Interesting zombie blogs

There has been a lot of interest in my Zombie Protocol series so far, and for that I want to thank everyone who has wandered into the Dark Laboratory (even if you were just looking for the little girls' room).  Zombies are always hot around Comic Con, and there is always some new book or movie coming out.  It's easy to find pop culture information on the next apocalypse, but much harder to find scientific discussions about the phenomenon (hence, this blog!).

Tara Smith  had a nice piece yesterday in Science Blogs about teaching children science concepts using zombies as a backdrop (here's the link).  Disease transmission, pandemics, zombie bugs, neurobiology, and other biological topics were used to discuss what strategies the kids would use for different types of zombie outbreaks.  They also used watermelons as, um, volunteers, to compare the effectiveness of various weapons.  Where were these people when I was in school!!

Coolest logo EVER!

Anyhow, I'm also starting to include links on the right to various sources of zombie science.  Scott Kenemore's blog is doubly good as it also discusses new zombie books and movies.  He's also on the advisory board of the Zombie Research Society, which looks a little tongue-in-cheek but has some heavy hitter scientists on their Board. Coincidentally, Tara Smith is also on the Advisory Board.   Both sites have an occasional discussion about scientific literature and are worth a read.  If you know of other sites that have a scientific bent to zombie research, or any other topic that might be of interest to the Lab, please leave a note below.  Spam will, of course, be fed to the zombies...

The Zombie Protocol, Part 2

Bufotenine: Zombie Poison

With 'bath salts' in the news these days, there is suddenly a lot of interest in zombification.  If you recall, in part 1 of the Zombie Protocol, I described the role of tetrodotoxin in the creation of Haitian zombies.  There are, however, other ingredients in the zombie poison that have significant pharmacological activity.  Besides related TTX toxins from other species of fish, there can also be poisons from frogs, reptiles, and spiders.  The poison from the frog, Bufo marinus, is of particular interest as it contains bufotenine (5-hydroxy-N,N-dimethyltriptamine, or 5-OH-DMT).  Bufotenine is an alkaloid related to DMT and 5-MeO-DMT, as well as psilocin (one of the psychedelic molecules found in mushrooms).  It is also related to serotonin, a well-known neurotransmitter.

Serotonin: Important neurotransmitter

Therefore, it seems very plausible that bufotenine is present in the zombie powder to enhance the effect of the tetrodotoxin.  Remember from my last post that TTX is present at sub-lethal doses (in some studies, the amount of TTX is very low -- see Benedek and Rivier, Toxicon 27:473-480, 1989), so other components of the powder could be necessary to boost the effect of the mixture without killing the victim.  Psychoactive substances would also be very effective since the victim is likely to be conscious while under the influence of TTX.  I mentioned the Poe-like experience of being buried alive without any way to move or scream... now imagine that happening while having horrible hallucinations!  You talk about a bad trip!!

Seed pods from Mucuna pruriens (Wikapedia)

Plants known to be irritants were also added to some of the zombie powders.  In Wade's paper (abstract), he lists Urera baccifera, Dalechampia scandens, and Mucuna pruriens as some of the more common plants.  These plants all have irritating hairs or needles that cause contact dermatitis. In the case of M. pruriens, the hairs on the seed pods contain (interestingly enough) serotonin.  Why would irritants be important to a zombie poison?  Personally, I think this is one of the ingenious parts of the protocol... the irritants are there for delivery.  To pull off the zombification, a voodoo priest would have to administer this part of the poison without the victim's knowledge.  Poisoning food or using darts are quite tricky, but what about a chance encounter on the busy streets of Port-au-Prince?  You have the drug cocktail smeared on the end of a walking stick, or some other convenient device.  Following your target through the busy streets, you wait until you have a good opportunity.  He's distracted by a street vendor, so you bring the stick high and as you pass behind him, you gently poke him in the back of the neck.  He may not even notice but after you have left, he feels a burning sensation and scratches it.  The itching and burning intensifies until he scratches it raw.  The drugs are now able to enter the blood and begin the process of poisoning the victim.  He won't remember the chance encounter, but you know he will be at the hospital soon, and at the morgue not too long after that.

I know I promised zombie cucumbers in Part 2... but they will show up in Part 3, I promise.

T. gondii in the news again

     It's been bat shit crazy in the Not-So-Dark Laboratory (otherwise known as my day job), so my apologies for getting behind on the real crazy science.  One of my first posts (zombie science) talked about zombie science and the potential role of Toxoplasma gondii. T. gondii is a neurotropic protozoan parasite that has been linked to a variety of mental disorders. When rats are infected, they lose many of the behavioral adaptations that protect them from cats and aggressively try to get themselves eaten.  Sadly, the effect of T. gondii on humans seems more subtle. But there is a new paper out on the relationship between T. gondii and suicide, which provides stronger evidence that this parasite is a potentially serious global problem.

     Several recent studies have shown that T. gondii affects human behavior. A meta analysis published in 2007 (abstract) found that there is a correlation between schizophrenia and serum antibody levels against T. gondii.  The predictive power of the association was weak, since more that one third of the entire population is thought to be seropositive for T. gondii, and aside from a few ex-girlfriends and that driver on the 15 the other day, not all of them are crazy. One flaw with the meta-analysis is that it was not clear when the patients were infected relative to the onset of disease.  If they all got T. gondii after they became schizophrenic, then the association is meaningless.  The new study just published by Pedersen et al. (abstract) tries to account for that by measuring T. gondii antibody levels when women gave birth (using samples from heel-stick cards in the birth records) and then looking at the risk of depression and suicide later in their life. Seropositivity was 26.8% at delivery, consistent with the notion that 1/3 of the population has already been infected (remember that infants don't start making their own antibodies for about three months after birth, so these are the mom's antibodies).

From Pedersen et al, Arch. Gen. Psych. 2012.

Pedersen et al. found that seropositive women had a 1.53-fold greater risk of self-directed violence (ie, suicide) than seronegative.  Women with the highest antibody titers had nearly a 2-fold higher risk.  The risk is small, but significant and is also consistent with other recent studies on the relationship between T. gondii antibody titers and mental illness (for example, see this).  I'm generally not a big fan of these types of analyses, because the data could simply be a case of "true, true, and unrelated".  There is no mechanistic hypothesis for why antibodies against T. gondii would alter behavior, or whether the infection caused permanent damage to the brain.  However, evidence continues to accumulate that T. gondii infection can cause permanent changes in human behavior, and with billions of people having been infected at some point, it isn't too hard to see the beginnings of a zombie apocalypse.

