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At least 433 records · Page 24Linked to original sources
PubMed2004

EST-based analysis of gene expression in the porcine brain.

Since pig is an important livestock species worldwide, its gene expression has been investigated intensively, but rarely in brain. In order to study gene expression profiles in the pig central nervous system, we sequenced and analyzed 43,122 high-quality 5' end expressed sequence tags (ESTs) from porcine cerebellum, cortex cerebrum, and brain stem cDNA libraries, involving several different prenatal and postnatal developmental stages. The initial ESTs were assembled into 16,101 clusters and compared to protein and nucleic acid databases in GenBank. Of these sequences, 30.6% clusters matched protein databases and represented function known sequences; 75.1% had significant hits to nucleic acid databases and partial represented known function; 73.3% matched known porcine ESTs; and 21.5% had no matches to any known sequences in GenBank. We used the categories defined by the Gene Ontology to survey gene expression in the porcine brain.

Animals↗
PubMed2025

Whole genome study and construction of SHERLOCK detection method for endemic strains of Burkholderia pseudomallei in Hainan based on third-generation sequencing.

UNLABELLED: Burkholderia pseudomallei (Bp) is a gram-negative bacterium found in soil and surface water. It is also the pathogen that causes melioidosis disease in humans and animals. This study aimed to obtain the whole genome sequence of the endemic strain of Bp in Hainan, using third-generation sequencing (TGS) technology, and elucidate the genome structure, function, and genetic evolution. Additionally, the study aimed to achieve rapid and specific identification of these endemic strains using specific high-sensitivity enzymatic reporter unlocking (SHERLOCK) detection technology, providing a new strategy for the early diagnosis of melioidosis. Utilizing the PacBio platform for TGS technology, we completed whole genome sequencing of 16 Bp strains from Hainan. High-precision and complete genome sequences were obtained through quality control and genome assembly of the sequencing data. Additionally, we established a nucleic acid detection technology platform based on SHERLOCK, which could be completed from nucleic acid extraction to result reading within 1-2 hours, demonstrating good sensitivity and specificity (both are 100%). The lateral chromatography strip method does not require special equipment and holds promise as an immediate screening method for the early diagnosis of melioidosis. IMPORTANCE: Melioidosis is a highly pathogenic infectious disease caused by a gram-negative bacterium of Burkholderia pseudomallei (Bp). The traditional gold standard for diagnosing melioidosis is still isolation and culture from clinical samples. Although this method has high specificity, it has low sensitivity and is time-consuming, which often leads to misdiagnosis or missed diagnosis of melioidosis, affecting subsequent treatment. In this study, recombinase polymerase amplification technology and clustered regularly interspaced short palindromic repeats/Cas13a technology were combined to establish the Specific High-sensitivity Enzymatic Reporter Unlocking detection technology, which can achieve rapid and accurate identification of Bp, providing a new method for the early diagnosis of melioidosis.

Burkholderia pseudomallei↗
PubMedDate not supplied

Prediction of homology model quality with multivariate regression.

A new method has been developed for prediction of homology model quality directly from the sequence alignment, using multivariate regression. Hence, the expected quality of future homology models can be estimated using only information about the primary structure. This method has been applied to protein kinases and can easily be extended to other protein families. Homology model quality for a reference set of homology models was verified by comparison to experimental structures, by calculation of root-mean-square deviations (RMSDs) and comparison of interresidue contact areas. The homology model quality measures were then used as dependent variables in a Partial Least Squares (PLS) regression, using a matrix of alignment score profiles found from the Point Accepted Mutation (PAM) 250 similarity matrix as independent variables. This resulted in a regression model that can be used to predict the accuracy of future homology models from the sequence alignment. Using this method, one can identify the target-template combinations that are most likely to give homology models of sufficient quality. Hence, this method can be used to effectively choose the optimal templates to use for the homology modeling. The method's ability to guide the choice of homology modeling templates was verified by comparison of success rates to those obtained using BLAST scores and target-template sequence identities, respectively. The results indicate that the method presented here performs best in choosing the optimal homology modeling templates. Using this method, the optimal template was chosen in 86% of the cases, as compared to 62% using BLAST scores, and 57% using sequence identities. The method presented here can also be used to identify regions of the protein structure that are difficult to model, as well as alignment errors. Hence, this method is a useful tool for ensuring that the best possible homology model is generated.

