Volume V Issue I FALL 2019

JUST VOL V // ISSUE I // FALL 2019

LETTER FROM THE EDITOR-IN-CHIEF Dear Reader, I am thrilled to present to you with Volume V, Issue I 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, Associated Students of Madison 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.

UW-Madison's only undergraduate STEM research & communication journal

is RECRUITING for Spring 2020! editors | staff writers | designers and accepting submissions for:

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 start of my second 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,

research reports | editorials | photographs Helen Heo JUST Editor-in-Chief

www.justjournal.org | contact@justjournal.org JUST VOL V // ISSUE I // FALL 2019

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SPONSORS & PARTNERS EDITOR-IN-CHIEF Helen Heo MANAGING EDITORS Haley Dagenais Stephen Halada

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 Sciences [CALS]; The Wisconsin Institute for Discovery and the Associated Students of Madison (ASM) for financially supporting the production of JUST’s Spring 2019 issue. Thank you!

TABLE OF CONTENTS EDITORIALS From Detergent to Medicine...................6 Aadhshire Kasat

Green Roofs to Urban Heat Islands...........................................................9 Jackie Olson

On the Urban Beekeeping Trend...........................................................13 Anna Feldman

DIRECTOR OF FINANCE Aditya Singh

Right to Try....................................................................16

DIRECTOR OF MARKETING Tammy Zhong

Ryan Brown

DIRECTOR OF DESIGN Ashley Harris

PIXELS

WEBMASTER Cayman McKee

John Vandevender...........................................................................18

MARKETING ASSISTANT Jenny Lee

REPORTS

EDITORS OF CONTENT Ben Fordyce Haley Van Beek Jaitri Joshi Luke Zangl Mary Magnuson

Digging for Drugs in the Dirt......................................................................22 Sarah Uhm, Alayna Fender, Adriana Kotchkoski, Austin Trowbridge, Dr. Joshua D. Pultorak

Incidence Rate of Parkinsons Based on Gender............30

COPY EDITOR Mary Magnuson STAFF WRITERS Aadhishre Kasat, Head Staff Writer Aislen Kelly Ryan Brown Anna Feldman Parabhjot Singh

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Marie Bai 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.

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velopment of more efficient medicinal pathways. Conventionally, produc-tion of enantiomerically pure medicines with high yields was the most important criteria of drug development. Enantiomeric purity is the production of a certain substance in a specific configura-tion. This is a significant criterion because different configurations of the same product (enantio-mers) can have dramatically different biological effects in the physiological environments of human beings. Along with enantiomeric purity and high yields, today medicinal pathways are also de-signed to be more environmentally friendly and conscious of atom economy. Atom economy falls within the realm of green chemistry. A reaction is said to have high atom economy when most of the starting reagent is converted into the final desirable product. This is favorable because it mini-mizes wastes and consumption of resources. To meet these standards, companies use enzymes.

CHEMISTRY

This image depicts a man Pouring Liquid Laundry Detergent. Acquired from Shutter stock Image Library. Public Domain.

From Detergents to Medicines

Several chemical reactions in the human body result in the formation of synthons that are present in or serve as precursors for medicines. For instance, tryptamines (Fig 1.) are an important class of bioactive molecules

"I am the tiny capsule you take before eating icecream, or the solution you use to clean your contact lenses. Weekends after rough midterms, I help you remove wine stains from your favorite outfit.

Who am I?"

I

am the tiny capsule you take before eating ice-cream, or the solution you use to clean your contact lenses. Weekends after rough midterms, I help you remove wine stains from your favorite outfit. Who am I? Even though an average person uses several enzyme-containing products on a daily basis, a large portion of the population is still unaware of enzymes and their wide range of applica-tions.

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Enzymes are substances that catalyze biochemical reactions. They were traditionally known to have therapeutic applications and now have finally emerged in the field of modern medicine.

Today, research and development departments of pharmaceutical companies are becoming increas-ingly interested in the de-

that are present in serotonin and rizatriptan, both of which have medicinal use (Fig 2.). The synthetic production of tryptamine is a multistep reaction. However, in the body it can be produced in a one-step reaction by the enzyme tryptophan decarboxylase. The formation of tryp-tamine only serves as the starting point for the production of much more complex medicines. The synthetic production of those medicines, while being successful doesn’t

Enzymatic engineering has become possible because of our deepening knowledge of the genetic code. It is also a rapidly developing field because scientists are constantly developing newer tools for protein engineering and advancements in the screening process.

There are several advantages associated with the use of enzymes in synthetic transformations. The first step of biocatalysis in-volves the identification of an enzyme that performs the required transformation and results in the formation of the desired product. It is important to note that even before any engineering, enzymes are already great at performing their designated reaction. Which is to say they can already complete a certain transformation at a rate reasonable enough for life to exist.

They also complete the trans-formation in a non-toxic and efficient manner.

Enzymes are attractive molecules for the development of medicines because their activity and speci-ficity can be improved by making favorable mutations in the genetic sequence that encodes for the formation of the enzyme.

This is possible because changes in the DNA cause a change in the struc-ture and chemical composition of enzymes. These modifications can also make the enzyme more stable in its optimum reaction’s conditions, consequently increase its turnover numbers. The turno-ver number of an enzyme is the total number of reactions a certain enzyme can perform before be-coming non-functional. A higher turnover number advantageous because it is proportional to a higher rate of reaction. Engineered enzymes also have expanded substrate scope, which is benefi-cial characteristic in drug discovery, because it allows for the development of new medicines. This is possible because when a certain engineering enzyme catalyzes its specific transformation on newer and wider variety of substrates, a wider variety of products are also formed. These products can JUST VOL V // ISSUE I // FALL 2019

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EDITORIAL

EDITORIAL

By Aadhishre Kasat

conform to the principles of green chemistry, since it involves several steps. While the biocatalytic production of those medi-cines is green, it is also inefficient. This is because enzymes are highly specific molecules — they bind to a specific substrate and produce a very specific product. As a result of which a different enzyme would be required for every new modification. On the other hand, enzymatic engineering allows for a green and efficient production of the medicines because it enables a single enzyme to be used for all modifications of the pathway.

be further processed and used in the development of newer medicines with varying effects.

In addition, chemical complexity can also be added to products by using multi-enzyme cascades. Bio-catalysis can protect groups, thereby removing additional wasteful steps from chemical synthesis. Enzymology in medicine allows for applying biocatalysts in areas in which existing chemistry is underdeveloped or inefficient, such as C–H activation, reductive amination, and halogenation. The use of biocatalysts gives us access to previously restricted chemical spaces.

Despite the advances, there are several major obstacles we are yet to overcome. Enzyme engineer-ing is much faster than it was ten years ago, but changing 30-40 amino acids and screening tens of thousands of candidates still requires a large research team. Since the field is developing rapidly there are several engineering strategies that can be used for the identification and validation of en-zymatic variants. Which ones are the better strategies is still unclear.

References

ENVIRONMENT

McDonald, Allwin D., Lydia J. Perkins, and Andrew R. Buller. "Facile in vitro biocatalytic pro-duction of diverse tryptamines." ChemBioChem (2019).

Patel, Kartik R., Dhrubo Jyoti Sen, and Viraj P. Jatakiya. "Atom economy in drug synthesis is a playground of functional groups." Am. J. Adv. Drug Deliv 1 (2013): 73-83.

Devine, Paul N., et al. "Extending the application of biocatalysis to meet the challenges of drug de-velopment." Nature Reviews Chemistry 2.12 (2018): 409-421.

Schoemaker, Hans E., Daniel Mink, and Marcel G. Wubbolts. "Dispelling the myths--biocatalysis in industrial synthesis." Science 299.5613 (2003): 1694-1697.

School of Human Ecology Green Rooftop UW-Madison (Campus Planning & Landscape Architecture)

Green Roofs & Urban Heat Islands

By Jackie Olson

S This image depicts assorted pills. Acquired from Shutterstock Image Library. Public Domain.

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ustainable design centers around creating spaces that meet the needs of human use, support existing ecosystems and ensuring all life forms maintain the ability to satisfy their needs. During my studies in Landscape Architecture and my work at the Office of Sustainability, I have learned how we can create beautiful facilities through the principles of sustainable design, while maintaining the health of each site.

