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Friday, February 26, 2010

Why Johnny can’t read (but Jane can)


Reading is not a skill that comes naturally.  Unlike learning spoken language, which the human brain has evolved to absorb almost effortlessly, learning to read is a protracted and difficult process.  It involves the categorical association of arbitrary visual symbols with phonemes and also the ability to break words down into component phonemes.  It thus relies on an integration between visual and auditory processes, combining spatial and temporal information, within a learned linguistic context. The fact that reading is such a specialized and integrative skill may partly explain why it can be selectively impaired in people of otherwise normal intellectual abilities.

Dyslexia, as this type of selective reading difficulty is called, is quite common, affecting anywhere from 5% to 20% of children, depending on the criteria used in its diagnosis.  Cohort studies which directly tested all individuals have found that dyslexia is about twice as common in boys as in girls.  This is not unusual; many psychiatric disorders show a different rate of occurrence between the sexes, with autism, schizophrenia and attention-deficit hyperactivity disorder all being more common in males, while depression and some other non-psychiatric conditions, such as synaesthesia, are more common in females.  The study of dyslexia is providing some insights into how the interaction between predisposing factors and sex can influence phenotypic outcome. 

First, some clues as to what the underlying cause may be.  Dyslexia is highly heritable and in recent years a number of genes have been linked to it, through unbiased genetic mapping approaches.  Remarkably, all of these genes are involved in some way in controlling cell migration in the cerebral cortex.  This astonishing convergence parallels neuroimaging and neuropathology studies which have found a high rate of subtle malformations of the cerebral cortex in brains of dyslexic subjects.  These include small clusters of misplaced neurons (termed nodular heterotopia), as well as small infoldings of the cortical sheet (microgyria).  Disruption in the normally highly regular organization of the cortex may thus underlie this disorder. 

One hypothesis to explain how this occurs is that these heterotopia and microgyri disrupt communication between cortical areas in a way that impairs the integrative processes required for reading.  

A study from the lab of Christopher Walsh provides direct evidence supporting this idea.  He and his colleagues studied a rare, inherited condition, called periventricular nodular heterotopia, which is characterised by the presence of misplaced clusters of neurons within the white matter near the cerebral ventricles, and which is also associated with very high rates of dyslexia. Using diffusion tensor imaging to follow specific axonal tracts within the brain, the authors found that these heterotopia did indeed interrupt the structural connections that passed through the affected regions (while tracts in parts of the brain without heterotopia were normal). 

But the impact of such displaced neurons is not limited to the cortex itself.  A fascinating set of studies from Albert Galaburda and colleagues with an animal model of microgyria has revealed secondary consequences on other parts of the brain that connect to these regions, in particular the thalamus.  In young rats, very localized microgyria can be induced by application of a freezing probe to the surface of the brain (in this case to the parietal cortex).  These lesions resulted in defects in auditory processing, specifically of very fast temporal sequences, which may parallel some of the auditory processing defects seen in dyslexia, especially in phonemic processing.  The lesions also resulted in secondary changes in the medial geniculate nucleus, the auditory centre of the thalamus which connects to the parietal cortex.  Most remarkable, however, was that both the histological changes in the thalamus and the defects in auditory processing were much more pronounced in male rats than in females.  When females were treated with testosterone and lesioned they showed similar secondary effects to male rats. It is known that testosterone regulates proliferation, cell death and connectivity during development, but it is also involved in various processes of neuroplasticity, which may explain the differences in secondary effects observed here.

The sex difference in this case thus involves not the primary defect itself but the way in which the brain responds to it.  As many psychiatric disorders involve an interaction between primary neurodevelopmental insults and ongoing neuroplasticity processes, this mechanism may be of much wider relevance in explaining the different susceptibility of male and female brains to various conditions. 

