December 18th, 2018
Apoptosis! How Endoperoxides Could Be a Difference

Artemisinin – also known as Qinghoasu – is produced by the sweet wormwood tree Artemisia annua. For hundreds of years, unaware of its potential in treating cancer and malaria, the sweet wormwood tree was used in ancient Chinese medicine to treat fevers, which we now know were caused by the Malarial parasite. It wasn’t until 1972 that the Chinese scientist Youyou Too and her collaborators isolated the active anti-cancer and anti-malarial ingredient from Artemisia annua, Artemisinin. The active portion of Artemisinin is an oxygen-oxygen bond that forms free radicals when exposed to iron. These free radicals then disrupt cellular function, thereby inducing cell death. In the case of cancer cells, research has shown that most types of cancer cells have increased intake of iron compared to non-cancerous cells. As a result, iron reacts with Artemisinin, producing free radicals, inducing apoptosis, and causing cell death. Therefore, Artemisinin may also be effective when treating cancer. However, despite Artemisinin’s effect on cancer and malaria, there are disadvantages to its usage. Since Artemisinin constitutes less than only about 1% dry weight of the sweet wormwood plant it has limited availability in developing countries and it is very costly to extract. Additionally, the original Artemisinin molecule has trouble reaching its target due to its limited bioavailability. Therefore, we have synthesized analogues of Artemisinin that have the same oxygen-oxygen bond as the original Artemisinin molecule but are smaller and inexpensive to make. This Summer, my lab and I have been testing the novel analogues on A549 lung cancer, MCF7 breast cancer, BEAS-2B normal lung, and MCF10A normal mammary cell lines to see the effect of the analogues on inducing cell death. We have witnessed an increase in cell apoptosis in cancerous cells and not in normal cells and will continue testing the various analogues to find the one with the greatest efficacy at the lowest dose. 

Realities of Research

In my journey as a researcher, I have learned a lot about the advantages and downfalls of researching. Before entering Benedictine University, there was a stigma in my mind towards researching. I couldn’t imagine myself sitting in a lab because the idea of this sounded monotonous and unpleasing. Once I began researching, I realized the importance of it, making me love what I do now. Witnessing the novel drugs killing cancer cells was fascinating and exciting because I was able to make useful discoveries. Furthermore, I have gained knowledge on how to maintain various cancer and normal cell lines using proper cell culture protocol. I have seen just how easily cells can become contaminated and the headache involved with sterilizing everything and starting over. I have learned to follow safety protocols better to prevent future contamination. Additionally, I have become fluent in the usage of various lab equipment and techniques including the flow cytometer, absorbance reader, fluorescence microscope, Western Blotting, and protein assays. Having to perform some of these experiments multiple times due to errors I’ve made has helped me better my technique. Although not all the experiments I completed turned out how I wanted due to human error, the experiments that went correctly supported my original hypothesis.

Life of a Scientist

The day in the life of a scientist begins early in the morning. I wake up, get ready, and am in the lab by 9:00 am daily. Every Monday, Wednesday, and Friday I begin the day by placing media to feed the cells in the water bath. While the media is warming up, I check confluency of the cells to determine whether I need to split them or just feed them. From there, I feed or split cells, clean the hood, and continue with the rest of the day. I then go to my research mentor’s office to determine which experiments need to be completed first, conduct those experiments, and end the day discussing the results. The best part of being a student researcher is the flexibility. I can do so many unique experiments with the cells I am growing, allowing me to test various things simultaneously. Additionally, I have a phenomenal research team and we enjoy conversing with one another. The worst part of researching is the long hours spent in the lab. It does get exhausting to be in the lab all day, however, with my great research group I find ways to help the time pass by. Researching has shown me the importance of interdisciplinary work with the collaboration between the organic chemistry lab and my lab, as well as the importance of effective communication.

 

Mohammed U. Haq is a senior majoring in Health Science at Benedictine University in Lisle, IL. He is a 2018 Undergraduate Student Research Fellow (UGSRF) working in Dr. Jayashree Sarathy’s physiology lab at Benedictine University in Lisle, IL. Mohammed’s fellowship is funded by APS. After graduation, Mohammed plans to pursue a career in medicine with an interest in conducting research in medical school.
December 14th, 2018
Detrusor, Urothelium and Mitochodria – Oh My!

The summer of 2018, I worked under Dr. Johanna Hannan at Brody School of Medicine in order to study sex differences in bladder dysfunction and study the impact of obesity-induced bladder dysfunction. With one-third of Americans, aged 40 years or older, reporting to have some level of urinary incontinence, we know that bladder dysfunction is a common condition.1 Both males and females experience bladder dysfunction, but they can experience varying degrees of stress or urge incontinence, overactive bladder, and obstructed bladder. Overall, females experience greater urinary incontinence compared to males.1 Our other interest, obesity induced bladder dysfunction, is pertinent because an increased BMI correlates with a higher risk of urinary incontinence. The mechanism that obesity-induced bladder dysfunction occurs is poorly understood. Specifically, we looked at the urothelium, the inner lining of the bladder responsible for signaling, and the detrusor smooth muscle, which contracts the bladder to dispel urine. Our interest within these tissues were mitochondria, the powerhouse of the cell, responsible for creating ATP; mitochondria is a model indicator of cell health. To study the health of mitochondria, we measured mitochondrial respiration within mice urothelium and detrusor smooth muscle layers of the bladder. Different substrates were added to promote or inhibit certain pathways within oxidative phosphorylation so that differences in mitochondrial metabolism could be studied. We believe that impaired mitochondrial function is contributing to the decreased contraction and inflammation that leads to bladder dysfunction in obese men and women.