Urban development strains a variety of

EDITORIAL

EDITORIAL

"We can create beautiful facilities through the principles of sustainable design, while maintaining the health of each site." natural resources. Heavily industrialized societies create factories, buildings and other structures that release dangerous chemicals into our atmosphere, including greenhouse gases and pollutants. These structures also require significant energy extraction. By coupling development with sustainable design, we can mitigate the negative impacts of urban development.

Let’s take the example of a green roof.

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grasses, trees and other plants, which lowers the roof surface temperature and cools the surrounding air, while also improving stormwater management. Green roofs alleviate “urban heat islands,” which arise in areas where roads, buildings and other infrastructure replaces the natural ground. These “surfaces that were once permeable and moist” cause a heat influx in urban regions.

We want to foster healthy environments for ourselves and those around us. By doing so, we improve our overall condition and our interactions with each other and the natural world. Green roofs are often designed to be seen from the inside of the building. This allows people to interact with nature by introducing green space into the built environment. Studies have shown that humans who form connections to nature “benefit human physical and mental health and productivity, and reduce blood pressure and hospital stays” (EPA). In addition, green roofs provide comfortable spaces for workers and community members to relax, and feel at peace. We must keep our communities livable and enjoyable if we want to sustain the present and the future.

able environment by improving air quality in two ways: reducing the amount of energy needed to cool the building by reducing the emission of greenhouse gases through burning coal, and bearing plants that can filter toxic substances in our air.

Green roofs also provide structure for stormwater management. Conventional roofs typically use the same material throughout and do not have any features for stormwater retention, meaning water immediately runs off the roof and down the gutters, which can flood the surrounding area. Roof runoff eventually finds its way into a watershed, though it collects chemicals, particles and other materials on its way. Often, these materials harm the ecosystem.

For example, farms surround the downtown area of Madison, WI, which includes four lakes. When it rains, phosphorus from pesticides, manure, etc. trickles back into the lakes through runoff. These increased phosphorus levels disrupt food chains, allowing cyanobacteria (blue-green algae) to multiply in mass quantities.

These algal blooms harm animals living in the lake and any humans who swim during one of those times. Green roofs store stormwater in the plants’ root systems as well as act as a filter for the water that does run off.

How Green Roofs Work

Green roofs utilize a variety of materials, living and nonliving, to mitigate higher temperatures, provide cleaner air, hold and reduce stormwater runoff and provide visual relief. Green Roofs replace conventional roofs, which are commonly made of materials such as slate, rubber, and fiber cement.

Conventionally dark-colored roof materials absorb much of the heat radiation from sunlight. Green Roofs, in contrast, regulate a building’s internal temperature and lower air pollution. The physical structure of plants allows them to absorb heat energy from the sunlight which the roofing materials and surrounding air might otherwise absorb.

Since these plants reduce the temperature of the roof, the building consumes less energy.

The plants used on green roofs can also capture airborne pollutants and filter harmful gases. Green roofs allow the ecosystem to thrive and future generations to enter a liv10 JUST VOL V // ISSUE I // FALL 2019

Education Building

A great example of a green roof implementation exists right here at UW-Madison.

This vegetative rooftop sits on top of a parking lot behind the Education Building. This project addressed green infrastructure goals and met regulatory requirements (Campus Master Plan) by redeveloping an area on campus. The campus also wanted to reduce the environmental impact of stormwater runoff. As stated earlier, stormwater runoff contamination is a huge problem in Madison, WI. Here is an image of what the project looks like today.

Before this project, a parking lot occupied the space. By planting a green roof, the university improved the volume, rate and quality of stormwater runoff here. This site sits close to Lake Mendota, making it the perfect spot to reduce pollution. In addition, members of this university, including students, faculty, and

School of Human Ecology

Another example of a green roof on campus is the top of the School of Human Ecology Building, otherwise known as SoHE. This building underwent various renovations and expansions about a decade ago and was officially reopened to students and faculty in fall 2012. UW-Madison’s head landscape architect Gary Brown worked alongside Saiki Design firm to design the site. This green roof provides a unique view of Henry Mall and west campus, while also reducing the effect of urban heat islands, improving stormwater management and offering visual relief to the UW-Madison community. I find this location quite fitting for the roof, as the entire school is focused on Human Ecology and an increased exposure to green space has been scientifically proven to promote human health and well-being.

As seen in this photo, durable materials made to withstand time yet also contribute to the character of the gathering space make up the roof. Brown used “Ipe” decking on sections of the wood paneled floor, along with patterned squares of sedum plants along the perimeter of the roof and granite pavers throughout. Wooden benches and a pergola provide seating and a partially covered area to enjoy, while moveable tables and chairs allow for flexibility within the space. Native Wisconsin plants offer users the ability to engage with nature.

This green roof is very special to me and other Office of Sustainability student interns; we frequent this location to get a change of scenery and work outside, when the weather permits. I personally find this to be one of the best designed green roofs on campus in terms of flexible use, layout, orientation and plant choice/diversity. For those on campus who have not visited this site, I strongly encourage you to within the next month so you can reap the benefits this green roof so generously provides us.

Education Building Green Rooftop UW-Madison (2015 Campus Master Plan)

References: 1. Brown, Gary. “University of Wisconsin-Madison Green Infrastructure & Stormwater Management Plan.” UW-Madison Campus Planning & Landscape Architecture , 2010. Madison (WI): University of Wisconsin-Madison; [cited 2019 Sept 10]. Available from https://cpla.fpm.wisc.edu/wp-content/uploads/sites/20/2017/10/Appen-2-Green-Infrastructure-MP-2016-1019-low-res-1.pdf 2. “Heat Island Cooling Strategies.” EPA, Environmental Protection Agency, 12 Aug. 2016.

Madison (WI): University of Wisconsin-Madison; [cited 2019 Sept 10]. Available from https:// www.epa.gov/heat-islands/heat-island-cooling-strategies 3. “About Green Roofs.” Green Roofs for Healthy Cities. Madison (WI): University of Wisconsin-Madison; [cited 2019 Sept 10]. Available from https://greenroofs.org/about-green-roofs

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EDITORIAL

"A green roof is composed of a vegetative layer of shrubs, grasses, trees and other plants, which reduces the temperature of the roof surface and the surrounding air, and improves stormwater management."

other members of our Madison community use and adore the outdoor space at the Education Building. Students spend multiple hours per day in classrooms and libraries, so this Green Roof provides a space for students to enjoy nature, whether for academic or relaxation purposes.

ECOLOGY

Honey Bee, iStock Image Photo Library, Public Domain.

On the Urban Beekeeping TrendWhat is the Best Solution to the Pollinator Crisis?

"The role of bees in our diet expands far beyond producing honey; they pollinate plants which produce fruits, vegetables, and nuts we consume. "

O

ver the past few years, the slogan “Save the bees!” accompanied with sketches of bees and honeycombs has appeared on pins, tote bags, tee-shirts, etc. Despite this call to action, many people aren’t sure why bees are so important, what the threat to bees is, and how we can help save them.

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The role of bees in our diet expands far beyond producing honey. They pollinate plants which produce fruits, vegetables and nuts, which we consume. It is estimated that over 35% of the food we eat relies on pollinators including bees, as well as butterflies, birds, moths, wasps, beetles and bats, making them an essential part

as urban hives are kept as a hobby and by entrepreneurs who sell the honey and rent out the hives. [6] In Paris, pesticides have been prohibited throughout the city, and bee populations are monitored to ensure that there is enough pollen available to support them [7].

One article from The New York Times argued there are too many hives in Berlin, especially those belonging to people who don’t adequately care for them. Berlin has over 20 hives per square mile belonging to registered beekeepers, as well as an unknown number of hives kept by unregistered beekeepers. Some see beekeeping as an easy hobby that is beneficial for the environment, but do not invest proper time into caring for their hives. Berlin has “swarm catchers” who keep track of bees that leave their hives (presumably not well looked after) and create hives in other parts of the cities where they interfere with city life. Meanwhile, the rest of Germany has only about six hives per square mile. Brandenburg, the state surrounding Berlin, has less than three, according to Benedikt Polaczek, who teaches beekeeping at the Free University of Berlin, via The New York Times.

The ecosystems of cities with a large urban beekeeping culture such as Berlin may be suffering from having too many honeybees. Jonas Geldmann, a researcher at the University of Cambridge, told The New York Times “the honeybee is one of 20,000 species of bees” and in places with too many bees, they consume the nectar other wild insects depend on, harming local biodiversity.

