Volume IV Issue II SPRING 2019

JUST VOL IV // ISSUE II // SPRING 2019

LETTER FROM THE EDITOR-IN-CHIEF Dear Reader, I am thrilled to present to you with Volume IV, Issue II of the Journal of Undergraduate Science and Technology (JUST). This issue is a continuation of a campus-wide effort and a celebration of not only the extraordinary research that takes place on this campus, but the work conducted by undergraduates specifically. I would like to extend my sincerest thanks to the undergraduate researchers who submitted their work along with the faculty and staff who supported them. I would also like to express my gratitude for the JUST staff that have chosen to make JUST a part of their undergraduate experience and worked diligently to bring you this publication. Additionally, without the generous support of the Wisconsin Institute for Discovery, the Holtz Center for Science and Technology Studies, and the College of Agriculture and Life Sciences, the publication of this journal would not have been possible.

JUST’s mission has always been to support undergraduate researchers and make science accessible to broader audiences. At UW-Madison, we have been uniquely able to provide the opportunity for undergraduates to publish their work in a peer-reviewed journal and give students a glimpse into the publication process of an academic journal. On the other hand, our staff gain exceptional skills and experience the publication process from the perspective of a producer in a scholarly journal. We believe that these experiences are an invaluable supplement to a traditional undergraduate education, especially for those students who wish to continue research.

SCIENCE + SOCIETY: How to be creative and effective in a rapidly changing environment Integrated Studies in Science, Engineering, and Society Undergraduate Certificate Program (ISSuES) at UW–Madison

ISSuES offers undergraduate engineering and natural science students an opportunity to interact with the social sciences and humanities in a way that emphasizes the relationship between science, technology, engineering, and society.

For more information, visit sts.wisc.edu or speak to the certificate advisor at 608-263-2927.

As for the second part of our mission, I believe that scientific literacy is more important than ever in today’s advancing society. STEM topics have immersed themselves in all aspects of daily life, and all of our lives can only be enriched by a solid understanding of scientific thought. Effective communication of research and science is key to this. We are honored to be a small part in a much larger effort to make research and scientific achievement more accessible to non-expert communities beyond academia.

This issue will mark the end of my first year as the Editor-in-Chief of JUST. I am very excited to continue as Editor-in-Chief in the 2019-2020 academic year. I look forward to another fruitful year of research publication and science communication for JUST. As the continuing Editor-in-Chief, I hope to continue to grow the journal, increase readership, and aid JUST in making an impact on this campus.

In this issue of JUST, you will find a wide range of scientific disciplines represented both by our peer-reviewed reports and our shorter editorials, as well as the visual pleasure of scientific imagery.

Please join us in making it a tradition to recognize the incredible research and thoughtful written pieces presented by UW-Madison undergraduates, and in our larger pursuit to support science literacy.

Sincerely,

Helen Heo JUST Editor-in-Chief

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SPONSORS & PARTNERS EDITOR-IN-CHIEF Helen Heo MANAGING EDITORS Teja Karimikonda Aliyah Keval DIRECTOR OF FINANCE Aditya Singh

We would like to sincerely thank the Integrated Studies in Science, Engineering, and Society Undergraduate Certificate Program [ISSuES] at UW-Madison; The College of Agriculture and Life Sciencces [CALS]; and The Wisconsin Institute for Discovery for financially supporting the production of JUST’s Spring 2019 issue. Thank you!

Hae Rin Lee

The Importance of Model Organisms...........................................................12 Annika Peterson

PIXELS John Vandevender..........................................................................................................................16 Helen Heo......................................................................................................................................16

MARKETING ASSISTANT Annika Peterson

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Can We Be More Lactose Persistent?...........................................................10

Aadhishre Kasat

WEBMASTER Cayman McKee

STAFF WRITERS Annika Peterson, Head Staff Writer Hae Rin Lee Aislen Kelly Ryan Brown Aadhshire Kasat

Luke Zangl and Ryan Brown

X-Men May Not Just Be a Movie....................................................................14

DIRECTOR OF DESIGN Elizabeth Owens

COPY EDITOR Mary Magnuson

EDITORIALS Alleviating Vaccine Hesitancy: The Path to a Safer Future...................6

DIRECTOR OF MARKETING Tammy Zhong

EDITORS OF CONTENT Bailey Spiegelberg, Senior Kort Driessen, Senior Eric Leisten, Senior Haley Dagenais, Associate Stephen Halada, Associate Sydney Ring, Associate Reilly Mooney, Associate Madison Knobloch, Associate & Copy

TABLE OF CONTENTS

REPORTS Cholecystokinin Protects HP62 Human β-cells from Cytokine-induced Apoptosis...........................................................................................................20 Jiayin Tang, Arnaldo Henrique de Souza, and Dawn Belt Davis The Journal of Undergraduate Science and Technology (JUST) is an interdisciplinary journal for the publication and dissemination of undergraduate research conducted at the University of Wisconsin-Madison. Encompassing all areas of research in science and technology, JUST aims to provide an open-access platform for undergraduates to share their research with the university and the Madison community at large.

Effects of Urbanization on Algal Abundance, Diversity, and Succession on Dock Pilings within The Bocas Del Toro Archipelago............26 Alaina Eckert

Improving Clinical Training and Electrocardiograms Analysis Through Adaptive Artificial Intelligence..................................................32 Peter Rehani, Dr. Joshua Medow, Dr. Scott Hagen, Dr. John Webster, Quan Chen

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MEDICINE

This image depicts a 0.5ml vial of tetanus toxoid, reduced diphtheria toxoid, and acellular pertussis vaccine (DTaP), adjacent to which was the syringe that would be used to administer the vaccine to a selected recipient. CDC/Amanda Mills. Acquired from Public Health Image Library. Public Domain.

Alleviating Vaccine Hesitancy: The Path to a Safer Future "The decision to vaccinate places us at such a crossroads and for some, the choice is easy: vaccinate. But, for others, it is not so clear. Vaccine hesitancy is not a new phenomenon, yet it poses a great threat and must be dealt with in new and innovative ways."

T

he human mind is constantly working on the fly, adapting to situations, and processing new information while considering the old. When posed with opposing courses of action, the best we can do is weigh our options in an attempt to mitigate risk and maximize reward by using what is presented to us along with our past experiences.

However, clear cut realities can be blurred and muddled by misleading opinions and emotions.

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It is at this point that we may make the wrong choice, setting forth a course of events that not only puts the lives of our own children at risk, but the lives of others as well. The decision to vaccinate places us at such a crossroads and for some, the choice is easy: vaccinate. But, for others, it is not so clear. Vaccine hesitancy is not a new phenomenon, yet it poses a great threat and must be dealt with in new and innovative ways.

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EDITORIAL

EDITORIAL

By Luke Zangl and Ryan Brown

Since the days of Edward Jenner, the pionly one in a million, there are some who choose oneer scientist behind modern vaccination, stanto go against the physician and Centers for Disdard-of-care preventative medicines have been ease Control and Prevention (CDC) vaccine recat odds with the resistance, or those who feel ommendations. This is availability bias at work the risk of vaccination outweighs the benefit. As in its most potent form, especially in the modern medical advances continue to be made, anti-vacera: parents haven’t had exposure to the deadly cine sentiments follow closely behind, nipping diseases that recommended vaccine series protect at its coattails. Now, this anti-vaccine mentality against, but they are inundated with information does not appear to come from a place of malice on potential harmful side effects of vaccination. but is rooted in an innate parental desire to proFar too much credence is given to the risks of vide what is believed vaccination when the "...this anti-vaccine mentality does not choice should be clear. to be best for children.

The mindset of a paris not to say that appear to come from a place of malice This ent is a peculiar one, we shouldn’t ask quesbut is rooted in an innate parental tions and voice conespecially in this modern era of media and but we should desire to provide what is believed to be cerns, tabloid, with so many ask them with an best for children." questions to be asked open mind and engage and such a variety of the factual answers answers from sources of information, misinforprovided by healthcare professionals and governmation, and agenda. Given all the outlets of pament agencies devoted to understanding all there rental advice, it is easy to become overwhelmed is to know about vaccines. For some, these fears and unsure of the “best way to raise a child." At are alleviated by strong physician recommenthis point, in moments of uncertainty, the human dation, but for other parents, vaccine schedules mind falls back on heuristics of decision making, are altered or forgone completely. At this point, capable of bringing about flawed judgement with when a parent decides to not vaccinate or delay lasting effects. vaccination, dire downstream effects are put in

When the time comes to balance rational motion and often other unavoidable measures thinking and emotion, it is often easiest to rearise preventing parents from returning with a vert to the latter and change of heart about only rely on instinct. immunization.

These gut reactions,

There are varyfounded on informaing reasons why a tion that is placed at parent is unable to the forefront of one’s initially vaccinate or working memory, ofreturn to a healthcare ten fall prey to availprovider to vaccinate ability bias. This is of their child, some of most concern when which are legitimate. considering the state In a 2013 study of the of infection transmispreceding year, one in sion in the United three parents voiced States of America. some form of concern Due to advances in regarding vaccination Image depicts healthcare professional vaccinating baby. Public Domain. medical technology, [2]. In many cases, vaccination has been there are religious or deemed one of the most substantial advents in personal beliefs that prevent families from vaccirecent history [3] by eradicating or nearly eliminating. For example, traditional Amish families nating certain diseases and as such, has greatly do not practice vaccination, and in turn are left diminished the human fear of disease due to lack more susceptible to preventable disease contracof exposure. We have become accustomed to life tion than other sectors of the population. Over without the threat of fatal infection, and in dotime, vaccines have been deemed “unnatural” ing so have relocated our fears elsewhere. In this, and as such strike fear into some of those who vaccines have clearly become victims of their own prioritize a “natural” lifestyle, resulting in vacsuccess. Just as hearing of a rare shark attack cine hesitancy [3]. It seems somewhat inherent can discourage one from enjoying time at a beach, to have a general mistrust in the prescribed inwhen a parent hears about the chance of an exjection of an unknown solution of chemicals into treme reaction to a vaccine, even if that chance is your child, let alone multiple at once. Without

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EDITORIAL

an accessible fashion can be further amplified by instituting changes at the legislative level.

Recent changes in the state of California have shed a great deal of light on the power of government mandates to influence vaccination rates. In the face of decreased vaccination administration by percent of population, California passed measure AB2109 in 2014. This piece of legislation permitted the continued acknowledgement of personal and religious beliefs as excuse for not vaccinating children beginning at kindergarten, yet required physician signature confirming that the risks of not vaccinating were discussed with a parent if they desired to exercise personal belief exemption. Once in effect, a small drop in prevalence of unvaccinated children was observed [3]. Following up, the state of California passed SB277 in 2016 eliminating all exemptions (including religious belief exemptions), except for those medical in nature, upon school entrance.

From this legislation, an even more intense drop in prevalence of unvaccinated children upon admittance was observed [3]. An important aspect of the analysis of vaccination rates must be recognized at this point: vaccination rates are measured upon the entrance to kindergarten at age five. This is at an age far older than the maximum age recommended by the CDC for nearly all vaccines. If we want to properly address the vaccine hesitancy crisis, we must analyze it in a way that provides information from the most important demographics.

The measures passed in California set a promising precedent of legislation that when applied nationwide can greatly increase vaccination rates, hopefully to a level that permits acceptable herd immunity for each vaccine preventable disease. However, in response to the passage of SB277, there was an observed spike in parents claiming medical exemption [3]. This indicates that a subset of anti-vaccine parents continued to subvert entrance requirements by finding doctors to sign off on medical exemptions.

Knowing this emphasizes the necessity of interdisciplinary collaboration: legislation to require vaccinations and reach appropriate degrees of herd immunity, physicians to act in an ethical manner and not sign off on medical exemptions when they clearly are not the case, all healthcare professionals to provide strong yet compassionate recommendation for vaccination, education systems to reinforce the damage of unchecked disease, and researchers to better publicize the overwhelming benefits to vaccination and to explore demographics of interest.