Realities of Research

Working within a research lab is an experience you never forget. Life as a research scientist is different than what I had previously thought. It was not every day that I was running experiments; there were days where I read papers in order to understand and apply the results from the experiments. While we had originally believed that females would have decreased mitochondrial respiration because they had a higher prevalence of bladder dysfunction, the data obtained from an oxygraphy-2K (it measures oxygen within a chamber) showed that males actually had lower respiration. These results were found in the presence of a fatty acid which seems to impact male bladder metabolism. Though our hypothesis was proved wrong, our results are significant because they uncover novel information related to males having an impaired fatty acid metabolism.

The best part of working in a research lab was contributing to the field of science. Though our hypothesis was proved wrong, the data still had relevance to bladder dysfunction and how it impacts the population. Before our research, there was little to no information on bladder mitochondria in males and females. On the other hand, the worst part of research was when a machine would malfunction during the experiment. It not only compromised the results, but the tissue that was in it was also rendered compromised. Whenever this happened, there was always someone in the lab that I could ask for help. Also, this experience demonstrated that is it okay to ask for help – especially from people within the lab! They probably experienced the same problem and had their own tips and tricks to prevent it from happening again. Collaboration and discussion were encouraged in the lab; it is something I hope to continue to practice as I continue a career in science.

 

 

References

  1. org. (2018). American Urological Association – Bladder Health. [online] Available at: https://www.auanet.org/advocacy/bladder-health.

 

Hanna Kosnik is a junior at East Carolina University in Greenville, NC working towards majors in Biochemistry and Chemistry. She conducted research under Dr. Johanna Hannan in the Department of Physiology at Brody School of Medicine in Greenville, NC. Hanna is recipient of the 2018 Undergraduate Summer Research Fellowship (UGSRF) funded by the American Physiology Society (APS). After graduating, Hanna plans to pursue a career in medicine.
December 11th, 2018
My Summer of Science with the Ts65Dn Mouse

This summer I worked in a lab that studies the Ts65Dn mouse, which is an animal model for Down syndrome. Previous studies have shown that people with Down syndrome suffer from sleep apnea during the night, which exacerbates some of the cardiovascular and neurological deficits that are already associated with the disease.  My role this summer was to collect breathing and metabolic data from an older cohort of this strain as they were exposed to various gases that stressed their respiratory system. Other members of the lab collected data on the muscular and neurological functions of these mice. The overall goal of our work is to identify the causes of the deficits found within this strain of mouse. In the long run we are hopeful that the work we are doing could eventually lead to therapies for people with Down syndrome who suffer from sleep apnea.

 

Before my summer research started I had already been working in my current lab for about two semesters. I did not really have to adjust to much about the lab besides the fact that I was going in all day, every day. I was conducting my experiments using a barometric plethysmography technique. This technique involves placing mice in a chamber that records several respiratory outputs as air is pumped into and out of the chamber. Even though I was familiar with the technique that I used to collect breathing data, there were a few calibration issues that required some troubleshooting when I first began collection. Once those issues were fixed data collection went smoothly. In addition to conducting plethysmography experiments, I was working with the rest of my lab to harvest and freeze organs that were dissected from our mice in order to look at the specific proteins related to muscle function. We are currently working through analyzing and interpreting our data, but so far have found interesting results that lead us to believe that there is a neurological component that is modulating the deficits found in the Ts65Dn mouse.

Over the course of my summer, I realized that the life of a scientist varies every day. I was on a strict schedule during plethymosgraphy data collection because the mice needed to be tested during specific hours and within days of each other in order to attain accurate results. On the days that I was helping with harvesting and freezing organs, the work moved quickly because organs needed to be removed in a timely fashion in order for them to be viable for further testing. Once all of our mice were euthanized the work calmed down a bit and I was able to take my time analyzing my data, running stats and working through interpretation of statistical outputs. I really enjoyed watching the older members of our lab work through their experiments. I have learned a lot from them and it is helpful to be able to see what my life could look like if I continue down this path. I did struggle a bit at first with learning new techniques and how to run some statistical tests, but having to work through issues and figure those things out for myself has already made me a better scientist. I think that learning how to troubleshoot and work through experimental/statistical/interpretational issues on my own has been the most valuable part of my summer research experience.

