January 19th, 2018
The Life of a Summer Student Researcher

Childhood obesity has become a major health issue in the United States recently. Research studies show that childhood obesity is associated with an increased risk of declined renal function, which is defined as renal injury. Since renal hyperfiltration, which is when the kidneys are working more to than needed, this leads to excessive amounts of protein to be produced in the urine (proteinuria). This condition is typically associated with obesity. The current study will determine if we prevent the renal hyperfiltration, can we decrease proteinuria and/or renal injury?

Working on ground breaking research and being in an environment that is focused and effective was eye-opening for me. I enjoyed being around scientists that had similar mindsets and were able to help me develop a scientific mind of my own. It surprised me that everyone was so open and welcoming to a new unexperienced college student. But it made me more comfortable, especially when some of my experiment’s failed, but they were very helpful and encouraging throughout the entire process. During my time in the lab the initial study I worked on was not successful, so I was give the study I have now and the results that were collected were what my research host expected and it was a successful study that will be continued.

Being a scientists is like riding a roller coaster every day that you enter into the lab. Some days you are so busy with experiments that you forget to eat lunch. But then other days are very slow, because you are waiting on data or results. It is a great environment to learn not only about science but about yourself, how you manage time, how you interact with people, and how well you work alone. I think the best part about being a scientist is that every day is a new challenge because every day you are working on something that could possibly change the world. I think that worst part is that even if you do everything correctly the data can still not turn out how you want it. But overall I really enjoyed the team aspect of working in a lab. Everyone in the lab helps everyone on their projects and vice versa, because everyone wants to see the lab/experiments succeed because it could have a positive impact on the world.

Alyssa Pennington is a senior majoring in Chemistry at Jackson State University. Alyssa is working in Dr. Jan Michael Williams lab at the University of Mississippi Medical Center in Jackson, MS. Alyssa is a second year Short-Term Education Program for Underrepresented Persons (STRIDE) Fellow which is funded by the APS and a grant from the National Heart, Lung and Blood Institute (Grant # 1 R25 Hl115473-01). After graduation Alyssa plans to pursue a career in medicine and research.
January 16th, 2018
Investigating the Role of Piezo2 in Serotonin Release

At the mention of serotonin, people commonly think of its role as regulating anxiety, mood, and happiness in the brain. However, 95% of the body’s serotonin is produced by enterochromaffin (EC) cells present in the tissue lining of the gut (1) before being taken up by platelets that circulate it throughout the body (2). The EC cell releases serotonin in response to mechanical and chemical stimulation, and this release differs depending on whether the body has been fed or is fasted (3). Gut-derived serotonin is important for regulating:

  • Stretch and contraction of the gut
  • Gut sensation
  • The ability for platelets to clot blood
  • Bone development
  • Blood pressure
  • Metabolism (1)

There are still many questions regarding how the EC cell releases serotonin. Recent studies from Dr. Beyder’s laboratory of Mayo Clinic discovered that a mechanosensitive ion channel called Piezo2 is present in human and mouse EC cells, and that Piezo2 is important for serotonin release in EC cell models after a mechanical force like pressure is applied (4, 5). In the broad scheme of things, understanding the role of Piezo2 in EC cell serotonin release and in regulation of blood serotonin levels could lead to future drug developments for gastrointestinal diseases.

A visual representation of serotonin release from EC cells and platelet uptake. Reference: Modified from Mawe & Hoffman. Nature Rev Gastro & Hep. 2013. (1)

Dr. Beyder’s laboratory created a mouse model with the Piezo2 gene knocked out of the gastrointestinal tissue lining the gut. These knockout mice can be compared to the control mice that do not have the Piezo2 gene removed to see how Piezo2 affects the physiology of the gut. My aims for my ten week research fellowship were:

  • To determine the role of Piezo2 in mechanosensitive EC cell serotonin release from primary EC cells from fed and fasted epithelial Piezo2 knockout and control mice
  • To determine the role of Piezo2 in systemic serotonin levels in fed and fasted epithelial Piezo2 knockout and control mice

As is the case with most research, everything did not turn out as planned!

In the first few weeks of my research project, I had already faced unexpected complications. I was planning to collect cells from mouse colon and place them on a special plate with the proper nutrients and environment to help them grow into what is called a primary culture. However, we were not able to take pictures to show that the primary cultures were present since we did not have a microscope lens that could fit the wells in the plate. With the limited time I had for the project, my team decided to focus on measuring whole gut serotonin instead. This highlights how quickly a research project can change and the many approaches a scientist can take to solve a problem. There were also times I would try a new technique to yield better results, but would ultimately go back to the original technique. Overall, the general research process was to look back to see how an experiment was done in the past, and then complete a series of trial and error cycles from there to optimize the protocol for my specific project.

After collecting and preparing samples, I ran a test called an enzyme-linked immunosorbent assay (ELISA) that quantifies the amount of serotonin in a sample.

