Author Archives: Izzy Costanzo

Oh, the places I’ll go (next)!

With all the incredible experiences I’ve been able to have this summer, both inside the lab and out, it shouldn’t surprise me that everything came to an end so soon (time flies, and all that). It still feels unbelievable to me, though, just how much I was able to cram into my brain in such a brief yet rewarding amount of time. Today, I am able to run procedures and use machines I had never seen or heard of prior to BSURF without a second thought. (What I like to call my) scientist’s intuition for understanding and interpreting data has grown beyond my wildest dreams; I no longer rush to turn to Julia for help interpreting an electrophoresis gel image, I get to tell her what I think happened and we can discuss together next steps. My understanding of new procedures, (even the ever so complicated Western Blot) is high, and I can infer what each step is doing or make an educated guess that Julia can help confirm or adjust. My brain has never been this full of scientific aptitude, and my excitement to continue to apply what I’ve learned in lab settings has only grown.

My renewed excitement and passion for science is all thanks to a number of people who were able to guide me along the way throughout this program. Julia, my mentor, of course made every day easier than the last with her helpful explanations and direct answers to all of my questions. She is a fantastic graduate student to learn from, and inspires me every day to not be afraid of making mistakes, but to be excited to learn from them. Because what fun would research be if everything went perfectly, anyways? Dr. Tenor and Dr. Perfect are some of the kindest people I have ever met and their vast knowledge of the science world and community baffles me, but I aspire to know as much as they do someday. I am grateful to them for welcoming me into the lab and for showing me the way to a bright future in research. Of course, Emily, my lab partner in crime, was so much fun to spend time with this summer and I can’t wait to see her in lab again in the fall!

Lastly Dr. Grunwald and Dr. Harrell, many thanks to you for putting together such a wonderful summer research experience, especially for the sophomores who couldn’t do it last summer. My disappointment and anger at COVID-19 for ruining my summer last year was completely overshadowed but the scientific joy and gratitude I felt this summer, making up for lost time. Your commitment to promoting good science in undergraduates really shines through in all that you do, so thank you so much for the opportunity you’ve given me and the doors you have opened.

Though it’s sad to leave now that the program is over, I will hold onto all the knowledge, skills, and memories from these past weeks for a long time, many of which I’m sure I’ll carry with me after graduation, into my career, and beyond. So for the head start on a long journey through undergraduate, graduate school, job-hunting, etc., I am eternally grateful. See everyone in the fall!

Co-localization of Potential Nitrogen Catabolite Repression Transcription Factors in Cryptococcus Neoformans

Isabella Costanzo

Mentors: Julia Palmucci, Jennifer Tenor, Ph.D., John Perfect, M.D.

Department of Medicine, Division of Infectious Diseases

Cryptococcus neoformans, a ubiquitous infectious yeast, proliferates in cerebrospinal fluid (CSF) causing fungal meningitis in immunocompromised individuals. CSF is deficient in nitrogen, a necessary growth nutrient. To overcome this hostile growth environment and maximize fitness, many fungi employ nitrogen catabolite repression (NCR) in which genes required to break down unfavorable nitrogen sources are repressed when more favorable nitrogen sources are present. Other fungal genera like Saccharomyces and Aspergillus have well-studied NCR pathways, but NCR has not been thoroughly investigated in C. neoformans. Current research suggests that Tar1 and Ure3 in C. neoformans may be orthologous to NCR gene repressors in Aspergillus and Saccharomyces (NmrA and Ure2, respectively), and may regulate the GATA transcription factor Gat1—the master regulator of NCR. Tar1 and Ure3 proteins were fluorescently tagged with mCherry and transformed into both wild-type (H99) and Gat1::GFP backgrounds to determine the localization of these proteins during nitrogen replete and deplete conditions and co-localization with Gat1. Co-localization of Ure3 or Tar1 with Gat1 would indicate the C. neoformans NCR pathway is similar to Saccharomyces or Aspergillus, respectively. Negative results may indicate a unique regulatory system in C. neoformans. Further experimentation would examine additional NCR interactors and investigate its effect on C. neoformans on survival in CSF.

