Wheels of Time

Plants, animals (including humans), and even many bacteria have internal clocks that give the organism some sense of what time of day it is.  Chronobiology is the branch of science that studies the regulation and biological significance of these cyclic phenomena.

Plants have photochemical clocks that utilize photoreactive pigments such as cryptochrome to detect the length of the nighttime, allowing them to determine what time of year it is.  They use this information to correctly time critical plant events like dormancy (summer for deserts, winter for temperate regions) and flowering.  There is no point in wasting energy on flowering if other flowers and the proper pollinators aren't available!

Human life is largely constructed around the Circadian rhythm, which matches the 24 hour cycle of day and night.  Light is the primary means by which the Circadian rhythm is kept in time with, or 'entrained', to the cycle of the Sun; other minor 'Zeitgebers' (from the German for 'time-givers') include temperature and patterns of eating and social interaction.  Artificial lighting disturbs this normal cycle, as can disorders such as Delayed Sleep Phase Syndrome and Non-24-Hour Sleep Wake Syndrome.

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When left to my own devices, I regularly go to bed between 3 and 5am.  I often find myself wide awake long after everyone else is asleep.  My peak work productivity usually falls between 10pm-3am, and I am almost completely nonfunctional before 10am.  To force myself to be awake at the 'regular' times, I pull all-nighters at a frequency of about once per week so that I am awake in the morning, become exhausted, and can fall asleep at a 'normal' hour.

For these reasons, I've often wondered if I have either DSPS or Non-24.  Many sleep disorder practitioners recommend keeping a sleep journal, but a more practical way to analyze activity patterns is to make use of activity data that you already have on-hand.  For my analysis, I graphed several months of time-stamped text message and Google search records:

Though this is a graph of received text messages, texts are usually exchanged in volleys, so this should provide some quantitative sense of when I am awake.  The text message data will tend to underestimate my probability of consciousness during the hours when no one in their right mind is awake (3-8am).

This data is much more useful, because it includes a much larger sample size and is not reliant on the participation of others.

The International Classification of Sleep Disorders diagnostic criteria for DSPS include:

1.  ...a chronic or recurrent complaint of inability to fall asleep at a desired conventional clock time together with the inability to awaken at a desired and socially acceptable time.

5.  Sleep-wake logs and/or actigraphy monitoring for at least two weeks document a consistent habitual pattern of sleep onsets, usually later than 2 a.m.

6.  Occasional noncircadian days may occur (i.e., sleep is "skipped" for an entire day and night plus some portion of the following day)

7.  The symptoms do not meet the criteria for any other sleep disorder causing inability to initiate sleep or excessive sleepiness.

Bingo.

The Brewing Continues

Both fermenters are bubbling like crazy today; I don't have a rotameter hooked up, but I would guess that carbon dioxide production is peaking in excess of 100mL per minute.  Everything in the must is now suspended, buoyed by the bubbles rising from the yeast, which are now floating in a cloudy layer near the surface.

It's obvious enough that yeast produces a ton of carbon dioxide during anaerobic growth... but how?  Molecular oxygen is not present to act as the final electron acceptor in oxidative phosphorylation, so the TCA cycle (the source of the carbon dioxide produces by you right now) certainly isn't active.  For a closer look, we go to the basic (unbalanced) bioreaction stoichiometry:

C₆H₁₂O₆ (sugar) + CH_1.9O_0.51N_0.23 (protein) -> CH1_1.77O_0.49N_0.24 (biomass) + C₂H₆O (ethanol)

Most available protein is going to biomass (which has almost the exact same stoichiometric ratios as protein), along with some of the sugar.  Most of the sugar will go toward generating reducing equivalents (NADH) and energy carriers (ATP), in which it will be almost stoichiometrically converted to ethanol.  If you take a look at that stoichiometry:

C₆H₁₂O₆ (sugar) -> C₂H₆O (ethanol)

Glucose on the left has a 1:1 C:O ratio, whereas ethanol has a 2:1 C:O ratio.  Carbon dioxide has a 1:2 C:O ratio, so this is a likely solution for closing the mass balance!  Of course, this is all nasty global inference, which should be left to systems biology (fake science).

What really happens?  Pyruvate decarboxylase (using thiamine pyrophosphate [pictured] in an acid-catalyzed reaction) hacks pyruvate into acetaldehyde and carbon dioxide.  The acetaldehyde is converted into ethoxide by reaction with NADH, and is then protonated to yield ethanol.  This is 'Ethanolic Fermentation'.

