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JULS

Celebrating

Journal of Undergraduate Life Sciences

THE UNIVERSITY OF TORONTO’S th BIRTHDAY

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VOLUME 11 | ISSUE 1 | SPRING 2017


Sponsors The Journal of Undergraduate Life Sciences would like to thank the following sponsors for supporting the production of this year’s issue.

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University of Toronto Journal of Undergraduate Life Sciences (Print) juls.ca jps.library.utoronto.ca/index.php/juls

Journal of Undergraduate Life Sciences • Volume 11• Issue 1 • Spring 2017

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VOLUME 11 • ISSUE 1 • SPRING 2017

Table of Contents 06 07

About Us JULS Staff

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The Patient Practitioner: Understanding Acute Stress of Surgeons in the OR Moses Cook

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The Hirsch Index and Medical Imaging: Measuring the Academic Footprint of a Department Yang Zang and Runzhi Huang

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Trying to Gain Control Over an Uncontrollable Event: Reflection on Completing an Advance Directive Nicole Fogel

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Novel Support for the Emerging Paradigm of Genetic Convergence in Mammalian Echolocators Sam J.F. Walmsley

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Calreticulin Acts as a Critical Mediator of Bone and Heart Cell Differentiation Through Regulating the Nuclear Localization of Critical Transcription Factors Ponthea Pouramin and Michal Opas

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Effects of Short-Term High-Fat Diet Feeding On the Outcomes of Lyme Disease Infection in Mice Zoha Anjum

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Biological Versus Foster Antillean Manatee Calf: Who Receives more Social Interaction from Mother? Lucy Liu

Letter from the Editors

News

Research

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VOLUME 11 • ISSUE 1 • SPRING 2017

Reviews

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Weighing the Beneficial and Detrimental Consequences of Reactive Oxygen Species and their Role in Diseases Nidaa Rasheed, et al.

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The Psychological Impact of Type 2 Diabetes on Non-Diabetic Spouses Samantha Jagasar

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Examining Short and Long-Term Cardiovascular and Urological Outcomes of Living Kidney Donors Michael Lee, et al.

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Impacts of Mercury Poisoning on Brain-Derived Neurotrophic Factor: A Review of Recent Literature Mohammad-Masoud Zavvarian

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The Role of Transforming Growth Factor Beta in Allergic Asthma Pathogenesis Abi Kirubarajan, et al.

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Prenatally Stressed Rats Exhibit Behavior that Signals Depression when Evaluating Blood Glucose Metabolism Neda Jafarian

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O-GlcNAcylation in the Pathological Heart: A Nexus of Hypertrophic Circuitry? Ahmad Kamal

Interviews

76 79 Author Interviews Dr. Cynthia Goh

Journal of Undergraduate Life Sciences • Volume 11 • Issue 1 • Spring 2017

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JULS

Journal of Undergraduate Life Sciences On the Cover

Staff List

This year’s cover celebrates the University of Toronto’s 190th birthday. Featured is the front façade of University College (UC), one of the constituent colleges of the University. While the University of Toronto (formerly King’s College) was originally operated by the Church of England, UC was created specifically as an institution free of religious affiliation. This marked the beginning of an institution that is now home to diverse students from across the globe. Construction of the main UC building (depicted) was completed in 1859. Part of the building was destroyed in a fire in 1890, and subsequently rebuilt. Designated as a National Historic Site of Canada, the building has become one of the main faces of the University, sitting prominently in King’s College Circle and directly facing the University’s main gates. In many ways, the building itself reflects the history and legacy of the University, which JULS is proud to honor and celebrate this year.

Editorial Review Board Conan Chua Nikita Gupta Samuel Haile Stefan Jevtic Soomin Maeng

Anson Sathaseevan Utkarshna Sinha Clara Thaysen Alisa Ugodnikov Michelle Wan

Editors-in-Chief George Li

Tianyu Wang

Iva Avramov

Dhia Azzouz

Senior Editors

Michelle Liu

Jenny Xiao

Layout Managers Linwen Huang

Hanatu Tak

Rachel Chen

Martha Ghebreselassie

Review Board Managers

Layout Associates Andrew Cheon Joshua Efe Jordan Ho

Michael Lee Stella Song Michael Xu

News Managers Clara Hong

Faculty Review Board Dr. Mark Engstrom Dr. William Ju Dr. Uros Kuzmanov Dr. Deborah McLennan Dr. Arthur Mortha

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Dr. Alex Palazzo Dr. Sian Patterson Dr. Dana Philpott Dr. Ashley WaggonerDenton

Victoria Hue

External Affairs

Journal of Undergraduate Life Sciences • Volume 11 • Issue 1 • Spring 2017

Rouzbeh Ostadsharif Memar

Maria Raveendran


VOLUME 11 • ISSUE 1 • SPRING 2017

Letter from the Editors Dear Reader, It is with great pleasure that we present you with the 11th edition of the Journal of Undergraduate Life Sciences (JULS). As JULS enters its second decade of showcasing undergraduate research, the University of Toronto is celebrating its 190th year of operation. As we conclude our time as editors-in-chief, we would like to thank the many staff, professors, sponsors, and of course undergraduate researchers who have contributed countless hours to making this exemplary peer-reviewed publication a reality. We have dedicated this issue to celebrating the diversity of research and education that has occurred at the University of Toronto over the past 190 years. The University was first founded in 1827 as King’s College, the first institution of higher learning in the then colony of Upper Canada. Originally operated by the Church of England, it gained its current name after becoming a secular institution in 1850, seventeen years prior to the confederation of Canada. For almost two centuries, the University has been one of the major academic centers in Canada, home to an extensive list of groundbreaking discoveries, including the discovery of insulin, the T cell receptor, and stem cell research. We can’t help but be proud to realize that we are a part of this institution, and that the staff and undergraduate students we’ve worked with over this past year may go on to generate their own significant discoveries in the future. A new section our staff has worked hard to introduce in this issue of JULS is our “Life of an Undergraduate Researcher” series, which is available fully on our website as well as in part within this issue. While one of the main goals of JULS is to promote the work of undergraduate students to the academic community, a secondary goal of ours has always been to inspire and encourage incoming undergraduates to take on research of their own. Having both started as undergraduates looking for research opportunities, we realize how intimidating and foreign the world of academic research may seem. We previously featured interviews with established faculty members, whose experiences, while extremely valuable, may not have been as relatable to our readers. As such, our intention with this new series was to interview undergraduate researchers, including both JULS authors and staff. We hope that this will provide readers with unique and relatable stories, emboldening them to pursue their own academic interests outside of the classroom. Finally, we’d like to end off by thanking you for your readership. JULS exists to serve you, and we hope that you will find the contents presented herein valuable no matter what stage of your academic career you are in. Sincerely,

George Li & Tianyu Wang Editors-in-Chief, 2016-2017

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JULS

NEWS@UOFT

The Patient Practitioner: Understanding Acute Stress of Surgeons in the OR Moses Cook Stress is a complex phenomenon that affects everyone. Although the detrimental physiological and cognitive effects of chronic stress have been studied in literature, little is known about how acute stress pathways interact. Acute stress can have a negative impact on an individual’s technical skills when performing a task. What happens when a surgeon’s ability to adapt to acute stress affects their technical skills intraoperatively? Carol-Anne Moulton’s Lab at Toronto General Hospital is conducting a pilot study to understand a surgeon’s acute stress profile in the Operating Room (OR). I was given the opportunity to support the lab, and my colleague Sydney McQueen, in their endeavours. By researching the complexity of the human stress response, I learned that, like many things in life, there may be no clear answer that I could simply flip to at the end of a textbook. A delayed gratification only comes after persevering through many small successes and failures of finding a solution that I never knew when, nor if, would come. The complexity of acute stress arises from the fact that it can manifest through several physiological and cognitive pathways in the body. Occasionally, we can distinguish when we are acutely stressed whether it be shaky hands or an elevated heart rate but often times we can be stressed without consciously being aware of it. Thus, we are collecting surgeons’ intraoperative cortisol, a hormone that is released upon the interruption of an acutely stressful event. However, we are often unaware of such hormonal responses. We are also analyzing surgeons’ heart rate variability as a marker of acute stress. It has been shown that decreased variability can be a sign of low autonomic adaptability to a given stressful event, thus prompting an electrocardiogram (ECG) flag in a subject’s ECG tachogram . Finally, we are employing the State-Trait Anxiety Inventory, a survey assessing surgeons’ resource appraisal of perceived stressful incidents in the OR. The logistics and assembly of an OR can be very complex, so our team had to devise a methodology to safely extract physiological data from the surgeon without being intrusive. It also presented technical challenges as I was responsible for collecting and analyzing ECG data. Whether it was losing six hours of data collection or spending countless hours trying to understand what a fast Fourier transform was, the project definitely had its trials. Understanding a surgeon’s stress profile may allow for the development of tools or strategies to manage their stress in an effective way; thus not compromising patients’ well-beings. This, therefore, is the end goal. It has been demonstrated that stressful responses in surgeons are detrimental to their technical skills , thus showcasing the need for a deeper understanding.

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I had the best experience of my undergraduate years trying to overcome the challenges presented in this ongoing study. It was through these many trials that I learned to persevere and not lose sight of the end goal: understanding how stress can manifest in surgeons in the OR.

References

1. Castaldo, R et al: Acute mental stress assessment via short term HRV analysis in healthy adults: A systematic review with meta-analysis. Biomedical Signal Processing and Control, ISSN 1746-8094, 04/2015, Volume 18, pp. 370 - 377. 2. Wetzel CM, Black SA, Hanna GB, et al: The effects of stress and coping on surgical performance during simulations. Annals of surgery 251:171-176, 2010.

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NEWS@UOFT

The Hirsch Index and Medical Imaging: Measuring the Academic Footprint of a Department Yang Zang and Runzhi Huang A recent survey by the World Economic Forum placed the University of Toronto’s computer science program among the top 50 in the world [1]. Systems such as QS (QS World University Rankings, an annual publication of university rankings by Quacquarelli Symonds) are increasing their inclusion of citationbased scores such as the h-index to calculate their own scores. The h-index (or Hirsch Number) is a measure of the impact of academics on their institutions. It does so by combining the number of peerreviewed journals and the number of citations by other authors in comparable publications. For example, to receive an h-index of five, you must have published five research articles each of which must have been cited at least five times in other research articles. Over the summer of 2016, as a Research Opportunity Program project with Professor Pascal Tyrrell’s MiDATA lab in the department of Medical Imaging, we conducted a study to compare the h-index of medical imaging departments across Canada [2]. We also analyzed the h-index of departments in other disciplines to compare whether certain factors were true across these disciplines, or not. This study furthered analysis of h-index based on recently published work from our group [3]. Our study found a positive correlation between departmental h-index and world university rankings: as a medical imaging department’s h-index scored higher, so did the corresponding university’s rank. This is not surprising since the university’s ranking incorporates the h-index. However, the positive correlations be-

tween various university rankings and the h-indices of their medical imaging departments were found to be of less influence than the number of faculty members when comparing within a single discipline, which in our case was medical imaging (Figure 1, bottom left). This relationship also makes sense: a larger faculty means more publications, each of which has a chance at citation. Our finding supports previous research from our lab that showed an increase in the number of faculty within a specific department contributes to an increase in the department h-index over time when analyzed these departments individually. Interestingly, when we compared the correlation between departmental h-index and the number of faculty members across different disciplines, this positive relationship was lost (Figure 1, right). In this case, increasing the number of faculty members contributed very little, if at all, to a rise in h-index. These findings bring to light some of the limitations of the h-index. Firstly, it may not be appropriate to compare performance across disciplines, as they often differ by publication requirements and procedures and by publication type. For example, book chapters are not captured by standard citation databases (e.g. Scopus, Web of Science). These differences can lead to academics in the classical sciences appearing to out-perform academics in the social sciences and humanities. Secondly, the hindex does not account for co-authorship: a researcher included as a co-author with varying levels of contribution in many articles will therefore be favoured. Nevertheless, the h-index is an easy-toobtain, robust and increasingly well-recognized bibliometric measure of academic impact. When used appropriately – while accepting its limitations – the h-index remains a useful tool.

References

Figure 1.

1. Sam Shead (Business Insider), 2016. These are the best computer science schools in the world | World Economic Forum. World Economic Forum. Available at: https://www.weforum.org/ agenda/2016/07/these-are-the-best-computer-science-schools-inthe-world [Accessed January 29, 2017]. 2. Faculty of Arts and Science (UofT), 2016-2017 Research Opportunity Program — Current Students. Available at: http://www. artsci.utoronto.ca/current/course/rop [Accessed January 29, 2017]. 3. Tyrrell, P.N. et al., 2017. Departmental h-Index: Evidence for Publishing Less? Canadian Association of Radiologists Journal, 68(1), pp.10–15. Available at: http://www.ncbi.nlm.nih.gov/ pubmed/27742484 [Accessed January 29, 2017].

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JULS

NEWS@UOFT

Trying to Gain Control Over an Uncontrollable Event: Reflection on Completing an Advance Directive Nicole Fogel Even though the timing and possible causes of death cannot be controlled, medical advances have allowed patients to live longer, providing the opportunity for patients and their families to make certain end-of-life (EOL) care decisions. Stipulations regarding EOL health and personal care can be specified in an advance care planning (ACP) document, called an advance directive (AD), which aims to make decisions easier for the decision-maker(s) termed proxy(ies) by outlining the beliefs, values, and wishes of the patient when medical problems arise [2,6]. Even though specific stipulations are outlined in an AD, their ability to control EOL care and decisions is limited. There are problems associated with completing an AD (if they are even completed at all), such as the inability to predict specific medical illnesses and conflicts of interest among patients, families, and doctors. These problems are only apparent after one completes an AD him- or her-self; only then do the multitude of considerations present in and the complexity of the AD process become apparent. Here, I reflect on my personal experiences completing an advance directive from the University of Toronto Joint Centre for Bioethics, which includes instructional and proxy directives. I will explore the following topics, discussing my own experiences as well as providing support from published research and lectures: religion, organ donation, and proxy or family conflicts of interest. I conclude with a discussion of further problems associated with AD and offer possible suggestions to better ACP in the future. I first want to start off with some general comments about the experience completing an advance directive. When I first sat down to complete the AD from the University of Toronto Joint Centre for Bioethics, I was sorely under the impression that the process would be easy. Just filling out the initial proxy information on the first page created, for me, a whole slew of thoughts, and I immediately realized why completing an AD can be difficult. Questions of proxy values aligning with your values and the potential for proxy disagreements immediately came to mind. When prompted to fill out which particular proxies I wanted to make health or personal care decisions and if I wanted my wishes to be followed exactly or give leeway, I paused. It was extremely challenging to think about what I really wanted for the end of my life (especially since it is your own life and not someone else’s). I felt a certain vulnerability and was even hesitant about the finality of what I was writing specifically about being put on a ven-

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tilator in the case of a mild stroke, for example, or how I wanted my nutrition and hygiene handled. To me, this information was very personal and really detailed, and I was nervous that maybe these directions would not be followed or that they might be too outdated for future use. When completing the AD, I tried to be specific enough without being too specific because, to me, I understood that, realistically, not every specific situation could be accounted for nor could every specific wish be upheld. When talking to my proxy 1 and proxy 2 about what I had stipulated in my AD, there were certain topics that brought about some conflict and were discussed more in detail. First, when reading out my advance directive to my proxy 1 and proxy 2 (my parents), surprisingly, conflicting interests in religious beliefs were discussed most frequently. I wanted to make it clear in my AD that having a blood transfusion, for example, would not be a problem and also indicated that I am an atheist since I thought including this information might help proxies make better decisions in situations that weren’t specified in the AD, even postmortem decisions of burial (financial, school, and burial matters where not asked in the AD). I was not prepared for the reaction my mother solely provided; she was actually shocked to hear that I was an atheist and started questioning whether I felt lost in life not believing in a higher power or whether my upbringing could have changed this. My father is Jewish and my mother is Catholic, and I found this conversation interesting and quite unexpected, since my family never attended temple or church regularly growing up. Religion was simply not a big part of my life, so why did it matter in an EOL decision? In realizing that this might be a conflict of interest, previously unforeseen, I worried what the consequences of this might be for when it came time for my proxies to fulfill the wishes written in my AD. In fact, a recent review examined factors like location and religion that might affect EOL decisions in ICUs around the world [11]. Interestingly, it was found that religious people “choose more active life-sustaining measures than would nonreligious people”. This same result was observed in a study conducted in 2012 more specific to my situation, that examined differences in EOL and patient autonomy between religious and affiliated individuals in questionnaire responses among Protestants, Catholics, and Jews in European countries [3]. In talking with my proxy 1 and proxy 2, I noticed that my response to not be put on a ventilator or tube

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Trying to Gain Control Over an Uncontrollable Event: Reflection on Completing an Advance Directive

feeding in the case of permanent coma was different from that of my mother’s, who considers herself more religious. In contrast, she wanted these life-saving measures, since she believed that it was possible for the coma to be reversed, and that God would save her. This conversation brought to light the challenges to EOL care, due to differences in religion. In support of my feelings and concerns, Shinall et al. cite that understanding the patient’s wishes might be problematic when the family’s wishes differ from those of the patient [11]. However, it is worth mentioning that for my particular circumstance, my mother respected my decisions as an independent, mature, and educated adult. Knowing this did lessen my concern that my wishes might not be followed due to differences in religious beliefs. Discussing religious differences are necessary, as certain beliefs affect decisions in the case of physician-assisted suicide or euthanasia, which is less likely to be employed by religious persons, as they feel that human life is sacred [10]. Feelings towards organ donation might differ between the religious and non-religious individuals, as I experienced. The second major discussion point I noticed, when reading out my AD to proxy 1 and proxy 2 circled around organ donation. In my AD, I tried to make decisions for proxies easier by being specific about post-mortem consequences, such as whether or not I wanted my organs to be donated or not. In my AD, I stipulate that I would like to be an organ donor, a decision that stems from my university studies and agreement with philosophers who argue that there is nothing after death and so the body should be put to use for someone who needs them [4]. My mother, being more religious than I am, respected my decision, but did not agree with me. Interestingly, though she expressed a willingness to help someone else in need of an organ, she believed that God wanted to see her whole, and not violated or mutilated, when she ascended to heaven. This made me realize that for some, decisions are made on more than religious beliefs, but also on personal or family connections. As Souter et al. indicate, a sensitivity for cultural and religious beliefs is needed in EOL care and decision-making [12]; they cite that in some religions, organ donation before the moment when all brain wave activity has ceased would be unacceptable (S507). Delaney et al. write that an organ draft would violate the first amendment too, the right to religious freedom [4]. Being interested by this conversation with my mother, I wanted to research beyond the course material why religious beliefs affect organ donation beliefs. I found a link between the two in a recently published article in which the authors studied the discrepancy between the organ transplant waiting list and organ donors in an African American population in a cross-sectional study [8]. The willingness to donate was actually negatively correlated with religious beliefs. This finding relates to my personal experience, as my mother’s religiosity decreased her willingness to donate and became a reason for her not to donate, though this might be only one factor among many [2]. This study provided support that religion plays a major role in some people’s lives and influences their EOL decision-making and views on organ donation. Differences of belief may affect who patients appoint as their proxy or proxies in the AD. Aside from the religion and organ donation talk with my proxies, I found that deciding who to appoint as my proxies (and in what order) was a difficult decision that required me to truly reflect on my own interests and if they aligned with the proxies’ interests

News@UofT

as well as reflect on who would know me well enough to carry out my wishes and make appropriate decisions in this monumental moment in life. I knew I wanted to choose my parents, but not in what order. Placing my father or mother first as the decisionmaker might create unnecessary conflict and favoritism. I decided to place my father first because I thought that he would be less emotional, plus his business and legal background might influence his decisions. Surprisingly, establishing a hierarchy in this case did not create a conflict; my mother agreed that she too would want my father to be first for similar reasons. This part of the conversation made me realize that I should not underestimate my family’s response during times of crises. To support this feeling, it has been shown that better family functioning and higher levels of emotional support increased the probability of partaking in informal discussions and completion of ACP [9, 1]; therefore, understanding family dynamics is a crucial influence on how EOL decisions will be made. My mother was very open and talked freely about the issues discussed here, while my father actually became very upset that I was discussing this because I was his daughter. He refused to provide further comments. Although this was unexpected, I still felt that my proxy indications should remain the same, but questioned my perception of others. Particular to my situation, I have a little brother who is 14 years old now and thought about making him a proxy too. However, due to the uncertainty of when this AD would be used, I decided against it. This was a conflicting decision, since I would want all members of my immediate family to participate in the decision, but thought that even if my brother was 40 years old at the time when the AD was read, I would not want to burden him. This is because I feel that I am very close to him and this event would be very painful and emotional for him. Actually, a study by Khodyakov et al. found that sibling relationship quality and closeness declines after parental death [5]. This study provides great evidence that talking about EOL care with family is extremely necessary, especially to avoid unwanted conflicts or harmful relationships to those surviving the patient. Most importantly, what I have learned from this experience of completing my own AD is that the process is complex; in every section, there is something further to consider. From this experience, having space to write my health care and personal decisions was beneficial, as it allowed me to freely express my wishes; however, I felt that it was difficult to know what to stipulate for each section. For example, there were some questions to think about regarding how you would like your safety to be managed, but understanding what the safety section means might prevent one from accurately completing the section. It was difficult to be specific enough to allow proxies to make easier decisions, but to also be flexible in accounting for uncertain future events. I observed this in my own grandparents’ will, which they lent to me, and realized that being too vague would cause proxies to question what should be done in certain situations. According to Dr. Jonathon Breslin’s Lecture 9, phrases like “common accident” might be too vague and would require interpretation [2]. Another problem I observed while filling out the treatment table was that it is impossible to predict every specific situation (and medical advancement) that could arise [2]. It was difficult to imagine myself in each scenario; one’s feelings while filling out the AD might be different once that person is in the actual situation

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News@UofT

Trying to Gain Control Over an Uncontrollable Event: Reflection on Completing an Advance Directive

(similar to the AD lecture case-study). As Henry Perkins writes, AD “promise more control over future care than is possible” [6]. An additional source of confusion with the treatment table might be the use of technical terms. For laypersons, understanding exactly what having a ‘moderate stoke’, for example, entails symptomatically might be problematic when accurately completing the AD. Finally, allowing proxies to make decisions as a group or to side with the majority might cause controversies similar to being in a business group that has to decide on one proposal. Dr. Breslin in Lecture 9 also cites in his lecture that some people may just prefer to leave EOL decisions to proxies, ignoring their AD, and proxies might not even be aware of their selection [2]. Even in places where I have made specific stipulations, it might be difficult for proxies to know if they are carrying out the stipulations effectively [6]. Other barriers include: not completing an AD at all, not having documents readily accessible when needed, and not updating the document for a long time (i.e. I would probably change my mind from 20 years old to 50 years old) [2]. How accurately one can make predictions about future desires is a question often raised; the Margot Bentley case nicely illustrates this problem. To conclude, from my experience completing an advance directive, it is evident that there are many factors to consider that one doesn’t acknowledge until one partakes in this insightful process. Religious beliefs, organ donations, proxy selection, and other considerations like family relationships or dynamics should be taken into account when discussing advance care planning. I felt some comfort in knowing that I can indicate end-of-life choices for myself and was able to truly reflect on who I am as a person; however, I did not feel a total sense of control over my end-of-life decisions due to the realization that in an emotionally-pressured and fastpaced time, physicians and proxies might not even read or fully be able to implement the specific stipulations in the moment. After studying the many problems and limits associated with advance directives, I feel that my view of their reliability and credibility was negatively altered when completing the AD. After being through this process, I might not want to fill one out at all in reality. In order to address these problems, it might be best to have conflicts resolved by a team leader, as following one person might provide a clear line of reasoning in a chaotic time and might avoid confrontations among proxies. As suggested by Perkins, we should evolve our thinking about advance care planning to advance care preparing to help avoid confusion and non-reasonable decisions during end-of-life care, when uncertainties and difficulties arise [6]. In speaking with the Palliative Care Team at SickKids Hospital in Toronto, it was made clear that in practice, formal documents were not signed; rather, five wishes or points were talked through with patients and proxies (Rapoport). This hints at the ineffectiveness of AD in practical settings, for which the transfer of AD theory to practice needs further research. Death is an uncontrollable event, yet advance directives were implemented to try to gain control of an uncontrollable event and prevent disputes by outlining specific wishes of the patient [2]. However, there are complex historical, cultural/religious, and social factors to consider that might influence end-of-life decisions and interests, compelling philosophers to better understand human personal values and beliefs, which might try to further answer, in a broader sense, controversial discussions about the definition and determination of death.

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References

1. Boerner, Kathrin, Carr, Deborah, and Sara Moorman. “Family Relationships and Advance Care Planning: Do Supportive and Critical Relations Encourage or Hinder Planning?” The Journals of Gerontology, Series B, Psychological Sciences and Social Sciences 68 (2013) : 24656. Web. 2. Breslin, Jonathan. “Advance Directives.” University of Toronto. Ramsay Wright Laboratories, Toronto, ON. 12 November 2014. Lecture 9. ---. “Controversies in Organ Donation I.” University of Toronto. Ramsay Wright Laboratories, Toronto, ON. 19 November 2014. Lecture 10. 3. Bu¨low, Hans-Henrik et al. “Are religion and religiosity important to end-of-life decisions and patient autonomy in the ICU? The Ethicatt study.” Intensive Care Medicine 38 (2012) : 1126-1133. Web. 4. Delaney, James, and David Hershenov. “Why Consent May Not Be Needed For Organ Procurement.” The American Journal of Bioethics 9 (2009) : 3-10. Web. 5. Khodyakov, Dmitry, and Deborah Carr. “The Impact of Late-Life Parental Death on Adult Sibling Relationships: Do Parents’ Advance Directives Help or Hurt?” Research on Aging 31 (2009) : 495-519. Web. 6. Perkins, Henry. “Controlling Death: the False Promise of Advance Directives.” Annals of Internal Medicine 147 (2007) : 51-57. Web. 7. Rapoport, Adam. “Re: Advance Directives.” Message to Paediatric Advanced Care Team (PACT) Medical Director. 13 Nov. 2014. E-mail. 8. Robinson, Dana et al. “Understanding African American’s Religious Beliefs and Organ Donation Intentions.” Journal of Religious Health 53 (2014) : 1857-1872. Web. 9. Romain, Marc, and Charles Sprung. “Approaches to patients and families with strong religious beliefs regarding end-of-life care.” Current Opinion in Critical Care 20 (2014) : 668-72. Web. 10. Schuklenk U et al (2011). End-of-Life Decision-Making in Canada: The Report by the Royal Society of Canada Expert Panel on End-of-Life Decision-Making. Chapters 2 & 3 (pp. 21-51). 11. Shinall, Myrick, and Oscar Guillamondegui. “Effect of Religion on End-of-Life Care Among Trauma Patients.” Journal of Religion and Health (2014) : doi 10.1007/s10943-014-9869-4. Web. 12. Souter, Michael, and Gail Norman. “Ethical controversies at end of life after traumatic brain injury: Defining death and organ donation.” Critical Care Medicine 38 (2010) : S502-9. Web.

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JULS

RESEARCH

Novel Support for the Emerging Paradigm of Genetic Convergence in Mammalian Echolocators Sam J.F. Walmsley Corresponding Author: Sam J.F. Walmsley (sjf.walmsley@gmail.com)

Abstract

Recent studies have identified three cochlear cell-related genes that have undergone convergent evolution in species with welldeveloped high frequency hearing: cetacean and bat echolocators. These studies have added to a growing database supporting the existence of gene-level convergence, genetic similarity with functional but not phylogenetic proximity. To determine if this case of convergence extends to other genes and other echolocators, a new cochlear cell-related gene, DIAPH3, was analyzed through the construction of gene trees and tests of positive selection. In addition to the new gene, a lesser-known mammalian echolocator, the common shrew, Sorex araneus, was included in the analysis. Though not entirely conclusive, DIAPH3-generated gene topologies clustered the echolocators in a monophyletic group, which is not supported by clade-level phylogenetic trees. Tests of positive selection identified two broad areas of convergence within this gene. In contrast to previous findings, echolocating mammals beyond cetaceans and bats appear to show isomorphic genetic convergence with respect to the evolution of hearing genes. Keywords: Mammalian echolocation, DIAPH3, genetic convergence, auditory genetics, molecular evolution, sensory ecology, Bayesian statistics

Background

Echolocation is the perceptual phenomenon in which high-frequency sound waves are both emitted and received by an individual, so that the speed and frequency of return can be analyzed [1]. These qualities of received sound are then translated into representations of the organism’s external environment, affording non-visual spatial awareness. Indeed, many animals use acoustical cues to develop spatial maps of their environments, and to determine the location of objects (or other individuals) around them. While cetaceans and chiropterans are well known for their echolocation abilities, echolocation is actually used by a much broader group of organisms. For instance, shrews, tenrecs and tree shrews use echolocation for movement and navigation, though not for prey detection [2-4]. In addition to differences in ecological function of echolocation systems, there are also differences in the anatomical structures corresponding to emission and reception of high-frequency sound. For example, odontocetes (the toothed whales) have large, oil-filled “melons” which are thought to facilitate the focusing of sounds emitted from a nasal plug [5]. Echolocating bats have developed specialized facial appendages called “nose-leaves” designed to manage sound originating from the larynx [6]. Certain tenrecs of Madagascar even navigate using the high-frequency sounds produced by tongue clicks [6]. Thus, the anatomical structures supporting echolocation have clearly diversified within different environments.

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The distribution of echolocation within Mammalia indicates that it has arisen independently numerous times within the clade [3,6]. Identifying convergence, the independent evolution of similar characters in distantly related lineages, is important because it gives researchers the opportunity to study the effects of similar environmental features and thus, similar selection pressures on character evolution. There is ample evidence for the existence of convergence in a variety of different phenotypic traits from morphology and physiology through ecology and behavior. However, convergence at the level of genetics (specifically in the protein sequences derived from genes), once deemed very rare, is being detected more and more in a variety of taxa [7]. One of the best examples of genetic convergence comes from the prestin gene, which has been found to have a high level of protein similarity in certain echolocating bats and cetacean species [2,8]. Prestin codes for the expression of a motor protein that supports the amplification and reception of sound waves in mammals. Further studies suggested that this convergence is not limited to prestin, but applies to other hearing-related genes as well, specifically Tmc1 and Pjvk [9]. This study investigates another candidate gene for the complicated genetic basis of echolocation, diaphanous-related formin 3 (DIAPH3). Both Tmc1 and Pjvk have been linked to Drosophila and human cases of auditory neuropathy [9,10]. Auditory neuropathy results in deafness and is characterized by defects in inner-cochlear

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Novel Support for the Emerging Paradigm of Genetic Convergence in Mammalian Echolocators

Research Articles

hair cells, as opposed to outer hair cell function, which is where more common types of deafness emerge [10,11]. DIAPH3 has been newly linked to this auditory condition, and, following the success of previous neuropathic genes, was chosen to investigate the possibility of further convergence. The precise function of the DIAPH3encoded protein with respect to inner auditory cells is unknown, but the DIAPH3 protein belongs to a group of actin nucleation factors supporting cell structure and the vesicular transport of cellular materials [10]. DIAPH3 is a long gene (3633 nucleotides) and is found on chromosome 13 in humans, but there is variation in length, as well as genomic location between species [13]. Given the diversity in echolocation-related anatomy, the best candidates for genetic convergence in mammalian echolocation should code for structures relating to the first-order reception of auditory stimuli. These structures and their genetic foundations are much more likely to be similar across echolocating taxa than genes relating to group or species-specific anatomies. I hypothesize that perceptual capacities will favour structural similarity in hearing-related genes across mammalian species. Based on previous research on prestin, Tmc1, and Pjvk, I predict that (i) DIAPH3 sequences from two echolocating species (bat and dolphin) will cluster together on the gene tree and (ii) the relationship of the third echolocating species will be ambiguous, as in studies of other genes. Beyond the investigation of the genetic evolution of mammalian echolocation, this study endeavours to use bioinformatics tools to simultaneously further the understanding of the frequency of genetic convergence in biological organisms.

analysis was complete, the tree was visualized with Mega6 [15]. In regards to the Bayesian tree, ModelTest was first employed to find the best fitting model. MrBayes was then used in the construction of a Bayesian phylogenetic analysis [16,17]. The tree was constructed with the use of 500,000 generations and was run twice in completion. The burnin value was set to 25% with the standard use of three “hot chains” and one “cold chain”. The Markov chain was sampled every 100 cycles, as per standard settings. Once the MrBayes analysis was completed, the resulting Bayesian tree was visualized with FigTree (http://tree.bio.ed.ac.uk/software/figtree/) and was rooted with the platypus outgroup. Finally, the tree was adjusted to display posterior probabilities calculated in the analysis. PhyML was also used (with default settings) to construct a tree for use by positive selection models. Additional tree visualizations were done with iTOL [18,19].

Methods

Gene Tree In the final alignment, 36% (1074/2910) of the sites were invariant, with 62% (1795/2910) of the sites conserved. There were 410/2910 parsimony-informative sites, and 661/2910 singleton sites. The resulting gene tree did not reflect the species-based topology. Sorex araneus was erroneously placed in a clade with the cetacean and chiropteran species. Additionally, the carnivorans were displaced out of their original association with other Laurasiatheria. Finally, the rat was removed from the primates (humans and gorilla) such that the Euarchontoglires were separated. The bootstrap analysis found certain nodes with very high confidence (caniformia and primates both had bootstrap values of 100),) and others with low confidence (i.e. node separating Tursiops truncatus from chiroptera and Sorex araneus with bootstrap value 23). The Bayesian method (Figure 1 (b)) returned a tree topology that matched that of the maximum likelihood tree. There is strong evidence that the MrBayes analysis reached statistical convergence. The log plot showed no upward or downward trend with reference to the x-axis, the standard deviation of split frequencies sufficiently approached 0 (highest value = 0.004), and the potential scale reduction factor approached 1 (all reported as 1.000), all of which indicates successful convergence. This topology was supported with uniformly high posterior probabilities, the node of lowest confidence being that separating Tursiops truncatus from the chiroptera and Sorex araneus.

Taxonomic Coverage Species in this study were chosen to attain an approximate balance of echolocators and non-echolocators. Prototheria is the sister-group to the clade comprising the Metatheria and Eutheria (in which the echolocators are found) so the prototherian platypus served as a natural outgroup. Given discrepancies in length of transcripts, as well as the absence of small portions of the Tursiops truncatus, Sorex araneus, and Pteropus vampyrus sequences, trimming and re-organization of the transcripts was necessary prior to alignment. To reduce this problem as much as possible, I chose non-echolocating species with the most complete available transcripts.

Aligning Sequences with Mega6

All DIAPH3 transcripts used in this study were downloaded from the open-source ENSEMBL database [13]. BLAST was used to assist in the search for matching orthologous species transcripts [14]. Once stop codons had been removed (no premature stop codons were detected), the sequences were aligned by ClustalW with codons using Mega6 [15]. Mega 6 was also used to extract information pertaining to the degree of conserved vs. variable sites in the alignment [15]. While a significant degree of trimming was required, the final adjusted alignment contained 80.1% of the longest original sequence. This was deemed to be a sufficient amount of genetic material to proceed, especially given the substantial length of DIAPH3 transcripts.

Phylogenetic Analysis with DIAPH3 A maximum likelihood tree was constructed with the finalized alignment. The support for each node was examined using 500 bootstrap replications. Default specifications were used for the remaining test options, such as the Tamura-Nei model, and uniform rates among sites. Once the

Detecting and Locating Positive Selection The codeml program was run through PAML4.8 with the PhyMLgenerated phylogenetic topology in order to assess possible positive selection in the data [20]. The following nested, random-sites models were chosen to be compared via paired likelihood ratio tests: M0 (null) and M3, M1a (null) and M2a, M7 (null) and M8. With the PAML analysis completed, probable sites of positive selection were identified with the Bayes Empirical Bayes (BEB) analysis [21]. These sites were corrected to match positions in DIAPH3, accounting for sequence trimming and re-organization. Corrected sites of statistically significant omega values were then mapped and visualized.

Results

Random-sites Selection Model ModelTest found the Jukes-Cantor model to be the best fitting model (equal base frequencies and equal base rates) given the Akaike information criterion. Each of the likelihood ratio tests was

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Novel Support for the Emerging Paradigm of Genetic Convergence in Mammalian Echolocators

highly significant (p<0.001), such that all null models (M0, M1a, M7) were rejected. The M3 model had the highest ln-likelihood (-7510.04), very nearly matched by that of the M8 model (-7510.59), while the M0 model had the lowest ln-likelihood (7625.29). See (Table 1 for these results in detail). These findings were reified by the BEB analysis, which found positive selection to have occurred in the following sites: 292, 375, 378**, 635** for the M2a model, and 292, 375, 378**, 380, 393, 395, 446, 587, 635**, 636 for the M8 model (** refers to p<0.01, otherwise standard alpha p<0.05, Figure 2). Thus, in total, 9 sites were identified. All sites of positive selection were confined to 2 smaller, distinct, regions of interest, each occupying less than 15% of the entire sequence; these occurred between sites 292-446 and 587-636, respectively.

Discussion

Assessing Tree and PAML Analyses DIAPH3-derived topologies and Bayesian positive selection models suggest gene-level convergence among all of the mammalian echolocating species examined in this study. The gene tree placed all three echolocators (Sorex araneus, Tursiops truncatus, Myotis lucifugus) in a monophyletic group within the Laurasiatheria, with carnivores clustering as the sister-group to that clade. While placement of these echolocators within the Laurasiatheria agrees with previous species level studies [22], the relationships of the echolocators with respect to each other does not. This is exactly what would be expected if DIAPH3 were evolving convergently within the shrew, bat and dolphin clades. There are, however, several unexpected results based on my analysis of DIAPH3 sequences. First, why did the non-echolocating fruit bat, Pteropus vampyrus, cluster within the echolocation “clade”? If this is a genuine case of gene-level convergence in relation to echolocation function, the presence of P. vampyrus could be explained by a) the loss of echolocation in Old world fruit bats coupled with some remaining genetic vestiges [23], or simply that b) the analysis detected genuine phylogenetic signal within DIAPH3. This latter suggestion is supported by the fact that the sequences also correctly clustered Felis catus and Canis lupus familiaris, Gorilla gorilla and Homo sapiens, and placed Sorex araneus (Eulipotyphla) within Laurasiatheria [22], so clearly the gene does carry some signal that can be recovered in a phylogenetic analysis. This answer to this problem can be sought in future research by including one of (the few) echolocating fruit bats in a larger analysis. Second, it is odd that Rattus norvegicus did not cluster with primates in the typical Euarchontoglires clade. At the moment there is no obvious explanation for this discrepancy with respect to auditory function. Resolution of this problem awaits further description of the function of DIAPH3 variants in particular species; that is, in examining the relationship between gene sequence variability and functional changes. This misplacement might also reflect, in part, the incompleteness of the transcripts used in these analyses. At this point it is difficult to assess how much this affected results, given that DIAPH3 is yet to be analyzed in terms of threedimensional structure. However, given that the analysis identified sites of positive selection, it appears that crucially relevant domains of the gene were conserved in the alignment process. Given the high degree of statistical significance in each case of the PAML analysis, all of the alternative models (M3, M2a, M8)

16

a)

b)

Figure 1. a) Taxon-level relationships on reduced tree (following Foley et al 2016) [22]. b) Bayesian gene tree based on DIAPH3 sequences. Note the erroneous grouping of echolocating shrews with dolphins and bats to the exclusionw of carnivorans in the gene tree. Both trees were visualized with iTOL [19], and the names of echolocating species are in blue.

provided better fit while keeping excessive parameters in check. As the data was better modeled by a) the inclusion of multiple classes of omegas (in the case of M3) and b) the addition of a selection (dN/dS > >1) class, it is evident that some degree of positive selection is at play [21]. The identification of two regions of positively selected sites may be indicative of the presence of functional domains, though further protein analysis to determine the folded structure, such as crystallography, is necessary to confirm this hypothesis [24]. Assessing the Possibility of Genetic Convergence Genetic convergence is a relatively novel phenomenon, at least in empirical terms, and it is necessary to be clear about what is taken to be sufficient evidence for its occurence. Rokas and Carroll suggested that by ignoring tests of selection, attempts to describe such processes often fail in distinguishing genuine convergence from “non-adaptive homoplasy”, i.e. trait identity without adaptive function [25]. Thus, it is important to include tests of selection in studies of homoplasy in order to separate biologically meaningful convergence from otherwise stochastic effects. Indeed, convergent evolution at the level of genes is determined by 4 criteria: functional convergence, presence of parallel sites, common selective pressure at said parallel sites, and parallel substitutions resulting in the parallel functional changes [26]. Applying all of these criteria to this dataset is beyond the meth-

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Research Articles

Novel Support for the Emerging Paradigm of Genetic Convergence in Mammalian Echolocators

Model

np

In L

K

M0 M1a M2a M3 M7 M8

19 20 22 23 20 22

-7625.29 -7532.37 -7511.65 -7510.04 -7537.01 -7510.59

3.40018 3.37396 3.7918 3.77202 3.38498 3.77863

ω0/p 0.61452 0 0.16159 0 0.23297 0.53032

Parametersa ω1/q 1 1 0.555 0.17961 0.59873

Null

LRT

df

p

3.29978 2.58591

M1a M0

41.437 230.51

2 4

1.00496E-09 1.02765E-48

2.8805

M7

52.84

2

3.35704E-12

ω2/ωp

Table 1. Likelihood ratio tests for random-sites models (PAML) of mammalian DIAPH3.

odological scope of this paper given that the functional outcome of the apparent adaptive substitutions is unknown. However, the incorrect topology combined with very strong evidence of positive selection is a meaningful step towards building a rigorous case for adaptive genetic convergence in DIAPH3. If validated in further tests, the result would join a growing dataset of genetic convergence examples in which multiple loci seem to have converged on the same function [9]. This remains a relatively rare, or at least poorly detected evolutionary process [9]. More importantly, most cases of protein convergence described to date (e.g., rhodopsins in squid and primates; myoglobin in seals and cetaceans) have been limited to singular, small sequence changes [9,27,28]. Multi-locus genetic convergence is rare in the molecular evolution literature, although Davies et al suggested that it might be more common than previously thought [9]. While the results of this study should be qualified, by identifying a likely case of gene-level convergence, my findings add credence to the belief that genetic convergence is common in evolution. Interestingly, much in the way that cases of convergent evolution indicate “containment” of function by environment [29], cases of genetic convergence go one step further and imply containment of structure by function. If the results presented here are valid (i.e. reflect a genuine case of genetic convergence), the increasingly pow-

erful model surrounding the genetic basis of echolocation may indicate that there are fascinatingly narrow restrictions on genotypic design-space, at least for high-frequency hearing. Of course such design space is affected by, and must be viewed within the context of extra-genetic factors. For example, although spectral sensitivity tuning is driven by genetic specificity (such that one can even use gene-bioinformatics to analyze colour perception and estimate habitat conditions), individual history also plays a role in the development of visual abilities. Kröger et al. found this very effect, in which Aequidens pulcher (a common cichlid fish) reared under different light conditions developed dramatically different spectral sensitivities [30]. The importance of developmental effects are especially clear in the case of blind human individuals gaining the capacity for echolocation [31].