 

Brianna Eassa is a senior Biology major at Le Moyne College in Syracuse, NY. She is a 2018 Undergraduate Summer Research Fellow (UGSRF) working in Dr. Lara DeRuisseau’s lab at Le Moyne College. The UGSRF award is funded by the APS. In the future, Brianna hopes to continue to work in a lab setting in order to get more experience to learn what direction she wants to go towards when entering graduate school.

 

December 7th, 2018
Protecting the Miracle of Childbirth

The spectacular process of human reproduction is complex, time consuming, and, above all, fascinating! Much has been learned over the years dealing with the mechanisms of pregnancy in many of Earth’s lifeforms. The research on genetics, like uncovering the entire human genome, makes incredible strides toward fully grasping why certain physiological processes happen. However, there are still numerous question marks, specifically speaking about women’s health during pregnancy and after, that require research and understanding. Full efforts are being undertaken that aim to ultimately lead to safer pregnancies, better means of treating diseases, and developing new techniques.

Research Project

Preeclampsia is a disorder during pregnancy characterized by high blood pressure and excess protein excretion. Because the condition is not entirely understood, treatment options are far and few between for women suffering. Currently, the only remedy is a low-dose treatment of aspirin. The effects and mechanisms of this aspirin treatment is not completely understood either, so the purpose of my study is to attempt to demystify the workings of the treatment. Specifically, I am targeting human trophoblast cells, the major cell type involved in the development of the placenta, an organ that provides nutrition to the developing fetus. By varying different doses of aspirin, I am examining the changes, or lack of change, in the trophoblast DNA. If changes are observed, we will have knowledge on how and if aspirin will help women suffering from preeclampsia, which will ultimately lead to a safer pregnancy for both the mother and child.

Realities of Research

It has been an exciting experience working in a research lab. Not only have I learned valuable techniques, but I am directly impacting the future of medicine, even if it is in a seemingly small way. I have been surprised by the level of attention and precision that is addressed when conducting research. I always knew that attention to detail was important, but the extent to this precision that I have been performing has shocked me along with how these techniques were practiced. For example, RNA isolation is a delicate, yet simple process that requires attention and a good grasp on how to pipette well. If a step is skipped, such as forgetting to add the homogenate additive, then the RNA yield could be put at risk. It is too early in my research stages where results and conclusions can be made. Typically, one trial of cell growth requires one full week, so multiply one trial by the many that we are attempting and the overall experiment becomes lengthy.

Life of a Scientist

The day-to-day life of a young scientist has been exciting. While not all of the parts of my day are groundbreaking and entertaining, it is still a rewarding process. I usually begin my day with notebook entries, planning, and reading up on current events in my field. My research involves a fairly strict time schedule, so in the afternoon, the experimenting and cell ‘farming’ as I call it, can begin. I was surprised by the equipment that I have at my fingertips.  Nothing is more thrilling than looking through a $45,000 microscope or running a 6 well plate through a machine that you can’t even pronounce. The best part so far for me has been the adjusting to a real life laboratory. I have begun to entertain the idea of having my own lab in the future, and becoming familiar with how a lab is run has been a wonderful experience. The worst part has been the waiting that is required between experiments. It makes me wish I had a magic wand that would make the cells grow and be ready for testing at the flick of my wrist. It has been so wonderful working with everyone in my lab. I love the feeling of having an independent project, but still being under a larger umbrella of research with my coworkers where we can discuss information and findings.

 

Brandon Cooley is a junior at the University of Iowa where his is studying biology. His future plans involve graduation with his degree and enrolling in an MD/PhD program where he can further develop his researching skills while being present as a clinician in a hospital!
December 4th, 2018
What I Learned From 10 Weeks of Experimenting

My research project focuses on the Parkin protein found in the mitochondria of the cell. Mitochondria is essentially the energy source for cells. Without it, cells, organs and eventually the body would stop functioning. It is in high abundance in muscles that need energy to contract, such as the heart. Parkin helps in removing the damaged mitochondria from the cell, making room for healthy mitochondria to thrive. If Parkin is nonfunctioning, diseases such as type two diabetes and heart disease can arise.

My project analyzes Parkin expression in the hearts of four groups of rats: lean sized rats with a sensitivity to insulin, obese rats with insulin resistance, and two groups of both obese and lean rats that were placed on a diet. Overall, this will test the effect a restricted diet has on this proteins expression in the heart. We will monitor if something as simple as a diet will bring the protein back to normal levels. If the diet proves to restore protein levels, perhaps this will become an alternative to medication for specific heart diseases.

 

Realities of Research

I started research with the expectation that if I followed the protocol, everything should go right, and I would get results. I found to my surprise, that this was a huge misconception. Sometimes reagents expire, or machinery doesn’t work properly. Sometimes the building experiences a power outage. Although this can be a little disheartening, it made me appreciate the results I did obtain so much more. In our lab we had weekly duties/ chores, and everyone helped each other out. We had a very open and friendly atmosphere. If we had some downtime, we would help another person out with their experiment. This comes in handy when trying to balance starting an experiment over, all the while saving time. The results I ended up obtaining were not what we expected. When looking for my original protein the project was based on, we couldn’t get results. We then probed for another protein in the same pathway and obtained some data. This didn’t change the research question much because the proteins were in the same pathway.