Amid the journal clubs and experiments, this summer I learned about the collaborative efforts required to be successful as a scientist. I shared equipment and bench space with other researchers in the lab, so we had to coordinate a schedule so that everyone could complete their experiments. I enjoyed getting to know the other members of the lab, many with diverse backgrounds and specialties. Every week the department had a lab meeting where someone from each lab would take turns presenting research updates. It was a great opportunity to learn about other projects that related to the enteric nervous system and new adjustments made to fix problems. That being said, life as a researcher has its challenges. There were days I would spend hours preparing an experiment, only to made a mistake that could have been prevented if I would have asked the right person. If I had to give any advice from my summer experience, I would say don’t be afraid to ask for help – especially if you are feeling discouraged! Everyone in a lab develops his or her own expertise in something, which makes it impossible to know everything. But one of the beauties of science is that there is still a lot for us to learn, and sometimes the most frustrating failures can bring unexpected discoveries.

References

  1. Mawe GM, Hoffman JM. Serotonin signalling in the gut–functions, dysfunctions and therapeutic targets. Nature Reviews: Gastroenterology and Hepatology 10: 473-86, 2013; 10.1038/nrgastro.2013.105.
  2. Berger M, Gray JA, Roth BL. The expanded biology of serotonin. Annu Rev Med. 60: 355-66, 2009; 10.1146/annurev.med.60.042307.110802.
  3. Sumara G, Sumara O, Kim JK, Karsenty G. Gut-derived serotonin is a multifunctional determinant to fasting adaptation. Cell Metab. 16: 588-600, 2012; 10.1016/j.cmet.2012.09.014.
  4. Wang F, Knutson K, Alcaino C, Linden DR, Gibbons SJ, Kashyap PK, Grover M, Oeckler R, Gottlieb PA, Li HJ, Leiter AB, Farrugia G, Beyder A. Mechanosensitive ion channel Piezo2 is important for enterochromaffin cell response to mechanical forces. J Physiol. 595: 79-91, 2016; 10.1113/JP272718 PMCID: PMC27392819.
  5. Alcaino C, Knutson K, Gottlieb PA, Farrugia G, Beyder A. Mechanosensitive ion channel Piezo2 is inhibited by D-GsMTx4. Channels. 11: 245-253, 2017; 10.1080/19336950.2017.1279370.
Alecia Alto attends the University of Minnesota Rochester in Rochester, MN. She is a 2017 Undergraduate Summer Research Fellow (UGSRF) doing research in Dr. Arthur Beyder’s lab at Mayo Clinic in Rochester, MN.  After graduating with a bachelor or science in health sciences, she is considering a career as a physician scientist.

 

January 12th, 2018
Frozen In Time: The Story of a Tree Frog and its Potential to Help Humans Freeze Their Organs, and Possibly Themselves

I know what you’re thinking. What in the world does a tree frog have to do with humans freezing their organs? And yes, before beginning my undergraduate research project, I too would likely be thinking this exact same thought. But nature allows animals to do a very funny thing: adapt according to their environment. And the gray tree frog that you are itching to read about in the following paragraphs has adapted in one of the most extreme ways found in the animal kingdom: it has the ability to freeze itself and survive.

Credit: John White, University of California, Berkeley

My 2017 summer research project, funded through the American Physiological Society, is focused on Cope’s gray tree frog, Hyla chrysoscelis. H. chrysoscelis has developed a dramatic strategy to combat the cold during the winter months, known as freeze tolerance. Freeze tolerance permits this tree frog to withstand the cold by allowing it to convert 50-65% of its total fluid body water into ice. During its time spent frozen, Cope’s gray tree frog experiences fundamental changes in its physiology and biochemistry, including the abandonment of:

  • Blood circulation
  • Breathing
  • Nerve conduction
  • Brain activity
  • A heartbeat

Yes, you read that right. It has no heartbeat while it is frozen.

However, as wintertime comes to a close and external temperatures rise, the thawing process begins, vital signs reactivate, and normal life resumes for this frog. My research project for the summer is aimed at understanding a specific cellular process involved in the whole body freezing of H. chrysoscelis, which may provide future scientists with the capacity to not only freeze human organs in the future, but also freeze our entire bodies.

H. chrysoscelis experiences a variety of stresses during freezing and thawing, most of them being osmotic stresses. This is because the majority of the fluid surrounding the frog’s cells freezes, leaving a higher concentration of solutes outside the cell than prior to freezing. To combat this disruption of fluid homeostasis, H. chrysoscelis has evolved a transmembrane protein known as aquaglyceroporin HC-3 that acts as a channel for water and solutes to flow through, which helps to re-establish a fluid homeostatic equilibrium. In non-science terms, this protein, HC-3, helps to keep the cells of this frog from shriveling up like raisins or bursting open like a balloon with too much air.

