Volume 501 Number 7466
Sequenced from the start p.135
Four US studies are set to explore how genomic data can best help healthy and ill newborns. They must also settle some questions of ethics.
Under threat p.135
The grey wolf is at risk of losing its endangered status under US law.
Reality at risk p.136
Don’t treat a memoir as anything other than one person’s interpretation of events.
Grey wolves left out in the cold p.143
US plan to remove federal protection elicits howls of protest.
Physicists net fractal butterfly p.144
Decades-old search closes in on recursive pattern that describes electron behaviour.
Hackles rise over privatization plan p.145
UK Natural Environment Research Council proposes to cut four institutes loose, but scientists fear for long-term data.
Secrets of fracking fluids pave way for cleaner recipe p.146
Disclosure of chemicals used in hydraulic fracturing will empower green chemistry.
More cuts loom for US science p.147
Stalemate in Congress puts spending plans on hold.
Ultimate upgrade for US synchrotron p.148
Argonne lab banks on beam-bending magnets in bid for world’s most focused X-ray light source.
Taxonomy: The spy who loved frogs p.150
Physics: Quantum quest p.154
News & Views
Social science: The mathematics of murder p.170
Biological techniques: An embryonic view of tumour development p.171
Materials science: Boosting X-ray emission p.172
Immunology: Lipopolysaccharide sensing on the inside p.173
Geochemistry: Sulphur from heaven and hell p.175
Astrochemistry: Extracts of meteorite p.177
Computational biology: A recipe for ligand-binding proteins p.177
Social reward requires coordinated activity of nucleus accumbens oxytocin and serotonin p.179
In male mice oxytocin acts as a social reinforcement signal within the nucleus accumbens (NAc) core, where it elicits a presynaptically expressed long-term depression (LTD) of excitatory synaptic transmission in medium spiny neurons; deletion of oxytocin receptors from the dorsal raphe nucleus, which provides serotonergic innervation of the NAc, and blockade of NAc serotonin 1B receptors both prevent oxytocin-induced LTD and social reward.
RNAi screens in mice identify physiological regulators of oncogenic growth p.185
Here, the first genome-wide in vivo RNA interference screens in a mammalian animal model are reported: genes involved in normal and abnormal epithelial cell growth are studied in developing skin tissue in mouse embryos, and among the findings, β-catenin is shown to act as an antagonist to normal epithelial cell growth as well as promoting oncogene-driven growth.
Stimulated X-ray emission for materials science p.191
Resonant inelastic X-ray scattering and X-ray emission spectroscopy can be used to probe the energy and dispersion of the elementary low-energy excitations that govern functionality in matter: vibronic, charge, spin and orbital excitations. A key drawback of resonant inelastic X-ray scattering has been the need for high photon densities to compensate for fluorescence yields of less than a per cent for soft X-rays. Sample damage from the dominant non-radiative decays thus limits the materials to which such techniques can be applied and the spectral resolution that can be obtained. A means of improving the yield is therefore highly desirable. Here we demonstrate stimulated X-ray emission for crystalline silicon at photon densities that are easily achievable with free-electron lasers. The stimulated radiative decay of core excited species at the expense of non-radiative processes reduces sample damage and permits narrow-bandwidth detection in the directed beam of stimulated radiation. We deduce how stimulated X-ray emission can be enhanced by several orders of magnitude to provide, with high yield and reduced sample damage, a superior probe for low-energy excitations and their dispersion in matter. This is the first step to bringing nonlinear X-ray physics in the condensed phase from theory to application.
