CHLOROQUINE is thought to act against falciparum malaria by accumulating in the acid vesicles of the parasite and interfering with their function14. Parasites resistant to chloroquine expel the drug rapidly in an unaltered form, thereby reducing levels of accumulation in the vesicles5. The discovery that verapamil partially reverses chloroquine resistance in vitro 6 led to the proposal that efflux may involve an ATP-driven P-glycoprotein pump similar to that in mammalian multidrug-resistant (mdr) tumor cell lines. Indeed, Plasmodium falciparum contains at least two mdr-like genes7,8, one of which has been suggested to confer the chloroquine resistant (CQR) phenotype7,9,10. To determine if either of these genes is linked to chloroquine resistance, we performed a genetic cross between CQR and chloroquine-susceptible (CQS) clones of P. falciparum. Examination of 16 independent recombinant progeny indicated that the rapid efflux phenotype is controlled by a single gene or a closely linked group of genes. But, there was no linkage between the rapid efflux, CQR phenotype and either of the mdr-like P. falciparum genes or amplification of those genes. These data indicate that the genetic locus governing chloroquine efflux and resistance is independent of the known mdr-like genes.


The cellular and molecular mechanisms that enable us to sense cold are not well understood. Insights into this process have come from the use of pharmacological agents, such as menthol, that elicit a cooling sensation. Here we have characterized and cloned a menthol receptor from trigeminal sensory neurons that is also activated by thermal stimuli in the cool to cold range. This cold- and menthol-sensitive receptor, CMR1, is a member of the TRP family of excitatory ion channels, and we propose that it functions as a transducer of cold stimuli in the somatosensory system. These findings, together with our previous identification of the heat-sensitive channels VR1 and VRL-1, demonstrate that TRP channels detect temperatures over a wide range and are the principal sensors of thermal stimuli in the mammalian peripheral nervous system.


Missense mutations in the CIAS1 gene cause three autoinflammatory disorders: familial cold autoinflammatory syndrome, Muckle–Wells syndrome and neonatal-onset multiple-system inflammatory disease. Cryopyrin (also called Nalp3), the product of CIAS1, is a member of the NOD-LRR protein family that has been linked to the activation of intracellular host defence signalling pathways. Cryopyrin forms a multi-protein complex termed ‘the inflammasome’, which contains the apoptosis-associated speck-like protein (ASC) and caspase-1, and promotes caspase-1 activation and processing of pro-interleukin (IL)-1β (ref. 4). Here we show the effect of cryopyrin deficiency on inflammasome function and immune responses. Cryopyrin and ASC are essential for caspase-1 activation and IL-1β and IL-18 production in response to bacterial RNA and the imidazoquinoline compounds R837 and R848. In contrast, secretion of tumour-necrosis factor-α and IL-6, as well as activation of NF-κB and mitogen-activated protein kinases (MAPKs) were unaffected by cryopyrin deficiency. Furthermore, we show that Toll-like receptors and cryopyrin control the secretion of IL-1β and IL-18 through different intracellular pathways. These results reveal a critical role for cryopyrin in host defence through bacterial RNA-mediated activation of caspase-1, and provide insights regarding the pathogenesis of autoinflammatory syndromes.


Obesity is now so common within the world's population that it is beginning to replace undernutrition and infectious diseases as the most significant contributor to ill health. In particular, obesity is associated with diabetes mellitus, coronary heart disease, certain forms of cancer, and sleep-breathing disorders. Obesity is defined by a body-mass index (weight divided by square of the height) of 30 kg m(-2) or greater, but this does not take into account the morbidity and mortality associated with more modest degrees of overweight, nor the detrimental effect of intra-abdominal fat. The global epidemic of obesity results from a combination of genetic susceptibility, increased availability of high-energy foods and decreased requirement for physical activity in modern society. Obesity should no longer be regarded simply as a cosmetic problem affecting certain individuals, but an epidemic that threatens global well being.


The development and maintenance of an epithelium requires finely balanced rates of growth and cell death. However, the mechanical and biochemical mechanisms that ensure proper feedback control of tissue growth, which when deregulated contribute to tumorigenesis, are poorly understood. Here we use the fly notum as a model system to identify a novel process of crowding-induced cell delamination that balances growth to ensure the development of well-ordered cell packing. In crowded regions of the tissue, a proportion of cells undergo a serial loss of cell–cell junctions and a progressive loss of apical area, before being squeezed out by their neighbours. This path of delamination is recapitulated by a simple computational model of epithelial mechanics, in which stochastic cell loss relieves overcrowding as the system tends towards equilibrium. We show that this process of delamination is mechanistically distinct from apoptosis-mediated cell extrusion and precedes the first signs of cell death. Overall, this analysis reveals a simple mechanism that buffers epithelia against variations in growth. Because live-cell delamination constitutes a mechanistic link between epithelial hyperplasia and cell invasion, this is likely to have important implications for our understanding of the early stages of cancer development.


Viral replication usually requires that innate intracellular lines of defence be overcome, a task usually accomplished by specialized viral gene products. The virion infectivity factor (Vif) protein of human immunodeficiency virus (HIV) is required during the late stages of viral production to counter the antiviral activity of APOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G; also known as CEM15), a protein expressed notably in human T lymphocytes. When produced in the presence of APOBEC3G, vif-defective virus is non-infectious. APOBEC3G is closely related to APOBEC1, the central component of an RNA-editing complex that deaminates a cytosine residue in apoB messenger RNA. APOBEC family members also have potent DNA mutator activity through dC deamination; however, whether the editing potential of APOBEC3G has any relevance to HIV inhibition is unknown. Here, we demonstrate that it does, as APOBEC3G exerts its antiviral effect during reverse transcription to trigger G-to-A hypermutation in the nascent retroviral DNA. We also find that APOBEC3G can act on a broad range of retroviruses in addition to HIV, suggesting that hypermutation by editing is a general innate defence mechanism against this important group of pathogens.


PROGRESSIVE cerebral deposition of the amyloid β-peptide is an early and invariant feature of Alzheimer's disease. The β-peptide is released by proteolytic cleavages from the β-amyloid precursor protein (βAPP)1, a membrane-spanning glycoprotein expressed in most mammalian cells. Normal secretion of βAPP involves a cleavage in the β-peptide region2-3, releasing the soluble extramembranous portion4,5 and retaining a 10K C-terminal fragment in the membrane6. Because this secretory pathway precludes β-amyloid formation, we searched for an alternative proteolytic processing pathway that can generate β-peptide-bearing fragments from full-length β APP. Incubation of living human endothelial cells with a βAPP antibody revealed reinternalization of mature βAPP from the cell surface and its targeting to endosomes/lysosomes. After cell-surface biotinylation, full-length biotinylated βAPP was recovered inside the cells. Purification of lysosomes directly demonstrated the presence of mature βAPP and an extensive array of β-peptide-containing proteolytic products. Our results define a second processing pathway for βAPP and suggest that it may be responsible for generating amyloid-bearing fragments in Alzheimer's disease.


Cell growth, an increase in mass and size, is a highly regulated cellular event. The Akt/mTOR (mammalian target of rapamycin) signalling pathway has a central role in the control of protein synthesis and thus the growth of cells, tissues and organisms. A striking example of a physiological context requiring rapid cell growth is tissue repair in response to injury. Here we show that keratin 17, an intermediate filament protein rapidly induced in wounded stratified epithelia, regulates cell growth through binding to the adaptor protein 14-3-3σ. Mouse skin keratinocytes lacking keratin 17 (ref. 4) show depressed protein translation and are of smaller size, correlating with decreased Akt/mTOR signalling activity. Other signalling kinases have normal activity, pointing to the specificity of this defect. Two amino acid residues located in the amino-terminal head domain of keratin 17 are required for the serum-dependent relocalization of 14-3-3σ from the nucleus to the cytoplasm, and for the concomitant stimulation of mTOR activity and cell growth. These findings reveal a new and unexpected role for the intermediate filament cytoskeleton in influencing cell growth and size by regulating protein synthesis.


The three-dimensional structure of an HNF-3/fork head DNA-recognition motif complexed with DNA has been determined by X-ray crystallography at 2.5 Å resolution. This α/β protein binds B-DNA as a monomer, through interactions with the DNA backbone and through both direct and water-mediated major and minor groove base contacts, inducing a 13° bend. The transcription factor fold is very similar to the structure of histone H5. In its amino-terminal half, three α-helices adopt a compact structure that presents the third helix to the major groove. The remainder of the protein includes a twisted, antiparallel β-structure and random coil that interacts with the minor groove.


Chromosomal instability (CIN) is a hallmark of many tumours and correlates with the presence of extra centrosomes. However, a direct mechanistic link between extra centrosomes and CIN has not been established. It has been proposed that extra centrosomes generate CIN by promoting multipolar anaphase, a highly abnormal division that produces three or more aneuploid daughter cells. Here we use long-term live-cell imaging to demonstrate that cells with multiple centrosomes rarely undergo multipolar cell divisions, and the progeny of these divisions are typically inviable. Thus, multipolar divisions cannot explain observed rates of CIN. In contrast, we observe that CIN cells with extra centrosomes routinely undergo bipolar cell divisions, but display a significantly increased frequency of lagging chromosomes during anaphase. To define the mechanism underlying this mitotic defect, we generated cells that differ only in their centrosome number. We demonstrate that extra centrosomes alone are sufficient to promote chromosome missegregation during bipolar cell division. These segregation errors are a consequence of cells passing through a transient 'multipolar spindle intermediate' in which merotelic kinetochore-microtubule attachment errors accumulate before centrosome clustering and anaphase. These findings provide a direct mechanistic link between extra centrosomes and CIN, two common characteristics of solid tumours. We propose that this mechanism may be a common underlying cause of CIN in human cancer.


