Author Archives: Brennan Simon

A Reflection Most Cathartic

Hey readers! Well, after 8 short weeks of gratifying summer research, the time has come for me to graduate from the BSURF program. Yesterday I presented the poster I created detailing the research I performed this summer. It felt satisfying to show so many people the work I am so proud of completing. A lot of the people I talked to had little to no experience in cancer research, so it was good practice explaining my project from the most basic level. I also got into some really good conversations with other cancer researchers, and there was one particular PI that I talked to for a while who focused his research on one of the genes I identified as being upregulated in metastasis. It was a lot of fun walking around the poster session and seeing what other students had spent their time researching.

The poster session marked my last day of BSURF, so I think it’s fitting that for my last blog post I will reflect on the summer as a whole. First of all, I am so grateful to Jake (my primary mentor), Ben (another hugely helpful graduate student in the lab), Dr. Pendergast (my PI), Dr. Grunwald (program leader of BSURF), Anna (another program leader of BSURF), and everyone else who helped support my research this summer. That includes the funding provided by the Biological Summer Undergraduate Research Fellowship program as well as the NIH. So much planning went into creating this opportunity to grow as a researcher this summer, and I am extremely appreciative of it all.

As far as my research in the lab, I think I identified both a lot of answers and a lot of questions. My screen of potentially upregulated genes in metastasis revealed 8 ABL kinase-linked genes that are significantly upregulated in the brain metastatic line. I also determined that 5 of these genes were driven by the transcription factor TAZ, a known downstream target of ABL. Many of these genes were kinases or receptors whose upregulation I believe fit the phenotype of a brain metastasis. However, there are still questions to be answered, such as what is/are the transcription factors responsible for the upregulation of the other 3 genes, and what about these genes promoted metastasis? For my assessment of ABL in the EML4-ALK fusion cell line, I showed that although ABL is a functional part of the signal transduction pathway, knocking out ABL has no impact on cell viability. Although this means EML4-ALK isn’t a great model in which to study ABL, this was really useful information for the lab who had not explored this cancer type previously.

It’s also amazing to see how much I grew as a researcher this summer. I began this research experience with the main goal of becoming more independent in the lab. Through a fairly independent research project, a mentor that trusted me to work on my own, and personal perseverance, I was able to improve in my independence. I also learned multiple new techniques, most notably RT-qPCR which was a large part of my project. I also honed my Western Blot skills to the point where Ben trusted me to perform some of his blots for him (they came out very well). Furthermore, since I had never given a poster presentation before, I learned a lot about scientific communication, a skill repeatedly emphasized by Dr. Grunwald. Synthesizing my research into a single poster, and then a single abstract was difficult at first, but after practice much easier. I grew a lot as a scientist this summer, and although there is a long way to go before I’m ready for my future career as a cancer researcher, I am extremely proud of what I accomplished and what I learned this summer.

This is the end of my summer blog series! I hope you enjoyed following along as much as I enjoyed writing. Although it won’t beunder the BSURF title any more, I’m sure I’ll be writing things in the future, hopefully with the journal title “Nature” or “Cell” in there somewhere. See ya then!

-Brennan

Your gut bacteria will not be happy if you eat barbecue for 5 straight days

Hey readers! Welcome to another week of my blog. I submitted my poster for printing this afternoon, so for the next week I will be finishing a couple of experiments and practicing for our poster presentation on Friday. For this week’s blog post, I will be reflecting on one of the many faculty talks that I heard this summer through our program. My favorite of the talks has been Dr. Lawrence David’s. I loved hearing Dr. David’s faculty talk. His energy about the bacteria that live in our gut was infectious and his love for his field was clear. I have had a curiosity for the microbiome especially recently as more and more fundamental processes have been shown to be linked to the bacteria that live in our digestive tract. Dr. David is clearly a well-known figure in the field as some of his papers have been published in the top scientific journals.

