08 14 27
The Development of OSCER: A Conversation with Dr. Joseph Lauher Understanding the Probable Causes of Mass Extinction Events Expanding the Cyanobacterial Synthetic Biology Promoter Toolbox
Spring 2019 Volume 12
STAFF 2018-2019 Editor-in-Chief: Sahil Rawal ’19
Head of Cabinet: Peter Alsaloum ’19
Layout Chief: Dahae Julia Jun ’19
Managing Editors: Stephanie Budhan ’21 Rachel Kogan ’19
Cabinet: Jesse Pace ’20 Akshani Patel ’19 Jerin Thomas ’19
Assistant Layout Chief: Lauren Yoon ’20
Associate Editors: Nina Gu ’21 Samara Khan ’19 Bridgette Nixon ’21 Caleb Sooknanan ’20 Anna Tarasova ’19 Dan Walocha ’19
Webmaster: Thomas James ’19
Layout Editors: Priya Aggarwal ’21 Matthew Ng ’22 Faculty Advisors: Dr. John Peter Gergen Dr. Laura Lindenfeld Dr. Nicole Leavey
Copy Editors: Claire Garfield ’20 Nomrota Majumder ’21 Shrey Thaker ’22 Nita Wong ’21
Graduate Advisor: Amanda Ng
Writers: Priya Aggarwal ’21 Stephanie Budhan ’21 Annamaria Cavaleri ’22 Raymond Cheung ’22 Travis Cutter ’22 Sanket Desai ’19 Riya Gandhi ’22 Sultan Kaur ’21 Karthik Ledalla ’21 Natalie Lo ’21 Allan Mai ’20 Mariam Malik ’22 Jesse Pace ’20 Rideeta Raquib ’19 Kavindra Sahabir ’21 Ellie Teng ’21 Ruhana Uddin ’19 Nita Wong ’21 Nicole Zhao ’20
LETTER FROM THE EDITOR-IN-CHIEF Stony Brook Young Investigators Review is proud to release our 12th biannual publication. As always, we have spent the past semester working hard to provide an outlet for student research on campus, as well as giving students from various disciplines the opportunity to work and refine their writing. To celebrate the release of each issue, we host an honorary speaker at our colloquium, and in the past we have had speakers such as the Hero of the Planet, Dr. Sylvia Earle, the Founding Director of the Wyss Institute for Biologically Inspired Engineering at Harvard University, Dr. Donald Ingber, Dr. Arthur Horwich, whose research at Yale University led to the uncovering of chaperonin proteins, and Dr. Joshua Willis who is a Project Scientist at NASA’s Jet Propulsion Laboratory. In this issue, readers will find articles that range from topics like stroke detection in pre hospital conditions to pain in mental illnesses. We also have articles that discuss the latest research on Huntington’s Disease, as well as an interview with Professor Lauher of the Department of Chemistry here at Stony Brook University. We are also proud to host a primary research article from the Stony Brook iGEM team regarding their research over the past summer. This year, we are proud to be hosting Dr. Christopher J Rozell, who is currently a Professor in Electrical and Computer Engineering at the Georgia Institute of Technology. His research focuses on machine learning, and the use of neuro-technologies to increase the effectiveness of clinical therapies. 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 Alda Center for Communicating Science, as well as our faculty advisors, Dr. Peter Gergen, Dr. Laura Lindenfeld, Dr. Nicole Leavey, and our graduate advisor, Amanda Ng, for their guidance. Welcome to SBYIR. We sincerely hope you enjoy.
TABLE OF CONTENTS Spring 2019
Development of 08 The OSCER: A Conversation with Dr. Joseph Lauher
Organs on a Chip: The Modern Petri Dish By Rideeta Raquib ’19
By Nita Wong ’21
New Approaches to Treating Chorea and Huntington’s Disease By Sanket Desai ’19
Science Reviews Detection of Stokes in Pre-Hospital Conditions By Ruhana Uddin ’19
Role of Pain in 24 The Mental Illness By Sultan Kaur ’21
Understanding the Probable Causes of Mass Extinction Events By Travis Cutter ’22
Primary Research Expanding the Cyanobacterial Synthetic Biology Promoter Toolbox By Priya Aggarwal ’21, Natalie Lo ’21, Karthik Ledalla ’21, Stephanie Budhan ’21, J. Peter Gergen, Ph.D
RESEARCH HIGHLIGHTS Find more news online!
Fall 2018 Colloquium: A Look Back
By Jesse Pace ’20
Relationship Between Academic Environment and Mental Health By Raymond Cheung ’22
Figure 1 Dr. Dany Adams
This past fall, SBYIR had the honor of hosting Dr. Dany Adams of Tufts University as our esteemed guest speaker. As the Founding Editor-In-Chief of the new Mary Ann Liebert Inc. journal Bioelectricity and Life Science Manager at Akita Innovations, Dr. Adams has diverse expertise in both academia and industry. She is very passionate about promoting diversity in STEM and actively encourages female participation in science through her board position in the Rosalind Franklin Society. In addition to presenting her groundbreaking research which lies at the intersection of bioelectricity and cancer biology, Dr. Adams shared advice with aspiring scientists at all levels. Both undergradu-
ate students and Stony Brook University faculty had the opportunity to meet with Dr. Adams and asked questions about her career path and her plans for the future. Her previous participation with the Alan Alda Center for Communicating Science illustrates her dedication to spreading research and promoting science literacy. We would like to thank Dr. Adams for a wonderful lecture and eye-opening insights.
Researchers at the University of Munich and other universities analyzed the effect of a high-achieving environment on the mental health of students. Their work is vital because it provides insight into how a high-performing environment can harm the academic development and mental wellbeing of students. In the study, the researchers analyzed the responses of over 7,700 German school students with regards to their positive/negative emotions and academic achievement measured by test results. The researchers found that students’ academic achievement and positive emotions were positively related. In other words, students who performed better than the average performance of their peers often reported more positive emotions. Conversely, the opposite holds true for negative emotions; students who were in classes with top performers and performed below them often reported more negative emotions. These findings identify an essential relationship between one’s academic environ-
ment and his/her emotions, because the emotions may have a compounding effect that hinders a student’s academic achievement. For instance, a below-performing student may be further discouraged if surrounded by top performers. The study’s findings also contribute to knowledge about the development of fixed and growth mindsets in students. A fixed mindset is a perception that one’s intelligence is static and this study identifies how one’s environment may influence his/her perception towards his/her academic intelligence. Further exploration of the effect of academic environments is essential in ensuring the mental well-being of academically developing students.
References 1. P. Reinhard, et al., Happy fish in little ponds: Testing a reference group model of achievement and emotion. Journal of Personality and Social Psychology, (2019). doi: 10.1037/ pspp0000230. 2. Image retrieved from: https://pixabay.com/ en/book-reading-literature-classics-856151/
Figure 1 A student’s environment could dictate his emotional state.
Metabolic Reactions Activated During 58-Hour Fasting By Ellie Teng ’21
Fasting is an ancient component of numerous religions and cultures. Although it is used for weightloss purposes, there is still a heated debate surrounding its efficacy. A team of scientists from the Okinawa Institute of Science and Technology Graduate University and Kyoto University recently found that fasting comes with a myriad of health benefits. Blood samples from four healthy individuals who fasted for 34-58 hours were obtained and studied by the team. The level of metabolites was analyzed, and researchers found more than 30 previously unreported metabolites that increased significantly during fasting (1). A total of 44 metabolites increased between 1.50 to 60-fold in all four volunteers. The compounds purine and pyridine are responsible for gene expression, protein synthesis, transcriptional programming, and increasing antioxidant production. Fasting appeared to drive the metabolism of these compounds, resulting in an increase in ergothioneine and carnosine, two antioxidants that protect against free
radicals. These results suggest that fasting may promote protein formation. This is beneficial to fighting aging and harmful environmental factors. Scientists also observed a spike in organic acids and coenzymes during fasting, which indicated the activation of mitochondrial activity in tissues. Additionally, the Pentose Phosphate Pathway (PPP) metabolites and antioxidants levels were higher during fasting, but only in plasma. Significant products of the PPP include 6-phosphogluconate and glucose-6-phosphate, which both are essential for redox maintenance and nucleic acid synthesis. This discovery provided scientists with a deeper understanding of metabolism and could be used in further research involving weight loss or aging.
References 1. T. Teruya, et al., Diverse metabolic reactions activated during 58-hr fasting are revealed by non-targeted metabolomic analysis of human blood. Scientific Reports 9, 854 (2019). doi: https://doi.org/10.1038/s41598-018-36674-9. 2. Image retrieved from: https://www.pexels. com/photo/silhouette-of-women-on-lakeagainst-sky-248139/
Figure 1 Fasting for at least 34 hours boosts metabolism in healthy adults.
South Asians Have the Highest Rates of Heart Disease By Nicole Zhao ’20
Figure 1 Out of all the races and ethnicities, South Asians have the highest incidence of coronary artery calcium deposits, which is one of the major factors leading to heart disease.
Heart disease is the leading cause of death for both men and women worldwide. However, people of South Asian descent have a higher death rate from heart disease than any other group. Following a variety of diets, from omnivorous to vegetarian, South Asians are four times more likely to be diagnosed with heart disease even at normal body weight and tend to develop the pathology up to a decade earlier compared to the general population. A joint research project between the University of California, San Francisco and Northwestern University known as MASALA (The Mediators of Atherosclerosis in South Asians Living in America) aims to shed light on risk factors for the disease. In a recent study, the team found that the incidence and progression of coronary artery calcium in South Asians were highest when compared to four race/ethnic groups. Coronary artery calcification (CAC) is a condition characterized by hardened blood vessels, which restricts blood flow and increases the likelihood of a heart attack or stroke; CAC is a marker of atherosclerosis. In this study, a total of 698 South Asian participants were monitored for about five years and their coronary artery calcium levels were periodically scored via CT scans. It was found that the median annual CAC progression was 26 for men
and 13 for women. The CAC score thresholds were defined for risk prediction ranging from very low to very high-risk events (CAC 0; 1-100; 101-400; and >400). Therefore, an annual increase of 26 in one’s CAC score could substantially increase one’s risk for a cardiovascular event over a lifetime. Comparing these results with MESA, the Multi-Ethnic Study of Atherosclerosis, CAC incidence was similar in South Asian men and white, black, Latino men, but was significantly higher than in Chinese men. Moreover, it was found that diabetes, hypertension, and statin medication use, which is used to lower cholesterol levels, was higher in South Asian and white men. South Asian women’s calcium artery levels had no significant differences when compared to women of other races. This study is indubitably important as it reveals disparities in heart disease risk among races, which should be taken into account when determining cardiovascular disease risk and treatment options.
