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Scientists must push to preserve a small part of a large US survey that provides essential information on the ever-changing scientific workforce.
Pressures in all stages of the news-making process can lead to hype in science reporting.
Climate negotiations in Lima stumbled on transparency, but there is time to adjust.
The misuse of wolf research by Swedish politicians should be a warning to all biodiversity scientists, says Guillaume Chapron.
A coral-reef fish can match its scent to the odour of the surrounding reef, masking itself from predators.Harlequin filefish (Oxymonacanthus longirostris; pictured) live around reefs in the Pacific and Indian oceans and feed on particular species of coral. A team led by
Geoscientists have found possible evidence of two typhoons that, according to Japanese legend, wiped out invading Mongol fleets in the years 1274 and 1281.Jon Woodruff of the University of Massachusetts, Amherst, and his colleagues collected a 2,000-year-old sediment record from a coastal lake on
Hail storms can cause billions of dollars' worth of damage, but until now scientists have known little about the precise mass and shape of hail. A study has found that hailstones that are not perfectly spherical can sometimes travel faster and hit objects with greater
A study of primate and bacterial proteins involved in capturing iron from the blood has revealed an evolutionary arms race in the battle over this important nutrient.Matthew Barber and Nels Elde at the University of Utah in Salt Lake City focused on transferrin, a
One of the major Maya cities thrived in a tropical forest by using sophisticated agricultural, forestry and water-management techniques.David Lentz at the University of Cincinnati in Ohio and his colleagues surveyed modern forests at the site of Tikal in Guatemala, which was a bustling
Physicists have used lasers to increase the amount of energy that electrons gain per metre by more than two orders of magnitude compared with traditional accelerators.Conventional colliders can accelerate particles to much greater energies, but over many kilometres. Wim Leemans at Lawrence Berkeley National
Ibuprofen can be produced in minutes by mixing reagents as they flow through a series of connecting tubes.Synthesizing a substance in a continuous-flow process offers more control over reactions and allows less solvent to be used than batch production in flasks. But solid by-products
An insecticide banned in some areas for its effect on bees not only fails to kill certain pests, but also harms the predators that feed on them.Neonicotinoid insecticides are used on many crops, including soya-bean plants, on which pest slugs (Deroceras reticulatum)
Using a modest-sized ground-based telescope, astronomers have spotted a planet twice the size of Earth passing in front of its host star.Researchers typically study planets outside the Solar System using space telescopes or much larger telescopes on Earth, but studies with space telescopes are
Scientists, press officers and journalists online are pointing fingers in light of a paper that traces the origins of exaggerated claims in health news.
The week in science: Activists harm Nazca lines; Large Hadron Collider heads towards reboot; and Russia promises nuclear reactors for India.
A front-line report from Sierra Leone examines efforts to change hearts and minds in West Africa’s villages.
Birds get fresh perches in revamped tree of life built by vast collaboration.
Are geopolitical tensions destroying important links with the West, or can Russian research go it alone?
Comets, stem cells and cosmic dust are among the year's top stories.
Eruptions, comets and a see-through mouse all captured the imagination in 2014.
Ten people who mattered this year.
Attempts to exempt speculative theories of the Universe from experimental verification undermine science, argue George Ellis and Joe Silk.
Scaling up the recruitment of individuals who have recovered from infection deserves urgent consideration, argue Joshua M. Epstein, Lauren M. Sauer and colleagues.
Aaron Hirsh celebrates the 75th anniversary of the marine-biology classic by Ed Ricketts, the bohemian scientist who inspired John Steinbeck.
The landing of the Philae probe on comet 67P/Churyumov–Gerasimenko last month has led to speculation that comets might have delivered the building-block elements of life to Earth — an idea anticipated by the French astronomer Camille Flammarion more than a century ago in his 1880
Protected conservation areas face huge challenges globally (see J. E. M.Watsonet al. Nature515, 67–73;10.1038/515067a2014). But examples that are effectively funded and managed can be found in Namibia and in the Brazilian Amazon. In our
On 1 January 2015, a large new government office will take over Hungary's research-grant agency for basic science, OTKA. This will assume all budget management for research, development and innovation— destroying what the European Science Foundation has described as the agency's “high degree of
Conventional methods of flood protection such as levees are no longer adequate against the increased risk of flooding in Asian delta cities. We call for a multipronged approach that focuses on long-term, sustainable solutions to increase these cities' resilience to flooding (see also L.Giosan
One solution to the challenges posed by voluntary peer review (M.ArnsNature515, 46710.1038/515467a (2014) and see Nature515, 480–482;10.1038/515480a2014) might be to create a professional, independent body of reviewers
Discoverer of the tau lepton subatomic particle.
The Internet offers ways to broaden your contacts and assist you in your job search, says Peter Fiske.
All you want for Christmas.
The News Feature‘Ebola’s lost ward’ (Nature513, 474–477; 2014) incorrectly stated that nurse Veronica Koroma contracted Ebola.
An iron catalyst has been developed that mediates bond formation between a wide range of alkene reactants, opening up short synthetic routes to compounds that were previously accessible only through arduous pathways. See Article p.343
The development of RNA-based devices called toehold switches that regulate translation might usher in an era in which protein production can be linked to almost any RNA input and provide precise, low-cost diagnostics.
Intensive longitudinal sampling of malaria mosquitoes in the African semi-desert reveals that three morphologically indistinguishable species have distinctive strategies for surviving the dry season.
50 Years AgoDr. H. J. Kingsley and Dr. J. E. A. David of Bulawayo have described ... the case of a girl aged 22 months ... She appeared to be completely insensitive to pain ... She was admitted to hospital for investigation and was
New analysis reveals the conservation gains that could be achieved by expanding the global network of protected areas— but also how this may be undermined by land-use change and a lack of international coordination. See Letter p.383
Magnetoelectric materials allow magnetism to be controlled by an electric field. The discovery of an indirect path for switching electrical polarization in one such material brings this idea close to practical use. See Letter p.370
Crystal structures of the complete RNA polymerases from influenza A and B viruses provide insight into how these enzymes initiate RNA synthesis, and reveal targets for antiviral drug design. See Articles p.355 aamp; p.361
Extracts from selected News aamp; Views articles published this year.
Carbon–carbon (C–C) bonds form the backbone of many important molecules, including polymers, dyes and pharmaceutical agents. The development of new methods to create these essential connections in a rapid and practical fashion has been the focus of numerous organic chemists. This endeavour relies heavily on
Naturally occurring variations of Polycomb repressive complex 1 (PRC1) comprise a core assembly of Polycomb group proteins and additional factors that include, surprisingly, autism susceptibility candidate 2 (AUTS2). Although AUTS2 is often disrupted in patients with neuronal disorders, the mechanism underlying the pathogenesis is
The influenza virus polymerase transcribes or replicates the segmented RNA genome (viral RNA) into viral messenger RNA or full-length copies. To initiate RNA synthesis, the polymerase binds to the conserved 3′ and 5′ extremities of the viral RNA. Here we present the crystal structure of
Influenza virus polymerase uses a capped primer, derived by‘cap-snatching’ from host pre-messenger RNA, to transcribe its RNA genome into mRNA and a stuttering mechanism to generate the poly(A) tail. By contrast, genome replication is unprimed and generates exact full-length copies of the template. Here
Stars spend most of their lifetimes on the main sequence in the Hertzsprung–Russell diagram. The extended main-sequence turn-off regions—containing stars leaving the main sequence after having spent all of the hydrogen in their cores—found in massive (more than a few tens of thousands of solar masses), intermediate-age (about one to three billion years old) star clusters are usually interpreted as evidence of internal age spreads of more than 300 million years, although young clusters are thought to quickly lose any remaining star-forming fuel following a period of rapid gas expulsion on timescales of order 107 years. Here we report, on the basis of a combination of high-resolution imaging observations and theoretical modelling, that the stars beyond the main sequence in the two-billion-year-old cluster NGC 1651, characterized by a mass of about 1.7 × 105 solar masses, can be explained only by a single-age stellar population, even though the clusterhas a clearly extended main-sequence turn-off region. The most plausible explanation for the existence of such extended regions invokes a population of rapidly rotating stars, although the secondary effects of the prolonged stellar lifetimes associated with such a stellar population mixture are as yet poorly understood. From preliminary analysis of previously obtained data, we find that similar morphologies are apparent in the Hertzsprung–Russell diagrams of at least five additional intermediate-age star clusters, suggesting that an extended main-sequence turn-off region does not necessarily imply the presence of a significant internal age dispersion.
The technological appeal of multiferroics is the ability to control magnetism with electric field. For devices to be useful, such control must be achieved at room temperature. The only single-phase multiferroic material exhibiting unambiguous magnetoelectric coupling at room temperature is BiFeO3 (refs 4 and 5). Its weak ferromagnetism arises from the canting of the antiferromagnetically aligned spins by the Dzyaloshinskii–Moriya (DM) interaction. Prior theory considered the symmetry of the thermodynamic ground state and concluded that direct 180-degree switching of the DM vector by the ferroelectric polarization was forbidden. Instead, we examined the kinetics of the switching process, something not considered previously in theoretical work. Here we show a deterministic reversal of the DM vector and canted moment using an electric field at room temperature. First-principles calculations reveal that the switching kinetics favours a two-step switching process. In each step the DM vector and polarization are coupled and 180-degree deterministic switching of magnetization hence becomes possible, in agreement with experimental observation. We exploit this switching to demonstrate energy-efficient control of a spin-valve device at room temperature. The energy per unit area required is approximately an orderof magnitude less than that needed for spin-transfer torque switching. Given that the DM interaction is fundamental to single-phase multiferroics and magnetoelectrics, our results suggest ways to engineer magnetoelectric switching and tailor technologically pertinent functionality for nanometre-scale, low-energy-consumption, non-volatile magnetoelectronics.
The concerted motion of two or more bound electrons governs atomic and molecular non-equilibrium processes including chemical reactions, and hence there is much interest in developing a detailed understanding of such electron dynamics in the quantum regime. However, there is no exact solution for the quantum three-body problem, and as a result even the minimal system of two active electrons and a nucleus is analytically intractable. This makes experimental measurements of the dynamics of two bound and correlated electrons, as found in the helium atom, an attractive prospect. However, although the motion of single active electrons and holes has been observed with attosecond time resolution, comparable experiments on two-electron motion have so far remained out of reach. Here we show that a correlated two-electron wave packet can be reconstructed from a 1.2-femtosecond quantum beat among low-lying doubly excited states in helium. The beat appears in attosecond transient-absorption spectra measured with unprecedentedly high spectral resolution and in the presence of an intensity-tunable visible laser field. We tune the coupling between the two low-lying quantum states by adjusting the visible laser intensity, and use the Fano resonance as a phase-sensitive quantum interferometer to achieve coherent control of the two correlated electrons. Given the excellent agreement with large-scale quantum-mechanical calculations for the helium atom, we anticipate that multidimensional spectroscopy experiments of the type we report here will provide benchmark data for testing fundamental few-body quantum dynamics theory in more complex systems. They might also provide a route to the site-specific measurement and control of metastable electronic transition states that are at the heart of fundamental chemical reactions.
Microbial ecosystems can be sustained by hydrogen gas (H2)-producing water–rock interactions in the Earth’s subsurface and at deep ocean vents. Current estimates of global H2 production from the marine lithosphere by water–rock reactions (hydration) are in the range of 1011 moles per year. Recent explorations of saline fracture waters in the Precambrian continental subsurface have identified environments as rich in H2 as hydrothermal vents and seafloor-spreading centres and have suggested a link between dissolved H2 and the radiolytic dissociation of water. However, extrapolation of a regional H2 flux based on the deep gold mines of the Witwatersrand basin in South Africa yields a contribution of the Precambrian lithosphere to global H2 production that was thought to be negligible (0.009 × 1011 moles per year). Here we present a global compilation of published and new H2 concentration data obtained from Precambrian rocks and find that the H2 production potential of the Precambrian continental lithosphere has been underestimated. We suggest that this can be explained by a lack of consideration of additional H2-producing reactions, such as serpentinization, and the absence of appropriate scaling of H2 measurements from these environments to account for the fact that Precambrian crust represents over 70 per cent of global continental crust surface area. If H2 production via both radiolysis and hydration reactions is taken into account, our estimate of H2 production rates from the Precambrian continental lithosphere of 0.36–2.27 × 1011 moles per year is comparable to estimates from marine systems.
Protected areas are one of the main tools for halting the continuing global biodiversity crisis caused by habitat loss, fragmentation and other anthropogenic pressures. According to the Aichi Biodiversity Target 11 adopted by the Convention on Biological Diversity, the protected area network should be expanded to at least 17% of the terrestrial world by 2020 (http://www.cbd.int/sp/targets). To maximize conservation outcomes, it is crucial to identify the best expansion areas. Here we show that there is a very high potential to increase protection of ecoregions and vertebrate species by expanding the protected area network, but also identify considerable risk of ineffective outcomes due to land-use change and uncoordinated actions between countries. We use distribution data for 24,757 terrestrial vertebrates assessed under the International Union for the Conservation of Nature (IUCN)‘red list of threatened species’, and terrestrial ecoregions (827), modified by land-use models for the present and 2040, and introduce techniques for global and balanced spatial conservation prioritization. First, we show that with a coordinated global protected area network expansion to 17% of terrestrial land, average protection of species ranges and ecoregions could triple. Second, if projected land-use change by 2040 (ref. 11) takes place, it becomes infeasible to reach the currently possible protection levels, and over 1,000 threatened species would lose more than 50% of their present effective ranges worldwide. Third, we demonstrate a major efficiency gap between national and global conservation priorities. Strong evidence is shown that further biodiversity loss is unavoidable unless international action is quickly taken to balance land-use and biodiversity conservation. The approach used here can serve as a framework for repeatable and quantitative assessment of efficiency, gaps and expansion of the global protected area network globally, regionally and nationally, considering current and projected land-use pressures.
During the long Sahelian dry season, mosquito vectors of malaria are expected to perish when no larval sites are available; yet, days after the first rains, mosquitoes reappear in large numbers. How these vectors persist over the 3–6-month long dry season has not been resolved, despite extensive research for over a century. Hypotheses for vector persistence include dry-season diapause (aestivation) and long-distance migration (LDM); both are facets of vector biology that have been highly controversial owing to lack of concrete evidence. Here we show that certain species persist by a form of aestivation, while others engage in LDM. Using time-series analyses, the seasonal cycles of Anopheles coluzzii, Anopheles gambiae sensu stricto (s.s.), and Anopheles arabiensis were estimated, and their effects were found to be significant, stable and highly species-specific. Contrary to all expectations, the most complex dynamics occurred during the dry season, when the density of A. coluzzii fluctuated markedly, peaking when migration would seem highly unlikely, whereas A. gambiae s.s. was undetected. The population growthof A. coluzzii followed the first rains closely, consistent with aestivation, whereas the growth phase of both A. gambiae s.s. and A. arabiensis lagged by two months. Such a delay is incompatible with local persistence, but fits LDM. Surviving the long dry season in situ allows A. coluzzii to predominate and form the primary force of malaria transmission. Our results reveal profound ecological divergence between A. coluzzii and A. gambiae s.s., whose standing as distinct species has been challenged, and suggest that climate is one of the selective pressures that led to their speciation. Incorporating vector dormancy and LDM is key to predicting shifts in the range of malaria due to global climate change, and to the elimination of malaria from Africa.
The move of vertebrates to a terrestrial lifestyle required major adaptations in their locomotory apparatus and reproductive organs. While the fin-to-limb transition has received considerable attention, little is known about the developmental and evolutionary origins of external genitalia. Similarities in gene expression have been interpreted as a potential evolutionary link between the limb and genitals; however, no underlying developmental mechanism has been identified. We re-examined this question using micro-computed tomography, lineage tracing in three amniote clades, and RNA-sequencing-based transcriptional profiling. Here we show that the developmental origin of external genitalia has shifted through evolution, and in some taxa limbs and genitals share a common primordium. In squamates, the genitalia develop directly from the budding hindlimbs, or the remnants thereof, whereas in mice the genital tubercle originates from the ventral and tail bud mesenchyme. The recruitment of different cell populations for genital outgrowth follows a change in the relative position of the cloaca, the genitalia organizing centre. Ectopic grafting of the cloaca demonstrates the conserved ability of different mesenchymal cells to respond to these genitalia-inducing signals. Our results support a limb-like developmental origin of external genitalia as the ancestral condition. Moreover, they suggest that a change in the relative position of the cloacal signalling centre during evolution has led to an altered developmental route for external genitalia in mammals, while preserving parts of the ancestral limb molecular circuitry owing to a common evolutionary origin.
Brown adipose tissue (BAT) is specialized in energy expenditure, making it a potential target for anti-obesity therapies. Following exposure to cold, BAT is activated by the sympathetic nervous system with concomitant release of catecholamines and activation ofβ-adrenergic receptors. Because BAT therapies based on cold exposure or β-adrenergic agonists are clinically not feasible, alternative strategies must be explored. Purinergic co-transmission might be involved in sympathetic control of BAT and previous studies reported inhibitory effects of the purinergic transmitter adenosine in BAT from hamster or rat. However, the role of adenosine in human BAT is unknown. Here we show that adenosine activates human and murine brown adipocytes at low nanomolar concentrations. Adenosine is released in BAT during stimulation of sympathetic nerves as well asfrom brown adipocytes. The adenosine A2A receptor is the most abundant adenosine receptor in human and murine BAT. Pharmacological blockade or genetic loss of A2A receptors in mice causes a decrease in BAT-dependent thermogenesis, whereas treatment with A2A agonists significantly increases energy expenditure. Moreover, pharmacological stimulation of A2A receptors or injection of lentiviral vectors expressing the A2A receptor into white fat induces brown-like cells—so-called beige adipocytes. Importantly, mice fed a high-fat diet and treated with an A2A agonist are leaner with improved glucose tolerance. Taken together, our results demonstrate that adenosine–A2A signalling plays an unexpected physiological role in sympathetic BAT activation and protects mice from diet-induced obesity. Those findings reveal new possibilities for developing novel obesity therapies.
Gastric diseases, including peptic ulcer disease and gastric cancer, affect 10% of the world’s population and are largely due to chronic Helicobacter pylori infection. Species differences in embryonic development and architecture of the adult stomach make animal models suboptimal for studying human stomach organogenesis and pathogenesis, and there is no experimental model of normal human gastric mucosa. Here we report the de novo generation of three-dimensional human gastric tissue in vitro through the directed differentiation of human pluripotent stem cells. We show that temporal manipulation of the FGF, WNT, BMP, retinoic acid and EGF signalling pathways and three-dimensional growth are sufficient to generate human gastric organoids (hGOs). Developing hGOs progressed through molecular and morphogenetic stages that were nearly identical to the developing antrum of the mouse stomach. Organoids formed primitive gastric gland- and pit-like domains, proliferative zones containing LGR5-expressing cells, surface and antral mucous cells, and a diversity of gastric endocrine cells. We used hGO cultures to identify novel signalling mechanisms that regulate early endoderm patterning and gastric endocrine cell differentiation upstream of the transcription factor NEUROG3. Using hGOs to model pathogenesis of human disease, we found that H. pylori infection resulted in rapid association of the virulence factor CagA with the c-Met receptor, activation of signalling and induction of epithelial proliferation. Together, these studies describe a new and robust in vitro system for elucidating the mechanisms underlying human stomach development and disease.
Naive embryonic stem cells hold great promise for research and therapeutics as they have broad and robust developmental potential. While such cells are readily derived from mouse blastocysts it has not been possible to isolate human equivalents easily, although human naive-like cells have been artificially generated (rather than extracted) by coercion of human primed embryonic stem cells by modifying culture conditions or through transgenic modification. Here we show that a sub-population within cultures of human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) manifests key properties of naive state cells. These naive-like cells can be genetically tagged, and are associated with elevated transcription of HERVH, a primate-specific endogenous retrovirus. HERVH elements provide functional binding sites for a combination of naive pluripotency transcription factors, including LBP9, recently recognized as relevant to naivety in mice. LBP9–HERVH drives hESC-specific alternative and chimaeric transcripts, including pluripotency-modulating long non-coding RNAs. Disruption of LBP9, HERVH and HERVH-derived transcripts compromises self-renewal. These observations define HERVH expression as a hallmark of naive-like hESCs, and establish novel primate-specific transcriptional circuitry regulating pluripotency.
The nuclear envelope is a double membrane that separates the nucleus from the cytoplasm. The inner nuclear membrane (INM) functions in essential nuclear processes including chromatin organization and regulation of gene expression. The outer nuclear membrane is continuous with the endoplasmic reticulum and is the site of membrane protein synthesis. Protein homeostasis in this compartment is ensured by endoplasmic-reticulum-associated protein degradation (ERAD) pathways that in yeast involve the integral membrane E3 ubiquitin ligases Hrd1 and Doa10 operating with the E2 ubiquitin-conjugating enzymes Ubc6 and Ubc7 (refs 2, 3). However, little is known about protein quality control at the INM. Here we describe a protein degradation pathway at the INM in yeast (Saccharomyces cerevisiae) mediated by the Asi complex consisting of the RING domain proteins Asi1 and Asi3 (ref. 4). We report that the Asi complex functions together with the ubiquitin-conjugating enzymes Ubc6 and Ubc7 to degrade soluble and integral membrane proteins. Genetic evidence suggests that the Asi ubiquitin ligase defines a pathway distinct from, but complementary to, ERAD. Using unbiased screening with a novel genome-wide yeast library based on a tandem fluorescent protein timer, we identify more than 50 substrates of the Asi, Hrd1 and Doa10 E3 ubiquitin ligases. We show that the Asi ubiquitin ligase is involved in degradation of mislocalized integral membrane proteins, thus acting to maintain and safeguard the identity of the INM.
