Combining intracellular selection with protein-fragment complementation to derive A interacting peptides

Aggregation of the β-amyloid (Aβ) peptide into toxic oligomers is considered the primary event in the pathogenesis of Alzheimer's disease. Previously generated peptides and mimetics designed to bind to amyloid fibrils have encountered problems in solubility, protease susceptibility and the population of small soluble toxic oligomers oligomers. We present a new method that opens the possibility of deriving new amyloid inhibitors. The intracellular protein-fragment complementation assay (PCA) approach uses a semi-rational design approach to generate peptides capable of binding to Aβ. Peptide libraries are based on Aβ regions responsible for instigating amyloidosis, with screening and selection occurring entirely inside Escherichia coli. Successfully selected peptides must therefore bind Aβ and recombine an essential enzyme while permitting bacterial cell survival. No assumptions are made regarding the mechanism of action for selected binders. Biophysical characterisation demonstrates that binding induces a noticeable reduction in amyloid. Therefore, this amyloid-PCA approach may offer a new pathway for the design of effective inhibitors against the formation of amyloid in general

A Branched Kinetic Scheme Describes the Mechanochemical Coupling of Myosin Va Processivity in Response to Substrate

Myosin Va is a double-headed cargo-carrying molecular motor that moves processively along cellular actin filaments. Long processive runs are achieved through mechanical coordination between the two heads of myosin Va, which keeps their ATPase cycles out of phase, preventing both heads detaching from actin simultaneously. The biochemical kinetics underlying processivity are still uncertain. Here we attempt to define the biochemical pathways populated by myosin Va by examining the velocity, processive run-length, and individual steps of a Qdot-labeled myosin Va in various substrate conditions (i.e., changes in ATP, ADP, and Pi) under zero load in the single-molecule total internal reflection fluorescence microscopy assay. These data were used to globally constrain a branched kinetic scheme that was necessary to fit the dependences of velocity and run-length on substrate conditions. Based on this model, myosin Va can be biased along a given pathway by changes in substrate concentrations. This has uncovered states not normally sampled by the motor, and suggests that every transition involving substrate binding and release may be strain-dependent. © 2012 Biophysical Society

Solid State Joining of High Temperature Alloy Tubes for USC and Heat-Exchanger Systems

The principal objective of this project was to develop materials enabling joining technologies for use in forward looking heat-exchanger fabrication in Brayton cycle HIPPS, IGCC, FutureGen concepts capable of operating at temperatures in excess of 1000{degree}C as well as conventional technology upgrades via Ultra Super-Critical (USC) Rankine-cycle boilers capable of operating at 760{degree}C (1400F)/38.5MPa (5500psi) steam, while still using coal as the principal fossil fuel. The underlying mission in Rankine, Brayton or Brayton-Rankine, or IGCC combined cycle heat engine is a steady quest to improving operating efficiency while mitigating global environmental concerns. There has been a progressive move to higher overall cycle efficiencies, and in the case of fossil fuels this has accelerated recently in part because of concerns about greenhouse gas emissions, notably CO{sub 2}. For a heat engine, the overall efficiency is closely related to the difference between the highest temperature in the cycle and the lowest temperature. In most cases, efficiency gains are prompted by an increase in the high temperature, and this in turn has led to increasing demands on the materials of construction used in the high temperature end of the systems. Our migration to new advanced Ni-base and Oxide Dispersion Strengthened (ODS) alloys poses significant fabrication challenges, as these materials are not readily weldable or the weld performs poorly in the high temperature creep regime. Thus the joining challenge is two-fold to a) devise appropriate joining methodologies for similar/dissimilar Ni-base and ODS alloys while b) preserving the near baseline creep performance in the welded region. Our program focus is on solid state joining of similar and dissimilar metals/alloys for heat exchanger components currently under consideration for the USC, HIPPS and IGCC power systems. The emphasis is to manipulate the joining methods and variables available to optimize joint creep performance compared to the base material creep performance. Similar and dissimilar butt joints were fabricated of MA956, IN740 alloys and using inertia welding techniques. We evaluated joining process details and heat treatments and its overall effect on creep response. Fixed and incrementally accelerated temperature creep tests were performed for similar and dissimilar joints and such incremental creep life data is compiled and reported. Long term MA956-MA556 joint tests indicate a firm 2Ksi creep stress threshold performance at 850{degree}C with a maximum exposure of over 9725 hours recorded in the current program. A Larsen Miller Parameter (LMP) of 48.50 for a 2Ksi test at 850{degree}C was further corroborated with tests at 2Ksi stress at 900{degree}C yielding a LMP=48.80. Despite this threshold the joints exhibit immense temperature sensitivity and fail promptly when test temperature raised above 900{degree}C. In comparison the performance of dissimilar joints was inferior, perhaps dictated by the creep characteristics of the mating nickel-base alloys. We describe a parametric window of joint development, and post weld heat treatment (PWHT) in dissimilar joints with solid solution (IN601, IN617) and precipitate strengthened (IN740) materials. Some concerns are evident regarding the diffusion of aluminum in dissimilar joints during high temperature recrystallization treatments. It is noted that aggressive treatments rapidly deplete the corrosion protecting aluminum reservoir in the vicinity of the joint interface. Subsequently, the impact of varying PWHT has been evaluated in the context on ensuing creep performance

