A complement to the modern crystallographer's toolbox: Caged gadolinium complexes with versatile binding modes

A set of seven caged gadolinium complexes were used as vectors for introducing the chelated Gd3+ ion into protein crystals in order to provide strong anomalous scattering for de novo phasing. The complexes contained multidentate ligand molecules with different functional groups to provide a panel of possible interactions with the protein. An exhaustive crystallographic analysis showed them to be nondisruptive to the diffraction quality of the prepared derivative crystals, and as many as 50% of the derivatives allowed the determination of accurate phases, leading to high-quality experimental electron-density maps. At least two successful derivatives were identified for all tested proteins. Structure refinement showed that the complexes bind to the protein surface or solvent-accessible cavities, involving hydrogen bonds, electrostatic and CH-π interactions, explaining their versatile binding modes. Their high phasing power, complementary binding modes and ease of use make them highly suitable as a heavy-atom screen for high-throughput de novo structure determination, in combination with the SAD method. They can also provide a reliable tool for the development of new methods such as serial femtosecond crystallography. © 2014 International Union of Crystallography.Peer Reviewe

996-11 Direct Gene Transfer and Expression with Arterial Iontophoretic Catheter Delivery

Iontophoresis is a technique of molecular delivery which uses electric current to enhance movement of charged molecules into tissues. A porous balloon catheter was tested with a central silver chloride electrode capable of generating a potential gradient across the arterial wall using an adhesive patch placed on the skin to serve as the anode. We hypothesized that this catheter delivery system might effectively transfer negatively charged plasmid DNA into arterial cells in vivo.MethodsTo localize plasmid DNA arterial delivery, a 7 Fr iontophoretic porous balloon catheterwast,’aced into porcine carotid arteries underflouroscopic guidance. 15μg of 35S-Iabeled plasmid DNA (1.4×106 cpm/μg) expressing the heat stable human alkaline phosphatase (hAP) gene with an RSV promoter was infused through the balloon at 6 atm pressure. A constant current density of 2.5 mA/cm2 was maintained for 10 minutes. The ;35S-labeled plasmid DNA delivery was repeated on the contralateral carotid artery under identical conditions with the absence of electric current. 20 minutes after gene transfer, the arteries were fixed in situ and processed for autoradiography. To analyze gene transfer and expression, 8 porcine carotid arterial segments were subject to iontophoretic gene delivery for 10 minutes at 6 atm with a current density of 2.5 mA/cm2 using the RSV hAP plasmid (n=6) or control plasmid (n=2). Animals were sacrificed 5 days after gene delivery and the transfected arteries analyzed by PCR and heat stable alkaline phosphatase histochemistry.ResultsAutoradiography of the arteries which underwent ;35S-labeled plasmid delivery revealed minimal radiolabel in the luminal cells of the control artery in which current was not delivered. In contrast, significant amounts of radiolabel were present in the media and adventitia of the artery subject to current delivery. PCR analysis of the arterial segments studied 5 days after delivery confirmed gene transfer in all hAP segments and was negative in control arteries. Staining for heat stable recombinant alkaline phosphatase activity demonstrated recombinant protein expression in 5% of medial cells and 10% of adventitial cells in arteries which underwent hAP gene transfer. Control arteries were negative for hAP staining.ConclusionsIontophoretic catheter gone delivery can be used to perform direct plasmid DNA delivery with expression of recombinant protein in medial and adventitial cells

Towards freeform curved blazed gratings using diamond machining.

Concave blazed gratings greatly simplify the architecture of spectrographs by reducing the number of optical components. The production of these gratings using diamond-machining offers practically no limits in the design of the grating substrate shape, with the possibility of making large sag freeform surfaces unlike the alternative and traditional method of holography and ion etching. In this paper, we report on the technological challenges and progress in the making of these curved blazed gratings using an ultra-high precision 5 axes Moore-Nanotech machine. We describe their implementation in an integral field unit prototype called IGIS (Integrated Grating Imaging Spectrograph) where freeform curved gratings are used as pupil mirrors. The goal is to develop the technologies for the production of the next generation of low-cost, compact, high performance integral field unit spectrometers

