Genome-wide genetic analysis of polyploidy in yeast

Abstract: Polyploidy, increased sets of chromosomes, occurs during development, cellular stress, disease and evolution.

Abstract: Despite its prevalence, little is known about the physiological alterations that accompany polyploidy.

Abstract: We previously described ‘ploidy-specific lethality’, where a gene deletion that is not lethal in haploid or diploid budding yeast causes lethality in triploids or tetraploids.

Abstract: Here we report a genome-wide screen to identify ploidy-specific lethal functions.

Abstract: Only 39 out of 3,740 mutations screened exhibited ploidy-specific lethality.

Abstract: Almost all of these mutations affect genomic stability by impairing homologous recombination, sister chromatid cohesion, or mitotic spindle function.

Abstract: We uncovered defects in wild-type tetraploids predicted by the screen, and identified mechanisms by which tetraploidization affects genomic stability.

Abstract: We show that tetraploids have a high incidence of syntelic/monopolar kinetochore attachments to the spindle pole.

Abstract: We suggest that this defect can be explained by mismatches in the ability to scale the size of the spindle pole body, spindle and kinetochores.

Abstract: Thus, geometric constraints may have profound effects on genome stability; the phenomenon described here may be relevant in a variety of biological contexts, including disease states such as cancer.


Diverse and heritable lineage imprinting of early haematopoietic progenitors

Abstract: Haematopoietic stem cells (HSCs) and their subsequent progenitors produce blood cells, but the precise nature and kinetics of this production is a contentious issue.

Abstract: In one model, lymphoid and myeloid production branch after the lymphoid-primed multipotent progenitor (LMPP), with both branches subsequently producing dendritic cells.

Abstract: However, this model is based mainly on in vitro clonal assays and population-based tracking in vivo, which could miss in vivo single-cell complexity.

Abstract: Here we avoid these issues by using a new quantitative version of ‘cellular barcoding’ to trace the in vivo fate of hundreds of LMPPs and HSCs at the single-cell level.

Abstract: These data demonstrate that LMPPs are highly heterogeneous in the cell types that they produce, separating into combinations of lymphoid-, myeloid- and dendritic-cell-biased producers.

Abstract: Conversely, although we observe a known lineage bias of some HSCs, most cellular output is derived from a small number of HSCs that each generates all cell types.

Abstract: Crucially, in vivo analysis of the output of sibling cells derived from single LMPPs shows that they often share a similar fate, suggesting that the fate of these progenitors was imprinted.

Abstract: Furthermore, as this imprinting is also observed for dendritic-cell-biased LMPPs, dendritic cells may be considered a distinct lineage on the basis of separate ancestry.

Abstract: These data suggest a ‘graded commitment’ model of haematopoiesis, in which heritable and diverse lineage imprinting occurs earlier than previously thought.


Crowding induces live cell extrusion to maintain homeostatic cell numbers in epithelia

Abstract: For an epithelium to provide a protective barrier, it must maintain homeostatic cell numbers by matching the number of dividing cells with the number of dying cells.

Abstract: Although compensatory cell division can be triggered by dying cells, it is unknown how cell death might relieve overcrowding due to proliferation.

Abstract: When we trigger apoptosis in epithelia, dying cells are extruded to preserve a functional barrier.

Abstract: Extrusion occurs by cells destined to die signalling to surrounding epithelial cells to contract an actomyosin ring that squeezes the dying cell out.

Abstract: However, it is not clear what drives cell death during normal homeostasis.

Abstract: Here we show in human, canine and zebrafish cells that overcrowding due to proliferation and migration induces extrusion of live cells to control epithelial cell numbers.

Abstract: Extrusion of live cells occurs at sites where the highest crowding occurs in vivo and can be induced by experimentally overcrowding monolayers in vitro.

Abstract: Like apoptotic cell extrusion, live cell extrusion resulting from overcrowding also requires sphingosine 1-phosphate signalling and Rho-kinase-dependent myosin contraction, but is distinguished by signalling through stretch-activated channels.

Abstract: Moreover, disruption of a stretch-activated channel, Piezo1, in zebrafish prevents extrusion and leads to the formation of epithelial cell masses.

Abstract: Our findings reveal that during homeostatic turnover, growth and division of epithelial cells on a confined substratum cause overcrowding that leads to their extrusion and consequent death owing to the loss of survival factors.

Abstract: These results suggest that live cell extrusion could be a tumour-suppressive mechanism that prevents the accumulation of excess epithelial cells.


Direct observation of ligand recognition by T cells

Abstract: The activation of T cells through interaction of their T-cell receptors with antigenic peptide bound to major histocompatibility complex (MHC) on the surface of antigen presenting cells (APCs) is a crucial step in adaptive immunity.

Abstract: Here we use three-dimensional fluorescence microscopy to visualize individual peptide–I-Ek class II MHC complexes labelled with the phycobiliprotein phycoerythrin in an effort to characterize T-cell sensitivity and the requirements for forming an immunological synapse in single cells.

Abstract: We show that T cells expressing the CD4 antigen respond with transient calcium signalling to even a single agonist peptide–MHC ligand, and that the organization of molecules in the contact zone of the T cell and APC takes on the characteristics of an immunological synapse when only about ten agonists are present.

Abstract: This sensitivity is highly dependant on CD4, because blocking this molecule with antibodies renders T cells unable to detect less than about 30 ligands.


Crypt stem cells as the cells-of-origin of intestinal cancer

Abstract: Intestinal cancer is initiated by Wnt-pathway-activating mutations in genes such as adenomatous polyposis coli (APC).

Abstract: As in most cancers, the cell of origin has remained elusive.

Abstract: In a previously established Lgr5 (leucine-rich-repeat containing G-protein-coupled receptor 5) knockin mouse model, a tamoxifen-inducible Cre recombinase is expressed in long-lived intestinal stem cells.

Abstract: Here we show that deletion of Apc in these stem cells leads to their transformation within days.

Abstract: Transformed stem cells remain located at crypt bottoms, while fuelling a growing microadenoma.

Abstract: These microadenomas show unimpeded growth and develop into macroscopic adenomas within 3-5weeks.

Abstract: The distribution of Lgr5+ cells within stem-cell-derived adenomas indicates that a stem cell/progenitor cell hierarchy is maintained in early neoplastic lesions.

Abstract: When Apc is deleted in short-lived transit-amplifying cells using a different cre mouse, the growth of the induced microadenomas rapidly stalls.

Abstract: Even after 30weeks, large adenomas are very rare in these mice.

Abstract: We conclude that stem-cell-specific loss of Apc results in progressively growing neoplasia.


Microbial engineering for the production of advanced biofuels

Abstract: Advanced biofuels produced by microorganisms have similar properties to petroleum-based fuels, and can 'drop in' to the existing transportation infrastructure.

Abstract: However, producing these biofuels in yields high enough to be useful requires the engineering of the microorganism's metabolism.

Abstract: Such engineering is not based on just one specific feedstock or host organism.

Abstract: Data-driven and synthetic-biology approaches can be used to optimize both the host and pathways to maximize fuel production.

Abstract: Despite some success, challenges still need to be met to move advanced biofuels towards commercialization, and to compete with more conventional fuels.


Sperm chromatin proteomics identifies evolutionarily conserved fertility factors

Abstract: Male infertility is a long-standing enigma of significant medical concern.

Abstract: The integrity of sperm chromatin is a clinical indicator of male fertility and in vitro fertilization potential: chromosome aneuploidy and DNA decondensation or damage are correlated with reproductive failure.

Abstract: Identifying conserved proteins important for sperm chromatin structure and packaging can reveal universal causes of infertility.

Abstract: Here we combine proteomics, cytology and functional analysis in Caenorhabditis elegans to identify spermatogenic chromatin-associated proteins that are important for fertility.

Abstract: Our strategy employed multiple steps: purification of chromatin from comparable meiotic cell types, namely those undergoing spermatogenesis or oogenesis; proteomic analysis by multidimensional protein identification technology (MudPIT) of factors that co-purify with chromatin; prioritization of sperm proteins based on abundance; and subtraction of common proteins to eliminate general chromatin and meiotic factors.

Abstract: Our approach reduced 1,099 proteins co-purified with spermatogenic chromatin, currently the most extensive catalogue, to 132 proteins for functional analysis.

Abstract: Reduction of gene function through RNA interference coupled with protein localization studies revealed conserved spermatogenesis-specific proteins vital for DNA compaction, chromosome segregation, and fertility.

Abstract: Unexpected roles in spermatogenesis were also detected for factors involved in other processes.

Abstract: Our strategy to find fertility factors conserved from C. elegans to mammals achieved its goal: of mouse gene knockouts corresponding to nematode proteins, 37% (7/19) cause male sterility.

Abstract: Our list therefore provides significant opportunity to identify causes of male infertility and targets for male contraceptives.


Nanog safeguards pluripotency and mediates germline development

Abstract: Nanog is a divergent homeodomain protein found in mammalian pluripotent cells and developing germ cells.

Abstract: Deletion of Nanog causes early embryonic lethality, whereas constitutive expression enables autonomous self-renewal of embryonic stem cells.

Abstract: Nanog is accordingly considered a core element of the pluripotent transcriptional network.

Abstract: However, here we report that Nanog fluctuates in mouse embryonic stem cells.

Abstract: Transient downregulation of Nanog appears to predispose cells towards differentiation but does not mark commitment.

Abstract: By genetic deletion we show that, although they are prone to differentiate, embryonic stem cells can self-renew indefinitely in the permanent absence of Nanog.

Abstract: Expanded Nanog null cells colonize embryonic germ layers and exhibit multilineage differentiation both in fetal and adult chimaeras.

Abstract: Although they are also recruited to the germ line, primordial germ cells lacking Nanog fail to mature on reaching the genital ridge.

Abstract: This defect is rescued by repair of the mutant allele.

Abstract: Thus Nanog is dispensible for expression of somatic pluripotency but is specifically required for formation of germ cells.

Abstract: Nanog therefore acts primarily in construction of inner cell mass and germ cell states rather than in the housekeeping machinery of pluripotency.

Abstract: We surmise that Nanog stabilizes embryonic stem cells in culture by resisting or reversing alternative gene expression states.


Succinate is an inflammatory signal that induces IL-1β through HIF-1α

Abstract: Macrophages activated by the Gram-negative bacterial product lipopolysaccharide switch their core metabolism from oxidative phosphorylation to glycolysis.

Abstract: Here we show that inhibition of glycolysis with 2-deoxyglucose suppresses lipopolysaccharide-induced interleukin-1β but not tumour-necrosis factor-α in mouse macrophages.

