Rational Experimental Design Research Articles

Pathogenic determinants of the mucosally transmissible CXCR4-specific SHIV(SF33A2) map to env region.

Infection of rhesus macaques with chimeric simian-human immunodeficiency viruses (SHIV) is an established method to study AIDS pathogenesis and is increasingly used to assess the efficacy of vaccine and antiviral candidates. For these reasons, a detailed understanding of those molecular determinants, which confer pathogenic potential to SHIV viruses, should assist in both rational experimental design and interpretation of results. In this report, we describe the development and in vivo characterization of a pathogenic molecular clone, SHIVSF33A2, which contains an envelope sequence derived from the CXCR4-dependent isolate, HIV-1SF33. Proviral DNA, amplified from a rhesus macaque infected with the pathogenic isolate SHIVSF33A, was substituted into the corresponding region of the parental, nonpathogenic SHIVSF33 genome creating the molecular clone SHIVSF33A2. Coreceptor specificity of SHIVSF33A2 was determined to be CXCR4 specific. Naive rhesus macaques were productively infected after a single exposure to cell-free SHIVSF33A2 by either the intravenous (IV) or intravaginal (IVAG) routes. Animals infected with SHIVSF33A2 suffered a severe loss of peripheral CD4+ T cells and high acute plasma viremia with development of simian AIDS 9 months after inoculation. Sequence analysis identified 25 discreet amino acid changes within the V1-V5 regions of the envelope protein when compared with the nonpathogenic parental virus. These data indicate that domains within the HIV-1 envelope protein are sufficient to define pathogenic potential in the context of the SIVmac239 genome.

A Dynamical Systems Model to Simulate the Perturbation Kinetics of Gene Expression by Antisense Oligonucleotides

Antisense oligonucleotides owe their efficacy to an ability to induce RNase H-dependent suppression of RNA translation, for sufficient time to allow physiological proteolysis. The magnitude and time delay preceding the protein nadir concentration determine the extent and timing of maximum antisense oligonucleotide activity. Antisense oligonucleotide degradation underlies reversal of RNA downregulation. The kinetics of protein downregulation is therefore determined by the complex interaction of both ligand chemistry (nuclease stability, affinity and RNase H activation), and gene expression kinetics. Optimization of antisense oligonucleotide efficacy and experimental design requires understanding of these interactions. The kinetics of protein and RNA downregulation have therefore been simulated by analysing a two-compartment kinetic model incorporating RNase H-dependent transcript degradation. The system of nonlinear differential equations describing this model was solved numerically using Runge–Kutte integration. The timecourse solutions corresponding to the four state variables (RNA, protein, antisense/RNA heteroduplex and antisense oligonucleotide), were determined simultaneously. This allowed systematic in silico examination of the consequences of altering variables such as oligonucleotide concentration, affinity, and stability, or the scheduling of multiple transfections on RNA and protein perturbations. By providing a tool for examining antisense oligonucleotide action theoretically, this heuristic model should facilitate both the rational design and interpretation of antisense experiments.

Screening the molecular surface of human anticoagulant protein C: a search for interaction sites.

Protein C (PC), a 62 kDa multi-modular zymogen, is activated to an anticoagulant serine protease (activated PC or APC) by thrombin bound to thrombomodulin on the surface of endothelial cells. PC/APC interacts with many proteins and the characterisation of these interactions is not trivial. However, molecular modelling methods help to study these complex biological processes and provide basis for rational experimental design and interpretation of the results. PC/APC consists of a Gla domain followed by two EGF modules and a serine protease domain. In this report, we present two structural models for full-length APC and two equivalent models for full-length PC, based on the X-ray structures of Gla-domainless APC and of known serine protease zymogens. The overall elongated shape of the models is further cross-validated using size exclusion chromatography which allows evaluation of the Stokes radius (rs for PC = 33.15 A; rs for APC = 34.19 A), frictional ratio and axial ratio. We then propose potential binding sites at the surface of PC/APC using surface hydrophobicity as a determinant of the preferred sites of intermolecular recognition. Most of the predicted binding sites are consistent with previously reported experimental data, while some clusters highlight new regions that should be involved in protein-protein interactions.

Development of spontaneous autoimmune thyroiditis in Obese strain (OS) chickens.

