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The End of an Era

And just like that, B-SURF is over. These past six weeks I’ve learned a lot about myself and the things that interest me. I came into this program not knowing what I wanted to do in science, just that I wanted to be a scientist. After spending six weeks hearing about my colleagues’ struggles in the lab moving small amounts of colorless liquid from tube to tube, I’ve decided that wet-lab isn’t for me.
At the beginning of this program the general vibe I got from people was that they felt sorry that my lab work was all virtual. Now, after going into the lab twice to make nanoparticles, I can say that I prefer coding over staring at tubes any day. I also didn’t have to deal with the frustrations of cells/organisms dying on me the day before data was ready to be collected, or the monotony of pipetting samples for hours on end. Throughout this program I was constantly engaged with my work. It was up to me to design the software pipeline for my project. I was given a task by my mentor, and it was on me to implement the features he wanted. One of the challenges that I’ve struggled with most being a self-taught programmer was finding confidence in my coding abilities. I knew that I knew how to code and problem solve, but other than stock problems I had no means to apply my skills. This program was exactly the push that I needed to give me the confidence in myself that I can accomplish problems put in front of me. As the second half of summer, and a condensed semester of organic chemistry, looms ahead of me, I am excited to say that I will continue with my project in the Reker lab. I still need to implement a machine learning model that will hopefully accurately make predictions in nanoparticle formation for me. I’m excited to see what the future holds, and I’m thankful for this experience allowing me to narrow down my search for what I want to do.

Thanks and Gratitude

Reflecting on the past 8 weeks of BSURF, I feel extremely excited and confident about my choice of pursuing a career in research.

Working in Wray lab this summer has completely shifted my perspective on what it’s like to be a researcher in a lab:

  • The lab environment isn’t only cutthroat and isolating, but a community of researchers who share knowledge, support each other, and build off of each other’s ideas.
  • Novel research and discoveries do not take weeks, more like years or decades.
  • Not everyone is 100% sure on what they are doing; you will fail more than you succeed when it comes to research.

 

This summer, the learning curve was steep; every day I stepped out of the lab, I had learned more than my brain could ever retain, and I left with even more questions.  One of my goals was to always strive to take on challenges and learn new things outside of my comfort zone. My mentors taught me about dozens of new molecules, reagents, chemical reactions, and protocols. Some of my favorites were RNA extraction, qPCR, stem cell culturing, antibody staining, and fluorescent microscopy. Every day, I became more confident walking into the lab, setting up my experiments, working through the calculations, and trusting myself to carry out the procedure well. I started to ask more questions, try out new things that may not work 100%, and weigh in on decisions that influenced my projects’ trajectory. I greatly enjoyed the environment that my PI instilled in the lab and am grateful for his involvement and guidance throughout my project.

 

Outside of the lab, I learned a tremendous amount from the guest speakers we heard from every week. Their success, resilience, and love for what they do inspired me greatly. It gave me insight into what a career in science research would actually look like, which was a lot different from my previous assumptions.

 

Next semester, I intend to build upon my research in Wray lab through an independent study as I continue to solidify what my specific interests and passions are. Thank you BSURF and Wray Lab for being my introduction into the wonderful world of scientific research.

Interviewing Jonathan Behrens, PhD candidate

Jonathan Behrens, or Jonny, is my graduate student mentor at the Bernhardt Lab. This summer, he’s taught me valuable field work methods and skills. I was excited to learn that he spent 3.5 years as a community organizer when he was an undergraduate. As a fellow organizer, I was thrilled to find somebody else who’s had experience in organizing and wants to use that knowledge to help make science accessible and people-serving. He says he enjoyed the critical thinking skills that organizing requires. Additionally, it helped him build connections with the community and understand his own passions.

Jonny majored in chemistry and minored in environmental studies at UChicago. He worked at a laboratory during his time as an undergraduate, but he didn’t think about pursuing a PhD after graduating. Instead, he worked for a science policy thinktank for the federal government. As Jonny worked, he realized that systemic issues couldn’t be fixed overnight. He wanted to combine his interests in chemistry, environmental science, and community organizing to learn how contaminants get into waterways and find methods to meaningfully address these issues.

Jonny searched for researchers that were answering the questions he was interested in. That is how he met Emily Bernhardt, the lab’s PI. They hit it off well and Jonny became a new member of the Bernhardt lab, where we are now working together! In the future, Jonny says he wouldn’t mind being a professor, but he would prefer being a scientist working for the federal government to help inform policy.