Metazoans identify and eliminate bacterial pathogens in microbe-rich environments such as the intestinal lumen; however, the mechanisms are unclear. Host cells could potentially use intracellular surveillance or stress response programs to detect pathogens that target monitored cellular activities and then initiate innate immune responses. Mitochondrial function is evaluated by monitoring mitochondrial protein import efficiency of the transcription factor ATFS-1, which mediates the mitochondrial unfolded protein response (UPRmt). During mitochondrial stress, mitochondrial import is impaired, allowing ATFS-1 to traffic to the nucleus where it mediates a transcriptional response to re-establish mitochondrial homeostasis. Here we examined the role of ATFS-1 in Caenorhabditis elegans during pathogen exposure, because during mitochondrial stress ATFS-1 induced not only mitochondrial protective genes but also innate immune genes that included a secreted lysozyme and anti-microbial peptides. Exposure to the pathogen Pseudomonas aeruginosa caused mitochondrial dysfunction and activation of the UPRmt. C. elegans lacking atfs-1 were susceptible to P. aeruginosa, whereas hyper-activation of ATFS-1 and the UPRmt improved clearance of P. aeruginosa from the intestine and prolonged C. elegans survival in a manner mainly independent of known innate immune pathways. We propose that ATFS-1 import efficiency and the UPRmt is a means to detect pathogens that target mitochondria and initiate a protective innate immune response.
The neutralizing antibody response to influenza virus is dominated by antibodies that bind to the globular head of haemagglutinin, which undergoes a continuous antigenic drift, necessitating the re-formulation of influenza vaccines on an annual basis. Recently, several laboratories have described a new class of rare influenza-neutralizing antibodies that target a conserved site in the haemagglutinin stem. Most of these antibodies use the heavy-chain variable region VH1-69 gene, and structural data demonstrate that they bind to the haemagglutinin stem through conserved heavy-chain complementarity determining region (HCDR) residues. However, the VH1-69 antibodies are highly mutated and are produced by some but not all individuals, suggesting that several somatic mutations may be required for their development. To address this, here we characterize 197 anti-stem antibodies from a single donor, reconstruct the developmental pathways of several VH1-69 clones and identify two key elements that are required for the initial development of most VH1-69 antibodies: a polymorphic germline-encoded phenylalanine at position 54 and a conserved tyrosine at position 98 in HCDR3. Strikingly, in most cases a single proline to alanine mutation at position 52a in HCDR2 is sufficient to confer high affinity binding to the selecting H1 antigen, consistent with rapid affinity maturation. Surprisingly, additional favourable mutations continue to accumulate, increasing the breadth of reactivity and making both the initial mutations and phenylalanine at position 54 functionally redundant. These results define VH1-69 allele polymorphism, rearrangement of the VDJ gene segments and single somatic mutations as the three requirements for generating broadly neutralizing VH1-69 antibodies and reveal an unexpected redundancy in the affinity maturation process.
Chromosomal rearrangements have a central role in the pathogenesis of human cancers and often result in the expression of therapeutically actionable gene fusions. A recently discovered example is a fusion between the genes echinoderm microtubule-associated protein like 4 (EML4) and anaplastic lymphoma kinase (ALK), generated by an inversion on the short arm of chromosome 2: inv(2)(p21p23). The EML4–ALK oncogene is detected in a subset of human non-small cell lung cancers (NSCLC) and is clinically relevant because it confers sensitivity to ALK inhibitors. Despite their importance, modelling such genetic events in mice has proven challenging and requires complex manipulation of the germ line. Here we describe an efficient method to induce specific chromosomal rearrangements in vivo using viral-mediated delivery of the CRISPR/Cas9 system to somatic cells of adult animals. We apply it to generate a mouse model of Eml4–Alk-driven lung cancer. The resulting tumours invariably harbour theEml4–Alk inversion, express the Eml4–Alk fusion gene, display histopathological and molecular features typical of ALK+ human NSCLCs, and respond to treatment with ALK inhibitors. The general strategy described here substantially expands our ability to model human cancers in mice and potentiallyin other organisms.
Cancer is a multistep process that involves mutations and other alterations in oncogenes and tumour suppressor genes. Genome sequencing studies have identified a large collection of genetic alterations that occur in human cancers. However, the determination of which mutations are causally related to tumorigenesis remains a major challenge. Here we describe a novel CRISPR/Cas9-based approach for rapid functional investigation of candidate genes in well-established autochthonous mouse models of cancer. Using a KrasG12D-driven lung cancer model, we performed functional characterization of a panel of tumour suppressor genes with known loss-of-function alterations in human lung cancer. Cre-dependent somatic activation of oncogenic KrasG12D combined with CRISPR/Cas9-mediated genome editing of tumour suppressor genes resulted in lung adenocarcinomas with distinct histopathological and molecular features. This rapid somatic genome engineering approach enables functional characterization of putative cancer genes in the lung and other tissues using autochthonous mouse models. We anticipate that this approach can be used to systematically dissect the complex catalogue of mutations identified in cancer genome sequencing studies.
Eukaryotic genomes are folded into three-dimensional structures, such as self-associating topological domains, the borders of which are enriched in cohesin and CCCTC-binding factor (CTCF) required for long-range interactions. How local chromatin interactions govern higher-order folding of chromatin fibres and the function of cohesin in this process remain poorly understood. Here we perform genome-wide chromatin conformation capture (Hi-C) analysis to explore the high-resolution organization of the Schizosaccharomyces pombe genome, which despite its small size exhibits fundamental features found in other eukaryotes. Our analyses of wild-type and mutant strains reveal key elements of chromosome architecture and genome organization. On chromosome arms, small regions of chromatin locally interact to form‘globules’. This feature requires a function of cohesin distinct from its role in sister chromatid cohesion. Cohesin is enriched at globule boundaries and its loss causes disruption of local globule structures and global chromosome territories. By contrast, heterochromatin, which loads cohesin at specific sites including pericentromeric and subtelomeric domains, is dispensable for globule formation but nevertheless affects genome organization. We show that heterochromatin mediates chromatin fibre compaction at centromeres and promotes prominent inter-arm interactions within centromere-proximal regions, providing structural constraints crucial for proper genome organization. Loss of heterochromatin relaxes constraints on chromosomes, causing an increase in intra- and inter-chromosomal interactions. Together, our analyses uncover fundamental genome folding principles that drive higher-order chromosome organization crucial for coordinating nuclear functions.
The formation of R-loops is a natural consequence of the transcription process, caused by invasion of the DNA duplex by nascent transcripts. These structures have been considered rare transcriptional by-products with potentially harmful effects on genome integrity owing to the fragility of the displaced DNA coding strand. However, R-loops may also possess beneficial effects, as their widespread formation has been detected over CpG island promoters in human genes. Furthermore, we have previously shown that R-loops are particularly enriched over G-rich terminator elements. These facilitate RNA polymerase II (Pol II) pausing before efficient termination. Here we reveal an unanticipated link between R-loops and RNA-interference-dependent H3K9me2 formation over pause-site termination regions in mammalian protein-coding genes. We show that R-loops induce antisense transcription over these pause elements, which in turn leads to the generation of double-stranded RNA and the recruitment of DICER, AGO1, AGO2 and the G9a histone lysine methyltransferase. Consequently, an H3K9me2 repressive mark is formed and heterochromatin protein 1γ (HP1γ) is recruited, which reinforces Pol II pausing before efficient transcriptional termination. We predict that R-loops promote a chromatin architecture that defines the termination region for a substantial subset of mammalian genes.
Nature474, 502–505 (2011); doi:10.1038/nature09992Dr Michael Lynch has indicated that he no longer has confidence in the original data presented in this Letter, and would like to have his name removed as a co-author. Dr Ariel Fernández has
Nature511, 483–487 (2014); doi:10.1038/nature13473In this Letter, the ArrayExpress microarray data set accession number was wrongly given as E-MTAB-1524; the correct accession number is E-MTAB-1886. This has been corrected in the online versions of the paper.
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Endocytosis is a process by which molecules gain access to a cell. An unusual mode of endocytosis has now been shown to regulate cell signalling, and to be highjacked by bacterial toxins.
This study describes a fast, clathrin-independent endocytic pathway mediated by endophilin, dynamin and actin; the pathway is activated by ligand binding to a variety of cargo receptors, and endophilin-mediated endocytosis occurs primarily at the leading edges of cells where lamellipodin and the lipid PtdIns(3,4)P2 ensure endophilin targeting.
More than twenty types of retinal ganglion cells conduct visual information from the eye to the rest of the brain. Each retinal ganglion cell type tessellates the retina in a regular mosaic, so that every point in visual space is processed for visual primitives such as contrast and motion. This information flows to two principal brain centres: the visual cortex and the superior colliculus. The superior colliculus plays an evolutionarily conserved role in visual behaviours, but its functional architecture is poorly understood. Here we report on population recordings of visual responses from neurons in the mouse superior colliculus. Many neurons respond preferentially to lines of a certain orientation or movement axis. We show that cells with similar orientation preferences form large patches that span the vertical thickness of the retinorecipient layers. This organization is strikingly different from the randomly interspersed orientation preferences in the mouse’s visual cortex; instead, it resembles the orientation columns observed in the visual cortices of large mammals. Notably, adjacent superior colliculus orientation columns have only limited receptive field overlap. This is in contrast to the organization of visual cortex, where each point in the visual field activates neurons with all preferred orientations. Instead, the superior colliculus favours specific contour orientations within ∼30° regions of the visual field, a finding with implications for behavioural responses mediated by this brain centre.
Mast cells are primary effectors in allergic reactions, and may have important roles in disease by secreting histamine and various inflammatory and immunomodulatory substances. Although they are classically activated by immunoglobulin (Ig)E antibodies, a unique property of mast cells is their antibody-independent responsiveness to a range of cationic substances, collectively called basic secretagogues, including inflammatory peptides and drugs associated with allergic-type reactions. The pathogenic roles of these substances have prompted a decades-long search for their receptor(s). Here we report that basic secretagogues activate mouse mast cells in vitro and in vivo through a single receptor, Mrgprb2, the orthologue of the human G-protein-coupled receptor MRGPRX2. Secretagogue-induced histamine release, inflammation and airway contraction are abolished in Mrgprb2-null mutant mice. Furthermore, we show that most classes of US Food and Drug Administration (FDA)-approved peptidergic drugs associated with allergic-type injection-site reactions also activate Mrgprb2 and MRGPRX2, and that injection-site inflammation is absent in mutant mice. Finally, we determine that Mrgprb2 and MRGPRX2 are targets of many small-molecule drugs associated with systemic pseudo-allergic, or anaphylactoid, reactions; we show that drug-induced symptoms of anaphylactoid responses are significantly reduced in knockout mice; and we identify a common chemical motif in several of these molecules that may help predict side effects of other compounds. These discoveries introduce a mouse model to study mast cell activation by basic secretagogues and identify MRGPRX2 as a potential therapeutic target to reduce a subset of drug-induced adverse effects.
During endocytosis, energy is invested to narrow the necks of cargo-containing plasma membrane invaginations to radii at which the opposing segments spontaneously coalesce, thereby leading to the detachment by scission of endocytic uptake carriers. In the clathrin pathway, dynamin uses mechanical energy from GTP hydrolysis to this effect, assisted by the BIN/amphiphysin/Rvs (BAR) domain-containing protein endophilin. Clathrin-independent endocytic events are often less reliant on dynamin, and whether in these cases BAR domain proteins such as endophilin contribute to scission has remained unexplored. Here we show, in human and other mammalian cell lines, that endophilin-A2 (endoA2) specifically and functionally associates with very early uptake structures that are induced by the bacterial Shiga and cholera toxins, which are both clathrin-independent endocytic cargoes. In controlled in vitro systems, endoA2 reshapes membranes before scission. Furthermore, we demonstrate that endoA2, dynamin and actin contribute in parallel to the scission of Shiga-toxin-induced tubules. Our results establish a novel function of endoA2 in clathrin-independent endocytosis. They document that distinct scission factors operate in an additive manner, and predict that specificity within a given uptake process arises from defined combinations of universal modules. Our findings highlight a previously unnoticed link between membrane scaffolding by endoA2 and pulling-force-driven dynamic scission.
High-resolution data on ocean circulation during the last glacial cycle suggest that the formation of North Atlantic Deep Water and associated heat transport may be more stable than previously thought.
Using electron cryomicroscopy, the structure of the closed-state rabbit ryanodine receptor RyR1 in complex with its modulator FKBP12 is solved at 3.8 Å; in addition to determining structural details of the ion-conducting channel domain, three previously uncharacterized domains help to reveal a molecular scaffold that allows long-range allosteric regulation of channel activities.
Extreme, abrupt Northern Hemisphere climate oscillations during the last glacial cycle (140,000 years ago to present) were modulated by changes in ocean circulation and atmospheric forcing. However, the variability of the Atlantic meridional overturning circulation (AMOC), which has a role in controlling heat transport from low to high latitudes and in ocean CO2 storage, is still poorly constrained beyond the Last Glacial Maximum. Here we show that a deep and vigorous overturning circulation mode has persisted for most of the last glacial cycle, dominating ocean circulation in the Atlantic, whereas a shallower glacial mode with southern-sourced waters filling the deep western North Atlantic prevailed during glacial maxima. Our results are based on a reconstruction of both the strength and the direction of the AMOC during the last glacial cycle from a highly resolved marine sedimentary record in the deep western North Atlantic. Parallel measurements of two independent chemical water tracers (the isotope ratios of 231Pa/230Th and 143Nd/144Nd), which are not directly affected by changes in the global cycle, reveal consistent responses of the AMOC during the last two glacial terminations. Any significant deviations from this configuration, resulting in slowdowns of the AMOC, were restricted to centennial-scale excursions during catastrophic iceberg discharges of the Heinrich stadials. Severe and multicentennial weakening of North Atlantic Deep Water formation occurred only during Heinrich stadials close to glacial maxima with increased ice coverage, probably as a result of increased fresh-water input. In contrast, the AMOC was relatively insensitive to submillennial meltwater pulses during warmer climate states, and an active AMOC prevailed during Dansgaard–Oeschger interstadials (Greenland warm periods).
Interventions that delay ageing mobilize mechanisms that protect and repair cellular components, but it is unknown how these interventions might slow the functional decline of extracellular matrices, which are also damaged during ageing. Reduced insulin/IGF-1 signalling (rIIS) extends lifespan across the evolutionary specctrum, and in juvenile Caenorhabditis elegans also allows the transcription factor DAF-16/FOXO to induce development into dauer, a diapause that withstands harsh conditions. It has been suggested that rIIS delays C. elegans ageing through activation of dauer-related processes during adulthood, but some rIIS conditions confer robust lifespan extension unaccompanied by any dauer-like traits. Here we show that rIIS can promote C. elegans longevity through a program that is genetically distinct from the dauer pathway, and requires the Nrf (NF-E2-related factor) orthologue SKN-1 acting in parallel to DAF-16. SKN-1 is inhibited by IIS and has been broadly implicated in longevity, but is rendered dispensable for rIIS lifespan extension by even mild activity of dauer-related processes. When IIS is decreased under conditions that do not induce dauer traits, SKN-1 most prominently increases expression of collagens and other extracellular matrix genes. Diverse genetic, nutritional, and pharmacological pro-longevity interventions delay an age-related decline in collagen expression. These collagens mediate adulthood extracellular matrix remodelling, and are needed for ageing to be delayed by interventions that do not involve dauer traits. By genetically delineating a dauer-independent rIIS ageing pathway, our results show that IIS controls a broad set of protective mechanisms during C. elegans adulthood, and may facilitate elucidation of processes of general importance for longevity. The importance of collagen production in diverse anti-ageing interventions implies that extracellular matrix remodelling is a generally essential signature of longevity assurance, and that agents promoting extracellular matrix youthfulness may have systemic benefit.
Crust at many divergent plate boundaries forms primarily by the injection of vertical sheet-like dykes, some tens of kilometres long. Previous models of rifting events indicate either lateral dyke growth away from a feeding source, with propagation rates decreasing as the dyke lengthens, or magma flowing vertically into dykes from an underlying source, with the role of topography on the evolution of lateral dykes not clear. Here we show how a recent segmented dyke intrusion in the Bárðarbunga volcanic system grew laterally for more than 45 kilometres at a variable rate, with topography influencing the direction of propagation. Barriers at the ends of each segment were overcome by the build-up of pressure in the dyke end; then a new segment formed and dyke lengthening temporarily peaked. The dyke evolution, which occurred primarily over 14 days, was revealed by propagating seismicity, ground deformation mapped by Global Positioning System (GPS), interferometric analysis of satellite radar images (InSAR), and graben formation. The strike of the dyke segments varies from an initially radial direction away from the Bárðarbunga caldera, towards alignment with that expected from regional stress at the distal end. A model minimizing the combined strain and gravitational potential energy explains the propagation path. Dyke opening and seismicity focused at the most distal segment at any given time, and were simultaneous with magma source deflation and slow collapse at the Bárðarbunga caldera, accompanied by a series of magnitude M ggt; 5 earthquakes. Dyke growth was slowed down by an effusive fissure eruption near the end of the dyke. Lateral dyke growth with segment barrier breaking by pressure build-up in the dyke distal end explains how focused upwelling of magma under central volcanoes is effectively redistributed over long distances to create new upper crust at divergent plate boundaries.
Gene transcription in animals involves the assembly of RNA polymerase II at core promoters and its cell-type-specific activation by enhancers that can be located more distally. However, how ubiquitous expression of housekeeping genes is achieved has been less clear. In particular, it is unknown whether ubiquitously active enhancers exist and how developmental and housekeeping gene regulation is separated. An attractive hypothesis is that different core promoters might exhibit an intrinsic specificity to certain enhancers. This is conceivable, as various core promoter sequence elements are differentially distributed between genes of different functions, including elements that are predominantly found at either developmentally regulated or at housekeeping genes. Here we show that thousands of enhancers in Drosophila melanogaster S2 and ovarian somatic cells (OSCs) exhibit a marked specificity to one of two core promoters—one derived from a ubiquitously expressed ribosomal protein gene and another from a developmentally regulated transcription factor—and confirm the existence of these two classes for five additional core promoters from genes with diverse functions. Housekeeping enhancers are active across the two cell types, while developmental enhancers exhibit strong cell-type specificity. Both enhancer classes differ in their genomic distribution, the functions of neighbouring genes, and the core promoter elements of these neighbouring genes. In addition, we identify two transcription factors—Dref and Trl—that bind and activate housekeeping versus developmental enhancers, respectively. Our results provide evidence for a sequence-encoded enhancer–core-promoter specificity that separates developmental and housekeeping gene regulatory programs for thousands of enhancers and their target genes across the entire genome.
Mitotic cell division separates chromosome pairs into two genetically identical daughter cells. A study in fission yeast reveals that this separation is guided by the sequential activation of three phosphatase enzymes.
The finding that protein A of Staphylococcus aureus diverts the immune response so that it ineffectively responds to other structures from the bacterium explains the failure of ongoing attempts to develop working S. aureus vaccines.
An analysis reveals that the dragonfly's impressive ability to catch its prey arises from internal calculations about its own movements and those of its target— the first example of such predictions in invertebrates.
The extent to which coastal-ocean regions act as a sink for carbon dioxide has been enigmatic. An estimate based on more than 3 million observations suggests a smaller sink than was thought, concentrated at high latitudes.
Molecular diffusion of some enzymes is enhanced when they catalyse reactions, but the reason for this was obscure. Dissipation of heat generated by catalysis through the protein is now thought to propel the molecules.
This study tracks dragonfly head and body movements during high-velocity and high-precision prey-capture flights, and shows that the dragonfly uses predictive internal models and reactive control to build an interception trajectory that complies with biomechanical constraints.
The role of cellular metabolism in regulating cell proliferation and differentiation remains poorly understood. For example, most mammalian cells cannot proliferate without exogenous glutamine supplementation even though glutamine is a non-essential amino acid. Here we show that mouse embryonic stem (ES) cells grown under conditions that maintain naive pluripotency are capable of proliferation in the absence of exogenous glutamine. Despite this, ES cells consume high levels of exogenous glutamine when the metabolite is available. In comparison to more differentiated cells, naive ES cells utilize both glucose and glutamine catabolism to maintain a high level of intracellularα-ketoglutarate (αKG). Consequently, naive ES cells exhibit an elevated αKG to succinate ratio that promotes histone/DNA demethylation and maintains pluripotency. Direct manipulation of the intracellular αKG/succinate ratio is sufficient to regulate multiple chromatin modifications, including H3K27me3 and ten-eleven translocation (Tet)-dependent DNA demethylation, which contribute to the regulation of pluripotency-associated gene expression. In vitro, supplementation with cell-permeable αKG directly supports ES-cell self-renewal while cell-permeable succinate promotes differentiation. This work reveals that intracellular αKG/succinate levels can contribute to the maintenance of cellular identity and have a mechanistic role in the transcriptional and epigenetic state of stem cells.