References 1. A. Kanaya, et al., Incidence and progression of coronary artery calcium in South Asians compared with 4 race/ethnic groups. Journal of American Heart Association 8, (2019). DOI: 10.1161/JAHA.118.011053. 2. Image retrieved from: https://www. pexels.com/photo/bright-cardiac-cardiology-care-433267/
Use of Arithmetic Operations and Memory Processing Shown in Bees By Mariam Malik ’22
Some animals are mentally capable of understanding the concept of numbers, emotion, and even language. However, at RMIT University in Australia, an experiment performed on bees showed that they are not only able to understand the concept of numbers, but that they also comprehend arithmetic operations, such as addition and subtraction, with the use of colored symbols. To determine if some animals are capable of numerical cognition, Dr. Scarlett Howard and his research team chose honeybees as the experimental organism. Previous research stated that these animals are capable of learning various rules and ideas to solve conflicts and can distinguish between the idea of left/right, larger/smaller, front/behind, etc. When trained with rewards, bees have also shown an ability to count and distinguish between numbers. With this, the researchers’ goal was to train the bees to identify a common color, either yellow or blue, as a symbol of whether to use addition (blue) or subtraction (yellow), and then pick the correct result of the operation. Furthermore, the bees were trained to enter a Y-maze. First, they had to fly through an entrance hole that led them to a chamber where there would be a sample stimulus. Each color had its own maze and trial, since they represented two different operations. In the blue Y-maze,
for example, the sample stimulus in the chamber would show two blue shapes. Then they would fly through another hole to the decision chamber, with two holes on each side and different stimulus near each hole. With the blue Y-maze, one hole would show one blue shape and the other showed three blue shapes. In this experiment, the hole with three blue shapes was selected as the correct choice, since the blue symbolized addition and the sample stimulus had two blue shapes. The bees would then fly into the hole that had the correct answer fifteen centimeters away from it. For choosing the correct or incorrect answer, the bees were given a few drops of 50% sucrose solution or quinine solution, respectively. The results demonstrated a significant increase in the number of bees that answered correctly, showing that bees were able to add and subtract by one depending on the color of the sample stimulus before the decision chamber. The study showed that bees were mentally capable, despite their minuscule brains, of information processing, including the representation of numerical attributes and then using those representations in their working memory. From an ecological perspective, the bees’ ability to think arithmetically and remember is advantageous in remembering which characteristics of flowers (color, type, size) pro-
Figure 1 Bees’ arithmetic cognition and memory helps these organisms remember which flowers provide the highest nutritional value.
vide essential resources and which do not. This new finding highlights areas of research in the brains of insects, and implies that this combination of arithmetic and learning ability based on symbolism may be applicable to other species.
References 1. S. R. Howard, et al. Numerical cognition in honeybees enables addition and subtraction. Science Advances 5, (2019). doi: 10.1126/ sciadv.aav0961. 2. Image retrieved from: https://www.pexels. com/photo/animal-bees-bloom-blooming-553251/
Mind the Gap! Nanoparticles Increase Endothelial Leakiness By Riya Gandhi ’22
Although recent advancements in the field of nanomedicine are elucidating potential novel therapies for cancer, researchers have uncovered one major drawback called gap growth. Under the leadership of principal investigator Dr. Fei Peng, a recent study at the National University of Singapore has discovered that the introduction of nanomaterial into animal bodies may result in micro-sized gaps in the endothelial lining, which can spur metastasis in cancer patients. Dr. Peng et al. primarily focused on the effects of titanium dioxide, silica, and gold nanoparticles on interactions between VE-cadherin – a protein that maintains order at intercellular junctions and is especially important in endothelial cells – and adherens junctions. The scientists exposed the proteins to the nanoparticles through a dosage treatment and documented any changes at the end of approximately 30 minutes. They noticed that exposing titanium dioxide to the endothelial cells induced dose-dependent leakage and that increasing the dosage led to the creation of more gaps. Furthermore, when an analogous procedure was conducted on the blood of mice, the researchers discovered that human breast cancer genes in the mice had spread more than anticipated. Ultimately, they deduced that the use of nanoparticles in cancer treatment may result in major drawbacks such as intravasation and extravasation. Extravasation occurs when IV drugs inadvertently leak into healthy tissue. Intravasation, the influx of cancer cells through lymphatic or
Figure 1 Dark red liver with pale mestastasis nodules from from pancreatic cancer.
blood vessels, may follow, thereby exacerbating metastasis of cancer. Through this experiment, the scientists broadened their understanding of the implications of nanomedicine. Although it is extremely important for the field of medicine to generate improved treatments for cancer patients, it is equally, if not more, important to ensure that these therapies are not inflicting excessive harm on healthy tissues within the body. Before such procedures are implemented in clinical medicine, nanoparticle leakage must be addressed and controlled.
References 1. F. Peng, et al., Nanoparticles promote in vivo breast cancer cell intravasation and extravasation by inducing endothelial leakiness. Nature Nanotechnology, (2019). doi: 10.1038/s41565018-0356-z. 2. Image retrieved from: https://en.wikipedia. org/wiki/File:Secondary_tumor_deposits_in_ the_liver_from_a_primary_cancer_of_the_ pancreas.jpg
Fibrinogen Plays a Neurodegenera- The Effect of Diabetes on Fingernail tive Role in Alzheimer’s Disease Quality By Natalie Lo ’21
Alzheimer’s Disease (AD) is characterized by the formation of β-amyloid plaques (Aβ), microglial activation, and inflammation in the brain. Microglia are immune cells found in the central nervous system (CNS). In AD, the blood-brain barrier is disrupted, which leads to bleeding, vascular damage, and an increase in blood proteins. Currently, there is an unknown relationship between vascular dysfunction, proteins like fibrinogen (a blood coagulation protein), and Aβ. The pathogenesis of AD has captured the attention of a group of scientists at the Gladstone Institute of Neurological Disease in San Francisco. The group of researchers conducted in vivo 3D immunofluorescent labeling in 5XFAD mouse brains to examine Aβ deposits and fibrinogen. The study revealed that fibrinogen can be utilized as a detector for abnormalities in AD and links the protein as a trigger for characteristics of AD, such as cognitive impairment, microglia-dependent spine elimination, and dendrite loss in regions with Aβ deposits. In the CNS, fibrinogen activates the microglia by binding to
By Kavindra Sahabir ’21
CD11b-CD18, which can prevent the clearing of β-amyloid plaques and thus lead to further neurodegeneration. When the binding between fibrinogen and CD-11b is prevented genetically, the Aβ plaque and neurodegeneration decrease in the hippocampus. Furthermore, fibrinogen is enough to cause AD pathogenesis even without the presence of Aβ accumulation. This study reveals more about the pathogenesis of Alzheimer’s disease, leading the medical field one step closer to finding the underlying cause of AD, and ultimately to discovering potential treatments for AD that target fibrin and help diminish cognitive disorders.
References 1. M. Merlini, et al., Fibrinogen induces microglia-mediated spine elimination and cognitive impairment in an alzheimer’s disease model. Neuron 101, 1-10 (2019). doi:10.1016/j. neuron.2019.01.014l. 2. Image retrieved from: http://jem.rupress. org/content/204/3/571.figures-only
Figure 1 Fibrinogen affects microglia activation, which impacts Alzheimer’s Disease.
Figure 1 Diabetes is a disease affecting more than just blood sugar levels, as it affects tissue and organs in unpredictable ways.
In our current understanding, Type 2 diabetes (T2D) is associated with the need for sugar-free foods and blood sugar monitors. Beyond a high blood sugar level, however, it also causes chronic degradation and damage to nerves, joints, and other bodily tissues. A study performed by Dr. Silhota endeavored to determine whether the fingernail could be a useful site to measure the amount of tissue degradation, and provide an accessible way to determine tissue quality in people with T2D. In this experiment, nail quality was measured against three groups of middle-aged people. One group, which was the control, was classified as healthy with an average HbA1c level of 5.4. This level is the measure of hemoglobin (HbA1c) in the body, which is associated with blood sugar level. The two other groups were controlled diabetes (DC), because participants actively took care of their diabetes, and uncontrolled diabetes (UD), with average HbA1c levels of 6.6 and 8.4 respectively. The study found that nail quality, which was determined using mineral analysis, varied significantly between all three groups. In particular calcium content, as measured in relation to the control
group, was found to decrease by 6.3% in the DC group and 75% in the UD group. The disulfide bond content, which contributes to the stability of the fingernail, was found to decrease by 30% for the DC group and around 66% in the UD group in relation to the control group. These results show that an analysis of nail quality can actually serve as a method for determining tissue degradation in diabetic individuals. The results are particularly enticing, considering the trends in the decrease of the mineral quality of the nails correlated with increased HbA1c %. This supports the authors’ hypothesis and provides a novel method of assessing bone quality of people suffering from T2D, which may one day allow doctors and even patients to keep better track of the progression of diabetes.
References 1. P. Silhota, et al., Investigation of diabetic patient’s fingernail quality to monitor type 2 diabetes induced tissue damage. Scientific Reports, (2019). doi: 10.1038/s41598-01939951-3 2. Image retrieved from: https://pixabay.com/ photos/diabetes-blood-sugar-diabetic-528678/
The Unlikely Relation Between the Gut and Brain By Allan Mai ’20
With the high selectivity of the blood-brain barrier, it appears unlikely that microorganisms in the stomach could ever be able to reach the brain. However, past studies that have suggested a correlation between depression and specific gut bacteria and even a correlation between social behavior and gut bacteria activity have sparked substantial research regarding the “gut-brain” axis. Among these studies is the Belgian Flemish Gut Flora Project (FGFP), which was conducted under the direction of principal author Dr. Mireia Valles-Colomer at the Rega Institute for Medical Research of the Katholieke Universiteit Leuven. Researchers working on the FGFP used DNA sequencing to analyze the fecal microbiota of over 1,000 human participants. Next, researchers correlated different microbial taxa with each participant’s quality of life based on self-reported and physician-supplied diagnoses. Their results demonstrated that levels of the bacteria Coprococcus and Dialister were reduced in those with depression while the butyrate-producing Faecalibacterium was consistently associated with
higher quality-of-life indicators. The study also pointed to a positive correlation between quality of life and the potential ability of the gut bacteria to synthesize a breakdown product of dopamine. This breakdown product, known as 3,4-dihydroxyphenylacetic acid, serves as one of the strongest pieces of evidence of the gut-brain relation. Despite the implications of this study, the researchers stress that these preliminary studies are only correlational; their present research methods are unable to demonstrate that microbiota in the gut directly trigger certain activity within the brain. Furthermore, given that most current studies on the gut-brain relation utilize animal models, which are not necessarily representative of the human gutbrain relation, research remains to be done on the topic.
References 1. M. Valles-Colomer, et al., The neuroactive potential of the human gut microbiota in quality of life and depression. Nature Microbiology, (2019). doi: 10.1038/s41564-018-0337-x. 2. Image retrieved from: https://www.pexels. com/photo/close-up-of-microscope-256262/
Figure 1 The recent discovery of a gut-brain relation has considerably increased research interest in the topic.
CYFIP1 Gene Responsible For Movement Impairments in Autism By Annamaria Cavaleri ’22
Figure 1 This depicts a nerve cell (neuron) in the brain.
Researchers from Cardiff University in the UK discovered a link between the CYFIP1 gene and developmental movement impairments in autism. The group concluded that this genetic mutation leads to alterations in developing brain cells, causing motor problems linked to motor learning difficulties at a young age. This, however, may be able to be reversed through behavioral training. People with autism tend to experience difficulty with social interactions and communication. Additionally, they experience repetitive behaviors and movement disorders related to posture and coordination. It is known that the CYFIP1 gene mutation impacts the structural stability of cells in the brain. The team of researchers on this study found that the mutation affected the formation of spines of brain cells, which resulted in cell instability and caused motor problems to arise during the development of autism in the brain through microscopy. Researchers believe that through movement therapy, during the early developing stages of autism, the impact of this gene may be able to be reversed and movement impairments could be reduced. Movement therapy is a relatively new type of therapy that combines movement and music with positive reinforcement to treat those with autism.
