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We introduce Paircoil2, a new version of the Paircoil program, which uses pairwise residue probabilities to detect coiled-coil motifs in protein sequence data. Paircoil2 achieves 98% sensitivity and 97% specificity on known coiled coils in leave-family-out cross-validation. It also shows superior performance compared with published methods in tests on proteins of known structure.
Some sugars, specifically white beet sugar and raw cane sugars, possess off-flavors and off-odors. Although not necessarily the source, the presence of short-chain fatty acids serves as an indicator of an off-odor problem in sugar. Solid-phase microextraction (SPME) is used to collect the volatile compounds from the headspace of sugar. The temperature, moisture, and type of SPME fiber are varied to optimize recovery. Sugars analyzed in the absence of water using an incubation temperature of 70 degrees C with a divinylbenzene-carboxen-polydimethylsiloxane fiber yield the most reproducible results. Data from depletion analyses report a recovery level of 38% for the first injection. The semiquantitative analysis of butyric acid is accomplished using injected standards to develop a calibration curve.
We previously reported a cDNA selection method using DNA latex particles to identify expressed genes in specific regions of genomes and named this cDNA scanning method (Hayashida et al., 1995 Gene 155 161). We applied the cDNA scanning method to the YAC CIC3B1-S DNA on Arabidopsis thaliana chromosome 5, and constructed a region-specific sublibrary in which cDNAs for genes on the YAC CIC3B1-S DNA were concentrated. We isolated 545 cDNA clones from the sublibrary, and determined partial sequence of them to produce expressed sequence tags (ESTs) derived from the YAC region. In total, 74 nonredundant groups of cDNAs were obtained from 545 cDNA clones. Forty-seven percent of these EST clones had significant homology to functional proteins such as protein kinases, LON protease, nucleic acid binding protein and chloride channel protein. We compared the cDNA sequences isolated by the cDNA scanning method to the Arabidopsis genomic sequence corresponding to the YAC CIC3B1-S region, and found that 69% of the selected cDNAs are located in the region. We discuss the fidelity and efficiency of the cDNA scanning method for cloning region-specific cDNAs and its useful application in positional cloning.
Research into new methods to identify genes in anonymous genomic sequences has been going on for more than 15 years. Over this period of time, the field has evolved from the designing of programs to identify protein coding regions in compact mitochondrial or bacterial genomes, to the challenge of predicting the detailed organization of multi-exon vertebrate genes. The best program currently available perfectly locates more than 80% of the internal coding exons, and only 5% of the predictions do not overlap a real exon. Given such accuracy, computational methods are indeed very useful; however, they do not alleviate the need for experimental validation. If the performances are satisfactory for the identification of the coding moiety of genes (internal coding exons), the determination of the full extent of the transcript (5' and 3' extremities of the gene) and the location of promoter regions are still unreliable. As the human and mouse genome sequencing projects enter a production mode, the fully automated annotation of megabase-long anonymous genomic sequences is the next big challenge in bioinformatics.
Part I. Cocaine-Lidocaine Mixtures A rapid procedure based on second-derivative UV spectrophotometry is proposed for the simultaneous determination of cocaine and lidocaine. The optimum magnitudes are chosen in terms of response linearity, sensitivity, relative percent error, relative recovery data, and reproducibility. Part II. Cocaine-Procaine Mixtures A derivative spectrophotometric method based on second-derivative ultraviolet absorption spectra has been developed for the simultaneous determination of cocaine and procaine. Several derivative magnitudes have been investigated. A statistical analysis of the results is reported. Part III. Cocaine-Tetracaine Mixtures A simple method has been developed for the simultaneous determination of cocaine and tetracaine by measuring the amplitudes of the positive peak at 286.6 nm and the negative peak at 313.9 nm with respect to zero baseline. Part IV. Cocaine-Benzocaine Mixtures Derivative ultraviolet spectrophotometry has been used for the simultaneous determination of cocaine and benzocaine. Both cocaine and benzocaine can be assayed by measurement of, respectively, the peak-to-peak amplitude D261.4,277.4 and the peak-to-zero amplitude D299.4. The method is applied to simulated street samples of cocaine.
