Transgenic Rescue of the LARGEmyd Mouse: A LARGE Therapeutic Window?

LARGE is a glycosyltransferase involved in glycosylation of α-dystroglycan (α-DG). Absence of this protein in the LARGEmyd mouse results in α-DG hypoglycosylation, and is associated with central nervous system abnormalities and progressive muscular dystrophy. Up-regulation of LARGE has previously been proposed as a therapy for the secondary dystroglycanopathies: overexpression in cells compensates for defects in multiple dystroglycanopathy genes. Counterintuitively, LARGE overexpression in an FKRP-deficient mouse exacerbates pathology, suggesting that modulation of α-DG glycosylation requires further investigation. Here we demonstrate that transgenic expression of human LARGE (LARGE-LV5) in the LARGEmyd mouse restores α-DG glycosylation (with marked hyperglycosylation in muscle) and that this corrects both the muscle pathology and brain architecture. By quantitative analyses of LARGE transcripts we also here show that levels of transgenic and endogenous LARGE in the brains of transgenic animals are comparable, but that the transgene is markedly overexpressed in heart and particularly skeletal muscle (20–100 fold over endogenous). Our data suggest LARGE overexpression may only be deleterious under a forced regenerative context, such as that resulting from a reduction in FKRP: in the absence of such a defect we show that systemic expression of LARGE can indeed act therapeutically, and that even dramatic LARGE overexpression is well-tolerated in heart and skeletal muscle. Moreover, correction of LARGEmyd brain pathology with only moderate, near-physiological LARGE expression suggests a generous therapeutic window

Cosmogenic neutron production at the Sudbury Neutrino Observatory

Neutrons produced in nuclear interactions initiated by cosmic-ray muons present an irreducible background to many rare-event searches, even in detectors located deep underground. Models for the production of these neutrons have been tested against previous experimental data, but the extrapolation to deeper sites is not well understood. Here we report results from an analysis of cosmogenically produced neutrons at the Sudbury Neutrino Observatory. A specific set of observables are presented, which can be used to benchmark the validity of geant4 physics models. In addition, the cosmogenic neutron yield, in units of 10-4 cm2/(g·μ), is measured to be 7.28±0.09(stat)-1.12+1.59(syst) in pure heavy water and 7.30±0.07(stat)-1.02+1.40(syst) in NaCl-loaded heavy water. These results provide unique insights into this potential background source for experiments at SNOLAB

Cosmology of a Scalar Field Coupled to Matter and an Isotropy-Violating Maxwell Field

Motivated by the couplings of the dilaton in four-dimensional effective actions, we investigate the cosmological consequences of a scalar field coupled both to matter and a Maxwell-type vector field. The vector field has a background isotropy-violating component. New anisotropic scaling solutions which can be responsible for the matter and dark energy dominated epochs are identified and explored. For a large parameter region the universe expands almost isotropically. Using that the CMB quadrupole is extremely sensitive to shear, we constrain the ratio of the matter coupling to the vector coupling to be less than 10^(-5). Moreover, we identify a large parameter region, corresponding to a strong vector coupling regime, yielding exciting and viable cosmologies close to the LCDM limit.Comment: Refs. added, some clarifications. Published in JHEP10(2012)06

LARGE Expression Augments the Glycosylation of Glycoproteins in Addition to α-Dystroglycan Conferring Laminin Binding

Mutations in genes encoding glycosyltransferases (and presumed glycosyltransferases) that affect glycosylation and extracellular matrix binding activity of α-dystroglycan (α-DG) cause congenital muscular dystrophies (CMDs) with central nervous system manifestations. Among the identified genes, LARGE is of particular interest because its overexpression rescues glycosylation defects of α-DG in mutations of not only LARGE but also other CMD-causing genes and restores laminin binding activity of α-DG. It is not known whether LARGE protein glycosylates other proteins in addition to α-DG. In this study, we overexpressed LARGE in DG-deficient cells and analyzed glycosylated proteins by Western blot analysis. Surprisingly, overexpression of LARGE in α-DG-deficient cells led to glycosylation dependent IIH6C4 and VIA4-1 immunoreactivity, despite the prevailing view that these antibodies only recognize glycosylated α-DG. Furthermore, the hyperglycosylated proteins in LARGE-overexpressing cells demonstrated the functional capacity to bind the extracellular matrix molecule laminin and promote laminin assembly at the cell surface, an effect that was blocked by IIH6C4 antibodies. These results indicate that overexpression of LARGE catalyzes the glycosylation of at least one other glycoprotein in addition to α-DG, and that this glycosylation(s) promotes laminin binding activity