Directions for Further Research There is great potential for this line of research simply in replicating the above methods for other candidate genes. Given the high number of genes known to cause auditory neuropathy [11], it is likely that more incidences of genetic convergence are to be found. In addition, each new gene investigated will promote greater understanding of the mechanistic background of high-frequency hearing in various mammals. Integration of biochemistry and animal physiology in this regard is likely to be very informative. This is especially true with respect to identifying whether or not the apparent regions of positive selection (292-446, and 587-636) in DIAPH3 correspond to, or at least include, important functional domains. There is also potential for further research that includes other organisms capable of highfrequency hearing. Because of problems with incomplete or absent DIAPH3 transcripts for many relevant species, this study was limited to the three mammalian echolocators, only one of which that had not been studied in previous experiments. Future studies need to expand the DIAPH3 database, both within Mammalia and Figure 2. Graph displaying sites of positive selection in mammalian DIAPH3 transcripts as determined by BEB to non-mammalian echolocators Analysis with models M2a and M8. Categorical significance (p<0.05, p<0.01) in tests for positive selection as well (e.g., roosting oilbirds) [31]. (omega>1) is shown to identify regions of interest. Journal of Undergraduate Life Sciences • Volume 11 • Issue 1 • Spring 2017

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Novel Support for the Emerging Paradigm of Genetic Convergence in Mammalian Echolocators

Conclusion

The DIAPH3-constructed tree topologies and tests of positive selection are strongly suggestive of a genetic convergence in cochlear hair cell genes for mammalian echolocators. Contrary to previous studies finding no convergence with respect to noncetacean or chiropteran echolocators, the common shrew Sorex araneus extends this phenomenon to new taxa. These results add DIAPH3 to the growing list of genes (Prestin, tmc1, and pjvk) involved in the evolution of echolocation. Further research will be fruitful in furthering this fascinating and increasingly powerful case of convergent evolution. Availability of Supporting Data The data supporting the analysis and conclusions of this study remain available on the ENSEMBL database website. Accession numbers are listed in supplementary data, Table S1.

List of Abbreviations Used

BEB – Bayes Empirical Bayes analysis BLAST – Basic Local Alignment Search Tool DIAPH3 – diaphanous-related formin 3 PAML – Phylogenetic Analysis by Maximum Likelihood Pjvk – common alias for DFNB59 gene (deafness, autosomal recessive 59) Prestin – protein encoded by SLC26A5 gene (solute carrier family 26 (anion exchanger), member 5) Tmc1 – transmembrane cochlear-expressed gene 1

Acknowledgements

I would like to thank Dr. Belinda Chang and Sarah Dungan from the University of Toronto, both of whom provided conceptual guidance during the research process. I would also like to thank Dr. Deborah McLennan and the staff editors of JULS for their thorough commentary and helpful edits.

Overduin B, Parker A, Patricio M, Perry E, Pignatelli M, Harpreet SR, Sheppard D, Taylor K, Thormann A, Vullo A, Wilder SP, Zadissa A, Aken BL, Birney E, Harrow J, Kinsella R, Muffato M, Ruffier M, Searle SMJ, Spudich G, Trevanion SJ, Yates A, Zerbino DR, Flicek P.Cunningham F, Amode MR, Barrell D, Beal K, Billis K, Brent S, … Flicek P. Ensembl 2015. Nucleic Acids Research. 2014;42:662-669. 14. Altschul SF, Gish W, Miller W, Myers EW, Lipman, DJ. Basic local alignment search tool. J. Mol. Biol. 1990;215:403-410. 15. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Molecular Biology and Evolution. 2013;30:2725-2729. 16. Guindon S, Gascuel O. A simple, fast and accurate method to estimate large phylogenies by maximum-likelihood. Systematic Biology 2003;52:696-704. 17. Ronquist F, Huelsenbeck JP. MRBAYES 3: Bayesian phylogenetic inference under mixed models. Bioinformatics. 2003;19:1572-1574. 18. Guindon S, Dufayard JF, Lefort V, Anisimova M, Hordijk W, Gascuel O. New Algorithms and Methods to Estimate Maximum-Likelihood Phylogenies: Assessing the Performance of PhyML 3.0. Systematic Biology. 2010;59:307-321. 19. Letunic I, Bork P. Interactive Tree of Life (iTOL): an online tool for phylogenetic tree display and annotation. Bioinformatics. 2006;23:127-128. 20. Yang, Z. PAML 4: a program package for phylogenetic analysis by maximum likelihood. Molecular Biology and Evolution. 2007;24:1586-1591. 21. Yang, Z. Bayes Empirical Bayes Inference of Amino Acid Sites Under Positive Selection. Molecular Biology and Evolution. 2005;22:1107-1118. 22. Foley NM, Springer MS, Teeling EC. Mammal madness: is the mammal tree of life not yet resolved? Philosophical Transactions of the Royal Society B. 2016;371:20150140. 23. Springer MS. Phylogenetics: Bats United, Microbats Divided. Current Biology. 2013;23:2262-2267. 24. Wilson JH, Hunt T. Molecular Biology of the Cell, 4th Edition: A Problems Approach. New York: Garland Science, 2002. 25. Rokas A, Carroll SB. “Frequent and Widespread Parallel Evolution of Protein Sequences.” Molecular Biology and Evolution. 2008;25:1943-953. 26. Zhang J. “Parallel Adaptive Origins of Digestive RNases in Asian and African Leaf Monkeys.” Nature Genetics Nat Genet. 2006:819-823. 27. Morris, A., J. K. Bowmaker, and D. M. Hunt. The Molecular Basis of a Spectral Shift in the Rhodopsins of Two Species of Squid from Different Photic Environments. Proceedings of the Royal Society B: Biological Sciences. 1993;254: 233-240. 28. Romero-Herrera, A. E., H. Lehmann, K. A. Joysey, and A. E. Friday. Evolution of Myoglobin Amino Acid Sequences in Primates and Other Vertebrates. Molecular Anthropology. 1976:289-300. 29. Shapiro L. Adapted Minds. Naturalism, Evolution, and Intentionality. Ed. Jillian Scott McIntosh. Calgary, Alta., Canada: U of Calgary, 2001. 30. Kröger RHH, Knoblauch B, Wagner HJ. Rearing in different photic and spectral environments changes the optomotor response to chromatic stimuli in the cichlid fish Aequidens pulcher. Journal of Experimental Biology. 2003;106:1643-1648. 31. Stroffregen, Thomas A., Pittenger JB. “Human Echolocation as a Basic Form of Perception and Action.” Ecological Psychology. 1995;7:181-216.

References

1. Thomas JA, Moss C, Vater M. Echolocation in Bats and Dolphins. Chicago: U of Chicago, 2004. 2. Rossiter SJ, Zhang S, Liu Y. Prestin and high frequency hearing in mammals. Communicative and Integrative Biology. 2011;4:236-239. 3. Gould E. Evidence for Echolocation in the Tenrecidae of Madagascar. Proceedings of the American Philosophical Society. 1965;109:352-360. 4. Siemers BM, Shauermann G, Turni H, von Merten S. Why do shrews twitter? Communication or simple echo-based orientation. Biology Letters. 2009;5:593- 596. 5. “Cetacean Echolocation.” National Marine Mammal Laboratory. 01 Dec. 2015. http://www. afsc.noaa.gov/nmml/education/cetaceans/cetaceaechol.php 6. Fitch, T. “Production of Vocalizations in Mammals.” Encyclopedia of Language & Linguistics. 2006:115-21. 7. Liu Z, Qi F, Zhou X, Ren H, Shi P. Parallel Sites Implicate Functional Convergence of the Hearing Gene Prestin among Echolocating Mammals. Molecular Biology and Evolution 2014;31:2415-2424. 8. Parker J, Tsagkogeorga G, Cotton JA, Liu Y, Provero P, Stupka E, Rossiter SJ. Genome-wide signatures of convergent evolution in echolocating mammals. Nature. 2013; 502:228-231. 9. Davies KTJ, Cotton JA, Kirwan JD, Teeling EC, Rossiter SJ. Parallel signatures of sequence evolution among hearing genes in echolocating animals: an emerging model of genetic convergence. Nature Heredity. 2012;108:480-489. 10. Schoen CJ, Emery SB, Thorne MC, Ammana HR, Sliwerska E, Arnett J, Hortsch M, 11. Dror AA, Avraham KB. Hearing Impairment: A Panoply of Genes and Functions. Cell – Neuron. 2010;68:293-308. 12. Hannan F, Burmeister M, Lesperance MM. Increased Activity of Diaphanous Homolog 3 (DIAPH3)/diaphanous Causes Hearing Defects in Humans with Auditory Neuropathy and in Drosophila. Proceedings of the National Academy of Sciences. 2010;107:13396-13401. 13. Cunningham F, Amode MR, Barrell D, Beal K, Billis K, Brent S, Carvalho-Silva D, Clapham P, Coates G, Fitzgerald S, Gil L, Girón CG, Gordon L, Hourlier T, Hunt SE, Janacek SH, Johnson N, Juettemann T, Kähäri AK, Keenan S, Martin FJ, Maurel T, McLaren W, Murphy DN, Nag R,

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Novel Support for the Emerging Paradigm of Genetic Convergence in Mammalian Echolocators

Supporting Data Common Name

Scientific Name

Accession Number

Megabat

Pteropus vampyrus

ENSPVAT00000011544

Dolphin

Tursiops truncatus

ENSTTRG00000001490

Gorilla

Gorilla gorilla

ENSGGOT00000027141

Cat

Felis catus

ENSFCAT00000031017

Dog

Canis lupus familiaris

ENSCAFT00000007906

Microbat

Myotis lucifugus

ENSMLUT00000010044

Rat

Rattus norvegicus

ENSRNOT00000012167

Shrew

Sorex araneus

ENSSART00000004319

Human

Homo sapiens

ENST00000400324

Platypus

Ornithorhynchus anatinus

ENSOANT00000011152

Location GeneScaffold_1821: 55,738472,950 GeneScaffold_1573: 55,806613,554 Chromosome 13: 42,166,67942,571,640 Chromosome A1: 35,468,26435,970,636 Chromosome 22: 15,573,63216,078,565 Scaffold GL429841: 1,143,4981,447,021 Chromosome 15: 70,039,42470,349,983 GeneScaffold_3538: 50,085394,071 Chromosome 13: 59,665,58360,163,987 Chromosome 2: 50,376,61750,580,026

Transcript Length (AA)

Echolocation Capacity*

1189

No

1191

Yes

1109

No

1188

No

1191

No

973

Yes

1042

No

1116

Yes

1193

Rare

983

No

Table S1. Data for species included in study, with accession number corresponding to the specific transcript used in the analysis.

Canadian Studies Cognitive Science Health Studies Sexual Diversity Studies UC One

The founding college of the University of Toronto

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RESEARCH

Calreticulin Acts as a Critical Mediator of Bone and Heart Cell Differentiation Through Regulating the Nuclear Localization of Critical Transcription Factors Ponthea Pouramin1 and Michal Opas1 Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada.

1

Abstract:

The specification of Embryonic Stem (ES) cells into osteogenic and cardiogenic lines is dependent on the activity of transcription factors. Bone specification is regulated by the transcription factor Osterix (Osx) while heart specification is controlled by the transcription factors NFATc4 and Slug. Calreticulin (Crt), an endoplasmic reticulum calcium-binding protein, is an important regulator of both cardiogenic and osteogenic development. Although Crt regulates the expression of Osx and Slug, the post-translational regulation of these transcription factors during osteogenic and cardiogenic development have yet to be elucidated. We investigated if Crt controls the nuclear localization of the Osx, Slug, and NFATc4 transcription factors during ES cell differentiation. ES cells were differentiated under bone or cardiac differentiation conditions in Crt knockout and control cells. Fractionation was used to investigate the translocation of transcription factors and immunoprecipitation was used to determine interactions between Calcineurin (CaN) and Osx during osteogenic differentiation. We demonstrate that knocking out Crt reduces expression of CaN and Osx over the course of 21-days of ES differentiation. We further show that Osx interacts with CaN. Finally we show that knocking out Crt reduces the nuclear localization of Osx at D5 and D7 of ES differentiation. Likewise, during cardiac differentiation, we show that knocking out Crt reduces the nuclear localization of Slug at D5 and Slug and NFATc4 at D10. We conclude that Crt facilitates bone and heart cell specification in part through its regulation of Osx and Slug nuclear localization.

Introduction

Stem cells are of great interest for regenerative medicine for their unique ability renew themselves and differentiate into multiple cell types [1]. Embryonic Stem (ES) cells obtained from the inner cell mass of blastocysts are pluripotent [2], and have been used to create osteoblasts, and cardiomyocites [3,4]. Osteoblasts are important in bone metabolism and produce bone matrix protein to counterbalance osteoclasts [5,6]. Cardiomyocytes are the fundamental heart cell, which through uniform retraction cause the heart to beat [4].

ously demonstrated that Crt deficiency reduces osteogenic markers including BSP, Runx2, and Osterix. As a calcium chaperone, Crt plays an essential role in regulating the activity of Calcineurin (CaN), a serine/threonine phosphatase sensitive to calcium/calmodulin signalling [10,11]. Specifically, the relationship between CaN and Crt is established in cardiac stem cells, whereby the expression of CaN reverses embryonic lethality caused by Crt-deficient mice [12]. Furthermore, CaN is critical to osteoblast commitment by stem cells, and CaN-null mice demonstrate severe osteoporosis and reduced bone formation [13].

Calreticulin regulates ES Lineage Commitment. An emerging regulator of ES lineage commitment is the Endoplasmic Reticulum (ER) calcium buffering chaperone Calreticulin (Crt). Calreticulin plays an important role in calcium homeostasis and calcium signalling pathways, including regulating among other functions, calcium storage and cell-adhesion [7]. Under osteoblast differentiation conditions, Crt plays a critical role to inhibit PPARγ, an adipogenic transcription factor, and this inhibits a switch to adipogenesis [8]. Moreover, Yu [9] has previ-

Osterix regulates Osteoblast Differentiation. Osterix (Osx) is an important transcription factor is for osteoblast differentiation and controls the transcription of important osteoblast markers including osteocalcin, collagen Type 1A1, and osteopontin [14]. In Osx-null mice, bone stem cells were arrested in differentiation15, resulting in no osteoblast formation and no bone formation [16,17]. The molecular mechanism and regulation of Osx are currently not well understood. NFATc1 overexpression, another important

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Journal of Undergraduate Life Sciences • Volume 11 • Issue 1 • Spring 2017


Calreticulin Acts as a Critical Mediator of Bone and Heart Cell Differentiation Through Regulating the Nuclear Localization of Critical Transcription Factors

osteogenic transcription factor, upregulates Osx [15]. Furthermore, Osx interacts with NFATc1 to form a DNA-complex and promote gene expression [15]. This interaction might suggest that Osx and NFATc1 are similarly regulated. NFATc1 possesses a CaN binding LxVP motif, which facilitates its dephosphorylation by CaN [18]. Upon dephosphorylation, NFATc1 localizes to the nucleus to effect changes in gene expression. Interestingly, Osx also possesses a LxVP motif [19], which in Human Embryonic Kidney (HEK) cells also facilitated a CaN/Osx interaction [19]. Moreover, CaN and Osx are known co-localize the nucleus [20], an interaction dependant on the Osx LxVP motif [20]. These data indicate that CaN regulates Osx nuclear localization. It remains unclear whether this interaction is important for osteoblast formation. Calreticulin regulates cardiomyocyte differentiation. In addition to osteogenic differentiation, Crt is an important regulator of cardiac development [12,21], however the transcriptional mediators require further investigation. The NFAT family of proteins including NFATc4, are critical to cardiomyogenesis and are regulated by CaN [22]. Moreover, NFATs were downregulated in Crt-null mice [12]. In unpublished work, Opas et al. has demonstrated that Crt regulates Epithelial-Mesenchymal Transition (EMT) pathways, in turn regulating E-cadherin expression. In cancer cells, Crt has been shown to act through the transcription factor slug to regulate E-cadherin expression [23]. Unpublished work from Opas et al. shows that Slug’s promoter contains a predicted NFATc4 binding site. For these reasons, both NFATc4 and Slug exists as promising targets to investigate as downstream mediators of Crt signaling. Considering the emerging role of Crt in cardiogenic and osteogenic differentiation, this work investigated how Crt regulates the nuclear localization of Osx, Slug, and NFATc4. We hypothesized that Crt would be required for the nuclear localization of Osx during osteoblast differentiation, and Slug and NFATc4 during cardiac differentiation.

Materials and Methods ES cells and differentiation

To study osteogenic differentiation, R1 murine ES cells were used (J1 129/Sv mice, from Dr. Janet Rossant, Mount Sinai Hospital, Toronto, Canada), and G45 calreticulin knockout (KO) ES cells, (from Dr. Marek Michalak, Department of Biochemistry, University of Alberta) and cultured using the hanging drop method as previously described [21]. ES cells were suspended from petri dishes for three days and subsequently formed embryoid bodies (EBs). EBs were grown in suspension for two days. On D5 of growth, the EBs were plated on 0.1% gelatinized tissue culture plates with differentiation media. Osteoblast differentiation. Cells were plated with high glucose Dubecco modified Eagle’s medium (DMEM) supplemented with sodium pyruvate and L-glutamine (Multicell, Wisent), 20% Fetal Bovine Serum (FBS; Multicell, Wisent), nonessential amino acids, and β-mercaptoethanol. From D3-D5 retinoic acid (0.1 µM, Sigma-Aldrich) was supplemented into the differentiation medium. From D6, 50 µg/mL ascorbic acid and 10 mM β-glycerolphosphate were supplemented in the medium until D21. In addition, starting at D10, 0.1 µM dexamethasone (Sigma-Aldrich) was added into the medium. Cells were collected from D0-D21 of osteogenic differentiation. Cardiomyocyte differentiation. Cells were grown in high glucose

Research Articles

DMEM supplemented by 15% FBS, minimal essential medium nonessential amino acids, and β-mercaptoethanol. Cells were collected on D0-D14. For Ionomycin experiments, Ionomycin was added to the medium at 1µM for 1 hour prior to cell collection.

Subcellular Fractionation Subcellular fractionation was used to separate proteins present in the cytoplasm from those in the nucleus to measure nuclear translocation. R1 and Crt KO cells of osteogenic and cardiac mouse ES cell lineages were spun down, collected in the pellet fraction, and homogenized in STM (250 mM Sucrose, 50 mM Tris-HCL pH 7.4, 5mM MgCl2, 1 phosphatase inhibitor tablet, 10 μL of protease inhibitor) fractionation buffer and centrifuged. The nuclear contents formed a pellet while cytoplasm was retained within the supernatant for and subsequently purified. Nuclear Fraction. To enrich the nuclear fraction, for two rounds, the collected pellet (from above) was resuspended in STM buffer and centrifuged. Finally, the pellet was retained and resuspended in NET buffer (20mM Hepes pH 7.9, 5mM Mgcl2, 0.5M NaCl, 0.2 mM EDTA, 20% glycerol, 1% Triton 100x, 1 phosphatase inhibitor tablet, 10μL of protease inhibitor), cooled on ice, centrifuged, and the supernatant collected. Cytosolic Fractionation. To further purify the cytosolic fraction, the supernatant collected from the subcellular fractionation was centrifuged. Equal volume of cold acetone was added to the supernatant and incubated at -20°C to allow protein precipitation. The solution was centrifuged and the pellet was collected and resuspended in NET buffer.

Cell Lysis For whole cell protein analysis, cells were lysed in NET buffer, lysates were sonicated and centrifuged at room temperature. The supernatant was collected and used for downstream applications.

Western Blotting To identify changes in protein expression, we performed a standard western blot. Protein concentrations were calculated using a Bradford assay using Bio-Rad Protein Assay reagent. A total of 30μg was ran on a 10% Sodium dodecyl sulfate (SDS) Polyacrylamide gel and transferred onto nitrocellulose membrane. Washes were performed with PBS-T. The membrane was blocked in 5% skimmed milk powder in PBS-T (0.1% Tween20 in 1XPBS) at room temperature. Primary antibodies were diluted in PBS-T: 1:1000 rabbit α-GAPDH (Cell Signalling), rabbit α- Histone (H3) (AbCam) antibody, rabbit α-Slug (cell signalling) rabbit α-Osterix (Abcam), and rabbit α-NFATc4 (AbCam). Secondary antibodies were diluted 1:5000 in PBS-T. Protein visualization was carried out using Chemiluminescence, and equivalent exposures were used between KO and WT samples.

Immunoprecipitation R1 (wild type) osteogenic mouse ES cells were pre-cleared by washing in PBS, and centrifuged. The supernatant was removed and resuspended in Lysis buffer (150 mM NaCl, 1.0% NP-40, 50 mM Tris-HCl pH8.0, 50 mM NaF and 1 mM Na3PO4). The lysates were pre-incubated with rabbit α-Osterix (Abcam) or α-Calcineurin (Cell signalling) antibodies overnight at 4°C. The protein A/G plus agarose beads (Santa Cruz) were washed with PBS and incubated with the lysates that were pre-incubated with either with rabbit α-Osterix (Abcam) or α-Calcineurin (Cell signalling) antibodies. All experiments were performed in duplicates.

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Calreticulin Acts as a Critical Mediator of Bone and Heart Cell Differentiation Through Regulating the Nuclear Localization of Critical Transcription Factors

Results

Calreticulin regulates expression and localization of Osterix during osteogenic differentiation. We established a time-course protein expression pattern of Osx and CaN in R1 murine wild type (WT) and Crt knockout (KO) cells. Our preliminary data showed R1 cells express CaN relatively constantly beginning from D0 until a reduction at D19 and D21 (Figure 1A). By contrast, Osx expression levels began at D3 (low expression) and show a peak at D5-D7. Expressions levels were also prominent between days D14-D21. The Crt KO cells demonstrate a clear inhibition of both CaN and Osx (Figure 1B). Expression of CaN is almost completely inhibited. Likewise, Osx shows abrogated expression except for minor peaks at D5, and D7, and again at D17, and D19. The effects of Crt KO can be reversed by administration of Ionomycin (Figure 1C), a drug which releases intracellular calcium stores. Administration of Ionomycin rescued CaN expression, and increased Osx expression starting at D0. These preliminary data suggest that Crt may regulate CaN and Osx expression. The peaks in Osx expression at D5 and D7 point to these days in particular as interesting developmental days for subsequent analysis. Next, fractionation was used to determine if at D5 and

Figure 1: Crt regulates the expression of CaN and Osx. A western blot of whole cell lysates of R1 and CRT KO ES cells. A) A time course expression analysis of CaN and Osx in R1 wild type control cells show increased expression of CaN and Osx at day 5. B) A time course analysis of CaN and Osx in CRT KO cells demonstrates significantly reduced expression of CaN and Osx. C) Administration of Ionomycin in CRT KO cells rescues the phenotype, and strongly upregulates CaN and Osx. Images are representative from N=2 samples tested.

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Figure 2: Crt regulates Osx nuclear localization. Western Blotting and Fractionation of R1 and CRT KO cells nuclear and cytoplasmic fractions during Days 5 and 7 of osteogenic differentiation. Western blot probing for OSX, GAPDH, and H3 demonstrates that nuclear localization of Osx is impaired in CRT KO cells at Day 5 and Day 7. Likewise, relative protein expression levels of OSX and GAPDH in the cytoplasmic fraction show an increased or normal amount of Osx at days 5 and 7. Images are representative from N=2 samples tested.

Figure 3: CaN and Osx interact during osteoblast differentiation. Interaction of Calcineurin and Osterix during Day 5 and Day 7 of osteogenic differentiation. ES cells of Days 5 and 7 were immunoprecipitated with beads incubated with either an α-OSX or α-CaN antibody, which were subjected to western blot analysis and probed for OSX, and CaN with IgG as a control.

Figure 4: Crt regulates Slug nuclear localization at day 5 and day 10. Western Blotting and Fractionation of R1 and CRT KO cells nuclear and cytoplasmic fractions during Days 5 and 10 of cardiac differentiation. Western blot probing for NFATc4, Slug, GAPDH, and H3 demonstrates that nuclear localization of Slug is impaired in CRT KO cells at Day 5 and Day 10. Likewise, relative protein expression levels of Slug and GAPDH in the cytoplasmic fraction shows normal levels of Slug at days 5 and 10. Images are representative from N=2 samples tested.

Journal of Undergraduate Life Sciences • Volume 11 • Issue 1 • Spring 2017


Calreticulin Acts as a Critical Mediator of Bone and Heart Cell Differentiation Through Regulating the Nuclear Localization of Critical Transcription Factors

D7 during osteoblast differentiation, Crt regulated the nuclear translocation of Osx. In the nuclear fraction, when Crt is knocked out, Osx localization to the nucleus appeared to be reduced at D5 (Figure 2). Likewise, in the cytoplasmic fraction, at D5 we observed an increase of Osx protein, but no interpretable change during D7 (Figure 2). To test if Osx and CaN interaction occurred during D5 and D7 of osteoblast differentiation, we performed an immunoprecipitation. During both D5 and to a lesser extent, D7, the Osx pull-down precipitated an interaction with CaN (Figure 3). The CaN pull-down did not yield strong results. Calreticulin regulates both Slug and NFATc4 nuclear localization during cardiac differentiation. We also analyzed the nuclear localization of NFATc4 and Slug in Crt KO and WT cells. In Crt KO cells at D0 of differentiation, both NFATc4 and Slug protein levels were reduced in both the nucleus and the cytoplasm (Figure 4). During D5 of cardiac differentiation, Crt KO cells demonstrated reduced levels of Slug protein in the nucleus (Figure 4A). Likewise, a similar level of Slug protein in the cytoplasm was observed (Figure 4B). The translocation pattern of NFATc4 however is less consistent. At D5, there were increased protein levels in both the cytoplasm and nucleus (Figure 4). However, at D10, NFATc4 protein levels in the nucleus were reduced in Crt KO cells (Figure 4A). Together, this provides preliminary evidence that Crt regulates the localization of Slug at D5 and D10 during cardiac differentiation.

Discussion

This study investigated the role of Crt in facilitating the nuclear localization of the transcription factors Osx during osteoblast differentiation, and Slug and NFATc4 during cardiac differentiation. During osteoblast differentiation, Osx expression peaked at D5 and D7 of differentiation, suggesting that these points during development might be critical for osteogenesis. Knocking out Crt globally reduced levels of Osx and CaN, suggesting Crt plays a role in this signalling pathway. Interestingly, administration of Ionomycin, which increases cellular Ca2+ levels, is sufficient to rescue Osx and CaN levels, and even up-regulate Osx expression at earlier than normal time points (D0 in KO + Iono versus D5 in R1). This data is consistent with a model where calcium signalling, controlled by Crt, regulates Osx expression. However, we cannot rule out Ionomycin induced apoptosis, and in the future a cell viability assay should be performed. Our data has been supported by previous qPCR and immunofluorescence work which demonstrated knocking out Crt reduced expression of Osteoblast markers as early as D7 [9]. This work, through the generation of differentiation timeline, presents evidence that Crt regulates Osx earlier, beginning at D5, while also supporting D7 as an important day. Our work further investigates the role of Crt on Osx nuclear localization. While the data is perhaps difficult to interpret, in addition to reducing the overall levels of Osx, Crt KO reduced the amount of Osx present in the nucleus at D5. This was associated with a corresponding increase of Osx present in the cytoplasm observed in the day 5 experiment. While we cannot rule out that reduced nuclear localization of Osx is due to reduced Osx expression, the fact that cytoplasmic levels of Osx were approximately increased, or normal, suggests that Crt regulates Osx in part through facilitating its nuclear localization.

Research Articles

We also begin to delineate a possible mechanism responsible for regulating Osx nuclear localization during osteoblast differentiation. The movement of Osx into the nucleus has been shown to be regulated by its phosphorylation state [24]. Localization of Osx to the nucleus requires de-phosphorylation [24]. A candidate for Osx dephosphorylation is the phosphatase CaN which has been shown to facilitate Osx nuclear localization through interaction of the Osx LxVP motif [19]. Importantly, Crt is a known upstream regulator of CaN. We show that CaN complexes with Osx at D5 of differentiation. Considering that CaN and Osx expression levels are dependent on Crt, and calcium signalling, we provide evidence to support a model whereby Crt regulates Osx translocation through the activity of CaN. Future work could more precisely measure this interaction, by measuring the phosphorylation state and localization of Osx in the presence or absence of a CaN inhibitor. We also explored how Crt regulates cardiac cell differentiation. In particular, we looked at the transcription factor Slug, known to regulate E-cadherin expression during cardiac development. A genetic regulation between Crt and Slug has been reported in kidney cells, where overexpression of Crt increased expression of Slug [23]. Here, we investigated this relationship by knocking out Crt and show that at D0, D5, and D10 during cardiac differentiation, nuclear levels of Slug are reduced, and virtually ablated at D5 and D10. As cytoplasmic levels of Crt are relatively consistent between Crt KO and R1 controls, our data suggests Crt regulates the movement of Slug into the nucleus. We also explored the possibility that the expression and localization of Slug might be tied to NFATc4. Slug possesses NFATc4 binding sites in its promoter. Moreover, NFATc4 contains the CaN binding LXvP motif, which regulates its nuclear localization, linking its movement to Crt [25]. Our data concerning NFATc4 localization is inconclusive. Crt reduced NFATc4 nuclear localization at D0 and D10 during cardiac differentiation, but not at D5. This inconsistency could be due to the input of other factors. In addition to cardiac differentiation, NFATc4 has also been known to regulate differentiation to adipocytes [26]. Knockout of Crt in ES cells is known to increase adipogenesis [8]. It is possible that other pathways regulate NFATc4 nuclear localization as part of an adipocyte lineage commitment. It is interesting that specifically at D5, NFATc4 nuclear localization is increased, coinciding with an Osx expression peak during osteoblast commitment. This might indicate that D5 of differentiation is a critical time when Crt represses a switch to adipogenesis. Indeed, it has been shown that Crt exerts its adipocyte inhibitory activity early during development (D3) but not at later time points (D7, D9) [8]. Future work should explore Crt as a general differentiation repressor of adipogenesis. This work is preliminary in nature and a major limitation is the reliance on fractionation to identify localization changes. Between fractionation preparations there is often cross contamination between cytoplasmic and nuclear fractions. This makes precise analysis on localization patterns difficult. As a result, our fractionation data cannot be analyzed quantitatively and can largely only be read as a binary, large change or no change, result. Furthermore, due to our low sample sizes (N=2) for the whole cell experiments, we cannot reach statistical conclusions, and our data can only be interpreted through robust changes. Our Gapdh reference controls, however, were somewhat consistent allowing for some qualitative observations on repression and expression peaks.

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Calreticulin Title Acts as a Critical Mediator of Bone and Heart Cell Differentiation Through Regulating the Nuclear Localization of Critical Transcription Factors

Future work should add to, and quantify this data and also examine nuclear localization and expression through fluorescence intensity. Localization changes should be supported with functional changes in gene expression, as measured through qPCR. A luciferase assay of transcription factor targets would also be useful in elucidating when transcription factors are active in the nucleus. Collectively, our results show a role of Crt in regulating both cardiogenic and osteogenic differentiation pathways, and these pathways may play a role in disease. The role of Crt in promoting osteoblast formation could yield an important link to diseases such as osteoporosis, characterized by breakdown of osteoblasts [27]. Dysregulation of Crt and calcium signalling as a whole has been implicated in regulating heart fibrosis [28], and cancer [29]. Future studies should look at these diseases through the context of Crt mediated stem cell dysregulation. In conclusion we demonstrated that Crt regulates the expression on potentially nuclear localization of Osx during osteoblast differentiation of ES cells. In addition, we show that Crt regulates the nuclear localization of Slug during cardiomyocyte differentiation of ES cells.

phosphorylation status of transcription factor osterix. Biochem. Biophys. Res. Commun. 379, 440–444 (2009). 20. Shields, J. M. & Yang, V. W. Two potent nuclear localization signals in the gut-enriched Krüppellike factor define a subfamily of closely related Krüppel proteins. J. Biol. Chem. 272, 18504–7 (1997). 21. Papp, S., Dziak, E. & Opas, M. Embryonic stem cell-derived cardiomyogenesis: A novel role for calreticulin as a regulator. Stem Cells 27, 1507–1515 (2009). 22. Cripps, R. M. & Olson, E. N. Control of Cardiac Development by an Evolutionarily Conserved Transcriptional Network. Dev. Biol. 246, 14–28 (2002). 23. Hayashida, Y. et al. Calreticulin down-regulates E-cadherin gene expression via Slug in a Ca2+-dependent manner. Cancer Res. 66, (2014). 24. Artigas, N., Urena, C., Rodriguez-Carballo, E., Rosa, J. L. & Ventura, F. Mitogen-activated Protein Kinase (MAPK)-regulated Interactions between Osterix and Runx2 Are Critical for the Transcriptional Osteogenic Program. J. Biol. Chem. 289, 27105–27117 (2014). 25. Gal, M., Li, S., Luna, R. E., Takeuchi, K. & Wagner, G. The LxVP and PxIxIT NFAT motifs bind jointly to overlapping epitopes on calcineurin’s catalytic domain distant to the regulatory domain. Structure 22, 1016–27 (2014). 26. Ho, I. C., Kim, J. H., Rooney, J. W., Spiegelman, B. M. & Glimcher, L. H. A potential role for the nuclear factor of activated T cells family of transcriptional regulatory proteins in adipogenesis. Proc. Natl. Acad. Sci. U. S. A. 95, 15537–41 (1998). 27. Tanaka, Y., Nakayamada, S. & Okada, Y. Osteoblasts and Osteoclasts in Bone Remodeling and Inflammation. Curr. Drug Target -Inflammation Allergy 4, 325–328 (2005). 28. Kypreou, K. P. et al. Altered expression of calreticulin during the development of fibrosis. Proteomics 8, 2407–2419 (2008). 29. Lu, Y.-C., Weng, W.-C. & Lee, H. Functional Roles of Calreticulin in Cancer Biology. Biomed Res. Int. 2015, 1–9 (2015).

Acknowledgements

I am grateful to Dr. Opas for being supportive supervisor, and guiding me throughout the project. I would like to thank Dr. Fereshta Karmizahdeh for her advice, expertise, and encouragement, and Dr. Carlos Piquil for his support and technical expertise.

References

1. Bodnar, M. S., Meneses, J. J., Rodriguez, R. T. & Firpo, M. T. Propagation and maintenance of undifferentiated human embryonic stem cells. Stem Cells Dev. 13, 243–53 (2004). 2. Wobus, A. M., Rohwedel, J., Maltsev, V. & Hescheler, J. In vitro differentiation of embryonic stem cells into cardiomyocytes or skeletal muscle cells is specifically modulated by retinoic acid. Roux’s Arch. Dev. Biol. 204, 36–45 (1994). 3. Czekanska, E. M., Stoddart, M. J., Richards, R. G. & Hayes, J. S. In search of an osteoblast cell model for in vitro research. Eur. Cell. Mater. 24, 1–17 (2012). 4. Kehat, I. et al. Human embryonic stem cells can differentiate into myocytes with structural and functional properties of cardiomyocytes. J. Clin. Invest. 108, 407–14 (2001). 5. Karsenty, G. & Wagner, E. F. Reaching a genetic and molecular understanding of skeletal development. Dev. Cell 2, 389–406 (2002). 6. Karsenty, G. The complexities of skeletal biology. Nature 423, 316–8 (2003). 7. Michalak, M., Parker, J. M. R. & Opas, M. Ca2+ signaling and calcium binding chaperones of the endoplasmic reticulum. Cell Calcium 32, 269–278 (2002). 8. Szabo, E., Qiu, Y., Baksh, S., Michalak, M. & Opas, M. Calreticulin inhibits commitment to adipocyte differentiation. J. Cell Biol. 182, (2008). 9. Yu, Y. Osteogenic Differentiation from Mouse Embryonic Stem Cells the Role of Calreticulin. (University of Toronto, 2013). 10. Stern, P. H. The calcineurin-NFAT pathway and bone: intriguing new findings. Mol. Interv. 6, 193–6 (2006). 11. Mulero, M. C. et al. Inhibiting the calcineurin-NFAT (nuclear factor of activated T cells) signaling pathway with a regulator of calcineurin-derived peptide without affecting general calcineurin phosphatase activity. J. Biol. Chem. 284, 9394–401 (2009). 12. Mesaeli, N. et al. Calreticulin is essential for cardiac development. J. Cell Biol. 144, 857–68 (1999). 13. Crabtree, G. R. Calcium, calcineurin, and the control of transcription. J. Biol. Chem. 276, 2313–6 (2001). 14. Gao, Y., Jheon, A., Nourkeyhani, H., Kobayashi, H. & Ganss, B. Molecular cloning, structure, expression, and chromosomal localization of the human Osterix (SP7) gene. Gene 341, 101–10 (2004). 15. Koga, T. et al. NFAT and Osterix cooperatively regulate bone formation. Nat. Med. 11, 880–5 (2005). 16. Zhou, X. et al. Multiple functions of Osterix are required for bone growth and homeostasis in postnatal mice. Proc. Natl. Acad. Sci. U. S. A. 107, 12919–24 (2010). 17. Huang, W. & Olsen, B. R. Skeletal defects in Osterix-Cre transgenic mice. Transgenic Res. 24, 167–72 (2015). 18. Gal, M., Li, S., Luna, R. E., Takeuchi, K. & Wagner, G. The LxVP and PxIxIT NFAT motifs bind jointly to overlapping epitopes on calcineurin’s catalytic domain distant to the regulatory domain. Structure 22, 1016–27 (2014). 19. Okamura, H., Amorim, B. R., Wang, J., Yoshida, K. & Haneji, T. Calcineurin regulates

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Journal of Undergraduate Life Sciences • Volume 11 • Issue 1 • Spring 2017


JULS

RESEARCH

Effects of Short-Term High-Fat Diet Feeding On the Outcomes of Lyme Disease Infection in Mice Zoha Anjum Matrix Dynamics Group, Faculty of Dentistry, University of Toronto, Toronto, Ontario, Canada.

Abstract

Lyme disease is a common tick-borne bacterial infection in North America. The clinical manifestations of Lyme disease include inflammation of heart (carditis) and joints (arthritis). Previous studies from our laboratory have demonstrated that Lyme carditis is more severe and proportional to the bacterial DNA in the hearts of diet-induced obese mice. However, it is unknown if aggravated disease manifestations are caused by obesity or the composition of the diet used to induce obesity. To distinguish between obesityand diet-related effects on Lyme disease outcomes, we preconditioned mice on a high fat diet (HFD) for ten days instead of the standard protocol of 3-months duration. Our objective was to investigate effects of a short-term HFD on Lyme disease outcomes in a mouse model. Mice were preconditioned with a HFD or a standard rodent chow (ND) for ten days, and subsequently inoculated with the bacterium Borrelia burgdorferi. Four weeks post-inoculation, various organs were harvested and the number of bacterial DNA copies in the organs were measured using quantitative real-time PCR (qPCR). Short-term HFD feeding led to a rate of increase in body weight comparable to the weight gained after standard long-term HFD preconditioning. However, no difference in bacterial burden was observed between the two groups in the heart, ear, skin, bladder, liver, brains, lungs, knee and tibiotarsal joints. Short-term HFD feeding does not translate into an increased severity of infection in the murine model of Lyme disease, likely due to absence of immunological impairment in response to short-term consumption of HFD.

Introduction

In industrialized countries, the number of obese and overweight individuals is increasing at an alarming rate, partially due to an easily accessible energy-dense diet [1-4]. Obesity has been previously associated with metabolic conditions such as diabetes, heart disease, and stroke [1]. Moreover, recent research has established that obesity interferes with the host immune system, thus increasing the susceptibility of the host to infectious diseases [1,5]. For example, obesity is associated with a high occurrence of infections such as influenza, urinary tract infection, periodontitis, and surgical infections [1,4,5,7,8,9]. Possible mechanisms explaining the relationship between obesity and infections include abnormal levels of immune system mediators and impaired functioning of immune cells [1,4]. Obesity and associated pathologies may therefore impact the host immune status and predispose an individual to altered infections outcomes. One of the infections aggravated by obesity is Lyme disease as observed in mouse models [1,5]. Lyme disease is a vector-borne disease that is highly prevalent in North America and Europe [2,5,10,11]. The causative agent of Lyme disease, the bacterium Borrelia burgdorferi, is transmitted to humans and other mammals by Ixodes spp. ticks [2,5,10]. As the infection progresses and B. burgdorferi dissewminates from the inoculation site to the target

organs, several inflammatory pathologies such as Lyme carditis, Lyme arthritis, and neuroborreliosis may develop [2,12]. Lyme disease is also characterized by acute cutaneous inflammatory pathologies [2,12]. The clinical manifestations are specific to the genotype of the host and the bacteria, which makes Lyme disease prognosis hard to predict in an individual [12]. Lyme disease is usually treated with antibiotics, but the exact treatment depends on the symptoms and the stage of the disease. Approximately 10 % of Lyme disease patients experience fatigue and neurological complications even after completing the standard course of antibiotic treatment for Lyme disease [13,14]. This condition is known as post-treatment Lyme disease syndrome, and it is hypothesized to be caused by an autoimmune response to Lyme disease infection or persistent bacterial colonization [13,14]. The direct cause of the post-treatment Lyme disease syndrome and its risk factors have yet to be identified [14,15]. A high-caloric diet intake is the leading cause of obesity in humans; therefore, a diet-induced obesity (DIO) model is used to study Lyme disease and other infections in the context of obesity [1,3,4]. In mice, DIO has been shown to alter the Borrelia burgdorferi burden in different organs such as heart, blood, brain, and liver [5]. Additionally, DIO mice also display more severe cardiac inflammatory pathologies proportional to the increased number of bacterial DNA copies (referred to as bacterial burden)

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Effects of Short-Term High-Fat Diet Feeding On the Outcomes of Lyme Disease Infection in Mice

[5]. Impaired clearance of bacterial DNA from the organs is also associated with poor activity of macrophages in DIO mice [5]. Nonetheless, the exact underlying mechanisms of DIO and Lyme disease outcomes are unclear. As mentioned earlier, B. burgdorferi burden in HFD-fed DIO mice is elevated when compared to their counterparts fed a standard rodent chow (ND) [5]. It has not been established if the increase in bacterial burden is caused by obesity-related physiological/immunological pathologies (stemming from a long-term intake of energy-dense meals) or a HFD-feeding as HFD may provide a favorable environment for bacterial growth and/or dissemination. Therefore, to differentiate between the effects of a short-term HFD and DIO stemming from long-term HFD preconditioning, the mice were fed a HFD for ten days before substantial obesity-related physiological changes had developed [5]. Subsequent infectivity studies allowed us to explore the effects of short-term of HFD-feeding. Our study provides a foundation for epidemiological studies in the human population focused on exploring the relationship between obesity and Lyme disease. Furthermore, this study may deepen our understanding of the association between the composition of dietary intake and susceptibility to infectious diseases.

Methods

Ethics Statement The project was conducted according to the principles stated in the Guide to the Care and use of Experimental Animals by the Canadian Council on Animal Care. All the experimental procedures were approved by the University of Toronto Animal Care Committee. The bacterium, Borrelia burgdorferi was handled according to the guidelines of the University of Toronto, Public Health Agency of Canada, and Canadian Food Inspection Agency. The authors declare no conflicts of interest involved in this study.

Animal Work Four-week old female C3H/HeN mice were purchased from Charles River (Montréal, QC, Canada). Mice were housed in groups of four per cage under pathogen-free conditions. Immediately upon arrival, mice were assigned to one of the two groups with ten mice per group, and fed a high-fat-diet (HFD) or a standard rodent chow (normal diet, ND). Mice preconditioned on a HFD received 18.4%, 21.3% and 60.3% kcal from protein, carbohydrate, and fat, respectively, whereas ND-preconditioned mice consumed 20.1%, 69.8%, 10.2% kcal from protein, carbohydrate, and fat, respectively. After ten days of preconditioning, mice were infected with B. burgdorferi by subcutaneous injection in the dorsal midline section (104 bacteria in 100 µl of bacterial cultivation medium). Mice were sacrificed four weeks post-inoculation and the organs were harvested. The weight and non-fasting blood glucose of mice were measured at the time on infection and at the time of sacrifice.

Serum Collection & ELISA Blood was collected before the sacrifice by cardiac puncture. Serum was collected after spinning clotted blood for 5 minutes at 10,000 x g, and placed immediately in -80 ˚C until further experiments. Enzyme linked immunosorbent assay (ELISA) were performed to measure anti-B. burgdorferi IgGs produced by mice as described previously [16]. 1:200 dilutions of each mouse serum were used in technical duplicates, and pooled sera from normal uninfected mice and B. burgdorferi-infected mice were used as negative and positive controls, respectively, also in technical duplicates.