Abstract: A comprehensive metabolic map of lipopolysaccharide-activated macrophages shows upregulation of glycolytic and downregulation of mitochondrial genes, which correlates directly with the expression profiles of altered metabolites.

Abstract: Lipopolysaccharide strongly increases the levels of the tricarboxylic-acid cycle intermediate succinate.

Abstract: Glutamine-dependent anerplerosis is the principal source of succinate, although the ‘GABA (γ-aminobutyric acid) shunt’ pathway also has a role.

Abstract: Lipopolysaccharide-induced succinate stabilizes hypoxia-inducible factor-1α, an effect that is inhibited by 2-deoxyglucose, with interleukin-1β as an important target.

Abstract: Lipopolysaccharide also increases succinylation of several proteins.

Abstract: We therefore identify succinate as a metabolite in innate immune signalling, which enhances interleukin-1β production during inflammation.


Planar cell polarity signalling couples cell division and morphogenesis during neurulation

Abstract: Environmental and genetic aberrations lead to neural tube closure defects (NTDs) in 1 out of every 1,000 births.

Abstract: Mouse and frog models for these birth defects have indicated that Van Gogh-like 2 (Vangl2, also known as Strabismus) and other components of planar cell polarity (PCP) signalling might control neurulation by promoting the convergence of neural progenitors to the midline.

Abstract: Here we show a novel role for PCP signalling during neurulation in zebrafish.

Abstract: We demonstrate that non-canonical Wnt/PCP signalling polarizes neural progenitors along the anteroposterior axis.

Abstract: This polarity is transiently lost during cell division in the neural keel but is re-established as daughter cells reintegrate into the neuroepithelium.

Abstract: Loss of zebrafish Vangl2 (in trilobite mutants) abolishes the polarization of neural keel cells, disrupts re-intercalation of daughter cells into the neuroepithelium, and results in ectopic neural progenitor accumulations and NTDs.

Abstract: Remarkably, blocking cell division leads to rescue of trilobite neural tube morphogenesis despite persistent defects in convergence and extension.

Abstract: These results reveal a function for PCP signalling in coupling cell division and morphogenesis at neurulation and indicate a previously unrecognized mechanism that might underlie NTDs.


The challenge of new drug discovery for tuberculosis

Abstract: Tuberculosis (TB) is more prevalent in the world today than at any other time in human history.

Abstract: Mycobacterium tuberculosis, the pathogen responsible for TB, uses diverse strategies to survive in a variety of host lesions and to evade immune surveillance.

Abstract: A key question is how robust are our approaches to discovering new TB drugs, and what measures could be taken to reduce the long and protracted clinical development of new drugs.

Abstract: The emergence of multi-drug-resistant strains of M. tuberculosis makes the discovery of new molecular scaffolds a priority, and the current situation even necessitates the re-engineering and repositioning of some old drug families to achieve effective control.

Abstract: Whatever the strategy used, success will depend largely on our proper understanding of the complex interactions between the pathogen and its human host.

Abstract: In this review, we discuss innovations in TB drug discovery and evolving strategies to bring newer agents more quickly to patients.


Subunit Arrangement and Phenylethanolamine Binding in GluN1/GluN2B NMDA Receptors

Abstract: Since it was discovered that the anti-hypertensive agent ifenprodil has neuroprotective activity through its effects on NMDA (N-methyl-D-aspartate) receptors, a determined effort has been made to understand the mechanism of action and to develop improved therapeutic compounds on the basis of this knowledge.

Abstract: Neurotransmission mediated by NMDA receptors is essential for basic brain development and function.

Abstract: These receptors form heteromeric ion channels and become activated after concurrent binding of glycine and glutamate to the GluN1 and GluN2 subunits, respectively.

Abstract: A functional hallmark of NMDA receptors is that their ion-channel activity is allosterically regulated by binding of small compounds to the amino-terminal domain (ATD) in a subtype-specific manner.

Abstract: Ifenprodil and related phenylethanolamine compounds, which specifically inhibit GluN1 and GluN2B NMDA receptors, have been intensely studied for their potential use in the treatment of various neurological disorders and diseases, including depression, Alzheimer's disease and Parkinson's disease.

Abstract: Despite considerable enthusiasm, mechanisms underlying the recognition of phenylethanolamines and ATD-mediated allosteric inhibition remain limited owing to a lack of structural information.

Abstract: Here we report that the GluN1 and GluN2B ATDs form a heterodimer and that phenylethanolamine binds at the interface between GluN1 and GluN2B, rather than within the GluN2B cleft.

Abstract: The crystal structure of the heterodimer formed between the GluN1b ATD from Xenopus laevis and the GluN2B ATD from Rattus norvegicus shows a highly distinct pattern of subunit arrangement that is different from the arrangements observed in homodimeric non-NMDA receptors and reveals the molecular determinants for phenylethanolamine binding.

Abstract: Restriction of domain movement in the bi-lobed structure of the GluN2B ATD, by engineering of an inter-subunit disulphide bond, markedly decreases sensitivity to ifenprodil, indicating that conformational freedom in the GluN2B ATD is essential for ifenprodil-mediated allosteric inhibition of NMDA receptors.

Abstract: These findings pave the way for improving the design of subtype-specific compounds with therapeutic value for neurological disorders and diseases.


Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn's disease.

Abstract: Crohn's disease and ulcerative colitis, the two main types of chronic inflammatory bowel disease, are multifactorial conditions of unknown aetiology.

Abstract: A susceptibility locus for Crohn's disease has been mapped to chromosome 16.

Abstract: Here we have used a positional-cloning strategy, based on linkage analysis followed by linkage disequilibrium mapping, to identify three independent associations for Crohn's disease: a frameshift variant and two missense variants of NOD2, encoding a member of the Apaf-1/Ced-4 superfamily of apoptosis regulators that is expressed in monocytes.

Abstract: These NOD2 variants alter the structure of either the leucine-rich repeat domain of the protein or the adjacent region.

Abstract: NOD2 activates nuclear factor NF-kB; this activating function is regulated by the carboxy-terminal leucine-rich repeat domain, which has an inhibitory role and also acts as an intracellular receptor for components of microbial pathogens.

Abstract: These observations suggest that the NOD2 gene product confers susceptibility to Crohn's disease by altering the recognition of these components and/or by over-activating NF-kB in monocytes, thus documenting a molecular model for the pathogenic mechanism of Crohn's disease that can now be further investigated.


Human cardiovascular progenitor cells develop from a KDR+ embryonic-stem-cell-derived population

Abstract: The functional heart is comprised of distinct mesoderm-derived lineages including cardiomyocytes, endothelial cells and vascular smooth muscle cells.

Abstract: Studies in the mouse embryo and the mouse embryonic stem cell differentiation model have provided evidence indicating that these three lineages develop from a common Flk-1+ (kinase insert domain protein receptor, also known as Kdr) cardiovascular progenitor that represents one of the earliest stages in mesoderm specification to the cardiovascular lineages.

Abstract: To determine whether a comparable progenitor is present during human cardiogenesis, we analysed the development of the cardiovascular lineages in human embryonic stem cell differentiation cultures.

Abstract: Here we show that after induction with combinations of activin A, bone morphogenetic protein 4 (BMP4), basic fibroblast growth factor (bFGF, also known as FGF2), vascular endothelial growth factor (VEGF, also known as VEGFA) and dickkopf homolog 1 (DKK1) in serum-free media, human embryonic-stem-cell-derived embryoid bodies generate a KDRlow/C-KIT(CD117)neg population that displays cardiac, endothelial and vascular smooth muscle potential in vitro and, after transplantation, in vivo.

Abstract: When plated in monolayer cultures, these KDRlow/C-KITneg cells differentiate to generate populations consisting of greater than 50% contracting cardiomyocytes.

Abstract: Populations derived from the KDRlow/C-KITneg fraction give rise to colonies that contain all three lineages when plated in methylcellulose cultures.

Abstract: Results from limiting dilution studies and cell-mixing experiments support the interpretation that these colonies are clones, indicating that they develop from a cardiovascular colony-forming cell.

Abstract: Together, these findings identify a human cardiovascular progenitor that defines one of the earliest stages of human cardiac development.


Cancer-related inflammation

Abstract: The mediators and cellular effectors of inflammation are important constituents of the local environment of tumours.

Abstract: In some types of cancer, inflammatory conditions are present before a malignant change occurs.

Abstract: Conversely, in other types of cancer, an oncogenic change induces an inflammatory microenvironment that promotes the development of tumours.

Abstract: Regardless of its origin, 'smouldering' inflammation in the tumour microenvironment has many tumour-promoting effects.

Abstract: It aids in the proliferation and survival of malignant cells, promotes angiogenesis and metastasis, subverts adaptive immune responses, and alters responses to hormones and chemotherapeutic agents.

Abstract: The molecular pathways of this cancer-related inflammation are now being unravelled, resulting in the identification of new target molecules that could lead to improved diagnosis and treatment.


ESCRT-III recognition by VPS4 ATPases

Abstract: The ESCRT (endosomal sorting complex required for transport) pathway is required for terminal membrane fission events in several important biological processes, including endosomal intraluminal vesicle formation, HIV budding and cytokinesis.

Abstract: VPS4 ATPases perform a key function in this pathway by recognizing membrane-associated ESCRT-III assemblies and catalysing their disassembly, possibly in conjunction with membrane fission.

Abstract: Here we show that the microtubule interacting and transport (MIT) domains of human VPS4A and VPS4B bind conserved sequence motifs located at the carboxy termini of the CHMP1–3 class of ESCRT-III proteins.

Abstract: Structures of VPS4A MIT–CHMP1A and VPS4B MIT–CHMP2B complexes reveal that the C-terminal CHMP motif forms an amphipathic helix that binds in a groove between the last two helices of the tetratricopeptide-like repeat (TPR) of the VPS4 MIT domain, but in the opposite orientation to that of a canonical TPR interaction.

Abstract: Distinct pockets in the MIT domain bind three conserved leucine residues of the CHMP motif, and mutations that inhibit these interactions block VPS4 recruitment, impair endosomal protein sorting and relieve dominant-negative VPS4 inhibition of HIV budding.

Abstract: Thus, our studies reveal how the VPS4 ATPases recognize their CHMP substrates to facilitate the membrane fission events required for the release of viruses, endosomal vesicles and daughter cells.


Pif1 family helicases suppress genome instability at G-quadruplex motifs

Abstract: The Saccharomyces cerevisiae Pif1 helicase is the prototypical member of the Pif1 DNA helicase family, which is conserved from bacteria to humans.

Abstract: Here we show that exceptionally potent G-quadruplex unwinding is conserved among Pif1 helicases.

Abstract: Moreover, Pif1 helicases from organisms separated by more than 3 billion years of evolution suppressed DNA damage at G-quadruplex motifs in yeast.

