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Undergraduate Research Journal Division of Biological Sciences SALTMAN QUARTERLY VOL. 14

LETTER FROM THE EDITOR Dear Esteemed and Respected Reader,

In an effort to engage the UC San Diego community, Saltman Quarterly holds an annual photo contest. The winners of this contest have their images featured on the cover and interior pages of the journal. FRONT COVER: A Leafy Seadragon, Phycodurus eques, at Birch Aquarium stands out vividly against the black background, yet this creature survives on its ability to blend in and remain invisible. Photo by Jonathan Chu. INSIDE COVER: A Ruby-throated hummingbird, Archilochus colubris, drinks nectar from flowers in an Irvine suburbia. Photo by Tara Terpening. SALTMAN QUARTERLY

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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.

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In Saltman Quarterly Volume 14, you will find dazzling photography, eye-catching illustrations, paradigm-challenging feature articles, undergraduate research manuscripts, and an insight into the minds of the hardworking biology students at UC San Diego. You will feel the fruits of labor from over 200 SQ members and authors. You will benefit from over 365 days of daily group meetings, long nights in the office, and closely-met deadlines. The mission of our organization is to spread a passion for science and biology, and to communicate that science effectively to people around us. While our mission has stayed constant since our first publication in 2004, I have seen our reach and execution of ideas improve tremendously through this year. I became involved in Saltman Quarterly within the first three weeks of college. Despite being otherwise a very average freshman college student, writing for SQ was the most powerful support for my curiosity for science, and broadened my scope of research at UC San Diego. During my time as a student blogger, researching campus resources and our professors’ findings helped me realize the international scope of scientific thought. The discoveries being made by the fabulous researchers all over the world are not getting any simpler, and the public at large has a right to know about them, especially because we know science constantly

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changes over time. This year, more than any year in recent history, science is threatened by a national administration that focuses on façades, not facts, and on power without progress. Research across the board faces severe funding cuts, gutted endowments, and demolished programs meant for the well-being of our nation. I am more proud of our organization than ever for publishing boundary-pushing rhetoric in our Fall Newsletter, Science and Policy, and being one of most prominent UC San Diego organizations at the San Diego’s March for Science. In our near community, we mentor K-12 students as they work on their very own research projects and vibrantly support them when they stand on stage. Here on campus, we host bi-monthly Journal Clubs, inviting students from all over to listen and analyze seminal research papers, all led by faculty. We responded to the threats to science with the voice of its supporters, and our staff continues to create opportunities for others to learn more. In Saltman Quarterly Volume 14, you will find an eclectic variety of topics from transmitted diseases, to CRISPR, to coastal reserves, to stress on demographics, and many more student executed research projects. We have collected artwork and photography from the students all over campus who are passionate about nature’s wildlife and have showcased their art. Finally, we encouraged the Honors students to submit the best representation of their capstone projects to our journal to recognize their work. Finally, in Saltman Quarterly Volume 14, you will see the pride and joy in the year’s work done by our staff members, and I write this letter for them. Sincerely,

Rahul Nachnani Editor-in-Chief Saltman Quarterly 2016-17

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TABLE OF CONTENTS 04

SALTMAN DEDICATION “He was a Rebel Rouser.” Those were the words first used to describe Dr. Paul Saltman to me by one of his long-time colleagues, Dr. Gabriele Wienhausen. A lifelong scientific educator and former advisor to the Saltman Quarterly, her enthusiastic description juxtaposed the image of a dedicated biochemist against that of a charismatic revolutionary... BY FAREED J. DIBAZAR

FEATURES 08 Vector Skelter: Climate Change and the Challenges of Vector Borne Illnesses BY LAUREN BRUMAGE

Editing Out Malaria: Using CRISPR/Cas9 to Rethink Disease Transmission

MicroRNA: Discovery and its Role in Novel Therapeutic Techniques

Demystifying the UTI: New Perspective on Flora Flourishing Inside the Urogenital Tract

BY NEAL SHAH

BY PETER CHEW

BY ANIKA ULLAH

RESEARCH 30

Variation in Hymenoptera Species Richness by Land Use in Monteverde, Costa Rica

Relationship Between Psychosocial Stress and Intuitive Eating in Overweight Latino and African-American Youth

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BY MUKTA KELKAR

BY VENNIS HONG

Stress-Induced Tumor Progression in Melanoma BY JORDAN SETAYESH

SENIOR HONORS THESES The Division of Biological Sciences’ Senior Honors Theses 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.

STAFF

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PAUL SALTMAN “He was a rabble-rouser.” These words were used to describe Dr. Paul Saltman by one of his long-time colleagues, Dr. Gabrielle Wienhausen. A lifelong educator and former advisor to Saltman Quarterly, Wienhausen’s enthusiastic description juxtaposed the image of a dedicated biochemist against that of a charismatic revolutionary. There’s no question that Dr. Saltman was a prolific academic. Currently, in the UC San Diego’s Special Collection Archives, there is a lifetime of literature that attests to his achievements in nutritional research and education. It is easy to look back at his legacy and see the awards and honors that bear his name and the simple volume of literature he produced, but what what truly places Dr. Saltman into the realm of legend is the impact he left on people. His dedication to UC San Diego, biological work, and educational outreach is acknowledged not only by his students and colleagues, but by a global audience for his lifetime of achievements. How did Dr. Saltman earn the unusual title of “rabble-rouser?” His greatest commitment was to the integrity with which he pursued every endeavor. Any researcher working today is all too familiar with the never ending cycle of the looming pressure to perform, to publish breakthrough research and to secure more grants, a trend that was just as prescient now as it was during Dr. Saltman’s lifetime. Yet despite pressure to please, Dr. Saltman spoke of his scientific opinions truthfully, even when they went against the existing narrative. Reflecting on his time working with the Atomic Energy Commission before a Scripps-Caltech conference in 1959, Dr. Saltman once mused:

“At the end of the talks, I made a value judgement. I said that I thought there were better ways to use atomic energy than spewing it forth into the atmosphere while telling everybody it was good for them... I received an interesting letter from the Atomic Energy Commission shortly thereafter, asking me what my qualifications were to talk on such matters, and that after all, I had an Atomic Energy Commission grant, and shouldn’t I maybe shape up?”

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To think that a career researcher would risk losing his livelihood by saying such a provocative statement is just as radical now as it was then. Yet, Dr. Saltman pulled no punches, even as he joked that he was still just an associate professor at the time. Indeed, this act of questioning the agenda of his own sponsors would not be an isolated incident, but rather a hallmark of his career: advocating for the greater engagement of the global population in the sciences. He starred in the Academy Award-winning documentary “Why Man Creates,” a film which discusses the the nature of creativity and features a segment on the importance, as well as the shortcomings, of scientific research. On October 22, 1959, he guest starred on the live television game show, “You Bet Your Life,” playfully trading jabs with vaudeville comedian Groucho Marx as he relayed the role of ATP in causing muscle cells to twitch. During the same conversation with Mr. Marx on live television, Dr. Saltman criticized the lack of gender equality in the sciences at his place of employment, the University of Southern California.

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Dr. Saltman also famously toured China and Japan as a science educator, evaluating university programs and fostering dialogue during a time of dubious and tenuous relations between these countries and the United States during the Cold War era. This journal owes its existence to that same mentorship and support Dr. Saltman provided to undergraduates during his career. As “the rabble-rouser,” Dr. Saltman’s engagement with undergraduates and dedication to teaching is one of the fundamental reasons why undergraduates across the UC campuses are now actively present and engaged at all levels of research and scientific communication. His advocacy and discussions with the Biology department regarding undergraduate recruitment into research the groundwork for current surge of undergraduate engagement. Whereas even a decade ago undergraduate presence in high-level biological research was considered uncommon, it is a testament to Dr. Saltman’s legacy that he is directly responsible for the breadth of opportunities undergraduates now have at UC San Diego to engage in research and, more importantly, pass on what they have learned.

In an era where scientific engagement and public education are continuously threatened by misinformation and private agendas, it is heartening to consider the example one man left behind for others. Rather than avoiding scrutiny, Dr. Saltman lived boldly by challenging students, colleagues, and faculty to actively engage and educate the public. He confronted misinformation and ignorance not with cold candor, but with community engagement, education, and inclusion. In that regard, while the challenges of scientific communication continue to increase globally, the legacy of Dr. Paul Saltman, rabble-rouser, has never been more inspiring.

WRITTEN BY FAREED J. DIBAZAR Fareed is a General Biology and Political Science with a Specialization in International Relations major from Roger Revelle College. He will graduate in 2017.

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.

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FEATURES 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.

California Sea Lions, Zalophus californianus, at La Jolla Cove Photo by Ben Kong SALTMAN QUARTERLY

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FEATURES

VECTOR SKELTER:

CLIMATE CHANGE AND THE CHALLENGE OF

VECTOR BORNE ILLNESSES Tarik Benmarhnia, whose work will be discussed in this article, seek to elucidate the diverse mechanisms through which climate change may impact human health in order to determine how humanity may best prepare for the challenges to come.

LAUREN BRUMAGE STAFF WRITER Illustration by April Damon Photography by Evelyn Tong

Vector-borne illnesses are diseases that involve transmission of a pathogen to a host via a third-party vector species, such as a mosquito. Changes in temperature, precipitation, and humidity associated with climate change could expand the viable habitat zones for vectors, allowing the pathogens they transmit to infect more people. It is critical to generate predictive models regarding the potential impact of climate change on vector-borne illnesses so as to identify regions vulnerable to potential outbreaks. Once at-risk areas are determined, it is possible to craft public health policy plans that mitigate vulnerability. This article features information from an interview with Dr. Tarik Benmarhnia, an epidemiologist at UC San Diego who studies climate change and human health.

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Consider a disease that is insidiously asymptomatic in most patients but can be transmitted from unsuspecting mothers to their unborn children, a disease that can be transmitted by something as simple as a mosquito bite, but can cause some infants to be born with microcephaly. Microcephaly of the fetus, or underdevelopment of the brain, is one potential effect of the disease known as Zika virus. Zika has increasingly garnered the attention of public health officials and the general populace alike. However, Zika virus is only one member of the much broader category of vector-borne diseases that includes Chagas disease, schistosomiasis, dengue fever, and malaria. Currently, these illnesses and their vectors are largely restricted to the tropics, but this could change in the future as the effects of climate change intensify. Climate change has the capacity to alter not only temperatures but also humidity levels, rainfall amounts, and the prevalence as well as intensity of severe weather events — all of which can impact vector distribution and the health services infrastructure. Researchers, including Dr.

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Vectors and Climate Specificity

The communicable diseases that are perhaps most intimately connected to climate change are vector-borne illnesses. Vector-borne illnesses are characterized by the transmission of a pathogen to a host via a carrier species (the vector). One of the most common vectors is the mosquito, although different species of mosquito transmit different vector-borne diseases. Aedes aegypti transmits Zika virus, dengue fever, and chikungunya while mosquitoes in the genus Anopheles are known to transmit malaria. The aforementioned Zika virus can cause symptoms ranging from microcephaly in infants to headache and rash in adults. Fever and nausea are more common with dengue fever, chikungunya, and malaria than they are with Zika. Vector-borne diseases can prove fatal; for instance, the Centers for Disease Control and Prevention cites a death toll of 438,000 worldwide from malaria in 2015. But why are vector-borne illnesses so sensitive to climate and climate change? According to the European Centre for Disease Prevention and Control (ECDC), as arthropods bereft of internal temperature regulation, many vectors are easily affected by changes in temperature. This restricts vector-borne diseases to regions with the appropriate climate, such as the tropics and other relatively warm locations. However, climate change threatens to disrupt the currently established zones of these diseases. Latitudes north and south of the tropics will experience higher temperatures and humidity levels, giving vectors the chance to infiltrate new regions.

An Epidemiological Approach

To garner more insight on how climate change may impact vectors and vector-borne pathogens, I interviewed Dr. Tarik Benmarhnia, joint assistant professor of the UC San Diego Department of Family Medicine and Public Health and the Scripps Institution of Oceanography. Dr. Benmarhnia conducts research in climate science, epidemiology, and the link between environment and health. He is particularly interested in how different populations may experience different health consequences due to climate change. Generating predictive models is a central component of Dr. Benmarhnia’s work. These models integrate historical data related to climate pattern and incidence of disease outbreaks with projected climate conditions to predict where and to what extent a particular disease might increase in prevalence. However, many uncertainties plague the process of generating a predictive model. Dr. Benmarhnia believes that the “most critical uncertain[ties]” are “behavioral…[and] sociodemographic changes.” Human behavior is complex and intrinsically linked to environmental circumstances, so it is difficult to evaluate and quantify precisely how it will change over time. As Dr. Benmarhnia stipulates, climate change could significantly alter city organization and distribution of people. In a survey of the potential effects of climate change on populations and disease, scientists from the University of Ghana cite

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FEATURES FIGURE 1 Climate change threatens to cause more extreme weather events including severe rainstorms and flooding. This can modify habitats suitable to vectors; for instance, stagnant pools of water are common breeding grounds for mosquitoes.

virus and dengue fever. Therefore, these diseases could become more widespread in California. In recent years, the spread of vector-borne illnesses to new regions has already been observed. The ECDC has observed outbreaks of West Nile fever (in Romania during 1996-1997) and chikungunya (in Italy in 2007). It is possible that similar outbreaks will occur in the future. The spread of vector-borne illnesses is especially threatening to low-income areas with poor housing. In the presence of urban crowding and absence of adequate shelter and clean water, humans are more exposed to vectors in their environment and are therefore more susceptible to vector-borne illnesses.

Looking Forward a statistical estimate from the 2006 Stern Review on The Economics of Climate Change: Up to one billion people may be forced to migrate by 2050 due to changing climate. Shifting population densities in urban centers and migration between different areas could facilitate the spread of vectors within a crowded urban center or to novel regions. Therefore, other data and statistical uncertainties such as changes in human behavior must somehow be incorporated in the predictive model to paint a clearer picture of potential outcomes.

Incomplete Data

Another complicating factor in predicting disease spread is that existing data and current research are regionally biased as well as incomplete. Malaria has been the subject of much epidemiological research about climate change whereas dengue fever has been largely neglected in comparison. Additionally, much of the regional analysis work done on how climate change may impact vector-borne illnesses has focused on Europe, Africa, and Asia. Latin America, on the other hand, has been the focal point of a vanishingly small amount of such research. For this reason, Dr. Benmarhnia stresses the importance of 10 SALTMAN QUARTERLY

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expanding research on climate change and vector-borne illnesses in Latin America. The last disparity Dr. Benmarhnia cites is the clear contrast between research done in lower-income countries and research done in higher-income countries. With fewer resources to spare for investigation and research, lower-income countries sometimes struggle to collect adequate data. It is possible that other, as yet unexplored, disparities exist that hinder analysis. It is clearly difficult to predict the potential changes in vector distribution over time due to climate change. Nevertheless, it is important to do this work and diversify existing research. According to Dr. Benmarhnia, regardless of how humanity changes its environmental impact now, there will be consequences from climate change in the coming decades. Therefore, through examining potential alterations in the range of vector-borne illnesses, we can prepare and seek to prevent the worst consequences. What, then, are some of the possibilities? Due to changing rainfall patterns in Northern California, the mosquito Aedes aegypti could become more common in the state. As previously stipulated, this mosquito is the carrier of Zika

FIGURE 2 The symptoms of vectorborne diseases are quite varied and can be mistaken as signs of other illnesses. While many of the symptoms do not seem severe, they can become lifethreatening.

