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PubMed2000

Phylogenetic analysis of Pasteurella multocida subspecies and molecular identification of feline P. multocida subsp. septica by 16S rRNA gene sequencing.

Pasteurella multocida is commonly found in the oral cavity of cats and dogs. In humans it is known as an opportunistic pathogen after bites from these animals. Phenotypic identification of P. multocida based on biochemical reactions is often limited and usually only done on a species level, even though 3 subspecies are described. For molecular taxonomy and diagnostic purposes a phylogenetic analysis of the three subspecies of P. multocida based on their 16S rRNA (rrs) gene sequence was therefore carried out. We found P. multocida subsp. septica on a distinguished branch on the phylogenetic tree of Pasteurellaceae, due to a 1.5% divergence of its rrs gene compared to the two other, more closely related subspecies multocida and gallicida. This phylogenetic divergence can be used for the identification of P. multocida subsp. septica by rrs gene determination since they form a phylogenetically well isolated and defined group as shown with a set of feline isolates. Comparison to routine phenotypic identification shows the advantage of the sequence-based identification over conventional methods. It is therefore helpful for future unambiguous identification and molecular taxonomy of P. multocida as well as for epidemiological investigations.

Animals↗
PubMed1994

Identification of Bacillus strains isolated from milk and cream with classical and nucleic acid hybridization methods.

A total of 529 bacterial strains have been isolated from milk and cream sampled at different sites in a dairy production plant under conditions selective for aerobic sporeforming bacteria. Identification with classical methods based on morphological, physiological and biochemical criteria showed Bacillus licheniformis to be the most frequently occurring Bacillus sp. The investigation also revealed 62 unidentified strains. Classical identification methods were time consuming (3-7 d), lacked specificity and--because of their dependence on phenotypic gene expression--sometimes produced ambiguous results. Consequently, a colony hybridization method developed for the identification of B. licheniformis strains and using nonradioactive labelled 23S rRNA targeted oligonucleotide probes was also used. Identification of B. licheniformis with both classical and hybridization methods revealed diverging identification results for 70 strains.

Animals↗
PubMed1981

Comparison of the Microbact-12E and 24E systems and the API-20E system for the identification of Enterobacteriaceae.

The Microbact-24E and the Microbact-12E systems are two new miniaturized identification systems for the identification of organisms belonging to the family Enterobacteriaceae. These two systems were compared to the API-20E system for the identification of 352 fresh clinical isolates of Enterobacteriaceae. All three systems were easy to use and came complete with computerized profile registers to assist with final identification of the isolates. The Microbact-24E identified 98%, the API-20E and MB-12E identifying 94.3% and 88.6% respectively. Where different identifications were obtained with the Microbact-24E and API-20E conventional biochemical tests, motility and serology were performed. The Microbact-24E system proved to be a very accurate and convenient means of identifying members of the family Enterobacteriaceae.

Bacteriological Techniques↗
PubMed1999

Impairment of olfactory identification in obsessive-compulsive disorder.

BACKGROUND: Olfactory identification ability has been associated with processing in the orbitofrontal cortex (OFC), an area that has been implicated in the pathophysiology of obsessive-compulsive disorder (OCD). Although olfactory sensitivity is normal in patients with OCD, no study has investigated olfactory identification in this disorder. METHODS: A group of 20 subjects with OCD and 23 age- and education-matched controls performed a standardized test of olfactory identification. They also performed computerized tests of spatial memory span, spatial working memory and spatial recognition memory that have been shown previously to be sensitive to cognitive deficits in patients with OCD. RESULTS: Performance on the olfactory identification task, spatial recognition task and spatial span task was significantly worse in the OCD group than controls. CONCLUSIONS: While impairment in spatial cognition is consistent with previous studies of OCD, its significance for brain-behaviour models of OCD is unclear. However, the finding of abnormal olfactory identification in patients with OCD is consistent with the hypothesis that there is a disruption to processing at the level of the OFC in the disorder.

