Analysis techniques for microarray time-series data.

We address possible limitations of publicly available data sets of yeast gene expression. We study the predictability of known regulators via time-series analysis, and show that less than 20% of known regulatory pairs exhibit strong correlations in the Cho/Spellman data sets. By analyzing known regulatory relationships, we designed an edge detection function which identified candidate regulations with greater fidelity than standard correlation methods. We develop general methods for integrated analysis of coarse time-series data sets. These include 1) methods for automated period detection in a predominately cycling data set and 2) phase detection between phase-shifted cyclic data sets. We show how to properly correct for the problem of comparing correlation coefficients between pairs of sequences of different lengths and small alphabets. Finally, we note that the correlation coefficient of sequences over alphabets of size two can exhibit very counterintuitive behavior when compared with the Hamming distance.     

New techniques for DNA sequence classification.

DNA sequence classification is the activity of determining whether or not an unlabeled sequence S belongs to an existing class C. This paper proposes two new techniques for DNA sequence classification. The first technique works by comparing the unlabeled sequence S with a group of active motifs discovered from the elements of C and by distinction with elements outside of C. The second technique generates and matches gapped fingerprints of S with elements of C. Experimental results obtained by running these algorithms on long and well conserved Alu sequences demonstrate the good performance of the presented methods compared with FASTA. When applied to less conserved and relatively short functional sites such as splice-junctions, a variation of the second technique combining fingerprinting with consensus sequence analysis gives better results than the current classifiers employing text compression and machine learning algorithms.     

Comparative evaluation of techniques for the harmonic analysis of human motion data

Four mathematical techniques for the estimation of the Fourier coefficients of pseudoperiodic non-exact discrete functions were submitted to comparative evaluation. These techniques were devised within the following basic procedures: (1) numerical harmonic analysis applied either directly or after redistribution of the data points through some interpolation procedure, so that they be evenly spaced in time and exactly fit on cycle period; (2) fitting of the empirical data with an analytical model followed by the calculation of the Fourier integrals of this model. The evaluation was carried out with special reference to the use of these techniques for processing human motion photogrammetric data. The following criteria were used: (1) accuracy of the Fourier coefficient estimates, (2) capability of yielding information about this accuracy, (3) a priori information needed regarding the statistical properties of the experimental error, (4) factors concerning implementation in digital computers. Practical information was obtained for the non-specialist user with regard to the choice of one technique among the several possible in particular circumstances.     

New method for the analysis of flow cytometric data

A numerical method for deriving the fractions of cells in different phases of the cell cycle from a single observed DNA histogram is presented. The observed histogram is regarded as a polluted version (containing allocation errors) of the true histogram. A mathematical model is used to describe the pollution process. A theoretical histogram, representing the true histogram, is constructed so that G1 cells are put into one channel and G2M cells into another; the distribution of S cells in between is approximated with a set of harmonic functions. This theoretical histogram is subsequently disturbed with Gaussian dispersion functions to stimulate the pollution, yielding a predicted histogram. Using a maximum likelihood estimation technique, the model parameters are adjusted iteratively, matching the predicted histogram to the actually observed one. With the final parameter values substituted, the corresponding final theoretical histogram is regarded as a reliable reconstruction of the true histogram. From the latter, the required percentages can be read directly. The advantage of this approach over other mathematical analysis methods is that it allows a wide range of different, continuous distributions for relatively few model parameters (thus featuring flexibility and realism and a diminished risk of encountering computational problems). In addition, estimation errors providing a measure of accuracy can be obtained. To test the method, it was used to analyze various observed histograms from the literature that have been obtained by either simulation or actual flow cytometric measurements. The method appeared to perform well, as compared to the reported results of several other methods of analysis applied to the same data.     

Electrophysiological analysis of retrieval of conditioned taste aversion. Physiological techniques and data processing.

