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PubMedDate not supplied

Autofluorescence removal, multiplexing, and automated analysis methods for in-vivo fluorescence imaging.

The ability to image and quantitate fluorescently labeled markers in vivo has generally been limited by autofluorescence of the tissue. Skin, in particular, has a strong autofluorescence signal, particularly when excited in the blue or green wavelengths. Fluorescence labels with emission wavelengths in the near-infrared are more amenable to deep-tissue imaging, because both scattering and autofluorescence are reduced as wavelengths are increased, but even in these spectral regions, autofluorescence can still limit sensitivity. Multispectral imaging (MSI), however, can remove the signal degradation caused by autofluorescence while adding enhanced multiplexing capabilities. While the availability of spectral "libraries" makes multispectral analysis routine for well-characterized samples, new software tools have been developed that greatly simplify the application of MSI to novel specimens.

Algorithms↗
PubMed1997

A Peptide from the Male Accessory Gland in Leptinotarsa decemlineata: Purification, Characterization and Molecular Cloning.

Our interest in the male accessory glands (MAGs) of Leptinotarsa decemlineata was raised recently by our finding that certain cells produce a secretory substance that is recognized by one of our monoclonal antibodies (MAC-18), developed for the immunohistochemical demonstration of peptidergic neurons in the brain. We undertook to isolate this substance, presumably a peptide, to find out more about its role in the post-mating physiology of the recipient of this peptide, the mated female. This paper describes the purification and chemical characterization of the immunoreactive peptide from 100 pairs of male accessory glands. The peptide was purified by two subsequent reversed-phase-HPLC runs, and fractions were analyzed on Western blots that were immunostained by MAC-18. This indicated the presence of an 8 kDa peptide in the MAG. Partial analysis of the N-terminal amino acids by automated Edman degradation revealed a sequence of 40 amino acid residues. To obtain the full amino acid sequence of this peptide, the technique of reverse transcriptase PCR (3'RACE) was used. A PCR product of 350 bp was obtained, which encoded the 3'-end of the mRNA. After cloning and sequencing, this product contained most of the genetic information of the MAG peptide. The PCR product was also used as a probe for screening a cDNA library constructed from mRNA extracted from MAGs. The nucleotide sequence coding for the signal peptide was elucidated by 5'RACE. The cDNA and 5'RACE clones were analyzed and sequenced. The sequence of the cDNA clone contained an insert of 411 bp, which agreed well with the mRNA size measured by Northern blotting. Translation of the DNA sequences confirmed the data from partial amino acid sequence analyses and also predicted the remainder of the amino acid sequence. The entire peptide, designated Led-MAGP, consists of 74 residues; its mass was calculated and confirmed by mass spectrometry at 7971 Da. The peptide contains seven imperfect hexa-repeats, and this hexa-repeat sequence shows remarkable similarity to the hexa-repeat section of the chicken prion protein. The physiological function of the peptide has yet to be determined, but the hexa-repeat motif has recently been identified as the signal that induces internalization of the prion protein by coated-pit mediated endocytosis. Possible implications for the control of reproductive activities in L. decemlineata are discussed. Copyright 1997 Elsevier Science Ltd. All rights reserved

Journal Article↗
PubMed1991

Identification of drugs in physiological fluids following on-line liquid chromatographic purification and analysis.

This paper describes the application of an automated algorithm for drug identification following the liquid chromatographic on-line separation and purification of drugs in physiological fluids. Two hundred fifty-seven (257) drugs and 45 drug metabolites were assembled for testing. Two internal standards were used which monitored different columns in the multi-step analytical separation. Retention reproducibility over a nine month period was 4 p. cent or less for most drugs. The algorithm for matching unknown peaks against the stored library utilized relative retention, lambda maxima, normalized spectral overlays, wavelength ratios and zero-intercepts from the second derivative in a combined forward-reverse search. Most of the drugs which could not be unambiguously identified were sedatives which eluted in the first minutes of the chromatogram. Diasteromers were partially resolved. Repetitive analysis of 200-400 micrograms/l of amphetamine, diphenhydramine, imipramine and morphine were evaluated to establish an absorption cutoff of 12 mAbs, above which at least 95% of peaks were correctly identified. This corresponded to a signal-to-noise ratio of 12 for the scanning UV detector which was evaluated.