Stereoinversion of tertiary alcohols to tertiary-alkyl isonitriles and amines p.195
The SN2 reaction (bimolecular nucleophilic substitution) is a well-known chemical transformation that can be used to join two smaller molecules together into a larger molecule or to exchange one functional group for another. The SN2 reaction proceeds in a very predictable manner: substitution occurs with inversion of stereochemistry, resulting from the ‘backside attack’ of the electrophilic carbon by the nucleophile. A significant limitation of the SN2 reaction is its intolerance for tertiary carbon atoms: whereas primary and secondary alcohols are viable precursor substrates, tertiary alcohols and their derivatives usually either fail to react or produce stereochemical mixtures of products. Here we report the stereochemical inversion of chiral tertiary alcohols with a nitrogenous nucleophile facilitated by a Lewis-acid-catalysed solvolysis. The method is chemoselective against secondary and primary alcohols, thereby complementing the selectivity of the SN2 reaction. Furthermore, this method for carbon–nitrogen bond formation mimics a putative biosynthetic step in the synthesis of marine terpenoids and enables their preparation from the corresponding terrestrial terpenes. We expect that the general attributes of the methodology will allow chiral tertiary alcohols to be considered viable substrates for stereoinversion reactions.
Changes in North Atlantic nitrogen fixation controlled by ocean circulation p.200
In the ocean, the chemical forms of nitrogen that are readily available for biological use (known collectively as ‘fixed’ nitrogen) fuel the global phytoplankton productivity that exports carbon to the deep ocean. Accordingly, variation in the oceanic fixed nitrogen reservoir has been proposed as a cause of glacial–interglacial changes in atmospheric carbon dioxide concentration. Marine nitrogen fixation, which produces most of the ocean’s fixed nitrogen, is thought to be affected by multiple factors, including ocean temperature and the availability of iron and phosphorus. Here we reconstruct changes in North Atlantic nitrogen fixation over the past 160,000 years from the shell-bound nitrogen isotope ratio (15N/14N) of planktonic foraminifera in Caribbean Sea sediments. The observed changes cannot be explained by reconstructed changes in temperature, the supply of (iron-bearing) dust or water column denitrification. We identify a strong, roughly 23,000-year cycle in nitrogen fixation and suggest that it is a response to orbitally driven changes in equatorial Atlantic upwelling, which imports ‘excess’ phosphorus (phosphorus in stoichiometric excess of fixed nitrogen) into the tropical North Atlantic surface. In addition, we find that nitrogen fixation was reduced during glacial stages 6 and 4, when North Atlantic Deep Water had shoaled to become glacial North Atlantic intermediate water, which isolated the Atlantic thermocline from excess phosphorus-rich mid-depth waters that today enter from the Southern Ocean. Although modern studies have yielded diverse views of the controls on nitrogen fixation, our palaeobiogeochemical data suggest that excess phosphorus is the master variable in the North Atlantic Ocean and indicate that the variations in its supply over the most recent glacial cycle were dominated by the response of regional ocean circulation to the orbital cycles.
Retardation of arsenic transport through a Pleistocene aquifer p.204
Groundwater drawn daily from shallow alluvial sands by millions of wells over large areas of south and southeast Asia exposes an estimated population of over a hundred million people to toxic levels of arsenic. Holocene aquifers are the source of widespread arsenic poisoning across the region. In contrast, Pleistocene sands deposited in this region more than 12,000 years ago mostly do not host groundwater with high levels of arsenic. Pleistocene aquifers are increasingly used as a safe source of drinking water and it is therefore important to understand under what conditions low levels of arsenic can be maintained. Here we reconstruct the initial phase of contamination of a Pleistocene aquifer near Hanoi, Vietnam. We demonstrate that changes in groundwater flow conditions and the redox state of the aquifer sands induced by groundwater pumping caused the lateral intrusion of arsenic contamination more than 120 metres from a Holocene aquifer into a previously uncontaminated Pleistocene aquifer. We also find that arsenic adsorbs onto the aquifer sands and that there is a 16–20-fold retardation in the extent of the contamination relative to the reconstructed lateral movement of groundwater over the same period. Our findings suggest that arsenic contamination of Pleistocene aquifers in south and southeast Asia as a consequence of increasing levels of groundwater pumping may have been delayed by the retardation of arsenic transport.
