Fall 2011

The Stony Brook

Young Investigators Review UNDERGRADUATE JOURNAL OF SCIENCE

Graphene

The Future of Technology

Reviews: Gene Therapy and Chemoselective Vector Modification Features: Discovering Topology with Dr. John Milnor Research highlights: Poliovirus: A Promising Candidate for Oncolytic Treatment Student research: The Crystallization of Riboswitches: Potential Targets for Antibiotics Vo l u m e 3 Fall 2011

Sponsorship

Thank you to the following Stony Brook departments for their funding and support!

• • • • •

The Department of Biochemistry and Cell Biology The Department of Physics The Department of Neurobiology The Department of Biology The Department of Ecology and Evolution

Contents

In This Issue

On the Cover

Graphene, a carbon atom monolayer of incredible strength and conductive properties, is poised to be a major player in the future of technology. Turn to page 19 to read Zaki Shafi’s review of this new Nobel Prize-winning material.

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9 Discovering Topology with

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People

19 President Noel Joseph B.S. in Pharmacology, 2012

Senior Editors Polina Pinkhasova Olesya Levsh Faye Marie Vassel

Vice President Zaki Shafi

Staff Writers Faye Marie Vassel Zaki Shafi Olesya Levsh Polina Pinkhasova Contributing Writers Karen Bulaklak Rana Said Artem Serganov Amy Patel Maha Mamoor Nadya Peresleni Kazi Ullah Layout Editors Sam Kilb Nadya Peresleni

Dr. John Milnor

Micro RNA and its Tremendous Role in Cancer

Poliovirus: A Promising Candidate for Oncolytic Treatment of Neuroblastoma

Editor-in-Chief Nadya Peresleni B.S. in Biochemistry, 2011

Associate Editors Zaki Shafi Rob Castellano Steven Leigh Stephanie Jones Malack Hamade Ida Li

Exploring Gustation and Olfaction with Dr. Fontanini

Web Editor Maria R. Stupnikov Copy Editors Sam Kilb Yousra Yusuf Advisory Board Dr. Robert Haltiwanger Dept. of Biochemistry and Cell Biology Dr. Harvard Lyman Dept. of Biochemistry and Cell Biology A special thank you to Isaiah Schuster, YIR President 2009-2010, for his involvement with the journal and its staff throughout the entire academic year, and to Alexander Chamessian, YIR founder and President, 20082009, for his continual support.

Cover article: Graphene: The Possibilities are Endless

Gene Therapy and the Power of Chemoselective Vector Modification

A Scientific Breakthrough

The Crystallization of Ribo- switches: Potential Targets for Antibiotics

Enhancement of Reactivation of Murine Gammherpesvirus 68 from Latency Through NF-kB Inhibition

Death Associated with Activation of MAP and PI3 Kinases: The Role of TRAF4

Inhibitors of Serine Proteinases, Matrix Metallo- proteinases and Histone Deacetylases: Thermorubin, COL-308, Myricetin, and Tellimagrandin

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25 Farewell to Diabetes: 27 31

36 Ethanol-Induced Epithelial Cell 41

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Notes

Visit us on the Web:

www.younginvestigators.com The Stony Brook Young Investigators Review, Fall 2011

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Letter from the Editor Dear Stony Brook students, faculty and staff, I am very proud to present the third issue of YIR to the Stony Brook University community. This publication is the result of much dedication, effort and diligence from a small group of core members committed to the endurance and success of Stony Brook’s first and only undergraduate science journal. Through a lot of hard work, we’ve been able to overcome the continual transitions as our members graduate, and pursue the mission set forth by the journal’s founders three years ago.

Our campus is full of talented undergraduates who are either involved in research or thinking about doing so. Most students are working in a laboratory for the first time and are at the very beginning of their research careers. Doing research is an exciting and enriching educational experience, and our goal at YIR is to showcase the potential of Stony Brook’s young researchers to their fellow peers and the rest of the campus community. We often hear students respond to our requests for their contributions by modestly exclaiming that their results are not significant enough. I would like to emphasize that we do not expect to be receiving student articles on new scientific breakthroughs (although those are always welcome). Our purpose is to showcase the work of individuals who retain their excitement and passion for finding the answers to scientific questions despite the inevitable difficulties that emerge in the course of conducting experiments. Hopefully, the work done by these students will inspire those not yet involved in research to do so, and raise their awareness of the importance of scientific inquiry.

Our journal provides an excellent opportunity for students thinking about research or medicine as a career to hone their science writing and analytical skills. Along the way, YIR offers students a chance to get to know the academic community on a more personal level. YIR members not only interview, but seek advice from and forge relationships with faculty members.

Stony Brook professors and graduate students are a tremendous resource to undergraduates, and getting to know them can be a very rewarding experience.

If you think you would like to join us or contribute your research reports, please contact us as soon as you can find a way to do so. We see YIR as an integral part of our academic undergraduate scientific community. The exchange of research results in scientific journals opens the door for collaboration and creatiivty; we want Stony Brook students to be able to do the same for each other. All of us at YIR believe that by coming together in a joint effort to share and publish research news, students can greatly enrich our university’s undergraduate academic life.

I would like to express gratitude to Dr. Robert Haltiwanger

and the Biochemistry Department for supporting our journal over the past three years, and to Dr. Lorna Role and the Department of Neurobiology and Behavior and the Departments of Physics, Biology, Chemistry, and Ecology and Evolution. I would also like to personally thank all of our writers and eidtors.

A special thank you goes out to Isaiah Schuster, the previous president of YIR, for his continuing support and to Alexander Chamessian, the founder of YIR, for his advice and encouragement.

I look forward to seeing student applications, questions, or requests for more information in our mailbox at younginvestigators@gmail.com.

Thank you for reading the third issue of YIR! Sincerely, Nadya Peresleni Editor-in-Chief B.S. in Biochemistry, Class of 2011

President’s Message The story of Stony Brook’s success can be attributed to the many esteemed faculty members conducting cutting edge research within our walls.

The fruits of their labor are exhibited in the vital discoveries made at Stony Brook.

Examples include Magnetic Resonance Imaging (MRI) and the discovery of the cause for Lyme disease. Stony Brook researchers have been honored with many prestigious awards including the Nobel Prize, the Fields Medal in Mathematics, the NIH Pioneer Award, and the M.W. Beijerinck Virology Prize .

Stony Brook undergraduates are some of the brightest minds in academia, and spending only a few hours at the Stony Brook labs will allow you to see the dedication undergraduate students have for their research. Whether it be running a QPCR or running a Western Blot, Stony Brook undergraduates take pride in their research However, a common problem for undergraduates conducting research is the lack of opportunities to showcase their work to fellow peers and faculty. Oftentimes, the only eyes to ever see a student’s research are supervising faculty members and those deemed in charge to review the student’s research. Consequently and unfortunately, students’ research endeavors often go unnoticed by their fellow peers and other faculty. It is from this impetus that the Stony Brook Young Investigators Review was established and continues to grow.

The principal ambition of our journal is to ensure that the research conducted by undergraduates does not go unnoticed by faculty and, most importantly, by fellow peers. We strive to foster an environment in which students may present their research in a forum that encourages constructive feedback. Accordingly, we publish a journal once a year that encompasses all disciplines of scientific research conducted at Stony Brook. Through print and online publication, as well as holding symposiums featuring speakers who have revolutionized their respective fields, such as Dr. Robert Weinberg and Nobel Laureate Dr. Martin Chalfie, we hope to attract students from various academic backgrounds as well as those who may have no yet been exposed to laboratory work. We extend an invitation to undergraduate researchers from all fields of science with the end goal of publishing in a journal that is disseminated to the student body.

As president, it has been an honor to continue the hard work of my predecessors in “spreading the scientific word” and having the privilege to help exhibit the exceptional research being done by my fellow peers in their respective fields.

I would like to thank our writers and editors who make the issue a success. I would also like to thank the Departments of Biochemistry, Neurobiology and Behavior, Physics, Chemistry, Biology, and Ecology and Evolution for all their contributions

to our journal. Additionally I would like to thank Dr. Lorna Role and Dr. Robert Haltiwanger for offering guidance and support.

Respectfully yours, Noel S. Joseph B.S. in Pharmacology (2012) President

features

Fall 2011

Exploring Gustation and Olfaction with Dr. Fontanini

The OFC-BLA-INS circuit loop undergoes maturation during the development of an individual from preadolescence to adulthood. The slow development of this circuit accounts for the difference in behavior toward rewarding and aversive stimuli during these changing stages. By studying the process of maturation we can understand why adolescence is associated with a heightened risk of developing addictions and eating disorders [2].

Maha Mamoor, ‘14 To draw a scenario, during the adolescence stage, which is Introduction characterized by hormonal, physiological, neural, and behavioral alterations, adolescent rats exhibit elevations in risk-taking The taste of your and novelty-seeking behavior [6]. They have shown favorite birthday cake to be more prone to novelty seeking and less prone to or the sensation of taste aversion. Current research aims to explain how burning hot sauce on hedonic value changes in adolescent rats over time your tongue can leave and how it can be compared to abnormal processing a lasting emotional of hedonics in humans. For instance, depressed teenimpression. Such imagers tend to exhibit anhedonia, which is the inability pressions and emoto experience pleasurable emotions [2]. This may, in tional responses are turn, give rise to potential targets for early pharmaexactly what got Dr. cological and behavioral therapies [2]. The nature and Alfredo Fontanini, of amount of what we consume is influenced by brain the Neurobiology and reward circuits, which generate “liking” and “wantBehavior Department ing” for foods. Dysfunction in reward circuits might at Stony Brook, inter- Dr. Fontanini received his PhD at the University of contribute to the recent rise of obesity and eating disested in doing research Brescia Medical School in Italy, where he studied orders. Dr. Fontanini’s work can potentially provide on gustation and olfac- neuroscience. insights into eating disorders such as anorexia, which tion. is the third most common chronic illness among adoDespite the common misconception, gustation and olfac- lescents [1]. tion do go hand in hand. Dr. Fontanini and his lab conduct research on how the brain responds to chemical stimuli, and Taste and Smell: How One Sense Affects the how these responses are enriched with emotions. The results of Other the research that Dr. Fontanini and his group have done led him to receive the 2010 Presidential Early Career Award for Scien“It is a beautiful relationship between the two of them. The tists and Engineers (PECASE), which recognizes scientists at fact that we study only taste or only olfaction is one of the bigthe beginning of their research careers who show outstanding gest misconceptions in our field,” says Dr. Fontanini about taste success and potential. and smell. He presents the following illustration: An individual

Scientific Basis

The goal of Dr. Fontanini’s research is to uncover and control the neural mechanisms that regulate emotions associated with sensory perception. The term “hedonic value” describes the goodness or badness of a food. This is a subjective perception that can be objectively measured in animals by looking at facial expressions of pleasure and disgust. Taste is an ideal sensory system for studying how processing of sensory components and hedonic value are associated. The relationship can be seen in the anatomy of the gustatory system, where sensory areas and reward processing regions are connected [2]. The circuit loop responsible for the processing of tastes involves the orbitofrontal cortex (OFC), insular cortex (INS) and the basolateral nucleus of the amygdala (BLA). Interactions between the OFC and BLA are responsible for processing hedonic value and passing this information to the insular lobe. The INS is responsible for processing stimulus-specific integration of senses and emotions [2].

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first approaches a food through olfaction and proceeds to consume it. The result is a gustatory sensation on the tongue. Then, at the first exhalation, the air in the mouth is pushed back into the nose, which again engages olfaction. The relationship between taste and smell is very intimate. The smell of an odor that has no association with the taste (i.e. the smell of a strawberry and a bitter flavor) will cause the two sensations to conflict and potentially depress each other. But the smell of an odor with its complementary taste (the smell of strawberry and a sweet flavor) will cause a synergistic interaction, which enhances the perception psychologically and results in a more pleasurable experience. Thus, taste and smell are constantly influencing each other, even when we do not realize that these senses are interacting.

Obesity The appearance and the context that surrounds food can discourage or encourage overeating. Dr. Fontanini presents a

The Stony Brook Young Investigators Review, Fall 2011

features the brain reveal that they are very strongly connected with the emotional centers of the brain, namely with the amygdala.

Attention, Expectation, and Distinction

Local field potentials in gustatory cortex in response to a gustatory stimulus and spectogram of response frequencies.

simple scenario: when you consume candy you experience a taste that is “over the top” and very satisfying. Since you like the way it tastes, you also get used to the visual appearance of what you are eating. This accomplishes two things: you get used to an “over the top” taste, such as excessive sweetness, and an appearance that is completely unnatural. The solution to the problem of unhealthy eating is to get people conditioned to healthy foods, which have less excessive taste and a natural appearance.

Encouraging healthy choices early on in life will help an individual’s development into a healthy adult. Since the OFCBLA-INS loop undergoes maturation, we can program it to prefer natural and healthy choices as early as the preadolescent stage.

Learning to dislike An issue encountered by many college students over the age of twenty one is alcohol intoxication, which may lead to extreme sickness through alcohol poisoning. Experiencing this can, in turn, lead to the development of an aversion to alcoholic beverages. This is an example of associative learning, termed Conditioned Taste Aversion, which has a profound meaning for our survival. Dr. Fontanini presents an example of this: If you are put into the wild and taste something novel that makes you sick, your body will signal to your brain that whatever you just tasted is potentially poisonous. This signal will create an aversion for the food that caused the illness. This is a way to ensure you do not give the poison a second chance to kill you by consuming it again. The same process of aversion has been observed in laboratory animal studies [4].

How do the factors of attention level and expectation modulate processing of taste processing and gustatory cortical activity? Levels of attention tremendously change the way tastes are perceived. If a person pays more attention to what he tastes, he or she can then better discriminate the basic tastes - sweet, sour, salty, bitter, and umami. For instance, in experiments conducted with mice, the animals make the most efficient distinction on whether the odor is dangerous or not, accurately discerning whether something is a predator or prey. If, however, the mouse is not paying attention, its main concern is whether the odor is pleasant or not. Once it makes this distinction, the brain becomes activated, causing the animal to become attentive and discriminate whether the smell represents an item that is both good and physiologically necessary, such as a salty food. It is the ability to discriminate between different aspects of information that changes depending on one’s state of attention [2].

A Big Challenge for Behavioral Neuroscientists How do we convert expectations to quantitative data? How do we objectively measure neurological processing? The answer to these questions lies in the careful analysis of behavior. One can examine if an animal likes or dislikes something based on a few different measures, including how much it consumes of the substance (motivation) and which substance it prefers. This can be done by observing oro-facial reactions, which are “funny faces” mice make when they are given something to taste. When they dislike what they tasted, mice engage in gaping behavior, which involves attempting to remove the substance from their mouth with their paws. If they like the substance, they lick their

Remembrance of Things Past The renowned, French author Marcel Proust wrote seven volumes of remembrance and memories that all began with the smell of a Madeleine known as “Remembrance of Things Past” [5]. Both odor and taste are very powerful in evoking emotions through the OFC-BLA-INA circuit in the brain. Taste, even in its simplest form, is a very emotional sensory system. Studies on the interaction between the olfactory and gustatory areas of

lips.Local field potentials and action potentials from c a nmultiple neurons in a rat brain. into

The Stony Brook Young Investigators Review, Fall 2011

One g o more

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features detailed examination by counting the number of times the tongue protrudes. These are all tools used for measuring liking.

For expectation, operant behaviors can be used to tell whether an animal is expecting someLab logo, designed by Bren Bataclan. thing or not. For instance, a standard task (called go/no-go) relies on two auditory tones: one that predicts sucrose and one that predicts quinine. The animal has to press the lever to get sucrose when the sound associated with this substance comes up. When the quinine tone sounds and the animal happens to press the lever, it receives quinine, which is bitter and distasteful. Once these associations are learned, the animal will press the lever to the sucrose tone only and will avoid pressing the lever to the quinine tone. Based on these experiments, it can be observed that the animal was correctly expecting sucrose because it pressed the lever, and was correctly expecting quinine because it did not press the lever.

The Future of Science and Health “Preference” is a concept that plays a huge role in taste perception. Why do I like something? Why do you not like the same thing? Why does the brain like one thing, and not something else? Although Dr. Alfredo Fontanini believes these are not factors that need to be controlled, they do become necessary concerns when healthy foods are disregarded and poor foods are preferred. How an individual’s diet during development shapes his or her preferences is Dr. Fontanini’s main concern. Perhaps if we make healthier choices when we are younger, we can carry on those practices into adolescence and adulthood. Our brain is shaped by experience, and it is critical to encourage and understand which experiences may benefit us in the long run.

Recognition

Fall 2011

“This job can be terribly difficult and yet incredibly fun, so you need to see that fun in this job and choose a lab that you like, an environment that you think is exciting, and let this wave of excitement carry you over the difficulties.”

“Do not believe there is only one way to be successful- the definition of success is highly subjective and up to every individual.”

Dr. Alfredo Fontanini defines success as doing well in his profession and having a balanced, happy family life.

References 1. “Eating Disorder Statistics.” South Carolina Department of Mental Health. Web. 09 Feb. 2011. http://www.state.sc.us/ dmh/anorexia/statistics.htm>.

2. Fontanini, Alfredo. “Development of hedonic processing in the orbitofrontal-amygdalo-insular system.” Proposal. 3 Jan. 2011.

3. “Palatability – Definition and More from the Free MerriamWebster Dictionary.” Dictionary and Thesaurus – MerriamWebster Online. Web. 09 Feb. 2011. <http://www.merriamwebster.com/dictionary/palatability?show=1&t=1297294418>.

4. Yamamoto T, Fujimoto Y, Shimura T, Sakai N. 1995. “Conditioned taste aversion in rats with excitotoxic brain lesions.”

Neurosci Res. 22(1):31-49.

5. Pines, Maya. “The Vivid World of Odors.” Howard Hughs Medical Institute\Biomedical Research & Science Education (HHMI). Web. 09 Feb. 2011. <http://www.hhmi.org/senses/ d110.html >.

6. Presidential Early Career Award for Scientists and Engineers. Web.09 Feb. 2011. http://www.er.doe.gov/accomplishments_awards/pecase/pecase.htm>.

7. Spear, Linda P., Varlinskaya, Elena I, Doremus-Fitzwater, Tamara L. 2010. “Motivational Systems in Adolescence: Possible Implications for Age Differences in Substance abuse and other risk-taking behaviors.” Brain and Cognition. 72(1): 114-23.

As a result of writing this article, Maha discovered an interest in neurobiology and was accepted to work in Dr. Fontanini’s lab. Congratulations Maha!

A letter from the White House informed Dr. Alfredo Fontanini that he received the Presidential Early Career Award for Scientists and Engineers. This award is given to “outstanding scientists and engineers who show exceptional potential for leadership at the frontiers of scientific knowledge during the 21st century” [6]. Dr. Fontanini believes that the PECASE “is a great honor that will propel our research tremendously.” Dr. Fontanini notes that receiving such an award from the White House is very rewarding, but also a great responsibility.

Advice from the Scientist Himself “Follow whatever fires you, whatever gets you excited.”

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The Stony Brook Young Investigators Review, Fall 2011

features Discovering Topology with Dr. John Milnor Polina Pinkhasova, ‘12 Editor: Rob Castellano, ‘11

Introduction John Milnor, credited for introducing the field of differential topology, emerged as a leader of topological research at Princeton University in the time period spanning 1950 to 1970.

Since then, the field has reached great heights, generating unpredictable results, solutions to seemingly impossible problems and the ability to ask new questions, all as a result of a joint effort of mathematicians worldwide. John Milnor holds both a Bachelor’s Degree and Ph.D. from Princeton. He is the recipient of the world’s most prestigious awards in mathematics.

These include the Fields Medal, the Wolf Prize, the Leroy P. Steele Prize in several distinctions, the National Medal of Science, and most recently the Abel Prize, nicknamed “Nobel for Math”, which was awarded on May 24th, 2011 in Oslo, Norway.