Cross-Roll Flow Forming of ODS Alloy Heat Exchanger Tubes For Hoop Creep Enhancement

Mechanically alloyed oxide dispersion strengthened (ODS) Fe-Cr-Al alloy thin walled tubes and sheets, produced via powder processing and consolidation methodologies are promising materials for eventual use at temperatures up to 1200 C in the power generation industry, far above the temperature capabilities of conventional alloys. Target end-uses range from gas turbine combustor liners to high aspect ratio (L/D) heat exchanger tubes. Grain boundary creep processes at service temperatures, particularly those acting in the hoop direction, are the dominant failure mechanisms for such components. The processed microstructure of ODS alloys consists of high aspect ratio grains aligned parallel to the tube axis, a result of dominant axial metal flow which aligns the dispersoid particles and other impurities in the longitudinal direction. The dispersion distribution is unaltered on a micro scale by recrystallization thermal treatments, but the high aspect ratio grain shape typically obtained limits transverse grain spacing and consequently the hoop creep response. Improving hoop creep in ODS-alloy components will require understanding and manipulating the factors that control the recrystallization behavior, and represents a critical materials design and development challenge that must be overcome in order to fully exploit the potential of ODS alloys. The objectives of this program were to (1) increase creep-strength at temperature in ODS-alloy tube and liner components by 100% via, (2) preferential cross-roll flow forming and grain/particle fibering in the critical hoop direction. The research program outlined was iterative and intended to systematically (i) examine and identify post-extrusion forming methodologies to create hoop strengthened tubes, to be (ii) evaluated at 'in-service' loads at service temperatures and environments. Our report outlines the significant hoop creep enhancements possible via secondary cross-rolling and/or flow-forming operations. Each of the secondary processes i.e. hot rotary forming and ambient-temperature flow forming exhibited improvement over the base-line hoop-creep performance. The flow formed MA956 tubes exhibited performance superior to all other rolling/forming variants. At the conclusion of this program 2ksi creep-test exposure for flow formed materials exceeded 7300 hours, 7694 hours and 4200 hours for creep tests operating at 950 C, 975 C and 1000 C respectively. The Larsen-Miller parameter for these improvised flow-formed tubes now exceeds 54.14, i.e., better than ever recorded previously. The creep performance enhancement in cross-rolled MA956 material samples versus the base creep property is elucidated. At least 2-3 orders of magnitude of improvement in creep rates/day and concomitant increases in creeplife are demonstrated for the flow formed tubes versus the base reference tests

Co-populated Conformational Ensembles of β(2)-Microglobulin Uncovered Quantitatively by Electrospray Ionization Mass Spectrometry

Ordered assembly of monomeric human β(2)-microglobulin (β(2)m) into amyloid fibrils is associated with the disorder hemodialysis-related amyloidosis. Previously, we have shown that under acidic conditions (pH <5.0 at 37 °C), wild-type β(2)m assembles spontaneously into fibrils with different morphologies. Under these conditions, β(2)m populates a number of different conformational states in vitro. However, this equilibrium mixture of conformationally different species is difficult to resolve using ensemble techniques such as nuclear magnetic resonance or circular dichroism. Here we use electrospray ionization mass spectrometry to resolve different species of β(2)m populated between pH 6.0 and 2.0. We show that by linear deconvolution of the charge state distributions, the extent to which each conformational ensemble is populated throughout the pH range can be determined and quantified. Thus, at pH 3.6, conditions under which short fibrils are produced, the conformational ensemble is dominated by a charge state distribution centered on the 9+ ions. By contrast, under more acidic conditions (pH 2.6), where long straight fibrils are formed, the charge state distribution is dominated by the 10+ and 11+ ions. The data are reinforced by investigations on two variants of β(2)m (V9A and F30A) that have reduced stability to pH denaturation and show changes in the pH dependence of the charge state distribution that correlate with the decrease in stability measured by tryptophan fluorescence. The data highlight the potential of electrospray ionization mass spectrometry to resolve and quantify complex mixtures of different conformational species, one or more of which may be important in the formation of amyloid