Chickens of the Obese strain (OS) are known to develop spontaneous autoimmune thyroiditis (SAT) in the first 2-3 weeks post-hatching, but onset and severity of SAT for this period among sublines of OS chickens has not yet been analysed in detail. In the present paper, we described the kinetics of SAT in age-matched OSB13B13C, OSB5B5C, OSB15B15INN and OSB5B5INN chicken sublines. The results revealed no thyroid infiltration in OS fetuses at 20th ED of the analysed sublines. Mononuclear cell infiltration of thyroid was first noted in 2-4-day-old chickens, followed by aggravation of thyroiditis in 4-week-old OSB13B13C and OSB15B15INN chickens and in 5-week-old OSB5B5C birds. Interestingly, two subpopulations of OSB5B5INN chickens were found with different kinetics of SAT development: one with degree of SAT lower than 40%, was designated "low responders" and the other, with similar degrees of SAT as the other three sublines, was characterized as "high responders". Our results allow an age-dependent prediction of SAT development among OS chickens and the rational design of animal experiments, particularly for assessing the relevance of therapeutic interventions.

SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling.

Comparative protein modeling is increasingly gaining interest since it is of great assistance during the rational design of mutagenesis experiments. The availability of this method, and the resulting models, has however been restricted by the availability of expensive computer hardware and software. To overcome these limitations, we have developed an environment for comparative protein modeling that consists of SWISS-MODEL, a server for automated comparative protein modeling and of the SWISS-PdbViewer, a sequence to structure workbench. The Swiss-PdbViewer not only acts as a client for SWISS-MODEL, but also provides a large selection of structure analysis and display tools. In addition, we provide the SWISS-MODEL Repository, a database containing more than 3500 automatically generated protein models. By making such tools freely available to the scientific community, we hope to increase the use of protein structures and models in the process of experiment design.

Large-scale protein modelling and integration with the SWISS-PROT and SWISS-2DPAGE databases: the example of Escherichia coli.

Knowledge-based molecular modelling of proteins has proven useful in many instances, including the rational design of mutagenesis experiments, but it has generally been limited by the availability of expensive computer hardware and software. To overcome these limitations, we developed the SWISS-MODEL server for automated knowledge-based protein modelling. The SWISS-MODEL server uses the Brookhaven Protein Data Bank as a source of structural information and automatically generates protein models for sequences which share significant similarities with at least one protein of known three-dimensional structure. We have now used the software framework of the server to generate large collections of protein models, and established the SWISS-MODEL Repository, a new database for automatically generated and theoretical protein models. This repository is directly integrated with the SWISS-PROT and SWISS-2DPAGE databases through the ExPASy World Wide Web server (URL is http://expasy.hcuge.ch). Here we present an illustration of this process by an application to the Escherichia coli sequences.

Rational experimental design for bioanalytical methods validation illustration using an assay method for total captopril in plasma

Generally, bioanalytical chromatographic methods are validated according to a predefined programme and distinguish a pre-validation phase, a main validation phase and a follow-up validation phase. In this paper, a rational, total performance evaluation programme for chromatographic methods is presented. The design was developed in particular for the pre-validation and main validation phases. The entire experimental design can be performed within six analytical runs. The first run (pre-validation phase) is used to assess the validity of the expected concentration-response relationship (lack of fit, goodness of fit), to assess the specificity of the method and to assess the stability of processed samples in the autosampler for 30 h (benchtop stability). The latter experiment is performed to justify overnight analyses. Following approval of the method after the pre-validation phase, the next five runs (main validation phase) are performed to evaluate method precision and accuracy, recovery, freezing and thawing stability and over-curve control /dilution. The design is nested, i.e., many experimental results are used for the evaluation of several performance characteristics. Analysis of variance (ANOVA) is used for the evaluation of lack of fit and goodness of fit, precision and accuracy, freezing and thawing stability and over-curve control/ dilution. Regression analysis is used to evaluate benchtop stability. For over-curve control/ dilution, additional to ANOVA, also a paired comparison is applied. As a consequence, the recommended design combines the performance of as few independent validation experiments as possible with modern statistical methods, resulting in optimum use of information. A demonstration of the entire validation programme is given for an HPLC method for the determination of total captopril in human plasma.

Membrane Topology of the Human Na+/Glucose Cotransporter SGLT1

The membrane topology of the human Na+/glucose cotransporter SGLT1 has been probed using N-glycosylation scanning mutants and nested truncations. Functional analysis proved essential for establishment of signal-anchor topology. The resultant model diverges significantly from previously held suppositions of structure based primarily on hydropathy analysis. SGLT1 incorporates 14 membrane spans. The N terminus resides extracellularly, and two hydrophobic regions form newly recognized membrane spans 4 and 12; the large charged domain near the C terminus is cytoplasmic. This model was evaluated further using two advanced empirically-based algorithms predictive of transmembrane helices. Helix ends were predicted using thermo-dynamically-based algorithms known to predict x-ray crystallographically determined transmembrane helix ends. Several considerations suggest the hydrophobic C terminus forms a 14th transmembrane helix, differentiating the eukaryotic members of the SGLT1 family from bacterial homologues. Our data inferentially indicate that these bacterial homologues incorporate 13 spans, with an extracellular N terminus. The model of SGLT1 secondary structure and the predicted helix ends signify information prerequisite for the rational design of further experiments on structure/function relationships.