Dr. Milnor currently serves as Co-Director of the Institute for Mathematical Sciences at Stony Brook. His most famous work involves the concept of exotic spheres, which shows that there are at least 7 differentiable structures on the 7 dimensional sphere (S7) [1]. For this work, he was awarded the Fields Medal by the Clay Mathematics Institute in 1962, the highest prize in mathematics. Milnor further proved the existence of more than 7 differentiable structures on the 7 dimensional sphere (in total, 15 differentiable structures). These results, though very significant, are only a fraction of his mathematical accomplishments.

Topological Principles Understanding Dr. Milnor’s discovery involves understanding the concept of homeomorphism. Homeomorphism is the concept of bending and twisting one space into another and then back again. The two spaces are considered topologically equivalent, having all of the same properties. For instance, a square frame and a hula-hoop are homeomorphic to each other since you can deform one into the other in a continuous motion.

A baseball and a bead with a hole in it are not homeomorphic since you cannot perform the same continuous motion to convert one into the other. The idea of diffeomorphism involves the same motions as homeomorphism, but is limited to only smooth surfaces and smooth movements; meaning the existence of infinitely many derivates of these spaces and maps. A question was posed whether two surfaces could be found that are both smooth and homeomorphic yet not diffeomorphic. A differentiable structure is a structure on our space that makes it smooth. Dr. Milnor’s result showed that this conjecture is false in dimension 7. He writes, “there exists a differentiable structure on S7 not diffeomorphic to the standard one”, referring to the concept of exotic spheres [2].

Dr. Milnor received the Abel Prize in 2011 for his highly influential work in the mathematical fields of topology, geometry and algebra.

In order to fully appreciate the concept of exotic spheres, it is necessary to understand space and manifolds. There is one space for each dimension. One-dimensional space is a line, two-dimensional space is a plane, three-dimensional space is the usual 3-space, and the higher dimensional spaces are generalizations of these. A manifold is a space that when you zoom in on it, it looks like a “simple” space. In other words, locally it is “flat” and “simple,” though globally it can be very different and complicated. Examples of manifolds are a circle, a sphere, a sphere with an opening, a sphere with several openings, a donut, or figures of similar nature. Some manifolds have a property known as simple connectedness, which informally means the space has no “holes.” For example, a sphere has no holes, but a donut does. In dimension two, the sphere is the only manifold that is simply connected [2]. In the early 20th century, French mathematician Henri Poincaré questioned if spheres are the only simply connected manifolds in all dimensions. Solutions to the Poincare Conjecture were proved in several dimensions by mathematicians worldwide. Dimensions one and two were already solved in Poincaré’s time. The conjecture was proved in dimensions greater than four by Stephen Smale in 1961. It was then proved in dimension four by Michael Freedman in 1982.

Most recently Grigori Perelman proved the conjecture in dimension three, completing the conjecture for all dimensions [3].

At the start of Dr. Milnor’s time at Princeton, differing opinions existed of the definition of a concept of a manifold. Dr. Milnor explained: “A manifold is an object whose pieces look like pieces of Euclidean space but may be curved or shaped in some strange way like a donut or something more complicated.

Some theories studying manifolds assumed they were differentiable; they were defined by differential equations. Other theo-

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features ries allowed for manifolds with corners or very ragged surfaces.

There was a general belief that one definition was convenient in certain cases, and another definition was convenient in others.

Sometime in the 1950’s when I was trying to study examples of manifolds, I suddenly hit a snag: In one way, I proved that a certain manifold could exist, and in another, I proved that it could not. This was very baffling. Then I realized after a lot of worry that I had been passively assuming that it did not matter which definition one used. The manifold in question could exist if you allowed angles and strange shapes; it could not exist if everything was smooth and differentiable. This was a shock to me and many other people. They somehow never asked a question or imagined that such a thing was possible. In a way it started a new industry with the goal of trying to understand these things.”

Influences and Great Beginnings There were several important influences on Dr. Milnor’s important accomplishments. At the roots of his beginnings in topology were his teachers at Princeton, Ralph Fox and Norman Steenrod. René Thom, a French mathematician, also had a large influence. However, the area of topology had many different people working in it and many were influential. Therefore, it is hard to list only a few. Jean-Pierre Serre, another French mathematician, made very important contributions, and Friedrich Hirzerbruch from Germany did as well.” Dr. Milnor described his time at Princeton at the start of a new area of mathematics as a very exciting time. Certain questions were being asked by mathematicians that no one thought to ask before and new ideas developed on how to answer these questions. “Suddenly,” Dr. Milnor commented, “great human efforts were being made to attain a goal; it was certainly a very wonderful time.”

Applications of Topological Research A concern that troubles those studying the applied sciences is the possible applications of mathematical theories. A popular,

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Fall 2011

primary understanding of the sequence of studies is that mathematics underlies physics, while physics underlies chemistry and chemistry underlies the biological sciences. Mathematics is the strong fundament, at the bottom of the pyramid. Dr. Michael Freedman of the University of California, Santa Barbara, a Fields Medalist famous for his proof of the Poincare Conjecture in dimension four, explains the general work of mathematicians: “Theirs is a way of thinking that thrives by disdaining the need for practical applications. Let the applications come later by accident - they always do” [4].

The main applications are rooted in physics. For instance, linear algebra, operator theory, and group theory contribute to the theory of special relativity; differential geometry to general relativity; algebraic topology and algebraic geometry to quantum fields and the physics of elementary particles, among many others [5].

Mathematical Biology Dr. Milnor’s recent lecture at the Institute for Advanced Study at Princeton entitled Geometry of Growth and Form, based on a book of the same title by D’Arcy Thompson, explored the nature of organisms through dimensional analysis [6]. The discussion demonstrated how organisms can be compared based on their physical characteristics. An organism can grow and retain its form while varying in size, a phenomenon that can be understood from a topological standpoint. One of the central questions posed was the possibility of a conformal transformation (a map that preserves angles) of one closely related species to another without losing any of the species’ considerable corresponding features [7]. When do such transformations exist?

Within the lecture, Dr. Milnor discussed a paper by Xianfeng Gu of the Computer Science Department at Stony Brook and Shing-Tung Yau of Harvard University, on conformal mapping of the brain surface to a sphere [8]. This application can be considered an extremely useful tool in medical imaging. Mathematical methods can be used to more efficiently analyze and

The Stony Brook Young Investigators Review, Fall 2011

features compare brain images. With respect to the field, Dr. Milnor commented: “Mathematical biology is a relatively new subject, though statistics was used to study genetics for many years.

Certainly it is a very exciting field, especially data-mining and understanding how the many facts of the human genome give clues to many diseases.”

Mathematical Creativity When David Hilbert, a renowned German mathematician, was informed that one of his students was to leave the field of mathematics with a goal of studying poetry, Hilbert replied with positivity that the student was not creative enough to be a true mathematician. It is curious to understand the inconspicuous creativity involved in the study of advanced mathematics.

When does math stop being math and start being an art form?

When does the simple logic of equations stop being enough in generating proofs? According to Dr. Milnor, there is no clear distinction: “Math, when it is well done, tends to be an art form.

There is the distinction of math done out of curiosity or math done to solve some practical problem. In either case it can be done in an elegant or beautiful way. Sometimes there are questions and ideas that are important yet no one has found a nice way of doing them. There may be proofs that are just very ugly and that is always an unfortunate situation.”

Education and Legacy Stony Brook University graduate mathematics has been recognized among the top national programs in the field. National as well as worldwide interactions with the established faculty have helped produce phenomenal alumni and progressive students. As of May 2011, 6% of Stony Brook students graduate with a Bachelor’s degree in mathematics. Alan Tucker, a professor in the mathematics department at Stony Brook has argued to revitalize the math major known to his father’s (Albert Tucker) and Milnor’s generation dating to Princeton. He notes that the rigorous mental training of math majors produces a broad range of success in fields ranging from economics to the natural sciences [9].

The quality of the Stony Brook mathematical training can be reflected in Dr. Milnor’s position at the university. Dr. Milnor commented: “I was at the Institute for Advanced Study [at Princeton University] for many years, which was an idyllic place with perfect working conditions, but I found myself getting more and more isolated. I was working by myself and missed having students and giving classes. When I had an opportunity to start a small institute here at Stony Brook, it seemed like a very nice idea to me.” It is important to underscore the achievements of a professor by the success of his students, whom Dr. Milnor was able to produce at Stony Brook. “I have been very lucky in having some excellent graduate students here at Stony Brook”, he says. Among them are Jan Kiwi, Associate Professor at Pontificia Universidad Catolica de Chile; Saeed Zakeri, Associate Professor at Queens College and Graduate Center of CUNY; and Rodrigo Perez, Assistant Professor at Cornell University. Michael Spivak, who received his Ph.D. under the

supervision of Dr. Milnor, is well known for publishing multiple textbooks.

Research Techniques How is mathematical research carried out? A series of steps involves investigating specific properties, determining their legitimacy in several scenarios, generating a conjecture, and finally attempting to prove these properties. With no more materials necessary than a blackboard and chalk, established theories have come into existence However, mathematicians working at the peak of John Milnor’s career, such as Stephen Smale, Dennis Sullivan, William Thurston, Rene Thom, and Michael Freedman were all faced with a new crucial tool: computer incorporation. While some were enthusiastic and saw computers as a way to facilitate research, the general consensus was rather hesitant.

As Leslie G. Valiant, a fellow applied mathematician and theoretical computer scientist said at the International Congress of Mathematicians in 1986 regarding computers, “I should clarify my own position, I don’t use computers either” [4].

Dr. Milnor, on the other hand, conveyed his enthusiasm regarding the practical use of computers for simple tasks. “I am completely addicted. In the old days, just for the process of writing a paper I would have something typed up and I would scribble corrections all over it. The press secretary would have to change it again and a few times the secretary would get very grouchy. It is a different world now; we can make all the changes we want easily.” Dr. Milnor, however, does warn about the dangers of completely entrusting computers with respect to results. Computers do only as much as they are instructed to do, therefore, Dr. Milnor uses them with discretion. “There is a field called experimental mathematics in which you try to gather evidence about a problem which you cannot really solve,” Dr. Milnor said. “Computers are especially important to me in the area of dynamical systems when trying to figure out how some mathematical system changes with time. It is really complicated to work all the details out on paper. Thus, you try to work out what happens experimentally on a computer. There is always a danger of not knowing whether the computer model really grasps the original situation, so you have to be very cautious. Nowadays, solutions generated by computers are accepted as proof, but some people do not trust them. Classical proofs, if they are very complicated, can easily have mistakes as well.

Therefore, no matter what kind of proof it is you have to be very cautious and examine the conjectures very carefully.”

Recognition In 2011, Dr. Milnor received the highly prestigious Leroy P. Steele Prize for Lifetime Achievement award. “It is very gratifying. As I said at the ceremony, it is wonderful to get a prize for doing what you like to do.”

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features References 1. Milnor, John W. Topology from the Differentiable Viewpoint. Princeton, NJ: Princeton UP, 1997. Print.

2. Milnor, John. “Differentiable Structures on Spheres.” American Journal of Mathematics 81.4 (1959): 962-72. JSTOR. Web. <http://www.jstor.org/stable/2372998>.

3. Milnor, John. “Differential Topology Forty-Six Years Later.”

Notices of the American Mathematical Society 58.6 (2011): 804-09. Web. 20 Sept. 2011.

4. Gleick, James. “Reporter’s Notebook - But Aren’t Truth And Beauty Supposed To Be Enough? - NYTimes.com.” The New York Times - Breaking News, World News & Multimedia. 12 Aug. 1986. Web. 13 Sept. 2011.

5. Friedman, Yaakov. 2000. “A New Model for Physics Based on Mathematics of Symmetric Domains.” Jerusalem College of Technology, Jerusalem. Lecture.

6. Milnor, John. “Geometry of Growth and Form: Commentary on D’Arcy Thompson.” Lecture. Institute for Advanced Study, Princeton, Princeton. Institute for Advanced Study. <www.ias. edu>. Web. 13 Sept. 2011.

7. “Using a Computer to Visualise Change in Biological Organisms.” School of Mathematics and Statistics. School of Mathematics and Statistics University of St Andrews, Scotland JOC/EFR, Jan. 2000. Web. 13 Sept. 2011.

8. Gu, X., Y. Wang, T.F. Chan, P.M. Thompson, and S.-T. Yau. “Genus Zero Surface Conformal Mapping and Its Application to Brain Surface Mapping.” IEEE Transactions on Medical Imaging 23.8 (2004): 949-58. Print.

9. Tucker, Alan. “Revitalizing the 1960 Mathematics Major.”

Notices of the AMS 58.5 (2011): 704-05. Print.

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reviews Micro RNA and its Tremendous Role in Cancer Olesya Levsh, ‘13

Colorectal cancer is the second leading cause of death from cancer in the Western world. Gastric cancer is the second leading cause of death for men, and the third for women.

The fifth leading cancer-related cause of death is cancer of the liver, while pancreatic cancer has been deemed the fourth [1].

Among these numbers, hope dawns in the form of a prominent figure emerging in the field of oncology, and, though it is tiny, it is revolutionizing both our understanding of carcinogenesis and our ability to diagnose various cancers in phenomenal ways. Thanks to micro RNAs (miRNAs), scientists have been able to pinpoint patterns of expression so precisely that they have stumbled upon what may essentially be fingerprints for various types of cancers, complete with developmental history of the tumor. Shedding a new light on carcinogenic pathways gone awry, miRNA has helped spur progress in treatment as well as diagnosis.

A miRNA is a short (21 to 25 nucleotides), non-coding, negative regulator of gene expression. These molecules have the power to either destroy or inhibit the translation of messenger RNA, or mRNA, into protein. The mechanism by which this occurs involves one strand of a double-stranded miRNA entering an enzyme called RNA-induced silencing complex, or RISC, where it base pairs with a strand of mRNA and causes either its destruction or its inhibition. The difference in action is determined by the degree of mismatch between the base pairs of the two strands, and because this is the case, it is possible for each miRNA to target multiple genes for regulation [2].

These tiny, non-coding genes, discovered only two decades ago, are estimated to regulate over a third of all coding genes [1].

The link between miRNA and gene expression is the very factor that implicates miRNA in its involvement with cancer.

Cancer, as most know it, is a virulent mass of cells that does not cease replicating – but at the cellular level, it is much more complicated. For cells to become cancerous, they must ignore signals that tell them to undergo apoptosis (programmed cell death), checkpoints in the mitotic cycle, and a multitude of other feedback mechanisms. This provides cancerous cells with what are known to be their hallmark characteristics: unrestrained replicative potential and immortality. As long as cancerous cells continue to replicate and exhaust nutrients, such as oxygen, they will not die. And, unlike most cells, cancerous cells will never reach a phase of cellular senescence, or inability to undergo mitosis. They will trudge onward, dividing stubbornly regardless of what goes wrong – even if it is something as serious as improper DNA replication.

What could be the reason that these cells are so out of control? There is no simple answer. Responsible for this uncontrolled behavior is an amalgamation of several things, among which is gene expression. Research has linked miRNAs to gene amplification, deletion, and mutation – all of which contribute to the development of cancer. And because a single miRNA has the potential to affect many mRNAs, small changes in the

Figure 1. The cellular mechanism for production of miRNA.

expression of miRNAs can cause large changes in a cell’s protein diversity. For example, miR-100 is a potential target of the HOXA1 (HomeoboxA1) and IGF1R (insulin-like growth factor 1 receptor) genes, and has been shown to target the Plk1 gene, involved in regulation of mitosis [3].

The more researchers are delving into the mystery behind these miRNAs, the more they find that the proposed scenario makes sense. Typically, as cancerous cells become more harmful, they also become less differentiated. Experimentally, it has been shown that levels of miRNA are elevated when cells differentiate – in fact, cellular levels of miRNA are an accurate representation of differentiation, and within a cancerous cell, miRNA levels are lower than those within a healthy cell.

All of these findings point to an inverse relationship between miRNA expression and cell differentiation [4]. Since it is important to determine the origin of a tumor before treating

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it, miRNA has been utilized quite efficiently thus far – some- when present in high amounts, it reduces the rate of cell protimes, the deregulation of a single miRNA is enough to dis- liferation and increases cell sensitivity to radiation treatment, tinguish between closely related cancers, as Dr. Jingfang Ju of establishing it as an ideal candidate for therapy.

In hepatocellular carcinoma, one of the most important Stony Brook University’s Department of Pathology has found and best-understood miRNAs is the downregulated miR-122; [1]. In fact, Dr. Ju was the first researcher to discover that p53, it induces apoptosis, and also inhibits cancer growth, progresone of the most important regulatory genes in cancer, controls sion, and invasion by targeting a checkpoint miRNA expression [5]. He demonstrated that, in the mitotic cycle. Like miR-145 of gasin vitro, upregulation of certain miRNAs octric cancer, it increases cellular sensitivity curred when p53 was absent, and that levels to drugs, and is consequently a promising were low when p53 was overexpressed [6]. therapeutic target.

Like colorectal cancer, Amazingly, miRNA patterns were sucboth of these cancers also contain various cessfully used to not only distinguish between oncogenic miRNAs that serve similar funcdevelopmental origins of various carcinogenic tions: deregulating growth pathways and tissues, but even between those within the intensifying proliferation [1]. same type of tissue. Lu et al. collected bone Various ways to incorporate knowlmarrow samples from patients with acute lymedge of miRNAs and their functions into phoblastic leukemia and used miRNA analytreatment are presently being investigated. sis to successfully cluster them according to Given that countless problems leading to developmental lineage, proving that “miRNA carcinogenesis are spurred by a downreguexpression patterns encode the developmental lation of miRNAs, the logical approach is history of human cancers” [4]. to stimulate expression.

In a study where These are the facts, but how can we make cancerous bladder cells were treated with these miRNAs work for us? There are over a 5-Aza-CdR, an inhibitor of DNA meththousand miRNAs, and the network between ylation, and 4-phenylbutyric acid, a comthem and various mRNAs must be unimaginpound that restores and stabilizes wild type ably complex. It is the perfect labyrinth – perprotein structure, miR-127 expression was haps even impossible to solve. Rather than atstimulated, thereby causing inhibition of tempting to document this web of connections, the oncogenic BCL6 protein. scientists have taken a different approach: obTreatment with just 5-Aza-CdR was serving the miRNAs individually. enough to stimulate expression of miR-148 Colorectal cancers in various patients have and several other miRNAs useful in prevenshown consistency with regard to the downtion of metastasis.

Furthermore, some gene regulation of miR-145 and miR-192 and uptherapies using viral vectors containing the regulation of miR-20, miR-21, miR-31, and gene of interest are showing auspicious remiR-99b, suggesting a conserved pathway for sults.

When expression of miR-26a is ineach of these miRNAs. Upon further observatroduced into cancerous cells through such tion, miR-145 was shown to inhibit cell proa vector, the result is cellular apoptosis and liferation, specifically by inhibiting the insulin inhibition of proliferation [7]. receptor substrate 1 (IRS-1) protein, which is In prostate cancer, miR-205 and miRcrucial in mitogenic pathways. The upregulat31, two downregulated miRNAs, have been ed, and therefore oncogenic, miR-21 has been pinpointed to increase cellular sensitivity to associated with metastases, as well as high apoptosis induced by chemotherapy, and are tumor resistance to chemotherapy and a low currently valuable targets for treatment [8]. chance of survival.

Regarding non-small cell lung carcinoma, Therefore, observation of the many dereguchemotherapy often fails because it causes lated miRNAs in cancer patients can provide selection for resistant cells; forcing expresinsight into the development of the tumor and sion of miRNAs that increase sensitivity to even the likely prognosis. Additionally, miRchemotherapy is an encouraging solution NA circulating throughout the plasma can be [9]. used in relatively non-invasive tests for diagIn the foreseeable future, miRNAs are nosis. For example, the presence of downregulikely to become exceptional tools for both lated miR-92 is unique to colorectal cancer, Figure 2.

The secondary structure of the diagnosis and treatment of cancer. and can thus be used to distinguish it from miR-122, 85 nucleotides in length.

