issue.21 Reassigning sense codons presents many challenges and disadvantages, such as the aforementioned “ambiguous intermediate” theory. Another is an increase in the likelihood of mutations, because in the case of only one codon per amino acid, even a single nucleotide change would cause a change in amino acid. A lesser-known method to incorporate ncAAs involves the creation of new nucleotides. A group in 2019 was able to synthesize two artificial nucleotides, dNaM and dTPT3, creating an unnatural base pair (UBP) which could then be used to create ad-

ditional sense codons in a genetically modified organism (GMO), also known as a semi-synthetic organism (SSO). Out of nine new codons that were identified as stable within the DNA, three were found to be mutually orthogonal (orthogonal with respect to one another) within the SSO, increasing the total number of codons that could be decoded to 67. However, they discovered that codons with the UBP in the first position were decoded inefficiently compared to those with it in the second or third position.22 Another obstacle arises in that much of the materials required for replication, transcription, and translation have to be imported into the organism of interest via specific transporters. Each of these methods have their fair share of challenges, many of which can be overcome with the quadruplet codon system. The quadruplet codon system has unique advantages which bypass many of the issues with the alternate solutions described, like stop codon suppression or reassigning codons. It consists of four times the number of codons that are originally available in the human body, does not need to reassign existing codons to other functions, and does not require processes that would damage the cell such as the deletion of release factors. However, a significant problem with this system is competition with triplet codons. This means that the ribosome will use tRNAs that respond to triplet codons and code for canonical amino acids, instead of using orthogonal tRNAs that read quadruplet codons and incorporate ncAAs.23 Since the ribosome and the tRNAs that respond to triplet codons are part of the natural machinery of cells, it is difficult to incorporate quadruplet codons. This problem was first approached in 2005, when two scientists designed an orthogonal ribosome by duplicating ribosomes from E. coli. The scientists modified the ribosomes such that they would read orthogonal mRNAs (which could not be read by native ribosomes) in conjunction with the native ribosomes decoding the native mRNAs.24 However, these ribosomes were not suitable when it came to incorporating ncAAs.25 In 2007, Wang and colleagues expanded on the idea of orthogonal and natural ribosomes working in tandem.26 An orthogonal ribosome known as ribo-X, as well as an orthogonal mRNA, and an orthogonal tRNA and aaRS pair were used, all within E. coli. Using the amber codon, they were able to increase the efficiency of the site-specific incorporation of ncAAs from 20% to 60% for a gene with one amber codon, and 1% to 20% for a gene with two. Suppressing the amber codon prevented the release factor from terminating translation without having to remove it from the cells and potentially harm the bacterium. In fact, it is hypothesized that the basis for this increase in efficiency was decreased interaction between ribo-X and the release factor. The group also speculated that it may be possible to use the orthogonal ribosomes and mRNAs to create new genetic code by using tRNAs that work well specifically with the orthogonal ribosomes.26 In 2010, another group of scientists, most of whom worked on ribo-X, developed another orthogonal ribosome, ribo-Q1, which could translate an orthogonal mRNA.27 This ribosome was not only able to decode the amFigure 2. Competition between native tRNAs and orthogonal tRNAs. Oftentimes, the ber codon but was also able to decode several native tRNA will beat out the orthogonal tRNA in the ribosome and read the codon in a other quadruplet codons. By using an orthogonal tRNA and aaRS pair, the group was able to triplet manner instead of the desired quadruplet manner. Republished with permission.23 incorporate ncAAs in response to two of these

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new quadruplet codons.27 These experiments summarize many of the major studies that have been done in regards to orthogonal ribosomes, which are proving to be an extremely viable solution to competition between triplet and quadruplet codons. The quadruplet codon system has come a long way since it was first tested. Many major developments have been made to overcome potential setbacks and implement the system into mammalian cells. It can be used to introduce a multitude of ncAAs into natural systems, whose applications include, but are not limited to, gene editing, cancer therapy, vaccination, and post-translational modifications of proteins. This system is very new in the field of protein research, and much of it has yet to be explored. Still, it has high potential to expand the genetic code. Although its limitations are being challenged, scientists are researching ways to overcome them through experimentation. Many new ncAAs are being developed, and if we can efficiently incorporate them into the human body, we can potentially improve the function and structure of proteins. The quadruplet codon system is proving to be the best option for this purpose, and further experiments and studies should be carried out to identify its full potential.

ACKNOWLEDGEMENTS I would like to thank my mentor, Ryan Boyman, who works as a healthcare consultant at Simon-Kucher. He acted as my guide in narrowing down my research to a specific topic, helping collate sources and giving feedback on my work. I am very grateful to him for all the help and guidance I received. I would also like to acknowledge ACS Publications as well as John Wiley and Sons for granting permission to use these figures.

REFERENCES 1. Wagner, I. and Musso, H. New naturally occurring amino acids. Angewandte Chemie International Edition in English, 22(11), pp.816-828 (1983). 2. Noren, C. J., Anthony-Cahill, S. J., Griffith, M. C., and Schultz, P. G. A General Method For Site-Specific Incorporation Of Unnatural Amino Acids Into Proteins. Science 244, no. 4901, 182-188 (1989). 3. Mayer, C. Selection, Addiction And Catalysis: Emerging Trends For The Incorporation Of Noncanonical Amino Acids Into Peptides And Proteins In Vivo. Chembiochem 20, no. 11, 1357-1364 (2019). 4. Lopatniuk, M., Myronovskyi, M., and Luzhetskyy, A. Streptomyces albus: A New Cell Factory for Non-Canonical Amino Acids Incorporation into Ribosomally Synthesized Natural Products. ACS Chemical Biology, 12(9), 2362-2370 (2017).

12. Kohno, T., and Roth, J. A Salmonella frameshift suppressor that acts at runs of a residues in the messenger RNA. Journal Of Molecular Biology, 126(1), 37-52 (1978). 13. Moore, B., Persson, B., Nelson, C., Gesteland, R., and Atkins, J. Quadruplet codons: implications for code expansion and the specification of translation step size. Journal Of Molecular Biology, 302(1), 281 (2000). 14. Anderson, J. C. et al. An expanded genetic code with a functional quadruplet codon. Proceedings Of The National Academy Of Sciences, 101(20), 7566-7571 (2004). 15. Hankore, E. et al. Genetic Incorporation of Noncanonical Amino Acids Using Two Mutually Orthogonal Quadruplet Codons. ACS Synthetic Biology, 8(5), 1168-1174 (2019). 16. Niu, W. An Expanded Genetic Code in Mammalian Cells with a Functional Quadruplet Codon. ACS Chemical Biology, 8(7), 1640-1645 (2013). 17. Gubbens, J., Kim, S., Yang, Z., Johnson, A., and Skach, W. In vitro incorporation of nonnatural amino acids into protein using tRNACys-derived opal, ochre, and amber suppressor tRNAs. RNA, 16(8), 1660-1672 (2010). 18. Mukai, T. et al. Codon reassignment in the Escherichia coli genetic code. Nucleic Acids Research, 38(22), 8188-8195 (2010). 19. O'Donoghue, P. et al. Near-cognate suppression of amber, opal and quadruplet codons competes with aminoacyl-tRNAPyl for genetic code expansion. FEBS Letters, 586(21), 3931-3937 (2012). 20. Heinemann, I. et al. Enhanced phosphoserine insertion during Escherichia coli protein synthesis via partial UAG codon reassignment and release factor 1 deletion. FEBS Letters, 586(20), 3716-3722 (2012). 21. Zeng, Y., Wang, W., and Liu, W. Towards Reassigning the Rare AGG Codon in Escherichia coli. Chembiochem, 15(12), 1750-1754 (2014). 22. Fischer, E. et al. New codons for efficient production of unnatural proteins in a semisynthetic organism. Nature Chemical Biology, 16(5), 570-576 (2020). 23. Chatterjee, A., Lajoie, M., Xiao, H., Church, G., and Schultz, P. A Bacterial Strain with a Unique Quadruplet Codon Specifying Non-native Amino Acids. Chembiochem, 15(12), 1782-1786 (2014). 24. Rackham, O., and Chin, J. A network of orthogonal ribosome·mRNA pairs. Nature Chemical Biology, 1(3), 159-166 (2005). 25. Chen, I., and Schindlinger, M. Quadruplet codons: One small step for a ribosome, one giant leap for proteins. Bioessays, 32(8), 650-654 (2010). 26. Wang, K., Neumann, H., Peak-Chew, S., and Chin, J. Evolved orthogonal ribosomes enhance the efficiency of synthetic genetic code expansion. Nature Biotechnology, 25(7), 770-777 (2007). 27. Neumann, H., Wang, K., Davis, L., Garcia-Alai, M., and Chin, J. Encoding multiple unnatural amino acids via evolution of a quadruplet-decoding ribosome. Nature, 464(7287), 441-444 (2010).

5. Ma, J. et al. Versatile strategy for controlling the specificity and activity of engineered T cells. Proceedings Of The National Academy Of Sciences, 113(4), E450-E458 (2016). 6. Elsässer, S., Ernst, R., Walker, O., and Chin, J. Genetic code expansion in stable cell lines enables encoded chromatin modification. Nature Methods, 13(2), 158164 (2016). 7. Xue, L., Prifti, E., and Johnsson, K. A General Strategy for the Semisynthesis of Ratiometric Fluorescent Sensor Proteins with Increased Dynamic Range. Journal Of The American Chemical Society, 138(16), 5258-5261 (2016). 8. Chin, J. Expanding and reprogramming the genetic code. Nature, 550(7674), 53-60 (2017). 9. Yourno, J., and Kohno, T. Externally Suppressible Proline Quadruplet CCCUU. Science, 175(4022), 650-652 (1972). 10. Bossi, L., and Roth, J. Four-base codons ACCA, ACCU and ACCC are recognized by frameshift suppressor sufJ. Cell, 25(2), 489-496 (1981). 11. Yourno, J. Externally Suppressive +1 “Glycine” Frameshift: Possible Quadruplet Isomers for Glycine and Proline. Nature New Biology, 239(94), 219-221 (1972).

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Meta-analysis of the Pacific Oyster Microbiome: Characterizing MicrobiomeEnvironment Associations and Core Tissue Microbiomes INTRODUCTION Oysters are an integral part of many marine ecosystems. They provide food for several species of snails and crabs and act as natural water filters, preventing algal blooms and nutrient-induced oxygen depletion in aquatic ecosystems (eutrophication). Additionally, oyster reefs provide a habitat for several small fish and crustacean species. Lastly, they promote underwater vegetation growth, providing greater coastal resistance to erosion and protection against storms. In recognition of the significance of oysters in promoting marine biodiversity, countries such as the United States and Australia have invested millions of dollars in the restoration of oyster reefs along the Pacific, Atlantic, and Indian ocean coasts. A microbiome is a consortium of bacteria, fungi and other microbes associated with a host organism, such as the Pacific Oyster. By evaluating differences in the microbiome of various oyster tissue types from multiple geographic regions, our goal is to develop a deeper understanding of the factors that influence oyster microbiome composition and diversity and the core microbial taxa that may play an important role in oyster health. Numerous papers have indicated that doing so could help identify interactions between taxa and potentially their influence on oyster community composition as well. We chose to analyze the Pacific Oyster (Crassostrea gigas) microbiome, as this species dominates global shellfish production and is a prominent member of Pacific Ocean coastal ecosystems. Several individual studies have characterized Pacific Oyster microbiomes from multiple regions; however, a meta-analysis has not previously been conducted. In this analysis, we have compiled data from 10 studies representing several countries, multiple oyster tissue types, and in some cases experimental manipulations such as infection and translocation. A meta-analysis was conducted by combining data from multiple studies as by doing so it is

Yash Garodia [1]; Rachel E. Diner, JD, PhD [2]; PI: Jack A. Gilbert, PhD [3] [1] UC San Diego, B.S. General Biology, Class of 2022, Revelle College [2] UC San Diego, Postdoctoral Scholar, Gilbert Lab UC San Diego, La Jolla, CA, USA [3] Department of Pediatrics and Scripps Institution of Oceanography, UC San Diego, La Jolla, CA, USA

often possible to identify more reliable trends than by comparing the results of individual analyses. We utilized the open-source microbial analysis platform Qiita to gain new insight into Pacific Oyster microbiomes and to generate a tool for the scientific community, enabling future studies of new testable hypotheses. After reading papers utilized in the meta-analysis, we hypothesize that bacteria belonging to the Mycoplasmataceae and Spirochaetes family will be common dominant taxa across all geographic regions. Several papers have attributed oyster microbiome fluctuations to environmental changes. These studies found that some tissue types are more affected than others, particularly finding that gut taxonomic evenness and abundance varied the most by tissue type. Therefore, we also hypothesize that the core microbiome, particularly that of the gut, would be very small. A small core microbiome suggests a significant level of variation in taxonomic abundance and diversity for a given tissue type between samples.

METHODS We identified peer reviewed studies associated with Pacific oysters (Crassostrea gigas, also known as Magallana gigas) and their microbiome using relevant search terms in Google Scholar and Connected Papers, a citation-based paper identification tool. Studies were then inspected individually, and those that did not use 16S rRNA Illumina amplicon sequencing were excluded

Figure 1. Beta diversity heatmap of all Pacific Oyster microbiome samples using the Bray Curtis dissimilarity metric and hierarchical clustering. Axis numbers refer to individual Pacific Oyster microbiome samples in the meta-analysis. Yellow represents less similar samples with higher dissimilarity values (maximum = 1), and dark violet represents more similar samples with lower dissimilarity values (minimum = 0).

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manuscript from the analysis. Studies were also excluded if sequences were unavailable publicly or by author correspondence. Data were obtained from 10 studies and processed in the open-source microbial analysis platform Qiita.12 For each study, a metadata document was created using all supplementary information provided in the paper. The primary variables were sample name, research project, sample site, country of sampling, the hypervariable region of the 16S rRNA gene sequenced, sampling season, month and year, and additional variables were added to account for all unique environmental conditions and treatments that samples in different studies were subject to (such as infection and translocation). All raw data were downloaded as FASTQ files from the European Nucleotide Archive. Based on the sample name and the run prefix for the raw data files, a preparation information file was created. The metadata, preparation information, and raw data were then uploaded to Qiita for further processing and made available for public use (Analysis ID: 46943). The metadata variables were kept constant for a consistent meta-analysis. Each study was uploaded as a per-sample FASTQ without barcodes, and demultiplexed using the ‘Split Libraries Fastq’ command with a Phred quality score offset of 33. All sequences were trimmed to a length of 250 base pairs, followed by closed-reference OTU picking against the SILVA 119 database. This gave rise to a BIOM feature table for each study, which was combined for subsequent meta-analysis. The combined feature table underwent taxonomy-based feature table filtering to remove sequences annotated as mitochondria, chloroplasts, eukaryotic species, archaea, and any unknown or unclassified species. Beta diversity and Kruskal-Wallis pairwise beta group significance tests were performed on the filtered table to determine whether the difference in microbiome between samples was significant. Bray-Curtis dissimilarity was used as a metric for beta diversity, the results of which were also graphed on a Principal Coordinate Analysis (PCoA) plot (Figure 2). Alpha diversity analysis was then performed on the filtered table, using Shannon’s diversity index as the metric, the results of which were displayed on a box plot (Figure 3). The next part of the analysis focused on visualizing the microbiome and potentially identifying a core microbiome for each tissue type. The filtered feature table underwent sample filtering to produce a separate table for each tissue. The tables were collapsed by taxonomy to the family level (level 4) to quantify the relative abundance of each taxa in the samples. To identify the core microbiome of a tissue type, we ran the ‘identify core features’ process on the feature table after collapsing the filtered table according to taxonomy. We then looked at the various taxa found in 100%, 95%, and 90% of samples for each tissue type respectively, and considered 95% to be the benchmark for classifying a taxon as ‘core’. King and colleagues defined ‘core’ to be “an OTU that was present in at least all but one replicate,” but considering the large variability in number of samples for each tissue type, we considered this to be an impractical definition for this study. The high accuracy levels associated with Illumina amplicon sequencing have allowed the definition of core to extend to taxa that are found in 100% of samples, but to account for variability in the large number of different samples being analyzed, we reduced the threshold to 95%.

ta-analysis. The tissue types evaluated were gut, gill, whole oyster homogenate, extrapallial fluid, mantle, main cavity, adductor muscle, feces, and hemolymph. All samples are either from the west coast of the United States (California), Australia, the Netherlands, France, or Germany. Illumina HiSeq sequencing platforms were used for sequencing extrapallial fluid and main cavity samples, whereas MiSeq sequencers were used for all other tissue types. Although it was included in this study due to a different sequencing approach, the main cavity has been excluded from all further analyses as only one sample was available.