Abstract: The G-quadruplex-induced damage generated in the absence of Pif1 helicases led to new genetic and epigenetic changes.

Abstract: Furthermore, when expressed in yeast, human PIF1 suppressed both G-quadruplex-associated DNA damage and telomere lengthening.


Lysyl oxidase is essential for hypoxia-induced metastasis

Abstract: Metastasis is a multistep process responsible for most cancer deaths, and it can be influenced by both the immediate microenvironment (cell–cell or cell–matrix interactions) and the extended tumour microenvironment (for example vascularization).

Abstract: Hypoxia (low oxygen) is clinically associated with metastasis and poor patient outcome, although the underlying processes remain unclear.

Abstract: Microarray studies have shown the expression of lysyl oxidase (LOX) to be elevated in hypoxic human tumour cells.

Abstract: Paradoxically, LOX expression is associated with both tumour suppression and tumour progression, and its role in tumorigenesis seems dependent on cellular location, cell type and transformation status.

Abstract: Here we show that LOX expression is regulated by hypoxia-inducible factor (HIF) and is associated with hypoxia in human breast and head and neck tumours.

Abstract: Patients with high LOX-expressing tumours have poor distant metastasis-free and overall survivals.

Abstract: Inhibition of LOX eliminates metastasis in mice with orthotopically grown breast cancer tumours.

Abstract: Mechanistically, secreted LOX is responsible for the invasive properties of hypoxic human cancer cells through focal adhesion kinase activity and cell to matrix adhesion.

Abstract: Furthermore, LOX may be required to create a niche permissive for metastatic growth.

Abstract: Our findings indicate that LOX is essential for hypoxia-induced metastasis and is a good therapeutic target for preventing and treating metastases.


Identification of human brain tumour initiating cells

Abstract: The cancer stem cell (CSC) hypothesis suggests that neoplastic clones are maintained exclusively by a rare fraction of cells with stem cell properties.

Abstract: Although the existence of CSCs in human leukaemia is established, little evidence exists for CSCs in solid tumours, except for breast cancer.

Abstract: Recently, we prospectively isolated a CD133+ cell subpopulation from human brain tumours that exhibited stem cell properties in vitro.

Abstract: However, the true measures of CSCs are their capacity for self renewal and exact recapitulation of the original tumour.

Abstract: Here we report the development of a xenograft assay that identified human brain tumour initiating cells that initiate tumours in vivo.

Abstract: Only the CD133+ brain tumour fraction contains cells that are capable of tumour initiation in NOD-SCID (non-obese diabetic, severe combined immunodeficient) mouse brains.

Abstract: Injection of as few as 100 CD133+ cells produced a tumour that could be serially transplanted and was a phenocopy of the patient's original tumour, whereas injection of 105 CD133- cells engrafted but did not cause a tumour.

Abstract: Thus, the identification of brain tumour initiating cells provides insights into human brain tumour pathogenesis, giving strong support for the CSC hypothesis as the basis for many solid tumours, and establishes a previously unidentified cellular target for more effective cancer therapies.


Anthropometric reference data for international use: recommendations from a World Health Organization Expert Committee.

Abstract: The World Health Organization (WHO) convened an Expert Committee to reevaluate the use of anthropometry at different ages for assessing health, nutrition, and social wellbeing.

Abstract: The Committee's task included identifying reference data for anthropometric indexes when appropriate, and providing guidelines on how the data should be used.

Abstract: For fetal growth, the Committee recommended an existing sex-specific multiracial reference.

Abstract: In view of the significant technical drawbacks of the current National Center for Health Statistics (NCHS)/WHO reference and its inadequacy for assessing the growth of breast-fed infants, the Committee recommended the development of a new reference concerning weight and length/height for infants and children, which will be a complex and costly undertaking.

Abstract: Proper interpretation of midupper arm circumference for preschoolers requires age-specific reference data.

Abstract: To evaluate adolescent height-for-age, the Committee recommended the current NCHS/WHO reference.

Abstract: Use of the NCHS body mass index (BMI) data, with their upper percentile elevations and skewness, is undesirable for setting health goals; however, these data were provisionally recommended for defining obesity based on a combination of elevated BMI and high subcutaneous fat.

Abstract: The NCHS values were provisionally recommended as reference data for subscapular and triceps skinfold thicknesses.

Abstract: Guidelines were also provided for adjusting adolescent anthropometric comparisons for maturational status.

Abstract: Currently, there is no need for adult reference data for BMI; interpretation should be based on pragmatic BMI cutoffs.

Abstract: Finally, the Committee noted that few normative anthropometric data exist for the elderly, especially for those > 80 y of age.

Abstract: Proper definitions of health status, function, and biologic age remain to be developed for this group.


Diverse interventions that extend mouse lifespan suppress shared age-associated epigenetic changes at critical gene regulatory regions

Abstract: BACKGROUND Age-associated epigenetic changes are implicated in aging.

Abstract: Notably, age-associated DNA methylation changes comprise a so-called aging "clock", a robust biomarker of aging.

Abstract: However, while genetic, dietary and drug interventions can extend lifespan, their impact on the epigenome is uncharacterised.

Abstract: To fill this knowledge gap, we defined age-associated DNA methylation changes at the whole-genome, single-nucleotide level in mouse liver and tested the impact of longevity-promoting interventions, specifically the Ames dwarf Prop1 df/df mutation, calorie restriction and rapamycin.

Abstract: RESULTS In wild-type mice fed an unsupplemented ad libitum diet, age-associated hypomethylation was enriched at super-enhancers in highly expressed genes critical for liver function.

Abstract: Genes harbouring hypomethylated enhancers were enriched for genes that change expression with age.

Abstract: Hypermethylation was enriched at CpG islands marked with bivalent activating and repressing histone modifications and resembled hypermethylation in liver cancer.

Abstract: Age-associated methylation changes are suppressed in Ames dwarf and calorie restricted mice and more selectively and less specifically in rapamycin treated mice.

Abstract: CONCLUSIONS Age-associated hypo- and hypermethylation events occur at distinct regulatory features of the genome.

Abstract: Distinct longevity-promoting interventions, specifically genetic, dietary and drug interventions, suppress some age-associated methylation changes, consistent with the idea that these interventions exert their beneficial effects, in part, by modulation of the epigenome.

Abstract: This study is a foundation to understand the epigenetic contribution to healthy aging and longevity and the molecular basis of the DNA methylation clock.


Extracellular vesicles for drug delivery.

Abstract: Extracellular vesicles (EVs) are cell-derived membrane vesicles, and represent an endogenous mechanism for intercellular communication.

Abstract: Since the discovery that EVs are capable of functionally transferring biological information, the potential use of EVs as drug delivery vehicles has gained considerable scientific interest.

Abstract: EVs may have multiple advantages over currently available drug delivery vehicles, such as their ability to overcome natural barriers, their intrinsic cell targeting properties, and stability in the circulation.

Abstract: However, therapeutic applications of EVs as drug delivery systems have been limited due to a lack of methods for scalable EV isolation and efficient drug loading.

Abstract: Furthermore, in order to achieve targeted drug delivery, their intrinsic cell targeting properties should be tuned through EV engineering.

Abstract: Here, we review and discuss recent progress and remaining challenges in the development of EVs as drug delivery vehicles.


Effects of soy isoflavones and phytate on homocysteine, C-reactive protein, and iron status in postmenopausal women.

Abstract: BACKGROUND Soy protein or its components may protect against the atherosclerotic cardiovascular disease (CVD) risk factors total homocysteine (tHcy), C-reactive protein (CRP), and excess body iron, which generally increase with menopause.

Abstract: OBJECTIVE The primary objective of this study was to determine the independent effect of the soy protein components isoflavones and phytate on CVD risk factors in postmenopausal women.

Abstract: The secondary objective was to identify factors [blood lipids, oxidative stress indexes, serum ferritin, plasma folate, plasma vitamin B-12, and body mass index (BMI)] contributing to tHcy and CRP concentrations.

Abstract: DESIGN In a double-blind, 6-wk study, 55 postmenopausal women aged 47-72 y were randomly assigned to 1 of 4 soy protein (40 g/d) isolate treatments: native phytate and native isoflavone (n = 14), native phytate and low isoflavone (n = 13), low phytate and native isoflavone (n = 14), or low phytate and low isoflavone (n = 14).

Abstract: We measured iron indexes, tHcy, CRP, and BMI.

Abstract: RESULTS Soy protein with native phytate significantly reduced tHcy (P = 0.017), transferrin saturation (P = 0.027), and ferritin (P = 0.029), whereas soy protein with native isoflavones had no effect on any variables.

Abstract: At baseline, BMI was highly correlated with tHcy (r = 0.39, P = 0.003) and CRP (r = 0.55, P < 0.0001), whereas HDL cholesterol was correlated with CRP (r = -0.30, P = 0.02).

Abstract: Multiple regression analysis showed that LDL cholesterol and BMI contributed significantly (R2= 19.9%, P = 0.003) to the overall variance in tHcy.

Abstract: CONCLUSION Consuming phytate-rich foods and maintaining a healthy weight may reduce atherosclerotic CVD risk factors in postmenopausal women.


Replication Fork Stability Confers Chemoresistance in BRCA-deficient Cells

Abstract: Cells deficient in the Brca1 and Brca2 genes have reduced capacity to repair DNA double-strand breaks by homologous recombination and consequently are hypersensitive to DNA-damaging agents, including cisplatin and poly(ADP-ribose) polymerase (PARP) inhibitors.

Abstract: Here we show that loss of the MLL3/4 complex protein, PTIP, protects Brca1/2-deficient cells from DNA damage and rescues the lethality of Brca2-deficient embryonic stem cells.

Abstract: However, PTIP deficiency does not restore homologous recombination activity at double-strand breaks.

Abstract: Instead, its absence inhibits the recruitment of the MRE11 nuclease to stalled replication forks, which in turn protects nascent DNA strands from extensive degradation.

Abstract: More generally, acquisition of PARP inhibitors and cisplatin resistance is associated with replication fork protection in Brca2-deficient tumour cells that do not develop Brca2 reversion mutations.

Abstract: Disruption of multiple proteins, including PARP1 and CHD4, leads to the same end point of replication fork protection, highlighting the complexities by which tumour cells evade chemotherapeutic interventions and acquire drug resistance.


Plasma Corin as a Predictor of Cardiovascular Events in Patients With Chronic Heart Failure.

Abstract: OBJECTIVES The aim of this study was to determine the prognostic value of plasma corin in patients with chronic heart failure (CHF).

Abstract: BACKGROUND In recent years, accumulating evidence has indicated that corin plays a critical role in regulating blood pressure and cardiac function.