My hypothesis demands a week long experiment, involving a variety of techniques that enable me to begin with the blood of this frog, fresh from its brachial artery, and shrink all the way down to the molecular level to analyze HC-3, the protein that I suspect to be a vital player in the freezing process. The first part of my experiment requires culturing red blood cells from Hyla chrysoscelis over a period of 48 hours, which in non-science terms translates to: keep 90% of these cells alive over a period of two days. Sounds simple, right? Well, after the first two weeks of failed attempts in maintaining proper viability of the cells in culture, I was left devastated, demoralized, and with numerous flasks of cells contaminated with who-knows-what. Science, without mercy, and in just a period of two weeks, showed me just how difficult it would be to successfully complete my week long experiment by halting me after just the first two days.

As the summer has progressed, I have finally achieved viable cell cultures for the full 48 hours through troubleshooting and controlling for one variable at a time, leading me to the conclusion that a certain reagent I was adding to the media my cells were kept in had been contaminated earlier in the summer. Currently, I am now focusing on more demanding techniques, such as western blotting and immunocytochemistry, while still always encountering new obstacles to overcome and problems to solve. New to research, and even newer to researching full time, I have come to enjoy the independence and autonomy that I’ve found in the laboratory. Every day is a new opportunity, and a new challenge, demanding my full attention and effort to successfully complete my experiments for the day. Through the few successes and many failures of my summer of research thus far, I have gained a deeper appreciation for science and continue to aspire to engage in scientific research throughout the rest of my professional career.

References

  1. White, John. “Hyla chrysoscelis, Cope’s Gray tree frog.” University of California, Berkley Regents (2006).
Dante Pezzutti is a rising senior majoring in Pre-Medicine at the University of Dayton in Dayton, Ohio. He is a 2017 Undergraduate Summer Research Fellow (UGSRF) working in Dr. Carissa Krane’s laboratory at U.D. over the summer. Dante’s fellowship is funded through the American Physiological Society. After graduation, Dante plans to attend medical school to become a physician, and also aspires to engage in bench-to-bedside translational research as a medical doctor.
January 9th, 2018
Research Project Narrated for My Parents

My summer research project, which will span much of the next year, revolves around Classical Ferroportin Disease. Firstly, before explaining the minutia of Classical Ferroportin Disease it is important to know what ferroportin is; ferroportin is an iron transport protein that is responsible for exporting iron out of cells. Classical Ferroportin Disease is a loss of function disease, meaning that a mutation has occurred in the protein that makes ferroportin less efficient [1]. Seeing as though ferroportin is exporting iron out of cells, patients who have Classical Ferroportin disease can get iron into their cells, but their cells struggle to get iron back out of the cells. This ferroportin malfunction results in a net accumulation of iron in cells above what is physiologically healthy; macrophages of the liver and spleen become particularly iron overloaded because they metabolize a lot of iron. The disease can present clinically as anemia because the body is unable to acquire the adequate iron it needs for red blood cell production since the iron is being sequestered in the liver and spleen macrophages with no way to be properly exported [2]. The disease can also cause diseases of the liver such as fibrosis and cirrhosis because iron overload of liver cells is detrimental to liver health and functioning.

It is known that Classical Ferroportin exhibits autosomal dominant inheritance; my summer research project is interested in shedding light upon how Classical Ferroportin Disease is inherited via an autosomal dominant route. There are two models to explain the autosomal dominance that classical ferroportin disease exhibits. One model is a haploinsufficiency model, the other is a dominant-negative effect model [1]. These terms may sound complex but they are fundamentally simple. The haploinsufficiency model can simply be thought of in terms of dosage. When you make a cake the recipe may call for five eggs, if you were to only put one egg into the cake, said cake would turn out abysmally. Haploinsufficiency is exactly the same way; you inherit one copy of ferroportin from each parent. You need two good copies to be a well baked cake and make enough functional ferroportin protein, but if you only get one good copy and one mutant copy then you will be an abysmal cake with not enough functional ferroportin to properly export iron. All in all, in the haploinsufficiency model you don’t have enough functional ferroportin to keep up with the export rates needed to be healthy. The second model, the dominant-negative effect, is a little more complex but still fundamentally simpler than the name implies. This model is best envisioned as an arts and crafts project. Your task is to make a sphere out of two objects and super glue. THE TWO OBJECTS MUST COME TOGETHER TO CREATE THE PRODUCT. In the working art studio you’d be provided with two hemispheres and super glue; you would with ease construct the sphere and happily go about your day. In the art studio run by people lacking infant geometry knowledge, you are provided with one hemisphere, one cylindrical object, and super glue. No matter how hard you try with the materials provided you cannot construct the desired spherical object. Out of frustration you glue the block to the sphere permanently and leave full of rage to throw the mushroom shaped object at a wall.  The complete sphere, representing a healthy person without ferroportin disease, can be made when you are provided with two good hemispheres, one healthy ferroportin gene from each parent. The second art model, where the person is forced to construct a mushroom shaped object, represents Classical Ferroportin Disease via a dominant-negative effect. The person was forced to glue the hemisphere, a healthy ferroportin gene product, to a cylinder, a mutant ferroportin gene product, creating a non-sphere and thus a ferroportin protein unable to export iron. They key distinction between the haploinsufficiency model and the dominant-negative model is that in the haploinsufficiency diseased state you simply do not have enough of the good product, whereas in the dominant negative model the bad product hinders the good product from working all together. Overall my research project revolves around trying to figure out which model Classical Ferroportin Disease follows and my tools to do so are Xenopus laevis oocytes, human mRNA injections, confocal fluorescent microscopy, and scanning electron microscopy. This project falls into a huge sector of molecular biology and physiology that is cellular transport and it will aid in the treatment and understanding of one type of a family of diseases that fall under the name of Hemochromatosis.

