Research Blogging - Medicine - English

Neuroscientists should study Zombie Ants

2012-05-25T15:32:00Z

Zombie ant controlled by fungus (source)The fungus-controlled zombie ant is one of natures' greatest wonders. A fungus (e.g. O. Unilateralis) is inhaled by an ant (e.g. Camponotus Leonardi), and begins to grow inside its body. Eventually the fungus infests the brain of the ant, causing it to drunkenly wander, periodically convulse, climb up a leaf and clamp down on its ridge. Once the ant is securely in place, the fungus devours the brain and innards of the ant and grows out the back of its head often (but not always) releasing its spores onto the ground below. Un-freaking-believable, right?As if this wasn't amazing enough, it's not like it is only one fungus species that infects only one ant species. There are many of these fungi and they infect many different kinds of insect, but somehow maintain a species specificity. In other words, fungus#1 can infect SpeciesX, but not SpeciesY, and Fungus#2 infects SpeciesY, but not SpeciesQ, and so forth. So WHY does this happen? and HOW has no one looked at the brain cells of these ants? Though no one has looked at the brains of these ants, Last year a paper painstakingly characterized their behavior under 'fungi control'. The most interesting characteristics are:The ants display a 'drunkard's walk' (the author's words)The ants periodically spasm and fall down (if they are above ground level)The ants clamp down on the underside main vein of a leaf (never the side of the leaf, never the top) Interestingly they all bite down on the leaf around solar noon.Figure 1, Hughes et al., 2011This figure shows the behavior of several ants. Each ant was observed during the time of the horizontal blue bar. The black vertical lines and 'spasms' which caused the ants to fall down (gray stars), and the red triangles are when the ant bit down on the leaf ridge. Because we have no idea how the fungus is manipulating the ant, let's wildly speculate.1. The Drunken Walk:Why: The reason for this is not clear. The ant doesn't go far, so the non-directional walking could be to keep it close to more ants.How: The mechanism is also not clear, but usually an ants directional walking could be following a pheromone trail. The fungus could presumably cause random walking by confusing the ants ability to sense pheromones. It could possibly even cause 'hallucinatory' pheromone sensing.2. The Periodic Spasms: Why: The authors speculate that the purpose of these spasms is to keep the ant near the ground. The infect ants spend much more time on the ground level than the uninfected ants, and the spasms are often followed by a fall.How:A fungus could essentially cause a seizure in the ants brain by manipulating potassium or calcium channels. On the other hand, I suppose the fungus could be acting directly on the muscles, causing them to twitch in an uncontrolled way. 3. The Clamping: Why: This has an obvious function, to root the ant for ultimate fungal growth and dispersion. How: First of all, biting and even walking on leaves is not something these ants normally do. So the fungus isn't just hijacking a behavior that the ant already has, it's basically creating a new one. The correlation with solar noon indicates that a light or heat signal could contribute to the trigger, but basically nothing else is known about it. Interestingly, the clamping does not always have to be one single event either. A few of the ants clamped down on the leaf vein more than once. The authors of this paper spend time discussing fungi's direct effect on the mandible muscles of the ant.Figure 3 Hughes et al., 2011They show that the mandible muscles of the normal ant are fat and healthy (B), but the muscles of the infected ant are separated and reduced in size (C). Though this image is of an ant at the moment of biting, the authors suggests that the deterioration of the mandible muscle might be to prevent re-opening of the clamp. They do not speculate on how the clamp is initiated in the first place, or why it occurs at noon. So please, fellow neuroscientists, somebody stain these brains! It's just too fascinating to resist exploration. What proteins are altered? What is the receptor composition of behaviorally-specific neurons? Are the dendrites differently shaped? And who knows what sort of great advances might be hidden in these brain-controlling fungi. The magic of optogenetics comes from lowly light-sensitive bacteria, just think of the possibilities hidden in brain-controlling fungus. To be fair, some neuroscience has been done on parasitic brain control, but it is very limited. In fact it is limited to basically one histological study about parasitic worms who infest crickets and cause them to drown themselves (the subject of a future blog post). However, suicide-crickets are no zombie-ants and the exact mechanisms of the interaction © TheCellularScaleHughes DP, Andersen SB, Hywel-Jones NL, Himaman W, Billen J, & Boomsma JJ (2011). Behavioral mechanisms and morphological symptoms of zombie ants dying from fungal infection. BMC ecology, 11 (1) PMID: 21554670...