New techniques suitable for electrophysiological investigation of retrieval of conditioned taste aversion (CTA) were developed. The gustatory discrimination apparatus consisted of two parallel drinking spouts (20 mm apart) equipped with photoelectric lick sensors. The spouts contained water or the aversive fluid (0.15 M LiCl), respectively, and their position could be rapidly (50 ms) interchanged with a reversive electromotor. Licking at either spout and the position of the fluids were recorded on one channel of a tape recorder. Unit activity was picked up with tungsten microelectrodes (40 micrometers) inserted into the brain of a freely moving rat with a head-mounted microdrive system (weight 2 g), fixed to an implanted guiding cannula. The electrode was connected through a head-carried FET signal follower to a wide band integrated circuit amplifier and the unit activity was recorded in the other channel of the tape recorder. The records were evaluated using an off-line computer program (LINC 8) consisting of a spike detection subroutine followed by amplitude histogram analysis. The mean (M) and SD values were computed for uni- or bimodal amplitude distributions of the principal spike components. Spike falling within the M+/-2 SD range of the selected parameter were identified as single units and used for construction of post stimulus histograms triggered by licking or by spout switching.     

New approach to the statistical analysis of cardiovascular data.

Fourier-based approaches to analysis of variability of R-R intervals or blood pressure typically compute power in a given frequency band (e.g., 0.01-0.07 Hz) by aggregating the power at each constituent frequency within that band. This paper describes a new approach to the analysis of these data. We propose to partition the blood pressure variability spectrum into more narrow components by computing power in 0.01-Hz-wide bands. Therefore, instead of a single measure of variability in a specific frequency interval, we obtain several measurements. The approach generates a more complex data structure that requires a careful account of the nested repeated measures. We briefly describe a statistical methodology based on generalized estimating equations that suitably handles this more complex data structure. To illustrate the methods, we consider systolic blood pressure data collected during psychological and orthostatic challenge. We compare the results with those obtained using the conventional methods to compute blood pressure variability, and we show that our approach yields more efficient results and more powerful statistical tests. We conclude that this approach may allow a more thorough analysis of cardiovascular parameters that are measured under different experimental conditions, such as blood pressure or heart rate variability.     

Advances in techniques for ecdysteroid analysis

Summary

Recent improvements in chromatographic methods for ecdysteroids are reviewed. Minor improvements in liquid chromatography have increased the separation and range of compounds which can be accomodated, some useful techniques have been introduced in thin layer chromatography, and with new radioactivity scanners and direct mass spectrometry from TLC, this technique retains an important place among the tools of the ecdysonist. Supercritical fluid chromatography has been suggested as a rapid and more sensitive alternative to liquid chromatography. In mass spectrometry, fast atom bombardment has proved most useful, though coupling HPLC through thermospray and ptasmaspray methods has been attempted, but as yet, these techniques lack sensitivity. New solid phase immunoassay methods using colorimetric rather than radioactivity measurements promise to give a better means of analysis of small amounts of ecdysteroids, capable of use in the presence of radiolabeled compounds. A solid phase separation of C-20, 22 diol ecdysteroids with immobilized phenylboronic acid has been reported.     

Regression approaches for microarray data analysis.

A variety of new procedures have been devised to handle the two-sample comparison (e.g., tumor versus normal tissue) of gene expression values as measured with microarrays. Such new methods are required in part because of some defining characteristics of microarray-based studies: (i) the very large number of genes contributing expression measures which far exceeds the number of samples (observations) available and (ii) the fact that by virtue of pathway/network relationships, the gene expression measures tend to be highly correlated. These concerns are exacerbated in the regression setting, where the objective is to relate gene expression, simultaneously for multiple genes, to some external outcome or phenotype. Correspondingly, several methods have been recently proposed for addressing these issues. We briefly critique some of these methods prior to a detailed evaluation of gene harvesting. This reveals that gene harvesting, without additional constraints, can yield artifactual solutions. Results obtained employing such constraints motivate the use of regularized regression procedures such as the lasso, least angle regression, and support vector machines. Model selection and solution multiplicity issues are also discussed. The methods are evaluated using a microarray-based study of cardiomyopathy in transgenic mice.     

New techniques for whole-mount NADPH-diaphorase histochemistry demonstrated in insect ganglia.

