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Let’s Science From Home!

If things were normal, this would be Week 5 of BSURF. But, a pandemic and a summer class later, here I am, sitting at home, typing up my first blog post. Crazy, isn’t it?

This year’s BSURF program will be drastically different from previous years. Instead of being on campus with the other BSURF fellows, I’ll be working remotely for the next two months, going to Zoom meetings instead of going into the lab. Not all of it is bad news though. There are certain luxuries that come with working from home, such as setting your own schedule, spending time with family, and being able to eat (!) while working. 

Before I get further, a little about my project: under the guidance of Dr. Allen, my PI and mentor,  I’ll be deep-diving into the datasets of two separate studies. The first is the Pan-Cancer Analysis of Whole Genomes (PCAWG) study, which is an international collaboration that yielded groundbreaking research on cancer drivers in non-coding regions of the human genome. In essence, they took over 2,600 whole genome sequences of various cancer types and located potential cancer drivers (read: mutations that cause cancer) in non-coding regions. The second study is by Dr. Gersbach, who is a professor at Duke. His lab recently produced a dataset of “essential elements” in the human genome that regulate normal cell growth. My goal is to intersect these two datasets to see if there are any common hits between them. To put it simply, we’re looking for cause and effect. If, for example, there is a certain essential element that contains many cancer drivers, it would be the first step to confirming the validity of that cancer driver.  Eventually, the hope is to map non-coding cancer drivers to a phenotypic impact.

For the rest of this summer, I have two goals in mind. First, I want to become comfortable with research and the adventure it represents. In more objective terms, I hope to become familiar with the computational knowledge and techniques required to do research in the field of genetics. More importantly, I want to be able to embrace the daunting challenges and uncertainties that come with doing research. Second, I hope to build meaningful and long-lasting relationships with mentors and peers alike. I’m excited to be open-minded and have conversations, whether that’s about research projects, or about anything, really.

Already, research has proven to be an eye-opening experience. Whether it’s deep-diving into literature or emailing for help, I’ve quickly learned research is by no means a linear process. In reading my first paper, I had to read up on three other ones just to understand what was going on. And that’s what I’m coming to really enjoy. There are endless paths to choose from and avenues to explore, and while there will certainly be challenges to embrace, I’ll be ready to adapt, readjust, and push ahead as readily as ever.

Stay tuned, and welcome to the blog!

My workspace from home!

If You Don’t Know, Now You Know

This summer has been an amazing experience. I had no idea that I would learn as much as I did when I first started, and I am so glad to have developed a better understanding for the world of science and found my place within it. And I am so thankful to have done it alongside such wonderful people in my lab and in BSURF, thank you all so much!

One of the biggest things I learned or came to realize is how big the world of science is. From the faculty talks, chalk talks, and the poster session I got to hear about the incredibly diverse and important research brilliant people are doing just here at Duke. Previously I had this notion that once you really get fundamental understanding of a topic, you just move on. But this summer I learned there are always more questions to ask and always new perspectives to take. I learned this while working on my own research project, as I needed to approach my problem differently and ask new questions as I learned more. I also learned that science is an incredibly slow process. Everyone says it, but you don’t fully understand it until you’re waiting for the results you really really want to see and they don’t come. But luckily, research doesn’t stop just because BSURF is over. These eight weeks have been a great introduction, but there’s so much more to discover!

The biggest take away for me this summer was that the research you are doing is useless unless you communicate it. Before this summer, I often overlooked this portion of research and thought hands on work in the lab was most important. But I soon realized that posters, papers, and presentations give your work meaning and direction and make it impactful. I am so grateful for the opportunity to both observe and practice this communication of science, and I hope to only get better from here. Again, I am so glad I was apart of this program this summer. I will surely never look at science or fruit flies the same again.

The End But Not Really The End

This summer I was fortunate enough to spend eight weeks conducting research in the Bejsovec Lab. In addition to this research, I had opportunities both explore Durham and learn from faculty as they shared their research journeys with us. Beyond learning about these journeys, BSURF allowed me to think deeply and critically about how I want to spend my remaining years at Duke and the type of person who I wish to become by the end of my undergraduate career.

