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Sample Volume (sample + volume)
Kinds of Sample Volume Selected AbstractsClinical applications of laser scanning cytometryCYTOMETRY, Issue 3 2002Attila Tárnok Abstract This study reviews existing and potential clinical applications of laser scanning cytometry (LSC) and outlines possible future developments. LSC provides a technology for solid phase cytometry. Fluorochrome-labeled specimens are immobilized on microscopic slides that are placed on a conventional epifluorescence microscope and analyzed by one or two lasers. Data comparable to flow cytometry are generated. In addition, the position of each event is recorded, a feature that allows relocalization and visualization of each measured event. The major advantage of LSC compared with other cytometric methods is the combination of two features: (a) the minimal clinical sample volume needed and (b) the connection of fluorescence data and morphological information for the measured event. Since the introduction of LSC, numerous methods have been established for the analysis of cells, cellular compartments, and tissues. Although most cytometric methods use only two or three colors, the characterization of specimens with up to five fluorochromes is possible. Most clinical applications have been designed to determine ploidy and immunophenotype; other applications include analyses of tissue biopsies and sections, fluorescence in situ hybridization, and the combination of vital and nonvital information on a single-cell basis. With the currently available assays, LSC has proven its wide spectrum of clinical applicability in slide-based cytometry and can be introduced as a standard technology in multiple clinical settings. Cytometry (Clin. Cytometry) 50:133,143, 2002. © 2002 Wiley-Liss, Inc. [source] Near-infrared dyes for six-color immunophenotyping by laser scanning cytometryCYTOMETRY, Issue 3 2002Andreas O.H. Gerstner Abstract Background To adequately analyze the complexity of the immune system and reduce the required sample volume for immunophenotyping in general, more measurable colors for the discrimination of leukocyte subsets are necessary. Immunophenotyping by the laser scanning cytometer (LSC), a slide-based cytometric technology, combines cell detection based on multiple colors with their subsequent visualization without the need for physical cell sorting. In the present study, the filter setting of the LSC was adapted for the measurement of the far-red emitting dye cyanine 7 (Cy7), thereby increasing the number of measurable commercially available fluorochromes. Methods The optical filters of the LSC were replaced,photomultiplier (PMT) 3/allophycocyanin (APC): 740-nm dichroic long pass, and 670-/55-nm bandpass; PMT 4/Cy7: 810-/90-nm bandpass. Peripheral blood leukocytes were stained directly by fluorochrome-labeled antibodies or by indirect staining. The tandem dyes of Cy7 (phycoerythrin [PE]-Cy7, APC-Cy7) and the fluorochromes fluorescein isothiocyanate (FITC), PE, PE-Cy5, and APC were tested alone and in different combinations. Results With the new filter combination and tandem fluorochromes, Cy7 was measurable at 488-nm (argon laser) or 633-nm (helium-neon laser) excitation. Resolution was in the range of FITC for PE-Cy7 but approximately 30% lower for APC-Cy7; spillover into the respective donor fluorochrome channel for both tandem dyes was prominent. A six-color panel for leukocyte subtyping was designed. Conclusions With this adaptation, it is possible to measure the tandem conjugates PE-Cy7 and APC-Cy7. This new setup opens the way for six-color immunophenotyping by LSC. Cytometry 48:115,123, 2002. © 2002 Wiley-Liss, Inc. [source] Relationship between Slow Coronary Flow and Left Atrial Appendage Blood Flow VelocitiesECHOCARDIOGRAPHY, Issue 1 2007Recep Demirbag M.D. Aims: This study was undertaken to assess whether slow coronary flow (SCF)is related to low left atrial appendage (LAA) blood flow velocities. Methods: Study subjects consist of 44 patients with SCF and 11 volunteer subjects with normal coronary angiogram. The diagnosis of SCF was made using the TIMI frame count method. The blood flow velocities were obtained by placing a pulsed-wave Doppler sample volume inside the proximal third of the LAA. Results: The mean LAA emptying velocities (MEV)were significantly lower in patients than control subjects (34.5 ± 9.9 cm/sec vs 84.0 ± 12.1 cm/sec; P < 0.001). In bivariate analysis, significant correlation was found between MEV, and systolic pulmonary venous flow, mean TIMI frame count, deceleration time, and isovolumetric relaxation time (P < 0.05). By multiple linear regression analysis, mean TIMI frame count (ß=,0.865, P < 0.001) was identified as independent predictors of MEV. Conclusion: This study indicates that SCF phenomenon may be related to low LAA blood flows. [source] Precautions to improve the accuracy of quantitative determinations of biomarkers in clinical diagnosticsELECTROPHORESIS, Issue 16 2010Nasim Ghasemzadeh Abstract Although protein biomarkers have a great potential as biomarkers for diagnosis of diseases, they are seldom used in hospitals. There are many reasons for this, for instance, the difficulties to (i) find a biomarker for which the concentration in body fluids clearly differs between patients and healthy subjects, (ii) attain purification of the biomarker close to 100%, which is required for production of conventional protein antibodies as well as artificial gel antibodies for selective capture of a biomarker, (iii) design a standard curve for rapid and accurate determination of the concentration of the biomarker in the body fluid because of adsorption of the biomarker onto vials, pipettes, etc., (iv) determine accurately the sample volume delivered by a pipette, (v) avoid polymerization of the biomarker upon storage and to decide whether it is in the form not only of monomers, but also of dimers, trimers, etc., in the native state, (vi) determine the