Algorithms↗
PubMed2003

Cell and organ printing 1: protein and cell printers.

We have developed several devices for positioning organic molecules, molecular aggregates, cells, and single-cell organisms onto solid supports. These printers can create stable, functional protein arrays using an inexpensive technology. The cell printer allows us to create cell libraries as well as cellular assemblies that mimic their respective position in organs. The printers are derived from commercially available ink-jet printers that are modified to dispense protein or cell solutions instead of ink. We describe here the modifications to the print heads, and the printer hardware and software that enabled us to adapt the ink-jet printers for the manufacture of cell and protein arrays. The printers have the advantage of being fully automated and computer controlled, and allow for the high-throughput manufacture of protein and cell arrays.

Animals↗
PubMed1998

Packing of sidechains in low-resolution models for proteins.

BACKGROUND: Atomic level rotamer libraries for sidechains in proteins have been proposed by several groups. Conformations of side groups in coarse-grained models, on the other hand, have not yet been analyzed, although low resolution approaches are the only efficient way to explore global structural features. RESULTS: A residue-specific backbone-dependent library for sidechain isomers, compatible with a coarse-grained model, is proposed. The isomeric states are utilized in packing sidechains of known backbone structures. Sidechain positions are predicted with a root-mean-square deviation (r.m.s.d.) of 2.40 A with respect to crystal structure for 50 test proteins. The rmsd for core residues is 1.60 A and decreases to 1.35 A when conformational correlations and directional effects in inter-residue couplings are considered. CONCLUSIONS: An automated method for assigning sidechain positions in coarse-grained model proteins is proposed and made available on the internet; the method accounts satisfactorily for sidechain packing, particularly in the core.

Animals↗
PubMed2005

Neutron analysis of spent fuel storage installation using parallel computing and advance discrete ordinates and Monte Carlo techniques.

In the United States, the Nuclear Waste Policy Act of 1982 mandated centralised storage of spent nuclear fuel by 1988. However, the Yucca Mountain project is currently scheduled to start accepting spent nuclear fuel in 2010. Since many nuclear power plants were only designed for -10 y of spent fuel pool storage, > 35 plants have been forced into alternate means of spent fuel storage. In order to continue operation and make room in spent fuel pools, nuclear generators are turning towards independent spent fuel storage installations (ISFSIs). Typical vertical concrete ISFSIs are -6.1 m high and 3.3 m in diameter. The inherently large system, and the presence of thick concrete shields result in difficulties for both Monte Carlo (MC) and discrete ordinates (SN) calculations. MC calculations require significant variance reduction and multiple runs to obtain a detailed dose distribution. SN models need a large number of spatial meshes to accurately model the geometry and high quadrature orders to reduce ray effects, therefore, requiring significant amounts of computer memory and time. The use of various differencing schemes is needed to account for radial heterogeneity in material cross sections and densities. Two P3, S12, discrete ordinate, PENTRAN (parallel environment neutral-particle TRANsport) models were analysed and different MC models compared. A multigroup MCNP model was developed for direct comparison to the SN models. The biased A3MCNP (automated adjoint accelerated MCNP) and unbiased (MCNP) continuous energy MC models were developed to assess the adequacy of the CASK multigroup (22 neutron, 18 gamma) cross sections. The PENTRAN SN results are in close agreement (5%) with the multigroup MC results; however, they differ by -20-30% from the continuous-energy MC predictions. This large difference can be attributed to the expected difference between multigroup and continuous energy cross sections, and the fact that the CASK library is based on the old ENDF/B-II library. Both MC and SN calculations were run in parallel on a BEOWULF PC-cluster (eight processors). Timing results indicate that the SN calculation yielded a detailed dose distribution at over 318,426 points in -164 h. Unbiased continuous energy MC required 214 h to calculate dose rates with a 1% relative error in only 18 regions on the surface of the cask. The biased A3MCNP calculations yields dose rates with -0.8% relative error in only 2.5 h on one processor. This study demonstrates that a parallel code, such as the 3-D parallel SN transport code, PENTRAN can solve a complex large problem, such as the storage cask, accurately and efficiently. Moreover, this calculation was performed on a relatively inexpensive PC-cluster. Possible inadequacies of the CASK cross section library still need to be evaluated.

