De novo mutations revealed by whole-exome sequencing are strongly associated with autism

Multiple studies have confirmed the contribution of rare de novo copy number variations to the risk for autism spectrum disorders. But whereas de novo single nucleotide variants have been identified in affected individuals, their contribution to risk has yet to be clarified. Specifically, the frequency and distribution of these mutations have not been well characterized in matched unaffected controls, and such data are vital to the interpretation of de novo coding mutations observed in probands. Here we show, using whole-exome sequencing of 928 individuals, including 200 phenotypically discordant sibling pairs, that highly disruptive (nonsense and splice-site) de novo mutations in brain-expressed genes are associated with autism spectrum disorders and carry large effects. On the basis of mutation rates in unaffected individuals, we demonstrate that multiple independent de novo single nucleotide variants in the same gene among unrelated probands reliably identifies risk alleles, providing a clear path forward for gene discovery. Among a total of 279 identified de novo coding mutations, there is a single instance in probands, and none in siblings, in which two independent nonsense variants disrupt the same gene, SCN2A (sodium channel, voltage-gated, type II, α subunit), a result that is highly unlikely by chance.     

Immune self-reactivity triggered by drug-modified HLA-peptide repertoire

Human leukocyte antigens (HLAs) are highly polymorphic proteins that initiate immunity by presenting pathogen-derived peptides to T?cells. HLA polymorphisms mostly map to the antigen-binding cleft, thereby diversifying the repertoire of self-derived and pathogen-derived peptide antigens selected by different HLA allotypes. A growing number of immunologically based drug reactions, including abacavir hypersensitivity syndrome (AHS) and carbamazepine-induced Stevens-Johnson syndrome (SJS), are associated with specific HLA alleles. However, little is known about the underlying mechanisms of these associations, including AHS, a prototypical HLA-associated drug reaction occurring exclusively in individuals with the common histocompatibility allele HLA-B*57:01, and with a relative risk of more than 1,000 (refs?6, 7). We show that unmodified abacavir binds non-covalently to HLA-B*57:01, lying across the bottom of the antigen-binding cleft and reaching into the F-pocket, where a carboxy-terminal tryptophan typically anchors peptides bound to HLA-B*57:01. Abacavir binds with exquisite specificity to HLA-B*57:01, changing the shape and chemistry of the antigen-binding cleft, thereby altering the repertoire of endogenous peptides that can bind HLA-B*57:01. In this way, abacavir guides the selection of new endogenous peptides, inducing a marked alteration in 'immunological self'. The resultant peptide-centric 'altered self' activates abacavir-specific T-cells, thereby driving polyclonal CD8 T-cell activation and a systemic reaction manifesting as AHS. We also show that carbamazepine, a widely used anti-epileptic drug associated with hypersensitivity reactions in HLA-B*15:02 individuals, binds to this allotype, producing alterations in the repertoire of presented self peptides. Our findings simultaneously highlight the importance of HLA polymorphism in the evolution of pharmacogenomics and provide a general mechanism for some of the growing number of HLA-linked hypersensitivities that involve small-molecule drugs.     

Reactive oxygen species have a causal role in multiple forms of insulin resistance

Insulin resistance is a cardinal feature of type 2 diabetes and is characteristic of a wide range of other clinical and experimental settings. Little is known about why insulin resistance occurs in so many contexts. Do the various insults that trigger insulin resistance act through a common mechanism? Or, as has been suggested, do they use distinct cellular pathways? Here we report a genomic analysis of two cellular models of insulin resistance, one induced by treatment with the cytokine tumour-necrosis factor-alpha and the other with the glucocorticoid dexamethasone. Gene expression analysis suggests that reactive oxygen species (ROS) levels are increased in both models, and we confirmed this through measures of cellular redox state. ROS have previously been proposed to be involved in insulin resistance, although evidence for a causal role has been scant. We tested this hypothesis in cell culture using six treatments designed to alter ROS levels, including two small molecules and four transgenes; all ameliorated insulin resistance to varying degrees. One of these treatments was tested in obese, insulin-resistant mice and was shown to improve insulin sensitivity and glucose homeostasis. Together, our findings suggest that increased ROS levels are an important trigger for insulin resistance in numerous settings.     

