Bacterial RNA and small antiviral compounds activate caspase-1 through cryopyrin/Nalp3

Abstract: Missense mutations in the CIAS1 gene cause three autoinflammatory disorders: familial cold autoinflammatory syndrome, Muckle–Wells syndrome and neonatal-onset multiple-system inflammatory disease.

Abstract: 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.

Abstract: 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.

Abstract: Here we show the effect of cryopyrin deficiency on inflammasome function and immune responses.

Abstract: 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.

Abstract: 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.

Abstract: Furthermore, we show that Toll-like receptors and cryopyrin control the secretion of IL-1β and IL-18 through different intracellular pathways.

Abstract: 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.


Control of cortical GABA circuitry development by Nrg1 and ErbB4 signalling

Abstract: Schizophrenia is a complex disorder that interferes with the function of several brain systems required for cognition and normal social behaviour.

Abstract: Although the most notable clinical aspects of the disease only become apparent during late adolescence or early adulthood, many lines of evidence suggest that schizophrenia is a neurodevelopmental disorder with a strong genetic component.

Abstract: Several independent studies have identified neuregulin 1 (NRG1) and its receptor ERBB4 as important risk genes for schizophrenia, although their precise role in the disease process remains unknown.

Abstract: Here we show that Nrg1 and ErbB4 signalling controls the development of inhibitory circuitries in the mammalian cerebral cortex by cell-autonomously regulating the connectivity of specific GABA (γ-aminobutyric acid)-containing interneurons.

Abstract: In contrast to the prevalent view, which supports a role for these genes in the formation and function of excitatory synapses between pyramidal cells, we found that ErbB4 expression in the mouse neocortex and hippocampus is largely confined to certain classes of interneurons.

Abstract: In particular, ErbB4 is expressed by many parvalbumin-expressing chandelier and basket cells, where it localizes to axon terminals and postsynaptic densities receiving glutamatergic input.

Abstract: Gain- and loss-of-function experiments, both in vitro and in vivo, demonstrate that ErbB4 cell-autonomously promotes the formation of axo-axonic inhibitory synapses over pyramidal cells, and that this function is probably mediated by Nrg1.

Abstract: In addition, ErbB4 expression in GABA-containing interneurons regulates the formation of excitatory synapses onto the dendrites of these cells.

Abstract: By contrast, ErbB4 is dispensable for excitatory transmission between pyramidal neurons.

Abstract: Altogether, our results indicate that Nrg1 and ErbB4 signalling is required for the wiring of GABA-mediated circuits in the postnatal cortex, providing a new perspective to the involvement of these genes in the aetiology of schizophrenia.


Obesity as a medical problem.

Abstract: 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.

Abstract: In particular, obesity is associated with diabetes mellitus, coronary heart disease, certain forms of cancer, and sleep-breathing disorders.

Abstract: 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.

Abstract: 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.

Abstract: Obesity should no longer be regarded simply as a cosmetic problem affecting certain individuals, but an epidemic that threatens global well being.


Nonlinear Elasticity in Biological Gels

Abstract: Unlike most synthetic materials, biological materials often stiffen as they are deformed.

Abstract: This nonlinear elastic response, critical for the physiological function of some tissues, has been documented since at least the 19th century, but the molecular structure and the design principles responsible for it are unknown.

Abstract: Current models for this response require geometrically complex ordered structures unique to each material.

Abstract: In this Article we show that a much simpler molecular theory accounts for strain stiffening in a wide range of molecularly distinct biopolymer gels formed from purified cytoskeletal and extracellular proteins.

Abstract: This theory shows that systems of semi-flexible chains such as filamentous proteins arranged in an open crosslinked meshwork invariably stiffen at low strains without the need for a specific architecture or multiple elements with different intrinsic stiffnesses.


Live-cell delamination counterbalances epithelial growth to limit tissue overcrowding

Abstract: The development and maintenance of an epithelium requires finely balanced rates of growth and cell death.

Abstract: However, the mechanical and biochemical mechanisms that ensure proper feedback control of tissue growth, which when deregulated contribute to tumorigenesis, are poorly understood.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: We show that this process of delamination is mechanistically distinct from apoptosis-mediated cell extrusion and precedes the first signs of cell death.

Abstract: Overall, this analysis reveals a simple mechanism that buffers epithelia against variations in growth.

Abstract: 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.


Broad antiretroviral defence by human APOBEC3G through lethal editing of nascent reverse transcripts

Abstract: Viral replication usually requires that innate intracellular lines of defence be overcome, a task usually accomplished by specialized viral gene products.

Abstract: 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.

Abstract: When produced in the presence of APOBEC3G, vif-defective virus is non-infectious.

Abstract: APOBEC3G is closely related to APOBEC1, the central component of an RNA-editing complex that deaminates a cytosine residue in apoB messenger RNA.

Abstract: 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.

Abstract: 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.

Abstract: 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.


Generation of cell polarity in plants links endocytosis, auxin distribution and cell fate decisions

Abstract: Dynamically polarized membrane proteins define different cell boundaries and have an important role in intercellular communication—a vital feature of multicellular development.

Abstract: Efflux carriers for the signalling molecule auxin from the PIN family are landmarks of cell polarity in plants and have a crucial involvement in auxin distribution-dependent development including embryo patterning, organogenesis and tropisms.

Abstract: Polar PIN localization determines the direction of intercellular auxin flow, yet the mechanisms generating PIN polarity remain unclear.

Abstract: Here we identify an endocytosis-dependent mechanism of PIN polarity generation and analyse its developmental implications.

Abstract: Real-time PIN tracking showed that after synthesis, PINs are initially delivered to the plasma membrane in a non-polar manner and their polarity is established by subsequent endocytic recycling.

Abstract: Interference with PIN endocytosis either by auxin or by manipulation of the Arabidopsis Rab5 GTPase pathway prevents PIN polarization.

Abstract: Failure of PIN polarization transiently alters asymmetric auxin distribution during embryogenesis and increases the local auxin response in apical embryo regions.

Abstract: This results in ectopic expression of auxin pathway-associated root-forming master regulators in embryonic leaves and promotes homeotic transformation of leaves to roots.

Abstract: Our results indicate a two-step mechanism for the generation of PIN polar localization and the essential role of endocytosis in this process.

Abstract: It also highlights the link between endocytosis-dependent polarity of individual cells and auxin distribution-dependent cell fate establishment for multicellular patterning.


Congenital leptin deficiency is associated with severe early-onset obesity in humans.

Abstract: The extreme obesity of the obese (ob/ob) mouse is attributable to mutations in the gene encoding leptin, an adipocyte-specific secreted protein which has profound effects on appetite and energy expenditure.

Abstract: We know of no equivalent evidence regarding leptin's role in the control of fat mass in humans.

Abstract: We have examined two severely obese children who are members of the same highly consanguineous pedigree.

Abstract: Their serum leptin levels were very low despite their markedly elevated fat mass and, in both, a homozygous frame-shift mutation involving the deletion of a single guanine nucleotide in codon 133 of the gene for leptin was found.

Abstract: The severe obesity found in these congenitally leptin-deficient subjects provides the first genetic evidence that leptin is an important regulator of energy balance in humans.


Targeting of cell-surface β-amyloid precursor protein to lysosomes: alternative processing into amyloid-bearing fragments

Abstract: PROGRESSIVE cerebral deposition of the amyloid β-peptide is an early and invariant feature of Alzheimer's disease.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: After cell-surface biotinylation, full-length biotinylated βAPP was recovered inside the cells.

