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We describe the hardware and software of a general-purpose interface that permits a personal computer (PC) running MS-DOS to control cytometric devices, e.g., a scanning stage, shutters, and focus motor attached to a microscope. This note explains how to use the interface to convert the ZONAX microscope attachment controller from 8080-based hardware control to PC-based control.
Recommendations are made for hardware and software capabilities that will permit a level of performance of comparative genomic hybridization (CGH) analysis on metaphase chromosomes that is comparable to the best current practice. Guidelines for interpreting the results of CGH analysis in terms of chromosomal gains or losses are also presented.
BACKGROUND: The analysis of cells from multiple experimental groups by multiparameter flow cytometry leads to the generation of complex data sets, for which adequate analysis tools are not commonly available. We report here that software designed for transcriptomics applications can be used in multiparameter flow cytometry. METHODS: Lymphocytes isolated from nine different mouse organs were stained and subjected to 10-parameter flow cytometry. The resulting data set contained 594 different T cell subsets per organ per mouse and was organized into a so-called flow cytometry array (FCA). RESULTS: Computation of a hierarchical tree revealed that lymph nodes and spleen were populated by similar T cell subsets, while T cells from peripheral organs displayed a diverse subset composition. Furthermore, organ-specific T cell subsets were identified. CONCLUSIONS: This new FCA concept in flow cytomics proved to be a valuable tool for the fast and unbiased analysis of complex multiparameter flow cytometry data sets. It can be used for assessing disease progression and therapeutic intervention, and for the association of disease-related biomarkers on the protein level.
We present a newly developed software called "QSNPlite" that comprehensively interprets the data of SSCP and sequencing analyses obtained from capillary array electrophoresis systems used in the quantitative characterization of SNPs. QSNPlite assists in the genotyping of individuals with SNPs and in estimating the allele frequencies of SNPs using pooled DNA. We show that this estimation is accurate (mean absolute error, 1.4%) by comparing the results of the pooled analysis using QSNPlite with the true frequencies based on the allele counting after performing individual genotypings. The QSNPlite program runs on Windows XP and can be used to determine the allele frequencies of SNPs among a large number of individuals, such as in association studies of disease-responsible genes using the candidate gene approach.
Hydroxyapatite, the major component of bone, demonstrates significant reactivity with metals. Knowledge of spatial structure and energy data of the molecule helps understand the binding of metals by hydroxyapatite and elucidate the chemical and physical properties of such complexes. We used HyperChem software (Hypercube Inc.) to analyze the structure of hydroxyapatite when the central calcium atom is replaced by one of the metal ions (Mg, Cu, Zn, Fe, Cr, Mn) marked by us in bone. Our results show that hetero-ionic exchange affects composition and leads to deformation of hydroxyapatite crystals. Replacement was accompanied by changes in bond lengths between oxygen and calcium atoms in the hydroxyapatite molecule and by displacement of groups of atoms surrounding the central calcium atom. The use of molecular modeling as a computational tool enabled a preliminary and theoretical understanding of chemical structure without the need for laboratory tests.
An ash study was performed to analyze the accuracy of forearm measurements at the one-third site of three dual x-ray absorptiometry (DXA) systems using 20 cadavers. A Hologic QDR-2000, a Hologic QDR-1000/W, and a Lunar DPX-L system were used. The correlations between ash weight and DXA BMC were excellent for the three instruments (r > 0.97, p < 0.001), with accuracy errors < 5.2%. To perform a forearm cross-calibration of bone mineral content (BMC) and density (BMD) between the scanners, 10 healthy volunteers were additionally scanned at the distal one-third radius. The correlations among the DXA machines were excellent (r > 0.95); the absolute BMC and BMD values were significantly different between the two Hologics and the Lunar machine. The slope and intercept of both the BMC and BMD between the two Hologic systems were close to unity. In conclusion, the DXA forearm software packages provide accurate methods for assessing bone mineral content and density. The conversion of data among different manufacturers should be performed by careful cross-calibration measurements.
The identification and structure elucidation of drug metabolites is one of the main objectives in in vitro ADME studies. Typical modern methodologies involve incubation of the drug with subcellular fractions to simulate metabolism followed by LC-MS/MS or LC-MS(n) analysis and chemometric approaches for the extraction of the metabolites. The objective of this work was the software-guided identification and structure elucidation of major and minor buspirone metabolites using capillary LC as a separation technique and ion trap MS(n) as well as electrospray ionization orthogonal acceleration time-of-flight (ESI oaTOF) mass spectrometry as detection techniques. Buspirone mainly underwent hydroxylation, dihydroxylation and N-oxidation in S9 fractions in the presence of phase I co-factors and the corresponding glucuronides were detected in the presence of phase II co-factors. The use of automated ion trap MS/MS data-dependent acquisition combined with a chemometric tool allowed the detection of five small chromatographic peaks of unexpected metabolites that co-eluted with the larger chromatographic peaks of expected metabolites. Using automatic assignment of ion trap MS/MS fragments as well as accurate mass measurements from an ESI oaTOF mass spectrometer, possible structures were postulated for these metabolites that were previously not reported in the literature.