Directly Imaging Rocky Planets from the Ground

Over the past three decades instruments on the ground and in space have discovered thousands of planets outside the solar system. These observations have given rise to an astonishingly detailed picture of the demographics of short-period planets, but are incomplete at longer periods where both the sensitivity of transit surveys and radial velocity signals plummet. Even more glaring is that the spectra of planets discovered with these indirect methods are either inaccessible (radial velocity detections) or only available for a small subclass of transiting planets with thick, clear atmospheres. Direct detection can be used to discover and characterize the atmospheres of planets at intermediate and wide separations, including non-transiting exoplanets. Today, a small number of exoplanets have been directly imaged, but they represent only a rare class of young, self-luminous super-Jovian-mass objects orbiting tens to hundreds of AU from their host stars. Atmospheric characterization of planets in the <5 AU regime, where radial velocity (RV) surveys have revealed an abundance of other worlds, is technically feasible with 30-m class apertures in combination with an advanced AO system, coronagraph, and suite of spectrometers and imagers. There is a vast range of unexplored science accessible through astrometry, photometry, and spectroscopy of rocky planets, ice giants, and gas giants. In this whitepaper we will focus on one of the most ambitious science goals --- detecting for the first time habitable-zone rocky (<1.6 R_Earth) exoplanets in reflected light around nearby M-dwarfsComment: 8 pages, 1 figure, Astro2020 Science White Pape

SUMO chain formation is required for response to replication arrest in S. pombe

SUMO is a ubiquitin-like protein that is post-translationally attached to one or more lysine residues on target proteins. Despite having only 18% sequence identity with ubiquitin, SUMO contains the conserved betabetaalphabetabetaalphabeta fold present in ubiquitin. However, SUMO differs from ubiquitin in having an extended N-terminus. In S. pombe the N-terminus of SUMO/Pmt3 is significantly longer than those of SUMO in S. cerevisiae, human and Drosophila. Here we investigate the role of this N-terminal region. We have used two dimensional gel electrophoresis to demonstrate that S. pombe SUMO/Pmt3 is phosphorylated, and that this occurs on serine residues at the extreme N-terminus of the protein. Mutation of these residues (in pmt3-1) results in a dramatic reduction in both the levels of high Mr SUMO-containing species and of total SUMO/Pmt3, indicating that phosphorylation of SUMO/Pmt3 is required for its stability. Despite the significant reduction in high Mr SUMO-containing species, pmt3-1 cells do not display an aberrant cell morphology or sensitivity to genotoxins or stress. Additionally, we demonstrate that two lysine residues in the N-terminus of S. pombe SUMO/Pmt3 (K14 and K30) can act as acceptor sites for SUMO chain formation in vitro. Inability to form SUMO chains results in aberrant cell and nuclear morphologies, including stretched and fragmented chromatin. SUMO chain mutants are sensitive to the DNA synthesis inhibitor, hydroxyurea (HU), but not to other genotoxins, such as UV, MMS or CPT. This implies a role for SUMO chains in the response to replication arrest in S. pomb

Moltemplate: A Tool for Coarse-Grained Modeling of Complex Biological Matter and Soft Condensed Matter Physics

Coarse-grained models have long been considered indispensable tools in the investigation of biomolecular dynamics and assembly. However, the process of simulating such models is arduous because unconventional force fields and particle attributes are often needed, and some systems are not in thermal equilibrium. Although modern molecular dynamics programs are highly adaptable, software designed for preparing all-atom simulations typically makes restrictive assumptions about the nature of the particles and the forces acting on them. Consequently, the use of coarse-grained models has remained challenging. Moltemplate is a file format for storing coarse-grained molecular models and the forces that act on them, as well as a program that converts moltemplate files into input files for LAMMPS, a popular molecular dynamics engine. Moltemplate has broad scope and an emphasis on generality. It accommodates new kinds of forces as they are developed for LAMMPS, making moltemplate a popular tool with thousands of users in computational chemistry, materials science, and structural biology. To demonstrate its wide functionality, we provide examples of using moltemplate to prepare simulations of fluids using many-body forces, coarse-grained organic semiconductors, and the motor-driven supercoiling and condensation of an entire bacterial chromosome

Electrified Aircraft Propulsion (EAP) Educational Briefing

This is an educational briefing package for Electrified Aircraft Propulsion and Power (EAPP); this presentation will brief on NASA needs and challenges in Electrified Aircraft Propulsion and Power as well as the SBIR program and proposal guidance