RESULTS

The dispersion for all variables with the exception of tissue type was considered statistically significant (Table 1). This could be because most studies with available data were focused towards specific tissue types, such as gut or gill. Other tissue types, such

A total of 1,896 oyster samples from several anatomical and geographical sites were analyzed from 10 studies for this me-

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Beta Diversity Across All Samples Hierarchical clustering of pairwise beta diversity comparisons using the Bray Curtis dissimilarity metric across all samples revealed that most samples were highly dissimilar to each other, with limited clustering of certain sample types (Figure 1). Most dissimilarity values lie in the distance range of 0.9 to 0.95. The average distance was 0.9435, with the maximum distance being 1.0000 and lowest being 0.0566. Beta Diversity Group Significance and Pairwise Beta Diversity Analysis Within Sample Types In order to validate the meta-analysis, PERMDISP and PERMANOVA Beta Diversity group significance analyses were conducted to look at the key determining factors driving the differences in results. The variables looked at were the research project, tissue type, country, and type of oyster (control or not). A p-value <0.05 suggests that the variable has a statistically significant influence on the data obtained. Table 1: PERMDISP Beta Diversity Group Significance Results for Different Variables

Table 2: PERMANOVA Beta Diversity Group Significance Results for Different Variables

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Figure 2. Principal coordinate analysis depicting variation of Pacific Oyster microbiome community composition by tissue type. Bray-Curtis principal coordinate analysis plot illustrates variation among Pacific Oyster microbiome samples harvested from eight tissue types from ten studies with collections across five countries spanning three continents. Color-coding illustrates microbial community similarity by tissue type.

Figure 3. Principal coordinate analysis depicting variation of Pacific Oyster microbiome community composition by country. Bray-Curtis principal coordinate analysis plot, color-coded according to the country of sampling, was generated. Microbial composition of eight types of Pacific Oyster tissues were determined via 16S rRNA marker gene sequencing.

as extrapallial fluid and mantle, were less common; therefore, the dispersion of tissue types was not necessarily a driving factor. The difference in composition between samples for each variable shown through the PERMANOVA results was statistically significant (Table 2). Although the influence of dispersion and composition on the basis of research projects is significant, the remarkably higher F and pseudo F value (Tables 1 and 2) for the ‘country’ variable suggests that, while study may influence the data, the geographic region is the main driving factor for the results. This validates the data, as it suggests that differences in study design or 36

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hypervariable region sequenced, for instance, are not the primary drivers of the differences observed among samples. A beta diversity group significance boxplot using the Bray-Curtis dissimilarity metric was used to evaluate whether the difference in microbiomes across different tissue types was significant. Mean distances were at or close to 1 for all tissue types in comparison to each other. The level of variation in distance between the gill and mantle tissue was higher than between all other tissue types, indicating that the gill and mantle microbiome may have greater similarity compared to other samples. sqonline.ucsd.edu

A pairwise beta diversity group significance PERMDISP test was conducted on the samples according to tissue type to generate a statistical metric that quantifies the level of dispersion between these different tissue types. A p-value <0.05 is considered statistically significant, and comparison between tissue types assumes that the groups are similarly dispersed. Despite the boxplots showing relative similarity between the gill and the mantle microbiome, the pairwise analysis indicated a significant difference between them. There were no significant differences between the adductor and gut or gill, the feces and mantle or whole oyster, and the extrapallial fluid and feces, hemolymph, mantle, or whole oyster samples. All other pairwise combinations were significantly dispersed from each other. While these differences in group homogeneity may impact group statistical comparisons and should be appropriately considered, PERMANOVA non-parametric tests under similar assumptions. A p-value <0.05 indicates that the difference in composition between the two tissue types is statistically significant. Therefore, apart from extrapallial fluid, all compared sample types were statistically different. PCoA plots generated based on the Bray Curtis dissimilarity matrix indicated that the percentage of variance explained ranged from 4-8% for each axis, with a total of 19.45% explained by the three primary axes shown (Figures 2 and 3). Tissue types (denoted by color in Figure 2) formed distinct clusters in some cases. Data points on the whole oyster, mantle, adductor, gut, and gill were widely dispersed, suggesting higher microbiome variability in these tissue types. Hemolymph and fecal samples were clustered together and away from other sample types, suggesting that they were most likely to have a distinct but robust microbiome composition. Extrapallial fluid samples were centrally located on the graph and close to all tissue types, suggesting that it may share features with other tissue microbiomes. While tissue type was a strong driver of microbiome beta diversity, geographic location of the study also played a large role. Samples often clustered together based on geographic re-

gion (Figure 3) and in some cases this more strongly influenced community similarity than sample type (Figure 2); for example, digestive gland samples from southern California were more similar to other southern California tissue samples than to digestive gland samples from other geographic regions (Figures 2 and 3). Likewise, the adductor, gut and mantle samples from Australia were more similar to each other than other samples of the same tissue type from different regions (Figures 2 and 3). Additionally, the distance between samples from Germany and other countries in Europe has a greater difference in microbiome relative to the USA or Australia than the difference between samples from the USA and Australia (Figure 3). There is no significant difference in microbial abundance between samples from the Netherlands and Germany (Figure 3). Samples from France are well scattered, being close to gut samples from the United States, Australia and Germany, as well as adductor samples (Figure 3). Beta diversity group significance tests confirmed community similarities based on tissue type and geographic location. We observed these group differences in the PCOA plot, and also saw significant differences upon running the group pairwise PERMANOVA significance tests.

Alpha Diversity An alpha diversity plot based on the Shannon diversity metric was created to investigate the richness of species in the microbiome within each tissue type, and its respective variation (Figure 4). This is a metric that multiplies the proportion of species with the natural log of the respective proportions to account for both evenness and abundance of microbial species when determining richness. While abundance solely refers to the number of different species present in each tissue type, evenness refers to how similar the abundance of each species is across geographic regions. All tissue types except for extrapallial fluid had significant variation in diversity. In this case, Shannon entropy refers to the level of uncertainty as a result of variation in diversity results, considering that samples collected from each site were random. The majority

Figure 4. Alpha Diversity plot. The plot shows the level of bacterial diversity as determined by 16S rRNA marker gene sequencing within samples of each tissue type of the Pacific Oyster. Oysters were collected across a geographic range spanning five countries across three continents.

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of them had mean Shannon entropy in the range of 4.5-7, suggesting a moderate to high general level of variation in biodiversity within tissue types. The greatest range in entropy values were observed in gut samples. Extrapallial fluid had the greatest mean entropy, followed by whole oyster and hemolymph. The gut had the lowest entropy, followed by the adductor. This suggests that extrapallial fluid had the greatest variety of species in its microbiome, whereas the lowest was the gut, followed by the adductor.

Core Microbiome Rhodobacteraceae, Flavobacteriaceae and Alteromonadaceae were the most common bacterial families and formed the ‘core’ microbiota (represented in >95% of all samples). Alteromonadaceae was only found to be a core taxon in 6 of the 9 tissue types investigated. Tissue types displayed a varied range of taxa in their core microbiomes, with some such as the hemolymph having 13 core taxa. Most of the tissue types followed a linear or exponential pattern of decreasing taxonomic features relative to the fraction of samples, except for extrapallial fluid. Extrapallial fluid had the largest core microbiome, with 86 taxa being identified in 95% samples of each tissue type. This is much higher than fecal samples, which have a feature count of 14 and are the second highest. With two features in 95% of the samples, the gut had the smallest core microbiome. The only core taxa belonged to the Mycoplasmataceae and Rhodobacteraceae family; while Rhodobacteraceae was core for all samples, Mycoplasmataceae was only a core taxon in whole oyster and gut samples. Vibrionaceae was a core taxon in mantle, hemolymph, extrapallial fluid and fecal samples. Rhodobacteraceae and Flavobacteriaceae were found to be in the core microbiome for all tissues. Some taxonomic groups were notably common and of relatively high abundance. Mycoplasmataceae, Spirochaetaceae, and Vibrionaceae, for instance, were among the top ten bacteria in terms of frequency among samples from all geographical and anatomical sites. However, these bacteria were not a part of the core microbiome. While Spirochaetaceae dominated the mantle and gill microbiome frequencies, it is interesting to note that in both tissues, they were not found in more than 35% of samples. Mycoplasmataceae were the most frequent taxa in hemolymph and gut samples, with its frequency being more than double the second most frequent taxa in the gut. Unlike the Spirochaetaceae dominance in gill and mantle tissues, Mycoplasmataceae was found in almost all gut samples and in more than 90% of hemolymph samples. Vibrionaceae had the highest frequency in adductor muscles, followed by Spirochaetaceae. While Vibrionaceae was found in 80% of the samples, Spirochaetaceae was in less than half of all samples. Rhodobacteraceae were the most frequent taxa in whole oyster and fecal samples and were found in all samples for both tissues. The extrapallial fluid had the most unique frequent taxa, with Corynebacteriaceae being the most common in extrapallial fluid. These taxa were not in the list of most frequent taxa for any other sample types.

DISCUSSION The results of this meta-analysis suggest that oyster microbiomes vary significantly based on geographic region. Samples from the same continent were similar, indicated by clusters from the same region on the PCoA plot (Figure 3). Although individual factors such as temperature and salinity were not reported in the studies, the varying taxonomic abundances across geographic regions may be linked to environmental variability. Samples from 38

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the United States and Australia were closer to each other than they were to the Netherlands and Germany (Figure 2). Oyster samples for the former were obtained from sites next to the Pacific Ocean and the latter obtained from Wadden Sea, suggesting that oysters from similar environments tend to cluster together. A major contributor to this could be food sources. For instance, an analysis conducted by Simons and colleagues displayed a significant rise in the relative abundance of Vibrio in oyster fecal samples upon consumption of Tetraselmis algae.14 Additionally, environmental conditions such as pH and salinity have previously been shown to influence animal microbiomes. For example, studies have shown variations in ocean acidification at different regions affecting nutrient cycling and influencing the oyster microbiome. Different geographic regions may also feature genetically distinct Pacific oyster populations. Oyster host genetics have displayed a role in resistance to viral infection and may also influence host-microbiome interactions. Thus, distinct environmental and/or host genetic factors are likely linked to geographic distribution and have a significant impact on the oyster microbiome composition. Disentangling the interconnected effects of host genetics and environmental variability on microbiome composition is an important focus for future research. Tissue types also significantly influence taxonomic composition. Gut samples showed the highest levels of variation (Figure 2) and the smallest core microbiome, influencing geographical clustering as well. For instance, as seen in the PCoA plot (Figure 3), samples from France did not cluster with other samples from Europe (i.e. Germany and Netherlands), as they were all gut samples. As tissue type was highly scattered (Figure 2), if samples of a tissue type with a larger core microbiome were collected from France and analyzed, they would be more likely to be similar to samples from Germany and the Netherlands than their Pacific counterparts. Previous studies have shown that a shift in environmental conditions or specific microbial taxa can impact oyster health. This could explain the observed differences in the microbiome composition for certain tissue types in the present study, although the exact mechanism that causes this change is unknown and is an important question to investigate in future research. Investigation of core microbiomes and dominant microbial taxa among tissue types also supported characteristic signals within specific oyster microbial communities. Rhodobacteraceae, Flavobacteriaceae, and Alteromonadaceae were the most common families of taxa in the study, found in 95% of all samples. Studies have speculated that species belonging to the Flavobacteriaceae family have a specialized role in the decomposition of organic matter, and that species belonging to the Alteromonadaceae family play a role in oyster larvae development. Some genera belonging to the Rhodobacteraceae family, such as Roseobacter, have been found to play a major role in low-level nutrient enrichment for the establishment of microhabitats and cell-cell communication, suggesting that these microbes can play a role in the establishment of oyster reef microhabitats. While other studies have acknowledged the abundant presence of such families in oysters, the roles of Rhodobacteraceae and Flavobacteriaceae bacteria in oysters are yet to be understood and explored. Although the results indicate that tissue types have similar microbial features, the low number of core taxa in sample types such as gut, gill, and adductor samples make it difficult to define a core microbiome in these tissue types. This suggests that environmental changes and other factors may have a greater influence on these specific microbiomes, but less influence on other tissue sqonline.ucsd.edu

or sample types which are more stable. Additionally, although extrapallial fluid has the largest core microbiome, it is important to note that the low number of samples were all from the same sample site and study, making it difficult to compare the stability of its microbiome to other sample types with greater representation. Likewise, the main cavity sample type is only represented by one sample. More research would therefore have to be conducted on low variability or underrepresented sample types to identify their potential core microbiomes. Analysis of the 10 most frequent taxa for each tissue type revealed that although many dominant taxa were not part of the core microbiome for all tissue types, they may still play an important role in microbial ecology and potentially oyster health. For instance, Spirochaetaceae is the most frequently observed family of taxa in mantle tissues overall, even though it is only found in 69 of the 122 samples analyzed. This finding is supported by other studies and could be an indicator of infection. A study conducted by Matsuyama and colleagues on the Akoya pearl oyster (Pinctada fucata martensii) microbiome showed that genes of bacteria belonging to phylum Spirochaetes were only found in the mantle of oysters infected with Akoya Oyster Disease (AOD), an infection that stunts pearl oyster growth and causes significant oyster mortality every year.27 This suggests an important functional role for these microbes, despite the high variability in microbiome composition observed across studies, which may be related to study-specific factors including environmental variables and infection status. We also observed abundance correlations between several potentially ecologically important microbial taxa. For example, tissues with high Mycoplasmataceae frequency tend to have a relatively lower Vibrionaceae frequency, even though the number of samples in which they are found does not change largely. In gut samples, for instance, while the family Mycoplasmataceae has a frequency of 2,007,689, Vibrionaceae only has a frequency of 267,134. Studies have shown that taxa belonging to the Vibrionaceae family found in other sample types as well are associated with oyster mortality and cause vibriosis in humans, suggesting that these taxa could be potential keystone indicators of oyster health and their pathogenicity towards humans. King and colleagues conducted groundbreaking research in 2018 on the pacific oyster microbiome in Australia that displayed Mycoplasma to be dominant in the adductor muscles of healthy oysters, as well as a surge in Vibrio concentrations in diseased oysters.24 This study reinforces the idea that Vibrio-Mycoplasma ratios could potentially be key indicators of oyster health. However, further research must be conducted to understand the relationship between these taxa, and the mechanisms they use to protect or infect oysters.

