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PubMed2003

A Bayesian molecular interaction library.

We describe a library of molecular fragments designed to model and predict non-bonded interactions between atoms. We apply the Bayesian approach, whereby prior knowledge and uncertainty of the mathematical model are incorporated into the estimated model and its parameters. The molecular interaction data are strengthened by narrowing the atom classification to 14 atom types, focusing on independent molecular contacts that lie within a short cutoff distance, and symmetrizing the interaction data for the molecular fragments. Furthermore, the location of atoms in contact with a molecular fragment are modeled by Gaussian mixture densities whose maximum a posteriori estimates are obtained by applying a version of the expectation-maximization algorithm that incorporates hyperparameters for the components of the Gaussian mixtures. A routine is introduced providing the hyperparameters and the initial values of the parameters of the Gaussian mixture densities. A model selection criterion, based on the concept of a 'minimum message length' is used to automatically select the optimal complexity of a mixture model and the most suitable orientation of a reference frame for a fragment in a coordinate system. The type of atom interacting with a molecular fragment is predicted by values of the posterior probability function and the accuracy of these predictions is evaluated by comparing the predicted atom type with the actual atom type seen in crystal structures. The fact that an atom will simultaneously interact with several molecular fragments forming a cohesive network of interactions is exploited by introducing two strategies that combine the predictions of atom types given by multiple fragments. The accuracy of these combined predictions is compared with those based on an individual fragment. Exhaustive validation analyses and qualitative examples (e.g., the ligand-binding domain of glutamate receptors) demonstrate that these improvements lead to effective modeling and prediction of molecular interactions.

Algorithms↗
PubMed2002

The Criticism Management System for the Cochrane Library.

In April 1997, the Cochrane Collaboration implemented the Criticism Management System for The Cochrane Library, allowing readers to electronically submit criticisms on Cochrane reviews and protocols. As a result of resource constraints and user feedback, the Criticism Management System evolved through three different versions. Major problems have included difficult access to the system, a lack of automation within the system, a low number of submissions (171 criticisms submitted per 1,388 reviews), and a low response rate to these criticisms by authors of reviews (26 criticisms have received published responses). These low figures suggest that users of The Cochrane Library are not commenting on the published reviews or not using the Criticism Management System to make their criticisms known to the authors. An easier-to-use system and better ways of encouraging feedback from readers and responses from reviewers are needed in the future.

Databases, Bibliographic↗
PubMed2002

Automated main-chain model building by template matching and iterative fragment extension.

An algorithm for the automated macromolecular model building of polypeptide backbones is described. The procedure is hierarchical. In the initial stages, many overlapping polypeptide fragments are built. In subsequent stages, the fragments are extended and then connected. Identification of the locations of helical and beta-strand regions is carried out by FFT-based template matching. Fragment libraries of helices and beta-strands from refined protein structures are then positioned at the potential locations of helices and strands and the longest segments that fit the electron-density map are chosen. The helices and strands are then extended using fragment libraries consisting of sequences three amino acids long derived from refined protein structures. The resulting segments of polypeptide chain are then connected by choosing those which overlap at two or more C(alpha) positions. The fully automated procedure has been implemented in RESOLVE and is capable of model building at resolutions as low as 3.5 A. The algorithm is useful for building a preliminary main-chain model that can serve as a basis for refinement and side-chain addition.

Algorithms↗
PubMed1994

DNA sequencing: modular primers for automated walking.

Here we describe our progress in the development of the technology of DNA sequencing by primer walking based on the modular primer approach, which eliminates the primer synthesis bottleneck. Modular primers are assembled from 5-mers, 6-mers, or 7-mers selected from a presynthesized library of as few as 1000 oligonucleotides. This technology is expected to reduce the time per walk by a factor of 20 to 50, and the cost of DNA sequencing by a factor of 5 to 15. Both time and expense will be saved not only on the primer synthesis per se but, more importantly, as a result of the closed-end automation of the complete cycle of walking sequencing, where no human intervention will be required between the walks. Such closed-end automation has until now been precluded due to the need to synthesize a new primer for each walk. As a part of the closed cycle automation development, this report deals with modular primers used for sequencing with fluorescent dye terminators. The success rate and the quality of automated sequencing with modular primers are found similar to those with the conventional 17-20-base-long primers. One of the advantages of the described technique is simple purification without any proteins that need to be removed.

Base Sequence↗
PubMed1993

Backbone-dependent rotamer library for proteins. Application to side-chain prediction.