Computer-Aided Design↗
PubMed2005

Molecular classification of human gliomas using matrix-based comparative genomic hybridization.

Gliomas are the most frequent primary brain tumors and comprise a group of morphologically, biologically and clinically heterogeneous neoplasms. The different glioma types are associated with distinct genetic aberrations, which may provide useful information for tumor classification as well as prediction of prognosis and response to therapy. To facilitate the molecular classification of gliomas, we established a genomic microarray that consists of bacterial artificial chromosome (BAC) and P1-derived artificial chromosome (PAC) clones representing tumor suppressor genes, proto-oncogenes and chromosomal regions frequently gained or lost in gliomas. In addition, reference clones distributed evenly throughout the genome in approximately 15 Mbp intervals were spotted on the microarray. These customized microarrays were used for matrix-based comparative genomic hybridization (matrix CGH) analysis of 70 gliomas. Matrix CGH findings were validated by molecular genetic analyses of candidate genes, loss of heterozygosity studies and chromosomal CGH. Our results indicate that matrix CGH allows for the sensitive and specific detection of gene amplifications as well as low-level copy number gains and losses in clinical glioma samples. Furthermore, molecular classification based on matrix CGH data closely paralleled histological classification and was able to distinguish with few exceptions between diffuse astrocytomas and oligodendrogliomas, anaplastic astrocytomas and anaplastic oligodendrogliomas, anaplastic oligodendrogliomas and glioblastomas, as well as primary and secondary glioblastomas. Thus, matrix CGH is a powerful technique that allows for an automated genomic profiling of gliomas and represents a promising new tool for their molecular classification.

Adult↗
PubMed2003

Comparisons and validation of statistical clustering techniques for microarray gene expression data.

MOTIVATION: With the advent of microarray chip technology, large data sets are emerging containing the simultaneous expression levels of thousands of genes at various time points during a biological process. Biologists are attempting to group genes based on the temporal pattern of their expression levels. While the use of hierarchical clustering (UPGMA) with correlation 'distance' has been the most common in the microarray studies, there are many more choices of clustering algorithms in pattern recognition and statistics literature. At the moment there do not seem to be any clear-cut guidelines regarding the choice of a clustering algorithm to be used for grouping genes based on their expression profiles. RESULTS: In this paper, we consider six clustering algorithms (of various flavors!) and evaluate their performances on a well-known publicly available microarray data set on sporulation of budding yeast and on two simulated data sets. Among other things, we formulate three reasonable validation strategies that can be used with any clustering algorithm when temporal observations or replications are present. We evaluate each of these six clustering methods with these validation measures. While the 'best' method is dependent on the exact validation strategy and the number of clusters to be used, overall Diana appears to be a solid performer. Interestingly, the performance of correlation-based hierarchical clustering and model-based clustering (another method that has been advocated by a number of researchers) appear to be on opposite extremes, depending on what validation measure one employs. Next it is shown that the group means produced by Diana are the closest and those produced by UPGMA are the farthest from a model profile based on a set of hand-picked genes. AVAILABILITY: S+ codes for the partial least squares based clustering are available from the authors upon request. All other clustering methods considered have S+ implementation in the library MASS. S+ codes for calculating the validation measures are available from the authors upon request. The sporulation data set is publicly available at http://cmgm.stanford.edu/pbrown/sporulation

Algorithms↗
PubMed2003

"Multi-component reactions : emerging chemistry in drug discovery" 'from xylocain to crixivan'.

