I am now officially past the five week mark and half way done with my internship! Time has definitely flown here at the Human Performance Lab and looking back over the last several weeks, I’m amazed at how much I’ve learned and how generous everyone at the lab has been with their time, expertise, and bodily fluids (see previous posts). As I’ve described in previous posts, most of my time thus far has been spent learning new techniques, helping out with the various protocols the lab is running, and working on my GR project. Despite now having generated any data at this point, I feel that I have been able to do a significant amount of background research, which has allowed me to write a comprehensive introduction to my project (minus my findings of course) and develop a pretty solid plan for when the ELISA kits arrive and I can actually start analyzing samples. As I had mentioned in a previous post, one concern had been that since the samples consisted of frozen buffy coat with lots of red blood cells mixed in, the hemoglobin that would be freed (thawing blood cells tends to hemolyze them) might interfere with the ELISA by binding to the GR antibodies. However, after a lengthy talk with manufacturer of the kit, I believe this shouldn’t be a problem. While I have yet to test this theory, I was assured that for whatever reason, the GR antibody complex would fluoresce at a unique wavelength making identification accurate possible.
In other news this week, I learned how to use the lab’s new human DNA isolation/quantification kit had the opportunity to test out some new sweat patches that the lab is trying to incorporate into one of the heat tolerance studies because they might offer a much less invasive way to measure caffein and chemokines than sticking subjects with a needle and taking blood. Basically, I just jogged for an hour in the heat chamber to ensure they had plenty of sweat to work with. Looking forward, Josh has assured me my kits are on the way and I will try to have my PI (primary investigator) take a look at my abstract and introduction drafts so they are ready all set when poster time comes around.

Who knew the military loved to party so much. This morning I walked into the lab only to be told I would be attending the Navy’s Hospital Corpsman Birthday! (I think they turned 131 or something) Anyways, there was lots of delicious cake and solemn ceremony and a good time was had by all.
In my last post, I mentioned that I had gotten a very weak signal for my western blot. After discussing it with my PI and a couple of post-docs, it was decided that I either didn’t have enough buffy coat (leukocytes), which make up less than 1% of blood by volume or I had hemolyzed the erythrocytes and made most of my white blood cells sink because they were now heavier. To overcome these challenges, I decided to try two different techniques. The first was to “snap” freeze fresh blood immediately after spinning in our centrifuge at 3000rmp for 15min and removing the plasma layer. The idea was that I could then use a scalpel and razor blade to scrap off the top layer of white blood cells leaving them relatively intact and free of any contamination from the erythrocytes. However, scraping this precious layer off proved much easier said than done because, despite being stored in a -80C freezer, it melted after about 15sec. The second technique yielded a much bigger and cleaner layer of white blood cells. In order to effectively separate the white blood cells from the red ones, I used a “Serum Separator Tube” which is essentially a vacutainer filled with some kind of goo that weighs less than red blood cells and more than plasma and white blood cells. The buffy coat layer was a little thicker than I expected (think slightly thicker than jello) and it remains to be seen whether it will yield a stronger signal than I got last weekend. However, while a strong signal would be very encouraging and show that the western is working properly, the samples I will be using for the GR ELISA have already been frozen and I don’t think it would be possible to transfer them to Serum Separator Tubes without hemolyzing them so I might have to come up with yet another technique that would allow me to extract the buffy coat relatively uncontaminated with red blood cells from frozen epi-tubes.
In other news, I finished my Lab Animal Medicine training so am now officially allowed to take sample from mice if I run out of human donors (I think I’ve stuck everybody at least once who works here) and I have begun to set up my own bench space where I will run the GR ELISA once it arrives and I have a reliable technique to extract buffy coat from the samples.

I titled this post practice/plan because this week I’ve basically spent all my time working on the western blots (dry runs for my GR assay) and starting to hone in on on the context I will be looking at GR in. I developed the first batch of blots on Monday and Tuesday using a new chemiluminescence technique that was pretty neat but yielded a very weak signal for both gels.

