February 26th, 2019
Sex Differences in Asthma

Research Project

This summer, I was involved in research with Dr. Silveyra at Penn State College of Medicine. We looked at microRNAs, which are small pieces of RNA involved in various processes in the cell, including regulation of how much of a certain protein is made. MicroRNA-106a is a microRNA that influences asthma in mouse lung immune cells, which cause inflammation. miRNA-106a does so by preventing an anti-inflammatory protein, IL-10, from being made. Because we knew that more adult females have asthma than adult males, we hypothesized that miRNA-106a would also be expressed differently between males and females. To investigate this hypothesis, we allowed several mice to develop asthma by repeatedly exposing them to house dust mites for 5 weeks. We then exposed the mice, along with additional mice that did not develop asthma as a control, to either ozone or filtered air for 3 hours. After that, we harvested the lungs and extracted the RNA from the tissue. We separated out miRNA-106a from the total RNA and determined its relative amount compared to the amount of another RNA that is always present in cells. We discovered several differences in how much miRNA-106a was present between males and females within treatment groups. Along with other factors, these differences in miRNA-106a levels in mice may play a role in the differences seen in human asthma, which affects about 8% of all people in the US. It could even lead to a new treatment for asthma that is specific to men or women.

Realities of Research

Doing research full time in a lab was more enjoyable than I expected. Each day looked different for me in terms of the tasks I needed to complete. I learned several new techniques, including RNA extraction and real time PCR, and I used these techniques very frequently throughout the summer. During the experiments, we had to repeat our real time PCR plates several times due to a lot of random error, and we had to repeat the RNA extraction for several samples because we didn’t get the amount of RNA we wanted. It was a long process, with many setbacks, but we finally got results from our experiments. We did see differences in the amount of miRNA-106a between males and females, but we did not see the differences that we expected. Because of this, we understood that miRNA-106a may not be causing all the differences seen between men and women with asthma, but other factors may cause the differences as well.

Life of a Scientist

As said earlier, the part I most appreciated about my summer as a scientist was that each day brought new tasks. However, experiments can take a long time to complete, with several setbacks and problems that need to be addressed along the way. There is no instant gratification; I had to work for every bit of data I had. But it is still rewarding when one can publish or present research at a conference, and other scientists listen and ask questions. To be a scientist, one must know how to work as a team and communicate clearly so everyone understands what their role is in the lab. I felt that my team was very good at doing that, and our lab environment was better for it. My lab team was a huge help to me in teaching me techniques, assisting me in carrying them out, and helping me to make sense of my results. All in all, the life of a scientist, though with many obstacles, is rewarding, and I would encourage anyone to check it out.

Rachel Steckbeck is a junior at Messiah College in Mechanicsburg, PA. She is a 2018 Undergraduate Summer Research Fellow (UGSRF), and worked in Dr. Silveyra’s lab at Penn State College of Medicine in the summer of 2018 for 10 weeks. UGSRF is funded by the American Physiological Society. In the future, Rachel hopes to attend medical school and work at a local hospital or practice.
February 19th, 2019
Ways to reduce Myocardial Reperfusion Injury

Research Project

This summer I am working in the Department of Anesthesiology at Stanford University. The main aim of my assigned project is to study the role of TRPV-1 (Transient Receptor Potential Vanilloid-1) in myocardial reperfusion injury. TRPV-1 is considered as a main channel that mediates cellular responses to different stimuli such as pain and heat. Regulation of TRPV-1 is important because it controls mitochondrial membrane potential and reperfusion injury. Modulation of TRPV-1 interaction site with calcineurin is essential to reduce the injury. My main objective for the summer is to study the effect of previously synthesized peptide drugs on the interaction of TRPV-1 with calcineurin. We hope that this project will help to reduce damage from cardiac reperfusion injury since it is responsible for up to 50% of myocardial infarct size.

Realities of Research

Stanford was my dream and I spent this summer in this dream. As I had an inadequate knowledge in the real physiological lab before, I was lucky to have an opportunity to experience many new techniques and skills. The most captivating experience was rats. Unfortunately, my home university does not have animal resources and students are deprived of working with animals. Hence I am planning to pursue PhD next year, I was very anxious about my incompetence and inadequacy. Luckily, my assigned project for the summer required me to isolate fresh cardiomyocytes from adult rats and perform different cell death assays on them. I was very excited about upcoming surgeries but forgot about one significant nuance- rats itself. Rats were afraid of me, so do I. Ironically, my fear made them even more stressful, so eventually I ended up with zero cells. I repeated surgeries dozens of time forcing myself to be braver and bolder. After continuous failure, I got used to them and I am obtaining enough cells these days to continue further. Now, I understand that any skill can be acquired with determination and perseverance unless it is highly desired.

Life of a Scientist

People do the science for many reasons. For some, it gives a prestige and high social status, but for some, it gives a hope for the better future. When I first told my family a decision of becoming a scientist and pursuing an academia, I was warmly welcomed with benevolent joy about my safe future. For sure, being a scientist seems to be the most auspicious occupation for women hence it requires neither intense physical nor intellectual activity. That was when my first fairytale illusions about the science career have started. Years later, after experiencing both flukes and failures I understood that life of a scientist indeed requires something very special- struggle with Yourself.