Amino Acid Sequence↗
PubMedDate not supplied

A survey of potential problems and quality control in peptide synthesis by the fluorenylmethoxycarbonyl procedure.

The routine production of peptides by manual or automated solid-phase synthesis protocols has gained widespread usage among a variety of biological scientists as new synthetic procedures have been introduced over the past several years. We report here a detailed analysis of Fmoc synthesis procedures to identify problematic reactions and to evaluate analytical procedures for monitoring the quality of peptides during and after synthesis. The results of these studies demonstrate double additions of particular amino acids during single coupling cycles, frequent incomplete deblocking of peptides by standard piperidine reactions, and the failure of the Kaiser ninhydrin test to detect free amino groups of certain amino acids at the N-terminus of synthetic peptides. These results suggest the need for more careful monitoring of Fmoc synthesis reactions than previously recognized or recommended in standard protocols. We demonstrate the utility of plasma desorption mass spectrometry (PDMS) in analyzing peptide products and recommend a minimum sequence of analytical quality control including HPLC and PDMS.

Amino Acid Sequence↗
PubMed1992

Evaluation of the sequence template method for protein structure prediction. Discrimination of the (beta/alpha)8-barrel fold.

A multiple alignment of five (beta/alpha)8-barrel enzymes has been derived from their structure. The eight beta-strands and eight alpha-helices of the (beta/alpha)8-barrel are correctly aligned and the equivalenced residues in these regions fulfil similar structural roles. Each beta-strand has a central core of usually four residues, two residues contribute side-chains to the barrel core and the other two residues are involved in beta-strand/alpha-helix contacts. However, the fold imposes no constraints on the volumes of the residues at either a local or global level: the volume of the beta-barrel core varies between 1088 A3 in glycolate oxidase and 1571 A3 in taka-amylase. Sequence motifs derived from the multiple alignment were scanned against a database of 124 protein sequences, including 17 (beta/alpha)8-barrel enzymes. The results were evaluated in terms of the discrimination of (beta/alpha)8-barrel sequences and the quality of the alignments obtained. One motif was able to identify the top 12% of high scoring sequences as forming (beta/alpha)8-barrels with 50% accuracy and the bottom 50% of sequences as not being (beta/alpha)8-barrel proteins with 100% accuracy. However, in most instances the alignments were poor. The reasons for this are discussed with reference to the (beta/alpha)8-barrel proteins and the sequence motif method in general.

Alcohol Oxidoreductases↗
PubMed2003

High-throughput generation of sequence indexes from T-DNA mutagenized Arabidopsis thaliana lines.

A pipeline has been created for the characterization of Arabidopsis thaliana mutants by generating flanking sequence tags (FSTs) and optimized for economic, high-throughput production. The GABI-Kat collection of T-DNA mutagenized A. thaliana plants was used as a source of independent transgenic lines. The pipeline included robotized extraction of genomic DNA in a 96-well format, an adapter-ligation PCR method for amplification of plant sequences adjacent to T-DNA borders, automated purification and sequencing of PCR products, and computational trimming of the resulting sequence files. Data quality was significantly improved by (i) restriction digestion of the adaptor-ligation products to reduce trivial sequences caused by co-amplification of fragments derived from the free plasmid, and (ii) the design of the adaptor primers for the second amplification step to enhance selective generation of single PCR fragments, even from lines with multiple T-DNA insertions. Gel-purification was avoided by including these steps, the number of amplification reactions per line was reduced from four to three, and the percentage of lines that yielded at least one FST was increased from 66% to 86%. More than 58,000 FSTs have been submitted to GenBank and are available at http://www.mpiz-koeln.mpg.de/GABI-Kat/.

Arabidopsis↗
PubMed1995

[Development of a validation method for readers of ELISA microplates].