Abstract: Purification of lysosomes directly demonstrated the presence of mature βAPP and an extensive array of β-peptide-containing proteolytic products.

Abstract: 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.


A keratin cytoskeletal protein regulates protein synthesis and epithelial cell growth

Abstract: Cell growth, an increase in mass and size, is a highly regulated cellular event.

Abstract: 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.

Abstract: A striking example of a physiological context requiring rapid cell growth is tissue repair in response to injury.

Abstract: 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.

Abstract: Mouse skin keratinocytes lacking keratin 17 (ref.

Abstract: 4) show depressed protein translation and are of smaller size, correlating with decreased Akt/mTOR signalling activity.

Abstract: Other signalling kinases have normal activity, pointing to the specificity of this defect.

Abstract: 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.

Abstract: These findings reveal a new and unexpected role for the intermediate filament cytoskeleton in influencing cell growth and size by regulating protein synthesis.


Co-crystal structure of the HNF-3/fork head DNA-recognition motif resembles histone H5

Abstract: 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.

Abstract: 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.

Abstract: The transcription factor fold is very similar to the structure of histone H5.

Abstract: In its amino-terminal half, three α-helices adopt a compact structure that presents the third helix to the major groove.

Abstract: The remainder of the protein includes a twisted, antiparallel β-structure and random coil that interacts with the minor groove.


A Mechanism Linking Extra Centrosomes to Chromosomal Instability

Abstract: Chromosomal instability (CIN) is a hallmark of many tumours and correlates with the presence of extra centrosomes.

Abstract: However, a direct mechanistic link between extra centrosomes and CIN has not been established.

Abstract: 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.

Abstract: 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.

Abstract: Thus, multipolar divisions cannot explain observed rates of CIN.

Abstract: 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.

Abstract: To define the mechanism underlying this mitotic defect, we generated cells that differ only in their centrosome number.

Abstract: We demonstrate that extra centrosomes alone are sufficient to promote chromosome missegregation during bipolar cell division.

Abstract: 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.

Abstract: These findings provide a direct mechanistic link between extra centrosomes and CIN, two common characteristics of solid tumours.

Abstract: We propose that this mechanism may be a common underlying cause of CIN in human cancer.


Haematopoietic stem cells and early lymphoid progenitors occupy distinct bone marrow niches

Abstract: 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.

Abstract: This raises the fundamental question of whether HSCs and restricted progenitors reside within distinct, specialized niches or whether they share a common niche.

Abstract: Here we assess the physiological sources of the chemokine CXCL12 for HSC and restricted progenitor maintenance.

Abstract: 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.

Abstract: Conditional deletion of Cxcl12 from haematopoietic cells or nestin-cre-expressing cells had little or no effect on HSCs or restricted progenitors.

Abstract: Deletion of Cxcl12 from endothelial cells depleted HSCs but not myeloerythroid or lymphoid progenitors.

Abstract: Deletion of Cxcl12 from perivascular stromal cells depleted HSCs and certain restricted progenitors and mobilized these cells into circulation.

Abstract: Deletion of Cxcl12 from osteoblasts depleted certain early lymphoid progenitors but not HSCs or myeloerythroid progenitors, and did not mobilize these cells into circulation.

Abstract: 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.


Broad neutralization coverage of HIV by multiple highly potent antibodies

Abstract: Broadly neutralizing antibodies against highly variable viral pathogens are much sought after to treat or protect against global circulating viruses.

Abstract: Here we probed the neutralizing antibody repertoires of four human immunodeficiency virus (HIV)-infected donors with remarkably broad and potent neutralizing responses and rescued 17 new monoclonal antibodies that neutralize broadly across clades.

Abstract: Many of the new monoclonal antibodies are almost tenfold more potent than the recently described PG9, PG16 and VRC01 broadly neutralizing monoclonal antibodies and 100-fold more potent than the original prototype HIV broadly neutralizing monoclonal antibodies.

Abstract: The monoclonal antibodies largely recapitulate the neutralization breadth found in the corresponding donor serum and many recognize novel epitopes on envelope (Env) glycoprotein gp120, illuminating new targets for vaccine design.

Abstract: Analysis of neutralization by the full complement of anti-HIV broadly neutralizing monoclonal antibodies now available reveals that certain combinations of antibodies should offer markedly more favourable coverage of the enormous diversity of global circulating viruses than others and these combinations might be sought in active or passive immunization regimes.

Abstract: Overall, the isolation of multiple HIV broadly neutralizing monoclonal antibodies from several donors that, in aggregate, provide broad coverage at low concentrations is a highly positive indicator for the eventual design of an effective antibody-based HIV vaccine.


Coordination of Rho GTPase activities during cell protrusion

Abstract: The GTPases Rac1, RhoA and Cdc42 act together to control cytoskeleton dynamics.

Abstract: Recent biosensor studies have shown that all three GTPases are activated at the front of migrating cells, and biochemical evidence suggests that they may regulate one another: Cdc42 can activate Rac1 (ref. 8), and Rac1 and RhoA are mutually inhibitory.

Abstract: However, their spatiotemporal coordination, at the seconds and single-micrometre dimensions typical of individual protrusion events, remains unknown.

Abstract: Here we examine GTPase coordination in mouse embryonic fibroblasts both through simultaneous visualization of two GTPase biosensors and using a 'computational multiplexing' approach capable of defining the relationships between multiple protein activities visualized in separate experiments.

Abstract: We found that RhoA is activated at the cell edge synchronous with edge advancement, whereas Cdc42 and Rac1 are activated 2 micro-m behind the edge with a delay of 40 s. This indicates that Rac1 and RhoA operate antagonistically through spatial separation and precise timing, and that RhoA has a role in the initial events of protrusion, whereas Rac1 and Cdc42 activate pathways implicated in reinforcement and stabilization of newly expanded protrusions.


Understanding mechanisms underlying human gene expression variation with RNA sequencing

Abstract: 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.

Abstract: 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.

Abstract: We sequenced RNA from 69 lymphoblastoid cell lines derived from unrelated Nigerian individuals that have been extensively genotyped by the International HapMap Project.

Abstract: 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.

Abstract: Using the genotypes from the HapMap project, we identified more than a thousand genes at which genetic variation influences overall expression levels or splicing.

Abstract: 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.

Abstract: Our results illustrate the power of high-throughput sequencing for the joint analysis of variation in transcription, splicing and allele-specific expression across individuals.


Mesenchymal and haematopoietic stem cells form a unique bone marrow niche

Abstract: The cellular constituents forming the haematopoietic stem cell (HSC) niche in the bone marrow are unclear, with studies implicating osteoblasts, endothelial and perivascular cells.

Abstract: Here we demonstrate that mesenchymal stem cells (MSCs), identified using nestin expression, constitute an essential HSC niche component.

Abstract: 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.

Abstract: Nestin+ MSCs are spatially associated with HSCs and adrenergic nerve fibres, and highly express HSC maintenance genes.

Abstract: These genes, and others triggering osteoblastic differentiation, are selectively downregulated during enforced HSC mobilization or β3 adrenoreceptor activation.

Abstract: 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.

Abstract: 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.

Abstract: 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.


Response to self antigen imprints regulatory memory in tissues

Abstract: 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.

Abstract: The mechanisms by which tissues and the immune system communicate to establish and maintain immune homeostasis are currently unknown.