Fixation-resistant NADPH-diaphorase (NADPHd) activity is used widely as a marker for nitric oxide synthase (NOS). In frozen sections, NADPHd histochemistry yields high anatomic definition. In whole-mounts, however, poor penetration of the reagents, background staining, and tissue opacity severely limit its application. Here we report a combination of new methods that significantly improves whole-mount NADPHd staining. We demonstrate these methods in the thoracic ganglia of a large insect, the locust Schistocerca gregaria, in which NADPHd has been analyzed previously using both whole-mounts and serial section reconstructions. The penetration of the staining reagents was markedly improved after fixation in methanol/formalin compared to phosphate-buffered formaldehyde. Methanol/formalin also reduced nonspecific NADPHd and enhanced the selective staining. Penetration was further enhanced by incubation regimens that exploit the temperature- or pH-dependence of NADPHd. In combination with methanol/formalin fixation, this permitted staining to develop evenly throughout these comparatively large invertebrate ganglia. These improvements were complemented by a new clearing technique that preserves the NADPHd staining, gives excellent transparency, and avoids distortion of specimen morphology. The new methods revealed the three-dimensional architecture of NADPHd expression in locust ganglia in unprecedented detail and may similarly improve whole-mount detection of NADPHd in other invertebrate and vertebrate preparations.     

Real-time three-dimensional ultrasound methods for shape analysis and visualization.

Real-time three-dimensional (RT3D) ultrasound is a relatively new imaging modality that uses a special ultrasound transducer consisting of a matrix array of elements. The array electronically steers an ultrasound beam to interrogate a 3D volume of tissue. The real-time nature of RT3D ultrasound differentiates it from reconstructed 3D ultrasound, in which a conventional ultrasound transducer is moved mechanically through the third dimension. RT3D ultrasound is considerably faster than reconstructed 3D ultrasound, making it suitable for capturing continuous rapid motion such as that of the beating heart. Although RT3D ultrasound has not yet found widespread clinical use, these scanners are presently employed in more than 20 locations worldwide, primarily for cardiac research. The author helped develop the RT3D ultrasound technology as well as specialized analysis and visualization methods for the resulting data. In developing such methods, it has been necessary to consider the physical and mathematical processes by which the ultrasound data are collected. Difficulties arise because of high noise, variation in contrast and intensity between scans, ultrasound's nonrectilinear coordinate system, and the anisotropic nature of the echoes themselves. This article reviews these specific difficulties and provides solutions that are applicable to generalized analysis and visualization of RT3D ultrasound data. Some of the methods presented can also be applied to other imaging modalities with nonrectilinear coordinates.     

Multimegapixel images in histopathology.

OBJECTIVE: To describe methods and procedures for the assembly of very large scale microscopic image arrays. STUDY DESIGN: Microscopic imagery was recorded on different video microphotometers, equipped either with a three-chip CCD Sony MD 760 (Park-ridge, New Jersey, U.S.A.), a COHU vidicon (San Diego, California, U.S.A.) or a PROGRES camera (JenOptik, Jena, Germany), yielding image tiles of 512 x 470, 512 x 470 or 1,496 x 1,120 pixels, respectively. The slide was moved while mounted on a Maerzheuser scanning stage with 0.1-micron precision, under computer control. The MERGE software. (Optical Sciences Center, University of Arizona, Tucson, Arizona, U.S.A.) was written in C and currently implemented on a Sun. Ultra Sparc 2 computer (Sun Microsystems, Palo Alto, California, U.S.A.). RESULTS: The MERGE program allows the assembly of very large scale digitized image arrays preserving exact tile alignment such that even within a single nucleus, highly precise registration is maintained. Images up to 150 megapixels have been assembled, although most practical applications required assembly of only 60-300 tiles. CONCLUSION: The single limiting effect of assembling very large image arrays is the problem of angular misalignment between CCD scan line orientation and scanning stage travel direction. For misalignment of even less than 1 degree, very large arrays need substantial tile overlap. For object areas extending over only 5-10 mm, the effects can be controlled.     