The results, either positive or negative, of the ELISA tests performed on blood donors to detect viral markers, are always validated by reading microplates on photometers. In Rennes, these microplates can be read on MR 7000 (Dynatech) or IEMS (Labsystems) photometers. This step, the last one in the technical sequence, conditions the results. Regular checking of the photometers therefore may enhance the quality of the testing sequence. To check the quality of the photometers, we use a 96-well microplate, the "Universal Calibration Test Plate" marketed by BIO-TEK Instruments USA. The plate includes 8 blank wells, 6 wells equipped with glass filters (3 stained and 3 unstained). This plate comes with a table showing the optical density in relation to the wavelength. We first verify the repeatability of each photometer, and secondly, verify accuracy. The plate also permits verifying the linearity of each photometer. In view of the results, we were able to state that both photometers were accurate, since the results obtained were compatible with an optical density of +/- 0.024 +/- 1%. Do. The tests are repeatable, with a variation coefficient below 0.5%, linearity was verified (r: 0.999 for both photometers). Reading the plate only takes a few minutes and permits daily control of the photometers. It only requires that the test results be reported on a control card to follow-up possible deviations, and technical maintenance can thus be carried out immediately.

Calibration↗
PubMedDate not supplied

Experimental study on HASTE sequences: impacts of parameters on liver imaging.

This study investigates the impacts of effective TE (TEeff), echo spacing and echo train lengths (ETLs) on the imaging quality of HASTE sequences for liver imaging through a slit and a liver phantom. The HASTE sequence with a short echo spacing demonstrated higher CNR for a phantom simulating hepatocellular carcinoma (HCC) than that with long echo spacing. With a long echo spacing, the HASTE sequence showed higher spatial resolution for hemangiomas and metastasis. The improvement of spatial resolution along with the increase in ETLs (imaging matrix) was more prominent for hemangioma than for HCC.

Carcinoma, Hepatocellular↗
PubMed1999

A general approach to single-nucleotide polymorphism discovery.

Single-nucleotide polymorphisms (SNPs) are the most abundant form of human genetic variation and a resource for mapping complex genetic traits. The large volume of data produced by high-throughput sequencing projects is a rich and largely untapped source of SNPs (refs 2, 3, 4, 5). We present here a unified approach to the discovery of variations in genetic sequence data of arbitrary DNA sources. We propose to use the rapidly emerging genomic sequence as a template on which to layer often unmapped, fragmentary sequence data and to use base quality values to discern true allelic variations from sequencing errors. By taking advantage of the genomic sequence we are able to use simpler yet more accurate methods for sequence organization: fragment clustering, paralogue identification and multiple alignment. We analyse these sequences with a novel, Bayesian inference engine, POLYBAYES, to calculate the probability that a given site is polymorphic. Rigorous treatment of base quality permits completely automated evaluation of the full length of all sequences, without limitations on alignment depth. We demonstrate this approach by accurate SNP predictions in human ESTs aligned to finished and working-draft quality genomic sequences, a data set representative of the typical challenges of sequence-based SNP discovery.

Algorithms↗
PubMed2003

The Drosophila melanogaster genome sequencing and annotation projects: a status report.

The sequence and genome annotations of Drosophila melanogaster were initially published in late 1999 and early 2000. Since then, the Berkeley Drosophila Genome Project (BDGP) and FlyBase have improved the quality of the sequence and reviewed the annotations by hand, respectively, to produce an account of the fruit fly genome that is of the highest quality. This review discusses the main features of this process, both from the point of view of the biology revealed in the end result and in the development of software that has been central to this genome sequencing and annotation project.

Animals↗
PubMed1999

Analysis and perception of spectral 1/f characteristics of amplitude and period fluctuations in normal sustained vowels.

Two kinds of fluctuations are always observed in the steady parts of normal sustained vowels. One is amplitude fluctuation, defined as the cyclic changes of maximum peak amplitudes. The other is period fluctuation, defined as the cyclic changes of pitch periods. The primary purpose of this paper is to present quantitative descriptions of amplitude and period sequences obtained from normal sustained vowels. These fluctuation sequences consisted of maximum peak amplitudes or pitch periods extracted successively from 512 consecutive pitch periods in the steady part. Results of the frequency analysis indicated that their frequency characteristics seemed to be subject to the spectral 1/f power law. In order to investigate the possibility that the frequency characteristics of the fluctuation sequences influence the voice quality of sustained vowels, psychoacoustic experiments were conducted. Amplitude and period sequences evaluated in the experiments were spectral 1/f0 (white noise), 1/f, 1/f2, and 1/f3 sequences, respectively. The experimental results indicated that the subjective voice quality of synthesized sustained vowels could reflect the differences in the frequency characteristics of the fluctuation sequences.

