Drosophila Reporter Vectors Compatible with ΦC31 Integrase Transgenesis Techniques and Their Use to Generate New Notch Reporter Fly Lines

Complex spatial and temporal regulation of gene activity is fundamental to development and homeostasis. The ability to decipher the DNA sequences that accurately coordinate gene expression is, therefore, of primary importance. One way to assess the functions of DNA elements entails their fusion to fluorescent reporter genes. This powerful approach makes it possible to visualize their regulatory capabilities when reintroduced into the developing animal. Transgenic studies in Drosophila have recently advanced with the introduction of site-specific, ΦC31 integrase–mediated approaches. However, most existing Drosophila reporter vectors are not compatible with this new approach and have become obsolete. Here we describe a new series of fluorescent reporter vectors optimized for use with ΦC31 transgenesis. By using these vectors to generate a set of Notch reporter fly lines, we demonstrate their efficacy in reporting the function of gene regulatory elements

Three-Axis Fiber-Optic Body Force Sensor for Flexible Manipulators

This paper proposes a force/torque sensor structure that can be easily integrated into a flexible manipulator structure. The sensor's ring-like structure with its hollow inner section provides ample space for auxiliary components, such as cables and tubes, to be passed through and, hence, is very suitable for integration with tendon-driven and fluid-actuated manipulators. The sensor structure can also accommodate the wiring for a distributed sensor system as well as for diagnostic instruments that may be incorporated in the manipulator. Employing a sensing approach based on optical fibers as done here allows for the creation of sensors that are free of electrical currents at the point of sensing and immune to magnetic fields. These sensors are inherently safe when used in the close vicinity of humans and their measuring performance is not impaired when they are operated in or nearby machines, such as magnetic resonance imaging scanners. This type of sensor concept is particularly suitable for inclusion in instruments and robotic tools for minimally invasive surgery. This paper summarizes the design, integration challenges, and calibration of the proposed optical three-axis force sensor. The experimental results confirm the effectiveness of our optical sensing approach and show that after calibrating its stiffness matrix, force and momentum components can be determined accurately

Tribological properties of duplex plasma oxidised, nitrided and PVD coated Ti-6Al-4V

The authors gratefully acknowledge financial support from the UK Technology Strategy Board under Technology Programme project TP/22076, for underpinning research carried out at Sheffield University, on which the work presented in this paper was partially based.Sequential triode plasma oxidation and nitriding have been used to provide enhanced load support for physical vapour deposited (PVD) hard coatings. The diffusion process has been designed to maximise process efficiency and coating adhesion, thereby significantly improving the tribological properties of the Ti–6Al–4V alloy — particularly at high contact pressures. This has been demonstrated using unlubricated linear reciprocating-sliding ball-on-plate wear tests and micro-scratch adhesion testing. Also, surface micro- profilometry, nano/micro-indentation hardness testing, scanning electron microscopy (SEM), energy- dispersive X-ray spectroscopy (EDX), X-ray diffraction (XRD), and glow-discharge optical emission spectroscopy (GDOES) data are presented to corroborate the effect of the several plasma diffusion processes and duplex diffusion/coating combinations discussed here. The results presented show that the novel processing technique developed permits the use of oxygen diffusion in order to obtain relatively large case depths in shorter treatment times without compromising the adhesion strength of subsequently deposited PVD layers.peer-reviewe

Synthetic studies toward the brasilinolides: controlled assembly of a protected C1-C38 polyol based on fragment union by complex aldol reactions.

