Inhibierung des pathomolekularen Mechanismus einer t(4;11)-assoziierten Leukämie

Taspase1 stellt die bisher einzige Typ2-Asparaginase mit proteolytischer Aktivität dar. Das wichtigste Substrat der Taspase1 ist das MLL-Protein, einem Homolog des Trithorax- Proteins aus Drosophila melanogaster, das auch dort eine wichtige Rolle bei Differenzierungsprozessen spielt. Bei Patienten mit einer t(4;11)-Translokation ist Taspase1 maßgeblich an der Ausbildung einer t(4;11)-assoziierten Leukämie beteiligt. Die Inhibierung der proteolytischen Aktivität der Taspase1 könnte daher einen Ansatzpunkt für eine neuartige Krebstherapie darstellen. Aufgrund der ungewöhnlichen Eigenschaften von Taspase1 ist es bisher nicht gelungen einen selektiven Inhibitor für das katalytische Zentrum der Taspase1 zu identifizieren. Unter nativen Bedingungen (ca. 50 mM NaCl) befindet sich Taspase1 bereits in einem nahezu vollständig inhibierten Zustand, da im katalytischen Zentrum der Taspase1 ein Chloridion komplexiert ist. Dieses Chloridion wird einzig und allein nach Interaktion mit dem natürlichen Substrat MLL aus dem katalytischen Zentrum verdrängt, was zu einer kurzfristigen Aktivierung der Taspase1 führt. Nach Ablauf der hydrolytischen Spaltung des Substrates nimmt das Chloridion wieder seine Position im katalytischen Zentrum ein. Unter diesen Bedingungen ist aus sterischen Gründen die Bindung eines potentiellen Inhibitors im katalytischen Zentrum nicht möglich. Durch Herstellung von Mutanten der Taspase1 und deren Substrats konnte der Mechanismus der katalytischen Spaltung durch Taspase1 aufgeklärt werden. Dabei erwiesen sich drei Aminoäuren als essentiell für die Hydrolyse. Interessanterweise ist die Anwesenheit des Substrates, insbesondere des Aspartates an Position Sieben der cleavage sites CS1 bzw. CS2 notwendig um den katalytischen Prozess zu starten. Das negativ geladene Aspartat, verdrängt zunächst das Chloridion von seiner Position und aktiviert dadurch das katalytische Zentrum (Rotation von Threonin 234). Erst dadurch wird Threonin 234 zu einer katalytisch aktiven Aminosäure und kann einen nukleophilen Angriff auf die Peptidbindung zwischen Aspartat und Glycin des Substrates durchführen. Die Hydrolyse wird dabei durch die OH-Gruppe des Serins 252 durch Wechselwirkung mit dem Carboxylsauerstoff unterstützt. Durch Mutation beider Aspartate an Position sieben im artifiziellen Substrat 2CL zu Glycin oder Lysin führte zu einem vollständigen Verlust der hydrolytischen Spaltung an CS1 und zu einem starken Rückgang der hydrolytischen Spaltung an CS2. Die Mutationen T234D und S252D der Taspase1 führten beide zum vollständigen Verlust der katalytischen Spaltung, sowohl in cis, als auch in trans. Unter Verwendung des Taspase1-Aktivitätsassays konnte der transkriptionelle Regulator MLL4 als potentielles Substrat der Taspase1 identifiziert werden

Faster Goal-Oriented Shortest Path Search for Bulk and Incremental Detailed Routing

We develop new algorithmic techniques for VLSI detailed routing. First, we improve the goal-oriented version of Dijkstra's algorithm to find shortest paths in huge incomplete grid graphs with edge costs depending on the direction and the layer, and possibly on rectangular regions. We devise estimates of the distance to the targets that offer better trade-offs between running time and quality than previously known methods, leading to an overall speed-up. Second, we combine the advantages of the two classical detailed routing approaches - global shortest path search and track assignment with local corrections - by treating input wires (such as the output of track assignment) as reservations that can be used at a discount by the respective net. We show how to implement this new approach efficiently

The MOSAiC ice floe: sediment-laden survivor from the Siberian shelf

In September 2019, the research icebreaker Polarstern started the largest multidisciplinary Arctic expedition to date, the MOSAiC (Multidisciplinary drifting Observatory for the Study of Arctic Climate) drift experiment. Being moored to an ice floe for a whole year, thus including the winter season, the declared goal of the expedition is to better understand and quantify relevant processes within the atmosphere–ice–ocean system that impact the sea ice mass and energy budget, ultimately leading to much improved climate models. Satellite observations, atmospheric reanalysis data, and readings from a nearby meteorological station indicate that the interplay of high ice export in late winter and exceptionally high air temperatures resulted in the longest ice-free summer period since reliable instrumental records began. We show, using a Lagrangian tracking tool and a thermodynamic sea ice model, that the MOSAiC floe carrying the Central Observatory (CO) formed in a polynya event north of the New Siberian Islands at the beginning of December 2018. The results further indicate that sea ice in the vicinity of the CO (<40 km distance) was younger and 36 % thinner than the surrounding ice with potential consequences for ice dynamics and momentum and heat transfer between ocean and atmosphere. Sea ice surveys carried out on various reference floes in autumn 2019 verify this gradient in ice thickness, and sediments discovered in ice cores (so-called dirty sea ice) around the CO confirm contact with shallow waters in an early phase of growth, consistent with the tracking analysis. Since less and less ice from the Siberian shelves survives its first summer (Krumpen et al., 2019), the MOSAiC experiment provides the unique opportunity to study the role of sea ice as a transport medium for gases, macronutrients, iron, organic matter, sediments and pollutants from shelf areas to the central Arctic Ocean and beyond. Compared to data for the past 26 years, the sea ice encountered at the end of September 2019 can already be classified as exceptionally thin, and further predicted changes towards a seasonally ice-free ocean will likely cut off the long-range transport of ice-rafted materials by the Transpolar Drift in the future. A reduced long-range transport of sea ice would have strong implications for the redistribution of biogeochemical matter in the central Arctic Ocean, with consequences for the balance of climate-relevant trace gases, primary production and biodiversity in the Arctic Ocean