Abstract: 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.

Abstract: 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.

Abstract: In fact, these diseases often spontaneously resolve, despite persistent tissue autoantigen expression.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: Thus, Treg cells function to confer ‘regulatory memory’ to the target tissue.

Abstract: 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.


Reprogramming of human somatic cells to pluripotency with defined factors

Abstract: Pluripotency pertains to the cells of early embryos that can generate all of the tissues in the organism.

Abstract: Embryonic stem cells are embryo-derived cell lines that retain pluripotency and represent invaluable tools for research into the mechanisms of tissue formation.

Abstract: 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.

Abstract: 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.

Abstract: Human iPS cells resemble embryonic stem cells in morphology and gene expression and in the capacity to form teratomas in immune-deficient mice.

Abstract: 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.


Haematopoietic stem cells do not asymmetrically segregate chromosomes or retain BrdU

Abstract: Stem cells are proposed to segregate chromosomes asymmetrically during self-renewing divisions so that older (‘immortal’) DNA strands are retained in daughter stem cells whereas newly synthesized strands segregate to differentiating cells.

Abstract: Stem cells are also proposed to retain DNA labels, such as 5-bromo-2-deoxyuridine (BrdU), either because they segregate chromosomes asymmetrically or because they divide slowly.

Abstract: However, the purity of stem cells among BrdU-label-retaining cells has not been documented in any tissue, and the ‘immortal strand hypothesis’ has not been tested in a system with definitive stem cell markers.

Abstract: Here we tested these hypotheses in haematopoietic stem cells (HSCs), which can be highly purified using well characterized markers.

Abstract: We administered BrdU to newborn mice, mice treated with cyclophosphamide and granulocyte colony-stimulating factor, and normal adult mice for 4 to 10 days, followed by 70 days without BrdU. In each case, less than 6% of HSCs retained BrdU and less than 0.5% of all BrdU-retaining haematopoietic cells were HSCs, revealing that BrdU has poor specificity and poor sensitivity as an HSC marker.

Abstract: Sequential administration of 5-chloro-2-deoxyuridine and 5-iodo-2-deoxyuridine indicated that all HSCs segregate their chromosomes randomly.

Abstract: Division of individual HSCs in culture revealed no asymmetric segregation of the label.

Abstract: Thus, HSCs cannot be identified on the basis of BrdU-label retention and do not retain older DNA strands during division, indicating that these are not general properties of stem cells.


Circadian Clocks in Human Red Blood Cells

Abstract: Circadian (∼24 hour)

Abstract: clocks are fundamentally important for coordinated physiology in organisms as diverse as cyanobacteria and humans.

Abstract: All current models of the molecular circadian clockwork in eukaryotic cells are based on transcription-translation feedback loops.

Abstract: Non-transcriptional mechanisms in the clockwork have been difficult to study in mammalian systems.

Abstract: We circumvented these problems by developing novel assays using human red blood cells, which have no nucleus (or DNA) and therefore cannot perform transcription.

Abstract: 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.

Abstract: 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).

Abstract: Moreover, these rhythms are entrainable (that is, tunable by environmental stimuli) and temperature-compensated, both key features of circadian rhythms.

Abstract: 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.


G-protein-coupled receptor of Kaposi's sarcoma-associated herpesvirus is a viral oncogene and angiogenesis activator.

Abstract: 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).

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: 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.


Therapeutic potential of GSK-J4, a histone demethylase KDM6B/JMJD3 inhibitor, for acute myeloid leukemia

Abstract: PURPOSE Acute myeloid leukemia (AML) is a heterogeneous disease with poor outcomes.

Abstract: 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.

Abstract: Here, we investigated whether targeting KDM6B, the demethylase of tri-methylated histone H3 lysine 27 (H3K27me3), has a therapeutic potential for AML.

Abstract: 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.

Abstract: RNA-sequencing was performed to reveal the underlying mechanisms of inhibiting KDM6B for the treatment of AML.

Abstract: RESULTS Here we observed that the mRNA expression of KDM6B was up-regulated in AML and positively correlated with poor survival.

Abstract: 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.

Abstract: GSK-J4 treatment significantly induced cell apoptosis and cell-cycle arrest in Kasumi-1 cells, and displayed a synergistic effect with cytosine arabinoside.

Abstract: Notably, injection of GSK-J4 attenuated the disease progression in a human AML xenograft mouse model in vivo.

Abstract: 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.

Abstract: ChIP-qPCR validated an increased enrichment of H3K27me3 in the transcription start sites of these HOX genes.

Abstract: CONCLUSIONS In summary, our findings suggest that targeting KDM6B with GSK-J4 has a therapeutic potential for the treatment of AML.


Structure of the proton-gated urea channel from the gastric pathogen Helicobacter pylori

Abstract: Half the world's population is chronically infected with Helicobacter pylori, causing gastritis, gastric ulcers and an increased incidence of gastric adenocarcinoma.

Abstract: Its proton-gated inner-membrane urea channel, HpUreI, is essential for survival in the acidic environment of the stomach.

Abstract: The channel is closed at neutral pH and opens at acidic pH to allow the rapid access of urea to cytoplasmic urease.

Abstract: Urease produces NH(3) and CO(2), neutralizing entering protons and thus buffering the periplasm to a pH of roughly 6.1 even in gastric juice at a pH below 2.0.

Abstract: Here we report the structure of HpUreI, revealing six protomers assembled in a hexameric ring surrounding a central bilayer plug of ordered lipids.

Abstract: Each protomer encloses a channel formed by a twisted bundle of six transmembrane helices.

Abstract: The bundle defines a previously unobserved fold comprising a two-helix hairpin motif repeated three times around the central axis of the channel, without the inverted repeat of mammalian-type urea transporters.

Abstract: Both the channel and the protomer interface contain residues conserved in the AmiS/UreI superfamily, suggesting the preservation of channel architecture and oligomeric state in this superfamily.

Abstract: Predominantly aromatic or aliphatic side chains line the entire channel and define two consecutive constriction sites in the middle of the channel.

Abstract: Mutation of Trp 153 in the cytoplasmic constriction site to Ala or Phe decreases the selectivity for urea in comparison with thiourea, suggesting that solute interaction with Trp 153 contributes specificity.

Abstract: The previously unobserved hexameric channel structure described here provides a new model for the permeation of urea and other small amide solutes in prokaryotes and archaea.


Drosophila oocyte localization is mediated by differential cadherin-based adhesion.

Abstract: In a Drosophila follicle the oocyte always occupies a posterior position among a group of sixteen germline cells.

Abstract: 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.

Abstract: Here we show that the homophilic adhesion molecule DE-cadherin mediates oocyte positioning.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: Among the DE-cadherin positive follicle cells, the oocyte preferentially contacts those cells that express higher levels of DE-cadherin.

Abstract: 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.

Abstract: This is, to our knowledge, the first in vivo example of a cell-sorting process that depends on differential adhesion mediated by a cadherin.


Somatic sex identity is cell-autonomous in the chicken

Abstract: In the mammalian model of sex determination, embryos are considered to be sexually indifferent until the transient action of a sex-determining gene initiates gonadal differentiation.

Abstract: Although this model is thought to apply to all vertebrates, this has yet to be established.

Abstract: Here we have examined three lateral gynandromorph chickens (a rare, naturally occurring phenomenon in which one side of the animal appears male and the other female) to investigate the sex-determining mechanism in birds.

