Bad day in the lab…

Some days in science, things just don’t go the way you plan.  Today was a good example of that.  You can have a very planned, thought-out experiment and then it all goes to **** (insert expletive of your choice).

We recently observed that an enzyme digestion kit that we were using was cleaving some of the antigens (cell surface markers) off our cells.  In particular, CD4 and CD8, both of which are rather important for being able to identify T cells.  Since we’re trying to establish whether a certain type of T cell is present in the brain when there’s a viral infection, these are, well, crucial.

The problem is that the enzymes are very good at chewing up cell debris and myelin and other brain-related gunk that just doesn’t separate out well otherwise, and we get a cleaner preparation.  The bad thing is that if it’s trimming off CD4 and CD8, we can’t trust it to not remove other potentially important markers.  Today’s experiment was to try to shorten the incubation times with the enzyme to limit the damage so that we could still get a clean preparation and less or no antigen (marker) loss.

After carefully setting up enzyme incubations of 20, 15, 10, 5, and 0 minutes (no enzyme digestion), we cultured half of the 20-minute cells to see if they would/could re-express some of those markers.  Next, ALL the sample sets were separated by a centrifuge technique.

That was the idea, anyway.  The reality: two of the tubes wedged against the post in the centrifuge.  This prevented the buckets from swinging out all the way.  The tubes had varying degrees of cell separation from “ewww, wtf is that?!” to “hey, that actually almost looks normal but really dirty.”  It took 4 hours to get from start to total experiment fail.  It actually was not the first time this happened, but the third.

There are these little rubber feet in the bottom of the centrifuge tube holders. These are important because the centrifuge is spinning these tubes at 1200 x g, and some tubes will break at that speed, especially without some cushioning.  Your bones would probably break too.  If we take them out, the standard-sized tubes fit but could potentially shatter under the force (thus spinning all our cells all over the inside of the centrifuge).  If we leave them in, the tubes might wedge against the post again.  We tried a slightly different (shorter) tube, but they were such a tight fit they were hard to get back out, which is no good either.

Sometimes experiments fail because you mess up.  Sometimes they fail because your equipment messes up.

I am NOT doing the happy data dance today.

Immunology in SPACE!

wpid-screenshot_2015-01-10-09-52-05_1.jpg

On Saturday, this Tweet came across my feed and rather caught my attention.  The cell picture is what caught my attention first, closely followed by the text: T-cell in Microgravity.

My Ph.D. is in Biomedical Sciences, and I studied Immunology for my research.  In particular, I was looking at the immune system in the brain and how it responded to viral infections of the brain.  I found this concept immediately arresting because SPACE! I love space and I’ve always wanted to go into space and if NASA (or someone) offered me a chance of reaching Mars with a 50% chance at surviving on the surface I would go immediately.  But with regard to the immune system, this did not immediately make sense to me: why would anyone study the effects of microgravity on the immune system? How would anyone come up with such an idea?  Is it plausible to think that there might be some effects?

Even going back to the beginnings of the space program, we worried about astronaut health, but this was more due to the fact that medical care was… rather far away.  Your immune system is all you’ve got, and it’s pretty good at defending you.  But could microgravity (or zero-gravity) affect how well it functions? Perhaps.

I suppose the first thing to consider is that you’re stuck in a closed environment with other people.  Germs quickly become community property and are likely pretty universal, but what if someone has a latent viral infection (like, say, chicken pox) and someone else isn’t immune?  What if some freaky-weird space virus gets on the space suits and infects the crew?  These aren’t particularly plausible concerns (especially the second), but you do have to postulate that somehow, some new pathogen might get into the environment.  Maybe your experiment escapes and you have to worry about a mutant rampant fungus.  I don’t know.  But let’s assume that this pathogen gets into the body somehow.  What then?

The immune system has several sort of generic sets of cells that patrol for invaders.  If and when you get an infection, these cells are supposed to migrate toward the lymph node that serves that tissue.  For example, if you eat the pathogen, these immune cells should probably hit your cervical (neck) lymph nodes.  Except that fluid travel on Earth is used to having GRAVITY.  So, could microgravity disrupt that?  Possibly.

Once these first-defense immune cells arrive in the lymph nodes, their job is to activate T cells.  Physical contact is required for this, and there are lots of molecules involved in holding the immune cells together for a brief period to activate them.  That junction/border between the T cell and its activator is called the immunological synapse. Could that be affected by microgravity?  Again, maybe.  I’m not certain this is plausible because I imagine those interactions sort of like Velcro, and I’m not sure that they’re going to be gravity-dependent.

The T cells are then supposed to replicate and differentiate.  Gravity-dependent? Harder to say.  We do know that microgravity conditions affect bone and muscle cell growth and differentiation 1-3.  That makes sense because our bones and muscles on Earth are pretty gravity-dependent; weight-bearing exercise increases both muscle mass and bone density.  Perhaps more surprisingly, though, additional research shows that microgravity also affects male reproductive cells (in mice) 4 and even how sensitive cancer cells are to chemotherapy 5.  These studies lend some credence to the idea that immune system function might be affected by microgravity!

