Identification of novel interferon responsive protein partners of human leukocyte antigen A (HLA-A) using cross-linking mass spectrometry (CLMS) approach

The interferon signalling system elicits a robust cytokine response against a wide range of environmental pathogenic and internal pathological signals, leading to induction of a subset of interferon-induced proteins. We applied DSS (disuccinimidyl suberate) mediated cross-linking mass spectrometry (CLMS) to capture novel protein–protein interactions within the realm of interferon induced proteins. In addition to the expected interferon-induced proteins, we identified novel inter- and intra-molecular cross-linked adducts for the canonical interferon induced proteins, such as MX1, USP18, OAS3, and STAT1. We focused on orthogonal validation of a cohort of novel interferon-induced protein networks formed by the HLA-A protein (H2BFS-HLA-A-HMGA1) using co-immunoprecipitation assay, and further investigated them by molecular dynamics simulation. Conformational dynamics of the simulated protein complexes revealed several interaction sites that mirrored the interactions identified in the CLMS findings. Together, we showcase a proof-of-principle CLMS study to identify novel interferon-induced signaling complexes and anticipate broader use of CLMS to identify novel protein interaction dynamics within the tumour microenvironment

Superpixel Graph Label Transfer with Learned Distance Metric

We present a fast approximate nearest neighbor algorithm for semantic segmentation. Our algorithm builds a graph over superpixels from an annotated set of training images. Edges in the graph represent approximate nearest neighbors in feature space. At test time we match superpixels from a novel image to the training images by adding the novel image to the graph. A move-making search algorithm allows us to leverage the graph and image structure for finding matches. We then transfer labels from the training images to the image under test. To promote good matches between superpixels we propose to learn a distance metric that weights the edges in our graph. Our approach is evaluated on four standard semantic segmentation datasets and achieves results comparable with the state-of-the-art

A NEW METHOD TO ASSESS FUNCTIONAL ACTIVITY OF SERUM COMPLEMENT SYSTEM

Complement system is an important component of innate immunity, providing primary protection against pathogens invading the body. In addition, it was shown that the complement system is associated with many diseases, not only autoimmune and infectious, but also mental disorders. In this regard, it is necessary to develop affordable and fast method of measuring activity of the complement system in real-time mode. We present a new semi-automated method for assessment of serum complement activity. The assay is based on cytolytic action of complement system upon the ciliate organism Tetrahymena pyriformis. This method consists in repeated counting of live Tetrahymena motile cells by means of specially developed Biolat device, which consists of two video cameras, light sources, and movable round plate. The plate has two rows of holes. The device also includes microprocessor control unit based on AutoCiliata software, intended for control of operation module and counting the surviving cell. The calculations are based on fixation of two sequential video-frames, with subsequent software image processing. Cell death events were observed upon incubation in triethanolamine (TEA) buffer containing 5% of blood serum. We have also compared complement activity in different buffers, i.e., standard medium for culturing of ciliates, Veronal-Medinalum buffer, and the TEA buffer. TEA buffer was found superior to the Veronal buffer when applied in the test system. The time of cell death in the TEA-buffered medium containing 5% serum was < 15 minutes for all the sera studied. The parameters denoting serum complement activity were as follows: a half-life time for the moving cells (TLD50), and a similar value for 100% cell inactivation (1/TLD50, functional activity of the complement system, ACS). The sensitivity of this assay was calculated from dependencies between TLD50 and ACS, and actual serum concentrations. We have suggested an opportunity for evaluation of an integral complement activity, and interrelations between the intensity of synthesis and consumption of its major effector proteins. In the course of this study, we have tested different concentrations of Ca++ and Mg++ ions in the incubation buffer, with optimal physiological concentrations of2.5 mMand1.5 mM, respectively. We have also estimated statistical precision characteristics for pre-analytical and analytical steps of the method. The average coefficients of variation (CV) were 3.9% and 2.7%, respectively, thus satisfying the reliability criteria in research. A short performance time of the study suggests its potential application in clinical practice, including online examination regimens. A method for semi-automatic measurement of serum complement activity could be applicable in daily clinical practice, including the online performance

Single-Cell Analysis of Ploidy and Centrosomes Underscores the Peculiarity of Normal Hepatocytes

Polyploidization is the most well recognized feature of the liver. Yet, a quantitative and behavioral analysis of centrosomes and DNA content in normal hepatocytes has been limited by the technical challenges of methods available. By using a novel approach employing FISH for chromosomes 18, X and Y we provide, for the first time, a detailed analysis of DNA copies during physiological development in the liver at single cell level. We demonstrate that aneuploidy and unbalanced DNA content in binucleated hepatocytes are common features in normal adult liver. Despite the common belief that hepatocytes contain 1, 2 or no more than 4 centrosomes, our double staining for centrosome associated proteins reveals extranumerary centrosomes in a high percentage of cells as early as 15 days of age. We show that in murine liver the period between 15 days and 1.5 months marks the transition from a prevalence of mononucleated cells to up to 75% of binucleated cells. Our data demonstrate that this timing correlates with a switch in centrosomes number. At 15 days the expected 1 or 2 centrosomes converge with several hepatocytes that contain 3 centrosomes; at 1.5 months the percentage of cells with 3 centrosomes decreases concomitantly with the increase of cells with more than 4 centrosomes. Our analysis shows that the extranumerary centrosomes emerge in concomitance with the process of binucleation and polyploidization and maintain α-tubulin nucleation activity. Finally, by integrating interphase FISH and immunofluorescent approaches, we detected an imbalance between centrosome number and DNA content in liver cells that deviates from the equilibrium expected in normal cells. We speculate that these unique features are relevant to the peculiar biological function of liver cells which are continuously challenged by stress, a condition that could predispose to genomic instability