degree of possible glycosylation and amidation of the biomarker and (vii) decide whether glycosylation and amidation positively or negatively affects the possibilit to use the protein as a biomarker. In this article, we discuss in quantitative terms the difficulties (iii,vii) and how to overcome them, which also may help to overcome the difficulty (ii), which in turn minimizes difficulty (i). [source] A parylene-based dual channel micro-electrophoresis system for rapid mutation detection via heteroduplex analysis,ELECTROPHORESIS, Issue 18 2008Sertan Sukas Abstract A new dual channel micro-electrophoresis system for rapid mutation detection based on heteroduplex analysis was designed and implemented. Mutation detection was successfully achieved in a total separation length of 250,,m in less than 3,min for a 590,bp DNA sample harboring a 3,bp mutation causing an amino acid change. Parylene-C was used as the structural material for fabricating the micro-channels as it provides conformal deposition, transparency, biocompatibility, and low background fluorescence without any surface treatment. A new dual channel architecture was derived from the traditional cross-channel layout by forming two identical channels with independent sample loading and waste reservoirs. The control of injected sample volume was accomplished by a new u-turn injection technique with pull-back method. The use of heteroduplex analysis as a mutation detection method on a cross-linked polyacrylamide medium provided accurate mutation detection in an extremely short length and time. The presence of two channels on the microchip offers the opportunity of comparing the sample to be tested with a desired control sample rapidly, which is very critical for the accuracy and reliability of the mutation analyses, especially for clinical and research purposes. [source] Microautosamplers for discrete sample injection and dispensationELECTROPHORESIS, Issue 9 2005Chun-Wei Huang Abstract Microfluidic systems show considerable potential for use in the continuous reaction and analysis of biosamples for various applications, such as drug screening and chemical synthesis. Typically, microfluidic chips are externally connected with large-scale autosamplers to inject specific volumes of discrete samples in the continuous monitoring and analysis of multiple samples. This paper presents a novel microelectromechanical system (MEMS)-based autosampler capable of performing the discrete injection and dispensation of variable-volume samples. This microdevice can be integrated with other microfluidic devices to facilitate the continuous monitoring and analysis of multiple biosamples. By means of electroosmotic focusing and switching controlled by the direct application of electric sources on specific fluid reservoirs, a precise sample volume can be injected into the specified outlet port. Fluorescence dye images verify the performance of the developed device. An injection-and-washing scheme is developed to prevent cross-contamination during the continuous injection of different samples. This approach renders feasible the injection of several discrete samples using a single microchip. Compared to its large-scale counterparts, the developed microautosampler is compact in size, has low fabrication costs, is straightforward to control, and most importantly, is readily integrated with other microfluidic devices (e.g., microcapillary electrophoresis chips) to form a microfluidic system capable of the continuous monitoring and analysis of bioreactions. The proposed microautosampler could be promising towards realizing the micrototal analysis system (,-TAS) concept. [source] Roxarsone and transformation products in chicken manure: Determination by capillary electrophoresis-inductively coupled plasma-mass spectrometryELECTROPHORESIS, Issue 7-8 2005Charlita G. Rosal Abstract The determination of the animal feed additive roxarsone (3-nitro-4-hydroxyphenylarsonic acid) and six of its possible transformation products (arsenite, arsenate, monomethylarsonate, dimethylarsinate, 3-amino-4-hydroxyphenylarsonic acid, and 4-hydroxyphenylarsonic acid) in chicken manure was investigated using capillary electrophoresis-inductively coupled plasma-mass spectrometry (CE-ICP-MS). Initial method development was conducted using ultraviolet (UV) detection for ruggedness and time efficiency. Separation of these seven arsenic species was effected using a 20,mM phosphate buffer at pH 5.7. The CE-ICP-MS limits of detection in terms of As for each of the species was in the low µg·L,1 range, corresponding to absolute detection limits in the range 20,70,fg As (based on a 23,nL injection). Overall, the method developed in this study provides high selectivity and low limits of detection (1,3,µg·L,1 or low-ppb, based on As), uses small sample volume (low nL), and produces minimal wastes. [source] Integrated selective enrichment target , a microtechnology platform for matrix-assisted laser desorption/ionization-mass spectrometry applied on protein biomarkers in prostate diseasesELECTROPHORESIS, Issue 21-22 2004Simon Ekström Abstract The performance of a miniaturized sample processing platform for matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS), manufactured by silicon microfabrication, called integrated selective enrichment target (ISET) technology was evaluated in a biological context. The ISET serves as both sample treatment device and MALDI-MS target, and contains an array of 96 perforated nanovials, which each can be filled with 40 nL of reversed-phase beads. This methodology minimizes the number of sample transfers and the total surface area available for undesired adsorption of the analytes in order to provide high-sensitivity analysis. ISET technology was successfully applied for characterization of proteins coisolated by affinity chromatography of prostate-specific antigen (PSA) from human seminal fluid. The application of ISET sample preparation enabled multiple analyses to be performed