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PubMed2001

Fluorescent microsphere-based readout technology for multiplexed human single nucleotide polymorphism analysis and bacterial identification.

Large-scale human genotyping requires technologies with a minimal number of steps, high accuracy, and the ability to automate at a reasonable cost. In this regard, we have developed a rapid, cost-effective readout method for single nucleotide polymorphism (SNP) genotyping that combines an easily automatable single-tube allele-specific primer extension (ASPE) with an efficient high throughput flow cytometric analysis performed on a Luminex 100 flow cytometer. This robust technique employs an ASPE reaction using PCR-derived target DNA containing the SNP and a pair of synthetic complementary capture probes that differ at their 3' end-nucleotide defining the alleles. Each capture probe has been synthesized to contain a unique 25-nucleotide identifying sequence (ZipCode) at its 5' end. An array of fluorescent microspheres, covalently coupled with complementary ZipCode sequences (cZipCodes), was hybridized to biotin-labeled ASPE reaction products, sequestering them for flow cytometric analysis. ASPE offers both an advantage of streamlining the SNP analysis protocol and an ability to perform multiplex SNP analysis on any mixture of allelic variants. All steps of the assay are simple additions of the solutions, incubations, and washes. This technique was used to assay 15 multiplexed SNPs on human chromosome 12 from 96 patients. Comparison of the microsphere-based ASPE assay results to gel-based oligonucleotide ligation assay (OLA) results showed 99.2% agreement in genotype assignments. In addition, the microsphere-based multiplex SNPs assay system was adapted for the identification of bacterial samples by both ASPE and single base chain extension (SBCE) assays. A series of probes designed for different variable sites of bacterial 16S rDNA permitted multiplex analysis and generated species- or genus-specific patterns. Seventeen bacterial species representing a broad range of gram-negative and gram-positive bacteria were analyzed within 16 variable sites of 16S rDNA sequence. The results were consistent with the published sequences and confirmed by direct DNA sequencing.

Alleles↗
PubMed1992

Planned versus attained design in phase II clinical trials.

The standard phase II trial problem is to decide whether or not to continue the testing of a new agent (or combination). Typically, one tests the null hypothesis HO: p = pO against the alternative HA: p = pA, where p is the probability of response. There is available a variety of two-stage phase II designs, including optimal designs according to various criteria. Practical considerations in the conduct of multicentre trials, however, make it difficult to follow designs precisely. We investigate several approaches to adapting stopping rules when the attained sample size is not the planned size. We find that a simple approach of testing HA: p = pA at the 0.02 level at the first stage and HO: p = pO at the 0.055 level at the second stage works well across a variety of powers, pOs and pAs.

Decision Making↗
PubMed1982

Polymorphism of the haptoglobin peptides by isoelectric focusing electrophoresis and isoelectric point determinations.

In this investigation, the authors developed two new procedures: a micromethod for haptoglobin purification and the isoelectric focusing electrophoresis on slab polyacrylamide gel for peptide subtyping. These technics are adapted to the study of large sample series for population genetic surveys. The improvements obtained enabled us to disclose in an easy and highly reproducible way the Hp alpha and alpha 2 peptide chains. Electrophoretic separation of the alpha 2 FS, SS, and FF chains were greatly improved. Their frequencies estimated in a sample already investigated by the conventional PAGE presented higher values than previously described. New Hp alpha and alpha 2 mutants were also detected. For the first time, isoelectric points of the Hp peptides were determined; the values obtained are discussed with regard to their known amino acid structure.

Electrophoresis, Polyacrylamide Gel↗
PubMed1990

Sugar receptors of the stromal cell layer in human long-term bone marrow cultures: their presence, modulatory responses to changes in the microenvironment and potential role in cellular adhesion.