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Determination of Bacterial Burden in Target Organs DNA was isolated from heart, skin, ear, bladder, knee joint, tibiotarsal joint, brain, liver, and lungs of sacrificed animals using PureLink Genomic DNA Mini kit, according to the instructions of the manufacturer (ThermoFisher Scientific). Numbers of copies of B. burgdorferi flaB DNA sequence were measured by quantitative real-time PCR (qPCR) with the isolated genomic DNA, as reported previously (16). The number of flaB (B. burgdorferi-specific gene) DNA copies in each sample was determined using a standard curve, comprising of technical duplicates of tenfold serial dilutions of plasmid DNA with known number of flaB DNA copies (107 – 1 copies). Each sample was run in technical triplicates, and the average flaB DNA copy number (bacterial burden) was normalized to 1 microgram of total isolated DNA to control for potential discrepancies in DNA extraction efficiency (Figure 4 A-I).

Statistical Analysis Statistical analysis, to measure the difference in the bacterial burden (bacterial DNA copies) between the two groups, was performed using GraphPad Prism v.6.0 software (GraphPad Software, La Jolla, CA).

Results

Impact of Short-Term HFD Feeding On Weight and Glucose After ten days of preconditioning (Ti), HFD-treated mice weighed 15% more compared to ND-treated mice (p<0.05; Figure 1). At the time of sacrifice (Tf ), HFD mice weighed 23% more than ND mice (p<0.05; Figure 1). Non-fasting blood glucose levels were also measured at the time of infection and sacrifice. There was no significant difference in the blood glucose levels between the two diet groups (p>0.05; Figure 2). B. burgdorferi-Specific IgGs in Inoculated Mice To confirm B. burgdorferi infection in mice, we performed an ELISA on the serum collected four weeks after bacterial inoculation. The bacterial substrate used to perform ELISA was from the same bacterial growth stage (logarithmic phase of growth) as the cultures used to inoculate mice. Surprisingly, out of the twenty mice inoculated with B. burgdorferi, only eleven developed B. burgdorferi-specific IgGs response: six of them were from the HFD group and five were from the ND group (Figure 3). A qPCR analysis was performed on the heart, ear, skin, bladder, and knee-joint in mice that did not develop a B. burgdorferi-specific IgG response, and no bacterial burden was detected. Therefore, we concluded that there was no infection in some mice. Thus, the mice with no detectable Borrelia burgdorferi IgGs were excluded from the following analysis of bacterial burden, in order to prevent distortion of possible differences between HFD and ND groups by negative values (Figure 3B). Bacterial Burden Numbers at Target Organs In B. burgdorferiInfected Mice The number of B. burgdorferi flaB gene copies in different organs was measured by performing qPCR using B. burgdorferispecific primers. There was no significant difference in bacterial burden in the heart (Figure 4A), skin (Figure 4B), ear (Figure 4C), bladder (Figure 4D), knee-joint (Figure 4E), tibiotarsal joint (Figure 4F), liver (Figure 4G), lungs (Figure 4H), and brains (Figure 4I) between the HFD- and ND-preconditioned mice.

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Research Articles

Figure 1: Weights of HFD- preconditioned and ND- preconditioned mice: The weights of mice at the time of infection (Ti) and at the time of sacrifice (Tf) are shown. HFD- preconditioned mice and ND- preconditioned mice are represented as black circles and white triangles, respectively. 15% and 23% show the difference between HFD- and ND- fed mice at different points of infection. Asterisk represents statistically significant comparison (p<0.001) between HFD-treated and ND-treated mice, as determined by unpaired t-test.

Figure 2: Non-fasting blood glucose of HFD-treated and ND-treated mice. The non-fasting glucose levels of mice at the time of infection (Ti) and at the time of sacrifice (Tf) are shown. HFD- preconditioned mice and ND- preconditioned mice are represented as black circles and white triangles, respectively. NS stands for not significant (p > 0.05), as determined by unpaired t-tests.

Discussion

(CCL2), CCL3 and IL-8, consequently promoting inflammation in the local tissue/organ [17]. Previously, Staphylococcus aureus infection was reported to be aggravated in presence of DIO, possibly due to immunoglobin class switch impairment and defective adaptive immune system [18,19]. Furthermore, DIO also exacerbates periodontitis, caused by the bacterium Porphyromonas gingivalis. In DIO, infiltration and activation of macrophages may be compromised, consequently increasing the severity of periodontitis and other infections [9,20,21]. As previously reported, B. burgdorferi infection worsens in DIO mice [5]. In the presence of DIO, an increase in bacterial burden in heart, liver, blood, brain, and patella/knee-joint was observed [5]. Following a long-term HFD preconditioning, neutrophil and macrophage activity was severely hampered and the production of cytokines was also affected in B. burgdorferiinfected mice [16]. However, our study suggests that a short-term HFD of ten days does not impact the host system enough for the Lyme disease infection severity to aggravate. Further experiments on immune system mediators are needed to quantify the differences in infection between the long-term and short-term HFD. In addition to the immune system impairment, alterations in the microbiome in obesity may also contribute to altered infection outcomes [22]. DIO resulting from a HFD diet disrupts the gut microbiome, favoring the presence of one bacterial species over

The objective of this study was to investigate if the short-term HFD has the same effects on B. burgdorferi infection as a long-term HFD, and if elevated B. burgdorferi burden observed following three months of HFD preconditioning was due to obesity or the composition of the diet (HFD) [5]. Following the short-term HFD preconditioning, there was no statistically significant difference in the B. burgdorferi burden between the HFD- and ND-fed mice in all examined organs (heart, skin, bladder, ear, knee-joints, tibiotarsal joints, liver, lungs, and brain; Figure4). This suggests that the elevated bacterial burden observed after a long-term HFD preconditioning stems from obesity-related physiological changes, not from a HFD intake. These organs, in specific, were selected as they are known to be colonized by B. burgdorferi after inoculation [5,16]. Obesity is a metabolic condition often linked with physiological and immunological changes, and it may lead to a compromised immune system and chronic low-grade inflammation [1,3,4]. The altered Lyme disease outcomes following a long-term HFD preconditioning may stem from an impaired immune system function. Exact underlying mechanisms are mostly unclear; however, abnormal macrophage and neutrophil function and suppression of antiinflammatory cytokines may contribute to the worse outcomes of infectious disease in the context of obesity [9]. Obesity leads to an excessive production of cytokines such as IL-1B, TNFa, MCP-1

Figure 3: Quantification of B. burgdorferi-specific IgG antibody in the serum of mice four weeks after infection. Serum was diluted to 1:200 (dilution), and tested against whole cell lysate of B. burgdorferi strain that was used for infection. HFD- preconditioned and ND- preconditioned mice are shown in black circles and white triangles, respectively, and the bars represent median with 95 % confidence intervals. Dotted lines represent the lower threshold for absorbance values (405 nm with the reference wavelength of 492 nm) as determined by the sum of the average and three standard deviations of the negative control (A405 = 0.0873). Serum of seropositive mice used for bacterial burden quantification. NS: p > 0.05 as determined by parametric Welch’s test.

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Research Articles

Effects of Short-Term High-Fat Diet Feeding On the Outcomes of Lyme Disease Infection in Mice

Figure 4: Bacterial burden in tissues of individual mice. A. Heart; B. Skin; C. Ear; D. Bladder; E. Knee joint; F. Tibiotarsal joint; G. Liver; H. Lungs; & I. Brain. Bacterial burden was determined as flaB DNA copy numbers normalized to one microgram of the total isolated DNA from the respective tissue. HFD-preconditioned mice are shown as black circles and ND-treated mice as white triangles, respectively. Bars display medians with 95 % confidence intervals. All data were log transformed before statistical analyses. NS – no significant differences between HFD and ND groups (p > 0.05), as determined by parametric Welch’s test (4B-4I) or non-parametric Mann-Whitney test (4A).

the other [22]. The composition of diet may also affect the immune responses in the intestines of the host over time [22]. Following the HFD preconditioning in mice, there was high oxidative stress caused by an increase in number of a native gut bacterium, Lactobacillus reuteri, subsequently leading to the impairment of gut immune responses [22]. The relationship between obesity and infectious disease outcomes is multi-faceted, and further studies are required to explore the underlying mechanisms. In the present study, the HFD mice had gained 15% more weight than their ND counterparts by the time of infection (ten days of HFD-preconditioning), and 23% more weight by the time of sacrifice (four weeks after infection) (Figure 1). According to the standard preconditioning protocol, DIO mouse models are expected to gain more than 25% weight compared to ND-treated mice throughout the diet span [11]. In the previous DIO studies conducted by Zlotnikov et al. and Pětrošová et al., the weight gained by HFD-fed mice at the time of infection and at the time of sacrifice was 26% and 38%, respectively [5,16]. Therefore, the weight gained by mice in our study may be comparable to the results previously reported in the literature [5,11,16]. Additionally, non-fasting blood glucose levels were measured at the time of infection (Ti) and sacrifice (Tf), where no difference was observed between the two treatment groups (Figure 2). These results are also similar to what Pětrošová et al. and Zlotnikov et al. reported in their studies [5,16]. Despite the significant weight gain, short-

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term HFD feeding does not lead to hyperglycemia in mice in the present study. One limitation of our study is the small sample size. The ELISA results indicated that nine out of the twenty initially infected mice did not develop B. burgdorferi-specific IgGs post-infection. The lysates used for ELISA were from the same strain that was initially used for infection. The strains were grown to logarithmic phase and used for inoculation and preparation of ELISA lysate. Therefore, the differences in the stage of bacterial growth when the culture was harvested for inoculation and lysate preparation may not be an appropriate factor explaining the absence of antibody response in some mice. To further test the infection status, we performed a qPCR analysis on the heart, ear, skin, bladder, and knee-joint tissues in mice that did not develop a B. burgdorferi-specific IgG response. No bacterial burden was observed; therefore, we concluded that there was no infection in some mice. Thus, the mice with no detectable Borrelia burgdorferi IgGs were excluded from the following analysis of bacterial burden to prevent distortion of possible differences between HFD and ND groups (Figure 3). The absence of B. burgdorferi-specific IgGs can be explained by a variety of factors. Use of a lower dose of bacteria may have led to lack of B. burgdorferi-specific IgGs development [16]. Alternatively, the bacteria might have undergone antigenic shift; thus, delaying the process of seroconversion [23, 24]. Repeating the present study with a larger sample size may provide more statistical power to our results, and further support our conclusions. Another experiment

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Effects of Short-Term High-Fat Diet Feeding On the Outcomes of Lyme Disease Infection in Mice

to consider would be an additional control group of long-term HFD feeding to exclude all possible confounders. This may provide more insight into the relationship between HFD and Lyme disease.

Conclusions & Future Experiments

The DIO animal models have been used to study the intersection of immunology and microbiology for a number of pathogens. Our results suggest that the elevated bacterial burden observed in DIO B. burgdorferi-infected mice may be due to obesity-related pathologies, not the HFD intake. Future studies will focus on investigating the underlying mechanisms of the increased severity of B. burgdorferi infection in the context of DIO, specifically from the perspective of the host-pathogen interactions. We aim to explore if B. burgdorferi outer membrane proteins have the ability to interact with host molecules such as adipokines (cytokines produced by adipose tissue) as they are abundant in obese individuals. Investigating the function of B. burgdorferi proteins and how they interact with the host may further elaborate on the relationship between obesity and Lyme disease.

Research Articles

80:4409–4416. 20. Huang X, Yu T, Ma C, Wang Y, Xie B, Xuan D & Zhang J. Macrophages play a key role in the obesity-induced periodontal innate immune dysfunction via nucleotide-binding oligomerization domain-like receptor protein 3 pathway. J. Periodontol. 2016 Oct; 86 (10): 1195-1205. 21. Zhou Q, Leeman SE, Amar S. Signaling mechanisms in the restoration of impaired immune function due to diet-induced obesity. Proc Natl Acad Sci U.S.A. 2011 Feb; 108(7): 2867-2872. 22. Sun J, Qiao Y, Qi C, Jiang W, Xiao H, Shi Y, Le G. High-fat-diet-induced obesity is associated with decreased anti-inflammatory Lactobacillus reuteri sensitive to oxidative stress in mouse Peyer’s patches. Nutrition. 2016 Feb; 32: 265-272. 23. Songe MM, Thoen E, Evensen O & Skaar I. In vitro passages impact on virulence of Saprolegnia parasitica to Atlantic salmon, Salmo salar L. parr. J Fis Dis. 2014 Sept; 37(9): 825-834. 24. Liang FT, Yan J, Mbow LM, Sviat SL, Gilmore RD, Mamula M, Fikrig E. Borrelia burgdorferi changes its surface antigenic expression in response to host immune responses. Infect Immun. 2014 Oct; 72 (10): 5759-5767.

References

1. Huttunen R & Syrjanen J. Obesity and the risk and outcome of infection. Int. J of Obes. 2013 Mar; 37: 333-340. 2. Smith BG, Cruz Jr. AI, Milewski MD, Shapiro ED. Lyme disease and the orthopaedic implications of Lyme arthritis. J Am Acad Orthop Surg. 2011Feb; 19(2):91-100. 3. Dhurandhar NV, Bailey D, Thomas D. Interaction of obesity and infections. Obes Rev. 2015 Dec;16:1017–1029. 4. McNelis JC, Olefsky JM. Macrophages, immunity, and metabolic disease. Immunity. 2014 Jul; 41:36–48. 5. Zlotnikov N, Javid A, Ahmed M, Tang TT, Eshghi A, Arya A, et al. Infection with the Lyme disease pathogen suppresses innate immunity in mice with diet-induced obesity. Cell Microbiol [Internet]. 2016 Oct [cited 2016 Nov 1] Epub. Available from: http://onlinelibrary.wiley.com/ doi/10.1111/cmi.12689/epdf 6. Festa A, D’Agostino R jr, William K, Karter AJ, Mayer-Davis EJ, Tracy RP, et al. The relation of body fat mass and distribution to markers of chronic inflammation. Int J Obes Relat Metab Disord. 2011 Oct; 10: 1407-15 7. Kwong JC, Campitelli MA, Rosella LC. Obesity and respiratory hospitalizations during influenza seasons in Ontario, Canada: a cohort study. Clin Infect Dis. 2011 Sept; 53 (4): 413-21. 8. Mobley D, Baum N. The obesity epidemic and its impact on urologic care. Rev Urol. 2015; 17(3): 165-70 9. Amar S, Zhou Q, Shaik-Dasthagirisaheb Y, Leeman S. Diet-induced obesity in mice causes changes in immune responses and bone loss manifested by bacterial challenge. Proc Natl Acad Sci U.S.A. 2007 Dec; 104 (51):20466–20471. 10. Elsner RA, Hastey CJ, Oslen KJ, Baumgarth N. Suppression of long-lived humoral immunity following Borrelia burgdorferi infection. PLoS Pathog. [Internet] 2015 Jul [Cited 2016 Nov 1]; 11(7) e1004976. Available from: http://journals.plos.org/plospathogens/article?id=10.1371/ journal.ppat.1004976 11. Wang C-Y, Liah JK. A mouse model of diet-induced obesity and insulin resistance. Methods Mol Biol. 2012; 821:421-433. 12. Bockenstedt LK & Wormser GP. Unraveling Lyme disease. Arthritis Rheumatol. 2014 Sep; 66 (9):2313-2323. 13. Wright WF, Riedel DJ, Talwani R, Gilliam BL. Diagnosis and Management of Lyme Disease. Am Fam Physician. 2012 Jun; 85 (11): 1086-1093. 14. Wormser GP & Schwartz I. Antibiotic treatment of animals infected with Borrelia burgdorferi. Clin Microbiol Rev. 2009 Jul; 22(3):387-95. 15. Samuels DS, Radolf JD. Borrelia: Molecular Biology, Host interaction and pathogenesis. 1st ed. Poole: U.K. Horizon; c2010. 16. Pětrošová H, Eshghi H, Anjum Z, Zlotnikov N, Cameron CE & Moriarty TJ. Front Microbiol. [Internet]. 2017 Feb [Cited 2016 Nov 1]. Available from: http://journal.frontiersin.org/ article/10.3389/fmicb.2017.00292/abstract 17. Ray I, Mahata SK. De RK. Obesity: An immunometabolic perspective. Front. Endocrinol. [Internet]. 2016 Dec [Cited 2016 Nov 1]; 7: 157. Available from: http://journal.frontiersin.org/ article/10.3389/fendo.2016.00157/full. 18. Farnsworth CW, Shehatou CT, Maynard R, Nishitani K, Kates SL, Zuscik MJ, et al. A humoral immune defect distinguishes the response to Staphylococcus aureus infections in mice with obesity and type 2 diabetes from that in mice with type 1 diabetes. Infect Immun. 2015 Jun; 83(6): 2264-2274. 19. Yano H, Kinoshita M, Fujino K, Nakashima M, Yamamoto Y, Miyazaki H et al. Insulin treatment directly restores neutrophil phagocytosis and bactericidal activity in diabetic mice and thereby improves surgical site Staphylococcus aureus infection. Infect Immun. 2012 Dec;

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RESEARCH

Biological Versus Foster Antillean Manatee Calf: Who Receives More Social Interaction from Mother? Lucy Liu Department of Physiology, University of Toronto, Toronto, Ontario, Canada.

Abstract

For most species, social interaction is essential for the developmental process of the offspring, and its decline seems to be correlated with age. In captivity where there is an increase in cross-fostering, it is important for conservation purposes that the foster mother is providing sufficient social interactions with her adopted child. The objective of this paper is to examine the social interaction provided by the Antillean mother manatee to the younger foster calf and older biological calf. Social interaction between a mother Antillean manatee, and her biological and foster calf was assessed via morning and evening observations for a period of 4 days. Social interaction was measured in terms of i) the distance between the mother and the calves and duration spent in each distance via the Closeness Meter (CM), ii) synchronization displacement iii) who takes initiative (mother vs calves) to increase intimacy, and iv) any other specific intimate behaviours between the mother and calves. Both calves spent the longest time in the High Association level with the mother, 89% for the foster calf, and 66% for the biological. Compared with the foster calf, the biological also had twice as more Low Association time, and followed the mother 40% of the time. Although limited, the mother chose to only initiate grooming and hugging behaviour with her own calf. There also appears to be a difference in social interactions transitioning from morning to evening, but more research is required to confirm this. Overall, compared with the biological calf, the foster calf spent longer time in closer proximity, and followed the mother twice as more. This may be due to the fact that the foster calf is younger, and thus is more dependent on the mother. However, further study is needed to determine if there was maternal preference for her own kin in initiation of specific intimate behaviours.

Introduction

The Antillean Manatee (Trichechus manatus manatus), a subspecies of the West Indian Manatee is a large herbivorous aquatic mammal that possess a streamline body which is grey in colour. They are considered to be an endangered species and are found mainly in the warm coastal and inland waters of southeastern US and situating in Central and South America, as well as the eastern coasts of Mexico [1]. In general manatees are considered to be semi-social animals [2], and spend most of their day relaxing (2-12 hours) and feeding (6-8 hours), with the rest of the day spent travelling and socializing. Previous studies suggest that social behaviours include synchronized displacement, remaining close to one another, group feeding and resting, kissing, hugging, and nuzzling, among others [3,4]. Manatees generally breed all year round, but mate every 2-5 years due to their 12 months gestation period [3]. In the wild, although the calf can be weaned after its first year, the mother-calf bond can persist up to two years [5]. During this time, calves not only depend on the mother for feeding but also acquire knowledge of migration routes, resting and

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feeding areas, and warm water refuges. In the wild the mother usually nurses one calf at a time, however, in captivity, the incidence of non-offspring nursing increases [6]. Past research in captivity suggest the strongest social contact among manatees was between a mother manatee and her calf, and that social skills and social recognition abilities were developed by the manatee calf through interactions with the mother and other manatees [4]. Thus, it is important for conservation purposes that, the surrogate mother is devoting sufficient social interactions with her foster calf. This is particularly critical for those manatees planned to be released back into the wild as research suggest that social interactions may contribute to the development of vital skills important for survival [7]. To date, there has only been research conducted in captivity on interactions between groups of manatees [8,4], and between orphaned calves [5]. Little is known about the mother-calf bond in the context of cross-fostering. Therefore, the objectives of this study was to investigate i) the overall social interaction between a mother manatee and her foster and biological calf, and ii) if social interactions were correlated with times of day.

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Biological versus foster Antillean manatee calf: Who receives more social interaction from mother?

Methods

Research Articles

Results

Background This study was conducted on a group of Antillean manatees in a natural but captive facility for 4 days in May 2015. Daily observations were made in the morning (approximately between 7:30-10:00am) and evening (approximately between 4:30-7:30pm). Data collection stopped during times of feeding and during session times with the tourists, and any disturbances which could affect the results were noted. Study subjects include i) the mother who is 20 years old and is approximately 296.3 cm in length, ii) the older female biological calf who is a one-year-old, and iii) the younger female foster calf who is three months old. Throughout the study, the mother will be notated as M, the biological calf as B/o, and the foster calf as F/y.

Section 1: Overall association between mother and calves There was a significant difference between the two calves, F/y and B/o on all three levels (High, Med, Low) of association (p= 4.25E-14). Both calves spent the most time in High Association, followed by Low Association and finally Med Association. Compared with B/o, F/y spent approximately 1.5 times longer in High Association (p=2.0E-04). On the other hand, the time B/o spent in Med Association more than triples that of F/y’s time

A

Analysis Social interactions between the mother and the two calves were assessed via i) the proximity and duration between the mother and the two calves ii) synchronized displacement iii) who initiated the intimacy i.e. took the initiative to move close iv) manifestations of specific intimate behaviours such as kissing, hugging, and grooming. Proximity was measured using a Closeness Meter (CM) which assessed the distance between the mother and calves using the mother’s body length as a reference (Table 1). Duration of time spent at each CM distance was measured using a stop watch. The overall social interaction, morning, and evening percentages were calculated by dividing the amount of time spent at each CM distance by the total collection time, total time spent in the morning and evenings, respectively. CM values were further grouped into three types of associations: High (CM 3 + CM 4), Med (CM=2) and Low (CM 0+ CM 1) for an overall picture of the findings. The synchronization displacement behaviour of the calves was calculated using the number of times each calf followed the mother divided by the total number of times the mother moved. In addition, the amount of times the mother or calves moved close to increase the CM value (i.e. CM 3 -> CM 4) divided by the total times a change in CM value occurred was calculated to determine who initiated intimacy more. The Excel Chi Squared Test was used for statistical analysis to see if results were significant. Social Interaction Proximity-Closeness Meter (CM) High Association CM=4 CM= 3 Med Association CM= 2 Low Association CM=1 CM=0 Other Behaviour Synchronized Displacement Specific Intimate Behaviour Kissing Hugging Grooming

B

C

Description Mother and calf are touching; body to body contact Distance < ¼ of mother’s body length Distance = ½ of mother’s body length Distance = 1 mother body length Distance > 2 mother body length Movement of manatees either simultaneously or one following another Mouth to mouth contact Flipper to body contact Eating algae off one another

Table 1. Social interactions of three captive Antillean manatees.

Figure 1. A) Overall association between M and F/y as a percentage of total time. B) Overall association between M and B/o as a percentage of total time. C) Overall CM comparison between F/y and B/o as a percentage of total time.

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Research Articles Biological versure foster Antillean manatee calf: Who receives more social interaction from mother?

(p=5.62E-06), and time spent in Low Association more than doubles compared with F/y (p=1.77E-07). Overall, greatest time was spent at a distance of CM=3 between the mother and the two calves (Figure 1C). There was also a significant difference between the two calves on each CM level (0-4) (p= 6.60E-15), with F/y spending more time in CM levels of 3 and 4, while B/o spent more time in CM levels of 0-2.

A

BB

Figure 2. A) Association between M and F/y as a percentage of time spent in the morning and evening. B) Association between M and B/o as a percentage of time spent in the morning and evening.

Section 2: Differences in association levels between morning and evening In both the morning and evening, F/y spent greater time in High Association compared to B/o; 35% higher in the morning (p=7.41E-06), and 14% higher in the evening (p=0.07). On the other hand, B/o had higher time spent in both Med and Low Association levels in both morning and evening compared to F/y; 7% greater (p=0.001), and 28% greater (p=7.54E-09) in the morning respectively, 11% greater (p=0.0002), and 3% greater (p=0.30) in the evening. For both calves, while there was a significant difference between their individual association levels with M in the morning versus the evening (F/y: p=1.53E-05; B/o: p=5.92E-14), that significance only persisted for the Med and Low Association levels. Both Med and Low Association increased for F/y during the evening compared to the morning by 5% (p =0.03) and 7% (p=0.01) respectively (Figure 2A).

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For B/o, while the Med Association increased in the evening by 7% (p= 0.02), Low Association time decreased by 18 % (p =9.90E-05) (Figure 2B). Furthermore, there was not much difference between the time spent during morning and evening at CM=3 for both calves, (F/y: p=0.96, B/o: p=0.46). While, time spent at CM=4 decreased by 10% going from morning to evening for F/y(p=0.002), it increased by 3% for B/o (p=0.21). At the same time, both calves had an increase of time spent at CM=0 going from morning to evening, 9% increase for F/y (p=9.16E-05), and 4% increase for B/o (p=0.23). Section 3: Mother and calf behaviours Whenever the mother moved, F/y followed her twice as more times compared with B/o (p=0.01). In particular, the mother never followed either calf; it is always the calf following the mother. The frequency of synchronized displacement decreased by 28% from the morning to the evening for F/y (p=0.13), while for B/o it decreased by only 17% (p=0.38) (Figure 3A). There was a significant difference between who initiated the closeness. Overall, M tried to initiate intimacy or moved close to each calf only 11% of the time, while each calf moved close to M 88% of the time (p=2.56E-14). F/y moved closer to M 7% times more compared to B/o (p=0.60), while M moved closer to B/o 10% times more compared to F/y (p=0.13) (Figure 3B). Furthermore, F/y tried to increase the intimacy level 13 times in the morning and 23 times in the evening, while B/o tried 19 times in the morning and only 17 times in the evening. M tried to increase the intimacy level with F/y only during the evening, while she tried it with B/o during both the morning and evening. Out of the limited times where M moved close to the calves, 77% of the time was when the calves were at a distance of CM<3, and 22% when CM=3 (p=0.39). At a CM<3 both F/y and B/o moved close to M 83% of the time while, M moved close to both 16% of the time (Figure 3C, D). In addition, at a distance of CM=3, M never moved close to F/y to increase CM=3 to CM=4, however she did move close to B/o 16% of the time. Moreover, since the other specific intimate behaviours were so limited in appearance, they were reported here in terms of frequency of occurrence. F/y groomed M 6 times while B/o did it 5 times. M groomed B/o once but never for F/y. F/y kissed M twice, both times in the evening, but B/o never showed such behaviour in the morning or evening. M hugged B/o 3 times in the evening, but never hugged F/y in the morning or evening. Overall there was a significant difference (p=0.04) between M’s choice of initiating the specific intimate behaviour with F/y versus B/o. Section 4: Disturbance Disturbances which may have influenced the results include other manatees, humans, and trainers. However, the duration of each factor in possibly influencing the mother-calf interaction was less than 10% of the total data collection time: other manatees (8.8%), humans (6.7%), and trainers (1.2%). Whenever other manatees were present, both F/y and B/o would decrease their CM value. It can also be noted that other manatees have the most significant effect on the calves, i.e. CM value changed 93% of the time (p=5.74E-06) for F/y and 94% of the time (p= 4.46E-07) for B/o during other manatee presence. In particular, both calves increased time spent with other manatees during the evening compared with the morning (F/y 6min vs 22 min p= 0.02; B/o 15min vs 18min p=0.60). On the other hand, whenever humans were present, both calves would increase their CM value 17% of the time. During the presence of trainers, F/y and B/o were indifferent.

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Biological versus foster Antillean manatee calf: Who receives more social interaction from mother?

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B

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D D

Research Articles

Figure 3. A) F/y and B/o’s frequency of synchronized displacement with the mother during the morning versus the evening. B) Frequency of initiation of intimacy between the mother and F/y and B/o. C) Frequency of initiation of intimacy between the mother and F/y at distance of CM<3 and CM=3. D) Frequency of initiation of intimacy between the mother and B/o at distance of CM<3 and CM=3.

Discussion

Section 1: Overall association between mother and calves In the wild, calves usually remain close to the mother, travelling just parallel to the mother’s side [9]. This may account for the fact that both calves had the greatest duration spent in High Association. The age difference between the two calves may also account for F/y spending more time in closer proximity with M compared to B/o. As calves grow older, they become less dependent on their mothers both nutritionally and behaviourally [10]. Thus, the higher Low Association level between M and B/o may be attributed to this notion. In addition, other studies of captive and wild primates demonstrate that a decrease in close contact with the mother as the young matures, is replaced by both a temporal and spatial increase in exploration of the surrounding area [11]. This may also be applied to B/o. Furthermore, since the likelihood of having twins is low in manatees [3], perhaps the mother is only able to expend sufficient energy to care for only one calf at a time, which may also explain for why B/o had more Low Association time with M. Compared to the wild, where mostly close interaction was seen, limited amounts of Med interaction was witnessed for B/o, and especially F/y. This suggests since the captivity environ-

ment reduces much of natural threats posed such as predation, thus, the mother could afford to let the calf stay in a position where she is neither too close nor too far. Close interaction of mothercalf observed in the wild may account for the fact that the greatest time was spent at distance of CM=3 for both calves, compared with other CM values. A higher CM=3 duration was also more pronounced in F/y than B/o. A similar study researching the spatial relationship between the mother-calf pair in right whales revealed analogous results. The authors found that calves who were within four months old remained in close proximity defined as remaining within ¼ of mother’s body length, while yearling calves initiated more leaves [12]. Finally, the touching behaviour CM=4 was seen to be higher for F/y than B/o. This suggests this behaviour may decrease as a consequence of age. Section 2: Differences in association levels between morning and evening Although there was no significant difference in High Association going from morning to evening for the two calves, it is interesting to note that F/y decreased her association with M in the evening while B/o increased hers. For F/y, the decrease in High

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Biological versure foster Antillean manatee calf: Who receives more social interaction from mother?

Association was replaced by a significant increase in both Med and Low Association. This may be attributed to the fact that F/y associated with other manatees in the evening more compared with the morning. This finding is consistent with another study conducted on Antillean manatees with a 15-year-old mother and calf that is six months old, where it was hypothesized that the calf associated with other manatees for the development of social recognition [4]. For B/o, a greater High Association witnessed in the evening can be connected to a significant decrease of Low Association time. This suggests that perhaps the mother chooses to associate more with one calf during different periods of day. However, more research is needed to confirm this notion. Section 3: Mother and calf behaviours Greater incidences of synchronized displacement were seen for F/y compared with B/o. In a similar study involving whales, calves followed the mother wherever she went, however, as they grew older, synchronized displacement gradually weakened [12]. For B/o, this suggests that as she matures and becomes increasingly aware of the locations of the mother, she does not need to continually follow M. It is also possible that since B/o grew up in captivity she has little sense of environmental danger, and thus does not feel the need to follow the mother to ensure her own safety. Furthermore, since B/o is used to living in an enclosed environment, she may be aware that the mother can only be within this area, and thus does not worry about getting lost or being unable to find the mother. Finally, although, the result did not show significance, synchronized displacement decreased from morning to evening for both calves. A previous study of Amazonian Manatees implies that perhaps there is a difference in activity level as greater displacement behaviours was seen during the day [13]. Alternatively, this result may be explained by the greater association time with other manatees in the enclosure. Another Antillean manatee study found that manatees interacted with different individuals going from day to night [4]. It is also interesting to observe the increase in CM value was due to F/y and B/o’s efforts, rather than M moving closer to the calves. F/y also moved frequently towards the mother more than B/o. This finding is comparable to the research conducted on two orphan Amazonian manatees, where social interactions were mainly initiated by the younger calf [7]. This suggests that younger calves like to seek attention from older conspecifics. Furthermore, the fact that M chose to initiate intimacy 55% more with the calves at CM<3 compared to CM=3 suggests the mother takes distance into account. In the wild, staying close to calves allows the mother to protect them from danger, thus in captivity perhaps M also feels that there are more threats at a greater distance, and therefore would move closer to her calves on more occasions at CM<3. In addition, M never tried to increase CM from 3 to 4 with F/y but did so with B/o 16%. Another study involving right whales found the opposite result; in their case, the mother approached her infant calf more than her yearling calf [12]. Since manatees are a different species than whales, and the times where M tried to increase intimacy were so limited, more research is needed to determine if the mother manatee indeed favours her own child. Specific intimate behaviours noted in this study were, kissing, hugging, and grooming, however they were rarely seen during

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the data collection period. Both calves groomed M but M only groomed B/o. M never initiated kissing with either calf; it was always F/y initiating the behaviour whilst B/o never did so. In another study done with manatees also revealed that the kissing behaviour was rarely seen between not only the mother and calf but also among other manatees [4]. This suggests that maybe kissing in general was not a common behaviour amongst Antillean manatees. The previous study, however, did show the mother frequently hugging her own biological calf who was 6 months old [4]. In this study, M initiated the hugging behaviour exclusively with B/o. If intimacy behaviour does decline with age, then one would expect M to engage in such behaviour more frequently with F/y rather than B/o. The before mentioned study was conducted from Feb-May, while this study was done exclusively in May, so perhaps seasonality also has an effect on this behaviour. Overall, the limited times where M engaged in specific intimate behaviour only with B/o suggests for possible maternal preference for her own kin. Differential treatment also depends on if the mother is able to discriminate between kin and nonkin [14]. Unfortunately, there is not much research regarding manatees in this area. However, past research did indicate that in the wild, there have been reported cases of mother manatees adopting orphaned calves [3], this suggests that perhaps they are capable of kin recognition. Nonetheless, more research is required to elucidate this hypothesis. Moreover, the nursing behaviour was not seen in this study between M and calves, probably due to the fact that the trainers provide the milk for the calves. It is also interesting to note that both hugging and kissing occurred exclusively during the evening, this parallels with the other manatee study suggesting that the evening is where most social interactions amongst groups of manatees occur [4]. Section 4: Disturbance and limitations Since this study was conducted in the early morning and during the evening, each disturbance constitutes less than 10% of the total collection time. Nonetheless, it is interesting to point out that other manatees were the factor that contributed most to changing the CM values for the calves, and that humans had little effect on the mother-calf association. This suggests that in captivity, the manatees may be used to human presence, and thus do not view them as a threat. Also, the fact that M allows association of her calves with other manatees suggests that she does not see them as a threat either. The biggest limitation of this study was that the age difference between the two calves poses as a significant confounder to the results. Other limitations include limited study period, and that data points were collected solely based on observation. Future studies should be conducted with biological and foster calves of similar age to detect whether maternal preference exists, and follow the mother-calf pair for one to two years until the calves are weaned to detect changes in social interactions. Furthermore, additional variables could also be used to measure social interactions such as synchronized breathing, and vocalization. In particular, there is empirical evidence suggesting individual vocal recognition of the West Indian manatee [15], and that vocalization is important for maintaining contact between the mother and calf [16]. Thus, it would be interesting to study this factor in the context of cross-fostering, and see if differences would arise in the mother-calf pairs.

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Biological versus foster Antillean manatee calf: Who receives more social interaction from mother?

Research Articles

Conclusion

Overall, compared with B/o, F/y had the highest contacts with M, in terms of the association levels and synchronized displacement. This may reflect on the idea that there is a decline in social interaction as the calf ages, and becomes more independent from the mother. With respect to other measures of social interaction, such as who took the initiative to move close, and the engagement of specific intimate behaviours such as hugging, it appears that M did indeed show a preference towards her own kin. However, due to the limited frequencies of such behaviour displayed by M, further research is still required to confirm this notion. A significant difference in social interaction between the morning and evening was noted for some interactions, such as in the association level between the mother and calf pairs, but not in others, like synchronized displacement. Finally, other manatees in the enclosure seemed to contribute to a change in the CM values, which suggest for the importance of group interactions. 3

­

Acknowledgements

This work was done as part of the physiology field course PSL379, under the wonderful mentorship of Dr. Carin Wittnich and Dr. Jim Goltz.

References

1. Craig D. Manatees. Science Weekly. 2007; 23(14): E1-8. 2. Barnett C. Reviews Hartman D. Ecology and behavior of the manatee (Trichechus manatus) in florida. J Mammal. 1980; 61(3): 577-579. 3. Hartman DS. Ecology and behavior of the manatee (Trichechus manatus) in Florida. Ithaca, N.Y.: American Society of Mammalogists Special Publication; 1979. 4. Hénaut Y, Lopez S, Machkour-M’rabet S, Morales-Vela B, Winterton P, Delfour F. Activities and social interactions in captive Antillean manatees in Mexico. Mammalia. 2010; 74(2): 141-146. 5. Arévalo-Sandi AR, Castelblanco-Martínez DN. Interactions between calves of Amazonian manatees in Peru: a study case. Acta biol. Colomb. 2016; 21(2): 355-364. 6. Packer C, Lewis S, Pusey A. Comparative analysis of non-offspring nursing. Anim Behav. 1992; 43(2): 265-281. 7. Anderson PK. Habitat, niche, and evolution of sirenian mating systems. J Mamm Evol. 2002; 9(1-2): 55-98. 8. Harper J, Schulte B. Social interactions in captive female Florida manatees. Zoo Biol. 2005; 24(2): 135-144. 9. Marmontel M. The West Indian manatee in the Caribbean and northern south Atlantic. Nairobi: United Nations Environment Programme; 1996. 10. Bonde RK. Population genetics and conservation of the Florida manatee: past, present and future. [PhD Dissertation]. University of Florida. 2009. 11. Struhsaker T. Social behaviour of mother and infant vervet monkeys (Cercopithecus aethiops). Anim Behav. 1971;19(2): 233-250. 12. Taber S, Thomas P. Calf development and mother-calf spatial relationships in southern right whales. Anim Behav. 1982;30(4): 1072-1083. 13. Mercadillo-Elguero MI, Castelblanco-Martínez DN, Padilla-Saldívar JA. Behavioral patterns of a manatee in semi-captivity: implications for its adaptation to the wild. J Mar Anim Ecol. 2014; 7(2): 31-41. 14. Todrank J, Heth, G. (2001). Rethinking cross-fostering designs for studying kin recognition mechanisms. Anim Behav. 2001; 61(2): 503-505. 15. Sousa-Lima R, Paglia A, Fonseca G. Gender, age, and identity in the isolation calls of Antillean manatees (Trichechus manatus manatus). Aquat Mamm. 2008; 34(1): 109-122. 16. O’shea T, Poché L. Aspects of underwater sound communication in Florida manatees (Trichechus manatus latirostris). J Mammal. 2006; 87(6): 1061-1071.

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JULS

REVIEWS

Weighing the Beneficial and Detrimental Consequences of Reactive Oxygen Species and their Role in Diseases Nidaa Rasheed1a, Dhia Azzouz1a, Cheng Min Sung1a, Filip Dinic1a, Jon Babi1a, Kevin Liu1a, Seung Gwan Ryoo1a, Darius Hung1a, Natalie J. Galant2b, Imre. G. Csizmadia3a,c Department of Chemistry, University of Toronto, Toronto, Ontario M5S 3H6, Canada Department of Medical Biophysics, University of Toronto, Ontario Department of Chemistry and Chemical Informatics, University of Szeged, Boldogasszony sgt.6.,H-6725 Szeged, Hungary

a

b c

Abstract

Reactive oxygen species (ROS) are a group of reactive molecules and natural by-products of many biochemical reactions within the body. While evidence has shown that the generation of ROS in vivo can result in multiple harmful effects, their potential benefits have been explored less often. ROS production is linked to many human diseases, as it is known to oxidize critical macromolecules and is mutagenic. An increasing body of evidence supports the beneficial roles of ROS within a human body, such as antibacterial agents, redox regulators, and potential antineoplastic agents. The following review will explore both benefits and detriments of ROS production, in addition to investigating their role in illness and their use in developing better models for disease treatment.

Introduction

Reactive oxygen species (ROS) are chemically reactive molecules containing oxygen and unpaired electrons known as free radicals. One free radical can give rise to a range of reactive oxygen species, the most common examples being superoxide anion (O2-), hydrogen peroxide (H2O2), and hydroxyl radical (•OH) [1]. The formation of •OH from H2O2 is catalyzed by iron ions in a reaction called the Fenton reaction [2]. O2•- can be converted into H2O2 and further to •OH, in a reaction called the Haber-Weiss reaction [3]. In the human body, ROS are formed as by-products of natural metabolic processes within the human body. While there are sources of exogenous ROS production, such as smoke, air pollutants, ultraviolet (UV) radiation, and xenobiotic compounds [4], most ROS are generated endogenously. The major sources of endogenous generation [5,6] of ROS NADPH oxidase (NOX) complexes are found in cell membranes, mitochondria, peroxisomes, and the endoplasmic reticulum. Endogenous production of ROS is dominantly formed within mitochondria through a series of incomplete reduction of O2 through the electron transport chain, especially under hypoxia conditions [7]. The formation of adenosine triphosphate (ATP) by mitochondria involves the electron transport chain, which features an oxygen molecule as the last destination for an electron [7]. In normal conditions, the oxygen is reduced to produce water; however, in about 0.1–2% of electrons, the oxygen is instead prematurely and incompletely reduced to give the superoxide radical (•O2-) [8,9]. Other enzymatic systems also produce ROS, including cytochrome P450, lipoxy

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genase, cyclo-oxygenase, the NADPH oxidase complex, xanthine oxidase, and peroxisomes [10]. Elevated levels of ROS have been shown to cause oxidative damage to host biomolecules and induce deleterious effects to the host genome. This damaging process, known as oxidative stress, has been shown to contribute to development of illness, particularly age-related diseases. [11,12] During times of physical stress on the body, including UV exposure or physical exercise, the metabolic rate increases, which in turn acts to increase the production of ROS in multiple parts of the body, such as in the blood, skeletal muscle, heart, brain, and liver, among others [13-15]. An increase in ROS levels or a decrease in cellular antioxidant capacity overwhelms the antioxidant defense system, causing oxidative stress. Oxidative stress has been associated with damage to nucleic acids, proteins, and lipids, and has been implicated in carcinogenesis, neurodegeneration, atherosclerosis, diabetes, aging, and tumor metastasis. [16,17] The human body maintains homeostasis by restricting ROS production to appropriate subcellular locations, times, levels, and durations. [17] Oxidative stress causes ROS-mediated damage which can also cause cell death or damage to DNA and mutagenesis [17]. Aside from mutagenic and tumorigenic characteristics of ROS, they also have multiple beneficial functions. Evidences suggests that intracellular ROS are an important component of intracellular signaling cascades that regulate vascular tone, insulin synthesis, cell proliferation, differentiation, and migration [18]. Moreover, studies suggest that ROS have beneficial roles as antibacterial agents and regulators in the human body. Furthermore, they may

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Weighing the Beneficial and Detrimental Consequences of Reactive Oxygen Species and their Role in Diseases

be useful in cancer therapies. Finally, new studies have shown that the production of ROS by increased physical activity has promising benefits to health, and this will be evaluated at length in the review. Therefore, while evidence has shown that the generation of ROS can have harmful effects on the body [2], they may also have beneficial effects, which have not been thoroughly considered. Therefore, this review article examines the benefits and detriments of ROS, and their role in medicine.