Arguably as vital as understanding the potential consequences of climate change for vector-borne illnesses is understanding what changes we can make to resist negative outcomes. Dr. Benmarhnia believes that it is important for public health officials to collaborate with urban planners and entomologists to minimize habitats, such as stagnant pools of water, conducive to insect vectors in cities. Improvements in urban planning have previously been shown to reduce the incidence of vector-borne illnesses; for instance, according to the ECDC, dengue fever used to be found in Europe, but it disappeared due to the advent of extensive piped water systems. Furthermore, if housing is improved, then people will be less exposed to their environment. Health services worldwide must be strengthened since, as Dr. Benmarhnia notes, an outbreak can cripple an already fragile health services infrastructure. This outcome has been seen recently — in 2013-2016, during the West African Ebola epidemic — where inadequate medical services facilitated and prolonged the outbreak according to an analysis by a group of scientists from York University in Canada. Accessible, high-quality health care is imperative to halting an outbreak before it spirals out of control.

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“

REGARDLESS OF HOW HUMANITY CHANGES ITS ENVIRONMENTAL IMPACT NOW, THERE WILL BE CONSEQUENCES FROM CLIMATE CHANGE IN THE COMING DECADES.” -DR. TARIK BENMARHNIA

Climate change presents a critical challenge for epidemiologists. Generating predictive frameworks is a task fraught with uncertainties and confounding variables, but epidemiologists are problem solvers whose key task is, in Dr. Benmarhnia’s words, to “dissect mechanisms” through which processes occur. Disentangling the various factors that influence the spread of vector-borne illnesses is a necessary first step toward addressing underlying societal vulnerabilities to these diseases. Global improvements to health services, monitoring of current and emerging infectious diseases, and interdisciplinary collaboration will help mitigate these vulnerabilities

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as well as facilitate the proposal of appropriate adaptation strategies. Perhaps of utmost importance is international collaboration among researchers. By collaborating and combining regional expertise, as well as expanding research to include neglected regions such as Latin America, the quality of research will improve and empower humanity to prepare against the spread of vector-borne diseases.

References

Benmarhnia, T. Interview. 09 Dec 2016.

de Souza DK, Owusu PN, Wilson MD. 2012. Impact of climate change on the geographic scope of diseases. In: Chhetri Netra, editor. Human and social dimensions of climate change. InTechOpen. p. 245-264 [Internet]. Aedes aegypti. European Centre for Disease Prevention and Control. Mosquitoes. Centers for Disease Control and Prevention. Malaria. Centers for Disease Control and Prevention; [cited 2017 Jan 26].

Vector-borne diseases. European Centre for Disease Prevention and Control. Ali H, Dumbuya B, Hynie M, Idahosa P, Keil R, Perkins P. 2016. The social and political dimensions of the Ebola response: global inequality, climate change, and infectious disease. In: Leal Filho W, Azeiteiro UM, Alves F, editors. Climate change and health. Springer. p. 151-169 [Internet]. Githeko AK, Lindsay SW, Confalonieri UE, Patz JA. 2000. Climate change and vector-borne diseases: a regional analysis. World Health Organization [Internet]. Altizer S, Ostfeld RS, Johnson PTJ, Kutz S, Harvell CD. 2013. Climate change and infectious diseases: from evidence to a predictive framework. Science [Internet].

WRITTEN BY LAUREN BRUMAGE Lauren is a Molecular Biology major from Thurgood Marshall College. She will graduate in 2019.

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EDITING OUT MALARIA:

FEATURES

USING CRISPR/CAS9 TO RETHINK

DISEASE TRANSMISSION

NEAL SHAH STAFF WRITER Illustration by Varsha Rajesh

One of the newest and most promising developments in biology has been CRISPR/ Cas9, a genome editing technology allowing scientists to edit DNA with stunning precision. At UCSD, Dr. Valentino Gantz uses CRISPR/Cas9 to modify the germline of mosquitoes so that their progeny would become malaria resistant, and continue to pass this resistance down for generations. However, with such a powerful technology, ethical and ecological questions are often asked with this research.

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Lauded as 2015’s “Breakthrough of the Year” by Science, CRISPR/Cas9 has quickly emerged as one of the most exciting and powerful genetic technologies of the past decade, and scientists have already begun to explore its applications. In 2014, Chinese researchers edited the wheat genome to make wheat grains resistant to a common disease, and later that year, CRISPR/Cas9 was used to fix a mutation in a gene associated with a tyrosinemia, a human metabolic disease (Wang et al. 2014). Earlier this year, Scottish researchers genetically engineered pigs to protect them from an infection that causes breeding problems in females and breathing problems in young pigs, while Chinese researchers introduced a gene to create cattle resistant to tuberculosis. With widespread applications in the agricultural, food, and health industry, it’s creating quite the buzz in the science world — and for good reason. What exactly is CRISPR/ Cas9, and why is it being hailed as the newest “darling” of genetic research?

CRISPR/Cas9

CRISPR/Cas9 is a genome editing technology that allows researchers to edit and modify DNA with incredible precision. It consists of two main components. First, is a CRISPR (Clustered

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Regularly Interspersed Short Palindromic Repeats) RNA sequence, which serves as a molecular “guide,” and second is the Cas9 protein (CRISPR associated protein), which serves as molecular “scissors.” The CRISPR RNA sequence binds to a longer strand of non-coding RNA called tracrRNA (trans-activating CRISPR RNA) to form a complex called guide RNA. This guide RNA complex binds to the target complementary sequence on the DNA strand, and the Cas9 enzyme follows, cutting the genome across both strands of the DNA at the exact points designated by the RNA complex. Scientists can design specific guide RNA sequence to introduce mutations and deletions at precise locations of interest. With CRISPR/Cas9, scientists can “knock out” a specific gene, which makes it nonfunctional, or can edit a gene to modify its function. This requires a few more steps. In order to edit a particular sequence of DNA, it is necessary to take advantage of the cell’s own complex repair system called homology-directed repair, which uses a DNA template to synthesize the DNA fragment cut out by Cas9. Therefore, a designed DNA repair template is also introduced into the cell, so the cell’s repair machinery can synthesize the modified fragment of the gene complementary to the sequence introduced via the DNA repair template. This slightly more complicated mechanism allows scientists to not just knock out a gene, but to modify its functions.

Active Genetics

Dr. Valentino Gantz at UC San Diego applies CRISPR/Cas9 technology to create gene-drive systems to combat malaria. “Gene-drive” is a molecular technique to introduce a particular gene into a population and increase its prevalence in

“

ANY TIME THERE’S A CONTROVERSIAL APPLICATION THAT MIGHT HAVE ETHICAL CONCERNS, IT SHOULD BE DISCUSSED AND DELIBERATED UPON.” -DR. VALENTINO GANTZ

the population, thereby “driving” the gene into future populations. Using CRISPR/ Cas9, he introduces an anti-malaria gene in the germline of Anopheles stephensi, an Asian mosquito that is a known malaria vector, ensuring both copies of its chromosome contains this mutation. By genetically turning the genotype homozygous, the gene-drive method ensures the mosquito passes both copies of the modified gene to its progeny. Because both chromosomes have been modified, and the trait is homozygous, in theory, 100% of the progeny should contain the anti-malaria gene, instead of 50%. Therefore, Dr. Gantz’s method bypasses the traditional model of Mendelian inheritance and “drives” the gene into a population. The results corroborate the hypothesis — the gene has successfully been carried through four generations of mosquitoes, with 99% of the progeny expressing the modified gene. Though just a few generations in, the

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FEATURES

FIGURE 1 Mechanism illustrating Cas9 endonuclease cleaving genomic DNA at precise base pair after complementary binding by guide RNA.

FIGURE 2 Upon CRISPR/Cas9 genome editing, over 99% of future Anopheles stephensi populations carried the malaria resistant gene. Photo courtesy of Gantz Lab.

research, and claims that, “Any time there’s a controversial application that might have ethical concerns, it should be discussed and deliberated upon.” He believes such technology should not be implemented in the natural world unless there is a deep understanding of genedrive both scientifically and ethically. As for Dr. Gantz’s gene-drive research now, he sticks to controlled studies in enclosed populations of mosquitoes. He is particular about keeping these mosquito populations tightly confined in the laboratory, and continuing to understand the molecular underpinnings of gene-drive before even thinking about applying it in nature.

Conclusion

potential effects are certainly thrilling. The ability to drive an anti-malaria geneto-be through generations of malaria vectors is a step in the right direction to establish populations of mosquitoes unable to transmit the disease. Dr. Gantz hopes to apply this research in the future to numerous other other vectorborne diseases and create populations of organisms unable to transmit many of the world’s most dangerous diseases. “It’s a very basic mechanism,” he said. “If you can get a few [DNA] breaks in your way, you’re hijacking an endogenous system to your advantage; therefore, you should be able to do this in any system.”

Ethical Concerns

With such an immensely powerful technology, there are significant ethical and moral dilemmas associated with driving a gene into a population. Opponents of this research argue that 14 SALTMAN QUARTERLY

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biologically altering natural populations of species via gene-editing could lead to unforeseen consequences in the balance of our ecosystems. This could result in irreversible damage to both the nature of our ecosystems and creatures that reside in it must be deeply understood and carefully deliberated before applying it. In addition, some skeptics are concerned that when a gene is driven through the population via natural breeding of organisms in our ecosystem, how can it be controlled, and what if something goes wrong? Often times in science, consequences of seemingly harmless procedures or experiments do not appear until much later. If such an event occurs with a modified gene, it would be nearly impossible to stop the dissemination of the deleterious gene throughout a population, especially one that has an almost guaranteed chance of passing the trait onto its progeny. In an extreme

scenario like this, the consequences could be particularly damning, as gene drive has the potential to impact large populations of organisms. In an age of such rapid scientific development, our understanding of innovative, powerful, scientific technology outpaces our understanding of its ethical implications. This is exemplified by the fact that just a few months ago, activists and officials in the United Nations seriously considered imposing a moratorium on gene-drive research until they had a better understanding of its underpinnings and implications. Though the motion to impose the moratorium was eventually shut down, it highlights that these ethical fears and concerns are real and must be taken into deep consideration, especially with research of this nature. Dr. Gantz completely understands the potential ethical dilemmas and concerns among skeptics who are wary of applying this

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Gene-drive is a powerful aspect of CRISPR/Cas9 genome editing, and its possibilities to improve our ecosystem, combat disease transmission, and help restore endangered species, is truly revolutionary. The ability to drive a gene through a population with such alarming efficiency is met with both incredible excitement and a level of skepticism in how this technology need or need not be applied. These ethical dilemmas must be discussed and deliberated upon, along with proper oversight to ensure this research is applied carefully, and does truly benefit the world. With CRISPR/Cas9 being such a captivating and impactful tool, it is necessary to have innovative scientists tap into its power to effectively combat world problems, such as malaria. Fortunately, talented researchers right here at UC San Diego, such as Dr. Gantz, are pushing the boundaries of what could be done with CRISPR/Cas9 to introduce exciting ways to change how we approach disease transmission. The promises of these results and potential reproducibility with various organisms besides mosquitoes

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are truly exciting advances to combat diseases, as it eradicates a virus from its means of transmission: organisms. The application of CRISPR/Cas9 to introduce anti-viral genes into disease carrying vectors can possibly change populations of disease-carriers in the world — and lower transmission risks for generations to come.

References

Carlos, Naia. 2016. “Conservation vs. Genetics: Scientists Debate the Ethics of DNA Research, Gene Drives.” Nature World News. Accessed March 9, 2017. Gantz, Valentino et al. 2015. “Highly efficient Cas9-mediated gene drive for population modification of the malaria mosquito Anopheles stephensi.” Ran, F. Ann et al. 2013. “Double Nicking by RNA-Guided CRISPR Cas9 for

Enhanced Genome Editing Specificity.” Cell. Wang, Yanpeng et al. 2014. “Simulataneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew.” Nature. Webber, Bruce L. et al. 2015. “Opinion: Is CRISPR-based gene drive a biocontrol silver bullet or global conservation threat?”

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

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MICRORNA:

ITS DISCOVERY AND ROLE IN NOVEL

THERAPEUTIC TECHNIQUES PETER CHEW STAFF WRITER Illustration by Connie Mach Photography by Daniel Melnick

The human genome is vast, but only 1.5% of it codes for the construction of proteins. The other 98.5% of DNA is a veritable ocean of biological code that we have only begun to decipher. This non-coding DNA has been found to contain, among other regulatory elements, segments coding for microRNA, which are extremely small singlestrand RNA transcripts that inhibit the expression of DNA products, such as protein and other RNA. Normal organismal development is heavily dependent on microRNA mechanisms. Many congenital defects and autoimmune diseases, such as rheumatoid arthritis, have been linked to improper microRNA expression. At UC San Diego, researchers, including Dr. Amy Pasquinelli and Dr. Li-Fan Lu, study microRNA targets and the mechanisms by which they work. In the wake of their efforts, biochemical companies are beginning to develop experimental microRNA-based therapies.

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At almost three billion nucleotides long, the human genome encodes almost all of our biological functions. Most of these functions are carried out through proteins, which are constructed from messenger RNA (mRNA) transcripts of DNA sequences. The DNA sequences from which mRNA copies from are deemed “coding” sequences, and are understandably crucial to maintaining life. However, as scientists began to sequence the human genome, they discovered that only a miniscule portion of the genome actually encodes protein. This was puzzling to biologists; how could such a complex organism such as a human run on so few coding sequences? Even simpler organisms, such as bacteria, had a higher protein-coding ratio in their genomes. After further study, however, it became apparent that the rest of the human genome held the secrets to this paradox. Complexity in the form of extensive regulatory mechanisms was revealed, and many new biomolecules that regulate genes and gene expression were discovered. One such discovery were microRNAs, small and inconspicuous molecules that affect a wide range of human biological systems.

The Discovery of microRNA

While one of nature’s most common use of RNA molecules is in the production of proteins, scientists discovered RNAs that have specialized functions in gene regulation. One type of specialized RNA, termed

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“microRNA,” plays an important role in the suppression of gene expression by preventing the manufacturing of the proteins for which they encode. The first microRNA molecule was discovered in 1993, when both Dr. Victor Ambros’s and Dr. Gary Ruvkun’s projects converged on a very small single-stranded RNA molecule that was crucial for normal roundworm (Caenorhabditis elegans) development. Researchers in both labs had noted that mutations in the lin-4 gene resulted in adult roundworms retaining larval characteristics, such as underdeveloped outer female sex organs. Since roundworms are hermaphroditic, the offspring could not be expelled from the parent’s body in the usual manner. Instead, the larvae would accumulate within the parent’s body and eventually consume the parent from the inside out. When the product of the lin-4 gene was identified, it was found not to be a protein, but a 21-nucleotide RNA sequence that was complementary to the mRNA transcript of another gene, lin-14. A single-stranded microRNA molecule produced from lin-4 bound to the 3’ untranslated region (“tail end”) of a lin-14 mRNA molecule, catalyzing its degradation. Thus, microRNA acted as an incredibly precise inhibiting agent that prevented certain genes from expressing their gene product. This downregulation of mRNA, as demonstrated later by Ruvkun’s lab, was enough to implement the precise temporal control needed for proper roundworm growth.

As Told At Ground Zero: microRNA

At first, the small mRNA-interfering molecule was considered to be an oddity that was confined to roundworm physiology. Dr. Amy Pasquinelli, who leads a lab specializing in basic scientific research on microRNA at UC San Diego, recounted her time as a postdoctoral student in Ruvkun’s laboratory and the moments when they realized the ubiquity of microRNA, highlighting its potential therapeutic use in fighting human diseases. As additional research into microRNA within C. elegans continued for the next

six years, more genes, such as let-7, were discovered and associated with crucial developmental stages. By 1999, the Human Genome Project, an effort to sequence and identify all genes within the human genome, began to publish a database of human genes. In order to search through the sea of information produced by the Project, the Basic Local Alignment Search Tool (BLAST) was developed to help researchers identify specific DNA sequences, and homologous (similar) occurrences in the genomes of other organisms. Once Ruvkun’s lab had published the paper announcing let-7’s discovery, Ruvkun and his team noticed that a sequence in the human genome was, strikingly, almost identical to the let-7 gene found in the roundworms they studied. A serendipitous search with BLAST had highlighted this fact, and now Ruvkun wanted to confirm whether let-7 microRNA was truly present and expressed in humans. The task fell upon Pasquinelli to perform experiments called Northern Blots, which are designed to detect the presence of RNA molecules, on human tissue samples. After the Northern Blots were finished, it was evident that the let-7 gene was not only conserved in the human genome, but also actively expressed as microRNA in humans. She recalls telling this to Ruvkun shortly after a staff Christmas dinner, “I went up to Gary and said, ‘Guess what? I think that let7 is in humans!’, he went ‘What! Really?’… We realized that this was a very exciting discovery, and that it really told the world that these little, weird, small RNA’s were not just an oddity of worms but were present in probably all kinds of organisms.”

