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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.

Protecting Hearts

This summer, I received an American Physiological Society Short-Term Research Education Program to Increase Diversity in Health-Related Research (APS STRIDE) fellowship. This fellowship, funded by APS and a grant from the National Heart, Lung and Blood Institute, allowed me to do 10 weeks of research at the University of Missouri. I focused on how aldosterone can decrease adenosine-induced coronary vasodilation, an important cardioprotective mechanism. Aldosterone is a steroid hormone which is associated with activation of the renin-angiotensin-aldosterone system (RAAS) (Klabunde, 2016), and adenosine is an important compound which has numerous roles in the body. This concept becomes important during heart attacks when adenosine would ideally increase dilation (widening) of heart blood vessels to increase blood delivery to damaged areas (Chen et al., 2013). Increased RAAS activation increases levels of aldosterone which ultimately decreases this protective mechanism, and the infarcted areas do not receive the blood they need in order to be protected. I will be exploring how aldosterone performs this action and whether it acts on one of the two main adenosine receptors in question: A_2A and A_2B. Individuals with high RAAS activation have higher levels of plasma aldosterone as well as a higher risk for heart attack, so understanding this pathway can be beneficial for those individuals. I used mice as an animal model and I am hoping that a better understanding of this mechanism can help humans with heart damage.

We increased plasma aldosterone in the mice by implanting osmotic mini pumps that infused aldosterone over a course of 4 weeks. Afterwards, we dissected coronary artery rings and measured vasodilatory (vessel widening) responses to a few vasodilators and specific agonists of the A_2A and A_2Breceptors. This was done by mounting the rings in a muscle force measuring machine (wire myograph) and collecting data on their isometric tension production. We followed up these experiments with PCR (Polymerase Chain Reaction used to amplify segments of DNA), DHE staining (Dihydroethidium used to detect reactive oxygen species), and western blotting (identification and measurement of proteins).

Spending the summer as a STRIDE fellow was a really amazing experience for me. I’ve always been very passionate and interested in research and this summer was a reaffirmation of that. I enjoyed being involved in the project and working as a part of a team. This fellowship was incredibly helpful because we were able to network with other fellows from all over the country. We also completed regular assignments that strengthened our critical thinking, writing, and communication skills. I express my sincerest thanks to APS and the Bender lab for this opportunity!

References

Chen, J. F., Eltzschig, H. K., & Fredholm, B. B. (2013, April). Adenosine receptors as drug targets–what are the challenges? Retrieved July 28, 2017, from https://www.ncbi.nlm.nih.gov/pubmed/2353593
Klabunde, R. E. (2016, December 8). Renin-Angiotensin-Aldosterone System. Retrieved July 28, 2017, from http://www.cvphysiology.com/Blood%20Pressure/BP015

Maloree Khan is a senior majoring in Biochemistry at the University of Missouri. She is a 2017 APS STRIDE fellow working in Dr. Shawn Bender’s Lab at the University of Missouri Department of Biomedical Sciences. Khan’s fellowship is funded by the APS and a grant from the National Heart, Lung and Blood Institute.

Science That Gets Your Blood Racing

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

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

Realities of Research

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

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

References

High Blood Pressure Fact Sheet [Online]. Centers for Disease Control and Prevention. Centers for Disease Control and Prevention: 2016. https://www.cdc.gov/dhdsp/data_statistics/fact_sheets/fs_bloodpressure.htm [14 Jul. 2017].
Miao CY and Li ZY. The role of perivascular adipose tissue in vascular smooth muscle cell growth. British Journal of Pharmacology 165(3): 643-658, 2012.
Ayala‐Lopez N, Martini M, Jackson WF, Darios E, Burnett R, Seitz B, Fink GD, Watts SW. Perivascular adipose tissue contains functional catecholamines [Online]. Pharmacology Research & Perspectives: 2014. http://onlinelibrary.wiley.com/doi/10.1002/prp2.41/full [14 Jul. 2017].
CD® IGS Rat Crl:CD(SD) [Online]. CD® IGS Rat | Charles River. http://www.criver.com/products-services/basic-research/find-a-model/cd-igs-rat?loc=US [14 Jul. 2017].

