Author Archives: Ashley Alcantar Magana

In my first blog post (written what seems like both eons and mere days ago), I emphasized the role that uncertainty plays in becoming a more active learner and better scientist. Instead of shying away from uncertainty, I expected to become comfortable with it, to the point that when I left BSURF, I would do so with a greater appreciation for challenges and my own capabilities.

Indeed, over the course of the program, I found myself in situations that ultimately strengthened my resolve about my academic and professional goals. Through the faculty talks, the chalk talks, the grad/MSTP student panel, and the poster sessions, I was able to hear about only a fraction of the science going on around me here at Duke. While the thought is a little overwhelming when it comes to the breadth of opportunities for engaging said science, it’s also heartening. I have been able to narrow down what concepts I want to continue to investigate, the types of questions I want to ask, and the community I want to be surrounded by. Thanks to my time in my lab, I have learned some of the technical skills that will enable my scientific pursuits and furthered my comprehension of cell biology. As such, I greatly appreciate my time in BSURF and hope to continue this growth throughout the rest of my time at Duke.     

Although I enjoyed all of the faculty talks, Dr. West’s in particular resonated with me. Before arriving at Duke, I didn’t know what an MD-Phd dual degree was. Even after learning a bit about it last semester, I was still unsure about whether it is something I could see myself doing, especially when going into medicine has been my plan for some time. As such, Dr. West’s insight has answered some of the questions I had about this path and whether it’s suited for me. I appreciated that she delineated the intricacies of medical, graduate, and MSTP applications, as well as some of the characteristics that might make someone a good fit for each type of program. Her comments about the Duke MSTP and what it entails are what emphasized for me that this pursuit, while very admirable, is not what I envision for myself at the moment. I would like to learn and contribute to the research advancing scientific and medical knowledge, but my primary desire is to be able to serve and care for patients. After Dr. West’s talk, I feel more reassured about my next steps and the research I will participate in throughout the rest of my education. Going along with this idea of uncertainty, I also appreciated Dr. West’s explanation of her own introduction to research and determining what she wanted to specialize in during her undergrad and graduate career. Her research about neuronal regulation of synaptic maturation during development and its implications sounded fascinating to me, and overall her talk gave me a lot to think about.

Mentors: Daichi Shonai, Scott Soderling, PhD, Cell Biology

Interrogation of endogenous proteins is pivotal to the study of cell types and disease etiology. CRISPR-Cas9-based approaches allow the insertion of DNA sequences into genes of interest (GOI), providing an opportunity to label endogenous proteins and thus advance proteomics. One such CRISPR-based method is homology-independent universal genome engineering (HiUGE), which utilizes adeno-associated virus (AAV) vectors to deliver both the payload lacking gene-specific sequences and the gene-specific gRNA that integrates the payload into the GOI. While universally compatible with genome targets accessible by CRISPR-Cas9, the question remains of whether a double- versus single-oriented payload or HiUGE donor might affect the target efficiency of this strategy. As such, we transfected NIH3T3-Cas9 cells with different HiUGE donors, conducted immunofluorescence (IF) staining to enable epitope tagging, and quantified tag expression to test the hypothesis that double-oriented HiUGE donors would demonstrate greater or even double the efficiency of their single-oriented counterparts. As we continue comparison between single-oriented HA and double-oriented HA-HA, KI D620N/WT VPS35 and KI WT/WT VPS35, and UltraID and Dual-UltraID, preliminary results show that although the difference across constructs varies, the dual-oriented donors not only successfully integrated despite larger size, but they also correspond to increased expression of tagged endogenous proteins.

Last week’s chalk talks presented a valuable opportunity for all 0f us to learn about the science we are contributing to as individuals and as a group. One such talk was Vidita’s discussion on the potential role of POSTN in ovarian cancer cells’ sensitivity to chemotherapy. She began her talk with an explanation of the stages to ovarian cancer diagnosis and treatment, including the diagnosis of the tumor, treatment using platinum-based chemotherapy, and remission. For the purposes of her and her lab’s research, this also included recurrence of the tumor, tumor resistance to the platinum-based chemotherapy, and progression of the cancer. In order to gain a better understanding of the factors involved in this tumor recurrence and resistance to chemotherapy, her research focuses on the tumor microenvironment – specifically, how the presence of POSTN in the tumor microenvironment affects the chemosensitivity of ovarian tumors. This microenvironment consists of non-tumor cells, extracellular matrix, and blood vessels. In humans, the POSTN gene encodes the periostin protein, which functions as a ligand to support the adhesion and migration of epithelial cells (or cells that line major cavities of the body). It has been reported that large amounts of periostin are found in the microenvironment of recurring tumors, which lends support to the hypothesis that this protein does indeed contribute to chemotherapy resistance in cancerous cells by supporting the progression of the cancer.