The Zombie Protocol, Part 1

     As I mentioned in my previous post, some reports of zombies are more likely observations of people suffering from mental illness, long-term drug abuse, or both.  Given the loose definition of a zombie as somebody "acting strange" who had been missing and presumed dead, one wonders how many urban dwellers and street people in this country might be considered zombies.  I was at a conference in Barbados a few years back and walked past a homeless guy on Broad St. in Bridgetown.  He rose out of a pile of belongings to approach me, his legs stiff and his arms trembling. He was a pitiful sight, and communicated using grunts and various hand gestures.  He wound up following me for a block and even after I crossed the street and doubled back, he was still behind me.  I told everyone back home that I had seen a zombie, no doubt fulfilling my obligation to keep these legends alive.

  But what about "real" zombies? Is there a way to bring someone back from the dead?  Does the Zombie Protocol exist?  Without a doubt, the answer to this is 'yes'.  Although the legend of the zombie has existed in Haiti for generations, there is enough physical evidence to suggest that the process of zombification is likely rooted in scientific fact.  Please ignore the Hollywood ideas of brain-sucking zombies, and think instead about a circus act.  How would somebody make a zombie without learning all that dark magic (which, by all accounts, is very difficult)?  You fake it, but you fake it well.

Tetrodotoxin structure: Note the guanidido group (NH2+)
 group on the left.

In Wade Davis' excellent article The Ethnobiology of the Haitian Zombi (abstract), he lists the composition of zombie poisons from five separate locations around Haiti.  Although there were many differences, based on what was available at the different geographical locations, there were several key ingredients.  One of the most important was the puffer fish (Diodon hystrix, Diodon holacanthus, and others).  Puffer fish in the region have very high levels of tetrodotoxin (TTX), which is, of course, the same toxin found in Japanese fugu fish.  Tetrodotoxins are extremely toxic molecules and act by blocking Na ion channels, presumably through the positively charged guanidido group, which competes for the Na binding site in the channel.  Without Na exchange, nerve impulses do not propagate which leads to paralysis, as well as cardiac and respiratory failure. The LD50 of TTX is estimated to be about 5 ug/kg in humans (less than1 mg of TTX), so what if a person ingested something a little less than that?  Respiratory function decreases, blood pressure drops to near zero, pupils are fixed and dilated, the body is paralyzed, but maybe, just maybe, they won't actually die.  This is the first trick of the Voodoo priests, namely, to make the family think the victim is dead by titrating the amount of puffer fish in the poison to near the LD50.
     What is interesting, and also horrifying, is that TTX does not usually affect cognitive function.  This means that the victim falls ill and becomes "dead" while remaining conscious.  They hear the doctors discussing the death, hear the family wailing in grief, hear the preparations for burial, and then find themselves in a box, quiet and still.  Imagine the terror of being buried alive but having no way to move, scream, or otherwise respond to the panic.  The fear is completely trapped in the victim's mind..  Poe couldn't have scripted that much better...

Next up in the Zombie Protocol, toad toxins and zombie cucumbers...

Two articles on zombification

While I was doing research for my first book, I read a lot of articles on zombies.  Not the brain-eating type that are often found in movies, but real reports of zombies.  It was sometimes difficult to differentiate a true case from an urban legend ("I swear I saw a zombie walk right into that bar!") or a case of mistaken identity ("I thought it was a zombie, but it turned out to be just my mother-in-law...").  However, I ran across a very interesting article published in 1997 in the Lancet, a highly respected medical journal, on clinical findings from several reported zombies (here's the reference... a search will turn up the pdf elsewhere).  The paper, published by Roland Littlewood, from University College, London and Chavannes Douyon, a Haitian doctor, makes a fairly shocking claim. Although the process of zombification has been attributed to both poisoning or sorcery, the empirical data from Haiti suggest that people demonstrating behaviors consistent with a zombie are on the order of a thousand cases per year.  This is truly staggering, if accurate.

The Lancet article studies three reported cases of zombies during 1996-1997.  Patients FI and WD, although described by relatives as becoming ill and dying prior to their return months or even years later, were probably not dead to begin with.  Both patients suffered from mental illness and it seems more likely that they had simply wandered away from their homes and were taken in by other families.  For FI, the Lancet study concluded that she suffered from catatonic schizophrenia, a syndrome that could be considered zombie-like by locals.  WD likely suffered from organic brain syndrome and epilepsy.  The Lancet article speculated that these conditions were due to an unspecified period of anoxia.  Perhaps WD had been buried alive?  Unlike FI's coffin, which was filled with stones, WD's family refused to open the grave to Littlewood and Douyon.

Patient MM was a 31-year old female who had disappeared at age 18 after attending a service for a friend who had been zombified.  She fell ill shortly after and died.  Thirteen years later, she reappeared in the town and claimed to have been a zombie slave at a distant farm.  Littlewood and Douyon found her to be of very low intelligence but were unable to diagnose a more specific mental illness.  Upon return to the town near her captivity, locals recognized her as a zombie and several people argued over their claim to her.  Interestingly, she had a round, 1cm scar on her back, similar to a scar found on patient WD.  What were these scars from?

Unfortunately, they ultimately state that none of the three were true zombies, and that much of their behavior could be explained by mental illness.  However, one of the references they cite is the now classic paper by Wade Davis, published in the Journal of Ethnopharmacology in 1983, titled "The ethnobiology of the Haitian zombi" (abstract).  Is that a cool title or what! In this little gem, he provides a pretty detailed 'zombie protocol' and this forms the basis for Davis' book The Serpent and the Rainbow.  In my next post, I'll discuss his findings and several other classic zombie manuscripts...

Zombie cockroaches

Just when you thought the zombie apocalypse couldn't get any worse... now we have roaches to worry about!  Fortunately, zombie roaches in the wild are not aggressive, brain-sucking insects. It turns out that the wasps that make the zombies are what we should really be concerned about.

