Enzymatic catalysis of anti-Baldwin ring closure in polyether biosynthesis

Polycyclic polyether natural products have fascinated chemists and biologists alike owing to their useful biological activity, highly complex structure and intriguing biosynthetic mechanisms. Following the original proposal for the polyepoxide origin of lasalocid and isolasalocid and the experimental determination of the origins of the oxygen and carbon atoms of both lasalocid and monensin, a unified stereochemical model for the biosynthesis of polyether ionophore antibiotics was proposed. The model was based on a cascade of nucleophilic ring closures of postulated polyepoxide substrates generated by stereospecific oxidation of all-trans polyene polyketide intermediates. Shortly thereafter, a related model was proposed for the biogenesis of marine ladder toxins, involving a series of nominally disfavoured anti-Baldwin, endo-tet epoxide-ring-opening reactions. Recently, we identified Lsd19 from the Streptomyces lasaliensis gene cluster as the epoxide hydrolase responsible for the epoxide-opening cyclization of bisepoxyprelasalocid A to form lasalocid A. Here we report the X-ray crystal structure of Lsd19 in complex with its substrate and product analogue to provide the first atomic structure-to our knowledge-of a natural enzyme capable of catalysing the disfavoured epoxide-opening cyclic ether formation. On the basis of our structural and computational studies, we propose a general mechanism for the enzymatic catalysis of polyether natural product biosynthesis.     

Phase transitions in the assembly of multivalent signalling proteins

Cells are organized on length scales ranging from ?ngstr?m to micrometres. However, the mechanisms by which ?ngstr?m-scale molecular properties are translated to micrometre-scale macroscopic properties are not well understood. Here we show that interactions between diverse synthetic, multivalent macromolecules (including multi-domain proteins and RNA) produce sharp liquid-liquid-demixing phase separations, generating micrometre-sized liquid droplets in aqueous solution. This macroscopic transition corresponds to a molecular transition between small complexes and large, dynamic supramolecular polymers. The concentrations needed for phase transition are directly related to the valency of the interacting species. In the case of the actin-regulatory protein called neural Wiskott-Aldrich syndrome protein (N-WASP) interacting with its established biological partners NCK and phosphorylated nephrin, the phase transition corresponds to a sharp increase in activity towards an actin nucleation factor, the Arp2/3 complex. The transition is governed by the degree of phosphorylation of nephrin, explaining how this property of the system can be controlled to regulatory effect by kinases. The widespread occurrence of multivalent systems suggests that phase transitions may be used to spatially organize and biochemically regulate information throughout biology.     

Expression and self-assembly of Heterocapsa circularisquama RNA virus-like particles synthesized in Pichia pastoris

Heterocapsa circularisquama RNA virus(HcRNAV) is the first single-stranded RNA virus to be characterized that infects dinoflagellates.The ability of HcRNAV coat protein(HcRNAV CP) to self-assemble into virus-like particles(VLPs) in vitro suggested that heterologous expression was possible,and that the VLPs might be ideal nanocontainers for the targeted delivery of genes and chemicals.In this paper,we report the expression of a codon-optimized HcRNAV 109 CP gene in Pichia pastoris and the production of self-assembled HcRNAV VLPs using large-scale fermentation.The HcRNAV 109 CP gene was synthesized according to the codon preference of P.pastoris and cloned into a pPICZA vector.The recombinant plasmid pPICZA-CPsyns was transformed into P.pastoris by electroporation.The resulting yeast colonies were screened by PCR and analyzed for protein expression by SDS polyacrylamide gel electrophoresis.After large-scale fermentation,the yield of HcRNAV CPsyns reached approximately 2.5 g L 1 within 4 d.The HcRNAV VLPs were purified using PEG precipitation followed by cesium chloride density gradient ultracentrifugation,and were subsequently analyzed using UV spectrophotometry and transmission electron microscopy.Fluorescence dye-labeled myoglobin was loaded into the cages of the HcRNAV VLPs and the encapsulation was confirmed by fluorescence spectroscopy.The results point to the possible utilization in pharmacology or nanotechnology of HcRNAV VLPs produced by P.pastoris fermentation.     

Recent advances in hydrogen storage technologies based on nanoporous carbon materials

Hydrogen is a promising energy carrier that can potentially facilitate a transition from fossil fuels to sustainable energy sources without producing harmful by-products. Prior to realizing a hydrogen economy, however, viable hydrogen storage materials must be developed. Physical adsorption in porous solids provides an opportunity for hydrogen storage under low-stringency conditions. Physically adsorbed hydrogen molecules are weakly bound to a surface and, hence, are easily released. Among the various surface candidates, porous carbons appear to provide efficient hydrogen storage, with the advantages that porous carbon is relatively low-cost to produce and is easily prepared. In this review, we summarize the preparation methods, pore characteristics, and hydrogen storage capacities of representative nanoporous carbons, including activated carbons, zeolite-templated carbon, and carbide-derived carbon. We focus particularly on a series of nanoporous carbons developed recently: metal–organic framework-derived carbons, which exhibit promising properties for use in hydrogen storage applications.     