I believe my summer experience influenced my perceptions about research in a positive way. Before this summer experience, I never had a clear idea of what the life of a researcher looks like, how slow science could be, but at the same time how rewarding failures and successes can be. I believe among the many experiences I’ve had these past eight weeks, the people that I’ve met and the memories created are the best parts of this summer in addition to learning how to conduct research and how to communicate science.

This summer has allowed me to become more certain of the career I would like to pursue in addition to showing me the multitude of routes that I can take to achieve my ultimate end goal. Furthermore, the advice given to us throughout the past eight weeks has not only made me wiser, but certain that the end of BSURF isn’t truly an end.

I would like to thank BSURF and the Bejsovec Lab for providing me an amazing opportunity to learn and observe science, as well as my fellow BSURF-ers for an amazing summer experience.

I Know Why the Caged Bird Sings

One of my favorite parts of the program this summer has been hearing about the work that so many renown scientists are doing here at Duke. It has been really wonderful getting personable insight into the biological field of research, stories of unique paths into research, and advice for those of us just starting out. I have been able to take away so many words of wisdom and cool stories from every talk, so I want to thank the faculty for their time and willingness to share their stories with us.

A presentation that really interested me was by Dr. Steve Nowicki who talked to us about categorical perception and the evolution of animal signals, focusing in on birds. I feel like as humans, we are so consumed by our own perception and how we view the world that we forget all organisms on the planet perceive the world in their own special way. He talked about how female birds while choosing mates categorically sort out information that is relevant to the fitness of the male birds. It was also really interesting to me how a single note change in song or a slight discoloration in the beak could be consequence of a long combination of environmental stressors and genes that would ultimately affect female choice.

This talk was really interesting to me because the topic intersects a lot of things I enjoyed learning about in class, like sexual dimorphisms and sexual selection in biology and perception and attention in neuroscience. This really unique research shows me how you can find your own niche in biological research and ask questions only you would think to ask, while investigating something you really enjoy. I’m looking forward to learning more from professors in the biology department about their research.

All Roads Lead to Research

Very few times in life do we get a chance to hear from people who are at the very top of their field— intelligent, communicative, innovative, paradigm-shifting individuals who have improved the lives of millions with their work. Dr. Robert Lefkowitz, one of Duke’s two active Nobel Laureates, is one of those people. His lab’s discovery of G-protein coupled receptors has led to the creation of nearly ⅓ of all pharmaceuticals in circulation today; nearly all of us know someone who has benefitted from his discovery. This summer, my peers and I had the opportunity to listen to him speak about his life, career, and research.

 

However, if you had asked Dr. Lefkowitz following his undergraduate degree if he dreamed of becoming a nobel laureate, he would’ve emphatically answered, “No, my goal is to become a practicing physician and care for my patients.” Dr. Lefkowitz always dreamed of becoming a doctor, but when he was completing his training in the late 1960s, the United States Military implemented a “doctor draft”, meaning that he would have to spend multiple years in a branch of the military, presumably with a deployment to the war-torn country. Dr. Lefkowitz heavily weighed his options, and realized that he could apply for a position at the NIH to do scientific research, a position at the time considered a branch of the United States Military, and thus eligible to fulfill his military commitment. Coming from the Columbia School of Physicians and Surgeons, Dr. Lefkowitz was able to secure one of the treasured spots for medical trainees and avoid deployment to a combat zone.

 

It was here at the NIH that Dr. Lefkowitz inadvertently discovered his love for academic research. He completed his obligation and returned to medical training, but while practicing medicine, thought to himself, “I really miss data” and realized that he had to get back involved in research of some sort, which led him to further his academic training and eventually make a Nobel Prize-winning, life-saving discovery later in his career.

 

Dr. Lefkowitz’s research is compelling and highly applicable to everyday medicine, but what I found more interesting from his talk was the unconventional path he took towards becoming a scientist. At the age of 19, as a rising sophomore in college, it’s getting to the time in which I’ll have to declare a major and start preparing for medical school, graduate school, or whichever other path I may take.  Dr. Lefkowitz’s life story was a nice reminder that I don’t have to worry too much— if I work hard and follow what I find interesting, it’s never too late to chase a dream, no matter how lofty it may be.

Working 9 to 5!

