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PubMed2006

Development of an at-line method for the identification of angiotensin-I inhibiting peptides in protein hydrolysates.

A fast at-line method was developed for the identification of ACE inhibiting (ACEI) peptides in protein hydrolysates. The method consists of activity measurements of fractions collected from a two-dimensional HPLC fractionation of the peptide mixture followed by MS identification of the peptides in the inhibiting fractions. The inhibition assay is based on the inhibiting effect of ACEI peptides on the hydrolytic scission of the substrate Hippuric acid-His-Leu (HHL) during the ACE-catalysed hydrolysis reaction. A fast LC method was developed for the quantification of Hippuric acid (H) and Hippuric acid-Histidine-Leucine (HHL), allowing a large number of fractions to be analysed within a reasonable time period. The method is sensitive and uses only standard laboratory equipment. The limit of detection is 0.34 microM for the known ACEI peptide IPP. This is sufficiently sensitive for the identification of only moderately active peptides and/or ACEI peptides present at low concentrations. The relative standard deviation of the inhibition assay was 12% measured over a time period of 2 months. The IC50 value of IPP measured with the assay was 5.6 microM, which is comparable to the values of 5 microM and 5.15 microM reported in literature for the standard Matsui method. The assay was successfully applied in the identification of ACEI peptides in enzymatically hydrolysed caseinate samples. Two new, not earlier published ACEI peptides were identified; MAP (beta-casein f102-104) and ITP (alpha-s2-casein f119-121) with IC50 values of 3.8 microM and 50 microM, respectively.

Angiotensin I↗

PubMed2004

Fractionation of peptides and identification of proteins from Saccharomyces cerevisiae in proteomics with the use of reversed-phase capillary liquid chromatography and pI-based approach.

The aim of the work was to explore the identification of proteins from Saccharomyces cerevisiae using combined capillary reversed-phase liquid chromatography (RPLC) and in-solution isoelectric focusing (sIEF) for fractionation of peptides prior to mass spectrometry analysis. That method was proved to be the alternative separation method for complex mixtures of protein tryptic digests in proteomics. Analysis of the identification of peptides was performed with the use of electrospray ionization-ion trap tandem mass spectrometry (ESI-IT-MS/MS). First, the sIEF fractionation was carried out prior to separation and mass spectrometry identification by nano-LC/ESI-MS/MS instrument. The proposed approach based on sIEF and nano-LC/ESI-MS/MS analysis was proved to be an efficient and accurate alternative fractionation method of complex protein digests and can be considered as the useful tool for identification of proteins. Moreover, analytical information from that approach can be considered as the additional source of database matching constraint and can be valuable tool for analytical and bioinformatics studies of peptides fractionation in proteomics. Based on the MS/MS results obtained with ESI-IT-MS/MS instrument, 851 proteins from S. cerevisiae were identified. However, after careful analysis of the data reduction in number of proteins to 126 was obtained. Those results are discussed and interpreted in the view of the evaluation method used.

Animals↗

PubMed2005

Establishment of a Fast Chemical Identification System for screening of counterfeit drugs of macrolide antibiotics.

A Fast Chemical Identification System (FCIS) consisting of two colour reactions based on functional groups in molecules of macrolide antibiotics and two TLC methods was developed for screening of fake macrolide drugs. The active ingredients could be extracted from their oral preparations by absolute alcohol. Sulfuric acid reaction as a common reaction of macrolides was first used to distinguish the macrolides from other types of drugs and then 16-membered macrolides and 14-membered ones were distinguished by potassium permanganate reactions depending on the time of loss of colour in the test solution; after which a TLC method carried out on a GF(254) plate (5 cm x 10 cm) was chosen to further identification of the macrolides. The mobile phase A consisting of ethyl acetate, hexane and ammonia (100:15:15, v/v) was used for the identification of 14-membered macrolides, and the mobile phase B consisting of trichloromethane, methanol and ammonia (100:5:1, v/v) was used for the identification of 16-membered ones. A suspected counterfeit macrolide preparation can be identified within 40 min. The system can be used under different conditions and has the virtues of robustness, simplicity and speed.

Ammonia↗

PubMed2006

Determination of new retention indices for quick identification of essential oils compounds.

