Author Archives: Chad Munger

Logistics and Heterogeneity

The faculty seminars were always brimming with new information, and not just on the speakers’ fields. Each offered advice to us as young scientists. The multitude of career paths by the faculty demonstrated the unpredictability of the future and helped to limit one of my greatest fears, wasting time working on a path that I will never follow to the end. Every talk reinforced the heterogeneity of the people who practice science, the types of research performed, and the methods used to answer questions.

The work in the lab itself was tremendously educational. I learned so many techniques from the ground-up. I gained easy skills that are necessary for basic biology, and others that are very unique for this project and require a lot of dexterity and practice. The work in the lab also made me realize how much work beyond the experiments is performed. Time spent on planning how a test should be run, adapting on the fly, and organizing the materials needed was just as critical to understanding how science is performed as actually doing experiments.

I wish I could say I have a definitive career path after performing research. I have enjoyed my time immensely and do plan to continue research in biology. However, I haven’t yet caught the ‘research bug’ and I still want to participate in other opportunities in similar disciplines. This mirrors the advice of what a lot of speakers pointed to. Experiment with experiences. Look for that potential path. Regardless of my path in the future, I am confident that my research experiences this summer, and in the future, will be extremely impactful on my ways of thinking.

Thank you so much to everyone who organized BSURF, especially Dr. G and Jason. Also, thank you to my lab mentor, Marguerita Klein, for having the patience to teach me so much, and to my P.I, Dr. Bohórquez, for allowing me to participate in a great lab. Finally, thank you to my peers for a great eight weeks and I hope to meet with you all again during the year.

Considering Research Environments and Science Policy

Dr. Charlie Gersbach’s seminar was one of the most interesting talks presented during the BSURF program. What really struck me about his path to where he is now was his consideration between the environments of working in industry versus academia. He described how academia provides flexibility to study what you wish, but at the cost of spending a lot of time applying for funding. On the other hand, industry can provide funding, but for projects that are often more repetitive or lacking stimulation. It made me realize that applying what is discovered in research to the real world requires an adaptation from the mindset that was performing research, to a focus on optimization. I am considering studying biotechnology and, if I go down that path, that choice is going to be critical. Even now, there is no clear answer and I think I will need more experience in both environments for an effective consideration.

His talk also brought up the state of public policy when it comes to genetic engineering and gene editing. I was surprised to hear that editing the DNA of food does not make it a GMO. Instead, ‘foreign DNA’ must be introduced. It demonstrated that, regardless about how one feels about GMOs, there is a gap between public policy and science that needs to be resolved. This requires people trained in the sciences and who have the ability to communicate effectively to those who implement policy.

Zeno’s Plasmids

Going into the program, I certainly expected experiments to completely fail. I expected them to return completely unexpected results. I expected them to be unidentifiably wrong in some way. But what I did not expect is for the experiments to tease me with how close they were to what I wanted.
For much of the summer, my major goal has been to confirm the identity of several plasmids. In order to do so, the plasmids need to first be isolated from bacteria and resuspended in solution. Then restriction enzymes are used to cut up the DNA into single strands that can be visualized on gels in order to see if the expected bands appear.
The first step of isolating the DNA started off well enough. I was able to produce a solution of at least 0.1 micrograms of DNA per microliter. The problem was that two of the plasmids just wouldn’t surpass this threshold. One only reached 0.09 micrograms per microliter, while the other only reached 0.07 micrograms per microliter. I attempted each at least three times, and each time, it was so close, but so far. It was not a complete failure, but just not enough success to warrant using the solution in future experiments.
Also, when using the restriction enzymes to confirm the identity of the plasmids, it seemed as if only a fraction of each run would work, even though there doesn’t seem to be a reason one would work, while another wouldn’t. I started with ten plasmids. Two were confirmed during the first run. Four on the next run. Two during the third attempt. Only one was confirmed during the fourth attempt. The final confirmation required streaking, culturing, purifying, and digesting all over again in. It reminded me of Zeno’s dichotomy paradox that only allowed a weary runner to travel half the whole distance, then half of the fraction remaining, and so on, never reaching the finishing line.
Despite taking much longer than anticipated to confirm the plasmids, there was a great amount of satisfaction after accomplishing something that took that much patience. Additionally, it was much faster to see the application of the labor as two of the plasmids were transfected into HEK 293 cells and shown to express in vibrant reds and greens.
What I have definitely learned through this experience is that the best accomplishments cannot occur suddenly or without trial. Being able to do something immediately, without effort or thought just breeds complacency with the result. The best joys of research have been at the end of periods of exacerbation.
While I am quite confident that my project will produce results within the next week and a half, I am hesitant to admit so since it might jinx the effort. Just as I have learned to trust but verify results, I have also learned to hope for a good outcome, but be prepared for an unexpected result.

pH and bNAps

All of the chalk talks last week were extremely interesting. It was a great opportunity to understand what everyone was researching.

