Analyse der Chromosomenverteilung in menschlichen Fibroblasten mittels 3D-Vielfarben Fluoreszenz in situ Hybridisierung

In human diploid fibroblasts (HDFs) the cell nucleus is oval in shape, quite large in xy-diameter (10-20µm), but flat in z-direction (5µm). In these nuclei chromosome territories typically lie side by side or slightly above each other. The question whether these arrangements are ordered or variable has yielded conflicting answers. We hybridised an improved 7-fluorochrome MFISH probe set on 3D-preserved cell nuclei, fixed with buffered 4% paraformaldehyde. A LEICA wide field microscope with an 8-filter-wheel and an automated z-step motor was used for imaging of the 7 fluorochromes plus DAPI. Multicolor images from nuclei were taken as serial sections in z-direction. After deconvolution, a specifically developed Program goldFISH Saracoglu K. et al. 2001 was used to classify the images according to the labelling scheme. The classification algorithm corresponds to the procedure previously used for metaphase spreads, now adopted to 3-D studies of chromosome territory arrangements in the cell nucleus. The analysis of 30 G0-fibroblast nuclei and 30 prometaphase rosettes revealed a pronounced variability of chromosome territory neighbourhoods, as described by Allison D. C. et al. 1999, but in contrast to Nagele R. et al. 1995. However we noted a distinct radial order: small chromosomes were located close to the centre while large chromosomes were positioned towards the nuclear rim. This non-random radial positioning could also be observed in prometaphase rosettesDie Erforschung von Chromosomenpositionen im menschlichen Zellkern führte zu kontroversen Ergebnissen: In Lymphozyten konnte eine radiale Chromosomenposition entdeckt werden, die von der Gendichte der Chromosomen abhing (Boyle A. L. et al. 1990; Cremer M. et al. 2001). Genreiche Chromosomen lagen zentraler im Zellkern als genarme. In Fibroblasten wurde zwar ebenfalls eine radiale Verteilung gefunden, jedoch korreliert diese besser mit der genomischen Größe der Chromosomen. Hier lagen kleinere Chromosomen zentraler als größere (Sun et al. 2000). Darüberhinaus finden einige Publikationen eine starr gegenüberliegende und fixierte nachbarschaftliche Anordnung (Nagele et al. 1995), andere hingegen eine zufällige Nachbarschaft (Allison und Nestor 1999). Weitfeldmikroskoptechnik, Dekonvolution und Klassifikation: Ein Großteil der Arbeit betraf die Etablierung und das Testen eines 3D-Mikroskopie- und Dekonvolutions-System für M-FISH Präparate. Ein Leica Weitfeld-Epifluoreszenzmikroskop wurde mit einem Filterrad für acht Bandpassfilter und einem z-Schrittmotor für den Mikroskoptisch ausgestattet. Die Software zu Steuerung wurde in Zusammenarbeit mit Leica Microsystems angepasst. Nach Aufnahme der Bildstapel wurden sie der Dekonvolution unterzogen, um sie auf die Qualität konfokaler Bilder anzuheben. Für die Dekonvolution war es nötig die richtigen Programme und Parameter herauszufinden. ReFISH-Präparate konnten mit einem konfokalen Mikroskop aufgenommen werden, jedoch war es nötig das Präparat nach der ersten Hybridisierung erneut zu waschen und zu denaturieren, um den zweiten, komplementären Sondensatz zu hybridisieren. Die zwei nacheinander aufgenommenen Bildstapel mussten mit einem Alignmentprogramm aneinander angeglichen werden. Schließlich wurde es durch die Erweiterung des Karyotypisierungsprogramms auf 3 Dimensionen möglich, Chromosomenterritorien im Zellkern automatisch zu bestimmen. Das Programm klassifiziert die Regionen anhand des Markierungsschemas und weist jedem erkannten Chromosomenterritorium eine Klassifikationsfarbe zu. Die Schwerpunkte der Chromosomenterritorien wurden in eine Ausgabetabelle geschrieben. Die daraus berechneten Abstände und Winkel liessen eine Aussage bezüglich der Chromosomentopologie zu. Chromosomenpositionen in Fibroblasten: In allen Experimenten an G0-Fibroblasten und Prometaphaserosetten wur-de in den Kernen eine größenabhängige, radiale Verteilung der Chromoso-menterritorien gefunden, wobei kleinere Chromosomen allgemein zentraler lagen als größere. Eine starre Ordnung, wie sie in Nagele et al. (1995) pro-pagiert wurde, konnte nicht nachvollzogen werden. Vielmehr stellte sich heraus, dass die nachbarschaftliche Anordnung der Chromosomen im Zell-kern weitgehend zufällig is

Three-Dimensional Maps of All Chromosomes in Human Male Fibroblast Nuclei and Prometaphase Rosettes