ROS Leads to DNA Mutations and RNA Damage Resulting in Diseases

There are several mechanisms by which increases in ROS causes DNA damage. For example, guanine residues can be modified into 8-oxoguanine, which can then mis-pair with a thymine residue and result in G-T and C-A substitutions. [16] Moreover, increased levels of 8-oxoguanine detected in body fluids are associated with cancer. [19] Finally, oxidation of guanine in telomeres negatively impacts recombination and increases telomere loss [20]. This results in a higher chance of DNA damage, disease from telomere removal and earlier senescence of the cell line. [21] For another example, guanine residues can also be nitrated by peroxynitrate into 8-nitroguanine, which is also linked to cancer [19]. Therefore, ROS induces DNA damage, and as a result, may cause oncogenic mutations, resulting result in aberrant cell growth and the development of cancer [6]. Mitochondrial ROS production associated with cancer initiation can be induced by hypoxia and glucose deprivation [7]. The vast production of mitochondrial ROS following hypoxia results in elevated levels of hypoxia-inducible transcription factors (HIF-1α and HIF-2α) [8, 9]. HIF-1α expression results in the activation of vascular endothelial growth factor (VEGF), which helps in tumor growth and cancer metastasis [22,23]. HIF-2α expression results in activation of the epidermal growth factor receptor signaling pathway, which also helps in tumor growth and cancer metastasis, such as head and neck cancer [24]. Cross-links are another mechanism of DNA damage, which forms between nucleotides in the same strand [25]. These crosslinks are mutagenic, and inhibit transcription and DNA replication [26]. Cross-links can also form between thymine and associated histones, which disrupts transcription regulation and chromatin remodeling. [27] Depending on the affected proteins and pathways, these mutations may have adverse effects on a cell [28]. One current theory of aging holds that its major cause is the accumulation of DNA damage by ROS [19]. Furthermore, ROS can modify the transcriptome of a cell by oxidation of RNA bases [29]. RNA is not heritable, therefore its mutation will not be passed down to the next generation. But, it is more especially prone to oxidation, due to its single-stranded structure and the lack of protection by the nuclear membrane [30]. Thus, although RNA damage is not usually carcinogenic, it accounts for a larger portion of dysfunctional cellular proteins [31]. The same mechanisms of oxidation appear in RNA as in DNA, but with increased frequency [32]. High mRNA and rRNA oxidation is a feature of individuals affected by Alzheimer’s disease [24]. 8-hydroxyguanosine, produced by a similar mechanism as 8-oxoguanine, occurs in early stages of Parkinson’s disease and ALS [33]. Additionally, ROS has been implicated in many other psychiatric disorders [34], such as Autism [35]. ROS has also been implicated in anxiety [36], bipolar disorder [37,38] and sleep ap-

Review Articles

nea [39]. In the case of schizophrenia, upregulation of NOX2 has been reported [40]. High levels of ROS in the brain may result in neurotoxicity [41,42]. Microglia activation by amyloid results in increased levels of oxidative stress resulting in neuronal damage and can result in dementia [43]. In fact, ROS has been reported to contribute to Alzheimer’s disease [44] and Parkinson’s disease [33, 45]. Furthermore, RNA oxidation is an early event in pathogenesis of a plethora of common diseases, such as epilepsy and atherosclerosis, although the exact mechanisms are still unknown [45].

ROS Enhances Protein and Lipid Oxidation that Prompt Neurological and Cardiovascular Diseases

Aside from DNA mutations and RNA damage, ROS can also change the structure and function of proteins, causing many human diseases. For example, they can oxidize amino acid side chains and protein backbones, and they can cause cross-links between amino acid side chains. This may cause a loss of activity of protein or cause the protein to form toxic aggregates in the cell [46]. When proteins are oxidized, human cells naturally degrade them so that they do not cause harm. However, not every oxidation-damaged protein can be successfully degraded. When this happens, the protein may be incompletely cleaved into small, toxic fragments that aggregate and may result in neurodegenerative diseases. [47] There are specialized redox-sensing proteins in a human cell that detects ROS using cysteine oxidation. [48] The Trx-ASK1 redox sensor is a protein complex that is dissociated by ROS possibly by the binding of the TNF associated factor 2 (TRAF2) and ASK1 homo-oligomerization, which initiates the ASK1-JNK pathway for TNF-R1-mediated apoptosis. [49,50] The pathway causes release of apoptotic signals, inducing cell death [51]. An implication of such oxidation is cataract formation, which accounts for 47.8% of causes of blindness [52], and it is the result of protein aggregation [52]. The oxidation of DNA, proteins and/or lipid peroxidation is involved in cataract formation [28]. Some forms of hearing loss are also the result of excessive oxidative stress [53]. NOX3 is present in large amounts in the inner ear and has been reported in being involved in hearing loss [54]. In addition to nucleic acids and proteins, ROS may oxidize fatty acids. Similarly, this oxidation has significant implications, especially in cardiovascular disease and stroke. [55] In atherosclerosis, low-density lipoproteins (LDL) are oxidized by ROS into oxidized low-density lipoprotein (OxLDL). Under inflammatory conditions, adhesion molecules are released, and monocytes differentiate into macrophages, producing scavenging receptors that bind OxLDL. Then, macrophages intake OxLDL; these OxLDLloaded macrophages (foam cells) produced increased amounts of ROS themselves, which in turn causes more LDL oxidation, and more foam cells. Moreover, foam cells secrete matrix metalloproteinases (MMPs) which degrade the fibrous cap formed by atherosclerosis. If the blood clot dislodges and is carried to the brain by arteries, it can result in an ischemic stroke. [56] Atherosclerosis [36], rheumatoid arthritis [56], lupus erythematosus [38] and cystic fibrosis [39] have also been associated with excessive neutrophil extracellular trap (NET) formation caused by excessive oxidative bursts from over-activated neutrophils. The inability to produce an oxidative burst results in immunodeficiency resulting in severe and repeating infections [57]. This is due to the inability of host immune cells, macrophages and neutrophils to kill bacteria. Chronic

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Review Articles

Weighing the Beneficial and Detrimental Consequences of Reactive Oxygen Species and their Role in Diseases

granulomatous disease (CGD) patients lack NADPH oxidase NOX2-induced ROS production [1]. Furthermore, cardiovascular cells express NOX1, NOX2, NOX4 and NOX5 [58]. While ROS plays important biological roles in cardiovascular cells, excessive ROS has been implicated in cardiovascular disease [59]. ROS is involved in hypertension pathophysiology [60]. Upregulation of NOX1 and NOX2 are correlated with hypertension [61].

Excessive ROS Inhibits Tumor Growth and Increases Apoptosis that Can Be Used in Cancer Treatment

Tumor cells often express increased amount of ROS due to oncogenic signaling pathways that promote cell proliferation, growth and differentiation, metabolism, and cell survival, which is partly from the increased signaling of NADPH oxidase complex and hypoxia-related ROS [62]. However, tumor cells also possess elevated antioxidant levels, suggesting that strict redox balancing is necessary for cancer cell functioning [63]. Interestingly, ROS not only promotes tumor-functioning events, but also induces apoptosis signaling pathways. Therefore, a novel challenge for treating cancer with ROS includes fine-tuning tumor pathways to apoptotic induction [63]. Apoptosis is usually the result of an escalated oxidative stress in mitochondria; this generates cytochrome C release, an irreversible event that leads to caspase activation and eventually cell death [64]. Although strictly regulated, high levels of ROS in tumor cells promote cancer functioning. Excessive and disproportionate increases of intracellular ROS are able to induce arrest of tumor growth, senescence, and apoptosis. In particular, this can be achieved by administering treatments that disrupt ROS scavenging, disrupting antioxidant proteins, administering cancer chemotherapy, or administering drugs that generate ROS [33]. One of the first drugs developed to generate ROS is procarbazine, which, when oxidized in aqueous environment, gives rise to hydrogen peroxide (H2O2) [65]. Today, procarbazine is used in several combinatory treatments for cancers. For example, a BEACOPP (bleomycin, etoposide, doxorubicin, cyclophosphamide, vincristine, procarbazine and prednisone) regimen is used in Europe for patients with Hodgkin’s lymphoma. Moreover, a PCV (procarbazine, lomustine (CCNU) and vincristine) regimen is administered to patients with anaplastic astrocytoma and oligodendroglioma [66]. Other drugs favoring apoptotic-ROS signaling include buthionine sulfoximine (BSO) and motexafin gadolinium (MGd) [44]. BSO leads to increased sensitivity for oxidative cytotoxic effect of platinum compounds and alkylators [67]. In contrast, MGd reduces oxygen into ROS and oxidizes protein thiols, which renders them unable to act as antioxidants for ROS and results in more apoptosis [64]. Senescence is an important, unrecoverable terminal differentiation activated by several different stimuli such as oncogene activation and telomere dysfunction [60]. Senescence induction limits tumor growth in both mouse and human cells[4]. Although mechanisms are not well understood, ROS plays a role in the induction of senescence, which promotes tumor suppression [63]. Under low stress conditions, mitogens inhibit retinoblastoma tumor suppressor (Rb), which inhibits transcriptional factor E2F. Therefore, an increase in mitogen signaling increases E2F and cell proliferation [12]. ROS levels are regulated with activation of E2F and transcription of genes involving ROS metabolism [25]. In contrast, under high cellular stress, activation of anti-proliferation

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proteins p53 and p16 induces an increase of Rb and inhibition E2F, resulting in accumulation of ROS and senescence [60].

ROS Enhances Anti-Microbial Activity and Promotes Pathogen Elimination

ROS have roles in mediating viral infections using apoptosis as a defense mechanism. Elevated levels of ROS have been shown to induce apoptosis in host cells which decreases the spread of viral infection [74]. NADPH oxidase is an enzyme necessary for respiratory burst, the process resulting in elevated oxygen consumption when a microbe is recognized by phagocytes and engulfed [75]. One study found that ROS produced in the phagosome cause oxidative damage to phagocytosed pathogens, which causes the respiratory burst that reduces viral infection [76,77]. Another study found that VSV-infected cells treated with paraquat, a drug that induces oxidative stress, reduced viral amounts in cells. This demonstrates the role of ROS in viral progression [78]. Moreover, Chikungunya virus, an arbovirus transmitted by mosquitos, causes oxidative stress that inhibits mTOR, a nutrient sensor, which causes a cease in cell growth and is linked to autophagy [79]. Thus, this is an example of a ROS-dependent mechanism against a virus via the use of autophagy to inhibit the apoptosis of host cell causing a decrease in the number of virally infected cells due to the apoptosis mediated propagation of infection in cell culture [80]. Furthermore, ROS promotes inflammation through activating signaling proteins that induce immunopathology in infected tissues. This is shown to enhance host immunity by preventing pathogen-induced pro-inflammatory cytokines [81,82].

Benefits of ROS During Exercise

While early studies of ROS have focused on oxidative damage, further research has shown that ROS heavily participates in redox regulation, and are known to function as regulators and initiators in establishing redox homeostasis in vivo [68]. Moreover, recent studies have found that ROS may promote recovery from muscle damage [69], improve sensitivity to insulin levels [70], and mediate widening and tightening of blood vessels (vasodilation) [71]. It is becoming increasingly clear that exercise level and intensity differentiates between ROS serving beneficial or detrimental roles to muscle repair and recovery. ROS play a role in cell signaling and regulation of redox-sensitive processes; therefore, they play an important role in regulation of processes involving redox in muscular repair. [72] Consequently, as ROS has been shown to cause some detrimental effects on human health, there has now been an increased use of antioxidants. The food industry markets antioxidants as protection against free radicals. However, many studies have found that antioxidants may promote cellular damage, hinder recovery of muscle damage, and increase tissue damage [73].

Conclusion and Future Directions

The literature establishes many detriments and benefits of ROS on human health. Indeed, there is extensive evidence that ROS have negative impacts in the form of oxidative stress, which has been implicated in cancer and mutagenesis. However, recent studies have demonstrated that ROS also have beneficial roles in many occasions, such as in cancer treatment and as antibacterial agents. ROS leads to DNA mutations and RNA changes as well as lipid oxidation that cause various neurological disorders or other

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diseases. On the contrary, ROS has benefits in cancer treatment, pathogen elimination and exercise. However, while it is evident that ROS can be harnessed in order to prevent or cure diseases like cancer, one must consider the reactivity associated with ROS. The risks involved with using ROS as a medical treatment are not well understood. For example, issues such as ROS monitoring, targeting, and transport need to be studied further. Future research is needed to understand how to minimize these risks in order to use ROS as effective medical treatments.

References

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Chronic granulomatous disease (CGD) and complete myeloperoxidase deficiency both yield strongly reduced dihydrorhodamine 123 test signals but can be easily discerned in routine testing for CGD. Clinical chemistry, 53(5), 890-896. 36. Mozzini, C., Garbin, U., Pasini, A. M. F., & Cominacini, L. (2016). An exploratory look at NETosis in atherosclerosis. Internal and Emergency Medicine, 1-10. 37. Corsiero, E., Pratesi, F., Prediletto, E., Bombardieri, M., & Migliorini, P. (2016). NETosis as Source of Autoantigens in Rheumatoid Arthritis. Frontiers in Immunology, 7. 38. Carmona-Rivera, C., Zhao, W., Yalavarthi, S., & Kaplan, M. J. (2014). Neutrophil extracellular traps induce endothelial dysfunction in systemic lupus erythematosus through the activation of matrix metalloproteinase-2. Annals of the rheumatic diseases, annrheumdis-2013. 39. Rahman, S., & Gadjeva, M. (2014). Does NETosis contribute to the bacterial pathoadaptation in cystic fibrosis?. Frontiers in immunology, 5, 378. 40. Waris, G., & Ahsan, H. (2006). Reactive oxygen species: role in the development of cancer and various chronic conditions. Journal of carcinogenesis, 5(1), 14. 41. Therade-Matharan, S., Laemmel, E., Duranteau, J., & Vicaut, E. (2004). Reoxygenation after hypoxia and glucose depletion causes reactive oxygen species production by mitochondria in HUVEC. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology, 287(5), R1037-R1043. 42. Semenza, G. L. (2000). Expression of hypoxia-inducible factor 1: mechanisms and consequences. Biochemical pharmacology, 59(1), 47-53.   43. Liao, D., Corle, C., Seagroves, T. N., & Johnson, R. S. (2007). Hypoxia-inducible factor-1α is a key regulator of metastasis in a transgenic model of cancer initiation and progression. Cancer research, 67(2), 563-572. 44. Laughner, E., Taghavi, P., Chiles, K., Mahon, P. C., & Semenza, G. L. (2001). HER2 (neu) signaling increases the rate of hypoxia-inducible factor 1α (HIF-1α) synthesis: novel mechanism for HIF-1-mediated vascular endothelial growth factor expression. Molecular and cellular biology, 21(12), 3995-4004. 45. Welsh, S. J., Bellamy, W. T., Briehl, M. M., & Powis, G. (2002). The Redox Protein Thioredoxin-1 (Trx-1) Increases Hypoxia-inducible Factor 1α Protein Expression Trx-1 Overexpression Results in Increased Vascular Endothelial Growth Factor Production and Enhanced Tumor Angiogenesis. Cancer research, 62(17), 5089-5095. 46. Wang, X., & Schneider, A. (2010). HIF-2α-mediated activation of the epidermal growth factor receptor potentiates head and neck cancer cell migration in response to hypoxia. Carcinogenesis, 31(7), 1202-1210. 47. Lassègue, B., & Griendling, K. K. (2010). NADPH oxidases: functions and pathologies in the vasculature. Arteriosclerosis, thrombosis, and vascular biology, 30(4), 653-661. 48. Abe, J. I., & Berk, B. C. (1998). Reactive oxygen species as mediators of signal transduction in cardiovascular disease. Trends in cardiovascular medicine, 8(2), 59-64. 49. Touyz, R. M. (2004). Reactive oxygen species and angiotensin II signaling in vascular cells: implications in cardiovascular disease. Brazilian Journal of Medical and Biological Research, 37(8), 1263-1273. 50. Touyz, R. M., & Schiffrin, E. L. (2004). Reactive oxygen species in vascular biology: implications in hypertension. Histochemistry and cell biology, 122(4), 339-352. 51. Matsuno, K., Yamada, H., Iwata, K., Jin, D., Katsuyama, M., Matsuki, M., ... & YabeNishimura, C. (2005). Nox1 Is Involved in Angiotensin II–Mediated Hypertension A Study in Nox1-Deficient Mice. Circulation, 112(17), 2677-2685. 52. Murdoch, C. E., Alom-Ruiz, S. P., Wang, M., Zhang, M., Walker, S., Yu, B., ... & Shah, A. M. (2011). Role of endothelial Nox2 NADPH oxidase in angiotensin II-induced hypertension and vasomotor dysfunction. Basic research in cardiology, 106(4), 527-538. 53. Brieger, K., Schiavone, S., Miller Jr, F. J., & Krause, K. H. (2012). Reactive oxygen species: from health to disease. Swiss medical weekly, 142, w13659. 54. Napoli, E., Wong, S., & Giulivi, C. (2013). Evidence of reactive oxygen species-mediated damage to mitochondrial DNA in children with typical autism. Molecular autism, 4(1), 1. 55. Rammal, H., Bouayed, J., Younos, C., & Soulimani, R. (2008). Evidence that oxidative stress is linked to anxiety-related behaviour in mice. Brain, Behavior, and Immunity, 22(8), 1156-1159. 56. Yumru, M., Savas, H. A., Kalenderoglu, A., Bulut, M., Celik, H., & Erel, O. (2009). Oxidative imbalance in bipolar disorder subtypes: a comparative study. Progress in NeuroPsychopharmacology and Biological Psychiatry, 33(6), 1070-1074. 57. Steckert, A. V., Valvassori, S. S., Moretti, M., Dal-Pizzol, F., & Quevedo, J. (2010). Role of oxidative stress in the pathophysiology of bipolar disorder. Neurochemical research, 35(9), 1295-1301. 58. Nair, D., Dayyat, E. A., Zhang, S. X., Wang, Y., & Gozal, D. (2011). Intermittent hypoxiainduced cognitive deficits are mediated by NADPH oxidase activity in a murine model of sleep apnea. PLoS One, 6(5), e19847.

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59. Behrens, M. M., & Sejnowski, T. J. (2009). Does schizophrenia arise from oxidative dysregulation of parvalbumin-interneurons in the developing cortex?. Neuropharmacology, 57(3), 193-200. 60. Abraham, A. G., Condon, N. G., & Gower, E. W. (2006). The new epidemiology of cataract. Ophthalmology Clinics, 19(4), 415-425. 61. Perry, R. E., Swamy, M. S., & Abraham, E. C. (1987). Progressive changes in lens crystallin glycation and high-molecular-weight aggregate formation leading to cataract development in streptozotocin-diabetic rats. Experimental eye research, 44(2), 269-282. 62. Babizhayev, M. A. (2011). Mitochondria induce oxidative stress, generation of reactive oxygen species and redox state unbalance of the eye lens leading to human cataract formation: disruption of redox lens organization by phospholipid hydroperoxides as a common basis for cataract disease. Cell biochemistry and function, 29(3), 183-206. 63. Banfi, B., Malgrange, B., Knisz, J., Steger, K., Dubois-Dauphin, M., & Krause, K. H. (2004). NOX3, a superoxide-generating NADPH oxidase of the inner ear. Journal of Biological Chemistry, 279(44), 46065-46072. 64. Sorce, S., & Krause, K. H. (2009). NOX enzymes in the central nervous system: from signaling to disease. Antioxidants & redox signaling, 11(10), 2481-2504. 65. Knight, J. A. (1997). Reactive oxygen species and the neurodegenerative disorders. Annals of Clinical & Laboratory Science, 27(1), 11-25. 66. Block, M. L., Zecca, L., & Hong, J. S. (2007). Microglia-mediated neurotoxicity: uncovering the molecular mechanisms. Nature Reviews Neuroscience, 8(1), 57-69. 67. Benzi, G., & Moretti, A. (1995). Are reactive oxygen species involved in Alzheimer’s disease?. Neurobiology of aging, 16(4), 661-674. 68. Devasagayam T, Tilak JC, Boloor KK, Sane Ketaki S, Ghaskadbi Saroj S, Lele RD (October 2004). “Free Radicals and Antioxidants in Human Health: Current Status and Future Prospects”. Journal of Association of Physicians of India (JAPI). 52: 796. 69. Rodriguez DA, Kalko S, Puig-Vilanova E, Perez-Olabarria M, Falciani F, Gea J, et al. Muscle and blood redox status after exercise training in severe COPD patients. Free radical biology & medicine. 2012 Jan 1;52(1):88-94. PubMed PMID: 22064359. Epub 2011/11/09. eng. 70. Smuder AJ, Kavazis AN, Min K, Powers SK. Exercise protects against doxorubicin-induced oxidative stress and proteolysis in skeletal muscle. Journal of applied physiology (Bethesda, Md : 1985). 2011 Apr;110(4):935-42. PubMed PMID: 21310889. Pubmed Central PMCID: 3075128. Epub 2011/02/12. eng. 71. Salo DC, Donovan CM, Davies KJ. HSP70 and other possible heat shock or oxidative stress proteins are induced in skeletal muscle, heart, and liver during exercise. Free radical biology & medicine. 1991;11(3):239-46. PubMed PMID: 1937141. Epub 1991/01/01. eng. 72. Falone S, D’Alessandro A, Mirabilio A, Petruccelli G, Cacchio M, Di Ilio C, et al. 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Journal of Undergraduate Life Sciences • Volume 11 • Issue 1 • Spring 2017


JULS

REVIEWS

The Psychological Impact of Type 2 Diabetes on Non-Diabetic Spouses Samantha Jagasar Department of Psychology, University of Toronto, Toronto, Ontario, Canada.

Abstract

Type 2 diabetes (T2D) is the sixth leading cause of death in Canada and its global disease burden continues to grow, claiming approximately 1.5 million lives annually. It is a chronic condition that is physiologically and emotionally debilitating to both patients and their family. This review focuses on the psychological impact of T2D on the patient’s non-diabetic spouse as they are frequently involved in disease management. Recent literature finds that non-diabetic spouses tend to experience an increased risk of anxiety, depression, marital tension, and decreased marital enjoyment. This psychological distress is associated with negative appraisals of T2D, increased frequency of symptoms, increased control over diabetes management, and long disease duration (>11.60 years). Earlier treatment interventions including diabetes education and counselling services may reduce the negative impact of this disease on the non-diabetic spouse. Future studies should broaden the demographics of the participant population and investigate methods to identify and alter negative appraisals of T2D. Such research studies may improve the psychological well-being of the non-diabetic spouse.

Background

Disease Burden of Type 2 Diabetes Type 2 diabetes (T2D) is a chronic disease where the body is unable to regulate blood glucose concentration due to deficient insulin production or improper use of insulin [1]. This debilitating disease is the sixth leading cause of death in Canada as it claims over one million lives each year [1]. T2D impacts the physiological and psychological well-being of those living with the disease. In addition to a change in diet and regular exercise, daily management of this disease involves frequent blood glucose monitoring, diabetes medication and/or insulin injections with every meal. Maintaining this disease is burdensome as blood glucose concentrations may fluctuate from day to day, and prolonged exposure to high blood glucose concentrations may lead to an increased risk of cardiovascular disease, diabetic retinopathy, and chronic kidney disease [2-4]. Although the potential physiological consequences of this disease are detrimental, the psychological burden is equally inimical. Studies focusing on the psychological complications of T2D indicate that patients have an increased risk of experiencing depression, anxiety, and feelings of hopelessness [5-7]. Those with diabetes tend to score higher on the Beck Hopelessness Scale (BHS), a self-report inventory that measures loss of motivation, feelings about the future, and expectations, than participants with epilepsy, glaucoma, HIV, and chronic daily headaches [5]. In addition, 61% of participants believed that their diabetes contributed to their symptoms of depression, which include anxiety about health complications and hopelessness from the inability to control their illness [7]. It is evident that T2D has a negative psychological effect

on the individuals diagnosed with this chronic disease. However, their spouses are also involved in disease management and tend to exhibit similar psychological effects. The onset of severe health conditions, including diabetes, can lead to a decline in the physical and mental well-being of the affected individual’s spouse [16]. This finding was based on an 8-item version of the Center for Epidemiologic Studies Depression Scale (CES-D), which included sleep disturbance, loneliness, and sadness to measure depression in the healthy spouse. The results indicate that the spouses of those who have been affected with a chronic illness tend to have higher scores on the CES-D than those with healthy spouses [16]. Current Literature This review will explore the psychological burden of T2D on the non-diabetic spouse of the patient. This research is valuable as recent literature suggests that these non-diabetic spouses experience psychological distress in the form of increased anxiety, depressive symptoms, marital tension, and decreased marital enjoyment. This psychological distress is associated with negative appraisals of T2D, increased frequency of T2D symptoms [10,11,13,16-18], increased control over their spouse’s disease management [8,9,12,14,15], and long disease duration [9,13]. Although this novel area of research has a few limitations (i.e. narrow sample population, use of subjective measures), it is apparent that the burden of T2D is shared by both spouses. As a result, it is important to provide support for the non-diabetic spouse to alleviate the negative effects that this disease has on their mental health and relationship. For the purposes of this review paper, the spouse without

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The Psychological Impact of Type 2 Diabetes on Non-Diabetic Spouses

T2D will be referred to as “non-diabetic spouse” and the patient with T2D will be referred to as “spouse”.

Search Methodology

The literature search was conducted using PsycINFO and PubMed, which were accessed through University of Toronto libraries. The search included the following terms: “diabetes AND (spouse OR partner) AND (stress OR depression OR anxiety)”. The search was limited to peer reviewed, scholarly journal articles, published after 2010. Seventy articles were generated and then screened for relevance by review of abstracts. Articles were included if they investigated the impact of T2D on a non-diabetic spouse of an individual with T2D. In total, 11 articles were relevant and were included in the review.

Correlates of Psychological Distress

The Frequency and Duration of Diabetes-Related Symptoms The frequency of diabetes-related symptoms in the spouse is linked to the experience of psychological distress by the non-diabetic spouse. A study found that diabetic symptoms such as poor blood glucose readings were correlated with an increase in spousal tension and a decrease in marital enjoyment [13]. After analyzing the self-reported symptoms recorded in the non-diabetic spouse’s electronic diary, this study noted that on days where few diabetesrelated symptoms occurred, the non-diabetic spouses found interactions with their spouse to be more pleasurable. Likewise, on days where many symptoms of diabetes were present, the non-diabetic spouses found the interactions to be less pleasurable [13]. It is apparent that the frequency of diabetes-related symptoms is correlated to the experience of tension and decreased marital enjoyment. Another study found that non-diabetic spouses of those who had T2D for a long period of time (>11.60 years) experienced less pleasurable interactions with their spouse on days when there were more diabetes-related symptoms than usual. However, this finding was not present when the spouses’ diabetes was of shorter duration (<11.60 years) [9]. Based on the stress accumulation hypothesis, it is suggested that the longer the disease duration, the more sensitized the non-diabetic spouse will be to their partner’s illness. Ultimately, the strain of coping with this chronic stress will accumulate and decrease marital enjoyment over time [9]. Another study suggests that the longer the disease duration, the more routinely involved the non-diabetic spouse will be in monitoring their partner’s health. Over time, they may feel that their efforts are unnoticed, causing a strain on the relationship [13]. It is apparent that the frequency of symptom presentation may lead to an increase in spousal tension and is a mediator for the relationship between disease duration and decreased marital enjoyment for the non-diabetic spouse. This psychological distress may lead to a decline in the general well-being of the non-diabetic spouse. Earlier treatment interventions including diabetes education and counselling services should be provided to reduce the distress that the non-diabetic spouse experiences. Appraisals of T2D In addition to the frequency of symptoms, negative appraisals of T2D have been positively correlated with psychological distress in non-diabetic spouses. A study finds that non-diabetic spouses experienced more anxiety when their spouse did not perceive T2D

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as a serious condition compared to the non-diabetic spouses of those who perceive T2D as more burdensome [10]. It is suggested that the non-diabetic spouse’s anxiety stems from an understanding that their spouse underestimates the severity T2D and may expose themselves to more health consequences [10]. The route to diagnosis of T2D also affects the non-diabetic spouse’s appraisal of their spouse’s T2D. The screening method detects potential symptoms of T2D, indicates suspicion of the presence of the disease and may eventually lead to a diagnosis, whereas a diagnosis through clinical symptoms confirms the presence or absence of T2D. The non-diabetic spouses of those diagnosed through a screening method were more concerned about their spouse’s illness and perceived more T2D health complications for their spouse compared to those of spouses diagnosed through clinical symptoms [17]. Initially, the screening method may lead to more anxiety and concern for their spouse’s health as there is more uncertainty of whether T2D is present. The overarching finding from these studies is that negative appraisals of T2D frequently lead to anxiety, depressive symptoms, and emphasis on the consequences of diabetes in non-diabetic spouses. As a result, additional support should be provided to the non-diabetic spouses to positively alter their cognitive appraisal in order to reduce anxiety during the screening process. Exerting Control over T2D Management Due to the non-diabetic spouse’s fear that the complications of T2D will progress, they tend be actively involved in illness management [18]. However, exerting control over their spouse’s diabetes management may lead to increased levels of stress [9]. Diabetes management involves adhering to the complex treatment regimen of health behaviours, which includes taking oral medication or insulin injections, and maintaining an appropriate diet. When the non-diabetic spouse attempts to control their spouse’s diabetes management, the non-diabetic spouse frequently experiences more personal and marital stress [9]. This psychological distress was more prevalent if their spouse neglects diabetes management guidelines, were ill for a long time, responds negatively to their involvement, or possesses comorbid health conditions [8, 9]. Furthermore, spouses who do not expect their non-diabetic spouse to be involved in their diabetes management react with greater hostility, which increases marital stress [14]. Although they are frequently involved in diabetes management, exerting too much pressure on their spouse can lead to psychological and marital distress for the non-diabetic spouse [14]. However, when the couples share responsibility for diabetes management, there is a significant decrease in stress for the spouse, thus lowering martial tension for both parties [15]. As a result, counselling and diabetes education services aimed at promoting a shared approach to diabetes management may reduce the strain of T2D on both partners.

Discussion

The overall body of research regarding the psychological impact of T2D on the patient’s loved ones suggests that the nondiabetic spouses tend to experience diabetes-related, psychological distress. More specifically, the frequency of diabetic symptoms is correlated with increased spousal tension and depressive symptoms, and decreased marital enjoyment [9,13]. Interestingly, this

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The Psychological Impact of Type 2 Diabetes on Non-Diabetic Spouses

distress increases on days when more symptoms are present as well as when the spouse has a longer disease duration (>11.60 years) [9,13]. This finding suggests that an earlier treatment intervention, aimed at providing support to both spouses (i.e. diabetes education, counselling), may reduce this psychological distress. Anxiety and depressive symptoms also tend to increase when the non-diabetic spouse perceives more health complications regarding their spouse’s illness, especially when the route to diagnosis involved a screening method [10,12,17]. As a result, counselling and diabetes education services should be offered for the non-diabetic spouse in order to provide further diabetes education and alter their negative appraisal of T2D. Although non-diabetic spouses often assist their spouse in diabetes management, attempts to take control typically lead to greater marital stress and diabetesrelated distress [8,9,14,15]. However, promoting a shared approach when managing diabetes may reduce the strain of T2D on both partners [15]. Unfortunately, the novel subsection of literature regarding the impact of T2D on the spouses of those affected is not without limitations. For instance, many of these studies obtain participant data by primarily using subjective measures, including daily dairy entries and questionnaires [8-15,17,18]. This self-reported data may be influenced by the participant’s memory as well as their emotions at the time the information is recorded. In addition, current research typically focuses on a narrow participant population consisting of non-diabetic spouses who are primarily Caucasian [8,9,11-16], over the age of 60 [8-10,12-17], and have been married to their spouse for 29 years on average [8,9,11-16,18]. This sampling is problematic given that the impact of T2D on the non-diabetic spouse may not be generalizable to those of different ethnicities or age cohorts.

Conclusion and Future Directions

Based upon the current literature, the spouses of those with T2D tend to be actively involved in disease management and experience the psychological distress that accompanies it. It is important to implement support systems for these non-diabetic spouses to mitigate the negative impact that this chronic disease may have on their relationship and mental health. For instance, future research studies should incorporate physiological measures of stress or investigate the non-diabetic spouse’s implicit attitudes toward T2D in order to accurately assess their automatic, appraisal of their partner’s illness. Together with support systems consisting of counselling and diabetes education, negative appraisals may be identified and altered to help them view their spouse’s illness positively, thus reducing the psychological burden. In addition, future studies should broaden their population samples to include couples from varying ethnic backgrounds, age groups, and those who have been married for varying lengths of time to increase the generalizability of this research. These future studies may lead to the development of earlier interventions to treat the psychological distress experienced by the non-diabetic spouse, as well as the affected individual, in order to promote healthy adjustment and adaptation to T2D.

Review Articles

2. Aleo, C. L., Murchison, A. P., Dai, Y., Hark, L. A., Mayro, E. L., Collymore, B., & Haller, J. A. (2015). Improving eye care follow-up adherence in diabetic patients with ocular abnormalities: The effectiveness of patient contracts in a free, pharmacy-based eye screening. Public Health, 129(7), 996-999. doi:http://dx.doi.org/10.1016/j.puhe.2015.05.012 3. Thomas, N., & Bryar, R. (2013). An evaluation of self-management package for people with diabetes at risk of chronic kidney disease. Primary Health Care Research and Development, 14(3), 270-280. doi:http://dx.doi.org/10.1017/S1463423612000588 4. Tovar, E., & Clark, M. C. (2015). Knowledge and health beliefs related to heart disease risk among adults with type 2 diabetes. Journal of the American Association of Nurse Practitioners, 27(6), 321-327. doi:http://dx.doi.org/10.1002/2327-6924.12172 5. Innamorati, M., Lester, D., Balsamo, M., Erbuto, D., Ricci, F., Amore, M., . . . Pompili, M. (2014). Factor validity of the beck hopelessness scale in Italian medical patients. Journal of Psychopathology and Behavioral Assessment, 36(2), 300-307. doi:http://dx.doi.org/10.1007/ s10862-013-9380-3 6. Smith, K. J., & Schmitz, N. (2014). Association of depression and anxiety symptoms with functional disability and disability days in a community sample with type 2 diabetes. Psychosomatics: Journal of Consultation and Liaison Psychiatry, 55(6), 659-667. doi:http:// dx.doi.org/10.1016/j.psym.2014.05.015 7. Tanenbaum, M. L., Ritholz, M. D., Binko, D. H., Baek, R. N., Shreck, M. S. E., & Gonzalez, J. S. (2013). Probing for depression and finding diabetes: A mixed-methods analysis of depression interviews with adults treated for type 2 diabetes. Journal of Affective Disorders, 150(2), 533539. doi:http://dx.doi.org/10.1016/j.jad.2013.01.029 8. August, K. J., Rook, K. S., Parris Stephens, M. A., & Franks, M. M. (2011). Are spouses of chronically ill partners burdened by exerting health-related social control? Journal of Health Psychology, 16(7), 1109-1119. doi:http://dx.doi.org/10.1177/1359105311401670 9. August, K. J., Rook, K. S., Franks, M. M., & Parris Stephens, M. A. (2013). Spouses’ involvement in their partners’ diabetes management: Associations with spouse stress and perceived marital quality. Journal of Family Psychology, 27(5), 712-721. doi:http://dx.doi.org/10.1037/ a0034181 10. Dimitraki, G., & Karademas, E. C. (2014). The association of type 2 diabetes patient and spouse illness representations with their well-being: A dyadic approach. International Journal of Behavioral Medicine, 21(2), 230-239. doi:http://dx.doi.org/10.1007/s12529-013-9296-z 11. Franks, M. M., Hemphill, R. C., Seidel, A. J., Stephens, M. A. P., Rook, K. S., & Salem, J. K. (2012). Setbacks in diet adherence and emotional distress: A study of older patients with type 2 diabetes and their spouses. Aging & Mental Health, 16(7), 902-910. doi:http://dx.doi.org/1 0.1080/13607863.2012.674486 12. Franks, M. M., Sahin, Z. S., Seidel, A. J., Shields, C. G., Oates, S. K., & Boushey, C. J. (2012). Table for two: Diabetes distress and diet-related interactions of married patients with diabetes and their spouses. Families, Systems, & Health, 30(2), 154-165. doi:http://dx.doi.org/10.1037/ a0028614 13. Iida, M., Stephens, M. A. P., Franks, M. M., & Rook, K. S. (2013). Daily symptoms, distress and interaction quality among couples coping with type 2 diabetes. Journal of Social and Personal Relationships, 30(3), 293-300. doi:http://dx.doi.org/10.1177/0265407512455308 14. Rook, K. S., August, K. J., Stephens, M. A. P., & Franks, M. M. (2011). When does spousal social control provoke negative reactions in the context of chronic illness? The pivotal role of patients’ expectations. Journal of Social and Personal Relationships, 28(6), 772-789. doi:http:// dx.doi.org/10.1177/0265407510391335 15. Stephens, M. A. P., Franks, M. M., Rook, K. S., Iida, M., Hemphill, R. C., & Salem, J. K. (2013). Spouses’ attempts to regulate day-to-day dietary adherence among patients with type 2 diabetes. Health Psychology, 32(10), 1029-1037. doi:http://dx.doi.org/10.1037/a0030018 16. Valle, G., Weeks, J. A., Taylor, M. G., & Eberstein, I. W. (2013). Mental and physical health consequences of spousal health shocks among older adults. Journal of Aging and Health, 25(7), 1121-1142. doi:http://dx.doi.org/10.1177/0898264313494800 17. Woolthuis, E. P. K., de Grauw, Wim J. C., Cardol, M., van Weel, C., Metsemakers, J. F. M., & Biermans, M. C. J. (2013). Patients’ and partners’ illness perceptions in screen-detected versus clinically diagnosed type 2 diabetes: Partners matter! Family Practice, 30(4), 418-425. doi:http://dx.doi.org/10.1093/fampra/cmt003 18. Zimmermann, T., Herschbach, P., Wessarges, M., & Heinrichs, N. (2011). Fear of progression in partners of chronically ill patients. Behavioral Medicine, 37(3), 95-104. doi:http:// dx.doi.org/10.1080/08964289.2011.605399

References

1. Canadian Diabetes Association. (2017). Types of diabetes. Retrieved from http://www. diabetes.ca/about-diabetes/types-of-diabetes

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Examining Short and Long-Term Cardiovascular and Urological Outcomes of Living Kidney Donors Michael Lee1,2, Yue Wang2,3, Olusegun Famure2,4, S. Joseph Kim2,4 Division of Nephrology, Faculty of Health Science, McMaster University, Hamilton, ON, Canada Multi-Organ Transplant Student Training Program, Toronto General Hospital, University Health Network, Toronto, ON, Canada 3 Faculty of Arts and Science, University of Toronto, Toronto, ON, Canada 4 Kidney Transplant Program, Toronto General Hospital, University Health Network, Toronto, ON, Canada 1 2

Abstract

With an organ shortage crisis, transplant centres are increasingly relying on living donation to drive transplantation rates. However, conflicting evidence has emerged questioning the risks associated with living donation, specifically a higher long-term risk for adverse cardiovascular and urological outcomes. As more donors come forward, understanding such risks is important in creating a more informed healthcare system. Articles published between April 1996 and April 2016 on the short- and long-term cardiovascular and urological outcomes associated with living kidney donors (LKD) were selected from OVID MEDLINE®. From an initial 861 articles, 36 remained relevant after title and abstract review. Most urological articles analysed glomerular filtration rate, nephron size or nephrosclerosis as a measure of remaining renal function and long-term end-stage renal disease (ESRD) risk. One such article by Lam NN et al. suggested a positive association between nephrectomy and ESRD requiring dialysis or transplantation at 10 years. However, known associations between LKD and development of hypertension mentioned by Garg AX et al may confound this relationship. Another article studied post-donation pregnancies and discovered a significantly higher risk for both adverse maternal and renal outcomes. Cardiovascular-related articles, on the other hand, could not comment on or found no difference in cardiovascular risks. Overall, short follow-up times, homogeneous populations and small donor pools may impede applicability to the Canadian

Introduction

Overview Over recent years, kidney transplant donation is becoming increasingly more common amongst LKDs due to its higher survivability and lower comorbidities associated with living kidney donation [1]. However, literature regarding short and long-term cardiovascular and urological outcomes of LKDs is poorly elucidated due to insufficient follow-up times, with a stronger emphasis towards kidney transplant recipients. As current literature contains variability in conclusions regarding cardiovascular and urological outcomes and possible risk factors, we conducted a review to reinforce and clarify concerns for LKD’s amongst the medical community. Definition of Cardiovascular-Outcome Our definition of a cardiovascular outcome consists of all major adverse cardiovascular events (MACE); namely, ischaemic heart disease requiring angioplasty or bypass, congestive heart failure, myocardial infarction, angina, stroke, abdominal aortic aneurysm repair and peripheral vascular bypass surgery [2–5]. Diagnosis of hypertension, though a known cause of cardiovascular disease, is secondary to MACE [5].

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Definition of Urological-Outcome Our definition of a urological outcome consists of diagnosis of chronic kidney disease requiring dialysis or kidney transplantation-most notably end-stage renal disease (ESRD), glomerulosclerosis, glomerulonephritis, urolithiasis, reduced GFR (< 90 mls/ min/1.73m2), hyperuricemia and proteinuria [4–24]. Diagnosis of gout is secondary to the outcomes listed above [14].

Literature Search Methodology

Search Methodology OVID Medline® was the main database searched to find articles relating cardiovascular and urological outcomes in LKD. The search was performed June 1, 2016 and included all articles after January 1, 1980. The exclusion criteria contained all articles unrelated to “Living Donors”/ “Kidney”/ “Nephrectomy”/ “Cardiovascular Diseases”/ “Cardiovascular System” / “Creatinine”/ “Kidney Diseases” or “Kidney Function Tests”. Any outcomes in the title and abstract that were unrelated to urological, renal or cardiovascular outcomes were also subject to exclusion. Case studies and comments regarding a previously conducted study were also excluded. A total of 26 articles were included after considerations.

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Examining Short and Long-Term Cardiovascular and Urological Outcomes of Living Kidney Donors

Records identified through database searching (n = 861) Unrelated to urological, renal or cardiovascular outcomes of donors (n = 607) Potential articles after title analysis (n = 254) Inaccessible full text Missing abstract (n = 122) Potential articles after abstract analysis (n = 132) Population < 500 Comments and case reports Articles included in the review (n = 26)

Figure 1. Study Flow Diagram (PRISMA flowchart).

Cardiovascular-Outcomes of Living Kidney Donors

Major Adverse Cardiac Events (MACE) and Hypertension Of the four articles relevant to cardiovascular disease, three mention no significant increase in a risk for MACE. Reese et al. conducted the same study with two sets of data from the Organ Procurement and Transplantation Network/United Network for Organ Sharing (OPTN/UNOS) and Medicare Claims (n = 3368). They matched on race and sex, neighbourhood poverty and categorized on ≥ 55 years (P = 0.70) and ≥ 60 years (P = 0.72) with a median follow-up of 7.8 years [2]. The second study by Patel et al. collected data on 4586 patients and also claimed an overall insignificant mortality rate of 0.44% from MACE [3]. As a result of the low incidence rate, the authors were unable to determine any pre-operative risks and long-term consequences of living kidney donation for the donors. The third article by Garg et al. was a more robust study than the other two as it matched 5 (n = 1278): 1 (n = 6359) on living donors and controls with a mean follow-up of 6.2 years. At the conclusion of the study, the investigators found no significant difference of MACE between donors and controls (1.3% vs 1.7%; HR 0.7, 95% [CI] 0.4-1.2, P = 0.225) [5]. The last article by Lam et al. did not analyse an association regarding MACE other than reporting hospitalization of a LKD by “cardiovascular diseases” requiring acute dialysis [4]. It is well known that the effects of cardiovascular risk factors generally longer to manifest as MACE, these four studies seem to suggest that there is no relationship between MACE and living kidney donation. However, the article by Garg et al. does link hypertension with MACE as a risk factor in the causal pathway and may be important to understanding of the long-term implications of living kidney donation and development of MACE, but with an

Review Articles

overall mean short follow-up time of 6.2 years, the long-term effects of hypertension on the cardiovascular system may not manifest as MACE until much later than the current follow-up lengths [2,3,5]. An important secondary outcome that is known to lead to MACE is hypertension, which is defined as any systolic BP > 140 mmHg, or diastolic BP > 90 mmHg or use of anti-hypertensive medication, by Mjoen et al. The article written by Garg et al. mentions a statistically significant association between living kidney donation and diagnosis of hypertension (16.3% vs 11.9%, HR 1.4, 95% [CI] 1.2-1.7, P < 0.001) with no significant difference between genetically related and unrelated donors [5]. Similarly, Mjoen et al. also found a significant increase in the mean blood pressure and prevalence at one (124.3 ± 14.2/77.9 ± 8.2 mmHg (n = 649)) and five (127.2 ± 15.4/78.8 ± 8.3 mmHg (n = 247)) years post-nephrectomy when compared to pre-nephrectomy (122.5 ± 10.6/7.6 ± 7.5 mmHg (n = 908)) [19]. Uncomplicated hypertension was prevalent and statistically at 2.6% (n = 17) and increased to 11.7% (n = 76) and 27.1% (n = 67) 1 and 5 years post-nephrectomy respectively [19]. Though they did not calculate a measure of association, Rizzari et al. and Elsherbiny et al. had similar findings with a prevalence of 22.4% and 21.8% (Rizzari) and 11% (Elsherbiny) and the seeming overall conclusion is that there is a significant relationship between living kidney donation and hypertension. It is important to note, however, that these articles make no mention of cardiovascular disease or MACE with the exception of Garg et al. and this is something worth observing in the future.

