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The detection of occult liver metastases of colorectal carcinoma.

Despite undergoing an apparently curative surgery, over 50% of colorectal cancer patients will die within 5 years, the majority within the first 2 years from disseminated disease. Occult liver metastases are reported to be present in approximately 30% of patients and they predominantly determine the likelihood of dying from disseminated disease. The question is how to detect these occult hepatic metastases. This article reviews some of the alternative techniques, such as dynamic scintigraphy, functional computerized tomography, and duplex colour Doppler sonography (DCDS), which have been developed to predict which patients harbor these occult metastases. More recently, DCDS measurements of the Doppler perfusion index (DPI: ratio of hepatic arterial to total liver blood flow) have been shown to be highly accurate in the detection of these occult liver metastases. In addition, irrespective of Dukes stage, patients with abnormal DPI were shown to have a very poor outcome while for those with normal DPI, the prognosis was excellent. If the data are reproduced, the administration of adjuvant chemotherapy should be based on DPI status rather than Dukes stage.

Adenocarcinoma↗

Does the donor-acceptor concept work for designing synthetic metals? III. Theoretical investigation of copolymers between quinoid acceptors and aromatic donors.

Homopolymers of quinoxaline (QX), benzothiadiazole (BT), benzobisthiadiazole (BBT), thienopyrazine (TP), thienothiadiazole (TT), and thienopyrazinothiadiazole (TTP) and copolymers of these acceptors with thiophene (TH) and pyrrole (PY) were investigated with density functional theory. Theoretical band-gap predictions reproduce experimental data well. For all but six copolymers, band-gap reductions with respect to either homopolymer are obtained. Four of the acceptors, BBT, TP, TT, and TTP, give rise to copolymers with band gaps that are smaller than that of polyacetylene. BBT and TTP copolymers with PY in 1:2 stoichiometry are predicted to be synthetic metals. Band-gap reductions result from upshifts of HOMO energies and much smaller upshifts of LUMO values. The smallest band gaps are predicted with TTP, since changes in LUMO energies upon copolymerization are particularly small. The consequence of the small interactions between LUMO levels of donor and acceptor are vanishingly small conduction bandwidths.

Metals↗

New dimensions in Hirsutism.

The following is a review of a satellite symposium held at the British Medical Laser Association Meeting, in November 2005. Prof. V.A. Randall gave a comprehensive overview of hormones and hair growth, followed by an in-depth discussion of hirsutism, the therapeutic options, treatment and trends, by Dr. S. Lanigan. Dr. I. Hamzavi concluded the symposium with a presentation of the latest work on eflornithine in combination with laser hair removal. Data were reproduced with the speakers' permission.

Androgens↗

Mechanical effects of ionic replacements in articular cartilage. Part I: The constitutive model.

A three-phase multi-species electro-chemo-mechanical model of articular cartilage is developed that accounts for the effect of two water compartments, namely intra-fibrillar water stored in between collagen fibrils and extra-fibrillar water covering proteoglycans. The collagen fibers constitute the solid phase while intra-fibrillar water and dissolved NaCl and CaCl(2) on one hand and extra-fibrillar water, ions Na(+), Ca(2+) and Cl(-) and proteoglycans on the other hand, form the two fluid phases. The complete picture that includes time-dependent mass transfers between the two fluid phases, diffusion of water and ions and electrical flow emerges from the Clausius-Duhem inequality but it is deferred to further study. The analysis is restricted to equilibrium states. The present work complements the mechanical model developed in Loret and Simões (Mech Material 36(5-6): 515-541, 2004a) where the presence of the sole NaCl was considered. In its current version, the model can handle mechanical and chemical loadings and unloadings involving the two salts, NaCl and CaCl(2). In order to reproduce experimental data, the shielding effects are made cation-dependent. Strong orientation of collagen fibers parallel to the joint surface implies anisotropic mechanical properties. Electro-chemo-mechanical couplings result in a chemistry-dependent apparent tensile Poisson's ratio, that increases to large values as the solution gets fresher. The model captures these aspects as well. The features of the model are first exposed in an infinitesimal strain context. Subsequently, large strains that typically occur in uniaxial traction under deionized water are accounted for, and a nonlinear anisotropic hyper-elastic behavior is developed. Parametric identification and simulations of actual loading processes are described in a companion paper, Loret and Simões (Biomech Model Mechanobiol, in press, DOI 10.1007/s10237-004-0063-6).

