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PubMed2005

Multistage isoelectric focusing in a polymeric microfluidic chip.

This paper reports a protocol that improves the resolving power of isoelectric focusing (IEF) in a polymeric microfluidic chip. This method couples several stages of IEF in series by first focusing proteins in a straight channel using broad-range ampholytes and then refocusing segments of the first channel into secondary channels that branch from the first one at T-junctions. Experiments demonstrate that several fluorescent proteins that had focused within a segment of the straight channel in the first stage were refocused at significantly higher resolution due to the shallower pH gradient and higher electrical field gradient. Two variants of green fluorescent protein from the second-stage IEF fractionation were further separated in a third stage. Three stages of IEF were completed in less than 25 min at electric field strengths ranging from 50 to 214 V/cm.

Dimethylpolysiloxanes↗

PubMed1990

Measurement of protein biomass by Fourier transform infrared-photoacoustic spectroscopy.

A relatively new analytical technique, Fourier transform infrared-photoacoustic spectroscopy (FTIR-PAS), provides spectra of bacteria, fungi, and other microorganisms in solid states not suitable for conventional absorption spectroscopy. In this paper the feasibility of quantitative measurement of protein biomass on solid substrates by FTIR-PAS is examined and discussed. By measuring photoacoustic absorption bands from amide groups in the protein of microorganisms, the increase in biomass that occurs during growth was monitored directly and accurately. Incorporation of polyacrylonitrile into the sample as an internal standard was shown to be a convenient method for improving both the reliability and the range of detection by photoacoustic spectroscopy. Results of FTIR-PAS measurements of known quantities of microbial mass in simulated growth experiments suggest that the technique may be especially suitable for assays of microorganisms used in solid-state biosyntheses of drugs, hormones, and other biological agents.

Acoustics↗

PubMed1997

Processing of color- and noncolor-coded signals in the gourami retina. II. Amacrine cells.

The same set of stimuli and analytic methods that was used to study the dynamics of horizontal cells () was applied to a study of the response dynamics and signal processing in amacrine cells in the retina of the kissing gourami, Helostoma rudolfi. The retina contains two major classes of amacrine cells that could be identified from their morphology: C and N amacrine cells. C amacrine cells had a two-layered dendritic field, whereas N cells had a monolayered dendritic field. Both types of amacrine cell were tracer-coupled but coupling was more extensive in the N amacrine cells. Responses from C amacrine cells lacked a DC component and had a small linear component that was <10% in terms of mean square error (MSE); the second-order component often accounted for >50% of the modulation response. The C amacrine cells did not show any characteristic color coding under any stimulus condition. Most responses of N cells to a pulsatile stimulus consisted of a series of depolarizing transient potentials and steady illumination did not generate any DC potential in these cells. The response to a white-noise modulated input was composed of well-defined first- and second-order components and, possibly, higher-order components. The response evoked by a red or green white-noise-modulated stimulus given alone was not color coded. Modulated red illumination in the presence of a green illumination elicited a color-coded response from >70% of N amacrine cells. Color information was carried not only by the polarity but also by the dynamics of the first-order component. No convincing evidence was obtained to indicate that the second-order component might be involved in color processing. Some N amacrine cells produced a well-defined (second-order) interaction kernel to show that the temporal sequence of red and green stimuli was a parameter to be considered. In a complex cell such as an amacrine cell, responses evoked by a pulsatile stimulus given in darkness and by modulation of a mean luminance could be very different in terms of their characteristics. It was not always possible to predict the response evoked by one stimulus from observing the cell's response to another stimulus. This is because, in N cells, a flash-evoked (nonsteady state) response is composed largely of nonlinear components whereas a modulation (steady state) response is composed of linear as well as nonlinear components.

Animals↗

PubMed1978

Chromatographic determination of angiotensin-converting enzyme and angiotensinase activity.

