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PubMed2004

The use of acoustic microscopy to study the mechanical properties of glass-ionomer cement.

OBJECTIVES: The aim of the study was to investigate the relationship between microstructure, acoustic and mechanical properties of hardened dental cement samples, prepared with different powder/water ratios. METHODS: Glass-ionomer dental cement samples, prepared with a standard amount of cement powder and different amounts of water have been examined after being hardened. Surface microstructure and ultrasound, longitudinal and shear velocities were obtained with a scanning acoustic microscope. Conditional effective elastic modulus and Poisson's ratio have been calculated using longitudinal and shear sound velocity values. Then on the same samples elastic modulus and microhardness have been determined by standard tests. Additional samples have been used to determine compressive strength. RESULTS: Density; conditionally instantaneous elastic modulus; high-elasticity modulus and compression strength of the samples decrease when large amounts of water were used for their preparation. At the same time porosity and microhardness of the cement matrix increase. Acoustic parameters and parameters of elasticity, calculated on the basis of sound velocity, demonstrated changes, similar to those obtained in standard mechanical tests. SIGNIFICANCE: The established relation between microstructure, acoustic and mechanical parameters demonstrated a high capacity of acoustic microscopy application for non-destructive characterization of dental materials. A particular advantage of the acoustic microscopy is the opportunity to evaluate microstructure and mechanical properties on the same sample.

Compressive Strength↗
PubMed1995

Polyethylene membrane as a sample support for direct matrix-assisted laser desorption/ionization mass spectrometric analysis of high mass proteins.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) of high mass proteins (> 100,000 Da), directly deposited on polyethylene membranes, is demonstrated. The spectral quality obtained, using standard sample preparation conditions, is equal or superior to that obtained with metal sample stages. Compared to the use of poly(vinylidene difluoride) transfer membranes, this material allows the acquisition of excellent quality MALDI mass spectra from high-mass proteins with a standard UV laser. This gain in capability is not at the expense of either mass accuracy or signal reproducibility; both approach that obtained with standard sample preparations on stainless steel. In addition, for the applications shown, the use of PE as a sample support reduces the severe ion suppression effects typically observed in the MALDI analysis of high-mass mixtures. This permits more precise mass measurements to be made via the use of internal calibration and is illustrated by the mass measurement of a chimeric mouse/human antibody (MW approximately 150,000 Da) by coaddition of bovine albumin dimer (MW approximately 130,000 Da).

Animals↗
PubMed2024

Integrative Multi-PTM Proteomics Reveals Dynamic Global, Redox, Phosphorylation, and Acetylation Regulation in Cytokine-Treated Pancreatic Beta Cells.

Studying regulation of protein function at a systems level necessitates an understanding of the interplay among diverse posttranslational modifications (PTMs). A variety of proteomics sample processing workflows are currently used to study specific PTMs but rarely characterize multiple types of PTMs from the same sample inputs. Method incompatibilities and laborious sample preparation steps complicate large-scale physiological investigations and can lead to variations in results. The single-pot, solid-phase-enhanced sample preparation (SP3) method for sample cleanup is compatible with different lysis buffers and amenable to automation, making it attractive for high-throughput multi-PTM profiling. Herein, we describe an integrative SP3 workflow for multiplexed quantification of protein abundance, cysteine thiol oxidation, phosphorylation, and acetylation. The broad applicability of this approach is demonstrated using cell and tissue samples, and its utility for studying interacting regulatory networks is highlighted in a time-course experiment of cytokine-treated β-cells. We observed a swift response in the global regulation of protein abundances consistent with rapid activation of JAK-STAT and NF-κB signaling pathways. Regulators of these pathways as well as proteins involved in their target processes displayed multi-PTM dynamics indicative of complex cellular response stages: acute, adaptation, and chronic (prolonged stress). PARP14, a negative regulator of JAK-STAT, had multiple colocalized PTMs that may be involved in intraprotein regulatory crosstalk. Our workflow provides a high-throughput platform that can profile multi-PTMomes from the same sample set, which is valuable in unraveling the functional roles of PTMs and their co-regulation.

