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At least 379 records · Page 21Linked to original sources
PubMed1997

High-resolution echo-planar imaging of the brain: is it suitable for routine clinical imaging?

To assess the value of echo-planar imaging (EPI) for ultra-fast routine clinical diagnostic MRI, we compared four different EPI sequences with conventional T2-weighted spin-echo images on a commercial clinical imager. We examined 25 randomly selected patients who posed different clinical questions. The images were interpreted by two experienced neuroradiologists blinded as to the sequence used. Image quality and diagnostic certainty were evaluated and the main diagnosis established from the EPI study was compared to that obtained from the T2-weighted images. Finally, EPI- and T2-based diagnoses were compared with the diagnosis resulting from a complete MRI examination. Apart from one sequence that was generally rated low as regards both diagnostic certainty and image quality, the EPI sequences were comparable to each other, but inferior to the T2-weighted images. However, two EPI sequences gave better diagnostic results than T2-weighted images compared to the full MRI examination. Gradient-echo EPI was particularly sensitive to haemorrhagic lesions. All normal cases were correctly identified on EPI studies. Only two pathological cases were missed; both had isolated cranial nerve lesions. The absence of false-positive results and the high sensitivity to ischaemic and mass lesions mean that EPI can be used for ultra-fast screening. However, from these initial studies, EPI seems unsuitable for neuroradiological investigation of patients who may have subtle lesions whose detection requires either special sequences or administration of contrast medium. EPI can nevertheless be used in addition to high-resolution T1-weighted images and may replace T2-weighted spin-echo sequences for special indications.

Adolescent↗
PubMed2026

Oxford Nanopore Sequencing of Clinical DNA for Identification and Comparative Genomic Analysis of Erysipelothrix piscisicarius.

The genus Erysipelothrix comprises facultative anaerobic, nonspore-forming, gram-positive bacteria that can cause skin infections and severe diseases such as septicemia and endocarditis in humans. Although E. rhusiopathiae is the primary pathogen, other species may also be involved, necessitating accurate identification. However, 16S rDNA sequencing lacks sufficient resolution to differentiate among Erysipelothrix species. In this study, we used Oxford Nanopore Technology (ONT) to directly sequence low-quality DNA extracted from heart valve tissue of a 66-year-old female patient with a fatal case of septicemia and aortic endocarditis. In contrast to 16S rDNA Illumina sequencing and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), which incorrectly identified the pathogen as E. rhusiopathiae, direct sequencing via ONT precisely identified E. piscisicarius as the cause of infection. About 1.47 Mb genome was retrieved from nanopore direct sequencing. Within the E. piscisicarius genome, we detected genes associated with virulence. Phylogenetic analysis showed that our strain clustered with a human-derived E. piscisicarius strain from China and swine-derived strains from Brazil. In conclusion, this study demonstrated that ONT can be used to sequence low-quality DNA extracted directly from patient specimens, obtain a draft bacterial genome, and reliably distinguish between pathogenic species.

Aged↗
PubMed2024

Optimized methods for the targeted surveillance of extended-spectrum beta-lactamase-producing Escherichia coli in human stool.

Understanding transmission pathways of important opportunistic, drug-resistant pathogens, such as extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli, is essential to implementing targeted prevention strategies to interrupt transmission and reduce the number of infections. To link transmission of ESBL-producing E. coli (ESBL-EC) between two sources, single-nucleotide resolution of E. coli strains, as well as E. coli diversity within and between samples, is required. However, the microbiological methods to best track these pathogens are unclear. Here, we compared different steps in the microbiological workflow to determine the impact different pre-enrichment broths, pre-enrichment incubation times, selection in pre-enrichment, selective plating, and DNA extraction methods had on recovering ESBL-EC from human stool samples, with the aim to acquire high-quality DNA for sequencing and genomic epidemiology. We demonstrate that using a 4-h pre-enrichment in Buffered Peptone Water, plating on cefotaxime-supplemented MacConkey agar and extracting DNA using Lucigen MasterPure DNA Purification kit improves the recovery of ESBL-EC from human stool and produced high-quality DNA for whole-genome sequencing. We conclude that our optimized workflow can be applied for single-nucleotide variant analysis of an ESBL-EC from stool.IMPORTANCEDrug-resistant infections are increasingly difficult to treat with antibiotics. Preventing infections is thus highly beneficial. To do this, we need to understand how drug-resistant bacteria spread to take action to stop infection and transmission. This requires us to accurately trace these bacteria between different sources. In this study, we compared different laboratory methods to see which worked best for detecting extended-spectrum beta-lactamase (ESBL)-producing E. coli, a common cause of urinary tract or bloodstream infections, from human stool samples. We found that enriching stool in a nutrient broth for 4 h, then plating the bacterial suspension on antibiotic-selective MacConkey agar, and finally extracting DNA from the bacteria using a specific DNA purification kit resulted in improved recovery of ESBL E. coli and high-quality DNA. Sequencing multiple isolates from stool allowed us to distinguish unambiguously and at high resolution between different variants of ESBL E. coli present in stool.