Dr. David described his research most basically as “designing meals and feeding them to people.” It’s amazing to me that such a simple experimental setup can yield such clinically relevant data. One of the experiments Dr. David described involved feeding participants either barbecue or a plant based diet for 5 days. He monitored the composition of their microbiome throughout the experiment and compared how their microbiome changed. It was amazing to see how the barbecue group’s microbiomes were significantly altered after just 5 days, whereas the plant group’s microbiome hardly shifted and returned to normal levels quickly. Dr. David also mentioned that the participants that had the barbecue were in general more cranky. I probably would be too if I only ate barbecue for a week, and maybe the microbiome is part of the reason why.

Dr. David also talked a lot about the importance of the Human Genome Project and how it impacted microbiome research. After the HGP, the cost of DNA sequencing dropped, and continues to drop, allowing microbiotic sequencing studies to be cost efficient. The decrease in cost vitalized the field of microbiome research, leading to a lot of the discoveries that have since come out. Dr. David also mentioned some of his clinical trials that are currently underway. One of these involves feeding participants fiber supplements to try to strengthen the diversity of the microbiome. He said to us that a lot of the yogurts and other probiotics that claim to be beneficial to our microbiome probably have no effect on our microbiome, but a diet high in fiber likely does. This was news to me, as I had always believed that yogurt (which contains live cultures of bacteria) helps seed diverse populations of bacteria in the gut.

It was a pleasure listening to Dr. David’s talk. In addition to the exciting science, Dr. David shared a lot of his opinions on careers in science, graduate school, and research. His faculty talk was especially enjoyable and memorable. Since this is the last week of my summer research through BSURF, I only have one more blog post which will be a reflection on the summer. See you then!

-Brennan

A Concrete Abstract

Hey readers! This week’s blog post is a draft of my abstract for my summer project. With our poster presentation in less than two weeks, my projects are beginning to wrap up, so I wrote this draft to encapsulate the work I’ve done so far in a concise paragraph:

There are current inadequacies in the understanding and treatment of brain metastases. This study explored the role of ABL kinase, a non-receptor tyrosine kinase, in lung to brain metastasis with the goal of identifying novel targets for anti-metastatic therapy. We explored this concept using two approaches: 1) assessing the role of ABL in an EML4-ALK fusion cell line (unexplored to date) and 2) screening genes linked to ABL and examining their role in promoting metastasis. These questions were explored in vitro, either in the EML4-ALK fusion cell line or in a brain metastatic-derived cell line (respectively for each approach). Many properties of the EML4-ALK fusion line were found to be impacted by ABL, identifying ABL as an important, and targetable, regulator of this cancer subtype. In comparison to the parental cell line, multiple genes were shown to be upregulated in the brain metastatic-derived cell line. Furthermore, many of these targets were significantly attenuated in an ABL knockdown line. These results implicate ABL as an influential part of the EML4-ALK fusion cancer type, as well as establish novel mechanisms of ABL-regulated metastasis.

Next week I’ll be reflecting on one of the faculty talks we have had so far this summer. See ya then!

