SQ Vol 17 (2019-2020) by Saltman Quarterly

Undergraduate Research Journal Volume 17 2019â&#x20AC;&#x201C;20 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 Willet (Tringa semipalmata) at Salt Creek Beach in November 2019. Photo by Bridget Spencer INSIDE COVER: Diver cleaning the main viewing tank at the Birch Aquarium. Photo taken on Feb 25, 2019. Photo by Michael Endow

Generously underwritten by

THE SALTMAN FAMILY & supported by

LETTER FROM

the editor

Dear Reader, Just last quarter, our editors and I were in the SQ office meeting with writer after writer to work on their articles. In retrospect, the hours we spent together to connect with our writers and to comment on their drafts was an incredible privilege. Cut to the final weeks to get ready for print, and everyone I looked forward to seeing each week is now on my computer screen. The COVID-19 pandemic has undoubtedly cast a shadow over many people—not only on the medical professionals and researchers working tirelessly at the front lines to understand the virus on both a human and scientific level, but also on those waiting patiently indoors for the outbreak curve to flatten. This ever-changing landscape highlights the importance of biological research and the necessity for clear science communication. It has been remarkable to witness how this unsettling situation has brought out the best in SQ; that during this unprecedented time, our team channeled their undimmed passion and adaptability into our community and into Volume 17 of Saltman Quarterly. With the print timelines planned to tee only to suddenly be derailed, I am impressed by what was accomplished. The end of winter quarter saw our production team editing final drafts despite the school shutting down and closing our office; meanwhile, our community outreach team tested video conferencing platforms to engage with local San Diegans from the safety of their homes. This creativity is palpable in our initiatives to create new communication methods that match the already dynamic discipline of biology. We have explored different ways to engage the public, whether it is through our video project The SQueeze, our social media campaign Viral Virtual Updates, or our student perspective piece “Quotes from Quarantine;” while some have succeeded and others are still in progress, as scientists-in-training, we carry the spirit of experimentation by adapting when our results don’t match our hypotheses. With new initiatives underway, I’m also excited to introduce the work that continues the SQ legacy. In Volume 17, our writers, illustrators, photographers, and editors curated a

selection of article subjects from treatment-resistant cancer stem cells to microbe communication, as well as research submissions ranging from a review paper on DNA barcoding to a manuscript detailing the many variables that affect tank bromeliads. These topics not only showcase our team members’ various passions, but also reflect the reality of the eclectic field of biology. This dynamic discipline relies on diverse methods to communication, and SQ is passionate about finding new avenues to share our content. Our SQ Online team produced a variety of articles, from skin regeneration in wound healing to a blog series that breaks down the science behind novel experiences. In addition, the quarterly SQ Insider featured engaging themes like interspecies interactions, forensic science, and aging. Last but not least, our content could not have gained the visibility it had without our publicity team and webmasters, who used social media and our online platform to engage a larger audience of readers. After three years at SQ, I feel privileged to lead a staff united in combining our passion for biology with the skill of communicating the progress of the field in the form of an award-winning, visually stunning journal. And when stayat-home isolation stresses the need for connection, I am grateful for the SQ community and the support we bring to each other during this exceptionally difficult time. This year’s Saltman Quarterly is dedicated to our staff and also to you, the reader; good communication relies on both the speaker and the active listener. As my year as Editor-inChief has come to a close, I look forward to joining you as a loyal reader and supporter of SQ, and I hope you enjoy the journal as much as I have in the past year. Best,

Emma Huie

Editor-in-Chief, Saltman Quarterly 2019-20

TABLE of CONTENTS SALTMAN DEDICATION 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.

FEATURES Targeting Cancer Stem Cells: Progress in a Sisyphean Battle Talking the Tiny Talk: How Microbes Communicate Development of Dilated Cardiomyopathy: One Molecule at a Time HAND's Grasp on the Brain: HIV and Neurocognitive Disorder Axonal Pathfinding: From Development to Regeneration

by Ingrid Heumann by Eleanor Wang by Lilit Vardanyan by Kaz Nuckowski by Nandita Rangu

RESEARCH, BREVIAS, & REVIEWS

Effects of Canopy Cover, Water, and Habitat Size on Micro-Algae Richness and Abundance in Tank Bromeliads (Guzmania lingulata) in Monteverde, Costa Rica The Role of NEDD4 Protein in Cardiac Function and Disease The Role of B-Cell Lymphoma 6 in the Proliferation and Differentiation of Skeletal Muscle Tracking Cell Lineages through Barcoding Evasion and Modulation of Immune Responses by Mycobacterium Tuberculosis: Implications for Vaccine Development

by Edward Y. Chen by Tiana Huynh by Brian M. Gutierrez by Breanna Lam by Colin J. Mann

SENIOR HONORS THESES

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.

STAFF

52 2

Meet the members of the 2019-2020 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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PAUL SALTMAN

hese are scary, uncertain times. Scientific questions are at the forefront of every discussion as everyone tries to not only understand the novel virus behind COVID-19, but also navigate through our changed world. Now more than ever, we see the importance of accurate science and its representation in all aspects of society, including politics, social life, the economy, and of course, healthcare. Dr. Paul Saltman’s career in science education was focused on preparing the next generation of UC San Diego students to face the problems of today. Dr. Saltman began his career in chemistry after graduating from CalTech with a bachelor's and then a PhD in biochemistry. After 14 years as a faculty member at the University of Southern California, he joined the Division of Biology at UC San Diego in 1967, where he spent the next 32 years educating, inspiring, and engaging with his students as a professor of biology and the provost of Revelle College. He was beloved; Robert Dynes, UC San Diego chancellor at the time, said "Paul was a teacher who inspired every student with whom he came in contact. His love of learning, his enthusiasm for science, [and] his communication skills, made him a role model for students and faculty alike. My tenure here has been enriched by knowing him—by his advice, his personal integrity, his passion for teaching and for life." One of Dr. Saltman’s most significant contributions was the series of interdisciplinary classes, Frontiers of Science, which he developed for non-science majors. He believed in introducing scientific questions to students of all majors,

knowing that the teachers, politicians, businesspeople, and engineers of tomorrow would need to learn how to approach the ever-present debates around science and healthcare. He approached teaching with the goal of inspiring students so that they themselves would be excited to learn more. He wanted students to ask questions about their world and to pursue their own inquiries. In the Frontiers of Science course, Dr. Saltman would challenge his students to think critically about the relationship between science and society, asking questions like: “Can you read a newspaper story about the FDA, the pill, cancer, or mercury pollution and make an analysis of the situation based on what you know about science? If you were to make your choice as a voter, how would you vote?” His passion and talent for teaching were recognized by countless students. He received a number of awards, including the first Career Teaching Award from the San Diego Division of the Academic Senate and the Excellence in Teaching awards from Revelle, Muir, Warren, and Marshall Colleges. He was also recognized by his alma mater, CalTech, with the Distinguished Alumnus Award. Dr. Saltman advocated for scientific literacy far beyond just the university classroom. His initial interest in metabolism made him a proponent of nutrition education for the general populace. He authored the well-received UC San Diego Nutrition Book, a text intended to introduce the reader to the current scientific understanding of nutrition and what that means for one’s own dietary choices. He made multiple appearances on national television and in

Saltman Quarterly does best what Dr. Saltman loved most: inspiring undergraduates to communicate and engage with science in every way.

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“I became a teacher to make people more well, to share with students the pleasure of learning just as other people have shared with me.” –Dr. Paul Saltman

various publications, presenting to audiences and journalists without jargon and ensuring that scientific information, especially about nutrition, was accurately and palatably conveyed to the general public. Throughout his life, Dr. Saltman emphasized the importance of communicating scientific findings in their ethical and social contexts, enabling people to make educated choices about their own lives. Saltman Quarterly is one branch of Dr. Saltman’s long-lasting legacy at UC San Diego. Started by students after Dr. Saltman’s passing, this growing program hopes to carry forward his enthusiasm for sharing science with the broader community, beginning with the students and faculty at our university. Our two magazines, Saltman Quarterly and Under the Scope, as well as our website, quarterly SQ Insider, publicity team, and community outreach programs, bring together scientists, writers, artists, editors, photographers, and many others on our campus. We hope to deliver the stories of biology research being conducted here, as well as around the world, and convey them to readers of both scientific and non-scientific backgrounds alike. My own experience as head of the Review Board has introduced me to the enthusiastic nature of our undergraduate class. Our meetings are a tangible continuation of Dr. Saltman’s legacy, as each generation of Saltman Quarterly staff is bonded by a mutual passion for science and is strengthened with each publication we produce. Review Board engages undergraduate students with their peers’ research by having them edit the research manuscripts

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that are to be published in the Saltman Quarterly journal. It creates a community where students dig deep into science, past the surface level of textbook introductions, and think critically about the research being done. Many of our discussions begin with “Who is this research addressed to?,” “What is the importance of this research?,” and “Was the information conveyed in a clear, concise, and accessible manner?” We continually ask these questions because we recognize the importance of combining good storytelling without diminishing scientific accuracy or integrity. Every branch of our organization finds new ways to relay scientific learning to the world, and we are all incredibly excited to be a part of it. Dr. Saltman’s legacy at UC San Diego is forever immortalized in Saltman Quarterly, as we share his passion for science education, interdisciplinary problem-solving, and an inquisitive approach to everything in life. The challenges we face each day will differ from the ones before, but Dr. Saltman’s inspiration and guidance will remain with us all as we continue to learn and grow into our future.

WRITTEN BY GAYATHRI KALLA Gayathri is a BS/MS student from Thurgood Marshall College studying Microbiology in Dr.Kit Pogliano’s lab. She will graduate in 2020.

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A striped surgeonfish (Acanthurus lineatus) swimming amongst corals at the Birch Aquarium. Photo by Sam Zilberman

F E AT U R E S

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

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targeting cancer stem cells:

progress in a sisyphean battle written by

ingrid heumann

illustrated by

corly huang

features

Cancer stem cells (CSCs) are a subpopulation of cells in a tumor. CSCs exhibit resistance to cancer therapy and can thrive without a matrix, giving them the ability to initiate metastasis. Dr. David Cheresh’s laboratory has created LM609, an antibody that targets a CSC-specific protein called αvβ3. Antibody binding triggers a cytotoxic response from tumor associated macrophages (TAMs), immune cells within the tumor niche. Action by TAMs results in the death of CSCs. Used in combination with a different cancer drug, LM609 antibody administration significantly reduces tumor volume in mice models, illustrating that targeting CSCs impedes tumor growth. Cancer is a condition infamous for its lethality and variation. A diagnosis of certain cancers guarantees death, while others can be eradicated over time. With such disparity, the search for cures and conserved characteristics of various cancers is an elusive and ongoing process. The traditional view of cancer, born out of research efforts in the 20th century, characterized the disease as a ‘monoclonal’ condition, in which a build-up of mutations in a single cell allowed for profuse proliferation and tumorigenesis.1 This theory necessarily supports the conception of a homogenous cancer tumor, composed of an unvarying mass of cells with identical mutations. Current characterizations of tumors, however, illustrate that they are not uniform but instead consist of many different cell types per tumor, including differentiated and undifferentiated cells. This tumor heterogeneity fundamentally contradicted the monoclonal cancer theory and has forced it to give way to other hypotheses.

an introduction to cancer stem cells One new characterization of cancer is the cancer stem cell (CSC) theory. This conception proposes that CSCs, a subpopulation of undifferentiated tumor cells, can initiate metastasis, the migration of cancer cells to different locations in the body. CSCs are capable of living outside of the primary tumor site, and thus can trigger a metastatic cascade, in which the body becomes compromised by the spread of cancer cells. In addition to

metastasis, CSCs have been implicated in stimulating “angiogenesis,” in which local blood vessels extend towards the growing tumor, increasing its blood and nutrient supply. CSCs can also release stem cell factors that cause nearby tumor cells to display more stem-like characteristics. As such, CSCs can promote the growth of other stem-like cancer cells within the tumor, producing a mélange of tumor cells, which fall between the categories of pure stem cells and completely differentiated cells. The mixed population of CSCs, stem-like cancer cells, and fully differentiated tumor cells culminates in a heterogeneous tumor that affirms contemporary characterizations of cancer. CSC formation has been associated with many initiating factors, including inflammation, drug treatment, and stressful growth environments. The particular connection between CSCs and an inflammatory microenvironment, or niche, reflects a highly established link between inflammation and cancer. Inflammation itself is not harmful; it is simply the body’s generic, ephemeral response to combat pathogens and heal injuries. However, chronic inflammation, in which inflammation persists and can even damage surrounding tissues, has been dubbed a ‘cancer-inducing niche’ because it can prime cells for cancerous, unchecked growth.1 Under such prolonged conditions of inflammation, cells behave as though they are in a wound and upregulate specific genes, including those that code for the growth and proangiogenic factors involved in the healing process. Normally, these factors promote tissue repair, but they can also encourage tumor growth and development through proliferation and angiogenesis, respectively. This dual nature of the body’s wound response can be exploited

by cancer cells, which hijack the natural healing process to nourish the tumor, treating it like an inflammatory wound.

from cell to cancer stem cell Research into CSCs is an evolving topic at UC San Diego, specifically in the laboratory of Dr. David Cheresh. Dr. Cheresh explained that, just as the environment of a normal wound is prone to various stresses (such as infection), which must be overcome for complete recovery, the tumor microenvironment also encounters obstacles to growth. For cancer cells, one of the main sources of stress is cancer therapy, which is administered to eradicate the tumor. While cancer therapies may demonstrate significant tumor shrinkage, they can also promote the reprogramming of tumor cells, in which some cells de-differentiate into more primitive, robust cell forms that exhibit less sensitivity to treatment. This process can be likened to a cell regression, in which differentiated cells lose some of their specificity but increase their survival capacity. These reprogrammed cells become uniquely situated to survive in the harsh cancer niche, which is characterized by low nutrients, hypoxic conditions, and an acidic pH. As Dr. Cheresh says, such cells “believe they are in a wound and act accordingly. They lose their specialized behavior and gain a degree of drug resistance.” Over the course of this cell reprogramming, cell resilience increases and gene expression patterns change such that certain stem cell markers become upregulated. Reprogramming under stress thus constitutes an adaptation of normal tumor cells into de-differentiated CSCs. This relationship SALTMAN QUARTERLY

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Combination Treatment

Single Treatment

Cancer Cell

Cancer Cell Treatment

Treatment 1

vs Cancer cell mutates.

Treatment ineffective.

Treatment 2

figure 1. Basic schematic showing an

ineffective single drug treatment (left) and a more effective combination therapy using two drugs (right).

establishes cancer therapies as an ironic architect of CSC enrichment. In addition to promoting proliferation and differentiation, CSCs play a direct role in metastasis by spreading cancer cells throughout the body. CSCs themselves have enhanced migratory capabilities because they can survive and proliferate without a matrix; they also can encourage the migration of and subsequent invasion by differentiated tumor cells by decreasing cellular adhesion.2 Dr. Cheresh emphasized that together these capabilities allow CSCs to promote metastasis, in which they initiate tumorigenesis at a distinct site from the primary tumor.

targeting the tumor's plasticity To successfully obliterate CSCs, Dr. Cheresh’s laboratory, along with many others, uses a form of treatment called combination therapy, in which the administration 8

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Subsequent therapy prevents cancer cell mutation. of one drug is followed by the use of another. This treatment targets the tumor’s adaptive ability, or its capacity to enrich its CSC population in response to cancer treatments. The goal of a multiple drug treatment is to inhibit the different pathways that the tumor may use, thus reducing its ability to grow in the presence of administered drugs (i.e. decreasing its drug resistance). CSC research in Dr. Cheresh’s lab centers around αvβ3, a cell surface integrin and receptor. Early on in this research, some CSCs were found to express high levels of αvβ3, a discovery that aligns with the integrin’s long-standing association with cancer progression. This preliminary finding allowed Dr. Cheresh and his laboratory to use αvβ3 expression as an indicator of CSC populations.3 Further investigations showed a correlation between high αvβ3 expression and increased populations of tumor-associated

macrophages (TAMs), immune cells that are highly enriched within the tumor niche and aid in its growth and immunosuppressive capacities. Because of their close proximity to the tumor, if TAMs could be reprogrammed to attack rather than support the tumor, this could severely obstruct tumor expansion. Along this line of thought, the Cheresh Lab developed a mouse antibody they called LM609, which targets αvβ3 on mice-grown human CSCs. LM609 binds to αvβ3 and bridges it with the TAM immune cells, stimulating TAMs to kill αvβ3-bearing cells. As most CSCs express αvβ3, the LM609 antibody essentially recruits TAMs to target therapy-resistant CSCs within the tumor niche. Dr. Cheresh has characterized his laboratory’s research as a way of “arming the body’s immune system” against cancer, a procedure in which TAM immune or effector cells are given the ability to recognize and destroy therapy-resistant CSCs. This result is primarily achieved through an immune response called antibody-dependent cellular cytotoxicity (ADCC), in which immune cell (TAM) receptors recognize and bind to an antibody (LM609), which is simultaneously bound to αvβ3 on the tumor cell. Once this coupling occurs, TAMs release cytotoxic factors that kill the αvβ3-expressing tumor cells. This specific form of mediated cell death is of great importance because CSCs are known to express high levels of CD47, which is recognized by immune cells as a “don’t eat me signal”.3 Elevated CD47 thus allows CSCs to evade phagocytosis, the major process of mediated cell death that macrophages use to destroy tumor cells. Dr. Cheresh elucidated that ADCC essentially becomes the main mechanism of action through which CSCs can be targeted. Additionally, depletion of macrophages from mice rendered the antibody’s therapeutic abilities ineffective, illustrating that LM609 functions in a macrophage (TAM)-dependent fashion. This work illustrates that TAMs can be induced to combat tumor growth by targeting the αvβ3-bearing CSCs.3 Recently, in a novel combination treatment, the LM609 antibody was adminissqonline.ucsd.edu

tered to mice in conjunction with erlotinib, an FDA-approved drug that inhibits the epidermal growth factor receptor (EGFR) and shrinks tumors that depend on EGF. Interestingly, erlotinib administration alone was found to initially decrease tumor size, indicating that the drug contained a preliminary capacity to hinder tumor growth. Over time, however, as tumor cells became resistant to erlotinib, both αvβ3 expression and CSC levels increased, suggesting that erlotinib administration led to a subsequent enrichment in CSCs. This can be explained by the fact that tumor cells, acting under woundlike conditions, simply classified erlotinib as a form of stress. In response, it appears that a large fraction of the tumor population underwent reprogramming in which cells de-differentiated to increase survival capacity, resulting in an elevation of CSCs. By comparing the effects produced by erlotinib treatment alone, the combination treatment of erlotinib followed by LM609 was found to be quite effective at reducing tumor volume. The success of this combination treatment rests on the fact that the mechanism of action for LM609 is enhanced by erlotinib. In essence, erlotinib administration results in a reconstitution of the tumor itself, in which αvβ3-bearing cells, the targets of TAMs, become enriched.3 Consequently, TAMs have an increased ability to reduce tumor growth because they can kill the expanded CSC population within the tumor niche.

The increased capability of LM609 to stimulate TAM-mediated tumor cell death is thus dependent on the initial administration of a drug like erlotinib. The interplay between these two drugs illustrates the utility of combination therapy in that the synergy of erlotinib and LM609 far surpasses the effects either treatment has alone.

looking to the future In essence, CSCs represent a novel target for cancer therapeutics, as these cells are responsible for metastasis and therapy resistance within the tumor niche. By removing CSCs, it becomes possible to eliminate the core regenerative abilities of the tumor that contribute to its resilience against standard cancer therapeutics. The Cheresh Lab is currently investigating ways to take their antibody "from the bench to the bedside" by creating a version of LM609 for human use. Dr. Cheresh explained that their aim is to accomplish in humans the same breakthrough of tumor reduction that was achieved in the mouse models. Cancer’s ability to mutate and renew itself has long presented a virtually insurmountable obstacle for cancer treatment; that, however, may change as cancer research continues to extend the frontiers of knowledge, and therapies begin to target the tumor’s dynamic nature. One possible method through which the tumor can lose

its regenerative properties is the extermination of CSCs, an area of investigation that is constantly expanding. The boulder may yet reach the top of the hill.

references 1. Said AM and Seno M. 2019. Conversion of Stem Cells to Cancer Stem Cells: Undercurrent of Cancer Initiation. Cancers, MDPI, doi:10.3390/cancers11030345. 2019.11.03. 2. Prager, BC, Xie Q, Bao S, Rich JN. 2018. Cancer Stem Cells: The Architects of the Tumor Ecosystem. Cell Stem Cell, vol. 18, no. 1, 2016, doi:10.1016/j.stem.2015.12.014. 3. Wettersten HI, Weis, SM, Pathria P, Schalscha T, Minami T, Varner JA, Cheresh DA. 2019. Arming Tumor-Associated Macrophages to Reverse Epithelial Cancer Progression. American Association for Cancer Research, doi:10.1158/0008-5472.CAN-19-1246. 2019.08.08.

written by

ingrid heumann

Ingrid is a Biochemistry and Cell Biology and Humanities major from Roger Revelle college. She will graduate in 2021.

Antibody Dependent Cellular Cytotoxicity (ADCC) LM609

αvβ3

Cancer Stem Cell

LM609 binds with αvβ3

TAM

TAM attacks αvβ3 bearing cells

figure 2. The schematic shows a rough procession of the events following LM609 administration: LM609

recognizes and binds αvβ3 on the surface of CSCs; this binding then stimulates nearby TAMs to attack cells that contain αvβ3.