Bioassays to Monitor Taspase1 Function for the Identification of Pharmacogenetic Inhibitors

Background: Threonine Aspartase 1 (Taspase1) mediates cleavage of the mixed lineage leukemia (MLL) protein and leukemia provoking MLL-fusions. In contrast to other proteases, the understanding of Taspase1's (patho)biological relevance and function is limited, since neither small molecule inhibitors nor cell based functional assays for Taspase1 are currently available. Methodology/Findings: Efficient cell-based assays to probe Taspase1 function in vivo are presented here. These are composed of glutathione S-transferase, autofluorescent protein variants, Taspase1 cleavage sites and rational combinations of nuclear import and export signals. The biosensors localize predominantly to the cytoplasm, whereas expression of biologically active Taspase1 but not of inactive Taspase1 mutants or of the protease Caspase3 triggers their proteolytic cleavage and nuclear accumulation. Compared to in vitro assays using recombinant components the in vivo assay was highly efficient. Employing an optimized nuclear translocation algorithm, the triple-color assay could be adapted to a high-throughput microscopy platform (Z'factor = 0.63). Automated high-content data analysis was used to screen a focused compound library, selected by an in silico pharmacophor screening approach, as well as a collection of fungal extracts. Screening identified two compounds, N-[2-[(4-amino-6-oxo-3H-pyrimidin-2-yl)sulfanyl]ethyl]benzenesulfonamideand 2-benzyltriazole-4,5-dicarboxylic acid, which partially inhibited Taspase1 cleavage in living cells. Additionally, the assay was exploited to probe endogenous Taspase1 in solid tumor cell models and to identify an improved consensus sequence for efficient Taspase1 cleavage. This allowed the in silico identification of novel putative Taspase1 targets. Those include the FERM Domain-Containing Protein 4B, the Tyrosine-Protein Phosphatase Zeta, and DNA Polymerase Zeta. Cleavage site recognition and proteolytic processing of these substrates were verified in the context of the biosensor. Conclusions: The assay not only allows to genetically probe Taspase1 structure function in vivo, but is also applicable for high-content screening to identify Taspase1 inhibitors. Such tools will provide novel insights into Taspase1's function and its potential therapeutic relevance

Quantifying Sea Ice Formation Rates In The Laptev Sea By Means Of ENVISAT SAR Scenes And Airborne Ice Thickness Measurements

The Laptev Sea is considered as one of the most significant sites of net ice production in the Arctic Ocean. However, the few existing satellite and model-based studies on ice formation rates in the Laptev Sea show large differences in ice flux estimates. These discrepancies can be explained by the use of different sensor systems, models, observation periods and the lack of observational data. The latter gap may be filled by the so called electromagnetic (EM) Bird of the Sea Ice Group at the Alfred Wegener Institute. The Bird is an airborne system that utilizes the contrast of electrical conductivity between sea water and sea ice to determine the ice thickness along the flight path. Together with the EM-Bird ice thickness measurements, polynya area estimates obtained from ENVISAT SAR satellite images were used to quantify seasonal ice export and production rates in the Laptev Sea. The approach yielded a total ice volume of 81 km³ that originates from polynyas in the eastern Laptev Sea in 2008 (9 % of the net annual ice formation in the Laptev Sea). Our results imply that the contribution of polynyas to the net sea ice formation in the Laptev Sea is indeed higher than suggested by previous studies

Improved non-destructive 2D and 3D X-ray imaging of leaf venation

Background: Leaf venation traits are important for many research fields such as systematics and evolutionary biology, plant physiology, climate change, and paleoecology. In spite of an increasing demand for vein trait data, studies are often still data-limited because the development of methods that allow rapid generation of large sets of vein data has lagged behind. Recently, non-destructive X-ray technology has proven useful as an alternative to traditional slow and destructive chemical-based methods. Non-destructive techniques more readily allow the use of herbarium specimens, which provide an invaluable but underexploited resource of vein data and related environmental information. The utility of 2D X-ray technology and microfocus X-ray computed tomography, however, has been compromised by insufficient image resolution. Here, we advanced X-ray technology by increasing image resolution and throughput without the application of contrast agents. Results: For 2D contact microradiography, we developed a method which allowed us to achieve image resolutions of up to 7 µm, i.e. a 3.6-fold increase compared to the industrial standard (25 µm resolution). Vein tracing was further optimized with our image processing standards that were specifically adjusted for different types of leaf structure and the needs of higher imaging throughput. Based on a test dataset, in 91% of the samples the 7 µm approach led to a significant improvement in estimations of minor vein density compared to the industrial standard. Using microfocus X-ray computed tomography, very high-resolution images were obtained from a virtual 3D–2D transformation process, which was superior to that of 3D images. Conclusions: Our 2D X-ray method with a significantly improved resolution advances rapid non-destructive bulk scanning at a quality that in many cases is sufficient to determine key venation traits. Together with our high-resolution microfocus X-ray computed tomography method, both non-destructive approaches will help in vein trait data mining from museum collections, which provide an underexploited resource of historical and recent data on environmental and evolutionary change. In spite of the significant increase in effective image resolution, a combination of high-throughput and full visibility of the vein network (including the smallest veins and their connectivity) remains challenging, however