Estimation of both the potency and efficacy of alpha7 nAChR agonists from single-concentration responses

The assessment of functional properties is a crucial step in the screening of potential new drug candidates. The development of moderate to high throughput electrophysiological recording systems such as OpusXpress (Molecular Devices) has facilitated the process of testing new drugs to a large degree. However, while the simple screening of multiple drugs at a single concentration identifies "hits" and "misses", the generation of full concentration-response studies is still a bottleneck in drug development. The alpha7 nicotinic acetylcholine receptor displays a unique concentration dependence of response kinetics which permits estimates of EC50 and Imax values for experimental drugs to be generated from single-concentration responses. This method is based on the analysis of 13 different concentration-response studies utilizing either human or rat alpha7 nAChR. Each experimental response was first normalized to an ACh control, and then a transformation of the pooled data was generated which, based on the relationship between the net charge and peak current to their respective EC50 values defined the "functional concentration" (the test concentration relative to the EC50 for the given agonist). At low functional concentrations, net charge is large relative to peak current amplitude and at higher functional concentration this relationship reverses. For any single-concentration response, the ratio of net charge to peak current can be used to estimate functional concentration. Efficacy can then be estimated by comparing the observed (net charge) response to the expected value for a full agonist at the estimated functional concentration. This extended analysis, combined with automated recording methods, should greatly increase the efficiency with which promising new drug candidates can be characterized.     

Modeling and simulation of molecular biology systems using petri nets: modeling goals of various approaches

Petri nets are a discrete event simulation approach developed for system representation, in particular for their concurrency and synchronization properties. Various extensions to the original theory of Petri nets have been used for modeling molecular biology systems and metabolic networks. These extensions are stochastic, colored, hybrid and functional. This paper carries out an initial review of the various modeling approaches based on Petri net found in the literature, and of the biological systems that have been successfully modeled with these approaches. Moreover, the modeling goals and possibilities of qualitative analysis and system simulation of each approach are discussed.     

Magnetic resonance imaging using a clinical whole body system: an introduction to a useful technique in small animal experiments.

A clinical whole body magnetic resonance imaging (MRI) system with high resolution coils was used to obtain non-invasive images of the living rat. The results demonstrate the feasibility of the set-up and the advantages of this new imaging technique: detailed information, no extra costs, longitudinal studies without killing animals and simple anaesthesia. It is concluded that in small animal experimentation, this use of high resolution coils in whole body magnetic resonance systems may be particularly helpful in establishing effects of experimental procedures. Whenever non-invasive visualization is required, especially in longitudinal animal studies, e.g. biomaterial research or tumour investigation, this use of MRI will offer challenging possibilities.     

A mathematical model for experimental gene evolution

The purpose of this paper is to determine the optimal mutation rate for random mutagenesis procedures used to make mutant libraries for subsequent screening. When the mutation rate is low, the probability of achieving a rare beneficial mutation is low. When the mutation rate is high, the probability of producing lethal mutations which result in loss of function is also high. We demonstrate that between these two extremes, an optimal mutation rate exists for experimental gene improvement. This rate depends strongly on the number of simultaneous mutations required for a beneficial change of the gene, but only weakly on the number of possible lethal mutations. This model predicts that when mutagenesis is performed at the optimum mutation rate, at least 63% (1--e(-1)) of the cloned genes in a mutant library will be non-functional.     

The Design of Fermentation and Biological Assay Procedures for Assessment of Penicillin Production in Populations of Aspergillus nidulans

The design of shake flask fermentations and biological assay procedures for the assessment of penicillin titres in populations of Aspergillus nidulans strains is described. Consideration of the amount and stage of replication and the arrangement of the culture filtrates on the assay plate led to the adoption of a general procedure where strains are replicated in 2, 3 or 4 flasks, depending upon the precision required. Each flask is tested using only one inhibition zone and the zones for one replicate set of strains are completely randomized on one assay plate. This experimental design does not require a high degree of statistical competence for interpretation of the results and it may be easily adapted to accommodate a variety of experimental sizes or to allow any required level of discrimination between strains to be achieved. The general approach and principles may be applied to many large scale, low precision assays carried out in fermentation research.     