Humans↗
PubMed2002

Use of high flip angle in T1-prepared FAST sequences for myocardial perfusion quantification.

This study reports on the first use of high flip angle and radio-frequency (RF) spoiling in T1-prepared fast acquisition in steady state (FAST) sequence for myocardial perfusion in patients. T1 dynamic range was measured in vitro with a FAST, an RF FAST and a snapshot fast low-angle shot (FLASH) sequences with a 90 degrees flip angle. Myocardial perfusion was then measured twice in 6 patients during the same MR session. The RF FAST and FLASH, but not the FAST sequence, demonstrated an extended T1 dynamic range; however, the FLASH images were degraded by artifacts not present on the RF FAST images. The myocardial perfusion indices K1 (first-order transfer constant from the blood to the myocardium for the Gd-DTPA) and Vd (distribution volume of Gd-DTPA in myocardium) did not differ significantly between the two injections. K1 was 0.48+/-0.12 ml/min g(-1) and Vd was 12.5+/-2.9%. With an extended T1 dynamic range and the sensitivity required for myocardial perfusion quantification, the RF FAST sequence with a 90 degrees flip angle outperformed the snapshot FLASH sequence in terms of image quality and the FAST sequence in terms of contrast dynamic range.

Adult↗
PubMed2000

Acute neuromedical and neurosurgical admissions. Standard and ultrafast MR imaging of the brain compared with cranial CT.

PURPOSE: To evaluate the role of standard and ultrafast MR brain imaging and compare the information with CT. MATERIAL AND METHODS: This was a prospective study of 114 patients with acute neurological symptoms and signs. CT brain examinations consisted of axial non-enhanced images. MR imaging consisted of standard spin-echo/fast spin-echo sequences and a series of rapid techniques including echoplanar and single shot fast spin-echo sequences. RESULTS: Using standard MR methods, 41% of the patients had all five sequences of good technical quality, while using ultrafast methods 81% of the patients had good technical quality examinations in all five sequences. In 3% of the cases, ischaemic stroke was incorrectly reported on CT. In 24% of the cases, MR gave extra diagnostic information not reported on CT and in a further 8%, one neuroradiologist reported the abnormality in agreement with the MR, whilst the other neuroradiologist reported the CT as normal. In 2 cases, subarachnoid haemorrhage was missed on MR. Subarachnoid haemorrhage was not shown on the ultrafast sequences. CONCLUSION: MR can be used to image acute neurological admissions with a high success rate, particularly using ultrafast methods. In many cases, MR provided extra information of direct clinical relevance not shown on CT.

Acute Disease↗
PubMed1996

A polynomial-time algorithm for a class of protein threading problems.

This paper presents an algorithm for constructing an optimal alignment between a three-dimensional protein structure template and an amino acid sequence. A protein structure template is given as a sequence of amino acid residue positions in three-dimensional space, along with an array of physical properties attached to each position; these residue positions are sequentially grouped into a series of core secondary structures (central helices and beta sheets). In addition to match scores and gap penalties, as in a traditional sequence-sequence alignment problem, the quality of a structure-sequence alignment is also determined by interaction preferences among amino acids aligned with structure positions that are spatially close (we call these 'long-range interactions'). Although it is known that constructing such a structure-sequence alignment in the most general form is NP-hard, our algorithm runs in polynomial time when restricted to structures with a 'modest' number of long-range amino acid interactions. In the current work, long-range interactions are limited to interactions between amino acids from different core secondary structures. Dividing the series of core secondary structures into two subseries creates a cut set of long-range interactions. If we use N, M and C to represent the size of an amino acid sequence, the size of a structure template, and the maximum cut size of long-range interactions, respectively, the algorithm finds an optimal structure-sequence alignment in O(21C NM) time, a polynomial function of N and M when C = O(log(N + M)). When running on structure-sequence alignment problems without long-range intersections, i.e. C = 0, the algorithm achieves the same asymptotic computational complexity of the Smith-Waterman sequence-sequence alignment algorithm.

