Volume III Issue I FALL 2017 by JUST Journal

Volume III Issue I

Gene overexpression and heart fibrosis A mouse model

Tired of Being Tired? p. 6

p. 22

Making The Modern Tomato p. 8

The Science Behind Gender Identity p. 13

Nanoparticles: Small Particles, Big Potential p. 10

LETTER FROM THE EDITOR IN CHIEF Dear Reader, I am exceedingly pleased to present to you Volume III, Issue I of the Journal of Undergraduate Science and Technology (JUST). JUST is truly a campus wide effort and 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 board members— some of whom have been with us since JUST’s founding in Fall 2015—that have chosen to make JUST part of their undergraduate experience. 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 non-expert audiences. On campus, we have been uniquely able to provide the opportunity for undergraduates to publish their work in a peer reviewed journal, and to teach them about how the publication process works. On the other side, our undergraduate editors and staff are able to learn critical skills and experience the process from the perspective of an academic journal. We believe that these experiences are a strong supplement to a traditional undergraduate education, especially for those students who wish to pursue graduate education in research. As for the second part of our mission, I believe that scientific literacy is more important than ever in today’s global society. Science isn’t just a topic for the ivory tower anymore; all lives can be enriched by a solid understanding of scientific thought. People need to have the skills to gather information, analyze their options, and ultimately make the best possible decisions in their lives. Better communication of research and science is key to this. We are honored to be a part of making research and scientific achievement more accessible to non-expert communities beyond academia. In many ways, the space we occupy on campus mirrors the tenets of the Wisconsin Idea: that the influence of the university should better people’s lives outside of the classroom and across the state. We believe that by helping to train the next generation of researchers and assisting in the dissemination of scientific knowledge, JUST is helping to realize and advance the Wisconsin Idea. 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. I encourage you to read carefully and explore the content of our talented undergraduate researchers and writers.

All the best,

Evan Cory Editor in Chief

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SPONSORS & PARTNERS

Volume III Issue I Fall 2017 Editor in Chief Evan Cory

Managing Editors Jieun Heo Meng Lou We would like to sincerely thank CALS, the Associated Students of Madison, the Wisconsin Institute for Discovery, and the Holtz Center for Science and Technology Studies for financially supporting the production of JUSTâ&#x20AC;&#x2122;s issue. Thank you!

Director of Finance Aditya Singh

Director of Marketing Teja Karimikonda

Director of Design Margaret Seybold

Webmaster

Cayman McKee

Editors of Content Bailey Spiegelberg Greg Zilberg Lekha Nelavelli Madelyn Goedland Markayle Schears Rachel Gruenke Tammy Zhong

Copy Editor

Madison Knobloch

Malik Anderson, WUD Publications Committee Director Jim Rogers, WUD Publications Committee Advisor Iffat Bhuiyan, Wisconsin Union President

Staff Writers

Alina Dillahunt Annika Peterson

All images courtesy of creative commons (CC0) or Margaret Seybold.

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TABLE OF CONTENTS { EDITORIALS }

p. 9

Neuroscience . . . . . . . . . . . . . . . . . . . . . . 6 Teja Karimikonda

Agronomy . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Annika Peterson

Nanomedicine . . . . . . . . . . . . . . . . . . . 1 0 Meng Lou

Neuroendocrinology . . . . . . . . . . . 1 3 Alina Dillahunt

{ REPORTS } Mouse gene overexpression regulates inflammatory proteins and causes heart fibrosis..............................22 Erika Henningsen, Tetsuya Takimoto, Giangela Stokes, Sarah Lewis, Akihiro Ikeda

The Gibbs Paradox ............30 Aniket Sudeep Dalvi , Chinmaya Bhargavay

{ PIXELS } Nikki Noughani Natalia Lucero . . . . . . . . . . . . . . . . . . 1 8 Celia Glime

Comparison of treatments for exercise-induced bronchoconstriction ...........34 Matthew Kuik, Tom Schneider, Bryan Jackler

Kaylyn Freeman . . . . . . . . . . . . . . . . 1 9

p. 18

p. 19

p. 10

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// Neuroscience

Tired of Being Tired? Pulling all-nighters has become a common occurrence for many college students as they prepare last-minute for midterms or rush to finish that 10-page essay. What many are not aware of is the fact that sleep deprivation can have some serious negative effects such as anxiety and depression. Sleep is a necessity for a healthy human body, which is probably why it is recommended you spend 1/3 of your life sleeping.

hey say that eight hours of sleep each night is the commonly accepted parameter of a good night’s sleep; however, most students would agree that this statement is more of a dream than a reality. Many students stay up until 2 or 3 a.m. finishing up assignments or studying for tests. Repeating this process night after night results in poor sleep for students. Justin Pope reports that college health officials are realizing that healthy sleep habits can alleviate some issues ailing students: anxiety, depression, physical health problems, and of course academic troubles [1]. A time-consuming activity that every individual across the globe participates in is sleep. If an individual lived 80 years, approximately 27 years will have been spent sleeping, assuming eight hours of sleep each night. This is a lot of time which prompts the question: how long can we stay awake? Randy Gardner set the commonly cited record for most time an individual voluntarily resisted sleep in 1964 when he was 17 years old for a science fair project. He did not sleep for 264 hours straight,

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approximately 11 days [2]! Although the purpose of sleep and why the urge to rest is so strong at specific times—like when your TA is explaining polynomials—is still unknown, studies have shown sleep deprivation to be correlated to poorer health and decreased

"A time-consuming activity that every individual across the globe participates in is sleep. If an individual lived 80 years, approximately 27 years will have been spent sleeping, assuming eight hours of sleep each night.

cognitive performance [3]. Though strong parallels are drawn between sleep deprivation and body decline, such as immune system dysfunction, sickness, and elevated blood pressure, the consensus appears to be that these negative effects do not seem permanent [2]. But this does not mean that sleep deprivation

should be taken lightly. Both sleep quality and duration have been shown to have a direct impact on health [4]. Many studies show that sleep deprivation impairs learning and memory processes, both of which play crucial roles in an individual’s academic performance [4]. In addition to poor academic performance, sleep deprivation has also been linked with obesity. A study involving fourteen healthy individuals subjected to either four nights of healthy sleep or sleep deprivation found that those who were deprived of sleep felt hungrier and consumed twice as much fat and protein as the control group. These food cravings were explained through amplified endocannabinoid levels, endogenous lipid-based neurotransmitters, which were involved in food cravings and led to hunger pangs [5]. Is this where the freshman 15 comes from? Though it is simple to cite lack of sleep as an excuse for poor academic performance and health, it is more relevant to ask why so many college students suffer from sleep deprivation. Students staying up late to study who have early morning classes are more likely to not attend their classes as they may not wake to their alarms. In this case, they may lose even more sleep catching up on what they may have missed by not attending class. This cycle creates an endless feedback loop which could potentially be rectified through better understanding of the necessity of sleep. Students can in essence, be shooting themselves in their own foot as a study showed that sleep deprived students performed poorly though they believed themselves to have higher levels of performance [4]. With introspection, students can create schedules which accommodate their schoolwork as well as

hour libraries across campus [6]. As a longterm initiative, the benefits of good sleep practices and how to go about obtaining them should presented to the students. For example, housing fellows within the dorms could present this information within

GOOD SLEEP HABITS [7] • Schedule enough time for sleep • Set aside your electronics for the night • Try to set regular routine bedtimes and rise times • Spend some time exercising each day (though not late at night) • Do not nap in the middle of the day • Wind down towards bedtime • Deal with problems early in the evening

one of the house meetings. Universities should educate their students both on the importance of sleep and on the detrimental effects of sleep deprivation. Furthermore,

"With

introspection, students can create schedules which accommodate their schoolwork as well as any extracurricular activates in a manner that allows them to get a good night’s sleep each night.

-- Teja Karimikonda REFERENCES 1. Pope J. Colleges wake up to notion that better sleep means better grades. Washington Times. 2012; Available from: http://www.washingtontimes.com/ news/2012/sep/3/colleges-wake-up-tonotion-that-better-sleep-means/. 2. Hadhazy A. How long can we stay awake. BBC. 2015; Available from: http:// www.bbc.com/future/story/20150220how-long-can-we-stay-awake. 3. Saper CB, Scammell TE, Lu J. Hypothalamic regulation of sleep and circadian rhythms. Nature. 2005 Oct 27;437(7063):1257-63. 4. Curcio G, Ferrara M, De Gennaro L. Sleep loss, learning capacity and academic performance. Sleep medicine reviews. 2006 Oct 31;10(5):323-37. 5. Bromwich J. Poor sleep gives you the munchies, study says. New York Times. 2016; Available from: http://www.nytimes. com/2016/03/05/science/sleep-eatingcraving-food.html. 6. Akhtar A. Michigan library opens nap station for students. Washington Times. 2014; Available from: http://www. washingtontimes.com/news/2014/may/12/ michigan-library-opens-nap-station-forstudents/. 7. Wilson S, Nutt D. Good sleep habits. Psychiatry. 2007;7(6):301-4.

any extracurricular activates in a manner that allows them to get a good night’s sleep each night. Universities can also take measures to address the issue of sleep deprivation. University of Michigan introduced an interesting initiative to tackle this issue. They designed “napping centers” within libraries, as it is common to see many students falling asleep in the 24-

students should be encouraged to speak with their professors if they find themselves experiencing sleep-deprivation for an extension or some other solution as sleep deprivation both impacts the health of the student and their academic performance as well. Though this number may vary by individual, everyone should strive to sleep eight quality hours each day as sleep is a necessity for a healthy human body.

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// Agronomy

Have you ever wondered why tomatoes from the store taste so bland compared to ones you buy at the farmer’s market? The modern tomato has a rich and colorful history going from domestication to mass production; plant breeding is at its core. As unassuming as it may seem, a simple tomato has behind it complex agricultural research and techniques that make these juicy summer fruits what they are today.

or many Midwesterners, devouring delicious tomatoes that ripen near the end of the summer is a highlight of the year. There are endless ways to enjoy summer’s bounty, from elaborate dishes to just some salt and pepper embellishment. Many varieties of juicy, ripe tomatoes can be found in backyards, farmers markets, and grocery stores. All of the tomatoes we enjoy come from a single species, Solanum lypcopersium. The history of this crop is long, with many changes in shape, color, and geographic location over time. Even now, its identity is still changing! The tomatoes we enjoy today didn’t exist thousands of years ago. At first, South Americans domesticated the wild ancestors of the modern tomato [1]. Since cultivation proved more efficient than gathering crops, they selected seeds from the plants that best

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Making the Modern Tomato

fit their needs. For example, they picked and grew easy-to-cultivate and pleasant tasting tomatoes more often. Nowadays, this selection process continues in modern fields and research labs. Plant breeders continue to choose traits that improve our crops, but now they use genetic methods to identify genes that cause certain characteristics.

where key changes took place in the evolution of the plant genes [1]. The process of modern breeding has drastically changed the tomato in the last 100 years [1]. Farmers and plant breeders have selected for larger fruits with a pleasant shape and color, and varieties that are easy to harvest. Because breeding focuses on qualities like yield and size, another key

"The process of modern breeding has drastically changed the

tomato in the last 100 years. Farmers and plant breeders have selected for larger fruits with a pleasant shape and color, and varieties that are easy to harvest.