 

 

Chang, B., Katzir, T., Liu, T., Corriveau, K., Barzillai, M., Apse, K., Bodell, A., Hackney, D., Alsop, D., Wong, S., & Walsh, C. (2007). A structural basis for reading fluency: White matter defects in a genetic brain malformation Neurology, 69 (23), 2146-2154 DOI: 10.1212/01.wnl.0000286365.41070.54

 

Rosen GD, Herman AE, & Galaburda AM (1999). Sex differences in the effects of early neocortical injury on neuronal size distribution of the medial geniculate nucleus in the rat are mediated by perinatal gonadal steroids. Cerebral cortex (New York, N.Y. : 1991), 9 (1), 27-34 PMID: 10022493

 

Sunday, February 21, 2010

Psst!… Pass it on! Cortical communication via the thalamus

Many neuroscientists think of the thalamus – a compact structure lying right in the centre of the brain – simply as a relay station, where sensory information from the periphery converges and is then passed on to the cortex.  The cortex is thought to be the site of perception and cognition, with different cortical areas specialized to subserve different functions.  Communication between cortical areas can be mediated by axonal tracts running in the white matter of the cortex.  This leads readily to the view that once information reaches the cortex it is processed and integrated with other information about the external world and internal states entirely within the cortex, resulting in conscious perception or some kind of motor (or emotional) output.  In most neuroimaging studies, for example, the focus is solely on activity in the cortex and the thalamus (and other lower brain areas) are explicitly ignored.

A new study by S. Murray Sherman and colleagues demonstrates unequivocally that cortical areas can also pass information indirectly via the thalamus.  It has been known for some time that communication between thalamus and cortex is bidirectional.  In fact, the thalamus receives far more inputs from the cortex than it does from the periphery.  The circuits between thalamus and cortex can be broken down into two main types – those that drive the activity of their target neurons (whether in thalamus or cortex) and those that act more to modulate the activity of their targets, especially their temporal responsiveness.  These pathways can be distinguished based on their neurochemical profiles, the types of synapses that they form and, in the case of projections from thalamus to cortex, the layers which they innervate.  Driving connections from thalamus project with quite precise topography to layers 4 and 6, while modulatory connections project more diffusely within layers 1 and 5. 

These modulatory connections from the thalamus are essential mediators of communication between cortical areas, due to their crucial role in the synchronization of ongoing neuronal oscillations – rhythmic patterns of activity of local ensembles of neurons.  Areas where these rhythms are oscillating in phase with each other are more responsive to signals from each other – this is, in effect, a kind of frequency modulation – when one cortical area sends out a signal, it is picked up selectively by those areas that are tuned to the same frequency.  This tuning can be mediated by corticothalamocortical loops (where the corticothalamic connection is driving and the thalamocortical connection is modulatory).  In this scheme, however, the information itself is transferred via direct cortical connections. 

The new study shows that even if these cortical connections are severed, information can still be transferred from one cortical area to another if corticothalamocortical circuits remain intact (in this case both the corticothalamic and the thalamocortical connections are driving).  In a thalamocortical slice preparation from the mouse brain, the authors showed, using a new optical imaging technique, that activity in secondary somatosensory cortex (S2) could be elicited by activating primary somatosensory cortex (S1).  There is nothing remarkable in that – what was remarkable was that when the direct connections between these cortical areas were severed that the activation of S2 was almost as potent.  This activation depended on an intact corticothalamocortical loop – subsequent disruption of these circuits abolished activation of S2.  There are thus most likely two routes of communication between these cortical areas – one direct and one via the thalamus. 

These findings reinforce the important point that the function of the cortex cannot be divorced from that of the thalamus.  Perception is not simply a matter of passing information along a hierarchy of processing stations – this would leave the question of who is receiving the final information.  It can be envisaged instead as a process of reiterative comparison of top-down predictions with bottom-up information, much of which may be mediated by reverberating activity in corticothalamocortical circuits.

 

Theyel, B., Llano, D., & Sherman, S. (2009). The corticothalamocortical circuit drives higher-order cortex in the mouse Nature Neuroscience, 13 (1), 84-88 DOI: 10.1038/nn.2449

Friday, February 19, 2010

Noisy genes and the limits of genetic determinism

Why are genetically identical monozygotic twins not phenotypically identical?  They are obviously much more similar than people who do not share all their DNA, but even in outward physical appearance are not really identical.  And when it comes to psychological traits or psychiatric disorders, they can be quite divergent (concordance between monozygotic twins for schizophrenia for example is only around 50%).  What is the source of this phenotypic variance?  Why are the effects of a mutation often variable, even across genetically identical organisms?

“Nurture” has been the answer proffered by many, but there is good evidence that environmental or experience-dependent effects can not explain all the extra phenotypic variance and in most cases contribute very little to it.  (See post on “Nature, nurture and noise” on June 24th, 2009 for more on this: http://wiringthebrain.blogspot.com/2009/06/nature-nurture-and-noise.html).