Skylar’s Stem Cells

This past week, I was completely in awe of all the amazing research being conducted through the BSURF program this summer. Everyone did an incredible job explaining their projects and practicing the communication part of science that is so important. While all the talks left me with some fascinating pieces of science to think about, one of the presentations that really stuck with me was Skylar’s chalk talk about iPSCs and HESCs.

My personal research interests intersect a lot with the ethical concerns in the scientific community, in the way scientific research is conducted, presented, and applied in practical settings. Hearing her talk about this different way of using induced pluripotent stem cells to avoid the ethical concerns of utilizing human embryonic stem cells I thought addressed a very interesting question that arises a lot in scientific, especially biology, research that I don’t think gets talked about enough.

The second part of her project was just as intriguing to think about: comparing neural progenitor cells in chimps and humans to see how regulation in the genes of interest in these cells differ. The whole time I was listening, I kept thinking about what a neat application of science to focus on. The hybrid model, to me, was especially interesting in the way that it helped to control for and eliminate potential confounding factors, like their (chimp and human) environment. I can’t wait to see what new things were discovered at Skylar’s poster presentation, and I am definitely looking forward to hearing more about her amazing stem cell research.

Practically Perfect in Every Way

My first stop on my way into the lab is my little lab bench/office space to drop off my backpack. Then, I always go to the shared office space (where there is usually fresh food someone brought) to check in with my bench mentor, Julia. She and I go over the plan for the day, and we both write it down on a sticky note to keep track of our “to-do list.” The actual details of the plan vary from day to day, but, most often, it includes me returning to my bench to first prepare some PCR samples and sticking them in the thermocycler. Then comes the waiting.

There tends to be a lot of inactive time in the lab when Julia and I discuss the project, next steps, what’s actually happening while we’re waiting, etc., but usually this time gives me the opportunity to update my lab notebook. I spend a lot of time organizing it, taping in important pictures of gels we’ve run, writing down data and results and next steps. As someone who likes to keep a physical planner and bullet journal on hand for organizational purposes, the physical lab notebook is the perfect way for me to stay organized, and I make sure that it is kept pristine and well-detailed.

About 30 minutes before the PCR is done, I head over to the main lab space to pour a gel to run with the PCR products so we can image the gel and see if the products are the right size. This doesn’t take too long, and usually by the time I’m done, the PCR is nearly ready. After the samples are ready, I take them over to the shared lab space to add the loading buffer and wait for the gel to finish setting up. Carefully, I load the samples to a finished gel sitting in a buffer solution, then I hit start and let the gel run for about a half hour. This waiting is more anxious, because we’re so close to seeing if our reactions worked. Then it’s time to image the gel, print out pictures, and Julia and I discuss what the next step is.

For the first few weeks, it was often more PCR and more gel-running, but now we are getting to an even more exciting part of our project where I’ll get to learn new procedures, and hopefully perform a biolistics transformation within the week. Even though I’ve grown a lot in terms of becoming adept at performing many of the day-to-day lab procedures on my own, every day is a new adventure in the lab, and each hour is an opportunity for me to learn something exciting.

The Best of Both Worlds

On February 14, 1978, a patient with underlying Hodgkin’s disease received a lumbar puncture to collect cerebrospinal fluid for further testing. The doctor working that night examined the fluid and identified the H99 strain of Cryptococcus neoformans for the very first time, which is still being used in various lab work (including my own) today. This physician who isolated this novel strain is none other than my PI, Dr. Perfect, and given his vivid recount of the details from that night, it appears as though for him the historic event feels like it occurred only yesterday, despite it being the start of a nearly 50 year career working with Crypto.

Dr. Perfect described a path into the science community that felt very relatable to my own experiences thus far; a career decision solidified in middle school, summer undergraduate research, inspirations from famous philanthropic scientists, etc. However Dr. Perfect’s overall philosophies were what struck me as both the most valuable and the most intriguing takeaways from the interview. His original idealism and goals of wanting to help or “save the world” grew into skepticism as he emerged into the clinical setting, where politics can make such a feat tricky for even the most optimistic of scientists. However, this has never hindered his commitment to good science. The toughest part about clinical research currently, he suggests, is the cost and ability to afford the necessary infrastructure for a successful system. Aside from the logistics of it all, Dr. Perfect calls attention to the ever growing issue of researcher and patient interface — the trust, or lack thereof — that gets snuffed out quickly in today’s society saturated with misinformation. The ethical concerns on the researchers’ end, combined with mistrust on the patients’ end work symbiotically to hinder the progress of science, but Dr. Perfect’s long and illustrious career indicates that important progress is not halted completely.

