Use of a novel micro-fluidic device to create arrays for multiplex analysis of large and small molecular weight compounds by surface plasmon resonance

There is an increasing demand to develop biosensor monitoring devices capable of biomarker profiling for predicting animal adulteration and detecting multiple chemical contaminants or toxins in food produce. Surface plasmon resonance (SPR) biosensors are label free detection systems that monitor the binding of specific biomolecular recognition elements with binding partners. Essential to this technology are the production of biochips where a selected binding partner, antibody, biomarker protein or low molecular weight contaminant, is immobilised. A micro-fluidic immobilisation device allowing the covalent attachment of up to 16 binding partners in a linear array on a single surface has been developed for compatibility with a prototype multiplex SPR analyser.

The immobilisation unit and multiplex SPR analyser were respectively evaluated in their ability to be fit-for-purpose for binding partner attachment and detection of high and low molecular weight molecules. The multiplexing capability of the dual technology was assessed using phycotoxin concentration analysis as a model system. The parent compounds of four toxin groups were immobilised within a single chip format and calibration curves were achieved. The chip design and SPR technology allowed the compartmentalisation of the binding interactions for each toxin group offering the added benefit of being able to distinguish between toxin families and perform concentration analysis. This model is particularly contemporary with the current drive to replace biological methods for phycotoxin screening.

SOCS2 regulates T helper type 2 differentiation and the generation of type 2 allergic responses

The incidence of allergy and asthma in developed countries is on the increase and this trend looks likely to continue. CD4(+) T helper 2 (Th2) cells are major drivers of these diseases and their commitment is controlled by cytokines such as interleukin 4, which are in turn regulated by the suppressor of cytokine signaling (SOCS) proteins. We report that SOCS2(-/-) CD4(+) T cells show markedly enhanced Th2 differentiation. SOCS2(-/-) mice, as well as RAG1(-/-) mice transferred with SOCS2(-/-) CD4(+) T cells, exhibit elevated type 2 responses after helminth antigen challenge. Moreover, in in vivo models of atopic dermatitis and allergen-induced airway inflammation, SOCS2(-/-) mice show significantly elevated IgE, eosinophilia, type 2 responses, and inflammatory pathology relative to wild-type mice. Finally, after T cell activation, markedly enhanced STAT6 and STAT5 phosphorylation is observed in SOCS2(-/-) T cells, whereas STAT3 phosphorylation is blunted. Thus, we provide the first evidence that SOCS2 plays an important role in regulating Th2 cell expansion and development of the type 2 allergic responses.

Advances in biosensor-based analysis for antimicrobial residues in foods

Biosensors are used for a large number of applications within biotechnology, including the pharmaceutical industry and life sciences. Since the production of Biacore surface-plasmon resonance instruments in the early 1990s, there has been steadily growing use of this technology for the detection of food contaminants (e.g., veterinary drugs, mycotoxins, marine toxins, food dyes and processing contaminants). Other biosensing technologies (e.g., electrochemical and piezoelectric) have also been employed for the analysis of small-molecule contaminants. This review concentrates on recent advances made in detection and quantification of antimicrobial compounds with different types of biosensors and on the emergence of multiplexing, which is highly desirable as it increases sample analysis at lower cost and in less time. (C) 2010 Elsevier Ltd. All rights reserved.

The relationship between cement fatigue damage and implant surface finish in proximal femoral prostheses

The majority of cemented femoral hip replacements fail as a consequence of loosening. One design feature that may affect loosening rates is implant surface finish. To determine whether or not surface finish effects fatigue damage accumulation in a bone cement mantle, we developed an experimental model of the implanted proximal femur that allows visualisation of damage growth in the cement layer. Five matt surface and five polished surface stems were tested. Pre-load damage and damage after two million cycles was measured. Levels of pre-load (shrinkage) damage were the same for both matt and polished stems; furthermore damage for matt vs. polished stems was not significantly different after two million cycles. This was due to the large variability in damage accumulation rates. Finite element analysis showed that the stress is higher for the polished (assumed debonded) stem, and therefore we must conclude that either the magnitude of the stress increase is not enough to appreciably increase the damage accumulation rate or, alternatively, the polished stem does not debond immediately from the cement. Significantly (P = 0.05) more damage was initiated in the lateral cement compared to the medial cement for both kinds of surface finish. It was concluded that, despite the higher cement stresses with debonded stems, polished prostheses do not provoke the damage accumulation failure scenario. (C) 2003 IPEM. Published by Elsevier Ltd. All rights reserved.