An analytical method utilizing an automatic amino acid analyzer is described for the separation, identification, and measurement of 5 to 50 nmol of angiotensin I, angiotensin II, [Des-Phe8]angiotensin II, Phe-His-Leu, His-Leu, isoleucine, leucine, tyrosine, and phenylalanine. Aminex A-5 cation-exchange resin (0.9 x 15 cm) is sequentially eluted with three sodium citrate buffers: pH 3.25, 0.2 N; pH 4.85, 0.54 N, and pH 6.5, 0.39 N at 60 and 80 degrees C. Reaction with ninhydrin is used for detection. This chromatographic system was used to determine angiotensin-converting enzyme activity and the angiotensinase activity of rabbit brain endopeptidase B. In each assay, the unhydrolyzed substrate and both products were measured simultaneously in one step without pretreatment of the hydrolysate. Products were recovered in 1:1 molar ratios and the overall recovery of an hydrolyzed substrate of products was quantitative.

Amino Acids↗

PubMed2002

Atmospherc mercury deposition during the last 270 years: a glacial ice core record of natural and anthropogenic sources.

Mercury (Hg) contamination of aquatic ecosystems and subsequent methylmercury bioaccumulation are significant environmental problems of global extent. At regional to global scales, the primary mechanism of Hg contamination is atmospheric Hg transport. Thus, a better understanding of the long-term history of atmospheric Hg cycling and quantification of the sources is critical for assessing the regional and global impact of anthropogenic Hg emissions. Ice cores collected from the Upper Fremont Glacier (UFG), Wyoming, contain a high-resolution record of total atmospheric Hg deposition (ca. 1720-1993). Total Hg in 97 ice-core samples was determined with trace-metal clean handling methods and low-level analytical procedures to reconstruct the first and most comprehensive atmospheric Hg deposition record of its kind yet available from North America. The record indicates major atmospheric releases of both natural and anthropogenic Hg from regional and global sources. Integrated over the past 270-year ice-core history, anthropogenic inputs contributed 52%, volcanic events 6%, and background sources 42%. More significantly, during the last 100 years, anthropogenic sources contributed 70% of the total Hg input. Unlike the 2-7-fold increase observed from preindustrial times (before 1840) to the mid-1980s in sediment-core records, the UFG record indicates a 20-fold increase for the same period. The sediment-core records, however, are in agreement with the last 10 years of this ice-core record, indicating declines in atmospheric Hg deposition.

Air Pollutants↗

PubMed2006

A simple method to measure effective catalase activities: optimization, validation, and application in green coffee.

Oxidative metabolism in coffee cherries during maturation appears to be regulated by the timely expression of redox enzymes such as catalase (CAT), peroxidase (POD), and polyphenoloxidase (PPO). Among these enzymes, CAT is suspected to contribute significantly in setting the redox status of the healthy cherry and the processed bean. The initial redox status of the green bean might further control the nature and dynamics of reactions induced by roasting and eventually quality aspects of the end product. In this respect, Arabica (Coffea arabica) and Robusta (Coffea canephora) typically differ by their cup coffee flavor profiles. We developed an assay that allowed us to screen numerous green coffee samples for effective CAT activities. The proposed assay, which monitors CAT activities by online oxygen sensing in green coffee crude suspensions incubated with H2O2, seeks to integrate potential effects of endogenous inhibitors and activators. After optimization and validation of the assay, 23 Arabicas, 23 Robustas, and 8 Arabustas were analyzed. Nearly all Arabicas (22 of 23) harbored high CAT activity levels, whereas all Robustas harbored low ones. Arabustas performed like Arabicas of the lower CAT activity range. The traditional spectrophotometric assay did not reveal these specificities. Because of its simplicity, our assay might be valuable for assessing effective CAT activities in various plant tissues.

Catalase↗

PubMed1990

Patterns of affectivity in the transition to adolescence.

The present paper reports the findings of a factor analytic investigation of adolescent affect in a cross-sectional sample of 483 male and female fifth through ninth graders. Following the ESM method, adolescents carried electronic pagers and self-report booklets for one week. Students were paged seven times daily and completed self-report forms after each signal describing their moods and feelings. Principal components analysis yielded two internally consistent factors which were virtually identical to the positive and negative affect dimensions described in the literature on adult emotion. Contrary to findings reported in the developmental literature, arousal did not emerge as a dimension of adolescent affect. Further, the bipolar structure obtained for the entire sample, positive and negative affect, consistently emerged in separate analyses of adolescent gender and school groups. MANOVA analysis of estimated positive and negative affect scores indicated that while the underlying dimensions of adolescent affect were comparable to those observed for adults, variation along those dimensions may have been related to the social transitions (e.g., schooling) which punctuate adolescence.