Abstract: These studies demonstrated that gynandromorph birds are genuine male:female chimaeras, and indicated that male and female avian somatic cells may have an inherent sex identity.

Abstract: To test this hypothesis, we transplanted presumptive mesoderm between embryos of reciprocal sexes to generate embryos containing male:female chimaeric gonads.

Abstract: In contrast to the outcome for mammalian mixed-sex chimaeras, in chicken mixed-sex chimaeras the donor cells were excluded from the functional structures of the host gonad.

Abstract: In an example where female tissue was transplanted into a male host, donor cells contributing to the developing testis retained a female identity and expressed a marker of female function.

Abstract: Our study demonstrates that avian somatic cells possess an inherent sex identity and that, in birds, sexual differentiation is substantively cell autonomous.


Centrosome polarization delivers secretory granules to the immunological synapse

Abstract: 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.

Abstract: 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.

Abstract: However, the final steps of secretion have remained unclear.

Abstract: 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.

Abstract: Actin and IQGAP1 are cleared away from the synapse, and granules are delivered directly to the plasma membrane.

Abstract: 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.


Deubiquitination of p53 by HAUSP is an important pathway for p53 stabilization

Abstract: The p53 tumour suppressor is a short-lived protein that is maintained at low levels in normal cells by Mdm2-mediated ubiquitination and subsequent proteolysis.

Abstract: Stabilization of p53 is crucial for its tumour suppressor function.

Abstract: However, the precise mechanism by which ubiquitinated p53 levels are regulated in vivo is not completely understood.

Abstract: By mass spectrometry of affinity-purified p53-associated factors, we have identified herpesvirus-associated ubiquitin-specific protease (HAUSP) as a novel p53-interacting protein.

Abstract: HAUSP strongly stabilizes p53 even in the presence of excess Mdm2, and also induces p53-dependent cell growth repression and apoptosis.

Abstract: Significantly, HAUSP has an intrinsic enzymatic activity that specifically deubiquitinates p53 both in vitro and in vivo.

Abstract: In contrast, expression of a catalytically inactive point mutant of HAUSP in cells increases the levels of p53 ubiquitination and destabilizes p53.

Abstract: These findings reveal an important mechanism by which p53 can be stabilized by direct deubiquitination and also imply that HAUSP might function as a tumour suppressor in vivo through the stabilization of p53.


Defective tryptophan catabolism underlies inflammation in mouse chronic granulomatous disease

Abstract: Half a century ago, chronic granulomatous disease (CGD) was first described as a disease fatally affecting the ability of children to survive infections.

Abstract: Various milestone discoveries have since been made, from an insufficient ability of patients’ leucocytes to kill microbes to the underlying genetic abnormalities.

Abstract: 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.

Abstract: Patients with CGD also suffer from chronic inflammatory conditions, most prominently granuloma formation in hollow viscera.

Abstract: The precise mechanisms of the increased microbial pathogenicity have been unclear, and more so the reasons for the exaggerated inflammatory response.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: Yet, this condition can be reverted by reactivating the pathway downstream of the superoxide-dependent step.


Experimental and theoretical study of mitotic spindle orientation

Abstract: The architecture and adhesiveness of a cell microenvironment is a critical factor for the regulation of spindle orientation in vivo.

Abstract: Using a combination of theory and experiments, we have investigated spindle orientation in HeLa (human) cells.

Abstract: Here we show that spindle orientation can be understood as the result of the action of cortical force generators, which interact with spindle microtubules and are activated by cortical cues.

Abstract: We develop a simple physical description of this spindle mechanics, which allows us to calculate angular profiles of the torque acting on the spindle, as well as the angular distribution of spindle orientations.

Abstract: Our model accounts for the preferred spindle orientation and the shape of the full angular distribution of spindle orientations observed in a large variety of different cellular microenvironment geometries.

Abstract: It also correctly describes asymmetric spindle orientations, which are observed for certain distributions of cortical cues.

Abstract: We conclude that, on the basis of a few simple assumptions, we can provide a quantitative description of the spindle orientation of adherent cells.


CHROMOTHRIPSIS FROM DNA DAMAGE IN MICRONUCLEI

Abstract: Genome sequencing has uncovered a new mutational phenomenon in cancer and congenital disorders called chromothripsis.

Abstract: Chromothripsis is characterized by extensive genomic rearrangements and an oscillating pattern of DNA copy number levels, all curiously restricted to one or a few chromosomes.

Abstract: The mechanism for chromothripsis is unknown, but we previously proposed that it could occur through the physical isolation of chromosomes in aberrant nuclear structures called micronuclei.

Abstract: Here, using a combination of live cell imaging and single-cell genome sequencing, we demonstrate that micronucleus formation can indeed generate a spectrum of genomic rearrangements, some of which recapitulate all known features of chromothripsis.

Abstract: These events are restricted to the mis-segregated chromosome and occur within one cell division.

Abstract: We demonstrate that the mechanism for chromothripsis can involve the fragmentation and subsequent reassembly of a single chromatid from a micronucleus.

Abstract: Collectively, these experiments establish a new mutational process of which chromothripsis is one extreme outcome.


DNA-binding factors shape the mouse methylome at distal regulatory regions

Abstract: Methylation of cytosines is an essential epigenetic modification in mammalian genomes, yet the rules that govern methylation patterns remain largely elusive.

Abstract: To gain insights into this process, we generated base-pair-resolution mouse methylomes in stem cells and neuronal progenitors.

Abstract: Advanced quantitative analysis identified low-methylated regions (LMRs) with an average methylation of 30%.

Abstract: 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.

Abstract: LMRs are occupied by DNA-binding factors and their binding is necessary and sufficient to create LMRs.

Abstract: A comparison of neuronal and stem-cell methylomes confirms this dependency, as cell-type-specific LMRs are occupied by cell-type-specific transcription factors.

Abstract: 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.


Molecular basis of RNA-dependent RNA polymerase II activity

Abstract: RNA polymerase (Pol) II catalyses DNA-dependent RNA synthesis during gene transcription.

Abstract: There is, however, evidence that Pol II also possesses RNA-dependent RNA polymerase (RdRP) activity.

Abstract: Pol II can use a homopolymeric RNA template, can extend RNA by several nucleotides in the absence of DNA, and has been implicated in the replication of the RNA genomes of hepatitis delta virus (HDV) and plant viroids.

Abstract: Here we show the intrinsic RdRP activity of Pol II with only pure polymerase, an RNA template–product scaffold and nucleoside triphosphates (NTPs).

Abstract: Crystallography reveals the template–product duplex in the site occupied by the DNA–RNA hybrid during transcription.

Abstract: RdRP activity resides at the active site used during transcription, but it is slower and less processive than DNA-dependent activity.

Abstract: RdRP activity is also obtained with part of the HDV antigenome.

Abstract: The complex of transcription factor IIS (TFIIS) with Pol II can cleave one HDV strand, create a reactive stem-loop in the hybrid site, and extend the new RNA 3′ end.

Abstract: Short RNA stem-loops with a 5′ extension suffice for activity, but their growth to a critical length apparently impairs processivity.

Abstract: The RdRP activity of Pol II provides a missing link in molecular evolution, because it suggests that Pol II evolved from an ancient replicase that duplicated RNA genomes.


Bacteria activate sensory neurons that modulate pain and inflammation

Abstract: Nociceptor sensory neurons are specialized to detect potentially damaging stimuli, protecting the organism by initiating the sensation of pain and eliciting defensive behaviours.

