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A Conversation with Dr. Nancy Goroff
The Impact of the World Trade Center Attacks on Pollutant Levels
The Synthesis of 3’ Difluorovinyltaxoids (SB-T-12854)
Volume 9 Fall 2017
staff 2017-2018 Editor-in-Chief: Sahil Rawal ’19
Layout Chief: Danielle Espine ’18
Head of Cabinet: Peter Alsaloum ’19
Managing Editors: Rachel Kogan ’19 Jenna Mallon ’18
Assistant Layout Chief: Dahae Jun ’19
Cabinet: Benjamin Kerner ’18 Jesse Pace ’20 Jerin Thomas ’19
Associate Editors: Stephanie Budhan ’21 Samara Khan ’19 Bridgette Nixon ’21 Lillian Pao ’18 Anna Tarasova ’19 Nina Gu ’21
Layout Editor: Lauren Yoon ’21 Webmaster: Ronak Kenia ’18
Faculty Advisors: Dr. Peter Gergen Dr. Laura Lindenfeld Kathryn Fullam Graduate Advisor: Amanda Ng
Writers: Kunwar Ishan Sharma ’20 Sarika Hira ’18 Anurekha Ravikumar ’18 Rideeta Raquib ’19 Caleb Sooknanan ’20 Anam Rabbani ’18 Andrew Kim ’19 Muhsena Rahman ’18 Meenu Johnkutty ’21 Maryna Mullerman ’20 Meghan Bialt-DeCelie ’19 Ramanjot Singh ’19 Gene Yang ’19 Matthew Lee ’21
Copy Editors: Nita Wong ’21 Nomrota Majumder ’21 Aaradhana Natarajan ’20 Caleb Sooknanan ’20 Daniel Walocha ’19
Letter from the staff Formed in 2008 by a group of undergraduates with the intent of pushing the boundaries of scientific literature, Stony Brook Young Investigators Review (SBYIR) was created with the intention to provide an outlet for student research on campus. Since then, we have grown to publish eight issues, and have worked tirelessly with students and faculty to increase our presence on campus. In the past, we have invited several prominent scientists to speak at our biannual colloquia, including the Queen of Carbon Science, Dr. Mildred Dresselhaus, Hero of the Planet, Dr. Sylvia Earle, Dr. Donald Ingber, the Founding Director of the Wyss Institute for Biologically Inspired Engineering at Harvard University, and Dr. Arthur Horwich, whose research at Yale University led to the uncovering of chaperonin proteins. SBYIR has recently had the opportunity to formally partner with the Alan Alda Center for Communicating Science, which focuses on helping scientists more effectively communicate their work, marking a new era for our organization. To commemorate this new beginning, we have decided to rebrand our logo, a symbol of our partnership. In our ninth issue, readers will find articles that range from topics such as obesity and Parkinson’s disease to an interview with Dr. Nancy Goroff, a Stony Brook researcher and chair of the Chemistry Department. We also present articles on up and coming science technology, such as biophotonic imaging and the use of 3D printing to create cancer models for treatment. Readers will gain the opportunity to delve into primary research completed by undergraduates at the university, as Sarika Hira and Kunwar Ishan Sharma present their findings on 3’ difluorovinyltaxoids and pollutant levels in the Bronx post-9/11 respectively. To celebrate the release of this issue, we are humbled to host speaker Emily Kinser, a Master Innovator at IBM. Her work at IBM helped in the creation of Watson, and our student body is thrilled to hear about the future of artificial intelligence. None of this could be possible without the help of our staff members and writers who worked countless hours to create this publication. Furthermore, we would like to thank our partners at the Alan Alda Center for Communicating Science and the College of Engineering and Applied Sciences for their constant support, as well as our faculty advisors, Dr. Peter Gergen, Dr. Laura Lindenfeld, and Kathryn Fullam, for their guidance. This issue could not have been published without the help of our generous donors, so we also thank the Departments of Biomedical Engineering, Undergraduate Biology, and Undergraduate Biochemistry. Welcome to SBYIR. We sincerely hope you enjoy.
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TABLE OF CONTENTS Fall 2017
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interview A Conversation with Dr. Nancy Goroff By Muhsena Rahman ’18
Volume 9
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Models for Medicine: Modern Applications of 3D Printing in Cancer Treatment By Caleb Sooknanan ’20
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science reviews The Benefits And Applications of Terahertz Biophotonics Imaging
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Recent Advances in Parkinson’s Disease By Anam Rabbani ’18
By Anurekha Ravikumar ’18
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A Golden Age: Utilizing Gold Nanoparticles in Cancer Imaging and Therapy
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By Rideeta Raquib ’19
primary research The Impact of the World Trade Center Attacks on Pollutant Levels and Asthma-Related Emergency Department Visits in the Bronx
By Kunwar Ishan Sharma ’20
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The Multifacted Health Effects of Obesity By Andrew Kim ’19
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The Synthesis of 3’ Difluorovinyltaxoids (SB-T-12854) By Sarika Hira ’18
RESEARCH HIGHLIGHTS Find more news online! Visit us at sbyireview.com
The impact of physical activity intervention in schools on child motor skills
Fighting the Avian Leukosis Virus through Genome Modification
By Ramanjot Singh ’19 Previous research has shown that quality physical education may improve children’s focus on tasks. Dr. Ryan Burns of the University of Utah decided to further these findings by analyzing the effects of enhanced physical activity on children’s gross motor skills. He hypothesized that installing a Comprehensive School Physical Activity Programming (CSPAP) would significantly advance their motor skills. For 36 weeks, CSPAP’s physical activity leaders worked with the physical education personnel at five schools to improve their physical education programs, and added two to three ten-minute activity breaks during the school day. Dr. Burns then used the third edition Test for Gross Motor Development (TGMD3) to compare the motor skill levels of each child before the intervention to after the intervention. The total sample size for this study was 959 children ranging from first grade to sixth grade. The TGMD-3 pre-and-post intervention results indicated significant increases in gross motor skills. For example, children showed an increase in both loco-
By Matthew Lee ’21 motor, general movement such as walking and running, and ball skills, with an overall total motor skill increase. These results indicate that increased physical activity indeed improves cognitive and motor performance. Despite the significant results, there are several limitations to the study that may have influenced the data received. The researchers neglected to have a control group, and only sampled low-income children from a small concentrated area in the Western region of the United States. The limitations constrain the generalization of the results; nevertheless, the results are considered legitimate enough to support further studies on the use of CSPAP. References 1. Burns, R., et al., School physical activity programming and gross motor skills in children. American Journal of Health Behavior 41, 591598 (2017). doi:10.5993/AJHB.41.5.8. 2. Image retrieved from: https://pixabay.com/ en/baseball-little-league-children-92382/
Fig 1. A child playing baseball, a sport that enhances motor skill development.
Fig 1. Brood of chickens in a hen house.
The avian leukosis virus subgroup J (ALV-J) has plagued the poultry industry since its initial recognition about 30 years ago. The economic hardships caused by decreases in poultry yield make developing resistance mechanisms a topic of interest. Recently, a team led by Dr. Hong Jo Lee from Seoul National University decided to take the novel approach of editing the chicken genome in order to induce ALV-J resistance. To do this, the team used CRISPR-Cas9, a tool used to edit the genome through excision and insertion by vectors. The team targeted the Na+/ H+ exchange 1 (chNHE1) receptor on fibroblasts, a type of connective tissue, which is specifically required for ALV-J to gain entry to the host cell. Four different types of vectors were used as experimental groups; two had puromycin resistance while the other two featured neomycin resistance. Both puromycin and neomycin are antibiotics, however, puromycin inhibits protein synthesis while neuomycin prevents bacterial growth. Cells were infected using an infectious ALV-J vector with green fluorescent protein (GFP) expressing capabilities. Untreated and infected wild type fibroblasts were used with a control. The response from the virus was telling. Cells with premature stop codons in the chN-
HE1 transcript demonstrated almost no GFP expression, indicating complete resistance. Cells that were tailored with a tryptophan deletion at position 38 (Trp38) showed significantly less GFP expression than cells without the deletion. This indicates that tryptophan deletion may prove to reduce the level of infection. Techniques in bioinformatics were then utilized to discover that the localized region with Trp38 represented a major binding interface for ALV-J. Thus, removing Trp38 in tandem with other important amino acid residues that form crucial conformation structures could prevent ALV-J infection in poultry. The development of a system to combat avian diseases requires further research, as the chNHE1 receptor is highly conserved in chickens. However, the newly developed, precise CRISPR-Cas9 technique appears to be promising. References 1. H. Jo Lee, et. al., Precise gene editing of chicken Naþ/Hþ exchange type 1 (chNHE1) confers resistance to avian leukosis virus subgroup J (ALV-J). Developmental and Comparative Immunology 77, 340-349 (2017). doi: https://doi.org/10.1016/j.dci.2017.09.006 2. Image retrieved from: https://pixabay.com/ en/chicken-laying-hens-stall-624977/
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PoIsonous Frogs: Evolution of Epibatidine Resistance
Controlling Mitochondria to Stop the Clocks
By Gene Yang ’19 Animals that use toxins as an anti-predator defense usually evolve a method of resistance, often at a high physiological cost, to prevent self-intoxication. Poisonous frogs, a broad polyphyletic group within the order Anura, often use one such method known as target site insensitivity, which is the alteration of the molecular target of the toxin to disallow the toxin from binding. Researchers from University of Texas and Harvard University studied epibatidine resistance in poisonous frogs and discovered the specific amino acid replacements in the toxin’s molecular target: nAChR. nAChR makes resistance possible—a result that provides insight into the evolution of toxin defense. Epibatidine is a toxin that binds and activates the nicotinic acetylcholine receptor (nAChR), a common receptor protein that normally responds to the neurotransmitter acetylcholine (ACh). The researchers began by sequencing the genes of the three genera of poison frogs known to utilize epibatidine (Oophaga, Ameerega, and Epipedobates), and then compared those genes to those of 19 other lineages of poisonous frogs. This resulted in the identification of unique amino acids found in the nAChR of epibatidine-wielding poison frogs that
By Meghan Bialt-DeCelie ’19 were not present in non-epibatidine lineages. Site-directed mutagenesis, which is the intentional change of a specific DNA sequence, was first used to express wild-type nAChR and epibatidine-resistant nAChR in Xenopus laevis oocytes. Both the wild-type nAChR groups and epibatidine-resistant nAChR groups were then exposed to ACh and epibatidine. The results revealed that a single amino acid replacement in nAChR, from serine to cysteine (S108C), was responsible for decreased epibatidine sensitivity. However, this convergently evolved mutation also decreased acetylcholine sensitivity, and further amino acid replacements unique to each lineage were needed to restore nAChR functionality. The amino acid replacement S108C demonstrated that this convergent evolution mutation provides a strong selective advantage. References: 1. R.D. Tarvin, et al., Interacting amino acid replacements allow poison frogs to evolve epibatidine resistance. Science 357, 1261-1266 (2017). doi: 10.1126/science.aan5061. 2. Image retrieved from: https://upload. wikimedia.org/wikipedia/commons/e/e8/ Flickr_-_ggallice_-_Pleasing_poison_frog.jpg
Fig 1. The poison frog, Ameerega bassleri, is one of the three studied lineages of frogs that evolved resistance to epibatidine, a toxin lethal in microgram-doses.
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Fig 1. Scientists from UCLA controlled expression of genes Drp1 and Atg1 in fruit flies to promote breakdown and removal of damaged mitochondria.
The respiratory function of the mitochondrion, the energy producing organelle found in the cell, can decline over time. This is because of how the mitochondria enlarge and assume a more elongated shape. Typically, that mitochondrion will eventually break down and get removed during processes called mitochondrial fission and mitophagy, respectively. Accumulation of the ineffective mitochondria and inability to remove them are major signs of aging. Researchers led by Dr. Anil Rana from the University of California, Los Angeles extended the life of middle-aged Drosophila flies by controlling the expression of several genes that mediate mitochondrial dynamics. Specifically, they upregulated Dynamin-related protein 1 (Drp1), a protein responsible for mitochondrial fission, for a week in middle-aged Drosophila. The researchers were also able to extend the lifespan of fruit flies by knocking down genes responsible for Mitofusion proteins (Mfn) that typically allow mitochondria to fuse into larger, dysfunctional morphologies. Atg1, a gene responsible for autophagy of mitochondria, was also studied. The importance of mitochondrial morphology and dynamics were further exhibited when Atg1 was knocked out and the life-extending effects of Drp1 were increased. This demonstrated that
the expression of some genes and the suppression of others involved in mitochondrial maintenance can impact life span. These findings will allow for the future development of drugs that may control mitochondrial fission and autophagy. This will not only increase the lifespan patients, but may also improve their quality of life during those years. References: 1. A. Rana, et al., Promoting Drp1-mediated mitochondrial fission in midlife prolongs healthy lifespan of Drosophila melanogaster. Nature Communications 8, 448 (2017). doi:10.1038/ s41467-017-00525-4. 2. Image retrieved from: https://commons.wikimedia.org/wiki/File:Mitochondria.svg
HABITUAL Gamers MAY HAVE Learning ADVANTAGE
NEW MODEL TO DETERMINE INFLUENCES OF HUMAN VIABILITY
By Meenu Johnkutty ’21
By Maryna Mullerman ’20 Human viability is the survival of individuals after birth, and more research is needed to understand how associated genetic factors affect human survival and life expectancy. Dr. Hakhamanesh Mostafavi and researchers at Columbia University in New York developed a method to recognize genetic variants that influence human survival. The proposed method would provide information about human fitness in the environment and the cost of longer lifespan. The team analyzed participants from the Genetic Epidemiology Research on Adult Health and Aging (GERA) and UK Biobank. They focused on individuals of different ages and sexes while testing for allele frequency differences. The researchers hypothesized that an allele’s frequency should be the same for all individuals if the allele does not affect viability. The study divided the participants into specific age bins to understand sex-linked and age-linked factors. The researchers also investigated the trends of allele frequencies while considering parental age of death. They examined the nicotine receptor gene CHRNA3, located at a locus involving a significant change in allele frequency regarding a father’s age of death. The model was also adopted to determine changes in the frequency of multiple genetic variants. The trends of 42 polygenic traits were investigated in a Genome-wide Association Study (GWAS), whose
Fig 1. A German study recently revealed that video gamers may have an advantage over non video gamers when learning.