Jewel Wasp (A. compressa)
Image courtesy of Morphbank.net

The Jewel Wasp (Ampulex compressa) has an elegant and extremely sophisticated method to secure a good home for its eggs.  It attacks and injects a cocktail of neurotransmitters into the brain of a roach, effectively turning it into the slave of the wasp (Educated Earth has a good video of this process, see here).  The wasp then drags the roach to a suitable location and lays its eggs in the abdomen. The roach is hypokinetic for several days until the larvae hatch and then they slowly consume the host.  Interestingly, the larvae eat the internal organs in a way that maximizes the lifespan of the roach.  It's a morbid process, but how the wasp achieves this zombification is truly incredible.

The wasp actually stings the roach twice.  The first sting is to the thorax and produces a mild and very transient anesthetic that paralyzes the front legs.  A study by Moore et al. (abstract) showed that the active components of the first sting activate GABA receptors (GABA itself, and receptor agonists beta-alanine and taurine).  During the minute or so of action, the wasp then stings the roach again, but in a very precise location within the brain.  In a sense, the wasp is acting like a brain surgeon, who needs their patient to remain still while a precise operation is done.  This second injection is a cocktail of neuroactive compounds that have very specific effects on the roach's brain.  After the anesthetic wears off, the roach grooms itself excessively and then becomes lethargic.  It is not paralyzed, just sluggish.  When the first studies on A. compressa were done, there was a great deal of controversy over whether the venom was delivered to the central nervous system, or directly to the site of action in the brain.  In an elegant study by Haspel et al. (abstract), they milked the venom from wasps and injected them with radiolabeled carbon (14C).  This meant that the fresh venom produced by the wasp would contain the radiolabel.  Haspel could then visualize the location of the venom in the roach brain by taking pictures of brain slices.  Those of you who think that would be cool as hell, raise your hand!!

  Anyhow, it was found that the neurotoxin attacks the brain directly, with the most venom at the supra-esophageal ganglia (SupEG) and the sub-esophageal ganglia (SEG).  In insects, these areas are thought to provide "higher order" brain function.  A recent study by Gal et al.(abstract) found that the venom affects the drive to initiate and maintain walking.  Other motor skills (like flying, swimming, self-righting) were not affected.  Based on the electrochemical difference between stung and unstung wasps, they concluded that stung wasps had a deficit in the ability to "reach a decision" to walk.  There must be human versions of this wasp because I swear, there are days when I have trouble reaching the decision to get off the couch.

A follow-up study by Gal et al in 2010 (PLoS article) made another very interesting discovery.  Roaches who had their brain ganglia removed were stung for a much longer period of time (196 sec) than normal roaches (39 sec).  Why?  Because the wasp was actively seeking this part of the brain and couldn't find it!  What does it search for?  A specific morphology?  A defined depth within the brain?  Nobody knows.  Furthermore, why is it so important for the wasp to target this region so specifically?  The answer is also still open to speculation but this has zombie written all over it.  The wasp needs the roach to be compliant to move it into the proper position for incubation of the larvae, but resist the urge to flee for several days.  So, the venom basically takes away whatever "freewill" the roach might have had.

Octopamine
 (image courtesy of Wikidocs)

So, what is this supernatural cocktail?  Not a whole lot is known about the composition of the venom (another great project for biohackers or kids looking for obscure science fair projects). I will post more details later but one key component appears to be an antagonist of the octopamine receptor.  Octopamine is a monoamine neurotransmitter that is specific to invertebrates and OA levels are known to be associated with the hypokinetic state.  Several years ago, Rosenberg et al. (abstract) gave roaches a octopamine receptor agonist and found that motor activity in stung subjects was at least partially restored.  Other receptor agonists and antagonists had little effect.  This is certainly one clue about how A. compressa makes zombie cockroaches but for now, the rest of the process is all voodoo.

Resveratrol: One step forward, two steps back

Resveratrol is in the news again but this time for all the wrong reasons.  No, it didn't just break up with a pop star or get busted for breaking probation.  Stunning allegations from the University of Connecticut suggest that a significant percentage of Dipak Das' (UConn professor and Director of the Cardiovascular Research Center) scientific research on RES may suffer from scientific fraud (see here for just one article).  I looked at some of the (60,000 page!) report and it looks like much of the fraud was based on images of Western blots that had been altered or fabricated.  I have long complained about figures of Western blots in various publications where only the bands of interest are shown with no molecular weight markers or anything.  This is like buying a car based on a picture you see on the internet.  Yes, there is a band there but you have no idea how good the antibody is that you are using to probe with, if the protein runs at the right place on the gel, etc.  In Das' case, it looks like random bands were just pasted on there.  No bueno, pal, no bueno.

Does this mean that resveratrol is now demoted to a worthless contamination in an otherwise tasty glass of wine?  Um, no.  I'll admit I have read some of Das' stuff and it has influenced some of my opinions about RES, but there are a whole host of researchers out there that have demonstrated how RES impacts biological pathways and (in my opinion) there is very clear evidence that it has a significant effect if the dose is high enough.  Unfortunately, cases like this place a stigma on research involving RES and could hinder progress towards understanding the physiological benefit of this molecule.

Ok, so now on to better news.  My favorite wine goddess maker, Kerith Overstreet from Bruliam Wines, has a new blog post on the cardioprotective properties of wine. It's pretty funny (you can check it out here) but in it she highlights not RES, but oligomeric proanthocyanidins (OPCs).  If you recall an early post I made on the magic of winemaking (here), you may recognize OPC as another term for polymerized flavinoids, which include tannins such as catechin.  Most OPCs originate from the grape skin, so the amount of OPC in any given bottle can vary dramatically.  The final levels depend, in part, on how long the grape skins are left in the fermentation since it is the rising alcohol content that ultimately extracts the monomeric proanthocyanidins from the grape.  Therefore, craft is a big variable in determining the benefit of wine to the drinker (we are all counting on you, Kerith!) Interestingly, Das was involved in a company called Dry Creek Nutrition, that was trying to purify and sell proanthocyanidins.  In light of the Das debacle, maybe OPCs are the new RES!

Discovery of an upside down, carnivorous plant in Brazil

I've always been a fan of carnivorous plants.  Pitcher plants, Venus flytraps... very cool.  An article just published in PNAS describes a very bizarre plant that has been recently characterized as carnivorous.  The flowering plant, from the genus Philcoxia, is a pathetic specimen.  Found in dry savannas of the cerrado in central Brazil, the stems are leafless, which means it resembles many of my potted plants here at the house.