Urological-Outcomes of Living Kidney Donors

Reduced GFR and End-Stage Renal Disease (ESRD) Due to a generally low incidence of ESRD found in LKDs it is often hard to find a large enough population to estimate risk factors. However, a few of the articles suggest a genetic predisposition to ESRD for the LKD. Articles written by Wafa et al., Rosenblatt et al. and Fehrman-Ekholm et al. all found a low incidence rate (0.4%, 0.33%, 0.21%) with populations of n = 2000, 1195 and 2801 respectively [11–13]. However, all of the LKDs in these articles who developed ESRD were first-relatives of the patients who had a transplant [11–13]. In addition, Rosenblatt et al. found that three of the four patients who developed ESRD had also newly developed hypertension post-nephrectomy [12]. All three authors hypothesised that there is a possible link between de novo hypertension, progressive proteinuria, cardiovascular disease, intercurrent bacterial or viral infection, obesity, diabetes mellitus, hyperuricemia and eclamptic toximeia as all possible contributing factors for developing ESRD [11–13]. Other studies by Muzaale et al. and Mueller et al. suggested that risk of onset of ESRD did not increase after 7.6 years and 12.2 years of follow-up, but their follow up does not extend as far as the previous three of at least 33, 15.5 and 20 years at least and possibly did not see the long term effects of possible risk factors leading up towards ESRD. A cause for concern would include the inherent risk of a reduction in GFR associated with donation [8]. Living kidney donation would inherently decrease GFR to approximately 60ml/ min/1.73m2 and increase lifetime risk of ESRD by 50%; this acceleration of GFR loss is equivalent to 7.5 years of diabetic or aggressive non-diabetic kidney diseases [8]. If such a patient already begins with a lower premorbid GFR they are two to three times more likely than the mean population value to develop ESRD as a

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Review Articles

Examining Short and Long-Term Cardiovascular and Urological Outcomes of Living Kidney Donors

result of an already decreased baseline [8]. Other studies contained conflicting information regarding GFR rates post donation. Two studies found a fractional rise in GFR as a result of the remaining nephron hypertrophy within the first three years post donation [19,20], while a third found no temporary increase in GFR but a continual decrement of 10ml/min/1.73m2 up to ten years post donation [23]. Gout and Urological Outcomes Gout is caused by an increase in uric acid accumulation in the blood that leads to puffiness especially in the lower extremities of patents [14]. Lam et al. found a higher rate of diagnoses of acute kidney failure (rate ratio 12.5; 95% [CI] 1.5-107.0) and chronic kidney disease (rate ratio 5.0; 95% [CI] 2.1-11.7) when compared to a sample of LKD without gout and as a result the incidence of gout may predispose a LKD to renal failure [14].

Conclusion

Though a large proportion of the scientific community has established that hypertension is a risk factor for cardiovascular disease; studies have not been able to establish a definitive timeline for clinicians as to when the risk of hypertension is high enough as to cause MACE. Short follow-up times and a low incidence of MACE during the study period has not allowed most studies to draw significant conclusions. As per urological outcomes, there may be a link between an intrinsic nephron hypertrophy, due to nephrectomy, leading to de novo hypertension and other risk factors for a long-term progression to ESRD. Many of these studies only focused on one aspect of LKD with a homogenous population. This warrants further study into a more diverse population representative of the Canadian population as a database at the Toronto General Hospital renal transplantation program would provide. Its Comprehensive Renal Transplant Research Information System (CoReTRIS) contains comprehensive variables relevant to studies regarding cardiovascular and urological outcomes and utilization of such a database may contribute to current literature.

Donation: A Single-Center Experience. Transplant. Proc. 40, 1315–1318 (2008). 13. Fehrman-Ekholm, I. et al. Living Kidney Donors Developing End-Stage Renal Disease. Transplant. Proc. 38, 2642–2643 (2006). 14. Lam, N. N. et al. Gout after living kidney donation: correlations with demographic traits and renal complications. Am. J. Nephrol. 41, 231–40 (2015). 15. Lentine, K. L. et al. Race, Relationship and Renal Diagnoses After Living Kidney Donation. Transplantation 99, 1723–1729 (2015). 16. Thomas, S. M. et al. Risk of Kidney Stones With Surgical Intervention in Living Kidney Donors. Am. J. Transplant. 13, 2935–2944 (2013). 17. Rizzari, M. D., Suszynski, T. M., Gillingham, K. J., Matas, A. J. & Ibrahim, H. N. Outcome of living kidney donors left with multiple renal arteries. Clin. Transplant. 26, E7-11 (2012). 18. Rule, A. D. et al. Association of Kidney Function and Metabolic Risk Factors With Density of Glomeruli on Renal Biopsy Samples From Living Donors. Mayo Clin. Proc. 86, 282–290 (2011). 19. Mjoen, G. et al. One- and five-year follow-ups on blood pressure and renal function in kidney donors. Transpl. Int. 24, 73–77 (2011). 20. Doshi, M., Garg, A. X., Gibney, E. & Parikh, C. Race and renal function early after live kidney donation: an analysis of the United States Organ Procurement and Transplantation Network Database. Clin. Transplant. 24, E153–E157 (2010). 21. Ibrahim, H. N. et al. Pregnancy outcomes after kidney donation. Am. J. Transplant 9, 825–34 (2009). 22. Heimbach, J. K. et al. Obesity in Living Kidney Donors: Clinical Characteristics and Outcomes in the Era of Laparoscopic Donor Nephrectomy. Am. J. Transplant. 5, 1057–1064 (2005). 23. Garg, A. X. et al. Proteinuria and reduced kidney function in living kidney donors: A systematic review, meta-analysis, and meta-regression. Kidney Int. 70, 1801–1810 (2006). 24. Nobakht Haghighi, A. et al. A 4-year follow-up of living unrelated donors of kidney allograft in Iran. Iran. J. Kidney Dis. 9, 273–8 (2015).

References

1. Fehrman-Ekholm, I. Living Donor Kidney Transplantation. Transplant. Proc. 38, 2637–2641 (2006). 2. Reese, P. P. et al. Mortality and cardiovascular disease among older live kidney donors. Am. J. Transplant 14, 1853–61 (2014). 3. Patel, N. et al. Renal function and cardiovascular outcomes after living donor nephrectomy in the UK: quality and safety revisited. BJU Int. 112, E134–E142 (2013). 4. Lam, N. et al. Acute dialysis risk in living kidney donors. Nephrol. Dial. Transplant. 27, 3291–3295 (2012). 5. Garg, A. X. et al. Cardiovascular Disease and Hypertension Risk in Living Kidney Donors: An Analysis of Health Administrative Data in Ontario, Canada. Transplantation 86, 399–406 (2008). 6. Elsherbiny, H. E. et al. Nephron hypertrophy and glomerulosclerosis and their association with kidney function and risk factors among living kidney donors. Clin. J. Am. Soc. Nephrol. 9, 1892–902 (2014). 7. Lam, N. N., Lentine, K. L. & Garg, A. X. End-stage renal disease risk in live kidney donors: what have we learned from two recent studies? Curr. Opin. Nephrol. Hypertens. 23, 592–6 (2014). 8. Steiner, R. W., Ix, J. H., Rifkin, D. E. & Gert, B. Estimating Risks of De Novo Kidney Diseases After Living Kidney Donation. Am. J. Transplant. 14, 538–544 (2014). 9. Muzaale, A. D. et al. Risk of End-Stage Renal Disease Following Live Kidney Donation. JAMA 311, 579 (2014). 10. Mueller, T. F. & Luyckx, V. A. The Natural History of Residual Renal Function in Transplant Donors. J. Am. Soc. Nephrol. 23, 1462–1466 (2012). 11. Wafa, E. W. et al. End-stage renal disease among living-kidney donors: single-center experience. Exp. Clin. Transplant. 9, 14–9 (2011). 12. Rosenblatt, G. S., Nakamura, N. & Barry, J. M. End-Stage Renal Disease After Kidney

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Impacts of Mercury Poisoning on BrainDerived Neurotrophic Factor: A Review of Recent Literature Mohammad-Masoud Zavvarian Department of Human Biology, University of Toronto, Toronto, Ontario, Canada.

Abstract

Mercury poisoning is a serious concern due to its devastating effects on human health and development. It is often a result of overconsumption of seafood that are obtained from contaminated aquatic ecosystems. Mercury is able to bioaccumulate in the aquatic food web in the form of methylmercury, and once digested by humans, it can enter the circulatory system. Physiological processes transfer and convert methylmercury into many forms, each of which has a unique effect on human health. Elemental mercury and methylmercury are both able to pass the brain blood barrier, and can cause various neurological deficits. In the brain, one of the targets that is affected is brain-derived neurotrophic factor (BDNF), which is essential for growth and development. In this review, the impacts of different forms of mercury on BDNF, as well as possible detoxification pathways, will be analyzed. This includes the potential processes by which elemental mercury can change the geometric confirmation of BDNF and the mechanisms involved in the alteration of BDNF expression pattern by methylmercury.

Introduction

Mercury (Hg) is a toxic metal that can have serious physiological impact on most multicellular organisms [1]. In humans, mercury can cause weight loss, weakness, apathy, acute and chronic renal failure, damage to GI track, lung damage, stomatitis, gastroenteritis, autoimmune disorders, tremor, psychological and brain damage, and impairments in the development of the nervous system [2]. Mercury poisoning is mainly caused by consumption of contaminated marine food or by occupational exposure in industrial complexes. The safe limit for consumption of mercury, defined by the World Health Organization, is 0.5 g/Kg of sea food [3]. Due to industrial pollution, the level of mercury is higher in various sites around the world [4], which is why its impact on human health needs to be better understood. This review analyzes the impact of different chemical states of mercury on both developing and adult nervous systems. In particular, it looks at the impact of mercury on brain-derived neurotrophic factor (BDNF), which is a growth stimulus that plays an important role in neurogenesis, synaptogenesis, prevention of apoptosis, and memory formation [5]. Since mercury is known to cause abnormal brain development, it can be hypothesized that some of these effects are mediated through the alteration of BDNF function. Because the impact of mercury on BDNF is still not perspicuous, this review relies on the recent literature to assess our current understanding of the role of BDNF in mercury poisoning.

Chemical State

Examples

Elemental

Mercury metal (Liquid and vapour form)

Inorganic

Mercuric (Hg2+) ions, Mercurous (Hg+) ions

Organic

Methylmercury, Ethylmercury, Phenylmercury

Table 1. The

chemical states of mercury.

Exposure to Mercury

Mercury exists in three different chemical states: elemental mercury, inorganic mercury, and organic mercury (Table 1). Elemental mercury is the metallic form of mercury with zero oxidation state. It is liquid at room temperature; however, it is able to form vapour particles due to its volatile nature [2]. Inorganic mercury is present in two forms: mercurous (Hg1+) and mercuric (Hg2+), both of which are usually bound to other ions [2]. Organic mercury is mercury bound to one or more carbon atoms with the most well known of these being methylmercury [2]. In nature, methylmercury is produced by the methylation of inorganic mercury occurring in microorganisms found in aquatic sediments [7]. Exposure to elemental mercury can be either in its liquid form or vapour form. Unlike liquid mercury, which is not well absorbed, vapour form of elemental mercury can be easily absorbed

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Impacts of Mercury Poisoning on Brain-Derived Neurotrophic Factor: A Review of Recent Literature

through lungs and mucosal membranes [3]. Industrial workers are mainly affected by this type of exposure. When elemental mercury is absorbed through lungs, it enters the blood circulation and is transported into various parts of body, including the brain [3,6]. Inorganic mercury in the form mercurous ions is poorly absorbed; however, it is readily absorbed in the form of mercuric ions [3]. Predominantly, humans are exposed to organic mercury, in the form of methylmercury by overconsumption of fish from contaminated aquatic ecosystems [6]. In such environments, vapour mercury is deposited from the atmosphere, which then travels to the benthic level of aquatic ecosystems [2]. Microorganisms in these areas can convert inorganic mercury into methylmercury [8]. Methylmercury is not toxic to these organisms, as they have evolved mechanisms to protect themselves against methylmercury [2]. However, once these organisms are consumed by higher trophic levels species, methylmercury can be stored in their tissues, and cause physiological damage [2]. Methylmercury is able to bioaccumulate, and it has larger impact on higher trophic levels, such as various fish strains that are consumed by humans [22]. If humans consume these aquatic species, methylmercury will be transferred into humans. It is important to note that the human digestive track can absorb methylmercury, which leads to its deposition in human tissues [4]. Once elemental mercury enters the bloodstream thorough the lungs, it is converted to mercuric and mercurous ions by catalase and oxidative enzymes found in red blood cells [6]. Inorganic mercury is not able to cross lipid membranes due to its hydrophilic nature [3,6]. Mercuric ions are able to bind to glutathione, metallothionein, and sulfhydryl group on erythrocytes, as well as being suspended in plasma. Mercuric ions are stored in placenta, amniotic fluid, fetal tissues, and many other organs, but cannot cross the brain blood barrier efficiently [2,3]. Inside the brain, methylmercury and elemental mercury can be converted into mercuric ions. The liver plays an important role in mercury circulation because its excretes methylmercury into bile, which is later eliminated from the body [2]. (Figure1)

Figure 1: Overview of entrance, regulation, and impacts of mercury on the human body. Mercury enters human body main in form vaporized elemental form through the lungs, and in form methylmercury and mercuric ions through the GI track. It can enter the blood circulation, where it can either bind to thiol groups of erythrocytes or be transported to different organs. Two important organs that are effected are the brain and utero (embryo). In both of these organs, mercury leads to reduction in the amount of BDNF expressed, and possibly altering the structure of BDNF. A portion of methylmercury is excreted from the body by formation of bile in the liver, which is then excreted as feces.

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Effect of mercury on nervous system

It has been observed that methylmercury can induce cell death by apoptosis or necrosis [7]. These effects arise because of disturbances in calcium homeostasis in the brain and the generation of reactive oxidative species in mitochondria [7]. In vivo studies have identified several biological functions in the brain that are affected by mercury poisoning. These include, inhibition of glutamate uptake, increased level of hydroperoxide in the cerebellum, increased extracellular glutamate levels, increased responsiveness of the NMDA receptors (glutamate-gated ion channels [19]) in cortical neurons, reduction of glutathione (GSH) levels in the entire brain, higher level of activated caspase-3 in hippocampus, and reduced neuronal precursor activation [7,20]. It is important to note that individuals who are exposed to methylmercury early in life have a lower IQ compared to normal individuals [7]. These individuals have impaired speech, motor function, and visuospatial perception [7].

Brain-Derived Neurotrophic Factor (BDNF)

Neurotrophins are a class of signalling proteins responsible for synaptic plasticity, neuronal growth, axonal targeting, and maturation of synapses. Two important neurotrophins include brainderived neurotrophic factor (BDNF) and nerve growth factor (NGF) [10]. BDNF is a pro-survival signalling molecule that plays an important role in proper brain functioning and development (Figure 2A). BDNF is especially crucial for axonal growth during developmental ages [21]. BDNF also regulates synaptic plasticity and memory formation in the adult brain [21]. Upon its release from target cells, it is able to bind to Tropomyosin receptor kinase B (TrkB) receptors of neurites, which leads to activation of a series of downstream molecules that ultimately change the genomic expression patterns [5] (Figure 2B). Through its interaction with TrkB receptors, BDNF promotes the release of neurotransmitters in presynaptic neurons and enhances the ion channels in postsynaptic neurons (Figure 3). Furthermore, BDNF is important in protecting the hippocampus from ischemic damage by increasing antioxidant enzymes [21]. Deficit in BDNF function has been linked to many neurological disorders, such as Alzheimer’s disease [21]. Conformational Changes Exposure to divalent ions, such as Zn2+, Cu2+, Pd2+, Hg2+, causes conformational changes in neurotrophins, inhibiting their biological effects [10]. Although the exact conformational changes of BDNF in presence Hg2+ is unknown, it has been reported that the N-terminal domain of BDNF, which is essential for its interaction with TrkB receptors, binds to divalent ions such as zinc (Zn2+) and copper (Cu2+) [11,12]. Zinc forms a tetrahedral geometry, bound to an amino group, an imidazole, and two water molecules [11]. In contrast, copper forms a different tetrahedral geometry bound to an amino group, a carboxylate group, and two amide nitrogens [12]. As a result, zinc increases the proliferative effect of BDNF, whereas copper reduces this proliferative effect [11]. This suggests that copper and zinc ions have unique binding properties to BDNF. It has been hypothesized that Hg2+ can have similar conformational affects on BDNF, due to its divalent nature, although the exact mechanism is yet to be determined [10]. It is important to note that conformational analysis of BDNF in the presence of methylmercury indicate that unlike Hg2+, methylmercury does not cause any geometric changes in BDNF molecule [10].

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Impacts of Mercury Poisoning on Brain-Derived Neurotrophic Factor: A Review of Recent Literature

Figure 2. A) The 3 dimensional structure of BDNF visualized by PDB [17]. B) The mechanism by which BDNF binds to TrkB and initiate a cascade of signaling molecules throughout the cell that ultimately leads to change in gene expression. Binding BDNF to TrkB (a tyrosine kinase) phosphorylates various sites of this receptor that leads to activation of MAPK/Erk pathway (important for growth and differentiation of cells), PI3K pathway (important for survival of the cell), protein lipase C (leads to enhanced calmodulin kinase (CamK) activity, important in synaptic plasticity). Picture taken from Autry & Monteggia (2012) [5].

Expression changes Thus far, most of our understanding regarding the expression changes of BDNF due to mercury poisoning is derived from animal models. In rodents, there are nine promoters that regulate BDNF gene expression. Each of these promoters are followed by an exon [5]. These exons are spliced together to translate into BDNF precursor, also known as pro-BDNF. This precursor is turned into active BDNF by plasminogen activator [5]. In mice, methylmercury exposure is associated with decrease in BDNF mRNA expression in dentate gyrus [13]. This is because, methylmercury promotes a long-term repressive state at the BDNF promoter regions. The epigenetic changes include DNA hypermethylation, a decrease in H3 acetylation at the promoter 4, and an increase in histone H3K27 tri-methylation [13]. The hypermethylation of DNA leads to silencing of gene. Since histone acetylation of lysine 9 in H3 leads to active chromatin structure, a decrease in acetylation will lead to deactivation of the gene [13]. In humans, in utero exposure to methylmercury is associated with decreased serum BDNF in concentration dependent manner. This pattern appears to be sex-dependent, since methylmercury appears to mainly affect female subjects [14]. In addition, exposure to methylmercury causes reduction in the number of TrkB receptors [10]. Surprisingly, it has been reported that astrocytes have protective roles against methylmercury by increasing expression of BDNF and nerve growth factor (NGF) when treated treated with methylmercury, which helps them avoid neuronal cell death [15]. The exact mechanism by which astrocytes increase their BDNF and NGF productions is still unknown and requires further research.

Potential Treatments

Treatment of mercury poisoning involves the administration of chelator agents. There are two potential chelators that are used, including 2,3-dimercapto-1-propanesulfonate (DMPS) and dimercaptosuccinic acid (DMSA). These chelator agents have lower side effects compared to other chelators, which allows for extended chelation therapy [16]. In addition, it was shown that administration of fluoxetine in mice was able to reverse to the effects of methylmercury on promoter 4 of the BDNF gene [13].

Review Articles

Figure 3: Expression of BDNF gene in an adult human brain. This indicates the regions that are likely to be affected by mercury poisoning due alteration of BDNF function. The left picture illustrates the cortical regions and the right picture illustrates the subcortical regions. Red indicates regions where BDNF is highly expressed, and green indicates regions where BDNF is poorly expressed. The data was visualized by Allen Brain Atlas [18].

Conclusion

Mercury poising is a serious concern due to its detrimental effects on the nervous system and brain development. One particular protein affected by mercury poisoning is BDNF. Evidence suggests that mercuric ions can interfere with the interaction of BDNF and TrkB receptors. Also, methylmercury causes reductions in the expression of both BDNF and TrkB. In future studies, the structure of BDNF in the presence of mercuric ions and its interaction with TrkB must be analyzed using structure elucidation techniques.

Acknowledgments:

This study acknowledges and appreciates the contributions of Dr. Alistair Dias and HMB437H1F for supervising this project. I would also like to thank Dr. Brad Bass, Dr. Franco Taverna, Dr. William Ju, and Dr. Colleen Dockstader for encouraging me to pursue a path in research.

References:

1. Bernhoft RA. Mercury Toxicity and Treatment: A Review of the Literature. Journal of Environmental and Public Health. 2012;2012: 1–10. 2. Clarkson TW, Magos L. The Toxicology of Mercury and Its Chemical Compounds. Critical Reviews in Toxicology. 2006;36(8): 609–662. 3. Azevedo BF, Furieri LB, Pec¸anha FM, Wiggers GA, Vassallo PF, Sim˜oes MR, Rossoni L, Stefanon I, Alonso MJ, Salaices M, et al. Toxic Effects of Mercury on the Cardiovascular and Central Nervous Systems. Journal of Biomedicine and Biotechnology. 2012;2012: 1–11. 4. Gochfeld M. Cases of mercury exposure, bioavailability, and absorption. Ecotoxicology and Environmental Safety. 2003;56(1): 174–179. 5. Autry AE, Monteggia LM. Brain-Derived Neurotrophic Factor and Neuropsychiatric Disorders. Pharmacological Reviews. 2012;64(2): 238–258. 6. Echeverria D, Woods JS, Heyer NJ, Rohlman DS, Farin FM, Bittner AC, et al. Chronic low-level mercury exposure, BDNF polymorphism, and associations with cognitive and motor function. Neurotoxicology and Teratology. 2005 Nov;27(6):781–96. 7. Santos AAD, Hort MA, Culbreth M, López-Granero C, Farina M, Rocha JB, Aschner M. Methylmercury and brain development: A review of recent literature. Journal of Trace Elements in Medicine and Biology. 2016;38: 99–107. 8. Takemoto T, Ishihara Y, Ishida A, Yamazaki T. Neuroprotection elicited by nerve growth factor and brain-derived neurotrophic factor released from astrocytes in response to methylmercury. Environmental Toxicology and Pharmacology. 2015;40(1): 199–205. 9. Kim M-K, Zoh K-D. Fate and Transport of Mercury in Environmental Media and Human Exposure. Journal of Preventive Medicine & Public Health. 2012;45(6): 335–343. 10. Colquhoun A, Eibl JK, Krol KM, Chan HM, Ross GM. Conformational analysis of the effects of methylmercury on nerve growth factor and brain derived neurotrophic factor. Environmental Toxicology and Pharmacology. 2009;27(2): 298–302. 11. Travaglia A, Mendola DL, Magrì A, Pietropaolo A, Nicoletti VG, Grasso G, Malgieri G, Fattorusso R, Isernia C, Rizzarelli E. Zinc(II) Interactions with Brain-Derived Neurotrophic Factor N-Terminal Peptide Fragments: Inorganic Features and Biological Perspectives. Inorganic Chemistry. 2013;52(19): 11075–11083. 12. Travaglia A, Mendola DL, Magrì A, Nicoletti VG, Pietropaolo A, Rizzarelli E. Copper, BDNF and Its N-terminal Domain: Inorganic Features and Biological Perspectives. Chemistry - A European Journal. 2012;18(49): 15618–15631.

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13. Onishchenko N, Karpova N, Sabri F, Castrn E, Ceccatelli S. Long-lasting depression-like behavior and epigenetic changes of BDNF gene expression induced by perinatal exposure to methylmercury. Journal of Neurochemistry. 2008;106(3): 1378–1387. 14. Spulber S, Rantamaki T, Nikkila O, Castren E, Weihe P, Grandjean P, Ceccatelli S. Effects of Maternal Smoking and Exposure to Methylmercury on Brain-Derived Neurotrophic Factor Concentrations in Umbilical Cord Serum. Toxicological Sciences. 2010;117(2): 263–269. 15. Takemoto T, Ishihara Y, Ishida A, Yamazaki T. Neuroprotection elicited by nerve growth factor and brain-derived neurotrophic factor released from astrocytes in response to methylmercury. Environmental Toxicology and Pharmacology. 2015;40(1): 199–205. 16. Aaseth J, Jacobsen D, Andersen O, Wickstrøm E. Treatment of mercury and lead poisonings with dimercaptosuccinic acid and sodium dimercaptopropanesulfonate. A review. Analyst. 1995;120(3): 853–854. 17. Berman H, Westbrook J, Feng Z, Gilliland G, Bhat T, Weissig H, Shindyalov I, Bourne P. The Protein Data Bank. Nucleic Acids Research. 2000;28: 235-242. 18. Hawrylycz MJ, Lein ES, Guillozet-Bongaarts AL, Shen EH, Ng L, Miller JA, Lagemaat LNVD, Smith KA, Ebbert A, Riley ZL, et al. An anatomically comprehensive atlas of the adult human brain transcriptome. Nature. 2012;489(7416): 391–399. 19. Paoletti P, Bellone C, Zhou Q. NMDA receptor subunit diversity: impact on receptor properties, synaptic plasticity and disease. Nature Reviews Neuroscience. 2013;14(6): 383–400. 20. Sakaue M, Maki T, Kaneko T, Hemmi N, Sekiguchi H, Horio T, et al. Potentiation of Methylmercury-Induced Death in Rat Cerebellar Granular Neurons Occurs by Further Decrease of Total Intracellular GSH with BDNF via TrkB in Vitro. Biological and Pharmaceutical Bulletin. 2016;39(6): 1047–1054. 21. Binder DK, Scharfman HE. Brain-derived Neurotrophic Factor. Growth Factors. 2004;22(3): 123–31. 22. Ynalvez R, Gutierrez J, Gonzalez-Cantu H. Mini-review: toxicity of mercury as a consequence of enzyme alteration. BioMetals. 2016;29(5): 781–8.

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Journal of Undergraduate Life Sciences • Volume 11 • Issue 1 • Spring 2017


JULS

REVIEWS

The Role of Transforming Growth Factor Beta in Allergic Asthma Pathogenesis Abi Kirubarajan, BHSc (c)1, John-Paul Oliveria, BSc, PhD(c)1, Gail Gauvreau, PhD1* Department of Medicine, Division of Respirology, McMaster University, Hamilton, Ontario

1

*Corresponding Author: Gail Gauvreau, PhD (gauvreau@mcmaster.ca)

Abstract

Transforming growth factor beta (TGF-β) is a key fibrogenic and immunomodulatory cytokine, with nuanced roles in both initiation and resolution of inflammation, as well as airway remodelling. TGF-β has been implicated in allergic asthma as asthmatics present high TGF-β levels within bronchoalveolar lavage fluid, as well as overexpression of TGF-β mRNA in bronchial biopsies. However, due to the contradictory effects of the cytokine on inflammation, the precise role of TGF-β in allergic asthma remains unknown. While this review will focus on pro- and anti-inflammatory functions, it is important to note that TGF-β is a key cytokine in airway remodeling. The release of TGF-β by structural cells and fibroblasts contributes to subepithelial fibrosis, in addition to thickening of the basement membrane, increased vascularity through angiogenesis, and increased volume of submucosal glands. These changes lead to asthma’s clinical presentations of narrowed airway lumens and edematous secretions. In its pro-inflammatory role, TGF-β induces the chemotaxis of both innate and adaptive immune cells, including neutrophils, macrophages, B lymphocytes, and mast cells. Following chemotaxis, TGF-β activates cell degranulation, releasing histamine, serine proteases, and IL-4 to cause alveolar epithelial damage. Furthermore, TGF-β has been implicated in eosinophilic inflammation and airway hyperresponsiveness due to increased lymphocyte proliferation, as well as increased survival of T lymphocytes upon TGF-β stimulation. However, TGF-β also demonstrates anti-inflammatory roles within allergy, due to the decrease of B cell proliferation and promotion of IgA class switching. In addition, TGF-β is released by T regulatory cells to suppress Th2 response and cytokine production. TGF-β1 in specific inhibits IL-1 and IL-2-dependent T cell activation and differentiation into T helper 17 cells. Finally, TGF-β can also downregulate the expression of cytokine receptors such as IL-2R. This review will conclude with a discussion comparing these contradictory roles and current gaps in literature, as well as potential therapeutic implications.

Background

Asthma Asthma is a chronic inflammatory disease of the airways, particularly within the structural conducting zone (i.e. bronchi and bronchioles) [1]. Asthma is a complex syndrome involving airway hyperresponiveness (AHR), edema, and remodeling, contributing to symptoms such as recurrent wheezing, tightness within the chest, and difficulty breathing [2]. The field of asthma has been of growing interest for researchers as the prevalence of asthma has risen globally, due to both improvements of diagnostic testing as well as changes in environmental stimuli [3]. As of 2014, 8.5% of Canadians over the age of twelve have been diagnosed with asthma, with higher numbers in the male population [3]. As symptom assessment is often subjective due to self-reporting protocols, diagnosis of asthma is often conducted through spirometric measurements. A ratio of forced expiratory volume in 1 second (FEV1) to forced vital capacity (FVC) of ≤ 70% for a predicted demographicmatched value is indicative of airway obstruction [4,5]. In addition, subsequently increasing doses of methacholine are inhaled until a 20% decrease (PC20) in FEV1 is measured [4,5]. A positive

methacholine challenge is defined by a PC20 of ≤8 mg [4]. If there is an observed increase in FEV1 of ≥12% from baseline, the airway obstruction can be classified as reversible; alternatively, an observed FEV1 of ≥10% after inhalation of a short-acting beta-agonist can also be used to determine the extent of AHR reversibility [4,5]. Moreover, in addition to the variability of inflammatory pathways and clinical presentations, asthma diagnosis is further complicated as its exact cause is currently unknown; researchers associate the etiology of asthma with complex relationships between genetic and environmental influences [1,5]. Allergic Asthma Allergic asthma (AA) is the most prevalent subtype of asthma, contributing to 60-90% of asthma cases in 2014 [6,7]. AA diagnosis is more common in adolescent males, while non-allergic asthma is more commonly diagnosed in older females [7]. In addition, AA is often considered to be more mild than non-allergic asthma, with less severe complications resulting from symptoms [7]. In order to receive a diagnosis of AA, patients must demonstrate the aforementioned symptoms of asthma, in addition to allergic sensitization [7]. Allergic sensitization can be concluded through positive skin prick

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test or demonstration of elevated serum immunoglobulin (IgE) levels in response to an allergen [7]. In allergic asthmatics, allergen inhalation can induce IgE-mediated inflammatory processes, resulting in abnormal increases of Cluster of Differentiation (CD) 4+ T cells and eosinophils [8,9]. Type 2 cytokines, such as interleukin (IL)-4, and IL-13, can further upregulate allergic inflammation through increasing maturation of IgE-producing B cells through co-stimulatory interactions with CD40 ligands [8,9]. This type 2 inflammatory response also contributes to airway smooth muscle hyperplasia and hypertrophy, in addition to downregulating the opposing type 1 inflammatory response [10]. Allergic asthmas are unable to sufficiently enact anti-inflammatory pathways within the airways in order to mediate the immune response [5,9,10].

Transforming Growth Factor Beta

Introduction Transforming growth factor beta (TGF-β) is a key fibrogenic and immunomodulatory cytokine in allergic asthma pathogenesis [11, 12]. Due to its heterogeneous roles in both inflammation and airway remodeling, extensive research has been conducted both in vitro and in vivo to establish both its function and possible therapeutic implications. TGF-β is a member of the transforming growth factor superfamily of cytokines, which include transforming growth factor alpha (TGF-α) [13]. While both TGF-β and TGF-α are both implicated in polypeptide growth, TGF-β plays a larger role in inflammatory diseases such as allergic asthma due to its extensive functions in tissue regeneration, cell differentiation, and immunoregulation [11,14]. TGF-β and Allergic Asthma TGF-β has been implicated in allergic asthma pathogenesis, as asthmatics have high circulating TGF-β levels within bronchoalveolar lavage (BAL) fluid in comparison to healthy controls [15,16]. Research has also demonstrated that the bronchial biopsies of asthmatics demonstrate overexpression of TGF-β mRNA and proteins, signifying higher levels of TGF-β within bronchial epithelium in addition to BAL fluid [16]. In addition, while the role of TGF-β is allergy has not yet been fully substantiated, it has been shown that TGF-β levels increase significantly 24 hours following positive segmental bronchoprovocation after allergen exposure [17]. Even without asthma-related symptoms (such as wheezing or recurrent cough), TGF-β expression increases following repeated low dose exposure of allergens in healthy patients [18]. Together, these findings suggest that TGF-β plays a role in allergic asthma, however, due to the various effects of the cytokine on both inflammation initiation and resolution, as well airway remodeling, the precise balance of the roles of TGF-β in allergic asthma remains unknown [15]. Isoforms of TGF-β TGF-β has three prevalent isoforms: TGF-β1, TGF-β2, and TGF-β3 [19]. The peptide structures of the three isoforms are highly similar, with 70-80% of the primary sequences homologous. However, post-transcriptional alternative splicing within the C-terminus of the TGF-β structure results in three different functions throughout the body [20]. TGF-β1, the most prevalent isoform of TGF-β, plays a large role in the immune system in both the initiation and resolution of the inflammation [21]. Conversely,

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TGF-β2 modulates embryonic development through cell differentiation, while the effects of TGF-β3 include cell adhesion and formation of the extracellular matrix [15, 21]. This paper will largely focus on TGF-β1’s role in allergic asthma pathogenesis, though TGF-β3’s function in cell adhesion will also be discussed in the context of airway remodeling. Structure of TGF-β TGF-β has a dimeric structure consisting of a cysteine knot, connected through a tertiary structure of intramolecular disulfide bonds [1, 22]. Eight cysteine residues within TGF-β form disulfide bonds for the characteristic knot structure of the TGF-β superfamily [1, 22]. The final ninth cysteine residue forms a disulfide bond with a separate TGF-β to produce a homodimer structure [1, 22]. The region between the fifth and sixth cysteines is highly variable, leading to both receptor binding and specificity, as well as the separate three isoform structures upon alternative splicing [23, 24]. (Figure 1)

Figure 1. Structure of TGF-beta.

Synthesis and Secretion of TGF-β Due to the potential therapeutic implications of TGF-β, its synthesis pathway has been highly studied, leading to the sequencing of its pre-pro-structure. The TGF-β precursor consists of a N-terminus pre-region acting as a signaling peptide (20-30 amino acid residues), which is necessary for secretion from the cell, in addition to a pro-region latency associated peptide (LAP) [25,26]. The C-terminus of the pre-pro-TGF-β comprises of the mature peptide that will be released following proteolytic cleavage from the cleavage protein furin, which is also expressed within the precursory TGF-β structure [27]. Following synthesis, the homodimer of TGF-β interacts with the LAP to form the Small Latent Complex (SLC), a protein-based complex which remains in the cell until bound by the Latent TGF-β-Binding Protein (LTBP) to form the Large Latent Complex (LLC) [25,26]. The LLC is later secreted to the ECM. The multiple steps of complex formation allow for greater regulation and specificity of the TGF-β release, due to its potent actions within the body [26,34]. (Figure 2) Cell-Mediated TGF-β Release While TGF-β was first isolated from platelets, previous literature has established that many cell types can produce the cytokine,

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The Role of Transforming Growth Factor Beta in Allergic Asthma Pathogenesis

Review Articles

can also lead to increased bronchospasm due to smooth musclemediated hyperplasia and hypertrophy, as well as hypercontractility due to increased sensitivity to contractile stimuli [39]. This airway remodeling supplements the inflammatory pathways within allergic asthma to exaggerate airway narrowing and cause symptoms such as recurrent wheezing and shortness of breath [35-39].

Figure 2. Steps of synthesis and secretion of TGF-beta. Hayashi H, Sakai T. Biological significance of local TGF-β activation in liver diseases. Frontiers in physiology. 2012 Feb 6;3:12.

including both inflammatory cells in the bronchial mucosa, and structural cells within the airway wall [28, 29]. Pattern recognition receptors, in particular toll-like receptors detecting lipopolysaccharide, can stimulate mast cells and monocyte-lineage cells to release TGF-β for both pro- and anti-inflammatory purposes [30,31]. In addition, circulating cytokines such as tumour necrosis factor alpha, IL-1-beta, and interferon gamma can stimulate mast cells, as well as B and T lymphocytes to secrete TGF-β [32]. Conversely, eosinophils are predominantly stimulated by hyaluronan (a nonsulfated glycosaminoglycan hypothesized to play a role in virulence) to release TGF-β, in addition to the stimulation and receptor recognition of type 2 cytokines such as IL-4, IL-5, IL-9, and IL-13 [33]. Structural cells, such as fibroblasts, epithelial cells, and smooth muscle cells, are stimulated by transforming nuclear factor alpha, IL-1, pH changes, and reactive oxygen species (such as superoxide anion and hydrogen peroxide) [35]. In vivo immunohistochemistry and in situ hybridization has showed that epithelial damage can stimulate these structural cells to release TGF-β for the purposes of airway remodeling [35]. However, it is important to note that not many TGF-β-activating pathways are fully elucidated, and further research must be conducted to understand activating factors for TGF-β – specifically the roles of pH changes, integrins, and proteases to trigger structural cell cytokine release. (Figure 3) [38]

Airway Remodelling Role of TGF-β

While this paper will focus on the pro- and anti-inflammatory roles of TGF-β within allergic asthma, it is important to note that TGF-β is a key cytokine in airway remodeling [35-38]. The release of TGF-β by structural cells and fibroblasts contributes to increased subepithelial fibrosis, in addition to thickening of the basement membrane, increased vascularity through angiogenesis, increased volume of submucosal glands and secretions, and increased collagen and matrix deposition [35-38]. These changes within the airway lead to the clinical presentation of narrowed airway lumens and increased edematous submucosal secretions, in addition to reduction of baseline airway capacity [35-38]. Furthermore, TGF-β pathways

Figure 3. Variable stimuli for TGF-beta release.

Pro-Inflammatory Role of TGF-β

Role in Chemotaxis and Cell Degranulation TGF-β induces the chemotaxis of both innate and adaptive immune cells, including neutrophils, macrophages, B lymphocytes, and mast cells, allowing infiltration to the bronchial mucosa and subsequent inflammation [28]. According to Sporn et al, at concentrations of 0.1 to 10 pg/mL, TGF-β potently induces directed monocyte migration in vivo, concurrent with the expression of high-affinity TGF-β receptors present on the monocytes (Kd of 1-10 pM) [40]. (Figure 4) TGF-β-induced chemotaxis increases in a dose-dependent manner, at rates comparable to that of IL-8 for neutrophils, until a plateau and subsequent decrease. This decrease is common with high doses of chemoattractants (such as IL-8 or N-formylmethionyl-leucyl-phenylalanine), in order to modulate the immune reaction and provide a specific inflammatory response [40]. Following chemotaxis, TGF-β activates cell degranulation for mast cells, eosinophils, and neutrophils [28,41]. This degranulation releases pro-inflammatory mediators such as histamine, serine proteases, lysosomal enzymes, IL-4, and reactive oxygen species (ROS), contributing to bronchial and alveolar epithelial damage and inflammation [42]. In particular, the role of TGF-β in the release of ROS has been well-documented within allergic asthma pathogenesis. Innate immune cells typically release reactive oxygen species (ROS) via degranulation in order to facilitate respiratory bursts against extracellular pathogens [43,44]. TGF-β can induce nicotinamide adenine dinucleotide phosphate (NADPH) oxidase within inflammatory cells to reduce O2 into superoxide anions and hydrogen peroxide, thus producing damaging free radicals [45-47]. TGF-β contributes to this oxidative stress by both activating and differentiating granular cells [48]. While ROS can indeed be an effective defense against external pathogens within the lungs, it is important to note that respiratory bursts can also contribute to chronic lung injury through creation of oxidative stress, as this imbalance between free radicals and endogenous antioxidants can damage both bronchial and alveolar epithelium [44-47]. In turn, this ROS-

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pathways [50]. However, it is important to note that TGF-β’s role in lymphocyte-driven pathways is largely anti-inflammatory, which will be elucidated in the next section.

Anti-Inflammatory Pathways of AM

Figure 4. Dose-dependent chemotactic effects of TGF-beta on monocytes. Wahl SM, Hunt DA, Wakefield LM, McCartney-Francis N, Wahl LM, Roberts AB, Sporn MB. Transforming growth factor type beta induces monocyte chemotaxis and growth factor production. Proceedings of the National Academy of Sciences. 1987 Aug 1;84(16):5788-92.

mediated oxidative stress influences adaptive immunity through production of pro-inflammatory cytokines via induced gene expression through activation of the nuclear kappa-light-chain enhancer of activated B cells (NF-κB) pathway [49]. This increased production of pro-inflammatory cytokines can subsequently cause airway eosinophilia, in addition to inducing positive feedback loops for prolonged innate immune cell activation through specific cytokine receptors [45-47]. Role in Th2 cell-mediated Eosinophilic Inflammation Furthermore, while TGF-β is traditionally associated with dampening Th2 cell-mediated eosinophilic inflammation, TGF-β has been implicated in the hallmarks of eosinophilic inflammation and AHR due to increased lymphocyte proliferation, as well as increased survival of T lymphocytes upon TGF-β stimulation [32,49]. Particularly regarding immature T lymphocytes, TGF-β can prolong the lymphocyte response, as well as prolong the release and half-life of pro-inflammatory cytokines such as IL-4 and IL-5 [28,32]. Depending on the differentiation state of the cell, TGF-β can also prolong the survival of eosinophils within bronchial and alveolar mucosa, in addition to increasing the proliferation of eosinophils to initiate inflammatory responses via IL-5-mediated

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Inhibition of B and T cell Response TGF-β is typically inhibitory of B lymphocyte-driven inflammation [51-54]. TGF-β decreases B cell proliferation through inducing the transcription factor ld3 to induce expression of cyclin-dependent kinase inhibitor 12, which regulates cell cycle progression [51,53]. This also represses regulatory genes such as c-myc and ATM, preventing the growth of B cells [51,53]. In addition, TGF-β blocks B cell activation and promotes IgA class switching in both murine and human models, proving to have an inhibitory effect on antibody pathways [52]. TGF-β also decreases B cell survival through inducing apoptosis of immature or resting B cells through the aforementioned c-myc regulatory gene [52,54]. For this reason, TGF-β is released by regulatory B and T cells in order to resolve inflammatory responses and contribute to immunoregulation [51-54]. As a positive feedback mechanism, TGF-β can also induce the differentiation of B cells into regulatory B cells, allowing for the release of IL-10 to supplement the cytokine’s antiinflammatory effects [51-54]. In addition, TGF-β is released by T regulatory cells to suppress Th2 response and cytokine production [28,55,56]. TGF-β induces CD4+ T cells to induced T regulatory cells. TGF-β1 in specific prevents the activation of quiescent helper T cells and cytotoxic T cells, in addition to inhibiting IL-1 and IL-2-dependent T cell activation and differentiation into proinflammatory T helper 17 cells [28,55,56]. Finally, TGF-β can also downregulate the protein expression of cytokine receptors, such as IL-2 receptors, to downregulate the inflammatory responses associated within allergic asthma to contribute to resolution of the adaptive response [28]. Inhibition of Alveolar Macrophage Response Alveolar macrophages have receptors specific to circulating TGF-β; upon receptor recognition, alveolar macrophages can be deactivated in their response of innate immunity [28,57-61]. TGF-β inhibits the lipopolysaccharide-induced production of tumor necrosis factor alpha, IL-1 alpha, and IL-1 beta [57]. It takes 12-16 hours for TGF-β to induce this suppressive effect in vivo through translational mechanisms [57]. Western blot experiments demonstrated that this inhibition did not affect mRNA release of preformed cytokines (tumor necrosis factor alpha, specifically), the release of preformed cytokine, nor cytokine degrading – leading the locus of interference to be hypothesized at the translationallevel [28,61]. Furthermore, TGF-β induces the factor IKBa, which inhibits NF-κB activation, preventing the formation of pro-inflammatory cytokines and defensins, as well as macrophage activation [28,57-60]. TGF-β also suppresses interferon gammastimulated nitric oxide production, typically released by alveolar macrophages in pro-inflammatory pathways [61]. This is done through decreased stability and translated of inducible nitric oxide synthase (iNOS) mRNA, in addition to increased degradation of iNOS protein [62]. (Figure 5)

Future Directions

While TGF-β remains a promising target for therapeutic

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The Role of Transforming Growth Factor Beta in Allergic Asthma Pathogenesis

Figure 5. TGF-beta suppresses iNOS induction through dose-dependent mechanisms in soluble and particulate compartments. Vodovotz Y, Bogdan C, Paik J, Xie QW, Nathan C. Mechanisms of suppression of macrophage nitric oxide release by transforming growth factor beta. The Journal of experimental medicine. 1993 Aug 1;178(2):605-13.

intervention due to its roles in both pro- and anti-inflammatory pathways, further research must explore the relationship between TGF-β and its methods of activation for separate pathways [28]. In addition to establishing a more substantial timeframe for its actions, research must clarify the roles that its isoforms play in a pharmacokinetic and pharmacodynamic context regarding halflife. Finally, further research must be conducted regarding polymorphisms of TGF-β expressions [62,63]. Studies of asthmatics have revealed that polymorphisms of TGF-β (specifically the 509T variant on haplotype 1) can act as markers for diagnosis – the internal validity of these conclusions, as well as the feasibility of their implications, must be further elucidated in order to establish any potential clinical practices [62,63].