Adult↗
PubMed2000

Comparison of dual spin echo echo planar imaging (SE_EPI), turbo spin echo with fat suppression and conventional dual spin echo sequences for T(2)-weighted MR imaging of focal liver lesions.

The performance of T(2)-weighted spin-echo version of echo planar imaging (SE_EPI), conventional spin echo (SE) and fat-suppressed turbo spin-echo (TSE_SPIR) sequences for the detection of focal liver lesions was evaluated. Twenty patients that were included in our study, had CT examinations prior to the MR study and were scheduled for surgery for removal of liver lesions. All patients had intraoperative sonographic examinations. Qualitative and quantitative analysis of the images was performed. Overall image quality of SE_EPI sequences was better than SE (p<0.001) and similar to TSE_SPIR sequences. There were fewer motion and ghost artifacts on SE_EPI and TSE_SPIR images compared to SE images (p<0.001). Susceptibility artifacts were statistically equivalent on SE_EPI and SE images (p<0.001) while chemical shift artifacts were equally observed on SE and SE_EPI sequences. Overall image quality of EPI-SE and TSE_SPIR sequences was better compared to SE sequences. There was no significant difference in the number of lesions detected by each of the three sequences. Quantitative analysis showed that liver/lesion contrast-to-noise ratio (CNR) and signal-to-noise ratio (SNR) of liver, lesion, spleen was higher on TSE_SPIR sequences (p<0.001) while SE_EPI and SE sequences showed non-significant differences (p>0.05). SE_EPI sequences of the liver resulted in fewer artifacts and shorter acquisition times than SE sequences. They provide a diagnostic performance similar to TSE_SPIR and better than that of SE sequences.

Adult↗
PubMed2002

Quo vadis dialysis membrane?

The development of dialysis membranes is closely related to the development of dialysis as a routine therapy for patients with kidney failure. Without having membranes and dialyzers available as commodity products, the treatment of more than 1 million uremic patients worldwide would be impossible. Several transition periods can be identified: a change in membrane geometry from flat sheet to capillaries, a shift in market appreciation from cellulose to synthetic polymers, and from low-flux to high-flux dialyzers. This shift is supported by the notion that convective therapies using high-flux membranes allow the removal of large-molecular-weight solutes. From a historical background, three eras of perception can be identified for both membrane and dialysis development. First, the period of survival when nephrologists had to focus on techniques for blood access and availability of membranes. Second, the period of issues dedicated to rather specific features of membranes and dialysis therapy such as dose of dialysis, reuse, sterilization, and membrane biocompatibility. And third, the period of quality tops this sequence with a complicated approach: the principal area of interest from the medical community has switched to issues such as quality of life, morbidity, mortality, therapy standards, and cost-effectiveness. New membrane developments should focus on this situation.

Biocompatible Materials↗
PubMed1996

Experiment files and their application during large-scale sequencing projects.

The data for large scale sequencing projects are passed through several processing steps prior to assembly, and post-assembly processing generally requires knowledge of more than just the sequence of each reading. We address here the problem of providing data to individual programs and of combining all the tasks into a single process. The solution comprises two components: a file format (experiment file format) that stores information about readings, and a script (PREGAP) that controls the creation and use of experiment files by the processing programs. PREGAP can take a batch of data from a variety of sequencing instruments, gather information about each reading, and then scan the reading to select the 3' end of the good quality data, mark sequencing vector, other cloning vector sequences, and Alu segments. The results of all these operations are added to the experiment file for each reading, ready for processing by the assembly program. Experiment files also provide a mechanism for using alternative assembly engines with our package.

Sequence Analysis↗
PubMed2002

A new method for digital video documentation in surgical procedures and minimally invasive surgery.