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FEATURES The implication of the ubiquity of microRNAs excited the scientists the most. Not only did they confirm the existence of a new biomolecule in humans, but they also opened the floodgates to a new wave of interest in noncoding regulatory DNA sequences, the study of changes in gene expression that does not involve mutations in DNA sequences. Many researchers sought to learn more about regulatory biomolecules produced from non-coding DNA, which was traditionally considered “junk” DNA, in order to harness their potential to modulate important bodily responses to disease. Dr. Pasquinelli continues to conduct basic research on microRNA, examining various targets for microRNA interaction in many different scenarios. One such project being run in her lab seeks to discover how certain microRNA activity increases only when an organism is under stress, which highlights the complexity of post-transcriptional regulation. The knowledge generated from Pasquinelli’s lab is then utilized by other researchers seeking to apply microRNA techniques to specific biological systems and diseases.

FIGURE 1 Student researchers in Dr. Lu’s lab run experiments to study immune system behavior in various model organisms.

microRNA and Its Applications In Immunology

“

WE REALIZED THAT THIS WAS A VERY EXCITING DISCOVERY — AND THAT IT REALLY TOLD THE WORLD THAT THESE LITTLE, WEIRD, SMALL RNA’S WERE NOT JUST AN ODDITY OF WORMS BUT WERE PRESENT IN PROBABLY ALL KINDS OF ORGANISMS.” -DR. LI-FAN LU 18 SALTMAN QUARTERLY

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Post-transcriptional regulatory mechanisms like microRNA are of great interest to those who seek to change how our bodies react to disease. Dr. Li-Fan Lu studies immunology at UC San Diego and researches microRNA techniques as potential agents in regulating immune responses to disease. Two of the disease types that interest him are cancer and autoimmune diseases. Both involve the immune system working to our disadvantage — cancer deactivates immune system responses in order to allow malignant tumors to propagate, while autoimmune diseases involve an overactive immune system damaging healthy tissue. One of Dr. Lu’s main areas of focus is on microRNAs and their effect on regulatory T cells. Regulatory T cells, or Treg cells, have an indispensable function in the human immune system — to prevent unnecessary

immune responses from damaging healthy body cells. Treg cells are heavily dependent on microRNA activity; when microRNA pathways are unavailable and no microRNA is present, Treg cells often lose their inhibitory function. This can be seen in autoimmune diseases such as lupus and multiple sclerosis. When a lower-than-normal concentration of miR-146a is present in Treg cells, their ability to rein in the response of helper T cells is impaired, which may lead to autoimmunity. Various types of cancer, such as leukemia, have also been linked to microRNA aberrations. Often, the decreased concentration of tumor-suppressing microRNAs is associated with cancer onset; if a microRNA that prevents cancer-causing genes from being expressed is deleted, an individual will be at much greater risk of developing tumors. On the flip side, when “proto-oncogenic” microRNAs that promote cell immortality are expressed in greater concentrations due to dysregulation, this can lead to cancer as well. Cancer also has another terrible trick up its sleeve: It may eventually begin to spoof Treg cells. These “rogue” Treg cells secrete the same inhibitory signal molecules as normal ones do, further preventing our immune system from acting upon the tumor cells. The mechanisms through which various microRNAs may influence the workings of the immune system are complex. It has been observed that microRNA-mediated responses undergo very precise, location-sensitive regulation. This is evident in microRNA’s heavy influence in the formation of anatomical structures in C. elegans and other organisms. Our current understanding does not encompass the diversity of interactions that microRNA may have with the body. However, this fact has not deterred scientists from experimenting with novel treatments that take advantage of microRNA mechanisms. If proven safe and effective, microRNA therapeutics will offer medical science powerful tools to combat difficult-to-treat ailments. One example of a microRNA-based drug that has completed Phase I clinical trials is RG-101, an anti-microRNA oligonucleotide designed by Regulus Therapeutics in La Jolla, CA. This drug is designed to bind to and inactivate miR-122 in the liver. Recall that microRNA is a single-stranded molecule that must pair with a closely-matched mRNA sequence in order to inactivate the

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FIGURE 2 The biosynthesis of microRNA, from transcription within the nucleus to mRNA targeting in the cytoplasm. This process was gradually elucidated in the early 2000’s.

mRNA. When RG-101 itself binds with miR-122, the microRNA molecule can no longer bind with mRNA, thus inactivating miR-122 and increasing the concentration of the miR-122 target. Regulus aims to use RG-101 to disrupt the Hepatitis C virus (HCV) lifecycle within liver cells. The company hopes that their drug will be accepted into regular use alongside other antiviral drugs used to treat Hepatitis C, as microRNA pathways can target even strains of HCV that have mutated to become resistant to common antiviral drugs. While Hepatitis C is currently treatable, it nevertheless provides an excellent case study for the maturation of microRNA treatments in general. If such treatments succeed, treatments for diseases such as cystic fibrosis and cancer may soon follow. More research and clinical trials must be done before these treatments are to be released to the public, but the ability to treat some of the most widespread public health concerns in developed countries is within our reach.

Impact

Research of microRNA mechanisms has given medicine a new and potentially powerful system to treat autoimmune diseases and cancer. Experimental drugs that utilize microRNA to target and suppress malfunctioning genes are projected to have uses ranging from alleviating inflammation in rheumatoid arthritis, to boosting our body’s natural cancer defenses and preventing its onset. By deciphering the non-protein coding parts of

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the human genome, we can begin to unlock Pasquinelli AE. (2015). MicroRNAs: herthe full potential of gene therapeutics in alds of the noncoding RNA revolution. improving the lives of individuals with RNA. diseases currently considered incurable. Bagga, S., Bracht, J., Hunter, S., Massirer, K., Holtz, J., Eachus, R., & Pasquinelli, A. E. (2005). Regulation by let-7 and lin-4 References miRNAs results in target mRNA degraLee, H M, et al. “Progress and Challenge dation. Cell, 122, 553–563. of MicroRNA Research in Immunity.” PubMed, U.S. National Library of Medi- Hogan, Daniel J. et al. “Anti-miRs Competitively Inhibit microRNAs in Argonaute cine, 12 June 2014. Complexes.” Ed. Sebastien Pfeffer. PLoS Liston, A, et al. “MicroRNA in the AdapONE 9.7 (2014): e100951. PMC. Web. 1 tive Immune System, in Sickness and in Feb. 2017. Health.” National Center for Biotechnology Information, U.S. National Library of Medicine, May 2010. WRITTEN BY Lu, Li-Fan et al. “Function of miR-146a in PETER CHEW Controlling Treg Cell-Mediated Regulation of Th1 Responses.” Cell 142.6 Peter is a Molecular Biology (2010): 914–929. PMC. Web. 18 Feb. major from Earl Warren College. He will graduate in 2018. 2017.

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FEATURES

DEMYSTIFYING THE UTI:

NEW PERSPECTIVES ON FLORA FLOURISHING

INSIDE THE UROGENITAL TRACT

ANIKA ULLAH STAFF WRITER Illustration by Michelle Martinez Photography by Alexis Padilla

Until recently, the urinary tract infection, a disease affecting nearly one in three women globally by the age of 24, was thought to be caused by the invasion of a single, foreign, pathogenic bacterium into one’s otherwise sterile urinary tract. However, contemporary advances in metagenomics have revealed that urine is not sterile, and, like other environments of the body, the healthy urogenital tract has its own distinct microbiota that collectively play a role in mediating the state of the urinary tract. The significance of such a discovery is its implication that the current clinical practice of prescribing antibiotics for all UTIs may not represent the best therapy, considering that it would diminish the presence of protective microbes in the bladder and contribute to the steadily increasing phenomenon of antibiotic resistance. Furthermore, recent studies have illuminated the possible influence of diet, immunity, estrogen levels, stress, genetic predispositions, and comorbid diseases such as diabetes or obesity in mediating the onset of UTI.

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Hourly trips to the bathroom, a burning sensation served with a side of crippling pain, and the far too familiar sight of empty cranberry juice bottles lining your bedside table — three circumstances all attributed to a certain visitor. Regardless of how hostile you are to this visitor, it seems to brazenly show up at your doorstep, unannounced and undeterrable. Meet the urinary tract infection (UTI). As one of the most common type of infection in the human body, UTIs unapologetically send approximately 150 million people worldwide to the emergency room and clinic every year. Of these patients, approximately 80% are women. UTI persists as a dominant issue of women’s health; studies show that one in three women contract at least one UTI by the time they reach the age of 24. The good news is that UTI is technically curable. A promptly prescribed 10-day course of neatly packaged antibiotics usually does the trick. However, an inconspicuous danger breeds with each

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cursory antibiotic prescription. What happens when the first-rank clinical decision is for doctors to prescribe broad spectrum antibiotics for each and every UTI occurrence? What issues arise when unnecessary antibiotic treatment for a misidentified UTI occurs in hospitals 39% of the time? Antibiotic resistance increases the likelihood of a recurrent UTI, and women suffer. As harmless as a UTI may initially seem, if ineffectively treated, the infection could spread up to the kidneys and into the bloodstream, causing severe organ damage or even death. According to WIRED, “From 2000 to 2010, the proportion of UTIs resistant to [the commonly prescribed antibiotic] ciprofloxacin (cipro) rose from 3% to over 17%, and that percentage is steadily rising.” Another clinical practice that has contributed to the rise of antibiotic resistance is the prescription of antibiotics as a preventative measure for chronic UTIs — research suggests that doing so actually has conflicting short term effects on preventing recurrent UTIs and increases the risk of antibiotic resistance. These issues, coupled with globally ascending rates of antibiotic-resistant Escherichia coli (E. coli) in food and the environment, reveal that the exclusive use of antibiotics for UTI treatment is an ominous shortterm fix to a long-term issue and illuminates the dire need for alternative therapies. Until recent technological innovations and the surge of interest in microbiome research, a combination of issues, such as the underfunding of women’s health research, urban myths, and the incorrect scientific belief that urine is sterile, drove the construction of four be-all-end-all causes of UTI: engaging in excessive sexual activity, not urinating after sexual inter-

course, wearing tight undergarments, and spreading bacteria from the anus to the urethra by “wiping from back to front.” Thus, sexist stigma shrouding UTI is illuminated: that these four culturally and clinically accepted sole causes of UTI culminate in the depiction of women as contracting UTIs due to “promiscuous behavior” or supposedly preventable behavioral choices that “lack common-sense.” While these factors do constitute a few ways through which UTI-causing bacteria can spread to the urethra, they are by no means scientifically supported as the exclusive or most significant causative factors of UTI. The fallacy of attributing UTI to purely behavioral causes is evident in the phenomenon that 30-50% of women who experience a UTI have a recurrent one within the next year, even after making conscious behavioral changes to avoid the four aforementioned causes. In fact, research has actually revealed that with each UTI incidence, a patient is at an increasingly elevated risk for a recurrent UTI. There is clearly something veiled in the grander pathological scheme of UTI. Perhaps easy access to antibiotics and lack of viable technology previously subdued biomedical interest in a deeper understanding of UTI pathology. However, increasing resistance to an alarming variety of contemporary antibiotics is now necessitating a closer examination of non behavioral UTI causes in the search for knowledge that can inform the creation of alternative therapies or shape preventive education.

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FEATURES FIGURE 1 A visual representation of a few suspected contributing factors to UTI onset: excessive antibiotic usage, altered hormone levels, diabetes, obesity, and poultry raised with antibiotics. Photo by Anika Ullah.

Recent developments in the field of metagenomics, which studies bacterial populations that inhabit, regulate, and interact with various environments of the body, reveal a cutting-edge discovery; like the healthy gut, skin, and vagina, the urogenital tract has its own resident microbiome — a signature composition of various microbial populations — that maintains urogenital health. Prior to this discovery, the urogenital tract and urine were considered to be sterile entities, and UTI was viewed as a disease simply caused by the introduction of foreign bacteria into the bladder due to behavioral choices or physiological consequences. However, the implications of these advances in microbiome studies reveal that UTI is actually characterized by the “dysbiosis,” or microbial imbalance, of already existing bacteria in the urinary tract. Even more surprising is the revelation that dysbiosis can be influenced by an array of risk factors such as diet, immunity, stress, diminished estrogen levels, genetic predispositions, and comorbid diseases such as diabetes, obesity, and rheumatoid arthritis. 22 SALTMAN QUARTERLY

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For example, a malfunctioning immune system, which is a harbinger of common autoimmune diseases such as diabetes and multiple sclerosis, has been linked to UTIs,. It becomes easier to contract a UTI if the body’s natural defense mechanism cannot eradicate E. coli in the bladder, or if the immune system accidentally uproots protective bacteria that exist in the bladder and normally assist in the beneficial regulation of urogenital health. A few clinical studies have demonstrated that estrogen deficiency in postmenopausal women contributes to UTI incidence, and that estrogen-boosting therapies have been clinically effective in treating recurrent UTIs. Estrogen has been supported to play a role in protecting the lining of the bladder from pathogenic E. coli uptake and increasing protective bacterial populations of vaginal lactobacilli, which have been linked to suppressing UTI. Other studies have highlighted the dependence of pathogenic E. coli on gluconeogenesis, which is why high glucose content in the bladder, specifically in

the context of diabetes, puts patients at higher risk for UTI as a result of pathogenic E. coli consuming and thriving off of this glucose. When contemplating the impact of diet on UTIs, many instinctually recall the iconic bittersweet ‘cure’ that is cranberry juice. Surprisingly, multiple clinical studies evaluating the effectiveness of cranberry juice or cranberry supplements for UTI treatment deem that there is no conclusive scientific support that such remedies can treat UTIs. Conversely, an unexpected yet scientifically supported culprit behind increasing recurrent UTIs exists in the amorphous plastic-wrapped form sitting in aisle four of your local supermarket: poultry. The epidemic of excessive antibiotic usage in the American meat industry is exponentially increasing the amount of antibiotic resistant E. coli in poultry — the very poultry that 92% of American consumers regularly purchase and consume. Some of these E. coli colonize and remain in your gut microbiome, while others are excreted. The increase in concentration of these pathogenic bacteria in proximity to the urogenital region can escalate the probability of resistant E. coli traveling up the urethra and inducing UTI. As a deeper and more multifaceted understanding of UTIs coalesces at the forefront of human microbiome research, the extent of clinical variation and unpredictability in causative factors of UTI is starting to make more sense, and previous notions regarding the causes of UTIs are rapidly becoming antiquated. Why else are the antiquated assumptions of UTI risk factors problematic aside from the cultural backlash women

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FIGURE 2 A schematic of the network of factors that cause UTI via dysbiosis of the gut or vaginal microbiome. These factors are thought to be interconnected, and are currently being elucidated.