It turns out that the leaves of Philcoxia are actually underground! Most plants would find this adaptation ridiculous.  The primary function of a leaf is to capture sunlight and produce energy through photosynthesis.  What kind of freakish plant would put the leaf underground where there is no light?  A murderous plant, of course.

What Pereira et al. show in the PNAS paper (abstract) is that the leaves of Philcoxia have evolved to trap and eat nematodes in the soil.  The data to support this comes from an elegant study using nematodes that had been fed nitrogen-15 (15N), an isotope of natural nitrogen.  Releasing the nematodes in the vicinity of the plant, they measured the change in 15N in the leaves after two days.  They show that the absorbed 15N increases from nearly undetectable levels before the experiment to about 15% of all nitrogen content by Day 2.  They talk in the methods about how they extensively washed and dried the leaves to remove all traces of 'nematode remains', but the greatest risk in this experiment is that the 15N they are measuring is simply from the residue of dead worms on the leaves.  Nevertheless, they also show that the leaves are covered  with a sticky sap and that enzymes on the surface are available to break down the corpse for consumption, as found in other carnivorous plants. Interestingly, the glands that produce the sap are also similar to those found on other carnivorous plants.  So, it certainly seems plausible that another plant has found its way to the dark side.  Better call NCIS (that's Nematode Criminal Investigative Service).

Maybe I'll just start telling people that the dead plants I have around my house are rare Philcoxia from central Brazil and that they are supposed to look dead.  Maybe I'll tell them that I have re-engineered them to eat, um, larger prey. I wonder if carnivory could be engineered into grass?  That would keep the neighbor's dog off of the yard!! Ahh, the possibilities...

Bioluminescent Light Bulbs?

There is a news blurb going around about using flasks of bioluminscent bacteria to light your house.  I mentioned this type of thing in the Foxfire post, but Philips has been working on this for some time.  The lighting system consists of a wall of hand-blown glass flasks, coupled to methane lines that are sourced locally (ie, from the users trash).  The bacteria have been engineered to glow when methane is present, so users can control the lighting.  Although this is impractical for general use at the moment, it is probably the beginning of the next revolution in lighting. Some of the articles talk about the most likely first application, which would be for safety lighting near buildings or on roadways.  I think it would be cool to have trees or shrubs engineered to glow as well... it would be awesome to have glowing bushes near our front walkway!  If you want to see how the bioluminscent bacteria fit into the Microbial Home, see the link at Philips' web site ( link), it's pretty trippy stuff.  If you want to try your hand at growing bioluminescent algae at home (or for a cool science fair project!), here is a good starting place (link).  If you want to see what a team from Cambridge University did for their iGEM project last year (hint: it's on making bioluminscent products) check out this link.

  Philips is not the only company looking at commercializing bioluminescence for non-medical applications.  A company called BioLume (link), based in Research Triangle Park, NC is trying to put bioluminescent proteins in food.  Yeah, that's right.  Glowing food.  They use examples like candy and alcohol as likely products, as well as makeup.  Sounds like a Rave Gone Wild! The company has IP around many different bioluminescent proteins (mostly luciferases) found in marine life.  I imagine that they formulate it in a way that the enzyme becomes active when there is a change in the environment.  They mention a calcium-induced reaction of a enzyme-substrate fusion, as well as fusions with fluorescent proteins.  I'm sure that the proprietary chemistry and photophysics involved in these products is really cool!  I do hope the metabolized product is non bioluminscent... there is nothing more scary that glowing pee!

Red wine in a pill: Metabolic effects of resveratrol in humans

Remember back in July I talked about a future where simply popping a couple of resveratrol tablets gave the same benefit as a walk around the block?  That future may be closer than we think!  A recent paper in the journal Cell Metabolism describes the results of a very small study of resveratrol in humans and the data is pretty exciting.  Let me repeat that caveat... this was a small study!  Still, the metabolic effects described in this work clearly emphasize the need for further research on this and other magical polyphenols.

The paper published by Timmers et al. (abstract) is the first to study the metabolic effects of resveratrol (RES) in a clinical setting.  Eleven obese men were given either RES (at 150mg/day) or placebo for 4weeks, followed by a 4-week washout and then the treatment was switched (this is known as a crossover study).  Patients and doctors were both blind as to what treatment was being administered and during treatment, a variety of metabolic tests were conducted.  There is a lot of data in the paper (and it looks like the pdf is free, so check it out yourself!) but let me hit a few highlights:

1) Patients taking RES show an increase in mitrochondrial efficiency, particularly in fatty acid oxidation of muscle fats, and decreased levels of triglycerides and glucose levels.  Significantly, these changes are seen at the gene level, suggesting that it is the overall metabolic pathway that is improved, not just a downstream clearance of metabolic markers.  A similar pattern of changes has been noted in athletes undergoing endurance training (they reference Dube et al, 2008 and Meex et al, 2010).  This is fairly consistent with the Momken paper I blogged about back in July, ie, RES acts like an exercise mimetic.

2) Changes in glucose and insulin levels are modest.  Timmers et al. report a statistically significant drop in serum glucose and insulin levels in the patients taking RES, but this effect is pretty modest.  There is also a shift in peak glucose and insulin levels after a liquid test meal, suggesting some changes in glucose homeostasis, but they could not draw definitive conclusions.  If you recall, the rat data from Momken et al. was also pretty weak with regard to insulin/glucose levels.

3) RES also showed other health benefits:  This study also demonstrated a significant effect of RES on lowering systolic blood pressure and mean arterial blood pressure, as well as decreases in resting energy expenditure and sleeping metabolic rate.  The later two effects are also seen in studies of calorie restriction and further illustrate the metabolic changes induced by RES.  Calorie restriction is also associated with increased lifespan in animals, so these observations may be pointing to another possible health benefit of RES.  They also observed a decrease in markers of inflammation, further suggesting an overall improvement in health. Although this is interesting, I still think the catechins are the more potent player here... I'd like to think that as the flavinoids polymerize during aging, the wine gets better and better for your heart. It would be interesting to see how some of these molecules perform in a study similar to this.