Abstract: Bacterial infections produce pain by unknown molecular mechanisms, although they are presumed to be secondary to immune activation.

Abstract: 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.

Abstract: Mechanical and thermal hyperalgesia in mice is correlated with live bacterial load rather than tissue swelling or immune activation.

Abstract: 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.

Abstract: 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.

Abstract: Thus, bacterial pathogens produce pain by directly activating sensory neurons that modulate inflammation, an unsuspected role for the nervous system in host-pathogen interactions.


E2F1-3 Switch from Activators in Progenitor Cells to Repressors in Differentiating Cells

Abstract: In the established model of mammalian cell cycle control, the retinoblastoma protein (Rb) functions to restrict cells from entering S phase by binding and sequestering E2f activators (E2f1, E2f2 and E2f3), which are invariably portrayed as the ultimate effectors of a transcriptional program that commit cells to enter and progress through S phase.

Abstract: Using a panel of tissue-specific cre-transgenic mice and conditional E2f alleles we examined the effects of E2f1, E2f2 and E2f3 triple deficiency in murine embryonic stem cells, embryos and small intestines.

Abstract: We show that in normal dividing progenitor cells E2f1-3 function as transcriptional activators, but contrary to the current view, are dispensable for cell division and instead are necessary for cell survival.

Abstract: In differentiating cells E2f1-3 function in a complex with Rb as repressors to silence E2f targets and facilitate exit from the cell cycle.

Abstract: The inactivation of Rb in differentiating cells resulted in a switch of E2f1-3 from repressors to activators, leading to the superactivation of E2f responsive targets and ectopic cell divisions.

Abstract: Loss of E2f1-3 completely suppressed these phenotypes caused by Rb deficiency.

Abstract: This work contextualizes the activator versus repressor functions of E2f1-3 in vivo, revealing distinct roles in dividing versus differentiating cells and in normal versus cancer-like cell cycles.


K-Ras(G12C) inhibitors allosterically control GTP affinity and effector interactions

Abstract: 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.

Abstract: 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.

Abstract: Oncogenic mutations result in functional activation of Ras family proteins by impairing GTP hydrolysis.

Abstract: With diminished regulation by GTPase activity, the nucleotide state of Ras becomes more dependent on relative nucleotide affinity and concentration.

Abstract: This gives GTP an advantage over GDP and increases the proportion of active GTP-bound Ras.

Abstract: Here we report the development of small molecules that irreversibly bind to a common oncogenic mutant, K-Ras(G12C).

Abstract: These compounds rely on the mutant cysteine for binding and therefore do not affect the wild-type protein.

Abstract: 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.

Abstract: 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.

Abstract: Our data provide structure-based validation of a new allosteric regulatory site on Ras that is targetable in a mutant-specific manner.


Cyclin A Regulates Kinetochore-Microtubules to Promote Faithful Chromosome Segregation

Abstract: The most conspicuous event in the cell cycle is the alignment of chromosomes in metaphase.

Abstract: Chromosome alignment fosters faithful segregation through the formation of bi-oriented attachments of kinetochores to spindle microtubules.

Abstract: 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.

Abstract: How robust error correction is achieved in prometaphase to ensure error-free mitosis remains unknown.

Abstract: Here we show that k-MT attachments in prometaphase cells are considerably less stable than in metaphase cells.

Abstract: The switch to more stable k-MT attachments in metaphase requires the proteasome-dependent destruction of cyclin A in prometaphase.

Abstract: Persistent cyclin A expression prevents k-MT stabilization even in cells with aligned chromosomes.

Abstract: By contrast, k-MTs are prematurely stabilized in cyclin-A-deficient cells.

Abstract: Consequently, cells lacking cyclin A display higher rates of chromosome mis-segregation.

Abstract: Thus, the stability of k-MT attachments increases decisively in a coordinated fashion among all chromosomes as cells transit from prometaphase to metaphase.

Abstract: Cyclin A creates a cellular environment that promotes microtubule detachment from kinetochores in prometaphase to ensure efficient error correction and faithful chromosome segregation.


Induced pluripotent stem cells from a spinal muscular atrophy patient

Abstract: Spinal muscular atrophy is one of the most common inherited forms of neurological disease leading to infant mortality.

Abstract: Patients have selective loss of lower motor neurons resulting in muscle weakness, paralysis and often death.

Abstract: 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.

Abstract: Here we report the generation of induced pluripotent stem cells from skin fibroblast samples taken from a child with spinal muscular atrophy.

Abstract: 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.

Abstract: 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.

Abstract: As such, it represents a promising resource to study disease mechanisms, screen new drug compounds and develop new therapies.


A role for mitochondria in NLRP3 inflammasome activation

Abstract: An inflammatory response initiated by the NLRP3 inflammasome is triggered by a variety of situations of host ‘danger’, including infection and metabolic dysregulation.

Abstract: 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.

Abstract: Here we show that mitophagy/autophagy blockade leads to the accumulation of damaged, ROS-generating mitochondria, and this in turn activates the NLRP3 inflammasome.

Abstract: 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.

Abstract: Notably, both ROS generation and inflammasome activation are suppressed when mitochondrial activity is dysregulated by inhibition of the voltage-dependent anion channel.

Abstract: This indicates that NLRP3 inflammasome senses mitochondrial dysfunction and may explain the frequent association of mitochondrial damage with inflammatory diseases.


Break-induced telomere synthesis underlies alternative telomere maintenance

Abstract: Homology-directed DNA repair is essential for genome maintenance through templated DNA synthesis.

Abstract: Alternative lengthening of telomeres (ALT) necessitates homology-directed DNA repair to maintain telomeres in about 10–15% of human cancers.

Abstract: How DNA damage induces assembly and execution of a DNA replication complex (break-induced replisome) at telomeres or elsewhere in the mammalian genome is poorly understood.

Abstract: Here we define break-induced telomere synthesis and demonstrate that it utilizes a specialized replisome, which underlies ALT telomere maintenance.

Abstract: DNA double-strand breaks enact nascent telomere synthesis by long-tract unidirectional replication.

Abstract: Proliferating cell nuclear antigen (PCNA) loading by replication factor C (RFC) acts as the initial sensor of telomere damage to establish predominance of DNA polymerase δ (Pol δ) through its POLD3 subunit.

Abstract: Break-induced telomere synthesis requires the RFC–PCNA–Pol δ axis, but is independent of other canonical replisome components, ATM and ATR, or the homologous recombination protein Rad51.

Abstract: Thus, the inception of telomere damage recognition by the break-induced replisome orchestrates homology-directed telomere maintenance.


Innate immunity induced by composition-dependent RIG-I recognition of hepatitis C virus RNA

Abstract: 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).

Abstract: Hepatitis C virus (HCV) is an RNA virus that replicates in the liver, and infects 200 million people worldwide.

Abstract: Infection is regulated by hepatic immune defences triggered by the cellular RIG-I helicase.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: 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.


Generation of transgenic non-human primates with germline transmission

Abstract: The common marmoset (Callithrix jacchus) is increasingly attractive for use as a non-human primate animal model in biomedical research.

Abstract: It has a relatively high reproduction rate for a primate, making it potentially suitable for transgenic modification.

Abstract: 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.

Abstract: 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.

Abstract: Notably, we achieved germline transmission of the transgene, and the transgenic offspring developed normally.

Abstract: 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.