Coming to the lab every day and continuously repeating the same routine experiments over and over again can frustrate even the nerds. But for me, this is a real beauty of the science. When the experiments don’t work for weeks and the moment when you start quitting, surprisingly everything begins to work by lucky chance. You get happy and renew the cycle from the very beginning without even a tiny memory of those hapless days. Ironically, the cycle goes on further and combo of successful/unsuccessful experiments continue to form a chain reaction in your life. I agree with Mare Curie that “A scientist in the laboratory is not only a technician: he is also a child placed before natural phenomena which impress him like a fairy tale”. If you still believe in miracles and get amazed by the natural laws, follow your passion and just chase the science!

 

Reference

Hurt, C.M., Lu, Y., Stary, C.M., Piplani, H., Small, B.A., Urban, T.J., Qvit, N., Gross, G.J., Mochly-Rosen,D.,  Gross, E.R. Transient Receptor Potential Vanilloid 1 Regulates Mitochondrial Membrane Potential and Myocardial Reperfusion Injury. J Am Heart Assoc 5(9), 2016.

Zhuldyz Zhanzak is a senior majoring in Molecular Biology at Nazarbayev University, Kazakhstan. She is a 2018 Undergraduate Research Excellence Fellowship (UGREF) Fellow working in Dr. Eric Gross lab at Stanford University School of Medicine, CA. Zhuldyz’s fellowship is funded by the APS. Following the graduation, Zhuldyz plans to pursue PhD in immunology and follow a career as a scientist in the academia.
February 12th, 2019
Hypertension Prevention: does it also improve motor cognitive function?

Research Project

This summer, I’m studying the effects of inspiratory muscle strength training on motor and cognitive function in middle-aged to older adults. Motor function is the ability to move the muscles in your body, and cognitive function is your brain’s ability to perform tasks. Inspiratory muscle strength training, or IMST, is a relatively understudied technique of exercising the muscles you use to breathe in. By breathing in, or inspiring, against a resistance with a small device that looks similar to an inhaler, you can make these muscles stronger and hopefully improve many bodily and cellular functions, including motor-cognitive function. What is currently known about IMST is that it can significantly improve blood pressure in healthy adults (DeLucia, De Asis and Bailey, 2018), but its effects on adults with high blood pressure have not been tested yet. The IMST study on the large scale is focusing on the possible blood pressure and cardiovascular benefits of IMST in adults with high blood pressure, and I’m focusing my research and data analysis specifically on the motor cognitive functions that we also test.

Because a decline in motor-cognitive (shortened to motor-cog) function is highly correlated to mortality (death) risk, there is a large scientific effort to evaluate the effectiveness of various forms of intervention to improve these variables. Motor-cognitive decline is characterized by dementia and immobility, which are not only independent predictors of mortality risk, but large influential factors on perceived quality of life for older adults. It is well established that a consistent aerobic exercise routine will effectively prevent motor-cognitive decline in older adults, but because of its heavy burden, it is not frequently adhered to. Finding other effective practices of improving motor and cognitive function that are more adherable will greatly improve the quality of life for aging individuals and lower their mortality risk.

Realities of Research

Figure 1: Formaldehyde used for preserving endothelial cells

So far, I have found that doing clinical research in a lab is full of excitement and surprises! Beginning at the start of the fellowship, clinical interactions and the IMST study became only two of my many responsibilities. I have been trained on wet lab procedures such as cell collection and isolation, as well as blood processing to assess certain chemicals in the blood. These tasks can be tedious, and are very time and method sensitive. I can spend two hours doing the cell collection and isolation for them to later not give any helpful results when I analyze them. Experiencing frustrating aspects like this is helping me develop the very important skill of patience. I have to use patience in many aspects of my work; wet lab mishaps are not the only issues one can encounter during a typical day of research. I am working in one of the most dynamic physiology labs in the country, with some of the brightest in the field, but despite this we all run into our fair share of hiccups. For example, our huge datasheet for our study got deleted and we spent several days trying to find it. Also, our freezer logging software crashed and left us without decades of logged biological sample information!

Figure 2: Freezing plasma in liquid nitrogen for later analysis

 

 

On the other side of my work, I have to use patience with the clinical subjects. When working with more stubborn older subjects, I find it’s essential to remain patient to maintain professionalism, and to represent our lab in the most positive way possible. I’ve had difficult interpersonal interactions with some of the subjects, but remaining patient and working through issues and questions with them upholds the highest standards of human research we have as an institution. Anyways, our IMST study has (despite some problems) been very on-track and is moving along at a quick pace. We just received more funding from the NIH through a grant that will support us through the next two years of research. Woohoo!

