January 11th, 2019
The Effect of Acidic or Neutral Environment on the Mitochondrial Morphology of Human Kidney Cells

This summer, I had the opportunity to investigate the effect of environmental acidity on the mitochondrial shape of human kidney cells.

African Americans have a much higher chance of getting kidney disease than European Americans because they frequently carry risk mutations in a gene called APOL1. Two mutations, termed as G1 and G2, have been discovered only in African American populations. Those who carry two copies of G1 or G2 variants are more likely to develop chronic kidney disease (CKD). Thirteen percent of African Americans carry two copies of APOL1 variants, and these two variants contribute to at least 70% of CKD in this population (Science, 2010). However, only 20% of African Americans with two copies of APOL1 gene variants eventually develop CKD. Therefore, we believe there must be a “second stressor” working together with the APOL1 variants to cause CKD. Prior studies at Wake Forest School of Medicine have concluded that APOL1 G1 and G2 variants induced malfunction of the mitochondrion, a critical organelle providing energy for normal cell activity (Ma, JASN 2017). An acidic environment may have additional negative effects on cells carrying defective APOL1 G1 or G2 variants, for two reasons. The first reason is that the protein encoded by the APOL1 gene variants is sensitive to acid and an acidic environment affects its function (Thomson and Finkelstein, PNAS 2015). The second reason is that kidney tissue accumulates more acid than other tissues in the body and presents an acidic environment that may affect the function of APOL1 protein or potentially be that “second stressor”.

This mitochondrial network image was taken with confocal microscopy during the 2018 APS undergraduate summer research program

Therefore, we investigated how the difference in environmental acidity level, defined by pH value, affects the mitochondrial shape of cultured human kidney cells — that is whether the mitochondrial pattern is normal or fragmented in the cells exposed to environments of different acidity (=different pH). We performed confocal microscopy to scan serial images of mitochondria from human kidney cells expressing normal APOL1 G0 and G1/G2 variants, and used Fiji software to measure the relative mitochondrial length. These experiments were done when APOL1 expression levels were comparable among cells of different APOL1 genotypes, and the cell viability was intact when APOL1 was moderately expressed. We found that an acidic environment enhanced the negative effect of APOL1 risk variants on the mitochondrial pattern of kidney cells. According to these preliminary experiments, an acidic environment appears to elicit more mitochondrial fragmentation, which typically suggests that mitochondria may not be working properly.

Our preliminary studies suggest that the effect of environmental acidity in the kidney may be important for understanding how APOL1 variants pose a high risk for CKD in the African American community. Understanding why products of APOL1 gene variants, which are expressed around body, damage only the kidney and how and why kidney disease develops in those individuals who carry G1 and G2 variants of this gene will have a huge impact on the African American community and help in the fight against kidney disease. This study is also a part of the larger project to identify other possible “second stressors” to APOL1 gene associated kidney disease, and relatively high environmental acidity of the kidney may be one of those.

 

Realities of Research

When I first came into the lab, my mind was filled with awe and admiration because I saw how dedicated the lab members were every day. I have found that scientific research is such a complex process. In order for a research project to be successful, every step of the experiment should be planned ahead of time and in minute detail. My mentor and lab instructor have been so considerate. They discussed every aspect of the experiment, such as when to seed cells, when to add doxycycline, when to purify and extract RNA from cells, etc. This allowed me to perform the experiments efficiently and saved me a lot of time so that I would not make too many mistakes and have to start all over again. Without their instruction and guidance, I cannot imagine how I could have done the cell culture (including doxycycline controlled gene overexpression), taken images on the confocal microscope, isolated RNA from cultured cells, and run RT-PCR all in a short period of ten weeks. The procedures were overwhelming to someone like me who had no previous experience with cell experiments. After the first two weeks of “playing” with cells, I realized it is delicate work requiring patience and fine motor skills. For example, I struggled opening and closing the cap of the falcon tube only with one hand or pipetting minute amounts of fluid for PCR experiments. After several rounds of practice, I felt comfortable performing the task. Now, I have a true understanding of “practice makes perfect”. To analyze the mitochondrial patterns, we used a new technique called Fiji/MiNA software to measure the length of the mitochondria of kidney cells. Based on literature, Fiji/MiNA software was used to measure the length of mitochondria in neuron cells. We adopted this software and successfully adjusted the settings to apply to mitochondrial lengths in kidney cells and accurately captured the small fragments of mitochondria, which made the measurement more precise. Thanks to my mentor and lab instructor, their dedication and precision greatly influenced me. From time to time, my mentor praised me for my contributions to the study. As a team, we have been able to complete the study and obtain our results as we expected.