If these measures are not undertaken, then the grip of vaccine hesitancy will persist.

The detriments of an under-vaccinated popula-

tion have recently been spotlighted in lieu of the most recent measles outbreak and reinforce the need for instituted change. In the decade before 1963, the year the measles vaccine was introduced, it is estimated that 3 to 4 million people in the United States contracted measles each year, 500 of which died from the disease annually [4]. In the year 2000, measles was declared eliminated.

Since then, a slight enough decrease in measles vaccine administration has resulted in cases such as the 2014 Disneyland outbreak and the current national outbreak. A disease thought to be eliminated is now reemerging, and those who are not vaccinated have the highest risk of infection [3].

As determined in the Disneyland outbreak, and as will likely be the case in the current outbreak, the immensely high degree of international travel puts the United States and essentially every other country at risk of cross contamination from areas of low vaccination and high disease prevalence. Without sufficiently high levels of immunization, there is no other way to effectively resist many vaccine-preventable infections.

By inhabiting a progressing global society, the necessity of vaccination becomes increasingly clear. Fear of minimal threats that vaccines pose must be mitigated through rational discourse to promote their near-universal usage. The much needed shift in mentality of a select portion of the public can only be achieved through interdisciplinary cooperation. By having all tendrils of government, health, education, and research extend toward public perception of vaccination, appropriate levels of immunity can be reached at a community level and individual lives can be saved.

References: 1. US Department of Health and Human Services. Centers for Disease Control and Prevention (2011). Ten Great Public Health Achievements. Atlanta.

2. Wheeler M. and Buttenheim A. Parental vaccine concerns, information source, and choice of alternative immunization schedules. Hum Vaccin Immunother. 2013 Aug 1; 9(8): 17821789.

3. Jones, M 2017, Vaccine Refusal and Public Health, presentation, Wednesday Nite @ the Lab, University of Wisconsin- Madison, delivered September 27, 2017.

4. US Department of Health and Human Services. Centers for Disease Control and Prevention (2018). Measles (Rubeola). Atlanta. ●

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EDITORIAL

proper understanding of those chemicals, it is not

To properly address the crisis of vaccine hard to imagine why a parent doesn’t feel comhesitancy, public opinion must be considered at fortable in vaccinating their child. There are also all fronts. Parents, who only wish to provide the issues related to vaccine access: cost of vaccinabest for their child, are caught in the middle of tion, insurance plans of patients, availability of this turmoil and are at the mercy of the inforimmunization stock and of appointments at clinmation that they are exposed to, correct or incorics. If local providers are unable to administer rect. The dissemination of well-researched advice a vaccination due to inventory and the patient must be propagated from all directions toward lives a busy lifestyle, unable to return to the clinthose who have to make the decision to vaccinate. ic in the near future, it is easy for the patient to It is the education system’s responsibility to shed continue to push off vaccination, leaving themlight on the times of immense mortality from disselves and the community at risk for that much ease and emphasize the privilege of vaccination longer of a time. Now, there are some individuals that too many people take for granted. An awarewho are at great risk from the immune response ness of the extreme pain and suffering that can that is mounted from vaccine administration itbe caused by preventable illness must be made self. A fraction of a percent of the population reto overtake availability bias, which for some, inports that they are unable to receive one vaccine clines them to overestimate the perceived risks or another due to being immunocompromised or of vaccination. If the public is shown the damallergic to the vaccine [3]. These are the individage that illnesses can wreak on the human body, uals who have no agency over their susceptibility hopefully they will be jolted out of their lull of to disease and are the complacency. On an"Optimally, all individuals would other front, physicians reason why the rest of the population needs healthcare providbe immunized, but as of the current and to be vaccinated. ers must exercise the situation this is not possible. There will power that they wield Responsibility to protect those who public sentiment always be those with compromised over are medically unable and be well trained in immune systems, allergies, or those effectively communito receive vaccines falls on the shoulders with patients. who receive a vaccine not of absolute cating of the general popuThe recommendation efficacy. Due to this reality, it becomes of health professionals lous. Epidemiologists often cite the basic be overstated, the duty of the rest of society to act cannot reproductive number but most effective inas immunological barriers defending teractions come from of an infectious disease when discussing place genuine interagainst vaccine preventable diseases aaction the ability to protect and care for the to protect at-risk communities." non-immunized inpatient’s wellbeing dividuals. Variables and understanding. considered when scientists determine this are Healthcare providers must be taught to convey characteristics such as mode of infectivity and their well-informed knowledge in unambiguous estimated levels of immunized host populations. ways so as to leave their patients with an ease The latter of the two is often known as the degree of mind in the decision to vaccinate. It must be of herd immunity, a statistical phenomenon that emphasized that physicians resist the urge to hinges the protection of nonimmune individuals talk down to those who see them and meet their on the likelihood that they will encounter a carpatients from a place of respect. Moreover, the rier of disease. Optimally, all individuals would general public needs to be continually reaffirmed be immunized, but as of the current situation in their decision to vaccinate. This support can be this is not possible. There will always be those provided in the form of persistent research and with compromised immune systems, allergies, or publication surrounding the efficacy and safety of those who receive a vaccine not of absolute efficastandard-of-care vaccinations. If studies are done cy. Due to this reality, it becomes the duty of the to emphasize the benefits of vaccination and the rest of society to act as immunological barriers detriments of low vaccination rates and results defending against vaccine preventable diseases of these explorations are made more accessible, to protect at-risk communities. The sentiment then the public can know their health is of utof vaccine hesitancy works in stark contrast to most concern and be constantly reassured of the forming competent herd immunity, which reindecision to vaccinate. These proposals to modify forces the necessity of a nationwide campaign to education systems, update healthcare training emphasize the importance of vaccination. programs, and publicize cutting edge research in

NUTRITION

Pouring Milk from a ceramic bottle to a glass. Public Domain.

Can We Be More Lactose Persistent?

By Hae Rin Lee

T

oday, we eat a lot of dairy products: milk, yogurt, cheese, ice cream, you name it. Despite the large consumption of the dairy products, a large portion of populations worldwide are lactose intolerant. I happen to be one of them. It made me wonder why so many people are lactose intolerant, so I began to search for an answer.

According to the U.S. National Library of Medicine, lactose intolerance defined as an impaired ability to digest lactose, a sugar found in milk and other dairy products. Lactose is digested

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by an enzyme called lactase which is produced by cells in the lining of the small intestine. Normally, babies are initially born with the ability to make lactase since their diet relies on breast milk or infant formulas, unless they have a rare genetic disorder that prohibits production of lactase. However, as babies grow up and move away from consuming milk as the sole source of food, they gradually lose the ability to make up lactase with the decreased expression of the LCT gene that regulates the lactase production. As such,

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EDITORIAL

EDITORIAL

Despite the prevalence of the gradual lactose intolerance in adults, there are some populations less affected than others. Could it then be based on cultural differences?

roughly 75% of the world’s adult human popuin our guts. The microorganisms in probiotics in lation become lactose intolerant [1]. However, the colon can be selectively metabolized through despite the prevalence of the gradual lactose infermentation by specific lactic acid producing tolerance in adults, there are some populations bacteria, which can alter the lactose metabolism less affected than others. a beneficial way that decreases discomfort. Many

Could it then be based on cultural difpopular dairy products like yogurts can be a good ferences? If some communities eat dairy prodsource of probiotics, promoting easier and benucts more than others, could they bypass this eficial digestion of lactose. However, it is not a decreased activity of lactase production? There permanent or long-term change like the lactose has been lots of research conducted in order to persistent gene [4]. The good news is that there answer this more properly and there seems to be are many ongoing research projects and clinical cultural correlations between lactose intolerance trials to determine a more effective way to conand persistence. The theories of increased lactose trol the lactose digestion all around the world. persistence on certain cultures are based on the So maybe in future, lactose intolerance could be profound nutritional benefits of dairy products. a more minor discomfort that can be easily conFor example, the North-Western part of Europe quered. experience the highest frequency of the lactose

While we are on the quest to solving the persistence phenotypes- while the South-East enigma of lactose intolerance, we can still enjoy account for 15 to 54%, the North-West Europe some dairy products. There are many commercial accounted for 89 to 96% [2]. The spread of lactase dairy products on the market that contain lower persistence alleles in Northern Europe seems to concentrations of lactose, like yogurt or lactose correlate with the need to consume more milk free milk. All of us may not be the champions of than other communities as a source of energy, lactose persistence genetically, but we all know which promoted inhow to tolerate the creased calcium and "The lactose intolerance certainly limits discomfort and turn it vitamin D absorption a still enjoyable your milk consumption physiologically, into to help prevent osteopart of our lives. malacia (softening of but does not deprive you of your right bones due to deficiento enjoy it unless serious discomforts References: cy in vitamin D and 1. Silanikove, N., Leitcalcium) in low-sunmay arise. As lactose persistence found ner, G., & Merin, U. light regions. People The Interreways to smear through the Northern (2015). in these regions may lationships between have experienced a Europe, many of us also figured out Lactose Intolerance spread of the lactase and the Modern Dairy how to tolerate it differently. " persistence allele in Industry: Global Perpositive correlation spectives in Evoluwith the selection for denser bone buildup for the tional and Historical Backgrounds. Nutrients, promoted absorption of calcium and vitamin D 7(9), 7312-7331. doi:10.3390/nu7095340 [3].

2. Gerbault, P., Liebert, A., Itan, Y., Powell, A.,

Then, should we be sad that we don’t Currat, M., Burger, J., . . . Thomas, M. G. have this allele and remain lactose intolerant?

(2011). Evolution of lactase persistence: An Despite the fact that many of us are still lactose example of human niche construction. Philintolerant, you can still see the continued success osophical Transactions of the Royal Society of dairy industries around the world. The lactose B: Biological Sciences, 366(1566), 863-877. intolerance certainly limits your milk consumpdoi:10.1098/rstb.2010.0268 tion physiologically, but does not deprive you of 3. Silanikove, N., Leitner, G., & Merin, U. (2015). your right to enjoy it unless serious discomforts The Interrelationships between Lactose Intolmay arise. As lactose persistence found ways to erance and the Modern Dairy Industry: Globsmear through the Northern Europe, many of al Perspectives in Evolutional and Historical us also figured out how to tolerate it differentBackgrounds. Nutrients, 7(9), 7312-7331. ly. The one popular mechanism in both clinical doi:10.3390/nu7095340 and commercial markets is probiotic products 4. Szilagyi, A. (2015). Adaptation to Lactose in or dairy products with a lower lactose content.

Lactase Non Persistent People: Effects on InThe idea behind this is called the colonic adaptolerance and the Relationship between Dairy tation by the microbiome. This is rather simple: Food Consumption and Evalution of Diseasthe gradual and regular consumption of probiotic es. Nutrients, 7(8), 6751-6779. doi:10.3390/ products could change the metabolism of lactose nu7085309. ●

BIOLOGY

Drosophila. March 24, 2017. Credit Katja Schulz.

The Importance of Model Organisms Many important discoveries in human biology that affect how we treat disease and understand human development were not made by studying the human body.

I

nstead, these discoveries were made by studying the biological processes of organisms like mice, fish, and worms. Model organisms are species that are extensively studied to understand biological phenomena that provide insight into the functions of other organisms. Researching these organisms explores the basic biology and chemistry of life [1]. Why choose to study a model organisms instead of the organism of interest?

Research of human biology, development, or ge12 JUST VOL IV // ISSUE II // SPRING 2019

netics on humans is complicated and poses ethical challenges in many situations. In humans it is very difficult to separate the effects of a gene from the effects of the environment. However, with models, variables of interest can be engineered and confounding variables are more easily controlled [2].

