Development of a minor groove binder assay for real-time one-step RT-PCR detection of swine vesicular disease virus

The design and development of a 5' conjugated minor groove binder (MGB) probe real-time RT-PCR assay are described for rapid, sensitive and specific detection of swine vesicular disease virus (SVDV) RNA. The assay is designed to target the 2C gene of the SVDV genome and is capable of detecting 2 x 10(2) copies of an RNA standard per reaction. It does not detect any of the other RNA viruses that cause vesicular disease in pigs, or the human enterovirus, Coxsackie B5 virus (CVB5) which is closely related antigenically to SVDV. The linear range of this test was from 2 x 10(2) to 2 x 10(8) copies/mu l. The assay is rapid and can detect SVDV RNA in just over 3.5 h including the time required for nucleic acid extraction. The development of this assay provides a useful tool for the differential diagnosis of SVD or for the detection of SVDV in research applications. This study demonstrates the suitability of MGB probes as a real-time PCR chemistry for the diagnosis of swine vesicular disease. (C) 2010 Elsevier B.V. All rights reserved.

Development of an Arcellacean (testate lobose amoebae) based transfer function for sedimentary phosphorous in lakes.

Arcellacea (testate lobose amoebae) communities were assessed from 73 sediment-water interface samples collected from 33 lakes in urban and rural settings within the Greater Toronto Area (GTA), Ontario, Canada, as well as from forested control areas in the Lake Simcoe area, Algonquin Park and eastern Ontario. The results were used to: (1) develop a statistically rigorous arcellacean-based training set for sedimentary phosphorus (Olsen P (OP)) loading; and (2) derive a transfer function to reconstruct OP levels during the post-European settlement era (AD1870s onward) using a chronologically well-constrained core from Haynes Lake on the environmentally sensitive Oak Ridges Moraine, within the GTA. Ordination analysis indicated that OP most influenced arcellacean assemblages, explaining 6.5% (p < 0.005) of total variance. An improved training set where the influence of other important environmental variables (e.g. total organic carbon, total nitrogen, Mg) was reduced, comprised 40 samples from 31 lakes, and was used to construct a transfer function for lacustrine arcellaceans for sedimentary phosphorus (Olsen P) using tolerance downweighted weighted averaging (WA-Tol) with inverse deshrinking (RMSEPjack-77pp; r2jack = 0.68). The inferred reconstruction indicates that OP levels remained near pre-settlement background levels from settlement in the late AD 1970s through to the early AD 1970s. Since OP runoff from both forests and pasture is minimal, early agricultural land use within the lake catchment was as most likely pasture and/or was used to grow perennial crops such as Timothy-grass for hay. A significant increase in inferred OP concentration beginning ~ AD 1972 may have been related to a change in crops (e.g. corn production) in the catchment resulting in more runoff, and the introduction of chemical fertilizers. A dramatic decline in OP after ~ AD 1985 probably corresponds to a reduction in chemical fertilizer use related to advances in agronomy, which permitted a more precise control over required fertilizer application. Another significant increase in OP levels after ~ AD 1995 may have been related to the construction of a large golf course upslope and immediately to the north of Haynes Lake in AD 1993, where significant fertilizer use is required to maintain the fairways. These results demonstrate that arcellaceans have great potential for reconstructing lake water geochemistry and will complement other proxies (e.g. diatoms) in paleolimnological research.

Development of primary human nasal epithelial cell cultures for the study of cystic fibrosis pathophysiology

Cultured primary epithelial cells are used to examine inflammation in cystic fibrosis (CF). We describe a new human model system using cultured nasal brushings. Nasal brushings were obtained from 16 F508del homozygous patients and 11 healthy controls. Cells were resuspended in airway epithelial growth medium and seeded onto collagen-coated flasks and membranes for use in patch-clamp, ion transport, and mediator release assays. Viable cultures were obtained with a 75% success rate from subjects with CF and 100% from control subjects. Amiloride-sensitive epithelial Na channel current of similar size was present in both cell types while forskolin-activated CF transmembrane conductance regulator current was lacking in CF cells. In Ussing chambers, cells from CF patients responded to UTP but not to forskolin. Spontaneous and cytomix-stimulated IL-8 release was similar (stimulated 29,448 ± 9,025 pg/ml; control 16,336 ± 3,308 pg/ml CF; means ± SE). Thus nasal epithelial cells from patients with CF can be grown from nasal brushings and used in electrophysiological and mediator release studies in CF research.