Non-chondritic sulphur isotope composition of the terrestrial mantle p.208
Core–mantle differentiation is the largest event experienced by a growing planet during its early history. Terrestrial core segregation imprinted the residual mantle composition by scavenging siderophile (iron-loving) elements such as tungsten, cobalt and sulphur. Cosmochemical constraints suggest that about 97% of Earth’s sulphur should at present reside in the core, which implies that the residual silicate mantle should exhibit fractionated 34S/32S ratios according to the relevant metal–silicate partition coefficients, together with fractionated siderophile element abundances. However, Earth’s mantle has long been thought to be both homogeneous and chondritic for 34S/32S, similar to Canyon Diablo troilite, as it is for most siderophile elements. This belief was consistent with a mantle sulphur budget dominated by late-accreted chondritic components. Here we show that the mantle, as sampled by mid-ocean ridge basalts from the south Atlantic ridge, displays heterogeneous 34S/32S ratios, directly correlated to the strontium and neodymium isotope ratios 87Sr/86Sr and 143Nd/144Nd. These isotope trends are compatible with binary mixing between a low-34S/32S ambient mantle and a high-34S/32S recycled component that we infer to be subducted sediments. The depleted end-member is characterized by a significantly negative δ34S of −1.28 ± 0.33‰ that cannot reach a chondritic value even when surface sulphur (from continents, altered oceanic crust, sediments and oceans) is added. Such a non-chondritic 34S/32S ratio for the silicate Earth could be accounted for by a core–mantle differentiation record in which the core has a 34S/32S ratio slightly higher than that of chondrites (δ34S = +0.07‰). Despite evidence for late-veneer addition of siderophile elements (and therefore sulphur) after core formation, our results imply that the mantle sulphur budget retains fingerprints of core–mantle differentiation.
Computational design of ligand-binding proteins with high affinity and selectivity p.212
The ability to design proteins with high affinity and selectivity for any given small molecule is a rigorous test of our understanding of the physiochemical principles that govern molecular recognition. Attempts to rationally design ligand-binding proteins have met with little success, however, and the computational design of protein–small-molecule interfaces remains an unsolved problem. Current approaches for designing ligand-binding proteins for medical and biotechnological uses rely on raising antibodies against a target antigen in immunized animals and/or performing laboratory-directed evolution of proteins with an existing low affinity for the desired ligand, neither of which allows complete control over the interactions involved in binding. Here we describe a general computational method for designing pre-organized and shape complementary small-molecule-binding sites, and use it to generate protein binders to the steroid digoxigenin (DIG). Of seventeen experimentally characterized designs, two bind DIG; the model of the higher affinity binder has the most energetically favourable and pre-organized interface in the design set. A comprehensive binding-fitness landscape of this design, generated by library selections and deep sequencing, was used to optimize its binding affinity to a picomolar level, and X-ray co-crystal structures of two variants show atomic-level agreement with the corresponding computational models. The optimized binder is selective for DIG over the related steroids digitoxigenin, progesterone and β-oestradiol, and this steroid binding preference can be reprogrammed by manipulation of explicitly designed hydrogen-bonding interactions. The computational design method presented here should enable the development of a new generation of biosensors, therapeutics and diagnostics.
De novo mutations in epileptic encephalopathies p.217
Epileptic encephalopathies are a devastating group of severe childhood epilepsy disorders for which the cause is often unknown. Here we report a screen for de novo mutations in patients with two classical epileptic encephalopathies: infantile spasms (n = 149) and Lennox–Gastaut syndrome (n = 115). We sequenced the exomes of 264 probands, and their parents, and confirmed 329 de novo mutations. A likelihood analysis showed a significant excess of de novo mutations in the ∼4,000 genes that are the most intolerant to functional genetic variation in the human population (P = 2.9 × 10−3). Among these are GABRB3, with de novo mutations in four patients, and ALG13, with the same de novo mutation in two patients; both genes show clear statistical evidence of association with epileptic encephalopathy. Given the relevant site-specific mutation rates, the probabilities of these outcomes occurring by chance are P = 4.1 × 10−10 and P = 7.8 × 10−12, respectively. Other genes with de novo mutations in this cohort include CACNA1A, CHD2, FLNA, GABRA1, GRIN1, GRIN2B, HNRNPU, IQSEC2, MTOR and NEDD4L. Finally, we show that the de novo mutations observed are enriched in specific gene sets including genes regulated by the fragile X protein (P < 10−8), as has been reported previously for autism spectrum disorders.