Survey research in conservation biology

We present systematic arguments for the necessity of field survey in conservation biology. Preservation of biological diversity has become a major challenge in conservation biology, but to comprehend diversity, ecologists have to obtain information on what units the 'diversity' of different parts of the world consists of, where these units are, and how they respond to natural and human-induced environmental change. To reach this end, systematic survey procedures need to be developed that incorporate data collecting, data analysis and conclusions about distributional patterns as well as management recommendations into an iterative process that is corrected as experience accumulates. The appropriate survey design depends on the task and needs to be fixed separately in each case; developing long-term observational systems is no less challenging a task than developing experimental systems in laboratory research, or modeling systems in theoretical research. We conclude the paper with five principles of ecological survey. A common denominator of these principles is the need to make explicit decisions at each step so that errors and insufficiency can be corrected later.     

Inhibition of autoxidation of egg yolk phosphatidylcholine in homogenous solution and in liposomes by oxidized ubiquinone

The aim of this study was to obtain a quantification of the antioxidant activity of ubiquinone. To this purpose the oxidation of egg yolk phosphatidylcholine both in solvent and in liposomes initiated by an azocompound has been studied either in the absence or in the presence of ubiquinone-3, using alpha-tocopherol as a reference antioxidant. The two experimental systems gave similar results. In the presence of ubiquinone-3 the oxidation rate was reduced with respect to control experiments but was faster than that in the presence of alpha-tocopherol. The amount of ubiquinone required to decrease the autoxidation rate was so high as to prevent detection of the induction period. The stoichiometric factor was greater than 2 and the rate constant of inhibition was two orders of magnitude lower than that of alpha-tocopherol. It is concluded that high concentrations of ubiquinone are required to exhibit significant antioxidant activity. A possible mechanism compatible with the stoichiometric factor larger than 2 for the inhibiting effect of ubiquinone is also suggested.     

Scalable Session Locking for a Distributed File System

File systems provide an interface for applications to obtain exclusive access to files, in which a process holds privileges to a file that cannot be preempted and restrict the capabilities of other processes. Local file systems do this by maintaining information about the privileges of current file sessions, and checking subsequent sessions for compatibility. Implementing exclusive access in this manner for distributed file systems degrades performance by requiring every new file session to be registered with a lock server that maintains global session state. We present two techniques for improving the performance of session management in the distributed environment. We introduce a distributed lock for managing file access, called a semi-preemptible lock, that allows clients to cache privileges. Under a semi-preemptible lock, a file system creates new sessions without messages to the lock manager. This improves performance by exploiting locality – the affinity of files to clients. We also present data structures and algorithms for the dynamic evaluation of locks that allow a distributed file system to efficiently manage arbitrarily complex locking. In this case, complex means that an object can be locked in a large number of unique modes. The combination of these techniques results in a distributed locking scheme that supports fine-grained concurrency control with low memory and message overhead and with the assurance that their locking system is correct and avoids unnecessary deadlocks.     

A bioseparation apparatus with high-pressure fluid injection and fluid sampling

A novel apparatus in which fluids may be injected and sampled at high pressure is described. Bioseparation applications of the apparatus were demonstrated in three model systems: (1) lambdaDNA was eluted under pressure from an anion exchange column into a low-salt (0.25 M) buffer, thereby eliminating conventional time-consuming desalting procedures required for downstream analysis of the DNA; (2) RNA was separated under pressure from a RNA/DNA mixture, thereby enabling rapid differential preparation of nucleic acids; and (3) an antibody was purified from a protein mixture by affinity capture at one pressure and dissociation from the antigen binding partner at a second pressure, thereby enabling the immunoreactivities of both antibody and antigen to be preserved during the separation process.     