DIABETES WEAKENED IMMUNE SYSTEM

URINARY GLUCOSE GENETICS + BEHAVIOR

OBESITY GUT MICROBIOTA SHIFT

ESTROGEN DYSBIOSIS

POULTRY CONSUMPTION ANTIBIOTIC RESISTANCE

MENOPAUSE

VAGINAL MICROBIOTA SHIFT

UTI

experience? Perhaps it is because such assumptions portray UTI as something that can only be cured downstream from infection with antibiotics, which may have been the most effective treatment a decade ago. However, with the steady rise of antibiotic resistance and promising alternative therapies, directing the totality of research time and funds into antibiotic therapies will come to no avail. Further research for causes of UTI is needed, considering how it could bring important changes in preventative education and clinical practice for UTI without contributing to the global epidemic of antibiotic resistance. This shift of clinical care away from antibiotics will help not only the patient, but society at large, considering the estimated $1.5 billion dollars spent on UTI treatment annually, and the fact that hospitals are high risk sites for E. coli induced infections. According to Rob Knight, a renowned microbiologist, the future of research on microbiome influenced diseases such as UTI depends on a systems-based approach

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that considers key players that shape the microbiome, such as metabolite production, environmental factors, and clinical data. While scientists are working to further understand and create novel therapies for UTI, we can work to bring the ideologies behind the bench to the bedside by actively reshaping general and patient education for UTI. As scientists work to combat antibiotic resistance, we can lead the crusade against the stigmas that seem to drive women to under report UTI incidence.

“Urinary tract infection: pathogenesis and outlook.” Trends in Molecular Medicine 22.11 (2016): 946-957. Whiteside, S. A., Razvi, H., Dave, S., Reid, G., & Burton, J. P. (2015). The microbiome of the urinary tract—a role beyond infection. Nature Reviews Urology, 12(2), 81-90. doi:10.1038 Brubaker, L., & Wolfe, A. (2016). The urinary microbiota: a paradigm shift for bladder disorders? Current Opinion in Obstetrics and Gynecology, 28(5), 407412. doi:10.1097

References

A. Al-Badr, G. Al-Shaikh. Recurrent urinary tract infections management in women: a review. Sultan Qaboos Univ. Med. J., 13 (2013), pp. 359–367 Knight, Rob. “CMI Workshop Opening.” Personal interview. 18 Jan. 2017. Foxman, Betsy. “Epidemiology of urinary tract infections: incidence, morbidity, and economic costs.” The American journal of medicine 113.1 (2002): 5-13. McLellan, Lisa K., and David A. Hunstad.

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

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A Balinese long-tailed monkey, Macaca fascicularis, at the Indonesian Monkey Forest in Ubud, Bali. Photo by Laurel Bowling

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

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Variation in Hymenoptera Species Richness by Land Use in Monteverde, Costa Rica Mukta Kelkar | Advisors: Emilia Triana, M.S. and Federico Chinchilla, Ph.D.

RESEARCH Estación Forest

Life Monteverde

Figure 1 A Venn diagram describing the overlap of morphospecies among the three main study sites. The number in the center is the number of morphospecies shared by all three sites, the numbers in the white sectors describe the number of morphospecies shared by two overlapping sites, and the numbers in the outermost colored sectors describe the number of morphospecies unique to each site. There were four morphospecies that were present at all three sites and five that were present at only Life Monteverde and the backyard garden.

Humans reshape their environment wherever they live, modifying existing ecosystems and sometimes creating new ones. Insect species, in this case hymenopterans, have specific requirements of their environment. I investigated Hymenoptera species richness in agricultural, urban, and forested land in Monteverde, Costa Rica. There I collected insects at three different sites, counted all the insects, and identified and counted the hymenopteran morphospecies, which are groups of organisms categorized by morphology and not traditional taxonomy. I then tested the hypotheses that the forest would have the greatest proportion of hymenopteran insects to all insects, that urban areas would have the lowest species richness, and that there would be variation in richness among different parts of the agricultural land. My findings suggest that agricultural land has the highest richness of dominant species and that agroforestry is good for hymenopterans. Dominant species are those that much more abundant than the other species present. My findings imply that urban areas support less species richness and thus have potential implications for environmental management of agricultural and urban areas.

Introduction

It is both interesting and important to examine how humans interacting with and changing the environment influences biodiversity. When humans develop land, that area will take many years to return to its natural state of being, if it ever does return. Thus, understanding how development affects native ecosystems allows people to make informed and effective decisions regarding conservation methods1. In many cases, habitat loss due to both urbanization and agriculture has caused local extinctions of some animal species and reduced species abundance 1,2. However, in some species, which are often non-native, moderate urbanization actually increases species richness3. Hymenoptera is the insect order that includes bees, ants, and wasps. Hymenopterans live in both natural and manmade ecosystems. A rich population of hymenopterans is critical to any ecosystem because plants depend on them for ecosystem services, like pollination, and the other animals depend on the plants. The four largest categories of Hymenoptera are phytophages, parasitoids, predators, and pollinators4. Phytophages use leaf litter, growing leaves, or seeds for food. Many of these Hymenoptera form mutualistic relatioships with the organisms on which they feed. Parasitoids lay eggs inside of other organisms and their presence is correlated with increasing host diversity4,5. Predators have two subgroups; some have specialized habitats and prey on hosts while others are social and gather food for their larvae. The fourth category, pollinators, overlaps with the others. Hymenopterans transport pollen between plants, and thus are key for both plant life cycles and the life cycles of organisms that depend on plants. Consequently, pollinators are necessary for all ecosystems, including forests and farms4.

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Location/Site

Hymenoptera Individuals

Total Insects

Percentage Hymenoptera

Life Monteverde

330

15%

Life Monteverde -Crops

116

16%

Life Monteverde -Forest

123

17%

Life Monteverde -Coffee

10%

Estación Biológica Forest

108

25%

Urban Garden

631

13%

Table 1 Number and percentage of hymenopterans at every site and subsite.

Hymenoptera diversity is related to the way people use the land, though not necessarily in a negative way. Parasitoid Hymenoptera in agroforests — agricultural lands in which the farmers conserve some of the trees — are influenced by the distances between said forests; the smaller the distances, the greater the species richness6. In a study on trap-nesting bees and wasps conducted in coffee farms on Indonesia, the researchers found that while social bee abundance decreased with land use intensity, the abundance of solitary bees and wasps increased7. Furthermore, in San Francisco Bay, California, bee visitation to plants in urban and agricultural ecosystems was higher than in the natural forest8. In coastal Ecuador, researchers found that rice fields, pasture land, coffee agroforests, abandoned coffee agroforests, and native forest fragments varied spatially and tempo-

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Backyard Garden rally in Hymenoptera diversity. They also found that habitat specificity decreased when habitat disturbance increased9. Additionally, some Hymenoptera, specifically pollinators, can be used as bioindicators of environmental degradation caused by human activity. Examples of degradation include disease, habitat modification, pesticide presence, and introduced parasites. Pollinators play a key role in plant reproduction, and thus are an essential part of most terrestrial ecosystems. Pollinators are good bioindicators because they constantly sample their environment in the process of pollination10. Honeybees are widely used pollinators in agriculture because they are well understood and easy to transport11. While most organisms can serve as bioindicators, pollinators are especially useful because they play a critical role in natural and agricultural productivity and are intertwined with landscape ecology10. I examined and compared the species richness of Hymenoptera in an organic coffee farm, an urban garden, and a secondary forest. A secondary forest is one that has previously been damaged by human activity but has since recovered enough that the past damage is not apparent. These three sites provide exemplary examples of different kinds of development: agricultural, urban, and natural. By examining the species richness at these three sites, I was able to test if Hymenoptera species richness was different in the various kinds of land use in and around Monteverde, Costa Rica. Species richness is the most commonly used measure of biodiversity3. My questions were as follows: Is the percentage of hymenoptera to non-hymenoptera individuals different by land use? Is the species richness of Hymenoptera different by land use? Is species richness different in different parts — the coffee plants, the crop garden, and the forest — of the

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same coffee farm? I hypothesized thatthe forest, being the most natural environment, would contain the greatest percentage of hymenopterans, and for this reason I proposed that the same would be true of the species richness. Furthermore, the makeup of the flora on the coffee farm would correlate to the richness of the fauna.

Materials and Methods

Data were collected from three different sites: Life Monteverde in Cañitas, a backyard garden in Cerro Plano, and the forest at the Estación Biológica. All three sites are located around Monteverde, Costa Rica. Life Monteverde consists of a coffee farm, some forest, and a crop garden. Equal effort was devoted to each part of the farm, and insects from each part of the farm were stored separately. The backyard garden has grasses, flowering plants, and herbs. The forest is thickly vegetated and has some hiking trails. Insects were collected from Life Monteverde on 13 May 2016 and 17 May 2016, from the forest around the Estación on 14 May 2016 and 16 May 2016, and from the backyard garden on 20 May 2016. On 13 May 2016 insects were collected from 1100 to 1400, and on all other days insects were collected between 900 and 1200. The weather was sunny on all collection days at Life Monteverde and the Estación Forest and was rainy during collection at the backyard garden. Insects were collected by swiping a 1-foot-diameter butterfly net over the vegetation for 60 second intervals. To ensure comparability both among the three sites and within Life Monteverde, the thirty 60 second intervals of data collection at Life Monteverde were evenly divided among three subsites. Insects were collected in ten 60 second intervals in each of the three subsites so the total effort at Life Monteverde was equal to the effort at the other two sites. After the 60 second interval,

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Crops

Coffee

Figure 2 A Venn diagram describing the overlap of morphospecies among the three subsites in Life Monteverde. The number in the center is the number of morphospecies shared by all three subsites, the numbers in the white sectors describe the number of morphospecies shared by two overlapping subsites, and the numbers in the outermost colored sectors describe the number of morphospecies unique to each subsite. There were no morphospecies unique to the coffee, crops and coffee share one morphospecies, forest and coffee share one, and all three subsites share two morphospecies.

Forest insects were transferred from the net to a sandwich bag, placed in a freezer for 5 minutes, and then moved to a small jar with 70% ethanol. This process was conducted 30 times per site. To identify the insects, hymenopterans were separated fromnon-hymenopterans using a dissecting scope. Then, the number of hymenopterans and non-hymenopterans were counted for each site. The hymenopterans were then separated into morphospecies. Morphospecies are groups of organisms categorized by morphology instead of traditional taxonomy. The characteristics of every morphospecies are decided by the researcher based on observations. Characteristics of families within the order Hymenoptera were used as a guide for defining morphospecies. Finally, the number of individuals of each morphospecies were counted. I conducted two sets of comparisons. First, I compared the forest, the farm, and the backyard garden to each other. Second, I compared the three collection sites within the farm Life Monteverde itself. I used the True Diversity Index to quantify overall species richness (q=0), richness of common species (q=1), and richness of dominant species (q=2) at every site and subsite15. I used a Chi-Square Test for Variation to see if there was a difference in richness between each site.

each of the urbanized areas (Figure 1). There was no difference in morphospecies overlap among Life Monteverde, the backyard garden, and the forest (χ2=4.571, df=2 p=0.1017; Figure 1). Within Life Monteverde, there were no morphospecies that were unique to the coffee plants: all species in the coffee plants overlapped with the crops, the forest, or both (Figure 2). The True Diversity Index describes three kinds of diversity. Q=0 represents overall richness, which is the total number of species. Q=1 represents richness of common species, which takes in to consideration both richness and abundance. Q=2 represents the dominance, which is the number of dominant species. Dominant species are those that are much more abundant than the other species. My three study sites had a difference in dominance (χ2=7.812, df=2, p=0.0201; Figure 3). Life Monteverde had the greatest number of dominant species, the forest had the second greatest, and the urban garden had the fewest (Figure 3). There was no difference either in species richness between the three sites (χ2=3.714, df=2, p=0.156; Figure 3) or in diversity (χ2 =3.859, df=2, p=0.1452; Figure 3). There was no difference in overall species richness at Life Monteverde (χ2=2.24, df=2, p=0.3263; Figure 4). There was also no difference when examining the diversity (χ2=2.026, df=2, p=0.3631; Figure 4) nor the number of dominant species (χ2=2.562, df=2, p=0.2778; Figure 4).

r d Results

In total I collected 159 hymenopterans, which belonged to 24 morphospecies. There was no difference in percentage of hymenopterans at each of the three sites (χ2=4.61801, df =2, p=0.099; Table 1). When comparing the three subsites at Life Monteverde, there was no difference between the percentage of Hymenoptera (χ2=2.1731, df=2, p=0.337; Table 1). Among the three main study sites, there were four morphospecies that were present at all four sites, five morphospecies shared between Life Monteverde and the backyard garden, and one species shared between Estación Forest and

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Discussion

I found that there is a difference in the richness of the dominant species among the three land uses. The farm Life Monteverde had the greatest richness of dom nant species, followed by the Estación Forest, followed by the home garden. High primary productivity is associated with high biodiversity, especially in developed areas3. Thus Life Monteverde may have high primary productivity, which could be an explanation for the high level of dominance. Additionally, dominance may vary seasonally, possibly depending on the

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plants in bloom12,13,14. Since I collected samples over a one-week period, I cannot account for seasonality. It is interesting that there was a q=2 value that was lower than expected in the True Diversity Index for both the comparison between rural, urban, and forest and the comparison within Life Monteverde. Normally, the lines connecting q=0, q=1, and q=2 run approximately parallel to each other and don’t intersect14. In Figure 3 The True Diversity Index calculated for Life Monteverde, my homestay’s garden, and the True Diversity Index for all three the forest around the Estación. There is a difference in the species richness of dominant species sites, the backyard garden dominance (q=2) between the three sites. q=0 signifies overall species richness, q=1 signifies diversity, and drops suddenly. In the True Diversity q=2 signifies dominance. Index for Life Monteverde, the crop garden dominance drops suddenly. The irregularities in both True Diversity Indexes may be related to changing seasonal diversity, especially considering I collected insects only at the start of the rainy season12,13,14. this study is loss of small insects in transfer from the net to In addition, the coffee portion of the farm had no the ethanol. Some insects were too small to see, got stuck in morphospecies that were unique to it. It is possible that the the gaps in the net or the rims of the sandwich bag, or were forest and the crops are needed for the presence of hycrushed in the process of transferring them from the net to menopterans in the coffee plants, and that the hymenopterthe plastic bag to the jar of ethanol. Another possible source ans are flying between the coffee and other parts of the of error is the weather. Since it rained during collection farm. Life Monteverde is primarily a coffee farm. Since at the backyard garden, the weather was not comparable hymenopterans pollinate the coffee and may be supported between study sites, and the rain may have affected which by the forest and the crops, it is possible that the farm would insects were active during collection in the garden. be less productive if the farmers cleared the forest and the In summary, I detected a difference in the richness of crop garden. There was no significant difference in percentdominant species between agricultural land, urban land, age of Hymenoptera among Life Monteverde, the backyard and forested land. The agricultural land had the highest garden, and the Estación forest. I also found no difference richness of dominant species and the urban land had the between the overall species richness and the diversity belowest. This may be an indicator that urban land does not tween the sites. This may be because both of my developed support as much diversity as agricultural and forested land, sites — Life Monteverde and the home garden — had a lot which has potential implications for urban planning and of plants, and thus had habitat for hymenopterans. Addienvironmental management. Finally, my results show that tionally, the lack of difference in overall species richness the forest and crop areas of Life Monteverde share hyand diversity may indicate that all three habitats are equality menopterans with the coffee areas, and that it is possible the suitable for hymenopteans. Finally, there was no difference coffee areas have few to no hymenoptera unique to them. in either percentage or richness within the farm Life MonThis may be an indicator that maintaining some forest when teverde. The sections of Life Monteverde that I collected establishing a farm is not only environmentally friendly but from were within the flight range of a single stingless bee16. is actually good for the productivity of the farm. The distance between the sections was also very close to the Future studies should collect information about the plant flight range of a parasitoid wasp17. Therefore, it’s possible species in each type of land use in addition to the Hymethat no difference occurred between thesub-sites within noptera species and measure the size of the experimental Life Monteverde because the hymenopteran populations plots. Information about the plant species present in each overlapped across the sites that I studied. type of land would help decipher what about urban land My study was observational and so it is not possible to — the lack of vegetation, the type of vegetation, or neither infer a causal relationship between land use and species — supports less diversity. Data regarding the plot size would dominance. One possible source of methodological error in help determine if habitat area is related to species richness.