CONCLUSIONS & FUTURE DIRECTIONS The Pacific oyster microbiome is a complex system that remains poorly understood. The purpose of this meta-analysis was to draw on several recent studies in order to identify major factors that may influence tissue-specific oyster microbiomes from diverse regions and the potential importance of microbial taxa in oyster health. We observed that both geographic variability and sample type influenced taxonomic abundance and community composition. Additionally, variables rarely examined in oyster microbiome studies, including the environment and host genetics, may play a significant role in oyster microbiome variability. As these studies were limited to only four countries, in order to have a greater understanding of the effect of geographic location,

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samples from regions such as Japan, New Zealand, and other European countries must be analyzed as well in order to thoroughly understand this field. More studies on infected, translocated, and heat-shocked oysters must be conducted to identify potential links between such factors and the microbiome. Furthermore, environmental factors such as temperature, salinity, and nutrient concentrations must be quantified. This way, differential abundance tools such as Songbird and DEICODE can be used to visualize change in taxonomic abundance in accordance with various environmental factors, helping us understand the extent to which each factor influences the abundance of specific microbiota. The results of this study reinforce previous suggestions that particular core or abundant microbial taxa may play a role in determining oyster health. Further research must be conducted to understand their mechanisms and individual effects on the microbiome; this could focus on keystone taxa which could be additional indicators of oyster health. Looking at the ratio of relative abundance between two species within oysters could potentially help with identifying oyster symbionts and pathogens, possibly making it an exceptional indicator of oyster health. To extend understanding of how oyster microbiomes are linked to human health, metadata collected during future oyster microbiome analyses could be extended to include quantification of human pathogens, particularly those of marine origin such as Vibrio spp., which are known to accumulate in oysters. A significant amount of financial investment is therefore needed, combined with an indepth understanding of host-microbe interactions and microbial dynamics, to make accurate conclusions on the oyster microbiome and bridge the knowledge gaps there are today.

ACKNOWLEDGEMENTS We would like to thank UC San Diego PhD student Carolina Carpenter for her assistance in validating the meta-analysis.

REFERENCES 1.Office of Habitat Conservation. “Oyster Reef Habitat.” NOAA, 27 July 2020, www.fisheries.noaa.gov/national/habitat-conservation/oyster-reef-habitat. 2. Caruso, Lisa. “CBF Welcomes Fiscal 2021 Oyster Restoration Funds in Bill Approved by House Appropriations Committee.” Chesapeake Bay Foundation, 13 July 2020, www.cbf.org/news-media/newsroom/2020/ federal/cbf-welcomes-fiscal-2021-oyster-restoration-funds-in-bill-approved-by-house-appropriations-committee.html#:~:text=The%20 Corps%20will%20use%20the,restoration%20budget%20is%20%245%20 million. 3. The Nature Conservancy.“Rebuilding Australia’s Lost Shellfish Reefs.” The Nature Conservancy Austraila, 1 June 2020, www.natureaustralia. org.au/what-we-do/our-priorities/oceans/ocean-stories/restoring-shellfish-reefs. 4. Brennan, Dan. “Oysters: Are They Good for You? Pros and Cons, Nutrition Information, and More.” WebMD, WebMD, 29 Sept. 2020, www. webmd.com/diet/oysters-good-for-you#:~:text=Oysters%20are%20 a%20rich%20source,be%20more%20effective%20than%20supplements. 5. Pimentel, Zachary T., et al. “Microbiome Analysis Reveals Diversity and Function of Mollicutes Associated with the Eastern Oyster, Crassostrea Virginica.” MSphere, 2021. 6. King, Gary M., et al. “Analysis of Stomach and Gut Microbiomes of the Eastern Oyster (Crassostrea Virginica) from Coastal Louisiana, USA.” PLoS ONE, vol. 7, no. 12, 2012.

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7. Boettcher, Katherine J., et al. “Roseovarius Crassostreae Sp. Nov., a Member of the Roseobacter Clade and the Apparent Cause of Juvenile Oyster Disease (JOD) in Cultured Eastern Oysters.” International Journal of Systematic and Evolutionary Microbiology, vol. 55, no. 4, 2005, pp. 1531–1537. 8. “Oysters and Vibriosis.” Centers for Disease Control and Prevention, Centers for Disease Control and Prevention, 22 June 2021, www.cdc.gov/ foodsafety/communication/oysters-and-vibriosis.html. 9. Wendling, Carolin C et al. “Persistence, seasonal dynamics and pathogenic potential of Vibrio communities from Pacific oyster hemolymph.” PloS one vol. 9,4 e94256. 11 Apr. 2014. 10. Harris, Jill. “Aquatic Invasive Species Profile: Pacific Oyster, Crassostrea Gigas (Thunberg, 1793) .” University of Washington, Dec. 2008. 11. Gonzalez, Antonio, et al. “Qiita: Rapid, Web-Enabled Microbiome Meta-Analysis.” Nature Methods, vol. 15, no. 10, 1 Oct. 2018, pp. 796– 798. 12. King, William L., et al. “Variability in the Composition of Pacific Oyster Microbiomes across Oyster Families Exhibiting Different Levels of Susceptibility to OSHV-1 Μvar Disease.” Frontiers in Microbiology, vol. 10, 2019.

24. Dang, Hongyue, et al. “Cross-Ocean Distribution of Rhodobacterales Bacteria as Primary Surface Colonizers in Temperate Coastal Marine Waters.” Applied and Environmental Microbiology, vol. 74, no. 1, 17 Dec. 2020, pp. 52–60. 25. Laroche, Olivier, et al. “Understanding Bacterial Communities for Informed Biosecurity and Improved Larval Survival in Pacific Oysters.” Aquaculture, vol. 497, 2018, pp. 164–173. 26. Arfken, Ann, et al. “Denitrification Potential of the Eastern Oyster Microbiome Using a 16S Rrna Gene Based Metabolic Inference Approach.” PLOS ONE, vol. 12, no. 9, 2017. 27. Matsuyama, Tomomasa, et al. “A Spirochaete Is Suggested as the Causative Agent of AKOYA Oyster Disease by Metagenomic Analysis.” PLOS ONE, vol. 12, no. 8, 2017. 28. Petton, Bruno, et al. “The Pacific Oyster Mortality Syndrome, a Polymicrobial and Multifactorial Disease: State of Knowledge and Future Directions.” Frontiers, Frontiers, 18 Feb. 2021, www.frontiersin.org/articles/10.3389/fimmu.2021.630343/full. 29. Froelich, Brett A., and Rachel T. Noble. “Vibrio Bacteria in Raw Oysters: Managing Risks to Human Health.” Philosophical Transactions of the Royal Society B: Biological Sciences, vol. 371, no. 1689, 2016.

13. Hernandez-Agreda, Alejandra, et al. “Defining the Core Microbiome in Corals’ Microbial Soup.” Trends in Microbiology, vol. 25, no. 2, Feb. 2017, pp. 125–140.

30. King, William L., et al. “Characterisation of the Pacific Oyster Microbiome during a Summer Mortality Event.” Microbial Ecology, vol. 77, no. 2, 10 July 2018, pp. 502–512.

14. Simons, Ariel Levi, et al. “High Turnover of Faecal Microbiome from Algal Feedstock Experimental Manipulations in the Pacific Oyster (Crassostrea Gigas).” Microbial Biotechnology, vol. 11, no. 5, 2018, pp. 848–858.

31. Morton, James T., et al. “Establishing Microbial Composition Measurement Standards with Reference Frames.” Nature Communications, vol. 10, no. 1, 2019

15. Stevick, Rebecca J., et al. “Functional Plasticity in Oyster Gut Microbiomes along a Eutrophication Gradient in an Urbanized Estuary.” Animal Microbiome, vol. 3, no. 1, 2021.

32. Martino, Cameron, et al. “A Novel Sparse Compositional Technique Reveals Microbial Perturbations.” MSystems, vol. 4, no. 1, 12 Feb. 2019.

16. Scanes, Elliot et al. “Climate Change Alters the Haemolymph Microbiome of Oysters.” Marine Pollution Bulletin, vol. 164, Mar. 2021, p. 111991. 17. Bernatchez, S. et al. “Seascape genomics of eastern oyster (Crassostrea virginica) along the Atlantic coast of Canada” Evol Appl. 2019; 12: 587– 609. 18. de Lorgeril, J. et al. “Immune-suppression by OsHV-1 viral infection causes fatal bacteraemia in Pacific oysters” Nat Commun 9, 4215 (2018). 19. Pathirana, E., et al. “The Role of Tissue Type, Sampling and Nucleic Acid Purification Methodology on the Inferred Composition of Pacific Oyster (Crassostrea Gigas) Microbiome.” Journal of Applied Microbiology, vol. 127, no. 2, 2019, pp. 429–444. 20. King, William L., et al. “Oyster Disease in a Changing Environment: Decrypting the Link between Pathogen, Microbiome and Environment.” Marine Environmental Research, vol. 143, Jan. 2019, pp. 124–140. 21. De Lorgeril, Julien, et al. “Immune-Suppression by OSHV-1 Viral Infection Causes Fatal Bacteraemia in Pacific Oysters.” Nature Communications, vol. 9, no. 1, 11 Oct. 2018. 22. Fernández-Gómez, Beatriz, et al. “Ecology of Marine Bacteroidetes: A Comparative Genomics Approach.” The ISME Journal, vol. 7, no. 5, 10 Jan. 2013, pp. 1026–1037. 23. Dubé, Caroline Eve, et al. “Microbiome of the Black-Lipped Pearl Oyster Pinctada Margaritifera, a Multi-Tissue Description With Functional Profiling.” Frontiers, Frontiers, 5 Jan. 2019, www.frontiersin.org/ articles/10.3389/fmicb.2019.01548/full#B67.

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The Effect of the Amino-Terminal Fragment (ATF) on the Activity and Inhibition of Urokinase-Type Plasminogen Activator (uPA) Harriet J. Song [1], Constanza Torres-Paris [2], and Elizabeth A. Komives*

[1] Division of Biological Sciences, University of California San Diego, Earl Warren College, Molecular and Cell Biology Major, Class of 2022, La Jolla, California, United States, [2] Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California, United States

ABSTRACT Urokinase-type plasminogen activator (uPA) is a serine protease responsible for cleaving and activating inactive plasminogen to its active form, plasmin, which is involved in fibrinolysis, angiogenesis, and extracellular matrix degradation. Plasmin, in turn, is a uPA zymogen activator. Hence, the plasmin-uPA system is a positive feedback loop. The catalytic activity of uPA is influenced by the orientation of its catalytic triad and the substrate-binding affinity at the uPA’s specificity pocket. The enzyme consists of an amino-terminal fragment (ATF) connected to the uPA protease domain via a disulfide bond and a disordered linker. We wanted to examine the effect the ATF has on the catalytic activity of the uPA protease. We hypothesized that the presence of the ATF enhances the activity, increases the inhibitory capacity (how much inhibitor uPA can tolerate), and alters the dynamics of the uPA protease. In this study, the activity and inhibition of full-length human uPA (huPA) with the ATF, huPA with an N-terminal polyhistidine tag replacing the ATF (huPA protease with N-terminal His-tag), and huPA without ATF entirely (huPA protease), were compared with and without the inhibitor benzamidine. Our results showed that the catalytic efficiency (kcat/Km) of full-length huPA and huPA protease with N-terminal His-tag are fourfold and threefold higher than that of huPA protease, respectively. Furthermore, the uPA inhibitor, benzamidine’s IC50 (inhibitor concentration needed to inhibit uPA to half its uninhibited activity) for the full-length huPA and the huPA protease with N-terminal His-tag are fivefold and twofold higher than that of the protease domain only, respectively. These results suggest that the presence of the ATF enhances the catalytic activity of huPA and reduces benzamidine’s capacity to inhibit huPA. Moreover, the N-terminal His-tag, like the ATF, seems to partially enhance huPA activity. By scrutinizing the significance of the ATF to the activity of huPA, we can introduce more research questions about how the uPA-plasmin system can be regulated in our physiological systems.

INTRODUCTION Urokinase-type plasminogen activator (uPA) is a serine protease responsible for cleaving and activating the inactive plasminogen to active plasmin. The plasmin-uPA system is largely involved in regulating the degradation of the extracellular matrix and fibrin blood clots as well as angiogenesis, which is the formation of new blood vessels.1,2 Deregulation of the uPA-plasmin system can contribute to tumor progression caused by factors such as tumor angiogenesis.3,4 uPA consists of an N-terminal Epidermal Growth Factor-like (EGF-like) domain, a Kringle domain, a disordered linker region, and a protease domain. The EGF-like domain and the Kringle domain collectively comprise the amino-terminal fragment (ATF).5 The linker region connects the ATF to the C-terminal catalytic protease domain of the uPA (Fig 1). In its zymogen form, uPA is one single chain. Active uPA after plasmin cleavage consists of two chains: the ATF-linker and the catalytic protease, which are held together via a disulfide bond.2 Previous hydrogen-deuterium exchange mass spectrometry (HDX-MS) data from our lab revealed that although the ATF is distant from the protease domain, its presence makes regions of the protease more rigid (less dynamic). Higher rigidity in certain uPA regions indicate that the regions are more “stuck” in a certain conformation. Thus, the presence of the ATF may play a role in the dynamic allostery, or the modulation of conformational dynamics of the uPA protease from a distance. Additionally, our lab also showed that when the ATF is present, uPA shows an increase in catalysis (Fig 2). In the crystal structure of the protease domain of uPA (PDB: 1EJN), we can see the catalytic triad (His57, Asp102 and Ser195) which is responsible for cleaving substrates and the approximate specificity pocket region (around Asp189) which is where substrates would bind. The activity of uPA results from both the catalytic efficiency of the catalytic triad and the substrate-binding affinity at the specificity pocket. Thus, the presence of the ATF seems to affect both of the mentioned regions of the protease domain. As most of the current research on uPA scrutinizes the uPA protease domain only, we wanted to investigate whether the presence of the ATF alters the activity and inhibitory capacity of huPA. Understanding how the ATF influences the activity and inhibition of huPA would allow us to better understand and pose more questions regarding the role of the ATF in huPA. While huPA activity results from both the activity of the catalytic triad and substrate binding specificity at the specificity pocket, inhibition assays will depict the ATF’s particular effects on the

Figure 1: 2-D domain cartoon depiction of huPA. (a) full-length huPA, (b) huPA protease, (c) huPA protease with N-terminal His-tag.

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Figure 2: Previously done activity assays of huPA with the S-2444 and Glu-plasminogen substrates, done side-by-side. The rates of hydrolysis of (a) synthetic chromogenic uPA substrate S-2444 and (b) uPA substrate Glu-plasminogen by 10nM of full-length huPA and huPA protease after preincubation with the indicated concentrations of S-2444 (0-500 μM) and Glu-plasminogen (0-10 μM) are shown.

inhibitor and substrate binding specificity pocket.6 We examined full-length huPA which consists of the entire ATF-linker-protease and compared it to huPA protease which consists of only the linker-protease. In addition, we also examined huPA protease with an N-terminal His-tag in the place of the ATF to see whether the activity and inhibition of the enzyme are altered. We hypothesize that the ATF can stabilize the uPA protease by increasing its rigidity and determining its dynamics. Particularly, with the ATF, the uPA catalytic triad would more likely be in the active state conformation, thus it would be more frequent and effective at cleaving its substrate. In the case of the huPA protease, we hypothesize that because there is no ATF present, the protease domain responsible for catalysis is more dynamic (frequent movements), so the catalytic triad is less frequently in the correct conformation for substrate cleavage. Furthermore, the ATF may play a role in increasing the substrate specificity and affinity of uPA’s substrate-binding pocket in the uPA protease domain, making full-length huPA more selective.

