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

End-to-end Simulation of the SCALES Integral Field Spectrograph

We present end-to-end simulations of SCALES, the third generation thermal-infrared diffraction limited imager and low/med-resolution integral field spectrograph (IFS) being designed for Keck. The 2-5 micron sensitivity of SCALES enables detection and characterization of a wide variety of exoplanets, including exoplanets detected through long-baseline astrometry, radial-velocity planets on wide orbits, accreting protoplanets in nearby star-forming regions, and reflected-light planets around the nearest stars. The simulation goal is to generate high-fidelity mock data to assess the scientific capabilities of the SCALES instrument at current and future design stages. The simulation processes arbitrary-resolution input intensity fields with a proposed observation pattern into an entire mock dataset of raw detector read-out lenslet-based IFS frames with calibrations and metadata, which are then reduced by the IFS data reduction pipeline to be analyzed by the user.Comment: 13 pages, 8 figures, Society of Photo-Optical Instrumentation Engineer

Translesion synthesis in mammalian cells

DNA damage blocks the progression of the replication fork. In order to circumvent the damaged bases, cells employ specialized low stringency DNA polymerases, which are able to carry out translesion synthesis (TLS) past different types of damage. The five polymerases used in TLS in human cells have different substrate specificities, enabling them to deal with many different types of damaged bases. PCNA plays a central role in recruiting the TLS polymerases and effecting the polymerase switch from replicative to TLS polymerase. When the fork is blocked PCNA gets ubiquitinated. This increases its affinity for the TLS polymerases, which all have novel ubiquitin-binding motifs, thereby facilitating their engagement at the stalled fork to effect TLS

ATR-mediated phosphorylation of DNA polymerase η is needed for efficient recovery from UV damage

DNA polymerase η (polη) belongs to the Y-family of DNA polymerases and facilitates translesion synthesis past UV damage. We show that, after UV irradiation, polη becomes phosphorylated at Ser601 by the ataxia-telangiectasia mutated and Rad3-related (ATR) kinase. DNA damage–induced phosphorylation of polη depends on its physical interaction with Rad18 but is independent of PCNA monoubiquitination. It requires the ubiquitin-binding domain of polη but not its PCNA-interacting motif. ATR-dependent phosphorylation of polη is necessary to restore normal survival and postreplication repair after ultraviolet irradiation in xeroderma pigmentosum variant fibroblasts, and is involved in the checkpoint response to UV damage. Taken together, our results provide evidence for a link between DNA damage–induced checkpoint activation and translesion synthesis in mammalian cells

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

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

The Versatile Molecular Complex Component LC8 Promotes Several Distinct Steps of Flagellar Assembly

LC8 is present in various molecular complexes. However, its role in these complexes remains unclear. We discovered that although LC8 is a subunit of the radial spoke (RS) complex in Chlamydomonas flagella, it was undetectable in the RS precursor that is converted into the mature RS at the tip of elongating axonemes. Interestingly, LC8 dimers bound in tandem to the N-terminal region of a spoke phosphoprotein, RS protein 3 (RSP3), that docks RSs to axonemes. LC8 enhanced the binding of RSP3 N-terminal fragments to purified axonemes. Likewise, the N-terminal fragments extracted from axonemes contained LC8 and putative spoke-docking proteins. Lastly, perturbations of RSP3’s LC8-binding sites resulted in asynchronous flagella with hypophosphorylated RSP3 and defective associations between LC8, RSs, and axonemes. We propose that at the tip of flagella, an array of LC8 dimers binds to RSP3 in RS precursors, triggering phosphorylation, stalk base formation, and axoneme targeting. These multiple effects shed new light on fundamental questions about LC8-containing complexes and axoneme assembly