Myocardial infarction (MI), a leading cause of death around the world, displays a complex pattern of inheritance. When MI occurs early in life, genetic inheritance is a major component to risk. Previously, rare mutations in low-density lipoprotein (LDL) genes have been shown to contribute to MI risk in individual families, whereas common variants at more than 45 loci have been associated with MI risk in the population. Here we evaluate how rare mutations contribute to early-onset MI risk in the population. We sequenced the protein-coding regions of 9,793 genomes from patients with MI at an early age (≤50 years in males and ≤60 years in females) along with MI-free controls. We identified two genes in which rare coding-sequence mutations were more frequent in MI cases versus controls at exome-wide significance. At low-density lipoprotein receptor (LDLR), carriers of rare non-synonymous mutations were at 4.2-fold increased risk for MI; carriers of null alleles at LDLR were at even higher risk (13-fold difference). Approximately 2% of early MI cases harbour a rare, damaging mutation in LDLR; this estimate is similar to one made more than 40 years ago using an analysis of total cholesterol. Among controls, about 1 in 217 carried an LDLR coding-sequence mutation and had plasma LDL cholesterol ggt; 190 mg dl−1. At apolipoprotein A-V (APOA5), carriers of rare non-synonymous mutations were at 2.2-fold increased risk for MI. When compared with non-carriers, LDLR mutation carriers had higher plasma LDL cholesterol, whereas APOA5 mutation carriers had higher plasma triglycerides. Recent evidence has connected MI risk with coding-sequence mutations at two genes functionally related to APOA5, namely lipoprotein lipase and apolipoprotein C-III (refs 18, 19). Combined, these observations suggest that, as well as LDL cholesterol, disordered metabolism of triglyceride-rich lipoproteins contributes to MI risk.
The widespread reorganization of cellular architecture in mitosis is achieved through extensive protein phosphorylation, driven by the coordinated activation of a mitotic kinase network and repression of counteracting phosphatases. Phosphatase activity must subsequently be restored to promote mitotic exit. Although Cdc14 phosphatase drives this reversal in budding yeast, protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) activities have each been independently linked to mitotic exit control in other eukaryotes. Here we describe a mitotic phosphatase relay in which PP1 reactivation is required for the reactivation of both PP2A-B55 and PP2A-B56 to coordinate mitotic progression and exit in fission yeast. The staged recruitment of PP1 (the Dis2 isoform) to the regulatory subunits of the PP2A-B55 and PP2A-B56 (B55 also known as Pab1; B56 also known as Par1) holoenzymes sequentially activates each phosphatase. The pathway is blocked in early mitosis because the Cdk1–cyclin B kinase (Cdk1 also known as Cdc2) inhibits PP1 activity, but declining cyclin B levels later in mitosis permit PP1 to auto-reactivate. PP1 first reactivates PP2A-B55; this enables PP2A-B55 in turn to promote the reactivation of PP2A-B56 by dephosphorylating a PP1-docking site in PP2A-B56, thereby promoting the recruitment of PP1. PP1 recruitment to human, mitotic PP2A-B56 holoenzymes and the sequences of these conserved PP1-docking motifs suggest that PP1 regulates PP2A-B55 and PP2A-B56 activities in a variety of signalling contexts throughout eukaryotes.
The concept of germ layers has been one of the foremost organizing principles in developmental biology, classification, systematics and evolution for 150 years (refs 1, 2, 3). Of the three germ layers, the mesoderm is found in bilaterian animals but is absent in species in the phyla Cnidaria and Ctenophora, which has been taken as evidence that the mesoderm was the final germ layer to evolve. The origin of the ectoderm and endoderm germ layers, however, remains unclear, with models supporting the antecedence of each as well as a simultaneous origin. Here we determine the temporal and spatial components of gene expression spanning embryonic development for all Caenorhabditis elegans genes and use it to determine the evolutionary ages of the germ layers. The gene expression program of the mesoderm is induced after those of the ectoderm and endoderm, thus making it the last germ layer both to evolve and to develop. Strikingly, the C. elegans endoderm and ectoderm expression programs do not co-induce; rather the endoderm activates earlier, and this is also observed in the expression of endoderm orthologues during the embryology of the frog Xenopus tropicalis, the sea anemone Nematostella vectensis and the sponge Amphimedon queenslandica. Querying the phylogenetic ages of specifically expressed genes reveals that the endoderm comprises older genes. Taken together, we propose that the endoderm program dates back to the origin of multicellularity, whereas the ectoderm originated as a secondary germ layer freed from ancestral feeding functions.
Recent studies have shown that the diffusivity of enzymes increases in a substrate-dependent manner during catalysis. Although this observation has been reported and characterized for several different systems, the precise origin of this phenomenon is unknown. Calorimetric methods are often used to determine enthalpies from enzyme-catalysed reactions and can therefore provide important insight into their reaction mechanisms. The ensemble averages involved in traditional bulk calorimetry cannot probe the transient effects that the energy exchanged in a reaction may have on the catalyst. Here we obtain single-molecule fluorescence correlation spectroscopy data and analyse them within the framework of a stochastic theory to demonstrate a mechanistic link between the enhanced diffusion of a single enzyme molecule and the heat released in the reaction. We propose that the heat released during catalysis generates an asymmetric pressure wave that results in a differential stress at the protein–solvent interface that transiently displaces the centre-of-mass of the enzyme (chemoacoustic effect). This novel perspective on how enzymes respond to the energy released during catalysis suggests a possible effect of the heat of reaction on the structural integrity and internal degrees of freedom of the enzyme.
Titan, the largest satellite of Saturn, exhibits extensive aeolian, that is, wind-formed, dunes, features previously identified exclusively on Earth, Mars and Venus. Wind tunnel data collected under ambient and planetary-analogue conditions inform our models of aeolian processes on the terrestrial planets. However, the accuracy of these widely used formulations in predicting the threshold wind speeds required to move sand by saltation, or by short bounces, has not been tested under conditions relevant for non-terrestrial planets. Here we derive saltation threshold wind speeds under the thick-atmosphere, low-gravity and low-sediment-density conditions on Titan, using a high-pressure wind tunnel refurbished to simulate the appropriate kinematic viscosity for the near-surface atmosphere of Titan. The experimentally derived saltation threshold wind speeds are higher than those predicted by models based on terrestrial-analogue experiments, indicating the limitations of these models for such extreme conditions. The models can be reconciled with the experimental results by inclusion of the extremely low ratio of particle density to fluid density on Titan. Whereas the density ratio term enables accurate modelling of aeolian entrainment in thick atmospheres, such as those inferred for some extrasolar planets, our results also indicate that for environments with high density ratios, such as in jets on icy satellites or in tenuous atmospheres or exospheres, the correction for low-density-ratio conditions is not required.
A defining feature of vertebrates (craniates) is a pronounced head that is supported and protected by a robust cellular endoskeleton. In the first vertebrates, this skeleton probably consisted of collagenous cellular cartilage, which forms the embryonic skeleton of all vertebrates and the adult skeleton of modern jawless and cartilaginous fish. In the head, most cellular cartilage is derived from a migratory cell population called the neural crest, which arises from the edges of the central nervous system. Because collagenous cellular cartilage and neural crest cells have not been described in invertebrates, the appearance of cellular cartilage derived from neural crest cells is considered a turning point in vertebrate evolution. Here we show that a tissue with many of the defining features of vertebrate cellular cartilage transiently forms in the larvae of the invertebrate chordate Branchiostoma floridae (Florida amphioxus). We also present evidence that during evolution, a key regulator of vertebrate cartilage development, SoxE, gained new cis-regulatory sequences that subsequently directed its novel expression in neural crest cells. Together, these results suggest that the origin of the vertebrate head skeleton did not depend on the evolution of a new skeletal tissue, as is commonly thought, but on the spread of this tissue throughout the head. We further propose that the evolution of cis-regulatory elements near an ancient regulator of cartilage differentiation was a major factor in the evolution of the vertebrate head skeleton.
Therapy-related acute myeloid leukaemia (t-AML) and therapy-related myelodysplastic syndrome (t-MDS) are well-recognized complications of cytotoxic chemotherapy and/or radiotherapy. There are several features that distinguish t-AML from de novo AML, including a higher incidence of TP53 mutations, abnormalities of chromosomes 5 or 7, complex cytogenetics and a reduced response to chemotherapy. However, it is not clear how prior exposure to cytotoxic therapy influences leukaemogenesis. In particular, the mechanism by which TP53 mutations are selectively enriched in t-AML/t-MDS is unknown. Here, by sequencing the genomes of 22 patients with t-AML, we show that the total number of somatic single-nucleotide variants and the percentage of chemotherapy-related transversions are similar in t-AML and de novo AML, indicating that previous chemotherapy does not induce genome-wide DNA damage. We identified four cases of t-AML/t-MDS in which the exact TP53 mutation found at diagnosis was also present at low frequencies (0.003–0.7%) in mobilized blood leukocytes or bone marrow 3–6 years before the development of t-AML/t-MDS, including two cases in which the relevant TP53 mutation was detected before any chemotherapy. Moreover, functional TP53 mutations were identified in small populations of peripheral blood cells of healthy chemotherapy-naive elderly individuals. Finally, in mouse bone marrow chimaeras containing both wild-type and Tp53+/− haematopoietic stem/progenitor cells (HSPCs), the Tp53+/− HSPCs preferentially expanded after exposure to chemotherapy. These data suggest that cytotoxic therapy does not directly induce TP53 mutations. Rather, they support a model in which rare HSPCs carrying age-related TP53 mutations are resistant to chemotherapy and expand preferentially after treatment. The early acquisition of TP53 mutations in the founding HSPC clone probably contributes to the frequent cytogenetic abnormalities and poor responses to chemotherapy that are typical of patients with t-AML/t-MDS.
The African Genome Variation Project presents genotyping and whole-genome data from individuals across sub-Saharan Africa, giving insight into population history and guiding future genomic studies on the continent.
The discovery that the neural navigation system of the mammalian brain acts in three dimensions sheds light on how mammals orient themselves in complex environments.
Bladder-cancer cells have been found to release prostaglandin E2 when they are killed by chemotherapy. Paradoxically, this molecule stimulates the proliferation of surviving cancer stem cells, leading to tumour repopulation.
A study of freely moving bats provides new insights into how the brain encodes a three-dimensional neural compass; neurons were identified encoding the three Euler rotation angles of the head (azimuth, pitch, and roll) and recordings from these head-direction cells revealed a toroidal model of spatial orientation mapped out by cells tuned to two circular variables (azimuth× pitch).
The African Genome Variation Project contains the whole-genome sequences of 320 individuals and dense genotypes on 1,481 individuals from sub-Saharan Africa; it enables the design and interpretation of genomic studies, with implications for finding disease loci and clues to human origins.
In human societies, cultural norms arise when behaviours are transmitted through social networks via high-fidelity social learning. However, a paucity of experimental studies has meant that there is no comparable understanding of the process by which socially transmitted behaviours might spread and persist in animal populations. Here we show experimental evidence of the establishment of foraging traditions in a wild bird population. We introduced alternative novel foraging techniques into replicated wild sub-populations of great tits (Parus major) and used automated tracking to map the diffusion, establishment and long-term persistence of the seeded innovations. Furthermore, we used social network analysis to examine the social factors that influenced diffusion dynamics. From only two trained birds in each sub-population, the information spread rapidly through social network ties, to reach an average of 75% of individuals, with a total of 414 knowledgeable individuals performing 57,909 solutions over all replicates. The sub-populations were heavily biased towards using the technique that was originally introduced, resulting in established local traditions that were stable over two generations, despite a high population turnover. Finally, we demonstrate a strong effect of social conformity, with individuals disproportionately adopting the most frequent local variant when first acquiring an innovation, and continuing to favour social information over personal information. Cultural conformity is thought to be a key factor in the evolution of complex culture in humans. In providing the first experimental demonstration of conformity in a wild non-primate, and of cultural norms in foraging techniques in any wild animal, our results suggest a much broader taxonomic occurrence of such an apparently complex cultural behaviour.
The manufacture of geometric engravings is generally interpreted as indicative of modern cognition and behaviour. Key questions in the debate on the origin of such behaviour are whether this innovation is restricted to Homo sapiens, and whether it has a uniquely African origin. Here we report on a fossil freshwater shell assemblage from the Hauptknochenschicht (‘main bone layer’) of Trinil (Java, Indonesia), the type locality of Homo erectus discovered by Eugène Dubois in 1891 (refs 2 and 3). In the Dubois collection (in the Naturalis museum, Leiden, The Netherlands) we found evidence for freshwater shellfish consumption by hominins, one unambiguous shell tool, and a shell with a geometric engraving. We dated sediment contained in the shells with 40Ar/39Ar and luminescence dating methods, obtaining a maximum age of 0.54 ± 0.10 million years and a minimum age of 0.43 ± 0.05 million years. This implies that the Trinil Hauptknochenschicht isyounger than previously estimated. Together, our data indicate that the engraving was made by Homo erectus, and that it is considerably older than the oldest geometric engravings described so far. Although it is at present not possible to assess the function or meaning of the engraved shell, this discovery suggests that engraving abstract patterns was in the realm of Asian Homo erectus cognition and neuromotor control.
Cytotoxic chemotherapy is effective in debulking tumour masses initially; however, in some patients tumours become progressively unresponsive after multiple treatment cycles. Previous studies have demonstrated that cancer stem cells (CSCs) are selectively enriched after chemotherapy through enhanced survival. Here we reveal a new mechanism by which bladder CSCs actively contribute to therapeutic resistance via an unexpected proliferative response to repopulate residual tumours between chemotherapy cycles, using human bladder cancer xenografts. Further analyses demonstrate the recruitment of a quiescent label-retaining pool of CSCs into cell division in response to chemotherapy-induced damages, similar to mobilization of normal stem cells during wound repair. While chemotherapy effectively induces apoptosis, associated prostaglandin E2 (PGE2) release paradoxically promotes neighbouring CSC repopulation. This repopulation can be abrogated by a PGE2-neutralizing antibody and celecoxib drug-mediated blockade of PGE2 signalling. In vivo administration of the cyclooxygenase-2 (COX2) inhibitor celecoxib effectively abolishes a PGE2- and COX2-mediated wound response gene signature, and attenuates progressive manifestation of chemoresistance in xenograft tumours, including primary xenografts derived from a patient who was resistant to chemotherapy. Collectively, these findings uncover a new underlying mechanism that models the progressive development of clinical chemoresistance, and implicate an adjunctive therapy to enhance chemotherapeutic response of bladder urothelial carcinomas by abrogating early tumour repopulation.
Contusive spinal cord injury leads to a variety of disabilities owing to limited neuronal regeneration and functional plasticity. It is well established that an upregulation of glial-derived chondroitin sulphate proteoglycans (CSPGs) within the glial scar and perineuronal net creates a barrier to axonal regrowth and sprouting. Protein tyrosine phosphataseσ (PTPσ), along with its sister phosphatase leukocyte common antigen-related (LAR) and the nogo receptors 1 and 3 (NgR), have recently been identified as receptors for the inhibitory glycosylated side chains of CSPGs. Here we find in rats that PTPσ has a critical role in converting growth cones into a dystrophic state by tightly stabilizing them within CSPG-rich substrates. We generated a membrane-permeable peptide mimetic of the PTPσ wedge domain that binds to PTPσ and relieves CSPG-mediated inhibition. Systemic delivery of this peptide over weeks restored substantial serotonergic innervation to the spinal cord below the level of injury and facilitated functional recovery of both locomotor and urinary systems. Our results add a new layer of understanding to the critical role of PTPσ in mediating the growth-inhibited state of neurons due to CSPGs within the injured adult spinal cord.
Most haematopoietic cells renew from adult haematopoietic stem cells (HSCs), however, macrophages in adult tissues can self-maintain independently of HSCs. Progenitors with macrophage potential in vitro have been described in the yolk sac before emergence of HSCs, and fetal macrophages can develop independently of Myb, a transcription factor required for HSC, and can persist in adult tissues. Nevertheless, the origin of adult macrophages and the qualitative and quantitative contributions of HSC and putative non-HSC-derived progenitors are still unclear. Here we show in mice that the vast majority of adult tissue-resident macrophages in liver (Kupffer cells), brain (microglia), epidermis (Langerhans cells) and lung (alveolar macrophages) originate from a Tie2+ (also known as Tek) cellular pathway generating Csf1r+ erythro-myeloid progenitors (EMPs) distinct from HSCs. EMPs develop in the yolk sac at embryonic day (E) 8.5, migrate and colonize the nascent fetal liver before E10.5, and give rise to fetal erythrocytes, macrophages, granulocytes and monocytes until at least E16.5. Subsequently, HSC-derived cells replace erythrocytes, granulocytes and monocytes. Kupffer cells, microglia and Langerhans cells are only marginally replaced in one-year-old mice, whereas alveolar macrophages may be progressively replaced in ageing mice. Our fate-mapping experiments identify, in the fetal liver, a sequence of yolk sac EMP-derived and HSC-derived haematopoiesis, and identify yolk sac EMPs as a common origin for tissue macrophages.
T-helper type 17 (TH17) cells that produce the cytokines interleukin-17A (IL-17A) and IL-17F are implicated in the pathogenesis of several autoimmune diseases. The differentiation of TH17 cells is regulated by transcription factors such as RORγt, but post-translational mechanisms preventing the rampant production of pro-inflammatory IL-17A have received less attention. Here we show that the deubiquitylating enzyme DUBA is a negative regulator of IL-17A production in T cells. Mice with DUBA-deficient T cells developed exacerbated inflammation in the small intestine after challenge with anti-CD3 antibodies. DUBA interacted with the ubiquitin ligase UBR5, which suppressed DUBA abundance in naive T cells. DUBA accumulated in activated T cells and stabilized UBR5, which then ubiquitylated RORγt in response to TGF-β signalling. Our data identify DUBA as a cell-intrinsic suppressor of IL-17 production.
Using electron cryomicroscopy, the structure of the rabbit RyR1 calcium channel is determined at 6.1 Å resolution in the closed state and 8.5 Å in the open state, revealing how calcium binding to the EF-hand of RyR1 regulates channel opening and facilitates calcium-induced calcium release.
Using electron cryomicroscopy, the closed-state structure of rabbit RyR1 is determined at 4.8 Å resolution; analysis confirms that the RyR1 architecture consists of a six-transmembrane ion channel with a cytosolic α-solenoid scaffold, and suggests a mechanism for Ca2+-induced channel opening.
Angelman syndrome is a single-gene disorder characterized by intellectual disability, developmental delay, behavioural uniqueness, speech impairment, seizures and ataxia. It is caused by maternal deficiency of the imprinted gene UBE3A, encoding an E3 ubiquitin ligase. All patients carry at least one copy of paternal UBE3A, which is intact but silenced by a nuclear-localized long non-coding RNA, UBE3A antisense transcript (UBE3A-ATS). Murine Ube3a-ATS reduction by either transcription termination or topoisomerase I inhibition has been shown to increase paternal Ube3a expression. Despite a clear understanding of the disease-causing event in Angelman syndrome and the potential to harness the intact paternal allele to correct the disease, no gene-specific treatment exists for patients. Here we developed a potential therapeutic intervention for Angelman syndrome by reducing Ube3a-ATS with antisense oligonucleotides (ASOs). ASO treatment achieved specific reduction of Ube3a-ATS and sustained unsilencing of paternal Ube3a in neurons in vitro and in vivo. Partial restoration of UBE3A protein in an Angelman syndrome mouse model ameliorated some cognitive deficits associated with the disease. Although additional studies of phenotypic correction are needed, we have developed a sequence-specific and clinically feasible method to activate expression of the paternal Ube3a allele.
Members of the dynein family, consisting of cytoplasmic and axonemal isoforms, are motors that move towards the minus ends of microtubules. Cytoplasmic dynein-1 (dynein-1) plays roles in mitosis and cellular cargo transport, and is implicated in viral infections and neurodegenerative diseases. Cytoplasmic dynein-2 (dynein-2) performs intraflagellar transport and is associated with human skeletal ciliopathies. Dyneins share a conserved motor domain that couples cycles of ATP hydrolysis with conformational changes to produce movement. Here we present the crystal structure of the human cytoplasmic dynein-2 motor bound to the ATP-hydrolysis transition state analogue ADP.vanadate. The structure reveals a closure of the motor’s ring of six AAA+ domains (ATPases associated with various cellular activites: AAA1–AAA6). This induces a steric clash with the linker, the key element for the generation of movement, driving it into a conformation that is primed to produce force. Ring closure also changes the interface between the stalk and buttress coiled-coil extensions of the motor domain. This drives helix sliding in the stalk which causes the microtubule binding domain at its tip to release from the microtubule. Our structure answers the key questions of how ATP hydrolysis leads to linker remodelling and microtubule affinity regulation.
