Tag Archives: UGSRF

Regulation of Salinity Stress Tolerance in the European Green Crab, Carcinus maenas

Throughout the summer of 2017, I worked in the invertebrate physiology lab under Markus Frederich, investigating how the enzyme AMPK affects the salinity stress tolerance of European green crabs. Using crabs collected from the Maine intertidal areas, which often experience variances in salinity and temperature, I focused on injecting and activating this enzyme. By doing so in settings of different salinities or temperatures I was able to see how the enzyme plays a role in ion regulation and stress tolerance, giving insight into what makes this species such  successful invaders.

Participating in this research experience has been extremely valuable. I have been able to acquire lab skills which are imperative to my future as a scientist, while also receiving a unique opportunity to develop a very strong understanding of a very specific topic that is very exciting. Coming into my summer research experience, I felt nervous and overwhelmed thinking that I had so much to learn in so little time, but very quickly I got into a comfortable position of diligent work, where I learn something new every day. Scientific research involves patience, and while everything might go right the first time, I have learned that it may not be because of a mistake, but that a different approach needs to be taken.

Being a scientist is a surreal experience because it is such a meticulous practice. The smallest things can cause large alterations in data, and knowing that makes me appreciate the practices of scientific research that much more. Every day involves thinking outside the box, and a true dedication of time. The best part is learning something new every day, and while it may be exhausting to sometimes go multiple days with nothing making sense or things not working, there comes a moment where something new is discovered, which is extremely rewarding. I am truly appreciative of the lab that I work in because everyone is willing to help each other, working together to answer questions, and learn from one another. My advisor specifically has been very influential in making this summer experience one of the best things I have ever done, and has been a strong influence for my love for research.

 

Pierce Lancor attends the University of New England in Biddeford, ME. She is a 2017 Undergraduate Summer Research Fellow (UGSRF) and worked in Dr. Markus Frederich’s lab over the summer at the University of New England. After graduating, she plans to continue her education and research toward a masters and PhD in zoology.
Kidney Gene Per1 Regulation of Blood Pressure

This summer I had the great opportunity to work as a fellow within the American Physiology Societies Summer Undergraduate Research Fellowship program. I was able to study under Dr. Michelle Gumz who focuses on circadian rhythms in the kidney at the University of Florida. Circadian rhythms help us wake up and brush our teeth in the morning and fall asleep the same time every evening. Four distinct classes of genes called Per, Cry, Bmal, and Clock, regulate these rhythms. Our lab studies the regulation of Per1 and its effects on blood pressure as a circadian rhythm. Mean arterial blood pressure decreases at night when humans sleep, called “dipping” and peeks when we first wake in the morning (Solocinski, et al., 2016). My research project is designed to study the effects of removing the Per1 gene globally from the 129/sv/s1s4 mice background. After the mice are developed we inserted a radio-telemetry device to monitor their hourly blood pressure. With that data we run specific statically analysis, including cosinor analysis. Cosinor analysis is a program to studies the mesor, amplitude, period, and acrophase of the mean arterial pressure (MAP) of each animal. The mesor, midline-estimating statistic of rhythm, is the midpoint on the y-axis and based on the distribution of MAP across the circadian rhythm. The amplitude is the distance between the mesor and the maximum/minimum point of oscillation. The period is the measure of a full circadian wave. The acrophase is the distance along the cycles peaks or crests that fits the measurement of the circadian rhythm. Each variable allows us to observe the MAP in a circadian pattern to determine the variation between WT and KO 129/sv/s1s4 mice. After I complete the analysis of the blood pressure data from the 129/sv/s1s4 mice we will then run western blot and immunohistochemistry analysis to observe the different proteins within the kidney.

Working in a biochemistry research lab everything is measured in micro values and we work with small portions of protein and DNA to test for specific genes. The process of discovering new proteins expressed in wild type or knockout kidneys is an exciting experience. We develop new conditions for western blots, which is a detection process for select proteins within a sample by using antibodies. Each antibody needs individual concentrations and washing methods to discover the correct ratio to produce a viable band of protein. In addition to study the changes within different proteins levels it is also important to observe the physiological response within the animal models. Measuring hourly blood pressure points allows us as researchers to recognize how the animal is adjusting towards the global removal of the Per1 gene. Once we understand the physiological response and how the body adapts to gene changes we can then apply this result to human studies. Blood pressure regulation is a major contributor to heart disease. If we are able to better comprehend the circadian clock gene regulation within the kidney we could improve cardiovascular outcome.

