Exosomes released from breast cancer carcinomas stimulate cell movement

For metastasis to occur cells must communicate with to their local environment to initiate growth and invasion. Exosomes have emerged as an important mediator of cell-to-cell signalling through the transfer of molecules such as mRNAs, microRNAs, and proteins between cells. Exosomes have been proposed to act as regulators of cancer progression. Here, we study the effect of exosomes on cell migration, an important step in metastasis. We performed cell migration assays, endocytosis assays, and exosome proteomic profiling on exosomes released from three breast cancer cell lines that model progressive stages of metastasis. Results from these experiments suggest: (1) exosomes promote cell migration and (2) the signal is stronger from exosomes isolated from cells with higher metastatic potentials; (3) exosomes are endocytosed at the same rate regardless of the cell type; (4) exosomes released from cells show differential enrichment of proteins with unique protein signatures of both identity and abundance. We conclude that breast cancer cells of increasing metastatic potential secrete exosomes with distinct protein signatures that proportionally increase cell movement and suggest that released exosomes could play an active role in metastasis

Proteomics approaches to fibrotic disorders

This review provides an introduction to mass spectrometry based proteomics and discusses several proteomics approaches that are relevant in understanding the pathophysiology of fibrotic disorders and the approaches that are frequently used in biomarker discovery

Proteomic, biomechanical and functional analyses define neutrophil heterogeneity in systemic lupus erythematosus

Funder: NHLI FoundationFunder: NIHR Imperial Biomedical Research Centre; FundRef: http://dx.doi.org/10.13039/501100013342Funder: National Heart Lung and Blood InstituteFunder: Medical Research Council; FundRef: http://dx.doi.org/10.13039/501100000265Funder: National Institute of Biomedical Imaging and Bioengineering; FundRef: http://dx.doi.org/10.13039/100000070Funder: Gates Cambridge ScholarshipFunder: NIH/OXCAM FellowshipObjectives: Low-density granulocytes (LDGs) are a distinct subset of proinflammatory and vasculopathic neutrophils expanded in systemic lupus erythematosus (SLE). Neutrophil trafficking and immune function are intimately linked to cellular biophysical properties. This study used proteomic, biomechanical and functional analyses to further define neutrophil heterogeneity in the context of SLE. Methods: Proteomic/phosphoproteomic analyses were performed in healthy control (HC) normal density neutrophils (NDNs), SLE NDNs and autologous SLE LDGs. The biophysical properties of these neutrophil subsets were analysed by real-time deformability cytometry and lattice light-sheet microscopy. A two-dimensional endothelial flow system and a three-dimensional microfluidic microvasculature mimetic (MMM) were used to decouple the contributions of cell surface mediators and biophysical properties to neutrophil trafficking, respectively. Results: Proteomic and phosphoproteomic differences were detected between HC and SLE neutrophils and between SLE NDNs and LDGs. Increased abundance of type 1 interferon-regulated proteins and differential phosphorylation of proteins associated with cytoskeletal organisation were identified in SLE LDGs relative to SLE NDNs. The cell surface of SLE LDGs was rougher than in SLE and HC NDNs, suggesting membrane perturbances. While SLE LDGs did not display increased binding to endothelial cells in the two-dimensional assay, they were increasingly retained/trapped in the narrow channels of the lung MMM. Conclusions: Modulation of the neutrophil proteome and distinct changes in biophysical properties are observed alongside differences in neutrophil trafficking. SLE LDGs may be increasingly retained in microvasculature networks, which has important pathogenic implications in the context of lupus organ damage and small vessel vasculopathy

What factors affect patients' recall of general practitioners' advice?