Intimate cellular contacts and coordinated supply of regulatory factors are required to maintain the still inexplicable dynamic equilibrium of hemopoiesis. To infer the potential participation of protein-carbohydrate interaction in this complex process, human long-term bone marrow cultures were initiated from eleven donors, and the adherent cell layer was characterized enzyme- and immunohistochemically. Utilizing an array of carrier-immobilized carbohydrate ligands and sulfated polysaccharides as probes, specific binding of various constituents of the carbohydrate chains of cellular glycoconjugates to the stromal cells was unmistakably disclosed. Biochemical analysis, employing glycocytologically effective ligands in affinity chromatography, corroborated this result. The extent of binding was markedly lower in the two samples, derived from leukemia patients. Pronounced adaptive responses for this characteristic followed changes in the culture microenvironment that are known to influence qualitative and quantitative aspects of hemopoiesis in vitro, namely omission of hydrocortisone and horse serum or addition of cytokines. Similarly, such adaptive modulation occurred on the level of accessible cell surface receptors, monitored by neoglycoenzymes. These binding sites can be involved in mediation of cellular interactions, as revealed in a model system by the interference of N-acetyl-D-galactosamine in cell adhesion. Overall, the results support the idea that glycobiological recognition may contribute to the functional integrity of the stromal cell layer as well as provide the basis for further analysis.

Bone Marrow↗
PubMed2001

Clinical and subclinical body dysmorphic disorder.

BACKGROUND: The aim of the study was to define the main demographic and clinical characteristics of Body Dysmorphic Disorder (BDD) and subclinical BDD (sBDD) in a sample derived by a screening survey done on a population of individuals referring to aesthetical medicine centers. METHOD: 487 subjects referring to hospital centers for aesthetical medicine were administered the SCID-I and the Yale-Brown Obsessive-Compulsive Scale adapted for BDD (BDD-YBOCS). The sample was thus sub-divided in three sub-samples: 1) BDD, 2) sub-clinical BDD, and 3) controls. The main demographic and clinical variables were considered and compared between the BDD and the sBDD samples. RESULTS: As previously reported, the prevalence of BDD and sBDD was 6.3% and 18.4%, respectively. The most frequent comorbid diagnosis in both BDD and sBDD patients and their relatives was Obsessive-Compulsive Disorder (OCD). A higher severity of symptoms was found in male BDD patients, while no gender-related differences were found in the sBDD group. Suicidal ideation was found in 12.1% of the sBDD and in 49.7% of the BDD patients. CONCLUSIONS: These results support the hypothesis of BDD and sBDD belonging to the OCD spectrum, and appear to advise long-term follow-up studies on the course and the prognosis of sBDD.

Adolescent↗
PubMed1988

An automated Western blot analysis using the phastsystem.

We have developed a rapid, automated method for preparing Western blots of very small amounts of proteins, utilizing a commercially available electrophoresis system (Phastsystem, Pharmacia). This system has been adapted to transfer to nitrocellulose experimental samples that were initially separated in the same system by gradient-sodium dodecyl sulfate gel electrophoresis. The developing unit of the Phastsystem has permitted automation of all the necessary steps including incubation with antibodies, saturation of nonspecific binding sites, and washing. The total elapsed time for protein separation, transfer, and staining is about 6 to 7 h.

Animals↗
PubMed1991

On-sequencer pyridylethylation of cysteine residues after protection of amino groups by reaction with phenylisothiocyanate.

Cysteine residues in polypeptides are not easily identified during automated N-terminal sequence analysis. Reaction of cysteine side chains with 4-vinylpyridine and identification as the pyridylethylated phenylthiohydantion derivative (PE-PTH-Cys) were proposed. However, after this reaction a desalting step is necessary. If limited sample amounts do not allow this desalting step, on-sequencer pyridylethylation is an alternative, although preview of the consecutive amino acid is usually observed in this case. We describe an on-sequencer procedure that avoids such preview formation by derivatizing the peptide with phenylisothiocyanate (PITC) prior to reaction with 4-vinylpyridine. The pyridylethylation is performed in the cartridge of the sequencer after immobilization of the protein or peptide on a polybrene-coated glass fiber filter and thiocarbamylation with PITC. Preview caused by N-alkylation is not observed and PE-PTH-Cys is detected in much higher yields than usual. The procedure reported here is significantly shortened, optimized to reduce side products, and avoids losses during sample handling. It can easily be adapted to any automated version of the sequencers.