Realities of Research

Experimental design is key; hard work pays handsomely in the form of pretty graphs; don’t start to genotype at 3 PM’ and experiments will be repeated a frustrating amount of times until those variance bars look pretty.

The importance of well thought out experimental design became a frustrating reality about 5 weeks into my project. I had been tirelessly performing experiments for weeks in a way that could only be analyzed via a one-way ANOVA. An ANOVA is a commonly used statistical test used to analyze data; all statistical tests require an individualized experimental design in order to be analyzed via a specific test. One day at lunch my PI and I were discussing my progress and he looks at me worriedly and says “How have you been setting up your experiments and how have you been analyzing the data?” He then went on to inform me that my experimental design had a critical weak point and that a one-way ANOVA would not provide the insight needed to make a strong claim on my hypothesis. So the rest of the lunch was spent redesigning my experimental approach so that I could run a two-way ANOVA, yet just another statistical test. This taught me that experimental design is worth troubling your mind over for a long while because good data is meaningless unless it is masterfully collected with a specific statistical analysis in mind.

A lab reality surprise I encountered was how rewarding good data and graphs can be. I grew up playing baseball and soccer and I excelled at them, therefore I know the feeling of hitting a homerun and scoring a goal. They are full body highs encompassed with an adrenaline surge and confidence boost. I was surprised how close to those feelings I got when I got my first very usable data set and graphs. It’s very comparable to sports because you train in much the same methodical way for so long so that when you finally see results it feels amazing. Hard work does in fact pay handsomely with good data and graphs.

Another lab reality is planning your day saves your dinner date. I learned this the hard way multiple times when I started an experiment, genotyping, or prep way too late in the day.  It has become increasingly apparent that planning is your best friend in the lab. Fifteen minutes of thoughtful planning at the start of the day may save you hours each day. AKA don’t hold off on very pressing genotyping until 3 PM unless you plan to be in the lab until 9 PM.

Lastly, a frustrating lab reality is that some experiments will have to be repeated so many times you that start to worry you’ll be running the same experiment with the same experimental groups forever, or even worse you fear they will never give you interpretable data…. gasp. This is a frustrating reality that can never truly be realized until you have suffered first-hand through running the same experiment for weeks on end. It always gets better though; see baseball homerun feelings above.

Life of a Scientist

The most striking part of the day to day of a scientist is how social it is. I always assumed scientists were reclusive and withdrawn. I was wrong, outright wrong. The lab I work in is bustling with life. Intellectual exchange and idea bouncing occurs incessantly as we pipet about running assays. We come together at lunch and effortless fill the air with debate and jovial disputes. But the chit chat goes beyond academics, which I was most surprised about. Beyond bouncing ideas off each other and discussing relevant academic papers, a plethora of enticing conversations about extracurricular activities also buzz loud. We talk about music, the best coffee places, the trendiest foods, sports, and philosophy. Science is inescapably social in a beautiful way. Another thing I love about being a scientist is that your schedule is decently malleable as long as you get plan well. You are able to catch lunch with a friend or leave a little early one day if you make up for that time and work somewhere else; I am trying to get at the fact that science is not a strict 9-5 because as long as you get your work done no one is going to hound you about when you do it. This is a huge plus because it plays very well into an adequate work-life balance.

References

  1. Hal Drakesmith, Elizabeta Nemeth, Tomas Ganz, Ironing out Ferroportin, Cell Metabolism, Volume 22, Issue 5, 2015, Pages 777-787
  2. Mayr, Roman et al. “Ferroportin Disease: A Systematic Meta-Analysis of Clinical and Molecular Findings.” Journal of Hepatology5-3, 2010
John Bonamer is a senior majoring in Medical Sciences at the University of Cincinnati in Cincinnati, Ohio. He is a UGSRF fellow working in Dr. Bryan Mackenzie’s lab at the University of Cincinnati. John’s Fellowship is funded by the APS. After graduation John plans to pursue an MD/PhD with special interest in Urology.
January 5th, 2018
My Summer Experience Observing Changes to Sarcomeres with Titin Regulation

Muscles are comprised of repeating units called sarcomeres, which can be seen as bands in myofibrils. Prior research has shown that the consistency of the lengths of these bands correlates to muscle strength – the more consistent the bands, the stronger the muscle. My project focuses on the relationship between the consistency of these sarcomeres and titin, the longest known protein which is proposed to maintain sarcomere length. Our lab developed an inducible skeletal muscle specific Bmal1 knockout mouse that demonstrates a to a longer titin isoform. By altering titin and comparing the variance of lengths of these sarcomeres to a control group, I hope to demonstrate a correlation between sarcomere length and the expression of titin. My project takes a histological approach to study changes in sarcomere length and A-band centrality in the skeletal muscle by staining for alpha actinin and myosin. These images are overlaid and peak fluorescence is measured. Knowledge from this experiment would contribute to knowledge about titin and could eventually lead to more targeted drugs or therapies. Results of my experiment thus far indicate that there is a trend toward a longer sarcomere length and no significant change in variance, but there are still other stains and trials I would like to do to strengthen my findings. If my hypothesis is supported then my project would support the assertion that titin has a structural role in the sarcomere.