The emergence of catalysis in early genetic polymers such as RNA is considered a key transition in the origin of life, pre-dating the appearance of protein enzymes. DNA also demonstrates the capacity to fold into three-dimensional structures and form catalysts in vitro. However, to what degree these natural biopolymers comprise functionally privileged chemical scaffolds for folding or the evolution of catalysis is not known. The ability of synthetic genetic polymers (XNAs) with alternative backbone chemistries not found in nature to fold into defined structures and bind ligands raises the possibility that these too might be capable of forming catalysts (XNAzymes). Here we report the discovery of such XNAzymes, elaborated in four different chemistries (arabino nucleic acids, ANA; 2′-fluoroarabino nucleic acids, FANA; hexitol nucleic acids, HNA; and cyclohexene nucleic acids, CeNA) directly from random XNA oligomer pools, exhibiting in trans RNA endonuclease and ligase activities. We also describe an XNA–XNA ligase metalloenzyme in the FANA framework, establishing catalysis in an entirely synthetic system and enabling the synthesis of FANA oligomers and an active RNA endonuclease FANAzyme from its constituent parts. These results extend catalysis beyond biopolymers and establish technologies for the discovery of catalysts in a wide range of polymer scaffolds not found in nature. Evolution of catalysis independent of any natural polymer has implications for the definition of chemical boundary conditions for the emergence of life on Earth and elsewhere in the Universe.
Filamentous actin (F-actin) is the major protein of muscle thin filaments, and actin microfilaments are the main component of the eukaryotic cytoskeleton. Mutations in different actin isoforms lead to early-onset autosomal dominant non-syndromic hearing loss, familial thoracic aortic aneurysms and dissections, and multiple variations of myopathies. In striated muscle fibres, the binding of myosin motors to actin filaments is mainly regulated by tropomyosin and troponin. Tropomyosin also binds to F-actin in smooth muscle and in non-muscle cells and stabilizes and regulates the filaments there in the absence of troponin. Although crystal structures for monomeric actin (G-actin) are available, a high-resolution structure of F-actin is still missing, hampering our understanding of how disease-causing mutations affect the function of thin muscle filaments and microfilaments. Here we report the three-dimensional structure of F-actin at a resolution of 3.7 Å in complex with tropomyosin at a resolution of 6.5 Å, determined by electron cryomicroscopy. The structure reveals that the D-loop is ordered and acts as a central region for hydrophobic and electrostatic interactions that stabilize the F-actin filament. We clearly identify map density corresponding to ADP and Mg2+ and explain the possible effect of prominent disease-causing mutants. A comparison of F-actin with G-actin reveals the conformational changes during filament formation and identifies the D-loop as their key mediator. We also confirm that negatively charged tropomyosin interacts with a positively charged groove on F-actin. Comparison of the position of tropomyosin in F-actin–tropomyosin with its position in our previously determined F-actin–tropomyosin–myosin structure reveals a myosin-induced transition of tropomyosin. Our results allow us to understand the role of individual mutations in the genesis of actin- and tropomyosin-related diseases and will serve as a strong foundation for the targeted development of drugs.
Photosystem II, a photosynthetic protein complex, is prone to X-ray damage during crystallography. A high-resolution structure of the undamaged complex now offers a detailed view of its catalytic centre.
Human cancers, including breast cancers, comprise clones differing in mutation content. Clones evolve dynamically in space and time following principles of Darwinian evolution, underpinning important emergent features such as drug resistance and metastasis. Human breast cancer xenoengraftment is used as a means of capturing and studying tumour biology, and breast tumour xenografts are generally assumed to be reasonable models of the originating tumours. However, the consequences and reproducibility of engraftment and propagation on the genomic clonal architecture of tumours have not been systematically examined at single-cell resolution. Here we show, using deep-genome and single-cell sequencing methods, the clonal dynamics of initial engraftment and subsequent serial propagation of primary and metastatic human breast cancers in immunodeficient mice. In all 15 cases examined, clonal selection on engraftment was observed in both primary and metastatic breast tumours, varying in degree from extreme selective engraftment of minor (llt;5% of starting population) clones to moderate, polyclonal engraftment. Furthermore, ongoing clonal dynamics during serial passaging is a feature of tumours experiencing modest initial selection. Through single-cell sequencing, we show that major mutation clusters estimated from tumour population sequencing relate predictably to the most abundant clonal genotypes, even in clonally complex and rapidly evolving cases. Finally, we show that similar clonal expansion patterns can emerge in independent grafts of the same starting tumour population, indicating that genomic aberrations can be reproducible determinants of evolutionary trajectories. Our results show that measurement of genomically defined clonal population dynamics will be highly informative for functional studies using patient-derived breast cancer xenoengraftment.
Safe and powerful energy storage devices are becoming increasingly important. Charging times of seconds to minutes, with power densities exceeding those of batteries, can in principle be provided by electrochemical capacitors—in particular, pseudocapacitors. Recent research has focused mainly on improving the gravimetric performance of the electrodes of such systems, but for portable electronics and vehicles volume is at a premium. The best volumetric capacitances of carbon-based electrodes are around 300 farads per cubic centimetre; hydrated ruthenium oxide can reach capacitances of 1,000 to 1,500 farads per cubic centimetre with great cyclability, but only in thin films. Recently, electrodes made of two-dimensional titanium carbide (Ti3C2, a member of the ‘MXene’ family), produced by etching aluminium from titanium aluminium carbide (Ti3AlC2, a ‘MAX’ phase) in concentrated hydrofluoric acid, have been shown to have volumetric capacitances of over 300 farads per cubic centimetre. Here we report a method of producing this material using a solution of lithium fluoride and hydrochloric acid. The resulting hydrophilic material swells in volume when hydrated, and can be shaped like clay and dried into a highly conductive solid or rolled into films tens of micrometres thick. Additive-free films of this titanium carbide ‘clay’ have volumetric capacitances of up to 900 farads per cubic centimetre, with excellent cyclability and rate performances. This capacitance is almost twice that of our previous report, and our synthetic method also offers a much faster route to film production as well as the avoidance of handling hazardous concentrated hydrofluoric acid.
Photosynthesis converts light energy into biologically useful chemical energy vital to life on Earth. The initial reaction of photosynthesis takes place in photosystem II (PSII), a 700-kilodalton homodimeric membrane protein complex that catalyses photo-oxidation of water into dioxygen through an S-state cycle of the oxygen evolving complex (OEC). The structure of PSII has been solved by X-ray diffraction (XRD) at 1.9 ångström resolution, which revealed that the OEC is a Mn4CaO5-cluster coordinated by a well defined protein environment. However, extended X-ray absorption fine structure (EXAFS) studies showed that the manganese cations in the OEC are easily reduced by X-ray irradiation, and slight differenceswere found in the Mn–Mn distances determined by XRD, EXAFS and theoretical studies. Here we report a ‘radiation-damage-free’ structure of PSII from Thermosynechococcus vulcanus in the S1 state at a resolution of 1.95 ångströms using femtosecond X-ray pulses of the SPring-8 ångström compact free-electron laser (SACLA) and hundreds of large, highly isomorphous PSII crystals. Compared with the structure from XRD, the OEC in the X-ray free electron laser structure has Mn–Mn distances that are shorter by 0.1–0.2 ångströms. The valences of each manganese atom were tentatively assigned as Mn1D(iii), Mn2C(iv), Mn3B(iv) and Mn4A(iii), based on the average Mn–ligand distances and analysis of the Jahn–Teller axis on Mn(iii). One of the oxo-bridged oxygens, O5, has significantly longer distances to Mn than do the other oxo-oxygen atoms, suggesting that O5 is a hydroxide ion instead of a normal oxygen dianion and therefore may serve as one of the substrate oxygen atoms. These findings provide a structural basis for the mechanism of oxygen evolution, and we expect that this structure will provide a blueprint for the design of artificial catalysts for water oxidation.
The TRIM37 (also known as MUL) gene is located in the 17q23 chromosomal region, which is amplified in up to∼40% of breast cancers. TRIM37 contains a RING finger domain, a hallmark of E3 ubiquitin ligases, but its protein substrate(s) is unknown. Here we report that TRIM37 mono-ubiquitinates histone H2A, a chromatin modification associated with transcriptional repression. We find that in human breast cancer cell lines containing amplified 17q23, TRIM37 is upregulated and, reciprocally, the major H2A ubiquitin ligase RNF2 (also known as RING1B) is downregulated. Genome-wide chromatin immunoprecipitation (ChIP)-chip experiments in 17q23-amplified breast cancer cells identified many genes, includingmultiple tumour suppressors, whose promoters were bound by TRIM37 and enriched for ubiquitinated H2A. However, unlike RNF2, which is a subunit of polycomb repressive complex 1 (PRC1), we find that TRIM37 associates with polycomb repressive complex 2 (PRC2). TRIM37, PRC2 and PRC1 are co-bound to specific target genes, resulting in their transcriptional silencing. RNA-interference-mediated knockdown of TRIM37 results in loss of ubiquitinated H2A, dissociation of PRC1 and PRC2 from target promoters, and transcriptional reactivation of silenced genes. Knockdown of TRIM37 in human breast cancer cells containing amplified 17q23 substantially decreases tumour growth in mouse xenografts. Conversely, ectopic expression of TRIM37 renders non-transformed cells tumorigenic. Collectively, our results reveal TRIM37 as an oncogenic H2A ubiquitin ligase that is overexpressed in a subset of breast cancers and promotes transformation by facilitating silencing of tumour suppressors and other genes.
DEAD-box RNA helicases are vital for the regulation of various aspects of the RNA life cycle, but the molecular underpinnings of their involvement, particularly in mammalian cells, remain poorly understood. Here we show that the DEAD-box RNA helicase DDX21 can sense the transcriptional status of both RNA polymerase (Pol) I and II to control multiple steps of ribosome biogenesis in human cells. We demonstrate that DDX21 widely associates with Pol I- and Pol II-transcribed genes and with diverse species of RNA, most prominently with non-coding RNAs involved in the formation of ribonucleoprotein complexes, including ribosomal RNA, small nucleolar RNAs (snoRNAs) and 7SK RNA. Although broad, these molecular interactions, both at the chromatin and RNA level, exhibit remarkable specificity for the regulation of ribosomal genes. In the nucleolus, DDX21 occupies the transcribed rDNA locus, directly contacts both rRNA and snoRNAs, and promotes rRNA transcription, processing and modification. In the nucleoplasm, DDX21 binds 7SK RNA and, as a component of the 7SK small nuclear ribonucleoprotein (snRNP) complex, is recruited to the promoters of Pol II-transcribed genes encoding ribosomal proteins and snoRNAs. Promoter-bound DDX21 facilitates the release of the positive transcription elongation factor b (P-TEFb) from the 7SK snRNP in a manner that is dependent on its helicase activity, thereby promoting transcription of its target genes. Our results uncover the multifaceted role of DDX21 in multiple steps of ribosome biogenesis, and provide evidence implicating a mammalian RNA helicase in RNA modification and Pol II elongation control.
Horizontal gene transfer allows organisms to rapidly acquire adaptive traits. Although documented instances of horizontal gene transfer from bacteria to eukaryotes remain rare, bacteria represent a rich source of new functions potentially available for co-option. One benefit that genes of bacterial origin could provide to eukaryotes is the capacity to produce antibacterials, which have evolved in prokaryotes as the result of eons of interbacterial competition. The type VI secretion amidase effector (Tae) proteins are potent bacteriocidal enzymes that degrade the cell wall when delivered into competing bacterial cells by the type VI secretion system. Here we show that tae genes have been transferred to eukaryotes on at least six occasions, and that the resulting domesticated amidase effector (dae) genes have been preserved for hundreds of millions of years through purifying selection. We show that the dae genes acquired eukaryotic secretion signals, are expressed within recipient organisms, and encode active antibacterial toxins that possess substrate specificity matching extant Tae proteins of the same lineage. Finally, we show that a dae gene in the deer tick Ixodes scapularis limits proliferation of Borrelia burgdorferi, the aetiologic agent of Lyme disease. Our work demonstrates that a family of horizontally acquired toxins honed to mediate interbacterial antagonism confers previously undescribed antibacterial capacity to eukaryotes. We speculate that the selective pressure imposed by competition between bacteria has produced a reservoir of genes encoding diverse antimicrobial functions that are tailored for co-option by eukaryotic innate immune systems.
Pathogenic mycobacteria induce the formation of complex cellular aggregates called granulomas that are the hallmark of tuberculosis. Here we examine the development and consequences of vascularization of the tuberculous granuloma in the zebrafish–Mycobacterium marinum infection model, which is characterized by organized granulomas with necrotic cores that bear striking resemblance to those of human tuberculosis. Using intravital microscopy in the transparent larval zebrafish, we show that granuloma formation is intimately associated withangiogenesis. The initiation of angiogenesis in turn coincides with the generation of local hypoxia and transcriptional induction of the canonical pro-angiogenic molecule Vegfaa. Pharmacological inhibition of the Vegf pathway suppresses granuloma-associated angiogenesis, reduces infection burden and limits dissemination. Moreover, anti-angiogenic therapies synergize with the first-line anti-tubercular antibiotic rifampicin, as well as with the antibiotic metronidazole, which targets hypoxic bacterial populations. Our data indicate that mycobacteria induce granuloma-associated angiogenesis, which promotes mycobacterial growth and increases spread of infection to new tissue sites. We propose the use of anti-angiogenic agents, now being used in cancer regimens, as a host-targeting tuberculosis therapy, particularly in extensively drug-resistant disease for which current antibiotic regimensare largely ineffective.
Certain structural elements allow messenger RNAs not usually processed by the protein-synthesis apparatus to be translated. It now seems that they also control the expression of genes involved in embryonic development.
A new class of fatty acid— found in food and synthesized by mammalian tissues — enhances glucose uptake from the blood and reduces inflammation, suggesting that these fats might be used to treat diabetes.
An experiment shows that although bank employees behave honestly on average, their dishonesty increases when they make decisions after having been primed to think about their professional identity.
The finding that intestinal viruses can substitute for intestinal bacteria to promote the health of their mammalian hosts raises the possibility that viruses in the gut may be beneficial in some circumstances.
Specialized ribosomes (with a particular protein composition) carry out translation of specific transcripts; analysis of Hox mRNA translation in mice reveals that unique RNA structural elements within their 5′ UTRs, including internal ribosome entry sites and translation inhibitory elements, are responsible for this specialized mode of translation.
Trust in others’ honesty is a key component of the long-term performance of firms, industries, and even whole countries. However, in recent years, numerous scandals involving fraud have undermined confidence in the financial industry. Contemporary commentators have attributed these scandals to the financial sector’s business culture, but no scientific evidence supports this claim. Here we show that employees of a large, international bank behave, on average, honestly in a control condition. However, when their professional identity as bank employees is rendered salient, a significant proportion of them become dishonest. This effect is specific to bank employees because control experiments with employees from other industries and with students show that they do not become more dishonest when their professional identity or bank-related items are rendered salient. Our results thus suggest that the prevailing business culture in the banking industry weakens and undermines the honesty norm, implying that measures to re-establish an honest culture are very important.
Intestinal microbial communities have profound effects on host physiology. Whereas the symbiotic contribution of commensal bacteria is well established, the role of eukaryotic viruses that are present in the gastrointestinal tract under homeostatic conditions is undefined. Here we demonstrate that a common enteric RNA virus can replace the beneficial function of commensal bacteria in the intestine. Murine norovirus (MNV) infection of germ-free or antibiotic-treated mice restored intestinal morphology and lymphocyte function without inducing overt inflammation and disease. The presence of MNV also suppressed an expansion of group 2 innate lymphoid cells observed in the absence of bacteria, and induced transcriptional changes in the intestine associated with immune development and type I interferon (IFN) signalling. Consistent with this observation, the IFN-α receptor was essential for the ability of MNV to compensate for bacterial depletion. Importantly, MNV infection offset the deleterious effect of treatment with antibiotics in models of intestinal injury and pathogenic bacterial infection. These data indicate that eukaryotic viruses have the capacity to support intestinal homeostasis and shape mucosal immunity, similarly to commensal bacteria.
Obesity-linked insulin resistance is a major precursor to the development of type 2 diabetes. Previous work has shown that phosphorylation of PPARγ (peroxisome proliferator-activated receptor γ) at serine 273 by cyclin-dependent kinase 5 (Cdk5) stimulates diabetogenic gene expression in adipose tissues. Inhibition of this modification is a key therapeutic mechanism for anti-diabetic drugs that bind PPARγ, such as the thiazolidinediones and PPARγ partial agonists or non-agonists. For a better understanding of the importance of this obesity-linked PPARγ phosphorylation, we created mice that ablated Cdk5 specifically in adipose tissues. These mice have both a paradoxical increase in PPARγ phosphorylation at serine 273 and worsened insulin resistance. Unbiased proteomic studies show that extracellular signal-regulated kinase (ERK) kinases are activated in these knockout animals. Here we show that ERK directly phosphorylates serine 273 of PPARγ in a robust manner and that Cdk5 suppresses ERKs through direct action on a novel site in MAP kinase/ERK kinase (MEK). Importantly, pharmacological inhibition of MEK and ERK markedly improves insulin resistance in both obese wild-type and ob/ob mice, and also completely reverses the deleterious effects of the Cdk5 ablation. These data show that an ERK/Cdk5 axis controls PPARγ function and suggest that MEK/ERK inhibitors may hold promise for the treatment of type 2 diabetes.
The prominent and evolutionarily ancient role of the plant hormone auxin is the regulation of cell expansion. Cell expansion requires ordered arrangement of the cytoskeleton but molecular mechanisms underlying its regulation by signalling molecules including auxin are unknown. Here we show in the model plant Arabidopsis thaliana that in elongating cells exogenous application of auxin or redistribution of endogenous auxin induces very rapid microtubule re-orientation from transverse to longitudinal, coherent with the inhibition of cell expansion. This fast auxin effect requires auxin binding protein 1 (ABP1) and involves a contribution of downstream signalling components such as ROP6 GTPase, ROP-interactive protein RIC1 and the microtubule-severing protein katanin. These components are required for rapid auxin- and ABP1-mediated re-orientation of microtubules to regulate cell elongation in roots and dark-grown hypocotyls as well as asymmetric growth during gravitropic responses.
MicroRNAs are short non-coding RNAs expressed in different tissue and cell types that suppress the expression of target genes. As such, microRNAs are critical cogs in numerous biological processes, and dysregulated microRNA expression is correlated with many human diseases. Certain microRNAs, called oncomiRs, play a causal role in the onset and maintenance of cancer when overexpressed. Tumours that depend on these microRNAs are said to display oncomiR addiction. Some of the most effective anticancer therapies target oncogenes such as EGFR and HER2; similarly, inhibition of oncomiRs using antisense oligomers (that is, antimiRs) is an evolving therapeutic strategy. However, the in vivo efficacy of current antimiR technologies is hindered by physiological and cellular barriers to delivery into targeted cells. Here we introduce a novel antimiR delivery platform that targets the acidic tumour microenvironment, evades systemic clearance by the liver, and facilitates cell entry via a non-endocytic pathway. We find that the attachment of peptide nucleic acid antimiRs to a peptide with a low pH-induced transmembrane structure (pHLIP) produces a novel construct that could target the tumour microenvironment, transport antimiRs across plasma membranes under acidic conditions such as those found in solid tumours (pH approximately 6), and effectively inhibit the miR-155 oncomiR in a mouse model of lymphoma. This study introduces a new model for using antimiRs as anti-cancer drugs, which can have broad impacts on the field of targeted drug delivery.
Oxidative stress promotes genomic instability and human diseases. A common oxidized nucleoside is 8-oxo-7,8-dihydro-2′-deoxyguanosine, which is found both in DNA (8-oxo-G) and as a free nucleotide (8-oxo-dGTP). Nucleotide pools are especially vulnerable to oxidative damage. Therefore cells encode an enzyme (MutT/MTH1) that removes free oxidized nucleotides. This cleansing function is required for cancer cell survival and to modulate Escherichia coli antibiotic sensitivity in a DNA polymerase (pol)-dependent manner. How polymerases discriminate between damaged and non-damaged nucleotides is not well understood. This analysis is essential given the role of oxidized nucleotides in mutagenesis, cancer therapeutics, and bacterial antibiotics. Even with cellular sanitizing activities, nucleotide pools contain enough 8-oxo-dGTP to promote mutagenesis. This arises from the dual coding potential where 8-oxo-dGTP(anti) base pairs with cytosine and 8-oxo-dGTP(syn) uses its Hoogsteen edge to base pair with adenine. Here we use time-lapse crystallography to follow 8-oxo-dGTP insertion opposite adenine or cytosine with human pol β, to reveal that insertion is accommodated in either the syn- or anti-conformation, respectively. For 8-oxo-dGTP(anti) insertion, a novel divalent metal relieves repulsive interactions between the adducted guanine base and the triphosphate of the oxidized nucleotide. With either templating base, hydrogen-bonding interactions between the bases are lost as the enzyme reopens after catalysis, leading to a cytotoxic nicked DNA repair intermediate. Combining structural snapshots with kinetic and computational analysis reveals how 8-oxo-dGTP uses charge modulation during insertion that can lead to a blocked DNA repair intermediate.