Taken together, this paper highlights some of the metabolic effects of RES in humans and may offer some insight into the health benefits of this polyphenol.  Much like the rat study, however, this is a very high dose (the equivalent of >100 glasses of wine per day) and so who knows if there are long term side effects at this dose.  The fact that they see statistical significance with only eleven patients is also very surprising.  Clinical studies usually need hundreds, or even thousands of patients to provide enough statistical power to draw conclusions like this.  Personally, I would find taking a pill much less satisfying that enjoying a nice glass of Cabernet. Since that glass of Cab is a veritable grab bag of Redox goodies, I think it is also very likely that there are many other 'good' polyphenols in wine that scientists haven't studied as rigorously as RES.  So as dozens of trick-or-treaters descended upon our neighborhood on Halloween night, I had to raise my glass to our ancient ancestors who discovered the wonderful winemaking process, and the scientists who now try and tease apart how it does what it does. I may have also stolen a chocolate or two... in the interests of science, you know.

Halloween winemaking magic at Bruliam Wines

Just in time for Halloween, Kerith Overstreet at Bruliam Wines has a great blog post this week about working with her spooky 2011 harvest (beating it into submission, actually).  Sounds like a challenging year!  She describes the redox chemistry that goes on during the early fermentation process and actually has a graph from her lab!  I first blogged about the magic of winemaking after Kerith's great talk on the subject (here is my post) and even she refers to the process as magic in her latest post (but she also refers to wife swapping, Alanis Morissette, and Hanukkah miracles, so who knows what state of mind she was in as she wrote this).  Anyhow, a very fun, informative read directly from the mysterious front lines of winemaking.  Enjoy it with a glass of good Cabernet, the official wine of the dead.

Keriths latest blog post:  (link)

21st Century mummy

Just a quick pointer to a cool article on a recent attempt at mummification (here's the link). Stephen Buckley, a chemist at York Univeristy in England, has spent two decades studying how ancient Egyptians made mummies.  He studied tissue samples and chemical traces left on canopic jars in an effort to reproduce the method.  He then tested the process in his shed, using pig's legs as a proxy for human flesh (there's a DIYbio project for you!).  I'm not sure if this guy is married, but even here in the Dark Lab, this work would be pushing the limits.  Anyhow, this year he felt that he was ready for prime time.  He placed an ad looking for suitable volunteers and the lucky person was... Alan Billis, a London cab driver.  Terminally ill with lung cancer, he went through the mummification process after he died. By all accounts, it was a success and the body will be kept for at least a year to study.  Hopefully, Alan is hanging out with a bunch of cool, Egyptian princesses.  Thousands of years from now, archaeologists will argue over whether our society placed a high value on cab drivers, or if Mr. Billis was simply a member of the ruling elite.  They will come up with grand theories on how he lived, how he died, and why he was the only surviving mummy of the period.  Should make for an interesting read.



Cat mummy at the British Museum (link)



To me, it is truly amazing that with all of today's technology, it is difficult to reproduce the mummification procedure.  The Egyptians likely had years of empirical data to build from and since it was considered a sacred ritual for the upper class, there was significant motivation for young priests to be good at making a mummy.  I can see a room full of young mummification interns, trying to preserve rats or some other suitable test animal.  After months of work, the mentor unwraps the package, only to find a rancid, decomposing corpse. "Aw, man," the student whines (or whatever the angsty teen expression was at that time).  He then slouches his way over to the stone quarry.

A gene important for creating zombie caterpillars

Advancements in zombie science are coming fast and furious!  An email from a colleague (and fellow reader) noted that I missed a recent article in the journal Science concerning zombies.  The article, titled "A Gene for an Extended Phenotype", seemed pretty innocuous, however, after going back and reading it more carefully it turned out to be a pretty cool discovery.

First, a bit about zombie caterpillars.  There have been several documented reports of zombie-like behavior in moth larvae.  This report is focused on the infection of the gypsy moth (Lymantria dispar) by a baculovirus (known as LdMNPV).  During the various stages of molting, larvae typically hang out on the ground and away from The Very Hungry Birdie, but climb up into the trees at night to feed on leaves.  After infection by the baculovirus, their behavior changes (noticing a pattern here?).  As the virus replicates and ravages the inside of the caterpillar, the infected host climbs up into the leaves during the daylight hours and eventually dies.  The body then liquefies, and virus-laden particles rain down on the uninfected victims below.  Yeah, you can't script horror much better than that.

Ok, so Hoover et al. (from Penn State, see abstract) were interested in identifying which genes were important for the change in behavior.  To do this, they infected caterpillars with wild type baculovirus, as well as virus that had been genetically engineered to be missing certain genes.  The caterpillars were placed in 1 liter soda bottles equipped with a fiberglass screen for climbing (in true DIYbio fashion!!).  Interestingly, when a gene called egt was removed, the caterpillars died at ground level, suggesting that the behavioral control of the virus had been altered.  To make sure it wasn't an artifact of the mutated virus, they re-engineered the mutated constructs so that the egt gene was present again and the zombie caterpillars climbed up the mesh and died.  It would appear that the egt gene in the virus has evolved to make the caterpillars engage in high-risk behavior, and to place the caterpillars in a location where rain/gravity/hungry birdies can maximize viral spread. Genius.

The next step is to figure out the mechanism.  Interestingly, Hoover et al. mention that the egt gene encodes an enzyme which deactivates a hormone (20-hydroxyecdysone) involved in the process of molting.  It is intriguing that the virus blocks the molting process in order to give itself time to replicate inside the host, but it was hard for me to understand how the modification of the hormone leads to behavioral changes.  Perhaps entomologists already know that part.  Coincidentally, the hormone is also reported to have a variety of biological effects in humans, even though we do not molt and lack the endogenous receptor. What would the modified enzyme do in a human?  Until we find this stuff out, it's probably a good idea to check the trees around your house, particularly if you hear a dripping sound... and if your neighbor has been missing awhile.

Yes, there are zombies all around us.