on a limited sample volume, which resulted in the discovery that prolactin inducible protein (PIP) was coisolated from the samples. [source] Ultra-trace analysis of multiple endocrine-disrupting chemicals in municipal and bleached kraft mill effluents using gas chromatography,high-resolution mass spectrometryENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 2 2008Michael G. Ikonomou Abstract A comprehensive gas chromatographic,high-resolution mass spectrometric (GC-HRMS),based method was developed that permitted the simultaneous determination of 30 estrogenic endocrine-disrupting chemicals (EDCs) and related compounds, including surfactants, biogenic and synthetic steroids, fecal sterols, phytoestrogens, and plasticizers, in wastewater. Features of the method include low sample volume (,40 ml), optimized Florisil® cleanup to minimize matrix interferences and optimized analyte derivatization to improve sensitivity via GC-HRMS. Detection limits were in the low- to mid-ng/L range, and recoveries were greater than 60% for most target analytes. This new method allows for high throughput analysis of many organic wastewater contaminants in a complex matrix with relative standard deviation of less than 15% for most measurable compounds. The applicability of the method was demonstrated by examining wastewater samples from different origins. Compounds such as di(2-ethylhex-yl)phthalate, cholesterol, cholestanol, and other cholesterol derivatives were measured in much higher concentrations in untreated sewage and were reduced substantially in concentration by the treatment process. However, steroidal compounds, particularly estrone (E1), 17,-estradiol (E2), and estriol (E3), as well as plant sterols (except stigmastanol), were greater in the treated municipal wastewater versus the untreated effluent. Plant and fungi sterols, stigmastanol and ergosterol, were found largely associated with bleached kraft mill effluent (BKME) as compared to the municipal effluents. [source] Semen quality in fertile US men in relation to geographical area and pesticide exposureINTERNATIONAL JOURNAL OF ANDROLOGY, Issue 1 2006Shanna H. Swan Summary We conducted the first US study to compare semen quality among study centres using standardized methods and strict quality control. We present data on semen quality in partners of 493 pregnant women recruited through prenatal clinics in four US cities during 1999,2001. Sperm concentration, semen volume and motility were determined at the centres and morphology was assessed at a central laboratory. While between-centre differences in sperm morphology and sample volume were small, sperm concentration and motility were significantly reduced in Columbia, MO (MO) relative to men in New York, NY, Minneapolis, MN and Los Angeles, CA; total number of motile sperm was 113 × 106 in MO and 162, 201 and 196 × 106 in CA, MN and NY respectively. Differences among centres remained significant in multivariate models that controlled for abstinence time, semen analysis time, age, race, smoking, history of sexually transmitted disease and recent fever (all p -values <0.01). We hypothesized that poorer sperm concentration and motility in MO men relative to other centres might be related to agricultural pesticides that are commonly used in the mid-west. We investigated this hypothesis by conducting a nested case,control study within the MO cohort. We selected 25 men in this cohort for whom all semen parameters (concentration, % normal morphology and % motile) were low as cases and an equal number of men for whom all semen parameters were within normal limits as controls. We measured metabolites of eight non-persistent, current-use pesticides in urine samples the men had provided at the time of semen collection. Pesticide metabolite levels were elevated in cases compared with controls for the herbicides alachlor and atrazine, and for the insecticide diazinon (2-isopropoxy-4-methyl-pyrimidinol) (p -values for Wilcoxon rank test = 0.0007, 0.012, and 0.0004 for alachlor, atrazine and diazinon respectively). Men with higher levels of alachlor or diazinon were significantly more likely to be cases than men with low levels [odds ratios (OR) = 30.0, 16.7 for alachlor and diazinon respectively], as were men with atrazine over the limit of detection (OR = 11.3). These associations between current-use pesticides and reduced semen quality suggest that agricultural chemicals may have contributed to the reduced semen quality seen in fertile men from mid-Missouri. [source] Comparison of ATP and in vivo bioluminescence for assessing the efficiency of immunomagnetic sorbents for live Escherichia coli O157:H7 cellsJOURNAL OF APPLIED MICROBIOLOGY, Issue 6 2002W. Sun Aims:,To develop methods to assess the efficiency of immunomagnetic separation (IMS). Methods and Results:,The capturing efficiency of biosorbents for Escherichia coli O157:H7, constructed using streptavidin-coated magnetic beads and biotinylated antibodies, was tested using both in vivo and ATP bioluminescence. Both methods were suitable for the enumeration of bacteria captured by the biosorbents. The level of both ATP and in vivo bioluminescence depended on the media used, but was unaffected by the magnetic beads. The capture efficiency depended on time and sample volume, but did not depend on the length of spacer arm of the biotinylation agent. For cell concentrations of , 105 cfu ml,1, in a 1-ml sample volume, nearly 80,85% recovery of the pathogen was observed after 0·5 h of incubation. For an 11-ml sample containing 104 cfu ml,1, maximum recovery (50% of cells) was achieved only after 2 h incubation. Conclusions:,The detection limit of an ATP-based bioluminescent assay for E. coli O157:H7 was reduced by 1 log cycle after optimization of IMS. The bioluminescent methods could be used for screening and testing the affinity of antibodies or other affinity elements of biosorbents towards live bacterial cells. Significance and Impact of the Study:,Bioluminescent