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PubMed1994

Profile enhancement and cephalometric landmark identification.

The reproducibility of two soft tissue landmarks (SN, V) and two anterior bony landmarks (A, ANS) was determined by three observers for three cephalometric techniques. The three techniques were aimed at soft tissue profile enhancement either by standard exposure control (technique 1), a hand-held metal shield covering the profile (technique 2), or a brass wedge in the collimator (technique 3). For each technique, the sample was restricted to 20 subjects with a skeletal convexity greater than +4 mm. The four landmarks were identified three times with 7-day intervals between readings. The figure-of-merit (or mean radius) method was used to assess the probability of "hitting" a target (landmark) area. The mean radius from the sample mean point of impact (MPI) ranged from 0.585 mm to 1.758 mm. For a specific landmark, the difference was never greater than 0.5 mm when grouped by observer and technique. No technique excelled in overall consistency for the identification of anterior bony and soft tissue landmarks. Techniques 1 and 3 produced the most consistent identification of points ANS and SN, but with no significant difference between the two techniques. Interacting factors prevented any recommendation regarding a preferential technique for the identification of points A and V. There is statistically no reason to recommend the use of a handheld, metal profile shield for more consistent landmark identification.

Analysis of Variance↗

PubMed1997

Olfactory identification deficit in relation to schizotypy.

Past studies have shown that schizophrenic men tend to be impaired on the olfactory identification task. We examined individual differences in the olfactory function in relation to schizotypy. Healthy individuals who might carry latent liability for schizophrenia participated in olfactory identification and acuity tasks. Psychometrically ascertained schizotypic men showed deficits on the olfactory identification task but not on the olfactory acuity task. The olfactory identification function partly depended on the menstrual cycle in women. But further systematic research is needed to clarify the possible role of estrogen.

Adult↗

PubMed2003

Rapid identification of Trichophyton tonsurans by PCR-RFLP analysis of ribosomal DNA regions.

BACKGROUND: Culture morphology of Trichophyton (T.) tonsurans, an emerging pathogen of dermatophytosis in Japan, varies widely and species level identification is sometimes very difficult. Reliable molecular markers are expected to be introduced for their identification. OBJECTIVE: The present study was conducted to evaluate the efficacy of restriction fragment length polymorphism (RFLP) analysis of PCR amplified ribosomal (r) DNA including internal transcribed spacers (ITS), as an identification tool. METHODS: Total cellular DNA was extracted from 26 Japanese isolates of T. tonsurans, along with several taxa of the members in the T. mentagrophytes complex, T. rubrum, T. violaceum and Epidermophyton floccosum, using a mini-preparation method. PCR amplicons were digested with restriction enzymes Mva I or Hinf I, then electrophoresed on 5% polyacrylamide gel. RESULTS: The banding profiles were observed about 8 h from initiating DNA extraction. Intraspecies polymorphism was not detected among T. tonsurans isolates, and their profiles obtained using Mva I digestion were clearly different from those of the other dermatophyte species. The restriction profiles evaluated from nucleotide sequence of the regions by a computer analysis were compatible with the electrophoresed profiles on gel. CONCLUSION: PCR-RFLP analysis is a rapid and reliable tool for the identification of T. tonsurans.

Adolescent↗

PubMed2003

PCR-based identification of common dermatophyte species using primer sets specific for the DNA topoisomerase II genes.

BACKGROUND: We have determined nucleotide sequences of the DNA topoisomerase II genes of the dermatophyte species, and conducted a PCR-based identification system using species-specific primers for the nucleotide sequences. OBJECTIVE: To identify the major dermatophytes, Trichophyton rubrum, T. mentagrophytes, T. violaceum, M. gypseum, M. canis and E. floccosum, by PCR amplifications at the species level, without determining the nucleotide sequence. METHODS: For PCR-based identification of the major dermatophyte species, a common primer set (dPsD1) for these species and species-specific primer sets (PsT and PsME) for each species were designed based on the genomic sequences of the DNA topoisomerase II genes of the dermatophytes, and tested for their specificities in PCR amplifications. The method consisted of amplification of the genomic DNA topoisomerase II gene by the common primer set, followed by a second PCR with the primer sets consisting of species-specific primers for each dermatophyte species. RESULTS: Using dPsD1, a DNA fragment of 3390 bp was amplified from the genomic DNA of all the dermatophyte species. In the subsequent nested PCR using species-specific primer sets (PsT and PsME), both sets amplified unique sizes of PCR products, all of which corresponded to a species of the dermatophytes even in the presence of other fungal DNA. CONCLUSION: We demonstrate that the PCR-based identification targeting the DNA topoisomerase II gene is rapid and simple, and is available as a tool for the identification of the major dermatophyte species.

