All The (Incredibly) Small Things

Last week felt oddly empty. Maybe it was because I wasn't in the lab, or that I wasn't constantly thinking of (hopefully) funny things to put into my blog. (Or maybe, I actually missed blogging in general. What a weird concept to think about.) Either way, my break was relaxing. Mentally, at least. It was pretty physically challenging with soccer games, so I suppose these balance each other out.

On a separate - but not any less important - topic, the decisions are coming, the decisions are coming! I'd like to thank all the "reach" schools I applied to for waiting until the last moment to release their decisions (some will be released as late as March 31st in the evening). I guess this is all a lesson in patience. 

Since not much new has happened, I've decided to go back to the basics for this blog post and describe how the influenza virus works. I don't claim to be an expert in this subject, so bear with me.

Sadly, I didn't draw this. (Surprising, right?)
 Credit to: http://www.nature.com/nbt/journal/v28/n3/fig_tab/nbt0310-239_F1.html

In replication, the virus first binds to a sialic acid-contating receptor on the host cell, which triggers the endocytosis of the virus. On a side note, do you recall that the types of the flu are labeled as HXNY, where X and Y are numbers? (If you didn't, now you do.) The "H" part of this - the hemagglutinin (HA) part - is responsible for the binding of the virus onto the host cell. There are around 18 known variations of the HA, although only a few, such as H 1, 2, and 3, are common in humans.

Once the virus is engulfed, it's inside an endosome that has a pH of about 5 or 6. Because of this low pH, the M2 channels in the virus allow protons to enter the virus, which then changes the virus's conformation, essentially activating it. This is the step that my lab is trying to prevent. Block the M2 channels, block the proton uptake - and therefore block viral replication. (In theory, anyways.)

The pH also allows the virus to release its eight single-stranded "negative-sense" RNA into the cytoplasm and, eventually, nucleus. In this stage, new HAs can be formed when two different strands of the influenza virus, such as a human and avian strand, infect the same host. The RNAs are mixed, and the viruses that are produced contain RNA from both sources. The "negative-sense" means that the RNA cannot be directly made into proteins; first, it must be translated into "positive-sense" RNA. (At least this naming makes sense.)

The host cell makes more "negative-sense" viral RNA. The virus even prevents the host cell from making its regular mRNA, so the host cell is basically converted into a virus-producing factory for the rest of its short lifespan. After enough viruses are produced, the cell breaks down. Poor cell.

Viruses are produced when they "bud" off of the host cell. In doing so, the RNA is enclosed in the host cell's membrane, along with the HAs and neuraminidases (NAs) - the "N" part of HXNY - that the host cell also produced. (You totally remembered these too, right?) After the "budding," the virus is still attached to the cell through the sialic acid. The NA cleaves the sialic acid, releasing the virus, which can then go on to infect more cells. (More viruses. Yay.)

And that's the process. Simple enough, right? If not, I'm always open for questions. In the meantime, good luck to anyone who's waiting for more college decisions!

Until next time!

- Lauren

Shake It Out

These last few weeks have been filled with learning experiences. I think one of the biggest lessons I've learned is that the bus waits for no one. Although this lesson was a hard pill to swallow, I've (mostly) accepted my fate of missing the bus. Fortunately for me, the bus comes every 15 minutes. This, however, doesn't make me feel any better when I'm about one minute from the bus stop and the bus suddenly pulls into the bus stop, waits ten seconds (if I'm lucky), then continues on its path.

Also, I've taken the lack of comments from my blog group on my last post to mean that they don't mind if my blog posts get to be a bit longer, or if I decide to start posting more often. Thanks, guys!

As for new experiences this week (since missing the bus isn't exactly new):

My cousin and I in bubble balls.

Enough said on that topic.