Adult↗
PubMed2003

Left hippocampal volume loss in Alzheimer's disease is reflected in performance on odor identification: a structural MRI study.

The very high sensitivity and specificity of odor identification tasks in discriminating between Alzheimer's patients and normals suggests that they reflect the presence of underlying neuropathology. Significant neuropathological changes are seen in areas critical to processing olfactory information, even in the early stages of Alzheimer's disease (AD). The current study was designed to investigate whether performance on olfactory tasks (odor threshold and odor identification) was related to volumetric MRI measures of mesial temporal areas central to olfactory information processing and important in the neuropathology of AD. Participants were 8 male and 5 female patients with probable AD, and 10 male and 12 female normal age-matched controls, diagnosed at the UCSD Alzheimer's Disease Research Center. The study investigated correlations between volumetric measures of hippocampus, the parahippocampal gyrus and the amygdala, and the psychophysical measures of olfactory function. Robust relationships were observed between mesial temporal lobe volumes and olfactory functional measures. The finding of a strong relationship between left hippocampal volume and performance on the odor identification task (r = .85) is compatible with a left-hemisphere superiority for verbally mediated olfactory tasks. The findings suggest a neural substrate for the breakdown in functional performance on verbally mediated odor identification tasks in Alzheimer's disease and suggest the utility of quantitative MRI measures and psychophysical performance in the assessment of AD. These results support the potential clinical utility of inclusion of odor identification tests in diagnostic batteries for detecting AD.

Aged↗
PubMed2003

MultiTag: multiple error-tolerant sequence tag search for the sequence-similarity identification of proteins by mass spectrometry.

The characterization of proteomes by mass spectrometry is largely limited to organisms with sequenced genomes. To identify proteins from organisms with unsequenced genomes, database sequences from related species must be employed for sequence-similarity protein identifications. Peptide sequence tags (Mann, 1994) have been used successfully for the identification of proteins in sequence databases using partially interpreted tandem mass spectra of tryptic peptides. We have extended the ability of sequence tag searching to the identification of proteins whose sequences are yet unknown but are homologous to known database entries. The MultiTag method presented here assigns statistical significance to matches of multiple error-tolerant sequence tags to a database entry and ranks alignments by their significance. The MultiTag approach has the distinct advantage over other sequence-similarity approaches of being able to perform sequence-similarity identifications using only very short (2-4) amino acid residue stretches of peptide sequences, rather than complete peptide sequences deduced by de novo interpretation of tandem mass spectra. This feature facilitates the identification of low abundance proteins, since noisy and low-intensity tandem mass spectra can be utilized.

Alcohol Dehydrogenase↗
PubMed2004

Fluorogenic derivatization reagents suitable for isolation and identification of cysteine-containing proteins utilizing high-performance liquid chromatography-tandem mass spectrometry.

The fluorogenic derivatization reagents with a positive charge, 4-(dimethylaminoethylaminosulfonyl)-7-chloro-2,1,3-benzoxadiazole (DAABD-Cl) and 7-chloro-2,1,3-benzoxadiazole-4-sulfonylaminoethyltrimethylammonium chloride (TAABD-Cl), are proposed for use in proteomics studies. Following derivatization of protein mixtures with these reagents, a series of standard processes of isolation, digestion, and identification of the proteins were performed utilizing high-performance liquid chromatography-fluorescence detection and tandem mass spectrometry with the probability-based protein identification algorithm. Both DAABD and TAABD derivatives were detected fluorometrically at the femtomole level and showed more than 100-fold improvement in sensitivity compared to the underivatized original compounds with an electrospray ionization ion trap mass spectrometer analysis. The modification of the MASCOT database search system memorized with the fragment information of a DAABD-attached Cys residue allowed the identification of the proteolytic peptide fragments of the derivatized bovine serum albumin (BSA) with an estimated 38% sequence coverage of BSA. Utilizing DAABD-Cl as a derivatization reagent, identification of several proteins was also possible in a soluble extract of Caenorhabditis elegans (10 microg of protein). Consequently, for identification of proteins in the complex matrixes of proteins, DAABD-Cl could be a more appropriate reagent than ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate as reported previously.

