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July 2013 Archive

Posted by unknown on July 15th, 2013  ⟩  0 comments

There are many perks to working in an undergraduate lab. You get to make great networking connections, work on different projects, and be a part of cutting edge research. I had a great project that worked on bone-marrow derived stem cells (MSCs) that we successfully differentiated into endothelial cells (ECs). This was very exciting for me to be a part of as it was my first year working in a lab, outside of the long hours of lab duty you're assigned to as a biology major. Nevertheless the technique that we used was a nice blend of old practices mixed with some new ideas.

To differentiate our MSCs we used growth factors like FGF, EGF and VEGF to coax them along the path of turning into endothelial cells. They also were plated with different combinations of basal lamina components to assist them in their transition. We had previously differentiated MSCs in vivo with the help of all of the cross-talk between the cells. However, this is what made our task interesting to me. Our goal was to try to determine the key players that were working within the body to differentiate stem cells to whatever cell type was needed, in our case endothelial cells. So after we decided on the components we wanted to test on the cells, we had to decide which model to use: suspension or coating. If you've ever seen the hashtag #overlyhonestmethods, then I think this would fall nicely into that category. We were trying for the suspension model, but since the gel didn't quite set-up we had a “trap” model. This is basically to say we trapped our cells somewhere between the plate and our newly created network. Nevertheless this “new technique” seemed to work out great as our cells differentiated nicely into ECs. This was tested via X-gal staining with a lovely blue to show we had positive cells.

However, our next goal was to implant the cells to see if they could actually form vasculature in the body. It was great that we had positive results, but if it couldn't translate into something therapeutically significant then the excitement would quickly diminish for me. The goal here was simply to see if their dual-nature of being part stem cell and part endothelial cell would allow them to hone in on the site of injury and also start the repair. So we needed an in vivo model to test out our new hypothesis. When looking at our options, we decided to use luciferin. Now, this is where my inner child/geek kind of exploded. As a kid I was fascinated with lightening bugs and their tiny little glowing bodies. I would run around catching them for hours, while trying to figure out how in the world they were able to glow without being plugged in. They apparently seemed to catch the eye of the scientific community as well, as we now use their glow in cell culture. You see, it was great to have positive cells, but we needed to watch their progression inside the body which is not possible with staining. Therefore the new goal was to transfect our differentiated MSCs with luciferase and watch their movement inside the body.

While there are other options to watch living cells, like RFP or GFP, with luciferin we did not need to use a special microscope to view the cells. This was an added bonus for me, since you had to have a key to get in to use the very expensive fluorescent microscope, and I did not. So again this left us with a great model for viewing our living cells, without having to hassle with scheduling for the microscope. The results are still pending, but it was definitely cool to get to work with one of my favorite childhood toys while still being a part of cutting edge research. I would say that makes for a great day any day.

Deanna Tiek
Vanderbilt Class of 2014
Deanna is an intern at Goldbio during the summer of 2013.

Category Code: 79108

Posted by Chris on July 18th, 2013  ⟩  0 comments

In the world of cancer, in which the word ‘frightening’ takes on entirely new perspectives, perhaps none is more frightening than brain cancer. Although brain cancer is not one of the leading types of cancer per capita, the brain’s absence of pain receptors can hide the cancer’s growth and effects and it is often left undiagnosed until it has grown dangerously large. Maybe our fear has to do with the turbid violation of our innermost self, our control center; or maybe it is those subtle (or not so subtle changes) that make us question whether we’re losing our mind or if there is actually something physically wrong. Regardless, in our world of microscopic monsters, brain cancer is one of the most spine-chilling, nerve-wracking thoughts we have to deal with.