However, many aspects of miRNA remain other diseases with comparable symptoms [1]. enigmatic – for example, the method by Various cases of gastric cancer also show which these molecules are downregulated in various cancers similarities in deregulation that seem to be, for the most part, continues to confound scientists. universal. The downregulated miR-415 is a prime example –

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The Stony Brook Young Investigators Review, Fall 2011

reviews While there seems to be no obvious change in any of the proteins that process miRNA, they are still somehow being under-expressed, implying a separate pathway for regulation that has yet to be discovered [4]. Though it is difficult to predict the types of pros and cons these tiny miRNAs may bring into the field of medicine, the venture – given more time and inquiry – is filled with substantial promise.

References 1. Song, B. and Ju, J. (2010) Impact of miRNAs in gastrointestinal cancer diagnosis and prognosis. Expert Reviews in Molecular Medicine, 12, e33. doi:10.1017/S1462399410001663.

2. Meltzer, P.S. (2005) Small RNAs with big impacts. Nature, 435, 745-746. doi:10.1038/435745a.

3. Boni, V., et al. (2010). Role of primary miRNA polymorphic variants in metastatic colon cancer patients treated with 5-fluorouracil and irinotecan. The Pharmacogenomics Journal. doi: 10.1038/tpj.2010.58.

4. Lu, J., et al. (2005). MicroRNA expression profiles classify human cancers. Nature, 435, 834-838. doi:10.1038/nature03702.

5. Andrea Schmidt. (September 29, 2009). Jingfang Ju, Ph.D., Associate Professor. Retrieved March 11, 2011, from http:// www.stonybrookmedicalcenter.org/pathology/faculty/ju.

6. Xi, Y., Edwards, J.R., and Ju, J. (2007). Investigation of miRNA biology by bioinformatics tools and impact of miRNAs in colorectal cancer – regulatory relationship of c-Myc and p53 with miRNAs. Cancer Informatics, 3, 245-253.

7. Kelly, T.K., Carvalho D.D.D., and Jones, P.A. (2010). Epigenetic modifications as therapeutic targets. Nature Biotechnology, 28, 1069-1078. doi:10.1038/nbt.1678.

8. Bhatnagar, N., et al. (2010). Downregulation of miR-205 and miR-31 confers resistance to c h e m o t h e r a p y - i n d u c e d apoptosis in prostate cancer cells. Cell Death and Disease, 1, e105. doi:10.1038/cddis.2010.85 9. Lin, P.Y., Yu S.L., Yang, P.C. (2010). MicroRNA in lung cancer. British Journal of Cancer, 103, 1144-1148. doi:10.1038/ sj.bjc.6605901.

As a result of writing this review, Olesya developed an interest in miRNA and was accepted to a summer research program at Pennsylvania State University during the summer of 2011 to work on a new miRNA project. Congratulations Olesya!

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Poliovirus: A Promising Candidate for Effective Oncolytic Treatment of Neuroblastoma Amy Patel, ‘12

Have we finally developed a method to fight cancerous tumors, our callous rivals? Not exactly, but we have found an alternative to the adverse effects of chemotherapy and radiation therapy: oncolytic viruses. As infectious agents, viruses have been clinically proven to infect and lyse cancer cells, and have thus become a promising new class of antitumor therapy. Following viral replication, the engineered vectors act to destruct tumor cells and stimulate antitumor immune responses. While research has demonstrated successful results in animal models, efficacy on human subjects has been more restricted. Poliovirus is a recent addition to the list of oncolytic viruses that have therapeutic values. Interestingly, poliovirus has been proven to replicate efficiently in almost all tested tumor cell lines, making it an attractive approach for developing new treatment strategies against multiple cancers. [1] Poliovirus causes poliomyelitis, or polio, an illness that was first recognized in England in 1789, and reported in the U.S. in 1843. As the illness swept through developed nations in the Northern Hemisphere, the average age of the affected also increased, and consequently its severity. In 1952, paralytic cases radically elevated to about 21,000 in the U.S. A decline in polio cases occurred only after the certification of the poliovirus vaccine in the 1950s. Currently, this disease is almost extinct and only smatterings of cases are reported yearly throughout the world [2].

Poliovirus enters its host through ingestion and multiplies as it moves through the digestive tract. Unable to dissolve in the acidic conditions, it may stay intact and cause infection [2]. This non-enveloped, positive-stranded RNA enterovirus, a member of the family of Picornaviridae, may target motor neurons of the Central Nervous System (CNS), lead to poliomyelitis, and eventually death [3].

A virus can cause cell carcinogenesis by promoting harmful genetic alterations within the cellular DNA [4]. Because

viruses are an established cause of cancer, the introduction of these daunting infectious agents to an organism to impede tumor growth may seem unrealistic and doubtful. One may ask whether dangers such as paralysis by poliovirus invasion have been taken into account, or if vaccinations against polio given during childhood interfere with the ability of poliovirus to kill tumor cells in vaccinated children. Scientists were able to effectively account for these potential complications by designing experiments that tested each factor and resolved each issue [4].

This resulted in the development of highly attenuated polioviruses that would be suitable for tumor therapy without being noxious to healthy tissue. Additionally, an immunocompetent animal model resembling a human who has been vaccinated against polio was developed, which would allow for the examination of the oncolytic proficiency of neuroattenuated viruses [3]—viable viruses that have a marked reduction in their virulence towards the host [4].

Defining neuroblastoma and approaches towards its treatment Neuroblastoma is a form of cancer in which malignant cells form in the nervous tissue of the embryo or fetus. It occurs most often in infants and young children and has become a challenge for pediatric oncologists. [3,4] Resistance to conventional therapies, such as radiation therapy and chemotherapy, motivated researchers to find a new therapeutic approach to treat this tumor. Scientists proposed the introduction of a novel attenuated poliovirus as a promising oncolytic treatment of human neuroblastoma, even in the presence of polio immunity.

Replication of the virus in neuroblastoma cells of mice, Neuro2aCD155, which expressed the gene for the primate poliovirus receptor CD155, resulted in the elimination of neural tumors without causing paralysis or death. Furthermore, re-inoculation of Neuro-2aCD155 did not show signs of tumor growth in mice cured of neuroblastoma, indicating that the destruction of tumor cells by poliovirus may induce a potential antitumor immune response. [3]

Successfully attenuating poliovirus[4]

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After several trials, scientists in the laboratory of Dr. Cello at Stony Brook University found that a stable attenuation of poliovirus could be produced by the interception of the cisacting replication element (cre) into the “spacer region”. This spacer region is located between two domains, the cloverleaf and internal ribosomal entry site (IRES), in the 5’-nontranslated region (NTR), as shown in Figure 1. The native cre, a stem-loop structure, was duplicated and inactivated by 3 point mutations. Both mono-crePV and dual-crePV can replicate in human neuroblastoma, SK-N-MC, cells. However, it is less The Stony Brook Young Investigators Review, Fall 2011

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Figure 1. Structure of the PV1(M) and A133Gmono-crePV genome.

probable for a cre element deletion to occur in mono-crePV than in dual-crePV because the deletion of a single cre element, when there is only one to start off with, results in loss of viability. Furthermore, injecting either mono-crePV or dual-crePV into CD155 transgenic mice showed a strong phenotype of attenuation (Table 1), but neurovirulent variants of mono-crePV were never isolated from infected animals, which revealed mono-crePV as a good candidate for further investigations.

After finding that the cause of increased poliovirus replication in mice Neuro-2a cells was a point mutation (A133G), the transition was genetically engineered into domain II of the 5â&#x20AC;&#x2122;-NTR in mono-crePV. However, this increase in replication capabilities occurred in conjunction with an increase in neuropathogenicity (Table 1). [3] The A133G mutation increased poliovirus replication not only in Neuro-2a cells in mice, but also in human neuroblastoma cells, SH-SY5Y and SK-N-MC. Conversely, a major flaw of A133Gmono-crePV was revealed when two of the four mice treated with A133Gmono-crePV died due to paralysis. Geneticists later found that this could be prevented by immunizing the mice. [3]

Following PV1(M) administration, scientists established a Neuro-2aCD155 tumor in the immunized mice. When the tumor had reached a volume of 170mm3, intratumoral injections of either PBS or A133Gmono-crePV were randomly given to immunized mice for four consecutive days. While all PBS-treated mice were euthanized due to extreme tumor growth by day 8, the 12 mice injected with A133Gmono-crePV showed great regression in tumor size. The A133Gmono-crePV-treated mice also did not show any signs of paralysis and resulted in a mean tumor volume of 128.8mm3. However, only 9 mice showed an absence of recurrent tumors by day 180, as indicated in Figure 3. When the two mice with recurrent tumors were treated with A133Gmono-crePV again, they showed extreme tumor growth and were euthanized. Western blot analysis showed that the expression of CD155, when compared to non-recurrent tumors, was low in the residual and recurrent tumor cells. [3]

Re-challenge of neuroblastoma-cured mice Scientists hypothesized the existence of acquired antineuroblastoma immunity and tested this hypothesis by rechallenging mice that showed complete regression with Neuro-2aCD155 cells. None of these re-challenged mice showed signs of tumor growth, which enabled them to conclude that oncolytic therapy by A133Gmono-crePV leads to an antitumor response that is independent of A133Gmono-crePV after about 6 months. [3]

Poliovirus-mediated tumor treatment The current treatments for neuroblastoma in children are radiation therapy and chemotherapy [4]. Novel therapeutic strategies are essential to replace these noxious treatments. Poliovirus was chosen as the commendable candidate because it is benign, as long as accidental invasion of the CNS is avoided. As

The Treatment The sequence of events leading to the therapy of Neuro-2aCD155 tumors in mice is presented in Figure 2. Three times a week, CD155 tgA/J mice were immunized with mono-crePV, causing the formation of high titers of neutralizing antibodies against the virus. Neurovirulent poliovirus type I (PV1(M)) was administered to both the immunized and control mice to observe antibody capability of protection from paralysis. All of the conCD155 tumors trol mice died within 5 days of PV1(M) Figure 2. Graphic representation of A133Gmono-crePV therapy on Neuro-2a in CD155 tgA/J mice with immunity against poliovirus. injection [3].

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described above, this novel attenuated oncolytic poliovirus was proven to be an effective treatment for neuroblastoma in mice [3]. The antitumor immune response evoked by the infection and lysis of neuroblastoma cells was significant. A recent study showed that intratumoral inoculation of attenuated poliovirus strain into Neuro-2aCD155 tumors in immunocompetent mice induces a systemic cytotoxic CD8+T cell-mediated immunity against neuroblastoma [5]. The animal model used in this study may hold as a guide for oncolytic treatment of human neuroblastoma. Scientists are hoping to enter an exploratory trial in humans in the near future [4].

References 1. 2011 American Society of Gene & Cell Therapy. “Oncolytic Virotherapy: Molecular Therapy.” Nature Publishing Group : Science Journals, Jobs, and Information. 15 Mar. 2011. <http:// www.nature.com/mt/webfocus/oncolytic/index.html>.

2. Hoyle, Brian. “Poliomyelitis.” Gale Encyclopedia of Neurological Disorders. 2005. Retrieved June 14, 2011 from Encyclopedia.com: http://www.encyclopedia.com/doc/1G2-3435200283. html.

3. Toyoda H, Yin J, Mueller S, Wimmer E, Cello J. 2007. Oncolytic treatment and cure of neuroblastoma by a novel attenuated poliovirus in a novel poliovirus-susceptible animal model.

Cancer Res 67(6):2857-2864.

4. Jeronimo Cello, Department of Molecular Genetics and Microbiology. Stony Brook University, Stony Brook, NY. Personal communication. March 16, 2011.

5. Toyoda H, Wimmer E, Cello J. 2011. Oncolytic poliovirus therapy and immunization with poliovirus-infected cell lysate induces potent antitumor immunity against neuroblastoma in vivo. International Journal of Oncology 38: 81-87.

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Figure 3 Elimination of established neuroblastoma tumor implants in CD155 tgA/J mice with A133Gmono-crePV.

The Stony Brook Young Investigators Review, Fall 2011

reviews Graphene: The possibilities are endless Zaki Shafi, ‘12

When Alexander Parkes introduced Parkesine, the first human-made plastic, at the Great International Exhibition of 1862 in London, little did the world know that this new class of synthetic polymers would single-handedly metamorphosize humanity’s existence [1]. For proof, look around you; or rather, look at yourself. There will undoubtedly be at least four different plastics on your person and every single person around you: belts, cell phones, pens, anything. From car tires to grocery bags to Teflon military armor, plastic is a god among synthetic materials, present in literally thousands upon thousands of products necessary for daily living.

While plastics reign in the field of structural matters, electronic mobility prays to another god: silicon. The use of silicon for its remarkable semiconductive properties has monopolized the Information Technology industry and shrunk the global network. The modern world simply cannot function without the applications of silicon in transistors and computer chips; without it, Moore’s Law would never be realized. IT has grown tremendously on the utilization of this single vital element in every single electronic device dealing with data transfer and manipulation. Silicon and plastic rule the modern world.

But now comes along a Nobel Prize winning discovery to conquer both thrones. On December 10, 2010, Andre Geim and Konstantin Novoselov received the Nobel Prize in Physics “for groundbreaking experiments regarding the two-dimensional material graphene.” [2] The two Russian physicists first isolated graphene in 2004 by simply sticking pencil lead between two sticky sides of Scotch tape and peeling it apart, layer by layer. In just six years, graphene, a single layer of pencil lead, is now a leader in atomic, thermal, electrical, optical,

and physical properties. It sweeps the board with its range of remarkable properties; it is a goldmine not just for applications, but theoretical research as well. [3] Graphene is essentially a monolayer of graphite. More explicitly, it is a single atomic plane of sp2 hybridized carbons arranged in a hexagonal honeycomb lattice structure with each carbon bonding to three others. It looks a bit like chicken wire extending infinitely in two dimensions. Of course, empirically, it has a third-dimensional component on the scale of atomic diameters, but from the point of view of encircling electron clouds, this atomic width is so insignificantly minute that it is mathematically negligible. So for all intents and purposes, graphene lies merely in two dimensions in our x, y, z world of triads. But a two dimensional material in itself is not very impressive. There are carbon allotropes existing with even fewer dimensionalities; from the perspectives of mathematical modeling and electron clouds, fullerenes are considered zero dimensional point particles and nanotubes lie in one dimension as a simple line. But the grandest of the allotrope family is the material acting in two dimensions: graphene. [3] Graphene is the strongest material ever tested in science. Measurements show that it has an elastic breaking strength 200 times that of steel with an intrinsic strength of 130 gigapascals. Its strength comes from its elastic properties. When pulled along its dimensional axis, it is extremely difficult to tear. Graphene has a second and third order elastic stiffness of 340 N m-1 and 690 N m-1, respectively. Its breaking strength is 42 N m-1 and has a Young’s modulus of 1.0 terapascals. In the more visual terms of Columbia’s Fu Foundation of Engineering researchers, “It would take an elephant, balanced on a pencil, to break through a sheet of graphene the thickness of Saran Wrap.” [4]

Massless Dirac Fermions

Graphene is the building block of 0-D fullerenes and 1-D nanotubes.

The real beauty of graphene lies in its electronic properties; its semiconductive capabilities can surpass that of silicon a dozen times over. But in addition to superior conductance, graphene’s electrons behave very unusually.

Carbon atoms have four valence electrons. While three of these in graphene are used for carbon to carbon bonds, the fourth electron exists in perpendicular p orbitals. Graphene’s sp2 hybridization allows for these p orbital electrons to move about the entire plane as their field. Without the structural restrictions of three-dimensional materials, these electrons essentially have no scattering. Ergo, graphene’s electrons are highly delocalized and have great mobility. [8] The most interesting feature of graphene arises from the generation of quasiparticles as a result of the charge carrier’s (electrons and electron holes) interactions with the “periodic potential of graphene’s honeycomb lattice” when graphene

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reviews is studied as a semiconductor [3]. The single most exciting feature of graphene is the classification of these quasiparticles as “massless Dirac fermions” [3,5,6,7]. To understand this concept, the following definitions are necessary.

Quasiparticles are excitations that behave as particles; pockets of energy with momentum, such as photons, phonons and fermions. Fermions are quasiparticles that have a halfinteger spin and can occupy only a single quantum state at a time in accordance with the Pauli Exclusion Principle. A Dirac fermion is simply a fermion with a respective antiparticle.

An electron is classified as a Dirac fermion because it has a positron as its respective antiparticle. Most fermions have a corresponding antiparticle and so most fermions are classified as Dirac fermions. [3,5,6,7] In most materials, Erwin Schrodinger’s wave equation efficiently models quantum behavior, but graphene’s fermions, exhibiting relativistic behavior, follows the Dirac relativistic quantum mechanical wave equation. Momentum and mass have an intrinsic relation with the energy of particles and quasiparticles. From Schrodinger’s equation, the following relation relates a particle’s energy, mass, and momentum: E = p2/2m.

This equation holds only when studying non-relativistic phenomena. However, fermions in graphene, electrons and electron holes, travel at relativistic speeds. [3,6,7] From the Dirac equation, the relation between mass, momentum, and energy for particles moving at the speed of light is given by E = ±√(m2c4+p2c2) [5]. Graphene is a massless relativistic quasiparticle (no rest mass is the proper term) and so the equation simplifies to E = ±pc [6][7]. Graphene has a Fermi velocity of c/300 ≈ 106ms-1[3,6]. And so the c, the speed of light, is replaced by vf, the Fermi velocity of graphene, to give E = ±√(px2+py2)vf. [5,6,7,8]. This equation relates energy, momentum (no mass for graphene’s fermions) and the position of the fermions in space. The graph of this equation is shown below on the right. The relation on the left shows the situation for “normal” materials whose mass results in an energy gap in contrast with graphene’s massless wonders.

The zero band gap is significant for semiconductor applications and quantum relativistic studies. As shown in the graph on the left, other semiconductive materials, whose quasiparticles have mass, have a band gap separating top and bottom energy curves. This signifies the amount of energy that must be supplied to ‘jump’ an electron from the valence to the conducting band. But for graphene, there is no band gap.

Note the unique cone structure of the energy graph that is so different from most semiconductive materials.

The Dirac point leads to endless possibilities for theoretical physicists, experimentalists and innovative engineers.

Aside from the numerous applications possible for engineers, graphene is commonly used for “tabletop experiments” by physicists [3]. To study quantum relativity, there is now no need to expend vast amounts of energy on multimillion dollar particle accelerators, because graphene’s fermions are already travelling at relativistic speeds, even at room temperature; a truly monumental opportunity for science. Physicists all over the world can now explore Einstein’s theory of relativity simply with scotch tape and some pencil lead. In our very own

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The band structure of a representative three-dimensional solid (left) is parabolic, with a band gap between the lower-energy valence band and the higher-energy conduction band. The energy bands of two-dimensional graphene (right) are smooth-sided cones, which meet at the Dirac point.(http://www.lbl.gov/Science-Articles/ Archive/assets/images/2008/May/23-Fri/hires/Graphene-band-gaps.jpg)

community, Prof. Xu Du of the Physics & Astronomy Department is currently working on this revolutionary molecule. In his own words, his primary research focus is “to develop a deep understanding of the emerging materials and to explore their applications in new technologies.” For the past three years, all of Prof. Xu Du’s publications have been experimental reports about graphene.

Dr. Du’s experiments rely on the newfound capability to isolate a single crystal layer of graphene and study it in a suspended form. If the layer lies on a surface, it is easily disturbed, its lattice structure slightly skews and disorders are observed; obtained data is unrepresentative of intrinsic graphene. But in a suspended form, the material can be studied without disturbances.

Dr. Du focuses mainly on studying graphene’s electrical properties. More specifically, he focuses on four aspects. The first is the physics of Dirac fermions and understanding their behavior under induced magnetic fields, as well as the scattering of the fermions as they traverse the lattice field. The second aspect is an exploration into the superconductive applications by studying the “superconducting proximity effect and graphene based balometers.” [12] The third facet of his work is in collaboration with Prof. Matthew Dawber, who teaches honors Classical Physics here at Stony Brook. This work focuses on ferroelectric devices, whose electrical and magnetic properties can be altered by changing microscopic structure. Prof.