Studies of higher-order chromatin arrangements are an essential part of ongoing attempts to explore changes in epigenome structure and their functional implications during development and cell differentiation. However, the extent and cell-type-specificity of three-dimensional (3D) chromosome arrangements has remained controversial. In order to overcome technical limitations of previous studies, we have developed tools that allow the quantitative 3D positional mapping of all chromosomes simultaneously. We present unequivocal evidence for a probabilistic 3D order of prometaphase chromosomes, as well as of chromosome territories (CTs) in nuclei of quiescent (G0) and cycling (early S-phase) human diploid fibroblasts (46, XY). Radial distance measurements showed a probabilistic, highly nonrandom correlation with chromosome size: small chromosomes—independently of their gene density—were distributed significantly closer to the center of the nucleus or prometaphase rosette, while large chromosomes were located closer to the nuclear or rosette rim. This arrangement was independently confirmed in both human fibroblast and amniotic fluid cell nuclei. Notably, these cell types exhibit flat-ellipsoidal cell nuclei, in contrast to the spherical nuclei of lymphocytes and several other human cell types, for which we and others previously demonstrated gene-density-correlated radial 3D CT arrangements. Modeling of 3D CT arrangements suggests that cell-type-specific differences in radial CT arrangements are not solely due to geometrical constraints that result from nuclear shape differences. We also found gene-density-correlated arrangements of higher-order chromatin shared by all human cell types studied so far. Chromatin domains, which are gene-poor, form a layer beneath the nuclear envelope, while gene-dense chromatin is enriched in the nuclear interior. We discuss the possible functional implications of this finding

The GRAVITY+ Project: Towards All-sky, Faint-Science, High-Contrast Near-Infrared Interferometry at the VLTI

The GRAVITY instrument has been revolutionary for near-infrared interferometry by pushing sensitivity and precision to previously unknown limits. With the upgrade of GRAVITY and the Very Large Telescope Interferometer (VLTI) in GRAVITY+, these limits will be pushed even further, with vastly improved sky coverage, as well as faint-science and high-contrast capabilities. This upgrade includes the implementation of wide-field off-axis fringe-tracking, new adaptive optics systems on all Unit Telescopes, and laser guide stars in an upgraded facility. GRAVITY+ will open up the sky to the measurement of black hole masses across cosmic time in hundreds of active galactic nuclei, use the faint stars in the Galactic centre to probe General Relativity, and enable the characterisation of dozens of young exoplanets to study their formation, bearing the promise of another scientific revolution to come at the VLTI.Comment: Published in the ESO Messenge

Impacts of electric vehicle charging systems on distribution networks

Abweichender Titel laut Übersetzung der Verfasserin/des VerfassersMan erhofft sich durch die Elektromobilität einen wesentlichen Beitrag zur Senkung der Treibhausgasemissionen. Zur Zeit wird in vielen Gebieten geforscht und entwickelt (Fahrzeuge, Motoren, Antriebsstränge, Speichertechnologien, etc.). Die vorliegende Arbeit beschäftigt sich mit der Ladeinfrastruktur für batteriegespeiste Elektroautos. Dabei werden auf Basis eines dichtverbauten Wohngebiets die Auswirkungen elektrischer Mobilität auf das bestehende Niederspannungs-Verteilnetz untersucht.Anhand von Netzdaten wird ein Matlab-Modell zur Simulation verschiedener Szenarien erstellt. Dabei werden Ladestationen unterschiedlicher Leistung mit variablen Durchdringungsraten von Elektrofahrzeugen herangezogen. Die Ergebnisse von Lastflussanalysen mit Elaplan liefern mit Normen und Vorschriften eine Bewertung der Beeinflussungen. Zu den untersuchten Auswirkungen zählen Spannungshaltung, Kabelbelastungen, Oberwellenspannungen, Spannungsunsymmetrien, Spannungsänderungen und Flicker. Es stellt sich heraus, dass sich die Elektromobilität im Allgemeinen in das Verteilnetz integrieren lässt, wobei aber abhängig von der Art und Anzahl der Ladestationen Grenzen gegeben sind. Der Betrieb von Schnellladestationen führt rasch zu Engpässen und erfordert einen erhöhten Aufwand an Planung und Steuerung.11

An optimized probe set for the detection of small interchromosomal aberrations by use of 24-color FISH.