(Isn't mechanistic biochemistry so much more satisfying?)

Smell That?

The return of the earthy scents is one of my favorite parts of the transition from winter back into spring.  Of all of the flavors, the scent of rain is the most unmistakable.

One of the primary contributors to this distincitive smell is the bicyclic organic compound shown here, geosmin.  According to Wikipedia, it is produced by a number of soil bacteria such as the Actinomycetes (which also have a variety of biotechnological uses), and is also responsible for the earthy taste of beets and bottom-dwelling freshwater fish.  The human nose can detect geosmin at vanishingly low parts-per-trillion concentrations.

Ahhh... the sweet smell of microbial metabolic biochemistry.

Anatomy of an Exam

Yesterday, I took the least satisfying exam I have ever had the misfortune of encountering.  (Note that this isn't a case of sour grapes on my part; I scored fine, and my cumulative marks are safely in the 'good range'.)

The Content
Some of the questions on this 25-question, multiple-choice molecular genetics final included:

Q: "Which domain(s) recognize(s) methylated histone tails?"
a) Chromo domains    b) Bromo domains     c) Both Chromo and Bromo domains

Q: "Yeast Gal4 is one of the most well-characterized transcription factors in eukaryotes. What type of activating region does Gal4 have?"
a) Gln-rich region      b) Acidic region      c) Pro-rich

Q: "6S RNA in E. coli cells downregulates gene expression in the stationary phase.  What protein is the target for 6S RNA binding?"
a) Large ribosomal subunit    b) Sigma70 RNA polymerase holoenzyme    c) Aminoacyl-tRNA synthetase

The answers are a, a, and... it doesn't matter; the minutiae addressed in this exam are completely irrelevant to understanding the course topic, to the point of this being discernible to someone unaffiliated with the biological sciences.  The professor has wasted his one opportunity to determine whether students understood the important concepts covered in the course, in favor of testing for the ability to memorize thousands of unrelated facts.  Asking questions such as these in molecular biology is akin to testing a geography student on their ability to match countries with their capitol cities, instead of on their knowledge of, say, geography's effects on the development of civilization.  This only reinforces my suspicion that many professors do not know what the main concepts are in the courses that they are assigned to teach.  This is probably a side-effect of the extremely narrow focus of most professors' research.

The Format
Exam format is also at issue.  Four multiple choice, 25-question exams are the only method of evaluation in this 400-level junior/senior class.  While I understand that this is partially due to the course's size (120+ students), this limitation does not prevent the questions themselves from being much better than they are.  All 25 questions had only three possible answers, turning the whole affair into something of a stochastic crapshoot.  It's not like we pay a lot for this or anything.  I really hope this professor is a class-A researcher.

With such a simple format, proofreading also would have been fairly straightforward and much appreciated:
     "Answer which one determines the copy number of plasmid."
     a) an origin of replication      b) selection marker      c) multiple clorning site

It's Not That Hard
Writing decent exam questions that actually address concepts is not difficult; as an unpaid undergraduate TA last spring, I came up with:

"In the DNA, G can be converted to 8-oxo-G and 5-methyl-C to T by various biochemical mechanisms.  What is a 'failsafe glycosylase'?  How would you design a failsafe glycosylase to address the two base conversions described above?"

"Consider the structure of RNA versus that of DNA.  How do their structural differences reflect their different functions in the cell?"

"The Gibbs free energy is used to evaluate the thermodynamics of biochemical transformations in living organisms.  How is this thermodynamic potential an appropriate choice for this application?"

"The partial hydrolysis of a nucleotide triphosphate into a nucleotide monophosphate and a pyrophosphate is only moderately favorable (X kJ/mol), while the hydrolysis of pyrophosphate is more favorable (Y kJ/mol).  How does a difference of (Y-X) kJ/mol influence the equilibrium position of these reactions?  Why is this important in the process of DNA synthesis?"

The topics of these questions have a lot of overlap with the material covered in this molecular genetics course, and I think they're sufficient to illustrate the point.

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So what do you think: do I have a point here?
Why is it so hard to get your money's worth at a university these days?

The World of Isoprenes

While I was poking around the web this morning looking for some decent metabolic maps, I came upon this really excellent map of isoprenoid biosynthesis. I've seen a couple maps of similar scope, but I like how this one highlights the metabolites that people are likely to be familiar with (essential oils, vitamins) and shows how the isoprene subunit is the root of synthesis of so many disparate biological products. Isoprenes aren't just for plants - the retinal that functionalizes rhodopsin to enable us to perceive light and the cholesterol that forms the basis for the synthesis of the steroid hormones are both isoprene derivatives.