assays provide an easy way to optimize conditions for the capture of bacteria by biosorbents in real time. [source] Application of ultrasonic shear rheometer to characterize rheological properties of high protein concentration solutions at microliter volumeJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 6 2005Atul Saluja Abstract The purpose of this work was to conduct preliminary rheological analysis on high protein concentration solutions by using the technique of ultrasonic shear rheometry at megahertz frequencies. The work was aimed at establishing the viability of the technique for analyzing protein solution rheology as well as obtaining an initial understanding of the effect of solution conditions on solution rheology of a model protein. Bovine serum albumin (BSA) was used for this study, and rheological analysis was conducted at 20 ,L sample volume between pH 2.0 and 9.0 at different ionic strengths at 25°C using 5 and 10 MHz quartz crystals. Significant differences in storage modulus among solutions at pH 5.0, 7.0, and 9.0 could only be detected at 10 MHz, and the errors associated with measurements were smaller as compared to those at 5 MHz for all the solutions studied. Solutions at pH 2.0 and 3.0 showed a time-dependent change in solution rheology. For solutions at pH 5.0, 7.0, and 9.0, which did not show time dependence in solution rheology, loss modulus data at lower concentrations correlated well with the dilute solution data in the literature. At higher concentrations, pH 5.0 solutions exhibited a higher loss modulus than pH 7.0 and pH 9.0 solutions. Storage modulus decreased with increasing ionic strength, unlike loss modulus, which did not show any change, except at pI of protein when no effect was observed. The results show the potential of high frequency rheometry for analyzing subtle differences in rheology of pharmaceutically relevant protein solutions at microliter volume. © 2005 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 94:1161,1168, 2005 [source] Automated stir plate (bar) sorptive extraction coupled to high-performance liquid chromatography for the determination of polycyclic aromatic hydrocarbonsJOURNAL OF SEPARATION SCIENCE, JSS, Issue 14 2010Chunhe Yu Abstract Automated methods of PDMS/,-CD/divinylbenzene-coated stir plate sorptive extraction (SPSE) coupled to HPLC-fluorescence detector were reported for the first time. Three automation modes, static SPSE, circular flow SPSE and continuous flow SPSE, were evaluated and critically compared with stir bar sorptive extraction by using six polycyclic aromatic hydrocarbons as model analytes. It was found that the operable sample volume for circular flow SPSE and continuous flow SPSE was larger than that for static SPSE. Under the same extraction conditions, continuous flow SPSE exhibited the highest extraction efficiencies in all automated modes and manual stir bar sorptive extraction for the target compounds. Compared with the manual operation (approximately 5,10,min), automated SPSE required a relatively short time (117,180,s) to finish sampling, washing and sample loading. Besides being labor-saving and time-saving, automated SPSE has other advantages, such as no time limit and non-attended operation. The proposed continuous flow PDMS/,-CD/divinylbenzene-coated SPSE-HPLC-fluorescence detector was successfully applied to environmental water analysis. [source] Determination of trace organophosphorus pesticides in water samples with TiO2 nanotubes cartridge prior to GC-flame photometric detectionJOURNAL OF SEPARATION SCIENCE, JSS, Issue 14 2010Yunrui Huang Abstract This article described a new method for the sensitive determination of organophosphorus pesticides in water samples using SPE in combination with GC-flame photometric detection. In the procedure of method development, TiO2 nanotubes were used as SPE adsorbents for the enrichment of organophosphorus pesticides from water samples. Several factors, such as eluent and its volume, sample pH, sample volume, sample flow rate, and concentration of humic acid, were optimized. Under the optimal conditions, the proposed method had good linear ranges as 0.1,40,,g/L for each of them, LOD of 0.11, 0.014, and 0.0025,,g/L, and LOQs of 0.37, 0.047, and 0.0083,,g/L for chlorpyrifos, phorate, and methyl parathion, respectively. The proposed method was validated with real environmental water samples and the spiked recoveries were over the range of 86.5,115.1%. All these results indicated that TiO2 nanotubes, as a new SPE adsorbent, would be used widespread for the preconcentraiton and determination of environmental pollutants in the future. [source] Highly sensitive determination of tetrabromobisphenol A and bisphenol A in environmental water samples by solid-phase extraction and liquid chromatography-tandem mass spectrometryJOURNAL OF SEPARATION SCIENCE, JSS, Issue 11 2010Ru-Song Zhao Abstract Using bamboo-activated charcoal as SPE adsorbent, a novel SPE method was developed for the sensitive determination of tetrabromobisphenol A and bisphenol A in environmental water samples by rapid-resolution LC-ESI-MS/MS. Important parameters influencing extraction efficiency, including type of eluent, eluent volume, sample pH, volume and flow rate, were investigated and optimized. Under the optimal extraction conditions (eluent: 8,mL methanol, pH: 7; flow rate: 4,mL/min; sample volume: 100,mL), low LODs (0.01,0.02,ng/mL), good repeatability (6.2,8.3%) and wide linearity range (0.10,10,ng/mL) were obtained. Satisfied results were achieved when the proposed method was applied to determine the two target compounds in real-world environmental water samples with spiked recoveries over the range of 80.5,119.8%. All these facts indicate that trace determination of tetrabromobisphenol A and bisphenol A in real-world environmental water samples can be realized by bamboo-activated charcoal SPE-rapid resolution-LC-ESI-MS/MS. [source] Optimisation of the headspace-solid phase