A Designated Query Protocol for Serverless Mobile RFID Systems with Reader and Tag Privacy

Recently, a new type of Radio Frequency IDentification (RFID) system with mobile readers is introduced. In such a system, it is more desirable for mobile readers to identify tags without a back-end server, and thus it is frequently referred as a serverless mobile RFID system. In this paper, we formalize a serverless mobile RFID system model and propose a new encryption-based system that preserves the privacy of both tags and readers in the model. In addition, we define a new adversary model for the system model and show the security of the proposed system. Throughout comparisons between ours and the other alternatives, we show that our proposed system provides a stronger reader privacy and robustness against a reader forgery attack than the competitors.     

A phosphatase complex that dephosphorylates gammaH2AX regulates DNA damage checkpoint recovery

One of the earliest marks of a double-strand break (DSB) in eukaryotes is serine phosphorylation of the histone variant H2AX at the carboxy-terminal SQE motif to create gammaH2AX-containing nucleosomes. Budding-yeast histone H2A is phosphorylated in a similar manner by the checkpoint kinases Tel1 and Mec1 (ref. 2; orthologous to mammalian ATM and ATR, respectively) over a 50-kilobase region surrounding the DSB. This modification is important for recruiting numerous DSB-recognition and repair factors to the break site, including DNA damage checkpoint proteins, chromatin remodellers and cohesins. Multiple mechanisms for eliminating gammaH2AX as DNA repair completes are possible, including removal by histone exchange followed potentially by degradation, or, alternatively, dephosphorylation. Here we describe a three-protein complex (HTP-C, for histone H2A phosphatase complex) containing the phosphatase Pph3 that regulates the phosphorylation status of gammaH2AX in vivo and efficiently dephosphorylates gammaH2AX in vitro. gammaH2AX is lost from chromatin surrounding a DSB independently of the HTP-C, indicating that the phosphatase targets gammaH2AX after its displacement from DNA. The dephosphorylation of gammaH2AX by the HTP-C is necessary for efficient recovery from the DNA damage checkpoint.     

'Designer atoms' for quantum metrology

Entanglement is recognized as a key resource for quantum computation and quantum cryptography. For quantum metrology, the use of entangled states has been discussed and demonstrated as a means of improving the signal-to-noise ratio. In addition, entangled states have been used in experiments for efficient quantum state detection and for the measurement of scattering lengths. In quantum information processing, manipulation of individual quantum bits allows for the tailored design of specific states that are insensitive to the detrimental influences of an environment. Such 'decoherence-free subspaces' (ref. 10) protect quantum information and yield significantly enhanced coherence times. Here we use a decoherence-free subspace with specifically designed entangled states to demonstrate precision spectroscopy of a pair of trapped Ca+ ions; we obtain the electric quadrupole moment, which is of use for frequency standard applications. We find that entangled states are not only useful for enhancing the signal-to-noise ratio in frequency measurements--a suitably designed pair of atoms also allows clock measurements in the presence of strong technical noise. Our technique makes explicit use of non-locality as an entanglement property and provides an approach for 'designed' quantum metrology.     

A keratin cytoskeletal protein regulates protein synthesis and epithelial cell growth

Cell growth, an increase in mass and size, is a highly regulated cellular event. The Akt/mTOR (mammalian target of rapamycin) signalling pathway has a central role in the control of protein synthesis and thus the growth of cells, tissues and organisms. A striking example of a physiological context requiring rapid cell growth is tissue repair in response to injury. Here we show that keratin 17, an intermediate filament protein rapidly induced in wounded stratified epithelia, regulates cell growth through binding to the adaptor protein 14-3-3sigma. Mouse skin keratinocytes lacking keratin 17 (ref. 4) show depressed protein translation and are of smaller size, correlating with decreased Akt/mTOR signalling activity. Other signalling kinases have normal activity, pointing to the specificity of this defect. Two amino acid residues located in the amino-terminal head domain of keratin 17 are required for the serum-dependent relocalization of 14-3-3sigma from the nucleus to the cytoplasm, and for the concomitant stimulation of mTOR activity and cell growth. These findings reveal a new and unexpected role for the intermediate filament cytoskeleton in influencing cell growth and size by regulating protein synthesis.