Real-time single-molecule imaging reveals a direct interaction between UvrC and UvrB on DNA tightropes

Nucleotide excision DNA repair is mechanistically conserved across all kingdoms of life. In prokaryotes, this multi-enzyme process requires six proteins: UvrA?D, DNA polymerase I and DNA ligase. To examine how UvrC locates the UvrB? DNA pre-incision complex at a site of damage, we have labeled UvrB and UvrC with different colored quantum dots and quantitatively observed their interactions with DNA tightropes under a variety of solution conditions using oblique angle fluorescence imaging. Alone, UvrC predominantly interacts statically with DNA at low salt. Surprisingly, however, UvrC and UvrB together in solution bind to form the previously unseen UvrBC complex on duplex DNA. This UvrBC complex is highly motile and engages in unbiased one-dimensional diffusion. To test whether UvrB makes direct contact with the DNA in the UvrBC?DNA complex, we investigated three UvrB mutants: Y96A, a b-hairpin deletion and D338N. These mutants affected the motile properties of the UvrBC complex, indicating that UvrB is in intimate contact with the DNA when bound to UvrC. Given the in vivo excess of UvrB and the abundance of UvrBC in our experiments, this newly identified complex is likely to be the predominant form of UvrC in the cell. © 2013 The Author(s)

Spontaneous entanglement leakage of two static entangled Unruh-DeWitt detectors

Two entangled two-level Unruh-DeWitt detectors, which are in rest, spontaneously loose entanglement when at least any one of them is not isolated from the environment quantum fields. For eternal interaction between the detectors and environment, the spontaneous emission from the detectors' exited states and vacuum fluctuations of field influence this negative effect. Consequently, it suggests that two entangled qubits become less communicated during their free-fall towards the black hole horizon.Comment: Minor modification, to appear in Phys. Rev.

Plastic Deformation in Laser-Induced Shock Compression of Monocrystalline Copper

Copper monocrystals were subjected to shock compression at pressures of 10–60 GPa by a short (3 ns initial) duration laser pulse. Transmission electron microscopy revealed features consistent with previous observations of shock-compressed copper, albeit at pulse durations in the µs regime. The results suggest that the defect structure is generated at the shock front. A mechanism for dislocation generation is presented, providing a realistic prediction of dislocation density as a function of pressure. The threshold stress for deformation twinning in shock compression is calculated from the constitutive equations for slip, twinning, and the Swegle-Grady relationship

Maintenance of Fertility in the Face of Meiotic Drive

Selfish genetic elements that gain a transmission advantage through the destruction of sperm have grave implications for drive male fertility. In the X-linked meiotic drive system (SR) of a stalk-eyed fly, we found that SR males have greatly enlarged testes and maintain high fertility despite the destruction of half of their sperm, even when challenged with fertilizing large numbers of females. Conversely, we observed reduced allocation of resources to the accessory glands that probably explains the lower mating frequency of SR males. Body size and eye span were also reduced, which are likely to impair viability and precopulatory success. We discuss the potential evolutionary causes of these differences between drive and standard males

Selective antagonism of cJun for cancer therapy

The activator protein-1 (AP-1) family of transcription factors modulate a diverse range of cellular signalling pathways into outputs which can be oncogenic or anti-oncogenic. The transcription of relevant genes is controlled by the cellular context, and in particular by the dimeric composition of AP-1. Here, we describe the evidence linking cJun in particular to a range of cancers. This includes correlative studies of protein levels in patient tumour samples and mechanistic understanding of the role of cJun in cancer cell models. This develops an understanding of cJun as a focal point of cancer-altered signalling which has the potential for therapeutic antagonism. Significant work has produced a range of small molecules and peptides which have been summarised here and categorised according to the binding surface they target within the cJun-DNA complex. We highlight the importance of selectively targeting a single AP-1 family member to antagonise known oncogenic function and avoid antagonism of anti-oncogenic function