My final blog post. Wow.
I went from having not ever used a pipette to using all sorts of lab equipment, talking about antifungal targeting and gene phenotypes, and conversing with post docs about Cryptococcus! I’ve been introduced to the science world this summer, and with so many resources and tools that have helped me along the way, I just feel extremely thankful to have been apart of such a generous program. I now feel like an insider.
I think back to the start of it when I was feeling really worried about my lack of experience and limited knowledge. It feels like so long ago. Working in the lab made me realize how much of science is a collaborative effort. There’s mentors, peers, and journals to turn to. And because there’s bound to be an area you’re not the most confident in, it’s expected you will receive help and sometimes even give help yourself. One thing I always hear is that everyone is still always learning, even having been in the field for 20+ years. It’s just made me have so much to look forward to in terms of what more I will be exposed to in the coming years and the people and perspectives I will be given the opportunity to see and hear.
This little, enriching taste of what it’s like to work in the lab has enabled me to confirm that this line of work- asking questions and discovering more about life on Earth little by little- is exactly what I want to do. Seriously identifying this passion has been another substantial piece to my enjoyment this summer. I can literally envision myself in a hospital lab working in micro and running tests on patients’ samples. So, again, I want to say that I really enjoyed myself this summer and am extremely thankful to Dr. Harrell and Dr. Grunwald for providing me with this opportunity. Certainty feels really good.
Jennifer Tenor, PhD, John Perfect, PhD
Emily Prudot Gonzalez
Department of Medicine
Cryptococcus, a fungal pathogen, can be inhaled through pigeon excretes and multiple trees, which leads to the life threatening disease, cryptococcal meningitis. The growing emergence of cryptococcal anti-fungal resistance has made it difficult for clinics to treat cryptococcal meningitis, especially with limited antifungals. In deleting genes and growing them in conditions with increasing concentrations of the anti-fungal, fluconazole, we were able to target the deletion strains with fluconazole sensitivity and fluconazole resistance. The deletion strains were then defined along with their cellular or functional processes through the gene ontology, FungiDB. This allowed us to observe a measurable amount of membrane-related deletion strains and hypothetical proteins in both fluconazole resistant and sensitive phenotypes. Mitochondria-related deletions strains also have a considerably high concentration in the fluconazole resistant phenotype. These findings allow us to investigate the possible defense mechanisms within the mitochondria when met with high concentrations of fluconazole as well as create a starting point in researching the role of hypothetical proteins in the cell. Further down the line, the hopes are to use these learned mechanisms to develop a drug target and treat cryptococcal meningitis.
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.
Ever since I saw this self-care YouTube video by some random woman saying it’s ~self-care~ to wake up early enough so you don’t have to rush to get ready + have some time to yourself, I’ve woken up at 7-7:30AM everyday- minus weekends. So, I start my day off at 7 and go into lab around 10-11AM depending on the day. We have several back-to-back imaging days, so I tend to stay by the BM3-BC Colony Processing Robot, or as I call it, the phenotyping robot, as it takes images of plates with growing crypto. in it. While the robot does its thing, I take note of the start and end times and identify deletion strains from previous sets of plates that have already been imaged. I identify these strains using Fungidb which is a nice deletion library that has an extensive description of each strain and contributing papers. Occasionally I’ll run into several hypothetical proteins which is exciting because it gives an opportunity for an additional project to dig deeper from what we’ve found to learn more about the strain. After imaging of a set of plates is complete, which can take 50 minutes to 2 hours depending on the size of the set, I mark for growth on plates with 32 or greater micrograms per milliliter of FCN. I don’t find too many, as 32 is already a generally high concentration. Sometimes I think about the gap between 32 μg/ml and 64 μg/ml and wonder if it’d be worth repeating this experiment, only zooming into those concentrations. We’ve only seen two strains that have been able to grow at 64 μg/ml, which although that’s a good thing, it’s still kind of threatening and scary to me as 64 μg/ml is such a large concentration. I’d definitely like to study more about those two strains as well and its genes to learn more about how it’s able to withhold such high concentrations of FCN. The data inserting process will take much of my time this week because I’m observing so many deletion strains (~4700) and I’m also learning how to use Excel, so that’s fun. Later this week I expect we will begin the process for confirming the FCN resistant strains.
