Sq vol13

Undergraduate Research Journal Volume 13 2015–16 sq.ucsd.edu

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: The unique Rosa hybrid cultivar, also known as ‘Paul Ecke’s Jr’, during springtime at the San Diego Botanical Gardens. Photo by Michelle Bulterys.

INSIDE COVER: Kanab is a North American Grey Wolf, Canis lupis lycaon, and a current resident of a wildlife park in Camp Verde, Arizona. Photo by Erika Nilsson.

LETTER FROM THE EDITOR

B

iology is the study of life, and biologists are its intrepid explorers.

They map out interactions between proteins and the genome or investigate the relationship between cognition and neuroscience. UC San Diego biologists are no different; they study a wide range of biological questions ranging from coral reef ecology to plant immune systems. It is my privilege to present to you the Saltman Quarterly research journal, which gives you a snapshot of the research discoveries made the talented students and faculty of the Division of Biological Sciences at UC San Diego.

In 2004, Saltman Quarterly was a purely academic research journal. Our staff wrote editorials and edited research articles and reviews. Now, Saltman Quarterly has evolved into a comprehensive undergraduate biology communications organization.

We have retained our research editors and writers, but we also recruit photographers and illustrators, outreach volunteers and review board members along with bloggers and designers. Each of our staff members has unique backgrounds in biology, varied scientific interests, and different perspectives on communicating science. I am immensely proud of the work our staff mem-

bers do to create a research journal that is representative of the numerous branches that make up biology.

Saltman Quarterly is founded upon the ideals and legacy of Dr. Paul Saltman, a man who has inspired me to delve into the field of scientific communication as a career. Dr. Saltman was a UC San Diego biology professor who taught nutrition and often headed to the beach to surf before teaching his 8 a.m. class. He was also a staunch advocate for undergraduates who promoted undergraduate involvement in teaching as well as research. This year in the annual tribute to our namesake, we consider how his legacy is carried on through the work of current undergraduate instructional assistants.

Dr. Saltman also promoted increased scientific literacy. He recognized the importance of understanding scientific progress in the context of a rapidly developing and interconnected world and where scientific advances could be shared at the click of a button.

As our world has become more connected, so has our science. This year, Saltman Quarterly has published a quarterly – the SQ Insider – that discusses hotly contested topics in the public that happen to be mul-

tidisciplinary in scope. Our fall quarter SQ Insider, my personal favorite, focused on the intersection of LGBTQIA issues with biology while the spring quarter SQ Insider focused on biological impacts of climate change. In this volume of the Saltman Quarterly research journal, you can read about advances in antiviral immune responses, learn about the benefits of composting, and critique theories surrounding the neurological basis of intelligence. The scientific jungle has suddenly become so much larger, and it is my hope that our research journal can serve as a guidebook to help you navigate a portion of that jungle here at UC San Diego.

To the reader, I hope you read, learn, and discover. I am humbled and deeply honored to present to you the 13th edition of our Saltman Quarterly research journal.

Jacky Lu Editor-in-Chief Saltman Quarterly 2015-16

Generously underwritten by

THE SALTMAN FAMILY and supported by

The views expressed in this publication are solely those of Saltman Quarterly, its principal members and the authors of the content of this publication. While the publisher of this publication is a registered student organization at UC San Diego, the content, opinions, statements, and views expressed in this or any other publication published and/or distributed

by Saltman Quarterly are not endorsed by and do not represent the views, opinions, policies, or positions of the ASUCSD, GSAUCSD, UC San Diego, the University of California and the Regents or their officers, employees, or agents. The publisher of this publication bears and assumes the full responsibility and liability for the content of this publication.

TABLE OF CONTENTS

SALTMAN DEDICATION 4

FEATURES

8 12 16 20 24 2

SALTMAN QUARTERLY

DECODING THE NETWORK OF DISEASE

by Neal Shah

GLOBAL ALLIANCE AGAINST ANTIVIRAL RESISTANCE

by Karen To

WALTZING OUR WAY TO CREATIVITY AND COGNITION

by Anika Ullah

ECOLOGICAL MODELING: A NOVEL MEANS OF APPROACHING CORAL REEF PRESERVATION

by Ishan Goyal

PRETTY BUT FIERCE: PLANT EDITION

by Michelle Lee

VOL. 13

sqonline.ucsd.edu

RESEARCH EFFECT OF COMPOST TEA ON GROWTH OF LACTUCA SATIVA SEEDLINGS

by Brianna Egan

RHO-GEF SOLO REGULATES EPITHELIAL COLLECTIVE MIGRATION VIA ADHESION COMPLEX PROTEIN PLAKOGLOBIN

by Daniel Jun Rindner

DEVELOPING AN EDUCATIONAL INTERVENTION TO IMPROVE GENETIC RISK ASSESSMENT IN YOUNG BREAST CANCER SURVIVORS

by Deepika Suresh

30 37 43

REVIEW: INTELLIGENT NETWORKS

by Erika Johannessen

sq.ucsd.edu

49

SENIOR HONORS THESES

54

STAFF

66

SALTMAN QUARTERLY

VOL. 13

3

PAUL SALTMAN

INNOVATIVE IDEAS Getting to Know the Man

It’s not hard to guess what we as students do best: we learn. Equipped with caffeine jitters and notebooks, we go to class, take notes, and spend wee hours of the night poring over structures and mechanisms. Our mastery of this skill set is what has made us so successful in this textbook jungle. It’s how we got into UC San Diego, and it has kept us afloat when our course responsibilities pile up. However, it’s not often that these roles are reversed, and the student becomes the teacher. Only when we find ourselves responsible for the education of our peers do we begin to understand all that teaching requires: self-confidence, thorough knowledge of the material, and the ability to effectively communicate that knowledge to others.

These skills are tremendously important for our future careers, and no one understood that more than Dr. Paul Saltman.

4

SALTMAN QUARTERLY

VOL. 13

Dr. Paul Saltman began his teaching career at the University of Southern California, but it was at UC San Diego where he made the biggest impact. Although his research was largely dedicated to scientific understanding of nutrition, his true ambition was to share his love of science with anyone who was willing to listen and learn. He advocated for the involvement of undergraduate students in the classroom as Instructional Assistants (IAs) and in the laboratory as researchers at a time when such ideas were not as prevalent. Dr. Saltman continually challenged not only his students, but also the institution of higher learning to become “purveyors against mediocrity” and to continually strive for academic excellence. Saltman Quarterly is as much a tribute to him as it is a celebration of the accomplishments of our peers.

When I researched more about our journal’s namesake in the Mandeville Collection at Geisel Library, it became blatantly obvious to me that Dr. Saltman was not only an incredible scientist, he was also outspoken with his opinions. I fumbled through heaps of biochemical research alongside social commentaries on the role of the university and interviews about educating San Diego’s communities. During this time, I discovered one underlying theme from the plethora of Dr. Saltman’s works: the importance of teaching.

A self-proclaimed “teaching junkie,” Dr. Saltman was more than qualified to comment on the topic. During his time as an educator, he received the Excellence in Teaching award from both USC and from UC San Diego’s Revelle, Muir, Warren, and Marshall Colleges. He believed that good teachers “[...]convey their delight of learning, their job of discovery, their wealth of knowledge, and their love of life.” The simple relaying of information from an instructor to the student audience is not enough. One frank quote from Dr. Saltman describes his attitude towards this concept: “If you say that teaching is the transmission of facts and knowledge, I say we shoot all of the teachers today; they’re useless.”

Students as Teachers Dr. Saltman not only recognized how important it was for students to have great teachers in their lives, but also how necessary it was for students to have the opportunity to become great teachers, themselves.

With this in mind, he was a pivotal character in the implementation of undergraduate biology students as Instructional Assistants (IAs).

With the influx of biology students enrolling in UC San Diego during the 1970s, there were simply not enough graduate students to fill IA positions for the upcoming school year. IAs are essential to the smooth functioning of a classroom as they allow students to seek assistance on a one-on-one basis — a luxury not afforded to classes with 500 students and one professor. Dr. Saltman proposed a solution to

sqonline.ucsd.edu

With time comes confidence. As the quarter progressed, Harvey this issue. He believed that undergraduates who had shown exemplary found himself truly enjoying teaching. “It’s a lot of fun, and you learn skill in certain courses possessed the knowledge and charisma necso much,” Harvey notes, “and if you like what you are teaching, you essary to educate their peers. Not only would this benefit the biology basically get an hour per week to talk about what you love.” Saadi, student body as a whole, but it would allow the student to step into the Harvey, and Kuwamoto not only gained a thorough knowledge of role of teacher. On this matter, Dr. Saltman stated, “not only do the the material, they learned it from a different perspective and became TAs become knowledgeable about the strategies and tactics of good stronger, more confident public speakers. Additionally, Kuwamoto teaching, but they also learn, by teaching, of their own limitations and comments that some knowledge gained from this teaching experience have options to grow and become strong.” lasts a lifetime: “I will forever know what a phytochemical is, and it Since then, many UC San Diego undergraduate students have exdoesn’t get any better than that.” perienced the challenges and benefits associated with teaching their As undergraduates, the opportunity to peers. As an “A” student, Seth Harvey hone these skills would not have been posthought it would be easy to step into his role as a teacher. However, after He believed that good teachers sible without Dr. Saltman’s intervention. His his first lecture as an IA, he realized unrelenting faith in our capabilities as stu“[...] convey their delight of there is a tremendous difference bedents and his challenge for us to take charge learning, their job of discovery, of our futures is immortalized in the use of tween being the student jotting down undergraduate students as IAs. In the world notes and being the person providing their wealth of knowledge, of academia, Dr. Saltman realized it is easy the material; teaching the material and their love of life.” to be sheep. It is effortless to follow the flock, required a much more intimate re- Dr. Paul Saltman but not as simple to be the one to guide it. lationship with the information proAnd instead of allowing the student body to vided. On the first day, Harvey felt enter the world unprepared, he armed students with the tools of con“unprepared, unprofessional, and flustered” in front of his peers, and was afraid of not knowing enough of the information he needed to fidence and determination necessary to navigate themselves outside communicate. Tanzim Saadi shared this same experience, claiming, of a college campus. With this newly established skill set, we are ready “If I was my own student, I probably wouldn’t have shown up again.” to stand in front of our peers, clear our throats, and become living Tyler Kuwamoto, who experienced the same shaky start, realized that embodiments of Dr. Saltman’s ideals. Let’s just hope we can get our it’s okay to not be an expert. Regarding his section and about the diffistudents as excited about glycolysis as he was able to. culties with answering tough questions, Kuwamoto says, “I may have tried to give a somewhat correct answer from the archives of my noggin but after awhile […] the students realize you’re an undergraduate WRITTEN BY KATRINA FORREST too. You look up the information, you get back to them, everybody Katrina is a Microbiology Biology major from John Muir College.

She will graduate in 2016. is happy.”

Saltman Quarterly would like to especially thank the Saltman family for its generosity and support. Its contributions have allowed us 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 our surrounding communities.

sq.ucsd.edu

SALTMAN QUARTERLY

VOL. 13

5

A red-tailed monkey, Cercopithecus ascanius, at the San Diego Zoo. Photo by Megan Lao

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 ground-breaking work accomplished by researchers affiliated with the UC San Diego campus.

FEATURES

DECODING THE NETWORK OF DISEASE

Neal Shah Staff Writer

Illustration by April Damon

FEATURE

One of the most important consequences of the Human Genome project was the development of a new field called systems biology — the study of how individual biological components interact within a larger system, such as the cell, and how these interactions change during the course of disease. At UC San Diego, Dr. Trey Ideker uses a computational, systems biology approach to elucidate protein networks that are better indicators of metastatic cancers than just a single molecule. The implications of this research can help characterize metastatic versus indolent cancers, and network-based biomarkers as a whole can potentially achieve a higher level of accuracy in disease diagnosis.

I

n 2003, the completion of the Human Genome Project spurred the development of a new approach towards biology that seeks to understands how proteins and the genome, an organism’s complete set of DNA, interact with each other within the living cell.

This newly developed field, called systems biology, synthesizes computational and quantitative approaches, such as constructing intricate biological webs, protein-protein interaction networks, signaling networks, and epigenetic regulatory networks, to understand how the individual parts of a biological system interact and relate with each other. Thus, instead of understanding how genes and proteins function in isolated conditions, systems biology focuses on the complex interactions between genes, proteins, and the environment within the actual cell, and how these networks change during the course of disease.

At UC San Diego, many systems biologists are trying to decipher the “wiring” of a cell (the biological interactions that give a cell its functionality), and what goes wrong when the cell is damaged. Dr. Trey Ideker, Director of the San Diego Center for Systems Biology and Professor of Medicine at UC San Diego, addresses these fundamental questions by modeling molecular interaction networks in cells and observing how they change during pathogenesis.

es, nor for more complex changes within a certain disease, such as predicting whether a cancer is metastatic. The problem with detecting a single molecular biomarker by just looking at changes in gene expression or by seeking a specific molecule is that this approach doesn’t paint the whole picture of what’s happening in the cell. For instance, a seemingly insignificant change in gene expression for a single protein may be overlooked, when, in actuality, the larger downstream pathway involving this single protein could vary greatly from patient to patient.

“We need to move on from looking at a single gene as a catch-all to see if a patient has a good outcome or a bad outcome,” says Dr. Ideker, “it’s not a single gene, but a circuit, in the cell.” With this principle in mind, the idea that the components within a cell are intricately interconnected like wires in a circuit, Dr. Ideker employs a systems bi-

Classical Biology Detailed pathways

Research in the Ideker Lab A common way of assessing disease onset is to look for specific biological markers, called “biomarkers.” Within the context of disease, a molecular biomarker is a biological substance, such as a genetic sequence, protein, or hormone, that is indicative of a certain disease or disease outcome. Traditionally, researchers look at gene or mRNA expression patterns in diseased cells in comparison to healthy cells and aim to locate a biomarker that will signal the onset or predict the severity of a particular disease.

Though successful for some diseases, such as the discovery of the HER2/neu oncogene as a biomarker for certain breast cancers, the single molecule approach to biomarker discovery is not highly accurate for many types of diseassq.ucsd.edu

Small-scale modeling

Prediction

Hypothesis

Single-molecule experiments Genome-wide experiments

Prediction Abstract networks

Measurements

Network inference Network integration

Systems Biology

Figure 1 Classical biological discourse versus systems biology discourse. Systems biology focuses on larger network interactions between biological components in a system.

SALTMAN QUARTERLY

VOL. 13

9

Figure 2 Computational representation of network-based biomarker, showing the protein-protein interactions falling on a common pathway.

ology approach to biomarker identification, interpreting profiles of patients suffering from metastatic breast cancer, the Ideker lab identified various mutations and expression large sets of intracellular data to improve disease diagnochanges and analyzed them to see if the resulting mutated sis, particularly cancer metastasis. A metastasized cancer or overexpressed proteins were part of common pathways. refers to the spread of the cancer to different locations By using this data in conjunction throughout the body, and often with established protein interacbecomes much harder to treat therapeutically. By seeking to map “We need to move on from tion networks of tumor cells, he subnetworks of larger out more accurate biomarkers, looking at a single gene as a identified signaling pathways in metastatDr. Ideker hopes to better predict catch-all to see if a patient ic breast cancer cells versus the the onset of particular cancers. indolent, non-metastatic breast has a good outcome or cancer cells. These subnetworks Methods a bad outcome,” says Dr. served as the network-based bioInstead of focusing on finding Ideker, “it’s not a single gene, markers, and his approach showed a higher diagnostic accuracy than a single protein or genomic inbut a circuit, in the cell.” the traditional single-gene/protein dicator which would constitute markers. The data suggest that a a biomarker, he analyzes gene systems biology approach to medexpression networks and proicine may be able to more accurately diagnose certain distein-protein interaction data to find “network-based bioeases at the molecular level than the traditional approach. markers.” Network-based biomarkers encompass different networks and expression patterns that better reflect both the molecular and interactional changes undergoing in a Further Steps metastasized tumor. For instance, tumors may not have the same mutations, but could have those different muAlready showing promising results, Dr. Ideker’s research tations fall in the same protein networks. Therefore, it has much potential to directly reach patients suffering may be more efficient to search for what pathways are from various diseases beyond just cancer. Though the altered in cancers rather than what specific mutations network-based biomarkers are currently being applied to arise. To elucidate these biomarkers, Dr. Ideker overlays study metastatic cancers, he is working closely with otha cancer patient’s gene expression profile with published er biologists at UC San Diego to expand this approach to protein-protein interaction maps to search for signaling different diseases. pathways that are commonly perturbed, allowing him to identify pathways that may be predictive of a particular For instance, the Ideker lab, in conjunction with the Moores metastatic cancer.

Cancer Center, is also working on a project to improve diagnosis for aggressive versus indolent chronic lymphocytic Recently, he applied this methodology to find netleukemia, a common blood cancer in adults, and on anothwork-based biomarkers that would better diagnose meter project to identify the altered pathways that affect macuastatic breast cancer. By analyzing the gene expression lar degeneration and neuronal development.

10

SALTMAN QUARTERLY

VOL. 13

sqonline.ucsd.edu

A

B

NGRF

CAV1

SYT1

Conclusion

TAF1C TP53

E2F1

VEGF

PIN1 NGRF

JUN

UBE2A

CHUK

FGF5

AHR ESR1

YWHAZ

EGF

TP53

C PTK2B

RAD51 TEP1

VEGF

RPL5

SSTR3

JAK2 ERBB2

UBE2A

VEGF8

F

SERPINA3

RN111

USP4 ERBB4

DAB2 CDC27

D MAP2K5

HESX1

RIT1

SMARCAS

HABP2 E2F1 COL4A4

DCTN2 SMAD3

SMAD2

E

ONECUT1

COL4A2

G

SHC1

BRCA1

IFRD1 E2F5

I

E2F4

SMAD4

NRAS SOS1 EWSR1

DAB2 RASL12

H

AKAP13

RACGAP1

PZP

PIK3CA

BAD

PZP

CXCL12

J

CCL2

K BNMT3A

NFYC USP4

NMI

PSMC6

ALK INPPL1

TFAP2B

TP73 TUBA8

This research with a therapy-based focus, like that of Dr. Ideker’s, is a promising step towards personalized medicine, where each patient is treated uniquely according to his or her conditions. A downstream application of this research could be large-scale analysis of gene and mRNA expression profiles and interaction networks varying from patient to patient, which could lead to personalized disease prognosis and targeted therapy. Given Dr. Ideker’s early results, a highly accurate means to identify disease at the molecular level, before the onset of physical symptoms, may not just be a distant goal in the future, but an imminent reality.

SHC1 KRAS

References

FRS2

ARHGEF7

NRAS PIK3CA

Figure 3 Network-based biomarkers of cancer’s common traits.

Nodes and spokes represent protein-protein interactions, and the color of each node corresponds to the color scale, which depicts the gene’s expression pattern in metastatic vs. non-metastatic cancer.

Furthermore, Dr. Ideker is the co-director of a large, ambitious research endeavor by faculty from UC San Diego and UC San Francisco, called the “Cancer Cell Map Initiative”, to decipher how a cancer cell is wired, and how all of its components interact with one another. “Cancer is a disease of pathways and networks,” said Dr. Ideker, and “the key idea of systems biology is to know how genes are linked in larger biological systems.” Though we currently have limitless biological data on different types of mutations present in cancers, it is necessary to connect all this genomic data by understanding how it relates to one another and how they interact. This initiative seeks to finally draw out the wiring of the cancer cell, mapping the circuits of cancer. It is a potentially revolutionary new project which can provide novel insight into cancer progression previously not understood.

sq.ucsd.edu

Modern biology has been revolutionized by the advent of genomic technologies, that continue to surpass expectations. We are at a stage now where we can generate data at an immensely large scale, but the limiting factor is our ability to interpret the data into something of significance.

Systems biology integrates these vast amounts of genomic, mRNA, or protein data and seeks to construct complex models to understand how each part of the cell interacts within the larger system.

GHR

MYC FOS

PSMC6 CDC25A

RABAC1

IL17A IL17RA

GSTT1

STAT5A

PDGFRB

SHC1

HRAS

PRCC

TFAP2B

MYC

CYP1A1

TP73

PZP

CXCL13

BRCA1 ESR1

GOLT1B

MMP9

IL8RB

CAV1

RPS25

CDH5

VTN

FEATURE

1.Chuang, Han-Yu et al. “Network-Based Classification of Breast Cancer Metastasis.” Molecular Systems Biology 3 (2007): 140. PMC. Web. 5 Apr. 2016.

2. Chuang, Han-Yu, Matan Hofree, and Trey Ideker. “A Decade of Systems Biology.” Annual Review of Cell and Developmental Biology 26 (2010): 721–744. PMC. Web. 5 Apr. 2016.

3. LaFee, Scott. “UC San Diego, UC San Francisco Launch New Cancer Cell Mapping Initiative.” UC San Diego Health. 29 January 2015

WRITTEN BY NEAL SHAH Neal is a Molecular Biology major from Thurgood Marshall College. He will graduate in 2018.

SALTMAN QUARTERLY

VOL. 13

11

GLOBAL ALLIANCE AGAINST ANTIVIRAL RESISTANCE

Karen To Staff Writer

Illustration by Connie Mach

FEATURE

Viruses rapidly multiply and acquire mutations that may increase their resistance to current drug treatments. The swift adaptations of viruses is one of the main reasons why antiviral resistance has become a rising global health issue. The World Health Organization reports that only 33 out of the 133 countries it has surveyed has implemented comprehensive programs to combat the issue. Researchers like Dr. Elina Zuniga at UC San Diego are currently searching for solutions by analyzing methods that viruses use to subvert the host’s immune system.

Through new discoveries in the laboratory, novel treatments can hopefully be developed to effectively target viruses.

V

iruses may be small, but their rapid reproOf the patients confirmed for the flu who had antibiotductive ability and high mutation rates make ic records on hand, 30 percent were prescribed one of them difficult to combat. They easily acquire three common antibiotics while only 16 percent were resistance to antiviral drugs, which is an isprescribed antiviral drugs. This low percentage of antivisue that researchers hope to solve ral prescriptions indicates that cliniby studying their complex adaptaare not effectively distinguish“Research is an endeavor cians tions. Viruses succeed in evading ing the use of antiviral drugs from that is accomplished by host immune systems due to ranthat of antibiotics. Treating viral indom mutations that enable them to fections with antibiotics, which have the combined effort of outcompete other viruses through no effect on such maladies, would many scientists at different only worsen the drug resistance crinatural selection. For example, mutations in the protein coating of the laboratories, so I’m satisfied sis. As a consequence of improperly influenza virus lead to new strains antibiotics, treatment with the idea that our studies administering that subvert current flu vaccines. As resistance could potentially build up are helping to pave the a consequence, the influenza virus against bacteria while viruses remain continues to persist, killing around way towards the common unaffected and continue to multiply 250,000 to 500,000 people each year. and mutate. Researchers are contingoal of for improving With the combined efforts of the uously developing new medications World Health Organization (WHO) to rectify the threat that viruses pose, immune responses which stresses the need for educatand the Centers for Disease Control against viral infections.” ing medical personnel on the proper and Prevention (CDC), we can raise social awareness and support reuse of both types of medication for -Dr. Elina Zuniga searchers such as Dr. Elina Zuniga. the improvement of healthcare.

