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Item Open Access Quantitative passive soil vapor sampling for VOCs- part 2: laboratory experiments(Royal Society of Chemistry, 2014-01-24) McAlary, Todd; Groenevelt, Hester; Seethapathy, Suresh; Sacco, Paolo; Crump, Derrick; Tuday, Michael; Schumacher, Brian; Hayes, Heidi; Johnson, Paul; Gorecki, TadeuszControlled laboratory experiments were conducted to demonstrate the use of passive samplers for soil vapor concentration monitoring. Five different passive samplers were studied (Radiello, SKC Ultra, Waterloo Membrane Sampler, ATD tubes and 3M OVM 3500). Ten different volatile organic compounds were used of varying classes (chlorinated ethanes, ethanes, and methanes, aliphatics and aromatics) and physical properties (vapor pressure, solubility and sorption). Samplers were exposed in randomized triplicates to concentrations of 1, 10 and 100 ppmv, with a relative humidity of ∼80%, a temperature of ∼24 °C, and a duration of 30 minutes in a chamber with a face velocity of about 5 cm min−1. Passive samplers are more commonly used for longer sample durations (e.g., 8 hour workday) and higher face velocities (>600 cm min−1), so testing to verify the performance for these conditions was needed. Summa canister samples were collected and analyzed by EPA Method TO-15 to establish a baseline for comparison for all the passive samplers. Low-uptake rate varieties of four of the samplers were also tested at 10 ppmv under two conditions; with 5 cm min−1 face velocity and stagnant conditions to assess whether low or near-zero face velocities would result in a low bias from the starvation effect. The results indicate that passive samplers can provide concentration measurements with accuracy (mostly within a factor of 2) and precision (RSD < 15%) comparable to conventional Summa canister samples and EPA Method TO-15 analysis. Some compounds are challenging for some passive samplers because of uncertainties in the uptake rates, or challenges with retention or recovery.Item Open Access A Simple Model System Enabling Human CD34+ Cells to Undertake Differentiation Towards T Cells(Public Library of Science (PLoS), 2013-07-23T00:00:00Z) Lapenna, Antonio; B-Lynch, C.; Kapeni, C.; Aspinall, RichardChannelling the development of haematopoietic progenitor cells into T lymphocytes is dependent upon a series of extrinsic prompts whose temporal and spatial sequence is critical for a productive outcome. Simple models of human progenitor cells development depend in the main on the use of xenogeneic systems which may provide some limitations to development. Here we provide evidence that a simple model system which utilises both human keratinocyte and fibroblast cell lines arrayed on a synthetic tantalum coated matrix provides a permissive environment for the development of human CD34⁺ haematopoietic cells into mature CD4⁺ or CD8⁺ T lymphocytes in the presence of Interleukin 7 (IL-7), Interleukin 15 (IL-15) and the Fms-like tyrosine kinase 3 ligand (Flt-3L). This system was used to compare the ability of CD34(+) cells to produce mature thymocytes and showed that whilst these cells derived from cord blood were able to productively differentiate into thymocytes the system was not permissive for the development of CD34(+) cells from adult peripheral blood. Our study provides direct evidence for the capacity of human cord blood CD34(+) cells to differentiate along the T lineage in a simple human model system. Productive commitment of the CD34⁺ cells to generate T cells was found to be dependent on a three-dimensional matrix which induced the up-regulation of the Notch delta-like ligand 4 (Dll-4) by epithelial cells.Item Open Access A double-blind, placebo-controlled randomised trial evaluating the effect of a polyphenol-rich whole food supplement on PSA progression in men with prostate cancer—the UK NCRN Pomi-T study(Nature Publishing Group, 2014) Thomas, R.; Williams, M.; Sharma, H.; Chaudry, A.; Bellamy, PatriciaBACKGROUND: Polyphenol-rich foods such as pomegranate, green tea, broccoli and turmeric have demonstrated anti-neoplastic effects in laboratory models involving angiogenesis, apoptosis and proliferation. Although some have been investigated in small, phase II studies, this combination has never been evaluated within an adequately powered randomised controlled trial. METHODS: In total, 199 men, average age 74 years, with localised prostate cancer, 60% managed with primary active surveillance (AS) or 40% with watchful waiting (WW) following previous interventions, were randomised (2:1) to receive an oral capsule containing a blend of pomegranate, green tea, broccoli and turmeric, or an identical placebo for 6 months. RESULTS: The median rise in PSA in the food supplement group (FSG) was 14.7% (95% confidence intervals (CIs) 3.4–36.7%), as opposed to 78.5% in the placebo group (PG) (95% CI 48.1–115.5%), difference 63.8% (P¼0.0008). In all, 8.2% of men in the FSG and 27.7% in the PG opted to leave surveillance at the end of the intervention (w2 P¼0.014). There were no significant differences within the predetermined subgroups of age, Gleason grade, treatment category or body mass index. There were no differences in cholesterol, blood pressure, blood sugar, C-reactive protein or adverse events. CONCLUSIONS: This study found a significant short-term, favourable effect on the percentage rise in PSA in men managed with AS and WW following ingestion of this well-tolerated, specific blend of concentrated foods. Its influence on decision-making suggests that this intervention is clinically meaningful, but further trials will evaluate longer term clinical effects, and other makers of disease progression.Item Open Access Growth Of Salmonella Enteritidis And Salmonella Typhimurium In The Presence Of Quorum Sensing Signalling Compounds Produced By Spoilage And Pathogenic Bacteria(Elsevier Science B.V., Amsterdam, 2014-06-10) Dourou, Dimitra; Ammor, Mohammed Salim; Skandamis, Panagiotis N.; Nychas, George-John E.The effect of acylated homoserine lactones (AHLs) and autoinducer-2 (AI-2) signalling compounds present in the cell-free culture supernatants (CFS), of Pseudomonas aeruginosa, Yersinia enterocolitica-like GTE 112, Serratia proteamaculans 00612, Y. enterocolitica CITY650 and Y. enterocolitica CITY844, on the growth of two Salmonella Enteritidis and two S. Typhimurium strains was assessed though monitoring of changes in conductance of the medium. Detection times (Tdet), area and slope of conductance curves were recorded. Except for P. aeruginosa 108928, which was not found to produce AI-2, all other strains produced both AHLs and AI-2. Thereafter, aliquots (20% in the final volume) of these CFS were transferred into NZ Amine broth inoculated with ca. 103CFU/ml of stationary phase cultures of each Salmonella strain. While the CFS of P. aeruginosa induced a shorter detection time, i.e. acceleration of the metabolic activity, the CFS of the other microorganisms increased the detection time of Salmonella strains compared to control samples (i.e. without CFS). Results indicate that the growth of Salmonella may be affected by the presence of Quorum sensing (QS) signalling compounds and/or other novel signals existing in CFS, produced by other bacterial species and confirm the complexity of bacterial communication.Item Open Access Application of multiple response optimization design to quantum dot-encoded microsphere bioconjugates hybridization assay(Elsevier Science B.V., Amsterdam, 2011-07-01T00:00:00Z) Thiollet, Sarah; Bessant, Conrad M.; Morgan, Sarah L.The optimization of DNA hybridization for genotyping assays is a complex experimental problem that depends on multiple factors such as assay formats, fluorescent probes, target sequence, experimental conditions, and data analysis. Quantum dot-doped particle bioconjugates have been previously described as fluorescent probes to identify single nucleotide polymorphisms even though this advanced fluorescent material has shown structural instability in aqueous environments. To achieve the optimization of DNA hybridization to quantum dot-doped particle bioconjugates in suspension while maximizing the stability of the probe materials, a nonsequential optimization approach was evaluated. The design of experiment with response surface methodology and multiple optimization response was used to maximize the recovery of fluorescent probe at the end of the assay simultaneously with the optimization of target-probe binding. Hybridization efficiency was evaluated by the attachment of fluorescent oligonucleotides to the fluorescent probe through continuous flow cytometry detection. Optimal conditions were predicted with the model and tested for the identification of single nucleotide polymorphisms. The design of experiment has been shown to significantly improve biochemistry and biotechnology optimization processes. Here we demonstrate the potential of this statistical approach to facilitate the optimization of experimental protocol that involves material science and molecular biology.Item Open Access A membrane-based ELISA assay for the herbicide Isoproturon in soil samples(Taylor & Francis, 2012-01-31T00:00:00Z) Baskeyfield, Damian E. H.; Davis, Frank; Magan, Naresh; Tothill, Ibtisam E.A membrane based enzyme linked immunosorbent assay (MELISA) for the detection of a common herbicide, isoproturon is described. A heterogeneous competitive ELISA was the format chosen for isoproturon detection. An immunoassay system with a horseradish peroxidase (HRP) labeled polyclonal antibody preparation was developed and characterized before suitable sensitivity and selectivity for isoproturon were attained. After development as a microtiter plate