Abstract: This model will be valuable to many fields of biomedical research.


Induction of human neuronal cells by defined transcription factors

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: These methods may facilitate robust generation of patient-specific human neurons for in vitro disease modelling or future applications in regenerative medicine.


PAAR-repeat proteins sharpen and diversify the Type VI secretion system spike

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: Neither the exact protein composition of the T6SS organelle nor the mechanisms of effector selection and delivery are known.

Abstract: 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.

Abstract: 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.

Abstract: We demonstrate that PAAR proteins are essential for T6SS-mediated secretion and target cell killing by Vibrio cholerae and Acinetobacter baylyi.

Abstract: 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.


Replication stress activates DNA repair synthesis in mitosis

Abstract: Oncogene-induced DNA replication stress has been implicated as a driver of tumorigenesis.

Abstract: Many chromosomal rearrangements characteristic of human cancers originate from specific regions of the genome called common fragile sites (CFSs).

Abstract: CFSs are difficult-to-replicate loci that manifest as gaps or breaks on metaphase chromosomes (termed CFS ‘expression’), particularly when cells have been exposed to replicative stress.

Abstract: The MUS81–EME1 structure-specific endonuclease promotes the appearance of chromosome gaps or breaks at CFSs following replicative stress.

Abstract: Here we show that entry of cells into mitotic prophase triggers the recruitment of MUS81 to CFSs.

Abstract: The nuclease activity of MUS81 then promotes POLD3-dependent DNA synthesis at CFSs, which serves to minimize chromosome mis-segregation and non-disjunction.

Abstract: We propose that the attempted condensation of incompletely duplicated loci in early mitosis serves as the trigger for completion of DNA replication at CFS loci in human cells.

Abstract: Given that this POLD3-dependent mitotic DNA synthesis is enhanced in aneuploid cancer cells that exhibit intrinsically high levels of chromosomal instability (CIN+) and replicative stress, we suggest that targeting this pathway could represent a new therapeutic approach.


A specific amyloid-β protein assembly in the brain impairs memory

Abstract: Memory function often declines with age, and is believed to deteriorate initially because of changes in synaptic function rather than loss of neurons.

Abstract: Some individuals then go on to develop Alzheimer's disease with neurodegeneration.

Abstract: Here we use Tg2576 mice, which express a human amyloid-β precursor protein (APP) variant linked to Alzheimer's disease, to investigate the cause of memory decline in the absence of neurodegeneration or amyloid-β protein amyloidosis.

Abstract: Young Tg2576 mice (< 6 months old) have normal memory and lack neuropathology, middle-aged mice (6–14 months old) develop memory deficits without neuronal loss, and old mice (> 14 months old) form abundant neuritic plaques containing amyloid-β (refs 3–6).

Abstract: We found that memory deficits in middle-aged Tg2576 mice are caused by the extracellular accumulation of a 56-kDa soluble amyloid-β assembly, which we term Aβ*56 (Aβ star 56).

Abstract: Aβ*56 purified from the brains of impaired Tg2576 mice disrupts memory when administered to young rats.

Abstract: We propose that Aβ*56 impairs memory independently of plaques or neuronal loss, and may contribute to cognitive deficits associated with Alzheimer's disease.


Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death

Abstract: Inflammatory caspases (caspase-1, -4, -5 and -11) are critical for innate defences.

Abstract: Caspase-1 is activated by ligands of various canonical inflammasomes, and caspase-4, -5 and -11 directly recognize bacterial lipopolysaccharide, both of which trigger pyroptosis.

Abstract: Despite the crucial role in immunity and endotoxic shock, the mechanism for pyroptosis induction by inflammatory caspases is unknown.

Abstract: Here we identify gasdermin D (Gsdmd) by genome-wide clustered regularly interspaced palindromic repeat (CRISPR)-Cas9 nuclease screens of caspase-11- and caspase-1-mediated pyroptosis in mouse bone marrow macrophages.

Abstract: GSDMD-deficient cells resisted the induction of pyroptosis by cytosolic lipopolysaccharide and known canonical inflammasome ligands.

Abstract: Interleukin-1β release was also diminished in Gsdmd−/− cells, despite intact processing by caspase-1.

Abstract: Caspase-1 and caspase-4/5/11 specifically cleaved the linker between the amino-terminal gasdermin-N and carboxy-terminal gasdermin-C domains in GSDMD, which was required and sufficient for pyroptosis.

Abstract: The cleavage released the intramolecular inhibition on the gasdermin-N domain that showed intrinsic pyroptosis-inducing activity.

Abstract: Other gasdermin family members were not cleaved by inflammatory caspases but shared the autoinhibition; gain-of-function mutations in Gsdma3 that cause alopecia and skin defects disrupted the autoinhibition, allowing its gasdermin-N domain to trigger pyroptosis.

Abstract: These findings offer insight into inflammasome-mediated immunity/diseases and also change our understanding of pyroptosis and programmed necrosis.


Role of corin in trophoblast invasion and uterine spiral artery remodelling in pregnancy

Abstract: In pregnancy, trophoblast invasion and uterine spiral artery remodelling are important for lowering maternal vascular resistance and increasing uteroplacental blood flow.

Abstract: 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.

Abstract: 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.

Abstract: Unexpectedly, corin expression was detected in the pregnant uterus.

Abstract: Here we identify a new function of corin and ANP in promoting trophoblast invasion and spiral artery remodelling.

Abstract: We show that pregnant corin- or ANP-deficient mice developed high blood pressure and proteinuria, characteristics of pre-eclampsia.

Abstract: In these mice, trophoblast invasion and uterine spiral artery remodelling were markedly impaired.

Abstract: Consistent with this, the ANP potently stimulated human trophoblasts in invading Matrigels.

Abstract: In patients with pre-eclampsia, uterine Corin messenger RNA and protein levels were significantly lower than that in normal pregnancies.

Abstract: Moreover, we have identified Corin gene mutations in pre-eclamptic patients, which decreased corin activity in processing pro-ANP.

Abstract: 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.


Metabolic rescue in pluripotent cells from patients with mtDNA disease

Abstract: 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.

Abstract: Mutations in mtDNA can cause fatal or severely debilitating disorders with limited treatment options.

Abstract: Clinical manifestations vary based on mutation type and heteroplasmy (that is, the relative levels of mutant and wild-type mtDNA within each cell).

Abstract: Here we generated genetically corrected pluripotent stem cells (PSCs) from patients with mtDNA disease.

Abstract: 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.

Abstract: 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.

Abstract: Furthermore, somatic cell nuclear transfer (SCNT) enabled replacement of mutant mtDNA from homoplasmic 8993T>G fibroblasts to generate corrected Leigh-NT1 PSCs.

Abstract: 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.

Abstract: Moreover, genetically rescued patient PSCs displayed normal metabolic function compared to impaired oxygen consumption and ATP production observed in mutant cells.

Abstract: 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.


The yeast Pif1p helicase removes telomerase from telomeric DNA

Abstract: Telomeres are the physical ends of eukaryotic chromosomes.

Abstract: Genetic studies have established that the baker's yeast Pif1p DNA helicase is a negative regulator of telomerase, the specialized reverse transcriptase that maintains telomeric DNA, but the biochemical basis for this inhibition was unknown.

Abstract: Here we show that in vitro, Pif1p reduces the processivity of telomerase and releases telomerase from telomeric oligonucleotides.