Life of a Scientist

Living as a scientist this summer has been a truly rewarding and educational experience. Going from a very part-time volunteer in the lab doing data entry, to a full-time member of the lab team with much more responsibilities has been an eye-opening transition. I am a much more integral part of the team, and I have to be much more accountable than I was before. I went from only having one task to focus on at a time, to having three-four or more, including clinical visits, grant reviewing, abstract writing, journal club presentations, and reading physiology literature. Balancing and prioritizing my lab tasks is difficult and stressful, and has shown me the less-glamorous side of basic research that you don’t realize until you experience it for yourself. On the other hand, I have been supported in so many ways I never expected, and I love working in a team-based environment. As a non-traditional community college student, I always felt like an outsider and thought I would never be fully accepted into the scientific/academic world. I was proven wrong. Our lab has endless support and an open-door policy when it comes to questions and concerns, which encourages communication. Every member of my lab team has been so helpful in educating me in physiology and research topics I’m unfamiliar with, and has given me all the opportunities I could ask for to be successful at this early stage of my research career. In fact, I should have at least two publications by the end of next year, and probably three by the time I finish my undergraduate studies. This amazing experience has strongly encouraged me to further pursue my career in physiological research.

 

Sources:

DeLucia, C., De Asis, R. and Bailey, E. (2018). Daily inspiratory muscle training lowers blood pressure and vascular resistance in healthy men and women. Experimental Physiology, 103(2), pp.201-211.

Makinzie Hamilton is an undergraduate junior studying Integrative Physiology and Philosophy at the University of Colorado, Boulder. She is working at UCB under Dr. Douglas Seals at the Integrative Physiology of Aging Laboratory, which focuses on improving cardiovascular aging. She is funded through grants from the STRIDE fellowship from the APS and The National Heart and Lung Association (Grant #1 R25 HL115473-01). After graduating in 2020, she hopes to pursue a dual MD/PhD degree and do clinical research regarding infectious disease and tropical medicine. In her free time, she enjoys studying true crime, cooking, painting, and fishing.
February 8th, 2019
Targeting NHE3 in the Hfe mouse model of hemochromatosis

Research Project

Over the summer I have been working in Dr. Bryan Mackenzie’s lab at the University of Cincinnati College of Medicine in the Pharmacology and Systems Physiology department. My focus has been on finding novel therapeutic targets in the treatment and/or prevention of iron overload disorders. Hemochromatosis is just one of several common hereditary disorders that result in iron overload and subsequent liver disease, heart damage, and a slew of other health disorders across several body systems. Since there is no regulated mechanism for iron excretion, iron homeostasis of the body is regulated by iron absorption. The main mechanism by which non-heme iron (the majority of iron humans consume from their diets) is absorbed from the small intestine is the divalent metal ion transporter-1 (DMT1) (3). DMT1 is already a validated therapeutic target in hereditary hemochromatosis. More recently, the Mackenzie lab identified a role for the Na+/H+ exchanger-3 (NHE3) in intestinal absorption of non-heme iron by DMT1.

 

Intestinal iron uptake (redrawn from reference 1)

The Mackenzie lab demonstrated that NHE3 generates the driving force for iron absorption via DMT1 by transporting protons into the small intestine (2). My project this summer has built upon this prior knowledge using an Hfe knockout mouse model. Rendering this single gene inoperative in mice results in a very good model of iron overload. We are then able to test the hypothesis that iron overload in the Hfe KO mouse can be corrected by ablation of NHE3. The idea is that without NHE3, the driving force for DMT1-mediated iron uptake is reduced, thereby reducing iron absorption. If the data supports this hypothesis, we can conclude that ablating NHE3 prevents iron loading in the Hfe mouse model of hereditary hemochromatosis. Even further, we can reveal intestinal NHE3 as a potential therapeutic target in the treatment of iron overload.

Realities of Research

Throughout the summer I have learned countless basic research techniques and have mastered various skills that are good to possess in the Mackenzie Lab. I have learned how to ween and care for our colonies of mice. I now know how to genotype them using tail clippings and polymerase chain reaction (PCR). This step is especially important because it determines which mice are our experimentals, or the mice we collect blood and tissue samples from on experiment days. I was placed in charge of the mouse experiments, learning how to effectively set-up and delegate responsibilities. Being my first full-time experience in a research lab, I appreciated this opportunity to take charge and have ownership over such a large task. I also had the task of running serum iron assays and non-heme iron assays on freezers full of samples that have been collected. This task proved to be challenging and slightly stressful at times, as I found errors in our established assay protocols and analytical procedures. For example, some mice had very little blood collected on experiment days, meaning I had very little serum to run the assays. When the protocols had detrimental errors, I felt I had wasted irreplaceable serum at times. Luckily, I have corrected our protocols and have finally organized/assayed most of our samples. I have also not only learned how to operate a software program called SigmaPlota to analyze my data, but have learned how to edit our labs established transformations within this software. This summer full of learning techniques, troubleshooting, and analyzing my data has been an invaluable experience. I have transformed into a proficient, independent, and productive lab member in just a few months’ time.

Life of a Scientist

I have enjoyed my day-to-day life in the lab this summer. I was pleased to learn that a 9-to-5 workday is certainly possible, with proper planning. I also understood that some late nights/early mornings are unavoidable, especially when things didn’t go as planned and required a little extra time. I discovered that the environment in the lab was much more relaxed than I anticipated. I was able to control my day to day activities with little intervention, unless I needed assistance or extra guidance. This assistance from other lab members is something I really appreciated. Although everyone has their own projects and own experiments to run, a helping hand was always there when I needed it. I liked this collaborative, team-based environment and believe it greatly increased all I was able to learn in the lab this summer. The only con in the life of a scientist that I can think of is the down time that sometimes occurs, as I’m the type of person that likes to stay busy. Luckily, I was able to fill my time by reviewing relevant literature in the field or discussing my progress with other lab members. Overall, I really enjoyed getting to experience the life of a scientist for a summer and look forward to finishing up my project/exploring new directions in the lab.

