The Telomeres

Hello and welcome to the final blog!

This week, we resolved our issues, made great headway, and ate some lunch while we were at it.

Firstly, we definitely corrected the issue from last week of high density v. low density. I plated a lower density experiment to be analyzed, and the results have been interesting (but that’s for the presentation). Luckily, we were able to use the high density results for the good of the project, instead of letting it go to waste. The higher density, although it did not show quite as much of a reduction in the MYC protein which actually drives the cancer and causes proliferation, showed a definite reduction in the IRF4 protein, which is the protein the drug was meant to target. If you remember, in previous blogs I mentioned that IRF4 drove the production of MYC in multiple myeloma. So, the purpose of the drug was to target IRF4, reducing it so that MYC would subsequently be reduced and the cancer would die off. So, what we saw is that the drug was working—but that, in high density settings, there still remained enough IRF4 for the MYC to be produced and the cancer to continue its proliferation.

So, when we began experimenting on the lower density cells, we saw a dramatic decrease in cell proliferation after 6 days of drug treatment (a promising sign that MYC was knocked down). However we also got some strange results after putting the cells through some flow cytometry, which analyzes each cell in a sample individually. And those results should be revealed in the presentation.

Until then, thank you for reading!

Angela

It's a Friday

Hello and welcome back!

This week at the Mayo Clinic has been quite eye-opening, especially when one realizes that one has been conducting an experiment wrong for the last two weeks.

Nevertheless, at the start of the week, everything was going smoothly. We were repeating the western blot, which had a strange result (it showed no effect on the cell lines) and we were midway through the RNA Analysis.

By the second day, the western blot was ready to be put into a gel to be read and we were using the PCR machine to make more RNA for later testing.

And then, come Friday, we had finally finished the Western Blot and had already put the RNA into the gel, when we decided to look back at the protocol and I realized that everything was wrong.

Well, not everything. I exaggerate. But, it happens to be a slight problem when you plate the cells at 5 times the concentration they’re meant to be at. If you recall, one of the first problems the experiment had run into when we were first analyzing it was that the drug did not work under high concentrations of cells. The cells are meant to be at a low concentration of 50K cells/ml and I, in all my infinite wisdom, had been plating the most recent cells at 250K cells/ml.

Oops.

Luckily, it’s easily solved and doing the experiments a third time over just means I’ll have a more experienced hand, right?

Right.

Thank you for reading!

Angela

Lab, lab, LAB!

Hello and welcome back to the blog!

This week, I did much more of the same: RNA and protein analysis, excel graphs, and presentation slides. Our results have been very consistent so far, and we’re seeing a confirmation of what we expect to be happening theoretically in every step of the DNA to protein synthesis. The graphs, by far, are still the most time consuming part—both in the way of examining the data and plotting it. Did you know about the log scale function? I do now…

But, as a small change of events, I was able to go to one of the lab meetings this week. Usually, the lab meetings are on Fridays and are a short presentation of our work between the 5 of us in the lab. So, I should have known something was odd when I found out there was a lab meeting Monday, instead. But, at the time, I didn’t. And when I arrived at the lab meeting and there were buckets of food outside the door, I should have thought that was odd as well. But, I didn’t and I happily picked up a veggie sandwich. It was really only when I opened the door to the meeting room and 6 times the amount of people I was used to seeing that I knew something was up.

Evidently, this would be the mother ship of meetings: a discussion by all the scientists working on Multiple Myeloma in the Mayo Clinic Labs. A lot of the information that was transferred between this groups passed over my head: but what little I did gather was incredible and encouraging. There are many new connections to be made about multiple myeloma that will allow us to understand it more. It was more than exciting to see so many so passionate about this one field, and to witness the joining of like, experienced minds.

Thank you for reading!

Angela

Don't Cell Out

Hello, and welcome back!

This week, we found some of our results and everything in the way of the lab seems to be going smoothly and consistently.

Things in the way of presentation, however, are not going so smoothly.

To start off, the drug titration showed the same results as the test on the drug proliferation. It seems that the cells tend to drop off at a drug concentration of around 1uM.

The western blot also showed consistent data. We saw that the MYC protein was reduced once the active drugs were added, compared to the inactive drugs. Unfortunately, we will have to repeat the experiment since the staining did not turn out quite as clearly as we would have liked.

And finally, the analysis of the RNA is in progress, and coming along smoothly.