Glioblastoma multiforme (GBM) is a type of brain cancer which is technically rare, occurring in only 2-3 cases per 100,000, but represents just over half of all brain tissue tumors. The survival odds are bleak, with a median of only 1-2 years with typical care (surgery, radiation and chemotherapy) and 3 months without. GBM tend to form in the cerebral white matter and grow very quickly and may often cross bilaterally to the opposite hemisphere, but rarely extends beyond the central nervous system. The symptoms may include seizures, headaches, nausea and vomiting, but may also include memory loss or personality changes. GBM is traditionally difficult to treat for various reasons: including the brain’s susceptibility to damage from traditional care, the GBM tumor cells are resistant to some forms of traditional care, the brain has limited self-repairing abilities, and many drugs lose efficacy crossing (or just cannot cross) the blood-brain barrier.

Perhaps one of the biggest recent discoveries in cancer research has been Cancer Stem Cells (CSCs). CSCs are cancer cells which also possess characteristics associated with standard stem cells, specifically the ability to give rise to all cell types found in the tumor. They are believed to be tumor-forming, or tumorigenic, and may be one of the principle reasons why GBMs are so resistant to most chemo drugs. Their existence also explains many of the tendencies of some cancers, including metastasis and reoccurrence after seemingly successful treatments. Several developmental pathways have been implicated in CSC, including NOTCH and Sonic Hedgehog (SHH) and therapeutic recombinant proteins are being designed in attempt to target the undifferentiated cell populations in tumors. Among the more popular of these is from the super family of TGF-β. Within that family, a company called Genelux out of California is looking into a potential GBM CSC regulator with a growth factor called BMP4 (Bone Morphogenetic Protein-4).

In their recent publication, Rohit Duggal and associates expressed BMP4 in GBM models, showed significant tumor regression and a simply amazing increase in long-term survival of mice who had been inoculated with GBM and then treated with a VACV virus encoding BMP4. In the process, they utilized the firefly luciferase gene and some Luciferin to document the progression of GBM tumors in vivo mice brains, as compared to serum-grown glioma tumor line. With the bioluminescent marker encoded in the cancer cells, it becomes clear exactly how these cancers grow and spread in our system. I think this picture below sums up exactly why GBM cancer is so incredibly frightening.

GBM Cancer - Luciferase

This research is an early, proof of concept, experiment. There are many regulatory hurdles and trials before something of this nature could ever see medical treatment. Yet it provides hope; Hope that as we continue to discover more about how cancer originates and how it proliferates, we will learn how to treat this awful disease; Hope that we can beat this through scientific research; Hope that there is life after cancer and we will all know that someday. And if there is one thing that every cancer patient needs, it is Hope.

 
 

Duggal, R., Geissinger, U., Zhang, Q., Aguilar, J., Chen, N. G., Binda, E., Vescovi, A. & Szalay, A. A. (2013). Vaccinia virus expressing bone morphogenetic protein-4 in novel glioblastoma orthotopic models facilitates enhanced tumor regression and long-term survival. Journal of translational medicine, 11(1), 155.

Category Code: 88221 88241

Posted by Chris on July 25th, 2013  ⟩  0 comments

Tuberculosis is a common, and often lethal, infectious disease which has circulated since the dawn of mankind. It is caused by the bacteria, Mycobacterium tuberculosis; a small, aerobic, nonmotile bacillus, which tends to lodge in the pulmonary system and from where it then spreads through coughing or sneezing respiratory fluids through the air. It has been found in the bones of Egyptian mummies and was once the cause of nearly 25% of all deaths in Europe. TB remains the world’s second most lethal, infectious disease (behind HIV) and has a significantly high rate of occurrence in African, South American and Asian countries, where the rate of death is anywhere from 250-3000+ per 100,000. The World Health Organization (WHO) estimates that roughly 1/3 of the world’s population have been infected with TB (although 90-95% remain asymptomatic) and about 1.5 million people die from it every year. The resurgence of the disease, and its drug-resistant varieties, led the WHO to declare it a global health emergency in 1993. Tuberculosis is also one of the major research areas to which the Bill and Melinda Gates Foundation fund every year, which awards nearly $800M (total) to researchers through its Global Health Program.