I appreciated that Vidita began with a brief description of the motives underlying  her lab’s research, namely the interrogation of the role of the tumor microenvironment in ovarian tumor recurrence. She also did a good job of incorporating and explaining terminology that would perhaps be unfamiliar to a general audience. It would be interesting to see what the next step would be for these findings, particular in terms of treatments and application to other cancers.

Every morning I arrive at the Soderling Lab with an idea of what my goals and steps are for the day ahead. At some point within the past week, my bench mentor and I would have discussed the experiments, protocols, and concepts I would be working with, as well as any questions I might have had for him. Still, if I am unsure about where to start, I only have to ask and thus be on my way.

Some days, this means starting off in the cell culture room, beginning a days-long protocol for transfecting some NIH3T3/Cas9 cells underneath the HEK cell hood. Or, I might be observing my handiwork from days prior with the microscope, ensuring the cells look healthy. At my station, I may be gathering reagents to run a PCR, or gathering a marker, my epitubes of purified DNA, a 10uL pipette, and some TE Buffer to measure DNA concentration on the 3rd floor of the Nanaline Duke building. I might find myself in the laser room to observe the results of my immunofluorescent staining from a day or so ago, imaging my samples and preparing to quantify the cells I can see. This leads to me practicing my Excel skills in order to evaluate the consequent data, creating graphs and running statistical tests. There are also the occasions on which I visit the “mouse house”, or GSRBII, where I suit up in PPE and get to see in vitro experiments become in vivo ones, or where I attend in-house lectures and presentations by grad students and visiting scholars.

Some days, I am running one experiment after another only to realize that it already past 4pm. Others, I am performing a single task that requires my attention the entirety of the day. Regardless, I have learned something new and check in with my mentor about what’s next before heading out for the day.

The Chair of Cell Biology at Duke University Medical Center, George Barth Geller Distinguished Professor for Research in Molecular Biology, Professor of Cell Biology and Neurobiology, and Director of the Transgenic Mouse Facility, Dr. Scott Soderling began his research journey while completing his undergraduate degree at Pacific Lutheran University. As a school with strong biology and business departments, and a close proximity to Mount Rainier conducive to pursuing mountain climbing, it wasn’t until a particular genetics class that Dr. Soderling knew which of the two majors to declare. Indeed, through an introduction to gene mapping using drosophila with a gene mutation, Dr. Soderling became fascinated with discovery – being the first to see something that has existed for millions of years. Ergo, he majored in biology with a concentration in biochemistry, going on to pursue a PhD in the department of Pharmacology at the University of Washington in a lab that studied cell communication, particularly signal transduction through the identification of enzymes impacting this process. Through this intersection of molecular biology and bioinformatics, Dr. Soderling concentrated on the role of cAMP and cGMP in the activation of kinase-mediated phosphorylation and thus cell signaling. During his postdoc at the Vollum Institute, where Dr. Soderling wanted to understand the impact of cell spatial organization and communication on physiology, he identified the WRP protein as being imperative for the WAVE signaling network, the latter of which directs actin reorganization in neurons (Soderling et al., 2002). Additionally, Dr. Soderling saw a clear link between what he was studying and human health when, in the case of a patient with an intellectual disability whose X chromosome and chromosome 3 had been translocated, there was a link between this phenomenon and a mutation in the gene for WRP. 

Today, Dr. Soderling and his lab focus on the neurobiology of neurodegenerative, developmental, and psychiatric disorders through proteomics, mouse models, and genetics. Although the job requires hard work to ensure success and presents challenges when grants or papers are rejected, Dr. Soderling believes the genuine enjoyment he derives from his career surpasses any such tribulations. In fact, this, combined with the freedom to be creative with his academic pursuits, being able to work with smart people from all over the world, opportunities for travel, and watching students and postdocs grow in the lab, is what makes him feel fortunate about the path he’s chosen. For Dr. Soderling, some of the best advice he’s ever received came from his father, who suggested that he choose between academia and industry according to whichever path offered the most options afterwards. Now, his own advice for students considering a career in science or research is to choose that which inspires the most passion and love, for if so, hard work won’t feel like work at all. 