Powerpoint, however, has not been so easy. And Excel, its analytical cousin, has been equally ornery. I’ve been attempting to create diagrams and graphs that would present the data with the elegance it deserves, and, unfortunately for everyone, the best I could do was this:

Obviously, this isn’t going to work. So, I’ve been trying to enlist the help of this strange thing called “google” to get me to a better diagram. Unfortunately, every time I google “cells”, I’m either taken back to fifth grade science or a man in black and white stripes. On the bright side, I have a few more weeks to become a professional in the art of Office.

Until then, thank you for reading!

Angela

Juggling

Hello and welcome back!

This week I learned to juggle… that is, I learned to juggle multiple new experiments and strange new jobs.

To start off, though it may seem a little unrelated, I’ve begun working at McDonald’s. It’s been interesting to say the least. Luckily, it hasn’t affected my schedule at Mayo Clinic, although it has altered my outlook a little bit. I think I can appreciate the slow, relaxed, meticulous pace of the lab more now that I can compare it to swearing customers, burning French fries, and toppling ice cream cones. However, as different as it may seem, I’ve been able to draw some parallels between the two; like the fact that in either work place I’m constantly sanitizing. In one job I’m growing bacteria, and in another I’m getting rid of it…and I honestly couldn’t tell you which one is which.

But, regarding juggling experiments at the Mayo Clinic, I’ve now got 3 active experiments going at the same time. One experiment is the drug titration, which determines the progress of cell proliferation. Another is the analysis of the RNA produced by each cell line. And the last is the western blot, to determine the protein production. All three steps will be the final determination of what our conclusion to the experiment will be. The end is approaching, and it’s exciting stuff.

The results from last week’s drug titration also came in, and it seems that there may be some off target effects coming from the inactive version of the drug. The inactive versions are meant to act as a control, having the same effect on the cells as those that have not been treated. However, when we reviewed the results, in some cases the cells treated with the inactive drug grew even more than those with no drug. This may hint that the drugs are having some unwanted side effects. But, we’ve decided to run the test again to make sure that our results are more certain.

Until then, thank you for reading!

Angela

Experiments: the endless cycle

Hello and welcome back!

The results are in! And they’re consistent—something I hadn’t realized was so important to experiments until now. After putting the U266 (the cell line that reacted to the drug, even though we didn’t expect it to) through a drug titration (an experiment to see at what concentration the cells are affected) it showed marked signs of inhibited growth when a drug concentration of 1 uM was added. This is good news, since it not only verifies that we are following the drug company’s protocol but also that I am not horrible at lab experiments.

Unfortunately for me, I must’ve done too well—because this week I had to extend the drug titration to all four cell lines. That means 16 different drug concentrations, 160 wells, and 800,000 cells. My poor thumbs.

We’ve also begun to look at the protein and RNA synthesis of the treated cell lines. The ASO’s are meant to decrease production of IRF4 by inhibiting its RNA production. So, we should expect to see a marked decrease in RNA and protein production in cell lines treated with the active drugs. It will also be interesting to see the results of the cell lines that did not react to the drug. It may give us a clearer insight as to why the drug has no effect. We will be using a western blot to determine protein production (something I’ve heard about in Biology class but have never tried). It’s bound to be interesting!

Until then, thank you for reading!

Angela

A solution

Hello and welcome back,

The results of the new experiment are in, and are filled with surprises. After conducting a MTS assay on all four cell lines, we were able to see the effect of the drug on cell proliferation by measuring the ATP produced. Though we did not know exactly what to expect, we did have the hypothesis that the U266 cell line would be resistant to the ASO’s because it does not produce MYC, and—we thought—would not be dependent on IRF4 because of the lack of MYC. However, we saw that the ATP production of U266 was cut in half when administered the active ASO’s. This hints that U266 may still be dependent on IRF4, despite a lack of MYC production. In contrast, the FR4, which does produce MYC, was not affected by the ASO’s at all—a very unexpected turn of events. We will be examining these cell lines in depth in the upcoming weeks to determine just why this is the case. Interestingly, the KMS11 and the XG7 reacted as expected.

But, now that we’ve answered the question of which cell lines are reacting, we’ve decided to test another direction of the experiment: determining at what concentration of the drug the cells become sensitive. So, we’ve set up drug titration experiment, whereby we’ve plated one cell line (in the same reduced concentration of 5K cells/100 ul) with varying concentrations of all the ASO drugs (active and inactive).

