Digital pulse width selection circuit Patent

Development of electric circuit for production of different pulse width signal

Methods for high-dimensonal analysis of cells dissociated from cyropreserved synovial tissue

© 2018 The Author(s). Background: Detailed molecular analyses of cells from rheumatoid arthritis (RA) synovium hold promise in identifying cellular phenotypes that drive tissue pathology and joint damage. The Accelerating Medicines Partnership RA/SLE Network aims to deconstruct autoimmune pathology by examining cells within target tissues through multiple high-dimensional assays. Robust standardized protocols need to be developed before cellular phenotypes at a single cell level can be effectively compared across patient samples. Methods: Multiple clinical sites collected cryopreserved synovial tissue fragments from arthroplasty and synovial biopsy in a 10% DMSO solution. Mechanical and enzymatic dissociation parameters were optimized for viable cell extraction and surface protein preservation for cell sorting and mass cytometry, as well as for reproducibility in RNA sequencing (RNA-seq). Cryopreserved synovial samples were collectively analyzed at a central processing site by a custom-designed and validated 35-marker mass cytometry panel. In parallel, each sample was flow sorted into fibroblast, T-cell, B-cell, and macrophage suspensions for bulk population RNA-seq and plate-based single-cell CEL-Seq2 RNA-seq. Results: Upon dissociation, cryopreserved synovial tissue fragments yielded a high frequency of viable cells, comparable to samples undergoing immediate processing. Optimization of synovial tissue dissociation across six clinical collection sites with ~ 30 arthroplasty and ~ 20 biopsy samples yielded a consensus digestion protocol using 100 μg/ml of Liberase™ TL enzyme preparation. This protocol yielded immune and stromal cell lineages with preserved surface markers and minimized variability across replicate RNA-seq transcriptomes. Mass cytometry analysis of cells from cryopreserved synovium distinguished diverse fibroblast phenotypes, distinct populations of memory B cells and antibody-secreting cells, and multiple CD4+ and CD8+ T-cell activation states. Bulk RNA-seq of sorted cell populations demonstrated robust separation of synovial lymphocytes, fibroblasts, and macrophages. Single-cell RNA-seq produced transcriptomes of over 1000 genes/cell, including transcripts encoding characteristic lineage markers identified. Conclusions: We have established a robust protocol to acquire viable cells from cryopreserved synovial tissue with intact transcriptomes and cell surface phenotypes. A centralized pipeline to generate multiple high-dimensional analyses of synovial tissue samples collected across a collaborative network was developed. Integrated analysis of such datasets from large patient cohorts may help define molecular heterogeneity within RA pathology and identify new therapeutic targets and biomarkers

Defining inflammatory cell states in rheumatoid arthritis joint synovial tissues by integrating single-cell transcriptomics and mass cytometry

© 2019, The Author(s), under exclusive licence to Springer Nature America, Inc. To define the cell populations that drive joint inflammation in rheumatoid arthritis (RA), we applied single-cell RNA sequencing (scRNA-seq), mass cytometry, bulk RNA sequencing (RNA-seq) and flow cytometry to T cells, B cells, monocytes, and fibroblasts from 51 samples of synovial tissue from patients with RA or osteoarthritis (OA). Utilizing an integrated strategy based on canonical correlation analysis of 5,265 scRNA-seq profiles, we identified 18 unique cell populations. Combining mass cytometry and transcriptomics revealed cell states expanded in RA synovia: THY1(CD90) + HLA-DRA hi sublining fibroblasts, IL1B + pro-inflammatory monocytes, ITGAX + TBX21 + autoimmune-associated B cells and PDCD1 + peripheral helper T (T PH ) cells and follicular helper T (T FH ) cells. We defined distinct subsets of CD8 + T cells characterized by GZMK + , GZMB + , and GNLY + phenotypes. We mapped inflammatory mediators to their source cell populations; for example, we attributed IL6 expression to THY1 + HLA-DRA hi fibroblasts and IL1B production to pro-inflammatory monocytes. These populations are potentially key mediators of RA pathogenesis

Cultural engagement and the economic performance of the cultural and creative industries: an occupational critique

This article presents a new critical engagement with the concept of Cultural and Creative Industries (CCIs), focusing on the rationale for grouping occupations and industries under this label. We show how the definition of ‘creativity’ used to demonstrate CCIs’ economic performance remains contested and variable, particularly with regard to the inclusion of specific parts of the IT sector. In demonstrating the importance of IT to the economic narrative regarding CCIs, we then unfold a related critique, exploring patterns in cultural consumption within CCI occupations. We demonstrate how some CCI workers have distinctively high cultural consumption, others reflect their broader social class, and some, including IT workers, show lower than expected consumption. Overall, we question the coherence of the prevailing CCI category, particularly in government policy, and suggest a new mode of ‘cultural’ occupational analysis for the sociology of CCIs

Documented spatial data set containing the subdivision of the basins into groundwater systems and subsystems, the selected locations per subsystem and a description of these sites, available data and projected additional measurements and equipment

The establishment of tools for trends analysis in groundwater is essential for the prediction and evaluation of measures taken within context of the Water Framework Directive and the draft Groundwater Directive. This report describes the spatial data sets which will be used for the purpose of detection, aggregation and extrapolation of temporal trends in groundwater quality. Trend analysis methods will be applied and tested at various scales and in various hydrogeological situations. The report contains a description of the studied sub-basins in TREND 2, including information on hydrogeology, land use and pressures, available data and projected additional measurements. Major differences between the sub-basins and the data sets are described to examine consequences for the work on trend detection. One of the challenges for TREND 2 is to define criteria for the application of various statistical and deterministic trend approaches for a range of hydrogeological conditions, spatial scales and types of groundwater monitoring. An overview of these conditions, scales and monitoring types is provided in the present report.FP6 Integrated Project AquaTerra Integrated Modelling of the river-sediment-soil-groundwater system; advanced tools for the management of catchment areas and river basins in the context of global change (Project no. 505428 - GOCE