An alternative source of variation is intrinsic to the developmental programme itself.  In particular, small, random fluctuations in the expression of genes at various times during development can have large effects on the phenotypic outcome.  A new study in Nature by Raj and colleagues directly illustrates this point for the first time and highlights several important principles of developmental systems. 

They studied the effects of mutations in components of a genetic network involved in the specification of a small number of intestinal cells in the nematode, Caenorhabditis elegans.  This is the perfect organism for such studies, as the cells in question are individually identifiable and generated in an invariant pattern in wild-type animals.  Mutations in one of the components led to an incompletely penetrant mutant phenotype: some animals made intestinal cells and others did not (even though all had the identical geneotype). 

To determine whether noise in gene expression could explain this diversity the authors directly measured the precise number of messenger RNA molecules being transcribed from the genes encoding other components of this developmental pathway in particular cells of each embryo and correlated these measurements with phenotypic outcome.  They showed that the expression of one of these genes in particular became highly variable in the mutant background.  If, by chance, the level of expression crossed a particular threshold it turned on the master gene responsible for intestinal cell specification and these cells were generated.  If the levels did not cross the threshold then the cells were not generated.  In this way, a bimodal phenotypic distribution can arise from an identical starting genotype. 

This study illustrates several important principles of complex regulatory systems that apply not just to developmental and genetic networks but also to neuronal networks.  First, a certain amount of noise is a normal part of the system – a feature, not a bug – that increases robustness to external variation.  Developmental systems are normally buffered, however, to reduce noise in gene expression and to absorb its effects.  This buffering can be disrupted when individual components of a regulatory system are removed; this is why when genes are mutated, one expects (and always sees) not just a change in phenotype but an increase in phenotypic variability.  The effects of stochastic fluctuations in expression levels of various genes can lead to a continuous distribution of phenotypic outcomes or, as in this case, dramatically different phenotypes.  Interlocking positive and negative feedback loops can generate extremely discrete thresholds, where once a certain level of a component is reached it will reinforce its own expression and shift the network into a different state.  Such bistability is a common feature of complex systems and is sometimes taken advantage of to generate phenotypic diversity or plasticity. 

This study elucidates a molecular mechanism of intrinsic variation in developmental systems and shows that it can have a large effect on the eventual phenotype, even in genetically identical organisms.  No matter how precise the recipe, you can’t bake the same cake twice. 


Raj, A. (2010). Variability in Gene Expression Underlies Incomplete Penetrance in C. Elegans: Using Single Molecules To Study the Development of Single Cells Biophysical Journal, 98 (3), 14-14 DOI: 10.1016/j.bpj.2009.12.087

Monday, February 15, 2010

What’s in a name? Genetic overlap between major psychiatric disorders

The criteria used to assign patients to specific psychiatric disease categories are set out in the Diagnostic and Statistical Manual of Mental Disorders, published by the American Psychiatric Association.  (There is also a World Health Organisation equivalent, the International Classification of Disease).  Every so often, these criteria are revised to reflect new research and changing concepts of disease.  The APA has just released a draft of preliminary revisions to the current diagnostic criteria (available at http://www.dsm5.org/) as part of the preparations for the fifth release (DSM-5), due out in 2013. 

The specific diagnosis given to any patient who shows up with a spectrum of symptoms has major implications not only for their clinical treatment but also for insurance, education, employment and many other aspects of their lives.  Given the authority and influence of this tome for clinical practice as well as research purposes, it is timely to consider how genetics is, or is not, informing the diagnostic criteria. 

One example where this question is especially relevant is to debates that have been going on for over a century about the underlying validity of the distinction between schizophrenia and bipolar disorder (and the various names each has gone under over that time).  DSM-IV currently defines these disorders in such a way that they are effectively mutually exclusive, though they share many individual symptoms.  Certain combinations of these symptoms co-occur often enough to warrant a label for the syndrome.  One could argue that the distinction between these two categories is quite valid and clinically useful, in terms of prognosis and treatment.  (Though see “Madness Explained: Psychosis and Human Nature” by Richard P. Bentall for a contrary viewpoint).  On the other hand, the definitions have been explicitly designed in order to separate them at either end of a spectrum, with the intervening spaces filled in with a variety of other categories, such as schizotypal personality disorder or schizoaffective disorder.  While the ends of the spectrum might be quite different, each move along it might be more dimensional than categorical.  