 

References

  1. Mackenzie B, Garrick MD. Iron Imports. II. Iron uptake at the apical membrane in the intestine. American Journal of Physiology-Gastrointestinal and Liver Physiology289, 2005.
  1. Shawki A, Anthony SR, Nose Y, Engevik MA, Niespodzany EJ, Barrientos T, Öhrvik H, Worrell RT, Thiele DJ, Mackenzie B. Intestinal DMT1 is critical for iron absorption in the mouse but is not required for the absorption of copper or manganese. American Journal of Physiology-Gastrointestinal and Liver Physiology309, 2015.
  1. Shawki A, Engevik MA, Kim RS, Knight PB, Baik RA, Anthony SR, Worrell RT, Shull GE, Mackenzie B. Intestinal brush-border Na /H exchanger-3 drives H -coupled iron absorption in the mouse. American Journal of Physiology-Gastrointestinal and Liver Physiology311, 2016.
Sydney Stone is a junior majoring in Medical Sciences at the University of Cincinnati in Cincinnati, OH. She is a 2018 Short-Term Research Education Program to Increase Diversity in Health-Related Research Fellow (STRIDE) working in Dr. Bryan Mackenzie’s lab at the University of Cincinnati College of Medicine. Her summer of research was supported by APS and a grant from the National Heart, Lung, and Blood Institute (Grant #1 R25 HL115473-01). Upon graduation, Sydney plans to pursue an MD/PhD or MD/MS in order to become a physician scientist. She hopes to combine proficiency in physiology with invaluable access to patients in order to follow her passion for preventing and curing human disease.
February 5th, 2019
Life Behind the Lab Coat: Part 2

Are you curious about the world of science? Do you crave to find new and exciting things about physiology? Well, I do!  Last summer, I had the opportunity to work in a research lab at the University of South Florida Morsani College of Medicine. Furthermore, I was able to renew my APS STRIDE fellowship and continue to work in Drs. Jessica Dominguez Rieg and Timo Rieg’s lab.

Research Project

During my time at the lab, my research project expanded on my work from last summer involving a transporter called the sodium/hydrogen exchanger (NHE). NHE is used to exchange sodium for hydrogen across a cell’s membrane.  There are several different isoforms of NHE; however, my project focused on the isoform NHE3. When NHE3 is genetically deleted from a mouse, called a whole animal knockout, they absorb or reabsorb sodium from the intestine or kidney. Consequently, the NHE3 knockout mice have severe diarrhea, low blood volume, and low blood pressure1. Furthermore, since NHE3 transports hydrogen, it also plays an important role in acid-base regulation. Moreover, NHE3 is also significant for balancing fluid and the regulation of blood pressure.  Last year, my studies concluded that the inducible deletion of NHE3 only in the intestine causes fluid accumulation in the intestine and metabolic acidosis.

This year, my research project remained focused on the role of intestinal versus renal NHE3 and its effects on phosphate homeostasis and blood pressure regulation. Moreover, my research is significant as nothing like this has been done before!  Additionally, there are currently drugs on the market that block NHE3, one example is Tenapanor, which helps to lower blood pressure or high blood phosphate levels; however, the mechanisms of how they work are unclear. As of last year, I saw significant changes in acid-base regulation. Hopefully, this year I will be able to determine how NHE3 affects phosphate homeostasis. Furthermore, I hope I am able to produce more data in order to help answer these ongoing questions about NHE3 and its effects on phosphate homeostasis and blood pressure regulation. Thus far, I have been conducting physiological experiments to determine changes in fluid and food intake, collecting blood and urine for further analysis.  My project is still being conducted but I am excited to see the results!

Life of a Scientist

I love being part of a research lab! Working with people who share the same passion and love discovering new things is amazing! Although I love doing research, the hours can be long. Having to work 8 plus hours a day during the week can be tiring and then having to come in again on the weekends, but I’ll tell you it’s all worth it! One thing I learned is experiments are a process and take time. Last year I thought working in a lab would be similar to being in the labs at the university that were required for your major, but they’re not. Even though they are very different, I still loved every moment I came into work! I am so thankful for this opportunity that APS and my mentors allowed me to expand my knowledge and skills. I am forever grateful for this experience.

 

Citations:

  1. McDonough AA. Mechanisms of proximal tubule sodium transport regulation that link extracellular fluid volume and blood pressure. Am J Physiol Regul Integr Comp Physiol 298: R851–R861, 2010. doi:10.1152/ajpregu.00002.2010.
Alexandria Valdez is a Senior majoring in Biomedical Science and minoring in Psychology at the University of South Florida in Tampa, FL. Alexandria is a 2018 second year Short-Term Research Education Program to Increase Diversity in Health-Related Research (STRIDE) Fellow working in Dr. Jessica Dominguez Rieg and Dr. Timo Rieg’s lab at the University of South Florida Morsani College of Medicine in Tampa, FL. Alexandria’s fellowship is funded by the APS and a grant from the National Heart, Lung and Blood Institute (Grant #1 R25 HL115473-01). Upon graduation, Alexandria wishes to pursue a career in biomedical science as a research scientist.
February 1st, 2019
Take My Breath Away- A Summer of Asthma Research

Research Project

Pathology of asthma.