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TALKING THE TINY TALK: how microbes communicate

written by

eleanor wang

illustrated by

cristina corral

features Microbes live everywhere, from our gut to the depths of the ocean. These tiny organisms play big roles in shaping their surroundings by interacting with each other, with other living beings, and with the physical environment. While there are innumerable methods of microbial communication, taking a closer look at specific ones and understanding how they work can provide us with a better understanding of the role microbes play in the world.

The small is often associated with the simple. One individual, unicellular bacterium pales in comparison to a full human being with complex organ systems and trillions of cells. Big things, however, can come in small packages. Perhaps individual microorganisms don’t have room for muscles, bones, or guts, but the microbial world is anything but simple. Rather than considering bacteria as unicellular organisms living as isolated beings, we should remember that these tiny creatures live within larger communities—they are not alone. In doing so, we can begin to understand the ways they function and affect the world. Microbes live almost anywhere imaginable, from our skin and guts to the soil and ocean. Everywhere these microscopic creatures live, they perform important tasks to influence the health of their habitat. For instance, marine cyanobacteria and algae fix carbon dioxide and per-

form most of the photosynthesis on earth.1 Moreover, microbes living in the human gut have been found to affect susceptibility to cancer, Alzheimer’s disease, depression, inflammatory bowel disease, and other major human diseases.2 Whether we are looking at the ocean or the large intestine, most microbial communities have relatively high species diversity, and these different species perform different functions, interact with other microbes in different ways, and have different effects on the rest of the world. To understand how changes in the composition of microbes in our gut might cause or prevent disease, it is important to understand how microbial communities are formed and shaped in the first place. While this article will focus on specific examples of bacterial communication, it is important to keep in mind that there are many more ways, both discovered and undiscovered, in which bacteria communicate with each other and with other spe-

cies. This excludes interactions between bacteria and other kingdoms, such as fungi and viruses, and host-microbe interactions (the “host” may be a plant or animal). Much like how humankind is often at war for limited resources, bacteria also compete against each other to occupy a physical space or for specific nutrients. In the context of human disease, bacterial competition can be important in preventing pathogenic bacteria from colonizing the body and causing disease. Friendly commensal bacteria that typically reside in the gut can outcompete less friendly bacteria like Salmonella and pathogenic strains of Escherichia coli. Bacterial competition is multifaceted, but one of many competition mechanisms is the type VI secretion system (T6SS).3,4 Bacteria use T6SS to fight each other: one bacterium, the “donor,” punctures its victim, known as the recipient, with a syringe-like structure and injects a

figure 1.

Abstract depiction of one bacterium using T6SS to mediate lysis of another.

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variety of protein toxins that target and degrade different cell structures within the recipient. T6SS is particularly important for shaping bacterial communities where bacteria live in close proximity with one another. The regulatory mechanisms, components, and effects of T6SS are highly complex, but Dr. Alistair Russell, an Assistant Professor in UC San Diego’s Division of Biological Sciences, investigated the mechanisms of T6SS during his graduate studies and has extensive experience and expertise in the field. He published several important papers throughout his career detailing mechanisms and pathways by which T6SS operates. For instance, his 2011 Nature article describes the finding that Pseudomonas aeruginosa, a bacterium commonly known for its ability to infect cystic fibrosis patients, delivers proteins into the recipient that target and degrade the bacterial cell wall.5 The same paper identifies that P. aeruginosa itself is immune to the effects of these cell wall-degrading proteins. The fact that these proteins target and degrade other species but not P. aeruginosa itself implicates T6SS as a mechanism for bacterial antagonism. In 2012, Dr. Russell published a paper identifying the same and very sim-

ilar cell wall-degrading proteins in a variety of bacterial species.6 These cell wall-degrading proteins were also found together with similar “immunity” proteins that had protected P. aeruginosa from degrading itself. These findings suggested that T6SS is applied to different bacterial species in the community, allowing these different species to duel each other. While T6SS is primarily associated with competition, other potential functions include, but are not limited to, cell-to-cell signaling, the killing of “non-cooperators,” biofilm remodelling, and the lysis of phage-infected bacteria. How would a bacterium use T6SS to perform these complex functions? This question has not been fully answered yet, but in an interview, Dr. Russell discussed a paper which demonstrated that bacteria use T6SS to eliminate members of the community that refuse to “cooperate.”7 In the study of interest, it was found that Burkholderia thailandensis, a soil-dwelling species, evolved so that T6SS is coupled to quorum sensing. During quorum sensing, small molecules are produced and secreted to signal to other bacteria and regulate gene expression. It was previously unclear how bacteria could evolve such a system, as those who do not produce quorum sensing molecules would still benefit from the work of

bacteria producing the molecules. Therefore, these “non-producing” bacteria could be considered “social cheats” that are not cooperating with other members of their species and are undesirable. By using quorum sensing to activate T6SS, B. thailandensis is able to eliminate the cheaters and maintain social order. T6SS is present in bacteria from a wide range of environments, and while it is only one of many mechanisms for microbial communication, understanding T6SS helps us understand multiple aspects of microbial communities. As quoted from an interview with Dr. Russell, “microbial communities are incredibly important in the carbon cycle, and a million other things, so understanding interactions between microbes is going to be especially important to our understanding of our changing world.” The UC San Diego Biodynamics lab, led by Dr. Jeff Hasty and Dr. Lev Tsimring, recently published a paper studying a phenomenon mediated by T6SS.8 As the title “Flower-like patterns in multi-species bacterial colonies” suggests, the study examines how T6SS mediates the formation of flower-like bacterial colonies. Essentially, a mixture of motile and

figure 2.

Interactions between two different bacterial species can influence the morphology of colony formation.

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non-motile bacterial species battle it out with T6SS and end up forming large flower-shaped colonies. The lab generated mathematical models to simulate the formation of these beautiful patterns, as being able to predict the community dynamics of different species helps reinforce our understanding of how different bacterial species interact with one another. If T6SS is capable of mediating such a variety of phenomena, we can only imagine the complexity underlying other forms of microbial communication. While quite different from T6SS, electrochemical signaling is a form of microbial communication that has only recently been characterized in bacterial communities. Electrical signaling in cells is often associated with the image of an action potential propagating down the axon of a neuron, induced by the influx of sodium ions, the efflux of potassium ions, and the subsequent reestablishment of membrane potential by the sodium-potassium pump. Incredibly, the Süel Lab at UC San Diego recently discovered

that bacteria can use ion channels to conduct long-range electrical signals. The lab’s 2015 paper, published in Nature, identifies that ion channels in bacterial biofilm communities allow for the propagation of potassium for the coordination of metabolic states.9 An interview with Dr. Süel highlighted the fact that there are still many important and interesting questions to be answered about electrochemical signaling in bacteria. Studying these processes requires using the right tools to answer the right questions. For instance, current studies of the human microbiome are heavily based on DNA sequencing methods. DNA sequences can help us identify the species present in a given population, and identifying existing genes within those populations allows us to generate predictions about how these bacterial species and communities might function. Validation of these predictions, however, is much more difficult and depends heavily on the system being studied. Electrical signals cannot be detected and measured through a DNA sequence. Analysis of the colony

growth and change over time involves microscopy and mathematical modelling. Studies of T6SS dynamics and its effects on bacterial community formation also involved a wide variety of experimental techniques. The question of how bacteria interact with each other and the surrounding world is not an easy one to answer. It is one that requires collaborative efforts between not only microbiologists but also chemists, physicists, and engineers. T6SS represents but a small fraction of microbial “communication” mechanisms. There are theoretically ten different secretion systems with different functions and purposes.10 Bacteria, and microbes in general, while invisible, are ubiquitous in our world. Their microscopic actions have a big effect on other living systems. Knowing how one bacterium attacks another, or how bacterial communities exchange nutrients, can lead us to understand more about different factors that drive changes in the environment and in human health. There is much more than meets the eye, quite literally.

written by

eleanor wang

references: 1. Hutchins, D. A., Fu, F., (2017). “Microorganisms and ocean global change”. Nature Microbiology, 2, 17058. https://doi.org/10.1038/nmicrobiol.2017.58 2. Knight, R., Callewaert, C., Marotz, C., Hyde, E. R., Debelius, J. W., McDonald, D., Sogin, M. L. (2017). “The Microbiome and Human Biology”. Annual Review of Genomics and Human Genetics, 18:1, 65-86. https://doi.org/10.1146/annurev-genom-083115-022438

Eleanor Wang is a Biochemistry and Cell Biology major from Thurgood Marshall college. She will graduate in 2021.

3. Stubbendieck, R. M., Straight, P. D. (2016). “Multifaceted Interfaces of Bacterial Competition”. Journal of Bacteriology 198(16), 2145-2155. DOI: 10.1128/JB.00275-16 4. Russell, A. B., Brook Peterson, S., Mougous, J. D. (2014). “Type VI secretion system effectors: poisons with a purpose”. Nature Reviews Microbiology, 12, 137-148. https://doi.org/10.1038/nrmicro3185 5. Russell, A. B., Hood, R., Bui, N. et al. (2011). “Type VI secretion delivers bacteriolytic effectors to target cells”. Nature 475, 343–347. https://doi.org/10.1038/nature10244 6. Russell, A. B., Singh, P., Brittnacher, M., et. al. (2012). “A Widespread Bacterial Type VI Secretion Effector Superfamily Identified Using a Heuristic Approach”. Cell Host & Microbe, 11:5, 538-549. https://doi.org/10.1016/j.chom.2012.04.007. 7. Majerczyk, C., Schneider, E., Greenberg, P. (2016). “Quorum sensing control of Type VI secretion factors restricts the proliferation of quorum-sensing mutants”. eLife. https://doi.org/10.7554/eLife.14712 8. Xiong, L., Cao, Y., Cooper, R., et al. (2020). “Flower-like patterns in multi-species bacterial colonies”. eLife. https://doi. org/10.7554/eLife.48885 9. Prindle, A., Liu, J., Asally, M. et al. (2015). “Ion channels enable electrical communication in bacterial communities”. Nature 527, 59–63. https://doi.org/10.1038/nature15709 10. Green, E. R., Mecsas, J. “Bacterial Secretion Systems: An Overview”. Microbiology Spectrum, 4(1):VMBF-0012-2015. http:// dx.doi.org/10.1128/microbiolspec.VMBF-0012-2015

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development of dilated cardiomyopathy one molecule at a time

written by lilit vardanyan 14

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illustrated by ellis zhang sqonline.ucsd.edu

features Among many cardiac diseases, one of the most common and devastating is dilated cardiomyopathy (DCM). The diseased heart is unable to adequately pump blood throughout the entire body because of its weakened left ventricle.1 Since a molecular cause is not yet found, UC San Diego researcher Dr. Stephan Lange studies the molecular development of DCM. Although the project is in its early stages, he has identified various small-molecule inhibitors and a possible molecular switch that causes the development of DCM.

Muscles: they move our bodies, transport the food we eat through our intestines, and help us breathe. A specialized version of this essential collection of tissues is the cardiac muscle, set apart to perform one of the most integral functions of life: keeping our hearts beating. Consequently, cardiomyopathy—an abnormality of the heart muscle—poses a serious threat to people impacted by the condition. Dr. Stephan Lange, a professor and researcher at the UC San Diego School of Medicine, explores specific mechanisms and pathways to understand the development of a specific variety of cardiomyopathy, dilated cardiomyopathy (DCM), at a molecular level.2 DCM is one of the most common causes of heart failure, affecting people of all ages. It is characterized by the heart’s inability to provide enough blood to the r e s t of the body. As the left ventricle— the strongest chamber of the heart—is stretched and thinned, it is unable to pump blood with the same strength and efficiency as a healthy heart. For many patients, signs and symptoms of this disease

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include fatigue, shortness of breath, chest pain, heart murmurs, and edema, which is the fluid build-up in the legs and abdomen.1 Although DCM displays similar symptoms in many patients, its cause varies from case to case. According to Dr. Lange, this condition may stem from either a familial trait, a missing gene, or environmental conditions. Since the underlying problem is so different for each patient, it is important to dig deeper. Knowing that biomedical research was the right path for him, Dr. Stephan Lange was always fascinated with the intricate microscopes and riveted by the idea of understanding the inner workings of a cell. His love for and interest in DCM development is rooted in his first research experience as an undergraduate; years later, Dr. Lange joined Dr. Ju Chen’s laboratory at UC San Diego to continue his journey as a postdoctoral fellow. Today, with his own team of researchers, he furthers his exploration by looking at small-molecule inhibitors along with sarcomeric and cardiac muscle-associated proteins to decode the molecular mechanisms that lead to the development of DCM. In the process of identifying a possible molecular signaling pathway that leads to the development of DCM, Dr. Lange and his group revealed the immensely important functions of the Ankrd gene, specifically the Ankrd1 and Ankrd2 genes, which express the cardiac adriamycin-responsive proteins, CARP1 and CARP2.2 Proteins—complex polymers—play essential roles in biological pathways and processes, and their absence or malfunction can be absolutely detrimental. The CARP protein, complexed with a protein kinase C alpha (PKCalpha), plays a crucial role in the normal development and function of cardiac muscle cells, as its presence can be both beneficial and detrimental for the heart. It has been observed that when the heart is in a stressful situation, there is an overproduction and upregulation of

CARP protein, and the complex can either help the muscle overcome the strain in the short term or harm the heart if the stress is chronic. The intercalated discs of the muscle cells, which allow the uniform contraction of the heart tissue, are the locations of the concentration and chronic activation of PKCalpha. According to Dr. Lange, this could be the “molecular switch” when the activation of PKCalpha is either acute and helps the heart overcome stress, or is chronic and damages the muscle cell.3 Given this observation, a new question arose: would inhibiting the expression of the Ankrd gene result in a healthy cardiac cell? Answering this question requires definitively identifying the “molecular switch” and finding an inhibitory factor; Dr. Lange and his team have a long journey ahead. Using several different techniques, their research began with screening for small molecules in cardiac cells that could efficiently block the expression of CARP protein. One of the techniques was harvesting and isolating heart muscle tissue from a living organism to make induced pluripotent stem cells (iPS cells), and reprogram them to become cardiac muscle cells. After many challenges, Dr. Lange and his team identified several small molecules that can inhibit the Ankrd gene; however, hundreds and thousands more need to be tested before a definitive molecule is established as an effective and useful inhibitor. Although the screening project is in its earlier stages, by characterizing specific disease-related post-translational modifications in Ankrd1/CARP1, the researchers already have leads on what the process of the “molecular switch” could be. Thus far, with the help of the UC San Diego proteomics core and its head, Dr. Majid Ghassemian, Dr. Lange’s team used proteomics-based approaches to identify some post-translational changes; they verified and characterized these changes using biochemical and cellSALTMAN QUARTERLY

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ANKRD

transcription

figure 1.

the inhibition The Ankrd gene produces the CARP protein through transcription and translation. The small molecule can act as an inhibitor of CARP production, thus acting as a “molecular switch”.

translation CARP

biological methods. The identification of a post-translational modification as part of the switch will certainly benefit the screening of small molecules as well, as the group will have a better idea of how to approach the inhibition of the CARP protein. Dr. Lange mentions that, “it becomes more 16

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interesting when another piece of the puzzle becomes available.” As this is an ongoing project in the Lange lab, there is still more to be discovered. Testing for CARP inhibition in single cells can clarify whether a specific small molecule can one day become the

basis of a possible treatment for patients with DCM. For now, however, the molecules that were found to inhibit the Ankrd gene will be tested in a small animal that is used as a model. Inhibitory molecules are tested in an animal model in order to confirm that results in single cells are also observed in actual living organisms. Checking for efficacy, the Lange group must also confirm that the inhibition is doing exactly what they want it to do, and not causing any harmful side effects. Dr. Lange states that, “there must be a follow-up to see what the [consequences] of the inhibition of CARP protein formation may be, because if the inhibition causes the cardiac muscle to stop contracting, then that will not be good.” Looking to the future, randomized clinical trials will directly impact patient lives; however, this project is nowhere near that stage, as many years and possibly decades of work must be done. Dr. Lange describes the process of turning basic research into a clinical trial as “the unicorn everyone wants to find.” However, these challenges do not stop the Lange lab from expanding to human cardiac cell models. sqonline.ucsd.edu

figure 2.

from basic research to the hearts of patients As information about small molecules is passed down from researchers to physicians, this drawing shows the evolution and translation of basic research into medicine.

With the help of a collaborator, the group gets access to very valuable, human cardiac muscle cells from patients who undergo heart transplants. This gives the researchers an opportunity to study human samples and see whether their results and predictions in single cells and mice are comparable to people. Through a different collaborator and fellow researcher in London, U.K., Dr. Lange implemented his studies in a small population of human heart cell donors from Sydney, Australia and the findings were an amazing advancement. According to Dr. Lange, “of all the molecular markers identified [that] go wrong in [our] animal model, in about fifty percent of the patients, [we] see the same thing happening.” This was a huge contribution to the field, because as he describes, “the signaling pathway we identified [that] is activated and leads to the development of the disease might be active in 50% of the population. That is actually [a significant proportion], considering it is such a devastating disease.” sqonline.ucsd.edu

These findings are exciting, not only because the group can correlate its laboratory research to actual human tissue, but also because their work could be on its way to making a big discovery in the future. DCM is one of the most common causes of heart failure; it continues to affect 36 out of 100,000 adults, and patients often require heart transplantation.4 Dr. Lange and his team are in search of what can one day give patients with DCM another chance at a healthy life. Every finding, although a small piece of the bigger puzzle, is a victory and an encouragement to move forward.

3. Lange S, Gehmlich K, Lun AS, Blondelle J, Hooper C, Dalton ND, Alvarez EA, Zhang X, Bang M-L, Abassi YA, et al. MLP and CARP are linked to chronic PKCα signalling in dilated cardiomyopathy. Nature Communications. 2016 [accessed 2020 Jan 11];7(1). doi:10.1038/ ncomms12120 4. Mahmaljy H. Dilated Cardiomyopathy. StatPearls [Internet]. 2019 Dec 12 [accessed 2020 Apr 12]. https://www.ncbi.nlm.nih.gov/books/ NBK441911

references

1. Dilated cardiomyopathy. Mayo Clinic. 2018 Mar 9 [accessed 2020 Feb 15]. https:// www.mayoclinic.org/diseases-conditions/ dilated-cardiomyopathy/symptoms-causes/ syc-20353149 2. Mechanisms and signaling pathways important for the development of dilated cardiomyopathy (DCM). Stephan Lange - DCM Research. [accessed 2020 Apr 12]. http://slangelab.ucsd. edu/dcm.html

written by

lilit vardanyan Lilit is a Human Biology major from Thurgood Marshall college. She will graduate in 2021.