Recent genetic findings are much more congruent with a dimensional distinction than a categorical one and suggest substantial shared etiology between these disorders.  Surprisingly, they also demonstrate overlapping risk for other major psychiatric disorders such as attention deficit hyperactivity disorder, autism, epilepsy and mental retardation (or intellectual disability).

First, very large-scale epidemiological studies have confirmed that psychiatric disorders tend to co-occur generally within families.  Thus, if a person has a first-degree relative with schizophrenia, while their risk of schizophrenia is ten times higher than the average person’s, their risk of bipolar disorder or autism or epilepsy is also dramatically increased.  If these disorders are caused by single mutations (and the field seems to be slowly moving to acceptance of this model), then the implication is that any such mutation may result in a spectrum of symptoms in one individual, which leads to a diagnosis of disorder A, and in a different spectrum in another individual, which leads to a diagnosis of disorder B. 

This model is borne out by the analysis of the effects of mutations in a growing number of specific loci.  These include mutations in single genes such as CNTNAP2, NRXN1, and DISC1, as well as co-called copy number variants (CNVs) – deletions or duplications of chunks of a chromosome that may contain more than one gene – including recurrent CNVs at 1q21.1, 15q11.2, 15q13.3, 16p11.2, 22q11.2 and about thirty others.  In all these cases, such mutations are associated with increased risk not to just one disorder but to many.

These findings raise some crucially important questions: why can so many mutations result in the same disorder (or spectrum of symptoms)? And, conversely, why can one mutation result in quite different spectra of symptoms in different individuals?  The mapping from genotype to phenotype is not direct, at least if phenotype is defined by clinical category.  Clearly, genetic interactions with other mutations in each person’s genome are likely to have an important influence.  Just as clearly, these cannot be the sole determinants of the eventual phenotype as monozygotic twins are often discordant for psychiatric disorders.  While environment and experience may influence phenotype, it is also likely that intrinsic developmental variation plays an important part (See post: “Nature, nurture and noise” on June 24th, 2009 for more on this).  All of these factors are likely to contribute to an individual’s developmental trajectory and eventual phenotypic end-point. 

These findings obviously raise some doubts about the clinical validity of the diagnostic categories.  Perhaps they are overly and arbitrarily defined constructs of limited usefulness that actually hamper medical practice and research.  Alternatively, they may describe truly distinct phenotypic end-points reflecting real differences in current pathophysiology, despite the similar genotypic starting point.  For example, one patient with psychosis may share more in common with another patient with psychosis (and a different genetic etiology) than they do with another carrier of the same mutation who does not have psychosis. 

The draft recommendations for DSM-5 include the welcome addition of dimensional criteria that can be applied across diagnoses to give a quantitative measure of specific symptoms.  Genetics and neuroscience will continue to offer greater insights into pathogenic and pathophysiological mechanisms over the next few years – it will be interesting to see how these can be incorporated into the final version of DSM-5 and what their influence will be on clinical practice.  

 

LICHTENSTEIN, P., YIP, B., BJORK, C., PAWITAN, Y., CANNON, T., SULLIVAN, P., & HULTMAN, C. (2009). Common genetic determinants of schizophrenia and bipolar disorder in Swedish families: a population-based study The Lancet, 373 (9659), 234-239 DOI: 10.1016/S0140-6736(09)60072-6

 

Rzhetsky, A., Wajngurt, D., Park, N., & Zheng, T. (2007). Probing genetic overlap among complex human phenotypes Proceedings of the National Academy of Sciences, 104 (28), 11694-11699 DOI: 10.1073/pnas.0704820104

 

Sebat J, Levy DL, & McCarthy SE (2009). Rare structural variants in schizophrenia: one disorder, multiple mutations; one mutation, multiple disorders. Trends in genetics : TIG, 25 (12), 528-35 PMID: 19883952 

Monday, February 8, 2010

Bad to the bone; altered connections in the brains of psychopaths

The manipulative con-man.  The guy who lies to your face, even when he doesn’t have to.  The child who tortures animals.  The cold-blooded killer.  