Abstract: The released telomerase is enzymatically active because it is able to lengthen a challenger oligonucleotide.

Abstract: In vivo, overexpression of Pif1p reduces telomerase association with telomeres, whereas depleting cells of Pif1p increases the levels of telomere-bound Est1p, a telomerase subunit that is present on the telomere when telomerase is active.

Abstract: We propose that Pif1p helicase activity limits telomerase action both in vivo and in vitro by displacing active telomerase from DNA ends.


Small regulatory RNAs inhibit RNA Polymerase II during the elongation phase of transcription

Abstract: Eukaryotic cells express a wide variety of endogenous small regulatory RNAs that regulate heterochromatin formation, developmental timing, defence against parasitic nucleic acids and genome rearrangement.

Abstract: Many small regulatory RNAs are thought to function in nuclei.

Abstract: For instance, in plants and fungi, short interfering RNA (siRNAs) associate with nascent transcripts and direct chromatin and/or DNA modifications.

Abstract: To understand further the biological roles of small regulatory RNAs, we conducted a genetic screen to identify factors required for RNA interference (RNAi) in Caenorhabditis elegans nuclei.

Abstract: Here we show that the gene nuclear RNAi defective-2 (nrde-2) encodes an evolutionarily conserved protein that is required for siRNA-mediated silencing in nuclei.

Abstract: NRDE-2 associates with the Argonaute protein NRDE-3 within nuclei and is recruited by NRDE-3/siRNA complexes to nascent transcripts that have been targeted by RNAi.

Abstract: We find that nuclear-localized siRNAs direct an NRDE-2-dependent silencing of pre-messenger RNAs (pre-mRNAs) 3' to sites of RNAi, an NRDE-2-dependent accumulation of RNA polymerase (RNAP) II at genomic loci targeted by RNAi, and NRDE-2-dependent decreases in RNAP II occupancy and RNAP II transcriptional activity 3' to sites of RNAi.

Abstract: These results define NRDE-2 as a component of the nuclear RNAi machinery and demonstrate that metazoan siRNAs can silence nuclear-localized RNAs co-transcriptionally.

Abstract: In addition, these results establish a novel mode of RNAP II regulation: siRNA-directed recruitment of NRDE factors that inhibit RNAP II during the elongation phase of transcription.


Cdk1 is sufficient to drive the mammalian cell cycle

Abstract: Unicellular organisms such as yeasts require a single cyclin-dependent kinase, Cdk1, to drive cell division.

Abstract: In contrast, mammalian cells are thought to require the sequential activation of at least four different cyclin-dependent kinases, Cdk2, Cdk3, Cdk4 and Cdk6, to drive cells through interphase, as well as Cdk1 to proceed through mitosis.

Abstract: This model has been challenged by recent genetic evidence that mice survive in the absence of individual interphase Cdks.

Abstract: Moreover, most mouse cell types proliferate in the absence of two or even three interphase Cdks.

Abstract: Similar results have been obtained on ablation of some of the activating subunits of Cdks, such as the D-type and E-type cyclins.

Abstract: Here we show that mouse embryos lacking all interphase Cdks (Cdk2, Cdk3, Cdk4 and Cdk6) undergo organogenesis and develop to midgestation.

Abstract: In these embryos, Cdk1 binds to all cyclins, resulting in the phosphorylation of the retinoblastoma protein pRb and the expression of genes that are regulated by E2F transcription factors.

Abstract: Mouse embryonic fibroblasts derived from these embryos proliferate in vitro, albeit with an extended cell cycle due to inefficient inactivation of Rb proteins.

Abstract: However, they become immortal on continuous passage.

Abstract: We also report that embryos fail to develop to the morula and blastocyst stages in the absence of Cdk1.

Abstract: These results indicate that Cdk1 is the only essential cell cycle Cdk.

Abstract: Moreover, they show that in the absence of interphase Cdks, Cdk1 can execute all the events that are required to drive cell division.


Calorie restriction extends Saccharomyces cerevisiae lifespan by increasing respiration

Abstract: Calorie restriction (CR) extends lifespan in a wide spectrum of organisms and is the only regimen known to lengthen the lifespan of mammals.

Abstract: We established a model of CR in budding yeast Saccharomyces cerevisiae.

Abstract: In this system, lifespan can be extended by limiting glucose or by reducing the activity of the glucose-sensing cyclic-AMP-dependent kinase (PKA).

Abstract: Lifespan extension in a mutant with reduced PKA activity requires Sir2 and NAD (nicotinamide adenine dinucleotide).

Abstract: In this study we explore how CR activates Sir2 to extend lifespan.

Abstract: 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.

Abstract: We discuss how this metabolic strategy may apply to CR in animals.


Mosaic PPM1D mutations are associated with predisposition to breast and ovarian cancer

Abstract: Improved sequencing technologies offer unprecedented opportunities for investigating the role of rare genetic variation in common disease.

Abstract: However, there are considerable challenges with respect to study design, data analysis and replication.

Abstract: 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.

Abstract: 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).

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: Our results have implications for the detection and management of breast and ovarian cancer risk.

Abstract: 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.


Immunogenicity of induced pluripotent stem cells

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: In contrast to B6 ESCs, teratomas formed by B6 ViPSCs were mostly immune-rejected by B6 recipients.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: 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.


Direct conversion of human fibroblasts to multilineage blood progenitors

Abstract: As is the case for embryo-derived stem cells, application of reprogrammed human induced pluripotent stem cells is limited by our understanding of lineage specification.

Abstract: Here we demonstrate the ability to generate progenitors and mature cells of the haematopoietic fate directly from human dermal fibroblasts without establishing pluripotency.

Abstract: Ectopic expression of OCT4 (also called POU5F1)-activated haematopoietic transcription factors, together with specific cytokine treatment, allowed generation of cells expressing the pan-leukocyte marker CD45.

Abstract: These unique fibroblast-derived cells gave rise to granulocytic, monocytic, megakaryocytic and erythroid lineages, and demonstrated in vivo engraftment capacity.

Abstract: We note that adult haematopoietic programs are activated, consistent with bypassing the pluripotent state to generate blood fate: this is distinct from haematopoiesis involving pluripotent stem cells, where embryonic programs are activated.

Abstract: These findings demonstrate restoration of multipotency from human fibroblasts, and suggest an alternative approach to cellular reprogramming for autologous cell-replacement therapies that avoids complications associated with the use of human pluripotent stem cells.


Mical links semaphorins to F-actin disassembly

Abstract: How instructive cues present on the cell surface have their precise effects on the actin cytoskeleton is poorly understood.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: Mical enzymes perform reduction–oxidation (redox) enzymatic reactions and also contain domains found in proteins that regulate cell morphology.

Abstract: However, nothing is known of the role of Mical or its redox activity in mediating morphological changes.

Abstract: Here we report that Mical directly links semaphorins and their plexin receptors to the precise control of actin filament (F-actin) dynamics.

Abstract: We found that Mical is both necessary and sufficient for semaphorin–plexin-mediated F-actin reorganization in vivo.

Abstract: Likewise, we purified Mical protein and found that it directly binds F-actin and disassembles both individual and bundled actin filaments.

Abstract: 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.

Abstract: 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.


Novel Foxo1-dependent transcriptional programs control Treg cell function

Abstract: Regulatory T (Treg) cells, characterized by expression of the transcription factor forkhead box P3 (Foxp3), maintain immune homeostasis by suppressing self-destructive immune responses.

