Ratiometric and nonratiometric Ca2+ indicators for the assessment of intracellular free Ca2+ in a breast cancer cell line using a fluorescence microplate reader

Transporters of Ca2+ are potential drug targets and Ca2+ is a useful signal in the assessment of G-protein-coupled receptor activation. Assays involving the assessment of intracellular Ca2+ using microplate readers most often use Ca2+ indicators which do not exhibit a spectra shift on Ca2+ binding (e.g. fluo-3). Indicators that do exhibit a spectral shift upon Ca2+ binding (e.g. fura-2) offer potential advantages for the calibration of intracellular Ca2+ levels. However, experimental limitations may limit the use of ratiometric dyes in microplate readers capable of screening. In this study, we compared the assessment of intracellular Ca2+ in adherent breast cancer cells using ratiometric and nonratiometric Ca2+ indicators. Our results demonstrate that both fluo-3 and fura-2 detect ATP dose-dependent increases in intracellular Ca2+ in the MCF-7 breast cancer cell line and that some of the limitations in the use of fura-2 appear to be overcome by the use of glass bottom microplates. The calibrated intracellular Ca2+ levels derived using fura-2 are consistent with those from microscopy and cuvette-based studies. Fura-2 may be useful in microplate studies, where cell lines with different properties are compared or where screening treatments lead to differences in the number of cells or dye loading. (C) 2003 Elsevier B.V. All rights reserved.

Precautionary principles: a jurisdiction-free framework for decision-making under risk

Fundamental principles of precaution are legal maxims that ask for preventive actions, perhaps as contingent interim measures while relevant information about causality and harm remains unavailable, to minimize the societal impact of potentially severe or irreversible outcomes. Such principles do not explain how to make choices or how to identify what is protective when incomplete and inconsistent scientific evidence of causation characterizes the potential hazards. Rather, they entrust lower jurisdictions, such as agencies or authorities, to make current decisions while recognizing that future information can contradict the scientific basis that supported the initial decision. After reviewing and synthesizing national and international legal aspects of precautionary principles, this paper addresses the key question: How can society manage potentially severe, irreversible or serious environmental outcomes when variability, uncertainty, and limited causal knowledge characterize their decision-making? A decision-analytic solution is outlined that focuses on risky decisions and accounts for prior states of information and scientific beliefs that can be updated as subsequent information becomes available. As a practical and established approach to causal reasoning and decision-making under risk, inherent to precautionary decision-making, these (Bayesian) methods help decision-makers and stakeholders because they formally account for probabilistic outcomes, new information, and are consistent and replicable. Rational choice of an action from among various alternatives-defined as a choice that makes preferred consequences more likely-requires accounting for costs, benefits and the change in risks associated with each candidate action. Decisions under any form of the precautionary principle reviewed must account for the contingent nature of scientific information, creating a link to the decision-analytic principle of expected value of information (VOI), to show the relevance of new information, relative to the initial ( and smaller) set of data on which the decision was based. We exemplify this seemingly simple situation using risk management of BSE. As an integral aspect of causal analysis under risk, the methods developed in this paper permit the addition of non-linear, hormetic dose-response models to the current set of regulatory defaults such as the linear, non-threshold models. This increase in the number of defaults is an important improvement because most of the variants of the precautionary principle require cost-benefit balancing. Specifically, increasing the set of causal defaults accounts for beneficial effects at very low doses. We also show and conclude that quantitative risk assessment dominates qualitative risk assessment, supporting the extension of the set of default causal models.

Changes to porcine blastocyst vitrification methods and improved litter size after transfer

The objective was to improve the protocol that was used to obtain the first reported piglets from transferred vitrified and warmed zona-intact blastocysts. Blastocysts were collected from superovulated sows and gilts, centrifuged to polarize lipid, vitrified, warmed and cultured for 24 h or transferred immediately. Removing the zona pellucida after warming increased the number of cells in the surviving blastocysts (zona-free 60.8 +/- 4.3, zona-intact 39.1 +/- 2.8; P < 0.05). Thinning the zona pellucida produced similar results to zona removal. Changing the basal medium of the vitrification and warming solutions from modified PBS to phosphate buffered NCSU-23 increased the number of cells (44.7 +/- 2.2 versus 56.0 +/- 3.9, respectively; P < 0.05). Reducing the plunge temperature of the liquid nitrogen from - 196 degrees C to less than -204 degrees C improved the embryo survival rate (61.9% versus 82.9%, respectively; P < 0.05). These modifications were incorporated into the vitrification protocol that was used to vitrify and warm 105 blastocysts (that were subsequently transferred into four recipients). Three recipients became pregnant, farrowing three litters (average litter size, 5.3; 18.8% embryo survival in farrowing sows). Changing the warming protocol to using sucrose rather than ethylene glycol resulted in a trend towards improved embryo survival (73.5% versus 91.2%) but this was not statistically significant. Incorporating this modification, 203 blastocysts were vitrified, warmed and transferred into seven recipients. Five became pregnant and 36 fetuses were recovered (average litter size 7.2; 24.8% embryo survival in pregnant sows) at Day 40 of pregnancy. In conclusion, changes made to the vitrification protocol improved pregnancy rate and in vivo embryo survival compared to an earlier study using the original protocol. (c) 2005 Elsevier Inc. All rights reserved.