The field of infectious diseases is fascinating mainly because it “cuts across all the clinical areas,” connecting inherently to a wide variety of specialties. Dr. Perfect believes that this field will require more attention in the coming years, and the spotlight is well-deserved, as is evident based on the current onslaught of research brought on by the pandemic (Dr. Perfect notes that he has now been alive through two pandemics, HIV being the first). His particular work connects easily from the clinical side to the research lab, and with each side being so intensive, his career is akin to having 2 simultaneous full time jobs. While the lab research is more mechanistic, studying the “in the woods stuff,” its applications are just as exciting in the clinical perspective, offering a “30,000 foot view;” both sides equally “tricky but extraordinarily important.”

The vast depth of his experience cannot be overstated, which is why his parting advice rings so poignantly in the young ears of a prospective graduate student in the coming years. For potential medical school applicants, when faced with that one brutal yet telling question, “why medical school,” Dr. Perfect suggests that the only correct answer should be a desire to help people, and a want to improve their health. For students all across Duke’s campus, he warns not to squander the plethora of opportunities readily available, and to “optimize your time at a research university.” Lastly, to all young and aspiring scientists, he offers his own sentiments as proof that a career in science is a life worth pursuing. Whether you are discovering new strains of fungal pathogens in the Duke Hospital, or pipetting clear liquids into clear tubes for PCR reactions that may (or may not) work, “at times, it’s just excitement!”

The Path(ogen) Less Traveled

Before I was dropped off at college, I distinctly remember the ominous warning from my mother: be careful, don’t get meningitis! The disease is known for occurring in infants and college students, but I didn’t really know what it was…until this summer, now that I’m studying it. While there are several pathogens that are able to cause meningitis, Dr. Perfect’s lab studies Cryptococcus neoformans, or the fungal pathogen readily found in the environment causing cryptococcal meningitis. Though its environmental ubiquity seems alarming, Crypto. is actually an opportunistic infection, meaning it only causes disease when it is able to take advantage of weakened immune systems, such as those with HIV/AIDS, transplant recipients on immunosuppressants, or possibly even survivors of COVID-19. Even so, the prevalence of cryptococcal meningitis is significantly higher in poorer countries with less access to necessary treatments and supplies. Duke Hospital’s own survival rate for Crypto. infections is around 80%, while the global survival rate is only 50% (skewed largely by the populations lacking in adequate healthcare resources). Despite this frightening statistic, research on Crypto. is largely underfunded due to the fact that cryptococcal meningitis is a noncommunicable disease, or it cannot be spread person to person via a cough or a sneeze the way that the flu, COVID-19, or other common diseases can.

Luckily, Dr. Perfect’s lab is doing the hard work, spending every day working towards understanding Crypto. and trying to find new ways to target it through drugs and other methods to help prevent or cure future cryptococcal meningitis infections. This is where I come in (with my bench mentor/graduate student Julia, of course). One of the most interesting things about Crypto. is where it causes infection: the brain/spinal cord. A vast majority of other pathogens struggle significantly with the lack of nutrients and overall harsh environment of cerebral spinal fluid in the central nervous system, which is why Crypto. is such an intriguing fungus; where others fail at mere survival, Crypto. seems to thrive in this nutrient-deficient environment, hence its deadly survival rates. One project I am helping work with Julia to look at this summer is one particular set of genes that may be influencing this growth. These genes regulate nitrogen catabolism, or the identifying and processing of available nitrogen sources for use. Nitrogen catabolite repression (NCR) is the name for the regulation of these genes via certain regulators based on the nitrogen sources available (normal, easily metabolized sources results in a negative regulation — turning the genes off — and sources that require several steps to break down cause a positive regulation response — activating the genes encoding for certain necessary enzymes to break the nitrogenous compounds down –). Our first steps so far have been looking at two potential NCR genes, tagging them with a fluorescent tag (we are using mCherry, but for comparison it’s like GFP), and looking for any interactions between the proteins based on localization (if the proteins are co-localizing, we will see a certain fluorescent pattern showing them near each other). Knowing if these genes work together to regulate nitrogen catabolism is critical for moving forward in the overall investigation of identifying these regulation pathways in Crypto.