Algorithms↗
PubMed2001

BAliBASE (Benchmark Alignment dataBASE): enhancements for repeats, transmembrane sequences and circular permutations.

BAliBASE is specifically designed to serve as an evaluation resource to address all the problems encountered when aligning complete sequences. The database contains high quality, manually constructed multiple sequence alignments together with detailed annotations. The alignments are all based on three-dimensional structural superpositions, with the exception of the transmembrane sequences. The first release provided sets of reference alignments dealing with the problems of high variability, unequal repartition and large N/C-terminal extensions and internal insertions. Here we describe version 2.0 of the database, which incorporates three new reference sets of alignments containing structural repeats, trans-membrane sequences and circular permutations to evaluate the accuracy of detection/prediction and alignment of these complex sequences. BAliBASE can be viewed at the web site http://www-igbmc.u-strasbg. fr/BioInfo/BAliBASE2/index.html or can be downloaded from ftp://ftp-igbmc.u-strasbg.fr/pub/BAliBASE2 /.

Algorithms↗
PubMed2006

PDA v.2: improving the exploration and estimation of nucleotide polymorphism in large datasets of heterogeneous DNA.

Pipeline Diversity Analysis (PDA) is an open-source, web-based tool that allows the exploration of polymorphism in large datasets of heterogeneous DNA sequences, and can be used to create secondary polymorphism databases for different taxonomic groups, such as the Drosophila Polymorphism Database (DPDB). A new version of the pipeline presented here, PDA v.2, incorporates substantial improvements, including new methods for data mining and grouping sequences, new criteria for data quality assessment and a better user interface. PDA is a powerful tool to obtain and synthesize existing empirical evidence on genetic diversity in any species or species group. PDA v.2 is available on the web at http://pda.uab.es/.

Algorithms↗
PubMed2006

QualitySNP: a pipeline for detecting single nucleotide polymorphisms and insertions/deletions in EST data from diploid and polyploid species.

BACKGROUND: Single nucleotide polymorphisms (SNPs) are important tools in studying complex genetic traits and genome evolution. Computational strategies for SNP discovery make use of the large number of sequences present in public databases (in most cases as expressed sequence tags (ESTs)) and are considered to be faster and more cost-effective than experimental procedures. A major challenge in computational SNP discovery is distinguishing allelic variation from sequence variation between paralogous sequences, in addition to recognizing sequencing errors. For the majority of the public EST sequences, trace or quality files are lacking which makes detection of reliable SNPs even more difficult because it has to rely on sequence comparisons only. RESULTS: We have developed a new algorithm to detect reliable SNPs and insertions/deletions (indels) in EST data, both with and without quality files. Implemented in a pipeline called QualitySNP, it uses three filters for the identification of reliable SNPs. Filter 1 screens for all potential SNPs and identifies variation between or within genotypes. Filter 2 is the core filter that uses a haplotype-based strategy to detect reliable SNPs. Clusters with potential paralogs as well as false SNPs caused by sequencing errors are identified. Filter 3 screens SNPs by calculating a confidence score, based upon sequence redundancy and quality. Non-synonymous SNPs are subsequently identified by detecting open reading frames of consensus sequences (contigs) with SNPs. The pipeline includes a data storage and retrieval system for haplotypes, SNPs and alignments. QualitySNP's versatility is demonstrated by the identification of SNPs in EST datasets from potato, chicken and humans. CONCLUSION: QualitySNP is an efficient tool for SNP detection, storage and retrieval in diploid as well as polyploid species. It is available for running on Linux or UNIX systems. The program, test data, and user manual are available at http://www.bioinformatics.nl/tools/snpweb/ and as Additional files.

Base Sequence↗
PubMed2000

Characterization of five novel human genes in the 11q13-q22 region.