Behavior analysis and neuroscience

Darwin proposed a powerful functional principle-natural selection-to interpret phylogenetic diversity and complexity. Nevertheless, some 70 years elapsed before even biologists embraced his account. The triumph of natural selection required two additional factors: (a) biological mechanisms that implemented the functional principle (i.e. genetics); and (b) quantitative procedures that traced its cumulative effects (i.e. population genetics). Thorndike and, later, Skinner proposed a functional principle-selection by reinforcement-that interpreted ontogenetic diversity and complexity. This principle has been substantially refined by subsequent experimental and theoretical work and now provides an equally powerful functional account. However, a purely functional principle has once again not persuaded most scientists (apart from the behavior-analytic minority) that complex behavior can be understood by a selection principle. If the history of ontogeny recapitulates the history of phylogeny, the biological mechanisms that implement selection by reinforcement must be discovered and quantitative techniques that trace its effects must be devised. In short, the triumph of reinforcement may await the integration of behavior analysis with the neurosciences and the development of coordinated quantitative procedures. This article identifies specific interrelations with neuroscience that form the basis for a behavior-analytically faithful interpretation of reinforcement using neural-network techniques. Implications for the architecture of networks and for the learning algorithm are emphasized.     

Reorganization of functional networks in mild cognitive impairment

Whether the balance between integration and segregation of information in the brain is damaged in Mild Cognitive Impairment (MCI) subjects is still a matter of debate. Here we characterize the functional network architecture of MCI subjects by means of complex networks analysis. Magnetoencephalograms (MEG) time series obtained during a memory task were evaluated by synchronization likelihood (SL), to quantify the statistical dependence between MEG signals and to obtain the functional networks. Graphs from MCI subjects show an enhancement of the strength of connections, together with an increase in the outreach parameter, suggesting that memory processing in MCI subjects is associated with higher energy expenditure and a tendency toward random structure, which breaks the balance between integration and segregation. All features are reproduced by an evolutionary network model that simulates the degenerative process of a healthy functional network to that associated with MCI. Due to the high rate of conversion from MCI to Alzheimer Disease (AD), these results show that the analysis of functional networks could be an appropriate tool for the early detection of both MCI and AD.     

Analytical ultracentrifugation combined with X-ray and neutron scattering: Experiment and modelling

Analytical ultracentrifugation and solution scattering provide different multi-parameter structural and compositional information on proteins. The joint application of the two methods supplements high resolution structural studies by crystallography and NMR. We summarise the procedures required to obtain equivalent ultracentrifugation and X-ray and neutron scattering data. The constrained modelling of ultracentrifugation and scattering data is important to confirm the experimental data analysis and yields families of best-fit molecular models for comparison with crystallography and NMR structures. This modelling of ultracentrifugation and scattering data is described in terms of starting models, their conformational randomisation in trial-and-error fits, and the identification of the final best-fit models. Seven applications of these methods are described to illustrate the current state-of-the-art. These include the determination of antibody solution structures (the human IgG4 subclass, and oligomeric forms of human IgA and its secretory component), the solution structures of the complement proteins of innate immunity (Factor H and C3/C3u) and their interactions with macromolecular ligands (C-reactive protein), and anionic polysaccharides (heparin). Complementary features of joint ultracentrifugation and scattering experiments facilitate an improved understanding of crystal structures (illustrated for C3/C3u, C-reactive protein and heparin). If a large protein or its complex cannot be crystallised, the joint ultracentrifugation-scattering approach provides a means to obtain an overall macromolecular structure.     

Wiener analysis of nonlinear systems using Poisson-Charlier crosscorrelation

Nonlinear systems with event-sequence input, such as are often encountered in neurophysiology, may be experimentally tested with all possible input sequences by stimulation with a Poisson process eventsequence. A complete predictive model of the system's response may be constructed from this data with the Wiener expansion based on the Poisson-Charlier polynomials. Here it is shown how this formulation leads to an efficient method for the evaluation of unknown systems by crosscorrelation, generalizing previous methods. The basic statistical properties of the procedure are demonstrated and the length of experiment required for accurate estimation of the model is computed. The procedure is translated into digital algorithms and the analogous procedures for white noise analysis are presented.     

