Dalius Matuzevičius

Proteomic Analysis of Stromal Cells Derived from the Dental Pulp of Human Exfoliated Deciduous Teeth

Human dental pulp derived from exfoliated deciduous teeth has been described as a promising alternative source of multipotent stem cells. While these cells share certain similarities with mesenchymal stem-like cells (MSC) isolated from other tissues, basically they are still poorly characterized. In this study, for the first time, a proteomic map of abundantly expressed proteins in stromal cells derived from the dental pulp of human exfoliated deciduous teeth (SHED) was established. We also analyzed proteomic signatures of 2 clonal strains derived from SHEDs by single-cell cloning. The SHEDs were established from enzyme-disaggregated deciduous dental pulp from 6-year-old children. They had typical fibroblastoid morphology and high colony-forming efficiency index (16.4%). Cloning was performed at the second passage using limiting dilution in a 96-well plate (0.3 cell/well). Differentiation assessment revealed strong osteogenic but no adipogenic potential of the SHEDs in either clonal strain. The cells expressed characteristic antigens of MSC-like cells, including CD73, CD90, CD105, CD146, and did not express hematopoietic markers CD14, CD34, and CD45, as assessed with FACS analysis. For proteomic studies, cytosolic and nuclear proteins were analyzed with 2-dimensional gel electrophoresis (2-DE) and identified using matrix-assisted laser desorption/ionization (MALDI)-time of fl ight (TOF)-mass spectrometry (MS). All proteins were identified with high level of confidence (the lowest sequence coverage was 27%). Identification of highly expressed proteins in SHEDs revealed proteomic profiles very similar to that of MSC-like cells derived from other tissues. We also found a high degree of similarity between proteomic signatures of primary SHEDs and clonal cell strains. Thus, our data confirm a close resemblance between SHEDs and MSC-like cells from other tissues and may serve as starting point for creating-comprehensive proteomic maps.

Feature selection for segmentation of 2-D electrophoresis gel images

Two-dimensional gel electrophoresis is the powerful technique used by biochemists to resolve and visualize protein samples.Commonly gels produced from several samples are analyzed in order to detect changes of protein expression. Thus computer-aided gel image analysis for protein spot detection became the main step in the whole process.Nevertheless accurate automatic spot detection is still difficult due to large variations in spot shape, image background and various inevitable artifacts. In this paper we investigate features of two-dimensional electrophoresis gel images.We look for those image features that will yield good results of protein spot detection done by the Feedforward Multilayer Neural Network. Feature comparison and spot segmentation results are presented and indicate that rotational symmetry features empowers segmentation of saturated and overlapped protein spots.

Alpha-Dystrobrevin and its associated proteins in human promyelocytic leukemia cells induced to apoptosis

Dystrobrevin is a dystrophin-related component of the dystrophin-associated protein complex (DAPC). Using alpha-dystrobrevin as indicator, we aimed to elucidate the interaction network of the DAPC with other proteins during apoptosis of promyelocytic HL-60 cells. The precise role(s) of DBs are not known, but we and others have shown that they play a role in intracellular signal transduction and cellular organization. Apoptosis was induced with etoposide in the absence or presence of Z-VAD to block caspase activity, and we then followed the cellular distribution of α-DB and its association with other proteins, using confocal imaging and cell fractions analyses after immune-precipitation with anti-α-DB and mass spectrometry. Confocal imaging revealed distinct spatial relocalizations of α-DB between the cell membrane, cytosol and nucleus after induction of apoptosis. The expression levels of the identified proteins were evaluated with computer-assisted image analysis of the gels. We thus identified associations with structural and transport proteins (tropomyosin, myosin), membrane (ADAM21, syntrophin), ER-Golgi (TGN51, eIF38) and nuclear (Lamins, ribonucleoprotein C1/C2) proteins. These results suggest that apoptosis-induction in HL-60 cells involves not only classical markers of apoptosis but also a network α-DB-associated proteins at the cell membrane, the cytoplasm and nucleus, affecting key cellular transport processes and cellular structure.

Histone modifications patterns in tissues and tumours from acute promyelocytic leukemia xenograft model in response to combined epigenetic therapy.