Everything’s routine now. The moment I open the door to the Bryan Research Building, a quick rush of AC floods over me. I click the “^” button, step in, click the “4” button, step out. I round the corner, smile and wave to Grace in front of me, plop down at my desk, turn right to say hi to my mentor Jenny. I check the board, scanning the pinned schedule that Jenny and I write on the past Fridays. It will be a mixture of different procedures: burr hole injection surgeries, perfusions, brain slicing, mounting brain slices, imaging, and coding, respectively. Everything must be done in order, and each step takes time, leading to weeks worth of waiting to obtain data. 

Each procedure is intricate and cannot be rushed. 

I usually begin my week with the first procedure: burr hole injection surgeries. I carefully drill a hole into an anesthetized mouse’s skull in order to inject fluorescent tags called tdTomato. After a mouse receives a viral injection in its brain, it takes at least two weeks for the virus to be expressed, or visible through a microscope.

The snowball effect begins.

After two weeks, the mouse is ready to be perfused, and the mouse’s brain must sit in PFA overnight. After a night, the mouse’s brain is rinsed with PBS three separate times in 15 minute intervals. After approximately an hour, the mouse’s brain must be submerged in 30% sucrose for at least a day. After a couple days, the brain is be manually sliced into delicate, thin slices and placed in PBS once more. After the brain is divided, the brain slices are meticulously mounted onto glass slides where they must properly dry overnight. After a night, the researcher must make time to image every brain slice under a microscope connected to a camera. After taking pictures of the injected brain slices, the data must be analyzed in old or new MatLab code. 

After this long process to collect data from one mouse, something could have gone wrong at any stage. The injection may have been too deep or too far to the left, the brain may have been damaged during perfusion, the brain could have been sliced at the wrong angle, the injected area may have been physically stretched out during mounting, and more. Science is slow, and I never understood why researchers say this so often until now. 

Despite the weeks it takes to collect data from one mouse, I’ve learned to appreciate all researchers who have brilliant ideas and work day and night to generate data that may or may not significant. I take Dr. Glickfeld’s words to heart: “We don’t ever hope to see a certain result.” Anything that comes out of experiments will contribute to science in some way. 

I have a rhythm at work now where I can easily come into lab to get into a flow. I’m sad to think that BSURF only has three more weeks left, but I hope to hop back unto the Glickfeld Lab once the school year starts. I love the work here, and even though I don’t have a lot of neurobiology background, I am happy to learn something new everyday. I can’t wait to see what these next three weeks will bring! 

Fly With Me

A day in the life of me in the lab is like a 90s sitcom, where you can predict what’s going to happen but each episode brings it’s own laughs. Each day in the lab is essentially a step in my experiment process, with it’s own victories and defeats, which keeps me looking forward to what each day brings.

First things first, I get to lab and check on my fly babies. I have around 20 vials of flies to take care of so I go in and make sure they’re alive and well. If it’s getting too crowded or if they need new food I flip the flies into new vials. I then collect whatever line of flies I need for dissection and take them to the dissection scopes. Dissection has come to be my favorite part of the experiments I do. After a lot of practice, I think I’ve gotten to a place where fly brain dissection has become far less stressful and actually enjoyable. I grab my forceps and the buffer solution and dissect the brains I need to, at a rate of about 15 flies per hour. I have to dissect a line in about an hour to prevent the brain tissue from deteriorating. I then fix the brains in a PFA solution and wash them thoroughly.

The next step in the process is antibody staining to target the DIP-alpha expression in the brains. We usually put the brains in primary antibody overnight in a 4 degree C fridge, so if this is where I am in the process on a given day, I will come in and get the brains from the fridge and wash them thoroughly to remove the primary antibody. After that I’ll put them in secondary antibody, which will ultimately give us the fluorescence we need to identify DIP-alpha expression under the microscope. This staining process occurs for 2 hours at room temperature and is followed by yet another cycle of washing.

Finally, all the dissection and staining and washing leads up to the defining part of the experiment: imaging. This is where we can see if any of our work paid off. This part involves mounting the brains neatly on a slide and using a confocal microscope to view the brains and any specific signals from the antibody staining. I take pictures of the data and use the results to determine which flies I need to dissect next, what antibody I should change, and start the process all over again.

A day in the life

Dear Diary,

Today was a pretty typical day in lab.  I completed a phosphorylation assay using 10% DMSO to make sure that the assay works with DMSO.

