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PubMed2005

Detection of rubella immunoglobulin M from dried venous blood spots using a commercial enzyme immunoassay.

Confirmation of the diagnosis is critical to disease control in measles elimination and rubella control programs. In countries with limited infrastructure and laboratory capacity, collection, transport, and testing of venous blood samples may be difficult. We report the adaptation of a commercial enzyme immunoassay for the detection of rubella immunoglobulin M (IgM) in dried venous blood (DVB). We used 60 DVB, prepared at the time of venous blood collection, from the enhanced measles/rubella surveillance program and 28 DVB prepared using donor red blood cells spiked with serum, which had been tested as part of a rubella outbreak. Adaptations of the manufacturer's protocol included variations in incubation times and washing procedures. Optical densities were corrected for kit variation as recommended by the manufacturer, but no further adjustment was needed to compare serum and DVB results. Counting equivocal results as positive, the sensitivity of the DVB compared with serum for the categorization of rubella IgM as positive or negative was 96.7% (95% confidence interval [CI], 83.3-100%) and the specificity was 100% (95% CI, 93.7-100%).

Antibodies, Viral↗
PubMed2005

Entamoeba histolytica: analysis of the trophozoite proteome by two-dimensional polyacrylamide gel electrophoresis.

The Entamoeba histolytica genome project carried out at TIGR and the Sanger Institute has produced a near-complete set of deduced open reading frame sequences. These data provide strong support for the identification of signals from proteomic analyses such as two-dimensional electrophoresis by protein sequencing and/or mass spectrometric methods. To carry out an initial investigation of the E. histolytica proteome, appropriate sample preparation and silver staining protocols were adapted. After preparation of protein extracts from E. histolytica HM-1:IMSS trophozoites, solubilized proteins were separated in the first dimension in IPG (immobilized pH gradient) strips depending on their pI and subsequently in the second dimension according to their molecular weight by SDS-polyacrylamide gel electrophoresis. Of the more than 1500 protein spots visualized, several landmark spots were isolated from the gels and identified by either tryptic cleavage and subsequent MALDI-TOF mass spectrometry or by protein sequencing.

Animals↗
PubMed2004

Development and validation of a gas chromatography-mass spectrometry method for the simultaneous determination of buprenorphine, flunitrazepam and their metabolites in rat plasma: application to the pharmacokinetic study.

Buprenorphine (BUP), a synthetic opioid analgesic, is frequently abused alone, and in association with benzodiazepines. Fatalities involving buprenorphine alone seem very unusual while its association with benzodiazepines, such as flunitrazepam (FNZ), has been reported to result in severe respiratory depression and death. The quantitative relationship between these drugs remain, however, uncertain. Our objective was to develop an analytical method that could be used as a means to study and explore, in animals, the toxicity and pharmacological interaction mechanisms between buprenorphine, flunitrazepam and their active metabolites. A procedure based on gas chromatography-mass spectrometry (GC-MS) is described for the simultaneous analysis of buprenorphine, norbuprenorphine (NBUP), flunitrazepam, N-desmethylflunitrazepam (N-DMFNZ) and 7-aminoflunitrazepam (7-AFNZ) in rat plasma. The method was set up and adapted for the analysis of small plasma samples taken from rats. Plasma samples were extracted by liquid-liquid extraction using Toxi-tubes A. Extracted compounds were derivatized with N,O-bis-(trimethylsilyl)trifluoroacetamide (BSTFA), using trimethylchlorosilane (TMCS) as a catalyst. They were then separated by GC on a crosslinked 5% phenyl-methylpolysiloxane analytical column and determined by a quadrupole mass spectrometer detector operated under selected ion monitoring mode. Excellent linearity was found between 0.125 and 25 ng/microl plasma for BUP, 0.125 and 12.5 ng/microl for NBUP and N-DMFNZ, 0.125 and 5 ng/microl for FNZ, and between 0.025 and 50 ng/microl for 7-AFNZ. The limit of quantification was 0.025 ng/microl plasma for 7-AFNZ and 0.125 ng/microl for the four other compounds. A good reproducibility (intra-assay CV=0.32-11.69%; inter-assay CV=0.63-9.55%) and accuracy (intra-assay error=2.58-12.73%; inter-assay error=0.83-11.07%) were attained. Recoveries were 71, 67 and 81%, for BUP, FNZ and N-DMFNZ, respectively, and 51% for NBUP and 7-AFNZ, with CV ranging from 5.4 to 13.9%, and were concentration-independent. The GC-MS method was successfully applied to the pharmacokinetic study of BUP, NBUP, FNZ, DMFNZ and 7-AFNZ in rats, after administration of BUP and FNZ.

