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doi: 10.1038/500121a

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doi: 10.1038/500121b

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doi: 10.1038/500129a

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doi: 10.1038/500130a

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doi: 10.1038/500131a

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doi: 10.1038/500132a

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doi: 10.1038/500135a

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doi: 10.1038/500136a

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doi: 10.1038/500138a

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doi: 10.1038/500154a

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doi: 10.1038/nature12462

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doi: 10.1038/nature12461

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doi: 10.1038/500158a

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doi: 10.1038/500159a

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doi: 10.1038/500160a

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doi: 10.1038/nature12429

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doi: 10.1038/nature12346

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doi: 10.1038/nature12450

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Saturn’s moon Enceladus emits a plume of water vapour and micrometre-sized ice particles from a series of warm fissures located near its south pole. This geological activity could be powered or controlled by variations in the tidal stresses experienced by Enceladus as it moves around its slightly eccentric orbit. The specific mechanisms by which these varying stresses are converted into heat, however, are still being debated. Furthermore, it has proved difficult to find a clear correlation between the predicted tidal forces and measured temporal variations in the plume’s gas content or the particle flux from individual sources. Here we report that the plume’s horizontally integrated brightness is several times greater when Enceladus is near the point in its eccentric orbit where it is furthest from Saturn (apocentre) than it is when near the point of closest approach to the planet (pericentre). More material therefore seems to be escaping from beneath Enceladus’ surface at times when geophysical models predict its fissures should be under tension and therefore may be wider open.

doi: 10.1038/nature12371

p.185

Monitoring a mechanical object’s motion, even with the gentle touch of light, fundamentally alters its dynamics. The experimental manifestation of this basic principle of quantum mechanics, its link to the quantum nature of light and the extension of quantum measurement to the macroscopic realm have all received extensive attention over the past half-century. The use of squeezed light, with quantum fluctuations below that of the vacuum field, was proposed nearly three decades ago as a means of reducing the optical read-out noise in precision force measurements. Conversely, it has also been proposed that a continuous measurement of a mirror’s position with light may itself give rise to squeezed light. Such squeezed-light generation has recently been demonstrated in a system of ultracold gas-phase atoms whose centre-of-mass motion is analogous to the motion of a mirror. Here we describe the continuous position measurement of a solid-state, optomechanical system fabricated from a silicon microchip and comprising a micromechanical resonator coupled to a nanophotonic cavity. Laser light sent into the cavity is used to measure the fluctuations in the position of the mechanical resonator at a measurement rate comparable to its resonance frequency and greater than its thermal decoherence rate. Despite the mechanical resonator’s highly excited thermal state (104 phonons), we observe, through homodyne detection, squeezing of the reflected light’s fluctuation spectrum at a level 4.5 ± 0.2 per cent below that of vacuum noise over a bandwidth of a few megahertz around the mechanical resonance frequency of 28 megahertz. With further device improvements, on-chip squeezing at significant levels should be possible, making such integrated microscale devices well suited for precision metrology applications.

doi: 10.1038/nature12307

p.190

The growth and reduction of Northern Hemisphere ice sheets over the past million years is dominated by an approximately 100,000-year periodicity and a sawtooth pattern (gradual growth and fast termination). Milankovitch theory proposes that summer insolation at high northern latitudes drives the glacial cycles, and statistical tests have demonstrated that the glacial cycles are indeed linked to eccentricity, obliquity and precession cycles. Yet insolation alone cannot explain the strong 100,000-year cycle, suggesting that internal climatic feedbacks may also be at work. Earlier conceptual models, for example, showed that glacial terminations are associated with the build-up of Northern Hemisphere ‘excess ice’, but the physical mechanisms underpinning the 100,000-year cycle remain unclear. Here we show, using comprehensive climate and ice-sheet models, that insolation and internal feedbacks between the climate, the ice sheets and the lithosphere–asthenosphere system explain the 100,000-year periodicity. The responses of equilibrium states of ice sheets to summer insolation show hysteresis, with the shape and position of the hysteresis loop playing a key part in determining the periodicities of glacial cycles. The hysteresis loop of the North American ice sheet is such that after inception of the ice sheet, its mass balance remains mostly positive through several precession cycles, whose amplitudes decrease towards an eccentricity minimum. The larger the ice sheet grows and extends towards lower latitudes, the smaller is the insolation required to make the mass balance negative. Therefore, once a large ice sheet is established, a moderate increase in insolation is sufficient to trigger a negative mass balance, leading to an almost complete retreat of the ice sheet within several thousand years. This fast retreat is governed mainly by rapid ablation due to the lowered surface elevation resulting from delayed isostatic rebound, which is the lithosphere–asthenosphere response. Carbon dioxide is involved, but is not determinative, in the evolution of the 100,000-year glacial cycles.

