Progress!

As anyone following these posts may know, I’ve had plenty of difficulty gathering the RNA that I need to finish the “wet lab” portion of my research project. To highlight in the most succinct way possible, I am collecting RNA from Mycobacterium bovis-BCG (BCG) that I have exposed to the human-derived antioxidant, Glutathione (GSH), so as to understand how Mycobacterium tuberculosis (Mtb) responds and ultimately survives what should be a fatal reductive stress encountered within the granulomas in lungs of patients suffering from this pervasive disease. BCG is used for my studies because it shares nearly 100% of its DNA with Mtb, and because BCG is considered a BSL-2 organism (which is allowable in the type of labs present within Pace University).

In my last post, I highlighted the difficulties of conducting Undergraduate Research while carrying the burden of a full-time course load and a part-time job with extracurricular responsibilities to tend to as well. I also mentioned that obtaining the pure, high yield RNA required for my analysis is the most difficult, and often discouraging part of my project. But, I also said, “as a scientist and driven individual, the only solution is to push forward,” and I’m surely glad that I did. Because…

I FINALLY HAVE MY RNA!

This means my wet lab work is essentially finished. I only have my transcriptional analysis to work through, which will be a demon in and of itself. But, I know that if I was able to persevere through the mental anguish of obtaining RNA from Mycobacteria (a notoriously difficult achievement), then the analysis can be done as well.

I have learned what my niche is in the spectrum of biology-related careers.

Knowing that I can work through a problem and come out as the “winner” when the odds are stacked against me is the key lesson I have learned in working through this project. By thinking critically, troubleshooting my failures, and feeling what victory in this field feels like, I have found again my desire to continue on with projects of the like and get my PhD in this field so that my passion can someday help those who are, or those who know someone who is, impacted by pathogens evasive to the treatments available to the public.

Blog #2

This semester has presented itself as one of the most challenging four months of my undergraduate career. From attending school with a full-time course load, working full-time at a coffee shop, conducting my research, working as a lab technician, and helping BIO 101 students as a discussion leader, it goes without saying that I have had a monumentally stressful Fall.

It has been difficult to keep on track with my research, as I have often found excuses to put off a growth trial and RNA extraction until the following week. However, as slow as it may be coming along, my research continues to move forward toward the final few remaining extractions. One of the most difficult problems I have encountered with my research is obtaining a high enough yield of RNA that is usable for sequencing and analysis. Often times, I will perform an extraction and visualize the RNA on a gel, only to find out (through NanoDrop spectrophotometer analysis) that my yield is not high enough, and the sample cannot be used. As I have, at that point in time, already invested 4 days of work into the trial, one could imagine how frustrating this is.

But, as a scientist and driven individual, the only solution is to push forward. As of now, I have all of my control and 8 mM GSH mid-log BCG RNA samples completed, and I am two-thirds of the way completed with my NRP BCG RNA extractions.

Unfortunately, until I have all my samples collected, we will not be sending them out for sequencing. Therefore, I have no analyzable data. This is, in one sense, discouraging, but in another sense, it reveals one of my favorite aspects about scientific research: unbiased analysis. I am glad we don’t have my preliminary data, for I fear that if I were to examine the mid-log BCG RNA transcriptome before being capable of unraveling the mystery of the NRP transcriptome, my analysis would be skewed toward biases obtained during the mid-log analysis. I would fear a tunnel-visioning of my analysis toward my preconceived notions of what I should see, rather than an inquisitive and removed analysis of the results I do see. Therefore, I feel the lack of data in my case is a blessing in disguise. By the time my third blog post arrives, however, brace yourselves for some data. I expect that I will have finished my extractions by the end of this month, and we will have sequencing results shortly thereafter.

Reflections on Research

At the beginning of the Summer Undergraduate Research Program, I hadn’t started work on the project described in the first blog post.  However, it was my lofty intention to not only finish the project by the end of summer, but also to have interpreted the data I collected.

That being said, I have no interpretable data. Yet.

In my experience, Mycobacterium bovis-BCG is quite the picky organism to grow, which is counterintuitive for me to say because Tuberculosis (the organism with which I’m using BCG as a model for) is such a hearty and difficult-to-treat disease.  Sometimes, the culture I start at the beginning of the week is at mid-log by day 5 or day 6 of growth, which is “on track” with how it’s supposed to grow, and by that day I’d be able to dilute the culture and begin my GSH growth trial and have my RNA extracted by the end of the ninth day since the original inoculation.  However, at rather random times, it seems BCG likes to throw a curveball. Sometimes, the bacteria will take much longer to grow than I expect, and that will throw me off schedule with RNA extractions. Ideally, it should take one whole week, or at most eight days, from the start to finish an extraction and visualize my RNA on an agarose gel, but because of the curveballs, I’ve spent up to two weeks on growing bacteria.

Alongside that, for reasons Dr. Kelly and I have not yet elucidated, the addition of GSH to 8mM changes the opacity of our growth media, which originally skewed the spectrophotometric reading. Because of this, I was not collecting enough cells to extract the large amounts of RNA from my experimental group that I need to send off for NextGen Sequencing.  Just recently, we have updated our experimental procedure to combat this problem, but I have yet to implement it to determine if this will affect the yield of RNA I’m able to extract from the 8mM GSH cultures. I am optimistic it will, however.

I have not yet started work on my NRP samples, unfortunately. This is because growing NRP takes 100 days, and due to the huge investment in time, I wanted to wait until we solved the “opacity change” problem before I began. I’m currently growing a new sample of BCG, and once I test out our updated procedure, I will begin working on NRP and will finally have the data I so desperately want!