Analgesics, Opioid↗
PubMed2005

Standardization of quantitative tests for preclinical detection of neuromotor dysfunctions in pediatric neurotoxicology.

In the neurotoxicology pediatric domain, few neuromotor tests are specifically designed to be sensitive enough for the early detection of subtle deficits in voluntary and involuntary movements. In research and clinical domains, an effort is done to objectify or quantify the qualitative aspects of a movement (pattern of movement) in predicting neurological problems. This study aimed to standardize quantitative motor measures initially developed for adults and adapted to the evaluation of preschoolers. The sample consisted of 110 healthy children aged 4-6. The following quantitative neuromotor tests were selected: alternating movements and pointing movements (DOCO Microsystèmes Inc., Montréal, Canada), postural tremor, postural sway and simple reaction time (Danish Product Development Ltd., Snekkersten, Denmark). Validation measures included global motor tasks and a neurological examination. Results indicate adequate test-retest reliability and complementarities amongst the selected voluntary and involuntary measures. Both the feasibility and relevance of quantitative neuromotor tests in preschool aged children were established. Results also provide a representation of intra-individual and inter-individual variability within this population. Lastly, the results highlight the importance of developmental factors, behavioral factors and testing conditions in the neuromotor evaluation of young children. The proposed tests could help in the early detection of children at risk for motor dysfunctions following neurotoxic exposure. The tests can also be used for the follow up of various conditions relating to motor functions (cerebral palsy, muscular dystrophy, preterm infants) and in the evaluation of the effects of medication.

Attention↗
PubMed2004

Seroprevalences for ovine enzootic abortion in Switzerland.

Our aim was to assess the seroprevalence of Chlamydophila (Cd) abortus (Chlamydia psittaci serotype 1), denoted ovine enzootic abortion (OEA), in the Swiss sheep population. A competitive enzyme-linked immunosorbent assay (cELISA) was adapted for the investigation of pooled serum samples (pool approach) and receiver-operator characteristic (ROC) analysis was applied to define the cut-off of the pool approach. At a cut-off value of 30% inhibition, the flock-level pooled sensitivity and specificity were 92.9% and 97.6% when compared to classifying the flock based on individual-animal samples. Subsequently, sera from 775 randomly selected flocks out of 11 cantons of Switzerland were investigated using the pool approach. The cantons included in the study represented 72% of the Swiss sheep flocks and 76% of Swiss sheep population. Antibodies against Cd. abortus were found in almost 19% (144) of the 775 examined sheep flocks. Test prevalences were adjusted for the imperfect test characteristics using the Rogan-Gladen estimator and Bayesian inference. Seroprevalence was highest (43%) in the canton Graubunden. In the remaining 10 cantons the seroprevalence ranged from 2 to 29%. The cELISA in combination with testing pooled sera and statistical methods for true prevalence estimation provided a good survey tool at lower costs and time when compared to other approaches.

Abortion, Veterinary↗
PubMed2004

Relationship between baseline cortisol, social functioning and depression: a mediation analysis.

Both elevated cortisol secretion and low social support have been commonly found in depressed patients, but their respective roles in depression remain unclear. In fact, it may not be a lack of social support but a failure to obtain it that is important. The present study used mediation analysis to study the interrelationships among cortisol, social functioning and depression. Sixty healthy volunteers were recruited from the community. Depression and social functioning were measured by the Beck Depression Inventory and the Social Adaptation Self-evaluation Scale, respectively. Salivary samples were collected to measure the cortisol. Using mediation analysis, it was found that elevated cortisol secretion was a vulnerability factor for low social functioning, leading to higher depression scores. Hypercortisolaemia may be a predisposing factor and may interact with a low level of social functioning leading to depression.

Adult↗
PubMed1981

Sensory evaluation of drinking water by consumer panels.

