Release of Histone Lysine Methyltransferases from Rat Brain Chromatin by Nuclease Digestion

The histone lysine methyltransferases catalyze the transfer of methyl groups from S-adenosyl-L-methionine to specific ε-N-lysyl residues in the N-terminal regions of histones H3 and H4. These enzymes are located exclusively within the nucleus and are firmly bound to chromatin. The chromosomal bound enzymes do not methylate free or loosely bound histones. However, histones H3 and H4 associated within the nucleosomes are methylated. The enzymes were extracted by limited digestion (12-16%) of chromosomal DNA from rapidly proliferating rat brain chromatin with micrococcal nuclease. The enzymes were further purified by gel filtration, ammonium sulfate fractionation and DEAE-cellulose chromatography. The histone methyltransferases were resolved into two distinct fractions by Sepharose 6B-100 and DEAE-cellulose chromatography. One enzyme fractionated by DEAE-cellulose chromatograpy was specific for histone H3, while the other enzyme was specific for histone H4. Histone H3 lysine methyltransferase was shown to methylate only the lysyl residues of chromosomal bound or soluble histone H3. The molar ratio of mono- to di- to trimethyllysine in the soluble system was 1.0:2.1:1.0, while the ratio with chromosomal bound histone H3 was 1.9:1.0:0.08. The histone H4 lysine methyltransferase which was detectable in the crude nuclease digest, was extremely labile loosing all activity upon further purification. The enzyme specific for histone H4 after DEAE-cellulose chromatography methylated only arginyl residues in histone H4 and would not methylate lysyl residues in histone H4. The pH optimum for histone H3 lysine methyltransferase with soluble rat brain histone H3 as substrate was 8.5 with little variation from pH 8.2 to 8.7. The pH optimum for histone H4 arginine methyltransferase with soluble histone H4 as the methyl acceptor was 7.5 with little variation from pH 7.3 to 7.8. After DEAE-cellulose chromatography both enzymes were extremely unstable. Complete removal of DNA by DNAase I digestion resulted in the complete loss of enzyme activity. However, when the enzymes remained associated with DNA fragments they were quite stable. Indicating that the enzymes require DNA for stability and/or activity. The requirement for DNA may only be important to prevent hydrophobic interactions involving the enzyme with itself and/or other non-histone chromosomal proteins

Iterative Reconstruction Methods for Cosmological X-Ray Tomography

We consider the imaging of cosmic strings by using Cosmic Microwave Background (CMB) data. Mathematically, we study the inversion of an X-ray transform in Lorentzian geometry, called the light ray transform. The inverse problem is highly ill-posed, with additional complexities of being large-scale and dynamic, with unknown parameters that represent multidimensional objects. This presents significant computational challenges for the numerical reconstruction of images that have high spatial and temporal resolution. In this paper, we begin with a microlocal stability analysis for inverting the light ray transform using the Landweber iteration. Next, we discretize the spatiotemporal object and light ray transform and consider iterative computational methods for solving the resulting inverse problem. We provide a numerical investigation and comparison of some advanced iterative methods for regularization including Tikhonov and sparsity-promoting regularizers for various example scalar functions with conormal type singularities.Comment: 22 pages; codes for this paper will be made available at https://github.com/lonisk1/CMB_InvProb once the revision process is complet

Range restricted iterative methods for linear discrete ill-posed problems

Linear systems of equations with a matrix whose singular values decay to zero with increasing index number, and without a significant gap, are commonly referred to as linear discrete ill-posed problems. Such systems arise, e.g., when discretizing a Fredholm integral equation of the first kind. The right-hand side vectors of linear discrete ill-posed problems that arise in science and engineering often represent an experimental measurement that is contaminated by measurement error. The solution to these problems typically is very sensitive to this error. Previous works have shown that error propagation into the computed solution may be reduced by using specially designed iterative methods that allow the user to select the subspace in which the approximate solution is computed. Since the dimension of this subspace often is quite small, its choice is important for the quality of the computed solution. This work describes algorithms for three iterative methods that modify the GMRES, block GMRES, and global GMRES methods for the solution of appropriate linear systems of equations. We contribute to the work already available on this topic by introducing two block variants for the solution of linear systems of equations with multiple right-hand side vectors. The dominant computational aspects are discussed, and software for each method is provided. Additionally, we illustrate the utility of these iterative subspace methods through numerical examples focusing on image reconstruction. This paper is accompanied by software