Du’s final area of focus is the development of graphene-based electronics and sensors.

Applications Measurements of graphene’s electronic properties reported by groups such as Prof. Du’s are in accordance with the Dirac theory and establish graphene as one of the most electrically conductive materials tested. The significance of the Dirac point and a zero band gap will lead to the replacement of silicon and the renewal of Moore’s Law. For some time, engineers have predicted the end of Moore’s Law due to structural limitations of silicon for electronics. But now, graphene has the potential to replenish the electronics industry and lead to innovations

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In addition to its outstanding physical and electrical properties, graphene also has supreme thermal properties. Its thermal conductance far surpasses silicon. Thermal conductance is important because heat is constantly generated in transistors and other electronic devices. Efficiency of electronics directly relates to how rapidly generated heat can dissipate.

[9] Graphene’s supreme electrical properties in conjunction with great thermal conductance and mechanical strength make it an ideal new vessel for continued electronic evolution and its subsequent rise to claim the throne of plastic and silicon.

Graphene has a final unique and magical property: it is optically conductive. Researchers have recently demonstrated graphene’s ability to conduct photons and become electrically charged. However, graphene, having a thickness of only one atom, has about a 97% transparency which prevents efficient photon capture [10]. Regardless, this remarkable feature cannot be dismissed and can lead to widespread applications, such as light emitting diodes, paper-thin touch-screen devices that recharge by absorbing light, and infinitely more.

A complete coverage of graphene’s possible commercial applications is quite impossible as human innovation knows no bounds. A material with outstanding atomic, electrical, optical, thermal and physical properties really has no limits. The most pressing and obvious application is the immediate replacement of silicon. However, that covers just one of its five remarkable features. Dr. Michio Kaku, Professor of Theoretical Physics at the City College of New York and a well-known communicator of science, does best in summarizing the future implications of graphene: “Potential applications for the material include the replacing of carbon fibers in composite materials to eventually aid in the production of lighter aircraft and satellites; replacing silicon in transistors; embedding the material in plastics to enable them to conduct electricity; graphene-based sensors could sniff out dangerous molecules; increasing the efficiency of electric batteries by use of graphene powder; optoelectronics; stifferstronger-lighter plastics; leak-tight, plastic containers that keep food fresh for weeks; transparent conductive coatings for solar cells and displays; stronger wind turbines; stronger medical implants; better sports equipment; supercapacitors; improved conductivity of materials; high-power high frequency electronic devices; artificial membranes for separating two liquid reservoirs; advancements in touchscreens; LCD’s; OLED’s; graphene nanoribbons could be a way to construct ballistic transistors; and nanogaps in graphene sheets may potentially provide a new technique for rapid DNA sequencing.” [11]

References 1. Stephen Fenichell, Plastic: The Making of a Synthetic Century, HarperBusiness, 1996, ISBN 0887307329 p. 17 2. “The 2010 Nobel Prize in Physics - Press Release”. Nobelprize.org. 7 Jan 2011 http://nobelprize.org/nobel_prizes/physics/laureates/2010/press.html.

3. Geim, A.K., and Novoselov, K.S, The Rise of Graphene, Nature, v. 6, pg. 181-191 (March 2007).

4. Lee, C., Wei, X., Kysar, J. W, Measurement of the elastic properties and intrinsic strength of monolayer graphene, Science, v. 321, no. 5887 pg. 385-388 ( July 2008).

5. Nomuar, K. and McDonald, A.H., Quantum Transport of Massless Dirac Fermions, American Physical Society, PRL 98, 076602 (February 2007).

6. Charlier, J.C., Eklund, P.C., Zhu, J., and Ferrari, A.C, Electron and Phonon Properties of Graphene: Their Relationship with Carbon Nanotubes, from Carbon Nanotubes: Advanced Topics in the Synthesis, Structure, Properties and Applications, Ed. A. Jorio, G. Dresselhaus, and M.S. Dresselhaus. Berlin/ Heidelberg: Springer-Verlag 2008.

7. Geim, A.K. and Novoselov, K.S, Two-dimensional gas of massless Dirac fermions in graphene, Nature 438 (7065): 197–200. (2005).

8. Nomura, K., Koshino, M., and Ryu, S., Topological Delocalization of Two-Dimensional Massless Dirac Fermions, The American Physical Society, PRL 99, 146806 (October 2007).

9. Balandin, A.A, Superior Thermal Conductivity of SingleLayer Graphene, American Chemical Society, v. 8(3) pg. 902907 (February 2008).

10. Falkovsky, L.A, Optical properties of Graphene, IOP Publishing, Journal of Physics: Conference Series 129 012004 (2008).

11. Kaku, Michio. “Graphene Will Change the Way We Live.”

Dr. Kaku’s Universe. Big Think, 06 Oct 2010. Web. 4 Dec 2010.

12. Du, Xu. “Xu Du Research Group.” Stony Brook Department of Physics and Astronomy. Stony Brook University, n.d, Web. 10 Apr 2011.

Dr. Kaku’s insightful list covers just the most obvious applications of graphene. We can safely assume that this list will grow tenfold in the coming years.

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Gene therapy and the Power of Chemoselective Vector Modification Faye Vassel, ‘11

Gene Therapy: What is it, and how does it work?

In the last forty years, advances in our understanding of genes and their functions have set the stage for scientists to tackle genetic disorders by employing a host of genetic manipulations. By targeting deregulated genes, scientists have been able to utilize molecular techniques to successfully alter an organism’s genetic material to fight or prevent disease. Gene therapy is a technique commonly employed by researchers to correct defective genes that may lead to abnormal cell function. Specifically, it is an experimental treatment that involves introducing genetic material (DNA or RNA) into a person’s cells to fight disease [1]. In general, therapeutic genes cannot be delivered directly to target cells. To address this issue, carrier molecules called “vectors” are used to deliver modified genes to their target cells. Currently, viruses, such as retroviruses and adenoviruses, are the most common types of vectors that are genetically altered to carry therapeutic genes. Because viruses are unable to replicate on their own, they have evolved ways of invading and delivering their genes to host cells in a pathogenic manner that results in viral gene replication. In viral-mediated gene therapy, scientists take advantage of this capability and manipulate the virus genome to remove disease-causing genes and insert therapeutic genes [2]. Researchers are studying a variety of ways to treat diseases—including hemophilia, HIV, and cancer—using this technique. Some viral-mediated gene therapies replace disease-causing mutated genes with healthy copies, while others are used to inactivate, or “knock out,” a mutated gene that is functioning improperly.

illness. Conditions range from the common cold to pneumonia.

Adenoviral infections are generally self-limiting, yet in some cases adenoviruses have been observed to cause other illnesses, such as gastroenteritis and conjunctivitis.

Though adenoviruses function in nature as pathogens, researchers have shown that when their viral genome is manipulated correctly, replication-defective or conditionally replicating viruses have proven to be potent gene delivery systems. In most adenovirus-mediated gene therapies, specific modifications are made to viral fiber proteins to direct HAdV to targeted cell types.

The appeal of adenovirus mediated gene therapy: Developments and setbacks in the field The advantages of using recombinant HAdV vectors in gene therapy are immense, for HAdV vectors have proven to be powerful viral delivery systems. These advantages include: relative ease of production, genome stability, an ability to transduce dividing and non-dividing cells with high efficiency, low level of vector genome integration, and well-defined virus biology [4]. Adenoviral host cell entry is mediated via fiber proteins attached to knob regions that protrude from the vertex of the viral icosahedron. Once inside the host cell, the adenoviral

What are adenoviruses?

In recent years, researchers conducting viral mediated gene therapy studies have primarily used retroviruses or adenoviruses to deliver the desired therapeutic genes to target cells. These viral vectors differ in whether they alter the cell’s DNA permanently or temporarily and in how efficiently they transfer genes to the cells they recognize and are able to infect [1]. The viruses used in viral mediated gene therapies are chosen according to the goals of the research study.

Adenovirus mediated gene therapies typically use human adenovirus type 5 (Human Ad5) as gene transfer vectors due to their low pathogenic potential and ease of production. The wild type Human Adenovirus (HAdV ) is a non-enveloped virus comprised of a double-stranded linear 36 kb genome packaged into an icosahedral protein capsid, of approximately 90-100 nm [3].

Each viral particle has a fiber protein that protrudes from the vertex of the icosahedron. While there are at least 52 immunologically distinct types of adenoviruses that can cause human infections, adenoviruses most commonly cause respiratory

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Schematic of adenoviral replication in a host cell. (www.liebertonline.com)

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Illustration of the two-step “click” labeling process Isaac Carrico’s group uses to selectively modify adenoviral particles. (http://pubs.acs.org)

vector enters the nuclear pore complex (NPC), where the viral particle disassembles and the viral DNA is able to enter the nucleus and replicate. In the early stages of replication, the E1, E2, E3, and E4 promoters use viral and host cell machinery to mediate the replication of viral DNA [5]. Over the last two decades, a variety of HAdV vectors have been designed based on modifications of the E1, E2, E3, and E4 genes in the viral genome. The E1 region encodes for gene products that are directly involved in the replication of the virus, whereas the E2 region encodes for proteins that provide the machinery for viral DNA replication. The gene products encoded by the E3 region modulate the immune response of infected cells. The proteins encoded by the E4 region regulate the metabolism of virus messenger RNA and provide functions that promote virus DNA replication and shut-off of host protein synthesis.

Adenoviral vector molecular modifications commonly fall into three categories. “First generation” adenoviral vectors were the first HAdV gene transfer vectors and have a deletion of the E1 gene region, and, at times, a deletion of the E3 gene region.

These adenoviral vector modifications are appealing because they can mediate strong short-term transgene expression in immunocompetent animals [4]. “Second generation” viral gene modifications further expand the capacity of adenoviral vectors by deleting E1 gene regions as well as inactivating or deleting E2 or E4 regions. These vectors may allow for prolonged transgene expression and lower host cell immune responses. The most commonly used adenoviral vectors are known as “third generation” adenovirus vectors. With “third generation” adenovirus vectors, researchers delete all viral coding sequences. As a result, “third generation” adenovirus vectors have a capacity to hold up to 36 kilobases of foreign DNA [5]. Most importantly, because “third generation” adenovirus vectors lack viral genes, their in-cell toxicity and immunogenicity are greatly reduced, resulting in significantly longer transgene expression.

While significant progress has been achieved with gene therapy studies that manipulate the adenovirus vector genome, researchers using the HAdV vector system still face the issue of safely and efficiently expressing these vectors in vivo. One

hurdle with using HAdV vectors arises from the induction of a strong innate immune response, which may be caused by HAdV vectors interacting with antigen-presenting cells, like macrophages [4]. This often results in the destruction of the HAdV gene transfer vectors long before target cell modifications occur. Another hurdle that researchers face when genetically reengineering HAdV vectors arises from the lack of specific details about the viral protein structure and viral protein assembly. Without more structural information, researchers who employ genetic manipulations to modify HAdV vectors risk compromising viral production and/or infectivity. Also, because HAdV binds to coxsackie and adenovirus receptors (CAR), which are present on a host of cell types, many genetic manipulations fail to generate HAdV vectors with dedicated specificity for a particular cell line [4]. Consequently, it is evident that most of the issues surrounding efficient and effective HAdV mediated gene delivery emerge from the fact that the majority of targeting approaches rely on genetic manipulation of viral surface proteins.

Current advances To overcome issues inherent to adenovirus genetic manipulations, such as immunogenicity, disrupted virion assembly, and limitations in adenoviral vector functionality, researchers have turned to the field of chemical biology. It has been recognized that the chemical modification of viral particles with synthetic polymers, also known as the bio-orthogonal chemical reporter strategy, has proven to be a promising way to overcome in vivo hurdles typically associated with adenovirus gene transfer vectors. One particularly significant advantage of chemical modifications of HAdV vectors is that all modifications are made after the production and purification of viral vectors. Currently, the majority of chemical modifications utilize Polyethylene Glycol (PEG) molecules. PEG molecules are appealing tags because their chemical composition masks the agent from the host immune system, thus reducing the immune response. In addition to this, PEG molecules have been observed to pro-

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reviews long agent circulatory time. Though PEG chemical modifications can be made on a variety of molecular targets because these modifications are dependent upon the altering naturally occurring residues, they are less precise than alterations made via viral genetic manipulations and thus may not be as efficient [4]. Other chemical modifications commonly made to viral particles include the incorporation of unnatural amino acids into simple viral protein assemblies and the modification of natural residues on the surfaces of viruses or virus-like particles with azides or alkynes. (While these molecules readily facilitate imaging and drug delivery applications, their relatively nonspecific construction prohibits infection and gene delivery into mammalian cells [6].

Researchers in Dr. Isaac Carrico’s lab at Stony Brook University have circumvented this issue by using a two-step “click” labeling process. “Click chemistry” is a term coined by K. Barry Sharpless of The Scripps Research Institute. It describes the method used to generate substances quickly and efficiently by joining small molecules together. A staple characteristic of click reactions is general insensitivity to oxygen and water. The latter allows click chemistry to be used in a variety of reactions, from those that involve drug-discovery to those that use click chemistry to generate small chemical reporters that can be used to modify biological molecules. In response to questions about the increased use of click chemistry mediated reactions, Dr. Carrico said: “Click chemistry is appealing to both chemists and biologists alike because of its relative ease and efficiency”[6]. The “click” labeling process employed by the Carrico group labels adenoviral particles during production by metabolically incorporating unnatural azido sugars to viral fiber proteins [7]. Importantly, the Carrico group has shown that subsequent chemical modifications can be made to the viral vectors, which allow for an array of viral effector functionality. The Carrico group has used click chemistry to generate chemoselective adenoviral vectors that are capable of selectively targeting breast cancers cells. To accomplish this, the Carrico group incorporates azido sugars, N-azidoacetylglucosamine (GlcNaz), to specific serine (Ser109) residues on the adenoviral fiber protein.

Dr. Carrico’s group incorporates azide chemical reporters into vectors using a variation of the Straudinger ligation, a reaction that has been used to modify glycans by covalently attaching molecular probes to azide-bearing biomolecules [7].

The modified Staudinger ligation reaction, conducted by the Carrico group to produce chemoselective adenoviral vectors, employs a copper catalyst and is known as “Copper-catalyzed [3+2] azide-alkyne cycloaddition”. The primary advantages of copper-catalyzed click-labeling over the Staudinger ligation are its faster rate of reaction, selectivity and easily obtainable reagents. Particularly, copper catalyzed click-labeling is advantageous because the introduced azide allows for chemoselective modification of full infective adenovirus particles with flurophores, peptides, and other small molecular targets [8].

By taking advantage of these properties, the Carrico group has been able to produce adenoviral vectors modified with folate, a molecule that can be used to chemoselectively target breast cancer cells, where folate receptors are frequently overexpressed.

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What is also quite remarkable is that click chemistry-mediated folate modification has even enabled efficient gene delivery to murine breast cells, which are normally refractive to adenoviral infection [7]. The primary disadvantage of copper-catalyzed “click” labeling is the cellular toxicity of the metal catalyst [8].

However, Dr. Carrico’s group and others have started to overcome issues of metal catalyst toxicity by using “strain-promoted cycloaddition,” which activates azides via the use of ring strain.

While “strain-promoted cycloaddition” is currently not as fast as copper-catalyzed click labeling, it is as selective and thus is proving to be a more attractive alternative.

In summary, the relative ease and specificity of click labeling demonstrates that adenoviruses can be chemoselectively labeled and subsequently used as effective gene transfer vectors.

The non-perturbing nature of the two-step process employed by the Carrico group suggests that in the future, researchers may be able to use click chemistry to safely and selectively label a host of viral gene transfer vectors without compromising the efficiency of viral production or infectivity. Most importantly, the fact that the Carrico group has shown that chemical-mediated adenoviral modifications are extremely potent and potentially more effective than genetic modifications suggests that, with more research, viral-mediated therapies may be designed to be used safely and effectively in translational research as tools to combat debilitating human diseases.

References 1. “Gene Therapy for Cancer: Questions and Answers - National Cancer Institute.” Comprehensive Cancer Information - National Cancer Institute. Web. 20 Jan. 2011. <http://www.cancer.gov/ cancertopics/factsheet/Therapy/gene>.

2. “Gene Therapy.” Oak Ridge National Laboratory. Web. 20 Jan. 2011. <http://www.ornl.gov/sci/techresources/Human_Genome/ medicine/genetherapy.shtml>.

3. “Gene Therapy Adenoviral Vectors Explained.” Gene Therapy Net - News, Clinical Trials, Viral Vectors and Patient Information. Web. 20 Jan. 2011. <http://www.genetherapynet.com/viralvectors/adenoviruses.html>.

4. Kreppel F. and Kochanek S. 2008. Modification of adenovirus gene transfer vectors with synthetic polymers: A scientific review and technical guide. The American Society of Gene Therapy, 16(1): 16-29.

5. “Adenovirus.” 17.14. Gene Therapy Review. 04 Sept. 2008.

Web. 25 Jan. 2011. <http://www.genetherapyreview.com/genetherapy-education/gene-transfer-vectors/1-viral-vectors/2-adenovirus.html>.

6. Carrico, Isaac. Personal Interview. 14 December 2010.

7. Banerjee P., Ostapchuk P., Hearing P., and Carrico I. (2010).

Chemoselective attachment of small molecule effector functionality to human adenoviruses facilitates gene delivery to cancer cells.

Journal of The American Chemical Society, 132(39): 1361513617.

8. Prescher J. and Bertozzi C. (2005). Chemistry in living systems.

Nature Chemical Biology, 1(1): 13-20.

Congratulations to Faye on her acceptance to graduate school in biochemistry at MIT, Fall 2011!

The Stony Brook Young Investigators Review, Fall 2011

reviews Farewell to Diabetes: A Scientific Breakthrough Kazi Ullah, ‘14 and Malack Hamade, ‘13

Piercing an index finger to test for hyperglycemia is a tale familiar to every diabetic. Depending on the severity and type of diabetes, the frequency of monitoring blood sugar levels can vary anywhere from once a week to ten times a day with every diabetic. The burden of living with such an invasive disease coupled with the staggering number of a reported 350 million people who suffer from it worldwide has encouraged extensive investment in diabetic research. [1] While the cause at the heart of the problem is essentially one, initiatives in researching diabetes have spurred from many fields including genetics, neurobiology and cell differentiation, to name a few. Chronicled in this article are the latest of these initiatives, revolutionary approaches to what may be the next big discoveries in curing diabetes since the extraction of insulin in 1921.

Overview A simple sugar, glucose is obtained from our diets and circulates through our bloodstream as an accessible energy source for our cells. In order for cells to metabolize glucose, the pancreas must first secrete insulin, a hormone that induces the transcription and insertion of GLUT family transporters into cell membranes. These transporters then allow glucose molecules to diffuse across the plasma membrane of cells to be metabolized.

When regulated, glucose is essential for the body’s metabolic processes and everyday functions. If left to accumulate in the bloodstream, glucose becomes the root cause of severe symptoms such as ketoacidosis, blindness and renal failure—all characteristic of diabetes.

Diabetes Mellitus is marked by the sufferer’s inability to properly process insulin. In type I diabetes, the patient’s T-cells attack the insulin-producing beta-cells in the islets of Langerhans in the pancreas, depleting the patient of an insulin source.

This type is also referred to as insulin-dependent diabetes and is often seen in children and young adults. The other type, type II diabetes, is the more common form of diabetes in the United States; it is a condition in which the cells of the body grow increasingly irresponsive to the insulin being produced. Type II diabetic cells are said to be insulin resistant. Comorbidity with obesity is usually the case, if not the cause, of type II diabetes.