The myosin stain allowed me to see if there were any differences in the location in the A-band in relation to the Z-line.

The alpha actinin stain labels the z-lines, allowing the sarcomere to be visualized and measured.

Realities of Research

Research in Dr. Esser’s lab was very different from what I had anticipated. Generally, people think of scientific research as solitary, but what I have found is there is a lot of collaboration in academic research. When we encountered an error in the RNA sequencing program of the model, and we reached out to other people at the university in order to help understand and correct this error. Additionally, we borrowed equipment such as the cryostat (a machine used to cut muscle at cold temperatures) and microscopes from other labs. Lab meetings were also a useful part of my experience because they allowed for brainstorming when I faced challenges. In addition to the RNA sequencing obstacle, there was also a problem with a control antibody stain because there was fluorescence in an unexpected region. After discussing the problem, we came up with the hypothesis that the secondary stain for one of the regions was attaching to the conjugated antibody of another region, which was why we were seeing the unexpected fluorescence. To correct this, I applied the antibodies in two steps instead of combining them. All of these obstacles were easier to overcome because I had people around to assist me, an invaluable asset that I did not expect.

Day-to-Day of a Scientist

I look forward to coming in to the lab because I like the challenge of overcoming the unforeseen obstacles. Designing ways to solve problems is definitely my favorite part of working in a lab. The production of ideas about how an experiment or procedure should work and testing the methods is exciting. While these obstacles can be a nice challenge, they can also become tedious. The problem with the RNA sequencing was especially frustrating because it was out of our hands. Despite these complications, my research was overall very enjoyable. I am very appreciative of the APS for allowing me to pursue my interests this summer, and would encourage others to do a program because the experience was very rewarding.

Joseph Mijares, also known as Robby, is a junior at the University of Florida in Gainesville, FL. He is grateful to be a 2017 Undergraduate Summer Research Fellow in Dr. Karyn Esser’s Lab at the University of Florida.  Robby plans on applying to several MD and MD-PhD programs after his junior year, with hopes of attending after graduation.
December 19th, 2017
Post Spinal Cord Injury Female Mice have a Higher Concentration of Glial Cells

In the laboratory this summer, I studied one of the myriads of cell responses that occur in the spinal cord post-injury. The cells that I studied were microglia, which play a role in cleaning up debris and acting as an immune response. It has been found that post-SCI (spinal cord injury) female mice have better functional recovery than their male counterparts. Scientists studying SCI’s are currently researching possible causes for this difference in healing. My project focused on whether male or female mice have a higher microglia response post injury. The tissue I analyzed was collected from male and female mice 42 days post-moderate SCI. I first had to stain the spinal cord sections with eriochrome cyanine, which shows the degree of degeneration the spinal cord underwent post injury. Immunohistochemistry was then performed on the tissue, which is a form of staining that binds fluorescent antibodies to the cells you are looking for. Because of the fluorescence that is attached to the cell, you can then use light to illuminate the microglia and then image the cells. To get the most accurate comparison, I selected a portion of the spinal cord that I could identify in every animal and quantified the cells in just that area. My data concluded that female mice have significantly (p: .0187) less microglia.

Realities of Research

This was my first time taking part in research in a professional lab setting and there were a few things that surprised me, but for the most part I knew relatively what I was getting into. I think the most surprising thing was that there was a lot of sitting around and waiting for either results or for my slides to dry. As a student that works better when pressurized, I feel that this aspect will be the biggest barrier keeping me from going into a research career. There were also a lot of issues that I had with the tissue itself and aspects that hindered my ability to analyze my results.

My responsibilities changed from day to day, whether I was sitting at a desk and reading for hours straight or doing a stain. I also spent a lot of time learning about lab equipment and how to safely complete an experiment. I think the best part was when I was able to work by myself and take ownership of my own project. However, there were many times that I was lost or confused and really needed help. The worst part of this research was all of the down time that I had. Working as a lab team was very interesting because there were always people around that had something interesting to contribute to my project. However, there were also issues with finding the specific person that could help you with a part of your project.