One of the most fascinating talks was Ajile’s presentation. I had never heard of ‘broad neutralizing antibodies’ before. They are antibodies that can attach to, and neutralize HIV. Unfortunately, these proteins do not maintain their function in the human body. This leads to Ajile’s research question. How can the degradation of bNAbs be prevented? She is testing the effect of pH on bNAps. By understanding the conditions that bNAps need to survive, there are all kinds of future research avenues. Can similar antibodies be engineered to not degrade but maintain function? If pH turns out not to be a serious cause of the degradation, then what is the main cause?

The point that I most appreciate about her research topic is its importance and urgency. Knowing how bNAbs degrade and how they can be maintained could create an entirely new HIV treatment that, if it cannot prevent HIV, can at least limit the symptoms.

Becoming a Morning Person

Lab starts everyday at 7:00. On Mondays, a general lab meeting lasts until 9:00 in a room shared by the labs on the same floor. During these meetings, each person describes what they have done in the past week and what they plan to do during the upcoming week. Sometimes, someone presents results from a multi-week project or summarizes information learnt through classes and scientific literature. After the meeting, if it’s a Tuesday, Wednesday, or Thursday, I attend the morning BSURF seminar. After that, I plan the day’s goals with my mentor. I then work until noon at which point I look for a stopping point to get lunch. After returning thirty minutes later, I work and read literature until at least three o’clock and take the bus back to the apartments.

Most of my days thus far have been focused on confirming DNA plasmids that were shipped to the lab. I streak bacterial stabs received and wait overnight to see single colonies develop on plates. The next day, I culture them in flasks with LB broth overnight. On the third day, I isolate the DNA from the bacteria in the cultures. On the fourth day, I use restriction enzymes to cut the DNA so that I can compare the bands that appear during gel electrophoresis to predicted bands. My other main task thus far has been transfecting Human Embryonic Kidney cells with pre-GRASP and post-GRASP in order to confirm that they are expressed. This required splitting and maintaining tissue cultures and performing lipofection. During the time that I am not confirming plasmids or transfecting cells, I have observed procedures being performed so that I can understand exactly what the protocols are demanding.

The lab occupies three benches of a larger room. The bench I mainly occupy is for work with plasmids. The other two benches are for dissections, microscopes, and dissociations. There is a room that is shared with another lab that has a hood to work in, an EVOS machine for imaging, and two incubators.

Hoping for a Glowing Connection

The Bohórquez Lab focuses on the connection between the gut and the brain, especially how the brain perceives what the gut feels. Team members have performed a neurotracing using a monosynaptic rabies virus. The modified rabies virus they used could not move transynaptically because of a lack of its envelope glycoprotein rabG. They could permit the virus to jump a single synapse by expressing rabG in the target cell. By expressing rabG in PYY-expressing cells (often used as an indicator of being an enteroendocrine cell), they found that the virus could jump from PYY-expressing cells to efferent neighboring neurons. This strongly suggests a synaptic connection between enteroendocrine cells and the efferent neurons.

My project’s goal is to confirm this result using mammalian GFP reconstitution across synaptic partners (mGRASP). This technique uses constructs named pre-mGRASP and post-mGRASP. Pre-mGRASP uses a neurexin fragment to present half of a GFP fragment, while post-mGRASP uses neuroligin-1 to present its half of GFP. Normally, neurexin and neuroligin-1 connect across synapses, but in mGRASP, these two proteins reached across synapses to connect the halves of GFP. When connected, the protein fluoresces under the correct wavelength of light.

The aim of my project is to express one of these two constructs in cultured enteroendocrine cells and the other in cultured nodose neurons. The plan is to then co-culture the two groups of cells to see if there is GFP fluorescence. The constructs also express separate fluorescent proteins, mCerulean and dTomato. They are used to confirm that the cells are transfected with the constructs, even if there is not fluorescence from the reconstitution of GFP across a synapse.

The Importance of Opportunity and Reading: An Interview with Dr. Bohórquez

Dr. Bohórquez is an Assistant Professor of Medicine and Assistant research Professor in Neurobiology at Duke University. He refers to himself as a “gut-brain neuroscientist” because he studies the way that the GI tract connects with the brain. Becoming a scientist was not a sudden transition as there was a gradual progression to his current position.

Dr. Bohórquez entered college already attracted to the idea of attaining a PhD. The fundamental reason he was attracted to science was curiosity, and he saw the way that PhD’s, “had the ability to lead a group in developing a project or doing research,” and the independence that they had. The combination of investigation and freedom was extremely enticing.