MATERIAL AND METHODS Purification of Full-Length huPA, huPA protease, and huPA protease with N-terminal His-tag. To test whether the addition of the ATF impacted catalytic ability, we made the enzyme using the following approach. Fulllength huPA, huPA protease, and huPA protease with N-terminal His-tag were expressed as inclusion bodies in BL21 (DE3) competent E. coli cells. To properly refold the proteins and form the correct disulfide bonds, the E. coli cell pellets were resuspended in sonication buffer (0.5 M NaCl; 50 mM Tris-HCl pH 8; 1 mM EDTA pH 8; 10% glycerol by volume; 1 mM β-mercaptoethanol; 1% Triton X by volume) and sonicated on ice (amplitude 50). 300 µg DNAse I, 300 µL 1 M MgCl2, and 300 µL 1 M MnCl2 were added to the lysed cells, which were then incubated while rocking at room temperature. The sonicated cell lysate was treated with DNAse for 45 minutes and then centrifuged at 17,000 RCF, 4 °C for 15 minutes. The pellets were washed once with a modified sonication buffer with 0.25% Triton X by volume, and once with another modified sonication buffer without Triton X. With each wash, the pellet was resuspended and centrifuged at 17,000 RCF, 4 °C for 10 minutes. The washed inclusion bodies were then resuspended in denaturing buffer 1 (6 M Urea, 100 mM NaCl; 50 mM Tris-HCl pH 8; 1 mMradation during storage). When ready for activity and inhibition assays, fresh huPA proteins were size excluded with a Superdex 75 column equilibrated with 1X PBS at pH 7.4. Fractions collected for the corresponding huPA protein peak were pooled together, concentrated, and used for activity and inhibition assays.

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Activity Assays with Chromogenic Substrates To evaluate the activity of full-length huPA, huPA protease, and huPA protease with N-terminal His-tag, 10 nM enzyme was incubated for 10 minutes in 1X PBS pH 7.4 with various concentrations of substrates at 37 °C using 96-well microtiter plates. After adding huPA, the initial velocities of the chromogenic substrate cleavage resulting in p-nitroaniline (pNA) formation were monitored for 10 minutes at 37 °C at an absorbance of 405 nm in a microplate reader. The two huPA substrates used were S-2444 and Glu-plasminogen. S-2444 (Pyro-Glu-Gly-Arg-pNA) (DiaPharma): The concentrations of S-2444 used were 0 µM to 500 µM. When S-2444 is hydrolyzed by huPA, pNA is produced. The product absorbs light at 405 nm, thus pNA formation can be monitored with absorbance at 405 nm to quantify the initial velocity of the huPA enzymes. The absorbance data was converted to the velocity of product formation (initial velocity of enzyme); the maximum reaction rate (Vmax) and Michaelis constant (Km) values were determined by fitting the data to the Michaelis-Menten kinetics equation in GraphPad Prism. The turnover number (kcat) and the catalytic efficiency of enzymes (kcat/Km) were determined with the corresponding Vmax and Km values. Glu-plasminogen (Haematologic Technologies): The concentrations of Glu-plasminogen used ranged from 0 µM to 10 µM. The chromogenic substrate for active plasmin is S-2251 (DiaPharma). When Glu-plasminogen is activated to plasmin by huPA, plasmin hydrolyzes S-2251 and produces pNA. pNA formation was monitored with absorbance at 405 nm to quantify the initial velocity of plasmin and therefore the initial velocity of the huPA enzymes. Inhibition Assays with Benzamidine and UK-122 Benzamidine: To evaluate how full-length huPA, huPA protease, and huPA protease with N-terminal His-tag are inhibited by competitive inhibitors, 10 nM huPA enzymes were incubated with various concentrations of the inhibitor at 37 °C in 1X PBS for 10 minutes on 96-well microtiter plates. Then, S-2444 was added to a final concentration of 75 µM in the reaction. The initial velocities of the chromogenic substrates cleavage to pNA by huPA enzymes were monitored for 10 minutes at 37 °C with an absorbance of 405 nm in the microplate reader. The IC50 values for the huPA constructs were calculated using GraphPad Prism by fitting the normalized initial velocity data of the huPA enzymes to the equation Dose-response – Inhibition: [inhibitor] vs. normalized response – variable slope. The normalized data (percent activity of the huPA proteins) were calculated based on the initial velocity of the huPA proteins when no inhibitor was added (100 percent activity).

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Table 1: S-2444 and Glu-plasminogen activity assay kinetic constants for the huPA proteins are analyzed according to Michaelis-Menten kinetics equation on GraphPad Prism. The table shows ± standard errors of experimental duplicates. kcat and kcat/Km error are calculated with an error propagation calculator (University of British Columbia).

UK-122 inhibition: UK-122 was prepared in dimethyl sulfoxide (DMSO). 10 nM huPA enzymes were incubated with different concentrations of UK-122 at 37 °C in 1X PBS, 5% DMSO, and 10% BSA for 10 minutes. As UK-122 is colored, a control well was implemented using all the reagents that comprise the experimental wells, except the huPA enzymes. The absorbances from the control wells were subtracted from every corresponding experimental well.

DATA AND RESULTS Activity and inhibition assay analyses were performed with both full-length huPA (with ATF) and huPA protease (without ATF). Our lab has previously shown that with the S2444 and Glu-plasminogen huPA substrates, there appear to be differences between the activity of full-length huPA and huPA protease. The difference is more apparent in the Glu-plasminogen activity assay, where the Vmax of the huPA protease showed an approximate sixfold decrease in comparison to that of the full-length huPA (Fig 2b). Additionally, the kcat/Km value of full-length huPA is approximately fourfold higher than that of huPA protease (Table 1b). On the other hand, the rate of the hydrolysis of full-length huPA is approximately one and a half times lower than that of huPA protease in the S-2444 activity assay (Table 1a). The activity assays showcase enzyme differences in both the catalytic triad dynamics (how often the triad is in the correct catalytic orientation to hydrolyze the substrate) and the substrate binding specificity at the specificity pocket. Thus, it is worth investigating how the two protein constructs differ specifically at the specificity pocket with inhibition assays. As inhibition assays focus on binding of inhibition substrates at the specificity pocket, we used this method to compare full-length huPA and huPA protease to elucidate the effect of ATF on the huPA protease domain. sqonline.ucsd.edu

Activity Assays with Chromogenic Substrate S-2444 and Substrate Glu-plasminogen Activity assays of full-length huPA and huPA protease with N-terminal His-tag were performed with the chromogenic substrate S-2444 and substrate Glu-plasminogen (Fig 3a-3b). The activity of the full-length huPA and huPA protease with N-terminal His-tag are approximately identical. The kcat/Km values of fulllength huPA and huPA protease with N-terminal His-tag are very similar in both the S-2444 and Glu-plasminogen activity assays. The kcat/Km value of huPA protease is more than threefold lower than that of huPA protease with N-terminal His-tag (Table 1a1b). When superimposed, we can clearly see the activity of huPA protease with N-terminal His-tag is only slightly lower than the activity of full-length huPA (Fig 3c). Inhibition Assays with uPA Inhibitor Benzamidine Inhibition assays of full-length huPA, huPA protease, and huPA protease with N-terminal His-tag were performed with S-2444 as the chromogenic substrate to measure huPA activity, and benzamidine as the competitive inhibitor to monitor inhibition (Fig 4). The corresponding IC50 values, the concentration of benzamidine required for the enzyme to be inhibited by 50% at 75 μM S-2444, are indicated in Table 2. Full-length huPA has the highest IC50, approximately twofold higher than the IC50 of huPA protease with N-terminal His-tag and approximately fivefold higher than that of huPA protease. Based on the inhibition assay curves, we can see that the inhibition of full-length huPA has a higher percent activity (~25%) than huPA proteases (~15%). Although the IC50 of huPA protease is higher than that of huPA protease with N-terminal His-tag, the inhibition curves plateau at similar percent activity values.

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Figure 3: Activity assays of huPA with the S-2444 and Glu-plasminogen substrates conducted side-by-side. The rates of hydrolysis of (a) synthetic chromogenic uPA substrate S-2444 and (b) uPA substrate Glu-plasminogen by 10nM of full-length huPA and huPA protease with N-terminal His-tag after preincubation with the indicated concentrations of S-2444 (0-500 μM) and Glu-plasminogen (0-10 μM) are shown. Although not directly comparable (not done side-by-side), rates of hydrolysis of Glu-plasminogen for full-length huPA (Figure 2), is superimposed in (c) with huPA protease, and huPA protease with N-terminal His-tag for visualization.

Figure 4: Inhibition of full-length huPA, huPA protease, and huPA protease with N-terminal His-tag by protease inhibitor, benzamidine. Rates of hydrolysis of S-2444 (75 μM) by 10 nM of huPA after preincubation with the indicated concentrations of benzamidine (0 – 1.5 mM) are shown. The rates of hydrolysis/activity of the huPA enzymes were normalized to the rates of hydrolysis in the absence of the inhibitor benzamidine (100% activity), resulting in the percent activity of the enzymes at different benzamidine concentrations.

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Table 2: IC50 of the huPA proteins for benzamidine are analyzed according to the GraphPad Prism equation Dose-response – Inhibition: [inhibitor] vs. normalized response – variable slope. The table shows ± standard errors of experimental duplicates.

Inhibition Assays with uPA Inhibitor UK-122 UK-122 is a larger and more specific competitive inhibitor than benzamidine.7 Inhibition assays of full-length huPA and huPA protease with N-terminal His-tag using UK-122 as the competitive inhibitor and S-2444 as the chromogenic substrate were performed and are in progress of standardization. We started the UK-122 inhibition assay with full-length huPA with a low range of UK-122 concentration (0 to 500 nM) because the IC50 of uPA for UK-122 is 200 nM.7 However, the range of UK-122 was too low and narrow, as full-length huPA activity only went down to approximately 75% (Fig 5a). Next, we broadened the UK-122 concentration range and performed the inhibition assay with huPA protease with N-terminal His-tag (Fig 5b) and obtained its IC50 for UK-122 (Table 2).

DISCUSSION

Previous experiments with Glu-plasminogen activity assays demonstrated that full-length huPA has higher activity than huPA protease (unpublished data by Constanza Torres Paris). These findings evoked the research question regarding the effects ATF may have on the activity (catalytic triad dynamics and substrate affinity at the specificity pocket) and inhibition (inhibition and substrate binding affinity at the specificity pocket) of huPA. On the other hand, the differences between full-length huPA and huPA protease were not as apparent in S-2444 activity assays. The differences in the results from the Glu-plasminogen and S-2444 activity assays provoke questions as to why different substrates show different uPA activity changes in the uPA constructs used in this study. Plasminogen (molecular weight: 88,000 g/mol) is a much bigger substrate for uPA than S-2444 (molecular weight: 489.9 g/mol). We are interested in investigating whether the presence of the ATF impacts the catalytic triad orientation and the specificity pocket, as well as the ability to identify these effects. The Glu-plasminogen and S-2444 activity assays both showed no significant difference between the hydrolysis rates of the full-length huPA and huPA protease with N-terminal His-tag (Fig 3). For the Glu-plasminogen assay in particular, the activity of huPA protease with N-terminal His-tag is more than threefold greater than the activity of huPA protease, which has no His-tag in the place of the ATF. The data suggests that having the N-terminal His-tag in the place of the ATF influenced the activity and inhibition of huPA. In other words, when the His-tag is attached to the N-terminal side of the huPA protease in place of the ATF, huPA activity is improved. This is shown by an increase in Vmax and kcat/Km in comparison to huPA protease. It is possible that the ATF enhances the efficiency of the uPA protease domain through an allosteric effect on the protease domain via the linker. However, the effect of the N-terminal His-tag on huPA protease is not apparent in the results obtained from S-2444 activity assays. This may be due to the differences in the substrates S-2444

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and Glu-plasminogen, as plasminogen is a much bigger huPA substrate than S-2444. The presence of the N-terminal ATF and N-terminal His-tag may have contributed to a higher substrate binding affinity for plasminogen, the natural substrate of huPA. The enhancement of specificity may not have been as apparent for the synthetic chromogenic substrate S-2444. However, since activity assays look at both the catalytic triad efficiency and the specificity pocket, the differences between full-length huPA and huPA protease may have resulted from a difference in the catalytic efficiency of the catalytic triad as well. Thus, inhibition assays may be able to tell us more about what is happening at the specificity pocket of the uPA protease. Inhibition assays using benzamidine as the inhibitor and S-2444 as the huPA substrate were performed. It was hypothesized that full-length huPA will bind weaker to benzamidine because it is likely more specific to its substrates than a general serine protease inhibitor. According to the IC50 values in Table 2, full-length huPA requires a fivefold higher concentration of benzamidine than huPA protease to have its activity inhibited by 50%. huPA protease with N-terminal His-tag also requires more than twofold more benzamidine than huPA protease to inhibit its activity by 50%. These findings suggest that full-length huPA binds weaker to the competitive inhibitor benzamidine than the other two uPA constructs. Interestingly, huPA protease with N-termial His-tag also requires more than twofold more benzamidine than huPA protease to inhibit its activity by 50%. These findings suggest that full-length huPA binds weaker to the competitive inhibitor benzamidine than the other two uPA constructs. Interestingly, huPA protease with N-terminal His-tag experienced less inhibition than huPA protease. Collectively, these inhibition assays suggest that the presence of the ATF enhances the substrate binding affinity for S-2444 more than the presence of the His-tag at the specificity pocket of the huPA protease domain. Benzamidine-Glu-plasminogen inhibition assays were unfeasible because benzamidine will inhibit the plasmin when plasminogen is activated.1 Thus, we will not be able to measure huPA’s activity if plasmin cannot hydrolyze its chromogenic substrate, S-2251, to show uPA activity (Fig 2b). Benzamidine is a very small inhibitor in comparison to plasminogen, the natural substrate for huPA. Moreover, benzamidine is also a very broad inhibitor with low specificity to huPA. The findings from benzamidine inhibition assays provoke the question of whether other inhibitors with variations in sizes and specificity will show similar results when investigating the impact of ATF on uPA. UK-122 is a synthetic inhibitor which was reported to have a high specificity for uPA, with an IC50 of 0.2 μM.2 The IC50 obtained from our experiments on huPA protease, however, was 96 μM (reported as 0.10 ± 0.01 mM in Table 2). Our results show that UK-122 binds weaker to both full-length huPA and huPA protease compared to what was previously reported. In addition, the

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binding between UK-122 and full-length huPA was too weak to calculate an IC50. We were surprised to find a discrepancy between our results and the previously reported IC50 of 0.2 μM.7 It is worth noting that when UK-122 was first synthesized and tested, purified full-length huPA was reportedly used by American Diagnostica.7 Based on our experiments we learned that active full-length huPA quickly undergoes autocatalytic cleavage into huPA protease by cleaving off the ATF. Thus, the experimenters who developed and tested UK-122 could have been testing the inhibitor with huPA protease instead of full-length huPA; we plan to measure the UK-122 inhibition assays in the future to evaluate these findings. Moreover, we will test UK-122 for inhibition with Glu-plasminogen as the substrate because UK-122 is reported to be specific for uPA and does not inhibit plasmin at the UK-122 concentrations we will be using in our assays.7 It is possible that UK-122 is a better inhibitor against plasminogen than against S-2444. As all of the assays done in this project were performed with freshly purified huPA proteins, the findings discovered in this project push for future investigations into the differences between full-length huPA and huPA protease.