Broad and deep tumour genome sequencing has shed new light on tumour heterogeneity and provided important insights into the evolution of metastases arising from different clones. There is an additional layer of complexity, in that tumour evolution may be influenced by selective pressure provided by therapy, in a similar fashion to that occurring in infectious diseases. Here we studied tumour genomic evolution in a patient (index patient) with metastatic breast cancer bearing an activating PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha, PI(3)Kα) mutation. The patient was treated with the PI(3)Kα inhibitor BYL719, which achieved a lasting clinical response, but the patient eventually became resistant to this drug (emergence of lung metastases) and died shortly thereafter. A rapid autopsy was performed and material from a total of 14 metastatic sites was collected and sequenced. All metastatic lesions, when compared to the pre-treatment tumour, had a copy loss of PTEN (phosphatase and tensin homolog) and those lesions that became refractory to BYL719 had additional and different PTEN genetic alterations, resulting in the loss of PTEN expression. To put these results in context, we examined six other patients also treated with BYL719. Acquired bi-allelic loss of PTEN was found in one of these patients, whereas in two others PIK3CA mutations present in the primary tumour were no longer detected at the time of progression. To characterize our findings functionally, we examined the effects of PTEN knockdown in several preclinical models (both in cell lines intrinsically sensitive to BYL719 and in PTEN-null xenografts derived from our index patient), which we found resulted in resistance to BYL719, whereas simultaneous PI(3)K p110β blockade reverted this resistance phenotype. We conclude that parallel genetic evolution of separate metastatic sites with different PTEN genomic alterations leads to a convergent PTEN-null phenotype resistant to PI(3)Kα inhibition.
TP53 is commonly altered in human cancer, and Tp53 reactivation suppresses tumours in vivo in mice (TP53 and Tp53 are also known as p53). This strategy has proven difficult to implement therapeutically, and here we examine an alternative strategy by manipulating the p53 family members, Tp63 and Tp73 (also known as p63 and p73, respectively). The acidic transactivation-domain-bearing (TA) isoforms of p63 and p73 structurally and functionally resemble p53, whereas theΔN isoforms (lacking the acidic transactivation domain) of p63 and p73 are frequently overexpressed in cancer and act primarily in a dominant-negative fashion against p53, TAp63 and TAp73 to inhibit their tumour-suppressive functions. The p53 family interacts extensively in cellular processes thatpromote tumour suppression, such as apoptosis and autophagy, thus a clear understanding of this interplay in cancer is needed to treat tumours with alterations in the p53 pathway. Here we show that deletion of the ΔN isoforms of p63 or p73 leads to metabolic reprogramming and regression of p53-deficient tumours through upregulation of IAPP, the gene that encodes amylin, a 37-amino-acid peptide co-secreted with insulin by the β cells of the pancreas. We found that IAPP is causally involved in this tumour regression and that amylin functions through the calcitonin receptor (CalcR) and receptor activity modifying protein 3 (RAMP3) to inhibit glycolysis and induce reactive oxygen species and apoptosis. Pramlintide, a synthetic analogue of amylin that is currently used to treat type 1 and type 2 diabetes, caused rapid tumour regression in p53-deficient thymic lymphomas, representing a novel strategy to target p53-deficient cancers.
Chronic stress can cause depression in some individuals, but leaves others untouched. Engagement of a molecular pathway controlling the production of tiny RNA snippets might help to explain the difference.
Two studies find that an intracellular quality-control mechanism called autophagy is regulated by nuclear receptor proteins that govern the expression of autophagy genes.
Hereβ-catenin, which has been implicated in neurological and psychiatric diseases, including depression, is shown to mediate resilience to chronic stress in mice through induction of Dicer and microRNAs in nucleus accumbens, a key brain reward region.
The spindle assembly checkpoint (SAC) maintains genomic stability by delaying chromosome segregation until the last chromosome has attached to the mitotic spindle. The SAC prevents the anaphase promoting complex/cyclosome (APC/C) ubiquitin ligase from recognizing cyclin B and securin by catalysing the incorporation of the APC/C co-activator, CDC20, into a complex called the mitotic checkpoint complex (MCC). The SAC works through unattached kinetochores generating a diffusible‘wait anaphase’ signal that inhibits the APC/C in the cytoplasm, but the nature of this signal remains a key unsolved problem. Moreover, the SAC and the APC/C are highly responsive to each other: the APC/C quickly targets cyclin B and securin once all the chromosomes attach in metaphase, but israpidly inhibited should kinetochore attachment be perturbed. How this is achieved is also unknown. Here, we show that the MCC can inhibit a second CDC20 that has already bound and activated the APC/C. We show how the MCC inhibits active APC/C and that this is essential for the SAC. Moreover, this mechanism can prevent anaphase in the absence of kinetochore signalling. Thus, we propose that the diffusible ‘wait anaphase’ signal could be the MCC itself, and explain how reactivating the SAC can rapidly inhibit active APC/C.
Autophagy is an evolutionarily conserved catabolic process that recycles nutrients upon starvation and maintains cellular energy homeostasis. Its acute regulation by nutrient-sensing signalling pathways is well described, but its longer-term transcriptional regulation is not. The nuclear receptors peroxisome proliferator-activated receptor-α (PPARα) and farnesoid X receptor (FXR) are activated in the fasted and fed liver, respectively. Here we show that both PPARα and FXR regulate hepatic autophagy in mice. Pharmacological activation of PPARα reverses the normal suppression of autophagy in the fed state, inducing autophagic lipiddegradation, or lipophagy. This response is lost in PPARα knockout (Ppara−/−, also known as Nr1c1−/−) mice, which are partially defective in the induction of autophagy by fasting. Pharmacological activation of the bile acid receptor FXR strongly suppresses the induction of autophagy in thefasting state, and this response is absent in FXR knockout (Fxr−/−, also known as Nr1h4−/−) mice, which show a partial defect in suppression of hepatic autophagy in the fed state. PPARα and FXR compete for binding to shared sites in autophagic gene promoters, with opposite transcriptional outputs. These results reveal complementary, interlocking mechanisms for regulation of autophagy by nutrient status.
Lysosomal degradation of cytoplasmic components by autophagy is essential for cellular survival and homeostasis under nutrient-deprived conditions. Acute regulation of autophagy by nutrient-sensing kinases is well defined, but longer-term transcriptional regulation is relatively unknown. Here we show that the fed-state sensing nuclear receptor farnesoid X receptor (FXR) and the fasting transcriptional activator cAMP response element-binding protein (CREB) coordinately regulate the hepatic autophagy gene network. Pharmacological activation of FXR repressed many autophagy genes and inhibited autophagy even in fasted mice, and feeding-mediated inhibition of macroautophagy was attenuated in FXR-knockout mice. From mouse liver chromatin immunoprecipitation and high-throughput sequencing data, FXR and CREB binding peaks were detected at 178 and 112 genes, respectively, out of 230 autophagy-related genes, and 78 genes showed shared binding, mostly in their promoter regions. CREB promoted autophagic degradation of lipids, or lipophagy, under nutrient-deprived conditions, and FXR inhibited this response. Mechanistically, CREB upregulated autophagy genes, including Atg7, Ulk1 and Tfeb, by recruiting the coactivator CRTC2. After feeding or pharmacological activation, FXR trans-repressed these genes by disrupting the functional CREB–CRTC2 complex. This study identifies the new FXR–CREB axis as a key physiological switch regulating autophagy, resulting in sustained nutrient regulation of autophagy during feeding/fasting cycles.
Lung diseases such as chronic obstructive pulmonary disease and pulmonary fibrosis involve the progressive and inexorable destruction of oxygen exchange surfaces and airways, and have emerged as a leading cause of death worldwide. Mitigating therapies, aside from impractical organ transplantation, remain limited and the possibility of regenerative medicine has lacked empirical support. However, it is clinically known that patients who survive sudden, massive loss of lung tissue from necrotizing pneumonia or acute respiratory distress syndrome often recover full pulmonary function within six months. Correspondingly, we recently demonstrated lung regeneration in mice following H1N1 influenza virus infection, and linked distal airway stem cells expressing Trp63 (p63) and keratin 5, called DASCp63/Krt5, to this process. Here we show that pre-existing, intrinsically committed DASCp63/Krt5 undergo a proliferative expansion in response to influenza-induced lung damage, and assemble into nascent alveoli at sites of interstitial lung inflammation. We also show that the selective ablation of DASCp63/Krt5in vivo prevents this regeneration, leading to pre-fibrotic lesions and deficient oxygen exchange. Finally, we demonstrate that single DASCp63/Krt5-derived pedigrees differentiate to type I and type II pneumocytes as well as bronchiolar secretory cells following transplantation to infected lung and also minimize the structural consequences of endogenous stem cell loss on this process. The ability to propagate these cells in culture while maintaining their intrinsic lineage commitment suggests their potential in stem cell-based therapies for acute and chronic lung diseases.
The human genome is arguably the most complete mammalian reference assembly, yet more than 160 euchromatic gaps remain and aspects of its structural variation remain poorly understood ten years after its completion. To identify missing sequence and genetic variation, here we sequence and analyse a haploid human genome (CHM1) using single-molecule, real-time DNA sequencing. We close or extend 55% of the remaining interstitial gaps in the human GRCh37 reference genome—78% of which carried long runs of degenerate short tandem repeats, often several kilobases in length, embedded within (G+C)-rich genomic regions. We resolve the complete sequence of 26,079 euchromatic structural variants at the base-pair level, including inversions, complex insertions and long tracts of tandem repeats. Most have not been previously reported, with the greatest increases in sensitivity occurring for events less than 5 kilobases in size. Compared to the human reference, we find a significant insertional bias (3:1) in regions corresponding to complex insertions and long shorttandem repeats. Our results suggest a greater complexity of the human genome in the form of variation of longer and more complex repetitive DNA that can now be largely resolved with the application of this longer-read sequencing technology.
DNA methylation is an important epigenetic modification that is essential for various developmental processes through regulating gene expression, genomic imprinting, and epigenetic inheritance. Mammalian genomic DNA methylation is established during embryogenesis by de novo DNA methyltransferases, DNMT3A and DNMT3B, and the methylation patterns vary with developmental stages and cell types. DNA methyltransferase 3-like protein (DNMT3L) is a catalytically inactive paralogue of DNMT3 enzymes, which stimulates the enzymatic activity of Dnmt3a. Recent studies have established a connection between DNA methylation and histone modifications, and revealed a histone-guided mechanism for the establishment of DNA methylation. The ATRX–DNMT3–DNMT3L (ADD) domain of Dnmt3a recognizes unmethylated histone H3 (H3K4me0). The histone H3 tail stimulates the enzymatic activity of Dnmt3a in vitro, whereas the molecular mechanism remains elusive. Here we show that DNMT3A exists in an autoinhibitory form and that the histone H3 tail stimulates its activity in a DNMT3L-independent manner. We determine the crystal structures of DNMT3A–DNMT3L (autoinhibitory form) and DNMT3A–DNMT3L-H3 (active form) complexes at 3.82 and 2.90 Å resolution, respectively. Structural and biochemical analyses indicate that the ADD domain of DNMT3A interacts with and inhibits enzymatic activity of the catalytic domain (CD) through blocking its DNA-binding affinity. Histone H3 (but not H3K4me3) disrupts ADD–CD interaction, induces a large movement of the ADD domain, and thus releases the autoinhibition of DNMT3A. The finding adds another layer of regulation of DNA methylation to ensure that the enzyme is mainly activated at proper targeting loci when unmethylated H3K4 is present, and strongly supports a negative correlation between H3K4me3 and DNA methylation across the mammalian genome. Our study provides a new insight into an unexpected autoinhibition and histone H3-induced activation of the de novo DNA methyltransferase after its initial genomic positioning.
Biotin-dependent carboxylases are widely distributed in nature and have important functions in the metabolism of fatty acids, amino acids, carbohydrates, cholesterol and other compounds. Defective mutations in several of these enzymes have been linked to serious metabolic diseases in humans, and acetyl-CoA carboxylase is a target for drug discovery in the treatment of diabetes, cancer and other diseases. Here we report the identification and biochemical, structural and functional characterizations of a novel single-chain (120 kDa), multi-domain biotin-dependent carboxylase in bacteria. It has preference for long-chain acyl-CoA substrates, although it is also active towards short-chain and medium-chain acyl-CoAs, and we have named it long-chain acyl-CoA carboxylase. The holoenzyme is a homo-hexamer with molecular mass of 720 kDa. The 3.0 Å crystal structure of the long-chain acyl-CoA carboxylase holoenzyme from Mycobacterium avium subspecies paratuberculosis revealed an architecture that is strikingly different from those of related biotin-dependent carboxylases. In addition, the domains of each monomer have no direct contact with each other. They are instead extensively swapped in the holoenzyme, such that one cycle of catalysis involves the participation of four monomers. Functional studies in Pseudomonas aeruginosa suggest that the enzyme is involved in the utilization of selected carbon and nitrogen sources.
The mammalian taste system is responsible for sensing and responding to the five basic taste qualities: sweet, sour, bitter, salty and umami. Previously, we showed that each taste is detected by dedicated taste receptor cells (TRCs) on the tongue and palate epithelium. To understand how TRCs transmit information to higher neural centres, we examined the tuning properties of large ensembles of neurons in the first neural station of the gustatory system. Here, we generated and characterized a collection of transgenic mice expressing a genetically encoded calcium indicator in central and peripheral neurons, and used a gradient refractive index microendoscope combined with high-resolution two-photon microscopy to image taste responses from ganglion neurons buried deep at the base of the brain. Our results reveal fine selectivity in the taste preference of ganglion neurons; demonstrate a strong match between TRCs in the tongue and the principal neural afferents relaying taste information to the brain; and expose the highly specific transfer of taste information between taste cells and the central nervous system.
Inactivation of APC is a strongly predisposing event in the development of colorectal cancer, prompting the search for vulnerabilities specific to cells that have lost APC function. Signalling through the mTOR pathway is known to be required for epithelial cell proliferation and tumour growth, and the current paradigm suggests that a critical function of mTOR activity is to upregulate translational initiation through phosphorylation of 4EBP1 (refs 6, 7). This model predicts that the mTOR inhibitor rapamycin, which does not efficiently inhibit 4EBP1 (ref. 8), would be ineffective in limiting cancer progression in APC-deficient lesions. Here we show in mice that mTOR complex 1 (mTORC1) activity is absolutely required for the proliferation of Apc-deficient (but not wild-type) enterocytes, revealing an unexpected opportunity for therapeutic intervention. Although APC-deficient cells show the expected increases in protein synthesis, our study reveals that it is translation elongation, and not initiation, which is the rate-limiting component. Mechanistically, mTORC1-mediated inhibition of eEF2 kinase is required for the proliferation of APC-deficient cells. Importantly, treatment of established APC-deficient adenomas with rapamycin (which can target eEF2 through the mTORC1–S6K–eEF2K axis) causes tumour cells to undergo growth arrest and differentiation. Taken together, our data suggest that inhibition of translation elongation using existing, clinically approved drugs, such as the rapalogs, would provide clear therapeutic benefit for patients at high risk of developing colorectal cancer.
The incompleteness of the fossil record obscures the origin of many of the more derived clades of vertebrates. One such group is the Ichthyopterygia, a clade of obligatory marine reptiles that appeared in the Early Triassic epoch, without any known intermediates. Here we describe a basal ichthyosauriform from the upper Lower Triassic (about 248 million years ago) of China, whose primitive skeleton indicates possible amphibious habits. It is smaller than ichthyopterygians and had unusually large flippers that probably allowed limited terrestrial locomotion. It also retained characteristics of terrestrial diapsid reptiles, including a short snout and body trunk. Unlike more-derived ichthyosauriforms, it was probably a suction feeder. The new species supports the sister-group relationships between ichthyosauriforms and Hupehsuchia, the two forming the Ichthyosauromorpha. Basal ichthyosauromorphs are known exclusively from south China, suggesting that the clade originated in the region, which formed a warm and humid tropical archipelago in the Early Triassic. The oldest unequivocal record of a sauropterygian is also from the same stratigraphic unit of the region.
ATP-binding cassette (ABC) transporters translocate substrates across cell membranes, using energy harnessed from ATP binding and hydrolysis at their nucleotide-binding domains. ABC exporters are present both in prokaryotes and eukaryotes, with examples implicated in multidrug resistance of pathogens and cancer cells, as well as in many human diseases. TmrAB is a heterodimeric ABC exporter from the thermophilic Gram-negative eubacterium Thermus thermophilus; it is homologous to various multidrug transporters and contains one degenerate site with a non-catalytic residue next to the Walker B motif. Here we report a subnanometre-resolution structure of detergent-solubilized TmrAB in a nucleotide-free, inward-facing conformation by single-particle electron cryomicroscopy. The reconstructions clearly resolve characteristic features of ABC transporters, including helices in the transmembrane domain and nucleotide-binding domains. A cavity in the transmembrane domain is accessible laterally from the cytoplasmic side of the membrane as well as from the cytoplasm, indicating that the transporter lies in an inward-facing open conformation. The two nucleotide-binding domains remain in contact via their carboxy-terminal helices. Furthermore, comparison between our structure and the crystal structures of other ABC transporters suggests a possible trajectory of conformational changes that involves a sliding and rotating motion between the two nucleotide-binding domains during the transition from the inward-facing to outward-facing conformations.
Human immunodeficiency virus type 1 (HIV-1) assembly proceeds in two stages. First, the 55 kilodalton viral Gag polyprotein assembles into a hexameric protein lattice at the plasma membrane of the infected cell, inducing budding and release of an immature particle. Second, Gag is cleaved by the viral protease, leading to internal rearrangement of the virus into the mature, infectious form. Immature and mature HIV-1 particles are heterogeneous in size and morphology, preventing high-resolution analysis of their protein arrangement in situ by conventional structural biology methods. Here we apply cryo-electron tomography and sub-tomogram averaging methods to resolve the structure of the capsid lattice within intact immature HIV-1 particles at subnanometre resolution, allowing unambiguous positioning of all α-helices. The resulting model reveals tertiary and quaternary structural interactions that mediate HIV-1 assembly. Strikingly, these interactions differ from those predicted by the current model based on in vitro-assembled arrays of Gag-derived proteins from Mason–Pfizer monkey virus. To validate this difference, we solve the structure of the capsid lattice within intact immature Mason–Pfizer monkey virus particles. Comparison with the immature HIV-1 structure reveals that retroviral capsid proteins, while having conserved tertiary structures, adopt different quaternary arrangements during virus assembly. The approach demonstrated here should be applicable to determine structures of other proteins at subnanometre resolution within heterogeneous environments.
Immunoglobulins protect against disease to a considerable extent by activating complement and stimulatory immunoglobulin crystallizable fragment receptors (Ig FcRs), and aggregating microbial pathogens. Yet IgG1, the predominant murine serum Ig isotype, cannot activate complement by the classical pathway, binds more avidly to an inhibitory than to stimulatory FcRs, and has limited ability to aggregate pathogens. In these regards, it resembles human IgG4 (ref. 4). We hypothesized that limited ability to activate effector mechanisms might protect against immune complex immunopathology. Here we show that IgG1-deficient (γ1−) mice, immunized with a potent antigen, develop lethal renal disease soon after they begin to produce antigen-specific antibody, whereas similarly immunized wild-type mice remain healthy. Surprisingly, renal disease in this model is complement and FcR independent and results from immune complex precipitation in glomerular capillaries, as in some cryoglobulinaemic humans. IgG3, which self-associates to form large immune complexes, accounts for more than 97% of the mouse Ig in this cryoglobulin; furthermore, glomerular disease develops when mice are injected with IgG3 anti-trinitrophenyl(TNP) monoclonal antibody followed by a TNP-labelled protein. Renal disease is prevented in both active and passive immunization models by antigen-specific IgG1; other isotypes are less potent at preventing disease. These observations demonstrate the adaptive significance of Ig isotypes that poorlyactivate effector mechanisms, reveal an immune-complex-dependent, complement- and FcR-independent nephrotoxic mechanism, and suggest that isotypes that poorly activate effector mechanisms may be useful for inhibiting immune complex immunopathology.
Next-generation sequencing of human tumours has refined our understanding of the mutational processes operative in cancer initiation and progression, yet major questions remain regarding the factors that induce driver mutations and the processes that shape mutation selection during tumorigenesis. Here we performed whole-exome sequencing on adenomas from three mouse models of non-small-cell lung cancer, which were induced either by exposure to carcinogens (methyl-nitrosourea (MNU) and urethane) or by genetic activation of Kras (KrasLA2). Although the MNU-induced tumours carried exactly the same initiating mutation in Kras as seen in the KrasLA2 model (G12D), MNU tumours had an average of 192 non-synonymous, somatic single-nucleotide variants, compared with only six in tumours from the KrasLA2 model. By contrast, the KrasLA2 tumours exhibited a significantly higher level of aneuploidy and copy number alterations compared with the carcinogen-induced tumours, suggesting that carcinogen-induced and genetically engineered models lead to tumour development through different routes. The wild-type allele of Kras has been shown to act as a tumour suppressor in mouse models of non-small-cell lung cancer. We demonstrate that urethane-induced tumours from wild-type mice carry mostly (94%) Kras Q61R mutations, whereas those from Kras heterozygous animals carry mostly (92%) Kras Q61L mutations, indicating a major role for germline Kras status in mutation selection during initiation. The exome-wide mutation spectra in carcinogen-induced tumours overwhelmingly display signatures of the initiating carcinogen, while adenocarcinomas acquire additional C ggt; T mutations at CpG sites. These data provide a basis for understanding results from human tumour genome sequencing, which has identified two broad categories of tumours based on the relative frequency of single-nucleotide variations and copy number alterations, and underline the importance of carcinogen models for understanding the complex mutation spectra seen in human cancers.