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HAND's Grasp on the Brain: HIV and Neurocognitive Disorder written by kaz nuckowski illustrated by diana presas

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features HIV is a virus that can lead to AIDS, and then death unless treated. Modern medicine allows people with HIV to live longer; however, HIV-associated neurocognitive disorder (HAND) is an observed effect of long-term infection. Dr. Ronald Ellis’ research on the neurological complications of HIV revealed that neurocognitive decline has a legacy effect, meaning neurological damage caused by HIV persists even in patients on effective antiretroviral therapies. More research into treating HIV and HAND must be conducted to determine how HIV impacts the brain and which treatments can mitigate neurocognitive decline. Imagine surviving exposure to a lethal virus, only to live with a chronic condition that impedes your ability to return to your previous quality of life. Human immunodeficiency virus (HIV), infamous for causing the AIDS crisis of the 1980s, may lead to neurocognitive deterioration, which can be debilitating for patients. To stay healthy, patients must adhere to a strict medication regimen, but they may forget to do so because of potential neurological damage caused by HIV itself. Furthermore, many individuals with HIV might not have access to consistent medical care, especially in marginalized communities. Forgetting to take medication or attend doctor’s appointments can greatly hinder the treatment process. This ongoing cycle is one that many people with HIV will contend with for the rest of their lives, and scientists are in the process of understanding the relationship between HIV infection and cognitive deterioration. HIV attacks a person’s CD4+ T-cells, which communicate with other types of immune cells to coordinate immune responses.1 Once T-cell count is diminished to an extremely low level, the individual’s body has a reduced ability to combat other infections. That leads to acquired immunodeficiency syndrome (AIDS), a disease that leaves the patient vulnerable to a

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plethora of opportunistic infections. Since its development in the early 1990s, antiretroviral drugs reduce the amount of virus in the blood to slow the progression of the HIV infection. There are several types of antiviral medications that interfere with the viral life cycle at different stages, such as viral fusion with CD4+ T-cells and integration into the genome of the host cell.6 Combined antiretroviral therapy (cART) is the most effective way to manage HIV to date, since it uses multiple antiretroviral drugs to minimize drug resistance.7 Back when effective treatments for HIV did not exist, the infection progressed rapidly to AIDS and then death; patients passed away before the effects of dementia and neurocognitive decline could be observed and studied.4 Today, because cART significantly extends life expectancy, there are more observations of neurocognitive degeneration directly related to their HIV infection, also known as HIV-associated neurocognitive disorder (HAND). HAND is correlated with two main areas of functional limitations: decreased cognitive set-shifting abilities and prospective memory loss. Set-shifting is the ability to intentionally change focus between different kinds of information responses.12 Prospective memory loss is a reduced ability to remember an action one intends to do. In people with HIV, prospective memory loss brought with HAND often causes the most detriment by inhibiting patients’ ability to remember to follow their treatment plan and maintain their health, career, and finances. Both of these neurocognitive dysfunctions make it more challenging for individuals living

with HIV to do activities that are essential to daily life. HIV’s exact process of affecting patients’ brains is unknown, which is especially concerning given that HAND is observed in about 50% of individuals with HIV, including those who are receiving treatment.11 However, the current theory of HAND pathology is multifactorial. One proposed mechanism is related to the theory that infected immune cells in the brain may survive cART and form reservoirs of HIV-infected macrophages in the brain, causing damage indirectly through inflammation.8 In treated individuals with HIV, reservoirs of HIV in blood plasma, the GI tract, the cerebrospinal fluid, and lymph nodes can linger, as well as viral proteins; both can trigger persistent immune activation.2,3 This impacts a myriad of body systems—crucially, the nervous system, where neuroglia play an important role in combating potential infection in the brain and an overabundance of immune activators and mediators can become neurotoxic.3 Ultimately, this results in neurocognitive degeneration, which presents itself as rapid-onset, severe dementia caused by the virus penetrating the blood brain barrier to attack neuroglial cells.3 HIV infections have also been shown to cause epigenetic aging, including deregulation of gene expression, which may increase the incidence of neurocognitive disorders in younger individuals who would otherwise be relatively unlikely to develop such a severe level of cognitive dysfunction.9,10 The relationship between HIV and neurocognition has interested many researchers, including Dr. Ronald Ellis, a clinical neurologist specializing in HIV and HAND at UC San Diego Health and School of Medicine. The main objective of Dr. Ellis’ research is to relate different severity levels of HAND with imaging of varying levels of brain abnormalities, and more broadly, to assess whether or not SALTMAN SALTMANQUARTERLY QUARTERLY

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there is a legacy effect involved in HAND. A longitudinal study was designed to compare the effects of HAND in individuals with undetectable viral loads—mainly those on cART—to the entire study cohort, including individuals who do not exhibit cognitive degeneration or are incompletely virally suppressed. Individuals were classified on the spectrum of HAND based on the observed damage to their brain matter as well as their exhibited degree of functional limitations. By comparing the subset of virally suppressed individuals on cART to those with some level of HIV, the researchers could investigate a potential legacy effect. If there is no legacy effect, the brain damage caused by HIV infection would reverse after cART treatment. However, if there is a legacy effect, the next step would be to determine the extent of the white and grey matter damage and determine whether brain matter damage changes over time. The study used complementary data collection methods to assess negative impacts on brain tissue. At a micro scale, the presence or absence of key biomarkers in different regions of the brain provided insight into the extent that neuron structure and function persisted in the presence of inflammation; at a macro scale, structural MRI results indicated the extent and nature of damage to white and grey matter. White matter, in particular, is considered the ‘superhighway’ of the brain because it is essential for rapid communication between different regions of grey matter in the brain; the destruction of white matter inhibits cognitive coordination.14 In tandem, these two imaging perspectives provided a comprehensive picture of levels of brain damage. Results showed lower levels of biomarkers that signify neuronal integrity, and higher levels of biomarkers that indicate inflammation and damage to the cell membrane, especially in cases of more severe degeneration.13 Additionally, researchers saw that patients exhibiting cognitive impairment had a greater proportion of abnormal white matter and unhealthier white and grey matter.13 Statistically significant levels of white and grey matter damage did in fact persist. All of this evidence strongly suggests that a legacy effect is indeed present; in other words, brain matter damage and 20 20

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abnormalities persist even with effective and the trajectory of functional limitations cART. This opens the door to more re- for people with HIV. Young people living search, which is needed to establish why with HIV in particular are already more there is a legacy effect. susceptible to other neurocognitive disWhite matter damage persisted in indi- orders like Parkinson’s and Alzheimer’s.3 viduals on older antiretroviral therapies, so Future research at UC San Diego and beDr. Ellis and his colleagues hypothesized yond on HAND will examine the prognothat white matter damage would similar- sis of accelerated epigenetic aging in oldly persist in individuals on cART. Though er populations and study how the natural cART protects T-cells by reducing viral deregulation of DNA expression can be load, it cannot sufficiently protect the im- compounded by HIV. Additional research mune system alone, especially in the face should also be done to find what causes of chronic inflammation. This persistent immune acfigure 1. different classes of antiretrotivation is thought to be a result of HIV targeting inviral drugs interfere with different key flammation-regulating prostages of the viral life cycle teins.15 Broadly, the fact that This could be through 1) blocking receptors on cell immune dysfunction and insurfaces and inhibiting proteins that are required for flammation persist with viral viral fusion, or 2) preventing RNA from integrating load reduction strongly sugto host DNA by eliminating reverse transcriptases or gests that other factors are at mimicking nucleotides. play, and that HIV likely targets the immune system in a more comprehensive manprevents virus ner than scientists originally from attaching suspected. In the clinical setting, a legacy effect means that even individuals placed on cART as soon as they are infected are at risk for developing HAND. The functional limitations that result from HAND will not be reduced by effective cART alone. This must be taken into account when determining the most appropriate medical care for current and future individuals with HIV, and research is being conducted to find additional treatments for the effects of HAND itself. Dr. Ellis himself is currently working on phase II drug trials for tesamorelin, a medication that showed promise in slowing the progression of cognitive deterioration, and could be a promising method of action to reduce neurocognitive degeneration in people with HIV.16 prevents integration The presence of a legacy efof viral DNA fect opens new questions on how HAND will affect aging sqonline.ucsd.edu sqonline.ucsd.edu

the deregulation of the immune response, which could point to potential treatments. If chronic inflammation causes neurocognitive degeneration, treatments that reduce the activity of inflammatory pathways may be the first step to preventing HAND. Overall, HAND has yet to be deciphered. Dr. Ellis’ work is crucial because it demonstrates that treatments for HIV, such as cART, are not the end of the story for individuals with HIV. Furthermore, the incidence of neurocognitive disease underscores the importance of social support systems, which are essential for helping people with HIV manage their healthcare and navigate life successfully. Doctors must also be aware of the implications of HAND and work with individuals living with HIV to minimize functional neurocognitive impairment. Living with HIV is a lifelong struggle; many of the impacts of this virus are not problems we have solved: they are problems we are continuing to understand. written by

kaz nuckowski Kaz is a Human Biology major from John Muir college. They will graduate in 2023.

references:

1. Cichocki M. What Are CD4 T-Cells and Why Are They Important? 2019 Dec 10 [accessed 2020 Feb 14]. https://www.verywellhealth.com/what-are-cd4-t-cells-49354 2. Hileman CO, Funderburg NT. Inflammation, Immune Activation, and Antiretroviral Therapy in HIV. Current HIV/AIDS Reports. 2017;14(3):93–100. doi:10.1007/s11904-0170356-x 3. Amor S, Puentes F, Baker D, Valk PVD. Inflammation in neurodegenerative diseases. Immunology. 2010;129(2):154–169. doi:10.1111/j.1365-2567.2009.03225.x 4. Levine AJ, Quach A, Moore DJ, Achim CL, Soontornniyomkij V, Masliah E, Singer EJ, Gelman B, Nemanim N, Horvath S. Accelerated epigenetic aging in brain is associated with pre-mortem HIV-associated neurocognitive disorders. Journal of NeuroVirology. 2015;22(3):366–375. doi:10.1007/s13365-0150406-3 5. CDC. About HIV/AIDS. 2019 Dec 2 [accessed 2020 Feb 14]. https://www.cdc.gov/hiv/ basics/whatishiv.html.

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normal brain

HAND affected brain

figure 2. abnormal and degraded white matter is a pathological characteristic of dementia In HAND, persistent inflammation contributes to white matter degradation.2 6. Canto MCD. AIDS-Dementia-Complex: pathology, pathogenesis and future directions. 1989;10(3):277–287.

neurocognitive disorders. Neurology: Clinical Practice. 2015;5(3):224–231. doi:10.1212/ cpj.0000000000000117

7. IPM Admin. How ARV-based Microbicides Work. International Partnership For Microbicides. 2010 Oct 1 [accessed 2020 Feb 14]. https://www.ipmglobal.org/why-microbicides/ how-arv-based-microbicides-work

12. Tsutsumimoto K, Makizako H, Shimada H, Doi T, Suzuki T. Set-Shifting Ability Is Associated with Gray Matter Volume in Older People with Mild Cognitive Impairment. 2015;5(3):395–403.

8. Pirrone V, Thakkar N, Jacobson JM, Wigdahl B, Krebs FC. Combinatorial Approaches to the Prevention and Treatment of HIV-1 Infection. Antimicrobial Agents and Chemotherapy. 2011;55(5):1831–1842. doi:10.1128/aac.0097610

13. Alakkas A, Ellis RJ, Watson CW-M, Umlauf A, Heaton RK, Letendre S, Collier A, Marra C, Clifford DB, Gelman B, et al. White matter damage, neuroinflammation, and neuronal integrity in HAND. Journal of Neurovirology. 2019;25(1). doi:10.1007/s13365-018-0682-9

9. Dementia Australia. HIV associated dementia. Dementia Australia. 2014 Aug 4 [accessed 2020 Feb 15]. https://www.dementia.org.au/ about-dementia/types-of-dementia/aids-related-dementia

14. Fields RD. Change in the Brains White Matter. Science. 2010;330(6005):768–769. doi:10.1126/science.1199139

10. Cohen RA, Seider TR, Navia B. HIV effects on age-associated neurocognitive dysfunction: premature cognitive aging or neurodegenerative disease? Alzheimer's Research & Therapy. 2015;7(1). doi:10.1186/s13195-015-0123-4 11. Rumbaugh JA, Tyor W. HIV-associated

15. Vijayan KKV, Karthigeyan KP, Tripathi SP, Hanna LE. Pathophysiology of CD4 T-Cell Depletion in HIV-1 and HIV-2 Infections. 2017;8. 16. Ellis RJ. Phase II Trial of Tesamorelin for Cognition in Aging HIV-Infected Persons. ClinicalTrials.gov. 2019 Mar 22 [accessed 2020 Feb 15]. https://www.clinicaltrials.gov/ct2/ show/NCT02572323 SALTMAN QUARTERLY

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axonal pathfinding:

from development to regeneration

written by nandita rangu illustrated by shae galli

features With a steadily increasing aging population, there is a heightened emphasis on understanding axonal regeneration following central nervous system injuries. Researching axonal pathfinding, which occurs during early neural development, can provide better insight of axonal regeneration. Researchers at UC San Diego are studying how molecular guidance cues and their receptors control regeneration following central nervous system injuries. Their research will hopefully play a role in the development of regenerative medicine that could potentially help patients struggling with central nervous system injuries.

At this moment, you are one of 7.8 billion people in the world, and that number is only increasing.¹ Despite the world being so vast and diverse, moments and memories are shared across thousands of miles. Through communication, humans work together despite their distances and differences to solve the world’s impending problems and ensure future development. Similarly, there are eighty-six billion neurons within the human brain. These eighty-six billion neurons work together by making direct, physical connections to ensure proper development and function. With so many neurons, one thing is for sure: communication is key. Each neuron has a thin, hairlike projection, called an axon, which transmits signals sent to different neurons, muscles, and glands. Axons relay information by making connections with their appropriate targets, such as a specific muscle or tissue, through the process of axonal pathfinding. Growth cones on the tips of axons guide the growth of axons by responding to mechanical and chemical signaling cues.² Axonal pathfinding predominantly occurs during early neuronal development, but it also occurs during axonal regeneration following central nervous system damage. Therefore, axonal pathfinding is essential to understanding regeneration following different types of central nervous system injuries. Axonal pathfinding was first introduced in the late 19th century by Santiago Ramón y Cajal, the father of modern neuroscience and the first person to visualize axonal growth.³ While studying chicken embryos, Cajal noticed that axons

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had specific branching patterns as a result of directed growth. He believed that gradients of chemical guidance cues were responsible for guiding growth, and he hypothesized that axons contained coneshaped structures that interpreted these cues.³ The structures that Cajal hypothesized eventually became known as growth cones and were later identified in chicken embryos by Cajal’s student Francisco Tello.⁴ Cajal’s hypothesis about chemically guided axonal growth is now known as neurotrophic theory, and has prompted new questions about how developmental neuroscience works on a molecular level. Since Cajal first introduced neurotrophic theory, there was a great deal of debate over whether or not axonal growth was primarily guided by chemical guidance cues, because no one had been able to identify these cues. This debate came to an end in the 1950s when Rita Levi-Montalcini discovered nerve growth factor.⁵ Further research showed that chemical guidance cues can either inhibit or promote axonal growth, and the strength of a specific guidance cue is based on the location of that particular neuron within the nervous system.³,⁵ Varying concentrations of specific chemical guidance cues, known as expression gradients, are likely involved in directing appropriate axonal growth, which is essential for proper development. Cajal’s neurotrophic theory now serves as the accepted explanation for directed axonal growth, and more research is currently being done on the guidance cues involved in axonal pathfinding. Over the years, various guidance cues involved in multiple stages of early development have been discovered. One of the first axon guidance molecules discovered was the Wnt family of signaling proteins. Wnt proteins provide necessary directional information for axonal growth during early development and are essential for the formation of neural connec-

tions between the brain and the spinal cord.⁶ Cells secrete Wnt family proteins in specific expression gradients and Wnt proteins bind to receptors found on the axon fibers. Depending on which receptors Wnt binds to, the resulting signalling pathway creates either attractive or repulsive responses; this mechanism is used to navigate axons to their respective targets. Dr. Yimin Zou, a researcher and member of the neurobiology faculty at UC San Diego, specializes in studying the Wnt family of signaling proteins. He was one of the first researchers to study the pathways that control Wnt signaling in relation to axonal turning based on the growth cone’s interpretation of chemical signaling. While The Zou Lab is working to understand the role of Wnt signaling during development, they are also interested in understanding the role of Wnt signaling in axon regeneration following nerve injury. Prior research showed the importance of a Wnt receptor termed Ryk, which interprets Wnt as a repulsive signaling molecule. When Wnt is bound to Ryk, axonal growth is inhibited.⁷ This inhibition of axonal growth could prevent neurons from regenerating in the instance of trauma and explain why it is extremely difficult to recover from a spinal cord injury as an adult. Therefore, inhibiting this signaling pathway by blocking Ryk may be extremely beneficial in the future of regenerative medicine. To understand the relationship between the Wnt-Ryk signaling pathway and axonal regeneration, Zou’s lab created a Ryk knockout mouse model, which is a genetically modified mouse with no functional Ryk receptor.

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= Wnt proteins = Ryk receptor

figure 1. 1a (above): When Wnt proteins bind to the Ryk receptor, axonal growth is inhibited.

= Wnt proteins = Ryk receptor = inhibited Ryk

In order to determine whether or not inhibiting the Wnt-Ryk pathway would lead to increased regeneration, the Ryk knockout mice received a dorsal spinal cord injury and were given tasks to test different functions. The mice were previously trained to complete complex forelimb reaching tasks, and their recovery of forelimb reaching ability post-injury was tested after 12 weeks of recovery and rehabilitation. The forelimb reaching ability of the Ryk knockout mouse model recovered about 20% more of the pre-injury ability than the control mouse.⁷ Imaging of the spinal cord tissue showed an increased density of axons and increased axonal branching in Ryk knockout mice, which is indicative of increased connectivity and potential regeneration.⁷ This strongly suggests that the improved neuronal circuitry is due to decreased Wnt-Ryk signaling. Therefore, blocking Ryk function through the use of antibodies, proteins used by the immune system to target foreign substances, may be a potential therapy that could help people regain motor function following a spinal cord injury.

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1b (below): When the Ryk receptor is inhibited, Wnt proteins no longer bind to the Ryk receptor. This enables axonal growth to continue. Therefore, inhibiting the Wnt-Ryk signalling pathway could be useful in regenerative medicine.

The Zou lab hopes to further their research by looking for ways to promote axonal growth while removing the inhibition of axonal growth. Through a better understanding of axonal pathfinding in early neural development, it may be possible to better promote axonal regeneration. This knowledge can later be applied to better understand axonal regeneration, and potentially help patients with spinal cord injuries. UC San Diego researcher Dr. Binhai Zheng also studies how axons respond to injury, but through a different approach: in vivo imaging. In vivo imaging describes a variety of techniques that enable the visualization of dynamic molecular interactions as they occur in living cells.⁸ This differs from the previous method of visualization, immunohistochemistry, which involved the staining of animal tissues post mortem. Because axonal regeneration is considered a dynamic process best studied within an organism, in vivo imaging is thought to be a more comprehensive way of studying axonal responses to spinal cord injury. The Zheng lab previously found that some axons follow complex branching patterns that enable a single neuron to connect with multiple targets. Specialized axonal branching patterns are thought to be critical in the formation and func-

tioning of neuronal circuits. After understanding the location of an injury and the branch structure of the injured axon, the Zheng lab used in vivo imaging to visualize axonal responses to localized axonal injury. They specifically studied whether an injury prior to, directly at, or after the branch point—the location at which the axon splits into multiple branches— impacted axonal response.⁸ Since axons have to interact with various targets, they have multiple branches, including main, ascending, and descending branches. The main branch serves as the axon’s connection to its own cell body, the ascending branches target neurons in the brain, and the descending branches target muscles. The Zheng lab used a specialized laser that can localize an injury to a single axon in vivo in order to see how location of injury impacted whether axons chose to regenerate or degenerate. Their experiments show that regeneration occurred in about 90% of neurons within 5 days of a main axon injury.⁸ This is because an injury to the main axon ultimately separates the axon from its source of nutrients— the cell body. In contrast, injury in either the ascending or descending branches led to regeneration in only about 10% of neurons within 5 days of a main axon injury.⁸ Zheng’s lab also found that double branch injury in both ascending and descending branches increased the rate of regeneration to approximately 70%.⁸ This phenomenon is likely due to the fact that axons with a spared ascending or descending branch have partial functionality, whereas axons with a double branch injury lose most, if not all, of their functionality, making regeneration more likely to occur. It is still unknown how neurons ultimately regenerate or degenerate based on the situation at hand, but Dr. Zheng believes that it could be a result of various molecular signaling pathways, such as the Wnt-Ryk signaling pathway that the Zou Lab studies. The research at the Zheng lab provides a fundamental understanding of responses to axonal injury. Through further studies on axonal responses to central nervous system injuries, it may be possible to apply this knowledge to promote axonal regeneration in patients with spinal cord injuries.

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main branch injury

single branch injury

double branch injury

branch point

rapid regeneration

In the future, the Zheng lab hopes to shift towards understanding how intrinsic factors, or factors within neurons, impact axonal regeneration. Specifically, they are studying how a pair of kinases, Drk and Lzk, contribute to this axonal regeneration and regulate neuronal interactions with astrocytes, supporting central nervous system cells. Through further research on axonal regeneration, the Zheng lab hopes to better understand responses to central nervous system injuries in humans. The steady increase of the aging population worldwide made regenerative medicine more prominent than ever. According to the U.S. Census, approximately 77 million people in the U.S. will be older than 65 by 2034.⁹ There are currently about 291,000 people in the U.S. living with a spinal cord injury and approximately 17,730 new cases are expected every year.¹⁰ Although aging impacts everyone, those with spinal cord injuries show signs of aging earlier and are more prone to secondary conditions, like bone loss.¹¹ With an increasing aging population more people are likely to experience spinal cord injuries from falls or other similar injuries, understanding axonal pathfinding and regeneration has become a significant goal of researchers. According to Dr. Zou, understanding how “our developmental knowledge can be applied to help patients” will be essential to the development of therapies that can potentially help those suffering from a central nervous system injury. sqonline.ucsd.edu

moderate regeneration

limited regeneration

figure 2.

The location of an axonal injury, specifically in relation to the branch point, impacts the ability of an axon to regenerate. The amount of axonal regeneration following an injury depends on the functionality of the injured axon.

references:

1. Chamie, J. 2020. World Population: 2020 Overview. YaleGlobal Online: Yale University; [cited 2020 April 11]. https://yaleglobal.yale. edu/content/world-population-2020-overview 2. Tamariz E, Varela-Echavarría A. 2015. The discovery of the growth cone and its influence on the study of axon guidance. Frontiers in Neuroanatomy [Internet]. [cited 7 February 2019]; 9(51). https://www.ncbi.nlm.nih.gov/ pmc/articles/PMC4432662/ 3. Sotelo, C. 2004. The Neurotrophic Theory of Santiago Ramon y Cajal. IBRO History of Neuroscience. 4. Raper J, Mason C. 2010. Cellular Strategies of Axonal Pathfinding. Cold Spring Harbor Perspectives in Biology [Internet]. [cited 2019 February 7]; 2(9). https://www.ncbi.nlm.nih. gov/pmc/articles/PMC2926747/ 5. Aloe L. 2004. Rita Levi-Montalcini: the discovery of nerve growth factor and modern neurobiology. Trends in Cell Biology [Internet]. [cited 15 March 2019]; 14(7): 395-399. https://www.sciencedirect.com/science/article/pii/S0962892404001436 6. Onishi K, Hollis E, Zou Y. 2014. Axon guidance and injury — lessons from Wnts and Wnt signaling. Current Opinion in Neurobiology. 27: 232-240. 7. Hollis E, Ishiko N, Yu T, Lu C, Haimovich A, Tolentino K, Richman A, Tury A, Wang S, Pessian M, Jo E, et al. 2016. Ryk controls

remapping of motor cortex during functional recovery after spinal cord injury. Nature Neuroscience. 8. Zheng B, Lorenzana A, Ma L. 2019. Understanding the axonal response to injury by in vivo imaging in the mouse spinal cord: A tale of two branches. Experimental Neurology. 318: 277-285. 9. United States Census Bureau. 2018. Older People Projected to Outnumber Children for First Time in U.S. History 10. National Spinal Cord Injury Statistical Center. Spinal Cord Injury Facts and Figures at a Glance [Internet]. 2019. University of Alabama at Birmingham; [cited 2020 April 10]. 11. Model Systems Knowledge Translation Center. Aging and SCI [Internet]. [cited 2020 April 10]. Available from: https://msktc.org/ sci/factsheets/aging-and-sci

written by

nandita rangu Nandita is a Neurobiology major from Eleanor Roosevelt college. She will graduate in 2023.