Amino Acid Sequence↗
PubMed1991

An ion-exchange chromatography procedure for the isolation and concentration of basic amino acids and polyamines from complex biological samples prior to high-performance liquid chromatography.

The original objective of this study was to develop a selective and sensitive method for the analysis and quantification of basic amino acids from biological samples via reversed-phase high-performance liquid chromatography. Using various previously described techniques for the separation of amino acids, we were unsuccessful in measuring levels of histidine, arginine, ornithine, and lysine in biological samples due to the presence of interfering compounds. A "cleanup" procedure for the isolation of the basic amino acids using a weakly acidic cation exchange resin, Biorex-70 (Bio-Rad), is described in detail. Upon separation from the bulk of the neutral and acidic amino acids, the basic amino acids were subjected to precolumn fluorescence derivatization using 9-fluorenylmethyl chloroformate (FMOC) and the fluorescent derivatives were separated by RP-HPLC. The advantages of this method over previously described amino acid analysis techniques are (i) isolation and stable recovery (greater than 95%) of the desired basic amino acids, (ii) sensitivity of detection (low pmol range), (iii) complete resolution of derivatized amino acids via HPLC, (iv) limited amount of sample required for analysis, and (v) samples readily concentrated by lyophilization or rotoevaporating. This ion-exchange cleanup procedure was also adapted for the analysis of polyamines in concentrated culture media samples and proved additionally advantageous by eliminating the use of costly C-18 extraction columns required by previously described techniques.

Amino Acids, Diamino↗
PubMed1995

Evaluation of oxidant stress in chronic hemodialysis patients: use of different parameters.

Patients with chronic renal failure, particularly those undergoing regular dialysis treatment (RDT) are candidates for free radical damage. It is difficult to quantitate free radicals because of their short half-lives and reactive nature. Therefore, indirect methods measuring products of lipid peroxidation and protein oxidation are preferred. The present study displays a profile of lipid peroxidation and protein oxidation parameters, which are more sensitive and specific than the widely used method measuring thiobarbituric acid reactive substances (TBARS), adapted to the plasma and erythrocyte samples of RDT patients. We have observed increased levels of plasma and erythrocyte lipid peroxidation and also demonstrated increased protein oxidation in erythrocyte membranes of RDT patients.

Blood Proteins↗
PubMed1988

A microtiter cell-culture assay for the determination of anti-human immunodeficiency virus neutralizing antibody activity.

A microtiter cell-culture assay is described for measuring neutralizing antibody activity directed against the human immunodeficiency virus (HIV). The assay relies upon inhibition of HIV-mediated cell killing of infected MT-4 lymphoid cells. The assay exhibits comparable sensitivity to two other methods used for such measurements, is relatively rapid, may be adapted for screening large numbers of samples and involves minimal handling of infectious virus.

Antibodies, Viral↗
PubMed1987

New automated high-performance liquid chromatographic analysis of cyclosporin A and G in human serum.

An automated isocratic high-performance liquid chromatographic (HPLC) method is described for the determination of cyclosporin A and G in human serum. This method involves the use of an automated solid-liquid extraction procedure following rapid protein precipitation with acetonitrile. The use of a disposable C8 extraction cartridge allows a good recovery of cyclosporine (87%) from serum and a detection limit of 20 ng/ml with good reproducibility using 0.5 ml of sample. This method can also be adapted to whole blood measurements. The choice of a 3-micron cyano analytical column and of the mobile phase hexane-isopropanol (85:15) permitted a low column temperature (50 degrees C), a low flow-rate (0.6 ml/min) and a short run time (14 min). This method allows the accurate and fast routine monitoring of cyclosporine by HPLC, which is particularly important in hepatic transplantations.

