Semiautomated preparation of 3,5,6-trichloro-2-pyridinol in human urine using a Zymate XP laboratory robot with quantitative determination by gas chromatography-negative-ion chemical ionization mass spectrometry.
A rapid and sensitive semiautomated method was developed for quantitation of the chlorpyrifos metabolite 3,5,6-trichloro-2-pyridinol (TCP) in human urine. A Zymark Zymate XP laboratory robotics system was used to mix urine samples, transfer aliquots, add the stable-isotope-labeled TCP internal standard (13C2- or 13C2,15N-), and liberate conjugates of TCP from urine via acid hydrolysis. Samples were manually extracted into toluene, derivatized, and analyzed by gas chromatography-negative-ion chemical ionization mass spectrometry. Determination of the metabolic TCP was performed by selected ion monitoring of the dichloropyridinol fragment ions: m/z 161 for TCP and m/z 165 for 13C2-TCP or m/z 168 for 13C2,15N-TCP. Interday precision and accuracy were demonstrated over 3 years of analyses using the 13C2-TCP internal standard, with an average recovery from fortified urine samples of 93+/-12% (N = 54, concentration range 1-140 ng/mL). The method was found to be linear over the range of 0.5 to 200 ng/mL, and the limit of detection for TCP in urine was estimated to be 0.2 ng/mL with a limit of quantitation of 1 ng/mL. The effect of solids distribution on the concentration of TCP in the thawed urine samples was examined, and the results indicated that homogeneous distribution is critical for quantitation. The precision and accuracy of the automated method with respect to the transfer of homgeneous urine aliquots and delivery of internal standard yielded equivalent or improved results over the manual techniques. Overall, this method is more simple than existing methodologies, and it yields results with improved precision, accuracy, and sensitivity over previously developed methods. (+info)
Identification of Epichloe endophytes in planta by a microsatellite-based PCR fingerprinting assay with automated analysis.
Epichloe endophytes are a group of filamentous fungi that include both sexual (Epichloe) and asexual (Neotyphodium) species. As a group they are genetically diverse and form both antagonistic and mutualistic associations with temperate grasses. We report here on the development of a microsatellite-based PCR system for fingerprinting this group of fungi with template isolated from either culture or infected plant material. M13mp19 partial genomic libraries were constructed for size-fractionated genomic DNA from two endophyte strains. These libraries were screened with a mixture of DIG-labeled dinucleotide and trinucleotide repeat probes. Positive clones were sequenced, and nine unique microsatellite loci were identified. An additional microsatellite was serendipitously identified in the 3' untranscribed region of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase gene from N. lolii Lp19. Primers were designed for each locus and a panel of endophytes, from different taxonomic groupings, was screened to determine the degree of polymorphism. On the basis of these results a multiplex assay was developed for strain identification with fluorescently labeled primers for five of these loci. Using this system the size of the products amplified can be precisely determined by automated analysis, and an allele profile for each strain can be readily generated. The assay was shown to resolve endophyte groupings to the level of known isozyme phenotype groupings. In a blind test the assay was used successfully to identify a set of endophytes in planta. A reference database of allele sizes has been established for the panel of endophytes examined, and this will be expanded as new strains are analyzed. (+info)
Understanding adverse events: human factors.
(1) Human rather than technical failures now represent the greatest threat to complex and potentially hazardous systems. This includes healthcare systems. (2) Managing the human risks will never be 100% effective. Human fallibility can be moderated, but it cannot be eliminated. (3) Different error types have different underlying mechanisms, occur in different parts of the organisation, and require different methods of risk management. The basic distinctions are between: Slips, lapses, trips, and fumbles (execution failures) and mistakes (planning or problem solving failures). Mistakes are divided into rule based mistakes and knowledge based mistakes. Errors (information-handling problems) and violations (motivational problems) Active versus latent failures. Active failures are committed by those in direct contact with the patient, latent failures arise in organisational and managerial spheres and their adverse effects may take a long time to become evident. (4) Safety significant errors occur at all levels of the system, not just at the sharp end. Decisions made in the upper echelons of the organisation create the conditions in the workplace that subsequently promote individual errors and violations. Latent failures are present long before an accident and are hence prime candidates for principled risk management. (5) Measures that involve sanctions and exhortations (that is, moralistic measures directed to those at the sharp end) have only very limited effectiveness, especially so in the case of highly trained professionals. (6) Human factors problems are a product of a chain of causes in which the individual psychological factors (that is, momentary inattention, forgetting, etc) are the last and least manageable links. Attentional "capture" (preoccupation or distraction) is a necessary condition for the commission of slips and lapses. Yet, its occurrence is almost impossible to predict or control effectively. The same is true of the factors associated with forgetting. States of mind contributing to error are thus extremely difficult to manage; they can happen to the best of people at any time. (7) People do not act in isolation. Their behaviour is shaped by circumstances. The same is true for errors and violations. The likelihood of an unsafe act being committed is heavily influenced by the nature of the task and by the local workplace conditions. These, in turn, are the product of "upstream" organisational factors. Great gains in safety can ve achieved through relatively small modifications of equipment and workplaces. (8) Automation and increasing advanced equipment do not cure human factors problems, they merely relocate them. In contrast, training people to work effectively in teams costs little, but has achieved significant enhancements of human performance in aviation. (9) Effective risk management depends critically on a confidential and preferable anonymous incident monitoring system that records the individual, task, situational, and organisational factors associated with incidents and near misses. (10) Effective risk management means the simultaneous and targeted deployment of limited remedial resources at different levels of the system: the individual or team, the task, the situation, and the organisation as a whole. (+info)
Computerized tailored feedback to change cognitive determinants of smoking: a Dutch field experiment.