The Quantitative Selected Ion Monitoring Processing System (QSIMPS) is a collection of software and hardware which was designed with the capacity to analyze 30,600 samples per year in support of pharmacokinetic studies. On a per sample basis, QSIMPS was designed to inject a sample into the GC, control the GC divert valve, collect selected ion monitoring data, identify the peaks for the drug and one metabolite and each compound's reference standard, fit the peaks to a relevant chromatographic model, calculate chromatographic features of merit, calculate the peak heights and ratio of the drug and its reference standard and the metabolite and its reference standard, and, using calibration data, convert the ratio to an amount of drug. On a per tray (batch) basis, QSIMPS was designed to fit all the peak height ratios from the calibration standards to either a linear equation, or a generalized nonlinear isotope dilution equation, report a statistical analysis of the fit, and, using aliquot factors, convert the measured amount of drug into concentrations. On a per project basis, QSIMPS prints reports summarizing statistical data on the calibration standards and the quality assurance samples, and prints reports presenting the concentration data as a function of, for examples, subject, drug treatment, time postdose, etc., along with other ancillary data such as subject sex, weight, species, etc. In addition, QSIMPS can fit the concentration data to a number of common pharmacokinetic model-derived equations, and report the resulting pharmacokinetic parameters along with a statistical comparison of the parameters.
This paper describes a new quantitation method called AQSES for short echo time magnetic resonance spectra. This method is embedded in a software package available online from www.esat.kuleuven.be/sista/members/biomed/new/ with a graphical user interface, under an open source license, which means that the source code is freely available and easy to adapt to specific needs of the user. The quantitation problem is mathematically formulated as a separable nonlinear least-squares fitting problem, which is numerically solved using a modified variable-projection procedure. A macromolecular baseline is incorporated into the fit via nonparametric modelling, efficiently implemented using penalized splines. Unwanted components such as residual water are removed with a maximum-phase FIR filter. Constraints on the phases, dampings and frequencies of the metabolites can be imposed. AQSES has been tested on simulated MR spectra with several types of disturbance and on short echo time in vivo proton MR spectra. Results show that AQSES is robust, easy to use and very flexible.
This article describes proton MR spectroscopic analysis of cerebrospinal fluid of 167 children suffering from meningitis and 24 control cases. Quantification of 12 well-separated and commonly observed cerebrospinal fluid metabolites viz., beta-hydroxybutyrate, lactate, alanine, acetate, acetone, acetoacetate, pyruvate, glutamine, citrate, creatine/creatinine, glucose (total) and urea was carried out using Bruker's NMRQUANT software with respect to a known concentration of sodium-3-(trimethylsilyl)-2,2,3,3-d4-propionate (TSP), serving as an external reference. The assignment of urea in CSF is reported for the first time by NMR. The presence of cyclopropane, observed for the first time in tuberculous meningitis overall in 85.1% of cases, acts as a finger-print marker for the differential diagnosis. Multivariate discriminant function analysis was carried out for the proton MR-detected metabolite information and the clinical symptoms data of the meningitis and control cases to find the important descriptors for classification, followed by a re-validation of the entire database. It was found that the control could be differentiated from the disease group with a success rate of 96.4%, followed by the differential diagnosis of tuberculous meningitis with a corresponding value of 77.2%. Excluding the presence of cyclopropane, bacterial meningitis could be classified 84.4% correct and viral meningitis with a rate of 83.3%. It is proposed that the NMR spectroscopic information, along with other routine clinical features, may serve as an additional diagnostic tool for the differential diagnosis of meningitis in children.
A software tool, Sweet Substitute, is described, which assists tandem mass spectrometry (MS/MS)-based glycosylation characterization from within a tryptic digest. The algorithm creates a virtual nanoelectrospray-quadrupole time-of-flight style-MS/MS spectrum of any user-defined N-linked glycan structure. An empirical peak height modeling routine is implemented in the program. By comparing the theoretical MS/MS data with the deconvoluted and deisotoped experimental MS/MS data, the user is able to quickly assess whether a proposed candidate oligosaccharide structure is a plausible one.
Images obtained from high-throughput mass spectrometry (MS) contain information that remains hidden when looking at a single spectrum at a time. Image processing of liquid chromatography-MS datasets can be extremely useful for quality control, experimental monitoring and knowledge extraction. The importance of imaging in differential analysis of proteomic experiments has already been established through two-dimensional gels and can now be foreseen with MS images. We present MSight, a new software designed to construct and manipulate MS images, as well as to facilitate their analysis and comparison.
The MultiTag method (Sunyaev et al., Anal. Chem. 2003 15, 1307-1315) employs multiple error-tolerant searches with peptide sequence tags (Mann and Wilm, Anal. Chem. 1994, 66, 4390-4399) for the identification of proteins from organisms with unsequenced genomes. Here we demonstrate that the error-tolerant capabilities of MultiTag increased the number of peptide alignments and improved the confidence of identifications in an EST database. The MultiTag outperformed conventional database searching software that only utilizes stringent matching of tandem mass spectra to nucleotide sequences of ESTs.