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RESEARCH

As one of the most prestigious public schools for the biological sciences, UC San Diego offers a unique undergraduate experience with enriching opportunities, such as handson laboratory experience. Through original research manuscripts and review papers, the Research section showcases the current understanding of various fields in biology.

Sally Lightfoot Crab (Grapsus (Grapsus grapsus)) on Volcanic rocks grapsus in the Galapago Islands. Photo by Moises Goodman

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manuscript

Effects of Canopy Cover, Water, and Habitat Size on Micro-Algae Richness and Abundance in Tank Bromeliads (Guzmania lingulata) in Monteverde, Costa Rica abstract The ubiquitous presence of micro-algae lends insight into the habitat requirements of these photoautotrophic organisms. Specifically, the presence of algae in tank bromeliads offers a unique model for investigating habitat structures. This study aimed to determine the relative impacts of abiotic factors and biotic factors on algae abundance and richness. The phytotelmata of bromeliads are shaped by rainfall, sunlight, and nutrient inputs. The community structure of these aquatic ecosystems is further influenced by trophic interactions between microscopic flora and fauna. Thirty-four Guzmania lingulata (Bromeliaceae) were sampled over a two-week period in the pre-montane wet forest of Monteverde, Costa Rica. Canopy cover, tank water volume, and plant morphologies were measured to see if gradients in these physical attributes will affect algae richness and abundance. Thirty morphospecies of algae were identified and algae were discovered in all samples. We hypothesized that the increasing amounts of these environmental factors will provide more resources to increase algae productivity as reflected in algal biomass. However only the number of bromeliad leaves predicted algal biomass, with greater numbers of leaves leading to declines in algal biomass. It is suggested that algae possess certain plasticity to adapt to the extreme conditions and high local disturbances of the cloud forest understory. Also, the symbiosis of the two photoautotrophs may have presented a competition scenario. However, the open habitat structure of the bromeliads may allow high levels of dispersal that support algae abundance.

Edward Y. Chen [1], Mauricio García-C. [2]

[1] UC San Diego, Roger Revelle College, General Biology, 2020 [2] The Monteverde Institute, Monteverde, Puntarenas, Costa Rica

ter into the phytotelmata can provide resources and nutrients that can initiate a food web.8 Canopy opening, besides providing light, is also associated with nutrient input that can further support algae growth in lentic aquatic systems.3 Studies have shown that the amount of these food resources to algae consumers vary depending on the level of shade to the bromeliad.8,13 Algae in unshaded plants constitute a high energetic resource due to their nutritionally important compounds such as sterols, fatty acids, and organic carbon.12 Water availability and habitat size can further aid in the establishment of primary producer algae which become food resources for aquatic consumers such as insect larvae.25 Such physical properties as well as biotic interactions with algae can shape their biodiversity and reflect ecosystem function and stability and can determine whether the system is favorable to be colonized by algae; a phenomenon known as the “green condition”.9,23 The input of such energy and nutrients could linearly increase productivity and biomass of algae as described in

Figure 1. G. lingulata of the Bromeliaceae family in the

tropical pre-montane wet forest of Monteverde, Puntarenas, Costa Rica. Photos were taken between Nov. 18—Nov. 29, 2019.

introduction Algae are common photoautotrophic organisms that can colonize aquatic systems for growth and reproduction.36 In these bodies of water, environmental factors are known to have an impact on the productivity of algae.13 One such factor is the amount of light the algal cells are exposed to and available for their photosynthesis. The reason is that light irradiance is correlated with a higher photosynthetic response in algae.32 Studies have also suggested that other physical factors are important regulators of the relative impacts of biotic and abiotic factors on algae.18,21 These factors include habitat size, water, and nutrient availability, and they are especially relevant to algae growth in phytotelmata systems of tropical rainforests.9 Tank bromeliads are commonly used as model systems for examining the interactions of environmental factors on algae and other organisms.30 Bromeliads contain discrete communities that can be used to individually assess different physical parameters on community function.3 A bromeliad’s phytotelma is a temporary cavity of water that can host a variety of organisms, and thus can maintain biodiversity.31 Direct contribution of organic matsqonline.ucsd.edu

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the complementary study by Reiners (1986). This can lead to an overall equilibrium state of the system.6,26 The equilibrium state is when the community and habitat attributes are constant enough to maintain diversity and abundance with increasing productivity.15 The complementary model and equilibrium state are often functions of diversity because it allow the proper interactions to take place within an ecosystem.6 In nature, these confined metacommunities often provide transparency and consistency to their processes within. The ability of algae to establish in bromeliads indicates the presence of ecological interactions, but more research is needed to see if these community functions can apply at the spatial scale of the bromeliad.30 This raises the central question of how different amounts of canopy cover and other physical attributes such as water volume and plant size will affect the richness and abundance of algal communities in tank bromeliads. If algae species richness increases with community function, this may support the complementary hypothesis in which the community function is a result of increased energetic input into the system and can lead to its stability and diversity.6

Figure 2. 4 out of the 30 morphospecies of micro-algae

found from 30 G. lingulata collected between Nov. 18Nov.29, 2019, Estación Biológica Monteverde, Puntarenas, Costa Rica.

materials and methods Sampling was done in the forest surrounding the Monteverde Biological Station in Monteverde, Puntarenas, Costa Rica from November 18th to November 29th in 2019. The location is classified as a premontane wet forest at an elevation of 1540 meters above sea level.11 Thirty-four G. lingulata (Bromeliaceae) were chosen and collected from for their abundance in the cloud forest (Figure 1). At the beginning of each sampling, a convex Model A densiometer (Forestry Suppliers Inc., Bartlesville, OK, USA) was used to measure the amount of canopy cover above the bromeliad. Three canopy readings were taken for each bromeliad from the north, west, and east cardinal points in relation to the bromeliad. Next, plant morphologies such as the height of the bromeliad (23.06cm ± 7.90cm), the diameter (36.59cm ± 14.55cm), and the height above the ground of the plant (108.09cm ± 42.59cm) were measured. Then, the number of leaves on the bromeliad were counted (16.15 ± 5.12). Next, a pipette was used to siphon all the water from the phytotelma as well as between the leaves of the bromeliad. The water volume was measured with a graduated cylinder to the nearest cubic centimeter, and was inspected for visible presence of mosquito larvae. A sample of that water was mixed and pipetted into a 1.5mL centrifuge tube for analysis in the laboratory. The remainder of the water was deposited back into the plant to prevent desiccation of the ecosystem. In the lab, the test tube sample was swirled, and then a midstream droplet was delivered on the slide and analyzed at 100x magnification under an Olympus CX31 compound microscope. A scanning of the whole cover slide was done to search for micro-algae across the sample. The algae were categorized by morphospecies, and the individual count of the algae was recorded. The algae were first characterized as colonial, unicellular, or filamentous. The distinguishing factor that aided in such identification was the shape of the cell wall and the overall distinct morphology. Then the identification of algae was confirmed by the presence of chloroplasts. The shape and distribution of the chloroplasts were

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further categorized as even or clumped to further distinguish between morphospecies. The only exception were diatoms, which were categorized using different criteria with the aid of an identification guide.33 For example, for diatoms, symmetry was noted. If there was symmetry, the presence of an intact silica cell wall was noted. A catalog of morphospecies was kept during the study with pictures of the algae from each sample (Figure 2). Micro-algae were present in all the samples. The Spearman’s rank correlation test was used to determine correlation coefficients and p-values between canopy cover, water volume, plant height, diameter, height above the ground and algae richness and abundance. A Kruskal-Wallis Chi-squared test was done to test for the difference in samples with and without mosquito larvae presence. All statistical tests were performed in R.24

results In total, 30 morphospecies of freshwater microalgae were characterized from 34 bromeliad water samples. The cumulative number of species across the samples showed that it does not flatten out after 34 samples (Figure 3). Overall, the average algae species richness per sample was 5.38 ± 1.44 and the average number of individual algae species per sample was 18.53 ± 14.36. The average percentage of canopy cover of the bromeliads was 90.27% ± 5.51%. The amount of canopy cover above the bromeliad did not have an effect on the richness (Spearman’s correlation, r = 0.10, p = 0.56) (Figure 4) or abundance (Spearman’s correlation, r = 0.16, p = 0.37) (Figure 5) of algae in the bromeliad tanks. The average volume of water inside the tank bromeliads was 5.68mL ± 3.43mL,

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Adaptations to aquatic systems in bromeliads are often dependent on the biotic factors as well as the physical environment such as sufficient water, light and nutrients.29 This study aimed to find the relative effects of canopy cover, water volume, and habitat size on algae biomass and diversity. Results showed that several of the habitat variables had no effect on the abundance or richness of algae, contrary to the original hypothesis. Even though habitat parameters may have had trivial effects, the algae’s survival may be owed to their special adaptations as a result of the influence of non-equilibrium dynamics such as disturbances and dispersal to the system.1 A disturbance is a change in the environmental conditions that could lead to a strong change in the ecosystem.35 The lack of consistent environmental conditions in the bromeliad conditions may have led to a deviation from the stable state, but with an overall positive effect for the algae.15 The variation in canopy cover did not contribute to changes in algae abundance likely due to the low light environment the algae were living in that selected for certain adaptations.16 Furthermore, the presence of canopy cover could have contributed to input of detritus which led to turbid conditions in the tank bromeliad water that further reduced the light influx to the algal cells.16,18 However, some algae are known to colonize areas with light limitations in order to avoid desiccation.34 Light limitation is a stressor that could result in a difference in fundamental light-harvesting mechanisms between phytoplankton and that of higher vascular plants.27 Unicellular phytoplankton can alter photosynthetic apparatus to respond to low photon flux while maintaining low energetic costs.27 This could explain the ability of algae to survive in the cloud forest conditions. Having said that, water levels did not significantly alter the abundance or richness of algae due to the adaptability of algae to

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Cumulative number of species

Samples

Figure 3. Cumulative number of species. The plots

show the number of new species found per every sample. A total of 34 species found over the sampling period of Nov. 18-Nov. 29, 2019 around the Monteverde Biological Station.

Canopy cover vs. Algae species richness

Number of species

discussion

Species accumulation curve

Canopy cover (%)

Figure 4. The amount of canopy cover above the bromeliads as a percentage in relation to algae morphospecies richness defined as the number of species per sample. Data from 30 G. lingulata collected between Nov. 18-Nov. 29, 2019, Estación Biológica Monteverde, Puntarenas, Costa Rica.

Canopy cover vs. Algae abundance Number of individuals

but the amount of water in bromeliad tanks did not affect algae richness (Spearman’s correlation, r = -0.09, p = 0.61) (Figure 6) or abundance (Spearman’s correlation, r = -0.07, p = 0.69) (Figure 7). The other habitat factors, including the height of the bromeliad plant, did not have an effect on the amount of algae richness (Spearman’s correlation, r = -0.03, p = 0.88) or algae abundance (Spearman’s correlation, r = -0.12, p = 0.51). Nor was there an effect of bromeliad diameter on algae richness (Spearman’s correlation, r = -0.03, p = 0.89) or algae abundance (Spearman correlation, r = -0.18, p = 0.30). The average height of the bromeliad from the ground was 102.09cm ± 42.59cm, but the height from the ground of the bromeliad did not affect the algae richness (Spearman’s correlation, r = -0.19, p = 0.27) or the algae abundance (Spearman’s correlation, r = 0.13, p = 0.45). The average number of leaves per bromeliad was 16.15 ± 5.12, and an increasing number of leaves on the bromeliads led to a decrease in abundance of algae present (Spearman’s correlation, r = -0.37, p = 0.03) (Figure 8). In terms of analyzing the potential trophic interactions between macroinvertebrates and algae, nine out of the 34 samples collected contained mosquito larvae. However, mosquito larvae presence did not influence the mean algae species richness (Kruskal-Wallis test, p = 0.14) or algae abundance (Kruskal-Wallis test, p = 0.44).

Canopy cover (%)

Figure 5. The amount of canopy cover above the bromeliad as a percentage in relation to algae abundance defined as the number of individuals per sample. Data from 30 G. lingulata collected between Nov.

18-Nov. 29, 2019, Estación Biológica Monteverde, Puntarenas, Costa Rica.

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Number of species

Bromeliad tank volume vs. Algae species richness

Tank water volume (mL)

Figure 6. The amount of water in the phytotelmata of the bromeliads in mL with relation to number of algae morphospecies as defined as the number of species per sample. Data from 30 G. lingulata collected between Nov. 18-Nov. 29, 2019, Estación Biológica Monteverde, Puntarenas, Costa Rica.

Number of individuals

Bromeliad tank volume vs. Algae abundance

Tank water volume (mL)

Figure 7. The amount of water in the phytotelmata of the bromeliads in mL with relation to total algae abundance defined as the number of individuals per sample. Data from 30 G. lingulata collected between Nov.

18-Nov. 29, 2019, Estación Biológica Monteverde, Puntarenas, Costa Rica.

Number of individuals

Number of leaves vs. Algae abundance

desiccated environments.19 The tank bromeliads sampled had an average water volume of 5.68mL which could be low enough to often subject the phytotelmata to drought conditions.4 The amount of disturbances to the micro-algae could have led to an evolutionary adaptation to low water conditions. Algae have a mucilage that could delay desiccation and help in absorbing moisture.17 Thus, their ability to remain dormant during unfavorable conditions presented an advantage to survive in harsh conditions.19 There was a significant negative correlation between the number of leaves and the algae abundance. The dependence of algae on nutrients could have led to competition with the bromeliad leaves, but still allowed for the co-existence of the algae in the bromeliads.4,15 Non-equilibrium dynamics may explain how algae and bromeliads can co-exist without competitively excluding one another. The open habitat structures of bromeliads may bring regional scale disturbances into play and subject the habitat to non-equilibrium dynamics, such as dispersal and local extinctions.10 Non-equilibrium theory states that ecological systems are seldom stable or linear, and are often influenced by disturbances, heterogeneity, and the existence of multiple stable states.1 The ubiquity of algae despite the low light and water conditions could be due to their strong dispersal ability through air, water, and airborne vectors.14,20 One study described the reproduction behavior of algae as opportunistic forms that happen to find habitats suitable for growth for restricted periods.28 Furthermore, the richness of algae can be a result of intermediate levels of disturbances such as droughts that allow the population of algae to persist because no one competitor can dominate if the system is subject to disturbances that lead to rapid successions.5,6,28 In addition to algae’s fast turnover rate of about 1.4 days, their rapid succession can maintain their ability to re-colonize and co-exist with the bromeliads.6,28 The resulting adaptations of algae are reflective of the phenotypic plasticities of organisms as a response to such variability in habitat extremes.7 Also, the lack of control from biotic factors could be an example of non-equilibrium coexistence.22 Biotic factors could have also influenced algae biomass because it is known that algae are often subjected to herbivory by larvae of insects such as mosquitoes.2 However, the samples with larvae did not show significant difference in terms of average algae richness or abundance possibly due to a lack of larvae presence. Larvae in larger quantities could have altered the algae biomass because the study by Carrias and colleagues (2014) suggested that biotic factors like herbivory from macroinvertebrates could overall have a larger impact on algae biomass than abiotic factors. The study by DeAngelis and Waterhouse (1987) characterized the impacts of biotic factors as a deviation from equilibrium known as the biotic feedback instability.

conclusion Number of bromeliad leaves

Figure 8. The number of leaves per bromeliad with relation to the total algae abundance defined as the number of individuals per sample. Data from 30 G.

lingulata collected between Nov. 18-Nov. 29, 2019, Estación Biológica Monteverde, Puntarenas, Costa Rica.

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The species accumulation curve revealed the amount of micro-algal diversity in the limited aquatic environments of the G. lingulata. The apparently trivial effects of the physical environments on algae have been described in literature as the “phytoplankton paradox”.28 The open structure of the bromeliad system allowed non-equilibrium dynamics to influence the algal com-

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munity structure. Traditional deterministic processes may not be suitable for describing highly complex spatial systems such as those of bromeliads.6 Further research with direct manipulations on bromeliads while controlling parameters may reveal if bromeliads can ever achieve a stable state. However, the reality of many ecosystems does not contain a rigid boundary, and oftentimes it is necessary to consider the transient nature of ecosystems in order to understand their true workings.6,35

acknowledgements Laboratory space for this study was provided by the Estación Biológica Monteverde. Text was reviewed by advisors Mauricio García-C. and Lucia Vargas, and peer reviewed by Madeline Tafoya. This study is also dedicated to instructors Frank Joyce, Federico Chinchilla, Félix Salazar, and JP Monge as well as the EAP classmates of Fall 2019 for their guidance and support. Thanks to the Santamaria family for their hospitality during field work. Lastly, thanks to the Monteverde Institute for hosting the research symposium and sponsoring this program. This research was conducted under the Minae/Sinac (Costa Rica) research permit; RESOLUCION N°: M-P-SINAC-PNI-ACAT-075-2019.

references 1. Briske, D., Illius D., A. W., and Anderies J. M. Nonequilibrium ecology and resilience theory. Rangeland Systems Springer Series on Environmental Management. Springer, New York, NY (2017). 2. Brouard, O. et al. Are algae relevant to the detritus-based food web in tank-bromeliads? PLoS ONE 6(5), e20129 (2011). https://doi.org/10.1371/journal. pone.0020129 3. Brouard, O. et al. Understorey environments influence functional diversity in tank-bromeliad ecosystems. Freshwater Biology 57, 815–823 (2012). 4. Carrias, J.-F et al. Two coexisting tank bromeliads host distinct algal communities on a tropical inselberg. Plant Biology 16, 997–1004 (2014). 5. Connell, J. H. Diversity in tropical rain forests and coral reefs. Science 199, 1302– 1310 (1978).

15. Lengyel, E., Padisák J., and Stenger-Kovács C. Establishment of equilibrium states and effect of disturbances on benthic diatom assemblages of the Torna-stream, Hungary. Hydrobiologia 750, 43–56 (2014). 16. Lopez, L. C. S., Alves R. R. D. N., and Rios R. I. Micro-environmental factors and the endemism of bromeliad aquatic fauna. Hydrobiologia 625, 151–156 (2009). 17. Lütz, C., Seidlitz H. K., and Meindl U. Physiological and structural changes in the chloroplast of the green alga Micrasterias denticulata induced by UV-B simulation. UV-B and Biosphere 128, 55-64 (1997). 18. Marino, N. A. C., Guariento R. D., Dib V., Azevedo F. D., and Farjalla V. F. Habitat size determine algae biomass in tank-bromeliads. Hydrobiologia 678, 191–199 (2011). 19. Nayaka, S., Toppo K., and Verma S. Adaptation in algae to environmental stress and ecological conditions. Plant Adaptation Strategies in Changing Environment. Springer, Singapore (2017). 20. Novis, P. M., Beer T., and Vallance J. New records of microalgae from the New Zealand alpine zone, and their distribution and dispersal. New Zealand Journal of Botany 46, 347–366 (2008). 21. Petermann, J. S., Kratina P., Marino N. A. C., Macdonald A. A. M., and Srivastava D. S. Resources Alter the Structure and Increase Stochasticity in Bromeliad Microfauna Communities. Plos One 10(3), e0118952 (2015). https://doi. org/10.1371/journal.pone.0118952. 22. Pickett, S. T. A. Non-equilibrium coexistence of plants. Bulletin of the Torrey Botanical Club 107, 238 (1980). 23. Pires, A. P. F., Leal J. D. S., and Peeters E. T. H. M. Rainfall changes affect the algae dominance in tank bromeliad ecosystems. Plos One 12(4), e0175436 (2017). https://doi.org/10.1371/journal.pone.0175436 24. R Development Core Team. R: A language and environment for statistical computing. http://www.R-project.org/ (Accessed December 13, 2019). 25. Rangel, J. V., Araújo R. E., Casotti C. G., Costa L. C., Jr. W. P. K., and Moretti M. S.. Assessing the role of canopy cover on the colonization of phytotelmata by aquatic invertebrates: an experiment with the tank-bromeliad Aechmea lingulata. Journal of Limnology 76(2), 230-239 (2016). 26. Reiners, W. A. Complementary Models for Ecosystems. The American Naturalist 127, 59–73 (1986). 27. Richardson, K., Beardall J., and Raven J. A. Adaptation of unicellular algae to irradiance: an analysis of strategies. New Phytologist 93, 157–191 (1983). 28. Richerson, P., Armstrong R., and Goldman C. R. Contemporaneous Disequilibrium, a New Hypothesis to Explain the "Paradox of the Plankton". Proceedings of the National Academy of Sciences 67, 1710–1714 (1970).

6. Deangelis, D. L., and Waterhouse J. C. Equilibrium and nonequilibrium concepts in ecological models. Ecological Monographs 57, 1–21 (1987).

29. Schneider, D. W., and Frost T. M. Habitat duration and community structure in temporary ponds. Journal of the North American Benthological Society 15, 64–86 (1996).

