Development of high-yield autofluorescent protein microarrays using hybrid cell-free expression with combined Escherichia coli S30 and wheat germ extracts

<p>Abstract</p> <p>Background</p> <p>Protein-based microarray platforms offer considerable promise as high-throughput technologies in proteomics. Particular advantages are provided by self-assembling protein microarrays and much interest centers around analysis of eukaryotic proteins and their molecular interactions. Efficient cell-free protein synthesis is paramount for the production of self-assembling protein microarrays, requiring optimal transcription, translation, and protein folding. The <it>Escherichia coli </it>S30 extract demonstrates high translation rates but lacks the protein-folding efficiency of its eukaryotic counterparts derived from rabbit reticulocyte and wheat germ extract. In comparison to <it>E. coli</it>, eukaryotic extracts, on the other hand, exhibit slower translation rates and poor overall protein yields. A cell-free expression system that synthesizes folded eukaryotic proteins in considerable yields would optimize <it>in vitro </it>translation for protein microarray assembly.</p> <p>Results</p> <p>Self-assembling autofluorescent protein microarrays were produced by <it>in situ </it>transcription and translation of chimeric proteins containing a C-terminal Green Fluorescent Protein tag. Proteins were immobilized as array elements using an anti-GFP monoclonal antibody. The amounts of correctly-folded chimeric proteins were quantified by measuring the fluorescence intensity from each array element. During cell-free expression, very little or no fluorescence was observed from GFP-tagged multidomain eukaryotic plant proteins when <it>in vitro </it>translation was performed with <it>E. coli </it>S30 extract. Improvement was seen using wheat germ extract, but fluorescence intensities were still low because of poor protein yields. A hybrid <it>in vitro </it>translation system, combining S30 and wheat germ extracts, produced high levels of correctly-folded proteins for most of the constructs that were tested.</p> <p>Conclusion</p> <p>The results are consistent with the hypothesis that the wheat germ extract enhances the protein folding capabilities of the <it>in vitro </it>system by providing eukaryotic ribosomes and chaperones and, at the same time, the <it>E. coli </it>S30 extract, which includes an ATP regeneration system, translates the polypeptides at high rates. This hybrid cell-free expression system allows the facile production of high-yield protein arrays suitable for downstream assays.</p

Control of rotorcraft retreating blade stall using air-jet vortex generators

A series of low-speed wind tunnel tests were carried out on an oscillating airfoil fitted with two rows of air-jet vortex generators (AJVGs). The airfoil used had an RAE 9645 section and the two spanwise arrays of AJVGs were located at x/c=0.12 and 0.62. The devices and their distribution were chosen to assess their ability to modify/control dynamic stall; the goal being to enhance the aerodynamic performance of helicopter rotors on the retreating blade side of the disc. The model was pitched about the quarter chord with a reduced frequency (k) of 0.1 in a sinusoidal motion defined by a=15o+10sin_ t. The measured data indicate that, for continuous blowing from the front row of AJVGs with a momentum blowing coefficient (C &#956;) greater than 0.008, modifications to the stalling process are encouraging. In particular, the pitching moment behavior exhibits delayed stall and there is a marked reduction in the normal force hysteresis

Possible Jurassic age for part of Rakaia Terrane: implications for tectonic development of the Torlesse accretionary prism

