PITX1 is a regulator of TERT expression in prostate cancer with prognostic power

Simple Summary Most prostate cancer is of an indolent form and is curable. However, some prostate cancer belongs to rather aggressive subtypes leading to metastasis and death, and immediate therapy is mandatory. However, for these, the therapeutic options are highly invasive, such as radical prostatectomy, radiation or brachytherapy. Hence, a precise diagnosis of these tumor subtypes is needed, and the thus far applied diagnostic means are insufficient for this. Besides this, for their endless cell divisions, prostate cancer cells need the enzyme telomerase to elongate their telomeres (chromatin endings). In this study, we developed a gene regulatory model based on large data from transcription profiles from prostate cancer and chromatin-immuno-precipitation studies. We identified the developmental regulator PITX1 regulating telomerase. Besides observing experimental evidence of PITX1′s functional role in telomerase regulation, we also found PITX1 serving as a prognostic marker, as concluded from an analysis of more than 15,000 prostate cancer samples. Abstract The current risk stratification in prostate cancer (PCa) is frequently insufficient to adequately predict disease development and outcome. One hallmark of cancer is telomere maintenance. For telomere maintenance, PCa cells exclusively employ telomerase, making it essential for this cancer entity. However, TERT, the catalytic protein component of the reverse transcriptase telomerase, itself does not suit as a prognostic marker for prostate cancer as it is rather low expressed. We investigated if, instead of TERT , transcription factors regulating TERT may suit as prognostic markers. To identify transcription factors regulating TERT , we developed and applied a new gene regulatory modeling strategy to a comprehensive transcriptome dataset of 445 primary PCa. Six transcription factors were predicted as TERT regulators, and most prominently, the developmental morphogenic factor PITX1. PITX1 expression positively correlated with telomere staining intensity in PCa tumor samples. Functional assays and chromatin immune-precipitation showed that PITX1 activates TERT expression in PCa cells. Clinically, we observed that PITX1 is an excellent prognostic marker, as concluded from an analysis of more than 15,000 PCa samples. PITX1 expression in tumor samples associated with (i) increased Ki67 expression indicating increased tumor growth, (ii) a worse prognosis, and (iii) correlated with telomere length

Technology and feminism : a strange couple

The "gender digital divide" constitutes a prolific research program that compares the differences between women and men in access to Information and Communication Technologies (ICT). Nevertheless, those using feminist socio-constructivist perspectives argue for the need to pay attention, not only to "access," but also to "design," in addition to considering social relations as something that is coded within technological artifacts. From this perspective, gender constitutes an integral part of technological production. This paper explores the co-constitution of gender and technology, considering a specific action-research experience. It is argued that the re-signification of gendered and technological codes drifts through: a) the opening of gendered and technological codes; b) the production of new cultural imaginaries that question hegemonic representations of gender; and c) the production of new subjectivities through the reorganization of socio-technical practices to develop performative acts that transform patriarchal relationsLa "brecha digital de género" constituye un prolífico programa de investigación que compara las diferencias entre mujeres y hombres en el acceso a las Tecnologías de Información y Comunicación. Las perspectivas socio-constructivistas feministas, sin embargo, abogan por la necesidad de prestar atención no sólo al "acceso", sino también al "diseño", y consideran las relaciones sociales como elementos codificados en el interior los artefactos tecnológicos. Desde esta perspectiva, el género constituye una parte integral de la producción tecnológica. Este trabajo explora la constitución conjunta de género y tecnología a partir de una experiencia de investigación-acción específica. Se argumenta que la resignificación de los códigos de género y tecnológicos se desplaza a través de: a) la apertura de los códigos de género y tecnológicos; b) la producción de nuevos imaginarios culturales que cuestionan las representaciones hegemónicas de género; y c) la producción de nuevas subjetividades a través de la reorganización de las prácticas socio-técnicas para el desarrollo de actos performativos que transforman las relaciones patriarcales

Localization microscopy with multiple colors and its application in biological samples