Proteomics↗
PubMed1991

High-performance liquid chromatographic assay with electrochemical detection for azithromycin in serum and tissues.

High-performance liquid chromatographic methods using reversed-phase chromatography and electrochemical detection have been developed for the quantitation of azithromycin in serum and tissues of laboratory animals and humans. Serum sample preparation involved addition of internal standard, alkalinization, and solvent extraction. Tissue sample preparation involved Polytron homogenization in acetonitrile containing internal standard, evaporation of the supernatant, alkalinization of the residue, and solvent extraction. Serum samples were chromatographed on an alkylphenyl-bonded silica column eluted with pH 6.8-7.2 mobile phase with a dual-electrode coulometric detector operated in the oxidative screen mode. Serum and tissue samples were chromatographed on a gamma RP-1 alumina column with pH 11 mobile phase with a glassy carbon amperometric detector. Recovery of azithromycin was 87% from serum and 85% from tissues. Linear standard curves were prepared in serum over two concentration ranges (0.01-0.20 and 0.20-2.0 micrograms/ml) and in tissues over several concentration ranges (0.1-2, 1-10, 10-100, and 100-1000 micrograms/g). In serum and tissues, intra- and inter-assay precision ranged from 1 to 8% and 4 to 11%, respectively. The tissue assay has been applied to liver, kidney, lung, spleen, muscle, fat, brain, tonsil, lymph nodes, eye, prostate and other urological tissues, and gynecological tissues.

Azithromycin↗
PubMed2003

Optimization and validation of a rapid method to determine citrate and inorganic phosphate in milk by capillary electrophoresis.

Quantification of phosphate and citrate compounds is very important because their distribution between soluble and colloidal phases of milk and their interactions with milk proteins influence the stability and some functional properties of dairy products. The aim of this work was to optimize and validate a capillary electrophoresis method for the rapid determination of these compounds in milk. Various parameters affecting analysis have been optimized, including type, composition, and pH of the electrolyte, and sample extraction. Ethanol, acetonitrile, sulfuric acid, water at 50 degrees C or at room temperature were tested as sample buffers (SB). Water at room temperature yielded the best overall results and was chosen for further validation. The extraction time was checked and could be shortened to less than 1 min. Also, sample preparation was simplified to pipet 12 microl of milk into 1 ml of water containing 20 ppm of tartaric acid as an internal standard. The linearity of the method was excellent (R2 > 0.999) with CV values of response factors <3%. The detection limits for phosphate and citrate were 5.1 and 2.4 nM, respectively. The accuracy of the method was calculated for each compound (103.2 and 100.3%). In addition, citrate and phosphate content of several commercial milk samples were analyzed by this method, and the results deviated less than 5% from values obtained when analyzing the samples by official methods. To study the versatility of the technique, other dairy productssuch as cream cheese, yogurt, or Cheddar cheese were analyzed and accuracy was similar to milk in all products tested. The procedure is rapid and offers a very fast and simple sample preparation. Once the sample has arrived at the laboratory, less than 5 min (including handling, preparation, running, integration, and quantification) are necessary to determine the concentration of citric acid and inorganic phosphate. Because of the speed and accuracy of this method, it is promising as an analytical quantitative testing technique.

Animals↗
PubMed1994

Isolation of myosin heavy chain from small skeletal muscle samples by preparative continuous elution gel electrophoresis: application to measurement of synthesis rate in human and animal tissue.

A convenient procedure for the isolation of milligram quantities of myosin heavy chain (MHC) from small samples of muscle tissue using preparative gel electrophoresis is described. Application of the methodology to measurements of the fractional synthesis rates (ks) of MHC using skeletal muscle biopsy samples from humans and cardiac tissue from pigs following continuous intravenous infusions of stable isotope (L-[1-13C]-leucine) is demonstrated. The sensitivity and reproducibility of the determination of stable isotope enrichment are also defined. In addition, measurements of the ks of MHC in skeletal muscle of rats in vivo using radioisotope-tracer methodologies are described.