[1]

Hope In 2006, a study identified two mutations in the trpv1 gene as potential causes of autoimmune diabetes. Defects in neurons expressing trpv1 gene led to minimal secretion of substance P—a compound critical to islet cell regulation of insulin—from the nerve terminals of the pancreas. When comparing the nerves

(which express the TRPV1 receptor) of diabetes-prone mice to that of healthy mice, it was found that the nerves in diabetesprone mice did not produce sufficient amounts of Substance P. This inability to produce Substance P led to hyper-insulinemia and consequently, insulin resistance and islet cell destruction in autoimmune diabetes. [2] Direct injection of Substance P into the pancreas kept nonobese mice healthy for up to three weeks, and some even for months. The suggestion was promising: restoring the function of the TRPV1 neuron could result in the reversal of autoimmune diabetes.

While genetic observations were critical to the findings in the aforementioned study, a team of researchers at Stony Brook University took genetic practices even further by demonstrating a complete reversal of hyperglycemia in diabetic mice subjects through the use of induced-pluripotent stem cells. An induced pluripotent stem cell, or iPS cell, is created from an adult cell such as a liver, stomach or other mature cell through the introduction of genes that reprogram the cell and virtually transform it into an embryonic stem cell. This enables the iPS cell to transform from an undifferentiated state to any of the 220 types of cells in the human body. [3] In the study, iPS cells were differentiated into insulin-secreting beta-like cells; cells triggered by the presence of glucose.

The beta-like cells were transplanted into the portal veins of thirty mice—half of them type I diabetic and the other half type II—and allowed to engraft. The hyperglycemia in both mouse models was corrected and by the 12th week, 15 mice were able to remain “normoglycemic.” Although thirteen mice died in total by the end of the study, six of them were intentionally killed for histological purposes. And of the surviving mice, only two had relapsed into hyperglycemia after 8 weeks, an extremely promising statistic. [4] In the four months the type I diabetic mice were followed, glucose levels dropped rapidly and remained stable post-transplantation [4]. In the type II diabetic mouse models, the transplanted iPS cell-derived beta-like cells increased the amount of insulin significantly. An average 4.35-fold increase in insulin levels was observed in treated mice compared to levels in untreated mice indicating that transplanted cells are able to prevent insulin resistance and beta-cell failure. Conclusively, the study hinted that an iPS cell-derived beta-like cell therapy is able to correct for a hyperglycemic phenotype by restoring insulin secretion and consequentially, normal glucose levels [4].

An immunology study conducted in 2003 used splenocytes, splenic white blood cells, to eliminate autoimmunity and restore normal glycemic levels in mice. Splenocytes impede T-cells from attacking islets by presenting partially or fully matched MHC Class I antigens. The antigen specific-cytotoxic

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reviews REPORTS T-cells latch onto these MHC Class I antigens of the splenocyte and are directed against attacking the beta-cells of the pancreas. This has the potential to reverse diabetes by inducing the reappearance of insulin-secreting islets in the pancreas. Of the 12 non-obese diabetic mice injected with live splenocytes, 11 remained normogylcemic for more than 26 weeks after the onset of diabetes, while 11 of 13 mice that received irradiated splenocytes also remained normoglycemic for greater than 27 weeks. The diabetic mice treated with the irradiated splenocytes demonstrated restoration of normal glucose levels and even islet regeneration, however at a slower rate than that of the live splenocytes control. Overall, both instances of splenocytes were able to yield newly functional islet cells, an amazing feat nevertheless [5].

Next time you pop a seemingly innocuous piece of candy or lollipop into your mouth, remember that such a luxury eludes 8.3% of the population. Yet hope has been shed on this suffering contingency with the advent of modern medical techniques.

While regular diet, daily exercise and insulin supplementation have until now been the best way for regulating diabetes, it is the audacity of medical research that promises to produce longterm solutions and the hope of a tomorrow free of lancing.

References 1. 3.”Diabetes Overview.” National Diabetes Information Clearinghouse. Web. 17 Apr. 2011. <http://diabetes.niddk.nih. gov/dm/pubs/overview/>.

2. Razavi R, Chan Y, Afifiyan FN et al. 2006. TRPV1+ sensory neurons control beta cell stress and islet inflammation in autoimmune diabetes. Cell. 127(6):1123-35.

3. “What Are iPS Cells?” University of Wisconsin System. Web. 18 Apr. 2011. <stemcells.wisc.edu/pdf/What_Are_IPS_Cells. pdf>.

4. Alipio Z, Liao W, Roemer EJ, et al. 2010. Reversal of hyperglycemia in diabetic mouse models using induced-pluripotent stem (iPS)-derived pancreatic beta-like cells. Proc Natl Acad Sci U S A. 107(30):13426-31 5. Kodama, Shohta. 2003. “Islet Regeneration During the Reversal of Autoimmune Diabetes in NOD Mice.” Science Magazine 302 Cytostem. Web. 18 Apr. 2011.

6. Bour-Jordan, Helene, and Jeffrey Bluestone. 2006. Sensory Neurons Link the Nervous System and Autoimmune Diabetes.

Cell. 127(6):1097-9.

7. “Diabetes - PubMed Health.” National Center for Biotechnology Information. Web. 17 Apr. 2011. <http://www.ncbi.nlm. nih.gov/pubmedhealth/PMH0002194/>.

8. “Diagnosis of Diabetes.” Cleveland Clinic. Web. 17 Apr. 2011.<http://my.clevelandclinic.org/disorders/Diabetes_Mellitus/hic_Diagnosis_of_Diabetes.aspx

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The Crystallization of Riboswitches: Potential Targets for Antibiotics Artem Serganov, â&#x20AC;&#x2DC;12

Supervisor: Dr. Dinshaw J. Patel, Ph.D., Department of Structrual Biology Memorial Sloan-Kettering Cancer Center 1275 York Avenue, New York, NY 10065

Introduction Widespread antibiotic resistance in bacteria is rapidly developing, making many modern antibiotics ineffective in interfering with the functions of proteins essential for cellular processes in bacteria. This necessitates the development of novel classes of antibiotics specific for new molecular targets. The recent discoveries of RNA interference and other RNA-based regulation systems highlight the importance of RNA in controlling gene expression. Riboswitches are the latest addition to the growing field of RNA-based control elements [1,2] with the unique ability to sense concentrations of metabolites without protein assistance and to direct expression of genes involved in the synthesis and transport of these molecules.

Riboswitches are RNA sequences typically located in the 5´ untranslated regions (UTR) of messenger RNAs (mRNA) [3]. Riboswitches usually consist of sensing domains, which specifically bind to metabolites, and expression platforms, which carry gene expression signals. Riboswitches exist in two alternative conformations: Bound or not bound to a metabolite, determining whether gene expression is turned on or off [4]. In the metabolite-bound form the ribosome binding site (RBS) of some riboswitches

becomes inaccessible to ribosomes and gene expression is turned off (Figure 1a).

When the riboswitch is in a non-bound state, the ribosome can bind to the RBS and proceed with translation. Depending on the expression platform, riboswitches can control gene expression by translational (via RBS) or transcriptional (via transcription termination) (Figure 1b) mechanisms.

Riboswitches can interact with different types of small molecules including co-enzymes, amino acids, nucleobases, magnesium cations, and sugar derivatives [5,6]. Since riboswitches control expression of genes essential for viability of many pathogenic bacteria, and since they have not yet been found in humans, they represent potentially good targets for new antibiotics5. Moreover, riboswitches are naturally adept at binding small druglike molecules that can be used as the foundation for the design of new antibiotics. Bacteria and humans share many metabolites, therefore, to avoid toxicity in humans, metabolite-like drugs have to be different from the natural version but nevertheless capable of binding to the riboswitches and shutting down gene expression.

Interestingly, several metabolite-like compounds such as pyrithiamine7 and L-aminoethylcysteine [8,9], utilized for years to combat microbes, likely target

riboswitches.

Although new metabolite-like drugs can be found by screening large combinatorial libraries, this process can be facilitated by knowing three-dimensional (3-D) structures of riboswitches bound to their ligands. Many laboratories have already begun efforts to determine the 3-D structures of various riboswitch-ligand complexes, primarily through X-ray crystallography. However the progress of these studies is slow [6,10]. One of the major obstacles in X-ray crystallography is crystallization, which first requires extensive trial-and-error screenings of crystallization conditions, and then necessitates very careful optimization of initial conditions. Therefore, the goal of my project was to produce crystals of two riboswitches specific to S-adenosylmethionine (SAM) and pre-queuosine1 (preQ1) for the determination of their 3-D structures.

The SAM-II riboswitch specifically recognizes SAM and negatively controls expression of the bacterial metA gene, which encodes the protein that catalyzes the first step of methionine synthesis [11] (Figure 2a,b). Interestingly, three riboswitches (SAM-I, SAM-II and SAM-III) with different sequences and secondary structures recognize SAM1115, potentially featuring similar SAM binding pockets. If a SAM-like drug is

Figure 1. Example of riboswitch-mediated control of gene expression. In both cases, translation repression (a) and transcription termination (b), the binding of a metabolite (red M) stabilizes the riboswitch structure.

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Figure 4. Representative NMR spectra of the preQ1b riboswitch binding to its ligand.

Arrows indicate characteristic changes in the spectra.

Figure 3. (a) Structure of riboswitch that controls expression of B. subtillis queCDEF gene. (b) Structure of preQ. (c,d,e,f) Predicted secondary structures of preQ1a-b, respectively. Species from which the riboswitch constructs were derived are listed prioer to each construct.

designed based on common binding rules for all SAM riboswitches, it may potentially target all three riboswitches, reducing the chances of bacteria developing antibiotic resistance. Since the structure of the SAM-I riboswitch was determined by another laboratory [16] and because the SAM-III riboswitch is narrowly distributed amongst bacteria [13], I focused my research on the SAM-II riboswitch.

The preQ1 riboswitch recognizes preQ1, anintermediate in queuosine biosynthesis (Figure 3a, b) [17]. Queuosine is a hypermodified nucleotide occupying the wobble position of the anticodon of certain transfer RNAs. The preQ1 riboswitch is located in the 5´ UTR of the queCDEF operon that is involved in preQ1 synthesis in a wide range of bacteria. Both the SAM-II and the preQ1 riboswitches have a common theme in their secondary structures. In contrast to other riboswitches, they are built around a stem-loop structure that may interact with the 3´ region of the riboswitch. Such secondary structures suggest a novel type of 3-D structure and metabolite binding in riboswitches, which may be important for drug design.

Materials and Methods In vitro transcription was chosen to generate large quantities of homogenous

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SAM-II RNA for crystallization. DNA templates for in vitro transcription were prepared from chemically synthesized DNA oligonucleotides that were annealed together and cloned into a modified plasmid that carried the T7 promoter [18,19]. The plasmid was transformed into the XL1-Blue strain of E. coli, which was used in large scale plasmid DNA purification to generate preparative quantities of DNA for in vitro transcription reactions [18]. PreQ1 RNA was short enough to be efficiently synthesized through chemical means and was purchased from Dharmacon.

Riboswitch-ligand complexes were formed by mixing RNA with SAM or preQ1 in a buffer containing D2O and deuterated potassium acetate. The ligand was added to RNA to reach a 1 to 1 molar ratio while performing NMR spectroscopy until spectrum changes were no longer observed.

For crystallization, each RNA-ligand complex was mixed with the same volume of reservoir solution (from commercial sparse matrix kits) and crystals were grown by vapor diffusion in either hanging or sitting drop formats [20]. Crystals were shot by an X-ray beam with test frames recorded at three crystal orientations. Data sets for were also collected at the Advanced Photon Source (APS) (Chicago, IL).

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Results SAM-II constructs were designed for crystallization using published software to determine secondary structures in the riboswitch [21,22]. Two of the best-folding SAM-II riboswitches were chosen for the study. The first construct, 58 nucleotide long SAM-IIa, incorporates the central evolutionary conserved part of the sensing domain, while the second construct, 83 nucleotide long SAMIIb, contains a hairpin on the 3´ end of the sequence. The preQ1 riboswitch has the smallest metabolite-sensing domain, which, according to predictions [17], does not have a pronounced secondary structure. Four preQ1 RNA constructs, ranging from 34-57 nucleotides long, were designed for crystallization. NMR spectroscopy was used to monitor formation of complexes between riboswitches and their ligands in a 1 to 1 proportion.

For example, the imino proton spectrum of free preQ1b RNA (Figure 4) shows four well resolved peaks and the addition of the ligand formed new peaks, representing changes in riboswitch base-pairing. Titration stopped after changes in the peaks were no longer observed.

Formation of crystals requires macromolecules to be present in an oversaturated solution, which is achievable by a technique called vapor diffusion.

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Figure 5. Representative pictures of crystals of SAM-II and preQ1 riboswitch-ligand complexes.

Figure 6. Representative diffraction images of preQ1 riboswitch-ligand crystals showing the improving diffraction limits as optimization of conditions continues. Arrows indicate diffraction limits. (a, b) Diffraction images of preQ1d crystals. (c) Diffraction image of crystal generated from a complex of methylated preQ1c and its ligand.

Each macromolecule, however, requires unique crystallization solutions that are unpredictable, thereby necessitating large scale trial-and-error for crystallization.

For initial screening of crystallization conditions, riboswitch-ligand complexes with different concentrations were tested at two temperatures and with different crystallization solutions from commercial kits.

Approximately 600-1,800 different conditions for each riboswitch-ligand complex were tested for crystal formation, summing up to over 6,000 different conditions. Initial crystallization hits were found for all tested complexes: 24 for the SAM-II and 102 for the preQ1 constructs.

Most of the hits were crystalline-like material; nevertheless, promising crystals were found for all cases (Figure 5). Crystallization of the preQ1a-ligand complex resulted in the highest success rate with 3.8 % of tested conditions producing crystals, while other riboswitch-ligand

complexes had a success rate of 1-2 %.

Optimization of crystallization conditions is necessary to grow larger and better diffracting crystals. Careful optimization of initial hit crystal conditions, such as varying the reagent concentrations and pH, yielded eleven conditions with moderate quality crystals: one each for SAM-IIa, SAM-IIb, and preQ1a, and four each for the preQ1c and preQ1d riboswitches. The best crystals generated from these optimizations showed the following diffraction limits: SAM-IIb, 8.0 Å; preQ1a, 6.6 Å; preQ1c, 5.8 Å; and preQ1d, 6.5 Å (Fig. 6a, b as examples of diffraction images).

Simultaneously during the traditional optimization of crystal conditions, an alternative approach was pursued for the preQ1c-ligand complex, the most promising construct for the generation of high quality crystals. During chemical synthesis of preQ1c RNA, a hydrogen atom in the 2´-hydroxyl group was replaced with a methyl group in one of the nucleotides of

Figure 7. Modified uridine monophosphate used for crystallization of preQ1c.

Green shading shows 2’-O-methylation Potential Se substitution is indicated in red.

the helix (Figure 7). It was thought that a single methyl group would not affect helix formation, but would introduce a hydrophobic patch in the RNA backbone and could promote formation of novel packing interactions during crystallization. Four variants of the preQ1c RNA were prepared with single substitutions at nucleotides A-3, U-4, A-19, or U-20 (Figure 3e). However, crystals for the modified RNA-ligand complexes were not reproducible in previously optimized conditions, requiring new crystal screenings to find initial hits. Constructs with methylated A-3 and U-4 failed to produce crystals in 1056 tested conditions, while the constructs with modifications at A-19 and U-20 produced crystals in 29 of 672 tested conditions.

X-ray testing of a crystal produced from modified U-20 preQ1c showed a resolution limit of 3.1 Å (Figure 6c), the best obtained results of the project. Furthermore, resolution limits better than 3.5 Å are typically considered sufficient

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REPORTS for 3-D structure determination by X-ray crystallography, signifying that the obtained X-ray data may help determine the 3-D structure of the preQ1 riboswitch.

Further analysis of crystal data suggested stacking conformations of RNA-ligand complexes that were different from previously observed stacking interactions for non-methylated RNA. This suggests that crystallization of RNA with 2’-O-methylated nucleotides generates hydrophobic patches, reorganizing RNA in different conformations. 2’-O-methylations can also be used to acquire phase information for structure determination by replacing the oxygen with a selenium atom. Such a substitution would permit application of single- and multiple-wavelength anomalous dispersion (SAD and MAD) techniques to the selenium atom, simplifying the daunting task of 3-D structure determination.

Conclusions As a unique RNA system for gene expression control, riboswitches stand out as potential targets for antimicrobial drugs because they control essential genes in a wide range of bacteria and can recognize small drug-like molecules. With the help of 3-D structures, these natural metabolites may be redesigned to modern drug standards. The riboswitch-ligand complexes for both SAM-II and preQ1 were successfully crystallized, completing the primary goal of the project. Careful optimization of crystal conditions led to the generation of preQ1 crystals that diffracted at 3.1 Å, a resolution suitable for determination of the 3-D structure. Additional work on the preQ1 riboswitch will likely reveal novel metabolite binding rules, which may contribute to the design of future riboswitch-targeting antimicrobial drugs.

References

1. Mironov, A.S. et al. Sensing small molecules by nascent RNA: a mechanism to control transcription in bacteria. Cell 111, 747-756 (2002).

2. Winkler, W. et al. Thiamine derivatives bind messenger RNAs directly to regulate bacterial gene expression. Nature 419, 952-956 (2002).

3. Breaker, R.R. in The RNA World (eds.

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Gesteland, R.F., Cech, T.R. & Atkins, J.F.) 89-108 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 2006).

4. Nudler, E. & Mironov, A.S. The riboswitch control of bacterial metabolism.

Trends Biochem. Sci. 29, 11-17 (2004).

5. Blount, K.F. & Breaker, R.R. Riboswitches as antibacterial drug targets.

Nat. Biotechnol. 24, 1558-1564 (2006).

6. Dann, C.E., 3rd et al. Structure and mechanism of a metal-sensing regulatory RNA. Cell 130, 878-892 (2007).

7. Sudarsan, N. et al. Thiamine pyrophosphate riboswitches are targets for the antimicrobial compound pyrithiamine.

Chem. Biol. 12, 1325-1335 (2005).

8. Blount, K.F. et al. Antibacterial lysine analogs that target lysine riboswitches.

Nat. Chem. Biol. 3, 44-49 (2007).

9. Sudarsan, N. et al. An mRNA structure in bacteria that controls gene expression by binding lysine. Genes Dev. 17, 26882697 (2003).

10. Wakeman, C.A. et al. Structural features of metabolite-sensing riboswitches.

Trends Biochem. Sci. 32, 415-424 (2007).

11. Corbino, K.A. et al. Evidence for a second class of S-adenosylmethionine riboswitches and other regulatory RNA motifs in alpha-proteobacteria. Genome Biol. 6, R70 (2005).

12. Epshtein, V. et al. The riboswitchmediated control of sulfur metabolism in bacteria. Proc. Natl. Acad. Sci. U S A 100, 5052-5056 (2003).

13. Fuchs, R.T. et al. The SMK box is a new SAM-binding RNA for translational regulation of SAM synthetase. Nat. Struct. Mol. Biol. 13, 226-233 (2006).

14. McDaniel, B.A. et al. Transcription termination control of the S box system: direct measurement of S-adenosylmethionine by the leader RNA. Proc. Natl.

Acad. Sci. U S A 100, 3083-3088 (2003).

15. Winkler, W.C. et al. An mRNA structure that controls gene expression by binding S-adenosylmethionine. Nat. Struct. Biol. 10, 701-707 (2003).

16. Montange, R.K. & Batey, R.T. Structure of the S-adenosylmethionine riboswitch regulatory mRNA element. Nature 441, 1172-1175 (2006).

17. Roth, A. et al. A riboswitch selective for the queuosine precursor preQ1 contains an unusually small aptamer domain. Nat. Struct. Mol. Biol. 14, 308-317 (2007).

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18. Sambrook, J. et al. Molecular cloning: a laboratory manual. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1989).

19. Vieira, J. & Messing, J. Production of single-stranded plasmid DNA. Methods Enzymol. 153, 3-11 (1987).

20. McPherson, A. Introduction to Macromolecular Crystallography (WileyLiss, Wilmington, 2003).

21. Griffiths-Jones, S. et al. Rfam: annotating non-coding RNAs in complete genomes. Nucleic. Acids. Res. 33, D121124 (2005).