Although haematopoietic stem cells (HSCs) are commonly assumed to reside within a specialized microenvironment, or niche, most published experimental manipulations of the HSC niche have affected the function of diverse restricted progenitors. This raises the fundamental question of whether HSCs and restricted progenitors reside within distinct, specialized niches or whether they share a common niche. Here we assess the physiological sources of the chemokine CXCL12 for HSC and restricted progenitor maintenance. Cxcl12(DsRed) knock-in mice (DsRed-Express2 recombined into the Cxcl12 locus) showed that Cxcl12 was primarily expressed by perivascular stromal cells and, at lower levels, by endothelial cells, osteoblasts and some haematopoietic cells. Conditional deletion of Cxcl12 from haematopoietic cells or nestin-cre-expressing cells had little or no effect on HSCs or restricted progenitors. Deletion of Cxcl12 from endothelial cells depleted HSCs but not myeloerythroid or lymphoid progenitors. Deletion of Cxcl12 from perivascular stromal cells depleted HSCs and certain restricted progenitors and mobilized these cells into circulation. Deletion of Cxcl12 from osteoblasts depleted certain early lymphoid progenitors but not HSCs or myeloerythroid progenitors, and did not mobilize these cells into circulation. Different stem and progenitor cells thus reside in distinct cellular niches in bone marrow: HSCs occupy a perivascular niche and early lymphoid progenitors occupy an endosteal niche.


Understanding the genetic mechanisms underlying natural variation in gene expression is a central goal of both medical and evolutionary genetics, and studies of expression quantitative trait loci (eQTLs) have become an important tool for achieving this goal. Although all eQTL studies so far have assayed messenger RNA levels using expression microarrays, recent advances in RNA sequencing enable the analysis of transcript variation at unprecedented resolution. We sequenced RNA from 69 lymphoblastoid cell lines derived from unrelated Nigerian individuals that have been extensively genotyped by the International HapMap Project. By pooling data from all individuals, we generated a map of the transcriptional landscape of these cells, identifying extensive use of unannotated untranslated regions and more than 100 new putative protein-coding exons. Using the genotypes from the HapMap project, we identified more than a thousand genes at which genetic variation influences overall expression levels or splicing. We demonstrate that eQTLs near genes generally act by a mechanism involving allele-specific expression, and that variation that influences the inclusion of an exon is enriched within and near the consensus splice sites. Our results illustrate the power of high-throughput sequencing for the joint analysis of variation in transcription, splicing and allele-specific expression across individuals.


The cellular constituents forming the haematopoietic stem cell (HSC) niche in the bone marrow are unclear, with studies implicating osteoblasts, endothelial and perivascular cells. Here we demonstrate that mesenchymal stem cells (MSCs), identified using nestin expression, constitute an essential HSC niche component. Nestin+ MSCs contain all the bone-marrow colony-forming-unit fibroblastic activity and can be propagated as non-adherent ‘mesenspheres’ that can self-renew and expand in serial transplantations. Nestin+ MSCs are spatially associated with HSCs and adrenergic nerve fibres, and highly express HSC maintenance genes. These genes, and others triggering osteoblastic differentiation, are selectively downregulated during enforced HSC mobilization or β3 adrenoreceptor activation. Whereas parathormone administration doubles the number of bone marrow nestin+ cells and favours their osteoblastic differentiation, in vivo nestin+ cell depletion rapidly reduces HSC content in the bone marrow. Purified HSCs home near nestin+ MSCs in the bone marrow of lethally irradiated mice, whereas in vivo nestin+ cell depletion significantly reduces bone marrow homing of haematopoietic progenitors. These results uncover an unprecedented partnership between two distinct somatic stem-cell types and are indicative of a unique niche in the bone marrow made of heterotypic stem-cell pairs.


Immune homeostasis in tissues is achieved through a delicate balance between pathogenic T-cell responses directed at tissue-specific antigens and the ability of the tissue to inhibit these responses. The mechanisms by which tissues and the immune system communicate to establish and maintain immune homeostasis are currently unknown. Clinical evidence suggests that chronic or repeated exposure to self antigen within tissues leads to an attenuation of pathological autoimmune responses, possibly as a means to mitigate inflammatory damage and preserve function. Many human organ-specific autoimmune diseases are characterized by the initial presentation of the disease being the most severe, with subsequent flares being of lesser severity and duration. In fact, these diseases often spontaneously resolve, despite persistent tissue autoantigen expression. In the practice of antigen-specific immunotherapy, allergens or self antigens are repeatedly injected in the skin, with a diminution of the inflammatory response occurring after each successive exposure. Although these findings indicate that tissues acquire the ability to attenuate autoimmune reactions upon repeated responses to antigens, the mechanism by which this occurs is unknown. Here we show that upon expression of self antigen in a peripheral tissue, thymus-derived regulatory T cells (Treg cells) become activated, proliferate and differentiate into more potent suppressors, which mediate resolution of organ-specific autoimmunity in mice. After resolution of the inflammatory response, activated Treg cells are maintained in the target tissue and are primed to attenuate subsequent autoimmune reactions when antigen is re-expressed. Thus, Treg cells function to confer ‘regulatory memory’ to the target tissue. These findings provide a framework for understanding how Treg cells respond when exposed to self antigen in peripheral tissues and offer mechanistic insight into how tissues regulate autoimmunity.


Pluripotency pertains to the cells of early embryos that can generate all of the tissues in the organism. Embryonic stem cells are embryo-derived cell lines that retain pluripotency and represent invaluable tools for research into the mechanisms of tissue formation. Recently, murine fibroblasts have been reprogrammed directly to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) to yield induced pluripotent stem (iPS) cells. Using these same factors, we have derived iPS cells from fetal, neonatal and adult human primary cells, including dermal fibroblasts isolated from a skin biopsy of a healthy research subject. Human iPS cells resemble embryonic stem cells in morphology and gene expression and in the capacity to form teratomas in immune-deficient mice. These data demonstrate that defined factors can reprogramme human cells to pluripotency, and establish a method whereby patient-specific cells might be established in culture.


Circadian (∼24 hour) clocks are fundamentally important for coordinated physiology in organisms as diverse as cyanobacteria and humans. All current models of the molecular circadian clockwork in eukaryotic cells are based on transcription-translation feedback loops. Non-transcriptional mechanisms in the clockwork have been difficult to study in mammalian systems. We circumvented these problems by developing novel assays using human red blood cells, which have no nucleus (or DNA) and therefore cannot perform transcription. Our results show that transcription is not required for circadian oscillations in humans, and that non-transcriptional events seem to be sufficient to sustain cellular circadian rhythms. Using red blood cells, we found that peroxiredoxins, highly conserved antioxidant proteins, undergo ∼24-hour redox cycles, which persist for many days under constant conditions (that is, in the absence of external cues). Moreover, these rhythms are entrainable (that is, tunable by environmental stimuli) and temperature-compensated, both key features of circadian rhythms. We anticipate that our findings will facilitate more sophisticated cellular clock models, highlighting the interdependency of transcriptional and non-transcriptional oscillations in potentially all eukaryotic cells.


The Kaposi's sarcoma-associated herpesvirus (KSHV/HHV8) is a gamma-2 herpesvirus that is implicated in the pathogenesis of Kaposi's sarcoma and of primary effusion B-cell lymphomas (PELs). KSHV infects malignant and progenitor cells of Kaposi's sarcoma and PEL, it encodes putative oncogenes and genes that may cause Kaposi's sarcoma pathogenesis by stimulating angiogenesis. The G-protein-coupled receptor encoded by an open reading frame (ORF 74) of KSHV is expressed in Kaposi's sarcoma lesions and in PEL and stimulates signalling pathways linked to cell proliferation in a constitutive (agonist-independent) way. Here we show that signalling by this KSHV G-protein-coupled receptor leads to cell transformation and tumorigenicity, and induces a switch to an angiogenic phenotype mediated by vascular endothelial growth factor, an angiogenesis and Kaposi's-spindle-cell growth factor. We find that this receptor can activate two protein kinases, JNK/SAPK and p38MAPK, by triggering signalling cascades like those induced by inflammatory cytokines that are angiogenesis activators and mitogens for Kaposi's sarcoma cells and B cells. We conclude that the KSHV G-protein-coupled receptor is a viral oncogene that can exploit cell signalling pathways to induce transformation and angiogenesis in KSHV-mediated oncogenesis.


PURPOSE Acute myeloid leukemia (AML) is a heterogeneous disease with poor outcomes. Despite increased evidence shows that dysregulation of histone modification contributes to AML, specific drugs targeting key histone modulators are not applied in the clinical treatment of AML. Here, we investigated whether targeting KDM6B, the demethylase of tri-methylated histone H3 lysine 27 (H3K27me3), has a therapeutic potential for AML. METHODS A KDM6B-specific inhibitor, GSK-J4, was applied to treat the primary cells from AML patients and AML cell lines in vitro and in vivo. RNA-sequencing was performed to reveal the underlying mechanisms of inhibiting KDM6B for the treatment of AML. RESULTS Here we observed that the mRNA expression of KDM6B was up-regulated in AML and positively correlated with poor survival. Treatment with GSK-J4 increased the global level of H3K27me3 and reduced the proliferation and colony-forming ability of primary AML cells and AML cell lines. GSK-J4 treatment significantly induced cell apoptosis and cell-cycle arrest in Kasumi-1 cells, and displayed a synergistic effect with cytosine arabinoside. Notably, injection of GSK-J4 attenuated the disease progression in a human AML xenograft mouse model in vivo. Treatment with GSK-J4 predominantly resulted in down-regulation of DNA replication and cell-cycle-related pathways, as well as abrogated the expression of critical cancer-promoting HOX genes. ChIP-qPCR validated an increased enrichment of H3K27me3 in the transcription start sites of these HOX genes. CONCLUSIONS In summary, our findings suggest that targeting KDM6B with GSK-J4 has a therapeutic potential for the treatment of AML.


In a Drosophila follicle the oocyte always occupies a posterior position among a group of sixteen germline cells. Although the importance of this cell arrangement for the subsequent formation of the anterior-posterior axis of the embryo is well documented, the molecular mechanism responsible for the posterior localization of the oocyte was unknown. Here we show that the homophilic adhesion molecule DE-cadherin mediates oocyte positioning. During follicle biogenesis, DE-cadherin is expressed in germline (including oocyte) and surrounding follicle cells, with the highest concentration of DE-cadherin being found at the interface between oocyte and posterior follicle cells. Mosaic analysis shows that DE-cadherin is required in both germline and follicle cells for correct oocyte localization, indicating that germline-soma interactions may be involved in this process. By analysing the behaviour of the oocyte in follicles with a chimaeric follicular epithelium, we find that the position of the oocyte is determined by the position of DE-cadherin-expressing follicle cells, to which the oocyte attaches itself selectively. Among the DE-cadherin positive follicle cells, the oocyte preferentially contacts those cells that express higher levels of DE-cadherin. On the basis of these data, we propose that in wild-type follicles the oocyte competes successfully with its sister germline cells for contact to the posterior follicle cells, a sorting process driven by different concentrations of DE-cadherin. This is, to our knowledge, the first in vivo example of a cell-sorting process that depends on differential adhesion mediated by a cadherin.