Maleeha Ahmad is a senior majoring in Genomics and Molecular Genetics at Michigan State University in East Lansing, MI. Her Summer 2017 Undergraduate Research Excellence Fellowship (UGREF) gave her the opportunity to continue working in Dr. Stephanie Watts’s lab at Michigan State University, where she has been conducting research for the past three years. Maleeha’s fellowship is funded by the APS. After graduation, she plans to attend medical school and be involved in clinical research.

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.

A Study into the Effects of Physical Activity and Endothelial Function into Mitigating Preeclampsia

Preeclampsia is a pregnancy-related disease in which complications inflict harm onto the mother and/or baby, and in severe cases may lead to death. These complications are known to affect 2-8% of all pregnancies. My research has two aims focused on uncovering the methodology behind preeclampsia so that it may be diagnosed prior to delivery and successfully combated against. The first relationship I am looking at is one between physical activity levels and the risk of developing preeclampsia. I have theorized that an increase in physical activity levels during pregnancy will cause a decrease in the risk of developing preeclampsia. This will be assessed by the Pregnancy Physical Activity Questionnaire and postpartum medical reports stating if any diagnoses of preeclampsia were present. Preeclampsia is marked by high blood pressure, so the second aim will look at the relationship between the development of preeclampsia and endothelial dysfunction. The endothelium is the layer of cells that line organs, cavities, and especially blood vessels, the heart, and the lymphatic system. I will be looking at how fast blood flows between two points on the body, a measurement called Pulse-Wave Velocity, that will indicate the stiffness of the arteries. Other measurements will also be collected to help assess arterial stiffness, and thus endothelial dysfunction.

I started into this research lab my sophomore year. What I expected was being in a setting similar to my advanced biology lab courses where we ran experiments such as western-blots, PCR reactions, and such. Though my lab does have this component, I chose to be involved in the clinical side of my research, where I would go to see human participants in the hospital. I really enjoyed interacting with human subjects because it made my work feel more personal and translatable to human life. I have learned a lot of new techniques since my first day, such as collecting images of the arterial walls contracting and relaxing as blood flows and also recording blood pressures by listening to the sound of the heart. What surprised me, however, was how difficult it could be to collect clear vascular measurements since a lot of our work required participants to lie still and awake for 2.5 hours, and occasionally not swallow. At times like those, the vascular imaging data would become too distorted or imprecise for the analysis software later. Thus I learned how to help communicate these needs to the participant in a way that still kept them calm and comfortable but help bring clarity and accuracy to the data. Luckily, we have not ran into any major challenges in our study, and so our research question has remained the same.

What have you learned about life as a scientist?

What I’ve learned about the life of a scientist is that your study makes your schedule, in most cases. Though my role in my lab is more flexible than my more experienced peers who tend to be involved in more than one study, my weekly schedule is still created by the availability of the participants. This was sometimes a great thing, such as when your participants come in on time and everything runs smoothly. But it can also have cons, such as participants never showing up, canceling at the last minute, or having scheduled visits at painfully early hours like 7 a.m. What has made it continuously worth it has been getting to learn something new every day not just from my scientific peers, but also from the participants who volunteer their time to helping us hopefully discover more answers and treatments. Being a part of these efforts to understand preeclampsia and mitigate its impact on up to 8% of pregnant women has been one of my most valuable involvements and I hope to continue to grow in this area and as an aspiring scientist.

Rumbidzai Majee is a senior majoring in Human Physiology/Pre-med track at the University of Iowa in Iowa City, IA. She is a 2017 STRIDE fellow working in Dr. Gary Pierce’s lab at the University of Iowa in Iowa City, IA. Rumbidzai’s fellowship is funded by the APS and a grant from the National Heart, Lung and Blood institute (Grant #1 R25 HL115473-01). After graduation, Rumbidzai plans to pursue a career as a physician scientist in the area of Obstetrics and Gynecology or Dermatology.