Of course, this sounds easy enough. But. It. Took. Forever. 40 wells, 16 stocks of drugs, and 96 pipette tips later, the plate is set to sit in the incubator for another 6 days before I can go through another 40 tips and a tube of cell titer-glo to see the results.

Until then, thank you for reading. Happy pipetting!

Angela

Killing it

 

Hello and welcome back!

Firstly, I’d like to calmly inform you that…

THE EXPERIMENT WAS A SUCCESS!!!!!!!!!!!!!!! THE CELLS ARE REALLY REALLY DEAD BUT IN A GREAT WAY!!!!!! LOOK AT MY 12 WELL PLATE WITH ALL THE DEAD CELLS IN IT!!!!!!!!!!!

 

 

Ahem.

So, from this experiment we can conclude that the drugs are, in fact, working, which is a good start. This was, however, only tested in one cell line.

This week, I began applying the same experimental conditions to other cell lines with different genetic variables. One of these cell lines (the one with the results above) is especially sensitive to IRF4, while another is not. So, the next experiment will hopefully show the drug’s effect on the one cell line but not on the other. More cell lines are being tested and if the hypothesis hold true in this next experiment, we may be able to answer more questions about those cell lines.

 

So, in preparation for the next experiment, I’ve plated 48 wells of cells via pipette (I’m not sure if you know how much thumb work that requires, but let me just say that challenging me to a thumb war should not be taken lightly) with the placebo and drug. We will be testing the effect of the drug by counting the live cells, for now. Later, we hope to test the mRNA and protein production. We may even throw in a titration of the drug. Who knows? Endless possibilities when it comes to killing myeloma cells.

 

However, there is some bad news. Now, two lines of my cells are dying. It seems there may be a pattern here… I’ve replaced them with fresh cell lines, and hopefully they will live to die in the next experiment.

Regardless, thank you for reading.

Until next week,

Angela

 

Rebellious Cell Lines

http://www.calpaclab.com/science-jokes/

Hello all and welcome to the blog!

 

My week at the lab has been full of promise (and, maybe some progress). Much has been learned from last week, and I am beginning to think that next week may be even better.

 

Let me start off by giving the status of the sad cell line from last week: dying, as ever. But, I am now aware of multiple factors that may have led to its slow demise.

1.      This cell line especially likes crowding. My experiment does not.

2.      This cell line has been growing poorly for everyone at the lab, perhaps hinting that the cell line itself is having an issue.

3.      The cell counter is poor at reading viability.

 

So, luckily for us, the cells may not be dying off quite as dramatically as we’d originally thought. If anything, it may just be a case of teen-rebellion on this growing cell line’s part. After all, it can be difficult for pubescent cell lines to deal with new changes in their petri dish like finding glutamine out of nowhere in their fetal plex media.

 

But, just to help them get more situated, I’ve spun down the cells and put them in some fresh media, so hopefully we will see some improvement next week.

 

More good news: the experiment is finally showing signs of working. Our cell lines are reacting to the drug as we’d predicted. This time, it might just be a good thing that they’re dying. But, I should only know the official results of the test on Monday.

 

Until then, thank you for reading!

Angela

A Somber Start

A small piece of ice which lived in a test tube fell in love with a Bunsen burner.
"Bunsen, my flame! I melt whenever I see you!" said the ice.
The Bunsen burner replied, "It's just a phase you're going through."

(http://www.calpaclab.com/science-jokes/)

 

Good morning and welcome back!

 

It’s been a crazy time at the lab, with no signs of stopping. To begin with, we’ve hit a bump in the road with our experiment. Having received unexpected results, we’ve decided to back up a little and try to understand what went wrong and to recreate the biotech company’s experiment to make sure that we are able to arrive at the same conclusion. Despite our trial and error, the biotech company has been great at collaborating and helping us continue with the experiment.

 

In addition to this small bump, one of the cell lines is dying. And this time, it wasn’t on purpose. This particular cell line tends to thrive in crowded conditions, so we suspect that its sudden demise is due to our dilution of it (which the experiment required). No worries, we have a second batch ready to go and we will be holding a bleach funeral for the sad first batch.