doi: 10.1038/nature12374

p.194

Ninety per cent of marine organic matter burial occurs in continental margin sediments, where a substantial fraction of organic carbon escapes oxidation and enters long-term geologic storage within sedimentary rocks. In such environments, microbial metabolism is limited by the diffusive supply of electron acceptors. One strategy to optimize energy yields in a resource-limited habitat is symbiotic metabolite exchange among microbial associations. Thermodynamic and geochemical considerations indicate that microbial co-metabolisms are likely to play a critical part in sedimentary organic carbon cycling. Yet only one association, between methanotrophic archaea and sulphate-reducing bacteria, has been demonstrated in marine sediments in situ, and little is known of the role of microbial symbiotic interactions in other sedimentary biogeochemical cycles. Here we report in situ molecular and incubation-based evidence for a novel symbiotic consortium between two chemolithotrophic bacteria—anaerobic ammonium-oxidizing (anammox) bacteria and the nitrate-sequestering sulphur-oxidizing Thioploca species—in anoxic sediments of the Soledad basin at the Mexican Pacific margin. A mass balance of benthic solute fluxes and the corresponding nitrogen isotope composition of nitrate and ammonium fluxes indicate that anammox bacteria rely on Thioploca species for the supply of metabolic substrates and account for about 57 ± 21 per cent of the total benthic N₂ production. We show that Thioploca–anammox symbiosis intensifies benthic fixed nitrogen losses in anoxic sediments, bypassing diffusion-imposed limitations by efficiently coupling the carbon, nitrogen and sulphur cycles.

doi: 10.1038/nature12365

p.199

A major unsolved problem in mammalian evolution is the origin of Allotheria, including Multituberculata and Haramiyida. Multituberculates are the most diverse and best known Mesozoic era mammals and ecologically resemble rodents, but haramiyids are known mainly from isolated teeth, hampering our search for their phylogenetic relationships. Here we report a new haramiyid from the Jurassic period of China, which is, to our knowledge the largest reported so far. It has a novel dentition, a mandible resembling advanced multituberculates and postcranial features adapted for arboreal life. Our phylogenetic analysis places Haramiyida within crown Mammalia, suggesting the origin of crown Mammalia in the Late Triassic period and diversification in the Jurassic, which contrasts other estimated divergence times of crown Mammalia. The new haramiyid reveals additional mammalian features of the group, helps to identify other haramiyids represented by isolated teeth, and shows again that, regardless of various phylogenetic scenarios, a complex pattern of evolution involving many convergences and/or reversals existed in Mesozoic mammals.

doi: 10.1038/nature12353

p.203

Some evolutionary innovations may originate non-adaptively as exaptations, or pre-adaptations, which are by-products of other adaptive traits. Examples include feathers, which originated before they were used in flight, and lens crystallins, which are light-refracting proteins that originated as enzymes. The question of how often adaptive traits have non-adaptive origins has profound implications for evolutionary biology, but is difficult to address systematically. Here we consider this issue in metabolism, one of the most ancient biological systems that is central to all life. We analyse a metabolic trait of great adaptive importance: the ability of a metabolic reaction network to synthesize all biomass from a single source of carbon and energy. We use novel computational methods to sample randomly many metabolic networks that can sustain life on any given carbon source but contain an otherwise random set of known biochemical reactions. We show that when we require such networks to be viable on one particular carbon source, they are typically also viable on multiple other carbon sources that were not targets of selection. For example, viability on glucose may entail viability on up to 44 other sole carbon sources. Any one adaptation in these metabolic systems typically entails multiple potential exaptations. Metabolic systems thus contain a latent potential for evolutionary innovations with non-adaptive origins. Our observations suggest that many more metabolic traits may have non-adaptive origins than is appreciated at present. They also challenge our ability to distinguish adaptive from non-adaptive traits.

doi: 10.1038/nature12301

p.207

The HeLa cell line was established in 1951 from cervical cancer cells taken from a patient, Henrietta Lacks. This was the first successful attempt to immortalize human-derived cells in vitro. The robust growth and unrestricted distribution of HeLa cells resulted in its broad adoption—both intentionally and through widespread cross-contamination—and for the past 60 years it has served a role analogous to that of a model organism. The cumulative impact of the HeLa cell line on research is demonstrated by its occurrence in more than 74,000 PubMed abstracts (approximately 0.3%). The genomic architecture of HeLa remains largely unexplored beyond its karyotype, partly because like many cancers, its extensive aneuploidy renders such analyses challenging. We carried out haplotype-resolved whole-genome sequencing of the HeLa CCL-2 strain, examined point- and indel-mutation variations, mapped copy-number variations and loss of heterozygosity regions, and phased variants across full chromosome arms. We also investigated variation and copy-number profiles for HeLa S3 and eight additional strains. We find that HeLa is relatively stable in terms of point variation, with few new mutations accumulating after early passaging. Haplotype resolution facilitated reconstruction of an amplified, highly rearranged region of chromosome 8q24.21 at which integration of the human papilloma virus type 18 (HPV-18) genome occurred and that is likely to be the event that initiated tumorigenesis. We combined these maps with RNA-seq and ENCODE Project data sets to phase the HeLa epigenome. This revealed strong, haplotype-specific activation of the proto-oncogene MYC by the integrated HPV-18 genome approximately 500 kilobases upstream, and enabled global analyses of the relationship between gene dosage and expression. These data provide an extensively phased, high-quality reference genome for past and future experiments relying on HeLa, and demonstrate the value of haplotype resolution for characterizing cancer genomes and epigenomes.