Behavioral training is an option given to early age patients who experience difficulties in motor learning. This is crucial for the work toward future prevention of autism related symptoms before its full onset. Future longitudinal studies may be done with testing behavioral training techniques and observing possible reduction of autism symptoms.
References 1. S. Bachmann, et al., Behavioral training rescues motor deficits in Cyfip1 haploinsufficiency mouse model of autism spectrum disorders. Translational Psychiatry 9, (2019). doi: 10.1038/s41398-018-0338-9. 2. Image retrieved from: https://pixabay.com/ en/nerve-cell-neuron-brain-neurons-2213009/
The Development of OSCER: A Conversation with Dr. Joseph Lauher By Nita Wong â&#x20AC;&#x2122;21 8
This upcoming semester, over 1,500 undergraduate students will use the Organic Seawolf Center for Education and Research (OSCER) course management system to communicate with instructors and teaching assistants, submit prelecture quizzes and workshop exercises, access lecture slides, and view their exam scores. However, OSCER’s formal description as an online course management system barely scratches the surface of its capabilities. OSCER Sketch Plus, a supplemental feature of this program, enables students to write out and submit curved-arrow reaction mechanisms. OSCER SYN provides feedback regarding the feasibility of these reactions, and even predicts the products of featured synthetic reactions. Behind the software that forms the instructional foundation of multiple introductory chemistry courses at Stony Brook University – from CHE 321 and 322 (Organic Chemistry), CHE 152, 331, and 332 (Molecular Science), to CHE 133 and 134 (General Chemistry Laboratory) – is a member of the Department of Chemistry: Dr. Joseph Lauher. While pursuing his PhD at Northwestern University and throughout his tenure at Stony Brook University, Dr. Lauher’s research focused on the accurate prediction of the three-dimensional crystal structure of both inorganic and organic molecules and solids. Over the last seven years, however, his focus has gradually and completely shifted to the development and expansion of OSCER. To start off, can you talk about your career path? How did you develop an interest in structural chemistry research? When I went for my PhD at Northwestern University, I was in a group whose main interest was the x-ray determination of molecular structure. The group was more focused on inorganic chemistry, but I ended up doing a lot of work with organic chemistry. Over the years, even though my specific interest in chemistry evolved quite a bit, my general interest was always focused on the structure of molecules. How did your interest in programming begin? How did that translate into the initial idea behind OSCER? I became interested in computer programming very early on. When I was an undergraduate, the computer science major didn’t even exist yet, so I never took a computer science course. Over the years, I did a lot of programming; when I worked with crystallography, I wrote a graphics program to display colored images of molecules. Most of the early programs I wrote soon became dated by fancy commercial programs, so that area of work eventually died out. When the web started to become more widely available, I was one of the earliest people to use it to post notes and such. When Blackboard came along, professors began trying to do quizzes on there. Trying to do chemistry on Blackboard was extremely difficult because you couldn’t do structures. I thought to myself, “We’ve got to be able to do better than this.” And that’s how OSCER began. Can you describe the initial writing process for OSCER? There wasn’t really an initial writing process for OSCER, because I never had an organized plan for its development. Obviously, there was an enormous learning curve for me; I’m
not a computer scientist. When I started this, I had to learn to do server site programming. I knew how to set up a webpage using HTML, but adding functionality to a server – I had to teach myself all that. If I were to start over, I would have a more concrete idea of what the finished product would look like. In programming, they have these established “best practices,” and I didn’t and don’t use any of those. I made mistakes, and I changed things accordingly. Can you give us an idea of OSCER in its early stages? There’s been some form of OSCER around for around six or seven years. OSCER, as we recognize it today, is an outgrowth of a webpage that didn’t feature much interactivity. In fact, OSCER didn’t start off with its own drawing program; we had to get the figures from an external source, which limited our versatility, especially in comparison to what we do now. Even so, it was already much easier to do chemistry on our own system. We started out with MarvinSketch, a commercial chemistry graphics program. Because MarvinSketch is based on Java, which is difficult to run on the web due to security holes, we eventually had to drop that. We then tried out a program that was not Java-based but relied on vendors surfacing it. Every time you wanted to generate a structure, you had to go through the vendors to have it checked. That caused a lot of problems, so during that year, I decided to make my own. We started writing OSCER Sketch, and that has been our primary activity for the past three years. It keeps getting more powerful, but the main focus has been to keep the system from collapsing. How has your vision for OSCER changed over the years of its development? We’re definitely using OSCER in ways we couldn’t in the beginning as the program becomes more powerful. For example, OSCER is currently also used for general chemistry lab, and that’s set up so the students can complete their pre-labs, which involve analyzing distinct sets of data. That would be enormously difficult to achieve on Blackboard. What has been the most difficult aspect of OSCER’s development? One of the most complicated aspects is dealing with all the different hardware components. If I just needed OSCER to work on my own PC, it would be a lot easier than to develop a system that works on smartphones, tablets, PCs and Macs, and on different browsers. For example, this week, the webcams quit working. It wasn’t working on the Macs, but it did work on phones and tablets. On PCs, it didn’t work on Chrome or Firefox, but it did work on Microsoft Edge. If I were a web developer, I would be constantly reading about these updates and be on top of the situation. However, because I’m not a web developer, this came as a surprise. So there’s the aspect of having to constantly keep up with the updates of technology and learn to troubleshoot the problems that arise as a result. Can you describe the process of adding new synthetic reactions to OSCERSYN? How do you determine which reactions to add, and how do you program them into the system? My goal is to have all the reactions that would be covered
in a standard introductory course. The program isn’t smart in the sense that it’s able to look at the features of a molecule and determine how it’s going to react. There’s two components to the smarts; it’s like a baby artificial intelligence. The first module – molecular analysis based on the functional group database – looks at the molecule and sorts it by functional group on different levels of intricacy. The program considers the amount of carbons in the compound first in order to flag it with a basic categorization. Then, the program looks at factors such as the position of hydrogens and the presence of double bonds to give the molecule a more sophisticated and accurate categorization. The second module, the reaction database, allows the program to take the information it already knows about the molecule and consider its possible transformations in response to known reactions. In this way, the program can integrate information about a molecule with known reaction paths to predict the products of a certain mechanism. Both modules – the functional group and the reaction databases – essentially use their own programming language. Because of this, OSCER SYN can take into account factors such as electron density, stability, and space constraints. If there are conflicting factors – say space versus stability – OSCER SYN can also predict multiple products to account for both and arrange them in the order of favorability. That also reflects reality, since mixtures of products are common in the chemistry lab. How do you plan to expand OSCER’s capabilities in both the near and long-term future? I figure that society as a whole, and definitely education, are shifting to more and more online. We’re starting to teach organic online this summer, and the university really wants courses to go online to save money. One of the most promi-
I’d like to make this program — as well as OSCER-SYN — available to the world.
nent issues with making a purely virtual course is that it decreases interaction with the instructional staff, whether it be the faculty or, even more importantly, the TAs. But students still need feedback on their work. So I would really like OSCER to be able to evaluate students’ reaction mechanisms. OSCER SYN is already working that way. As long as you stay within reasonably close to the course database, it functions properly, and that’s part of it. The other part, however, is the mechanistic component, which I think, given time, could really be exciting. Right now, I could ask a problem in which I provide students with the original structure and the final structure and ask students to draw the arrows from here to there, and the magic arrow can tell whether the intermediates are self-consistent, but it can’t tell whether the mechanism in its entirety is reasonable. I’ve started this: when you write a mechanism on OSCER, the system generates warnings on the bottom of the page. If you form a primary carbocation, for example, it’ll warn you that you’ve formed a highly unstable intermediate. What I want to do, however, which I think is doable but will be somewhat complicated, is to have each step evaluated in terms of whether it breaks any basic rules of chemistry. However, because there are alternate mechanisms that can occur in certain cases, in terms of writing a program, you can never have the correct mechanism. Realizing this mechanistic program – a system that would allow students to insert any mechanism they can dream of and determine whether the mechanism is reasonable – that’s the goal. I’d also like to make this program – as well as OSCER SYN – available to the world. But I’ll have to work on eliminating errors first. Having errors internally is one thing, but I don’t want to put out something that’s incorrect publicly.
Detection of Strokes in Pre-Hospital Conditions By Ruhana Uddin ’19
Figure 1 The image above illustrates what a blood vessel looks like in the brain during an ischemic stroke. The clot is visible and blocks the flow of blood to the remainder of the brain.
Introduction Strokes, also known as cerebrovascular accidents (CVA), are the second most common cause of death and lead to 5.5 million deaths worldwide each year (1). In the United States alone, about 800,000 strokes occur per year. Although other diseases and ailments have comparable statistics, what makes strokes so dangerous is their time-sensitivity: failure to provide treatment immediately after the stroke can lead to motor and behavioral deficits, such as altered mental status, changes in speech, and a myriad of other effects depending on which part of the brain is damaged (2). Since CVAs can have such detrimental effects, administering treatment as soon as possible is vital to ensuring that the patient walks away with minimal impairments. However, in the case of a stroke, it can take significant time for the patients to arrive at the hospitals; often times, the long-term consequences of the stroke have already set in by the time patients arrive at the hospital. A proposed solution to this issue is implementing prehospital treatments of strokes that are carried out before the patient arrives at the hospital – for example, sending physicians out to the patients or introducing new forms of technology for stroke diagnosis and treatment. Background Strokes can be divided into two different types: ischemic and hemorrhagic. Ischemic strokes develop when a blood vessel in the brain is blocked due to a blood clot, thus compromising blood flow in the brain. An ischemic stroke is the more common type of stroke that people present with (3). A hemorrhagic stroke, on the other hand, is less common and develops when a blood vessel in the brain ruptures. A stroke is considered a time-sensitive-disease because as soon as a stroke occurs, the blood flow to and within the brain is altered; cells do
not receive an adequate amount of blood, and as result, these same cells do not obtain enough oxygen. Because of this lack of oxygen, a stroke causes rapid cell death, which in turn leads to detrimental impairments (3). There are various risk factors that increase the chances of stroke occurrence, such as alcohol and tobacco use, unhealthy diet, and high cholesterol. Stroke presentations in patients are not homogenous: some people may experience symptoms that others do not, making stroke detection particularly difficult. Stroke symptoms include facial or limb numbness, slurred or changes in speech, and trouble with coordination and headaches (3). Current treatment of strokes includes the use of a tissue plasminogen activator, which disintegrates the clot and restores blood flow in the brain (1). Plasminogen activators initiate the conversion of plasminogen into the enzyme plasmin. Once plasmin is activated, it starts breaking down the fibrin mesh that keeps blood clots together (4). Once the clot breaks down, blood flow is restored. Tissue plasminogen activator is the only FDA-approved and therefore the most common form of treatment for stroke; however, it must be administered within four to five hours after a stroke’s onset and can only be given to a patient through an intravenous drip (3). This is where the issue arises, because paramedics and EMTs must not only be able to accurately diagnose a stroke in a patient but also be able to successfully administer this drug to relieve the clot – and all within a limited window of time. Innovative Prehospital Stroke Diagnosis Tool There are various ways to confirm a stroke in a patient, but deciding which is the most accurate and efficient is still widely debated. One option that has been proposed for early stroke detection is the use of transcranial ultrasounds on pa-
tients before they arrive at the hospital (1). This is a portable ultrasound that has been utilized to diagnose cardiac conditions and has the potential to accurately detect strokes. This ultrasound device can detect when a clot has lysed, regardless of the presence of the tissue plasminogen activator. Currently, strokes are detected via CT scans, a process that allows tissue visualization based off of their differing densities, after which a tissue plasminogen activator can be administered (5). Tissue plasminogen activator cannot be used for certain types of strokes, such as a hemorrhagic. During a hemorrhagic stroke, a vessel is leaking out blood. In order to counteract it and halt the leakage, a solution with a clotting treatment is used. A plasminogen activator, however, will do the opposite and continue to promote the leakage. Therefore, the CT is crucial in ensuring that a stroke is not an intracranial hemorrhage. However, CTs cannot be performed en route to the hospital as the machines require an immense amount of space, are not mobile, and require the patient to remain still. Transcranial ultrasounds, comparatively, improve catheter placement, trauma assessment, cardiac arrest detection, and more recently, stroke diagnosis. These ultrasound machines can also be used in strenuous situations – for example, in helicopters and moving ambulances. In 2011, a 49-year-old man was treated for a stroke using a transcranial ultrasound in a helicopter during patient transport; this inspired ultrasound-use during transport of other potential stroke patients (1). This case sheds light on the device’s potential – particularly the machine’s mobility and its ability to address the patient’s needs before he or she arrives at the hospital. In addition to improving devices to detect strokes, groups of individuals known as mobile stroke treatment units (MSTU) may be used to deliver treatment to stroke patients before the individual is rushed to the hospital (6). These mobile units are able to reach patients faster (as opposed to having the patient transported to the Emergency Department). Each unit has a vascular neurologist that can administer thrombolysis treatment, which is required to break down the clot. When the effectiveness of these units was tested, 99 out
of 100 patients were treated successfully. However, there were errors in the execution such as video disconnection or delayed imaging times (6). Fortunately, these errors did not affect patient care and did not last longer than sixty seconds. The treatment, including CT and intravenous thrombolysis, was also completed faster than if it would have been in a hospital since the required equipment arrived to the patient at a quicker rate. Thus, MSTU may be a feasible and cost-effective solution for diagnosing and treating strokes. Future Implications Research on early diagnosis in potential stroke patients will allow strokes to be treated quickly and efficiently, making it possible to treat strokes before the onset of deficits and thereby improve patients’ quality of life post-ailment. Furthermore, an earlier and more accurate detection of strokes will allow physicians to utilize different treatments that may not have been implemented in the past due to time constraints. If these pre-hospital treatments are shown to be effective and successful, other studies can be performed to fine-tune stroke diagnoses and ultimately save lives.
