Context dependence between subdomains in the DNA binding interface of the I-CreI homing endonuclease

Homing endonucleases (HE) have emerged as precise tools for achieving gene targeting events. Redesigned HEs with tailored specificities can be used to cleave new sequences, thereby considerably expanding the number of targetable genes and loci. With HEs, as well as with other protein scaffolds, context dependence of DNA/protein interaction patterns remains one of the major limitations for rational engineering of new DNA binders. Previous studies have shown strong crosstalk between different residues and regions of the DNA binding interface. To investigate this phenomenon, we systematically combined mutations from three groups of amino acids in the DNA binding regions of the I-CreI HE. Our results confirm that important crosstalk occurs throughout this interface in I-CreI. Detailed analysis of success rates identified a nearest-neighbour effect, with a more pronounced level of dependence between adjacent regions. Taken together, these data suggest that combinatorial engineering does not necessarily require the identification of separable functional or structural regions, and that groups of amino acids provide acceptable building blocks that can be assembled, overcoming the context dependency of the DNA binding interface. Furthermore, the present work describes a sequential method to engineer tailored HEs, wherein three contiguous regions are individually mutated and assembled to create HEs with engineered specificity

Using a structural and logics systems approach to infer bHLH–DNA binding specificity determinants

Numerous efforts are underway to determine gene regulatory networks that describe physical relationships between transcription factors (TFs) and their target DNA sequences. Members of paralogous TF families typically recognize similar DNA sequences. Knowledge of the molecular determinants of protein–DNA recognition by paralogous TFs is of central importance for understanding how small differences in DNA specificities can dictate target gene selection. Previously, we determined the in vitro DNA binding specificities of 19 Caenorhabditis elegans basic helix-loop-helix (bHLH) dimers using protein binding microarrays. These TFs bind E-box (CANNTG) and E-box-like sequences. Here, we combine these data with logics, bHLH–DNA co-crystal structures and computational modeling to infer which bHLH monomer can interact with which CAN E-box half-site and we identify a critical residue in the protein that dictates this specificity. Validation experiments using mutant bHLH proteins provide support for our inferences. Our study provides insights into the mechanisms of DNA recognition by bHLH dimers as well as a blueprint for system-level studies of the DNA binding determinants of other TF families in different model organisms and humans.National Institute of General Medical Sciences (U.S.) (DK068429)National Institute of General Medical Sciences (U.S.) (HG003985)European Union (PROSPECTS HEALTH-F4-2008-201648

Iron isotope systematics in estuaries : the case of North River, Massachusetts (USA)

Author Posting. © The Author(s), 2009. This is the author's version of the work. It is posted here by permission of Elsevier B.V. for personal use, not for redistribution. The definitive version was published in Geochimica et Cosmochimica Acta 73 (2009): 4045-4059, doi:10.1016/j.gca.2009.04.026.Recent studies have suggested that rivers may present an isotopically light Fe source to the oceans. Since the input of dissolved iron from river water is generally controlled by flocculation processes that occur during estuarine mixing, it is important to investigate potential fractionation of Fe-isotopes during this process. In this study, we investigate the influence of the flocculation of Fe-rich colloids on the iron isotope composition of pristine estuarine waters and suspended particles. The samples were collected along a salinity gradient from the fresh water to the ocean in the North River estuary (MA, USA). Estuarine samples were filtered at 0.22 μm and the iron isotope composition of the two fractions (dissolved and particles) were analyzed using high resolution MC-ICP-MS after chemical purification. Dissolved iron results show positive δ56Fe values (with an average of 0.43 ± 0.04 ‰) relative to the IRMM-14 standard and do not display any relationships with salinity or with percentage of colloid flocculation. The iron isotopic composition of the particles suspended in fresh water is characterized by more negative δ56Fe values than for dissolved Fe and correlate with the percentage of Fe flocculation. Particulate δ56Fe values vary from -0.09‰ at no flocculation to ~ 0.1‰ at the flocculation maximum, which reflect mixing effects between river-borne particles, lithogenic particles derived from coastal seawaters and newly precipitated colloids. Since the process of flocculation produces minimal Fe-isotope fractionation in the dissolved Fe pool, we suggest that the pristine iron isotope composition of fresh water is preserved during estuarine mixing and that the value of the global riverine source into the ocean can be identified from the fresh water values. However, this study also suggests that δ56Fe composition of rivers can also be characterized by more positive δ56Fe values (up to 0.3 per mil) relative to the crust than previously reported. In order to improve our current understanding of the oceanic iron isotope cycling, further work is now required to determine the processes controlling the fractionation of Fe isotopes during continental run-off.This study was supported by the National Science Foundation (OCE 0550066) to O. Rouxel and Edward Sholkovitz