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diversity of trap-nesting Hymenoptera in adjacent agroforestry. Journal of Animal Ecology, 75(2), 315-323 (2006). 7. Klein, A. M., I. Steffan-Dewenter, D. Buchori, , & T. Tscharntke. Effects of land- use intensity in tropical agroforestry systems on coffee flower-visiting and trap-nesting bees and wasps. Conservation biology, 16 (4), 1003-1014 (2002). 8. Leong, M., C. Kremen, & G. K. Roderick. Pollinator interactions with yellow starthistle (Centaurea solstitialis) across urban, agricultural, and natural landscapes. PloS one, 9 (1), e86357 (2014). 9. Tylianakis, J. M., A. M. Klein, Figure 4 The True Diversity Index calculated for the subsites within Life Monteverde: the crop A. M., & T. Tscharntke. Spatiotemgarden, the forest, and the coffee plants. There was no difference in species richness between poral variation in the diversity of these subsites. q=0 signifies overall species richness, q=1 signifies diversity, and q=2 signifies Hymenoptera across a tropical habitat dominance. gradient. Ecology, 86(12), 3296-3302 (2005). 10. Kevan, P. G. Pollinators as bioindicators of the state of the environment: species, activity and diversity. AgriculAcknowledgements ture, Ecosystems & Environment, 74(1), 373-393. (1999). A sincere thank you to the Estación Biológica, Life 11. Free, J.B. Insect Pollination of Crops, 2nd ed. AcaMonteverde Farm, and my homestay family for allowing me demic Press, London, 684 pp. (1993). collect insects on their land. I would also like to thank Emilia 12. Sääksjärvi, I. E., S. Haataja, S. Neuvonen, I. D. Triana for helping me with my project every step of the way, Gauld, R. Jussila, J. Salo, & A. M. Burgos. High local species staying calm when nothing was working, and sharing her richness of parasitic wasps (Hymenoptera: Ichneumonidae; incredible enthusiasm for insects. Thank you to Federico Pimplinae and Rhyssinae) from the lowland rainforests of Chinchilla for helpful comments on my writing. Thank you Peruvian Amazonia. Ecological Entomology, 29(6), 735-743 to The Monteverde Institute and UCEAP Costa Rica for (2004). providing funding and resources for this project. 13. Ackerman, J. D. Diversity and seasonality of male euglossine bees (Hymenoptera: Apidae) in Central Panama. References Ecology, 274-283. (1983) 1. McKinney, M. L. Urbanization, biodiversity, and 14. Rezende Diniz, I., & K. Kitayama. Seasonality of conservation the impacts of urbanization on native species vespid species (Hymenoptera: Vespidae) ina central Braare poorly studied, but educating a highly urbanized human zilian cerrado. Revista de Biologia Tropical, 46(1), 109-114 population about these impacts can greatly improve species (1998). conservation in all ecosystems. BioScience, 52(10), 883-890 15. Jost, L. Partitioning diversity into independent alpha (2002). and beta components. Ecology, 88(10), 2427-2439 (2007). 2. Connor, E. F., J. Hafernik, J. Levy, V. L. Moore, & J. 16. Araújo, E. D., M. Costa, J. Chaud-Netto, & H. G. K. Rickman. Insect conservation in an urban biodiversity Fowler. Body size and flight distance in stingless bees (Hyhotspot: the San Francisco Bay Area. Journal of Insect Conmenoptera: Meliponini): inference of flight range and possiservation, 6(4), 247-259 (2002). ble ecological implications. Brazilian Journal of Biology, 64 3. McKinney, M. L. Effects of urbanization on species (3B), 563-568 (2004). richness: a review of plants and animals. Urban ecosystems, 17. Wanner, H., H. Gu, & S. Dorn. Nutritional value 11(2), 161-176 (2008). of floral nectar sources for flight in the parasitoid wasp, 4. Ugalde, J. A. Avispas, abejas y hormigas Costa Rica Cotesia glomerata. Physiological Entomology, 31(2), 127-133 Wasps, bees and ants. National Institution of Biodiversity, (2006). Santo Domingo, Heredia, Costa Rica. (2002). 5. Tylianakis, J. M., T. Tscharntke, & A. M. Klein. Diversity, ecosystem function, and stability of parasitoid-host interactions across a tropical habitat gradient. Ecology, 87 WRITTEN BY MUKTA KELKAR (12), 3047-3057 (2006). Mukta is an Ecology, Behavior and Evolution major from John 6. Klein, A., I. N. G. O. L. F. Steffan-Dewenter, & T. Muir College. She will graduate in 2017. Tscharntke. Rain forest promotes trophic interactions and

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RESEARCH Relationship between Psychosocial Stress and Intuitive Eating in Overweight Latino and African-American Youth Vennis Hong1 | Co-authors: Claudia Toledo-Corral, Ph.D. , Marc J. Weigensberg, M.D., Michael I. Goran, Ph.D.

Intuitive eating (IE) is the practice of eating in accordance with physiological cues and natural hunger signals. It is characterized by 1) no restrictions on food choices, 2) eating strictly for physical reasons, and 3) responding to physiological hunger and satiety cues. While IE has been identified as a potential longterm strategy for reducing obesity rates, little is currently known about factors that determine intuitive eating choices and behaviors. The objectives of this study were to investigate the relationship between psychosocial stress and IE and to determine whether cortisol is mediating this relationship. This cross-sectional study included a one-time survey assessing participants’ stress levels and IE behaviors. Urinary free cortisol, salivary cortisol, and serum cortisol measures were collected through an overnight inpatient stay. The participants and data for this study were from the DREAM (Diabetes Risk due to Ectopic Adiposity in Minority youth) study conducted at the University of Southern California. Participants (N = 183) include African Americans and Latinos between the ages of 8 and 17 (µ = 14.87, ơ = 2.61). Participants were overweight or obese (BMI at the 85th percentile and higher; for BMI, µ = 30.83, ơ = 7.16) but otherwise healthy. Multivariate linear regressions revealed significant associations between perceived stress and IE (β = -0.023, p = <0.001). No significant associations were found between stressful life events and IE. While salivary cortisol at 22:00 hours was associated with IE (β = -0.079, p = 0.020), cortisol was not a mediator in the relationship between stress and IE. Greater levels of perceived stress were associated with less IE habits, suggesting that, in minority children and adolescents, IE may be influenced by psychosocial stressors but that the relationship might not mediated by cortisol. Understanding characteristics that facilitate IE may contribute to the successful strategies of weight management programs for overweight and obese minority youth.

Introduction

With nearly a third of the U.S. child and adolescent population identified as overweight or obese, pediatric obesity is a public health epidemic. African Americans and Latinos are dispropotionately affected, with nearly 40% of African-American and Latino youth identified as overweight or obese1. The Black and Hispanic populations are expected to increase by 42% and 114.8%, respectively, between 2014 and 2060. The problem of minority health disparities calls for attention as it poses a cost to society 2,3. Unfavorable eating behaviors, such as emotional overeating, have been found to be associated with body mass index (BMI) and obesity status, hence the growing interest in studying various patterns of eating4. Little is known, however, about psychosocial factors that influence intuitive eating choices. Our study may, therefore, be beneficial in expanding the current knowledge on intuitive eating. Stress and Intuitive Eating Intuitive eating is defined by three essential facets: 1) no restrictions on food choices, 2) eating strictly for physical reasons, 3) responding to physiological hunger and satiety cues5,6. Intuitive eating habits have been associated with fewer disordered eating behaviors, such as chronic dieting and binge eating, as well as lower BMI. For this reason, intuitive eating is recognized as an effective, long-term strategy for weight management7,8.

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Evidence has long suggested a relationship between stress and eating behavior. In fact, many studies have found that stress is linked to unhealthy behaviors, such as emotional overeating, extreme weight control, and bulimia nervosa 9, 10, 11 . Although little research has assessed a relationship between psychosocial stress and intuitive eating behaviors, Järvelä-Reijonen et al. (2016) found that high perceived stress was associated with less intuitive eating in a working age population in Finland12. Cortisol, Stress, and Eating Behaviors A hypothesized physiological mechanism facilitating the relationship between stress and intuitive eating is the cortisol response. Cortisol is a hormone that is regulated and released by the hypothalamic-pituitaryadrenal (HPA) axis upon exposure to stress13. The current literature on the association between psychosocial stress and cortisol levels is inconsistent. While some studies have found that stress is positively associated with cortisol levels14,15, others have found that stress is actually negatively associated cortisol output16. Studies have also suggested a relationship between cortisol and amount of food intake. In fact, a randomized controlled trial done by George, Khan, Briggs, and Abelson (2010) found that elevated cortisol levels (exogenously injected) were directly related to higher food intake17. Studies have also found that participants with blunted cortisol morning responses tend to have higher food intake, reduced satiety responsiveness, and

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Table 1 Summary of

participants’ cortisol measures by ethnicity and sex.

more emotional eating 18. Although no studies have investigated the relationship between cortisol and intuitive eating, the evidence that currently exists on the associations between cortisol and other eating behaviors suggests that cortisol may be correlated to intuitive eating behavior. Current Study To the best of our knowledge, psychosocial stress and intuitive eating is a relatively unexplored area of study, and the limited literature that exists in this area focuses on adult populations outside of the United States. This study, therefore, aims to investigate the relationship between psychosocial stressors and intuitive eating in overweight and obese African-American and Latino youth. Furthermore, because few studies on the relationship between cortisol and intuitive eating choices has been done, this study assesses the mediating role of cortisol in the relationship between psychosocial stress and intuitive eating. We hypothesize that greater levels of perceived stress and more exposure to stressful life events are associated with less intuitive eating. We also predict that cortisol plays a mediating role in the relationship between psychosocial stress and cortisol.

m Methods

Recruitment A group of overweight but healthy African American and Latino boys and girls (N = 183) ages 8 to 17 took part in the Diabetes Risk due to Ectopic Adiposity in Minority Youth (DREAM) study. Participants were recruited from Los Angeles County community health clinics, health fairs, and by word

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of mouth. The following criteria were met for inclusion in the study: 1) either African-American or Latino ethnicity (all four grandparents of African-American or Latino descent), 2) ages 8 to 17, and 3) BMI at the 85th percentile according to Centers for Disease Control and Prevention standards. Participants were excluded if they had Type I or Type II diabetes, took medications, or had any other conditions that affected insulin activity. Both the participants and their parents completed a written informed consent and assent process. Procedures Participants made two visits to the Clinical Trials Unit at the USC University Hospital. During the first visit, a medical history and physical examination were conducted. Pubertal stage (by Tanner scale), vital signs, and body composition, such as BMI and body fat via DEXA, were assessed as well. Participants were asked to complete questionnaires about their stress levels, feelings, and eating habits. If the questionnaires were not completed during the first visit, they were done either during the second visit or by phone with a staff member. During the second visit, participants were admitted in the evening for an overnight stay as inpatients. They were served dinner and a snack before beginning an overnight fast at 20:00 hours. Urine and saliva samples were collected at bedtime at 22:00 hours. Participants were then woken up at 05:30 hours to collect urine and saliva a second time. Saliva samples were collected again at 05:45 hours and 06:00 hours. Fasting blood was drawn at 08:00 hours. Psychosocial Stress Measures Participants completed surveys that measured their level of perceived stress, their exposure to stressful life events, and their overall psychological well-being. Perceived Stress. The Perceived Stress Scale (PSS) was used to

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measure participants’ level of perceived stress. The survey contained 14 questions, each of which included five answer choices that offered a spectrum from “never” to “very often.” Example questions include “In the last month, how often have you been upset because of something that happened unexpectedly?” and “In the last month, how often have you felt that you were on top of things?” A higher score indicates more perceived stress with 0 as the minimum score and 56 as the maximum score19. Stressful Life Events. The Life Events Scale (LES) was used to measure participants’ exposure to stressful life events. The survey contained a list of 66 positive and negative stressful life events which were categorized into school, family, peer, and personal events. Examples of stressful life events include getting bad grades, experiencing a death in the family, being teased, and learning something new20. Participants were asked to fill in the circle next to the events to which they were exposed. A composite score was taken for each category of the LES as well as for the entire scale. Higher scores indicate more exposure to stressful life events, with 0 as the minimum score and 66 as the maximum score. Stressful life events data were missing for 6 participants. Intuitive Eating Measure Table 2 Results of the linear regression of stress on intuitive eating Intuitive eating was measured using the Intuitive Eating Scale (IES), a list of 30 statements with which participants are asked the extent to which they and is defined as the increase in salivary cortisol at 05:30 hours agree21. Example statements in the IES include “I seldom eat and 06:00 hours. 168 observations were collected for NCR, and unless I notice I am physically hungry” and “There are certain 161 observations were collected for CAR. foods I really like, but I try to avoid them so that I won’t gain Statistical Analysis weight.” Average score per question was used for this study, Normality of cortisol variables was assessed through Shapwith 0 as the minimum score and 5 as the maximum score. iro-Wilk test of normality. Non-normal variables (urinary free Intuitive eating data were missing for 12 participants. cortisol and salivary cortisol at 22:00 hours) were natural log Cortisol Measures tran sformed for analysis. ANOVA and chi-square tests were Measures for HPA axis activity included urinary free cortiused to assess mean and frequency of variables by ethnicity sol, salivary cortisol, and serum cortisol. Urinary free cortisol and sex. A multivariate linear regression analysis was used to was assayed using Coat-A-Count radioimmunoassay and assess the effects of perceived stress and stressful life events corrected for body surface area. Urinary free cortisol data were on intuitive eating. To assess the mediating role of cortisol, a collected from 136 of the 183 participants. Saliva samples were multivariate linear regression of each stress measure was done collected using the Salivette system by Sarstedt, which involves on each cortisol measure. A multivariate linear regression of placing a cotton swab in the mouth for two minutes. They were each cortisol measure was then done on intuitive eating. All then processed by centrifuging for 10 minutes at 2500 rpm regression models were adjusted for age, sex, ethnicity, percent and frozen at -70ºC until assayed. Salivary and serum cortisol body fat, and SES. Data were analyzed using SPSS version 23.0 were assayed using an automated enzyme immunoassay. The for Windows. inter-assay coefficient of variation for the assay kit was 11.2%, Results and the intra-assay coefficient of variation was 8.2%. 180 Participant Characteristics observations were collected for salivary cortisol at 22:00 hours, Table 1 summarizes participant characteristics by ethnicity and 171 observations at 05:30 hours, and 169 observations at 06:00 sex. Participants consist of more Latinos than African Amerhours. Nocturnal cortisol rise (NCR) is a measure of overicans (115 vs. 68), and more males than females (109 vs. 74). night cortisol activity and is defined as the increase in salivary Females are, overall, older than males (µ = 16.11 vs. µ = 14.02, cortisol from 22:00 hours to 05:30 hours. Cortisol awakening p = <0.001) and are, thus, at a more advanced Tanner pubertal response (CAR) is a measure of the waking HPA axis activity

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Table 3 Table 3

notes the linear regression of cortisol on different psychosocial stresses.

stage than males (µ = 4.82 vs. µ = 3.40, p = <0.001). The average age and Tanner pubertal stage of the participants re 14.87 and 3.98, respectively. African Americans have higher Hollingshead SES scores than Latinos (µ = 31.23 vs. µ = 20.97, p = <0.001), while the average Hollingshead score for the participants is 30.50. African American females have a significantly higher average BMI than male participants (p = <0.001), while the average BMI of the participants is 30.83. Average BMI percentile is 93.88, and an average of 28.9% of participants are prediabetic. Psychosocial Stress and Intuitive Eating Table 1 summarizes participants’ stress and intuitive eating measures by ethnicity and sex. The average perceived stress score for all participants is 23.69 out of a maximum score of 56. The a verage numbers of school-related, family-related, peer-related, and personal stressful life events experienced are 3.13, 6.81, 3.86, and 5.76, respectively. Latina females experienced significantly more family-related (µ = 9.79, p = <0.001) and personal (µ = 8.00, p = <0.01) stressful life events than other participants. The average intuitive eating score of the participants is 3.61 out of 5. Cortisol Table 1 summarizes participants’ cortisol measures by ethnicity and sex. The average urinary-free cortisol, salivary cortisol at 22:00 hours, and salivary cortisol at 05:30 hours for the participants are 5.06, 0.16, and 0.84, respectively. Latina females have significantly higher salivary cortisol at 06:00 hours than the other participants (µ = 2.13, p = <0.001), while the average salivary cortisol at 06:00 hours of all participants is 1.67. Latino males have significantly higher serum cortisol levels than African American males (11.56 vs 9.67 , p = 0.012), while the average serum cortisol level of the participants is 10.57. Average NCR of the participants is 0.70. Latina females 34 SALTMAN QUARTERLY

