Tytus Bernas

MicroRNAs 10a and 10b are potent inducers of neuroblastoma cell differentiation through targeting of nuclear receptor corepressor 2

MicroRNAs function as negative regulators of posttranscriptional gene expression, having major roles in cellular differentiation. Several neuroblastoma cell lines can be induced to undergo differentiation by all-trans-retinoic acid (ATRA) and are used for modeling signaling pathways involved in this process. To identify miRNAs contributing to differentiation, we profiled 364 loci following ATRA treatment of neuroblastoma cell lines and found miR-10a and miR-10b to be highly overexpressed in SK-N-BE, LAN5 and SHSY-5Y. Ectopic overexpression of these miRNAs led to a major reprogramming of the transcriptome and a differentiated phenotype that was similar to that induced by ATRA in each of these cell lines. One of the predicted downregulated miR-10a/b targets was nuclear receptor corepressor 2 (NCOR2), a corepressor of gene transcription, which is known to suppress neurite outgrowth. NCOR2 was experimentally validated as a direct target of miR-10a/b, and siRNA-mediated inhibition of this mRNA alone resulted in neural cell differentiation. Moreover, induction of differentiation could be blocked by ectopic upregulation of NCOR2 using an expression construct lacking the miR-10a/b 3′ untranslated region target site. We conclude that miR-10a/b has major roles in the process of neural cell differentiation through direct targeting of NCOR2, which in turn induces a cascade of primary and secondary transcriptional alterations, including the downregulation of MYCN.

Minimizing photobleaching during confocal microscopy of fluorescent probes bound to chromatin: role of anoxia and photon flux

Exposure to light can destroy the ability of a molecule to fluoresce. Such photobleaching limits the use of fluorescence and confocal microscopy in biological studies. Loss of fluorescence decreases the signal‐to‐noise ratio and so image resolution; it also prevents the acquisition of meaningful data late during repeated scanning (e.g. when collecting three‐dimensional images). The aim of this work was to investigate the role of oxygen in the photobleaching of fluorophores bound to DNA in fixed cells, and to explore whether anoxia could minimize such bleaching. Anoxia significantly reduced bleaching rates and changed the order of reaction of both propidium iodide (an intercalator) and chromomycin A3 (a minor‐groove binder) bound to DNA; it afforded the greatest protection at low photon fluxes. However, it had no effect on the bleaching of the green fluorescent protein (GFP) covalently attached to a histone and so bound to DNA, probably because the protein shielded the chromophore from oxygen. Bleaching of all three fluorophores depended on photon flux. Practical ways of minimizing bleaching were examined, and examples of three‐dimensional images of DNA marked by propidium and GFP (collected under standard and optimized conditions) are presented.

A numerical recipe for accurate image reconstruction from discrete orthogonal moments

Recursive procedures used for sequential calculations of polynomial basis coefficients in discrete orthogonal moments produce unreliable results for high moment orders as a result of error accumulation. This paper demonstrates accurate reconstruction of arbitrary-size images using full-order (orders as large as the image size) Tchebichef and Krawtchouk moments by calculating polynomial coefficients directly from their definition formulas in hypergeometric functions and by creating lookup tables of these coefficients off-line. An arbitrary precision calculator is used to achieve greater numerical range and precision than is possible with software using standard 64-bit IEEE floating-point arithmetic. This reconstruction scheme is content and noise independent.

Reduction of a tetrazolium salt, CTC, by intact HepG2 human hepatoma cells: subcellular localisation of reducing systems

Cell-mediated reduction of tetrazolium salts, including MTT, XTT, MTS, NBT, NTV, INT, in the presence or absence of intermediate electron carriers is used as a convenient test for animal or bacterial cell viability. Bioreduction of tetrazolium is considered an alternative to a clonogenic assay and a thymidine incorporation assay. However, correlation between clonogenic potential and capacity to reduce tetrazolium has not been demonstrated convincingly. Moreover, despite a wide use of tetrazolium viability assays, the mechanism and subcellular localisation of reducing systems or species in viable intact cells have not been fully elucidated. We report evidence indicating that a tetrazolium salt CTC can be reduced in the presence as well as in the absence of an electron carrier by viable HepG2 human hepatoma cells. CTC-formazan is formed within or at the outer surface of plasma membranes. We hypothesise that in the presence of an electron carrier the electron donors active in the reduction of CTC are located in the intracellular compartment, as well as in plasma membranes. However, in the absence of an electron carrier, the reduction occurs primarily via a plasma membrane-associated enzymatic system or species.