Arthrodermataceae↗

PubMed2003

Left-lateralized fMRI activation in the temporal lobe of high repressive women during the identification of sad prosodies.

We investigated with fMRI whether different lateralization types of cortical activation in prosodic tasks are caused by individually different stress-related coping strategies. After healthy women had been classified as high or low repressive they performed four different identification tasks with acoustically presented speech material while being in the MR scanner. The two materials presented in blocks were emotionally irrelevant CV syllables and adjectives with a mix of different prosodic intonations. Sad and happy intonations had to be targeted by two affective identification tasks in the same adjective mixtures. For testing stimulus-material effects the phoneme /a/ had to be identified both in the syllables and the adjectives. This design allowed us to test influences of coping strategies and affective tasks on cortical activation in both hemispheres. Results showed no differences in global cortical lateralization as a function of high or low repressiveness and no global support for either the valence hypothesis or the right-hemisphere hypothesis of emotional processing. However, we observed differences in auditory and speech cortex. In accordance to the construct of repression/sensitization, high repressive women showed larger left, low repressive women larger right hemisphere activation during the identification of sad intonations. Thus, differences in stress-related coping strategies may not lead to general differences in cortical lateralization, but may depend on specific elicitors and task-relevant brain areas. In contrast, the identification of happy intonations led to strong and right-lateralized global cortical activation independent of coping strategies which complies with the right-hemisphere hypothesis of emotional processing. In addition, this may reflect general cognitive and arousal effects of task difficulty as well as auditory cue-specific attentional effects.

Adaptation, Psychological↗

PubMed1991

Comparative evaluation of five agglutination techniques and a new miniaturized system for rapid identification of methicillin-resistant strains of Staphylococcus aureus.

The speciation of methicillin-resistant Staphylococcus aureus (MRSA) poses a significant diagnostic problem when rapid identification methods such as slide agglutination tests, are used, because of the high proportion of false-negative reactions. 150 perfectly identified MRSA strains were tested on 5 commonly used agglutination reagents ("Bacto staph latex test", "Monostaph", "Pastorex staph", "Staphaurex", and "Staphyslide test") in comparison with a new micromethod ("RAPIDEC staph") which detects a type of staphylocoagulase within 2 hours by a fluorescence test. The "RAPIDEC staph" reagent enabled identification of all the MRSA while the agglutination tests gave poorer results: "Monostaph" correctly identified 64.6% of strains, "Staphyslide", 59.3%, "Bacto staph latex test", 44.6%, "Pastorex staph", 38.6% and "Staphaurex", 28.6%. These results show that agglutination slide tests are not reliable enough for the identification of MRSA which are more and more encountered in hospital wards. The authors recommend not to use slide agglutination methods. They suggest the tube test for coagulase which is the reference technique, although it is time-consuming and not well standardized. The results of this evaluation encourage the use of the "RAPIDEC staph" reagent since it is an easy-to-use, reliable technique for the rapid identification of Staphylococcus aureus.

Agglutination Tests↗

PubMed1996

Comparison of BBL Crystal ANR ID Kit and API rapid ID 32 A for identification of anaerobic bacteria.