Animals↗
PubMed2004

Identification of bacteria using tandem mass spectrometry combined with a proteome database and statistical scoring.

Detection and identification of pathogenic bacteria and their protein toxins play a crucial role in a proper response to natural or terrorist-caused outbreaks of infectious diseases. The recent availability of whole genome sequences of priority bacterial pathogens opens new diagnostic possibilities for identification of bacteria by retrieving their genomic or proteomic information. We describe a method for identification of bacteria based on tandem mass spectrometric (MS/MS) analysis of peptides derived from bacterial proteins. This method involves bacterial cell protein extraction, trypsin digestion, liquid chromatography MS/MS analysis of the resulting peptides, and a statistical scoring algorithm to rank MS/MS spectral matching results for bacterial identification. To facilitate spectral data searching, a proteome database was constructed by translating genomes of bacteria of interest with fully or partially determined sequences. In this work, a prototype database was constructed by the automated analysis of 87 publicly available, fully sequenced bacterial genomes with the GLIMMER gene finding software. MS/MS peptide spectral matching for peptide sequence assignment against this proteome database was done by SEQUEST. To gauge the relative significance of the SEQUEST-generated matching parameters for correct peptide assignment, discriminant function (DF) analysis of these parameters was applied and DF scores were used to calculate probabilities of correct MS/MS spectra assignment to peptide sequences in the database. The peptides with DF scores exceeding a threshold value determined by the probability of correct peptide assignment were accepted and matched to the bacterial proteomes represented in the database. Sequence filtering or removal of degenerate peptides matched with multiple bacteria was then performed to further improve identification. It is demonstrated that using a preset criterion with known distributions of discriminant function scores and probabilities of correct peptide sequence assignments, a test bacterium within the 87 database microorganisms can be unambiguously identified.

Algorithms↗
PubMed1998

Modification of cysteine residues by alkylation. A tool in peptide mapping and protein identification.

Although mass spectrometric peptide mapping has become an established technique for the rapid identification of proteins isolated by polyacrylamide gel electrophoresis (PAGE), the results of the identification procedure can sometimes be ambiguous. Such ambiguities become increasingly prevalent for proteins isolated as mixtures or when only very small amounts of the proteins are isolated. The quality of the identification procedure can be improved by increasing the number of peptides that are extracted from the gel. Here we show that cysteine alkylation is required to ensure maximal coverage in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) peptide mapping of proteins isolated by PAGE. In the described procedure, alkylation was performed prior to electrophoresis to avoid the adventitious formation of acrylamide adducts during electrophoresis. In this way, homogeneous alkylation was obtained with three different alkylating reagents (4-vinylpyridine, iodoacetamide, acrylamide). Cysteine alkylation was also used as a tool for the identification of cysteine-containing peptides. Using a 1:1 mixture of unlabeled acrylamide and deuterium-labeled acrylamide ([2,3,3'-D3]acrylamide), the proteins of interest were alkylated prior to electrophoretic separation. Peptide mixtures produced by trypsin digestion of the resulting protein bands were analyzed by MALDI-TOF MS, and the cysteine content of the peptides was inferred from the isotopic distributions. The cysteine content information was readily obtained and used to improve the protein identification process.

Acrylamide↗
PubMed1999

Identification of polar toxicants in industrial wastewaters using toxicity-based fractionation with liquid chromatography/mass spectrometry.