Lastly, when asked if he wanted to share any embarrassing or funny moments he experienced in the lab, Dr. Soderling mentioned a practical joke his fellow lab members played on him while he was a postdoc. As he was reviewing data he had just gotten back for his paper on WAVE1 and the novel WRP protein, he received an email from an alleged professor at another research institute whose work mirrored his own. The implications of such an email were enough to make the blood drain from Dr. Soderling’s face. His lab mates let him sweat for a moment, before bursting out in laughter and confessing that it had been a ruse. Although the moment inspired a mixture of stress and embarrassment, Dr. Soderling laughs while telling the story.

A sincere thank you to Dr. Soderling for his time and the opportunity to learn about his own experiences with science and research.

One of the focuses of the Soderling Lab is the mechanisms underlying disrupted synaptic connections, abnormalities that play a role in the onset of neurodegenerative and psychiatric disorders. To accomplish this, the lab has developed a combination of proteomic and CRISPR approaches, such as the CRISPR-Cas9-based Homology-independent Universal Genome Engineering (HiUGE) method. HiUGE allows efficient and convenient alteration of endogenous proteins through adeno-associated virus (AAV) vectors of insertional DNA sequences that can merge into the part of the genome specified by guide-RNA vectors (Gao et al., 2019). As demonstrated in the following Graphical Abstract from Gao et al., HiUGE provides a simplified way to modify proteins in vivo and in vitro for the study of gene and proteins functions, thus making it easier to investigate the proteins and synaptic disruptions involved in diseases such as Parkinson’s (2019).

Parkinson’s disease (PD) is a progressive, age-associated neurodegenerative movement disorder often characterized by a resting tremor, slow movement, and difficulty maintaining posture due to the loss of dopamine-producing neurons in the midbrain. The etiology of the disease continues to be studied, with advances in the identification of gene mutations specific to familial PD encouraging further inquiry into the mechanisms by which these mutations induce it. One such PD-linked gene is the vacuolar protein sorting 35 ortholog (VPS35), with the VPS35 D620N mutation having been identified as pathogenic (Williams et al., 2017).

Together, HiUGE and PD are the foundation for my research project this summer. I will be investigating the factors that may improve the efficacy of the former while simultaneously employing HiUGE to observe the latter in vitro (and possibly in vivo). Currently, I have three research questions that include the following:

1. Are dual-oriented HiUGE-donors more efficient than single-oriented ones for gene expression?
2. What is the effect of knock in WT/DG20N VPS35 – a dual-oriented HiUGE-donor – in vitro (and potentially in vivo)?
3. Does UltraID produce biotinylation of alpha synuclein comparable to TurboID?

The first question relates to whether a DNA vector or construct created through the HiUGE method might produce double the expression of a specific gene if its epitope is dual-oriented versus single-oriented. The second question involves a dual-oriented HiUGE donor with healthy and PD-linked DNA fragments being introduced in vitro to a cell culture and potentially in vivo with mouse models. This second question also involves partially testing the efficacy of the dual-oriented HiUGE donor. Lastly, the third question has ramifications as to whether labeling proteins such as alpha synuclein and others potentially linked to PD in the synapses can be equally if not more effective through the use of the enzyme UltraID, a smaller molecule as opposed to the currently used TurboID.

I look forward to continuing to learn as I seek the answers to these and other questions in the Soderling Lab this summer.

What type of experiments will you be conducting? What organisms will you be working with? What will your research be contributing to? 

These were the sorts of questions I received from friends and family in the weeks leading up to the start of B-SURF, and my answers always varied. At the time, it was because I was still a bit unsure myself about what to expect. I could give an overview about the scope of the research being conducted at the Soderling Lab – the focus on neuronal circuits and the neurological disorders driven by genetic mutations affecting protein function – and the techniques implemented there – mouse models and CRISPR-based methods – but in terms of what my daily routine would be, that was wherein the uncertainty lay.

Now, one week into the program, my answers continue to change. And that’s okay. Every day in the lab has been a new experience; one day I will be transferring bacteria cell cultures to new Petri dishes and the next I will be running a polymerase chain reaction (PCR), allowing me to develop skills I can apply for years to come. I have had the opportunity to attend lectures given by visiting scholars and grad students within the Department of Cell Biology at the Medical School, broadening my horizons as to the field of neurobiology. I have made mistakes and asked questions, coming to terms with my gaps in knowledge and areas of improvement. Most importantly, I learn something new everyday, and regardless of how basic or complex the concept, it reminds me of why I decided to apply to B-SURF in the first place. During the next seven weeks, I expect to continue to ask questions and be exposed to new ideas, welcome uncertainty, and be prepared to leave the program with new answers.