microextraction for organomercury and organotin compound determination in sediment and biotaJOURNAL OF SEPARATION SCIENCE, JSS, Issue 4 2008Alejandra Delgado Abstract Headspace solid-phase microextraction was optimised for the simultaneous preconcentration of methylmercury (MeHg+), monobutyltin, dibutyltin, tributyltin, monophenyltin (MPhT), diphenyltin (DPhT), and triphenyltin (TPhT) from sediments and biota. Extraction time (3,24 min), extraction temperature (20,90°C), desorption time (1,10.4 min), desorption temperature (152,260°C), and sample volume (5,22 mL) were simultaneously optimised, while variables such as fibre type (30 ,m polydimethylsiloxane, PDMS), pH (acetic acid/sodium acetate, HOAc/NaOAc, 2 mol/L, pH , 4.8), the concentration of the derivatisation agent (sodium tetraethylborate, NaBEt4, 0.1% m/v), and the ionic strength (fixed by the buffer solution) were kept constant. The variables were optimised according to the experiments proposed by the MultiSimplex program and the responses were considered in order to establish the optimum conditions. The repeatability (relative standard deviation, RSD, 5,20.6%) and limits of detection (LODs, 0.05,0.97 ng/g) of the overall method were also estimated. The lowest precisions were obtained for DPhT and TPhT. The optimised preconcentration method was applied to the determination of MeHg+, butyl- and phenyltins in certified reference materials (IAEA-405 MeHg+ in estuarine sediment, BCR-646 butyl- and phenyltins in marine sediment, BCR-463 MeHg+ in tuna fish, DOLT-2 MeHg+ in dogfish liver, and BCR-477 butyltins in mussel tissue) by GC with microwave-induced plasma/atomic-emission detection. [source] Bipolar Fatigue Caused by Field Screening in Pb(Zr,Ti)O3 CeramicsJOURNAL OF THE AMERICAN CERAMIC SOCIETY, Issue 12 2007Nina Balke Bipolar cycling of lead zirconate titanate ceramics can lead to massive material damage in regions close to the electrode. The damaged region can be identified by color changes, and the microstructure in this region shows signs of interface melting. This damaged region can screen the sample volume from the applied voltages and reduced fields are applied to the undamaged part of the sample. This has two effects. The first one is that the bulk is effectively subjected to smaller fields, but the measured parameters are assigned to the applied field, yielding apparent fatigue curves. The second effect is that with further cycling, field screening protects the bulk of the sample from fatigue due to the reduced effective fields. If the damaged region is mechanically removed and the ferroelectric hystereses are measured again, nearly unfatigued parameters are obtained. [source] 31P CP/MAS NMR of polycrystalline and immobilized phosphines and catalysts with fast sample spinningMAGNETIC RESONANCE IN CHEMISTRY, Issue 6 2003S. Reinhard Abstract Cross-polarization (CP) at fast magic angle spinning (MAS) frequencies leads to a splitting of the Hartmann,Hahn (HH) matching profile into a centerband and additional bands of higher orders. The matching profiles differ with the substance categories. Therefore, signal intensity is usually lost, when e.g. the routine standard NH4H2PO4 is used for optimizing the 1H,31P HH match prior to measuring phosphines and their metal complexes in polycrystalline or immobilized form. Here, a variety of model compounds, such as Ph2PCH2CH2PPh2 and (CO)2Ni(PPh3)2, which can be used as 31P CP standards for analogous substances or materials are presented. Investigating the influences of MAS frequency, contact time, 1H pulse power and sample volume on the matching profiles of the model compounds leads to general trends. Thereby, a new strategy for measuring difficult samples with CP at high MAS rates has been developed: their optimum CP parameters are derived from the most intense maxima in the HH matching profiles of the corresponding model compounds. This new strategy is compared with variations of a conventional ramp sequence. Although the latter generally provide smaller signal half-widths, the new strategy leads to higher signal intensities. The new method was successfully applied to polycrystalline and immobilized phosphines and catalysts. Copyright © 2003 John Wiley & Sons, Ltd. [source] Delaunay Tessellation Field Estimator analysis of the PSCz local Universe: density field and cosmic flowMONTHLY NOTICES OF THE ROYAL ASTRONOMICAL SOCIETY, Issue 1 2007Emilio Romano-Díaz ABSTRACT We apply the Delaunay Tessellation Field Estimator (DTFE) to reconstruct and analyse the matter distribution and cosmic velocity flows in the local Universe on the basis of the PSCz galaxy survey. The prime objective of this study is the production of optimal resolution 3D maps of the volume-weighted velocity and density fields throughout the nearby universe, the basis for a detailed study of the structure and dynamics of the cosmic web at each level probed by underlying galaxy sample. Fully volume-covering 3D maps of the density and (volume-weighted) velocity fields in the cosmic vicinity, out to a distance of 150 h,1 Mpc, are presented. Based on the Voronoi and Delaunay tessellation defined by the spatial galaxy sample, DTFE involves the estimate of density values on the basis of the volume of the related Delaunay tetrahedra and the subsequent use of the Delaunay tessellation as natural multidimensional (linear) interpolation grid for the corresponding density and velocity fields throughout the sample volume. The linearized model of the spatial galaxy distribution and the corresponding peculiar velocities of the PSCz galaxy sample, produced by Branchini et al., forms the input sample for the DTFE study. The DTFE maps reproduce the high-density supercluster regions in optimal detail, both their internal structure as well as their elongated or flattened shape. The corresponding velocity flows trace the bulk and shear flows marking the region extending from the Pisces,Perseus supercluster, via the Local Superclusters, towards the Hydra,Centaurus