That map was put together by a real visionary, Dr. Donald Nicholson. Awardee of the ridiculously prestigious "Special Life Member" status in the IUBMB, his minimaps and animaps are changing the way people think about (and teach!) the chemistry of metabolism. His stated goal: "To Make Metabolism Meaningful, Wonderful and FUN." Bravo, sir!

For the stressed, the body-builders, the guys and gals out there who feel the effects of steroids every day: a fantastic map of steroidogenesis, courtesy Wikipedia. Bet you're glad you learned organic chemistry, right? It's never too late!

Powerpoint Magic

Powerpoint has some shortcomings, but it can be used to make some really spectacular presentations:

This was done entirely with Powerpoint; though I generated the molecular models in CN3D, the labels, the emphasis masking, everything else was Powerpoint-only (no Photoshop required!). It took me quite a while to put this slide together, but it can be done very rapidly once you know the process.

First, I pasted the molecular model image, then cropped and resized to cover the entire image. Next, I de-emphasized the area around the focus points of my image by creating a large grey oval, making it mostly transparent, and applying an edge-softening effect to the shape. The hard part was creating the focus points: Powerpoint does not have a function that allows you to 'cut holes' in shapes that you've made, so I had to devise a workaround to get the enzyme cofactors of interest to shine through the de-emphasis mask:

1. Create the 'emphasis ovals' by selecting a dashed, red outline and no fill
2. Copy the entire background molecular model image
3. Crop the background image to the area of interest, so it has exactly the same height and width as the emphasis oval
4. Select the cropped image and 'save as picture' to an easily-accessible directory
   
5. Repeat (2) and (3) for each emphasis oval
6. For each emphasis oval, switch the fill to 'picture', selecting the pictures you just cropped and saved

Next, I just added a bunch of arrows and some informative labels (again applying the 'softening' effect to the textboxes to make them blend in better) and I was done!

When you've inserted an image as a 'fill' on a shape, you can apply sorts of effects to the shape, change it to freeform, edit the defining points, and get some really neat results: link

Powerpoint is an often-abused artform... I saw some pretty horrific things on an industry internship a few years ago. It's a visual aid, so keep the text to the minimum! If your Powerpoint doesn't look like anyone else's, you're probably on the right track.

Laser Flash Photolysis

Discovering biochemistry was a big deal for me.

In highschool, I loved the systematic complexity of biology and the quantitative, mechanistic focus of chemistry. The latter won out and I headed to college in Chemical Engineering, but I made the decision near the end of my freshman year to pick up Biochemistry and Molecular Biology as a second major.

Photolyase: a light-activated enzyme that repairs UV-associated DNA damage

So many times, I came within a few minutes of dropping it. The problem: molecular biology. No offense, molecular biologists, but you ran out of fresh ideas about twenty years ago. There are a finite number of ways to do PCR, and you're really scraping the bottom of the bucket at this point (and are you still enjoying all that pipetting?). I had to wade through six credits of molecular biology of increasing irrelevance before I could break into the 400-levels, and only a mild interest in microbiology kept me at it.

Finally, BMB 401 - Introduction to Biochemistry. This is the first biology course I encountered which listed organic chemistry as a prerequisite, and it made all the difference. The chemistry, the physics, the mechanistic focus, the quantitative analysis - it was all there. The Biochemistry of Metabolism pulled me in a little further, and an excellent course in Analytical Biochemistry introduced me to the good stuff: UV-Vis, NMR, EPR, Circular Dichroism, &etc. Spectroscopy uses very basic physical interactions to 'see' the unseeable, providing an amazing look into the structure and function of the molecular machines that make biology go. Those who dislike math, physics, organic and physical chemistry need not apply.

I chose to overview an experimental technique termed "Laser Flash Photolysis" for a paper and presentation project in Graduate Chemical Kinetics. It's a pretty awesome method that's already scored two Nobel prizes in chemistry (1967 and 1999) that uses ultrafast pulses of laser light to cause and 'observe' chemical reactions on the timescales at which they actually occur (nano-, pico-, and femtoseconds).

My Laser Flash Photolysis review is available here. Definitely check out the 1967 Nobel lecture on 'Immeasurably Fast Reactions' that's referenced in the review - it's a little technical, but a good read on the initial development of the technique.