doi: 10.1038/nature12064

p.212

The extraction of directional motion information from changing retinal images is one of the earliest and most important processing steps in any visual system. In the fly optic lobe, two parallel processing streams have been anatomically described, leading from two first-order interneurons, L1 and L2, via T4 and T5 cells onto large, wide-field motion-sensitive interneurons of the lobula plate. Therefore, T4 and T5 cells are thought to have a pivotal role in motion processing; however, owing to their small size, it is difficult to obtain electrical recordings of T4 and T5 cells, leaving their visual response properties largely unknown. We circumvent this problem by means of optical recording from these cells in Drosophila, using the genetically encoded calcium indicator GCaMP5 (ref. 2). Here we find that specific subpopulations of T4 and T5 cells are directionally tuned to one of the four cardinal directions; that is, front-to-back, back-to-front, upwards and downwards. Depending on their preferred direction, T4 and T5 cells terminate in specific sublayers of the lobula plate. T4 and T5 functionally segregate with respect to contrast polarity: whereas T4 cells selectively respond to moving brightness increments (ON edges), T5 cells only respond to moving brightness decrements (OFF edges). When the output from T4 or T5 cells is blocked, the responses of postsynaptic lobula plate neurons to moving ON (T4 block) or OFF edges (T5 block) are selectively compromised. The same effects are seen in turning responses of tethered walking flies. Thus, starting with L1 and L2, the visual input is split into separate ON and OFF pathways, and motion along all four cardinal directions is computed separately within each pathway. The output of these eight different motion detectors is then sorted such that ON (T4) and OFF (T5) motion detectors with the same directional tuning converge in the same layer of the lobula plate, jointly providing the input to downstream circuits and motion-driven behaviours.

doi: 10.1038/nature12320

p.217

The inner ear contains sensory epithelia that detect head movements, gravity and sound. It is unclear how to develop these sensory epithelia from pluripotent stem cells, a process that will be critical for modelling inner ear disorders or developing cell-based therapies for profound hearing loss and balance disorders. So far, attempts to derive inner ear mechanosensitive hair cells and sensory neurons have resulted in inefficient or incomplete phenotypic conversion of stem cells into inner-ear-like cells. A key insight lacking from these previous studies is the importance of the non-neural and preplacodal ectoderm, two critical precursors during inner ear development. Here we report the stepwise differentiation of inner ear sensory epithelia from mouse embryonic stem cells (ESCs) in three-dimensional culture. We show that by recapitulating in vivo development with precise temporal control of signalling pathways, ESC aggregates transform sequentially into non-neural, preplacodal and otic-placode-like epithelia. Notably, in a self-organized process that mimics normal development, vesicles containing prosensory cells emerge from the presumptive otic placodes and give rise to hair cells bearing stereocilia bundles and a kinocilium. Moreover, these stem-cell-derived hair cells exhibit functional properties of native mechanosensitive hair cells and form specialized synapses with sensory neurons that have also arisen from ESCs in the culture. Finally, we demonstrate how these vesicles are structurally and biochemically comparable to developing vestibular end organs. Our data thus establish a new in vitro model of inner ear differentiation that can be used to gain deeper insight into inner ear development and disorder.

doi: 10.1038/nature12298

p.222

DNA methylation is a heritable epigenetic modification involved in gene silencing, imprinting, and the suppression of retrotransposons. Global DNA demethylation occurs in the early embryo and the germ line, and may be mediated by Tet (ten eleven translocation) enzymes, which convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Tet enzymes have been studied extensively in mouse embryonic stem (ES) cells, which are generally cultured in the absence of vitamin C, a potential cofactor for Fe(ii) 2-oxoglutarate dioxygenase enzymes such as Tet enzymes. Here we report that addition of vitamin C to mouse ES cells promotes Tet activity, leading to a rapid and global increase in 5hmC. This is followed by DNA demethylation of many gene promoters and upregulation of demethylated germline genes. Tet1 binding is enriched near the transcription start site of genes affected by vitamin C treatment. Importantly, vitamin C, but not other antioxidants, enhances the activity of recombinant Tet1 in a biochemical assay, and the vitamin-C-induced changes in 5hmC and 5mC are entirely suppressed in Tet1 and Tet2 double knockout ES cells. Vitamin C has a stronger effect on regions that gain methylation in cultured ES cells compared to blastocysts, and in vivo are methylated only after implantation. In contrast, imprinted regions and intracisternal A particle retroelements, which are resistant to demethylation in the early embryo, are resistant to vitamin-C-induced DNA demethylation. Collectively, the results of this study establish vitamin C as a direct regulator of Tet activity and DNA methylation fidelity in ES cells.