Development and validation of a fast monoclonal based disequilibrium enzyme-linked immunosorbent assay for the detection of triphenylmethane dyes and their metabolites in fish

Malachite Green (MG), Crystal Violet (CV) and Brilliant Green (BC) are antibacterial, antifungal and antiparasitic agents that have been used for treatment and prevention of diseases in fish. These dyes are metabolized into reduced leuco forms (LMG, LCV, LBG) that can be present in fish muscles for a long period. Due to the carcinogenic properties they are banned for use in fish for human consumption in many countries including the European Union and the United States. HPLC and LC-MS techniques are generally used for the detection of these compounds and their metabolites in fish. This study presents the development of a fast enzyme-linked immunosorbent assay (ELISA) method as an alternative for screening purposes. A first monoclonal cell line producing antibodies to MG was generated using a hybridoma technique. The antibody had good cross-reactivates with related chromatic forms of triphenylmethane dyes such as CV, BC, Methyl Green, Methyl Violet and Victoria Blue R. The monoclonal antibody (mAb) was used to develop a fast (20 min) disequilibrium ELISA screening method for the detection of triphenylmethanes in fish. By introducing an oxidation step with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) during sample extraction the assay was also used to detect the presence of the reduced metabolites of triphenylmethanes. The detection capability of the assay was 1 ng g(-1) for MG, LMG, CV, LCV and BC which was below the minimum required performance limit (MRPL) for the detection method of total MG (sum of MG and LMG) set by the Commission Decision 2004/25/EC (2 ng g(-1)). The mean recoveries for fish samples spiked at 0.5 MRPL and MRPL levels with MG and LMG were between 74.9 and 117.0% and inter- and intra-assay coefficients of variation between 4.7 and 25.7%. The validated method allows the analysis of a batch of 20 samples in two to three hours. Additionally, this procedure is substantially faster than other ELISA methods developed for MG/LMG thus far. The stable and efficient monoclonal cell line obtained is an unlimited source of sensitive and specific antibody to MG and other triphenylmethanes. (C) 2011 Elsevier B.V. All rights reserved.

Endocrine disrupting effects of zearalenone, alpha- and beta-zearalenol at the level of nuclear receptor binding and steroidogenesis.

The mycotoxin zearalenone (ZEN) is a secondary metabolite of fungi which is produced by certain species of the genus Fusarium and can occur in cereals and other plant products. Reporter gene assays incorporating natural steroid receptors and the H295R steroidogenesis assay have been implemented to assess the endocrine disrupting activity of ZEN and its metabolites -zearalenol (-ZOL) and -zearalenol (-ZOL). -ZOL exhibited the strongest estrogenic potency (EC50 0.022 ± 0.001 nM), slightly less potent than 17- estradiol (EC50 0.015 ± 0.002 nM). ZEN was ~70 times less potent than -ZOL and twice as potent as -ZOL. Binding of progesterone to the progestagen receptor was shown to be synergistically increased in the presence of ZEN, -ZOL or -ZOL. ZEN, -ZOL or -ZOL increased production of progesterone, estradiol, testosterone and cortisol hormones in the H295R steroidogenesis assay, with peak productions at 10 M. At 100 M, cell viability decreased and levels of hormones were significantly reduced except for progesterone. -ZOL increased estradiol concentrations more than -ZOL or ZEN, with a maximum effect at 10 M, with -ZOL (562 ± 59 pg/ml) > -ZOL (494 ± 60 pg/ml) > ZEN (375 ± 43 pg/ml). The results indicate that ZEN and its metabolites can act as potential endocrine disruptors at the level of nuclear receptor signalling and by altering hormone production.