In the lab, Tuberculosis can also be difficult to screen, with a 16-20 hour replication (compared to under an hour for most other bacteria), and can take 3-4 weeks to form on solid media before any in vivo testing can be done. This can be a significant impediment for fast, de novo antibiotic research and Nuria Andreu, from the Department of Medicine at Imperial College London, and associates wanted to use luciferase to change the score!

That’s not to say that bioluminescence hasn’t been used on TB in the past. Researchers have been using BLI on TB strains for almost 20 years, but not necessarily with the live strain of M. tuberculosis, or with in vivo imaging in live mice. Andreu wanted to create a reporter gene with FFluc in a virulent M. tuberculosis strain. First, the FFluc would need to be modified to red-shift the signal so that the signal would be more thermostable. This was was accomplished by mutating 6 amino acids in the sequence to develop a signal that shifted the emission signal from 560nm to 620nm. Second, they had to develop an integrase-free reporter into M. tuberculosis to stabilize FFluc reporter signal over successive generations. The integrase-free reporter responded with greater than 99% retention of the reporter gene after 3 months of in vitro growth, compared with just 60% retention rate for the parent strain!

Ultimately, this study shows great progress in creating a virulent reporter strain of M. tuberculosis which can be useful in drug research. Andreu was able to detect the presence of the bacteria in the lungs of live mice after only 2 weeks post infection (105 cfu), and as few as 103 cfu were detectable ex vivo in both lungs and spleens of the mice after dissection. Most importantly, they were able to make a rapid assessment of antibiotic efficacy, treating the diseased mice with Isoniazid (an organic compound often used as the first line medication in treatment of tuberculosis). They were able to see a nearly 9-fold decrease in the BLI signal for treated mice versus the control group after only 7 days of treatment.

TB-Luciferase Drug Treatment isoniazid

Ultimately, this looks to be a great system for early lab development of new antibiotics. As resistance in TB continues to spread, we need new forms of treatment in order to stem the tide of resurgence and begin to eliminate the disease in the poorer sections of the world where TB remains rampant and the death toll remains high. Hopefully, this new reporter will help in developing those new drugs and we can begin to put an old disease to rest.

 
 

Andreu, N., Zelmer, A., Sampson, S. L., Ikeh, M., Bancroft, G. J., Schaible, U. E., Wiles, S., & Robertson, B. D. (2013). Rapid in vivo assessment of drug efficacy against Mycobacterium tuberculosis using an improved firefly luciferase. Journal of Antimicrobial Chemotherapy.

Category Code: 88241 88231

Posted by unknown on July 29th, 2013  ⟩  0 comments

Summer is quickly coming to an end, which means that the start to the school year is just around the corner. This can be quite a stressful time for teachers and students alike, especially if you are just starting college or transferring to a new school. Luckily for me, I'll be returning to “Vandy” for my fourth and final year so the “back-to-school stress” will have no effect on me. It's actually more excitement than anything as I'll finally be back at my home away from home with some of the most amazing people you'll ever meet. Anyway, I will also be joining a new lab, which can be a little intimidating. Nevertheless, I'm sure that this feeling of anxiety or nervousness is something that my new PI will share as well. It was quite obvious when I had my interview that my PI was a little unsure about letting an undergrad into her lab. She would constantly tell me that they had never allowed an undergrad to join, and that she didn't know of any undergrads in her colleagues' labs either. So it was quite understandable that she would be a little apprehensive about letting a student come in and work on very expensive projects with really no “real lab” experience. However, this feeling was definitely mutual as I was beyond nervous that I would just mess everything up and waste thousands of dollars of her hard-earned grant money. There were certain kinks that we had to work through to make the experience the best it could possibly be for both parties involved.