BBL Crystal ANR ID Kit and the API System rapid ID 32 A are miniaturized identification systems for anaerobes using enzymatic tests. The incubation period of both systems is 4 hours. A comparative evaluation of the BBL Crystal Identification System Anaerobe ID Kit (Becton Dickinson Microbiology Systems, Cockeysville, USA) with anaerobes grown on Columbia and Schaedler agar plates (Becton Dickinson Microbiology Systems, Cockeysville, USA) and the API System rapid ID 32 A (BioMérieux SA, Lyon, France) with bacteria grown on Columbia agar (Becton Dickinson Microbiology Systems, Cockeysville, USA) which is recommended by the manufacturer as cultivation medium, was performed with 207 mostly fresh clinical anaerobe isolates, including 104 gram-negative bacilli, 12 gram-negative cocci, 15 gram-positive cocci, 14 gram-positive sporeforming bacilli and 62 representatives of gram-positive non-sporeforming bacilli. With supplemental testing the Crystal system with inocula from Columbia and Schaedler agar and API inoculates from Columbia agar identified to genus level 144 (69.6%), 152 (73.4%) and 109 (52.7%) isolates, respectively. Misidentification to genus level was found by Crystal from Columbia and Schaedler agar and by API from Columbia agar in 17 (8.2%), 15 (7.3%) and 12 (5.8%) isolates, respectively. 36 isolates were not determined to species level by classical anaerobic methods or the systems only identified to genus level. 26 anaerobes were not included in the database of the Crystal or API system. From the remaining 145 clinical isolates with supplemental testing, Crystal from Columbia and Schaedler agar plates correctly identified 91 (62.8%) and 102 (70.3%), respectively, and API, 69 (47.6%) isolates. For the correct identification to genus and species level of the 207 clinical isolates tested, the Crystal system from Columbia and Schaedler agar and API system from Columbia agar required supplemental testing, as specified by the manufacturer, for 39 (27.1%), 34 (22.4%) and 14 (12.8%) isolates, respectively. Among the 207 clinical isolates tested, 27 had been frozen and 26 had been lyophilized. In a comparative evaluation, the fresh isolates showed a slightly to significantly better identification rate than the frozen or lyophilized specimens in all three tests. The individual reproducibility of the Crystal ANR ID, which had been tested before the accuracy study was performed, ranged from 90.8% to 100%. The overall reproducibility was determined to be 97.3%. Time consumption studies and cost analysis did not show a significant difference between both systems, but Crystal ANR ID was found to be easier to use than API rapid ID 32.

Bacteria, Anaerobic↗

PubMed2001

Identification of confounders in the assessment of the relationship between lead exposure and child development.

PURPOSE: To explore the best approach to identify and adjust for confounders in epidemiologic practice. METHODS: In the Port Pirie cohort study, the selection of covariates was based on both a priori and an empirical consideration. In an assessment of the relationship between exposure to environmental lead and child development, change-in-estimate (CE) and significance testing (ST) criteria were compared in identifying potential confounders. The Pearson correlation coefficients were used to evaluate the potential for collinearity between pairs of major quantitative covariates. In multivariate analyses, the effects of confounding factors were assessed with multiple linear regression models. RESULTS: The nature and number of covariates selected varied with different confounder selection criteria and different cutoffs. Four covariates (i.e., quality of home environment, socioeconomic status (SES), maternal intelligence, and parental smoking behaviour) met the conventional CE criterion (> or =10%), whereas 14 variables met the ST criterion (p < or = 0.25). However, the magnitude of the relationship between blood lead concentration and children's IQ differed slightly after adjustment for confounding, using either the CE (partial regression coefficient: -4.4; 95% confidence interval (CI): -0.5 to -8.3) or ST criterion (-4.3; 95% CI: -0.2 to -8.4). CONCLUSIONS: Identification and selection of confounding factors need to be viewed cautiously in epidemiologic studies. Either the CE (e.g., > or = 10%) or ST (e.g., p < or = 0.25) criterion may be implemented in identification of a potential confounder if a study sample is sufficiently large, and both the methods are subject to arbitrariness of selecting a cut-off point. In this study, the CE criterion (i.e., > or = 10%) appears to be more stringent than the ST method (i.e., p < or = 0.25) in the identification of confounders. However, the ST rule cannot be used to determine the trueness of confounding because it cannot reflect the causal relationship between the confounder and outcome. This study shows the complexities one can expect to encounter in the identification of and adjustment for confounders.