Biopunks help solve structure of key viral enzyme

I've been meaning to blog about the program FoldIt for some time.  It's a program out of David Baker's lab (link to University of Washington) where users can manipulate protein structures to improve folding but in a unique twist, the improvement is 'scored', just like a game.  A problem is posed on the website and thousands of players compete to see who can find the best solution. They also have regular competitions to see how well players can predict the structure of a protein from just the amino acid sequence (the Holy Grail of the protein folding world).  I've played on and off for a couple of years and it's pretty fun... but it's a lot like my day job, so if I take the time to play a video game it is usually something like Rock Band.  Still, FoldIt is a pretty easy game to play (the number of rules is limited and the GUI is very intuitive) so the real challenge is in the player's ability to use logic and their skill in 3D visualization. I'll have a more detailed post about it and its less-familiar cousin EteRNA (the RNA equivalent of FoldIt) later. When you are ready to play, click here (it's free, easy, and fun... really!)

This week in the journal Nature Structural and Molecular Biology (I have had several rejection letters from this fine journal) is an article (abstract) describing the use of FoldIt to solve a scientific problem at least ten years old!  Crystallographers have been trying to determine the structure of the monomeric form of MPV protease. MPV stands for Mason Pfizer Virus, a virus that causes an AIDS-like syndrome in monkeys.  Although it is not HIV per se, the protease is a key enzyme in the production of a mature virus and so developing drugs using this structure could be useful in developing an HIV therapy.  This protein was one of those rare cases where good crystals could be grown but interpreting the x-ray data back into the final 3D picture of the protein could not be done. So, researchers looked to crowd-sourcing as a means to solve this problem.  For three weeks, FoldIt players had the chance to optimize a 3D model, based on an NMR structure of the monomer.  Teams competed to see who could come up with the best solution (based on how well the different structural pieces of the protein fit together).  After all that tweaking and optimizing, more than a million different models were created.  Crystallographers used the best of these models as a starting point and one of them successfully generated a solution to the x-ray data using molecular replacement.

Who were the people who provided the key to success?  The top groups were listed as co-authors by their team name (FoldIt Contenders Group and FoldIt Void Crushers Group) and by a few handles listed in the article (spvincent, grabhorn, mimi) so not much is known about these folks, but I'll bet many of them had no knowledge of crystallography or biochemistry and probably had no clue what this enzyme did.  However, the fact that thousands of players worked together on this problem and were ultimately successful highlights the power of crowd-sourcing and that top-flight science can be accomplished through a "game".  I think this paper will also motivate others, both players and scientists, to leverage the power of biopunks!

How about a GFP cat to go with your GFP beagle?



The GFP cat:
From Figure 2 of Wongsrikeao et al.



Well, that didn't take long.  I blogged last month about a paper describing GFP beagles that were able to glow under the control of a tetracycline promotor.  Now, a new Nature Methods paper describes a GFP cat!  The basic point of the paper (here's the abstract) was to demonstrate gamate-targeted transgenesis in cats and to use this ability to make a transgenic feline model for HIV research.  The HIV part was interesting, as they introduced the gene for a protein from the rhesus macaque known as TRIM5.  I studied TRIM5 pretty extensively when I was doing HIV research as it is a species-specific restriction factor that is effective at stopping HIV replication.  Cats don't have an analogous TRIM protein (that we know of) so by introducing this protein into the cat, researchers can study the transmission of FIV (the cat version of HIV).

The cool part was the GFP expression.  Wongsrikeao et al. wanted to see if they could introduce multiple genes, and since GFP is a convenient marker, they could also study the presence of the transgenes in progeny cats.  As I mentioned in the Beagle post, fluorescent proteins have been introduced in animals previously (see here and here for cats) but the efficiency in the present work is better.  I'm certainly no expert on transgenics, but the general trend over the last few years is clear... we are moving from the realm of the nearly impossible to the land of the challenging but doable.  Glowing cats, dogs, hamsters, bunnies, you name it are going to be popping up in your pet store relatively soon.  Lost your dog?  No problem... just look for the glowing blob in the woods.  How about a government program to develop a GFP opossum?  Think of how much road kill would be eliminated if you could see these animals before they got up close and personal with your tire.  Think about how cool the forest would be if all of the little woodland creatures glowed bright green. Think about how easy it would be to hunt.  I wonder how screwed up the food chain would get. I guess we would have to make GFP plants for the little GFP bunnies to hide in. Man, this would make a really cool dystopian/biopunk story!

Can gut bacteria make zombies?



L. rhamnosus: Not a zombie-
producing bacteria...yet.



If T. gondii weren't enough to worry about, now there is evidence that bacteria in our gut can influence brain function.  Lactobacillus and other probiotic organisms have long been speculated to have beneficial in vivo effects, and are most commonly known for improving the health of the digestive tract.  L. acidophilus is probably the most widely known, since it is used to make yogurt, but there are many other types of lactobacilli with alleged health benefits ranging from lowered cholesterol to improved mood.  Some of these benefits are speculative, and for many years any benefit (such as improved gut health) was thought to be due to local effects or secreted chemicals.  However, the impact of these bacterial colonies may be much more far-reaching.

A Proceeding of the National Academy of Sciences (PNAS) paper published by Heujtz et al. last January (abstract) showed that microbial colonization in the mouse gut led to the activation of signaling pathways involved with motor control and emotional response.  This was the latest in a growing body of literature that suggests gut bacteria could influence how we think and act.  Now, in the August edition of PNAS, Bravo and colleagues take this one step further (abstract).  They show that Lactobacillus rhamnosus can directly influence the expression of GABA receptors in the brain.  GABA is the primary neurotransmitter for regulating many physiological and psychological activities in humans.  For example, caffeine inhibits GABA and results in an overall increase in neurotransmitter activity.  In contrast, alcohol and sedatives tend to increase GABA activity, leading to reduced neurotransmitter activity. Bravo et al. showed that feeding mice L. rhamnosus reduced GABA expression in some areas of the brain, while increasing it in others.  The overall effect was to make the mice more calm.  Here in the Dark Lab, we would test this by subjecting the rats to endless episodes of Jersey Shore, Barney and Friends, and The Jonas Brothers, and then asking how long it takes before they fall into convulsions.  Bravo measured stress-induced hyperthermia (rise in core body temperature from stress) after a battery of different tests, including  forced swimming and mazes (less barbaric than listening to the Barney jingle, but presumably effective at producing stress) and showed that the L. rhamnosus-fed animals exhibited less stress during these activities.

