Fluorescent microplate-based analysis of protein-DNA interactions I:immobilized protein

A simple protein-DNA interaction analysis has been developed using a high-affinity/high-specificity zinc finger protein. In essence, purified protein samples are immobilized directly onto the surface of microplate wells, and fluorescently labeled DNA is added in solution. After incubation and washing, bound DNA is detected in a standard microplate reader. The minimum sensitivity of the assay is approximately 0.2 nM DNA. Since the detection of bound DNA is noninvasive and the protein-DNA interaction is not disrupted during detection, iterative readings may be taken from the same well, after successive alterations in interaction conditions, if required. In this respect, the assay may therefore be considered real time and permits appropriate interaction conditions to be determined quantitatively. The assay format is ideally suited to investigate the interactions of purified unlabeled DNA binding proteins in a high-throughput format.

Fluorescent microplate-based analysis of protein-DNA interactions II:immobilized DNA

A simple protein-DNA interaction analysis has been developed using both a high-affinity/high-specificity zinc finger protein and a low-specificity zinc finger protein with nonspecific DNA binding capability. The latter protein is designed to mimic background binding by proteins generated in randomized or shuffled gene libraries. In essence, DNA is immobilized onto the surface of microplate wells via streptavidin capture, and green fluorescent protein (GFP)-labeled protein is added in solution as part of a crude cell lysate or protein mixture. After incubation and washing, bound protein is detected in a standard microplate reader. The minimum sensitivity of the assay is approximately 0.4 nM protein. The assay format is ideally suited to investigate the interactions of DNA binding proteins from within crude cell extracts and/or mixtures of proteins that may be encountered in protein libraries generated by codon randomization or gene shuffling.

Studies of recognition and selectivity by fluorescent polymers imprinted with N1-benzylidene pyridine-2-carboxamidrazones

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Market orientation of service networks:direct and indirect effects on sustained competitive advantage

Market orientation is an organization-wide concept that helps explain sustained competitive advantage (SCA). Since networks become ever more important, especially in the service sector, there is need to expand the concept of MO to a network setting. In line with Narver and Slater (1990), the concept of Market Orientation of Networks (MONW) is developed. This study indicates how MONW relates to the resource-based view (RBV) of the firm and the industrial organization (IO) view in explaining SCA. It is argued that MONW has direct and indirect effects on SCA. More precisely, the antecedent effect of MONW to resources and industry structure is considered.

Performance, emission and combustion characteristics of an indirect injection (IDI) multi-cylinder compression ignition (CI) engine operating on neat jatropha and karanj oils preheated by jacket water

Renewable non-edible plant oils such as jatropha and karanj have potential to substitute fossil diesel fuels in CI engines. A multi-cylinder water cooled IDI type CI engine has been tested with jatropha and karanj oils and comparisons made against fossil diesel. The physical and chemical properties of the three fuels were measured to investigate the suitability of jatropha and karanj oils as fuels for CI engines. The engine cooling water circuit and fuel supply systems were modified such that hot jacket water preheated the neat plant oil prior to injection. Between jatropha and karanj there was little difference in the performance, emission and combustion results. Compared to fossil diesel, the brake specific fuel consumption on volume basis was around 3% higher for the plant oils and the brake thermal efficiency was almost similar. Jatropha and karanj operation resulted in higher CO 2 and NO x emissions by 7% and 8% respectively, as compared to diesel. The cylinder gas pressure diagram showed stable engine operation with both plant oils. At full load, the plant oils gave around 3% higher peak cylinder pressure than fossil diesel. With the plant oils, cumulative heat release was smaller at low load and almost similar at full load, compared to diesel. At full load, the plant oils exhibited 5% shorter combustion duration. The study concludes that the IDI type CI engine can be efficiently operated with neat jatropha (or karanj) oil preheated by jacket water, after small modifications of the engine cooling and fuel supply circuits. © 2012 Elsevier Ltd.