References: 1. T. Hölscher, et al., Prehospital stroke diagnosis and treatment in ambulances and helicopters—a concept paper. The American Journal of Emergency Medicine 31, 743-747 (2013). doi: 10.1016/j.ajem.2012.12.030. 2. D. Leifer, Stroke. Encylclopedia of Neuroscience 10, 573-578 (2009). doi:10.1016/B978-008045046-9.00613-6. 3. The University of Texas Health Science Center at Houston. Stroke symptoms, prevention, and clinics. McGovern Medical School Neurology, (2008). 4. H. Lijnen and D. Rijken, T-plasminogen activator. Handbook of Proteolytic Enzymes 3, 2946-2952 (2013). doi.org/10.1016/B978-0-12-382219-2.00646-3. 5. S. Waldman, Computed tomography. Pain Review, 739-761 (2009). doi/10.1016/B978-1-4160-5893-9.X0001-9. 6. A. Itrat, et al., Telemedicine in prehospital stroke evaluation and thrombolysis. Taking stroke treatment to the doorstep. Jama Neurology 73, 162-168 (2016). doi:10.1001/jamaneurol.2015.3849. Images Retrieved From: 1. http://www.strokecenter.org/patients/about-stroke/ischemic-stroke/ 2. https://www.hanscom.af.mil/News/Photos/igphoto/2001708571/
Figure 2 Strokes, also known as traumatic brain injury, are most often caused by motor vehicle accidents and result in cognitive deficits if not treated in time.
Understanding the Probable Causes of Mass EByxtinction E vents Travis Cutter â&#x20AC;&#x2122;22
Figure 1 Researchers determined that prehistoric volcanic activity could be identified by analyzing mercury content in sediment deposits.
Introduction It is nearly impossible to picture almost all life on Earth being wiped out in what is, geologically, a very short amount of time. However, five separate catastrophes nearly eradicated all life throughout Earth’s history (1). Naturally, these events led to extraordinarily significant changes for life on the planet. Recently, two teams of researchers decided to examine the Devonian and Ordovician periods in novel ways. A new method of utilizing the earth’s mercury content at various time periods, which the researchers used to acquire their findings, was created along with the studies. The researchers ultimately uncovered that each event was almost certainly caused by volcanic eruptions, and due to the recent nature of the methods used, these findings represent only the earliest developments in this new era of understanding mass extinction events (2,3). Background Given that the Earth is over 4 billion years old, it is important to organize this voluminous amount of time into manageable sections. The largest divisions are the four eons, which are then divided into eras that are further split into periods. The Triassic, Jurassic, and Cretaceous are all periods, for example (4). The Devonian and Ordovician ended 65 million years ago, while the Ordovician and Devonian periods ended over 440 and 350 million years ago, respectively. The respective ends of each of these periods were marked by mass extinction events that were similarly catastrophic to the Cretaceous event. By analyzing various sediment deposits, researchers suggested that volcanic eruptions caused each of the two extinctions. Since volcanic eruptions were the dominant method for the disbursement of mercury, the presence of mercury in the geological record indicated ancient volcanic events (2). The Late Ordovician Mass Extinction Before 2017, geochronological dating, wherein the history of the earth is timelined, was accomplished by using isotopes of zirconium, aluminum, and strontium, as well as data based on various minerals (3). However, this changed when a team of researchers led by Professor David S. Jones from Amherst College performed a study discussing mercury deposits, paving the way for new insights into mass extinction events. The team studied sediment deposits from volcanic formations in Wangjiawan, China, as well as formations from the Monitor Range in Nevada. By choosing these sites for study, researchers were able to sample from two ancient continents. During the Ordovician period, there were two major continents: Laurentia, which was the location of the Monitor Range site, and Gondwana, which is represented by the Wangjiawan site. As such, the researchers presented a more accurate depiction of what was occurring during the Ordovician period on a global
scale, rather than if they had taken samples from only one of the historical continents (2). The researchers used a mercury analyzer to determine the mercury contents of each sample; another machine called an elemental analyzer was used to find their carbonate contents. Finding the carbon content was especially important since mercury content per carbon measures environmental mercury buildup. For both regions, the team plotted the level of mercury, measured in parts per billion, over time, as well as the mercury content per the percent of organic carbon content over time. This data displays an abnormal spike in mercury content during the Hirnantian age, which was the last age of the Ordovician period. From this, the researchers suggested that the Ordovician extinction event was caused by volcanic activity, and further added that their success in utilizing new methods may spawn more accurate studies for other mass extinction events (2). The Frasnian-Famennian Biotic Crisis A team of researchers from the University of Silesia and the University of Leeds, led by Grzegorz Racki, conducted a study examining the Frasnian-Famennian biotic crisis, which occurred during the Devonian period. This team of researchers studied various sediment samples from Lahmida in Morocco, Kahlleite in Germany and Syv’yu in Russia, all of which were representatives of the Laurussia continent that existed during the Devonian period (3). This team of researchers expanded upon the methods developed by Jones and his team. While Racki also analyzed the mercury and carbon contents over time, he also collected data on aluminum and molybdenum so that the values of mercury and clay minerals could be plotted against the organic carbon content. By plotting the data in a similar way, researchers discovered significant spikes in mercury content towards the end of the Frasnian age and around the beginning of the Famennian age, indicating that volcanism may have been an important factor in the mass extinction event. Volcanic activity had not been considered an active contributor to this event prior to the publication of this study, suggesting the potential for geochronological dating to clarify or change our understanding of Earth’s geographical events. This study also lends further credence to a point emphasized in Jones’s research: the causes of the other extinction events could be linked to volcanic activity through this new mercury dating system, and mass extinction events may need to be reexamined (3). Conclusion It is perhaps surprising that accurate looks at the most ancient of events have been produced so recently. Nevertheless, these geochronological dating methods developed by
David Jones and other researchers in the field have already shown great promise, and it has provided much insight into the Devonian and Ordovician periods and the extinctions that marked their ends. While it is well established that the Cretaceous extinction was caused by an asteroid impact, there are still two other major extinction events to be studied with these new methods. Could the Triassic or Permian mass extinctions have been caused by similar volcanic activity? The causes of those events are not universally agreed upon, and since the new mercury analysis methods have yet to be applied to these events, it is impossible to know. It is, however, wholly possible that modern understanding of lifeâ&#x20AC;&#x2122;s history on Earth will be readjusted to fit these new avenues of study.
References 1. D. Raup and J. Sepkoski, Mass extinctions in the marine fossil record. Science 215, 1501-1503 (1982). 2. D. Jones, et al., A volcanic trigger for the late ordovician mass extinction? Mercury data from South China. Geology 45, 631-634 (2017). doi: 10.1130/G38940.1. 3. G. Racki, et al., Mercury enrichments and the Frasnian-Famennian biotic crisis: a volcanic trigger proved? Geology 46, 543-546 (2018). doi: 10.1130/G40233.1. 4. GSA Geologic Time Scale. The Geological Society of America, (2018). Images Retrieved From: 1. https://www.pexels.com/photo/erupting-lava-during-daytime-73830/ 2. https://commons.wikimedia.org/wiki/File:Geologic_time_scale.jpg
Figure 2 Researchers analyzed the Devonian and Ordovician periods, both of which occurred prior to the emergence and destruction of the dinosaurs in the Triassic and Cretaceous periods, respectively.