-Brennan

Take a Walk in My Lab Shoes

What’s up readers? This week I’ll be sharing a typical day’s routine from my time in the lab. My days start at 9 in the morning when I usually get to lab. I like to bike to work in the mornings from my apartment to wake myself up. My morning schedule completely depends on what experiments I ran the night before, but usually my mornings consist of completing Western Blots or harvesting RNA from my cells. Western Blots require an overnight antibody incubation step, so on days where I run Westerns, the next morning’s first step is to complete it with the necessary washing, secondary antibody incubation, and image development. On the mornings that I’m not developing blots, I often have RNA harvesting to do. One of my most common experiments is comparing RNA levels between different cell treatments using Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR). These experiments often require treating cells with certain drugs, some that inhibit our kinase of interest, others that inhibit other kinases, and others that act as control drugs. These incubations are often 24 hours, but can be as long as 72 hours depending on the drugs used. On days that I harvest RNA, I usually set up the RT-qPCR experiment. This involves an intermediate step known as the cDNA synthesis which converts the sample RNA into DNA for the PCR reaction using reverse transcriptase. After this I set up the RT-qPCR reaction. The entire procedure takes about half the day to set up, but it yields really good data. By the time I’ve finished developing my blots or setting up an RT-qPCR, it’s usually around lunch time. Most of the time I pack a lunch, but sometimes I’ll head to the Hospital Cafe or an off campus restaurant for lunch with some other members of the lab. If I eat in the lab, I like to analyze data or read papers during my lunch. I’ve found that there’s always more to read, and especially since my project is relatively open-ended, there are lots of papers out there that would aid my search for new mechanisms of Abl kinase mediated metastasis. After lunch is usually when I finish my tissue culture work for the day. I keep around 6 cell lines growing at all times, and to keep these cells healthy and happy, I have to dilute them for growing space and replace the media which they grow on every other day. Most of the lines I keep are lung cancer lines, but I also keep a line of 293T cells for the virus experiments that I occasionally do. In addition to regular upkeep of these cells, on most days I need to set up cell experiments. Often these are drug treatments, but they also include virus transductions, cell viability screens, and trans-well migration assays. For these other experiments, I have to count the cells that I have using a (very carcinogenic) dye, calculate the correct dilution ratios, plate a precise number of cells, and if I’m applying drugs, dilute the drugs to appropriate amounts. My tissue culture work can take as little as 30 minutes when I only need to split my cells, but it can take upwards of two hours on particularly busy days (usually fridays). After tissue culture, it’s often time to call the day quits. But if there’s extra time, I will usually run a Western Blot gel of do a quick cDNA synthesis to get a little ahead for the next day. I’ll usually bike home from work, relax for a little, cook dinner, and head to bed to rest up for the next day! My days can be pretty diverse depending on the experiments I need to complete. I love the variety of my schedule. Each day brings new challenges and new data to show to my lab-mates and to put on my poster. Although the 9 to 5 research life is long, I love my days spent in the lab. I’m nearing the end of my summer research, so in the coming weeks I’ll be finishing my research and preparing my poster. Can’t wait to check back in next week!

-Brennan

Microglia: The Coolest Team (of Cells) in the ACC (Not the Atlantic Coast Conference)

What’s up readers! It’s officially the halfway point of my time in the BSURF program, but it feels like it just started. My research is progressing quickly and I have a lot of exciting data to share next time I give an update on my project. However, for this week’s blog post, I will reflect on a fellow BSURFer’s Chalk Talk. Over the past week each of us spoke for 8 minutes about our projects in the lab using just the whiteboard and words to paint the picture of our research. I found myself curious throughout Kat’s talk, which was titled “Development, Microglia, and the ACC.” Kat works in the Bilbo Lab which studies neuron development in the brain with a focus on microglia. According to Kat’s Chalk Talk, microglia make up around 15% of the cells in the brain. They are critical to immune function in the brain, and their misregulation can lead to a variety of mental diseases. The Bilbo Lab has contextualized their research around Autism Spectrum Disorder (ASD), and they study the possible environmental factors that influence microglia development and contribute to Autism. A conclusion has been made in the field that genes alone cannot alone cause Autism, so it is extremely likely that environmental factors have a significant impact on a child’s likeliness to develop ASD. These factors can include heavy metals, chemicals, and pollution. Kat’s project focuses on the impact of pollution, which they simulate by exposing mice to diesel fuel. Kat’s project is to create a developmental timeline of microglia development in the Anterior Cingulate Cortex (ACC) in wild-type mice and mice exposed to diesel. She hypothesizes that the mice exposed to diesel will experience earlier neuronal synapse development than wild-type mice, but after the standard pruning process, fewer overall synapses will remain. The backbone of this hypothesis is that microglia moderate the timeline of synapse formation. Kat has begun creating this timeline of development by imaging slices of mouse brains under a high powered fluorescent microscope, counting the synapses seen, and plotting them on charts. She even mentioned that the images she creates are 3-dimensional, so I can’t wait to see them! This work seems very relevant to the modern world, where rates of ASD diagnosis are growing. I am so excited to see the final results of her project and see whether her hypothesis is true. Her talk was insightful and engaging, with plenty of drawings of microglia and depictions of charts. I enjoyed listening to her Chalk Talk!