References:

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Transforming growth factor-β and its role in asthma. Pulmonary pharmacology & therapeutics. 2003 Aug 31;16(4):181-96. 30. Ten Dijke P, Yamashita H, Ichijo H, Franzen P, Laiho M, Miyazono K, Heldin CH. Characterization of type I receptors for transforming growth factor-beta and activin. Science. 1994 Apr 1;264(5155):101-4. 31. Medzhitov R. Toll-like receptors and innate immunity. Nature Reviews Immunology. 2001 Nov 1;1(2):135-45. 32. Beutler B. Inferences, questions and possibilities in Toll-like receptor signalling. Nature. 2004 Jul 8;430(6996):257-63. 33. Letterio JJ, Roberts AB (1998). “Regulation of immune responses by TGF-beta”. Annu. Rev. Immunol. 16: 137–61. doi:10.1146/annurev.immunol.16.1.137. PMID 9597127 34. Ohkawara Y, Tamura G, Iwasaki T, Tanaka A, Kikuchi T, Shirato K. Activation and transforming growth factor-β production in eosinophils by hyaluronan. American journal of respiratory cell and molecular biology. 2000 Oct 1;23(4):444-51. 35. Dallas SL, Miyazono K, Skerry TM, Mundy GR, Bonewald LF. Dual role for the latent transforming growth factor-beta binding protein in storage of latent TGF-beta in the extracellular matrix and as a structural matrix protein. The Journal of cell biology. 1995 Oct 15;131(2):539-49. 36. Nelson HS, Davies DE, Wicks J, Powell RM, Puddicombe SM, Holgate ST. Airway remodeling in asthma: new insights. Journal of allergy and clinical immunology. 2003 Feb 28;111(2):215-25 37. Chakir J, Shannon J, Molet S, Fukakusa M, Elias J, Laviolette M, Boulet LP, Hamid Q. Airway remodeling-associated mediators in moderate to severe asthma: effect of steroids on TGF-β, IL-11, IL-17, and type I and type III collagen expression. Journal of Allergy and Clinical Immunology. 2003 Jun 30;111(6):1293-8. 38. Makinde T, Murphy RF, Agrawal DK. The regulatory role of TGF-β in airway remodeling in asthma. Immunology and cell biology. 2007 Jul 1;85(5):348-56. 39. Halwani R, Al-Muhsen S, Al-Jahdali H, Hamid Q. Role of transforming growth factor–β in airway remodeling in asthma. American journal of respiratory cell and molecular biology. 2011 Feb;44(2):127-33. 40. Ebina M, Takahashi T, Chiba T, Motomiya M. Cellular hypertrophy and hyperplasia of airway smooth muscles underlying bronchial asthma: a 3-D morphometric study. American Review of Respiratory Disease. 1993 Sep;148(3):720-6. 41. Wahl SM, Hunt DA, Wakefield LM, McCartney-Francis N, Wahl LM, Roberts AB, Sporn MB. Transforming growth factor type beta induces monocyte chemotaxis and growth factor production. Proceedings of the National Academy of Sciences. 1987 Aug 1;84(16):5788-92. 42. Gruber BL, Marchese MJ, Kew RR. Transforming growth factor-beta 1 mediates mast cell chemotaxis. The Journal of Immunology. 1994 Jun 15;152(12):5860-7. 43. Bloemen K, Verstraelen S, Van Den Heuvel R, Witters H, Nelissen I, Schoeters G. The allergic

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cascade: review of the most important molecules in the asthmatic lung. Immunology letters. 2007 Oct 31;113(1):6-18. 44. Lohmann-Matthes , M . L . , Steinmuller , C . & Franke-Ullmann , G . Pulmonary macrophages . Eur. Respir. J. 7, 1678 – 1689( 1994 ). 45. Forman HJ, Torres M. Reactive oxygen species and cell signaling: respiratory burst in macrophage signaling. American journal of respiratory and critical care medicine. 2002 Dec 15;166(supplement_1):S4-8. 46. Joseph M, Tonnel AB, Capron A, Voisin C. Enzyme release and superoxide anion production by human alveolar macrophages stimulated with immunoglobulin E. Clinical and experimental immunology. 1980 May;40(2):416. 47. Kinnula VL, Everitt JI, Whorton AR, Crapo JD. Hydrogen peroxide production by alveolar type II cells, alveolar macrophages, and endothelial cells. American Journal of Physiology-Lung Cellular and Molecular Physiology. 1991 Aug 1;261(2):L84-91. 48. Rahman I, Gilmour PS, Jimenez LA, MacNee W. Oxidative stress and TNF-a induce histone Acetylation and NF-кB/AP-1 activation in Alveolar epithelial cells: Potential mechanism In gene transcription in lung inflammation. In Oxygen/Nitrogen Radicals: Cell Injury and Disease 2002 (pp. 239-248). Springer US. 49. Kaul N, Forman HJ. Activation of NFκB by the respiratory burst of macrophages. Free Radical Biology and Medicine. 1996 Dec 31;21(3):401-5. 50. Sanjabi S, Zenewicz LA, Kamanaka M, Flavell RA. Anti-inflammatory and pro-inflammatory roles of TGF-β, IL-10, and IL-22 in immunity and autoimmunity. Current opinion in pharmacology. 2009 Aug 31;9(4):447-53. 51. Ohno I, Nitta Y, Yamauchi K, Hoshi H, Honma M, Woolley K, O’Byrne P, Tamura G, Jordana M, Shirato K. Transforming growth factor beta 1 (TGF beta 1) gene expression by eosinophils in asthmatic airway inflammation. American journal of respiratory cell and molecular biology. 1996 Sep;15(3):404-9. 52. Li, Ming O.; Wan, Yisong Y.; Sanjabi, Shomyseh; Robertson, Anna-Karin L.; Flavell, Richard A. (2006-01-01). “Transforming Growth Factor Beta- Review of Immune Responses” Review of Immunology. 24 (1): 99–146. 53. Roes, Jürgen; Choi, B. Ken; Cazac, Balthazar B. (2003-06-10). “Redirection of B cell responsiveness by transforming growth factor β receptor”. Proceedings of the National Academy of

Sciences. 100 (12): 7241–7246. 54. Patil, Supriya; Wildey, Gary M.; Brown, Thomas L.; Choy, Lisa; Derynck, Rik; Howe, Philip H. (2000-12-08). “Smad7 Is Induced by CD40 and Protects WEHI 231 B-lymphocytes from Transforming Growth Factor-β-induced Growth Inhibition and Apoptosis”. Journal of Biological Chemistry. 275 55. Arsura, Marcello; Wu, Min; Sonenshein, Gail E. (1996-07-01). “TGFβ1 Inhibits NF-κB/Rel Activity Inducing Apoptosis of B Cells: Transcriptional Activation of IκBα”. Immunity. 5 (1): 31–40 56. Eisenstein, Eli M.; Williams, Calvin B. (2009-05-01). “The Treg/Th17 Cell Balance: A New Paradigm for Autoimmunity”. Pediatric Research. 65(5 Part 2): 26R–31R 57. Morishima, Noriko; Mizoguchi, Izuru; Takeda, Kiyoshi; Mizuguchi, Junichiro; Yoshimoto, Takayuki (2009-08-14). “TGF-β is necessary for induction of IL-23R and Th17 differentiation by IL-6 and IL-23” 58. Kubiczkova, Lenka; Sedlarikova, Lenka; Hajek, Roman; Sevcikova, Sabina (2012-09-03). “TGF-β – an excellent servant but a bad master”. Journal of Translational Medicine. 59. Smythies, Lesley E.; Sellers, Marty; Clements, Ronald H.; Mosteller-Barnum, Meg; Meng, Gang; Benjamin, William H.; Orenstein, Jan M.; Smith, Phillip D. (2005-01-03). “Human intestinal macrophages display profound inflammatory anergy despite avid phagocytic and bacteriocidal activity”. Journal of Clinical Investigation 60. Wahl, Sharon M (February 2007). “Transforming growth factor-β: innately bipolar”. Current Opinion in Immunology. 19 (1): 55–62. 61. Ma J, Chen T, Mandelin J, Ceponis A, Miller NE, Hukkanen M, Ma GF, Konttinen YT. Regulation of macrophage activation. Cellular and Molecular Life Sciences CMLS. 2003 Nov 1;60(11):2334-46. 62. Vodovotz Y, Bogdan C, Paik J, Xie QW, Nathan C. Mechanisms of suppression of macrophage nitric oxide release by transforming growth factor beta. The Journal of experimental medicine. 1993 Aug 1;178(2):605-13. 63. Hobbs K, Negri J, Klinnert M, Rosenwasser LJ, Borish L. Interleukin-10 and transforming growth factor-β promoter polymorphisms in allergies and asthma. American journal of respiratory and critical care medicine. 1998 Dec 1;158(6):1958-62. 64. Buckova D, Izakovicova Holla L, Benes P, Znojil V, Vacha J. TGF-b1 gene polymorphisms.

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Prenatally Stressed Rats Exhibit Behavior that Signals Depression when Evaluating Blood Glucose Metabolism Neda Jafarian Department of Human Biology, University of Toronto, Toronto, Ontario, Canada.

A prenatal stress model of depression was chosen for animal studies to evaluate depression-like behavior changes initiated the hyperactivity of the HPA axis and blood glucose levels increase. The biological processes were measured in the context of the impact of the stress. The adult animals did not show immediate changes after prenatal stress but instead the changes progressed slowly. The slow progression of change in the adults allowed time for the researchers to observe the biological responses to stress. This knowledge allowed the researchers to evaluate adverse characteristics overtime. Prenatal stress events were observed to have an impact on the degree of glycolysis in the frontal cortex and in the hippocampus as well as affecting the lactate levels in the frontal cortex and the function of tri-carboxylic acid cycle in the hippocampus of the rats. The changes occurred at the metabolic level but currently the physiological impact cannot be predicted. The results demonstrated that elevated brain glucose levels are not caused by glycolysis inhibition. In addition, the measurements showed that brain glycolysis inhibition was not evident because pyruvate and lactate levels showed no change in concentration. The authors of the chosen primary articles, which their work has been criticized and analyzed in this paper, have recommended more research on the topic to try to assess the physiological importance of differences in the metabolism without and with prenatal stress. Key words: prenatal stress model brain glucose metabolism; HPA axis; glycolysis inhibition Tri-Carboxylic Cycle; lactate levels; pyruvate dehydrogenase; depression

Background

The data from several research studies have indicated that pathogenesis of depression is influenced by glucose metabolism [7]. On the other hand Campbell and MacQueen (2004) learned that when depressive symptoms improved no correlation was observed with improved metabolic parameters or glucose. Lustman et al. (2000) observed diabetes, depression, and source of resistance to insulin and glucose intolerance disorder in patients with increased cortisol levels. Depression in a high number of patients exhibited hyperinsulinemia and insulin resistance is produced with onset of glucose intolerance [12]. Three major hypotheses for the observed link between diabetes and depression have been presented in the research community: the neuro-biochemical-changes that are associated with diabetes are the primary cause of depression; or the onset of depression is linked to psychological and social factors associated with the diabetes treatment or the disease; or since diabetes onset is associated with many factors, depression exhibits as an independent risk factor [2]. Serotonin when abnormally regulated is one of the major pathogenesis of depression [8]. Note the biological perspective shows multi-level connections between diabetes and depression such as the lower concentrations of primary serotonin observed with alterations in neurotransmitter and endocrine activities; insulin’s hypoglycemic effect is impacted by the “stimulation of the pro-

duction of glucocorticoids, growth hormone and glucagon” because they “work counter-regulationally against insulin’s hypoglycemic effect” [8]. The “serotonin hypothesis of depression” hypothesizes that the Waptic and post-synaptic areas is a highly significant for start of depression as pathology, not a transient sadness [2]. A prenatal stress model of depression was chosen because depression-like behavior changes take place when hyperactivity of the HPA axis and blood glucose levels increase. Therefore the biological processes were measured in context of the impact of the stress. The adult animals did not show immediate changes after prenatal stress but instead the changes progressed slowly. The slow progression of change in the adults allowed time for the researchers to observe the biological responses to stress. This knowledge allowed the researchers to evaluate adverse characteristics overtime. Dekta et al. 2013 have evaluated the data available and noted one reason that diabetes and depression are comorbidities may be “metabolic disturbances” as a result of excessive glucocorticoid action. The same animal model was used for evaluating glucocorticoid activity in the brain was used in the present experiment evaluating brain glucose metabolism for depression [4]. The glycolytic enzymes, two tri-carboxylic enzymes and two products of glycolysis were assessed in the current research. The activity of a significant enzyme in the pentose phosphate pathway (PPP), glucose-6-phosphate dehydrogenase, was also evaluated.

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Prenatally stressed rats exhibit behavior that signals depression when evaluating blood glucose metabolism

Figure 1. Bélanger, Allaman and. Magistrett (2011) illustrated in vitro findings (above) when determining the end product of glycolysis. They found that lactate, not both lactate and pyruvate, resulted from reactions in vitro with glucose-6-phosphate dehydrogenase [1]. The research demonstrated that glutamine synthetase is an astrocyte specific. Glucose is synthesized into glutamate due to processes of the astrocytes [10].

Summary of Major Results

The prenatally stressed rats showed statistically significant higher immobility and lower time spent climbing measurements than the control group. The stressed rats demonstrated behavior that signaled depression. Several processes that are linked to glucose metabolism take place in different cell compartments. The influence of disturbed neuronal and glial function is known with regards to depression. The results were similar to other published research that indicated some increase in glycolysis while at the same time some decrease is observed in the tri-carboxylic cycle during depression. The results demonstrated that elevated brain glucose levels are not caused by glycolysis inhibition. The measurements showing that brain glycolysis inhibition was not evident based on the observation that pyruvate and lactate levels showed no change in concentration. The glycolytic enzyme pyruvate kinase shWowed a decrease in both the frontal cortex and the hippocampus for acute stress. Meanwhile the second glycolytic enzyme observed, hexokinase did not show any change for either the hippocampus or the frontal cortex. The third glycolytic enzyme observed, phosphofructokinases showed the largest number of location activities increased during acute stress in the frontal cortex and the hippocampus. One of the products glycolysis pyruvate showed group dependent change in the frontal cortex and increased change for acute stress in the hippocampus. The other product observed, lactate behaved differently in the frontal cortex with an increase during prenatal stress, and meanwhile lactate showed an increase in glucose treatment in the hippocampus.

Conclusions and Discussion

The research of Detka et al. (2015) added knowledge to topic of using an animal model to evaluate the glucose metabolism enzymes with reference to depression. The only similar research addresses a different stage of the metabolism process [14]. For example, DeVry et al. (2016) evaluated how the TrkB receptor is moderator of behavior from the hippocampus and affects the nucleus accumbens (NAc). Only a few research studies are available perhaps due to the difficulty in identifying changes in the brain when the subjects exhibit distinctive behavior towards vulner-

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able or resistant to stress. The current research under discussion was motivated by the indication that the “brain glucose uptake and glycolysis are rather increased and the Krebs cycle and/or oxidative phosphorylation are rather decreased in depression” [4]. Kurek et al. (2015) also noted the potential of brain glucose metabolism disruption as an impact on pathological depression. The current study demonstrated using the prenatally stressed model of depression with rats as the subject that frontal cortex and hippocampus glucose concentration became elevated [4]. Interestingly no decrease in the key glycolytic enzymes accompanied the increased glucose concentrations indicating no link with a decrease glycolytic process [4]. Therefore the most probable link was with the increased glucose uptake or gluconeogenesis [4]. The findings led to further research by Kurek et al. (2015) to evaluate the degree that permanent changes were caused by prenatal stress. The focus of the research was on glucose transporters (GLUTs) expression to learn about the effect of prenatal stress specifically on the glucose transporter GLUT1, GLUT3 and GLUT4 [7]. During the same experiment a control group of rats and a prenatally stressed group of rats two other factors were evaluated a) acute immobilization stress and b) oral glucose administration [7]. Related to the current research under discussion, the researchers observed that the glucose transporter GLUT1 was identified in higher concentrations [7]. The experimental results indicated that the increased concentration of all three glucose transporters, especially GLUT1 influenced higher levels of glucose in the hippocampus and in the frontal cortex [7]. Kurek et al. (2015) noted that prenatal stress did initiate depression behaviors (long-term) and permanent changes were observed for the glucose transporters concentrations. Human studies on changes in depression behavior were carried out in 2011 on over 480 diabetes patients. The purpose of the experiment was to evaluate the education level of the patient and the effect on glucose monitoring. Interestingly the results demonstrated improved “association with H1bA1c and with glucose” Of particular interest in reference to the current study the experiment demonstrated no depression symptoms dependency on “improved metabolic parameters or glucose” [2]. Other research discussed here showed the independent characteristic of depression behaviors under experimental conditions.

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Figure 2. Graph A shows the impacts of acute stress, prenatal stress and glucose administration in the frontal lobe glucose-6 phosphate activity (The values are the mean ±SEM). The frontal cortex experiment demonstrated acute stress that was statistically significance for several cases. Graph B Shows the same impacts but on the hippocampus glucose-6-phosphate activity (The values used are the mean ±SEM). The hippocampus demonstrates statistically significant effect of prenatal stress and of glucose administration. The two experimental groups, the control and the prenatally stressed rats underwent 1 hour of acute stress or were administered 1g/kg, po. *p < 0.05 versus the control group; the control group was not treated with acute stress, prenatal stress or glucose loading; #p < 0.05 versus the appropriate control group. [4]. (SEM = Standard Error of the Mean; p = probability; g/kg, po is grams per kilogram administered glucose)

Dekta et al. (2015) hypothesized that the metabolic changes could be a way to balance the strong glycolysis with the weaker phosphorylation or the weaker Kreb’s cycle. Another hypothesis offered by the researchers was the possibility that prenatal stress has a positive effect on metabolic processes by acting as making provision for protection of aging hippocampal cells so the cells are not easily damaged [4]. The glucose metabolic change markers changed when negative factors were applied in the prenatally stressed rats when the cells were aging but not under basal conditions [4]. The research by DeVry et al. (2016) has the potential to fill in some gaps that the other research discussed here does not yet fill. The hippocampus is influenced by effects similar to an antidepressant when is in the presence of the Brain- derived neurotrophic factor (BDNF) [6]. Interestingly pro-depressant effects are stimulated in the NAc when BDNF is present. The NAc population of young cells of the neuron increased when TrkB was overexpressed. On the other hand the overexpression of TrkB in the same location decreased despair and basal corticosterone levels [6]. Affective did not change when TrkB was overexpressed in the hippocampus but the astrocyte cell population increased [6]. The research of Bélanger et al. (2011) is exciting in the context of the current research because of the implications on brain studies and the regulatory processes that are operating in the astrocyte or the neuron. Since the isolation of the astrocyte researchers recognized that the neuron and the astrocyte are both important for implementing the mechanisms that allow delivery (both spatially and temporally) of energy substrates. Bélanger et al. (2011) recognized cooperation between the neuron and astrocyte in order to enhance the brain energy metabolism at the cellular level. The major energy substrate is glucose but other substrates that are blood derived are also used in specific situations [10]. Lactate is the energy substrate that increases when individuals or subjects in experiments are carrying out strong exertions in physical activities [1]. Glucose6-phophate is not limited to one specific metabolic pathway

but follows three main pathways PPP; through the production of glycolysis that then produces lactate or becomes involved in mitochondrial metabolism, or the pathway of glycogenesis in astrocytes [1]. The production of carbon dioxide and water through oxidization is only a very basic description of the chemistry because glucose metabolic intermediates are formed across the range of possibilities in order to produce glutamate, pyruvate, lactate and acetate for energy [1]. The finding that lactate seems likely to be the only product of glycolysis has implications on further animal studies using animal depression models and investigating brain glucose metabolism [1, 13]. Further knowledge about the processes in the astrocytes will expand investigations in order to encompass the neuronastrocyte collaborations.

Criticisms and Future Directions

A better understanding of the role of glycolysis in depression would be a result of identifying the natural product of glycolysis in vivo. Data is only available for in vitro studies at this time. The end product of glycolysis in the past was assumed to be pyruvate and lactate, but in vitro research studies show that pyruvate may only be an intermediary to the production of lactate as the only key product [1,4,13]; see Figure 2. The ability to narrow the research to the connection between glucose, serotonin and the synaptic and post-synaptic areas of the brain are highly relevant to the study of depression in humans. The ability to treat depression similarly to diabetes is one possibility by measuring natural serotonin levels and then tailoring the daily dose of an anti-depressant to an individual needs is one possibility. Because depression is so often comorbid with diabetes self-administrating a medicine to relieve both problems in an individual would significantly improve their quality of life. An increased capability of offering quality health care and reduction in health care costs will be two of the major results. For example chronic physical illness with comorbid depression

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Prenatally stressed rats exhibit behavior that signals depression when evaluating blood glucose metabolism

lead to poor health outcomes including death, greater incidence of seasonal flu and diabetes, and increased use of medical services [2,5]. Depression experienced in individuals as diabetes comorbid depression is identified with poor outcomes ranging from reduced glycogenic control, higher risk of dementia, greater number of complications with diabetes and even to mortality [5]. Breznoscakova and Nagyova (2013) also emphasize that depression and diabetes impact the rapid growth of obesity in countries of high and low economic status. Depression negatively influences diabetic patients to maintain healthy diets, but since depression is not easy to diagnose too many diabetics are depressed and receiving no therapy. Evaluating the two illnesses in terms of cost let Elspeth et al. (2014) to determine that depression comorbidity with chronic disease leads to from 33 percent to 169 percent higher monthly average health costs. Demand for emergency care increases potentially by as much as 50 percent when patients with chronic pain were also depressed; other studies have confirmed that depression comorbid with physical disease increases the need for emergency care leading to higher health costs [5]. The future of research on the connection between brain glucose metabolism and depression shows potential for developing a solution to relieve the distress of depression for millions of people worldwide.

References

1. Bélanger M, Allaman I, Magistretti PJ (2011) Brain Energy Metabolism: Focus on AstrocyteNeuron Metabolic Cooperation. Cell Metabolism, 14(6):724-738. 2. Breznoščáková D, Nagyova I (2013) Depression and Glucose Metabolism (Diabetes Mellitus), Mood Disorders, Prof. Nese Kocabasoglu (Ed.) 3. Campbell S, MacQueen G (2004) The role of the hippocampus in the pathophysiology of major depression. Journal of Psychiatry and Neuroscience. 29(6):17–426. 4. Dekta J., Kurek A, Kucharczyk M, Glombik K, Basta-Kaim A, Kubera M, Lason W, Budziszewska B (2015) Neuroscience. 250:198-208. 5. Elspeth A, Guthrie EA, Dickens C, Blakemore A, Watson J, Chew-Graham C, Lovell K, Afzal C, Kapur N, Barbara Tomenson B (2014) Depression predicts future emergency hospital admissions in primary care patients with chronic physical illness, Journal of Psychosomatic Research. In Press (available online) 6. De Vry J, van Mierlo T, Martínez-Martínez P, Losen M, Temel Y, Boere J, Kenis G, Steckler T, Steinbusch HWM, De Baets M, Prickaerts J. (2016) TrkB in the hippocampus and nucleus accumbens differentially modulates depression-like behavior in mice. Behavioural Brain Research. 296(1):15-25. 7. Kurek A, Detka J, Basta-Kaim A, Kubera M, Leśkiewicz M, Lasoń W, Budziszewska B, (2015) The concentrations of brain glucose transporters in an animal model of depression. Pharmacological Reports. 65(S1):124-125. 8. Kuzmiakova M, Horacek J, Andel M (1999) The relationship between central serotonergic activity and insulin sensitivity in healthy volunteers. Phsyconeuroendocrinology. 24(8): 785-797. 9. Lustman PJ, Williams MM, Sayuk GS, Nix BD, Clouse RE (2007) Factors influencing Glycaemic Control in Type 2 Diabetes During Acute-and Maintenance-Phase Treatment of Major Depressive Disorder with Bupropion. Diabetes Care. 30: 459-466. 10. Magistretti PJ (2008) Brain energy Metabolism. In fundamental Neuroscience, L.R. Squire, D. Berg, F.E. Bloom, S. du Lac, A. Ghosh, and N.C. Spitzer (Eds.). San Diego: Academic Press. pp. 271-293. 11. Masi G, Brovedani P (2011) The hippocampus, neurotrophic factors and depression: possible implications for the pharmacotherapy of depression. CNS Drugs. 25(11):913-31. 12. Okamura F, Tashiro A, Utumi A, Imai T, Suchi., Tamura D, Sato Y, Suzuki S, Hongo, M. (2000) Insulin resistance in patients with depression and its changes during the clinical course of depression: minimal model analysis. Metabolism. 49:1255-1260. 13. Schurr A, Payne RS (2007) Lactate, not pyruvate, is neuronal aerobic glycolysis end product: An in vitro electrophysiological study, Neuroscience. 147(3):613-619. 14. Tagliari B, Noschang CG, Ferreira AGK, Feska LR, Wannmacher CMD, Dalmaz C, Wyse ATS (2010) Chronic variable stress impairs energy metabolism in prefrontal cortex and hippocampus of rats: prevention by chronic antioxidant treatment. Metabolic Brain Disease. 25(2):169-176.

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O-GlcNAcylation in the Pathological Heart: A Nexus of Hypertrophic Circuitry? Ahmad Kamal1,2 Department of Physiology, Faculty of Medicine, University of Toronto, Toronto, ON Institute of Neuropathology, University Hospital of Zurich, Zurich, CH-8091, Switzerland

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Abstract

The heart is a highly plastic organ, growing or shrinking in accordance with biological demands. It responds to physiological stimuli such as pregnancy and exercise as well as pathological stimuli such as hypertension and aortic stenosis. However the latter induce a form of cardiac hypertrophy concomitant with structural, functional, electrical, and metabolic dysregulations that ultimately culminate in heart failure, a leading cause of mortality. The association between metabolic remodelling and cardiac disease is a subject of intense ongoing investigation, as a growing body of evidence is suggesting a bidirectional causality. The hexosamine biosynthetic pathway (HBP) is a metabolically-linked biochemical cascade that results in the addition of an O-linked β-N-acetylD-glucosamine (O-GlcNAc) to a plethora of proteins to alter their subcellular localization, stability and activity. Numerous studies reported an upregulation of key enzymes in the HBP subsequent to cardiac stress such as ischemia/reperfusion; O-GlcNAcylation has also been shown to mediate myriad cardioprotective and cardiotoxic effects via mechanisms that remain largely unknown. The goal of this review is to elucidate the role of O-GlcNAcylation in cardiac hypertrophy, and to dissect the mechanisms underlying its effects on the associated remodelling in order to glean insight into novel potential therapeutic targets.

Introduction

The chronological path of the heart is littered with a multitude of stresses that elicit biological changes within an astounding range: the dynamic growth-shrinkage window of the heart exceeds 100% [1]. To overcome an acute hemodynamic overload, the heart utilizes length-dependent activation (Frank-Starling mechanism), a limited intrinsic mechanism of the myocardium that enhances cross-bridge formation to increase contractility [2,3]. Neurohumoral signalling is a chemical inotropic mechanism that can also be recruited to compensate for an insufficiency in contractility [3]. However, chronic inciting stimuli, such as biophysical and biochemical stresses, trigger cardiac remodelling at the structural, electrical, functional and metabolic levels [4]. Structural changes include hypertrophy and atrophy, which describe an increase or decrease in myocardial mass, respectively, and can be either pathologically- or physiologically-induced [1]. A change in cardiac mass occurs mainly at the cardiomyocytic level, where a postnatal cardiomyocyte may increase in size due to a change in protein synthesis or stability [1,5-7]. Based on the law of LaPlace, which states that heightened systolic wall stress can be offset by wall thickening, it has been argued that hypertrophy is a compensatory mechanism induced to surmount a hemodynamic burden [8,9]. Nevertheless, pathologically hypertrophy (PaH) has been linked to multiple adverse clinical conditions, and recent evidence challenges the notion that it has a necessary compensatory role [10-14]. Therefore, recent investigations are seeking to eliminate hypertrophy rather than attenuate or reverse it in hopes of preventing its progression to cardiac decompensation.

O-linked β-N-acetyl-D-glucosamination (O-GlcNAcylation) is a type of post-translational modification that glycosylates proteins by attaching the nucleotide sugar uridine 5’-diphosphate-N-acetylD-glucosamine (UDP-O-GlcNAc) to serine or threonine residues [15]. In turn, UDP-GlcNAc is synthesized by the hexosamine biosynthetic pathway (HBP), a cascade that branches off glycolysis [15]. This glycosylation, often considered a signalling system, has been shown to modulate cardiac function by mass-regulating large portions of the proteome and transcriptome during diseased conditions [15-19]. Therefore, being a possible nexus of the cardiac stress response, this glycosylation appears to be a tempting avenue for therapeutic manipulation in the effort to eliminate PaH altogether. This review will present a monographic overview of cardiac hypertrophy, mainly the pathological form, before integrating a sum of studies on the role of O-GlcNAcylation in the development of the pathology. The aim of this work is to elucidate the current state of the literature on the role of O-GlcNAcylation in cardiac hypertrophy and to tease out the molecular mechanisms governing its effects in order to glean insight into novel potential therapeutic targets.

1. Cardiac Hypertrophy

1.1. Classification 1.1.1. Pathological and Physiological Hypertrophy. Although cardiac hypertrophy is usually classified in a dichotomous fashion (physiological or pathological), it has been proposed that the two types constitute the extremes of a continuum with multi-parameter gradients: the collective subtypes of both classes can be ordinally arranged based on the combined spectra of mi-

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O-GlcNAcylation in the Pathological Heart: A Nexus of Hypertrophic Circuitry?

tochondrial biogenesis, metabolic substrate utilization (glucose or fatty acids), and fetal gene program expression [20]. Physiological hypertrophy (PhH) is triggered by postnatal development, pregnancy and exercise [1]. By contrast, PaH is instigated by pressureoverload (e.g. hypertension and aortic stenosis), volume-overload (e.g. valvular insufficiency), neurohumoral stress (e.g. adrenergic signalling), electrophysiological stress (e.g. progressively increasing tachycardia and cessation of sustained tachycardia), myocardial infarction, and other cardiomyopathy-causing conditions (e.g. genetic mutations and diabetes mellitus) [21-29]. Hypertrophy can be further subdivided by the aforementioned inciting stimuli. Therefore, giving their diversity, it is imperative to delineate the biological processes underlying each subtype of hypertrophy before studying the manner with which a cellular process, such as O-GlcNAcylation, could modulate them. 1.1.2. Geometric Morphology. Cardiac hypertrophy can also be classified based on the change in morphological architecture, a variable dependent on the nature of the inciting stimulus rather than its time course (chronic or intermittent) [30,31]. The heart can hypertrophy in a concentric or eccentric manner, both of which lead to an increase in mass. Concentric geometry is distinguished by an increase in relative wall thickness without a considerable change in chamber volume due to a parallel deposition of sarcomeres and a consequent lateral growth of the cardiomyoctyes [1]. Eccentric geometry is characterized by an increase in chamber volume accompanied by little or no change in relative wall thickness due to a serial deposition of sarcomeres and a consequent longitudinal growth of the cardiomycotyes [1]. Stimuli that elevate systolic wall stress tend to induce concentric hypertrophy (e.g. hypertension) while those that elevate diastolic wall stress produce the eccentric form (e.g. valvular insufficient) [32]. However, eccentric hypertrophy in a pathological milieu is concomitant with a decline in relative ventricular wall thickness as opposed to the proportional cardiac enlargement observed in PhH [33]. Of note is the fact that recent evidence, reporting no significant cardiac remodelling subsequent to resistance training, challenges the long-standing notion that resistance training and endurance training induce concentric and eccentric hypertrophy, respectively (the Morganroth hypothesis) [34]. Nonetheless, characterization by geometric morphology remains clinically relevant as it is of high prognostic value: pathologically-induced eccentric left ventricular hypertrophy confers a greater risk of developing systolic dysfunction [35]. 1.2. Instigating and Impelling Factors In this work, the main pathways and molecular alterations underlying the functional derangements observed in PaH will only be briefly visited; merely the relevant specific pathways will be examined. There is a vast array of stimuli that can provoke the development of cardiac hypertrophy, and in this section, only the key biophysical and biochemical factors that initiate or augment it will be discussed. A number of these stimuli were a crucial research milestone in the effort of deciphering the complex signalling network involved, while others were directly implicated in the hypertrophic response. 1.2.1. Biophysical Stimuli. Although one of the major ini-

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tiating stimuli of cardiac hypertrophy is biomechanical stress concomitant with a hemodynamic burden, the molecular basis of mechanosensing in the heart remains elusive. One study reported an activation of an inward Ca2+ current subsequent to mechanical perturbation in chick cardiomyocytes in vitro, and this ionic influx was abrogated upon treatment with the stretch-activated channel blockers streptomycin and gadolinium suggesting a role for these channels in mechanosensing [36-39]. Owing to accumulating evidence, it has been also strongly suggested that RTPC channels are a substantial component of the mechanosensing mechanism in the heart: they have been showing to re-induce the fetal gene program and to play a pivotal role in the development of pressure-overload and agonist-induced hypertrophy [40]. Moreover, the upregulation of integrin proteins α1, α5, α7B, β1A, and β1D during hypertrophy in rodents and the implication of β1 integrins in normal cardiac function and pressure-overload and neurohumorally-induced hypertrophy unveil another important class of proteins that participate in the mechanosensing process [41]. In addition, considerable evidence points to sarcomeric (e.g. Z disc proteins and titin) and cytoskeletal (e.g. desmin) proteins as mechanosensors relevant in the context of cardiac hypertrophy [41-44]. Hence, although the above proteins and channels may constitute a single cellular cascade, a plausible explanation is that mechanosensation in the heart is mediated by multiple cellular components, segregated by sensing threshold, to elicit various responses. Biomechanical stress also stimulates myriad biochemical cascades (mechanotransduction) that leads to a heightened expression of a number of autocrine and paracrine hypertrophic or hypertrophy-related signals. Interestingly, pressure-overload and volume-overload hypertrophy invoke the upregulation of different genes [45]. For instance, a study demonstrated that pressureoverload and volume-overload both stimulate and elevation of atrial natriuretic peptide mRNA, but only the former upregulates β-myosin heavy chain and skeletal α-actin and downregulates the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) [46]. Similarly, only pressure-overload upregulates transforming growth factor-β3 (TGF-β3) and insulin-like growth-factor-1 (IGF-1) (solely in cardiomyocytes) [46]. In cardiomyocytes, only volumeoverload downregulates acidic fibroblast growth factor [46]. In fact, the subtypes of PhH, likewise, display different transcriptional profiles (e.g. pregnancy and exercise) [47]. 1.2.2. Biochemical Stimuli. In additional to biomechanical stress, a large number of other factors have also been directly linked to cardiac hypertrophy such as vasoactive substances (e.g. angiotensin II, endothelin-1 and prostaglandin F2α), cytokines (e.g. cardiotrophin-1, leukemia inhibitory factor, and tumor necrosis factor α). Growth factors 9e.g. fibroblast growth factors, IGF-1, and TGF-β1), and hormones (e.g. growth hormone, thyroid hormone, and catecholamines) [48]. Furthermore, there is evidence that mechanical stress can enhance the biosynthesis and secretion of angiotensin II and endothelin-1 as well as activate angiotensin II receptors irrespective of the ligand [49-51]. Studies on a number of animal models (including human) demonstrate that IGF-1 is secreted preferentially in PhH and that angiotensin II (synthesis and bioactivity), endothelin-1 and catecholamine release is specific to PaH [52-59]. Downstream of the hypertrophic stimuli is a plethora of

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O-GlcNAcylation in the Pathological Heart: A Nexus of Hypertrophic Circuitry?