Adolescent↗

PubMed2004

The international suicide prevention trial (interSePT): rationale and design of a trial comparing the relative ability of clozapine and olanzapine to reduce suicidal behavior in schizophrenia and schizoaffective patients.

Suicidal behavior in patients with psychotic disorders represents a seriously undertreated life-threatening condition. The International Suicide Prevention Trial (InterSePT) is the first large-scale, prospective study designed to evaluate the potential of antipsychotic medications to reduce suicidal behaviors in patients with schizophrenia or schizoaffective disorder who are known to be at high risk for suicide. The unique challenges to study design and the solutions identified for the InterSePT study are described. These challenges included defining suicidal behavior in patients with psychosis, endpoint selection, determination of analytic strategy, and development of scales to assess suicidal behavior. Given the life-threatening nature of suicidal behavior, ethical considerations required that the design minimize suicide attempts and deaths. While the study focused primarily on treatment of suicide, opportunities were used to collect data in related areas of interest, including suicide risk factors, other efficacy measures (e.g., Positive and Negative Syndrome Scale, Covi Anxiety Scale, Calgary Depression Scale), adverse events, pharmacoeconomics, and pharmacogenetics. Because of the complexity of the design issues, a steering committee, suicide monitoring board, and publication committee were established to assist with their management.

Adolescent↗

PubMed2004

Fractal-based scaling and scale-invariant dispersion of peak concentrations of crop protection chemicals in rivers.

A new regulatory approach is needed to characterize peak pesticide concentrations in surface waters over a range of watershed scales. Methods now in common use rely upon idealized edge-of-field scenarios that ignore scaling effects. Although some watershed-scale regulatory models are available, their complexity generally prevents them from being used duringthe pesticide registration decision process, even though nearly all exposure to both humans and aquatic organisms can occur only at this scale. The theory of fractal geometry offers a simpler method for addressing this regulatory need. Mandelbrot described rivers as "space-filling curves" (Mandelbrot, B. B. The Fractal Geometry of Nature; Freeman: New York, 1983), a class of fractal objects implying two useful properties we exploit in this work. The first is a simple power-law relationship in which log-log plots of maximum daily concentrations as a function of watershed area tend to be linear with a negative slope. We demonstrate that the extrapolation of such plots down to smaller watersheds agrees with edge-of-field concentrations predicted using the Pesticide Root Zone Model, but only when the modeling results are properly adjusted for use intensity within the watershed. We also define a second useful property, "scale-invariant dispersion", in which concentrations are well described by a single analytical solution to the convective--dispersion equation, regardless of scale. Both of these findings make it possible to incorporate the effect of watershed scale directly into regulatory assessments.

Environment↗

PubMed2005

In-line monitoring of moisture content in fluid bed dryers using near-IR spectroscopy with consideration of sampling effects on method accuracy.

In-line near-IR moisture monitoring of the dynamic, fluid bed drying environment has been reported in recent years by several research groups; however, analytical figures of merit with regard to prediction accuracy are discussed in only a subset of this work, and issues with sampling and sample presentation are scarcely addressed at all. In this study, experiments were performed at 65-, 300-, and 600-L drying scales using several different sampling configurations in an effort to better understand and improve in-line near-IR method accuracy. Findings from this work demonstrate that process heterogeneity plays a major role in determination of apparent prediction accuracy. This aspect is general to all in-line measurements and plays an especially important role in solids and slurry systems that are prone to heterogeneity. In addition to experimental results, simulations based on these findings and sampling theory demonstrate an interesting paradox: depending on the sampling configuration employed, the method with the smallest apparent error is not necessarily optimal for process monitoring and control. Furthermore, sampling configuration influences the number of samples necessary to define an adequate calibration set. Finally, process understanding that was gained as a result of temporally rich, in-line measurements will be presented.