and the Shapley concentration. The most outstanding and unique feature of the DTFE maps is the sharply defined radial outflow regions in and around underdense voids, marking the dynamical importance of voids in the local Universe. The maximum expansion rate of voids defines a sharp cut-off in the DTFE velocity divergence probability distribution function. We found that on the basis of this cut-off DTFE manages to consistently reproduce the value of ,m, 0.35 underlying the linearized velocity data set. [source] Downregulation of pro-inflammatory cytokines by lupeol measured using cytometric bead array immunoassayPHYTOTHERAPY RESEARCH, Issue 1 2010Sheikh Fayaz Ahmad Abstract The objective of the study was to investigate the activity of Lupeol (LUP) on proinflammatory and anti-inflammatory cytokines in the pleural exudate from male swiss albino mice. We applied Cytometric bead array technology for simultaneously measurement of these cytokines in pleurisy induced mice treated with lupeol in graded oral doses. Cytometric bead array uses the sensitivity of amplified fluorescence detection by flowcytometer to measure soluble analytes in a particle based immune assay. This assay can accurately quantitate 5 cytokines in a 50 microlitre sample volume. Oral administration of LUP at doses of 25, 50, 100 and 200 mg/kg p.o. produced dose related inhibition of IL-2, IFN-gamma and TNF- , in the pleural exudate with the most significant effect at 100 mg/kg oral dose. LUP had a non significant inhibitory effect on the levels of IL-4 and IL-5. Copyright © 2009 John Wiley & Sons, Ltd. [source] Rapid and sensitive on-line liquid chromatographic/tandem mass spectrometric determination of an ethylene oxide-DNA adduct, N7-(2-hydroxyethyl)guanine, in urine of nonsmokersRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 5 2008Chih-Chun Jean Huang Ethylene oxide (EtO) is classified as a known human carcinogen. The formation of EtO-DNA adducts is considered as an important early event in the EtO carcinogenic process. An isotope-dilution on-line solid-phase extraction and liquid chromatography coupled with tandem mass spectrometry method was then developed to analyze one of the EtO-DNA adducts, N7-(2-hydroxyethyl)guanine (N7-HEG), in urine of 46 nonsmokers with excellent accuracy, sensitivity and specificity. The merits of this method include small sample volume (only 120,µL urine required), automated sample cleanup, and short total run time (12 minutes per sample). This method demonstrates its high-throughput capacity for future molecular epidemiology studies on the potential health effects resulting from the low-dose EtO exposure. Copyright © 2008 John Wiley & Sons, Ltd. [source] Development of a low volume plasma sample precipitation procedure for liquid chromatography/tandem mass spectrometry assays used for drug discovery applicationsRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 15 2005Xiaoying Xu The demand for high sensitivity bioanalytical methods has dramatically increased in the drug discovery stage; in addition, there has been a growing trend of reducing the sample volume that is required for these assays. A sensitive high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) procedure has been developed and tested to meet these needs. The assay requires only a low plasma sample volume (10,µL) and employs a protein precipitation procedure using a 1:6 plasma/acetonitrile ratio. The supernatant is injected directly into the LC/MS/MS system using the selected reaction monitoring (SRM) procedure for detection. A generic HPLC gradient based on a methanol/water mobile phase with a flow rate set to 0.8,mL/min was used. The test method showed very good linearity between 0.1,1000,ng/mL (R2,=,0.9737), precision (%RSD,=,6,9), accuracy (%RE,=,,2) and reproducibility (%RSD,=,11). A drug discovery IV/PO study was assayed using both the new low volume method and our standard volume (50,µL) method. The correlation of the two sets of data from the two methods was excellent (R2,=,0.9287). This new assay procedure has been successfully used in our laboratory for over 100 different rat or mouse discovery PK studies. Copyright © 2005 John Wiley & Sons, Ltd. [source] Let them fly or light them up: matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry and fluorescence in situ hybridization (FISH),APMIS, Issue 11-12 2004BIRGITTA SCHWEICKERT This review focuses on clinical bacteriology and by and large does not cover the detection of fungi, viruses or parasites. It discusses two completely different but complementary approaches that may either supplement or replace classic culture-based bacteriology. The latter view may appear provocative in the light of the actual market penetration of molecular genetic testing in clinical bacteriology. Despite its elegance, high specificity and sensitivity, molecular genetic diagnostics has not yet reached the majority of clinical laboratories. The reasons for this are manifold: Many microbiologists and medical technologists are more familiar with classical microbiological methods than with molecular biology techniques. Culture-based methods still represent the work horse of everyday routine. The number of available FDA-approved molecular genetic tests is limited and external quality control is still under development. Finally, it appears difficult to incorporate genetic testing in the routine laboratory setting due to the limited number of samples received or the lack of appropriate resources. However, financial and time constraints, particularly in hospitals as a consequence of budget cuts and reduced length of stay, lead to a demand for significantly shorter turnaround times that cannot be met by culture-dependent diagnosis. As a consequence, smaller laboratories that do not have the technical and personal equipment required for molecular genetic amplification techniques may adopt alternative methods such as fluorescence in situ hybridization (FISH) that combines easy-to-perform