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have a significantly higher CAR than the rest of the participants (p = <0.001), while the average CAR for all participants is 0.86. Associations between Psychosocial Stress and Intuitive Eating Results of the linear regression of stress on intuitive eating are shown in Table 2. After adjusting for age, sex, ethnicity, percent fat, and SES, a linear regression analysis reveals a negative association between perceived stress and intuitive eating (β = -0.017, p = <0.01). No significant associations are observed between stressful life events and intuitive eating. Cortisol as a Potential Mediator After adjusting for age, sex, ethnicity, percent fat, and SES, a linear regression analysis of each stress measure on each cortisol measure reveals no significant relationship between stress and cortisol (Table 3). A regression of each cortisol measure on intuitive eating reveals a negative association between cortisol at 22:00 hours and intuitive eating (β = -0.079, p = 0.038, Table 4). No other significant association is found between other cortisol measures and intuitive eating. Cortisol, therefore, does not appear to play a mediating role in the relationship between stress and intuitive eating.

d Discussion

The primary objective of this study was to investigate the impact of psychosocial stress on intuitive eating behaviors in overweight and obese Latino and African American children and adolescents. After adjusting for age, sex, ethnicity, SES, and percent body fat, perceived stress was negatively associated with intuitive eating. Stressful life events, however, were not associated with intuitive eating. Our finding that the interaction between stressful life events and intuitive eating was not statistically significant disagreed with our hypothesis. We believe this relationship was not statistically significant because the stressful life events measure

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did not take into account protective factors against psychosocial stress, such as resilience, coping methods, social support, and overall well-being. Our finding suggests that measures of the number or types of psychosocial stressors alone are insufficient in predicting the impact of psychosocial stress on behavioral outcomes. For instance, participants who have experienced many stressful life events may not necessarily be stressed if they are resilient and have a strong support network. On the other hand, our finding that perceived stress was negatively associated with intuitive eating aligned with our hypothesis. Protective factors against psychosocial stress, such as resilience and coping, are taken into account through the measure of perceived stress, which may explain why we found a significant association with perceived stress but not stressful life events. The results of this investigation suggest that experiencing more psychosocial stress may contribute to less intuitive eating habits. Our finding that perceived stress was negatively associated with intuitive eating was consistent with many studies that have found that psychosocial stress negatively impacts eating behaviors. In a population of overweight and obese adults in Finland, Järvelä-Reijonen et al. (2016) found that exposure to more psychosocial stress was associated with less intuitive eating12. Perceived stress has also been found to be positively associated with bulimic behaviors, extreme weight control behaviors such as vomiting and taking diet pills, and binge eating7,22. The secondary objective of this study was to asTable 4 A regression of each cortisol measure on intuitive eating. sess cortisol as a mediator in the relationship between psychosocial stress and intuitive eating. After adjusting for age, sex, ethnicity, SES, and percent body fat, salivary findings regarding the relationship between psychosocial stress cortisol at 22:00 hours was negatively associated with intuitive and cortisol may be due to differences in research methodoleating. However, because no association was found between ogy. Karlén et al. (2011), for instance, used hair cortisol and psychosocial stress and cortisol, cortisol does not appear to be serious life events within the last three months14. Armbruster a mediator in the relationship between psychosocial stress and (2012), on the other hand, collected stressful life events using intuitive eating. Nonetheless, we argue that further research semi-structured interviews, induced psychosocial stress using is needed to draw more meaningful conclusions regarding the Trier Social Stress Test, and obtained salivary cortisol cortisol as a biological mediator in the relationship between measures after onset of stress sessions16. Our finding that psychosocial stress and intuitive eating. psychosocial stress was not correlated with overnight salivary, Our finding that cortisol was not a statistically significant serum, and urinary free cortisol, thus, adds to the current body mediator in the relationship between psychosocial stress and of knowledge on psychosocial stress and cortisol but does not intuitive eating contradicted our hypothesis. Our finding that clarify their relationship. no association existed between psychosocial stress and cortisol Although cortisol was not found to be a statistically signifagreed with Olstad et al. (2016), who found similar results in icant mediator in the relationship between psychosocial stress a population of socially disadvantaged women and children23. and intuitive eating, our finding that salivary cortisol at 22:00 Previous studies, however, have found that psychosocial stress hours was negatively associated with intuitive eating suggests was associated with flatter diurnal cortisol rhythms24,25 and elethe need for further research in cortisol as a biological medivated cortisol levels13,16. We believe that a relationship was not ator in this relationship. This finding was in agreement with a found between psychosocial stress and cortisol because cortisol previous finding that blunted diurnal responses were associmeasures were taken over only one night. This short timeframe ated with higher food intake in a population of female college may have been insufficient in detecting a relationship between students17. This finding also agreed with our hypothesis as psychosocial stressors and cortisol activity. These inconsistent cortisol levels are typically at their lowest prior to sleep at night time in healthy individuals. To the best of our knowledge, SALTMAN QUARTERLY sqonline.ucsd.edu VOL. 14 35

our finding is the first reported finding on the relationship between cortisol and intuitive eating behaviors. We believe an association with intuitive eating was found for salivary cortisol at 22:00 hours but not for other cortisol measures because, in youth, alterations in cortisol levels at one time point may be an early indicator of changes that are to come through maturation26. In other words, while salivary cortisol at 22:00 hours may be the only cortisol measure that is currently associated with intuitive eating in this age group, cortisol measures at other time points may also experience alterations as participants mature. Nonetheless, alterations in cortisol levels at one time point does not imply that cortisol measures at all other time points must also follow suit, as an alteration in only one time point is sufficient to cause changes to diurnal rhythms. Due to the ambiguity of our finding regarding the relationship between cortisol and intuitive eating, we argue that more investigations need to be done to determine whether cortisol may be a biological mediator. The strengths of this study include, first, that it is one of the first to explore the impact of psychosocial stress on intuitive eating, an understudied eating behavior. Second, it is also one of few eating behavior studies that focus on children and adolescents, particularly Latinos and African Americans who are disproportionately affected by obesity. Some limitations of the study include, first, that causality could not be established due to the cross-sectional design of the study. Second, our findings may be confounded by resilience, coping strategies, and overall well-being due to the lack of data on these variables. Third, the findings of this study may not be generalizable to White and Asian populations, populations outside of Los Angeles, and overweight or obese populations who have Type I or Type II diabetes and/or other conditions that affect insulin activity. Finally, due to the reliance on surveys, this study is subject to self-report errors. Future studies should further investigate the impact of psychosocial stress on intuitive eating by including measures that take into account participants’ overall wellbeing, coping strategies, and lifestyle. Future studies should also include a White comparison group to determine whether minority status plays a role in this relationship. In addition, future studies should investigate other hormones that may act as biological mediators in the relationship between psychosocial stress and intuitive eating, including but not limited to catecholamines, vasopressin, prolactin, insulin, ghrelin, leptin, thyroid hormones, and melatonin27.

c Conclusion

This study finds that perceived stress was associated with less intuitive eating choices and behaviors in overweight and obese Latino and African American youth, which is consistent with the current literature on eating behaviors. Contrary to a recent finding from another study, however, cortisol is not a mediator in this relationship. Future studies should further explore factors that influence intuitive eating choices and habits, which may contribute to the successful strategies of weight management programs for overweight and obese minority youth.

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RESEARCH

Acknowledgements

The study was funded by NIH Grant P60 MD00254 (M.I. Goran & M.J. Weigensberg) and CDC Cooperative Agreement Number 3U50MN000025.

References

1. Ogden CL, Carroll MD, Kit BK, et al. Prevalence of Obesity and Trends in Body Mass Index Among US Children and Adolescents, 1999-2010. JAMA. 2012;307(5):483. doi:10.1001/ jama.2012.40. 2. Cook BL, Liu Z, Lessios AS, Loder S, Mcguire T. The Costs and Benefits of Reducing Racial-Ethnic Disparities in Mental Health Care. Psychiatr Serv. 2015;66:389-396. doi:10.1176/appi.ps.201400070. 3. Colby SL, Ortman JM. Current Population Reports. 2015. 4. Vollmer RL, Adamsons K, Foster JS, Mobley AR. Association of fathers’ feeding practices and feeding style on preschool age children’s diet quality, eating behavior and body mass index. Appetite. 2015;89:274-281. doi:10.1016/j.appet.2015.02.021. 5. Tribole E, Resch E. Intuitive Eating : A Recovery Book for the Chronic Dieter: Rediscover the Pleasures of Eating and Rebuild Your Body Image. St. Martin’s Paperbacks; 1996. 6. Tylka TL, Wilcox JA. Are Intuitive Eating and Eating DisorderSymptomatology Opposite Poles of the Same Construct? J Couns Psychol. 2006;53(4):474-485. doi:10.1037/00220167.53.4.474. 7. Denny KN, Loth K, Eisenberg ME, Neumark-Sztainer D. Intuitive eating in young adults. Who is doing it, and how is it related to disordered eating behaviors? Appetite. 2013;60:1319. doi:10.1016/j.appet.2012.09.029. 8. Anderson LM, Reilly EE, Schaumberg K, Dmochowski S, Anderson DA. Contributions of mindful eating, intuitive eating, and restraint to BMI, disordered eating, and meal consumption in college students. Eat Weight Disord. 2016;21(1):83-90. doi:10.1007/s40519-015-0210-3. 9. Goldschmidt AB, Wonderlich SA, Crosby RD, et al. Ecological momentary assessment of stressful events and negative affect in bulimia nervosa. J Consult Clin Psychol. 2014;82(1):30-39. doi:10.1037/a0034974. 10. Loth K, van den Berg P, Eisenberg ME, Neumark-Sztainer D. Stressful Life Events and Disordered Eating Behaviors: Findings from Project EAT. Vol 43.; 2008. doi:10.1016/j. jadohealth.2008.03.007. 11. Richardson AS, Arsenault JE, Cates SC, Muth MK. Perceived stress, unhealthy eating behaviors, and severe obesity in low-income women. Nutr J. 2015;14:122. doi:10.1186/s12937015-0110-4. sqonline.ucsd.edu

12. Järvelä-Reijonen E, Karhunen L, Sairanen E, et al. High perceived stress is associated with unfavorable eating behavior in overweight and obese Finns of working age. Appetite. 2016;103:249-258. doi:10.1016/j.appet.2016.04.023. 13. Cohen S, Kessler R, Gordon L. Strategies for Measuring Stress in Studies of Psychiatric and Physical Disorders. New York: Oxford University Press; 1995. Accessed October 27,2016. 14. Karlén J, Ludvigsson J, Frostell A, Theodorsson E, Faresjö T. Cortisol in hair measured in young adults - a biomarker of major life stressors? BMC Clin Pathol. 2011;11:12. doi:10.1186/1472-6890-11-12. 15. Stawski RS, Cichy KE, Piazza JR, Almeida DM. Associations among daily stressors and salivary cortisol: Findings from the National Study of Daily Experiences. Psychoneuroendocrinology. 2013;38(11):2654-2665. doi:10.1016/j.psyneuen.2013.06.023. 16. Armbruster D, Mueller A, Strobel A, et al. Children under stress - COMT genotype and stressful life events predict cortisol increase in an acute social stress paradigm. Int J Neuropsychopharmacol. 2012;15(9):1229-1239. doi:10.1017/ S1461145711001763. 17. George SA, Khan S, Briggs H, Abelson JL. CRH-stimulated cortisol release and food intake in healthy, non-obese adults. Psychoneuroendocrinology. 2010;35(4):607-612. doi:10.1016/j.psyneuen.2009.09.017. 18. Lumeng JC, Miller A, Peterson KE, et al. Diurnal cortisol pattern, eating behaviors and overweight in low-income preschool-aged children. Appetite. 2014;73:65-72. doi:10.1016/j. appet.2013.10.016. 19. Cohen S, Kamarck T, Mermelstein R. A global measure of perceived stress. J Health Soc Behav. 1983;24(4):385-396. http://www.ncbi.nlm.nih.gov/pubmed/6668417. Accessed July 11, 2016. 20. Booker CL, Gallaher P, Unger JB, Ritt-Olson A, Johnson CA. Stressful life events, smoking behavior, and intentions to smoke among and multiethnic sample of sixth graders. Ethn Health. 2004;9(4):369-397. doi:10.1080/1355785042000285384. 21. Hawks S, Merrill RM, Madanat HN. The Intuitive Eating Scale: Development and Preliminary Validation. Am J Heal

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Educ Goodman. 2013;35(2). 22. Kwan MY, Gordon KH. The effects of social support and stress perception on bulimic behaviors and unhealthy food consumption. Eat Behav. 2016;22:34-39. doi:10.1016/j. eatbeh.2016.03.024. 23. Olstad DL, Ball K, Wright C, Abbott G, Brown E, Turner AI. Hair cortisol levels, perceived stress and body mass index in women and children living in socioeconomically disadvantaged neighborhoods: the READI study. Stress. 2016;3890(June):1-10. doi:10.3109/10253890.2016.1160282. 24. De Vente W, Van Amsterdam JGC, Olff M, Kamphuis JH, Emmelkamp PMG. Burnout Is Associated with Reduced Parasympathetic Activity and Reduced HPA Axis Responsiveness, Predominantly in Males. Biomed Res Int. 2015;2015. doi:10.1155/2015/431725. 25. Hajat A, Diez-Roux A, Franklin TG, et al. Socioeconomic and race/ethnic differences in daily salivary cortisol profiles: The Multi-Ethnic Study of Atherosclerosis. Psychoneuroendocrinology. 2010;35:932-943. doi:10.1016/j.psyneuen.2009.12.009. 26. Joseph D, Chong NW, Shanks ME, et al. Getting Rhythm: How Do Babies Do It? Arch Dis Child Fetal Neonatal. 2015;100:F50-F54. http://fn.bmj.com/content/fetalneonatal/100/1/F50.full.pdf. Accessed March 25, 2017. 27. Ranabir S, Reetu K. Stress and hormones. Indian J Endocrinol Metab. 2011;15(1):18-22. doi:10.4103/2230-8210.77573.

WRITTEN BY VENNIS HONG Vennis is a Public and Global Health major from John Muir College. She will graduate in 2017.

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REVIEW Stress-Induced Tumor Progression in Melanoma

p c

Jordan Setayesh | Physiology & Neuroscience

Psychological Support and Treatment Outcomes

Introduction

There is a long history of scientists and health professionals linking experiences of the mind to the conditions of the body. One specific example of an aspect of human psychology that influences health is chronic stress. Many studies have linked environmental stress and cancer treatment outcomes3,4,5. One type of cancer that is susceptible to environmental stress is melanoma. When someone experiences chronic stress, the body releases catecholamines, such as epinephrine (E) and norepinephrine (NE)1. These are hormones that bind to β-adrenergic receptors (β-ARs) (Figure 1)2. The β-adrenergic system has been shown to be an integral aspect of pro-tumorigenic pathways in melanoma cancers. Therefore, tumor growth can be supplemented by a pathway induced by chronic stress2. β-blockers are molecules that can disrupt the β-adrenergic system and have been shown to counteract the pro-tumorigenic effects of the β-adrenergic system2. Additionally, the amount of perceived external stress correlates with the outcome of melanoma treatment. Thus, the application of β-blockers and psychological stress treatment is a promising supplemental strategy to suppress the tumor progression of melanoma cancers.