immunoassay, the system was transferred to an affinity membrane sorbent based ELISA where the isoproturon/ovalbumin conjugate was immobilized on commercial membranes. Different porosities and immobilization conditions were utilized to optimize the MELISA, including sensitivity, selectivity, and stability studies. This enabled detection of isoproturon in the range 0.5ngml-1-20µgml-1, with an LLD90 of 0.5ngml-1. The use of acetonitrile extracts from soil samples was found to not overly impair the performance of the MELISA. Good correlation between ELISA and HPLC was obtained for extracts from spiked soil samples.Item Open Access Biochemical and textural ripeness assessment of avocado fruit from different origins(Ishs; 1999, 2010-10-06T00:00:00Z) Landahl, Sandra; Meyer, Marjolaine D.; Terry, Leon A.; Herppich, W. B.Stage of ripeness and quality of avocado is notoriously difficult to assess by conventional methods. Texture is a very important determinant of avocado fruit quality and can change radically during storage. The difficulties in determining avocado quality are related, in part, to the spatial heterogeneity of fruit characteristics during ripening. The aim of this study was to assess the discriminatory capabilities of physiochemical properties to determine ripeness of imported 'Hass' avocado fruit. Fruit were stored at 12°C and sampled at regular intervals. Using a previously unreported method, the texture of different slices from individual fruit was measured during ripening. Maximum load, elasticity and viscosity of fruit tissue were measured using a universal testing machine fitted with a 500N or 5N load cell. The same tissue slice was then further processed prior to subsequent quantification of non-structural carbohydrates (NSCs) and fatty acid methyl esters using standard high performance liquid chromatography coupled to evaporative light scattering detection and gas chromatography coupled to flame ionisation detection, respectively. Spatio-temporal differences in maximum load, elasticity, viscosity, NSCs and fatty acid composition were found in avocados from different origins. Results of measured texture and target analytes were used to differentiate avocado fruit into definable groups using partial least squares discriminant analysis.Item Open Access Textural, biochemical and micro-structural changes in mesocarp tissue of imported avocado from Peru during ripening(2010-12-31T00:00:00Z) Landahl, Sandra; Terry, Leon A.Avocado (Persea americana Mill.) fruit is a valuable product and notorious for the difficulties encountered in determining quality. Typically the degree of ripeness of many climacteric fruits, such as avocado, is measured by assessing flesh firmness. The aim of the presented work was to elucidate the temporal and spatial changes in texture, biochemistry and micro-structure in different avocado tissues from the same fruit. Fruit were first treated with ethylene and then ripened at 12°C. Samples were taken four times over 10 days storage. Maximum load and viscoelasticity of horizontally-cut slices from fruit (n=24) imported from Peru were measured during ripening. These texture parameters were measured using an Instron 5542 universal testing machine fitted with a 500 N or 5 N load cell. Non-structural carbohydrates (NSCs; viz. sucrose, mannoheptulose, perseitol) and fatty acid methyl esters from the same samples were identified and quantified using standard HPLC coupled to evaporative light scattering detection and gas chromatography coupled to flame ionisation detection, respectively. Samples taken from adjacent mesocarp tissue slices were examined: each specimen included the sclerenchymatic exocarp, fleshy mesocarp and endocarp including the coat of the seed. An analysis of variance was performed to elucidate the change of parameters during fruit ripening. NSC content and textural properties changed during ripening and showed spatial heterogeneity within individual fruit. Micro-structural changes were evident during the latter stages of ripening and coincided with fruit softening and degradation in mannoheptulose. These findings might be used to enhance quality monitoring of imported avocado fruit.Item Open Access MRMaid: the SRM assay design tool for Arabidopsis and other species(2012-07-20T00:00:00Z) Fan, Jun; Mohareb, Fady R.; Jones, Alexandra M. E.; Bessant, Conrad M.Selected reaction monitoring (SRM), sometimes called multiple reaction monitoring (MRM), is becoming the tool of choice for targeted quantitative proteomics in the plant science community. Key to a successful SRM experiment is prior identification of the distinct peptides for the proteins of interest and the determination of the so-called transitions that can be programmed into an LC-MS/MS to monitor those peptides. The transition for a given peptide comprises the intact peptide m/z and a high intensity product