Our procedures for western blots and immunohistochemistry can take two to three days to see results. It can be concerning when we do not produce correct results, but as a lab we make adjustments and start the protocol over again. Through the optimizing process it can take weeks to produce a blot with results from our Per1 knockout mice population. However, once we optimize the antibodies we can test multiple samples to gain data for publication. Working alongside Dr. Gumz and her PhD students, it creates a reliable environment for undergraduate students learning basic scientific procedures. Working with a team allows me to make mistakes and learn how to correct different procedures to perfect western blot, immunohistochemistry, and telemetry protocols. The life of a scientist can be very rewarding when you are able to optimize protocols and receive results that can determine different gene regulations. Within the Nephrology Department at the University of Florida we attend weekly meetings to discuss other research from a variety of department labs. This allows me to learn about different lab protocols and apply their procedures to our current studies to produce ideal results. This summer research fellowship was a wonderful opportunity for me to improve as an undergraduate researcher and make connections with students across the country that are also interested in pursuing science.

References

  1. Solocinski, K., Holzworth, M., Wen, X., Cheng, K., Lynch, I. J., Cain, B. D., . . .Gumz, M. L. (2016). Desoxycorticosterone pivalate-salt treatment leads to non-dipping hypertension in Per1 knockout mice. Acta Physiologica,220(1), 72-82. doi:10.1111/apha.12804
Amber Miller is a rising senior majoring in Health Science at the University of Florida in Gainesville, Fl. She is a 2017 student in the Undergraduate Summer Research Fellowship that is funded by the APS. Amber works in Dr. Michelle Gumz’s lab at the University of Florida that focuses on circadian genes and its effects on blood pressure. She is currently applying to medical school this summer with plans to become an academic physician in the field of physiology.
Getting Salty About Hypertension

About 30% of the world’s population has hypertension due to a recent surge of salt in our diets (1). According to the American Heart Association, the average American eats more than 3,400 milligrams of sodium daily, while the ideal limit is 1,500 mg per day for most adults (2). So, how does your body know when you have ingested salt? Your brain has specialized cells that monitor the amount of sodium (and other ions or substances). Some of these cells are located in the Organum Ventral Lamina Terminus (OVLT). OVLT is one of a few special section of the brain that lacks a complete blood brain barrier that allows substances in the blood to pass more freely from the bloodstream to the brain. So, this section of the brain essentially has the equivalent of “x-ray vision”; it can “see” the particles in the blood, such as salt, and send messages throughout the body to initiate responses. One of these responses may be to increase blood pressure, which is known as salt-sensitive hypertension. Salt-sensitive hypertension refers to an increase in arterial blood pressure to an increase of salt intake.

 

Figure 1. Rat diagram with optogenetic set-up.

My project utilized a new innovative tool known as optogenetics to inhibit or “turn off” OVLT neurons during dehydration and acute NaCl loads. Optogenetics works by shining a light at different wavelengths at a region of the brain where a light activated or inhibited ion channel was injected via a virally-mediated delivery system. In my experiments, water intake, urine output, and blood pressure (via telemetry) were measured in rats dehydrated overnight or infused with a concentrated NaCl solution with or without the laser on to inhibit OVLT neurons. I found that when these neurons were inhibited, they would decrease the volume of water intake of the rats. Upon further investigation, if OVLT continues to prove to be a major contributor to salt-sensitive hypertension, we hope more methods will be developed and implemented in humans suffering from this disease.

 

Figure 2. Leica Vibratome used to take serial sections of brain to determine injection location. Credit: Dr. Sean D. Stocker’s Lab, University of Pittsburgh Medical Center

Research is a very rewarding occupation. Although experiments can be long and time sensitive, the outcome is worthwhile. As long as you are careful and perform experiments honestly, any result can be used to learn. Further, I was amazed by the techniques I used to complete my experiments. The science and technology behind them is very interesting and innovative. The use of optogenitics is relatively new and entails complex surgeries and sciences. Unfortunately, with complexity comes consequences, and I have hit some bumps in the road. The technology used on the rats was very fragile and could not be fixed if broken. In addition, since it is not possible to alter salt concentrations without altering other aspects of the rat, we had to test the secondary stimuli to see what affect they had on the rats’ behavior. I am not finished collected data yet for this summer, but the results appear promising. In addition, I still need to test more animals and determine if the injected virus hit OVLT in order to define my conclusions.