An efficient and novel method for the identification of toxic compounds in industrial wastewater was developed. In the first step, the samples collected were tested for toxicity using the recently developed ToxAlert 10 system based upon luminescence inhibition of freeze-dried Vibrio fischeri. In the second step, sequential solid-phase extraction (SSPE) and liquid chromatography/mass spectrometry (LC/MS) for compound identification were employed to isolate and identify compounds in the waters. Average recoveries ranging from 72 to 95% were obtained using the SSPE methodology for ubiquitous analytes such as poly(ethylene glycol)s, nonylphenol and alcohol polyethoxylates, phenols, linear alkylbenzenesulfonates, and benzene- and naphthalenesulfonates. In the third step, all the extracts obtained after SSPE followed by LC/MS identification were tested again with the ToxAlert system. The procedure was applied to influent and effluent samples of a sewage treatment plant (STP) and to a raw tannery effluent that constitutes the main type of influent in the receiving waters of the STP. This method has shown that, despite the complexity of the untreated tannery wastewaters with an average total organic carbon (TOC) value of 1960 mg of C/L, the biological treatment of the STP eliminates compounds that inhibit luminiscence of V. fischeri. In the final step, the chemical toxicity of the tentatively identified chemicals was tested to identify the toxicants in the waters. Comparison of the toxicities of the sample, the extracts, and individual components has shown that diverse classes of pollutants were responsible for toxicity, as all fractions of toxic samples gave significant bioluminescence inhibition values. Toxicity of the two intermediate-polarity SSPE fractions was attributed to alcohol ethoxylates, nonylphenol ethoxylates, bis(2-ethylhexyl)phthalate, and linear alkylbenzenesulfonates. In the most nonpolar and most polar fractions, identification of the compounds responsible for toxicity was unclear. By the toxicity-based fractionation, followed by LC/MS methodology, it was feasible to identify between 1.4 and 7.5% of the TOC, thus expanding the number of toxicants identified in these complex wastewaters as compared to those identified by conventional gas chromatography/mass spectrometric (GC/MS) methods. When artificial water samples were reconstituted using similar concentrations of the chemicals detected in the wastewaters, nonsynergetic toxicity effects were observed for all analytes with the exception of 2,6-naphthalenedisulfonate (2,6-NPS), which promoted the bioluminescence inhibition. The toxicity-directed identification was successful for the STP's samples and showed 1400 times higher toxicity for the raw tannery wastewaters as compared to the mixed industrial and domestic wastewaters by applying the Weibull model.

Chromatography, Liquid↗
PubMed2000

An algorithm for automated bacterial identification using matrix-assisted laser desorption/ionization mass spectrometry.

An algorithm for bacterial identification using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is being developed. This mass spectral fingerprint comparison algorithm is fully automated and statistically based, providing objective analysis of samples to be identified. Based on extraction of reference fingerprint ions from test spectra, this approach should lend itself well to real-world applications where samples are likely to be impure. This algorithm is illustrated using a blind study. In the study, MALDI-MS fingerprints for Bacillus atrophaeus ATCC 49337, Bacillus cereus ATCC 14579T, Escherichia coli ATCC 33694, Pantoea agglomerans ATCC 33243, and Pseudomonas putida F1 are collected and form a reference library. The identification of test samples containing one or more reference bacteria, potentially mixed with one species not in the library (Shewanella alga BrY), is performed by comparison to the reference library with a calculated degree of association. Out of 60 samples, no false positives are present, and the correct identification rate is 75%. Missed identifications are largely due to a weak B. cereus signal in the bacterial mixtures. Potential modifications to the algorithm are presented and result in a higher than 90% correct identification rate for the blind study data, suggesting that this approach has the potential for reliable and accurate automated data analysis of MALDI-MS.

Algorithms↗
PubMed2000

Identification of protein vaccine candidates from Helicobacter pylori using a preparative two-dimensional electrophoretic procedure and mass spectrometry.