Overall, Jonny is a grounded, results-driven scientist who wants to use his research to address systemic issues such as climate change and environmental degradation.

 

 

Impact of Low PD-1 Expression on Chronic Pain-Induced Depression and Anxiety

Programmed Cell Death Protein 1 (PD-1) is primarily recognized for its role in immunomodulation, where it functions as an inhibitory regulator of the immune response. However, recent research has started to examine PD-1’s involvement in neuromodulation. This project explores PD-1’s role within the context of chronic pain-induced anxiety and depression. The chronic pain model was established using a Spared Nerve Injury (SNI) on PD-1 KO and Wild Type (WT) mice, and verified using pain quantification tests. The resulting anxiety and depression were measured using several behavioral tests. The behavioral tests measuring anxiety did not find significant differences between the KO and WT mice at two weeks, which was expected as anxiety-like behaviors often take 6-8 weeks to appear in chronic pain models. We plan on addressing this limitation by performing a long-term version of this study. We did find notable differences in depressive behaviors between the two groups, in which the KO mice displayed lower levels of depression. This suggests that anti-PD-1 treatments may have a protective effect against depression. The preliminary results from this project will provide the basis for a continuation of research, leading to a greater understanding of PD-1’s role in pain-induced anxiety and depression.

Constructing Electronic Devices for Recording Cell-Specific Neural Signaling

Jenna White

Mentors: Isaac Weaver, Sasha Burwell, Michael Tadross, PhD.

Department of Biomedical Engineering

Neurological diseases such as Parkinson’s have been found to alter electrical and chemical signaling in the brain, but it is unknown how these diseases affect neural signaling due to insufficient technologies for neural recording. It is hypothesized that if the fabrication of devices featuring transparent electrodes with appropriate dimensions is feasible, these devices would allow for recordings of individual neurons. This project involves the creation of devices using glass wafers layered with a conductor, utilizing the method of photolithography (exposing the wafers to UV light) for patterning electrodes on the conductor and patterning a layer of insulator added on top of the conductor. These wafers were then diced into separate devices, and wells for holding neurons were added onto each device. Despite errors in constructing these devices, over half of the devices created yielded well-aligned electrodes with a diameter similar to that of a neuronal soma. This suggests that the dimensions of each electrode allow for the isolation of signals from a single cultured neuron in vitro. The fabrication of these devices has shown feasibility, and with future improvements such as increased mechanical stability, these devices show potential for cell-specific neural recording.

Analysis of Hydrogen Bond Formation in Molecular Dynamics Simulations Predicts Formation of Self-Aggregating Nanoparticles

Joe Laforet

Mentors: Zilu Zhang, Dr. Daniel Reker PhD

Department of Biomedical Engineering, Duke University

Co-aggregating nanoparticles can stabilize drugs with more than 90% drug loading capacity. While machine learning can be productively employed to identify nanoparticles, this approach requires large datasets. Simulations provide an opportunity to design nanoparticles without prior data generation, but this method has not yet shown sufficient accuracy. Here, I will develop a novel simulation-based approach that achieves productive accuracy of nanoparticle predictions. By pairing a predictive machine learning model and molecular dynamics simulation software, we analyzed hydrogen bond formation in simulations and used our findings to identify pairs of interest. We compared our predictions against already known data and found that the presence of hydrogen bonding indicates higher likeliness of nanoparticle formation in more than 75% of analyzed pairs. Using this analysis protocol, we plan to analyze and predict other small antiviral nanoparticle formulations aimed at targeting viral diseases such as COVID-19.

Introgression between I.cordatotriloba and I.lacunosa

Lola Adewale

Mentors: Jonathan Colen, Mark Rausher, PhD

Department of Biology 

Recent research has shown that introgression between species through hybridization is common. Despite this, some traits are seen to resist gene flow between species in sympatric environments. One organism that this is seen in is the morning glory. When I.lacunosa and I.cordatotriloba are present in the same area, lac populations are seen to stay practically the same while cord populations are seen to change. Furthermore, we commonly see limb color resist introgression but a loss of throat color when these species are in sympatry. This renders I.lac and I.cord good model organisms to study species boundaries and gene flow. This project is asking two questions with these species. One being how often does pink I.cord sire offspring, the other being what is the recombination rate between the limb color and throat color gene. While research is still ongoing, preliminary data has shown that these two genes have a low but noticeable recombination rate.

Does reduced microglial MyD88 signaling increase voluntary ethanol consumption and anxiety-like responses in a mouse model of binge drinking?