psychopath

Psychopaths are characterised by an absence of empathy and poor impulse control, with a total lack of conscience.  About 1% of the total population can be defined as psychopaths, according to a detailed psychological profile checklist.  They tend to be egocentric, callous, manipulative, deceptive, superficial, irresponsible and parasitic, even predatory.  The majority of psychopaths are not violent and many do very well in jobs where their personality traits are advantageous and their social tendencies tolerated.  However, some have a predisposition to calculated, “instrumental” violence; violence that is cold-blooded, planned and goal-directed.  Psychopaths are vastly over-represented among criminals; it is estimated they make up about 20% of the inmates of most prisons.  They commit over half of all violent crimes and are 3-4 times more likely to re-offend.  They are almost entirely refractory to rehabilitation.  These are not nice people. 

So how did they get that way?  Is it an innate biological condition, a result of social experience, or an interaction between these factors?  Longitudinal studies have shown that the personality traits associated with psychopathy are highly stable over time.  Early warning signs including “callous-unemotional traits” and antisocial behaviour can be identified in childhood and are highly predictive of future psychopathy.  Large-scale twin studies have shown that these traits are highly heritable – identical twins, who share 100% of their genes, are much more similar to each other in this trait than fraternal twins, who share only 50% of their genes.  In one study, over 80% of the variation in the callous-unemotional trait across the population was due to genetic differences.  In contrast, the effect of a shared family environment was almost nil.  Psychopathy seems to be a lifelong trait, or combination of traits, which are heavily influenced by genes and hardly at all by social upbringing.

The two defining characteristics of psychopaths, blunted emotional response to negative stimuli, coupled with poor impulse control, can both be measured in psychological and neuroimaging experiments.  Several studies have found decreased responsiveness of the amygdala to fearful or other negative stimuli in psychopaths.  They do not seem to process heavily loaded emotional words, like “rape”, for example, any differently from how they process neutral words, like “table”.  This lack of response to negative stimuli can be measured in other ways, such as the failure to induce a galvanic skin response (heightened skin conduction due to sweating) when faced with an impending electrical shock.  Psychopaths have also been found to underactivate limbic (emotional) regions of the brain during aversive learning, correlating with an insensitivity to negative reinforcement.  The psychopath really just doesn’t care.  In this, psychopaths differ from many people who are prone to sudden, impulsive violence, in that those people tend to have a hypersensitive negative emotional response to what would otherwise be relatively innocuous stimuli. 

What these two groups have in common is poor impulse control.  This faculty relies on the part of the brain called the prefrontal cortex, most particularly the orbitofrontal cortex.  It is known that lesions to this part of the brain impair planning, prediction of consequences, and inhibition of socially unacceptable behaviour – the cognitive mechanisms of “free won’t”, rather than free will.  This brain region is also normally activated by aversive learning, and this activation is also reduced in psychopaths.  In addition, both the prefrontal cortex and the amygdala show substantial average reductions in size in psychopaths, suggesting a structural difference in their brains. 

These findings have now been united by a recent study that directly analysed connectivity between these two regions.  Using diffusion tensor imaging (see post of August 31st 2009), Craig and colleagues found that a measure of the integrity of the axonal tract connecting these two regions, called the uncinate fasciculus, was significantly reduced in psychopaths.  Importantly, connectivity of these regions to other parts of the brain was normal.  These data thus suggest a specific disruption of the network connecting orbitofrontal cortex and amygdala in psychopaths, the degree of which correlated strongly with the subjects’ scores on the psychopathy checklist. 

All of these findings are pointing to a picture of psychopathy as an innate, genetically driven difference in connectivity between parts of the brain that normally drive empathy, conscience and impulse control.  Not a fault necessarily, and not something that could be classified as a disease or that is always a disadvantage.  At a certain frequency in the population, the traits of psychopathy may be highly advantageous to the individual.

This conclusion has serious ethical and legal implications.  Could a psychopath mount a legal defense by saying “my brain made me do it”?  Or my “genes made me do it”?  Is this any different from saying my rotten childhood made me do it?  Psychopaths know right from wrong – they just don’t care.  That is what society calls “bad”, not “mad”.  But if they are constitutionally incapable of caring, can they really be blamed for it?  On the other hand, if violent psychopaths are a continuing danger to society and completely refractory to rehabilitation, what is to be done with them?  Perhaps, as has been proposed in the UK, people with the extreme psychopathic personality profile (or maybe in the near future even a specific genetic profile?) should be monitored or segregated even before they commit a crime. 