Animals↗
PubMed2006

Detection of central nervous system tissue on meat and carcass-splitting band saw blade surfaces using modified fluorescent glial fibrillary acidic protein enzyme-linked immunosorbent assay sampling and extraction procedures.

This study was conducted to determine optimal buffer pH, extraction procedure, and temperature for detecting central nervous system (CNS) tissue on meat surfaces and on carcass-splitting band saw blades using swab sampling. Glial fibrillary acidic protein (GFAP) is restricted to CNS tissue and has been used as a marker for CNS tissue presence in meat products. Sample preparation, extraction procedure, and extraction temperature of glial fibrillary acidic protein fluorescent enzyme-linked immunosorbent assay (GFAP F-ELISA) were modified to detect CNS tissue on meat surfaces and on carcass-splitting band saw blades. Maximum GFAP recovery was observed with an extraction buffer pH of 7.4. Extracting samples at room temperature by vortexing for 30 s in 1 ml of extraction buffer (phosphate-buffered saline [pH 7.4] plus 0.05% sodium dodecyl sulfate) consistently provided detection of GFAP on meat surfaces contaminated with 500 microg of spinal cord suspension per 50 cm2 and on carcass-splitting band saw blades contaminated with 20 microg of spinal cord suspension per 50 cm2. Recovery of GFAP was not affected by storing samples overnight at 4 degrees C. The current studies demonstrate the effectiveness of modified sampling procedures and preparations, sample extraction buffer pH, and extraction temperatures. These modifications introduced to the original F-ELISA sampling protocol result in asensitive and repeatable assay for detection of CNS tissue on meat surfaces and on carcass-splitting band saw blades.

Animals↗
PubMed2000

Quantification of polycyclic aromatic hydrocarbons in the NIST standard reference material (SRM1649A) urban dust using thermal desorption GC/MS

A thermal desorption GC/MS technique has been developed for the quantification of polycyclic aromatic hydrocarbons (PAHs) in airborne particulate matter using the NIST Standard Reference Material (SRM1649a) Urban Dust. The technique was developed using standard linearity tests in order to establish optimum sample weights and optimum desorption and chromatographic parameters. This direct analysis technique eliminates the use of solvents in the sample preparation (reducing volatile component losses) and also significantly reduces the sample preparation time (no extraction procedure). The technique has been shown to give linearity in terms of the overall TIC response as well as for a prominent series of n-alkanes (C20-C33) and 10 NIST priority PAHs, 8 of which have been quantified. The technique is reported to be uniquely sensitive (PAH concentrations 2-6 mg kg(-1)) and reproducible (MW = 178-228 SD < or =0.228 mg kg(-1), < or =7%; MW = 252 SD < or =0.922 mg kg(-1), < or =33%) over the range of sample weights (1-5 mg). Such sample weights illustrate that the technique can be equally applied to the analysis of airborne particulate samples collected over short time periods (24-48 h) using only commonly used low-volume collection devices.

Journal Article↗
PubMed2002

Association of a photoreceptor-specific tetraspanin protein, ROM-1, with triton X-100-resistant membrane rafts from rod outer segment disk membranes.