A lot of my summer has been spent investing hours and hours of time and metaphorical blood, sweat and tears into solving the many problems I encountered with my project, but there is one positive note which I’m extremely proud of: after my first attempt, I extracted RNA and visualized it on a gel successfully! Once I put the gel in our UV box to look for the 18S and 28S bands of rRNA to determine whether I even had RNA or not, I jumped up and down when I saw the two distinctive bands (I’m not exaggerating there, I really jumped up and down).  To me, that was worth all the time I spent.

This summer project has humbled me greatly.  One of the biggest lessons I learned is that research is a huge investment, mentally, physically, and emotionally. All I wanted this summer was to finish this project and submit data to Dr. Kelly, and no words can describe how frustrating it is to watch your experiments—which I’ve already invested a week’s worth of work into—flush down the drain week in and week out. That last sentence was hypothetical, of course, for I would not flush live BSL-2 bacteria down the train, but you get my point. But, to feel a strong urge to triumph over hardships and come back into the lab with a smile on my face the next day knowing it’s the beginning of a new attempt is always a huge relief on the psyche.

This project has also cemented the idea that I want to get my Ph.D. in microbiology (or perhaps even mycobacteriology). For a while, I was between emphases in the whole of biology, and while originally I was settling with Physical Therapy as my emphasis, I switched to a Ph.D. mindset once I began working with Marcy. For a while, I felt disillusioned because I was unsure with my decision to work toward Physical Therapy, and after I had an internship in the field, I decided I wanted to pursue different avenues in biology. That scared me, because I’m jumping head first into my senior year at Pace University and I was worried that I didn’t have my life together. However, the past year I spent working with Marcy has provided a solid ground for me to grow on, and for that I can’t thank her enough.

Determining the Molecular Mechanisms behind Reductive Stress Resistance by Persistent BCG through Genetic Profiling

The World Health Organization (WHO) recently reported that approximately one-third of the world’s population are thought to be infected with Mycobacterium tuberculosis (1). At least one half of those infected individuals are infected with drug/multi-drug resistant strains. Although the global incidence of tuberculosis has been slowly declining since 2003, approximately nine million individuals contracted the disease with mortality rates approaching 20% in 2013. To meet the lofty goals of the new WHO End TB Strategy to end the global TB epidemic, there is a great need develop more effective therapies and vaccines to treat and prevent the disease. One way to do this is to better characterize how mycobacteria respond to the environments they may encounter within the host.

M. tuberculosis infects an individual through inhalation of airborne bacilli, and settles in the inner lining of the lungs, where they are then engulfed by macrophages present in alveoli. The mycobacteria avoid the innate antimicrobial response and persist inside the macrophage. Latent TB infection initiates after the infected macrophages are quarantined inside a tubercle, where the anoxic conditions force the mycobacteria to respond by entering a non-replicative persistence (NRP) state (2). It is hypothesized that, within this tubercle, the cells are exposed to reactive oxygen intermediates (ROIs) and reactive nitrogen intermediates (RNIs), which have shown mycobactericidal effects during early infection (3). In order to avoid oxidative damage as a result of ROI and RNI exposure, immune response cells produce glutathione (GSH), an important compound in the cell-mediated immune response detoxification system. GSH controls oxidative balance within the host cell’s cytoplasm in the presence of oxidizing agents (ROIs and RNIs).

In the coming Summer months, I will center my research of Mycobacterium bovis-BCG (a common model organism belonging to the M. tuberculosis complex, MTCB, 4) on the phenomenon that persistent BCG (BCG in the NRP state) is resistant to killing by reductive stress. Reductive stress is defined as an abnormal increase in reducing equivalents (including GSH, MSH, NADH, NADPH; 5). By performing genetic profiling experiments on persistent BCG (attained through the Wayne Model; 6), using GSH is the reductive stressor, I hope to gain insight into the molecular mechanisms with which persistent BCG resists the normal damages associated with reductive stress.
Experiments performed by fellow researchers Raheem Lawrence and Neil Patel have provided evidence through extensive growth trials that physiological levels of GSH (8 mM) negatively impact the viability and growth of BCG. These phenomena were not detected with NRP BCG. Intracellular ATP in active BCG increased to 2.3 uM after 4 days of GSH exposure, and by day 5 they markedly decreased to near 0 uM. This decline coincided with decrease in active BCG viability. The levels of intracellular ATP in NRP BCG remained low throughout the trial, ranging between 0.02 uM to 0.2 uM. Thiol content analysis in active BCG showed an increase of intracellular thiol from 0.81 uM to 4.6 uM, which HPLC analysis identified as mycothiol (MSH), which is theorized to be the mycobacterial equivalent to glutathione. However, these same analyses in NRP BCG showed that MSH content of the persistent bacteria did not increase over the same time frame, hinting at the reductive stress resistance in the NRP state noted earlier in this proposal. It has now become a priority to elucidate the molecular means for the phenomena Lawrence and Patel have uncovered.

In order to deterime the means by which such a strong resistance to reductive stress may exist in NRP BCG, I will analyze a global transcriptional response of BCG. RNA will be isolated in cultures of persistent BCG which are both exposed and unexposed to GSH, and these transcriptional responses will be compared with mid-log BCG as my control group. In order to extract RNA from these cultures, I will follow a procedure that Josie Blair, Dr. Kelly’s previous research assistant, developed to attain high-quality samples. All RNA will be purified with a combined Trizol/Qiagen RNeasy/phenol-chloroform extraction procedure. Once collected, I will work together with the Center for Applied Genomics at Rutgers University in Newark in order to profile and analyze the transcription responses by both samples of persistent BCG, and I hope to eventually identify novel genes accountable for the stress response.