doi: 10.1038/nature12362

p.227

The newly emergent Middle East respiratory syndrome coronavirus (MERS-CoV) can cause severe pulmonary disease in humans, representing the second example of a highly pathogenic coronavirus, the first being SARS-CoV. CD26 (also known as dipeptidyl peptidase 4, DPP4) was recently identified as the cellular receptor for MERS-CoV. The engagement of the MERS-CoV spike protein with CD26 mediates viral attachment to host cells and virus–cell fusion, thereby initiating infection. Here we delineate the molecular basis of this specific interaction by presenting the first crystal structures of both the free receptor binding domain (RBD) of the MERS-CoV spike protein and its complex with CD26. Furthermore, binding between the RBD and CD26 is measured using real-time surface plasmon resonance with a dissociation constant of 16.7 nM. The viral RBD is composed of a core subdomain homologous to that of the SARS-CoV spike protein, and a unique strand-dominated external receptor binding motif that recognizes blades IV and V of the CD26 β-propeller. The atomic details at the interface between the two binding entities reveal a surprising protein–protein contact mediated mainly by hydrophilic residues. Sequence alignment indicates, among betacoronaviruses, a possible structural conservation for the region homologous to the MERS-CoV RBD core, but a high variation in the external receptor binding motif region for virus-specific pathogenesis such as receptor recognition.

doi: 10.1038/nature12328

p.232

Manipulation of the gut microbiota holds great promise for the treatment of inflammatory and allergic diseases. Although numerous probiotic microorganisms have been identified, there remains a compelling need to discover organisms that elicit more robust therapeutic responses, are compatible with the host, and can affect a specific arm of the host immune system in a well-controlled, physiological manner. Here we use a rational approach to isolate CD4+FOXP3+ regulatory T (T_reg)-cell-inducing bacterial strains from the human indigenous microbiota. Starting with a healthy human faecal sample, a sequence of selection steps was applied to obtain mice colonized with human microbiota enriched in T_reg-cell-inducing species. From these mice, we isolated and selected 17 strains of bacteria on the basis of their high potency in enhancing T_reg cell abundance and inducing important anti-inflammatory molecules—including interleukin-10 (IL-) and inducible T-cell co-stimulator (ICOS)—in T_reg cells upon inoculation into germ-free mice. Genome sequencing revealed that the 17 strains fall within clusters IV, XIVa and XVIII of Clostridia, which lack prominent toxins and virulence factors. The 17 strains act as a community to provide bacterial antigens and a TGF-β-rich environment to help expansion and differentiation of T_reg cells. Oral administration of the combination of 17 strains to adult mice attenuated disease in models of colitis and allergic diarrhoea. Use of the isolated strains may allow for tailored therapeutic manipulation of human immune disorders.

doi: 10.1038/nature12331

p.237

Cellular metabolism converts available nutrients into usable energy and biomass precursors. The process is regulated to facilitate efficient nutrient use and metabolic homeostasis. Feedback inhibition of the first committed step of a pathway by its final product is a classical means of controlling biosynthesis. In a canonical example, the first committed enzyme in the pyrimidine pathway in Escherichia coli is allosterically inhibited by cytidine triphosphate. The physiological consequences of disrupting this regulation, however, have not been previously explored. Here we identify an alternative regulatory strategy that enables precise control of pyrimidine pathway end-product levels, even in the presence of dysregulated biosynthetic flux. The mechanism involves cooperative feedback regulation of the near-terminal pathway enzyme uridine monophosphate kinase. Such feedback leads to build-up of the pathway intermediate uridine monophosphate, which is in turn degraded by a conserved phosphatase, here termed UmpH, with previously unknown physiological function. Such directed overflow metabolism allows homeostasis of uridine triphosphate and cytidine triphosphate levels at the expense of uracil excretion and slower growth during energy limitation. Disruption of the directed overflow regulatory mechanism impairs growth in pyrimidine-rich environments. Thus, pyrimidine homeostasis involves dual regulatory strategies, with classical feedback inhibition enhancing metabolic efficiency and directed overflow metabolism ensuring end-product homeostasis.

doi: 10.1038/nature12445