If scientists want to learn about a metabolic process that only occurs in eukaryotes, a mouse or worm works as a model organism. Sin-

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EDITORIAL

By Annika Peterson

gle-celled yeasts are advantageous in the study food supply. There are many historical and reof processes conserved across all life [2].Many cent discoveries that have been made using moduseful model organisms share similar traits such el organisms. Will this type of research continue as small size, short generation time, the ability to to be useful as we understand more of the fundabe easily manipulated, and the ability to survive mental mechanisms of biology? As other methods in a controlled environment. of study such as computer modeling become more

Model organisms have led to many imsophisticated, the use of model organisms may portant discoveries in science. C. elegans, a small decrease [1]. However, considering the enormous nematode, was the first animal to have its geamount of unanswered questions in basic biolonome completely sequenced in 1998. Not only gy, research using model organisms will remain was this an achievement, it also provided a modan integral part of science [6]. Understanding bael for sequencing that was used in the Human sic biology will continue to benefit society, so it is Genome Project. The C. elegans genome is a tool important that research using model organisms that is still used today to understand the funccontinue. tion of eukaryotic genes [3]. Furthermore, studies of single-celled yeast have led to discoveries References: into how cells divide—information that is benefi1. Using Research Organisms to Study Health cial to cancer research in humans [1]. and Disease. NIH National Institute of Gen Researchers use model organisms to uneral derstand basic biology in Medical Sciences. Novemresearch around the world, ber 2018. Available from: including at University of https://www.nigms.nih.

Wisconsin-Madison.

Regov/education/Pages/modsearcher Dr. Jeff Hardin elorg_factsheet.aspx. Citat UW-Madison uses C. eled March 2, 2019. egans to study molecular 2. Reed L, Baer F, Edison processes that control cell S. Considerations When movements and adhesions Choosing a Genetic Model in embryonic development.

Organism for MetabolomThis research may shed ics Studies. Current Opinlight on mechanisms of canion in Chemical Biology. cer metastasis and events 2017; 36: 7-14 that lead to common birth 3. Wilson R. How the defects. A recent study Worm Was Won: the C. elpublished by the Hardin egans Genome Sequencing Lab characterized protein Project. Trends in GenetSORB-1. It is involved in ics. 1999; 15: 51-58 focal adhesions, structures 4. Loveless T, Qadota that transmit force between H, Benian M, Hardin J. the extracellular matrix Caenorhabditis elegans Arabidopsis. October 16, 2014. Credit Frost Museum. and cytoplasm of cells [4].

SORB-1 Localizes to InteResearch using C. elegans can contribute to ungrin Adhesion Sites and is Required for Orderstanding of basic cellular process including ganization of Sarcomeres and Mitochondria in cellular development and wound repair [4].

Myocytes. Molecular Biology of the Cell. 2017;

Dr. Andrew Bent is another researcher 28: 3621-3633 at UW-Madison who uses model organisms to 5. Briggs G, Adams-Phillips L, Keppler B, Zebell understand basic biology. His research into plant S, Arend K, Apfelbaum A, Smith J, Bent, A. disease resistance uses the plant Arabidopsis A Transcriptomics Approach Uncovers Novel thaliana to understand the plant immune sysRoles for Poly(ADP-ribosyl)ation in the Bastem. Recent research in the Bent Lab developed al Defense Response in Arabidopsis thaliana. knowledge of poly(ADP-ribosyl)ation (PARP) in PLOS One. 2017; 12: 1-30 the basal defense response of Arabidopsis. PARP 6. Hunter P. The Paradox of Model Organisms. is part of the plant response to biotic stress and European Molecular Biology Organization. can help understand how plants resist disease [5].

2008; 9: 717-720. â—?

Research using model organisms can help understand plants, animals, and microbes and lead to discoveries that impact our everyday lives through life-saving medicine and a stable

and as a result, lead to the dramatic increase in life expectancy. Children will be stronger and smarter, and will have a better chance of succeeding in life. Children can also be given genes that their parents don’t possess, therefore allowing for a more diverse and varied human population.

While CRISPR has been designed in a manner to allow for imaginations to run wild, it is important to note that unlike the artistic disciplines, here, the stakes are too high. Firstly, during experiments, mistakes are possible. While CRISPR can make babies stronger and smarter, the smallest of mistakes can make them the opposite, hence causing damage to the overall gene pool.

Secondly, children would have no say in the way their genome is being designed.

This demonstrates the absence of informed consent.

However, many argue that parents have the right to make this decision for the child, and it is practically impossible to gain informed consents from embryos.

GENETICS

Genetc engineering. Creative commons.

X-Men May Not Just Be a Movie

By Aadhishre Kasat

uring low points in our lives, many of us find ourselves tempted to compare ourselves to the straight-A student in our math class, or perhaps our attractive neighbor who has a date lined up every night.

We may have dreamt at some point to have the ability to alter or maybe even completely replace a certain characteristic of ourselves. It may be the color of our eyes or perhaps our intelligence. The reality of such fantasies is now closer than ever. Researchers have termed genetic makeup that can be altered or can be chosen to provide the desired genome as “designer babies”.

In fact, there are three techniques that have been developed to enable designer babies, all of which require the genetic manipulation of fertilized eggs before in-vitro fertilization (the fertilization of eggs outside the body). First, Preimplantation Genetic Diagnosis: this technique doesn’t involve gene manipulation, rather, it involves the selection of the most desirable embryo out of all 14 JUST VOL IV // ISSUE II // SPRING 2019

the viable embryos. This technique is especially advantageous because it allows for the screening of life-threatening diseases. Second, Transcription Activator-like Effector nucleases (TALEN’s): these are enzymes that can be designed to replace targeted parts of the DNA helix. This technique has been traditionally used to produce genetically modified wheat, tomatoes, and fuel. Finally, the revolutionary CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats). CRISPR is analogous to performing a surgery, except the surgery is being performed on the DNA sequence.

Certain unfavorable DNA stretches can be identified, for example, the DNA mutation that causes sickle cell anemia. Scientists then use their “special scissors” to snip the unfavorable gene sequence from the genome. Ultimately a “special syringe” is used to fill in the gap with a healthier gene sequence.

The benefits of CRISPR are clear. It would definitely reduce genetic and inherited diseases,

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EDITORIAL

D

cial distinctions delineating enhanced individuals from unenhanced individuals.

The door to this Pandora's box has already been cracked ajar in China and the United Kingdom. Such acceptances around the globe are pushing the rest of the countries further towards the acceptance of human genetic modifications. Very soon governments may also begin to encourage genetically modified humans for the simple reason of lower healthcare costs. There are several labs around the globe that want to utilize the technology of CRISPR for developing treatments and solutions for existing deadly diseases. However, if this technology is used for human genetic modification and if it leads to a problematic situation, the whole field would have to pay that price. This means that researchers previously practicing CRISPR simply for the development of medication would have to discontinue. This could potentially ruin the possibility of discovering and employing valuable treatFurthermore, many parments. ents would lean in the

I believe that our direction of making the desire to control and ma“better child”, hence causnipulate all elements of naing a loss of individuality, ture could eventually lead because everyone will be to our downfall if we hasttempted to adopt the generily jump into the ambiguic ideal. It is imperative to ity of this technology. It is note that most genes have crucial for us to learn more multiple functions, and if about the working of the changed in an incorrect human genome before we manner could have wildly attempt to alter it. Above unpredictable consequencall it is necessary for us to es, such as the developweigh the ethical concerns ment of new diseases and associated with the prac“What Is CRISPR?” CB Insights Research, 7 Feb. 2019, vulnerabilities. The most tice of CRISPR for human www.cbinsights.com/research/what-is-crispr/. daunting ethical concern, in genetic modification. my opinion, is the determination of good versus References: bad. Who will determine what characteristics are 1. “The Embryo Project Encyclopedia.” Temperaconsidered good and which are considered bad? ture-Dependent Sex Determination in Reptiles And on what basis will these determinations be | The Embryo Project Encyclopedia, Arizona made? The social argument against designer baState University. School of Life Sciences. Cenbies is that if this technology becomes a realistic ter for Biology and Society. Embryo Project Enand accessible medical practice, then it would cyclopedia., embryo.asu.edu/pages/ethics-decreate a division between those that can afford signer-babies. the service and those that cannot. Therefore, the 2. “What Are the Ethical Concerns about Gewealthy would be able to afford the selection of nome Editing?” National Human Genome desirable traits in their offspring, while those of Research Institute (NHGRI), www.genome. lower socioeconomic standing would not be able gov/27569225/what-are-the-ethical-concernsto access the same options. As a result, economic about-genome-editing/. ● divisions may grow into genetic divisions, with so-

PIXELS

John Vandevender: Pictured is Earth's only moon. The moon orbits at an average distance of 382,500 km from the Earth.

[Photo pictured on cover].

where science and art collide

John Vandevender: A backcountry lake in Ontario, Canada.

This area is home to various mammals including Black Bear (Ursus americanus), Moose (Alces americanus), and Whitetail Deer (Odocoileus virginianus).

Helen Heo: Mitotic mouse neural stem cells with CRISPRed H2B-mCherry and human vimentin overexpression

16 JUST VOL IV // ISSUE II // SPRING 2019

John Vandevender: Have you ever thought how wild it is that spiders make these webs?! This spider web was photographed in Lenawee Co. Michigan. Spiders Rule!

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Cholecystokinin Protects HP62 Human β-cells from Cytokine-induced Apoptosis.............................20 β-cell apoptosis caused by cytokine stress, especially Interleukin (IL)-1β, tumor necrosis factor (TNF)-α and interferon (IFN)-γ, is involved in the mechanism of diabetes. Cholecystokinin (CCK), a peptide hormone released due to obesity stress, has been shown to protect β-cells from cytokine-induced apoptosis on the INS-1 rat cell line and the Min6 mouse cell line. Whether CCK reduces human β-cells apoptosis caused by cytokine stress remains unclear. Using the HP62 human pancreatic β-cell line, we determined whether CCK increased β-cell living percentage under cytokine stress by conducting trypan blue cell viability assay. We also examined whether CCK treatment altered cytokine stress-related gene expression. C/EBP homologous protein (CHOP), immunoglobulin protein (BIP), X-box binding protein 1 (XBP1) and signal transducer and activator of transcription 1 (STAT1) were chosen to do real-time PCR (RT-qPCR). Investigating the protective effect of CCK evaluates its potential efficacy as a therapeutic target for diabetes. Based on the cell viability assay, CCK treatment resulted in increased cell viability compared to the vehicle.

Under cytokine stress, the presence of CCK significantly reduced β-cell apoptosis for 24hour treatment. No significant increase of living cell percentage was detected for 48-hout treatment. Therefore, CCK protects human β-cells from cytokine-induced apoptosis, but the protective effect is time dependent. No significant gene expression changes of CHOP, BIP, XBP1 and STAT1 in response to CCK treatment were found in the study. the same individual of some species and very similar flowering dates for the same individuals of other species.

This indicates that the relationship between climate and flowering time is species-dependent. Future data collection will continue to shed light on this relationship.

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Algal growth, blooms, and diversity have been widely documented as indicators of increased nutrients in bodies of water. Consequently, increased coastal urbanization such as dock pilings can cause nutrient-rich agricultural and commercial waste runoff. However, it is not widely determined if algae remains the dominant organism after long-term urbanization. The effects of urbanization on algal abundance, diversity, and succession on dock pilings was observed within the Bocas del Toro archipelago (09°15'60.00" N, 82°14'60.00" W) between April 14th-18th, 2018. Average algal abundance differed significantly between sites; however, contrary to the initial hypothesis, rural sites had a higher algal abundance (56.3% cover) than urban sites (36.2%). The difference in algal abundance on old (41.2%) and new (48.1%) pilings was not significant. Within the archipelago, average algal diversity did not differ significantly between urban, rural and new sites and similar species of algae were found throughout sites. These findings reveal that urbanization is likely not the only factor impacting algal communities within the archipelago, and dock pilings can support a viable ecosystem for many organisms. This research and further investigation lead to a greater understanding of how primary producers and their ecological role are impacted by increased coastal urbanization.

