Improvement in reliability of probabilistic test of significant differences in GeneChip experiments.

A probabilistic test (FUMI theory) for GeneChip experiments has been proposed for selecting the genes which show significant differences in the gene expression levels between a single pair of treatment and control. This paper describes that the reliability of the judgment by the FUMI theory can be enhanced, when the selected genes are referred to biomolecular-functional networks of a commercial database. The genes judged as being differently expressed are grouped into a cluster in the biomolecular networks. It is also demonstrated that false positive genes have a trend in the networks to be isolated from each other, and also away from the clustered genes, since the false positive genes are randomly selected.     

Identification of critical genes in microarray experiments by a Neuro-Fuzzy approach

Gene expression profiling by microarray technology is usually difficult to interpret into a simpler pattern. One approach to resolve the complexity of gene expression profiles is the application of artificial neural networks (ANNs). A potential difficulty in this strategy, however, is that the non-linear nature of ANN makes it essentially a 'black-box' computation process. Addition of a fuzzy logic approach is useful because it can complement ANN by explicitly specifying membership function during computation. We employed a hybrid approach of neural network and fuzzy logic to further analyze a published microarray study of gene responses to eight bacteria in human macrophages. The original analysis by hierarchical clustering found common gene responses to all bacteria but did not address individual responses. Our method allowed exploration of the gene response of the host to individual bacterium. We implemented a two-layer, feed-forward neural network containing the principle of 'competitive learning' (i.e. 'winner-take-all'). The weights of the trained neural network were fed into a fuzzy logic inference system. A new measurement, called the impact rating (IR) was also introduced to explore the degree of importance of each gene. To assess the reliability of the IR value, a bootstrap re-sampling method was applied to the dataset and a confidence level for each IR was obtained. Our approach has successfully uncovered the unique features of host response to individual bacterium. Further, application of gene ontology (GO) annotation to the genes of high IR values in each response has suggested new biological pathways for individual host-pathogen interactions.     

Comparison of sample-labeling techniques in DNA microarray experiments

Usage of DNA microarrays for gene expression analysis has become a common technique in many research laboratories and industry. Several target-labeling techniques have been devised to reduce the amount of RNA required for microarray experiments. In order to facilitate comparison and sharing of microarray data across the laboratories, it is crucial to determine the relative affects of these different sample-labeling techniques on the final results obtained from these experiments. We have compared two labeling methods designed for small RNA samples, an enzyme-based tyramide method (TSA) and a nucleic acid-based dendrimer method, to a more typical direct-labeling method that requires larger amounts of RNA. We observed comparable levels of reproducibility between replicate spots, with all the techniques. The dendrimer method resulted in a minimum number of spots (0.08%) that showed differential labeling due to a bias in the dyes used but resulted in highest background with only 71.4% of the spots measurable (above background) as compared to 93.3% for the TSA technique and 79.7% for the direct-labeling method. The results from differential labeling experiments showed that the dendrimer method performed better than the TSA method in detecting the same set of differentially expressed genes as observed with the direct method. Overall, our results show that the dendrimer method performs better than the TSA method. Differential labeling experiments using the TSA method show a non-linearity in the data at high intensities, leading to skewing of a portion of the data.     

Particle distribution in a microporous material: experiments with zeolite A.

The theory on particle distribution and exchange equilibria in a microporous material is applied to experimental ion-exchange data involving zeolite Na-A and zeolite K-A, with silver ions as the exchanging species. The presented method enables direct evaluation of the measured data and consideration of nonequivalent particle sites. The isotherms of the K+ versus Ag+ exchange in zeolite K-A rise much more steeply, at low exchange degrees, than those of the Na+ versus Ag+ exchange in zeolite Na-A. This result implies a different course of the ion-exchange reactions. Spectroscopic measurements on dehydrated, partly silver-exchanged zeolites Na-A and K-A do indeed show that in zeolite Na-A, the Na+ ions occupying four-ring positions are exchanged faster for Ag+ than the Na+ ions occupying eight- and six-ring positions, while in zeolite K-A the exchange does not start with the four-ring ion but with six-ring ions, followed by the four-ring ion. These findings are consistent with the results obtained from evaluation of the ion-exchange data. The resulting thermodynamic quantities significantly differ from published reference values, which we suggest should be revised.     

Electrochemical evidences and consequences of significant differences in ions diffusion rate in polyacrylate-based ion-selective membranes

The origin and effect of surface accumulation of primary ions within the ion-selective poly(n-butyl acrylate)-based membrane, obtained by thermal polymerization, is discussed. Using a new method, based on the relation between the shape of a potentiometric plot and preconditioning time, the diffusion of copper ions in the membrane was found to be slow (the diffusion coefficient estimated to be close to 10(-11) cm(2) s(-1)), especially when compared to ion-exchanger counter ions--sodium cations diffusion (a diffusion coefficient above 10(-9) cm(2) s(-1)). The higher mobility of sodium ions than those of the copper-ionophore complex results in exposed ion-exchanger role leading to undesirably exposed sensitivity to sodium or potassium ions.     