Dr. Zuniga directs a research laboratory at UC San Diego focused on illuminating new viral pathways to improve Virology Immunology Research antiviral treatments. at UC San Diego

Past Studies Reveal the Threat of Antiviral Resistance To analyze the issue of antiviral resistance on a global scale, the WHO surveyed 133 countries in 2013 through 2014. They discovered that only 34 of the numerous countries have implemented comprehensive plans to tackle the issue of drug resistance. Without the implementation of concrete guidelines, the misuse of antiviral drugs in these countries will persist. A study performed during the 2012-2013 flu season by the CDC, provides insight into this phenomenon. It revealed that clinicians were under-prescribing antiviral drugs and may have over-prescribed antibiotics to treat patients infected by influenza.

sq.ucsd.edu

Working to combat antiviral drug resistance, Dr. Elina Zuniga cites statistical data on the 500 million people afflicted with chronic hepatitis B and C (HBV and HCV) as the drive behind the research she conducts. She oversees a laboratory at UC San Diego that studies how the mammalian immune system responds to chronic viral infections in an effort to elucidate viral pathways that can be targeted in treatments. She recognizes that “the immune system is so powerful, but viruses evolve rapidly.”

Aware of the arms race between the mammalian immune system and rapidly evolving viruses, Professor Zuniga is confident that “[as] researchers, we can rationalize new interventions to be able to cope with the fast evolution of microbes that allow them to evade the immune system.”

SALTMAN QUARTERLY

VOL. 13

13

Figure 1 Type 1 interferon (IFN-1) production by white blood cells (macrophages, conventional dendritic cells, and plasmacytoid dendritic cells) drops rapidly during chronic stages of infection by persistent viruses such as lymphocytic choriomeningitis virus (LCMV) in mice, and human immunodeficiency virus (HIV).

Dr. Zuniga approaches her research with three main questions on virus-host interactions. The first question focuses on how the host immune system keeps viruses in check while avoiding excessive immune responses that could end in the host’s death. The second question is focused on elucidating mechanisms that proliferating viruses use to suppress immune responses. The third question centers on how the lab can incorporate their newfound discoveries into tackling chronic autoimmune diseases, such as systemic lupus erythematosus, in which the body’s immune system attacks healthy tissue, causing inflammation of skin, joints, and organs.

Ongoing Research of the Zuniga Lab Currently, Professor Zuniga’s lab is evaluating immune responses in mice to continually replicating viruses, such as lymphocytic choriomeningitis virus (LCMV). Collaborative research is done with other laboratories focusing on other viruses, such as human immunodeficiency virus (HIV) in humans and simian immunodeficiency virus (SIV) in primates, to confirm conservation of pathways mapped out in the mice. The Zuniga lab investigates both the adaptive and innate branches of the mammalian immune system. Innate immunity responds to a broad range of foreign substances, or antigens that present themselves in the body. Adaptive immunity, on the other hand, targets specific pathogens and involves the secretion of antibodies that bind to antigens, marking them as foreign.

To discern how viruses affect innate immune responses, production of type 1 interferons (IFN-1) were measured during the early and late stages of chronic LCMV infection. Interferons, which are named for their ability to inhibit viral replication, are secreted by white blood cells 14

SALTMAN QUARTERLY

VOL. 13

(leukocytes) that aid both immune systems. The researchers in the Zuniga group focused on a protein from LCMV in mice, which Professor Zuniga states, “is able to suppress IFN-1 — an occurrence that we are trying to understand by interacting with different host proteins.” The researchers noted a sudden drop of IFN-1 production between the early and late stages of chronic infection. The difference in IFN-1 production is speculated to be due to viral suppression of the innate pathways. While IFN-1 secretions declined, other innate responses during chronic infection become heightened. In chronic HCV infection in humans, a type of leukocyte called macrophages become hyper-responsive. This leads to the severe liver inflammation that is characteristic of this infection.

Host Immune System Adapts to Coexist with Persistent Viruses Notably, a fascinating immune response that the Zuniga group observed in their research on chronic LCMV infection in mice is the exhaustion of CD8+ T cells. T cells (or T lymphocytes) are a type of leukocyte that work alongside another leukocyte subtype known as B lymphocytes in the adaptive immune system. When a specific pathogen is detected, B and T cells are stimulated to replicate and form potent memory cells, which are a hallmark of acquired immunity. These memory cells lead to a faster immune response and increased antibody concentrations upon re-encountering the same pathogen. As such, when CD8+ T cell responses are robust, they offer protection against disease in patients infected by HIV, HCV and HBV. However, during the study, the researchers noticed that CD8+ T cell responses were ineffective in chronic viral infections. They described the lack of response shown by CD8+ T cells as exhaustion because some functions still operate to keep the sqonline.ucsd.edu

FEATURE

virus infection in check. However, the CD8+ T cells weren’t working at their maximal potential.

There appears to be a similar pattern of exhaustion in cells of the innate immune system as well, according to studies performed by the Zuniga laboratory. This hypo-responsiveness was particularly seen in innate cells that are specialized to produce massive amounts of antiviral interferons. With this new insight, Professor Zuniga notes that “all the cells of the innate immune system are somehow being modulated and maybe recalibrated or re-educated to learn how to live with these lifelong passengers that are persistent viruses.” Because continuous antigen secretion is observed in both chronic infections and tumors, the Zuniga group believes that a better understanding of how immune cells adapt to severe viral infections can elucidate new methods to improve anti-tumor immunity. A further look into the complex mechanisms of T cells and the phenomenon of their exhaustion may help researchers achieve an endeavor of this scale.

Figure 3 The lymphatic system acts as a transportation highway that feeds B and T lymphocytes into the body while filtering out dead cells and pathogens.

The Vision of the Zuniga Lab The study holds many promises, but the race against the rapid evolution of viruses and their evasive methods is still ongoing. In the future, Professor Zuniga hopes, “that [her laboratory] can contribute to uncovering pathways that can be targeted to improve immune responses against viral infections and improve the lives of people.” However, in tackling a global issue such as antiviral resistance, Professor Zuniga stresses the importance of the contributions made by other researchers worldwide. “Research is an endeavor that is accomplished by many people at different labs, so

I’m also satisfied with the idea that our contribution may just pave the way for future research to improve immune responses against viral infections.” Whichever route the researchers at the Zuniga laboratory decide to embark on next, their past, present, and future contributions are vital in the ongoing battle against antiviral resistance.

References 1. Havers, Fiona, Swathi Thaker, Jessie R. Clippard, Michael Jackson, Huong Q. McLean, Manjusha Gaglani, Arnold S. Monto, Richard K. Zimmerman, Lisa Jackson, Josh G. Petrie, Mary Patricia Nowalk, Krissy K. Moehling, Brendan Flannery, Mark G. Thompson, and Alicia M. Fry. “Clinical Infectious Diseases.” Use of Influenza Antiviral Agents by Ambulatory Care Clinicians During the 2012–2013 Influenza Season. N.p., 16 July 2014. Web. 18 Apr. 2016.

2. World Health Organization. “Worldwide country situation analysis: response to antimicrobial resistance.” (2015).

Figure 2 The mammalian immune system is divided into two branches. Innate immunity protects against a broad range of pathogens. Adaptive immunity targets antigens to neutralize specific pathogens.

sq.ucsd.edu

3. Zuniga, Elina I., Monica Macal, Gavin M. Lewis, and James A. Harker. “Innate and Adaptive Immune Regulation During Chronic Viral Infections.” - Annual Review of Virology, 2(1):573. N.p., 2 Sept. 2015. Web. 18 Apr. 2016.

WRITTEN BY KAREN TO Karen is a Physiology and Neuroscience major from Roger Revelle College. She will graduate in 2017.

SALTMAN QUARTERLY

VOL. 13

15

WALTZING OUR WAY TO CREATIVITY AND COGNITION

Anika Ullah Staff Writer

Illustration by Grace Lo

FEATURE

With today’s increasingly innovative advances in research, individuals such as biologists, artists, physicists, engineers, dancers, cognitive scientists, and dreamers in each and every field of academia strive to understand phenomena in their realms of expertise. What if these experts in diverse fields convened to synthesize their knowledge towards the exploration of a universal question?

What drives creativity and imagination in the human body?

At University of California, San Diego, Dr. David Kirsh at the Arthur C. Clarke Center for Human Imagination is one researcher who traversed into the fields of dance, psychology, and cognitive science in order to investigate the relationship between the human body, cognition, and creativity.

Could using the physical body be a key to unlocking a normally inaccessible realm of our minds?

U

niversity of California, San Diego: a well esthe theory of embodied cognition, which is the idea that teemed public research institution characterwe think with our bodies and not just our brains. It is well ized by an iconic spaceship-structured library, established in the field of neuroscience that motor resoa plethora of modernist architecture, and an nance — perhaps more widely recognized as muscle memunmistakable tilted house perched precariously atop the ory — plays an undeniably monumental role in learning roof of the famed Jacobs School of Engineering building. and performing certain movements. Everyone knows the However, the unconventional architecture at UC San Dipervasive idiom about practice: “practice makes perfect,” ego isn’t the only thing that appears to have warped its not “sitting and thinking about it makes perfect.” But is way into the present — our cutthat all there is to so-called perThe physical anchor is crucial fection? Kirsh hypothesizes that ting-edge interdisciplinary research tackles themes that are “bodies are involved in cognition in enabling people to travel seemingly light years ahead of us. in more ways than motor resobetween the realms Specifically in the universe of cognance and common coding” and nitive neuroscience, UC San Diego suggests that the body can actually of imagination and reality ranks first in the nation and perbe viewed as a tool that our minds with ease and to manifest sists to be at the forefront of innomay use to conjure and imagsuch novel possibilities vative cognitive science research. ine novel possibilities of a task at One lab at UC San Diego that purhand. The fundamental biological in their actions. sues this unique interdisciplinary processes that constitute this pheapproach is the Imaginarium Lab at the Arthur C. Clarke nomenon of motor resonance help explain how the body Center for Human Imagination. The Clarke Center is can be used as a tool to unlock an increased and varied comprised of researchers from the fields of biology, visual form of imagination. arts, physics, dance, neuroscience, literature, engineering, and medicine, who have convened to work together and Kirsh’s study involved teaching 10 dancers a short dance understand the neuroscience behind human imagination routine for 10 minutes, then having each dancer return to and creativity. the studio to perform the dance for evaluation of individual baseline performance. Afterward, dancers practiced By synthesizing viewpoints and knowledge from a variety independently for 10 minutes in one of three randomly asof disciplines, the Clarke Center tackles the supposedly signed conditions: ‘full-out’ [complete physical repetition intractable question of what triggers creativity and innoof the dance], ‘marking’ [hand motions and other halfvation in the human mind, and explores how these pheformed gestures that inaccurately simulated parts of the nomena can be linked to aspects of human behavior such routine], and ‘simulate in the head’ (Figure 2). Each dancas kinematic movement and visual cues. Dr. David Kirsh er was then individually regraded. After the first round, is one researcher at the Imaginarium lab who decided to the dancers swapped practice conditions and were taught analyze the contingency of imagination and learning on a second phase of the dance routine that was similar in duthe physical body through a study involving dance, psyration and complexity to the first, and the evaluation prochology, and cognitive science. Kirsh’s study is based on cess was repeated. Each dancer’s performance was graded

sq.ucsd.edu

SALTMAN QUARTERLY

VOL. 13

17

according to technique, memory, timing, and dynamics (ex. force, speed, emotionality, and intentionality), first by the teacher in real time and later by two independent experts who reviewed the video frame by frame. The results of the study were astounding: marking was the most effective method of practicing. Marking yielded maximum improvement in the categories of memory, technique, and timing. Furthermore, the study discovered that marking and full-out practice led to “substantially more learning than mental simulation across all dimensions,” and “in many cases practice by mental simulation led to a decrease in performance” (Figure 3).

So, what does it all mean? How could practicing movements that are essentially incorrect physical representations lead to a significantly better performance than practicing a complete and accurate rendition of a learned task? According to various neurophysiological studies, the answer lies in the capability of marking to stimulate the motor resonance system associated with a certain task, especially when the task has been performed previously in full.

Figure 1 Marking unlocks the imagination!

When a movement is performed, a lasting association between the motor and sensory neurons involved in the movement is left behind. According to Prinz’s common coding theory, which delineates that there is a “common code” or “language” used by sensory and motor neurons to couple perception and action together, one can access these sets of associated neurons by perceiving an action or partaking in it. Neurophysiological evidence for this theory lies in the discovery of the activation of mirror neuron

regions “in the ventral premotor cortex, dorsal premotor cortex, and intraparietal cortex of humans when watching an individual perform.” Therefore, the phenomenon of marking aligns with this theory in how a person is able to imagine and call upon a perceptual experience and its related neuronal memory with the crutch of their physical, half formed movements.

Full-out

Marking

Simulate in the Head

Figure 2 Experimental conditions. Subjects practiced mastering a phase in one of three conditions. they marked the phase, practiced it full out, or lay on their back and mentally simulated dancing the phrase. After being evaluated they had a five-minute rest, changed conditions, and were then taught a new phrase. In this way all subjects practiced in each condition.

18

SALTMAN QUARTERLY

VOL. 13

sqonline.ucsd.edu

So then why is marking more effective than full-out practice if both activate the same set of commonly coded neurons? According to Kirsh’s study, it is because the act of marking requires the dancer to imagine their half formed, inaccurate gestures as ideal movements. This partially imagined nature of marking creates room for the imagination of alternate possibilities that have never been experienced, which results in beneficial change in that common code. When marking, the dancers were found to be thinking of the key movements of the dance rather than the details associated with each movement. Since the dancers didn’t expend mental energy concerning themselves with the details of the sequence, they were able to free their imaginations to experiment with various renditions of the dance in their minds in order to improve their practice of the dance. By imagining and imposing these constructed alternate perceptual experiences unto one’s mind, one is able to revise the pre existing associations of the motor– sensory system and improve the performance of the dance itself, as depicted in the results of the study.

However, it is important to remember that just imagination alone cannot accomplish this feat— one must have some kind of physical anchor to support the process of imposing the imagined possibility unto the previously created tangible experience. In Kirsh’s study, dancers were able to tether themselves to reality by leaving the basic structure of the dance movements to muscle memory through marking and thus focus their mental energy on improving imagined renditions of the dance. This phenomenon of using a physical anchor to “interactively sketch” across dimensions can additionally be noted in fields apart from dance—musicians often use foot tapping to anchor themselves to the basic structure of a song while allowing their minds to imagine various harmonies and improvised variations of the song at hand. With the aid of foot-tapping, the musician is then empowered to implement their imagined improvisation unto the song and effectively change the actual performance of the song. Thus, the anchor is crucial in enabling a person to travel between the realms of imagination and reality with ease and to manifest such novel imagined possibilities in their actions. Essentially, we can leave the basic structure of a task to our bodies and rely on our imaginations to improve the details!

The primary discovery of Kirsh’s study is that using bodies to work “across modalities can reshape the conceptualization of something (e.g., a dance phrase) beyond its origins in one modality. [...] Bodies can extend the range of thought by leading us to new ideas that are far from the sensory-specific ways we encountered these ideas originally,” whereas passively perceiving denies the opportunity for imagination. Kirsh’s exploration of the relationship between mind, body, and cognition bears universal relevance,

sq.ucsd.edu

FEATURE

Figure 3 Table shows the mean improvement from practice (the learning delta) as measured on a 5-point scale.

Improvement was best for marking, less for full-out and negative for mental simulation.

considering that his discoveries can be applied to realize deeper understanding of novel ways to inspire and utilize our own creativity in every realm of human life. Perhaps selective engagement with the body can be introduced to more scientifically minded fields of study, classroom settings, or even learning of everyday tasks to augment not only our imaginations but also reality.

References 1. Kirsh, David. “Embodied Cognition and the Magical Future of Interaction Design.” ACM Transactions on Computer Human Interaction 20.2 (2013): 1-34

WRITTEN BY ANIKA ULLAH Anika is a Human Biology and Visual Arts double major from Thurgood Marshall College. She will graduate in 2018.

SALTMAN QUARTERLY

VOL. 13

19

ECOLOGICAL MODELING: A NOVEL MEANS OF APPROACHING CORAL REEF PRESERVATION

Ishan Goyal Staff Writer

Illustration by April Damon

FEATURE

While globalization and industrial advances mark progress for mankind, it comes at a grave cost to the wellbeing of the environment, specifically marine ecosystems such as coral reefs.

As a target of countless human stressors such as pollution and overfishing, the health of coral reefs is rapidly depleting and becoming an increasing subject of concern. The Sandin Lab observes coral reefs using a three dimensional image mapping technique called photomosaics.

By building a repository of this spatial representation of the reef, the Sandin Lab can accurately track coral reef health and organism interactions in various locations over time.

T

he increasing rate of industrialization, coupled cies, coral reefs cover less than two percent of the ocean with the intensifying effects of global climate bottom. In addition, many drugs and treatments for dischange, present the pressing challenge of finding eases such as arthritis, human bacterial infections, viral efficient methods to preserve the environment. infections, and cancer are being developed from coral reef The National Oceanic and Atmospheric Administration animals. With the evident significance of coral reef ecosys(NOAA) released figures showing that approximately 1.4 tems, the Sandin Lab aims to directly focus their research billion pounds of pollution enter the ocean every year. regarding coral reef health on tangible problems in the Further adding to the breadth of this problem, humans ecological preservation community. have released over 83,000 unique synthetic industrial compounds into the environment in the past century alone.

Given their interest in tackling issues in the manageThese modern industrial pollutants include several highment industry, the Sandin Lab is tasked with addressing ly persistent and harmful compounds that are known to a wide array of problems. PhD student Brian Zglicynski contribute to the greenhouse effect, described the Sandin Lab as “a specincluding perfluorocarbons (teflon) trum of differently skilled individuals and polybrominated diphenlyethers Due to the vast biodiversity including theoretical ecologists and (flame retardants). Unfortunately, fish experts collaborating on multiple and medical potential a major repository for these global scales and aspects.� In addition, the of coral reef ecosystems, lab partners with various nonprofit pollutants is the ocean, making it essential to direct research efforts toorganizations, such as OneReef, to the Sandin Lab focuses wards the preservation of the biodithese organizations’ scientiftheir research on tangible utilize versity in marine ecosystems such as ic advisories in conservation agreeproblems to find new coral reefs. By observing and mapments. The lab also collaborates with ping the interactions of organisms government agencies like the NOAA methods of preserving within these ecosystems and the efto analyze their monitoring data. In this environment. fects of human stressors, researchers order to approach these quandaries attempt to test and validate reliable involving coral reef management, the indicators of coral reef health and establish clear metrics Sandin Lab focuses their research efforts upon collecting for coral reef management. detailed and interactive pictures of coral reef ecosystems called photomosaics. They then use these biological maps to analyze the spatial interactions between species to furMore about the Sandin Lab ther answer questions regarding the coral reef health, sustainability, and management.

The Sandin Lab of the Scripps Institute of Oceanography of UC San Diego is headed by Dr. Stuart Sandin and facilitates detailed studies of coral reef population dynamics and species interactions in order to develop accurate models for ecological conservation. The NOAA reports that coral reefs alone support more species per unit area than any other marine environment, including an average of about 4000 different species of fish, 800 species of hard corals, and millions of undiscovered species. Despite the fact that they foster such a large portion of oceanic spe-

sq.ucsd.edu

Understanding Ecological Interactions with Photomosaics In conjunction with their efforts to understand the influence of humans on the marine biodiversity, the Sandin Lab strives to look into how species at different trophic levels, or positions on the food chain, interact with each other. This research is related to the perplexing patterns of SALTMAN QUARTERLY

VOL. 13

21

Figure 1 A trophic diagram depicts the structural interactions between simple organisms, such as phytoplankton, and complex predators.

productivity that support predator-heavy reef communities. Thermodynamic constraints assure that a ‘topheavy’ trophic structure can only be supported through a ‘bottom-heavy’ pattern of productivity, with lower trophic level organisms growing more quickly than the apex predators. Understanding the competitive interactions between benthos organisms, the flora and fauna found near the sediment, allows researchers to build mathematical models of how a community will respond to external stimuli. By studying the relationships and levels of these organisms, the Sandin Lab hopes to test basic hypotheses relating assemblage of organisms to the overall biodiversity of the ecosystem.

One of the newer and upcoming technologies that the Sandin Lab strives to use with their research is photomosaics. Postdoctoral fellow Yoan Eynaud explains the

22

SALTMAN QUARTERLY

VOL. 13

concept of photomosaics as “taking countless high resolution two-dimensional images and using a software called Agisoft to process the images and generate a three-dimensional spatial dataset.” The software optimizes the model by using between 600-800 million points from the individual photos to create a comprehensive representation of the coral reef. Analyzing the benthos assemblage with languages such as R helps provide a statistical understanding of the spatial ecology amongst these organisms. By building a vast repository of these photomosaics at different locations, the Sandin Lab can track changes in the structure, health, and organism interactions in response to environmental stressors over time. The lab can ultimately construct comprehensive models using Python and Matlab that evaluate these changes and draw ties between how external conditions affect the coral reef on a holistic level.

sqonline.ucsd.edu

FEATURE

Unfished Atoll

Pacific Ocean

Northern Line Islands

Fished Atoll

Kingman Palnyra Teraina Tabuaeran Kiritimati

Equator Jarvis

Figure 2 The Sandin Lab collects data during expansive expeditions throughout the Northern Line Islands.

The Sandin Lab Expeditions In particular, the Sandin Lab studies islands and atolls (ring-shaped reefs) in the remote central Pacific to understand the impact of human industrialization on marine ecosystems. Selecting this area of uninhabited islands provides the unique opportunity to study the ecology of baseline reef ecosystems or reefs in the absence of local human stressors such as fishing and pollution. The Sandin Lab also observes the more densely populated Caribbean coral reefs to study how human activities can affect coral growth or be managed to improve coral growth and recovery. One of the lab’s more recently established goals is the 100 Island Challenge, which involves visiting at least five different sites on 100 unique islands to collect information about the various coral reef ecosystems.

The group’s most recently documented expedition in 2013 was to the Southern Line Islands, a series of uninhabited islands 1000 miles south of the Hawaiian archipelago.

The Sandin Lab had previously observed that, in general, reef fishery productivity and thus overall biodiversity was lower in systems that were heavily fished. This led them to primarily investigate how reef ecosystem productivity of the overall biomass varies across natural conditions.

Eynaud discusses the usefulness of the data gathered during these expeditions as a means of comparing and archiving how systems react over time to environmental stressors such as El Niño and human interactions.

Studies by the Sandin group in the Millennium Atoll, one of the few remaining untouched coral reef ecosystems in the world, further demonstrate the negative effects of human presence. Species that are heavily exploited throughout the Pacific and potentially classified as endangered,

sq.ucsd.edu

such as the blacktip reef shark and Napoleon wrasse, were found in great abundance in this environment. This stark difference in biodiversity was also observed amongst the flora and fauna of the islands themselves. This information indicates that the detrimental effects of human interactions isn’t limited to the coral reefs. For example, the Pisonia grandis, a flowering tree common to the natural archipelago surroundings, was replaced with nonnative coconut palms in many locations prone to human interactions. While the expeditions require an immense amount of forethought and training, the Sandin Lab has established a great means of periodically collecting data and tracking change across countless coral reef environments.

References 1. Wood, Chelsea L., et al. “Fishing drives declines in fish parasite diversity and has variable effects on parasite abundance.” Ecology 95.7 (2014): 1929-1946.

