Monoclonal antibody-based ELISA for the quantification of nitrofuran metabolite 3-amino-2-oxazolidinone in tissues using a simplified sample preparation

A sensitive and specific monoclonal ELISA for the determination of tissue bound furazolidone metabolite 3-amino-2-oxazolidinone (AOZ) is described. The procedure enables the detection of AOZ in matrix supernatant after homogenisation, protease treatment, acid hydrolysis and derivatisation of AOZ released from the tissue by o-nitrobenzaldehyde. The formed p-nitrophenyl 3-amino-2-oxazolidinone (NPAOZ) is determined by ELISA calibrated with matrix-matched standards in the concentration range of 0.05-5.0 mu g l(-1). The assay was validated according to criteria set down by Commission Decision 2002/657/EC for the performance and validation of analytical methods for chemical residues. Detection capability, set on the basis of acceptance of no false negative results, was 0.4 mu g kg(-1) for shrimp, poultry, beef and pork muscle. This sensitivity approaches the established confirmatory LC-MS/MS able to quantify tissue-bound AOZ at levels as low as 0.3 mu g kg(-1). An excellent correlation of results obtained by ELISA and LC/MS-MS within the concentration range 0-32.1 mu g kg(-1) was found in the naturally contaminated shrimp samples (r = 0.999, n = 8). A similar con-elation was found for the incurred poultry samples within the concentration range of 0-10.5 mu g kg(-1) (r = 0.99, n = 8). (c) 2005 Elsevier B.V All rights reserved.

Asymmetric information without common priors: an indirect evolutionary analysis of quantity competition

The common prior assumption justifies private beliefs as posterior probabilities when updating a common prior based on individual information. We dispose of the common prior assumption for a homogeneous oligopoly market with uncertain costs and firms entertaining arbitrary priors about other firms' cost-type. We show that true prior beliefs can not be evolutionarily stable when truly expected profit measures (reproductive) success.

The indirect effects of biofuels and what to do about them:The case of grass biomethane and its impact on livestock

Grass biomethane surpasses the 60% greenhouse gas (GHG) savings relative to the fossil fuel replaced required by EU Directive 2009/28/EC. However, there are growing concerns that when the indirect effects of biofuels are taken into account, GHG savings may become negative. There has been no research to date into the indirect effects of grass biomethane; this paper aims to fill that knowledge gap. A causal-descriptive assessment is carried out and identifies the likely indirect effect of a grass biomethane industry in Ireland as a reduction in beef exports to the UK. Three main scenarios are then analyzed: an increase in indigenous UK beef production, an increase in beef imported to the UK from other countries (EU, New Zealand and Brazil), and a decrease in beef consumption leading to increased poultry consumption. The GHG emissions from each of these scenarios are determined and the resulting savings relative to fossil diesel vary between -636% and 102%. The significance of the findings is then discussed. It is the view of the authors that, while consideration of indirect effects is important, an Irish grass biomethane industry cannot be held accountable for the associated emissions. A global GHG accounting system is therefore proposed; however, the difficulty of implementing such a system is acknowledged, as is its probable ineffectualness. Such a system would not treat the source of the problem - rising consumption. The authors conclude that the most effective method of combating the indirect effects of biofuels is a reduction in general consumption. © 2011 Society of Chemical Industry and John Wiley & Sons, Ltd.

A simple bioanalytical method for the quantification of antiepileptic drugs in dried blood spots

