Just a Small Town Girl, Living in a Snowy World

This week, I saw snow. I know snow isn't exactly uncommon (especially on the East Coast right now), but where I live, even rain is a Facebook event. And snow? That'll blow up your feeds for the entire day. I admired the snow for a few minutes, but the novelty wore off as soon as I went outside. Snow is cold; I am not. Unfortunately for me, I had to walk in this weather from the bus stop to the house. I'm not looking forward to doing this again next week. Fortunately, I had a nice warm coat so I didn't get too cold (except for my nose). I'd like to thank the parental units (again) for getting me said coat.

Weather aside, I'd like to think that this week went pretty well (in the lab, at least), although I was a bit dubious in the beginning. I'll use this post to explain more in detail what I'm doing, so bear with me (or skip until the end if you really want. I won't know the difference).

First, I got four small vials and had to clean them. To this end, I took them to the fume hood and washed them with acetone, chloroform, ethanol, and petroleum ether (oh my). Why I was trusted with these chemicals, I'll never know. But the lab is still in one piece! For now, anyways. Then, I turned around and saw this lovely sight.

"If you kill these slices, be prepared to donate your own brain cells for today's experiments."

Ah, I feel welcomed already. On a side note, I was able to see the last frog surgery on Monday. They wanted to collect oocytes for further testing of the M2 channel. It's similar to what I'm doing, but they use mRNA to make the M2 channels, while I'm artificially making liposomes with M2 channels.

Next, I add around 150 μL of lipids, which are suspended in chloroform, to each vial. After placing parafilm (how is that stuff so stretchy?) on three of the vials, I place the other vial on the rotavapor, which spins the vial while pumping a continuous stream of nitrogen gas. Eventually, the chloloform is gone, and the lipids are stuck to the end of the vial. I repeat this with the other three vials. When I'm not using the vials, I always put parafilm over them; the oxygen in the air can react with the lipids, making them unreliable in the testing phase. It'd be rather unfortunate if I labored over these lipids and liposomes for hours, only to have the liposomes be leaky during the test (although, I'm sure this'll happen eventually).

Then, I add around 180 μL of both chloroform and wildtype M2 (meaning that the M2 channel has not mutated) to two of the vials. And then I repeat the rotavapor phase, but I sit there longer, staring off into space, because the M2 channels were suspended in methanol. Methanol takes much longer to evaporate off, although adding the chloroform makes it go a bit faster for some reason. Now that I think about it, I could probably read during this time. Or I could be efficient in making more. Decisions, decisions....

After adding about 150 μL of internal buffer to each vial, I put them on the vortexer and sonicator, and then repeat this until there aren't any more lipids on the sides of the vials. Finally, I store these mixtures in the fridge. Viola! We have liposomes. And thus, my day one of this process is complete. (I know, I know. I'm sorry, this is going to be a long post.)

On day two, my fingers get a work out. I have to heat up the internal buffer, the vials, and the extruder (this sounds kind of ominous) to 57 degrees Centigrade. However, before I heat the extruder up, I have to assemble it. To do this, I wash the pieces with methanol, then place filter supports, a 0.2μ filter, and more filter supports, into the chamber. Here's what it looks like when it's all assembled with the syringes.

It's not so scary now, is it?

The heating just makes the extruding process easier (or so I'm told). In this process, I first put some internal buffer into the syringe on the right. Then, I push that syringe in, and the liquid (somewhat magically, to me at least) goes into the syringe on the left. I repeat this ten more times, so that the liquid ends in the syringe on the left. Because of this, the filter absorbs some liquid and makes extrusion easier (easier is better, trust me).

After I remove the internal buffer solution, I put all of one vial into the right syringe. Usually, I use a vial without the wildtype M2 channels first. Next, I'll push that through the filter 21 times, so, again, the mixture ends on the left, and I'll deposit that into an Eppendorf tube (such a big name for a small vial. The picture actually shows the final products after extrusion, when the mixtures are in the Eppendorf tubes.). Since I have another vial that is also a blank (that is, it has no M2 channel), I can filter that through as well. Then, I repeat this whole heating-and-assembling-extruder process (which is harder than it seems, since the filters and filter supports are so small), just so I can extrude the mixtures that have the wildtype M2 channels. (And, let me tell you, extruding liposomes with the M2 channel is a lot more difficult.) Finally, I let the liposomes sit for about a day in room temperature.

Why do we extrude the liposomes, you might ask (well, I asked, anyways). Although we can get better signals (I'll explain later) from bigger liposomes, having liposomes completely different sizes makes the signals a bit unreliable. By extruding the liposomes, they will be roughly the same, small size, so this makes it easier to replicate the experiment. 

And all of this prepwork leads up to day three! You'd think that we'd have some really awesome experiment that takes a while to run and is extremely satisfying, but that's not exactly the case. For the test, we use the pH meters I showed you in an earlier post. These pH meters don't actually read the pH, though; they read a voltage, which can then (somehow) be converted to a pH. 

First, I add 3 mL (pretty much the only measurement that isn't in μLs) of external buffer and a stir bar (small magnets that spin based on a current going through a coil of wire) to a different, smaller vial. The actual test only lasts about 10 minutes per sample. I start by adding 30 μLs of 0.1 M HCl and 30 μLs of one sample to the external buffer (and I add 30 μLs of a drug solution to two of the mixutres), then I start the pH meter. Throughout the test, I add valinomycin, CCCP, 0.001 M HCl (twice), valinomycin, and CCCP. I'll explain what these molecules do and why we add them in a later post.

Finally (for real this time), I upload the results into a floppy disk (this was, actually, my first time using a floppy disk. Nice to know that I'm learning how to use old technology as well as new technology.), so I can transfer them to a different computer that has a magical excel spreadsheet that makes sense of all the numbers. Again, I'll explain more about this later.