The redundancy of sequences in dbEST has approached a level where contiguous cDNA sequences of genes can be assembled, without the need to physically handle the clones from which the ESTs are derived. This is termed EST based in silico gene cloning. With the availability of sequence chromatogram files for a subset of ESTs, the quality of EST sequences can be ascertained accurately and used in contig assembly. In this report, we performed a study using this approach and isolated five novel human genes, C11orf1-C11orf5, in the 11q13-q22 region. The full open reading frames of these genes were determined by comparison with their orthologs, of which four mouse orthologs were isolated (c11orf1, c11orf2, c11orf3 and c11orf5). These genes were then analyzed using several proteomics tools. Both C11orf1 and C11orf2 are nuclear proteins with no other distinguishing features. C11orf3 is a cytoplasmic protein containing an ATP/GTP binding site, a signal peptide located in the N-terminus and a similarity to the C. elegans protein "Probable ARP 2/3 complex 20kD subunit." C11orf4 is a peptide which displays four putative transmembrane domains and is predicted to have a cytoplasmic localization. It contains signal peptides at the N- and C-termini. C11orf5 is a putative nuclear protein displaying a central coiled coil domain. Here, we propose that this purely EST-based cloning approach can be used by modestly sized laboratories to rapidly and accurately characterize and map a significant number of human genes without the need of further sequencing.

Animals↗
PubMed1998

Exonuclease enhances hybridization efficiency: improved direct cycle sequencing and point mutation detection.

Solution hybridization is an essential step in sequencing and some point mutation detection methods. In practice, this hybridization is hampered resulting in the need of additional purification of the amplification products. The use of T7 gene 6 exonuclease may lead to efficient production of single-stranded DNA. In this study, the effect of pretreatment with exonuclease on direct cycle sequencing and point mutation detection was analyzed. Exonuclease-treated products were directly cycle sequenced without further purification. This resulted in highly efficient quality improvement for sequencing allowing detection of heterozygotes. Point mutation detection by Point-EXACCT (exonuclease-amplification coupled capture technique) demonstrated detection of one cell containing a mutation in an excess of 75000 wild type cells. Exonuclease-enhanced detection methods offer simple, rapid detection strategies that are easily adaptable for widespread clinical laboratory use. With the use of exonuclease, the detection of heterozygosity using fluorescent cycle sequencing is becoming more reliable. The high sensitivity of Point-EXACCT due to the use of exonuclease makes it a highly promising method for large-scale screening of (pre)malignant changes in patients with a high risk for developing cancer.

DNA Mutational Analysis↗
PubMed2003

[A new method for EST clustering].

We developed an EST (expressed sequence tag) clustering method, ESTClustering, to generate high-quality unique expressed sequence based on large-scale EST sequencing. The method uses consensus sequences to sequence analyze with megablast and assemble each cluster with phrap in clustering process. The clustering strategy can efficiently identify gene family and alternate splicing forms of expressed sequences. It can also reduce the adverse effects caused by sequence errors. The ESTClustering method tends to provide more expressed gene forms comparing with the UniGene clustering method of the National Center for Biotechnology Information. Analysis of the 112,256 ESTs of Arabidopsis with ESTClustering produced 23,581 EST clusters. Among these Arabidopsis EST clusters, 13,597 have corresponding genome coding sequences and this number is close to the number of genes predicted with Arabidopsis ESTs. Using this clustering method, a total of 147,191 rice ESTs were clustered into 33,896 groups.

Algorithms↗
PubMed1991

Improvements in the chain-termination method of DNA sequencing through the use of 7-deaza-2'-deoxyadenosine.

Significant improvements in the quality of DNA sequencing data have been shown when deoxyadenosine triphosphate (dATP) is replaced by 7-deaza-2'-deoxyadenosine triphosphate (c7dATP). The use of c7dATP in conjunction with 7-deaza-2'-deoxyguanosine triphosphate (c7dGTP) further decreases anomalies in electrophoretic mobility which are caused by compressions involving G and/or A residues. This effect is observed for both isotope-based and fluorescence-based sequencing approaches. Replacing dATP with c7dATP also results in a higher degree of uniformity in the frequency of chain termination reactions, when such terminations involve the incorporation of fluorescence-labeled dideoxynucleotides by T7 polymerase. These improvements in the gel-resolution and distribution of chain-terminated DNA products result in higher accuracy in both manual and automated base assignment.

Adenosine Triphosphate↗
PubMed2002

A Drosophila full-length cDNA resource.

BACKGROUND: A collection of sequenced full-length cDNAs is an important resource both for functional genomics studies and for the determination of the intron-exon structure of genes. Providing this resource to the Drosophila melanogaster research community has been a long-term goal of the Berkeley Drosophila Genome Project. We have previously described the Drosophila Gene Collection (DGC), a set of putative full-length cDNAs that was produced by generating and analyzing over 250,000 expressed sequence tags (ESTs) derived from a variety of tissues and developmental stages. RESULTS: We have generated high-quality full-insert sequence for 8,921 clones in the DGC. We compared the sequence of these clones to the annotated Release 3 genomic sequence, and identified more than 5,300 cDNAs that contain a complete and accurate protein-coding sequence. This corresponds to at least one splice form for 40% of the predicted D. melanogaster genes. We also identified potential new cases of RNA editing. CONCLUSIONS: We show that comparison of cDNA sequences to a high-quality annotated genomic sequence is an effective approach to identifying and eliminating defective clones from a cDNA collection and ensure its utility for experimentation. Clones were eliminated either because they carry single nucleotide discrepancies, which most probably result from reverse transcriptase errors, or because they are truncated and contain only part of the protein-coding sequence.