This study reports the isolation and characterization of a Triton X-100-resistant membrane fraction from homogenates of rod outer segment (ROS) disk membranes purified free of the surrounding plasma membrane. A portion of the ROS disk membrane was found to be resistant to Triton X-100 extraction at 4 degrees C. This detergent-resistant fraction was isolated as a low buoyant density band on sucrose density gradients and exhibited an increase in light scattering detected at 600 nm. Biochemical analysis of the Triton X-100-resistant fraction showed it to be enriched in cholesterol and sphingomyelin relative to phospholipid and in phospholipid relative to protein compared with the soluble fraction. The Triton X-100-resistant membranes described herein did not arise simply from partial solubilization of the ROS disk membranes because detergent-treated low buoyant density fractions isolated from homogenates with octyl glucopyranoside had cholesterol and sphingomyelin content indistinguishable from that of solubilized ROS disk homogenates. Analysis of proteins associated with the Triton X-100-resistant fraction showed it to be enriched in the rim-specific protein ROM-1 and caveolin; surprisingly, the fusion protein peripherin/rds (where rds is retinal degeneration slow), also localized to the disk rim, was entirely absent from the membrane raft domain. The lipid profiles of the Triton X-100-resistant membranes were virtually identical in preparations homogenized in either the light or dark. Slightly more ROM-1 was recovered from samples prepared in the light (23%) than from samples prepared in the dark (13%), but peripherin/rds could not be detected in either preparation. When the Triton X-100-resistant membranes were treated with methyl-beta-cyclodextran to deplete membrane cholesterol, the resultant membranes contained slightly lower levels of ROM-1, specifically in the dimeric form. Cholesterol depletion also resulted in the collapse of the large caveolin complex to monomeric caveolae. The results presented herein characterize a pool of ROM-1, a photoreceptor tetraspanin protein, that may play a regulatory role in peripherin/rds-dependent fusion.

Animals↗
PubMed1993

Use of the polymerase chain reaction to detect Bordetella pertussis in patients with mild or atypical symptoms of infection.

Nasopharyngeal aspirates and nasopharyngeal swabs from 177 children exhibiting mild to severe clinical symptoms of whooping cough were tested by the polymerase chain reaction (PCR) and culture for the presence of Bordetella pertussis. In the PCR analysis amplifications of samples prepared with and without DNA extraction were compared. In 26% of samples prepared without DNA extraction, the PCR was found to be inhibited, whereas no inhibition was detected after DNA extraction. Twelve percent (21/177) of the samples were positive in both culture and the PCR, and an additional 49% (87/177) of the samples were positive exclusively in the PCR. Thirty-eight percent (8/21) of culture-positive patients and 63% (55/87) of the patients in whom infection was detected only by PCR had mild or atypical clinical symptoms. Of these groups 26% (5/19) and 50% (39/78), respectively, had been fully vaccinated with three or more doses of diphtheria, tetanus and pertussis vaccine.

Bacteriological Techniques↗
PubMed2004

Extraction methods and chemical standardization of botanicals and herbal preparations.

Botanicals and herbal preparations are medicinal preparations, containing a single or two or more medicinal plants. The focus of this review paper is on the analytical methodologies, which included the combination of sample preparation tools and chromatographic techniques for the chemical standardization of marker compounds or active ingredients in botanicals and herbal preparations. The common problems and key challenges in the chemical standardization of botanicals and herbal preparations were discussed. As sample preparation is the most important step in the development of analytical methods for the analysis of constituents present in botanicals and herbal preparations, the strength and weakness of different extraction techniques are discussed. For the analysis of compounds present in the plant extracts, the applications of common chromatographic techniques, such as HPLC, CE, HRGC/MS, HPLC/MS and HPLC/MS/MS are discussed. The strength, weakness and applicability of various separation tools are stated. Procedures for the identification of marker or active compounds in plant extracts, using HPLC/MS, were proposed. Finally, the effects of batch-to-batch variation of the medicinal plants are investigated and discussed.

Chromatography↗
PubMed2001

Investigation of atrazine metabolism in river sediment by high-performance liquid chromatography/mass spectrometry.