Learning is an everyday occurrence that extends beyond the traditional classroom setting, whether this means quickly memorizing a bus route or remembering a colleague’s number. A recent study led by Dr. Sabrina Schenk of Ruhr University in Bochum, Germany revealed that video gamers may have an edge in learning over non-video gamers. In this study, researchers explored categorization learning, defined as the ability to recognize and sort objects based on certain characteristics. Fifteen right-handed video gamers who reported playing more than 15 hours of video games a day and 15 right-handed non-gamers who reported gaming less than four hours a day participated in the study. Questionnaires screened potential participants for the study. Those who met the established criteria were ultimately chosen to participate. The participants did not have any current or past mental illness and had either normal or corrected vision. To test their theory, the researchers administered the Weather Prediction Task, which examines subjects’ ability to respond to cues based on associations learned at the start of the experiment. The task’s main objective is to test the process of learning probabilities –in this case, the chance of a certain weather pattern happening. Participants sought to master the patterns associated with certain types of weather; after each round of testing, the researchers informed the subjects about the accuracy of their pat-
tern-weather associations. Based on this feedback and multiple rounds of association-weather matching, participants gradually learned the correct associations. While the subjects were taking the test, MRI (magnetic resonance imaging) machines recorded brain activity. After the study was completed, the researchers administered questionnaires that determined the extent of the knowledge that subjects had gained from the Weather Prediction Task. The results revealed that video gamers had acquired more knowledge about the cue card combinations than non video gamers had. Additionally, MRIs showed that throughout the task, video gamers had higher activation in the hippocampus, an area of the brain related to memory and learning. This study is the first to link positive task performance in the Weather Prediction Task to hippocampal activation. The researchers suggest that these findings are relevant to cognitive training and neuropsychological rehabilitation, especially in regards to neural plasticity and the cognitive limitations that come with age. In light of these benefits, gamers can certainly argue for a better reputation: they’re not only having fun, but also training their brains.
polygenic score was calculated to investigate associations between polygenic traits of evolutionary importance. With the GERA participants’ age distribution, the method was useful for identifying allele frequency trends in middle-aged individuals, as the study successfully determined trends of the two alleles e4 and e3 for the APOE gene. The e4 allele for the APOE gene increased the risk of cardiovascular diseases and Alzheimer’s disease, while the e3 allele seemed to have a protective effect. The GWAS showed that delayed puberty in both males and females, along with delayed age at first birth (AFB) in females, was associated with higher chances of survival. However, detained puberty and AFB affect biological fitness in the environment. The researchers’ method provides a new approach to investigating genetic variables and their effects on human survival and life expectancy. This affords researchers new tools to explore genetic longevity in humans. References: 1. H. Mostafavi, et. al., Identifying genetic variants that affect viability in large cohorts. PLOS Biology 15, (2017). doi: e2002458. 2. Image retrieved from: https://static.pexels. com/photos/109919/pexels-photo-109919.jpeg
References: 1. S. Schenk, et. al., Games people play: How video games improve probabilistic learning. Behavioural Brain Research 335, 208-214 (2017). doi: 10.1016/j.bbr.2017.08.027. 2. Image retrieved from: https://www.pexels.com/photo/blur-close-up-device-display-442576
Fig 1. Human viability depends on many genetic variants, including those of evolutionary importance.
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A Conversation with Dr. Nancy Goroff By Muhsena Rahman ’18
The Chair of the Department of Organic Chemistry at Stony Brook Graduate School, Dr. Nancy Goroff is a leading researcher in the field of organic semiconductors. Her research probes the interface between organic chemistry and materials science. Dr. Goroff earned her A.B. in Chemistry from Harvard University, and her Ph.D. from the University of California, Los Angeles. Throughout Dr. Goroff’s time at Stony Brook, she has held many positions, including Assistant Professor, Associate Provost, and Interim Dean of the Graduate School. While she primarily serves as Principal Investigator of the Goroff Research Group, she also serves as a research mentor for both undergraduate and graduate students. How did you develop an interest in organic chemistry? In high school, I liked general chemistry a lot. It was straightforward to me, and I enjoyed doing problem sets more than writing papers. I didn’t actually become interested in organic chemistry until I got to college. What initially sparked my fascination with organic chemistry were the patterns and the visual aspects of it, because to me, it was very similar to art. I have always been interested in art, specifically drawing. I could always draw a pretty good likeness of what was in front of me, but I could never draw or create something from my own imagination. Organic chemistry gives me the power to create things that I’ve imagined. I can come up with a molecule, draw it out on paper, and figure out how to make something that’s only previously existed in my mind or in the minds of other chemists. If you look at the research I do, a lot of the molecules that we focus on are pretty unusual-looking, and
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that’s reflective of my interest in the aesthetics and the art of organic chemistry. I know the main focus of your research is organic semiconductors. Can you talk a little more about why semiconductors are important? We think of materials as being in three classes: insulators, semiconductors, and conductors. Insulators do not conduct electricity; anything that has a charge can move through it given a high enough voltage, but with an insulator, the voltage that you would need to make a charge move through it is much, much higher. Conductors are things like metals where there is a free flow of electrons, so you can put in a charge and it can move with very low resistance. Semiconductors are in between conductors and insulators, charge can move through with a medium amount of resistance. The reason why they are important is that you can use them to make transistors, which can then become the basis for computer chips, LEDs, and many different optical and electronic technologies. What is the main goal of your research into semiconductors? We look at materials that are organic, meaning that they are carbon-based, that have the potential to be semiconductors. We can determine this through the band gap, which is the energy that it takes to promote electrons from the ground state to the excited state. We want materials where the band gap is small enough that you can create a device from them. However, the main focus of our research isn’t the application of the compounds. We’re more interested in push-
ing the limits of organic structure. The materials that we’ve made so far aren’t likely to end up in anyone’s household or cell phone, primarily because of issues with their chemical stability and solubility. Our primary motivation in making these compounds is to take what we already know about structure, including chemical and physical properties, and broaden that knowledge. Once we succeed in making these molecules, we try to understand their properties. The purpose comes more from a structural standpoint than an application standpoint. Do you know where you want to see your research in ten years? This is a question that I’m still very much trying to figure out! I honestly don’t know where the research will take us. We keep learning new things that put us on paths that we didn’t expect to go on. I think what will be important in the future is keeping the focus on the discovery of new structures and creating things that are atypical to what’s already out there. But we never know exactly where the chemistry will take us, which I think is relevant for a lot of researchers out there.
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You have to do something that you love so that you can stay excited through the hard times of solving the next problem.
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Do you have any advice for students doing research here at Stony Brook? I would say to accept that failure is a big part of doing research. I always tell my students that you have to be really excited about the basis for your project because the nature of research is that you spend a lot of your time on things that don’t end up working. And once you finally do get something to work and you’ve mastered that technique, you have to immediately move on to the next step. You’re constantly spending time on things that you don’t know how to do, because the things that are already known aren’t the basis for a good research question. I think it’s important to use the energy and motivation that you get from solving one problem towards taking on the next big struggle in your experiment, because that’s the only thing that’s going to keep you going. I think it’s also important to do research in an area that you’re personally passionate about, not something that you think would look good to a medical school or graduate school review committee. You have to do something that you love so that you can stay excited through the hard times of solving the next problem.
which is something that I really enjoy. What advice would you give to women wanting to go into science? I would say that we definitely need more women in science. Our department has quite a few women, but I know that this isn’t necessarily the case at other universities. I suppose my main advice would be to try and create your own path, and to not try and fit into somebody else’s box. You’re going to do your best work if you work hard at something that you love. There are definitely some people that you have to deal with that you would rather not deal with, but I don’t think that should be the main focus here. That’s not the main story of my life, or any other woman’s life. I don’t think that having to deal with difficult people should be something that defines you or sets you back. Do you think that you have reached a level of success and fulfillment that you are happy with? I do think that I am successful, but I also think that I am still on my own personal journey towards success. To be successful as a professor at a research university, you have to possess so many different types of skills: research skills, teaching skills, people skills, and time management skills. I definitely feel like I’ve been able to develop these skills to a level that I am happy with. And I can absolutely say that I love all parts of my job. I love teaching. I love mentoring graduate students. I love developing research ideas. I don’t even mind writing papers. With what I do now, I spend much more time writing papers than I do with anything that looks like a problem set, which is ironic, because when I first went into science I hated writing papers. It turns out that when I am writing about my research I have a story to tell that no one else knows yet, which I think is one of the most fulfilling things about my job. I’m just happy to be doing research and to be helping our students, both undergraduate and graduate, gain the skills they need to help them be successful in the future. References 1. M. Rahman, Interview with Dr. Nancy Goroff. Rec September 2017. MP3 2. Image retrieved from http://www.stonybrook.edu/happenings/facultystaff/ chemistry-professors-honored-with-acs-prizes/
In addition to being a researcher, you’re also a mentor for the graduate students and junior faculty in your department, as well as the students in your lab. What do you like about being a mentor? Mentoring is one of my biggest motivators. Even though it’s been years since I’ve taught an undergraduate class, I still teach research classes for my research students. It’s really rewarding seeing my students and colleagues progress and learn under my guidance. I also think that I learn as much, if not more, from my students and colleagues as they learn from me,
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THE BENEFITS AND APPLICATIONS OF TERAHERTZ BIOPHOTONICS IMAGING By Anurekha Ravikumar ’18
Terahertz (THz) radiation, also known as the submillimeter wave, is a type of electromagnetic radiation with a frequency range between 0.1 THz and 10 THz. The THz gap encompasses frequencies invisible to the naked eye and shares properties of both the microwave and infrared regions of the electromagnetic spectrum (1, 2). As opposed to other imaging modalities such as x-rays, THz radiation is intrinsically safe, non-destructive, and non-invasive. As a result of these properties, THz pulses are used in biomedical imaging to pinpoint tissues of interest (2). Specifically, THz pulse imaging spectrometry utilizes powerful lasers and detectors to image porcine tissues and show contrast between muscle and fat, as well as differentiate between regions of healthy skin and basal cell carcinoma in vitro (1). Besides imaging, THz radiation is also used in a variety of other fields including near-field microscopy, homeland security, and food and agriculture quality control (3). One of the greatest advantages of THz imaging is its use of non-ionizing radiation. Ionizing radiation, which includes x-ray and gamma rays, poses a great threat to health; in strong doses, it can change the genetic composition of cells. Non-ion-
izing radiation, on the other hand, is less harmful. A second benefit of THz imaging is that a wide range of wavelengths, between 1000 GHz and 4 THz, can be used to obtain information. This is a much wider range than those used for other more common imaging modalities, such as x-rays. Another beneficial feature is that THz wavelengths are comparable to the size of the molecules and tissue cells present in the skin, and thereby produces high-quality images. Furthermore, time-domain information is used to generate 3D images in THz imaging. This differs from frequency-domain imaging in that the scanning process takes much less time. In comparison to optical imaging and infrared radiation, THz also has less scatterings due to the size of the wavelengths used. Scattered radiation occurs when rays are deflected off objects and is an issue in imaging because it reduces image contrast. Thus, THz images have a higher signal-to-noise ratio and can be more helpful in diagnosis. Furthermore, THz imaging is unique in its capability to analyze the hydration profile of biological tissues. For example, the optical path length, the penetration of radiation through different tissues, in THz radiation can be used to measure the depth, or thickness, of different layers of skin (4).
