A loss-of-function RNA interference screen for molecular targets in cancer

The pursuit of novel therapeutic agents in cancer relies on the identification and validation of molecular targets. Hallmarks of cancer include self-sufficiency in growth signals and evasion from apoptosis; genes that regulate these processes may be optimal for therapeutic attack. Here we describe a loss-of-function screen for genes required for the proliferation and survival of cancer cells using an RNA interference library. We used a doxycycline-inducible retroviral vector for the expression of small hairpin RNAs (shRNAs) to construct a library targeting 2,500 human genes. We used retroviral pools from this library to infect cell lines representing two distinct molecular subgroups of diffuse large B-cell lymphoma (DLBCL), termed activated B-cell-like DLBCL and germinal centre B-cell-like DLBCL. Each vector was engineered to contain a unique 60-base-pair 'bar code', allowing the abundance of an individual shRNA vector within a population of transduced cells to be measured using microarrays of the bar-code sequences. We observed that a subset of shRNA vectors was depleted from the transduced cells after three weeks in culture only if shRNA expression was induced. In activated B-cell-like DLBCL cells, but not germinal centre B-cell-like DLBCL cells, shRNAs targeting the NF-kappaB pathway were depleted, in keeping with the essential role of this pathway in the survival of activated B-cell-like DLBCL. This screen uncovered CARD11 as a key upstream signalling component responsible for the constitutive IkappaB kinase activity in activated B-cell-like DLBCL. The methodology that we describe can be used to establish a functional taxonomy of cancer and help reveal new classes of therapeutic targets distinct from known oncogenes.     

Endothelial tubes assemble from intracellular vacuoles in vivo

The formation of epithelial tubes is crucial for the proper development of many different tissues and organs, and occurs by means of a variety of different mechanisms. Morphogenesis of seamless, properly patterned endothelial tubes is essential for the development of a functional vertebrate circulatory system, but the mechanism of vascular lumenization in vivo remains unclear. Evidence dating back more than 100 years has hinted at an important function for endothelial vacuoles in lumen formation. More than 25 years ago, in some of the first endothelial cell culture experiments in vitro, Folkman and Haudenschild described "longitudinal vacuoles" that "appeared to be extruded and connected from one cell to the next", observations confirmed and extended by later studies in vitro showing that intracellular vacuoles arise from integrin-dependent and cdc42/Rac1-dependent pinocytic events downstream of integrin-extracellular-matrix signalling interactions. Despite compelling data supporting a model for the assembly of endothelial tubes in vitro through the formation and fusion of vacuoles, conclusive evidence in vivo has been lacking, primarily because of difficulties associated with imaging the dynamics of subcellular endothelial vacuoles deep within living animals. Here we use high-resolution time-lapse two-photon imaging of transgenic zebrafish to examine how endothelial tubes assemble in vivo, comparing our results with time-lapse imaging of human endothelial-cell tube formation in three-dimensional collagen matrices in vitro. Our results provide strong support for a model in which the formation and intracellular and intercellular fusion of endothelial vacuoles drives vascular lumen formation.     

Optical turbulence profiling with single star SCIDAR technique

In order to characterize optical turbulence, we have developed a single star SCIDAR （SSS） for measurement of the distribution of Cn^2 with height. The SSS consists of a 40 cm telescope and a CCD camera for fast sampling of stellar scintillation pattern. Spatio- temporal auto and cross-correlation functions of the single star images are computed, providing vertical profiles of optical turbulence intensity C2（h） and wind speed V（h）. Using this new SSS experiment, profiles of turbulence can be obtained from the ground to the top of atmosphere, allowing the determination of seeing, isoplanatic angle and coherence time. Detailed characteristics of atmospheric optical turbulence are important for active and passive imaging, astronomical site testing, adaptive optics, laser communications, target tracking and designation, and laser beam control. We plan to improve the robotization of the SSS to be able to use it routinely even under harsh weather and altitude conditions that we expect to encounter on the high Tibetan plateau or at Dome A in Antarctica. SSS will also be applied for the site testing campaign of the future Chinese extremely large telescope.     