Irene Jonathan

Mentors: Julia Dziabis, Staci Bilbo, Ph.D.

Department of Psychology and Neuroscience

Microglia are immune cells of the brain and can be activated through toll-like receptors. When alcohol is consumed in excess, microglia produce inflammatory mediators. Zeroing in on the toll-like receptor adaptor molecule MyD88, our lab’s preliminary studies suggest that reduced microglial MyD88 signaling (dampening of inflammation) increases voluntary ethanol consumption. We hypothesized that the mice with altered microglial MyD88, Cre+, would drink more than the controls (Cre-) over a 6 week period. The Cre+ mice would be more anxious as well as having less cognitive flexibility compared to all other groups. To simulate chronic alcohol consumption, we utilized a drinking in the dark paradigm, where Cre+ and Cre- mice were exposed to alcohol 4 days a week for 6 weeks, and the amount consumed was tracked daily. Afterward, behavioral tests, such as Elevated Plus Maze, Light-Dark Box, and Barnes Maze were conducted when the animals were going through withdrawal. Our findings suggest that inhibiting the microglial MyD88-dependent pathway does not increase drinking in the Cre+ group compared to the controls, but overall females consumed more ethanol than males. Further exploration of the mechanisms underlying microglial inflammatory signaling and their relationship to excess alcohol consumption is an area of interest for future projects.

Allelic imbalance in chimpanzee and human neural progenitors.

Skylar Montague

Mentor: Micah Daily, Greg Wray, Ph.D

Biology Department, Duke University, Durham, North Carolina

Humans and Chimpanzees share 98% of their DNA, yet these minor differences cause vast phenotypic changes, such as humans having billions of more neurons in their brain. The specific differences in neural progenitor (NP) gene expression that pioneers these changes remains unknown. We tested for allelic imbalance in various genes expressed in chimpanzee and human neural progenitor cells, such as the CCNG1 gene and Sox1 gene. Human-Chimpanzee hybrid induced pluripotent stem cells were differentiated into neural progenitor cells to test for allele specific expression. Unique primers specific to human or chimpanzee genes were developed and qPCR was conducted to test for differences in allele expression of genes in humans and chimpanzee neural progenitors. Eventually, we would expect to uncover allelic imbalances in genes that regulate the cell cycle in neural progenitors and the transition of NP into neurons, astrocytes, or oligodendrocytes. These imbalances would give insight into some of the genes that drive the neuronal differences between humans and chimpanzees. These genes can be further investigated to uncover the specific mutations in the coding or non-coding regions that are responsible for the allelic imbalances that have evolved between chimpanzees and humans.

Where is cell division located in the wing imaginal discs of caterpillars?

Misaki Foster

Mentor: H. Frederik Nijhout, Ph.D.

Biology Department, Duke University, Durham, North Carolina

Developmental biologists are looking to answer the question of how tissues grow to their species-specific size, and one way to begin to answer that question is by analyzing how patterns of cell division change over place and time. In this study I analyzed the patterns of mitosis and DNA synthesis in developing insect wings, the wing imaginal disks. The wing imaginal discs of caterpillars during their final larval instar are dissected and removed before staining with Hoechst and EdU so as to be able view and analyze mitosis and DNA synthesis, respectively, using a fluorescence microscope. Nuclei undergoing cell division are manually digitized and run through MATLAB programs to create a contour map that conveys where mitosis occurs the most. Overlay images are created to capture and highlight areas where DNA is being synthesized by the cells within the imaginal disc. Each method is repeated throughout different growth stages to determine how the patterns of cell division change as the wings grow and change shape. It is hypothesized that, in any given stage, mitosis will mostly occur in the areas where DNA synthesis does not occur, and vice versa. The results of this study show that cell division is not homogeneously spread across the growing wing, but occurs in ever-changing discrete regions.

Urban Stream Ecology: Watershed development negatively impacts aquatic insect diversity and ecosystem health in urban rivers

Author: Xitlali Ramirez

Mentors: Jonathan Behrens, Emily Bernhardt, PhD

Department of Biology

Impervious surfaces in urban development dramatically increase river discharge and contaminant presence in urban rivers, thereby creating a turbulent environment for aquatic insects and the river ecosystem. Durham’s Ellerbe Creek (EC) and New Hope Creek (NHC) watersheds vary significantly in urbanization, with EC being % developed and NHC being 7.9% developed, however the effects of this urbanization on aquatic insects and river ecosystem health have yet to be measured in Durham. I hypothesize that overall aquatic insect biodiversity and the abundance of pollution-sensitive orders will be lower at EC than at NHC.  I calculate the biodiversity and abundance of aquatic insects at two EC sites and one NHC site using sticky traps and make a qualitative water quality assessment. I compare that data to precipitation and discharge data collected by the USGS and our sensors.  EC’s Glenn Stone site has the highest aquatic insect abundance among the three sites. However, NHC’s site shows more diversity and more resilient species among the sites, suggesting that pollution is highly likely at EC and highly unlikely at NHC. Further quantitative assessments of water quality and river ecosystem health at these sites are needed to inform urban river conservation in Durham.