Tada! I found some results. And an old computer.

Okay, that was a mouthful! It was a good review for me, too. Somehow, I managed to replicate this process mostly by myself, and my results were good! (This was, of course, after I transferred the wrong data and accidentally got a flat pH of about 3. But you live and you learn.) So now I'm trusted to run actual experiments (because this was just to see if I could do it), and I have to do it with six vials. I had four vials this time: one blank without drug, one blank with drug, one wildtype M2 without drug, and one wildtype M2 with drug. I'll need two more so I can test drugs on a mutant strain of the virus. (And, since we have colored tape, I decided to make them rainbow colored to keep track of them. Because I could.)

Let me know if you have any questions, because I sure do (but this isn't really unusual).

Until next time!

- Lauren

P.S. After I wrote this, I learned that most of my concentrations and numbers were off. Thankfully, it's not my fault, since this is what I was told. But most of my numbers will change, although the process will remain the same, so I'm not going to edit what I've already written.

All About That Place

Good news: I was officially approved by a BYU committee, so I can work in the lab now. More good news: I've taken a bus back to the house a few times, and I haven't gotten lost (yet). If you had told me last week that I'd be taking the bus back from BYU, I would've scoffed. I've never taken a bus by myself before, let alone wander a city that I've barely been in for a week. However, this week was full of new experiences, and I'd like to think that I've learned how to use public transportation (ie. bus drivers are not mind readers - you actually have to pull the yellow wire to request a stop. Who knew?)... at least a little bit. I'd like to thank the bus drivers for helping me throughout this process. Additionally, I managed to walk through buildings on a college campus without feeling like I stuck out. All in all, I'd say it's been a successful week.

On a different note, I finally started learning my way around the lab. The names of the equipment are apt; the vortexer mixes solutions in vials by creating a mini vortex, and the sonicator mixes solutions in submerged vials by sending sound waves through water (and, as the person training me said, it literally sounds like a dying bat). But don't let this simplicity fool you. Even though the name may be easy, using the equipment is not. Especially when the equipment you're using has, for all intents and purposes, a pressurized cannon connected to it. For those skeptics out there, I give you this proof:

No, it's not a death ray, although it looks like one. It's called a rotavapor.

That tank is filled with compressed nitrogen. It's chained to the wall because, if it fell over, it'd literally blow through a wall or two. Or maybe three. I'm actually going to be using this piece of equipment (yay, lucky me). The rotavapor rotates the vial while pumping in nitrogen gas, allowing the solvent to evaporate off and leaving the lipid evenly distributed. I think I'll post about my experiment process later, since it's rather lengthy. However, I will say that working in a lab requires patience. Preparing the liposomes took hours, and the actual test at the end took minutes. Most of those minutes, furthermore, were all controls, so those results didn't mean much, since they were expected.

I've enjoyed working in the lab so far. I'm a bit nervous for next week, however. My uncle says I'm going to replicate this mostly by myself, although I will receive help if I ask for it. My uncle is also the one that pushed me to take the bus. I think his plan is to make me more independent (and it might actually be working). Maybe I'll use this independence to walk over to the UVU library. Unfortunately, I don't think the library will allow me to check out any books unless I have a card - which I don't have. But my cousin has one....

Until next time!

- Lauren

First Thing's First, I'm a Chemist

I'm happy to say that I arrived in Utah in one piece! Unfortunately, my brain didn't like waking up at 4 am, and I consequently forgot to pack my laptop charger. I knew I'd forget something, and I'd like to thank my parents for shipping my charger to me.

On another note, yesterday I was introduced to the lab that I'll be working in, and,despite running on three to four hours of sleep, I managed to remember most of the equipment names. I'm particularly fascinated by the pH meters, of which there are four. Sadly, three of the meters are broken. On the other hand, three of the meters are broken. This means that I can practice using these pH meters without too much stress, especially since the bulb on the end of the meters is extremely fragile and thin (in the order of nanometers). I'm excited to begin working in a lab with actual equipment, since I didn't get a chance to do many experiments in high school.

Here's the counter-top that I'll mostly be working on, complete with the pH meters and Windows 2000.

In addition to this lab space, I'm also supposed to wear a lab coat while in the lab. Now, I look like an actual chemist, but I don't quite feel like one yet. This may be because of the paperwork that I've had to complete - I basically had to sign my life away. No big deal. In addition to the waivers, I just completed an online course in lab safety, which was helpful. I learned about the plethora of chemicals that could main and/or kill me (including a strain of influenza that was decently lethal in the early 1900s. Luckily for me, that virus isn't being used for testing at this time). I don't think I'll start working on my project until early next week because a committee at BYU needs to officially authorize me to use the equipment. 

Nevertheless, I've researched more on the mechanism of the influenza a virus and its usual vaccines. Most vaccines are manufactured months in advanced, and they target viruses with specific glycoproteins (hemagglutinin and neuraminidase). The influenza viruses are named based on these glycoproteins; for example, H1N1 and H5N1 are both influenza a  viruses, but with different humagglutinin molecules. Most vaccines are ineffective over long periods  because these glycoproteins mutate so rapidly. However, the M2 protein channels are consistent in the influenza a virus, and so a drug that blocks this channel would (hopefully) prevent all mutations of the influenza a virus from replicating.

I hope that I'll be able to work in the lab next week, so I can start making my own liposomes and testing some of the amantadine derivatives. I'll post later on the actual mechanisms (on both the influenza replication process and drug blockage) once I understand it more. For now, I'll continue waiting for approval, learning, and looking for a library (you can take me away from my books at home, but you can't take books away from me).

Until next time!
- Lauren