Genome-wide association studies combined with data from epigenomic maps for immune cells have been used to fine-map causal variants for 21 autoimmune diseases; disease risk tends to be linked to single nucleotide polymorphisms in cell-type-specific enhancers, often in regions adjacent to transcription factor binding motifs.
Site-specific gene addition can allow stable transgene expression for gene therapy. When possible, this is preferred over the use of promiscuously integrating vectors, which are sometimes associated with clonal expansion and oncogenesis. Site-specific endonucleases that can induce high rates of targeted genome editing are finding increasing applications in biological discovery and gene therapy. However, two safety concerns persist: endonuclease-associated adverse effects, both on-target and off-target; and oncogene activation caused by promoter integration, even without nucleases. Here we perform recombinant adeno-associated virus (rAAV)-mediated promoterless gene targeting without nucleases and demonstrate amelioration of the bleeding diathesis in haemophilia B mice. In particular, we target a promoterless human coagulation factor IX (F9) gene to the liver-expressed mouse albumin (Alb) locus. F9 is targeted, along with a preceding 2A-peptide coding sequence, to be integrated just upstream to the Alb stop codon. While F9 is fused to Alb at the DNA and RNA levels, two separate proteins are synthesized by way of ribosomal skipping. Thus, F9 expression is linked to robust hepatic albumin expression without disrupting it. We injected an AAV8-F9 vector into neonatal and adult mice and achieved on-target integration into∼0.5% of the albumin alleles in hepatocytes. We established that F9 was produced only from on-target integration, and ribosomal skipping was highly efficient. Stable F9 plasma levels at 7–20% of normal were obtained, and treated F9-deficient mice had normal coagulation times. In conclusion, transgene integration as a 2A-fusion to a highly expressed endogenous gene may obviate the requirement for nucleases and/or vector-borne promoters. This method may allow for safe and efficacious gene targeting in both infants and adults by greatly diminishing off-target effects while still providing therapeutic levels of expression from integration.
T-cell immunoglobulin domain and mucin domain-3 (TIM-3, also known as HAVCR2) is an activation-induced inhibitory molecule involved in tolerance and shown to induce T-cell exhaustion in chronic viral infection and cancers. Under some conditions, TIM-3 expression has also been shown to be stimulatory. Considering that TIM-3, like cytotoxic T lymphocyte antigen 4 (CTLA-4) and programmed death 1 (PD-1), is being targeted for cancer immunotherapy, it is important to identify the circumstances under which TIM-3 can inhibit and activate T-cell responses. Here we show that TIM-3 is co-expressed and forms a heterodimer with carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1), another well-known molecule expressed on activated T cells and involved in T-cell inhibition. Biochemical, biophysical and X-ray crystallography studies show that the membrane-distal immunoglobulin-variable (IgV)-like amino-terminal domain of each is crucial to these interactions. The presence of CEACAM1 endows TIM-3 with inhibitory function. CEACAM1 facilitates the maturation and cell surface expression of TIM-3 by forming a heterodimeric interaction in cis through the highly related membrane-distal N-terminal domains of each molecule. CEACAM1 and TIM-3 also bind in trans through their N-terminal domains. Both cis and trans interactions between CEACAM1 and TIM-3 determine the tolerance-inducing function of TIM-3. In a mouse adoptive transfer colitis model, CEACAM1-deficient T cells are hyper-inflammatory with reduced cell surface expression of TIM-3 and regulatory cytokines, and this is restored by T-cell-specific CEACAM1 expression. During chronic viral infection and in a tumour environment, CEACAM1 and TIM-3 mark exhausted T cells. Co-blockade of CEACAM1 and TIM-3 leads to enhancement of anti-tumour immune responses with improved elimination of tumours in mouse colorectal cancer models. Thus, CEACAM1 serves as a heterophilic ligand for TIM-3 that is required for its ability to mediate T-cell inhibition, and this interaction has a crucial role in regulating autoimmunity and anti-tumour immunity.
Lantibiotics are a class of peptide antibiotics that contain one or more thioether bonds. The lantibiotic nisin is an antimicrobial peptide that is widely used as a food preservative to combat food-borne pathogens. Nisin contains dehydroalanine and dehydrobutyrine residues that are formed by the dehydration of Ser/Thr by the lantibiotic dehydratase NisB (ref. 2). Recent biochemical studies revealed that NisB glutamylates Ser/Thr side chains as part of the dehydration process. However, the molecular mechanism by which NisB uses glutamate to catalyse dehydration remains unresolved. Here we show that this process involves glutamyl-tRNAGlu to activate Ser/Thr residues. In addition, the 2.9-Å crystal structure of NisB in complex with its substrate peptide NisA reveals the presence of two separate domains that catalyse the Ser/Thr glutamylation and glutamate elimination steps. The co-crystal structure also provides insights into substrate recognition by lantibiotic dehydratases. Our findings demonstrate an unexpected role for aminoacyl-tRNA in the formation of dehydroamino acids in lantibiotics, and serve as a basis for the functional characterization of the many lantibiotic-like dehydratases involved in the biosynthesis of other classes of natural products.
For many decades comparative analyses of protein sequences and structures have been used to investigate fundamental principles of molecular evolution. In contrast, relatively little is known about the long-term evolution of species’ phenotypic and genetic properties. This represents an important gap in our understanding of evolution, as exactly these proprieties play key roles in natural selection and adaptation to diverse environments. Here we perform a comparative analysis of bacterial growth and gene deletion phenotypesusing hundreds of genome-scale metabolic models. Overall, bacterial phenotypic evolution can be described by a two-stage process with a rapid initial phenotypic diversification followed by a slow long-term exponential divergence. The observed average divergence trend, with approximately similar fractions of phenotypic properties changing per unit time, continues for billions of years. We experimentally confirm the predicted divergence trend using the phenotypic profiles of 40 diverse bacterial species across more than 60 growth conditions. Our analysis suggests that, at long evolutionary distances, gene essentiality is significantly more conserved than the ability to utilize different nutrients, while synthetic lethality is significantly less conserved. We also find that although a rapid phenotypic evolution is sometimes observed within the same species, a transition from high to low phenotypic similarity occurs primarily at the genus level.
Establishing the hippocampal cellular ensemble that represents an animal’s environment involves the emergence and disappearance of place fields in specific CA1 pyramidal neurons, and the acquisition of different spatial firing properties across the active population. While such firing flexibility and diversity have been linked to spatial memory, attention and task performance, the cellular and network origin of these place cell features is unknown. Basic integrate-and-fire models of place firing propose that such features result solely from varying inputs to place cells, but recent studies suggest instead that place cells themselves may play an active role through regenerative dendritic events. However, owing to the difficulty of performing functional recordings from place cell dendrites, no direct evidence of regenerative dendritic events exists, leaving any possible connection to place coding unknown. Using multi-plane two-photon calcium imaging of CA1 place cell somata, axons and dendrites in mice navigating a virtual environment, here we show that regenerative dendritic events do exist in place cells of behaving mice, and, surprisingly, their prevalence throughout the arbour is highly spatiotemporally variable. Furthermore, we show that the prevalence of such events predicts the spatial precision and persistence or disappearance of place fields. This suggests that the dynamics of spiking throughout the dendritic arbour may play a key role in forming the hippocampal representation of space.
Plant stem cells in the shoot apical meristem (SAM) and root apical meristem are necessary for postembryonic development of aboveground tissues and roots, respectively, while secondary vascular stem cells sustain vascular development. WUSCHEL (WUS), a homeodomain transcription factor expressed in the rib meristem of the Arabidopsis SAM, is a key regulatory factor controlling SAM stem cell populations, and is thought to establish the shoot stem cell niche through a feedback circuit involving the CLAVATA3 (CLV3) peptide signalling pathway. WUSCHEL-RELATED HOMEOBOX 5 (WOX5), which is specifically expressed in the root quiescent centre, defines quiescent centre identity and functions interchangeably with WUS in the control of shoot and root stem cell niches. WOX4, expressed in Arabidopsis procambial cells, defines the vascular stem cell niche. WUS/WOX family proteins are evolutionarily and functionally conserved throughout the plant kingdom and emerge as key actors in the specification and maintenance of stem cells within all meristems. However, the nature of the genetic regime in stem cell niches that centre on WOX gene function has been elusive, and molecular links underlying conserved WUS/WOX function in stem cell niches remain unknown. Here we demonstrate that the Arabidopsis HAIRY MERISTEM (HAM) family of transcription regulators act as conserved interacting cofactors with WUS/WOX proteins. HAM and WUS share common targets in vivo and their physical interaction is important in driving downstream transcriptional programs and in promoting shoot stem cell proliferation. Differences in the overlapping expression patterns of WOX and HAM family members underlie the formation of diverse stem cell niche locations, and the HAM family is essential for all of these stem cell niches. These findings establish a new framework for the control of stem cell production during plant development.
The gastrointestinal tracts of mammals are colonized by hundreds of microbial species that contribute to health, including colonization resistance against intestinal pathogens. Many antibiotics destroy intestinal microbial communities and increase susceptibility to intestinal pathogens. Among these, Clostridium difficile, a major cause of antibiotic-induced diarrhoea, greatly increases morbidity and mortality in hospitalized patients. Which intestinal bacteria provide resistance to C. difficile infection and their in vivo inhibitory mechanisms remain unclear. Here we correlate loss of specific bacterial taxa with development of infection, by treating mice with different antibiotics that result in distinct microbiota changes and lead to varied susceptibility to C. difficile. Mathematical modelling augmented by analyses of the microbiota of hospitalized patients identifies resistance-associated bacteria common to mice and humans. Using these platforms, we determine that Clostridium scindens, a bile acid 7α-dehydroxylating intestinal bacterium, is associated with resistance to C. difficile infection and, upon administration, enhances resistance to infection in a secondary bile acid dependent fashion. Using a workflow involving mouse models, clinical studies, metagenomic analyses, and mathematical modelling, we identify a probiotic candidate that corrects a clinically relevant microbiome deficiency. These findings have implications for the rational design of targeted antimicrobials as well as microbiome-based diagnostics and therapeutics for individuals at risk of C. difficile infection.
One of the primary challenges of our time is to feed a growing and more demanding world population with reduced external inputs and minimal environmental impacts, all under more variable and extreme climate conditions in the future. Conservation agriculture represents a set of three crop management principles that has received strong international support to help address this challenge, with recent conservation agriculture efforts focusing on smallholder farming systems in sub-Saharan Africa and South Asia. However, conservation agriculture is highly debated, with respect to both its effects on crop yields and its applicability in different farming contexts. Here we conduct a global meta-analysis using 5,463 paired yield observations from 610 studies to compare no-till, the original and central concept of conservation agriculture, with conventional tillage practices across 48 crops and 63 countries. Overall, our results show that no-till reduces yields, yet this response is variable and under certain conditions no-till can produce equivalent or greater yields than conventional tillage. Importantly, when no-till is combined with the other two conservation agriculture principles of residue retention and crop rotation, its negative impacts are minimized. Moreover, no-till in combination with the other two principles significantly increases rainfed crop productivity in dry climates, suggesting that it may become an important climate-change adaptation strategy for ever-drier regions of the world. However, any expansion of conservation agriculture should be done with caution in these areas, as implementation of the other two principles is often challenging in resource-poor and vulnerable smallholder farming systems, thereby increasing the likelihood of yield losses rather than gains. Although farming systems are multifunctional, and environmental and socio-economic factors need to be considered, our analysis indicates that the potential contribution of no-till to the sustainable intensification of agriculture is more limited than often assumed.
Protein poly(ADP-ribosyl)ation (PARylation) has a role in diverse cellular processes such as DNA repair, transcription, Wnt signalling, and cell death. Recent studies have shown that PARylation can serve as a signal for the polyubiquitination and degradation of several crucial regulatory proteins, including Axin and 3BP2 (refs 7, 8, 9). The RING-type E3 ubiquitin ligase RNF146 (also known as Iduna) is responsible for PARylation-dependent ubiquitination (PARdU). Here we provide a structural basis for RNF146-catalysed PARdU and how PARdU specificity is achieved. First, we show that iso-ADP-ribose (iso-ADPr), the smallest internal poly(ADP-ribose) (PAR) structural unit, binds between the WWE and RING domains of RNF146 and functions as an allosteric signal that switches the RING domain from a catalytically inactive state to an active one. In the absence of PAR, the RING domain is unable to bind and activate a ubiquitin-conjugating enzyme (E2) efficiently. Binding of PAR or iso-ADPr induces a major conformational change that creates a functional RING structure. Thus, RNF146 represents a new mechanistic class of RING E3 ligases, the activities of which are regulated by non-covalent ligand binding, and that may provide a template for designing inducible protein-degradation systems. Second, we find that RNF146 directly interacts with the PAR polymerase tankyrase (TNKS). Disruption of the RNF146–TNKS interaction inhibits turnover of the substrate Axin in cells. Thus, both substrate PARylation and PARdU are catalysed by enzymes within the same protein complex, and PARdU substrate specificity may be primarily determined by the substrate–TNKS interaction. We propose that the maintenance of unliganded RNF146 in an inactive state may serve to maintain the stability of the RNF146–TNKS complex, which in turn regulates the homeostasis of PARdU activity in the cell.
Reproduction in jawed vertebrates (gnathostomes) involves either external or internal fertilization. It is commonly argued that internal fertilization can evolve from external, but not the reverse. Male copulatory claspers are present in certain placoderms, fossil jawed vertebrates retrieved as a paraphyletic segment of the gnathostome stem group in recent studies. This suggests that internal fertilization could be primitive for gnathostomes, but such a conclusion depends on demonstrating that copulation was not just a specialized feature of certain placoderm subgroups. The reproductive biology of antiarchs, consistently identified as the least crownward placoderms and thus of great interest in this context, has until now remained unknown. Here we show that certain antiarchs possessed dermal claspers in the males, while females bore paired dermal plates inferred to have facilitated copulation. These structures are not associated with pelvic fins. The clasper morphology resembles that of ptyctodonts, a more crownward placoderm group, suggesting that all placoderm claspers are homologous and that internal fertilization characterized all placoderms. This implies that external fertilization and spawning, which characterize most extant aquatic gnathostomes, must be derived from internal fertilization, even though this transformation has been thought implausible. Alternatively, the substantial morphological evidence for placoderm paraphyly must be rejected.
Organohalide chemistry underpins many industrial and agricultural processes, and a large proportion of environmental pollutants are organohalides. Nevertheless, organohalide chemistry is not exclusively of anthropogenic origin, with natural abiotic and biological processes contributing to the global halide cycle. Reductive dehalogenases are responsible for biological dehalogenation in organohalide respiring bacteria, with substrates including polychlorinated biphenyls or dioxins. Reductive dehalogenases form a distinct subfamily of cobalamin (B12)-dependent enzymes that are usually membrane associated and oxygen sensitive, hindering detailed studies. Here we report the characterization of a soluble, oxygen-tolerant reductive dehalogenase and, by combining structure determination with EPR (electron paramagnetic resonance) spectroscopy and simulation, show that a direct interaction between the cobalamin cobalt and the substrate halogen underpins catalysis. In contrast to the carbon–cobalt bond chemistry catalysed by the other cobalamin-dependent subfamilies, we propose that reductive dehalogenases achieve reduction of the organohalide substrate via halogen–cobalt bond formation. This presents a new model in both organohalide and cobalamin (bio)chemistry that will guide future exploitation of these enzymes in bioremediation or biocatalysis.
Hepatitis A virus (HAV) remains enigmatic, despite 1.4 million cases worldwide annually. It differs radically from other picornaviruses, existing in an enveloped form and being unusually stable, both genetically and physically, but has proved difficult to study. Here we report high-resolution X-ray structures for the mature virus and the empty particle. The structures of the two particles are indistinguishable, apart from some disorder on the inside of the empty particle. The full virus contains the small viral protein VP4, whereas the empty particle harbours only the uncleaved precursor, VP0. The smooth particle surface is devoid of depressions that might correspond to receptor-binding sites. Peptide scanning data extend the previously reported VP3 antigenic site, while structure-based predictions suggest further epitopes. HAV contains no pocket factor and can withstand remarkably high temperature and low pH, and empty particles are even more robust than full particles. The virus probably uncoats via a novel mechanism, being assembled differently to other picornaviruses. It utilizes a VP2‘domain swap’ characteristic of insect picorna-like viruses, and structure-based phylogenetic analysis places HAV between typical picornaviruses and the insect viruses. The enigmatic properties of HAV may reflect its position as a link between ‘modern’ picornaviruses and the more ‘primitive’ precursor insect viruses; for instance, HAV retains the ability to move from cell-to-cell by transcytosis.
The mammary gland is composed of multiple types of epithelial cells, which are generated by mammary stem cells (MaSCs) residing at the top of the hierarchy. However, the existence of these multipotent MaSCs remains controversial and the nature of such cells is unknown. Here we demonstrate that protein C receptor (Procr), a novel Wnt target in the mammary gland, marks a unique population of multipotent mouse MaSCs. Procr-positive cells localize to the basal layer, exhibit epithelial-to-mesenchymal transition characteristics, and express low levels of basal keratins. Procr-expressing cells have a high regenerative capacity in transplantation assays and differentiate into all lineages of the mammary epithelium by lineage tracing. These results define a novel multipotent mammary stem cell population that could be important in the initiation of breast cancer.
The germinal centre (GC) reaction supports affinity-based B-cell competition and generates high-affinity bone-marrow plasma cells (BMPCs). How follicular T-helper (TFH) cells regulate GC selection is not clear. Using competitive mixed chimaera, we show here that, beyond the role in promoting TFH development, ICOSL (inducible T-cell co-stimulator ligand, also known as ICOSLG) is important for individual B cells to competitively participate in the GC reaction and to develop into BMPCs. Using intravital imaging aided by a calcium reporter, we further show that ICOSL promotes an‘entangled’ mode of TFH–B-cell interactions, characterized by brief but extensive surface engagement, productive T-cell calcium spikes, and B-cell acquisition of CD40 signals. Reiterated entanglement promotes outer-zone co-localization of outcompeting GC B cells together with TFH cells, affording the former increased access to T-cell help. ICOSL on GC B cells is upregulated by CD40 signals. Such an intercellular positive feedback between contact-dependent help and ICOSL-controlled entanglement promotes positive selection and BMPC development, as evidenced by observations that higher-affinity B-cell receptor variants are enriched in the ICOSLhigh fraction, that numerically disadvantaged ICOSL-deficient GC B cells or BMPCs exhibit strong affinity compensation in competitive chimaera, and that when GC competition proceeds without ICOSL, selection of high-affinity variants in otherwise normal GC reactions is impaired. By demonstrating entanglement as the basic form of GC TFH–B-cell interactions, identifying ICOSL as a molecular linkage between T–B interactional dynamics and positive selection for high-affinity BMPC formation, our study reveals a pathway by which TFH cells control the quality of long-lived humoral immunity.
The mechanisms that underlie the origin of major prokaryotic groups are poorly understood. In principle, the origin of both species and higher taxa among prokaryotes should entail similar mechanisms—ecological interactions with the environment paired with natural genetic variation involving lineage-specific gene innovations and lineage-specific gene acquisitions. To investigate the origin of higher taxa in archaea, we have determined gene distributions and gene phylogenies for the 267,568 protein-coding genes of 134 sequenced archaeal genomes in the context of their homologues from 1,847 reference bacterial genomes. Archaeal-specific gene families define 13 traditionally recognized archaeal higher taxa in our sample. Here we report that the origins of these 13 groups unexpectedly correspond to 2,264 group-specific gene acquisitions from bacteria. Interdomain gene transfer is highly asymmetric, transfers from bacteria to archaea are more than fivefold more frequent than vice versa. Gene transfers identified at major evolutionary transitions among prokaryotes specifically implicate gene acquisitions for metabolic functions from bacteria as key innovations in the origin of higher archaeal taxa.
The semi-conservative centrosome duplication in cycling cells gives rise to a centrosome composed of a mother and a newly formed daughter centriole. Both centrioles are regarded as equivalent in their ability to form new centrioles and their symmetric duplication is crucial for cell division homeostasis. Multiciliated cells do not use the archetypal duplication program and instead form more than a hundred centrioles that are required for the growth of motile cilia and the efficient propelling of physiological fluids. The majority of these new centrioles are thought to appear de novo, that is, independently from the centrosome, around electron-dense structures called deuterosomes. Their origin remains unknown. Using live imaging combined with correlative super-resolution light and electron microscopy, we show that all new centrioles derive from the pre-existing progenitor cell centrosome through multiple rounds of procentriole seeding. Moreover, we establish that only the daughter centrosomal centriole contributes to deuterosome formation, and thus to over ninety per cent of the final centriole population. This unexpected centriolar asymmetry grants new perspectives when studying cilia-related diseases and pathological centriole amplification observed in cycling cells and associated with microcephaly and cancer.