Development of a solid-phase extraction coupling chemiluminescent enzyme immunoassay for determination of organophosphorus pesticides in environmental water samples

Solid-phase extraction (SPE) and direct competitive chemiluminescence enzyme immunoassay (dcCL-EIA) were combined for the detection of organophosphorus pesticides (OPs) in environmental water samples. dcCL-EIA based on horseradish peroxidase labeled with a broad-specificity monoclonal antibody against OPs was developed, and the effects of several physicochemical parameters on dcCL-EIA performance were studied. SPE was used for the pretreatment of water samples to remove interfering substances and to concentrate the OP analytes. The coupling of SPE and dcCL-EIA can detect seven OPs (parathion, coumaphos, phoxim, quinalphos, triazophos, dichlofenthion, and azinphos-ethyl) with the limit of quantitation below 0.1 ng/mL. The recoveries of OPs from spiked water samples ranged from 62.5% to 131.7% by SPE-dcCL-EIA and 69.5% to 112.3% by SPE-HPLC-MS/MS. The screening of OP residues in real-world environmental water samples by the developed SPE-dcCL-EIA and their confirmatory analysis using SPE-HPLC-MS/MS demonstrated that the assay is ideally suited as a monitoring method for OP residues prior to chromatographic analysis.

Development of a five-plex flow cytometric immunoassay for the simultaneous detection of six coccidiostats in feed and eggs

Coccidiostats are the only veterinary drugs still permitted to be used as feed additives to treat poultry for coccidiosis. To protect consumers, maximum levels for their presence in food and feed have been set by the European Union (EU). To monitor these coccidiostats, a rapid and inexpensive screening method would be a useful tool. The development of such a screening method, using a flow cytometry-based immunoassay, is described. The assay uses five sets of colour-coded paramagnetic microspheres for the detection of six selected priority coccidiostats. Different coccidiostats, with and without carrier proteins, were covalently coupled onto different bead sets and tested in combination with polyclonal antisera and with a fluorescent-labelled secondary antibody. The five optimal combinations were selected for this multiplex and a simple-to-use sample extraction method was applied for screening blank and spiked eggs and feed samples. A very good correlation (r ranging from 0.995 to 0.999) was obtained with the responses obtained in two different flow cytometers (Luminex 100 and FLEXMAP 3D). The sensitivities obtained were in accordance with the levels set by the EU as the measured limits of detection for narasin/salinomycin, lasalocid, diclazuril, nicarbazin (4,4'-dinitrocarbanilide) and monensin in eggs were 0.01, 0.1, 0.5, 53 and 0.1 µg/kg and in feed 0.1, 0.2, 0.3, 9 and 1.5 µg/kg, respectively.

The development of effective biomarkers for Alzheimer's disease:a review

OBJECTIVE: There is a widely recognised need to develop effective Alzheimer's disease (AD) biomarkers to aid the development of disease-modifying treatments, to facilitate early diagnosis and to improve clinical care. This overview aims to summarise the utility of key neuroimaging and cerebrospinal fluid (CSF) biomarkers for AD, before focusing on the latest efforts to identify informative blood biomarkers. DESIGN: A literature search was performed using PubMed up to September 2011 for reviews and primary research studies of neuroimaging (magnetic resonance imaging, magnetic resonance spectroscopy, positron emission tomography and amyloid imaging), CSF and blood-based (plasma, serum and platelet) biomarkers in AD and mild cognitive impairment. Citations within individual articles were examined to identify additional studies relevant to this review. RESULTS: Evidence of AD biomarker potential was available for imaging techniques reflecting amyloid burden and neurodegeneration. Several CSF measures are promising, including 42 amino acid ß-amyloid peptide (Aß(42) ); total tau (T-tau) protein, reflecting axonal damage; and phosphorylated tau (P-tau), reflecting neurofibrillary tangle pathology. Studies of plasma Aß have produced inferior diagnostic discrimination. Alternative plasma and platelet measures are described, which represent potential avenues for future research. CONCLUSIONS: Several imaging and CSF markers demonstrate utility in predicting AD progression and determining aetiology. These require standardisation before forming core elements of diagnostic criteria. The enormous potential available for identifying a minimally-invasive, easily-accessible blood measure as an effective AD biomarker currently remains unfulfilled. Copyright © 2012 John Wiley &amp; Sons, Ltd.