An increasing number of publications on the dried blood spot (DBS) sampling approach for the quantification of drugs and metabolites have been spurred on by the inherent advantages of this sampling technique. In the present research, a selective and sensitive high-performance liquid chromatography method for the concurrent determination of multiple antiepileptic drugs (AEDs) [levetiracetam (LVT), lamotrigine (LTG), phenobarbital (PHB)], carbamazepine (CBZ) and its active metabolite carbamazepine-10,11 epoxide (CBZE)] in a single DBS has been developed and validated. Whole blood was spotted onto Guthrie cards and dried. Using a standard punch (6. mm diameter), a circular disc was punched from the card and extracted with methanol: acetonitrile (3:1, v/v) containing hexobarbital (Internal Standard) and sonicated prior to evaporation. The extract was then dissolved in water and vortex mixed before undergoing solid phase extraction using HLB cartridges. Chromatographic separation of the AEDs was achieved using Waters XBridge™ C18 column with a gradient system. The developed method was linear over the concentration ranges studied with r=0.995 for all compounds. The lower limits of quantification (LLOQs) were 2, 1, 2, 0.5 and 1. µg/mL for LVT, LTG, PHB, CBZE and CBZ, respectively. Accuracy (%RE) and precision (%CV) values for within and between day were

Quantification of interpoint topographic correlations of threshold values in glaucomatous visual fields

Purpose: The authors sought to quantify neighboring and distant interpoint correlations of threshold values within the visual field in patients with glaucoma. Methods: Visual fields of patients with confirmed or suspected glaucoma were analyzed (n = 255). One eye per patient was included. Patients were examined using the 32 program of the Octopus 1-2-3. Linear regression analysis among each of the locations and the rest of the points of the visual field was performed, and the correlation coefficient was calculated. The degree of correlation was categorized as high (r > 0.66), moderate (0.66 = r > 0.33), or low (r = 0.33). The standard error of threshold estimation was calculated. Results: Most locations of the visual field had high and moderate correlations with neighboring points and with distant locations corresponding to the same nerve fiber bundle. Locations of the visual field had low correlations with those of the opposite hemifield, with the exception of locations temporal to the blind spot. The standard error of threshold estimation increased from 0.6 to 0.9 dB with an r reduction of 0.1. Conclusion: Locations of the visual field have highest interpoint correlation with neighboring points and with distant points in areas corresponding to the distribution of the retinal nerve fiber layer. The quantification of interpoint correlations may be useful in the design and interpretation of visual field tests in patients with glaucoma.

Homogenous growth of gold nanocrystals for quantification of PSA protein biomarker

We report on another alternative sensing platform for the detection of protein biomarker (PSA–ACT complex) based on homogenous growth of Au nanocrystals in solution phase. The immuno-recognition event is translated into the gold nanoparticle growth signal which can be intuitively recognized by an unaided eye, or quantitatively measured by an UV–vis spectrophotometric analysis. Surface plasmonic signature and kinetics of the Au nanogrowth in the homogenous phase containing of HAuCl4, AA, and CTAB have also been studied to provide suitable parameters for the immunoassay. As a result, detection limit of PSA–ACT complex was determined to be 10 fM. The result indicated that this is a very sensitive, robust, simple, and economic strategy to detect protein biomarkers, and it has great potential to detect other biological interactions.

Coexpressing shRNA with fluorescence tags for quantification of cell migration studies

Understanding migration of cells has many implications in human physiology; some examples include developmental biology, healing, immune responses and tissue remodeling. On the other hand, invasive migration by tumor cells is pathological and is a major cause of mortality amongst cancer sufferers. Cell migration assays have been widely used to quantify potentially metastatic genes. In recent years, the use of RNAi has significantly increased the tools available in cell migration research due to its specific gene targeting for knockdown. The inability to ensure 100% transfection/transduction efficiency reduces the sensitivity of cell migration assays because cells not successfully transfected/transduced with the RNAi are also included in the calculations. This study introduces a different experimental setup mathematically expressed in our named normalized relative infected cell count (N-RICC) that analyses cell migration assays by co-expressing retrovirally transduced shRNA with fluorescence tags from a single vector. Vectors transduced into cells are visible under fluorescence, thus alleviating the problems involved with transduction efficiency by individually identifying cells with targeted genes. Designed shRNAs were targeted against a list of potentially metastatic genes in a highly migratory breast cancer cell line model, MDA-MB-231. We have successfully applied N-RICC analysis to show greater sensitivity of integrin alpha5 (ITGA5) and Ras homologue A (RhoA) in cell metastasis over conventional methods in scratch-wound assays and migration chambers assays.