Majickal Elixirs

I don't have to go much farther than my Google Search History to find inspiration for something to document - I read Wikipedia like regular people read the Sports section. While most of my recent searches are stupid and pedestrian ("How long until doxycycline works?", I wondered at 12:42pm), there are a few good ones in here. Today's topic: 'elixir terpin hydrate', a medicine of years past.

Terpin hydrate is derived by high-temperature acid treatment of "oil of turpentine", which is composed mostly of alpha- and beta-pinene. These compounds are termed "terpenes", but they are more often known in science as "isoprenoids"; they share a common biosynthetic pathway in plants and some insects. Traditional biochemistry obsesses over lipids, carbohydrates, proteins, and amino acids... this is a very animal-centric perspective, and I think that isoprenoids are only excluded because they don't have a connection with the publicly well-known Big Three in nutrition (fat, carb, protein). They are the major component in all essential oils and most natural resins.

A medicine called "Elixer Terpin Hydrate" was available from the beginning of this century right up until the early 90's, and was a popular expectorant used to treat bronchitis. My parents remember the stuff fondly, but the FDA pulled it off the market because there was no indication that the active ingredient actually did anything. The original version was loaded with codeine and contained more alcohol than vodka... so that could explain why it was so popular.

See "Patent Medicine" for a fascinating look at the not-so-distant past of the pharmaceutical industry.

A Tale of Two Antibiotics, Part Two

Doxycycline, often marked as vibramycin, is a semisynthetic tetracycline derivative that Pfizer has been pushing since 1967. It works by a mechanism similar to that of the macrolides such as azithromycin, but it binds to a different portion of the ribosome and inhibits a different step in protein synthesis (but with the same result). Because it has been around much longer than azithromycin and it does not have features that would prevent the development of resistance (taken twice a day for ten days; half life = 18 hours), it is no longer effective on some types of bacteria. However, it is still very potent against a broad spectrum of infections, including anthrax, black plague, Legionnaires' disease, syphilis, lime disease, and malaria. Tetracyclines must be taken a minimum of one hour before or two hours after eating to be well-absorbed, are inactivated by high blood levels of calcium, magnesium, or iron, and can cause severe photosensitivity, birth defects, and hepatotoxicity.

But it works!

And hey, that's what you get for getting sick in the first place.

A Tale of Two Antibiotics, Part One

Ah, modern medicine...

Two antibiotics that I've had some recent experience with: the pharmacokinetically-curious macrolide, azithromycin, and the venerable (but still rather potent) tetracycline, doxycycline.

Azithromycin, commonly called 'Zithromax' or 'Z-Pak', is currently very popular as a broad-spectrum antibiotic that kills both Gram-positive (hard cell wall) and Gram-negative (outer membrane) bacteria. It does this by binding to the bacterial ribosome and interfering with the elongation step in protein synthesis, leading to cell degradation and death. Because bacterial and human ribosomes have slightly different structures, only bacterial cells are affected. Azithromycin is 'semisynthetic'; it is produced commercially by chemically-modifying erythromycin, another antibiotic that is produced by one species of actinomycete bacteria.

The odd thing about azithromycin: it is usually given as a single (rather gigantic) one-gram dose in powder form, but it remains effective for up to a week(!) afterwords. Like all macrolide antibiotics and many other drugs, azithromycin is involved in enterohepatic circulation: it is absorbed very readily in the small intestine and transported directly to the liver via the hepatic portal vein, where it is excreted in bile back into the duodenum (first section of the small intestine). For the chemical engineers out there, the hepatic portal-gall bladder-bile duct loop is just a recycle stream, and because it's running at a very high recycle ratio and the rate of azithromycin breakdown in the body is so low, it's easy to see how azithromycin can remain at therapeutic levels in the body for a long time on a single dose. Another form of recycle circulation further improves its half-life and effectiveness: azithromycin is preferentially taken up by white blood cells, which actively transport it to the site of infection and release it while consuming bacteria. Ion trapping and azithromycin's high lipid solubility keep its concentration at the infected site many times higher than in the blood plasma. Combined with its long half-life (68 hours) in the body and the fact that the patient doesn't have the option to discontinue the drug when they start feeling better, this helps to prevent the infecting bacteria from developing antibiotic resistance.

NEVERTHELESS, any given antibiotic will only kill certain bacteria. Azithromycin didn't work for me, so a week later I was back for a second round.