I started out the day by stopping the dephosphorylation reaction that ran overnight.  The reaction mixture included dephosphorylated HipA, MnCl2, lambda-phosphatase, and buffer solution.  I began by isolating the dephosphorylated HipA using a Ni-affinity chromatography column.  After the protein was isolated, I needed to concentrate it up because a specific concentration is required for the phosphorylation assay.  I centrifuged 3 mLs of the elution fraction at a time, concentrating the protein.  Next, I needed to buffer exchange since the buffer used for the dephosphorylation reaction is different from the buffer required for the phosphorylation assay, so I centrifuged the protein three more times, diluting it by a factor of five with the new buffer each time, resulting in a 125-fold dilution.  I checked the concentration of the protein and found it to be 0.71 mg/mL, indicating approximately 70% yield, which is pretty good.  I left my protein in the fridge and headed to lunch.

After lunch, I began the phosphorylation assay.  I have already done this assay twice, but today I needed to try it with 10% DMSO to make sure that it gives the same results as without DMSO.  The compounds that I will test later are kept in 10% DMSO, so it is important to make sure that this will not affect my results and that the usual assays still work with DMSO.  I diluted my protein to a concentration of 0.05 mg/mL in assay buffer and put 500 uL of this dilution into one tube, and 450 uL of this dilution into a second tube.  I also added 50 uL of 100% DMSO to the second tube to create a concentration of 10% DMSO to mimic what the compounds to be tested later are stored in.  I removed 20 uL samples from each tube to serve as my controls.  Then I added 5 uL of ATP to each tube and incubated them at 37°C, beginning phosphorylation.  I removed 20 uL samples from each tube after five minutes, fifteen minutes, thirty minutes, forty-five minutes, an hour, and two hours.  After removing the samples, I immediately heated them at 99°C for five minutes to denature the protein and stop the reaction.  I then added loading dye and stored them in the fridge for later when I will run the gel that will show me my results.  The purpose of removing samples at different times is to demonstrate how HipA auto-phosphorylates in the presence of ATP over time.  I will compare the results of the protein with and without 10% DMSO to determine if the DMSO affected HipA’s auto-phosphorylation or if it affects the phosphostain procedure that I will use to visualize the gel.  If the two gels look the same, I am safe to use this procedure with the test compounds.  If the gels do not look the same and the DMSO does in fact affect the assay, I will have some troubleshooting to do before I can test out the compounds.

Overall, a usual, but rewarding, day in lab.  I can’t wait to stain the gels and see what my results are.

Sincerely,

Caroline

Microglia and Mice and Disorder, Oh My!

Going into week four of BSURF I was so excited to hear about all the great work my peers are doing in the lab. As the summer continues, I’m gaining a more holistic view of what science is and what scientific research looks like. From this week alone, I have learned so much about what biological research here at Duke encompasses, from studying the ultrasonic vocalizations of mice in helium to making mutant enzymes to degrade plastic. I would just like to thank everyone for sharing the stories of their research this week, you all did a great job!

One project that really stood out to me was Kat Beben’s. She is working in Dr. Staci Bilbo’s lab this summer and is characterizing the development of microglia in the anterior cingulate cortex (ACC) of mice. Kat outlined the full goal of Dr.Bilbo’s lab: understanding the prenatal factors (genes, the environment, maternal stress) that can lead to autism spectrum disorder. Through previous research, the lab has come to identify microglia, the immune cells of the brain, as being important for synaptic pruning and refinement, making their disfunction detrimental to biological systems, notably resulting in an autistic phenotype, as shown in mice. The lab is asking if combined prenatal stressors alter this synapse formation and refinement. They are specifically looking at the ACC, since this cortical region is involved with emotional processing, learning, and memory. As Kat works to characterize the normal microglial development in mice, hopefully we can better understand how various stressors disrupt this pathway.

I found it really interesting how the meticulous study and manipulation of such a small system can have such a large and significant impact. In my experience in learning about neuroscience, the function and influence of microglia has often been overlooked, but it is clearly not to be taken lightly. I really admire the work this lab is doing, and it reinforces to me the importance of biological research. As Dr. G reminds us, science isn’t just moving colorless liquids from one vial to the next, although I’m sure we’ve all done this. It’s about discovering more and more about our universe, one question at a time. I can’t wait to see what we uncover!