With the recent emergence of combinatorial chemistry and high-speed parallel synthesis for drug discovery applications, the multi-component reaction (MCR) has seen a resurgence of interest. Easily automated one-pot reactions, such as the Ugi and Passerini reactions, are powerful tools for producing diverse arrays of compounds, often in one step and high yield. Despite this synthetic potential, the Ugi reaction is limited by producing products that are flexible and peptide-like, often being classified as 'non drug-like'. This review details developments of new, highly atom-economic MCR derived chemical methods, which enable the fast and efficient production of chemical libraries comprised of a variety of biologically relevant templates. Representative examples will also be given demonstrating the successful impact of MCR combinatorial methods at different stages of the lead discovery, lead optimization and pre-clinical process development arenas. This will include applications spanning biological tools, natural products and natural product-like diversity, traditional small molecule and 'biotech' therapeutics respectively. In particular, this review will focus on applications of isocyanide based MCR (IMCR) reactions.

Animals↗
PubMed2001

Highly sensitive and selective fluorescence assays for rapid screening of endothelin-converting enzyme inhibitors.

The highly potent vasoconstrictor peptide endothelin (ET) is generated from an inactive precursor, big endothelin (bET), by endothelin-converting enzyme (ECE). ECE is a phosphoramidon-sensitive zinc metallopeptidase, which is closely related to neprilysin (neutral endopeptidase). It is possible that compounds which inhibit the formation of ET may be used as new drugs for the treatment of cardiovascular diseases. Such an approach requires a fast, simple and selective assay to measure ECE activity, allowing rapid screening of inhibitors. We describe here two new ECE substrates based on the concept of 'intramolecularly quenched fluorescence' which may fulfill this aim. One, S(1) [Pya(21)-Nop(22)-bET-1(19--35)], is the (19--35) fragment of the natural peptide big-ET-1(1--38), which is modified by introducing the fluorescent amino acid, pyrenylalanine (Pya), in position 21 and a quencher, p-nitrophenylalanine (Nop), in position 22. The second substrate (S(2)) is a small peptide, Ac-Ser-Gly-Pya-Lys-Ala-Phe-Ala-Nop-Gly-Lys-NH(2), from a biased substrate peptide library. The recombinant, hECE-1c, cleaved both Pya(21)-Nop(22)-bET-1(19--35) and the natural substrate selectively between residues 21 and 22, whereas cleavage occurred between alanine and phenylalanine in the small peptide. In both cases, this generated intense fluorescence emission. The synthesis and kinetic parameters of these substrates are described. These assays, which can be used directly on tissue homogenates, are the most sensitive and selective described to date for ECE, and are easily automated for a high-throughput screening of inhibitors.

Amino Acid Sequence↗
PubMed2005

JAtlasView: a Java atlas-viewer for browsing biomedical 3D images and atlases.

BACKGROUND: Many three-dimensional (3D) images are routinely collected in biomedical research and a number of digital atlases with associated anatomical and other information have been published. A number of tools are available for viewing this data ranging from commercial visualization packages to freely available, typically system architecture dependent, solutions. Here we discuss an atlas viewer implemented to run on any workstation using the architecture neutral Java programming language. RESULTS: We report the development of a freely available Java based viewer for 3D image data, descibe the structure and functionality of the viewer and how automated tools can be developed to manage the Java Native Interface code. The viewer allows arbitrary re-sectioning of the data and interactive browsing through the volume. With appropriately formatted data, for example as provided for the Electronic Atlas of the Developing Human Brain, a 3D surface view and anatomical browsing is available. The interface is developed in Java with Java3D providing the 3D rendering. For efficiency the image data is manipulated using the Woolz image-processing library provided as a dynamically linked module for each machine architecture. CONCLUSION: We conclude that Java provides an appropriate environment for efficient development of these tools and techniques exist to allow computationally efficient image-processing libraries to be integrated relatively easily.