While it is crucial that these debates are informed by good science, these issues have no clear-cut answers.  They will be resolved on a pragmatic basis, weighing the behaviour that society is willing to tolerate versus the rights of the individual, whatever their brains look like, to define their own moral standards. 

 

 

Craig, M., Catani, M., Deeley, Q., Latham, R., Daly, E., Kanaan, R., Picchioni, M., McGuire, P., Fahy, T., & Murphy, D. (2009). Altered connections on the road to psychopathy Molecular Psychiatry, 14 (10), 946-953 DOI:

Tuesday, February 2, 2010

Instant Neurons

A new study from Marius Wernig and colleagues at Stanford University has succeeded in transforming fibroblast cells directly into neurons. In the end, it was far simpler than expected. The identities of different cell types are known to be established during development by particular combinations of transcription factors, which, by controlling the expression of large numbers of target genes, define the genetic and biochemical profile of each cell type. It was thought that this profile of gene expression was then locked into place by chromatin proteins which bind to DNA and fix it into an active or inactive conformation. Over development, the passage from an early embryonic cell, with full potential to generate any cell type, to more differentiated cells, was believed to be unidirectional and irreversible.

The ability to clone an animal, by transferring the nucleus of a differentiated cell into a fertilised, enucleated oocyte, showed that, in fact, the profile of gene expression could be reversed, wiped clean and restored to a pristine, undifferentiated state. Groundbreaking experiments over the last few years, led by Shinya Yamanaka and colleagues, have established an even simpler method to take a differentiated somatic cell and convert it back to an undifferentiated state, a so-called induced pluripotent stem cell (iPS cell). This is achieved by forcing the expression of a set of transcription factors that define the stem cell fate – in essence, driving the biochemical profile to a stem cell pattern. Auto- and cross-regulatory interactions between these transcription factors and others in the cell ensure a stable transformation of cell identity. So, one can take a differentiated cell and reverse the direction of development, driving it back to an undifferentiated state. This is obviously a hugely powerful technique as the iPS cells can then be differentiated in vitro into many other cell types, for research or clinical purposes.

What was not clear, however, was how general this strategy might be. Was there something special about the stem cell fate or could the same approach be used to convert one differentiated cell-type directly into another, without having to go via a stem cell intermediate? Wernig and colleagues have now shown that this can be done.

They forced fibroblasts, derived from either embryonic or adult mice, to express a combination of three transcription factors which characterise a general neuronal fate, by transfecting them with lentiviruses carrying these genes. These genes, Ascl, Brn2 and Mytl1, were defined from an initial set of nineteen candidates based on genes known to be important in neuronal differentiation during development. Transfection with these genes very efficiently, over a period of several days, transformed the fibroblasts into neurons. They express neuronal proteins, show a neuronal morphology, elaborate axons and dendrites, have characteristic electrical membrane properties, fire action potentials and even synapse with each other. By all possible criteria, these are neurons. In particular, 99% of them seem to be glutamatergic and many express markers characteristic of cortical neurons.

One can expect a flurry of follow-on studies using different combinations of transcription factors to drive cells into a variety of more specific neuronal subtypes – likely to include dopaminergic neurons, motor neurons, photoreceptors and many others – as well as non-neuronal cell types.

There is every reason to expect the same approach will work with human cells, as was the case with the iPS cell technology. This will offer a rapid and very efficient method to generate a large supply of neurons from individuals with their own genotype. These are most obviously of possible use in cell replacement therapies for neurodegenerative disease, though the prospect of this in clinical practice remains some years off.

The value for clinical research is also immense, and likely more immediate. The ability to grow neurons with the genotypes of patients with psychiatric or neurological disorders, for example, offers a kind of virtual biopsy – access to neurons of the patient previously unobtainable. This will allow analysis of the possible genetic defects in each patient and their cellular consequences, likely to be invaluable in elucidating pathogenic mechanisms at a molecular level.

The future is now.


Vierbuchen, T., Ostermeier, A., Pang, Z., Kokubu, Y., S├╝dhof, T., & Wernig, M. (2010). Direct conversion of fibroblasts to functional neurons by defined factors Nature DOI: 10.1038/nature08797


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