In addition, Geldmann stated diseases spread quicker among managed species like the honeybee than among wild species. Furthermore, the domestic honeybee only pollinates 15% of the world’s food supply that requires pollination, while other wild pollinators pollinate 80% [8]. “The honeybee is not a native” to the United States, Williams said. Instead, it came from Italy, while “at least 200 species of wild bees pollinate” locally in the United States.

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EDITORIAL

EDITORIAL

By Anna Feldman

of our food system [1].

The honeybee population has been suf fering from colony collapse disorder (CCD) in the northern hemisphere, where honeybees have been disappearing from their hives. Potential causes for CCD include pesticides, various pathogens, parasites and habitat degradation from people clearing out natural landscapes for commercial use [2], as well as climate change and air pollution [3].

Prominent studies hypothesize that pesticides are the most detrimental factor of CCD [4 and 5]. Common crops such as wheat, corn, and sunflowers are commonly treated with pesticides that often contain neonicotinoids, a substance which studies have shown to be highly toxic to honeybees [4]. Williams says that neonicotinoids have a very significant (negative) effect on pollinators.

The pesticides diffuse through plant tissue and contaminate the nectar and pollen.

Honeybees and other pollinators collect the contaminated nectar and sometimes bring it back to the hive to store it, infecting the rest of the hive [2]. Current pesticide authorization procedures in many countries require testing whether the doses of pesticides in fields are below lethal levels for honeybees [2]. Even sublethal exposure to neonicotinoids is very harmful because it affects the memory of bees. The resulting brain damage interferes with their complex communication about where to gather pollen and nectar, therefore misleading other bees in their altered state, and inhibiting their ability to make it back to their hive [1].

In order to combat the bee population decline, campaigns promoted by environmental activist groups as well as the governments of some countries, such as Germany, have been encouraging people to keep honeybee hives, even those who live in cities [6]. In large metropolises such as Paris, Berlin, and New York, urban beekeeping has become very popular. Many people enjoy keeping urban beehives to feel more connected with nature in concrete jungles [4].

Classes on beekeeping have seen large turnouts

However, other pollinators do not receive as much attention as honeybees. “I found out gnats pollinate today!” Erik Iverson, the executive chief director of WARF (Wisconsin Alumni Research Foundation) who assisted Williams at the Wisconsin Science Festival, said at the festival. Many pollinator insects, birds, and bats face the same issues as the honeybee, but Williams said the focus of colony collapse disorder is only on one species. Therefore, Geldmann argued, increasing the population of only the honeybee may not be the best course of action. Instead, he proposed increasing pollinator plants in city parks and using less pesticides in them [6].

The countryside, on the other hand, could support more hives but it is not the best host for pollinators currently. Industrial farmers tend to have large fields of only one or very few different plants, and pollinators such as bees require a variety of plants to pollinate to be healthy. [4 and 5]. A 2012 USDA report said soybeans and corn account for over 50% of all cropland in the United States. Dr. Jon Lundgren, formerly a researcher at the USDA and now an organic farmer, said bee health in the countryside would improve with farmers minimizing pesticides and diversifying their crops” [4].

Williams similarly advised the public to “restore diversity in the landscapes through gardening” and “selectively garden to favor hummingbirds, other birds, bees” and other pollinators. He said while gardening in Wisconsin won’t restore the former wild oak savannah landscape, it is important to create a sustainable landscape now.

His goal is to “restore diversity for stabilizing the long-term.” Williams said it’s important to consider whether the emphasis should be on going back to native ecosystems or fulfilling specific objectives as they come up.

There is a variety of solutions to the pollinator crisis. Urban beekeeping, if the hives are properly taken care of, is an effective way to rejuvenate the dipping honeybee population. Additionally, while honeybees are an important pollinating concern, they are not the only endangered pollinators.

Reducing the use of pesticides, banning neonicotinoids, planting pollinator-friendly plants, creating more green spaces in cities and diversifying crops in the countryside are proactive steps to take to support wild pollinators and therefore sustain our food system for the future.

If you have access to some outdoor space, use it to plant some pollinator-friendly wildflowers — you’ll get a pretty view and support your local ecosystem!

A bat pollinating a flower

Madison resident Leonie Wieben creating smoke to make the bees drowsy in order to open her family’s beehives at their property outside of Spring Green, WI

References: 1. Natural Resources Conservation Service. (n.d.). Available from https://www.nrcs.usda. gov/wps/portal/nrcs/main/national/plantsanimals/pollinate

3. Leonard R. J., Pettit T. J., Irga P., Mcarthur C., & Hochuli D. F. Acute exposure to urban air pollution impairs olfactory learning and memory in honeybees. Ecotoxicology. 2019. doi: 10.1007/s10646-019-02081-7

4. Cameron, C. The Rise of the City Bee — How Urbanites Built the 21st ... 7 November 2017.

Available from https://daily.jstor.org/risecity-bee-urbanites-built-21st-century-apiculture/

7. Rubin, A. J. Paris Bees at Work from Notre-Dame to the Luxembourg Gardens. 24 August 2014. Available from https://www. nytimes.com/2018/08/24/world/europe/ bees-paris.html?action=click&module=RelatedCoverage&pgtype=Article®ion.

8. Prescott-Allen, R., & Prescott-Allen, C. How Many Plants Feed the World? Conservation Biology. 1990; 4: 365-374. doi: 10.1111/j.15231739.1990.tb00310.x

Honey bee, Pixabay image library, public domain

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EDITORIAL

2. Henry, M., Beguin, M., Requier, F., Rollin, O., Odoux, J.-F., Aupinel, P., … Decourtye, A. A Common Pesticide Decreases Foraging Success and Survival in Honey Bees. Science. 2012; 336: 349-350. doi: 10.1126/science.1215039

5. Pasquale G. D., Salignon M., Conte Y. L., Belzunces L. P., Decourtye A., Kretzschmar A., Alaux, C. Influence of Pollen Nutrition on Honey Bee Health: Do Pollen Quality and Diversity Matter? PLoS ONE. 2013; 8. doi: 10.1371/journal.pone.0072016 6. Schuetze, C. F., Karasz, P. Bees Swarm Berlin, Where Beekeeping Is Booming. 11 August 2019. Available from https://www.nytimes. com/2019/08/11/world/europe/berlin-beesswarm.html.

BIOETHICS

Pills. Creative commons.

Right to Try

By Ryan Brown

T

he drug approval process in the United States is particularly long and cumbersome. This is largely because of a push in the last 100 years to focus on evidence-based medicine and move away from “snake oil” therapies that dominated the early 1900s. Today, evidence-based medicine requires that every drug must go through a rigorous testing process before reaching market to ensure that patients cannot be fooled into taking ineffective therapies.

In order to create these safety and efficacy standards for each approved drug, a process has been developed that averages over 10 years to complete. This, in turn, creates new ethical dilemmas on how to efficiently get treatments to patients that have no available treatment options in the clinic. Such patients, often faced with no alternatives,

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may be interested in taking a higher-risk, unproven or partially-tested therapies that may prove a chance for better health outcomes. This has led to the commonly called “Right to Try” movement, spearheaded by libertarian groups such as the Goldwater Institute aiming to expand access of therapies for those who desire it. The movement has been wildly successful and resulted in a bill that was recently approved by Donald Trump in late 2018. What is particularly interesting about such Right to Try legislation is that the FDA already has an “expanded access pathway” that approves requests to use investigational therapies in cases where patients have no viable options.

This pathway approves 99.7 percent of all expanded access requests submitted for patients that have immediately

verse events occur. Small companies are especially concerned about this as it could possibly reduce the chance of an eventual FDA approval.

Additional concerns around these manufactures stem from the fact that companies would no longer need the FDA’s approval before charging for their therapies.

Furthermore, insurance companies may not be interested in covering unproven therapies along with the cost for delivery of that treatment. They also would not be required to cover for any costs associated with side effects that occur due to such unproven therapies [3]. All of this moves the entire burden of cost to lie upon the patient.

The broadening of expanded access programs could also reduce patients' interest in partaking in clinical trials. This is because they are aware that there is always a chance that they could get a placebo instead of an actual therapy. In order to avoid this, patients could use this new pathway to directly get access to a drug and avoid the chance that they don’t receive the treatment. This is problematic as it could possibly undermine the current research framework used for drug testing and approval.