Hughes DP, Andersen SB, Hywel-Jones NL, Himaman W, Billen J, & Boomsma JJ. (2011) Behavioral mechanisms and morphological symptoms of zombie ants dying from fungal infection. BMC ecology, 11(1), 13. PMID: 21554670

Friday Fun: A Week in the Life of a Grad Student

2012-05-25T13:15:02Z

As a graduate student, I don't punch a clock or fill out a time card. Although I have a faculty advisor, I don't have a boss - no one is keeping count of my hours. Most of my work time is spent doing a variety of tasks related to research or teaching, and these often change from week to week. I love the freedom and diversity of the academic life, but the lack of structure means that at the end of the week I'm often unsure of how exactly I spent my time. I like to get a good nights' sleep, so that certainly helps... but for the other 15 or so hours in my day, what exactly am I doing? This question intrigues me and for the past several months I've been thinking I should keep a log of my happenings to see how my days are spent. So I finally did it back in April. I created a word doc dedicated to the cause, and updated it every time I changed tasks Monday through Friday. I also kept a quick count of how much time I spent working on the weekend. It wasn't the most ordinary week with canceled meetings and a weekend visit to my hometown. But really, is any week "ordinary"? So I figured I'd just go ahead and do it while I was motivated. Read More->...

Coker, B. (2011) Freedom to surf: the positive effects of workplace Internet leisure browsing. New Technology, Work and Employment, 26(3), 238-247. DOI: 10.1111/j.1468-005X.2011.00272.x

Brain Connectivity Alterations in OCD

2012-05-25T11:51:41Z

Obsessive compulsive disorder (OCD) is an anxiety disorder characterized by intrusive thoughts interfering with everyday activities. The prevalence of OCD is estimated to be between 1 to 2 percent of the population. Despite several approved drug treatments and the availability of behavioral therapy, many individuals with OCD find their symptoms resistant to treatment. A better understanding of the underlying pathophysiology of OCD is needed for the development of more effective treatment.A recent brain imaging study provides some additional insight into the connectivity alterations found in those with OCD. In this study, 30 subjects with OCD were compared to a group of 32 control subjects using functional magnetic resonance imaging. The subjects were studied for functional connectivity of the default mode network (DFM) as well as the fronto-parietal network (FPN).The default mode network is the working network of brain regions typically active "at rest" when no specific brain-related task is being performed. You might think of this as what brain regions are in synchronization with each other during rest. We know that individuals with OCD commonly find their resting brain state is disrupted by their intrusive obsessional thoughts. This makes study of the default mode network promising for understanding OCD.The key findings in OCD from this study were:Reduction in the typical negative correlation between the DFM and the FPNAltered connectivity between the anterior insula and multiple DFM regions including the parahippocampus, medial frontal cortex, posterior cingulate cortex, and the posterior inferior parietal lobeIncreased connectivity of the FPN with regions outside the DFM including the thalamus, lateral frontal cortex and somatosensory/motor brain regionsThe authors note "Though speculative, the current findings of attenuated negative connectivity between anterior insula and DMN suggest a potential neural basis for the difficulty OCD patients may have in efforts to disengage from internal mental processes in order to respond to more appropriately salient external information related to potential risk (such as that informing them that dreaded events have not or will not occur)."The authors also note their study may not represent findings specific to OCD but may be related to broader anxiety conditions. This will need to be explored by contrasting differences (and similarities) across OCD with other anxiety disorders such as generalized anxiety disorder, panic disorder, social anxiety disorder and PTSD.This study is indicative of the evolving conceptualization of many mental disorders. Many mental disorders appear to not be due to a single neurotransmitter abnormality or a localized brain lesion but appear to be abnormalities of brain circuitry. Functional brain connectivity imaging of the DMN holds promise as a tool to better understand these circuitry alterations.Photo of female adult black bear from Lake Istasca State Park from the author's files.Stern, E., Fitzgerald, K., Welsh, R., Abelson, J., & Taylor, S. (2012). Resting-State Functional Connectivity between Fronto-Parietal and Default Mode Networks in Obsessive-Compulsive Disorder PLoS ONE, 7 (5) DOI: 10.1371/journal.pone.0036356...