Trust the Process

When most people talk about flies, it’s usually in disgust followed by swift swatting motions. But when Dr. Pelin Volkan talks about flies, it’s with fascination followed by swift strategies to answer the next scientific question. My principal investigator first started studying molecular biology and genetics at Bogazici University in Turkey and began to get interested in neuroscience. When she went to the University of North Carolina for her PhD, she began to study the nervous system of Drosophila and continued this study during her post doc at the University of California, Los Angeles. At the time, the olfactory system of Drosophila was just being described and peaked Dr. Volkan’s interest in the development and evolution of brain and behavior. This influenced the main work she does now at her lab at Duke, asking how a hard-wired system maintains plasticity, ultimately, what are the nature and nurture factors that influence this organism’s brain?

Dr. Volkan’s approach to doing science is really quite insightful. She notes that you almost never stick with the plan and that your study and goals will change with the problems or circumstances that you encounter. And importantly, chance favors the prepared mind. She told me she really enjoys the academic environment as you are frequently bombarded with new perspectives that open up new questions. She notes that science is not static, it’s progressive, and we need innovative and creative thinkers to continue to propel science forward. Dr. Volkan describes the most rewarding part of doing science is getting a crystal clear answer to one of these questions, but as she explained to me that very rarely happens.

But she also enjoys the chase, the process of discovery and the thrill of seeking answers. This is what drives her, and many other researchers, to press on, failed experiment through failed experiment to find an answer. She enjoys relaying this notion of taking joy in the process to her students in her favorite class here at Duke, her lab course in which students take on projects connected with the lab and experience what it’s like to do science. I am so grateful for this experience to work with Dr. Volkan who has already inspired me to press on and trust the process. 

Great Expectations

Summer is finally here, and along with it comes the heat, humidity and high, high hopes. As I begin my research experience in the Volkan lab, I hope to gain a new understanding of scientific research, take a closer look into the field of neurobiology, and find my place within it.

For the next 8 weeks, I expect to do science. This being my first research experience, I have never done science experiments outside the scope of classroom objectives. I have always walked into chemistry or biology lab with the goal of getting the lab done and getting the answers right. I am excited this summer to take a step away from that and begin to reason and question like a scientist. With the help of my mentor, I am already beginning to understand for my research project the important questions to ask that will lead to important conclusions or even more interesting questions. In essence, I would like to begin to think about science in a refreshing and novel way.

I also expect to learn more about the field of neurobiology and how I would like to be involved with it. I have always enjoyed learning about neuroscience in the classroom but I would really like to see how scientists go about uncovering the mysteries of the brain. In the lab, our model organisms are Drosophila, fruit flies, and we look at the mechanisms of olfactory neuronal development in the hopes of gaining some insight into the development of the complex circuitry of the human brain. Within the first week, I have already found it very interesting how scientists have approached the difficult subject in such innovative ways and employed genetic techniques to manipulate biological systems to learn something new. While working in the lab, I hope to use these techniques myself to contribute some meaningful data to the incredible work that those in the lab have already done.

Finally, I expect to discover how I fit into the world of research. During her faculty talk this week, Dr. Sheila Patek posed some important questions for us to consider that I would like to answer myself, such as what is important to me about research and what it means for research to have an impact. I hope through my work this summer, I come to find meaning and fulfillment in the lab, alongside my peers.

Good Bye BSURF!

I can honestly say that applying for this program was one of the best decisions I could have ever made. BSURF not only provided me the chance to conduct proper research, it also gave me the chance to consider the multiple pathways I could take in science. Going into this program I thought that I was almost positive as to what I wanted to do in the future. After seeing talks from several difference professors, and other faculty, my mind was definitely opened up to the   various possibilities science has to offer. As a result I’m considering exploring other interest that I have.

Another big aspect of the program that I liked and frustrated me at the same time was that it was extremely challenging! The intensive and complicated research I was conducting was not something I was use to, but I would not have a wanted it any other way. I believe the challenge that was presented to me helped me learn to adapt to tough situations and gain something from the experience.

Overall I loved my BSURF experience and I plan on using all the knowledge and experience that I gained from this opportunity in the near future. BSURF gave me an amazing  lab and I will definitely be continuing my research  with it.