22. Zuker, M. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31, 34063415 (2003).

23. Price, S.R. et al. Crystallization of RNA-protein complexes. I. Methods for the large-scale preparation of RNA suitable for crystallographic studies. J. Mol. Biol. 249, 398-408 (1995).

24. Klepper, F. et al. Robust Synthesis and Crystal-Structure Analysis of 7-Cyano-7-deazaguanine (PreQ0 Base) and 7-(Aminomethyl)-7-deazaguanine (PreQ1 Base). Helvetica Chimica Acta 88, 2610-2616 (2005).

25. COLLABORATIVE COMPUTATIONAL PROJECT. The CCP4 suite: programs for protein crystallography.

Acta Cryst. D 50, 760-763 (1994).

26. Schwalbe, H. et al. Structures of RNA switches: insight into molecular recognition and tertiary structure. Angew. Chem.

Int .Ed Engl. 46, 1212-1219 (2007).

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Enhancement of Reactivation of Murine Gammaherpesvirus 68 from Latency through NF-kB inhibition Karen Bulaklak, ‘12 Principle Investigator: Dr. Laurie Krug, Ph.D. Department of Molecular Genetics and Microbiology Stony Brook University, Stony Brook, NY 11794

Introduction Herpesviruses are double-stranded DNA viruses that establish life-long infections in their hosts. The gammaherpesvirus subfamily of herpesviruses is lymphotropic and is a leading cause of infection-associated cancers in AIDS patients. The human gammaherpesvirus, Epstein-Barr Virus (EBV ), has been linked to the development of multiple lymphomas such as Burkitt’s lymphoma, and Kaposi’s sarcoma-associated herpesvirus (KSHV ) is the causative agent of the neoplasm Kaposi’s sarcoma. Like other herpesviruses, the gammaherpesvirus lifecycle is characterized by intervals of latency and lytic replication. During latency, the viral genome persists within the host cell, but expression of viral genes is limited and no virus particles are made.

In this quiescent state, the virus is undetectable by the host and can maintain a chronic infection, thus occluding an effective therapeutic approach. In a process called reactivation, the latent virus can switch to a program of lytic replication under the right circumstances, initiating a cascade of viral gene expression that ends in infectious virus production. The signaling events that trigger this latent to lytic switch are an active area of investigation [1].

Gammaherpesviruses have developed a number of different strategies to persist within their natural hosts. Latency is essential in these techniques and allows the virus to evade the host innate and adaptive immune response. Furthermore, specific viral gene products modulate host signals conducive to latency. Recent studies indicate that the nuclear factor kappa B (NF-kB) signaling pathway is critical for gammaherpesvirus latency in vivo [6,7,8]. The NF-kB transcription factors

are important players in cell survival and inflammation. These proteins include NF-kB1 (p105 and p50), NF-kB2 (p100 and p52), c-Rel, RelB, and RelA (p65), which dimerize within the cytoplasm and are sequestered by inhibitory molecules, called IkBs. In two distinct pathways, different NF-kB-activating signals initiate the phosphorylation and subsequent degradation of the IkBs, either through an IKKk-dependent (canonical pathway) or IKKk-dependent (alternative pathway) manner. The released NF-kB dimers then regulate the expression of multiple genes such as in the inflammatory response. EBV and KSHV lymphomas have constitutively active NF-kB, and studies in vitro show that inhibition of this pathway leads to cell death. The virus may target this pathway to facilitate the survival, proliferation and differentiation of B cells, promoting pathogenesis and tumor formation [10]. Inhibitors of NFkB have also been shown to delay tumor progression of EBV+ and KSHV+ lymphomas in vivo, which suggests that the

pathway may be a good target for treating gammaherpesvirus-related lymphomas [5].

NF-kB inhibitors also lead to reactivation in EBV- and KSHV-positive B cell lymphomas [2,5]. We hypothesize that this pathway promotes latency of the gammaherpesviruses by repressing lytic gene expression. Since we cannot manipulate the virus-host interactions of EBV and KSHV latent B cell infections in vivo, we use murine gammaherpesvirus 68 (MHV68), a naturally occurring mouse pathogen closely related to human gammaherpesviruses. Introduction of MHV68 in mice leads to a robust latent infection in the spleen, which recapitulates many aspects of EBV and KSHV infection. The loss of NF-kB protein p50 also enhances virus replication in MHV68-positive mice, which further suggests that NF-kB plays a role in establishing latency [7]. Here we examined the role of NF-kB signaling in various cell lines infected with MHV68, including the A20-HE mature murine B cell line,

Figure 1: The role of NF-κB inhibitors in reactivation. The NF-κB pathway is important for latency and inhibits lytic replication in vivo (top). Reactivation may result from inhibition of the pathway through treatment with Bay11 or MG132, or upregulation of lytic gene expression through TPA treatment (bottom).

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REPORTS which is latently infected with a recombinant MHV68 encoding a hygromycin resistance protein fused to an enhanced green fluorescent protein [3]. We also explored other murine cell lines, such as the MHV68-infected lymphoma cell line, S11, and de novo infected (M12) B cells to examine reactivation from latency [9].

In MHV68, the replication and transcription activator, RTA (encoded by ORF50), is sufficient to disrupt latency and drive lytic replication [11]. TPA (12-O-tetradecanoyl-phorbol-13-acetate) is proposed to upregulate RTA expression in a protein kinase C-dependent manner [4]. Sodium butyrate (NaBut) has also been used to induce reactivation in KSHV cells. In this study, we treated latent cultures with TPA and NF-kB inhibitors, alone or in combination, and examined for reactivation.

We have screened Bay11-7082, which prevents IKK-dependent degradation of NF-kB inhibitor, IkBk, and has been shown to increase lytic replication in KSHV- and EBV-positive B lymphocytes [2]. Additional NF-kB inhibitory drugs were utilized, including MG132, a proteasome inhibitor that blocks the degradation of the NF-kB inhibitor IkBk and consequently prevents NF-kB activation.

We aim to apply these reactivation conditions to identify viral genes or other aspects of the virus life cycle that are impacted by NF-kB signaling. Further understanding of this pathway will give us better insight into a critical host-virus interaction that influences chronic infection and lymphomagenesis by gammaherpesviruses.

Methods Induction Experiments: A20-HE, M12 and S11 cell lines were grown at 37째C in RPMI media, supplemented with 10% fetal bovine serum, 5% L-glutamine, 5% penicillin/streptomycin and 50 uM betamercaptoethanol. A20-HE cells were maintained using 300 ug/mL hygromycin until the day before induction. All cell lines were subcultured 1:3 on the day before induction. On the day of induction, cells were seeded into 12-well plates at 106 cells/well and the appropriate drug combinations were added. Cells were in-

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cubated at 37째C and harvested at the 24or 48-hour time points to extract DNA and virus for quantitative PCR and viral plaque assays, respectively. Drug toxicity was also examined with Trypan Blue staining.

1. S11 cells: Lymphoma B cells from an infected mouse that are latently infected with MHV68 [9]. Final concentrations of TPA and NaBut were 20 ng/mL and 4 mM, respectively, at 24 hours after treatment.

2. MHV68-infected M12 cells: Mature murine B cells newly infected with MHV68 for four days. Before treatment, cells were split 1:4 into 6-well plates and grown in either fresh or conditioned (previously used) media. Final concentration of TPA was 20 ng/ml for 24 hours.

3. A20-HE2 cells: Mature murine B cells latently infected with MHV68. Final concentration of TPA was 20 ng/mL. MG132 concentrations were 1 uM and 5 uM for 24 hours.

4. A20-HE2 cells: As described above.

Final concentrations of TPA and BAY-11 were 20 ng/mL and 40 uM, respectively, at 48 hours.

De novo Infection of M12 cells (Spinoculation): One day prior to infection, M12 cells were subcultured 1:3. On the day of infection, cells were seeded at 2x106 cells/ mL per well in 12-well plates. Polybrene (8 ug/mL) was added to each milliliter of cells. Concentrated recombinant YFPMHV68 virus (MOI 10) was then applied and the plates were spun at 2500 RPM for 60 minutes. After centrifugation, 10% RPMI was added to bring the final volume to 1 mL. Drug Toxicity. To evaluate drug toxicity in HE and S11 cells, an aliquot of cells was stained with Trypan Blue and counted using a hemacytometer. The number of live cells in treated samples was graphed as the percentage of the number of cells in the untreated sample. Toxicity in M12 cells was measured using flow-cytometry.

Quantitative PCR. DNA from untreated and treated cells was purified using the Qiagen DNeasy Kit. Samples were loaded at 200 ng and quantitative PCR was performed using ORF50 and GAPDH primers to determine the concentration of viral and cellular DNA, respectively, for each treatment. The ratios

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of ORF50/GAPDH were compared to uninduced cells as a measure of fold induction.

Plaque Assays: 3T12 cells were previously seeded at 1.8x105 cells/well in 6-well plates. Virus was extracted from uninduced and induced cells through three consecutive cycles of freeze-thawing and bead disruption. Two dilutions of the cell/supernatant mixture were made (101 and 10-2) and 200 uL aliquots of each dilution were added to the 3T12 cells. The plates were swirled every 15 minutes for one hour and methylcellulose was then added. Cells were incubated at 37째C for one week. The number of plaque-forming units per milliliter (viral titer) was calculated for each condition.

Western Blot: Cells were lysed with complete RIPA buffer supplemented with 10 uL proteinase inhibitor cocktail (1:100, Sigma) and 100 uM PMSF and quantitated with the Bio-Rad DC protein assay. Samples were heated to 100째C and resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Resolved proteins were transferred to nitrocellulose and identified using a polyclonal mouse antibody for lytic antigen. Immobilized antibodyantigen was detected using horseradish peroxidase-conjugated secondary antibody and exposed to film. GAPDH was used as a loading control.

Results and Discussion

To determine the role of the NF-kB pathway in the gammaherpesvirus lifecycle, various treatment combinations of lytic activators TPA and NaBut, in combination with NF-kB inhibitors MG132 and Bay11, were used to induce reactivation in three different MHV86-positive murine B cell lines. Following a 24-48 hour post-treatment incubation period, cell viability, increases in viral DNA and infectious virus were considered in determining the optimal conditions for lytic replication.

S11 cells can be induced to reactivate.

Trypan blue assays suggest that the viability of S11 cells was decreased with TPA and/or NaBut treatment (Figure 2A).

At most, the NaBut-treated culture had 60% live cells in contrast to TPA+NaButtreated cells, which had about 30% viabil-

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ity. In addition to higher viability, NaBut treated samples also had greater levels of viral (ORF50) DNA compared to the mock (Figure 2B). TPA-treated samples did not differ from the mock in ORF50 content. Cells induced with NaBut were positive for lytic protein in an immunoblot, confirming reactivation took place (data not shown). Although S11 cells can be induced, a high level of spontaneous reactivation occurred, even in untreated samples (3.3x106 PFU/mL, data not shown). Therefore, it would be difficult to distinguish the effect of NF-ÎşB inhibitors in these cells. This stresses the need for a cell line that displays limited gene expression before induction and an increase in viral DNA after exposure to lytic activators.

De novo infected M12 cells are induced by TPA. We next examined establishment of latency and reactivation in the newly infected B cell line, M12.

Flow-cytometry analysis showed that about 20-25% of cells are capable of being infected, as the expression levels of YFP increases significantly in de novo infected M12 cells for both fresh and conditioned media samples (Figure 3). Following TPA induction, there is no change in YFP expression for fresh media samples, but a slight increase was observed in cells grown in conditioned media. In an immunoblot of M12 lysates, lytic protein is present in all infected samples (Figure 4). However, a stronger positive signal is present in conditioned media TPAtreated infected sample compared to the uninduced. Fresh media infected samples produced less lytic antigen compared to cells grown in conditioned media. These results suggest that reactivation in conditioned media is slightly enhanced.

However, the M12 cells do not exhibit a strong induction with TPA, and therefore may not be the best model for evaluating reactivation stimuli.

Reactivation of MHV68 by MG132 treatment is dose-dependent. We also explored the latently infected mature B cell line, A20-HE. The HE cells were treated with MG132, a proteasome inhibitor that may inhibit NF-kB by blocking the degradation of its inhibitor, IkBk. Trypan blue assays conducted with MG132treated A20-HE cells indicate that cell viability is largely compromised, with less

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Figure 2: Evaluation of TPA and NaBut in S11 cells. A. At 24 hours, treatment of S11 cells with either TPA or NaBut had an adverse effect on cell viability. B. NaBut increased viral copy number by 1.5-fold compared to the mock. TPA alone and in combination with NaBut did not increase ORF50 levels.

Figure 3: Treatment of M12 cells with TPA. De novo infected cells cultured in conditioned media exhibited a significant increase in YFP expression compared to uninfected samples. YFP expression increases slightly after TPA treatment with cells grown in conditioned media.

Figure 4: Immunoblot of TPA-treated M12 lysates. In cells seeded with conditioned media, viral antigen is observed upon de novo infection. This is further enhanced with TPA treatment. In contrast, there is no difference in infected cells with or without TPA stimulation when seeded with fresh media.

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Figure 5: Evaluation of MG132 A. Treatment of HE cells with MG132 is extremely toxic at 24 hours post-treatment, with a survival rate of less than 20%. B. MG132 causes a dose-dependent increase in viral copy-number

than 20% live cells (Figure 5A). However, application of TPA and increasing concentrations of MG132 produced about 4 to 7-fold greater levels of ORF50 DNA compared to the mock, a much greater increase in viral copy number in comparison to the previous two cell lines. MG132 in combination with 5 uM of MG132 in particular showed the greatest increase in viral copy number (Figure 5B). TPA and 1 uM MG132 contained the lowest levels of ORF50 DNA, with no corresponding differences in viability. No plaques were observed and therefore viral titer could not be determined. The cell toxicity of MG132 may be linked to the drugâ&#x20AC;&#x2122;s nonspecific degradation of proteins within the cell, leading to the disruption of multiple signaling pathways that lead to cell death. Therefore, MG132 would not be

an ideal reagent to test NF-kB inhibition in the context of reactivation.

Additional induction experiments were conducted in which MG132 was removed after 2 hours (data not shown).

These experiments revealed increased cell viability but no notable changes in viral DNA (0.94-fold increase in ORF50 DNA).

Treatment with TPA and Bay11 increases viral DNA 4-fold. Treatment of HE cells with TPA, Bay11 and in combination was conducted in triplicate to determine statistical significance. Based on Trypan blue viability staining, cells treated with TPA have decreased cell viability by about 50%, while application of Bay11 does not impact viability (Figure 6A). Quantitative PCR analysis indicates that treatment with TPA+Bay11 yields

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a significant 4-fold increase in ORF50 content compared to TPA alone (Figure 6B). By itself, TPA increases reactivation in comparison to the mock, while Bay11 samples have no changes in viral DNA levels. In samples treated with TPA and Bay11 together, higher levels of infectious virus as well as a positive result for lytic antigen complement this increase in viral DNA (Figure 6C, 7).

Unfortunately, the plaque assay results varied slightly between replicates and conditions are still being optimized in order to confirm the significance of this enhancement. The immunoblot of treated cell lysates reveals the importance of the 48 hours post-treatment timepoint. While no lytic antigen is detected for any treatment at 24 hours, there is a faint positive signal for TPA and a strong positive signal for TPA+Bay11 at 48 hours post-treatment compared to the mock (Figure 7).

This data suggests that more lytic protein is produced with TPA and Bay11 together and reactivation has been successfully initiated. As a NF-kB specific inhibitor, Bay11 appears to work synergistically with TPA by inducing the transcription activator RTA and removing the signal for IKK-dependent degradation of IkBk. Thus, Bay11 may be a better approach for observing NF-kBâ&#x20AC;&#x2122;s role in the switch from latency into lytic replication. In comparison to S11 and M12 cells, induced A20-HE cells show the greatest fold-increase in viral DNA ver-

Figure 6: Evaluation of Bay11. A. At 48 hours post-treatment, there is no significant change in HE cell viability in samples treated with Bay11 and TPA in combination compared to the mock. B. The combination of TPA and Bay11 increases viral copy number as compared to TPA alone (p=0.05). C. There appears to be a greater level of infectious virus with the combination of treatments in contrast to the treatments alone.

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REPORTS derived from murine gammaherpesvirus68-infected mice. Journal of Virology.

70(9):6516-8.

10. Webster GA, Perkins ND. 1999.

Transcriptional cross talk between NFkappaB and p53. Molecular and Cellular Biology. 19(5):3485-95.

11. Wu TT, Usherwood EJ, Stewart JP, Nash AA, Sun R. 2000. Rta of murine gammaherpesvirus 68 reactivates the complete lytic cycle from latency. Journal of Virology. 74(8):3659-67.

Figure 7: Immunoblot of TPA and/or Bay11-treated HE cell lysates. At 48 hours, there is an increase in lytic antigen with TPA treatment (lane 8 versus lane 3). Combination with Bay11 further increases lytic antigen (lane 8 versus lane 10).

sus uninduced samples, showing a better delineation between the latent and lytic state. Therefore, we have continued using this cell line to evaluate optimal reactivation conditions.

Conclusions In this study, we aimed to optimize cell culture conditions to induce lytic replication in MHV68-infected B cell lines. For the latently infected A20-HE cells, TPA-stimulated reactivation was enhanced with the addition of NF-kB inhibitors, supporting our model whereby NF-kB activation promotes latency by repressing lytic gene expression. The S11 and M12 cell lines can also be induced to reactivate; however, because there is a great deal of spontaneous reactivation, they may not be ideal for studying the role of NF-kB in reactivation. In the future, we plan to explore additional cell lines, further validate our findings and identify the viral genes that are regulated

by NF-kB signaling.

References 1. Barton E, Mandal P, Speck SH. 2011.

Pathogenesis and host control of gammaherpesviruses: lessons from the mouse.

Annual Review of Immunology. 29:35197.

2. Brown HJ, Song MJ, Deng H, Wu TT, Cheng G, Sun R. 2003. NF-kappaB inhibits gammaherpesvirus lytic replica-

tion. Journal of Virology. 77(15):8532-40.

3. Forrest JC, Speck SH. 2008. Establishment of B-cell lines latently infected with reactivation-competent murine gammaherpesvirus 68 provides evidence for viral alteration of a DNA damagesignaling cascade. Journal of Virology.

82(15):7688-99.

4. Gradoville L, Kwa D, El-Guindy A, Miller G. 2002. Protein kinase C-independent activation of the Epstein-Barr virus lytic cycle. Journal of Virology.

76(11):5612-26.

5. Keller SA, Hernandez-Hopkins D, Vider J, Ponomarev V, Hyjek E, Schattner EJ, Cesarman E. 2006. NF-kappaB is essential for the progression of KSHV- and EBV-infected lymphomas in vivo. Blood.

107(8):3295-302.

6. Krug, LT, JM Moser, SM Dickerson, and SH Speck. 2007. Inhibition of NFkB activation in vivo impairs establishment of gammaherpesvirus latency. PLoS Pathogens 3(1): e11 7. Krug, LT, CM Collins, LM Gargano, SH Speck. 2009. NF-kB p50 plays distinct roles in the establishment and control of murine gammaherpesvirus 68 latency. Journal of Virology. 83:4732-4748 8. Krug, LT, E Torres-Gonzรกlez, Q Qin, D Sorescu, M Rojas, A Stecenko, SH Speck, AL Mora. 2010. Inhibition of NF-kappa B signaling reduces virus load and gammaherpesvirus-induced pulmonary fibrosis. American Journal of Pathology. 177: 608-621 9. Usherwood EJ, Stewart JP, Nash AA. 1996. Characterization of tumor cell lines The Stony Brook Young Investigators Review, Fall 2011

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Ethanol-Induced Epithelial Cell Death Associated with Activation of MAP and P13 Kinases: The Role of TRAF4 Nadya Peresleni, ‘11 and Dr. Victor Romanov, PhD Principle Investigator: Dr. Ghassan Samara, MD Department of Surgery Stony Brook University, Stony Brook, NY 11794

Introduction Tobacco and alcohol are the main risk factors for oral cancer development [1]. Prolonged and excessive alcohol intake results in numerous disorders in the human body, including negative effects on oral mucosa, which can result in the development of oral cancer [2-4]. The mortality rate associated with oral cancer in the United States is approximately 8,000 deaths per year, in comparison to melanoma and cervical cancer, which account for approximately 7,000 and 5,000 deaths per year, respectively. These numbers have not improved for decades. The

importance of studying the mechanisms of neoplastic transformation leading to oral cancer is evident. [1] It has been shown that ethanol in low concentrations (0.13-4%) induces apoptotic cell death in different types of cells, changes gene expression, activates signaling pathways such as NF-kB, and changes mitogen-activated protein kinase (MAPK) activity and cytokine production [5,6].