The classical methods of chromatographic identification of compounds were based on calculation of retention indices by using different stationary phases. The aim of the work was to differentiate essential oils extracted from different plant species by identification of some of their major compounds. The method of identification was based on the calculation of new retention indices of essential oils compounds fractionated on a polar chromatographic column with temperature programming system. Similar chromatograms have been obtained on the same column for one plant family with two different temperature gradients allowing the rapid identification of essential oils of different species, sub-species or chemotypes of Citrus, Mentha and Thymus.

Algorithms↗

PubMed2004

In vivo identification of sentinel lymph nodes for clinical stage I non-small cell lung cancer for abbreviation of mediastinal lymph node dissection.

BACKGROUND: We previously reported that an identification of sentinel lymph node (SN) with a techenetium-99m (99mTc) tin colloid by ex vivo counting, i.e. the radio-activity of dissected lymph nodes, was a reliable method of establishing the first site of nodal metastasis in non-small cell lung cancer [J. Thorac. Cardiovasc. Surg. 124(2002)486]. However, for SN navigation surgery, SN should be identified before lymph node dissection (in vivo) but not after that (ex vivo). In order to reduce mediastinal lymph node dissection for clinical stage I non-small cell lung cancer (NSCLC) by SN navigation surgery, the SN identifications for hilar lymph nodes by ex vivo counting, and for mediastinal lymph nodes by in vivo, were evaluated. METHODS: Intra-operative SN identification using 99mTc tin colloid was conducted on 104 patients with clinical stage I NSCLC who had had major lung resections with mediastinal lymph node dissections. The hilar SNs were identified by ex vivo counting (after lung resection) and the mediastinal SNs were identified by in vivo counting (before lymph node dissection). To evaluate the accuracy of mediastinal SN identification by in vivo counting, it was compared with the data by ex vivo counting. RESULTS: SNs were identified in 84 patients (81%). SNs were identified at the hilum by ex vivo counting in 78 patients (93%) and at the mediastinum by in vivo counting in 40 patients (48%). While 15 patients had lymph node metastases, i.e. N1 in six and N2 in nine, the SNs could be found to have metastases during operation in 13 of the 15 patients (87%). The in vivo counting of the mediastinum missed out the mediastinal SNs identified by ex vivo counting in four of the 84 patients (5%). CONCLUSION: If the hilar SNs identified by ex vivo counting and the mediastinal SNs identified by in vivo counting had no metastases, then mediastinal lymph node dissection could be abbreviated for patients with clinical stage I NSCLC.

Aged↗

PubMed2003

Identification of foodborne bacteria by infrared spectroscopy using cellular fatty acid methyl esters.

Identification of bacterial species by profiling fatty acid methyl esters (FAMEs) has commonly been carried out by using a 20-min capillary gas chromatographic procedure followed by library matching of FAME profiles using commercial MIDI databases and proprietary pattern recognition software. Fast GC (5 min) FAME procedures and mass spectrometric methodologies that require no lipid separation have also been reported. In this study, bacterial identification based on the rapid (2 min) infrared measurement of FAME mixtures was demonstrated. The microorganisms investigated included Gram positive bacteria Staphylococcus aureus, Listeria monocytogenes, Bacillus anthracis, and Bacillus cereus, and Gram negative bacteria from the family Enterobacteriacae: Yersinia enterocolitica, Salmonella typhimurium, Shigella sonnei, and Escherichia coli (four strains of E. coli), and non-Enterobacteriacae: Vibrio cholerae, Vibrio vulnificus, and Vibrio parahemolyticus. Foodborne bacterial mixtures of FAMEs were measured by using an attenuated total reflection (ATR)-Fourier transform infrared (FTIR) spectroscopic procedure and discriminated by multivariate analysis. Results showed that the Enterobacteriacae could be discriminated from the vibrios. The identification was at the level of species (for the Bacillus and Vibrio genera) or strains (for the E. coli species). A series of bacterial FAME test samples were prepared and analyzed for accuracy of identification, and all were correctly identified. Our results suggest that this infrared strategy could be used to identify foodborne pathogens.