Once the T cells are activated, they’re supposed to migrate back to the site of infection following a chemical trail left by the first defense cells.  That may or may not be gravity-dependent as well.  Then, on arrival, they must physically interact with and identify infected cells: more Velcro.  Finally, they have to kill the infected cells.  Along the way, some of these T cells are converted (differentiate) into memory cells, in case your body sees that same pathogen again.

Now, here’s what the Principal Investigator (PI) has to say about her work:

I think this research is tremendously exciting and I wish I were in that lab and participating!  It can be hard to justify and explain why these types of studies are important.  I’m probably never going into space.  I doubt many of you who read this (or your children) are, either. But what if? As we explore and learn more and take our first tentative steps into that out there, being informed is good.  And what if, just what if, we learn from such experiments that T cells grown in microgravity can aggressively attack and kill cancer cells? What value might that have?  I am so excited about this payload and I can’t wait to read the results (which will be a while. Science requires patience.)

 References

  1. PLoS One. 2013 Oct 7;8(10):e76710. doi: 10.1371/journal.pone.0076710. eCollection 2013.
  2. Cell Cycle. 2013 Sep 15;12(18):3001-12. doi: 10.4161/cc.26029. Epub 2013 Aug 14.
  3. Aviakosm Ekolog Med. 2012 Sep-Oct;46(5):64-7. Russian.
  4. PLoS One. 2010 Feb 4;5(2):e9064. doi: 10.1371/journal.pone.0009064.
  5. Neurosci Lett. 2009 Sep 29;463(1):54-9. doi: 10.1016/j.neulet.2009.07.045. Epub 2009 Jul 21.

Random Mutations and Cancer in the news

Very recently in the news, there have been a lot of stories about how most cancers (2/3) are due to random mutation. This has been very widely broadcast and published and written about, and I debated whether to join the bandwagon. People tend to ascribe great significance to disease and/or illness. In the modern U.S.A., we seem to believe that we can somehow prevent everything bad from happening to us, to our health. The preponderance of dietary and lifestyle books are generally intended to help us with just that.

I will admit that my first thought on hearing about this story was “Oh thank god.” It has bothered me for a long time that the general public talks about finding “a cure for cancer.” It’s painfully evident that cancer is not just ONE THING. We have progressed from naming and treating cancers based on what organ or tissue they were found in to being able to identify what organ or tissue type they originated from (or are). Our therapies have advanced from chemotherapy and radiation therapy to specific monoclonal antibodies that target and destroy very specific cancer cells. Our understanding, detection, and treatment of various forms of cancer is much more nuanced today than it was even a decade ago.

But what causes cancer? Mostly, mutation. And biologically, there are only a two ways to get mutations: inherit them from your biological parents, or acquire them randomly throughout your life. Okay, so what can cause mutation? Well, radiation, smoking, and according to the media, pretty much everything in our lives is carcinogenic. Air? If it has secondhand smoke! Water? What about the pollutants and BPA? Nothing is safe!

Mutation is an inevitable consequence of LIVING. We know that mutations in our cells accummulate throughout our lives. Some mutations are good–otherwise we’d never have neat traits like Elizabeth’s Taylor’s infamous eye color. Some mutations are bad, like the ones that cause cancers. Some mutations don’t matter much. So who cares if you have a mutation in an important heart gene if that occurs in your toenail-producing cells? That gene wasn’t being used there anyway!

To put this in further perspective, if we look at a particular gene called APC, the functional part of this gene is 8,475 DNA bases long (2015). A loss of just one DNA base (#41 in particular) leads to a form of colorectal cancer known as Familial Adenomatous Polyposis (FAP)(Miyoshi et al., 1992). Although this particular form of cancer is inherited, there are 8,474 other potential places along this gene where a different mutation can cause the exact same form of cancer. That’s a lot of potential “causes” for just this one particular cancer. What caused that initial mutation? That is hard to say–and that is what we attribute to “bad luck.”

You have a trillion cells in your body. That’s 1,000,000,000,000. That’s a lot of cells. Most of them divide every now and then. Your skin, for example, renews much faster than your brain cells (which can actually divide). Your bones grow at a different rate. Some cells never truly get replaced (that’s usually scar tissue). The longer we live, the more these cells have to renew and replace (thus causing those stem cell divisions mentioned in most of the write-ups). Every time they do, more mutations arise just due to mistakes, kind of like genetic typos (have you spotted the one in this post?). The DNA copying is pretty accurate, but it still makes about 120,000 mistakes per cell. Fortunately, your cells also have some proofreading mechanisms that clean up those “typos”, reducing that ultimately to about one permanent mutation per cell each time it divides. (Pray, 2008) Most of the time those mutations are pretty harmless (do you need that lung gene in your eyelashes? Didn’t think so). However, the more often your cells have to divide, the better the chances that one of them will hit the jackpot and get a really (not-awesome) cancer mutation. Even then, don’t panic: the immune system actually probably cleans up most of those before they ever become problematic.