molecular hybridization with microscopy, a technique familiar to every microbiologist. FISH is hence one of the technologies presented here. For large hospital or reference laboratories with a high sample volume requiring massive parallel high-throughput testing we discuss matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) of nucleic acids, a technology that has evolved from the post-genome sequencing era, for high-throughput sequence variation analysis (1, 2). [source] Quantitive evaluation of macromolecular crystallization experiments using 1,8-ANS fluorescenceACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2010David Watts Modern X-ray structure analysis and advances in high-throughput robotics have allowed a significant increase in the number of conditions screened for a given sample volume. An efficient evaluation of the increased amount of crystallization trials in order to identify successful experiments is now urgently required. A novel approach is presented for the visualization of crystallization experiments using fluorescence from trace amounts of a nonspecific dye. The fluorescence images obtained strongly contrast protein crystals against other phenomena, such as precipitation and phase separation. Novel software has been developed to quantitatively evaluate the crystallization outcome based on a biophysical metric correlated with voxel protein concentration. In >1500 trials, 85.6% of the successful crystallization experiments were correctly identified, yielding a 50% reduction in the number of `missed hits' compared with current automated approaches. The use of the method in the crystallization of three previously uncharacterized proteins from the malarial parasite Plasmodium falciparum is further demonstrated. [source] Simultaneous assay of sildenafil and desmethylsildenafil in neonatal plasma by ultra-performance liquid chromatography,tandem mass spectrometryBIOMEDICAL CHROMATOGRAPHY, Issue 2 2010Bregje C. Witjes Abstract Sildenafil is used to treat pulmonary hypertension in neonatal and pediatric patients. Pharmacokinetic studies in these patients are complicated by the limited sample volume. We present the validation results of an assay method to quantitate sildenafil and desmethylsildenafil simultaneously in 50,µL of plasma. Deuterated sildenafil was used as an internal standard. After liquid,liquid extraction, analytes were separated on an ultra-performance liquid chromatography (UPLC)-column and quantified via tandem mass spectrometry. The calibration range was linear, with acceptable accuracy and a precision of <15% for both compounds. The lower limits of quantification were 1,ng/mL. Matrix effects were present, but inter-plasma batch variability was under 12%. The method was successfully applied to samples from a pharmacokinetic study into sildenafil pharmacokinetics in neonates, making maximum use of the limited number and amount of plasma samples available. Copyright © 2009 John Wiley & Sons, Ltd. [source] LC-MS/MS determination of 2-(4-((2-(2S,5R)-2-Cyano-5-ethynyl-1-pyrrolidinyl)-2-oxoethylamino)-4-methyl-1-piperidinyl)-4-pyridinecarboxylic acid (ABT-279) in dog plasma with high-throughput protein precipitation sample preparationBIOMEDICAL CHROMATOGRAPHY, Issue 11 2007Joseph Kim Abstract As an effective DPP-IV inhibitor, 2-(4-((2-(2S,5R)-2-Cyano-5-ethynyl-1-pyrrolidinyl)-2-oxoethylamino)-4-methyl-1-piperidinyl)-4-pyridinecarboxylic acid (ABT-279), is an investigational drug candidate under development at Abbott Laboratories for potential treatment of type 2 diabetes. In order to support the development of ABT-279, multiple analytical methods for an accurate, precise and selective concentration determination of ABT-279 in different matrices were developed and validated in accordance with the US Food and Drug Administration Guidance on Bioanalytical Method Validation. The analytical method for ABT-279 in dog plasma was validated in parallel to other validations for ABT-279 determination in different matrices. In order to shorten the sample preparation time and increase method precision, an automated multi-channel liquid handler was used to perform high-throughput protein precipitation and all other liquid transfers. The separation was performed through a Waters YMC ODS-AQ column (2.0 × 150 mm, 5 µm, 120 Å) with a mobile phase of 20 mm ammonium acetate in 20% acetonitrile at a flow rate of 0.3 mL/min. Data collection started at 2.2 min and continued for 2.0 min. The validated linear dynamic range in dog plasma was between 3.05 and 2033.64 ng/mL using a 50 µL sample volume. The achieved r2 coefficient of determination from three consecutive runs was between 0.998625 and 0.999085. The mean bias was between ,4.1 and 4.3% for all calibration standards including lower limit of quantitation. The mean bias was between ,8.0 and 0.4% for the quality control samples. The precision, expressed as a coefficient of variation (CV), was ,4.1% for all levels of quality control samples. The validation results demonstrated that the high-throughput method was accurate, precise and selective for the determination of ABT-279 in dog plasma. The validated method was also employed to support two toxicology studies. The passing rate was 100% for all 49 runs from one validation study and two toxicology studies. Copyright © 2007 John Wiley & Sons, Ltd. [source] High-throughput determination of atrasentan in human plasma by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometryBIOMEDICAL CHROMATOGRAPHY, Issue 9 2005Perry G. Wang Abstract Atrasentan (A-147627) is an endothelin antagonist receptor being developed at Abbott Laboratories for the treatment of prostate cancer. A quick and sensitive method for the determination of atrasentan in human plasma has been developed and validated using high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry. A dual-column, single mass spectrometer system is used to provide a reliable and routine means to increase sample throughput. The analytical