Metabolite Profiling of Alzheimer's Disease Cerebrospinal Fluid

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by progressive loss of cognitive functions. Today the diagnosis of AD relies on clinical evaluations and is only late in the disease. Biomarkers for early detection of the underlying neuropathological changes are still lacking and the biochemical pathways leading to the disease are still not completely understood. The aim of this study was to identify the metabolic changes resulting from the disease phenotype by a thorough and systematic metabolite profiling approach. For this purpose CSF samples from 79 AD patients and 51 healthy controls were analyzed by gas and liquid chromatography-tandem mass spectrometry (GC-MS and LC-MS/MS) in conjunction with univariate and multivariate statistical analyses. In total 343 different analytes have been identified. Significant changes in the metabolite profile of AD patients compared to healthy controls have been identified. Increased cortisol levels seemed to be related to the progression of AD and have been detected in more severe forms of AD. Increased cysteine associated with decreased uridine was the best paired combination to identify light AD (MMSE>22) with specificity and sensitivity above 75%. In this group of patients, sensitivity and specificity above 80% were obtained for several combinations of three to five metabolites, including cortisol and various amino acids, in addition to cysteine and uridine

A Unifying Mechanism for Mitochondrial Superoxide Production during Ischemia-Reperfusion Injury.

Ischemia-reperfusion (IR) injury occurs when blood supply to an organ is disrupted--ischemia--and then restored--reperfusion--leading to a burst of reactive oxygen species (ROS) from mitochondria. It has been tacitly assumed that ROS production during IR is a non-specific consequence of oxygen interacting with dysfunctional mitochondria upon reperfusion. Recently, this view has changed, suggesting that ROS production during IR occurs by a defined mechanism. Here we survey the metabolic factors underlying IR injury and propose a unifying mechanism for its causes that makes sense of the huge amount of disparate data in this area and provides testable hypotheses and new directions for therapies.Work in our laboratories is supported by the Medical Research Council (UK) and the British Heart Foundation. E.T.C. is supported by a Human Frontiers Science Program fellowship.This is the author accepted manuscript. The final version is available from Cell Press via http://dx.doi.org/10.1016/j.cmet.2015.12.00

MAVEN H- Data (2014-2023)

These data were primarily obtained from the Neutral Gas and Ion Mass Spectrometer (NGIMS), Solar Wind Ion Analyzer (SWIA), and Solar Wind Electron Analyzer (SWEA) onboard the Mars Atmosphere and Volatile EvolutioN (MAVEN) spacecraft. These data sets were derived from these three instruments' data products and utilized in the article Characterizing Precipitation Behaviors of H- in the Martian Atmosphere, which is being submitted to JGR: Space Physics. Additional data sets include the list of orbits examined, relevant cross sections mentioned throughout the manuscript, and solar wind conditions derived from MAVEN data & an artificial neural network

Report on validation of the stochastic traffic simulation (Part A): Deliverable D6.23

This document is intended to give an overview of the human subject study in a driving simulator that was conducted by the Chair of Traffic and Transportation Psychology (Verkehrspsychologie – VPSY) of the Technische Universität Dresden (TUD) to provide the Chair of Automotive Engineering (Lehrstuhl Kraftfahrzeugtechnik – LKT) of TUD with the necessary input for the validation of a stochastic traffic simulation, especially for the parameterization, consolidation, and validation of driver behaviour models. VPSY planned, conducted, and analysed a driving simulator study. The main purpose of the study was to analyse driving behaviour and gaze data at intersections in urban areas. Based on relevant literature, a simulated driving environment was created, in which a sample of drivers passed a variety of intersections. Considering different driver states, driving tasks, and traffic situations, the collected data provide detailed information about human gaze and driving behaviour when approaching and crossing intersections. The collected data was transferred to LKT for the development of the stochastic traffic simulation

Report on integration of the stochastic traffic simulation: Deliverable D5.13

As part of the AutoDrive project, the OpenPASS framework is used to develop a cognitive-stochastic traffic flow simulation for urban intersection scenarios described in deliverable D1.14. This framework was adapted and further developed. The deliverable D5.13 deals with the construction of the stochastic traffic simulation. At this point of the process, the theoretical design aspects of D4.20 are implemented. D5.13 explains the operating principles of the different modules. This includes the foundations, boundary conditions, and mathematical theory of the traffic simulation

Report on validation of the stochastic traffic simulation (Part B): Deliverable D6.23

This document is intended to give an overview of the validation of the human subject study, conducted in the driving simulator of the Chair of Traffic and Transportation Psychology (Verkehrspsychologie – VPSY) of the Technische Universität Dresden (TUD), as well of the validation of the stochastic traffic simulation developed in the AutoDrive project by the Chair of Automotive Engineering (Lehrstuhl Kraftfahrzeugtechnik – LKT) of TUD. Furthermore, the evaluation process of a C-AEB (Cooperative-Automatic Emergency Brake) system is demonstrated. The main purpose was to compare the driving behaviour of the study participants and the driving behaviour of the agents in the traffic simulation with real world data. Based on relevant literature, a validation concept was designed and real world data was collected using drones and stationary cameras. By means of qualitative and quantitative analysis it could be shown, that the driving simulator study shows realistic driving behaviour in terms of mean speed. Moreover, the stochastic traffic simulation already reflects reality in terms of mean and maximum speed of the agents. Finally, the performed evaluation proofed the suitability of the developed stochastic simulation for the assessment process. Furthermore, it could be shown, that a C-AEB system improves the traffic safety for the chosen test-scenarios