Abstract: 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.

Abstract: However, whether Foxo proteins act beyond the Treg-cell-commitment stage to control Treg cell homeostasis and function remains largely unexplored.

Abstract: Here we show that Foxo1 is a pivotal regulator of Treg cell function.

Abstract: Treg cells express high amounts of Foxo1 and display reduced T-cell-receptor-induced Akt activation, Foxo1 phosphorylation and Foxo1 nuclear exclusion.

Abstract: 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.

Abstract: 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.

Abstract: These findings show that the evolutionarily ancient Akt–Foxo1 signalling module controls a novel genetic program indispensable for Treg cell function.


Functional screening identifies miRNAs inducing cardiac regeneration

Abstract: In mammals, enlargement of the heart during embryonic development is primarily dependent on the increase in cardiomyocyte numbers.

Abstract: Shortly after birth, however, cardiomyocytes stop proliferating and further growth of the myocardium occurs through hypertrophic enlargement of the existing myocytes.

Abstract: As a consequence of the minimal renewal of cardiomyocytes during adult life, repair of cardiac damage through myocardial regeneration is very limited.

Abstract: Here we show that the exogenous administration of selected microRNAs (miRNAs) markedly stimulates cardiomyocyte proliferation and promotes cardiac repair.

Abstract: We performed a high-content microscopy, high-throughput functional screening for human miRNAs that promoted neonatal cardiomyocyte proliferation using a whole-genome miRNA library.

Abstract: Forty miRNAs strongly increased both DNA synthesis and cytokinesis in neonatal mouse and rat cardiomyocytes.

Abstract: 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.

Abstract: After myocardial infarction in mice, these miRNAs stimulated marked cardiac regeneration and almost complete recovery of cardiac functional parameters.

Abstract: The miRNAs identified hold great promise for the treatment of cardiac pathologies consequent to cardiomyocyte loss.


Specificity of sensory–motor connections encoded by Sema3e–Plxnd1 recognition

Abstract: Spinal reflexes are mediated by synaptic connections between sensory afferents and motor neurons.

Abstract: The organization of these circuits shows several levels of specificity.

Abstract: Only certain classes of proprioceptive sensory neurons make direct, monosynaptic connections with motor neurons.

Abstract: 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.

Abstract: 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.

Abstract: However, determinants of fine synaptic specificity here, as in most regions of the central nervous system, have yet to be defined.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: 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.


A chromatin remodelling complex involved in transcription and DNA processing.

Abstract: The packaging of the eukaryotic genome in chromatin presents barriers that restrict the access of enzymes that process DNA.

Abstract: To overcome these barriers, cells possess a number of multi-protein, ATP-dependent chromatin remodelling complexes, each containing an ATPase subunit from the SNF2/SWI2 superfamily.

Abstract: Chromatin remodelling complexes function by increasing nucleosome mobility and are clearly implicated in transcription.

Abstract: Here we have analysed SNF2/SWI2- and ISWI-related proteins to identify remodelling complexes that potentially assist other DNA transactions.

Abstract: We purified a complex from Saccharomyces cerevisiae that contains the Ino80 ATPase.

Abstract: The INO80 complex contains about 12 polypeptides including two proteins related to the bacterial RuvB DNA helicase, which catalyses branch migration of Holliday junctions.

Abstract: The purified complex remodels chromatin, facilitates transcription in vitro and displays 3' to 5' DNA helicase activity.

Abstract: Mutants of ino80 show hypersensitivity to agents that cause DNA damage, in addition to defects in transcription.

Abstract: These results indicate that chromatin remodelling driven by the Ino80 ATPase may be connected to transcription as well as DNA damage repair.


Oncogenic pathway signatures in human cancers as a guide to targeted therapies

Abstract: 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.

Abstract: 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.

Abstract: Various studies have also demonstrated the potential for using gene expression profiles for the analysis of oncogenic pathways.

Abstract: Here we show that gene expression signatures can be identified that reflect the activation status of several oncogenic pathways.

Abstract: 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.

Abstract: Combining signature-based predictions across several pathways identifies coordinated patterns of pathway deregulation that distinguish between specific cancers and tumour subtypes.

Abstract: 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.

Abstract: Predictions of pathway deregulation in cancer cell lines are also shown to predict the sensitivity to therapeutic agents that target components of the pathway.

Abstract: 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.


CTCF mediates methylation-sensitive enhancer-blocking activity at the H19/Igf2 locus.

Abstract: The Insulin-like growth factor 2 (Igf2) and H19 genes are imprinted, resulting in silencing of the maternal and paternal alleles, respectively.

Abstract: This event is dependent upon an imprinted-control region two kilobases upstream of H19 (refs 1, 2).

Abstract: On the paternal chromosome this element is methylated and required for the silencing of H19 (refs 2-4).

Abstract: On the maternal chromosome the region is unmethylated and required for silencing of the Igf2 gene 90 kilobases upstream.

Abstract: We have proposed that the unmethylated imprinted-control region acts as a chromatin boundary that blocks the interaction of Igf2 with enhancers that lie 3' of H19 (refs 5, 6).

Abstract: This enhancer-blocking activity would then be lost when the region was methylated, thereby allowing expression of Igf2 paternally.

Abstract: Here we show, using transgenic mice and tissue culture, that the unmethylated imprinted-control regions from mouse and human H19 exhibit enhancer-blocking activity.

Abstract: Furthermore, we show that CTCF, a zinc finger protein implicated in vertebrate boundary function, binds to several sites in the unmethylated imprinted-control region that are essential for enhancer blocking.

Abstract: Consistent with our model, CTCF binding is abolished by DNA methylation.

Abstract: This is the first example, to our knowledge, of a regulated chromatin boundary in vertebrates.


Broad and potent neutralization of HIV-1 by a gp41-specific human antibody

Abstract: Characterization of human monoclonal antibodies is providing considerable insight into mechanisms of broad HIV-1 neutralization.

Abstract: Here we report an HIV-1 gp41 membrane-proximal external region (MPER)-specific antibody, named 10E8, which neutralizes ∼98% of tested viruses.

Abstract: An analysis of sera from 78 healthy HIV-1-infected donors demonstrated that 27% contained MPER-specific antibodies and 8% contained 10E8-like specificities.

Abstract: In contrast to other neutralizing MPER antibodies, 10E8 did not bind phospholipids, was not autoreactive, and bound cell-surface envelope.

Abstract: The structure of 10E8 in complex with the complete MPER revealed a site of vulnerability comprising a narrow stretch of highly conserved gp41-hydrophobic residues and a critical arginine or lysine just before the transmembrane region.

Abstract: Analysis of resistant HIV-1 variants confirmed the importance of these residues for neutralization.

Abstract: The highly conserved MPER is a target of potent, non-self-reactive neutralizing antibodies, suggesting that HIV-1 vaccines should aim to induce antibodies to this region of HIV-1 envelope glycoprotein.


Disruption of the Hepcidin/Ferroportin Regulatory System Causes Pulmonary Iron Overload and Restrictive Lung Disease

Abstract: Emerging evidence suggests that pulmonary iron accumulation is implicated in a spectrum of chronic lung diseases.

Abstract: However, the mechanism(s) involved in pulmonary iron deposition and its role in the in vivo pathogenesis of lung diseases remains unknown.

Abstract: 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.

Abstract: 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.

Abstract: These findings implicate iron in lung pathology, which is so far not considered a classical iron-related disorder.