This property is used to detect and distinguish the coagulation zone from the stasis zone in a burnt region of skin (4). Since no other imaging modality has the capacity to effectively measure hydration levels of the skin, researchers have developed protocols for skin burn diagnosis using THz radiation. According to the American Burn Association, approximately 450,000 burn injuries receive medical treatment in the US every year. The clinical course of treatment varies for burns of differing severity (5). Some injuries cannot heal without surgical and skin grafting procedures, while others may recover over a 2-3 week period after the burn. As a result, diagnosing the severity of these burn injuries becomes key to correctly treating them and expediting the healing process. The current accuracy rate of clinical assessment of burn injuries is only 64%, likely due to the inefficiency of diagnostic methods. Because different levels of skin burns show different levels of water absorption, the capability of THz to distinguish between skin water absorption levels makes it useful in burn diagnosis. Severe burns can develop to full-thickness, meaning that the injury has spread across the entire area, penetrating both the epidermis and dermis layers of the skin; sensory nerves are destroyed and sensation in the burn area is lost. Partial-thickness burns, on the other hand, are less severe than full-thickness burns and, consequently, are more easily treated. By measuring skin water absorption levels, THz methods can be used to distinguish between partial-thickness burns that will heal naturally and full-thickness burns that require surgical intervention (5). THz imaging, therefore, is a non-invasive way to analyze the depth and nature of a burn without conducting a biopsy. The high absorption of THz radiation by water molecules provides sensitive signal contrast for imaging. Dr. Zachary Taylor and his colleagues at UCLA recently conducted a study on reflective THz imaging of biological tissues that demonstrated the potential of THz imaging. The researchers used a reflective pulsed-imaging system to obtain high-resolution images of porcine skin tissues with superficial second-degree burns. The images showed minimal degradation in quality even when the imaged sample was covered with ten layers of dry gauze. This is important considering that burned areas are frequently covered with gauze for protection. Thus, a high-quality image of an injury covered with gauze could still be achieved using THz technology. The images also demonstrated high contrast between burned and unburned tissues using THz reflectivity and produced a high signal-tonoise ratio. Furthermore, in the THz images the researchers were able to observe a “halo” surrounding the burned areas that showed the degree of severity of the burn injury. Dr. Taylor also collected data from each high-quality pixel of in vitro samples using a reflective THz system, which has led to the recent development of real-time in vivo THz medical imaging systems (6). The sensitivity of THz radiation to water content also makes it a useful imaging modality in the field of ophthalmology. The pathology of a number of corneal diseases such as glaucoma, Fuchs’ Dystrophy, keratoconus, and corneal graft rejection, among others, is characterized by varying tissue water content and intraocular pressure. THz imaging, therefore, is being explored as a non-invasive and non-contact modality for early diagnosis of these conditions (7). One potential issue with using THz imaging to measure corneal tissue water con-
tent is that such a method would require knowing the thickness of the corneal tissue. However, this can be resolved by the narrow range of corneal thickness found in vivo. Furthermore, the accessibility of corneal tissue, compared to other tissues located deeper in the body, makes it an ideal candidate for THz imaging (7). There are multiple benefits to using THz imaging in diagnosis and analysis, including its non-invasive nature and speed. Its sensitivity to water absorption in tissues makes it particularly useful in the diagnosis of various conditions including burns and corneal disease. Furthermore, the size of the equipment used in THz imaging is continually shrinking; this improves its ease of accessibility for use in clinical trials (8). Thus, due to its intrinsic and physical properties, THz imaging has great diagnostic potential; researchers expect that THz technology will dramatically improve the accuracy of clinical assessment of burn injuries in the future (8). References 1. E. Pickwell, V.P. Wallace, Biomedical applications of terahertz technology. Journal of Physics D: Applied Physics 39, (2006). doi: 10.1088/0022-3727/39/17/ R01/meta. 2. A. Pawar, D. Derle, Terahertz technology and its applications. Drug Invention Today 5, 157-163 (2013). doi: 10.1016/j.dit.2013.03.009 3. M. Tonouchi, Cutting-edge THz technology. Nature Photonics 1, (2007). doi: 10.1038/nphoton.2007.3. 4. S. Fan, et. al., The growth of biomedical terahertz research. Journal of Physics D: Applied Physics 47, (2014). doi: 10.1088/0022-3727/47/37/374009/meta. 5. M. Sumetsky, Surface Nanoscale Axial Optomechanics. OSA Technical Digest, (2017). doi: 10.1364/CLEO_SI.2017.SF2L.4 6. Z. Taylor, et al., THz medical imaging: in vivo hydration sensing. IEEE Transactions on Terahertz Science and Technology 1, 201-219 (2011). doi: 10.1109/ TTHZ.2011.2159551. 7. Z. Taylor, et. al., THz and mm-wave sensing of corneal tissue water content. IEEE Transactions on Terahertz Science and Technology 5, 170-196 (2015). doi:10.1109/TTHZ.2015.2392619. 8. M. Arbab, et. al., Terahertz spectroscopy for the assessment of burn injuries in vivo. Journal of Biomedical Optics 18, (2013). doi: 10.1117/1.JBO.18.7.077004 9. Image retrieved from https://en.wikipedia.org/wiki/Terahertz_radiation
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A Golden Age:
Utilizing Gold Nanoparticles in Cancer Imaging and Therapy By Rideeta Raquib ’19
Image retrieved from https://www.pexels.com/search/line/
Introduction Nanotechnology involves the design and application of devices that control scale within the nanometer range (1). X-rays and nanoparticles interact with atoms of elements with high atomic numbers in cell cultures to yield photoelectric absorption and produce photoelectrons that can emit lethal energy in close vicinity, resulting in greater cell death. As a result, nanoparticles can be utilized in cancer treatment to more effectively deliver radiation therapy to the target tumor site without harming the healthy tissues. A nanoparticle treatment that can be used for this type of radiation therapy consists of gold nanoparticles. Among the most researched nanoparticles, including cadmium selenide quantum dots and carbon nanotubes, gold nanoparticles (AuNPs) have been at the center of attention due to their outstanding biocompatibility. Atomic force microscopy and electron microscopy enable the direct imaging of AuNPs and the control over properties such as size, thus presenting a new prospect in nanotechnology. The enhanced radiative properties of AuNPs allow for a more effective method of targeting tumors, specifically ovarian, breast, pancreatic, and lung cancers (2, 3). Currently, researchers are considering the usage of AuNPs in cancer imaging, such as spectroscopic tumor detection, and cancer treatment, such as photothermal therapy, because of their non-toxicity and ability to be safely excreted from the body (4). Spectroscopic Tumor Detection Spectroscopy, a tool widely used in medical imaging, uses light to measure electromagnetic radiation. When a metal is exposed to light, free electrons oscillate or vibrate in the metal, leading to a dipole oscillation along the electric field of light. The maximum amplitude of oscillation at a specific
frequency, referred to as surface plasmon resonance (SPR), generates a strong absorption of incident light. The SPR band intensity is dependent upon factors that affect the electron charge density on the particle’s surface, such as its shape and size. This absorption value is noticeably high in plasmon nanoparticles, especially gold and silver (4). As a result of the exhibition of surface plasmon resonance, gold displays intense SPR bands in the visible region, around 520 nm in wavelength. This allows for strong visible bands to form when observed with the spectrometer. In addition to the absorption of light, the vibration of the electrons cause photons to be emitted in the form of scattered light in a shifted frequency from incident light, referred to as Raman scattering. This scattering is about six times higher than the magnitude in an AuNP. SPR allows for an increase in the Raman scattering of adjacent molecules in a phenomenon called surface enhanced Raman scattering (SERS). This property of AuNPs can be applied non-invasively to detect the presence of cancer, as illustrated in a study, done by S. Feng et al., which analyzed various urine samples (5). The application of AuNPs as a noninvasive diagnostic tool is also plausible via analysis of urinary nucleosides, which are biomarkers of several cancers, including lung, breast, and colon cancers. Dr. Shangyuan Feng and his research team conducted a study at the Key Laboratory of OptoElectronic Science and Technology for Medicine in the Fujian Province, China to explore the use of AuNPs as cancer biomarkers in urine samples. In this experiment, the urinary modified nucleosides were extracted from the urine samples by running the centrifuged urine through a glass column of phenylboronic gel. After washing and further extraction protocols such as rotary evaporation, the AuNPs were added to the purified nucleosides. A Raman spectrometer was utilized to measure the SERS spectra or the colored bands that resulted from the Raman scat-
tering of the AuNPs. These protocols were repeated for urine samples from 62 nasopharyngeal cancer patients, 55 esophageal cancer patients, and 52 healthy volunteers, and the differences in SERS peaks were then analyzed to detect the presence of cancer. Among all the SERS peaks for the urine samples, a significant distinction in SERS peaks between the nasopharyngeal and esophageal cancer samples were detected. This indicated that the SERS of urinary nucleosides in cancer patients generated greater wavelength bands in comparison to healthy individuals, thus setting a standard gradient that can improve cancer detection (5). SERS is a novel way of diagnosing cancer under noninvasive conditions. There is a positive correlation between the scattering to absorption ratio and the size of the nanoparticles: the larger the size of the nanoparticles, the greater the scattering exhibited. For imaging purposes, a stronger scattering is preferable because a greater excitation in photons will lead to a higher intensity in radiation with wavelengths in the near-infrared regions. This will translate into a clearer image of the target region or tumor; thus, larger nanoparticles can be applied to attain high scattering (5). Photothermal Therapy Photothermal therapy is a technique by which photon energy is converted to heat energy to induce cellular damage. AuNPs have the ability to absorb light at an immensely greater intensity than organic dye molecules do. Through a series of non-radiative processes, AuNPs are able to convert absorbed light to heat as the electron collisions in the particles allow for the generation of heat. Subsequently, via convection, the heat is passed off into the surrounding media. This deems AuNPs as viable candidates for photothermal contrast agents. A contrast agent refers to a substance that enables the distinction between one structure and another. In this case, the tumor would stand out from its surrounding tissue. Tools, such as pulsed and continuous wave lasers, can be used in tandem with AuNPs to achieve SPR absorption in the visible region (5). However, lasers are only capable of penetrating a limited area of soft tissue, such as the ones present in skin cancer, and cannot be employed in deep tumors. Hence, this technique is primarily valuable for eradicating small tumors. The heat efficiency of pulse lasers is relatively low, as heat is lost after one excitation. The employment of continuous wave (CW) lasers is more efficient in terms of accumulation of heat in order to induce cancer cell death by hyperthermia or evaporation. AuNPs with surface plasmon resonance are beneficial particularly because the absorption
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of light can reach the wavelength of the near infrared region (NIR), approximately 800 nm, which is the ideal wavelength for tissue penetration (6). At this wavelength, hemoglobin absorption decreases and water absorption rises, enabling the tissue to be more vulnerable to infiltration. Dr. James Hainfeld and his team of researchers at the University of Waterloo in Ontario, Canada utilized NIR lasers where AuNPs were irradiated with the lasers and injected intravenously into murine tumors. It was found that the small AuNPs, about 1 to 15 nm in size, injected into the tumor could aggregate and be NIR absorptive, hence increasing the efficacy of targeting the tumor site (7). Antibodies are proteins generated by the immune system to target and infiltrate harmful or foreign substances in the body with high specificity. Antibody-conjugated AuNPs are thereby designed to bind to tumor cells. The clustering of AuNPs in the tumor site facilitate the amplified NIR absorbance and production of heat via hyperthermia. Taking these findings into consideration, the researchers tested the effectiveness of diversely prepared AuNP constructs in mice by injecting them with A431 cells mixed with AuNPs. A431 cells are a part of a human cell line that mimics cancerous cells and expresses high amounts of growth factors. In this study, the mice were exposed to NIR and the AuNPs accumulated in tumor cells, which caused them to be destroyed as the light was converted to heat. Some constructs demonstrated 100 percent elimination of tumors. The next step in this field of research is to carry out human clinical trials to compare chemotherapy and photothermal therapy and analyze whether both therapies together would cause a synergistically greater effect (8). Overall, further research and clinical trials need to be conducted to understand the applicability of AuNPs in thermal therapy. Some studies indicate that approximately 5000 nanoparticles must be delivered to one cell to induce coagulative necrosis or cell death (8). However, there is the issue of clearance that must be factored into these trials to design the AuNPs with the appropriate size and surface coating. The ideal AuNPs must not only absorb light in the NIR region and induce heat production, but also effectively eliminate these particles from the body.
Biocompatibility and Clearance The biocompatibility, or ability of cells to uptake AuNPs without resulting in any toxic outcomes or harsh immune responses, and clearance — expulsion of the particles from the body — are essential points to consider prior to the application of AuNPs for cancer treatment (9). Several factors, such as size or surface material, affect the localization of AuNPs in tumor cells. Dr. Jihyun Lee of the department of Radiation Oncology at the University of Texas investigated different side effects and solutions to toxicity concerns that illustrate how the application of AuNPs can be improved to ensure safe removal from the body. The study first lists the characteristics of AuNPs that affect localization in tumor cells. AuNPs ranging from 2 to 6 nm and those that are coated with tiopronin can be taken up by the cytoplasm and nucleus of cells, whereas AuNPs that are 15 nm in size are found in the cytoplasm and do not enter the nucleus, indicating that smaller AuNPs tend to have greater penetration. The tiopronin coating, which is a thiol drug, has the ability to bind to urinary cysteine to help excrete the particles from the body (10). The next portion of the study focused on the concept of clearance. It was found that AuNPs with a positive charge enable the molecules to bind to plasma proteins, causing the reticuloendothelial system (RES) or macrophage system to engulf the nanoparticles, interfering with renal clearance from the body. Particles that were small, approximately 5 to 6 nm, were cleared at a higher magnitude. Most AuNPs are coated with polyethylene glycol (PEG) to enhance their biocompatibility. PEG acts like a cloak to prevent the nanoparticles from being taken up by the RES (10). Substituting PEG with smaller materials, such as cysteine, is a plausible approach to ensuring a greater rate in renal clearance. Cysteine, an uncharged molecule, will not bind to plasma proteins and thus can be eliminated smoothly.
Fig 2. AuNPs with varying properties, such as size or coating can be manipulated for greater biocompatibility.
Image retrieved from https://ninithi.wordpress.com/2015/07/23/a-new-nanogold-based-cancertreatment-burns-cancer-cells-from-within/
Dr. Lee and his team also addressed the cytotoxicity of AuNPs, which were found to have minimal toxic effects. In one of the experiments discussed in the study, the AuNPs were coated with various materials, such as biotin, cysteine, and cetrimonium bromide (CTAB), and were left to incubate in human leukemia cell cultures. The particles had no effect on the mortality of the cell, but CTAB in particular was discovered to induce cytotoxicity, thus the researchers were able to narrow down the coating materials to ones that are safe. Another experiment observed the internalization of AuNPs in rabbit models and no acute toxicity was observed in a 24-hour period. In the end, further testing of diverse coating materials and varying AuNP sizes, as well as clinical trials, are essential to ensure that AuNPs can be administered safely for cancer treatment (9). Conclusion As new technologies emerge and oncology treatments evolve, gold nanoparticles can be employed to improve cancer imaging and therapy. The ability of AuNPs to obtain SPR absorption has enabled the production and testing of lasers, specifically NIR lasers that can infiltrate tumors by inducing hyperthermia. Their radioactive properties have also enabled efficient visualization of cancer cells, which can help scientists optimize drug delivery to target tumors. The enhancement of Raman scattering via AuNPs, which can produce distinctive peaks via spectroscopy, is another approach that has the potential to be employed in cancer diagnostics. The manipulation of AuNP properties to prevent cytotoxicity and ensure clearance from the body is being researched extensively and is relaying positive results. Overall, research in oncology and nanotechnology is evolving, and the implementation of nanoparticles poses a potential advancement for tackling cancer. References 1. S. Jain, D. Hirst, and J. O’Sullivan. Gold nanoparticles as novel agents for cancer therapy. The British Journal of Radiology 85, 101-113 (2012). doi: http://doi.org/10.1259/ bjr/59448833 2. M. Ali, et al., Nuclear membrane-targeted gold nanoparticles inhibit cancer cell migration and invasion. American Chemical Society Nano 11, 3716-3726 (2017). 3. R. King, et al., An overview of current practice in external beam radiation oncology with consideration to potential benefits and challenges for nanotechnology. Cancer Nanotechnology 8, (2017). doi: 10.1186/s12645-017-0027-z 4. X. Huang and M. El-Sayed, Gold nanoparticles: optical properties and implementations in cancer diagnosis and photothermal therapy. Journal of Advanced Research 1, 13-28 (2010). 5. S. Feng, et al., A noninvasive cancer detection strategy based on gold nanoparticle surface-enhanced raman spectroscopy of urinary modified nucleosides isolated by affinity chromatography. Biosensors and Bioelectrics 91, 616-622 (2017). 6. C. Ayala-Orozco, et al., Au nanomatryoshkas as efficient near-infrared photothermal transducers for cancer treatment: benchmarking against nanoshells. American Chemical Society Nano 8, (2014). doi: 10.1021/nn501871d 7. J. Hainfield, et al., Infrared-transparent gold nanoparticles converted by tumors to infrared absorbers cure tumors in mice by photothermal therapy. PLoS ONE, (2014). 8. R. Kumar, et al., Abstract B41: gold nanoparticles based platforms for localized radiosensitization in cancer radiation therapy. American Association for Cancer Research, (2017). 9. J. Lee, et al., Gold nanoparticles in breast cancer treatment: promise and potential pitfalls. Cancer Left 347, 46-53 (2014). 10. S. Naahidi, et al., Biocompatibility of engineered nanoparticles for drug delivery. Journal of Controlled Release 166, 182-194 (2013).