Cytotoxic T lymphocytes (CTLs) destroy virally infected and tumorigenic cells by releasing the contents of specialized secretory lysosomes—termed ‘lytic granules’—at the immunological synapse formed between the CTL and the target. On contact with the target cell, the microtubule organizing centre of the CTL polarizes towards the target and granules move along microtubules in a minus-end direction towards the polarized microtubule organizing centre. However, the final steps of secretion have remained unclear. Here we show that CTLs do not require actin or plus-end microtubule motors for secretion, but instead the centrosome moves to and contacts the plasma membrane at the central supramolecular activation cluster of the immunological synapse. Actin and IQGAP1 are cleared away from the synapse, and granules are delivered directly to the plasma membrane. These data show that CTLs use a previously unreported mechanism for delivering secretory granules to the immunological synapse, with granule secretion controlled by centrosome delivery to the plasma membrane.


Half a century ago, chronic granulomatous disease (CGD) was first described as a disease fatally affecting the ability of children to survive infections. Various milestone discoveries have since been made, from an insufficient ability of patients’ leucocytes to kill microbes to the underlying genetic abnormalities. In this inherited disorder, phagocytes lack NADPH oxidase activity and do not generate reactive oxygen species, most notably superoxide anion, causing recurrent bacterial and fungal infections. Patients with CGD also suffer from chronic inflammatory conditions, most prominently granuloma formation in hollow viscera. The precise mechanisms of the increased microbial pathogenicity have been unclear, and more so the reasons for the exaggerated inflammatory response. Here we show that a superoxide-dependent step in tryptophan metabolism along the kynurenine pathway is blocked in CGD mice with lethal pulmonary aspergillosis, leading to unrestrained Vγ1+ γδ T-cell reactivity, dominant production of interleukin (IL)-17, defective regulatory T-cell activity and acute inflammatory lung injury. Although beneficial effects are induced by IL-17 neutralization or γδ T-cell contraction, complete cure and reversal of the hyperinflammatory phenotype are achieved by replacement therapy with a natural kynurenine distal to the blockade in the pathway. Effective therapy, which includes co-administration of recombinant interferon-γ (IFN-γ), restores production of downstream immunoactive metabolites and enables the emergence of regulatory Vγ4+ γδ and Foxp3+ αβ T cells. Therefore, paradoxically, the lack of reactive oxygen species contributes to the hyperinflammatory phenotype associated with NADPH oxidase deficiencies, through a dysfunctional kynurenine pathway of tryptophan catabolism. Yet, this condition can be reverted by reactivating the pathway downstream of the superoxide-dependent step.


Methylation of cytosines is an essential epigenetic modification in mammalian genomes, yet the rules that govern methylation patterns remain largely elusive. To gain insights into this process, we generated base-pair-resolution mouse methylomes in stem cells and neuronal progenitors. Advanced quantitative analysis identified low-methylated regions (LMRs) with an average methylation of 30%. These represent CpG-poor distal regulatory regions as evidenced by location, DNase I hypersensitivity, presence of enhancer chromatin marks and enhancer activity in reporter assays. LMRs are occupied by DNA-binding factors and their binding is necessary and sufficient to create LMRs. A comparison of neuronal and stem-cell methylomes confirms this dependency, as cell-type-specific LMRs are occupied by cell-type-specific transcription factors. This study provides methylome references for the mouse and shows that DNA-binding factors locally influence DNA methylation, enabling the identification of active regulatory regions.


Nociceptor sensory neurons are specialized to detect potentially damaging stimuli, protecting the organism by initiating the sensation of pain and eliciting defensive behaviours. Bacterial infections produce pain by unknown molecular mechanisms, although they are presumed to be secondary to immune activation. Here we demonstrate that bacteria directly activate nociceptors, and that the immune response mediated through TLR2, MyD88, T cells, B cells, and neutrophils and monocytes is not necessary for Staphylococcus aureus-induced pain in mice. Mechanical and thermal hyperalgesia in mice is correlated with live bacterial load rather than tissue swelling or immune activation. Bacteria induce calcium flux and action potentials in nociceptor neurons, in part via bacterial N-formylated peptides and the pore-forming toxin α-haemolysin, through distinct mechanisms. Specific ablation of Nav1.8-lineage neurons, which include nociceptors, abrogated pain during bacterial infection, but concurrently increased local immune infiltration and lymphadenopathy of the draining lymph node. Thus, bacterial pathogens produce pain by directly activating sensory neurons that modulate inflammation, an unsuspected role for the nervous system in host-pathogen interactions.


Somatic mutations in the small GTPase K-Ras are the most common activating lesions found in human cancer, and are generally associated with poor response to standard therapies. Efforts to target this oncogene directly have faced difficulties owing to its picomolar affinity for GTP/GDP and the absence of known allosteric regulatory sites. Oncogenic mutations result in functional activation of Ras family proteins by impairing GTP hydrolysis. With diminished regulation by GTPase activity, the nucleotide state of Ras becomes more dependent on relative nucleotide affinity and concentration. This gives GTP an advantage over GDP and increases the proportion of active GTP-bound Ras. Here we report the development of small molecules that irreversibly bind to a common oncogenic mutant, K-Ras(G12C). These compounds rely on the mutant cysteine for binding and therefore do not affect the wild-type protein. Crystallographic studies reveal the formation of a new pocket that is not apparent in previous structures of Ras, beneath the effector binding switch-II region. Binding of these inhibitors to K-Ras(G12C) disrupts both switch-I and switch-II, subverting the native nucleotide preference to favour GDP over GTP and impairing binding to Raf. Our data provide structure-based validation of a new allosteric regulatory site on Ras that is targetable in a mutant-specific manner.


The most conspicuous event in the cell cycle is the alignment of chromosomes in metaphase. Chromosome alignment fosters faithful segregation through the formation of bi-oriented attachments of kinetochores to spindle microtubules. Notably, numerous kinetochore-microtubule (k-MT) attachment errors are present in early mitosis (prometaphase), and the persistence of those errors is the leading cause of chromosome mis-segregation in aneuploid human tumour cells that continually mis-segregate whole chromosomes and display chromosomal instability. How robust error correction is achieved in prometaphase to ensure error-free mitosis remains unknown. Here we show that k-MT attachments in prometaphase cells are considerably less stable than in metaphase cells. The switch to more stable k-MT attachments in metaphase requires the proteasome-dependent destruction of cyclin A in prometaphase. Persistent cyclin A expression prevents k-MT stabilization even in cells with aligned chromosomes. By contrast, k-MTs are prematurely stabilized in cyclin-A-deficient cells. Consequently, cells lacking cyclin A display higher rates of chromosome mis-segregation. Thus, the stability of k-MT attachments increases decisively in a coordinated fashion among all chromosomes as cells transit from prometaphase to metaphase. Cyclin A creates a cellular environment that promotes microtubule detachment from kinetochores in prometaphase to ensure efficient error correction and faithful chromosome segregation.


Spinal muscular atrophy is one of the most common inherited forms of neurological disease leading to infant mortality. Patients have selective loss of lower motor neurons resulting in muscle weakness, paralysis and often death. Although patient fibroblasts have been used extensively to study spinal muscular atrophy, motor neurons have a unique anatomy and physiology which may underlie their vulnerability to the disease process. Here we report the generation of induced pluripotent stem cells from skin fibroblast samples taken from a child with spinal muscular atrophy. These cells expanded robustly in culture, maintained the disease genotype and generated motor neurons that showed selective deficits compared to those derived from the child’s unaffected mother. This is the first study to show that human induced pluripotent stem cells can be used to model the specific pathology seen in a genetically inherited disease. As such, it represents a promising resource to study disease mechanisms, screen new drug compounds and develop new therapies.


An inflammatory response initiated by the NLRP3 inflammasome is triggered by a variety of situations of host ‘danger’, including infection and metabolic dysregulation. Previous studies suggested that NLRP3 inflammasome activity is negatively regulated by autophagy and positively regulated by reactive oxygen species (ROS) derived from an uncharacterized organelle. Here we show that mitophagy/autophagy blockade leads to the accumulation of damaged, ROS-generating mitochondria, and this in turn activates the NLRP3 inflammasome. Resting NLRP3 localizes to endoplasmic reticulum structures, whereas on inflammasome activation both NLRP3 and its adaptor ASC redistribute to the perinuclear space where they co-localize with endoplasmic reticulum and mitochondria organelle clusters. Notably, both ROS generation and inflammasome activation are suppressed when mitochondrial activity is dysregulated by inhibition of the voltage-dependent anion channel. This indicates that NLRP3 inflammasome senses mitochondrial dysfunction and may explain the frequent association of mitochondrial damage with inflammatory diseases.


Innate immune defences are essential for the control of virus infection and are triggered through host recognition of viral macromolecular motifs known as pathogen-associated molecular patterns (PAMPs). Hepatitis C virus (HCV) is an RNA virus that replicates in the liver, and infects 200 million people worldwide. Infection is regulated by hepatic immune defences triggered by the cellular RIG-I helicase. RIG-I binds PAMP RNA and signals interferon regulatory factor 3 activation to induce the expression of interferon-α/β and antiviral/interferon-stimulated genes (ISGs) that limit infection. Here we identify the polyuridine motif of the HCV genome 3′ non-translated region and its replication intermediate as the PAMP substrate of RIG-I, and show that this and similar homopolyuridine or homopolyriboadenine motifs present in the genomes of RNA viruses are the chief feature of RIG-I recognition and immune triggering in human and murine cells. 5′ terminal triphosphate on the PAMP RNA was necessary but not sufficient for RIG-I binding, which was primarily dependent on homopolymeric ribonucleotide composition, linear structure and length. The HCV PAMP RNA stimulated RIG-I-dependent signalling to induce a hepatic innate immune response in vivo, and triggered interferon and ISG expression to suppress HCV infection in vitro. These results provide a conceptual advance by defining specific homopolymeric RNA motifs within the genome of HCV and other RNA viruses as the PAMP substrate of RIG-I, and demonstrate immunogenic features of the PAMP–RIG-I interaction that could be used as an immune adjuvant for vaccine and immunotherapy approaches.