Next week I will share a Day In The Life of my time in the lab. I’ll get to share some of the experiments I conduct on a weekly basis and show all you readers how I have been spending my summer! See ya then!

-Brennan

Dr. Pendergast’s Polymorphic Path to PI

What’s up readers? Welcome to Week 3 of my summer research blog. For this week’s stimulating content I interviewed Dr. Pendergast, the PI of the lab I’m researching in. Dr. Pendergast has a great story about how she got to current position as a PI at Duke. I really enjoyed hearing how her career panned out since I am thinking about a similar path.

As an undergraduate, Dr. Pendergast majored in chemistry and minored in molecular biology. She was always interested in pursuing academic research as a career, so she completed graduate school in California and did a postdoc in Dr. Owen Witte’s lab at UCLA. She found an interest in tyrosine kinases at UCLA, and has since continued studying these cell messengers at Duke. Her lab focuses on a specific kinase, the ABL kinase, and its role in cancer metastasis. It was interesting to hear how the direction of her research has changed through her time at Duke. She began researching the ABL kinase in the context of leukemia, and she even used developmental biology experiments to understand the kinase’s functions in cancer. Later she focused more on solid tumors and metastasis, and finally settling on brain metastasis as the primary focus of the lab which is where the lab is now. The non-undergraduate members of her lab are composed of four graduate students and one senior scientist. When I asked her why she doesn’t have any postdocs in her lab, Dr. Pendergast explained that she loves the energy that graduate students have in the lab and that training graduate students is one of her favorite aspects of being a PI. She also told me that the senior scientist in the lab brings really valuable experience to the lab. The combination of old and young makes the lab a vibrant and successful environment. For a fun conclusion to the interview, I asked Dr. Pendergast if there had been any disasters in the lab. Thankfully, she said she has never had any major accidents in the lab, but one student from another lab nearby did accidentally stab herself with a pipette tip! The student was alright afterwards. It was really informative to hear Dr. Pendergast’s path from her time as an undergraduate to PI at Duke. Maybe my path will follow a similar route and I’ll end up as a PI somewhere too. Over the next week I’ll present a chalk talk of my project to the rest of the BSURF program and then reflect on someone else’s for next week’s blog post. See ya then!

-Brennan

Don’t inject cancer cells into your heart if you don’t want brain cancer: a PSA

Hello readers! Welcome to Week 2 of my science blog about my research experience. This week I get to talk about the science that my lab is exploring as well as my specific project. To start, I’ll begin with some background:

Lung cancer causes the most deaths per year out of all cancer types. Nearly 150,000 men and women are projected to die due to lung cancer in 2019 in America alone (American Cancer Society). Furthermore, lung to brain metastasis (the spreading of cancer from the lung to the brain) is a common occurrence and particularly poor prognosis for patient survival. My lab studies the mechanisms of brain metastases by exploring the signaling machinery that goes awry in cancer. We are focusing on a specific kinase (a protein that causes chemical shifts in other proteins, acting like a master signaler) and its role in metastasis. Jake and I are using a line of cancer cells that naturally metastasize to the brain for our research. Jake made this line earlier by injecting cancer cells into the hearts of mice, isolating tumors that spread to the brain, and repeating this process until the cancers reliably migrated to the brain. I think that’s a really elegant way to design a cancer line that is specific to our research.