CONCLUSION Our results showed that the kcat/Km, of the full-length huPA is fourfold higher than that of the huPA protease alone, while the huPA protease with N-terminal His-tag had a kcat/Km threefold higher than that of huPA protease. On the other hand, the IC50 of benzamidine on full-length huPA is fivefold higher than that of the huPA protease alone, while the IC50 of huPA protease with an N-terminal His-tag is twofold higher than that of the huPA protease alone. These results indicate that the presence of the ATF does affect huPA activity. In particular, the ATF enhances catalytic activity and reduces the capacity of benzamidine to inhibit uPA. Interestingly, the N-terminal His-tag appears to serve a role in partially stabilizing the protease in the place of the ATF. Studying the ATF’s effect on uPA activity gives insight as to how uPA activates plasmin. This observation facilitates an improved comprehension of plasmin’s involvement in the corresponding physiological processes such as regulating the degradation of the extracellular matrix and fibrin blood clots. Additionally, uPA activity is closely related to unregulated involvement of plasmin in angiogenesis, a significant pathological phenomenon under study. In the future, we plan to reproduce and optimize the inhibition and activity assays with UK-122 and new uPA inhibitors and activators, including ATP (inhibitor) and polyphosphates (activator), that are present in natural blood clot environments. By looking at more inhibitors with different sizes and uPA specificities, we can take a closer look at how the ATF influences the specificity pocket of huPA. After comparing the inhibition assay data from full-length huPA and huPA protease, we also plan to perform HDX-MS to scrutinize the change in dynamics between the various huPA constructs and map where these compounds are binding to the uPA.

periential Learning Scholars (TRELS) Quarterly Program as well as the Undergraduate Research Hub at UCSD.

REFERENCES 1. Andreasen, P. A., Kjoller, L., Christensen, L. & Duffy, M. J. The urokinase-type plasminogen activator system in cancer metastasis: a review. Int J Cancer 72, 1–22 (1997). 2. Andreasen, P. A., Egelund, R. & Petersen, H. H. The plasminogen activation system in tumor growth, invasion, and metastasis. Cell Mol Life Sci 57, 25–40 (2000). 3. Dano, K. et al. Plasminogen activators, tissue degradation, and cancer. Adv Cancer Res 44, 139–266 (1985). 4. Deryugina, E. I., & Quigley, J. P.. Cell surface remodeling by plasmin: a new function for an old enzyme. Journal of biomedicine & biotechnology, 2012, 564259. 5. Barinka, C. et al.. Structural basis of interaction between urokinase-type plasminogen activator and its receptor. Journal of molecular biology, 363(2), 482–495 (2006). 6. Hedstrom, L.. Serine protease mechanism and specificity. Chemical Reviews, 102(12), 4501–4523 (2002). 7. Zhu, M. et al.. Identification of a novel inhibitor of urokinase-type plasminogen activator. Molecular Cancer Therapeutics, 6(4), 1348–1356 (2007). 8. Kromann-Hansen, T. et al.. Ligand binding modulates the structural dynamics and activity of urokinase-Type plasminogen activator: A possible mechanism of plasminogen activation. PLoS ONE, 13(2), 1–16 (2018). 9. Kromann-Hansen, T., Lund, I. K., Liu, Z., Andreasen, P. A., Høyer-Hansen, G., & Sørensen, H. P.. Allosteric inactivation of a trypsin-like serine protease by an antibody binding to the 37- and 70-Loops. Biochemistry, 52(40) (2013) 10. Alves, N. J., & Kline, J. A.. Comparative study on the inhibition of plasmin and delta-plasmin via benzamidine derivatives. Biochemical and Biophysical Research Communications, 457(3), 358–362 (2015). 11. Papaleo, E. et al.. The role of protein loops and linkers in conformational dynamics and allostery. Chemical Reviews, 116(11), 6391–6423 (2016). 12. Sperl, S. et al.. (4-Aminomethyl)phenylguanidine derivatives as nonpeptidic highly selective inhibitors of human urokinase. Proceedings of the National Academy of Sciences of the United States of America, 97(10), 5113–5118 (2000).

ACKNOWLEDGEMENTS I acknowledge the UC San Diego Komives Lab. Specifically, this work was supervised and supported by my Principal Investigator Dr. Elizabeth Komives and graduate student mentor Constanza Torres Paris. This work was funded by the Undergraduate Research Scholarships (URS) – Eureka! Scholarship for Biological Sciences Majors, which was sponsored by Dr. Wendy Kwok. Additionally, I would like to acknowledge the Triton Research & Ex46

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Identifying Drivers of Neuropathic Pain in Arthritis ASIM MOHIUDDIN

Neurobiology Major, Eleanor Roosevelt PI: Dr. Maripat Corr, UC San Diego Department of Rheumatology, Allergy & Immunology

Rheumatoid arthritis (RA) is a debilitating autoimmune disease that causes painful inflammation within joint tissue. Despite the success of modern therapeutics in treating detectable inflammation, patients with RA often experience lingering neuropathic pain. Neuropathic pain is a chronic burning sensation that results from a disruption of the somatosensory nervous system

and extensive nerve damage. This study focuses on identifying potential connections between neuropathic pain and the inflammatory response that is characteristic of RA. These connections were determined and analyzed using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) program application. Genes previously identified as relating to human disease in both mouse models and human genes were first entered into STRING. Based on the quantity and strength of the connections generated, several genes were identified as potential critical intersection points for the development of neuropathic pain. The assessment included several characteristics of the genes such as physical proximity, pathway relation, co-expression, and their history of co-mention in previous PubMed abstracts. The genes identified in this study as critical intersection points for neuropathic pain development can be further studied to develop possible therapeutics against RA.

INTRODUCTION

Figure 1. Two genes were chosen from each functional group for having the largest number and strongest correlative connections. In the image above, the genes were remapped independently to reveal their cross genomic interactions. The genes depicted in this STRING virtual model are TRPV1 and SCN10A (purple), IL10 and TLR4 (yellow), POMC and CCR2 (blue), BDNF and COMT (green), and SOD2 and TGFB1 (red). The red and green connecting lines represent predicted interactions between genes while blue and pink lines represent known interactions.

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Rheumatoid arthritis (RA) is an autoimmune disease where the immune system causes joint inflammation, potentially resulting in permanent damage and deformity.1 This form of arthritis affects women three times more often than men, and in severe cases, it disrupts organ functionality.1 An estimated 1.3 million people, making up one percent of the world’s population, are afflicted with this disease.1 Common symptoms of RA include joint swelling and tactile pain on the limbs. The chronic pain characteristic of RA does not necessarily correlate with detected levels of joint swelling. There is a range in the levels of joint swelling in relation to pain that patients experience. Specifically, the onset of pain prior to swelling is a main complaint from RA patients.2 The pain induced by RA is often described as gnawing or aching, descriptions which have often been associated with nociceptive pain caused by tissue damage. Joint structures are lined with nociceptive neurons, whose cell bodies are located in the dorsal root ganglion. During the inflammatory process, immune cells release proinflammatory cytokines while nociceptive cells release calcitonin gene-related peptides which sensitize the primary afferent neuron, which relays the pain signal to the CNS. As a result, patients experience amplified pain in the joints and spinal column. This mechanism outlines the onset and effects of nociceptive pain.4 In contrast to this nociceptive pain, many RA patients also use descriptors that are typical of neuropathic pain: the SALTMAN QUARTERLY

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perception of pain in the absence of tissue damage or nociceptive input. Neuropathic pain is usually caused by damage to central and peripheral nerves and causes sensations such as burning or prickling.4 Unlike nociceptive pain, the mechanism for neuropathic pain is not yet completely understood; however, it is assumed to be caused by a lesion or disease affecting the somatosensory nervous system, thus leading to the perception of pain even in the absence of pain stimuli. It is maladaptive (impairs daily motor functions) and persists with minimal or undetected peripheral inflammatory pathology. Patients are diagnosed with neuropathic pain using a combination of sensory tests and clinical expertise.5 Symptoms include several abnormal sensations such as allodynia, or pain caused by a stimulus that does not induce pain under normal conditions. The neuropathic element of pain in chronic RA has been previously well documented; therefore, this project aims to investigate the role of the neuropathic element of pain in the larger scheme of an inflammatory response. Specific antibodies target the synovium (soft tissue that lines joint cavities) and cartilage, leading to the progression of RA. Recent studies show that epigenetic disorders such as abnormal histone modification and DNA methylation contribute to the generation of rheumatoid arthritis synovial fibroblasts (RASF).3 RASF lines the joint and secrete degrading enzymes, proinflammatory cytokines, and chemokines that generate inflammation.2 This inflammation activates osteoclasts, or cells that break down bone tissue, which can cause permanent bone damage. Current treatment options for RA include non-steroidal anti-inflammatory drugs which block cyclooxygenase, an enzyme which is used to make prostaglandins. Prostaglandins respond to injury in the body and cause inflammation. Consequently, blocking cyclooxygenase relieves inflammation. Another treatment option includes disease-modifying antirheumatic drugs. This class of drugs works to treat RA symptoms by suppressing the body's immune and inflammatory responses. The mechanisms of these drugs often operate by inhibiting the transcription of specific genes in inflammatory and immune pathways. The wide range of RA symptoms makes it challenging to discover therapeutics that address both pain and swelling. Although several effective anti-inflammatory treatments have been developed, current therapies targeted at effectively treating inflammation inadequately address the accompanying chronic pain patients’ experiences.

METHODOLOGY In this project we aimed to investigate the role of the neuropathic element of pain in the larger scheme of an inflammatory response. A literature search was conducted utilizing the PubMed search engine for known genes associated with inflammation, neuropathic pain, nociceptive pain, and proteins in these pathways. The following terms were used: inflammation, neuropathic pain, immune response, and nociceptive pain. The immune pathways are incredibly complex and consist of many cellular messenger cascades including cytokine mediators, phosphorylation complexes, and a variety of cross-genomic interactions. Due to this inherent complexity, the literature provided an abundance of relevant genes. These genes included promoters, as well as growth factors, transcription factors, cytokines, and a variety of other ligands. All of the ligands we found work together in the cell signaling of nociceptive pain, neuropathic pain, inflammation, and immune response. A collection of 150 genes were compiled from multiple articles and then entered into STRING, an open access 48

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biological database and web-based resource of known and predicted interactions between proteins (https://string-db.org/). The program provided a plethora of information including background information on each protein, co-expression rates, relevant pathways, and tissue expression. STRING then ranked the most highly connected genes. From this initial screening, candidate genes that were observed to have the highest densities of correlations with other genes were selected and re-entered into STRING for further analysis. More specific analysis of the relationship between these genes may assist in identifying the larger role these genes may play in the pathology of RA, and more specifically neuropathic pain, as the pathologies of these genes overlap with those proven to be drivers of RA and neuropathic pain.

RESULTS From our compiled list of 150 genes, the program identified 5 main clusters of proteins coded by the genes. The functional clusters are genes which impact inflammation, neuropathic pain, cellular growth, and immune response. The fifth category, termed “other,” identifies genes which characteristically differ from the specified categories. Connections between two genes are indicated by a line drawn between the two. The two most correlated genes from each group (highest ranked by STRING) were chosen and mapped to see the quality and quantity of the cross-genomic interactions. The genes identified were BDNF & COMT (cellular growth), POMC & CCR2 (immune response), TRPV1 & SCN10A (neuropathic pain), IL10 & TLR4 (inflammation), and SOD2 & TGFB1 (other). The selected genes were entered into STRING a second time to narrow the potential critical links between them. As seen in Figure 1, the correlative strength and quantity of connections varies. IL10, BDNF, and TRPV1 were the most connected genes shown. STRING shows that these genes are involved in the defense response of the human body, such as cytokine binding, with an abundance of genes being expressed in dendritic cells. Dendritic cells (which function as antigen presenting cells in the immune system) can induce a primary immune response in otherwise inactive T-lymphocytes and therefore play an important role in relaying information between the innate and adaptive immune system.

DISCUSSION This project was conducted by entering pain-associated genes intThe goal of this study aimed to investigate the role of the neuropathic element in the larger scheme of an inflammatory response with the overarching goal of developing therapeutics for neuropathic pain in RA. Through this study, several genes were identified as potential targets for therapeutics. In recent years, researchers have developed monoclonal antibodies, or laboratory-made antibodies aimed to bolster the immune system defense, directed against cytokines to reduce disease burden particularly in inflammation and pain. However, the treatment has not completely arrested long-term bone destruction in RA.6 Many of these monoclonal antibodies target individual cytokines. Additionally, our research did not identify the tumor necrosis factor (TNF) as a key variable in RA induced neuropathic pain, indicating that its correlative strength and quantity of connections did not merit a high enough ranking in STRING. TNF is the most commonly targeted cytokine by existing RA drug therapies, so it is unusual this gene would not show up in our meta-analysis. A possible explanation is that TNF is involved sqonline.ucsd.edu

in nociceptive pain and inflammation but not neuropathic pain. The following are the highlighted target genes of this study. Our search uncovered type I interferon receptor (IFNAR1) and IL10 as significant to RA. In standard immune responses, type I interferons have an important role in immune defense. However, RA patients demonstrate persistent activation of the type I interferon pathway. Overactivation of this pathway has been shown to increase inflammatory response.7 These results indicate that IL10 has anti-inflammatory and immunoregulatory roles that suggest a potential therapeutic role in RA. The Janus kinase (JAK) inhibitors reduce the downstream signaling pathways for many cytokine receptors including the type I interferon pathway and the IL10 receptor, thus regulating the pathways which cause inflammation. However, these kinase inhibitors are only effective in roughly two-thirds of RA patients.7 Type I interferon (IFN) expression in the joints of arthritic mice is regulated by interferon regulatory factors (IRF) 3 and 7. These IRFs can be translocated to the nucleus by a variety of immune stimuli including toll-like receptor ligands (a class of pattern recognition proteins used to identify foreign molecules in the body), which induce an immune response if a virus is detected in the body.8 In the literature, IFNAR1deficient mice exhibited osteopenia (weakening of the bones) with increased production of osteoclasts which degrade bone, indicating that IFN signaling may decrease osteoclast differentiation. Receptor activator of nuclear factor kappa-Β ligand (RANKL) maintains bone homeostasis through c-Fos-dependent induction of IFN-β.8 As a regulatory loop, IFN-β strongly inhibits the osteoclast differentiation by interfering with the RANKL-induced expression of c-Fos.9 This regulatory loop can be a target for future therapeutics and should be studied further. Osteoclasts have been implicated in sustaining the signals for neuropathic pain. Additionally, several target genes identified in the search have been explored as possible targets for therapeutics in existing literature. Brain-derived neurotrophic factor (BDNF) is a neurotrophin with functions related to neuronal survival and proliferation processes as well as inflammation. BDNF is also an important central pain mediator. Severe RA patients reportedly express high levels of BDNF; however, BDNF expression has been shown to decrease in response to anti-TNF treatment.10 Another target is transforming growth factor (TGF) β1, which is expressed in the rheumatoid synovium. TGF-β1 contributes to the progression of inflammation and joint destruction in RA, an effect specific to arthritic synovial fibroblasts.5 Again, TGF-β1 can be a future target for therapeutic study and more research should be conducted on its mechanisms. Finally, transient receptor potential vanilloid subtype 1 (TRPV1) is best known for its function in nociception, especially in response to heat and inflammatory compounds. The literature suggests that it serves a similar function in joint afferent neurons, which encode information of joint movement, position, and nociception. Animals with Trpv1-/- exhibit lower levels of pain. When present, TRPV1 may therefore play a role in facilitating the joint damage and swelling that is characteristic of arthritis and therefore should be a target for future therapeutic study.11

CONCLUSIONS

of new, effective, and specialized strategies for RA pain therapeutics. Moving forward, we would like to study the role of these new target genes in RA inflammatory pathways as well as acute and chronic pain in mouse models in order to further elucidate the relationship between tactile pain and paw swelling. Finally, we would also like to explore gender differences between mice with these genetic deficiencies to better understand the gender disparity in RA diagnoses.