Improving Clinical Training and Electrocardiograms Analysis Through Adaptive Artificial Intelligence.........................................32 Computerized signal analysis and interpretation have long been lauded as the next generation of medical equipment technology. However, none have looked to understand key points where machines are able to detect features that humans cannot and improve the process. Here, we attempt to develop a method to understand key problem areas within medical signals and scans that lead to misdiagnosis for training clinicians. Initial attempts are being designed with de-identi ed Electrocardiogram (ECG) signals: the base structure employs an 'ideal' student through a Hybrid Convolutional Neural Network (CNN). This CNN dynamically selects the highest accuracy arti cial learning technique to provide the best response both for classi cation of the signal in question and for the student's input data.

From there, immediate visual feedback is provided to the student and metadata summaries are made available to professors and teachers, systematically creating a pro le of common misconceptions with associations to clinically relevant signal features.To the best of my knowledge, this is the first cost-effectiveness analysis for evaluating genetic testing of pregnant women for G6PD as a means of malaria eradication in Thai-Myanmar border refugee camps.

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REPORTS

Effects of Urbanization on Algal Abundance, Diversity, and Succession on Dock Pilings Within the Bocas del Toro Archipelago................................26

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Cholecystokinin Protects HP62 Human β-cells from Cytokine-induced Apoptosis Jiayin Tang1, Arnaldo Henrique de Souza2,3, Dawn Belt Davis4

Department of Integrative Biology, University of Wisconsin-Madison, Madison, Wisconsin; 2School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin; 3Department of Medicine, Division of Endocrinology, University of Wisconsin-Madison, Madison, Wisconsin; 4William S. Middleton Memorial Veterans Hospital, Madison, Wisconsin 1

ABSTRACT β-cell apoptosis caused by cytokine stress, especially Interleukin (IL)-1β, tumor necrosis factor (TNF)-α and interferon (IFN)-γ, is involved in the mechanism of diabetes. Cholecystokinin (CCK), a peptide hormone released due to obesity stress, has been shown to protect β-cells from cytokine-induced apoptosis on the INS-1 rat cell line and the Min6 mouse cell line. Whether CCK reduces human β-cells apoptosis caused by cytokine stress remains unclear. Using the HP62 human pancreatic β-cell line, we determined whether CCK increased β-cell living percentage under cytokine stress by conducting trypan blue cell viability assay. We also examined whether CCK treatment altered cytokine stress-related gene expression. C/EBP homologous protein (CHOP), immunoglobulin protein (BIP), X-box binding protein 1 (XBP1) and signal transducer and activator of transcription 1 (STAT1) were chosen to do real-time PCR (RT-qPCR). Investigating the protective effect of CCK evaluates its potential efficacy as a therapeutic target for diabetes. Based on the cell viability assay, CCK treatment resulted in increased cell viability compared to the vehicle. Under cytokine stress, the presence of CCK significantly reduced β-cell apoptosis for 24-hour treatment. No significant increase of living cell percentage was detected for 48-hout treatment. Therefore, CCK protects human β-cells from cytokine-induced apoptosis, but the protective effect is time dependent. No significant gene expression changes of CHOP, BIP, XBP1 and STAT1 in response to CCK treatment were found in the study.

β-cells are important in regulating body glucose metabolism. When β-cells fail to produce an adequate amount of insulin to regulate blood glucose levels, diabetes can develop. There are two major types of diabetes, Type 1 and Type 2. Type 1 diabetes (T1D) is characterized by inflammation and progressive insulin-producing β-cell loss.

Inflammation can lead to β-cell destruction, function suppression, and regeneration inhibition [1]. β–cell destruction due to inflammation can range from 60-100% [2]. Reduced β-cell mass causes the loss of insulin production. Type 2 diabetes (T2D) is characterized by an increased insulin requirement caused by insulin resistance. Possible cellular stresses that lead to insulin resistance, such as oxidative stress and endoplasmic reticulum stress (ER stress), all relate to inflammation and may directly induce inflammatory responses, which negatively affect the function of β-cells [3].

Thus, T2D can be viewed as an auto-inflammatory disease [4]. Inflammation, which reduces β-cell 20 JUST VOL IV // ISSUE II // SPRING 2019

mass and damages β-cell function, is involved in the pathogeneses of both T1D and T2D.

Cytokines can cause inflammation. Interleukin (IL)-1β, tumor necrosis factor (TNF)-α and interferon (IFN)-γ are some of cytokines that are involved in diabetes mechanisms [5]. Op de Beeck and Eizirik [2] showed that local inflammatory cytokines, especially IL-1β, TNF-α and IFN-γ, lead to the decrease of β-cell insulin secretion in patients with T1D. The function damage eventually causes β-cell death in response to cytokine stress [2]. For patients with T2D, increased rate of β-cell apoptosis is found due to elevated circulating levels of cytokines in their bodies [3]. Cytokine stress leads to gene expression changes via inflammation, which further causes β-cell apoptosis. Extensive activation of signal transducer and activator of transcription 1 (STAT1) caused by cytokine exposure increases β-cells apoptosis in rats [5]. Cytokines can induce ER stress to cause cell apoptosis. For INS-1 cells, an insulin secreting β-cell rat-derived cell line, MIN6 cells, a mouse insulinoma cell line, and

(SV40) [11]. It is used to study cytokine-mediated regulation of adhesion molecule expression on the surface of β-cells. The HP62 cell line is used as a surrogate model resembling the pancreatic β-cells in vitro. HP62 cells were cultured in medium RPMI-1640 supplemented with 10% Fetal bovine serum (FBS), 2mM L-glutamine, 5ug/ml transferrin (Sigma), 10-8 M hydrocortisone (Sigma) and antibiotics (1% penicillin/streptomycin).

Cells were cultured in the media at 37°C. The media was changed every 48 hours. Cells were passaged every week and/or when the cells had obtained 70% confluence. Cells were harvested using 0.05% trypsin.

CCK Treatment:

Cells were seeded in 12-well culture plates with seeding density 0.2x106 cells per well.

Cells were cultured in the media and incubated at 37◦C for 48 hours before treatments. Stable form of exogenous CCK-8 (Sigma) was administered at 100 nM concentration to the HP62 cell line [10]. Cells were incubated at 37◦C for either 24 or 48 hours after introduction of treatments.

Time range helped to determine whether the effect of CCK treatment was time dependent.

Cytokines Treatment:

Cells were seeded in 12-well culture plate with seeding density 0.2x106 per well. Cells were then allowed to incubate for 48 hours before a cytokine cocktail was added. The cytokine cocktail containing IL-1β (75 U/ml), IFN-γ (750 U/ml) and TNF-α (1000 U/ml) was administered to the HP62 cell line [12]. Cells were incubated at 37◦C after introduction of cytokines for either 24 or 48 hours.

Group design:

Cells were divided into four treatment groups: vehicle, CCK-treated only, cytokine-treated only, combination of CCK and cytokines.

METHODS

Cell Viability and Death Assays:

The percentage of dead cells was measured by trypan blue viability assay. 0.4% trypan blue dye (BIO-RAD, #1450013) was added to a small portion of the cell suspension to obtain a 1 to 2 dilution [13]. The counting slides (BIO-RAD, #1450011) and automated cell counter (BIORAD, TC10) were used to do the viability assay.

Counting was done within five minutes after adding the trypan blue dye.

Models of Human β-cells:

The HP62 cell line is derived from transfection of human islets with simian virus 40

RT-qPCR:

Total RNA from each group was extracted using RNeasy kit (Qiagen). Complementary JUST VOL IV // ISSUE II // SPRING 2019

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INTRODUCTION

human islets, cytokines significantly increase the expression of ER stress markers, such as activating transcription factor 4 (Atf4), binding immunoglobulin protein (BIP), and X-box binding protein 1 (XBP1) [6]. C/EBP homologous protein (CHOP), related to ER stress-mediated apoptosis, can also be induced by cytokines, IL-1β and IFN-γ, in rat β-cells [7].

β-cell-derived Cholecystokinin (CCK), a peptide hormone released due to obesity stress, protects cells against acute apoptotic stressors, such as cytokines. Lavine et al. [8] showed that when the expression of CCK mRNA was increased by 200-fold in the INS-1 cell model, 27% decrease of β-cell death and 40% reduction of cytokine-induced activation of caspase, a group of protease enzymes resulting in cell death, were found. The protective effect of CCK on rat β-cells is significant. In addition, Ning et al. [9] found that, based on mouse β-cells, via CCK1 receptors, CCK-8s reduced β-cell death and increased insulin secretion. Percentage of β-cell apoptosis decreased from 80% to less than 50% [9]. The significant protect effect of CCK is also indicated by other studies. Using islets from CCKLacZ- ob/ ob mice with CCK deficiency, Lavine et al. [10] showed that when 100 nM of CCK-8 was present, there was 39% reduction in cell apoptosis. These results based on animal models indicate that CCK has an anti-apoptotic effect on β-cells and directly reduces β-cell apoptosis under stress.

Although previous studies showed that CCK can protect rat and mouse β-cells from cytokine-induced apoptosis, the effect of CCK on human β-cells is not well studied. Based on the protective effect of CCK on animal cell models, we expect that the presence of CCK reduced cytokine-induced apoptosis on human β-cells by down-regulating cytokine sensitive gene expression. The primary purpose of this study is to explore whether CCK protects human β-cells from cytokine-induced apoptosis and whether CCK treatment alters expression of genes related to cytokine stress. We aim to determine the protective effect of CCK on increasing living cell percentage under cytokine stress and identifying possible gene expression changes that occurred in response to CCK treatment. Understanding the protection mechanism of CCK on human β-cells creates a chance to identify potential anti-apoptotic methods and evaluate potential efficacy of CCK as a therapeutic target for diabetes.

DNA (cDNA) of each treatment was made using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). This cDNA was used as a template for the real-time PCR (RT-qPCR) to determine the gene expression alternation after treatments. The reference gene beta-actin was used to normalize data of mRNA quantification of target genes. Genes, CHOP, BIP, XBP1 and STAT1, and reagent CYBR green were used for RT-qPCR. Primers of each gene are shown in Table 1.

Statistics:

The difference of living cell percentage was evaluated by one-way ANOVA test followed

by Tukey test for different treatment groups.

Based on the results of the one-way ANOVA, gene expression was evaluated using an independent-sample t-test. Confidence intervals for all data were set at 95%.

RESULTS

CCK reduced cytokine-induced HP62 human β-cell apoptosis for 24-hour treatments

Cytokines lowered the cell viability for 24-hour (avg=85.56, SEM=1.18) but not for 48hour (avg= 89.40, SEM=1.39) treatments compared to the vehicle (avg=93.88, SEM=0.82 for 24 hours and avg=91.70, SEM=1.25 for 48 hours, Fig. 1 and 2). When CCK was present, cytokines had a reduced negative effect on the cell viability of HP62 human β-cells. Shown in Fig. 1 and 2, the group treated with both CCK and cytokines had higher percentage of living cells (avg=92.87, 22 JUST VOL IV // ISSUE II // SPRING 2019

Gene expression was not significantly altered by CCK treatment

Since significant difference was detected between the group treated with cytokines-only and the one treated with both CCK and cytokines for 24-hour treatments, RT-qPCR was only conducted for 24-hour treatments. There was no significant difference for all gene expression between the group treated with CCK-only and the vehicle (p=0.13 for CHOP, p=0.92 for XBP1, p=0.80 for BIP, p=0.07 for STAT1, Fig. 3). Cytokines up-regulated gene expression for STAT1 (p=0.21 for CHOP, p=0.11 for XBP1, p=0.09 for BIP, p<0.01 for STAT1, Fig. 3) compared to the vehicle. When CCK co-existed with cytokines, there was no significant difference for all gene expression (p=0.81 for CHOP, p=0.34 for XBP1, p=0.46 for BIP, p=0.60 for STAT1, Fig. 3) compared to the cytokines-only group.