Rational design of linear calibration experiments for the quantitative estimation of chlorophyll a using high-performance liquid chromatography, atomic absorption spectrometry and electronic absorption spectrometry

There is considerable difference in published molar absorption coefficients for chlorophyll a. This paper involves estimating the molar absorption coefficient in acetone by calibrating against measured Mg using AAS. Chlorophyll can then be routinely estimated using peak areas in diode array detector HPLC. In order to account for the main errors in the estimates, designed linear calibration experiments are required, involving 14 measurements at five levels, with four instrumental and five dilution replicates. Three errors namely, lack-of-fit, dilution and instrumental errors can be calculated for each type of measurement, guiding the experimenter as to where problems may arise in the experimental procedure.

A mutation data matrix for transmembrane proteins

The widely used Mutation Data Matrix (MDM), is an amino acid comparison matrix calculated from a study of the exchange probabilities (or odds) derived from an analysis of the evolutionary changes seen in groups of very similar proteins. In this work, a mutation data matrix is calculated for membrane spanning segments. This new mutation data matrix is found to be very different from matrices calculated from general sequence sets which are biased towards water-soluble globular proteins, and the differences are discussed in the context of specific structural requirements of membrane spanning segments. This new matrix will help improve the accuracy of integral membrane protein sequence alignments, and could also be of use in the rational design of site directed mutagenesis experiments for this class of proteins.

Model of lactose repressor core based on alignment with sugar-binding proteins is concordant with genetic and chemical data.

Using primary sequence similarity to arabinose-binding protein, D-glucose/D-galactose-binding protein, and ribose-binding protein (Vyas, N. K., Vyas, M. N., and Quiocho, F. A. (1991) J. Biol. Chem. 266, 5226-5237; Mowbray, S. L., and Cole, L. B. (1992) J. Mol. Biol. 225, 155-175), the core domain (residues 62-323) of the bacterial regulatory protein lac repressor has been aligned to these sugar-binding proteins of known structure. Although the sequence identity is not striking, there is strong overall homology based on two separate matrix scoring systems (minimum base change per codon (MBC/C) and amino acid homology per residue (AAH/R)) (mean score: MBC/C < 1.25, AAH/R > 5.50; random sequences: MBC/C = 1.45, AAH/R = 4.46). Similarly, the predicted secondary structure of the repressor exhibits excellent agreement with the known secondary structures of the sugar-binding proteins. Using this primary sequence alignment, the tertiary structure of the core domain of the lac repressor has been modeled based on the known structures of the sugar-binding proteins as templates. While the structure deduced for the repressor is hypothetical, the model generated allows a comparison between the predicted tertiary arrangement and the wealth of genetic and chemical data elucidated for the repressor. Important residues involved in operator and sugar binding and in protein assembly have been identified using genetic methods, and placement of these residues in the model is consistent with their known function. This approach, therefore, provides a means to visualize the core domain of the lac repressor that allows interpretation of genetic and chemical data for specific residues and rational design of future experiments.

Model of lactose repressor core based on alignment with sugar-binding proteins is concordant with genetic and chemical data

Using primary sequence similarity to arabinose-binding protein, D-glucose/D-galactose-binding protein, and ribose-binding protein (Vyas, N. K., Vyas, M. N., and Quiocho, F. A. (1991) J. Biol. Chem. 266, 5226-5237; Mowbray, S. L., and Cole, L. B. (1992) J. Mol. Biol. 225, 155-175), the core domain (residues 62-323) of the bacterial regulatory protein lac repressor has been aligned to these sugar-binding proteins of known structure. Although the sequence identity is not striking, there is strong overall homology based on two separate matrix scoring systems (minimum base change per codon (MBC/C) and amino acid homology per residue (AAH/R)) (mean score: MBC/C 5.50; random sequences: MBC/C = 1.45, AAH/R = 4.46). Similarly, the predicted secondary structure of the repressor exhibits excellent agreement with the known secondary structures of the sugar-binding proteins. Using this primary sequence alignment, the tertiary structure of the core domain of the lac repressor has been modeled based on the known structures of the sugar-binding proteins as templates. While the structure deduced for the repressor is hypothetical, the model generated allows a comparison between the predicted tertiary arrangement and the wealth of genetic and chemical data elucidated for the repressor. Important residues involved in operator and sugar binding and in protein assembly have been identified using genetic methods, and placement of these residues in the model is consistent with their known function. This approach, therefore, provides a means to visualize the core domain of the lac repressor that allows interpretation of genetic and chemical data for specific residues and rational design of future experiments.