The common marmoset (Callithrix jacchus) is increasingly attractive for use as a non-human primate animal model in biomedical research. It has a relatively high reproduction rate for a primate, making it potentially suitable for transgenic modification. Although several attempts have been made to produce non-human transgenic primates, transgene expression in the somatic tissues of live infants has not been demonstrated by objective analyses such as polymerase chain reaction with reverse transcription or western blots. Here we show that the injection of a self-inactivating lentiviral vector in sucrose solution into marmoset embryos results in transgenic common marmosets that expressed the transgene in several organs. Notably, we achieved germline transmission of the transgene, and the transgenic offspring developed normally. The successful creation of transgenic marmosets provides a new animal model for human disease that has the great advantage of a close genetic relationship with humans. This model will be valuable to many fields of biomedical research.


Somatic cell nuclear transfer, cell fusion, or expression of lineage-specific factors have been shown to induce cell-fate changes in diverse somatic cell types. We recently observed that forced expression of a combination of three transcription factors, Brn2 (also known as Pou3f2), Ascl1 and Myt1l, can efficiently convert mouse fibroblasts into functional induced neuronal (iN) cells. Here we show that the same three factors can generate functional neurons from human pluripotent stem cells as early as 6 days after transgene activation. When combined with the basic helix-loop-helix transcription factor NeuroD1, these factors could also convert fetal and postnatal human fibroblasts into iN cells showing typical neuronal morphologies and expressing multiple neuronal markers, even after downregulation of the exogenous transcription factors. Importantly, the vast majority of human iN cells were able to generate action potentials and many matured to receive synaptic contacts when co-cultured with primary mouse cortical neurons. Our data demonstrate that non-neural human somatic cells, as well as pluripotent stem cells, can be converted directly into neurons by lineage-determining transcription factors. These methods may facilitate robust generation of patient-specific human neurons for in vitro disease modelling or future applications in regenerative medicine.


The bacterial type VI secretion system (T6SS) is a large multicomponent, dynamic macromolecular machine that has an important role in the ecology of many Gram-negative bacteria. T6SS is responsible for translocation of a wide range of toxic effector molecules, allowing predatory cells to kill both prokaryotic as well as eukaryotic prey cells. The T6SS organelle is functionally analogous to contractile tails of bacteriophages and is thought to attack cells by initially penetrating them with a trimeric protein complex called the VgrG spike. Neither the exact protein composition of the T6SS organelle nor the mechanisms of effector selection and delivery are known. Here we report that proteins from the PAAR (proline-alanine-alanine-arginine) repeat superfamily form a sharp conical extension on the VgrG spike, which is further involved in attaching effector domains to the spike. The crystal structures of two PAAR-repeat proteins bound to VgrG-like partners show that these proteins sharpen the tip of the T6SS spike complex. We demonstrate that PAAR proteins are essential for T6SS-mediated secretion and target cell killing by Vibrio cholerae and Acinetobacter baylyi. Our results indicate a new model of the T6SS organelle in which the VgrG-PAAR spike complex is decorated with multiple effectors that are delivered simultaneously into target cells in a single contraction-driven translocation event.


In pregnancy, trophoblast invasion and uterine spiral artery remodelling are important for lowering maternal vascular resistance and increasing uteroplacental blood flow. Impaired spiral artery remodelling has been implicated in pre-eclampsia, a major complication of pregnancy, for a long time but the underlying mechanisms remain unclear. Corin (also known as atrial natriuretic peptide-converting enzyme) is a cardiac protease that activates atrial natriuretic peptide (ANP), a cardiac hormone that is important in regulating blood pressure. Unexpectedly, corin expression was detected in the pregnant uterus. Here we identify a new function of corin and ANP in promoting trophoblast invasion and spiral artery remodelling. We show that pregnant corin- or ANP-deficient mice developed high blood pressure and proteinuria, characteristics of pre-eclampsia. In these mice, trophoblast invasion and uterine spiral artery remodelling were markedly impaired. Consistent with this, the ANP potently stimulated human trophoblasts in invading Matrigels. In patients with pre-eclampsia, uterine Corin messenger RNA and protein levels were significantly lower than that in normal pregnancies. Moreover, we have identified Corin gene mutations in pre-eclamptic patients, which decreased corin activity in processing pro-ANP. These results indicate that corin and ANP are essential for physiological changes at the maternal–fetal interface, suggesting that defects in corin and ANP function may contribute to pre-eclampsia.


Mitochondria have a major role in energy production via oxidative phosphorylation, which is dependent on the expression of critical genes encoded by mitochondrial (mt)DNA. Mutations in mtDNA can cause fatal or severely debilitating disorders with limited treatment options. Clinical manifestations vary based on mutation type and heteroplasmy (that is, the relative levels of mutant and wild-type mtDNA within each cell). Here we generated genetically corrected pluripotent stem cells (PSCs) from patients with mtDNA disease. Multiple induced pluripotent stem (iPS) cell lines were derived from patients with common heteroplasmic mutations including 3243A>G, causing mitochondrial encephalomyopathy and stroke-like episodes (MELAS), and 8993T>G and 13513G>A, implicated in Leigh syndrome. Isogenic MELAS and Leigh syndrome iPS cell lines were generated containing exclusively wild-type or mutant mtDNA through spontaneous segregation of heteroplasmic mtDNA in proliferating fibroblasts. Furthermore, somatic cell nuclear transfer (SCNT) enabled replacement of mutant mtDNA from homoplasmic 8993T>G fibroblasts to generate corrected Leigh-NT1 PSCs. Although Leigh-NT1 PSCs contained donor oocyte wild-type mtDNA (human haplotype D4a) that differed from Leigh syndrome patient haplotype (F1a) at a total of 47 nucleotide sites, Leigh-NT1 cells displayed transcriptomic profiles similar to those in embryo-derived PSCs carrying wild-type mtDNA, indicative of normal nuclear-to-mitochondrial interactions. Moreover, genetically rescued patient PSCs displayed normal metabolic function compared to impaired oxygen consumption and ATP production observed in mutant cells. We conclude that both reprogramming approaches offer complementary strategies for derivation of PSCs containing exclusively wild-type mtDNA, through spontaneous segregation of heteroplasmic mtDNA in individual iPS cell lines or mitochondrial replacement by SCNT in homoplasmic mtDNA-based disease.


Calorie restriction (CR) extends lifespan in a wide spectrum of organisms and is the only regimen known to lengthen the lifespan of mammals. We established a model of CR in budding yeast Saccharomyces cerevisiae. In this system, lifespan can be extended by limiting glucose or by reducing the activity of the glucose-sensing cyclic-AMP-dependent kinase (PKA). Lifespan extension in a mutant with reduced PKA activity requires Sir2 and NAD (nicotinamide adenine dinucleotide). In this study we explore how CR activates Sir2 to extend lifespan. Here we show that the shunting of carbon metabolism toward the mitochondrial tricarboxylic acid cycle and the concomitant increase in respiration play a central part in this process. We discuss how this metabolic strategy may apply to CR in animals.


Improved sequencing technologies offer unprecedented opportunities for investigating the role of rare genetic variation in common disease. However, there are considerable challenges with respect to study design, data analysis and replication. Using pooled next-generation sequencing of 507 genes implicated in the repair of DNA in 1,150 samples, an analytical strategy focused on protein-truncating variants (PTVs) and a large-scale sequencing case–control replication experiment in 13,642 individuals, here we show that rare PTVs in the p53-inducible protein phosphatase PPM1D are associated with predisposition to breast cancer and ovarian cancer. PPM1D PTV mutations were present in 25 out of 7,781 cases versus 1 out of 5,861 controls (P = 1.12 × 10−5), including 18 mutations in 6,912 individuals with breast cancer (P = 2.42 × 10−4) and 12 mutations in 1,121 individuals with ovarian cancer (P = 3.10 × 10−9). Notably, all of the identified PPM1D PTVs were mosaic in lymphocyte DNA and clustered within a 370-base-pair region in the final exon of the gene, carboxy-terminal to the phosphatase catalytic domain. Functional studies demonstrate that the mutations result in enhanced suppression of p53 in response to ionizing radiation exposure, suggesting that the mutant alleles encode hyperactive PPM1D isoforms. Thus, although the mutations cause premature protein truncation, they do not result in the simple loss-of-function effect typically associated with this class of variant, but instead probably have a gain-of-function effect. Our results have implications for the detection and management of breast and ovarian cancer risk. More generally, these data provide new insights into the role of rare and of mosaic genetic variants in common conditions, and the use of sequencing in their identification.


Induced pluripotent stem cells (iPSCs), reprogrammed from somatic cells with defined factors, hold great promise for regenerative medicine as the renewable source of autologous cells. Whereas it has been generally assumed that these autologous cells should be immune-tolerated by the recipient from whom the iPSCs are derived, their immunogenicity has not been vigorously examined. We show here that, whereas embryonic stem cells (ESCs) derived from inbred C57BL/6 (B6) mice can efficiently form teratomas in B6 mice without any evident immune rejection, the allogeneic ESCs from 129/SvJ mice fail to form teratomas in B6 mice due to rapid rejection by recipients. B6 mouse embryonic fibroblasts (MEFs) were reprogrammed into iPSCs by either retroviral approach (ViPSCs) or a novel episomal approach (EiPSCs) that causes no genomic integration. In contrast to B6 ESCs, teratomas formed by B6 ViPSCs were mostly immune-rejected by B6 recipients. In addition, the majority of teratomas formed by B6 EiPSCs were immunogenic in B6 mice with T cell infiltration, and apparent tissue damage and regression were observed in a small fraction of teratomas. Global gene expression analysis of teratomas formed by B6 ESCs and EiPSCs revealed a number of genes frequently overexpressed in teratomas derived from EiPSCs, and several such gene products were shown to contribute directly to the immunogenicity of the B6 EiPSC-derived cells in B6 mice. These findings indicate that, in contrast to derivatives of ESCs, abnormal gene expression in some cells differentiated from iPSCs can induce T-cell-dependent immune response in syngeneic recipients. Therefore, the immunogenicity of therapeutically valuable cells derived from patient-specific iPSCs should be evaluated before any clinic application of these autologous cells into the patients.


