Monthly Archives: October 2018

Dietary Fiber: Why Your Parents Told You to Eat More of It

Histologic section of intestinal tissue isolated from healthy mice stained to visualize intestinal epithelial cells. Credit: Lance Peterson, Theresa Alenghat, and David Artis

The epithelium is a layer of cells that separates the inside of the human body from the external environment. In the gastrointestinal (GI) tract, these cells are known as enterocytes and must form a barrier against harmful pathogens present in the gut lumen, while at the same time aiding in the digestion and absorption of nutrients. It is important that all these functions of the epithelium are tightly controlled to maintain homeostasis. Dysregulation of these complex processes has been shown to lead to diseases such as inflammatory bowel diseases (IBDs) which affect over 1 million US residents (Kaplan, 2015). IBDs, which include Crohn’s disease and ulcerative colitis, are characterized by chronic inflammation of the GI tract leading to abdominal pain, weight-loss, fever and a loss of quality of life. While the exact cause of the disease remains incompletely understood, we know that the integrity of the barrier of our GI tract is crucial in IBD prevention (Martini, Krug, Siegmund, Neurath, & Becker, 2017).


At a young age, we are often told to eat our vegetables and that fiber is good for our digestive health, but what does that entail? Recently, we have shown that a dietary fiber known as rhamnogalacturonan (RGal) enhances gut barrier function. Furthermore, we have shown that RGal decreases disease severity in a mouse model of colitis. However, how RGal improves intestinal barrier function remains incompletely understood. My project over the summer aimed to characterize the mechanism through which RGal enhances epithelial barrier function. Specifically, my project aimed to evaluate the role of intracellular proteins known as protein kinase Cs (PKCs) in the modulation of barrier function in an intestinal epithelial cell line in response to RGal. Our lab used an apparatus called the Ussing Chamber to measure epithelial barrier permeability. In my project, I will treat my cells with various chemical inhibitors of PKCs in Ussing Chambers and then determine barrier permeability to small ions in response to RGal. If PKCs are involved in the modulation of barrier permeability in response to RGal, chemical inhibition of PKC will block the beneficial effect of RGal on barrier function. By understanding signalling pathways that enhance barrier function in inflammatory diseases in the GI tract, we have the potential to use dietary fibers such as RGal to leverage these pathways to treat active IBD.


There are two realities of research that I was able to experience this summer. First, I think one of the most rewarding things about my research is that we sometimes did not obtain the results that we expected to. Although this may seem counterintuitive, unexpected results in my project were always the most interesting because I was not only able to observe my supervisor’s stunned reaction, but those results were the ones that allowed us to come up with an alternative hypothesis and steer the project in a completely different direction than we initially planned. I think that the experiments that generate unexpected results are my favorite thing about science. Secondly, I think that the most important thing for people to realize about the day-to-day lives of scientists is that finding the cure for cancer or any other major disease does not happen every day. While understanding the bigger picture in the context of a particular disease and the rationale behind the experiments that we conduct is important and keeps us focused, the things that we study day-to-day often involve understanding the physiological role of a particular cellular protein or defining a cell signalling pathway. Although learning cell signalling pathways may sound a little less exciting than curing cancer, a single cell signalling experiment contributes to the overall body of knowledge which eventually leads to the development of a therapy.


This summer, I was incredibly fortunate to work with the people that I did. First, my supervisors Dr. MacNaughton and Dr. Baggio really allowed me to discover my passion for science. Every day, I am able to see their excitement about my work and their devotion to educating the next generation of scientists. Secondly, my lab mates were some of the most knowledgeable, supportive, and enthusiastic scientists that I know. Five years from now, I will not only remember the science from this summer, but I will still remember our debates about fruit with meat in salads, our arguments about whether or not the word ‘meth’ should be allowed in Scrabble (it shouldn’t), our common frustrations about failed western blots and our disagreements about how to pronounce words like ‘drama’ or ‘garage’.


Judie Shang attends the University of Calgary in Alberta, Canada where she is majoring in Biomedical Sciences. She is an Undergraduate Research Excellence Fellow (UGREF) and is working over the summer with Dr. Wallace MacNaughton at the University of Calgary where she is studying the effect of dietary fibre on the intestinal epithelium. After graduation, she plans to attend graduate school to study mucosal immunology.
My “Diary”al Journey