Experimental investigation of the ZrBClH CVD system

The ZrBClH system was experimentally studied by LPCVD using gas mixtures of BCl 3 , ZrCl 4 and H 2 . A rational experimental design was adopted and the results showed that the deposition rate and the surface morphology depended strongly on the reduction of boron trichloride by hydrogen. The kinetics were detailed and a deposition diagram was established. The experimental diagram is in good agreement with the thermodynamic predictions. From the characterization of deposits it resulted that zirconium diboride is the only compound which can be synthesized in the system. Depending on the experimental conditions, it can either crystallize in isotropic form or in three types of textures, and can either be accompanied by free boron or not. It also showed the existence of an amorphous domain which is constituted by boron with a small quantity of zirconium (<5%). Das ZrBClH System wurde ausgehend von Gas-mischungen von BCl 3 , ZrCl 4 und H 2 mittels LPCVD untersucht. Durch Anwendung einer rationellen Versuchsanordnung konnte gezeigt werden, daß die Abscheidungsgeschwindigkeit und die Oberflachenmorphologie in starken Maße von der Reduktion von Bortrichlorid durch Wasserstoff abhängig sind. Die Kinetik wird ausfurlich dargestellt und ein Abscheidungsdiagram erstellt, welches in guter Übereinstimmung mit thermodynamischen Betrachtungen ist. Aus der Charakterisierung der Abscheidungs-produkte folgt, daß Zirkoniumdiborid als einzige Verbindung in diesem System erhalten werden kann. In Abhängigkeit von den experimentellen Bedingungen kristallisiert die Substanz entweder in einer isotropen Form oder in drei Texturtypen und unter bestimmten Umständen kann nebenbei elementares Bor auftreten. Die Existenz einer amorphen Domäne aus Bor mit einem geringen Anteil von Zirkonium (<5%) wurde ebenfalls nachgewiesen. Une exploration paramétrique du système ZrBClH est effectuée sous pression réduite à partir de mélanges de BCl 3 , de ZrCl 4 et de H 2 . Un plan d'expérience rationnel montre que la vitesse de dépôt et la morphologie de surface des couches obtenues dépendent étroitement de la réduction de BCl 3 par l'hydrogène. La cinétique a été précisée, et un diagramme de dépôt a été établi. Ce dernier est en bon accord avec les prévisions thermodynamiques. De la caractérisation des dépôts, il ressort que le seul composé synthétisé est le diborure de zirconium. Suivant les conditions paramétriques, il peut cristalliser de façon isotrope ou présenter trois types de textures et être accompagné ou non de bore libre en plus ou moins grande quantité. Un domaine amorphe constitué de bore contenant moins de 5% de zirconium a également été mis en évidence.

A Straightforward Method for the Study of Drug Interactions: An Isobolographic Analysis Primer

The study of drug interactions presents investigators with questions about both experimental design and interpretation of results that easily can become complex and confusing. Among the advantages of the isobolographic method is that it leads to rational experimental design and renders both the descriptive and analytical considerations of results obtained relatively simple and straightforward. In particular, the descriptive use of the method is such that it permits experimenters to express compactly and unambiguously the salient features of the observed interaction.

Liability in Use of Investigational Drugs

RECENT DEVELOPMENTS, including the growth of clinical research centers, the new regulations of the Food and Drug Administration (FDA), and the publicity surrounding the reported adverse reactions to thalidomide have prompted this examination of the moral and legal entanglements facing the clinical investigator. Purpose of Clinical Investigator The primary purpose of clinical investigation is to produce scientifically reliable data which will serve to answer specific questions or, simply, to satisfy the investigator's scientific curiosity. Any such investigation, therefore, must be done within the framework of a rational experimental design. Moreover, since the purpose of a drug investigation is to acquire and interpret reliable data which may lead to the proper application of new therapeutic agents, it must include basic, clinical, and therapeutic studies—all of these are necessary if medicine is to advance in knowledge and wisdom. Finally, it is not necessarily the purpose of the clinical investigator to avoid controversy,