ACKNOWLEDGEMENTS

Thank you to Dr. Maripat Corr and lab members Peter Pham, Gwendallyn Stilson, and Valerie Hsu, Dr. Yaksh and his lab members, and the Undergraduate Research Scholarship Ledell family research program.

REFERENCES

1.G uo Q, Wang Y, Xu D, Nossent J, Pavlos NJ, Xu J. Rheumatoid arthritis: pathological mechanisms and modern pharmacologic therapies. Bone Res. 2018;6:15. 2. Elliott MJ, Maini RN, Feldmann M, et al. Repeated therapy with monoclonal antibody to tumour necrosis factor alpha (cA2) in patients with rheumatoid arthritis. Lancet.1994;344(8930):1125–1127. 3. Schwartz DM, Kanno Y, Villarino A, Ward M, Gadina M, O'Shea JJ. JAK inhibition as a therapeutic strategy for immune and inflammatory diseases. Nat Rev Drug Discov. 2017;17:78. 4. O'Shea JJ, Kontzias A, Yamaoka K, Tanaka Y, Laurence A. Janus kinase inhibitors in autoimmune diseases. Ann Rheum Dis 2013;72 Suppl 2:ii111–5. 5. Sweeney S.E., Kimbler T.B., Firestein G.S. Synoviocyte innate immune responses: II. Pivotal role of IFN regulatory factor 3. J. Immunol. 2010;184:7162–7168. 6. Troutman, Ty Dale et al. “Toll-like receptors, signaling adapters and regulation of the pro-inflammatory response by PI3K.” Cell cycle (Georgetown, Tex.) vol. 11,19 (2012): 3559-67. 7. Takayanagi, H., S. Kim, K. Matsuo, H. Suzuki, T. Suzuki, K. Sato, T. Yokochi, H. Oda, K. Nakamura, N. Ida, et al. 2002. RANKL maintains bone homeostasis through c-Fos–dependent induction of interferon-β. Nature. 416:744–749. 8. Takayanagi, H., Kim, S., & Taniguchi, T. (2002). Signaling crosstalk between RANKL and interferons in osteoclast differentiation. Arthritis research,4 Suppl 3(Suppl 3), S227–S232. 9. Kouskoff V, Signorelli K, Benoist C, Mathis D. Cassette vectors directing expression of T cell receptor genes in transgenic mice. J. Immunol. Methods. 1995;180:273–280. 10. Forsgren, Sture et al. “Measurements in the Blood of BDNF for RA Patients and in Response to Anti-TNF Treatment Help Us to Clarify the Magnitude of Centrally Related Pain and to Explain the Relief of This Pain upon Treatment.” International journal of inflammationvol. 2011 (2011): 650685. 11. Galindo T., Reyna J., Weyer A. (2018). Evidence for transient receptor potential (TRP) channel contribution to arthritis pain and pathogenesis. Pharmaceuticals11:105. 10.3390/ph11040105

This project was conducted by entering pain-associated genes into STRING to demonstrate that inflammatory genes are highly connected and involved in numerous biological pathways, therefore implicating multiple specific inflammatory mechanisms that may be associated with neuropathic pain. The findings from the virtual model also elucidated several new target genes of interest for further study. This is especially important for the development sqonline.ucsd.edu

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SENIOR HONOR 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.

Blueface angelfish, Pomacanthus xanthometopon, swimming at the Birch Aquarium in La Jolla. Photo by Sam Zilberman

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Senior Honor Theses

ECOLOGY, BEHAVIOR & EVOLUTION PI: Jennifer Smith, Ph.D., Scripps Institution of Oceanography, UCSD

Evaluating Conditions that Induce Tetrasporogenesis in the Red Seaweed Asparagopsis taxiformis

EMMA PALMER ROGER REVELLE COLLEGE ECOLOGY, BEHAVIOR & EVOLUTION MAJOR MARINE BIOLOGY MAJOR PI: Ryan F. Hechinger, Ph.D., Scripps Institution of Oceanography

Cercarial Birth Rates of Philophthalmid Trematode Parasites Infecting the California Horn Snail, Cerithideopsis Californica Parasitic trematodes form colonies of cooperating individuals inside their first intermediate host mollusk. Although these parasites have been identified as an attractive system to study basic sociobiological questions, a key hurdle is that observing the colony requires dissecting and killing the host, preventing long-term observations. However, as these colonies are mathematically equivalent to closed populations, we can use demographic modeling to explore colony dynamics. To help parameterize these models, we quantified the birth rate of trematode dispersive stages (cercariae) for three trematode species infecting the marine snail, Cerithideopsis californica, under different natural conditions. Using a generalized linear model, we generated an equation that uses trematode species, colony size, water temperature, and weather to estimate the average annual cercaria production for a given colony. These results will help parameterize colony demographic models, further opening the door to using trematodes as a model system in sociobiology.

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Asparagopsis taxiformis is a red, bromoform-producing seaweed that reduces methane emissions in ruminant livestock when used as a feed supplement. However, little is known about how to cultivate this seaweed in captivity. In order to cultivate this species as a feed supplement for livestock there is an urgent need to better understand the conditions that facilitate different portions of this alga's life cycle. The diploid tetrasporophyte is generally more abundant, but the larger, haploid gametophyte that arises from tetraspores could be used to selectively breed for traits such as increased JOHN MUIR COLLEGE bromoform production. The ideal conditions to induce spore formation and the next stage in the lifecycle are currently unknown. Here, we examined if and how variation in temperature (15, 18, and 21 degrees C) and daylength (6, 8, 10, 12, and 24 hours of light) influences spore production from vegetative fragments of the tetrasporophyte phase of this alga. Based on preliminary results, exposure to 24 hours of light seemed to stress the algae resulting in both spore formation and high rates of mortality. Further, we observed higher rates of spore production in cooler temperatures but more results are needed to increase confidence in these preliminary findings. We hope that these results will contribute to our understanding of how to commercially produce this seaweed as a methane mitigating supplement in livestock.

CHARLOTTE SUE

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

In Vitro CRISPR System for High-Throughput Target Validation Today, CRISPR-Cas9 targeted gene editing plays a major role in understanding diseases and investigating biology, with applications in the discovery of gene therapies or discernment of aberrant signaling pathways. High efficacy of Cas9-induced knockout is dependent on the targeting efficiency of a gRNA. Popular methods of gRNA validation include plasmid delivery of Cas9 and gRNAs, which is often onerous and inefficient, or ribonucleoprotein (RNP) delivery of Cas9 and sgRNAs, which can be prohibitively expensive; this is often followed by restriction fragment THURGOOD MARSHALL COLLEGE length polymorphism (RFLP) assays that are tedious and costly. Here, LITERATURE MINOR we report the development of a novel Cas9-expressing 293T cell line that allows for high-throughput validation of gRNAs, quantified by TIDE/Synthego-ICE analysis of Sanger sequencing. This method decreases the cost and variability raised by RNA production. Moreover, TIDE/Synthego-ICE allows for inexpensive indel efficiency calculations comparable to that of NGS—ultimately introducing a low-cost, high-throughput gRNA validation system.

ADAM AL-NIHMY

PI: Judith A. Varner, Ph.D., UCSD School of Medicine, Department of Pathology

Macrophage PI3Kgamma Regulates Fibrosis and Scar Tissue Development in Mice

Fibrosis is a pathological form of wound healing in which connective tissue replaces normal organ tissue in an unchecked manner, leading to extensive tissue remodeling and permanent scarring. Recently, it has been shown that chronic inflammation promotes fibrosis in a macrophage-dependent manner, potentially through macrophage PI3Kgamma (phosphoinositide 3-kinase gamma). Inhibition of this myeloid cell selective kinase reduces macrophage expression of pro-fibrotic factors, including TGFbeta and PDGF-BB, thereby suppressing fibrosis in mouse models of pancreatic cancer as well as in models of lung, liver, and cardiac fibrosis. Despite these results, PI3Kgamma regulated pro-fibrotic pathways have not been entirely elucidated. We EARL WARREN COLLEGE hypothesized that PI3Kgamma inhibition could prevent fibrosis and thereMUSIC MINOR fore scar formation by controlling macrophage expression of pro-fibrotic factors that act on fibroblasts to secrete collagenous extracellular matrices, which form scar tissue. We continue to investigate the molecular mechanisms in which macrophages stimulate fibroblast collagen production in vitro and in vivo.

NATHAN CHAN

SALTMAN QUARTERLY

VOL. 19

Senior Honor Theses

HUMAN BIOLOGY PI: Karl J. Wahlin, Ph.D., UC San Diego Health, Department of Ophthalmology

Direct Conversion of Müller Glial Cells into Retinal Neurons Using AAV

GRACE KIM ROGER REVELLE COLLEGE

Regenerating functional retinal neurons by direct conversion of existing cells in the eye is one approach that could address cell loss in many retinal diseases. Previous studies have shown that some species, such as zebrafish, can coax Müller glial cells into proliferating retinal progenitor cells and new retinal neurons upon injury. However, this regenerative capacity is insufficient in mammals. Thus, we are exploring how Müller glial cells derived from human retinal organoids can be induced to regenerate major retinal cell types and restore vision by using adeno-associated viruses (AAV), which can be used as delivery vehicles for the expression of transcription factor transgenes. Viral particles carrying a transgene cassette have been produced via HEK293 cells and used to successfully transduce astrocytes. Further efforts will be conducted to infect Müller glial cells with AAVs carrying the appropriate transgenes that recapitulate endogenous regeneration in mammals.

Developing Fluorescent Biomarkers to Study Human Retinal Development

DAPHNE PHAM THURGOOD MARSHALL COLLEGE

PI: Omar S. Akbari, Ph.D., Professor, Division of Biological Sciences, Cell and Developmental Biology Section, University of California, San Diego

Learning the Role of Myo-fem in Developing Precision Guided Sterile Insect Technique (PgSIT) System in Aedes albopictus

SANJANA SHARMA THURGOOD MARSHALL COLLEGE GLOBAL HEALTH MINOR

SALTMAN QUARTERLY

The Akbari lab has successfully developed a CRISPR-based technology termed precision-guided sterile insect technique (PgSIT) system in Aedes aegypti for mosquito population control and in turn limiting spread of vector-borne diseases. The PgSIT system targets essential genes for male sterility and female viability with the aim to mass produce healthy sterile males which in the wild can compete and mate with the wild-type females, to eliminate mosquito populations in multiple generations. Our team is working towards expanding the PgSIT approach in Aedes albopictus, a species of mosquito known to transmit deadly arboviruses such as dengue, Zika virus, yellow fever, and chikungunya. During the course of my training, I have successfully identified, and characterized the myosin heavy chain (MyoFem) gene, which codes for female flight muscle protein, in Aedes albopictus. I am also learning various bioinformatic tools to analyze and design guide RNAs to target genes of interest. Targeting Myo-Fem will interfere with the female’s flight ability resulting in flightless females. Female flight is extremely important for mating, anautogeny, and to take-off from water breeding sites after eclosion of female mosquitoes, making it an attractive candidate for PgSIT approach. VOL. 19

EARL WARREN COLLEGE PSYCHOLOGY MINOR PI: Nigel Calcutt, Ph.D., UCSD School of Medicine, Department of Pathology

Effects of HDAC6 Inhibitio on Paclitaxel-Induced Peripheral Neuropathy in Mice

PI: Karl J. Wahlin, Ph.D., UC San Diego Health, Department of Ophthalmology

Retinal degeneration (RD) is one of the leading causes of blindness, and human induced pluripotent stem cell (hiPSC) derived retinal organoids are useful tools for modeling retinal development. Genetically encoded fluorescent reporters facilitate live cell imaging, which helps validate retinal organoid models. I made two dual color reporters to identify early and late stages of development; cells that include reporters at both loci have been genotyped and Sanger sequence verified. The VSX2 reporter will be used to study the progression of retinal progenitors to bipolar cells during development, while NRL and LHX4 reporters will label photoreceptors. We are working to reprogram Müller glial cells into new photoreceptors, and these reporters will help us monitor the conversion of Müller glia into rods and cones, respectively. We aim to utilize these reporter systems to contribute to the development of translational tools and techniques for inducing intrinsic repair of the retina in humans.

RAKESH SHARMA NEMMANI

Chemotherapeutics such as paclitaxel stop tumors from proliferating by preventing microtubule assembly and mitotic division. However, microtubules also support axonal transport to nerve terminals, and chemotherapeutics can induce neurodegeneration, termed Chemotherapy Induced Peripheral Neuropathy (CIPN), in cancer patients. The histone deacetylase (HDAC) enzyme family regulates protein acetylation, with the HDAC6 subtype focused on deacetylation of cytosolic tubulin. HDAC6 inhibitors increase acetylated tubulin levels and stabilize microtubules. I therefore investigated efficacy of a HDAC6 inhibitor (Miralinc Pharma Inc.) against paclitaxel-CIPN. Mice were given vehicle or paclitaxel, with sub-groups (N=10/ group) given the HDAC6 inhibitor (20 or 60mg/kg/day or 60mg/kg/ day twice daily), gabapentin (60mg/ kg/day) or vehicle. Paclitaxel caused tactile allodynia and heat hypoalgesia. HDAC6 inhibition attenuated both disorders, with best efficacy at 20mg/kg/day. Gabapentin, the standard of care for CIPN, was also effective. These results support the potential of HDAC6 inhibitors to prevent CIPN and allow optimal use of chemotherapeutics.

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Senior Honor Theses

HUMAN BIOLOGY PI: Neil Chi, M.D., Ph.D., UCSD School of Medicine, Division of Cardiovascular Medicine

Multi-lineage Tracing Reveals that Neural Crest Cells do not Contribute to Ventricular Cardiomyocytes

KIROLLOS SAMIR TADROUSSE EARL WARREN COLLEGE PI: Kellie Breen Church, Ph.D., UCSD School of Medicine, Department of Obstetrics and Gynecology

The Role of Norepinephrine Neurons in the Locus Coeruleus in Stress-Induced Suppression of Luteinizing Hormone The Locus Coeruleus (LC) is part of the brainstem thought to regulate stress responses. I am investigating neurons in the LC during conditions known to suppress reproduction. Female C57/Bl6 mice were exposed to psychosocial stress paradigms: acute restraint stress (n=3), chronic restraint stress (n=3) or control (n=3). We also examined LC neurons following activation of another brainstem region (nucleus of the solitary tract [NTS]) using chemogenetics, which mimics most stress responses (activated: n=7, control: n=6). Immunohistochemistry was performed on neural tissue to label cFos, a marker for cell activation, and dopamine beta-hydroxylase (DBH), a marker for norepinephrine cells. We observed no significant differences in LC DBH cell activation in mice exposed to either restraint stress paradigm compared to controls. However, we found an increase in LC DBH cell activation in animals following activation of the NTS. Thus, the LC is regulated during NTS cell activation, but not in psychosocial stress.