Right to Try has remained politically popular, gaining the approval of over 40 states before federal regulation was introduced. However, it appears that the new law has done little to actually improve the already existing expanded access framework. Instead, it has brought up additional concerns while not identifying or addressing the underlying problems that exist when attempting to get patients in need innovate therapies.

References [1] Jarow, Jonathan P., et al. "Expanded access of investigational drugs: the experience of the Center of Drug Evaluation and Research over a 10-year period." Therapeutic innovation & regulatory science 50.6 (2016): 705-709.

[2] FDA https://www.fda.gov/downloads/AboutFDA/ ReportsManualsForms/Forms/UCM083533.pdf [3] Richardson, Elizabeth, “Health Policy Brief: Rightto-Tr y Laws,” Health Affairs, Updated April 9, 2015.

[4] Wong C.H., Siah K.W., and Lo A.W. (2018). Estimation of clinical trial success rates and related parameters.

Biostatistics. https://doi.org/10.1093/biostatistics/ kxx069 [5] Thomas D. W., Burns J., Audette J., Carrol A., Dow-Hygelund C., and Hay M. (2016). Clinical Development Success Rates 2006–2015. San Diego: Biomedtracker.

[6] Hay M., Thomas D. W., Craighead J. L., Economides C., and Rosenthal J. (2014). Clinical development success rates for investigational drugs. Nature Biotechnology 32, 40–51. https://doi.org/10.1038/nbt.2786 [7] "Examining Patient Access to Investigational Drugs."

October 03, 2017. http://docs.house.gov/meetings/IF/ IF14/20171003/106461/HHRG-115-IF14-Transcript20171003.pdf.

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EDITORIAL

EDITORIAL

"Right to Try has remained politically popular, gaining the approval of over 40 states before introducing federal regulation. But it appears that the new law has done little to actually improve the already existing expanded access framework."

life-threatening illnesses and cannot participate in clinical trials [1]. This new Right to Try legislation appears to remove the FDA from having to approve the use of such untested therapies outside the use of a clinical trial.

The benefits of such a pathway are exemplified by critics of the current expanded access pathway, who state that the process is too burdensome, discouraging individuals from applying. This is a valid concern as the FDA stated on their own forms that the process could take up to 100 hours to complete [2]. Furthermore, the form must be filled out either by the company providing the drug or the physician issuing it which may be another barrier especially considering the time needed for the form.

However, in 2015, the FDA created a new framework that significantly reduced this bureaucratic burden [3]. While this may have helped this framework, many believed that these various barriers still were too much of a burden for patients.

This debate also stems into a more philosophical one about freedom of choice. Many believe that patients should have complete autonomy to take risks within their own lives outside of the federal government’s involvement. On the other hand, opponents believe that these laws will undermine public safety and the medical research system at large. While it is still too early to determine these effects, these concerns can still be explored.

Therapies most commonly go through a three-phase process. Under the recent legislation of Right to Try, therapy requests can be made anytime after passing these Phase I trials [3]. Many opponents to Right to Try believe that this is too early for access as Phase I only tests in small groups of around 20-50 patients that are healthy and not in the drug’s targeted population. Furthermore, 66% of therapies pass Phase I trials, but most of these fail phase II or phase III trails for showing no indication of effectiveness. The chances of passing each phase varies for each drug type, for example cancer only gets 3% of all therapies approved, but vaccines have a success rate of 33% [4,5,6]. It is a concern that those looking to circumvent the system with no current options are likely to have low approval chances, especially during phase II or II trials.

Beyond ethical concerns of safety, many believe that this legislation does not actually address the real problem.

Current FDA Commissioner Dr. Scott Gottlieb stated that, “I think there is a perception, ...that there are certain companies and products that aren’t necessarily being offered under the current construct and the Right to Try legislation might provide more of an incentive and an opportunity. ...I don’t necessarily see that same opportunity because I think the biggest obstacle to offering drugs through expanded access is the supply constraints.” [7] Gottlieb recognizes that the FDA is lenient for patients with no options. The legislation implemented thus only pretends to solve the real problem. This is because the supply constraints reside with small, independent companies staffed by few individuals. It may be difficult for them to manufacture therapies for an increased number of patients. Furthermore, they may not be interested in providing the therapies altogether as they may be concerned about the additional liability associated when ad-

PIXELS

John Vandevender: A summer sunset over Lake Wabigoon, Ontario. As the sun dips below the horizon, blue light is dissipated.

Longer wavelengths of light remain, resulting in a sky full of red and orange hues.

where science and art collide

John Vandevender: A Grizzly Bear (Ursus arctos), cruising through a valley, stops to survey its surroundings. In 1975, the Grizzly Bear, with a Greater Yellowstone Ecosystem population in the low 100s, was designated a threatened species. Today, the population is over 700, making it a conservation success story.

John Vandevender: Honey bees (Apis mellifera) play an imperative role in the ecosystem.

From producing honey to pollinating numerous crops, humans would not last long without bees.

So the next time a bee is buzzing around your face, resist the urge to swat, and just say “thank you.�

John Vandevender: A mature Bighorn ram watches over the Badlands.

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While many studies have examined the effect of age on the incidence of Parkinson’s Disease (PD), very few have been carried out in attempts to understand differences in PD incidence due to biological sex of the population examined. In response to this deficit of research on the occurrence of PD with respect to the sex of patients, prior studies in the field were aggregated and analyzed to determine any difference in PD occurrence due to this particular demographic identifier. Google Scholar and PubMed were searched for studies on the topic. After filtering of eligible published works, findings from four studies were compiled and analyzed via an ANOVA test to determine if differences in PD incidence between males and females were significant. No significant difference was found. While there was no significant difference in PD occurrence between males and females, the trend towards higher PD incidence in men warrants further investigation with more controlled factors in order to draw a more definitive conclusion.

REPORT

The misuse of antibiotics selects for bacterial pathogens that are drug resistant, creating increased demand for new treatments. Soil is a prime environment to study for possible antibiotic-producing organisms due to its high concentration and diversity of microbial communities. Human activity such as agriculture, industrialization, and waste management cause environmental stress on soil microbes. This engenders enhanced interaction and competition between bacteria species, precipitating antibiotic production through increased horizontal gene transfer events and development of defense mechanisms. This study aims to differentiate the number of antibiotic-producing bacteria from areas with varying degrees of human activity- industrial, residential, and protected nature soil environments. Isolates from each location were screened against safe relatives of the multi-drug resistant ESKAPE pathogens. Any isolate that inhibited pathogen growth was characterized with differential testing and genetic sequencing. The data indicate that residential soil settings have the highest concentration of antibiotic-producing bacteria strains, supported by a p-value of 0.023.

The environmental stress in residential soil selects for the most competitive bacteria. These bacteria are more likely to produce antibiotics as a competitive defense mechanism. Further studies should be done focusing on what specific compounds or possible cell interactions lead to bacterial antibiotic production.

Incidence Rate of Parkinson’s Disease based on Gender.........................................30

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REPORTS

Digging for Drugs in Dirt: How Human Activity Impacts Soil Environments and Precipitates Antibiotic-Producing Bacteria.............................22

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Digging for Drugs in Dirt: How Human Activity Impacts Soil Environments and Precipitates Antibiotic-Producing Bacteria Sarah Uhm1, Alayna Fender2, Adriana Kotchkoski2, Austin Trowbridge2, Dr. Joshua D. Pultorak3 1Tiny Earth research group at the University of Wisconsin-Madison 2019 2Tiny Earth research group at the University of Wisconsin-Madison 2019 3Tiny Earth research group project coordinator at the University of Wisconsin-Madison 2019

ABSTRACT

The misuse of antibiotics selects for bacterial pathogens that are drug resistant, creating increased demand for new treatments. Soil is a prime environment to study for possible antibiotic-producing organisms due to its high concentration and diversity of microbial communities. Human activity such as agriculture, industrialization, and waste management cause environmental stress on soil microbes. This engenders enhanced interaction and competition between bacteria species, precipitating antibiotic production through increased horizontal gene transfer events and development of defense mechanisms. This study aims to differentiate the number of antibiotic-producing bacteria from areas with varying degrees of human activity- industrial, residential, and protected nature soil environments. Isolates from each location were screened against safe relatives of the multi-drug resistant ESKAPE pathogens. Any isolate that inhibited pathogen growth was characterized with differential testing and genetic sequencing. The data indicate that residential soil settings have the highest concentration of antibiotic-producing bacteria strains, supported by a p-value of 0.023. The environmental stress in residential soil likely selects for the most competitive bacteria. This antibiotic production may be a defense mechanism. Further studies should be done focusing on what specific compounds or possible cell interactions lead to bacterial antibiotic production.