The brasilinolides are an architecturally complex family of 32-membered macrolides, characterised by potent immunosuppressant and antifungal properties, which represent challenging synthetic targets. By adopting a highly convergent strategy, a range of asymmetric aldol/reduction sequences and catalytic protocols were employed to assemble a series of increasingly elaborate fragments. The controlled preparation of suitable C1-C19 and C20-C38 acyclic fragments 5 and 6, containing seven and 12 stereocentres respectively, was first achieved. An adventurous C19-C20 fragment union was then explored to construct the entire carbon chain of the brasilinolides. This pivotal coupling step could be performed in a complex boron-mediated aldol reaction to install the required C19 hydroxyl stereocentre when alternative Mukaiyama-type aldol protocols proved unrewarding. A protected C1-C38 polyol 93 was subsequently prepared, setting the stage for future late-stage diversification toward the various brasilinolide congeners. Throughout this work, asymmetric boron-mediated aldol reactions of chiral ketones with aldehydes proved effective both for controlled fragment assembly and coupling with predictable stereoinduction from the enolate component.We thank the EPSRC (EP/F025734/1) and Syngenta for support, the Isaac Newton–Mays Wild Research Fellowship at Downing College (M.P.H.), the Herchel Smith Postdoctoral Fellowships Fund at Cambridge (C.J.C.) and the Deutsche Akademie der Naturforser Leopoldina (F.A.M.; BMBF-LPD 9901/8-148) for additional funding, and the EPSRC National Mass Spectrometry Centre (Swansea) for mass spectra.This is the final published version of the article. It was originally published in Organic & Biomolecular Chemistry (Paterson I, Housden MP, Cordier CJ, Burton PM, Mühlthau FA, Loiseleur O, Organic & Biomolecular Chemistry, 2015,13, 5716-5733 doi:10.1039/C5OB00498E). The final version is available at http://dx.doi.org/10.1039/C5OB00498

An investigation into the effect of Triode Plasma Oxidation (TPO) on the tribological properties of Ti6Al4V

The authors gratefully acknowledge the UK Technology Strategy Board for financial support under the collaborative project LIB-TEC, project No TP 22076.Improving the tribological properties of titanium alloys has been the subject of extensive research for many years. A number of thermochemical processes have been developed for that purpose. In this study, surface hardening of Ti6Al4V is achieved by Triode Plasma Oxidation (TPO) which differs from conventional diode plasma treatments through the use of a third electrode; a negatively biased tungsten filament to enhance the ionisation levels in the plasma. The resultant surface generally consists of a top oxide layer with an oxy- gen diffusion zone lying immediately underneath it. The effects of process parameters such as substrate tem- perature, current density and oxygen partial pressure have been investigated. Surface hardness measurements at various indentation loads were carried out to assess the changes in hardness with depth across the diffusion layer. The hardness profiles obtained confirmed the gradual decrease in hardness with treatment depth and provided an indication of the thickness of the hardened layer produced. Ball-on-plate reciprocating sliding wear data and glancing angle XRD analyses of the oxidised samples are also presented. The results indicate that a harder and deeper case is achieved at both high substrate temperature and high oxygen partial pressure. Furthermore, XRD data show that the substrate temperature strongly affects the structure of the oxide layer produced. All TPO-treated samples exhibit significantly better wear performance compared to the untreated material.peer-reviewe

Evaluating the effects of PIRAC nitrogen-diffusion treatments on the mechanical performance of Ti-6Al-4V alloy

The authors would like to thank the European Regional Development Fund (Malta) for research equipment funded through the application of the project “Developing an Interdisciplinary Material Testing and Rapid Prototyping R&D Facility (Ref. no. 012)”. The authors are also greatly indebted to MATERAþ/ERA-NET Plus for funding support for this research (Project ESM-1935).Powder Immersion Reaction Assisted Coating (PIRAC) is a relatively simple nitrogen diffusion based process which has been proposed as a technique capable of considerable improvements in the tribological performance of ceramics and metals alike; however, the necessary exposure of the substrate material to high temperatures for several hours may have an adverse effect on the bulk properties of materials such as titanium alloys. The effect of PIRAC treatments on the bulk metallography and mechanical properties of Ti–6Al–4V has been studied. Following PIRAC nitrogen-diffusion treatment, studies using X-ray diffraction and cross-sectional microscopy have shown evidence of the formation of a thin (1.4 mm) TiN/Ti2N layer, together with the presence of some Ti3Al intermetallic phase. Semi- logarithmic S–N plots show a deleterious effect after PIRAC treatment in terms of material cyclic fatigue strength, particularly at higher treatment temperatures. Samples processed at 800 1C for 4 h however exhibit better fatigue performance than others treated at lower temperatures for longer nitriding times. Fractographic inspection has shown that fatigue cracks originate at (or near) the surface for the untreated Ti-alloy and from the subsurface regions following diffusion treatment, owing to the build-up of compressive stresses in the latter, which hinder crack propagation.peer-reviewe

Transcriptional dynamics elicited by a short pulse of notch activation involves feed-forward regulation by E(spl)/Hes genes.