Cartilage, Articular↗

Mechanical effects of ionic replacements in articular cartilage. Part II: Simulations of successive substitutions of NaCl and CaCl(2).

A three-phase multi-species electro-chemo-mechanical model of articular cartilage was developed in a companion paper, Loret and Simões (in Biomech Model Mechanobiol, in press, DOI 10.1007/s10237-004-0062-7). The model can handle mechanical and chemical loadings and unloadings involving the two salts, NaCl and CaCl(2). In order to reproduce experimental data, the shielding effects are made cation-dependent. In a tensile experiment, at constant axial strain, refreshment of the bath in contact with the cartilage is observed, and simulated, to induce a much different increase in tension depending on the order of the chemical sequence to which the cartilage is exposed. For example, the sequence dw (distilled water)-NaCl-dw-CaCl(2)-dw results in a decrease in tension. But the initial tension is recovered if the chemical sequence is pursued by NaCl-dw. Therefore, ionic replacements are essentially reversible, as evidenced when the chemical loading events respect a certain symmetry. Distinct shielding effects by cations sodium and calcium stem from two main features: (1) different free enthalpies of formation that represent different affinities of the proteoglycans for these ions and that result in an equilibrium constant not equal to 1; (2) distinct valences but approximately the same diameter, which results in a more efficient shielding by cations calcium. The model accounts also: (1) for the anisotropy of the mechanical properties that are due to the strong orientation of collagen fibers; (2) for large deformations that occur during uniaxial traction with deionized water.

Calcium Chloride↗

Early diagnosis of nontransmural myocardial infarction by two-dimensional echocardiography.

Thirty patients with chest pain syndromes were studied on admission by two-dimensional echocardiography (2DE) to confirm or exclude acute myocardial infarction (AMI). Twelve patients suffered nontransmural AMI and 18 patients had no AMI. There was no significant difference between these groups in background characteristics. 2DE studies were analyzed quantitatively and qualitatively. The most sensitive and specific technique for detecting AMI was qualitative analysis, using the presence of severe hypokinesis as the criterion for an abnormal study. Using this technique, 10 of 12 patients (83%) with AMI and all 18 patients (100%) without AMI were correctly identified. This analysis technique was highly reproducible. These data indicate that in patients with chest pain syndromes without diagnostic ECG findings of AMI, 2DE may provide a rapid, sensitive, specific tool to aid in the establishment of the correct diagnosis. In patients with nontransmural AMI, the presence of severe hypokinesis appears to be the best discriminator of myocardial infarction.

Adult↗

Reproducibility of quantitative two-dimensional echocardiography.

In order to assess reproducibility of quantitative planimetry, three physicians trained in two-dimensional echocardiography performed five successive studies on one another over 2 weeks (30 total studies). Then each physician traced each study (90 total tracings) for left ventricular and atrial volumes and ejection fraction by means of a modification of Simpson's rule, and left ventricular mass and average wall thickness by means of a truncated ellipsoid formula. Calculation of intertechnician variability, intertracer variability, and 95% confidence limits showed that measurements of volumes were less reproducible than measurements of ejection fraction, average wall thickness, and mass. Mean intertracer variability of 15% exceeded mean intertechnician variability of 11%; this disparity was magnified in the subject who was technically difficult to image. Ninety-five percent confidence limits were: ejection fraction +/- 7%, average wall thickness +/- 9%, left ventricular mass +/- 12%, left ventricular end-diastolic volume +/- 11%, stroke volume +/- 14%, left ventricular end-systolic volume +/- 15%, and left atrial volume +/- 19%. Reproducible planimetry data can be obtained in normal hearts with the use of a protocol for quantitative imaging and planimetry.