ion that can be monitored at a characteristic retention time (RT). To aid the design of SRM experiments, several online tools and databases have been produced to help researchers select transitions for their proteins of interest, but many of these tools are limited to the most popular model organisms such as human, yeast, and mouse or require the experimental acquisition of local spectral libraries. In this paper we present MRMaid1, a web-based SRM assay design tool whose transitions are generated by mining the millions of identified peptide spectra held in the EBI’s PRIDE database. By using data from this large public repository, MRMaid is able to cover a wide range of species that can increase as the coverage of PRIDE grows. In this paper MRMaid transitions for 25Arabidopsis thalianaproteins are evaluated experimentally, and found capable of quantifying 23 of these proteins. This performance was found to be comparable with the more time consuming approach of designing transitions using locally acquired orbitrap data, indicating that MRMaid is a valuable tool for targeted Arabidopsis proteomics.Item Open Access Phylogenetic and molecular characteristics of Eurasian H9 avian influenza viruses and their detection by two different H9-specific RealTime reverse transcriptase polymerase chain reaction tests(Elsevier Science B.V., Amsterdam., 2013-03-23T00:00:00Z) Slomka, M. J.; Hanna, A.; Mahmood, S.; Govil, J.; Krill, D.; Manvell, R. J.; Shell, W.; Arnold, M. E.; Banks, J.; Brown, I. H.Avian influenza viruses (AIVs) of the H9 haemagglutinin subtype are endemic in many Asian and Middle-East countries, causing mortality and morbidity in poultry. Consequently there is a need for accurate and sensitive detection of Eurasian H9 subtype viruses. Two H9 RealTime reverse transcriptase polymerase chain reaction (RRT-PCR) tests, developed by Monne et al. (2008) and Ben Shabat et al. (2010), were originally validated with a limited number of H9 specimens. In the present study, the two tests have been assessed using 66 diverse H9 isolates and 139 clinical specimens from six H9 poultry outbreaks in four geographically disparate Eurasian countries. The Monne et al. (2008) test was modified and successfully detected all H9 viruses from all three Eurasian H9 lineages. Bayesian analysis of the clinical specimens' results revealed this test to be more sensitive (97%) than the Ben Shabat et al. (2010) test (31%). The latter test detected most H9 isolates of the G1 lineage, but no isolates from other H9 lineages. Mismatches in the primer/probe binding sequences accounted for sensitivity differences between the two H9 RRT-PCRs. Genetic analysis of 34 sequenced H9 haemagglutinin genes showed the South Asian and Middle-East H9 isolates to belong to the H9 G1 lineage, and possessed residues that appear to preferably bind alpha 2,6-linked sialic acid receptors which indicate a potential for human infection. European H9s clustered phylogenetically in a broader geographical group that includes recent North American H9 wild bird isolates and contemporary Asian viruses in the Y439 H9 lineage.Item Open Access An in silico study of the differential effect of oxidation on two biologically relevant G-quadruplexes: Possible implications in oncogene expression(Public Library of Science (PLoS), 2012-08-22T00:00:00Z) Stebbeds, William Joshua David; Lunec, Joseph; Larcombe, L. D.G-quadruplex structures, formed from guanine rich sequences, have previously been shown to be involved in various physiological processes including cancer-related gene expression. Furthermore, G-quadruplexes have been found in several oncogene promoter regions, and have been shown to play a role in the regulation of gene expression. The mutagenic properties of oxidative stress on DNA have been widely studied, as has the association with carcinogenesis. Guanine is the most susceptible nucleotide to oxidation, and as such, G-rich sequences that form G-quadruplexes can be viewed as potential "hot-spots" for DNA oxidation. We propose that oxidation may destabilise the G-quadruplex structure, leading to its unfolding into the duplex structure, affecting gene expression. This would imply a possible mechanism by which oxidation may impact on oncogene expression. This work investigates the effect of oxidation on two biologically relevant G-quadruplex structures through 500 ns molecular dynamics simulations on those found in the promoter regions of the c-Kit and c-Myc oncogenes. The results show oxidation having a detrimental effect on stability of the structure, substantially destabilising the c-Kit quadruplex, and with a more attenuated effect on the c-Myc quadruplex. Results are suggestive of a novel route for oxidation-mediated oncogenesis and may have wider implications for genome stability.Item Open Access Experiences in Pattern Recognition for Machine Olfaction(American