Organs on a Chip: The Modern Petri Dish By Rideeta Raquib ’19
Introduction Of all the research dedicated to developing more effective drugs targeting cancer and other ailments, only ten percent have made it through extensive clinical trials. Clinical trials consist of four phases to ensure that the drug is safe in both the laboratory and human beings. Currently, cell cultures and mouse models are used to test drug efficacy and safety. However, these models do not fully highlight the dynamic environment of the human body, such as the pressure from blood flow. Hence, the majority of the drugs approved for clinical trials are ineffective when exposed to the human body. The in vitro models that researchers utilize today consist of Petri dishes or 3-dimensional (3D) models to test various drugs (1). The 3D models are a new advancement, which involves placing primary cells or stem cells into a 3D structure via a scaffold or a unique type of plate. This system enables the cells to establish cell-cell interactions and extracellular matrices, which imitate the cell polarization that is observed in epithelial cell microenvironments. Regardless of this improvement, the 3D model is fairly static and disregards the dynamic environment of various organs, such as the expansion of lungs as a result of breathing, or muscle contractions in the intestinal cells to move food or waste. To combat the conditions of a static environment, a new technology called the ‘Organ-on-a-Chip’, or OoC, has been developed to mimic the fluid and dynamic environments of various organs. Current Design & 3-D Printing Induced Microfluidics The term “microfluidics” refers to technology that focuses on the control and movement of fluids through a microchannel. This is usually established by confining the fluid to a small channel that is only a few millimeters in scale. The small
Figure 1 3D printing is revolutionizing the development of scientific models and microfluidics for research.
size is necessary to fit a group of networks into a chip. This reduces the cost, limits the waste of reagents, and enables the chip to be easily transported. The materials that are widely utilized to create microfluidic channels include polymethylmethacrylate (PMMA), polycarbonate (PC), and polystyrene (PS) (2). An injection molding method is used to insert molten material into a cast or mold, where it hardens and takes up its shape. It is preferred that the materials have low viscosity, so that they can better stick to the mold and produce more intricate structures. These structures may, in turn, be utilized to manufacture complex veins or miniature channels. There are various parameters and limitations to consider when engineering these channels via hot embossing or mold injection methods, including the melting point of the material, thermal expansion coefficient, and glass transition temperature. As previously discussed, the injection molding method is traditionally used to design the microfluidic channels, but the rise of 3D printing provides a more efficient means of manufacturing these channels (2). Various 3D printing techniques have been developed, such as stereolithography and electron beam melting. These techniques employ photocurable resin and photopolymers to construct the channels with high precision with respect to pore size and diameter. Specifically, stereolithography employs lasers that can be used to build diverse structures by coming into contact with liquid polymers, causing them to harden. Comparatively, electron-beam-melting uses the kinetic energy of the electrons from the beams, and converts this energy into heat. The heat generated causes the polymers to melt and ultimately fuse with one another. A difficult organ to model is multilayered skin, which is comprised of multiple dermis layers. This organ is especially difficult to model because the skin contains hair follicles and sweat glands that are not accounted for in cell cultures. This is where 3D printing can be used to create these layers according to the desired or specified thickness and patterns (3). Utilizing a microvalve mediated droplet printing method, Lee and her research team at Rensselaer Polytechnic Institute in New York developed a skin-on-a-chip model with two layers of keratinocytes, double stacks of dermal matrix layers, and fibroblasts layers (4). The microvalve mediated droplet printing device that was employed in the study consists of robotic arms or dispensers that can precisely place cells as droplets. This tool enables the researchers to place the cells with deliberate accuracy in order to imitate the diverse cell layers. In traditional seeding methods, which involves layering the cells on the plate, model shrinkage is a common problem. However, no shrinkage was observed in the printed model two weeks after formation. The engineering of the multilayered body structures on a chip provides a better representation of natural organs and circumvents animal testing, making the process more humane.
What Has Been Developed? One of the most complex organs in the human body is the brain. The brain is the executive organ that sends signals and enables humans to carry out vital functions involving both voluntary and involuntary movement. In addition, our behavioral and cognitive processes are mediated by the central nervous system. Humans have a very complex way of thinking, and animal models in research do not fully replicate the reactions of testable drugs on the human brain. A device that compensates for the human brain will enable scientists to gain a better understanding of neurodegenerative disease progression. Dr. Onur Kilic and his team at Johns Hopkins University created this device by utilizing pluripotent human cells, or embryonic stem cells that are able to generate any type of
Figure 2 The developmental processes affecting the human brain are being modeled and studied using Organ-on-a-Chip technology.
adult cell, to recreate the central nervous system and include a blood-brain barrier on a chip (5). Stem cells aid development by growing or proliferating. The expression or repression of certain genes enable these cells to go into a differentiated pathway or to become a specific type of cell. The study that utilized this device was used to observe the interaction between fetal progenitor, or stem cells, and differentiated cells. In nature, differentiation generally occurs during embryogenesis, or during the development of the embryo; however, in a laboratory setting, the differentiation is facilitated by inducing the expression or repression of genes necessary to possess the characteristics of a specific type of cell. Migration of neural progenitor cells in the tissue environment is a phenomenon that is not well understood; thus, the purpose of this study was to understand the underlying mechanism of such migrations to predict the occurrence of neural diseases, such as brain cancer. The manufactured multi-layer silicone device consisted of progenitor cells that were grown and differentiated over four weeks to neuronal clusters and axon bundles. In addition, an interface of microvascular endothelial cells was incorporated to mimic the blood-brain barrier. The model was used to observe the migration of progenitor cells in response to chemotactic cues, which are attractants or signals that cause cells to migrate. The model showed greater migration due to a chemotactic gradient of CXCL12, a chemokine, which is a type of cytokine or signaling molecule. This
signal is present usually during embryonic development, or in pathological regions. This chip is a convenient model of neural differentiation and cell migration; hence it can be applied for various studies to better understand neural behavior. Another study conducted by Dr. JiSoo Park and his research team at Korea University imitated in vitro Alzheimer’s disease on a chip or small model, where neurospheroids, neuron-like cells derived from pluripotent stem cells, were grown (6). The media flow was generated and altered to enable neural differentiation and produce a dynamic environment in the body. The detrimental implications of amyloid-β, which are misguided peptides involved in Alzheimer’s disease, and the effects of treatment with amyloid-β on neurospheroids were tested in the presence and absence of media flow. The study revealed that the media flow corresponded with better neurospheroid growth and was a more accurate representation of the actual physiological conditions of the human brain. The administration of amyloid-β in the dynamic environment showed lower neurospheroid viability or low growth when compared to static conditions; this illustrated that the 3D model on the microfluidic chip was a better representation of neurodegenerative diseases. From this, it is thought that these OoC devices will help to produce more accurate predictions when future drugs and therapies are tested. Another example of an organ on a chip is the lung-ona-chip, developed by Dr. Stucki and her team at the University of Berne, Switzerland. The lungs are constantly exposed to various volatile compounds and aerosols in the air. The effects of these compounds are often difficult to observe in mouse models or Petri dishes, as mouse lungs are too small and cell cultures can get contaminated. In addition, blood and oxygen flow is not modeled in current 3D systems. The OoC lung model is designed to mimic the structure and function of alveolar and capillary interfaces. There are two microchannels lined adjacent to each other and are divided by a porous membrane that contains fibronectin and collagen (2). The human epithelial cells from the alveoli are seeded on the top of the membrane, which imitates the air chamber, whereas the bottom side of the membrane contains pulmonary microvascular endothelial cells (7). In addition to the interface between alveoli and capillaries, the team also created the muscular rhythms facilitated by the diaphragm. This was done by connecting the
Figure 3 Unlike Organ-on-a-Chip technology, cell cultures can get contaminated, making it difficult to study physiological processes.
membrane with the cell monolayer to an electro-pneumatic setup, which is a tool that uses electric current to manipulate air pressure. The model represented the stretching of the lung wall during respiration due to movement of the diaphragm by establishing the changes in air pressure (7). The study further analyzed the effects of mechanical lung movement on alveolar epithelial barrier permeability, which is exposed to the air we breathe. Small hydrophilic molecules, like sodium with fluorescent markers, were used to trace the movement and entry of these molecules. There was a greater permeability of the hydrophilic molecules in the dynamic model versus the static model, which the researchers hypothesize was due to stretching of the cells and the junctions between. The researchers believe that this lung-on-a-chip model is useful in understanding toxicology and how harmful substances can embed into the epithelial barrier from the environment. Conclusions and Future Directions The human organ-on-a-chip technology opens a new way to test drug effectiveness and enhances artificial tissue construction, thereby moving the pharmaceutical and tissue engineering field forward. The microfluidic channels provide a way for scientists to precisely control fluid, or media, movement in order to create the dynamic environment of the human body and versatile organs, therefore advancing from the current in vitro and in vivo models. This technology can also be applied to cancer research to test metastasis, tumor growth, and chemotherapy drug efficacy. The next step is to connect different organs on chips and model the entire human body (2). In order to achieve this goal, it is necessary to have
diverse types of cells generated from stem cells, and the cells should be maintained for an extended period of time. Cancer cells can also be used in place of regular cells to understand the progress or metastasis of tumors, as well as the mechanism of angiogenesis. This research is currently ongoing, as the Wyss Institute at Harvard University is working to revolutionize this integration of bioengineering and medical research. This may be a turning point in the medical field resulting in the elimination of animal models, a more efficient pre-clinical trial process, and more accurate and ethical models of the human organs and body are provided for future research.
References 1. X. Hui, et al., A dynamic in vivo-like organotypic blood-brain barrier model to probe metastatic brain tumors. Scientific Reports 6, (2016). doi: 10.1038/ srep36670. 2. J. Hernandez, et al., Organs-on-a-chip module: a review from the development and applications perspective. Micromachines 9, (2018). doi: 10.3390/mi9100536. 3. Y. Hee-Gyeong, L. Hyungseok, and C. Dong-woo, 3D printing of organs-onchips. Bioengineering 10, (2017). doi: 10.3390/bioengineering4010010. 4. V. Lee, et al., Design and fabrication of human skin by three-dimensional bioprinting. Tissue Eng. Part C, Methods 20, 473-484 (2013). doi: 10.1089/ten. tec.2013.0335. 5. O. Kilic, et al., Brain-on-a-chip model enables analysis of human neuronal differentiation and chemotaxis. Lab Chip 16, 4152-4162 (2016). doi: 10.1039/ c6lc00946h. 6. J. Park, et al., Three-dimensional brain-on-a-chip with an interstitial level of flow and its application as an in vitro model of Alzheimerâ&#x20AC;&#x2122;s disease. Lab Chip 15, 141-150 (2015). doi: 10.1039/c4lc00962b. 7. A.O Stucki, et al., A lung-on-a-chip array with an integrated bio-inspired respiration mechanism. Lab Chip 15, 1302-1310 (2015). doi: 10.1039/c4lc01252f. Images retrieved from: 1. https://commons.wikimedia.org/wiki/File:Felix_3D_Printer_-_Printing_ Head_Cropped.JPG 2. https://commons.wikimedia.org/wiki/File:Human_brain_01.jpg 3. https://upload.wikimedia.org/wikipedia/commons/5/56/Agarplate_redbloodcells_edit.jpg 4. https://upload.wikimedia.org/wikipedia/commons/7/74/Lung-on-a-chipwyss.jpg
Figure 4 By combining different Organ-on-a-Chip models, the human body system can be studied in an accurate, in vitro way.
New Approaches to Treating Chorea and Huntington’s Disease By Sanket Desai ’19
Introduction & Background Huntington’s Disease (HD) is a neurological disorder that leads to the degradation of neurons and loss of function in parts of the brain. This disease afflicts 30,000 people in the United States and has no cure. However, in recent years, scientists have strived to create treatments that could alleviate the effects of HD. Many of these treatments manage involuntary movements, one of the most common symptoms of HD. HD is derived from a trinucleotide repeat of the CAG (cytosine-adenine-guanine) sequence in the IT15 gene (1). This gene is transcribed and translated into the huntingtin (htt) protein, of which its mutated form has many glutamate amino acids near the N-terminus, giving the protein abnormal function. Htt is involved in many pathways necessary for cellular function such as protein folding, transcription regulation, and transport of molecules within a cell. Therefore, abnormal function of the protein would adversely affect these pathways and possibly cause cell death (1). The effects of this mutation cause damage within the basal ganglia of the brain (2). Other parts of the brain are affected as well but to a lesser extent. The basal ganglia are connected to the motor cortex, the part of the brain that controls movement, through two pathways: the direct pathway and the
indirect pathway. HD affects these two pathways by causing neuronal death. When the direct pathway loses stimulatory neurons in the basal ganglia, less signaling occurs in the motor cortex. However, when the indirect pathway loses inhibitory neurons in the basal ganglia, pathways involved in movement are overstimulated. The destruction of both pathways by HD leads to chorea, or sudden, involuntary movements. Chorea is the target of many drugs and other treatments for HD (2). Current Treatments for Chorea Tetrabenzaline Tetrabenzaline (TBZ) is the first drug approved by the FDA for treatment of chorea (3). This drug binds to a receptor called central vesicular monoamine transporter type 2 (3). This transporter is responsible for the movement of dopamine, a neurotransmitter in the basal ganglia of the brain that helps control movement (4, 5). TBZ works by inhibiting this receptor, thereby reducing the amount of released dopamine (5). Dopamine release is targeted with this treatment because dopamine levels are elevated in early stages of HD and contribute to chorea (6). TBZ is effective because its main binding targets are parts of basal ganglia. However, this binding is reversible and thus, this drug does not have long-term effects (3).