Echocardiography↗

Steroid receptors analysis in human mammary tumors by isoelectric focusing in agarose.

A high resolution and quantitative method for isoelectric focusing has been developed to separate the isoforms of estrogen and progesterone receptors in human mammary tumor cytosols stabilized by sodium molybdate. Agarose gels (0.5%) were used. Six samples can be analyzed on one gel in about 2 h, and 35-microliters samples are sufficient to determine the estrogen receptor isoform pattern. The constant yields and the reproducibility of data allow a quantitative analysis of these receptors. Four estrogen receptor isoforms have been observed (pI 4.7, 5.5, 6, and 6.5), isoforms with pI 4.7 and 6.5 being present in all tumors. After incubation at 28 degrees C in high ionic strength, the comparison of isoelectric focusing and high-performance size exclusion chromatography patterns of estrogen receptor confirms the oligomeric structure of the pI 4.7 isoform and suggests a monomeric structure for the pI 6.5 isoform. Under the same conditions of analysis, only one progesterone receptor isoform has been detected with pI 4.7.

Aged↗

A radiometric assay for HIV-1 protease.

A rapid, high-throughput radiometric assay for HIV-1 protease has been developed using ion-exchange chromatography performed in 96-well filtration plates. The assay monitors the activity of the HIV-1 protease on the radiolabeled form of a heptapeptide substrate, [tyrosyl-3,5-3H]Ac-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2, which is based on the p17-p24 cleavage site found in the viral polyprotein substrate Pr55gag. Specific cleavage of this uncharged heptapeptide substrate by HIV-1 protease releases the anionic product [tyrosyl-3,5-3H]Ac-Ser-Gln-Asn-Tyr, which is retained upon minicolumns of the anion-exchange resin AG1-X8. Protease activity is determined from the recovery of this radiolabeled product following elution with formic acid. This facile and highly sensitive assay may be utilized for steady-state kinetic analysis of the protease, for measurements of enzyme activity during its purification, and as a routine assay for the evaluation of protease inhibitors from natural product or synthetic sources.

Amino Acid Sequence↗

Double mixing stopped-flow method for the study of equilibria and kinetics of dimer-tetramer association of hemoglobins: studies on Hb Carp, Hb A, and Hb Rothschild.

A double mixing stopped-flow method is described for studying the dimer-tetramer equilibria of oxyhemoglobins and the kinetics of association of unliganded dimers. The three hemoglobins studied were: Hb Carp, Hb A, and Hb Rothschild (Trp beta 37 (C3)----Arg). The new method reproduces the data obtained for oxyHb A by other established methods. In agreement with previous studies, the new method indicates little, if any, dissociation of oxyHb carp into dimers even in 2 M urea solutions (0.1 M Bis-Tris pH 7.0). OxyHb Rothschild, on the other hand, is extensively dissociated into dimers (K(Hb4L4 in equilibrium with 2Hb2) = 37.3 x 10(-6) M) and the rate constant for the association of deoxy dimers of Hb Rothschild is about one-tenth of the value for Hb A indicating that the deoxy tetramer of Hb Rothschild is at least 10 times more dissociated into dimers than deoxyHb A.

Animals↗

Solubilization and characterization of vitamin K epoxide reductase from normal and warfarin-resistant rat liver microsomes.

Two procedures have been developed for the solubilization of vitamin K epoxide reductase from rat liver microsomal membranes using the detergent Deriphat 160 at pH 10.8. The methods are applicable to both normal and Warfarin-resistant-strain rat liver microsomes and yield material suitable for further purification. The preparations retain dithiothreitol-dependent vitamin K quinone reductase activity as well as vitamin K epoxide reductase and are free of vitamin K-dependent carboxylase and epoxidase activities. Optimal epoxide reductase activity is obtained at 0.1 M KCl and pH 9 in the presence of sodium cholate. Artifactual formation of vitamin K metabolites was eliminated through the use of mercuric chloride to remove excess dithiothreitol prior to extraction and metabolite assay. Using the solubilized enzyme, valid initial velocities were measured, and reproducible kinetic data was obtained. The substrate initial velocity patterns were determined and are consistent with a ping-pong kinetic mechanism. The kinetic parameters obtained are a function of the cholate concentration, but do not vary drastically from those obtained using intact microsomal membranes. At 0.8% cholate, the enzymes solubilized from normal Warfarin-sensitive- and Warfarin-resistant-strain rat livers exhibit respective values of Vmax = 3 and 0.75 nmol/min/g liver; Km for vitamin K epoxide = 9 and 4 microM; and Km for dithiothreitol of 0.6 and 0.16 mM.