Autoanalysis↗
PubMed1985

Evaluation of CMV total antibody EIA. An enzyme immunoassay for detection of antibodies to cytomegalovirus.

The performance of an indirect enzyme-immunoassay (CMV Total Antibody EIA, Abbott Laboratories) for the detection of the presence or absence of antibodies to cytomegalovirus (CMV) was evaluated by investigators at five clinical sites. The interassay and intraassay coefficient of variation data showed that the EIA could be performed in a highly reproducible manner at all sites. The investigators tested a total of 2314 clinical samples for antibody to CMV by the CMV Total Antibody EIA and by an indirect hemagglutination assay (IHA). The overall results demonstrated that compared with IHA the sensitivity of the EIA was 97.9% (1096/1120) and the specificity was 93.6% (1096/1171). The clinical samples tested included sera, plasma, and whole blood. These results show a good agreement between the CMV Total Antibody EIA and the IHA. This EIA is a simple, objective, and reproducible method for the detection of antibodies to CMV in sera, plasma, or whole blood samples, and the total procedure is adaptable for batching and automation and takes only 3 hr of elapsed time.

Antibodies, Viral↗
PubMed2006

Biologically directed environmental monitoring, fate, and transport of estrogenic endocrine disrupting compounds in water: A review.

Endocrine disrupting compounds (EDCs) are contaminants that may be hormonally active at low concentrations and are emerging as a major concern for water quality. Estrogenic EDCs (e-EDCs) are a subclass of EDCs that, when organisms are exposed to them, function as estrogens. Given that there are numerous e-EDCs that can negatively affect humans and wildlife, general screening techniques like biologically based assays (BBAs) may provide major advantages by estimating the total estrogenic effects of many e-EDCs in the environment. These techniques may potentially be adapted for field portable biologically directed sampling and analyses. This article summarizes available BBAs used to measure estrogenic e-EDCs in the environmental samples and also presents results relating to fate and transport of e-EDCs. Estrogenic EDCs appear to be almost ubiquitous in the environment, despite low solubility and high affinity of organic matter. Potential transport mechanisms may include: (1) transport of more soluble precursors, (2) colloid facilitated transport, (3) enhanced solubility through elevated pH, and (4) the formation of micelles by longer-chain ethoxylates. Due to their persistent and ubiquitous nature, source control strategies for e-EDCs may reduce influent concentration to wastewater treatment plants so that the post treatment effluent will decrease concentrations to estrogenically inactive levels. Alternatively if source reduction is not possible, then more testing is needed on tertiary treatment technologies and treatment efficiencies for e-EDCs. There is still a need for research on remediation and restoration approaches for habitats disturbed by elevated e-EDC concentrations.

Animals↗
PubMed2006

Comparison of an acetonitrile extraction/partitioning and "dispersive solid-phase extraction" method with classical multi-residue methods for the extraction of herbicide residues in barley samples.

An acetonitrile/partitioning extraction and "dispersive solid-phase extraction (SPE)" method that provides high quality results with a minimum number of steps and a low solvent and glassware consumption was published in 2003. This method, suitable for the analysis of multiple classes of pesticide residues in foods, has been given an acronymic name, QuEChERS, that reflects its major advantages (quick, easy, cheap, effective, rugged, safe). In this work, QuEChERS method, which was originally created for vegetable samples with a high amount of water, was modified to optimise the extraction of a wide range of herbicides in barley. Then, it was compared with known conventional multi-residue extraction procedures such as the Luke method, which was simplified and shortened by eliminating the Florisil clean-up (mini Luke) and the ethyl acetate extraction, which involves a subsequent clean-up by gel permeation chromatography (GPC) and which is the official extraction method used by some of European authorities. Finally, a simple acetone extraction was carried out to check the differences with the other three methods. Extracts were analysed by gas chromatography-time-of-flight mass spectrometry (GC-TOF/MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Mini Luke was significantly more effective for the extraction of non-polar and medium-polar compounds, but the best recoveries for polar compounds were achieved by QuEChERS and ethyl acetate methods. QuEChERS was the only method that provided an overall recovery value of 60-70% for non-, medium- and polar compounds, with some exceptions due to co-eluted matrix interferences. Clean-up by dispersive SPE was effective and did not differ so much with ethyl acetate extracts considering that QuEChERS clean-up step is much easier and less time-consuming. As a conclusion, it resulted to be the most universal extraction method by providing a well-defined phase separation without dilution and achieving acceptable recoveries in average including the extraction of the always difficult acidic herbicides. However, recoveries were not as good as required for validation purposes suggesting that residues are prone to strong matrix interactions in dry samples as barley and further method adaptation incrementing solvent strength, extraction time or more acidic or basic conditions is needed in order to achieve a complete extraction.