Figure 1 Huntington’s Disease is caused by mutation in the htt protein.
Dopamine Antagonists Other classes of drugs such as dopamine antagonists, benzodiazepines, and glutamate antagonists can directly affect HD chorea (3). Antagonists are molecules that bind to receptors to prevent the neurotransmitter from binding and initiating a signal. Dopamine antagonists are antipsychotic drugs that are used often to treat chorea in HD patients. There
Figure 2 HD affects a series of basal ganglia circuits.
are two types of these antipsychotics: typical and atypical (3). Typical antipsychotics work by blocking the D2-receptor involved in dopamine signaling (7). Atypical antipsychotics work by blocking the 5-Ht2A receptor involved in serotonin and dopamine signaling (7). Typical antipsychotics have been shown to be ineffective against chorea in trials (3). An exception is seen in a clinical study by AN Barr at the University of Illinois regarding the effects of haloperidol, a type of typical antipsychotics. This study showed that patients who took haloperidol saw a 30% decrease in chorea (8). Atypical antipsychotics have been somewhat more successful, as more patients have seen a greater reduction on chorea than in trials with typical antipsychotics (3). Drugs of this category that were successful in treating chorea include olanzapine, risperidone, and quetiapine. Despite this success, they still have significant side effects (10). Benzodiazepines Anxiety is linked with the worsening of chorea, and benzodiazepines are mainly used as anxiety-reducing (anxiolytic) medication (9, 10). They work by binding to the GABA neurotransmitters in the brain (11). Binding to these neurotransmitters keeps these channels open, resulting in an influx of chlorine anions and an inhibitory signal. This results in less
brain activity, which reduces anxiety in the patient (10). Some benzodiazepines that have been proven to be effective against HD are short-acting drugs like Lorzepam and Alprazolam, and long-lasting drugs like Clonazepam and Diazepam (long-lasting) (12). The effects of long- acting drugs last for days are preferred to short-term ones that last minutes to hours (9). However, using these drugs for a long period of time can result in addiction, and if benzodiazepine intake is suddenly stopped, withdrawal syndrome can result (12). Glutamate Antagonists Glutamate antagonists and blockers are those that prevent glutamate release and binding to its target. These include amantadine, riluzole, remacemide, and ketamine (13). Amantadine is an anti-viral and anti-parkinsonian drug that can be used to treat HD chorea (14). Its mechanism of action is yet to be determined, but it is theorized that it inhibits the N-methyl-D-aspartate (NMDA) receptor, preventing glutamate from acting on an adjacent cell. The drugâ&#x20AC;&#x2122;s administration reduces chorea in patients at a dose of 400 mg/day or higher (3). However, there are significant side effects such as irritability, as seen in a study by JT Stewart at the Gainseville Veterans Administration Medical Center (15). Rizuoleâ&#x20AC;&#x2122;s mechanism of action is not exactly known, but it blocks glutamate release on the striatum and decreases activity of glutamate receptors (3). Additionally, it can modulate activity of voltage-gated sodium channels to prevent signaling (3). Riluzole causes a major reduction of chorea at 200 mg/day, but at lesser doses it does not have much of an effect. (16, 17). Remacemide is a drug that can be used to treat Parkinsonâ&#x20AC;&#x2122;s Disease, HD, and epilepsy (18). It also acts as an NMDA antagonist and, in its clinical trials, there was a reduction of chorea with limited side effects (19, 20). Finally, ketamine is used for anesthesia, as it has hypnotic and sedative effects (21). It works by blocking not only the NMDA receptor but also the HCN1 receptor and other
Figure 3 Tetrabenazine is a drug normally used for the treatment of hyperkinetic movement disorders.
neurotransmitter systems (21). Clinical trials by Dr. Mestre at the Hospital de Santa Maria have shown that it is not effective in reducing chorea (22). Future Treatments for Chorea Two novel drugs, OSU6162 and pridopidine, are being developed to treat chorea in HD. They work to modulate the dopamine levels in the brain (23). Researcher JP Rung at the University of Guthenburg experimented with these two drugs to determine which receptors in the brain are affected. (24). It
was found that the drugs’ actions target increased dopamine levels in the striatum in the early stages of HD. Additionally, they target both dopamine and serotonin receptors and will inhibit or stimulate them. Pridopidine’s effectiveness is currently being tested in clinical trials (25). Researchers found that these drugs cannot necessarily cure chorea but they are effective in modulating dopamine levels at different stages of HD. Since HD has high levels of dopamine in early-onset and low levels in the late stages, these drugs decrease dopamine levels in the beginning and increase them in the later stages (23).
ditionally, there are new drugs being developed that bind the same receptors mentions but they are more effective than the currently used drugs because they can detect neurotransmitter levels or have enhanced properties through different chemical structures. Finally, there are potential advancements in surgery to help treat those with chorea. Although there is no cure for HD chorea, these current treatments have been shown to be effective against this disease.
Deutetrabenazine Additionally, a novel drug called deutetrabenazine received FDA approval in 2017. This drug is a form of tetrabenazine that has deuterium atoms instead of hydrogen atoms at certain parts of the molecule (26). Two studies were conducted in order to compare the effects of tetrabenazine versus deutetrabenazine in reducing chorea. Researchers found that deutetrabenazine is more efficacious at lower daily doses, and has longer-lasting effects.
1. C. Landles and G.P. Bates, Huntingtin and the molecular pathogenesis of Huntington’s disease. Fourth in molecular medicine review series. EMBO Reports 5, 958-63 (2004). doi: 10.1038/sj.embor.7400250. 2. S. Liou, The basic neurobiology of huntington’s disease (text and audio). Huntington’s Outreach Project for Education At Stanford (2010). 3. S. Frank, Treatment of Huntington’s disease. Neurotherapeutics:The Journal of the American Society for Experimental NeuroTherapeutics 11,153-60 (2013). doi: 10.1007/s13311-013-0244-z. 4. J.L. Lanciego, N. Luquin, and J.A. Obeso, Functional neuroanatomy of the basal ganglia. Cold Spring Harbor Perspectives in Medicine 2, (2012). doi: 10.1101/ cshperspect.a009621. 5. G. Zheng, L.P. Dwoskin, and P.A. Crooks, Vesicular monoamine transporter 2: role as a novel target for drug development. The American Association of Pharmaceutical Scientists, (2006). doi: 10.1208/aapsj080478. 6. C. Cepeda, et al., The role of dopamine in Huntington’s disease. Progress in brain research 211, 235-54 (2014). doi:10.1016/B978-0-444-63425-2.00010-6. 7. Typical and atypical antipsychotic agents. GoodTherapy. 8. A.N. Barr, et al., Serum haloperidol concentration and choreiform movements in Huntington’s disease. Neurology 38, 84–88 (1988). doi: 10.1212/ WNL.38.1.84. 9. S.N. Tyagi, et al., Symptomatic treatment and management of Huntington’s disease: An overview. Global Journal of Pharmacology 4, 6-12 (2010). 10. C.E. Griffin, et al., Benzodiazepine pharmacology and central nervous system-mediated effects. The Ochsner Journal 13, 214-23 (2013). 11. A. Ogbru, Benzodiazepines. RxList. 12. Benzodiazepines. Huntington’s Disease News. 13. V.M. André, C. Cepeda, and M.S. Levine, Dopamine and glutamate in Huntington’s disease: a balancing act. CNS neuroscience & therapeutics 16,163-78 (2010). doi: 10.1111/j.1755-5949.2010.00134. 14. C. Chang & K. Ramphul, Amantadine. StatPearls [Internet]. Treasure Island (FL), 2018. 15. J. Stewart, Adverse behavioral effects of amantadine therapy in Huntington’s disease. Southern Medical Journal 80, (1987). doi: 10.1097/00007611198710000-00032. 16. Huntington Study Group,Dosage effects of riluzole in Huntington’s disease: a multicenter placebo-controlled study. Neurology 61, 1551–1556 (2003). 17. G.B. Landwehrmeyer, et al., Riluzole in Huntington’s disease: a 3-year, randomized controlled study. Annals of Neurology 62, 262–272 (2007). doi: 10.1002/ana.21181. 18. M.G. Cersósimo and F.E. Micheli, Antiglutamatergic drugs in the treatment of Parkinson’s disease. Handbook of Clinical Neurology 84, 127-136 (2007). doi: 10.1016/S0072-9752(07)84036-X. 19. Huntington Study Group, A randomized, placebo-controlled trial of coenzyme Q10 and remacemide in Huntington’s disease. Neurology 57, 397–404 (2001). 20. K Kieburtz, et al., A controlled trial of remacemide hydrochloride in Huntington’s disease. Movement Disorders 11, 273–277 (1996). doi: 10.1002/ mds.870110310. 21. J. Sleigh, M. Harvey, L. Voss, and B. Denny, Ketamine – more mechanisms of action than just NMDA blockade. Trends in Anaesthesia and Critical Care 4, 2-3 (2014). 76-81. doi: 10.1016/j.tacc.2014.03.002. 22. T Mestre, et al., Therapeutic interventions for symptomatic treatment in Huntington’s disease. Cochrane Database of Systematic Reviews 3, (2009) doi: 10.1002/14651858.CD006456.pub2. 23. S.L. Mason and R.A. Barker. Novel targets for Huntington’s disease: future prospects. Degenerative Neurological and Neuromuscular Disease 6, 25-36 (2016). doi:10.2147/DNND.S83808. 24. J.P. Rung, et al., Effects of (−)-OSU6162 and ACR16 on motor activity in rats, indicating a unique mechanism of dopaminergic stabilization. Journal of Neural Transmission (Vienna) 115, 899–908 (2008). doi: 10.1007/s00702-008-0038-3. 25. What are the phases of clinical trials? American Cancer Society,(2017). 26. J. Radke, FDA approves drug for Huntington’s Disease. Rare Disease Report,(2017). 27. Deep brain stimulation for movement disorders. Neurological Surgery. 28. V. Gonzalez, et al., Deep brain stimulation for Huntington’s disease: long-term results of a prospective open-label study. Journal of Neurosurgery 121, 114–122 (2014). doi: 10.3171/2014.2.JNS131722.