An Arnoldi-based preconditioner for iterated Tikhonov regularization

Many problems in science and engineering give rise to linear systems of equations that are commonly referred to as large-scale linear discrete ill-posed problems. These problems arise, for instance, from the discretization of Fredholm integral equations of the first kind. The matrices that define these problems are typically severely ill-conditioned and may be rank-deficient. Because of this, the solution of linear discrete ill-posed problems may not exist or be very sensitive to perturbations caused by errors in the available data. These difficulties can be reduced by applying Tikhonov regularization. We describe a novel "approximate Tikhonov regularization method" based on constructing a low-rank approximation of the matrix in the linear discrete ill-posed problem by carrying out a few steps of the Arnoldi process. The iterative method so defined is transpose-free. Our work is inspired by a scheme by Donatelli and Hanke, whose approximate Tikhonov regularization method seeks to approximate a severely ill-conditioned block-Toeplitz matrix with Toeplitz-blocks by a block-circulant matrix with circulant-blocks. Computed examples illustrate the performance of our proposed iterative regularization method

Project Management Competences by Teaching and Research Staff for the Sustained Success of Engineering Education

Projects have become an essential instrument for the success of universities. In a context of globalization and increasing complexity, they must sharpen their resourcefulness to face these challenges and adapt to this changing environment. To reach these objectives, they undertake a series of activities of a unique, concrete and temporary nature, not always technical but managerial ones. If universities work with people on projects in the production, transmission and dissemination of knowledge, then they link with society to solve its problems. For this reason, teaching and research staff (TRS) should promote a range of professional project management (PM) competences in different areas for the proper management of the projects in which they take part. Through a Delphi technique, a panel of twenty-four accredited teaching experts who are carrying out significant research and holding directive roles, measured the importance of acquiring and/or improving professional PM competences by their TRS. Consensus and stability reached after two rounds of consultation confirmed there are a series of crucial competences for the practice of relevant teaching and pioneer research. Results obtained are the basis for a gap plan that allows the TRS to participate in and/or lead university projects with greater self-confidence and personal motivation

Impact of Immunization Technology and Assay Application on Antibody Performance – A Systematic Comparative Evaluation

Antibodies are quintessential affinity reagents for the investigation and determination of a protein's expression patterns, localization, quantitation, modifications, purification, and functional understanding. Antibodies are typically used in techniques such as Western blot, immunohistochemistry (IHC), and enzyme-linked immunosorbent assays (ELISA), among others. The methods employed to generate antibodies can have a profound impact on their success in any of these applications. We raised antibodies against 10 serum proteins using 3 immunization methods: peptide antigens (3 per protein), DNA prime/protein fragment-boost (“DNA immunization”; 3 per protein), and full length protein. Antibodies thus generated were systematically evaluated using several different assay technologies (ELISA, IHC, and Western blot). Antibodies raised against peptides worked predominantly in applications where the target protein was denatured (57% success in Western blot, 66% success in immunohistochemistry), although 37% of the antibodies thus generated did not work in any of these applications. In contrast, antibodies produced by DNA immunization performed well against both denatured and native targets with a high level of success: 93% success in Western blots, 100% success in immunohistochemistry, and 79% success in ELISA. Importantly, success in one assay method was not predictive of success in another. Immunization with full length protein consistently yielded the best results; however, this method is not typically available for new targets, due to the difficulty of generating full length protein. We conclude that DNA immunization strategies which are not encumbered by the limitations of efficacy (peptides) or requirements for full length proteins can be quite successful, particularly when multiple constructs for each protein are used

FcRn Overexpression in Transgenic Mice Results in Augmented APC Activity and Robust Immune Response with Increased Diversity of Induced Antibodies

Our previous studies have shown that overexpression of bovine FcRn (bFcRn) in transgenic (Tg) mice leads to an increase in the humoral immune response, characterized by larger numbers of Ag-specific B cells and other immune cells in secondary lymphoid organs and higher levels of circulating Ag-specific antibodies (Abs). To gain additional insights into the mechanisms underlying this increase in humoral immune response, we further characterized the bFcRn Tg mice. Our Western blot analysis showed strong expression of the bFcRn transgene in peritoneal macrophages and bone marrow derived dendritic cells; and a quantitative PCR analysis demonstrated that the expression ratios of the bFcRn to mFcRn were 2.6- and 10-fold in these cells, respectively. We also found that overexpression of bFcRn enhances the phagocytosis of Ag-IgG immune complexes (ICs) by both macrophages and dendritic cells and significantly improves Ag presentation by dendritic cells. Finally, we determined that immunized bFcRn mice produce a much greater diversity of Ag-specific IgM, whereas only the levels, but not the diversity, of IgG is increased by overexpression of bFcRn. We suggest that the increase in diversity of IgG in Tg mice is prevented by a selective bias towards immunodominant epitopes of ovalbumin, which was used in this study as a model antigen. These results are also in line with our previous reports describing a substantial increase in the levels of Ag-specific IgG in FcRn Tg mice immunized with Ags that are weakly immunogenic and, therefore, not affected by immunodominance

The concept of a sports discipline thematic atlas on the example of the Atlas of archery

Gołębiowska, IzabelaKorycka-Skorupa, JolantaGołębiowska, Izabel