They have found the best ways to breed and grow plants to improve qualities like yield, disease resistance, and appearance. Researchers can also identify when and

quality—taste—has been lost. Some people may notice that tomatoes bought at a farmers’ market taste better than those purchased at a grocery store. Often, the

types of tomatoes sold at farmers’ markets are heirloom varieties, a term that is not well defined, but suggest a tomato variety that has been cultivated and passed down from grower to grower for at least 50 years [2]. On the other hand, tomatoes produced by modern breeding are brightly colored, easy to transport, and are available in supermarkets year-round. While widely available, they may not be as tasty as those found in the farmers’ market. Though researchers have concluded that the balance of acid to sugar affects the taste of a tomato, smell also plays a significant role. A diverse set of volatile compounds or molecules that we smell when they vaporize, affects the flavor of a tomato [2]. Even though volatile compounds change the way a tomato tastes, plant breeders did not take them into consideration [2]. A recent study explored this

recent study explored this through the comparison of heirloom, modern, and the closest wild (not cultivated) relatives to the tomato we know. In modern cultivars, lower levels of flavor volatiles are present, indicating less flavor in these tomatoes.

through the comparison of heirloom, modern, and the closest wild (not cultivated) relatives to the tomato we know. In modern cultivars, lower levels of flavor volatiles are present, indicating less flavor in these tomatoes. Another difference between older and modern varieties is the genes associated with sugar content. As breeding continued, the average fruit size increased at the expense of decreased sugar content and flavor [2]. Through these changes over time, yield and ease of harvest was increased while flavor decreased. Utilizing modern genetic technologies, researchers can understand how the genetic makeup of a crop causes the qualities we see and taste, and how it changes over time. At the University of Wisconsin-Madison, researchers do consider flavor in deciding what tomatoes to grow and research. An experiment is taking place that includes flavor as

one of its main considerations when evaluating tomato varieties. Dr. Julie Dawson in the Horticulture Department here at UWMadison studies tomatoes under organic or low input conditions. In other words, tomatoes grown with little use of pesticides or fertilizers. At harvest time of the most recent year of the study, Dawson’s team evaluated characteristics of the tomatoes for both yield and disease, but most importantly also taste. In the evaluation, quantitative measurements of sweetness and acidity were matched with qualitative assessments from the public and local chefs [3]. This unique way of measuring the taste of tomatoes gave new and useful information to farmers, plant breeders, and chefs alike. A recent event called Farm to Flavor featured this unique and innovative method of evaluating crops. Chefs prepared unique dishes with local farmers and researchers’ vegetables to celebrate and share this research with the public [4]. This research adds to the story of the modern tomato. The humble tomato has changed over hundreds of years to give us the delicious fruit we enjoy today. From domestication to modern genetic analysis, we have changed this crop to fit our needs. While production and resistance to disease increased, tomato flavor decreased. However, with the exciting recent work to understand the flavor of tomatoes, the future of tomatoes looks delicious. -- Annika Peterson REFERENCES 1. Blanca J, Montero-Pau, Sauvage C, Bauchet G, Eudald I, José Díez M et al. Genomic variation in tomato, from wild ancestors to contemporary breeding accessions. BMC Genomics 2015; 16 (257). 2. Tieman D, Zhu G, Resende Jr. M, Lin T, Cuong N, Bies D et al. A chemical genetic roadmap to improved tomato flavor. Science 2017; 355 (6323): 391-394. 3. Silva E, Dawson J. Tomato Variety Trials for Flavor, Quality and Agronomic Performance, to Increase High-value Direct Marketing Opportunities for Farmers and On-Farm Trialing Capacity. Sustainable Agriculture Research and Education. 2016 Available from: https://projects.sare.org/sare_project/lnc14357/?ar=2016. 4. Savidge, N. Chefs, farmers and UW scientists team up for flavorful produce. Wisconsin State Journal. 6 Dec 2015. Available from: http://host.madison.com/ wsj/news/local/education/ university/chefs-farmersand-uw-s cientists-te amup-for-flavorful-produce/ article_633a2f89-01be-5a12a237-98cbbef6a09d.html.

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// Nanomedicine

Nanoparticles: small particles, big potential Cancer is one of the most complex non-communicable diseases known to man. An umbrella term for many different forms of uncontrolled cell division, cancer has been combated with a diverse number of therapeutics, ranging from chemotherapy to immunotherapy. In the pinnacle of modern chemical engineering and biochemistry, nanoparticles emerge as a promising superweapon against cancer.

he apple has classically served as an embodiment of health and prosperity. Many wonder why the Greek Goddesses Hera, Athena, and Aphrodite fought so tenaciously over such a token? However, where there is a virtue there is also a deceptive counterpart, for the apple has been implemented just as many times in negative light. In the classical Snow White, the villain gifts a poisonous apple to kill the protagonist. Despite this, there is some truth to the “poisonous apple” metaphor. Its shiny red exterior is matched only by the sweetness of the fruit, tempting and deceptive by nature. The seeds, however, contain traces of cyanide. When consumed in excess, they can be lethal. The apple represents an interesting scenario-dependent duality in the context of the divine, it represents health; of villains, deadly poison. Nanoparticles, miniscule particles, invisible to the naked eye and capable of delivering cargo to cells in the body, mimic this double-edged nature, bringing us a step closer to a potential treatment to cancer. Cancer, simply defined, is an uncontrolled growth of cells; canonically, a manifestation of a multitude of hallmarks ranging from angiogenesis to upregulation of growth factors, to suppression of programmed cell death [1]. In all its complexity, cancer is ultimately one thing: a self-perpetuating problem. With the ability to not only divert large amounts of resources and expand its population, cancer presents a further degree of spontaneity with its capacity to migrate and metastasize to a new

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location [1]. Difficulties in treatment of cancer surgically stem from its mobility, contributing to the high frequency of relapses. New methods have been employed to fight the war against cancer that has plagued humanity for generations—methods that now begin to match the complexity of cancer cells themselves. Among the promising candidates of cancer treatment are Bi-specific T-cell

"New methods have been employed to fight the war against cancer that has plagued humanity for generations—methods that now begin to match the complexity of cancer cells themselves.

Engagers (BITE), which physically bring activated killer T-cells within cancer cell proximity to take advantage of T-cell cytotoxic behaviors. Additionally, chimeric antigen receptor T-cell therapy (CARs) is an approach that programs T-cells isolated from one’s blood to recognize and target cancer for a more personalized, living cancer therapeutic [2, 3]. With recent developments in chemistry and chemical engineering, manipulation and synthesis of nanoparticles may provide a new paradigm in cancer treatment. Classical cancer therapeutics generally involve various of chemotherapies, which in combination can create a potent method of fighting cancer. However, their efficacy is only matched by their potential to inflict collateral damage on healthy cells. In

of siRNA, RNA capable of gene-silencing via complementation with messenger RNA (mRNA), nanoparticles can perturb chemotherapy resistance mechanisms from a genetic standpoint. Combine this with classical chemicals capable of killing cancer cells, and the nanoparticles can disable the resistance mechanisms and deliver the final blow. It is a superweapon that beautifully takes the two simple tools already in place and optimizes them spatially and temporally. While nanoparticles are elementary in principle, implementation of such a therapeutic is susceptible to both chemical and biological challenges. How will the release of drug be staggered to give the siRNA enough time to silence genes? Paula Hammond of MIT offers an ingenious solution. A clever â&#x20AC;&#x153;layer-by-layerâ&#x20AC;? model of nanoparticles, composed of multiple layers intercalated with specific substrates, allows the chemotherapy drug to be enveloped by layers of siRNA [13]. This way, the internal architecture of the nanoparticle provides hierarchical control, allowing siRNA to disable resistance genes prior to a toxic chemical release. However, to minimize collateral damage of healthy cells, this potent superweapon must specifically

"In

recruiting a layer of water, the charged particles serve as an invisibility cloak for the nanoparticle, undetected by the immune system.

essence, one trades one dangerous ailment for a multitude of lesslethal problems. Chronic subjugation to certain chemotherapies has been linked to anemia, hypertension that contributes to heart disease, and infertility [4,5,6]. Chemotherapy also possesses the potential to initiate other effects, such as secondary cancers in the form of new neoplastic growths due to chemical treatment [7,8]. Nevertheless, perhaps the most concerning aspect of chemotherapy is cancerâ&#x20AC;&#x2122;s ability to develop resistance [9, 10]. This evolutionary paradigm manifests in the form of alterations in drug trafficking and drug efficacy. Anti-neoplasm drugs typically require internalization and functionality in order to be effective. Cancer cells exploit these requirements and develop resistance to drugs is two primary ways: i. decreasing the abundance of the drug through altered drug transport and enhanced efflux and ii. enzymatic deactivation of proteins required to activate the drug, rendering it impotent. Both mechanisms have been shown to be heavily rooted in genetics. MDR1, a gene encoding a promiscuous membrane transporter, is heavily upregulated in cancer cells, allowing increased efflux of therapeutic drugs [10, 11]. Similarly, downregulation of enzymes that convert non-toxic drugs into their

target cancer cells. Here, specific ligands that bind to aggressive tumor cells can be intercalated into the shell of the nanoparticle, promoting interaction of nanoparticles specifically with cancer cells [14]. Finally, given the tendency for the immune system to destroy foreign objects, how will the nanoparticles hide from the defensive innate immune system? By coating the nanoparticle with negatively charged compounds, the half-life of the nanoparticle is increased, and the charged compounds add an element of stealth [15]. In recruiting a layer of water, the charged particles serve as an invisibility cloak for the nanoparticle, undetected by the immune system. Already, this technology has been demonstrated to be a highly personalized and potent cancer therapeutic in mice [15].

"To

overcome this obstacle, nanoparticles capable of delivering cargos specifically into a cell, present an opportunity as the next wave of modern cancer therapeutics.

effective counterparts confers resistance to the drug (e.g. DCK and Arabinoside) [10, 12]. As such, cancer manages to evade death while healthy cells are susceptible to the toxicity of chemotherapy. In light of this information, cancer therapeutics have encountered a major hurdle. To overcome this obstacle, nanoparticles capable of delivering cargos specifically into a cell, present an opportunity as the next wave of modern cancer therapeutics. Through the use

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Currently, it proceeds to clinical trial in efforts of developing a cancer therapeutic that is both safe and effective. However, nanoparticles are not limited to cancer therapeutics; integration with CRISPRCas9 gene-editing technology has also allowed target-specific genetic manipulations [16]. For being so miniscule, nanoparticles have the potential to change the future. Nanoparticles, are the paradigm-shifting apples of the therapeutic world – with their high specificity for cancer cells and deadly combination of gene-silencing with poisonous toxins, they are a nightmare for cancer cells. However, just as apples act as a symbol for health, nanoparticle serves as a beacon of light in the dark and tumultuous abnormality that is cancer. -- Meng Lou REFERENCES 1. Hanahan L and Weinberg R. 2011. Hallmarks of Cancer: The Next Generation. Cell. 144(5):646-74 2. Ross S. et al. 2017. Bispecific T cell engager (BiTE®) antibody constructs can mediate bystander tumor cell killing. PLOS. 12(8): e0183390 . 3. Fesnak A and Levine B. 2016. Engineered T cells: the promise and challenges of cancer immunotherapy. Nature Reviews. 16: 566– 581. 4. Berger A. et al. 2010. Cancer-related fatigue". Journal of the National Comprehensive Cancer Network. 8 (8): 904–31. 5. Pai V and Nahata M. 2000. Cardiotoxicity of chemotherapeutic agents: incidence, treatment and prevention. Drug Safety. 22(4):263302. 6. Ajala T. et al. 2010. Fertility Preservation for Cancer Patients: A Review. Obstet Gynecol Int. 10. 7. Errico A. 2014. Retinoblastoma—chemotherapy increases the risk of secondary cancer. Nature Reviews Clinical Oncology 11: 623. 8. Rüther U. et al. 2000. Secondary Neoplasias following Chemotherapy, Radiotherapy, and Immunosuppression. Contributions to Oncology (Beiträge zur Onkologie). 55. 9. Zuccala E. 2016. Chemotherapy: Clocking up resistance. Nature Reviews Cancer. 10. Holohan C. et al. 2013. Cancer drug resistance: an evolving paradigm. Nature Reviews Cancer. 13: 714-26. 11. Housman G. et al. 2014. Drug Resistance in Cancer: An Overview. Cancers. 6(3): 1769–1792. 12. Flasshove M. et al. 1994. Structural analysis of the deoxycytidine kinase gene in patients with acute myeloid leukemia and resistance to cytosine arabinoside. Leukemia. 8(5):780-5. 13. Deng Z. et al. 2013. Layer-by-layer nanoparticles for systemic codelivery of an anticancer drug and siRNA for potential triplenegative breast cancer treatment. ACSNano. 7 (11): 9571–84. 14. Dreaden E. et al. 2014. Bimodal tumor-targeting from microenvironment responsive hyaluronan layer-by-layer (LbL) nanoparticles. ACSNano. 8(8):8374-82 . 15. Gu L. et al. 2017. A Combination RNAi-Chemotherapy Layer-by-Layer Nanoparticle for Systemic Targeting of KRAS/P53 with Cisplatin to Treat Non-small Cell Lung Cancer. Clinical Cancer Research. Hammond. 16. Lee K. et al. 2017. Nanoparticle delivery of Cas9 ribonucleoprotein and donor DNA in vivo induces homologydirected DNA repair. Nature Biomedical Engineering.