A common mass scale for satellite galaxies of the Milky Way

The Milky Way has at least twenty-three known satellite galaxies that shine with luminosities ranging from about a thousand to a billion times that of the Sun. Half of these galaxies were discovered in the past few years in the Sloan Digital Sky Survey, and they are among the least luminous galaxies in the known Universe. A determination of the mass of these galaxies provides a test of galaxy formation at the smallest scales and probes the nature of the dark matter that dominates the mass density of the Universe. Here we use new measurements of the velocities of the stars in these galaxies to show that they are consistent with them having a common mass of about 10(7) within their central 300 parsecs. This result demonstrates that the faintest of the Milky Way satellites are the most dark-matter-dominated galaxies known, and could be a hint of a new scale in galaxy formation or a characteristic scale for the clustering of dark matter.     

Visualizing transient events in amino-terminal autoprocessing of HIV-1 protease

HIV-1 protease processes the Gag and Gag-Pol polyproteins into mature structural and functional proteins, including itself, and is therefore indispensable for viral maturation. The mature protease is active only as a dimer with each subunit contributing catalytic residues. The full-length transframe region protease precursor appears to be monomeric yet undergoes maturation via intramolecular cleavage of a putative precursor dimer, concomitant with the appearance of mature-like catalytic activity. How such intramolecular cleavage can occur when the amino and carboxy termini of the mature protease are part of an intersubunit beta-sheet located distal from the active site is unclear. Here we visualize the early events in N-terminal autoprocessing using an inactive mini-precursor with a four-residue N-terminal extension that mimics the transframe region protease precursor. Using paramagnetic relaxation enhancement, a technique that is exquisitely sensitive to the presence of minor species, we show that the mini-precursor forms highly transient, lowly populated (3-5%) dimeric encounter complexes that involve the mature dimer interface but occupy a wide range of subunit orientations relative to the mature dimer. Furthermore, the occupancy of the mature dimer configuration constitutes a very small fraction of the self-associated species (accounting for the very low enzymatic activity of the protease precursor), and the N-terminal extension makes transient intra- and intersubunit contacts with the substrate binding site and is therefore available for autocleavage when the correct dimer orientation is sampled within the encounter complex ensemble.     

Editorial

Systemic Practice and Action Research -     

Cortical steroids: Configurations at C20 relative to C17

Zusammenfassung Sieben inaktive Begleit-Corticosteroide, die vonReichstein und anderen aus der Nebennierenrinde isoliert worden sind, enthalten ein Asymmetriezentrum in C₂₀. Es sind bisher sind noch keine Schlußfolgerungen bezüglich der Konfiguration der natürlichen Substanzen in diesem Zentrum relativ zum Rest des Moleküls gezogen worden.In der vorliegenden Abhandlung wird darauf hingewiesen, daß die Konfiguration an Hand der Betrachtung des Hinderniseffekts, welcher wahrscheinlich den sterischen Lauf der Reaktionen, die zur Partialsynthese der Verbindungen dieser Gruppe angewendet werden, reguliert, mit ziemlicher Sicherheit abgeleitet werden kann. Auf diese Weise sind die Konfigurationen aller sieben natürlichen Steroide festgesetzt und mit den Bezeichnungen der vorgeschlagenen Nomenklatur für die Beschreibung der Konfiguration in C₂₀ relativ zu der in C₁₇ unter Bezugnahme auf ein zyklisches Zwischenprodukt von festgesetzter geometrischer Orientierung ausgedrückt worden.Die Konfiguration in C₂₀ einiger synthetischer 20-Oxy-Verbindungen der 17-Isoallopregnan- und 17-Iso-⁵-pregnen-Reihe sind durch Vergleich mit den entsprechenden 17-Normal-Verbindungen mit Hilfe der Methode des Molekularrotationsunterschiedes nachBarton abgeleitet worden. Im Falle zweier epimerischer 17-Isoallopregnan-3,17,20-triole sind die optischen Eigenschaften nicht entscheidend und zwei Serien von experimentellen Tatsachen scheinen sich zu widersprechen.Experimentelles Beweismaterial, das erst nach der Fertigstellung dieser Abhandlung beigebracht worden war, zeigt, daß eine der Literaturangaben unrichtig ist, wie in der «Note added to proof» erwähnt worden ist.In der Abhandlung ist eine Reihe von Vorschlägen für die Standardisierung der Nomenklatur der Steroide eingeschlossen.