The first scientific project he encountered was during his senior year of undergrad. It was an evaluation of a new diet for piglets. The nutrition regimen’s goal was to make the transition from weaning to solid food did not cause severe problems. Previously, especially in industrial farming, there had been serious troubles with the transition. The project demonstrated the effectiveness of the new diet, but more importantly, he learned the most was that “he had the ability to test something in an animal… to create knowledge.” The investigative process that he learned was more important than the empirical data.

He also participated in an internship at a farm during his senior year. The cattle herd at the farm had a mortality rate of 2-3%. He realized that it was more effective economically and practically to implement a preventative solution, such as a change in nutrition, than a responsive solution, such as a veterinarian. He noted that this is similar to the way that medicine is adapting to the realization that “preventative is much better than responsive.” He decided to pursue a PhD in nutrition.

The connection between the brain and gut became important during a postdoc position. He was asked to take histological sections of a chicken’s intestine to compare the effects of two different diets on microvilli. When he saw the image, he “was captivated by it. By how beautiful the wall was. That’s where food becomes thought, where food becomes energy.” It was then that he began studying neurobiology as well. He saw two large benefits of studying neurobiology. Firstly, it has “been pushing the edge of science for the last fifteen years.” Secondly, “the brain is still a black box. How we think, imagine, dream is still a mystery.” His reading of ‘The Second Brain’ and ‘The Enteroendocrine System’ confirmed to him how little is known especially about the connection between the gut and brain.

This realization of an opportunity for research is a critical trait that Dr. Bohórquez values. “The people who are able to make progress in any field are those that have the ability to smell opportunity. Opportunity is an opening for making progress.” He saw that people were not studying the gut-brain connection from a ‘receptor-cell-nerve circuitry’ standpoint.

Beyond his research, Dr. Bohórquez reads and listens to audiobooks consistently. He feels that “communication is one of the biggest problems in science,” and not only can reading help to show how ideas are communicated, it can also provide context and therefore urgency for research. The jargon and density of scientific articles makes it difficult for both scientists and the public to understand what should be simple concepts to communicate.

Reading widely can also help to eliminate what he sees as the ‘lineage’ problem in science. Too often, people answer questions in a similar way to their mentors. This leads to an “inbreeding of knowledge” that limits a diversity of approaches and potentially limits available answers.

Understanding the context of research, through reading and experience, can also lead to an avoiding of “science for the sake of science.” Dr. Bohórquez worried about scientists who performed science without a goal in mind. While “in a world where resources were unlimited… you could do whatever you want with your time,” but “you have to have a purpose or you are wasting your time” when there are constraints on time and funding. He appreciates science with urgency, with a final goal.

The final subject that I discussed with Dr. Bohorquez was the way science is taught. He lamented that “unilateral teaching” failed to instill an imagination in students. The idea of questioning being a sign of disrespect just limits the benefit that a student can gain from a teacher. While “it is easier to lead without questions, it is not as effective.” Lessons should talk about the entire scope of a topic to engage the imagination of students. For example, teach the history and applications of genetics when learning the basics of the topic. Dr. Bohorquez wants to see knowledge be created, and he believes the recipe requires one-half knowledge and one-half imagination.

Trying to Transition from a Science Fan to a Scientist

 

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I have always been a fan of science. I read The Big Book of Knowledge as a child. I subscribed to Scientific American. But what I never have been is a scientist. The mindset of a fan of science is to accept what is written, memorize the remarkable facts, and marvel at the ability of science to answer questions about the cosmos. It is a role that requires no active participation beyond reading. However, a scientist has a different way of thinking that requires questioning, answering, and researching in particular ways. I expect this summer to show me the difference between being a fan of science and being an actual scientist.

The most important lesson that my experience could help me to learn is what questions to ask. Just accepting a laboratory result would not be performing science if the protocol were not reductively examined, the statistical significance of the data was ignored, or the potential contaminations were not considered. My lab experience will hopefully provide me exposure to the types of questions that will lead to appropriate consideration of the concerns listed, among others.

The second expectation that I have for my summer experience with BSURF is that I will learn a plethora of techniques. Understanding the current capabilities available provides a toolbox to answer questions. These procedures also provide intellectual building blocks for designing brand new methods of investigation that are adapted to answer brand new inquiries.

The final expectation that I have of my experience is that I will see what maintaining a lab requires. Interviewing my PI and observing the way my mentor spends her time will provide an insight into the process of grant/paper writing, material purchasing, and managing people.

Additional note:

The distinction between being a fan of science and being a scientist does not mean that one cannot be both. In fact, I believe that the majority of scientists are also fans of science sometimes.

Image Source:

Weinersmith, Zach. “How to Tell the Difference.” Comic. Saturday Morning Breakfast Cereal, 30 January 2010. Web. http://www.smbc-comics.com/?id=1777