361 research outputs found

    A human embryonic kidney 293T cell line mutated at the Golgi -mannosidase II locus

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    Disruption of Golgi -mannosidase II activity can result in type II congenital dyserythropoietic anemia and can induce lupus-like autoimmunity in mice. Here, we isolate a mutant human embryonic kidney (HEK) 293T cell line, called Lec36, that displays sensitivity to ricin that lies between the parental HEK 293T cells, whose secreted and membrane-expressed proteins are dominated by complex-type glycosylation, and 293S Lec1 cells, which only produce oligomannose-type N-linked glycans. The stem cell marker, 19A, was transiently expressed in the HEK 293T Lec36 cells, and in parental HEK 293T cells with and without the potent Golgi -mannosidase II inhibitor, swainsonine. Negative-ion nano-electrospray ionization mass spectra of the 19A N-linked glycans from HEK 293T Lec36 and swainsonine-treated HEK 293T cells were qualitatively indistinguishable and, as shown by collision-induced dissociation spectra, dominated by hybrid-type glycosylation. Nucleotide sequencing revealed mutations in each allele of MAN2A1, the gene encoding Golgi -mannosidase II: a point mutation in one allele mapping to the active site and an in-frame deletion of twelve-nucleotides in the other. Expression of wild-type but not the mutant MAN2A1 alleles in Lec36 cells restored processing of the 19A reporter glycoprotein to complex-type glycosylation. The Lec36 cell line will be useful for expressing therapeutic glycoproteins with hybrid-type glycans and provides a sensitive host for detecting mutations in human MAN2A1 causing type II congenital dyserythropoietic anemia

    The polo-like kinase 1 (PLK1) inhibitor NMS-P937 is effective in a new model of disseminated primary CD56+ acute monoblastic leukaemia

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    CD56 is expressed in 15–20% of acute myeloid leukaemias (AML) and is associated with extramedullary diffusion, multidrug resistance and poor prognosis. We describe the establishment and characterisation of a novel disseminated model of AML (AML-NS8), generated by injection into mice of leukaemic blasts freshly isolated from a patient with an aggressive CD56+ monoblastic AML (M5a). The model reproduced typical manifestations of this leukaemia, including presence of extramedullary masses and central nervous system involvement, and the original phenotype, karyotype and genotype of leukaemic cells were retained in vivo. Recently Polo-Like Kinase 1 (PLK1) has emerged as a new candidate drug target in AML. We therefore tested our PLK1 inhibitor NMS-P937 in this model either in the engraftment or in the established disease settings. Both schedules showed good efficacy compared to standard therapies, with a significant increase in median survival time (MST) expecially in the established disease setting (MST = 28, 36, 62 days for vehicle, cytarabine and NMS-P937, respectively). Importantly, we could also demonstrate that NMS-P937 induced specific biomarker modulation in extramedullary tissues. This new in vivo model of CD56+ AML that recapitulates the human tumour lends support for the therapeutic use of PLK1 inhibitors in AML

    iPSCORE: A Resource of 222 iPSC Lines Enabling Functional Characterization of Genetic Variation across a Variety of Cell Types.

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    Large-scale collections of induced pluripotent stem cells (iPSCs) could serve as powerful model systems for examining how genetic variation affects biology and disease. Here we describe the iPSCORE resource: a collection of systematically derived and characterized iPSC lines from 222 ethnically diverse individuals that allows for both familial and association-based genetic studies. iPSCORE lines are pluripotent with high genomic integrity (no or low numbers of somatic copy-number variants) as determined using high-throughput RNA-sequencing and genotyping arrays, respectively. Using iPSCs from a family of individuals, we show that iPSC-derived cardiomyocytes demonstrate gene expression patterns that cluster by genetic background, and can be used to examine variants associated with physiological and disease phenotypes. The iPSCORE collection contains representative individuals for risk and non-risk alleles for 95% of SNPs associated with human phenotypes through genome-wide association studies. Our study demonstrates the utility of iPSCORE for examining how genetic variants influence molecular and physiological traits in iPSCs and derived cell lines

    A cell topography-based mechanism for ligand discrimination by the T cell receptor.