Xenograft models are suitable for in vivo study of leukemias pathogenesis and the preclinical development of anti-leukemia agents but understanding of epigenetic regulatory mechanisms linking to adult cell functions in pathological conditions during different in vivo treatments is yet unknown. In this study, for the first time epigenetic chromatin modifications were characterized in tissues and tumours from murine xenograft model generated using the human acute promyelocytic leukemia (APL) NB4 cells engrafted in immunodeficient NOG mice. Xenografts were subjected to combined epigenetic treatment by histone deacetylase inhibitor Belinostat, histone methyltransferase inhibitor 3-DZNeaplanocin A and all-trans-retinoic acid based on in vitro model, where such combination inhibited NB4 cell growth and enhanced retinoic acid-induced differentiation to granulocytes. Xenotransplantation was assessed by peripheral blood cells counts, the analysis of cell surface markers (CD15, CD33, CD45) and the expression of certain genes (PML-RAR alpha, CSF3, G-CSFR, WT1). The combined treatment prolonged APL xenograft mice survival and prevented tumour formation. The analysis of the expression of histone marks such as acetylation of H4, trimethylation of H3K4, H3K9 and H3K27 in APL xenograft mice tumours and tissues demonstrated tissue-specific changes in the level of histone modifications and the APL prognostic mark, WT1 protein. In summary, the effects of epigenetic agents used in this study were positive for leukemia prevention and linked to a modulation of the chromatin epigenetic environment in adult tissues of malignant organism.

Application of human visual system models to LCD image analysis

Automated analysis of the segment LCD images may be utilized to detect hardware or software related problems of the device or LCD itself. The malfunction may be revealed through detection of faulty segments. Difficulties to automate image analysis arise from existing intensity and geometric distortions. We adopt concepts of biological visual systems to improve performance of computational system. In this paper we compare conventional and bio-inspired local feature detectors along with descriptors, and introduce a new feature matching approach based on evidence accumulation. Finally we construct specialized algorithm for alignment of LCD images. The performance of the proposed algorithm has been evaluated on the set of semi-synthetic images. The comparison with common image registration techniques shows elimination of image misalignments.

Vector model for mapping of visual space to subjective 4-D sphere

Here we present a mathematical model of binocular vision that maps a visible physical world to a subjective perception of it. The subjective space is a set of 4-D vectors whose components are outputs of four monocular neurons from each of the two eyes. Monocular neurons have one of the four types of concentric receptive fields with Gabor-like weighting coefficients. Next this vector representation of binocular vision is implemented as a pool of neurons where each of them is selective to the objects particular location in a 3-D visual space. Formally each point of the visual space is being projected onto a 4-D sphere. Proposed model allows determination of subjective distances in depth and direction, provides computational means for determination of Panums area and explains diplopia and allelotropia.

Epigenetic changes during hematopoietic cell granulocytic differentiation – comparative analysis of primary CD34+ cells, KG1 myeloid cells and mature neutrophils

BackgroundEpigenetic regulation is known to affect gene expression, and recent research shows that aberrant DNA methylation patterning and histone modifications may play a role in leukemogenesis. In order to highlight the co-operation of epigenetic mechanisms acting during the latter process it is important to clarify their potential as biomarkers of granulocytic differentiation.ResultsIn this study we investigated epigenetic alterations in human hematopoietic cells at a distinct differentiation stages: primary hematopoietic CD34+ cells, KG1 myeloid leukemic cells, whose development is stopped at early stage of differentiation, and mature neutrophils. We focused on the epigenetic status of cell cycle regulating (p15, p16) and differentiation related (E-cadherin and RARβ) genes. We found that the methylation level in promoter regions of some of these genes was considerably higher in KG1 cells and lower in CD34+ cells and human neutrophils. As examined and evaluated by computer-assisted methods, histone H3 and H4 modifications, i.e. H3K4Me3, H3K9Ac, H3K9Ac/S10Ph and H4 hyperAc, were similar in CD34+ cells and human mature neutrophils. By contrast, in the KG1 cells, histone H3 and H4 modifications were quite high and increased after induction of granulocytic differentiation with the HDAC inhibitor phenyl butyrate.ConclusionsWe found the methylation status of the examined gene promoters and histone modifications to be characteristically associated with the hematopoietic cell progenitor state, induced to differentiate myeloid KG1 cells and normal blood neutrophils. This could be achieved through epigenetic regulation of E-cadherin, p15, p16 and RARβ genes expression caused by DNA methylation/demethylation, core and linker histones distribution in stem hematopoietic cells, induced to differentiation KG1 cells and mature human neutrophils, as well as the histone modifications H3K4Me3, H3K9Ac, H3K9Ac/S10Ph and H4 hyperAc in relation to hematopoietic cell differentiation to granulocyte. These findings also suggest them as potentially important biomarkers of hematopoietic cell granulocytic differentiation and could be valuable for leukemia induced differentiation therapy.