The final point of the paper was what I found most interesting.  Some of the animals had the vagus nerve cut prior to the start of the experiment.  This nerve is a direct link between the gut and the brain and is responsible for transmitting signals about hunger and satiation.  In these animals,there was absolutely no effect from consuming L. rhamnosus.  No changes in GABA expression and no behavior changes versus the control animals.  This means that the probiotic bacteria that colonize in the gut might actually use this nerve to signal directly to the brain.  Although these bacteria appear to provide a health benefit, I can certainly imagine other strains that are more nefarious.  Yeah, I'm talking about zombies again.  This phenomenon is not all that different from what is seen in T. gondii or the zombie ants... microbial agents that manipulate the brain function of the host.  If a probiotic strain can mimic the effect of caffeine-overstimulation or, even worse, caffeine-deprivation, then that would be a whole new kind of scary.  Anyone who has seen me before that first cup of coffee knows what I'm talking about... it's bad enough to give a zombie nightmares!

Zombie rats are horny!

You may remember a very early post on zombies, where I talked about a parasite known as Toxoplasma gondii.  I just read a new article published in PLoS that is really mind-blowing.  It also should fall in the category of science known as what-kind-of-PhD-do-I-need-to-study-this-shit.  Very interesting, very strange, and very cool.  Here's the abstract to check out for yourself (abstract).  As you know, rats that are infected with T. gondii lose their fear of cats.  This is important for the parasite because part of its life-cycle occurs in the gut of the cat.  This report takes that one step further.  House et al. show that as part of this shift in rat behavior, the rats are actually attracted to cat urine.  Not attracted like "this smells like roses" but rather "yowsa, hot babes" (rats do say 'yowsa'... I've heard them).  Yes, the parasite changes the way the rat brain responds to the smell of cat urine.  Neurons in the ventromedial hypothalmus, dorsomedial part (mercifully abbreviated as VMHdm), which normally are active in response to fear, are silent when infected rats are exposed to urine.  In contrast, the area in the brain the DOES light up is the posterodorsal medial amygdala.  These are the same neurons that light up when rats watch porn (or are exposed to estrous females, but somehow that doesn't sound as sexy).  Their conclusion is that T. gondii makes rats think that if they follow the cat pee, they will get laid.  Although that may work in some dive bars, for these rats it ultimately leads to just a single dinner date.

These results are also consistent with other findings that show an increased level of dopamine in infected rats.  Dopamine is, of course, the primary molecule in behavioral reward so it seems reasonable that this pathway would also be effective at shifting rat behaviors.  They cite a 2006 paper by Webster et al. (abstract) where it was shown that dopamine receptor antagonists prevent the attraction to cat urine.  Could this receptor be the first therapeutic target for treating zombies?  I'll have to propose that at the next New Target meeting. 

The ability of T. gondii to alter brain function and behavioral response is incredibly cool and a little bit scary.  Why scary?  Because it is estimated that at least one third of the human population has been exposed to the zombie-inducing T. gondii (and no, it's not just pop stars and politicians).  Even subtle changes in behavior on that scale can lead to massive changes in society.  Although it is unclear how well this study translates to humans, it does suggest that in the event of a zombie apocalypse, it's probably not a good idea to hide out in the girls' bathroom.

Highlights from the Protein Society Symposium

A week ago, I attended the 25th annual symposium of the Protein Society in Boston.  I've gone to this meeting three times and this was the best yet.  Very, very cool stuff.  I also got to see a lot of old friends from grad school, including my graduate advisor.  It was great to hear him talk fondly about the 'good ol' days' (ie, when I was his student) as I distinctly remember them being less fond and more frantic.  I think finishing a PhD thesis is the mental equivalent of giving birth to an elephant while running a marathon, but for several obvious reasons I will never be able to test the hypothesis.  I also met some cool new people.  I talked politics with a girl from Poland in a loud Irish pub and met another girl who is some kind of flute prodigy from a well-known coffee empire.  At the reception, I met a guy from, well, some European country who had done a postdoc in San Diego and I knew many of the trails he had hiked.  We tried watching the Red Sox game from the 50th floor of the Prudential building... great view but when the outfielders look like fleas on a green dog, it's really hard to see what is happening.  We kind of made it up as we went along and since the Polish chick didn't know the game, it was all good.  He still owes me pictures of Fenway.  The poster session was crazy, with two overlapping sessions and the very friendly (but bored) vendor who bribed me with chocolate every time I passed her booth.  I was also a poster judge this year, so I missed most of that session, but tracked the presenters down later to ask questions.  It's a little awkward at the coffee breaks, since everyone is staring at the nametags trying to find people they want to talk to.  I would try and catch a glimpse of their badge over the rim of my coffee and hope they were not offended when I simply walked away.  It's a very unusual hierarchy at conferences.  There is absolutely no guessing about where you stand in the pecking order.

Anyhow, I can't talk about the things I found most exciting because I was there for work, and work stuff has to stay off the radar.  However, let me briefly describe two (not work-related) things that were pretty cool.  One was a talk by Della David at UCSF on protein aggregation as a part of aging.  I don't know a lot about this field, but one of her early slides really caught my attention.  She was discussing the role of protein "aging" in inducing aggregation using C. elegans (a worm) as a model.  As the worm aged, she showed that the concentration of over 400 different proteins increased in the insoluble fraction.  In simpler terms, if you take all of the proteins out of the worm, many of them are soluble but some fraction are in an aggregated form, which is not soluble.  Although the total amount of protein seemed fairly constant with age, the proportion of aggregated protein increased and seemed to disrupt the natural process of homeostasis.  Then came the kicker... to show that this was an active process (that is, controlled by a cellular system) she used a C. elegans that had been engineered to have a specific mutation in the Daf-2 receptor.  These worms had twice the life span of a normal worm.  Whoa!  Sign me up for that mutation! Imagine living 160 years!  It turns out that the fraction of aggregates is independent of the lifespan, suggesting that the process is controlled.  Two things here... Daf2, which is part of the insulin/IGF-1 signaling pathway, can regulate lifespan (possibly related to the observation that mice that eat less live longer?) and that protein aggregation as a result of aging could also be controlled.  Here is a review on insulin/IGF-1 signaling in aging (abstract) and here is David's recent open-access paper covering some of this story (PLOS paper).  Listen folks, please hurry up with this important work... I'm not getting any younger.