Common Genetic Variants Modulate Pathogen-Sensing Responses in Human Dendritic Cells

Little is known about how human genetic variation affects the responses to environmental stimuli in the context of complex diseases. Experimental and computational approaches were applied to determine the effects of genetic variation on the induction of pathogen-responsive genes in human dendritic cells. We identified 121 common genetic variants associated in cis with variation in expression responses to Escherichia coli lipopolysaccharide, influenza, or interferon-β (IFN-β). We localized and validated causal variants to binding sites of pathogen-activated STAT (signal transducer and activator of transcription) and IRF (IFN-regulatory factor) transcription factors. We also identified a common variant in IRF7 that is associated in trans with type I IFN induction in response to influenza infection. Our results reveal common alleles that explain interindividual variation in pathogen sensing and provide functional annotation for genetic variants that alter susceptibility to inflammatory diseases.National Human Genome Research Institute (U.S.) (Grant P50 HG006193)National Institutes of Health (U.S.). Pioneer Award (DP1 CA174427)Howard Hughes Medical InstituteNational Institutes of Health (U.S.) (Grant HG004037)National Institutes of Health (U.S.). Pioneer Award (DP1 MH100706)National Institutes of Health (U.S.) (Transformative R01 Grant R01 DK097768)W. M. Keck FoundationMcKnight FoundationMerkin, Richard N.Damon Runyon Cancer Research FoundationSearle Scholars ProgramSimons Foundatio

Genetic association study of QT interval highlights role for calcium signaling pathways in myocardial repolarization.

The QT interval, an electrocardiographic measure reflecting myocardial repolarization, is a heritable trait. QT prolongation is a risk factor for ventricular arrhythmias and sudden cardiac death (SCD) and could indicate the presence of the potentially lethal mendelian long-QT syndrome (LQTS). Using a genome-wide association and replication study in up to 100,000 individuals, we identified 35 common variant loci associated with QT interval that collectively explain ∼8-10% of QT-interval variation and highlight the importance of calcium regulation in myocardial repolarization. Rare variant analysis of 6 new QT interval-associated loci in 298 unrelated probands with LQTS identified coding variants not found in controls but of uncertain causality and therefore requiring validation. Several newly identified loci encode proteins that physically interact with other recognized repolarization proteins. Our integration of common variant association, expression and orthogonal protein-protein interaction screens provides new insights into cardiac electrophysiology and identifies new candidate genes for ventricular arrhythmias, LQTS and SCD

Synoviocyte-targeted therapy synergizes with TNF inhibition in arthritis reversal

Fibroblast-like synoviocytes (FLS) are joint-lining cells that promote rheumatoid arthritis (RA) pathology. Current disease-modifying antirheumatic agents (DMARDs) operate through systemic immunosuppression. FLS-targeted approaches could potentially be combined with DMARDs to improve control of RA without increasing immunosuppression. Here, we assessed the potential of immunoglobulin-like domains 1 and 2 (Ig1&2), a decoy protein that activates the receptor tyrosine phosphatase sigma (PTPRS) on FLS, for RA therapy. We report that PTPRS expression is enriched in synovial lining RA FLS and that Ig1&2 reduces migration of RA but not osteoarthritis FLS. Administration of an Fc-fusion Ig1&2 attenuated arthritis in mice without affecting innate or adaptive immunity. Furthermore, PTPRS was down-regulated in FLS by tumor necrosis factor (TNF) via a phosphatidylinositol 3-kinase–mediated pathway, and TNF inhibition enhanced PTPRS expression in arthritic joints. Combination of ineffective doses of TNF inhibitor and Fc-Ig1&2 reversed arthritis in mice, providing an example of synergy between FLS-targeted and immunosuppressive DMARD therapies.publishedVersio

Deconstruction of rheumatoid arthritis synovium defines inflammatory subtypes

Rheumatoid arthritis is a prototypical autoimmune disease that causes joint inflammation and destruction1. There is currently no cure for rheumatoid arthritis, and the effectiveness of treatments varies across patients, suggesting an undefined pathogenic diversity1,2. Here, to deconstruct the cell states and pathways that characterize this pathogenic heterogeneity, we profiled the full spectrum of cells in inflamed synovium from patients with rheumatoid arthritis. We used multi-modal single-cell RNA-sequencing and surface protein data coupled with histology of synovial tissue from 79 donors to build single-cell atlas of rheumatoid arthritis synovial tissue that includes more than 314,000 cells. We stratified tissues into six groups, referred to as cell-type abundance phenotypes (CTAPs), each characterized by selectively enriched cell states. These CTAPs demonstrate the diversity of synovial inflammation in rheumatoid arthritis, ranging from samples enriched for T and B cells to those largely lacking lymphocytes. Disease-relevant cell states, cytokines, risk genes, histology and serology metrics are associated with particular CTAPs. CTAPs are dynamic and can predict treatment response, highlighting the clinical utility of classifying rheumatoid arthritis synovial phenotypes. This comprehensive atlas and molecular, tissue-based stratification of rheumatoid arthritis synovial tissue reveal new insights into rheumatoid arthritis pathology and heterogeneity that could inform novel targeted treatments