DISCUSSION

Reducing β–cell apoptosis is important for treating diabetes [2, 3]. Results of this study indicate that CCK has a positive effect on preventing β–cell death. By comparing living cell percentage of the vehicle and the group treated with CCK only, we found that the presence of CCK led to 2 percent and 3.5 percent increase of living cells for 24-hour and 48-hour treatments, respectively. Significant difference between these two groups suggests that CCK has a positive effect on human β–cell viability. This result agrees with results of other studies based on mice. Using CCKWT-ob/ob mice, which has CCK expression, and CCKLacZ- ob/ob mice, which is null for CCK, Lavine et al. [10] found that CCK deficiency reduced β–cell mass significantly. 65% reduction in β–cell area was detected on CCKLacZ- ob/ob mice by week 10 compared to CCKWT-ob/ob mice. Since no changes in cell proliferation was found, CCK deficiency reduced β–cell mass by increasing β– cell death rate [10]. This is a possible explanation for the effect of CCK. CCK is involved in the protection mechanism of β–cells and it may also increase the amount of CCK receptors on β–cells.

As cells have more CCK receptors, CCK prevents cells from apoptosis to a greater extent. PCR test

about CCK receptor changes can be done in the future to examine gene expression changes of CCK receptor in response to CCK treatment.

β–cells apoptosis induced by cytokines is one of the major reasons that lead to β–cell failure [2, 3]. Dealing with damage effects of cytokines, especially IL-1β, IFN-γ and TNF-α, induced by invading immune cells and obesity is important for treating T1D and T2D [5]. The result of cell viability assay indicates that CCK protects β–cells from apoptosis when cells are not under stress and the protective effect is more significant when β–cells are under cytokine stress. Cytokines significantly lowered cell viability for 24-hour treatments compared to the vehicle. When CCK was present, the damage effect of cytokines on human β–cells was no longer significant. 100nM CCK used in the study decreased cell viability by 7% compared to cytokines-only group for 24-hour treatments. The positive effect was significant.

However, for 48-hour treatments, the protective effect was not significant, although the group treated with both CCK and cytokines decreased cell viability by 2% compared to the group treated with cytokines only. The result supports the hypothesis that the presence of CCK reduces cytokine-induced apoptosis for HP62 β–cells to some extent. However, the protective effect seems to be time-dependent. The protective effect of CCK shown in this study is also indicated by other studies that used animal models. Using Min6 cells with high expression of CCK, Lavine et al. [10] found that there was 38% increase of cytokine-induced apoptosis when CCK expression was down-regulated by 66%. The protective effect is much more notable in the mouse cell line. The difference could be due to different concentrations of cytokine cocktails used in different studies. Lavine et al. [10] used cytokines containing IL-1β (1000 U/ml) and TNF-α (5000 U/ml), while our study used cytokines containing IL-1β (75 U/ml), IFN-γ (750 U/ml) and TNF-α (1000 U/ ml). Different mechanisms involved in different models can be another reason, since HP62 cells may not respond similarly to Min6 cells. In addition, the concentration of CCK used in this study was based on the concentration used in the study conducted by Lavine et al. [10], in which 100 nM CCK-8 had shown the positive effect on reducing mouse β–cell death. Concentration that is optimal for mouse islets may not be appropriate for human cells. Concentration that allows CCK to show a protective effect under cytokine stress can also be different from the one that allows CCK to show a positive effect under no stress.

A large dosage can down regulate CCK receptor expression and a small dosage may not be able to significantly change the expression. When CCK

receptor expression is not at a proper level, the protective effect of CCK cannot be clearly demonstrated. Therefore, different concentrations of CCK can be tested in the future to further examine the effect of CCK on cytokine-induced β–cell apoptosis.

Shown in Fig. 3, there was no significant difference of gene expression between the vehicle and the CCK-only group. When cells were treated with cytokines only, all four genes were up-regulated (Fig. 3). This result roughly matches the one from the study conducted by Brozzi et al. [6], except for BIP expression. They [6] indicated that exposed human β–cell line EndoC-βH1 to cytokines significantly up regulated CHOP and XBP1 but not BIP. However, since different cell lines have different mechanisms, based on our data, BIP is actively involved in the mechanism of the HP62 cell line when cells are under cytokine stress. When CCK was present, no significant down-regulation of four genes was found compared to the cytokine-only group, which was different than our expectations. We hypothesized that the CCK protective effect could be shown by down-regulating expression of genes that were up-regulated by cytokine stress. To confirm there is no gene expression changes for CHOP, XBP1, BIP and STATI in response to CCK treatment, other techniques, such as western blot or immunofluorescence for specific proteins should be used in the future. Several reasons can explain this result. First, cytokine stress affects expression of numerous genes. By exposing purified rat β-cells or insulin-producing cells to IL-1β and/or IFN-γ for 1-24 hours, Cnop et al. [5] identified approximate 700 genes and expressed sequence tags that are sensitive to cytokine stress. The protective effect of CCK suggested by the cell viability assay may be due to the changes of other gene expression, not CHOP, XBP1, BIP and STAT1 used in the study. Second, although similarities exist between cell lines, such as Oleson et al. [12] found that STAT1 signaling pathway can be activated by cytokines in EndoC-βH1 cells and our study also shows significant increased expression of STAT1 for HP62, differences are indicated by studies for different cell lines. BIP and XBP1 which are highly up-regulated by cytokines in Min6 mouse cells are much less induced in rat cells after both 24 and 48-hour cytokine exposure [6]. They also showed that cytokines, combined IL-1β, TNF-α and IFN-γ, induced less expression of XBP1 in human islets compared to mouse and rat cells [6].

70-80% reduction of β–cell mass for T1D and 25-50% reduction for T2D were found at diagnosis time [5]. Finding a molecular target that can rescue β–cells from cytokine-induced apoptoJUST VOL IV // ISSUE II // SPRING 2019

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CCK-treated only group reduced HP62 human β-cell apoptosis compared to the vehicle

The results of one-way ANOVA tests for both 24-hour and 48-hour treatments indicated that living cell percentages were significantly different amongst the treatment groups (F(3,60) = 23.38, N=4, p < 0.01 and F(3,33) = 4.47, N=3, p < 0.01, respectively). For 24-hour treatments, according to Fig. 1, the group treated with CCK only had higher percentage of living cells (avg=95.92, SEM=0.56) than the vehicle (avg=93.88, SEM=0.82). The difference between two groups was significant (p=0.048). The result of 48-hour treatments showed a similar result.

Shown in Fig. 2, the group treated with CCK only had higher percentage of living cells (avg=95.20, SEM=0.87) than the vehicle (avg=91.70, SEM=1.25). The difference between two groups was significant (p=0.035). Based on results of 24-hour and 48-hour treatments, CCK-treated group had significantly reduced β-cell apoptosis compared to the vehicle.

SEM=1.09) for 24-hour treatments and for 48hour treatments (avg=91.86, SEM=0.96). The difference between the percentage of living cells for the group with cytokines-only and the one with both cytokines and CCK was significant (p<0.01) for 24-hour treatments. However, although β-cells had decreased apoptosis when CCK was present for 48-hour treatments, the difference between two groups was not significant (p=0.17).

sis is important for diabetes treatments. The protective effect of CCK shown in the cell viability assay makes it a potential target. However, the effect is only for the HP62 β-cell line and more replications are needed to confirm these positive results. Studies about human islets or other cell lines can be done to further examine the effect of CCK on cytokines-induced cell death. In ad-

dition, the effect of CCK could vary based on the testing environment. After confirming the effect of CCK in vitro using cell lines, in vivo experiments need to be done to evaluate the potential efficacy of CCK. Further investigation helps to develop a potential therapeutic target for diabetes.

Figure 3. Gene expression changes for treatments. The result was based on the average of HP62 passage 16, 17 and 18 (replication N=3). There was no significant difference of gene expression between the vehicle and the CCK-only group (p=0.13 for CHOP, p=0.92 for XBP1, p=0.80 for BIP, p=0.07 for STAT1). Cytokines up regulated gene expression (p=0.21 for CHOP, p=0.11 for XBP1, p=0.09 for BIP, p<0.01 for STAT1). When CCK co-existed with cytokines, there was no significant difference for all gene expression (p=0.81 for CHOP, p=0.34 for XBP1, p=0.46 for BIP, p=0.60 for STAT1) compared to the cytokines-only group.

FIGURES AND TABLES REFERENCES

Figure 1. Average of living cell percentage after 24-hour treatments for HP62 β-cell passage 16, 17, 18 and 19. CCK-8 (100 nM) and cytokines (75 U/ml IL-1β, 750 U/ml IFN-γ, 1000 U/ml TNF-α) were administered to the cells. Based on the average of replications (N=3), the group treated with CCK only had higher percentage of living cells than the vehicle (p=0.048). Cytokines lowered the cell viability, but the difference was not significant compared to the vehicle (p=0.23). When CCK was present, cytokines had a reduced negative effect on cell viability of HP62 human β-cells (p<0.01).

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Figure 2. Average of living cell percentage after 48-hour treatments for HP62 β-cell passage 17, 18 and 19. CCK-8 (100 nM) and cytokines (75 U/ml IL-1β, 750 U/ml IFN-γ, 1000 U/ml TNF-α) were administered to the cells. Based on the average of replications (N=3), the group treated with CCK only had higher percentage of living cells than the vehicle (p=0.035). Cytokines lowered the cell viability (p<0.01) compared to the vehicle. When CCK was present, β-cells had reduced apoptosis, but the difference was not significant(p=0.17).

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151: 3577-3588, 2010.

11. Vives M, Soldevila G, Alcalde L, Lorenzo C, Somoza N, Pujolborrell R. Adhesion molecules in human islet β-cells: De novo induction of ICAM-1 but not LFA-3. Diabetes. 40: 13821390, 1991.

12. Oleson BJ, Mcgraw JA, Broniowska KA, Annamalai M, Chen J, Bushkofsky JR, Davis DB, Corbett JA, Mathews CE. Distinct differences in the responses of the human pancreatic β-cell line EndoC-βH1 and human islets to proinflammatory cytokines. American Journal of Physiology-Regulatory Integrative and Comparative Physi. 309: 525-534, 2015.

13. Phillips HJ, Terryberry JE. Counting actively metabolizing tissue cultured cells. Exp Cell Res. 13: 341-347, 1957. ●

Effects of Urbanization on Algal Abundance, Diversity, and Succession on Dock Pilings Within the Bocas del Toro Archipelago Alaina Eckert

The School for Field Studies

ABSTRACT Algal growth, blooms, and diversity have been widely documented as indicators of increased nutrients in bodies of water. Consequently, increased coastal urbanization such as dock pilings can cause nutrient-rich agricultural and commercial waste runoff. However, it is not widely determined if algae remains the dominant organism after long-term urbanization. The effects of urbanization on algal abundance, diversity, and succession on dock pilings was observed within the Bocas del Toro archipelago (09°15'60.00" N, 82°14'60.00" W) between April 14th-18th, 2018. Average algal abundance differed significantly between sites; however, contrary to the initial hypothesis, rural sites had a higher algal abundance (56.3% cover) than urban sites (36.2%). The difference in algal abundance on old (41.2%) and new (48.1%) pilings was not significant. Within the archipelago, average algal diversity did not differ significantly between urban, rural and new sites and similar species of algae were found throughout sites. These findings reveal that urbanization is likely not the only factor impacting algal communities within the archipelago, and dock pilings can support a viable ecosystem for many organisms. This research and further investigation lead to a greater understanding of how primary producers and their ecological role are impacted by increased coastal urbanization.