 

Life of a Scientist

The Procedure I did was western blotting. It’s a two-day procedure: one long day (~10-12 hours) followed by a short day (~3 hours). I would usually catch the bus to lab at 10 am and leave anywhere from 8 pm to 4 am (a onetime occurrence). There are about six hours of waiting during the first day, so in this time I would make gels, refill buffers, work on assignments, or eat lunch. The best parts of lab work were learning new procedures and mastering them, obtaining results, and the friendships we made. I loved working as a team. Usually, one person would excel in one area and not in the other, so we became a unit, helping each other work our way towards results. The worst part was the unpredictable schedule. I became apprehensive to making plans, whether that be for scheduling doctor appointments or spending time with family and friends, because I didn’t know if I would need to start a part of an experiment over again and stay longer than expected.

 

Chelsy Cummings is attending the University of California Merced, Merced, CA. As a fellow in the STRIDE fellowship program, Chelsy is being hosted by Dr. Rudy M. Ortiz who is also a professor at the University of California Merced, Merced, CA. Chelsy’s future plans include studying abroad this fall. She is excited to be traveling to London to complete her studies and is looking forward to progressing her research project and presenting those findings at EB in Spring 2019. During that time, she will also be preparing for graduation and volunteering at Valley Children’s Hospital to acquire some experience in the medical field.
November 9th, 2018
Multisensory Control of Blood Pressure

When we go from a lying down position to standing up, our blood tends to pool in our legs and has to fight against the force of gravity to travel our brain. Overcoming the force of gravity is the greatest challenge the cardiovascular system faces. Luckily for us, our body is able to detect when such changes are to occur and can effectively change blood pressure to compensate for the change in posture. The body has many different sensory inputs that contribute to homeostatic regulation of blood pressure during these postural changes. The vestibular system, baroreceptors, chemoreceptors, and higher order brain centers (hypothalamus and cerebral cortex) are all examples of the body’s many sensory inputs (Yates et al. 2014). If these homeostatic responses perform inadequately, then conditions such as orthostatic hypotension can be the result (Yates et al. 2017). Orthostatic hypotension results from a drop in blood pressure due to a change in posture, where insufficient perfusion of blood with the brain occurs. My research project this summer focused on the cardiovascular responses that are necessary to maintain normal biological homeostasis during anticipated imposed postural changes. Homeostasis is defined as the tendency of biological systems to maintain relatively constant conditions in the internal environment while continuously interacting with and adjusting to change originating within or outside of the system (The Free Dictionary, n.d.).

In this study, my lab investigated the activity of neurons in the RVLM (rostral ventrolateral medulla), located in the brainstem, in awake adult female cats. There has been significant research to support the claim that neurons in the RVLM play in important role in blood pressure regulation in both animals and humans (Yates et al. 2017). RVLM neurons are necessary for cardiovascular responses to maintain homeostasis during postural movements. The neurons in the RVLM were identified by their changes in firing rate correlated with the cardiac cycle and their location. The experiments in the lab involved recording the activity of neurons in the RVLM during imposed 40º head-up tilts that were preceded by a light cue. The light cue occurred 10 seconds before the 40º tilt, and the heart rate and firing rate of neurons were determined for 5 second time periods before and during the tilts.

Our hypothesis is that in neurons that exemplify cardiac related activity there is an increase in neuronal activity prior to the onset of the 40º imposed tilt, in the time interval after the light stimulus as compared to before the light. This would indicate that when the animal is able to anticipate when the change in posture is about to occur, the animal’s cardiovascular response system will increase heart rate and blood pressure to prepare for the postural change. This would be an example of a feedforward mechanism rather than a feedback. In a feedback mechanism, the animal would have already undergone the change in posture and body positon in space before the cardiovascular response system could compensate heart rate and blood pressure.  This research project will ultimately further our knowledge of cardiovascular homeostasis and may lead to new treatment options for orthostatic hypotension and also a treatment for cognitive mechanisms that can cause changes in blood pressure such as stress and anxiety.

Figure 1: An example of the data that was collected and analyzed during one of the tilt table experiments. The x-axis shows the change in time as the table went from a nose down position, to a 40º head-up tilt. The neuronal firing was isolated along with the heart rate through the use of an EKG. The sun cartoon indicates when the light stimulus was shown with the five second time intervals appropriately labeled before and during the head-up tilt. Credit: Dr. Bill Yates’ Lab, UPMC Department of Otolaryngology.