<p>Abstract</p> <p>Background</p> <p>In order for patients to adhere to advice, provided by family doctors, they must be able to recall it afterwards. However, several studies have shown that most patients do not fully understand or memorize it. The aim of this study was to determine the influence of demographic characteristics, education, amount of given advice and the time between consultations on recalled advice.</p> <p>Methods</p> <p>A prospective survey, lasting 30 months, was conducted in an urban family practice in Slovenia. Logistic regression analysis was used to identify the risk factors for poorer recall.</p> <p>Results</p> <p>250 patients (87.7% response rate) received at least one and up to four pieces of advice (2.4 ± 0.8). A follow-up consultation took place at 47.4 ± 35.2 days. The determinants of better recall were high school (OR 0.4, 95% CI 0.15-0.99, p = 0.049) and college education (OR 0.3, 95% CI 0.10-1.00, p = 0.050), while worse recall was determined by number of given instructions three or four (OR 26.1, 95% CI 3.15-215.24, p = 0.002; OR 56.8, 95% CI 5.91-546.12, p < 0.001, respectively) and re-test interval: 15-30 days (OR 3.3, 95% CI 1.06-10.13, p = 0.040), 31-60 days (OR 3.2, 95% CI 1.28-8.07, p = 0.013) and more than 60 days (OR 2.5, 95% CI 1.05-6.02, p = 0.038).</p> <p>Conclusions</p> <p>Education was an important determinant factor and warrants further study. Patients should be given no more than one or two instructions in a consultation. When more is needed, the follow-up should be within the next 14 days, and would be of a greater benefit to higher educated patients.</p

A Mighty Small Heart: The Cardiac Proteome of Adult Drosophila melanogaster

Drosophila melanogaster is emerging as a powerful model system for the study of cardiac disease. Establishing peptide and protein maps of the Drosophila heart is central to implementation of protein network studies that will allow us to assess the hallmarks of Drosophila heart pathogenesis and gauge the degree of conservation with human disease mechanisms on a systems level. Using a gel-LC-MS/MS approach, we identified 1228 protein clusters from 145 dissected adult fly hearts. Contractile, cytostructural and mitochondrial proteins were most abundant consistent with electron micrographs of the Drosophila cardiac tube. Functional/Ontological enrichment analysis further showed that proteins involved in glycolysis, Ca2+-binding, redox, and G-protein signaling, among other processes, are also over-represented. Comparison with a mouse heart proteome revealed conservation at the level of molecular function, biological processes and cellular components. The subsisting peptidome encompassed 5169 distinct heart-associated peptides, of which 1293 (25%) had not been identified in a recent Drosophila peptide compendium. PeptideClassifier analysis was further used to map peptides to specific gene-models. 1872 peptides provide valuable information about protein isoform groups whereas a further 3112 uniquely identify specific protein isoforms and may be used as a heart-associated peptide resource for quantitative proteomic approaches based on multiple-reaction monitoring. In summary, identification of excitation-contraction protein landmarks, orthologues of proteins associated with cardiovascular defects, and conservation of protein ontologies, provides testimony to the heart-like character of the Drosophila cardiac tube and to the utility of proteomics as a complement to the power of genetics in this growing model of human heart disease

Barrier-to-Autointegration Factor Proteome Reveals Chromatin-Regulatory Partners

Nuclear lamin filaments and associated proteins form a nucleoskeletal (“lamina”) network required for transcription, replication, chromatin organization and epigenetic regulation in metazoans. Lamina defects cause human disease (“laminopathies”) and are linked to aging. Barrier-to-autointegration factor (BAF) is a mobile and essential component of the nuclear lamina that binds directly to histones, lamins and LEM-domain proteins, including the inner nuclear membrane protein emerin, and has roles in chromatin structure, mitosis and gene regulation. To understand BAF's mechanisms of action, BAF associated proteins were affinity-purified from HeLa cell nuclear lysates using BAF-conjugated beads, and identified by tandem mass spectrometry or independently identified and quantified using the iTRAQ method. We recovered A- and B-type lamins and core histones, all known to bind BAF directly, plus four human transcription factors (Requiem, NonO, p15, LEDGF), disease-linked proteins (e.g., Huntingtin, Treacle) and several proteins and enzymes that regulate chromatin. Association with endogenous BAF was independently validated by co-immunoprecipitation from HeLa cells for seven candidates including Requiem, poly(ADP-ribose) polymerase 1 (PARP1), retinoblastoma binding protein 4 (RBBP4), damage-specific DNA binding protein 1 (DDB1) and DDB2. Interestingly, endogenous BAF and emerin each associated with DDB2 and CUL4A in a UV- and time-dependent manner, suggesting BAF and emerin have dynamic roles in genome integrity and might help couple DNA damage responses to the nuclear lamina network. We conclude this proteome is a rich source of candidate partners for BAF and potentially also A- and B-type lamins, which may reveal how chromatin regulation and genome integrity are linked to nuclear structure