Hammerhead ribozymes were synthesized in which the tetranucleotide loop II was replaced by non-nucleotidic linkers of 7, 13, 17 and 19 atoms length. Ribozymes with 17 and 19 atom linkers, in combination with a 4 base pair stem II, had catalytic efficiencies which were 2 fold increased to that of the parent ribozyme with a tetranucleotide loop. Ribozymes with these linkers, but in combination with a 2 base pair stem II, showed a 2 fold decrease in catalytic efficiency when compared to the parent ribozyme. Prolonged preincubation in the presence of MgCl2 was required for hexaethylene glycol linker-modified ribozymes to obtain maximum activity and reproducible kinetic data.
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We describe MUSCLE, a new computer program for creating multiple alignments of protein sequences. Elements of the algorithm include fast distance estimation using kmer counting, progressive alignment using a new profile function we call the log-expectation score, and refinement using tree-dependent restricted partitioning. The speed and accuracy of MUSCLE are compared with T-Coffee, MAFFT and CLUSTALW on four test sets of reference alignments: BAliBASE, SABmark, SMART and a new benchmark, PREFAB. MUSCLE achieves the highest, or joint highest, rank in accuracy on each of these sets. Without refinement, MUSCLE achieves average accuracy statistically indistinguishable from T-Coffee and MAFFT, and is the fastest of the tested methods for large numbers of sequences, aligning 5000 sequences of average length 350 in 7 min on a current desktop computer. The MUSCLE program, source code and PREFAB test data are freely available at http://www.drive5. com/muscle.
DNA microarray is an important tool for the study of gene activities but the resultant data consisting of thousands of points are error-prone. A serious limitation in microarray analysis is the unreliability of the data generated from low signal intensities. Such data may produce erroneous gene expression ratios and cause unnecessary validation or post-analysis follow-up tasks. In this study, we describe an approach based on normal mixture modeling for determining optimal signal intensity thresholds to identify reliable measurements of the microarray elements and subsequently eliminate false expression ratios. We used univariate and bivariate mixture modeling to segregate the microarray data into two classes, low signal intensity and reliable signal intensity populations, and applied Bayesian decision theory to find the optimal signal thresholds. The bivariate analysis approach was found to be more accurate than the univariate approach; both approaches were superior to a conventional method when validated against a reference set of biological data that consisted of true and false gene expression data. Elimination of unreliable signal intensities in microarray data should contribute to the quality of microarray data including reproducibility and reliability of gene expression ratios.
We report on a new type of systematic annotation error in genome and pathway databases that results from the misinterpretation of partial Enzyme Commission (EC) numbers such as '1.1.1.-'. This error results in the assignment of genes annotated with a partial EC number to many or all biochemical reactions that are annotated with the same partial EC number. That inference is faulty because of the ambiguous nature of partial EC numbers. We have observed this type of error in multiple databases, including KEGG, VIMSS and IMG, all of which assign genes to KEGG pathways. The Escherichia coli subset of the KEGG database exhibits this error for 6.8% of its gene-reaction assignments. For example, KEGG contains 17 reactions that are annotated with EC 1.1.1.-. A group of three E.coli genes, b1580 [putative dehydrogenase, NAD(P)-binding, starvation-sensing protein], b3787 (UDP-N-acetyl-D-mannosaminuronic acid dehydrogenase) and b0207 (2,5-diketo-D-gluconate reductase B), is assigned to 15 of those reactions, despite experimental evidence indicating different single functions for two of the three genes. Furthermore, the databases (DBs) are internally inconsistent in that the description of gene functions for genes with partial EC numbers is inconsistent with the activities implied by reactions to which the genes were assigned. We infer that these inconsistencies result from the processing used to match gene products to reactions within KEGG's metabolic pathways. These errors affect scientists who use these DBs as online encyclopedias and they affect bioinformaticists who use these DBs to train and validate newly developed algorithms.