 

Realities of Research

I was extremely grateful to take part in this research opportunity this summer as it really allowed me to grow as an undergraduate researcher. The reality of research is that you get out of it what you put in. If you’re motivated and passionate about what you are doing, then I believe that anyone will be satisfied with the outcome of their experience. I became enamored with learning about physiology after completing Dr. Yates Honors Human Physiology class. After completing his course, I was eager to investigate what research was like on the physiological spectrum. Over a course of 10 weeks, I completed dozens of experiments and analyzed large amounts of data. We performed single-unit in vivo recordings using electrophysiological techniques and procedures. When experiments ran to completion and a respectable amount of usable data was collected, the day was considered successful. Another reality of research is that not every experiment is going to go as planned. There were experiments where either the animal wasn’t cooperating on the table, an electrode bent, or we just simply couldn’t isolate any good neurons to record from. When problems like these would occur, we would simply end the experiment and try again the next day. Because there are a great deal of experiments left to perform and more data analysis to complete, there are no final results or conclusions yet to be made. However, we do predict that the firing rate of the cardiac-related neurons will increase after the light stimulus is given, prior to the onset of the tilt.

Figure 2: The tilt table that is used in all of the single-unit in vivo recordings. During an actual experiment, the curtains would be drawn and the lights would be dimmed to allow for the light stimulus to appropriately work. The machines in the back are used to find and isolate a neuron in the RVLM and to change the position of the table. Credit: Dr. Bill Yates’ Lab, UPMC Department of Otolaryngology.

 

Life of a Scientist

I sincerely enjoyed my daily routine as a scientist in the Yates lab. I was able to set my own schedule and I developed meaningful friendships with everyone I worked with. It was a very-low stress working environment and most days I would follow the same routine. A huge part of my project this year was analyzing the data collected from the experiments. When I wasn’t running any experiments I would be on the computer analyzing large amounts of data. Analyzing data is something that a lot of undergraduate students have trouble with, mostly because it takes a lot of time to learn and pick up. I was very surprised by how quickly I was able to pick up the skill and it allowed the lab to make huge amounts of progress throughout the summer. My fondest memory of my time in the lab didn’t actually happen in the lab. On a Friday afternoon after work, everyone in the lab went out to a Pirates game at PNC Park in Pittsburgh. It was my first one and it was really fun to see everyone outside of the lab doing something together. Overall, I had a great summer and I’m very thankful that APS gave me the opportunity to see what full-time research was actually like. This summer experience was very rewarding and has motivated me to pursue my continued interest in biomedical research.

 

References

  1. Yates, B. J., P. S. Bolton, and V. G. Macefield. 2014. Vestibulo‐sympathetic responses. Compr. Physiol. 4:851–887.
  2. Patel, N.M., Baker, E.A.G., Wittman, S.R., Engstrom, I.C., Bourdages, G.H., McCall, A.A., Miller, D.M. and Yates, B.J. Cardiovascular adjustments during anticipated postural changes. Physiol. Rep. 6(1), e13554, 2018
  3. TheFreeDictionary.com. (2018). Homeostasis (Biology). [online] Available at: https://medical-dictionary.thefreedictionary.com/Homeostasis+(Biology)
John Bielanin is a rising senior majoring in Neuroscience and minoring in Chemistry, with a certificate in Conceptual Foundations of Medicine at the University of Pittsburgh in Pittsburgh, PA. He is a 2018 Undergraduate Summer Research Fellow working in Dr. Bill Yates’ lab in UPMC’s Department of Otolaryngology at the University of Pittsburgh. John’s Undergraduate Summer Research Fellowship (UGSRF) is funded by the American Physiological Society. Outside of school and work, John enjoys reading, writing music, and spending time outdoors. After graduation, John plans on applying to medical school while continuing to pursue research opportunities in his gap year.
November 6th, 2018
Novel Mechanisms of Preeclampsia Prevention via SGK1 and Corticosteroids

Preeclampsia, a hypertensive disorder in pregnancy, affects more than 6 million pregnancies per year worldwide. It is a dangerous condition during pregnancy which involves high blood pressure, proteinuria, and swelling. The Santillan lab has shown that single dose early of BMTZ early in pregnancy will reverse late pregnancy hypertension and proteinuria (1). The molecular mechanism by which this reversal occurs is unclear. One potential pathway involves serum/glucocorticoid regulated kinase 1 (SGK1), a serine/threonine kinase stimulated by corticosteroids. SGK1 dysregulation and human genetic variants in SGK1 have been associated with hypertension. The overall goal of this study is to determine whether SGK1 and its regulation play a role in preeclampsia.  If so, BMTZ has the potential to prevent preeclampsia in humans. Because mir-365 has been shown to decrease SGK-1 expression in human placentas from pregnancies with poor placentation, I will examine the effect of vasopressin and SGK1 in placental cells on mir-365 expression. In addition, it will be determined if placental mir-365a-3p is differentially expressed in human preeclampsia. This project is significant because it may help to determine how BMTZ protects from preeclampsia and whether BMTZ could be useful in humans.