Mechanochemical analysis of DNA gyrase using rotor bead tracking

DNA gyrase is a molecular machine that uses the energy of ATP hydrolysis to introduce essential negative supercoils into DNA. The directionality of supercoiling is ensured by chiral wrapping of the DNA around a specialized domain of the enzyme before strand passage. Here we observe the activity of gyrase in real time by tracking the rotation of a submicrometre bead attached to the side of a stretched DNA molecule. In the presence of gyrase and ATP, we observe bursts of rotation corresponding to the processive, stepwise introduction of negative supercoils in strict multiples of two. Changes in DNA tension have no detectable effect on supercoiling velocity, but the enzyme becomes markedly less processive as tension is increased over a range of only a few tenths of piconewtons. This behaviour is quantitatively explained by a simple mechanochemical model in which processivity depends on a kinetic competition between dissociation and rapid, tension-sensitive DNA wrapping. In a high-resolution variant of our assay, we directly detect rotational pauses corresponding to two kinetic substeps: an ATP-independent step at the end of the reaction cycle, and an ATP-binding step in the middle of the cycle, subsequent to DNA wrapping.     

Silent spread of H5N1 in vaccinated poultry

International debate on the merits of vaccinating poultry against the H5N1 influenza A virus has raised concerns about the possibility of an increased risk of between-flock transmission before outbreaks are detected. Here we show that this 'silent spread' can occur because of incomplete protection at the flock level, even if a vaccine is effective in individual birds. The use of unvaccinated sentinels can mitigate, although not completely eliminate, the problem.     

The receptors and cells for mammalian taste

The emerging picture of taste coding at the periphery is one of elegant simplicity. Contrary to what was generally believed, it is now clear that distinct cell types expressing unique receptors are tuned to detect each of the five basic tastes: sweet, sour, bitter, salty and umami. Importantly, receptor cells for each taste quality function as dedicated sensors wired to elicit stereotypic responses.     

A bottom-up approach to gene regulation

The ability to construct synthetic gene networks enables experimental investigations of deliberately simplified systems that can be compared to qualitative and quantitative models. If simple, well-characterized modules can be coupled together into more complex networks with behaviour that can be predicted from that of the individual components, we may begin to build an understanding of cellular regulatory processes from the 'bottom up'. Here we have engineered a promoter to allow simultaneous repression and activation of gene expression in Escherichia coli. We studied its behaviour in synthetic gene networks under increasingly complex conditions: unregulated, repressed, activated, and simultaneously repressed and activated. We develop a stochastic model that quantitatively captures the means and distributions of the expression from the engineered promoter of this modular system, and show that the model can be extended and used to accurately predict the in vivo behaviour of the network when it is expanded to include positive feedback. The model also reveals the counterintuitive prediction that noise in protein expression levels can increase upon arrest of cell growth and division, which we confirm experimentally. This work shows that the properties of regulatory subsystems can be used to predict the behaviour of larger, more complex regulatory networks, and that this bottom-up approach can provide insights into gene regulation.     

High resolution imaging using scanning ion conductance microscopy with improved distance feedback control

Microscopy is an essential technique for observation on living cells. There is currently great interest in applying scanning probe microscopy to image-living biological cells in their natural environment at the nanometer scale. Scanning ion conductance microscopy is a new form of scanning probe microscopy, which enables non-contact high-resolution imaging of living biological cells. Based on a scanned nanopipette in physiological buffer, the distance feedback control uses the ion current to control the distance between the pipette tip and the sample surface. However, this feedback control has difficulties over slopes on convoluted cell surfaces, which limits its resolution. In this study, we present an improved form of feedback control that removes the contribution of up to the third-order slope from the ion current signal, hence providing a more accurate signal for controlling the distance. We show that this allows faster and lower noise topographic high-resolution imaging.