The other talk I liked was by Ken Dill (a long-time favorite of mine and also from UCSF).  I'm used to him talking about transfer free energies and lattice models for proteins but this time he was talking about the stability of the proteome.  He (and others) have shown pretty convincingly that on a macro scale, protein stability is roughly dependent on the length of the protein.  (Seems simple but it has taken decades to model it in a way that makes physical sense).  Armed with this model, he determined the stability of the entire proteome and found that it is only marginally stable.  Over 500 proteins have stabilities less than 3 kcal/mol, which means they are barely folded and functional.  The implication of this result is that even slight increases in temperature can cause many of these proteins to unfold.  The resulting denaturation catastrophe overwhelms the cell and causes cell death.  This is the most plausible explanation yet for why slight increases in temperatures cause such problems (even for humans, an increase in body temp of 7-8 degrees can be fatal).  I asked him about the proteome of thermophilic bacteria and whether it might explain their ability to survive extreme temperatures and he said he is working on that now.  I'm guessing that might explain some of the adaptability, although the detailed mechanism is still a mystery.  For you DIYbio people out there, this model provides a pretty simple way to do this type of analysis yourself. The simplicity of the model, and the fact that minor ensemble changes can be magnified into major improvements for the organism tell me that life might be lurking everywhere there is an energy gradient (I'm looking at you, Titan).  On the flip side, it shows how sensitive life can be to slight changes in the environment. Here are the links to the articles (proteome stability and Dill's model)

  Dill also started off with a joke: "There are three kinds of mathematicians... those that can count, and those that can't."  Nothing like a geek joke to start off a talk... but hopefully his material will be better in San Diego next year.

GFP Beagles: Disease model or designer pet?

A recent paper in the journal genesis describes the production of transgenic beagles that glow when exposed to UV light (abstract).  The eGFP transgene was introduced into a beagle embryo using a similar (but much improved) technique that was used to clone Dolly the sheep.  Making a puppy that glows is not really new, as it has been demonstrated by the same group in 2009 (abstract) and others (in dogs, as well as other small mammals) but there are two cool things about the recent work.  First of all, they put the transgene under a promotor.  This means that the puppy does not glow green until the gene gets switched on, in this case by the drug doxycycline.  Feeding the dog low levels of doxycycline induced the expression of the GFP protein (green glow) and the effect could be turned off simply by removing the drug from the dog's diet.  This is pretty easy to do in small animals (like rats and mice) but pretty complicated in large animals. 

  In a separate article, also in genesis, they report the transmission of the transgene to offspring (abstract).  This was also interesting, as the GFP-containing females were totally fertile and had normal pregnancies and births.  The dads were wild type beagles so roughly 50% of the offspring carried the transgene.  This is consistent with stable germline transmission.  This result suggests that we are really not far off from having designer dogs.  The initial cloning will be hard (efficiency is still in the 1-5% range) but if the transgene is stable, simple breeding techniques should be enough to make zillions of glowing puppies!  Although this is a crude example, one can envision a vastly different world where hair color, facial and body features, and perhaps even personality traits could be engineered.  Genetic defects and disease determinants could be engineered out.  One could build the perfect dog.  You did realize I was still talking about dogs... right?

Foxfire: Chemistry of the undead



Ghostly mushrooms



I am currently approaching the half way mark in my current work-in-progress (WIP-2) and have been making particularly heavy use of foxfire.  I didn't start out to write about that... in fact, this book started out as a humorous middle grade adventure and quickly turned into a dark, YA biopunk.  Write what you know, I suppose.  One of the emerging themes is the struggle between the protagonists and the technologically-advanced fungi that exists in their (slightly dystopian) world.  So, I found it a little coincidental that a report came out a week ago by Marina Capelari and colleagues about a type of ghost mushroom that had been re-discovered in a Brazilian rainforest after being extinct for over 150 years (abstract in the journal Mycologia).  The mushroom, formerly known as Agaricus gardneri, is notable for its bright and constant bioluminescence.  To understand why this is unusual, here is a brief description of how foxfire comes to be:

Bioluminescence is generally accepted to come from a 2-step reaction.  A chemical called luciferin (L) is first reduced (to LH₂) and this reaction is catalyzed by an enzyme called reductase.  NADH is a molecule (di-nucleotide, actually) that is a cofactor in many redox reactions.  Its basic function is to move protons around (you're a geek if you noticed the chemistry pun).

L + 2NADH <--> LH₂ + 2NAD+

Reduced luciferin is then oxidized (to LO) by an enzyme called luciferase.  This process also produces a photon of light and is the source of the creepy glow.

LH₂ + O₂ <--> LO + H₂O+ LIGHT



Illudin S: Potential substrate for ghost fungi

Why am I cryptically showing fungal luciferin as L, instead of showing the chemical structure?  Could it be that my chemistry skills are so bad, I couldn't tell the difference between L and LH if my life depended on it?  Well, yes -- but it is also true that the luciferase substrate in fungi is not well characterized. The luciferin for A.gardneri is probably a member of the sesquiterpene family, most likely an illudin.  Some of these compounds have been studied as anticancer agents but the illudins tend to be extremely toxic (possibly another reason they are called ghost mushrooms!).  Interestingly, other luciferins (such as those found in fireflies, shrimp, etc) have totally different chemical structures, which gives them different biological properties and unique spectral characteristics (ie, different colors, brightness, etc).  Changes in the luciferin structure, amino acid substitutions in the active site of luciferase, and varying levels of oxygen or water can each contribute to changes in the emission of light.  What is unusual about A. gardneri is that unlike other species, the bioluminescence is almost constant.  In fireflies, the luciferin is released when they want to blink and in the case of other species, they light up only after contact (probably a means of self-defence).  So why does this mushroom glow all the time? No one knows yet.  The biochemistry of these things is almost as mysterious as seeing their eerie ghostly glow on some rotting tree stump at midnight.

However, it provides a great real-world example of the potential technology for my story.  It doesn't take much imagination to think that these mushrooms could be engineered to be very bright or to respond in controlled ways depending on environmental input.  A basic example from my WIP is that these types of fungi are used for lighting underground.  No electricity required, no pollution, and little maintenance.  They are almost the perfect type of lighting... or are they?  Anyhow, I thought it was a pretty clever idea early on until I found out that Ben Franklin used foxfire from mushrooms to light the inside of one of the first submarines.  Was there anything this guy didn't know about?  I guess he's going to have to go on my list of card-carrying biopunks.