Analysis of radicals and radical reaction products in cell signalling and biomolecular damage:the long hard road to gold-standard measures

The field of free radical biology and medicine continues to move at a tremendous pace, with a constant flow of ground-breaking discoveries. The following collection of papers in this issue of Biochemical Society Transactions highlights several key areas of topical interest, including the crucial role of validated measurements of radicals and reactive oxygen species in underpinning nearly all research in the field, the important advances being made as a result of the overlap of free radical research with the reinvigorated field of lipidomics (driven in part by innovations in MS-based analysis), the acceleration of new insights into the role of oxidative protein modifications (particularly to cysteine residues) in modulating cell signalling, and the effects of free radicals on the functions of mitochondria, extracellular matrix and the immune system. In the present article, we provide a brief overview of these research areas, but, throughout this discussion, it must be remembered that it is the availability of reliable analytical methodologies that will be a key factor in facilitating continuing developments in this exciting research area.

An investigation of thin amorphous carbon-based sputtered coatings for MEMS and micro-engineering applications

The work presented in this thesis describes an investigation into the production and properties of thin amorphous C films, with and without Cr doping, as a low wear / friction coating applicable to MEMS and other micro- and nano-engineering applications. Firstly, an assessment was made of the available testing techniques. Secondly, the optimised test methods were applied to a series of sputtered films of thickness 10 - 2000 nm in order to: (i) investigate the effect of thickness on the properties of coatingslcoating process (ii) investigate fundamental tribology at the nano-scale and (iii) provide a starting point for nanotribological coating optimisation at ultra low thickness. The use of XPS was investigated for the determination of Sp3/Sp2 carbon bonding. Under C 1s peak analysis, significant errors were identified and this was attributed to the absence of sufficient instrument resolution to guide the component peak structure (even with a high resolution instrument). A simple peak width analysis and correlation work with C KLL D value confirmed the errors. The use of XPS for Sp3/Sp2 was therefore limited to initial tentative estimations. Nanoindentation was shown to provide consistent hardness and reduced modulus results with depth (to < 7nm) when replicate data was suitably statistically processed. No significant pile-up or cracking of the films was identified under nanoindentation. Nanowear experimentation by multiple nanoscratching provided some useful information, however the conditions of test were very different to those expect for MEMS and micro- / nano-engineering systems. A novel 'sample oscillated nanoindentation' system was developed for testing nanowear under more relevant conditions. The films were produced in an industrial production coating line. In order to maximise the available information and to take account of uncontrolled process variation a statistical design of experiment procedure was used to investigate the effect of four key process control parameters. Cr doping was the most significant control parameter at all thicknesses tested and produced a softening effect and thus increased nanowear. Substrate bias voltage was also a significant parameter and produced hardening and a wear reducing effect at all thicknesses tested. The use of a Cr adhesion layer produced beneficial results at 150 nm thickness, but was ineffective at 50 nm. Argon flow to the coating chamber produced a complex effect. All effects reduced significantly with reducing film thickness. Classic fretting wear was produced at low amplitude under nanowear testing. Reciprocating sliding was produced at higher amplitude which generated three body abrasive wear and this was generally consistent with the Archard model. Specific wear rates were very low (typically 10-16 - 10-18 m3N-1m-1). Wear rates reduced exponentially with reduced film thickness and below (approx.) 20 nm, thickness was identified as the most important control of wear.

Formulation and process engineering of freeze-dried orally disintegrating tablets

Orally disintegrating tablets (ODTs) which are also referred to as orodispersible and fast disintegrating tablets, are solid oral dosage forms which upon placing on the tongue, disperse/disintegrate rapidly before being swallowed as a suspension or solution. ODTs are therefore easier and more convenient to administer than conventional tablets and are particularly beneficial for paediatric and geriatric patients, who generally have difficulty swallowing their medication. The work presented in this thesis involved the formulation and process development of ODTs, prepared using freeze-drying. Gelatin is one of the principal excipients used in the formulation of freeze-dried ODTs. One of the studies presented in this thesis investigated the potential modification of the properties of this excipient, in order to improve the performance of the tablets. As gelatin is derived from animal sources, a number of ethical issues surround its use as an excipient in pharmaceutical preparations. This was one of the motivations, Methocel™ and Kollicoat® IR were evaluated as binders as alternative materials to gelatin. Polyox™ was also evaluated as a binder together with its potential uses as a viscosity increasing and mucoadhesive agent to increase the retention of tablets in the mouth to encourage pre-gastric absorption of active pharmaceutical ingredients (APIs). The in vitro oral retention of freeze-dried ODT formulations was one property which was assessed in a design of experiments – factorial design study, which was carried out to further understand the role that formulation excipients have on the properties of the tablets. Finally, the novel approach of incorporating polymeric nanoparticles in freeze-dried ODTs was investigated, to study if the release profile of APIs could be modified, which could improve their therapeutic effect. The results from these studies demonstrated that the properties of gelatin-based formulations can be modified by adjusting pH and ionic strength. Adjustment of formulation pH has shown to significantly reduce tablet disintegration time. Evaluating Methocel™, in particular low viscosity grades, and Kollicoat® IR as binders has shown that these polymers can form tablets of satisfactory hardness and disintegration time. Investigating Polyox™ as an excipient in freeze-dried ODT formulations revealed that low viscosity grades appear suitable as binders whilst higher viscosity grades could potentially be utilised as viscosity increasing and mucoadhesive agents. The design of experiments – factorial design study revealed the influence of individual excipients in a formulation mix on resultant tablet properties and in vitro oral retention of APIs. Novel methods have been developed, which allows the incorporation of polymeric nanoparticles in situ in freeze-dried ODT formulations, which allows the modification of the release profile of APIs.