Incubating secondary antibodies for western

I plan to run at least two more practice gels, which will hopefully yield a stronger signal next week.
In other news, I’m finally starting to put together a plan for the rest of my time at HPL! Right now, its basically centered around a modest research project to correlate GR sensitivity/expression level to obesity and insulin resistance in African American (AA) subgroups. If I can find such a relationship, it would lend itself to a greater understanding of how HPA (hypothalamic-adrenal-pituary) dysregulation is related to insulin resistance and obesity since insulin is an antagonist to cortisol and both insulin resistance and obesity affect AA at higher rates than Caucasians (CA). This is essentially based on literature that says HPA dysregulation can result from chronic stress and AA experience disproportionate levels of chronic stress and insulin resistance. I am trying to see whether differential GR sensitivity can help link the two. In other words, can the hypothalamuses’ inability to respond to negative feedback inhibition be linked to insulin resistance through GR sensitivity. While GR sensitivity can be influenced by several things besides competing ligands such as insulin, I believe quantifying GR in different AA subgroups offers an excellent opportunity to discover a real relationship. Like I said in the last post, the GR kits are supposedly on the way so as soon as they arrive and I can prove to Joel my western technique is up to bar, things should start to happen. (Even though I will be using ELISAs to look at GR, westerns are similar enough in concept that Joel is letting me prove myself with them using estrogen receptors). If everything goes my way, I will be using a cohort of 100 AA, who will hopefully represent several subgroups (obese, normal weight, insulin resistant, chronically stressed..etc.)

One aspect of my internship this summer is a project that I will present to the lab staff in August. I have been informed that the only requirement of this project/presentation is relevancy to a current or prior research conducted by the HPL (Human Performance Lab). Because both heat related studies are ongoing, I have decided that, while I continue to help out with them, I will start focusing my attention on a recently concluded HPL study looking at the relationship between cortisol and obesity and start developing a presentation based on this research. Since this study has already been concluded and all samples have been collected, it presents the perfect opportunity to do a little original research (under Joel’s guidance). I expect more specifics next week but until then, I can tell you that we are going to be looking at GR (glucocorticoid recceptors) in plasma samples from the cortisol/obesity study. GR was proposed as a logical candidate for follow up research because it is an important cortisol receptor. Right now, our main objective is to isolate and characterize our GR receptors. The appropriate ELISA kits have been ordered and we started our first receptor isolation dry run today using a western blot to isolate and quantify nuclear ER (estrogen receptors) from blood I was able to get from a team member. (People are remarkably generous with their blood here). Hopefully I will be able to get a couple of western blot dry runs in before the actual kits arrive so I will be proficient enough to do them without Joel’s supervision. I said we started the western blot today because Joel told me that one of the best ways to reduce background is to incubate membrane and blocker (5% milk in this case) for 24hrs, which means we will be able to develop the membrane on Monday or Tuesday. The gel itself ran beautifully! Since we have been given the freedom to fine tune the protocol, we decided to try two methods of cell lysis: freeze thaw (3x) and sonication in order to break the ER free from the nuclear membrane. I must say the freeze thaws were particularly fun because we used dry ice to “snap” freeze the the samples after each thaw. And what a difference! the sonication lanes were almost clear while the freeze thaw lanes were muddles with cell debris! The transfer to the nitrocellulose membrane very quickly (7 minutes) using a new transfer machine although the gel marker lanes still has some visible bands, which might have indicated that not all the proteins were transferred to the nitrocellulose. We then submerged the membrane in milk for blocking in order to prevent non-specific binding of our antibodies and prepared the primary and secondary antibodies for development after the weekend. We are hoping that if we get clean results, we can use a similar protocol in the coming weeks with the new kits to isolate and quantify GR.

Other than working on the western blot, this weeks highlights included two presentations on the the effects of preventing the dimerization of GR in mice and my first real lab meeting, where everyone shared progress reports on whatever they had been individually working on this week and we discussed a protocol issue with the heat tolerance study. Apparently the ASCM guidelines meant to screen for diabetes mandate no food intake for 8hrs prior to the measuring of fasting glucose levels, a key biomarker collected prior to the VO2 max test. The issue, as I understood it, was that subjects’ max scores might be significantly influenced by this rule. In the end it was decided with the input of one of the lab physicians that fasting glucose levels could be accurately measured after only 4 hrs of fasting and that stressing our subjects to the fainting point was not necessary.