As the DNA sequence of the human genome is now nearly finished, the main task of genome research is to elucidate gene function and regulation. DNA methylation is of particular importance for gene regulation and is strongly implicated in the development of cancer. Even minor changes in the degree of methylation can have severe consequences. An accurate quantification of the methylation status at any given position of the genome is a powerful diagnostic indicator. Here we present the first assay for the analysis and precise quantification of methylation on CpG positions in simplex and multiplex reactions based on matrix-assisted laser desorption/ ionisation mass spectrometry detection. Calibration curves for CpGs in two genes were established and an algorithm was developed to account for systematic fluctuations. Regression analysis gave R(2) >or= 0.99 and standard deviation around 2% for the different positions. The limit of detection was approximately 5% for the minor isomer. Calibrations showed no significant differences when carried out as simplex or multiplex analyses. All variable parameters were thoroughly investigated, several paraffin-embedded tissue biopsies were analysed and results were verified by established methods like analysis of cloned material. Mass spectrometric results were also compared to chip hybridisation.
Gap closure is a challenging phase in microbial random shotgun genome sequencing projects, particularly since genome assemblies are often complicated by the presence of repeat elements, insertion sequences and other similar factors that contribute to sequence misassemblies. While it is well recognized that the conservation of genetic information between microbial genomes, combined with the exponential increase in available microbial sequences, can be exploited to increase the efficiency of gap closure, we lack the computational tools to aid in this process. We describe here a new tool, MGView, which was developed to create a graphical depiction of the alignment of a set of microbial contigs against a completed microbial genome. The results of our assembly of the Staphylococcus aureus RF122 genome show that MGView enables a considerable reduction in time and economic cost associated with closure. Together, the results also show that the application of MGView not only enables a reduction in fold-coverage requirements of the random shotgun sequence phase, but also provides interesting insights into differences in gene content and organization between finished and unfinished microbial genomes.
In order to reach a world-wide consensus on the normal range of left (LV) and right ventricular (RV) ejection fraction (EF) at rest and during exercise, pooled data of 1200 normal subjects from 28 leading centres in the field of nuclear cardiology (68% of those contacted) was analysed. Weighted mean normal values for LVEF at rest were 62.3 +/- 6.1% (ISD) with a lower limit of normal of 50% and for RVEF 52.3 +/- 6.2% (N = 365) with a lower limit of normal of 40%. During exercise, LVEF increased in 475 subjects by +8.0 EF% (range 3-15%), a normal increase being accepted to be greater than or equal to 5% over a normal resting value for both LVEF and RVEF. Subgroup analysis of results at rest revealed no significant differences regarding selection of normal subjects (based on normal catheterization findings vs. normal volunteers with low probability of disease), age or sex. During exercise, however, significantly larger increases in LVEF measurements were noted for men versus women (P less than 0.01), for normal volunteers versus subjects selected as 'normals' based on a normal coronary angiogram (P less than 0.001) and for younger versus older subjects (P less than 0.001). Data on reproducibility and variability showed that radionuclide angiocardiography can be considered to be a reliable method today. No consensus was found for measurements of regional LV function or wall motion mainly because of differences in methodology used. These normal values may serve as general guidelines for future applications of these techniques but factors which may influence the normal range as defined and discussed in this study should be recognized.
An improved antibody modelling algorithm has been developed which incorporates significant improvements to the earlier versions developed by Martin et al. (1989, 1991), Pedersen et al. (1992) and Rees et al. (1996) and known as AbM (Oxford Molecular). The new algorithm, WAM (for Web Antibody Modelling), has been launched as an online modelling service and is located at URL http://antibody.bath.ac.uk. Here we provide a summary only of the important features of WAM. Readers interested in further details are directed to the website, which gives extensive background information on the methods employed. A brief description of the rationale behind some of the newer methodology (specifically, the knowledge-based screens) is also given.