Experimental investigation of performance, emission and combustion characteristics of an indirect injection multi-cylinder CI engine fuelled by blends of de-inking sludge pyrolysis oil with biodiesel

De-inking sludge can be converted into useful forms of energy to provide economic and environmental benefits. In this study, pyrolysis oil produced from de-inking sludge through an intermediate pyrolysis technique was blended with biodiesel derived from waste cooking oil, and tested in a multi-cylinder indirect injection type CI engine. The physical and chemical properties of pyrolysis oil and its blends (20 and 30 vol.%) were measured and compared with those of fossil diesel and pure biodiesel (B100). Full engine power was achieved with both blends, and very little difference in engine performance and emission results were observed between 20% and 30% blends. At full engine load, the brake specific fuel consumption on a volume basis was around 6% higher for the blends when compared to fossil diesel. The brake thermal efficiencies were about 3-6% lower than biodiesel and were similar to fossil diesel. Exhaust gas emissions of the blends contained 4% higher CO2 and 6-12% lower NOx, as compared to fossil diesel. At full load, CO emissions of the blends were decreased by 5-10 times. The cylinder gas pressure diagram showed stable engine operation with the 20% blend, but indicated minor knocking with 30% blend. Peak cylinder pressure of the 30% blend was about 5-6% higher compared to fossil diesel. At full load, the peak burn rate of combustion from the 30% blend was about 26% and 12% higher than fossil diesel and biodiesel respectively. In comparison to fossil diesel the combustion duration was decreased for both blends; for 30% blend at full load, the duration was almost 12% lower. The study concludes that up to 20% blend of de-inking sludge pyrolysis oil with biodiesel can be used in an indirect injection CI engine without adding any ignition additives or surfactants.

Synthesis and characterization of dual-functionalized core-shell fluorescent microspheres for bioconjugation and cellular delivery

The efficient transport of micron-sized beads into cells, via a non-endocytosis mediated mechanism, has only recently been described. As such there is considerable scope for optimization and exploitation of this procedure to enable imaging and sensing applications to be realized. Herein, we report the design, synthesis and characterization of fluorescent microsphere-based cellular delivery agents that can also carry biological cargoes. These core-shell polymer microspheres possess two distinct chemical environments; the core is hydrophobic and can be labeled with fluorescent dye, to permit visual tracking of the microsphere during and after cellular delivery, whilst the outer shell renders the external surfaces of the microspheres hydrophilic, thus facilitating both bioconjugation and cellular compatibility. Cross-linked core particles were prepared in a dispersion polymerization reaction employing styrene, divinylbenzene and a thiol-functionalized co-monomer. These core particles were then shelled in a seeded emulsion polymerization reaction, employing styrene, divinylbenzene and methacrylic acid, to generate orthogonally functionalized core-shell microspheres which were internally labeled via the core thiol moieties through reaction with a thiol reactive dye (DY630-maleimide). Following internal labeling, bioconjugation of green fluorescent protein (GFP) to their carboxyl-functionalized surfaces was successfully accomplished using standard coupling protocols. The resultant dual-labeled microspheres were visualized by both of the fully resolvable fluorescence emissions of their cores (DY630) and shells (GFP). In vitro cellular uptake of these microspheres by HeLa cells was demonstrated conventionally by fluorescence-based flow cytometry, whilst MTT assays demonstrated that 92% of HeLa cells remained viable after uptake. Due to their size and surface functionalities, these far-red-labeled microspheres are ideal candidates for in vitro, cellular delivery of proteins, as described in the accompanying paper.