Abstract: METHODS We enrolled 1,148 consecutive CHF patients in a prospective cohort study and explored the association between plasma corin levels and clinical prognosis using multivariate Cox regression analysis.

Abstract: RESULTS Patients with low corin levels (<458 pg/ml) were more likely to be women and to be hypertensive.

Abstract: Low corin was found to be associated with an increase in New York Heart Association (NYHA) functional class and N-terminal pro-B-type natriuretic peptide (NT-proBNP) levels, and a decrease in left ventricular ejection fraction (LVEF) and the estimated glomerular filtration rate (eGFR).

Abstract: Multivariate Cox regression analysis suggested that log corin was an independent predictor of major adverse cardiac event(s) (MACE) (hazard ratio: 0.62; 95% confidence interval: 0.39 to 0.95), together with age, diabetes, NYHA functional class, LVEF, eGFR, and log NT-proBNP.

Abstract: In addition, log corin was also a significant predictor for cardiovascular death (p = 0.041) and heart failure rehospitalization (p = 0.015) after adjustment for clinical variables and established biomarkers of adverse prognosis.

Abstract: The Kaplan-Meier survival curves showed that low corin was a significant predictor of MACE in patients with NT-proBNP levels above and below the median.

Abstract: CONCLUSIONS Our study demonstrates that plasma corin is a valuable prognostic marker of MACE in patients with CHF, independent of established conventional risk factors.


Cryo-EM structures of Tau filaments from Alzheimer’s disease brain

Abstract: Alzheimer's disease is the most common neurodegenerative disease, and there are no mechanism-based therapies.

Abstract: The disease is defined by the presence of abundant neurofibrillary lesions and neuritic plaques in the cerebral cortex.

Abstract: Neurofibrillary lesions comprise paired helical and straight tau filaments, whereas tau filaments with different morphologies characterize other neurodegenerative diseases.

Abstract: No high-resolution structures of tau filaments are available.

Abstract: Here we present cryo-electron microscopy (cryo-EM) maps at 3.4-3.5 Å resolution and corresponding atomic models of paired helical and straight filaments from the brain of an individual with Alzheimer's disease.

Abstract: Filament cores are made of two identical protofilaments comprising residues 306-378 of tau protein, which adopt a combined cross-β/β-helix structure and define the seed for tau aggregation.

Abstract: Paired helical and straight filaments differ in their inter-protofilament packing, showing that they are ultrastructural polymorphs.

Abstract: These findings demonstrate that cryo-EM allows atomic characterization of amyloid filaments from patient-derived material, and pave the way for investigation of a range of neurodegenerative diseases.


Modulation of the proteoglycan receptor PTPσ promotes recovery after spinal cord injury

Abstract: Contusive spinal cord injury leads to a variety of disabilities owing to limited neuronal regeneration and functional plasticity.

Abstract: It is well established that an upregulation of glial-derived chondroitin sulphate proteoglycans (CSPGs) within the glial scar and perineuronal net creates a barrier to axonal regrowth and sprouting.

Abstract: Protein tyrosine phosphatase σ (PTPσ), along with its sister phosphatase leukocyte common antigen-related (LAR) and the nogo receptors 1 and 3 (NgR), have recently been identified as receptors for the inhibitory glycosylated side chains of CSPGs.

Abstract: Here we find in rats that PTPσ has a critical role in converting growth cones into a dystrophic state by tightly stabilizing them within CSPG-rich substrates.

Abstract: We generated a membrane-permeable peptide mimetic of the PTPσ wedge domain that binds to PTPσ and relieves CSPG-mediated inhibition.

Abstract: Systemic delivery of this peptide over weeks restored substantial serotonergic innervation to the spinal cord below the level of injury and facilitated functional recovery of both locomotor and urinary systems.

Abstract: Our results add a new layer of understanding to the critical role of PTPσ in mediating the growth-inhibited state of neurons due to CSPGs within the injured adult spinal cord.


A gp130–Src–YAP module links inflammation to epithelial regeneration

Abstract: Inflammation promotes regeneration of injured tissues through poorly understood mechanisms, some of which involve interleukin (IL)-6 family members, the expression of which is elevated in many diseases including inflammatory bowel diseases and colorectal cancer.

Abstract: Here we show in mice and human cells that gp130, a co-receptor for IL-6 cytokines, triggers activation of YAP and Notch, transcriptional regulators that control tissue growth and regeneration, independently of the gp130 effector STAT3.

Abstract: Through YAP and Notch, intestinal gp130 signalling stimulates epithelial cell proliferation, causes aberrant differentiation and confers resistance to mucosal erosion.

Abstract: gp130 associates with the related tyrosine kinases Src and Yes, which are activated on receptor engagement to phosphorylate YAP and induce its stabilization and nuclear translocation.

Abstract: This signalling module is strongly activated upon mucosal injury to promote healing and maintain barrier function.


Hemoglobin concentration in white, black, and Oriental children: is there a need for separate criteria in screening for anemia?

Abstract: The concentration of hemoglobin in blacks was found to be 0.5 to 1.0 g/dl lower than that of income-matched whites in several large surveys.

Abstract: This difference could be a racial characteristic of blacks, or it might be due to a higher frequency of genetic traits such as thalassemia minor and hemoglobinopathies, or to environmental factors such as iron deficiency.

Abstract: To help in making this distinction, we analyzed the data from multiphasic examinations (1973 to 1975) on 1718 white, 741 black, and 315 Oriental healthy, nonindigent children between 5 and 14 years of age.

Abstract: In the entire population, the median hemoglobin concentration averaged 0.5 g/dl lower in blacks than in whites of both sexes (t test, P less than 0.001).

Abstract: The differences still averaged 0.5 g/dl (P less than 0.001) after exclusion of all those with abnormal hemoglobin by electrophoresis (Hgb S and C) and those whose mean corpuscular volume was more than 5% below the normal mean for age (to exclude iron deficiency or thalassemia minor).

Abstract: The data strengthen the impression that blacks normally have a concentration of hemoglobin averaging about 0.5 g/dl less than in whites.

Abstract: If this is the case, about 10% of normal blacks will be mistakenly designated anemic, if the same norms are applied.


Divergent reprogramming routes lead to alternative stem-cell states

Abstract: Pluripotency is defined by the ability of a cell to differentiate to the derivatives of all the three embryonic germ layers: ectoderm, mesoderm and endoderm.

Abstract: Pluripotent cells can be captured via the archetypal derivation of embryonic stem cells or via somatic cell reprogramming.

Abstract: Somatic cells are induced to acquire a pluripotent stem cell (iPSC) state through the forced expression of key transcription factors, and in the mouse these cells can fulfil the strictest of all developmental assays for pluripotent cells by generating completely iPSC-derived embryos and mice.

Abstract: However, it is not known whether there are additional classes of pluripotent cells, or what the spectrum of reprogrammed phenotypes encompasses.

Abstract: Here we explore alternative outcomes of somatic reprogramming by fully characterizing reprogrammed cells independent of preconceived definitions of iPSC states.

Abstract: We demonstrate that by maintaining elevated reprogramming factor expression levels, mouse embryonic fibroblasts go through unique epigenetic modifications to arrive at a stable, Nanog-positive, alternative pluripotent state.

Abstract: In doing so, we prove that the pluripotent spectrum can encompass multiple, unique cell states.


Autophagy mediates degradation of nuclear lamina

Abstract: Macroautophagy (hereafter referred to as autophagy) is a catabolic membrane trafficking process that degrades a variety of cellular constituents and is associated with human diseases.

Abstract: Although extensive studies have focused on autophagic turnover of cytoplasmic materials, little is known about the role of autophagy in degrading nuclear components.

Abstract: Here we report that the autophagy machinery mediates degradation of nuclear lamina components in mammals.

Abstract: The autophagy protein LC3/Atg8, which is involved in autophagy membrane trafficking and substrate delivery, is present in the nucleus and directly interacts with the nuclear lamina protein lamin B1, and binds to lamin-associated domains on chromatin.

Abstract: This LC3-lamin B1 interaction does not downregulate lamin B1 during starvation, but mediates its degradation upon oncogenic insults, such as by activated RAS.

Abstract: Lamin B1 degradation is achieved by nucleus-to-cytoplasm transport that delivers lamin B1 to the lysosome.

Abstract: Inhibiting autophagy or the LC3-lamin B1 interaction prevents activated RAS-induced lamin B1 loss and attenuates oncogene-induced senescence in primary human cells.

Abstract: Our study suggests that this new function of autophagy acts as a guarding mechanism protecting cells from tumorigenesis.


Pro-resolving lipid mediators are leads for resolution physiology

Abstract: Advances in our understanding of the mechanisms that bring about the resolution of acute inflammation have uncovered a new genus of pro-resolving lipid mediators that include the lipoxin, resolvin, protectin and maresin families, collectively called specialized pro-resolving mediators.

Abstract: Synthetic versions of these mediators have potent bioactions when administered in vivo.

Abstract: In animal experiments, the mediators evoke anti-inflammatory and novel pro-resolving mechanisms, and enhance microbial clearance.

Abstract: Although they have been identified in inflammation resolution, specialized pro-resolving mediators are conserved structures that also function in host defence, pain, organ protection and tissue remodelling.

Abstract: This Review covers the mechanisms of specialized pro-resolving mediators and omega-3 essential fatty acid pathways that could help us to understand their physiological functions.


ZMYND11 links histone H3.3K36me3 to transcription elongation and tumour suppression

Abstract: Recognition of modified histones by ‘reader’ proteins plays a critical role in the regulation of chromatin.

Abstract: H3K36 trimethylation (H3K36me3) is deposited onto the nucleosomes in the transcribed regions after RNA polymerase II elongation.

Abstract: In yeast, this mark in turn recruits epigenetic regulators to reset the chromatin to a relatively repressive state, thus suppressing cryptic transcription.

Abstract: However, much less is known about the role of H3K36me3 in transcription regulation in mammals.

Abstract: This is further complicated by the transcription-coupled incorporation of the histone variant H3.3 in gene bodies.

Abstract: Here we show that the candidate tumour suppressor ZMYND11 specifically recognizes H3K36me3 on H3.3 (H3.3K36me3) and regulates RNA polymerase II elongation.

Abstract: Structural studies show that in addition to the trimethyl-lysine binding by an aromatic cage within the PWWP domain, the H3.3-dependent recognition is mediated by the encapsulation of the H3.3-specific ‘Ser 31’ residue in a composite pocket formed by the tandem bromo–PWWP domains of ZMYND11.