Anatomy↗
PubMed2005

Polymer-assisted solution-phase chemical library synthesis.

Polymer-assisted solution-phase (PASP) synthesis has become a prevalent method for the parallel synthesis of chemical libraries. PASP methodology enables the chemist to prepare libraries of compounds more quickly and efficiently than previously. PASP technology encompasses a number of purification techniques for intermediate and final product purification. This methodology continues to progress, providing the practitioner with a broad range of ingenious purification methods, enabling multi-step syntheses of complex compounds. This review covers significant new advances in the use of tagged-reagents/substrates, resin capture-release techniques and PASP multi-step synthesis, some of which incorporate microwave heating and automation.

Anthracenes↗
PubMed1994

Recent advances in drug design methods: where will they lead?

Drug design methods have made significant new advances over the last ten years, mainly in the areas of molecular modelling. In more recent times important developments in theory have led to a different type of modelling becoming possible, the so-called de novo or automated design algorithms. In this new method the programs perform much of the chemist's thinking, in finding appropriately sized chemical groups to fit into a target site. However this is a combinatoric problem which has no general analytical solution; it is ripe for optimization. Other advances, such as combinatorial chemical synthesis and screening, will dramatically influence the search for new lead structures for target sites, which at present are poorly understood. Already these methods are being applied to peptide libraries. Peptides do not make good drug compounds because of their poor bioavailability; further, their flexibility reduces their affinity. In some cases peptide backbones can be removed and replaced with rigid non-peptide scaffolds.

Drug Design↗
PubMed2005

High-content screening and profiling of drug activity in an automated centrosome-duplication assay.

Maintenance of centrosome number is essential for cell-cycle progression and genomic stability, but investigation of this regulation has been limited by assay difficulty. We present a fully automated image-based centrosome-duplication assay that is accurate and robust enough for both careful cell-biology studies and high-throughput screening, and employ this assay in a series of chemical-genetic studies. We observe that a simple cytometric profiling strategy, which is based on organelle size, groups compounds with similar mechanisms of action; this suggests a simple strategy for excluding compounds that undesirably target such activities as protein synthesis and microtubule dynamics. Screening a library of compounds of known activity, we found unexpected effects on centrosome duplication by a number of drugs, most notably isoform-specific protein kinase C inhibitors and retinoic acid receptor agonists. From a 16 320-member library of uncharacterized small molecules, we identified five potent centrosome-duplication inhibitors that do not target microtubule dynamics or protein synthesis. The analysis methodology reported here is directly relevant to studies of centrosome regulation in a variety of systems and is adaptable to a wide range of other biological problems.

Animals↗
PubMed1996

Automated solid-phase synthesis of cyclic peptides bearing a side-chain tail designed for subsequent chemical grafting.

Recent developments in allyl chemistry and palladium solubilization allow automated continuous-flow solid-phase synthesis of cyclic or branched peptides, with specific side-chain cleavage and on-line cyclization. In this paper, we adapted the method to the synthesis of cyclic peptides bearing an anchoring tail on a side chain of the cycle. Side products were obtained with the standard procedure and an additional washing step had to be introduced in the synthesis protocol to remove side products resulting from the palladium allyl cleavage step. The method is illustrated by the automated synthesis of cyclo[-DVal-Arg-Gly-Asp-Glu (-epsilon Ahx-Cys-NH2)-] which contains the Arg-Gly-Asp adhesion motif (RGD) recognized by cellular integrins. The tail of the peptide was designed with a thiol at the carboxylic end to allow subsequent grafting by covalent attachment. Such tailed cyclic peptides can be grafted on different supports for new applications in biomaterial design, cell adhesion assays, affinity chromatography, immunization, vaccine development, ELISA kits, and the building of libraries of conformationally constrained peptides.

Amino Acid Sequence↗
PubMedDate not supplied

High-throughput screening assay for the tunable selection of protein ligands.