Pathogenic bacteria such as pneumonia, tuberculosis, and gonorrhea are beginning to exhibit high degrees of antibiotic resistance, causing these previously treatable diseases to become untreatable [1]. Antibiotic resistance arises from genetic mutations in individual bacteria, which eventually replicate and precipitate an evolved population [1]. Resistance transmission occurs via horizontal gene transfer between bacteria.

But resistance spreads at a faster rate due to human activity such as the misuse of antibiotics, antibiotic use in pets and livestock, and antibiotic pollution of the environment [2]. For example, up to 90% of the antibiotics given to livestock are excreted and dispersed through fertilizer, groundwater, and surface runoff [3]. This leads to antibiotic pollution of the environment at a massive scale, which precipitates the spread of antibiotic resistance.

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Multidrug resistance, seen in bacteria such as the six ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species), further limits treatment options. The resistance exhibited by the ESKAPE pathogens pose a massive threat to public health as they are the leading cause of nosocomial infections in the world [4]. In the United States alone, two million people contract an antibiotic-resistant infection each year, and 23,000 of those people die [2].

This precipitates a high demand for novel antibiotics. Bioactive microorganisms are known to often produce antibiotic compounds [4]. Therefore, environments that encourage microbial bioactivity and diversity are ideal sources for new antibiotic production. A prime example of such an environment is soil.

A heightened level of antibiotic resistant bacteria is often accompanied by a high level of antibiotic-producing bacteria. This circumstance commonly occurs in residential soil. Extended use of fertilizing agents and the presence of animal feces in residential areas promotes horizontal gene transfer, which subsequently increases both antibiotic resistant bacteria and antibiotic-producing bacteria [5]. Plasmid-mediated antibiotic-producing genes are often transferred from one bacterium to another, allowing antibiotic resistant bacteria to coexist with antibiotic-producing bacteria as they continuously evolve to compete. Antibiotic resistant bacteria may also produce antibiotics against other bacteria [5]. This microbial competition throughout residential soil consequently leads to more bacterial antibiotic-production as a competitive defense mechanism.

This study aims to identify which soil environments best enhance the occurrence of antibiotic-producing bacteria. Soil was sampled from three different environments, industrial, residential, and protected nature. Industrial was defined as soil locations experiencing prolonged heavy metal presence. Residential was defined as suburban neighborhoods comprising primarily of houses and apartment complexes. Protected nature acted as a control and was defined as areas of nature that had been legally preserved for at least ten years. It was hypothesized that industrial and residential sites would yield the highest levels of antibiotic-production due to heightened environmental stress and bacterial interaction and competition.

METHODS Soil sampling, Culturing, and Master Plates

Industrial soil samples were collected from the UW-Madison Chemistry Building construction site and the Charter Street Heating and Cooling Plant. Residential soil samples were from the apartment complexes of College Station and Campus Village. Protected nature samples were from the Lakeshore Nature Preserve and Owen Conservation Park. All samples were collected within a five-mile radius on February 12th, 2019 at a soil depth of three inches. The air temperature during time of collection for each sample was -1.67°C. All samples were approximately ten milliliters and stored at 4°C.

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INTRODUCTION

One teaspoon of soil contains approximately 100 million to 1 billion bacteria [1]. Such an abundance of bacteria causes a high degree of interaction and competition, encouraging microbial bioactivity [5]. These bioactive compounds can be extremely effective antibiotics, as approximately 70-80% of antibiotics currently used in clinical settings are derived from soil isolates [4].

Human activity can also further enhance the bioactivity of soil bacteria. For example, environmental factors such as agricultural runoff and soil compaction alter competitive relationships within microbial communities [5].

Human activity increases interactions and competition between soil bacteria. The conversion of natural habitats into industrialized and residential locations causes soil compaction and nutrient degradation [6]. Compaction and nutrient degradation limit resources due to the reduction of available space, air, and water.

This forces soil bacteria to interact and compete more than usual. Heightened interspecific bacterial interaction often increases the possibility of horizontal gene transfer. This also increases the likelihood of antibiotic resistance genes being shared amongst microbial populations.

Conversely, more competition may prompt bacteria to produce antibiotics as a survival mechanism against other strains. These antibiotics act through competitive mechanisms such as inhibition of cell wall synthesis [7]. This phenomenon of bioactivity is more prevalent in areas that experience high levels of human activity, such as industrial and residential locations.

Soil pollution causes shifts in bacterial bioactivity. Industrial soil experiences higher levels of pollution, primarily from heavy metals.

Heavy metal exposure induces unique changes in bacterial genetic regulation [8]. For example, mercury pollution alters functional bacteria genes linked to methylation and reduction [8].

This selects for more competitive bacteria that are able to withstand high levels of environmental stress. These bacteria may also be more likely to produce antibiotics as a competitive defense mechanism. Heavy metal contamination of soil has also been linked to increased horizontal gene transfer events to disseminate genes that provide adaptations to the environmental stress [9].

Human activity therefore has a lasting impact on the bioactivity of soil microbial communities, which results in altered levels of antibiotic production.

One gram of soil was added to ten milliliters of sterile water, then the sample was serially diluted. Dilutions of 1:100, 1:1000, and 1:10000 were plated onto LB nutrient agar for each sample. These plates were incubated at 37°C for three days. Sixteen to twenty individual and isolated bacterial colonies were chosen from the serially diluted plates of each sample location and plated onto a new, master plate using sterile inoculation loops. These plates were then incubated as previously described.

Screen for isolate antibiotic production – Spread/Patch technique

The ESKAPE pathogens of focus were Klebsiella pneumonia, Staphylococcus aureus, and Acinetobacter baumannii. Escherichia coli, Staphylococcus epidermidis, and Acinetobacter baylyi were used for this experiment as they are the corresponding safe relatives of the three listed ESKAPE pathogens. These alternatives were used to prevent any possible exposure to the real ESKAPE pathogens, which have high levels of contagion and mortality. For every master plate, a new plate covered in each safe relative was created. The colonies from the master plates were patched onto the new plates in the same orientation via sterile inoculation loop.

The following procedures were carried out on isolates that exhibited a zone of inhibition against at least one of the three safe relatives of the tested ESKAPE pathogens. A zone of inhibition was defined as a clear region surrounding an isolate where the tested ESKAPE was unable to grow.

Standard PCR bead protocol according to the Tiny Earth Research Guide [1] was used to amplify the 16S rRNA segment of each antibiotic-producing isolate. The 16S rRNA gene was chosen as it has conserved regions, which allow it to be easily identified, and non-conserved regions, which is where species differentiation and genetic mutation is seen. The primers used were 27F and 1492R. Standard gel electrophoresis protocol according to the Tiny Earth Research Guide [1] was followed. The DNA segment of interest, the 16S rRNA gene, yields a band around 1500-bp. Standard DNA sequencing preparation 24 JUST VOL V // ISSUE I // FALL 2019

Characterization Tests

The antibiotic-producing isolates were further analyzed via MacConkey agar differential medium, motility tests, starch hydrolysis tests, and interactions with eukaryotic cells. The MacConkey agar differentiated between lactose fermenters and non-lactose fermenters. Standard MacConkey Agar protocol according to the Tiny Earth Research Guide was used [1]. Standard motility test protocol according to the Tiny Earth Research Guide was used to determine if the antibiotic-producing bacteria were motile or non-motile [1]. Standard starch test protocol according to the Tiny Earth Research Guide was used to determine if the bacterial isolate hydrolyzed starch from its environment or not [1].

All antibiotic-producing strains were co-cultured with eukaryotic cells. The eukaryotic cells used were two fungal isolates, Colletotrichum trifolii and Fusarium oxysporum. This determined if the isolate was antagonistic to eukaryotic cells, which would make it unsafe for human use.