A Novel Suite of Biosensors to Investigate Vinculin Coordination of Collective Cell Migration

Benjamin Johns

Mentors: T. Curtis Shoyer, Brenton Hoffman, Ph.D.

Department of Biomedical Engineering

Collective cell migration (CCM) features a group or chain of linked cells moving together with the same speed in the same direction. This multicellular process is critical in physiological events such as wound healing and morphogenesis. The coordination of forces crucial to CCM is achieved by connecting the actin cytoskeletons of adjacent cells together at dynamic cadherin cell-cell contacts. However, how this mechanical linking regulates CCM at the molecular level remains poorly understood. We hypothesize that the actin-binding protein vinculin mediates these connections to control the speed and coordination of CCM. To test this, we are fusing vinculin to the fluorescent protein mScarletI and creating versions with point mutations that affect specific vinculin protein-protein interactions. With this suite of biosensors, we can visualize vinculin localization and dynamics through fluorescence microscopy and examine vinculin’s effects on CCM speed and coordination through a migration assay. Specifically, we expect that the actin-binding mutant vinculin will alter CCM speed and coordination. Identifying and characterizing these key molecular players of CCM would both greatly advance our understanding of this biological process and possibly provide future targets for therapeutic and tissue engineering purposes.

Carnivorous Plants in Research

On the final day for ChalkTalks, most of the presenters- including myself- focused on ecology in our research. Personally, out of all of the amazing presentations that occurred over the three days, the one that stuck out to me the most was Ali’s talk about her pitcher plants.

She drew three different species of the carnivorous plants on the white board, one was small and wide without a lid to cover the top opening, another was taller and narrower with a lid to cover the opening, and a mix between the two with a medium size and a lid that partially covered the top opening of the pitcher plant. What her lab noticed was that there is a larger and more diverse bacterial population in the short pitcher plant without the lid, and the rainwater that is collected allows the diverse bacteria to thrive. The bacteria living within the plant also breaks down any insect that falls in the pitcher, thus maintaining a commensalistic relationship. In the taller species with the lid, less rainwater enters the pitcher, so less bacteria can thrive in the environment. However, because of this, more of the energy from the insects that become victim to the tall pitcher plant can be obtained instead of it having to share with the bacteria colonies. It is thought that the hybrid species that shares traits with the tall and the short plants houses an environment somewhere in between.

I am eager to hear more about Ali’s findings with her project and to learn more about these intriguing carnivorous plants.

How I’m Probably Slowly Ruining My Vision

At the Nijhout lab, I work with imaginal discs, which are about half a millimeter in size on average. From the time that I get there around 10-11am to the time that I leave the lab around 6pm, I’m either looking under the microscope or staring at a computer.

My typical lab days have become steady enough that I’ve figured out how to stick to a comfortable schedule. Because my imaginal disc batches have to sit overnight, the first thing I do in the late morning is rinse them out and continue adding and washing out different chemicals while working under the microscope. After the procedure is complete and some time has passed, I set the batch onto microscope slides and add coverslips. I then move to a different room that houses a larger microscope with higher magnifications so I can analyze either mitosis or DNA synthesis in the stained discs on the computer. If I feel that I have enough time before 2pm rolls around, I’ll select more caterpillars for dissections, stain them, and let them incubate for 2 hours while I go off to lunch and/or discuss with my PI what my next steps for the project should be. When I come back, I “fix” the discs with formaldehyde (still working under the microscope) and put them in the refrigerator overnight so that I can repeat the process the next day.

It might seem monotonous or boring to some people, but it makes my day to see the stained imaginal discs under the high powered microscope and learn more about my research. I am glad to come into work everyday to continue learning something new.

Habitual and Goal-Directed Behavior

I really enjoyed listening through everyone’s chalk talks last week. As more people kept presenting, I felt like I was able to tap into so many different subfields and essential questions in biological research. Biology extends from observing the neural behavior in our brains, to the engineering of medical technology, to even the measuring of insect populations in streams. I could go on, but we were all there those three days.