 

With all this, something good was bound to happen. And it took form in understanding. On Friday mornings, the lab staff gather to hold a meeting in order to discuss their experiments. Dr. Bergsagel presented his findings regarding patients who were going through the clinical trial. It was only then that I realized what a host of treatment myeloma patients must go through. Multiple myeloma is not curable as of today. Part of this is because every person reacts differently to the drugs given to them. There are about 5-10 different genetic groupings for myeloma, and there are double that many drugs to treat it. So, each patient had to go through at least 4 different phases of drugs, some with good reactions and others not. Each patient receives a trial-and-error of cocktail drugs that hopefully have an effect. Through this, it became clear to me that individualized medicine (medical treatment that is fitted specifically to each person) may hold the brightest future for myeloma. With so many different genetic lines that can lead to myeloma, which all differ in treatment, it becomes almost crucial for us to study myeloma in terms of genetics. And now understanding this more, I feel certainly blessed to have been given the opportunity to help begin this journey.

 

Until then, thank you for reading.

Angela

"What are we going to do today, Brain?" "The same thing we do every day, Pinky. Try to take over the world!"

"Biology is the only science in which multiplication is the same thing as division."

 

Hello, and welcome back!

 

It’s my first official week working on my Senior Research Project over at the Scottsdale Mayo Clinic, and I’m already learning and growing. Literally. I’ve got myeloma cell lines growing out of petri dishes like chia seeds on a clay Scooby Doo.

The uncanny similarities….

 

Backing up a bit, I began the week discussing our battle plans against these myeloma cell lines. Armed with some pipettes and a little bit of trypan blue, the lab has teamed up with a pharmaceutical company in order to look at other ways to manipulate myeloma. We have decided to test antisense oligonucleotides (ASO’s), which attach to mRNA to stop its manufacture of protein, specifically for myeloma, IRF4. Recent studies have shown that IRF4 may be the “Achilles Heel” in the Trojan war against myeloma. Myeloma seems to feed off of the production of IRF4, and an arrow to the production of IRF4 may kill myeloma cells entirely without harming normal cells. So, the question remains, can ASO’s be this arrow? And, will all types of myeloma be subject to the predicted effects? If not, why?

 

With these technical plans set firmly in the future, my present is momentarily absorbed by the certainty of two things:

1.      The equation (M₁)(V₁)=(M₂)(V₂) is not to be underestimated

2.      Pipetting requires serious deltoid muscles

 

Despite my aching arms, working at the lab has definitely been the highlight of my week. It’s been a whirlwind of warming media, using acronyms, looking under microscopes, and being totally amazed by the advancement of science.

 

I hope to see you again next week!

Until then, thank you and happy reading!

 

-Angela (or “The (M₁)(V₁)=(M₂)(V₂) girl”)

 

The Basics: ATCG

“How do you eat DNA spaghetti?”

You use a replication fork. -Anonymous

 

 

Hello and welcome to the blog!

My name is Angela Hemesath. As a senior at BASIS Scottsdale in Arizona, I am participating in one of BASIS’s unique programs: the Senior Research Project. The Senior Research Project (SRP) is an opportunity for BASIS Seniors to use their third trimester to explore a research question through an internship.

With the help of my Faculty Adviser, Mr. John Nishan, and my On-Site Mentors, Dr. Dan Riggs, Mrs. Victoria Garbitt, and Dr. P. Leif Bergsagel, I have been given the opportunity to intern at the Mayo Clinic in Scottsdale (linked on the right). My research will focus largely on the genetic components of Multiple Myeloma, a cancer of the plasma cells (“Diseases and Conditions Multiple Myeloma”). Studies have shown that the malfunction of a certain gene, called the MYC gene, may be heavily involved in causing Multiple Myeloma (Dib). Through my work at the lab, I hope to answer the following questions: “Can the distance of one gene indeed affect another? Is multiple myeloma actually caused by the translocation and subsequent dysregulation of the MYC gene? Will a genetic study of medicine lead to individualized care?”          

I hope you follow me on my journey and I hope that I can entertain you a little along the way!

Best wishes,

Angela Hemesath

 

If you’d like to read more on my SRP, my proposal is linked on the right.

 

Sources:

Dib, Amel, Ana Gabrea, Oleg K. Glebov, P. Leif Bergsagel, and W. Michael Kuehl. “Characterization of MYC Translocations in Multiple Myeloma Cell Lines.” Nih.com. NCBI, 3 Sep. 2009. Web. 16 Dec. 2014.

“Diseases and Conditions Multiple Myeloma.” Mayoclinic.org. Mayo Clinic, n.d. Web. Oct.         2014.