Microbial degradation processes play an important role in chemical water clearance taking place in river sediments. Bacteria remove not only easily degradable organic species, but various xenobiotics as well, producing clear and xenobiotic free water for bank-filtered wells. Atrazine is a widely used herbicide, and it is one of the most common xenobiotics present in Danube water. In this study the pathway and kinetics of atrazine metabolism of sedimental microbiota were studied. Samples were collected from river sediment and from pure microbial growth cultures. An analytical scheme including sample preparation, chromatography and mass spectrometry was developed and optimised. Solid-phase extraction (SPE) was found to be satisfactory for sample preparation. For qualitative analysis of samples both reversed-phase and normal-phase high-performance liquid chromatography/mass spectrometry (HPLC/MS) methods were developed and used. Selectivity, detection limits and accuracy of the two methods were compared. Using this analytical scheme, the full atrazine metabolism of the organism Comamonas acidovorans was explored. Altogether, 12 metabolites were identified from the original compound to the urea end product. Detection limits in the range of 50 ng L(-1)-1 microg L(-1) were obtained for different metabolites.

Atrazine↗
PubMed1979

Determination of bile acids in serum by capillary gas-liquid chromatography.

A glass capillary column and an appropriate relatively simple procedure for sample preparation have been developed for determination of serum bile acids. Sample preparation involved extraction with Amberlite XAD-2, solvolysis of sulfates, enzymatic hydrolysis with cholylglycine hydrolase, methylation and silylation. Because of complete chromatographic separation of bile acid trimethylsilylether derivatives from cholesterol on the capillary column, an additional step for elimination of cholesterol could be omitted. Trimethylsilylether derivatives were separated on a 20 meter x 0.3 mm i.d. glass capillary column covered with a crystal layer of barium carbonate and coated with polyethyleneglycol 20,000 as liquid phase according to Grob, K. and Grob, G. (1976) J. Chromatogr.125, 471--485, and Grob, K., Grob, G. and Grob, Jr., K., (1977) Chromatographia 10, 181--187. Overall recovery of the major human conjugated bile acids ranged from 86 to 89%. Reproducibility of bile acid determination was satisfactory in both normal and pathological serum with elevated bile acid concentrations (coefficient of variation 7.6 to 10.0%). The mean concentrations of cholic, deoxycholic, chenodeoxycholic and lithocholic acid in the serum of healthy subjects were 0.9, 1.0, 1.7 and 0.2 mumol/l in males, and 1.0, 0.8, 1.4 and 0.2 mumol/l in females.

Adolescent↗
PubMed2001

Dependencies of the radiation sensitivity of human tooth enamel in EPR dosimetry.

The EPR dose response of tooth enamel was determined for human molars collected in Egypt. The influence of age, gender and residence of the tooth donors as well as tooth position and sample preparation on EPR sensitivity and its variability over the enamel samples was investigated. The EPR sensitivity and its variability were found to depend only on the sample preparation procedure. The variability in EPR sensitivity of enamel from Egyptian teeth was maximally 10% and the mean sensitivity was in good agreement with that of German teeth.

Age Factors↗
PubMed1999

Improved progenitor assay standardization using peripheral blood progenitor cells from a donor treated with granulocyte-colony-stimulating factor.

BACKGROUND: Progenitor assays are the principal method for evaluating hematopoietic cell function. The magnitude of assay variability and the assay steps contributing to variability were determined, and modifications intended to increase assay consistency were evaluated. STUDY DESIGN AND METHODS: Assays were performed using a serum-free progenitor assay medium with cells plated at 5.0 x 10(4) and 1.0 x 10(5) cells per plate. A peripheral blood progenitor cell component collected from a normal donor after administration of granulocyte-colony-stimulating factor was divided into identical aliquots. Each experiment involved at least 5 technologists, each performing assays in duplicate on five aliquots, with each person scoring all assay plates. Three sample preparation methods were tested: 1) ficoll mononuclear cell enrichment and sample dilution, 2) sample dilution without ficoll separation, and 3) sample dilution without ficoll separation, with cell counts performed before and after each dilution step, dilution volumes calculated on the basis of each cell count, automated electronic pipettors used in dilution steps, and colony frequency calculated on the basis of cell counts from the final specimen. RESULTS: Global variability for colony-forming units-granulocyte-macrophage, represented by the percentage of CV for all specimens and all technologists, was 89.6 percent at 5.0 x 10(4) cells per plate and 81.3 percent at 1.0 x 10(5), when ficoll separation was used. Subjective differences in scoring plates did not account for most of the variability observed, as results for any individual plate read by multiple technologists had a mean CV of 15.6 percent and 19.7 percent at the two plating concentrations. Method 3 resulted in the greatest improvement, reducing CV to 24.4 percent at 5.0 x 10(4) cells per plate and to 24.2 percent at 1.0 x 10(5) cells per plate. Similar results were obtained for erythroid-burst-forming units. CONCLUSIONS: Baseline assay results were extremely inconsistent. Interindividual differences in colony interpretation did not contribute significantly to assay variability, although sample preparation and plating did. Improved control over cell concentration decreased assay variability by 70 to 73 percent.