Development of the vitellaria of the liver fluke, Fasciola hepatica in the rat host

The development of the vitellaria of Fasciola hepatica within the liver of its rat host was studied by means of whole-mount stained preparations and transmission electron microscopy, together with light and electron immunocytochemistry using an antibody to vitelline protein B, an eggshell precursor protein synthesized by F. hepatica. No vitelline cells could be identified in flukes recovered from the liver parenchyma, by any of the methods used. In contrast, follicles were present in flukes at the earliest time of recovery from the bile duct, namely, 5 weeks 3 days post-infection. The vitellaria in these flukes formed a row of small follicles on either side of the body. Development of the follicles was rapid: by 6 weeks 3 days, the vitellaria resembled those in the adult fluke and eggs were present in the uterus. Immunolabelling was confined to the shell protein globules in the vitelline cells, confirming the packaging of the eggshell protein within the shell globule clusters.

Development of an in vitro model for study of the efficacy of ischemic preconditioning in human skeletal muscle against ischemia-reperfusion injury

Ischemia-reperfusion (I/R) injury causes skeletal muscle infarction and ischemic preconditioning (IPC) augments ischemic tolerance in animal models. To date, this has not been demonstrated in human skeletal muscle. This study aimed to develop an in vitro model to investigate the efficacy of simulated IPC in human skeletal muscle. Human skeletal muscle strips were equilibrated in oxygenated Krebs-Henseleit-HEPES buffer (37 degrees C). Aerobic and reperfusion phases were simulated by normoxic incubation and reoxygenation, respectively. Ischemia was simulated by hypoxic incubation. Energy store, cell viability, and cellular injury were assessed using ATP, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), and lactate dehydrogenase (LDH) assays, respectively. Morphological integrity was assessed using electron microscopy. Studies were designed to test stability of the preparation (n = 5-11) under normoxic incubation over 24 h; the effect of 1, 2, 3, 4, or 6 h hypoxia followed by 2 h of reoxygenation; and the protective effect of hypoxic preconditioning (HPC; 5 min of hypoxia/5 min of reoxygenation) before 3 h of hypoxia/2 h of reoxygenation. Over 24 h of normoxic incubation, muscle strips remained physiologically intact as assessed by MTT, ATP, and LDH assays. After 3 h of hypoxia/2 h of reoxygenation, MTT reduction levels declined to 50.1 +/- 5.5% (P <0.05). MTT reduction levels in HPC (82.3 +/- 10.8%) and normoxic control (81.3 +/- 10.2%) groups were similar and higher (P <0.05) than the 3 h of hypoxia/2 h of reoxygenation group (45.2 +/- 5.8%). Ultrastructural morphology was preserved in normoxic and HPC groups but not in the hypoxia/reoxygenation group. This is the first study to characterize a stable in vitro model of human skeletal muscle and to demonstrate a protective effect of HPC in human skeletal muscle against hypoxia/reoxygenation-induced injury.

Development of a rapid low cost fluorescent biosensor for the detection of food contaminants

<p>A prototype fluorescent based biosensor has been developed for the antibody based detection of food related contaminants. Its performance was characterised and showed a typical antibody binding signal of 200-2000 mV, a short term noise of 9.1 mV, and baseline slope of -0.016 mV/s over 4 h. Bulk signal detection repeatability (n=23) and reproducibility (n=3) were less than 2.4%CV. The biosensor detection unit was evaluated using two food related model systems proving its ability to monitor both binding using commercial products and inhibition through the development of an assay. This assay development potential was evaluated by observing the biosensor's performance whilst appraising several labelled antibody and glass slide configurations. The molecular interaction between biotin and an anti-biotin antibody was shown to be inhibited by 41% due to the presence of biotin in a sample. A food toxin (domoic acid) calibration curve was produced, with %CVs ranging from 2.7 to 7.8%, and a midpoint of approximately 17 ng/ml with further optimisation possible. The ultimate aim of this study was to demonstrate the working principles of this innovative biosensor as a potential portable tool with the opportunity of interchangeable assays. The biosensor design is applicable for the requirements of routine food contaminant analysis, with respect to performance, functionality and cost. (C) 2012 Elsevier B.V. All rights reserved.p>