BACKGROUND: Documentation of surgical procedures is limited to the accuracy of description, which depends on the vocabulary and the descriptive prowess of the surgeon. Even analog video recording could not solve the problem of documentation satisfactorily due to the abundance of recorded material. By capturing the video digitally, most problems are solved in the circumstances described in this article. METHODS: We developed a cheap and useful digital video capturing system that consists of conventional computer components. Video images and clips can be captured intraoperatively and are immediately available. The system is a commercial personal computer specially configured for digital video capturing and is connected by wire to the video tower. Filming was done with a conventional endoscopic video camera. A total of 65 open and endoscopic procedures were documented in an orthopedic and a thoracic surgery unit. The median number of clips per surgical procedure was 6 (range, 1-17), and the median storage volume was 49 MB (range, 3-360 MB) in compressed form. The median duration of a video clip was 4 min 25 s (range, 45 s to 21 min). Median time for editing a video clip was 12 min for an advanced user (including cutting, title for the movie, and compression). The quality of the clips renders them suitable for presentations. CONCLUSION: This digital video documentation system allows easy capturing of intraoperative video sequences in high quality. All possibilities of documentation can be performed. With the use of an endoscopic video camera, no compromises with respect to sterility and surgical elbowroom are necessary. The cost is much lower than commercially available systems, and setting changes can be performed easily without trained specialists.

Documentation↗
PubMed2006

ANGLE: a sequencing errors resistant program for predicting protein coding regions in unfinished cDNA.

In the process of making full-length cDNA, predicting protein coding regions helps both in the preliminary analysis of genes and in any succeeding process. However, unfinished cDNA contains artifacts including many sequencing errors, which hinder the correct evaluation of coding sequences. Especially, predictions of short sequences are difficult because they provide little information for evaluating coding potential. In this paper, we describe ANGLE, a new program for predicting coding sequences in low quality cDNA. To achieve error-tolerant prediction, ANGLE uses a machine-learning approach, which makes better expression of coding sequence maximizing the use of limited information from input sequences. Our method utilizes not only codon usage, but also protein structure information which is difficult to be used for stochastic model-based algorithms, and optimizes limited information from a short segment when deciding coding potential, with the result that predictive accuracy does not depend on the length of an input sequence. The performance of ANGLE is compared with ESTSCAN on four dataset each of them having a different error rate (one frame-shift error or one substitution error per 200-500 nucleotides) and on one dataset which has no error. ANGLE outperforms ESTSCAN by 9.26% in average Matthews's correlation coefficient on short sequence dataset (< 1000 bases). On long sequence dataset, ANGLE achieves comparable performance.

Algorithms↗
PubMed1997

Protein fold recognition by prediction-based threading.

In fold recognition by threading one takes the amino acid sequence of a protein and evaluates how well it fits into one of the known three-dimensional (3D) protein structures. The quality of sequence-structure fit is typically evaluated using inter-residue potentials of mean force or other statistical parameters. Here, we present an alternative approach to evaluating sequence-structure fitness. Starting from the amino acid sequence we first predict secondary structure and solvent accessibility for each residue. We then thread the resulting one-dimensional (1D) profile of predicted structure assignments into each of the known 3D structures. The optimal threading for each sequence-structure pair is obtained using dynamic programming. The overall best sequence-structure pair constitutes the predicted 3D structure for the input sequence. The method is fine-tuned by adding information from direct sequence-sequence comparison and applying a series of empirical filters. Although the method relies on reduction of 3D information into 1D structure profiles, its accuracy is, surprisingly, not clearly inferior to methods based on evaluation of residue interactions in 3D. We therefore hypothesise that existing 1D-3D threading methods essentially do not capture more than the fitness of an amino acid sequence for a particular 1D succession of secondary structure segments and residue solvent accessibility. The prediction-based threading method on average finds any structurally homologous region at first rank in 29% of the cases (including sequence information). For the 22% first hits detected at highest scores, the expected accuracy rose to 75%. However, the task of detecting entire folds rather than homologous fragments was managed much better; 45 to 75% of the first hits correctly recognised the fold.

Algorithms↗