7. Denslow, J. Tropical rainforest gaps and tree species diversity. Annual Review of Ecology and Systematics 18, 431–451 (1987).

30. Srivastava, D. S. et al. Are natural microcosms useful model systems for ecology? Trends in Ecology & Evolution 19, 379–384 (2004).

8. Farjalla, V. F. et al. Terrestrial support of aquatic food webs depends on light inputs: a geographically-replicated test using tank bromeliads. Ecology 97, 2147–2156 (2016).

31. Torreias, S. R. D. S., and Ferreira-Keppler R. L. Macroinvertebrates inhabiting the tank leaf terrestrial and epiphyte bromeliads at Reserva Adolpho Ducke, Manaus, Amazonas. Brazilian Archives of Biology and Technology 54, 1193–1202 (2011).

9. Gessner, M. O., Inchausti P., Persson L., Raffaelli D. G., and Giller P. S. Biodiversity effects on ecosystem functioning: insights from aquatic systems. Oikos 104, 419–422 (2004). 10. He, F., Gaston K. J., Connor E. F., and Srivastava D. S. The local–regional relationship: immigration, extinction, and scale. Ecology 86(2), 360–365 (2005). 11. Holdridge, L. R., and Tosi Jr. J.A. Life Zone Ecology. Tropical Science Center, San Jose, Costa Rica (1967). 12. Kaufman, M. G. et al. Importance of algal biomass to growth and development of Anopheles gambiae Larvae. Journal of Medical Entomology 43, 669–676 (2006). 13. Kratina, P., Petermann J. S., Marino N. A. C., Macdonald A. A. M., and Srivastava D. S. Environmental control of the microfaunal community structure in tropical bromeliads. Ecology and Evolution 7, 1627–1634 (2017). 14. Kristiansen, J. Dispersal of freshwater algae — a review. Biogeography of Freshwater Algae 336, 151–157 (1996).

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32. Torremorell, A. et al. Annual patterns of phytoplankton density and primary production in a large, shallow lake: the central role of light. Freshwater Biology 54, 437–449 (2009). 33. University of Colorado Boulder. Taxa by Morphology. Diatoms of North America. https://diatoms.org/ (Accessed December 13, 2019). 34. Wehr, J. D., and Sheath R. G. Habitats of Freshwater Algae. Freshwater Algae of North America. Academic Press, San Diego, CA (2003). 35. White, P., and Pickett S. Natural Disturbance and Patch Dynamics: An Introduction. The Ecology of Natural Disturbance and Patch Dynamics. Academic Press, San Diego, CA (1985). 36. Wu, N., Schmalz B., and Fohrer N. Distribution of phytoplankton in a German lowland river in relation to environmental factors. Journal of Plankton 33(5), 807820 (2010).

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The Role of NEDD4 Protein in Cardiac Function and Disease Tiana Huynh [1], PI: Ju Chen, Ph.D. [2] [1] UC San Diego, Biochemistry and Cell Biology, M.S. 2020 [2] UC San Diego School of Medicine

background Neuronal precursor cell-expressed developmentally downregulated 4 (NEDD4) gene is a member of the NEDD4 family of “Homologous to the E6-AP Carboxyl Terminus” (HECT) E3 ubiquitin ligases. While NEDD4 is highly expressed in cardiomyocytes, or cardiac muscle cells, its specific role in these cells remains unknown. To study the role of NEDD4 adult cardiomyocytes, a Nedd4 floxed mouse model was generated and crossed with tamoxifen-inducible aMHC-MerCreMer mice to generate an inducible Nedd4 cardiomyocyte-specific knockout mouse model (Nedd4 icKO). In this study, comprehensive molecular and cardiac physiology studies were performed on Nedd4 icKO mice. Our results show that Nedd4 icKO mice displayed cardiomyopathy after tamoxifen-induced gene deletion. Utilizing the Nedd4 icKO mice model, it will be possible to gain significant insight into the mechanisms by which the loss of NEDD4 in adult cardiomyocytes results in cardiomyopathy. Ultimately, this could help identify new potential therapeutic targets for human cardiomyopathy.

when the heart is under stress from external pressures, making them strong indicators of cardiac stress.1 For that reason, we measured ANF and BNP levels within cardiac tissue using qRT-PCR. As expected, Nedd4 icKO mice 2 weeks and 5 weeks post-tamoxifen injection show a significant increase in both ANF and BNP levels when compared to the levels of Nedd4 control mice, indicating that the heart’s homeostasis has been disrupted and is undergoing cardiac stress (Figure 1, F and G). In order to observe the morphology of the heart, histological studies - including hematoxylin and eosin staining - were performed on Nedd4 control and icKO mice 10 weeks post-tamoxifen injection. This time point was used, instead of 2 weeks or 5 weeks post-tamoxifen injection, in order to allow the heart to develop any phenotypic changes. The studies revealed an enlarged heart in Nedd4 icKO mice when observed via microscope (Figure 1H).

results

In conclusion, the generation of inducible Nedd4 cardiomyocyte-specific knockout mice have shown that tamoxifen-induced gene deletion does result in enlarged hearts and decreased cardiac function. However, much is still unknown with regards to NEDD4’s specific role in ubiquitin-mediated protein quality control and the mechanism that leads to cardiomyopathy. Our findings potentially allow us to identify new therapeutic targets for human cardiomyopathy, but further research is required in order to gain significant insight behind this mechanism.

To validate our Nedd4 icKO mouse model, NEDD4 mRNA levels of both Nedd4 control and icKO mice were measured 2 weeks and 5 weeks post-tamoxifen injection. Levels of mRNA were measured with quantitative real-time polymerase chain reaction (qRT-PCR). At both time points, Nedd4 icKO mice showed a decrease in NEDD4 mRNA levels when compared to that of Nedd4 control mice (Figure 1, B and D). Western blot analysis performed on protein lysates from Nedd4 control and icKO mice at 2 weeks and 5 weeks post-tamoxifen injection also revealed a decrease in protein levels in both groups of Nedd4 icKO mice when compared to the levels of Nedd4 control mice (Figure 1, C and E). Atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) are members of the natriuretic peptide family that are produced and secreted from the atria.1 They play an important role in maintaining cardiac homeostasis and display increased levels

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conclusion

references 1. Bold, M. K. D., & Bold, A. D. (2009). Natriuretic Peptides. Encyclopedia of Neuroscience, 15–18. doi: 10.1016/b978-008045046-9.01438-8 2. Chan, J. K. (2014). The Wonderful Colors of the Hematoxylin–Eosin Stain in Diagnostic Surgical Pathology. International Journal of Surgical Pathology,22(1), 12-32. doi:10.1177/1066896913517939

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brevia

Figure 1. (A) Generation of inducible Nedd4 cardiomyocyte-specific knockout mice using a tamoxifen-induced Cre-Lox

recombination method. (B) qRT-PCR analysis of NEDD4 mRNA levels 2 weeks post-tamoxifen injection using. (C) Western blot analysis of NEDD4 protein levels 2 weeks post-tamoxifen injection. (D) qRT-PCR analysis of NEDD4 mRNA levels 5 weeks post tamoxifen-injection. (E) Western blot analysis of NEDD4 protein levels 5 weeks post-tamoxifen injection. (F) qRT-PCR analysis of cardiac stress markers 2 weeks post-tamoxifen injection. (G) qRT-PCR analysis of cardiac stress markers 5 weeks posttamoxifen injection. (H) Microscopic images of Nedd4 control and icKO mice. (Scale bar = 1mm) (I) H&E staining of Nedd4 control and icKO mice. (Scale bar = 2mm)

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brevia

The Role of B-Cell Lymphoma 6 in the Proliferation and Differentiation of Skeletal Muscle introduction B-Cell Lymphoma 6 (BCL6) is a transcription factor that represses gene expression through the recruitment of the SMRT/ NCOR corepressor complex to its BTB domain, which serves as a motif for transcriptional regulation.1 BCL6 plays an important role in the differentiation of B-cells in their germinal centers, but not much is known about the function of BCL6 in skeletal muscle. BCL6 remodels the chromatin in the B-cell germinal centers and this mechanism is similar in the skeletal muscle.2 Here we investigate the role of BCL6 in skeletal muscle development by the use of a BCL6 specific inhibitor, FX1, during myoblast proliferation or fusion.3 This gene is known to play a role in adipocyte reduction and hepatic lipid metabolism.4 The data shows that BCL6 is a regulator in the development of skeletal muscle.

methods The C2C12 immortalized cell line of mouse skeletal myoblasts was used. Each experiment contained 9 negative controls with Dimethyl Sulfoxide (DMSO, 0.2%) and 9 drug-treated groups with FX1 (25uM). Crystal Violet Assay was used to stain the cells and absorbance readings at 590nm were used to determine the number of cells in each well. Two variations of this study, DMSO and FX1 treatments, were added for 3 days. Before maximum confluency was met on Day 3, a Crystal Violet Assay was conducted. To assess BCL6â&#x20AC;&#x2122;s mediation of myoblast fusion, cells became con-

Brian Manalansan Gutierrez [1], [2]; Ryan Welch, Ph.D. [2]; Weiwei Fan, Ph.D. [2]; Annette Atkins, Ph.D. [2]; Ruth Yu, Ph.D. [2]; Michael Downes, Ph.D. [2]; PI: Ronald Evans, Ph.D. [2] [1] UC San Diego, Biology M.S. 2020 [2] Gene Expression Laboratory, Salk Institute for Biological Sciences, La Jolla, California, USA

fluent before inducing them to differentiate by serum withdrawal (2% horse serum in DMEM) and were exposed to either DMSO or FX1 for 7 days. A Jenner-Giemsa stain was conducted to measure the extent of differentiation. Verification of developmental effects was seen from the failure of multinucleated myotube formation as the cells progressively differentiated up to 7 days.

results Crystal Violet Assay revealed a drastic decrease in the proliferation rate with BCL6 inhibition by FX1 when compared to the control group (Figure 1a). The figure shows an absorbance reading of 0.75 for the control group and 0.6 for the FX1 group (p < 0.0001). The less dense the area, the less proliferation. A Jenner-Giemsa stain indicated that FX1 completely blocked myoblast fusion (Figure 1b). Comparison of the two images in the figure revealed that there is a two-fold increase in the amount of myotubes found in the vehicle group. This data indicated that BCL6 is a crucial regulator of myoblast proliferation and differentiation in vitro.

discussion These in vitro results support that BCL6 is a crucial regulator in the development of skeletal muscle. Blocking the SMRT/NCOR corepressor complex from BCL6 causes an impairment of skeletal muscle development. Our results indicate that BCL6 may serve as a therapeutic target to treat muscle wasting or enhance healing if utilizing either a BCL6 agonist or genetic overexpression. Knowing that BCL6 plays a role in adipocyte reduction and hepatic lipid metabolism, BCL6 activity may also affect metabolic activity in the skeletal muscle. Once the full role of BCL6 is understood, possible therapies can be used to further resolve issues such as muscular dystrophy.

references 1. Weimin, C., Polo, J.M., Melnick, A. B-cell lymphoma 6 and the molecular pathogenesis of diffuse large B-cell lymphoma. Curr Opin Hematol. USA 15, 381-390 (2008).

Figure 1. (A) Crystal Violet Assay conducted on C2C12 myoblasts

showing the control group (left) and the drug-treated group (right). (B) Jenner-Giemsa stain displays the multinucleated myotubes. Drugtreated group (right) has a lack of myotubes formed compared to the vehicle group (left). n = 3, p < 0.0001 determined by 2-tailed student t-test. Statistical significance of two-tailed student t-tests between the two groups were determined by GraphPad Prism (Version 7).

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2. Puvion-Dutilleul, F., Souquere-Besse, S., and Albagli-Curiel, O. The relationship between BCL6 bodies and nuclear sites of normal and halogenated DNA and RNA synthesis. Microscopy Research and Technique. France 61, 389-407 (2003). 3. Cardenas, M.G., Yu, W., Beguelin, W., Teater, M.R., Geng, H. et al. Rationally designed BCL6 inhibitors target activated B cell diffuse large B cell lymphoma. J Clin Invest. USA 126, 3351-3362 (2016). 4. LaPensee, C.R., Lin, G., Dent, A.L., Schwartz, J. Deficiency of the transcriptional repressor B cell lymphoma 6 (Bcl6) is accompanied by dysregulated lipid metabolism. PLOS. USA. 9, e97090.

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review

Tracking Cell Lineages Through Barcoding introduction In grocery stores, barcodes are used to distinguish one product from another. In a similar way, DNA barcodes–short sequences of differing nucleotides incorporated into the genome–can be used by scientists to distinguish different organisms from one another.1 When comparing organisms, scientists insert unique DNA barcodes that differ for each species, giving insight to whether two organisms are from the same species. Therefore, DNA barcoding has been a very useful tool for evolutionary biologists, and has even led to the “Barcode of Life Initiative,” a project that seeks to identify new species.1 More recently, researchers have been using barcodes to understand how individual cells from an organism are able to differentiate. In these cases, individual cells are tagged with barcodes that allow the cells to be distinguished.2 Tracking cells and their changes has been difficult in the past because cells need to be fixed to be tagged and observed via most optical assays, making it difficult to study their dynamics. The new technologies available have allowed for more creative ways of introducing barcodes and observing them in situ. The feasibility of tracking individual cell lineages may lead to knowledge on how cells interact and, more importantly, how their dysfunction can lead to maladies.3 By understanding the populations of cells that are present in a healthy body and how the composition of may cells change, scientists can develop methods to combat these unwanted cellular changes.

genome editing leads to advancements in barcoding Using barcoding, along with optical assays and next-generation sequencing techniques, scientists have been able to study intriguing questions in recent years, like how cells from zebrafish embryos progress over time and how individual mice cell lineages differ.4,5 Advancements in genome editing technology, such as CRISPR/Cas9, have allowed scientists to optimize DNA barcoding by introducing site-directed mutations with an unprecedented level of specificity. These mutations can be used to create unique barcodes that help track cellular lineages.4,5,6

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Breanna Lam [1]

[1] UC San Diego, Roger Revelle College, Molecular and Cell Biology, 2020

Specifically, CRISPR-Cas9 technology has led to the development of ScarTrace. ScarTrace is a method of tracking cell types and their cloning history. It is based on insertions, also known as scars, that the CRISPR-Cas9 system introduces in the absence of a homologous repair template.7 This technology is powerful because the CRISPR-Cas9 insertions vary in both size and sequence, creating diversity in regards to “scarring” the cells.7 From these scars, scientists are able to determine changes that occur in a subset of cells over time through sequencing and optical assays. These optical assays can help determine which populations of cells are present and whether the population increases or decreases over time. For example, ScarTrace was used to determine that a small population of embryonic cells led to the development of haematopoietic cells in kidney marrow.5 This use of ScarTrace shows the promise it has in tracking individual cells and studying development. Advancements in genome editing have also led to mSCRIBE, which records molecular events, such as DNA mutations, in mammalian cells.6 mSCRIBE takes advantage of genome editing technologies by introducing a guide RNA that targets a specific site in the gene and using an endonuclease to cut and mutate it repeatedly so that a unique barcode is created. Unlike ScarTrace which utilizes non-dynamic scarring, the barcodes are mutated repeatedly so that mutations on the gene accumulate, allowing biological and molecular events to be tracked over time. mSCRIBE is promising since it has shown that the activation of pathways, such as an immune response to inflammation, can be tracked, ultimately leading to a better understanding of the body’s response to pathogens.6 Although these systems have improved barcodes and cellular tracking, they have not been extensively studied in human cells or those of different organisms because the technology and tools are relatively new. Further optimization of these systems could potentially provide the platform and tools needed to observe intracellular dynamics in situ.

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identifying barcodes in situ

the future of barcoding

In addition to these studies, scientists have also been developing new techniques to identify barcodes and track cells in situ, without the use of sequencing.4,8 Distinguishing barcodes without the use of sequencing is important because it would allow for an easier method of screening.4 Moreover, although sequencing provides valuable information about a cell, tracking cells through sequencing alone would be tedious since cells acquire random mutations and would need to be sequenced continuously to reveal changes, resulting in a need for an in situ method of tracking that allows the cell to be seen in its cellular environment.2 Most notably, the Elowitz Lab at the California Institute of Technology has developed two novel systems, MEMOIR and Zombie, that allow for simple in situ detection of different barcodes through optical analysis.8 The MEMOIR system incorporates both a barcode and a scratchpad. The scratchpad, similar to a DNA barcode, is a genetic recording element that is introduced using CRISPR and helps distinguish cells from one another.4 The scratchpad changes stochastically because of CRISPR technologies, and the changes help identify whether changes have been made to the specific cell. These barcode and scratchpad regions together have made it easier to distinguish individual cells from one another, undergoing detection using a technique known as fluorescence in situ hybridization (FISH).4 Similarly, barcodes are used in the Zombie system to distinguish individual cells from one another. They are transcribed by phage RNA polymerases, which amplify the barcodes, before being identified using FISH.8 FISH allows barcodes to be phenotypically distinguished from one another, making it unnecessary to individually sequence the cells. Remarkably, Zombie produced high accuracy detection of DNA barcodes as short as 20-bp, serving as an improved version of MEMOIR. Unlike MEMOIR and the stochastic deletions it created through CRISPR technologies, the Zombie system is able to accurately give readouts of base edits. Furthermore, experiments demonstrated Zombie’s ability to function in other organisms, such as chick embryos and adult mouse brain tissue, showing promise for the use of Zombie in a wide variety of organisms.8 The improvement of Zombie could lead to significant progress in the field of developmental biology, paving the way for a comprehensive understanding of cell lineages.

Although many researchers are continuing to revolutionize the way barcoding is used in studying how cells develop, which could demonstrate the processes cells go through as a person ages, many challenges still lie ahead. With Zombie, challenges, such as developing an extensive library of barcodes that can be identified by Zombie, will need to be overcome before we are able to fully take advantage of the system to track cell lineages in the human body.8 As scientists continue to come up with creative ways of tracking barcodes, they will be able to answer important questions, like how cells are able to differentiate and what paths cells take over time. Moreover, the development of new technologies will allow for cheaper and more efficient identification of barcodes, and will enable them to be used more commonly in research studies as a tool for tagging and identifying a cell of interest. Ultimately, being able to track cell lineages and understand how they differentiate will not only provide insight into the mechanisms that underlie normal development, but also those that cause abnormal development and pathogenesis.

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references 1. Savolainen Vincent, Cowan Robyn S, Vogler Alfried P, Roderick George K and Lane Richard. Towards writing the encyclopaedia of life: an introduction to DNA barcoding. Phil. Trans. R. Soc. B. 360, 1805-11 (2005) 2. Kebschull, J.M., Zador, A.M. Cellular barcoding: lineage tracing, screening and beyond. Nat Methods 15, 871–879 (2018) 3. Mott MC, Gordon JA, Koroshetz WJ The NIH BRAIN Initiative: Advancing neurotechnologies, integrating disciplines. PLoS Biol 16, 11 (2018) 4. Frieda, K., Linton, J., Hormoz, S. et al. Synthetic recording and in situ readout of lineage information in single cells. Nature 541, 107–111 (2017) 5. Alemany, A., Florescu, M., Baron, C. et al. Whole-organism clone tracing using single-cell sequencing. Nature 556, 108–112 (2018) 6. Perli, S. D., Cui, C. H. & Lu, T. K. Continuous genetic recording with self-targeting CRISPR-Cas in human cells. Science 353, aag0511 (2016). 7. Junker, J. et al. Massively parallel clonal analysis using CRISPR/Cas9 induced genetic scars. BioRxiv (2016). 8. Askary, A., Sanchez-Guardado, L., Linton, J.M. et al. In situ readout of DNA barcodes and single base edits facilitated by in vitro transcription. Nat Biotechnol (2019).

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review

Evasion and Modulation of Immune Responses by Mycobacterium Tuberculosis: Implications for Vaccine Development the global mTB pandemic Mycobacterium tuberculosis (mTB) continues to pose a significant threat to human health on a global scale. Endemic to many developing countries in South America, Africa, and Asia, about 25% of the world’s population is thought to be infected, with about 10 million new infections each year.1 In 2018 alone, the World Health Organization (WHO) estimates that over 1.6 million people died as a result of mTB infection, making it the single most deadly human infectious agent.1 An estimated 90% of infections result in asymptomatic bacterial latency, a state of dormancy that may eventually give rise to pathological infection upon re-activation of the bacteria. Active infection is characterized by rapid bacterial growth and granuloma formation, typically within the lung, and carries a mortality rate of

Colin J. Mann [1]

[1] UC San Diego, John Muir College, Microbiology, 2020

66% without treatment.1 Recent rise in drug-resistant (DR-TB) and multiple-drug-resistant (MDR-TB) tuberculosis has prompted further research efforts into the development of novel therapeutics and prophylactic vaccines. Extensively-drug-resistant (XDR-TB) tuberculosis poses an even more formidable challenge as it is resistant to the two most effective first line anti-TB drugs, isoniazid and rifampicin, and carries a 60% mortality rate even with advanced treatment.2,3 Efforts to develop a novel prophylactic vaccine have remained stagnant, hampered by mTB’s complex mechanisms of immune evasion and modulation. Thus, emerging approaches will have to carefully consider and exploit weaknesses revealed by host-pathogen interactions.