Bile acid diarrhea is seen in approximately 10 million Irritable Bowel Syndrome (IBS) patients, and about 86% of Crohn’s patients. Furthermore, there are 90 million patients worldwide annually that have Bile Acid-Associated Diarrhea (BAD). Despite this high prevalence, the underlying mechanism associated with BAD is not completely understood. The primary goal of our research is to identify the mechanism behind bile acid-induced tight junction dysfunction that results in diarrhea. Bile is produced by the liver, released into the duodenum where it helps emulsify fat. Once its job is done, about 95% of bile acid is recycled back to liver, and less than 5% enters the colon. The primary bile acids enter the colon, where anaerobic bacteria convert the primary bile acids to secondary bile acids, and they aid with normal secretion of fluid. However, if the bile acid reabsorption is hindered, as in cases like Crohn’s disease and Celiac disease, there is an excess amount of bile acids that accumulate in the colon, resulting in fluid loss and diarrhea. It has been shown by us and others that bile acids with two hydroxyl groups, such as chenodeoxycholate (CDCA), can alter tight junctions when present in the luminal surface and stimulate chloride secretion when present in the basolateral surface to result in diarrhea. However, the bile acid with one hydroxyl group, lithocholate (LCA), does not alter secretion. In fact, it helps attenuate the barrier dysfunction caused by CDCA. So, how does CDCA travel from lumen to the basolateral surface? This summer, in collaboration with an organic chemist, Dr. Rubush, at my university, we tested the hypothesis that CDCA loosens tight junctions to travel paracellularly to reach basal surface.  We synthesized and tested fluorescently tagged CDCA and LCA in order to visually track their movement in experiments. Our results thus far, show that there is a time dependent increase in CDCA content in the basolateral chamber of the transwell. I will continue to test if LCA behaves similarly. The reason for the yin and yang in bile acid action could be due to it structural variation, the lack of a hydroxyl group in the 7th position.  Therefore, we are synthesizing 7, methyl CDCA to compare its effects on tight junctions with that of CDCA and LCA.

Being part of a research laboratory has made me realize that the process of doing science is filled with many unknowns and surprises.  From having to deal with slow growing cells and lack of the expected band in a Western blot to problems getting the methyl group on the correct hydroxyl group and trying to increase the yield in chemistry lab, I was exposed to the challenges in day-to-day activities in a scientific lab. For example, a minor problem with the air conditioner in our school resulted in the decomposition of an intermediate reactant in the synthesis of methyl CDCA, which set us back by a week or so.  Suddenly, our colon cell line started to grow slowly and this made us change the direction of our research temporarily and focus on performing ELISA assays on samples frozen previously.

My day-to-day life as a researcher consists of a combination of planned activities, such as making buffers, media and feeding cells every Monday, Wednesday, and Friday, and designing and running experiments on confluent cells at other times. At first, I thought research would be very repetitive with rigid timings and I would be alone doing experiments independently in a lab.  To my surprise, my research timings were very flexible, and I collaborated with students and professors of different departments at my university. In fact, along with organic chemistry students in the summer research program at Benedictine University who synthesized the tagged bile acids, we gave a collaborative, interdisciplinary research presentation which was very well received by faculty and peers from the College of Science. As with life in general, there were good days when we got great data, and bad days when we had contaminated cells; but overall it is a very invigorating experience!


Ugne Dinsmonaite is a senior majoring in Biology at Benedictine University in Lisle, IL. She is a 2017 APS Short-Term Research Education Program to Increase Diversity in Health-Related Research (STRIDE) Fellow working in Dr. Jayashree Sarathy’s lab at Benedictine University in Lisle, IL. Ugne’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, Ugne plans to pursue a career in dentistry with research in craniofacial pathobiology.
Learning to Become a Researcher

When people or animals feel threatened, their sympathetic nervous system, a.k.a. ‘fight-or-flight’ system, releases chemicals that increase their blood pressure and heart rate to prepare for fighting or fleeing danger.  Unfortunately, when someone is obese or eats a chronically high-fat diet, their fight-or-flight system can be in an almost permanent state of overdrive.  This can place too much strain on the heart and blood vessels, potentially leading to hypertension (high blood pressure) and subsequent cardiovascular disease such as a heart attack or stroke.  My research project for the summer was to identify specific pathways in the mouse brain that influence the fight-or-flight response.  More specifically, I aimed to determine how inhibition of the dorsomedial hypothalamus (an area of the brain) by neuropeptide-Y (a brain-specific chemical messenger) leads to decreased activity in the fight-or-flight system.  By determining how various chemicals and pathways in the body and brain influence the fight-or-flight system, we may be able to find new treatments for people who have hypertension, hopefully increasing their longevity by decreasing their risk for serious conditions like heart attack or stroke.


Working in a research lab is simply amazing.  There is an almost endless amount of techniques, equipment, and software available to learn how to use.  This summer I have learned how to perform immunohistochemistry, how to use a confocal microscope, and how to utilize different analysis software programs to interpret results from fluorescent images.  If time permits, I may even learn how to perform microinjection surgery on a mouse and how to use RNAscope to complement my immunohistochemistry experiments.


Two things that surprised me about working in a research lab were how time-consuming experiments can be, and how expensive research supplies are.  For instance, it takes a minimum of sixteen days post-injection before the mouse brains are ready for me to begin processing them.  The brains must then be frozen, sectioned, immunohistochemically treated, mounted onto slides, then imaged, all of which adds up to around thirty hours of processing for a set of three or four brains.  Additionally, much of the processing utilizes expensive solutions and equipment, such as the $400 primary antibody used in the immunohistochemistry, or the fluorescent microscope which costs around $55/day to use for imaging.  This experience helped me to realize the importance of organization, precision, and time-management when conducting an experiment, since any mistake could result in hundreds of dollars wasted and countless hours lost.  Thankfully the experiments I’ve conducted so far this summer have turned out great, and I look forward to starting my next large batch of experiments next week.