Journal Article↗

PubMed1998

A taxonomy of adolescent health: development of the adolescent health profile-types.

OBJECTIVES: The aim of this study was to develop a taxonomy of health profile-types that describe adolescents' patterns of health as self-reported on a health status questionnaire. The intent was to be able to assign individuals to mutually exclusive and exhaustive groups that characterize the important aspects of their health and need for health services. METHODS: Cluster analytic empirical methods and clinically based conceptual methods were used to identify patterns of health in samples of adolescents from schools and from clinics that serve adolescents with chronic conditions and acute illnesses. Individuals with similar patterns of scores across multiple domains were assigned to the same profile-type. Results from the empirical and conceptually based methods were integrated to produce a practical system for assigning youths to profile-types. RESULTS: Four domains of health (Satisfaction, Discomfort, Risks and Resilience) were used to group individuals into 13 distinct profile-types. The profile-types were characterized primarily by the number of domains in which health is poor, identifying the unique combinations of problems that characterize different subgroups of adolescents. CONCLUSIONS: This method of reporting the information available on health status surveys is potentially a more informative way of identifying and classifying the health needs of subgroups in the population than is available from global scores or multiple scale scores. The reliability and validity of this taxonomy of health profile-types for the purposes of planning and evaluating health services must be demonstrated. That is the purpose of the accompanying study.

Acute Disease↗

PubMedDate not supplied

Evaluation of the VIDAS Listeria (LIS) immunoassay for the detection of Listeria in foods using demi-Fraser and Fraser enrichment broths, as modification of AOAC Official Method 999.06 (AOAC Official Method 2004.06).

A multilaboratory study was conducted to compare the VIDAS LIS immunoassay with the standard cultural methods for the detection of Listeria in foods using an enrichment modification of AOAC Official Method 999.06. The modified enrichment protocol was implemented to harmonize the VIDAS LIS assay with the VIDAS LMO2 assay. Five food types--brie cheese, vanilla ice cream, frozen green beans, frozen raw tilapia fish, and cooked roast beef--at 3 inoculation levels, were analyzed by each method. A total of 15 laboratories representing government and industry participated. In this study, 1206 test portions were tested, of which 1170 were used in the statistical analysis. There were 433 positive by the VIDAS LIS assay and 396 positive by the standard culture methods. A Chi-square analysis of each of the 5 food types, at the 3 inoculation levels tested, was performed. The resulting average Chi square analysis, 0.42, indicated that, overall, there are no statistical differences between the VIDAS LIS assay and the standard methods at the 5% level of significance.

Animals↗

PubMed2006

Novel biophysical techniques for investigating long-term cell adhesion dynamics on biomaterial surfaces.

Cell adhesion on biomaterial surface is crucial for the regeneration and function of clinically viable cell and tissues. In turn, the cellular phenotypes, following the mechanochemical transduction of adherent cells on biomaterials, are directly correlated to the biophysical responses of cells. However, the lack of an integrated bio-analytical system for probing the cell-substrate interface poses significant obstacles to understanding the behavior of cells on biomaterial surface. We have developed a novel method, based on the principle of confocal reflectance interference contrast microscopy (C-RICM) that has enabled us to study the biomechanical deformation of cells on biomaterial surfaces. In this article, we would like to describe our recent development of the C-RICM system that integrates a confocal fluorescence microscope, phase contrast microscope and GFP expression system. We shall demonstrate the system by determining the adhesion contact kinetics, initial deformation rate, cytoskeleton structures of adherent cells on extracellular matrices (e.g., collagen and fibronectin) and biodegradable polymer (e.g., poly(lactic acid)) during long-term culture. We shall demonstrate that this unique approach could provide valuable biophysical information necessary for designing optimized biomaterial surfaces for cell/tissue regeneration applications.

Animals↗

PubMed2001

Long-term evaluation of electrospray ionization mass spectrometric analysis of glycated hemoglobin.