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THE MULTIFACETED HEALTH EFFECTS OF OBESITY By Andrew Kim ’19
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Introduction The body mass index (BMI) is the most commonly used indicator of a person’s body fat-to-weight ratio. BMI can be found by dividing a person’s weight in kilograms by the square of their height in meters; a BMI of approximately 30 or higher is the main criterion for obesity. Although obesity is often associated with external factors such as unhealthy eating habits and a lack of physical activity, some aspects of obesity may have non-environmental origins. One internal factor involves inheriting certain genes that may increase an individual’s chances of becoming obese (1). The increasing prevalence of obesity can also increase the prevalence of other conditions, indicating that obesity may play a role in a person’s overall health profile. Cardiovascular disease is a major type of illness connected to obesity. In 2011, a research team at Jackson State University compared Alabama, Louisiana, Mississippi, and Tennessee — all of which have the highest rates of obesity and cardiovascular disease in the United States — to Colorado, which has the lowest rates. Compared to the aforementioned southern states, Colorado had the lowest rates of high blood pressure, stroke rates, and myocardial infarction. Through statistical analysis, the researchers determined that obesity was responsible for the difference in high blood pressure prevalence between the two groups (2). The statistical study reveals the need to examine the correlation between obesity and other illnesses. This in turn has spurred research attempting to link obesity with neurodegenerative and respiratory diseases, as well as certain types of cancer. Neurodegenerative Diseases The onset of neurodegenerative disease is sudden and unpredictable. Although no clear clinical pathways have been established to prevent the development of these diseases, there are precautionary measures that can be taken to reduce the risk of neurodegenerative disease. One of these measures is limiting caloric intake. The degree of oxidative stress in a person’s body, which can be a contributor to neurodegenerative disease, has been correlated with obesity. Oxidative stress occurs when the body is unable to account for the excess oxygen radicals entering the body. There is an increase in reactive oxygen species (ROS) when the amount of calories consumed exceeds the number of calories used, inducing oxidative stress. The influx of ROS inhibits the mitochondria’s ability to produce ATP. Prior studies have indicated that impaired mitochondrial function is often a factor in many neurodegenerative disorders. Thus, caloric overconsumption results in increased oxidative stress that can account for neurodegenerative disease development. The Laboratory of Neurobiology of Inflammatory and Metabolic Processes at the University of Southern Santa Catarina conducted a bibliographic review searching for references between March and October of 2016 to link obesity with an increased incidence of neurodegenerative diseases. The research suggests that complications in the central nervous system may
be related to high caloric intake, ultimately leading to higher susceptibility to neurodegenerative diseases such as Parkinson’s disease (PD). The defining feature of PD is a loss of dopaminergic neurons, which causes a decrease in the amount of dopamine transported to the striatum. The striatum is responsible for the control of motor functions; this is why PD causes posture instability, stiffness, and bradykinesia, or notably sluggish movements. The degeneration of the dopaminergic system is a consequence of oxidative stress, mitochondrial dysfunction, and neuroinflammation, all of which can increase with obesity and be moderated by limiting caloric consumption (3). Furthermore, prior research has found obesity to influence the rate of endoplasmic reticulum (ER) stress. The Laboratory of Nutrition and Biochemistry at the Pontifical Xavierian University discovered that both obesity and neurodegenerative diseases activate similar cellular responses to ER stress. The elements of ER stress include protein misfolding, lipid metabolism impairment, and inflammatory responses, all of which have been shown to be present in neurodegenerative diseases and obesity. High levels of free fatty acids (FFA), caused by a diet high in fat, have been found to increase ER stress. Researchers studied tissue from obese individuals and determined that ER stress is modulated by three factors: increased levels of FFA, increased levels of ROS, and dysregulation of calcium homeostasis. Cellular processes of the liver, brain, pancreatic, and endothelial tissue in vitro all showed similar metabolic responses to the three factors. The permeability transition pore, a protein formed in the inner membrane of the mitochondria, opens in response to the changes in the inflow of FFA and the concentration gradient. This results in calcium dysregulation, with apoptosis of the cells following shortly after. Apoptosis of necessary cells can lead to damage to the central nervous system, giving rise to neurodegenerative diseases including PD and Alzheimer’s Disease (AD) (4). Respiratory Diseases In addition to its adverse effects on the nervous system, obesity has been shown to negatively impact the respiratory system. A research group at the Children’s Hospital of Philadelphia observed the correlation between respiratory disease and obesity by examining recent studies on both topics. One of these studies compares the effects of asthma in a group of obese adolescents to those from a group of lean adolescents over the course of four years. Given this time period, the group with the obese individuals exhibited a 44% higher risk of needing an emergency department visit due to asthmatic complications. This is because the obese group displayed a greater resistance to budesonide, a medicine used to treat asthma attacks. The lean subjects showed an improvement in forced expiratory volume in response to the budesonide, whereas the obese group did not. A separate study determined a link between Obstructive Sleep Apnea Syndrome (OSAS) and obesity. For children
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between the ages of 2 and 18, gender, race, and age all had a role in the presence of OSAS. However, obesity was ultimately the greatest contributing factor when conducting a statistical analysis. Because fat deposits obstruct the upper airway, recognition of hypercapnia, the presence of high levels of CO2 in the blood during sleep, is hindered in obese children. Smaller nasopharyngeal airway volumes have been suggested to indicate OSAS in children. The smaller passageway reduces an individual’s ability to breathe properly, frequently leading to irregular breathing patterns. If enlarged, the tonsils and adenoids would be surgically removed for patients suffering from OSAS, as these organs would block the upper respiratory tract. For both the obese and healthy individuals, the ability to breathe properly improved, but the improvement was more pronounced in the non-obese children (5).
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Obesity is a condition affecting about a third of the US population, as well as a large proportion of individuals in other countries.
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Cancer The number of deaths due to breast cancer is large and continually growing, with about 40,000 deaths each year. This high mortality rate shows the importance of studying ways to prevent the development of this disease. Recently, research has found a potential correlation between breast cancer and obesity. Leptin, a signal molecule produced by fat cells, is overexpressed in breast cancer tumors. This molecule accelerates the proliferation of mitogen-activated protein kinases and phosphatidylinositol 3-kinases. These two proteins are critical for the overproliferation of cells, which often leads to the formation of tumors (6). To combat the rise of tumor cells, Dr. Javier Menendez and his team at the Catalan Institute of Oncology conducted a study on monounsaturated fats to demonstrate that diet changes may lower the effect of tumorigenesis, or the formation of tumors. The researchers determined that olive oil, which contains high levels of monounsaturated fats, targets breast tumors by inducing apoptosis in cells expressing the tyrosine kinase HER2. Tumors that express this kinase are usually more aggressive than those that do not. Blocking the effects of tyrosine kinase reduces the chances of tumor cell formation. To illustrate this, the researchers used two groups of breast-cancer-induced mice. One group was fed a high-corn diet, whereas the other was fed a diet rich in olive oil. The mice that consumed the olive oil displayed lower proliferation of tumor cells, as well as lower DNA damage compared to the group that was fed the corn diet (7). This shows that consumption of monounsaturated fats may lower the chances of contracting breast cancer. Diet modifications are important in lowering the risk for both obesity and cancer.
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As previously noted, breast cancer occurs due to the rapid formation of tumors. Tumor surfaces contain estrogen receptors, suggesting that the tumors are dependent on estrogen for development. When comparing the hormone levels of obese and lean groups of women, the obese subjects tend to express high levels of the enzyme aromatase, which is responsible for estrogen production, compared to those of lean individuals in several studies. However, if aromatase is overexpressed, it can develop into tumors. After trying to decrease the amount of aromatase in these women, aromatase inhibitors proved less effective in the obese group (8). Conclusion Obesity is a condition affecting about a third of the US population, as well as a large proportion of individuals in other countries. Although it is commonly acknowledged that obesity has negative effects on the body, the diversity and severity of these effects are not always discussed. There is evidence suggesting a correlation between obesity and many types of diseases, such as cardiovascular diseases, respiratory diseases, and cancer. Due to its genetic component, obesity’s development may not be possible to prevent completely. However, there are precautionary measures that individuals predisposed to obesity can undertake to reduce the risks associated with the disease. Additionally, educating at-risk populations about the possible correlates of obesity and the importance of leading a healthy lifestyle is vital to maintaining and eventually reducing rates of obesity and the diseases associated with it. References: 1. C. Ogden, Overweight & obesity statistics. National Institute of Diabetes and Digestive and Kidney Diseases, (2017). 2. L. Akil and H. Ahmed, Relationships between obesity and cardiovascular diseases in four southern states and Colorado. Journal of Health Care for the Poor and Undeserved 22, (2011). doi: 10.1353/hpu.2011.0166 3. G. Rezin, et al., The impact of obesity on neurodegenerative diseases. Life Sciences 182, 22-28 (2017). doi: 10.1016/j.lfs.2017.06.002 4. G. Barreto, et al., Astrocytes and endoplasmic reticulum stress: a bridge between obesity and neurodegenerative diseases. Progress in Neurobiology, (2017). doi: 10.1016/j.pneurobio.2017.08.001 5. I. Tapia and M. Xanthopoulos, Obesity and common respiratory diseases in children. Paediatric Respiratory Reviews 23, 68-71 (2017). doi: 10.1016/j. prrv.2016.10.002 6. R. Robison, et al., Mechanics behind breast cancer prevention – focus on obesity, exercise and dietary fat. Asian Pacific Journal of Cancer Prevention 14, 2207-2212 (2013). 7. A. Segura-Carretero, et al., Extra-virgin olive oil polyphenols inhibit HER2 (erbB-2)-induced malignant transformation in human breast epithelial cells: relationship between the chemical structures of extra-virgin olive oil secoiridoids and lignans and their inhibitory activities on the tyrosine kinase activity of HER2. International Journal of Oncology 34, 43-51 (2009). doi: 10.3892/ijo_00000127 8. K. Brown and C. Gérard, Obesity and breast cancer – role of estrogens and the molecular underpinnings of aromatase regulation in breast adipose tissue. Molecular and Cellular Epidemiology, (2017). doi: 10.1016/j.mce.2017.09.014
Models for Medicine:
Modern Applications of 3D Printing in Cancer Treatment by caleb sooknanan '20
Introduction Three-dimensional (3D) printing, also known as additive manufacturing, is a process by which a solid object is created from a digital file by accumulating layers of material to create the object. First introduced in the 1980s, this technology is now one of the leading methods for fabricating customizable products and is widely used in different fields and practices. 3D printing has become especially helpful in the medical field, as it can be applied towards designing intricate, patient-specific models from medical images obtained from computed axial tomography (CAT) and magnetic resonance imaging (MRI) (1). All of these advantages increase the potential for commercially available medical products and develop a basis for novel research areas such as tissue and organ printing. 3D printing technology has even been used to facilitate treatments for cancer types such as malignant liver tumors, skin cancer, and colorectal cancer, thereby providing a new method of relief for patients worldwide. 3D Malignant Liver Tumor Templates Malignant liver tumors — characterized as cancerous tumors — are growths on the liver that can yield negative health effects such as nausea, vomiting, and abdominal swelling. These liver tumors are categorized as primary tumors, which originate in the liver, and metastatic tumors, which start in a region other than the liver and spread throughout the body. Radioactive 125I-seed implantation, a therapeutic treatment, involves inserting needles into patients to transfer radioactive material. The resulting radiation is selective enough to kill cancerous tumors without damaging other body regions. However, lung tissue obstruction, along with liver shakiness in the body due to breathing, makes it difficult for patients to receive proper implantation. Dr. Tao Han and researchers at the General Hospital of Shenyang Military Region in China conducted a study to determine whether radioactive seed implantation would be improved with 3D printing templates (2). The researchers conducted the study among two groups: Group A contained 15 liver cancer patients who would receive template-assisted treatment, and Group B, which contained 25 tumor patients who would be treated without the assistance of a template. To designate specific templates for Group A, the researchers first tagged operational areas and converted them into digital stereolithography (STL) files, or computer-aided files that facilitate 3D printing. The researchers then utilized computerized tomography (CT) scans to determine the optimal dosage levels amongst each patient. This methodology allowed a 3D printing system to create the templates, which would help organize the implantation particles along a cloth
and thereby simulate the path of particle implantation into the body. During the path simulation, the researchers asked patients to hold their breath for two seconds to prevent significant liver displacement. The patients in Group B underwent the same procedures as those involving Group A, with the exception of the 3D printing systems and templates (2). All patients in Groups A and B were successfully treated with particle implantation; however, the treatment duration in Group A was shorter than that in Group B, with average operation times being 42.5 minutes and 67.6 minutes, respectively. From these results, the researchers suggest that radioactive seed implantation may be a more effective treatment when conducted under the guidance of 3D-printed templates. Limitations of the study included the small sample size, the retrospective nature of the study, and an occasional lack of compliance among patients (2). Nevertheless, the study demonstrates how 3D printing could be used for accurate liver cancer treatments and dose distributions. Further research is needed to understand the benefits of 3D-printed templates among larger and more randomized study populations. 3D-Printed Skin Cancer Brachytherapy Skin cancer is a common cancer found in patients. Brachytherapy is a therapeutic procedure that is often used to treat skin cancer by implanting radioactive seeds into the cancerous regions of a patient’s body. Brachytherapy allows researchers to design custom molds that can pinpoint regions of injection, but such molds are frequently made by hand and are thereby susceptible to error. Dr. Meritxell Arenas and researchers at Hospital Universitari Sant Joan de Reus in Tarragona, Spain designed custom brachytherapy molds with 3D printing software to investigate the clinical workflow associated with 3D printing and compare the effectiveness of 3D-printed molds with that of handmade molds (3). The researchers utilized a 3D surface scanner to digitally render the surface of a patient and develop a visual representation of the cancerous regions to create the new brachytherapy molds. 3D modeling software was then applied towards conforming the model to the patient’s specific anatomy and creating the spaces necessary for seed implantation. The proper configuration of treatment would allow the researchers to take a computerized tomography (CT) scan of the 3D-printed mold and proceed with the implantation, accordingly. The researchers compared the labor costs and expected durations of 3D-printed brachytherapy treatment with those of conventional procedures. The duration of 3D-printed brachytherapy treatment was 6.25 hours, while the duration of the conventional pro-
cess was approximately 9.5 hours. Additionally, the labor costs of 3D-printed treatment and conventional treatment were 152.5 and 211.5 hours, respectively (3). A 49.5% difference in material costs was especially evident between a manual and 3D-printed mold, which would cost $374 and $186, respectively. The results suggest that 3D-printed brachytherapy would be a quicker and more effective way of treating skin cancer patients in clinical settings. Therefore, more intricate molds could be designed by 3D printers, thereby reducing the possibility of errors in treatment. More work is needed to optimize the number and position of implantation spaces, and the researchers aspire to design more personalized scanning methods to detect tumors within the skin and other regions. 3D Colorectal Cancer Metastasis Assessments Colorectal cancer (CRC) is one of the most widespread forms of cancer, with the liver being the primary area of metastasis. Current therapies cannot fully determine treatments for solid tumors due to measurement complications. 3D measurements can address these difficulties and thus provide better visualizations of human anatomy; hence, more 3D-printed models could be used to assess tumor growth in clinical environments. Dr. Ye Ra Choi and researchers at Boramae Medical Center in Korea designed 3D-printed liver tumor models from patient data. This investigation was conducted to evaluate the accuracy and reliability of liver metastasis analysis from 3D-printed models (4). The study comprised a retrospective analysis of twenty CRC patients who had undergone chemotherapy and computerized tomography (CT) scans. Tumor models were created for each patient from CT scan results and were applied towards the formation of STL files, which can be recognized by conventional 3D printers. Moreover, the STL files were converted into a printable format and physically rendered with silicone and graphite powder casts. Two radiologists measured the volume of each 3D printing model using 3-dimensional ultrasound (US), and with CT as a reference, the researchers compared the differences between the obtained CT and US tumor volumes. The reference volume from the CT images was 7.42 Âą 5.76 mL, while the volume of 3D printed models maintained an average of 7.44 mL. The tumor volume measurements obtained from 3D US for the first and second radiologist were 7.18 mL and 8.31 mL, respectively (4). Therefore, the tumor volume measurements from 3D US demonstrated a significant degree of correlation with the CT tumor volumes, with a correlation coefficient of ~0.978. The researchers suggest that 3D-printed models could become more practical in assessing tumor positions and corresponding treatments. Some limitations of the study included the prolonged length of time needed for data acquisition and image interpretation, as well as the possibility for measurement errors made by the radiologists. Also, the small viewing window of liver tumors made it difficult to render large lesions in one digital file (4). Nevertheless, the researchers successfully demonstrated how 3D-printed tumor models could be used in CRC patients with metastatic conditions. However, more work is needed to devise better volumetric treatment responses, especially among patients with different cancer types.