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Neuroendocrinology \\

The Science Behind Gender Identity

Science has shown that gender is not a duality; there are intersex individuals, individuals with hormonal disorders, and people whose identities do not match their assigned sex. Transgender individuals' rights are threatened by politicians proposing bills that donâ&#x20AC;&#x2122;t align with the science of gender identity. This can lead the public to be swayed to also overlook scientific evidence as well, which can be dangerous for those who are alienated.

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ecently, U.S. politicians in 16 states have proposed bills governing which bathroom transgender individuals are allowed to use. All attempted bills have failed and been met with great opposition. North Carolina’s legislature managed to pass a piece of legislation nicknamed the “bathroom bill,” but it was later repealed [1]. There are various beliefs and opinions surrounding transgender individuals’ identities. Some believe that transgender individuals should be able to use the bathroom of the gender they identify with, or the bathroom they feel most comfortable using. Others suggest that sharing a bathroom with a transgender person may make it unsafe for cisgender individuals, those who identify with their assigned sex. However, there is no evidence that transgender individuals have harmed a cisgender person in a bathroom [2]. Legislation on transgender bathroom use can affect how individuals perceive each other and themselves. Therefore, it can have a significant impact on the lives of the target population. When a lawmaker proposes a bills that does not align with scientific findings, the public may be swayed to also discount or overlook scientific evidence. Therefore, bathroom bills and other legislation impacting transgender individuals should be constructed from research in the fields of social psychology, biology, and endocrinology, instead of scientifically unsupported opinions. Gender is complex and there are many exceptions to the dual categorization system of male and female. Canonically, a genetic female, possessing

XX chromosomes, will have more female hormones like estrogen and progesterone; a genetic male with XY genotype will have more testosterone. Through differences

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in hormones, chromosomal sex usually leads to corresponding body appearance. However, there are several cases where the chromosomal sex of an individual does not match their hormone production due to various naturally occurring disorders. One such abnormality is congenital adrenal hyperplasia, resulting in females with more male hormones. Another, androgen deficiency, is where a male will lack male hormones and have a more female appearance [3]. Many believe that gender categories are limited

"Through

differences in hormones, chromosomal sex usually leads to corresponding body appearance. However, there are several cases where the chromosomal sex of an individual does not match their hormone production due to various naturally occurring disorders.

to physical appearance; however, between .01% to 1.7% of infants are born intersex with ambiguous genitalia [4]. Whether it is due to hormonal disorders or being born intersex, many people do not fit within the dual categorization that is implied within bathroom bill legislations. Early in life, brain formation is influenced by surging hormones. Depending on whether the hormone estradiol is let into the brain early on, a brain becomes “masculinized” or “feminized” [5, 6]. Differences in brain structure are not functionally understood, but some differences do exist between the “feminized” and the “masculinized” brain. The relay center for motor systems, a structure called the basal ganglia stria terminalis, is larger in females than in males. Additionally, estrogen is associated with the brain’s communication between hemispheres across the corpus callosum to process language, whereas testosterone is associated with language processing on one side of the brain [7]. Researchers have observed that brain structures in male to female transgender individuals can be significantly different from a typical male and more comparable to female counterparts, suggesting that brain

structures may match their identity instead of their assigned sex [5, 8]. In fact, Dick Swaab, an expert in sex hormones and neurobiology, has found through various brain studies that gender identity is likely formed in the womb when the first surge of hormones occurs. He also found little evidence supporting the impact of social environment on gender identity [5, 9] . Concomitantly, this presents evidence that there is a biological response to gender identity. In fact, many believe that once gender identity is established, it is irreversible, confirming that gender identity should be considered over assigned sex, especially in proposed legislations regarding transgender individuals [9, 10]. From an early age, children have been shown to have different ways of playing that arise from hormone levels, categorized as male or female typical [11]. Females more traditionally play with dolls and draw, while males typically participate in rough and tumble play, and construct with blocks. However, females with more androgens, or male hormones, partake in male typical play, suggesting that hormones also have

"Researchers

have observed that brain structures in male to female transgender individuals can be significantly different from a typical male and more comparable to female counterparts, suggesting that brain structures may match their identity instead of their assigned sex.

an affect on the behavior of an individual. Individually, one may fall anywhere on a spectrum of male or female hormones within their body, again lessening support for a strict, dual categorization of female

and male [11]. Complex behaviors such as math skills, verbal skills, motor skills, and physical abilities, are less strongly linked to

making decisions that can have wide ranging effects on the public opinions and transgender individuals. -- Alina Dillahunt

"In fact, many believe that once gender identity is established, it is irreversible, confirming that gender identity should be considered over assigned sex, especially in proposed legislations regarding transgender individuals.

hormones than play [8]; socialization and hormones both play a role in the different abilities found between genders. Men tend to perform better in spatial tasks and target motor skills [8, 12]. Women generally excel in fine motor skills, verbal abilities, and perceptual speed. Social environment

affects the way children develop, the entities they choose to like, and the activities and subjects in school they seek out [13]. Parents often start socializing their children based on gendered stereotypes, often about type of play, sports, and math and writing performance [14]. Teachers continue this socialization in the classroom where boys are seen as more competent and rambunctious, and girls are expected to be quiet and polite. This leads to teachers calling on male students more often, and giving them more attention in the classroom [9]. Socialization plays a large role in one’s gender identity, and is another element that complicates a dual categorization of gender. Dividing gender into two rigid categories does not fully encompass every individual, whether that be because they have a hormonal disorder, they were born intersex, or their assigned sex does not match their identified sex. When making legislation, gender should be treated as a dual concept, and must be representative of all individuals, not just those that fit into social stereotypes. Legislators must take care to understand the research before

REFERENCES 1. Kralik, J. (2017, July 28). "Bathroom Bill" Legislative Tracking. 2. Bianco, Marcie (April 2, 2016). "Statistics Show Exactly How Many Times Trans People Have Attacked You in Bathrooms". 3. Beatty, W. W. (1979). Gonadal hormones and sex differences in nonreproductive behaviors in rodents: Organizational and activational influences. Hormones and Behavior, 12(2), 112-163. doi:10.1016/0018-506x(79)90017-5. 4. Blackless, M., Charuvastra, A., Derryck, A., Fausto-Sterling, A., Lauzanne, K. and Lee, E. (2000), How sexually dimorphic are we? Review and synthesis. Am. J. Hum. Biol., 12: 151–166. doi:10.1002/(SICI)15206300(200003/04)12:2<151::AIDAJHB1>3.0.CO;2-F. 5. Bao, A., & Swaab, D. F. (2011). Sexual differentiation of the human brain: Relation to gender identity, sexual orientation and neuropsychiatric disorders. Frontiers in Neuroendocrinology, 32(2), 214-226. doi:10.1016/j.yfrne.2011.02.007. 6. Bakker, J., Mees, C. D., Douhard, Q., Balthazart, J., Gabant, P., Szpirer, J., & Szpirer, C. (2006). Alpha-fetoprotein protects the developing female mouse brain from masculinization and defeminization by estrogens. Nature Neuroscience, 9(2), 220-226. doi:10.1038/nn1624. 7. Luders, E., Thompson, P. M., & Toga, A. W. (2010). The Development of the Corpus Callosum in the Healthy Human Brain. Journal of Neuroscience, 30(33), 10985-10990. doi:10.1523/ jneurosci.5122-09.2010. 8. Kimura, D. (2003). Sex differences in the brain. Scientific American-American Edition-, 287, 32-37. 9. Teacher Bias. (n.d.). The SAGE Encyclopedia of Psychology and Gender. 10. Bao, A., & Swaab, D. F. (2010). Sex Differences in the Brain, Behavior, and Neuropsychiatric Disorders.

The Neuroscientist, 16(5), 550-565. doi:10.1177/107385841037700. 11. Henderson, B. A. and Berenbaum, S. A. (1997), Sex-typed play in opposite-sex twins. Dev. Psychobiol., 31: 115–123. doi:10.1002/ (SICI)1098-2302(199709)31:2<115::AIDDEV4>3.0.CO;2-N. 12. Blum, D. (1997). Sex on the brain: The biological differences between men and women. Penguin. 13. McGurk, H. (1993). Childhood social development: contemporary perspectives. Hove: Lawrence Erlbaum. 14. Eccles, J. S., Jacobs, J. E., & Harold, R. D. (1990). Gender Role Stereotypes, Expectancy Effects, and Parents Socialization of Gender Differences. Journal of Social Issues, 46(2), 183-201.

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// PIXELS Nikki Noughani

The first week of July I had the incredible opportunity to go to Kitt Peak in Arizona and work on one of the telescopes owned by UW Madison. My team spent five nights observing various galaxies. Those I observed were particularly of interest because they were galaxies where the center spun in the opposite direction as the surrounding arms. It made my summer remarkable, and I am so thankful for having had the opportunity to go. And, hopefully, my current work on variable stars will have me returning next summer to observe my own discoveries!

Natalia Lucero

Natalia Lucero captured this close-up of a neotropical jumping spider poised on the edge of the pages of a book on a farm in Costa Rica. The spiderâ&#x20AC;&#x2122;s prominent eyes are uniquely structured for estimating the distance and direction of motion of prey, giving them remarkably accurate pouncing skills.*

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Celia Glime

This photo illustration depicts a range of colors produced in test tubes by three chemical reactions: cobalt, hydrochloric acid, and deionized water; copper chloride hexahydrate, ammonia, and deionized water; and copper chloride hexahydrate, deionized water, and sodium hydroxide.*

Kaylyn Freeman, Cengiz Lab

In a mouse, the presence of a protein called tyrosine kinase B (colored green in this image of mouse brain cells), protects neurons (colored blue) that might otherwise be damaged by lack of oxygen. UW Madison studies show female mice are more able to produce these protective proteins, hinting at the potential of a drug to protect newborns deprived of oxygen by pregnancy complications.*

*Printed with permission from UW Madison 2017 Cool Science Image Contest

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REPORT | Mouse TMEM135 overexpression exhibits protein regulation causing chronic inflammation and fibrosis of the heart

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The overexpression of mitochondrial transmembrane protein Tmem135 in mice causes mitochondrial dysfunction and fibrosis of the heart, but it is unknown which regulatory proteins influence this pathway to cause these effects. Here, we observed the modulation of numerous inflammatory pathways to link the effects of Tmem135 overexpression to inflammation and the immune system, thereby increasing understanding of how mitochondrial dysfunction leads to fibrosis. A culmination of current knowledge of these topics and Tmem135 will be discussed by following the molecular pathway of mitochondrial dysfunction through change in the expression of inflammatory enzymes such as Phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K), protein kinase B (PKB or AKT), mitogen-activated protein kinase 8 (MAPK8 or JNK), mitogen-activated protein kinase 14 (MAPK14 or p38), nuclear factor kappa-light-chain-enhancer of activated B cells (Nf-ÎşB), and beta actin (Î˛-actin). The protein concentrations for these inflammatory enzymes have been analyzed through western blotting.