Helicobacter pylori is an important human gastric pathogen for which the entire genome sequence is known. This microorganism displays a uniquely complex pattern of binding to complex carbohydrates presented on host mucosal surfaces and other tissues, through adhesion molecules (adhesins) on the microbial cell surface. Adhesins and other membrane-associated proteins are important targets for vaccine development. The identification and characterization of cell-surface proteins expressed by H. pylori is a prerequisite for the development of vaccines designed to interfere with bacterial colonization of host tissues. However, identification of membrane proteins is difficult using a traditional proteomics approach employing 2D-PAGE. We have used a novel approach in the identification of microbial proteins that employs a rapid preparative two-dimensional electrophoretic separation followed by mass spectrometry and database searches. No pre-enrichment of bacterial membranes was required. The entire process, from sample preparation to protein identification, can be completed in less than 18 hours, and the presence of proteins can be monitored after both the first- and second-dimensional separations using mass spectrometry. We were able to identify 40 proteins from a detergent-solubilized H. pylori preparation; over one-third of these were membrane or membrane-associated proteins. A functionally characterized low-abundance membrane protein, the Leb-binding adhesin, was found in this group. The use of this rapid 2D electrophoretic separation in proteomic studies of H. pylori is expected to speed up the identification of expressed virulence proteins and vaccine targets in this and other microbial pathogens.

Amino Acid Sequence↗
PubMedDate not supplied

Peptide identification using vectors of small fragment ions.

Traditionally, peptide identification using fragmentation spectra relies on extracting the maximum amount of information from spectra. Using different combinations of small ion masses, we show that identifying a small number of fragment ions in a spectrum is sufficient for peptide identification. We consider y2-, y3-, b2-, and b3-ions and find the combination of b2-y2 to be sufficient for many peptides. Adding either the y3- or the b3-ion increases specificity and allows reliable peptide identification in the human proteome. Fragmentation spectra and peptides are represented as n-dimensional vectors, where n is given by the number of fragment ions considered, and the peptide mass. The identification score is given by the Euclidian distance between the spectra and the matching peptide in n-dimensional space. We show that this approach, using minimal information, allows for precise and fast peptide identification.

Humans↗
PubMed2001

Eyewitness identification in actual criminal cases: an archival analysis.

This study analyzed 271 actual police cases in order to address several prevalent issues in the eyewitness literature. Suspect identification (SI) rates were obtained for 289 photographic lineups, 258 field showups, 58 live lineups, and 66 lineup identifications preceded by earlier identifications. SI rates were assessed for 3 levels of extrinsic evidence: no extrinsic evidence, evidence of minimal probative value, and evidence of substantial probative value. The SI rates for the photographic lineups were assessed as a function of delay, same vs. cross-race conditions, witness type, and weapon presence. SI rates declined significantly over time; SI rates were significantly greater for the same-race condition. SI rates were much greater for field showups than photographic lineups, 76% vs. 48%. The SI rates for the field showups did not vary as a function of eyewitness conditions. The relation between confidence and suspect/foil identifications for the live lineups was significant and moderately high. The utility of archival identification studies for eyewitness testimony research is discussed.

Adolescent↗
PubMed1998

Identification accuracy of children versus adults: a meta-analysis.

Identification accuracy of children and adults was examined in a meta-analysis. Preschoolers (M = 4 years) were less likely than adults to make correct identifications. Children over the age of 5 did not differ significantly from adults with regard to correct identification rate. Children of all ages examined were less likely than adults to correctly reject a target-absent lineup. Even adolescents (M = 12-13 years) did not reach an adult rate of correct rejection. Compared to simultaneous lineup presentation, sequential lineups increased the child-adult gap for correct rejections. Providing child witnesses with identification practice or training did not increase their correct rejection rates. Suggestions for children's inability to correctly reject target-absent lineups are discussed. Future directions for identification research are presented.

Adolescent↗
PubMed2004

Identification of species of the genus Candida by analysis of the 5.8S rRNA gene and the two ribosomal internal transcribed spacers.