Bias↗

PubMed1997

Identification of peroxisome proliferator-activated receptor ligands from a biased chemical library.

BACKGROUND: The peroxisome proliferator-activated receptors (PPARs) were cloned as orphan members of the nuclear receptor superfamily of transcription factors. The identification of subtype-selective ligands for PPARalpha and PPARgamma has led to the discovery of their roles in the regulation of lipid metabolism and glucose homeostasis. No subtype-selective PPARdelta ligands are available and the function of this subtype is currently unknown. RESULTS: A three-component library was designed in which one of the monomers was biased towards the PPARs and the other two monomers were chosen to add chemical diversity. Synthesis and screening of the library resulted in the identification of pools with activity on each of the PPAR subtypes. Deconvolution of the pools with the highest activity on PPARdelta led to the identification of GW 2433 as the first high-affinity PPARdelta ligand. [3H]GW 2433 is an effective radioligand for use in PPARdelta competition-binding assays. CONCLUSIONS: The synthesis of biased chemical libraries is an efficient approach to the identification of lead molecules for members of sequence-related receptor families. This approach is well suited to the discovery of small-molecule ligands for orphan receptors.

Binding, Competitive↗

PubMed2006

The influence of visual and nonvisual attributes in visual object identification.

To elucidate the role of visual and nonvisual attribute knowledge on visual object identification, we present data from three patients, each with visual object identification impairments as a result of different etiologies. Patients were shown novel computer-generated shapes paired with different labels referencing known entities. On test trials they were shown the novel shapes alone and had to identify them by generating the label with which they were formerly paired. In all conditions the same triad of computer-generated shapes were used. In one condition, the labels (banjo, guitar, violin) referenced entities that were both visually similar and similar in terms of their nonvisual attributes within semantics. In separate conditions we used labels (e.g., spike, straw, pencil or snorkel, cane, crowbar) that referenced entities that were similar in terms of their visual attributes but were dissimilar in terms of their nonvisual attributes. The results revealed that nonvisual attribute information profoundly influenced visual object identification. Our patients performed significantly better when attempting to identify shape triads whose labels referenced objects with distinct nonvisual attributes versus shape triads whose labels referenced objects with similar nonvisual attributes. We conclude that the nonvisual aspects of meaning must be taken into consideration when assessing visual object identification impairments.

Aged↗

PubMed2002

Identification of components of protein complexes using a fluorescent photo-cross-linker and mass spectrometry.

This study describes a novel method for improving the specific recognition, detection, and identification of proteins involved in multiprotein complexes. The method is based on a combination of coimmunoprecipitation, chemical cross-linking, and specific fluorescent tagging of protein components in close association with one another. Specific fluorescent tagging of the protein complex components was achieved using the cleavable, fluorescent cross-linker sulfosuccinimidyl 2-(7-azido-4-methylcoumarin-3-acetamido) ethyl-1,3'-dithiopropionate (SAED). Following dissociation and separation by SDS-PAGE, the fluorescently tagged proteins are then visualized by UV illumination, excised, and, following in-gel digestion, identified by mass spectrometry. In this study, a complex of the HIV-envelope protein gp120 and its cellular receptor CD4 was used as a model system. The sensitivity of detection of fluorescent SAED-labeled proteins in SDS gels, and the sensitivity of the mass spectrometric identification of fluorescent proteins after in-gel digestion, is in the range of a few hundred femtomoles of protein. This sensitivity is comparable to that achieved with silver-staining techniques, but fluorescence detection is protein independent and no background interference occurs. Furthermore, fluorescence labeling is significantly more compatible with mass spectrometric identification of proteins than is silver staining. The first application of this strategy was in the investigation of the mechanism of spermiation, the process by which mature spermatids separate from Sertoli cells. For the coimmunoprecipitation experiment, an antibody against paxillin, a protein involved in spermatid-Sertoli cell junctional complexes, was used. More components of the paxillin protein complex were visible by fluorescence detection of SAED-labeled proteins than were visible on comparable silver-stained gels. Mass spectrometric analysis of the fluorescently labeled proteins identified integrin alpha6 precursor as a protein associated in a complex with paxillin. The identification of integrin alpha6 precursor was confirmed by Western blot analysis and verifies the applicability of this novel approach for identifying proteins involved in protein complexes.