Abstract: Chromatin immunoprecipitation followed by sequencing shows a genome-wide co-localization of ZMYND11 with H3K36me3 and H3.3 in gene bodies, and its occupancy requires the pre-deposition of H3.3K36me3.

Abstract: Although ZMYND11 is associated with highly expressed genes, it functions as an unconventional transcription co-repressor by modulating RNA polymerase II at the elongation stage.

Abstract: ZMYND11 is critical for the repression of a transcriptional program that is essential for tumour cell growth; low expression levels of ZMYND11 in breast cancer patients correlate with worse prognosis.

Abstract: Consistently, overexpression of ZMYND11 suppresses cancer cell growth in vitro and tumour formation in mice.

Abstract: Together, this study identifies ZMYND11 as an H3.3-specific reader of H3K36me3 that links the histone-variant-mediated transcription elongation control to tumour suppression.


Autocrine BDNF–TrkB signalling within a single dendritic spine

Abstract: Brain-derived neurotrophic factor (BDNF) and its receptor TrkB are crucial for many forms of neuronal plasticity, including structural long-term potentiation (sLTP), which is a correlate of an animal’s learning.

Abstract: However, it is unknown whether BDNF release and TrkB activation occur during sLTP, and if so, when and where.

Abstract: Here, using a fluorescence resonance energy transfer-based sensor for TrkB and two-photon fluorescence lifetime imaging microscopy, we monitor TrkB activity in single dendritic spines of CA1 pyramidal neurons in cultured murine hippocampal slices.

Abstract: In response to sLTP induction, we find fast (onset < 1 min) and sustained (>20 min) activation of TrkB in the stimulated spine that depends on NMDAR (N-methyl-d-aspartate receptor) and CaMKII signalling and on postsynaptically synthesized BDNF.

Abstract: We confirm the presence of postsynaptic BDNF using electron microscopy to localize endogenous BDNF to dendrites and spines of hippocampal CA1 pyramidal neurons.

Abstract: Consistent with these findings, we also show rapid, glutamate-uncaging-evoked, time-locked BDNF release from single dendritic spines using BDNF fused to superecliptic pHluorin.

Abstract: We demonstrate that this postsynaptic BDNF–TrkB signalling pathway is necessary for both structural and functional LTP.

Abstract: Together, these findings reveal a spine-autonomous, autocrine signalling mechanism involving NMDAR–CaMKII-dependent BDNF release from stimulated dendritic spines and subsequent TrkB activation on these same spines that is crucial for structural and functional plasticity.


Whole genomes redefine the mutational landscape of pancreatic cancer

Abstract: Pancreatic cancer remains one of the most lethal of malignancies and a major health burden.

Abstract: We performed whole-genome sequencing and copy number variation (CNV) analysis of 100 pancreatic ductal adenocarcinomas (PDACs).

Abstract: Chromosomal rearrangements leading to gene disruption were prevalent, affecting genes known to be important in pancreatic cancer (TP53, SMAD4, CDKN2A, ARID1A and ROBO2) and new candidate drivers of pancreatic carcinogenesis (KDM6A and PREX2).

Abstract: Patterns of structural variation (variation in chromosomal structure) classified PDACs into 4 subtypes with potential clinical utility: the subtypes were termed stable, locally rearranged, scattered and unstable.

Abstract: A significant proportion harboured focal amplifications, many of which contained druggable oncogenes (ERBB2, MET, FGFR1, CDK6, PIK3R3 and PIK3CA), but at low individual patient prevalence.

Abstract: Genomic instability co-segregated with inactivation of DNA maintenance genes (BRCA1, BRCA2 or PALB2) and a mutational signature of DNA damage repair deficiency.

Abstract: Of 8 patients who received platinum therapy, 4 of 5 individuals with these measures of defective DNA maintenance responded.


Human oocytes reprogram adult somatic nuclei of a type 1 diabetic to diploid pluripotent stem cells

Abstract: The transfer of somatic cell nuclei into oocytes can give rise to pluripotent stem cells that are consistently equivalent to embryonic stem cells, holding promise for autologous cell replacement therapy.

Abstract: Although methods to induce pluripotent stem cells from somatic cells by transcription factors are widely used in basic research, numerous differences between induced pluripotent stem cells and embryonic stem cells have been reported, potentially affecting their clinical use.

Abstract: Because of the therapeutic potential of diploid embryonic stem-cell lines derived from adult cells of diseased human subjects, we have systematically investigated the parameters affecting efficiency of blastocyst development and stem-cell derivation.

Abstract: Here we show that improvements to the oocyte activation protocol, including the use of both kinase and translation inhibitors, and cell culture in the presence of histone deacetylase inhibitors, promote development to the blastocyst stage.

Abstract: Developmental efficiency varied between oocyte donors, and was inversely related to the number of days of hormonal stimulation required for oocyte maturation, whereas the daily dose of gonadotropin or the total number of metaphase II oocytes retrieved did not affect developmental outcome.

Abstract: Because the use of concentrated Sendai virus for cell fusion induced an increase in intracellular calcium concentration, causing premature oocyte activation, we used diluted Sendai virus in calcium-free medium.

Abstract: Using this modified nuclear transfer protocol, we derived diploid pluripotent stem-cell lines from somatic cells of a newborn and, for the first time, an adult, a female with type 1 diabetes.


A three-dimensional human neural cell culture model of Alzheimer’s disease

Abstract: Alzheimer’s disease is the most common form of dementia, characterized by two pathological hallmarks: amyloid-β plaques and neurofibrillary tangles.

Abstract: The amyloid hypothesis of Alzheimer’s disease posits that the excessive accumulation of amyloid-β peptide leads to neurofibrillary tangles composed of aggregated hyperphosphorylated tau.

Abstract: However, to date, no single disease model has serially linked these two pathological events using human neuronal cells.

Abstract: Mouse models with familial Alzheimer’s disease (FAD) mutations exhibit amyloid-β-induced synaptic and memory deficits but they do not fully recapitulate other key pathological events of Alzheimer’s disease, including distinct neurofibrillary tangle pathology.

Abstract: Human neurons derived from Alzheimer’s disease patients have shown elevated levels of toxic amyloid-β species and phosphorylated tau but did not demonstrate amyloid-β plaques or neurofibrillary tangles.

Abstract: Here we report that FAD mutations in β-amyloid precursor protein and presenilin 1 are able to induce robust extracellular deposition of amyloid-β, including amyloid-β plaques, in a human neural stem-cell-derived three-dimensional (3D) culture system.

Abstract: More importantly, the 3D-differentiated neuronal cells expressing FAD mutations exhibited high levels of detergent-resistant, silver-positive aggregates of phosphorylated tau in the soma and neurites, as well as filamentous tau, as detected by immunoelectron microscopy.

Abstract: Inhibition of amyloid-β generation with β- or γ-secretase inhibitors not only decreased amyloid-β pathology, but also attenuated tauopathy.

Abstract: We also found that glycogen synthase kinase 3 (GSK3) regulated amyloid-β-mediated tau phosphorylation.

Abstract: We have successfully recapitulated amyloid-β and tau pathology in a single 3D human neural cell culture system.

Abstract: Our unique strategy for recapitulating Alzheimer’s disease pathology in a 3D neural cell culture model should also serve to facilitate the development of more precise human neural cell models of other neurodegenerative disorders.


Mesenchymal-endothelial-transition contributes to cardiac neovascularization

Abstract: Endothelial cells contribute to a subset of cardiac fibroblasts by undergoing endothelial-to-mesenchymal transition, but whether cardiac fibroblasts can adopt an endothelial cell fate and directly contribute to neovascularization after cardiac injury is not known.

Abstract: Here, using genetic fate map techniques, we demonstrate that cardiac fibroblasts rapidly adopt an endothelial-cell-like phenotype after acute ischaemic cardiac injury.

Abstract: Fibroblast-derived endothelial cells exhibit anatomical and functional characteristics of native endothelial cells.

Abstract: We show that the transcription factor p53 regulates such a switch in cardiac fibroblast fate.

Abstract: Loss of p53 in cardiac fibroblasts severely decreases the formation of fibroblast-derived endothelial cells, reduces post-infarct vascular density and worsens cardiac function.

Abstract: Conversely, stimulation of the p53 pathway in cardiac fibroblasts augments mesenchymal-to-endothelial transition, enhances vascularity and improves cardiac function.

Abstract: These observations demonstrate that mesenchymal-to-endothelial transition contributes to neovascularization of the injured heart and represents a potential therapeutic target for enhancing cardiac repair.


Estimation of optimal serum concentrations of 25-hydroxyvitamin D for multiple health outcomes.

Abstract: Recent evidence suggests that vitamin D intakes above current recommendations may be associated with better health outcomes.

Abstract: However, optimal serum concentrations of 25-hydroxyvitamin D [25(OH)D] have not been defined.

Abstract: This review summarizes evidence from studies that evaluated thresholds for serum 25(OH)D concentrations in relation to bone mineral density (BMD), lower-extremity function, dental health, and risk of falls, fractures, and colorectal cancer.

Abstract: For all endpoints, the most advantageous serum concentrations of 25(OH)D begin at 75 nmol/L (30 ng/mL), and the best are between 90 and 100 nmol/L (36-40 ng/mL).

Abstract: In most persons, these concentrations could not be reached with the currently recommended intakes of 200 and 600 IU vitamin D/d for younger and older adults, respectively.

Abstract: A comparison of vitamin D intakes with achieved serum concentrations of 25(OH)D for the purpose of estimating optimal intakes led us to suggest that, for bone health in younger adults and all studied outcomes in older adults, an increase in the currently recommended intake of vitamin D is warranted.

Abstract: An intake for all adults of > or =1000 IU (25 microg) [DOSAGE ERROR CORRECTED] vitamin D (cholecalciferol)/d is needed to bring vitamin D concentrations in no less than 50% of the population up to 75 nmol/L. The implications of higher doses for the entire adult population should be addressed in future studies.


Cohesin-dependent globules and heterochromatin shape 3D genome architecture in S. pombe

Abstract: Eukaryotic genomes are folded into three-dimensional structures, such as self-associating topological domains, the borders of which are enriched in cohesin and CCCTC-binding factor (CTCF) required for long-range interactions.

Abstract: How local chromatin interactions govern higher-order folding of chromatin fibres and the function of cohesin in this process remain poorly understood.

Abstract: Here we perform genome-wide chromatin conformation capture (Hi-C) analysis to explore the high-resolution organization of the Schizosaccharomyces pombe genome, which despite its small size exhibits fundamental features found in other eukaryotes.

Abstract: Our analyses of wild-type and mutant strains reveal key elements of chromosome architecture and genome organization.

Abstract: On chromosome arms, small regions of chromatin locally interact to form 'globules'.