Integrating Cytomics and Proteomics

Systems biology along with what is now classified as cytomics provides an excellent opportunity for cytometry to become integrated into studies where identification of functional proteins in complex cellular mixtures is desired. The combination of cell sorting with rapid protein-profiling platforms offers an automated and rapid technique for greater clarity, accuracy, and efficiency in identification of protein expression differences in mixed cell populations. The integration of cell sorting to purify cell populations opens up a new area for proteomic analysis. This article outlines an approach in which well defined cell analysis and separation tools are integrated into the proteomic programs within a core laboratory. In addition we introduce the concepts of flow cytometry sorting to demonstrate the importance of being able to use flow cytometry as a cell separation technology to identify and collect purified cell populations. Data demonstrating the speed and versatility of this combination of flow cytometry-based cell separation and protein separation and subsequent analysis, examples of protein maps from purified sorted cells, and an analysis of the overall procedure will be shown. It is clear that the power of cell sorting to separate heterogeneous populations of cells using specific phenotypic characteristics increases the power of rapid automated protein separation technologies.

Confocal Fluorescence Imaging of Photosensitized DNA Denaturation in Cell Nuclei

The double‐stranded helical structure of DNA is maintained in part by hydrogen bonds between strands and by stacking interactions between adjacent purine and pyrimidine bases in one strand. The transition (denaturation) from a double‐stranded (ds) to a single‐stranded (ss) form can be induced in isolated DNA or fixed cells by exposure to elevated temperatures, alkali or acids, aprotic or nonpolar solvents or some drugs. We report here that DNA denaturation can occur in situ in cell nuclei as a result of interaction between light and an intercalated dye, acridine orange or ethidium bromide. This DNA photodenaturation was probed using metachromatic properties of acridine orange and imaged by fluorescence confocal microscopy. Furthermore, an empirical kinetic model was developed to separate changes of acridine orange luminescence intensities caused by photobleaching from those that were a result of DNA denaturation. We investigated the influence of oxygen on these phenomena and propose a mechanism by which photodenaturation may occur.

Loss of image quality in photobleaching during microscopic imaging of fluorescent probes bound to chromatin

Prolonged excitation of fluorescent probes leads eventually to loss of their capacity to emit light. A decrease in the number of detected photons reduces subsequently the resolving power of a fluorescence microscope. Adverse effects of fluorescence intensity loss on the quality of microscopic images of biological specimens have been recognized, but not determined quantitatively. We propose three human-independent methods of quality determination. These techniques require no reference images and are based on calculation of the actual resolution distance, information entropy, and signal-to-noise ratio (SNR). We apply the three measures to study the effect of photobleaching in cell nuclei stained with propidium iodide (PI) and chromomycin A3 (CA3) and imaged with fluorescence confocal microscopy. We conclude that the relative loss of image quality is smaller than the corresponding decrease in fluorescence intensity. Furthermore, the extent of quality loss is related to the optical properties of the imaging system and the noise characteristics of the detector. We discuss the importance of these findings for optimal registration and compression of biological images.

Quadratic form: A robust metric for quantitative comparison of flow cytometric histograms