OBJECTIVES: To investigate the importance of activation of the transcription factor, nuclear factor-kappaB (NF-kappaB) by interleukin-1beta (IL-1beta) and tumour necrosis factor-alpha (TNF-alpha) in the pathogenesis of osteoarthritis (OA) and assess its suitability as a target for therapy by determining its role in the induction of the cytokine IL-6 and the degenerative enzymes, matrix metalloproteinase (MMP)-1 and MMP-3 in vitro. METHODS: Three distinct cellular models, derived from primary OA tissue, were employed, namely, fibroblast-like synoviocytes (OA-SF); co-cultures containing phenotypic macrophage-like and fibroblast-like cells (OA-COCUL); and primary OA synovial tissue explants (OA-EXP). These were treated with specific inhibitors of IL-1beta, TNF-alpha and NF-kappaB to assess their differential role in the production of pathologically relevant mediators, specifically IL-6, MMP-1, MMP-3 and the tissue inhibitor of metalloproteinases-1 (TIMP-1), which were quantified by enzyme-linked immunosorbent assay. RESULTS: Inhibition of NF-kappaB by a novel agent, RO100 at a dose of 0.1 microM, exerted significant (P < 0.05) repression of IL-6, MMP-1 and MMP-3 production in OA-SF. Notably, neither TIMP-1 production nor cell viability was significantly affected at the dose tested. These data were reproduced in OA-EXP, which might be considered as having greater physiological relevance. Interestingly, comparable efficacy was noted using IL-1beta and TNF-alpha neutralizing antibodies in OA-COCUL. CONCLUSIONS: We have demonstrated that a novel pharmacological inhibitor of NF-kappaB, RO100 inhibits pathological mediators of OA progression with equivalent efficacy as established IL-1beta and TNF-alpha neutralizing strategies. Our findings highlight a potential for developing NF-kappaB targeted therapeutics for positively regulating disease activity and improving clinical outcome in OA.
An improvement of the Local Effect Model (LEM) is presented which takes clustered DNA damage into account. Single strand breaks (SSBs) and double strand breaks (DSBs) are distributed stochastically onto the DNA molecule and additional DSBs are recorded. Consideration of this additional damage leads to a modification of the underlying photon survival curve at high doses. As a consequence of the new approach, the ratio of maximum relative biological effectiveness (RBE) values to minimum RBEs is increased. This can be understood in terms of a higher radiation effect resulting from the cluster damage at high local doses. We find that the extended LEM including cluster effects reproduces experimental data for V79 cells significantly better than the original LEM.
Electron-beam computed tomography (EBCT) is a relatively new imaging modality that provides transaxial cardiac images with excellent spatial and temporal resolution. At present EBCT is used primarily to detect and quantify the extent of coronary calcification. In autopsy studies, increasing areas of classification are associated with the increasing likelihood of more advanced atherosclerosis, but the relationship is not linear, with large confidence limits, and EBCT may underestimate the total plaque burden. Compared with angiography, EBCT has a high sensitivity to detect coronary classification and a high negative predictive value to rule out a significant stenosis. But it may miss atherosclerotic plaque, including unstable plaque. Recent studies have aimed at improving the characterization of individual plaques by EBCT. Despite validation against autopsy and angiography, the clinical utility of EBCT still is not well defined. Additional data on reproducibility and better consensus on criteria for abnormality for coronary calcification are advised.
The recording of visual evoked potentials (VEPs) is an important means of obtaining reproducible, quantitative data on the function of the anterior visual pathways. In this review, the technical aspects of recording VEPs are briefly discussed, components of the VEPs are described, and the clinical uses of VEPs are considered. It is concluded that VEPs are useful in providing information concerning the functional integrity of the anterior visual pathways. They are especially useful in evaluating patients with visual symptoms but no objective findings on examination and in patients without visual symptoms but with diseases that are known to involve the visual pathways commonly and subclinically. Lastly, the utility and limitations of VEPs in various neurological disorders are summarized, bearing in mind the widespread availability of advanced neuroimaging techniques.