WRITTEN BY ISHAN GOYAL Ishan is a Bioinformatics major from Roger Revelle College.

He will graduate in 2018.

SALTMAN QUARTERLY

VOL. 13

23

Michelle Lee Staff Writer

Illustration by Grace Lo

PRETTY BUT FIERCE: PLANT EDITION

FEATURE

For a while, it has been a mystery of how plants are able to adapt to their environments so readily. Surviving for about 700 million years, in the hottest and coldest climates on Earth, plants have acquired many evolutionary adaptations, including a robust immune system. The molecular basis of plant immunity and its functions have puzzled many scientists, until this last century, when plant immunity research has blossomed. As researchers like Dr. Huffaker at UC San Diego continue to search for answers, many novel discoveries can be made, even those that can affect our daily lives and our distant future.

P

lants may be brainless, but they are much more perceptive than they appear; they are able to sense their surroundings, react by activating their immune responses, and signal other organisms to come protect them. Through evolutionary adaptations such as their immune systems, they have been able to survive on the planet for about 700 million years.

During this time, they have survived the hottest and coldest climates on Earth and the most dangerous environments known to “Through humankind (Anissimov).

known to resist disease, plants weren’t thought to be capable of activating a systemic immune response the way animals can. In the last couple decades, scientist Clarence Ryan from Washington State University demonstrated that plants are capable of systemic immune responses when attacked and that this required activity of a peptide hormone. Subsequently, plant researchers have discovered that plants are able to recognize specific chemicals from foreign organisms, such as bacteria, caterpillars, and destrucevolutionary tive microbes, so they can trigger adaptations such as their their immune responses and protect The abundance of plants allows them immune systems, [plants] themselves. With these discoveries, to be a reliable source for many humany scientists began to study the man needs and activities. Most have been able to survive sophisticated molecular mechanisms importantly, they are one of the prithat drive plant immunology. Ason the planet for about mary sources of food in our diet and sistant Professor Alisa Huffaker, in 700 million years.” livestock. The world’s food supply the Division of Biological Sciences - Dr. Alisa Huffaker comes from 150 species of plants that at UC San Diego, is currently studyare actively cultivated for food and ing the contributions of hormones cattle (“Facts & Figures on Food and Biodiversity.”). In addiscovered during her graduate work in Clarence Ryan’s dition, they also serve as raw materials for many pharmalaboratory, the Plant Elicitor Peptides (PEPs). ceuticals, and much of medical research relies on plants and plant extracts. The chemistry of plants allow us to Main Molecules Behind It All: PEPs have products such as coffee, alcohol, and other drugs like pseudoephedrine. Furthermore, plant-derived commodiPEPs are small protein hormones that help turn on and ties also play a big role in society; for example, products of amplify immune responses during attacks to limit incotton are prevalently used for clothing, and wood is used fections and mitigate the damages; however, the specific for lumber products and for shelter and warmth, particmolecular mechanisms by which they activate plant imularly in the rural areas of the world. Without plants, the munity is not yet well-understood. Dr. Huffaker’s research food web would disintegrate and our economy and health on this phenomenon may help to uncover how PEPs would be in danger. Hence, understanding plant immuregulate plant immunity and help develop new methnity and finding optimal ways for plants to defend themods to fight against pests and diseases plaguing crops, a selves can help with not only their survival, but also ours. serious problem that currently affects our global market.

Within the last century, scientists have discovered much about how plant immunity works. In the 1940s, Harold Henry Flor became the first scientist to study the integration of the host and parasite genetic systems (Raven). He demonstrated that plants could inherit resistance to certain microbes. Also known as the gene-for-gene model, this proposition led to the identification of many genes involved in plant immunity. Although some plants were

sq.ucsd.edu

All higher plants have families of PEP hormones that regulate their immune responses; some PEPs activate defenses that help protect against disease-causing pathogens, while others turn on responses that fight off pests such as caterpillars. As various PEPs appear to have differing functions, Dr. Huffaker studies the signaling and defense strategies mediated by individual PEPs and by exogenous elicitors, which are compounds originating

SALTMAN QUARTERLY

VOL. 13

25

Figure 1 Rough diagram of the timeline of plant evolution. Symbolic representations of plants that could have existed during each era. Plant evolution goes back to about 600 million years ago, during the pre-Cambrian era. Throughout the years, plants have acquired many adaptations.

from pathogens that also stimulate the plant defense system. Her goal is to decode PEP-induced response specificity and identify molecular components that can be used to enhance resistance against a variety of attackers, including pathogens and herbivores (“Huffaker Lab”).

A facet of her research is how PEPs coordinate plant interactions with other organisms in their environment.

For instance, one suite of responses regulated by PEPs occurs when caterpillars chew on plants. Upon herbivory, PEPs promote signaling in leaves that results in release of a complex volatile blend of chemicals with a fragrance that smells like perfume or flowers. Some chemicals in this scent act as an antiseptic to protect the wound site against potential microbial invaders. Others are perceived by neighboring plants in the vicinity as a warning signal to activate their own immune systems. Still others attract beneficial organisms in the community such as parasitoid wasps. Wasps that are recruited to the plant by the PEP-induced chemical blend act as plant bodyguards by laying their eggs inside the caterpillars, thus killing them.

Administering PEPs to plants as an immune-booster attracts wasps and also causes leaves to accumulate proteins and chemicals that directly limit caterpillar growth. Currently several members of her lab are researching newly discovered transcription factors and other proteins activated by PEPs to directly regulate expression of the genes required for these immune response (“Huffaker Lab”).

Huffaker’s lab analyzes these plant immune responses using a wide variety of bioassays, transcriptional profil-

26

SALTMAN QUARTERLY

VOL. 13

ing, gas chromatography and mass spectrometry. Recently in collaboration with the lab of another UC San Diego Biology faculty member, Dr. Steven Briggs, the Huffaker lab identified many new candidate proteins involved in signal transduction regulating plant immunity through comprehensive screening of proteins with rapidly altered phosphorylation patterns in response to PEPs. The Huffaker lab has discovered that plants with mutations in genes encoding many of these proteins display abnormal immune responses. Currently several members of her lab are researching the molecular mechanisms by which these newly identified signaling proteins are activating and fine-tuning plant immunity.

Significance of PEPS in Our World Just like us, plants can get very sick without a robust immune system, and there is a wide range of diseases that affect plants. Fungi are the largest group of pathogens can cause harm to plants by numerous mechanisms. Fungi cannot produce their own food through photosynthesis, so like us, many obtain nutrients from plants. Some fungi can actively penetrate plant tissue, while others opportunistically enter plants through wounds or are introduced by insects. Some fungi destroy so much leaf tissue that plants cannot photosynthesize to maintain their growth; they essentially starve and fail to reproduce or die (“Plant Pathology.”). Other microbial pathogens (both fungi and bacteria) invade the plant vascular system, disabling their ability to transport water and nutrients, thus killing the plant through dehydration. sqonline.ucsd.edu

FEATURE

As PEPs amplify immune responses against a variety of these invaders in all plants, we may be able to use them to help fight off these different diseases. PEP-activated innate immune responses help plants strengthen their cell wall, fortifying it to create a better barrier, and stimulate production of potent antimicrobial compounds.

One fungal pathogen that Dr. Huffaker aims to leverage PEP-induced immune responses against is Aspergillus flavus, which attacks corn plants. When the fungus colonizes ears of corn, it can produce dangerous toxins called aflatoxins that are harmful to both animals and people.

In the US, Aspergillus flavus infection causes economic losses to farmers that can’t sell their corn due to aflatoxin contamination. In developing countries, the impact is even more severe, as people still eat the contaminated corn due to shortages of food resources. Aflatoxins are lethal in large doses and long-term exposure is the number one cause of liver cancer worldwide. Furthermore, present-day weather conditions pose an even greater health risk: corn is especially susceptible to Aspergillus flavus during drought and heat waves, which are increasingly common due to global climate change (“Huffaker Lab”).

Improving plant resistance to this pathogen and others

Figure 3 Diagram of how PEPs function. Dr. Huffaker at UC San Diego is researching Pep-induced response specificity and the molecular components that can be used to enhance resistance against a variety of attackers.

is critical to maintaining a safe food supply, especially in developing countries. Dr. Huffaker’s research program is uncovering basic molecular mechanisms regulating plant immunity and resistance that will facilitate sustainable agriculture by helping minimize food shortage and contamination and curbing the use of pesticides and fungicides.

References 1. Anissimov, Michael, and Bronwyn Harris. “What Is the Evolutionary History of Plants?” WiseGeek. Conjecture, n.d. Web 2. “Facts & Figures on Food and Biodiversity.” Facts & Figures on Food and Biodiversity. IDRC Communications, n.d. Web. 13 Apr. 2016.

3. “Huffaker Lab.” Personal interview. 30 Nov. 2015.

4. “Plant Pathology.” CMG Garden Notes. Colorado State University Extension, n.d. Web. 5. Raven, Peter H., and George B. Johnson. Biology. Boston: McGraw-Hill, 2002. Print.

Figure 2 Aspergillus flavus infected corn. Aflatoxins, which are produced by infected corn, are lethal in large doses and long-term exposure. Currently, the number one cause of liver cancer worldwide, this fungus poses a great risk in underdeveloped countries.

sq.ucsd.edu

WRITTEN BY MICHELLE LEE Michelle is a Biochemistry/Cell Biology and Psychology double major from Thurgood Marshall College.

She will graduate in 2016.

SALTMAN QUARTERLY

VOL. 13

27

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 hands-on laboratory experience. Through original research manuscripts and review papers, the Research section showcases the current understanding of various fields in biology.

RESEARCH

An Ibis, Geronticus calvus, at the San Diego Zoo. Photo by Megan Lao

Effect of compost tea on growth of Lactuca sativa seedlings Brianna Egan| Advisors: Frank Joyce, Ph.D and Sofía Arce Flores, M.S. Compost tea (CT) is prepared by placing compost into water to allow the soluble microbes and nutrients to pass from solid compost (SC) into liquid. This study evaluated the effect of compost tea on lettuce (Lactuca sativa) growth compared to SC treatments and a control of water alone. I “brewed” six types of compost teas from three compost types (i) cattle manure-based vermicompost, (ii) goat manure and carbon-based thermophilic compost, and (iii) human manure-based thermophilic compost, and each for two different time periods, 24-hours and 72-hours. I applied the treatments every two days to a total of 96 potted lettuce plants. I then measured the leaf length, width, and leaf number every three days. I also analyzed the properties of the pre- and post-treatment soils and the liquid treatments. I found a significant effect of treatment type on leaf length and leaf width after 18 days of plant growth. Specifically, leaf length increase was significantly higher in plants with Goat CT (for both the 24-hour and 72-hour “brew” varieties) and control, than for the Goat SC counterparts. With the exception of one treatment, all treatments of SC and CT experienced greater leaf width increase than control, although these results were not significant. The components of the CT were indeed different from the water used for control plants: Potassium and phosphorus levels increased or remained constant in all post-treatment 72-hour CT soils, whereas these nutrients declined in control soil. Additionally, the electrical conductivity and total dissolved solids of all CTs were significantly higher than for water. Thus cations and other particles had effectively passed through from solids into liquids. The effect of treatments on plant growth was likely due to the repeated application of soil nutrients and beneficial microorganisms administered by CT.

Introduction

Soil is composed of a rich network of bacteria, fungi, nematodes, and other organisms. Pesticides, fertilizers, and other applications rob the soil of its nutrients and often decimate its microbiota. The process of creating compost from decomposed plant and animal waste ensures that nutrients and beneficial microorganisms can be captured as organic matter. Compost can be added to soil and to plant roots to encourage growth, deter pathogens, and improve the soil structure (Ingham 2000). Compost provides crop-growers with a way to grow plants without the use of agrochemicals.

Compost tea (CT) is a water-based extract of compost that is applied to plants as a soil drench and foliar spray. CT retains much of the beneficial microbiological and chemical components found in solid compost (SC). These factors include mineral nutrients that support increased growth, plant hormones such as humic and fulvic acids, and microorganisms such as bacteria and fungi (Edwards et al.

2006). After placing SC and water into a bucket or a commercial CT system, the resulting dark liquid is strained from the solids, diluted with water, and then applied to crops. High quality compost contains a large biomass and diversity of beneficial bacteria and fungi that, in turn, translates to high quality CT. 30

SALTMAN QUARTERLY

VOL. 13

When compared to SC, CT is easier to transport from far distances and can be more efficiently applied onto crops (Arancon et al. 2007). Additionally, CT may be advantageous in that it can be applied throughout the plant’s growth as a repeated biofertilizer and supplement, rather than just one time at the soil surface. However, CT does not store as well

as SC does: While SC remains stable and viable for months after maturation, CT may begin to populate with pathogens if stored for more than one month (Naidu et al. 2010). The possibility of producing pathogens can be mitigated by using high quality compost that does not carry pathogens and by maintaining aerobic conditions during the brewing process to Figure 1: A Latin Square design randomizes treatments to reduce variability introduced by placement in rows and columns. For my design, “A” was control, “B” was cattle manure compost, “C” was goat manure compost, and “D” was human manure compost. I had six total Latin Square plots; two plots for each treatment parameter (24-hour CT, 72-hour CT, SC).

sqonline.ucsd.edu

RESEARCH

Figure 2: Leaf length growth for goat compost treatments and control. On day 10 of growth, the mean leaf length of plants with 72-hour Goat CT was significantly greater than that of control plants (t30=-2.598, p=0.0144). On day 18, the mean leaf length of plants with 72-hour Goat CT and control were both significantly greater than that of plants with 1/3 Goat SC (t14=-5.030, p=0.0002 and t30=5.450, p<0.0001, respectively). ensure that pathogens do not develop (Ingham 2000).

In many studies, CT functions as a liquid supplement and fertilizer for cultivation: Pant et al. (2009) noted increased plant height, leaf area, and leaf number of pak choi plants treated with CT compared to control. Edwards et al. (2006) found significantly greater germination rates and leaf area growth on tomato plants treated with CT compared to control. Furthermore, CT enriches the microbial diversity of plant soil: Pant et al. (2009) and Arancon et al. (2007) both performed microbial analyses and found greater abundance and activity of bacterial and fungal populations in plants treated with CT than in those that were not. In one study, Pant et al. (2012) investigated several CT types from different compost sources—chicken manure-based vermicompost tea, chicken manure-based thermophilic tea, and food waste vermicompost tea—and demonstrated that tea quality and effectiveness can be predicted based on SC quality and CT microbial activity. As evidenced by past studies, CT can be made from a variety of composts (plant or animal waste, vermicompost or thermosq.ucsd.edu

philic), ratios of compost to water (from 1:3 to 1:200), brew time periods (from 12 hours to 3 weeks), and brew methods (aerated or non-aerated).

Many previous studies have focused on CT’s ability to suppress plant diseases, including Verticillium in tomato plants (Edwards et al. 2006) and powdery mildew in rose plants (Scheuerell and Mahaffee 2002). The beneficial microorganisms present in CT are thought to reduce disease by out-competing pathogenic microorganisms for resources on the plant leaves (Ingham 2000).

In all, there are few systematic studies on the benefits of CT on nutritional quality and growth of crops. Additionally, there are no studies assessing the effect of human waste-based compost tea on plant growth. My study seeks to address these gaps, posing the question: How will plant growth differ when treated with (i) compost tea versus control (water), and (ii) compost tea versus solid compost? I hypothesize that CT has a high concentration of nutrients and microorganisms that would benefit the growth of plants. I predict that CT will improve plant growth

due to its higher microbial activity and nutrient content compared to water. I also predict that CT will be more effective than SC on plant growth as it is added repeatedly to the soil, rather than one time.

Materials and Methods

Study Site and Setup My study was hosted at Life Monteverde sustainable coffee farm in Cañitas, Costa Rica from November 17th to December 6th, 2015. I used three different compost types to brew compost tea, with each compost type brewed for 24 hours and 72 hours for a total of six tea treatments. The three compost types were (i) cattle manure vermicompost from a dairy farm in La Cruz, Costa Rica, (ii) goat manure, carbon, and whey thermophilic compost mixture from Life Monteverde, and (iii) human waste and sawdust thermophilic compost from Life Monteverde’s composting toilet. Rainwater from a rainwater collection tank onsite was applied for control replicates and for solid compost replicates.

On November 17th, 2015 I obtained 100 Lactuca sativa lettuce seedlings from a hydroponic farm in Monteverde, Costa Rica.

SALTMAN QUARTERLY

VOL. 13

31

The seedlings were of uniform appearance, and were purchased and delivered from a nursery in San Jose, Costa Rica. I chose to cultivate lettuce as a model crop due to its fast growth time. On November 18th, 2015 I collected soil from a horticulture plot at Life Monteverde and placed the soil in planter bags. I used this soil to pot the seedlings undergoing CT treatments and those serving as control. I used the three compost types in a ratio of one-part compost to two-parts soil in bags for seedlings undergoing SC treatments.

Experimental Design I interspersed the seedling bags in a Latin Square design with two replicates of 4x4 plots for each of three treatment categories (24-hour CT, 72-hour CT, and 1:3 SC). I chose a Latin Square design to minimize the effect from confounding variables such as shade and wind that could form a gradient along the area. As such, in a total of six 4x4 plots, each CT and SC treatment had 8 replicates. Four control plants were present in each plot, with a total of 24 replicates of control (Figure 1). Five days after potting the seedlings, I moved all bags from a garden area outdoors and into a greenhouse to maximize light exposure and to avoid damage from wind and rain.

I brewed CT using the open-air Bucket Method (Ingham 2000): I placed one-part compost to two-parts rainwater in buckets for both 24-hour and 72-hour periods.

During these brew periods I stirred the CT brew every 4 hours during the daytime

to maintain aeration. After the brew was complete, I strained out the dark liquids using cotton pillowcases and dilute to 1:5 compost tea to rainwater. I then applied the treatments every two days to seedling bags as a foliar spray and a root drench. For the initial application of CT I used 24-hour CT for all seedlings. I created fresh brews of CT for all subsequent applications.

Data Analysis Every three days, I measured the number of leaves, the length of the longest leaf from end to tip, and the width of the widest leaf at the broadest part. Over 18 days of growth I had a total of five days of plant measurements (November 19th, November 22nd, November 25th, November 28th, and December 6th 2015). I used LaMotte colorimetric soil analysis kits to analyze pH and the soil macronutrients nitrogen, phosphorus, and potassium. I analyzed soil macronutrient levels for pre- and post-treatment soil samples from control plants and plants with 72-hour teas. I chose to sample soil from 72-hour tea treatments because longer brew times theoretically allow for populations of microorganisms to rise and for concentrations of macronutrients to be higher (Ingham 2000). The soil analysis kits measured nutrients in units of lb/acre, which I then converted to kg/ ha and g/m2 to make more applicable to the scale of my study. The kits measured at a precision of ±20-40 kg/ha or ±2-4 g/ m2. For liquid CT and rainwater analysis, I obtained a volume of at least 500 mL of

the liquid, and then used a YSI 5500 device and an ExStik meter to measure the pH, electrical conductivity, total dissolved solids, and dissolved oxygen in the liquids.

I collected all chemical waste at the Monteverde Institute to be disposed of at the University of Costa Rica.

I performed within subjects (or repeated measures) ANOVA analyses for leaf height, leaf width and leaf number measurements across treatments and days. I conducted post-hoc paired samples t-tests to compare certain treatments with the control. Finally, I used one-way ANOVA tests with Tukey HSD tests to analyze the significant differences between pH, electrical conductivity, total dissolved solids, and dissolved oxygen of teas.

Results

Plant Growth There was a significant effect of treatment type on leaf length (ANOVA Wilks’ Lambda=0.491 F36=1.81, p=0.004). Specifically, leaf lengths of 72-hour Goat CT plants (=10.26 cm, SD=1.89 cm) were significantly greater than those of control (=8.98 cm, SD=0.92 cm) on day 10 of growth (t30= -2.598, p=0.0144). On day 18 of growth, the mean leaf length of plants with 72-hour Goat CT (=14.36 cm, SD=1.43 cm) and control (=13.63 cm, SD=1.05 cm) were both significantly greater than that of plants with 1/3 Goat SC (=11.38 cm, SD=0.88 cm) (t14=-5.030, p=0.0002 and t30=5.450, p<0.0001, respec-

Figure 3a: Mean leaf length increase is defined as the increase in length from initial to final measurements (day 1 to day 18). Means that have no superscript in common are significantly different from each other (Tukey’s HSD, p<0.05).

There is a significant effect of treatment type on leaf length increase (ANOVA F9,86=3.7615, p=0.0005). Specifically, leaf length increase of plants with 1/3 Goat SC was significantly less than plants with 72-hour and 24-hour Goat CT and control plants (p<0.0001).

32

SALTMAN QUARTERLY

VOL. 13

sqonline.ucsd.edu

RESEARCH tively). This growth is illustrated by Figure 2, which compares the leaf lengths over 18 days of growth for plants with treatments of 72-hour Goat CT and 1/3 Goat SC, as well as control.

I calculated mean leaf increase from the difference between the final and initial leaf lengths. There was a significant relationship between treatment type and leaf length increase (ANOVA F9,86=3.7615, p=0.0005). Control plants experienced greater mean leaf length increases than did all SC treatments. Additionally, plants with 24-hour and 72-hour CTs resulted in greater leaf length increases than control, with the exception of 72-hour cattle CT, which was lower than control (Figure 3a). Leaf length increase of plants with 1/3 Goat SC (=5.25, SD=1.01) was significantly lower than with its counterpart plants to which 24-hour (=8.06, SD=1.42) and 72hour Goat CT (=8.34, SD=0.83) were applied, as well as with control plants (=7.66, SD=1.14) (Tukey HSD p<0.0001). The 1/3 Goat SC plants were also significantly lower in leaf length increase to 24-hour Cattle CT (=8.11, SD=0.89), 24-hour Human CT (=7.88, SD=0.78) and 72-hour Human CT plants (=7.83, SD=2.03) (Tukey HSD p<0.0001). Thus for the goat compost trials, SC resulted in shorter plants than control and both brew periods of CT. Mean leaf widths across treatments and days were also statistically significant (ANOVA Wilksâ&#x20AC;&#x2122; Lambda=0.502 F36=1.61, p=0.0180), although significant relation-

ships were among pairs that are not of particular interest, such as between 72-hour Goat CT and 1/3 Human SC. For mean leaf width increase, all 72-hour CTs experienced greater leaf width increases than did their 24-hour CT counterparts and their SC counterparts, with the exception of 72-hour Cattle CT, which had a lower increase than 24-hour Cattle CT (Figure 3b). All treatments (1/3 SC, 24-hour CT, 72-hour CT) experienced greater mean leaf width increases than did the control (=2.22, SD=0.82), with the exception of 24-hour Human CT, which was lower than control. However, treatments were not statistically different for leaf width increase.

Similarly, leaf number did not differ between treatments.

Plants treated with 72-hour Goat CT revealed the largest increase in leaf length growth, while plants treated with 72-hour Human CT revealed the largest increase in leaf width growth.