Animals↗

PubMed2004

Impact of ion trap tandem mass spectra variability on the identification of peptides.

Peptide identification based on tandem mass spectrometry and database searching algorithms has become one of the central technologies in proteomics. At the heart of this technology is the ability to reproducibly acquire high-quality tandem mass spectra for database interrogation. The variability in tandem mass spectra generation is often assumed to be minimal, and peptide identifications are typically based on a single tandem mass spectrum. In this paper, we characterize the variance of scores derived from replicate tandem mass spectra using several database search algorithms and demonstrate the effects of spectral variability on the correct identification of peptides. We show that the variance associated with the collection of tandem mass spectra can be substantial leading to sizable errors in search algorithm scores ( approximately 5-25% RSD) and ultimately incorrect assignments. Processing strategies are discussed to minimize the impact of tandem mass spectra variability on peptide identification.

Algorithms↗

PubMed2004

An alternative to tandem mass spectrometry: isoelectric point and accurate mass for the identification of peptides.

The traditional approach to the identification of peptides in complex biological samples integrally involves the use of tandem mass spectrometry to generate a unique fragmentation pattern in order to accurately assign its identity to a particular protein. In this article we describe the theoretical basis for a new paradigm for the identification of peptides and proteins. This methodology employs the use of accurate mass and peptide isoelectric point (pI) as identification criteria, and represents a change in focus from current tandem mass spectrometry-dominated approaches. A mathematical derivation of the false positive rate associated with accurate mass and pI measurements is presented to demonstrate the utility of the technique. The equations for calculation of the experimental false positive rate allow for the determination of the validity of the data. The false positive rate issue examined in detail here is not restricted to accurate mass-based approaches, but also has application to the tandem mass spectrometry community as well. The theoretical proteomes of Escherichia coli and Rattus norvegicus are used to evaluate the efficacy of this approach. The power of the technique is demonstrated by analyzing a series of peptides with the same monoisotopic masses but with differing isoelectric points. Finally, the speed of algorithm when combined with the experimental peptide analysis has the potential to rapidly accelerate the protein identification process.

Algorithms↗

PubMed2004

Increased identification of peptides by enhanced data processing of high-resolution MALDI TOF/TOF mass spectra prior to database searching.

This paper presents application of sequential enhanced data processing procedures to high-resolution tandem mass spectra for identification of peptides using the Mascot database search algorithm. A strategy for (1) selection of fragment ion peaks from MS/MS spectra, (2) utilization of improved mass accuracy of the precursor ions, and (3) wavelet denoising of the mass spectra prior to fragment ion selection have been developed. The number of peptide identifications obtained using the enhanced processing was then compared with that obtained using software provided by the instrument manufacturer. Approximately 9000 MS/MS spectra acquired by the Applied Biosystems 4700 TOF/TOF MS instrument were used as a model data set. After application of the new processing, an increase of 33% unique peptides and 22% protein identifications with at least two unique peptides were found. The influence of the processing on the percentage of false positives, estimated by searching against a randomized database, was estimated to increase false positive identifications from 2.7 to 3.9%, which was still below the 5% error rate specified in the Mascot search. These data processing approaches increase the amount of information that can be extracted from LC-MS analysis without the necessity of additional experiments.

Algorithms↗

PubMed2005

Detection of botulinum neurotoxin A in a spiked milk sample with subtype identification through toxin proteomics.