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The mammalian heart comprises various specialized cell types derived from different progenitors during early cardiac development. Recent studies using Wnt1-Cre to label neural crest cells (NCCs) revealed that NCCs contribute to significant amounts of cardiomyocytes (CMs) in the ventricles. However, conventional CreloxP mediated genetic lineage tracing methods have limitations including the lack of promoter specificity and Wnt1-Cre exhibiting ectopic expression, thus resulting in Cre-leakage and potentially spurious conclusions. To address this problem, we have used a NCC mouse line, Sox10-Cre, and an ectoderm-marker mouse, Sox2-CreERT2, to examine whether NCCs contribute to CMs in the ventricles. We also used a dual lineage tracing approach using Cre and Dre promoters to confirm the contribution of NCCs to CMs lineage. Overall, our results do not support that NCCs can contribute to CMs in the ventricles.

MI HUE TRAN ELEANOR ROOSEVELT COLLEGE PSYCHOLOGY MINOR

PI: Amir Zarrinpar, M.D., Ph.D., UC San Diego, Department of Medicine

The Role Of Microbial Bile Salt Hydrolase In Atherosclerosis

KIMBERLY YIN SIXTH COLLEGE

Gut microbiome driven dysregulation of lipid metabolism contributes to atherosclerosis, a major cause of cardiovascular disease characterized by the formation of plaques in artery walls. Our aim is to assess the role of bile salt hydrolase (BSH) overexpression on lipid metabolism and plaque formation in Ldlr-/- mice, an atherosclerosis model, using engineered native bacteria (ENB). However, we first have to determine whether ENB can engraft in this model. To determine the extent of colonization along the gut, we cultured intestinal tissues and stool from Ldlr-/- mice that were gavaged with either ENB with and without BSH. ENB colonized the entire gut, with high concentrations in the ileum and cecum for 12 and 22 weeks after gavage, demonstrating that it can engraft in the lumen of an atherosclerosis mouse model. We will use these mice to determine the effects of BSH overexpression on how microbiome functions drive atherosclerosis.

PI: Michael Karin, Ph.D., UCSD School of Medicine, Department of Pharmacology

Synergistic Inhibition of PDAC Growth by Macropinocytosis Inhibition and Chemotherapy

Pancreatic Ductal Adenocarcinoma (PDAC) is one of the most lethal malignancies. Current chemotherapy treatment is unable to significantly increase the life expectancy of PDAC patients and the relapse of PDAC is partly due to the resistance to chemotherapy of PDAC stem cells (Lambert A, et al. Semin Oncol. 2021). Recently, it was reported that macropinocytosis plays an important role in the survival and proliferation of PDAC through the uptake of extracellular fluid droplets containing proteins and other macromolecules (Su H, et al. Cancer Cell. 2021). In our study, we examined the effect of combining existing chemotherapeutic drugs with macropinocytosis inhibitors on PDAC cell lines. Intriguingly, macropinocytosis inhibitor only showed a synergistic effect with chemotherapeutic drug THURGOOD MARSHALL COLLEGE in soft agar 3D spheroid formation assay, but not under 2D culture conditions. We are currently investigating whether this synergistic effect is due to suppression of cancer stem cells by inhibition of macropinocytosis.

MANDY XUAN LIN ZHU

SALTMAN QUARTERLY

VOL. 19

Senior Honor Theses

BIOINFORMATICS

PI: Cole Ferguson, M.D., Ph.D., UCSD School of Medicine, Department of Pathology

Ubiquitin Signaling Regulates Epigenetic State in the Developing Mammalian Brain Inherited neurodevelopmental disorders result from deregulation of diverse molecular pathways, however the cellular mechanisms of disease pathogenesis often remain undefined. We investigated mouse models of intellectual disability resulting from mutation of the Anaphase Promoting Complex (APC), a major E3 ubiquitin ligase that controls degradation of key nuclear substrates in post-mitotic neurons. Proteomics analysis in brain of mutant mice showed dramatic increase in the kinase Aurora B (AurkB), and functional studies revealed elevation of its product phosphorylated histone 3 (H3S10ph), suggesting an unrecognized role for the APC in chromatin regulation. To explore epigenetic state in mutant neurons at activated and repressed loci, we employed the method CUT&RUN. Mutant neurons exhibited striking attenuation of both H3K27me3 and H3k27ac, which are associated with heterochromatin and euchromatin, respectively. Follow-up studies will explore chromatin conformation and gene expression in mutant neurons. Our findings uncover a novel role for ubiquitin signaling in epigenetic regulation during neurodevelopment. THURGOOD MARSHALL COLLEGE

LENA KROCKENBERGER

COMPUTER SCIENCE MINOR

MICROBIOLOGY PI: Matthew Daugherty, Ph.D., UCSD Division of Biological Sciences, Molecular Biology

Characterizing New Host-virus Arms Races Between Host Proteins and Viral Proteases The cell intrinsic immune system rapidly responds to viral infection through recognition of pathogenic molecular patterns. The immediate response of this intrinsic system is subject to immense evolutionary pressure as it must adapt to subdue the viral presence but also evade viral antagonism of host factors. One subset of this intrinsic system are the interferon stimulated genes (ISGs); this group includes proteins such as MxA, TRIM5, TRIM34, and others. We have recently discovered that proteases from viral families including Picornaviridae and Coronaviridae can cleave human MxA. This data suggest that these viruses are subject to MxA’s antiviral properties and that MxA is rapidly evolving to escape viral protease-specific degradation. My project identifies MxA’s effectiveness against picorna- and coronaviruses and determine show viral inhibitory strategies hamper MxA’s antiviral properties as well as identifies other ISGs in similar host-pathogen conflicts.

MILES ROBERT CORLEY ROGER REVELLE COLLEGE MARINE SCIENCE MINOR

MOLECULAR & CELL BIOLOGY PI: Anne Hiniker, M.D., Ph.D., UCSD School of Medicine, Department of Pathology

Optimizing a Chemical-Genetic Approach to Define Substrates of PKCα in Alzheimer’s Disease Protein kinase Cα (PKCα) is a serine/threonine kinase that binds to second messengers Ca2+ and diacylglycerol, causing a series of downstream events that suppress cell proliferation. Recently, gain of function missense mutations in PKCα resulting in increased kinase activity have been identified in some patients with Alzheimer’s disease (AD), a neurodegenerative disease characterized by amyloid-β (Aβ) protein aggregates and synaptic degeneration. To understand the mechanism of PKCα-mediated AD pathogenesis, this project seeks to identify kinase substrates of AD-associated PKCα mutants (PKCα-AD). To achieve this, we are applying a chemical genetic kinase-substrate mapping approach to PKCα-AD and PKCα, which involves mutating PKCα’s “gatekeeper” residue, a conserved bulky amino acid, to a small non-polar amino acid to allow for specific ATP analogs that cannot be utilized by most endogenous kinases. Using an optimized in vitro kinase assay, we are testing conditions necessary for PKCα-AD and PKCα substrate tracing.

MICHELLE LIU EARL WARREN COLLEGE COGNITIVE SCIENCE MINOR

SALTMAN QUARTERLY

VOL. 19

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Senior Honor Theses

GENERAL BIOLOGY PI: Jack A. Gilbert, Ph.D., UCSD School of Medicine, Department of Pediatrics and Scripps Institute of Oceanography

Microbes in Space: Characterizing Virulence of Staphylococci from the International Space Station using In-Vivo Nematode Models and High-Throughput Screening Approaches

JACOB PORATH THURGOOD MARSHALL COLLEGE PI: Anne Hiniker, M.D., Ph.D., UCSD School of Medicine, Department of Pathology

Rab38 Modifies LRRK2 Cellular Localization LRRK2 is a kinase which is commonly mutated in individuals with Parkinson’s disease. It is known to have substrates in the Rab GTPase family but LRRK2-Rab interactions are not well characterized. It has been shown previously that overexpressed Rab29 can direct LRRK2 to the cell membrane and increase its kinase activity. Here, we show that the Rab29 homolog Rab38 has a strong effect on LRRK2 localization endogenously in B16-F10 mouse melanocytes. LRRK2 localizes to the pericentriolar region of these cells endogenously but this LRRK2 localization is not seen in the absence of Rab38. When Rab38 levels are lowered by siRNA knockdown, LRRK2 is diffusely cytoplasmic. Additionally, LRRK2 is diffusely cytoplasmic in the LRRK2 D2017A & T1348N mutations which both eliminate LRRK2 kinase activity. This is the first reported instance of endogenous Rab38 regulating LRRK2 localization.

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YASH GARODIA

The built environment of the International Space Station (ISS) is non-sterile, with extensive microbial diversity identified, some of which are opportunistic pathogens. These could pose serious risks to crew health, as the harsh conditions in outer space could result in mutations that increase virulence. ROGER REVELLE COLLEGE Bacteria belonging to the Staphylococci family, in particular, have been frequently identified. Using isolates from the Microbial Tracking-1 expedition, I am investigating whether virulence genotype matches observed phenotype. For this, I have selected five staphylococci strains collected from the dining area on the ISS to create bacterial suspensions. Using fluorescent stains and a Caenorhabditis elegans model, I am investigating the virulence of these isolates. Our current results indicate increased virulence in strains, indicated by increased killing of C. elegans, suggesting that microbial isolates from the ISS may indeed pose a risk to human health. Our future work will validate these findings and determine virulence enhancing mechanisms. PI: Yunde Zhao, Ph.D., UCSD Division of Biology, Cell and Developmental Biology

The Roles of Gretchen Hagen 3 (GH3) Group III Genes in Hormone Homeostasis and Plant Development

JUNJIE HU EARL WARREN COLLEGE

Gretchen Hagen 3 (GH3) plays essential roles in conjugating active plant hormones to amino acids. GH3 genes can be divided into three groups based on their sequence homology. Group I is involved in jasmonic acid (JA) homeostasis. Group II functions in auxin inactivation. Group III was proposed to play a role in stress response. However, the exact functions of Group III GH3 have remained unclear. Arabidopsis genome contains ten group III GH3 genes that likely have overlapping functions. I used CRISPR-Cas9 gene editing technology to specifically delete GH3 genes. I have generated single mutants for the 10 GH3 genes. The mutant lines were confirmed by genotyping and DNA sequencing. I am combining the single mutants into multiple knockout mutants through crosses and genotyping. Characterization of the gh3 mutant lines will reveal the roles of the GH3 genes in stress-induced salicylic acid biosynthesis and plant disease resistance.

PI: Alexandra Jazz Dickinson, Ph.D., UCSD Section of Cell and Developmental Biology, Division of Biology

The Secondary Metabolite Itaconate Mediates the Development of Roots and Responses to Environmental Stress in Plants Itaconate is a small carboxylic acid metabolite derived from the Citric Acid Cycle metabolite cis-aconitate. The functions of itaconate as a cell reprogrammer is well known in mammalian cells, however, its function in plants is relatively unknown. To understand the role of itaconate in plants, Arabidopsis Thaliana were treated with exogenous itaconate. After experimentation, it was determined that itaconate can significantly inhibit plant growth by reducing overall root development. Notably, itaconate treated seedlings are significantly more variable in size than control seedlings. To understand this variability, itaconate resistant (IR) individuals were grown for harvest and their offspring were characterized. IR seedlings had enhanced drought and salt tolerance on average. To simulate agricultural conditions, itaconate treatments were then conducted in media absent of sucrose. Results showed impaired root development, suggesting the itaconate signaling overlaps with that of sucrose. Ultimately, this shows itaconate is a potent regulator of plant development.

AMMAN KLAIR THURGOOD MARSHALL COLLEGE CHEMISTRY MINOR

SALTMAN QUARTERLY

VOL. 19

Senior Honor Theses

NEUROBIOLOGY PI: Yishi Jin, Ph.D., UCSD Biological Sciences, Neurobiology

Investigation of Dosage and Duration of DLK Expression on Neuron Death

QIANYI PU ROGER REVELLE COLLEGE

A key signaling molecule in pro-regenerative and pro-apoptotic neuronal responses is the Dual Leucine Kinase (DLK). DLK is expressed in many cell types in the nervous system, including glutamatergic neurons, and prior studies have suggested DLK may be involved in the neuron death associated with neurodegenerative diseases such as Alzheimer’s disease. In this senior honor thesis research, I investigated how dosage and duration of DLK signaling contribute to cell death. I used immunofluorescence to visualize the CA1 pyramidal layer in mice with varying levels of endogenous and transgenic DLK through use of conditional knockout, overexpression, and the combination of knockout and overexpression. My results show DLK dependent cell death is enhanced with greater levels of DLK. My current studies aim to investigate how age affects this phenotype.

Ectopic Overexpression of Three Transcription Factors Involved in Hair Cell Specification and Differentiation to Induce Direct Conversion of Cochlear Hair Cells from mESCs

SAMEEHA RASHID SIXTH COLLEGE

Neuromodulatory Dopaminergic Projection in Olfactory Bulb During Olfactory Learning

JOHN MUIR COLLEGE NEUROBIOLOGY MAJOR DEVELOPMENTAL PSYCHOLOGY MAJOR

SALTMAN QUARTERLY

The olfactory bulb (OB) is the first site of the central nervous system that receives odorant information, and it receives centrifugal projections from multiple brain regions, including the primary olfactory cortex and neuromodulation regions. Previous neuroanatomy tracing studies has shown that OB receives direct cholinergic, norepinephrine, and serotonin projection input. However, it remains unclear about the existence of a direct project from dopamine neurons in VTA/SN to the olfactory bulb and how they could affect OB circuits and changes in behavior. This project aims to identify the existence of dopaminergic projection in OB and examine their functional contribution to olfactory learning. We will use anterograde/retrograde virus and transgenic mice to identify and locate the dopaminergic projection and image the neuromodulation projection in rodents’ olfactory bulbs using two-photon microscopy during an olfactory discrimination task.

VOL. 19

THURGOOD MARSHALL COLLEGE GLOBAL HEALTH MINOR

Investigating Roles of Protein Translation in C. Elegans Motor Circuit

PI: Takaki Komiyama, Ph.D., UCSD Division of Biological Sciences and UCSD School of Medicine

YUXUAN YUE

BANGYAN HU PI: Yishi Jin, Ph.D., Division of Biological Sciences, Section of Neurobiology

PI: Rick A. Friedman, M.D., Ph.D., UCSD School of Medicine, Department of Surgery

Mammals cannot regenerate mature cochlear hair cells (HCs), sensory cells within the ear that send auditory information to the brain. Damage to these hair cells can result in hearing loss. Previous attempts in creating a mouse cochlea organoid succeeded in creating HC-like structures but not fully functional HCs. We need an in-vitro model to culture functional HCs in a more reproducible and robust manner. Only then, can we design a disease-specific model for hearing impairments. In our lab, we have created a reporter polycistronic construct that contains Neurog1, Atoh1, and Pou4f3, three specific transcription factors involved in hair cell specification and differentiation. Through ectopic overexpression of these genes in mouse differentiated cochlear cells, we hope to directly convert postnatal cochlear non-sensory cells (mESCs) into functional HCs. Our findings will help elucidate HC development and how to proceed in making an in-vitro cochlea organoid.