Future Surgical Treatments Treatments for HD and more specifically chorea may also come in the form of surgical treatments such as Deep Brain Stimulation (DBS) (23). DBS features the insertion of an electrode in the brain that sends a of pulse of signals to over-
Figure 4 Deep brain stimulation of the nucleus basalis of Meynert is a current surgical treatment offered for HD.
shadow the abnormal signals (27). This treatment is already being used for other movement disorders such as Parkinson’s (27). A study by V. Gonzalez at the University of Montpellier showed that when the internal pallidus of the brain was stimulated, there was a decrease in chorea (28). However, this treatment may worsen other symptoms associated with HD such as heart conditions. Conclusion HD is a debilitating disease that primarily affects the brain and causes numerous ailments such as chorea. There are numerous drugs that have a diverse set of mechanisms to treat chorea by binding to the dopamine, glutamate, or GABA receptors. Benzodiazepines, glutamate antagonists, and dopamine antagonists have been shown to be efficacious in some drugs and trials, but not all drugs such as typical antipsychotics. Ad-
Images retrieved from: 1. https://commons.wikimedia.org/wiki/File:Huntington%27s_disease_ (5880985560).jpg 2. https://upload.wikimedia.org/wikipedia/commons/9/9e/Basal_ganglia_circuits.svg 3. https://commons.wikimedia.org/wiki/File:Tetrabenazine_structure.svg 4. https://en.wikipedia.org/wiki/Deep_brain_stimulation#/media/File:Deep_ Brain_St
Figure 1 A representation of a depressed person.
The Role of Pain in Mental Illness By Sultan Kaur ’21
Introduction Pain, defined by The International Association for the Study of Pain as “an unpleasant sensory or emotional experience associated with actual or potential tissue damage,” alternates between emotional and physical forms (1). Mental illnesses, such as depression and anxiety, and detrimental behaviors, such as substance abuse and smoking, have previously been linked to physical pain. Physical pain can worsen mental illness, which can, in turn, exacerbate physical pain, creating a vicious cycle. Many studies label this cycle as a bidirectional relationship with both aspects simultaneously affecting each other. The physical pain associated with mental illness can exist in the form of migraine headaches or musculoskeletal pain, both of which coincide with the symptoms of anxiety and depression. The fear-avoidance model explains where this pain originates, and why it may occur as strongly as it does in certain patients. This model relies on the psychology of an individual who transitions from acute, or mild pain to chronic, or long-term pain. A hyperbolic fear of the pain’s potential is the key factor in this model. In other words, fear drives isolation and lethargy that may potentially help individuals circumvent pain in the future if they encounter the sensation in the future. This model works to combat “pain catastrophizing,” or the human response of expecting only the worst outcome, which can be detrimental to patients who already have symptoms of depression and anxiety (2). Connection Between Pain and Emotion There exists a strong association between physical and chronic pain and severe emotional sadness. These two factors, in addition to low motivation, are key manifestations of depression. Previous studies found that someone with major depression is three times more likely to suffer from migraines than someone who is not depressed (3). Moreover, someone with a migraine is five times more likely to exhibit a depressive episode. In a population study of 845 adults conducted by the Mayo Clinic College of Medicine, Dr. Hooten found that participants with back or neck pain were twice as likely to have a depressive episode than participants without spinal pain. The study also found that individuals with severe depression were four times more likely to develop spinal pain than individuals who were only minorly depressed. This bidirectional relationship is hypothesized to link emotional processing functions in the brain to pain stimuli processing. More specifically, it states that the the pain-processing region in depressed individuals may line up with their emotional processing (2). There also seems to exist an extensive relationship between physical pain and anxiety. Like depression, anxiety has been shown to double the incidence of migraines, and individuals with migraines are two to three times more likely to develop panic disorders, or post-traumatic stress disorder
when compared to individuals without regular migraine episodes (2). In one study, researchers injected high-anxiety Wistar Kyoto rats with monosodium acetate, a pain-inducing model used to mimic osteoarthritis pain, and monitored them for anxiety symptoms. There was greater pain and higher anxiety in injected rats when compared to the normally anxious Sprague-Dawley rats, which were injected with the same monosodium acetate. This data was cross-referenced with a population study, which found that high anxiety levels over the course of a year correlates with frequent knee pain (4). Another study by the International Association for the Study of Pain showed that for patients who reported minimal pain, anxiety increased less rapidly, growing slowly over the course of three years (1). Anxiety and depression are also connected to certain detrimental behaviors such as substance abuse and smoking. Substance abuse in the form of opioids and cannabis can be instigated by chronic pain. Through various clinical observations in the International Study of Pain, Hooten found that patients with chronic pain are two to three times more likely to exhibit substance abuse than patients without chronic pain. In addition, patients with a history of substance abuse tend to experience chronic pain. Although smoking is generally on the decline, smokers tend to report chronic and severe pain, and depression can be a factor affecting its severity. A proposed explanation is that smoking activates the nicotinic acetylcholine receptors in the central nervous system for morphine, which releases pleasure stimuli (2). Smokers become addicted to the morphine so they are eventually forced to smoke more to achieve the same level of pleasure stimuli, which dampens the stimuli of normal life and deepens their depression and pain. In some cases, addiction, pain-related disorders, emotional pain, and mental health problems can culminate in suicidal thoughts. Suicide is a frequently occurring symptom of patients suffering from chronic pain (2). Methods of Treatment There are various ways to alleviate symptoms of pain and mental illness. Hooten suggests that cognitive behavioral therapy, acceptance, and commitment therapy are helpful. CBT uses a variety of techniques and mental exercises, particularly ones that help control undesirable behaviors. Commitment therapy is based on meditation and mindfulness strategies of accepting the pain that exists, or coming to terms with its existence and learning to cope and move forward from it (2). Exercise is another strategy to dampen depressive symptoms. In Doyne’s study conducted at the University of Rochester, 40 women with depression were chosen through a mass media format followed a fitness program to understand if aerobic, running, and non-aerobic exercise such as weight lifting can help significantly improve depressive symptoms.
All 40 women were between the ages of 18 and 35, and diagnosed to have a form of depression using the Research Diagnostic Criteria. There was no significant difference found between aerobic and non-aerobic exercise, though exercise did decrease overall symptoms of depression (5). There are various existing hypotheses regarding why exercise is so effective in lowering the symptoms from distraction to endorphins, but there is insufficient data to support any of them. However, because exercise has been shown to effectively improve depressive symptoms, it may be used to decrease pain, since the two appear to be linked. Some of the medicinal treatments prescribed for both pain and mental illnesses are identical. For example, serotonin-norepinephrine reuptake inhibitors, which prevent the reuptake of norepinephrine and serotonin, are activated in descending inhibitory pathways. Thus, they relieve pain and stimulate the body by enhancing excitatory neural processes. The inhibitor itself is effective in treating neck and back pain as well as depression symptoms (2). Additionally, the medication behaves as a partial sedative with fewer side effects than many other antidepressant medications (3). Conclusion There is a bidirectional relationship between pain, mental health disorders like depression and anxiety, and risky behaviors such as substance abuse. Current treatments available for pain-related symptoms include behavioral therapy, exercise, and medication. Future research on mental disorders will increase efficacy of treatment options and bring us closer to liberating the patients from emotional and physical pain.
References 1. E. Heer, et al., Pain as a risk factor for common mental disorders. Results from the Netherlands mental health survey and incidence study-2: a longitudinal, population-based study. Pain 159, 712-718 (2018). doi: 10.1097/j. pain.0000000000001133. 2. M. Hooten, Chronic pain and mental health disorders. Mayo Clinic Proceedings 91, 955 â&#x20AC;&#x201C; 970 (2016). doi: https://doi.org/10.1016/j.mayocp.2016.04.029. 3. Depression and pain. Harvard Medical School, (2009). 4. L. Craft and F. Perna, The benefits of exercise for the clinically depressed. Primary care Companion to the Journal of Clinical Psychiatry 6, 104-111(2004). 5. J. Burston, et al., The impact of anxiety on chronic musculoskeletal pain and the role of astrocyte activation. Pain 160, 658 -669 (2019). doi: 10.1097/j. pain.0000000000001445. Image Retrieved From: 1. https://www.nbcnews.com/better/health/can-your-instagram-photos-revealyou-re-depressed-ncna794041
Expanding the Cyanobacterial Synthetic Biology Promoter Toolbox Priya Aggarwal ’21, Natalie Lo ’21, Karthik Ledalla ’21, Stephanie Budhan ’21, J. Peter Gergen, Ph.D.
ABSTRACT Though the genetic engineering of photosynthetic organisms could be used as a solution for the massive amounts of carbon dioxide that is being released into the atmosphere, their full potential is yet untapped. This is mostly due to a lack of prior research, slow growth rates, and a complicated transformation procedure. Specifically, the promoters which regulate the transcription of DNA are poorly understood in cyanobacteria. This study aimed to characterize four promoters that are native to the strain, Synechococcus elongatus PCC 7942 — two strong constitutive promoters (Pcpc and Pcpc560), a ferrous-ion repressible promoter (PidiA), and a high-light inducible promoter (PpsbA2) using bacterial transformation and a luminescence marker. Our research successfully characterized the expression of Pcpc and Pcpc560 and laid the groundwork for future research into inducible systems.
Introduction Synthetic biology, the intersection of biology and engineering, involves genetically engineering microbiological organisms to to produce products that can be used to resolve issues concerning medicine, energy, and the environment (1,2,3). Inserting the human insulin gene into Escherichia coli, for example, facilitated the mass production of the insulin protein (1). Most current research in synthetic biology uses bacteria and yeast (Saccharomyces cerevisiae) as host organisms or chassis. These are the most commonly used chassis because their biological systems are well understood, they have fast growth rates, and have the ability to efficiently take up DNA (4). One such chassis is cyanobacteria, or photosynthetic bacteria. These bacteria were responsible for the “Great Oxygenation Event” nearly 2.4 billion years ago, when the carbon dioxide (CO2) content of the Earth’s atmosphere decreased dramatically and oxygen was introduced into the atmosphere (5). In 2017 alone, humans were responsible for the release of about 32.5 gigatons of CO2 in the atmosphere (6). As cyanobacteria have a precedent for causing mass decreases in atmospheric carbon dioxide levels, many studies have identified them as a potential solution to remove CO2 from the atmosphere and make Earth carbon-neutral or even a carbon-sink (2). Working with cyanobacteria can be difficult due to its relatively slow growth rates and complicated transformation procedure associated with this chassis, as well as the lack of prior research of its native systems (7). Promoters, which regulate the transcription of expression of genes in bacteria are poorly understood in cyanobacteria. For example, most studies with cyanobacteria that involve the introduction of promoters that are native to other bacteria result in issues such as poor systematic control (7). In our work, we sought to characterize promoters that are native to cyanobacteria Synechococcus elongatus (S. elongatus) PCC 7942. These promoters include two strong constitutive (or constantly-expressed) promoters (Pcpc and Pcpc560) and two inducible/repressible promoters (PpsbA2 and PidiA). Pcpc, PpsbA2, and PidiA are all native to S. elongatus PCC 7942, whereas Pcpc560 is from a closely related species of cyanobacteria Synechocystis sp. 6803 (8,9,10). The psbA2 and idiA promoters are especially powerful. The former is high-light inducible, which is especially potent in photosynthetic systems, and the latter is ferrous-ion repressible, which allows the promoter to be expressed only under nutrient-deficient conditions (9,10). Nonetheless, there is little quantifiable data that assesses the relative expression of these four promoters under certain conditions. By using a luciferase enzyme which produces light upon reacting with its substrate, the activity of these promoters can be tracked by measuring luminescence (9).
Methods Promoter DNA, or constructs, were designed using public genome data for Synechococcus elongatus and Synechocystis sp. 6803. Using Snap-
Figure 1 Cyanobacterial chassis, such as Synechococcus elongatus PCC 7942, have photosynthetic cabilities, but their native genetic systems are poorly understood.