β-Blockers and Tumor Progression

The β-adrenergic system plays a vital role in melanoma tumor progression, so the use of β-blockers to inhibit melanoma tumor progression and metastasis has promise. Characteristic melanoma phenotypes, such as tumor growth and metastasis, are promoted by activation of the β-adrenergic system2 (Figure 2). By treating melanoma cancers cells with epinephrine and norepinephrine, many studies have demonstrated that malignant tumors express significantly higher amounts of β1- and β2-ARs2 ,4. The amount of β-AR receptors expressed is a common metric for quantifying the degree to which the β-adrenergic system activates processes that promote tumor progression2 ,4. Similarly, increased tumor growth in carcinoma cells (cancers originating from skin cells) has a significant correlation with increased activation of the β-adrenergic system4. Thus, both tumor growth and metastasis are promoted by increased activation of the β-adrenergic system. By blocking the function of the β-adrenergic system, the ability to inhibit tumor progression will likely be achievable. Evidence for this claim is supported by the success of β-blockers in clinical trials. In mice, the β-blocker propranolol was demonstrated to inhibit tumor growth caused by treatment of melanoma

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In addition to a biochemical approach to treating stressinduced tumor progression in metastatic melanoma, psychological treatment can also inhibit melanoma tumor progression via the same B-adrenergic pathway. Cancer treatment can be a significant life stressor, and a person’s mental health and attitude during treatment can affect treatment outcomes. Studies demonstrate that longer survival times are associated with patients who perceive the aim of the treatment to be cured, do not express anger, and report a higher quality of life9. Unsurprisingly, expressing anger, reporting a low quality of life, and not viewing the purpose of treatment to be cured are all chronic stressors that activate the β-adrenergic system. Since B-adrenergic activation is correlated with tumor progression, such activation of the B-adrenergic system is likely responsible for the association between the aforementioned variables and longer survival

Figure 1 β-ARs are transmembrane proteins. They function through a G-protein coupled receptor. When epinephrine or norepinephrine binds to the β-AR, the G-protein coupled receptor dissociates, and one domain binds to adenylyl cyclase, which produces cAMP from ATP. This process causes increased heart rate, dilation of skeletal muscle blood vessels, and the breakdown glycogen to glucose3.

cancer cells with E and NE5 (Figure 3). Propranolol appears to be the most effective β-blocker for melanoma cancers. It is an ideal candidate drug because the few side effects associated with its administration are well documented and manageable5. The significant correlation between β-blocker administration and inhibition of tumor growth has also been identified in humans through the analysis of cancer treatment outcomes. Treatment with β-blockers has been shown to not only increase survival time of patients with metastatic melanoma tumors but also the survival rate6 ,7 . Clinical trials with thousands of patients have yielded similar results that point to disruption of the β-adrenergic receptors as a key component to supplementing melanoma cancer treatment6 ,7. Overall, the inhibition of β-adrenergic system has the potential to supplement traditional cancer treatments by decreasing the probability of metastasis and suppressing tumor growth; additionally, these effects can also be synergistically supplemented by psychological treatment for cancer patients.

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melanoma cancers. Thus, the best way to maximize the aforementioned inhibition of melanoma tumor progression by reduced stress response is through psychological support during treatment. Although the most effective psychological support will vary by individual, a stable and sensitive environment can synergistically suppress the β-adrenergic system with β-blockers to improve survival chances of melanoma patients.

Conclusions

It is clear that the bulk of scientific research points to the combinatory treatment of melanoma with β-blockers and psychological support as an effective way to both decrease the likelihood of melanoma tumor metastasis and inhibit tumor progression. These effects can serve as supplemental treatments in order to increase the success rate of standard melanoma treatments. The need for a holistic approach in cancer treatment is evident with the increasing number of melanoma patients each year. The gaps in the research, however, are clear. Melanoma is a complex disease, and different β-blockers could be useful in different stages and types of melanoma. Thus, understanding the appropriate application of specific β-blockers in specific situations is necessary to maximize the efficacy and reliability of the

Figure 2 This figure illustrates the stages of tumor progression. The initial mutation develops into hyperplasia, which is when the size of an organ or tissue increases due to an increased rate of reproduction by cells. Dysplasia is the presence of abnormal types of cells in the tissue. However, after these two stages progress, in situ cancer develops. In this stage the cancer is still localized to one place in the body. The stage between in situ cancer and invasive cancer is what is commonly referred to as tumor progression in the literature8.

times. Murine (mouse) models with different stress tolerances have been utilized to illustrate a similar concept. After being exposed to chronic stressors and inoculated with melanoma, mice with high stress tolerances have been shown to exhibit less tumor growth and higher rates of remission than mice with low stress tolerance1. High and low stress tolerance was distinguished by taking the most extreme responses to stressors and making them progenitors of a mouse line1. Since the only difference between these mice was the nature of their response to chronic stress, differences in cancer outcomes are most likely due to differences in activation of the β-adrenergic system11. Therefore, the the ability to cope with stress can potentially increase the success of treatment outcomes in

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treatment. Similarly, the mechanism through which external psychological stressors activate of the β-adrenergic system during cancer treatment or other tumor promoting pathways is still unclear. The current information available now, however, is sufficient to extend the time-period where standard cancer treatments can be effective. The synergistic efforts of biochemical and psychological approaches can suppress tumor growth and provide patients and doctors with more time to treat melanoma in patients. This ability is essential in both melanoma and other quickly progressing cancers. Most tumors are not deadly until metastasis and angiogenesis (the formation of blood vessels) occur. SALTMAN QUARTERLY

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Figure 3This is an example of common experimental data that shows the ability of propranolol to inhibit tumor progression. The stressed mice clearly experienced much quicker tumor progression than the non-stressed mice. However, with the administration of propranolol, the amount of tumor progression was limited to that in the control mice11.

Ultimately, preventing tumor progression is as useful as cancer prevention, and the lethal effects of cancer are eliminated nearly simultaneously with elimination of tumor growth. References 1. Colucci, Roberta, and Moretti, Silvia. “The role of stress and beta-adrenergic system in melanoma: current knowledge and possible therapeutic options.” Journal of Cancer Research and Clinical Oncology. 142.5, 1021-129 (2016). 2. Moretti, Silvia. “Beta-adrenoceptors are upregulated in human melanoma and their activation releases protumorigenic cytokines and metalloproteases in melanoma cell lines.” Laboratory Investigation. 93, 279-290 (2013). 3. “Encyclopedia Britannica.” G-protein coupled receptor, last modified May 11, 2013, accessed January 4, 2017, https:// www.britannica.com/science/G-protein-coupled-receptor. 4. Yang, Eric et al. “Norepinephrine up-regulates the expression of vascular endothelial growth factor, matrix metalloproteinase (MMP)-2, and MMP-9 in nasopharyngeal carcinoma tumor cells” Cancer Research. 66.21, 10357-10364 (2006). 5. Deng, Duo-Hua, et al. “Exogenous norepinephrine attenuates the efficacy of sunitinib in a mouse cancer model.” Journal of Experimental and Clinical Cancer Research. 33.1, (2014). accessed January 3, 2017. http://dx.doi.org/ 10.1186/1756-9966-33-21. 6. Lemeshow, Stanley, et al. “Beta-Blockers and survival among Danish patients with malignant melanoma: a

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population-based cohort study.” Cancer Epidemiology, Biomarkers, and Prevention. 20.10, 2273-2279 (2011). 7. De Giorgi, Vincenzo, et al. “Treatment with β-Blockers and reduced disease progression in patients with thick melanoma.” Arch Internal Medicine. 171.8, 779-781 (2011). 8. “The Biology of Cancer.” accessed January 3, 2017. http://sphweb.bumc.bu.edu. 9. Macloone, Jordana, Menzies, Scott, Meiser, Bettina, Mann, Graham, Kasparian, Nadine. “Psycho-educational interventions for melanoma survivors: a systematic review.” Psycho-oncology. 22, 444-456. (2013). 10. Ragan, Agnieszka, et al . “Chronic mild stress facilitates tumor growth in mouse lines selected for high and low stress-induced analgesia.” The International Journal on the Biology of Stress. 16.5, 571-80 (2013). 11. Lamkin, Donald, et al. “Chronic stress enhances progression of acute lymphoblastic leukemia via β-adrenergic signaling.” Brain, Behavior, and Immunity. 26.4, 635-641 (2012).

WRITTEN BY JORDAN SETAYESH Jordan is a Physiology and Neuroscience major from Roger Revelle College. He will graduate in 2019.

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Students in the Biology Honors program are required to complete a written thesis detailing their scientific research progress. The Senior Honors Theses section, which presents the abstracts of their individual theses, highlights the achievements of the accomplished undegraduate researchers in the class of 2017.

SENIOR

HONORS THESES

California Sea Lions, Zalophus californianus, resting at La Jolla Cove. Photo by Ben Kong 42 SALTMAN QUARTERLY

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The Division of Biological Sciences Senior Honors Theses 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 2016-17 academic year.

The Role of Astrocytes in the Regulation of Blood-Brain Barrier Properties in Brain Endothelial Cells

Tamara Chan

Earl Warren College, Physiology & Neuroscience Major, Psychology Minor

Jiaying Chen Sixth College, Molecular Biology Major, Accounting Minor

The blood-brain barrier (BBB) is a set of properties belonging to endothelial cells of the central nervous system. Its function is to reduce the transport of ions and harmful molecules between the blood and brain. Previous research has shown the importance of astrocytes, a type of glial cell, in mediating communication between neurons and blood vessels in the context of blood flow, as well as in the maintenance of BBB properties. However, most work has been done in vitro in a static context. My project aims to study astrocytic regulation of BBB properties in a dynamic context in an in vivo system. I am using transgenic mice that express Gq-DREADDs (Designer Receptors Exclusively Activated by Designer Drugs) to selectively activate astrocytes via intracellular calcium influx, and then purifying endothelial cells to detect BBB changes. Preliminary results show that Gq activation of astrocytes upregulates tight junction proteins and downregulates leukocyte adhesion molecules, both of which indicate strengthening of the BBB. Future directions include performing in vitro experiments to identify a mechanism by which this is happening.

THESIS

Urocortin 2 Effects on Liver Metabolic Gene Expression in Mice with Insulin Resistance Urocortin 2 (Ucn2) is an endogenous hormone belonging to the corticortropin-releasing-factor family. Adeno-associated virus mediated Ucn2 gene transfer (GT) increases insulin sensitivity and glucose disposal, resulting in normalization of glucose homeostasis in insulin-resistant (IR) mice fed with a high fat diet (HFD). To determine the mechanism for the Ucn2 effects, alterations in mice-liver gene expression were examined using focused gene arrays and RT-PCR. M â&#x20AC;&#x2039; ice that received Ucn2 GT prior to HFD prevented IR development, with five genes (Abca1, Akt1, CEBPB, Lepr, and G6PC) being up-regulated and eight genes (Foxa2, Slc27a5, Slc2a2, Frs3, Map2k1, Pik3r1, Raf1, and Sorbs1) demonstrating down-regulation compared against saline injected HFD-fed control mice. In HFD mice that already developed IR, Ucn2 GT was able to up-regulate the expression of two genes (Abca1 and Acox1) and down-regulate four genes (Fabp1, Ras2, Adrb3, and Dok2.) Our findings demonstrate that Ucn2 improves glucose homeostasis in HFD-fed mice by modulating metabolic gene expression in liver.

Roger Revelle College, Biochemistry & Cell Biology Major

PI: Mei Hua Gao, Ph.D., UC San Diego School of Medicine, Department of Medicine

PI: Richard Daneman, Ph.D., UC San Diego School of Medicine, Departments of Neurosciences and Pharmacology

Molecular Mechanisms Regulating Scrunching in Dugesia japonica

Interrogation of the Cryptic Biosynthetic Gene Cluster IPF38-51 in Microcystis aeruginosa PCC 7806 using Improved Genetic Tools and Methods

The ability to sense noxious stimuli and trigger an escape mechanism to avoid further damage is key to animal survival. It is known that scrunching, a locomotion gait in Dugesia japonica, is one such escape mechanism that helps planarians evade noxious stimuli, including capsaicin, low pH, and high heat. In other organisms, these kinds of noxious stimuli are sensed by transient receptor potential channels (TRPs) that then induce a pain response. We hypothesized that homologs of TRPs in D. japonica mediate scrunching. Through RNA interference-mediated knockdown, we assayed the role of the known TRP genes in D. japonica in regulating scrunching via quantitative behavioral assays. We verified gene knockdown through in situ hybridization. Our results so far are inconclusive and neither confirm nor negate our hypothesis.

Microcystis aeruginosa, a bloom-forming and toxin-producing cyanobacterial species, has caused harmful algal blooms across the world. The most threatening feature of this species is its production of the potent hepatotoxin microcystin, which causes local ecosystem issues and safety concerns on drinking-water. Although M. aeruginosa has been extensively studied, two of its biosynthetic gene clusters are still enigmatic and their products have yet to be identified. The focus of this project is to study the cryptic gene cluster, IPF38-51, which was shown to be regulated by external microcystin levels in a microcystin-deficient mutant strain. To identify the secondary metabolites produced by this gene cluster and to understand its regulation, a strain with a knockout mutation of the IFP48 gene and a strain harboring the yellow fluorescent protein driven by the upstream region of the IPF51 gene are being produced. Methods and tools for the genetic manipulation of M. aeruginosa are also being further developed.

Youngju Choi

PI: Eva-Maria Schoetz Collins, Ph.D., UC San Diego, Division of Biological Sciences, Section of Cell and Developmental Biology

Arya V. Dadhania John Muir College, Physiology & Neuroscience Major

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THESIS

Kappa-Opioid Stimulation Following Chronic Cardiac Pressure Overload Benefits Cardiac Function

Mugdha A. Joshi

Roger Revelle College, Human Biology Major, Latin Literature and Music Minors

The stimulation of kappa (κ)-opioid receptors has been shown to protect the heart from ischemic injury. The hypothesis of this study was that κ-opioid receptor stimulation would reduce pressure overload-induced heart failure caused by transverse aortic constriction (TAC). Mice underwent either TAC or sham surgery two weeks post-TAC, the κ-opioid receptor agonist U50488H or vehicle (V) was administered daily via intraperitoneal injections. Heart function was monitored via echocardiography before TAC and at 2, 4, 6, 8, and 10 weeks post-TAC. Analysis showed preserved cardiac function in TAC+U50 animals, as compared to TAC+V animals. Eleven weeks post-TAC transmission electron microscopy showed preserved mitochondrial structure in TAC+U50 animals, while TAC+V animals showed swollen mitochondria. Immunohistochemical analysis showed intracellular and reduced connexin-43 localization in TAC+V hearts, compared to distinct intercalated disc localization in TAC+U50 hearts. Based on these data, we suggest U50448H as a potential novel treatment for patients in heart failure. PI: Hemal Patel, Ph.D., UC San Diego School of Medicine, Department of Anesthesiology

Characterization of Rat Forelimb and Hindlimb Corticospinal Tract Regeneration into Neural Progenitor Cell Graft After Upper Cervical Spinal Cord Injury Spinal cord injury is a very salient obstacle for many people and much effort has been allocated to research mechanisms to re-establish post injury mobility. Our lab demonstrated robust regeneration of the corticospinal tract (CST) axons into neural progenitor cell (NPC) graft placed in cervical spinal cord injury locations. Whether the forelimb or hindlimb CST axons regenerate into NPC graft at cervical spinal cord is unknown. Forelimb or hindlimb CST neurons in adult rats were labeled retrogradely via injection of AAV-CRE vectors either into the lower cervical cord or middle lumbar cord, respectively. This activated CRE-dependent red fluorescent protein (RFP) expression in cortex. All rats were then subject to a C3 dorsal column lesion and received rat embryonic day 14 spinal cord derived NPC graft. Preliminary studies have indicated substantial regeneration of hindlimb CST, as compared to forelimb CST axons into an upper cervical graft after spinal cord injury. A more detailed characterization of such regeneration is underway. Characterization of CST regeneration into NPC graft is important for understanding its potential functions in restoring skilled voluntary motor movement after spinal cord injury.

