De novo generation of a non-segmented negative strand RNA virus with a bicistronic gene

Reverse genetics has facilitated the use of non-segmented negative strand RNA viruses (NNSV) as vectors. Currently, heterologous gene expression necessitates insertion of extra-numeral transcription units (ENTUs), which may alter the NNSV polar transcription gradient and attenuate growth relative to wildtype (Wt). We hypothesized that rescuing recombinant Sendai Virus (rSeV) with a bicistronic gene might circumvent this attenuation but still allow heterologous open reading frame (ORF) expression. Therefore, we used a 9-nucleotide sequence previously described with internal ribosome entry site (IRES) activity, which, when constructed as several repeats, synergistically increased the level of expression of the second cistron [Chappell, S.A., Edelman, G.M., Mauro, V.P., 2000. A 9-nt segment of a cellular mRNA can function as an internal ribosome entry site (IRES) and when present in linked multiple copies greatly enhances IRES activity. Proc. Natl. Acad. Sci. U.S.A. 97, 1536-1541]. We inserted the Renilla luciferase (rLuc) ORF, preceded by 1, 3 or 7 IRES copies, downstream of the SeV N ORF in an infectious clone. Corresponding rSeVs were successfully rescued. Interestingly, bicistronic rSeVs grew as fast as or faster than Wt rSeV. Furthermore, SeV gene transcription downstream of the N/rLuc gene was either equivalent to, or slightly enhanced, compared to Wt rSeV. Importantly, all rSeV/rLuc viruses efficiently expressed rLuc. IRES repetition increased rLuc expression at a multiplicity of infection of 0.1, although without evidence of synergistic enhancement. In conclusion, our approach provides a novel way of insertion and expression of foreign genes in NNSVs. (C) 2008 Elsevier B.V. All rights reserved.

Phase-Transfer Catalysis in Segmented Flow in a Microchannel: Fluidic Control of Selectivity and Productivity

Precise control over the interfacial area of aqueous and organic slugs in segmented flow in a microchannel reactor provides an attractive means to optimize the yield and productivity of a phase-transfer-catalyzed reaction. Herein, we report the selective alkylation of phenylacetonitrile to the monoalkylated product in a microchannel of 250-mu m internal diameter operated in a continuous and solvent-free manner in the slug-flow regime. The conversion of phenylacetonitrile increased from 40% to 99% as a result of a 97% larger slug surface-to-volume ratio when the volumetric aqueous-to-organic phase flow ratio was raised from 1.0 to 6.1 at the same residence time. The larger surface-to-volume ratio significantly promoted catalyst phase transfer but decreased selectivity because of the simultaneous increase of the rate of the consecutive reaction to the dialkylated product. There exists all Optimum flow ratio with a maximum productivity. Conversion and selectivity in the microchannel reactor were both found to be significantly larger than in a stirred reactor.

Thermoforming carbon fibre-reinforced thermoplastic composites

The use of carbon fibre composites is growing in many sectors but their use remains stronger in very high value industries such as aerospace where the demands of the application more easily justify the high energy input needed and the corresponding costs incurred. This energy and cost input is returned through gains over the whole life of the product, with for example, longer maintenance intervals for an aircraft and lower fuel burn. Thermoplastic composites however have a different energy and cost profile compared to traditional thermosets with notable differences in recyclability, but this profile is not well quantified or documented. This study considers the key process control parameters and identifies an optimal window for processing, along with the effect this has on the final characteristics of the manufactured parts. Interactions between parameters and corresponding sensitivities are extracted from the results.

The prediction of process-induced deformation in a thermoplastic composite in support of manufacturing simulation

Digital manufacturing techniques can simulate complex assembly sequences using computer-aided design-based, as-designed' part forms, and their utility has been proven across several manufacturing sectors including the ship building, automotive and aerospace industries. However, the reality of working with actual parts and composite components, in particular, is that geometric variability arising from part forming or processing conditions can cause problems during assembly as the as-manufactured' form differs from the geometry used for any simulated build validation. In this work, a simulation strategy is presented for the study of the process-induced deformation behaviour of a 90 degrees, V-shaped angle. Test samples were thermoformed using pre-consolidated carbon fibre-reinforced polyphenylene sulphide, and the processing conditions were re-created in a virtual environment using the finite element method to determine finished component angles. A procedure was then developed for transferring predicted part forms from the finite element outputs to a digital manufacturing platform for the purpose of virtual assembly validation using more realistic part geometry. Ultimately, the outcomes from this work can be used to inform process condition choices, material configuration and tool design, so that the dimensional gap between as-designed' and as-manufactured' part forms can be reduced in the virtual environment.