Segmentation of two- and three-dimensional data from electron microscopy using eigenvector analysis

An automatic image segmentation method is used to improve processing and visualization of data obtained by electron microscopy. Exploiting affinity criteria between pixels, e.g., proximity and gray level similarity, in conjunction with an eigenvector analysis, the image is subdivided into areas which correspond to objects or meaningful regions. Extending a proposal by Shi and Malik (1997, Proceedings of the IEEE conference on Computer Vision and Pattern Recognition, pp. 731-737) the approach was adapted to the field of electron microscopy, especially to three-dimensional application as needed by electron tomography. Theory, implementation, parameter setting, and results obtained with a variety of data are presented and discussed. The method turns out to be a powerful tool for visualization with the potential for further improvement by developing and tuning new affinity.     

Small-scale techniques for the analysis of recombinant plasmids.

Using the cloning of part of the T-DNA of pTiC58 from Agrobacterium tumefaciens as an example, techniques are described which enable recombinant plasmids to be mapped and used as hybridization probes. In all cases the starting material is a colony of cells grown on an agar plate which is then subjected to lysis by lysozyme and Triton X-100 in volumes of the order of 300 microliters thus eliminating the need for handling and centrifuging liquid cultures under restrictive containment conditions.     

Pathogenic variability of downy mildew (Sclerospora graminicola) on pearl millet. II. Statistical techniques for analysis of data

Different cultivars of pearl millet were exposed to infection by asexual propagules of Sclerospora graminicola in Polythene tunnels. Subsequent incidence of disease was measured on one-month old seedlings. The data consisted of binomial proportions based on differing total numbers of seedlings. It is shown how to utilise the logit link in the GLIM statistical package for analysis to compare variety performances. In addition the possibilities of interplot interference and intraplot variation were investigated using GLIM. Interplot interference occurred infrequently and did not affect the rank order of the cultivars in terms of their response to the fungus infection. Intraplot variation was also shown to be non significant in spite of proximity of some plants to the disease spreader plants. Once these potentially confounding phenomena were eliminated the cultivars could be compared directly with impunity. GLIM was also used to confirm that the type of symptom expression was a stable feature of each host genotype.     

New techniques in fast time-resolved structure determination.

New techniques in fast time-resolved X-ray crystallography provide a different approach to understanding the structural basis of protein function. Two biological systems have been studied as part of the refinement of these techniques, and have actually spurred new ideas in time-resolved structural studies. The dissociation of carbon monoxide from carbon-monoxy myoglobin has earlier been investigated over a time range spanning 18 orders of magnitude (femtoseconds to hours) using spectroscopic methods. Rapid time-resolved determination of the entire myoglobin structure made it possible to determine both the position of the CO after photodissociation and the entire globin structure, over a time range from nanoseconds to milliseconds, during which the heme and globin relax and the carbon monoxide rebinds. Photoactive yellow protein, a relative newcomer to biophysical research, has a fully-reversible photocycle containing several spectrally distinct intermediates. Identifying and solving the structures of each intermediate is the initial goal in time-resolved studies on this protein and will contribute to a greater understanding of the biological process of light driven signal transduction.     

New techniques for physical mapping of the human genome.

We describe improvements in techniques and strategies used for making maps of the human genome. The methods currently used are changing and evolving rapidly. Today's techniques can produce ordered arrays of DNA fragments and overlapping sets of DNA clones covering extensive genomic regions, but they are relatively slow and tedious. Methods under development will speed the process considerably. New developments include a range of applications of the polymerase chain reaction, enhanced procedures for high resolution in situ hybridization, and improved methods for generating, manipulating, and cloning large DNA fragments. More detailed genetic and physical maps will be useful for finding genes, including those associated with human diseases, long before the complete DNA sequence of the human genome is available.     

信息物质的化学分析技术

化学生态学研究中最常应用的化学分析技术是气相色谱-质谱联用技术(GC-MS)和高压液相色谱技术(HPLC)。本文根据实践经验和体会,重点介绍样品制备要点、GC-MS和HPLC的基本原理、如何选择化学分析技术、信息化学物质定性和定量分析的要点以及示例等。