Sterols are essential biological molecules in the majority of life forms. Sterol reductases includingΔ14-sterol reductase (C14SR, also known as TM7SF2), 7-dehydrocholesterol reductase (DHCR7) and 24-dehydrocholesterol reductase (DHCR24) reduce specific carbon–carbon double bonds of the sterol moiety using a reducing cofactor during sterol biosynthesis. Lamin B receptor (LBR), an integral inner nuclear membrane protein, also contains a functional C14SR domain. Here we report the crystal structure of a Δ14-sterol reductase (MaSR1) from the methanotrophic bacterium Methylomicrobium alcaliphilum 20Z (a homologue of human C14SR, LBR and DHCR7) with the cofactor NADPH. The enzyme contains ten transmembrane segments (TM1–10). Its catalytic domain comprises the carboxy-terminal half (containing TM6–10) and envelops two interconnected pockets, one of which faces the cytoplasm and houses NADPH, while the other one is accessible from the lipid bilayer. Comparison with a soluble steroid 5β-reductase structure suggests that the reducing end of NADPH meets the sterol substrate at the juncture of the two pockets. A sterol reductase activity assay proves that MaSR1 can reduce the double bond of a cholesterol biosynthetic intermediate, demonstrating functional conservation to human C14SR. Therefore, our structure as a prototype of integral membrane sterol reductases provides molecular insight into mutations in DHCR7 and LBR for inborn human diseases.
Intracellular ISG15 is an interferon (IFN)-α/β-inducible ubiquitin-like modifier which can covalently bind other proteins in a process called ISGylation; it is an effector of IFN-α/β-dependent antiviral immunity in mice. We previously published a study describing humans with inherited ISG15 deficiency but without unusually severe viraldiseases. We showed that these patients were prone to mycobacterial disease and that human ISG15 was non-redundant as an extracellular IFN-γ-inducing molecule. We show here that ISG15-deficient patients also display unanticipated cellular, immunological and clinical signs of enhanced IFN-α/β immunity, reminiscent of the Mendelian autoinflammatory interferonopathies Aicardi–Goutières syndrome and spondyloenchondrodysplasia. We further show that an absence of intracellular ISG15 in the patients’ cells prevents the accumulation of USP18, a potent negative regulator of IFN-α/β signalling, resulting in the enhancement and amplification of IFN-α/β responses. Human ISG15, therefore, is not only redundant for antiviral immunity, but is a key negative regulator of IFN-α/β immunity. In humans, intracellular ISG15 is IFN-α/β-inducible not to serve as a substrate for ISGylation-dependent antiviral immunity, but to ensure USP18-dependent regulation of IFN-α/β and prevention of IFN-α/β-dependent autoinflammation.
The adoption of a new form of tool use has been observed to spread along social-network pathways in a chimpanzee community. The finding offers the first direct evidence of cultural diffusion in these animals in the wild.
The spliceosome enzyme complex removes intron sequences from RNA transcripts to form messenger RNA. The crystal structure of a lasso-shaped RNA suggests a mechanism for this splicing process.
This study determines the structure of a branched lariat RNA, providing insights into rearrangement of the intron between the two steps of RNA splicing.
The response of the terrestrial carbon cycle to climate change is among the largest uncertainties affecting future climate change projections. The feedback between the terrestrial carbon cycle and climate is partly determined by changes in the turnover time of carbon in land ecosystems, which in turn is an ecosystem property that emerges from the interplay between climate, soil and vegetation type. Here we present a global, spatially explicit and observation-based assessment of whole-ecosystem carbon turnover times that combines new estimates of vegetation and soil organic carbon stocks and fluxes. We find that the overall mean global carbon turnover time is years (95 per cent confidence interval). On average, carbon resides in the vegetation and soil near the Equator for a shorter time than at latitudes north of 75° north (mean turnover times of 15 and 255 years, respectively). We identify a clear dependence of the turnover time on temperature, asexpected from our present understanding of temperature controls on ecosystem dynamics. Surprisingly, our analysis also reveals a similarly strong association between turnover time and precipitation. Moreover, we find that the ecosystem carbon turnover times simulated by state-of-the-art coupled climate/carbon-cycle models vary widely and that numerical simulations, on average, tend to underestimate the global carbon turnover time by 36 per cent. The models show stronger spatial relationships with temperature than do observation-based estimates, but generally do not reproduce the strong relationships with precipitation and predict faster carbon turnover in many semi-arid regions. Our findings suggest that future climate/carbon-cycle feedbacks may depend more strongly on changes in the hydrological cycle than is expected at present and is considered in Earth system models.
Analyses in mice and humans indicate that non-caloric artificial sweeteners may promote obesity-associated metabolic changes by changing the function of the bacteria that colonize the gut.
Non-caloric artificial sweeteners (NAS), widely used food additives considered to be safe and beneficial alternatives to sugars, are shown here to lead to the development of glucose intolerance through compositional and functional changes in the gut microbiota of mice, and the deleterious metabolic effects are transferred to germ-free mice by faecal transplant; NAS-induced dysbiosis and glucose intolerance are also demonstrated in healthy human subjects.
Rapid industrialization and urbanization in developing countries has led to an increase in air pollution, along a similar trajectory to that previously experienced by the developed nations. In China, particulate pollution is a serious environmental problem that is influencing air quality, regional and global climates, and human health. In response to the extremely severe and persistent haze pollution experienced by about 800 million people during the first quarter of 2013 (refs 4, 5), the Chinese State Council announced its aim to reduce concentrations of PM2.5 (particulate matter with an aerodynamic diameter less than 2.5 micrometres) by up to 25 per cent relative to 2012 levels by 2017 (ref. 6). Such efforts however require elucidation of the factors governing the abundance and composition of PM2.5, which remain poorly constrained in China. Here we combine a comprehensive set of novel and state-of-the-art offlineanalytical approaches and statistical techniques to investigate the chemical nature and sources of particulate matter at urban locations in Beijing, Shanghai, Guangzhou and Xi’an during January 2013. We find that the severe haze pollution event was driven to a large extent by secondary aerosol formation, which contributed 30–77 per cent and 44–71 per cent (average for all four cities) of PM2.5 and of organic aerosol, respectively. On average, the contribution of secondary organic aerosol (SOA) and secondary inorganic aerosol (SIA) are found to be of similar importance (SOA/SIA ratios range from 0.6 to 1.4). Our results suggest that, in addition to mitigating primary particulate emissions, reducing the emissions of secondary aerosol precursors from, for example, fossil fuel combustion and biomass burning is likely to be important for controlling China’s PM2.5 levels and for reducing the environmental, economic and health impacts resulting from particulate pollution.
Magnetoresistance is the change in a material’s electrical resistance in response to an applied magnetic field. Materials with large magnetoresistance have found use as magnetic sensors, in magnetic memory, and in hard drives at room temperature, and their rarity has motivated many fundamental studies in materials physics at low temperatures. Here we report the observation of an extremely large positive magnetoresistance at low temperatures in the non-magnetic layered transition-metal dichalcogenide WTe2: 452,700 per cent at 4.5 kelvins in a magnetic field of 14.7 teslas, and 13 million per cent at 0.53 kelvins in a magnetic field of 60 teslas. In contrast with other materials, there is no saturation of the magnetoresistance value even at very high applied fields. Determination of the origin and consequences of this effect, and the fabrication of thin films, nanostructures and devices based on the extremely large positive magnetoresistance of WTe2, will represent a significant new direction in the study of magnetoresistivity.
β-Thalassaemia major (β-TM) is an inherited haemoglobinopathy caused by a quantitative defect in the synthesis of β-globin chains of haemoglobin, leading to the accumulation of free α-globin chains that form toxic aggregates. Despite extensive knowledge of the molecular defects causing β-TM, little is known of the mechanisms responsible for the ineffective erythropoiesis observed in the condition, which is characterized by accelerated erythroid differentiation, maturation arrest and apoptosis at the polychromatophilic stage. We have previously demonstrated that normal human erythroid maturation requires a transient activation of caspase-3 at the later stages of maturation. Although erythroid transcription factor GATA-1, the master transcriptional factor of erythropoiesis, is a caspase-3 target, it is not cleaved during erythroid differentiation. We have shown that, in human erythroblasts, the chaperone heat shock protein70 (HSP70) is constitutively expressed and, at later stages of maturation, translocates into the nucleus and protects GATA-1 from caspase-3 cleavage. The primary role of this ubiquitous chaperone is to participate in the refolding of proteins denatured by cytoplasmic stress, thus preventing their aggregation. Here we show in vitro that during the maturation of human β-TM erythroblasts, HSP70 interacts directly with free α-globin chains. As a consequence, HSP70 is sequestrated in the cytoplasm and GATA-1 is no longer protected, resulting in end-stage maturation arrest and apoptosis. Transduction of a nuclear-targeted HSP70 mutant or a caspase-3-uncleavable GATA-1 mutant restores terminal maturation of β-TM erythroblasts, which may provide a rationale for new targeted therapies of β-TM.
The polycomb repressive complex 2 (PRC2) exerts oncogenic effects in many tumour types. However, loss-of-function mutations in PRC2 components occur in a subset of haematopoietic malignancies, suggesting that this complex plays a dichotomous and poorly understood role in cancer. Here we provide genomic, cellular, and mouse modelling data demonstrating that the polycomb group gene SUZ12 functions as tumour suppressor in PNS tumours, high-grade gliomas and melanomas by cooperating with mutations in NF1. NF1 encodes a Ras GTPase-activating protein (RasGAP) and its loss drives cancer by activating Ras. We show that SUZ12 loss potentiates the effects of NF1 mutations by amplifying Ras-driven transcription through effects on chromatin. Importantly, however, SUZ12 inactivation also triggers an epigenetic switch that sensitizes these cancers to bromodomain inhibitors. Collectively, these studies not only reveal an unexpected connection between the PRC2 complex, NF1 and Ras, but also identify a promising epigenetic-based therapeutic strategy that may be exploited for a variety of cancers.
Caspase-4 and caspase-11 are shown to be the direct sensors for cytoplasmic lipopolysaccharide in humans and mice, respectively, mediating inflammatory cell death in intracellular bacterial infection.
The balance between stem cell self-renewal and differentiation is controlled by intrinsic factors and niche signals. In the Drosophila melanogaster ovary, some intrinsic factors promote germline stem cell (GSC) self-renewal, whereas others stimulate differentiation. However, it remains poorly understood how the balance between self-renewal and differentiation is controlled. Here we use D. melanogaster ovarian GSCs to demonstrate that the differentiation factor Bam controls the functional switch of the COP9 complex from self-renewal to differentiation via protein competition. The COP9 complex is composed of eight Csn subunits, Csn1–8, and removes Nedd8 modifications from target proteins. Genetic results indicated that the COP9 complex is required intrinsically for GSC self-renewal, whereas other Csn proteins, with the exception of Csn4, were also required for GSC progeny differentiation. Bam-mediated Csn4 sequestration from the COP9 complex via protein competition inactivated the self-renewing function of COP9 and allowed other Csn proteins to promote GSC differentiation. Therefore, this study reveals a protein-competition-based mechanism for controlling the balance between stem cell self-renewal and differentiation.Because numerous self-renewal factors are ubiquitously expressed throughout the stem cell lineage in various systems, protein competition may function as an important mechanism for controlling the self-renewal-to-differentiation switch.
The connection between an altered gut microbiota and metabolic disorders such as obesity, diabetes, and cardiovascular disease is well established. Defects in preserving the integrity of the mucosal barriers can result in systemic endotoxaemia that contributes to chronic low-grade inflammation, which further promotes the development of metabolic syndrome. Interleukin (IL)-22 exerts essential roles in eliciting antimicrobial immunity and maintaining mucosal barrier integrity within the intestine. Here we investigate the connection between IL-22 and metabolic disorders. We find that the induction of IL-22 from innate lymphoid cells and CD4+ T cells is impaired in obese mice under various immune challenges, especially in the colon during infection with Citrobacter rodentium. While innate lymphoid cell populations are largely intact in obese mice, the upregulation of IL-23, a cytokine upstream of IL-22, is compromised during the infection. Consequently, these mice are susceptible to C. rodentium infection, and both exogenous IL-22 and IL-23 are able to restore the mucosal host defence. Importantly, we further unveil unexpected functions of IL-22 in regulating metabolism. Mice deficient in IL-22 receptor and fed with high-fat diet are prone to developing metabolic disorders. Strikingly, administration of exogenous IL-22 in genetically obese leptin-receptor-deficient (db/db) mice and mice fed with high-fat diet reverses many of the metabolic symptoms, including hyperglycaemia and insulin resistance. IL-22 shows diverse metabolic benefits, as it improves insulin sensitivity, preserves gut mucosal barrier and endocrine functions, decreases endotoxaemia and chronic inflammation, and regulates lipid metabolism in liver and adipose tissues. In summary, we identify the IL-22 pathway as a novel target for therapeutic intervention in metabolic diseases.
The pluripotency factor Lin28 inhibits the biogenesis of the let-7 family of mammalian microRNAs. Lin28 is highly expressed in embryonic stem cells and has a fundamental role in regulation of development, glucose metabolism and tissue regeneration. Overexpression of Lin28 is correlated with the onset of numerous cancers, whereas let-7, a tumour suppressor, silences several human oncogenes. Lin28 binds to precursor let-7 (pre-let-7) hairpins, triggering the 3′ oligo-uridylation activity of TUT4 and TUT7 (refs 10, 11, 12). The oligoU tail added to pre-let-7 serves as a decay signal, as it is rapidly degraded by Dis3l2 (refs 13, 14), a homologue of the catalytic subunit of the RNA exosome. The molecular basis of Lin28-mediated recruitment of TUT4 and TUT7 to pre-let-7 and its subsequent degradation by Dis3l2 is largely unknown. To examine the mechanism of Dis3l2 substrate recognition we determined the structure of mouse Dis3l2 in complex with an oligoU RNA to mimic the uridylated tail of pre-let-7. Three RNA-binding domains form an open funnel onone face of the catalytic domain that allows RNA to navigate a path to the active site different from that of its exosome counterpart. The resulting path reveals an extensive network of uracil-specific interactions spanning the first 12 nucleotides of an oligoU-tailed RNA. We identify three U-specificity zones that explain how Dis3l2 recognizes, binds and processes uridylated pre-let-7 in the final step of the Lin28–let-7 pathway.
Homeodomain proteins, described 30 years ago, exert essential roles in development as regulators of target gene expression; however, the molecular mechanisms underlying transcriptional activity of homeodomain factors remain poorly understood. Here investigation of a developmentally required POU-homeodomain transcription factor, Pit1 (also known as Pou1f1), has revealed that, unexpectedly, binding of Pit1-occupied enhancers to a nuclear matrin-3-rich network/architecture is a key event in effective activation of the Pit1-regulated enhancer/coding gene transcriptional program. Pit1 association with Satb1 (ref. 8) andβ-catenin is required for this tethering event. A naturally occurring, dominant negative, point mutation in human PIT1(R271W), causing combined pituitary hormone deficiency, results in loss of Pit1 association with β-catenin and Satb1 and therefore the matrin-3-rich network, blocking Pit1-dependent enhancer/coding target gene activation. This defective activation can be rescued by artificial tethering of the mutant R271W Pit1 protein to the matrin-3 network, bypassing the pre-requisite association with β-catenin and Satb1 otherwise required. The matrin-3 network-tethered R271W Pit1 mutant,but not the untethered protein, restores Pit1-dependent activation of the enhancers and recruitment of co-activators, exemplified by p300, causing both enhancer RNA transcription and target gene activation. These studies have thus revealed an unanticipated homeodomain factor/β-catenin/Satb1-dependent localization of target gene regulatory enhancer regions to a subnuclear architectural structure that serves as an underlying mechanism by which an enhancer-bound homeodomain factor effectively activates developmental gene transcriptional programs.
CHARGE syndrome is a multiple anomaly disorder in which patients present with a variety of phenotypes, including ocular coloboma, heart defects, choanal atresia, retarded growth and development, genitourinary hypoplasia and ear abnormalities. Despite 70–90% of CHARGE syndrome cases resulting from mutations in the gene CHD7, which encodes an ATP-dependent chromatin remodeller, the pathways underlying the diverse phenotypes remain poorly understood. Surprisingly, our studies of a knock-in mutant mouse strain that expresses a stabilized and transcriptionally dead variant of the tumour-suppressor protein p53 (p5325,26,53,54), along with a wild-type allele of p53 (also known as Trp53), revealed late-gestational embryonic lethality associated with a host of phenotypes that are characteristic of CHARGE syndrome, including coloboma, inner and outer ear malformations, heart outflow tract defects and craniofacial defects. We found that the p5325,26,53,54 mutant protein stabilized and hyperactivated wild-type p53, which then inappropriately induced its target genes and triggered cell-cycle arrest or apoptosis during development. Importantly, these phenotypes were only observed with a wild-type p53 allele, as p5325,26,53,54/− embryos were fully viable. Furthermore, we found that CHD7 can bind to the p53 promoter, thereby negatively regulating p53 expression, and that CHD7 loss in mouse neural crest cells or samples from patients with CHARGE syndrome results in p53 activation. Strikingly, we found that p53 heterozygosity partially rescued the phenotypes in Chd7-null mouse embryos, demonstrating that p53 contributes to the phenotypes that result from CHD7 loss. Thus, inappropriate p53 activation during development can promote CHARGEphenotypes, supporting the idea that p53 has a critical role in developmental syndromes and providing important insight into the mechanisms underlying CHARGE syndrome.
Mice deficient in the EDA protein lack normal tooth features. Restoring EDA in embryonic teeth at increasing doses has now been found to recover these dental features in a stepwise pattern that mimics evolution.
Gradual changes that occur to mammalian tooth morphology across evolutionary time were modelled in vitro and in vivo by modulation of signalling pathways in the mouse, and computer modelling was used to provide further analysis of the parameters influencing tooth morphology.
A comparison of colorectal cancer and normal cells from 103 patients identifies dozens of genes that are differently expressed in tumour cells as a result of altered regulation of transcription.
The cis-regulatory effects responsible for cancer development have not been as extensively studied as the perturbations of the protein coding genome in tumorigenesis. To better characterize colorectal cancer (CRC) development we conducted an RNA-sequencing experiment of 103 matched tumour and normal colon mucosa samples from Danish CRC patients, 90 of which were germline-genotyped. By investigating allele-specific expression (ASE) we show that the germline genotypes remain important determinants of allelic gene expression in tumours. Using the changes in ASE in matched pairs of samples we discover 71 genes with excess of somatic cis-regulatory effects in CRC, suggesting a cancer driver role. We correlate genotypes and gene expression to identify expression quantitative trait loci (eQTLs) and find 1,693 and 948 eQTLs in normal samples and tumours, respectively. We estimate that 36% of the tumour eQTLs are exclusive to CRC and show that this specificity is partially driven by increased expression of specific transcription factors and changes in methylation patterns. We show that tumour-specific eQTLs are more enriched for low CRC genome-wide association study (GWAS) P values than shared eQTLs, which suggests that some of the GWAS variants are tumour specific regulatory variants. Importantly, tumour-specific eQTL genes also accumulate more somatic mutations when compared to the shared eQTL genes, raising the possibility that they constitute germline-derived cancer regulatory drivers. Collectively the integration of genome and the transcriptome reveals a substantial number of putative somatic and germline cis-regulatory cancer changes that may have a role in tumorigenesis.
Giving monkeys antiretroviral therapy from just three days after exposure to simian immunodeficiency virus does not prevent a subsequent rebound of viral replication, suggesting that viral reservoirs are established early.
Net primary production is affected by temperature and precipitation, but whether this is a direct kinetic effect on plant metabolism or an indirect ecological effect mediated by changes in plant age, plant biomass or growing season length is unclear— this study develops metabolic scaling theory to be able to answer this question and applies it to a global data set of plant productivity, concluding that it is indirect effects that explain the influence of climate on productivity, which is characterized by a common scaling relationship acrossclimate gradients.
The viral reservoir represents a critical challenge for human immunodeficiency virus type 1 (HIV-1) eradication strategies. However, it remains unclear when and where the viral reservoir is seeded during acute infection and the extent to which it is susceptible to early antiretroviral therapy (ART). Here we show that the viral reservoir is seeded rapidly after mucosal simian immunodeficiency virus (SIV) infection of rhesus monkeys and before systemic viraemia. We initiated suppressive ART in groups of monkeys on days 3, 7, 10 and 14 after intrarectal SIVMAC251 infection. Treatment with ART on day 3 blocked the emergence of viral RNA and proviral DNA in peripheral blood and also substantially reduced levels of proviral DNA in lymph nodes and gastrointestinal mucosa as compared with treatment at later time points. In addition, treatment on day 3 abrogated the induction of SIV-specific humoral and cellular immune responses. Nevertheless, after discontinuation of ART following 24 weeks of fully suppressive therapy, virus rebounded in all animals, although the monkeys that were treated on day 3 exhibited a delayed viral rebound as compared with those treated on days 7, 10 and 14. The time to viral rebound correlated with total viraemia during acute infection and with proviral DNA at the time of ART discontinuation. These data demonstrate that the viral reservoir is seeded rapidly after intrarectal SIV infection of rhesus monkeys, during the‘eclipse’ phase, and before detectable viraemia. This strikingly early seeding of the refractory viral reservoir raises important new challenges for HIV-1 eradication strategies.