Abstract: This feature requires a function of cohesin distinct from its role in sister chromatid cohesion.

Abstract: Cohesin is enriched at globule boundaries and its loss causes disruption of local globule structures and global chromosome territories.

Abstract: By contrast, heterochromatin, which loads cohesin at specific sites including pericentromeric and subtelomeric domains, is dispensable for globule formation but nevertheless affects genome organization.

Abstract: We show that heterochromatin mediates chromatin fibre compaction at centromeres and promotes prominent inter-arm interactions within centromere-proximal regions, providing structural constraints crucial for proper genome organization.

Abstract: Loss of heterochromatin relaxes constraints on chromosomes, causing an increase in intra- and inter-chromosomal interactions.

Abstract: Together, our analyses uncover fundamental genome folding principles that drive higher-order chromosome organization crucial for coordinating nuclear functions.


A temporal shift in the circuits mediating retrieval of fear memory

Abstract: Fear memories allow animals to avoid danger, thereby increasing their chances of survival.

Abstract: Fear memories can be retrieved long after learning, but little is known about how retrieval circuits change with time.

Abstract: Here we show that the dorsal midline thalamus of rats is required for the retrieval of auditory conditioned fear at late (24 hours, 7 days, 28 days), but not early (0.5 hours, 6 hours) time points after learning.

Abstract: Consistent with this, the paraventricular nucleus of the thalamus (PVT), a subregion of the dorsal midline thalamus, showed increased c-Fos expression only at late time points, indicating that the PVT is gradually recruited for fear retrieval.

Abstract: Accordingly, the conditioned tone responses of PVT neurons increased with time after training.

Abstract: The prelimbic (PL) prefrontal cortex, which is necessary for fear retrieval, sends dense projections to the PVT.

Abstract: Retrieval at late time points activated PL neurons projecting to the PVT, and optogenetic silencing of these projections impaired retrieval at late, but not early, time points.

Abstract: In contrast, silencing of PL inputs to the basolateral amygdala impaired retrieval at early, but not late, time points, indicating a time-dependent shift in retrieval circuits.

Abstract: Retrieval at late time points also activated PVT neurons projecting to the central nucleus of the amygdala, and silencing these projections at late, but not early, time points induced a persistent attenuation of fear.

Abstract: Thus, the PVT may act as a crucial thalamic node recruited into cortico-amygdalar networks for retrieval and maintenance of long-term fear memories.


Derivation of novel human ground state naive pluripotent stem cells

Abstract: Mouse embryonic stem (ES) cells are isolated from the inner cell mass of blastocysts, and can be preserved in vitro in a naive inner-cell-mass-like configuration by providing exogenous stimulation with leukaemia inhibitory factor (LIF) and small molecule inhibition of ERK1/ERK2 and GSK3β signalling (termed 2i/LIF conditions).

Abstract: Hallmarks of naive pluripotency include driving Oct4 (also known as Pou5f1) transcription by its distal enhancer, retaining a pre-inactivation X chromosome state, and global reduction in DNA methylation and in H3K27me3 repressive chromatin mark deposition on developmental regulatory gene promoters.

Abstract: Upon withdrawal of 2i/LIF, naive mouse ES cells can drift towards a primed pluripotent state resembling that of the post-implantation epiblast.

Abstract: Although human ES cells share several molecular features with naive mouse ES cells, they also share a variety of epigenetic properties with primed murine epiblast stem cells (EpiSCs).

Abstract: These include predominant use of the proximal enhancer element to maintain OCT4 expression, pronounced tendency for X chromosome inactivation in most female human ES cells, increase in DNA methylation and prominent deposition of H3K27me3 and bivalent domain acquisition on lineage regulatory genes.

Abstract: The feasibility of establishing human ground state naive pluripotency in vitro with equivalent molecular and functional features to those characterized in mouse ES cells remains to be defined.

Abstract: Here we establish defined conditions that facilitate the derivation of genetically unmodified human naive pluripotent stem cells from already established primed human ES cells, from somatic cells through induced pluripotent stem (iPS) cell reprogramming or directly from blastocysts.

Abstract: The novel naive pluripotent cells validated herein retain molecular characteristics and functional properties that are highly similar to mouse naive ES cells, and distinct from conventional primed human pluripotent cells.

Abstract: This includes competence in the generation of cross-species chimaeric mouse embryos that underwent organogenesis following microinjection of human naive iPS cells into mouse morulas.

Abstract: Collectively, our findings establish new avenues for regenerative medicine, patient-specific iPS cell disease modelling and the study of early human development in vitro and in vivo.


Predicting immunogenic tumour mutations by combining mass spectrometry and exome sequencing

Abstract: Human tumours typically harbour a remarkable number of somatic mutations.

Abstract: If presented on major histocompatibility complex class I molecules (MHCI), peptides containing these mutations could potentially be immunogenic as they should be recognized as ‘non-self’ neo-antigens by the adaptive immune system.

Abstract: Recent work has confirmed that mutant peptides can serve as T-cell epitopes.

Abstract: However, few mutant epitopes have been described because their discovery required the laborious screening of patient tumour-infiltrating lymphocytes for their ability to recognize antigen libraries constructed following tumour exome sequencing.

Abstract: We sought to simplify the discovery of immunogenic mutant peptides by characterizing their general properties.

Abstract: We developed an approach that combines whole-exome and transcriptome sequencing analysis with mass spectrometry to identify neo-epitopes in two widely used murine tumour models.

Abstract: Of the >1,300 amino acid changes identified, ∼13% were predicted to bind MHCI, a small fraction of which were confirmed by mass spectrometry.

Abstract: The peptides were then structurally modelled bound to MHCI.

Abstract: Mutations that were solvent-exposed and therefore accessible to T-cell antigen receptors were predicted to be immunogenic.

Abstract: Vaccination of mice confirmed the approach, with each predicted immunogenic peptide yielding therapeutically active T-cell responses.

Abstract: The predictions also enabled the generation of peptide–MHCI dextramers that could be used to monitor the kinetics and distribution of the anti-tumour T-cell response before and after vaccination.

Abstract: These findings indicate that a suitable prediction algorithm may provide an approach for the pharmacodynamic monitoring of T-cell responses as well as for the development of personalized vaccines in cancer patients.


In vivo genome editing using Staphylococcus aureus Cas9

Abstract: The RNA-guided endonuclease Cas9 has emerged as a versatile genome-editing platform.

Abstract: However, the size of the commonly used Cas9 from Streptococcus pyogenes (SpCas9) limits its utility for basic research and therapeutic applications that use the highly versatile adeno-associated virus (AAV) delivery vehicle.

Abstract: Here, we characterize six smaller Cas9 orthologues and show that Cas9 from Staphylococcus aureus (SaCas9) can edit the genome with efficiencies similar to those of SpCas9, while being more than 1 kilobase shorter.

Abstract: We packaged SaCas9 and its single guide RNA expression cassette into a single AAV vector and targeted the cholesterol regulatory gene Pcsk9 in the mouse liver.

Abstract: Within one week of injection, we observed >40% gene modification, accompanied by significant reductions in serum Pcsk9 and total cholesterol levels.

Abstract: We further assess the genome-wide targeting specificity of SaCas9 and SpCas9 using BLESS, and demonstrate that SaCas9-mediated in vivo genome editing has the potential to be efficient and specific.


Effects of exercise intensity on cardiovascular fitness, total body composition, and visceral adiposity of obese adolescents.

Abstract: BACKGROUND Little is known about how the intensity of exercise influences cardiovascular fitness and body composition, especially in obese adolescents.

Abstract: OBJECTIVE Our goal was to determine the effects of physical training intensity on the cardiovascular fitness, percentage of body fat (%BF), and visceral adipose tissue (VAT) of obese adolescents.

Abstract: DESIGN Obese 13-16-y-olds (n = 80) were assigned to 1) biweekly lifestyle education (LSE), 2) LSE + moderate-intensity physical training, or 3) LSE + high-intensity physical training.

Abstract: The intervention lasted 8 mo.

Abstract: Physical training was offered 5 d/wk, and the target energy expenditure for all subjects in physical training groups was 1047 kJ (250 kcal)/session.

Abstract: Cardiovascular fitness was measured with a multistage treadmill test, %BF with dual-energy X-ray absorptiometry, and VAT with magnetic resonance imaging.

Abstract: RESULTS The increase in cardiovascular fitness in the high-intensity physical training group, but not in the moderate-intensity group, was significantly greater than that in the LSE alone group (P = 0.009); no other comparisons of the 3 groups were significant.

Abstract: Compared with the LSE alone group, a group composed of subjects in both physical training groups combined who attended training sessions >or=2 d/wk showed favorable changes in cardiovascular fitness (P < 0.001), %BF (P = 0.001), and VAT (P = 0.029).

Abstract: We found no evidence that the high-intensity physical training was more effective than the moderate-intensity physical training in enhancing body composition.

Abstract: CONCLUSIONS The cardiovascular fitness of obese adolescents was significantly improved by physical training, especially high-intensity physical training.

Abstract: The physical training also reduced both visceral and total-body adiposity, but there was no clear effect of the intensity of physical training.


Low methionine ingestion by rats extends life span.

Abstract: Dietary energy restriction has been a widely used means of experimentally extending mammalian life span.

Abstract: We report here that lifelong reduction in the concentration of a single dietary component, the essential amino acid L-methionine, from 0.86 to 0.17% of the diet results in a 30% longer life span of male Fischer 344 rats.

Abstract: Methionine restriction completely abolished growth, although food intake was actually greater on a body weight basis.

Abstract: Studies of energy consumption in early life indicated that the energy intake of 0.17% methionine-fed animals was near normal for animals of their size, although consumption per animal was below that of the much larger 0.86% methionine-fed rats.

Abstract: Increasing the energy intake of rats fed 0.17% methionine failed to increase their rate of growth, whereas restricting 0.85% methionine-fed rats to the food intake of 0.17% methionine-fed animals did not materially reduce growth, indicating that food restriction was not a factor in life span extension in these experiments.

Abstract: The biochemically well-defined pathways of methionine metabolism and utilization offer the potential for uncovering the precise mechanism(s) underlying this specific dietary restriction-related extension of life span.


Epicatechin and a cocoa polyphenolic extract modulate gene expression in human Caco-2 cells.

Abstract: We performed a functional genomic analysis to study the effect of epicatechin and polyphenolic cocoa extract in the human colon adenocarcinoma cell line Caco-2.

Abstract: The specific Human Hematology/Immunology cDNA arrays by Clontech, containing 406 genes in duplicate, were used.

Abstract: The differentially expressed genes were classified according to their level of expression, calculated as the ratio of the value obtained after each treatment relative to control cells, with a statistical significance of P < 0.05 (upregulated: ratio > 1.5; downregulated: ratio < 0.6).