 

Upon arriving, I was most surprised by the dynamic of the lab. All lab members are working on separate projects, but with related goals. Work is mostly independent, yet the goal is interdependent.  Working with a lab team was a fulfilling experience and helped me gain a better understanding of my project. I was able to learn new things and make better sense of my project through communicating with my team members. The best part of my research is the technology and innovation associated with it. One of my favorites is the way we analyzed water intake by applying basic principles of circuits. During experiments, the rats are in cages with metal floor, plastic walls, and water bottles with metal spouts. A wire connected the floor to the spout of the water bottle, creating an incomplete circuit. When the rat takes a drink, it completes the circuit because it is touching both the metal spout and floor. This causes a voltage to occur, which is recorded on a computer application. This data is used to determine when the rats were drinking, and it can approximate water intake because duration of time at the water bottle spout correlates with the amount of water the rats ingested. In contrary, the worst part of my research was the delicacy of the animals. It is very disheartening when the head caps fall off the rats and no longer can be used for experiments. Another incommodious aspect was the time sensitivity of experiments. I have to give the rats time between experiments, so some days were much slower than others. All in all, the positive aspects of research have outweighed the negative, thus far. It has further peaked my interest in a future of biomedical research, and I would recommend anyone interested in the sciences with a love of problem solving to engage in research!

References

  1. Choi HY, Park HC, Ha SK. Salt Sensitivity and Hypertension: A Paradigm Shift from Kidney Malfunction to Vascular Endothelial Dysfunction. Electrolytes & Blood Pressure 13:7-16, 2015 [14 July 2017].
  2. How Much Sodium Should I Eat Per Day? [Online]. American Heart Association. https://sodiumbreakup.heart.org/how_much_sodium_should_i_eat?utm_source=SRI&utm_medium=HeartOrg&utm_term=Website&utm_content=SodiumAndSalt&utm_campaign=SodiumBreakup [14 July 2017].
Rachel Maile is a junior majoring in Biological Systems Engineering and minoring in Biomedical Engineering at Virginia Polytechnic Institute and State University (Virginia Tech) in Blacksburg, VA. She is a 2017 Undergraduate Summer Research Fellow (UGSRF) working in Dr. Sean D. Stocker’s lab at the University of Pittsburgh Medical Center in Pittsburgh, PA. Rachel’s fellowship is funded by the APS and has enabled her to further her appreciation for research. After graduate, Rachel plans to perform biomedical research, using her engineering background and physiological expertise.
A Summer of Fibrosis

This summer I have had the unique opportunity to work on a project that involves human heart tissue, an opportunity provided to me by the American Physiological Society and the Campbell Muscle Lab at The University of Kentucky.  My project focused on heart failure, a topic that is vital to many Americans, in fact, the American Heart Association showed that approximately 5.7 million adults in the United States suffer from heart failure. My study looked specifically at the varying amounts of collagen based on sex and heart failure status. It has been researched and shown that there is an increase in fibrosis when a patient has heart failure. Fibrosis is a term that describes tough fibers that replace damaged cardiac tissue in a process known as cardiac remodeling; an example of fibrosis is collagen, the protein that we stained for. The unique part to this study is the comparison of men and women, regarding the amount of collagen present in the heart tissue. This idea stems from a previous experiment conducted in the lab that used a process called Nanostring to determine the expression of genes related to heart disease and failure in both failing and non-failing human hearts. This study showed that there were genes related to collagen that had sex specific differences. Although the experiment might not be revolutionary, it will aid in bridging the gap that currently exists in research regarding the physiological difference between men and women. Although, we do not have enough data to draw conclusions yet, we are already thinking about future directions for our project!

The Realities of Research

Research is an imperfect process to say the least, and in my opinion that is what makes it wonderful. Things do not always go as anticipated when conducting research. It might not always yield the expected results, but it was at the very least a learning experience. One of the largest realities I had to face was that just because a protocol is well established does not mean that it is easy to learn, or unalterable. The protocol for my experiment is one that is well established and has been for many years. That would ideally make the project simple, right? Wrong. At the beginning, there were numerous problems, most associated with a steep learning curve. Part of my job is to cut tissue, and there were many rat heart samples that had to be cut before I could begin working with human tissue. Once the cutting was mastered, there was still a problem with the staining aspect of the protocol.  The protocol written seemed clear, but the results of the stains were not up to par. So, we worked to adjust the protocol in order to obtain samples that were suitable enough to analyze. This steep learning curve was rough, but it made for a great application of problem solving skills.

The Daily Life of a Scientist

The daily life of a scientist is quite interesting, especially in my lab. Sometimes the day to day life of a scientist can be a little monotonous. I would come in and cut the tissue, stain the tissue, and image the samples, but just when things were getting a little boring, we would get a call on what we call the bat phone. The bat phone rings whenever there is a heart transplant or an organ donor, who is unable to donate his or her heart. We are then able to take the heart and store it for future research for our lab or any other that wants to use human samples. We are on call 24/7 and there are times where I must leave an experiment to collect a heart. Although this seemed inconvenient at times, such as 2 AM, it was great to have such a unique opportunity. The daily life of a scientist in the Campbell Muscle Lab is exciting, challenging, and unique to say the least.