Rotational moulding of thermoplastic polyester urethanes

Various grades of Thermoplastic Polyurethane (TPU) supplied by Bayer were studied to determine their suitability for the rotational moulding process. Following grinding, parts were produced using a variety of peak internal air temperatures and cooling rates. The tensile and impact properties of these parts were then analysed and it was found that both the grade and moulding conditions had a large bearing on the quality and mechanical strength of the part produced.

Efficient and selective hydrogen peroxide-mediated oxidation of sulfides in batch and segmented and continuous flow using a peroxometalate-based polymer immobilised ionic liquid phase catalyst

The peroxometalate-based polymer immobilized ionic liquid phase catalyst [PO4{WO(O-2)(2)}(4)]@PIILP has been prepared by anion exchange of ring opening metathesis-derived pyrrolidinium-decorated norbornene/ cyclooctene copolymer and shown to be a remarkably efficient system for the selective oxidation of sulfides under mild conditions. A cartridge packed with a mixture of [PO4{WO(O-2)(2)}(4)]@PIILP and silica operated as a segmented or continuous flow process and gave good conversions and high selectivity for either sulfoxide (92% in methanol at 96% conversion for a residence time of 4 min) or sulfone (96% in acetonitrile at 96% conversion for a residence time of 15 min). The immobilized catalyst remained active for 8 h under continuous flow operation with a stable activity/selectivity profile that allowed 6.5 g of reactant to be processed (TON = 46 428) while a single catalyst cartridge could be used for the consecutive oxidation of multiple substrates giving activity-selectivity profiles that matched those obtained with fresh catalyst.

High Strength Thermoplastic Bonding for Multi-channel, Multi-layer Lab-on-Chip Devices for Ocean and Environmental applications

A solvent-vapour thermoplastic bonding process is reported which provides high strength bonding of PMMA over a large area for multi-channel and multi-layer microfluidic devices with shallow high resolution channel features. The bond process utilises a low temperature vacuum thermal fusion step with prior exposure of the substrate to chloroform (CHCl3) vapour to reduce bond temperature to below the PMMA glass transition temperature. Peak tensile and shear bond strengths greater than 3 MPa were achieved for a typical channel depth reduction of 25 µm. The device-equivalent bond performance was evaluated for multiple layers and high resolution channel features using double-side and single-side exposure of the bonding pieces. A single-sided exposure process was achieved which is suited to multi-layer bonding with channel alignment at the expense of greater depth loss and a reduction in peak bond strength. However, leak and burst tests demonstrate bond integrity up to at least 10 bar channel pressure over the full substrate area of 100 mm x 100 mm. The inclusion of metal tracks within the bond resulted in no loss of performance. The vertical wall integrity between channels was found to be compromised by solvent permeation for wall thicknesses of 100 µm which has implications for high resolution serpentine structures. Bond strength is reduced considerably for multi-layer patterned substrates where features on each layer are not aligned, despite the presence of an intermediate blank substrate. Overall a high performance bond process has been developed that has the potential to meet the stringent specifications for lab-on-chip deployment in harsh environmental conditions for applications such as deep ocean profiling.

Modelling the nonlinear behaviour and fracture process of AS4/PEKK thermoplastic composite under shear loading

The accurate determination of non-linear shear behaviour and fracture toughness of continuous carbon-fibre/polymer composites remains a considerable challenge. These measurements are often necessary to generate material parameters for advanced computational damage models. In particular, there is a dearth of detailed shear fracture toughness characterisation for thermoplastic composites which are increasingly generating renewed interest within the aerospace and automotive sectors. In this work, carbon fibre (AS4)/ thermoplastic Polyetherketoneketone (PEKK) composite V-notched cross-ply specimens were manufactured to investigate their non-linear response under pure shear loading. Both monotonic and cyclic loading were applied to study the shear modulus degradation and progressive failure. For the first time in the reported literature, we use the essential work of fracture approach to measure the shear fracture toughness of continuous fibre reinforced composite laminates. Excellent geometric similarity in the load-displacement curves was observed for ligament-scaled specimens. The laminate fracture toughness was determined by linear regression, of the specific work of fracture values, to zero ligament thickness, and verified with computational models. The matrix intralaminar fracture toughness (ply level fracture toughness), associated with shear loading was determined by the area method. This paper also details the numerical implementation of a new three-dimensional phenomenological model for carbon fibre thermoplastic composites using the measured values, which is able to accurately represent the full non-linear mechanical response and fracture process. The constitutive model includes a new non-linear shear profile, shear modulus degradation and load reversal. It is combined with a smeared crack model for representing ply-level damage initiation and propagation. The model is shown to accurately predict the constitutive response in terms of permanent plastic strain, degraded modulus as well as load reversal. Predictions are also shown to compare favourably with the evolution of damage leading to final fracture.