In the last decade, attempts have been made to improve the efficacy of minimal interventions by tailoring them to individual features. In the development of these tailored interventions, it is important to know which information in interventions is essential. Most smoking cessation interventions contain information on the outcomes of quitting and skills to be used in a quit attempt. The present study was designed to assess the cognitive changes caused by both sorts of information. Therefore, 246 smokers who were planning to quit within 6 months were randomly assigned to three different conditions. In the first condition, the respondents received a computer-generated tailored letter on the outcomes of smoking cessation. In the second condition, the respondents received a computer-generated tailored letter containing self-efficacy enhancing information, mainly on skills. In both conditions, the contents of the letters were based on the pre-test scores of the participants. Participants in the control condition did not receive any cessation information. The results show that information on the outcomes of quitting changed expected outcomes while information on coping skills changed self-efficacy expectations, in comparison with the control condition. Comparing both experimental conditions, information on the outcomes led to changes in expected outcomes, whereas information on coping skills did not lead to higher self-efficacy expectations than information on the outcomes of quitting. It is concluded that the hypothesized effects were partly verified. (+info)
Long-term effectiveness of computer-generated tailored feedback in smoking cessation.
Although tailored interventions consisting of only a few pages of information lead to more quitting than no intervention in the short term, the long-term efficacy of a single tailored intervention still has to be proven. In the present study smokers were reactively recruited and randomly allocated to one of four intervention conditions: (1) outcome information, (2) self-efficacy enhancing information, (3) both sorts of information or (4) no information. Smokers in the three experimental groups received computer-generated tailored feedback containing the condition-specific information, by mail. The results from the 14 months follow-up can be summarized as follows. Compared to the no information condition, all three experimental conditions led to significantly more smokers who had engaged in 24-h quit attempts. However, no experimental condition led to more 7-day quitting than the no information condition. With regard to continuous abstinence, the experimental condition offering a combination of outcome information and self-efficacy enhancing information had a significant effect, compared to the no information condition. It is concluded that a minimal six-page tailored intervention can be beneficial in supporting smokers to quit smoking, even after 14 months. (+info)
Molecular diversity of cell-matrix adhesions.
In this study we have examined for molecular heterogeneity of cell-matrix adhesions and the involvement of actomyosin contractility in the selective recruitment of different plaque proteins. For this purpose, we have developed a novel microscopic approach for molecular morphometry, based on automatic identification of matrix adhesions, followed by quantitative immunofluorescence and morphometric analysis. Particularly informative was fluorescence ratio imaging, comparing the local labeling intensities of different plaque molecules, including vinculin, paxillin, tensin and phosphotyrosine-containing proteins. Ratio imaging revealed considerable molecular heterogeneity between and within adhesion sites. Most striking were the differences between focal contacts, which are vinculin- and paxillin-rich and contain high levels of phosphotyrosine, and fibrillar adhesions, which are tensin-rich and contain little or no phosphotyrosine. Ratio imaging also revealed considerable variability in the molecular substructure of individual focal contacts, pointing to a non-uniform distribution of phosphotyrosine and the different plaque constituents. Studying the quantitative relationships between the various components of the submembrane plaque indicated that the levels of vinculin, paxillin and phosphotyrosine in adhesion sites are positively correlated with each other and negatively correlated with the levels of tensin. Tyrosine phosphorylation of focal contacts was highly sensitive to cellular contractility, and was diminished within 5 minutes after treatment with the kinase inhibitor H-7, an inhibitor of actomyosin contractility. This was followed by the loss of paxillin and vinculin from the focal adhesions. Tensin-rich fibrillar adhesions were relatively insensitive to H-7 treatment. These findings suggest a role for contractility in the generation of matrix adhesion diversity. (+info)
Automated analysis of interatomic contacts in proteins.
MOTIVATION: New software has been designed to assist the molecular biologist in understanding the structural consequences of modifying a ligand and/or protein. RESULTS: Tools are described for the analysis of ligand-protein contacts (LPC software) and contacts of structural units (CSU software) such as helices, sheets, strands and residues. Our approach is based on a detailed analysis of interatomic contacts and interface complementarity. For any ligand or structural unit, these software automatically: (i) calculate the solvent-accessible surface of every atom; (ii) determine the contacting residues and type of interaction they undergo (hydrophobic-hydrophobic, aromatic-aromatic, etc.); (iii) indicate all putative hydrogen bonds. LPC software further predicts changes in binding strength following chemical modification of the ligand. AVAILABILITY: Both LPC and CSU can be accessed through the PDB and are integrated in the 3DB Atlas page of all PDB files. For any given file, the tools can also be accessed at http://www.pdb.bnl. gov/pdb-bin/lpc?PDB_ID= and http://www.pdb.bnl. gov/pdb-bin/csu?PDB_ID= with the four-letter PDB code added at the end in each case. Finally, LPC and CSU can be accessed at: http://sgedg.weizmann.ac.il/lpc and http://sgedg.weizmann.ac.il/csu. (+info)
MIAH: automatic alignment of eukaryotic SSU rRNAs.
SUMMARY: MIAH is a WWW server for the automatic alignment of new eukaryotic SSU rRNA sequences to an existing alignment of 1500 sequences. AVAILABILITY: http://chah.ucc.ie/MIAH Contact : (+info)