Figure

1. Disruption of major histocompatibility complex (MHC) class II presentation by M. tuberculosis. The various steps in the MHC class II processing and presentation pathway that are known or postulated to be influenced by M. tuberculosis infection are illustrated. (A) New synthesis of MHC class II molecules is blocked by toll like receptor 2 (TLR2) signaling because of mycobacterial products such as the 19 kDa lipoprotein. (B) Intracellular trafficking of MHC class II is disrupted by the suppression of cathepsin S, which is because of induction of interleukin (IL) 10 by mycobacterial infection. (C) Generation of peptide antigens for loading onto MHC class II in relevant endocytic compartments (MIIC) is also inhibited by several effects of mycobacterial infection, including inhibition of phagosome– lysosome fusion, neutralization of phagosomal pH by urease and by blockade of recruitment of the vacuolar proton ATPase. (D) A proposed inhibition of autophagy and autophagic vacuole formation also eliminates a potential source of antigenic peptides that can load MHC class II molecules. (E) The reduction of peptide antigen availability and incomplete cleavage of MHC class II associated invariant chain (Ii) resulting from cathepsin S suppression result in a reduced transport of stable peptide loaded MHC class II molecules to the APC 9 surface. Baena (2009) Tissue Agents

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Figure 2. Delayed onset of adaptive immunity in tuberculosis compared with other infections. In acute, resolving infections, T-cell responses are initiated 3 – 5 days after initial infection; they peak 7 – 8 days after infection, and subsequently contract to establish memory populations. After aerosol infection with M. tuberculosis, T-cell responses are not initiated until 9 – 11 days after infection, peak several weeks after infection, and bacteria are not eliminated. LCMV, lymphocytic choriomeningitis virus. Urdahl (2011) Mucosal Immunology11

mTB pathenogenesis and critical structural features Although tuberculosis was identified nearly 200 years ago, a detailed understanding of its pathogenesis is only now on the horizon. New technologies, such as single-cell sorting, next-generation sequencing, and high-resolution electron microscopy, have provided researchers with tools to investigate how the bacterium interacts with the host immune system. It is known that mTB is most commonly transmitted in aerosolized particles produced when an actively infected person coughs, talks or even sings. As little as ten of these bacteria can initiate infection as they migrate deep within lung parenchyma, where they predominantly infect alveolar macrophages.4 M. tuberculosis possesses unique structural features that allow it to invade these primary sites and avoid detection by the innate immune system. The “waxy” cell wall of mTB is composed of a high-density array of mycolic acid derivatives such as trehalose monomycolate (TMM) and trehalose dimycolate (TDM), the latter of which is a central component of “cord factor”.5,6 Cord factor receives its name from its interwoven filamentous structure which interlocks with lipoarabinomannan (LAM), an abundant glycolipid within the mycobacterial outer cell wall, to promote bacterial growth into long rope-like colonies. The extensive cord factor lattice forms an extremely strong cell wall that is resistant to the degradative action of phagocytic lysosomes.5,6 This can allow the bacterium to reside within the macrophage and avoid immune detection (Figure 1C).

immune evasion and cytokine manipuation promote persistent infection Along with these unique structural features, mTB also possesses several immune evasion mechanisms. One example is ESAT-6, a cytolytic protein involved in disrupting the membrane of phagolysosomes, allowing escape and residence within the cytosol of macrophages.7 This cytosolic translocation is significant for virulence as it prevents further exposure to lysosomes and provides a more conducive environment for bacterial growth. Of note, absence of ESAT-6 and associated genes encoded by the re-

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gion of difference 1 (RD1) is the primary source of attenuation in the Bacillus Calmette-Geurin (BCG) strain of M. Bovis used for mTB vaccination.8 Furthermore, mTB downregulates antigen presentation pathways, specifically transcription and assembly of major histocompatibility complexes (MHC). These MHC complexes are important proteins on the plasma membrane of antigen presenting cells (APCs) which present mTB-specific peptides to B- and T-cells to induce their activation. While the mTB cell wall prevents lysosomal degradation and processing of antigen for loading on MHC, the 19kDa secreted protein, mTB39, binds TLR2 to prevent NfKb-induced MHC-II transcription (Figure 1A).9 Moreover, mTB stimulates the release of IL-10, which acts in an autocrine and paracrine manner to inhibit the proteolytic activity of cathepsin B in endosomes and prevent generation of MHC-loading peptides (Figure 1B).9 These mechanisms are then compounded by a downregulation of interferon-gamma (IFN-γ) in natural killer (NK) cells and T-cells, partially through inhibition of IL-12 signaling.9,10 This reduction of IFN-γ not only downregulates MHC transcription, but also restricts intracellular killing by phagocytes and generation of antigenic peptides (Figure 1E). The considerable decrease of bacterial killing and antigen presentation causes a significant delay in adaptive activation and T-cell-based immunity. A 2011 review illustrates this effect and discusses how mTB arrives in the draining lymph node as late as 15 days post-infection (Figure 2).11 This delayed and gradual recruitment of effector T-cells contrasts typical acute resolving infections which rapidly activate adaptive responses within 2-3 days. This provides ample time for the bacteria to establish a primary infection and dormant population. The lack of robust local memory responses is in fact one of the major reasons why vaccination fails to prevent mTB latency. Thus, an effective vaccine will likely require a tissue-targeting approach, such as aerosol immunization, to bolster defenses at the primary point of contact. In addition to several mechanisms for avoiding immune detection, mTB is also able to re-focus immune responses through modulation of local cytokines.9,10,11 Multiple studies have revealed a significant increase in immuno-suppressive cytokines, such as IL-10, and an upregulation of regulatory T-cells (TRegs).9,10,11 This is similar to the immune modulation seen in squamous cell carci-

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references 1. “Global Tuberculosis Report 2018.” World Health Organization, World Health Organization, 26 Sept. 2018, www.who.int/tb/publications/global_report/en/. 2. Alexander, P. E. and P. De. "The Emergence of Extensively Drug-Resistant Tuberculosis (Tb): Tb/Hiv Coinfection, Multidrug-Resistant Tb and the Resulting Public Health Threat from Extensively Drug-Resistant Tb, Globally and in Canada." Can J Infect Dis Med Microbiol 18, no. 5 (Sep 2007): 289-91. https://dx.doi. org/10.1155/2007/986794. 3. Chang, K. C. and W. W. Yew. "Management of Difficult Multidrug-Resistant Tuberculosis and Extensively Drug-Resistant Tuberculosis: Update 2012." Respirology 18, no. 1 (Jan 2013): 8-21. https://dx.doi.org/10.1111/j.1440-1843.2012.02257.x.

Figure 3. Macrophage and T-cell activation, killing of TB bacilli and

granuloma formation. Macrophages are activated by TB bacilli and produce cytokines and T-cell activation. Activated macrophages are mycobactericidal, but a few bacilli escape. The cell activation induces lymphocyte recruitment orchestrated by chemokines leading to the formation of granulomas which contain the bacilli. Antibody neutralization of TNF or IFN or T-cell depletion result in dissolution of the granuloma structure, rescue of surviving bacilli 16 with dissemination of infection. Quesniaux (2012) InTech

4. Cohen, S. B., B. H. Gern, J. L. Delahaye, K. N. Adams, C. R. Plumlee, J. K. Winkler, D. R. Sherman, M. Y. Gerner, and K. B. Urdahl. "Alveolar Macrophages Provide an Early Mycobacterium Tuberculosis Niche and Initiate Dissemination." Cell Host Microbe 24, no. 3 (Sep 12 2018): 439-46 e4. https://dx.doi.org/10.1016/j. chom.2018.08.001. 5. Wolfe, L. M., S. B. Mahaffey, N. A. Kruh, and K. M. Dobos. "Proteomic Definition of the Cell Wall of Mycobacterium Tuberculosis." J Proteome Res 9, no. 11 (Nov 5 2010): 5816-26. https://dx.doi.org/10.1021/pr1005873. 6. Brennan, P. J. "Structure, Function, and Biogenesis of the Cell Wall of Mycobacterium Tuberculosis." Tuberculosis (Edinb) 83, no. 1-3 (2003): 91-7. https://dx.doi. org/10.1016/s1472-9792(02)00089-6. 7. Vankayalapati, R. and P. F. Barnes. "Innate and Adaptive Immune Responses to Human Mycobacterium Tuberculosis Infection." Tuberculosis (Edinb) 89 Suppl 1 (Dec 2009): S77-80. https://dx.doi.org/10.1016/S1472-9792(09)70018-6.

noma (SCC) patients and severely impedes the ability of resident immune cells to respond to threats.12 Active infection also inhibits IL-12 release from activated APCs by an unknown mechanism. This manipulation of local cytokine environment produces a Th2-biased response and restricts Th1 differentiation, thereby undermining critical cell-mediated immunity (Figure 3).9,10,11 Structural investigations of mTB39 and Rv2660c revealed their role in this process, as their N-termini binds TLR2 to produce a non-canonical anti-inflammatory response by increasing IL-10 and suppressing IL-12 and IFN-γ.13,14

future directions for development of vaccines and therapeutics These investigations of host interactions with M. tuberculosis provide significant assets for the development of novel vaccines and therapeutics. Formulations consisting of critical mTB antigens, such as ESAT-6, Ag85, and mTB39 may be combined with potent adjuvants and regimen designs for a safer and more effective vaccine. One such vaccine candidate, M72, consists of a fusion protein of antigens mTB32 and mTB39 in a proprietary AS01-E adjuvant. Recent Phase IIb clinical trials of this M72 vaccine demonstrated 50% efficacy for the prevention of active TB, including re-activation in those with latent infections.15 This M72 study serves as an important proof of concept and illustrates the potential of rationally designed subunit vaccines for the prevention of active tuberculosis infection. Further exploration of host-pathogen interactions as well as optimization of immunization regimens is thus imperative for combating the global mTB epidemic.

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8. Hsu, T., S. M. Hingley-Wilson, B. Chen, M. Chen, A. Z. Dai, P. M. Morin, C. B. Marks, J. Padiyar, C. Goulding, M. Gingery, D. Eisenberg, R. G. Russell, S. C. Derrick, F. M. Collins, S. L. Morris, C. H. King, and W. R. Jacobs, Jr. "The Primary Mechanism of Attenuation of Bacillus Calmette-Guerin Is a Loss of Secreted Lytic Function Required for Invasion of Lung Interstitial Tissue." Proc Natl Acad Sci U S A 100, no. 21 (Oct 14 2003): 12420-5. https://dx.doi.org/10.1073/pnas.1635213100. 9. Baena, A. and S. A. Porcelli. "Evasion and Subversion of Antigen Presentation by Mycobacterium Tuberculosis." Tissue Antigens 74, no. 3 (Sep 2009): 189-204. https://dx.doi.org/10.1111/j.1399-0039.2009.01301.x. 10. Divangahi, Maziar. The New Paradigm of Immunity to Tuberculosis. Springer, 2013. 11. Urdahl, K. B., S. Shafiani, and J. D. Ernst. "Initiation and Regulation of T-Cell Responses in Tuberculosis." Mucosal Immunol 4, no. 3 (May 2011): 288-93. https:// dx.doi.org/10.1038/mi.2011.10. 12. Tallon, B. and J. Bhawan. "Foxp3 Expression Is Increased in Cutaneous Squamous Cell Carcinoma with Perineural Invasion." J Cutan Pathol 37, no. 11 (Nov 2010): 1184-5. https://dx.doi.org/10.1111/j.1600-0560.2009.01482.x. 13. Brennan, M. J. "The Enigmatic Pe/Ppe Multigene Family of Mycobacteria and Tuberculosis Vaccination." Infect Immun 85, no. 6 (Jun 2017). https://dx.doi. org/10.1128/IAI.00969-16. 14. Yihao, D., H. Hongyun, and T. Maodan. "Latency-Associated Protein Rv2660c of Mycobacterium Tuberculosis Augments Expression of Proinflammatory Cytokines in Human Macrophages by Interacting with Tlr2." Infect Dis (Lond) 47, no. 3 (Mar 2015): 168-77. https://dx.doi.org/10.3109/00365548.2014.982167. 15. Yihao, D., H. Hongyun, and T. Maodan. "Latency-Associated Protein Rv2660c of Mycobacterium Tuberculosis Augments Expression of Proinflammatory Cytokines in Human Macrophages by Interacting with Tlr2." Infect Dis (Lond) 47, no. 3 (Mar 2015): 168-77. https://dx.doi.org/10.3109/00365548.2014.982167. 16. Quesniaux, Valerie, Irene Garcia, Muazzam Jacobs, and Bernhard Ryffel. “Role of TNF in Host Resistance to Tuberculosis Infection: Membrane TNF Is Sufficient to Control Acute Infection.” InTech, 2012. https://doi.org/10.5772/31819.

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SENIOR HONORS THESES

BISP 196 is open to undergraduate biology majors who have an overall major GPA of 3.6 or higher, have senior standing, and commit to three consecutive quarters of research during their senior year. The program aims to increase faculty-student interactions and encourage more students studying biology to pursue independent research. These are the abstracts of all the exceptional research projects conducted by the undergraduates during the 2019-2020 academic year.

Assorted corals on view at the Birch Aquarium. Photo by Michael Endow 40

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Roger Revelle College Human Biology Major

SALAM ALLAHWERDY

PI: Jiun Do, M.D., Ph.D., UC San Diego School of Medicine, Dept. of Ophthalmology

Effects of Neural Stem Cells on Retinal Ganglion Cell Survival and Axonal Regeneration

Optic neuropathies cause vision loss by injuring retinal ganglion cells (RGCs), the cells responsible for transmitting visual signals to the brain. In the mammalian central nervous system, RGC axons do not spontaneously regenerate. Furthermore, RGCs die following injury. Improving RGC survival and axonal regeneration is critical for restoring vision. Here, we investigate whether human-derived neural stem cells (NSCs) can promote the survival and regeneration of RGCs. We use in vitro cultures of primary, postnatal rat RGCs and in vivo rat models of optic neuropathy. We found that NSCs directly co-cultured with RGCs enhanced axonal extension compared to RGCs cultured in isolation. To assess the indirect interactions between NSCs and RGCs, RGCs were cultured in NSC-conditioned media (NCM). We found that RGCs cultured in NCM increased RGC survival. Our results indicate that NSCs are capable of supporting RGCs survival and regeneration, suggesting a potential role for NSCs in vision restorative therapies.

John Muir College Human Biology Major

FILIPE ARAUJO-HOFFMAN

PI: Matthew Hangauer, Ph.D., Assistant Professor, UC San Diego School of Medicine, Dept. of Dermatology

Melanoma Cells Enter a Persister Cell State to Evade CD8 T Cell-Mediated Cytotoxicity

Tumors often relapse following initial therapy responses, leading to patient mortality. How cells change from a therapy sensitive state to a resistant state is poorly understood, particularly in the context of immunotherapy. Here, we investigated whether melanoma cells can enter into a persister cell state to evade CD8+ T cell-mediated cytotoxicity. We transduced primary human CD8+ T cells with retrovirus expressing a T cell receptor specific to a melanoma differentiation antigen, ESO-1. We then performed in vitro co-culture of A375 (ESO-1 positive) melanoma cells and these ESO-1 specific TCR T cells with T cells replenished every three days. We found that after initial melanoma cell death a residual population of melanoma cells survive through a reversible mechanism and eventually begin to regrow. Our results indicate that a persister state may arise to evade CD8 T cell-mediated cytotoxicity, but further studies are necessary to characterize the pathways leading to cytotoxicity resistance.

Roger Revelle College Microbiology Major Japanese Studies Minor

ERIKA BARRALL

PI: William Joiner, Ph.D., UC San Diego School of Health Sciences, Dept. of Pharmacology

Endogenous Ly6 Proteins in HEK-tsa Cells Limit Assembly and Alter the Pharmacology of Alpha7 nAChRs

Human embryonic kidney (HEK) cells are commonly used to assay the properties of transfected ion channels and receptors. In the present study we hypothesized that members of the Ly6 protein family are endogenously expressed in HEK cells where they may regulate nicotinic acetylcholine receptors (nAChRs). Using RNAseq we confirmed the presence of 10 different Ly6 transcripts in HEK cells. Next, using RNA knockdown or overexpression of these transcripts, we found that a subset of endogenously expressed Ly6 genes inhibits the maximal response of nAChRs to agonist. We also found that this inhibition is opposed by a chaperone of nAChRs called NACHO. Our results suggest that the properties of nAChRs and perhaps other receptors may be heavily influenced by the specific cell types in which these proteins are studied. This hypothesis may explain why drugs screens can yield candidate molecules with different efficacies in different cellular contexts.

Earl Warren College Biochemistry Major

ELENA DEMEESTER

PI: Adam Engler, Ph.D., Sanford Consortium, Dept. of Bioengineering

The Effect of the 9p21 Locus on Expression of Contractile Versus Synthetic Phenotype of Vascular iPSC Derived Smooth Muscle Cells

Coronary artery disease (CAD) remains one of the leading causes of death in the US and represents a mechanistically complex disease. GWAS studies have identified SNPs within the 9p21 locus that are correlated to increased susceptibility towards CAD. The SNPs are located within a non-coding region hypothesized to play a role in cell cycle regulation. Vascular Smooth muscle cells (VSMCâ&#x20AC;&#x2122;s) are of particular interest and are shown to exhibit 2 phenotypes: synthetic (weakly adherent) and contractile (strongly adherent). The exact mechanism that regulates VSMCs plasticity is still unknown and could play a key role in the development of CAD. Utilizing a shear assay, we have isolated 2 distinct populations: weakly and strongly adherent and have shown that they correspond to the synthetic (more proliferative) and contractile phenotypes. We have also shown that patients with risk/risk phenotypes show higher penetrance of the synthetic phenotype that could be leading to their higher risk of developing CAD.

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Roger Revelle College Biochemistry and Cell Biology Major Chemistry Minor

CARLA ESPINOZA

PI: Rachel Dutton, Ph.D., UC San Diego Biological Sciences

Detecting Horizontal Gene Transfer in the Cheese Rind Microbiome

Horizontal gene transfer (HGT) involves the transfer of genes from one organism to another. As microbes acquire foreign genes through HGT, they can attain novel functions and evolve to survive in a new environment. I investigated HGT occuring in cheese rinds by analyzing long read sequences of the microbial community. My results show possible HGT of a chitin-binding region occurring between the eukaryote Debaryomyces hansenii and prokaryote Brevibacterium linens. Binding chitin, which is prevalent in the cheese rind, may increase the fitness of these organisms by allowing them to extract a rich source of carbon from their environment. One mechanism for HGT is through transduction; therefore phage sequences extracted from the cheese rind were also analyzed to identify the role phage may play in HGT. Future research will focus on knocking out the identified HGT regions and comparing the fitness of the mutants to the wildtype microbes to assess how HGT may affect the fitness of their new hosts.

Roger Revelle College General Biology w/ spec in Bioinformatics & Applied Mathematics Majors

YI FU

PI: Graham Mcvicker, Ph.D., Salk Institute for Biological Studies

Using Convolutional Neural Network to Enhance Chromatin Looping Signals From Low Sequencing Depth HiC-Seq Data

Inside the nucleus, chromosomes form complex 3D structures which mediate promoter-enhancer interaction, insulation, and other forms of regulation. Hi-C Seq is a high-throughput sequencing technology that captures genome-wide chromosome conformation. High-coverage Hi-C is usually needed to calculate where chromatin loops are forming, but requires very expensive high-depth sequencing, ultimately limiting studies. HiCPlus is a recently published tool that trains Convolutional Neural Network models and imputes Hi-C interaction matrix from sparser sequencing data, allowing for loop calling from low coverage data. However, parameters important for loop calling in the context of our Leukemia patient cohort are not known. In this thesis, hyperparameters of the HiCPlus models (training sample size, training duration, training source, normalization type, etc.) and a few loop prediction algorithms are examined to see how they affect the accuracy of Hi-C imputation and loop calling. Preliminary results indicate that hyperparameters need to be fine-tuned to achieve optimal loop calling.

John Muir College Bioengineering (Biotechnology) & General Biology Majors

FANGCHAO HE

PI: John T. Chang, M.D., UC San Diego School of Medicine, Dept. of Medicine

Heterogeneity of Immune cells in Crohn’s Disease Revealed by Single-Cell RNA Sequencing Analyses

Inflammatory bowel disease (IBD) encompasses a spectrum of complex intestinal disorders characterized by dysregulated host innate and adaptive immune responses to gut microbiota in genetically susceptible hosts. Although many studies aimed at uncovering the cellular and molecular basis of IBD have been undertaken, the vast majority of published transcriptional datasets have relied on gene expression analyses of bulk intestinal tissue. Here we elucidated key components and cellular states of the gastrointestinal mucosal and peripheral immune systems in health and Crohn’s disease (CD) through single-cell RNA-sequencing integrated with protein detection. Substantial heterogeneity among immune cells was observed, along with alterations in the immune systems of CD patients compared to that of healthy individuals, insights that could only have been revealed by analyses performed at the single-cell level.

Thurgood Marshall College Biochemistry and Cell Biology Major

TINGFEI HU

PI: Renate B. Pilz , M.D., UC San Diego School of Medicine, Dept. of Medicine

Characterization of Myosin Binding Subunit-85

Cardiac contraction is modulated by the phosphorylation state of myosin light chain (MLC), which is controlled by the activities of MLC kinase and MLC phosphatase. The importance of MLC phosphorylation is shown by studies demonstrating that reduced MLC phosphorylation leads to cardiac failure. MBS85, a little-understood MLC phosphatase targeting subunit found in cardiac myocytes, is homologous to MYPT1 found in smooth muscle cells; the latter is thought to induce MLC de-phosphorylation by targeting the catalytic subunit, PP1cδ, to myosin. MBS85 and MYPT1 contain a conserved phosphorylation site for protein kinase G-1α (PKG1α); MYPT1 phosphorylation by PKGIα on this site increases MLC phosphatase activity. We hypothesize that MBS85 phosphorylation by PKG1α will regulate MLC phosphatase activity in the heart. Our preliminary data show that PKG1α phosphorylates MBS85 in vitro. Our studies will provide insights into the functions of MBS85 in the heart.