Conclusion Although cancer remains a prevalent disease, 3D printing is now an asset for cancer treatments, especially those regarding seed implantation and tumor detection models. The technology elicits better patient data analysis and more accurately renders cancerous locations within the body. Additionally, it can be used to more effectively design treatment templates for malignant liver tumors, address skin cancer brachytherapy, and improve colorectal cancer treatments. The technology’s limitations vary among the studies conducted in this area of medicine, however, the advances provide significant evidence that 3D printing is an emerging tool for treatments. As demonstrated by non-specific 3D-printed templates, tumor scanning methods, and volumetric responses, more research is needed to personalize 3D printing among specific patients. Current breakthroughs suggest that such treatments will become more available with the progression of technology. References: 1. C. Liaw and M. Guvendiren, Current and emerging applications of 3D printing in medicine. International Society of Biofabrication 9 (2017). doi: 10.1088/17585090/aa7279 2. T. Han, et al., Therapeutic value of 3-D printing template-assisted 125I-seed implantation in the treatment of malignant liver tumors. OncoTargets and Therapy 10, 3277–3283 (2017). doi: 10.2147/OTT.S134290 3. M. Arenas, et al., Individualized 3D scanning and printing for non-melanoma skin cancer brachytherapy: a financial study for its integration into clinical workflow. Journal of Contemporary Brachytherapy 9, 270-276 (2017). doi: 10.5114/ jcb.2017.68134 4. Y. Choi, et al., Therapeutic response assessment using 3D ultrasound for hepatic metastasis from colorectal cancer: Application of a personalized, 3D-printed tumor model using CT images. PLoS ONE 12 (2017). doi: 10.1371/journal.pone.0182596
Recent Advances in Parkinson’s Disease By Anam Rabbani ’18
Introduction Parkinson’s disease (PD) is a chronic and progressive neurodegenerative disorder that affects nearly one million people in the United States. Although the symptoms and progression of the disease vary from person to person, Parkinson’s generally affects an individual’s ability to move. Researchers are currently developing methods to slow down the progression of Parkinson’s disease. This includes development of new medications, such as novel receptor antagonists and utilization of induced pluripotent stem cells (iPSCs). Additionally, researchers are currently searching for the cause of Parkinson’s disease by exploring both the potential genetic and mitochondrial influences on the illness’s development. There are four main signs of altered physical motion, commonly referred to as primary motor symptoms, that determine the stage of the disease’s progression (1). The first sign is the presence of a tremor. The tremor consists of a shaking or oscillating movement usually while the muscles are at rest. In the early stages of the disease, the majority of individuals experience a resting tremor in the hand, foot, or on one side of the jaw or face. The tremor often spreads to the other side of the body as the disease progresses. The second sign is the presence of bradykinesia, or a general reduction in spontaneous movement. This can give the appearance of abnormal stillness, as well as decreased facial expressivity. Bradykinesia can also cause difficulty with repetitive movements. Because of this, people experiencing bradykinesia often have problems performing everyday tasks like buttoning a shirt or cutting food (1). The third sign of Parkinson’s is muscular rigidity or stiffness. Individuals with PD most commonly experience painful
tightness of the neck, shoulders, or legs. This can lead to a significantly decreased range of motion as the disease progresses. The fourth primary motor symptom is postural instability. An individual with postural instability has lost some of his or her ability to maintain an upright posture, and will topple backwards if disturbed even slightly. Postural instability can lead to a tendency to sway backwards when standing, turning, or getting up from a chair (1). In addition to these motor symptoms, PD can also cause nonmotor symptoms, such as a softer voice, diminished sense of smell, mood disturbances, sleep disorders, and cardiac arrhythmias. These symptoms vary widely from person to person (1). Although the exact cause of Parkinson’s is unknown, researchers have identified several main changes that take place in the brain when an individual develops the disease. Parkinson’s patients usually have low levels of dopamine in the basal ganglia, a group of neuronal structures located deep beneath the outer layer of the brain that is primarily responsible for motor control (1). The two regions of the basal ganglia affected by Parkinson’s are known as the substantia nigra and the striatum. A normal substantia nigra appears as a dark band due to the presence of dopaminergic neurons, which are pigmented neurons that use dopamine as a neurotransmitter. In Parkinson’s disease, the dopaminergic neurons in the pars compacta region of the substantia nigra progressively die, which leads to a loss of pigmentation in this region. In a normal brain, the pars compacta region of the substantia nigra is linked to the striatum, supplying the striatum with dopamine. However, in a patient with PD, the loss of dopaminergic neurons in the substantia nigra leads to a lack of dopamine in the striatum.
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One of the main functions of the striatum is the regulation of motor function, and thus the lack of dopamine in the striatum leads to loss of motor control in Parkinson’s patients (2). The cause of dopaminergic neuron death in the pars compacta is unknown. However, researchers have identified the presence of large aggregates of protein, called Lewy bodies, present in dopaminergic neurons of Parkinson’s patients. The primary component of Lewy bodies is the protein alpha-synuclein, whose function in the healthy brain is currently unknown. However, in patients with PD, the presence of large, abnormal aggregates of alpha synuclein indicates the presence of diseased dopaminergic neurons (1). Clinical treatment of Parkinson’s focuses mainly on replenishing depleted dopamine levels in the striatum. There are several classes of drugs that either provide the brain with dopamine or mimic dopamine’s effects on the brain (2). In terms of non-pharmacological remedies, deep brain stimulation (DBS) is currently being explored as an alternative treatment option (3). Although these treatments can help to manage symptoms of Parkinson’s, there is currently no cure for the disease. Because of this, researchers are currently exploring several promising therapies that could one day stop or even reverse the damaging effects of PD. Current Therapies Levodopa, also known as L-DOPA, is one of the most effective and commonly prescribed Parkinson’s medications (4). L-DOPA is converted from its initial form to dopamine after crossing the blood-brain barrier. This treatment is used instead of administering dopamine directly because dopamine itself cannot cross the blood-brain barrier. Levodopa is usually combined with either carbidopa or benserazide, two other types of organic molecules, in order to prevent it from being converted into dopamine in the body prematurely. If levodopa is converted to dopamine before crossing the blood-brain barrier, this can lead to nausea and dizziness. The use of carbi-
Fig 1. Surgical techniques can be employed to improve Parkinsonian symptoms. Image retrieved from https://en.wikipedia.org/wiki/File:Peds_DBS.jpg
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dopa or benserazide with levodopa can significantly reduce or even eliminate these side effects (4). Although levodopa is considered extremely effective at eliminating primary motor symptoms, its benefits tend to decrease as Parkinson’s progresses, causing physicians to prescribe increasingly greater dosages of the medication. However, higher dosages of levodopa usually lead to the onset of dyskinesia, or involuntary movements. Because of this, levodopa is usually not considered practical in terms of a long term treatment plan (4). In order to diminish the progressive side effects associated with levodopa, researchers are currently looking towards an alternate way to treat PD that involves dopamine agonists. Dopamine agonist drugs mimic the effects of dopamine on the brain, but do not provide the brain with any actual dopamine (5). There are two types of dopamine agonists - ergot derived and non-ergot derived. Ergot derived agonists are extracted from the Ergot fungus, while non-ergot derived agonists are synthesized from other alternative sources (6). Dopamine agonists tend to be less effective at reducing primary motor symptoms than levodopa, and also tend to have side effects such as hallucinations, confusion, and psychosis (6). Because of this, dopamine agonists are often used in conjunction with other drugs in the initial stages of PD (7). Dopamine agonists are usually not effective in individuals with advanced PD (8). MAO-B inhibitors provide the brain with dopamine in a different way than levodopa. These medications prevent the activity of the enzyme monoamine oxidase B, or MAO-B, which breaks down unused dopamine in the brain. In this way, MAO-B inhibitors allow dopamine to be more available for use by dopaminergic neurons in the substantia nigra. MAO-B inhibitors are usually used to enhance the effects of other medications, such as levodopa or dopamine agonists, in initial stages of PD. This class of drugs can significantly improve primary motor symptoms (6). A non-pharmacological treatment for Parkinson’s is Deep Brain Stimulation, or DBS. This technique stimulates the basal ganglia, using electrodes, to facilitate production of dopamine. This method is considered a favorable alternative to medications because of its lack of side effects. The Sotiropoulos research group found that patients undergoing DBS had a 46% improvement in their tremors, rigidity, and bradykinesia without significant side effects such as dyskinesia (9). However, this treatment can only be used in intermediate stages of Parkinson’s, because there have to be a certain number of active dopaminergic neurons present in order for DBS to be effective (9). In later stages of the disease, not enough viable neurons are present to be activated by DBS. Future Treatments The aforementioned treatments do not provide a cure to Parkinson’s; therefore, researchers are exploring several avenues that will potentially lead to a treatment that stops, or even reverses, the neurodegenerative effects of the disease (10). The first potential treatment involves iPSCs, or induced pluripotent stem cells, pioneered by the Yamanaka lab in Kyoto, Japan in 2006 (7). Dr. Yamanaka and his colleagues demonstrated that by introducing four genes that encode specific DNA transcription factors, an adult cell could be convert-
ed into a pluripotent stem cell. Pluripotent stem cells can divide indefinitely, and can give rise to every cell type in the body (7). Since these cells can be directly derived from adult cells, they can be made specifically for each patient, which means that an individual could have their own line of iPSCs. Researchers have been using several different approaches involving iPSCs to gain a better understanding of Parkinson’s. In a 2016 study done by Dr. Xiao and colleagues, researchers generated dopaminergic neurons from iPSCs in order to create a model for Parkinson’s. The main goal of this model was to discover what mechanism was causing the death of dopaminergic neurons in the substantia nigra. The study discovered that mitophagy, or degradation of the mitochondria of the cell, was an indicator of neuronal death. Researchers are now trying to use iPSCs to identify the cause of this mitophagy, as discovering the starting point to this mechanism could lead to alternative treatments for PD in the future (11). Another avenue of research involving iPSCs is screening of potential therapeutic molecules. In the same study done by Dr. Xiao in 2016, Coenzyme Q10, which was found to be depleted in Parkinson’s patients, was screened as a potential treatment option. Dopaminergic neurons were created via iPSCs derived from Parkinson’s patients, and it was found that treatment with Coenzyme Q10 reduced cell toxicity in Parkinson’s derived dopaminergic neurons. Rapamycin, which prevents mitochondrial degradation, was also found to reduce cell toxicity and cell death using the same methods as coenzyme Q10 (11). iPSCs are also being used for genetic analysis. Dr. Xiao and colleagues derived different dopaminergic neurons from many different Parkinson’s patients, and through a broad genetic analysis they were able to identify 28 possible gene variants that could be contributing to Parkinson’s disease (12). They aim to sequence these gene variants in the future to determine both how and why they contribute to the pathogenic dopaminergic neurons present in PD (12). One of the most promising uses for iPSCs is the possibility of neural transplantation. Recent attempts at transplanting dopaminergic neurons derived from iPSCs into the striatum of rodent and primate models have yielded encouraging results (13). In the rodent model, transplanted iPSCs successfully differentiated into dopaminergic neurons, and were able to alleviate motor symptoms. In the primate model, the transplanted iPSC derived neurons survived for six months post-surgery, although there was no behavioral improvement. In order to improve neuronal survival, researchers are currently looking into introducing neural progenitor cells, or NPCs, into the primate model instead of transplanting iPSCs that differentiate directly into dopaminergic neurons (14). A major drawback of iPSCs is the potential for tumorigenesis as many of the transcription factors used to create iPSCs from adult stem cells, such as KIf4 and MIc, are known oncogenes. Another factor that could lead to tumorigenesis in iPSCs is the effect of the disruption of the genome integrity. Researchers are concerned that the insertion of multiple new transcription factors into the genome could induce somatic mutations in the cells that differentiate from iPSCs (15). Therefore, iPSCs can only be applied in clinical trials once these concerns have been addressed and tumorigenesis can be prevented (13).