Induction of mouse germ-cell fate by transcription factors in vitro p.222
The germ-cell lineage ensures the continuity of life through the generation of male and female gametes, which unite to form a totipotent zygote. We have previously demonstrated that, by using cytokines, embryonic stem cells and induced pluripotent stem cells can be induced into epiblast-like cells (EpiLCs) and then into primordial germ cell (PGC)-like cells with the capacity for both spermatogenesis and oogenesis, creating an opportunity for understanding and regulating mammalian germ-cell development in both sexes in vitro. Here we show that, without cytokines, simultaneous overexpression of three transcription factors, Blimp1 (also known as Prdm1), Prdm14 and Tfap2c (also known as AP2γ), directs EpiLCs, but not embryonic stem cells, swiftly and efficiently into a PGC state. Notably, Prdm14 alone, but not Blimp1 or Tfap2c, suffices for the induction of the PGC state in EpiLCs. The transcription-factor-induced PGC state, irrespective of the transcription factors used, reconstitutes key transcriptome and epigenetic reprogramming in PGCs, but bypasses a mesodermal program that accompanies PGC or PGC-like-cell specification by cytokines including bone morphogenetic protein 4. Notably, the transcription-factor-induced PGC-like cells contribute to spermatogenesis and fertile offspring. Our findings provide a new insight into the transcriptional logic for PGC specification, and create a foundation for the transcription-factor-based reconstitution and regulation of mammalian gametogenesis.
The pluripotent genome in three dimensions is shaped around pluripotency factors p.227
It is becoming increasingly clear that the shape of the genome importantly influences transcription regulation. Pluripotent stem cells such as embryonic stem cells were recently shown to organize their chromosomes into topological domains that are largely invariant between cell types. Here we combine chromatin conformation capture technologies with chromatin factor binding data to demonstrate that inactive chromatin is unusually disorganized in pluripotent stem-cell nuclei. We show that gene promoters engage in contacts between topological domains in a largely tissue-independent manner, whereas enhancers have a more tissue-restricted interaction profile. Notably, genomic clusters of pluripotency factor binding sites find each other very efficiently, in a manner that is strictly pluripotent-stem-cell-specific, dependent on the presence of Oct4 and Nanog protein and inducible after artificial recruitment of Nanog to a selected chromosomal site. We conclude that pluripotent stem cells have a unique higher-order genome structure shaped by pluripotency factors. We speculate that this interactome enhances the robustness of the pluripotent state.
Mechanism of MEK inhibition determines efficacy in mutant KRAS- versus BRAF-driven cancers p.232
KRAS and BRAF activating mutations drive tumorigenesis through constitutive activation of the MAPK pathway. As these tumours represent an area of high unmet medical need, multiple allosteric MEK inhibitors, which inhibit MAPK signalling in both genotypes, are being tested in clinical trials. Impressive single-agent activity in BRAF-mutant melanoma has been observed; however, efficacy has been far less robust in KRAS-mutant disease. Here we show that, owing to distinct mechanisms regulating MEK activation in KRAS- versus BRAF-driven tumours, different mechanisms of inhibition are required for optimal antitumour activity in each genotype. Structural and functional analysis illustrates that MEK inhibitors with superior efficacy in KRAS-driven tumours (GDC-0623 and G-573, the former currently in phase I clinical trials) form a strong hydrogen-bond interaction with S212 in MEK that is critical for blocking MEK feedback phosphorylation by wild-type RAF. Conversely, potent inhibition of active, phosphorylated MEK is required for strong inhibition of the MAPK pathway in BRAF-mutant tumours, resulting in superior efficacy in this genotype with GDC-0973 (also known as cobimetinib), a MEK inhibitor currently in phase III clinical trials. Our study highlights that differences in the activation state of MEK in KRAS-mutant tumours versus BRAF-mutant tumours can be exploited through the design of inhibitors that uniquely target these distinct activation states of MEK. These inhibitors are currently being evaluated in clinical trials to determine whether improvements in therapeutic index within KRAS versus BRAF preclinical models translate to improved clinical responses in patients.