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REPORT | The Gibbs Paradox - Towards an axiomatic approach We discuss the Gibbs Paradox in statistical mechanics from an axiomatic perspective. We begin by introducing the paradox to the reader and discussing some past attempts. We then discuss one attempt that invokes the real gas scenario and uses the van der Waals constants to distinguish between gases. As seen in our example, the simple axiomatic requirements we propose are difficult to fulfill. In closing, we elucidate the relevance of the Gibbs Paradox and the ideas and concepts it motivated in the field of physics.

REPORT | Comparison of inhaled corticosteroids, mast cell stabilizing agents, and leukotriene receptor antagonists in the treatment of exercise-induced bronchoconstriction: A metaanalysis

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While many individuals suffer from asthma in their everyday lives, a significant number are misdiagnosed with this condition rather than the true source, exerciseinduced bronchoconstriction (EIB). Of the pharmacological methods used to treat EIB, none are specifically intended to protect those who suffer from the condition, but are instead identical to treatments prescribed to asthma patients. This meta-analysis was designed to compare these treatments and investigate which pharmacological method best manages the symptoms of EIB: inhaled corticosteroids, leukotriene receptor antagonists, or mast cell stabilizing agents. This will allow those diagnosed with the condition to make a more informed treatment decision. We found that the mean percent fall from baseline in percent Forced Expiratory Volume (%FEV) values for inhaled corticosteroids, leukotriene receptor antagonists, and mast cell stabilizing agents were improved by 10.205%, 6.544%, and 25.850%, respectively (SD = 4.618, 4.218, 8.651; p < 0.0001) compared to the placebo. Based on the studies analyzed, mast cell stabilizing agents were determined to be the most effective treatment for EIB. This may be due to the drugâ&#x20AC;&#x2122;s direct impact on the mast cells, which cause inflammation through the release of histamine. With mast cell stabilizing agents, those with EIB may be better able to control the symptoms of the condition, exercise for longer periods of time, and have improved lung function following strenuous activity. Our findings suggest that those who are diagnosed with EIB should consider using mast cell stabilizing agents as a method to best manage their symptoms following exercise.

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MOUSE TMEM135 OVEREXPRESSION EXHIBITS PROTEIN REGULATION CAUSING CHRONIC INFLAMMATION AND FIBROSIS OF THE HEART BY | Erika Henningsen, Tetsuya Takimoto, Giangela Stokes, Sarah Lewis, Akihiro Ikeda

ABSTRACT The overexpression of mitochondrial transmembrane protein Tmem135 in mice causes mitochondrial dysfunction and fibrosis of the heart, but it is unknown which regulatory proteins influence this pathway to cause these effects. Here, we observed the modulation of numerous inflammatory pathways to link the effects of Tmem135 overexpression to inflammation and the immune system, thereby increasing understanding of how mitochondrial dysfunction leads to fibrosis. A culmination of current knowledge of these topics and Tmem135 will be discussed by following the molecular pathway of mitochondrial dysfunction through change in the expression of inflammatory enzymes such as Phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K), protein kinase B (PKB or AKT), mitogen-activated protein kinase 8 (MAPK8 or JNK), mitogen-activated protein kinase 14 (MAPK14 or p38), nuclear factor kappa-light-chain-enhancer of activated B cells (Nf-ÎşB), and beta actin (Î˛-actin). The protein concentrations for these inflammatory enzymes have been analyzed through western blotting.

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INTRODUCTION | Mitochondrial dysfunction can be caused by the overexpression of transmembrane protein 135 (Tmem135) [1]. Overexpression of Tmem135 in transgenic mice causes mitochondrial dysfunction through the increase in mitochondrial fragmentation. Fragmentation is an indicator that more fission than fusion has occurred in these mitochondrial cells. Fission is comparable to mitochondrial division and fusion is comparable to a mitochondrial union. In contrast to the fission shown with overexpression of Tmem135, it was previously observed that mice with mutated Tmem135 experienced excessive mitochondrial fusion, resulting in elongated mitochondria and mitochondrial dysfunction. Therefore, both excessive mitochondrial fission and fusion lead to mitochondrial dysfunction. This is because both fragmentation and elongation of mitochondria lead to impaired respiration and increase the production of reactive oxygen species (ROS) [1]. ROS are created through incompletely reduced oxygen molecules in a redox reaction and are damaging to cells as they activate redox-sensitive transcription factors, ultimately causing tissue inflammation [2]. This inflammation can become malignant to cells as chronic inflammation is a constant stressor for cells. Excessive fission, excessive fusion, and the subsequent production of ROS contribute to the mitochondrial dysfunction resulting in chronic inflammation. When damage or inflammation occurs, signals throughout an organism report the disturbance. These signals are proteins and chemical mediators released by injured tissues. They are often cytokines, histamines, bradykinins, and prostaglandins that elicit an immune response by attracting macrophages and phagocytes to clean up and destroy any damaged cells via apoptosis, the process of cell destruction, which is often self-regulated within cells. This process should result in a reduction of cell inflammation in the affected areas as cell tissues are replaced (Figure 1) [3]. This typically allows the regenerative process to end without

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any long-lasting damage. Some components of inflammation are designed to destroy microbes, but this can harm other healthy tissues surrounding the inflamed region. For this reason, inflammation must be tightly regulated by the immune system. Chronic inflammation can be poisonous to surrounding healthy cell tissue and can be initiated by persistent and progressive damage, prolonged exposure to toxic agents such as ROS, or inappropriate inflammatory responses in autoimmunity. Injury that is initially acute inflammation can become chronic inflammation and both can lead to fibrosis (Figure 1).

FIGURE 1 | The pathways and progression of acute inflammation to resolution, chronic inflammation and fibrosis.

During regeneration of damaged cells, reconstruction attempts to be identical to previous cell and tissue architecture. However, if the restructuring of cells is complex or there is an excessive amount of tissue damage, the formation of scar tissue and continuation of inflammation can occur. This process is known as fibrosis. Scar tissue is the buildup of fibroblasts that become one large fibrosis scar. The scarring continues until the inflammation around these cells can be shut down [4]. Initially, the structure recreated

METHODS | Gene Identification and Analysis Through DAVID Previously researched and identified functions were expressed through Gene Ontology (GO) terms. A predetermined gene list composed from preliminary data regarding genes related to Tmem135 was analyzed through the Database for Annotation, Visualization, and Integrated Discovery (DAVID) to yield a functional analysis of these genes and to produce their GO terms. Important genes and pathways related to chronic inflammation and fibrosis were identified using the GO terms. Connections were made with

existing sample data which showed that gene expression increase or decrease was correlated to the overexpression of Tmem135. The gene expression was represented by twotailed, unpaired t-tests. These were performed by comparing transgenic (n=4) and wild-type (n=4) in log2 expression values. Those with a p-value of <0.05 were considered differentially expressed. To find different genes relating to inflammation and fibrosis, the subject areas of interleukins (IL), integrin alphas (Itga) and betas (Itgb), transforming growth factors (Tfg), fibroblast growth factors (Fgf), and toll-like receptors were studied. There are numerous different pathways that genes use to produce inflammation in cells. These pathways often have similar protein expression that can be experimentally tested to learn how they affect inflammation pathways when over or under-concentrated in comparison to control groups. To observe how Tmem135 overexpression affects expression in these proteins, the findings were then compared to the gene trends observed in the sample data. KEGG Pathways Search data from pathways altering Tmem135 expression with GO terms was related to inflammation centered around the integrin (Itg) family. Its pathways to inflammation were analyzed through the Kyoto Encyclopedia of Genes and Genomes (KEGG Pathways). The examined Itg pathways were centered between extracellular matrix accumulation and inflammation in transgenic mice. The pathways were analyzed for both their connections to inflammation as well as their connections to other inflammation pathways. To confirm the change of expression was due to Tmem135, these inflammatory pathways were compared and mutual proteins were chosen for experimentation. Western Blotting Tissues from 4 wild type mice and 4 transgenic mice overexpressing Tmem135 were pooled and homogenized in RIPA buffer with a protease inhibitor cocktail. Equal amounts of protein were subjected to SDS-PAGE using a 12% Bis-Tris gel and primary antibodies against Phosphatidylinositol-4,5bisphosphate 3-kinase (PI3K; Cell Signaling Technology), protein kinase B (AKT; Cell Signaling Technology), mitogenactivated protein kinase 14 (p38; Cell Signaling Technology), phosphorylated mitogen-activated protein kinase 14 (phosphorylated p38; Cell Signaling Technology), mitogenactivated protein kinase 8 (JNK; Cell Signaling Technology), nuclear factor kappa-light-chain-enhancer of activated B cells (Nf-κB; Santa Cruz Biotechnology), and β-actin (β-actin; Abcam). Rabbit (Cell Signaling Technology) or mouse (Cell Signaling Technology) secondary antibodies were used and protein concentrations were detected with a chemiluminescent agent (Amersham ECL Plus Western blotting detection system, General Electric, Buckinghamshire, UK) and exposed to X-ray film (Thermo Scientific, Rockford, IL). Novex Sharp Pre-Stained Protein Standard was chosen as a loading control because it expresses a wide range of molecular weight proteins. Western blot analyses were

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by fibroblasts heals and sustains areas containing damaged tissue, but over time the accumulation of fibroblasts and their byproducts can do more damage than good. This can be seen in cases of acute and chronic fibrosis. When tissue regeneration occurs in the heart, damaged muscle cells are reconstructed with fibroblasts and will have a strong healthy fibrous base, but after some time, numerous fibroblasts lead to contraction difficulties [5]. This stems from the combination of extracellular matrix (ECM) and collagen buildup that stiffen the fibrous muscle tissue in the heart. Mitochondrial dysfunction leads to inflammation and fibrosis according to Lewis, which explains why the amount of collagen found in transgenic mice that overexpress Tmem135 is extreme [6]. The ECM synthesized by fibroblasts holds collagen, a known stiffening agent. When the ECM is improperly reorganized, it can create an even larger fibrotic scar. The increase in collagen causes fibrotic scars to be stiffer than normal tissue and significantly affects myocardial muscle tissue. As noted, scar tissue in the heart is a major problem because it is very dense and prevents efficient heart muscle contraction [5]. A condition consistently found in transgenic mice with Tmem135 overexpression is hypertrophy. Hypertrophy is defined as the enlargement of the heart—specifically in the left ventricle—due to the buildup of fibroblasts that significantly reduces contractility and left ventricular blood volume ejection. This occurs due to the excessive creation of fibroblasts, which cause an enlargement of the left ventricle similar to swelling. The volume of the ventricle within the heart shrinks as the buildup of fibroblasts increases, resulting in reduced contractility and an increased risk of heart failure. This can also cause the organ as a whole to grow [6]. Because cardiovascular disease and heart failure are the leading causes of death in the United States, it is important to look at the pathways involved in diseased phenotypes of cardiomyopathy. We documented the protein expression of inflammatory pathways affected by Tmem135 in order to explore the connection between mitochondrial dysfunction and inflammation that causes fibrosis of the heart in transgenic mice at a molecular level. Thus, we investigated Tmem135’s connection to cardiovascular complications via expression analysis and tested the causal relationship of Tmem135 overexpression to mitochondrial dysfunction, which leads to chronic inflammation and hypertrophy from fibrosis.

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performed in duplicate experiments for each genotype (total eight mice assayed/genotype) and these X-ray images of protein concentrations were quantified with ImageJ and Fiji.