Another new treatment currently being explored is the use of LAG3, or Lymphocyte Activation Gene. This is a transmembrane protein that is involved in the transfer of alpha-synuclein, the primary component of Lewy bodies, between cells. Emerging evidence indicates that the pathogenesis of Parkinson’s disease may be due to cell-to-cell transmission of the misfolded alpha synuclein found in Lewy bodies (16). The most recently proposed mechanism states that LAG3 binds to the misfolded alpha synuclein with high affinity, initiating endocytosis of the mutated protein into the cell. It has been shown that LAG3 only binds to mutated forms of alpha-synuclein, and exhibits extremely minimal binding to the unmutated form. In addition, a lack of LAG3 delayed the loss of dopaminergic neurons in the substantia nigra. Therefore, LAG3 could be a promising target for inhibitory drugs. If a drug were developed that inhibited LAG3, the progression of PD could be slowed (16). Conclusion Although there are treatments for Parkinson’s disease, there is no cure. Ultimately, even with the use of current treatments, dopaminergic neurons continue to die, leading to the patient’s eventual death. Drugs that can increase levels of dopamine or mimic its effects can alleviate motor symptoms, but they do not help with the underlying issue of neuronal cell death. There are several treatments that are being explored in order to deal with the death of dopaminergic neurons. The use of iPSCs is very encouraging, as further research into neural transplants could lead to the widespread use of this therapy for the regeneration of functional dopaminergic neurons. In addition, LAG3 appears to be a very promising target for slowing the progression of PD. Although there is a lot of work that remains to be done, these new treatments show promising steps towards a cure for Parkinson’s disease. References: 1. R. Hauser, Parkinson Disease. (2017). 2. L. Marsili, et. al., Chapter twelve-treatment strategies in early parkinson’s disease. International Review of Neurobiology 132, 345-360 (2017). 3. E. Wolf, et. al., Long-term antidyskinetic efficacy of amantadine in parkinson’s disease. Movement Disorders 25(10), 1357-1363 (2010). 4. A. Espay, et. al., Optimizing extended-release carbidopa/levodopa in parkinson disease consensus on conversion from standard therapy. Neurology: Clinical Practice 7(1), 86-93 (2017). 5. R. Hauser, et. al., Longer duration of MAO-B inhibitor exposure is associated with less clinical decline in parkinson’s disease: an analysis of NET-PD LS1. Journal of Parkinson’s disease 7(1), 117-127 (2017). 6. R. Borgohain, et. al., Two year, randomized, controlled study of safinamide as add-on to levodopa in mid to late parkinson’s disease. Movement Disorders 29(10), 1273-1280 (2014). 7. K. Shannon, et. al., Efficacy of pramipexole, a novel dopamine agonist, as monotherapy in mild to moderate parkinson’s disease. Neurology 49(3), 724-728 (1997). 8. F. Stocchi, et. al., A randomized, double-blind, placebo-controlled trial of safinamide as add-on therapy in early parkinson’s disease patients. Movement Disorders 27(1), 106-112 (2012). 9. H. Akram, et. al., Subthalamic deep brain stimulation sweet spots and hyperdirect cortical connectivity in parkinson’s disease. NeuroImage, (2017). 10. R. Zanettini, et. al., Valvular heart disease and the use of dopamine agonists for Parkinson’s disease. New England Journal of Medicine 356(1), 39-46 (2007). 11. R. Katzenschlager R, et. al., Anticholinergics for symptomatic management of parkinson´s disease. Cochrane Database of Systematic Reviews, Issue 3 (2002). 12. F. Ory-Magne, et. al., Withdrawing amantadine in dyskinetic patients with parkinson disease The AMANDYSK trial. Neurology 82(4), 300-307 (2014). 13. M. Wernig, et. al., Neurons derived from reprogrammed fibroblasts functionally integrate into the fetal brain and improve symptoms of rats with parkinson’s disease. Proceedings of the National Academy of Sciences 105(15), 5856-5861 (2008). 14. B. Xiao, et. al., Induced pluripotent stem cells in parkinsons disease: scientific and clinical challenges. J Neurology, Neurosurgery, and Psychiatry 87(7), 697-702 (2016). 15. R. Pahwa, et. al., Amantadine extended release for levodopa-induced dyskinesia in Parkinson’s disease (EASED Study). Movement Disorders 30(6), 788-795 (2015). 16. X. Mao, et. al., Pathological α-synuclein transmission initiated by binding lymphocyte-activation gene 3. Science 353, (2016). doi: 10.1126/science.aah3374. 17. Cover image retrieved from: https://en.wikipedia.org/wiki/Dopamine
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The Impact of the World Trade Center Attacks on Pollutant Levels and Asthma-Related Emergency Department Visits in the Bronx Kunwar Ishan Sharma ’20, Sunit P. Jariwala, MD
ABSTRACT This study sought to demonstrate and explain the association between pollutant values and asthma-related emergency department visits (AREDV) in the New York City borough, of the Bronx, following the World Trade Center (WTC) attack. Using daily AREDV logs and pollutant levels from 1999 and 2002, median values from the two years were compared using a Mann-Whitney test to test for statistically significant differences. Daily values between 1999 and 2002 were compared seasonally in order to account for seasonal variations in AREDV and pollutant levels. There were significant increases in the winter and spring for AREDV and in the spring and summer for NO2. In addition, significant increases for median daily SO2 and O3 values between the years occurred in all four seasons. Consequentially, there was a significant increase in all of the variables for at least one season following the WTC attacks. Further multivariate analyses are needed to further examine the correlation between AREDV and pollutant values pre- and post-WTC attacks.
INTRODUCTION Many cities across the United States are considered hubs for developed infrastructure and technology and are home to large populations of people. The downside of these characteristics is the effect that pollution, from these masses of people using the infrastructure, has on air quality. Decreased air quality and increased environmental pollutants, such as NO2, SO2, and O3, are known to affect those with asthma (1-4). The New York City borough of the Bronx is a major example of this phenomenon, with one of the highest rates of asthma morbidity in the country (5). With this baseline of pollution existing on a consistent basis, the effect of disasters, such as the World Trade Center attacks, is likely to further increase pollution and negatively impact asthma patients. This investigation is aimed at understanding the association between the three major pollutants (NO2, O3, and SO2) and asthma-related emergency department (ED) visits in two Bronx hospitals before and after the World Trade Center attacks. METHODS Participants Given that the research was centered around the Bronx, it was essential to find patient ED visit data within this borough. For this reason, the study relied on two Bronx hospitals, Montefiore Moses and Montefiore Weiler, as sources for the asthma ED data. The participants of this study fell into all age groups. Since data was collected for two separate years, 1999 and 2002, two years before and one year after the attacks, the number of patients in each respective data set differs. For the timeframe before the attack, there was a total of 3,608 patients; for the timeframe after the attack, there was a total of 4,315 patients. These patients were selected on the criteria that they visited the emergency department during the designated timeline established for this study for asthma-related reasons.
study required the pollutant values in the Bronx were during the designated time period, so the National Climatic Data Center’s Bronx collection station was used to acquire data on NO2, SO2, and O3 levels. The specific timeline for analysis was 1999, two years before the WTC attacks, and 2002, one year after the attacks. The year 1999 was chosen as a control since no major disasters affected the Bronx area that year. This would allow for a more accurate baseline of pollutant values and asthma ED visits during a normal year. The year 2002 was chosen as the post WTC attack comparison since this year began only a few months after the attack. Thus, it would most likely account for the suspected environmental and asthma ED impacts. In order to account for seasonal variations of asthma exacerbations and pollutant levels, the data was broken up seasonally. This allowed for a closer analysis between specific months and the opportunity to see whether the attacks played a varied role on the data within the different seasons. With all of the data and the timeframe established, it was essential to have a means of interpreting differences between the two years. Thus, a Mann-Whitney statistical test was used to compare both pollutant and ED visit numbers between 1999 and 2002. A p-value of <0.05 was considered indicative of a statistical change in the respective numbers between the aforementioned years. RESULTS In terms of asthma-related emergency department visits (AREDV), there were observable differences between the two respective years for certain seasons. For instance, there was a statistically significant (p < 0.05) increase in ED visits between 1999 and 2002 during the winter (1999: 11; 2002: 17; p < 0.0001) and spring (1999: 9; 2002: 12; p < 0.0001) (Table 1; Figure 1). AREDV levels increased during the winter, especially in January and mid-February from 2002 to 1999 (Figure 1). As the seasons continued, there was a slight, non-significant increase during the summer and a significant decrease in the fall.
Data Collection and Analysis In order to access patient data, Montefiore’s Clinical Looking Glass software, which keeps track of patient ED visits, was used. Furthermore, the
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In terms of seasonal NO2, there were significant increases in the spring (1999: 0.037 ppm; 2002: 0.046 ppm; p = 0.039) and summer months (1999: 0.033 ppm; 2002: 0.044 ppm; p = 0.0002) (Table 1; Figure
2). The highest NO2 increases were found in early- and mid-August (Figure 2). On the other hand, there were significant decreases during the winter and fall months. In terms of seasonal SO2, there were significant increases in all four seasons between 1999 and 2002: winter (1999: 0.017 ppm; 2002: 0.026 ppm), spring (1999: 0.009 ppm; 2002: 0.013 ppm), summer (1999: 0.006 ppm; 2002: 0.095 ppm), and fall (1999: 0.01 ppm; 2002: 0.016 ppm) (Table 1, Figure 3). SO2 levels increased in late January and early March from 1999 to 2002 (Figure 3). Seasonal O3 exhibited the same values as seasonal SO2, where all four seasons showed significant increases between 1999 and 2002: winter (1999: 0.009 ppm; 2002: 0.014 ppm), spring (1999: 0.024 ppm; 2002: 0.035 ppm), summer (1999: 0.025 ppm; 2002: 0.0485 ppm) and fall (1999: 0.009 ppm; 2002: 0.016 ppm) (Table 1, Figure 4). The fall 2002 O3 values lie above the 1999 values for most of the season (Figure 4).
DISCUSSION Between 1999 and 2002, there were statistically significant increases in pollutant values and AREDV in at least two seasons. There are several pertinent correlations between the timing of the attacks and the timing of the variations in the data. For instance, NO2 values increased significantly in the summer, despite NO2 levels being historically lower in the summer months (6). In past analyses, NO2 values were found to decrease in the summer, so this NO2 increase indicates that the WTC attacks could have led to these variations. It is also important to note that NO2 is often referred to as “WTC Dust,” which encompasses a variety of pollutants and particulates that arose in the environment after the attacks. In terms of SO2, there is no definite cause for the uncharacteristic increases in levels during
Figure 1. 1999 and 2002 AREDV values (Spring)
Figure 2. 1999 and 2002 NO2 (ppm) values (Summer)
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Figure 3. 1999 and 2002 SO2 (ppm) values (Winter)
Figure 4. 1999 and 2002 O3 (ppm) values (Fall)
the non-winter months (6,7). The timing of the WTC attacks offers an analogous explanation for the increases in SO2. The association between the attacks and O3 is not as obvious, but O3 is known to be a common byproduct of NO2. In other words, with regard to this relationship to NO2 and the fact that O3 increased in all four seasons following the attacks, it is probable that the attacks impacted O3 presence. In terms of AREDV, with increases in the two seasons following the attacks, it appears that the pollutant variations, possibly caused by the attacks, played a role in these increases.
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Although these relationships display correlations between the factors, the results do not imply direct causation. However, this type of analysis may be important in terms of understanding the effects of the WTC attack on asthma patients, as well as understanding how to prepare for future disasters and mitigate their malignant effects. By understanding the correlation between these disasters and increases in pollutant values and ED visits, health organizations and hospitals can better predict influxes of asthma ED patients after disasters.
While the correlation between pollutant levels and asthma ED visits uncovered by this study may be beneficial to public health professionals, there are many components left to explore. Firstly, other environmental factors, such as particulate matter, pollen count, ambient temperature, and others, were likely affected in the same way these pollutant values were. Thus, by investigating the levels of these environmental factors in the same way that these pollutant values were investigated, it may be possible to gain an even greater understanding of the disaster’s implications on the environment. In addition, there are a few ways to further investigate the specifics of this research. Since only correlations were uncovered, instead of direct
Title
1999 – Median
2002 – Median
P-value
Winter NO2
.05250
.04200
.0004
Spring NO2
.03650
.04600
.0392
Summer .03300 NO2
.04350
.0002
Fall NO2
.05200
.03600
<.0001
Winter SO2
.01700
.02600
<.0001
Spring SO2
.00900
.01300
<.0001
Summer .00600 SO2
.00950
<.0001
Fall SO2 .01000
.01600
<.0001
Winter O3
.00900
.01400
<.0001
Spring O3
.02400
.03500
<.0001
Summer O3
.02500
.04850
<.0001
Fall O3
.00900
.01600
<.0001
Winter 11 AREDV
17
<.0001
Spring 9 AREDV
12
<.0001
Summer 6 AREDV
7
.0881
Fall 13 AREDV
12
.0329
causation, further statistical tests, such as multivariate analyses, are necessary to more accurately examine the relationship between pollutants and AREDV during the established timeframe. Furthermore, the generalizability of the results could be improved by increasing the sample size to include patients from multiple hospitals across New York City. However, this study is unique in that it is the first of its kind to investigate the impact of a modern-day disaster on the association between pollutant levels and asthma ED visits. This research can not only help scientists and policymakers better understand the environment but can also help predict how changes within it have affected the population. References 1. E. Marino, et. al., Impact of air quality on lung health: myth or reality? Therapeutic Advances in Chronic Diseases 6, 286-298 (2015). doi: 10.1177/2040622315587256. 2. S. Vieira, The health burden of pollution: the impact of prenatal exposure to air pollutants. International Journal of Chronic Obstructive Pulmonary Disease 10, 1111-1121 (2015). doi: 10.2147/ COPD.S40214. 3. D. Jenerowicz, et. al., Environmental factors and allergic diseases. Annals of Agricultural and Environmental Medicine 19, 475-481 (2012). 4. S. Lazarus, et. al., The leukotriene receptor antagonist zafirlukast inhibits sulfur dioxide-induced bronchoconstriction in patients with asthma. American Journal of Respiratory and Critical Care Medicine 156, 1725-1730 (1997). doi: 10.1164/ajrccm.156.6.9608006. 5. P. Sheffield, et. al., Ambient ozone exposure and children’s acute asthma in New York City: a case-crossover analysis. Environmental Health 14, (2015). doi: 10.1186/s12940-015-0010-2. 6. S. Jariwala, et. al., Association between tree pollen counts and asthma ED visits in a high-density urban center. Journal of Asthma 48, 442-448 (2011). doi: 10.3109/02770903.2011.567427. 7. New York City Department of Health and Mental Hygiene, The New York Community Air Survey: results from Winter Monitoring 2008–2009. Department of Health and Mental Hygiene, (2009).
Table 1. NO2 (ppm), SO2 (ppm), O3 (ppm), and AREDV median values pre and post WTC attack.
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The Synthesis of 3’ Difluorovinyltaxoids (SB-T-12854) Sarika Hira ’18, Yaozhong Zhang, B.S., Iwao Ojima, Ph.D.
INTRODUCTION
of which had a different reactivity, as shown in Figure 1 (10).
Cancer, the leading cause of death in the United States, is a group of diseases caused by the uncontrollable division of abnormal cells in the body. Several treatment options are available, including chemotherapy, radiation therapy, surgery, and transplantation. However, despite the huge investment and advancement in clinical and basic research over the decades, cancer remains one of the most challenging diseases to effectively cure (1). Conventional chemotherapy uses Food and Drug Administration (FDA) approved cytotoxic agents, such as paclitaxel and docetaxel, to slow or stop the growth of rapidly dividing cancer cells in the body. However, they have undesirable side effects due to the lack of tumor specificity and susceptibility to multi-drug resistance (MDR), which leads to systemic toxicity, including bone marrow damage (2,3). Additionally, it is ineffective in controlling and eradicating cancer stem cells (CSCs) (4). Since rapidly proliferating cancer cells overexpress cancer specific receptors to promote uptake of vitamins and nutrients, these receptors can be used as targets for cancer specific delivery of cytotoxic agents through receptor mediated endocytosis (5)(6). The synthesis of anticancer drugs for this type of targeted tumor therapy is needed to significantly mitigate the adverse effects associated with cancer chemotherapy (1). Paclitaxel (Taxol®) is a chemotherapy agent that belongs to a class called plant alkoids, which is used for the treatment of breast, ovarian, lung, and other types of solid tumor cancers (7). For the production of paclitaxel, two major subunits 10-Deacetylbaccatin III (10-DAB) and beta-lactam: (3R,4S)-N-benzol-3-O-(1’-ethoxylethoxyl)-4-phenyl-azetidin-2-one, were coupled together using the “Ojima-Holton coupling” method, developed in 1992, in which Ojima and Holton accomplished a semi-synthesis of paclitaxel using beta-lactam and 10-DAB (8). However, in clinical treatment, Taxol® shows little efficacy in treating melanoma, pancreatic, gastric, brain, and renal cancers (9).