This summer I had the pleasure of working with Dr. Silveyra and her research team at the Penn State College of Medicine investigating the mechanisms behind adult asthmatic females being more susceptible to air pollution than males. Research has shown that upon exposure to an air pollutant like ozone, females that already have a respiratory disease, such as asthma, are much more likely to develop worsening asthma as well as other respiratory problems. I believe this study is very important in today’s world, where the majority of ozone can be found in cities and industrialized areas that have very high populations. Therefor a female with asthma living in a populated city risks the possibility of her asthma worsening or developing other complications.  When people think of asthma, they usually think of shortness, of breath, trouble breathing, and wheezing. These symptoms are all due to a person’s airways becoming inflamed and an air pollutant like ozone could trigger this. The body expresses proteins called cytokines and when certain types of these cytokines are expressed, they can lead to inflammation like we see in asthma. In recent years a special type of RNA called microRNA (miRNA) has been found to play a key role in regulating the expression of these inflammatory cytokines. My research team and I were particularly interested in miR-712, which is a miRNA known to play a role in regulating inflammatory cytokines. Our idea was that if this miRNA is expressed differently between males and females, this could explain why asthmatic females are more susceptible to air pollutants than males.  This study could lead to the development of treatments specific for males and females.

Realities of Research

This was the first real research project I had ever participated in, so it was pretty intimidating in the beginning. However, as time went on I learned new techniques and began to understand the project and why it was important and that got me really excited to be doing research. The part that surprised me the most is how things that are so small like miRNA can play such a huge part in disease and keeping us healthy. Along the way I had to learn new techniques like how to extract the miRNA’s and how to determine how much was being expressed. In the end the results were a little surprising. Yes, we saw higher levels of miR-712 expressed in asthmatic females that had been exposed to ozone, but we also saw even higher levels of it in asthmatic males that had been exposed to ozone. This may have been due to the fact that male mice typically react quicker to the asthma model we used and that it may take females longer to actually “develop” asthma. I think in the future this model may need to be changed a little bit, but that is all a part of doing research.

Life of a Scientist

I found day-to-day life in the lab to be very exciting. There was always something to be doing and it was nice to know that everything you were doing in a day was for a purpose. One thing that surprised me was how long some procedures can actually take. Procedures I would read about in textbooks always seemed like they were quick and then you have your results, but there is actually a lot of hours that go into some procedures. The worst part was exposing the mice to ozone, because it was a three-hour long exposure and it would get pretty boring. The absolute best part was getting to work with my research team. Every single one of them was so helpful and knowledgeable and I am so grateful for them.

 

Citations

  1. Fuentes, N., Roy, A., Mishra, V., Cabello, N. and Silveyra, P. (2018). Sex-specific microRNA expression networks in an acute mouse model of ozone-induced lung inflammation. Biology of Sex Differences, 9(18).
Ashley Weaver is a rising senior at Penn State University in State College, Pennsylvania majoring in immunology and infectious disease. She is a 2018 Short-Term Research Education Program to Increase Diversity in Health-Related Research (STRIDE) fellow working in Dr. Patricia Silveyra’s lab at the Penn State College of Medicine in Hershey, Pennsylvania. Her fellowship is funded by APS and a grant from the National Heart, Lung, and Blood Institute (Grant #1 R25 HL115473-01). 
After graduating, Ashley plans to pursue a career in clinical lab work for government organizations and then to pursue her masters in immunology.
January 29th, 2019
The Fascinating Relationship Between Inflammation and Respiratory Plasticity

Research Project

I am grateful to say that this summer I had the opportunity to work with Dr. Irene C. Solomon at Stony Brook University, in the Department of Physiology and Biophysics, as an awarded fellow from the American Physiological Society’s Short-Term Research Education Program to Increase Diversity in Health-Related Research (STRIDE) Fellowship. Under the mentorship and guidance of Dr. Solomon, I was able to explore the scientific field of neuroinflammation and the underlying neural mechanisms controlling our central respiratory system.

Figure 1. Intermittent Hypoxia (IH) can induce respiratory motor plasticity, by increasing phrenic motor neuron and diaphragm activity

Acute Intermittent Hypoxia (AIH), also known as the delivery of low levels of oxygen in repetitive bouts, has been demonstrated to alter neural ventilatory control, and therefore, elicit different physiological changes within the respiratory system that can persist into normoxia (also otherwise known as normal O2/CO2/N2 levels).[1] These alterations are more formally known as neural plasticity, which is the nervous system’s response to varying, natural stimuli and/or the initiation and progression of diseases, injuries, and pathology.[2] What that means is that the nervous system attempts to compensate for the lower levels of oxygen by increasing the expression of neurotransmitters like serotonin and adenosine, which then affects the upregulation of specific growth/trophic factors within the Gq and Gs pathways involved in eliciting neural plasticity. More specifically, the G protein coupled receptors (GPCR), serotonin 5HT2 receptors and adenosine A2A receptors, are being targeted within the Gq and Gs, respectively.[3] Although there seems to be some interplay between these two pathways, the Gq pathway and the activation of the serotonin 5HT2 receptors appear to play a larger role in stimulating the physiological changes found during and after AIH.[4] The physiological changes that follow (i.e.increased phrenic motor neuron and diaphragm activity), are utilized within the central respiratory system to essentially breathe “better,” or in other words, consume more oxygen, in such hypoxic conditions.