The rapid spread of the use of new 24-color karyotyping techniques has preceded their standardization. This is best documented by the fact that the exact resolution limits have not yet been defined. Indeed, it is shown here that a substantial proportion of interchromosomal aberrations will be missed by all multicolor karyotyping systems currently in use. We demonstrate that both the sensitivity and the specificity of 24-color karyotyping critically depend on the fluorochrome composition of chromosomes involved in an interchromosomal rearrangement. As a solution, we introduce a conceptual change in probe labeling. Seven-fluorochrome sets that overcome many of the current limitations are described, and examples of their applications are shown. The criteria presented here for an optimized probe-set design and for the estimation of resolution limits should have important consequences for pre- and postnatal diagnostics and for research applications

Significance Levels for Pairwise Comparisons of 3D Distance Measurements Performed in 54 G0 Fibroblast Nuclei and in 50 SCD Model Nuclei

<div><p>Significance levels determined by the two-tailed K-S test are indicated by colors. Green, not significant, <i>p</i> > 0.01; yellow, <i>p</i> < 0.01; orange, <i>p</i> < 0.001; red, <i>p</i> < 0.0001. A minus (or plus) sign in a colored field indicates that the 3D CN–CT (A and B) or 3D CT–CT distance (C and D) indicated at the left (row) revealed a significantly shorter (or greater) mean radial distance than the CT indicated at the top (column).</p> <p>(A) Comparison of 3D CN–CT distances in G0 fibroblast nuclei.</p> <p>(B) Comparison of 3D CT–CN distances in fibroblast nuclei (vertical row) with 3D CN–CT distances in SCD model nuclei (horizontal row).</p> <p>(C) Comparison of 3D CT–CT distances between homologous chromosomes in G0 fibroblast nuclei.</p> <p>(D) Comparison of 3D CT–CT distances between homologous chromosomes in SCD model nuclei.</p></div

Radial Chromosome Positions Correlate with Chromosome Size in Quiescent Human Fibroblasts (G0)

<div><p>(A) Two-dimensional projections of the IGCs of CTs 1, 7, 11, 18, 19, and Y studied in 54 nuclei are represented by dots. Ellipses represent the 2D shape of a fibroblast nucleus normalized for shape and size and rotated so that the long axis of each nucleus evaluated lies on the abscissa. Projections of IGCs for all CTs are shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030157#sg002" target="_blank">Figure S2</a>. Note that we were not able to distinguish in nuclei between a “north” and “south” pole of the short axis or a “west” and “east” pole of the long axis. Either fibroblast nuclei do not possess such compass polarizations or we lack markers to recognize them. Accordingly, distance comparisons between IGCs located in different quadrants of the ellipse are not meaningful.</p> <p>(B) Cumulative 3D distance graphs of the CT distribution within a normalized nucleus taken from the data (A). The abscissa represents the normalized radial 3D distances of CTs 1, 7, 11, 18, 19, and Y from the center of the nucleus (CN; IGC of the DAPI-stained nucleus) to the IGC of a specific CT. The origin represents the CN, and “1” represents the nuclear periphery. The Ordinate represents the cumulative percentage of normalized 3D CN–CT distances. Cumulative graphs for the entire set of chromosomes are shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030157#sg004" target="_blank">Figure S4</a>.</p> <p>(C) Cumulative 3D distance graphs of PC distribution within a normalized prometaphase rosette. Abscissa and ordinate are as in (B), with PC being the IGC of a prometaphase chromosome and CR the center of the prometaphase rosette. IGC projections and cumulative graphs for all PCs are shown in Figures <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030157#sg003" target="_blank">S3</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030157#sg006" target="_blank">S6</a>, respectively.</p></div

Normalized Radial Chromosome Distances in G0 Fibroblast Nuclei, SCD Model Nuclei, and Prometaphase Rosettes

<div><p>(A) Normalized 3D radial CN–CT distances (filled squares) show a positive correlation with chromosome size (indicated by DNA content): CTs of small chromosomes were preferentially located in the center of the nucleus, whereas CTs of large chromosomes were found more often at the nuclear periphery. Open circles 1 and 21 indicate the endpoints of SCD model data simulating a statistical placement of CTs.</p> <p>(B) SCD model data indicate that geometrical constraints result in a reverse pattern of CT distributions, i.e., modeled small CTs show a significantly higher probability of being localized at the nuclear periphery, while modeled large CTs adopt a more internal localization.</p> <p>(C) In agreement with normalized 3D CN–CT distances, normalized 3D CR–PC distances also show a positive correlation with the DNA content or size of chromosomes: small PCs were preferentially located near the CR, large chromosomes at the rosette periphery.</p></div

Relative Spatial Distributions of Homologous Chromosomes of HSAs 7, 15, and 22

<p>Schematic outlines of fibroblast nuclei (as ellipses, A) and prometaphase rosettes (as circles, B) with normalized size and shape. The IGC of one randomly selected homolog was placed along the negative long axis. The IGC of the other homolog was marked at the corresponding nuclear position. Gray dots represent data obtained by confocal microscopy, open circles by wide-field microscopy. Angle measurements for all pairs of homologous chromosomes are presented in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030157#sg009" target="_blank">Figure S9</a> for G0 nuclei and in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030157#sg010" target="_blank">Figure S10</a> for rosettes.</p