Soil Nutrients Initial, pre-treatment soil macronutrient levels were the same across all samples because they came from the same soil source (N=0 g/m2, P=8.41 g/m2, K=13.45 g/m2) (Figure 4). Nitrogen (N) levels remained at trace levels between initial and final soil samples for control and all 72hour CT samples. Phosphorus (P) levels decreased in final control samples (P=2.80 g/m2) and remained constant in final 72hour Human CT, but increased in all other final CT samples (P=14.01 g/m2). Potassi-

um (K) levels remained constant in control samples but increased in all 72-hour CT samples (K=22.42 g/m2).

Compost Tea Properties In comparing the liquid properties of rainwater and CT as shown in Table 1, the pHs of Cattle CTs and Human CTs were acidic while the pHs of Goat CTs were basic. Both the 24-hour and 72-hour Human CTs were significantly more acidic than the Goat CTs (ANOVA F(6,8)=4.874, p=0.022). All 24-hour and 72-hour CTs had significantly greater electrical conductivity (ANOVA F(6,8)=21.730, p=0.0002) and total dissolved solids (ANOVA F(6,8)=22.930, p=0.0001) than control and Cattle CTs. Dissolved oxygen levels did not have significant differences among treatments.

All 72-hour teas were darker in color than their 24-hour counterparts. Additionally, I provided samples of Goat and Cattle CT to fellow student researchers for their study on natural alternatives to fungicides. After 24 hours of incubation at 25oC on plates with potato dextrose agar, both CT types developed microbial colonies: The 55.4 cm2 area of the plates were fully covered in bacterial colonies, significantly more so than the growth on control plates (Figure 5).

Discussion

Plant Growth The treatments in my experiment were statistically significant: Leaf length and

Figure 3b: Mean leaf width increase is defined as the increase in width from initial to final measurements (day 1 to day 18). Means that have no superscript in common are significantly different from each other (Tukeyâ&#x20AC;&#x2122;s HSD, p<0.05).

There is no significant effect of treatment type on leaf width increase (ANOVA F(9,86)=0.8646, p=0.5598). It is interesting to note that, with the exception of plants with 24-hour Human CT, all treatments result in higher mean leaf width increases than control plants.

sq.ucsd.edu

SALTMAN QUARTERLY

VOL. 13

33

leaf width measurements increased with CT treatments compared to control. In terms of leaf length (Figure 2), 72-hour Goat CT was most effective, and was statistically significant from control on day 10 and from its 1/3 SC counterpart. This suggests that 72-hour Goat CT stimulates the growth of plants. The 72-hour Goat CT was likely more effective than the 24hour Goat CT because its longer brew time allowed for beneficial microorganisms to form in greater numbers and for more nutrients to pass from solid to liquid (Ingham 2000). Goat CT may have been most effective due to higher microbial activity, as Pant et al. (2012) found that CT effectiveness can be predicted by microbial activity.

Other treatments did not reveal significant differences from control in measures of leaf length, leaf width, and the increase of both from initial to final measurements.

Instead, these treatments revealed general trends that could perhaps be strengthened by more replicates and a longer study period. These trends include: all CT treatments resulted in greater leaf length increase than did their SC counterparts, five of six CT treatments had greater leaf length increase than control, and all SC treatments had lower leaf length increase than control (Figure 3a). Also, all treatments, except for 24-hour Human CT, resulted in greater leaf width increase than control, and all 72-hour CT treatments had greater leaf width increase than their

SC counterparts (Figure 3b). These results suggest that CT is more effective than SC for the growth of lettuce seedlings, though this trend is not significant. In the context of this experiment, CT may have proved to have a stronger effect than SC because CT was applied as a repeated, rather than as a one-time, treatment to plants. SC may have proved to be poorer than control for leaf length increase because perhaps the 1/3 ratio of compost to soil was not the optimum ratio for lettuce growth. Previous studies employed commercial aeration systems to brew CT and to maintain high levels of dissolved oxygen. This study employed the open-bucket methodology for brewing CT, which is what one might find in a low-resource gardening and farming context. Perhaps greater aeration would result in more significant results with more available oxygen to sustain the microbial populations.

Soil Nutrients Certain nutrient levels such as phosphorus and potassium increased between pre- and post-treatment samples of CT plants (Figure 4). Phosphorus, unlike carbon and nitrogen, is stored mainly in soil, and occurs as phosphate ions or within soluble organic compounds (Gliessman 2000). Crop-growers may add various phosphate fertilizers to the soil because phosphate ions are quickly fixed by soil into less bio-available forms (Bohn et al.

1985). Phosphorus is later returned to the soil via animal excreta. However, with the

loss of human excreta to sewage, the cycle of returning phosphorus to its original soil becomes disrupted (Gliessman 2000).

As such, soil samples from control plants likely reduced in phosphorus levels because phosphate was fixed into insoluble compounds. Cattle and Goat CT soils likely increased in phosphorus levels because soluble phosphates were present in the teas due to the animal excreta-based compost.

Potassium cations become similarly fixed in soils. Thus, constant potassium fertilization is important for growing crops (Bohn et al. 1985). Cattle, Goat, and Human CT soils likely increased in potassium levels for similar reasons to phosphorus: soluble potassium cations were exchanged into the soil from the liquid teas. Nitrogen is stored mainly in the atmosphere and is actively absorbed by soil matter (Bohn et al. 1985), and is usually a limiting nutrient, especially in tropical soils (Gliessman 2000).

Nitrogen remained at trace levels between pre- and post-treatments, although it is unclear if nitrogen was present in the form of nitrites and ammonia in the CT itself, as I only analyzed solid soil samples.

Compost Tea Properties The electrical conductivity and total dissolved solids values of CTs (Table 1) reveal a significant difference from control, in that there are particles and cations present in the CT liquids that are not present in the control of water. In addition, the immediate growth of bacterial populations on incubated plates indicates that

Table 1: Summary table of properties of rainwater (control) and CT. Standard deviations are noted in parenthesis.

Means that have no superscript in common are significantly different from each other (Tukeyâ&#x20AC;&#x2122;s HSD, p<0.05).

34

SALTMAN QUARTERLY

VOL. 13

sqonline.ucsd.edu

RESEARCH Figure 4: All pre-treatment soil nutrient levels were the same because all samples used soil from the same source. Nitrogen (N), Phosphorus (P), and Potassium (K) levels changed or remained constant as indicated above.

I performed nutrient analysis using LaMotte soil kits; units of nutrients per area were converted from lb/acre to g/m2 to fit the scale and methodology of my study.

CT contains high microbial activity. CT is therefore significantly different in composition than water. The effect of CT on plant growth is thus related to the presence of dissolved nutrients and microorganisms in the liquid.

Conclusion

Composting effectively provides a way to convert plant and animal waste into resources and to return nutrients to the soil. Thus, applying compost and compost tea not only increases the nutrient levels of soil to stimulate plant growth, but also diverts waste from methane- and greenhouse gas-emitting landfills (Lou and Nair 2009). Compost tea provides a microbial and nutrient-rich supplement to plants with the added advantages of requiring less volume and weight than solid compost per plant, as well as the added utility in that it can be applied repeatedly during the time of the plant’s growth.

Human waste composting practices, as demonstrated with composting toilets, returns human waste into the nutrient cycle. The practice in developed countries of using large volumes of potable water to flush human excreta into sewage systems and later into the ocean is both resource-intensive and nutrient-inefficient; the influx of nutrients disrupt ocean ecosystems and become stored as sediments (Gliessman 2000). Composting toilets expend the lowest water, cost, energy, and carbon footprint, when compared to other alternative toilet technologies such sq.ucsd.edu

as toilets flushed with rainwater (Anand and Apul 2010). Of course, sound methodology must be in place to ensure proper processing of human compost to avoid human pathogens and antibiotic-contamination (Bohn et al. 1985).

When making compost teas for agricultural purposes, one can reference the USDA task force report (2004) on compost teas, which details optimum conditions for brewing CTs to prevent transmission of human pathogens on crops. Guidelines for CT usage on crops suggest the use of potable water and matured compost, as well as applying within a 90/120-day pre-harvest interval. In a comprehensive study, Ingram (2009) investigated the food-safety risks during SC and CT production and application to a field of strawberries.

Ingram identified the presence of Salmonella, Ecoli, and high amounts of fecal coliforms among composts widely available to U.S. crop-growers. Additionally, even with high amounts of aeration for CT, inoculated Salmonella and fecal coliforms persisted for 36 hours in brew systems.

Finally, Ingram tracked the population of E.coli in contaminated compost applied to strawberries as CT, but did not find E.coli on the surface of the fruits harvested four days after application.

The methodology of my study could be improved by systematizing the CT brewing process with commercially-available aeration systems, rather than relying on a simple bucket method. Further research, given the implications and growing usage

of CT, should investigate the proper protocol for applying CT on food crops that are to enter the fresh produce supply. Compost production processes should include routine foodborne pathogen testing and a refinement of methods to avoid culturing these harmful pathogens. Additional studies about human compost should rigorously evaluate the presence of enteric pathogens during the maturation process of the compost, and also track food safety risks in the application of human SC or CT on food crops. Human waste, an important and underutilized nutrient source is often also associated with the public’s reasonable fear and aversion, but more widespread development and testing of its properties can forge strong connections between waste management and sustainability.

Acknowledgments

I would firstly like to thank my advisors Sofía Arce Flores and Frank Joyce from the UCEAP Costa Rica program. They provided constant advice and assistance throughout the course of my independent project and allowed me to realize my goal to conduct an agro-ecology project during my time in Costa Rica. I am especially grateful to Guillermo Vargas Leitón for his generosity and support in providing me a study site—including the use of a greenhouse and various composts—at Life Monteverde. The farm was such a wonderful place to meet visitors from around the world and to learn from the passionate employees there about coffee cultivation SALTMAN QUARTERLY

VOL. 13

35

96-hour Goat CT, 40 mg/mL sample Incubated at 25° C for 24 hours

48-hour Cattle CT, 40 mg/mL sample Incubated at 25° C for 24 hours

Figure 5: Growth of bacterial colonies for 96-hour Goat CT and 48-hour Cattle CT on plates with potato dextrose agar.

Plates were fully covered in colonies after 24 hours of incubation at 25° C.

and sustainable tourism. Equipment such as buckets, plant bags, and soil analysis kits were provided by the University of California Education Abroad Program.

This research was funded in part by study abroad scholarships from the Division of Biology and Sixth College at the University of California, San Diego.

References

1. Anand, C., & Apul, D. S. (2011). Economic and environmental analysis of standard, high efficiency, rainwater flushed, and composting toilets. Journal of Environmental Management, 92(3), 419-428.

2. Arancon, N. Q., Edwards, C. A., Dick, R., & Dick, L. (2007). Vermicompost tea production and plant growth impacts. Biocycle, 48(11), 51.

3. Bohn, H. L., McNeal, B. L., & O’Connor, G. A. (1985). Soil Chemistry. A Wiley-Interscience Publication.

4. Edwards, C. A., Arancon, N. Q., & Greytak, S. (2006). Effects of vermicompost teas on plant growth and disease. Biocycle, 47(5), 28.

5. Gliessman, S. R. (2000). Agroecology: 36

SALTMAN QUARTERLY

VOL. 13

ecological processes in sustainable agriculture. CRC Press.

6. Ingham, E. R. (2000). The compost tea brewing manual. Corvallis, Oregon, USA: Unisun Communications.

7. Ingram, D. T. (2009). Assessment of foodborne pathogen survival during production and pre-harvest application of compost and compost tea. (Ph.D. Dissertation).

University of Maryland.

8. Lou, X. F., & Nair, J. (2009). The impact of landfilling and composting on greenhouse gas emissions–a review. Bioresource technology, 100(16), 3792-3798.

9. Naidu, Y., Meon, S., Kadir, J., & Siddiqui, Y. (2010). Microbial starter for the enhancement of biological activity of compost tea. Int. J. Agric. Biol, 12(1), 51-56.

10. National Organic Standards Board (NOSB) (2006). Crops Committee Recommendation for Guidance: Use of Compost, Vermicompost, Processed Manure, and Compost teas. U.S. Department of Agriculture.

11. Pant, A. P., Radovich, T. J., Hue, N. V., & Paull, R. E. (2012). Biochemical properties of compost tea associated with

compost quality and effects on pak choi growth. Scientia Horticulturae, 148, 138-146.

12. Pant, A. P., Radovich, T. J., Hue, N. V., Talcott, S. T., & Krenek, K. A. (2009).

Vermicompost extracts influence growth, mineral nutrients, phytonutrients and antioxidant activity in pak choi (Brassica rapa cv. Bonsai, Chinensis group) grown under vermicompost and chemical fertiliser. Journal of the Science of Food and Agriculture, 89(14), 2383-2392.

13. Scheuerell, S., & Mahaffee, W. (2002). Compost tea: principles and prospects for plant disease control. Compost Science & Utilization, 10(4), 313-338.

WRITTEN BY BRIANNA EGAN Brianna is a Human Biology major and Global Health minor from Sixth College. She will graduate in 2016.

sqonline.ucsd.edu

RESEARCH

Rho-GEF Solo regulates epithelial collective migration via adhesion complex protein plakoglobin Daniel Jun Rindner | Advisor: Kensaku Mizuno, Ph.D. This study aims to make clear the specific role of the Rho-family guanine nucleotide exchange factor (Rho-GEF) Solo (also named ARHGEF40 and Scambio) within living multicellular systems, specifically during epithelial collective migration. Knockdown of Solo by siRNAs in Type I Madin-Darby canine kidney (MDCKI) cells was found to lead to increased movement of migration fingers. Furthermore, Solo depletion led to the weakening of keratin intermediate filaments (IFs) as well as plakoglobin (γ-catenin) disorganization in migrating colonies at sites of cell-cell contact. This paper proposes a model where plakoglobin enlists the help of Solo in the mechanical stress-initiated self-strengthening of adhesion complexes.

Introduction

Collective migration is the fundamental basis of many biologically significant events, including embryogenesis, cancer metastasis, and wound healing. Furthermore, each process is morphologically very similar, making the understanding of cell movement relevant across many fields of biological and pathophysiological research.

During single cell migration, a polarized cell first initiates membrane protrusion in the direction of cell movement, during which leading edge actin filaments are reorganized and new focal adhesions are formed1,2. Simultaneously, the dissociation of cytoskeletal components to adhesion complexes and resulting detachment from substrate at the trailing edge of migratory cells generates the primary traction force which drives the cell body forward2,3,4. This process is conserved during collective migration most notably by select cells at the front-end migrating edge, called “leader” cells5, and has also been witnessed to a lesser degree in cells of the migrating body6,7. While being more physiologically significant8, the case of collective cell migration is much less understood. Many questions remain regarding how forces are generated, propagated, and received within the migrating body.

Do leading edge cells propagate the primary pulling force of collectively migrating bodies or do individual cells of the body’s entirety instead contribute equally to body migration by largely migrating of its own accord? Furthermore, what mechanisms exist to regulate this process? Trepat et al. reported that the main traction force of migrating cell bodies is generated many rows back of the migrating edge9. Still, a sq.ucsd.edu

separate study demonstrated that micromanipulator removal of leader cells in similarly migrating colonies dramatically disrupted cell movement10, suggestive of the cooperative necessity of both “leader” and “follower” cells as guiding and driving forces of collective migration.

Interestingly, in addition to substantial cell-substrate interactions, individual cells of collectively migrating bodies exert considerable physical force on surrounding cells during migration11. Such physical force is known to induce dramatic intercellular changes in a variety of biological systems, such as determination of a cell polarity and reorganization of the cytoskeleton12,13,14,15. Thus in addition to driving cell body movement, the physical interactions between cells are believed to act as an intracellular signaling mechanism for regulating collective cell migration. The process of converting physical stress into chemical signals (in what is referred to as mechanotransduction) can be thought to involve two “steps”. In the first, membrane associated mechanosensing complexes receive mechanical signals by inducing a conformational change in membrane-embedded or associated proteins16. In the second, this conformational change reveals a novel function of the complex that elicits and propagates some biochemical response16. One such example is the stretch-induced opening of human neuronal TRAAK K+ channels16,17. Recent studies have additionally pointed to cell-cell adhesion complexes as membrane-embedded hubs of mechanosensing potential and indicated their involvement in force-sensitive cytoskeletal remodeling18,19.

Cadherins-based focal adhesions and desmosomes are connected to the cytoskeleton via catenin-family proteins.

However, in addition to linking these two critical structural components, catenins are suspected to act as initial signaling proteins of adhesion complex-mediated mechanotransduction20. Initial experiments identified plakoglobin (also known as γ-catenin) as a structural component of both adherens junctions and desmosomes21, and mounting evidence since points to the emerging role of plakoglobin as a signaling protein. Plakoglobin is highly homologous to β-catenin22,23 and likely acts alongside β-catenin in Wnt signaling pathways involved in cytoskeletal organization24,25. Plakoglobin’s added importance comes from its tumor suppressor capabilities and was described to reduce cancer migration and invasiveness in various cell lines, hinting at its possible passive role in regulating cell motility26,27. Furthermore, force-initiated recruitment of keratin IFs was recently found to be plakoglobin-dependent and localized to cadherin adhesions under stress20. This study aimed to further elucidate which cytoskeletal remodeling pathways are involved in the mechanosensing function of plakoglobin.

A great deal has been discovered about the Rho family of small GTPases (extensively RhoA, RhoC, Rac1, and Cdc42) and their role in regulating cytoskeletal dynamics28,29,30. Rho has been found central in a range of cellular events including cell polarity determination and cell movement31,32,29. In addition, Wolf et al. found that cytokinetic RhoA activation is spatially regulated by desmosome and focal adhesion (FA) localizing Armadillo-fami SALTMAN QUARTERLY

VOL. 13

37

Materials and Methods

Cell lines and reagents YFP-tagged keratin 8/18 transformant Madin-Darby canine kidney-I (MDCKI) epithelial cells were used for all experiments. Cells were cultured at 37°C with 5% CO2 with high glucose Dulbecco’s modified Eagle’s medium containing L-Glutamine (Wako) supplemented with 10% fetal calf serum (Wako) and passaged at 70-80% confluence.

Collective and single cell migration Collagen-coated glass substrate collective migration was initialized by plating cells at mid-density (~7000 cells/17 mm 38

SALTMAN QUARTERLY

VOL. 13

(d og )# 2

So lo

si

Co nt ro l

(d

#1

o ol iS

(

3

)#

g do

s

Control

si

lo So

) og

si Solo (dog) #2

ly protein p007133. ROCK, a downstream protein of Rho, was discovered to direct K8/K18-regulated cell stiffening at FAs34 and artificially induced C3 transferase inhibition of Rho was moreover found to directly suppress force-induced FA assembly35. further implicating Rho’s possible role as an adhesion complex-coupled mechanosensing protein.

The activation of Rho is regulated by about 70 guanine nucleotide exchange factors (Rho-GEFs)36. However, the specificities of which, when, and how each of these Rho-GEF proteins activate their downstream target(s) are largely unknown. Notably, it was recently found that the RhoGEF Solo (also named ARHGEF40 and Scambio) enables the cyclic stretch-induced alignment of actin stress fibers perpendicular to force direction in endothelial cells through activation of RhoA37.

Moreover, Solo was found to have binding ability to keratins 8/18 and plakoglobin, hinting at a possible role in activating Rho during force-induced, desmosome-mediated IF remodeling38. In this paper, I discuss the role of Solo in collective migration and its function as a possible mechanosensing component of desmosomes via plakoglobin. I begin by showing that Solo activity is necessary for regulating collective migration speeds. I next show that Solo is responsible for maintaining cell shape in collectively migrating cell bodies.

Finally, I demonstrate that Solo acts upstream of plakoglobin, and is responsible for what is likely desmosomal plakoglobin accumulation at cell peripheries in response to physical cell stress.

Figure 1: Solo regulates degree of MDCKI collective cell migration. a. Western blot confirmation of siRNA viability. b. Collective migration of MDCKI YFP-K8/18 transformant cells cultured on 0.1 mg/mL collagen-coated glass dish. Red arrowheads indicate leader cells. Scale bar, 35 μm. c. Migration paths of 6 MDCKI YFP-K8/18 transformant cells. Leader cell migration shown in red. Graph and time-lapse still images show a 5-hour movement period. Arrows indicate migration direction. diameter glass-bottomed culture dish).

Cells were incubated for 24-48 hours before live viewing. Cells were plated at low density (~1000 cells/17 mm diameter glass-bottomed culture dish) on high-density collagen substrate for single cell migration experiments. For high-density collagen substrate collective migration experiments, cells were seeded in glass

cloning rings (Iwaki, Japan) at high density (4×104 cells/5 mm diameter ring) and incubated for 6 hours. Rings were then carefully removed to initiate radial finger growth and incubated for an additional 24 hours before fixation or live viewing.

Transfection and western blotting All siRNAs targeting dog Solo were designed using siDirect and custom made by sqonline.ucsd.edu

RESEARCH

Collectively Migrating Cell Speed (µm/hour)

a)

l ro nt o C

olo iS

(

Single Cell Speed (µm/hour)

s

b)

1

)#

g do

olo iS

2

)#

g do

(

olo iS

s

(

3

)#

g do

s

l ro nt o C

olo iS

(

1

)#

g do

s

Figure 2: Solo’s function during collective migration requires sites of cell-cell contact. a. Average collective migration speed of MDCKI on 1.6 mg/ml collagen substrates. Control and si Solo #1: n=2 ; si Solo #2 and si Solo #3: n=1. 10 cell average used for each experiment. b. Average single cell migration speeds of MDCKI on 1.6 mg/ml collagen substrates. n=2, 15 cell total, ≥5 cells per experiment. Statistical analysis was not performed due to inadequate sample size.

Sigma-Aldrich, and tested for knockdown effect 48 hours after transfection (Fig. 1).

Transfection was facilitated by RNAiMax reagent (Invitrogen). β-actin was detected by using mouse IgG1 monoclonal anti-β-actin clone AC-15 (Sigma-Aldrich) at a 1:1000 dilution, then using HRP-conjugated goat anti-mouse antibodies (GE Healthcare). Solo was detected using a rabsq.ucsd.edu

bit polyclonal antibody raised against dog and human Solo, then using HRP-conjugated goat anti-rabbit antibodies (GE Healthcare).

Time-lapse microscopy Migration experiments were performed on low-density collagen-coated glass and high-density fully polymerized collagen substrates. Collagen-coated glass bottom

plates (Rat-tail collagen Type I high concentration diluted to 0.1 mg/mL, Corning) were used where indicated. Fully polymerized collagen layers (Rat-tail collagen Type I-A diluted to 1.6 mg/mL, Cell Matrix) were also used where indicated. All collagen substrates were allowed to incubate in culture medium prior to cell seeding overnight to equilibrate substrate pH. All live cell imaging was performed with the Zeiss LSM 710 confocal microscope on a heated incubator stage with 5% CO2 at 37°C. Fluorescence microscopy Cell fixation was achieved using 4% paraformaldehyde in PBS and stored at4°C. Cell nuclei were stained using DAPI (4, 6-diamidino-2-phenylindole dihydrochloride) (Polysciences, Inc.) at a 1:20000 dilution. γ-catenin was stained and used to designate cell periphery using Mouse IgG2a γ-catenin (BD Biosciences) at a 1:1000 dilution and subsequent anti-mouse Alexa Fluor 568 (Invitrogen) at a 1:1000 dilution.

Fluorescent images were taken using the Leica DMI6000 B microscope. Cells were grown on fully polymerized collagen layers (rat-tail collagen Type I-A diluted to 1.6 mg/mL, Cell Matrix), as described above in collective and single cell migration.