 

Research in the lab can be very stressful. Things may not go as well as expected and troubleshooting is a process. Regardless, I had the opportunity to learn many new techniques that would help me in the future. I was surprised how research is comprised of so many different aspects. A little difference in one experiment may change the whole outcome. I learned a great set of skills like how to maintain a cell culture, perform an ELISA, BCA, and extract RNA. It took a while for me to start up on my experiments because I had to research some more background information to ensure I knew what I was doing. My experiments went smoothly, but it was later found that the drug I was using to treat the cells was not working in our mouse model; therefore, it may not be working with my cells as well. My project was put to a halt to first determine if the drug was correctly performing. The drug was aliquoted about a year ago and may have degraded. I would have to wait in order to determine whether I was able to continue or to start over. In the meantime, I worked with my mentor with small projects and learned useful techniques. Additionally, I worked on the second portion of my project involving whole placental tissues. The tissues were RNA prepped and analyzed via qPCR. The results showed that there was a significant difference with p-value of 0.016. This makes sense because Xu found that miR-365 negatively regulates IL-6 and it, in turn, is transcriptionally regulated by Sp1 and NF-κB. (2) So, transcriptional down-regulation of miR-365 should result in increased IL-6. This was interesting to hear, but we cell culture was needed to determine this and it was on standstill.

 

There were some busy days and other days there was a lot of down time. For example, one day there may be multiple tests to complete in a day, other days an experiment would consist of wait time. The most surprising part of participating in the lab is that I realized that a lot of the down time is used to write papers or grants. Research involves a great deal of writing to express the study to the public eye and document previous studies to help ongoing studies. I am appreciative of researchers because without those papers I would not have been able to understand my study without background information. Most days were very stressful trying to balance all of the work and trying to understand why a certain mechanism happened. My least favorite part during my time in the lab was working so hard on an experiment and in the end, not having it work out. The best part was working along with my mentor to learn new techniques and tests. I’m also glad that people around the lab worked well with one another and that they would take the time to reach out and teach me.

 

References:

  1. Santillan, M., Santillan, D., Scroggins, S., Min, J., Sandgren, J., Pearson, N., Leslie, K., Hunter, S., Zamba, G., Gibson-Corley, K. and Grobe, J. (2014). Vasopressin in Preeclampsia: A Novel Very Early Human Pregnancy Biomarker and Clinically Relevant Mouse Model. Hypertension, 64(4), pp.852-859.
  2. Xu et al. miR-365, a Novel Negative Regulator of Interleukin-6 Gene Expression, Is Cooperateively Regulated by Sp1 and NF-κB. Journal of Biochemistry 286: 21401-21412, 2011
Carolyn Lo is a junior majoring in Human Physiology and Biochemistry at the University of Iowa in Iowa City, Iowa. She is a 2018 Short-Term Research Education Program to Increase Diversity in Health-Related Research (STRIDE) Fellow working with Dr. Mark Santillan at the Carver College of Medicine in Iowa City, IA. Carolyn’s fellowship is funded by the APS and a grant from the National Heart, Lung and Blood Institute (NHLBI) (Grant #1 R25 HL115473-01). After graduation, Carolyn plans to pursue a doctorate degree in medicine.
October 26th, 2018
Dietary Fiber: Why Your Parents Told You to Eat More of It

Histologic section of intestinal tissue isolated from healthy mice stained to visualize intestinal epithelial cells. Credit: Lance Peterson, Theresa Alenghat, and David Artis

The epithelium is a layer of cells that separates the inside of the human body from the external environment. In the gastrointestinal (GI) tract, these cells are known as enterocytes and must form a barrier against harmful pathogens present in the gut lumen, while at the same time aiding in the digestion and absorption of nutrients. It is important that all these functions of the epithelium are tightly controlled to maintain homeostasis. Dysregulation of these complex processes has been shown to lead to diseases such as inflammatory bowel diseases (IBDs) which affect over 1 million US residents (Kaplan, 2015). IBDs, which include Crohn’s disease and ulcerative colitis, are characterized by chronic inflammation of the GI tract leading to abdominal pain, weight-loss, fever and a loss of quality of life. While the exact cause of the disease remains incompletely understood, we know that the integrity of the barrier of our GI tract is crucial in IBD prevention (Martini, Krug, Siegmund, Neurath, & Becker, 2017).

 

At a young age, we are often told to eat our vegetables and that fiber is good for our digestive health, but what does that entail? Recently, we have shown that a dietary fiber known as rhamnogalacturonan (RGal) enhances gut barrier function. Furthermore, we have shown that RGal decreases disease severity in a mouse model of colitis. However, how RGal improves intestinal barrier function remains incompletely understood. My project over the summer aimed to characterize the mechanism through which RGal enhances epithelial barrier function. Specifically, my project aimed to evaluate the role of intracellular proteins known as protein kinase Cs (PKCs) in the modulation of barrier function in an intestinal epithelial cell line in response to RGal. Our lab used an apparatus called the Ussing Chamber to measure epithelial barrier permeability. In my project, I will treat my cells with various chemical inhibitors of PKCs in Ussing Chambers and then determine barrier permeability to small ions in response to RGal. If PKCs are involved in the modulation of barrier permeability in response to RGal, chemical inhibition of PKC will block the beneficial effect of RGal on barrier function. By understanding signalling pathways that enhance barrier function in inflammatory diseases in the GI tract, we have the potential to use dietary fibers such as RGal to leverage these pathways to treat active IBD.