Stern, E., Fitzgerald, K., Welsh, R., Abelson, J., & Taylor, S. (2012) Resting-State Functional Connectivity between Fronto-Parietal and Default Mode Networks in Obsessive-Compulsive Disorder. PLoS ONE, 7(5). DOI: 10.1371/journal.pone.0036356

Cochrane and a Significantly Biased Review of Steroids for acute spinal cord injury

2012-05-25T08:00:00Z

An interesting relic of trauma care is the use of steroids for the treatment of acute spinal cord injury. As with The Golden Hour, there are people still promoting this idea. In the words of Monty Python, it’s not quite dead, yet. Who is promoting this idea? The Cochrane Collaboration. Here is a list of the papers evaluated in this 2012 update of the 2009 Cochrane Review, which was an update of the 2002 Cochrane Review. There has been no change in the references, since the most recent paper reviewed is from 2000....

Bracken MB. (2012) Steroids for acute spinal cord injury. Cochrane database of systematic reviews (Online). PMID: 22258943

Ask Not What You Can Do For Educational Technology, But What Educational Technology Can Do For You

2012-05-25T00:46:54Z

It’s irritating that people talk about educational technology in terms of iPads in the classroom when the real impact will come from pinpoint differentiation, instant student assessment, and a third thing that nobody talks about – improved simulations in speciality learning. For example, medical students who use virtual patients — an “interactive computer simulation of real-life [...]...

Consorti, F., Mancuso, R., Nocioni, M., & Piccolo, A. (2012) Efficacy of virtual patients in medical education: A meta-analysis of randomized studies. Computers , 59(3), 1001-1008. DOI: 10.1016/j.compedu.2012.04.017

Delayed ACL Reconstructions may be Associated with More Severe Meniscal and Chondral Lesions

2012-05-25T00:07:00Z

Anterior cruciate ligament (ACL) ruptures often coincide with meniscal and cartilaginous injuries. These ruptures are generally treated with surgical reconstruction or non-surgical treatment. Patients who chose to delay surgical treatment may be at greater risk for increasing the severity of the associated injuries; however, this has not been demonstrated in the literature. Therefore, Fok and Yau completed a retrospective, comparative study investigating (1) if delaying ACL reconstruction is associated with the number of meniscal and articular cartilage lesions, and (2) if ACL-deficient patients experiences greater frequency and magnitude of pain....

Fok AW, & Yau WP. (2012) Delay in ACL reconstruction is associated with more severe and painful meniscal and chondral injuries. Knee Surgery, Sports Traumatology, Arthroscopy. PMID: 22552616

UK vs. US in Media Reporting on Eating Disorders: Who Does it Better?

2012-05-24T23:50:19Z

Given the popularity of my post on how the media portrays eating disorders, I thought I’d do a follow-up post about a more recent and comprehensive study on media reporting of eating disorders. Shepherd & Seale (2010) wanted to build up on the findings of O’Hara & Clegg-Smith, but focusing on UK newspapers. In particular, they: compared UK and US media reporting of EDs tracked changes of in ED coverage over a 17-year period studied the differences between newspapers with different target audiences Shepherd & Seale reiterate much of what O’Hara & Clegg-Smith wrote: ED specialists and researchers understand that EDs are complex, multi-factorial diseases with complex genetic and environmental underpinnings, that they are often associated with many medical complications …...

Shepherd, E., & Seale, C. (2010) Eating disorders in the media: The changing nature of UK newspaper reports. European Eating Disorders Review, 18(6), 486-495. DOI: 10.1002/erv.1006

Validation of the Dime

2012-05-24T14:35:00Z

The current Annals of Emergency Medicine has a pair of editorials on the article I wrote about[1] in This is the Way to Bad Medicine back in January. Dr. Radecki also was critical of this paper.[2] There is another study that refers to the same question published in this issue, but I will write about that paper later. "These data raise the real question, Do such findings matter? By admitting more patients and ordering more CTs, do we improve outcomes? Or do we simply find more things that have little clinical importance? Indeed, in Menditto’s sample,8 only 1 patient needed craniotomy. In Kaen’s,14 none did.[3]"...