As far as my research goes, I am dividing my work into two projects. For one of the projects, I am replicating a number of experiments that Jake completed earlier in a different cell line. His results were really promising, and it would help the story immensely if we could show the same effects in a lung cancer cell type with a different “driver” mutation (the major contributor to the tumorigenic phenotype). This project is fairly straightforward since the path has already been laid out, and the process is going smoothly so far.

My second project is much less structured, at least so far. Earlier, Jake compared RNA sequencing data between our metastasizing cancer line and the “parental” line (the same cancer line without the repeated intracardiac injections) to find a list of about 100 genes that appeared to be upregulated in the metastazing line compared to the parental line. Jake and previous students explored some of these genes, but most of them haven’t been analyzed yet. My project is to pick some of these unexplored, upregulated genes and perform tests to determine their levels of upregulation, impact on metastasis, and any interesting new pathways they may be involved in. I chose about 30 of the genes and designed PCR primers for all of them in order to perform an RT-qPCR experiment to quantify their levels in the metastasis line and parental line. The preliminary results were really interesting. In fact, one of the genes appears to be upregulated over 70 times in the metastasis line! Multiple other genes were also upregulated, usually on the order of 2-4 times. These results will need to be repeated to confirm their legitimacy, but my next step is to focus on these genes and figure out why they are upregulated. My plan is to look at the transcription factors linked to these genes and try to trace a path back to the kinase we are focusing on. That would make a cool story!

I am really excited about my work so far. While I have run into speed bumps in different experiments on different days, in general the projects are going smoothly. I really hope I can make a significant contribution to Jake’s work and the work of the other members of the lab. Even if the data I collect doesn’t show anything interesting, I am still learning so much about the daily life of a researcher and what it means to be exploring cancer. Next week I will interview the PI of my lab, Dr. Pendergast, and share that here. See ya next week!

-Brennan

Brennan’s Super Ultra Radical FResearch (BSURF)

Hello readers! Welcome to my weekly stream of consciousness blog about my research experience as part of the BSURF program! I just completed my first week of research in the Pendergast Lab, and it has left me excited to begin week two. My first week was spent reviewing my two projects for the summer, getting acclimated to the lab, and planning my experiments for the next week. I also did some thinking and writing about what I hope to gain out of this summer experience. As someone with some previous research experience, I decided that my goal for the summer is to develop as an aspiring scientist. In more concrete terms, my goals are to achieve a higher level of independence in the lab by planning and completing my own experiments, as well as troubleshooting by myself first (before asking for help) when my experiments inevitably fail. I have already begun to develop a close relationship with my graduate student mentor, Jake, as well as many of the other members of the lab. When I can’t figure something out on my own, or when the guidance of a graduate student will aid my work (which I expect to be very frequent), I plan on using Jake and the other members for advice and help when I need. I hope that I can gather data and develop my skills as independently as I can while still successfully completing my two projects!

More about my projects. I am beyond excited to gather data in a largely unexplored territory in the field that we are studying. Earlier in the spring, Jake and some other members of the lab found a list genes that appear to be upregulated in some cases of  the brain metastasis line that we are studying along with the kinase that is the focus point of our research. Some of the targets have already been explored by other members of the lab, but there are still many left to be analyzed, so one of my projects is to dig deeper into some of those genes. I can’t wait to see what we can find out about the 30 or so genes I chose to investigate. Who knows, something really cool could be hiding there! My other project is to replicate a series of experiments that successfully worked earlier in a different cancer cell line. Hopefully this will be a relatively straightforward way to show that the promising results found earlier can be applied to other cancer cell types. With these two projects on my plate, I have plenty to plan and execute!

As I look forward into the next week, I feel nothing but excitement. I am already planning to learn two new techniques (at least), gather some impactful preliminary data, and work towards my goal of improving my autonomy in the lab. My next post will be a week from today, and I expect to have some interesting thoughts and new perspectives to share! See you then.

Pictures coming soon!

-Brennan