Completion of the entire hepatitis C virus life cycle in genetically humanized mice p.237
More than 130 million people worldwide chronically infected with hepatitis C virus (HCV) are at risk of developing severe liver disease. Antiviral treatments are only partially effective against HCV infection, and a vaccine is not available. Development of more efficient therapies has been hampered by the lack of a small animal model. Building on the observation that CD81 and occludin (OCLN) comprise the minimal set of human factors required to render mouse cells permissive to HCV entry, we previously showed that transient expression of these two human genes is sufficient to allow viral uptake into fully immunocompetent inbred mice. Here we demonstrate that transgenic mice stably expressing human CD81 and OCLN also support HCV entry, but innate and adaptive immune responses restrict HCV infection in vivo. Blunting antiviral immunity in genetically humanized mice infected with HCV results in measurable viraemia over several weeks. In mice lacking the essential cellular co-factor cyclophilin A (CypA), HCV RNA replication is markedly diminished, providing genetic evidence that this process is faithfully recapitulated. Using a cell-based fluorescent reporter activated by the NS3-4A protease we visualize HCV infection in single hepatocytes in vivo. Persistently infected mice produce de novo infectious particles, which can be inhibited with directly acting antiviral drug treatment, thereby providing evidence for the completion of the entire HCV life cycle in inbred mice. This genetically humanized mouse model opens new opportunities to dissect genetically HCV infection in vivo and provides an important preclinical platform for testing and prioritizing drug candidates and may also have utility for evaluating vaccine efficacy.
Pathogen blocks host death receptor signalling by arginine GlcNAcylation of death domains p.242
The tumour necrosis factor (TNF) family is crucial for immune homeostasis, cell death and inflammation. These cytokines are recognized by members of the TNF receptor (TNFR) family of death receptors, including TNFR1 and TNFR2, and FAS and TNF-related apoptosis-inducing ligand (TRAIL) receptors. Death receptor signalling requires death-domain-mediated homotypic/heterotypic interactions between the receptor and its downstream adaptors, including TNFR1-associated death domain protein (TRADD) and FAS-associated death domain protein (FADD). Here we discover that death domains in several proteins, including TRADD, FADD, RIPK1 and TNFR1, were directly inactivated by NleB, an enteropathogenic Escherichia coli (EPEC) type III secretion system effector known to inhibit host nuclear factor-κB (NF-κB) signalling. NleB contained an unprecedented N-acetylglucosamine (GlcNAc) transferase activity that specifically modified a conserved arginine in these death domains (Arg 235 in the TRADD death domain). NleB GlcNAcylation (the addition of GlcNAc onto a protein side chain) of death domains blocked homotypic/heterotypic death domain interactions and assembly of the oligomeric TNFR1 complex, thereby disrupting TNF signalling in EPEC-infected cells, including NF-κB signalling, apoptosis and necroptosis. Type-III-delivered NleB also blocked FAS ligand and TRAIL-induced cell death by preventing formation of a FADD-mediated death-inducing signalling complex (DISC). The arginine GlcNAc transferase activity of NleB was required for bacterial colonization in the mouse model of EPEC infection. The mechanism of action of NleB represents a new model by which bacteria counteract host defences, and also a previously unappreciated post-translational modification.
A type III effector antagonizes death receptor signalling during bacterial gut infection p.247
Successful infection by enteric bacterial pathogens depends on the ability of the bacteria to colonize the gut, replicate in host tissues and disseminate to other hosts. Pathogens such as Salmonella, Shigella and enteropathogenic and enterohaemorrhagic (EPEC and EHEC, respectively) Escherichia coli use a type III secretion system (T3SS) to deliver virulence effector proteins into host cells during infection that promote colonization and interfere with antimicrobial host responses. Here we report that the T3SS effector NleB1 from EPEC binds to host cell death-domain-containing proteins and thereby inhibits death receptor signalling. Protein interaction studies identified FADD, TRADD and RIPK1 as binding partners of NleB1. NleB1 expressed ectopically or injected by the bacterial T3SS prevented Fas ligand or TNF-induced formation of the canonical death-inducing signalling complex (DISC) and proteolytic activation of caspase-8, an essential step in death-receptor-induced apoptosis. This inhibition depended on the N-acetylglucosamine transferase activity of NleB1, which specifically modified Arg 117 in the death domain of FADD. The importance of the death receptor apoptotic pathway to host defence was demonstrated using mice deficient in the FAS signalling pathway, which showed delayed clearance of the EPEC-like mouse pathogen Citrobacter rodentium and reversion to virulence of an nleB mutant. The activity of NleB suggests that EPEC and other attaching and effacing pathogens antagonize death-receptor-induced apoptosis of infected cells, thereby blocking a major antimicrobial host response.