Whole-cell protein quantification using MS has proven to be a challenging task. Detection efficiency varies significantly from peptide to peptide, molecular identities are not evident a priori, and peptides are dispersed unevenly throughout the multidimensional data space. To overcome these challenges we developed an open-source software package, MapQuant, to quantify comprehensively organic species detected in large MS datasets. MapQuant treats an LC/MS experiment as an image and utilizes standard image processing techniques to perform noise filtering, watershed segmentation, peak finding, peak fitting, peak clustering, charge-state determination and carbon-content estimation. MapQuant reports abundance values that respond linearly with the amount of sample analyzed on both low- and high-resolution instruments (over a 1000-fold dynamic range). Background noise added to a sample, either as a medium-complexity peptide mixture or as a high-complexity trypsinized proteome, exerts negligible effects on the abundance values reported by MapQuant and with coefficients of variance comparable to other methods. Finally, MapQuant's ability to define accurate mass and retention time features of isotopic clusters on a high-resolution mass spectrometer can increase protein sequence coverage by assigning sequence identities to observed isotopic clusters without corresponding MS/MS data.
Rapid analysis of molecular mass distributions of triacylglycerol (TAG) mixtures and regioisomeric structures of selected molecular mass species is possible using ammonia negative ion chemical ionization mass spectrometry utilizing sample introduction by direct exposure probe. However, interpretation of spectra and calculation of results is time consuming, thus lengthening the total analysis time. To facilitate result calculation a software package (MSPECTRA 1.3) was developed and applied to automatic processing of triacylglycerol molecular mass distribution spectra and collision induced dissociation (CID) product ion spectra. The program is capable of identifying triacylglycerol molecular mass species possessing different ACN:DB (acyl carbon number:number of double bonds) ratios on the basis of m/z values of [M - H](-) ions. In addition to such identification the program also corrects spectra for abundances of naturally occurring (13)C isotopes and calculates relative proportions of triacylglycerol molecular species in the analyzed samples. If several replicate spectra are processed simultaneously the program automatically calculates an average and standard deviation of relative proportions of molecular species. In the case of CID spectra the program identifies fatty acid fragment ions [RCO(2)](-) and the corresponding [M - H - RCO(2)H - 100](-) ions, and calculates the relative proportions of ions in both groups. These proportions are then used automatically to calculate the fatty acid combinations comprising the parent triacylglycerol molecule and the regiospecific positions of fatty acids. Processing of several replicate product ion spectra simultaneously produces averaged proportions of regioisomers comprising the parent triacylglycerol molecular species and the standard deviation of the analysis. The performance of the program was tested by analyzing triacylglycerol samples of human milk, human milk substitutes, human chylomicron and cocoa butter, and by comparing results obtained by automated processing of the data with manually calculated results.
In order for national statistical offices to maintain the trust of the public to collect data and publish statistics of importance to society and decision-making, it is imperative that respondents (persons or establishments) be guaranteed privacy and confidentiality in return for providing requested confidential data. Consequently, for most survey and census data, disclosure limitation techniques must be applied before the data are ready for public release. For microdata, examples of methods that can be used to identify respondents include directly extracting identifying information from microdata files or indirectly identifying respondents by matching a given file with an external file. For tabular data, respondents may be identified directly from small cell counts or respondent contributions to heavily concentrated cells of magnitude data may be closely approximated by the cell value. Indirect disclosure is possible in tables through manipulation of additive tabular relationships between cell values and totals, e.g. manipulating rows and column totals in a two-dimensional table. Two-dimensional statistical tables are a staple of official statistics. This paper describes a desktop software system that for the first time implements within a single framework four standard disclosure limitation techniques for protecting tabular data in two-dimensional tables: complementary cell suppression, minimum-distance controlled rounding, unbiased controlled rounding, and controlled rounding subject to subtotals constraints, and a fifth, new method: controlled tabular adjustment, and summarizes the five methods.
Determining ionic concentrations in buffered solutions usually reduces to solving a set of simultaneous polynomial equations. Mathematica software offers a convenient method for doing this. Using buffering of Ca2+ by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) as an example, we provide a Mathematica script to estimate the apparent association constant. A second example shows how to calculate free ion concentrations when two ligands (Ca2+ and Mg2+) compete for one chelator (EGTA). Finally, the concentrations of all species are determined in a complex mixture containing Ca2+, EGTA, and calmodulin, a protein with four Ca(2+)-binding sites. Modifying the examples presented should allow analysis of most practical buffering problems.
A package of computer programs for analysis and visualization of three-dimensional human brain functional magnetic resonance imaging (FMRI) results is described. The software can color overlay neural activation maps onto higher resolution anatomical scans. Slices in each cardinal plane can be viewed simultaneously. Manual placement of markers on anatomical landmarks allows transformation of anatomical and functional scans into stereotaxic (Talairach-Tournoux) coordinates. The techniques for automatically generating transformed functional data sets from manually labeled anatomical data sets are described. Facilities are provided for several types of statistical analyses of multiple 3D functional data sets. The programs are written in ANSI C and Motif 1.2 to run on Unix workstations.