Outsourcing and offshoring engineering projects:understanding the value, sourcing models, and coordination practices

In this paper, we review recent developments in the field of outsourcing and offshoring and the implications for engineering management. We examine three aspects involved in outsourcing and offshoring, namely, sourcing models, coordination, and value extracted from outsourcing projects. We conclude that additional research is needed on recent trends in outsourcing and the impact of such change process on the practice of engineering management. © 2011 IEEE.

Adhesive wear of modern and traditional engineering coatings

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Excipient characterization and particle engineering to develop directly compressed orally disintegrating tablets

ODTs have emerged as a novel oral dosage form with a potential to deliver a wide range of drug candidates to paediatric and geriatric patients. Compression of excipients offers a costeffective and translatable methodology for the manufacture of ODTs. Though, technical challenges prevail such as difficulty to achieve suitable tablet mechanical strength while ensuring rapid disintegration in the mouth, poor compressibility of preferred ODT diluent Dmannitol, and limited use for modified drug-release. The work investigates excipients’ functionality in ODTs and proposes new methodologies for enhancing material characteristics via process and particle engineering. It also aims to expand ODT applications for modified drug-release. Preformulation and formulation studies employed a plethora of techniques/tests including AFM, SEM, DSC, XRD, TGA, HSM, FTIR, hardness, disintegration time, friability, stress/strain and Heckel analysis. Tableting of D-mannitol and cellulosic excipients utilised various compression forces, material concentrations and grades. Engineered D-mannitol particles were made by spray drying mannitol with pore former NH4HCO3. Coated microparticles of model API omeprazole were prepared using water-based film forming polymers. The results of nanoscopic investigations elucidated the compression profiles of ODT excipients. Strong densification of MCC (Py is 625 MPa) occurs due to conglomeration of physicomechanical factors whereas D-mannitol fragments under pressure leading to poor compacts. Addition of cellulosic excipients (L-HPC and HPMC) and granular mannitol to powder mannitol was required to mechanically strengthen the dosage form (hardness >60 N, friability <1%) and to maintain rapid disintegration (<30 sec). Similarly, functionality was integrated into D-mannitol by fabrication of porous, yet, resilient particles which resulted in upto 150% increase in the hardness of compacts. The formulated particles provided resistance to fracture under pressure due to inherent elasticity while promoted tablet disintegration (50-77% reduction in disintegration time) due to porous nature. Additionally, coated microparticles provided an ODT-appropriate modified-release coating strategy by preventing drug (omeprazole) release.

University-led innovative and inspirational engineering education

The UK Government and large employers have recognised the skills gap between learners leaving the education system and the requirements of employers. The current system is seen to be failing significant numbers of learners and has been accused of schooling but not educating our young people. University-led technical colleges are one part of the solution being developed to provide outstanding engineering education. This paper focusses on the learning experience that the Aston University Engineering Academy, the first University-led University Technical College (UTC), has created for entrants to the Engineering Academy in September 2012, when it opens in brand new buildings next to the University. The overall aim is to produce technically literate young people that have business and enterprise skills as well as insight into the diverse range of opportunities in Engineering and Technical disciplines. The project has brought University staff and students together with employers and Academy staff to optimise the engineering education that they will receive. The innovative model presented has drawn on research from across the world in the implementation of this new type of school, as well as educational practices from the USA and the Scandinavian countries. The resulting curriculum is authentic and exciting and expands the University model of problem-based learning and placements into the secondary school environment. The benefits of this close partnership for University staff and students, the employers and the Academy staff are expanded on and the paper concludes with a prediction of progression routes from the Academy.