Greywacke sandstone and argillite beds comprising Rakaia Terrane (Torlesse Complex) in mid Canterbury, South Island, New Zealand, are widely regarded as Late Triassic (Norian) in age based on the occurrence of Torlessia trace fossils, Monotis, and other taxa. This paleontological age assignment is tested using published 40Ar/39Ar mica and U-Pb zircon ages for these rocks and published and new zircon fission track (FT) ages. The youngest U-Pb zircon ages in the Rakaia Terrane rocks in mid Canterbury are Norian, whereas 10-20% of the 40Ar/39Ar muscovite ages are younger than Norian. Numerical modelling of these mica ages shows that they cannot have originated from partial thermal overprinting in the Torlesse prism if the thermal maximum was short-lived and early in the prism history (210-190 Ma), as commonly inferred for these rocks. The young component of mica ages could, however, be explained by extended residence (200-100 Ma) at 265-290deg.C in the prism. Early Jurassic (c. 189 Ma) zircon FT ages for sandstone beds from Arthur's Pass, the Rakaia valley, and the Hermitage (Mt Cook) are interpreted not to have experienced maximum temperatures above 210deg.C, and therefore cannot have been reduced as a result of partial annealing in the Torlesse prism. This is based on identification of a fossil Cretaceous, zircon FT, partial annealing zone in low-grade schists to the west, and the characteristics of the age data. The Early Jurassic zircon FT ages and the young component of 40Ar/39Ar mica ages are regarded therefore as detrital ages reflecting cooling in the source area, and constrain the maximum depositional age of parts of the Rakaia Terrane in mid Canterbury. The zircon FT data also show the initiation (c. 100 Ma) of marked and widespread Late Cretaceous cooling of Rakaia Terrane throughout Canterbury, which is attributed to uplift and erosion of inboard parts of the Torlesse prism due to continuing subduction accretion at its toe. The critical wedge concept is proposed as a new framework for investigating the development of the Torlesse Complex. The Rakaia Terrane may have formed the core of an accretionary wedge imbricated against the New Zealand margin during the Middle or Late Jurassic. Late Jurassic nonmarine sediments (e.g., Clent Hills Formation) accumulated upon the inner parts of the prism as it enlarged, emerged, and continued to be imbricated. Exhumation of Otago Schist from c. 135 Ma may mark the development of a balance (steady state) between sediments entering the prism at the toe and material exiting at the inboard margin. The enlargement of the area of exhumation to all of Canterbury from c. 100 Ma may reflect a dynamic response to widening of the prism through the accretion of Cretaceous sediments. The model of a dynamic critical wedge may help to explain the various expressions of the Rangitata Orogeny

Anonymous Single-Sign-On for n designated services with traceability

Anonymous Single-Sign-On authentication schemes have been proposed to allow users to access a service protected by a verifier without revealing their identity which has become more important due to the introduction of strong privacy regulations. In this paper we describe a new approach whereby anonymous authentication to different verifiers is achieved via authorisation tags and pseudonyms. The particular innovation of our scheme is authentication can only occur between a user and its designated verifier for a service, and the verification cannot be performed by any other verifier. The benefit of this authentication approach is that it prevents information leakage of a user's service access information, even if the verifiers for these services collude which each other. Our scheme also supports a trusted third party who is authorised to de-anonymise the user and reveal her whole services access information if required. Furthermore, our scheme is lightweight because it does not rely on attribute or policy-based signature schemes to enable access to multiple services. The scheme's security model is given together with a security proof, an implementation and a performance evaluation.Comment: 3

Development and evaluation of a high-throughput, low-cost genotyping platform based on oligonucleotide microarrays in rice

<p>Abstract</p> <p>Background</p> <p>We report the development of a microarray platform for rapid and cost-effective genetic mapping, and its evaluation using rice as a model. In contrast to methods employing whole-genome tiling microarrays for genotyping, our method is based on low-cost spotted microarray production, focusing only on known polymorphic features.</p> <p>Results</p> <p>We have produced a genotyping microarray for rice, comprising 880 single feature polymorphism (SFP) elements derived from insertions/deletions identified by aligning genomic sequences of the <it>japonica </it>cultivar Nipponbare and the <it>indica </it>cultivar 93-11. The SFPs were experimentally verified by hybridization with labeled genomic DNA prepared from the two cultivars. Using the genotyping microarrays, we found high levels of polymorphism across diverse rice accessions, and were able to classify all five subpopulations of rice with high bootstrap support. The microarrays were used for mapping of a gene conferring resistance to <it>Magnaporthe grisea</it>, the causative organism of rice blast disease, by quantitative genotyping of samples from a recombinant inbred line population pooled by phenotype.</p> <p>Conclusion</p> <p>We anticipate this microarray-based genotyping platform, based on its low cost-per-sample, to be particularly useful in applications requiring whole-genome molecular marker coverage across large numbers of individuals.</p

Rational isogenies from irrational endomorphisms

In this paper, we introduce a polynomial-time algorithm to compute a connecting $\mathcal{O}$-ideal between two supersingular elliptic curves over $\mathbb{F}_p$ with common $\mathbb{F}_p$-endomorphism ring $\mathcal{O}$, given a description of their full endomorphism rings. This algorithm provides a reduction of the security of the CSIDH cryptosystem to the problem of computing endomorphism rings of supersingular elliptic curves. A similar reduction for SIDH appeared at Asiacrypt 2016, but relies on totally different techniques. Furthermore, we also show that any supersingular elliptic curve constructed using the complex-multiplication method can be located precisely in the supersingular isogeny graph by explicitly deriving a path to a known base curve. This result prohibits the use of such curves as a building block for a hash function into the supersingular isogeny graph