The point of this article (most excellently summarized in the Medline link below(Day, 2015)) is that for about 2/3 of cancers diagnosed today, there is no family history of disease, and no obvious contributing lifestyle factors such as high-risk carcinogens or behaviors, or standing in a gamma ray chamber hoping to become the Hulk, or painting with radioactive materials. These cancers therefore are assumed to be NEW mutations.

Even here, our advancing technology is allowing us to do what we’ve never been able to do before: sequence our genetics to look for genetic risk factors such as the BRCA genes known to cause breast cancer and other similar specific mutations (screening for FAP is available as well). These tests are usually reserved for people with family histories of disease, but the gene might even run in our families but we never knew it because disease was never diagnosed. What this research really highlights is that most of us don’t HAVE a history of disease. We are patient zero.

 References

Day, Mary E. (2015). Random Mutations Responsible for About Two-Thirds of Cancer Risk: Study, HealthDay News. Retrieved from http://www.nlm.nih.gov/medlineplus/news/fullstory_150227.html

Gene, NCBI. (2015). APC adenomatous polyposis coli [ Homo sapiens (human) ] Available from NIH NCBI Gene Retrieved 03 January 2015 http://www.ncbi.nlm.nih.gov/gene?cmd=Retrieve&dopt=full_report&list_uids=324#reference-sequences

Miyoshi, Y., Nagase, H., Ando, H., Horii, A., Ichii, S., Nakatsuru, S., . . . Nakamura, Y. (1992). Somatic mutations of the APC gene in colorectal tumors: mutation cluster region in the APC gene. Hum Mol Genet, 1(4), 229-233.

Pray, Leslie A. (2008). DNA Replication and Causes of Mutation. Nature Education, 1(1), 214.

Protein, NCBI. (2014). adenomatous polyposis coli protein isoform a [Homo sapiens]. Available from NIH NCBI Protein Retrieved 03 January 2015 http://www.ncbi.nlm.nih.gov/protein/NP_001120983.2

 

 

For Tatyana Lobova… Cream of Tartar!

I just got home from my departmental Christmas party.  It was fun!  One of the strange questions I got was “what is Cream of Tartar and what is it for?”

Yes.  I have this in my spice cabinet.  Chemically, it  is potassium bitartrate:

Image from Wikipedia entry Potassium bitartrate
Image from Wikipedia entry Potassium bitartrate

 

 

 

 

From the chemical structure, you can see a three half OH groups (the half is the O).   The bitartrate ion shown here is therefore fairly alkaline.  When you throw this molecule into water, those 3 hydrogens pop right off (apparently), forming H+ ions.  That makes it an acid, and according to the illustrious Wikipedia entry, it creates a pH of about 3.557 in water.  Thus, tartrate and tartaric acid form a conjugate acid/base pair.

Some uses for cream of tartar in cooking:

  • Often used in meringue.  This helps to stabilize the egg whites as they foam during beating.  Note that Oregon State University has the pH of egg whites listed as about an 8, so they’re naturally alkaline.  Adding the cream of tartar shifts the pH and causes the egg proteins (albumin, mostly) to denature (solidify, in this case), so you get stiffer peaks that last longer.  You could get the same effect with vinegar or lemon juice, but that wouldn’t taste very good.
  • Also used in creating icing and smooth sugar solutions.  This chemically helps keep the sugar from forming regular crystals and solidifying.  It’s a geometry thing.
  • Ingredient in baking powder: the third ingredient of baking powder along with baking soda and corn starch.  Kind of makes you wonder why recipes call for both–usually, that has to do with pH again.  Baking soda neutralizes acids, including cream of tartar, when they’re mixed in liquid form (not as much happens in the dry, crystalline powder forms).
  • Leavening: this means it’s an ingredient that can contribute to rising/fluffiness.  Did you ever make a vinegar and baking soda volcano as a kid?  It’s the same chemistry here: acid + base = bubbles!  Those bubbles cause breads and other baked goods to rise without using yeast.  Unleavened breads usually refer to ones made without yeast, although some may still use these chemical leaveners (pita is a good example).
  • Sometimes used in whipped cream: what a waste!  Whipped cream is best enjoyed fresh.  However, it will denature milk proteins just as well as it will egg proteins.

 

Cookie Science 10: Finding the cookie difference | Student Science

https://student.societyforscience.org/blog/eureka-lab/cookie-science-10-finding-cookie
-difference?mode=blog&context=80

I really loved this post about cookie preference by @scicurious ! It does a grateful job of explaining how statistics work, evaluates preference between control and gluten-free cookies, and, well, COOKIES!