Daniel Kulinich

Thurgood Marshall College, Physiology & Neuroscience Major

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

Development of a Noninvasive Blood-Based Molecular Test for Monitoring Disease Progression in Huntington’s Disease (HD)

Aeri Kim Thurgood Marshall College, Biochemistry & Cell Biology Major

Huntington’s Disease (HD) is an inherited, neurodegenerative disorder characterized by cognitive, psychiatric, and motor dysfunction; however, the timing and rate of disease progression can be difficult to assess. Recently, cell-free (circulating) RNA (cf-RNA) has shown promise as a tissue-specific biomarker of cellular damage and degree of neurodegeneration. Working with a San Diego-based company, we propose to track changes in the cell-free transcriptome, as they correlate with disease progression. A small cohort of HD patients, of varying severity, and age-matched NC, was assessed by sequencing to determine initial feasibility of monitoring the cell-free transcriptome in HD patients while simultaneously evaluating neuronal specific targets for further assay development. Initial findings demonstrated the ability to identify neuronal transcripts in these patients and we are currently characterizing a larger cohort of samples across the three stages of HD, using our established protocol to identify gene targets that track with disease progression. PI: Jody Corey-Bloom, M.D., Ph.D., UC San Diego School of Medicine, Department of Neurosciences

Enhanced Wound Healing in Heparan Sulfate Deficient Mice Mammals have a limited ability to repair wounds from traumatic injury. As heparan sulfate regulates many of the cell signaling pathways important for wound repair and cell growth, we hypothesized that heparan sulfate biosynthetic mutant mice may have altered wound repair. To analyze wound repair, full-thickness dorsal skin excisional wounds were performed on two mouse models of heparan sulfate deficiency, genetic and pharmacological. In the genetic model of heparan sulfate deficiency, the mice lack copies of the EXT genes that encode for heparan sulfate biosynthetic copolymerase complex. In the pharmacological model, mice are treated with a competitive substrate for the heparan sulfate biosynthetic copolymerase complex. Both the pharmacological model and genetic model that had altered heparan sulfate biosynthesis closed their dorsal skin wounds faster than the wild-type mice. Biochemical analysis were performed to compare the variation of heparan sulfate biosynthesis.. Understanding how heparan sulfate functions in wound repair and regeneration can help enhance wound repair in patients.

Lu (Sophie) Li Thurgood Marshall College, Human Biology Major

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

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THESIS Neural Stem Cell Graft Differentation and Interaction with Host Central Nervous System After Spinal Cord Injury

Brian Lien

Eleanor Roosevelt College, Physiology & Neuroscience Major, Psychology Minor

Neural stem cells (NSCs) have the clinical potential to restore lost connections after spinal cord injury. In this study, human NSCs were transduced with green fluorescent protein (GFP) and embedded in fibrin matrices containing a growth factor cocktail. These human NSC grafts were then implanted into C5 lateral hemisection sites of spinal cord injury in immunodeficient rats. The subjects were euthanized at 1, 3, 6, 12, and 18 months post-implantation to study differentiation, migration, and maturation of graft-derived neurons and glia. We focused on the development of migrated glial cells from the NSC graft at 6, 12, and 18 months post-grafting by immunohistochemistry. The migrated human cells can be clearly observed at 6 months post- grafting and identified as glial progenitor cells at this early time point. Human cells migrated continuously at a rate of 2-3 mm per month and gradually matured as GFAP positive astrocytes. We also used a combination of immunohistochemistry and electron microscopy to observe how these migrating NSC-derived glial cells interacted with host neuron soma, axons, myelin, and blood vessels, over the same time period. Understanding how NSCs differentiate and interact with the host spinal cord will allow researchers and clinicians to optimize the use of NSC as a potential treatment for spinal cord injury, where clinical trials are currently ongoing. PI: Mark H. Tuszynski, M.D., Ph.D., UC San Diego School of Medicine, Department of Neurosciences

Sean Paknoosh John Muir College, Biochemistry & Cell Biology Major

Identifying the Mechanism of Action of New Antimicrobial Compounds Using Bacterial Cytological Profiling The emergence of multi-drug resistant bacteria and the decline in the number of new antibiotics creates an urgent need to discover antibiotics that act by novel mechanisms of action (MOA). We have developed a rapid and versatile platform, called Bacterial Cytological Profiling (BCP), which identifies drugs that act by novel MOA. BCP utilizes fluorescence microscopy to observe changes in cytological parameters of bacteria exposed to lethal concentrations of antibiotics. Antibiotics that inhibit targets in different pathways generate different cytological profiles. We screened a library of over 2,000 compounds against E. coli ΔtolC, determined the MOA of the hits, and identified five previously uncharacterized compounds active against both E. coli ΔtolC and Staphylococcus aureus. We also obtained compounds with unknown MOAs from collaborators, and identified three that inhibit peptidoglycan biosynthesis in B. subtilis. These molecules can be further developed as antibiotics for the treatment of infections caused by drug resistant bacteria.

Christine Peters

John Muir College, Biochemistry & Cell Biology Major, Global Health Minor

PI: Joe Pogliano, Ph.D., UC San Diego, Division of Biological Sciences, Section of Molecular Biology

Nuclear Factor of Activated T Cells (NFAT) and its Role in the Adult Drosophila Heart

Does Age Affect the Unfolded Protein Response Signal Transduction Pathway?

Heart disease is currently the leading cause of death in the U.S. Understanding the function of new genes and pathways in a simple model system may provide novel insights and lead to new therapeutic targets for heart disease in humans. In humans, calcineurinNFAT signaling has been implicated in regulation of the cardiac hypertrophic response. However, how this gene interacts with other molecular pathways in the heart is unknown. In order to investigate the role of NFAT in context of other signaling pathways, the singular function of NFAT in the heart was investigated, using the animal model system Drosophila melanogaster. Loss-of-function studies were conducted and the resulting heart phenotype was assessed. NFAT-RNAi lines were used to conduct gene knockdowns (KD) in a tissuespecific manner using the GAL4/UAS system. The flies were dissected to expose the beating heart tube; hearts were filmed and analyzed using Semi-Automated Optical Heartbeat Analysis (SOHA). Key cardiac performance indicators, such as heart period, fractional shortening, heart rate, stroke volume, and cardiac output, were measured. Obvious differences between NFAT gene knockdown and respective controls using a single RNAi KD line were not observed. However, this may be due to insufficient KD or due to compensation by changes in expression of other genes. We will examine the effects of KD using other independent NFAT RNAi lines, as well as possible non-autonomous effects by KD in other tissues. In addition, we will study the effect of NFAT KD in sensitized backgrounds, such as NFAT heterozygous mutants.

Folding and maturation of cell surface receptors and secreted proteins must occur in a cellular compartment called the endoplasmic reticulum (ER). In response to increased demands of ER functions (termed ER stress), a conserved signal transduction pathway called the unfolded protein response (or UPR) is activated. The UPR is conserved in a single cell eukaryote, yeast, S. cerevisiae, underscoring its importance. Thus far, most UPR studies have focused on young yeast cells and information on how aged cells respond to ER stress is limited. To understand the effect of age, I used a protocol to enrich aged yeast cells and tested their ability to activate UPR using a fluorescently tagged UPR reporter. As the importance of the UPR has been recognized for human diseases, understanding the impact of age on the UPR will provide an important step towards understanding age-exacerbated human diseases such as Alzheimer’s and Parkinson’s diseases.

Clarice Resso Sixth College, Human Biology Major

PI: Maho Niwa, Ph.D., UC San Diego, Division of Biological Sciences, Section of Molecular Biology

PI: Rolf Bodmer, Ph.D. and Karen Ocorr, Ph.D., Sanford Burnham Prebys Medical Discovery Institute

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Erika Sasaki

Earl Warren College, Biochemistry & Cell Biology Major and Politcal Science Minor

Rani Shiao

Roger Revelle College, Molecular Biology and Earth Sciences Majors, Political Science Minor

Small Molecule Target Identificaton and Validation in Plasmodium falcparum

Investigation of Kisspeptin Neurons as Mediators of Stress Induced Disruption of Reproductive Function

Today there is a significant need for new antimalarial drugs due to the prevalence of resistant parasite strains around the world. To create new antimalarial drugs, novel drug-able targets must be identified to reduce the risk of cross-resistance in Plasmodium falciparum, a protozoan parasite that causes malaria. Small molecules can be used to select for parasite resistance, which can then elucidate the target through SNVs and CNVs found through whole genome sequencing. MMV011903, MMV032967, MMV023949, and MMV676459 are small molecules discovered through phenotypic screening to have antimalarial activity. These compounds were used to evolve parent strains of Plasmodium falciparum at sub-inhibitory concentrations. MMV085203 resistant lines have been found to have mutations in a putative transporter, dihydrouridine synthase, and protein kinase on the second chromosome through whole genome sequencing. These targets were validated through CRISPR-Cas9 genome editing. By identifying novel targets in Plasmodium falciparum, more effective antimalarials can be produced and brought to the community at large.

In female mice, the treatment of the stress steroid, corticosterone, inhibits both estrous cyclicity and the luteinizing hormone (LH) surge required for ovulation. These functions are regulated by the hypothalamic-pituitary-gonadal (HPG) axis, which begins with GnRH signaling in the hypothalamus. We hypothesize that corticosterone inhibits this axis by acting directly on hypothalamic kisspeptin neurons. Kisspeptin neurons are key regulators of GnRH neurons and play an important role in regulating both LH pulsatility and the LH surge. Through immunofluorescence, we found expression of glucocorticoid-receptors (GR) within kisspeptin neurons, which confirmed that kisspeptin neurons can respond to corticosterone signaling. We are currently validating the knockdown of GR in kisspeptin neurons in the GR flox kisspeptin cre mouse model which, if validated, can be used to more thoroughly test the hypothesis. These findings can help us better understand the effects of corticosterone on the HPG axis and the mechanisms behind stress-induced disruption of reproductive function.

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

PI: Kellie B. Church, Ph.D., UC San Diego School of Medicine, Department of Reproductive Medicine

Evaluation of Neural Stem Cell Grafts on Pain Modulation Following Spinal Cord Injury

Age and Diabetes-Related Urogenital Muscle Atrophy and Fibrosis

Neural progenitor cell (NPC) grafts have demonstrated successful spinal integration in spinal cord injury (SCI) models, projecting axons over long distances and forming functional relays in the central nervous system to improve motor outcomes (Lu et al., 2012; Kadoya et al., 2016). However, because neuropathic pain remains a common detriment to the quality of life of SCI patients, careful screening of cell therapies is necessary to ensure that NPC implantation does not exacerbate nor produce pain states. To evaluate the effects of NPC grafts on neuropathic pain outcomes, behavioral responses to sensory stimuli were assessed in rats that underwent either a T3 transection or mild cervical lesion with or without graft. Immunohistological characterization of pain biomarkers in the cervical spinal cord was then conducted to correlate sensory data with glial proliferation and CGRP fiber sprouting. No significant differences in sensory pain outcomes nor biochemical analysis was detected between grafted and ungrafted injury models across tactile, heat, cold, and spontaneous pain modalities. These findings indicate that NPC therapies neither generate nor worsen post-SCI neuropathic pain states, supporting the feasibility of such grafting approaches for clinical translation.

Age/diabetes-related atrophy or fibrosis in urogenital muscles represents a significant risk factor for the development of incontinence/erectile dysfunction in geriatric populations. While the loss of muscle mass is a clear indication of dysfunction, little is known about the underlying molecular mechanisms of muscle degeneration. Rabbit and mouse (diabetic) models were used to establish a time course of age/diabetes-related urethral/penile muscle dysfunction and to study fibrotic mechanisms. Young and old rabbits as well diabetic mice were scarified to harvest urogenital tissue samples. Protein expression was determined using immunostaining and western blot analysis for its quantification. Immunological studies demonstrated upregulation of fibrotic and atrophy pathways involving TGF-Î˛ and downregulation of caveolin proteins confirming increased fibrogenic pathology in these samples. Urogenital muscle atrophy/fibrosis can greatly impact continence as well as sexual functions. Understanding of such mechanisms is important for the development of potential interventions. PI: Raj M. Rajasekaran, Ph.D., UC San Diego School of Medicine, Department of Surgery

Christopher I. Song Earl Warren College, Biochemistry & Cell Biology and Economics Majors

Sarah To Earl Warren College, Human Biology and Human Development Majors

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

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Identification and Classification of PACSIN-1 as a Novel Ligand for LRP1 in Schwann Cells

Curtis Triebswetter Sixth College, Physiology & Neuroscience Major

Low-density lipoprotein receptor-related protein 1 (LRP1) is an endocytic, cell signaling receptor for diverse ligands. LRP1 is up-regulated in Schwann cells (SC) after peripheral nervous system injury and orchestrates many elements of the SC repair program, including functional regeneration. To identify novel ligands responsible for regulating LRP1 signaling, injured peripheral nerves were immunoprecipitated with Fc-fusion proteins containing the extracellular ligand-binding domains of LRP1. Consecutive analysis by tandem mass spectrometry (MS/MS) revealed that Protein kinase C and casein kinase substrate in neurons 1 (PACSIN-1) may be involved in the LRP1-triggered SC repair program. To determine whether PACSIN-1 activates LRP1-dependent cell signaling, primary rat SC cultures were treated with recombinant human PACSIN-1 at various concentrations. Cells were then lysed and subjected to immunoblotting for ERK1/2 activation. Preliminary results indicate that depending on concentration, PACSIN-1 potentially acts as an agonist for LRP1-dependent cell signaling.

The Role of Gene PLD3 in the Immune System Enzymes within the phospholipase D (PLD) family have traditionally been known to catalyze the hydrolysis of phosphatidylcholine, which is a key component of the plasma membrane of mammalian cells. However, they may also possess other functions. Our research aims to uncover these functions, particularly of isoforms PLD3 and PLD4. I specifically studied PLD3 and employed the CRISPR-CAS9 system to create PLD3 mouse knockouts to help identify changes in their immune system. PLD3 is highly expressed in brain tissue, and genome-wide association studies have displayed an association between PLD3 variants and Alzheimerâ&#x20AC;&#x2122;s disease. My results confirm that my CRISPR constructs successfully knocked out mPLD3 in-vivo. Specifically, I observed a base insertion that resulted in a frameshift. This would effectively eliminate PLD3 protein function, likely due to a premature stop codon. Furthermore, ELISA testing suggests that PLD3 may affect IFN-1 response, which has been linked to neuroinflammation and Alzheimerâ&#x20AC;&#x2122;s disease. PI: David Nemazee, Ph.D., The Scripps Research Institute, Department of Immunology and Microbiology

PI: Wendy M. Campana, Ph.D., UC San Diego School of Medicine, Department of Anesthesiology

Armen Zeitjian Thurgood Marshall College, Human Biology Major

Novel Functions of Rad53 and Mec1 in S. cerevisiae at Telomeric Replication Forks in Maintaining Telomere Homeostasis

Waverly Tseng

Roger Revelle College, Biochemistry & Cell Biology Major

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Telomeres are the essential structures at the ends of linear chromosomes. One essential role is to protect the ends from DNA damage machinery. Recently, the Lundblad lab has become interested in the possibility that DNA damage machinery may also play a positive role in promoting telomere homeostasis, potentially by promoting replication through the duplex telomeric tract. Two major players in this pathway, Rad53 and Mec1, are important for DNA replication checkpoints. Based on previous work, we suspect these two proteins to be involved with telomeres. To this end, I am pursuing extensive mutagenesis of each protein by employing a novel, phenotype-based protocol developed in the Lundblad lab designed to rapidly detect separation-of-function mutations. By screening this panel of mutations for novel telomere-related phenotypes, I have found mutations that show such phenotypes that are currently undergoing further testing. Through further analysis of these mutations, I hope to elucidate the complexities of telomere homeostasis. PI: Victoria Lundblad, Ph.D., Salk Institute for Biological Studies

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