How instructive cues present on the cell surface have their precise effects on the actin cytoskeleton is poorly understood. Semaphorins are one of the largest families of these instructive cues and are widely studied for their effects on cell movement, navigation, angiogenesis, immunology and cancer. Semaphorins/collapsins were characterized in part on the basis of their ability to drastically alter actin cytoskeletal dynamics in neuronal processes, but despite considerable progress in the identification of semaphorin receptors and their signalling pathways, the molecules linking them to the precise control of cytoskeletal elements remain unknown. Recently, highly unusual proteins of the Mical family of enzymes have been found to associate with the cytoplasmic portion of plexins, which are large cell-surface semaphorin receptors, and to mediate axon guidance, synaptogenesis, dendritic pruning and other cell morphological changes. Mical enzymes perform reduction–oxidation (redox) enzymatic reactions and also contain domains found in proteins that regulate cell morphology. However, nothing is known of the role of Mical or its redox activity in mediating morphological changes. Here we report that Mical directly links semaphorins and their plexin receptors to the precise control of actin filament (F-actin) dynamics. We found that Mical is both necessary and sufficient for semaphorin–plexin-mediated F-actin reorganization in vivo. Likewise, we purified Mical protein and found that it directly binds F-actin and disassembles both individual and bundled actin filaments. We also found that Mical utilizes its redox activity to alter F-actin dynamics in vivo and in vitro, indicating a previously unknown role for specific redox signalling events in actin cytoskeletal regulation. Mical therefore is a novel F-actin-disassembly factor that provides a molecular conduit through which actin reorganization—a hallmark of cell morphological changes including axon navigation—can be precisely achieved spatiotemporally in response to semaphorins.


Regulatory T (Treg) cells, characterized by expression of the transcription factor forkhead box P3 (Foxp3), maintain immune homeostasis by suppressing self-destructive immune responses. Foxp3 operates as a late-acting differentiation factor controlling Treg cell homeostasis and function, whereas the early Treg-cell-lineage commitment is regulated by the Akt kinase and the forkhead box O (Foxo) family of transcription factors. However, whether Foxo proteins act beyond the Treg-cell-commitment stage to control Treg cell homeostasis and function remains largely unexplored. Here we show that Foxo1 is a pivotal regulator of Treg cell function. Treg cells express high amounts of Foxo1 and display reduced T-cell-receptor-induced Akt activation, Foxo1 phosphorylation and Foxo1 nuclear exclusion. Mice with Treg-cell-specific deletion of Foxo1 develop a fatal inflammatory disorder similar in severity to that seen in Foxp3-deficient mice, but without the loss of Treg cells. Genome-wide analysis of Foxo1 binding sites reveals ∼300 Foxo1-bound target genes, including the pro-inflammatory cytokine Ifng, that do not seem to be directly regulated by Foxp3. These findings show that the evolutionarily ancient Akt–Foxo1 signalling module controls a novel genetic program indispensable for Treg cell function.


In mammals, enlargement of the heart during embryonic development is primarily dependent on the increase in cardiomyocyte numbers. Shortly after birth, however, cardiomyocytes stop proliferating and further growth of the myocardium occurs through hypertrophic enlargement of the existing myocytes. As a consequence of the minimal renewal of cardiomyocytes during adult life, repair of cardiac damage through myocardial regeneration is very limited. Here we show that the exogenous administration of selected microRNAs (miRNAs) markedly stimulates cardiomyocyte proliferation and promotes cardiac repair. We performed a high-content microscopy, high-throughput functional screening for human miRNAs that promoted neonatal cardiomyocyte proliferation using a whole-genome miRNA library. Forty miRNAs strongly increased both DNA synthesis and cytokinesis in neonatal mouse and rat cardiomyocytes. Two of these miRNAs (hsa-miR-590 and hsa-miR-199a) were further selected for testing and were shown to promote cell cycle re-entry of adult cardiomyocytes ex vivo and to promote cardiomyocyte proliferation in both neonatal and adult animals. After myocardial infarction in mice, these miRNAs stimulated marked cardiac regeneration and almost complete recovery of cardiac functional parameters. The miRNAs identified hold great promise for the treatment of cardiac pathologies consequent to cardiomyocyte loss.


Spinal reflexes are mediated by synaptic connections between sensory afferents and motor neurons. The organization of these circuits shows several levels of specificity. Only certain classes of proprioceptive sensory neurons make direct, monosynaptic connections with motor neurons. Those that do are bound by rules of motor pool specificity: they form strong connections with motor neurons supplying the same muscle, but avoid motor pools supplying antagonistic muscles. This pattern of connectivity is initially accurate and is maintained in the absence of activity, implying that wiring specificity relies on the matching of recognition molecules on the surface of sensory and motor neurons. However, determinants of fine synaptic specificity here, as in most regions of the central nervous system, have yet to be defined. To address the origins of synaptic specificity in these reflex circuits we have used molecular genetic methods to manipulate recognition proteins expressed by subsets of sensory and motor neurons. We show here that a recognition system involving expression of the class 3 semaphorin Sema3e by selected motor neuron pools, and its high-affinity receptor plexin D1 (Plxnd1) by proprioceptive sensory neurons, is a critical determinant of synaptic specificity in sensory–motor circuits in mice. Changing the profile of Sema3e–Plxnd1 signalling in sensory or motor neurons results in functional and anatomical rewiring of monosynaptic connections, but does not alter motor pool specificity. Our findings indicate that patterns of monosynaptic connectivity in this prototypic central nervous system circuit are constructed through a recognition program based on repellent signalling.


The development of an oncogenic state is a complex process involving the accumulation of multiple independent mutations that lead to deregulation of cell signalling pathways central to the control of cell growth and cell fate. The ability to define cancer subtypes, recurrence of disease and response to specific therapies using DNA microarray-based gene expression signatures has been demonstrated in multiple studies. Various studies have also demonstrated the potential for using gene expression profiles for the analysis of oncogenic pathways. Here we show that gene expression signatures can be identified that reflect the activation status of several oncogenic pathways. When evaluated in several large collections of human cancers, these gene expression signatures identify patterns of pathway deregulation in tumours and clinically relevant associations with disease outcomes. Combining signature-based predictions across several pathways identifies coordinated patterns of pathway deregulation that distinguish between specific cancers and tumour subtypes. Clustering tumours based on pathway signatures further defines prognosis in respective patient subsets, demonstrating that patterns of oncogenic pathway deregulation underlie the development of the oncogenic phenotype and reflect the biology and outcome of specific cancers. Predictions of pathway deregulation in cancer cell lines are also shown to predict the sensitivity to therapeutic agents that target components of the pathway. Linking pathway deregulation with sensitivity to therapeutics that target components of the pathway provides an opportunity to make use of these oncogenic pathway signatures to guide the use of targeted therapeutics.


Emerging evidence suggests that pulmonary iron accumulation is implicated in a spectrum of chronic lung diseases. However, the mechanism(s) involved in pulmonary iron deposition and its role in the in vivo pathogenesis of lung diseases remains unknown. Here we show that a point mutation in the murine ferroportin gene, which causes hereditary hemochromatosis type 4 (Slc40a1C326S), increases iron levels in alveolar macrophages, epithelial cells lining the conducting airways and lung parenchyma, and in vascular smooth muscle cells. Pulmonary iron overload is associated with oxidative stress, restrictive lung disease with decreased total lung capacity and reduced blood oxygen saturation in homozygous Slc40a1C326S/C326S mice compared to wild-type controls. These findings implicate iron in lung pathology, which is so far not considered a classical iron-related disorder.


Haematopoietic stem cells (HSCs) and their subsequent progenitors produce blood cells, but the precise nature and kinetics of this production is a contentious issue. In one model, lymphoid and myeloid production branch after the lymphoid-primed multipotent progenitor (LMPP), with both branches subsequently producing dendritic cells. However, this model is based mainly on in vitro clonal assays and population-based tracking in vivo, which could miss in vivo single-cell complexity. Here we avoid these issues by using a new quantitative version of ‘cellular barcoding’ to trace the in vivo fate of hundreds of LMPPs and HSCs at the single-cell level. These data demonstrate that LMPPs are highly heterogeneous in the cell types that they produce, separating into combinations of lymphoid-, myeloid- and dendritic-cell-biased producers. Conversely, although we observe a known lineage bias of some HSCs, most cellular output is derived from a small number of HSCs that each generates all cell types. Crucially, in vivo analysis of the output of sibling cells derived from single LMPPs shows that they often share a similar fate, suggesting that the fate of these progenitors was imprinted. Furthermore, as this imprinting is also observed for dendritic-cell-biased LMPPs, dendritic cells may be considered a distinct lineage on the basis of separate ancestry. These data suggest a ‘graded commitment’ model of haematopoiesis, in which heritable and diverse lineage imprinting occurs earlier than previously thought.


Intestinal cancer is initiated by Wnt-pathway-activating mutations in genes such as adenomatous polyposis coli (APC). As in most cancers, the cell of origin has remained elusive. In a previously established Lgr5 (leucine-rich-repeat containing G-protein-coupled receptor 5) knockin mouse model, a tamoxifen-inducible Cre recombinase is expressed in long-lived intestinal stem cells. Here we show that deletion of Apc in these stem cells leads to their transformation within days. Transformed stem cells remain located at crypt bottoms, while fuelling a growing microadenoma. These microadenomas show unimpeded growth and develop into macroscopic adenomas within 3-5weeks. The distribution of Lgr5+ cells within stem-cell-derived adenomas indicates that a stem cell/progenitor cell hierarchy is maintained in early neoplastic lesions. When Apc is deleted in short-lived transit-amplifying cells using a different cre mouse, the growth of the induced microadenomas rapidly stalls. Even after 30weeks, large adenomas are very rare in these mice. We conclude that stem-cell-specific loss of Apc results in progressively growing neoplasia.