 

What a whirlwind

I can’t believe BSURF is over. I feel like I became so used to the 9 to 5 workdays it’s hard to believe they’re over for who knows how long. It’s surreal. I’m so thankful to have been given this opportunity to dedicate two full months to immersing myself in research. There was definitely a learning curve in the beginning as I had never been in a biology wet lab before. But by the end, I felt like I started to get a grasp on what I was doing and was working almost independently. We struggled in the beginning to get results and were forced to go back and optimize our procedure. After we did this, things really took off and we began making small findings, which was really exciting. I’m so happy that I got placed in the lab I’m in now. My mentor was such a good teacher and worked so hard to make sure I understood not only what I was doing but why it was important. My PI also worked hard to take an active role in my experience, teaching me the background information and checking in on my progress and how I was feeling about the whole process. I felt so supported in my lab and worked so hard to make them proud. There were many times when I was too hard on myself and my perfectionism hurt me. But wherever I struggled, my mentor and PI were there and ready to develop a plan to solve the problem.

A lot for me has changed this summer. I went into this program having ideas about my career choice that are slightly different from what I think I want to do now. This experience was incredible and allowed us to develop a realistic perspective on what doing research is like. It has confirmed for me that I love research. I love its hands-on nature, freedom, and flexibility. But I also realized how important other fields are to me too and how what I really wanted to pursue was an integration of biology with other fields of interest to me such as ethics and law. It wasn’t an easy realization to come too and in a way made things that much more unclear. It was scary but exciting. My heart will always be with biology and I still fully plan on continuing to take part in and engage with research. But I realized I needed to include some of my other interests and that I need to work to integrate them in some capacity in my career.

As for how this summer changed my view of science, I don’t think it has changed my view much. But I will say that knowing something and experiencing it are two very different things. To name a few: I knew research was challenging and filled with many obstacles. I also knew that producing interesting results is exciting in a way not much else it. I knew these things going into the summer, but I had never really experienced them myself in a real-world setting. This experience has allowed me to experience those things for myself rather than relying on what someone else tells me. I’m really proud of myself for my perseverance when I ran into obstacles and the final product I’m leaving this program with. It was truly an eye-opening experience and I’m so grateful to have had it and for everyone who dedicated so much time and energy to my development as a prospective member of the field of biology.

Regulation of System xCT (SLC7A11) by ABL Kinases in Human Lung Cancer

The ABL family of non-receptor tyrosine kinases, ABL1 and ABL2, are upregulated in non-small cell lung cancer and promote lung cancer metastasis. Recent work has shown that ABL kinases promote lung cancer brain metastasis and colonization. Global transcriptome analysis of targets controlled by ABL kinases in lung cancer cells revealed SLC7A11 as being differentially regulated in ABL inhibited cells. SLC7A11 encodes the plasma membrane antiporter system xCT which has been shown to be overexpressed in cancerous cells. System xCT exports glutamate and imports cystine, an intermediate molecule in the cellular oxidative stress response mechanism. Real-time polymerase chain reaction (RTPCR) verified a reduction in SLC7A11 mRNA levels following ABL pharmacologic inhibition or genetic knockdown. Continued work is being done to reveal a reduction in cellular protein levels through protein isolation and western blotting. FACS analysis will be deployed to determine whether membrane-localized levels of SLC7A11 decrease upon ABL inhibition. Additionally, a glutamate assay kit will be utilized to determine whether export of glutamate by xCT is reduced in ABL knockdown cells relative to control. The ultimate goal of this project is to understand if ABL kinases promote SLC7A11 expression in order to alter the brain microenvironment and promote tumor colonization.

Abstract and Progress

I’m happy to say in the past few weeks I’ve made some progress with my project. While the beginning was a pit rocky, after a few rounds of optimization and discovering we were using a faulty antibody results have started to come in and I couldn’t be more excited. What I’ve been told is that’s kind of how science goes and that a lot of results come in very quickly towards the end of a given amount of time. I’m excited to see what comes out of these last two weeks!