Image processing Cell movement and size were traced manually using the ImageJ software (NIH). Total distance was a summation of the linear distance traveled every 10 minutes and used to calculate approximate cell velocities.

Statistical analysis Statistical analysis was performed using Prism 6 (GraphPad Software, La Jolla, CA).

Results

Sites of cell-cell contact necessary for witnessing Solo-knockdown effect To understand Solo’s role in living systems, time-lapse images were taken of MDCKI collective cell migration. Knockdown of Solo by Solo-targeting siRNA transfection had no apparent effect on the mono-directional movement of migration fingers but markedly increased their migration speeds (Fig. 1). Furthermore, cells were cultured on differing substrates to mimic in vivo properties of the extracellular matrix and reduce possible phenotypic deviation due to irregular cell-substrate SALTMAN QUARTERLY

VOL. 13

39

adhesion conditions. Individual cell velocities were recorded of both collectively migrating and single cell bodies, revealing that migration speeds are significantly increased by Solo knockdown (Fig. 2). By contrast, single cell migration speeds were unaffected by Solo knockdown, suggesting that Solo plays a crucial role in collective migration at cell-cell adhesion sites.

Solo depletion inhibits local accumulation of plakoglobin along cell peripheries To identify Solo’s function at sites of cell-cell contact during collective migration, the effect of Solo knockdown on IF organization was examined. Solo depletion inhibited strengthening of keratin filaments, particularly at sites of cell-cell contact (Fig. 3). Furthermore, Solo knock-

Discussion

Coordinated activity of the cytoskeleton is central to a broad range of morpho-

gical events, including cell proliferation, polarization, and migration. As such, reorganization of stress fibers and intermediate filaments are both unequivocally necessary and tightly controlled within the cell. One of these controlling mechanisms is physical force sensing, which is now thought to involve cell-cell and cell-substrate adhesion complexes and subsequent mechanotransduction pathways. This study aimed to elucidate any relationship between the Rho signaling pathway, well known for its centrality to cytoskeletal remodeling, and emerging data indicating cell-cell adhesion complexes as centers of cellular force sensing. Here, I provide a novel link between the Rho-family of GTPases and adhesion complex protein plakoglobin through the

b)

Control

a)

down disrupted the tight, honeycomb-like attachment between cells of the monolayer and dramatically increased average cell size. Desmosome component protein plakoglobin was also discovered unable to accumulate neatly across cell membranes, including at sites of trans-cellular IF attachment, following Solo depletion. Solo knockdown also increased the presence of plakoglobin within the cell body. Interestingly, intracellular plakoglobin was also seen to co-localize with keratin filaments, evidence of an adhesion complex-independent link.

Cell Size(μm2)

si Solo (dog) #1

c)

C

)#1

ol

tr on

si S

olo

g (do

Figure 3: Solo responsible for contact-site plakoglobin accumulation and keratin filament strengthening and regulation of cell size. a. Equal intensity fluorescent images of MDCKI YFP-K8/18 migration fingers cultured on 1.6 mg/mL collagen. White arrowheads indicate sites of cell-cell contact. Scale bar, 35 μm. b. Non-adhesion complex plakoglobin accumulates endogenously around keratin filaments. Orange arrowheads indicate sites of high-density plakoglobin. c. Average cell size of migration fingers.

Data represents means ± s.d. of 50 cells of one experiment trial, ***p<0.0001.

40

SALTMAN QUARTERLY

VOL. 13

sqonline.ucsd.edu

RESEARCH specific Rho-GEF Solo. Although no distinction was made in this study between plakoglobin specific to a particular adhesion complex, evidenced Solo-dependent IF reorganization and Solo’s binding ability to keratin 8/18 suggests that Rho activity is likely linked to that of desmosomes, if not adherens junctions as well.

In this study, I first confirmed that Solo has biological significance by viewing the effects of Solo-depletion on epithelial collective migration, which is implicated in the important morphological events of embryogenesis and would healing, among others. Initial glass-substrate experiments revealed that Solo acts in inhibiting migration finger growth (Fig. 1), which would be crucial in regulating morphogenesis. To delineate phenotypic consequences of Solo knockdown specific to collectively migrating cells, migration speeds were then compared between collectively and independently migrating cells. Results showing that Solo knockdown has no evident impact on single cell speeds implied that viable cell-cell contact sites are necessary for Solo activity (Fig. 2). Previous studies have implicated Rho in adhesion complex-mediated mechanotransduction39, thus I aimed to establish Solo’s role as a Rho-GEF within this context.

Solo, like other Rho-GEFs, contains a common catalytic Dbl-homologous (DH) domain at its C-terminal region accounting for its Rho-GEF activity. However, this region alone was found inadequate to induce RhoA activation, and the presence of the full length of Solo including the N-terminal region was determined necessary for Rho-GEF activity37. Authors speculate that Solo’s DH domain could remain unavailable until mechanical force acting on both terminal ends induces a conformational change thereby “opening” Solo and revealing the DH catalytic region. Solo has binding capabilities to both keratin 8/18 and actin and was found to co-localize to areas of high keratin density38. Furthermore, collectively migrating cells were unable to organize plakoglobin and IFs at sites of cell-cell contact following knockdown of Solo (Fig. 3). Together, these results indicate that Solo’s mechanosensing function likely relies on a conformational change, and lies upstream of desmosome sq.ucsd.edu

strengthening. The approximately two-fold increase in cell size additionally indicates that Solo knockdown cells have lost their ability to maintain and strengthen IF and membrane contractility (Fig. 3), ultimately resulting in increased migration speeds.

However, whether Solo knockdown-induced plakoglobin disorganization is the reason for or merely a consequence of the apparent loss of cellular force sensing ability is unclear.

An idealistic model of Solo is as follows: Solo binds to desmosome plakoglobin and keratin at each terminal end, directly sensing mechanical force between them. This force induces a conformational change that reveals Solo’s DH domain, thereby activating Rho and initiating keratin turnover and desmosome strengthening. Interestingly, plakoglobin well within the cell body was also found to co-localize with keratin 8/18, hinting at the existence of “pre-assembled” mechanosensing units involving Solo (Fig. 3). This opens the possibility that Solo does not itself initiate mechanotransduction events, but rather acts primarily as a signal amplifier for other mechanotransduction pathways in response to cell stress.

Much more experimentation is necessary to concretely assert such claims, however the possibility of such a mechanotransduction mechanism remains nonetheless an exciting one.

Conclusion

It is likely that the Rho-GEF Solo is enlisted in adhesion complex mechanosensing to reorganize IFs and act in self-strengthening of cell-cell adhesion complexes in response to physical cell stress. siRNA knockdown of Solo in epithelial MDCKI dramatically increased movement of migration fingers and led to weakening of IFs and plakoglobin disorganization at sites of cell-cell contact. These results reveal Solo to be an integral regulator of collective cell motility via cell-cell adhesion sites. A fuller understanding of mechanotransduction and its role in regulating cell motility is critically important due to the highly integral nature of cell movement in biologically significant events such as embryogenesis, cancer metastasis, and wound healing, and the possibility of a direct interaction between Rho

and adhesion complex proteins would provide a beautifully seamless mechanism for physical force sensing.

Acknowledgments

I would first like to thank Dr. Kensaku Mizuno and Dr. Kazumasa Ohashi for their endless generosity both in and out of the laboratory, without whose guidance this work would not have been possible. I would also like to acknowledge the hard working members of the Mizuno Laboratory, with special thanks to Ryosuke Nishimura, Kouki Sakai, and Sachiko Fujiwara, whose continued morale and technical support proved invaluable to the making of this paper. This project was supported by the Ministry of Education, Culture, Science, Sports and Technology of Japan.

References

1. Raftopoulou, M., Hall, A. Cell migration: rho GTPases lead the way. Dev. Biol.

265, 23–32 (2004).

2. Friedl, P. and Wolf, K. Proteolytic interstitial cell migration: a five-step process.

Cancer Metastasis Rev. 28, 129-135 (2009).

3. Lauffenburger, D.A., Horwitz, A.F. Cell migration: A physically integrated molecular process. Cell 84, 359-369 (1996).

4. Friedl, P., Hegerfeldt, Y. and Tusch, M. Collective cell migration in morphogenesis and cancer. Int. J. Dev. Biol. 48, 441-449 (2004).

5. Haga, H., Irahara, C., Kobayashi, R., Nakagaki, T., Kawabata, K. Collective movement of epithelial cells on a collagen gel substrate. Biophys. J. 88(3), 2250-2256 (2005).

6. Farooqui, R., Fenteany, G. Multiple rows of cells behind an epithelial wound edge extend cryptic lamellipodia to collectively drive cell-sheet movement. J. Cell Sci. 118, 51-63 (2005).

7. Friedel, P. and Gilmour, D. Collective cell migration in morphogenesis, regeneration and cancer. Nat. Rev. Mol. Cell Biol.

10, 445-457 (2009).

8. Illina, O. and Friedl, P. Mechanisms of collective migration at a glance. Cell Sci. 122, 3203-3208 (2009).

9. Trepat, X., Wasserman, M. R., Angelini, T. E., Millet, E., Weitz, D. A., Butler, J. P., Fredberg, J. J. Physical forces during col SALTMAN QUARTERLY

VOL. 13

41

lective cell migration. Nat. Phys. 5, 426-430 (2009).

10. Yamaguchi, N., Mizutani, T., Kawabata, K., Haga, H. Leader cells regulate collective cell migration via Rac activation in the downstream signaling of integrin β1 and Pl3K. Scientific Reports 5:7656, 1-8 (2015).

11. Behrndt, M., Heisenberg, C.P. Spurred by resistance: mechanosensation in collective migration. Dev. Cell 22, 3-4 (2012).

12. Ingber, D.E. Cellular mechanotransduction: putting all the pieces together again. FASEB 20(7), 811-827 (2003).

13. Jaalouk, D.E., Lammerding, J. Mechanotransduction gone awry. Nat. Rev. Mol. Cell Biol. 10(1), 63-73 (2009).

14. Colombelli, J., Besser, A., Kress, H., Reynaud, E.G., Girard, P., Caussinus, E., Haselmann, U., Small, J.V., Schwarz, U.S., Stelzer, E.H. Mechanosensing in actin stress fibers revealed by a close correlation between force and protein localization. J. Cell Sci. 122(10), 1665-1679 (2009).

15. Vogel, V. and Sheetz, M. Local force and geometry sensing regulate cell functions. Nat. Rev. Mol. Cell Biol. 7, 265-275 (2006).

16. Hamill, O.P., Martinac, B. Molecular basis of mechanotransduction in living cells. Physiol. Rev. 81(2), 685-740 (2001).

17. Brohawn, S.G., Campbell, E.B., MacKinnon, R. Physical mechanism for gating and mechanosensitivity of the human TRAAK K+ channel. Nature 516, 126-130 (2014).

18. Geiger, B., Bershadsky, A., Pankov, R., Yamada, K.M. Transmembrane crosstalk between the extracellular matrix and the cytoskeleton. Nat. Rev. Mol. Cell Biol.

2, 793-805 (2001).

19. Aktary, Z. and Pasdar, M. Plakoglobin: Role in Tumorigenesis and Metastasis.

Int. J. Cell Biol. 2012, 1-14 (2012).

20. Weber, G.F., Bjerke, M.A., DeSimone, D.W. A mechanoresponsive cadherin-keratin complex directs polarized protrusive behavior and collective cell migration. Dev. Cell 22, 104-115 (2012).

21. Cowin, P., Kapprell, H.P., Franke, W.W., Tamkun, J., Hynes, R.O. Plakoglobin: a protein common to different kinds of intercellular adhering junctions. Cell 46(7), 1063-1073 (1986).

22. Butz, S., Stappert, J., Weissig, H., 42

SALTMAN QUARTERLY

VOL. 13

Kemler, R. Plakoglobin and β-catenin: distinct, but closely related. Science 257, 11421144 (1992).

23. McCrea, P.D., Turck, C.W., Gumbiner, B. A homolog of the armadillo protein in Drosophila (plakoglobin) associated with E-cadherin. Science 254(5036), 1359-1361 (1991).

24. Zhurinsky, J., Shtutman, M., BenZe’ev, A. Plakoglobin and β-catenin: protein interactions, regulation and biological roles. J. Cell Sci. 113, 3127-313 (2000).

25. Maeda, O., Usami, N., Kondo, M., Takahashi, M., Goto, H., Shimokata, K., Kusugami, K., Sekido, Y. Plakoglobin (gamma-catenin) has TCF/LEF family-dependent transcriptional activity in beta-catenin-deficient cell line. Oncogene 23(4), 964-972 (2004).

26. Nagashima, H., Okada, M., Hidai, C., Hosoda, H., Kasanuki, H., Kawana, M. The role of cadherin-catenin-cytoskeleton complex in angiogenesis: antisense oligonucleotide of plakoglobin promotes angiogenesis in vitro, and protein kinase C (PKC) enhances angiogenesis through the plakoglobin signaling pathway. Heart and Vessels, 12(12), 110–112 (1997).

27. Rieger-Christ, K.M., Ng, L., Hanley, R.S., Durrani, O., Ma, H., Yee, A.S., Libertino, J.A., Summerhayes, I.C. Restoration of plakoglobin expression in bladder carcinoma cell lines suppresses cell migration and tumorigenic potential. British J. Cancer 92, 2153-2159 (2005).

28. Bar-Sagi, D., Hall, A. Ras and Rho GTPases: a family reunion. Cell 103(2), 227-238 (2000).

29. Ridley, A.J. Rho GTPases and cell migration. J. Cell Sci. 114(15), 2713-2722 (2001).

30. Malliri, A., Collard, J.G. Role of Rho-family proteins in cell adhesion and cancer. Curr. Opin. Cell Biol. 15(5), 583589 (2003).

31. Yamana, N., Arakawa, Y., Nishino, T., Kurokawa, K., Tanji, M., Itoh, R.E., Monypenny, J., Ishizaki, T., Bito, H., Nozaki, K., Hashimoto, N., Matsuda, M., Narumiya, S. The Rho-mDia1 pathway regulates cell polarity and focal adhesion turnover in migrating cells through mobilizing Apc and c-Src. Mol. Cell Biol. 26(18), 6844-6858 (2006).

32. Georgiou, M. and Baum, B. Polar-

ity proteins and Rho GTPases cooperate to spatially organize epithelial actin-based protrusions. J. Cell Sci. 123, 1089-1098 (2010).

33. Wolf, A., Keil, R., Götzl, O., Mun, A., Schwarze, K., Lederer, M., Hüttelmaier, S., Hatzfeld, M. Armadillo protein p0071 regulates Rho signaling during cytokinesis. Nat. Cell Biol. 8(12), 1432-1449 (2006).

34. Bordeleau, F., Lapierre, M.M., Sheng, Y., Marceau, N. Keratin 8/18 regulation of cell stiffness-extraceullular matrix interplay through modulation of Rho-mediated actin cytoskeleton dynamics. PLoS ONE 6, 1-10 (2012).

35. Riveline, D., Zamir, E., Balaban, N.Q., Schwarz, U.S., Ishizaki, T., Narumiya, S., Kam, Z., Geiger, B., Bershadsky, A.D. Focal contacts as mechanosensors: externally applied local mechanical force induces growth of focal contacts by an mDia1-dependent and ROCK-independent mechanism. J. Cell Biol. 153, 11751185 (2001).

36. Cook, D.R., Rossman, K.L., Der, C.J. Rho guanine nucleotide exchange factors: regulators of Rho GTPase activity in development and disease. Oncogene 33, 40214035 (2014).

37. Abiko, H., Fujiwara, S., Ohashi, K., Hiatari, R., Mashiko, T., Sakamoto, N., Sato, M., Mizuno, K. Rho guanine nucleotide exchange factors involved in cyclic-stretch-induced reorientation of vascular endothelial cells. J. Cell Sci. 128, 1683-1695 (2015).

38. Fujiwara, S. Functional roles of RhoGEF Solo in regulation of actin and intermediate filament networks and mechanotransduction. Tohoku University (2015).

39. Todorović, V., Desai, B. V., Patterson, M. J. S., 2010. Plakoglobin regulates cell motility through Rho- and fibronectin-dependent Src signaling. J. Cell Sci. 123 (20), 3576–3586.

WRITTEN BY DANIEL JUN RINDNER Daniel is a Biochemistry and Cell Biology major and from Eleanor Roosevelt College.

He will graduate in 2016.

sqonline.ucsd.edu

RESEARCH

Developing an Educational Intervention to Improve Genetic Risk Assessment in Young Breast Cancer Survivors Deepika Suresh | Advisors: Irene Su, M.D., M.S.C.E. and Sally Dominick, Ph.D., M.P.H. Many breast cancer survivors in the United States are under the age of 45 and are at higher risk of carrying germline mutations that increase cancer risk. Professional society practice guidelines endorse referring these young breast cancer survivors for genetic risk assessment. The overall goal of this research project was to develop a web-based educational intervention to improve genetic risk assessment in young breast cancer survivors. We identified various barriers preventing young survivors from undergoing genetic risk assessment by conducting a literature review, key informant interviews and a review of professional society guidelines. Examples of these barriers include lack of healthcare provider referral, pre- and post-test anxiety, and insurance issues. Since addressing these barriers is an important step in encouraging young survivors to undergo risk assessment, educational content addressing these barriers was developed and incorporated into a web-based intervention. Our future direction is to test this web-based intervention in a randomized controlled trial.

Introduction

Ten percent of the 2.8 million breast cancer survivors in the U.S. are under the age of 45.1 Many of these young breast cancer survivors (YBCS) experience a myriad of survivorship issues, which adversely impact the survivorâ&#x20AC;&#x2122;s quality of life. One significant issue is hereditary cancer syndromes. YBCS are at higher risk of having a germline genetic mutation correlated with breast cancer risk, compared to older breast cancer survivors.2 It is estimated that greater than 10% of YBCS carry a germline cancer gene mutation.2 Assessing genetic risk in YBCS is important as genetic testing can bring to light predisposition for further cancers, such as ovarian cancer, and allow women to take preventative measures. Furthermore, these germline gene mutations can be inherited, meaning that YBCS risk passing on germline gene

mutations that increase cancer risk to their children. Having this knowledge can impact a womanâ&#x20AC;&#x2122;s decision on whether or not to have children in the future, whether and how to discuss testing with her children, or whether to use assisted reproduction to select out the mutation.

Genetic risk assessment includes genetic counseling with possible follow-up genetic testing. Genetic counseling provides YBCS and their families needed information on their risks of carrying genetic mutations that predispose them to breast and other cancers. After attending genetic counseling, genetic counselors can inform the YBCS on any specific genetic tests they recommend for the patient. For YBCS who undergo genetic testing, once the counselors receive test results, they can go through the results with the patient and allow them to make informed decisions about their healthcare and cancer risk. Despite poten-

tial benefits to YBCS, recent data show that only 34% of breast cancer patients are referred for genetic risk assessment.3 Our research group has developed a web-based Reproductive Health Survivorship Care Plan (SCP-R) intervention geared toward addressing four late effects (fertility concerns, birth control practices, sexual health dysfunction, and vasomotor symptoms) that are prevalent in many YBCS.4-7 We developed initial evidence-based content for these late effects and evaluated this content through YBCS focus groups. Based on YBCS feedback on a lack of information on hereditary cancer risk in our initial content, our group decided to add a genetic risk assessment module to the SCP-R. The overall goal of this module is to increase the number of YBCS who undergo genetic risk assessment. This paper focuses on the methods undertaken to develop the genetic risk assessment porFigure 1: Search terms for literature search.

sq.ucsd.edu

SALTMAN QUARTERLY

VOL. 13

43

Samples of the genetics content incorporated into the website Why should young breast cancer survivors undergo cancer genetics counseling?

Costs of cancer genetics counseling and testing

Table 1: Genetics content example tion of the SCP-R project. There were two primary objectives for developing the webbased genetics risk assessment content: 1) to identify barriers preventing YBCS from undergoing genetic risk assessment and 2) to develop content addressing the identified barriers in order to increase uptake of genetic risk assessment. We hypothesized that a multi-modal approach based on review of current research evidence, professional society guidelines and clinical expertise will allow us to generate evidence-based content that identifies and addresses these barriers in order to increase uptake of genetic risk assessment.

Materials and Methods

Identifying barriers to genetic risk assessment in YBCS A systematic literature review was conducted in order to identify perceived and real barriers preventing women from undergoing genetic risk assessment and gather evidence on how to overcome these bar44

SALTMAN QUARTERLY

VOL. 13

riers. The database systematically searched was Pubmed (1966-August 2015). The literature search was conducted by using a defined search strategy (Figure 1). Next, a search of professional society guidelines was undertaken. Websites of professional medical societies related to the care of YBCS were researched. Finally, key informant interviews were conducted with genetic counselors at the Moores UC San Diego Cancer Center Family Cancer Genetics Program. A structured interview guide was created ahead of the interviews.

Developing content for the Genetic Risk Assessment Survivorship Care Plan The genetics module content addressed the identified barriers and provided potential solutions to address these barriers.

Also the module described the genetic risks faced by YBCS and included ways to reduce risks of related cancers (e.g., risk-reducing removal of ovaries and fallopian tubes in BRCA 1 and BRCA2 carriers). The content was organized into three

sections: an abbreviated survivorship care plan in a question/answer format; a summary of professional society guidelines; and curated online resources for YBCS and their healthcare providers.

Following initial development, the genetics content was reviewed by a stakeholder panel (comprised of an oncologist, primary care physician, reproductive endocrinologist, sexual health therapist, health behaviorists, epidemiologists, and YBCS advocates) and genetic counselors for feedback on credibility and comprehension. The overall goal of this feedback and revisions was to render the content understandable and persuasive.

Results

Literature Review From the search terms, the titles and abstracts from 126 articles were reviewed; from these, 12 full text articles were included in the literature review. The results demonstrated that the main barriers to sqonline.ucsd.edu

RESEARCH

What type of Information is here?

What organization provides this resource?

Table 2a: Resources for Young Breast Cancer Survivors (YBCS) on genetic risk assessment and its many aspects. undergoing genetic risk assessment include the following: 1) healthcare providers not referring patients for genetic risk assessment when appropriate; 2) pre- and post-test anxiety and depression; 3) insurance issues; 4) lack of education; 5) worry that test results can be used against the patients.8-11 One main reason for limited referral by health care providers was lack of knowledge of current guidelines for referral to genetic counseling.12 Additionally, emotional concerns such as pre- and post-test anxiety were significant barriers preventing YBCS from seeking out genetic risk assessment. However, much anxiety resulted from a lack of knowledge about the process of genetic risk assessment and the numerous options given to those who chose to undergo it.13 A major concern for YBCS was cost and insurance. Many YBCS were concerned that genetic risk assessment is costly and were unaware that most insurance companies cover counseling, testing, and post-test procedures for this population.14 The lack of education on genetic risk assessment was observed as many YBCS were unaware of what genetic risk assessment entails and did not know the difference between genetic counseling and genetic testing.15 Finally, YBCS expressed concern on privacy of test results.8 Many YBCS believed that results of their genetic testing could be released and affect their employment opportunities or raise insurance prices. sq.ucsd.edu

Professional Societyâ&#x20AC;&#x2122;s Guidelines: In order to provide YBCS and their healthcare providers with the most up-todate and accurate information surrounding genetic risk assessment, a thorough review of the clinical guidelines on genetic risk assessment presented by professional societies was performed. The professional societiesâ&#x20AC;&#x2122; guidelines reviewed include those from the American Society of Clinical Oncology,16 the American Congress of Obstetricians and Gynecologists,17 National Comprehensive Cancer Network,18 and the National Society of Genetic Counselors.19 All of the above professional societies had similar guidelines for YBCS and how they should approach genetic risk assessment. The guidelines state that all patients diagnosed with breast cancer before age 45 should be referred to genetic counseling, where genetic testing panels can be recommended.16-19 Additionally, the guidelines state that all patients diagnosed with breast cancer before age 45 should undergo BRCA1/BRCA2 testing.16-19 Finally, the guidelines recommend that all genetic testing should occur with pre- and posttest genetic counseling.16-19 Often times, patients try to retrieve the test results alone without any form of counseling, which can often result in confusion or the misinterpretation of test results.13 Genetic Counselor Interviews To gain an additional perspective on why so many YBCS do not undergo genetic

risk assessment, we conducted structured interviews with 3 genetic counselors at the Moores UC San Diego Cancer Center.