Acinetobacter baylyi. The number of isolates that showed a zone of inhibition against at least one of these three pathogens are as follows, 12 out of 36 residential isolates, 6 out of 32 industrial isolates, and 2 out of 32 protected nature isolates (Fig. 1). A 2x3 Fisher Exact Test yielded a p-value of 0.023, indicating a statistically significant difference. The relationships between industrial/protected nature and industrial/ residential were not statistically significant, yielding p-values of 0.2565 and 0.270696 respectively. The relationship between residential and protected nature yielded a p-value of 0.006928.

PCR, Gel Electrophoresis, and DNA sequence analysis with BLAST

Gel electrophoresis yielded seven isolates with bands at 1500-bp, indicating the presence of the 16S rRNA gene, which was used to identify the species of each isolate. Residential and protected nature sample DNA was successfully amplified but the industrial samples yielded no band. All seven of the isolates sent in for analysis were successfully sequenced (Fig. 2). Each sequence was also successfully matched to a bacterial genus. Residential soil had three different genera, Bacillus, Lysinibacillus, and Pantoea.

Protected nature soil had one genus, Pseudomonas (Table #1). The top twenty matches for each isolate were all the same genus, indicating high confidence in the genus match.

Statistical Analysis

Characterization Tests

The data collected was primarily the number of bacteria isolates that showed a zone of inhibition when co-cultured with at least one of the three ESKAPE pathogens. Fisher Exact Test was used to analyze this data as it allows for comparison between the three environments. Any p-value less than 0.05 was considered a statistically significant result.

For the MacConkey agar tests, 13 out of the 20 antibiotic-producing isolates fermented lactose while 7 out of 20 did not ferment lactose (Fig. 3). All non-lactose-fermenting bacteria were from residential locations. Fermentation of lactose was indicated on the agar by the presence of a pink/red color in the bacteria, while lack of fermentation was defined as a lack of color. For the motility assessment, only the 9 samples used for PCR were tested. Of these isolates, 2 out of 9 were motile while the other 7 were nonmotile.

The motile isolates were found in residential and protected nature environments. Motility was defined as the presence of bacterial growth outside of the defined region it was originally placed in.

Of the 20 antibiotic-producing isolates, 6 hydrolyzed starch, 5 of these samples were from resi-

RESULTS Screen for isolate antibiotic production – Spread/Patch technique

Each isolate was co-cultured with Escherichia coli, Staphylococcus epidermidis, and

dential areas and one was from an industrial area (Fig. 3). Starch hydrolysis was defined as the lack of black pigmentation in the presence of iodine.

None of the 20 antibiotic-producing bacterial isolates exhibited any sort of antagonistic behavior against both species of fungal, eukaryotic cells (Fig. 3). The growth of the fungi was not inhibited at all by the presence of the bacterial isolates. The bacteria growth was also generally uninhibited by the presence of the fungi.

DISCUSSION

The hypothesis for this experiment was that industrial and residential soil settings would yield the highest concentration of antibiotic producing bacteria due to heightened environmental stress, which incentivizes microbial interaction and competition. Previous studies support this hypothesis, finding that industrial metal-contaminated soil and residential soil yields high levels of antibiotic-producing bacteria [5,8]. But the results indicate that residential soil has a far higher concentration of antibiotic-producing bacteria than both industrial and protected nature settings. Residential soil had double the amount of antibiotic-producing strains in comparison to industrial sites and six times the amount in comparison to nature protected sites (Fig. 1). The relationships between industrial/protected nature and industrial/residential were not statistically significant. But the p-value between residential and protected nature locations was 0.006928. This indicates that residential soil has a much higher incidence of antibiotic-producing bacteria than industrial areas.

The bacterial characterization tests indicate that residential soil harbors more diverse bacteria in terms of lactose fermentation and starch hydrolysis (Fig. 4). The genetic sequencing results also showed that residential soil harbored the most diverse bacterial genera. This heightened diversity likely contributes to the occurrence of antibiotic-producing bacteria. The high level of antibiotic production in the residential setting may also be attributed to concentrated and prolonged exposure to fertilizer as well as fecal matter. Such soil environments have been linked to an increased frequency of mobilized genetic elements and horizontal gene transfer [10].

The force behind this antibiotic-production is the enhanced bioactivity that occurs in

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PCR, Gel Electrophoresis, and DNA sequence analysis with BLAST

was followed according to the Tiny Earth Research Guide [1]. 27F primer was added to each sample to focus on the 16S rRNA gene. These samples were sent to the Wisconsin Institute for Discovery on April 11th, 2019 for sequencing.

The obtained nucleotide sequences were analyzed and ran through the BLAST website database to identify the isolate.

the presence of fertilizer and human waste. Previous studies have observed horizontal plasmid acquisition of genes between bacterial strains in residential soil, which enables antibiotic-producing bacteria and antibiotic resistant bacteria to coexist through a cycle of competition via synthesis of novel bioactive compounds [5]. This co-evolutionary mechanism creates a highly bioactive environment. Such an environment selects for the most competitive bacteria, therefore increasing the likelihood of antibiotic-production.

Perhaps the co-evolutionary mechanism of plasmid acquisition drives bacterial defense systems, which would increase both antibiotic resistance (to protect the bacteria from antibiotics) and antibiotic production (to protect the bacteria from other bacteria). This bioactivity suggests additional unknown processes of interaction between microbial populations and fertilizer and fecal matter. Long-term experiments observing bacteria in such environments may be able to ascertain the process that leads to this unique bioactivity.

This study sampled a very limited number of residential sites in only one part of the world. Therefore, a trend cannot be definitively confirmed. Further studies must be done with higher replicate numbers and in more varied geographical locations in order to determine if residential soil consistently has a greater number of antibiotic-producing bacteria. Another limitation is the very definition of “residential� as it may vary in different locations. For example, some residential areas may have formerly been industrial and then later developed into living areas. This study is also limited by the lack of analysis regarding the chemical makeup of each soil location. Future studies should include chemical soil analysis of each sample site to see if the presence of antibiotic producing bacteria is linked to a specific material. Examining the full microbial footprint to include organisms such as fungi may also yield a better understanding of communities with heightened antibiotic-producing bacteria.

The results indicate that residential soil environments best enhance the occurrence of antibiotic-producing bacteria. More specifically, environments with high levels of fertilizing agents and poor waste management. Residential areas with consistent lawn fertilization, high levels of pets, and extensive sewage lines in the soil

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may be a prime source of novel antibiotic-producing bacteria. But agricultural soil is also cyclically exposed to extremely high levels of fertilizer and feces (manure), likely making it a reservoir of diverse antibiotic-producing bacteria. Also, over 51% of land in the United States is used for agriculture, which is a massive, extensive area for possible antibiotic discovery [11]. The future of antibiotics therefore may lie in agricultural soil. But constant environmental stress may also inhibit bacterial diversity in the long-term.

Therefore, future studies should also investigate how bacterial diversity and the presence of antibiotic-producing bacteria change over time in consistently stressed environments.

Antibiotic resistance is driven by human activity such as the overuse and misuse of antibiotics, antibiotic use in pets and livestock, and antibiotic pollution of the environment [2].

But, antibiotic-production may also be driven by human activity. Residential environments arguably have the highest level of consistent human activity than any other location. This heightened human activity as expressed through fertilizing agents and the presence of concentrated waste products creates a bioactive environment that encourages antibiotic production [5]. Antibiotic resistance may be a growing issue, but the mechanisms that incite resistance also incite antibiotic production. Therefore, an increase in resistance is likely to be followed by an increase in antibiotic-production. A better understanding of this cycle will ultimately precipitate an enhanced ability to address the current antibiotic resistance crisis.

Fig 1. Proportion of Antibiotic-Producing Isolates. Stacked column bar graph showing the proportion of antibiotic producing strains for industrial, residential, and nature protected soil settings. The overall p-value between all three groups is 0.023. There is a statistically significant relationship between residential and protected-nature settings with a p-value of 0.006928.

ACKNOWLEDGEMENTS

Special thanks to Dr. Douglas Rouse and the Department of Plant Pathology at UW-Madison for supporting this research. The authors also greatly acknowledge WARF, the Wisconsin Institute for Discovery, and Tiny Earth for enabling and funding this project.

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FIGURES AND TABLES

Fig 3. Characterization of Antibiotic-Producing Isolates. Cluster bar graph arranged by soil sample location- industrial, residential, and protected nature. Each bar represents the number of antibiotic-producing isolates that fermented lactose, hydrolyzed starch, and showed antagonistic behavior toward eukaryotic cells.