 

Ubiquitination is a cellular response to damaged proteins caused by oxidative stress. But the mechanisms and targets of different ubiquitin structures have not been well characterized. This research investigated the ubiquitinating enzymes and relative targets related to K48 ubiquitination, a signal for protein degradation, and how the efficiency of relevant molecular machineries, the proteasome and deubiquitinating enzymes, is impacted by different levels of oxidative stress. This was done by using mutant yeast strains with ubiquitinating enzyme knockouts, exposing them to oxidative stress, and analyzing their K48 ubiquitin content with a western blot. Ribosomal isolation and K48 ubiquitin antibody tagging was used to examine ubiquitin targets. Proteasome and deubiquitinating enzyme activity was analyzed using a substrate that fluoresces when processed and tracking light levels emitted by stressed cell lysates. Our data suggests the K48 ubiquitination system involves multiple ubiquitinating enzymes and genes and the greater impact of oxidative stress on the deubiquitinating enzymes than previously anticipated. Improving our understanding of K48 ubiquitination in stressed cells could allow us to better understand diseases such as Parkinson’s and Alzheimer’s that are caused by protein aggregates in stressed cells, and potentially provide new targets for treatment to make cells more resistant to stress.

 

A Day in the Heitman Lab!

I have loved every moment of of my research project this summer. Although its been challenging adjusting to new terminology, vocabulary, and other hands on task, I could not ask for a better opportunity. My days in the lab have been pretty consistent for the most part, with few variations from the norm.

First I arrive around 10:30 where I go straight to my notebook to make sure I have documented everything I have done from previous days. Once I have done this I have a talk with my mentor, Ci, to understand what my next steps are in the project. Once we have had our discussion and I am sure that I understand what we are doing and the reasons why I begin setting up and preparing to run the experiment (most days this would be a PCR overlap). I write in my notebook every reagent that I will be using in order to ensure that I do not make any mistakes. Once I have completed this task, I begin conducting the experiment. I transfer the correct amounts of each primer and DNA template based on my master mix into PCR tubes.

Once I am sure that the correct reagents are in each tube I then place these tubes into a thermocycler (also known as a PCR machine). I program the machine based on the protocol of the polymerase that I am using. Once I confirm the time and temperature of each step, I start the machine to allow it to do its task of amplifying and constructing the DNA strands.

After the machine has finished amplifying the DNA strands, I take each PCR product and prepare it for gel electrophoresis. I do this by adding 5 micro-liters of 10x dye to each tube. Once this is finished I transfer each reagent into a separate well in the gel and run the gel in the electrophoresis machine.

Hopefully after doing this process I have successfully constructed and amplified the DNA strands that I need to continue my project. To confirm whether or not I have done this, I take a look at the gel using Ultraviolet light and see whether the position of the bands compared to the ladder matches my predicted outcome. If not, then I have to go back to the drawing board to see what went wrong. If so, then I will purify each PCR product that came out successfully and then continue on to the next step in the project. And that’s my day!

A Day in the Lab

Every day I spend in the lab is different. So it’s hard to choose a prototype of my day. The protocol for my lab work usually runs on a weekly basis and each day is an incremental step of completing a western blot in combination with optimizing previous western blot procedures and troubleshooting problems with antibodies and film resolution. I start my morning by doing prep-work, usually getting ice, labeling tubes, or turning on centrifuges, and thinking through what I have to get done for that day. On our lighter days of the western protocol, we conduct additional experiments that analyze K48 ubiquitination from different approaches. On these days, a lot of coordination is required to keep the timing correct on both experiments.

When people have a break in their work, around 12 PM, everyone in the lab goes for lunch together. Having lunch together is very important in my lab. They want to facilitate a culture that encourages us to just hang out outside of the lab in a more casual setting. It’s one of my favorite parts of the day. Besides the fact that by noon I’m starving and in need of my third coffee, I like to get a chance to talk to the members of my lab about their weekend and random topics without the immediate distraction of experiments in the background. The dynamics of the lab are great. Since it’s a relatively new lab, there are only 5 people in it: my PI, the lab manager, a postdoc, another summer undergraduate research assistant, and me. This has allowed us to get to know each other better due to such close contact. Some days we’ll take a break and go out for coffee and watch the world cup game that’s on. In the Silva lab, it’s not just about developing the ability to work with one another, but also developing relationships between lab members.