Influence of mass transfer on stable isotope fractionation

Biodegradation of contaminants is a common remediation strategy for subsurface environments. To monitor the success of such remediation means a quantitative assessment of biodegradation at the field scale is required. Nevertheless, the reliable quantification of the in situ biodegradation process it is still a major challenge. Compound-specific stable isotope analysis has become an established method for the qualitative analysis of biodegradation in the field and this method is also proposed for a quantitative analysis. However, to use stable isotope data to obtain quantitative information on in situ biodegradation requires among others knowledge on the influence of mass transfer processes on the observed stable isotope fractionation. This paper reviews recent findings on the influence of mass transfer processes on stable isotope fractionation and on the quantitative interpretation of isotope data. Focus will be given on small-scale mass transfer processes controlling the bioavailability of contaminants. Such bioavailability limitations are known to affect the biodegradation rate and have recently been shown to affect stable isotope fractionation, too. Theoretical as well as experimental studies addressing the link between bioavailability and stable isotope fractionation are reviewed and the implications for assessing biodegradation in the field are discussed.     

Organelle isolation: functional mitochondria from mouse liver, muscle and cultured fibroblasts

Mitochondria participate in key metabolic reactions of the cell and regulate crucial signaling pathways including apoptosis. Although several approaches are available to study mitochondrial function in situ are available, investigating functional mitochondria that have been isolated from different tissues and from cultured cells offers still more unmatched advantages. This protocol illustrates a step-by-step procedure to obtain functional mitochondria with high yield from cells grown in culture, liver and muscle. The isolation procedures described here require 1-2 hours, depending on the source of the organelles. The polarographic analysis can be completed in 1 hour.     

Development and Application of a Process Window for Achieving High-Quality Coating in a Fluidized Bed Coating Process

Next to the coating formulation, process conditions play important roles in determining coating quality. This study aims to develop an operational window that separates layering from agglomeration regimes and, furthermore, the one that leads to the best coating quality in a fluidized bed coater. The bed relative humidity and the droplet size of the coating aerosol were predicted using a set of engineering models. The coating quality was characterized using a quantitative image analysis method, which measures the coating thickness distribution, the total porosity, and the pore size in the coating. The layering regime can be achieved by performing the coating process at a certain excess of the viscous Stokes number (ΔSt _v). This excess is dependent on the given bed relative humidity and droplet size. The higher the bed relative humidity, the higher is the ΔSt _v required to keep the process in the layering regime. Further, it is shown that using bed relative humidity and droplet size alone is not enough to obtain constant coating quality. The changes in bed relative humidity and droplet size have been identified to correlate to the fractional area of particles sprayed per unit of time. This parameter can effectively serve as an additional parameter to be considered for a better control on the coating quality. High coating quality is shown to be achieved by performing the process close to saturation and spraying droplets small enough to obtain high spraying rate, but not too small to cause incomplete coverage of the core particles.     

Affinity chromatography techniques based on the immobilisation of peptides exhibiting specific binding activity

Affinity chromatography is one of the powerful techniques in selective purification and isolation of a great number of compounds. New challenges in scientific research, such as high-throughput systems, isolation procedures that allow to obtain a single substance from a complex matrix in high degree of purity, low costs and wide availability, have led to the discovery of new tailor-made synthetic recognition systems. In this review the design, synthesis, purification and characterisation of peptides with recognition properties are discussed. Applications of peptide ligands are described and analytical tools mentioned.     

A modular approach to the ECVAM principles on test validity

The European Centre for the Validation of Alternative Methods (ECVAM) proposes to make the validation process more flexible, while maintaining its high standards. The various aspects of validation are broken down into independent modules, and the information necessary to complete each module is defined. The data required to assess test validity in an independent peer review, not the process, are thus emphasised. Once the information to satisfy all the modules is complete, the test can enter the peer-review process. In this way, the between-laboratory variability and predictive capacity of a test can be assessed independently. Thinking in terms of validity principles will broaden the applicability of the validation process to a variety of tests and procedures, including the generation of new tests, new technologies (for example, genomics, proteomics), computer-based models (for example, quantitative structure-activity relationship models), and expert systems. This proposal also aims to take into account existing information, defining this as retrospective validation, in contrast to a prospective validation study, which has been the predominant approach to date. This will permit the assessment of test validity by completing the missing information via the relevant validation procedure: prospective validation, retrospective validation, catch-up validation, or a combination of these procedures.