BACKGROUND: Electrospray ionization mass spectrometry (ESIMS) has been successfully applied to the identification of hemoglobin (Hb) variants and the presence of glucose adducts (mass difference of 162 Da) on the separate Hb alpha and beta chains. To establish the potential of ESIMS as a routine and/or a reference method for the quantification of glycohemoglobin (HbA1c), we carried out a detailed evaluation over a 4-month period in a routine laboratory environment. METHODS: We optimized a procedure using ESIMS suitable for the routine quantitative analysis of HbA1c. We determined reliability and reproducibility over 4 months and assessed the potential for automated sample injection. We then compared values of 1022 blood samples from diabetic patients with a routine HPLC-based ion-exchange procedure (HA-8140; Menarini). RESULTS: Results of HbA1c measurement by ESIMS were available within 3 min. The analytical imprecision (CV) was 1.6-5.0% for both manual and automated injections. Data collection over the m/z 980-1400 range confirmed lower glycation of the alpha chain relative to the beta chain (0.66:1). Only one glycation was observed per globin chain. The overall glycohemoglobin (i.e., the average of alpha- and beta-chain glycations) measured by ESIMS (x) on 1022 blood samples was lower than by HPLC (y): y = 1.0432x + 0.4815. However, the ss-chain glycation measured by ESIMS was up to 20% higher than the value measured by ion-exchange HPLC and showed a close conformity, particularly at 5-10% HbA1c, with the ion-exchange Diabetes Control and Complications Trial (DCCT)-corrected and the United Kingdom National External Quality Assessment Scheme DCCT mean return values. CONCLUSIONS: ESIMS provides a precise measurement of HbA1c and, in particular, glycation of the beta chain. The method is robust and could be proposed as a procedure to substantiate HbA1c measurement and/or calibration.

Autoanalysis↗

PubMedDate not supplied

Rapid, sensitive, microscale determination of phosphate in water and soil.

A rapid and sensitive analysis of inorganic and organic phosphorus (P) is needed to analyze water and soil extracts at submicromolar concentrations. The proposed method, based on the complexation of malachite green with phosphomolybdate under acidic conditions, was adapted to a 96-well microtiter plate format, and was tested for matrix interferences using 15 soils and some common extractants, including water, KCI, CaCl2, NaOH, and HCl. The accuracy of P determination was affected when CaCl2 and HCl concentrations were greater than 0.1 M and when NaOH concentration exceeded 0.4 M. Potassium chloride concentration up to 1 M did not interfere with P determination. The molar absorptivity was 46 841 M(-1) cm(-1) and the reagent blank absorbance was 0.071+/-0.003 (n = 10). At the 99% confidence limit, the method detection limit was calculated to be 0.006 mg P L(-1). Recovery of added inorganic P in different types of soils and extracts ranged between 95 and 112% with an average of 102%. The proposed microplate method allows P to be determined rapidly in a wide range of soil types and extracts and requires limited volume (20-200 microL). The procedure uses limited quantities (40 microL) of two stable reagents (>1 yr), and generates low amounts of hazardous waste.

Chemistry Techniques, Analytical↗

PubMedDate not supplied

Alternative anaerobic enrichments to the bacteriological analytical manual culture method for isolation of Shigella sonnei from selected types of fresh produce.