Additionally, recent evidence from rodent studies, which have implemented different protocols/models for the acute administration of bacterial endotoxin lipopolysaccharide (LPS) — a toxic substance bound to bacterial cell wall, usually causing inflammation in different regions of the brain when

Possible mechanism, including the growth/trophic factor, BDNF, for motor plasticity following AIH in rats and humans

released via bacterium rupture or disintegration[1] — indicates that inflammatory processes can also alter ventilatory control, and subsequently, affect the level of respiratory plasticity that can be achieved. The interactions between LPS-induced inflammation, and accompanying neuroinflammation, and various aspects of ventilatory control, however, are not well understood. It is important to understand how LPS affects the respiratory system since LPS-induced neuroinflammation remains to be a compounding factor of many diseases, such as sepsis, acute lung injury (ALI), and/or acute respiratory distress syndrome (ARDS)[2], and serves as a cause of mortality across a wide variety of respiratory malfunctions (e.g. previously mentioned, OSA). Therefore, the fundamental hypothesis guiding this study is that inflammatory processes can impair multiple aspects of central respiratory neural control, resulting in a reduced ability to adequately and appropriately compensate for disease/pathology. The overall goals of my study are to (1) characterize and quantify the effects (basal ventilatory and respiratory-related neural activities) of moderate (11%O2) AIH in spontaneously breathing male rodents to the 2-hit LPS-induced neuroinflammation model, in which the rats will endure 5 cycles of 3 minutes of 11% O2, 2% CO2, and 87% N2 on and off, and (2) to characterize and quantify the chemical control of breathing via immunohistochemical analyses of various elements from collected tissue samples (e.g. respiratory brainstem cytokines expression, along with neural markers from the Gq pathway). 

Realities of Research

In the beginning of my fellowship, I spent a great deal of my time acquiring new methodological techniques and learning surgical procedures often used in conducting the physiological experiments that are run in lab. Some of the essential skills included establishing EMG signals using tongue and diaphragm electrodes, which is short for electromyography and is an electrodiagnostic medicine technique for evaluating and recording the electrical activity produced by skeletal muscles and the nerve cells that control them (motor neurons)[1], giving intraperitoneal injections (IP), intratracheal injections (IT)., and subcutaneous (sc) injections, conducting femoral and jugular cannulations, administering i.v. drug/saline supplements, and collecting tissue samples that were stored in liquid nitrogen. After acquiring the necessary skills, I was able to spend some time developing a thorough protocol that would help outline the experiments I would be running, and determine what my main hypothesis would be based off of previous findings from the lab and publications I read. I was also given the opportunity to develop my presentation skills as I would prepare and give a presentation on a paper during our bi-weekly lab meetings with the Collins Lab, who we extensively work with, and engage in meaningful conversations on new insights my fellow peers would bring up. I am currently going the data analysis of the rats’ BPM frequency and cyclic height (amplitude) for signs of either an increase or decrease in respiratory motor plasticity post-hypoxia. Immunohistochemical staining of the tissue samples collected will be used to further analyze respiratory brainstem cytokines expression for signs of neuroinflammation and potential decreases in neuroplasticity.

Life of a Scientist

Throughout my experience at the Solomon Lab, I have come to realize that the strange and mysterious world of scientific research is not one of only cut-throat competition and solitude, but a juxtaposition of individualized responsibilities and teamwork. There is something about research that, oddly enough, reminds me of my favorite childhood cartoon show, Scooby Doo. Just like how the Mystery Gang goes around in their fruity van looking for clues to solve the case and figure out the culprit of the crime, a research lab — surprisingly — works in a similar manner. See, being a part of the journey in trying to solve the big mystery is a team effort, unlike the stereotypical image most people conjure up in their minds of an old, crazy gray-haired scientist mixing chemicals together in an underground lab. Just as every member in the Mystery Gang was important to cracking the case, all members in a lab must be willing to cooperate and take on the smaller assignments in order to contribute to the larger picture and find the missing clues to the puzzle. And that’s what I love so much about scientific research: the camaraderie that forms among peers in a lab — sometimes even extending beyond the doors of your very own lab and with the people of the lab across yours. Overall, I am not only grateful for receiving the opportunity to delve deep into the world of physiology and neurobiology research, but also for the opportunity to meet such fantastic people who have helped me grow as a scientist and as an individual.