A backbone-dependent rotamer library for amino acid side-chains is developed and used for constructing protein side-chain conformations from the main-chain co-ordinates. The rotamer library is obtained from 132 protein chains in the Brookhaven Protein Database. A grid of 20 degrees by 20 degrees blocks for the main-chain angles phi, psi is used in the rotamer library. Significant correlations are found between side-chain dihedral angle probabilities and backbone phi, psi values. These probabilities are used to place the side-chains on the known backbone in test applications for six proteins for which high-resolution crystal structures are available. A minimization scheme is used to reorient side-chains that conflict with the backbone or other side-chains after the initial placement. The initial placement yields 59% of both chi 1 and chi 2 values in the correct position (to within 40 degrees) for thermolysin to 81% for crambin. After refinement the values range from 61% (lysozyme) to 89% (crambin). It is evident from the results that a single protein does not adequately test a prediction scheme. The computation time required by the method scales linearly with the number of side-chains. An initial prediction from the library takes only a few seconds of computer time, while the iterative refinement takes on the order of hours. The method is automated and can easily be applied to aid experimental side-chain determinations and homology modeling. The high degree of correlation between backbone and side-chain conformations may introduce a simplification in the protein folding process by reducing the available conformational space.

Amino Acid Sequence↗
PubMed1989

Sequencing of megabase plus DNA by hybridization: theory of the method.

A mismatch-free hybridization of oligonucleotides containing from 11 to 20 monomers to unknown DNA represents, in essence, a sequencing of a complementary target. Realizing this, we have used probability calculations and, in part, computer simulations to estimate the types and numbers of oligonucleotides that would have to be synthesized in order to sequence a megabase plus segment of DNA. We estimate that 95,000 specific mixes of 11-mers, mainly of the 5'(A,T,C,G)(A,T,C,G)N8(A,T,C,G)3' type, hybridized consecutively to dot blots of cloned genomic DNA fragments would provide primary data for the sequence assembly. An optimal mixture of representative libraries in M13 vector, having inserts of (i) 7 kb, (ii) 0.5 kb genomic fragments randomly ligated in up to 10-kb inserts, and (iii) tandem "jumping" fragments 100 kb apart in the genome, will be needed. To sequence each million base pairs of DNA, one would need hybridization data from about 2100 separate hybridization sample dots. Inevitable gaps and uncertainties in alignment of sequenced fragments arising from nonrandom or repetitive sequence organization of complex genomes and difficulties in cloning "poisonous" sequences in Escherichia coli, inherent to large sequencing by any method, have been considered and minimized by choice of libraries and number of subclones used for hybridization. Because it is based on simpler biochemical procedures, our method is inherently easier to automate than existing sequencing methods. The sequence can be derived from simple primary data only by extensive computing. Phased experimental tests and computer simulations increasing in complexity are needed before accurate estimates can be made in terms of cost and speed of sequencing by the new approach. Nevertheless, sequencing by hybridization should show advantages over existing methods because of the inherent redundancy and parallelism in its data gathering.

Algorithms↗
PubMed2006

Micro-colony array based high throughput platform for enzyme library screening.

Enzymes are becoming increasingly important tools for synthesizing and modifying fine and bulk chemicals. The availability of biocatalysts which fulfil the requirements of industrial processes is often limited. Recruiting suited enzymes from natural (e.g. metagenomes) and artificial (e.g. directed evolution) biodiversity is based on screening libraries of microbial clones expressing enzyme variants. However, exploring the complex diversity of such libraries needs efficient screening methods. Overcoming the "screening bottleneck" requires rapid high throughput technology allowing the analysis of a large diversity of different enzymes and applying different screening conditions. Facing these facts an efficient and cost effective method for high throughput screening of large enzyme libraries at the colony level was developed. Therefore, ordered high density micro-colony arrays were combined with optical sensor technology and automated image analysis. The system generally allows the simultaneous monitoring of enzyme activities reflected by up to 7000 micro-colonies spotted on a filter in the size of a micro-titer plate. A developed replica option also allows the analysis of clones under varying external conditions. The method was verified by a model screening using esterases and was proved to provide reliable enzyme activity measurements within single micro-colonies allowing the discrimination of activity differences in the range of 10-20%.

Calibration↗
PubMedDate not supplied

FLUFF-BALL, a template-based grid-independent superposition and QSAR technique: validation using a benchmark steroid data set.

The Flexible Ligand Unified Force Field (FLUFF) is a molecular mechanistic superposition algorithm utilizing a template structure, on top of which the ligand(s) are superimposed. FLUFF enables a flexible semiautomatic superimposition in which the ligand and the template are allowed to seek the best common conformation, which can then be used to predict the biological activity by Boundless Adaptive Localized Ligand (BALL). In BALL, the similarity of the electrostatic and van der Waals volumes of the template and ligand is evaluated using the template-based coordinate system which makes the FLUFF-BALL invariant as to the rotations and translations of the global coordinate system. When tested using the CBG (corticosteroid binding globulin) affinities of 31 benchmark steroids, the FLUFF-BALL technique produced results comparable to standard 3D-QSAR methods. Supplementary test calculations were performed with five additional data sets. Due to its high level of automation and high throughput, the FLUFF-BALL is highly suitable for use in drug design and in scanning of large molecular libraries.