Effect of Broccoli Sprouts and Live Attenuated Influenza Virus on Peripheral Blood Natural Killer Cells: A Randomized, Double-Blind Study

Enhancing antiviral host defense responses through nutritional supplementation would be an attractive strategy in the fight against influenza. Using inoculation with live attenuated influenza virus (LAIV) as an infection model, we have recently shown that ingestion of sulforaphane-containing broccoli sprout homogenates (BSH) reduces markers of viral load in the nose. To investigate the systemic effects of short-term BSH supplementation in the context of LAIV-inoculation, we examined peripheral blood immune cell populations in non-smoking subjects from this study, with a particular focus on NK cells. We carried out a randomized, double-blinded, placebo-controlled study measuring the effects of BSH (N = 13) or placebo (alfalfa sprout homogenate, ASH; N = 16) on peripheral blood mononuclear cell responses to a standard nasal vaccine dose of LAIV in healthy volunteers. Blood was drawn prior to (day-1) and post (day2, day21) LAIV inoculation and analyzed for neutrophils, monocytes, macrophages, T cells, NKT cells, and NK cells. In addition, NK cells were enriched, stimulated, and assessed for surface markers, intracellular markers, and cytotoxic potential by flow cytometry. Overall, LAIV significantly reduced NKT (day2 and day21) and T cell (day2) populations. LAIV decreased NK cell CD56 and CD158b expression, while significantly increasing CD16 expression and cytotoxic potential (on day2). BSH supplementation further increased LAIV-induced granzyme B production (day2) in NK cells compared to ASH and in the BSH group granzyme B levels appeared to be negatively associated with influenza RNA levels in nasal lavage fluid cells. We conclude that nasal influenza infection may induce complex changes in peripheral blood NK cell activation, and that BSH increases virus-induced peripheral blood NK cell granzyme B production, an effect that may be important for enhanced antiviral defense responses

Transcriptome and genome sequencing uncovers functional variation in humans

Summary Genome sequencing projects are discovering millions of genetic variants in humans, and interpretation of their functional effects is essential for understanding the genetic basis of variation in human traits. Here we report sequencing and deep analysis of mRNA and miRNA from lymphoblastoid cell lines of 462 individuals from the 1000 Genomes Project – the first uniformly processed RNA-seq data from multiple human populations with high-quality genome sequences. We discovered extremely widespread genetic variation affecting regulation of the majority of genes, with transcript structure and expression level variation being equally common but genetically largely independent. Our characterization of causal regulatory variation sheds light on cellular mechanisms of regulatory and loss-of-function variation, and allowed us to infer putative causal variants for dozens of disease-associated loci. Altogether, this study provides a deep understanding of the cellular mechanisms of transcriptome variation and of the landscape of functional variants in the human genome

Proliferation Index: A Continuous Model to Predict Prognosis in Patients with Tumours of the Ewing's Sarcoma Family