 

Life of a Scientist

Doing scientific research has always been my passion. Since my high school years, I longed that someday I could make new discoveries, which would eventually change people’s lives. Working in the lab, I have been so excited to learn new techniques needed to complete my project. I never get tired of tedious repeats of 200 scans of images. The best part of the fellowship is that I have a tremendous amount of guidance from my mentor to successfully complete the project in the time limit and obtain the results we are expecting. I have realized that scientific research not only requires patience and proper time management, but also requires thorough knowledge from a variety of disciplines such as physiology, anatomy, molecular/cell biology, etc. This is quite challenging for me as an undergraduate. I shared the frustrations of other fellows, whose experiments did not go as planned. They had to rely on trial and error, and were even unfortunate to find out that there was not enough knowledge to do the study in their lab and started moving in another direction. The worst part of my research project was that I had to spend days measuring and recording the mitochondrial lengths of human kidney cells after treatment with different pH solutions using software and manually enter the data first in Word, then into an Excel spread sheet. But overall, being a scientist especially working as a team member of this lab filled me with joy and pride as we were rewarded with a successful project.

 

DengFeng Li is a junior majoring in Biology at the University of North Carolina at Greensboro. He is a 2018 APS Undergraduate Summer Research Fellow working in Dr. Snezana Petrovic’s Lab at Wake Forest University School of Medicine. Li’s fellowship is funded by the APS and a grant from the National Institutes of Health.
January 8th, 2019
STRIDE, Statins, and Scientific Research: The Perfect Way to Spend Summer Vacation

After googling the definition of the word science, one will find that it means “the intellectual activity encompassing the study of the structure and behavior of the physical world through observations and experiments.” In simpler terms, science is about being curious and learning all that one can about something in order to better understand it. That is exactly what I have had the opportunity to do this past summer under the APS STRIDE fellowship program.

 

Research Project

 In scientific research, to start designing a project, one must first come up with or ask a question that nobody really knows the answer to. With that, something many people don’t know is that high cholesterol has no symptoms; therefore, many people do not even know they have high cholesterol levels. And those who are being diagnosed are being prescribed medication to lower their risk for developing heart disease and decreasing their chances for having a stroke (2). These medications being prescribed, called statins, are one of the most effective cholesterol-lowering drugs available. However, approximately 10-12% of patients taking statins develop muscle pain and dysfunction, which can be intensified with exercise (1). It is unclear as to how exercise and statins work in combination to yield these side effects. Yet, it is important to gain a better understanding as to how they work together to affect one’s health. Therefore, I have been currently researching the effects of the mixture of these two treatments in ApoE-/- mice, whom genetically have high cholesterol, in hopes of generating new insight as to how statins and exercise impact the health of individuals with hypercholesterolemia to contribute to the development of the most successful treatment options that decrease the severity of complications (3).

 

Realities of Research

This fellowship has allowed me to develop not only a greater understanding for the science behind the subject of the project itself, but also for the process and effort to perform and accomplish the project as a whole. What is fascinating about conducting research is that each day presents something different to be accomplished or overcome. I was most surprised by how much planning, preparation, and practice must be done prior to the actual start of a project. Whether it is acquiring supplies, matching up schedules, or deciding what types of experiments to conduct, it all takes time and dedication to ensure that the project runs smoothly. With that, I also had to learn and practice new techniques, such as injecting the mice with statin medication, training the mice, performing muscle dissections, and developing tissue samples to analyze protein levels. With what we’ve accomplished so far, there is no specific data that indicates a major difference between the effects of exercise alone and exercise in combination with statin in mice with high cholesterol. However, we hope to see some difference in muscle force between the two groups after we finish our experiments, in which we then plan to determine if there is a cellular basis that is being affected by the statin medication that is causing a difference. And if there’s no variance, then we know that this specific model doesn’t support our original theory. We would next look at a different element of force, such as endurance instead of strength. But the reality and beauty of research is that you never know what you’re going to find.

Life of a Scientist

Not knowing what to expect is one of the best and worst parts of the life of being a scientist. It is not a typical Monday through Friday 9:00am – 5:00pm job where you do the same thing every single day. You’re constantly learning new things and applying what you have learned to something new; you make connections from the past to the present to try and understand how concepts are related yet different. But even with these enlightening moments, there can be downsides, too. Challenges are thrown at you every day, whether it’s scheduling conflicts, flooding issues, or the results don’t turn out like you expected. Again, that’s where the art of science comes along, in that one must learn how to overcome these obstacles by becoming adaptable to every situation, thinking creatively to find a new route or how one can stay on their original path, and collaborating with others to share ideas as how to approach each step.

This summer has been about STRIDE, statins, and scientific research all of which have inspired me to never stop learning, to never stop questioning, and to never stop searching for an answer. It doesn’t matter if one is titled as a scientist or not, these are actions everyone should implicate into their lives to learn about themselves and their passions and to learn more about the world around them.