Algorithms↗
PubMed2000

Identification of pediococci by ribotyping.

Pediococci are among the most prevalent microbial contaminants in breweries and they can cause ropiness and the accumulation of high levels of diacetyl in beer. The accurate identification of pediococci is important, because different species do not possess equal spoilage potential. In this study, 18 Pediococcus strains, mainly of brewery origin, were first identified using phenotypical characterization (API 50 CHL and SDS-PAGE profiling), and then ribotyped using a RiboPrinterR System. Six Pediococcus type strains and three other Pediococcus strains were used as references. Ribotyping showed higher discriminative capacity than phenotypical identification methods. Strains could be identified to species level and in many cases, differentiated even at strain level using this genetic fingerprinting method. The identifications performed by ribotyping were confirmed by 16S rDNA sequencing of selected strains. Automated ribotyping was found to be a rapid and reliable method for identifying pediococci, but requires the construction of a comprehensive fingerprint library.

Bacterial Typing Techniques↗
PubMed2024

Reflective Evaluation of Next-Generation Sequencing Data during Early Phase Detection of the Delta Variant.

During the SARS-CoV-2 pandemic, next-generation sequencing (NGS) technologies like the Ion Torrent S5 and Illumina MiSeq, alongside advanced software, improved genomic surveillance in South Africa. This study analysed anonymized samples from the Eastern Cape using Genome Detective and NextClade, showing Ion Torrent S5 and Illumina MiSeq success rates of 96% and 94%, respectively. The study focused on genomic coverage (above 80%) and mutation detection (below 100), with the Ion Torrent S5 achieving 99% coverage compared to Illumina MiSeq's 80%, likely due to different primers used in amplification. The Ion Torrent S5 was more effective in sequencing varied viral loads, whereas Illumina MiSeq had difficulties with lower loads. Both platforms were adept at identifying clades, successfully differentiating between Beta (<45%) and Delta variants (<30%), despite minor discrepancies in assignments due to Illumina MiSeq's lower coverage, leading to a failure rate of up to 6%. Manual library preparation showed similar sample processing and clade identification capabilities for both platforms. However, differences in sequencing duration (3.5 vs. 36 hours), automation level, genomic coverage (80% vs. 99%), and viral load compatibility were noted, highlighting each platform's unique advantages and challenges in SARS-CoV-2 genomic surveillance. In conclusion, the Illumina MiSeq and Ion Torrent S5 platforms are both efficacious in executing whole-genome sequencing (WGS) via amplicons, facilitating precise, accurate, and high-throughput examinations of SARS-CoV-2 viral genomes. However, it is important to note the existence of disparities in the quality of data produced by each platform. Each system offers unique benefits and limitations, rendering them viable choices for the genomic surveillance of SARS-CoV-2.

Illumina MiSeq↗
PubMed1999

Application of in situ PCR to yeast cells for screening YAC libraries.

We have used in situ PCR technology in yeast cells with the ultimate goal of cloning and screening genomic yeast artificial chromosome (YAC) libraries. The target sequences in YAC clones were amplified "in situ" in yeast cells by the same set of microsatellite primers used in solution-based PCR screening. The method is fast and sensitive and obviates the steps required for individual isolation of DNAs from hundreds to thousands of YAC clones and thus has an advantage over conventional solution-based PCR screening. This approach can conceivably be applied to the products of automated robotic workstations.

Animals↗
PubMed2004

Automated yeast two-hybrid screening for nuclear receptor-interacting proteins.

High throughput analysis of protein-protein interactions is an important sector of hypothesis-generating research. Using an improved and automated version of the yeast two-hybrid system, we completed a large interaction screening project with a focus on nuclear receptors and their cofactors. A total of 425 independent yeast two-hybrid cDNA library screens resulted in 6425 potential interacting protein fragments involved in 1613 different interaction pairs. We show that simple statistical parameters can be used to narrow down the data set to a high confidence set of 377 interaction pairs where validated interactions are enriched to 61% of all pairs. Within the high confidence set, there are 64 novel proteins potentially binding to nuclear receptors or their cofactors. We discuss several examples of high interest, and we expect that communication of this huge data set will help to complement our knowledge of the protein interaction repertoire of this family of transcription factors and instigate the characterization of the various novel candidate interactors.

Automation↗
PubMed2005

A sensitive and rapid chemiluminescence ELISA for filamentous bacteriophages.