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RESULTS | Gene Identification and Analysis Through DAVID Inflammatory response was a major GO term found through DAVID analysis. Other important GO terms were cell proliferation and extracellular matrix organization due to their known influence on fibrosis. Analyzed research related to inflammation showed genes within the Itg family, among others, to be statistically significant in expression between wild type and transgenic mice. KEGG Pathways Analysis of the DAVID-produced genes yielded many interconnected pathways. When examining the pathway for Itg affecting inflammation, some of the pathways were up-regulated and inhibited different proteins. Phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) was a recurring protein in the examined pathways, making it an excellent experimental protein for western blot analysis. This protein is a component of actin cytoskeleton pathways (Figure 2), focal adhesion pathways (Figure 3), and the PI3K signaling pathways (Figure 4). In the Itg pathway, it has been observed that the enzyme PI3K relies on the expression of focal adhesion kinases (FAK). This then allows for the expression of PI3K activating phosphatidylinositol (3,4,5)-trisphosphate (PIP3), which in turn activates the expression of protein kinase B (AKT). AKT, another protein of interest that is

also known as PKB, is connected to both anti-apoptotic and apoptotic pathways. AKT can either activate nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB), causing anti-apoptotic results or inhibits B-cell (Bcl) proteins which causes apoptotic results. NF-kB is activated when AKT phosphorylates IÎşB kinase (IKK). IKK then indirectly promotes NF-kB. This promotion allows the transcription of pro-survival genes, which are genes that survive apoptosis. Further analysis of the KEGG pathways led to the examination of mitogen-activated protein kinase 8 (JNK) and mitogen-activated protein kinase 14 (p38). These proteins are related to AKT and are found in the MAPK signaling pathway (Figure 5). Additionally, JNK is also found in the PI3K signaling pathways (Figure 4). JNK and p38 are separately activated through phosphorylation conducted by the actions of the mitogen-activated protein kinase kinase (MKK) family which are in turn activated by AKT. JNK and p38, as a result, both have the ability to phosphorylate various proteins that cause proliferation, differentiation, inflammation, and apoptosis. This ability to up-regulate apoptosis leads us to believe that these protein concentrations will be lower in Tmem135 mice as previous research shows extreme levels of fibrosis may potentially be caused by the inability to complete apoptosis [7]. PI3K Western Blotting The experimental western blot analysis analyzed the Itg pathwayâ&#x20AC;&#x2122;s effect on Tmem135 and focused on the expression of PI3K, a protein found within this pathway. The antibody used for western blot analysis was against

FIGURE 2 | In the Itg pathway, it is observed that the enzyme PI3K relies on the expression of FAK. This then allows for the expression of PI3K activating PIP3 which in turn activates the expression of AKT (not shown).

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FIGURE 3 | In the Itg pathway, it is observed that the enzyme PI3K relies on the expression of FAK. This then allows for the expression of PI3K activating PIP3 which in turn activates the expression of AKT. AKT is also known as PKB and is connected to both anti-apoptotic and apoptotic pathways. AKT and can either activate NF-kB, causing anti-apoptotic results or inhibit Bcl-2 and Bcl-xL causing apoptosis (not shown).

REPORTS FIGURE 4 | In the Itg pathway, it is observed that the enzyme PI3K relies on the expression of FAK. This then allows for the expression of PI3K activating PIP3 which in turn activates the expression of AKT. AKT is also known as PKB and is connected to both anti-apoptotic and apoptotic pathways. AKT and can either activate NF-kB, causing anti-apoptotic results or inhibit Bcl-2 and Bcl-xL causing apoptosis. NF-kB is activated when AKT phosphorylates IKK. IKK then indirectly promotes NF-kB. This promotion of NF-kB allows the transcription of pro-survival genes, genes that survive apoptosis.

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FIGURE 5 | In the Itg pathway, it is observed that the enzyme PI3K relies on the expression of FAK. This then allows for the expression of PI3K activating PIP3 which in turn activates the expression of AKT. AKT is also known as PKB and is connected to both anti-apoptotic and apoptotic pathways. AKT and can either activate NF-kB, causing anti-apoptotic results. AKT can inhibit Bcl-2 and Bcl-xL (not shown) or activate JNK and p38 through the MKK family to cause apoptosis and inflammation. NF-kB is activated when AKT phosphorylates IKK. IKK then indirectly promotes NF-kB. This promotion of NF-kB allows the transcription of pro-survival genes, genes that survive apoptosis and inflammatory signaling.

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PI3K- p110Îą (C73F8). For all western blot experiments, we compared expression between control wild type mice and transgenic Tmem135 mice. PI3K was increased in transgenic mice overexpressing Tmem135 by approximately 28.8% (Figure 6). These results produced a p-value of 0.0003 and are therefore significant. AKT Western Blotting The antibody used for western blot analysis was

FIGURE 6 | Western blot analysis demonstrates the upregulation of PI3K proteins in transgenic mice overexpressing Tmem135 compared to their wild type counterparts through the analysis of the gray area produced by the chemiluminescent presence of PI3K proteins. PI3K proteins were significantly upregulated by 28.8% in transgenic mice with a p-value of 0.0003.

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against Akt (Ser473) (D9E). We observed the expression of AKT in both wild type and transgenic Tmem135 mice. Western blot data for AKT produced inconclusive results. Expression of AKT was not seen on the blots as neither the wild type nor transgenic mice produced any of AKT proteins within the heart. P38 Western Blotting The antibody used for this blot analysis was against p38Îą MAP Kinase. We observed the expression of p38 in both wild type and transgenic Tmem135 mice. Western blot data for p38 showed a greater protein concentration for wild type mice and a decreased expression level in transgenic mice overexpressing Tmem135. The data showed an average 43.96% higher concentration of p38 in the wild type mice (Figure 7). This data yielded a significant p-value of 0.0128. Phosphorylated p38 Western Blotting Similarly, phosphorylated p38 (Phospho-p38) was examined with western blot analysis. The primary antibody used was against Phospho-p38 MAPK (Thr180/Tyr182) (D3F9) XP. Expression of Phospho-p38 was observed in wild type and transgenic mice. The phosphorylated p38 protein was expressed in greater concentration in wild type mice and not as greatly expressed for transgenic mice overexpressing Tmem135 (Figure 8). The mean concentration difference was 9.87% and the data was shown to be significant with a

FIGURE 8 | Western blot analysis demonstrates the downregulation of phospho-p38 proteins in transgenic mice overexpressing Tmem135 compared to their wild type counterparts through the analysis of the gray area produced by the chemiluminescent presence of phospho-p38 proteins. Phospho-p38 proteins were significantly downregulated by 9.87% in transgenic mice with a p-value of 0.0151.

FIGURE 9 | Western blot analysis demonstrates the downregulation of JNK proteins in transgenic mice overexpressing Tmem135 compared to their wild type counterparts through the analysis of the gray area produced by the chemiluminescent presence of JNK proteins. JNK proteins were significantly downregulated by 9.4% in transgenic mice with a p-value of 0.0002.

FIGURE 10 | Western blot analysis demonstrates the upregulation of Nf-| B proteins in transgenic mice overexpressing Tmem135 compared to their wild type counterparts through the analysis of the gray area produced by the chemiluminescent presence of Nf-| B proteins. Nf-| B proteins were significantly upregulated by 25.88% in transgenic mice with a p-value of 0.03.

p-value of 0.0151. JNK Western Blotting The antibody used for western blot analysis was a phospho-SAPK/JNK antibody (CST:9251). We observed the expression of JNK in both wild type and transgenic Tmem135 mice. The western blot data showed wild type mice to have a greater mean concentration of JNK by 9.4% (Figure 9). This data was shown to be significant with a p-value of 0.0002. Nf-κB Western Blotting The antibody used for western blot analysis was against Nf-κB p65 (A) (sc-109). We observed the expression of Nf-κB in both wild type and transgenic Tmem135 mice. Western blot data for Nf-κB showed a greater protein concentration for transgenic mice who overexpressed Tmem135 compared to wild type mice. The data showed that the concentration of Nf-κB was an average of 25.88% higher in the transgenic mice (Figure 10). This data is significant with a p-value at 0.03. Beta Actin Western Blotting A beta-actin antibody (ab8226) was used. We

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FIGURE 7 | Western blot analysis demonstrates the downregulation of p38 proteins in transgenic mice overexpressing Tmem135 compared to their wild type counterparts through the analysis of the gray area produced by the chemiluminescent presence of p38 proteins. P38 proteins were significantly downregulated by 43.96% in transgenic mice with a p-value of 0.0128.

observed the expression of β-actin in both wild type and transgenic Tmem135 mice. The wild type western blot data had a greater mean concentration of β-actin than transgenic mice by 2.85% (Figure 11). This data was shown to be not significant with a p-value of 0.7255. Therefore, there is no significant difference in the concentration of β-actin in wild type mice and transgenic mice.

DISCUSSION | Initial research began in an attempt to gain an understanding of the different genes and processes altered by the overexpression of Tmem135. After establishing the relationship to inflammation with GO terms, previous research was examined for genes related to inflammation and cross checked with preliminary data that expressed changes in gene expression of Tmem135 mice. This research was limited as there was an incomplete list of proteins affecting the inflammatory process, but was still crucial to the foundation of our subject. Pathways were examined surrounding the genes and gene families of IL15, Itgb6, Bcl6b, Tgfb2, Tgfb3,

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FIGURE 11 | Western blot analysis demonstrates the insignificant downregulation of β-actin proteins in transgenic mice overexpressing Tmem135 compared to their wild type counterparts through the analysis of the gray area produced by the chemiluminescent presence of β-actin proteins. β-actin proteins were insignificantly downregulated by 2.85% in transgenic mice with a p-value of 0.726.

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Fgf 12-27, Tlr2, Chil1, TLr4, Tlr13, SPP1, and NLRc3. These genes all showed statistically significant changes in expression when comparing wild type and transgenic mice in preliminary data. However, the integrin (Itg) family was chosen as the main focus of this research. We chose the integrin family as the basis for our study because of their known connections to extracellular matrix (ECM), collagen, and the inflammatory process. Integrins are the primary receptors of ECM proteins, like collagen, that work to bind cells together, but if bound too tightly these cells can develop fibrotic tendencies and become immovable [8]. Therefore, integrins are extremely relevant to fibrotic research. Pathways surrounding the Itg family were researched and analyzed to search for connections to inflammation. Keywords and GO terms such as inflammation, apoptosis, cell proliferation, cell differentiation, and cytokines were treated as important. This research led to the analysis of the pathways titled “Actin Cytoskeleton”, “Focal Adhesion”, “PI3K-AKT Signaling Pathway”, and the “MAPK Signaling Pathway” (Figures 2-5). The focal adhesion and PI3K-AKT signaling pathways found in the KEGG database began with ECM accumulation, and as we knew ECM was related to fibrosis, we strongly focused on the proteins in these pathways that lead to changes in inflammation or apoptosis (Figures 3-4). If the proteins in the Itg pathways were significantly upregulated, a relationship to fibrosis and heart failure in transgenic Tmem135 mice could be determined. This could suggest that Tmem135 upregulates proteins that result in inflammation, creating the opportunity for excessive fibrosis and heart failure. We addressed this stipulation through western blot analysis of proteins within the Itg pathway. Our analysis of the KEGG pathways led us to test the proteins PI3K, AKT, p38, JNK, Nf-κB, and β-actin with western blot analysis. We predicted that PI3K and AKT would be enriched in in Tmem135 mice due to the proteins’ role in cell survival (Figure 4). Our results substantiated our prediction for PI3K as the concentrations found were significantly higher in the transgenic mice. (Figure 6). By contrast, results for AKT were inconclusive. AKT was expected to have a similar pattern to PI3K as they share a pathway (Figure 4). It may be beneficial to test phospho-