Here’s a visual to understand what I mean a bit better:
This is a plate with a concentration of 64 μg/ml, and there is growth in position F1. I’ve identified this to be a deletion named arginine biosynthesis by the Fungidb deletion library.
On Thursday of last week I interviewed Dr. Perfect in his office. It was really nice to see him in person. Much of what I learned described his position and general motivation behind it. Dr. Perfect is both a physician and a principal investigator, so he spends a lot of his time balancing the two. He’d always wanted to become a physician because the clinical connections and seeing different kinds of patients allowed him to see the relevance in the research he’s doing. Conducting or being apart of research is known to have a point where the purpose becomes a bit fuzzy, so he feels that having both roles really aids him in keeping himself focused.
He also gave some really valuable advice. Making the best of my college experience here at Duke would mean optimizing every opportunity around me to learn more and gain more experiences. This means building relationships with professors, staying productive over the summer, and always asking questions. He also added that going to graduate school or medical school will potentially be apart of my journey, but it is important to note that large motivation should be to genuinely want learn more and to help people. Especially in reference to graduate school, it takes much of your time and having a blurred vision of what you want to do isn’t an optimal mindset to have when entering grad or med school.
From all the bits of information my mentor has shared with me, some going over my head and some pasted on my head after mentally repeating it over and over, I know my project is about measuring the drug tolerance and resistance of Cryptococcus neoformans to different concentrations of Fluconazole (FCN). We’re using several strains of Crypto. to effectively take account of all possible outcomes.
At this point, I’ve grown the different strains of Crypto. in solid media (YPD + NAT 100 + CM 100) and liquid media (Liquid YPD + 1X Hogness), which took 3 days for each, and have inoculated the fungi cells in the solid media onto a new liquid media plate, which, after growth, will then be inoculated again onto the plates with different concentrations of FCN. I have only done this for the 96 well plates and plan to repeat this process with the 364 well plates. Once the inoculation onto the FCN concentrated plates begins, I will need to record the growth with a scanner for several days.
With the recent increase in Cryptococcus meningitis in several countries, this research will be a valuable contribution in discovering the most suitable method in treating Crypto. meningitis. It has already been found that the capsule size of the FCN-resistant isolates is not largely impacted by fluconazole in comparison to the sensitive isolates; thereby, sustaining its virulence. Still, in a 2016 study, the growth of those same FCN-resistant isolates at 37°C was observed to be reduced. This means that although the fungi is resistant to the drug, the isolates’ growth and its morphology is impacted by its Fluconazole resistance (Rossi 2016) . With this in mind, I plan to observe the severity of the changes in capsule size and morphology with the scans I get day after day.
Cryptococcus neoformans = something I had no clue about until I came here and became entirely flooded with interest in it
Coming into the lab the first time, I had no expectations. I knew a bit about the fungi I’d be researching and had already spoken with my project manager before then, but I also had never really done any research to have a sense of what it’s like to work in a lab. Ultimately, I feel it turned out to be a perfect fit. My project manager explains everything thoroughly and the research I’m doing easily keeps me super engaged. The readings I was given… are actually fun to read? This whole realization really soothed the uncertainty and skepticism I had about research, so it’s really nice to say I know this is what I want to do.
Because of the pretty quick and communicative relationship I formed with my project manager and mentor, I expect there to be a lot of comfortable bursts of passion in conversation, that much of my series of questions will be answered, and that I’ll obtain valuable advice. Just in the first day, I learned so much. I can’t imagine the kind of knowledge I’ll leave with on July 3rd. I also feel like there will be a much greater level of confidence and understanding of the scientific world. In my application, I wrote about desperately wanting to become an insider to this world. I’ve only been given two days of a taste of what it’s like, and I already feel so, so eager to know more.
Since my lab is solely on discovering more about C. neoformans, there are so many questions to ask. For example, the fungi is able to live in several unrelated environments that aren’t suitable for a living organism. It is unsure what makes it able to do this. The 7ish weeks that I’ll be here will be dedicated to measuring C. neoformans drug resistance and tolerance towards different concentrations of an antifungal agent, or drug, fluconazole (FCN). Still, I think that I’ll be captivated in finding out more about C. neoformans even after that time and will likely ask to continue working in Dr. Perfect, my principal investigator’s, lab.