A few blunders that I had to face focused mainly on what I already knew and my capacity to learn. I had already discussed all of my previous classes with my PI and about where I stood on basic lab techniques. However the rest of the crew didn't really know anything about me. One thing I will never forget is my first week there. I was doing a miniprep and after I had my sample I went to check the concentration on the nanodrop. I was just about ready to log off when another lab member came over and asked if I knew what DNA was. It was one of those moments where you just have to smile and respond politely. He of course didn't mean it in a negative way, but was just trying to gauge what I knew and what he needed to explain. In the end, I was not only working on my project but a side project of his as well. Needless to say it was somewhat awkward, but definitely necessary to let the others test your knowledge so that they can give you as much, or as little, help as you need.

Another thing that is important is patience. It's almost inevitable that we are going to mess something up along the way. Whether it is simply knocking over a test tube rack, or setting the centrifuge too high and losing all of our samples. We know that we'll mess up at some point, but it's important to remember that we are new at this. We are aware that the docs and post-docs who work in the lab can probably do most of their protocols from memory, but newbies need to be taught at least once. This is an area where putting in the extra 10-15 minutes can really be beneficial in the end. The whole purpose of undergrads working in the lab is to give us experience so that we can have a leg-up in grad school. So while most of us will try our best to remember that procedure you just finished in 5 minutes with a slew of almost illegible bottles, it's always helpful to have written instructions and a patient mentor. Fortunately for me, I had a great mentor, and have been thinking about a few simple things that my previous PI did that really made me feel like a part of the family.

One of the first things that my PI did was to lay out the whole project and explain what was expected of me. This was huge. At first I wasn't really sure what I was supposed to be doing, how much I should already know, or how many YouTube videos I needed to watch to be able to mimic different lab techniques. She was great at taking the extra time to really explain the procedures to me, and introducing me to the other lab members who were always more than willing to help with a protocol or question.

Another thing that my PI managed well were our lab meetings. A lot of my friends who also worked in labs either never attended lab meetings or only presented at the end of the year before our graded presentation. My PI always assigned me a day to present for the whole hour. Now, I can't say that this was always an exciting part of being in the lab, because having to talk about my project for an hour to people who are way smarter than me was a little bit of a daunting task. It was always quite stressful and nerve-wracking as they weren't really afraid of correcting mistakes. Nevertheless it did make me feel like a viable member of the lab, and even helped me with my presentation style and how to word things to sound more authoritative.

The last important thing to remember about year-long or semester-long students is that we like to know where our projects are headed. This is to say that it is important to know the significance or overall goal of our projects. We already know that we are not going to see the end result and that keeping us in the loop might seem like a waste of time, but knowing the impact of projects makes them more exciting, at least it does for me. If I'm engaged in a project, spending an extra hour, or weekend, in the lab doesn't seem as bad. It is one thing to get experience in tissue culture or bench work (which is still great), but knowing the big picture can help us connect the dots.

It was also nice when my PI would ask me where I saw the project going, or if I had any other expectations or ideas to add into the final goal. She was always great in keeping me involved and listening to my thoughts, even if it didn't mesh with her end goal. It at least let me know that she cared about my opinion and saw me as a member of the team, and not just an interloper. All of these factors really helped me feel more like a part of the family, and I can only hope that this next year works out just as well.

Deanna Tiek
Vanderbilt Class of 2014
Deanna is an intern at Goldbio during the summer of 2013.

Category Code: 79108

Posted by unknown on July 3rd, 2013  ⟩  0 comments

If you've been browsing around the blog, you might find this post (as well as the last few) to be a little different from the normal informative or product-related posts. Well I might as well introduce myself, I'm the summer intern and will be giving you a little insight into the life of a senior biology major. Feel free to ask questions or give feedback on topics that you would find interesting.

This past year I had the privilege to work in the medical center as a lab assistant for school credit. Before I go too far into the oh so interesting details of what my position entailed, let me give you a little advice if you are thinking about becoming a biology major: be persistent. The field is completely cut-throat as all science majors and pre-med students have to have research experience. For this reason you have to stick to your guns to get what you want. The lab I ended up working for told me they had never accepted an undergraduate student, and didn't really think it was the right time. Nevertheless, I ended up working there for the year and could not have imagined a better experience.