Self-trapping and excited state absorption in fluorene homo-polymer and copolymers with benzothiadiazole and tri-phenylamine

We thank the EPSRC [EP/J009318/1 and EP/J009016/1] for funding. MJP thanks the European Research Council (ERC) for funding under the European Union’s Seventh Framework Programme (FP7/2007-2013)/ERC Grant No. 258990.Excited state absorption (ESA) is studied using time-dependent density functional theory and compared with experiments performed in dilute solutions. The molecules investigated are a fluorene pentamer, polyfluorene F8, the alternating F8 copolymer with benzothiadiazole F8BT, and two blue-emitting random copolymers F8PFB and F8TFB. Calculated and measured spectra show qualitatively comparable results. The ESA cross-section of co-polymers at its maximum is about three times lower than that of F8. The ESA spectra are found to change little upon structural relaxation of the excited state, or change in the order of sub-units in a co-polymer, for all studied molecules. In all these molecules, the strongest ESA transition is found to arise from the same electronic process, exhibiting a reversal of the charge parity. In addition, F8PFB and F8TFB are found to possess almost identical electronic behaviour.Publisher PDFPeer reviewe

Absence of bias against smokers in access to coronary revascularization after cardiac catheterization

OBJECTIVE: Many consider smoking to be a personal choice for which individuals should be held accountable. We assessed whether there is any evidence of bias against smokers in cardiac care decision-making by determining whether smokers were as likely as non-smokers to undergo revascularization procedures after cardiac catheterization. DESIGN: Prospective cohort study. Subjects and setting. All patients undergoing cardiac catheterization in Alberta, Canada. MAIN MEASURE: Patients were categorized as current smokers, former smokers, or never smokers, and then compared for their risk-adjusted likelihood of undergoing revascularization procedures (percutaneous coronary intervention or coronary artery bypass grafting) after cardiac catheterization. RESULTS: Among 20406 patients undergoing catheterization, 25.4% were current smokers at the time of catheterization, 36.6% were former smokers, and 38.0% had never smoked. When compared with never smokers (reference group), the hazard ratio for undergoing any revascularization procedure after catheterization was 0.98 (95% CI 0.93-1.03) for current smokers and 0.98 (0.94-1.03) for former smokers. The hazard ratio for undergoing coronary artery bypass grafting was 1.09 (1.00-1.19) for current smokers and 1.00 (0.93-1.08) for former smokers. For percutaneous coronary intervention, the hazard ratios were 0.93 (0.87-0.99) for current smokers and 1.00 (0.94-1.06) for former smokers. CONCLUSION: Despite potential for discrimination on the basis of smoking status, current and former smokers undergoing cardiac catheterization in Alberta, Canada were as likely to undergo revascularization procedures as catheterization patients who had never smoked

Quantum resource estimates for computing elliptic curve discrete logarithms

We give precise quantum resource estimates for Shor's algorithm to compute discrete logarithms on elliptic curves over prime fields. The estimates are derived from a simulation of a Toffoli gate network for controlled elliptic curve point addition, implemented within the framework of the quantum computing software tool suite LIQ$Ui|\rangle$. We determine circuit implementations for reversible modular arithmetic, including modular addition, multiplication and inversion, as well as reversible elliptic curve point addition. We conclude that elliptic curve discrete logarithms on an elliptic curve defined over an $n$-bit prime field can be computed on a quantum computer with at most $9n + 2\lceil\log_2(n)\rceil+10$ qubits using a quantum circuit of at most $448 n^3 \log_2(n) + 4090 n^3$ Toffoli gates. We are able to classically simulate the Toffoli networks corresponding to the controlled elliptic curve point addition as the core piece of Shor's algorithm for the NIST standard curves P-192, P-224, P-256, P-384 and P-521. Our approach allows gate-level comparisons to recent resource estimates for Shor's factoring algorithm. The results also support estimates given earlier by Proos and Zalka and indicate that, for current parameters at comparable classical security levels, the number of qubits required to tackle elliptic curves is less than for attacking RSA, suggesting that indeed ECC is an easier target than RSA.Comment: 24 pages, 2 tables, 11 figures. v2: typos fixed and reference added. ASIACRYPT 201