Variable selection using probability density function similarity for support vector machine classification of high-dimensional microarray data

One problem with discriminant analysis of microarray data is representation of each sample by a large number of genes that are possibly irrelevant, insignificant or redundant. Methods of variable selection are, therefore, of great significance in microarray data analysis. To circumvent the problem, a new gene mining approach is proposed based on the similarity between probability density functions on each gene for the class of interest with respect to the others. This method allows the ascertainment of significant genes that are informative for discriminating each individual class rather than maximizing the separability of all classes. Then one can select genes containing important information about the particular subtypes of diseases. Based on the mined significant genes for individual classes, a support vector machine with local kernel transform is constructed for the classification of different diseases. The combination of the gene mining approach with support vector machine is demonstrated for cancer classification using two public data sets. The results reveal that significant genes are identified for each cancer, and the classification model shows satisfactory performance in training and prediction for both data sets.     

Replication of a DNA microarray

A mechanical method for efficient replication of DNA microarrays is described. The approach consists of three steps. First, a master DNA microarray consisting of single-stranded DNA elements is exposed to a solution containing the biotin-functionalized complement of each array element. Following hybridization, a replica surface modified with streptavidin is brought into contact with the master. This results in linking of the biotin-functionalized complement with the replica surface. Next, the replica is separated from the master, and the complementary strands are transferred to the replica surface. The resulting complementary DNA microarray contains position-coded sequences that mirror the information contained on the master DNA microarray. Multiple replicas can be prepared from a single master, the replicas efficiently hybridize only their complement, and DNA not labeled with biotin is not transferred to the replica surface.     

Electron-transfer reactions with significant changes in structure. Unsymmetrical crowded ethylenes

The electrochemical reduction mechanisms of xanthylideneanthrone, 6, thioxanthylideneanthrone, 7, 10-(diphenylmethylene)anthrone, 8, and 9-(diphenylmethylene)-9H-fluorene, 9, have been studied in dimethylformamide. The reduction of the first two compounds proceeds from folded forms of the neutral to twisted forms of the anion radical according to a square scheme. The data for reduction of 8 can be well accounted for by the same square scheme. However, one-step reduction with concerted electron transfer and structural change cannot be ruled out. Compound 9, whose fluorene ring system cannot fold, exists only in twisted forms in the neutral, anion radical, and dianion. Consequently, there are no major changes in structure upon reduction, and the compound is reduced in two reversible steps with the second complicated by rapid loss of the dianion that is probably due to protonation by components of the medium.     

Low-cost nanomanipulator for in situ experiments in a SEM.

Here, we describe the development of an inexpensive and versatile manipulation system for in situ experiments in a field emission scanning electron microscope based on a parallel-guiding plate-spring mechanism and low cost materials. The system has been tested for a wide range of applications, such as collecting, moving, and positioning particles, fabricating atomic force microscopy tips based on carbon nanotubes, and characterizing individual nanobjects. The nanomanipulation results demonstrate that there are many opportunities for the use of physical manipulation in the bottom-up approach to fabrication of nanodevices.     

Home-built integrated microarray system (IMAS). A three-laser confocal fluorescence scanner coupled with a microarray printer

Microarray technology covers the urgent need to exploit the accumulated genetic information from large-scale sequencing projects and facilitate investigations on a genome-wide scale. Although most applications focus on DNA microarrays, the technology has expanded to microarrays of proteins, peptides, carbohydrates, and small molecules aiming either at detection/quantification of biomolecules or investigation of biomolecular interactions in a massively parallel manner. Microarray experiments require two specialized instruments: An arrayer (or printer), for construction of microarrays, and a readout instrument (scanner). We have designed, constructed, and characterized the first integrated microarray system (IMAS) that combines the functions of a microarrayer and a three-laser confocal fluorescence scanner into a single instrument and provides excellent flexibility for the researcher. The three-axis robotic system that moves the printing head carrying multiple pins for arraying is also used for moving the microarray slide in front of a stationary optical system during scanning. Since the translation stages are the most expensive and crucial components of microarray printers and scanners, the proposed design reduces considerably the cost of the instrument and enhances remarkably its operative flexibility. Experiments were carried out at resolutions of 2.5, 5, 10, and 20 μm. The scanner detects 0.128 nmol L⁻¹ carboxyfluorescein (spots with diameters of 70 μm) corresponding to 1.8 molecules μm⁻². The linear range extends over 3.5 orders of magnitude (R ² = 0.997) and the dynamic range covers almost five orders of magnitude. DNA microarray model experiments were carried out, including staining with SYBR Green I and hybridization with oligonucleotides labeled with the fluorescent dyes Alexa 488, Alexa 594, and Alexa 633. Figure Lay-out of the home-built integrated microarray system (IMAS). For the first time, the functions of a microarrayer (printer) and a three-laser confocal fluorescence scanner are combined into a single instrument. The three-axis robotic system that moves the printing head for arraying is also used to move the microarray slide in front of a stationary optical system during scanning. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Enzymatic profiling system in a small-molecule microarray