Dynamic activity of signaling pathways, such as Notch, is vital to achieve correct development and homeostasis. However, most studies assess output many hours or days after initiation of signaling, once the outcome has been consolidated. Here we analyze genome-wide changes in transcript levels, binding of the Notch pathway transcription factor, CSL [Suppressor of Hairless, Su(H), in Drosophila], and RNA Polymerase II (Pol II) immediately following a short pulse of Notch stimulation. A total of 154 genes showed significant differential expression (DE) over time, and their expression profiles stratified into 14 clusters based on the timing, magnitude, and direction of DE. E(spl) genes were the most rapidly upregulated, with Su(H), Pol II, and transcript levels increasing within 5-10 minutes. Other genes had a more delayed response, the timing of which was largely unaffected by more prolonged Notch activation. Neither Su(H) binding nor poised Pol II could fully explain the differences between profiles. Instead, our data indicate that regulatory interactions, driven by the early-responding E(spl)bHLH genes, are required. Proposed cross-regulatory relationships were validated in vivo and in cell culture, supporting the view that feed-forward repression by E(spl)bHLH/Hes shapes the response of late-responding genes. Based on these data, we propose a model in which Hes genes are responsible for co-ordinating the Notch response of a wide spectrum of other targets, explaining the critical functions these key regulators play in many developmental and disease contexts

Enabling lightweight, high load aero-bearings

Environmental and commercial considerations are strongly driving research into weight saving in aircraft. In this research, innovative manufacturing processes were developed to produce lightweight titanium alloy bearings capable of withstanding high bearing pressures. This will enable the replacement of heavier conventional bearing materials with titanium alloy bearings of the same size thereby saving weight. Plasma processing and PVD coating techniques were refined and combined and a sound scientific understanding of the resulting novel processes developed to assure high performance, reliability and repeatability. These techniques were applied to test discs and small bearing (bush) samples, which were tested under progressively greater loads (pressures). FEA was also used to evaluate pressure distribution in a bush test assembly. The novel treatment has potential applications for many bearings and bearing surfaces throughout aircraft.peer-reviewe

Notch signaling coordinates ommatidial rotation in the Drosophila eye via transcriptional regulation of the EGF-Receptor ligand Argos

Abstract: In all metazoans, a small number of evolutionarily conserved signaling pathways are reiteratively used during development to orchestrate critical patterning and morphogenetic processes. Among these, Notch (N) signaling is essential for most aspects of tissue patterning where it mediates the communication between adjacent cells to control cell fate specification. In Drosophila, Notch signaling is required for several features of eye development, including the R3/R4 cell fate choice and R7 specification. Here we show that hypomorphic alleles of Notch, belonging to the Nfacet class, reveal a novel phenotype: while photoreceptor specification in the mutant ommatidia is largely normal, defects are observed in ommatidial rotation (OR), a planar cell polarity (PCP)-mediated cell motility process. We demonstrate that during OR Notch signaling is specifically required in the R4 photoreceptor to upregulate the transcription of argos (aos), an inhibitory ligand to the epidermal growth factor receptor (EGFR), to fine-tune the activity of EGFR signaling. Consistently, the loss-of-function defects of Nfacet alleles and EGFR-signaling pathway mutants are largely indistinguishable. A Notch-regulated aos enhancer confers R4 specific expression arguing that aos is directly regulated by Notch signaling in this context via Su(H)-Mam-dependent transcription