Gunkel M. Lokalisationsmikroskopie mit mehreren Farben und ihre Anwendung in biologischen Präparaten. Bielefeld (Germany): Bielefeld University; 2011.Fluorescence microscopy is an important method for studying biological structures. By intrinsic properties of the fluorophores it is possible to separate and individually localize their signals. These quantitative position information enable an improved structural resolution and a statistic analysis of the data. In this thesis, localization microscopy was applied for multiple spectral markers within the observed biological samples. Several algorithms to compensate mechanical and chromatic shift and to correct and analyze the position data were developed. For example, the relative distribution of a structural protein of the DNA (H2A) and a DNA remodeler protein (SNF2h) was examined. The experimental results were compared with random distributed signal positions within the same global structures. It could be shown that the real distributions are significantly different from the random ones. It is also possible to check the data for signal clusters and determine their properties. This was first tested on simulated distributions and later applied in the analysis of signal accumulations in biological structures like centromere protein clusters (CENP- A, CENP-B and CENP-C) within the human kinetochore. In addition, the axial position of the localization data within the structure can be determined with a precision of about 50nm. A model for the distribution of the fluorophores in tobacco mosaic virus structures was developed and fitted to the data. The width of these structures determined by electron microscopy to be 18nm could be confirmed. The mean localization accuracy in this case was 8nm. A new microscope setup was build to split the fluorescence signal emitted by the sample due to its different properties like direction of polarization or spectral range. Both parts of the signal could be imaged simultaneously on the same detector. This results in a shorter acquisition time for a two color measurement. Additionally, no mechanical drift between two measurements is apparent.Die Fluoreszenzmikroskopie ist eine wichtige Methode zur Erforschung biologischer Strukturen. Durch intrinsische Eigenschaften der Fluorophore ist es möglich, deren Signale voneinander zu trennen und individuell zu lokalisieren. Hierdurch erhält man quantitative Positionsinformationen, was zu einer Verbesserung der strukturellen Auflösung führt und die Möglichkeit statistischer Analysen eröffnet. In dieser Arbeit wurde die Lokalisationsmikroskopie für spektral verschiedene Markierungen innerhalb biologischer Präparate angewandt. Es wurden verschiedene Algorithmen entwickelt, um den mechanischen und chromatischen Versatz der erhaltenen Positionsdaten zu korrigieren und diese weiter zu analysieren. Beispielsweise wurde die relative Verteilung zwischen einem DNA-Strukturprotein (H2A) zu einem am DNA-Umbau beteiligten Protein (SNF2h) untersucht. Gleichzeitig wurden diese Verteilungen mit zufälligen Verteilungen von Signalpositionen innerhalb der gleichen globalen Strukturen verglichen. Dadurch konnte gezeigt werden, dass sich die Verteilungen signifikant von zufälligen Verteilungen unterscheiden. Die Fluoreszenzmikroskopie ist eine wichtige Methode zur Erforschung biologischer Strukturen. Durch intrinsische Eigenschaften der Fluorophore ist es möglich, deren Signale voneinander zu trennen und individuell zu lokalisieren. Hierdurch erhält man quantitative Positionsinformationen, was zu einer Verbesserung der strukturellen Auflösung führt und die Möglichkeit statistischer Analysen eröffnet. Weiterhin ist es möglich, die Daten auf Signalanhäufungen zu überprüfen und deren Eigenschaften zu bestimmen. Dies wurde erst an simulierten Verteilungen getestet und später unter anderem zur Analyse von Signalanhäufungen von Centromerproteinen (CENP-A, CENP-B und CENP-C) im menschlichen Kinetochor genutzt. Zusätzlich kann die axiale Position der Lokalisationsdaten innerhalb der Struktur auf etwa 50 nm genau bestimmt werden. Für die Verteilung der Fluorophore in Tabakmosaikviren wurde ein Modell entwickelt, welches an die experimentellen Daten angepasst wurde. Durch die Lokalisationsmikroskopie konnte die elektronenmikroskopisch bestimmte Breite dieser Struktur von 18 nm bestätigt werden. Die Positionsgenauigkeit der einzelnen Signalpositionen betrug in diesem Fall im Mittel 8 nm. Um das von der Probe emittierte Fluoreszenzsignal aufgrund verschiedener Eigenschaften, wie beispielsweise unterschiedliche Polarisationsrichtungen oder verschiedene Spektralbereiche, aufzuspalten und simultan auf einem einzigen Detektor abzubilden wurde ein neuer Mikroskopaufbau realisiert. Hierdurch kann die Aufnahme einer Zweifarbenmessung beschleunigt werden, da beide Farbkanäle gleichzeitig detektiert werden. Ein weiterer Vorteil ist, dass kein mechanischer Versatz zwischen beiden Messungen entsteht

Localisation Microscopy of Breast Epithelial ErbB-2 Receptors and Gap Junctions: Trafficking after γ-Irradiation, Neuregulin-1β, and Trastuzumab Application

In cancer, vulnerable breast epithelium malignance tendency correlates with number and activation of ErbB receptor tyrosine kinases. In the presented work, we observe ErbB receptors activated by irradiation-induced DNA injury or neuregulin- 1 β application, or alternatively, attenuated by a therapeutic antibody using high resolution fluorescence localization microscopy. The gap junction turnover coinciding with ErbB receptor activation and co-transport is simultaneously recorded. DNA injury caused by 4 Gray of 6 MeV photon γ -irradiation or alternatively neuregulin- 1 β application mobilized ErbB receptors in a nucleograde fashion—a process attenuated by trastuzumab antibody application. This was accompanied by increased receptor density, indicating packing into transport units. Factors mobilizing ErbB receptors also mobilized plasma membrane resident gap junction channels. The time course of ErbB receptor activation and gap junction mobilization recapitulates the time course of non-homologous end-joining DNA repair. We explain our findings under terms of DNA injury-induced membrane receptor tyrosine kinase activation and retrograde trafficking. In addition, we interpret the phenomenon of retrograde co-trafficking of gap junction connexons stimulated by ErbB receptor activation