The prognostic value of proliferation index (PI) and apoptotic index (AI), caspase-8, -9 and -10 expression have been investigated in primary Ewing's sarcoma family of tumours (ESFT). Proliferating cells, detected by immunohistochemistry for Ki-67, were identified in 91% (91/100) of tumours with a median PI of 14 (range 0–87). Apoptotic cells, identified using the TUNEL assay, were detected in 96% (76/79) of ESFT; the median AI was 3 (range 0–33). Caspase-8 protein expression was negative (0) in 14% (11/79), low (1) in 33% (26/79), medium (2) in 38% (30/79) and high (3) in 15% (12/79) of tumours, caspase-9 expression was low (1) in 66% (39/59) and high (3) in 34% (20/59), and caspase-10 protein was low (1) in 37% (23/62) and negative (0) in 63% (39/62) of primary ESFT. There was no apparent relationship between caspase-8, -9 and -10 expression, PI and AI. PI was predictive of relapse-free survival (RFS; p = 0.011) and overall survival (OS; p = <0.001) in a continuous model, whereas AI did not predict outcome. Patients with tumours expressing low levels of caspase-9 protein had a trend towards a worse RFS than patients with tumours expressing higher levels of caspase-9 protein (p = 0.054, log rank test), although expression of caspases-8, -9 and/or -10 did not significantly predict RFS or OS. In a multivariate analysis model that included tumour site, tumour volume, the presence of metastatic disease at diagnosis, PI and AI, PI independently predicts OS (p = 0.003). Consistent with previous publications, patients with pelvic tumours had a significantly worse OS than patients with tumours at other sites (p = 0.028); patients with a pelvic tumour and a PI≥20 had a 6 fold-increased risk of death. These studies advocate the evaluation of PI in a risk model of outcome for patients with ESFT

Metabolic Reconstruction for Metagenomic Data and Its Application to the Human Microbiome

Microbial communities carry out the majority of the biochemical activity on the planet, and they play integral roles in processes including metabolism and immune homeostasis in the human microbiome. Shotgun sequencing of such communities' metagenomes provides information complementary to organismal abundances from taxonomic markers, but the resulting data typically comprise short reads from hundreds of different organisms and are at best challenging to assemble comparably to single-organism genomes. Here, we describe an alternative approach to infer the functional and metabolic potential of a microbial community metagenome. We determined the gene families and pathways present or absent within a community, as well as their relative abundances, directly from short sequence reads. We validated this methodology using a collection of synthetic metagenomes, recovering the presence and abundance both of large pathways and of small functional modules with high accuracy. We subsequently applied this method, HUMAnN, to the microbial communities of 649 metagenomes drawn from seven primary body sites on 102 individuals as part of the Human Microbiome Project (HMP). This provided a means to compare functional diversity and organismal ecology in the human microbiome, and we determined a core of 24 ubiquitously present modules. Core pathways were often implemented by different enzyme families within different body sites, and 168 functional modules and 196 metabolic pathways varied in metagenomic abundance specifically to one or more niches within the microbiome. These included glycosaminoglycan degradation in the gut, as well as phosphate and amino acid transport linked to host phenotype (vaginal pH) in the posterior fornix. An implementation of our methodology is available at http://huttenhower.sph.harvard.edu/human​n. This provides a means to accurately and efficiently characterize microbial metabolic pathways and functional modules directly from high-throughput sequencing reads, enabling the determination of community roles in the HMP cohort and in future metagenomic studies.National Institutes of Health (U.S.) (U54HG004968

IL-33 Is Produced by Mast Cells and Regulates IgE-Dependent Inflammation

Background: IL-33 is a recently characterized IL-1 family cytokine and found to be expressed in inflammatory diseases, including severe asthma and inflammatory bowl disease. Recombinant IL-33 has been shown to enhance Th2-associated immune responses and potently increase mast cell proliferation and cytokine production. While IL-33 is constitutively expressed in endothelial and epithelial cells, where it may function as a transcriptional regulator, cellular sources of IL-33 and its role in inflammation remain unclear. Methodology/Principal Findings: Here, we identify mast cells as IL-33 producing cells. IgE/antigen activation of bone marrow-derived mast cells or a murine mast cell line (MC/9) significantly enhanced IL-33. Conversely, recombinant IL-33 directly activated mast cells to produce several cytokines including IL-4, IL-5 and IL-6 but not IL-33. We show that expression of IL-33 in response to IgE-activation required calcium and that ionomycin was sufficient to induce IL-33. In vivo, peritoneal mast cells expressed IL-33 and IL-33 levels were significantly lower within the skin of mast cell deficient mice, compared to littermate controls. Local activation of mast cells promotes edema, followed by the recruitment of inflammatory cells. We demonstrate using passive cutaneous anaphylaxis, a mast cell-dependent model, that deficiency in ST2 or antibody blockage of ST2 or IL-33 ablated the late phase inflammatory response but that the immediate phase response was unaffected. IL-33 levels in the skin were significantly elevated only during the late phase