The human senses play an important role in assessing the quality of food and of the environment. Particularly the chemical senses of taste and smell determine the pleasantness of foods and drinks and may provide a warning mechanism for the presence of more or less toxic contaminants. Even after the recent rapid development of powerful analytical techniques the human nose can easily detect trace amounts of chemicals at levels many times lower than the analytical detection limits. Although taste and odour assessment of drinking water has been practiced in many waterworks laboratories by small panels, relatively unreliable results were obtained and such sensory data played up till now only a minor role in the management of the water treatment plant. More sophisticated and reliable methods for sensory water quality evaluation are discussed. Special attention is given to the characteristics of the human senses of taste and smell, to which the methodology of sensory assessment should be better adapted. High numbers of observations on a sample are generally needed, for which purpose the use of large panels has to be realized. In this respect the help of large consumer panels is an alternative for the presently often used small laboratory groups. As an example the preliminary results of a large ongoing experiment of sensory water quality assessment by 2 consumer panels of 100 persons each in the Rotterdam area are discussed. Drinking water quality is judged at the consumer homes every week. The experiment started September 1979 and will be continued for one year.

Humans↗
PubMed2003

Techniques for studying the oxygen-sensitive transcription factor FNR from Escherichia coli.

A large variety of techniques can be adapted for use with oxygen-sensitive samples. The growth of cells and in vivo analyses, as well as protein purification and in vitro assays, can be executed either by performing necessary steps in anaerobic environments (ranging from simple closed containers to the anaerobic chamber) or by circumventing the need for anaerobiosis with the use of oxygen-resistant protein variants.

Biochemistry↗
PubMed2002

Rapid colorimetric screening test for gamma-hydroxybutyric acid (liquid X) in human urine.

A rapid colorimetric test for the detection of gamma-hydroxybutyric acid (GHB) is described. The ferric hydroxamate test for ester detection has been adapted to detect GHB in human urine samples from a healthy female and a healthy male subject. The assay can be performed within 5 min and with a GHB detection limit of 0.5 mg/ml when 0.3 ml of human urine is used and a GHB detection limit of 0.1 mg/ml when 1 ml of human urine is used. The colored complex indicating the presence of GHB is purple according to the assay conditions. Test results are free from the interference by alcohol, phenolic compounds and other biological chemicals under the assay conditions. In addition, the colorimetric test is free from the potential false-positive test result that could result from physiological concentrations of GHB.

Colorimetry↗
PubMed1994

Novelty seeking, risk taking, and related constructs as predictors of adolescent substance use: an application of Cloninger's theory.

This study tested derivations from Cloninger's (1987a) theory of substance use on a sample of 457 adolescents. A 67-item adaptation of the Tridimensional Personality Questionnaire (TPQ) and measures of 10 related constructs were administered together with measures of tobacco, alcohol, and marijuana use. Factor analysis indicated that the TPQ comprised nine subscales. An interaction for TPQ dimensions indicated that substance use was particularly elevated for persons with high novelty seeking, low harm avoidance, and low reward dependence. TPQ scales converged with constructs from other theoretical systems, being correlated with measures of behavioral undercontrol, risk taking, impulsiveness, anger, independence, life events, tolerance for deviance, and sensation seeking. Implications for substance abuse theory are discussed.

Adolescent↗
PubMed1996

Experimental analysis of internal rigid fixation osteosynthesis performed with titanium bone screw and plate systems.

The authors report the results of an experimental analysis performed on titanium miniplates and screws in order to gain a better understanding of dynamic forces in internal rigid fixation. Ten segments of bovine scapula were prepared. Osteotomies were carried out along the minor axis, following which five were fixed with four hole straight miniplates and the other five with six hole double-Y miniplates. Each sample was fastened in a special clamp adapted to a tension test machine and shearing force was applied. Force versus time was recorded and the 50 bone fragments were examined by a pathologist. On the basis of the test results, two simple computer models were developed. No significant difference was evident between the mechanical and computed tests. The most critical sections were located near the hole proximal to the osteotomy and the microscopic findings confirmed this. On the basis of the experimental results, the authors propose a new plate design in which the area subject to most stress, proximal to the bone section, would be of miniplate thickness, the distal aspect being thinner as in a microplate. It is suggested that this design would provide sufficient stability and a high degree of anatomical adjustment of the system.