In Vitro and In Vivo Effects of Natural Putative Secretagogues of Glucagon-Like Peptide-1 (GLP-1)

Glucagon-like peptide-1 (GLP-1) is an intestinal hormone with well-established glucose-lowering activity. The in vitro and in vivo actions of natural putative secretagogues of GLP-1 were investigated. The acute GLP-1 releasing activity of olive leaf extract (OLE), glutamine (GLN), alpha casein (ACAS), beta casein (BCAS) and chlorogenic acid (CGA) were assessed in STC-1 cells and C57BL/6 mice. All compounds except ACAS significantly increased acute in vitro GLP-1 secretion (66-386%; P

Holding eternity in an hour: A practical exploration of the arts in the health care of older people with dementia

This project involved creative artists working with older people with dementia and staff from two Belfast Health and Social Care Trust supported housing centres in a mixed programme of dance, painting, music and drama which culminated in an open workshop with relatives and friends of the tenants. The study steered away from traditional medical models of art/music/dance therapy where the participant is perceived as a ‘patient’ in favour of identifying the participant as a ‘student’ who avails of a life-long learning experience. A key premise was that access to the arts is a human right, especially in the context of advancing age and cognitive impairment. . According to one the tenants of Mullan Mews, the project served to ‘awaken - or reawaken - folk with dementia to the endless vista of possibility already in their lives if they will only look for it’. A phenomenographic analysis of video data generated by the project emphasises the importance of the individual experiences of participants in the programme. The evidence from these storylines gained strength from the development of a documentary-style film text that has proved successful in capturing and translating the live experience of the project participants into a supportive text that goes beyond the written word.

The potential for human exposure, direct and indirect, to the suspected carcinogenic triphenylmethane dye Brilliant Green from green paper towels

Triphenylmethanes - Malachite Green (MG), Crystal Violet (CV) and Brilliant Green (BC) are dyes with known genotoxic and carcinogenic properties. Apart from being illegally used in aquaculture for treatment of fish diseases they are also applied in industry such as paper production to colour paper towels widely used in hospitals, factories and other locations for hand drying after washing. The present study provides evidence that the triphenylmethane dye (BC) present in green paper towels can migrate through the skin even when the exposure time is short (30-300 s). The transfer of the dye from the towel to food (fish) was also studied and a high amount of colour was found to migrate during overnight exposure. The risk to humans associated with these two dye transfer studies was assessed using a 'margin of exposure approach' on the basis of the toxicological data available for the closely related dye MG and its metabolite Leucomalachite Green. The data indicated that the risk associated with the use of triphenylmethane containing paper towels is of a similar proportion to the risk associated with consumption of fish contaminated with these dyes due to the illegal application in aquaculture. (C) 2011 Elsevier Ltd. All rights reserved.

Development of a simple gel permeation clean-up procedure coupled to a rapid disequilibrium enzyme-linked immunosorbent assay (ELISA) for the detection of Sudan I dye in spices and sauces

Sudan dyes have been found to be added to chilli and chilli products for illegal colour enhancement purposes. Due to the possible carcinogenic effect, they are not authorized to be used in food in the European Union or the USA. However, over the last few years, many products imported from Asian and African countries have been reported via the Rapid Alert System for Food and Feed in the European Union to be contaminated with these dyes. In order to provide fast screening method for the detection of Sudan I (SI), which is the most widely abused member of Sudan dyes family, a unique (20 min without sample preparation) direct disequilibrium enzyme-linked immunosorbent assay (ELISA) was developed. The assay was based on polyclonal antibodies highly specific to SI. A novel, simple gel permeation chromatography clean-up method was developed to purify extracts from matrices containing high amounts of fat and natural pigments, without the need for a large dilution of the sample. The assay was validated according to the Commission Decision 2002/657/EC criteria. The detection capability was determined to be 15 ng g(-1) in sauces and 50 ng g(-1) in spices. The recoveries found ranged from 81% to 116% and inter- and intra-assay coefficients of variation from 6% to 20%. The assay was used to screen a range of products (85 samples) collected from different retail sources within and outside the European Union. Three samples were found to contain high amounts (1,649, 722 and 1,461 ng g(-1)) of SI by ELISA. These results were confirmed by liquid chromatography-tandem mass spectrometry method. The innovative procedure allows for the fast, sensitive and high throughput screening of different foodstuffs for the presence of the illegal colorant SI.