Growth of Gold Nanocrystals Incorporated with Magnetic Microbead-based Separation as a Tool for Quantification of Biological Interactions

Au nanoparticles (AuNPs) have been widely used not only as optical labels or ‘weight” labels for the detections of biorecognition events but also an amplifier of surface plasmon resonance biosensors. The intrinsic property of gold nuclei composing of a group of Au atoms to catalyze the reduction of metal ions on the NPs and thereby to enlarge the metallic nanoparticles is employed in different biosensing paths. In a solution containing Au+ ions (e.g. HAuCl4) and the Au clusters, hydrated electrons which are reduced from oxidation of reducers (H2O2, sodium citrate, ascorbic acid, or NaBH4) will be used to reduce the Au+ ion leading to the deposition of Au+ to the Au0 (Au clusters). The reaction will be catalyzed continuously by the Au0 until the Au+ ions and hydrated electrons are exhausted. As a result, the AuNPs will be grown and their optical properties are also changed. If the AuNP nanoclusters are used as probes, the color change will be dependent on amount of analytes, thus give a quantitative monitoring of the analytes.

In this study, we incorporate the use of magnetic beads with the nanocrystalline growth to quantify a target protein based on immunoreactions. Prostate specific antigen (PSA) is chosen as the target analyte because of its values in diagnosis of prostate cancer. A double-sandwiched immunoassay is performed by gold-tagged monoclonal PSA antibody-PSA antigen – magnetic bead-tagged polyclonal PSA antibody interactions. After the immunoreactions, the target analytes are preconcentrated and separated by the magnetic beads while the nanogrowth plays a role of colorimetric signal developer.

The result shows that this is a very sensitive, robust and excellent strategy to detect biological interactions. PSA antigen is detected at femtomolar level with very high specificity under the presence of undesired proteins of crude samples. Furthermore, the method also shows great potential to detect other biological interactions. More details will be described in our presentation.

Quantification of N-(glucitol)ethanolamine and N-(carboxymethyl)serine:two products of nonenzymatic modification of aminophospholipids formed in vivo

Chemical, nonenzymatic modification of protein and lipids by reducing sugars, such as glucose, is thought to contribute to age-related deterioration in tissue protein and cellular membranes and to the pathogenesis of diabetic complications. This report describes the synthesis and quantification of N-(glucitol)ethanolamine (GE) and N-(carboxymethyl)serine (CMS), two products of nonenzymatic modification of aminophospholipids. GE is the product of reduction and hydrolysis of glycated phosphatidylethanolamine (PE), while CMS is formed through reaction of phosphatidylserine (PS) with products of oxidation of either carbohydrate (glycoxidation) or lipids (lipoxidation). Gas chromatography/mass spectrometry procedures for quantification of the N,O-acetyl methyl ester derivatives of the modified head groups were developed. GE and CMS were quantified in samples of PE and PS, respectively, following incubation with glucose in vitro; CMS formation was dependent on the presence of oxygen during the incubation. Both GE and CMS were detected and quantified in lipid extracts of human red blood cell membranes. The content of GE, but not CMS, was increased in the lipids from diabetic compared to nondiabetic subjects. Measurement of these modified lipids should prove useful for assessing the role of carbonyl-amine reactions of aminophospholipids in aging and age-related diseases.