 

There are two realities of research that I was able to experience this summer. First, I think one of the most rewarding things about my research is that we sometimes did not obtain the results that we expected to. Although this may seem counterintuitive, unexpected results in my project were always the most interesting because I was not only able to observe my supervisor’s stunned reaction, but those results were the ones that allowed us to come up with an alternative hypothesis and steer the project in a completely different direction than we initially planned. I think that the experiments that generate unexpected results are my favorite thing about science. Secondly, I think that the most important thing for people to realize about the day-to-day lives of scientists is that finding the cure for cancer or any other major disease does not happen every day. While understanding the bigger picture in the context of a particular disease and the rationale behind the experiments that we conduct is important and keeps us focused, the things that we study day-to-day often involve understanding the physiological role of a particular cellular protein or defining a cell signalling pathway. Although learning cell signalling pathways may sound a little less exciting than curing cancer, a single cell signalling experiment contributes to the overall body of knowledge which eventually leads to the development of a therapy.

 

This summer, I was incredibly fortunate to work with the people that I did. First, my supervisors Dr. MacNaughton and Dr. Baggio really allowed me to discover my passion for science. Every day, I am able to see their excitement about my work and their devotion to educating the next generation of scientists. Secondly, my lab mates were some of the most knowledgeable, supportive, and enthusiastic scientists that I know. Five years from now, I will not only remember the science from this summer, but I will still remember our debates about fruit with meat in salads, our arguments about whether or not the word ‘meth’ should be allowed in Scrabble (it shouldn’t), our common frustrations about failed western blots and our disagreements about how to pronounce words like ‘drama’ or ‘garage’.

 

Judie Shang attends the University of Calgary in Alberta, Canada where she is majoring in Biomedical Sciences. She is an Undergraduate Research Excellence Fellow (UGREF) and is working over the summer with Dr. Wallace MacNaughton at the University of Calgary where she is studying the effect of dietary fibre on the intestinal epithelium. After graduation, she plans to attend graduate school to study mucosal immunology.
October 16th, 2018
My “Diary”al Journey

Bile acid diarrhea is seen in approximately 10 million Irritable Bowel Syndrome (IBS) patients, and about 86% of Crohn’s patients. Furthermore, there are 90 million patients worldwide annually that have Bile Acid-Associated Diarrhea (BAD). Despite this high prevalence, the underlying mechanism associated with BAD is not completely understood. The primary goal of our research is to identify the mechanism behind bile acid-induced tight junction dysfunction that results in diarrhea. Bile is produced by the liver, released into the duodenum where it helps emulsify fat. Once its job is done, about 95% of bile acid is recycled back to liver, and less than 5% enters the colon. The primary bile acids enter the colon, where anaerobic bacteria convert the primary bile acids to secondary bile acids, and they aid with normal secretion of fluid. However, if the bile acid reabsorption is hindered, as in cases like Crohn’s disease and Celiac disease, there is an excess amount of bile acids that accumulate in the colon, resulting in fluid loss and diarrhea. It has been shown by us and others that bile acids with two hydroxyl groups, such as chenodeoxycholate (CDCA), can alter tight junctions when present in the luminal surface and stimulate chloride secretion when present in the basolateral surface to result in diarrhea. However, the bile acid with one hydroxyl group, lithocholate (LCA), does not alter secretion. In fact, it helps attenuate the barrier dysfunction caused by CDCA. So, how does CDCA travel from lumen to the basolateral surface? This summer, in collaboration with an organic chemist, Dr. Rubush, at my university, we tested the hypothesis that CDCA loosens tight junctions to travel paracellularly to reach basal surface.  We synthesized and tested fluorescently tagged CDCA and LCA in order to visually track their movement in experiments. Our results thus far, show that there is a time dependent increase in CDCA content in the basolateral chamber of the transwell. I will continue to test if LCA behaves similarly. The reason for the yin and yang in bile acid action could be due to it structural variation, the lack of a hydroxyl group in the 7th position.  Therefore, we are synthesizing 7, methyl CDCA to compare its effects on tight junctions with that of CDCA and LCA.

Being part of a research laboratory has made me realize that the process of doing science is filled with many unknowns and surprises.  From having to deal with slow growing cells and lack of the expected band in a Western blot to problems getting the methyl group on the correct hydroxyl group and trying to increase the yield in chemistry lab, I was exposed to the challenges in day-to-day activities in a scientific lab. For example, a minor problem with the air conditioner in our school resulted in the decomposition of an intermediate reactant in the synthesis of methyl CDCA, which set us back by a week or so.  Suddenly, our colon cell line started to grow slowly and this made us change the direction of our research temporarily and focus on performing ELISA assays on samples frozen previously.