method involves liquid,liquid extraction and internal standard (A-166790). The plasma samples and internal standard are acidified with 0.3 m hydrochloric acid prior to being extracted into 1:1 (v[sol ]v) hexanes,methyl t -butyl ether. The organic extract was evaporated to dryness using heated nitrogen stream and reconstituted with mobile phase. Atrasentan and internal standard were separated with no interference in a Zorbax SB-C18 analytical column with 2.1 × 50 mm, 5 µm, and a Zorbax C8 guard column using a mobile phase consisting of 50:50 (v:v) acetonitrile,0.05 m ammonium acetate, pH 4.5, at a flow rate of 0.30 mL[sol ]min to provide 4 min chromatograms. For a 250 µL plasma sample volume, the limit of quantitation was approximately 0.3 ng[sol ]mL. The calibration was linear from 0.30 to 98.0 ng[sol ]mL (r2 > 0.995). A significant advantage of the method is the ability to employ parallel HPLC separations with detection by a single MS[sol ]MS system to provide sensitivity and selectivity sufficient to achieve robust analytical results with a lower limit of quantitation of 0.30 ng[sol ]mL and high throughput. Copyright © 2005 John Wiley & Sons, Ltd. [source] Biomedical applications of capillary electrophoresis with laser-induced fluorescence detectionBIOPHARMACEUTICS AND DRUG DISPOSITION, Issue 7-8 2001Ximena Páez Abstract Capillary electrophoresis (CE) is a high-efficiency analytical technique that has had a great impact as a tool in biomedical research, clinical and forensic practice in the last ten years. Only in one of the applications, the DNA analysis, it has had an explosive exponential growth in the last few years. This impact is expressed in an enormous amount of CE articles and many reviews. The CE advantages with respect to other analytical techniques: the required very small sample volume, rapid analysis, great resolution power and low costs, have made this technique ideal for the analysis of a numerous endogenous and exogenous substances present in biological fluids. The different modes of CE have been coupled to different detection techniques such as UV-absorbance, electrochemical, mass spectrometry and laser-induced fluorescence detection (LIFD) to detect different nature and molecular size separated analytes. This review focuses mostly on the applications of CE,LIFD, to measure drugs and endogenous neuroactive substances such as amino acids and monoamines, especially in microdialysis samples from experimental animals and humans. CE,LIFD trends are discussed: automated faster analysis with capillary array systems, resolution power improvement, higher detection sensitivity, and CE systems miniaturization for extremely small sample volume, in order to make CE easier and affordable to the lab bench or the clinical bed. Copyright © 2001 John Wiley & Sons, Ltd. [source] Separation of Pure and Immunoreactive Virus-Like Particles Using Gel Filtration Chromatography Following Immobilized Metal Ion Affinity ChromatographyBIOTECHNOLOGY PROGRESS, Issue 2 2001Yu-Shen Cheng A purification process was developed to obtain highly pure rVP2H particles, formed by a structural protein (VP2) of the infectious bursal disease virus (IBDV) with six additional histidine residues at its C-terminus. The ultimate goal was the development of an efficient subunit vaccine against IBDV infection. The particles within the infected High-Five (Hi-5) cell lysates were partially purified by employing immobilized metal ion (Ni2+) affinity chromatography (IMAC). The initial step could recover approximately 85% of immunoreactive rVP2H proteins but failed to separate the rVP2H particles from the free rVP2H proteins or its degraded products. To separate the particulate form from the free form of rVP2H, an additional step was added, which used either gel filtration chromatography or CsCl density gradient ultracentrifugation. Both were able to produce extremely pure rVP2H particles with a buoyant density close to 1.27 g/cm3. However, the former method can process a larger sample volume than does the latter. By integrating IMAC and gel filtration chromatography, 1 mg of extremely pure rVP2H particles was routinely obtained from a 500 mL Hi-5 cell culture broth. The separation of the particulate form from the free form of rVP2H proteins exposes their respective immunogenicity to induce the virus-neutralizing antibodies and the ability to protect chickens from IBDV infection. Additionally, the abundant quantities of pure rVP2H particles coupled with their uniform dimensions facilitates an understanding of higher order structure of the immunogenic particles and can therefore result in improved vaccines against the virus. [source] Photochemistry in Photonic Crystal Fiber NanoreactorsCHEMISTRY - A EUROPEAN JOURNAL, Issue 19 2010Jocelyn S. Abstract We report the use of a liquid-filled hollow-core photonic crystal fiber (PCF) as a highly controlled photochemical reactor. Hollow-core PCFs have several major advantages over conventional sample cells: the sample volume per optical path length is very small (2.8,nL,cm,1 in the fiber used), long optical path lengths are possible as a result of very low intrinsic waveguide loss, and furthermore the light travels in a diffractionless single mode with a constant transverse intensity profile. As a proof of principle, the (very low) quantum yield of the photochemical conversion of vitamin B12, cyanocobalamin (CNCbl) to hydroxocobalamin ([H2OCbl]+) in aqueous solution was measured for several pH values from 2.5 to 7.5. The dynamics of the actively induced reaction were monitored in real-time by broadband absorption spectroscopy. The PCF nanoreactor required ten thousand times less sample volume compared to conventional techniques. Furthermore, the enhanced sensitivity and optical pump intensity implied that even systems with very small quantum yields can be measured very quickly,in our experiments one thousand times faster than in a conventional cuvette. [source] | ||||||||||||||||||||||||||||||||||