Julie Wilson is a senior majoring in Biology and Chemistry at Baldwin Wallace University in Berea, OH. She is a 2017 Integrative Organismal Systems Physiology (IOSP) Fellow working with Dr. John C. Gensel at the University of Kentucky in Lexington, KY. Julie’s fellowship is funded by the APS and a grant from the National Science Foundation Integrative Organismal Systems (IOS) (Grant #IOS-1238831). After leaving Baldwin Wallace University, Julie plans to attend medical school and pursue a career in pediatrics working with queer and diverse youth.
December 15th, 2017
My Experience Interpreting Oxidative Stress and Inflammation in Hypoxia Using Lipid Metabolism

This summer I worked on a metabolomics project surrounding the effects that hypoxia, or deficient oxygenation, has on oxidative stress and inflammation. I used metabolomics, or the study of the functional molecules in the body, to interpret the molecular changes that eventually lead to physiological complications. Currently, we know that oxylipins, biological molecules that are metabolized from polyunsaturated fatty acids (PUFAs), are markers of oxidative stress and inflammation in conditions such as hypoxia (1). My lab extracted venous plasma from fetal and newborn sheep that were living at high-altitudes, as hypoxic conditions can be simulated by high-altitude living. By running tests that quantified the oxylipins in fetuses and newborns, I was able to distinguish which metabolites were prominently affected by hypoxic conditions. Later, I was able to find pathways, tracing how certain PUFAs were metabolized and from which PUFAs certain oxylipins were derived. Based on these relationships, I aimed to find possible roots for the oxidative stress and inflammation caused by chronic hypoxia.

These findings highlight our understanding of lipid metabolism as it is affected by high-altitude hypoxia. This study has the potential to help us develop treatments that target inflammatory pathways induced by pre and post-natal hypoxia. For instance, the enzymatic pathway CYP, which metabolizes PUFAs proved to play a large role in the production of oxylipins that were affected by hypoxia. Targeting this pathway early in the womb may help prevent lung dysfunction that may develop just after birth.

An area in my research that I found difficult was the dense literature. At first sight, it was intimidating‒ scientific jargon and compound nomenclature most of all. I realized that as I started connecting terms to function‒ associating oxylipins with potential roles in the body was now feasible. The truth is, it takes time and understanding to grasp the material, but the more I read and the more I searched, the less intimidating it all felt. Around the lab, there are several skills to master‒ several of which consist of success and failure. For instance, I had a hard time developing networks for my metabolites and working with statistical software early on in my research; this is now something I wish to improve. Often, I received results from my data that I did not expect and it reminded me how difficult it was to remain impartial. I ran into a list of questions that, over time, clarified and narrowed what in fact my research would delineate.

I could summarize the life of a scientist in one word: unpredictable. It surprised me how difficult consistency actually was with data. That being said, it is a huge task to filter data and focus on only a few aspects of it; everything seemed important. Moreover, a reliable, cooperative lab team is a vital component to a scientist’s life in the lab. While not everyone is specialized in the same subject or project, a team creates a supportive environment where we feed off of one another’s knowledge and work in collaboration for the interest of science.

References

  1. Gabbs M, Leng S, Devassy JG, Monirujjaman M, Aukema HM. Advances in Our Understanding of Oxylipins Derived from Dietary PUFAs. Adv Nutr 6: 513–540, 2015.
Vanessa Lopez is a junior Biochemistry major at Occidental College in Los Angeles, CA. She is a 2017 Short-Term Research Education Program to Increase Diversity in Health-Related Research (STRIDE) Fellow. She works with Dr. Sean Wilson in his lab at Loma Linda University in Loma Linda, CA. Vanessa’s fellowship is funded by the APS, as well as a grant from the National Heart, Lung and Blood Institute (Grant #1 R25 HL115473-01). Her research is also supported by NIH grants HD083132 [LZ], 1U24DK097154 [OF] through Dr. Wilson’s lab. She is interested in endocrinology and dietetics. Her plan is to go to medical school after graduation.
December 12th, 2017
Science That Gets Your Blood Racing

Our lab studies cardiovascular diseases such as high blood pressure (hypertension), which affects roughly one third of American adults and puts them at a higher risk for heart disease and stroke, both of which are leading causes of death in the United States [1]. One of our overarching questions addresses the role that the tissue surrounding our blood vessels, or “perivascular adipose tissue” (PVAT), plays in obesity-related hypertension. PVAT contains various types of cells, including adipocytes (fat cells), endothelial cells, macrophages, lymphocytes, fibroblasts, and more [2]. We have previously shown that PVAT contains functional norepinephrine, a signaling molecule that constricts blood vessels and thus increases blood pressure [3]. My project addresses where and how PVAT actually stores this norepinephrine using the PVAT surrounding normal rat mesenteric resistance vessels- the small arteries and veins that branch into the small intestine and are important for blood pressure regulation. We hypothesize that it is specifically the adipocytes in PVAT that store NE, and that they use the vesicular monoamine transporter (VMAT) to do so. A better understanding of this mechanism is important for the future development of treatments for obesity-related hypertension.