[reaction: see text] We have developed a microarray-based strategy for detection of three major classes of hydrolytic enzymes on the basis of their catalytic activities. This enables the sensitive detection of proteins not merely by their bindings but rather by their enzymatic activities. This may provide a valuable tool for screening, identification, and characterization of new enzymes in a high-throughput fashion.     

Strategies for immobilization of biomolecules in a microarray

Recent advances in the generation of peptide and protein microarrays are reviewed, with special focuses on different strategies available for site-specific immobilization of proteins and peptides.     

Development of a multiplex microarray microsystem

A hybrid multiplex microarray microsystem has been developed that consists of 32 individually addressable array reaction chambers, supporting the use of multichannel pipettes for addition of up to 8 samples simultaneously. Discrimination between Campylobacter jejuni and Campylobacter coli bacteria was observed in DNA samples containing Campylobacter spp., with the same specificity and sensitivity as when compared to a full-size microarray. The spinloaded multiplex microarray microsystem described provides a novel and convenient test format for simultaneous low-density microarray analysis and is universally adaptable to other DNA, protein or small molecule microarray based applications.     

Linking molecular models with ion mobility experiments. Illustration with a rigid nucleic acid structure

Ion mobility spectrometry experiments allow the mass spectrometrist to determine an ion's rotationally averaged collision cross section Ω_EXP. Molecular modelling is used to visualize what ion three‐dimensional structure(s) is(are) compatible with the experiment. The collision cross sections of candidate molecular models have to be calculated, and the resulting Ω_CALC are compared with the experimental data. Researchers who want to apply this strategy to a new type of molecule face many questions: (1) What experimental error is associated with Ω_EXP determination, and how to estimate it (in particular when using a calibration for traveling wave ion guides)? (2) How to generate plausible 3D models in the gas phase? (3) Different collision cross section calculation models exist, which have been developed for other analytes than mine. Which one(s) can I apply to my systems? To apply ion mobility spectrometry to nucleic acid structural characterization, we explored each of these questions using a rigid structure which we know is preserved in the gas phase: the tetramolecular G‐quadruplex [dTGGGGT]₄, and we will present these detailed investigation in this tutorial. © 2015 The Authors. Journal of Mass Spectrometry published by John Wiley & Sons Ltd.     

Improving the power to detect differentially expressed genes in comparative microarray experiments by including information from self-self hybridizations

Our ability to detect differentially expressed genes in a microarray experiment can be hampered when the number of biological samples of interest is limited. In this situation, we propose the use of information from self-self hybridizations to acuminate our inference of differential expression. A unified modelling strategy is developed to allow better estimation of the error variance. This principle is similar to the use of a pooled variance estimate in the two-sample t-test. The results from real dataset examples suggest that we can detect more genes that are differentially expressed in the combined models. Our simulation study provides evidence that this method increases sensitivity compared to using the information from comparative hybridizations alone, given the same control for false discovery rate. The largest increase in sensitivity occurs when the amount of information in the comparative hybridization is limited.     

An automated data collection system for membrane transport experiments : Part I. Test of onsager reciprocity

This work presents a direct experimental examination of the linear phenomenological flux equations from the thermodynamics of irreversible processes for membrane transport. Results are given which confirm Onsager reciprocity in a NaClH₂O—anion exchange membrane system with concentration and hydrostatic pressure differences. Data for isothermal, non-steady-state experiments are collected from a computerized and automated membrane transport apparatus. For the first time, the solvent and solute flux equations are solved simultaneously, and the four phenomenological transport coefficients are obtained from a single experiment using an ellipsoid algorithm for non-linear programming. It appears that the dependence of the L coefficients on the logarithmic mean transmembrane concentration, c?_se, is more important than the role of the water flux as an indicator of the limit of the linear region for membrane transport processes. Only in those experiments where c?_se was held nearly constant was Onsager reciprocity obtained.     

Biogenetically significant triterpenes in a species of meliaceae: Cabralea polytricha A.Juss.

Four triterpenes have been isolated from the fruits of Cabralea polytricha A.Juss. (Meliaceae). Two of them are new C₃₀ compounds whose structures have been established as the C-3, C-24 epimer of ocotillol-II and the C-24 epimer of ocotillone-II: 20S, 24S-epoxy-dammarane-3α,25-diol (IIa) and the corresponding 3-keto compound (IVa).