Based on these interviews, the counselors stated that the two most frequent barriers they heard from patients include: 1) misconception on the genetic risk assessment process and 2) lack of healthcare provider referral. The genetic counselors recognized that the YBCS population generally views genetic risk assessment as alack of healthcare provider referral. The genetic counselors recognized that the YBCS population generally views genetic risk assessment as a luxury that is unattainable for the general public. The counselors also discussed many misconceptions surrounding insurance coverage when it comes to genetic risk assessment. There is also frequent confusion surrounding popular services, such as direct-to-consumer genetic testing companies. Additionally, the counselors clarified the risks of genetic risk assessment, which include general anxiety, survivorâ&#x20AC;&#x2122;s guilt, and inconclusive results that can potentially cause the patient more harm than good.

Content Development for Genetic Risk Assessment SCP-R Section Based on the findings from the literature review, genetic counselor interviews, and professional society guidelines, a list of relevant topics and barriers was generated to address in the web-based genetics risk assessment content. The barriers SALTMAN QUARTERLY

VOL. 13

45

chosen for inclusion in the content were the following: lack of knowledge of professional society guidelines that endorse genetic risk assessment; lack of knowledge on what genetic risk assessment entails; financial costs and insurance coverage; and privacy of results.

To address these barriers, the content included: information on professional society recommendations; what one might expect when getting counseled and/or tested; the difference between genetic counseling and genetic testing; and information on the confidentiality of genetic risk assessment as well as the Genetic Information Nondiscrimination Act of 2008 (GINA).8,20 In addition, the importance of re-testing as new research emerges was noted. Table 1 provides a sample of the survivorship care content, formatted as questions and answers.

In addition to providing information in this format, action steps on specific actions needed in order to follow the module and improve the symptoms or change behavior were included. The action steps for the genetic risk assessment module include finding a cancer genetic counselor, discussing potential inherited risk during a genetic counseling session, deciding if genetic testing is right for them, and taking action to prevent future cancer risk in the case the YBCS discover they carry inherited cancer risks (Figure 2). Overall, the length of the Genetic Risk Assessment Survivorship Care Plan is one 8x11 inch page.

While key information was summarized in the survivorship care plan, there

was additional content for YBCS to explore online. This module included a resources table with online and offline resources for YBCS and their healthcare providers to reference when making decisions and taking action related to genetic risk assessment (Table 2).

Discussion

Genetic risk assessment has significant clinical implications for YBCS and their families, but is under-utilized in this population. Through a multi-modal approach, this study derived an educational intervention to provide education on genetic risk assessment in order to improve the likelihood of YBCS undergoing genetic risk assessment. A literature search, key informant interviews and review of professional guidelines were key to deriving the evidence-based content for YBCS and their healthcare providers.

The theoretical framework that was used in developing the Genetic Risk Assessment SCP-R intervention is the Health Belief Model. The Health Belief Model discusses several concepts regarding human behavior and the uptake of health services.21 One concept described in the Health Belief Model is that of “perceived barriers”. These barriers are obstacles that prevent someone from carrying out a certain action regarding their health. Other concepts addressed in the Health Belief Model are the “perceived susceptibility, severity, and benefits”. The “perceived susceptibility” is how significant one believes in their chances of getting a condition,

What type of Information is here?

such as hereditary cancer. Similarly, “perceived severity” is how serious one believes the consequences of the condition are. The “perceived benefits” are what one believes they can gain from up taking a health decision. The multi-modal approach taken to develop the educational content addressed these concepts of the Health Belief Model.

For example, the literature search found the evidence necessary to identify and highlight the “perceived barriers” related to genetic risk assessment. “Perceived susceptibility” and “perceived severity” were addressed by presenting the evidence from Professional Society Guidelines about why genetic risk assessment is recommended for the wellbeing of YBCS and their family members. Finally, “perceived benefits” were described in the content through evidence-based material outlining what YBCS gain from taking the steps necessary to undergo genetic risk assessment. Examples of these benefits include reducing future cancer risk through surgery and the ability to make informed decisions about family planning.

The literature search was significant in identifying barriers that prevent YBCS from undergoing genetic risk assessment.

The specific barriers found to be prevalent in a variety of articles were addressed in our developed genetics content. Since research demonstrated that education is the ideal method in overcoming the present barriers,8,22,23 our group decided to approach this issue through an educational intervention. Our content includes information on the procedure of genetic risk

What organization provides this resource?

Table 2b: Resources for Healthcare Providers (HCPs) on genetic risk assessments and its many aspects.

46

SALTMAN QUARTERLY

VOL. 13

sqonline.ucsd.edu

RESEARCH

Figure 2: These action steps are included in the content for study participants to follow and aid them in the process of receiving genetic risk assessment. assessment while addressing common concerns of cost, privacy of results, and emotional distress. Our aim is that by providing YBCS with concise, understandable information regarding genetic risk assessment, numerous misconceptions can be cleared and YBCS can make informed decisions on assessing cancer genetic risk.

Key informant interviews were critical to generating and revising content. Genetic counselor interviews provided practical information, such as their hands-on experience with insurance coverage. These interviews also corroborated findings in published studies on barriers and education as an intervention. With their clinical expertise, they were able to provide clarity and interpretation of the Genetic Information Nondiscrimination Act of 2008,21 which prohibits the use of genetic information in health insurance and employment.

The barriers addressed in our genetics content were driven by current data on this topic. An editorial was recently published that outlines why cancer genetics is important to revisit and include in cancer survivorship care. The article lists barriers preventing cancer patients from undergoing genetic risk assessment; these barriers include cost concerns, lack of healthcare provider referral and emotional distress, similar to the barriers we identified.24 However, this article also included as barriers disparities in various racial groups and the likelihood of each racial group to seek genetic risk assessment. It would be a unique addition to our content in the future to address these racial barriers.

Since our research showed that a significant barrier that prevents women from undergoing genetic risk assessment is a lack of referral from healthcare provid-

ers, our content includes resources about genetic risk assessment for healthcare providers. Knowing that cancer care is dependent on the healthcare provider as well as the patient, we developed content intended for both audiences. The resources are categorized by relevance for YBCS versus healthcare providers to make it easier for both audiences to find information geared toward them. The goal is that by providing this information and resources to the healthcare providers directly, the gap of knowledge will be improved and an increase of referrals for genetic risk assessment will occur.

Conclusion

Following diagnosis, YBCS have many decisions to make regarding their treatment plan and overall health. One significant decision is whether or not to receive genetic risk assessment, and sadly, many YBCS are choosing to not undergo this important assessment. Professional societies clearly recommend that all YBCS should seek genetic counseling and get tested forBRCA1/BRCA2 mutations. Yet, barriers including lack of health care provider referral, emotional distress, cost concerns, and a general lack of education prevent YBCS from seeking genetic risk assessment. By conducting a systematic literature search, reviewing professional society guidelines, and conducting key-informant interviews, our group developed a Genetic Risk Assessment SCP-R comprised of ev-

Figure 3. Reproductive Health Survivorship Care Plan (SCP-R) intervention design The SCP-R intervention is distributed to young breast cancer survivors (YBCS) and their healthcare providers (HCPs).

Both the YBCS and the HCPs take the actionable steps laid out in the SCP-R and later note improvements in the various symptoms through self-report questionnaires.

sq.ucsd.edu

SALTMAN QUARTERLY

VOL. 13

47

idence-based content and resources that aim to increase the uptake of genetic risk assessment in this population. The genetics module will be incorporated into the web-based reproductive health survivorship care intervention that will be tested in a randomized controlled trial (RCT) (Figure 3). The results from the RCT over the next three years will determine how effective the genetics module was in increasing uptake of genetic risk assessment in YBCS. Based on the data from the RCT, we plan to reassess the intervention and plan dissemination if effective.

Acknowledgments

This study was financially supported by an undergraduate research scholarship from the Doris Howell Foundation’s Fleet Family Foundation and by the California Breast Cancer Research Program Translational Award 200B-0 144. I would also like to thank Dr. Irene Su, my mentor and the Principal Investigator of my research group, and Dr. Sally Dominick for assisting me in the process of completing this project. Finally, I would like to thank the group of genetic counselors (Dr. Lisa Madlensky, Lauren Korty, Diana De Rosa, and Kimberly Forbes) who gave me great advice and information on all genetics aspects of this project.

References

1. American Cancer Society. Breast cancer facts & figures 2015-2016, American Cancer Society (2016).

2. Facing Our Risk of Cancer Empowered. BRCA testing for young breast cancer survivors. (2016).

3. National Comprehensive Cancer Network. Adherence patterns to National Comprehensive Cancer Network (NCCN) guidelines for referral to cancer genetic professionals. Gynecol Oncol 138, 109–114 (2015).

4. Cvancarova, M. et al. Reproduction rates after cancer treatment: experience from the Norwegian radium hospital. J Clin Oncol 27:334-43 (2009).

5. Gold, EB et al. Dietary factors and vasomotor symptoms in breast cancer survivors: the WHEL Study. Menopause 13, 423-33 (2006).

6. Safarinejad, MR Shafiei N, Safa48

SALTMAN QUARTERLY

VOL. 13

rinejad S. Quality of life and sexual functioning in young women with early-stage breast cancer 1 year after lumpectomy.

Psychooncology 22, 1242-8 (2013).

7. Su, HI et al. Antimullerian hormone and inhibin B are hormone measures of ovarian function in late reproductive-aged breast cancer survivors. Cancer 116, 592-9 (2010).

8. Anderson, B et al. Barriers and facilitators for utilization of genetic counseling and risk assessment services in young female breast cancer survivors. J Cancer Epidemiol (2012).

9. Vadaparampil ST et al. From Observation to Intervention: Development of a psychoeducational intervention to increase uptake of BRCA genetic counseling among high-risk breast cancer survivors. J Cancer Educ 29, 709-19 (2014).

10. Finlay E, et al. Factors determining dissemination of results and uptake of genetic testing in families with known BRCA1/2 mutations. Genet Test 12, 81-91 (2008).

11. Lerman C, Schwartz M. Adherence and psychological adjustment among women at high risk for breast cancer.

Breast Cancer Res Treat 28, 145-55 (1993).

12. Wood, ME et al. Quality of cancer family history and referral for genetic counseling and testing among oncology practices: A pilot test of quality measures as part of the American Society of Clinical Oncology Quality Oncology Practice Initiative. J Clin Oncology 32, 824-829 (2014).

13. Bernhardt, BA Biesecker, BB Mastromarino, CL. Goals, benefits, and outcomes of genetic counseling: Client and genetic counselor assessment. Am J Med Genet 94, 189-197 (2016).

14. Susan G. Komen. Learn about genetic testing for BRCA1 and BRCA2 mutations at Susan G. Komen. (2016).

15. Sheppard, VB et al. African American women’s limited knowledge and experiences with genetic counseling for hereditary breast cancer. J Genet Couns 23, 311-22 (2014).

16. Robson, ME et al. American Society of Clinical Oncology Policy statement update: Genetic and Genomic Testing for Cancer Susceptibility J Clin Oncol 5, 893901 (2015).

17. American Congress of Obste-

tricians and Gynecologists. Hereditary cancer syndromes and risk assessment ACOG, (2016).

18. National Comprehensive Cancer Network. NCCN clinical practice guidelines in genetic/familial high-risk assessment: breast and ovarian. (2016).

19. Berliner, JF A. Cummings, S. NSGC practice guideline: Risk assessment and genetic counseling for hereditary breast and ovarian cancer. J Genet Counsel 22, 155–163. (2013).

20. Chieng WS, Lee SC. Establishing a cancer genetics programme in Asia - the Singapore experience. Hered Cancer Clin Pract 4, 126-35 (2006).

21. Janz NK, Champion VL, Stretcher VJ. Health Behavior and Health Education.

Theory, Research,and Practice. (San Francisco, Jossey-Bass, 2002).

22. Chieng WS, Lee SC. Establishing a cancer genetics programme in Asia - the Singapore experience. Hered Cancer Clin Pract 4, 126-35 (2006).

23. Thompson, HS et al. Psychosocial predictors of BRCA counseling and testing decisions among urban African-American women. Cancer Epidemiol Biomarkers Prev 11, 1579-85 (2002).

24. Ruddy, KJ Risendal, BC Garber, JE, et al. Cancer survivorship care: An opportunity to revisit cancer genetics. J Clin Oncology (2015).

WRITTEN BY DEEPIKA SURESH Deepika is a Biochemistry and Cell Biology major from Revelle College. She will graduate in 2016.

sqonline.ucsd.edu

REVIEW

Intelligent Networks Erika Johannessen | Biophysics

Introduction

Stretching back to ancient Greek speculations on our rational nature, discussion of the features and limits of human intelligence has played a notable historic role in nearly all academic disciplines. Today, we have the advantage of approaching this question through neuroscience. There are currently two main theories for the neurological basis of intelligence: the neural efficiency hypothesis (NEH) and the parieto-frontal integration theory (P-FIT).

NEH asserts that more intelligent individuals require less mental effort to complete cognitive tasks due to energetic cost minimization of the brain, while P-FIT proposes a system of regions in the parietal and frontal cortices that work together to sample choices and make decisions.

Although both theories are incredibly important for understanding the neurological basis of intelligence, research shows that neither alone can account for all of the findings of intelligence variation within our species. In this short review, I develop the views of Ed Bullmore and Olaf Sporns to show that network theory provides a unifying framework in which to discuss the neural basis of intelligence.

Specifically, I argue that network theory can explain both the global transfer of information supported by P-FIT and the efficient local clustering backed by NEH.

Neural Efficiency

In 1988, Richard Haier used PET to reveal a negative correlation between regional cortical glucose levels and intelligent test scores, implying that more intelligent individuals use fewer resources during cognitive tasks.

However, this decrease of mental effort only holds true for tasks that require localized brain regions to complete (e.g. symbol recognition).5 The inverse is true when task difficulty increases: when faced with tasks that require recruitment of several brain regions (e.g., mental math), more intelligent people exhibit a larger desynsq.ucsd.edu

Figure 1: Parieto-frontal integration theory of intelligence. This figure shows the Brodmann Areas associated with intelligence and the arcuate fasciculus in yellow, which is the white matter tract connecting them. Adapted with permission from Dreary et al. 2010.8

chronization of neurons, which is thought to signal increasing cognitive effort.

For this reason, NEH has been criticized for only holding true for specific types of tasks.2 Alone, it is not a complete neurological theory of intelligence.

Parieto-Frontal Integration

The parieto-frontal integration theory is based on over 40 neuroimaging and lesion studies revealing a correlation between size and activity of parietal and frontal cortices and intelligence testing scores.4 In addition, studies reveal a critical role of thick white matter tracts, such as the arcuate fasciculus, which connects parietal and frontal regions. This connectivity pattern implies that communication between these areas fundamentally influences intelligence. The observed increase in activity of these areas directly contradicts the NEH and only provides structural similarities of more intelligent brains. Still, these commonalties do not provide an explanation for why this

organization exists nor how it is related to energy consumption in the brain.

Network Theory

Neural network theory describes the brain as a set of nodes (neurons) connect by edges (synapses).4 Using graph theoretical analysis of EEG, fMRI, and DTI data, Bullmore and Sporns showed that the brain has characteristics of a complex network that is neither perfectly ordered nor random. They hypothesized that the brain negotiates a trade-off between wiring cost (the energetic cost to make and maintain connections) and flexible information transfer (the ability to share information efficiently). In order to function flexibly, the brain must â&#x20AC;&#x153;spendâ&#x20AC;? energy to build and maintain active neural circuits. From a physics perspective, short distance connections are favorable to longer ones. However, if cost-minimization were the only factor in the development of the brain, it would be organized in a lat SALTMAN QUARTERLY

VOL. 13

49

Figure 2: The brain is a complex network. Graph theoretical analysis reveals that the brain is a complex network that is neither ordered nor random. This arrangement allows for modularity of local clusters of neurons to carry out specified functions efficiently.

In addition, central hubs allow information to be transferred across through more costly long distance tracts. Adapted with permission from Bullmore and Sporns 2012.8

tice topology, which would not allow for an efficient transfer of information across larger cortical regions.9 The actual topology of the brain is somewhere between a lattice and a random structure, containing both short-distance connections that give rise to modularity and longer distance connections that support fast communication across long distances.9 Thus, the brain spends some of its energy budget to facilitate flexible functionality. The features of a complex network that are present in the human connectome include small-world topology, modularity, and central hubs (Fig 3). The resulting neural topology has features that accommodate both NEH and P-FIT modeling, as this spatial configuration allows for an energetically favored minimization of cost while still allowing more costly long-range projections to exist.

The principles of network theory that outline the minimization of cost expenditure could help to substantiate the findings of the neural efficiency hypothesis.

In particular, the modularity of densely packed regions of neurons allows for the specification of function and a great deal of local efficiency.12 These clusters of neurons are not only close together in space, but have similar biochemical and biophysical properties which allow them to carry out specialized tasks in a synchronous 50

SALTMAN QUARTERLY

VOL. 13

manner. Modularity explains the findings of Haier’s original study in 1988, which showed low local metabolic rates indicating “densely packed” regions.5 Modular structure may also explains why simple tasks, particularly those only requiring recruitment of one brain area, obey the neural efficiency hypothesis while more complicated ones requiring activation of multiple modules do not.

Communication between hubs requires high cost and long-range connections, which opposes neural efficiency, but allows for better functionality and sharing of information required for difficult tasks.

These “expensive” tracts parallel P-FIT’s inclusion of the integrity long white matter tracts in intelligence scores. This suggests that the brain is capable of overcoming an energetic barrier in order to facilitate better communication and function. Interestingly, these central hubs appear to be most abundant in parietal and frontal cortices, suggesting that these regions compromise the central backbone of intelligence that is highlighted by P-FIT.

Conclusion

A small number studies have explored the relationship between complex network features of the brain and psychometrically measured intelligence, but none have cen-

tered on the changes in network dynamics with varying task difficulty.1,1,2 Analyzing variations in network dynamics over time is key to integrating NEH and P-FIT. In order to understand our brain’s capacity for problem solving, the relationship between network features that allow for regional efficiency should be discussed in tandem with the integration of information on a less cost-conservative global level. By providing a mathematical framework to model our connectome, network theory allows for a more complete picture of the neurological basis of human intelligence differences.

References

1. Langer, Nicolas, et al. “Functional Brain Network Efficiency Predicts Intelligence.” Human Brain Mapping. 33.6, 1393406 (2011).

2. Neubauer, Alijoscha, and Andreas Fink. “Intelligence and Neural Efficiency.”

Neuroscience and Biobehavioral Reviews 33, 1004-23 (2009).

3. Jung, Rex, and Richard Haier. “The Parieto-Frontal Integration Theory (P-Fit) of Intelligence.” Behavioral and Brain Sciences 30, 135-80 (2007).

4. Bullmore, Ed, and Olaf Sporns.

“Complex Brain Networks: Graph Theoretical Analysis of Structural and Funcsqonline.ucsd.edu

REVIEW

a) Small-World Attributes: In a small world-environment, any two neurons can communicate through a relatively small number of short-distance steps, thus enhancing information transfer.10

b) Modularity: Modules are spatially clustered neural populations that have similar biophysical properties, allowing them to carry out specialized tasks quickly and efficiently.11

c) Connector Hubs: Central hubs are neurons that have a high number of short distance connections with neighbors, making them the major intersections of information flow in the brain.12 Hubs play a major role in connection between modules, specifically for difficult tasks.

Figure 3: Features of a complex network tional Systems.” Nature Reviews Neuroscience 10.3, 186-98 (2009).

5. Haier, Richard, Benjamin Siegal, Keith Nuechterlein, Erin Hazlett, and Joseph Wu. “Cortical Glucose Metabolic Rate Correlates of Abstract Reasoning and Attention Studied with Positron Emission Tomography.” Intelligence 12, 199-217 (1988):.

6. Reichle, Erik D., Patricia A. Carpenter, and Marcel Adam Just. “The Neural Bases of Strategy and Skill in Sentence– Picture Verification.” Cognitive Psychology 40.4, 261-95. (2000).

7. Doppelmayr, M., W. Klimesch, K. Hödlmoser, P. Sauseng, and W. Gruber. “Intelligence Related Upper Alpha Desynchronization in a Semantic Memory Task.” Brain Research Bulletin 66, no. 2 171-77. (2005).

8. Deary, Ian J., Lars Penke, and Wensq.ucsd.edu

dy Johnson. “The Neuroscience of Human Intelligence Differences.” Nature Reviews Neuroscience Nat Rev Neurosci (2010).

9. Bullmore, Ed, and Olaf Sporns. “The Economy of Brain Network Organization.”

Nature Reviews Neuroscience 13, 336-49.

(2012).

10. Watts, Duncan, Stephen Strogatz.

“Collective Dynamics of ‘Small-World’ Networks.” Nature 393, 440-442. (1997).

11. Chen, Z. J., Y. He, P. Rosa-Neto, J. Germann, and A. C. Evans. “Revealing Modular Architecture of Human Brain Structural Networks by Using Cortical Thickness from MRI.” Cerebral Cortex 18.10, 2374-381. (2008).

12. Sporns, Olaf, Christopher J. Honey, and Rolf Kötter. “Identification and Classification of Hubs in Brain Networks.” PLoS ONE 2.10 (2007).

13. Li, Y., Y. Liu, J. Li, W. Qin, K. Li, C. Yu, and T. Jiang. “Brain Anatomical Network and Intelligence.” NeuroImage 47 (2009): 14. Heuvel, M. P. Van Den, C. J. Stam, R. S. Kahn, and H. E. Hulshoff Pol. “Efficiency of Functional Brain Networks and Intellectual Performance.” Journal of Neuroscience 29.23, 7619-624. (2009).

WRITTEN BY ERIKA JOHANNESSEN Erika is a Physics with Specialization in Biophysics major from Muir College. She will graduate in 2016.

SALTMAN QUARTERLY

VOL. 13

51

La Jolla Cove as the tide comes in.

Photo by Juan Jimenez

SENIOR

Students in the Biology Honors program are required to complete a written thesis detailing their scientific research progress. The Senior Honors Thesis section, which presents the abstracts of their individual theses, highlights the achievements of the accomplished undergraduate researchers in the class of 2016.