Abstract: Treatment with epicatechin decreased the expression of 21 genes and upregulated 24 genes.

Abstract: Upon incubation with the cocoa polyphenolic extract, 24 genes were underexpressed and 28 were overexpressed.

Abstract: The changes in expression for ferritin heavy polypeptide 1 (FTH1), mitogen-activated protein kinase kinase 1 (MAPKK1), signal transducer and activator of transcription 1 (STAT1), and topoisomerase 1 upon incubation with epicatechin, and for myeloid leukemia factor 2 (MLF2), CCAAT/enhancer binding protein gamma (C/EBPG), MAPKK1, ATP-binding cassette, subfamily c member 1 (MRP1), STAT1, topoisomerase 1, and x-ray repair complementing defective repair 1 (XRCC1) upon incubation with the cocoa polyphenolic extract were validated by RT-PCR.

Abstract: Changes in the messenger RNA levels for MAPKK1, STAT1, MRP1, and topoisomerase 1 upon incubation with either epicatechin or cocoa extract were further confirmed at the protein level by Western blotting.

Abstract: The changes in the expression of STAT1, MAPKK1, MRP1, and FTH1 genes, which are involved in the cellular response to oxidative stress, are in agreement with the antioxidant properties of cocoa flavonoids.

Abstract: In addition, the changes in the expression of C/EBPG, topoisomerase 1, MLF2, and XRCC1 suggest novel mechanisms of action of flavonoids at the molecular level.


An atlas of active enhancers across human cell types and tissues

Abstract: Enhancers control the correct temporal and cell-type-specific activation of gene expression in multicellular eukaryotes.

Abstract: Knowing their properties, regulatory activity and targets is crucial to understand the regulation of differentiation and homeostasis.

Abstract: Here we use the FANTOM5 panel of samples, covering the majority of human tissues and cell types, to produce an atlas of active, in vivo-transcribed enhancers.

Abstract: We show that enhancers share properties with CpG-poor messenger RNA promoters but produce bidirectional, exosome-sensitive, relatively short unspliced RNAs, the generation of which is strongly related to enhancer activity.

Abstract: The atlas is used to compare regulatory programs between different cells at unprecedented depth, to identify disease-associated regulatory single nucleotide polymorphisms, and to classify cell-type-specific and ubiquitous enhancers.

Abstract: We further explore the utility of enhancer redundancy, which explains gene expression strength rather than expression patterns.

Abstract: The online FANTOM5 enhancer atlas represents a unique resource for studies on cell-type-specific enhancers and gene regulation.


Precision microbiome reconstitution restores bile acid mediated resistance to Clostridium difficile

Abstract: The gastrointestinal tracts of mammals are colonized by hundreds of microbial species that contribute to health, including colonization resistance against intestinal pathogens.

Abstract: Many antibiotics destroy intestinal microbial communities and increase susceptibility to intestinal pathogens.

Abstract: Among these, Clostridium difficile, a major cause of antibiotic-induced diarrhoea, greatly increases morbidity and mortality in hospitalized patients.

Abstract: Which intestinal bacteria provide resistance to C. difficile infection and their in vivo inhibitory mechanisms remain unclear.

Abstract: Here we correlate loss of specific bacterial taxa with development of infection, by treating mice with different antibiotics that result in distinct microbiota changes and lead to varied susceptibility to C. difficile.

Abstract: Mathematical modelling augmented by analyses of the microbiota of hospitalized patients identifies resistance-associated bacteria common to mice and humans.

Abstract: Using these platforms, we determine that Clostridium scindens, a bile acid 7α-dehydroxylating intestinal bacterium, is associated with resistance to C. difficile infection and, upon administration, enhances resistance to infection in a secondary bile acid dependent fashion.

Abstract: Using a workflow involving mouse models, clinical studies, metagenomic analyses, and mathematical modelling, we identify a probiotic candidate that corrects a clinically relevant microbiome deficiency.

Abstract: These findings have implications for the rational design of targeted antimicrobials as well as microbiome-based diagnostics and therapeutics for individuals at risk of C. difficile infection.


Transcription initiation complex structures elucidate DNA opening

Abstract: Transcription of eukaryotic protein-coding genes begins with assembly of the RNA polymerase (Pol) II initiation complex and promoter DNA opening.

Abstract: Here we report cryo-electron microscopy (cryo-EM) structures of yeast initiation complexes containing closed and open DNA at resolutions of 8.8 Å and 3.6 Å, respectively.

Abstract: DNA is positioned and retained over the Pol II cleft by a network of interactions between the TATA-box-binding protein TBP and transcription factors TFIIA, TFIIB, TFIIE, and TFIIF.

Abstract: DNA opening occurs around the tip of the Pol II clamp and the TFIIE ‘extended winged helix’ domain, and can occur in the absence of TFIIH.

Abstract: Loading of the DNA template strand into the active centre may be facilitated by movements of obstructing protein elements triggered by allosteric binding of the TFIIE ‘E-ribbon’ domain.

Abstract: The results suggest a unified model for transcription initiation with a key event, the trapping of open promoter DNA by extended protein–protein and protein–DNA contacts.


Multiple mechanisms disrupt the let-7 microRNA family in neuroblastoma

Abstract: Poor prognosis in neuroblastoma is associated with genetic amplification of MYCN.

Abstract: MYCN is itself a target of let-7, a tumour suppressor family of microRNAs implicated in numerous cancers.

Abstract: LIN28B, an inhibitor of let-7 biogenesis, is overexpressed in neuroblastoma and has been reported to regulate MYCN.

Abstract: Here we show, however, that LIN28B is dispensable in MYCN-amplified neuroblastoma cell lines, despite de-repression of let-7.

Abstract: We further demonstrate that MYCN messenger RNA levels in amplified disease are exceptionally high and sufficient to sponge let-7, which reconciles the dispensability of LIN28B.

Abstract: We found that genetic loss of let-7 is common in neuroblastoma, inversely associated with MYCN amplification, and independently associated with poor outcomes, providing a rationale for chromosomal loss patterns in neuroblastoma.

Abstract: We propose that let-7 disruption by LIN28B, MYCN sponging, or genetic loss is a unifying mechanism of neuroblastoma development with broad implications for cancer pathogenesis.


Radiation and Dual Checkpoint Blockade Activates Non-Redundant Immune Mechanisms in Cancer

Abstract: Immune checkpoint inhibitors result in impressive clinical responses, but optimal results will require combination with each other and other therapies.

Abstract: This raises fundamental questions about mechanisms of non-redundancy and resistance.

Abstract: Here we report major tumour regressions in a subset of patients with metastatic melanoma treated with an anti-CTLA4 antibody (anti-CTLA4) and radiation, and reproduced this effect in mouse models.

Abstract: Although combined treatment improved responses in irradiated and unirradiated tumours, resistance was common.

Abstract: Unbiased analyses of mice revealed that resistance was due to upregulation of PD-L1 on melanoma cells and associated with T-cell exhaustion.

Abstract: Accordingly, optimal response in melanoma and other cancer types requires radiation, anti-CTLA4 and anti-PD-L1/PD-1.

Abstract: Anti-CTLA4 predominantly inhibits T-regulatory cells (Treg cells), thereby increasing the CD8 T-cell to Treg (CD8/Treg) ratio.

Abstract: Radiation enhances the diversity of the T-cell receptor (TCR) repertoire of intratumoral T cells.

Abstract: Together, anti-CTLA4 promotes expansion of T cells, while radiation shapes the TCR repertoire of the expanded peripheral clones.

Abstract: Addition of PD-L1 blockade reverses T-cell exhaustion to mitigate depression in the CD8/Treg ratio and further encourages oligoclonal T-cell expansion.

Abstract: Similarly to results from mice, patients on our clinical trial with melanoma showing high PD-L1 did not respond to radiation plus anti-CTLA4, demonstrated persistent T-cell exhaustion, and rapidly progressed.

Abstract: Thus, PD-L1 on melanoma cells allows tumours to escape anti-CTLA4-based therapy, and the combination of radiation, anti-CTLA4 and anti-PD-L1 promotes response and immunity through distinct mechanisms.


G-protein-independent coupling of MC4R to Kir7.1 in hypothalamic neurons

Abstract: The regulated release of anorexigenic α-melanocyte stimulating hormone (α-MSH) and orexigenic Agouti-related protein (AgRP) from discrete hypothalamic arcuate neurons onto common target sites in the central nervous system has a fundamental role in the regulation of energy homeostasis.

Abstract: Both peptides bind with high affinity to the melanocortin-4 receptor (MC4R); existing data show that α-MSH is an agonist that couples the receptor to the Gαs signalling pathway, while AgRP binds competitively to block α-MSH binding and blocks the constitutive activity mediated by the ligand-mimetic amino-terminal domain of the receptor.

Abstract: Here we show that, in mice, regulation of firing activity of neurons from the paraventricular nucleus of the hypothalamus (PVN) by α-MSH and AgRP can be mediated independently of Gαs signalling by ligand-induced coupling of MC4R to closure of inwardly rectifying potassium channel, Kir7.1.

Abstract: Furthermore, AgRP is a biased agonist that hyperpolarizes neurons by binding to MC4R and opening Kir7.1, independently of its inhibition of α-MSH binding.

Abstract: Consequently, Kir7.1 signalling appears to be central to melanocortin-mediated regulation of energy homeostasis within the PVN.

Abstract: Coupling of MC4R to Kir7.1 may explain unusual aspects of the control of energy homeostasis by melanocortin signalling, including the gene dosage effect of MC4R and the sustained effects of AgRP on food intake.


Role of ghrelin in the relationship between hyperphagia and accelerated gastric emptying in diabetic mice.

Abstract: BACKGROUND & AIMS Ghrelin is an orexigenic peptide with gastroprokinetic effects.

Abstract: Mice with streptozotocin (STZ)-induced diabetes exhibit hyperphagia, altered gastric emptying, and increased plasma ghrelin levels.

Abstract: We investigated the causative role of ghrelin herein by comparing changes in ghrelin receptor knockout (growth hormone secretagogue receptor [GHS-R](-/-)) and wild-type (GHS-R(+/+)) mice with STZ-induced diabetes.

Abstract: METHODS Gastric emptying was measured with the [(13)C]octanoic acid breath test.

Abstract: The messenger RNA (mRNA) expression of neuropeptide Y (NPY), agouti-related peptide (AgRP), and proopiomelanocortin was quantified by real-time reverse-transcription polymerase chain reaction.

Abstract: Neural contractions were elicited by electrical field stimulation in fundic smooth muscle strips.