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FIGURES AND TABLES

REFERENCES

Fig 2. Example of Sequenced 16S rRNA Gene. Snapshot of genetic sequencing data for the 16S rRNA gene for an antibiotic-producing isolate from a

Location of antibioticproducing isolate

Genus

Top Species Match

Match Confidence

Residential

Bacillus

97.16%

Residential

Bacillus

B. subtilis strain 629 B. sp. D2Pr67

Residential

Bacillus

B. sp. strain VII/5

98.01%

Residential

Lysinibacillus

L. sp. n-7

99.45%

Residential

Pantoea

P. sp. xBGRY4

87.96%

Protected Nature

Pseudomonas

88.19%

Protected Nature

Pseudomonas

P. grimontii strain BU106a P. fluorescent strain Lew-24

98.36%

Soils. In: Gogarten M, Gogarten J, Olendzenski L, editors. Horizontal Gene Transfer Methods in Molecular Biology, vol 532. Humana Press; 2009. pp 455-472.

10.

Aminov R. Horizontal gene exchange in environmental microbiota. Front Microbiol.

2011;2:158. doi:10.3389/fmicb.2011.00158 11.

Nickerson C, Ebel R, Borchers A, Carriazo F. Major Uses of Land in the United States, 2007. USDA Economic Research Service: 2011 Report No.: EIB-89.

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residential environment. Each letter on the top row denotes the nucleotide match to the color-corresponding peak below. The lack of overlapping peaks indicates higher confidence in each match. This is a species match of B. subtilis strain 629 with 97.16% confidence.

1. Hernandez S., Tsang T., Bascom-Slack C., Broderick N., Handelsman J. Tiny Earth A Research Guide to Student-sourcing Antibiotic Discovery. Acton-Massachusetts: XanEdu; 2018.

2. Centers for Disease Control and Prevention.

Antibiotic/Antimicrobial Resistance. 2018 Sep 10 [cited 20 April 2019]. In: CDC [Internet]. Available from: https://www.cdc.gov/ drugresistance/index.html 3. Ventola CL. The antibiotic resistance crisis: Part 1: causes and threats. P T. 2015;40(4):277– 283.

4. Santajit S, Indrawattana N. Mechanisms of Antimicrobial Resistance in ESKAPE Pathogens. Biomed Res Int. 2016. Article ID 2475067. doi: 10.1155/2016/2475067 5. Woappi Y. Emergence of Antibiotic-Producing Microorganisms in Residential Versus Recreational Microenvironments. British Microbiology Research Journal. 2013; 3(3): 280–294.

6. Stewart BA. Soil Degradation: A Global Threat. Advances in Soil Science. Lal R, Coninck F, Fausey N, Gupta R, Logan T, et al., editors. New York: Springer New York Inc. 1990.

7. Walsh C. Antibiotics: actions, origins, resistance. Washington, D.C.: ASM Press; 2003.

8. Liu Y-R, Delgado-Baquerizo M, Bi L, Zhu J, He J-Z. Consistent responses of soil microbial taxonomic and functional attributes to mercury pollution across China. Microbiome.

2018;6(1):183. doi:10.1186/s40168-0180572-7 9. Sobecky P, Coombs J. Horizontal Gene Transfer in Metal and Radionuclide Contaminated

97.48%

Table 1. 16S rRNA Genetic Sequencing Results. Table depicting the different genera conclusions and top species matches derived from DNA sequencing of the 16S rRNA gene followed by BLAST database analysis of results. The match confidence percentage refers to the confidence in the top species match.

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Incidence Rate of Parkinson’s Disease based on Gender Marie Bai ABSTRACT While many studies have examined the effect of age on the incidence of Parkinson’s Disease (PD), very few have been carried out in attempts to understand differences in PD incidence due to biological sex of the population examined. In response to this deficit of research on the occurrence of PD with respect to the sex of patients, prior studies in the field were aggregated and analyzed to determine any difference in PD occurrence due to this particular demographic identifier. Google Scholar and PubMed were searched for studies on the topic. After filtering of eligible published works, findings from four studies were compiled and analyzed via an ANOVA test to determine if differences in PD incidence between males and females were significant. No significant difference was found. While there was no significant difference in PD occurrence between males and females, the trend towards higher PD incidence in men warrants further investigation with more controlled factors in order to draw a more definitive conclusion.

INTRODUCTION

The exact biological causes of PD are unknown, but scientists believe that a combination of genetic and environmental factors can explain it. In terms of environmental factors, traumatic brain injuries, pesticide and herbicide exposure, exposure to metals and even areas of residence have been associated with an increased risk of developing PD [1]. These factors are varied and their interaction with genes can be quite complex to understand.

PD manifests as a challenge for people suffering from it as it impacts their quality of life and renders them incapable of being fully autonomous. Parkinson’s Disease manifests itself 30 JUST VOL V // ISSUE I // FALL 2019

through bradykinesia (slowness of movement) that is usually accompanied with tremor or rigidity. These movement symptoms are attributed to the malfunction of dopamine signaling, which is primary mechanism of movement coordination.

In addition to the changes to motor functions it causes, it can also create sleep problems, depression, pain and cognitive depression [2].

Multiple studies have established that the occurrence of the disease is elevated in older population [3]. From this research, programs intended to assist elderly individuals in coping with PD have been established. By exploring other demographic identifiers, further research and program implementation can be focused so as to specify areas of study and reach individuals most susceptible to PD, respectively.

Of the myriad of studies aimed at examining the occurrence of PD, few studies have been conducted over the course of the last decade to determine difference in PD incidence between males and females. These epidemiological differences could explain the higher PD incidence in men suggested by most studies. However, no definitive consensus has been reached on the correlation between PD and gender.

While the effect of age on the incidence of PD has been extensively explored throughout the

METHODS AND MATERIALS

Sorting through sources.

In compiling sources relevant to our hypothesis of interest, we searched PubMed and Google Scholar for key terms “Parkinson,” “sex,” and “gender.” The terms sex and gender were considered interchangeable for the purpose of this study, but not without ensuring the articles selected referred to gender in a purely biological context. This yielded 199 potential papers which were then further filtered by keywords “differences” and “variation.” The search spanned from November 1994 until May 2013 for studies involving at least 50 confirmed cases of PD, out of a total population of 100,000 person/year. We

arbitrarily set this number to 50 to have a sizable number of studies without having to sacrifice the quality of the data. We looked specifically for cases of PD and dismissed any other cases of Parkinsonism, such as drugs or trauma induced PD. We ensured the journals utilized for our purposes were ones which reported data on the gender of each individual and adjusted for age and gender using the direct method of standardization so as to prevent bias in populations where one gender or age group was more prevalent than another.

When reviewing selected papers, we realized that identifying a variable to measure PD occurrence in a population based on sex could prove difficult. We chose to use the incidence rate, which can be defined as the probability of occurrence of a given medical condition in a population at risk within a specified period (AREP, 2019). Therefore, all prevalent cases of PD were not considered.

Incidence rate being our common metric, it was eventually added as an additional key term searched to finally yield the 21 articles we reviewed before selecting the seven final articles used for this meta-analysis. Because of the scarcity of papers on the question, meta-analysis documents were included for comparison but were not used for data analysis. Only four studies were used for their data. Because the incidence rate for black males was abnormally high in the study

by Mayeux et al. and the sample size was smaller for other races, we obtained the incidence rate for white males and females. This allowed us to easily weight the data with the data from the other studies.

DATA ANALYSIS

We compiled raw data consisting of the number of males and females age adjusted incidence per 100,000 and pulled it onto a spreadsheet and organized by gender. We obtained an average for each group (males vs. female). We then used an ANOVA test to determine if there was a statistically significant difference between the two datasets. The threshold for a statistically significant result was set to p <0.05. If we found p to be less than that number, then we concluded that there was a statistically significant difference between the incidence rate for men versus women. In addition, we also created a forest plot to provide a clear visualization of the differences between the data for each article for men vs women. Because of the considerably large sample size of the Baldereschi study compared to the others, it was not included in the forest plot. The analysis was run on a personal computer using Excel.