HONORS THESES

THE DIVISION OF BIOLOGICAL SCIENCES SENIOR HONORS THESIS PROGRAM (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. Each student in the program has a faculty mentor who provides guidance throughout the year. During the spring quarter of each year, students in the program participate in a research showcase which gives them the opportunity to discuss their research with faculty and their fellow students. These are the abstracts of all the exceptional research projects conducted by undergraduates in the program during the 2015-16 academic year.

Effect of Stable and Fluctuating Low pH on Growth and Feeding Behavior of the limpet Tectura paleacea

Yaamini Venkataraman, John Muir College, General Biology and Environmental Systems: Environmental Policy Majors

It is common for marine invertebrates to experience altered metabolism and growth under experimental ocean acidification (OA) conditions. For the diverse animals living in nearshore surfgrass (Phyllospadix spp) habitats, however, reductions in local pH due to OA may be buffered by surfgrass photosynthesis, thereby causing inhabitants to experience daily fluctuations in pH that are not typically accounted for in OA experiments. We therefore exposed surfgrass limpets (Tectura paleacea) to both stable (without surfgrass) and fluctuating (with surfgrass) ambient and reduced pH treatments to determine the potential impact of realistic OA conditions on animal feeding behavior and growth. It is hypothesized that reduced pH will negatively impact animal growth and feeding, but to a lesser extent in fluctuating conditions. Results from this study will highlight the importance of considering local habitat dynamics when trying to determine the realistic impacts of future ocean conditions on marine organisms PI: Jennifer Taylor, Ph.D. Marine Biology Research Division, Scripps Institution of Oceanography

Six3: Role in Circadian Rhythms and Metabolism

Haley Oosterhouse, John Muir College, General Biology Major, Music Minor

54

SALTMAN QUARTERLY

The suprachiasmatic nucleus (SCN) of the hypothalamus coordinates circadian rhythms in the body. SIX3 is a homeodomain transcription factor and is required for normal brain and SCN neuron development. However, its role in adulthood is unknown. Deletion of Six3 in Six3Flox:SynapsinCre mice, a mouse model allowing specific deletion of Six3 in mature neurons, results in an abnormal metabolic phenotype, affecting body weight and fertility. Here, we examine the origin of the differing phenotypes in the Six3Flox:SynapsinCre mice. We first performed lineage tracing of SynapsinCre expression by tracing cells expressing RosaLacZ. We show that SynapsinCre is expressed only in certain structures of the brain, including the SCN, where it deletes Six3. We next investigated the effects on circadian rhythms of Six3Flox:SynapsinCre using running-wheel activity in male mice.

We show that the loss of Six3 in neurons leads to abnormal circadian rhythms, indicating that Six3 is critical for maintaining SCN output.

PI: Pamela Mellon, Ph.D., UC San Diego School of Medicine, Division of Reproductive Medicine

VOL. 13

sqonline.ucsd.edu

THESIS

M13 Phage Display of Thermus aquaticus Variable Protein Bio-macromolecular crystallography requires samples (e.g., proteins or RNA) that form large, welldefined crystals in order to determine molecular structures through X-ray diffraction. However, there are still many proteins that remain non-crystallizable, which can be due to their unstable structure or unfavorable entropy. Thermus aquaticus variable protein (TaqVP) can potentially aid in crystallizing these difficult proteins by acting as a crystallization chaperone. Thermostable proteins tend to crystallize better, and TaqVP has been crystallized and is thermostable up to 90°C. TaqVP can also potentially accommodate ~1014 different sequences, with a potential to bind many proteins.

TaqVP has been cloned into a phagemid vector for M13 phage display via the pIII coat protein, and its expression on the phage coat is being determined. If expression on the phage is successful, a phage library will be generated to select a TaqVP mutant that possesses high affinity and specificity for a crystallization candidate protein.

PI: Partho Ghosh, Ph. D., UC San Diego Department of Chemistry and Biochemistry

Adrian Bahn-Suh, Roger Revelle College, Biochemistry & Cell Biology Major

In Vivo 2-photon imaging of adult born dentate granule cells The growth and refinement of dendritic arbors are crucial steps in the formation of neuronal circuits; however, the processes that regulate dendrite morphogenesis are not yet fully understood. Some neuronal types undergo transient dendritic branch overgrowth, followed by extensive pruning. Here we studied the development of adult-born dentate granule cells (DGCs) in the mouse hippocampus using in vivo 2-photon imaging, following nascent dendrites over a period of several weeks. By knocking-down the expression of different proteins using shRNA we were able to study the effect of various molecular pathways on dendrite growth and pruning, thus obtaining novel insights on the process of dendrite morphogenesis.

Thanh Tran, Muir College, Biochemistry and Cell Biology Major, Global Health Minor

PI: Fred H. Gage, Ph.D., Salk Institute, Department of Neurobiology

CRISPR/Cas9 Mediated Double Mutant Generation of Tandem Genes VICTR and VICTL1 in Arabidopsis Thaliana The advent of CRISPR/Cas9 genomic editing has allowed for the creation of precise, targeted changes in the genome, and our goal is to utilize CRISPR/Cas9 genomic editing to characterize highly homologous tandem genes VICTR and VICTL1 in Arabidopsis thaliana plants. VICTR is a member of the Nucleotide Binding â&#x20AC;&#x201D; Leucine Rich Repeat (NB-LRR) immune receptor family and was identified by the Schroeder lab for its role in crosstalk between ABA plant-stress-hormone signaling and immune responses. Since NB-LRR genes are often functionally redundant and clustered in the genome, generating avictr victl double mutant could allow us to fully characterize this immune receptor gene. We transfected victl1 mutant plants with a CRISPR/Cas9 construct targeting the VICTR gene. The generated victr victl1 double mutants were confirmed by comparing them to victr mutant phenotypes, demonstrating the efficacy of CRISPR/Cas9 constructs in targeted mutagenesis in plants. Eliminating the CRISPR/Cas9 construct is in progress.

Vince Garin, John Muir College, Biochemistry and Cell Biology Major

PI: Julian Schroeder, Ph.D., UC San Diego, Department of Biology

sq.ucsd.edu

SALTMAN QUARTERLY

VOL. 13

55

Immune Priming of Day-Old Adult Apis mellifera against Nosema ceranae infection

Andrey Rubanov, Roger Revelle College, Human Biology Major

Apis mellifera, the Western honey bee, is an essential pollinator in a multitude of worldwide ecosystems. The honey bee occupies a prominent niche in the agricultural industry. Thus, preserving colony health is important. In particular, the microsporidian pathogen, Nosema ceranae, is a major contributor to poor colony health. A prophylactic remedy, immune priming, is to inoculate A. mellifera with heat-killed N. ceranae spores to strengthen the immune system for future exposure.

Bees that have been immune primed upon adult emergence and subsequently challenged with N. ceranae have demonstrated lower spore count levels upon death than bees not primed (5227 Âą11799 versus 90333Âą314469). However, this is based upon two trials and there may be an issue with immune primed bees having shorter lifespans, perhaps because of investing more in the activation of their Toll immune gene pathway. Therefore, more trials are needed.

PI: James Nieh, Ph.D., UC San Diego, Section Chair of Ecology, Behavior, and Evolution

The Role of Kisspeptin Signaling in Brown Adipose Tissue in Mice

Nuha Marooki, Roger Revelle College, Biochemistry and Cell Biology Major

The peptide kisspeptin and its receptor, Kiss1r, regulate reproduction by stimulating reproductive hormone secretion from the brain. Besides controlling reproduction, Kiss1r was also recently shown to regulate body weight (BW), metabolism, and glucose tolerance. Outside the brain, Kiss1r is found in the gonads, liver, pancreas, white adipose tissue, and brown adipose tissue (BAT). BAT is known for its high density of mitochondria, making it an important tissue for metabolic regulation.

Here, we tested the hypothesis that kisspeptin signaling in BAT influences BW, metabolism, and/or glucose tolerance. Comparing wild-type mice with conditional Kiss1r knockout in BAT, we found that absent Kiss1r in BAT does not alter reproduction, BW, metabolism, or glucose tolerance. This suggests that Kiss1r in the BAT does not have a requisite role in metabolic or glucose regulation, and the previously-observed obesity and metabolic dysfunction in global Kiss1r KOs are due to impaired kisspeptin signaling in another tissue(s).

PI: Alexander S. Kauffman, Ph.D., UC San Diego School of Medicine, Department of Reproductive Medicine

Factor Analysis of Temperament and Personality Traits in Bipolar Patients: Correlates with Comorbidity and Disorder Severity

Frank Qiu, Thurgood Marshall College, Physiology & Neuroscience and Mathematics Majors

Temperament and personality traits have been suggested as endophenotypes for bipolar disorder based on several lines of evidence. Previous work suggested an anxious-reactive factor identified across temperament and personality inventories that produced significant group discrimination.

We have further characterized this factor structure in a sample of 1195 bipolar patients. Dimension reduction identified 18 factors explaining 39% of the variance. The two largest factors reflected affective instability and general anxiety. All factors revealed specificity for associated clinical features in a predictable pattern. Subsequent cluster analysis identified one group with a strong lack of general anxiety and low affective instability represented. The remaining subjects could be distinguished into two groups by the presence of either more positive characteristics, including persistence, spirituality, expressivity, and humor, or more negative characteristics of depression and anxiety. These results suggest that cross-analyses of temperament and personality inventories may have utility for identifying clinical profiles in bipolar patients.

PI: Tiffany A. Greenwood, Ph.D., UC San Diego School of Medicine, Department of Psychiatry

56

SALTMAN QUARTERLY

VOL. 13

sqonline.ucsd.edu

THESIS

How do honey bees attract receivers when signaling with waggle and tremble dances?

Both waggle and tremble dances are vibratory signals known to elicit following behavior in neighboring bees. However, it is not known how tremble dances attract receivers. We trained honey bees (Apis mellifera) to a feeder where half were artificially attacked to elicit tremble dancing before being tracked back to the hive and filmed along with neighboring bees to capture both dancer and follower behavior. We found that waggle dancers attracted followers from a greater distance than tremble dancers, indicating waggle dancing to be more attractive than tremble dancing. We also found that, once near the dancer, a tremble follower would prefer to be oriented with her head facing the dancer’s head while a waggle follower would prefer to be oriented with her head perpendicular to the dancer’s abdomen. We are continuing to analyze our data, but these results provide the first insights into how tremble dancers broadcast their dance information.

Calvin Lam, Roger Revelle College, General Biology Major & Biophysics Major

PI: James Nieh, Ph.D., UC San Diego Biology, Section of Ecology, Behavior and Evolution

Sequences complementary to the miRNA 3’ end are necessary for specific miRNA targeting MicroRNAs (miRNAs), small RNAs 20-22 nucleotides long, post-transcriptionally regulate messenger RNAs (mRNAs) via imperfect base pairing. MiRNA families, such as the let-7 family in C. elegans, have identical seed sequences (nucleotides 2-8 from the 5’ end), which are known to be critical in miRNA targeting of mRNAs. Thus, families are thought to have overlapping targets.

However, lin-41 has been established to be regulated only by let-7, suggesting that there may be nonseed sequences that are important for specific miRNA-mRNA interaction. Using CRISPR/Cas9, we created a strain in which let-7 binding sites on the lin-41 3’UTR are modified at the region where the 3’ end of let-7 binds. Using a viability assay, we showed that this modification changes the specificity of lin-41. This work furthers our understanding of miRNA targeting by providing insight into how differences on the 3’ end of the miRNA can affect miRNA-mRNA interaction.

PI: Amy Pasquinelli, PhD, UC San Diego Division of Biological Sciences, Section of Molecular Biology

Jessica L. Huang, Roger Revelle College, Molecular Biology Major, Business Minor

The Role of Rab35 GTPase and endosomal trafficking in T-tubule remodeling T-tubules are specialized muscle cell plasma membrane invaginations critical for contraction, yet T-tubule formation and remodeling is poorly understood. We are studying these processes in Drosophila pupal abdominal muscles that disassemble and reassemble T-tubules during metamorphosis. We identified genes important to T-tubule remodeling, including several known regulators of endosomal trafficking and recycling. Specifically, Rab35 GTPase knockdown or dominant negative in muscle accelerated T-tubule disassembly but blocked reassembly with an accumulation of membrane vacuoles.

I showed that the Rab35 loss of function phenotype requires dynamin large GTPase activity, pointing to a shared and regulated endosomal pathway involved in T-tubule disassembly. Knockdown of other known Rab35 pathway components, such as CenB1A Arf6 GAP, or of the myopathy-relevant gene, mtm PI3-phosphatase, resulted in phenocopy of Rab35 depletion. My findings point to a role for regulated Rab35 endosomal recycling and its possible novel connection to an endosomal Mtm phosphoinositide pathway in regulating T-tubule remodeling.

Humphrey (Tzu-Han) Lin, Earl Warren College, Biochemistry and Cell Biology Major

PI: Amy Kiger, Ph.D. UC San Diego Department of Biological Sciences

sq.ucsd.edu

SALTMAN QUARTERLY

VOL. 13

57

Neurotransmitter Plasticity after Chronic Social Defeat

Christiana Stark, John Muir College, Molecular Biology Major

The underlying mechanism that allows one to cope with social stress is a process that may play a substantial role in the development of stress associated neuropsychiatric conditions. The response to stress may include neurotransmitter respecification in which a stressful stimulus causes neurons to synthesize a different neurotransmitter than before (Dulcis et al.,2013).In this project rats were exposed to an aggressor to serve as a model for stress. Following social defeat, neurotransmitter levels in various brain nuclei were quantified. The susceptible group, compared to resilient and controls, displayed significant neurotransmitter plasticity in the Raphe, an area associated with stress processing. We are investigating the molecular identity of neurons undergoing neurotransmitter plasticity by testing neurotransmitters expressed in the Raphe after social defeat. These changes may highlight differences in individual’s ability to cope with stress, which could provide insights into the mechanism behind stress related mood disorders and lead to advanced treatments.

PI: Davide Dulcis, Ph.D., UC San Diego School of Medicine, Department of Psychiatry

Role of Myelin Basic Protein in the Autoimmune Basis of Neuropathic Pain

Jake Leung, John Muir College, Human Biology Major

Neuropathic pain (NP) after peripheral nerve injury is a debilitating condition without effective treatments. Following nerve injury, Myelin Basic Protein (MBP), an integral component of myelin, is proteolyzed into smaller fragments. Several particular of the degraded (d)MBP fragments induce NP. Understanding the pathways responsible for generation and activity of dMBP in NP can translate to development of effective novel therapeutics. In this study, we hypothesized that dMBP is transported axonally from the site of peripheral nerve injury towards the central nervous system. A chronic constriction injury to sciatic nerve was utilized to induce NP in rats. The formation of dMBP in sciatic nerve segments was measured using conformation-specific antibody by immunoblotting and immunofluorescent imaging. Preliminary results support the hypothesis that dMBP released at the peripheral injury site is transported axonally along the damaged nervous system to sustain the chronic state of NP. PI: Veronica Shubayev, M.D., UC San Diego School of Medicine, Department of Anesthesiology

Structural Motifs of Ly6h That Facilitate Regulation of Nicotinic Acetylcholine Receptors

Clifford Liu Thurgood Marshall College, Biochemistry & Cell Biology and Sociocultural Anthropology Majors, Psychology Minor

58

SALTMAN QUARTERLY

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that contribute to cognition and pathophysiological conditions, such as Alzheimer’s disease. We have previously shown that several proteins in the Ly6 family are able to complex with and modulate nAChR activity. Since all Ly6 proteins are predicted to share a common tertiary structure with a three finger domain, we hypothesized that one or more of these fingers interacts with and regulates nAChR function. To test this hypothesis, we generated and assayed deletion constructs of Ly6h, an endogenous mammalian Ly6 protein required for normal intracellular trafficking of nAChRs and suppression of nAChR currents in the brain. Our data suggest that loop 3 is required for Ly6h to form stable complexes with both α7 and α4β2 nAChRs, and to suppress α4β2 function. These studies provide insight into regulation of neurotransmitter receptors by Ly6 proteins and suggest avenues to correct aberrant signaling in disease states.

PI: William Joiner, Ph.D., UC San Diego Department of Pharmacology

VOL. 13

sqonline.ucsd.edu

THESIS

TMZ-induced microRNA degradation upregulates DNA repair Glioblastoma is the most common form of primary brain tumor. It is an aggressively infiltrative tumor that is intrinsically resistant to DNA damaging agents, including conventional chemotherapy and radiation therapy. Our lab previously demonstrated that this resistance is mediated by the microRNA miR-181d. Here we demonstrate the molecular mechanism by which miR-181d regulates key DNA repair processes, including homologous recombination (HR). Using unbiased profiling experiments and miRNA binding site prediction algorithms, we found key HR genes as downstream affecters of miR-181d. Exogenous expression of 181d suppresses HR capacity in glioblastoma. Moreover, induction of DNA damage triggers degradation of miR-181d, causing up-regulation of HR and resistance to chemotherapy and radiation therapy. Consistent with these observation, key HR genes such as Rad 51, are significantly up-regulated in clinical glioblastoma specimens after chemotherapy. Our results indicated miR-181d is a master regulator miRNA that determines glioblastoma response to radiation therapy and chemotherapy.

Thien Nguyen, John Muir College, Neuroscience and Physiology Major, Sociology and Psychology Minors

PI: Dr. Clark Chen, M.D., Ph.D., UC San Diego School of Medicine, Department of Neurosurgery

The Effects of Hypoxia on the Proliferation and Differentiation of Trophoblast Stem Cells and Labyrinth Trophoblast Progenitors Trophoblast stem (TS) cells, derived from the polar trophectoderm, differentiate into all trophoblast lineage of the placenta and contribute to healthy placental development. Dysfunctional differentiation can be characteristic of placental insufficiency. Studies show that a prolonged hypoxic environment can adversely alter placental development, affecting cell proliferation and differentiation. Thus, we hypothesized that hypoxia affects proliferative trophoblast cells. To investigate the effects of hypoxia, mouse TS cells and two distinct TS cell subpopulations, Sca1+ and Epcam+, were independently cultured in hypoxic (1% O2) and normoxic chambers in proliferative and differentiating conditions.

Histological and qRTPCR analyses of cultures showed that hypoxia increased cell growth in proliferative conditions. In differentiating conditions, there was increased growth accompanied by prolonged expression of progenitor genes, particularly promoting differentiation to glycogen cells, shown by increased glycogen accumulation and elevated Aldh1a3 and Pcdh12gene expression. These results show that hypoxia alters both trophoblast cell proliferation and differentiation.

Priscilla Vu, Sixth College, Human Biology Major, Business Minor

PI: David Natale, Ph.D., UC San Diego School of Medicine, Department of Reproductive Medicine

sq.ucsd.edu

SALTMAN QUARTERLY

VOL. 13

59

Role of LST8 in mTOR Activity

Matthew Chan, Sixth College, Physiology and Neuroscience Major

The mechanistic target of rapamycin (mTOR) is a protein kinase that regulates cellular growth and proliferation. In mammals, mTOR exists in two complexes: mTORC1 and mTORC2. Both complexes contain the protein LST8, but the exact function of LST8 in the mTOR complex is unknown. Previous studies have shown that the level of Hsp70 associated to mTOR increases when bound LST8 levels decrease. Furthermore, attempts to use deuterium exchange mass spectrometry (DXMS) to analyze the structure mTOR in the absence of LST8 were unsuccessful.

Taken together, these studies suggest that LST8 is necessary for stable mTOR folding and full kinase activity. To test this hypothesis, we performed in vitro mTOR kinase reactions and found that LST8 association increases mTOR activity. These results demonstrate that LST8 plays a positive role in modulating mTOR function, and that LST8 may be chemotherapeutic target for treating tumorigenesis.

PI: Darren E. Casteel, Ph.D., UC San Diego School of Medicine

Foraging preferences of the invasive Argentine ant, Linepithema humile

Anna DiPaola, John Muir College, Ecology, Behavior and Evolution Major

The Argentine ant (Linepithema humile) disrupts native ant assemblages worldwide and displaces most native ants in the process. In invaded areas the Argentine ant dominates above-ground foraging, and its foraging preferences may affect arthropod food webs differently than the preferences of native ants. It remains unclear, however, whether the Argentine ant acts as a predator or a scavenger.

To learn more about this issue, I presented free-living Argentine ant colonies with different species of live and dead arthropods to test the extent to which the Argentine ants feeds on carrion versus live prey. Using these paired comparisons, I hypothesized that recruitment and rate of consumption would depend on the species of the prey item and whether it was alive or dead. Understanding the Argentine antâ&#x20AC;&#x2122;s foraging preferences helps us to clarify the ecological function of this abundant invader and to predict how arthropod community structure changes in response to invasion.

PI: David Holway, Ph.D., Division of Biological Sciences, Professor and Vice Chair of the Ecology, Behavior and Evolution section

Effects of Normal Paraneurial Tissues and Adhesions on Strain Gradients in Rat Sciatic Nerves

Rushil Patel, John Muir College, Physiology and Neuroscience Major

Stretching peripheral nerves during joint movement results in regional differences in their strain (deformation). Surgical repair often requires decompression of soft tissue adhesions (paraneurium) surrounding the nerve, and results in formation of new scar-like adhesions over time. The effects of paraneurium and scar-like adhesions on nerve biomechanics are unknown. To determine the role of these adhesions on the distribution of nerve strain, a rat sciatic nerve model was used. Strain was measured from digital images before and after decompression with the nerve relaxed and stretched based on knee and ankle configurations. After six weeks, the sciatic nerve was re-exposed and mechanical analysis repeated. Prior to decompression of paraneurial and scar-like adhesion formation, distal strain was significantly higher than proximal strain. After decompression, there was no significant difference between proximal and distal strains. Thus, both normal and surgically-induced strain adhesions created a similar strain gradient, which was eliminated by decompression.

PI: Partho Ghosh, Ph. D., UC San Diego Department of Chemistry and Biochemistry

60

SALTMAN QUARTERLY

VOL. 13

sqonline.ucsd.edu

THESIS

The Effect of PHLPP2 Removal in Mice on Cell Survival in the Brain A newly found serine/threonine phosphatase PHLPP can directly dephosphorylate several members of the AGC family of kinases, including Akt. There are two isoforms of PHLPP: PHLPP1 and PHLPP2. Our lab has demonstrated that removal of PHLPP1 in astrocytes increases Akt activity leading to protection from ischemic damage. However the role of PHLPP2 is unknown. Astrocytes have been isolated from global PHLPP2 knockout (KO) mice and cell death and the activation of signaling pathways following different stressors analyzed. Remarkably, cellular viability was higher in KO than wild-type astrocytes following metabolic stress and simulated ischemia, however Akt activity was unaltered. Thus, deletion of PHLPP2 seems to increase cell survival following stress; however its target is unknown. We will continue to investigate the protective effect of PHLPP2 deletion and the inflammatory response following wound injury in wild-type and KO astrocytes.

Findings will help identify new therapeutic targets for protection following brain injury.