Review Articles

Figure 1: The various types of biological stress that can evoke cardiac remodelling. F-6-P denotes fructose-6-phosphate; UDP-GlcNac, 5’-diphosphate-N-acetyl-D-glucosamine; O-GlcNAc transferase, UDP-GlcNAc:polypeptide β-N- acetylglucosaminyltransferase; AP, action potential; I¬NCX, sodium-calcium exchanger current; INHE, sodium-proton exchanger current; SERCA, sarcoplasmic/endoplasmic reticulum Ca2+ ATPase

pathways that lead to sarcomeric reorganization, gene expression alterations, and post-translational modifications [33]. Amongst The most implicated biochemical pathways are the IGF-1-PI3KAkt (physiological type) and the Gαq-GPCR (pathological type) signalling cascades [33]. PaH is also associated with alterations in the regulation of many protein kinases (e.g. MAPK, PKC and PKD) and Ca2+ handling proteins (e.g. calcineurin) [33]. Therefore, although the stimuli of both hypertrophy types may appear similar at the whole-organ level (e.g. increased afterload), the molecular factors instigating, mediating and impelling the growth of the heart are substantially different and highly complex. 1.3. Molecular and Electrical Remodelling 1.3.1. The Fetal Gene Program. Hypertrophy is accompanied by significant remodelling on multiple platforms (Figure 1). It is well established that PhH and PaH display a remarkable difference in their transcriptional profiles and molecular signatures [60,61]. The onset of the pathological form is accompanied by a transient activation of immediate-early genes (e.g. c-fos, c-myc and c-jun) and a subsequent re-induction of a suite of genes often designated the “fetal gene program” (FGP) [62-64]. The FGP facilitates a coordinated growth of cardiomyocytes by promoting the synthesis of certain proteins and altering the metabolism of the cell accordingly. It evokes multiple transcriptional alterations of genes encoding for contractile/cytoskeletal proteins (e.g. β-MHC/α-MHC ratio increase and upregulation of skeletal α-actin, ALC-1, and N2BA titin),

Ca2+-handling proteins (e.g. downregulation of SERCA2), peptide hormones (e.g. upregulation of ANP and BNP), and transcription factors (e.g. upregulation of GATA4, NKX2-5, MEF2, SP1, and HAND1/2) [64,65]. Although the current view holds a maladaptive role for the GFP in the adult heart due to its strong association with a decline in cardiac function, it has been argued that it may be a default transcriptional profile (much like a standard operating procedure) adopted to enhance cardioprotection in the context of stress [66,67]. For instance, although the change in the myosin heavy chain (MHC) composition towards the β-isoform reduces the shortening velocity, it also reduces energy consumption due to the lower ATPase activity of β-MHC compared to α-MHC [68]. Similarly, atrial essential light-chain myosin 1(ALC-1) and skeletal α-actin are associated with an increased contractility [65,69]. The hormones atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) also function as cardioprotectors by reducing the preload and afterload on the heart [70]. Conversely, although the reduction in shortening velocity is likely the initial foray into largescale adverse changes that facilitate the transition to heart failure [71]. The disruption in Ca2+ homeostasis is also detrimental, as the ion is highly toxic and intracellular levels are tightly regulated [71]. Therefore, the FGP likely functions as a cardioprotective mechanism during acute stress but may also be key to the modus operandi of the catastrophic transition from hypertrophy to heart failure. 1.3.2. Ionic Remodelling. Amongst the battery of detrimental

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changes discussed above, PaH also instigates an electrical remodelling of the heart underlain by an altered expression of sarcolemmal and sarcoplasmic ion channels and transporters. One of the main disruptions occurring in hypertrophy is a dysregulation of Ca2+ homeostasis [72]. Numerous studies agree that there is a prolongation of the action potential duration in hypertrophic hearts; however, it appears that the mechanism at play depends on the animal model used and the inciting hypertrophic stimulus. While it has been suggested that there is an inverse relationship between the magnitude of the L-type Ca2+ current (ICa-L) and the phenotypic severity of hypertrophy, other studies challenge the ab-initio notion that it is accompanied with an increased ICa-L [72,73]. For instance, in a feline animal model with aortic banding-induced hypertrophy (chronic pressure-overload), ICa-L density was unchanged but the inactivation kinetics of the L-type Ca2+ channel were slower, thereby protracting the action potential [74]. Conversely, the delayed rectifier K+ current was smaller with slower and faster activation and inactivation kinetics, respectively, also lengthening the action potential due to a reduced repolarizing current [74]. Another study reported no change in ICa-L density or channel kinetics accompanied by a decreased density of the inward rectifier K+ channels in spontaneously hypertensive rats [75]. Interestingly, the results of another study on spontaneously hypertensive rats are consistent with a trend of decreasing apparent ICa-L density as a function of age, albeit due to an increased in membrane capacitance without a parallel change in the absolute ICa-L or channel kinetics. The study also showed that a decreased in the transient outward K+ current (Ito) is responsive for the prolongation of the action potential duration [76]. In support, an increase in membrane capacitance has been previously described in a guinea pig model of aortic constrictioninduced hypertrophy [77]. Moreover, other studies suggested that the prolongation of the action potential duration is governed by a fine balance between Ito and ICa-L and that the voltage-gated Ca2+ channels are reverted to the fetal isoform post infarction [78,79]. This variability in the mechanisms of ionic remodelling led some investigators to conclude that ICa-L is increased in mild to moderate hypertrophy and unvaried or decreased in its severe forms [73]. However, a dependence on the type of hypertrophy is most likely a crucial determinant as well. Besides the alterations in ion channels, the hypertrophic remodelling also incorporates changes in ion transporters, which are essential to normal cardiac function. The sodium-calcium exchanger (NCX) is an electrogenic transporter that, under resting conditions, conducts one positive charge into the cell by exchanging a Ca2+ ion for three Na+ ions [72]. During phase 0 of the cardiac action potential or under excitotoxic conditions, NCX can function with a reverse directionality; this allows it to contribute positively to inotropy during systole and, significantly, to lusitropy during diastole [80-83]. In the compensated phase of a mouse model of pressure-overload hypertrophy, a study reported an upregulation of NCX1 mRNA and protein levels along with an apparently paradoxical decrease in NCX activity (INCX) [84]. On the contrary, other evidence points to the downregulation of NCX in a mouse model of neurohumorally-induced hypertrophy (enforced expression of Gαq) [85]. Regardless, the reduced activity of NCX in both models results in a consequent blunting of Ca2+ efflux, causing intracellular accumulation. Additionally, the activity of the sodium-proton exchanger (NHE) 1, an electroneutral transporter that extrudes pro-

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tons, is enhanced in the hypertrophic myocardium [86]. In turn, the increase in intracellular Na+ furthers the accumulation of Ca2+ in an NCX-mediated mechanism [86,87]. Due to a reduced expression of SERCA2 during hypertrophy, a depressed Ca2+ extrusion may result in the concentration of Ca2+ in the cytosol in lieu of the sarcoplasmic reticulum [64]. Ca2+ is a ubiquitous ion implicated in a vast array of cell signalling cascades; its effectors include mitogen-activated kinases, protein kinase C, and calcineurin, which are notorious for their pro-hypertrophic effects [72]. Collectively, the aforementioned evidence corroborates the centrality of Ca2+ homeostasis in the pathophysiology of hypertrophy. 1.3.3. Functional Aberration. As opposed to PhH, the pathological type is associated with necrosis, fibrosis, angiogenic dysregulation, endothelial dysfunction, and extracellular matrix derangements [33,88-91]. Fibrosis stiffens the myocardium and contributes significantly to the initiation and exacerbation of impairment in contractility and relaxation [1,33]. Along with the unfavored changes in the composition of the extracellular matrix, fibrosis also leads to a disruption in cardiac electrophysiology [92]. An excessive fibrillar collagen network promotes arrhythmogenicity due to anatomical uncoupling, the induction of a zigzag pattern of wavefront propagation, and the slowing of conduction owing to the activation of cardiomyocytic channels by a direct myofibroblastexerted mechanical force [92]. The depression in angiogenesis and the reduction in capillary density (concomitant with fibrosis) result in an ischemic state that aggravates cardiac function and possibly contributes to the progression of heart failure [33]. 1.4. Metabolic Remodelling Normally, the heart is a highly efficient pump that can circulate on average, about one ton of blood using only twice its weight of ATP every 2.4 hours [93]. With an ATP pool sufficient to energize less than 10 contractions, the myocardium evolved a promiscuous metabolic system that can shift is reliance onto various substrates [93]. In order of decreasing preference for utilization, these substrates include free fatty acids, glucose, ketones, pyruvate, lactate, amino acids, and cellular proteinaceous components [94]. In an adult non-pathological heart under resting conditions, 60-70% of biochemical energy is generated via fatty acid oxidation (FAO); the remaining fraction is produced symmetrically by the metabolism of glucose and lactate [33,95]. During fetal development, the contribution of the aforementioned substrates is reversed. For instance, in the fetal lapine heart ATP synthesis is fueled mainly by glycolysis (44) while FAO contributes to less than 15% of energy production; the contribution of glycolysis drops to 7% postnatally [96]. In a non-pathological heart under dynamic conditions, cellular metabolism can vary in accordance with fluctuations in the workload and the concentration of substrates in the bloodstream [33]. Therefore, in a condition such as PaH it follows that re-induction of the FGP results in a re-distribution of substrate-dependence towards that present in the fetal heart. 1.4.1. Shift in Metabolic Substrate. Metabolism is yet another dimension visited by the remodelling process pertinent to cardiac hypertrophy, albeit the relationship might have a bidirectional causality [97]. Consistent with other forms of remodelling, a plenitude of studies demonstrated that PaH is associated with the molding

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O-GlcNAcylation in the Pathological Heart: A Nexus of Hypertrophic Circuitry?

of the cardiac metabolic profile back into the fetal form. The hypertrophic heart displays a shift of substrate utilization towards glucose, a potentially protective transition against hypoxic conditions [98-102]. The underlying mechanism involves an accelerated glycolysis, as per isotopic labelling studies, and an inhibited FAO by the modulation of key enzymes that facilitate each process [103,104]. For instance, the key glycolysis-related proteins GLUT1, HK2, PFK, GAPDH, and PDK4 are all up-regulated in the hypertrophic heart [105]. In contrast, the FAO-related PPARα, PPARβ/δ, PPARγ co-activator-1, and CPT1 are downregulated, whereas PPARγ and its effector genes GPAT and GPDH are upregulated [105,106]. Such a bidirectional regulation of lipid catabolism and anabolism results in triglyceride accumulation in cardiomyocytes and consequent steatosis [105]. 1.4.2. Glycolytic Uncoupling. In the pathological heart, not only is there a change in substrate preference but also a change in substrate catabolism. In PaH, glycolysis favors glycogenolyticallyderived glucose over an exogenous supply and appears to be accelerated without a pari passu increase in pyruvate oxidation [107]. This metabolic incongruence has been termed “glycolytic uncoupling” and a study that sought to examine its teleological explanation reported that it functions as a cardioprotective mechanism against oxidative stress during chronic pressure-overload [108,109]. However, it is likely that this phenomenon is also a critical maneuver for the attenuation of the hypertrophy-associated disruption in Ca2+ homeostasis: glycolytically-derived ATP is preferentially utilized by ion pumps (e.g. SERCA) over that derived from oxidative phosphorylation presumably due to a functional compartmentalization of ATP synthesis [108,110]. In fact, enhancing glucose metabolism or uptake in genetically engineered mice protects them from cardiac dysfunction and dilatation in response to pressure-overload and ameliorates heart failure in clinical studies [111-113]. Depressing glucose utilization induces hypertrophy and exacerbates the prognosis of aortic constriction [114,115]. This led to the proposition that, similar to the changes in the composition of contractile proteins, the metabolic reversion to the fetal form during hypertrophy may potentially also be adaptive, at least in the short term [116]. 1.5. Untoward Clinical Sequelae 1.5.1. Heart Failure. Left ventricular hypertrophy is a sinister harbinger of morbidity and mortality, heralding imminent heart failure of almost all forms [117,118]. Heart failure is a debilitating complex clinical syndrome that exacts significant public and economic burdens and serves as an umbrella term encompassing several cardiovascular diseases that contribute to its pathogenesis [119]. It is characterized by a hemodynamic insufficiency, usually of myocardial origin, concomitant with a constellation of signs and symptoms such as fatigue, dyspnea and edema [120]. At present, heart failure afflicts about 5.7 million Americans over 20 years of age with a projected 46% increase in 2030 and, in 2012, its economic toll was $30.7 billion [121]. 1 in 9 deaths are associated with the syndrome and approximately 50% of its patients die within 5 years of diagnosis [121]. The molecular mechanisms mediating the culmination of PaH in heart failure are obscure and, currently, a subject of intense investigation. However, the transition process has been traditionally

Review Articles

subsumed into two major stages, the compensatory phase and the subsequent decompensatory phase [1]. During the former, the hypertrophic response normalizes wall stress to preserve the cardiac ejection fraction [1]. In the instance of an unremitting pathological stimulus, the ventricle becomes increasingly dilated—the decompensatory phase: ventricular dilation leads to an elevation in wall stress which reinforces the biomechanical hypertrophic stimulus initiating cell death and further ventricular dilation (a positive feedback loop) [122]. The fibrotic replacement of dead cardiomyocytes then exacerbates pump dysfunction and provides an arrhythmogenic substrate [122,123]. For example, chronic systemic hypertension induces left ventricular dilation which eventually gives rise to right ventricular dysfunction and global heart failure [124]. Cardiomyocytic degeneration and death are the hallmarks of the decompensatory phase in the failing heart. Cardiomyocytes die either via necrosis (or oncosis), apoptosis or autophagy [122]. The necrosis or ischemic core pathophysiological hypothesis proposes that due to an increased cellular cross-sectional area and reduced oxygenation of cardiac tissue (see 1.3.3. Functional Aberration), the mitochondria, situated at the core of the cell, become hypoxic causing cell death [122]. In the programmed death hypothesis, hypertrophy is associated with the activation of a stimulus-specific apoptotic pathway as opposed to the inhibition of a pro-survival pathway [122]. Finally, the autophagy hypothesis invokes that additional stresses (e.g. reduced oxygenation) on a cell with an accelerated protein turnover rate, as is the case in hypertrophy, can tip off the balance of the lysosomal and/or ubiquitin-proteasome systems towards autophagy resulting in cell death [122]. The evidence, relevant molecular mechanisms, and attendant strengths and weaknesses of each hypothesis has already been discussed in an exquisite review and, therefore, will not be discussed in this article [122]. 1.5.2. Arrhythmia. Hypertrophy is also an independent predictor of myocardial infarction, arrhythmia and sudden cardiac death [117]. There is a 3.4-fold and a 2.8-fold higher risk of developing supraventricular and ventricular tachycardia in patients with a left ventricular hypertrophy, respectively, which lent a strong impetus for mechanistic studies [125]. Since a heterogeneous prolongation of action potential duration is a well-documented repercussion of hypertrophic remodelling, it has been suggested to be the main mechanism underpinning the increased prevalence of early and delayed after depolarization arrhythmia [125]. As discussed above, fibrosis can also provide the substrate for a re-entrant arrhythmia via slowing and fractionating conduction [125]. Due to its numerous clinical sequelae, current research is aiming at inhibiting the progression or inducing the regression of PaH. An alternative therapeutic avenue is to contrast the molecular signatures of PhH and PaH in pursuance of a protracted physiological adaptation to a pathological state. Mainly because of the biophysical compensatory role the Law of LaPlace engraved for hypertrophy in the literature, only recently did the focus shift towards the elimination of hypertrophy in toto. In fact, numerous studies demonstrated that hemodynamic overload does not necessitate hypertrophic growth [126-129]. Thus, further investigation is warranted to elucidate the mechanisms of hypertrophic growth and the progression to heart failure. The urgency to develop new therapeutic strategies is highlighted due to the large contribution

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O-GlcNAcylation in the Pathological Heart: A Nexus of Hypertrophic Circuitry?

of these clinical phenomena to the mortality rate of the population despite the current treatments. For instance, recent reports demonstrated that O-GlcNAcylation imposes a significant effect on cardiac function, especially during pathological stress. The glycosylation seems to differentially promote cardioprotection or cardiotoxicity depending on certain parameters such as the time course of the pathology (acute vs. chronic) and the total levels of the glycosylated proteins (high vs. low) [15-19]. Therefore, being a substantial mechanism that casts influence on global cardiac function, its role and potential mechanistic pathways will be discussed in the next section.

2. O-GlcNAcylation

2.1. Biosynthesis and Regulation 2.1.1. O-GlcNAcylation as a Signalling System. Posttranslational modification is a rapid biological mechanism that is critical to the fine-tuned orchestration of almost all facets of cellular function. It involves a cleavage or covalent modification of proteins that alters their function, activity, subcellular localization and/or stability [18]. Examples of such modifications include acylation, acetylation, ubiquitination, methylation, thiolation, nitration, and glycosylation [18]. The latter classically occurs in the endoplasmic reticulum and the Golgi apparatus during the synthesis of glycoproteins and glycolipids, which are usually secreted or embedded in membranes [18]. Discovered by Torres and Hart in 1984, O-GlcNAcylation is a type of reversible O-linked glycosylation that occurs on serine and threonine residues [16,130]. However, O-GlcNAcylation differs from classical glycosylation in many aspects: O-GlcNAcylation modifies nucleocytoplasmic and mitochondrial proteins, is rapidly cycled and generally not elongated in metazoans, and lacks an identified consensus sequence [17]. In fact, its modification of over 1000 proteins, responsiveness to biological stimuli, importance to organismal function, and ability to regulate phosphorylation status (via competitive occupancy or steric hindrance) led to its classification as a signalling system [131,15]. Of note is that the accumulating evidence pointing to a reciprocal relationship between phosphorylation and O-GlcNAcylation trichotomizes signalling at a given serine or threonine residue (phosphorylation, O-GlcNAcylation or neither) which further convolutes the cellular interactome and confounds the results of serine/ threonine site-directed mutagenic approaches [131]. 2.1.2. The Hexosamine Biosynthetic Pathway and its Regulation. O-GlcNAcylation is characterized by the addition of the nucleotide sugar UDP-GlcNAc which is the product of the HBP [17]. The HBP, an accessory biochemical route to glucose metabolism, commences with the conversion of fructose-6-phosphate to glucosamine-6-phosphate by the rate-limiting enzyme L-glutamine-D-fructose 6-phosphate amidotransferase (GFAT) [17]. Fructose-6-phosphate is a glycolytic intermediate that is channelled into the HBP at an estimated rate of 2-5% [18]. Other substrates that feed into the HBP include glutamine, glucosamine, N-acetylglucosamine and acetyl-CoA, which links it to amino acid and lipid metabolism in addition to that of glucose [17,15]. After the generation of UDP-GlcNAc, the enzyme O-GlcNAc transferase (OGT) catalyzes the O-linked attachment of the sugar to serine and threonine residues [17]. It then follows that the O-GlcNAcome, defined as the collection of O-GlcNAcylated proteins at a given set

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of biological conditions, is proximally regulated by the functional balance between OGT and O-GlcNAcase (OGA)—the enzyme that catalyses the removal of the sugar moiety [132]. OGT has only three splice variants, namely nucleocytoplasmic (ncOGT), mitochondrial (mOGT) and short (sOGT) [15]. Similarly, OGA has a short isoform (sOGA) and a long isoform (lOGA) [15]. The large divergence of cellular effects from the activity of merely a few enzymes suggests that a substantially intricate regulatory system converges onto them so as to render the remarkable specificity required: for instance, the human phosphoproteome is directly controlled by at least 518 protein kinases and 255 phosphatases [133]. This nominates these two enzymes to being a bona fide nexus of cellular signalling. Unfortunately, the complex upstream regulation of O-GlcNAcylation remains largely unknown [15]. However, multiple studies reported that OGT expression, activity, localization and specificity can be modulated by a number of phosphoregulators and interacting proteins (e.g. tyrosine kinase, p38 mitogen-activated protein kinase, and myosin phosphatase) and by auto-O-GlcNAcylation [134-139]. OGT is particularly sensitive to intracellular UDP-GlcNAc, allowing it to act as a metabolic sensor as elaborated above; however, little is known about the regulation of the HBP enzymes upstream [18]. It is also thought that OGA is regulated by phosphorylation and protein-protein interactions, albeit the evidence is scarce [17]. For instance, one study reported that caspase-3 cleaves OGA without altering its enzymatic activity [140]. Due to the control of O-GlcNAcylation by a handful of enzymes, it is probable that the mainstay of regulation is via altering subcellular localization and/ or substrate specificity. Interestingly, OGT and OGA can form a single O-GlcNAczyme complex that likely accelerates the cycling of the glycosylation, which further substantiates the nature of O-GlcNAcylation as a signalling system [141]. 2.2. Role of O-GlcNAcylation in Cardiac Hypertrophy O-GlcNAcylation is omnipresent, exerting an influence on nuclear transport, gene expression, protein synthesis and degradation, signal transduction, cell structure, programmed death, and proliferation [19]. However, the overarching role of O-GlcNAcylation emerges as a broad cytoprotective mechanism that provides a basal enhancement of cellular viability in the context of various forms of stress [19]. For instance, it protects against oxidative stress, reduces inflammation, elevates heat tolerance, and attenuates Ca2+ overload [15]. Not surprisingly, aberrations in O-GlcNAc levels are associated with long-term cell death-related conditions such as cancer, neurodegenerative diseases, ageing and diabetes [19]. An ever-growing list of studies has demonstrated a fundamental role of the glycosylation in the pathological heart, and this has been examined in a number of excellent reviews [15-19]. However, the role of O-GlcNAcylation in cardiac hypertrophy specifically is relatively understudied, and given the strong association of left ventricular hypertrophy with pervasive high-mortality clinical phenomena, this topic will be dissected in the following sections. Inferring from its role in cell stress, O-GlcNAcylation could be the Achilles’ heel of hypertrophy and, by extension, of heart failure which would allow this work to lay novel avenues for potential inroads in combating the syndrome.

Journal of Undergraduate Life Sciences • Volume 11 • Issue 1 • Spring 2017


O-GlcNAcylation in the Pathological Heart: A Nexus of Hypertrophic Circuitry?

2.2.1. General Role 2.2.1.1. Global Cardiac Function. As outlined in 1.4. Metabolic Remodelling, a shift towards anaerobic glucose metabolism is one of the hallmarks of PaH. Conversely, in PhH, oxidative metabolism (fatty acids and glucose) is enhanced while glycolytic rates are decreased [142]. A logical corollary is that increased glycolytic flux can augment the HBP due to an increase in its substrate, fructose-6-phosphate [143]. In fact, numerous studies described a global increase in O-GlcNAcylation in response to pathological hypertrophic stimuli including senescence, diabetes and hemodynamic overload [144-148]. On the other hand, PhH is generally associated with unaffected or decreased O-GlcNAcylation [149151]. Such global changes in the O-GlcNAcome are evoked by the modulation of the expression, activity, or specificity of OGT and/or OGA or even the alteration of HBP flux by regulating its enzymes, such as GFAT [145]. Diabetic cardiomyopathy is a myocardial disorder that encompasses both metabolic dysregulation and cardiac dysfunction and is associated with hypertrophy and impending heart failure [152]. As O-GlcNAc is a major nutrient sensor and contributes significantly to cardiac physiology, its role in diabetic cardiomyopathy has been a subject of considerable research. A recent study by Marsh et al. explored this relationship by inducing PaH in a murine model of diabetes: it was shown that angiotensin II and phenylephrine increased the levels of the hypertrophic markers ANP and α-actin in non-diabetic mice, but not in their diabetic littermates [153]. This repression of hypertrophic signalling was mimicked in nondiabetic mice by inhibiting OGA or administering glucosamine or glucose, and it was partially abrogated in diabetic mice upon the inhibition of GFAT [153]. Conversely, in cultured neonatal rat cardiomyocytes, Ding et al. reported a hyperglycemia-induced upregulation of the fetal gene program and exacerbation of hypertrophy [154]. This response was abrogated by OGT siRNA and mimicked by the application of the OGA inhibitor PUGNac [154]. In addition to the different animal model Ding et al. used, they also studied the neonatal cardiomyocytes, which have been shown to be phenotypically and functionally different from terminally differentiated mature cardiomyocytes in a number of studies [154157]. Whereas the study by Marsh et al. utilized db/db mice with a fasting blood glucose concentration of 21 mM, Ding et al. exposed the rat neonatal cardiomyocytes to 30 mM of glucose for 72 hours [153-154]. This reveals that the chronic exposure to moderate hyperglycemia instills an O-GlcNAcylation-mediated basal level of cardioprotection against hypertrophic signalling in diabetes, albeit via only a limited number of pathways, since the hyperglycemialinked pro-apoptotic protein p53 had a similar expression in both cohorts, diabetic and non-diabetic, of the study by Marsh et al [153]. These studies also indicate a differential role of O-GlcNAc signalling depending on the time course of the stimulus. On the other end of the spectrum, the relationship between O-GlcNAcylation and exercise or PhH has also been a subject of substantial investigation. While the majority of the literature suggests that PhH or physical exertion is associated with a decrease in global O-GlcNAcylation, a study by Cox and colleagues demonstrated otherwise [158]. In this study, type 2 diabetic db/db and non-diabetic mice underwent a moderate treadmill exercise; after a 4-week exercise, the diabetic mice exhibited a decrease in global

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O-GlcNAcylation [158]. In contrast, a study by Bennett and colleagues reported that chronic exercise in mice improved diastolic function, reduced heart rate, and rescued the diabetes-induced increase in O-GlcNAcylation independent of the hyperglycemic state [159]. Of note is that as opposed to the db/db mice model and the treadmill exercise in the aforementioned study, Bennett et al. used the β-cell toxin streptozotocin to induce diabetes, and swimming, a more intense exercise, to study the relationship [159]. Other studies with opposite findings to that of Cox et al. have also utilized chronically swum mice to study the effect of PhH on global O-GlcNAcylation, but used non-diabetic mice as an animal model [149,151]. Contrariwise, in a form of compensated hypertrophy developed following Myc induction, O-GlcNAcylation levels were increased [160]. Therefore, in addition to calling attention to the dependence of O-GlcNAcylation on the experimental methodology, it is imperative to highlight the possibility that measuring global rather than protein-specific O-GlcNAcylation is futile: this type of glycosylation meets the requirements to being a signalling mechanism, and measuring its general levels parallels that of measuring total cellular phosphorylation in response to varying stimuli. 2.2.1.2. Compensation and Decompensation. O-GlcNAcylation has also gathered much attention after it has been shown to impose a significant effect on the heart during a hypoxic period, which is consistent with its role as a metabolic sensor. This led many studies to focus on how the glycosylation affects cell survival, proliferation and cell death, each of which contributes to the compensatory or decompensatory limbs of cardiac hypertrophy. In addition to its significance in the later period of the compensatory phase (i.e. ischemic core hypothesis), cell survival is also crucial during an episode of myocardial infarction, which is a very potent stimulus of hypertrophic growth: following an ischemic insult, the drop in cardiac output is counteracted by the activation of the sympathetic and renin-angiotensin-aldosterone systems which, along with the regional dilatation of the infarcted zone, subjects the noninfarcted zone to an elevated preload, afterload and neurohumoral stimulation [161,162]. In a recent study on bioengineered mice and isolated neonatal rat ventricular cardiomyocytes, Wang et al. identified spliced X-box binding protein 1 (Xbp1s) as the link between the HBP and the unfolded protein response in the milieu of ischemia [163]. The study first demonstrated that global O-GlcNAcylation and the transcripts of several HBP enzymes are prominently upregulated in the ischemic zone [163]. Second, they showed that Xbp1s not only promotes O-GlcNAcylation by upregulating several HBP enzymes, but also functions as an indispensable cardioprotector against ischemia/reperfusion injury (infarction area, contractility, fetal gene program expression, and hypertrophic growth) [163]. Thirdly, they reported that Xbp1s largely depends on GFAT1 to ameliorate cell death and enhance contractility (no observations on cardiac hypertrophy were reported) [163]. In all cases, Xbp1s and GFAT1 inhibition did not affect cardiac function under basal non-ischemic conditions [163]. Consistent with the cardioprotective role of O-GlcNAcylation in the context of myocardial infarction, Watson and colleagues reported a significant exacerbation of cardiac dysfunction in cardio-specific OGT-knockout mice following an ischemic insult but not a sham operation [164]. However, no significant hypertrophic growth was observed in hearts lacking OGT [164]. Therefore, the predominant role of O-GlcNAc signalling in

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O-GlcNAcylation in the Pathological Heart: A Nexus of Hypertrophic Circuitry?

acute myocardial infarctions emerges as a promoter of survival; however, its role in post-infarction hypertrophy remains elusive. Second, with regard to cellular proliferation, classical examples are Cyclin Ds which are members of the cyclin family of proteins that are known to regulate cell cycle progression [165]. They are implicated in almost all proliferative pathways and are mandatory for the completion of the G1 phase [165]. The upregulation of cyclin Ds in the hypertrophic heart is well-established (e.g. pressure-overload, volume-overload, post-myocardial infarction, and agonist-induced hypertrophy), and the inhibition of cyclin Ds or their downstream effectors Cdk4/6 has been shown to attenuate hypertrophy [165]. As expected considering the ubiquitous implication of O-GlcNAc signalling, the glycosylation has also been shown to interfere with cyclin expression: interestingly, OGT and OGA overexpressing cells both appear to downregulate cyclin D1 in vitro (various cell lines) [166]. In support, another study showed that altering the levels of OGT or OGA leads to the disruption of the mitotic spindle architecture [167]. These findings can be construed as that O-GlcNAcylation is a fine-tuning mechanism and a reserve signalling system that becomes increasingly relevant in periods of cellular stress. Hence, a substantial change in the O-GlcNAcome evoked by an alteration of OGA and/or OGT levels is indicative of elevated basal cellular stress which prevents the cell from progressing through the cell cycle. Recent evidence indicates that O-GlcNAcylation is also involved in regulating the response to DNA damage, lending further credence to above explanation [168]. However, in opposition, Ding et al. used OGT siRNA and OGA inhibition to demonstrate that O-GlcNAc signalling upregulates cyclin D2 in cultured rat neonatal cardiomyocytes [154]. As mentioned above (see 2.2.1.1. Global Cardiac Function) neonatal cardiomyocytes differ significantly from mature cardiomyocytes, and their mitogenic ability may alter the response of the cyclin proteins to O-GlcNAc signalling. Regardless, O-GlcNAcylation clearly plays a role in cell cycle progression; however, the collective evidence suggests that O-GlcNAcylation may promote the initiation of the cell cycle, resembling a growth response, but prevent complete progression possibly as a response to cellular stress. Lastly, with respect to cell death, another study was conducted on human fetal cardiomyocytes and reported that high glucose initiates caspase-3-mediated apoptosis concomitant with a temporal activation of p38 MAPK and nuclear O-GlcNAcylation [169]. Together with a report of caspase-3-mediated cleavage of OGA, the indirect findings point to the implication of the glycosylation in apoptosis [140]. In concordance, a study on rat cardiomyocytes demonstrated that a high glucose concentration leads to the O-GlcNAcylation of p53 followed by an increase in angiotensin II production and receptor accumulation (AT1) that culminates in a p38 MAPK-p53-mediated apoptosis [170]. This cascade was averted by using the non-specific O-glycosylation inhibitor benzyl 2-acetamido-2-deoxy-α-D-galactopyranoside [170]. Studies on other tissues also reported an HBP-dependent upregulation of angiotensinogen (angiotensin II precursor) by way of p38 MAPK activation [171,172]. Although the discussed evidence is inadequate to implicate the glycosylation in promoting apoptosis, they provide enough circumstantial evidence for further investigation. In agreement, Housley and colleagues identified the forkhead box protein (FoxO) 1, an important pro-apoptotic transcription factor, as a glucose-stimulated promoter of gluconeogenesis and oxidative cytoprotection that is targeted by O-GlcNAcylation

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in hepatocytes [173]. The study is germane to this review as it demonstrated that FoxO1 O-GlcNAcylation is stimulatory and is increased in diabetic rats [173]. Whereas gluconeogenesis is irrelevant to the heart, detoxification of reactive oxygen species via the upregulation of catalase and manganese superoxide dismutase is likely cardioprotective in the context of reperfusion injury as well as elevated oxidative metabolism [173]. FoxO proteins are also implicated in cell cycles, apoptosis, general proteolysis, and longevity, which further links the O-GlcNAcylation of FoxO1 to cardiac hypertrophy [173,174]. In fact, a study on cultured neonatal rat ventricular cardiomyocytes reported a FoxO1/3-driven reduction in calcineurin activity and upregulation of atrogin-1, an E3 ubiquitin ligase that promotes calcineurin proteolysis [175]. Consistent with the above, FoxO1 overexpression blunted angiotensin II-induced hypertrophy and FoxO3-null mice developed hypertrophy under basal conditions [175]. Interestingly, FoxO is inactivated by hemodynamic overload and PI3K/Akt-dependent agonists such as angiotensin II and phenylephrine [175]. Using the same animal model, another study reported a phenylephrine-mediated activation of FoxO1 [176]. The conflicting results are likely due to the different doses of phenylephrine used (10 μM vs. 50 μM, respectively): phenylephrine activation of Akt has been shown to be largely time course-dependent and dose-dependent [175-177]. Of note is that the activation of FoxO1 by O-GlcNAcylation is not necessarily equivalent to its classical disinhibition by dephosphorylation. However, since the mutation of select phosphorylation sites on FoxO accentuates its O-GlcNAcylation, it is probable that the protein is subject to reciprocal regulation by kinases and OGT as observed with certain proteins, but further investigation is required to validate this claim [173]. Regardless, FoxO proteins appear to be highly relevant to hypertrophy and their complex activation patterns and pro-apoptotic effects may contribute to the transition of neurohumorally-induced hypertrophy to heart failure. To further complicate matters, it is well-established that the overexpression of mOGT, the mitochondrially-targeted OGT isoform, is cytotoxic and triggers apoptosis [178]. In contrast, the O-GlcNAcylation of the voltage-gated anion channel is suggested to hamper the Ca2+ overload- and oxidative stress-induced formation of the mitochondrial permeability transition pore (mPTP) [179,180]. The mPTP is known to initiate an unregulated ingress and egress of large molecules thereby destroying the cellular energetic supply and triggering apoptosis [179]. Therefore, it seems that in the context of acute cellular stress, O-GlcNAcylation emerges, again, as a mechanism of cardioprotection. However, as suggested by Darley-Usmar and colleagues, under chronic pathological conditions such as diabetes, O-GlcNAcylation likely leads to cell death by way of modulating mitochondrial function [19]. 2.2.2. O-GlcNAcylation in Molecular Remodelling. From this subsection onwards, the role of O-GlcNAcylation will be discussed in the context of the specific types hypertrophic remodelling discussed in the first synoptic segment of this review. As briefly described in 1.3.1. The Fetal Gene Program, the FGP mediates largescale phenotypic changes in cardiomyocytes with onset of a pathological hypertrophic stimulus. The repressor element 1-silencing transcription factor (REST), also known as the neuron-restrictive silencer factor (NRSF), is a transcription regulator that complexes with mSin3A and various histone deacetylases (HDACs) to drive

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O-GlcNAcylation in the Pathological Heart: A Nexus of Hypertrophic Circuitry?

epigenetic alterations and chromatin remodelling thought to repress the FGP [181,182]. In fact, mice with a dominant negative mutant of REST exhibit dilated cardiomyopathy with eccentric hypertrophy concomitant with an increased risk of arrhythmia and sudden cardiac death [182]. Therefore, due to the importance of REST in regulating cardiac function, a series of studies examined the effect of a Western diet (increased simple carbohydrate and saturated fat consumption) on the heart in the context of O-GlcNAcylation and the REST complex. It was first established that a chronic 12-month Western diet in the rat increases O-GlcNAcylation of large proteins with no discernible abnormalities in the heart (e.g. hypertrophy, Ca2+ mishandling and cardiac dysfunction), although subtle remodelling was not examined [183]. The evidence suggests that the elevation in O-GlcNAcylation merely reflects the increase in the HBP substrate [183]. A later study demonstrated that OGT complexes with REST and mSin3A and glycosylates a number of HDACs in mice [150]. In addition, it was shown that acute exercise resulted in a reduction of OGT auto-O-GlcNAcylation and OGT/ REST interaction and a differential alteration of the glycosylation in the nuclear (small proteins) and cytoplasmic compartments [150]. Rationalized by their findings as well as the known exerciseinduced hypertrophy-initiating nuclear ejection of class II HDACs, the investigators then proposed a mechanism for the onset of PhH [150,184]. The mechanism suggests that a reduction in OGT autocatalysis dissociates it from the REST complex and induces the ejection of class II HDACs (repressors of hypertrophy) from the nucleus permitting the initiation of epigenetic modifications [150]. Hinging on the above results, a subsequent study demonstrated that a 2-week Western diet increases REST protein levels leading to a suppression of the FGP with a bias of nuclear O-GlcNAcylation towards larger proteins (as opposed to acute exercise) [181]. The study also reported a differential auto-O-GlcNAcylation of OGT isoforms [181]. A separate group of investigators studying db/db mice demonstrated moderate treadmill exercise reduced protein levels of mSin3A and HDAC1/2 and increased the associations of mSin3A and OGT with HDAC1 and HDAC2, respectively, without altering the expression of the FGP (e.g. skeletal α-actin, ANP, and BNP) [158]. While these findings re-inforce the role of O-GlcNAcylation and the REST/mSin3A/HDAC complex as proposed by Medford et al. above, they do not demonstrate their role in altering the expression of FGP during chronic exercise [150,158]. Therefore, O-GlcNAcylation is implicated in PhH and PaH and is affected by an intricate network of regulators that affect its specificity and, possibly, its localization in a manner dependent on the time course and nature of the stimulus (chronic or acute). Of note is that the presence of O-GlcNAcylated proteins in the nuclear or cytoplasmic compartments may reflect an OGT-induced translocation rather than a change in the localization of OGT itself. Besides the REST complex, a number of transcription factors that regulate hypertrophic remodelling have also been shown to be O-GlcNAcylated. For instance, the myocyte enhancer factor 2 (MEF2) family of transcription factors is a key participant in hypertrophic signalling [185]. MEF2-binding sequences have been found within the promoter regions of multiple contractile, hypertrophy-related and FGP-affected genes such as α-MHC, myosin light-chain 1/3 and 2v, skeletal α-actin, SERCA2a2, cardiac troponin T and C, desmin and dystrophin [185]. Whereas multiple members of the MEF2 family have already been identi-

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fied as targets of O-GlcNAcylation, only the functional significance of MEF2D1a modification has been elucidated: in skeletal muscle, O-GlcNAcylation of MEF2D1a inhibits its recruitment to the myogenin promoter thereby inhibiting myogenesis [186,187]. Moreover, NK-2 transcription factor related, locus 5 (NKX2-5/ Csx) is another O-GlcNAcylation target that promotes a number of cardiac fetal and hypertrophic genes; an increase in global O-GlcNAcylation is reported to decrease NKX2-5 expression and protein levels [185,188]. Thus far, together with the orchestrated gene silencing by OGT-mSin3A-mediated repression and histone deacetylation, the above findings imply that O-GlcNAcylation seemingly suppresses FGP expression [189]. However, further research is required to elucidate the effect of this glycosylation on the FGP (including unexamined genes such as MEF2A) during direct hypertrophic stimulation. 2.2.3. O-GlcNAcylation in Electrical and Functional Remodelling. The role of O-GlcNAcylation in cardiac electrical homeostasis is poorly understood; only its ability to regulate the capacitative Ca2+ entry (CCE), a.k.a. store-operated Ca2+ entry, is well-established. As a case in point, Pang et al. further supported the concept of diabetes-associated cardioprotection by demonstrating that hyperglycemia curbs Ca2+-dependent hypertrophy to about 40% in rat neonatal cardiomyocytes [190]. Specifically, the study showed that hyperglycemia attenuated the agonist-induced CCE in an HBP-dependent manner and also inhibited the translocation of nuclear factor of activated T-cells (NFAT)—a widely-recognized transcription factor implicated in many types of PaH (e.g. pressure-overload and volume-overload) [190-193]. The implication of CCE in agonist-induced hypertrophic signalling, including NFAT translocation, and its inhibition by O-GlcNAcylation, has been reported a number of times in the literature [194-196]. However, only recently was a mechanistic study able to delineate the cascade to the O-GlcNAcylated protein: Zhu-Mauldin et al. reported that the acute induction of CCE results in stromal interaction molecule 1 (STIM1) puncta formation; STIM1 is a well-documented mediator of CCE [197]. The application of glucosamine or inhibition of OGA led to an increased O-GlcNAcylation of STIM1 concomitant with the prevention of puncta formation and the attenuation of CCE [197]. In another perspective, a related study revealed that the HBP-mediated suppression of CCE inhibits the positive inotropy elicited by PLC-dependent hypertrophic agonists, such as phenylephrine [198]. Additionally, enforced expression of OGA is thought to rescue contractility, relaxation, Ca2+ transients, and sarcoplasmic Ca2+ sequestration in diabetic mice [199]. However, the negative inotropic effect could also be explained at the level of the contractile proteins; the O-GlcNAcylation of myofilaments has been shown to reduce their Ca2+ sensitivity (EC50) without altering their maximal force (Fmax) or cooperativity (Hill coefficient, nHill) [200,201]. Accordingly, although the inhibition of CCE by O-GlcNAcylation hinders hypertrophic development and curbs short-term Ca2+ overload, it seemingly dampens the compensatory component of hypertrophy as well. Interestingly, however, it has been suggested that a decrease in myofilament Ca2+ sensitivity may be adaptive as it contributes to a reduced diastolic tension [202]. As discussed in 1.3.3. Functional Aberration, fibrosis is strongly associated with cardiac hypertrophy and plays a key role in arrhythmogenic susceptibility and electrical dysregulation. A previous

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O-GlcNAcylation in the Pathological Heart: A Nexus of Hypertrophic Circuitry?

review has described a possible role of O-GlcNAcylation in cardiac fibrosis based on circumstantial evidence in glomerular mesangial cells involving HBP-dependent upregulation of TGF-β, a key participant in cardiac tissue remodelling and fibrosis [18,203,204]. More recent evidence reinforces the role of the glycosylation in mesangial cell fibrosis and also suggests the implication of fibroblast growth factor signalling in Drosophila [205,206]. However, another recent study conducted by Aguilar and colleagues directly addressed this issue in cultured rat cardiac fibroblasts and demonstrated that O-GlcNAcylation of Sp1 increases its recruitment to the collagen I α-1 promoter [207]. The study also showed that a decrease in O-GlcNAcylation likely mitigates fibrosis by reducing the protein levels of TGF-β, SMAD2/3, and collagen I and III and normalizing those of SMAD7 and arginase II [207]. Therefore, it appears that O-GlcNAcylation does indeed contribute to cardiac fibrosis, but further in vivo studies are required to ascertain this notion since fibrosis is a whole-organ form of hypertrophic remodelling and may not be faithfully recapitulated in vitro. 2.2.4. O-GlcNAcylation in Metabolic Remodelling. Similar to electrical and functional remodelling, studies on the role of O-GlcNAcylation in hypertrophic metabolic remodelling are lacking; however, a growing body of evidence is implicating it in the dysregulation and rewiring of metabolism in diabetes and cancer, respectively [208-210]. Based on the evidence from such studies, one might predict a potential role of the modification during cardiac hypertrophy. For instance, insulin resistance itself can lead to cardiomyopathy, independent of vascular disease: knocking down GLUT4 to 25% of its normal levels in the heart leads to the development of hypertrophy likely due to an increase in oxidative stress [211]. In turn, glucosamine infusion and GFAT overexpression have been shown to confer insulin resistance in multiple tissues such as muscle and fat [212-214]. The overexpression of OGT also abates insulin signalling and this effect is thought to be mediated by the O-GlcNAcylation of IRS-1 and IRS-2 and a reduction in GLUT4 translocation and Akt phosphorylation [19]. However, the involvement of the glycosylation appears to be more complex as the manipulation of OGA by pharmacological modulation and genetic bioengineering yields conflicting results in terms of insulin resistance [215]. A recent review suggested that a relationship of mutual dependence between the rival enzymes offers an explanation to this metabolic “OGT/OGA paradox” [215]. This notion is consistent with the fact that OGT and OGA form an O-GlcNAczyme that accelerates the recycling of the sugar moiety [141]. O-GlcNAcylation is also thought to play a role in the metabolic subsystems: from an anabolic perspective, the glycosylation appears to enhance gluconeogenesis (in the liver) and lipogenesis, but inhibit glycogenesis [215]. For example, O-GlcNAcylation of liver X receptor α increases the expression of the sterol regulatory element-binding protein 1c, an effector lipogenic gene [216]. Similarly, O-GlcNAcylation stabilizes carbohydrate responsive element-binding protein, a transcription factor of lipogenic genes [217]. An unbalanced enhancement of lipogenesis may lead to myocardial steatosis and consequent diastolic dysfunction; in fact, the enforced expression of OGA averts hepatic steatosis in diabetic mice [217,218]. On the other hand, O-GlcNAc addition to glycogen synthase and glycogenin is inhibitory suggesting that

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the glucose-stimulated glycosylation promotes the accumulation of intracellular glucose [219,220]. From a catabolic perspective, O-GlcNAcylation seemingly either retards or does not affect glycolysis. In the heart, glycolytic rate is not governed by a single enzyme; instead, the distribution of control on glycolytic steps varies dynamically depending on cellular conditions [221]. Mainly, three events limit glycolytic flux: glucose availability and the phosphofructokinase-1 and glyceraldehyde-3-phosphate dehydrogenase catalytic steps [221]. Glucose availability is dictated by its supply and phosphorylation, which are governed by glycogenolysis and GLUT4 flux (decreased during insulin resistance); and the hexokinase catalytic step, respectively [221]. The glyceraldehyde-3-phosphate dehydrogenase step is especially relevant during ischemic and oxidative stress [221]. Therefore, although OGT overexpression enhances the activity of hexokinase and lowers that of phosphoglycerate kinase and pyruvate kinase in tumor cells, one cannot draw a conclusion with regard to the effect on the overall flux [222]. However, using genetic bioengineering in a lung cancer cell line, it has been shown that OGT overexpression and OGA inhibition dampen glycolysis and lactate production, and that this effect is mediated by the activity-suppressing O-GlcNAcylation of phosphofructokinase-1 at serine 529 [222]. Conversely, glucosamine infusion of the intact heart and consequent elevation of intracellular O-GlcNAc does not seem to affect glycolytic flux or glucose oxidation but does decrease lactate and pyruvate oxidation [223]. The opposition in the findings is likely due to the difference in the approaches to elevating of O-GlcNAcylation, the time course of the experimental manipulation (chronic and acute, respectively) or the cell type and experimental conditions (in vitro and ex vivo, respectively). Therefore, no substantiated conclusion can be made with regard to the effect of O-GlcNAcylation on glycolysis in the heart. On another pertinent aspect of catabolism, studies are pointing to an O-GlcNAcylation-mediated augmentation of FAO. For example, Luo and colleagues found that chronic stimulation of the HBP by way of glucosamine treatment enhanced FAO in an AMPactivated protein kinase-dependent manner [224]. Mirroring these results, GFAT inhibition reduced FAO [224]. Likewise, an acute cardiac perfusion with glucosamine ex vivo increased palmitate oxidation and the membrane levels of FAT/CD36, a fatty acid transporter and, likely, a target of O-GlcNAcylalation [223,225]. In support of the above, a similar ex vivo study found that glucosamine enhances FAO in wild type but not spontaneously hypertensive rats; the findings of the study suggest that FAT/CD36 O-GlcNAcylation is playing a role [225]. In addition, glutamine also promotes FAO in an HBP-dependent manner [226]. Interestingly, however, recent evidence is implying an O-GlcNAcylation-mediated impairment of mitochondrial function (e.g. fragmentation and diminution of membrane potential) via an alteration in the respiratory chain complexes I, III and IV and translocation of dynamic-related protein 1 to the mitochondria [215]. These findings add a new twist to the role of the modification in metabolism and highlight the necessity of more studies that integrate multiple metabolic pathways. Therefore, the effect of O-GlcNAcylation on glucose metabolism and uptake requires further investigation in cardiac tissue: insulin resistance, which was demonstrated in other tissues, and the inhibition of glycolysis, which may be specific to tumor cells, reduce intracellular glucose, while parallel inhibition of glycogenesis

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O-GlcNAcylation in the Pathological Heart: A Nexus of Hypertrophic Circuitry?

causes accumulation, making any conclusion in this regard highly conjectural. In contrast, O-GlcNAcylation does appear to stimulate FAO which opposes the shift in metabolic substrate observed in hypertrophic hearts, but the significance of this phenomenon requires further investigation given a potential mitochondrial dysfunction occurring in conjunction. The implication of the glycosylation in the development of myocardial steatosis shows some promise and lays avenues for future research.