Stability and function of regulatory T cells is maintained by a neuropilin-1–semaphorin-4a axis p.252
Regulatory T cells (Treg cells) have a crucial role in the immune system by preventing autoimmunity, limiting immunopathology, and maintaining immune homeostasis. However, they also represent a major barrier to effective anti-tumour immunity and sterilizing immunity to chronic viral infections. The transcription factor Foxp3 has a major role in the development and programming of Treg cells. The relative stability of Treg cells at inflammatory disease sites has been a highly contentious subject. There is considerable interest in identifying pathways that control the stability of Treg cells as many immune-mediated diseases are characterized by either exacerbated or limited Treg-cell function. Here we show that the immune-cell-expressed ligand semaphorin-4a (Sema4a) and the Treg-cell-expressed receptor neuropilin-1 (Nrp1) interact both in vitro, to potentiate Treg-cell function and survival, and in vivo, at inflammatory sites. Using mice with a Treg-cell-restricted deletion of Nrp1, we show that Nrp1 is dispensable for suppression of autoimmunity and maintenance of immune homeostasis, but is required by Treg cells to limit anti-tumour immune responses and to cure established inflammatory colitis. Sema4a ligation of Nrp1 restrained Akt phosphorylation cellularly and at the immunologic synapse by phosphatase and tensin homologue (PTEN), which increased nuclear localization of the transcription factor Foxo3a. The Nrp1-induced transcriptome promoted Treg-cell stability by enhancing quiescence and survival factors while inhibiting programs that promote differentiation. Importantly, this Nrp1-dependent molecular program is evident in intra-tumoral Treg cells. Our data support a model in which Treg-cell stability can be subverted in certain inflammatory sites, but is maintained by a Sema4a–Nrp1 axis, highlighting this pathway as a potential therapeutic target that could limit Treg-cell-mediated tumour-induced tolerance without inducing autoimmunity.
Interactome map uncovers phosphatidylserine transport by oxysterol-binding proteins p.257
The internal organization of eukaryotic cells into functionally specialized, membrane-delimited organelles of unique composition implies a need for active, regulated lipid transport. Phosphatidylserine (PS), for example, is synthesized in the endoplasmic reticulum and then preferentially associates—through mechanisms not fully elucidated—with the inner leaflet of the plasma membrane. Lipids can travel via transport vesicles. Alternatively, several protein families known as lipid-transfer proteins (LTPs) can extract a variety of specific lipids from biological membranes and transport them, within a hydrophobic pocket, through aqueous phases. Here we report the development of an integrated approach that combines protein fractionation and lipidomics to characterize the LTP–lipid complexes formed in vivo. We applied the procedure to 13 LTPs in the yeast Saccharomyces cerevisiae: the six Sec14 homology (Sfh) proteins and the seven oxysterol-binding homology (Osh) proteins. We found that Osh6 and Osh7 have an unexpected specificity for PS. In vivo, they participate in PS homeostasis and the transport of this lipid to the plasma membrane. The structure of Osh6 bound to PS reveals unique features that are conserved among other metazoan oxysterol-binding proteins (OSBPs) and are required for PS recognition. Our findings represent the first direct evidence, to our knowledge, for the non-vesicular transfer of PS from its site of biosynthesis (the endoplasmic reticulum) to its site of biological activity (the plasma membrane). We describe a new subfamily of OSBPs, including human ORP5 and ORP10, that transfer PS and propose new mechanisms of action for a protein family that is involved in several human pathologies such as cancer, dyslipidaemia and metabolic syndrome.