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Parkinson's disease (PD) is a neurodegenerative disorder that manifests predominantly in dopamine-producing (“dopaminergic”) neurons in the substantia nigra of the brain [1]. Approximately 60,000 Americans are diagnosed with PD each year, and according to the Parkinson’s Foundation, nearly one million will be living with PD in the U.S. by 2020 [1]. Worldwide, this disease affects more than 10 million people, and studies have found that while the incidence of Parkinson’s disease increased with age, an estimated four percent of people with PD are diagnosed before the age of 50 [1].

literature, the gender as a biological contributor has not. Among the few studies that found gender differences in PD, differences were observed in the clinical manifestation of the disease and the level of neurotoxicity among others [4 and 5]. Findings in experimental animal models have found that estrogens protect neurons from various forms of injury, and the reported gender differences in PD could be related to the different level of circulating estrogens in men and women [4 and 5]. While this effect of estrogens has been observed in animal models, it has yet to be shown in humans [5].

Most studies on PD report on incidence instead of prevalence because prevalence data cannot make effectively make a distinction between the influence of incidence and survival, especially when studies conducted among clinical or referral series may include patients not representative of the total population with the disease [3]. Incidence is a measure of new cases of a disease over a specific period, while prevalence denotes of the proportion of a population with a disease or a particular condition at a specific point in time [6].

By aggregating relevant data in the field, we sought to determine the degree to which sex biologically predisposes an individual to developing PD. After examining trends in previous literature, we expect a higher proportion of men than women to be diagnosed with PD. In synthesizing data from a variety of sources in this meta-analysis, the increase in sample size will give credence to observed trends. Being able to draw a correlation between these variables, could help calibrate the effectiveness of outreach programs when it comes to a specific demographic and it could also help further medical research questions as to why one group may have a lower incidence rate of the disease.

While we are aware that the sample size from each study may have influenced the results in different ways, this bias was accounted for by averaging the “weight” of each data set so that a sample of 300 had the same impact as a sample of 3,000.

RESULTS

A table combining the data can be seen in Fig 1. The first study found an age adjusted incidence rate per 100,000 of 411 (95% Cl 253-570) for males and 217 (95% Cl 98-337) for females [6]. The Mayeux study’s age adjusted incidence rate was per 100,000 was 13.3 (95% Cl 6.7-20) for males and 11.8 (95% Cl 5.9-17.8) for females [7].

Van Deen et al. found an incidence rate of 19 (95% Cl 16.1-21.8) for males and women 9.9 (95% Cl7.6-12.2) [3]. Caslake et al. study revealed an age adjusted incidence rate of 21.1 (95% Cl 17.4-24.8) for males and 14.7 (95% Cl 11.6-17.8) for females [8]. The average incident rate for males was higher at 464.4 while the average incident rate for females was 253.4 out of 4,170 total participants. The ANOVA test (ran on all samples) returned a p-value of 0.65.

Based on the p-value of the ANOVA test, p= 0.65, we can conclude that there is no statistically significant difference between the incidence rate of PD in males and females.

However, because the individual and aggregate incidence rates show a larger incidence rate for males, further investigation is needed to explore this trend. While these studies found a trend with a higher incidence rate of PD in males, these results may be attributed to methodological differences in the studies. It is therefore important to recognize the limitations in the data findings for each study.

Although the data was adjusted for age, some studies such as Baldereschi et al. only focused on a specific portion of the population (65-84 years old). The results from such research cannot be generalized or extrapolated to all males and females. Another bias introduction has to do with the randomization of samples. Sample selection in some cases were not random. Mayeux et al. focused on a community in northern Manhattan and Van Deen et al. on a population from the Kaiser Permanente medical care program. Another component that may have influenced the results for these studies is the criteria used for identifying PD. Caslake et al. used patients having two or 32 JUST VOL V // ISSUE I // FALL 2019

ACKNOWLEDGEMENTS

I would like to thank Mr. Jon T. Breschak, Course and Assessment Coordinator in Dept. of Integrative Biology, UW-Madison College of Letters and Science.

REFERENCES 1. What is Parkinson's? Parkinson's Foundation.

Available from: https://parkinson.org/understanding-parkinsons/what-is-parkinsons 2. Symptoms of Parkinson’s: Men vs. Women.

Healthline. Available from: https://www. healthline.com/health/parkinsons-symptoms-men-women 3. Van Den Eeden S, M Tanner C, L Bernstein A, D Fross R, Leimpeter A, A Bloch D, et al. Incidence of Parkinson's Disease: Variation by Age, Gender, and Race/Ethnicity.

4. Shulman, L. “Gender Differences in Parkinsons Disease.” Gender Medicine 4, no. 1 (2007): 8–18. https://doi.org/10.1016/s15508579(07)80003-9.

5. Haaxma C, Bloem B, Borm G, et al. Gender differences in Parkinson’s disease. Journal of Neurology, Neurosurgery & Psychiatry 2007;78:819-824.

6. Incidence and Prevalence. Advanced Renal Education Program Available from: https:// www.advancedrenaleducation.com/content/ incidence-and-prevalence 7. Baldereschi M, Di Carlo A, Rocca W, Vanni P, Maggi S, Perissinotto E, et al. Parkinson’s disease and parkinsonism in a longitudinal study.

Neurology 55(9):1358.

8. Mayeux R, Cote L, Denaro J, Hemenegildo N, Mejia H, Tang M-X, et al. The Frequency of Idiopathic Parkinson's Disease by Age, Ethnic Group, and Sex in Northern Manhattan, 1988-1993. American Journal of Epidemiology 1995;142(8):820 - 827.

9. Caslake R, Taylor K, Scott N, Gordon J, Harris C, Wilde K, et al. Age-, gender-, and socioeconomic status-specific incidence of Parkinson's disease and parkinsonism in northeast Scotland: the PINE study. Parkinsonism Relat Disord 2013;19(5):515-21.

10.

Wright Willis A, Evanoff BA, Lian M, Criswell SR, Racette BA. Geographic and ethnic variation in Parkinson disease: a population-based study of US Medicare beneficiaries.

Neuroepidemiology 2010;34(3):143-151.

11.

Hemming J.P, Gruber-Baldini A, Anderson K, Fishman P, Reich S, Weiner W, et al. Racial and Socioeconomic Disparities in Parkinsonism. Archives of Neurology 2010;68(4):498-503.

12.

Chen H, Shrestha S, Huang X, Jain S, Guo X, Tranah GJ, Garcia ME, Satterfield S, Phillips C, Harris TB et al. Olfaction and incident Parkinson disease in US white and black older adults. Neurology 2017;89(14):14411447.

13.

Dahodwala N, Siderowf A, Xie M, Noll E, Stern M, Mandell DS. Racial differences in the diagnosis of Parkinson's disease. Movement disorders: official journal of the Movement Disorder Society 2009;24(8):1200-1205.

14.

Hirsch L, Jette N, Frolkis A, Steeves T, Pringsheim T. The Incidence of Parkinson's Disease: A Systematic Review and Meta-Analysis. Neuroepidemiology 2016;46(4):292-300.

15. Wooten GF, Currie LJ, Bovbjerg VE, Lee JK, Patrie J. Are men at greater risk for Parkinson’s disease than women? Journal of Neurology, Neurosurgery & Psychiatry 2004;75(4):637.

REPORT

REPORT

DISCUSSION

more cardinal motors signs as the identifying for diagnosing PD while some studies did not specify their definition of a PD diagnosis. Additionally, some studies used digital medical records which at the time weren’t common or accurate.

For all these reasons, we recommend that the subsequent experiments account for potential biases by using a large sample size, a randomized selection, an extensive study time, a specific identification of the PD diagnosis (one agreed upon by the medical corps). Patients identified should also have PD as their primary diagnosis and no underlying disease associated as to remove the echo of a secondary diagnosis in the results. A study conducted today would also benefit from the accuracy and the increase in the computerization of medical health records.

A study that returns definitive results on the difference in incidence rate of PD in males versus females will help scientists explore the possible causes of such difference, should the statistically significant difference be found.

With the varying causes of PD enumerated earlier, a difference in the incidence rate based on sex could help direct research to focus on sex-related genetic determinants of the disease. Further exploration of the disease will help drive decisions made by healthcare professionals and legislators to determine the appropriate allocation of resources to address the disease. Additionally, continued research on PD will lend itself to the development of assistance programs for populations at risk for the disease. These programs will be fundamental in delivering a more targeted approach to treatments, including preventative methods, education on the management of PD symptoms, and treatments for the afflicted demographic.

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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.