3. Conclusions

One of the salient conclusions that can be drawn from the literature is that O-GlcNAcylation is an atypical glycosylation that represents a novel form of signalling which seemingly affects countless cellular functions and, by extension, every aspect of cardiac hypertrophic remodelling. In so being, the glycosylation trichotomizes signalling at select serine/threonine residues incorporating a new dimension in the interactome and confounding certain site-directed mutagenic studies. This also strongly implies that O-GlcNAc-omicGlcNAcomic studies that solely examine the global levels of O-GlcNAcylation likely yield no precise information about the function of the signalling system in the context of a specific biological process. However, in general, this post-translational modification appears to function as a fine-tuning mechanism that maintains the cellular status quo through dynamic changes in nutrient availability. It also likely acts as a reserve signalling system that evokes multiple stress responses following the onset of various unfavorable conditions. Of note is that O-GlcNAcylation is not irrelevant or unimportant during rest conditions, as inhibiting the system during embryonic development is lethal, consistent with its effects on cell cycle progression [217]. More accurately, O-GlcNAcylation becomes increasingly important during acute cellular stress in cardiomyocytes, which undergo negligible mitotic division. The analyzed studies in this review imply that the role of O-GlcNAcylation in the heart is generally cardioprotective during acute stress but cardiotoxic during chronic stress, consistent with previous suggestions [16-19]. For instance, O-GlcNAcylation promotes cell survival during acute ischemia, Ca2+ overload, and likely mitochondrial swelling. Conversely, it promotes cell death during chronic hyperglycemia (e.g. FoxO activation), depresses contractile function (e.g. myofilament Ca2+ sensitivity), and enhances fibrosis, all of which potentially lead to heart failure in the long term. However, the issue is appreciably more complex than being explained as such. One example that complicates the matter is the likely inhibitory influence that O-GlcNAcylation imposes on the expression of the FGP, which itself is emerging as an acutely adaptive but chronically maladaptive phenomenon. The multiplex effects of the glycosylation on metabolism also lend credence to this notion: Although these effects might be explained by the variability in the experimental design of the studies from which they were concluded (e.g. tumor and non-cardiac tissues), if they were reproduced in a myocardial preparation, then O-GlcNAcylation might indeed be curbing the deviation of the cardiac metabolic profile from the adult form. Another case in point is the cardioprotection against hypertrophy that the glycosylation confers in diabetic subjects; such a condition is chronic but does not appear to promote a key predictor of heart failure—hypertrophy. Overall, sparse information exists about the role of O-GlcNAcylation in hypertrophy and the associated cardiac

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remodelling. However, based on the sum of studies examined, it can be concluded that the role of O-GlcNAcylation is highly context-dependent and cannot be summarized by a single general concept. This review emphasizes the need to appreciate the gravity of O-GlcNAcylation in cardiac hypertrophy, and summons more effort to decipher the regulatory network underlying the multifarious effects of this post-translational modification. The importance of such an approach would be to overcome the specificity conundrum impeding the potential translation of any advances to bedside: before resolving the modus operandi of the highly diverse, dynamic and responsive system that utilizes only a few enzymes, developing specific drugs would be extremely challenging. Other promising avenues include the further examination of the currently established O-GlcNAcylation targets to illuminate a previously unrecognized therapeutic approach in the context of specificity (e.g. OGT/REST complex interaction and OGT auto-O-GlcNAcylation). Moreover, investigators can screen an O-GlcNAcome database (such as the one developed by Wang et al.) using a gene ontology tool that can categorize by relevance to muscle hypertrophy before selecting proteins with a potential for therapeutic manipulation or that are closely related to other known O-GlcNAcylated proteins (e.g. MEF2A) [228-230].

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O-GlcNAcylation in the Pathological Heart: A Nexus of Hypertrophic Circuitry?

167. Tan EP, Caro S, Potnis A, Lanza C, Slawson C. O-linked N-acetylglucosamine cycling regulates mitotic spindle organization. J Biol Chem. 2013;288:27085-27099. doi: 10.1074/ jbc.M113.470187. 168. Zachara NE, Molina H, Wong KY, Pandey A, Hart GW. The dynamic stress-induced “O-GlcNAc-ome” highlights functions for O-GlcNAc in regulating DNA damage/repair and other cellular pathways. Amino Acids. 2011;40:793-808. doi: 10.1007/s00726-010-0695-z. 169. Li SY, Sigmon VK, Babcock SA, Ren J. Advanced glycation endproduct induces ROS accumulation, apoptosis, MAP kinase activation and nuclear O-GlcNAcylation in human cardiac myocytes. Life Sci. 2007;80:1051-1056. 170. Fiordaliso F, Leri A, Cesselli D, Limana F, Safai B, Nadal-Ginard B, Anversa P, Kajstura J. Hyperglycemia activates p53 and p53-regulated genes leading to myocyte cell death. Diabetes. 2001;50:2363-2375. 171. Hsieh TJ, Fustier P, Zhang SL, Filep JG, Tang SS, Ingelfinger JR, Fantus IG, Hamet P, Chan JS. 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Diabetes. 2004;53:1074-1081. 199. Hu Y, Belke D, Suarez J, Swanson E, Clark R, Hoshijima M, Dillmann WH. Adenovirusmediated overexpression of O-GlcNAcase improves contractile function in the diabetic heart. Circ Res. 2005;96:1006-1013. 200. Ramirez-Correa GA, Jin W, Wang Z, Zhong X, Gao WD, Dias WB, Vecoli C, Hart GW, Murphy AM. O-linked GlcNAc modification of cardiac myofilament proteins: a novel regulator of myocardial contractile function. Circ Res. 2008;103:1354-1358. doi: 10.1161/ CIRCRESAHA.108.184978. 201. Hedou J, Cieniewski-Bernard C, Leroy Y, Michalski JC, Mounier Y, Bastide B. O-linked N-acetylglucosaminylation is involved in the Ca2+ activation properties of rat skeletal muscle. J Biol Chem. 2007;282:10360-10369. 202. Varian KD. Cardiac myofilament calcium sensitivity in health and disease (doctoral dissertation). 2008. 203. Branton MH, Kopp JB. TGF-β and fibrosis. Microbes Infect. 1999;1:1349-1365. 204. Dobaczewski M, Chen W, Frangogiannis NG. Transforming growth factor (TGF)-β signaling in cardiac remodeling. J Mol Cell Cardiol. 2011;51:600-606. doi: 10.1016/j. yjmcc.2010.10.033. 205. Park MJ, Kim DI, Lim SK, Choi JH, Han HJ, Yoon KC, Park SH. High glucose-induced O-GlcNAcylated carbohydrate response element-binding protein (ChREBP) mediates mesangial cell lipogenesis and fibrosis: the possible role in the development of diabetic nephropathy. J Biol Chem. 2014;289:13519-13530. doi: 10.1074/jbc.M113.530139. 206. Mariappa D, Sauert K, Mariño K, Turnock D, Webster R, van Aalten DM, Ferguson MA, Müller HA. Protein O-GlcNAcylation is required for fibroblast growth factor signaling in Drosophila. Sci Signal. 2011;4:ra89. doi: 10.1126/scisignal.2002335. 207. Aguilar H, Fricovsky E, Ihm S, Schimke M, Maya-Ramos L, Aroonsakool N, Ceballos G, Dillmann W, Villarreal F, Ramirez-Sanchez I. Role for high-glucose-induced protein O-GlcNAcylation in stimulating cardiac fibroblast collagen synthesis. Am J Physiol Cell Physiol. 2014;306:C794-C804. doi: 10.1152/ajpcell.00251.2013. 208. Teo CF, Wollaston-Hayden EE, Wells L. Hexosamine flux, the O-GlcNAc modification, and the development of insulin resistance in adipocytes. Mol Cell Endocrinol. 2010;318:4453. doi: 10.1016/j.mce.2009.09.022. 209. Myslicki JP, Belke DD, Shearer J. Role of O-GlcNAcylation in nutritional sensing, insulin resistance and in mediating the benefits of exercise. Appl Physiol Nutr Metab. 2014;39:1205-1213. doi: 10.1139/apnm-2014-0122. 210. Jóźwiak P, Forma E, Bryś M, Krześlak A. O-GlcNAcylation and Metabolic Reprograming in Cancer. Front Endocrinol (Lausanne). 2014;5:145. doi: 10.3389/fendo.2014.00145. 211. Mellor KM, Bell JR, Ritchie RH, Delbridge LM. Myocardial insulin resistance, metabolic stress and autophagy in diabetes. Clin Exp Pharmacol Physiol. 2013;40:56-61. doi: 10.1111/j.1440-1681.2012.05738.x. 212. Hebert LF Jr, Daniels MC, Zhou J, Crook ED, Turner RL, Simmons ST, Neidigh JL, Zhu JS, Baron AD, McClain DA. Overexpression of glutamine:fructose-6-phosphate amidotransferase in transgenic mice leads to insulin resistance. J Clin Invest. 1996;98:930-936. 213. Hazel M, Cooksey RC, Jones D, Parker G, Neidigh JL, Witherbee B, Gulve EA, McClain DA. Activation of the hexosamine signaling pathway in adipose tissue results in decreased serum adiponectin and skeletal muscle insulin resistance. Endocrinology. 2004;145:2118-2128. 214. Virkamäki A, Daniels MC, Hämäläinen S, Utriainen T, McClain D, Yki-Järvinen H. Activation of the hexosamine pathway by glucosamine in vivo induces insulin resistance in multiple insulin sensitive tissues. Endocrinology. 1997;138:2501-2507. 215. Ruan HB, Singh JP, Li MD, Wu J, Yang X. Cracking the O-GlcNAc code in metabolism. Trends Endocrinol Metab. 2013;24:301-309. doi: 10.1016/j.tem.2013.02.002 216. Anthonisen EH, Berven L, Holm S, Nygård M, Nebb HI, Grønning-Wang LM. Nuclear receptor liver X receptor is O-GlcNAc-modified in response to glucose. J Biol Chem. 2010;285:1607-1615. doi: 10.1074/jbc.M109.082685.

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O-GlcNAcylation in the Pathological Heart: A Nexus of Hypertrophic Circuitry?

217. Guinez C, Filhoulaud G, Rayah-Benhamed F, Marmier S, Dubuquoy C, Dentin R, Moldes M, Burnol AF, Yang X, Lefebvre T, Girard J, Postic C. O-GlcNAcylation increases ChREBP protein content and transcriptional activity in the liver. Diabetes. 2011;60:1399-1413. doi: 10.2337/db10-0452. Epub 2011 Apr 6. 218. Rijzewijk LJ, van der Meer RW, Smit JW, Diamant M, Bax JJ, Hammer S, Romijn JA, de Roos A, Lamb HJ. Myocardial steatosis is an independent predictor of diastolic dysfunction in type 2 diabetes mellitus. J Am Coll Cardiol. 2008;52:1793-1799. doi: 10.1016/j. jacc.2008.07.062. 219. Parker GJ, Lund KC, Taylor RP, McClain DA. Insulin resistance of glycogen synthase mediated by o-linked N-acetylglucosamine. J Biol Chem. 2003;278:10022-10027. 220. Viep T, Chima-Okerke O, Le P, Khatra B. O-GlcNacylation of glycogenin inhibits glycogen synthesis. FASEB J. 2010;24:65915. 221. Depré C, Rider MH, Hue L. Mechanisms of control of heart glycolysis. Eur J Biochem. 1998;258:277-290. 222. Yi W, Clark PM, Mason DE, Keenan MC, Hill C, Goddard WA 3rd, Peters EC, Driggers EM, Hsieh-Wilson LC. Phosphofructokinase 1 glycosylation regulates cell growth and metabolism. Science. 2012;337:975-980. doi: 10.1126/science.1222278. 223. Laczy B, Fülöp N, Onay-Besikci A, Des Rosiers C, Chatham JC. Acute regulation of cardiac metabolism by the hexosamine biosynthesis pathway and protein O-GlcNAcylation. PLoS One. 2011;6:e18417. doi: 10.1371/journal.pone.0018417. 224. Luo B, Parker GJ, Cooksey RC, Soesanto Y, Evans M, Jones D, McClain DA. Chronic hexosamine flux stimulates fatty acid oxidation by activating AMP-activated protein kinase in adipocytes. J Biol Chem. 2007;282:7172-7180. 225. Lauzier B, Merlen C, Vaillant F, McDuff J, Bouchard B, Beguin PC, Dolinsky VW, Foisy S, Villeneuve LR, Labarthe F, Dyck JR, Allen BG, Charron G, Des Rosiers C. Post-translational modifications, a key process in CD36 function: lessons from the spontaneously hypertensive rat heart. J Mol Cell Cardiol. 2011;51:99-108. doi: 10.1016/j.yjmcc.2011.04.001. 226. Lauzier B, Vaillant F, Bouchard B, Legault JT, Dolinsky VW, Gelinas R, Dyck JRB, Chatham JC, Des Rosiers C. Glutamine modulates fatty acid metabolism in ex vivo working rat hearts via the hexosamine biosynthetic pathway. FASEB J. 2011;25:722.9 227. O’Donnell N, Zachara NE, Hart GW, Marth JD. Ogt-dependent X-chromosome-linked protein glycosylation is a requisite modification in somatic cell function and embryo viability. Mol Cell Biol. 2004;24:1680-1690. 228. Wang J, Torii M, Liu H, Hart GW, Hu ZZ. dbOGAP - an integrated bioinformatics resource for protein O-GlcNAcylation. BMC Bioinformatics. 2011;12:91. 229. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS, Eppig JT, Harris MA, Hill DP, Issel-Tarver L, Kasarskis A, Lewis S, Matese JC, Richardson JE, Ringwald M, Rubin GM, Sherlock G. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet. 2000;25:25-29. 230. Carbon S, Ireland A, Mungall CJ, Shu S, Marshall B, Lewis S; AmiGO Hub; Web Presence Working Group. AmiGO: online access to ontology and annotation data. Bioinformatics. 2009;25:288-289. doi: 10.1093/bioinformatics/btn615.

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Dr. Cynthia Goh Dr. Goh is a professor in the Department of Chemistry, the Institute of Medical Science, the Munk School of Global Affairs, and Director of the Impact Centre at the University of Toronto. Professor Goh has a diverse set of research interests, including fundamental studies of complex systems biomaterials, interfaces, probe microscopy, the development of new research instrumentation and nanotechnology. She invented the technique of diffraction-based sensing, a highly sensitive approach for the detection of biomolecules with applications in medical diagnostics and in drug discovery. She is also known for her interest in the translation of scientific discovery to technology and products, as well as the education of scientist-entrepreneurs. This has led to what is now known as Entrepreneurship101 at MaRS, the flagship entrepreneur training program of the MaRS Discovery District, as well as Techno, a one-month intensive training program specifically geared for university scientists intending to build a tech-based company. Interview conducted by JULS

“Learning is not just about being in your courses, learning is everywhere around you.” JULS: Why did you first decide to pursue the sciences? Was there a time or event that initially made you interested in science?

CG: I don’t know when it started because I’ve always been interested

in the world and what makes things happen, from the rocks to the plants to the stars. I grew up on a remote island where there was no television and electricity, so you could see the stars at night. Science is very natural, it’s about you wondering, “What’s up there? Why is that thing lighting up?”. So I think from a very early part of my life, I’ve been interested in science in general.

really understood the molecules and the atoms and you can control them, that gives you a lot of power to actually understand the world. I learned chemistry first when I stumbled into my mother’s old textbooks when I was in grade school and I thought, “Wow, it’s pretty cool that the properties of the world made sense if you only understood what the molecules did.”

JULS: You invented the technique of diffraction-based sensing for drug discovery and medical diagnostics, how did that come to be?

CG: I’m interested primarily in how molecules interact and creJULS: What was your undergraduate experience like and why did ate the desirable properties. So what a physical chemist would do you choose to pursue chemistry as field?

CG: I did my undergraduate studies in the Philippines. I come

from a remote island but I went to a big city to study and it was a question of chemistry, physics, biology, or math. In those days, and it is still true in the area where I am from, there weren’t as many programs, it was just physics, chemistry, math, biology, or you do engineering. I was more interested in the fundamental issues, so it was really only chemistry or physics. I thought, you know, if you

would be to design an experiment on how to measure that kind of interaction. I know a lot about optics and about designing instrumentation to measure interactions. But what is interaction, really? When you think about diagnostics, if you have an antibody in your system, all I need to do is partner an interaction to measure that you have antibodies in your blood. So diagnostics is a natural part of it. I know I can do many types of sensing, but what really inspired diffraction-based sensing is that one day I was in a meeting and I met somebody from the other side of campus, from

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the hospital. It turns out to be one of our really brilliant scientists who discovered the cystic fibrosis gene, Dr. Lap-Chee Tsui. He was complaining to me about how he didn’t have a way of detecting the SNPs for cystic fibrosis, and at that time gene chips were new. He said that he wanted one for cystic fibrosis but they wouldn’t build him one. So I told him that I could make something that detects it, but not in the same way. That’s when I invented the technique of diffraction-based sensing. I knew that it could be done, I just never really thought there was a need to do it because it’s not something I use in my lab. When he issued me a need, I told him I could do it, and so we built it from scratch and it worked. Later we realized that there are so many different ways of detecting DNA but not as many looking at proteins, so we zoomed in primarily for proteins.

“From a very early part of my life, I’ve been interested in science in general.” JULS: Much of the other work you do is with entrepreneurship.

How did you become started with that? Is it something you wanted to pursue in the beginning?

CG: No, when I grew up I was a pure scientist. I always thought that

the love of money was the root of all evil. But really, when I started diffraction-based sensing, I thought, there is a need for something that I know how to do. The question is, how do I get it out in the world? Even before that I thought a lot of the work I do had some relevance to products in the world, but I didn’t see the connection, how do I get it out? At that time I thought maybe I had to build a company myself. That was my first step into entrepreneurship because I wanted to show how I could get diffraction-based sensing out in order [for it] to become a really good medical diagnostic approach. I found was that it was a very interesting path, and I met a lot of really interesting people, and actually that got me into thinking that all students should have a little bit of exposure. Whether they want to be an entrepreneur or not, there are a lot of skills they could learn from an experience like this.

JULS: So speaking of entrepreneurship and students, you ran

entrepreneurship 101 at MaRS. Could you tell me about the program?

CG: I started that at the University of Toronto, so it was basically

a non-credit seminar series that we created. At the end of the year I challenged the students, and there were around 90 students coming to this non-credit seminar series. I gave them a challenge of applying what they had learned into something from their lab and make a 10-minute pitch. Four teams made a pitch and of those four teams, two are real companies now. They were successful. The year after I started it here at U of T, MaRS opened and the head of MaRS is a friend of mine, Ilse Treurnicht. She was my first investor, and she asked if MaRS could take part in this course. Well, it turned out that a lot of people wanted to come to this course and we didn’t have room at U of T for all those people so we just ran it at MaRS. In the first three years it was primarily students, but then there were more and more people from outside. MaRS had a lot of good people who could handle it so I eventually moved away. The focus

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has turned toward people in IT and non-students so it has changed flavours, but it’s been a hugely successful program.

JULS: What are some examples of student projects that have come out of it?

CG: We created the impact centre in 2010 when I started an

intensive training program for students with strong science and engineering backgrounds who wanted to build a company. We’ve had over 130 companies now. Some of the student projects are highly technical and some are simple. There was one project that came from my undergraduate class. These students were very interested in becoming entrepreneurs but they didn’t have as many technical skills. However, they had a passion, so we brought them into our intensive program. Their passion was to help visually impaired individuals so we taught them a lot of principles, how to analyze the problem and so on, and sent them to talk to potential customers about what the issues were, and they came up with a device. A visually impaired person has a cane, so they can sense the environment below the waist. However, they could get hit above the waist, so the students created a device that you wear, and it puts out an ultrasound so that when it encounters an obstacle coming near, it starts vibrating. It’s actually a very good device for the visually impaired and this is now on the market. They’ve learned how to produce it, how to manufacture it, how to get the money for it, etc. We’ve mentored them throughout the process. Another undergraduate student, a kinesiology student, was very conscious about posture, so he created a design on a shirt that you wear. It pulls up your muscles so that when you’re working, you can have good posture. These are simple projects, but we also have much more complicated projects. For example, there was a student in engineering whose PhD thesis was about damping systems for very tall buildings. There are a lot of theses that just end up in the library, but he pushed out to actually create a company. Now if you walk on Yonge street, just north of College, you’ll see a condo building going up that will be his first installation. There are a huge range of projects, which is why I tell students that there are many ways of pursuing entrepreneurship. It’s about what your passion is, what you are interested in, and knowing that you can do it.

JULS: Many students are interested in pursuing academia and

research, but they might not have thought about entrepreneurship or just didn’t know where to start. What advice would you have for them?

CG: There are many different programs now at the university, at

least 9 or 10 incubators if people really want to start up a company. However, if you’re not yet at the level of starting a company or even if you may never want to start a company, there are a lot of events. The first step might be to join an event, or at the very least begin with attending a talk and see whether it will inspire you, whether it resonates with you. The next stage is to attend an event like a hackathon or participate in an internship with a startup. Many places have a lot of interesting events going on throughout the year. At the minimum you should listen to a talk, but the next stage is to participate in one of the activities and then maybe you’ll know if you want to do more.

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Interviews

JULS: Lastly, do you have any advice for undergraduates looking to go into research?

CG: The University of Toronto is a research university so I always

advise students, if you can join a research group, you should. Only then will you get an idea of why we are one of the top universities in Canada and in the world. How do you get into research? Well that can be a difficult thing because sometimes they want you to have experience before they give you the experience. A way to get experience might be the ROP299 courses. If you can get into that, it’s one step into research. One thing you have to remember is that there are many different kinds of research. If you tried one and didn’t like it, that doesn’t mean you don’t like research, because there are many different types going on. If you have a home department, that’s the first step. Look at your home department, you can ask the faculty if there are any opportunities. Look at the faculty’s website to see if you are interested in what they are doing, and just contact the faculty and see whether they would have opportunities for you. Sometimes you may be able to volunteer at the beginning. They key is to start looking probably around January or February, the earlier the better. Think about what you can offer. I know people say, “I’m only a second year student, what do I know?” Well, actually, you do know something. You know something because of your courses, but you also probably know something because of your hobbies, your interests, or unusual circumstances. I’m a physical chemist and sometimes I ask people, “Do you know how to fix a bicycle or play with Lego? Because some of those skills may be relevant to what I would have you do in the lab”. I think in general for students, keep an active interest. Learning is not just about being in your courses, learning is everywhere around you, but be conscious that you’re learning because that is what you are going to have to sell to the faculty. The impact centre usually runs a research readiness program that allows a student who has never done research to learn about everything from safety issues to how to keep records, and we give them an introduction to a few skills. We are trying to develop that so when students go to knock at a professor’s door, they can say that they have a few skills already, aside from their courses, and they have been in a lab and are able to do this or that.

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because of my unfamiliarity with the field. After getting motivation from a friend, I applied for a work-study research position in my 2nd year, and this gave my research career a head-start. I embraced this position with an open mind and not too high expectations. Although the duties I performed as a beginner were not as sophisticated, I still learned a lot about research ethics, communication and intrapersonal skills during my time. It wasn’t until I took on my own project that I realized what scientific research entails. Research is about discovery! It’s about experimental failures that lead to success! Discoveries aren’t made overnight. The scientific knowledge we learn in our courses is the outcome of dedication and life worth of work of thousands of individuals. What’s even more interesting is that despite knowing a lot about science, we still don’t know what we don’t know!

Zoha Anjum Year of Study: 4th Year Program of Study: Double major in Physiology and Health & Disease

JULS: What is your current research project or what would you like your next research topic to be?

ZA: Lyme disease incidence has recently been on the rise. Due to

an energy-dense diet consumption, obesity rates have also been increasing in the industrialized world. In murine models, we found an aggravation of Lyme disease following a long-term consumption of a high fat diet. I am currently conducting a study on the relationship between Lyme disease and diet-induced obesity with an aim to identify molecular markers that may be involved in the pathogenesis of Lyme disease in obesity.

JULS: What inspired you to go into your current field? ZA: During my 2nd year, I completed a course an introductory

course in health & disease where I learned about the emerging and re-emerging infectious diseases. I was fascinated by how microbes exploit our normal physiological processes to cause infection. This experience motivated me to pursue research in infectious diseases and how our lifestyle may affect our susceptibility to developing infections. Aligning with my interests, I landed a work-study position in Dr. Tara Moriarty’s lab in the fall of 2015 and haven’t left ever since!

JULS: How did your undergraduate experience in science differ from your expectations?

“Research is about discovery! It’s about experimental failures that lead to success! ” JULS: What advice would you give to undergraduate students currently looking to get involved in research?

conducted by Tinafor Binesh Marvasti ZA: Get a mentor!Interview Don’t limit yourself! Apply positions that you

think you have no chance of getting, and reach out to your peers for help. Had I not applied for my very first work-study research position back in 2014, I would not have been able to have wonderful experiences and contribute to the research world. We all need to get our foot in the door and a little push towards what we want to do. Be your own advocate and make use of all the fantastic resources and opportunities available to you as a University of Toronto student. Also, don’t be afraid of cleaning glassware and filling pipette boxes as a beginner’s task. You have got to start somewhere at the bottom to make your way up, right?

JULS: What are your aspirations post-graduation? ZA: I aim to pursue a Master’s in Public Health with a specialization

in infectious disease epidemiology after graduation. I am very interested in using my animal studies in Lyme disease and obesity as a foundation to perform epidemiological studies in the human population. It would be interesting to see if there is an association between increasing incidence of obesity and Lyme disease. In case of presence of a correlation, this might be an opportunity to implement some public health programs to prevent the further increase in the incidence of Lyme disease.

JULS: What do you hope will happen next in science/your field of study?

ZA: I think the next best thing to happen in science would be development

of a computer software or a device that will allow us to predict future

ZA: I had no interest in research when I started university mainly epidemics such as Zika and Ebola before the severity escalates!

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Working in different research projects and being an undergraduate science student who is still learning was not only a great opportunity for me to practice, and improve, but also it was an outstanding experience that helped me think about my future personal and professional development plans. Although I had a strong background in the basics of health science and critical-thinking skills, one of the major transitions I had to make during my research involvement was moving from “textbook science” to “real world science.” In high school, the homework problems, projects, labs, etc. that were part of the courses were contrived and all had clear answers that students were expected to find. However, in undergraduate education and applications to practical science, a problem can have multiple correct solutions to meet the requirements.

“Science and IT have always fascinated me and the importance of understanding these two have never been more important than they are today. ”

Neda Jafarian Year of Study: 4th Year Program of Study: Neuroscience Major, double minor in Physiology and Biology

JULS: What advice would you give to undergraduate students currently looking to get involved in research?

JULS: What is your current research project or what would you like NJ: After being involved in research for over three years, I think I your next research topic to be?

NJ: I am currently working in a research team under supervision

of Dr. Shirazi at Ted Rogers School of Information and Technology. We work closely on a multidisciplinary research area and synthesize knowledge on HIV, IT and Big Data Analytics. We use information and Internet kiosk systems to provide medical information to HIV patients in developing countries.

JULS: What inspired you to go into your current field?

can narrow down the most useful advice to the following: First of all, DO NOT be afraid of taking on research that you’re not familiar with. Go after something that looks or sounds interesting to you, not necessarily a famous person’s lab. You’ll be soon surprised by how far it can take you down the road. Secondly, communicate well with your team, especially with the professor and learn from every person as much as possible. Research is really an extension of curriculum. Last but not least, do not cringe at the grunt work that is required during research. Sometimes something trivial can become of utmost importance.

NJ: Science and IT have always fascinated me and the importance JULS: What are your aspirations post-graduation? of understanding these two have never been more important than they are today. My current field of study is Neuroscience with a double minor in Physiology and Biology. During my second year at University of Toronto and my first research project at Ryerson, I became more interested in how science and technology feed off one another. Lastly, I enjoy the challenge of problem solving and believe that life science along with information technology would be a challenging – but rewarding – course of study.

JULS: How did your undergraduate experience in science differ from your expectations?

NJ: In general, the Science/Technology field has provided the

meaningful, challenging and cutting-edge work that I expected.

NJ: I wish to earn a degree of Master of Science in Management, later followed by a career in Health Management.

JULS: What do you hope will happen next in science/your field of study?

NJ: There are too many things that can happen over the next few years.

The most important one that I would like to happen is the development of Brain Computer Interface. BCI is a new technology that transmits signals directly to someone’s brain and helps them to see, hear or feel specific sensory inputs. For severely disabled people, development of BCI could be the most important technological breakthrough in decades.

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SJ: I would advise them to take advantage of every opportunity

out there. Apply for work-study positions and volunteer in labs on campus or at hospitals! Take courses that involve lab work and writing research articles or literature reviews. If you want to pursue a career in research, these experiences will enhance your skills and give you a competitive edge when applying to a post-graduate institution.

“As a person with type 1 diabetes, I’ve experienced the psychological impact that this disease can have on the diagnosed individual and their loved ones.” JULS: What are your aspirations post-graduation? SJ: After graduation, I plan on applying to a Master’s and then a

Samantha Jagasar Year of Study: 3rd Year Program of Study: Specialist (Co-operative) in Psychology

Ph.D. program in psychology. The ultimate goal is to pursue a career in teaching at a university or college, and continue contributing to research that investigates the relationship between chronic illness and mental health. Interview conducted by Tina Binesh Marvasti JULS: What do you hope will happen next in science/your field of study?

JULS: What is your current research project or what would you like SJ: I hope that more improvements will be made to the artificial your next research topic to be?

SJ: I am currently working with researchers at the Centre for

Addiction and Mental Health (CAMH) to investigate the relationship between fatty acid amide hydrolase (FAAH), an enzyme that regulates anandamide, and several neuropsychiatric conditions.

pancreas device system to improve the mental and physical health of those living with type 1 diabetes. More control of blood glucose levels and less needles would be nice!

JULS: What inspired you to go into your current field? SJ: As a person with type 1 diabetes, I’ve experienced the psychological impact that this disease can have on the diagnosed individual and their loved ones. This experience sparked my interest in psychology as I began to read scientific articles exploring the relationship between chronic illness and mental health. I have wanted to pursue a career in psychology ever since.

JULS: How did your undergraduate experience in science differ from your expectations?

SJ: I never expected to be contributing to research at CAMH as

an undergraduate student! Thanks to hard work, and University of Toronto’s co-op program and partnership with CAMH, I have this amazing opportunity.

JULS: What advice would you give to undergraduate students currently looking to get involved in research?

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INTERVIEWS

JULS: How did your undergraduate experience in science differ from your expectations?

KH: Typically, undergraduate science courses at the University of

Toronto deliver the bulk of their curriculum via presenting major studies that led us to where we are. However, such lectures tend to emphasize the experimental rationale, the methodology, and the results dismissing the numerous failures that littered the path from idea to publication. As I proceeded towards an actual research experience in a laboratory, I began to realize that a large amount of time is often spent on troubleshooting equipment and experimental design; though in a way that contributes to the magnitude of satisfaction upon success. I also found that, as opposed to laboratory courses, it was no longer sufficient to identify the problem and suggest solutions, but rather go one step further to solve it and re-direct the project towards new grounds in light of the new findings.

Kamal Hamid Year of Study: 2nd year MSc Program of Study: Undergraduate: HBSc, Physiology Specialist, University of Toronto, St. George Campus Graduate: MSc, Biology (concentration in Molecular and Cellular Biology, thesis in prion disease), Institute of Neuropathology, University Hospital of Zurich, University of Zurich

“During my undergraduate program, I studied a number of historic breakthroughs in my in my courses, which inspired me to pursue multiple research projects.” JULS: What advice would you give to undergraduate students currently looking to get involved in research?

KH: The University of Toronto is truly a research powerhouse that has

prion protein in the modulation of the expression, function, and subcellular localization of the metabotropic glutamate receptor 1 in the murine brain. I do not yet have a preference for an upcoming topic of research as my career goals do not lie in academia.

a heavy impact on the global perspective. In so being, I found that it makes every attempt to facilitate the participation of its students in the advancement of scholarly investigation. In my experience, professors at the institution have shifted their focus when assessing students to a constellation of traits portraying dedication, commitment and passion. I, therefore, encourage undergraduate students to never dismiss a potential opportunity on the basis of perceived inadequacy in any personal aspect but passion. Principal investigators merely require drive and competency, respectively; and at the University of Toronto, seldom is there a deficiency in the latter.

JULS: What inspired you to go into your current field?

JULS: What are your aspirations post-graduation?

JULS: What is your current research project or what would you like your next research topic to be?

KH: I am currently studying the physiological role of the cellular

KH: During my undergraduate program, I studied a number of KH: Upon completing my graduate degree, I plan to pursue my basic historic breakthroughs in my courses, which inspired me to pursue multiple research projects. During my fourth year, I spontaneously decided to attend a lecture by Prof. David B. Williams on his research on prions, which I found very interesting given what I had learned about them in my program. Somehow, the combination of Prof. Williams’ enthusiasm and the nature of his presentation of the results inspired me to further delve into the field. Several days later, I found myself considering applying to a globally renown laboratory studying prion disease at the University of Zurich.

studies in medicine where I eventually work towards a career as a clinician scientist.

JULS: What do you hope will happen next in science/your field of study? KH: Albeit too early, I hope to see researchers find evidence of pathomechanistic parallelism in all neurodegenerative diseases by elucidating transmissible spongiform encephalopathies, as prions are an excellent model for studying proteinopathies given their unique advantage of infectivity.

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you never know until you try. Take advantage of every opportunity that comes to you!

“Many people recognize the importance of the quality of life, but I believe the quality of death is just as significant.” JULS: What are your aspirations post-graduation? LL: I’m currently applying for some graduate school programs, while

working at an educational consulting company. Still waiting for results, fingers crossed, but I still have to wait to see where life takes me next!

JULS: What do you hope will happen next in science/your field of

Lucy Liu Year of Study: Graduated in 2016 Program of Study: Double major in Physiology and Human Biology, Immunology minor

study?

LL: I’m really looking forward to the development of a vaccine

appropriate for humans to combat the Zika virus, and later hopefully a cure to help those currently afflicted. Interview conducted by Tina Binesh Marvasti

JULS: What is your current research project or what would you like your next research topic to be?

LL: I am currently studying the quality of dying and death in rural

Ontario, and I hope my findings will help to improve end of life care for individuals suffering from terminal illnesses.

JULS: What inspired you to go into your current field? LL: Many people recognize the importance of the quality of life, but I

believe the quality of death is just as significant. I am a strong advocate for palliative care, and I sincerely hope that in the near future this service would be more readily available and individualized for both rural and aboriginal populations.

JULS: How did your undergraduate experience in science differ from your expectations?

LL: I originally thought I would specialize in one area of science, but it turns out that I enjoy taking courses from multiple disciplines.

JULS: What advice would you give to undergraduate students currently looking to get involved in research?

LL: I would strongly recommend students to try out different areas of

research whether it’s clinical or wet lab. Also, don’t let your marks hold you back from applying for research positions, as cheesy as it sounds,

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are run outside of these fields. Although computational chemistry is focused on using computer modeling programs in a dry lab setting, it provides the blueprints to future studies in drug binding, disease mechanisms and much more.

JULS: What advice would you give to undergraduate students currently looking to get involved in research?

NR: My biggest advice to undergraduate students would be to explore

a variety of research options and to have an open mind at the initial part of the process. Personally, aside of my current lab, I have worked in psychology, nutrition, clinical and neuroscience laboratories that not only allowed me to learn a diverse set of skills but also helped me discover what I was truly passionate about. Do not wait for a “perfect position” to appear, it will only hinder your chances of developing yourself as you close doors even before trying. Have courage and place yourself in positions outside of your comfort zone, as that is truly when you learn your potential and passion.

Nidaa Rasheed Year of Study: 4th year Program of Study: Double Major in Physiology and Neuroscience, Psychology Minor

JULS: What is your current research project or what would you like your next research topic to be?

“Although computational chemistry is focused on using computer modeling programs in a dry lab setting, it provides the blueprints to future studies in drug binding, disease mechanisms, and so much more.”

NR: I am currently working with Dr. Imre Csizmadia as a fourth JULS: What are your aspirations post-graduation? year thesis project student using computational chemistry in drug development and biomedicine. My current project looks at the structure-activity relationship of dicoumarol drugs and the effects on its antimicrobial activity against Staphylocous aureus (Staph Infection). My second project investigates the keto-enol tautomerism in Maple Syrup Urine Disease (branched-chain ketoaciduria) leading to the accumulation of toxic byproducts that can result in developmental delay and other health problems.

JULS: What inspired you to go into your current field? NR: I was accepted into the computational chemistry lab for CHM299 as part of the ROP299 (Research Opportunity Program), then continued as a thesis project student and Teaching Assistant for my third and fourth years. Initially I was inspired by having the liberty to work on an independent project of my choosing in the biomedical field and the ability to investigate diseases at a molecular setting.

NR: I aspire to become a physician-scientist who implements biomedical research into health care applications.

JULS: What do you hope will happen next in science/your field of study?

NR: I hope that computational chemistry is more appreciated as it

provides an opportunity to run theoretical studies that not only save time and effort for pharmaceutical and clinical studies, but also provide the chance to intertwine our growing use of technology in healthcare to enhance our potential to treat diseases.

JULS: How did your undergraduate experience in science differ from your expectations?

NR: Students tend to think research refers to only wet labs or working

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from your expectations?

MZ: Pursing science at undergraduate level is a two-way street.

Primarily, students are here to learn and improve their foundational knowledge. However, an important aspect of your learning is to be involved in research and contribute to the scientific community. By doing so, we will be more motivated and will be willing to work harder because we will have more insight in the significance of concepts learned in class.

JULS: What advice would you give to undergraduate students currently looking to get involved in research?

MZ: As a mentor for First-Year Learning Communities (FLC) and

MohammadMasoud Zavvarian Year of Study: 4th year Program of Study: Double major in Genomics and Neuroscience

as a peer advisor for Research Opportunities Program (ROP), I believe students should follow the fields that they enjoy the most, and embark on a path that they are willing to work tirelessly for. I also think having a broad knowledge by taking courses in different fields will help students to be able to contribute more effectively in their fields of study.

“I believe Interview geneticsconducted and neuroscience are by Tina Binesh Marvasti two rapidly expanding fields in life sciences. However, there are so many questions that are still unanswered.”

JULS: What is your current research project or what would you like JULS: What do you hope will happen next in science/your field of your next research topic to be?

study?

MZ: I have been involved in a number of research projects in the

MZ: I am looking forward to further advances in the domain of

fields of genetics and neurobiology. Currently, I am looking at how the brain pattern of patients with Generalized Anxiety Disorders differs from healthy controls. Specifically, I am focusing on P300 waveforms, which are being investigated for their abilities in lie detection devices or brain-computer interfering, and analyze how they can be used for diagnosis of anxiety disorders. Since anxiety disorders constitute the most common class of mental illness, this can have great clinical applications.

connectomics, which encompasses the development of an integrated map for the entire nervous system. This will have significant impact on our understanding of how gene regulations at specific parts of nervous system can lead to altered behavioral outcome. This can potentially help many of our patients with neurodegenerative and psychotic disorders.

JULS: What inspired you to go into your current field? MZ: I believe genetics and neuroscience are two rapidly expanding

fields in life sciences. However, there are so many questions that are still unanswered. With the new techniques that are being developed in these fields, our understanding will be significantly enhanced in future. This inspired me to learn more about genetics and neuroscience, and I am looking forward be able to contribute to these respected fields.

JULS: How did your undergraduate experience in science differ

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JULS: What is your current research project or what would you like

INTERVIEWS

PP: My aspirations include to continue my love of research and medicine.

“I think first understanding the mechanisms of specifying different cell types is critical towards creating effective therapies to use stem cells to regenerate tissue. ”

your next research topic to be?

JULS: What do you hope will happen next in science/your field of PP: My current research project used embryonic stem cells to study? investigate the role of the calcium chaperone Calreticulin towards regulating osteoblast and cardiac stem cell differentiation. We further looked downstream to identify transcription factors whose regulation was controlled by calreticulin activity. As for my next research project, I have become really interested in Multiple Sclerosis. There has been some recent stem work which showed great efficacy in clinical trials and I am excited by the utility of stem cells as a potential long term treatment.

PP: There is much excitement in the field of stem cell biology right now

with the successful creation of many important cell types, including pancreatic cells. The next step is to successfully transplant these cells into patients, and have them live long term. I think through better understanding the biology of how these cells are generated, the field will be successful in perhaps replacing, for example, damaged heart tissue following a heart attack.

JULS: What inspired you to go into your current field? PP: I have long been interested in the using stem cells to regenerate

damaged tissues. As a result, the Opas lab provided me an amazing opportunity to work on understanding the basic biology of how stem cells differentiate into various tissues. I think first understanding the mechanisms of specifying different cell types is critical towards creating effective therapies to use stem cells to regenerate tissue.

JULS: How did your undergraduate experience in science differ from your expectations?

PP: I didn’t expect the late long nights and working on weekends.

I was at the lab sometimes until midnight or even later because my experiment didn’t work the first time. Furthermore, during my first research experience, I had to make up my own project and read papers which was very different than any of my undergraduate courses. However, after some time, it became the best part of research, because there were so many possibilities.

JULS: What advice would you give to undergraduate students currently looking to get involved in research?

PP: Ask early, be persistent, and don’t get discouraged. There are

many labs that would love to have undergraduate researchers, so if you don’t get your first choice keep asking around. Also, science is exciting no matter your project, and is an amazing opportunity to learn new fields, so don’t be afraid to diversify yourself and try something new.

JULS: What are your aspirations post-graduation?

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JULS Samuel Walmsley Year of Study: Graduated Spring 2016 Program of Study: Ecology and Evolutionary Biology, Cognitive Science, Philosophy

JULS: What is your current research project or what would you like your next research topic to be?

SW: I’m currently working with GPS-collared wolves in Manitoba.

SW: One thing that’s often overlooked in ecological work is traditional

ecological knowledge, and particularly the understanding of any indigenous communities local to a study species in question. I think that integrating this vast source of knowledge into modern, paper-driven scientific efforts is important, not to mention informative!

“I fell in love with the lifestyle of a field biologist, and realized that the associated methodologies would allow me to answer really fascinating questions. ”

Fellow researchers and I are following them to identify sites where they’ve killed prey, bedded, and so on. I’m using this data to investigate social cohesion among individuals. Subsequently I hope to develop models of step-by-step wolf movement that include cognitive capacities (e.g. spatial memory) as an explanatory variable.

JULS: What inspired you to go into your current field? SW: On a whim, I applied for research excursion with Professor

James Thomson for the summer of 2014. This entailed living in the Rocky Mountain Biological Laboratory in Colorado, and catching bumblebees across altitudinal transects. I fell in love with the lifestyle of a field biologist, and realized that the associated methodologies would allow me to answer really fascinating questions.

JULS: How did your undergraduate experience in science differ from your expectations?

SW: Initially, I didn’t realize how important research experience

would be in terms of my understanding of science. I probably learned just as much from these projects as I did from coursework, if not more.

JULS: What advice would you give to undergraduate students currently looking to get involved in research?

SW: Look beyond U of T. I’ve been lucky to travel internationally

for a few different projects. In addition the fun of experiencing new places, I think that international collaboration and the immersion of oneself into new institutions is really valuable.

JULS: What are your aspirations post-graduation? SW: I’m in the process of deciding on a Master’s program in animal behaviour, either in Canada or Scotland.

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Journal of Undergraduate Life Sciences University of Toronto Call for Submissions The University of Toronto Journal of Undergraduate Life Sciences (JULS) is looking for submissions that showcase the research achievements of undergraduate life science students. We welcome manuscripts in the form of Research Articles or Reviews. Submissions must come from University of Toronto undergraduate students or undergraduate students outside of U of T who have conducted research for at least three months under the supervision of a faculty member at U of T. Research articles should present original research and address an area of the life sciences. Mini-reviews should focus on a specific scientific topic of interest or related to the research work of the author. Research articles should be between 2,000-3,000 words and mini-reviews between 1,500-2,000 words. All works must not have been previously submitted or published in another undergraduate journal. The deadline for submissions for each issue will appear on the JULS website at http://juls.ca.

Contact Us JULS is always looking for contributions from writers, artists, designers, and editors. Please contact JULS at juls@utoronto.ca if you are interested in joining the JULS team, or have any questions regarding any matter pertaining to the journal, or visit our websites: http://juls.ca or http://jps.library.utoronto.ca/index.php/juls. Share your thoughts on this yearâ&#x20AC;&#x2122;s issue on our facebook page: https://www.facebook.com/julsuoft.

Š 2017 University of Toronto Journal of Undergraduate Life Sciences (JULS). Authors retain all rights to their work published in JULS except the right to publish in another undergraduate journal. The content and/or opinions expressed in this publication are expressively those of the authors and do not necessarily reflect the views of the University of Toronto or JULS.


JULS

Journal of Undergraduate Life Sciences University of Toronto

Journal of Undergraduate Life Sciences: Spring 2017  

11th issue: Celebrating the University of Toronto's 190th Birthday