Abstract: RESULTS Diabetes increased plasma ghrelin levels to a similar extent in both genotypes.

Abstract: Hyperphagia was more pronounced in GHS-R(+/+) than in GHS-R(-/-) mice between days 12 and 21.

Abstract: Increases in NPY and AgRP mRNA expression were less pronounced in diabetic GHS-R(-/-) than in GHS-R(+/+) mice from day 15 on, whereas decreases in proopiomelanocortin mRNA levels were similar in both genotypes.

Abstract: Gastric emptying was accelerated to a similar extent in both genotypes, starting on day 16.

Abstract: In fundic smooth muscle strips of diabetic GHS-R(+/+) and GHS-R(-/-) mice, neuronal relaxations were reduced, whereas contractions were increased; this increase was related to an increased affinity of muscarinic and tachykinergic receptors.

Abstract: CONCLUSIONS Diabetic hyperphagia is regulated by central mechanisms in which the ghrelin-signaling pathway affects the expression of NPY and AgRP in the hypothalamus.

Abstract: The acceleration of gastric emptying, which is not affected by ghrelin signaling, is not the cause of diabetic hyperphagia and probably involves local contractility changes in the fundus.


Human serum 25-hydroxycholecalciferol response to extended oral dosing with cholecalciferol.

Abstract: BACKGROUND The cholecalciferol inputs required to achieve or maintain any given serum 25-hydroxycholecalciferol concentration are not known, particularly within ranges comparable to the probable physiologic supply of the vitamin.

Abstract: OBJECTIVES The objectives were to establish the quantitative relation between steady state cholecalciferol input and the resulting serum 25-hydroxycholecalciferol concentration and to estimate the proportion of the daily requirement during winter that is met by cholecalciferol reserves in body tissue stores.

Abstract: DESIGN Cholecalciferol was administered daily in controlled oral doses labeled at 0, 25, 125, and 250 micro g cholecalciferol for approximately 20 wk during the winter to 67 men living in Omaha (41.2 degrees N latitude).

Abstract: The time course of serum 25-hydroxycholecalciferol concentration was measured at intervals over the course of treatment.

Abstract: RESULTS From a mean baseline value of 70.3 nmol/L, equilibrium concentrations of serum 25-hydroxycholecalciferol changed during the winter months in direct proportion to the dose, with a slope of approximately 0.70 nmol/L for each additional 1 micro g cholecalciferol input.

Abstract: The calculated oral input required to sustain the serum 25-hydroxycholecalciferol concentration present before the study (ie, in the autumn) was 12.5 micro g (500 IU)/d, whereas the total amount from all sources (supplement, food, tissue stores) needed to sustain the starting 25-hydroxycholecalciferol concentration was estimated at approximately 96 micro g (approximately 3800 IU)/d.

Abstract: By difference, the tissue stores provided approximately 78-82 micro g/d.

Abstract: CONCLUSIONS Healthy men seem to use 3000-5000 IU cholecalciferol/d, apparently meeting > 80% of their winter cholecalciferol need with cutaneously synthesized accumulations from solar sources during the preceding summer months.

Abstract: Current recommended vitamin D inputs are inadequate to maintain serum 25-hydroxycholecalciferol concentration in the absence of substantial cutaneous production of vitamin D.


Intratumor heterogeneity and branched evolution revealed by multiregion sequencing.

Abstract: BACKGROUND Intratumor heterogeneity may foster tumor evolution and adaptation and hinder personalized-medicine strategies that depend on results from single tumor-biopsy samples.

Abstract: METHODS To examine intratumor heterogeneity, we performed exome sequencing, chromosome aberration analysis, and ploidy profiling on multiple spatially separated samples obtained from primary renal carcinomas and associated metastatic sites.

Abstract: We characterized the consequences of intratumor heterogeneity using immunohistochemical analysis, mutation functional analysis, and profiling of messenger RNA expression.

Abstract: RESULTS Phylogenetic reconstruction revealed branched evolutionary tumor growth, with 63 to 69% of all somatic mutations not detectable across every tumor region.

Abstract: Intratumor heterogeneity was observed for a mutation within an autoinhibitory domain of the mammalian target of rapamycin (mTOR) kinase, correlating with S6 and 4EBP phosphorylation in vivo and constitutive activation of mTOR kinase activity in vitro.

Abstract: Mutational intratumor heterogeneity was seen for multiple tumor-suppressor genes converging on loss of function; SETD2, PTEN, and KDM5C underwent multiple distinct and spatially separated inactivating mutations within a single tumor, suggesting convergent phenotypic evolution.

Abstract: Gene-expression signatures of good and poor prognosis were detected in different regions of the same tumor.

Abstract: Allelic composition and ploidy profiling analysis revealed extensive intratumor heterogeneity, with 26 of 30 tumor samples from four tumors harboring divergent allelic-imbalance profiles and with ploidy heterogeneity in two of four tumors.

Abstract: CONCLUSIONS Intratumor heterogeneity can lead to underestimation of the tumor genomics landscape portrayed from single tumor-biopsy samples and may present major challenges to personalized-medicine and biomarker development.

Abstract: Intratumor heterogeneity, associated with heterogeneous protein function, may foster tumor adaptation and therapeutic failure through Darwinian selection.

Abstract: (Funded by the Medical Research Council and others.).


Succession of microbial consortia in the developing infant gut microbiome.

Abstract: The colonization process of the infant gut microbiome has been called chaotic, but this view could reflect insufficient documentation of the factors affecting the microbiome.

Abstract: We performed a 2.5-y case study of the assembly of the human infant gut microbiome, to relate life events to microbiome composition and function.

Abstract: Sixty fecal samples were collected from a healthy infant along with a diary of diet and health status.

Abstract: Analysis of >300,000 16S rRNA genes indicated that the phylogenetic diversity of the microbiome increased gradually over time and that changes in community composition conformed to a smooth temporal gradient.

Abstract: In contrast, major taxonomic groups showed abrupt shifts in abundance corresponding to changes in diet or health.

Abstract: Community assembly was nonrandom: we observed discrete steps of bacterial succession punctuated by life events.

Abstract: Furthermore, analysis of ≈ 500,000 DNA metagenomic reads from 12 fecal samples revealed that the earliest microbiome was enriched in genes facilitating lactate utilization, and that functional genes involved in plant polysaccharide metabolism were present before the introduction of solid food, priming the infant gut for an adult diet.

Abstract: However, ingestion of table foods caused a sustained increase in the abundance of Bacteroidetes, elevated fecal short chain fatty acid levels, enrichment of genes associated with carbohydrate utilization, vitamin biosynthesis, and xenobiotic degradation, and a more stable community composition, all of which are characteristic of the adult microbiome.

Abstract: This study revealed that seemingly chaotic shifts in the microbiome are associated with life events; however, additional experiments ought to be conducted to assess how different infants respond to similar life events.


gamma-tocopherol, the major form of vitamin E in the US diet, deserves more attention.

Abstract: gamma-tocopherol is the major form of vitamin E in many plant seeds and in the US diet, but has drawn little attention compared with alpha-tocopherol, the predominant form of vitamin E in tissues and the primary form in supplements.

Abstract: However, recent studies indicate that gamma-tocopherol may be important to human health and that it possesses unique features that distinguish it from alpha-tocopherol.

Abstract: gamma-Tocopherol appears to be a more effective trap for lipophilic electrophiles than is alpha-tocopherol.

Abstract: gamma-Tocopherol is well absorbed and accumulates to a significant degree in some human tissues; it is metabolized, however, largely to 2,7,8-trimethyl-2-(beta-carboxyethyl)-6-hydroxychroman (gamma-CEHC), which is mainly excreted in the urine.

Abstract: gamma-CEHC, but not the corresponding metabolite derived from alpha-tocopherol, has natriuretic activity that may be of physiologic importance.

Abstract: Both gamma-tocopherol and gamma-CEHC, but not alpha-tocopherol, inhibit cyclooxygenase activity and, thus, possess antiinflammatory properties.

Abstract: Some human and animal studies indicate that plasma concentrations of gamma-tocopherol are inversely associated with the incidence of cardiovascular disease and prostate cancer.

Abstract: These distinguishing features of gamma-tocopherol and its metabolite suggest that gamma-tocopherol may contribute significantly to human health in ways not recognized previously.

Abstract: This possibility should be further evaluated, especially considering that high doses of alpha-tocopherol deplete plasma and tissue gamma-tocopherol, in contrast with supplementation with gamma-tocopherol, which increases both.

Abstract: We review current information on the bioavailability, metabolism, chemistry, and nonantioxidant activities of gamma-tocopherol and epidemiologic data concerning the relation between gamma-tocopherol and cardiovascular disease and cancer.


Disclosure of APOE genotype for risk of Alzheimer's disease.

Abstract: BACKGROUND The apolipoprotein E (APOE) genotype provides information on the risk of Alzheimer's disease, but the genotyping of patients and their family members has been discouraged.

Abstract: We examined the effect of genotype disclosure in a prospective, randomized, controlled trial.

Abstract: METHODS We randomly assigned 162 asymptomatic adults who had a parent with Alzheimer's disease to receive the results of their own APOE genotyping (disclosure group) or not to receive such results (nondisclosure group).

Abstract: We measured symptoms of anxiety, depression, and test-related distress 6 weeks, 6 months, and 1 year after disclosure or nondisclosure.

Abstract: RESULTS

Abstract: There were no significant differences between the two groups in changes in time-averaged measures of anxiety (4.5 in the disclosure group and 4.4 in the nondisclosure group, P=0.84), depression (8.8 and 8.7, respectively; P=0.98), or test-related distress (6.9 and 7.5, respectively; P=0.61).

Abstract: Secondary comparisons between the nondisclosure group and a disclosure subgroup of subjects carrying the APOE epsilon4 allele (which is associated with increased risk) also revealed no significant differences.

Abstract: However, the epsilon4-negative subgroup had a significantly lower level of test-related distress than did the epsilon4-positive subgroup (P=0.01).

Abstract: Subjects with clinically meaningful changes in psychological outcomes were distributed evenly among the nondisclosure group and the epsilon4-positive and epsilon4-negative subgroups.

Abstract: Baseline scores for anxiety and depression were strongly associated with post-disclosure scores of these measures (P<0.001 for both comparisons).

Abstract: CONCLUSIONS The disclosure of APOE genotyping results to adult children of patients with Alzheimer's disease did not result in significant short-term psychological risks.

Abstract: Test-related distress was reduced among those who learned that they were APOE epsilon4-negative.

Abstract: Persons with high levels of emotional distress before undergoing genetic testing were more likely to have emotional difficulties after disclosure.

Abstract: (ClinicalTrials.gov number, NCT00571025.)