[4] Sprague Dawley Rat. Photo Credit: Charles River

Realities of Research

When I first joined the Watts Lab a little over three years ago, I was a wide-eyed freshman who had never even used a micropipette before. Since then, my wonderful mentors have trained me in methods such as immunohistochemistry, brightfield and fluorescent microscopy, cell culture, handling and euthanizing rats, performing dissections, and isolating adipocytes from rat PVAT. More importantly, through these lab experiences, I have learned a scientific way of thinking and hypothesizing. This has been essential for my research project because it was incredibly challenging to develop a new protocol for the functional experiments using live adipocytes. It took over a year and endless troubleshooting for me to get to a point where I had a working procedure, and even longer to replicate the experiments to obtain a large enough sample size. One major obstacle was that freshly isolated adipocytes do not attach well to surfaces such as a microscope chamber slide. Additionally, one of the drugs I tested is only soluble in ethanol, which was toxic to the cells at most concentrations, so we had to find an alternative compound to use. Through trial and error, we have designed methods to effectively isolate, treat, and image PVAT adipocytes for this application. I am also very proud of the protocol we developed to quantify the fluorescent intensities of the cells I imaged, as it is crucial to analyze and present data in as objective and consistent a way possible. I was able to present our work in San Francisco at Council on Hypertension this past September, which was truly an incredible experience.

Conducting research in a lab has been one of the most rewarding adventures of my life. There is a certain thrill in knowing I am working to answer a question that no one else in the world is investigating in the same way. There are definitely setbacks that can be difficult to deal with, such as antibodies that do not work, cells that die for seemingly no reason, or not knowing how to begin designing an experiment you have in mind. However, this is why I love being a part of the scientific community- I have the opportunity to collaborate with the other scientists in our lab and department, or even at other institutions, to gain insight into how to better approach a research question. In addition to the hard work any researcher has to put into his or her own project, I believe it is this enthusiasm to help one another that ultimately allows all of us to be successful. Oh, and a little bit of luck never hurt anyone!

References

  1. High Blood Pressure Fact Sheet [Online]. Centers for Disease Control and Prevention. Centers for Disease Control and Prevention: 2016. https://www.cdc.gov/dhdsp/data_statistics/fact_sheets/fs_bloodpressure.htm [14 Jul. 2017]. 
  2. Miao CY and Li ZY. The role of perivascular adipose tissue in vascular smooth muscle cell growth. British Journal of Pharmacology 165(3): 643-658, 2012. 
  3. Ayala‐Lopez N, Martini M, Jackson WF, Darios E, Burnett R, Seitz B, Fink GD, Watts SW. Perivascular adipose tissue contains functional catecholamines [Online]. Pharmacology Research & Perspectives: 2014. http://onlinelibrary.wiley.com/doi/10.1002/prp2.41/full [14 Jul. 2017]. 
  4. CD® IGS Rat Crl:CD(SD) [Online]. CD® IGS Rat | Charles River. http://www.criver.com/products-services/basic-research/find-a-model/cd-igs-rat?loc=US [14 Jul. 2017].
Maleeha Ahmad is a senior majoring in Genomics and Molecular Genetics at Michigan State University in East Lansing, MI. Her Summer 2017 Undergraduate Research Excellence Fellowship (UGREF) gave her the opportunity to continue working in Dr. Stephanie Watts’s lab at Michigan State University, where she has been conducting research for the past three years. Maleeha’s fellowship is funded by the APS. After graduation, she plans to attend medical school and be involved in clinical research.
December 1st, 2017
Amniotic Membrane Supplementation in Rotator Cuff Reconstruction

Amniotic membranes have been of recent inquiry in the surgical world because of their composition of stem cells. These stem cells can differentiate into the desired type of cells in the body. In this study, amniotic membranes were placed on the insertion of the rotator cuff muscles after rotator cuff surgery to see how this affected recovery time. In order to determine this, the strength of the individual muscle fibers was determined using the single fiber isometric strength method. This method involves pulling out a single muscle fiber from a bundle of fibers obtained from the rat, tying it onto the apparatus and exposing it to high levels of calcium in order to allow for a contraction to occur. After determining the cross-sectional area and the peak isometric force, the specific force (the force per cross-sectional area) can be calculated in order to compare the relative strength of fibers from different samples. This study consisted of four different groups; uninjured control (no surgery performed), control repair (no amniotic membrane supplementation), injury only (no rotator cuff surgery performed), and experimental repair (surgery with amniotic membrane supplementation). There were six fibers obtained from each rat sample with 8 rats per group totaling 192 fibers. Once the specific force is determined for every group, comparisons were made to see if the amniotic membrane supplementation helped restore the specific force of the rats 4 weeks post intervention. This is applicable to human rotator cuff surgeries because it could help patients recovering from this surgery and decrease the recovery time. This would allow for these individuals to return to their normal activities more quickly. In addition, this method can be used in multiple different surgery sites to help improve recovery time.

Realities of Research

This project got frustrating at times because of the variable conditions of the fibers. There were some bundles that contained large, healthy fibers and some that contained extremely small and frail fibers (size comparison can be seen in images 1 and 2). While we did not know what groups these fibers were a part of because of the double-blind format, it was possible to infer which groups these fibers were from. Another interesting thing about this study is that it took three weeks to train for this technique. It started with learning how to tie the minuscule ties used to tie the fibers and then learning how to tie them on a practice machine. We practiced on separate machines because of the high probability we would break the real apparatus if we started on that. However, once data collection started, around 12-18 fibers were run each day and real progress was made. It was also interesting to see the variability of specific forces in a given bundle. This is something that we were not expecting, but was most definitely present. Luckily, the research question never changed, but there were many times that we would have to re-run fibers due slippage, ripping or any other issues that presented themselves.