Here, we describe a new protein-ligand binding assay that is amenable to high-throughput screening applications. The assay involves the use of SUPREX (stability of unpurified proteins from rates of H/D exchange), a new H/D exchange and mass spectrometry-based technique we recently developed for the quantitative analysis of protein-ligand binding interactions. As part of this work, we describe a new high-throughput SUPREX protocol, and we demonstrate that this protocol can be used to efficiently screen peptide ligands in a model combinatorial library for binding to a model protein system, the S-protein. The high-throughput SUPREX protocol developed here is generally applicable to a wide variety of protein ligands, including DNA, small molecules, metals, and other proteins. On the basis of the results of the model study in this work, one person with access to one MALDI mass spectrometer should be able to screen approximately 10 000 compounds per 24-h period using the protocol described here. With full automation and the use of a commercially available MALDI mass spectrometer optimized for high-throughput analyses, we estimate that the SUPREX-based assay described here could be used to screen on the order of 100 000 ligands per day.

Automation↗
PubMed2006

mGrid: a load-balanced distributed computing environment for the remote execution of the user-defined Matlab code.

BACKGROUND: Matlab, a powerful and productive language that allows for rapid prototyping, modeling and simulation, is widely used in computational biology. Modeling and simulation of large biological systems often require more computational resources then are available on a single computer. Existing distributed computing environments like the Distributed Computing Toolbox, MatlabMPI, Matlab*G and others allow for the remote (and possibly parallel) execution of Matlab commands with varying support for features like an easy-to-use application programming interface, load-balanced utilization of resources, extensibility over the wide area network, and minimal system administration skill requirements. However, all of these environments require some level of access to participating machines to manually distribute the user-defined libraries that the remote call may invoke. RESULTS: mGrid augments the usual process distribution seen in other similar distributed systems by adding facilities for user code distribution. mGrid's client-side interface is an easy-to-use native Matlab toolbox that transparently executes user-defined code on remote machines (i.e. the user is unaware that the code is executing somewhere else). Run-time variables are automatically packed and distributed with the user-defined code and automated load-balancing of remote resources enables smooth concurrent execution. mGrid is an open source environment. Apart from the programming language itself, all other components are also open source, freely available tools: light-weight PHP scripts and the Apache web server. CONCLUSION: Transparent, load-balanced distribution of user-defined Matlab toolboxes and rapid prototyping of many simple parallel applications can now be done with a single easy-to-use Matlab command. Because mGrid utilizes only Matlab, light-weight PHP scripts and the Apache web server, installation and configuration are very simple. Moreover, the web-based infrastructure of mGrid allows for it to be easily extensible over the Internet.

Computational Biology↗
PubMed2006

Natural products that reduce rotavirus infectivity identified by a cell-based moderate-throughput screening assay.

BACKGROUND: There is widespread interest in the use of innate immune modulators as a defense strategy against infectious pathogens. Using rotavirus as a model system, we developed a cell-based, moderate-throughput screening (MTS) assay to identify compounds that reduce rotavirus infectivity in vitro, toward a long-term goal of discovering immunomodulatory agents that enhance innate responses to viral infection. RESULTS: A natural product library consisting of 280 compounds was screened in the assay and 15 compounds that significantly reduced infectivity without cytotoxicity were identified. Time course analysis of four compounds with previously characterized effects on inflammatory gene expression inhibited replication with pre-treatment times as minimal as 2 hours. Two of these four compounds, alpha-mangostin and 18-beta-glycyrrhetinic acid, activated NFkappaB and induced IL-8 secretion. The assay is adaptable to other virus systems, and amenable to full automation and adaptation to a high-throughput format. CONCLUSION: Identification of several compounds with known effects on inflammatory and antiviral gene expression that confer resistance to rotavirus infection in vitro suggests the assay is an appropriate platform for discovery of compounds with potential to amplify innate antiviral responses.

Animals↗