 

McKenzie Temperly is a junior majoring in Health Sciences – Clinical & Applied and minoring in Chemistry at Drake University in Des Moines, IA. She is a 2018 Short-Term Research Education Program to Increase Diversity in Health-Related Research (STRIDE) Fellow working in Dr. Kimberly Huey’s lab at Drake University.  McKenzie’s fellowship is funded by the American Physiological Society and a grant from the National Heart, Lung, and Blood Institute (Grant #1 R25 HL115473-01). After graduation, McKenzie plans to attend medical school and pursue an M.D./Ph.D. degree. She is currently interested in specializing in general/orthopedic surgery or emergency medicine intermixed with biomedical research within her chosen field.
January 4th, 2019
Ozone: Protector or Pollutant?

We usually hear that we want more ozone in our atmosphere to protect us from the sun’s ultraviolet rays, but ozone isn’t always a good thing. That protective ozone is found in the stratosphere, while ozone on ground level is a harmful air pollutant caused by emissions from cars and factories. Ozone can do a lot of damage to human lungs, causing shortness of breath, coughing, inflammation and damage to airways, aggravation of lung diseases like asthma, and permanent lung damage. In response to ozone-induced injury, macrophages (immune cells which eat and break down viruses, bacteria, and dead cells) accumulate in the lung and contribute to inflammation and toxicity. Inflammation is important to get rid of any dangerous invaders or cell debris, but macrophages can also have damaging effects in the lungs.

We want to find out what can be done to reduce that inflammation and toxicity, so we are investigating valproic acid. Valproic acid is a fatty acid which has been shown to be anti-inflammatory and an antioxidant. My research project involves testing our hypothesis that valproic acid will reduce lung inflammation and toxicity caused by ozone-induced injury. To evaluate the effects of valproic acid on inflammation and toxicity, I stain thin slices of lung tissue by immunohistochemistry. In immunohistochemistry, the goal is to determine if alveolar macrophages are expressing markers of inflammation or toxicity – the more expression of a certain marker, the darker the macrophage should be stained. We expect that the lungs of mice treated with valproic acid will be less stained than the untreated if inflammation and toxicity are mitigated.

https://www.edf.org/health/why-smog-standards-are-important-our-health

Caption: Smog over LA. Ozone is the main component in smog.(1)

 

Realities of Research

Like the bad and good faces of ozone, doing a research in the lab is slow-going, but also rewarding. The pace is slow because I dedicate a lot of time to troubleshooting the immunohistochemistry process. For each marker of interest, the protocol needs to be optimized. This is time-consuming because it means going through the immunostaining process repeatedly, changing small details each time. It was especially frustrating when the results were not what we expected. When our controls were repeatedly turning out different from how they had looked in previous experiments, we had to figure out if it was the fault of the sample, a detail in the protocol, or the antibody. I’m currently still working on figuring out the discrepancy by testing samples from other labs and different antibodies. If it’s the samples that are faulty, we will put a hold on the immunohistochemistry until we can use the samples from an animal exposure we have planned in a couple of weeks. If the antibodies are the problem, we will order new ones. If I’m doing something in the protocol incorrectly, my research mentor will watch me go through the steps and find out. This complication has been slowing down our progress, but it’ll be rewarding to finally figure it out and get data.

Life of a Scientist

Even excluding the satisfaction of getting data, I feel like I’ve grown a huge amount working as a scientist this summer since it was completely new for me. It’s my first experience working full-time, in addition to taking place in the unique environment of a research lab. I was happily surprised by the amount of flexibility in schedule – each person comes in and leaves when they need to, depending on the work they need to get done that day. Some days are a typical 9 to 5, some might be much shorter, and some might go late into the night. It can become overwhelming meeting new people, catching up on literature, and learning new lab techniques. However, it’s also satisfying to soak up so much new information so quickly and see myself developing as a scientist and a student every week. In my experience so far, the best part of working full-time is the people I have been able to get to know. Seeing the lab tech, the faculty, the grad students, the undergrads, and the high school students every day gives me the chance to really learn about what they do inside and outside the lab. Because of them, coming into the lab every day is welcoming and exciting, which makes all the difference when I’m frustrated with my experiments. Working with them is easy and most of all, fun, and I’m grateful I was able to do research with such encouraging and friendly people.

 

References:

  1. Why smog standards are important for our health. (2018). Retrieved July 27,2018, from https://www.edf.org/health/why-smog-standards-are-important-our-health
Jordan Lee is a junior studying molecular biology and biochemistry at Rutgers University in New Brunswick, NJ. She is a 2018 Undergraduate Summer Research Fellow, funded by the APS. Jordan is working in Dr. Debra L. Laskin’s lab at the Ernest Mario School of Pharmacy at Rutgers. After graduation, she plans to continue doing research and exploring her interests in healthcare and science.
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.