Earlier, I was able to watch tests on oocytes (frog eggs). Since I'm not a student at BYU, I can't really work with living cells, but I'm still able to observe (and, of course, ask questions). My trainer, Kelly, injected oocytes with the full length M2 protein, then waited a few days for the cells to express the channels. His results will be a bit more accepted than mine, mostly because oocyte tests are common, while make-your-own-liposome tests are few and far in between. Anyways, moving on. When he was ready to test his oocytes, I followed him into the lab next door and saw this.

How come their death ray machine is better than our death ray machine? We have to step up our game.

I'm glad that I was able to see this experiment, though. Apparently Kelly has been working on this setup for months, and he's only started getting decent results recently. (And, as a side note, I think that we're eventually getting one of these in our lab. Yay for two death ray machines.)

After much more discussion, we've decided to change the M2 protein and lipid concentrations - again. I hope this is the last time, but I wouldn't be surprised if it was changed again while I'm gone for two weeks. (I'm going back to Arizona for some prior commitments, but I'll come back to the lab. And yes, in case you were actually worried, I will still be making blog posts) Naturally, we didn't decide on this until after I made my liposomes, so I'll make liposomes with the correct concentrations when I return.

Everything mostly ran smoothly this week, so I can't really complain. I learned, however, that chemicals and equipment tend to run out rather quickly, especially when you're not in the lab. So I spent some time on Thursday making new CCCP and valinomycin solutions, and I'm willing to bet that those will be gone upon my return. Such is life in the lab. I also learned that some substances just don't like to be dissolved. It turns out that most of the drugs I'm testing have hydrophobic groups.

This week, I think I've almost perfect my vial drying method. Since any water droplet in a test tube or vial could dilute our solutions, we have to shake them out and let them air dry until all visible water droplets are gone. The shaking includes vigorous amounts of arm shaking, along with a bit of wrist flicking. And the best part is, I haven't accidentally let go of the vial while drying it. (Yet.) (It's amazing how much our technology our methods use. It's almost as if I'm back at school.)

Because seven out of my eight minutes of actually testing liposomes is filled by me staring at the clock, waiting for the right time, I've come up with multiple ways to keep myself entertained. Sometimes, I'll try to clean up as much as I possibly can (or add pipette tips to the box) before my minute is up and I have to add something to the liposomes. Other times, it's as if I'm playing Simon Says, except it's Computer Says. I have to make sure I follow what's on the computer screen. To make things more difficult, the computer beeps at different intervals (it was used for similar testing, but the process has changed, and no one bothered to make the beeps on time). I think I'm winning this game of Computer Says so far.

Four out of six of my liposome samples this week were great. As for the other two.... The results from the S31N without drug and S31N with drug look exceedingly similar. Which isn't really that good. The drug is either horrible at blocking this mutant M2 channel, or there's something about the higher concentration of M2 proteins and lipids that's not ideal. Either way, I'm sure I'm going to be testing this same drug when I get back, just to see what the problem is.

I'm sure I'll have plenty of time to explain what "good" and "bad" results are later, but for now, I'll explain about pH meter "noise" (I'm sure I could explain both, but this post is getting to be longer than expected. Hopefully no one minds).

With "noise."

Without "noise."

Pictures, I think, speak louder than words. And maybe you can now appreciate the small scale that we're working on. Also, the numbers on the left are not the actual pH; they're a voltage of some sorts. The pH is somewhere in the 6 to 7 range, but the change in the voltage and the change in the pH are the same.

And happy early pi day! I wish I had been born tomorrow at 9:26 am.

Until next time!
- Lauren

P. S. I think I'm going to claim next week as my "spring break" since I'll be playing soccer all of next weekend, so if I don't post, that's why (I know, I know, I can sense your disappointment).

It's a Tall World After All

I learned earlier this week that my brother, sister, and mom are getting their motorcycle licenses. I leave for three weeks, and this is what happens.... I hope this means I'll get the car though. I could live with that.

On another note, it actually snowed this week. I can't even remember the last time I woke up and the ground was white. I never thought I'd say this, but I'm glad that it's back in the 40s again. Who knew that I could actually enjoy weather below 80 degrees (what is this blasphemy?).