Comparison of fluorescence distributions is a fundamental part of the analysis of flow cytometric data. This approach is applied to detect differences between control and test sample and thus analyze a biological response. Comparison of standard test samples over time provides an estimate of instrument stability for quality control. However, application of statistical methods of distribution comparison in flow cytometry is difficult owing to instrument noise and the complex shape of intensity distributions. We applied quadratic form (QF) as a mathematical metric for comparison of flow cytometry histograms. QF operates on histograms as vectors and calculates the total distance in an interbin manner using a matrix of distances between single histogram bins. Euclidean interbin distance and histograms normalized to unity were used. Critical values corresponding to 95% significance level were calculated using Monte‐Carlo simulation and single‐maximum Gaussian distributions populated with several numbers of events. The QF statistic was then validated for non‐Gaussian single‐maximum distributions and multiple‐maxima distributions. We determined that the critical values for Gaussian distributions depended on standard deviations and number of events in the compared histograms. A simple empirical function was constructed to characterize this dependence. Furthermore, it was verified that critical values (corresponding to 95% significance) for non‐Gaussian histograms were similar to values for the Gaussian histograms characterized by the same standard deviation. We applied the QF statistic to estimate the differences between histograms of DNA content (ploidy) in cells of old and young leaf tissue of Brassica campestris. Furthermore, we quantified differences in fluorescence intensity in immunostaining of human lymphocytes. Quadratic form (QF) provides a true (mathematical) metric for estimation of distance between flow cytometry histograms of arbitrary shape. QF can be applied as a statistical test for estimation of significance of the distance measure. The respective critical values depend only on the number of events and standard deviations of compared histograms and are not affected by distribution shape. Therefore, applications of QF do not require assumptions concerning distribution shape and can be easily implemented in practice. This notion was confirmed using empirical distributions of DNA content in plant tissue and distributions of immunofluorescence in human cells.

BH3-only proteins BIM and PUMA in the regulation of survival and neuronal differentiation of newly generated cells in the adult mouse hippocampus.

Neurogenesis persists in the adult hippocampus, where several thousand neurons are born every day. Most of the newly generated cells are eliminated by apoptosis, possibly because of their failure to integrate properly into neural networks. The BH3-only proteins Bim and Puma have been shown to mediate trophic factor withdrawal- and anoikis-induced apoptosis in various systems. We therefore determined their impact on proliferation, survival, and differentiation of adult-generated cells in the mouse hippocampus using gene-deficient mice. Wild-type, bim-, and puma-deficient mice showed similar rates of precursor cell proliferation, as evidenced by 5-bromo-2-deoxyuridine (BrdU)-incorporation. Deficiency in either bim or puma significantly increased the survival of adult-born cells in the dentate gyrus (DG) after 7 days. Consistently, we detected increased numbers of doublecortin (DCX)-positive and fewer terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelled-positive cells in the DG of bim- and puma-deficient mice. Bim and puma deficiency did not change early markers of neuronal differentiation, as evidenced by BrdU/DCX double-labelling. However, BrdU/NeuN double-labelling revealed that deficiency of bim, but not puma, accelerated the differentiation of newly generated cells into a neuronal phenotype. Our data show that Bim and Puma are prominently involved in the regulation of neuronal progenitor cell survival in the adult DG, but also suggest that Bim has an additional role in neuronal differentiation of adult-born neural precursor cells.

Application of lyophilization to prepare the nitrifying bacterial biofilm for imaging with scanning electron microscopy

Structure of bacterial biofilms may be investigated using several variants of scanning electron microscopy (SEM). We apply lyophilization to prepare nitrifying bacterial biofilm for conventional SEM imaging in high-vacuum mode (CSEM). We therefore replace standard biofilm fixation in glutaraldehyde cross-linking, ethanol dehydration, and critical-point drying (CPD) with less-invasive low-temperature drying by sublimation in vacuum. We compare this approach with: (1) standard preparation with glutaraldehyde fixation, ethanol dehydration, and CPD before CSEM, (2) cryo-sputter preparation of rapidly frozen biofilm in hydrated state (cryo-SEM), and (3) in situ observation without any sample pretreatment in environmental SEM. Combined imaging with these modalities revealed two distinct immobilization patterns on the polyurethane foam: (1) large irregular aggregates (flocs) of bacterial biofilm that exist as irregular biofilm fragments, rope-like structures, or biofilm layers on the foam surface; (2) biofilm threads adherent to the surface of polyurethane foam. Our results indicate that lyophilization was suitable for preservation of bacterial cells and many forms of structure of extracellular matrix. The lyophilized material could be imaged with high resolution (using CSEM) to generate structural information complementary to that obtained with other SEM techniques.