Filamentous bacteriophage (Ff) displayed random peptide and antibody libraries are widely used to identify specific, high affinity, binding ligands. A critical element in the identification of target-specific phages is to determine phage titers, not only at every round of selection, but also for normalizing phage titers of a set of individual clones for their comparative binding analysis. The conventional ELISA-based Ff titration methods require a minimum of 4-5 hr assay time and their lowest reported detection limit is approximately 50,000 particles/well. In this report, we present a sandwich ELISA that allows detection of approximately 1000 Ff particles/well in less than 2.5 hr. The values of correlation of coefficient (r2) for the curves at low phage concentrations (up to 106 TU/well) were about 0.999 in our ELISA. Experiments conducted at different temperatures suggest using 40 degrees C incubations when titering low phage concentration samples. Experiments were also conducted with conventional ELISA for comparison. Our ELISA method derives an advantage from using a chemiluminescence substrate that gives much larger signals and wide linear range of measurement, thus allowing discrimination between background and low Ff phage concentrations. In conclusion, the Ff titration method presented here is highly sensitive, rapid, and amenable to high throughput analysis.

Antibody Affinity↗
PubMed2002

Fluorescence imaging of mobility shifts: an expression cloning method for identification of cell signaling targets.

There is a need for a simple global approach to identify signaling targets that are posttranslationally modified in response to physiologic or pathologic stimuli within living cells. Reported here is a simple method, fluorescence imaging of mobility shifts (FIMS), which relies on in-gel detection of cell-expressed green fluorescent protein fusion proteins undergoing electrophoretic mobility shifts. This detection method is applied to a small pool cDNA library screening protocol. The readout is essentially a differential display of posttranslational modifications. Unlike biochemical approaches to identifying signaling targets, the screen is performed in living cells using standard methods for transient transfection. This enables detection of intracellular targets modified in response to either molecularly defined stimuli, such as growth factors or drugs, or complex pathologic stimuli, such as oxidative stress or hypoglycemia. FIMS is rapid, sensitive, inexpensive, and nonradioactive and easily adapted to automated high throughput methods, including capillary electrophoresis. The technique is sufficiently sensitive to easily detect fluorescent proteins expressed in a single well in 384-well format. FIMS is applicable to traditional cDNA library screening, but the method will be especially attractive for screening preselected collections of autofluorescent fusion proteins. A bonus of the technique is that examination of transfected cells by fluorescence microscopy provides immediate information about intracellular localization and stimulus-induced translocation of putative targets. We illustrate the utility of the technique with pilot screens for apoptotic and mitogenic targets modified by staurosporine and serum stimulation, respectively.

Animals↗
PubMed1994

The wavelet transform as a tool for recognition of biosignals.

The use of the wavelet transform as a signal analysis tool has been demonstrated by its successful application to the study of various signals. The first step in addressing pattern recognition problems is to define a representation that can be used for extracting the information content of signals. The sharp variation points of a signal amplitude are among the meaningful characterizations of the signal. The wavelet transform of the signal is found to be translation variant which makes it difficult for direct application in pattern recognition. However, the zero-crossings of a wavelet transform employing a particular class of wavelets can provide the translation invariant locations of the signal variation points. A zero-crossing representation augmented by the measure of the structure between the two consecutive zero-crossings has been studied by Stephane Mallat. On the basis of this representation, we demonstrate recognition of segments of biosignals embedded in streams of signals. The feasibility of employing zero-crossings of a wavelet transform as a tool in searching for a particular pattern class in the library of biosignals is explored.

Electrocardiography↗
PubMed1995

Determining educational needs for the biomedical library customer: an analysis of end-user searching in MEDLINE.

The analysis of CD Plus usage logs provides an opportunity to determine specific areas where searchers are "missing opportunities" by misusing, or failing to use, system specific features. This paper describes a methodology, based on transaction log analysis, to provide automated, continuous analysis of end-user searching. The data collected in this process can then be used to enhance and provide insight into the educational needs of users.

CD-ROM↗
PubMed1999

Initial assessment of gene diversity for the oomycete pathogen Phytophthora infestans based on expressed sequences.

A total of 1000 expressed sequence tags (ESTs) corresponding to 760 unique sequence sets were identified using random sequencing of clones from a cDNA library constructed from mycelial RNA of Phytophthora infestans. A number of software programs, represented by a relational database and an analysis pipeline, were developed for the automated analysis and storage of the EST sequence data. A set of 419 nonredundant sequences, which correspond to a total of 632 ESTs (63.2%), were identified as showing significant matches to sequences deposited in public databases. A putative cellular identity and role was assigned to all 419 sequences. All major functional categories were represented by at least several ESTs. Four novel cDNAs containing sequences related to elicitins, a family of structurally related proteins that induce the hypersensitive response and condition avirulence of P. infestans on Nicotiana plants, were among the most notable genes identified. Two of these elicitin-like cDNAs were among the most abundant cDNAs examined. The set also contained several ESTs with high sequence similarity to unique plant genes.

Actins↗