Tissue-Mimicking Geometrical Constraints Stimulate Tissue-Like Constitution and Activity of Mouse Neonatal and Human-Induced Pluripotent Stem Cell-Derived Cardiac Myocytes

The present work addresses the question of to what extent a geometrical support acts as a physiological determining template in the setup of artificial cardiac tissue. Surface patterns with alternating concave to convex transitions of cell size dimensions were used to organize and orientate human-induced pluripotent stem cell (hIPSC)-derived cardiac myocytes and mouse neonatal cardiac myocytes. The shape of the cells, as well as the organization of the contractile apparatus recapitulates the anisotropic line pattern geometry being derived from tissue geometry motives. The intracellular organization of the contractile apparatus and the cell coupling via gap junctions of cell assemblies growing in a random or organized pattern were examined. Cell spatial and temporal coordinated excitation and contraction has been compared on plain and patterned substrates. While the α-actinin cytoskeletal organization is comparable to terminally-developed native ventricular tissue, connexin-43 expression does not recapitulate gap junction distribution of heart muscle tissue. However, coordinated contractions could be observed. The results of tissue-like cell ensemble organization open new insights into geometry-dependent cell organization, the cultivation of artificial heart tissue from stem cells and the anisotropy-dependent activity of therapeutic compounds

Comparison of cell arrays and multi-well plates in microscopy-based screening

Multi-well plates and cell arrays enable microscopy-based screening assays in which many samples can be analysed in parallel. Each of the formats possesses its own strengths and weaknesses, but reference comparisons between these platforms and their application rationale is lacking. We aim to fill this gap by comparing two RNA interference (RNAi)-mediated fluorescence microscopy-based assays, namely epidermal growth factor (EGF) internalization and cell cycle progression, on both platforms. Quantitative analysis revealed that both platforms enabled the generation of data with the appearance of the expected phenotypes significantly distinct from the negative controls. The measurements of cell cycle progression were less variable in multi-well plates. The result can largely be attributed to higher cell numbers resulting in less data variability when dealing with the assay generating phenotypic cell subpopulations. The EGF internalization assay with a uniform phenotype over nearly the whole cell population performed better on cell arrays than in multi-well plates. The result was achieved by scoring five times less cells on cell arrays than in multi-well plates, indicating the efficiency of the cell array format. Our data indicate that the choice of the screening platform primarily depends on the type of the cellular assay to achieve a maximum data quality and screen efficiency

High-precision structural analysis of subnuclear complexes in fixed and live cells via spatially modulated illumination (SMI) microscopy. Chromosome Res

Abstract Spatially modulated illumination (SMI) microscopy is a method of wide field fluorescence microscopy featuring interferometric illumination, which delivers structural information about nanoscale architecture in fluorescently labelled cells. The first prototype of the SMI microscope proved its applicability to a wide range of biological questions. For the SMI live cell imaging this system was enhanced in terms of the development of a completely new upright configuration. This so called Vertico-SMI transfers the advantages of SMI nanoscaling to vital biological systems, and is shown to work consistently at different temperatures using both oil-and waterimmersion objective lenses. Furthermore, we increased the speed of data acquisition to minimize errors in the detection signal resulting from cellular or object movement. By performing accurate characterization, the present Vertico-SMI now offers a fully-fledged microscope enabling a complete three-dimensional (3D) SMI data stack to be acquired in less than 2 seconds. We have performed live cell measurements of a tet-operator repeat insert in U2OS cells, which provided the first in vivo signatures of subnuclear complexes. Furthermore, we have successfully implemented an optional optical configuration allowing the generation of high-resolution localization microscopy images of a nuclear pore complex distribution. Abbreviation

MicroRNAs influence the migratory ability of human umbilical vein endothelial cells

To identify miRNAs that are involved in cell migration in human umbilical vein endothelial cells (HUVECs), we employed RNA sequencing under high glucose incubation and text mining within the databases miRWalk and TargetScanHuman using 83 genes that regulate HUVECs migration. From both databases, 307 predicted miRNAs were retrieved. Differentially expressed miRNAs were determined by exposing HUVECs to high glucose stimulation, which significantly inhibited the migratory ability of HUVECs as compared to cells cultured in normal glucose. A total of 35 miRNAs were found as differently expressed miRNAs in miRNA sequencing, and 4 miRNAs, namely miR-21-3p, miR-107, miR-143-3p, and miR-106b-5p, were identified as overlapping hits. These were subjected to hub gene analysis and pathway analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG), identifing 71 pathways which were influenced by all four miRNAs. The influence of all four miRNAs on HUVEC migration was phenomorphologically confirmed. miR21 and miR107 promoted migration in HUVECs while miR106b and miR143 inhibited migration. Pathway analysis also revealed eight shared pathways between the four miRNAs. Protein–protein interaction (PPI) network analysis was then performed to predict the functionality of interacting genes or proteins. This revealed six hub genes which could firstly be predicted to be related to HUVEC migration