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JENNIFER HUANG

Thurgood Marshall College Human Biology Major Healthcareâ&#x20AC;&#x201D;Social Issues Minor

PI: John Ravits, M.D., UC San Diego School of Medicine, Dept. of Neuroscience

Sigma-1 Receptors in the Human Brainstem

Sigma-1 receptors (S1Rs) are chaperone proteins usually located on the mitochondria-associated membrane (MAM) of the endoplasmic reticulum (ER) and are thought to play a neuroprotective role by modulating ER stress proteins to promote cell survival. In cases of high ER stress or high concentration of agonists, S1Rs translocate from the MAM ER to the plasma membrane (PM). The roles of S1Rs have been studied in mice and other animal models, but they have yet to be well-evaluated in humans. Recently, a sigma-1 receptor agonist called Nuedexta has been approved to enhance bulbar function in amyotrophic lateral sclerosis (ALS). In this project, we will study S1Rs in motor neurons originating in the ambiguous nucleus of cranial nerves IX and X in the medulla oblongata and which innervate bulbar musculature. We will use immunohistochemistry and immunofluorescence to study expression in health and various disease phenotypes.

Earl Warren College Human Biology Major Political Science & Music Minors

EMMA HUIE

PI: Ralph Greenspan, Ph.D., UC San Diego Division of Biological Sciences, Dept. of Cognitive Sciences

Dopamineâ&#x20AC;&#x2122;s Role in Visual Trace and Delay Learning in Drosophila Melanogaster

Classical conditioning can be in the form of delay learning, where the biologically potent unconditioned stimulus (US) occurs during a neutral conditioned stimulus (CS) ends, or trace learning, where US occurs after CS ends. This behavior is observed in delay olfactory learning in D. melanogaster, which is an ideal model because of the many genetic tools available, and its simple neural network, and parallels to human genetic basis of disease. However, the presence of visual trace learning, the anatomical location associated with attention, and the related molecular process are unknown. In the current study, it was found that D. melanogaster can perform visual trace and delay learning, and that the central complex is closely related to that process. In addition, we also found that dopamine played a key role in the conditioning process. Investigating this can draw parallels to mammalian systems and give a better understanding of learning and attention.

Thurgood Marshall College General Biology Major

CAIRANG JIA

PI: Karl J. Wahlin, Ph.D., UC San Diego School of Medicine, Dept. of Ophthalmology

Retinal Ganglion Cell Targeting in a Human Stem Cell Based Retinal Model

Retinal ganglion cell (RGC) loss in glaucoma is a major cause of blindness. While previous research has shown that iPSCs can form RGCs in 2D and 3D, they are not exposed to their natural thalamic target, the lateral geniculate nucleus. Therefore, current models are likely to be inaccurate. Our long term goal is to integrate human 3D retinas with higher order brain tissues. The focus of my specific project will be to generate a triple color RGC reporter line that will label Brn3a, Brn3b and OPN4 target genes. This will be accomplished using CRISPR Cas9 gene-editing using customized targeting vectors that I have generated for HDR based gene integration. Upon differentiation into 3D retinas, these will be co-cultured with human cerebral organoids. I hypothesize that by co-culturing retinas with brain organoids we will be able to improved synaptic development and reduced RGC cell loss.

Sixth College Biochemistry and Cell Biology & Molecular Synthesis Major

WUJI JIANG

PI: Nicholas Spitzer, Ph.D., UC San Diego Division of Biological Sciences, Section of Neurobiology

Molecular Mechanism of Neurotransmitter Switching in a PTSD Mouse Model

Post-traumatic stress disorder (PTSD) is a psychological illness that is triggered by experiencing or witnessing sudden traumatic events. However, the molecular mechanism that mediates PTSD is still unknown. Here we established a mouse model of PTSD by intense electric foot-shock and show that neurotransmitter (NT) switching, a novel form of neuroplasticity, is involved in PTSD. We observed a loss of the inhibitory neurotransmitter GABA and gain of the excitatory transmitter glutamate in vivo in neurons in the lateral wings of the dorsal raphe nucleus (DRN) of PTSD mice. A corresponding increase in neuronal activity and phosphorylation of p38 mitogen activated protein kinase (MAPK) was observed in the same brain area. We hypothesize that NT switching is responsible for PTSD symptoms and that a signaling cascade involving p38 MAPK causes NT switching. Further studies will be conducted in order to uncover the causal relationship between p38 MAPK phosphorylation and PTSD symptoms.

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John Muir College General Biology Major

CHINMAY KALLURAYA

PI: Matthew Daugherty, Ph.D., UC San Diego Division of Biological Sciences

A Novel Phylogenomic Approach Vastly Increases Known Cases of Inter-Kingdom Horizontal Gene Transfer

Horizontal gene transfer (HGT) is an evolutionary mechanism that involves movement of genes between species by means other than from parent to offspring. A large gap in knowledge exists regarding HGT between bacteria and eukaryotes. Serendipitous discoveries have shown rare instances of HGT providing functions in immunity, metabolism and/or adaptation to new environments to the recipient eukaryote species. We reasoned that a comprehensive analysis for inter-kingdom HGT could substantially increase the number of known cases. We therefore developed an automated and unbiased phylogenetic-informed approach to discover inter-kingdom HGT that possesses the scaling and tractability needed to handle a continually growing genome database. Comparing a diverse panel of bacterial and eukaryotic genomes, we discovered over 100 novel instances of inter-kingdom HGT. These results demonstrate that the incidence of inter-kingdom HGT is more widespread than previously known and provide a catalogue of high-confidence HGT examples to support functional characterization of transferred genes.

Roger Revelle College Biochemistry and Cell Biology Major

ANDY KAO

PI: Randy Hampton, Ph.D., UC San Diego Division of Biological Sciences, Section of Cell and Developmental Biology

Structure Misfunction: An Analysis of Minimally Misfolded Quality Control Substrates

The misfolded substrates of protein quality control remain a poorly understood class of proteins. While elements such as hydrophobicity and degrons have been linked to certain quality control pathways in previous works, the search methods or model substrates used in those studies often reflect drastic structural alterations such as large truncations. In contrast, the most common protein folding errors encountered by the cell are more likely to be relatively minor missense mutations. To remedy this gap in knowledge we conducted a structure misfunction screen to create a collection of minimally misfolded quality control substrates. These substrates represent a more physiologically relevant class of quality control substrates, resulting from mere point mutations while also eliciting strong degradation and maintaining normal protein function. Through hydrogen deuterium exchange mass spectrometry and ANS hydrophobicity probing we are endeavoring to reveal the specific structural perturbations in these mutants which elicit quality control mechanisms.

FIROUZEH KHOOBCHEHR

Earl Warren College Microbiology Major

PI: Partha Ray Ph.D., UC San Diego Moores Cancer Center, Dept. of Surgery

Characterization of an Anti-KIT Aptamer

Aptamers are single-stranded DNA or RNA ligands selected by an in vitro process called SELEX (Systematic Evolution of Ligands by Exponential Enrichment) and bind their target proteins with high affinity and specificity, analogous to monoclonal antibodies. However, compared to antibodies, aptamer production is cost-efficient and faster. c-KIT is a receptor tyrosine kinase overexpressed in several cancers, e.g. Gastrointestinal Stromal Tumor, Mastocytosis. To investigate the binding of the anti-KIT aptamer, we established a stable transgenic cell line expressing c-KIT under Doxycycline-inducible promoter by using lentiviral-transduction. The construction of the cell line was tested by (1) Western blot, using an anti-KIT antibody. (2) Flow-cytometry by staining the cells with Fluorophore-conjugated anti-KIT aptamer. The anti-KIT antibody and a scrambled aptamer were used as a positive and negative control, respectively. The aptamer and antibody demonstrated comparable binding to the cell line expressing c-KIT and can be used as a biomarker for cancer cell detection.

Roger Revelle College Human Biology & Global Health Majors

ALLISON KRAMER

PI: Bradley Moore, Ph.D., Scripps Institution of Oceanography, Center for Marine Biotechnology and Biomedicine

Bacterial Remediation of the Environmental Neurotoxin Domoic Acid

Domoic acid is a neurotoxin produced by diatoms, unicellular eukaryotic microalgae. These algae are consumed by filter feeders, such as shellfish, which can bioaccumulate the toxin to high levels. Ingestion of these contaminated shellfish by humans, marine mammals, or birds leads to amnesic shellfish poisoning, which is characterized by memory loss or seizures. However, previous studies suggest certain mollusks contain bacteria that degrade domoic acid. The goal of this project is to isolate bacteria from marine sources, including clams and mussels, that are resistant to domoic acid and determine if and how they degrade it. For the purposes of experimentation, we primarily work with kainic acid, a related, safer, and more readily available molecule. We predict bacteria that can degrade kainic acid will also be able to do the same for domoic acid, possibly using it as a carbon or nitrogen source.

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John Muir College Molecular Biology Major

GAYATRI MAINKAR

PI: Mohit Jain, M.D., Ph.D., UC San Diego School of Medicine, Dept. of Medicine

Identification of Mediators of Pluripotency Maintenance and Cardiomyocyte Differentiation

Induced pluripotent stem cells (iPSCs) are widely used in biomedical research to produce patient-specific cardiomyocytes that accurately capture key features of several cardiac diseases. Although numerous signaling pathways that regulate differentiation into cardiomyocytes have been described, the role of signaling lipids within these pathways is unclear. An analysis of samples collected from different stem cells differentiating into cardiomyocytes as well as samples collected from the reprogramming of fibroblasts into iPSCs was done using liquid chromatography-mass spectrometry. It was shown that several bioactive lipids have signature expression patterns during the gain and loss of pluripotency. Interestingly, the inhibition of Cyp4A11, an enzyme involved in bioactive lipid synthesis, decreases the expression of fundamental pluripotency markers. The characterization of the role of unexplored metabolites in cardiomyocyte specification and pluripotency maintenance could potentially be useful in refining cellular models of cardiac diseases and heart development.

Thurgood Marshall College Molecular Biology Major

HIRSH MAKHIJA

PI: Cory Root, Ph.D., UC San Diego Division of Biological Sciences, Section of Neurobiology

The Effect of Anxiolytic and Anxiogenic Drugs on C-Fos Expression Within the Intercalated Cell Clusters of the Amygdala

The intercalated cells (ITC) of the amygdala are an understudied population of neurons that are thought to serve as an inhibitory gate on amygdala functions. They express the gene, FOXP2, are GABAergic and surround the Basal lateral amygdala throughout the rostral-caudal axis forming four distinct clusters. It has been proposed that they might regulate anxiety through inhibitory control of the amygdala. We hypothesized that they may be activated and inhibited by anxiolytic and anxiogenic drugs, respectively. A panel of anxiolytic and anxiogenic drugs was delivered to mice and ITC activation was measured using immunohistochemistry for c-fos, as a proxy for neural activity, and FOXP2. In general, anxiolytics and anxiogenics decreased and increased c-fos expression, respectively, relative to controls. Thus, anxiolytic and anxiogenic drugs affect ITC activation. Changes in c-fos expression varied between ITC clusters. It will be interesting to investigate the differing functions between ITC clusters in control of anxiety.

Roger Revelle College Molecular Biology Major

IPEK MIDILLIOGLU

PI: Amy Kiger, Ph.D., UC San Diego Division of Biological Sciences, Section of Cell and Developmental Biology

Role of Dynamin in T-tubule Maintenance and Remodeling

Transverse (T-) tubules are invaginations along the muscle cell membrane. T-tubules control synchronous sarcomere contraction essential for muscle function (Razzaq et al 2001). Moreover, T-tubule remodeling is required to maintain muscle membrane (Towler et al 2004). Dynamin is a large GTPase involved in membrane trafficking (Cowling et al 2012). Dominant mutations in dynamin 2 are associated with human myopathy. In Drosophila muscle, overexpression of dynamin disrupts T-tubules inappropriately, and we discovered that endogenous dynamin is required for wild type developmental muscle remodeling. To understand how dynamin may be regulated to mediate T-tubule remodeling, I explored the effects of dynamin mutant alleles on protein localization and the ability to induce T-tubule disruption when overexpressed in either Drosophila larval body wall muscle or adult flight muscle. My results establish approaches for studying dynamin requirements for T-tubule organization, and suggest that dynamin mutations analogous to human myopathy disease alleles may alter dynamin localization.

Eleanor Roosevelt College Human Biology Major

LINA MOHAMMED

PI: Xin Sun, Ph.D., UC San Diego School of Medicine, Dept. of Pediatrics

An Analysis of CDH Mouse Models and Mouse Lung Innervation

Project 1: Congenital Diaphragmatic Hernia (CDH) results from a hole in the diaphragm allowing abdominal organs to push into the chest cavity and impede lung development. Some genes that cause CDH might have essential lung functions. We generated lung specific conditional Kdm4a knockout mice and analyzed them for lung defects. They did not exhibit CDH phenotype and had normal differentiation. This suggests that Kdm4a is redundant in mice. Further research can attempt additional knockouts of multiple genes to counter gene redundancy and generate CDH models to study new possibilities for treatment. Project 2: Lung innervation determines the lungâ&#x20AC;&#x2122;s reaction to stressors such as allergens, but little is known about the lung as a sensory organ. I examined the vagal nerve innervation of PNECs and SMCs in the Nav1.8 cre line. Understanding innervation of these cells can help us understand reaction mechanisms in lung disease and utilize them for treatment.

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John Muir College General Biology Major Psychology Minor

NOORHAN MONTHER

PI: Don Cleveland, Ph.D., UC San Diego School of Medicine, Dept. of Cellular and Molecular Medicine

Elucidate Mechanism(s) that Regulate TDP-43 Cytoplasmic Mislocalization and its Toxicity

Amyotrophic Lateral Sclerosis (ALS) is the most common type of motor neuron disease which is characterized as the progressive loss of motor neurons. One landmark discovery in ALS research is that TDP-43, an RNA binding protein, forms pathological aggregate structures in 97% of ALS patients. This project involves generating a cellular disease model which replicates pathological features of TDP-43. These pathological features include nucleus to cytoplasmic TDP-43 mis-localization and TDP-43 protein aggregation. To monitor the localization of TDP-43, I have generated a cellular system that tags both alleles of TDP-43 with green fluorescent protein via a CRISPR-Cas9 mediated knock in method. The goal of this project is to elucidate the molecular pathways and mechanisms that regulate nucleus to cytoplasmic TDP-43 mis-localization and the toxicity caused by TDP-43 mis-localization.

John Muir College Human Biology & Public Health Major

WENSHUAI MU

PI: Dennis Carson, UC San Diego School of Medicine, Dept. of Medicine, Moores Cancer Center

Discovery of Vaccine Adjuvants that Affect the Immune Response

Vaccine adjuvants help create a stronger and prolonged pathogen specific immune responses. Through high throughput screening, we discovered a vaccine adjuvant candidate (compound42) that affects the activation pathways of NF-κB and Type I Interferon in antigen presenting cells. Compound42 is a small molecule that prolongs antigen-specific humoral immune response when used as co-adjuvant with toll-like receptors 4 (TLR4) ligand. After the immunization with inactivated influenza virus adjuvanted with TLR4 ligand, monophosphoryl lipid A(MPLA), and compound42, the mice were bled and dissected for spleen, lymph node, and muscle. The combination adjuvant, MPLA plus compound 42, significantly increased virus specific humoral responses. However, the flowcytometry data showed that there was no significant increase for monocytes, dendritic cells, and B cells after 1 day of vaccine injection. We are currently investigating immunologic parameters that correlate enhanced humoral immune responses by compound42.

EMILY PARIS

Roger Revelle College Biochemistry and Cell Biology Major Marine Science Minor

PI: Bradley S. Moore, Ph.D., Scripps Institution of Oceanography / Skaggs School of Pharmacy and Pharmaceutical Sciences, Center for Marine Biotechnology & Biomedicine

Domesticating Marine Bacteria in Micro-Orbs for Natural Product Discovery

The Great Plate Count Anomaly is an observation that less than 10% of microbial species in an environmental sample can be grown in culture. Traditional methods—such as agar and liquid culturing—contribute to this anomaly through lack of nutrients and environmental conditions required by certain microbes to survive. Growth of microbes in vitro is crucial for the study of microbial physiology, ecology and biochemistry, which could lead to more knowledge of biosynthetic pathways and possibly new drug discoveries. This project uses chitosan and alginate to encapsulate and grow new species of marine microbes in situ, allowing them to experience nutrient diffusion and a three-dimensional structure that mimics in their natural environments. By adjusting this encapsulation technique to seawater ecosystems, we were able to “capture” microbes from the marine sponge Tethya aurantium. Whether this technique provides the ability to culture novel microbes in comparison to traditional techniques is still being explored.

Roger Revelle College Psychology & Neuroscience Major

JANKI PATEL

PI: Matthew Banghart, UC San Diego Division of Biological Sciences, Section of Neurobiology

Molecular Identification of Neurons in the Descending Pain Modulatory Pathway that May Contribute to Opioid Analgesia

A descending midbrain circuit from the periaqueductal gray (PAG) to the rostroventral medulla (RVM) is capable of driving bidirectional modulation of incoming pain signals. The canonical model states that excitatory projections from the PAG to RVM are tonically suppressed by local inhibitory interneurons expressing the mu opioid receptors (MORs). Endogenous opioids and opioid-drugs cause analgesia in part by binding to these MORs and silencing the inhibitory interneurons, resulting in a net activation of PAG to RVM projections. Prior experiments, however, showed that silencing inhibitory neurons in the PAG to mimic opioid action in some cases caused hyperalgesia, rather than analgesia, suggesting that the current model of PAG microcircuitry is incomplete. We discovered a novel subset of excitatory and inhibitory projection cells that express MORs, which are not accounted for in the canonical model. This provides insight into the circuity of opioid action and may point to new drug targets for the development of safer analgesics.

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John Muir College Molecular Biology Major Psychology Minor

ANASTASIA PIMENTEL

PI: Jens Lykke-Andersen Ph.D., UC San Diego Division of Biological Sciences, Section of Molecular Biology

Examining the Roles of RNA Polymerases TENT4A and TENT4B in Non-Coding RNA Quality Control

RNA quality control is an important process by which cells are able to control which RNAs mature or are degraded. It is known in yeast that the RNA polymerase Trf4 tags non-coding RNAs (ncRNA) with post-transcriptional oligonucleotide tails which facilitates degradation via the nuclear RNA exosome. In humans, the RNA polymerases TENT4A and TENT4B are homologs and show preference for adenylation. However, the extent to which TENT4A/4B modify ncRNAs and which ncRNAs serve as their targets is not well understood. To address this, we performed knockdowns of TENT4A/4B protein via siRNA treatment in human T-REx-293 cell lines and are examining changes in ncRNA tail adenylation via global sequencing. To specifically assess the adenylating activity, exogenous genes containing wildtype or catalytic-mutant TENT4A/4B were created, inserted into T-REx-293 cells, and expressed during endogenous protein knockdown. Analyzing these data will provide a more complete picture of the roles these polymerases play in humans.

Roger Revelle College Human Biology Major

BERENICE RAMIREZ LEAL

PI: Joseph R. Ecker, Ph.D., The Salk Institute,HHMI and Laboratory of Plant Molecular and Cellular Biology

Identification of Protein-Protein Interactions in Arabidopsis Thaliana

Underlying every cellular process are proteins acting in complex with other proteins, yet most Protein-Protein Interactions (PPIs) have not been identified. CrY2H-seq is a Cre Recombinase reporter-mediated yeast two-hybrid assay that involves en masse mating of yeast two-hybrid (Y2H) libraries followed by next-generation sequencing to identify PPIs. Previously, this method was applied to an Arabidopsis ORF collection; however its use in screening cDNA-based libraries has not been tested. In this project, a cDNA-based yeast two-hybrid library was prepared from Arabidopsis seedlings, which involved growing and harvesting the plants, RNA extraction and reverse transcription, and downstream processing of the cDNAs for insertion into the Y2H plasmid by gap repair. How these cDNA-based Y2H libraries perform in CrY2H-seq will be assessed by the identification of experimentally known PPIs and new candidate PPIs. Results from this project have the potential to contribute to the understanding of a variety of plant processes and inform future CrY2H-seq cDNA screening.v

MONA ROSHAN

Thurgood Marshall College Biochemistry and Cell Biology Major

PI: Karl J. Wahlin, Ph.D, UCSD School of Medicine, Dept. of Ophthalmology

Directional Outgrowth of Human Retinal Ganglion Cells using a 3D Printed Scaffold

The loss of retinal ganglion cells (RGCs) causes vision loss in glaucoma and optic neuropathies. Pluripotent stem cells can be coaxed into becoming laminated 3D tissues with all major classes of retinal neurons, however, we have yet to integrate such structures with higher-order brain neurons. As a result, lab grown retinas fail to form proper synapses and RGCs eventually die. In order to develop better 3D models for studying RGC connections, my project is focused on developing an optic nerve model to study RGC axon outgrowth which will lead to improved retinal models. Our approach relies on the generation of RGC reporters that express fluorescent proteins coupled to the endogenous Brn3a, Brn3b, and OPN4 genes. By growing these reporters on micro-printed scaffolds in the presence of biomimetic growth factors and axon guidance cues, we seek to steer axons along an artificial scaffold and mimic an optic nerve.