Animals↗

A possible new mechanism of temperature dependence of electron transfer in photosynthetic systems.

A new theory for the electron transfer by the non-adiabatic process is formulated taking into account the origin shift and the frequency change of the vibration. The resultant formulas are quite similar to those of Jortner (Jortner, J. (1976) J. Chem. Phys. 64, 4860-4867) except that the free energy gap delta G is used instead of the energy gap delta E. By applying this theory to the photosynthetic electron transfer, the role of the remarkable temperature dependence of the electron transfer from cytochrome to P+ in Chromatium vinosum and the experimental data were reproduced very well using a small value of the coupling strength in contrast with the previous theory. This implies that proteins play a role to exclude many of the solvent molecules from the region of the electron transfer reaction between the donor and acceptor molecules. The negative activation process in the back electron transfer from QA- to P+, the very slow back electron transfer from I- to P+ and the solvent isotope effect on the cytochrome oxidation are also successfully explained by this new theory. It is shown that even a qualitative conclusion as to the molecular parameters obtained from the temperature dependence of the electron transfer is different between the present theory and that of Jortner.

Chromatium↗

Equilibrium binding of daunomycin and adriamycin to calf thymus DNA. Temperature and ionic strength dependence of thermodynamic parameters.

Absorbance and fluorescence quenching monitoring of the binding of the anthracyclines adriamycin (ADM) and daunomycin (DNM) to calf thymus DNA, provides reproducible binding data only when moderate drug/DNA molar ratios are used in the assays. Under these conditions, the fraction of DNA-bound drug, in equilibrium with free anthracycline, which can be reliably detected, ranged from 40-60% to 80-95% of the total added drug, depending upon ionic strength and temperature. Use of the neighbour exclusion model adequately fits such data and predicts that (i) the affinity of ADM for binding to the DNA is always higher than that corresponding to DNM, under similar experimental conditions, (ii) the binding constant for both drugs exhibits a strong salt and temperature dependence, and (iii) the exclusion parameter, indicative of the size of the anthracycline binding sites on the DNA, equals 3.1 +/- 0.4 and 3.3 +/- 0.4 base pairs for ADM and DNM, respectively, and is independent of salt concentration. The salt and temperature dependence of the binding constant is used to estimate the thermodynamic parameters involved in the interaction of the drugs with the DNA. Binding of the drugs is an exothermic process and the binding free energy arises primarily from a large negative enthalpy which, as the entropy, strongly depends upon ionic strength, and is much larger than predicted by polyelectrolyte theory. The enthalpy and entropy changes observed, appear to compensate each other over the entire range of salt concentrations used, and may arise from a complex variety of contributions, including salt-induced changes in secondary structure of the DNA, as indicated by circular dichroism techniques.

Animals↗

Recovery of malondialdehyde in urine as a 2,4-dinitrophenylhydrazine derivative analyzed with high-performance liquid chromatography.

Malondialdehyde (MDA) in urine was measured as a 2,4-dinitrophenylhydrazine (DNPH) derivative using high-performance liquid chromatography (HPLC) for the analysis. MDA standard coeluted with a peak obtained from rat urine after i.p. administration of MDA standard. This peak was also the only peak containing 14C after injection of a [14C]MDA standard, and was shown by mass spectrometry to contain 1-(2,4-dinitrophenyl)pyrazole, the derivative formed when MDA is treated with DNPH. Depending on the amount given (0.3-5.5 mumol), the recovery (after 24 h sampling period) in urine was 0.7-2.6%. This apparent non-linear kinetics may relate to several factors, such as dose-dependent metabolism. However, the peak urinary concentration approached the expected plasma concentration and reproducible recovery data were obtained, suggesting that MDA was passively excreted in a reasonably stable form. These data indicate that monitoring MDA excretion in urine can give useful information about lipid peroxidation in vivo.