References

  1. Moya EA and Powell FL. (2018) Serotonin and Adenosine G-protein Coupled Receptor Signaling for Ventilatory Acclimatization to Sustained Hypoxia. Front. Physiol. 9:860. 06 July 2018. DOI: 10.3389/fphys.2018.00860
  2. Fuller, D. D., & Mitchell, G. S. (2017, January). Respiratory neuroplasticity – Overview, significance and future directions. DOI: 10.1016/j.expneurol.2016.05.022. Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/27208699
  3. Figure 1. Dale, E. A., Mabrouk, F. B., & Mitchell, G. S. (2014, January 29). Unexpected Benefits of Intermittent Hypoxia: Enhanced Respiratory and Nonrespiratory Motor Function. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4073945/figure/F3/
  4. Figure 2. Dale, E. A., Mabrouk, F. B., & Mitchell, G. S. (2014, January 29). Unexpected Benefits of Intermittent Hypoxia: Enhanced Respiratory and Nonrespiratory Motor Function. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4073945/figure/F5/
  5. Moya EA and Powell FL. (2018) Serotonin and Adenosine G-protein Coupled Receptor Signaling for Ventilatory Acclimatization to Sustained Hypoxia. Front. Physiol. 9:860. 06 July 2018. DOI: 10.3389/fphys.2018.00860
  6.  Moya EA and Powell FL. (2018) Serotonin and Adenosine G-protein Coupled Receptor Signaling for Ventilatory Acclimatization to Sustained Hypoxia. Front. Physiol. 9:860. 06 July 2018. DOI: 10.3389/fphys.2018.00860
  7. Britannica, T. E. (2014, November 18). Endotoxin. Retrieved from https://www.britannica.com/science/endotoxin
  8. Hong Chen, Chunxue Bai & Xiangdong Wang (2010). The value of the lipopolysaccharide-induced acute lung injury model in respiratory medicine. Expert Review of Respiratory Medicine, 4:6, 773-783, DOI: 10.1586/ers.10.71
  9.  Electromyography (EMG). (2018, May 05). Retrieved from https://www.mayoclinic.org/tests-procedures/emg/about/pac-20393913

Rashi Bhatt is a sophomore majoring in Computational Neuroscience and Public Health, with a minor in Computational Medicine at Johns Hopkins University in Baltimore, MD. She is a Short-Term Research Education Program to Increase Diversity in Health-Related Research (STRIDE) Fellowship working in Dr. Irene C. Solomon’s lab at Stony Brook University in Stony Brook, NY. Rashi’s fellowship was funded by the APS and a grant from the National Heart, Lung and Blood Institute (Grant #1 R25 HL115473-01). After graduation, she plans on attending Johns Hopkins Bloomberg School of Public Health to receive her MPH and hopefully go on to attend medical school, and specialize in neurosurgery and neuro-imaging.

January 25th, 2019
Is Sodium Nitrite Supplementation the Solution to Aging Blood Vessels?

Research Project

With a quickly aging US population, research to help us live a longer life is on the forefront. Cardiovascular disease is the leading cause of mortality in the US, so finding ways to decrease cardiovascular disease risk could be the answer. At the Integrative Physiology of Aging Laboratory (IPA Lab) at the University of Colorado Boulder, we focus our research on finding ways to decrease this risk. A recent 5 year clinical research trial tested the effects of a sodium nitrite supplement on cardiovascular disease risk in middle aged and older adults. Sodium nitrite provides the body with a very important signaling molecule called nitric oxide, which specifically targets the blood vessels allowing them to dilate and increase blood flow. Nitric oxide production can decrease as a person ages. Past studies in the IPA Lab indicate that increases in nitric oxide are associated with improvements in the function of the blood vessels, thereby decreasing cardiovascular disease risk (1).  Now that the 5-year study is complete, myself and a team of researchers are analyzing endothelial cells collected from subjects. Endothelial cells are found, on the innermost layer of blood vessels and control dilation and constriction of the blood vessels. We are hoping to find mechanisms that sodium nitrate may be acting through to decrease cardiovascular disease risk in middle age and older adults.

Realities of Research

In a perfect world, research would flow without any issues. There would be no problems, no set backs, and no recollecting data. The reality is that this is impossible. There are always mishaps along the way, but the key is overcoming and problem solving these mishaps. Working in the IPA Lab over the past summer, I’ve learned that imaging endothelial cells is an imperfect art. Even if you complete the procedure correctly there are still some slides that don’t stain as well as others or have no cells on them to analyze. Our research team is constantly trying to improve our technique with endothelial cell staining and we have recently made changes to the collection procedure hoping to provide better staining images for the future.

Life of a Scientist

Over the past summer I have also gained insight on what the real life of a physiology researcher is. At times it can be frustrating if your procedure doesn’t work or you don’t receive the results you were expecting, but that’s part of the job. As a researcher it is essential to develop problem-solving skills to work past these frustrating times and to work as a team of researchers helping each other solve problems in the lab. At the same time though it can be one of the most rewarding careers. When you finally finish a paper and publish in a journal, you feel an overwhelming sense of accomplishment and feel as though you and your team of researchers have made a true impact on the future of science.

After staining the endothelial cell slides, I use a microscope to visualize the immunofluorescence of the cells.