My day-to-day life as a researcher consists of a combination of planned activities, such as making buffers, media and feeding cells every Monday, Wednesday, and Friday, and designing and running experiments on confluent cells at other times. At first, I thought research would be very repetitive with rigid timings and I would be alone doing experiments independently in a lab.  To my surprise, my research timings were very flexible, and I collaborated with students and professors of different departments at my university. In fact, along with organic chemistry students in the summer research program at Benedictine University who synthesized the tagged bile acids, we gave a collaborative, interdisciplinary research presentation which was very well received by faculty and peers from the College of Science. As with life in general, there were good days when we got great data, and bad days when we had contaminated cells; but overall it is a very invigorating experience!

 

Ugne Dinsmonaite is a senior majoring in Biology at Benedictine University in Lisle, IL. She is a 2017 APS Short-Term Research Education Program to Increase Diversity in Health-Related Research (STRIDE) Fellow working in Dr. Jayashree Sarathy’s lab at Benedictine University in Lisle, IL. Ugne’s fellowship is funded by the APS and a grant from the National Heart, Lung and Blood Institute (Grant #1 R25 HL115473-01). After graduation, Ugne plans to pursue a career in dentistry with research in craniofacial pathobiology.
October 12th, 2018
Learning to Become a Researcher

When people or animals feel threatened, their sympathetic nervous system, a.k.a. ‘fight-or-flight’ system, releases chemicals that increase their blood pressure and heart rate to prepare for fighting or fleeing danger.  Unfortunately, when someone is obese or eats a chronically high-fat diet, their fight-or-flight system can be in an almost permanent state of overdrive.  This can place too much strain on the heart and blood vessels, potentially leading to hypertension (high blood pressure) and subsequent cardiovascular disease such as a heart attack or stroke.  My research project for the summer was to identify specific pathways in the mouse brain that influence the fight-or-flight response.  More specifically, I aimed to determine how inhibition of the dorsomedial hypothalamus (an area of the brain) by neuropeptide-Y (a brain-specific chemical messenger) leads to decreased activity in the fight-or-flight system.  By determining how various chemicals and pathways in the body and brain influence the fight-or-flight system, we may be able to find new treatments for people who have hypertension, hopefully increasing their longevity by decreasing their risk for serious conditions like heart attack or stroke.

 

Working in a research lab is simply amazing.  There is an almost endless amount of techniques, equipment, and software available to learn how to use.  This summer I have learned how to perform immunohistochemistry, how to use a confocal microscope, and how to utilize different analysis software programs to interpret results from fluorescent images.  If time permits, I may even learn how to perform microinjection surgery on a mouse and how to use RNAscope to complement my immunohistochemistry experiments.

 

Two things that surprised me about working in a research lab were how time-consuming experiments can be, and how expensive research supplies are.  For instance, it takes a minimum of sixteen days post-injection before the mouse brains are ready for me to begin processing them.  The brains must then be frozen, sectioned, immunohistochemically treated, mounted onto slides, then imaged, all of which adds up to around thirty hours of processing for a set of three or four brains.  Additionally, much of the processing utilizes expensive solutions and equipment, such as the $400 primary antibody used in the immunohistochemistry, or the fluorescent microscope which costs around $55/day to use for imaging.  This experience helped me to realize the importance of organization, precision, and time-management when conducting an experiment, since any mistake could result in hundreds of dollars wasted and countless hours lost.  Thankfully the experiments I’ve conducted so far this summer have turned out great, and I look forward to starting my next large batch of experiments next week.

 

The day-to-day life of a scientist is highly variable based on my experience this summer.  During any one week I might complete a variety of different tasks based on the needs of my research project as well as the needs of my lab colleagues. While there are general deadlines to be met for certain things and some experiments that require assistance from others, for the most part I am free to schedule which tasks I will be working on for any given day.  One downside to working in research is that since certain equipment is too expensive for each lab to have one of their own, it must be purchased and shared by the whole department.  For instance, the fluorescent microscope that I use is a very popular tool for the type of research done in our department, so you must make a reservation in order to use it.  Unfortunately, if your imaging is taking longer than expected and you didn’t reserve enough time on the microscope to finish, you could end up waiting an entire week before another reservation is available.  Thankfully, with careful planning, this problem can usually be avoided.

 

Overall, working in research as part of a team with the members of my lab has been wonderful.  Each person has their own unique background in research, and since I’m the most junior member of the lab there is a wealth of knowledge I can learn from each of them.  I truly appreciate how much each of my lab colleagues is willing to teach me what they know, provide answers to my questions, and give me guidance for not only my research project, but for my education and career goals as well.

 

Alyssa Bonillas is a senior at Portland State University in Portland, OR, majoring in both Biology and Psychology.  She is a Hearst Fellow working in Dr. Virginia Brooks’ lab at the Oregon Health & Science University in Portland, OR.  Alyssa’s fellowship is funded by APS through a grant from the Hearst Foundation.  After graduation, Alyssa plans to further her education by completing an MD/PhD program, and continuing on to become a physician-scientist at an academic research institution.