Animals↗
PubMed2002

Quantitative dynamics of site-specific protein phosphorylation determined using liquid chromatography electrospray ionization mass spectrometry.

We have developed and validated a method that uses liquid chromatography/electrospray ionization-mass spectrometry to quantify site-specific protein phosphorylation. The method uses selected ion monitoring to determine the chromatographic peak areas of specific tryptic peptides from the protein of interest. The extent of phosphorylation is determined from the ratio of the phosphopeptide peak area to the peak area of an unmodified reference peptide that acts as internal standard, correcting for variations in protein amounts and peptide recovery in the digest preparation procedure. As a result, we refer to this protocol as the native reference peptide method. Mole of phosphate at the selected site per mole of protein is obtained from this ratio, using calibration curves of synthetic peptides to determine relative responses. Our method begins with protein separation by SDS-PAGE and is carried out on amounts of peptide produced by an in-gel digestion of single Coomassie blue-stained bands. To illustrate the utility of the method and provide validation, we used cardiac troponin I as analyte and monitored the time course of a protein kinase C betaII reaction. Those analyses appropriately demonstrate the time-dependent increase of phosphorylation at a PKC-preferred site, Ser44 in the peptide 41ISASPR45 and the concomitant consumption of the nonphosphorylated peptide. We believe that this method provides a novel tool to directly measure specific phosphorylation sites in proteins in different physiological states and expect that the method will be adaptable not only to a variety of samples types (i.e., culture cells, tissues, etc.) but to a variety of posttranslation modifications as well.

Animals↗
PubMed2002

Method detection limit determination and application of a convenient headspace analysis method for methyl tert-butyl ether in water.

Methyl tert-butyl ether (MTBE) is a common groundwater contaminant, introduced to the environment by leaking petroleum storage tanks, urban runoff, and motorized watercraft. In this study. a simplified (static) headspace analysis method was adapted for determination of MTBE in water samples and soil water extracts. The MDL of the headspace method was calculated to be 2.0 microg L(-1) by the EPA single-concentration design method(1) and 1.2 microg L(-1) by a calibration method developed by Hubaux and Vos (Hubaux, A.; Vos, G. Anal. Chem. 1970,42, 849-855). The MDL calculated with the Hubaux and Vos method was favored because it considers both a true positive and a false positive. The static headspace method was applied to analysis of a tap water sample and a monitoring well sample from a gasoline service station, a river sample, and aqueous extracts from soil excavated during removal of a leaking underground storage tank (LUST). The water samples examined in this study had MTTBE concentrations ranging from 6 to 19 microg L(-1). Aqueous extracts of a soil sample taken from the LUST site had 8 microg L(-1) MTBE.

Calibration↗
PubMed1998

In vivo evaluation of a closed loop monitoring strategy for induced paralysis.

OBJECTIVE: Reliable closed loop infusion systems for regulating paralysis level can be a great convenience to the anesthesiologists in automating their task. This paper describes the in vivo performance evaluation of a self-tuning controller that is designed to accommodate large variations in patient drug sensitivity, drug action delays and environmental interfering noise. METHODS: The infusion system was evaluated in six adult mongrel dogs. Following the manual induction of paralysis by an anesthesiologist, the controller regulated the infusion of vecuronium to maintain a desired level of paralysis. The integrated EMG response of the hypothenar muscle to a train-of-four stimulation of the ulnar nerve quantified the depth of paralysis. The controller's robustness was tested by contaminating the sensed twitch signal with electrocautery noise and electrode disconnection. RESULTS: The controller reached the initial level of paralysis of 100% in about 4.0 minutes and arrived at the desired level of 90% with an overshoot of 6.38% (+/-6.82). It maintained the desired level of paralysis with a 2.04% (+/-1.20) mean offset at 90% and 0.4% (+/-0.5) mean offset at 80% steady state level, respectively. The mean infusion rate to sustain 90% and 80% paralysis were 2.70 (+/-2.05) and 2.15 (+/-2.57) ((mg/kg)/min), respectively. CONCLUSIONS: The system adapted to a large variation in the sample subject drug sensitivity. It remained stable despite large amplitude disturbances and maintained the paralysis at the desired level following the removal of the disturbances.