Now something that I learned pretty early on is that every lab experience is very different. I have friends who sit and read data, run gels, do the dishes, and undertake really cool regeneration projects. So if you are just getting into the lab scene you have to realize that the field varies. Fortunately for me, I got the really cool projects. I had the opportunity to work on two new cutting edge technologies and basically worked on developing their protocols. When I first started in the lab I was not sure how the environment was going to pan out. It was all girls with the exception of a male senior researcher. I would be lying if I said I wasn't intimidated. Everyone in the lab knew way more about science than me, and you could tell they were always trying to figure out what exactly I knew. About the second week in, one of the other members of the lab asked me if I knew what DNA was. Needless to say, I of course said yes, and told them that I had been taking multiple science courses a semester and knew at least the basics. So after a month or so I really began to mesh with the other people in the lab, and even looked forward to getting in there just to see what I'd missed while I was in class. You almost become a weird family with everyone else (if you are fortunate enough to get a good group of people) because everyone is always helping each other out. I was especially grateful for this as I was still enrolled in 16 credit hours worth of classes, and could not possibly be in the lab 10 hours a day to be checking in on my experiments. The others were always more than happy to help me by starting or stopping my gels, blocking a Western, or thawing a reagent as soon as they got there. So for me, I was really lucky to get to be a part of such a cool group of scientists.

The other thing that makes science so great is that you can do something that have never been done before. My lab worked mostly in heart regeneration and diseases, or cancer research. I worked with mesenchymal stem cells (bone-marrow derived) to try to recreate vasculature, or blood vessels. This would not only be helpful in research to create mini organs to test products on, but also in human research to help patients who cannot undergo bypass surgery or have diabetes. So we had our target group in mind, and now had to figure out a way to differentiate our stem cells into endothelial cells (the cells that make blood vessels). Even though I was the undergrad, I suppose they liked me enough to let me work on the project on my own. This was both a terrifying and awesome feeling. I was so excited to get started, but at the same time if it didn't work it was going to be all my fault. This wouldn't be a big deal if all of the reagents I was playing around with weren't so dang expensive! (Another shameless Gold Bio plug!) It was always a game trying to figure out how to maximize reagents, while still having significant results. Nevertheless, a plan was made and it would take about 21 days to see the results.

This was by far the most agonizing part. There was nothing I could do to help along my little cells. I could only hope that by the end that they would turn sky blue with staining to show that they had indeed been transformed. So as the day arrived, we went to check them out under the microscope and take a few pictures if we could magnify them enough to get any hint of blue staining. This was not only important to show that the experiment worked, but I also had to give an hour long presentation about my experiment that would be way easier if they were blue. So with mounting anticipation we switched on the microscope and sure enough a blue field encompassed our screen. We even had to zoom out to show how the whole slide was blue. It was awesome. I don't even think excitement covers the feeling you get when your experiment works. It's one of those moments where you just have to do a little dance, snap a pic and make it the background on your phone. This way when someone makes the mistake of asking what in the heck your background is, you can brag a little bit while boring the other person to death. It's always a win-win. However, lab life isn't going to always work out. We had many projects give us conflicting data, or negative data. Negative data are just one of those things you always have to laugh at, like when you get an Rvalue of 0.056 (it's supposed to be 1). In fact, other people have found negative data to be quite informative apparently, as they have started a whole journal about negative results. You can check that out here. When an experiment doesn't work it just means that you need to take a small break or have another lab member try. Again, you will become a small family, so make sure you are willing to help out the other members because you never know when you'll need them to return the favor.

All in all, I'd say if you are even considering being a biology major or going into the sciences; stick with it and get involved. I had been teeter-tottering between being a special education teacher or going into research, but after working in the lab I can definitely say I'm hooked and in it for the long haul. It's definitely not easy, but after getting my first positive and significant results I definitely know that lab life is the right life for me.

Deanna Tiek
Vanderbilt Class of 2014
Deanna is an intern at Goldbio during the summer of 2013.