Key Words: Algal abundance, Urbanization, Diversity, Pilings, Bocas del Toro

At the current rate, the world population is proje At the current rate, the world population is projected to exceed 9.7 billion people by the year 2050 [1]. With many major cities neighboring the coast, development along coastal areas is predicted to significantly increase, resulting in 75% of the human population residing within 100 km of the coast by 2025 [2]. Coastal development, urbanization, and periodic anthropogenic disturbance can subsequently change organismal composition within an ecosystem [3,4]. These ecological changes negatively impact biodiversity, particularly species abundance and genetic diversity [5]. Due to the complex nature of urbanization and the wide range of effects and variables it encompasses, it is difficult to assume an overall impact within an area [5]. However, in many coastal ecosystems, urbanization threatens marine life and the fragile ecosystems it holds.

In the archipelago of Bocas del Toro, Panamá, ecological disturbance in the form of 26 JUST VOL IV // ISSUE II // SPRING 2019

banana and cacao plantations, subsistence farming, cattle rearing, urbanization, and unsuccessful waste management has caused increased nutrient runoff, particularly nitrogen and phosphorous, into Bahia Almirante and Laguna de Chiriqui [6]. Phosphate levels within Bahia Almirante and Laguna de Chiriqui have been recorded at 0.13 μM and 0.14 μM, respectively, 0.3 μM greater than outer lagoonal sites. These high phosphate levels also corresponded with high nitrate levels [6]. In addition, Laguna de Chiriqui has been recorded as having on average 2.5 times more silica than the exposed ocean surrounding the archipelago [7]. The human-induced nutrient rich waters around the archipelago have resulted in algal build up and the subsequent breakdown of algae by oxygen-consuming bacteria [8], giving way to an increased number of hypoxic or “deoxygenated zones” [9]. Algae are also detrimental to the surrounding ecosystem under nutrient-rich conditions, as blooms of toxic species and the resulting hypoxia can result in fish mortality and human sickness [9].

gal growth within the town. The purpose of the current study was to analyze both the effects of urbanization on algal growth and diversity as well as how piling age impacts algal growth within ecological succession. Specifically, ecological succession and urbanization contribution to algal growth, in a location where the effects of this combination could be clearly observed.

METHODS AND MATERIALS

Data were collected at 10 sites within the archipelago of Bocas del Toro, Panamá. Sites were chosen based on degree of urbanization of the location, age of the piling and piling material(Table 1, Fig. 1). In total, 90 pilings were sampled. Percent cover of algae was assessed with a 15 cm by 25 cm laminated quadrats and a grid overlay.

Quadrats were placed on the side of the pilings facing outward from the structure to which they are attached, and on one adjacent side. The top of the quadrat was positioned at depths of 0.5 m and 1.0 m below the lowest water mark on the piling, resulting in four measurements taken on each piling. If the sites were too shallow to collect data 1.0 m below the lowest water mark, then only the 0.5 m sample was recorded. Three perimeter pilings were selected from each side of the dock, resulting in nine pilings measured at each site. Pictures of each quadrat on each piling were taken using an underwater camera. A grid overlay with appropriate scaling was applied to each photo using ImageJ. A grid of points (15 cm x 25 cm = 375 pts) was used to determine percentage algal cover by counting the number of points over algal species. To determine species richness, algae species at depths between 0.5 m and 1.0 m below the lowest water mark were identified on each piling using STRI and NSF algal data and handbook [18,19]. This technique was used to gain a representative algal community sample at each location. The data were then divided by pertinence to species richness and percent cover, and algal species were identified visually.

The same quadrats used to determine percent algal cover were analyzed for algal succession.

Mean percentage cover and standard error were calculated for sites categorized by age and location. Site categories were further compared using ANOVA and Tukey tests. The statistical software, “Past” was used to analyze data.

RESULTS

Data concerning algal coverage between sites, as well as urban, rural, and abandoned percent cover were normally distributed and allowed for parametric analysis. However, data regardJUST VOL IV // ISSUE II // SPRING 2019

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INTRODUCTION

Algae are primary producers and, in a stable amount, are considered a foundational species because they produce oxygen and food for greater ecological consumption [10,11]. Limiting factors to algal growth and production include sunlight required for photosynthesis and nutrients, particularly nitrate, phosphate and silicate, which increase in areas of high ecological disturbance[11,12]. Human-accelerated eutrophication due to nutrient input is being exacerbated by the increased urbanization of Bocas del Toro. This is clearly seen within Bocas Town, the main hub of tourism located on Isla Colón with the archipelago. Maintaining and increasing this urban coastal infrastructure, particularly through structures built on pilings, can create opportunities for invasive or opportunistic species, like algae; to colonize and grow. These opportunistic species may grow in place of more complex marine organisms that comprise the climax community, as climax species, such as sponges and mussels, take longer to establish [4]. Transitions between these ecological communities take time with heterogeneous complex secondary pelagic algal communities generally replacing homogenous primary benthic communities, accompanying other climax species. [13,14] Locations with a larger ecological diversity are considered “healthier” because there is not one dominant species. As a result, algal diversity and abundance are important marine health indicator and can provide insight into how urbanization is impacting marine ecosystems on a larger scale.

Studies have attempted to analyze the ecological impacts of increased urbanization on short-term algal growth, particularly on sea walls and in artificial reefs. In Singapore algae was found to be a primary successor after ecological disturbance. [15] Additionally, Biofilms and green turf algae (filamentous algae) were found to precede species of red and brown algae after a period of time indicating that infrastructure age has an impact on species diversity and composition [15]. In Guam, algae was also recorded as a primary ecological successor [16]. In artificial reefs, filamentous algae is used to attract herbivorous fish and further coral growth, insinuating that there is a fundamental purpose for algae in the succession of climax species [17]. There have been few studies that have assessed algal dominance on a larger scale to determine if it is temporary. However, very little research has been done in the Bocas del Toro province to understand the posed theory that surrounding waters that are high in nutrients are in fact causing detrimental rapid algal growth. Due to the intense urbanization of Bocas Town, nutrient rich waters and increased infrastructure would increase al-

Fig. 2: Average percent algal cover between ten sites within the Bocas del Toro archipelago.

Table 2. ANOVA test p-values for percent cover of algae on pilings in urban, rural and abandoned sites when compared against each other.

P-values that were significant are bolded.

Fig. 1. Study sites within the Bocas del Toro archipelago of Panama. Rural sites are marked in pink and urban are green.

ing percent cover on old and new pilings were not normally distributed and not significantly different. An ANOVA revealed the 10 sites were significant against each other (p<0.0001) (F=59.17, df=274, N=275). Tukey tests showed that not all sites when paired together were significantly different, however 33 of the pairings yielded significant values. The abandoned pilings on Carenero (95.4% ± 0.33 ) had the largest average percent algae cover and Caribbean Nature (11.5% ± 0.32) the least (Fig. 2).

Further ANOVA analysis comparing urban, rural, and abandoned pilings indicated sites with these characteristics were significantly dif28 JUST VOL IV // ISSUE II // SPRING 2019

Fig. 3: Average percent algal cover on pilings in urban, rural, and abandoned sites. The error bar represents standard error.

DISCUSSION

While nutrient rich waters in highly urbanized areas, particularly around seawalls and docks, can provide breeding grounds for rapid and prolific algal growth [9,15], further investigation reveals there are multiple factors that influence this growth. The abandoned pilings on Isla Carenero had the largest average percent cover, with multiple pilings being covered in 100% algae. It was noted that the water in this area was calm with few waves, which may have contributed to increased algal abundance. Areas

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Table 1. Location of study sites within the archipelago of Bocas del Toro, Panama with their selected attributes indicated in parentheses.

ferent (p<0.001). Rural sites (56.3% ± 2.9) had a significantly higher average percent algal cover than both urban (36.2% ± 2.4) and abandoned pilings (40.0% ± 5.7) (Fig. 3). Urban sites were found to be significant against both rural (p<0.001) and abandoned sites (p = 0.05). A t-test revealed there was no significant difference in algal cover between old (41.2 % ± 2.6) and new (48.1% ± 2.7) dock pilings (p = 0.1).

Overall, 21 visually identifiable algal species were observed. Hostel Mamallena (urban, new) had the highest algal species diversity (n=9) and Blue Coconut (rural, old) had the lowest (n=2). There was no observable difference in average algae species composition between urban (5.00 ± 1.47 spp.), rural (4.25 ± 1.03), and abandoned (4.5 ± 1.50) settings. New pilings (5.75 ± 1.44 spp.) had a slightly higher average than older pilings (3.83 ± 0.60). The algal groups that were seen in the highest frequencies were: Chondrophycus papillosus (3 sites), Caulerpa sertularioides (4 sites), and crustose coralline algae of the order Corallinales (4 sites) and a filamentous brown algae of the class Phaeophyceae (10 sites) that grew on sponges. All other native species were identified only once or twice during the study. Species identification was limited by accurate visual identification.

with greater wave-exposure are shown to have an increased density of algal grazers in an area [20].

Lack of grazers in the area may have contributed to extreme algae abundance [21]. However, there are a number of factors such as the agricultural and commercial history of the site, its proximity to Bocas Town, and the health of nearby mangroves that could also be contributing to these results and should be studied further.

Caribbean Nature, one of the most remote and untouched sites visited during this study, yielded the least amount of algal cover across the 10 sites. This site is approximately 14 km away from Bocas Town, limiting nutrient runoff, and has been abandoned for approximately four years. Bordering this area are dense mangroves, which provide and protect fish breeding grounds and juvenile nurseries [22]. This rich neighboring ecosystem and fish habitat partnered with its distance from town may have led to the low levels of algae on the pilings at this site.

Contrary to the initial hypothesis and previous research, urban sites were shown to have a lower percentage of algae cover than rural sites [2,3] This was particularly interesting as most research indicates urban centers have nutrient rich waters with high nitrogen and phosphorus levels [9]. These results may have been influenced by indigenous communities living near rural areas. Often, indigenous communities are not able to safely treat or manage waste or agricultural runoff, leading to nutrient rich waters in the surrounding area. The results may also be explained by the relationship between algae and other organisms such as sponges. In many of the urban sites observed, sponges were more dominant compared to algae or were competing with them for space. Many interactions between algae and sponge species have been observed to be mutualistic; however, the nature of these specific interactions and the role sponges take in algal community growth are unknown [23]. It would be beneficial to assess the relationship between sponge and algal growth particularly in an urbanized setting in the future.

While previous research shows that algae is one of the first building blocks of a growing ecosystem [16], no significant difference was observed between old and new pilings in this study.

Newer pilings had a higher average algal cover by only 6.9%. These data insinuate that while algae may be dominant on these dock pilings, there are also other organisms that are part of this artificial habitat, even if there is no clear succession rate. Newer sites would be expected to have increased algal abundance because of the lack of climax species and relatively nutrient-rich waters from buildings on the coast. How-

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crucial primary producer and has a complex relationship with other organisms within an ecosystem. As seen in this study, algae is able remain at a stable population size in urbanized areas and near dock pilings. The increased tourism and urbanization in Bocas, while pressing, has also seemingly allowed new and diverse growth for algae and sponges. This paves the way for other climax species to inhabit these new artificial ecosystems. However, it is still important to monitor waste and natural runoff within the archipelago to sustain this balanced algal ecosystem. While levels are stable now and urbanization does not seem to immediately be causing harm, unhealthy algal growth can still occur. In the future, it would be beneficial to assess the relationship between algae, sponges, and other climax species within the archipelago, and the impact of different piling materials, such as wood, on algae, and the nutrient content within rural areas of the archipelago.