PI: Nicole H. Purcell, Ph.D., UC San Diego School of Medicine, Department of Pharmacology Biology

Sara Hakim, Roger Revelle College, Biochemistry & Cell Biology Major, Political Science Minor

Elucidating the Brainstem Circuitry Responsible for Mediating Motor Control of the Forelimb

Two brainstem systems that are postulated to mediate direct control of peripheral musculature from corticospinal neurons are the rubrospinal and reticulospinal tracts, which originate from the red and reticular nuclei, respectively. Our studies have utilized a viral intersectional approach to target genetically encoded axonal tracers to specific subsets of brainstem populations projecting to lower cervical segments responsible for the control of distal musculature. Our results demonstrate that these two key brainstem nuclei possess unique patterns of innervation to the spinal cord and are likely to modulate very distinct aspects of behavior. Ongoing studies will use the viral intersectional system to target retrograde transynaptic tracers (rabies virus) to specific subsets of reticulo and rubrospinal projections to identify monosynaptic inputs driving these neurons. Together, these studies will provide greater insight into the synaptic circuitry responsible for mediating fine motor control of the forelimb.

PI: Mark Tuszynski, M.D., Ph.D., UC San Diego School of Medicine, Department of Neurosciences

Eric Ovruchesky, Muir College, Neuroscience and Physiology Major

Neural Differentiation of Rat Embryonic Stem Cells Embryonic stem cells (ESCs) and ESC-derived neural stem cells (ES-NSCs)are promising potential therapies for repair of spinal cord injury (SCI). Rat ES-NSCs were therefore tested in vitro and in vivo for their ability to differentiate into the neurons and glia necessary for SCI repair. Rat ESCs were driven toward an NSC fate. Midway through the differentiation protocol, the cells tested positive for NSC markers and negative for pluripotency markers, confirming successful derivation of ES-NSCs. Some of these â&#x20AC;&#x2DC;intermediateâ&#x20AC;&#x2122; cells were allowed to complete differentiation in vitro, and eventually expressed markers of neurons and glia, confirming their progression beyond the NSC stage. In contrast,cells grafted into sites of rat SCI continued to express NSC markers nestin and vimentin, and failed to differentiate into neurons and glia. Thus, although successful differentiation of ES-NSCs was achieved in vitro, further experimentation is required to attain in vivo differentiation of ES-NSCs in SCI. PI: Ephron S. Rosenzweig, Ph.D., UC San Diego School of Medicine, Department of Neurosciences

sq.ucsd.edu

Arthi Rao, John Muir College, Biochemistry/ Cell Biology Major

SALTMAN QUARTERLY

VOL. 13

61

The effect of murine Schlafen proteins on HIV

Qingyue Zhou, Roger Revelle College, General Biology Major, Business Minor

Human Schlafen protein 11 has been proved to inhibit HIV, human immunodeficiency virus, in a codon-usage-based manner. The purpose of this project is to compare the anti-retroviral effect of murine Schlafen proteins to that of human Schlafen 11. Eight murine Schlafen DNAs which are mSlfn1, mSlfn2, mSlfn3, mSlfn5, mSlfn8, a shorter analog of mSlfn8, mSlfn9 and mSlfn10, along with HIV (pnl43) are transfected into 293T cells and harvested for western blot and qPCR. Human Schlafen 11 is used as positive control whereas wildtype GFP serves as the negative control. Western blot is probed for p24, which is a cleaved product of p55, a tag for HIV. The absence of bands near 24kDa on western blot would prove the inhibition on HIV by the murine Schlafen, and the level of mRNA quantified by qPCR would show whether the inhibition is pre or post transcription.

PI: Michael David, Pharm.D., Ph.D., UC San Diego, Division of Biological Sciences

Development of a High-Content Axon Regeneration Screening Assay

Philip Canete, Muir College, Physiology and Neuroscience Major

Genome-wide transcriptional gene expression analysis has gained major prominence in the last decade. This method of acquiring data has the great potential to generate whole gene regulation and interaction profiles for adult sensory neurons within an injury model in vivo. A major benefit of big data is the identification of hundreds to thousands of potential candidates, however these candidates must ultimately be subjected to further screening methods. We made use of cost efficient lipid based transfection methods and commercially available low volume siRNA libraries to develop a rapid method of screening hundreds of candidates in a high-content cost-efficient manner. We investigated two widely used lipid based transfection reagents Lipofectamine 2000 and RNAiMAX and found that both transfection reagents lead to comparable gene silencing efficiencies in adult sensory neurons with minimal negative impact on cell survival. This method is optimized for screening in 384-well optical plates utilizing pooled siRNA libraries.

PI: Mark Tuszynski, M.D., Ph.D., UC San Diego, Department of Neuroscience

The Exportin Crm1 Not Only Mediates Nuclear Export, But Also Pore Assembly

Yvonne Roca, John Muir College, Human Biology Major

The nuclear pore complex (NPC) is a multiprotein structure that controls communication between the nucleus and the cytoplasm. Transport through the NPC is mediated by import receptors (Importins) or export receptors (Exportins). Interestingly, Importins have also been shown to separately act in mitosis, specifically by regulating key mitotic events, such as spindle assembly and NPC assembly. Here we show that the Exportin Crm1 has a role in pore assembly. A novel way to test for such a role is to study the process in annulate lamellae (AL). AL are composed of stacked flattened cytoplasmic membranes containing regularly spaced pore structures that are identical in size, shape, and protein content to nuclear pores. We found that Crm1 negatively regulates AL formation in an in vitro system derived from Xenopus egg extracts. These results can now be extended to investigate the molecular mechanism by which Crm1 regulates AL and NPC pore formation.

PI: Douglass J. Forbes, Ph.D., Division of Biological Sciences, Section of Cell and Developmental Biology

62

SALTMAN QUARTERLY

VOL. 13

sqonline.ucsd.edu

THESIS

Passive acoustic monitoring of Cuvier’s beaked whale density in the Southern California Bight Cuvier’s beaked whales (Ziphius cavirostris) are deep-diving cetaceans found in offshore tropical to temperate waters globally. They are notoriously difficult to observe visually due to their prolonged dives. Passive acoustic monitoring provides a unique opportunity to observe Cuvier’s beaked whales in their environment. Our study used a combined total of nineteen years of continuous passive acoustic data recorded at six sites within the Southern California Bight to document presence of Cuvier’s beaked whales in this region. Echolocation frequency-modulated pulses were automatically detected and manually verified, and then two different counting methods for estimating animal density were applied. These counts were then used to determine animal density and spatiotemporal trends. Cuvier’s beaked whales were more abundant towards the southern and western parts of the Southern California Bight with a seasonal pattern of lower presence during the late summer, early fall months. These findings will improve our knowledge of beaked whale habitat and behavior and help inform conservation efforts.

Ellen Jacobs, Roger Revelle College, Evolution Major and Photography Minor

PI: Simone Baumann-Pickering Ph.D.,The Behavioral Acoustic Ecology Group at Scripps Institution of Oceanography

Visible root growth phenotype in known cadmium signaling and response Arabidopsis thaliana mutants Heavy metal contamination is problematic in agricultural lands since plants rapidly take up toxic heavy metals and metalloids, subsequently compromising the crop quality and the consumer’s health.

Whilst many elements of the chelation and transport pathways have been identified, the signaling network behind the robust gene regulation remains unknown. In this study, we have developed a cadmium-induced transcriptional reporter(SULTR1;2) line and have isolated cadmium response mutants. Three groups of mutants had been identified as either non-respondent to cadmium(NRC), super respondent to cadmium(SRC) and constitutive respondent to cadmium(CRC). Two nonrespondent mutants, nrc-1 and nrc-2 were previously mapped to γ-glutamylcysteine synthetase and glutathione synthetase respectively, and both showed reduced primary root growth upon heavy metal response. I found that the constitutive respondent to cadmium, crc-1, shows greatly improved primary root growth compared to control under sulfur-limited, heavy metal contaminated condition in the presence of selenium, which is chemically comparable to sulfur.

Ka Man Wong, Thurgood Marshall College, Biochemistry and Cell Biology Major

PI: Julian Schroeder, Ph.D, UC San Diego, Division of Biological Sciences

sq.ucsd.edu

SALTMAN QUARTERLY

VOL. 13

63

Tracing Neural Circuits Making a Version of the CaLexA Reporter System with a Higher Sensitivity to Calcium

Vaishali Talwar, Thurgood Marshall College, General Biology Major

Genetically encoded activity reporter systems allow us to identify and label active neurons in a model organism. Tracing neural circuits and elucidating the neurons responsible for various olfactory mechanisms is integral to our laboratory’s approach and much of this depends upon activity reporter systems. The CaLexA transcriptional activity reporter system uses a modified NFAT protein as well as a reporter gene such as GFP, (induced by the LexA/LexAop system) to label populations of active neurons. In the CaLexA system, the import of a modified version of the NFAT to the nucleus of a cell occurs upon sustained rise in intracellular calcium. This import of NFAT initiates the transcription of reporter genes such as GFP. We made a version of the CaLexA reporter with higher sensitivity to calcium. This was done by altering the calcineurin-binding site on the NFAT molecule, by site directed mutagenesis. The goal of constructing a more sensitive version of the CaLexA system is to detect relatively lower levels of neuronal activity in an organism. We now plan to express the original and more sensitive versions of the CaLexA systems in Drosophila brains, in order to compare the intensity of neuronal activity detected by each version of the CaLexA system.

PI: Jing Wang, PhD, Division of Biological Sciences, UC San Diego, Neurobiology Section

Decoding the Crosstalk Between a Chromatin Modifier and the Morphogenesis Checkpoint

Michael Hayes, Roger Revelle College, Molecular Biology Major

DNA is organized by wrapping around histone proteins to form chromatin. Post-translational modification of histones by enzymes, such as acetyltransferases, alters chromatin structure and accessibility of DNA to effectors in processes, such as gene expression and mitosis. One such acetyltransferase, Gcn5, interacts genetically with the kinase, Swe1, which regulates mitotic entry in the morphogenesis checkpoint. Specifically, loss of SWE1 alleviates stress sensitivities of gcn5Δ cells. The nature of this interaction is unknown. We found that two downstream kinases, Cdc28, the main cyclin dependent kinase in budding yeast, and Ipl1, which promotes chromosome biorientation, alleviate similar gcn5Δ stress sensitivities when mutated to mimic the phosphorylation environment resulting from loss of SWE1. This phenotype suggests functional interactions between Gcn5 and the morphogenesis checkpoint signaling cascade. To address the mechanism by which phospho-mutants of Cdc28 and Ipl1 rescue gcn5Δ phenotypes, we are examining levels of relevant histone modifications in these mutant strains.

PI: Lorraine Pillus, Ph.D., UC San Diego Division of Biological Sciences

Jerry Hsu, Roger Revelle College, Human Biology Major

The role of Senataxin in the modulation of C9ORF72 pathology in ALS Two autosomal dominant, gain of function mutations: L389S and R2136H in the senataxin (SETX) gene lead to ALS4, a juvenile onset form of amyotrophic lateral sclerosis. To examine the effects of these mutations, we used L389S knock-in and transgenic R2136H mice models, which we have already characterized. Cytotoxicity of these two mutations in primary cortical neurons was assessed using LDH-Cytotoxicity Colorimetric analysis and Propidium Iodide assay. This assay revealed that both mutations lead to considerable cell death in primary cortical neurons as well as primary cerebellar granule neurons. Currently we are studying if Senataxin can modulate C9ORF72 pathology in ALS. To address this we will check if knockdown of Senataxin exacerbates C9ORF72 pathophysiology.

PI: Dr. Albert La Spada, MD, PhD, UC San Diego Department of Pediatrics

64

SALTMAN QUARTERLY

VOL. 13

sqonline.ucsd.edu

THESIS

Identification of Plasmodium falciparum gametocytogenesis genes An important component of the malaria eradication campaign is eliminating transmission from the human to the mosquito. This process is governed by gametocytes, the sexual stages of Plasmodium falciparum. However, we know little about gametocyte biology and especially how the induction of gametocytogenesis is controlled at the molecular level. In order to investigate the cellular processes which control gametocytogenesis in P. falciparum parasites, 28 clonal parasite strains were isolated by limiting dilution and screened using high-content imaging. Three of those clones produced fewer gametocytes, and one of those clones possessed a nonsynonymous mutation in a transcription factor subunit by whole-genome sequence analysis. The other two clones had no detectable changes at the genome level and we hypothesize that they possess differences at the transcriptional level. Identification of genes involved in gametocytogenesis will advance our current understanding of P. falciparum sexual stages and provide new targets for transmissionblocking vaccines or drugs.

Alan Du, Revelle College, Molecular Biology Major

PI: Elizabeth Winzeler, Ph.D., UC San Diego School of Medicine, Department of Pediatrics

The role of TRPV ion channels in regulating scrunching behavior in Dugesia japonica Transient receptor potential (TRP) ion channels are a family of proteins that mediate response to a variety of stimuli in diverse taxa, including humans. The vanilloid TRP (TRPV) receptor responds to noxious stimuli such as capsaicin and heat. Here we show that TRPV mediates a distinct locomotive phenotype in the freshwater planarian Dugesia japonica, called scrunching. This characteristic pain response can be easily quantified by computational image analysis. D. japonica is an effective model organism for the study of TRPV channels and other sensory receptors because their tractable nervous system contains similar neurotransmitters and cell types to those of the vertebrate brain; they also readily absorb chemicals added to their aquatic environment. Thus, our research may aid in the future development of novel pharmaceuticals for pain treatment in humans.

PI: Eva-Maria Schoetz Collins, Ph.D, Department of Physics, Department of Cell and Developmental Biology, UC San Diego

Cambria Neal, John Muir College, Molecular Biology Major

The Role of Id2 in Transcriptional Regulation of Memory CD8+ T cells CD8+ T cells are essential components of the immune system that provide protection against intracellular pathogens and tumors. It is important to understand the transcriptional networks that guide CD8+ T cells to differentiate and maintain heterogeneous effector and memory populations following infection. The E protein transcription factors and their inhibitors, Id proteins, play vital roles in T cell differentiation and maintenance. Previously, Id2 has been shown to regulate effector CD8+ T cell differentiation and survival following infection. Id2 expression is maintained in memory populations; therefore, we hypothesize Id2 also functions in CD8+ T cell memory formation, maintenance and function. We have established an inducible knockout model, which gives us the capability to control the temporal deletion of Id2 and will allow us to evaluate Id2 regulation of E proteins in memory T cell populations. Understanding the unique transcriptional programs that define CD8+ T cell memory will allow for improved vaccine design.

PI: Ananda Goldrath, Ph.D., UC San Diego Division of Biological Sciences

sq.ucsd.edu

Marija Nadjsombati, Earl Warren College, Biochemistry and Cell Biology Major, International Studies â&#x20AC;&#x201C; History Major

SALTMAN QUARTERLY

VOL. 13

65

Neural activation changes during a computerized attention modification program for social anxiety

Christina Cui, John Muir College, Physiology and Neuroscience Major

Computerized attention modification programs (AMP) utilize the probe detection task to alleviate symptoms of anxiety disorders. While AMPs have been empirically proven to match the efficacy of established pharmacological and psychological interventions, little is known about the underlying neural mechanisms. Previous studies have demonstrated attenuated activation in the amygdala, insula, anterior cingulate cortex and increased activation in the prefrontal cortex while viewing emotional faces following treatment. Increased prefrontal cortex activation post-treatment was also correlated with decreased anxious reactivity to a stressor. However, research has yet to examine brain response throughout the course of AMPs. This study will utilize functional magnetic resonance imaging (fMRI) to look at neural changes during AMP training in fourteen individuals with high social anxiety symptoms while they implicitly learn to orient their attention away from threatening stimuli. It is hypothesized that AMPs mediate anxious responses via top down brain processes in the previously implicated regions.

PI: Dr. Charles Taylor, Ph.D. UC San Diego Department of Psychiatry

Modulation of antiviral signaling in Hepatitis B virus infection.

Zuhayr U. Haq, Sixth College, Molecular Biology Major

The Hepatitis B Virus (HBV) evades the immune system’s response and suppresses interferon production. Previous reports have demonstrated that the mitochondrial antiviral signaling protein (MAVS) is inhibited by HBV expression. However, the precise mechanism behind this inhibition is still elusive. We hypothesized that the Parkin protein exerts some effects on MAVS signaling. Upon knocking down Parkin in HBV infected cells, we observed an increase in ISRE expression. We next determined that the HBx protein is responsible for upregulated Parkin expression and may cripple MAVS signaling. This suggests that HBx is involved in HBV’s exploitation of Parkin, downregulating interferon production. In future experiments, the HBx defective genome of HBV will be used to explore the mechanism behind the MAVS-Parkin interplay. In addition, the MAVS signalosome will be examined to see how HBV or HBx disrupts MAVS’s interaction with partner molecules like TRAF2, 3, 5 and 6 in HBV infected cells.

PI: Aleem Siddiqui, Ph.D, Division of Infectious, Diseases at the Department of Medicine at UC San Diego

The role of the transmembrane protein Tmem2 in heart development

Parthiv Sheth, Earl Warren College, Biochemistry & Cell Biology Major, Psychology Minor

66

SALTMAN QUARTERLY

Migration of cardiac cells to the midline is crucial for heart development. The factors driving cardiac fusion, however, are not well understood. We have identified the transmembrane protein, Tmem2, as a novel regulator of cardiomyocyte migration. tmem2 mutants exhibit failed cardiac fusion, resulting in bilateral populations of cardiomyocytes. To determine Tmem2 function, we are examining its molecular characteristics. Domain analysis indicates that all four extracellular domains of Tmem2 are required for function. I am subsequently testing whether individual domains are functional. To do this, I am creating GFP-tagged constructs of Tmem2 protein domains. I will then assess whether overexpression of these domains rescues mutant defects as effectively as overexpression of full-length Tmem2. Additionally, we aim to identify Tmem2 protein interactors. My strategy is to immunoprecipitate Tmem2 and blot against candidate protein interactors. By elucidating the function of Tmem2, we hope to gain insight into the regulation of cardiac morphogenesis.

PI: Deborah Yelon, PhD., University of California, San Diego, Division of Biological Sciences

VOL. 13

sqonline.ucsd.edu

THESIS

Investigation into the Role of Circular RMST in the Regulation of Neurogenesis and Neural Development Using Neural Stem Cells and Neurons derived from Human Induced Pluripotent Stem Cells Advances in RNA detection technologies have elucidated the abundance of circular RNA (circRNA) present in the mammalian transcriptome, reigniting interest in the function of these previously overlooked genomic elements. circRNAs exhibit both tissue- and stage-specific expression patterns and appear to be enriched in the developing brain. RNA-Sequencing of neural stem cells and neurons, derived from human induced pluripotent stem cells, led to our novel finding that the circular isoform of rhabdomyosarcoma 2-associated transcript (RMST), a long noncoding RNA recently implicated in neurogenesis, is upregulated during neuronal differentiation. When compared to baseline expression in the induced pluripotent state, qPCR analyses revealed a significant 100-fold increase in circular RMST expression following 4 weeks of neuronal differentiation and a significant 50-fold increase following 7 weeks of neuronal differentiation. The upregulation of circular RMST, observed through RNA-Sequencing and qPCR, further support the hypothesis that circRNAs, like RMST, are involved in neurogenesis and neural development.

Kelsey Baron, Sixth College, Physiology & Neuroscience Major, Psychology Minor

PI: Shauna Yuan, M.D., UC San Diego School of Medicine, Department of Neurosciences

Jonathan Sun, Eleanor Roosevelt College, Biochemistry & Cell Biology Major, Economics Minor

PI: Kang Zhang, M.D., Ph.D., UC San Diego School of Medicine, Department of Ophthalmology

sq.ucsd.edu

SALTMAN QUARTERLY

VOL. 13

67

Bottom row (left to right): Alayna Brenman, Maryanne Vargas, Anita Narwan, Samantha Madala, Bisma Ajaz, Yuya Zhao, Sara Hakim, Amrit Narwan, Jacky Lu, Dr. Steven Wasserman, Hermila Torres, Yaamini Venkataraman, Arushi Atluri, Katrina Forrest, Maryanne Beckwith, Ellen Jacobs, Karen To, Eva Hu Middle row (left to right): Katherine Smith, Connie Mach, April Damon, Madalyn De Viso, Kristina Lapira, Lexy Hanna, Alexis Padilla, Madison Rae, Nicki Guivatchian, Lisa Chik, Samirah Martinez, Denice Belandres, Hanna Tran, Shuen Sun, Seerat Sekhon Top row (left to right): Sejal Patel, Callum Arras, Grace Lo, Jason Chien, Rachan Narala, Jaidev Bapat, Jordan Setayesh,Maxwell Ruckstuhl, Rahul Nachnani, Cade Oost, Kevin Chau, Siddhant Ambulkar, Dustin Phan

HEAD ADVISORS

STAFF ADVISORY BOARD

Steven Wasserman, Ph.D. Professor of Cell & Developmental Biology Hermila Torres Manager, do/bio Center

Madeleine Picciotto, Ph.D. Director, UCSD Writing Center

FACULTY ADVISORY BOARD

ALUMNI ADVISORY BOARD

Eric Allen, Ph.D Timothy Baker, Ph.D James Golden, Ph.D Suckjoon Jun, Ph.D Jill Leutgeb, Ph.D

Firooz Kabir Jasmine Chau Arjun Patel Edgar Villaruel

James Wilhelm, Ph.D Martin Yanofsky, Ph.D Elvira Tour, Ph.D David Holway, Ph.D

Maximo Prescott Editor-at-Large Florence Lambert

Special Thanks: Patera Design 68

SALTMAN QUARTERLY

VOL. 13

sqonline.ucsd.edu

STAFF Editor-in-Chief Jacky Lu

Research Design Editor Denny Bao

Blog Manager Christina Cui

Executive Editor Yaamini Venkataraman

Features Design Editor Dustin Phan

Head Production Editor Sejal Patel

Webmaster Maryanne Vargas

Bloggers Rachel Tran Julia Ramirez Katlin Neese Divya Sriram

Features Editor Rachan Narala

Online Editor Anna Nidhiry

Special Sections Editor Katrina Forrest

Publicity Chair Bisma Ajaz

Research Editor Rahul Nachnani

Publicity Committee Katherine Smith Lily Nguyen Catherine Nguyen Callum Arras Lisa Chik Seerat Sekhon

Head Technical Editor Hanna Tran Technical Editors Maxwell Ruckstuhl Denice Belandres Shuen Sun Sara Hakim Review Board Managers Amrit Narwan Shauheen Ladjevardi

Community Outreach Chair Siddhant Ambulkar Community Outreach Committee Lexius Waltar Kristina Lapira Kwang-Tao Chou Alexandrea Hanna

Staff Writers Neal Shah Michelle Lee Anika Ullah Karen To Ishan Goyal Head Illustrator/ Photographer Alayna Brenman Photographers Ellen Jacobs Malak Jaljuli Erika Nilsson Eva Hu Natalie Popescu Alexis Padilla Chandra Couzens Illustrators Connie Mach April Damon Grace Lo

REVIEW BOARD Aerie Kim Alexander Rinard Amanda Chong An Nguyen Anita Dev Callum Arras Crystal Choy Emily Liu Jadcha Gumban

Jason Chien Lisa Chik Madeleine Hill Maryanne Beckwith Na Kim Neeja Patel Sam Gurevich Samirah Martinez Stephanie Dong

Stephen Howell Swasthi Malladi Vidya Raghvendra Vineet Tummala Ye Hyang Kim Yogitha Chareddy Youngju Choi Yunlin Zhang Yuya Zhao

GET INVOLVED

Saltman Quarterly Research Journal Under the Scope Magazine SQ Insider Community Outreach SQ Online Biology Student Research Showcase

Saltman Quarterly Division of Biological Sciences University of California, San Diego sqonline.ucsd.edu sq.ucsd.edu