Institute of Physics, 2011) Bessant, Conrad M.Pattern recognition is essential for translating complex olfactory sensor responses into simple outputs that are relevant to users. Many approaches to pattern recognition have been applied in this field, including multivariate statistics (e.g. discriminant analysis), artificial neural networks (ANNs) and support vector machines (SVMs). Reviewing our experience of using these techniques with many different sensor systems reveals some useful insights. Most importantly, it is clear beyond any doubt that the quantity and selection of samples used to train and test a pattern recognition system are by far the most important factors in ensuring it performs as accurately and reliably as possible. Here we present evidence for this assertion and make suggestions for best practice based on these findings.Item Open Access MMpred: functional miRNA – mRNA interaction analyses by miRNA expression prediction(BioMed Central, 2012-11-14) Stempor, Przemyslaw A.; Cauchi, Michael; Wilson, PaulBackground: MicroRNA (miRNA) directed gene repression is an important mechanism of posttranscriptional regulation. Comprehensive analyses of how microRNA influence biological processes requires paired miRNA-mRNA expression datasets. However, a review of both GEO and ArrayExpress repositories revealed few such datasets, which was in stark contrast to the large number of messenger RNA (mRNA) only datasets. It is of interest that numerous primary miRNAs (precursors of microRNA) are known to be co-expressed with coding genes (host genes). Results: We developed a miRNA-mRNA interaction analyses pipeline. The proposed solution is based on two miRNA expression prediction methods – a scaling function and a linear model. Additionally, miRNA-mRNA anticorrelation analyses are used to determine the most probable miRNA gene targets (i.e. the differentially expressed genes under the influence of up- or down-regulated microRNA). Both the consistency and accuracy of the prediction method is ensured by the application of stringent statistical methods. Finally, the predicted targets are subjected to functional enrichment analyses including GO, KEGG and DO, to better understand the predicted interactions. Conclusions: The MMpred pipeline requires only mRNA expression data as input and is independent of third party miRNA target prediction methods. The method passed extensive numerical validation based on the binding energy between the mature miRNA and 3’ UTR region of the target gene. We report that MMpred is capable of generating results similar to that obtained using paired datasets. For the reported test cases we generated consistent output and predicted biological relationships that will help formulate further testable hypotheses.Item Open Access Removal of heavy metals using different polymer matrixes as support for bacterial immobilisation(Elsevier Science B.V., Amsterdam., 2011-07-01T00:00:00Z) Pires, Carlos; Marques, Ana P. G. C.; Guerreiro, Antonio R.; Magan, Naresh; Castro, Paula M. L.Great attention is focused on the microbial treatment of metal contaminated environments. Three bacterial strains, 1C2, 1ZP4 and EC30, belonging to genera Cupriavidus, Sphingobacterium and Alcaligenes, respectively, showing high tolerance to Zn and Cd, up to concentrations of 1000ppm, were isolated from a contaminated area in Northern Portugal. Their contribution to Zn and Cd removal from aqueous streams using immobilised alginate, pectate and a synthetic cross-linked polymer was assessed. In most cases, matrices with immobilised bacteria showed better metal removal than the non-inoculated material alone. For the immobilisation with all the polymers, 1C2 was the strain that increased the removal of Zn the most, whereas EC30 was the most promising for Cd removal, especially when combined with the synthetic polymer with up to a ca. 11-fold increase in metal removal when compared to the polymer alone. Removal of individual metals from binary mixtures showed that there was differential immobilisation. There was greater removal of Cd than Zn (removals up to 40% higher than those showed for Zn). The results show that metal contaminated environments constitute a reservoir of microorganisms resistant/tolerant to heavy metals that have the capacity to be exploited in bioremediation strategies.Capsule immobilisation of bacteria in the naturally occurring alginate and pectate and in a synthetic cross-linked polymer increased the Zn and Cd removal abilities from single and binary contaminated waters; the applications with the synthetic polymer were the most promising for Cd and Zn removal in single and binary mixtures.Item Open Access The Microbial Habitability of Weathered Volcanic Glass Inferred from Continuous Sensing Techniques(Mary Ann Leibert, 2011-09-16T00:00:00Z) Bagshaw, Elizabeth A.; Cockell, Charles