The day-to-day life of a scientist is highly variable based on my experience this summer.  During any one week I might complete a variety of different tasks based on the needs of my research project as well as the needs of my lab colleagues. While there are general deadlines to be met for certain things and some experiments that require assistance from others, for the most part I am free to schedule which tasks I will be working on for any given day.  One downside to working in research is that since certain equipment is too expensive for each lab to have one of their own, it must be purchased and shared by the whole department.  For instance, the fluorescent microscope that I use is a very popular tool for the type of research done in our department, so you must make a reservation in order to use it.  Unfortunately, if your imaging is taking longer than expected and you didn’t reserve enough time on the microscope to finish, you could end up waiting an entire week before another reservation is available.  Thankfully, with careful planning, this problem can usually be avoided.


Overall, working in research as part of a team with the members of my lab has been wonderful.  Each person has their own unique background in research, and since I’m the most junior member of the lab there is a wealth of knowledge I can learn from each of them.  I truly appreciate how much each of my lab colleagues is willing to teach me what they know, provide answers to my questions, and give me guidance for not only my research project, but for my education and career goals as well.


Alyssa Bonillas is a senior at Portland State University in Portland, OR, majoring in both Biology and Psychology.  She is a Hearst Fellow working in Dr. Virginia Brooks’ lab at the Oregon Health & Science University in Portland, OR.  Alyssa’s fellowship is funded by APS through a grant from the Hearst Foundation.  After graduation, Alyssa plans to further her education by completing an MD/PhD program, and continuing on to become a physician-scientist at an academic research institution.
Summer with GPER

GPER KO Kidney with damage

This summer I worked in Dr. Sarah Lindsey’s lab at Tulane University School of Medicine. Dr. Lindsey’s lab explores the role of the G protein-coupled estrogen receptor (GPER) in vascular protection, specifically in relation to postmenopausal cardiovascular disease. At the start of the summer, I was trained in the field of histological pathology. This involves the examination of disease in tissue through techniques like staining and microscopic analysis. I applied different stains to tissues from mice kidneys, hearts, and aortas, to assess the protective effects of GPER in cardiovascular and renal damage in hypertensive mice. We predicted that mice without GPER would show more damage in their tissues than the control mice due to the protective qualities of GPER, specifically its role in the maintenance of extracellular matrices. This project is one of many the lab is conducting investigating the protective characteristics of GPER to judge its potential as a drug target in postmenopausal cardiovascular disease. This research could help alleviate the high levels of arterial stiffening seen in postmenopausal women.


Working with all the great researchers, technicians, and students in the lab this summer has taught me a lot about lab techniques and scientific concepts, but has also opened my eyes to what it is really like to work in a research lab. One of the most important skills I learned to value is patience. Science is vast and ever-growing, but it isn’t fast. I realized that a summer worth of research wouldn’t equate to fulfilling a project in its entirety- it takes time to acquire tissue sections from animals, conduct trials, and troubleshoot errors during experiments. This also means that working together with other members of the lab is crucial. Everyone has different areas of expertise, and they are all willing to contribute their knowledge to other’s projects to make the process run smoothly. I also learned that even though you start a project with a probable hypothesis, it is possible for your findings to reflect something totally different. Regardless, all findings are valuable, even if unexpected.


“Having ownership over a project gives agency to make decisions and learn techniques on your own.”

Working in a research lab is the optimal learning environment due to the flexibility and creativity it allows. In my lab, there are undergraduate students, graduate students, post-doctoral fellows, technicians, and a PI. This allows for a hierarchy of knowledge and expertise that provides a very comfortable environment for asking questions and learning from others. There will always be someone around to help if needed, and other projects to follow and learn from. However, there is also the opportunity to work on a unique project of particular interest to you. Having ownership over a project gives agency to make decisions and learn techniques on your own. Additionally, weekly lab meetings allow everyone in the lab to understand what others are working on, ask questions, and offer suggestions. This structure is ideal for those who like to work autonomously when desired, yet still receive aid and feedback when needed. It is a pleasure to be surrounded by such smart, driven people every day.


Ines Aguerre is an undergraduate senior majoring in Neuroscience and Sociology at Tulane University in New Orleans, LA. Ines is conducting cardiovascular and renal research under the guidance of Dr. Sarah Lindsey, an Assistant Professor at Tulane School of Medicine, also in New Orleans, LA. Ines is a recipient of the STRIDE Fellowship, which is funded by the APS and a grant from the National Heart, Lung and Blood Institute (Grant #1 R25 HL115473-01). After graduating from Tulane University, Ines plans on attending medical school in the hopes of becoming an MD and conducting more research.