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AKT by western blotting instead of AKT. Phospho-AKT may be the only form of AKT used in this pathway. Nf-κB was also predicted to be upregulated in transgenic mice due to its correlation to cell proliferation, inflammation, and anti-apoptosis (Figure 5). Western blotting confirmed this prediction with a significant p-value of 0.03. Nf-κB is also a key protein in cancerous cells, which are able to withstand apoptotic cell tendencies, and may explain the prevalence of this protein in fibrotic cells [9]. Nf-κB was also interesting because of the varying molecular weight of the protein. In future testing, it would be interesting to investigate if other bands of Nf-κB are expressed differently between transgenic and wild type mice. The p80 band may be expressed at greater concentrations than the wild type due to the band strength of Tmem135 mice in our current tests. The ability to up-regulate apoptosis led us to predict that the protein concentrations of p38 and JNK would be lower in Tmem135 mice as previous research showed extreme levels of fibrosis that could potentially be attributed to the inability to complete apoptosis [7]. We find that p38, Pp38, and JNK were downregulated proteins in terms of concentration in transgenic mice when compared to wild type mice (Figures 9-11). As these proteins share a similar pathway to apoptosis and an inhibitory pathway to each other, this data confirms the reason for downregulation. This data suggests that Tmem135 causes anti-apoptotic protein expression or downregulation of apoptotic expression due to fibrotic tendencies. During chronic inflammation, dysfunctional cells would not be destroyed on cue and would prolong their inflammatory tendencies, causing fibrosis instead of a resolution to inflammation (Figure 1). β-actin was predicted to be downregulated because of the lack of cardiac muscle contraction in fibrotic Tmem135 mice as actin is a key player in muscle movement. Conversely, β-actin insignificantly downregulated in Tmem135 mice compared to wild type mice (Figure 11). As the concentrations of β-actin were similar between transgenic and wild type mice, β-actin may serve as a base protein where consistent results are seen between phenotypes. In closing, a Tmem135 overexpression in mice may serve as an effective model for studying aspects of inflammation, collagen accumulation, and fibrosis of the heart. Recently, many studies have begun to show further dangers of chronic and acute inflammation. The correlation of cell dysfunction to inflammation and fibrosis in the heart can help in understanding the damage that uncontrolled inflammation can cause in other tissues within the body. Most importantly, a better understanding of mitochondrial dysfunction, factors that cause inflammation, and the protein regulation involved in fibrosis can all be valuable in evolving future lifesaving research. REFERENCES | 1. Lee, W.H. Mouse Tmem135 mutation reveals a mechanism involving mitochondrial dynamics that leads to age-dependent retinal pathologies. eLife. 2016; 5:e19264.

30(1):e1-e8. 19. Negmadjanov U, Godic Z, Rizvi F, Emelyanova L, Ross G, Richards J et al. Tgf-beta 1-mediated differentiation of fibroblasts is associated with increased mitochondrial content and cellular respiration. Plos One. 2015; 10(4). 20. Strutz F, Zeisberg M. Renal fibroblasts and myofibroblasts in chronic kidney disease. Journal of the American Society of Nephrology. 2006; 17(11):2992-2998. 21. Su ZL, Yin JP, Wang T, Sun YK, Ni P, Ma R et al. Upregulated hmgb1 in eam directly led to collagen deposition by a pkc beta/erk1/2-dependent pathway: Cardiac fibroblast/ myofibroblast might be another source of hmgb1. Journal of Cellular and Molecular Medicine. 2014; 18(9):1740-1751.

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2. Lopez-Armada MJ, Riveiro-Naveira RR, Vaamonde-Garcia C, Valcarcel-Ares MN. Mitochondrial dysfunction and the inflammatory response. Mitochondrion. 2013; 13(2):106-118. 3. Schmidt-Bleek K, Kwee BJ, Mooney DJ, Duda GN. Boon ad bane of inflammation in bone tissue regeneration and its link with angiogenesis. Tissue engineering Part B, Reviews. 2015; 21(4):354-364. 4. Wynn TA. Cellular and molecular mechanisms of fibrosis. Journal of Pathology. 2008; 214(2):199-210. 5. Burlew BS, Weber KT. Connective tissue and the heart: Functional significance and regulatory mechanisms. Cardiology Clinics. 2000; 18(3):435-442. 6. Lewis, S. Manuscript in Progress. University of Wisconsin-Madison. 2017. 7. Gawlik, Kinga I. et al. Potent pro-Inflammatory and pro-fibrotic molecules, osteopontin and galectin-3, are not major disease modulators of laminin Îą2 chain-deficient muscular dystrophy. Scientific Reports. 2017; 7: 44059. 8. Alberts B, Johnson A, Lewis J, et al. Molecular Biology of the Cell. 4th edition. Garland Science, New York; 2002. 9. Karin M. Nf-kappa b as a critical link between inflammation and cancer. Cold Spring Harbor Perspectives in Biology. 2009; 1(5):14. 10. Abbas AK, Aster JC, Kumar V. Robbins Basic Pathology. Elsevier Inc, Canada; 2013. 11. Biernacka A, Frangogiannis NG. Aging and cardiac fibrosis. Aging and Disease. 2011; 2(2):158-173. 12. Cuneo AA, Autieri MV. Expression and function of anti-inflammatory interleukins: The other side of the vascular response to injury. Current Vascular Pharmacology. 2009; 7(3):267-276. 13. Frentzou GA, Drinkhill MJ, Turner NA, Ball SG, Ainscough JFX. A state of reversible compensated ventricular dysfunction precedes pathological remodelling in response to cardiomyocyte-specific activity of angiotensin ii type-1 receptor in mice. Disease Models & Mechanisms. 2015; 8(8):783-U418. 14. He Q, Zhou W, Xiong CJ, Tan G, Chen MH. Lycopene attenuates inflammation and apoptosis in postmyocardial infarction remodeling by inhibiting the nuclear factor-kappa b signaling pathway. Molecular Medicine Reports. 2015; 11(1):374-378. 15. Hummasti S, Hotamisligil GS. Endoplasmic reticulum stress and inflammation in obesity and diabetes. Circulation Research. 2010; 107(5):579-591. 16. Kong YP, Carrion B, Singh RK, Putnam AJ. Matrix identity and tractional forces influence indirect cardiac reprogramming. Scientific Reports. 2013; 3. 17. Liew FY, McInnes IB. Role of interleukin 15 and interleukin 18 in inflammatory response. Annals of the Rheumatic Diseases. 2002; 61:100-102. 18. Lijnen PJ, van Pelt JF, Fagard RH. Stimulation of reactive oxygen species and collagen synthesis by angiotensin ii in cardiac fibroblasts. Cardiovascular Therapeutics. 2012;

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THE GIBBS PARADOX - TOWARDS AN AXIOMATIC APPROACH BY | Aniket Sudeep Dalvi and Chinmaya Bhargava ABSTRACT

We discuss the Gibbs Paradox in statistical mechanics from an axiomatic perspective. We begin by introducing the paradox to the reader and discussing some past attempts. We then discuss one attempt that invokes the real gas scenario and uses the van der Waals constants to distinguish between gases. As seen in our example, the simple axiomatic requirements we propose are difficult to fulfill. In closing, we elucidate the relevance of the Gibbs Paradox and the ideas and concepts it motivated in the field of physics. the impermeable membrane will be equal to S12. A standard relation of the entropy of gas is described by the equation,

where ÎŠ represents the number of states of the system and kB is the Boltzmann constant. State, here, is defined as the condition of a system at a specific time which is specified by state variables like temperature and pressure. The Gibbs Paradox was originally considered by Josiah Willard Gibbs in his paper On the Equilibrium of Heterogeneous Substances. It involves the change of the entropy of mixing when moving from dissimilar gases to similar ones. This change is paradoxical to the continuous nature of entropy itself with respect to equilibrium and irreversibility in thermodynamic systems. Irreversibility here describes the nature of a process which cannot return to its original state. The entropy of an irreversible process always increases. Let us consider n1 and n2 moles of two dissimilar ideal gases 1 and 2 at constant temperature and pressure having volumes V1 and V2 at temperature T1 and T2 and pressure P1 and P2 respectively such that T1 = T2, and P1 = P2, present in a container separated by an impermeable membrane. Let these gases be at entropies S1 and S2, respectively. By the ideal gas law PV = nRT, where R is the Universal gas constant, we have,

Now, considering mole fraction of gas a, X = n1/n = V1/V and n = n1 + n2 we can rearrange equation (5) as,

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INTRODUCTION | In physics, there are two distinct paradoxes which are both known as the Gibbs paradox and are often confused with each other [1]. The one we discuss in this paper falls within the realm of thermodynamics. It addresses the fact that the entropy increase when combining two gases of different kinds is independent of the degree of similarity between the two kinds of gases, and that this entropy increase changes instantaneously at the transition from dissimilar to similar gases. Entropy here is defined as the measurement of disorder in a system. It is typically given by the Boltzmann equation,

Dividing these equations and using the given conditions, we get

Now the entropy when these two gases are mixed by removing

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which does not include any variables that point to the nature of the gases [2]. Hence, entropy S1 for gas 1 is given by and entropy S2 for gas 2 is given by therefore,

For the specific case of X = 1/2, i.e, taking equal amounts of gases, we get equation (8) as, which is non-zero. From equation (10) we can safely conclude that the change in entropy upon mixing two gases is independent of their nature. This, therefore, means that when mixing two similar gases, the change in entropy must be some finite value given by equation (10), which is zero only when X = 0 and (1 - X) = 0, that is, the mole fraction of either of the gases is zero. However, we know that the change in entropy of mixing two similar gases is zero. This can be deduced by the Boltzmann equation (equation (1)) as similar gases will have the same state variable. We thus have a paradox, as two ways of deduction give two distinct values. In this work, we propose an axiomatic approach towards resolving the Gibbs Paradox. The basis of the resolution is to incorporate variables that point to the nature of the gas into the calculation of the change in entropy in such a way that it vanishes for similar gases, providing continuity. A particular attempt in which the nature of the gases is parametrized using the van der Waals constants is worked as a negative example for implementing our axiomatic approach. PREVIOUS ATTEMPTS AT RESOLVING THE PARADOX |

One historically notable attempt at resolving the paradox was by realizing that if the two gases are composed of indistinguishable particles, they obey different statistics than if they are distinguishable. Since the distinction between the particles is discontinuous, so is the entropy of mixing. The resulting equation for the entropy of a classical ideal gas is known as the Sackur-Tetrode equation. This equation is an expression for the entropy of a monatomic classical ideal gas which incorporates quantum considerations that give a more detailed description of its regime of validity:

cubic equation using Mathematica yields,

where N is the number of particles in the gas, U is the internal energy of the gas, kB is the Boltzmann's constant, m is the mass of a gas particle, and h is Planck's constant. If equation (11) is used, the entropy value will have no difference after mixing two parts of the identical gases. Here, the term:

where

was introduced to resolve the paradox. This effectively makes the particles of the gas indistinguishable, as all the N! permutations of these N particle gas are identical and should be counted as one due to permutation symmetry. Rewriting this term using the Sterling Approximation for large N, ln(N!) = Nln(N) - N, the resulting equation is simplified to equation (11). However, this approach does not explore the possibility of resolving the paradox using properties of gases that distinguish them from each other. Our axiomatic resolution described below attempts to do so.

Now, as V explicitly depends on a and b which are unique for each gas, it implies that change in entropy (Î&#x201D;S) also explicitly depends on the nature of the gas (from equation (5)). Let us now look at another approach of resolving the paradox. Consider a function F defined as follows:

where a and b are the van der Waal's constants, which are unique for each gas, and describe the correction for molecular forces and the volume of one mole of the atoms or molecules respectively. Rearranging (13), we get which on further expansion gives, On transposing the terms on the right hand side to the left hand side, we get,

and

As volume V cannot be negative or complex, we get u = 1

where U is the energy of a purely mechanical system. This function F is known as the Free Energy of the system. Now, it has been previously established that and

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ATTEMPTED RESOLUTION USING VAN DER WAAL'S CONSTANTS | The mathematical outline of the paradox described earlier was based on the ideal gas equation. One potential attempt to resolve the paradox comes from considering the modified ideal gas equation for real gases, which is,

where uk can take three values:

where W is the external work done, Cp and Cv are the thermal capacities at constant pressure and volume respectively, and m is the constant of integration [3]. Substituting (23) and (24) in (22), we get Consider a function G defined as:

where F is the free energy of the system and G is defined as the Gibbs free energy of the system. As we know that CP = CV - R [3], substituting this and (25) in the expression for Gibbs free energy we get, Now, using van der Waal's corrections we get, Entropy as defined in the terms of G is,

which is a cubic equation of V. Solving for the roots of this

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Applying (29) to (28), we get

where M = (CPlogT - RlogP + a + RlogR + Rlogn). From equation (29) we have,

where a1, b1 and a2, b2 are the van der Waal's constants for both the gases respectively. The mixing rule used here to determine the van der Waal's constant of the mixture of the gases is a = Xa1 + (1 - X) a2 and b = Xb1 + (1 - X)b2, where X and (1 - X) are the mole fractions of the gases respectively, which in this case are equal to 1/2 as we take equal moles of each gas [4]. This equation also shows that S depends explicitly on van der Waals's constants a and b, therefore ******S, too, depends explicitly on the nature of the gas.