Figure 1: Structure of 10-DAB6
On protection of the more reactive C7 and C10 groups, the hydroxyl group at C13 reacted with different β-lactams via Ojima-Holton coupling to give the corresponding taxoids. In Scheme 1 the synthesis of paclitaxel displayed the coupling reaction between C7 and C10 protected baccatin III and beta-lactum.
MATERIALS AND METHODS Prior to synthesizing and forming the taxoid, 1H NMR and 13C NMR spectra were measured on Bruker 400 MHz, 500 MHz, or 700 MHz NMR spectrometers. Thin Layer Chromatography (TLC) analyses were performed on Sorbent Technologies aluminum-backed Silica G TLC plates (Sorbent Technologies, 200 μm, 20 cm × 20 cm), and were visualized with UV light and stained with sulfuric acid-EtOH, 10% phosphomolybdic acid (PMA)-EtOH, 10% vanillin-EtOH with 1% sulfuric acid, ninhydrin-butanol with 10% AcOH, or DACA stain. Column chromatography was carried out on silica gel 60 (Merck; 230-400 mesh ASTM). The chemicals, dichloromethane, methanol, ether, and tetrahydrofuran were dried in a 100 °C oven and distilled after being cooled to room temperature. 10-DAB III, obtained from Indena, S.p.A, Italy, structure revealed 4 hydroxyl groups in 10-DAB core at the C1, C7, C10 and C13 positions, each
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Scheme 1: β-Lactam approach by Ojima group (Adapated from Ref. 7.), pictured above
However, paclitaxel also displayed a multi-drug resistant phenomenon in the treatment of cancer. Due to this aspect, researchers have started focusing on developing some new-generation taxoids to attempt to find a potent, less toxic compound with better bioavailability and less drug resistance. A structural-activity relationship (SAR) was implemented to improve
the anti-cancer-drugs for conventional chemotherapy, namely 3’-diflurovinyltaxoids. In one instance, the FDA had approved a large number of fluorine-containing compounds. The C-F bond in medicinally active compounds has been found to possess desirable pharmaceutical properties such as higher metabolic stability, increased binding to target molecules, and enhanced membrane permeability (11). Additionally, highly potent fluoro-taxoids serve as the “warheads” or payloads for tumor targeting “molecular missiles” or tumor-targeting drug delivery systems. Therefore, to increase the potency by 2-3 orders of magnitude against drug-resistant cancer cell lines, the isobutenyl group at the C3’ position was modified by the attachment of two fluoride groups as shown in Figure 2 (12).
low oil. The crude was further purified by column chromatography on silica gel (gradient eluent: hexanes/ethyl acetate from 10/1 to 3/1) to give benzyl 2-(triisopropylsiloxy) acetate 1-3 as pale yellow oil. 2-(Triisopropylsiloxy) acetic acid (1-4) (15) Ethyl acetate was dried over anhydrous MgSO4, and the MgSO4 was subsequently removed by vacuum filtration to give dry ethyl acetate. Benzyl 2-(triisopropylsiloxy) acetate 1-3 (5.00 g, 15.5 mmol) in 80 mL dry ethyl acetate was added to a 250-mL round bottom flask. It was evacuated and flushed with nitrogen gas three times and 10% Pd/C (82.2 mg, 0.775 mmol) was then added. It was evacuated and flushed with nitrogen gas three times again. Afterwards the flask was evacuated and flushed with hydrogen gas three times, filled with hydrogen gas, and allowed to stir at room temperature for 11 hours. TLC monitored the reaction. Upon completion, the reaction mixture was filtered with the aid of celite to remove the catalyst, and washed with dry ethyl acetate. The filtrate was concentrated in vacuo to give crude 2-(triisopropylsiloxy) acetic acid 1-4 (3.60 g, 15.5 mmol) as pale yellow oil (over 100%, contain solvent ethyl acetate). This crude product was used in next step immediately without further purification. 2,5-Dioxopyrrolidin-1-yl 2-(triisopropylsiloxy)acetate (1-5)
Figure 2: Structure of SB-T-12854
EXPERIMENTAL PROCEDURES Synthesis of β-lactam Benzyl 2-hydroxyacetate (benzyl glycolate) (1-2) (14) To a 250-mL round-bottom flask 2-hydroxyaceticacid (glycolic acid) 1-1 (5.00 g, 65.7 mmol) was dissolved in 80 mL acetone. Triethylamine (7.25 g, 71.5 mmol) was added slowly within 20 minutes to the mixture. The reaction mixture was allowed to stir at room temperature for 30 minutes. Afterwards, benzyl bromide (10.2 g, 59.7 mmol) was added dropwise within 30 minutes. White precipitate came out immediately. After stirring at room temperature overnight, the white precipitate was removed by vacuum filtration. The filtrate was mixed with water (100 mL), and the mixture was extracted with ethyl acetate (3 x 100 mL). The combined organic layer was washed with brine (3 x 60 mL), and dried over anhydrous MgSO4. The MgSO4 was subsequently removed by vacuum filtration, and the filtrate was concentrated in vacuo to give benzyl 2- hydroxyacetate (benzyl glycolate) 1-2 (11.1 g, 66.8 mmol) as pale yellow oil. Benzyl 2-(triisopropylsiloxy) acetate (1-3) (15) 20 ml of DMF as solvent was added to 7.98 g of benzyl 2-hydroxyacetate (benzyl glycolate) 1-2 followed by the addition of imidazole (3.86 g, 56.8 mmol) in a 250-mL round-bottom flask. Afterwards, triisopropylsilyl chloride (10.9 g, 56.8 mmol) was added dropwise. White precipitate came out slowly. The reaction mixture was allowed to stir at room temperature overnight. The white precipitate was removed by vacuum filtration. Furthermore, the filtrate was mixed with water (50 mL), and it was extracted with ethyl acetate (3 x 50 mL). The combined organic layer was washed with brine (3 x 50 mL), and dried over anhydrous MgSO4. The MgSO4 was subsequently removed by vacuum filtration, and the filtrate was concentrated in vacuo to give crude benzyl 2-(triisopropylsilyloxy) acetate 1-3 as pale yel-
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCHCl) (9.09 g, 46.5 mmol) and N-hydroxysuccinimide (NHS) (5.46 g, 46.5 mmol) was added to a solution of crude 2-((triisopropylsilyl)oxy) acetic acid 1-4 (3.60 g, 15.5 mmol) in 40 mL Dichloromethane in a 100mL round-bottom flask. The reaction mixture was allowed to stir at room temperature overnight and was monitored by TLC. Upon completion, the reaction mixture was quenched with saturated ammonium chloride solution in water (50 mL), and extracted with DCM (3 x 50 mL). The combined organic layer was washed with brine (3 x 50 mL), and dried over anhydrous MgSO4. The MgSO4 was subsequently removed by vacuum filtration, and the filtrate was concentrated in vacuo to give crude 2,5-dioxopyrrolidin-1-yl 2-(triisopropylsiloxy) acetate 1-5 as pale yellow solid. (+)-(1R,2R)-1-Phenylcyclohexane-cis-1,2-diol (2-2) (16) Potassium ferricyanide (62.4 g, 190 mmol), anhydrous potassium carbonate (26.2 g, 190 mmol), methanesulfonamide (6.01 g, 63.2 mmol), potassium osmate dihydrate (70.0 mg, 0.190 mmol), (DHQD)2PHAL (981 mg, 1.26 mmol), 1- phenylcyclohexene (10.1 g, 63.8 mmol), and tert-butyl alcohol (54 mL) were added into 80 mL of water in a 250-mL flask once the stirring had already started. The slurry was stirred vigorously for two days and monitored by TLC. Upon completion, the reaction mixture was treated with ethyl acetate with stirring to dissolve the product. The organic layer was collected, washed with 2M KOH with vigorous shaking to remove methanesulfonamide, and then dried over anhydrous MgSO4. Once again, the MgSO4 was subsequently removed by vacuum filtration, and the filtrate was concentrated in vacuo to give crude (+)-(1R,2R)-1-phenylcyclohexane-cis-1,2-diol 2-2 (12.2 g, 63.5 mmol) as a white solid. (-)-(1R,2S)-trans-2-Phenyl-1-cyclohexanol (2-3) (16) A slurry of activated W-2 Raney nickel in wet ethanol was prepared (~80 mL settled Ra-Ni), and was added to a 500-mL 3-necked round-bottom flask with the aid of anhydrous ethanol in portions. Crude (+)-(1R,2R)-1-phenylcyclohexane-cis-1,2-diol 2-2 (11.7 g, 63.5 mmol) from the previous step in 60 mL anhydrous ethanol was then added. The reaction mixture was stirred vigorously by a mechanical stirring bar, and refluxed for
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3 hours. The reaction was monitored by TLC. Upon completion, it was allowed to cool to room temperature and transferred with anhydrous ethanol and filtered. The Raney nickel sludge was transferred with water to a specific waste container. The filtrate was concentrated and extracted with ethyl acetate (3 x 100 mL) and washed with brine (3 x 50 mL). The combined organic layer was dried over anhydrous MgSO4. The MgSO4 was subsequently removed by vacuum filtration, and the filtrate was concentrated in vacuo to give crude (-)-(1R,2S)-trans-2-phenyl-1-cyclohexanol 2-3 (9.93g, 56.4 mmol) as a pale yellow solid in 89% yield. The crude product was further purified by recrystallization with pentane to give (-)-(1R,2S)trans-2-phenyl-1- cyclohexanol 2-3 (7.72 g, 43.8 mmol) as a white solid.
acetate (3 x 50 mL). The combined organic layer was washed with brine (3 x 50 mL), and dried over anhydrous MgSO4. The MgSO4 was subsequently removed by vacuum filtration, and the filtrate was concentrated in vacuo to give crude 1-8 as yellow oil. The crude product was further carefully purified by column chromatography on silica gel (gradient eluent: hexanes/ethyl acetate from 50/1 to 4/1) to give (3R,4S)-1-(4-methoxyphenyl)-4-(2-methylprop-1-en-1-yl)-3-(triisopropylsiloxy)azetidin-2-one 1-8 as a pale yellow solid (1.16 g, 2.87 mmol) whose yield will be determined on the completion of the recrystallization.
(1R,2S)-trans-2-Phenylcyclohexyl-1-triisopropylsiloxyacetate (16, chiral ester) (17)
10-Deacetyl-7-triethylsilylbaccatin III (3-2) (19)
To a solution of (-)-(1R,2S)-trans-2-phenyl-1-cyclohexanol 2-3 (1.94 g, 11.1 mmol) and DMAP (1.62 g, 13.2 mmol) dissolved in DCM (100 mL) in a 250-mL round-bottom flask, was added 2,5-dioxopyrrolidin-1-yl 2-((triisopropylsilyl)oxy)acetate 1-5 (5.10 g, 15.5 mmol). The reaction was stirred at room temperature under inert conditions for 24 hours and monitored by TLC. Upon completion, the reaction was quenched with saturated ammonium chloride solution in water (50 mL), and extracted with ethyl acetate (3 x 100 mL). The combined organic layer was washed with brine (3 x 50 mL), and dried over anhydrous MgSO4. The MgSO4 was subsequently removed by vacuum filtration, and the filtrate was concentrated in vacuo to give crude (1R,2S)- trans-2-phenylcyclohexyl-1-triisopropylsiloxyacetate 1-6 as pale yellow oil. The crude product was further purified by column chromatography on silica gel (gradient eluent: hexanes/ethyl acetate from 100/1) to give (1R,2S)-trans-2-phenylcyclohexyl-1-triisopropylsiloxyacetate 1-6 as a colorless oil.