Animals↗
PubMed2005

Detection of Ki-ras mutations in tissue and plasma samples of patients with pancreatic cancer using PNA-mediated PCR clamping and hybridisation probes.

In the present study, we combined the PCR-clamping approach with melting curve analysis using mutant specific hybridisation probes and wild-type specific peptide nucleic acids (PNAs) to determine the genotypes of the most frequent point mutation in codon 12 of the proto-oncogene Ki-ras in tissue and plasma samples of patients with pancreatic cancer. The sensitivity of our assay was 1-5 x 10(-5). The melting curve analysis of tissue samples of four patients revealed two valine mutations, one none-valine mutation and one wild-type sequence. Ki-ras alterations were found in 28% of DNAs (18 out of 64) of nonrelated plasma samples of 10 patients with ductal adenocarcinoma of the pancreas. The valine mutation was the predominantly detected gene alteration (83%). Out of ten patients investigated, four patients (40%) became positive during clinical observation with respect to Ki-ras mutation. All four patients exhibited progressive disease and high levels of tumour marker CA 19-9. In conclusion, the one-step procedure discribed may be a useful clinical tool for analysing Ki-ras point mutations in tissue and plasmas samples. In addition, this method can be adapted for simultanous detection of multiple mutations and quantitation.

Carcinoma, Pancreatic Ductal↗
PubMed1992

Urinary cotinine fluoroimmunoassay for smoking status screening adapted to an automated analyser.

A polarization fluoroimmunoassay for cotinine, a major metabolite of nicotine, has been adapted for fully automated screening of urine samples on the Abbott TDx analyser. The method has sensitivity and specificity suitable for the discrimination of active smokers from non-smokers (including passive smokers) by application of a cut-off at 0.5 mg l-1 of total urinary cotinine. Most active smokers' urine gave results over 1 mg l-1, whereas apparent levels in non-smokers were 0.08 mg l-1 or lower. A result for one sample can be obtained in about 5 min and a throughput of 80 samples h-1 can be maintained for large-scale screening applications.

Adult↗
PubMed2004

A web application to manage a database for liquid nitrogen tanks.

Summary We have developed a software, Cell Database, for archiving records about cells stored in liquid nitrogen tanks. Once installed on a web server, the database is accessed through a standard web browser. This user-friendly and self-explanatory application is independent of computer platform and periodic upgrades of a commercial software. Our web application allows import of data from other database programs and adaptation to different tank formats, types of samples, and archiving needs.

Cells↗
PubMed2003

Glutamine synthetase from the marine cyanobacteria Prochlorococcus spp: characterization, phylogeny and response to nutrient limitation.

The regulation of glutamine synthetase (EC 6.3.1.2) from Prochlorococcus was previously shown to exhibit unusual features: it is not upregulated by nitrogen starvation and it is not inactivated by darkness (El Alaoui et al. (2001) Appl Environ Microbiol 67: 2202-2207). These are probably caused by adaptations to oligotrophic environments, as confirmed in this work by the marked decrease in the enzymatic activity when cultures were subjected to iron or phosphorus starvation. In order to further understand the adaptive features of ammonium assimilation in this cyanobacterium, glutamine synthetase was purified from two Prochlorococcus strains: PCC 9511 (high-light adapted) and SS120 (low-light adapted). We obtained approximately 100-fold purified samples of glutamine synthetase electrophoretically homogeneous, with a yield of approximately 30%. The estimated molecular mass of the subunits was roughly the same for both strains: 48.3 kDa. The apparent Km constants for the biosynthetic activity were 0.30 mM for ammonium, 1.29 mM for glutamate and 1.35 mM for ATP; the optimum pH was 8.0. Optimal temperature was surprisingly high (55 degrees C). Phylogenetic analysis of glnA from three Prochlorococcus strains (MED4, MIT9313 and SS120) showed they group closely with marine Synechococcus isolates, in good agreement with other studies based on 16 S RNA sequences. All of our results suggest that the structure and kinetics of glutamine synthetase in Prochlorococcus have not been significantly modified during the evolution within the cyanobacterial radiation, in sharp contrast with its regulatory properties.

Adenosine Triphosphate↗