The difference in cross sectional area for given fibers from different rats.

 

 

 

 

 

 

 

 

 

Day-to-Day Life of a Scientist

Life as a researcher was much more relaxed than I initially thought it would be. The arrival and departure time for each day was variable. While it was expected that you completed all of the work for the day, this could be done earlier or later in the day. This made it very low stress and everyone in the lab was extremely friendly and fun to be around. The best part of the research was definitely at the end of data collection when we were able to compare the specific forces and see if the amniotic membrane supplementation was effective. However, the worst part was definitely the struggles that I had with a specific rats sample that took me three days to get 6 good fibers run. I also had a great time in the collaborative lab meeting that was help in August with the entire research team. This gave an amazing opportunity to share results from multiple aspects of the project and see how all of the data fits together.

Jeffrey Kepple is a senior at Gonzaga University in Spokane, WA. He is a 2017 Undergraduate Summer Research Fellow (UGSRF) doing research in Dr. Chris Mendias’ lab at the University of Michigan in Ann Arbor, MI.  After graduating, he plans on pursuing an MD/PhD.
November 28th, 2017
It’s Getting Harder and Harder to Breathe! ~Maroon 5

This summer I have been hard at work in the Student Development Complex, here at Michigan Tech. It is a closed room with no windows, kind of odd for an exercise nut, but some of the best things are happening there! We have research going on testing the human body to its limits. Specifically, my research is testing the limits of our respiratory system during exercise. I believe that respiratory muscles are important in performance and if we can understand the limiting factors of the respiratory then we can help people with asthma, chronic obstructive pulmonary disease (COPD), and team and endurance athletes. I am taking members from our cross-country ski team and putting them through two arm cycling trials to failure, i.e. biking for your arms. One trial their whole body is 100% fresh, the second their respiratory muscles have been fatigued ~20%. During the trials, I track the time and all their respiratory and metabolic functions and compare them between the trials. I have had six people go through the tests and have seen a surprising decrease in time to failure when respiratory muscles were fatigued. Additionally, their breathing rate was low and volume high during the normal condition and it flipped during the fatigued condition, showing that when the muscles are already fatigued they work even harder trying to sustain performance. The next step in my study is finding out how and why this happens.

Tom Bye performing equipment tests while designing his protocol, Michigan Technological University

Here in the Upper Peninsula of Michigan it can get hot, for us at least. Our lab is air conditioned, unlike most buildings up here, so we can have a consistent climate for testing and it is the place to be somedays. Research in the lab is a blast! When I have subjects in for testing we keep the mood light and keep them determined with some tunes, conversation, and cheering. There are also pros and cons to the lab, because I have ski team commitments to come accomplish, my mornings arriving in the lab for set-up are as early as 6:15 AM and nights can be the same, ending late, crunching numbers or safety checking our laboratory. I have been learning a lot over the summer about different programs and technology we use in the lab. I’ve been fortunate enough to use ultrasound, metabolic carts, NIRS devices, and EMG. Currently my task at hand is to learn SigmaPlot, a graphing program that is Excel on steroids; I’m getting good at it! The main tests that I am running on subjects are maximum aerobic capacity and maximum upper-body exercise tolerance tests. Using flow meters I have also been able to test their basic respiratory function, one subject’s lungs could take in over 8 liters of air! A normal person his size would normally take in 5-6 liters of air. Our results are looking like we expect when we plot them out, but statistically only some are significant. The next step is to keep trucking on and see if the results stay consistent!

Tom Bye pushing a subject to their max during pilot data collection, Michigan Technological University

Our lab isn’t just me and my research host, we are a team of many undergraduate, graduate, and doctoral students! It is amazing to see how we transfer knowledge and share ideas with each other. It is great that I can go to a doctoral student (a goal of mine) and get info on schooling and my research. I have been in the lab a while now and teach some of the undergraduate and graduate students how the equipment works with their research project protocols. In a few weeks, we will have a testing blitz for my project and get five participants through five days of testing each! My worst experience so far is troubleshooting our metabolic carts and lab computer… It was supposed to take an hour and ended being the whole day. My favorite part of lab also surprises me too, it is the intensity that all the team members bring to the lab, but when we have a BBQ we are all relaxing and playing yard games.

Thomas Bye is a senior studying Exercise Science at Michigan Technological University in Houghton, MI. He is a research fellow in Dr. Steven Elmer’s laboratory at Michigan Technological University in Houghton, MI. He is supported by the American Physiology Society Undergraduate Summer Research Fellowship. After graduating, he plans on becoming a physical therapist and being involved with research in sports. Later in life, he would like to be a professor to pass on what he has learned.