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    The T cell receptor (TCR) initiates the elimination of pathogens and tumors by T cells. To avoid damage to the host, the receptor must be capable of discriminating between wild-type and mutated self and nonself peptide ligands presented by host cells. Exactly how the TCR does this is unknown. In resting T cells, the TCR is largely unphosphorylated due to the dominance of phosphatases over the kinases expressed at the cell surface. However, when agonist peptides are presented to the TCR by major histocompatibility complex proteins expressed by antigen-presenting cells (APCs), very fast receptor triggering, i.e., TCR phosphorylation, occurs. Recent work suggests that this depends on the local exclusion of the phosphatases from regions of contact of the T cells with the APCs. Here, we developed and tested a quantitative treatment of receptor triggering reliant only on TCR dwell time in phosphatase-depleted cell contacts constrained in area by cell topography. Using the model and experimentally derived parameters, we found that ligand discrimination likely depends crucially on individual contacts being ∼200 nm in radius, matching the dimensions of the surface protrusions used by T cells to interrogate their targets. The model not only correctly predicted the relative signaling potencies of known agonists and nonagonists but also achieved this in the absence of kinetic proofreading. Our work provides a simple, quantitative, and predictive molecular framework for understanding why TCR triggering is so selective and fast and reveals that, for some receptors, cell topography likely influences signaling outcomes.This work was funded by The Wellcome Trust, the UK Medical Research Council, the UK Biotechnology and Biological Sciences Research Council and Cancer Research UK. We thank the Wolfson Imaging Centre, University of Oxford, for access to their microscope facility. We would like to thank the Wellcome Trust for the Sir Henry Dale Fellowship of R.A.F. (WT101609MA), the Royal Society for the University Research Fellowship of S.F.L. (UF120277) and acknowledge a GSK Professorship (D.K.). We are also grateful to Doug Tischer (UCSF, US) and Muaz Rushdi (Georgia Tech, US) for their critical comments on the manuscript

    Simplified Models for LHC New Physics Searches

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    This document proposes a collection of simplified models relevant to the design of new-physics searches at the LHC and the characterization of their results. Both ATLAS and CMS have already presented some results in terms of simplified models, and we encourage them to continue and expand this effort, which supplements both signature-based results and benchmark model interpretations. A simplified model is defined by an effective Lagrangian describing the interactions of a small number of new particles. Simplified models can equally well be described by a small number of masses and cross-sections. These parameters are directly related to collider physics observables, making simplified models a particularly effective framework for evaluating searches and a useful starting point for characterizing positive signals of new physics. This document serves as an official summary of the results from the "Topologies for Early LHC Searches" workshop, held at SLAC in September of 2010, the purpose of which was to develop a set of representative models that can be used to cover all relevant phase space in experimental searches. Particular emphasis is placed on searches relevant for the first ~50-500 pb-1 of data and those motivated by supersymmetric models. This note largely summarizes material posted at http://lhcnewphysics.org/, which includes simplified model definitions, Monte Carlo material, and supporting contacts within the theory community. We also comment on future developments that may be useful as more data is gathered and analyzed by the experiments.Comment: 40 pages, 2 figures. This document is the official summary of results from "Topologies for Early LHC Searches" workshop (SLAC, September 2010). Supplementary material can be found at http://lhcnewphysics.or

    The remarkably low affinity of CD4/peptide-major histocompatibility complex class II protein interactions

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    The αβ T-cell co-receptor CD4 enhances immune responses more than one million-fold in some assays, and yet the affinity of CD4 for its ligand, peptide-major histocompatibility class II (pMHC II) on antigen-presenting cells, is so weak that it was previously unquantifiable. Here, we report that a soluble form of CD4 failed to bind detectably to pMHC II in surface plasmon resonance-based assays, establishing a new upper limit for the solution affinity at 2.5 mM. However, when presented multivalently on magnetic beads, soluble CD4 bound pMHC II-expressing B cells, confirming that it is active and allowing mapping of the native co-receptor binding site on pMHC II. Whereas binding was undetectable in solution, the affinity of the CD4/pMHC II interaction could be measured in two dimensions (2D) using CD4- and adhesion molecule-functionalized, supported lipid bilayers, yielding a 2D dissociation constant, Kd, of ~5000 molecules/μm2. This value is 2-3 orders of magnitude higher than previously measured 2D Kd values for interacting leukocyte surface proteins. Calculations indicated, however, that CD4/pMHC II binding would increase rates of T-cell receptor (TCR) complex phosphorylation by three-fold via the recruitment of Lck, with only a small, 2-20% increase in the effective affinity of the TCR for pMHC II. The affinity of CD4/pMHC II therefore appears to be set at a value that increases T-cell sensitivity by enhancing phosphorylation, without compromising ligand discrimination.This work was supported by the Wellcome Trust and the UK Medical Research Council. PJ was supported by grants from the Swedish Research Council (number: 623-2014- 6387 and 621-2014-3907). OD is supported by a Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society (Grant Number: 098363)