Macrophages activated by the Gram-negative bacterial product lipopolysaccharide switch their core metabolism from oxidative phosphorylation to glycolysis. Here we show that inhibition of glycolysis with 2-deoxyglucose suppresses lipopolysaccharide-induced interleukin-1β but not tumour-necrosis factor-α in mouse macrophages. A comprehensive metabolic map of lipopolysaccharide-activated macrophages shows upregulation of glycolytic and downregulation of mitochondrial genes, which correlates directly with the expression profiles of altered metabolites. Lipopolysaccharide strongly increases the levels of the tricarboxylic-acid cycle intermediate succinate. Glutamine-dependent anerplerosis is the principal source of succinate, although the ‘GABA (γ-aminobutyric acid) shunt’ pathway also has a role. Lipopolysaccharide-induced succinate stabilizes hypoxia-inducible factor-1α, an effect that is inhibited by 2-deoxyglucose, with interleukin-1β as an important target. Lipopolysaccharide also increases succinylation of several proteins. We therefore identify succinate as a metabolite in innate immune signalling, which enhances interleukin-1β production during inflammation.


Environmental and genetic aberrations lead to neural tube closure defects (NTDs) in 1 out of every 1,000 births. Mouse and frog models for these birth defects have indicated that Van Gogh-like 2 (Vangl2, also known as Strabismus) and other components of planar cell polarity (PCP) signalling might control neurulation by promoting the convergence of neural progenitors to the midline. Here we show a novel role for PCP signalling during neurulation in zebrafish. We demonstrate that non-canonical Wnt/PCP signalling polarizes neural progenitors along the anteroposterior axis. This polarity is transiently lost during cell division in the neural keel but is re-established as daughter cells reintegrate into the neuroepithelium. Loss of zebrafish Vangl2 (in trilobite mutants) abolishes the polarization of neural keel cells, disrupts re-intercalation of daughter cells into the neuroepithelium, and results in ectopic neural progenitor accumulations and NTDs. Remarkably, blocking cell division leads to rescue of trilobite neural tube morphogenesis despite persistent defects in convergence and extension. These results reveal a function for PCP signalling in coupling cell division and morphogenesis at neurulation and indicate a previously unrecognized mechanism that might underlie NTDs.


Crohn's disease and ulcerative colitis, the two main types of chronic inflammatory bowel disease, are multifactorial conditions of unknown aetiology. A susceptibility locus for Crohn's disease has been mapped to chromosome 16. Here we have used a positional-cloning strategy, based on linkage analysis followed by linkage disequilibrium mapping, to identify three independent associations for Crohn's disease: a frameshift variant and two missense variants of NOD2, encoding a member of the Apaf-1/Ced-4 superfamily of apoptosis regulators that is expressed in monocytes. These NOD2 variants alter the structure of either the leucine-rich repeat domain of the protein or the adjacent region. NOD2 activates nuclear factor NF-kB; this activating function is regulated by the carboxy-terminal leucine-rich repeat domain, which has an inhibitory role and also acts as an intracellular receptor for components of microbial pathogens. These observations suggest that the NOD2 gene product confers susceptibility to Crohn's disease by altering the recognition of these components and/or by over-activating NF-kB in monocytes, thus documenting a molecular model for the pathogenic mechanism of Crohn's disease that can now be further investigated.


The functional heart is comprised of distinct mesoderm-derived lineages including cardiomyocytes, endothelial cells and vascular smooth muscle cells. Studies in the mouse embryo and the mouse embryonic stem cell differentiation model have provided evidence indicating that these three lineages develop from a common Flk-1+ (kinase insert domain protein receptor, also known as Kdr) cardiovascular progenitor that represents one of the earliest stages in mesoderm specification to the cardiovascular lineages. To determine whether a comparable progenitor is present during human cardiogenesis, we analysed the development of the cardiovascular lineages in human embryonic stem cell differentiation cultures. Here we show that after induction with combinations of activin A, bone morphogenetic protein 4 (BMP4), basic fibroblast growth factor (bFGF, also known as FGF2), vascular endothelial growth factor (VEGF, also known as VEGFA) and dickkopf homolog 1 (DKK1) in serum-free media, human embryonic-stem-cell-derived embryoid bodies generate a KDRlow/C-KIT(CD117)neg population that displays cardiac, endothelial and vascular smooth muscle potential in vitro and, after transplantation, in vivo. When plated in monolayer cultures, these KDRlow/C-KITneg cells differentiate to generate populations consisting of greater than 50% contracting cardiomyocytes. Populations derived from the KDRlow/C-KITneg fraction give rise to colonies that contain all three lineages when plated in methylcellulose cultures. Results from limiting dilution studies and cell-mixing experiments support the interpretation that these colonies are clones, indicating that they develop from a cardiovascular colony-forming cell. Together, these findings identify a human cardiovascular progenitor that defines one of the earliest stages of human cardiac development.


The mediators and cellular effectors of inflammation are important constituents of the local environment of tumours. In some types of cancer, inflammatory conditions are present before a malignant change occurs. Conversely, in other types of cancer, an oncogenic change induces an inflammatory microenvironment that promotes the development of tumours. Regardless of its origin, 'smouldering' inflammation in the tumour microenvironment has many tumour-promoting effects. It aids in the proliferation and survival of malignant cells, promotes angiogenesis and metastasis, subverts adaptive immune responses, and alters responses to hormones and chemotherapeutic agents. The molecular pathways of this cancer-related inflammation are now being unravelled, resulting in the identification of new target molecules that could lead to improved diagnosis and treatment.


The ESCRT (endosomal sorting complex required for transport) pathway is required for terminal membrane fission events in several important biological processes, including endosomal intraluminal vesicle formation, HIV budding and cytokinesis. VPS4 ATPases perform a key function in this pathway by recognizing membrane-associated ESCRT-III assemblies and catalysing their disassembly, possibly in conjunction with membrane fission. Here we show that the microtubule interacting and transport (MIT) domains of human VPS4A and VPS4B bind conserved sequence motifs located at the carboxy termini of the CHMP1–3 class of ESCRT-III proteins. Structures of VPS4A MIT–CHMP1A and VPS4B MIT–CHMP2B complexes reveal that the C-terminal CHMP motif forms an amphipathic helix that binds in a groove between the last two helices of the tetratricopeptide-like repeat (TPR) of the VPS4 MIT domain, but in the opposite orientation to that of a canonical TPR interaction. Distinct pockets in the MIT domain bind three conserved leucine residues of the CHMP motif, and mutations that inhibit these interactions block VPS4 recruitment, impair endosomal protein sorting and relieve dominant-negative VPS4 inhibition of HIV budding. Thus, our studies reveal how the VPS4 ATPases recognize their CHMP substrates to facilitate the membrane fission events required for the release of viruses, endosomal vesicles and daughter cells.


Metastasis is a multistep process responsible for most cancer deaths, and it can be influenced by both the immediate microenvironment (cell–cell or cell–matrix interactions) and the extended tumour microenvironment (for example vascularization). Hypoxia (low oxygen) is clinically associated with metastasis and poor patient outcome, although the underlying processes remain unclear. Microarray studies have shown the expression of lysyl oxidase (LOX) to be elevated in hypoxic human tumour cells. Paradoxically, LOX expression is associated with both tumour suppression and tumour progression, and its role in tumorigenesis seems dependent on cellular location, cell type and transformation status. Here we show that LOX expression is regulated by hypoxia-inducible factor (HIF) and is associated with hypoxia in human breast and head and neck tumours. Patients with high LOX-expressing tumours have poor distant metastasis-free and overall survivals. Inhibition of LOX eliminates metastasis in mice with orthotopically grown breast cancer tumours. Mechanistically, secreted LOX is responsible for the invasive properties of hypoxic human cancer cells through focal adhesion kinase activity and cell to matrix adhesion. Furthermore, LOX may be required to create a niche permissive for metastatic growth. Our findings indicate that LOX is essential for hypoxia-induced metastasis and is a good therapeutic target for preventing and treating metastases.


The cancer stem cell (CSC) hypothesis suggests that neoplastic clones are maintained exclusively by a rare fraction of cells with stem cell properties. Although the existence of CSCs in human leukaemia is established, little evidence exists for CSCs in solid tumours, except for breast cancer. Recently, we prospectively isolated a CD133+ cell subpopulation from human brain tumours that exhibited stem cell properties in vitro. However, the true measures of CSCs are their capacity for self renewal and exact recapitulation of the original tumour. Here we report the development of a xenograft assay that identified human brain tumour initiating cells that initiate tumours in vivo. Only the CD133+ brain tumour fraction contains cells that are capable of tumour initiation in NOD-SCID (non-obese diabetic, severe combined immunodeficient) mouse brains. Injection of as few as 100 CD133+ cells produced a tumour that could be serially transplanted and was a phenocopy of the patient's original tumour, whereas injection of 105 CD133- cells engrafted but did not cause a tumour. Thus, the identification of brain tumour initiating cells provides insights into human brain tumour pathogenesis, giving strong support for the CSC hypothesis as the basis for many solid tumours, and establishes a previously unidentified cellular target for more effective cancer therapies.


The World Health Organization (WHO) convened an Expert Committee to reevaluate the use of anthropometry at different ages for assessing health, nutrition, and social wellbeing. The Committee's task included identifying reference data for anthropometric indexes when appropriate, and providing guidelines on how the data should be used. For fetal growth, the Committee recommended an existing sex-specific multiracial reference. In view of the significant technical drawbacks of the current National Center for Health Statistics (NCHS)/WHO reference and its inadequacy for assessing the growth of breast-fed infants, the Committee recommended the development of a new reference concerning weight and length/height for infants and children, which will be a complex and costly undertaking. Proper interpretation of midupper arm circumference for preschoolers requires age-specific reference data. To evaluate adolescent height-for-age, the Committee recommended the current NCHS/WHO reference. Use of the NCHS body mass index (BMI) data, with their upper percentile elevations and skewness, is undesirable for setting health goals; however, these data were provisionally recommended for defining obesity based on a combination of elevated BMI and high subcutaneous fat. The NCHS values were provisionally recommended as reference data for subscapular and triceps skinfold thicknesses. Guidelines were also provided for adjusting adolescent anthropometric comparisons for maturational status. Currently, there is no need for adult reference data for BMI; interpretation should be based on pragmatic BMI cutoffs. Finally, the Committee noted that few normative anthropometric data exist for the elderly, especially for those > 80 y of age. Proper definitions of health status, function, and biologic age remain to be developed for this group.