On an indirect integral equation approach for stationary heat transfer in semi-infinite layered domains in R3 with cavities

Regions containing internal boundaries such as composite materials arise in many applications.We consider a situation of a layered domain in IR3 containing a nite number of bounded cavities. The model is stationary heat transfer given by the Laplace equation with piecewise constant conductivity. The heat ux (a Neumann condition) is imposed on the bottom of the layered region and various boundary conditions are imposed on the cavities. The usual transmission (interface) conditions are satised at the interface layer, that is continuity of the solution and its normal derivative. To eciently calculate the stationary temperature eld in the semi-innite region, we employ a Green's matrix technique and reduce the problem to boundary integral equations (weakly singular) over the bounded surfaces of the cavities. For the numerical solution of these integral equations, we use Wienert's approach [20]. Assuming that each cavity is homeomorphic with the unit sphere, a fully discrete projection method with super-algebraic convergence order is proposed. A proof of an error estimate for the approximation is given as well. Numerical examples are presented that further highlights the eciency and accuracy of the proposed method.

Fluorescent probe:complexation of Fe3+ with the myo-inositol 1,2,3-trisphosphate motif

Natural myo-inositol phosphate antioxidants containing the 1,2,3-trisphosphate motif bind Fe3+ in the unstable penta-axial conformation.

The expression of P-glycoprotein does influence the distribution of novel fluorescent compounds in solid tumour models

Solid tumours display a complex drug resistance phenotype that involves inherent and acquired mechanisms. Multicellular resistance is an inherent feature of solid tumours and is known to present significant barriers to drug permeation in tumours. Given this barrier, do acquired resistance mechanisms such as P-glycoprotein (P-gp) contribute significantly to resistance? To address this question, the multicellular tumour spheroid (MCTS) model was used to examine the influence of P-gp on drug distribution in solid tissue. Tumour spheroids (TS) were generated from either drug-sensitive MCF7WT cells or a drug-resistant, P-gp-expressing derivative MCF7Adr. Confocal microscopy was used to measure time courses and distribution patterns of three fluorescent compounds; calcein-AM, rhodamine123 and BODIPY-taxol. These compounds were chosen because they are all substrates for P-gp-mediated transport, exhibit high fluorescence and are chemically dissimilar. For example, BODIPY-taxol and rhodamine 123 showed high accumulation and distributed extensively throughout the TSWT, whereas calcein-AM accumulation was restricted to the outermost layers. The presence of P-gp in TSAdr resulted in negligible accumulation, regardless of the compound. Moreover, the inhibition of P-gp by nicardipine restored intracellular accumulation and distribution patterns to levels observed in TSWT. The results demonstrate the effectiveness of P-gp in modulating drug distribution in solid tumour models. However, the penetration of agents throughout the tissue is strongly determined by the physico-chemical properties of the individual compounds.

An investigation of the fluorescence of Britishs bituminous coals:an analysis of fluorescent spectra of coal macerals

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IPOs, clustering, indirect learning and filing independently

IPO underpricing has been attributed to valuation uncertainty, which can be at least partially resolved by the indirect learning associated with IPO clustering [Benveniste, L.M., Ljungqvist, A., Wilhelm, W.J., Yu, X.Y., 2003. Evidence of information spillovers in the production of investment banking services. Journal of Finance 58, 577–608]. We examine why firms might choose not to issue their IPOs contemporaneously with clusters of similar firms, forgoing opportunities to learn from their peers. We find that the willingness to file an IPO without the benefit of indirect learning from peer firm IPOs is directly related to insiders’ needs for portfolio diversification and the firm’s need to raise capital.

A probabilistic indirect adaptive control for systems with input-dependent noise

A probabilistic indirect adaptive controller is proposed for the general nonlinear multivariate class of discrete time system. The proposed probabilistic framework incorporates input–dependent noise prediction parameters in the derivation of the optimal control law. Moreover, because noise can be nonstationary in practice, the proposed adaptive control algorithm provides an elegant method for estimating and tracking the noise. For illustration purposes, the developed method is applied to the affine class of nonlinear multivariate discrete time systems and the desired result is obtained: the optimal control law is determined by solving a cubic equation and the distribution of the tracking error is shown to be Gaussian with zero mean. The efficiency of the proposed scheme is demonstrated numerically through the simulation of an affine nonlinear system.