References

  1. Seals DR, Jablonski KL, Donato AJ. Aging and vascular endothelial function in humans. Clin Sci 120(9), 357-75 (2011)
Kayla Woodward is a senior majoring in Integrative Physiology at the University of Colorado in Boulder, CO. She is a 2018 Undergraduate Research Excellence Fellow (UGREF) working in Dr. Doug Seals’ Integrative Physiology of Aging Lab at the University of Colorado in Boulder, CO. Kayla’s fellowship is funded by the APS. After graduation this spring, Kayla plans to apply to medical school pursuing a career as physician and researcher.
January 22nd, 2019
Stem Cell Summer Research

Research Project

Throughout our lives we accumulate damage naturally in our day-to-day activities. Thankfully, our bodies have an incredible potential for repair. Damaged tissues might follow a sunny day at the beach without sunscreen or the micro-tears in muscles resulting from a hard workout, are repaired by our bodies via the orchestration of numerous cellular processes. In response to tissue damage, various proteins will signal to specific cells in the damaged tissue that have the potential to differentiate and repopulate damaged tissue, inducing a regenerative response. These specific cells are known as stem cells .

My research this summer focused on a protein complex that has reported roles during aging and in mediating repair in particular tissues: mTORC1. In muscle, mTORC1 has been shown to be crucial to the activation of muscle stem cells, which are normally dormant, so that they may repopulate the damaged muscle by replacing injured cells with new, healthy cells. Although the role of mTORC1 in tissue regeneration is well understood in muscle, its role during repair is unclear within many other organs, particularly the intestine. The inner lining of the intestine, known as the intestinal epithelium, experiences tremendous damage on a daily basis, in part, as a result of the mechanical stress to the intestinal cells from the passage of food. As such, the intestinal epithelium is one of the most proliferative tissues in the human body, having the capacity to turnover every 5-7 days. My research this summer aimed to understand the role mTORC1 in repairing the intestinal epithelium after injury, and also to understand how this role may differ from its activity in an uninjured context, during homeostasis. We hope that the results from this work will help us better understand the intestinal regenerative process in order to someday have the capability to pharmacologically enhance regeneration in injured individuals, or prevent it, in conditions such as cancer where tissue grows excessively quickly.

Realities of Research

One of the aims of my research project was to focus on the role of mTORC1 during homeostasis. I hypothesized that if the activity of mTORC1 was inhibited, stem cell activity in the intestinal epithelium would be impaired. After genetically manipulating mice so that mTORC1 activity was only depleted in the intestine, we saw that mice lost a dramatic amount of weight and became sickly. We also expected a change on the cellular level. For instance, we anticipated changes in the number of cells actively dividing in the intestinal epithelium as well as changes in the architecture of intestinal epithelial cells. In order to better visualize the number of cells actively proliferating within the intestinal epithelium, I used a cell staining technique called Edu staining which allowed me to visualize each cell undergoing division. Once imaged, I could then quantify the exact number of cells undergoing division within a set amount of time and compare the number of proliferating cells in mice in which mTORC1 was depleted, compared to control mice with normal mTORC1 activity . The Edu staining showed a decrease in the number of cells proliferating in mice depleted of mTORC1 activity, further supporting my hypothesis. In order to further characterize the changes that occur after obstruction of mTORC1 activity, I plan to determine whether intestinal stem cell number is affected following genetic mTORC1 depletion. I also plan to analyze the general structure of the epithelial tissue using a staining technique called H&E staining and quantify the number of cells undergoing programed cell death using cleaved caspase 3 staining.

Life of a Scientist

This past summer, I’ve learned a lot about what it means to do research. Primarily, I’ve discovered the necessity of having keen attention to detail. Prior to working in Dr. Samuelson’s lab, I hadn’t realized how many steps need to go correctly in order to obtain data from an experiment. For instance, to complete a Western Blot, which is an experimental technique used to separate and identify a specific protein from a sample containing hundreds or even thousands of proteins, each step in the process must be executed with acute accuracy and precision. First, the mice had to be treated with the proper experimental treatment. Following the harvesting of the tissue, the protein within had to be extracted and quantified extremely accurately. Finally, the Western Blot could be run, which also has potential for error. If even a single step within the entire time intensive process was completed inaccurately or improperly, the Western Blot likely wouldn’t work and the entire process would need to be redone.

As I near the end of my summer in Dr. Samuelson’s lab, one of my biggest takeaways from working in the lab is that a scientist is never done learning. Sitting in on lab meetings with a team of experienced scientists and PhD students has shown me that even experts in certain fields don’t have the answers to every question. During my ten weeks in the lab, each week was entirely different. While there were several core skills that I learned and expanded upon through practice throughout the whole summer, each week also came with new scientific techniques and new ways of thinking and approaching problems. This constant exposure to new information and lifelong learning is what excites me as a scientist.

Yasmine Abushukur is a senior at the University of Michigan studying both French and Bimolecular Science. This summer, she worked in Dr. Linda Samuelson’s lab thanks to funding from the APS Undergraduate Summer Research Fellowship. This upcoming school year, she plans to continue working under Dr. Samuelson’s guidance, studying gastrointestinal physiology. After graduation, she plans pursue a career as a physician-researcher.
January 11th, 2019
The Effect of Acidic or Neutral Environment on the Mitochondrial Morphology of Human Kidney Cells

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

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

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

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

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

 

Realities of Research

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

 

Life of a Scientist

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

 

DengFeng Li is a junior majoring in Biology at the University of North Carolina at Greensboro. He is a 2018 APS Undergraduate Summer Research Fellow working in Dr. Snezana Petrovic’s Lab at Wake Forest University School of Medicine. Li’s fellowship is funded by the APS and a grant from the National Institutes of Health.