BACKGROUND Age-associated epigenetic changes are implicated in aging. Notably, age-associated DNA methylation changes comprise a so-called aging "clock", a robust biomarker of aging. However, while genetic, dietary and drug interventions can extend lifespan, their impact on the epigenome is uncharacterised. To fill this knowledge gap, we defined age-associated DNA methylation changes at the whole-genome, single-nucleotide level in mouse liver and tested the impact of longevity-promoting interventions, specifically the Ames dwarf Prop1 df/df mutation, calorie restriction and rapamycin. RESULTS In wild-type mice fed an unsupplemented ad libitum diet, age-associated hypomethylation was enriched at super-enhancers in highly expressed genes critical for liver function. Genes harbouring hypomethylated enhancers were enriched for genes that change expression with age. Hypermethylation was enriched at CpG islands marked with bivalent activating and repressing histone modifications and resembled hypermethylation in liver cancer. Age-associated methylation changes are suppressed in Ames dwarf and calorie restricted mice and more selectively and less specifically in rapamycin treated mice. CONCLUSIONS Age-associated hypo- and hypermethylation events occur at distinct regulatory features of the genome. Distinct longevity-promoting interventions, specifically genetic, dietary and drug interventions, suppress some age-associated methylation changes, consistent with the idea that these interventions exert their beneficial effects, in part, by modulation of the epigenome. This study is a foundation to understand the epigenetic contribution to healthy aging and longevity and the molecular basis of the DNA methylation clock.


BACKGROUND Soy protein or its components may protect against the atherosclerotic cardiovascular disease (CVD) risk factors total homocysteine (tHcy), C-reactive protein (CRP), and excess body iron, which generally increase with menopause. OBJECTIVE The primary objective of this study was to determine the independent effect of the soy protein components isoflavones and phytate on CVD risk factors in postmenopausal women. The secondary objective was to identify factors [blood lipids, oxidative stress indexes, serum ferritin, plasma folate, plasma vitamin B-12, and body mass index (BMI)] contributing to tHcy and CRP concentrations. DESIGN In a double-blind, 6-wk study, 55 postmenopausal women aged 47-72 y were randomly assigned to 1 of 4 soy protein (40 g/d) isolate treatments: native phytate and native isoflavone (n = 14), native phytate and low isoflavone (n = 13), low phytate and native isoflavone (n = 14), or low phytate and low isoflavone (n = 14). We measured iron indexes, tHcy, CRP, and BMI. RESULTS Soy protein with native phytate significantly reduced tHcy (P = 0.017), transferrin saturation (P = 0.027), and ferritin (P = 0.029), whereas soy protein with native isoflavones had no effect on any variables. At baseline, BMI was highly correlated with tHcy (r = 0.39, P = 0.003) and CRP (r = 0.55, P < 0.0001), whereas HDL cholesterol was correlated with CRP (r = -0.30, P = 0.02). Multiple regression analysis showed that LDL cholesterol and BMI contributed significantly (R2= 19.9%, P = 0.003) to the overall variance in tHcy. CONCLUSION Consuming phytate-rich foods and maintaining a healthy weight may reduce atherosclerotic CVD risk factors in postmenopausal women.


Cells deficient in the Brca1 and Brca2 genes have reduced capacity to repair DNA double-strand breaks by homologous recombination and consequently are hypersensitive to DNA-damaging agents, including cisplatin and poly(ADP-ribose) polymerase (PARP) inhibitors. Here we show that loss of the MLL3/4 complex protein, PTIP, protects Brca1/2-deficient cells from DNA damage and rescues the lethality of Brca2-deficient embryonic stem cells. However, PTIP deficiency does not restore homologous recombination activity at double-strand breaks. Instead, its absence inhibits the recruitment of the MRE11 nuclease to stalled replication forks, which in turn protects nascent DNA strands from extensive degradation. More generally, acquisition of PARP inhibitors and cisplatin resistance is associated with replication fork protection in Brca2-deficient tumour cells that do not develop Brca2 reversion mutations. Disruption of multiple proteins, including PARP1 and CHD4, leads to the same end point of replication fork protection, highlighting the complexities by which tumour cells evade chemotherapeutic interventions and acquire drug resistance.


OBJECTIVES The aim of this study was to determine the prognostic value of plasma corin in patients with chronic heart failure (CHF). BACKGROUND In recent years, accumulating evidence has indicated that corin plays a critical role in regulating blood pressure and cardiac function. METHODS We enrolled 1,148 consecutive CHF patients in a prospective cohort study and explored the association between plasma corin levels and clinical prognosis using multivariate Cox regression analysis. RESULTS Patients with low corin levels (<458 pg/ml) were more likely to be women and to be hypertensive. Low corin was found to be associated with an increase in New York Heart Association (NYHA) functional class and N-terminal pro-B-type natriuretic peptide (NT-proBNP) levels, and a decrease in left ventricular ejection fraction (LVEF) and the estimated glomerular filtration rate (eGFR). Multivariate Cox regression analysis suggested that log corin was an independent predictor of major adverse cardiac event(s) (MACE) (hazard ratio: 0.62; 95% confidence interval: 0.39 to 0.95), together with age, diabetes, NYHA functional class, LVEF, eGFR, and log NT-proBNP. In addition, log corin was also a significant predictor for cardiovascular death (p = 0.041) and heart failure rehospitalization (p = 0.015) after adjustment for clinical variables and established biomarkers of adverse prognosis. The Kaplan-Meier survival curves showed that low corin was a significant predictor of MACE in patients with NT-proBNP levels above and below the median. CONCLUSIONS Our study demonstrates that plasma corin is a valuable prognostic marker of MACE in patients with CHF, independent of established conventional risk factors.


Alzheimer's disease is the most common neurodegenerative disease, and there are no mechanism-based therapies. The disease is defined by the presence of abundant neurofibrillary lesions and neuritic plaques in the cerebral cortex. Neurofibrillary lesions comprise paired helical and straight tau filaments, whereas tau filaments with different morphologies characterize other neurodegenerative diseases. No high-resolution structures of tau filaments are available. Here we present cryo-electron microscopy (cryo-EM) maps at 3.4-3.5 Å resolution and corresponding atomic models of paired helical and straight filaments from the brain of an individual with Alzheimer's disease. Filament cores are made of two identical protofilaments comprising residues 306-378 of tau protein, which adopt a combined cross-β/β-helix structure and define the seed for tau aggregation. Paired helical and straight filaments differ in their inter-protofilament packing, showing that they are ultrastructural polymorphs. These findings demonstrate that cryo-EM allows atomic characterization of amyloid filaments from patient-derived material, and pave the way for investigation of a range of neurodegenerative diseases.


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 phosphatase σ (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.


Inflammation promotes regeneration of injured tissues through poorly understood mechanisms, some of which involve interleukin (IL)-6 family members, the expression of which is elevated in many diseases including inflammatory bowel diseases and colorectal cancer. Here we show in mice and human cells that gp130, a co-receptor for IL-6 cytokines, triggers activation of YAP and Notch, transcriptional regulators that control tissue growth and regeneration, independently of the gp130 effector STAT3. Through YAP and Notch, intestinal gp130 signalling stimulates epithelial cell proliferation, causes aberrant differentiation and confers resistance to mucosal erosion. gp130 associates with the related tyrosine kinases Src and Yes, which are activated on receptor engagement to phosphorylate YAP and induce its stabilization and nuclear translocation. This signalling module is strongly activated upon mucosal injury to promote healing and maintain barrier function.


Pluripotency is defined by the ability of a cell to differentiate to the derivatives of all the three embryonic germ layers: ectoderm, mesoderm and endoderm. Pluripotent cells can be captured via the archetypal derivation of embryonic stem cells or via somatic cell reprogramming. Somatic cells are induced to acquire a pluripotent stem cell (iPSC) state through the forced expression of key transcription factors, and in the mouse these cells can fulfil the strictest of all developmental assays for pluripotent cells by generating completely iPSC-derived embryos and mice. However, it is not known whether there are additional classes of pluripotent cells, or what the spectrum of reprogrammed phenotypes encompasses. Here we explore alternative outcomes of somatic reprogramming by fully characterizing reprogrammed cells independent of preconceived definitions of iPSC states. We demonstrate that by maintaining elevated reprogramming factor expression levels, mouse embryonic fibroblasts go through unique epigenetic modifications to arrive at a stable, Nanog-positive, alternative pluripotent state. In doing so, we prove that the pluripotent spectrum can encompass multiple, unique cell states.


Macroautophagy (hereafter referred to as autophagy) is a catabolic membrane trafficking process that degrades a variety of cellular constituents and is associated with human diseases. Although extensive studies have focused on autophagic turnover of cytoplasmic materials, little is known about the role of autophagy in degrading nuclear components. Here we report that the autophagy machinery mediates degradation of nuclear lamina components in mammals. The autophagy protein LC3/Atg8, which is involved in autophagy membrane trafficking and substrate delivery, is present in the nucleus and directly interacts with the nuclear lamina protein lamin B1, and binds to lamin-associated domains on chromatin. This LC3-lamin B1 interaction does not downregulate lamin B1 during starvation, but mediates its degradation upon oncogenic insults, such as by activated RAS. Lamin B1 degradation is achieved by nucleus-to-cytoplasm transport that delivers lamin B1 to the lysosome. Inhibiting autophagy or the LC3-lamin B1 interaction prevents activated RAS-induced lamin B1 loss and attenuates oncogene-induced senescence in primary human cells. Our study suggests that this new function of autophagy acts as a guarding mechanism protecting cells from tumorigenesis.