Fatty Acids↗

PubMed2005

FT-IR spectroscopy as a tool for rapid identification and intra-species characterization of airborne filamentous fungi.

Identification of microfungi is time-consuming due to cultivation and microscopic examination and can be influenced by the interpretation of the macro- and micro-morphological characters observed. Fungal conidia contain mycotoxins that may be present in bioaerosols and thus the capacity for production of mycotoxins (and allergens) needs to be investigated to create a basis for reliable risk assessment in environmental and occupational hygiene. The present investigation aimed to create a simple but sophisticated method for the preparation of samples and the identification of airborne fungi by FT-IR spectroscopy. The method was suited to reproducibly differentiate Aspergillus and Penicillium species on the generic, the species, and the strain level. There are strong indications that strains of one taxon differing in metabolite production can be reliably distinguished by FT-IR spectroscopy (e.g. Aspergillus parasiticus). On the other hand, species from different taxa being similar in secondary metabolite production showed comparably higher similarities. The results obtained here can serve as a basis for the development of a database for species identification and strain characterization of microfungi. The method presented here will improve and facilitate the risk assessment in case of bioaerosol exposure, as strains with different physiological properties (e.g. toxic, non-toxic) could be differentiated. Moreover, it has the potential to significantly improve the identification of microfungi in various fields of applied microbiological research, e.g. high throughput screening in view of specific physiological properties, biodiversity studies, inventories in environmental microbiology, and quality control measures.

Air Microbiology↗

PubMed2006

16S rRNA sequencing in routine bacterial identification: a 30-month experiment.

Accurate identification of bacterial isolates is an essential task in clinical microbiology. Phenotypic methods are time-consuming and either fail to identify some bacteria such as Gram-positive rods entirely or at least fail to do so in some clinical situations. 16S rDNA sequencing is a recent method of identification which offers a useful alternative. In this study, we investigate the usefulness of this method for identifying a range of bacteria in a clinical laboratory under routine conditions. Over a period of 30 months, 683 isolates were obtained from clinical specimens, sequenced and analysed. For 568 of these isolates (83.1%), the sequence provided species level identification. For 108 isolates (15.8%), the identification was limited to the genus level, and for 7 isolates (1%), the sequence remained unidentifiable by 16S rDNA sequence analysis. For the isolates identified only to the genus level, the 16S rDNA approach failed to identify bacteria to the taxonomic level for 3 reasons: failure to differentiate between species in 72 isolates (66%), the lack of any closely related sequence in the database for 15 isolates (13.8%) and the presence of more than 1% of undetermined position in the sequence for 13 isolates (12%).

Bacteria↗

PubMed2003

Transgenic tumor models for carcinogen identification: the heterozygous Trp53-deficient and RasH2 mouse lines.

Genetically altered mouse models (GAMM) for human cancers have been critical to the investigation and characterization of oncogene and tumor suppressor gene expression and function and the associated cancer phenotype. Similarly, several of the mouse models with defined genetic alterations have shown promise for identification of potential human carcinogens and investigation of mechanisms of carcinogen-gene interactions and tumorigenesis. In particular, both the B6.129N5-Trp53 mouse, heterozygous for a p53 null allele, and the CB6F1-RasH2 mouse, hemizygous for the human H-ras transgene, have been extensively investigated. Using 26-week exposure protocols at or approaching the maximum tolerated dose, the summary results to date indicate the potential for GAMM to identify and, possibly, classify chemicals of potential risk to humans using short-term carcinogenicity experiments. This IWGT session focused on: (1) the development of recommendations for genetic/molecular characterization required in animals, tissues, and tumors before and after treatment for identification of presumptive human carcinogens based on the current state of knowledge, (2) identification of data gaps in our current state of knowledge, and (3) development of recommendations for research strategies for further development of our knowledge base of these particular models. By optimization of protocols and identification of significant outcomes and responses to chemical exposure in appropriate short-term mechanism-based genetically altered rodent models, strategies for prevention and intervention may be developed and employed to the benefit of public health.

Animals↗

PubMed2004

Automated human frontal lobe identification in MR images based on fuzzy-logic encoded expert anatomic knowledge.