Synthesis of the Baccatin Core
10-DAB III (254 mg, 0.466 mmol) and imidazole (160 mg, 2.33 mmol) were added to a 25-mL round bottom flask. The flask was purged with nitrogen gas and 10 mL anhydrous DMF was added. The mixture was dissolved and cooled to 0 °C in an ice bath. Then chlorotriethylsilane (0.4 mL, 2.33 mmol) was added drop wise. The reaction was monitored by TLC with a 1:1 eluent ratio of hexanes:ethyl acetate. This was stained with 5% sulfuric acid in ethanol to determine the purity of the product. Upon completion after 30 minutes, the reaction was quenched with 5 mL saturated ammonium chloride solution in H2O and diluted with 20 mL H2O. The aqueous layer was extracted with ethyl acetate (3 x 20 mL). The combined organic layer was washed with brine (3 x 20 mL), and dried over anhydrous MgSO4. The MgSO4 was subsequently removed by vacuum filtration, and the filtrate was concentrated in vacuo to give crude product 3-2 as a colorless oil. The crude product was further purified by column chromatography on silica gel (gradient eluent: hexanes/ethyl acetate from 4/1 to 1/1) to give 10- deacetyl-7-triethylsilylbaccatin III 3-2 as a white solid (562 mg, 0.853 mmol)
(E)-4-Methoxy-N-(3-methylbut-2-en-1-ylidene)aniline (1-7) (18) To a solution of recrystallized p-anisidine (0.47 g, 5.61 mmol) and anhydrous MgSO4 (0.90 g, 7.65 mmol) in DCM (20 ml) in a 250-mL round-bottom flask was added 3-methylbut-2-enal (0.38 g, 4.60 mmol) drop wise. The reaction mixture was allowed to stir at room temperature in a dark, inert atmosphere for three hours. The reaction was monitored via TLC. Upon completion, the reaction mixture was filtered to remove magnesium sulfate. The filtrate was evaporated in vacuo at room temperature in the dark to give crude (E)-4-methoxy-N-(3- methylbut-2-en-1-ylidene) aniline 1-7 (0.75 g, 3.85 mmol) as pale yellow oil in quantitative yield, which was then used immediately in the subsequent step without further purification. (3R,4S)-1-(4-Methoxyphenyl)-4-(2-methylprop-1-en-1-yl)-3 (triisopropylsiloxy)azetidin-2- one (1-8) (18) Dry THF (22 mL) was added to a 100-mL round bottom flask under inert conditions. It was cooled to -78 °C by an acetone/dry ice bath. Lithium Diisopropylamine (LDA) 2.0 M solution in THF/heptane/ethyl benzene) (10 mL, 3.07 mmol) was then added drop wise. To the mixture, a solution of chiral ester (1R,2S)-trans-2-phenylcyclohexyl-triisopropylsilyl-oxyacetate 1-6 (0.9 mg, 2.56 mmol) in dry THF (10 mL) was added drop wise. The reaction mixture was allowed to stir at -78 °C for another hour. A solution of crude imine (E)-4-methoxy-N-(3-methylbut-2-en-1- ylidene)aniline 1-7 (1.19 g, 6.31 mmol) in dry THF (15 mL) was then added very slowly at 5 ml/hr. The reaction mixture was allowed to stir at -78 °C for another 3 hr. Upon completion of the reaction, the mixture was quenched with a saturated solution of NH4Cl dissolved in water (30 mL), and extracted with ethyl
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10- N,N-Dimethylcarbamoyl -10-deacetyl-7-triethylsilylbaccatin III (3-3) (19) 10-deacetyl-7-triethylsilylbaccatin III 3-2 (307 mg, 0.466 mmol) was added to a 25-mL round-bottom flask and it was purged with nitrogen gas and 9 mL of freshly distilled anhydrous THF. The starting material was dissolved and cooled to -40 °C in an acetone/dry ice bath by adding a small amount of dry ice into acetone, and monitored using a thermometer. LiHMDS (0.69 mL, 0.69 mmol) was added drop wise, and then N,N-dimethylcarbamoyl chloride (0.6 mL, 0.93 mmol) was added drop wise. The reaction was monitored by TLC (Hexane/EA = 1/1, stain with 5% sulfuric acid in ethanol). Upon completion after 4 hours, the reaction was quenched with 5 mL saturated ammonium chloride solution and then diluted with 10 mL H2O. The aqueous layer was extracted with ethyl acetate (3 x 30 mL). The combined organic layer was washed with brine (3 x 30 mL), and dried over anhydrous MgSO4. The MgSO4 was subsequently removed by vacuum filtration, and the filtrate was concentrated in vacuo to give crude product as white solid. The crude product was further purified by column chromatography on silica gel (gradient eluent: hexanes/ethyl acetate from 10/1 to 1/1) to give 10-N,N-dimethylcarbamoyl-10- deacetyl-7-triethylsilylbaccatin III 3-3 as a white solid (320 mg, 0.438 mmol) whose yield will be determined once the product is weighed. RESULTS AND DISCUSSION β-Lactam 3’difluorovinyltaxoids are made using enantiopure β-lactam as well
as 10-DAB III. Enantiopure β-lactam is an important precursor that created by the synthesis of Whitesell’s chiral auxiliary as depicted in scheme 1.6 via Sharpless asymmetric dihydroxylation (AD), followed by chiral ester enolate - imine cyclocondensation. The synthesis of the chiral ester begins with benzyl protection of the commercially available glycolic acid. Crude benzyl glycolate 1-2 was generated in 72% yield when using triethylamine (TEA) as the base (Scheme 1.1). 1HNMR showed that the product was a pale yellow oil (14).
Scheme 1.4: Synthesis of activated ester 1-5 via EDC coupling
With activated ester 1-5 in hand, Whitesell’s chiral auxiliary was prepared in order to react with activated ester to form the chiral ester. First, crude (+)-(1R,2R)-1-phenylcyclohexane-cis- 1,2-diol 1-5 was obtained in quantitative yield via Sharpless asymmetric dihydroxylation (AD) reaction (Scheme 1.5). 1H NMR showed that the product was a pale yellow solid (16).
Scheme 1.1: Benzyl protection of glycolic acid
Benzyl glycolate 1-2 was then reacted with triisopropylsilyl chloride (TIPSCl) in the presence of imidazole. TIPS-Protected benzyl glycolate 1-3 was then generated in quantitative yield (Scheme 1.2). 1HNMR showed that the product was a pale yellow oil (15).
Scheme 1.5: Synthesis of diol 2-2 via Sharpless asymmetric dihydroxylation
Scheme 1.2: TIPS protection of benzyl glycolate 1-2
In this reaction, osmium tetroxide is a stereospecific oxidant that produces the diol from the alkene by a syn-addition. The reaction is carried out using three equivalents of potassium ferricyanide as oxidants and catalytic amount of osmium tetroxide. This oxidation reaction can be highly enantioselective in the presence of chiral ligands. (DHQD)2PHAL, one of the most effective ligands that will lead to an (R,R) configuration of the product, was used in this reaction. This ligand not only induced high enantioselectivity, but also accelerated the reaction. The presence of organic sulfonamide methanesulfonamide further made this reaction more effective by accelerating the hydrolysis of the osmate ester.
The benzyl group of TIPS-protected benzyl glycolate 1-3 was subsequently removed by hydrogenolysis under hydrogen gas in the presence of 10% Pd/C to afford crude product TIPS glycolic acid 1-4 in quantitative yield (Scheme 1.3). 1H NMR (400 MHz, CDCl3): δ 1.13 (m, 21H), 4.29 (s, 2H). The crude product was directly used in the next step without further purification, because the TIPS group is not very stable under acidic conditions and may fall off if not used at once.
Whitesell’s chiral auxiliary 2-3 was generated by selective reduction of the crude diol 2-2 from the previous step with Raney nickel in 69% yield after recrystallization with pentane (Scheme 1.6). Raney nickel is extremely pyrophoric when dry, and therefore must be kept in wet ethanol all the time. Aqueous slurry of Raney nickel is transferred into the flask with the aid of ethanol in portions. 1H NMR showed that the product was white solid crystals (16).
Scheme 1.6: Synthesis of Whitesell’s chiral auxiliary 2-3 by selective reduction of diol 1-5 Scheme 1.3: Benzyl deprotection of 1-3 by hydrogenolysis
The free carboxylic acid group in TIPS glycolic acid 1-4 was then activated by N- hydroxysuccinimide (NHS) via 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) coupling to give activated ester 1-5 in 33% yield (Scheme 1.4). 1H NMR showed that the product was a pale yellow oil (14).
Key intermediate chiral ester 1-6 for making the β-lactams was obtained by reacting Whitesell’s chiral auxiliary 2-3 with activated ester 1-5 in the presence of DMAP in DCM in 33 % yield (Scheme 1.7). 1H NMR showed that the product was a colorless oil (16). The purity of the starting materials is very important for the com-
29
pletion of this reaction in short time, and will affect the yield of this reaction to a large extent. The product need to be carefully purified by column chromatography and carefully dried on vacuum, as impurity or moisture in the chiral ester 1-6 may lead to the failure of the next key step: chiral ester enolate-imine cyclocondensation.
lectively acylate the C-10 position in the following step (Scheme 1.10). The structure of 10-deacetylbaccatin III contains four alcohols at C-1, C-7, C-10 and C-13, among which the C-7 alcohol is the most acidic one and could be selectively protected with chlorotriethylsilane (TESCl). The C-1 hydroxyl is sterically hindered by the benzoyl group at C-2 position, and does not compete with the C-7 alcohol. It was confirmed by the experiments that acylation of 10-DAB III 3-1 under different conditions gave C-7, C-10, and C-13 acylated products, leaving the C-1 hydroxyl group untouched (13). TLC carefully monitored the mono-TES protection of C-7 hydroxyl, so that multiple protections did not occur at the C-10 and C-13 positions. The reaction was done in 5 hours, and the desired 7-TES-10-DAB III 3-2 was obtained in 93% yield. 1H NMR showed that the product was a white solid (19).
Scheme 1.7: Synthesis of key intermediate chiral ester 1-6
The crude imine 1-7 was generated in quantitative yield in the condensation reaction of recrystallized p-anisidine and 3-methyl-2-butenal (Scheme 1.8). This is a condensation reaction between an aldehyde with a nitrogen nucleophile, and involves addition and elimination steps. The product of this reaction is an imine that is also known as a Schiff base. The reaction is reversible and the product is very reactive and unstable as it has a conjugated structure. Therefore, an excess of drying agent magnesium sulfate is used to prevent the hydrolysis of the product. The condensation reaction must be done in the dark, as the product is prone to isomerization. The resulting material was used immediately without further purification to prevent the product reverting to the starting material or isomerizing. 1H NMR showed that the product was a pale yellow oil (18).
Scheme 1.10: TES protection of C-7 position in 10-DAB III
For the synthesis of difluorovinyl taxoid SB-T-12854, after the C-7 alcohol of 10-DAB III was selectively protected, the C-10 position was selectively acylated with N,N-dimethylcarbamoyl chloride in the presence of LiHMDS (Scheme 1.11). The Rf value of the C-10 N,N-dimethylcarbamoyl acylated product 3-3 is very similar to the Rf value of the starting material 7-TES-10-DAB III 3-2. This reaction was carefully monitored by TLC and mass spectrometry to make sure all the starting material was converted to product. The reaction was finished within 4 hours at -40°C, and the desired product 3-3 was obtained. On weighing the product, the yield will be determined. 1H NMR showed that the product was a white solid (13).
Scheme 1.8: Synthesis of imine 1-8 by condesation reaction
With the chiral ester 1-6 and imine 1-7 in hand, the chiral ester enolate-imine cyclocondensation reaction, previously developed in the Ojima laboratory, was performed, affording enatiopure β-lactam 1-8. After the recrystallization was complete, the yield was determined (Scheme 1.9). The product was first purified by column chromatography, and then by recrystallization. 1H NMR showed that the product was a pale yellow solid (18). Scheme 1.11: Acylation of C-10 position in 3-2 with N,N-dimethylcarbamoyl chloride
FUTURE PLAN
Scheme 1.9: Synthesis of enantiopure β-Lactam 1-8 by chiral ester enolate-imine cyclocondensation
Baccatin Core The synthesis of 3’ difluorovinyltaxoid SB-T-12854 begins with triethylsilyl (TES) protection of the C-7 alcohol of 10-DAB III in order to se-
30
In the following months, the synthesis of the taxoid SB-T-12854 will be completed by adding the fluorine groups to the β-lactam molecule to make 3’ difluorovinyltaxoid, which could improve the anti-cancer drugs of conventional chemotherapy. This would create a highly potent fluoro-taxoid that serves as the “warhead” or payload for tumor targeting “molecular missiles” or tumor-targeting drug delivery systems, by increasing the potency by 2-3 orders of magnitude against drug resistant cancer cell lines. Additionally, future research will also attempt the synthesis of a polyethylene glycol (PEG) unit to connect the commercially available biotin molecule with the synthesized disulfide linker.
References 1. I. Ojima, Strategic incorporation of fluorine into taxoid anticancer agents for medicinal chemistry and chemical biology studies. J. Fluorine Chem. 198, 10-23 (2017) doi: 10.1016/j. jfluchem.2016.12.016. 2. E. Rowinsky, The development and clinical utility of the taxane class of antimicrotubule chemotherapy agents. Annu. Rev. Med. 48, 353–374(1997). doi: 10.1146/annurev.med.48.1.353. 3. M. Bissery, et. al., Docetaxel (Taxotere): a review of preclinical and clinical experience. Part I: Preclinical experience. Anticancer Drugs 6, 339–355 (1995). 4. P. Dalerba, et. al., Cancer stem cells: models and concepts. Annu. Rev. Med. 58, 267–284 (2007). doi: 10.1146/annurev.med.58.062105.204854. 5. O.C. Farokhzad, et.al., Targeted nanoparticle-aptamer bioconjugates for cancer chemotherapy in vivo . Proc. Nat. Acad. Sci. 103, 6315–6320 (2006). doi: 10.1073/pnas.0601755103 6. I. Ojima, et.al., Tumor-targeting drug delivery of new-generation taxoids. Future Med. Chem. 4, 33–50 (2012). doi: 10.4155/fmc.11.167. 7. McGuire, et.al., Cyclophosphamide and cisplatin compared with paclitaxel and cisplatin in patients with stage III and stage IV ovarian cancer. N. Engl. J. Med. 334, 1–6 (1996). doi: 10.1056/NEJM199601043340101 8. I. Ojima, et.al., New and Efficient Approaches to the Semisynthesis of Taxol and Its C-13 Side Chain Analogs by Means of β-Lactam Synthon Method, Tetrahedron, 48, 6985-7012 (1992). 9. E.K. Rowinsky and R.C. Donehower, Paclitaxel (taxol). N. Engl. J. Med. 332, 1004-1014.(1995). doi: 10.1056/NEJM199504133321507 10. W. P. McGuire, et. al., Cyclophosphamide and cisplatin compared with paclitaxel and cisplatin in patients with stage III and stage IV ovarian cancer. N. Engl. J. Med. 334, 1–6(1996).doi: 10.1056/NEJM199601043340101 11. K.L. Kirk, Fluorine in medicinal chemistry: Recent therapeutic applications of fluorinated small molecules. J. Fluorine Chem 127, 1013–1029 (2006). doi: 10.1016/j.jfluchem.2006.06.007 12. I. Ojima, et al., Design, Synthesis and Biological Evaluation of New Generation Taxoids. J. Med. Chem. 51, 3203–3221(2008). doi: 10.1021/jm800086e. 13. I. Ojima, et. al., Syntheses and structure-activity relationships of the second-generation antitumor taxoids: Exceptional activity against drug- resistant cancer cells. J Med Chem 39, 38893896(1996). doi: 10.1021/jm9604080. 14. M. Macrae, et.al., Nanoscale Ionic Diodes with Tunable and Switchable Rectifying Behavior. J Am Chem Soc 132, 1766-1767(2010). doi: 10.1021/ja909876h. 15. H. Yu, et. al., An inexpensive carbohydrate derivative used as a chiral auxiliary in the synthesis of alpha-hydroxy carboxylic acids. Tetrahedron 58, 7663-7679 (2002). 16. J. Gonzalez, et. al., Synthesis of (+)-(1S,2R)- and (-)- (1R,2S)-trans-2-phenylcyclohexanol via Sharpless asymmetric dihydroxylation (AD). Org Synth 79, 93-102(2003). doi: 10.15227/ orgsyn.079.0093. 17. I. Ojima, et. al., New and Efficient Approaches to the Semisynthesis of Taxol and Its C-13 SideChain Analogs by Means of Beta-Lactam Synthon Method. Tetrahedron 48, 6985-7012(1992). 18. I. Ojima, et. al., Syntheses and structure-activity relationships of taxoids derived from 14 beta-hydroxy-10-deacetylbaccatin III. J Med Chem 40, 267- 278 (1997). doi: 10.1021/jm960563e. 19. T. Mukaiyama, et.al., Asymmetric total synthesis of Taxol (R). Chem-Eur J. 5, 121-161(1999). doi: 10.1002/(SICI)1521-3765(19990104)5:1<121::AID-CHEM121>3.0.CO;2-O.
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