References

  1. Mozaffarian, Benjamin, Go, Arnett, Blaha, Cushman, . . . Turner. (2016). Heart Disease and Stroke Statistics—2016 Update: A Report From the American Heart Association. Circulation, 133(4), E38-E360.
Autumn Conger attends the University of Kentucky in Lexington, KY. She is a 2017 Undergraduate Summer Research Fellow (UGSRF) and worked in Dr. Kenneth Campbell’s lab at the University of Kentucky. Autumn plans to go to medical school and become a physician scientist. She hopes to help in bridging the gap between the fields of medicine and research.
Stemming Beyond the Brain

This summer, I am very thankful to be a recipient of the Undergraduate Summer Research Fellowship (UGSRF). This fellowship has given me the opportunity to conduct research under the guidance of Dr. Justin Grobe at the University of Iowa. My research project studies the role of AT1a receptors on the vasopressin-producing cells found in the hypothalamus region of the brain.

Vasopressin (AVP) is a hormone, produced in the magnocellular neurons within the hypothalamus, playing a key role in maintaining homeostasis. This hormone maintains homeostasis by increasing blood pressure through upregulation of water reabsorption and vasoconstriction. The AT1a receptor responds to high levels of angiotensin II, ANGII, by constricting the cells with AT1a as part of the renin-angiotensin-aldosterone system, RAAS. A previous study conducted in our lab has shown AT1a receptor localization on vasopressin-producing cells in the supraoptic nucleus of the hypothalamus1. In order to observe the role of AT1a receptors, the experiment compared the response of AT1aAVP-KO mice with control mice under the ICV infusion of ANGII or aCSF at 50ng/hr. Specifically, I am looking for responses in water-intake, urine output, blood pressure, and vasopressin secretion. This experiment will provide further insights to understand AVP secretion in response to brain RAAS and could potentially help prevent hypertension, exhibited patients with obesity, diabetes, and/or preeclampsia.

Figure 1: qPCR machine for gene expression analysis

I have learned many valuable skills from conducting research over the course of this summer thanks to my awesome mentors and principal investigator. They have devoted their time to teach me how to use Excel in analyzing the results of specific qPCR gene expressions and creating protocols for many experiments utilized in the lab. Nevertheless, there are many other essential skills that I have yet to learn such as interpreting results, reading scientific journals, and other lab protocols. They are important in all aspects of research but honing in these skills will require time and practice. For instance, several experiments that I performed yielded poor results and required additional extra time devoted towards troubleshooting the problem. Although frustrating at times, I have learned troubleshooting is one of the most important skills in research because not all the experiments will yield the desired results. While learning and refining my skills, the research project has progressed into the final stages towards completion and I am very excited to find out what results from the samples reveal regarding the role of AT1A receptors.

Figure 2: Tail-cuff system setup for blood pressure measurement

As a member of the lab during this summer, I have gained an insight into becoming a scientist. Every morning is devoted towards measuring the blood pressure in the mice cohorts, which need the measurements to be taken daily for three weeks prior to the surgery and two weeks after. The afternoon is spent performing qPCRs on specific genes from tissues collected earlier, analyzing data, learning new protocols, and/or reading publications pertaining to my research area. There are many basic aspects that scientists value greatly such as labeling tubes, checking the timer, and pipetting the exact amount of solutions. The focus on minute details is a trait developed over the years dedicated towards conducting research, and I have definitely noticed improvements in my attention to detail since I first began conducting research. In the next couple of weeks, I will have the opportunity to collect, organize, and analyze the results during the finals days of this experiment. I would like to again thank APS for the opportunity to conduct research at the University of Iowa this summer.

References

  1. Linggonegoro D, Sandgren J, Claflin K, Perschbacher K, Ni J, Pearson N, Pierce G, Santillan M, Grobe J. Physiological Significance of Angiotensin AT1A Receptors in Vasopressin-Producing Cells of the Supraoptic Nucleus. Experimental Biology.
  2. Guyton AC, Hall JETextbook of Medical Physiology. 10th ed. Philadelphia, PA: W.B. Saunders Company, 2010.
I am rising junior at the University of Iowa, majoring in biomedical engineering. I will be pursuing a biomechanics and biomaterials track with an interest in implant design and drug delivery. This summer, I had the opportunity to conduct research under my principal investigator, Dr. Justin Grobe, with the assistance of UGSRF fellowship funding by the American Physiological Society. My project investigated the role of AT1a receptors on the vasopressin-producing cell in cardiometabolic control.
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.

 

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.
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.
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.
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.