Menditto, V., Lucci, M., Polonara, S., Pomponio, G., & Gabrielli, A. (2012) Management of Minor Head Injury in Patients Receiving Oral Anticoagulant Therapy: A Prospective Study of a 24-Hour Observation Protocol. Annals of Emergency Medicine. DOI: 10.1016/j.annemergmed.2011.12.003

Clockworks: The Story of Drugs — Part 1

2012-05-24T11:15:00Z

In this installment, I will discuss why it is difficult to discover, design and develop a drug, in view of our current knowledge of physiology.With numerous, intertwined reactions happening, our body is a complex clockwork of biomachinery gears. What do you do, then, if some gears fail—that is, if you got sick? On one hand, it is a consolation that many gears are what biologists call 'redundant', which means that it's alright that a certain gear fails, because there are other gears that can take over its function. On the other hand, due to the intricacy of the gears, it is hard to pinpoint which gear is the problem, let alone fixing it. And the sheer number of gears: ICD-10 classifies tens of thousands diagnoses — tens of thousands ways the gears can fail — and those are only the ones we know; how about those we don't? Granted, some are not caused by our own gears failing, but by interferences of other, pesky gear systems: viruses, bacteria, misfolded proteins, errant microbiome, etc.; but the sense of magnitude is there.So we take drugs. In the past, the way we administer drugs is the equivalent of throwing various types of wrenches to the clockwork and then observe whether the gears are working again. Today we know more about the gears and the various shapes and teeth so that we are able to design a more sophisticated and targeted wrench, but we still don’t know enough.Our current thinking is that the machinery gears, mostly proteins, are of distinct shapes, or perhaps more fittingly, different tooth shapes. A drug has gotta fit into these various shapes. We think that if we can find 'keys' that fit to these 'locks' we can modulate that particular gear's activity: turn it up or down, switch it on or off. The key would fit into the lock, the lock would be induced to change shape / dissociate / do other curious stuff; which triggers happenings in the next gear in line. Like the dominoes falling off in line. Only that ‘the curious stuff’ may be more than a domino keeling over or a gear turning, but something that is more wackily messy, something like a Rube Goldberg contraption than a precise-looking clockwork, in this respect.How should we shape the serrated key? The first problem is scale. These gears are small — not microscopically, but nanoscopically so: To design the keys, we need the moulds, and so these very gears are the moulds. Structural biology to the rescue — X-rays crystallography/multidimensional NMR/cryo-electron microscopy can characterise the gears with varying resolutions.However, structural determination techniques face a big challenge: the interconnectedness of the system. You can't take out a gear out of context of the surrounding gears, examine it, hoping that your examinations will be valid and/or useful. Well, you sort of can. Sure, systems biology is important to gain a bird's-eye view of the whole shebang, but structural biologists routinely single out a gear and determine its structure to make it crystal-clear (heh heh) what its possible activation mechanism is, how it interacts with its activator/inhibitor, how it transduces signal to the next gear, and so on.The structural data, then, has to be taken with a grain of sodium chloride because essentially it is performed—in the physics equivalent of—in vacuo. That said, the gleaned information can be incredibly useful. Case in point: enfuvirtide. This anti-HIV-1 drug mimics a region—C-heptad repeats (CHR)—of the viral envelope glycoprotein, gp41, which is a crucial part of viral:cell membrane fusion machinery [1]. CHR is supposed to fold back to NHR like a hairpin (they are connected) and then Stuff happens, with gory details I will spare you from (Oh, fine: a channel is opened between HIV-1 membrane and that of your soon-to-be-infected T-cell; viral particles are pumped through and soon hijack your T-cell to become baby-virus-producing zombie until it bursts releasing said baby virions. Happy?) Now, the 'folding back' part is a mechanism that was uncovered through cleverly-devised experiments founded in structural studies. Thanks to this, we can deduce that if we somehow have a fake CHR, this 