Thurgood Marshall College Molecular Biology Major

PARAS SAHANI

PI: Maurizio Zanetti, M.D., UC San Diego School of Medicine, Moores Cancer Center

The Unfolded Protein Response: Linking Tumor Aneuploidy to Immune Dysregulation

Aneuploidy is present in nearly 90% of solid tumors and is considered a â&#x20AC;&#x153;hallmark of cancerâ&#x20AC;?. Our lab demonstrated that the unfolded protein response (UPR) triggers immune dysregulation in the tumor microenvironment. The new hypothesis is that aneuploidy could lead to UPR activation, and in turn immune dysregulation. To test it, we used two different models of aneuploidy in vitro: quasi-diploid human colorectal and ovarian carcinoma cancer cells treated with Reversine, and clonal cell lines derived from a cell-cell fusion event between murine melanoma cells and mouse embryonic fibroblasts. Both models of aneuploidy resulted in XBP1 splicing, indicating UPR activation. Analysis of macrophages treated with the conditioned media of aneuploid cells showed that gene expression of the pro-inflammatory cytokine IL-6 and the immune suppressive enzyme Arginase 1 was significantly induced. The results validate bioinformatic analysis of The Cancer Genome Atlas showing that the UPR links aneuploidy and immune dysregulation.

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SCOTT SUN

John Muir College Biochemistry and Cell Biology Major

PI: Enfu Hui, Ph.D., UC San Diego Division of Biological Sciences, Section of Cell and Developmental Biology

Comparison of the Phosphatase Recruiting Abilities of Siglec-9 and PD-1

Our immune cells are able to identify and destroy tumors, but tumors have evolved to hijack inhibitory molecules to evade destructive immunity. One such inhibitory molecule is Siglec-9, a type-I transmembrane receptor expressed on immune cells. Prior studies suggest that Siglec-9 can recruit SH2-containing phosphatases SHP-1 and SHP-2 through its intracellular domain which harbors phosphorylatable inhibitory tyrosine motifs. It is unclear if the Siglec-9:SHP-1/2 interactions are direct and if there is a preference for a specific phosphatase. Therefore, I sought to determine the mechanism behind Siglec-9â&#x20AC;&#x2122;s ability to recruit SHP-1 and SHP-2, using the structurally related, better studied inhibitory receptor PD-1 as a reference. Using membrane reconstitution assays with purified proteins, I found that Siglec-9 directly recruits both SHP-1 and SHP-2 with a preference to SHP-1, contrasting to the strong SHP-2 preference of PD-1. These results suggest that structurally related inhibitory receptors exhibit distinct phosphatase specificities.

Roger Revelle College Neuroscience and Physiology Major Cognitive Science Minor

ISAAC TANG

PI: Joseph Gerard Gleeson, M.D. UC San Diego School of Medicine, Dept. of Neuroscience

Intronic Pentanucleotide Repeat Expansions in Familial Adult Myoclonic Epilepsy

Recently, pentanucleotide repeats in the human genome were linked to Familial Adult Myoclonic Epilepsy (FAME). The particular repeat expansion linked to FAME2 consists of two tandem repeats: a (TTTTA)n expansion and a (TTTCA)n expansion, located in intron 1 of the STARD7 gene, both of which can extend from hundreds to thousands of repeats in length. During STARD7 transcription, RNA with these repeats is generated, which we hypothesize leads to accumulation of toxic repeat protein through RAN translation. Two methods will be implemented. In the first method, repeats were cloned and overexpressed in neural progenitor cells (NPCs). In the second method, FAME2 patient fibroblasts were reprogrammed then differentiated into NPCs. I am currently assessing for accumulation of RNA granules and toxic protein in these cells.

John Muir College General Biology w/ spec in Bioinformatics Major Computer Science Minor

DANIELLA VO

PI: Debashis Sahoo, Ph.D., UC San Diego School of Medicine, Dept. of Pediatrics, UC San Diego Jacobs School of Engineering, Dept. of Computer Science and Engineering

A Novel Tool: Boolean Implication Analysis Provides Insight Within Breast Milk Microbiomes

Microbiomes consist of bacteria, viruses, and other microorganisms that are present in habitats such as breast milk. Understanding the composition, functions and assembly of the human milk microbiota has important implications not only for the infant gut microbiota establishment, but also for maternal health. Here we show that using a novel method of Boolean implication analysis allows us to identify both symmetric and asymmetric relationships between microbe species. Our results demonstrate that this type of analysis can provide further insight into the relationships within the breast milk microbiome, thus allowing us to determine that factors such as alcohol and birth control may affect the breast milk microbiome. Independent validation of the logical relationships in other publicly available breast milk dataset will reveal potential universal relationships. These relationships would be clinically relevant, and the analyses may improve disease analysis in both mother and child in terms of accuracy and efficiency.

Thurgood Marshall College Biochemistry and Cell Biology Major Global Health & Chemistry Minors

ELEANOR WANG

PI: Hiutung Chu, Ph.D., UC San Diego School of Medicine, Dept. of Pathology

Characterization of Human Milk Oligosaccharide Metabolism by Infant Gut Bacteria

Breastfeeding has been shown to protect infants from a variety of infectious and inflammatory diseases. Human milk oligosaccharides (HMOs) are among the most abundant components of breastmilk, with high structural diversity. Although indigestible by humans, HMOs can be metabolized by bacterial members of the gut microbiome. The functional biology of HMOs includes inhibiting growth of pathogenic bacteria and promoting growth of beneficial commensal bacteria, however specific mechanisms that mediate these activities remain unclear. Here, we aim to characterize the metabolic activities of infant gut bacteria using HMOs isolated from human breast milk. We first identify a defined minimal medium appropriate for studying HMO metabolism by gut bacteria. We then analyze HMO metabolism by bacterial species using HPLC analyses. These studies will facilitate further mechanistic studies of bacterial metabolism and community development and provide insight into designing studies of more complex host-microbe interactions.

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Eleanor Roosevelt College Human Biology Major

LI WANG

PI: Ju Chen, Ph.D. UC San Diego School of Medicine

The Role of Eukaryotic Elongation Factor 1A2 in Developing Cardiomyocytes

Protein translation is the fundamental biosynthetic reaction whose regulation determines cell functions temporally and spatially. In eukaryotes, this process is mediated by the coordination among translation factors, transfer-RNAs (tRNAs), and ribosomes. In translation elongation, eukaryotic elongation factor 1A2 (eEF1A2) plays a canonical role of delivering aminoacylated tRNA to the A site of ribosome, enabling the stepwise addition of amino acids to the newly synthesized peptide. Recently, a study showed that patients with homozygous missense mutation p.P333L in eEF1A2 experienced severe dilated cardiomyopathy (DCM) via an unknown mechanism. Utilizing cardiac-specific constitutive eEF1A2 knock out and mutation knock-in mouse models along with various biochemical techniques, we showed that loss of eEF1A2 led to DCM without globally affecting translation, suggesting the mechanism was independent of its classic aminoacylated tRNA transfer function. Our results further suggested that eEF1A2 might play a specific role in regulating mitochondrial electron transport chain complex protein expression in heart.

Eleanor Roosevelt College Physiology and Neuroscience & Biochemistry Majors

SHUHE WANG

PI: Matthew Shtrahman, M.D., Ph.D., UC San Diego School of Medicine

Using Retrovirus to Induce Inhibitory Interneurons in Adult Hippocampus

Mesial temporal lobe epilepsy (mTLE) is the most common form of epilepsy in adults, and is characterized by pathology within the dentate gyrus (DG) in the hippocampus. Both human and animal studies of mTLE demonstrate dramatic alterations in the immature adult-born dentate granule cells (DGCs), which form pathological excitatory connections with neighboring DGCs. Studies in rodents show that this hippocampal hyperexcitability can be reversed via the introduction of inhibitory neuronal precursor cells into the hippocampus. To find out a safe and efficacious source of inhibitory precursor cells, we decided to use retrovirus (RV) to express inhibitory neuronal transcription factors in adult-born DGCs to test if we can reprogram them into interneurons. Preliminary data show that newborn cells infected with RV expressing SOX2, halts their development making them well-poised to be converted to inhibitory neurons with the addition of inhibitory transcription factors.

John Muir College Biochemistry and Cell Biology & Business Psychology Majors Biological Anthropology Minor

JUSTIN YU

PI: Shigeki Miyamoto, D.V.M., Ph.D., UC San Diego School of Medicine Dept. of Pharmacology

The Role of NEK7 in Regulation of Autophagy in Cardiomyocytes

The NIMA-related kinase 7 (NEK7) was originally discovered in mitosis. Interestingly, NEK7 expression is restricted to a subset of tissues including the brain and heart, which have limited proliferative capabilities. The role and significance of NEK7 in cardiomyocytes has however yet to be determined and we hypothesize that NEK7 regulates a non-mitotic cellular function in cardiomyocytes. We found that wild-type NEK7 expression increases LC3-II formation, an autophagic marker, in cardiomyocytes and this response is greater when a constitutively active mutant of NEK7 is expressed in NRVMs. Glucose deprivation induced autophagy is also increased by NEK7 expression. The mTOR signaling pathway, a major negative regulator of autophagy, is not inhibited by NEK7, suggesting mTOR signaling independent regulation of autophagy. We also found that constitutively active NEK7 inhibits the inflammasome activation while WT NEK7 enhances it. These results suggest that NEK7 activation induces autophagy and prevents activation of the inflammasome.

Earl Warren College General Biology Major Psychology Minor

JIE ZHANG

PI: Stephanie Dulawa, Ph.D., UC San Diego School of Medicine, Dept. of Psychiatry

Dopamine D2 Receptor Overexpression in the Nucleus Accumbens Core Induces Robust Weight Loss During Scheduled Fasting Selectively in Female Mice

Anorexia nervosa (AN) is an eating disorder observed predominantly in females. Yet, the neurobiological underpinnings of AN remain largely unclear. Recent studies suggest that AN should be re-conceptualized as metabo-psychiatric disorder and is associated with altered striatal dopaminergic signaling. Thereby, we virally overexpressed dopamine D2 receptors on indirect pathway neurons of the NAc core (D2R-OENAc) and tested mice of both sexes in the activity-based anorexia (ABA) paradigm, intraperitoneal glucose tolerance test (ipGTT), and Comprehensive Lab Animal Monitoring System (CLAMS). D2R-OENAc induced robust weight loss only in female mice during scheduled fasting, even without the wheel access on ABA. D2R-OENAc mice of both sexes showed glucose intolerance in the ipGTT. CLAMS suggested altered energy consumptions and utilizations by D2R-OENAc. Anterograde tracing confirms dense projections from NAc core D2R-expressing neurons to the lateral hypothalamus (LH). In conclusion, our results implicate the role of D2R-OENAc on metabolic homeostasis in this sexually dimorphic effect.

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Roger Revelle College Human Biology Major Cognitive Science Minor

ALEXANDER ZHANG

PI: Richard Daneman, Ph.D., UC San Diego School of Medicine, Dept. of Pharmacology

The Role of Pdlim1 in Endothelial Cells During Blood-Brain Barrier Dysfunction

The blood-brain barrier (BBB) regulates the movement of ions, molecules, and immune cells from blood into the central nervous system . This regulation is compromised in neurological conditions such as stroke, multiple sclerosis, seizures, and traumatic brain injury. We identified that the gene Pdlim1 is upregulated in endothelial cells during BBB dysfunction in mouse models of these four conditions. Its overexpression could be driving BBB dysfunction or promoting BBB repair. To differentiate between these two functions, we used in vitro luciferase assays to understand whether Pdlim1 transfection affected Wnt signaling (BBB maintenance) or NF-kB signaling (inflammation). We found that Pdlim1 transfection had no effect on Wnt signaling but decreased NF-kB signaling, suggesting that endothelial upregulation of Pdlim1 is protective during disease. We now plan to examine genes downstream of Pdlim1 to determine whether knockout or overexpression of Pdlim1 in vivo alters the expression of these genes in health and disease.

Earl Warren College Molecular Biology & Music (Performance) Majors

SHERRY ZHENG

PI: Andrew Chisholm, Ph.D., UC San Diego Division of Biological Sciences, Section of Cell and Developmental Biology

Patterning of the Extracellular Matrix in the C. Elegans Cuticle

The extracellular matrix (ECM) is a macromolecular network of non-cellular proteins that provides structural and biochemical support to surrounding cells. ECMs are required for tissue homeostasis, differentiation, and morphogenesis; however, the molecular mechanisms underlying ECM patterning remain poorly understood. In my thesis, I aim to dissect some of these mechanisms using the Caenorhabditis elegans cuticle as a tractable model of specialized ECM that exhibits a high degree of spatial and temporal patterning. I specifically focus on the biogenesis of cuticle structures called struts, which are distributed in regularly repeating arrays throughout the adult cuticle. The genes bli-1 and bli-2 encode cuticle collagens thought to be components of struts, and I am examining factors that may regulate strut biogenesis or function by studying other genes that interact genetically with bli mutants. I am also investigating genes required for strut patterning and examining the role of the epidermal cytoskeleton in strut organization.

Eleanor Roosevelt College General Biology Major

STEVEN ZHOU

PI: David Holway, Ph.D., UC San Diego Division of Biological Sciences, Section of Ecology, Behavior and Evolution

Effect of Pollinator Visitation on the Reproductive Success of Cultivated Squash (Cucurbita Pepo)

Apis mellifera, the European honey bee, is common and widespread across North America. Although the honey bee is competitive and abundant in southwestern United States, where it provides pollination services in agricultural systems, some evidence suggests that an over-visitation by Apis mellifera negatively affects plant reproductive performance. My project tests the relationship between pollinator diversity and the reproductive success of cultivated squash (Cucurbita pepo), which is visited by Apis mellifera as well as a number of native pollinator species. Video recordings of pollinators visiting squash flowers were obtained during the summer of 2019. Video data were transcribed to assess the relationship between different measures of visitation and seed production. We hypothesize that seed production increases with visitor evenness. The basis for this hypothesis stems from complementary effects that different pollinator taxa can have on plant reproductive performance.

Eleanor Roosevelt College Biochemistry and Cell Biology Major

RAYMOND H. ZURICH

PI: Jeffrey D. Esko, PhD., UC San Diego School of Medicine, Dept. of Cellular and Molecular Medicine

Enhanced Wound Induced Neogenesis through Heparan Sulfate Reduction

In normal mammalian wound healing, injuries to the dermis result in scars that lack hair and fat. However, if a dermal wound exceeds a critical size, a process termed Wound-Induced Neogenesis (WIN) can occur and regeneration of neogenic hair and fat is induced. It has been shown that Shh activation is necessary and sufficient to induce WIN. Based on previous observations that heparan sulfate deficiency (HSD) may alter Shh activation, we hypothesized that HSD can enhance WIN by increasing Shh activation. Using a systemic genetic model of HSD (Ext1+/-Ext2+/-), we tested whether mice with less heparan sulfate had enhanced WIN after an excisional dermal wound. We found increased WIN in HSD mice. We have preliminary data suggesting increased Shh activation in wounds of HSD mice. Our experiments suggest that inducing heparan sulfate deficiency can be a therapeutic avenue to enhance mammalian regeneration through activation of Shh during wound healing.

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DEVAVRAT BODAS

John Muir College Molecular Biology & Applied Mathematics Majors

PI: Amy E. Pasquinelli, Ph.D., UC San Diego Division of Biological Sciences

Regulation of the Heat Shock Response by miRNA-85

The microRNA (miR) pathway post-transcriptionally regulates various cellular processes by modulating a sequential cascade of genes in response to a genetic switch or an environmental stimulus. While the targets and biological functions of some miRNAs have been determined, the roles of most miRs are yet to be elucidated, specifically in development and stress. With predicted targets that include heat shock proteins and other chaperones, miR-85 may have a function in the broadly conserved heat stress response pathway. Caenorhabditis elegans worms lacking mir-85 have a reduced thermo-tolerance and an increased level of heat shock proteins, suggesting that this microRNA may play a critical role in the proper regulation of the heat shock pathway. This work investigates the mechanistic contribution of miR-85 in stress and in maintaining proteostasis and we hope this work will improve our understanding about how the microRNA pathway contributes to stress tolerance.

Thurgood Marshall College Human Biology Major Cognitive Science Minor

BAR YOSEF

PI: Gedeon Deรกk, Ph.D., UC San Diego Cognitive Development Lab, Dept. of Cognitive Science

Using Social and Personal Outcome Information to Guide Choices in a Dyadic Multi-Armed Bandit Paradigm

A central question in cognitive science is how people form and update mental representations of their own and conspecifics' actions. We describe a new Social Multi-Armed Bandit test of probabilistic learning. Participants see multiple action options ("arms"), each assigned different, unknown reward probabilities. In the social condition, participants take turns choosing arms on a touchscreen. In the control condition a non-interactive confederate touches the screen between the participant's turns. Arm choices and latencies are recorded to quantify strategies. Reward probabilities in the social condition differ between participants, so imitating a partner's actions reduces the participant's rewards. Visual fixations and electroencephalography (EEG) are recorded to explore individual differences in participants' propensity to fixate on their partner's actions, and possible correlations between EEG Event Related Potentials and choice behavior. Preliminary behavior analyses suggest that participants encode their partner's reward outcomes like their own, particularly in the first block of social condition trials.

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HEAD ADVISORS James Cooke, Ph.D. Assistant Teaching Professor of Neurobiology Jaime Estepa Student Engagement Coordinator Division of Biological Sciences

FACULTY ADVISORY BOARD Suckjoon Jun, Ph.D. Carolyn Kurle, Ph.D. Chih-Ying Su, Ph.D. Elvira Tour, Ph.D.

Special Thanks: Patera Design

Elsa Cleland, Ph.D. Jill Leutgeb, Ph.D. Lisa McDonnell, Ph.D.

Top Row: Angelita Rivera, Tania Gallardo, Bridget Spencer, Sam Zilberman, Hannah Abraham, Vickie Kuo, Anne Marie Berry, Shae Galli, Nancy Ji Middle Row: Lilit Vardanyan, Alexandra Casison, Yuxi Wei, Sathya Krishnasamy, Rebecca Chen, Soha Khalid, Diana Presas, Angela Wang, Anjali Iyangar, Nicole Adamson, Eleanor Wang, Ingrid Heumann, Varsha Mathew, Cristina Corral Bottom Row: Arya Natarajan, Zarina Gallardo, Shreya Shriram, Alejandro Dauguet, Olivia Gerald, Andra Thomas, Daniel Lusk, Nikhil Jampana, Julia Cheng, Salma Sheriff, Varsha Rajesh, Gayathri Kalla, Sharada Saraf, Michael Endow, Emma Huie

STAFF EDITOR-IN-CHIEF Emma Huie EXECUTIVE EDITOR Arya Natarajan EDITOR AT LARGE Sharada Saraf HEAD PRODUCTION EDITOR Zarina Gallardo RESEARCH DESIGN EDITOR Nicole Adamson FEATURES DESIGN EDITOR Anvitha Soordelu SPECIAL SECTIONS DESIGN EDITOR Tania Gallardo FEATURES EDITOR Nikhil Jampana STAFF WRITERS Eleanor Wang Ingrid Heumann Kaz Nuckowski Lilit Vardanyan Nandita Rangu

RESEARCH EDITORS Alejandro Dauguet Xaver Audhya HEAD TECHNICAL EDITOR Salma Sheriff TECHNICAL EDITORS Juliana Fox Lynn Nguyen Noorhan Amani Max Gruber Rebecca Chen REVIEW BOARD MANAGER Gayathri Kalla WEBMASTER Saksham Gupta WEB INTERN Toby Dizon ONLINE EDITOR Shreya Shriram ONLINE REPORTERS Mahima Advani Meena Kian Anjali Iyangar

PUBLICITY CHAIR Hanna Richkind PUBLICITY COMMITTEE Tu Anh Nguyen Clara Baek Sanjana Sharma Nancy Ji Elena Cheung Ann Truong Taryn Cornell Omar Abu-Hantash COMMUNITY OUTREACH CHAIR Olivia Gerald COMMUNITY OUTREACH COMMITTEE Tiffany Huynh Samantha Tuohey Belle Hilton Prithvi Setty Gautam Narayan Alexandra Babakanian Goonja Shah Martha Chow Teodora Moisa Alden Bugho Rhea Rajasingham BLOG MANAGER Daniel Lusk

REVIEW BOARD Leanne Dugan Sarah Burbach Sobyn Chung Swapnil Mittal Bindhu Hosuru Chetana Thapetta Dahye Hong

Matias F.G. Yuanshan Li Anthony Estrella Selena Lugosi Shaila Sarathy Shao-Ming Chin Yamila Salazar

Yuxi Wei Angela Wang Charles Xu Dorothy Tsai Ishrak Ramzan Nathan Jeon Janki Patel

BLOGGERS Mary Lombardi Angel Quiroa Lina Lew Neha Sahota HEAD ILLUSTRATOR Varsha Rajesh ILLUSTRATORS Cristina Corral Corly Huang Shae Galli Ellis Zhang Diana Presas Fatimah Khan Hannah Abraham Sara Kian Yichen Wang Phoebe Ahn Lu Yue Wang Angel Rivera HEAD PHOTOGRAPHERS Michael Endow Sam Zilberman PHOTOGRAPHERS Mark Allan Jacob Katie Clark Bridget Spencer Jason Dagoon Anne Marie Berry

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