The PCR amplification and subsequent restriction analysis of the ribosomal region spanning the internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene is applied to the identification of yeasts belonging to the genus Candida. This methodology has previously been used for the identification of some species of this genus, but in the present work this application has been applied to the identification and characterisation of a greater number of species of the genus Candida, with a special survey of species of clinical and biotechnological interest. Among the species of the genus Candida, the high variability observed, both in the length of the amplified region (ranging between 390 and 900 bp) and in their restriction patterns, allows the unequivocal identification to the species level, with the exception of the group of species that comprises C. membranifaciens, C. conglobata, C. atlantica, C. atmosphaerica, and C. oleophila, that required the sequencing of the D1/D2 domain of the 26S rRNA gene or the 5.8S-ITS region for their proper differentiation. The 5.8S-ITS restriction analysis also failed in the differentiation of species within the pairs C.aaseri/C.butyri,C.fructus/C.musae,C.santamariae var. santamariae / C. beechii and C. zeylanoides / C. krissii. In this case, the high sequence similarities obtained for their 26S D1/D2 domain and the 5.8S-ITS region indicate that each pair of species should be considered as a single species. The main purpose of this work is to generate a database for a high number of yeast species, of both biotechnological and clinical interest, and to facilitate their easy, fast, and reliable identification. The present work improves the database available online at the IATA web page (http://motor.edinfo.es/iata/) with the patterns of 75 species belonging to the genus Candida.

Animals↗
PubMed2002

X-linked recessive Menkes disease: identification of partial gene deletions in affected males.

Menkes disease is an X-linked recessive lethal disorder of copper metabolism, caused by defects in the ATP7A gene. Partial gene deletions comprise about 15% of the mutations causing Menkes disease. We have previously demonstrated identification of partial ATP7A deletions in patients by Southern blot analysis. In the present study, we report the use of three fast and reliable polymerase chain reaction (PCR)-based methods for the identification of partial ATP7A deletions in Menkes disease patients. First we demonstrate the use of multiplex PCR, a fast method for identification and rough localization of partial gene deletions, in which two exons of ATP7A are coamplified. Second, we present PCR amplification of genomic DNA across the deletion junctions, a method enabling identification of the deletion breakpoints and hence the exact size of the deletion. Finally, application of reverse transcription PCR (RT-PCR) for identification and localization of gene deletions at the cDNA level is demonstrated. By studying the mutation at the cDNA level the predicted effect of the mutation on the amino acid sequence and consequently the protein structure and function can be inferred. We demonstrate characterization of partial gene deletions in five patients, and in three of these we were able to determine the breakpoint sequences.

Adenosine Triphosphatases↗
PubMed2003

Identification of mutans streptococci by restriction fragment length polymorphism analysis of polymerase chain reaction-amplified 16S ribosomal RNA genes.

Mutans streptococci are frequently isolated from dental plaque and carious lesions. These bacteria have been identified by conventional methods such as biochemical and serologic tests followed by the isolation of colonies on the mitis-salivarius agar, which are sometimes inconsistent. Recently, species-specific polymerase chain reaction (PCR) has been reported to rapidly identify Streptococcus mutans and Streptococcus sobrinus. However, in the case of identification and classification into several species, e.g. within the group of mutans streptococci consisting of seven species, the identification using species-specific PCR seems somewhat inefficient because of need for the development and preparation of specific primers for each species. Therefore, in this study we developed a simple method using restriction fragment length polymorphism analysis of PCR-amplified 16S ribosomal RNA genes (16S rRNA genes PCR-RFLP) for the identification of seven different species included in the group of mutans streptococci. We amplified 16S rRNA gene sequences from genomic DNA samples by PCR using universal primers and digested the PCR products with the restriction endonucleases, HpaII and HaeIII. HpaII produced six RFLP patterns for eight reference strains, since the patterns for S. sobrinus, Streptococcus downei and Streptococcus ferus were similar. RFLP patterns produced with HaeIII could separate these three species. Furthermore, the RFLP patterns predicted from the 16S rRNA gene sequences in the GenBank database agreed with the actual RFLP patterns produced in the present study. The 16S rRNA sequence comparisons can be used to identify oral mutans streptococci; however, the identification by sequencing is sometimes difficult in large-scale studies and for small laboratories. Therefore, 16S rRNA genes PCR-RFLP, using HpaII and HaeIII, could be an alternative method for the identification of mutans streptococci, and may be applicable for large-scale studies on the cariogenicity of mutans streptococci.

DNA Primers↗