S.; Magan, Naresh; Wadham, Jemma L.; Venugopalan, T.; Sun, Tong; Mowlem, Matt; Croxford, Anthony J.Basaltic glasses (hyaloclastite) are a widespread habitat for life in volcanic environments, yet their interior physical conditions are poorly characterized. We investigated the characteristics of exposed weathered basaltic glass from a surface outcrop in Iceland, using microprobes capable of continuous sensing, to determine whether the physical conditions in the rock interior are hospitable to microbial life. The material provided thermal protection from freeze-thaw and rapid temperature fluctuations, similar to data reported for other rock types. Water activity experiments showed that at moisture contents less than 13% wet weight, the glass and its weathering product, palagonite, had a water activity below levels suitable for bacterial growth. In pore spaces, however, these higher moisture conditions might be maintained for many days after a precipitation event. Gas exchange between the rock interior and exterior was rapid (<10 min) when the rocks were dry, but when saturated with water, equilibration took many hours. During this period, we demonstrated the potential for low oxygen conditions within the rock caused by respiratory stimulation of the heterotrophic community within. These conditions might exist within subglacial environments during the formation of the rocks or in micro-environments in the interior of exposed rocks. The experiments showed that microbial communities at the site studied here could potentially be active for 39% of the year, if the depth of the community within the outcrop maintains a balance between access to liquid water and adequate protection from freezing. In the absence of precipitation, the interior of weathered basaltic glass is an extreme and life-limiting environment for microorganisms on Earth and other planets. Key Words: Basaltic glass-Palagonite-Oxygen sensing-Cryptoendoliths-Life in extreme environments.Item Open Access Diversity and distribution of sulphate-reducing bacteria in human faeces from healthy subjects and patients with inflammatory bowel disease(2012-06-01T00:00:00Z) Jia, Wenjing; Whitehead, Rebekah N.; Griffiths, Lesley; Dawson, Claire; Bai, Hao; Waring, Rosemary H.; Ramsden, David B.; Hunter, John O.; Cauchi, Michael; Bessant, Conrad M.; Fowler, Dawn P.; Walton, Christopher; Turner, Claire; Cole, Jeffrey A.The relative abundance of different groups of sulphate-reducing bacteria (SRB) in faecal DNA collected before and after therapy from patients suffering from Crohn's disease (CD), irritable bowel syndrome (IBS) or ulcerative colitis (UC) has been compared with that from healthy controls. Growth tests revealed that SRB were not more abundant in samples from patients with CD before treatment than in the healthy control group. For most of the 128 samples available, these preliminary results were confirmed using degenerate PCR primers that amplify the dsrAB gene. However, some samples from patients with CD before treatment contained a growth inhibitor that was absent from IBS or UC samples. In-depth sequencing of PCR-generated dsrB fragments revealed that the diversity detected was surprisingly low, with only eight strains of SRB and the sulphite-reducing bacterium, Bilophila wadsworthia, detected above the 0.1% threshold. The proportion of the two major species detected, B.wadsworthia and Desulfovibrio piger, was as high as 93.5% of the total SRB population in the healthy control group and lower in all patient groups. Four previously undescribed species were found: it is impossible to predict whether they are sulphate or sulphite-reducing bacteria.Item Open Access Construction and interrogation of enzyme microarrays using scanning electrochemical microscopy - optimisation of adsorption and determination of enzymatic activity.(Royal Society of Chemistry, 2011-12-21T00:00:00Z) Roberts, William St John; Davis, Frank; Collyer, Stuart D.; Higson, Seamus P. J.Scanning electrochemical microscopy (SECM) has been used to image and study the catalytic activity of horseradish peroxidase (HRP) immobilised in a patterned fashion onto glass slides. Microarrays of HRP islands could be deposited on amino-modified glass slides using glutaraldehyde crosslinking combined with the SECM being used as a micro-deposition device. The enzymatic activity of the immobilised enzyme on the surface was in the presence of its substrate observed to give rise to substantial positive feedback between the slide and the SECM microelectrode tip. Conversely when either blank slides - or slides coated with HRP which had been subsequently thermally denatured were utilised, these showed negative feedback effects. Various conditions such as enzyme concentration, incubation time and substrate concentration were systematically varied to optimise sensitivity. Regular arrays of HRP could be