As for new experiences, this weekend will be interesting. I can't remember the last time I had to change my clocks for daylight savings. What kind of nonsense is this? It just seems so inconvenient to change the time and lose an hour of sleep. It's not like the sun is staying in the sky for an extra hour or anything. I'm glad Arizona doesn't follow daylight savings - but then again, we get enough sun as it is. Why would we inconvenience ourselves for "more"? (Okay, maybe I'm a bit mad that I'm losing an hour of my day.)

I think my body has an internalized alarm clock, but I'm not sure yet. I'm not used to getting up by myself in the mornings since my mom usually wakes me up. (I know, I'm 18 years old, and my mom woke me up for school. And while I'm at it, I'll say that my dad still made my lunch. Sue me.). Whenever I set my alarm, I think I subconsciously get anxious that I'll miss the alarm, so I wake up around 30 minutes before it actually goes off. Does this happen to anyone else?

If there's one lesson I learned this week, though, it's that labs are not meant for short and/or small people (even if they're meant for working with small measurements). I mean, the pipettes can measure under 5 μL of fluid. I've never seen beakers so tiny either.

Aren't they so cute? Shh, don't tell Kelly (the person training me) that I said that. He thinks it's weird that people call beakers cute.

There are even smaller flasks than that. The two pieces on the left measure 25 mL. I couldn't find any dirty 10 mL ones to borrow for a picture. At least the lab has extra small gloves (but those are still too big. There's no winning. Sigh.). You would think that with all of these small things, that maybe there's be an extra small lab coat. But I guess that's too much to ask for. Don't worry, though; they still give some consideration to short people.

See? I get my own stool. It was just kind of  lonely so I claimed it.

Okay, I think I'm done with my (not so) passive aggressive ranting. Maybe. But I have good news! All my liposomes were good this week, and I had to make six vials instead of four. I'm two for two so far. I wasn't so sure about my S31N liposomes (which have mutated M2 channels that prevent amantadine from binding) because I left the dried lipids out for a few hours. I wasn't expecting to do that, but when I couldn't find any S31N protein, I had to ask for more. Which led to the S31N samples being put into the photospectrometer. Then I had to learn how to convert the absorbance into an actual concentration. I suppose I wouldn't learn as much if everything was easy.

On this note, I have more good news (surprising, right?): we received new pH meters on Wednesday.

This is where I test the liposomes. The computer is good for something, it turns out. It tells me when to add chemicals, and then beeps at the wrong time intervals. As long as I just look at the information and ignore the beeping, I'm fine.

However, I learned that new does not necessarily mean improved. One of the pH meters was too "noisy," meaning that there was too much fluctuation in its readings. And the other meter just didn't work. So I only had one pH meter to use. To test six different vials. Twice each. And the tests last about 10 minutes every time. Yay. When I think about it, the situation is kind of funny. We get new technology, and it doesn't work, but the older technology works just fine. Oh, the irony. (Maybe I should use floppy disks and Windows 2000 more often.)

Overall, I think this week was filled with too much waiting. I had to wait for the concentrations of the M2 proteins before I could resuspend my lipids in solution. I had to wait to see which concentrations of the lipids and proteins that I should use. I had to wait for the filter supports to arrive (which they did on Wednesday). (Insert more discussions on lipid and protein concentrations here. I think we're back to using what I originally had in my last post. That's what I used anyways.) I had to wait for more CCCP because I ran out in the middle of my experiments. Thankfully, we found some in the freezer. (Not before I put everything away, though.) But, since my liposomes ended up okay, I don't think I can complain much. I just hope that I can test a drug that'll be a decent block against the mutated viral proteins.

I always feel like I have so much to say (well, to write). Maybe I should start posting twice a week.... I'm not sure my blog group would be too happy with that. Oh, well.

Until next time!
- Lauren

P.S. Success! I still managed to finish this on Friday, even if it is late. I hope I can keep my goal to post on Fridays to be somewhat consistent.