'folding back' can be circumvented. And that's exactly what enfuvirtide is: a dummy gear that connects to NHR gear, but not connected to the gears down the road, thus—hip hip hurray—no zombie outbreak.This is kinda cheating though. Enfuvirtide is essentially free-roaming CHR region of gp41 and no rational design work was done. For example, peptidomimetic strategy could have been used to find something similar to enfuvirtide but is able to survive the gut—since a peptidic drugs like enfuvirtide won’t, so they have to be taken intravenously. But of course, HIV-1-infected individuals don’t have the luxury of time to wait for further work on enfuvirtide optimisation.Back to the 3D protein mould model built by structural determination techniques. Ideally, we can start building up the drug à la Lego bricks, fitting the chimaera into the protein-shape mould, right, right? Couldn't be more wrong. Enter multidimensional fitting. You see, a protein ain't like your Mom's muffin pan. Besides topology, there are other dimensions to fit—electrostatic charges, hydrophilicity/hydrophobicity, to name a few. The topology is not necessarily fixedly rigid either — playing with those poppin’ stick-and-ball molecular models may give us the illusion that proteins are rigid, but protein electron clouds are more like wobbly pudding (Mmmm, pudding...); plus, different environments (pH, oxidising level) may give rise to largely different topologies (e.g. due to different protonation states, broken disulphide bridge(s), etc.).One aspect of nanoscopic scale that is somewhat entangled with multiparameter fitting is that at this scale, there is evidence that quantum effects play an important role. Several examples of such systems have been studied like photosystems and birds’ navigation, but of more interest to a medicinal chemist would be tunnelling effect in certain enzymatic reactions. Who knows if quantum effects are more routinely utilised? Our view of physiology is biasedly mechanical—even my clockwork allegory evokes the mechanistic, so a paradigm shift may well be in order as more is known about the inner workings of such systems. If you blindly design a drug that target such systems, well, you will get an entangled mess—hopefully not the quantum kind. Moving on to pharmacokinetic restrictions: the human body imposes further restrictions from the non-negotiables (e.g. a drug cannot be too insoluble otherwise how can it dissolve in the bloodstream; a drug cannot have side effects outweighing its efficacy), to convenience (e.g. a drug is preferable to be ingested rather than injected). Our own physiology thus severely restricts the chemical space of entities that make up our drug candidate pool. As I mentioned earlier, enfuvirtide is a peptide, so it won’t survive stomach acidity and peptidases in the gut. Even with intravenous administration, it would have a short half-life due to blood proteases. All peptidic drugs—insulin is one—suffer from these problems. And for drugs targeting the central nervous system (CNS), they have to overcome another obstacle, the blood brain barrier.There seems to be some sort of patterns to the drug chemical space. To wit, some have observed that certain chemical scaffolds occur more frequently than others; they are so-called privileged scaffolds (e.g. benzodiazepines) [2] and an experienced medical chemist would be able to take a look at a chemical structure and decide whether it's 'drug-like' (while an inexperienced chemist like me would only know that a drug-like molecule can't be too small and simplistic, possesses some heteroatoms, usually has an aromatic ring or two—that's about it). Problem being, at its current state, drug-likeness is an empirical measure. No one has formulated a set of rules or equations to produce a predictive model. Lipinski's Rule of Five, for example, surveys already-existing drugs and look at the prevalent drug-like characteristics. Useful as rule of thumb; hardly predictive.Next is the issue of specificity. If you choose to take a drug topically, that’s fine and dandy because you can apply the drug locally to the area in need of treatment. But if you take a drug via oral/intravenous/other numerous administration routes, the drug is going to circulate in your bloodstream. How would you ensure that your wrench would reach, and affect only, the faulty gear? You can’t—not with certainty, at least—the wrench is going to wreck another gear, and that’s why you always ha...