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AXIOMATIC CONSIDERATIONS | Now, on considering real gases we know that the change in entropy is dependent on the nature of the gas. Thus, for similar gases such that a and b are the same, all the terms must get cancelled. This leads the entropy to be zero and averts the paradox. However, this does not occur as the above derived equation is nonlinear in n, that is, the highest power of n is not 1, contrary to a linear function which has a highest order of 1 for the variable of that function. This implies that thereby reinstating the Paradox. Thus, our first axiom is that in order to resolve the paradox it is necessary to have linearity in n such that Furthermore, our second axiom is that it is also necessary for the equation to be non-linear in a and b, such that, This ensures that they do not get cancelled for dissimilar gases, thereby nullifying their purpose. IMPLICATIONS AND IMPORTANCE | Up until now, this paper has discussed what the Gibbs Paradox is, past attempts at resolving the paradox and our axiomatic approach at resolving it. It is however, vital to understand the significance of the paradox and why analyzing it is important. The Gibbs Paradox played an extremely important role in the emergence of the concept of indistinguishable

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particles. These are particles that cannot be told apart from each other. If two indistinguishable particles interact in a process and are interchanged to form a new state, then this state is in no way different from the original state and must be considered the same state. It can be seen how the Gibbs Paradox fits this description, and the quantum mechanics approach uses this very principle to resolve the paradox. The theory of blackbody radiation postulated by Max Planck was another development that independently contributed to this concept of indistinguishable particles. Planck postulated the presence of photons that were indistinguishable from each other. This indistinguishability of particles has some important implications in the field of statistical mechanics as well. The primary one of these is the alteration in the partition function which describes the statistical properties of a system in a state of thermal equilibrium. Moreover, an important similarity that we can draw between the Boltzmann equation and quantum mechanics, is that both of them depend explicitly on states that play an important role in distinguishability in both cases respectively. Furthermore, the indistinguishability of particles had a profound impact on their statistical properties and behavior, playing an important role in the birth of quantum statistics. CONCLUSION | The Gibbs Paradox tells us that upon mixing two gases, a change in entropy that is independent of the nature of the gas occurs. This implies that even when mixing two same gases, there is a finite value of entropy change that is obtained, which evidently should not happen. This gives rise to the version of paradox considered here. We identify certain axiomatic requirements that are fundamental to solving the paradox. The first one is the requirement of linearity in the number of moles of the gases, and the second is non-linearity in the respective van der Waal constants of the gases. Although these requirements are simple, implementing them may not be straightforward, as is shown by our detailed example using the van der Waals gas constants. ACKNOWLEDGEMENTS | This work was carried out as a joint project at the Centre for Fundamental Research and Creative Education (CFRCE), Bangalore, India and the Poornaprajna Institute of Scientific Research (PPISR), Bangalore, India. We thank R. Srikanth for suggesting to us this problem to work on. We thank him, S. Aravinda, B. S. Ramachandra, B. R. Pratiti, Manogna Shastry, Vasudev Shyam, Madhavan Venkatesh and Stefan Westerhoff for related discussions, suggesting references, and reviewing certain versions of this paper.

REFERENCES | 1. Peters H. Demonstration and resolution of the Gibbs paradox of the first kind. 2013. Available from: arXiv:1306.4638v2. 2. Jaynes ET. The Gibbs Paradox. 1996. 3. Fermi E. Thermodynamics. 1936. 4. Newman A. Comparison of Mixing Rules for a van der Waals Gas Mixture. 2001.

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COMPARISON OF INHALED CORTICOSTEROIDS, MAST CELL STABILIZING AGENTS, AND LEUKOTRIENE RECEPTOR ANTAGONISTS IN THE TREATMENT OF EXERCISE-INDUCED BRONCHOCONSTRICTION A META-ANALYSIS BY | MATTHEW KUIK, TOM SCHNEIDER, BRYAN JACKLER

ABSTRACT While many individuals suffer from asthma in their everyday lives, a significant number are misdiagnosed with this condition rather than the true source, exercise-induced bronchoconstriction (EIB). Of the pharmacological methods used to treat EIB, none are specifically intended to protect those who suffer from the condition, but are instead identical to treatments prescribed to asthma patients. This meta-analysis was designed to compare these treatments and investigate which pharmacological method best manages the symptoms of EIB: inhaled corticosteroids, leukotriene receptor antagonists, or mast cell stabilizing agents. This will allow those diagnosed with the condition to make a more informed treatment decision. We found that the mean percent fall from baseline in percent Forced Expiratory Volume (%FEV) values for inhaled corticosteroids, leukotriene receptor antagonists, and mast cell stabilizing agents were improved by 10.205%, 6.544%, and 25.850%, respectively (SD = 4.618, 4.218, 8.651; p < 0.0001) compared to the placebo. Based on the studies analyzed, mast cell stabilizing agents were determined to be the most effective treatment for EIB. This may be due to the drug’s direct impact on the mast cells, which cause inflammation through the release of histamine. With mast cell stabilizing agents, those with EIB may be better able to control the symptoms of the condition, exercise for longer periods of time, and have improved lung function following strenuous activity. Our findings suggest that those who are diagnosed with EIB should consider using mast cell stabilizing agents as a method to best manage their symptoms following exercise.

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INTRODUCTION | Exercise-induced bronchoconstriction (EIB) is a distinct form of airway hyperresponsiveness, in which the airways narrow significantly and forcefully after bouts of strenuous exercise due to a range of bronchoconstrictor stimuli [1]. The severity of EIB is largely dependent upon the individual, the type of activity, and the environmental conditions in which the exercise is done. While common symptoms of EIB include coughing, wheezing, chest tightness, dyspnea, and fatigue, the extent of the symptoms can range from minor impairment in performance to severe bronchospasm and respiratory failure, although the latter is much less prevalent [2]. A bronchospasm is an acute, transient constriction of the muscles lining the bronchioles. There are three separate phases that EIB occurs in: severe bronchospasm, the refractory period, and minor bronchospasm. Because airway hyperresponsiveness is a key indicator of asthma, EIB is reported frequently in asthmatic patients. However, recent research has found that EIB is also prevalent in the absence of chronic asthma in populations of athletes and children [3]. It is estimated that EIB occurs in up to 90% of asthmatic patients; it also has an incidence of greater than 10% of the nominally non-asthmatic general population [2]. Another difference between asthma and EIB is the presence of mast cell activation following exercise. Mast cells are a type of white blood cell that release inflammatory histamines in response to certain stimuli. In patients with asthma, there is a distinct absence of mast cell activated inflammation, where no increase is observed in histamine or white blood

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cell count measured from bronchoalveolar lavage following exercise tests, while activation and inflammation are present for those with EIB [4]. Another key difference between the conditions is their effect on resting lung capacity. Those with chronic, uncontrolled asthma will normally wheeze while at rest and have abnormal peak airflow, whereas those with EIB have normal lung function and peak airflow at rest. Asthma is typically treated through long-term therapy with additional medications before or during exercise. By contrast, EIB can be treated with medication exclusively before periods of exercise, as symptoms only occur at a specific level of exertion [4]. The mechanism of EIB is not completely understood, and may be multifactorial; the main mechanisms that have been distinguished are airway cooling due to the intake of dry air and thermal reheating of the airways post-exercise. Reheating increases hydrostatic pressure in the capillaries, leading to a swelling of the airways. Similarly, breathing in dry air causes osmotic and thermal changes resulting in water loss from the airway surface. Receptors respond to this stimulus by producing excess mucus to rewet the pathways. The dryness also provokes coughing to remove material that is not being swept up by cilia in the bronchial tubes [2]. Airway exposure to environmental pollutants like chlorine in swimming pools can also place individuals at a higher risk for developing EIB. It is believed that environmental conditions such as these may act as allergic “triggers,” leading to bronchospasms by promoting inflammatory responses [2]. The pharmacotherapeutic agents used to prevent EIB are typically the same treatments used to control the

The purpose of this meta-analysis is to analyze and compare the effectiveness of ICS, LTRAs, and MCSA treatments on controlling the symptoms of EIB. This was assessed by analyzing studies that examined the effects of these drugs on the change in %FEV over the course of one minute in individuals diagnosed with EIB. FEV has become a popular method for analyzing pulmonary function as it offers a simple, noninvasive approach to objectively measure lung capability. The lower the %FEV fall value, the better the lungs are functioning due to reduced airway obstruction [10]. While it is generally understood that ICSs are recommended as the initial therapy to treat the symptoms of asthma and EIB in newly diagnosed patients, we believe that leukotriene modifiers will have a greater impact on managing the effects of EIB. This is because leukotriene modifiers act directly on G-protein coupled receptors which cause inflammation, allowing a preventative control of EIB instead of reducing the effects after an attack has occurred [11]. The mechanisms of an ICS treatment are meant to treat widespread inflammation through the repression of a multitude of genes in cells throughout the body, whereas LTRAs target specific receptors in the body to reduce mast cell response [5, 12]. Identifying the most effective pharmacological treatment method for EIB will enable patients to make a more informed decision about their treatment regiment. METHODS | Literary search: In order to identify studies that reported the effects of ICS, LTRA, and MCSA on the forced expiratory volume of individuals diagnosed with EIB, we utilized the electronic databases of PubMed and Web of Science. To generate a list of studies, we used keywords such as “inhaled corticosteroids,” “leukotriene receptor antagonists,” “mast cell stabilizing agents,” “exercise-induced bronchoconstriction,” “EIB,” and “FEV.” We also included articles found through searches in the citations of both review articles and studies that held relevant information and met the criteria as explained in the following Study Selection section. Study Selection: Of the studies found, we included only those that met the following criteria: studies of at least 15 subjects; the subjects had a minimum fall in %FEV of 10% after exercise; a standard exercise test was completed in the lab; reported dosage used in each trial; and reported both baseline and post-treatment mean FEV test results reported as “maximum fall in %FEV.” Studies were excluded if the dose of the drug being tested had been paired with another medicinal method or if more than 50% of the original subjects had dropped out of the study due to exacerbated asthma symptoms. Studies that utilized bias-reducing methodologies such as the doubledummy technique, where the placebo and treatment are administered blindly in alternating trials, were considered more relevant to our meta-analysis. Statistical Analysis: For each of our individual treatments, we found