Gene software, BioBrick prefix and suffix restriction enzyme sequences, 2030 basepair PCR overlap regions were affixed to the beginning and end of the sequence. To quantify the relative expression of the promoters, promoterless DNA coding for a luciferase enzyme, which luminesces in the presence of its substrate, decanal, was utilized. The four promoters were cloned into this promoterless DNA, also known as the promoterless luxAB vector pAM1414 (Addgene plasmid #40237). To amplify the promoter DNA, polymerase chain reactions (PCR) were employed. This PCR DNA was purified using the Monarch ® PCR and DNA Cleanup Kit. The PCR DNA was inserted into the luxAB vector pAM1414 using HiFi DNA Assembly, which utilizes PCR overlap to efficiently combine separate DNA molecules into a recombinant plasmid. The recombinant plasmid was subsequently used to transform DH5-alpha using standard transformation protocol. Bacteria were treated with standard concentrations of spectinomycin and grown in LB media at 37°C and 200
rpm. The PureYield™ Plasmid Miniprep System was then used to isolate the recombinant plasmid DNA. Successful transformation of the bacteria by the recombinant plasmid was confirmed by digesting the mini-prepped products with restriction enzymes and running the digest on a gel. Furthermore, the recombinant plasmid DNA were sent to the Stony Brook Genomics Core Facility for sequencing in order to further confirm successful transformation. After we confirmed the efficiency of the transformation, the recombinant plasmid DNA was used to transform cyanobacteria through double homologous recombination such that the construct of interest would be incorporated in the cyanobacterial genome. The cyanobacteria used were stock cultures of UTEX 2434 Synechococcus leopoliensis (also known as S. elongatus PCC 7942) that were donated by the UTEX Culture Collection of Algae and grown at 30°C and 5% CO2 using standard BG-11 media in 24 hour light conditions. The cyanobacteria were then transformed with plates of antibacterial concentrations (10 µg/ mL) of streptomycin and spectinomycin using standard cyanobacterial transformation protocols (9). Liquid cultures of transformed cyanobacteria were grown in a CO2 incubator at 33°C, 5% CO2, 90% humidity, and 120 µE/ m2/s of light. BG-11 media was modified to include 10 µg/mL of both spectinomycin and streptomycin, as well as 10 mM sodium bicarbonate. In order to test the expression of the four promoters, luciferase experiments were conducted following standard procedure (11). Decanal was added to cyanobacteria transformed with cpc, cpc560, psbA2, and idiA promoters. Luminescence was measured with a plate reader. This was performed during both the day and night to account for the influence of circadian rhythm of cyanobacteria. For the high-light experiment, after the addition of decanal which is the luciferase substrate, cyanobacteria were placed under 500 µE of high light for an hour to induce expression. To test the cyanobacteria transformed with the iron-repressible promoter (idiA), cyanobacteria were resuspended in BG-11 with 2.3 mM of 2,2-dipyridyl (iron chelating agent) donated by Dr. Jarrod French to induce expression; the luminescence was also measured with a plate reader after an hour. All assays were performed using three or more samples of the same batch. For statistical analyses, outliers were removed from the data set using the 1.5(IQR) rule and unpaired T-tests assuming unequal variances were performed using an alpha of 0.05. Results Gel electrophoresis and sequencing at each stage of the project indicated that we had a successful transformation for our plasmids, constructs, E. coli and cyanobacteria. The first luciferase experiment, conducted during the day, compared the expression of luciferase (measured by luminescence) in the two constitutive promoters (cpc and cpc560) and the non-transformed wild type (WT) (Figure 1). Both cpc and cpc-560 showed significant differences for daytime expression compared to wild type. Compared to cpc, cpc560 had a significantly higher expression (Figure 1). However, since cyanobacteria are photosynthetic, they are dependent on a circadian rhythm (11). Furthermore, cpc and cpc560 are promoters for phycocyanin, and are somewhat light-inducible as well (8). Therefore, though the strain was grown in 24-hour light conditions, differences in night and day expression of luciferase were checked using luminescence. Though cpc did not show any significant difference, cpc560 showed a significant difference between daytime and nighttime expression (Figure 2). After collecting baseline data regarding the constitutive promoters, we examined the standard expression of the high-light inducible psbA2 promoter and ferrous-ion repressible idiA promoter. Again, there was a significant difference in the expression of luciferase in cpc and cpc560 as opposed to WT (Figure 3). However, there was no significant difference in the expression of luciferase between WT, psbA2, and idiA. To test the inducible and repressible promoters, we exposed the
transformed cyanobacteria to high-intensity light and iron chelators. After exposing the promoters to 500 μE of light, the luciferase expression for cpc did not significantly increase. Interestingly, the luciferase expression of
Figure 2 (a) Standard expression of luciferase, measured by bioluminescence, in the daytime for WT and cpc and cpc-560 transformants. (b) Differences in the standard expression of luciferase in the day versus the night. (c) Standard expression of luciferase in the daytime for all transformants. Asterisks denote significant differences for all figures.
cpc560 increased, whereas luciferase coupled to psbA2 did not show any expression (Figure 4). After exposing the promoters to iron chelator, the luciferase expression for cpc did not change significantly. The expressions of cpc560, idiA, and psbA2 all decreased. However, it should be noted that even the psbA2 and idiA cyanobacteria that were not exposed to iron-chela-
tor for the hour incubation had significant expression of luciferase as opposed to WT (Figure 5). Discussion This research has better characterized key promoters useful in cyanobacterial synthetic biology research. This feature is important because compatibility of genetic parts with chassis organisms is often a major hurdle in
could be attributed in part to an inhibitory effect of red lights in the custom CO2 incubator (9). On the other hand, it could also be attributed to lower levels of double homologous repair, and thus lower levels of ploidy of psbA2 (12). Future experiments may wish to use quantitative reverse transcription-PCR (qRT-PCR) to quantify the levels of psbA2 expression. Although psbA2 yielded no significant data in our initial experiments, psbA2 did promote transcription of luciferase during the iron-chelating experiment, even in the control condition. For this experiment, all samples were kept in a biosafety cabinet under hood lighting for one hour; thus, it is possible that the lighting in this experiment better promoted the expression of psbA2. Furthermore, the iron-chelator experiments were not successful in that the expression of all constructs except cpc significantly decreased. Though the lack of significance for cpc can be explained by the small sample size, the decreasing trend for all the constructs, especially for cpc560, potentially suggests that the cyanobacteria were dying from prolonged exposure to iron-chelator agents. Future experiments should aim to narrow the time frame during which cyanobacteria are exposed to iron- chelator agent before assessing the extent to which they can be exposed to iron-depleted conditions before their viability is affected. The characterization of the constitutive promoters cpc and cpc560 for use in cyanobacterial research provides a foundation for much research in the environment and energy sector. Future research could use these promoters to create genetic circuits to regulate photosynthetic processes and bolster a mechanism for carbon sinking.
Acknowledgements We would like to acknowledge the full 2018 iGEM Team in addition to the authors: Matthew Mullin ’21, Matthew Lee ’21, Sara Vincent ’18, Ting-Ju (Woody) Chiang ’19, Robert Ruzic ’19, Lin Yu Pan ’20, Dominika Kwasniak ’20, Manvi Shah ’21, Lukas Velikov ’21, Jennifer Rakhimov ’21 We would also like to acknowledge our other advisors: Dr. Jarrod French, Dr. Gabor Balaszi, Dr. Steven Glynn, and Dr. Joshua Rest
Figure 3 (a) Effect of high-light induction on luciferase expression. (b) Effect of iron depletion on luciferase expression.
genetically modifying less-characterized chassis species, such as our own unique chassis S. elongatus PCC 7942. Therefore, this work on characterizing genetic parts expands the potential uses of cyanobacteria as a chassis. With regard to the promoters, we have identified cpc as a constitutive promoter for S. elongatus PCC 7942 that is able to promote transcription of coding sequences during both the daytime and nighttime under a modified 24 hour-light circadian rhythm. Furthermore, we have identified cpc560 as a stronger constitutive promoter as it consistently demonstrated greater expression than cpc. This is in agreement with literature that identifies it as a potential super-strong promoter due to the 14 identified transcription factor binding sites in the sequence (8). As such, cpc560 could be a powerful tool in synthetic biology research. We also confirmed that psbA2 and idiA are not typically expressed under standard conditions and so are not leaky promoters, making them powerful tools in genetic circuits. However, the finding that psbA2 is not typically expressed under normal conditions is not supported by the literature, which concludes that psbA2 has some weak constitutive expression (9). Furthermore, psbA2 was not activated by high-light conditions. This
1. N. Baeshen, et al., Cell factories for insulin production. Microbial Cell Factories 13, 141 (2014). doi: 10.1186/s12934-014-0141-0. 2. O. Adesina, et al., Embracing biological solutions to the sustainable energy challenge. Chem 2, 20-51 (2017). doi: 10.1016/j.chempr.2016.12.009. 3. P. Dvorak, et al., Bioremediation 3.0: engineering pollutant-removing bacteria in the times of synthetic biology. Biotechnology Advances 35, 845-866 (2017). doi: 10.1016/j.biotechadv.2017.08.001. 4. H. Chi, et al., Engineering and modification of microbial chassis for systems and synthetic biology. Synthetic and Systems Biotechnology 4, 25-33 (2019). doi: 10.1016/j.synbio.2018.12.001. 5. B.E. Schirrmeister, M. Gugger, and P.C.J. Donoghue, Cyanobacteria and the great oxygenation event: evidence from genes and fossils. Palaeontology 58, 769-785 (2015). doi: 10.1111/pala.12178. 6. N. Chestney, Global carbon emissions hit record high in 2017. Reuters: Environment, (2018). 7. D.C. Ducat, et al., Rerouting carbon flux to enhance photosynthetic productivity. Applied and Environmental Microbiology 78, 2660-2668 (2012). doi: 10.1128/AEM.07901-11. 8. J. Zhou, et al., Discovery of a super-strong promoter enables efficient production of heterologous proteins in cyanobacteria. Scientific Reports 4, (2014). doi: 10.1038/srep04500. 9. N. Tsinoremus, M. Schaefer, and S. Golden, Blue and red light reversibly control psbA expression in the cyanobacterium Synechococcus sp. strain PCC 7942. Journal of Biological Chemistry 263, 16143-16147 (1994). 10. K.P. Michel, E.K. Pistorius, and S.S. Golden, Unusual regulatory elements for iron deficiency induction of the idiA gene of Synechococcus elongatus PCC 7942. Journal of Bacteriology 183, 5015-5024 (2001). doi: 10.1128/JB.183.17.5015-5024.2001. 11. S.R. Mackey, et al., Detection of rhythmic bioluminescence from luciferase reporters in cyanobacteria. Methods in Molecular Biology 362, 115-129 (2007). doi: 10.1007/978-1-59745-257-1_8. 12. A.H. Ng, B.M. Berla, and H.B. Pakrasi, Fine-tuning of photoautotrophic protein production by combining promoters and neutral sites in the cyanobacterium Synechocystis sp. strain PCC 6803. Applied and Environmental Microbiology 81, 6857-6863 (2015). doi: 10.1128/AEM.01349-15. Images retrieved from: 1. Courtesy of Lin Yu Pan ‘20 2. Courtesy of Natalie Lo ‘21
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