Tonight I want to focus on Min Ju Lee’s research on habitual and goal-directed behavior observed through the change of brain circuits. She works with mice to measure these behaviors by having them in a cage and providing a stimuli to respond to. She explained that the stimulus, in this case, was a lever that would give the mice food when used. Overtime, the mice developed goal and habitual driven behavior. Goal driven behavior would look like the mice using the lever in order to get the food. There’s a purpose behind the action. Habitual driven behavior would be the mice using the lever just for the sake of it. There wouldn’t be a purpose behind the action.

Min Ju looks at the brain to identify these behaviors in the mice- the stratum, in particular. This is because the stratum can initiate or inhibit movement. The DMS tends to activate when the mice’s behavior is goal driven and the DLS activated when the mice’s behavior is habitual driven. Zach asked a really good question in the end that I felt like allowed Min Ju to elaborate more on the mice behavior. He asked how it is for certain than there won’t be any novel behavior? To this she answered that there’s a timeline for behavior and they work on the mice when they’ve passed that point of novel behavior. I thought that was really interesting because I didn’t think their minds would work as a step 1, step 2 kind of deal, but I guess they are just mice. Much of Min Ju’s work would help there be better understanding of where OCD and more comes from.

A New Approach to Gene Therapy

When listening to the Chalk-Talks this week, there was one that I remember immediately seeing the potential real-world/medical applications to and that was Nico Rey’s talk. Nico’s project provides a potential new mechanism through which gene therapy can be carried out. As spoken about in his chalk talk, the older model through which this was done was to introduce a subject to a viral vector that carries a functional form of the targeted gene so that cellular function  can be restored through the production of a functional form of the otherwise dysfunctional protein.

Rather than maintaining the use of a viral vector to introduce entire new genes, Nico’s project utilizes the splicing machinery native to the cells to replace the mutated/dysfunctional exons of the pre-mRNA from the target gene with a functional form of the exons thus rendering the protein and cellular function normal again.

The Importance of Water

I found Lali’s talk on her summer project very interesting, not only for the content, but also for the practical applications of her work. Water is an essential aspect of life, for both humans and other animals, and the quality of it has huge implications for everyone. I haven’t studied ecology much at all, but Lali broke her project down into something that was easy to understand by clearly outlining each component of it. While every project presented and discussed was important and had fairly clear benefits, I felt like Lali’s was one of the few that were centered on the Durham-Raleigh area, which made it really interesting to learn about. Her work incorporated ecology, environmental conservation, and social issues into a cohesive whole as it explored the water quality of two streams, one of which leads directly into a drinking water reservoir.

I also really liked that Lali’s project included fieldwork, and I enjoyed its innovative method of using semi-aquatic insects to indirectly measure the impact of urbanization and development on water and soil quality. Overall, I’m very interested in the long-term results of this project, and how its findings will be utilized in further work, whether that work is environmental research or reform. It certainly addresses a very important question and potential local issue.

Microbiomes in pitcher plants!

This week, I enjoyed listening to everyone as they presented and communicated their projects. One chalk talk that caught my interest was delivered by Ali and is titled “life in a pitcher.” This talk was about carnivorous pitcher plants and their relationship with the microorganisms that live in the water that accumulates in the plants. These organisms often take a bite out of the animals that land in the pitcher plant, but to my understanding they leave some leftovers for the plant. The plant also secretes digestive enzymes to help break down the animals. I was specifically intrigued by one of Ali’s research questions. I believe it was along the lines of “how does a pitcher plant’s latitudinal placement affect its enzyme secretion? The hypothesis is that the higher the latitude, the more energy the plants would have to conserve to stay alive. This makes sense because there is less sunlight available in higher latitudes compared to lower or mid-latitudes. Therefore, pitcher plants may decrease or completely stop secreting digestive enzymes to conserve energy. This would increase their dependency on microorganisms to break food down, even if those organisms take some food for themselves. 

 

I personally find this intriguing because the study of microbiomes and their relationships with the host is an exciting, emerging field. I think that it may be true that higher latitude pitcher plants may secrete less enzymes to conserve energy, but I wonder if having microorganisms that take some of their food is actually beneficial for them or if it’s a nuisance. Perhaps they prefer to conserve energy at higher latitudes by some other means and instead secrete more digestive enzymes. Perhaps the presence of these microorganisms is negligible for pitcher plants. Whatever the case is, I think it’s a very interesting topic to look into as it increases our understanding of microbiome-host relations.