'Free' surfactant in gastric aspirates and bronchoalveolar lavage in children with and without reflux oesophagitis

Aim: Dipalmitoylphosphatidycholine (DPPC) is the characteristic and main constituent of surfactant. Adsorption of surfactant to epithelial surfaces may be important in the masking of receptors. The aims of the study were to (i) compare the quantity of free DPPC in the airways and gastric aspirates of children with gastroesophageal reflux disease (GORD) to those without and (ii) describe the association between free DPPC levels with airway cellular profile and capsaicin cough sensitivity. Methods: Children aged < 14 years were defined as 'coughers' if a history of cough in association with their GORD symptoms was elicited before gastric aspirates and nonbronchoscopic bronchoalveolar lavage (BAL) were obtained during elective flexible upper gastrointestinal endoscopy. GORD was defined as histological presence of reflux oesophagitis. Spirometry and capsaicin cough-sensitivity test was carried out in children aged > 6 years before the endoscopy. Results: Median age of the 68 children was 9 years (interquartile range (IQR) 7.2). Median DPPC level in BAL of children with cough (72.7 mu g/mL) was similar to noncoughers (88.5). There was also no significant difference in DPPC levels in both BAL and gastric aspirates of children classified according to presence of GORD. There was no correlation between DPPC levels and cellular counts or capsaicin cough-sensitivity outcome measures. Conclusion: We conclude that free DPPC levels in the airways and gastric aspirate is not influenced by presence of cough or GORD defined by histological presence of reflux oesophagitis. Whether quantification of adsorbed surfactant differs in these groups remain unknown. Free DPPC is unlikely to have a role in masking of airway receptors.

Accelerated leap methods for simulating discrete stochastic chemical kinetics

Biologists are increasingly conscious of the critical role that noise plays in cellular functions such as genetic regulation, often in connection with fluctuations in small numbers of key regulatory molecules. This has inspired the development of models that capture this fundamentally discrete and stochastic nature of cellular biology - most notably the Gillespie stochastic simulation algorithm (SSA). The SSA simulates a temporally homogeneous, discrete-state, continuous-time Markov process, and of course the corresponding probabilities and numbers of each molecular species must all remain positive. While accurately serving this purpose, the SSA can be computationally inefficient due to very small time stepping so faster approximations such as the Poisson and Binomial τ-leap methods have been suggested. This work places these leap methods in the context of numerical methods for the solution of stochastic differential equations (SDEs) driven by Poisson noise. This allows analogues of Euler-Maruyuma, Milstein and even higher order methods to be developed through the Itô-Taylor expansions as well as similar derivative-free Runge-Kutta approaches. Numerical results demonstrate that these novel methods compare favourably with existing techniques for simulating biochemical reactions by more accurately capturing crucial properties such as the mean and variance than existing methods.

Making Fedora easier to implement with Fez: A free open source content model and workflow management front-end to Fedora

The University of Queensland, Australia has developed Fez, a world-leading user-interface and management system for Fedora-based institutional repositories, which bridges the gap between a repository and users. Christiaan Kortekaas, Andrew Bennett and Keith Webster will review this open source software that gives institutions the power to create a comprehensive repository solution without the hassle..

On compliance of business processes with business contracts

This paper addresses the problem of ensuring compliance of business processes, implemented within and across organisational boundaries, with the constraints stated in related business contracts. In order to deal with the complexity of this problem we propose two solutions that allow for a systematic and increasingly automated support for addressing two specific compliance issues. One solution provides a set of guidelines for progressively transforming contract conditions into business processes that are consistent with contract conditions thus avoiding violation of the rules in contract. Another solution compares rules in business contracts and rules in business processes to check for possible inconsistencies. Both approaches rely on a computer interpretable representation of contract conditions that embodies contract semantics. This semantics is described in terms of a logic based formalism allowing for the description of obligations, prohibitions, permissions and violations conditions in contracts. This semantics was based on an analysis of typical building blocks of many commercial, financial and government contracts. The study proved that our contract formalism provides a good foundation for describing key types of conditions in contracts, and has also given several insights into valuable transformation techniques and formalisms needed to establish better alignment between these two, traditionally separate areas of research and endeavour. The study also revealed a number of new areas of research, some of which we intend to address in near future.

Free-Surface Aeration and Momentum Exchange at a Bottom Outlet

This study aims to provide some new understanding of the air-water flow properties in high-velocity water jets discharging past an abrupt drop. Such a setup has been little studied to date despite the relevance to bottom outlets. Downstream of the step brink, the free-jet entrains air at both upper and lower air-water interfaces, as well as along the sides. An air-water shear layer develops at the lower nappe interface. At the lower nappe, the velocity redistribution was successfully modelled and the velocity field was found to be similar to that in two-dimensional wake flow. The results highlighted further two distinct flow regions. Close to the brink (Wex < 5000), the flow was dominated by momentum transfer. Further downstream (Wex > 5000), a strong competition between air bubble diffusion and momentum exchanges took place.

Assessing the competitiveness of loss allocation methods in a deregulated electricity market

This paper critically assesses several loss allocation methods based on the type of competition each method promotes. This understanding assists in determining which method will promote more efficient network operations when implemented in deregulated electricity industries. The methods addressed in this paper include the pro rata [1], proportional sharing [2], loss formula [3], incremental [4], and a new method proposed by the authors of this paper, which is loop-based [5]. These methods are tested on a modified Nordic 32-bus network, where different case studies of different operating points are investigated. The varying results obtained for each allocation method at different operating points make it possible to distinguish methods that promote unhealthy competition from those that encourage better system operation.

Experimental Verification of Decoherence-Free Subspaces

Using spontaneous parametric down-conversion, we produce polarization-entangled states of two photons and characterize them using two-photon tomography to measure the density matrix. A controllable decoherence is imposed on the states by passing the photons through thick, adjustable birefringent elements. When the system is subject to collective decoherence, one particular entangled state is seen to be decoherence-free, as predicted by theory. Such decoherence-free systems may have an important role for the future of quantum computation and information processing.

Calculation of line integrals in boundary integral methods

We propose quadrature rules for the approximation of line integrals possessing logarithmic singularities and show their convergence. In some instances a superconvergence rate is demonstrated.