Advances in our understanding of the mechanisms that bring about the resolution of acute inflammation have uncovered a new genus of pro-resolving lipid mediators that include the lipoxin, resolvin, protectin and maresin families, collectively called specialized pro-resolving mediators. Synthetic versions of these mediators have potent bioactions when administered in vivo. In animal experiments, the mediators evoke anti-inflammatory and novel pro-resolving mechanisms, and enhance microbial clearance. Although they have been identified in inflammation resolution, specialized pro-resolving mediators are conserved structures that also function in host defence, pain, organ protection and tissue remodelling. This Review covers the mechanisms of specialized pro-resolving mediators and omega-3 essential fatty acid pathways that could help us to understand their physiological functions.


Pancreatic cancer remains one of the most lethal of malignancies and a major health burden. We performed whole-genome sequencing and copy number variation (CNV) analysis of 100 pancreatic ductal adenocarcinomas (PDACs). Chromosomal rearrangements leading to gene disruption were prevalent, affecting genes known to be important in pancreatic cancer (TP53, SMAD4, CDKN2A, ARID1A and ROBO2) and new candidate drivers of pancreatic carcinogenesis (KDM6A and PREX2). Patterns of structural variation (variation in chromosomal structure) classified PDACs into 4 subtypes with potential clinical utility: the subtypes were termed stable, locally rearranged, scattered and unstable. A significant proportion harboured focal amplifications, many of which contained druggable oncogenes (ERBB2, MET, FGFR1, CDK6, PIK3R3 and PIK3CA), but at low individual patient prevalence. Genomic instability co-segregated with inactivation of DNA maintenance genes (BRCA1, BRCA2 or PALB2) and a mutational signature of DNA damage repair deficiency. Of 8 patients who received platinum therapy, 4 of 5 individuals with these measures of defective DNA maintenance responded.


The transfer of somatic cell nuclei into oocytes can give rise to pluripotent stem cells that are consistently equivalent to embryonic stem cells, holding promise for autologous cell replacement therapy. Although methods to induce pluripotent stem cells from somatic cells by transcription factors are widely used in basic research, numerous differences between induced pluripotent stem cells and embryonic stem cells have been reported, potentially affecting their clinical use. Because of the therapeutic potential of diploid embryonic stem-cell lines derived from adult cells of diseased human subjects, we have systematically investigated the parameters affecting efficiency of blastocyst development and stem-cell derivation. Here we show that improvements to the oocyte activation protocol, including the use of both kinase and translation inhibitors, and cell culture in the presence of histone deacetylase inhibitors, promote development to the blastocyst stage. Developmental efficiency varied between oocyte donors, and was inversely related to the number of days of hormonal stimulation required for oocyte maturation, whereas the daily dose of gonadotropin or the total number of metaphase II oocytes retrieved did not affect developmental outcome. Because the use of concentrated Sendai virus for cell fusion induced an increase in intracellular calcium concentration, causing premature oocyte activation, we used diluted Sendai virus in calcium-free medium. Using this modified nuclear transfer protocol, we derived diploid pluripotent stem-cell lines from somatic cells of a newborn and, for the first time, an adult, a female with type 1 diabetes.


Endothelial cells contribute to a subset of cardiac fibroblasts by undergoing endothelial-to-mesenchymal transition, but whether cardiac fibroblasts can adopt an endothelial cell fate and directly contribute to neovascularization after cardiac injury is not known. Here, using genetic fate map techniques, we demonstrate that cardiac fibroblasts rapidly adopt an endothelial-cell-like phenotype after acute ischaemic cardiac injury. Fibroblast-derived endothelial cells exhibit anatomical and functional characteristics of native endothelial cells. We show that the transcription factor p53 regulates such a switch in cardiac fibroblast fate. Loss of p53 in cardiac fibroblasts severely decreases the formation of fibroblast-derived endothelial cells, reduces post-infarct vascular density and worsens cardiac function. Conversely, stimulation of the p53 pathway in cardiac fibroblasts augments mesenchymal-to-endothelial transition, enhances vascularity and improves cardiac function. These observations demonstrate that mesenchymal-to-endothelial transition contributes to neovascularization of the injured heart and represents a potential therapeutic target for enhancing cardiac repair.


Recent evidence suggests that vitamin D intakes above current recommendations may be associated with better health outcomes. However, optimal serum concentrations of 25-hydroxyvitamin D [25(OH)D] have not been defined. This review summarizes evidence from studies that evaluated thresholds for serum 25(OH)D concentrations in relation to bone mineral density (BMD), lower-extremity function, dental health, and risk of falls, fractures, and colorectal cancer. For all endpoints, the most advantageous serum concentrations of 25(OH)D begin at 75 nmol/L (30 ng/mL), and the best are between 90 and 100 nmol/L (36-40 ng/mL). In most persons, these concentrations could not be reached with the currently recommended intakes of 200 and 600 IU vitamin D/d for younger and older adults, respectively. A comparison of vitamin D intakes with achieved serum concentrations of 25(OH)D for the purpose of estimating optimal intakes led us to suggest that, for bone health in younger adults and all studied outcomes in older adults, an increase in the currently recommended intake of vitamin D is warranted. An intake for all adults of > or =1000 IU (25 microg) [DOSAGE ERROR CORRECTED] vitamin D (cholecalciferol)/d is needed to bring vitamin D concentrations in no less than 50% of the population up to 75 nmol/L. The implications of higher doses for the entire adult population should be addressed in future 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.


Mouse embryonic stem (ES) cells are isolated from the inner cell mass of blastocysts, and can be preserved in vitro in a naive inner-cell-mass-like configuration by providing exogenous stimulation with leukaemia inhibitory factor (LIF) and small molecule inhibition of ERK1/ERK2 and GSK3β signalling (termed 2i/LIF conditions). Hallmarks of naive pluripotency include driving Oct4 (also known as Pou5f1) transcription by its distal enhancer, retaining a pre-inactivation X chromosome state, and global reduction in DNA methylation and in H3K27me3 repressive chromatin mark deposition on developmental regulatory gene promoters. Upon withdrawal of 2i/LIF, naive mouse ES cells can drift towards a primed pluripotent state resembling that of the post-implantation epiblast. Although human ES cells share several molecular features with naive mouse ES cells, they also share a variety of epigenetic properties with primed murine epiblast stem cells (EpiSCs). These include predominant use of the proximal enhancer element to maintain OCT4 expression, pronounced tendency for X chromosome inactivation in most female human ES cells, increase in DNA methylation and prominent deposition of H3K27me3 and bivalent domain acquisition on lineage regulatory genes. The feasibility of establishing human ground state naive pluripotency in vitro with equivalent molecular and functional features to those characterized in mouse ES cells remains to be defined. Here we establish defined conditions that facilitate the derivation of genetically unmodified human naive pluripotent stem cells from already established primed human ES cells, from somatic cells through induced pluripotent stem (iPS) cell reprogramming or directly from blastocysts. The novel naive pluripotent cells validated herein retain molecular characteristics and functional properties that are highly similar to mouse naive ES cells, and distinct from conventional primed human pluripotent cells. This includes competence in the generation of cross-species chimaeric mouse embryos that underwent organogenesis following microinjection of human naive iPS cells into mouse morulas. Collectively, our findings establish new avenues for regenerative medicine, patient-specific iPS cell disease modelling and the study of early human development in vitro and in vivo.


Human tumours typically harbour a remarkable number of somatic mutations. If presented on major histocompatibility complex class I molecules (MHCI), peptides containing these mutations could potentially be immunogenic as they should be recognized as ‘non-self’ neo-antigens by the adaptive immune system. Recent work has confirmed that mutant peptides can serve as T-cell epitopes. However, few mutant epitopes have been described because their discovery required the laborious screening of patient tumour-infiltrating lymphocytes for their ability to recognize antigen libraries constructed following tumour exome sequencing. We sought to simplify the discovery of immunogenic mutant peptides by characterizing their general properties. We developed an approach that combines whole-exome and transcriptome sequencing analysis with mass spectrometry to identify neo-epitopes in two widely used murine tumour models. Of the >1,300 amino acid changes identified, ∼13% were predicted to bind MHCI, a small fraction of which were confirmed by mass spectrometry. The peptides were then structurally modelled bound to MHCI. Mutations that were solvent-exposed and therefore accessible to T-cell antigen receptors were predicted to be immunogenic. Vaccination of mice confirmed the approach, with each predicted immunogenic peptide yielding therapeutically active T-cell responses. The predictions also enabled the generation of peptide–MHCI dextramers that could be used to monitor the kinetics and distribution of the anti-tumour T-cell response before and after vaccination. These findings indicate that a suitable prediction algorithm may provide an approach for the pharmacodynamic monitoring of T-cell responses as well as for the development of personalized vaccines in cancer patients.


BACKGROUND Little is known about how the intensity of exercise influences cardiovascular fitness and body composition, especially in obese adolescents. OBJECTIVE Our goal was to determine the effects of physical training intensity on the cardiovascular fitness, percentage of body fat (%BF), and visceral adipose tissue (VAT) of obese adolescents. DESIGN Obese 13-16-y-olds (n = 80) were assigned to 1) biweekly lifestyle education (LSE), 2) LSE + moderate-intensity physical training, or 3) LSE + high-intensity physical training. The intervention lasted 8 mo. Physical training was offered 5 d/wk, and the target energy expenditure for all subjects in physical training groups was 1047 kJ (250 kcal)/session. Cardiovascular fitness was measured with a multistage treadmill test, %BF with dual-energy X-ray absorptiometry, and VAT with magnetic resonance imaging. RESULTS The increase in cardiovascular fitness in the high-intensity physical training group, but not in the moderate-intensity group, was significantly greater than that in the LSE alone group (P = 0.009); no other comparisons of the 3 groups were significant. Compared with the LSE alone group, a group composed of subjects in both physical training groups combined who attended training sessions >or=2 d/wk showed favorable changes in cardiovascular fitness (P < 0.001), %BF (P = 0.001), and VAT (P = 0.029). We found no evidence that the high-intensity physical training was more effective than the moderate-intensity physical training in enhancing body composition. CONCLUSIONS The cardiovascular fitness of obese adolescents was significantly improved by physical training, especially high-intensity physical training. The physical training also reduced both visceral and total-body adiposity, but there was no clear effect of the intensity of physical training.