Alternative methods of reducing oxygen during anaerobic enrichment in the Bacteriological Analytical Manual (BAM) Shigella culture method were evaluated and compared to the current and less practical GasPak method. The alternative anaerobic methods included the use of reducing agents in Shigella broth and reducing culture container headspace volume to minimize atmospheric effects on oxygen concentration in Shigella broth during enrichment. The reducing agents evaluated were sodium thioglycollate, L-cystine, L-cysteine, titanium(III) citrate, and dithiothreitol, each at concentrations of 0.1, 0.05, and 0.01%. The use of Oxyrase for Broth with the enrichment medium (Shigella broth) was evaluated at concentrations of 10, 20 and 30 microL/mL. Recoveries of chill- and freeze-stressed S. sonnei strains 357 and 20143 were determined with each anaerobic method, including the GasPak method, using inoculation levels ranging from 10(0)to 10(3) cells. For each anaerobic method, strain, inoculation level, and stress type, 5 replicate enrichments were evaluated by streaking to MacConkey agar for isolation. The numbers of cultures with each method from which S. sonnei was isolated were used to compare the alternative anaerobic methods to the GasPak method. The alternative anaerobic method with which chill- and freeze-stressed S. sonnei strains 357 and 20143 were isolated most consistently was the use of Oxyrase for Broth in Shigella broth at a concentration of 20 microL/mL. This method was compared to the GasPak anaerobic method in evaluations on the recovery of S. sonnei strains 357 and 20143 from artificially contaminated test portions of parsley, cilantro, green onions, strawberries, carrots, and celery. A third anaerobic method included the use of 0.5 cm mineral oil overlay on cultures containing Oxyrase for Broth at concentrations of 20 microL/mL. Recovery rates of strain 357 were significantly greater (p < 0.05) with the GasPak method than with Oxyrase for Broth, with and without the 0.5 cm mineral oil overlay, for test portions of parsley, cilantro, and celery. When Oxyrase for Broth was used with Shigella broth, strain 357 was isolated at higher rates from all produce types, except cilantro, when 0.5 cm mineral oil overlay was applied to enrichment cultures. The use of mineral oil overlay with Oxyrase for Broth also improved recovery of strain 20143 from test portions of all produce types except green onion and strawberries. These differences were significant (p < 0.05) with parsley, carrots, and cilantro (1 of 2 evaluations). No statistically significant differences (p > 0.05) between the GasPak and Oxyrase for Broth anaerobic methods occurred when mineral oil overlay was used with Oxyrase for Broth. The use of Oxyrase for Broth with a 0.5 cm mineral oil overlay is a practical alternative for anaerobic enrichment with the BAM method in the analysis of some produce types. Differences in recovery among the different produce types and methods occurred between S. sonnei strains 357 and 20143, emphasizing the need for additional S. sonnei strains in future evaluations.

Anaerobiosis↗

PubMed2005

High throughput particle analysis: combining dielectrophoretic particle focussing with confocal optical detection.

A micro flow cytometer has been fabricated that detects and counts fluorescent particles flowing through a microchannel at high speed based upon their fluorescence emission intensity. Dielectrophoresis is used to continuously focus particles within the flowing fluid stream into the centre of the device, which is 40 microm high and 250 microm wide. The method ensures that all the particles pass through an interrogation region approximately 5 microm in diameter, which is created by focusing a beam of light into a spot. The functioning of the device was demonstrated by detecting and counting fluorescent latex particles at a rate of up to 250 particles/s. A mixture of three different populations of latex particle was used, each sub-population with a distinct level of fluorescent intensity. The device was evaluated by comparison with a conventional fluorescent activated cell sorter (FACS) and numerical simulation demonstrated that for 6 microm beads, and for this design of chip the theoretical throughput is of the order of 1000 particles/s (corresponding to a particle velocity of 10 mm s(-1)).

Biosensing Techniques↗

PubMed2001

Development and validation of a cholate binding capacity method for DMP 504, a bile acid sequestrant.

DMP 504, a highly cross-linked insoluble polymer, is a bile acid sequestrant developed by the DuPont Pharmaceuticals Company for serum cholesterol reduction. Since DMP 504 is insoluble, it was necessary to develop unique specific analytical methods to measure and control the quality of different lots of the drug. Since the mechanism of action of DMP 504 is believed to be by sequestration of bile acids, the in-vitro binding capacity of the polymer for cholic acid was chosen as a surrogate of in-vivo performance and used to assess potency of the compound. In this method, individual aliquots of DMP 504 at three different levels were incubated with a cholate solution of known concentration. The residual cholate solution was filtered and analyzed by a reversed-phase HPLC method using refractive index detection. When the bound cholate was plotted versus the mass of DMP 504, the resulting curve was linear. The slope of this curve is the cholate binding capacity of DMP 504. This method has been shown to be precise and robust. Precision of the method was shown to have an RSD of 2.0% with injection precision of 0.4% and stability of cholate solutions up to 73 h. It is also a unique binding capacity method due to its multi-point determination, and it has been shown to be a suitable quality control method for ensuring lot-to-lot consistency of drug substance.

Cholates↗