After lunch, we pick up where we left off on our experiments. I’m usually kept really busy while in the lab, running around mixing solutions and processing samples. More often than not, Latin music is being played in the lab while we work and sometimes big world cup games are streamed over the speaker. It’s a really good environment to work in and I’m really happy to be there.

Stage 4: A Quick Intermission

Although a life of research often leads to an extremely narrow field of expertise, reading and learning about new research and knowledge from other, sometimes completely unrelated, fields can be both exciting and intriguing. One such example is Ayana’s project on Cryptococcus neoformans, specifically on searching for links between the BZP4 gene and their virulence in humans.

C. neoformans is a very common yeast, often found clinging to the dirt and other plants and animals. While widespread, infections from the fungus is rare, as most people’s immune systems are capable of defending against the pathogen. However, occasionally, they opportunistically infect the lungs, which can then spread to the central nervous system where the fungi can cause meningitis or encephalitis- two very dangerous conditions. Therefore, Ayana’s lab is researching the pathology and potential factors that could help the development of some form of prevention or cure. Currently, her project revolves around the curious BZP4 gene within C. neoformans, as the gene has been previously known to fluctuate in gene expression levels in different conditions and upon knockout, the virulence of the fungi disappears.  Thus, she has decided to investigate its relationship to the virulence of the fungi by directly interfering with it function.

In Ayana’s first aim, she hopes to confirm the link between virulence and BZP4 in C. neoformans, by taking a BZP4-knockout strain and blasting it with the BZP4 gene to observe for a recovery in lethality. This is cool primarily because it involves the use of a gene gun, which is just a really awesome machine that exists apparently, transforming cells with DNA by blasting them with a gene-coated bullet- and it works! At some point in my life, I need to devise a transformation experiment that requires that machine just to see it in action. Furthermore, her work would further pinpoint a site of target for treatments of C. neoformans infections, which is a monumental success in the world of disease prevention and care. Additionally, it could demonstrate a stronger link between virulence and the BZP4 gene, as the gene’s interactions and expression post-transformation could be identical to pre-knockout levels which would indicate greater independence between the gene’s mere presence and virulence over the possibility of gene expression interactions and post-translational activity with nearby genes being the source of virulence.

In Ayana’s second aim, the goal is to determine if there a competitive advantage given by the BZP4 gene. To study this, she will be inoculating a BZP4+ strain of C. neoformans and a BZP4- strain in close proximity, and measuring their growth and interactions upon reaching one another. This will perhaps give some insight in the purpose of the BZP4 gene, as not much is known beyond the excitatory effects it has on melanin production, which at most can be suspected to boost tolerance to environmental oxidants such as UV radiation. This is also important because the results can be used to determine if virulent C. neoformans growth can be stymied by the introduction of nonvirulent BZP4- strains into important sites of infection. However, something I wondered in this component of the project but did not remember to ask was the activity of BZP4 in various environments. As antioxidants are so versatile and diverse, many function differently in different conditions, so I wonder if there are any environments where BZP4 activity is optimized, boosting the vitality of C. neoformans in that environment. Similarly, which environments is activity dampened in? This could perhaps explain what BZP4 is specialized for (or what the melanin is meant to do) and what pathway exactly that it manipulates in the body to cause so much damage. Studies like these fascinate me and remind me of just how essential research is to the field of medicine, even in straight biology settings. While not at all related to my research in Alzheimer’s disease, this project gave me some energy and exciting plans to bring to my work.

Weekly Updates

“A piece of my gel fell on the floor once and I didn’t notice, so renovations re-varnished the lab. Now it’s forever imprinted on the floor.”-Stuart Sundseth detailing one of many Western fails

“I accidentally threw my gel into the sink once.”-Stuart Sundseth

“I’ve torn a gel before”-Stuart Sundseth an hour before tearing another gel

“Have fun at the beach this weekend!”-Christine O’Connell
“Wow, you’re going to the beach again?”-Dang Nguyen
“Yeah, I left some potatoes in the cupboard”-Joan Wilson

“I feel…so dizzy…I think…I might fall over”-Hui Fang as she aggressively hoses the floor with liquid nitrogen

“And now I’ll just seal up this bad boy and throw him in the cold room for the-” *throws dead spider in box at Dang* “-night and let him blot since I can’t find the antibody.”-Stuart Sundseth
*graphic screaming*-Dang Nguyen