MOLECULAR & CELL BIOLOGY

Protein translation is mainly regulated by eukaryotic initiation factors (eIF1-6). As eIFs are often dysregulated in neurological disorders, it is important to understand how eIFsregulate translation in neurons. A subunit of the C. elegans eIF3 complex, EIF-3.G, has been shown to regulate activity-dependent translation in cholinergic motor neurons. EIF-3.G function involves a phosphatase NANP-1 and a protein with low-complexity domains LIN-66. My study focuses on the expression of these two genes and their interaction with EIF-3.G. I defined the genetic features underlying NANP-1 and LIN-66 function and studied the locomotor behaviors associated with loss of function in the two genes. I confirmed that NANP-1 is required for EIF-3.G function and that nanp-1 is expressed in the nervous system. I examined LIN-66 isoforms and their function in EIF3.G activity. My work has contributed to our understanding of how neurons control protein translation to regulate animal behavior.

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Senior Honor Theses

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

Femaleless Knockout Yields 100% Female Lethality in Anopheles gambiae, Enabling Novel Vector Control Technologies

KAMERON ALU THURGOOD MARSHALL COLLEGE CHEMISTRY MINOR PI: Joseph Gleeson, Ph.D., UCSD School of Medicine, Department of Neurosciences

Malaria is among the world's deadliest diseases, killing over half a million people annually. Precision-guided Sterile Insect Technique (pgSIT) is a genetic population-suppressing system that has potential to reduce the transmission of malaria when adapted to the African malaria vector, Anopheles gambiae. In pgSIT, sterile male mosquitoes are released into the wild to mate with monandrous wild type females. This sterilizes females, resulting in single-generation population suppression and reduced transmission of mosquito-borne disease. To exclusively generate sterile males for release, male fertility genes and female essential genes are targeted for CRISPR knockout. Here, we develop a pgSIT system in A. gambiae and characterize associated phenotypes, demonstrating its potential as a vector control technology.

Impact of Folic Acid on Transcriptome, Methylome, and Chromatin Accessibility Landscape in Mice

Folic acid (FA) supplementation has been shown to reduce the occurrence of meningomyelocele (MM) by as much as 70%. Although the mechanism by which FA reduces the occurrence of MM is unknown, it has been hypothesized that FA modifies the risk of MM through an epigenetic mechanism, thus altering gene expression. FA is a critical source of methyl groups for DNA and histone methylation, functioning through 5-methyltetrahydrofolate (5-MTHF). Thus, in this project, I aim to assess the effect of dietary FA on the transcriptome, methylome, and chromatin accessibility landscape in developing mouse embryos. By uncovering the effect of FA on the transcriptome and epigenome, I hope to uncover potential mechanisms by which FA reduces the incidence of MM and gain insight into treatments and eventual prevention of MM.

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REEMA APTE

ELEANOR ROOSEVELT COLLEGE CHEMISTRY MINOR

PI: Michael J. Sailor, Ph.D., Distinguished Professor of Chemistry and Biochemistry at UCSD

Hybrid Porous Silicon Microparticle and Polycaprolactone Drug Delivery System for Revascularization of the Anterior Cruciate Ligament Post Surgically

MADELINE BROWN ROGER REVELLE COLLEGE

100,000 Anterior Cruciate Ligament (ACL) reconstruction surgeries are performed in the U.S. per year. Although it is largely a successful procedure, patients endure a lengthy recovery time due to a lack of revascularization to the surgical graft. This project aims to improve the outcome of post-surgical recovery of ACL injuries by enhancing revascularization of the damaged ligament. Vascular Endothelial Growth Factor, or VEGF, is an endogenous protein that induces growth of blood vessels. Previous experimental studies have shown that local administration of VEGF to damaged tissues enhances revascularization. However, this approach can cause deleterious effects, which have been attributed to the abnormally high concentrations of the highly potent VEGF molecule that are present immediately after administration. This work focuses on a delivery system that can slowly release VEGF. The approach involves encapsulating VEGF into mesoporous silicon microparticles, then suspending these porous silicon particles inside a polymer scaffold.

PI: Tracy Handel, Ph.D., Skaggs School of Pharmacy and Pharmaceutical Sciences, Department of Pharmacology

Mutants of CCR10 Ligands CCL27 and CCL28 Reveal Receptor Trafficking Trends Chemokines are involved with cell migration in development and immune response and have been associated with a number of diseases. CCR10 is a chemokine receptor expressed on skin-homing T-cells. It’s ligands, CCL27 and CCL28, have not been widely studied and little is known of their structure-function. Through site-directed mutagenesis, our lab previously identified mutants with enhanced activity compared with wildtype. For example, mutant F-CCL27 (a Phe addition to the N-terminus of CCL27) exhibits super-agonist activity in cell migration and receptor internalization assays. To follow-up, our project uses Bioluminescence Resonance Energy Transfer (BRET) to study protein-protein interactions and characterize traditional receptor trafficking pathways. Initial BRET assays examining bystander β-arrestin association show greater potency of F-CCL27 compared with wildtype. Our aim is to further evaluate the effects of the mutants (F-CCL27 and N-terminal chimeras) on receptor trafficking such as with G-protein dissociation, CaaX internalization, and endosomal trafficking with Rabs.

AURA CELNIKER EARL WARREN COLLEGE ENVIRONMENTAL SYSTEMS MINOR

SALTMAN QUARTERLY

VOL. 19

Senior Honor Theses

MOLECULAR & CELL BIOLOGY PI: Dr. Calvin Yeang, M.D., Ph.D., Department of Medicine, Division of Cardiology

Defining the Inflammatory Monocyte and Cytokine Changes Associated with Lipoprotein Apheresis in Patients with Elevated Lipoprotein(a)

BRIAN DINH EARL WARREN COLLEGE POLITICAL SCIENCE MINOR

Elevated plasma lipoprotein(a) [Lp(a)] is a likely causal risk factor for CVD present in 20% of the population. It is also a largely undertreated risk factor, in part due to an incomplete understanding of which currently available therapies address Lp(a) mediated CVD risk. Lp(a) is the predominant lipoprotein carrier of pro-inflammatory oxidized phospholipids, and CVD risk attributed to Lp(a) is conditioned to systemic inflammation, suggesting that effective risk reduction in patients with elevated Lp(a) requires therapies that address inflammation. We aim to define the effect of Lp(a) and concurrent LDL-C lowering by lipoprotein apheresis (LA) on monocyte gene expression. We have enrolled 30 patients and collected peripheral blood mononuclear cells (PBMCs) before and after LA treatment. Monocytes from these patients will be purified and phenotyped by flow cytometry. RNA-sequencing of sorted monocytes and differential gene expression analysis of post- vs pre-LA monocytes from each patient will be performed.

Interaction Between PRL1 and SRC in the Promotion of TGFβ Signalling in Systemic Sclerosis

SOPHIE HAO EARL WARREN COLLEGE MOLECULAR & CELL BIOLOGY MAJOR HUMAN DEVELOPMENTAL SCIENCES MAJOR EUROPEAN STUDIES MINOR PSYCHOLOGY MINOR

PI: Xin Sun, Ph.D., UCSD School of Medicine, Department of Pediatrics

Mutation of tRNA Synthetase FARSB Causes Activation of the Integrated Stress Response and Additional Phenotypes Without Affecting Translation

JEFFREY KELLER ROGER REVELLE COLLEGE MOLECULAR & CELL BIOLOGY MAJOR POLITICAL SCIENCE: PUBLIC POLICY MAJOR

SALTMAN QUARTERLY

Amino-acyl tRNA synthetases are responsible for charging tRNAs with the appropriate amino acids prior to translation, but many are known to have additional, non-translational functions. In humans, specific mutations of phenylalanyl tRNA synthetase (FARSB) causes multi-organ interstitial disease without affecting phenylalanine charging. In order to investigate potential novel functions of FARSB, we modeled the human disease mutations in mice. In our model, compound mutations of FARSB lead to activation of the integrated stress response (ISR) at early post-natal stages. After activation of the ISR, mice experience significant alveolar fucosylation mediated by an over-expression of Fucosyltransferase 1 (FUT1) and experience a decrease in weight gain which persists to 35 weeks. At 21 weeks, mutant mice develop tertiary lymphoid structures at proximal airway branch points. The phenotypes characterized provide insight to continue investigating the non-translational functions of FARSB and how mutations of FARSB cause disease.

VOL. 18

SIXTH COLLEGE CHEMISTRY MINOR PSYCHOLOGY MINOR PI: Satchidananda Panda, Ph.D., Salk Institute of Biological Sciences, Department of Regulatory Biology

Synaptic Loss in the SCN Associated with Alzheimer’s disease

PI: Nunzio Bottini, M.D., Ph.D., UCSD School of Medicine, Department of Medicine, Division of Rheumatology, Allergy and Immunology

Systemic sclerosis is an autoimmune disease that is characterized by the thickening of the skin and internal organs called fibrosis. There is a critical need for anti-fibrotic therapies to reduce the progression of fibrosis in patients as most of them are diagnosed at the stage where steroids and immunosuppressants are ineffective. In previous research, our lab found that tyrosine phosphatase PRL1 is overexpressed in systemic sclerosis fibroblasts and promotes pro-fibrotic TGFβ signalling via enhancement of the halflife of the kinase SRC. It is also known that PRL1 gets phosphorylated and forms a stable phospho-cysteine intermediate, unlike other phosphatases. Furthermore, PRL1 is proposed to form a trimeric structure but the function remains unclear. My senior honor thesis project focuses on the details of the interaction between PRL1 and SRC, specifically studying the effects of phosphorylation and trimerization of PRL1.

BRIAN KHOV

Circadian disruption, and specifically sleep disruption, is an early indicator of Alzheimer’s disease (AD), occurring before the onset of more commonly known neurodegenerative symptoms such as memory loss. However, the anatomical basis for circadian disruption in the suprachiasmatic nucleus (SCN)—our central circadian clock that receives direct photic input from the retina—of those with Alzheimer’s disease is largely unknown. We obtained serial blockface scanning electron microscopy of 3-month and 8-month AD mouse models and observed evidence of the relationship between AD progression and disruption of the neuronal organization of the SCN associated with synaptic loss. We observed significant decreases in the volume of boutons with dendritic intrusions and axodendritic synapse frequency but a significant increase in bouton frequency on the axons. These preliminary results suggest that these changes in the connectomics of the SCN may be responsible for circadian disruption in Alzheimer’s disease patients.

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Senior Honor Theses

MOLECULAR & CELL BIOLOGY PI: Neil C. Chi, M.D., Ph.D., UCSD School of Medicine

Identification of Notch Ligand Responsible for Proepicardial Fate Induction in Zebrafish

SEBASTIAN ROHRER ROGER REVELLE COLLEGE CHEMISTRY MINOR PI: William Gerwick, Ph.D., Scripps Institution of Oceanography, Skaggs School of Pharmacy and Pharmaceutical Sciences

Mesodermal proepicardial and pacemaker-like myocardial cells arise from common Nkx2.5 progenitors in zebrafish (Ren et al. 2019). Our lab discovered inhibiting Notch signaling reduces proepicardial cell numbers while reciprocally increasing pacemaker cell numbers, suggesting Notch signaling regulates cell fate decisions between proepicardial and pacemaker cells. However, the ligand-receptor pair controlling this process remains unknown. We aim to identify the ligand responsible for Notch signaling during proepicardial cell fate decisions in zebrafish. We JOHN MUIR COLLEGE identified Notch ligand candidates through an in-situ expression screen POLITICAL SCIENCE MINOR and created mutants of these candidates, including jag1b and jag2b mutants. Using a tcf21 reporter and Islet1 immunostaining, we examined the number of proepicardial and pacemaker cells, respectively in these mutants, and discovered reduced tcf21+ proepicardial cell numbers in jag2b. These studies illuminate the Notch signaling components participating in proepicardial development and provide new insight into the regulation of epicardial and pacemaker cell production for future cardiac regenerative therapies.

APURV PRABHAKAR

Heterologous Expression of the Columbamide Biosynthetic Gene Cluster in Anabaena and Production of Novel Analogs Cyanobacteria are prolific producers of bioactive natural products, which constitute a promising source of new drug leads. Obtaining sufficient material for structure elucidation and bioassays from the native producers is a major bottleneck. Thus, we expressed the columbamide biosynthetic gene cluster (BGC) from native producer Moorena bouillonii in the model cyanobacterium Anabaena PCC 7120. The BGC was assembled in yeast from PCR products, sequence-verified, and transferred into Anabaena by conjugation from E. coli. Investigation of the engineered Anabaena revealed production of previously characterized as well as novel columbamides. We characterized new columbamide K, the w-dechlorinated analog of columbamide A, by comprehensive NMR experiments and HR-LCMS/MS. We present structures for new analogs I, J, L and M based upon 1H NMR and HR-LCMS/MS. These results show the enzymatic plasticity in the BGC and highlight heterologous expression as an efficient way to circumvent bottlenecks and generate novel chemical diversity for drug discovery.

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PI: Enfu Hui, Ph.D., UCSD Section of Cell and Developmental Biology

SHP2 Plays a Non-Catalytic Role in the PD-1 Inhibitory Axis of T-Cells

OLA MOSTAFA ROGER REVELLE COLLEGE EDUCATION STUDIES MINOR

The immune inhibitory receptor programmed death-1 (PD-1) inhibits T-cell activity by recruiting the tyrosine protein phosphatase SHP2. Studies have shown that PD-1 bound SHP-2 inhibits T-cell signaling through the de-phosphorylation of T-cell-antigen receptor (TCR) and costimulatory receptors that are responsible for cytokine production, cell survival and proliferation. More recent reports suggested that SHP2 has phosphatase-independent function in some immune receptors; however, it is not known whether SHP2 can contribute to PD1 signaling via a non-catalytic mechanism. Here, we provide evidence that phosphatase-dead SHP2 at least partially supports PD-1 inhibitory signaling in T-cells. This data suggests a non-catalytic function of SHP2 in the PD-1 axis. In addition, we show that the non-catalytic function of SHP2 still depends on its phosphatase domain. These results suggest a re-investigation and revision of the current model of PD-1:SHP2 signaling.

PI: David Cheresh, Ph.D., Distinguished Professor, Vice Chair of Pathology, Sanford Consortium for Regenerative Medicine, Moores Cancer Center

Therapeutic Antibodies Engaging Tumor Associated Macrophages for Combating Advanced Epithelial Cancer

A cell surface marker, integrin αvβ3, promotes drug resistance and metastasis in epithelial cancers. A humanized anti-αvβ3 antibody, etaracizumab, was developed to eliminate αvβ3+ cancer cells via natural killer (NK) cell-mediated antibody-dependent cellular-cytotoxicity (ADCC). However, the Cheresh lab recently found that αvβ3+ tumors are enriched with tumor-associated macrophages (TAMs) but not NK cells. Therefore, a new humanized anti-αvβ3 antibody (ABT101) was designed by changing etaracizumab’s immune receptor binding properties to favor TAM enROGER REVELLE COLLEGE gagement over NK cells. In vitro, we found ABT101 but not etaracizumab induces macrophage-mediated ADCC against αvβ3+ cancer cells. Our xenograft models also showed that ABT101 is more efficient in inhibiting αvβ3+ cancer growth and such ABT101 activity is diminished in macrophage depleted mice. This indicates that macrophage is the primary effector cell required for ABT101’s activity. These findings underline an innovative principle of “antigen-effector cell matching” to design antibodies that exploit TAM enrichment to inhibit cancer progression.

ZIQI YU

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Sara Jackrel, Ph.D. James Cooke, Ph.D. Lisa McDonnell, Ph.D. Alistair Russell, Ph.D. Ashley Juavinett, Ph.D. Chih-Ying Su, Ph.D. Elsa Cleland, Ph.D.

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