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mechanisms that induce asthma, even though it is known that they may not coincide. This leads to varying degrees of effectiveness when treating the symptoms of EIB alone [3]. There are currently many recommendations for treating EIB, the three most prominent being inhaled corticosteroids (ICS), leukotriene receptor antagonists (LTRA), and mast cell stabilizing agents (MCSA). It has been shown that inhaled corticosteroids (ICS) not only control the symptoms of EIB, but also improve pulmonary function. Corticosteroids are a part of the larger class of steroids called glucocorticoids, which bind to glucocorticoid receptors in a cell to regulate gene expression, including the repression of cytokines that cause inflammation [5]. Different ICSs will vary in their therapeutic and adverse effects depending upon the individual using the medicine because the mechanism for targeting only genes that produce therapeutic effects has yet to be found [5]. While ICSs have been used as an effective, long-term method of managing the symptoms of both asthma and EIB, there are many adverse effects that can occur if the corticosteroid begins to bind a receptor that affects an incorrect gene. These adverse effects include glaucoma, cataracts, reduced growth in children, osteoporosis in the elderly, and adrenal suppression [5]. The objective in prescribing ICS for a therapy treatment is to use the lowest dose possible, while also limiting the risk of treatment failure, which can vary individually. Leukotriene receptor antagonists (LTRAs), also called leukotriene modifiers, reduce the effects of EIB and protect airways from bronchospasms by blocking the actions of leukotrienes, making exposure and inhalation of various pollutants less likely to cause a bronchospasm. Leukotrienes are a class of inflammatory chemicals that are released from the body when contact with an allergen occurs [1]. In this analysis, the focus will be on Montelukast, the most commonly used LTRA. Leukotriene modifiers like Montelukast have shown no tolerance formation or exacerbation of rebound impaired of lung function after use of the drug has ceased [2]. However, there are side effects when taking Montelukast, ranging from psychological changes such as mood disorders and hallucinations to increased risk of upper respiratory infections and coughing [6]. The third pharmacological method in treating EIB is use of mast cell stabilizing agents (MCSA). One example is cromolyn sodium, which prevents mast cells from releasing cytokines, small proteins that attract histamines, causing the vasodilation and inflammation seen in EIB [2]. Cromolyn sodium is one of the most commonly used mast cell stabilizers for treating EIB; however, the exact mechanism that makes it effective at decreasing the symptoms of EIB is unknown [7]. Cromolyn sodium is fast-acting and can result in an inhibition in decrease of %FEV, a metric that reflects the degree of ease of a person’s breathing, as quickly as just one minute after initial treatment [8]. Inhaling cromolyn sodium has not shown any long-lasting negative side effects, but some milder side effects include a bad taste in the mouth, coughing, nausea, and throat irritation and dryness [9].

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the mean %FEV fall compared to mean baseline and placebo falls. Taking these values, we used a single-factor Analysis of Variance (ANOVA) test to find any statistically significant differences between the treatment types. We used an alpha level of 0.05 to determine statistical significance of the data. While the ANOVA test gave us a total comparative p-value for all three treatment types, a Tukey test was also completed to compare the data of two treatment types at a time in order to find if there were any significant differences between them. RESULTS | Inhaled Corticosteroids (ICS): When analyzing all ten studies focusing on ICS as the therapeutic treatment for EIB, there were a total of 493 participants, with an age range from 6 to 45 years old. The average length of the studies was 10.6 weeks. Nine of the ten used a double-blind procedure for the study. The mean fall in %FEV for all placebo trial groups was 21.68% (SD 6.69), while the mean fall in %FEV for treatment groups using ICS was 9.81% (SD 5.53). An ANOVA test produced a p-value of 0.0001, showing that there is a statistically significant difference between the % fall in FEV in the placebo-controlled group and in the ICS trial group, as seen in Figure 1.

FIGURE 2 | Differences in mean % FEV fall compared to placebo and LTRA. A p-value of 0.0390 was found, showing a statistically significant difference between placebo and treatment. Mean fall in %FEV from baseline for the placebo treatment was 22.66% (SD 8.18), while mean fall in %FEV from baseline for LTRA treatment was 16.39% (SD 6.92).

fashion and three were single-blind. Across these studies, the 99 total subjects ranged from ages 7 to 49 years old. Studies were as brief as one day and as lengthy as six weeks. Inhaling saline or substances that tasted similar to the MCSA were used as placebos in all studies. The mean fall in %FEV for the placebo group was 44.08% (SD 6.86%), while the mean fall in %FEV for the cromolyn sodium MCSA group was 18.21% (SD 5.34%) (Figure 3). An ANOVA test was run comparing fall in %FEV of the placebo to fall in %FEV after inhaling MCSA, and the p-value of this data set was 0.0013, showing significance.

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FIGURE 1 | Differences in mean % FEV fall compared between placebo and ICS. A p-value of 0.0001 was found, showing a statistically significant difference between placebo and treatment. Mean fall in %FEV from baseline for the placebo treatment was 21.68% (SD 6.69), while mean fall in %FEV from baseline for ICS treatment was 9.81% (SD 5.53). Error bars are representing the standard deviation.

Leukotriene Receptor Antagonist (LTRA): Of the six studies focusing on the LTRA drug Montelukast, five utilized double-blind, randomized crossover studies, and one used a double-blind non-crossover study. 252 people with ages ranging from 4 to 65 participated in the trials. Tests ranged from under 24 hours to over 8 weeks of treatment. The mean %FEV falls for placebo and LTRA Montelukast treatment groups were 22.66% (SD 8.18) and 16.39% (SD 6.92), respectively. Using ANOVA, a significant difference between the placebo and treatment groups was found, with a p-value of 0.0390 (Figure 2). Mast Cell Stabilizing Agents (MCSA): From the six placebo-tested studies that sought to quantify the effects of the mast cell stabilizing agent cromolyn sodium, three studies were conducted in a double-blind

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FIGURE 3 | Bar graph with standard deviations of mean % FEV fall comparing the placebo to MCSA treatment. The p-value for this data is 0.0013, showing significance. The mean fall in %FEV from baseline for the placebo treatment was 44.08% (SD 6.86), while the mean fall in %FEV from baseline for the MCSA treatment was 18.21% (SD 5.34).

Comparison: Our results have found that all three treatment types are effective in limiting the symptoms of EIB, but with varying degrees of effectiveness. The difference between placebo and treatment fall in %FEV was calculated for ICS (10.205%; SD 4.618), LTRA (6.544%; SD 4.218), and MCSA (25.850%; SD 8.651). The ANOVA run produced a p-value of <0.0001, showing that all three medicinal treatments reduced the effects of EIB by decreasing %FEV fall from baseline compared to placebo treatments (Figure 4). A Tukey test was then used to

determine significance between these three treatment groups by comparing each drug’s reduction in % fall in FEV in three separate ways; ICS:LTRA, ICS:MCSA, and LTRA:MCSA. It was found that ICS and LTRA were not significantly different; however, the differences in % fall of FEV using MCSA treatment were significantly higher than both ICS and LTRA treatments, where both comparisons had a p-value of <0.01 (Figure 4).

FIGURE 4 | Comparison of treatment types when change in % fall in FEV was found through subtractive treatment %FEV fall values from placebo %FEV fall values (placebo – treatment % fall in FEV). A significant difference exists when comparing the effectiveness of MCSA to ICS (p < 0.01) and MCSA to LTRA (p < 0.01).

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DISCUSSION | Our analysis finds MCSA as a more effective treatment for EIB than either ICS or LTRA. The MCSA treatment proved to be a significantly more effective pharmacological solution for EIB symptoms. This finding may be due to the fact that mast cells are the direct cause of inflammation in the lungs. Targeting mast cells may be important through the blocking of calcium ion flow, preventing the release of the inflammatory agent histamine [7]. When analyzing treatments of ICS for EIB, it was found that the maximal fall in %FEV was lower in the treatment group than in the placebo group. When comparing the effectiveness of ICS therapy to the placebo, there was a statistically significant difference (p-value = 0.0001) (Figure 1). These results were as we had expected, as ICS are used widely to repress the symptoms of inflammation for patients with asthma, as well as being prescribed for individuals with EIB. In the analysis of LTRA treatments for EIB, it was found that the mean %FEV fall was lower in the treatment group than the placebo group (Figure 2). This was expected as LTRAs, like Montelukast, are used actively in the pharmaceutical industry to treat asthma and EIB, supporting that LTRAs are a viable form of EIB reducer. Differences in the studies’ methodologies may have led to variability, such as the baseline FEV values, as differences among participants may show some importance in the responses to each treatment. An interesting difference between the three classes of drugs was the difference in duration of the treatment. Studies on ICS tended to focus primarily on long term analysis of its effect on pulmonary inflammation. Since it is a treatment

associated more with asthma than EIB, it was not studied with the sudden change in lung function associated with EIB. The MCSA and LTRA studies focused more on shorter term effects, as both are used as fast acting responders after exercise-induced airway obstruction has occurred. Even though these are all viable ways to treat EIB, as seen in the individual analyses for each treatment type, they are used in different ways. Our findings suggest that MCSA treatment may be a better alternative than ICS and LTRA for the control of EIB symptoms. We believe that this is primarily because MCSA blocks histamine release and consequently shuts down inflammation at its root [7]. Future studies may be able to help affirm our findings through a wider range of testing. Long term MCSA and LTRA treatment need to be further studied, as these studies are less prevalent. This testing could be useful in determining which treatment provides the most beneficial balance between cost, FEV outcome, and convenience for the patient. Additionally, a future study could attempt to negate the differences among individuals by giving each participant each treatment with proper washout periods in between trials. Age has not been studied sufficiently regarding FEV response, so it may be beneficial to study age groups in order to investigate the role of age between treatments and results. Similarly, studies on the differences between welltrained athletes and the general population are lacking, so it may be useful to compare elite athletes who compete in cold-environment sports to non-athletes who work in a low temperature setting. Our meta-analysis of various pharmacological treatments used to control the symptoms of EIB showed that MCSA provides the greatest relief following periods of strenuous exercise because it causes the maximum fall in %FEV. Due to the drug’s direct impact on cells that cause inflammation, those with EIB may find that MCSA can control their symptoms to a greater extent than their current medication. Our findings will help enrich understanding of not only the biological mechanisms that cause EIB, but also how these three treatment options interact with those mechanisms. Continued research will hopefully lead to an effective and convenient method to prevent or minimize the symptoms of EIB, a condition affecting a large percentage of the general public. REFERENCES | 1. Boulet LP, O'Byrne PM. Asthma and exerciseinduced bronchoconstriction in athletes. New England Journal of Medicine. 2015; 372(7):641-648. 2. Parsons JPM, Mastronarde JGM, FCCP. Exerciseinduced bronchoconstriction in athletes. Chest Journal. 2005; p. 3996-3974. 3. Weiler JM, Brannan JD, Randolph CC, Hallstrand TS, Parsons J, Silvers W, et al. Exercise-induced bronchoconstriction update-2016. Journal of Allergy and Clinical Immunology. 2016; 138(5):1292. 4. Hermansen CL, Kirchner JT. Identifying exercise-

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induced bronchospasm - Treatment hinges on distinguishing it from chronic asthma. Physician and Sportsmedicine. 2005; 33(12):25-30. 5. Gerber AN. Measuring safety of inhaled corticosteroids in asthma. Annals of Allergy Asthma & Immunology. 2016; 117(6):577-581. 6. Merck. Patient Information Singulair [Internet]. 2016. Merck Sharp & Dohme Corp; [cited 2017 Mar 3] Available from http://www.merck.com/product/usa/pi_ circulars/s/singulair/singulair_ppi.pdf. 7. Murphy S. Cromolyn sodium: basic mechanisms and clinical usage. Pediatr Asthma Allergy Immunol 1988; 2: 237-54. 8. Intal Inhaler package insert (RPRâ&#x20AC;&#x201D;US), Rev 9/97, Rec 2/99, 2016. 9. PDR Physicians' Desk Reference. 53rd Montvale, NJ: Medical Economics Company Inc; 1999; 53: 2589-91. 10. Arnold DH, Gebretsadik T, Abramo TJ, Hartert TV. Noninvasive testing of lung function and inflammation in pediatric patients with acute asthma exacerbations. Journal of Asthma. 2012; 49(1):29-35. 11. Kazani S, Sadeh J, Bunga S, Wechsler ME, Israel E. Cysteinyl leukotriene antagonism inhibits bronchoconstriction in response to hypertonic saline inhalation in asthma. Respiratory Medicine. 2011; 105(5):667-673. 12. Piliponsky AM, Gleich GJ, Bar I, Levi-Schaffer F. Effects of eosinophils on mast cells: a new pathway for the perpetuation of allergic inflammation. Molecular Immunology. 2002; 38(16-18):1369-1372.

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