TNF receptor-associated factor 4 (TRAF4) plays a known role as a signaling molecule involved in pathways related to cell death and survival [7]. It is an atypical member of the TRAF family of

adaptor proteins: Its expression is strongest during the process of development, it has an evolutionary conserved primary sequence and a regulation process that may differ from other TRAF proteins [8-9]. Most of the data obtained suggest that TRAF4 may be involved in events that are stressful for cells; recently it was shown that TRAF4 is overexpressed in human carcinomas [10]. TRAF4 has been shown to be involved in apoptosis, possibly as both a pro- and anti-apoptotic factor, and is a determined player in the MAP-kinase signal transduction pathway [9,11].

The goal of our research was to elu-

Figure 1. Cell survival and TRAF4 role in the cell survival after treatment with 4% EtOH for 4 h.

(A) Equal number of normal oral epithelial, HaCaT and II-4 cells were incubated with 4% of EtOH for 4 h. Cell viability was assayed using CellTiter-Blue Cell Viability kit (Promega Corp., Madison WI). The cell number percentage is represented as the mean± SEM of three independent experiments.* p<0.05 compared for EtOH only treated HaCaT cells.

(B) TRAF4 suppresses cell death induced by EtOH in HaCaT cells. Equal number of HaCaT cells, transfected with TRAF4 specific siRNA or control siRNA (scrambled) and untransfected cells were incubated with 4% EtOH for 4 h and cell viability was assayed. The cell number percentage is presented as the mean ± SEM of three independent experiments. * p<0.05 compared for EtOH only treated HaCaT cells.

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Materials and Methods

Cell culture and reagents. Spontaneously immortalized nontumorigenic human keratinocytes HaCaT and malignant cells (ras tumorigenic cell line) HaCaT-II-4 were cultured in DMEM (Dulbecco’s Modified Eagle’s Medium) supplemented with 10% heat-inactivated fetal bovine serum (FBS), glutamine (2mM), 1% penicillin and streptomycin at 37 at humidified atmosphere at 5% CO2. [12] The primary culture of human oral keratinocytes was provided by Dr. M. Simon, SUNY at Stony Brook, School of Dentistry. Cells were used for the experiments at ~80% of confluency.

Western blotting. Cells in 100 mm dishes were washed with ice-cold phosphate buffer solution (PBS), and directly lysed in sample buffer (Cell Signaling Technology, Beverly, MA). Western blots were performed with 30-50 µg of total protein. Primary antibodies (Cell Signalling Technology, Beverly, MA), secondary horseradish peroxidase-conjugated antibodies and chemiluminescent detection agent (ECL) (Amersham Biosciences Corp., NJ) were added to the memebranes according to manifactures instructions. TRAF4 and siRNAs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Blots were routinely stripped in buffer containing 1% beta-mercaptoethanol, 2% SDS, 62.5

mM Tris-HCL buffer (pH 6.7) for 1 h at 37C and reprobed.

Determination of cell viability after EtOH treatment. Cell viability was determined with CellTiter-Blue Cell Viability kit (Promega Corp., Madison, WI).

Typically cells were seeded at a density of 3000 cells per well in 96 well plates in full growth media or in serum-free media. EtOH was added in concentrations ranging from 0 to 4%. Incubation with EtOH lasted from 10min to 9 hrs.

Fluorescent substrate was placed after incubation with EtOH and/or inhibitors and after 1 h incubation at 37°C fluorescence at 590 nm was measured with a Fluorescence Multi-well Plate Reader Cyto-Fluor2 (PerSeptive Biosystems, Farmingham, MA).

RNA interference (RNAi) experiments.

Cells growing in 60-mm dishes were transfected with TRAF4-specific RNA duplex (Santa Cruz Biotechnology) using Lipofectamine 2000 (Invitorgen Corp). A fragment of RNA coding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a negative control under similar conditions.

Semiquantitative and real-time polymerase chain reaction (RT-PCR). Gene expression was accessed using reverse RTPCR analysis. These genes were selected with the respect to the magnitude of their EtOH response based on the comparison of microarray data for HaCaT cells pretreated for 4h with 4% of EtOH and untreated cells. cDNA was synthesized by reverse-transcribing of 1 µg of total

A

Apoptosis at 4 h (% of annexin N positive) cells)

cidate the molecular mechanisms of ethanol-induced cell death in oral epithelial cells. We exposed normal oral epithelial cells, HaCaT cells (spontaneously immortalized human keratinocyte cell line) and cells of the tumorigenic HaCaT-II-4 cell line to ethanol (EtOH) and determined cell toxicity and the resulting modulation of the MAP and PI3 kinase signal transduction pathways and the specific role of TRAF4 in these events.

One of the genes overexpressed in HaCaT and HaCaT-II-4 cells treated with EtOH was TRAF4 (evaluation of gene expression was performed by cDNA microarray analysis). RT-PCR analysis confirmed increased expression of TRAF4. The TRAF4 gene was then selected for further investigation of its

role in keratinocyte death induced by EtOH.

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B 3

Figure 2: Type of cell death activated by EtOH. (A) EtOH-induced apoptosis in HaCaT. Triplicate wells of HaCaT cells were treated with 0, 1, 2 and 4% EtOH in complete media for 4 h. To quantify the percent of apoptotic cells, cells resuspended in Annexin Buffer were analyzed on FACSort. Error bars represent ±SEM. (B) Accumulation of cleaved caspase-3 (CI.CASP3) and PARP after 4% EtOH treatment of HaCaT cells. HaCaT cells were incubated with 4% EtOH for the indicated time periods, and the cell lysates were subjected to Western blot analysis with the anti-CASP3 or anti-PARP antibodies.

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Figure 3. Erk 1/2, p38 and JNK phosphorylation in response to EtOH in oral epithelial cells (primary culture). Cells were incubated with 4% EtOH for the indicated time periods, and the cell lysates were subjected to Western blot analysis with anti-Erk, anti -p38 , anti- JNK, anti-phospho-Erk, anti-phospho-p38 or anti-phospho-JNK antibodies.

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RNA from HaCaT and II-4 cells treated and non-treated with 4% EtOH with Moloney murine leukemia virus (MMLV ) reverse transcriptase (Invitrogen, Carlsbad, CA) in the presence of 0.2 µM oligo-dT12-18 primer. cDNA product was used in a PCR with the housekeeping gene GAPDH as an internal control.

Primers were designed based on the GenBank sequences. PCR products sampled several cycles before the end of the exponential phase of amplification were quantified using Quantity One software (BioRad Laboratories, Hercules, CA). SYBR Green I (Molecular Probes, Eugene, OR) fluorescence readings acquired at 86°C was used for quantitation of real-time PCR. Each cDNA sample was analyzed in triplicate for quantitative assessment of RNA amplification. The comparative threshold cycle (Ct) method was used for relative quantification of target gene expression levels [13]. Calculations were normalized to the internal control GAPDH gene. An amlicon from pGL plasmid was used as an internal standard.

Statistical analysis. Results are expressed as mean ± standard error of at least three observations. Statistical significance was determined by use of Student’s t test and the SigmaStat program ( Jandel Scientific, San Rafael, CA).

Results and Discussion

Figure 4. Erk, p38 and JNK phosphorylation in response to EtOH. HaCaT cells, both transfected with TRAF4-specific siRNA or non-transfected, were incubated with 4% EtOH for the indicated time periods, and the cell lysates were subjected to Western blot analysis with either anti-p38 or anti-phospho-p38 antibodies, anti-Erk or anti-phosphoErk (pErk) antibodies, or anti-JNK or anti-phospho-JNK.

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We used primary oral epithelial cells to study cell survival and signal transduction under the action of EtOH. Since the results obtained with primary cells correlated well with the data obtained using HaCaT cells as a model of normal epithelial cells, we used HaCaT cell line as a basic experimental model in consecutive experiments.

RT-PCR analysis. TRAF4 elevation in EtOH-treated cells observed in microarray assay was confirmed by RTPCR that showed a 3.2 fold elevation of TRAF4 mRNA expression after 4 hours of 4% EtOH treatment in HaCaT and 2.8 fold in II-4 cells. Thus, the TRAF4 gene was selected for further investigation of its role in epithelial cell death induced by EtOH. Cell viability after exposure to 4% EtOH. To compare the effect of EtOH on cell viability of primary oral epithe-

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lial, HaCaT and II-4 cells, the cells were treated with 4% EtOH for 4 h. Only 57% of HaCaT cells survived, as compared to 82% of II-4 cells (Figure 1A). To reveal the prevalence of apoptotic cell death induced by EtOH, we counted the number of apoptotic cells after pretreating HaCaT cells with different concentrations of EtOH (Figure 2A). Over 50% of HaCaT cells pretreated with 4% EtOH for 4 h were apoptotic (Figure 2A). To confirm the presence of apoptotic cells after 4 h treatment of HaCaT cells with 4% EtOH, we observed accumulation of cleaved caspase-3 and poly ADP-ribose polymerase (PARP) (Figure 2B).

The role of TRAF4 in HaCaT cell survival after EtOH treatment. To evaluate the role of TRAF4 in EtOHinduced cell death, intact HaCaT cells and cells with transcriptionally silenced TRAF4 expression were treated with 4% EtOH for 4h (Figure 1B). TRAF4 protein expression was substantially lower in siRNA transfected cells than in nontransfected HaCaT and II-4 cells (data not shown). Cell viability was determined with CellTiter blue assay (Figure 1B).

To reveal the type of cell death activated

by EtOH, the number of apoptotic cells was determined after pretreating HaCaT cells with EtOH (Figure 2A). To confirm the finding that most of the cells died via apoptosis, we observed accumulation of cleaved caspase-3 and PARP after 4 h 4% EtOH treatment (Figure 2B). siRNA transcriptional silencing of TRAF4 expression in HaCaT cells increased EtOH-induced cell death (Figure 1B). 47% of EtOH-treated HaCaT cells died after 4 h. Inhibition of TRAF4 transcription with siRNA increased the number of dead cells by 17%. HaCaT

cells transfected with control (scrambled) siRNA did not display a change in cell survival rate (Figure 1B). This led to the conclusion that TRAF4 plays the role of a survival factor under the conditions of EtOH exposure. To understand the underlying molecular mechanisms we studied the role of TRAF4 in MAP and PI3 kinase signaling pathways, which are involved in cell death and survival.

MAP and PI3 kinase activation by EtOH in primary oral epithelial and HaCaT cells. MAPK signaling pathways are involved in many cellular functions, including cell growth, differentiation, development, and apoptosis [11].

They are, in general, subdivided into three different pathways: ERK, p38 kinase, and JNK signaling pathways. Akt and P13 are serine-threonine kinases involved in an anti-apoptotic signaling pathway [14, 15]. We evaluated the phosphorylation profile of all 3 MAPKs (ERK, JNK, and p38) and PI3/Akt after EtOH exposure.

To do this, we carried out Western blot analyses with phosphorylation-specific antibodies to detect the level of active (i.e., phosphorylated) Erk, p38 , c-jun and Akt, and non-phosphorylated antibodies, to quantitate the total level for each of the kinases. Activation of all 3 MAP kinase pathways and PI3/Akt kinase pathway by EtOH was observed in primary oral epithelial (Figure 3) and HaCaT cells (Figure 4, 5).

According to existing data, JNK activation is directly linked to cell death in many cell types; phosphorylated JNK has been found to be a pro-apoptotic factor [14]. Importantly, JNK signaling antagonizes the effect of the P13/Akt survival pathway [15].

In our experiments, the phosphory-

lation of Erk and p38 increased after EtOH exposure, reaching a maximum after 4h of treatment in both regular and TRAF4-inactivated HaCaT cells. In regular HaCaT cells, JNK was activated 1 hr after EtOH addition, while HaCaT cells with TRAF4 siRNA revealed a delay of JNK phosphorylation (Figure 4).

Akt was activated in less than 10 min., while in TRAF4-siRNA blocked cells, Akt activation was delayed (Figure 5).

Thus, TRAF4 siRNA-treated HaCaT cells incubated with 4% EtOH exhibited delays in JNK and Akt, but not Erk1/2 and p38 phosphorylation (Figure 4, 5).

The delay in JNK phosphorylation in HaCaT cells with inhibited TRAF4 expression may be explained by TRAF4â&#x20AC;&#x2122;s role in promoting cell death by JNK activation. The delay in Akt phosphorylation in EtOH-treated HaCaT cells with inhibited TRAF4 expression (Figure 5) revealed that in EtOH-treated cells, in addition to playing the role of a survival factor, TRAF4 may play a protective role in this experimental model by activating the Akt survival pathway.

Conclusions From our research it can be concluded that in oral keratinocytes, TRAF4 can integrate the Akt and JNK signaling pathways involved in cell death and survival during EtOH treatment, regulating the balance between cell fate alternatives.

Our data support the findings that cell life and death decisions are made by involving apoptotic and survival signaling through mammalian cell JNK and P13/ Akt pathways [15].

Overall, EtOH selectively induced cell death in over 50% of oral epithelial

Figure 5. EtOH triggers activation of Akt in HaCaT cells. HaCaT cells transfected with TRAF4 specific siRNA and non-transfected HaCaT cells were incubated with 4% EtOH for the indicated time periods, and the cell lysates were subjected to Western blot analysis with either anti-Akt or anti-phospho-Akt (Ser373) antibodies.

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REPORTS cells via apoptosis and, at a lesser extent, in transformed cells, which is in accordance with our previous laboratory hypothesis that EtOH permits selective growth of malignant cells (Samara, 2005, unpublished data). These processes are executed via activation of MAPK and P13/Akt kinase signaling pathways. It can be concluded that TRAF4 is a survival factor in EtOH-treated normal epithelial cells and interferes with crosstalk between the JNK and Akt signaling pathways.

References 1. CDC Oral Cancer Background Papers (2006) http://oralcancerfoundation.org/ cdc/index.htm 2. Thun, M.J., Peto R., Lopez A.D. et al.

(1997). Alcohol consumption and mortality among middle-aged and elderly U.S. adults. N Engl J Med, 337, 1705-14.

3. Cook, R. T. (1998) Alcohol abuse, alcoholism, and damage to the immune system-a review. Alcohol Clin Exp Res, 22, 1927-42.

4. Nelson, S. & Kolls, J. K. (2002) Alcohol, host defence and society. Nat Rev Immunol, 2, 205-9.

5. Hoek, J. B. & Pastorino, J. G. (2002) Ethanol, oxidative stress, and cytokineinduced liver cell injury. Alcohol, 27, 638.

6. Oak, S., Mandrekar, P., Catalano, D., Kodys, K. & Szabo, G. (2006) TLR2- and TLR4-mediated signals determine attenuation or augmentation of inflammation by acute alcohol in monocytes. J Immunol, 176, 7628-35.

7. Bradley, J. R. & Pober, J. S. (2001) Tumor necrosis factor receptor-associated factors (TRAFs). Oncogene, 20, 648291.

8. Abell, A. N., Granger, D. A. & Johnson, G. L. (2007) MEKK4 stimulation of p38 and JNK activity is negatively regulated by GSK3beta. J Biol Chem, 282, 30476-84.

9. Kedinger, V. & Rio, M. C. (2007) TRAF4, the unique family member. Adv Exp Med Biol, 597, 60-71.

10. Camillieri-Broet S., Cremer I., Marmey B., et al. (2007) TRAF4 overexpression is a common characteristic of human carcinomas. Oncogene 26, 142147.

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11. Krishna M and Narang H. (2008) Review: The complexity of mitogen-activated protein kinases (MAPKs) made simple. Cell Mol Life Sci, 65, 3525-3544.

12. Karen, J., Wang Y, Javaherian A. et al. (1999) 12-O-tetradecanoylphobrol13-acetate induces clonal expansion of potentially malignant keratinocytes in a tissue model of early neoplastic progression. Cancer Res. 59, 474-81.

13. Bustin S.A. (2000) Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol, 25, 169-193.

14. Guo, J., Sabri, A., Elouardighi, H., Rybin, V. & Steinberg, S. F. (2006) Alpha1-adrenergic receptors activate AKT via a Pyk2/PDK-1 pathway that is tonically inhibited by novel protein kinase C isoforms in cardiomyocytes. Circ Res, 99, 1367-75.

15. Sunayama, J., Tsuruta, F., Masuyama, N. & Gotoh, Y. (2005) JNK antagonizes Akt-mediated survival signals by phosphorylating 14-3-3. J Cell Biol, 170, 295304.

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Inhibitors of Serine Proteinases, Matrix Metalloproteinases and Histone Deacetylases: Thermorubin, COL-308, Myricetin, and Tellimagrandin Rana Said, ‘11 Advisor: Dr. Sanford Simon, Ph.D., Department of Biochemistry and Department of Pathology Stony Brook University, Stony Brook, NY 11794

Abstract

Introduction

Various natural products and their chemically modified derivatives have been reported to control acute and chronic inflammation as well as pathologically excessive cell proliferation and invasion in neoplastic tissues without risk of significant toxicity. A group of different compounds characterized by the presence of multiple keto and hydroxyl substituents and large hydrophobic domains have been tested for their ability to inhibit the enzymatic activities of zincdependent metalloproteinases of bacterial and mammalian origin, the proteolytic activity of the serine proteinase neutrophil elastase, and the amidolytic activity of class I and II histone deacetylases. The compounds investigated are Thermorubin, a highly conjugated antibiotic with multiple fused aromatic rings, COL-308, a nonantimicrobial chemically modified tetracycline, the flavonoid Myricetin, and the polyphenol Tellimagrandin. It is hypothesized that the inhibition of different types of enzymes by these compounds is attributed to both the compounds’ hydrophobic nature and the negative charges on ionized hydroxyl groups and enolic tautomers of keto groups which allow the inhibitors to interact with isolated positive charges buried in the hydrophobic environment of the enzymes in the vicinity of their active sites. Since the tested compounds inhibit multiple enzymes that are implicated in mechanisms of inflammation and tumor invasiveness without concomitant cytotoxicity, they may become attractive candidates for drug development programs directed towards reducing inflammatory tissue damage and minimizing progression of cancers.

Despite considerable progress in screening methods and rational drug design, the statistics of cancer-associated deaths have not shown a significant improvement over the last few decades.

Almost 89% of the anti-cancer drugs developed are designed to induce apoptosis in tumors by acting on a single target, whereas only a minor fraction of drugs target metastasis, the leading cause of cancer-associated death [1].

A persistent problem with the single target approach is that the complex systems regulating tumor cell function may conspire to reduce responsiveness to a drug while still maintaining those cell functions needed for survival. These sobering statistics prompted investigation of a novel drug design paradigm based on “natural multi-target treatments,” as described by Aggarwal in a special issue of Cancer Letters [2].

An underlying hypothesis for our research has been based on the principles outlined by Aggarwal (2008): the sum of multiple effects of a drug that acts on more than one target may be superior to the effects of another drug acting on a single target, even if that single target could be inhibited with greater potency.