ACKNOWLEDGEMENTS

I would like to thank Professor Carolyn Kovacs for her mentorship and guidance over the course of this study as well as the School for Field Studies in Bocas del Toro, Panama for materials and financial support. I thank R. Hill, B. Torjman, M. Correiro, L. Hamar, E. Scott, K.Sisson, and S. Martinez for their assistance in field work, and I would also like to thank my home institution, the University of Wisconsin-Madison, for their support in my study abroad.

REFERENCES [1] DESA, UN. World population projected to reach 9.7 billion by 2050. 2015. http://www. un.org/en/development/desa/news/population/2015-report.html [2] Bulleri, F., Chapman, M. G. The introduction of coastal infrastructure as a driver of change in marine environments. Journal of Applied Ecology. 2010; 47(1): 26-35.

[3] Connell, S. D. Urban structures as marine habitats: an experimental comparison of the composition and abundance of subtidal epibiota among pilings, pontoons and rocky reefs.

Marine Environmental Research. 2001; 52(2): 115-125.

[4] Airoldi, L., Bulleri, F. Anthropogenic disturbance can determine the magnitude of opportunistic species responses on marine urban infrastructures. PLoS One. 2011; 6(8): e22985.

[5] McKinney, M. L. Effects of urbanization on species richness: a review of plants and animals. Urban Ecosyst. 2008; 11(2): 161-176.

[6] Carruthers, T. J. B., Barnes, P. A., Jacome, G., Fourqurean, J. W. (2005) Lagoon scale processes in a coastally influenced Caribbean system: implications for the seagrass Thalassia testudinum. Caribbean Journal of Science. 2005; 41(3): 441-455.

[7] D'Croz, L., Rosario, J. B. D., Gondola, P. The effect of fresh water runoff on the distribution of dissolved inorganic nutrients and plankton in the Bocas del Toro Archipelago, Caribbean Panama. Caribb. J. Sci. 2005; 4(3): 414-429.

[8] Altieri, A. H., Harrison, S. B., Seemann, J., Collin, R., Diaz, R. J., Knowlton, N. Tropical dead zones and mass mortalities on coral reefs.

Proceedings of the National Academy of Sciences. 2017; 114(14): 3660-3665.

[9] Anderson, D. M., Glibert, P. M., Burkholder, J. M. Harmful algal blooms and eutrophication: nutrient sources, composition, and consequences. Estuaries Coast. 2002; 25(4): 704-726.

[10] Humann, P., Deloach, N., Wilk, L. Reef Coral Identification: Florida, Caribbean, Bahamas.

2002.

[11] Waller, M. E., Bramburger, A. J., Cumming, B. F. Bi-weekly changes in phytoplankton abundance in 25 tributaries of Lake St. Francis, Canada: evaluating the occurrence of nui-

sance and harmful algae. J. Great Lakes Res. 2016; 42(5): 1049-1059.

[12] Howell, E. T. Cladophora (green algae) and dreissenid mussels over a nutrient loading gradient on the north shore of Lake Ontario. J. Great Lakes Res. 2017; 44: 86-104.

[13] McGowan, S., Leavitt, P.R., Hal,l R.I., Anderson, N.J., Jeppesen, E., Odgaard, B.V. Controls of algal abundance and community composition during ecosystem state change.

Ecology. 2005; (8):2200-11.

[14] Williams, D.D., Tavares-Cromar, A., Coleman, J.R., Kushner, D.J., Happey-Wood, C.M. Colonization dynamics of algae in small artificial ponds. Canadian Journal of Botany.

1994;72(11):1654-65.

[15] Loke, L. H., Liao, L. M., Bouma, T. J., Todd, P. A. Succession of seawall algal communities on artificial substrates. Raffles Bull. Zool.

2016; 32: 1-10.

[16] Tsuda, R. T., Kami, H. T. Algal succession on artificial reefs in a marine lagoon environment in Guam. Journal of Phycology. 1973; 9(3): 260264.

[17] Clark, S., Edwards, A. J. An evaluation of artificial reef structures as tools for marine habitat rehabilitation in the Maldives. Aquat Conserv. 1999; 9(1): 5-21.

[18] STRI. Training in Tropical Taxonomy, Tropical Field Phycology Workshop. Smithsonian Tropical Research Institute. 2008; 1-52.

[19] NSF. NSF Pan-American Advanced Studies Institute, Advanced Methods in Tropical Phcology, National Science Foundation. 2009; 1-71.

[20] Leonard, G. H., Levine, J. M., Schmidt, P. R., Bertness, M. D. Flow-driven variation in intertidal community structure in a marine estuary.

Ecology. 1998; 79(4): 1395-1411.

[21] Williams I, Polunin N. Large-scale associations between macroalgal cover and grazer biomass on mid-depth reefs in the Caribbean.

Coral reefs. 2001 May 1;19(4):358-66.

[22] Mumby, P. J. et al. Mangroves enhance the biomass of coral reef fish communities in the Caribbean. Nature. 2004; 427(6974): 533.

[23] Ávila, E., Riosmena-Rodríguez, R., Hinojosa-Arango, G. Sponge–rhodolith interactions in a subtropical estuarine system. Helgol Mar Res. 2013; 67(2): 349. ●

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ever, the contrast between this study and previous research may be due to site outliers. The abandoned pilings on Carenero were recorded as having the highest algal abundance while Aqua Lounge and Caribbean Nature were recorded as having the least algal abundance between sites, but all were considered “old” sites. The extreme nature of these three sites within one category may have led to skewed results. This subject should be studied further at more sites around the archipelago as a larger sample size would limit the effect of outliers.

Algae diversity observations revealed newer sites had on average a more diverse array of algal species than older sites; however, this was only true of three of the four new sites studied. Species common to Bocas del Toro such as C.papillosus, C. sertularioides, crustose coralline algae (Rhodophyta) and a filamentous brown algae (Phaeophyta) that grows on sponges were consistently seen across many sites throughout the archipelago. Although there was some consistency among sites, many species were seen only once or twice during the study. This indicates that while there is high algal diversity in the Bocas del Toro archipelago, there is an unclear pattern of diversity between different pilings. This point would be supported by further research within the archipelago as algae identification and thorough examination at each site requires more observation time.

In summary, these results illustrate that while algal blooms can be of ecological concern, as seen in past studies, macroalgae remains a

Improving Clinical Training and Electrocardiograms Analysis Through Adaptive Artificial Intelligence Peter Rehani, Dr. Joshua Medow, Dr. Scott Hagen, Dr. John Webster, Quan Chen ABSTRACT Computerized signal analysis and interpretation have long been lauded as the next generation of medical equipment technology. However, none have looked to understand key points where machines are able to detect features that humans cannot and improve the process. Here, we attempt to develop a method to understand key problem areas within medical signals and scans that lead to misdiagnosis for training clinicians. Initial attempts are being designed with de-identified Electrocardiogram (ECG) signals: the base structure employs an 'ideal' student through a Hybrid Convolutional Neural Network (CNN). This CNN dynamically selects the highest accuracy artificial learning technique to provide the best response both for classification of the signal in question and for the student's input data. From there, immediate visual feedback is provided to the student and metadata summaries are made available to professors and teachers, systematically creating a pro le of common misconceptions with associations to clinically relevant signal features.

One of the largest fields of focus within Artificial Intelligence (AI) today is the application to medicine: the expansive datasets provide an ideal setting to train and create the next generation of tools to help clinicians. Signal analysis has been of interest to the field of medicine since the first attempts at Electrocardiogram (ECG) signal interpretations were made in 1959 [1].

Now common place within the field, most modern ECG machines are able to provide some degree of classification given a patient data set with the latest generations of smart watches offering Arrhythmia, or irregular rhythm detection [2]. Artificial intelligence as a field emerged at a similar time point with the foundation of the first computational model for intelligent systems arriving in the 1950s [3]. Michael A. Millenson, author of Demanding Medical Excellence: Doctors and Accountability in the Information Age, describes his interview with Homer Warner, father of the Health Evaluation through Logical Processing (or HELP) clinical monitor [4]. Warner explains that the system was primarily aimed to help physicians choose the optimal treatment method through accurate diagnosis, but was largely limited by the computational power available at the time. Today, a similar goal within the field remains as AI attempts to become the next stage of this revolution. Like HELP, the proposed tools 32 JUST VOL IV // ISSUE II // SPRING 2019

aim to supplement and aid the instructor and practitioner rather than replace. Allowing a doctor to provide more hands-on treatment vastly improves the quality of care. In areas where there is lower access to formally-trained medical care professionals, tools like these would be invaluable to 1-ensure that students and doctors in emerging countries are able to access a global standard of medical education and 2-reducing the stress placed on resources, improving efficiency within poverty stricken areas throughout the United States. In light of the extensive series of hospital closures, lower income areas are facing all-time highs in shortages of primary care staff , and in turn, increased rates of premature mortality{a study by Cheng et. al. in 2012 demonstrated a significant link be-tween socioeconomic status and life expectancy [5] While the issue of healthcare access inequalities cannot be solved by a simple computer algorithm, steps like these are the first toward a more comprehensive overhaul of systematic dispensaries in quality of care access across socioeconomic strata.

The original inspiration for this project came from the Stanford Machine Learning Group: in their landmark Nature Medicine publication, they describe cardiac arrhythmia detection and diagnosis at parity with board certified cardiologists [6]. Thus far, we have worked to adapt their implementation and devise an improved method of machine learning that seeks to continually im-

Atrial Fibrillation/A-B: Mis-coordination of atrial and ventricular contractions; often recognizable by irregular QRS intervals within the ECG. Atrial Flutter: Rapid atrial beats with a regular pattern; characterized by regular, rapid R-R intervals within the ECG. Supraventricular Tachycardia/SVT: Electrical impulse beginning at the AV node instead of the Sinus node; typically presents with a faster rate (>150 BPM) with merging of the T and P waves.

Additionally, as a means of differentiating SVT from other forms of tachycardia, narrow to wide QRS complex morphology is studied for constant rate (a consistent indicator of SVT specifically).

Ventricular Tachycardia/VT: Often found in heart attack patients complexities, VT is characterized by rapid beating of the ventricles. Specifically, in the presence of AV dissociation and fusion beats, VT is clearly differentiated from SVT. Bradycardia/AV Block: Typically characterized into 3 degrees; characteristically slow rate:

1st Degree: Delayed conductance with PR

interval of greater than 200msec

2nd Degree: T1 and T2 subtypes; main point of differentiation comes in the PR interval changes:

T1: With T1, there is progressive PR lengthening and a characteristically nar

row QRS that eventually drops out.

T2: T2 involves a constant PR interval with sudden non-conductance of the p-wave and changes within the r-wave leading to an eventual drop-off.

3rd Degree: Characterized by a lack of p-wave and lack of synchronization be tween atrial and ventricular impulses.

Premature Atrial Contraction/PAC: Early atrial ring leads to abnormal R-R intervals and merging of the P and T waves.

Premature Ventricular Contraction/PVC: Similarly to PAC, PVC is characterized by early ring or contraction of the ventricles leading to drops of the T wave.