Identification of human brain structures in MR images comprises an area of increasing interest, which also presents numerous methodological challenges. Here we describe a new knowledge-based automated method designed to identify several major brain sulci and then to define the frontal lobes by using the identified sulci as landmarks. To identify brain sulci, sulcal images were generated by morphologic operations and then separated into different components based on connectivity analysis. Subsequently, the individual anatomic features were evaluated by using fuzzy membership functions. The crisp decisions, i.e., the identification of sulci, were made by taking the maximum of the summation of all the membership functions. The identification was designed in a hierarchical order. The longitudinal fissure was extracted first. The left and right central sulci were then identified based on the left and right hemispheres. Next, the lateral sulci were identified based on the central sulci and hemispheres. Finally, the left and right frontal lobes were defined from the two hemispheres. The method was evaluated by visual inspection, comparison with manual segmentation, and comparison with manually volumetric results in references. The average Jaccard similarities of left and right frontal lobes between the automated and manual segmentation were 0.89 and 0.91, respectively. The average Kappa indices of left and right frontal lobes between the automated and manual segmentation were 0.94 and 0.95, respectively. These results show relatively high accuracy of using this novel method for human frontal lobe identification and segmentation.

Algorithms↗

PubMed2005

Redox proteomics identification of oxidized proteins in Alzheimer's disease hippocampus and cerebellum: an approach to understand pathological and biochemical alterations in AD.

Alzheimer's disease (AD) is characterized by the presence of neurofibrillary tangles, senile plaques and loss of synapses. There is accumulating evidence that oxidative stress plays an important role in AD pathophysiology. Previous redox proteomics studies from our laboratory on AD inferior parietal lobule led to the identification of oxidatively modified proteins that were consistent with biochemical or pathological alterations in AD. The present study was focused on the identification of specific targets of protein oxidation in AD and control hippocampus and cerebellum using a redox proteomics approach. In AD hippocampus, peptidyl prolyl cis-trans isomerase, phosphoglycerate mutase 1, ubiquitin carboxyl terminal hydrolase 1, dihydropyrimidinase related protein-2 (DRP-2), carbonic anhydrase II, triose phosphate isomerase, alpha-enolase, and gamma-SNAP were identified as significantly oxidized protein with reduced enzyme activities relative to control hippocampus. In addition, no significant excessively oxidized protein spots were identified in cerebellum compared to control, consistent with the lack of pathology in this brain region in AD. The identification of oxidatively modified proteins in AD hippocampus was verified by immunochemical means. The identification of common oxidized proteins in different brain regions of AD brain suggests a potential role for these oxidized proteins and thereby oxidative stress in the pathogenesis of Alzheimer's disease.

Aged↗

PubMed2005

Early, partly anticipatory, neural oscillations during identification set the stage for priming.

Perceptual priming is a fundamental long-term memory capability by which exposure to a stimulus improves later perceptual processing of that stimulus. A widespread hypothesis is that priming is the later result of perceptual learning during stimulus identification. Testing this hypothesis involves isolating priming without explicit memory, and appropriately measuring brain activity during initial experimental exposure to assess whether brain activity related to identification differs as a function of later priming. Here, we show, using magnetoencephalography (MEG), that words primed in a later test are distinguished from unprimed words at initial exposure by (a) more specific responses in perceptual brain areas, indicated by an early (within 240 ms after word onset) decrease in amplitude but increase in phase alignment of beta and gamma oscillations, and (b) improved coordination of responses across perceptual and higher brain areas in the same time window, indicated by an increase in interareal phase synchrony of alpha oscillations. The increase in interareal phase synchrony partly started already in the pre-stimulus period, approximately 60-80 ms prior to word onset, showing that the improved coordination of responses across areas was partly anticipatory. The anatomy and early timing of these patterns reveal a neural link between identification and long-term memory. The pre-stimulus findings additionally show that priming is related to the stimulus-specific anticipatory state of visual identification areas at initial exposure.

Adult↗

PubMed2006

Species identification of erythromycin-resistant Campylobacter isolates and optimization of a duplex PCR for rapid detection.