    Risk of breast cancer following exposure to tetrachloroethylene-contaminated drinking water in Cape Cod, Massachusetts: reanalysis of a case-control study using a modified exposure assessment

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    <p>Abstract</p> <p>Background</p> <p>Tetrachloroethylene (PCE) is an important occupational chemical used in metal degreasing and drycleaning and a prevalent drinking water contaminant. Exposure often occurs with other chemicals but it occurred alone in a pattern that reduced the likelihood of confounding in a unique scenario on Cape Cod, Massachusetts. We previously found a small to moderate increased risk of breast cancer among women with the highest exposures using a simple exposure model. We have taken advantage of technical improvements in publically available software to incorporate a more sophisticated determination of water flow and direction to see if previous results were robust to more accurate exposure assessment.</p> <p>Methods</p> <p>The current analysis used PCE exposure estimates generated with the addition of water distribution modeling software (EPANET 2.0) to test model assumptions, compare exposure distributions to prior methods, and re-examine the risk of breast cancer. In addition, we applied data smoothing to examine nonlinear relationships between breast cancer and exposure. We also compared a set of measured PCE concentrations in water samples collected in 1980 to modeled estimates.</p> <p>Results</p> <p>Thirty-nine percent of individuals considered unexposed in prior epidemiological analyses were considered exposed using the current method, but mostly at low exposure levels. As a result, the exposure distribution was shifted downward resulting in a lower value for the 90th percentile, the definition of "high exposure" in prior analyses. The current analyses confirmed a modest increase in the risk of breast cancer for women with high PCE exposure levels defined by either the 90th percentile (adjusted ORs 1.0-1.5 for 0-19 year latency assumptions) or smoothing analysis cut point (adjusted ORs 1.3-2.0 for 0-15 year latency assumptions). Current exposure estimates had a higher correlation with PCE concentrations in water samples (Spearman correlation coefficient = 0.65, p < 0.0001) than estimates generated using the prior method (0.54, p < 0.0001).</p> <p>Conclusions</p> <p>The incorporation of sophisticated flow estimates in the exposure assessment method shifted the PCE exposure distribution downward, but did not meaningfully affect the exposure ranking of subjects or the strength of the association with the risk of breast cancer found in earlier analyses. Thus, the current analyses show a slightly elevated breast cancer risk for highly exposed women, with strengthened exposure assessment and minimization of misclassification by using the latest technology.</p

    CCL28 Induces Mucosal Homing of HIV-1-Specific IgA-Secreting Plasma Cells in Mice Immunized with HIV-1 Virus-Like Particles

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    Mucosae-associated epithelial chemokine (MEC or CCL28) binds to CCR3 and CCR10 and recruits IgA-secreting plasma cells (IgA-ASCs) in the mucosal lamina propria. The ability of this chemokine to enhance migration of IgA-ASCs to mucosal sites was assessed in a mouse immunization model using HIV-1IIIB Virus-like particles (VLPs). Mice receiving either HIV-1IIIB VLPs alone, CCL28 alone, or the irrelevant CCL19 chemokine were used as controls. Results showed a significantly increased CCR3 and CCR10 expression on CD19+ splenocytes of HIV-1IIIB VPL-CCL28-treated mice. HIV-1 Env-specific IFN-γ, IL-4 and IL-5 production, total IgA, anti-Env IgA as well as gastro-intestinal mucosal IgA-secreting plasma cells were also significantly augmented in these mice. Notably, sera and vaginal secretions from HIV-1IIIB VLP-CCL28-treated mice exhibited an enhanced neutralizing activity against both a HIV-1/B-subtype laboratory strain and a heterologous HIV-1/C-subtype primary isolate. These data suggest that CCL28 could be useful in enhancing the IgA immune response that will likely play a pivotal role in prophylactic HIV vaccines
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