METHODS

The specific diseases of interest are outlined above. We have worked to improve detection by creating classification of components of a traditional ECG and then comparing abnormalities based on deviation from expected normal morphology to create predictive diagnoses. Python 3.6.7 has been used as the development environment for this work through an Anaconda package manager. Data collection has primarily utilized publicly available single lead ECG signal databases as a means of training the data. The nature of the project is therefore retrospective, and relies on training the model prior to inputting student data. A standard testing suite has been developed to train the CNN, and records are pulled from the Physiobank MIMC Databases which stands in full compliance with the Health Insurance Portability and Accountability Act (HIPPA) [9][10]. Additional independent cross validation will be performed through the Zio Monitor from iRhythym Technologies as presented by Hannun et. al. [6]. Annotations are provided with both training data sets, and serve as testing diagnoses for cross validation with board certified medical professionals. Tensor ow and Docker are being used as a means of creating and serving the ideal student model: both are being developed and implemented per Google's Tensor ow API [11]. Keras, a more streamlined version of Tensor ow, was not used as more granularity in model development was needed. Additionally, the model will be stored and trained on a Dell XPS 13 and then presented on a Samsung Galaxy Tab A Tablet running Android Pie for the student to input data. A visual description of the pipeline is available in Figure 1 (attached). Metadata calculations will occur by creating a cumulative score JUST VOL IV // ISSUE II // SPRING 2019

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INTRODUCTION

prove itself without human intervention as performed through what we are calling a Hybrid Convolutional Neural Network (CNN). A CNN can be thought of as taking in an input image or dataset, passing it through a series of layers that calculate various parameters about the features of the image, and finally producing a classification of what it may be. After this, an optimizer is used to try to minimize the error from the predicted to the actual classification [7]. This utilizes programming of ECG interpretations that are made through an implementation of Dubin's Rapid Interpretation of EKG's, a staple of modern medical education [8]. Certain metrics and analysis techniques inspired by real world diagnostic procedures have been implemented to enhance the accuracy of the model {a more in depth description of these techniques will be provided within the Methods section below. To brie y review common diagnoses and specify the scope of this project, the following common rhythm abnormalities have been of interest, with descriptions and diagnoses pulled from Durbin [8]:

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ure 2 (attached). The feedback is generated using the composite score from the aforementioned grading methodology. Over time, student's diagnostic procedures are pro led, allowing the system to be more personalized with continued usage and tailoring the feedback over time. To prevent abuse of the system in a guess-and-check fashion, the option exists for the instructor to limit the number of guesses and ensure that there is not an extreme point of wild min-max attempts. The system is able to detect the average number of guesses and tracks previous histories for similar diagnoses which allows for personalized feedback by tolerance adjustments. If a student is consistently under/over-volting, the algorithm encourages approximations closer to the correct value by adjusting slider tolerances and slowing down the indicator movement in the problem direction. A further explanation of this is provided in Figure 2.

Finally, if a value is entered that produces a redag in a clinical setting, we provide instructors the option to create an alert when a value like this is entered, making the response and ability to recognize these fatal mistakes far quicker.

RESULTS

The results of each algorithm compared against the aforementioned disease states are summarized below in Table 1. Specifically, Table 1 depicts the true positive rate, or percentage of correctly identified signals. Annotations were standardized and divided into the aforementioned testing criteria.

point for the given disease in question; however, given the structure of our proposed model, the best performing algorithm for a particular disease is used for diagnosis in the ideal model. Common problem areas in SVT and VT are similar to those seen in Hannun et. al. [6] were observed here: specifically of interest were the cardiologist diagnostic metrics observed, where board certified cardiologists were only able to attain an accurate diagnosis rate of 0.451 and 0.566 respectively. The CNN implemented in the aforementioned publication 0.488 and 0.541 respectively. Thus, there remains a significant degree of area to be covered in order to avoid misdiagnosis and improve detection in both of these common trouble areas. Other metrics were able to achieve a similar degree of accuracy to Hannun et. al. suggesting limited impact from the change of optimizer from the Adam's optimizer implemented by Hannun et. al. to the proprietary SGD optimizer implemented here.

Additionally, the criteria for selecting a disease state was placed at a stringent enough level to avoid a high false negative rate. This led to a true negative rate of greater than 95 percent across the board, which was desirable to avoid training the model to incorrectly classify data. From this, we can work to improve the metric of correctly identifying disease signals without compromise.

ACKNOWLEDGMENTS

Funding for our hardware has been purchased in part through a grant provided by the Doris Duke program from serving as a mentor in their 2018 summer program. Additional continued financial support has been generously provided by the Medow Laboratory in the Department of Neurosurgery at the University of Wisconsin, School of Medicine and Public Health. The primary author would like to extend a large thanks to the entire Medow Lab team for their encour-

agement and patience in teaching, development, and research. Special thanks to Dr. Medow himself for his experiences with problem formulation and solving in both Neurosurgery and machine learning, Dr. Hagan for providing his vast relevant clinical experiences in Cardiology, and Dr. Webster for his expertise in a celebrated career in Biomedical Engineering. Additional thanks to Quan, Zhe, and Chenxiao for their help and sanity checks on programming issues that arose, and a final extension of gratitude to Dr. David Page, Dr. Jerry Zhu, and the Doris Duke team for all that they do.

CONCLUSION

The model proposed in this paper is the tip of a vast array of options that remain to be explored. ECG diagnosis and analysis is one component of a comprehensive medical toolbox, especially with regard to data analysis. The eventual end goal for this system is to improve how instructors teach and streamline education next generation of doctors around the world. Clinically relevant medical training through the adaptation and application of AI within the field can serve as an aid in many under-served areas. As the project continues to grow and advance, we aim to reduce the burden placed on communities by increasing access to quality healthcare training. We hope that the exibility of the proposed model will allow it to be broadly applicable in multiple fields and easily expanded with advancements in medicine, technology, and their combination. Future focuses will also include utilizing the complexity of medical imaging data like CT, PET, and MRI scans for neurodegenerative disease research (including Alzheimer's, Parkinson's, and Schizophrenia). Ultimately, we aim to create a multifaceted pipeline that is ever expanding, with a dynamic approach to add more functionality down the line.

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based on the total accuracy of the diagnosis {this score is calculated as a net variance difference from the correct diagnosis to the reference image for the diagnosis input. All comparisons are made within the Fourier domain to reduce artifact interference and ensure accurate calculations are made. A proprietary Stochastic Gradient Descent Optimizer (SGD) is then deployed that attempts to minimize the variance across various machine learning methods. SGD was given preference over the faster optimizer options available, such as the Adam's optimizer deployed by Hunan et. al.

[6], due to its ability to avoid overstepping correct values and iterative approaches to ideal values.

The selection criteria for the machine learning algorithm is focused on applicability to the data set: the following methods have been implemented: Support Vector Machine (SVM), K nearest Neighbor Classification (KNNC), Random Forest (RF), Grid Search (GS). The method with the best performance for a given student is then selected using a neuro-evolution style process, and in turn advances. This differs again from the Adam's optimizer used by Hannun et. al. [6] as it will never overshoot the correct value and iteratively select enhanced detection/classification techniques over time, increasing efficiency dynamically [7]. The CNN then attempts to correlate the features detected by the ideal model to student responses.

Previous studies have found similar mistakes can be made between clinicians and trained models, so extra care has been taken to improve commonly missed signals (specifically differentiating SVT and VT) [6]. A series of metrics for this edge case have been developed that focus on the following: P wave morphology, QRS shape relative to rate (increasing spread is indicative of SVT), QRS duration (longer QRS typically indicates VT), Branch Bundle Block (BBB, often indicates SVT, especially with narrow initial complexes), fusion beats/AV dissociation (indicative of VT), and finally concordance between leads. These are often described as the gold standard for differentiating the two diagnoses, and despite being time consuming for non-experienced practitioners, a computer is able to make these comparisons within a matter of milliseconds. As with all applications of AI to a particular data set, cross validation is used as the best metric to prevent overfitting. Thus, within the testing dataset, we create a hybrid suite of the MIMIC-III and iRhythym signals to increase the cross application of the model. From this, a random selection of signals comprising between 20-30 percent of the total available is selected to train the model and the remainder is then used as at testing metric. Finally, a series of sliders provide immediate visual feedback to the students and allows them to slowly correct as seen in Fig-

Table 1: True Positive Classification Rate vs. Provided Diagnosis

DISCUSSION

As seen, the model shows promise for initial testing datasets. There is some degree of discrepancy between the optimal model at every JUST VOL IV // ISSUE II // SPRING 2019

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FIGURES AND TABLES

RATE Lower than goal

Goal

Higher than goal

ATRIAL OUTPUT Lower than goal

Goal

Higher than goal

VENTRICULAR OUTPUT Lower than goal

Goal

Higher than goal

OVERALL Closer to goal

Further from goal

Figure 2: Slider Feedback. A visual of the Android based visual feedback. A dynamic example can be found here: https://uwprod-my.sharepoint. com/personal/prehani_wisc_edu/Documents/test.avi. Multiple revisions have taken place; however, the general sentiment remains that there are bidirectional gradients for each of the parameters. In an example case, we could have a 6 month old patient with an AV canal repair. We understand that the cardiac output is low, leading to atrial capture at 4 mA and ventricular capture at 6 mA. An instructor may wish to set a certain requirement threshold for detection, which could be set in the app, say adding 5-10 mA to the minimum capture to ensure that the system has a degree of buffer. Finally, if a student routinely sets the capture values too low, the system will dynamically set the sliders to move more slowly to the correct direction, thus encouraging them to increase the capture value by higher increments.

Figure 1: A visual of the pipeline described. Clinical data is entered and then is displayed to the student as well as processed through the neural network.

After receiving the associated diagnosis, the pipeline takes in the student's response and delivers feedback to the student. Signal features are correlated to clinically relevant presentations, and a better assessment of common trouble areas can be made. As the student goes through more testing sets, the model pro les their performance and further adapts the feedback to target repeated problem areas. Additionally, the neuro-evolutionary feature of the model dynamically adapts the best strategy to detect the input patterns over time.

36 JUST VOL IV // ISSUE II // SPRING 2019

[1] L. Taback, E. Marden, H.l. Mason, H.v. Pipberger. Digital recording of electrocardiographic data for analysis by a digital computer. IRE Transactions on Medical Electronics (Volume: ME-6, Issue: 3), September, 1959. DOI: 10.1109/IRET-ME.1959.5007946.

[2] Apple Inc. Using Apple Watch for Arrhythmia Detection. December 2018.

[3] A. M. Turing. Computing Machinery and Intelligence. Mind 49:433-360. 1950.

[4] M. L. Millenson. Demanding Medical Excellence: Doctors and Accountability in the Informa-tion Age. University of Chicago Press, Chicago Illinois, 2000.

[5] Cheng ER, Kindig DA. Disparities in premature mortality between high- and low income US counties. Prev Chronic Dis 2012;9:110120.

DOI: http://dx.doi.org/10.5888/pcd9.110120 [6] A. Hannun, P. Rajpurkar, M. Haghpanahi, G. H. Tison, C. Bourn, M. P. Turakhia, A. Y. Ng. Cardiologist-level arrhythmia detection and classi cation in ambulatory electrocardiograms using a deep neural network Nature Medicine, January, 2019.

[7] I. Goodfellow, Y. Bengio, A. Courville. Deep Learning. The MIT Press, Cambridge, Mas-sachusetts, 2016.

[8] D. Dubin, MD. Rapid Interpretation of EKG's.

Cover Publishing Company, Tampa, Florida, 1984.

[9] Johnson AEW, Pollard TJ, Shen L, Lehman L, Feng M, Ghassemi M, Moody B, Szolovits P, Celi LA, and Mark RG. MIMIC-III, a freely accessible critical care database. Scienti c Data (2016). DOI: 10.1038/sdata.2016.35.

[10] Goldberger AL, Amaral LAN, Glass L, Hausdor JM, Ivanov PCh, Mark RG, Mietus JE, Moody GB, Peng C-K, Stanley HE. PhysioBank, PhysioToolkit, and PhysioNet: Compo-nents of a New Research Resource for Complex Physiologic Signals. Circulation 101(23):e215-e220 [Circulation Electronic Pages; http://circ.ahajournals.org/cgi/content/ full/101/23/e215]; 2005.

[11] Mart n Abadi, Paul Barham, Jianmin Chen, Zhifeng Chen, Andy Davis, Je rey Dean, et. al. Google Brain. TensorFlow: A System for Large-Scale Machine Learning. Proceedings of the 12th USENIX Symposium on Operating Systems Design and Implementation (OSDI '16). ISBN 978-1-931971-33-1. â&#x2014;?

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