This study describes the approach used to verify the species identity of 23 erythromycin-resistant Campylobacter isolates whose identity was initially determined based mainly on the results of the rapid hippurate hydrolysis test or the results of the API-Campy identification system. Species identification of the isolates investigated was confirmed by repeating hippurate hydrolysis using a modification of the rapid hydrolysis test, in addition to performing three genetic-based assays. The original identification was verified in 69.6% of the isolates. The remaining isolates showed discrepancies in identity as determined by results of the identification assays performed. A duplex PCR assay, targeting the hipO and aspA genes, indicated the existence of mixed cultures of C. jejuni and C. coli in the frozen stocks of two of these isolates.

Anti-Bacterial Agents↗

PubMed2005

Identification and differentiation of Staphylococcus carnosus and Staphylococcus simulans by species-specific PCR assays of sodA genes.

The aim of this study was to design species-specific PCR assays for rapid and reliable identification and differentiation of Staphylococcus (S.) carnosus and S. simulans strains. Two different sets of primers, targeting the manganese-dependent superoxide dismutase (sodA) gene of S. carnosus and S. simulans, respectively, were designed. Species-specificity of both sets of primers was evaluated by using 93 strains, representing 26 different species of the genus Staphylococcus, 3 species of the genus Kocuria (K.), 1 species of the genus Micrococcus (Mic.) and 1 species of the genus Macrococcus (Mac.) as reference. By using primers simF and simR the expected PCR fragment was obtained only when purified DNA from S. simulans strains was used. Amplification performed by using primers carF and carR produced a PCR fragment of the expected length, when DNA from strains of S. carnosus and S. condimenti were used as template. Nevertheless, DraI digestion of the carF/carR PCR fragment allowed a clear differentiation of strains of these two species. Species-specific PCR assays designed during this study, overcoming many of the limitations of the traditional identification procedures, can be considered a valid strategy for detection and identification of S. carnosus and S. simulans strains. The rapidity (about 4h from DNA isolation to results), the reliability and low cost of the PCR procedures established suggests that the methods may be profitably applied for specific detection and identification of S. carnosus, S. condimenti and S. simulans strains in starter cultures and meat products.

Bacterial Proteins↗

PubMed2004

Hazard identification for contaminants.

In recent years, the recognition of generation of large quantities of toxicants and their by-products due to the industrial and/or cultural activities and transport and their persistence in the environment and biological activities brings out the necessity and importance of their assessment of risk they pose to the ecosystems (e.g. aquatic environment-coastal waters, rivers, lakes and ground water). Indeed, understanding the impacts of contaminants on the environment, including the organisms which live in it, is rather complicated. Nevertheless, the need for protection of the scarce natural resources in the environment and wiser use of them brings the necessity and importance of focusing more attention to the issue. Accordingly the process of ecological risk assessment (ERA) has evolved rapidly since the Environmental Protection Agency (EPA) issued a framework for ecological risk assessment in 1992. The ecological risk assessment involves three stages in a continuous process: (1) problem formulation (problem identification-hazard identification), (2) the analysis of exposure and effects and (3) risk characterisation. Risk management follows the risk characterisation. Of these stages, problem identification is the most critical one which establishes the direction and scope of the ecological risk assessment. The stage involves identifying the actual environmental value(s) to be protected (assessment endpoints) and selecting ways in which these can be measured and evaluated (measurement endpoints). The accuracy of the risk estimation is largely based on the availability of the key information about the contaminant characteristics, ecosystem at risk and ecological effects and the less uncertainty associated with them. The key information required during this phase of the risk assessment process are as follows: (a) potential/actual contaminant of concern, (b) source of contaminant; current and historic use, (c) mode of action of the contaminant, (d) contaminant characteristics (e.g. physical/chemical properties and environmental behaviour, persistence in the ecosystem, transformation products and bioaccumulation), (e) ecosystem potentially at risk and (f) areas of uncertainty. Finally based on these information a conceptual model has to be developed to define the possible exposure and assessment scenarios. Herein, the aforementioned key issues concerning the problem-hazard identification stage of ecological risk assessment for contaminants have been briefly reviewed.

Chemical Phenomena↗

PubMed2005

Identification of Streptococcus spp. causing bovine mastitis by PCR-RFLP of 16S-23S ribosomal DNA.