Welcome to the New and Improved Undergraduate Researcher Blog!

Welcome to the newly revamped blog! Future posts are written by APS undergraduate summer research fellows to showcase their research and share their fellowship experiences with other undergraduates and the general public.

Every week, we will feature one or two entries from our fellows covering a range of research topics and sharing thoughts on the realities of research and perceptions of the life of a scientist. Please feel free to join in by commenting below each post!

To leave a comment, you must register first. You’ll be asked to provide an email address and create a password. Once your account is created, you will be redirected to fill in required community profile information. This information helps us better serve you by targeting resources relevant to your education level. You will not receive unsolicited emails from the LifeSciTRC.

Interested in becoming an APS Undergraduate Summer Research fellow? Visit the APS website to learn more.

This blog is just one way to engage with other APS Undergraduate Researchers. Also be sure to join us on social media: APS Undergraduate Researcher Facebook page and @APSEducation.

Disclaimer: Information and thoughts posted on the site are attributed to the individual user. APS, NSF, and NHLBI do not endorse any specific comment or post on this site. All discussions and related advice are not to be considered legal guidance.

Allison Hood is the Undergraduate Program Coordinator at the American Physiological Society. She is responsible for organizing and overseeing education projects aimed at fostering and promoting research and career development at the undergraduate level. Projects include the undergraduate summer research fellowship programs (UGSRF, UGREF, STRIDE and IOSP), the Barbara A. Horwitz/John M. Horowitz Outstanding Undergraduate Abstract Awards, the David S. Bruce Excellence in Undergraduate Research Awards, and undergraduate sessions at the annual Experimental Biology meeting. She joined the APS education team in 2013.

Welcome to the APS Undergraduate Researcher Blog!

The American Physiological Society (APS) and the Life Science Teaching Resource Community (LifeSciTRC) are pleased to bring you the APS Undergraduate (UG) Researcher Blog. This blog is dedicated to:

Providing an outlet for undergraduate researchers to share their fellowship experiences, discuss current physiological research, and share their career exploration and planning;
Encouraging undergrads to participate in the LifeSciTRC undergraduate researcher community, including taking leadership roles in the blog by learning how to write a blog post and comment on blog posts; and
Helping undergrads stay current in science content knowledge and professional development.

Interested? Monthly, we will provide discussion topics such as:

Science content/science stories
Society research stories related to comparative physiology topics
Careers in physiology
Tips on attending a national conference

Come read the blog, and leave your comments!

This blog is just one way to chat with other APS Undergraduate Researchers. Also be sure to join us on Facebook APS Undergraduate Researcher and Twitter APS Education

If you’d like to write a blog, email us at education@the-aps.org to sign up with a suggested topic. You only need to provide 2-3 paragraphs. You can cite it on your resume too! For a good example of a blog post, see the I Spy Physiology blog at http://ispyphysiology.com/.

Brooke Bruthers is Senior Program Manager for Diversity Programs at the American Physiological Society. Her main responsibilities include developing, organizing, and implementing education projects aimed at promoting diversity among physiologists and career advancement among physiologists from underrepresented groups. This includes several undergraduate summer research fellowship programs (STEP-UP, STRIDE, and IOSP), Minority Travel Fellowship Awards, Steven M. Horvath Professional Opportunity Awards, Porter Physiology Development Fellowship program, and the K-12 Minority Outreach Fellowship program. She works extensively with both the Porter Physiology Development and Minority Affairs and the Women in Physiology Committees on these projects and regularly attends meetings and conferences to give presentations about these programs.

Melinda Lowy is the Senior Program Manager for Higher Education Programs at the American Physiological Society. She is responsible for most of the awards and fellowship programs at the higher education level. At the undergraduate level, this includes several summer research fellowship programs, such as STEP-UP, STRIDE, and IOSP. She manages special orientation sessions, poster sessions and award programs at the Experimental Biology meeting for undergraduates, including a video contest. She also develops professional skills training courses, both live and online, for all levels.