Acetonitriles↗
PubMed2005

Mapping the dimensionality, density and topology of data: the growing adaptive neural gas.

Self-organized maps are commonly applied for tasks of cluster analysis, vector quantization or interpolation. The artificial neural network model introduced in this paper is a hybrid model of the growing neural gas model introduced by Fritzke (Fritzke, in Advances in Neural Information Processing Systems 7, MIT Press, Cambridge MA, 1995) and the adaptive resolution clustering modification for self-organized maps proposed by Firenze (Firenze et al., in International Conference on Artificial Neural Networks, Springer-Verlag, London, 1994). The hybrid model is capable of mapping the distribution, dimensionality and topology of the input data. It has a local performance measure that enables the network to terminate growing in areas of the input space that is mapped by units reaching a performance goal. Therefore the network can accurately map clusters of data appearing on different scales of density. The capabilities of the algorithm are tested using simulated datasets with similar spatial spread but different local density distributions, and a simulated multivariate MR dataset of an anatomical human brain phantom with mild multiple sclerosis lesions. These tests demonstrate the advantages of the model compared to the growing neural gas algorithm when adaptive mapping of areas with low sample density is desirable.

Algorithms↗
PubMed2006

A "sample-and-hold" pulse-counting integrator as a mechanism for graded memory underlying sensorimotor adaptation.

The mechanisms behind the induction of cellular correlates of memory by sensory input and their contribution to meaningful behavioral changes are largely unknown. We previously reported a graded memory in the form of sensorimotor adaptation in the electromotor output of electric fish. Here we show that the mechanism for this adaptation is a synaptically induced long-lasting shift in intrinsic neuronal excitability. This mechanism rapidly integrates hundreds of spikes in a second, or gradually integrates the same number of spikes delivered over tens of minutes. Thus, this mechanism appears immune to frequency-dependent fluctuations in input and operates as a simple pulse counter over a wide range of time scales, enabling it to transduce graded sensory information into a graded memory and a corresponding change in the behavioral output. This adaptation is based on an NMDA receptor-mediated change in intrinsic excitability of the postsynaptic neurons involving the Ca2+-dependent activation of TRP channels.

Acclimatization↗
PubMed2000

Solid-phase extraction for multiresidue analysis of organic contaminants in water.

To overcome the limitations of the detection systems associated with gas or liquid chromatography, a sample pretreatment is required with the objective to provide a sample fraction enriched with all the target analytes and as free as possible from other matrix components. There is now no doubt that solid-phase extraction (SPE) has now become the method of choice for carrying out simultaneously the extraction and concentration of many compounds in aqueous samples. Many recent applications of SPE to multiresidue analysis are reviewed with an emphasis on the importance of the choice of the sorbent and of the sample volume. SPE is particularly well adapted to multiresidue analysis including compounds from a wide range of polarity or characterized by various physico-chemical properties. However, SPE is not completely free from practical problems inherent to the nature of the compounds or to the coupling to the chromatographic systems. Many examples are reported to illustrate these problems which can in most cases be circumvented. New developments in SPE are also reviewed.

Chromatography, Liquid↗