Botulinum neurotoxin (BoNT) causes the disease botulism, which can be lethal if untreated. Rapid determination of exposure to BoNT is an important public health goal. Previous work in our laboratory focused on the development of Endopep-MS, a mass spectrometry-based endopeptidase method for detecting and differentiating BoNT in buffer. This method can rapidly determine the presence of BoNT in a sample and differentiate the toxin type of BoNT present but does not yield additional information about the subtype. We now describe here the application of Endopep-MS to detect BoNT A in a spiked milk sample. This work also describes subtype identification achieved through mass spectrometric analysis of the protein toxin itself and does not require the presence of DNA from the toxin-producing bacteria. Tryptic digests of A1 and A2 subtypes of BoNT were analyzed by mass spectrometry, and peptides unique to either the A1 or A2 subtype were subjected to tandem mass spectrometry analysis to confirm their identities. Finally, subtype identification through mass spectrometric analysis was performed on BoNT A isolated from spiked milk. In its entirety, this method would allow for analysis of BoNT with toxin type identification in a few hours and subtype identification within 24 h.

Amino Acid Sequence↗

PubMed1997

A strategy for rapid, high-confidence protein identification.

A procedure is described for rapid, high-confidence identification of proteins using matrix-assisted laser desorption/ionization tandem ion trap mass spectrometry in conjunction with a genome database searching strategy. The procedure involves excision of copper-stained bands or spots from electrophoretic gels, in-gel trypsin digestion of the proteins, single-stage mass spectrometric analysis of the resultant mixture of tryptic peptides, followed by tandem ion trap mass spectrometric analysis of selected individual peptides, and database searching of the relevant genomic database using the program PepFrag. The scheme provides sensitive, real-time protein identification as well as facile identification of modifications. A single operator can unambiguously identify 5-10 proteins/day from an organism whose genome is known at a level of > 0.5 pmol of protein loaded on a gel. The utility of the technique was demonstrated by the identification and characterization of a band from a human HTLV-I preparation and 11 different proteins from a yeast RNA polymerase II C-terminal repeat domain-affinity preparation. The technology has great potential for postgenome biological science, where it promises to facilitate the dissection and anatomy of macromolecular assemblages, the definition of disease state markers, and the investigation of protein targets in biological processes such as the cell cycle and signal transduction.

Amino Acid Sequence↗

PubMed1999

Full second-order chromatographic/spectrometric data matrices for automated sample identification and component analysis by non-data-reducing image analysis.

A data analysis method is proposed for identification and for confirmation of classification schemes, based on single- or multiple-wavelength chromatographic profiles. The proposed method works directly on the chromatographic data without data reduction procedures such as peak area or retention index calculation. Chromatographic matrices from analysis of previously identified samples are used for generating a reference chromatogram for each class, and unidentified samples are compared with all reference chromatograms by calculating a resemblance measure for each reference. Once the method is configured, subsequent sample identification is automatic. As an example of a further development, it is shown how the method allows identification of characteristic sample components by local similarity calculations thus finding common components within a given class as well as component differences between classes from the reference chromatograms. This feature is a valuable aid in selecting components for further analysis. The identification method is demonstrated on two data sets: 212 isolates from 41 food-borne Penicillium species and 61 isolates from 6 soil-borne Penicillium species. Both data sets yielded over 90% agreement with accepted classifications. The method is highly accurate and may be used on all sorts of chromatographic profiles. Characteristic component analysis yielded results in good agreement with existing knowledge of characteristic components, but also succeeded in identifying new components as being characteristic.

Chromatography↗

PubMed1999

Microorganism identification by mass spectrometry and protein database searches.

A method for rapid identification of microorganisms is presented, which exploits the wealth of information contained in prokaryotic genome and protein sequence databases. The method is based on determining the masses of a set of ions by MALDI TOF mass spectrometry of intact or treated cells. Subsequent correlation of each ion in the set to a protein, along with the organismic source of the protein, is performed by searching an Internet-accessible protein database. Convoluting the lists for all ions and ranking the organisms corresponding to matched ions results in the identification of the microorganism. The method has been successfully demonstrated on B. subtilis and E. coli, two organisms with completely sequenced genomes. The method has been also tested for identification from mass spectra of mixtures of microorganisms, from spectra of an organism at different growth stages, and from spectra originating at other laboratories. Experimental factors such as MALDI matrix preparation, spectral reproducibility, contaminants, mass range, and measurement accuracy on the database search procedure are addressed too. The proposed method has several advantages over other MS methods for microorganism identification.

Bacillus subtilis↗