Blood Donors↗
PubMed2004

A study of the damage on FIB-prepared TEM samples of AlxGa1-xAs.

The damage produced by focused ion beam (FIB) milling on a TEM sample of AlGaAs crystals has been studied. The damage observed on the sidewall of an AlGaAs transmission electron microscopy (TEM) sample was an amorphous layer. The thickness of the amorphous layer linearly increased with an increase in FIB accelerating voltage from 5 to 30 kV. The thickness of the amorphous layer of Al(x)Ga(1-x)As was constant at 3 nm and was independent of the Al concentration x when the accelerating voltage was below 5 kV. The thickness of the amorphous layer of Al(x)Ga(1-x)As decreased with an increase in Al concentration x when the accelerating voltage was above 5 kV. FIB milling at 5 kV effectively minimizes the thickness of the amorphous layer and also provides flat sidewalls on multilayer samples of Al(x)Ga(1-x)As that are prepared for TEM and scanning electron microscopy (SEM).

Aluminum↗
PubMed2005

Determination of HCN sampled from gasification product gases by headspace gas chromatography with atomic emission detector.

Nitrogen-containing fuels produce hydrogen cyanide when the fuel is gasified. The gas is poisonous and produces nitrogen oxides when it is burned. HCN is usually sampled into alkaline solutions and analysed using an ion selective electrode. The method is tedious and the electrode response is temperature-dependent. Samples are not stable and must be analysed immediately, and they contain ions which are poisonous to the electrode. Therefore a new gas chromatographic method was developed. In this new method HCN is released from the alkaline solutions with sulphuric acid in a headspace sampler and analysed by a gas chromatograph connected to an atomic emission detector. Measurements on carbon emission line 193.1 nm gave the limit of detection 0.05 mg CN-/l in the solution. The calibration curve was linear to 1000 mg CN-/l and the correlation was 0.997. The relative standard deviation of the calibration was 1.7% at the concentration of 5 mg CN-/l and 1.0% at 25 mg CN-/l. The developed headspace method allows automated analysis and it needs less sample preparations than the ion selective electrode method. This paper also reports the effect of sample preparation and storage time on the stability of the samples.

Journal Article↗
PubMed2003

Spatial profiling invertebrate ganglia using MALDI MS.

The ability of MALDI TOF MS to spatially map peptides and proteins directly from a tissue is an exciting advance to imaging mass spectrometry. Recent advances in instrumentation for MS have resulted in instruments capable of achieving several micron spatial resolution while acquiring high-resolution mass spectra. Currently, the ability to obtain high quality mass spectrometric images depends on sample preparation protocols that often result in limited spatial resolution. A number of sample preparation and matrix deposition protocols are evaluated for spatial profiling of Aplysia californica exocrine gland and neuronal tissues. Such samples are different from mammalian tissues, but make good targets for method optimization because of the wealth of biochemical information available on neuropeptide processing and distribution. Electrospray matrix deposition and a variety of freezing methods have been found to be optimum for these invertebrate tissues, with the exact protocols being tissue dependent.

Animals↗