Eckert, D., & Kim, P. (2001) Mechanisms of Viral Membrane Fusion and Its Inhibition. Annual Review of Biochemistry, 70(1), 777-810. DOI: 10.1146/annurev.biochem.70.1.777

Welsch, M., Snyder, S., & Stockwell, B. (2010) Privileged scaffolds for library design and drug discovery. Current Opinion in Chemical Biology, 14(3), 347-361. DOI: 10.1016/j.cbpa.2010.02.018

Marusyk A, Almendro V, & Polyak K. (2012) Intra-tumour heterogeneity: a looking glass for cancer?. Nature reviews. Cancer, 12(5), 323-34. PMID: 22513401

Getting to the root of Type II diabetes... with liquorice?

2012-05-24T04:34:02Z

Metabolism is a balancing act that gets harder with age (Picture of Philippe Petit on high wire, Notre-Dame Cathedral, Paris, 1971. picture: Cordisere) The liquorice root is full of surprises. Chewed as a breath freshener in Italy and a sweet in Sweden (and the north of England), this little brown stick has also been used as a remedy for mouth ulcers for thousands of years. New research has identified a natural chemical extracted from the liquorice root that may be used to treat Type II diabetes. Our metabolism is a delicate balance. Insulin, a hormone secreted by the pancreas, regulates levels of glucose and fatty acids in the blood by storing them out of the way in fat and muscle tissue. Some stored compounds can be converted back into glucose when the body needs energy. Wear and tear on this balance, as our cells age or through diet or stress, can overload our tissues with fatty acids. Fat and muscle cells become unable to soak up excess glucose and in some cases build a resistance to insulin, a hallmark of Type II diabetes. Recent drug-based therapies aimed to restore the metabolic balance by targeting the wiring of PPAR-gamma, a receptor protein in the nuclei of many fat cells. PPAR-gamma responds to fatty acids in digested food by activating genes to boost metabolism. The hope was to manipulate PPAR-gamma to lower the level of fatty acids and improve the cells' sensitivity to insulin. But there was a problem. The synthetic drug rosiglitazone triggers PPAR-gamma very strongly, successfully lowering blood glucose levels but also firing many other genes at the same time. Out of context, some of these genes were linked to unforeseen side-effects such as weight gain, fluid retention and heart disease. The liquorice root. Amorfruitins found at low levels inside might be extracted to treat Type II diabetes. (Picture: Ryan Opaz) In a recent study in PNAS, Christopher Weidner and colleagues investigated a natural alternative. Amorfruitins, extracted from the edible roots of Amorpha fruticosa (the indigo bush) and Glycyrrhiza foetida (a species of liquorice) are natural activators of PPAR-gamma. Amorfruitins were shown to influence glucose and fatty acid metabolism similarly to rosiglitazone but with more selective targeting of PPAR-gamma, respectful of its powerful role in controlling different sets of genes. The team, led by Sascha Sauer from Max Planck Institute for Molecular Genetics in Berlin showed that amorfruitins decreased insulin resistance in the fat cells of diabetic mice without any observed weight gain. Amorfruitins also reversed some of the genetic changes brought about by a high-fat diet. Dr Sauer said. “In view of the rapid spread of metabolic diseases like diabetes, it is intended to develop these substances further so that they can be used on humans in the future.” Sauer's team have begun to investigate how amorfruitins steer the wiring of PPAR-gamma so effectively. They found differences between the genes expressed by PPAR-gamma in response to rosiglitazone or amorfruitins. This is something of a smoking gun: a first step towards understanding what it is about liquorice, a legume, that gives amorfruitins their remarkable ability to correct wiring inside mammalian cells. What does this mean for me? It’s estimated that 190 million people are affected by Type II diabetes worldwide and that this figure will double over the next 20 years. This study shows not only a direct health benefit of a natural plant extract on metabolic diseases, but also suggests the mechanisms for how it might work inside mammalian cells. Sauer's team hope the edible nature of the liquorice root, will make it easier to obtain approval for the use of amorfruitins in humans. What does this mean for science? This study highlights the importance of "basic" cell biology research to support medicine: only after investigating how a drug works can we confidently predict what (side) effects it may have on the wiring inside our cells. The differences in gene expression patterns between natural and synthtic PPAR-gamma activators suggest clear differences in how they act inside the cell. This raises questions for future drug design approaches - what makes amorfruitins so subtle and selective? Can their mechanism be copied synthetically, maybe to target other important transcription factors? Reference: Weidner, C., de Groot, J., Prasad, A., Freiwald, A., Quedenau, C., Kliem, M., Witzke, A., Kodelja, V., Han, C., Giegold, S., Baumann, M., Klebl, B., Siems, K., Muller-Kuhrt, L., Schurmann, A., Schuler, R., Pfeiffer, A., Schroeder, F., Bussow, K., & Sauer, S. (2012). From the Cover: Amorfrutins are potent antidiabetic dietary natural products Proceedings of the National Academy of Sciences, 109 (19), 7257-7262 DOI: 10.1073/pnas.1116971109...

Weidner, C., de Groot, J., Prasad, A., Freiwald, A., Quedenau, C., Kliem, M., Witzke, A., Kodelja, V., Han, C., Giegold, S.... (2012) From the Cover: Amorfrutins are potent antidiabetic dietary natural products. Proceedings of the National Academy of Sciences, 109(19), 7257-7262. DOI: 10.1073/pnas.1116971109