Traditional microbiological methods for identification of Streptococcus spp. causing bovine mastitis have been demonstrated to be less than highly reliable. PCR-RFLP analysis of 16S-23S ribosomal DNA was used to characterise seven reference strains of streptococcal mastitis pathogens as well as four reference strains of other gram-positive, catalase-negative cocci of bovine origin to allow comparative identification of field isolates. RFLP analysis of PCR products, using a combination of two restriction endonucleases in single reactions (HaeIII and AluI, HaeIII and RsaI or AluI and RsaI) generated unique patterns for species of Streptococcus, Enterococcus and Lactococcus. One hundred field isolates of Streptococcus spp. collected from cows with clinical or subclinical mastitis were tested. Fifty-seven isolates, classified by conventional tests as S. uberis, were identified as 47 S. uberis and six S. parauberis by their unique RFLP patterns. The remaining four isolates had RFLP patterns distinct from the reference strains and two of these were identified as closely related to S. iniae and two to Aerococcus viridans by 16S rRNA sequencing. Conventional identification of 17 S. agalactiae and 26 S. dysgalactiae subsp. dysgalactiae was confirmed by RFLP. Use of a combination of restriction enzymes in a single tube enabled the rapid, accurate, cost effective and easily performed identification of all major streptococcal mastitis pathogens.

Animals↗

PubMed2004

Morphological identification of nematode larvae of small ruminants and cattle simplified.

A simplified system has been developed to facilitate identification of nematode larvae of the common nematodes of cattle, sheep and goats. Firstly, in addition to the characteristics conventionally used for identification (such as the shape of the cranial extremity and numbers of intestinal cells), the lengths of the infective sheath tails of infective larvae of each genus/species are related to that of Trichostrongylus spp. instead of using measurements for differentiation. For instance, if the mean length of the sheath tail (the distance the sheath extends caudad beyond the caudal tip of the larva) of Trichostrongylus spp. is assumed to be "X", then that of Haemonchus contortus is 2-2.7"X", and that of Oesophagostomum spp. from sheep is 4-7"X", etc. Secondly, by estimating the proportion of the sheath tail of a larva comprised of a terminal thin whip-like filament, identification is aided, particularly in those L3 of species that resemble one another closely, such as Chabertia ovina and Oesophagostomum venulosum or Oesophagostomum columbianum. After some practice with the system it is usually necessary to measure only one or two sheath tails of L3 in a mixed population, whereupon the identity of most of the remaining L3 can be estimated in relation to those measured, without a need for further measurements. The keys were found to facilitate differential larval identification and are particularly useful for training.

Animals↗

PubMed2006

Feature detection and letter identification.

Seeking to understand how people recognize objects, we have examined how they identify letters. We expected this 26-way classification of familiar forms to challenge the popular notion of independent feature detection ("probability summation"), but find instead that this theory parsimoniously accounts for our results. We measured the contrast required for identification of a letter briefly presented in visual noise. We tested a wide range of alphabets and scripts (English, Arabic, Armenian, Chinese, Devanagari, Hebrew, and several artificial ones), three- and five-letter words, and various type styles, sizes, contrasts, durations, and eccentricities, with observers ranging widely in age (3 to 68) and experience (none to fluent). Foreign alphabets are learned quickly. In just three thousand trials, new observers attain the same proficiency in letter identification as fluent readers. Surprisingly, despite this training, the observers-like clinical letter-by-letter readers-have the same meager memory span for random strings of these characters as observers seeing them for the first time. We compare performance across tasks and stimuli that vary in difficulty by pitting the human against the ideal observer, and expressing the results as efficiency. We find that efficiency for letter identification is independent of duration, overall contrast, and eccentricity, and only weakly dependent on size, suggesting that letters are identified by a similar computation across this wide range of viewing conditions. Efficiency is also independent of age and years of reading. However, efficiency does vary across alphabets and type styles, with more complex forms yielding lower efficiencies, as one might expect from Gestalt theories of perception. In fact, we find that efficiency is inversely proportional to perimetric complexity (perimeter squared over "ink" area) and nearly independent of everything else. This, and the surprisingly fixed ratio of detection and identification thresholds, indicate that identifying a letter is mediated by detection of about 7 visual features.

Adolescent↗