Amino Acid Sequence↗
PubMed2003

Breath-hold fast recovery fast spin echo versus conventional non-breath-hold fast spin echo T2-weighted MR imaging of focal liver lesions.

INTRODUCTION: We compare the breath-hold fast recovery fast spin echo (BHFRFSE) T2-weighted and non-breath-hold fast spin echo (NBHFSE) T2-weighted sequences in image quality and lesion characterisation of focal liver lesions. MATERIALS AND METHODS: Fat-suppressed T2-weighted magnetic resonance (MR) images obtained with the 2 sequences (BHFRFSE and NBHFSE) in 79 patients with 113 liver lesions were analysed retrospectively. The image quality and nature of the lesions were evaluated by 2 experienced radiologists. RESULTS: Based on receiver operating characteristic curve analysis, lesion characterisation was comparable for both sequences. The image quality of BHFRFSE was significantly better than that of NBHFSE. The NBHFSE missed 4 malignant lesions while BHFRFSE missed 2 malignant lesions. CONCLUSION: BHFRFSE performs similarly to NBHFSE in image quality and liver lesion characterisation.

Adult↗
PubMed1996

The Shannon information entropy of protein sequences.

A comprehensive data base is analyzed to determine the Shannon information content of a protein sequence. This information entropy is estimated by three methods: a k-tuplet analysis, a generalized Zipf analysis, and a "Chou-Fasman gambler." The k-tuplet analysis is a "letter" analysis, based on conditional sequence probabilities. The generalized Zipf analysis demonstrates the statistical linguistic qualities of protein sequences and uses the "word" frequency to determine the Shannon entropy. The Zipf analysis and k-tuplet analysis give Shannon entropies of approximately 2.5 bits/amino acid. This entropy is much smaller than the value of 4.18 bits/amino acid obtained from the nonuniform composition of amino acids in proteins. The "Chou-Fasman" gambler is an algorithm based on the Chou-Fasman rules for protein structure. It uses both sequence and secondary structure information to guess at the number of possible amino acids that could appropriately substitute into a sequence. As in the case for the English language, the gambler algorithm gives significantly lower entropies than the k-tuplet analysis. Using these entropies, the number of most probable protein sequences can be calculated. The number of most probable protein sequences is much less than the number of possible sequences but is still much larger than the number of sequences thought to have existed throughout evolution. Implications of these results for mutagenesis experiments are discussed.

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↗
PubMed2026

Indoor Air Quality (IAQ) analysis and 16S rRNA gene sequencing of indoor air pollutants in a 3000-capacity religious auditorium.

A thorough molecular and environmental evaluation of microbial isolates and indoor air quality (IAQ) at a place of worship is presented in this work. The study used a dual-methodological approach, including systematic environmental monitoring to assess occupant health concerns and 16S rRNA gene sequencing for taxonomic identification. Four different bacterial isolates were successfully identified by molecular analysis using BLAST and phylogenetic reconstruction: Bacillus tropicus (A11), Leclercia adecarboxylata (A12), Acinetobacter sp. (A13), and Escherichia coli (A14). The evolutionary position of isolate A13 indicated possible horizontal gene transfer, underscoring the existence of flexible, opportunistic pathogens within the indoor environment, whereas isolates A11 and A14 demonstrated significant genetic stability. Concurrently, six sessions of air quality monitoring showed a thermally demanding environment, with humidity (56.00-70.51%) and temperatures (29.21-33.79 °C) continuously surpassing ASHRAE guidelines. The average pollutant concentrations are within WHO and USEPA safety criteria, however, there were outliers that may suggest sporadic pollutant penetration (outdoor) or resuspension of dust. The results demonstrate a clear relationship between increased chemical pollutants (TVOCs) and reduced thermal comfort. To reduce the congregation's acute respiratory and cardiovascular risks, optimal ventilation systems and source-control measures are urgently needed, as evidenced by the presence of clinically relevant microorganisms combined with dangerous pollution levels.

Analysis↗