The neutrophil is the most proteolytically active inflammatory cell. Excessive infiltration of tissues with neutrophils could result in proteolytic destruction of connective tissues associated with inflammatory tissue damage as well as increased tumor metastatic spread. The two most destructive classes of proteinases in the neutrophil are the serine proteinases, especially human neutrophil elastase (HNE), and the matrix metallo-

proteinases (MMPs). HNE is a hydrolytic enzyme with the ability to degrade elastin, collagen, and fibronectin, leaving the tissues relatively defenseless against multiple mechanisms of injury. The release of HNE from neutrophils is apart of the cellular defense against invading pathogens; it may be triggered during the inflammatory response to blood clotting, complement activation, and mediators produced during infections. High levels of HNE damage extracellular matrix proteins of connective tissues and degrade multiple components of the immune response. MMPs are zinc endopeptidases capable of digesting the extracellular matrix, which allows them to play a role in removing damaged matrix components during wound healing and tissue repair.

Tumor cells, stromal cells, neutrophils, and macrophages secrete excess MMPs that contribute to the migration of tumor cells in metastatic cancer and to joint destruction in arthritis [3].

Histone deacetylases (HDACs) are enzymes that remove acetyl groups from the modified amino acid E-N-acetyl lysine found in a number of nuclear and cytosolic proteins. In the nucleus, the most frequently acetylated proteins are histones; their deacetylation contributes to chromatin condensation. In the cytosol, lysines may be acetylated on cytoskeletal proteins as well as on protein components of transcription factor activation cascades. The deacetylation of these proteins results in greater activation of the transcription factor Nf-kB, a major regulator of expression of inflammatory cytokines and chemokines.

Inhibitors of HDACs unravel the tightly wrapped chromatin in the nucleosome and promote selective transcrip-

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REPORTS tion and gene expression. They are used in cancer treatments to promote the expression of silenced genes that control cell cycle, differentiation, and apoptosis.

Additionally, inhibition of deacetylation of components of activation pathways for pro-inflammatory transcription factors such as Nf-kB by Class I and II HDACs downregulates the inflammatory response.

We have evaluated a group of different natural products and their derivatives that share the capacity to inhibit the following: 1) the proteolytic activity of neutrophil elastase; 2) the enzymatic activities of the MMPs and other zincdependent metalloproteinases of bacterial and mammalian origin; and 3) the amidolytic activity of class I and II histone deacetylases. These specific enzymes were selected because they conspire to cause tissue injury in inflammation and allow tumor cells to metastasize, in large part because they break down the surrounding connective tissue to facilitate invasiveness.

Thermorubin is a highly conjugated antibiotic with multiple fused aromatic rings. The bottom of the ring system is highly hydrophobic, while the top has keto groups that can enolize as well as hydroxyl groups, all of which can acquire partial negative charges. COL-308 [9-amino6-demethyl-4-de(dimethylamino)tetracycline] is a derivative of the parent compound doxycycline, a tetracycline antibiotic developed by Pfizer in the 1960s and used in treatment of chronic inflammatory diseases. Loss of the 4-dimethylamino group specifically eliminates all antimicrobial activity. The chemically modified tetracycline has multiple keto and hydroxyl substituents at the bottom of the fused ring system, while elimination of the amino and dimethyl amino groups results in a predominantly hydrophobic domain on the top of the molecule. [4, 5, 6] Both COL-308 and Thermorubin have similar linear arrays of oxygen atoms with hydrogens that can easily dissociate through a mechanism of tautomerization that gives the compounds a partial electronegative character and should enable them to bind to hydrophobic enzymes containing a positive charge in a hydrophobic domain. Myricetin is a ma-

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Figure 1: Chemical structures and properties of the inhibitors tested: (A) Thermorubin; (B) COL-308; (C) Myricetin; (D) Tellimagrandin

jor flavonoid found in several foods such as berries, grapes, onions, and red wine.

This natural product has one galloyl ring and hydroxyl substituents attached to two fused rings. It has anti-oxidant and anti-apoptotic activities [7]. The three hydroxyl groups on the galloyl ring can also undergo deprotonation, leaving the otherwise aromatic ring with partial negative charge. Tellimagrandin is an elligatannin, a bioactive polyphenol found in pomegranates, raspberries, strawberries and nuts. It has four galloyl rings (two are condensed) bound to glucose and has anti-oxidant and anti-inflammatory activities [8].

Since the compounds tested here were evaluated for their capacity to target multiple enzymes, we have considered that their potential pharmaceutical benefits may not be limited to inhibition of tumor cell proliferation and inflammatory tissue injury but may also extend to prevention of cancer and inflammation in healthy individuals.

Materials and Methods HNE was obtained from Athens Research and Technology (GA), an active

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form of the catalytic domain of MMP-9 was obtained from Anaspec Corp (CA), and a crude mixture of HDACs present in a lysate of HeLa cells was obtained from Active Motif (CA). The substrate MeOSuc-Ala-Ala-Pro-Val-pNA was obtained from Sigma-Aldrich (MO), the FRET-quenched 10-amino acid long peptide substrate for multiple MMPs was obtained from Anaspec, and a lysineacetylated peptide substrate derived from the p53 protein, and the Fluor-de-LysGreenâ&#x201E;˘ developer for detection of freed lysine E-amino groups were obtained from Enzo Life Sciences (PA). COL 308 was provided by Galderma, SA (France), Thermorubin (Gruppo Lepetit, Italy) was provided by Dr. Francis Johnson (Chemistry Department, Stony Brook University), Myricetin was obtained from SigmaAdrich, and Tellimagrandin was obtained from Nacalai Tesque ( Japan).

Amidolytic activity of HNE towards the tetrapeptide substrate MeOSuc-AlaAla-Pro-Val-p-Nitroanilide was assayed by release of p-nitroaniline and was followed by recording absorbance at 405 nm. Inhibition was seen as a lowered rate of OD increase.

MMP-mediated cleavage of a

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Figure 2: (A) Inhibition of neutrophil elastase amidolytic activity, expressed as normalized rate of p-nitroaniline formation, by 10, 20, 30, and 40uM Thermorubin and COL-308. (B) Dixon plots of the inverse normalized rate of amidolysis as a function of inhibitor concentration.

5-FAM/QXL520 Fluorescence Resonance Energy Transfer (FRET) -quenched peptide, a synthetic substrate for which multiple MMPs have high affinity, was followed by detection of the fluorescence of 5-FAM upon cleavage and was monitored at 490 nm excitation/520 nm emission. MMP-9 activity was detected as a time-dependent increase in relative fluorescence intensity generated from the cleavage of the FRET-quenched peptide. Inhibition of the activity of this MMP resulted in a slower rate of increase of fluorescence intensity.

A nuclear extract of HeLa cells was used as a source of HDAC activity. Time-dependent deacetylation of a peptide substrate derived from the p53 protein by the HDACs in the HeLa extract could be followed by reacting the deacetylated lysine side chains with the Fluor de Lysâ&#x201E;˘ developer (Fluor de Lys Green, BioMol Corp) to generate a fluorophore that emits at 530 nm with 485 nm excitation. Inhibition of HDACs was detected as decreased fluorescence intensity at 530 nm after a set incubation time.

Results Our laboratory has detected inhibition by all four compounds tested towards the amidolytic activity of neutrophil elastase; only results for COL-308

and thermorubin obtained by the first author are shown here (Figure 2). Thermorubin displayed somewhat higher inhibitory potency than COL-308, with 50% inhibition around the 30uM range. The dose dependence of this inhibitory activity was analyzed by the graphical method of Dixon: the nonlinear Dixon plots are consistent with a mixed mode of inhibition. All four inhibitors also decreased the rate of generation of fluorescence from cleavage of a FRET-quenched substrate after incubation with MMP-9 (Figure 3).

Inhibition of MMP-9 activity was dosedependent for three of the four inhibitors, with 50% inhibition reached at concentrations around 25 ÎźM. Tellimagrandin had the highest inhibition potency followed by Thermorubin and COL-308.

Inhibtion by Myricetin showed only limited dose dependence. Finally, we observed that all four compounds inhibited histone deacetylase activity, expressed as normalized relative fluoresence intensity detected after incubation with a crude HDAC preparation from lysed HeLa cells and substrate derived from the p53 protein, followed by a subsequent incubation with the developer (Figure 4). After incubation of the HeLa extract with the substrate in the presence of the inhibitors the fluorescence detected on subsequent incubation with the developer was decreased. Tellimagrandin was the most potent inhibitor of HDAC activity, followed by Thermorubin, Myricetin, and

COL-308; all the inhibitors showed 50% inhibition in the 10-4 M range.

Discussion All the compounds showed inhibitory activity with potencies, expressed as IC50 values, in the 10-5 - 10-4 M range in assays using enzymes implicated in mechanisms of inflammatory tissue injury and tumor invasiveness. While these potencies may be lower than those of some drugs with single targets that have nanomolar or picomolar IC50 values in selective in vitro studies, the cumulative effects of these natural agents may confer benefits comparable to those of more potent pharmaceuticals with reduced risk of toxicity. The ability of the compounds to inhibit multiple critical regulatory enzymes with lower potencies may also be beneficial in ensuring that the endogenous host defense systems against infection and cancer will not be impaired.

The interest in investigating and developing anti-inflammatory agents that can target multiple enzymes has been stimulated by a better understanding of the pivotal role of inflammation in seemingly unrelated diseases ranging from endocrine to neurologic to neoplastic disorders. The acute and chronic inflammatory responses involve the release of cytokines such as TNF-a, IL-1B, and VEGF, as well as HNE and the MMPs. Excessive unchecked inflammatory responses have

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REPORTS been implicated in autoinflammatory and autoimmune conditions, neurodegenerative diseases, and cancer metastasis [9].

As an example of the effects of agents inhibiting multiple steps in inflammation, the inhibitory effects of Myricetin on IL-1B induced production of inflammatory mediators (MMP-1 and IL-6) and activation of the MAP Kinase (MAPK) signaling pathway have been investigated in SW982 human synovial sarcoma cells, a line capable of secreting high levels of inflammatory cytokines and MMPs [7].

The goal of the study was to test the potential therapeutic and protective effect of Myricetin on rheumatoid arthritis, a chronic inflammatory disease. The results were consistent with our data in showing that Myricetin had anti-proteolytic activity and an inhibitory effect on IL-1B induced inflammatory mediators.

Furthermore, Myricetin down-regulated the MAPK signaling pathway involved in the activation of transcription factors that induce the synthesis of MMP-1 and cytokines.

Further investigations of the potential therapeutic effects of blocking cytokine-mediated inflammation have highlighted the significant connection between inflammation and diabetes.

Type II diabetes is a chronic disease in which production of IL-1B is triggered by the high levels of circulating glucose.

IL-1B-mediated inflammation progressively destroys B cells in the pancreas and causes the initiation of the “pre-diabetic” stage. Clinical evidence has shown that when IL-1B was blocked, diabetic patients required 66% less insulin to obtain the same level of glycemic control; agents that can contribute to downregulation of inflammatory cascades can therefore offer the potential to salvage B islet cells by reducing cytokine-mediated inflammation [10].

A more direct role of COL308 in reducing tissue injury associated with diabetes was demonstrated in a study in the Department of Oral Biology and Pathology at Stony Brook University conducted

by Drs. Lorne Golub, Hsi-Ming Lee, and Maria Ryan. These investigators showed that rats rendered diabetic through administration of streptozotocin or through the effects of homozygosity for the ob gene experienced aggressive bone and

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tooth loss resembling severe periodontitis. This iatrogenic periodontal disease was controlled effectively with the nonantimicrobial chemically-modified tetracycline COL-308 administered orally to the animals [11, 5, 12, 4]. Similar results have recently been obtained by Drs. Golub and Lee using analogs of thermorubin synthesized by Dr. Francis Johnson in the Department of Chemistry at Stony Brook University in unpublished studies. COL-308 has also been shown to inhibit VEGF secretion and endothelial cell angiogenesis induced by breast tumor cell lines by inhibiting the inflammatory pathway that results in Nf-kB activation [6, 13]. It remains for our laboratory to establish whether the effects of compounds like COL-308 and Thermorubin can be attributed to reductions in levels of final products of the Nf-kB pathway that could be achieved specifically through inhibition of HDACs. HDACs are known to remove an acetyl group from the protein STAT-1, resulting in its diminished capacity to block Nf-kB activation. It has been argued that HDAC inhibitors are effective protectors of acetylated STAT1, ensuring that it can efficiently prevent NF-kB activation and thereby reduce the expression of multiple inflammatory cytokines and mediators [14].

Histone Deacetylase Inhibitors have

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also been tested for their capacity to reduce inflammation of pancreatic islet tissues. Vorinostat (SAHA), a potent hydroxamic acid designed to target the essential zinc in HDACs, has been shown to reduce cytokine-mediated nitric oxide formation, thereby helping to protect isolated islet cells from diminished insulin secretion [15]. SAHA and other hydroxamate HDAC inhibitors such as Trichostatin A, have also been shown to significantly enhance neuroprotection and decrease B-amyloid production in animal models of neurodegenerative diseases such as Alzheimer’s disease [16].

Furthermore, results of clinical trials showed that many hematopoietic malignancies have been responsive to HDAC inhibitors [17]. Favorable responses were associated with low toxicity due to the ability of HDAC inhibitors to mediate anti-inflammatory and immunosuppressive actions while increasing the expression of pro-apoptotic genes silenced in malignant cells.

In recent years, there has been a great focus on natural dietary products that can be useful as chemopreventive or chemotherapeutic agents for many diseases including cancer. Both Myricetin and Tellimagrandin are “antioxidant” polyphenolic compounds and possess substantial anti-carcinogenic and anti-

Figure 3: Normalized inhibition of MMP-9 peptidolytic activity by 25, 50, 100, and 200 μM Thermorubin, COL-308, Myricetin, and Tellimagrandin. All the inhibitors showed 50% inhibition in the 25 μM range. Tellimagrandin had the highest inhibition potency followed

by Thermorubin and COL-308. Inhibtion by Myricetin showed only limited dose dependence.

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References 1. Liu, J. (2010) Rationale for the use of natural anti-inflammatory agents in cancer chemotherapy. N. Amer, J. Med. Sci. 3:160-166.

2. Aggarwal, B. (2008) The past, present and future of multi-targeted cancer treatment “naturally”: food for thought. Cancer Lett. 269:187-188 3. Mandal M., Mandal A., Das S., Chakraborti T., and Chakraborti S. (2003) Clinical implications of matrix metalloproteinases. Mol. Cell. Biol.

252:305-32.

4. Golub, L.M., McNamara, T.F., D’Angelo, G., Greenwald, R.A., and Ramamurthy, N.S. (1987) A non-antibacterial chemically-modified tetracycline inhibits mammalian collagenase activity.

Figure 4: Inhibition of HDAC activity by Thermorubin, COL-308, Myricetin, and Tellimagrandin at 100, 250, and 500 μM. Deacetylase activity was expressed as normalized rela- J. Dent. Res. 66:1310-1314.

5. Golub, L.M., Ramamurthy, N.S., tive fluoresence intensity detected after incubation with a crude HDAC preparation from lysed HeLa cells and substrate derived from the p53 protein, followed by a subsequent McNamara, T.F., Greenwald, R.A., and incubation with the developer. After incubation of the HeLa extract with the substrate in Rifkin, B.R. (1991) Tetracyclines inhibit the presence of the inhibitors the fluorescence detected on subsequent incubation with connective tissue breakdown: new therathe developer was decreased. Tellimagrandin was the most potent inhibitor of HDAC peutic implications for an old family of activity, followed by Thermorubin, Myricetin, and COL-308; all the inhibitors showed 50% drugs. Crit. Rev. Oral Biol. Med. 2:297inhibition in the 10-4 M range.

321. mutagenic activities [8]. Myricetin was pounds we have studied as well as other 6. Gu, Y., Lee, H.M., Roemer, E.J., found to suppress activation of the NF- related molecules, including some antibi- Musacchia, L., Golub, L.M., and Simon, kB pathway and to inhibit expression of otics and their derivatives, bioflavonoids S.R. (2001) Inhibition of tumor cell inNF-kB responsive genes such as COX-2, and polyphenols, which all have com- vasiveness by chemically modified tetranitric oxide, and MMP-9; excessive and mon chemical characteristics of hydro- cyclines. Curr. Med. Chem. 8:261-270. prolonged expression of these genes has phobicity and the presence of conjugated 7. Lee, Youg Soon (2010). Myricetin been linked with inflammation and tu- hydroxyl and keto groups, for manage- inhibits IL-1B induced inflammatory morigenesis. Furthermore, Myricetin was ment of autoimmune, autoinflammatory, mediators in SW982 human synovial also shown to inhibit phosphorylation and neurodegenerative diseases such as sarcoma cells.” Internat Immunopharm. of proteins of the MAPK family, result- rheumatoid arthritis, atherosclerosis, 1.10:812-814. ing in reduced expression and release of periodonditis, Alzheimer’s disease, Par- 8. Heber, D. (2009) Multi-targeted therMMP-2 and MMP-9, two gelatinolytic kinson’s disease, and both type I and type apy of cancer by ellagitannins. Cancer metalloproteinases typically associated II diabetes as well as in preventative and Lett. 269:262-268. with inflammatory and invasive processes therapeutic approaches to control cancer. 9. Dinarello, CA. (2010) Anti-inflammaThe compounds we tested show very low tory agents: present and future. Cell 140: [18]. cytotoxicity towards a number of primary 935-950. human cells in culture, and, in spite of 10. Larsen, C. M. (2009) Sustained efConclusion their limited solubility, can be detected in fects of IL-1 receptor antagonist treatThis study focused on testing direct the circulation after oral administration. ment in type 2 diabetes. Diabetes Care inhibition of HDACs and two of the This makes them attractive initial candi- 3:1663-1668. most destructive classes of proteinases, dates to serve as parent compounds from 11. Golub, L.M., Lee, H.M., Lehrer, G., MMP-9 and HLE, because they play key which future analogs may be developed et al. (1983) Minocycline reduces gingiroles in inflammation and cancer metas- for use as safe and effective anti-inflam- val collagenolytic activity during diabetes. Preliminary observations and a protasis by facilitating proteolytic degrada- matory and anti-cancer agents. posed new mechanism of action. J. Perio. tion of extracellular matrix proteins and Res. 18:516-526. breach of the basement membrane. Our 12. Rifkin, B.R., Vernillo, A.T., and Golresults, consistent with various other ub, L.M. (1993) Blocking periodontal studies, highlight the potential applicadisease progression by inhibiting tissuetions of the natural multi-targeting comThe Stony Brook Young Investigators Review, Fall 2011

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Notes REPORTS destructive enzymes: a potential thrapeutic role for tetracyclines and their chemfically-modified analogs. J. Periodontol.

64(suppl):819-827.

13. Kothari, M, and Simon, S.R. (2006) Chemically modified tetracyclines inhbit VEGF secretion by breast cancer cell lines. Cytokine 35:115-125.

14. Dinarello, CA (2006) Inhibitors of histone deacetylases as anti-inflammatory drugs. Ernst Schering Foundation Workshop 56:45-60.

15. Leoni, F. (2002) The antitumor histone deacetylase inhibitor suberoylanilide hydroxamic acid exhibits anti-inflammatory properties via suppression of cytokines. Proc. Natl. Acad. Sci. USA 99:2995-3000.

16. Larsen, L. (2007) Inhibition of Histone deacetylases prevents cytokine induced toxicity in beta cells. Diabetologia.

50:779-789.

17. Garcia-Manerio, (2008) Phase 1 study of the histone deacetylase inhibitor varinostat in patients with advanced leukemias and myelodysplastic syndromes.

Blood 111:1060-1066.

18. Nandakumar, V., Singh, T, and Katiyar, SK (2008) Multi-targeted prevention and therapy of cancer by proanthocyanidins. Cancer Lett. 269:378-387.

19. Kocer, S.S., Walker, S.G., Zerler, B., Golub, L.M., and Simon, S.R. (2005) Metalloproteinase inhibitors, nonantimicrobial chemically modified tetracyclines and Ilomastat, block Bacillus anthracis lethal factor activity in viable cells. Infect. Immun. 73:7548-7557.

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The Stony Brook Young Investigators Review, Fall 2011

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