These last two weeks, we have been assembling all the necessary pieces to create the tagged strains of Crypto. I first had to design primers that would work in a PCR reaction to amplify each of the 5 segments needed for the tagged strain (the 5’ UTR, for attaching to a plasmid backbone, the NAT antibiotic resistance cassette, for incentive to integrate the plasmid into Crypto., the promoter + gene itself, the mCherry fluorescent tag, and the 3’ UTR region to attach to the other end of the plasmid backbone). The PCR results so far have been mostly successful, with two stubborn fragments still left to successfully amplify (let’s just say I’ve run a LOT of electrophoresis gels this week). Once we know that the PCR worked, we cut out the bands and extracted the DNA from them to be saved until all the pieces are ready. This week, we are finally going to do a Gibson assembly to put all the puzzle pieces together.

These two weeks so far have been a whirlwind of reading papers and learning techniques and working towards an exciting goal; I feel like a supersaturated sponge, but I can’t wait to keep learning more!

My first (of many) electrophoresis gels showing the successful PCR reactions for gene 1: URE3. Our 2nd fragment had the best band, meaning it worked the best (NAT cassette), and was extracted, but most of the other four fragments had to be run again in a new PCR reaction.

This was the gel electrophoresis showing the PCR reactions for gene 2: TAR1. The brighter bands were the successful amplifications, and the ones that were very faint or didn’t show up at all in the column had to be run again. Here, we were able to gel extract from 1 and 2, and some from 3 and 4 (This was a vast improvement from the first URE3 gel, which was one of the first PCRs and gels I ran in the lab).


Sherlock Holmes, Disease Detective

Even before the pandemic cast its looming shadow over society as we knew it, diseases had always piqued my interest; first in middle school, where I first formulated my intense fascination with pathogens and the spread of disease (solidifying my early career goals of becoming an epidemiologist), to intriguing high school classes about the biotechnology techniques that can be used to investigate such diseases, to my college general microbiology course, which allowed me to culture, transform, and experience first-hand these pathogens with my own two gloved hands.

While this curiosity that I harbored was always omnipresent in my future academic and career goals, I likewise always felt a barrier between me and the incredible scientists I read about, the heroes developing vaccines, or discovering novel strains of various pathogens. Even in class, or in my beginning work with Dr. Tenor at the Perfect lab, I felt somewhat removed and distant from the research itself, and not just thanks to the Zoom format of our meetings. My whole life I’ve dreamed of being a “disease detective,” of exploring the origin of deadly pathogens and stopping the spread before they cause an outbreak, or worse, but if my dream was to be a Sherlock Holmes in the scientific community, I was his bumbling, newly acquired assistant Watson, unsure and still figuring out my role in the sleuthing process.

Despite a hesitant start last semester, being physically in the lab has already made a world of a difference. Asking questions comes more easily, visualizing processes and protocols makes more sense in context, and I am spending less time playing catch-up, and more time being only half a step behind my wonderful mentor Julia, the Sherlock to my Watson. The first three days in the lab have already exceeded my most ambitious expectations; in terms of learning, I feel as though my brain is already overflowing with useful knowledge to be applied down the road, additional tools to go into my scientific detective tool belt for future use. My hopes for this summer is to become a more active participant in these disease detective endeavors — to come up with my own theories about why Cryptococcus neoformans survives so well in the brain environment while other pathogens tend to fail, to investigate more deeply into the role of nitrogen catabolism and hypothesize how that can be utilized in antifungal treatments of C. neoformans infections. While I have always considered Watson to be an adorable sidekick in Sir Arthur Conan Doyles’ fast-paced adventures, my highest hopes for this summer is to grow as a scientist in terms of critical thinking, asking good and interesting questions, formulating exciting hypotheses, and making useful mistakes, all in the name of facilitating good scientific research; I aspire to become my own Sherlock Holmes, disease detective.