Microporous polycaprolactone matrices for drug delivery and tissue engineering:the release behaviour of bioactives having extremes of aqueous solubility

Microporous polycaprolactone (PCL) matrices loaded with hydrophobic steroidal drugs or a hydrophilic drug - pilocarpine hydrochloride - were produced by precipitation casting using solutions of PCL in acetone. The efficiency of steroid incorporation in the final matrix (progesterone (56 %) testosterone (46 %) dexamethasone (80 %)) depended on the nature of the drug initially co-dissolved in the PCL solution. Approximately 90 % w/w of the initial load of progesterone, 85 % testosterone and 50 % dexamethasone was released from the matrices in PBS at 37°C over 8 days. Pilocarpine hydrochloride (PH)-loaded PCL matrices, prepared by dispersion of powder in PCL solution, released 70-90 % of the PH content over 12 days in PBS. Application of the Higuchi model revealed that the kinetics of steroid and PH release were consistent with a Fickian diffusion mechanism with corresponding diffusion coefficients of 5.8 × 10-9 (progesterone), 3.9 × 10 -9 (testosterone), 7.1 × 10-10 (dexamethasone) and 22 × 10-8 cm2/s (pilocarpine hydrochloride). The formulation techniques described are expected to be useful for production of implantable, insertable and topical devices for sustained delivery of a range of bioactive molecules of interest in drug delivery and tissue engineering.

Immunological and biochemical techniques in the analysis of tear proteins

This study is concerned with the analysis of tear proteins, paying particular attention to the state of the tears (e.g. non-stimulated, reflex, closed), created during sampling, and to assess their interactions with hydrogel contact lenses. The work has involved the use of a variety of biochemical and immunological analytical techniques for the measurement of proteins, (a), in tears, (b), on the contact lens, and (c), in the eluate of extracted lenses. Although a diverse range of tear components may contribute to contact lens spoilation, proteins were of particular interest in this study because of their theoretical potential for producing immunological reactions. Although normal host proteins in their natural state are generally not treated as dangerous or non-self, those which undergo denaturation or suffer a conformational change may provoke an excessive and unnecessary immune response. A novel on-lens cell based assay has been developed and exploited in order to study the role of the ubiquitous cell adhesion glycoprotein, vitronectin, in tears and contact lens wear under various parameters. Vitronectin, whose levels are known to increase in the closed eye environment and shown here to increase during contact lens wear, is an important immunoregulatory protein and may be a prominent marker of inflammatory activity. Immunodiffusion assays were developed and optimised for use in tear analysis, and in a series of subsequent studies used for example in the measurement of albumin, lactoferrin, IgA and IgG. The immunodiffusion assays were then applied in the estimation of the closed eye environment; an environment which has been described as sustaining a state of sub-clinical inflammation. The role and presence of a lesser understood and investigated protein, kininogen, was also estimated, in particular, in relation to contact lens wear. Difficulties arise when attempting to extract proteins from the contact lens in order to examine the individual nature of the proteins involved. These problems were partly alleviated with the use of the on-lens cell assay and a UV spectrophotometry assay, which can analyse the lens surface and bulk respectively, the latter yielding only total protein values. Various lens extraction methods were investigated to remove protein from the lens and the most efficient was employed in the analysis of lens extracts. Counter immunoelectrophoresis, an immunodiffusion assay, was then applied to the analysis of albumin, lactoferrin, IgA and IgG in the resultant eluates.

Molecular and biochemical characterization of postharvest senescence in broccoli

Postharvest senescence in broccoli (Brassica oleracea L. var Italica) florets results in phenotypic changes similar to those seen in developmental leaf senescence. To compare these two processes in more detail, we investigated molecular and biochemical changes in broccoli florets stored at two different temperatures after harvest. We found that storage at cooler temperatures delayed the symptoms of senescence at both the biochemical and gene expression levels. Changes in key biochemical components (lipids, protein, and chlorophyll) and in gene expression patterns occurred in the harvested tissue well before any visible signs of senescence were detected. Using previously identified senescence-enhanced genes and also newly isolated, differentially expressed genes, we found that the majority of these showed a similar enhancement of expression in postharvest broccoli as in developmental leaf senescence. At the biochemical level, a rapid loss of membrane fatty acids was detected after harvest, when stored at room temperature. However, there was no corresponding increase in levels of lipid peroxidation products. This, together with an increased expression of protective antioxidant genes, indicated that, in the initial stages of postharvest senescence, an orderly dismantling of the cellular constituents occurs, using the available lipid as an energy source. Postharvest changes in broccoli florets, therefore, show many similarities to the processes of developmental leaf senescence.