The crystal structures of thalidomide and its derivatives bound to the E3 ligase subcomplex DDB1–CRBN are shown; these drugs are found to have dual functions, interfering with the binding of certain cellular substrates to the E3 ligase but promoting the binding of others, thereby modulating the degradation of cellular proteins.
Developmental enhancers initiate transcription and are fundamental to our understanding of developmental networks, evolution and disease. Despite their importance, the properties governing enhancer–promoter interactions and their dynamics during embryogenesis remain unclear. At the β-globin locus, enhancer–promoter interactions appear dynamic and cell-type specific, whereas at the HoxD locus they are stable and ubiquitous, being present in tissues where the target genes are not expressed. The extent to which preformed enhancer–promoter conformations exist at other, more typical, loci and how transcription is eventually triggered is unclear. Here we generated a high-resolution map of enhancer three-dimensional contacts during Drosophila embryogenesis, covering two developmental stages and tissue contexts, at unprecedented resolution. Although local regulatory interactions are common, long-range interactions are highly prevalent within the compact Drosophila genome. Each enhancer contacts multiple enhancers, and promoters with similar expression, suggesting a role in their co-regulation. Notably, most interactions appear unchanged between tissue context and across development, arising before gene activation, and are frequently associated with paused RNA polymerase. Our results indicate that the general topology governing enhancer contacts is conserved from flies to humans and suggest that transcription initiates from preformed enhancer–promoter loops through release of paused polymerase.
Rheumatoid arthritis is a chronic autoinflammatory disease that affects 1–2% of the world’s population and is characterized by widespread joint inflammation. Interleukin-1 is an important mediator of cartilage destruction in rheumatic diseases, but our understanding of the upstream mechanisms leading to production of interleukin-1β in rheumatoid arthritis is limited by the absence of suitable mouse models of the disease in which inflammasomes contribute to pathology. Myeloid-cell-specific deletion of the rheumatoid arthritis susceptibility gene A20/Tnfaip3 in mice (A20myel-KO mice) triggers a spontaneous erosive polyarthritis that resembles rheumatoid arthritis in patients. Rheumatoid arthritis in A20myel-KO mice is not rescued by deletion of tumour necrosis factor receptor 1 (ref. 2). Here we show, however, that it crucially relies on the Nlrp3 inflammasome and interleukin-1 receptor signalling. Macrophages lacking A20 have increased basal and lipopolysaccharide-induced expression levels of the inflammasome adaptor Nlrp3 and proIL-1β. As a result, A20-deficiency in macrophages significantly enhances Nlrp3 inflammasome-mediated caspase-1 activation, pyroptosis and interleukin-1β secretion by soluble and crystalline Nlrp3 stimuli. In contrast, activation of the Nlrc4 and AIM2 inflammasomes is not altered. Importantly, increased Nlrp3 inflammasome activation contributes to the pathology of rheumatoid arthritis in vivo, because deletion of Nlrp3, caspase-1 and the interleukin-1 receptor markedly protects against rheumatoid-arthritis-associated inflammation and cartilage destruction in A20myel-KO mice. These results reveal A20 as a novel negative regulator of Nlrp3 inflammasome activation, and describe A20myel-KO mice as the first experimental model to study the role of inflammasomes in the pathology of rheumatoid arthritis.
Myeloproliferative neoplasms (MPNs) are diseases caused by mutations in the haematopoietic stem cell (HSC) compartment. Most MPN patients have a common acquired mutation of Janus kinase 2 (JAK2) gene in HSCs that renders this kinase constitutively active, leading to uncontrolled cell expansion. The bone marrow microenvironment might contribute to the clinical outcomes of this common event. We previously showed that bone marrow nestin+ mesenchymal stem cells (MSCs) innervated by sympathetic nerve fibres regulate normal HSCs. Here we demonstrate that abrogation of this regulatory circuit is essential for MPN pathogenesis. Sympathetic nerve fibres, supporting Schwann cells and nestin+ MSCs are consistently reduced in the bone marrow of MPN patients and mice expressing the human JAK2(V617F) mutation in HSCs. Unexpectedly, MSC reduction is not due to differentiation but is caused by bone marrow neural damage and Schwann cell death triggered by interleukin-1β produced by mutant HSCs. In turn, in vivo depletion of nestin+ cells or their production of CXCL12 expanded mutant HSC number and accelerated MPN progression. In contrast, administration of neuroprotective or sympathomimetic drugs prevented mutant HSC expansion. Treatment with β3-adrenergic agonists that restored the sympathetic regulation of nestin+ MSCs prevented the loss of these cells and blocked MPN progression by indirectly reducing the number of leukaemic stem cells. Our results demonstrate that mutant-HSC-driven niche damage critically contributes to disease manifestation in MPN and identify niche-forming MSCs and their neural regulation as promising therapeutic targets.
The proton gradient is a principal energy source for respiration-dependent active transport, but the structural mechanisms of proton-coupled transport processes are poorly understood. YiiP is a proton-coupled zinc transporter found in the cytoplasmic membrane of Escherichia coli. Its transport site receives protons from water molecules that gain access to its hydrophobic environment and transduces the energy of an inward proton gradient to drive Zn(ii) efflux. This membrane protein is a well-characterized member of the family of cation diffusion facilitators that occurs at all phylogenetic levels. Here we show, using X-ray-mediated hydroxyl radical labelling of YiiP and mass spectrometry, that Zn(ii) binding triggers a highly localized, all-or-nothing change of water accessibility to the transport site and an adjacent hydrophobic gate. Millisecond time-resolved dynamics reveal a concerted and reciprocal pattern of accessibility changes along a transmembrane helix, suggesting a rigid-body helical re-orientation linked to Zn(ii) binding that triggers the closing of the hydrophobic gate. The gated water access to the transport site enables a stationary proton gradient to facilitate the conversion of zinc-binding energy to the kinetic power stroke of a vectorial zinc transport. The kinetic details provide energetic insights into a proton-coupled active-transport reaction.
‘Gain’ of supernumerary copies of the 8q24.21 chromosomal region has been shown to be common in many human cancers and is associated with poor prognosis. The well-characterized myelocytomatosis (MYC) oncogene resides in the 8q24.21 region and is consistently co-gained with an adjacent ‘gene desert’ of approximately 2 megabases that contains the long non-coding RNA gene PVT1, the CCDC26 gene candidate and the GSDMC gene. Whether low copy-number gain of one or more of these genes drives neoplasia is not known. Here we use chromosome engineering in mice to show that a single extra copyof either the Myc gene or the region encompassing Pvt1, Ccdc26 and Gsdmc fails to advance cancer measurably, whereas a single supernumerary segment encompassing all four genes successfully promotes cancer. Gain of PVT1 long non-coding RNA expression was required for high MYC protein levels in 8q24-amplified human cancer cells. PVT1 RNA and MYC protein expression correlated in primary human tumours, and copy number of PVT1 was co-increased in more than 98% of MYC-copy-increase cancers. Ablation of PVT1 from MYC-driven colon cancer line HCT116 diminished its tumorigenic potency. As MYC protein has been refractory to small-molecule inhibition, the dependence of high MYC protein levels on PVT1 long non-coding RNA provides a much needed therapeutic target.
What gives quantum computers that extra oomph over their classical digital counterparts? An intrinsic, measurable aspect of quantum mechanics called contextuality, it now emerges.
The finding that phosphoinositide-3-OH kinaseδ restrains the antitumour immune response by promoting the action of suppressive immune cells may broaden the applicability of drugs targeting this enzyme to multiple cancers.
Quantum computing promises advantages over classical computing for certain problems; now‘quantum contextuality’ — a generalization of the concept of quantum non-locality — is shown to be a critical resource that gives the most promising class of quantum computers their power.
The Eucalyptus grandis genome has been sequenced, revealing the greatest number of tandem duplications of any plant genome sequenced so far, and the highest diversity of genes for specialized metabolites that act as chemical defence and provide unique pharmaceutical oils; genome sequencing of the sister species E. globulus and a set of inbred E. grandis tree genomes reveals dynamic genome evolution and hotspots of inbreeding depression.
Large-scale single-cell RNA-seq of stimulated primary mouse bone-marrow-derived dendritic cells highlights positive and negative intercellular signalling pathways that promote and restrain cellular variation.
Inhibitors against the p110δ isoform of phosphoinositide-3-OH kinase (PI(3)K) have shown remarkable therapeutic efficacy in some human leukaemias. As p110δ is primarily expressed in leukocytes, drugs against p110δ have not been considered for the treatment of solid tumours. Here we report that p110δ inactivation in mice protects against a broad range of cancers, including non-haematological solid tumours. We demonstrate that p110δ inactivation in regulatory T cells unleashes CD8+ cytotoxic T cells and induces tumour regression. Thus, p110δ inhibitors can break tumour-induced immune tolerance and should be considered for wider use in oncology.
Undernourished children fall behind not only on growth, but also on maturation of their intestinal bacterial communities, according to a study comparing acutely malnourished and healthy Bangladeshi children.
The enzyme parkin is known to promote disposal of organelles called mitochondria that have suffered damage. The identification of an enzyme that opposes parkin demonstrates how a delicate balance is maintained in the cell.
Damaged mitochondria are removed by mitophagy, and defects in mitophagy are linked to Parkinson’s disease; here it is shown that USP30, a deubiquitinase localized to mitochondria, antagonizes mitophagy by removing the ubiquitin tags put in place by Parkin, USP30 inhibition is therefore potentially beneficial for Parkinson’s disease by promoting mitochondrial clearance and quality control.
Therapeutic food interventions have reduced mortality in children with severe acute malnutrition (SAM), but incomplete restoration of healthy growth remains a major problem. The relationships between the type of nutritional intervention, the gut microbiota, and therapeutic responses are unclear. In the current study, bacterial species whose proportional representation define a healthy gut microbiota as it assembles during the first two postnatal years were identified by applying a machine-learning-based approach to 16S ribosomal RNA data sets generated from monthly faecal samples obtained from birth onwards in a cohort of children living in an urban slum of Dhaka, Bangladesh, who exhibited consistently healthy growth. These age-discriminatory bacterial species were incorporated into a model that computes a‘relative microbiota maturity index’ and ‘microbiota-for-age Z-score’ that compare postnatal assembly (defined here as maturation) of a child’s faecal microbiota relative to healthy children of similar chronologic age. The model was applied to twins and triplets (to test for associations of these indices with genetic and environmental factors, including diarrhoea), children with SAM enrolled in a randomized trial of two food interventions, and children with moderate acute malnutrition. Our results indicate that SAM is associated with significant relative microbiota immaturity that is only partially ameliorated following two widely used nutritional interventions. Immaturity is also evident in less severe forms of malnutrition and correlates with anthropometric measurements. Microbiota maturity indices provide a microbial measure of human postnatal development, a way of classifying malnourished states, and a parameter for judging therapeutic efficacy. More prolonged interventions with existing or new therapeutic foods and/or addition of gut microbes may be needed to achieve enduring repair of gut microbiota immaturity in childhood malnutrition and improve clinical outcomes.
Transcriptional enhancers are crucial regulators of gene expression and animal development and the characterization of their genomic organization, spatiotemporal activities and sequence properties is a key goal in modern biology. Here we characterize the in vivo activity of 7,705 Drosophila melanogaster enhancer candidates covering 13.5% of the non-coding non-repetitive genome throughout embryogenesis. 3,557 (46%) candidates are active, suggesting a high density with 50,000 to 100,000 developmental enhancers genome-wide. The vast majority of enhancers display specific spatial patterns that are highly dynamic during development. Most appear to regulate their neighbouring genes, suggesting that the cis-regulatory genome is organized locally into domains, which are supported by chromosomal domains, insulator binding and genome evolution. However, 12 to 21 per cent of enhancers appear to skip non-expressed neighbours and regulate a more distal gene. Finally, we computationally identify cis-regulatory motifs that are predictive and required for enhancer activity, as we validate experimentally. This work provides global insights into the organization of an animal regulatory genome and the make-up of enhancer sequences and confirms and generalizes principles from previous studies. All enhancer patterns are annotated manually with a controlled vocabulary and all results are available through a web interface (http://enhancers.starklab.org), including the raw images of all microscopy slides for manual inspection at arbitrary zoom levels.
A unique property of many adult stem cells is their ability to exist in a non-cycling, quiescent state. Although quiescence serves an essential role in preserving stem cell function until the stem cell is needed in tissue homeostasis or repair, defects in quiescence can lead to an impairment in tissue function. The extent to which stem cells can regulate quiescence is unknown. Here we show that the stem cell quiescent state is composed of two distinct functional phases, G0 and an‘alert’ phase we term GAlert. Stem cells actively and reversibly transition between these phases in response to injury-induced systemic signals. Using genetic mouse models specific to muscle stem cells (or satellite cells), we show that mTORC1 activity is necessary and sufficient for the transition of satellite cells from G0 into GAlert and that signalling through the HGF receptor cMet is also necessary. We also identify G0-to-GAlert transitions in several populations of quiescent stem cells. Quiescent stem cells that transition into GAlert possess enhanced tissue regenerative function. We propose that the transition of quiescent stem cells into GAlert functions as an ‘alerting’ mechanism, an adaptive response that positions stem cells to respond rapidly under conditions of injury and stress, priming them for cell cycle entry.
Metabolism and ageing are intimately linked. Compared with ad libitum feeding, dietary restriction consistently extends lifespan and delays age-related diseases in evolutionarily diverse organisms. Similar conditions of nutrient limitation and genetic or pharmacological perturbations of nutrient or energy metabolism also have longevity benefits. Recently, several metabolites have been identified that modulate ageing; however, the molecular mechanisms underlying this are largely undefined. Here we show thatα-ketoglutarate (α-KG), a tricarboxylic acid cycle intermediate, extends the lifespan of adult Caenorhabditis elegans. ATP synthase subunit β is identified as a novel binding protein of α-KG using a small-molecule target identification strategy termed drug affinity responsive target stability (DARTS). The ATP synthase, also known as complex V of the mitochondrial electron transport chain, is the main cellular energy-generating machinery and is highly conserved throughout evolution. Although complete loss of mitochondrial function is detrimental, partial suppression of the electron transport chain has been shown to extend C. elegans lifespan. We show that α-KG inhibits ATP synthase and, similar to ATP synthase knockdown, inhibition by α-KG leads to reduced ATP content, decreased oxygen consumption, and increased autophagy in both C. elegans and mammalian cells. We provide evidence that the lifespan increase by α-KG requires ATP synthase subunit β and is dependent on target of rapamycin (TOR) downstream. Endogenous α-KG levels are increased on starvation and α-KG does not extend the lifespan of dietary-restricted animals, indicating that α-KG is a key metabolite that mediates longevity by dietary restriction. Our analyses uncover new molecular links between a common metabolite, a universal cellular energy generator and dietary restriction in the regulation of organismal lifespan, thus suggesting new strategies for the prevention and treatment of ageing and age-related diseases.
2-Oxoglutarate (2OG)-dependent oxygenases have important roles in the regulation of gene expression via demethylation of N-methylated chromatin components and in the hydroxylation of transcription factors and splicing factor proteins. Recently, 2OG-dependent oxygenases that catalyse hydroxylation of transfer RNA and ribosomal proteins have been shown to be important in translation relating to cellular growth, TH17-cell differentiation and translational accuracy. The finding that ribosomal oxygenases (ROXs) occur in organisms ranging from prokaryotes to humans raises questions as to their structural and evolutionary relationships. In Escherichia coli, YcfD catalyses arginine hydroxylation in the ribosomal protein L16; in humans, MYC-induced nuclear antigen (MINA53; also known as MINA) and nucleolar protein 66 (NO66) catalyse histidine hydroxylation in the ribosomal proteins RPL27A and RPL8, respectively. The functional assignments of ROXs open therapeutic possibilities via either ROX inhibition or targeting of differentially modified ribosomes. Despite differences in the residue and protein selectivities of prokaryotic and eukaryotic ROXs, comparison of the crystal structures of E. coli YcfD and Rhodothermus marinus YcfD with those of human MINA53 and NO66 reveals highly conserved folds and novel dimerization modes defining a new structural subfamily of 2OG-dependent oxygenases. ROX structures with and without their substrates support their functional assignments as hydroxylases but not demethylases, and reveal how the subfamily has evolved to catalyse the hydroxylation of different residue side chains of ribosomal proteins. Comparison of ROX crystal structures with those of other JmjC-domain-containing hydroxylases, including the hypoxia-inducible factor asparaginyl hydroxylase FIH and histone Nε-methyl lysine demethylases, identifies branch points in 2OG-dependent oxygenase evolution and distinguishes between JmjC-containing hydroxylases and demethylases catalysing modifications of translational and transcriptional machinery. The structures reveal that new protein hydroxylation activities can evolve by changing the coordination position from which the iron-bound substrate-oxidizing species reacts. This coordination flexibility has probably contributed to the evolution of the wide range of reactions catalysed by oxygenases.
The global shortening of messenger RNAs through alternative polyadenylation (APA) that occurs during enhanced cellular proliferation represents an important, yet poorly understood mechanism of regulated gene expression. The 3′ untranslated region (UTR) truncation of growth-promoting mRNA transcripts that relieves intrinsic microRNA- and AU-rich-element-mediated repression has been observed to correlate with cellular transformation; however, the importance to tumorigenicity of RNA 3′-end-processing factors that potentially govern APA is unknown. Here we identify CFIm25 as a broad repressor of proximal poly(A) site usage that, when depleted, increases cell proliferation. Applying a regression model on standard RNA-sequencing data for novel APA events, we identified at least 1,450 genes with shortened 3′ UTRs after CFIm25 knockdown, representing 11% of significantly expressed mRNAs in human cells. Marked increases in the expression of several known oncogenes, including cyclin D1, are observed as a consequence of CFIm25 depletion. Importantly, we identified a subset of CFIm25-regulated APA genes with shortened 3′ UTRs in glioblastoma tumours that have reduced CFIm25 expression. Downregulation of CFIm25 expression in glioblastoma cells enhances their tumorigenic properties and increases tumour size, whereas CFIm25 overexpression reduces these properties and inhibits tumour growth. These findings identify a pivotal role of CFIm25 in governing APA and reveal a previously unknown connection between CFIm25 and glioblastoma tumorigenicity.
Sulphur is an essential element for life and is ubiquitous in living systems. Yet how the sulphur atom is incorporated into many sulphur-containing secondary metabolites is poorly understood. For bond formation between carbon and sulphur in primary metabolites, the major ionic sulphur sources are the persulphide and thiocarboxylate groups on sulphur-carrier (donor) proteins. Each group is post-translationally generated through the action of a specific activating enzyme. In all reported bacterial cases, the gene encoding the enzyme that catalyses the carbon–sulphur bond formation reaction and that encoding the cognate sulphur-carrier protein exist in the same gene cluster. To study the production of the 2-thiosugar moiety in BE-7585A, an antibiotic from Amycolatopsis orientalis, we identified a putative 2-thioglucose synthase, BexX, whose protein sequence and mode of action seem similar to those of ThiG, the enzyme that catalyses thiazole formation in thiamine biosynthesis. However, no gene encoding a sulphur-carrier protein could be located in the BE-7585A cluster. Subsequent genome sequencing uncovered a few genes encoding sulphur-carrier proteins that are probably involved in the biosynthesis of primary metabolites but only one activating enzyme gene in the A. orientalis genome. Further experiments showed that this activating enzyme can adenylate each of these sulphur-carrier proteins and probably also catalyses the subsequent thiolation, through its rhodanese domain. A proper combination of these sulphur-delivery systems is effective for BexX-catalysed 2-thioglucose production. The ability of BexX to selectively distinguish sulphur-carrier proteins is given a structural basis using X-ray crystallography. This study is, to our knowledge, the first complete characterization of thiosugar formation in nature and also demonstrates the receptor promiscuity of the A. orientalis sulphur-delivery system. Our results also show that co-opting the sulphur-delivery machinery of primary metabolism for the biosynthesis of sulphur-containing natural products is probably a general strategy found in nature.
PTEN encodes a lipid phosphatase that is underexpressed in many cancers owing to deletions, mutations or gene silencing. PTEN dephosphorylates phosphatidylinositol (3,4,5)-triphosphate, thereby opposing the activity of class I phosphatidylinositol 3-kinases that mediate growth- and survival-factor signalling through phosphatidylinositol 3-kinase effectors such as AKT and mTOR. To determine whether continued PTEN inactivation is required to maintain malignancy, here we generate an RNA interference-based transgenic mouse model that allows tetracycline-dependent regulation of PTEN in a time- and tissue-specific manner. Postnatal Pten knockdown in the haematopoietic compartment produced highly disseminated T-cell acute lymphoblastic leukaemia. Notably, reactivation of PTEN mainly reduced T-cell leukaemia dissemination but had little effect on tumour load in haematopoietic organs. Leukaemia infiltration into the intestine was dependent on CCR9 G-protein-coupled receptor signalling, which was amplified by PTEN loss. Our results suggest that in the absence of PTEN, G-protein-coupled receptors may have an unanticipated role in driving tumour growth and invasion in an unsupportive environment. They further reveal that the role of PTEN loss in tumour maintenance is not invariant and can be influenced by the tissue microenvironment, thereby producing a form of intratumoral heterogeneity that is independent of cancer genotype.