Quantification of malondialdehyde and 4-hydroxynonenal adducts to lysine residues in native and oxidized human low-density lipoprotein

Malondialdehyde (MDA) and 4-hydroxynonenal (HNE) are major end-products of oxidation of polyunsaturated fatty acids, and are frequently measured as indicators of lipid peroxidation and oxidative stress in vivo. MDA forms Schiff-base adducts with lysine residues and cross-links proteins in vitro; HNE also reacts with lysines, primarily via a Michael addition reaction. We have developed methods using NaBH4 reduction to stabilize these adducts to conditions used for acid hydrolysis of protein, and have prepared reduced forms of lysine-MDA [3-(N epsilon-lysino)propan-1-ol (LM)], the lysine-MDA-lysine iminopropene cross-link [1,3-di(N epsilon-lysino)propane (LML)] and lysine-HNE [3-(N epsilon-lysino)-4-hydroxynonan-l-ol (LHNE)]. Gas chromatography/MS assays have been developed for quantification of the reduced compounds in protein. RNase incubated with MDA or HNE was used as a model for quantification of the adducts by gas chromatography/MS. There was excellent agreement between measurement of MDA bound to RNase as LM and LML, and as thiobarbituric acid-MDA adducts measured by HPLC; these adducts accounted for 70-80% of total lysine loss during the reaction with MDA. LM and LML (0.002-0.12 mmol/ mol of lysine) were also found in freshly isolated low-density lipoprotein (LDL) from healthy subjects. LHNE was measured in RNase treated with HNE, but was not detectable in native LDL. LM, LML and LHNE increased in concert with the formation of conjugated dienes during the copper-catalysed oxidation of LDL, but accounted for modification of <1% of lysine residues in oxidized LDL. These results are the first report of direct chemical measurement of MDA and HNE adducts to lysine residues in LDL. LM, LML and LHNE should be useful as biomarkers of lipid peroxidative modification of protein and of oxidative stress in vitro and in vivo.

Whole Slide Image Cytometry: a Novel Method for the Detection and Quantification of Abnormal DNA Contents in Barrett’s Oesophagus

Background: Barrett's oesophagus (BO) is a well recognized precursor of the majority of cases of oesophageal adenocarcinoma (OAC). Endoscopic surveillance of BO patients is frequently undertaken in an attempt to detect early OAC, high grade dysplasia (HGD) or low grade dysplasia (LGD). However histological interpretation and grading of dysplasia is subjective and poorly reproducible. The alternative flow cytometry and cytology-preparation image cytometry techniques require large amounts of tissue and specialist expertise which are not widely available for frontline health care.
Methods: This study has combined whole slide imaging with DNA image cytometry, to provide a novel method for the detection and quantification of abnormal DNA contents. 20 cases were evaluated, including 8 Barrett's specialised intestinal metaplasia (SIM), 6 LGD and 6 HGD. Feulgen stained oesophageal sections (1µm thickness) were digitally scanned in their entirety and evaluated to select regions of interests and abnormalities. Barrett’s mucosa was then interactively chosen for automatic nuclei segmentation where irrelevant cell types are ignored. The combined DNA content histogram for all selected image regions was then obtained. In addition, histogram measurements, including 5c exceeding ratio (xER-5C), 2c deviation index (2cDI) and DNA grade of malignancy (DNA-MG), were computed.
Results: The histogram measurements, xER-5C, 2cDI and DNA-MG, were shown to be effective in differentiating SIM from HGD, SIM from LGD, and LGD from HGD. All three measurements discriminated SIM from HGD cases successfully with statistical significance (pxER-5C=0.0041, p2cDI=0.0151 and pDNA-MG=0.0057). Statistical significance is also achieved differentiating SIM from LGD samples with pxER-5C=0.0019, p2cDI=0.0023 and pDNA-MG=0.0030. Furthermore the differences between LGD and HGD cases are statistical significant (pxER-5C=0.0289, p2cDI=0.0486 and pDNA-MG=0.0384).
Conclusion: Whole slide image cytometry is a novel and effective method for the detection and quantification of abnormal DNA content in BO. Compared to manual histological review, this proposed method is more objective and reproducible. Compared to flow cytometry and cytology-preparation image cytometry, the current method is low cost, simple to use and only requires a single 1µm tissue section. Whole slide image cytometry could assist the routine clinical diagnosis of dysplasia in BO, which is relevant for future progression risk to OAC.