assembled and when imaged, displayed lower limits of detection of 1.2 × 10(-12) mol ml(-1) of benzoquinone.Item Open Access A comparison of artificial neural networks and partial least squares modelling for the rapid detection of the microbial spoilage of beef fillets based on Fourier transform infrared spectral fingerprints(Elsevier Science B.V., Amsterdam, 2011-06-30T00:00:00Z) Panagou, Efstathios Z.; Mohareb, Fady R.; Argyri, Anthoula A.; Bessant, Conrad M.; Nychas, George-John E.A series of partial least squares (PLS) models were employed to correlate spectral data from FTIR analysis with beef fillet spoilage during aerobic storage at different temperatures (0, 5, 10, 15, and 20°C) using the dataset presented by Argyri etal. (2010). The performance of the PLS models was compared with a three-layer feed-forward artificial neural network (ANN) developed using the same dataset. FTIR spectra were collected from the surface of meat samples in parallel with microbiological analyses to enumerate total viable counts. Sensory evaluation was based on a three-point hedonic scale classifying meat samples as fresh, semi-fresh, and spoiled. The purpose of the modelling approach employed in this work was to classify beef samples in the respective quality class as well as to predict their total viable counts directly from FTIR spectra. The results obtained demonstrated that both approaches showed good performance in discriminating meat samples in one of the three predefined sensory classes. The PLS classification models showed performances ranging from 72.0 to 98.2% using the training dataset, and from 63.1 to 94.7% using independent testing dataset. The ANN classification model performed equally well in discriminating meat samples, with correct classification rates from 98.2 to 100% and 63.1 to 73.7% in the train and test sessions, respectively. PLS and ANN approaches were also applied to create models for the prediction of microbial counts. The performance of these was based on graphical plots and statistical indices (bias factor, accuracy factor, root mean square error). Furthermore, results demonstrated reasonably good correlation of total viable counts on meat surface with FTIR spectral data with PLS models presenting better performance indices compared to ANN.Item Open Access Modelling the relationship between environmental factors, transcriptional genes and deoxynivalenol mycotoxin production by strains of two Fusarium species(2011-01-06T00:00:00Z) Schmidt-Heydt, Markus; Parra, Roberto; Geisen, Rolf; Magan, NareshThe effect of changes in temperature/water activity (a(w)) on growth, deoxynivalenol (DON) production and trichothecene gene cluster expression (18 genes) for strains of Fusarium culmorum and Fusarium graminearum was studied. The expression data for six key transcription genes (TRI4, TRI5, TRI6, TRI10, TRI12 and TRI13) were analysed using multiple regression analyses to model the relationship between these various factors for the first time. Changes in a(w) and temperature significantly (p=0.05) affected growth and DON. Microarray data on expression of these genes were significantly related to DON production for both strains. Multi-regression analysis was done and polynomial models found to best fit the relationship between actual/predicted DON production relative to the expression of these TRI genes and environmental factors. This allowed prediction of the amounts of DON produced in two-dimensional contour maps to relate expression of these genes to either a(w) or temperature. These results suggest complex interactions between gene expression (TRI genes), environmental factors and mycotoxin production. This is a powerful tool for understanding the role of these genes in relation to environmental factors and enables more effective targeted control strategies to be developed.Item Open Access Ochratoxin A removal in synthetic media by living and heat-inactivated cells of Oenococcus oeni isolated from wines(Elsevier Science B.V., Amsterdam., 2010-01-31T00:00:00Z) Mateo, Eva M.; Medina-Vayá, Ángel; Mateo, Fernando; Valle-Algarra, Francisco M.; Pardo, Isabel; Jimenez, MisericordiaThe capacity of Oenococcus oeni to eliminate ochratoxin A (OTA) from synthetic media in different conditions was studied. Ten tested O. oeni strains removed OTA from the medium but with significant differences depending on the strain, incubation period, and initial OTA level in the medium. Mycotoxin reductions higher than 60% were recorded in 14-day cultures spiked with 2 mu g OTA/l. Toxin removal was independent of bacterial viability and culture medium composition. This is the first study carried out to study OTA removal dynamics by living and heat-inactivated cells of O. oeni. The results aim that this bacterium may be a very useful tool to control OTA in food and beverages. (C) 2009 Elsevier Ltd. All rights reserved.