Influence of a boundary on the development of a propeller wash

When vessels operate within harbours or over a density interface in an estuary, the seabed or interface may be close to the tip of the propeller blades. The presence of this boundary will have an effect on the propeller wash and this can affect the erosion of the boundary. The influence of such a boundary on the characteristics of a propeller wash was studied in experiments using a horizontal fixed boundary to confine a propeller jet. Detailed velocity measurements within the jet were obtained using a 3D Particle Image Velocimetry (PIV) system. The bottom stream of a propeller jet was found to expand at a faster rate due to the reduction in pressure beneath the jet caused by the suppression of the replacement fluid. The boundary was found to significantly increase the axial velocities close to it, and reduce the rate of decay of the maximum axial velocity due to the confinement, reducing the height of the jet. Three zones within the propeller wash were identified, the first being before the jet impacted the boundary, the second in which the boundary layer developed at the fixed boundary, followed by a fully developed boundary layer region. Predictive equations to estimate the influence of the boundary have been developed and are presented.

Development of a bovine collagen-apatitic calcium phosphate cement for potential fracture treatment through vertebroplasty

The aim of this study was to examine the potential of incorporating bovine fibres as a means of reinforcing a typically brittle apatite calcium phosphate cement for vertebroplasty. Type I collagen derived from bovine Achilles tendon was ground cryogenically to produce an average fibre length of 0.96 ± 0.55 mm and manually mixed into the powder phase of an apatite-based cement at 1, 3 or 5 wt.%. Fibre addition of up to 5 wt.% had a significant effect (P = 0.001) on the fracture toughness, which was increased by 172%. Adding =1 wt.% bovine collagen fibres did not compromise the compressive properties significantly, however, a decrease of 39-53% was demonstrated at =3 wt.% fibre loading. Adding bovine collagen to the calcium phosphate cement reduced the initial and final setting times to satisfy the clinical requirements stated for vertebroplasty. The cement viscosity increased in a linear manner (R = 0.975) with increased loading of collagen fibres, such that the injectability was found to be reduced by 83% at 5 wt.% collagen loading. This study suggests for the first time the potential application of a collagen-reinforced calcium phosphate cement as a viable option in the treatment of vertebral fractures, however, issues surrounding efficacious cement delivery need to be addressed. © 2012 Acta Materialia Inc.

Development of calcium phosphate cement for the augmentation of traumatically fractured porcine specimens using vertebroplasty

The study aim was to develop and apply an experimental technique to determine the biomechanical effect of polymethylmethacrylate (PMMA) and calcium phosphate (CaP) cement on the stiffness and strength of augmented vertebrae following traumatic fracture. Twelve burst type fractures were generated in porcine three-vertebra segments. The specimens were randomly split into two groups (n=6), imaged using microCT and tested under axial loading. The two groups of fractured specimens underwent a vertebroplasty procedure, one group was augmented with CaP cement designed and developed at Queen's University Belfast. The other group was augmented with PMMA cement (WHW Plastics, Hull, UK). The specimens were imaged and re-tested . An intact single vertebra specimen group (n=12) was also imaged and tested under axial loading. A significant decrease (p<0.01) was found between the stiffness of the fractured and intact groups, demonstrating that the fractures generated were sufficiently severe, to adversely affect mechanical behaviour. Significant increase (p<0.01) in failure load was found for the specimen group augmented with the PMMA cement compared to the pre-augmentation group, conversely, no significant increase (p<0.01) was found in the failure load of the specimens augmented with CaP cement, this is attributed to the significantly (p<0.05) lower volume of CaP cement that was successfully injected into the fracture, compared to the PMMA cement. The effect of the percentage of cement fracture fill, cement modulus on the specimen stiffness and ultimate failure load could be investigated further by using the methods developed within this study to test a more injectable CaP cement.