Application of k-means and MLP in the automation of matching of 2DE gel images

Critical information that is related to vital processes of the cell can be revealed comparing several two-dimensional electrophoresis (2DE) gel images. Through up to 10 000 protein spots may appear in inevitably noisy gel thus 2DE gel image comparison and analysis protocols usually involve the work of experts. In this paper we demonstrate how the problem of automation of 2DE gel image matching can be gradually solved by the use of artificial neural networks. We report on the development of feature set, built from various distance measures, selected and grounded by the application of self-organizing feature map and confirmed by expert decisions. We suggest and experimentally confirm the use of k-means clustering for the pre-classification of 2DE gel image into segments of interest that about twice speed-up the comparison procedure. We develop original Multilayer Perceptron based classifier for 2DE gel image matching that employs the selected feature set. By experimentation with the synthetic, semi-synthetic and natural 2DE images we show its superiority against the single distance metric based classifiers.

Histone Modifications Pattern Associated With a State of Mesenchymal Stem Cell Cultures Derived From Amniotic Fluid of Normal and Fetus-Affected Gestations

Human amniotic fluid (AF)‐derived mesenchymal stem cells (MSCs) sharing embryonic and adult stem cells characteristics are interesting by their multipotency and the usage for regenerative medicine. However, the usefulness of these cells for revealing the fetal diseases still needs to be assessed. Here, we have analyzed the epigenetic environment in terms of histone modifications in cultures of MSCs derived from AF of normal pregnancies and those with fetal abnormalities. The comparison of MSCs samples from AF of normal pregnancies (N) and fetus‐affected (P) revealed two distinct cultures by their proliferation potential (P I and P II). Cell populations from N and P I samples had similar growth characteristics and exhibited quite similar cell surface (CD44, CD90, CD105) and stemness markers (Oct4, Nanog, Sox2, Rex1) profile that was distinct in slower growing and faster senescent P II cultures. Those differences were associated with changes in 5‐Cyt DNA methylation and alterations in the expression levels of chromatin modifiers (DNMT1, HDAC1/2), activating (H4ac, H3K4me3), and repressive (H3K9me2/me3, H3K27me3) histone marks. MSCs isolated from AF with the genetic or multifactorial fetal diseases (P II samples) were enriched with repressive histone marks and H4K16ac, H3K9ac, H3K14ac modifications. This study indicates that differential epigenetic environment reflects a state of AF‐MSCs dependently on their growth, phenotype, and stemness characteristics suggesting a way for better understanding of epigenetic regulatory mechanisms in AF‐MSCs cultures in normal and diseased gestation conditions. J. Cell. Biochem. 118: 3744–3755, 2017.

Proteomic Analysis of Pollen and Blossom Honey from Rape Seed Brassica Napus L.

Abstract In the study, honey from oilseed rape Brassica napus L., and both hand-collected (winter rape Visby and Cult) and bee-collected pollen of oilseed rape were analyzed for their proteome content, in order to see if any plant proteins were present to allow the proteo-typing of the oilseed rape honey. Proteins were fractionated by two-dimensional gel electrophoresis (2DE), stained by Coomassie blue and then analyzed by mass spectrometry. All identified proteins were divided into few groups due to their biological function. In 2DE gels with separated proteins from blossom honey, only bee (Apis mellifera) main proteins (Major royal jelly protein 1-5 and Glucosidase) were found. So we analyzed all proteins using gel-free based analysis with the SYNAPT G2 high definition mass spectrometry. We identified proteins that were present in both oilseed rape pollen and honey (Bna, Polygalacturonase, Non-specific lipid-transfer protein, GAPDH and others). We believe that these proteins are important for the nutritional value of plant pollen-enriched honey and further research is required on honey and honeybee pollen protein.