Animals↗

Fragments of pituitary adenylate cyclase activating polypeptide discriminate between type I and II recombinant receptors.

Pituitary adenylate cyclase activating polypeptide (PACAP) analogues were tested for their ability to occupy the recombinant selective PACAP receptors (PACAP type I receptor) or the non-selective PACAP-vasoactive intestinal polypeptide (VIP) receptors (PACAP type II, VIP1 and VIP2 receptors) stably transfected and expressed in Chinese hamster ovary (CHO) cells. The synthetic analogues consisted of N- and/or C-terminally shortened peptides. Thus, peptides starting at amino acid 1, 2, 3 or 6 and terminating at amino acid 27, 29, 30, 32 or 38 were compared on the three receptors studied. The shortening of PACAP-(1-38) to PACAP-(1-27) was of little influence. However, in N-terminally deleted peptides the PACAP-38 derivatives were of higher affinity than the PACAP-27 fragments on PACAP type I and PACAP type II, VIP2 receptors but not on PACAP type II, VIP1 receptors. The presence of the sequence 28-32 was in all cases sufficient to reproduce the data obtained with the PACAP-38 analogues. PACAP-(3-32) is able to discriminate the PACAP type II, VIP2 subtype from the other two subtypes, and PACAP-(6-30), PACAP-(6-32) and PACAP-(6-38) can discriminate the PACAP type II, VIP1 receptors from the other two subtypes. These molecules may help in the quantitative detection of receptor subclasses in complex systems when two or more receptor subtypes are found.

Animals↗

Quantitative analysis of tylosin by column liquid chromatography.

A column liquid chromatographic method suitable for the quality control of tylosin A is described. The determination can be carried out on different C8 or C18 columns, using a mobile phase containing acetonitrile, 0.2 M tetrabutylammonium hydrogensulphate, 0.2 M phosphoric acid and water. The flow-rate is 1 ml/min and detection is performed at 280 nm. The method shows good selectivity towards the major components tylosin A, B, C and D and demycinosyltylosin. Minor degradation products, mainly observed in solutions, are also separated. The compositions of several standards are compared and results for a number of commercial samples are presented.

Calibration↗

Separation and determination of glycosaminoglycan disaccharides by micellar electrokinetic capillary chromatography for studies of pelt glycosaminoglycans.

Capillary electrophoresis based on cetyltrimethylammonium bromide micellar electrokinetic capillary chromatography (MECC) was developed for the separation and determination of glycosaminoglycan (GAG) disaccharide units without derivatization. The influence of changes in several separation conditions was studied, and the separation mechanisms are discussed. Tests of repeatability and linearity were performed for qualitative and quantitative evaluation of the method. The described procedure gives a rapid and efficient determination of GAG disaccharides. Samples of chondroitin sulphates and mink skin were treated with proteases, and the extent of protein cleavage was followed by free zone capillary electrophoresis. The result of the chondroitinase ABC treatment following the protease treatment was evaluated by the MECC method.

Animals↗

Analysis of dansyl amino acids in feedstuffs and skin by micellar electrokinetic capillary chromatography.

Micellar electrokinetic capillary chromatography (MECC) using sodium dodecyl sulphate (SDS) and sodium cholate have been used for analyses of 30 dansylated (Dns) amino acids. The influences of sample preparation, Dns/amino acid ratio, sample solvent composition, and separation conditions including voltage, temperature, pH and buffer composition were investigated. Complete separations of acidic and neutral amino acids were obtained within 45 min in the SDS system. The efficiency expressed as number of theoretical plates for the applied capillary 0.52 m long were between 210,000 and 343,000, and the repeatability was very good with relative standard deviations on relative migration times between 0.09 and 0.70% and on relative normalised peak areas (RNPAs) between 0.85 and 3.41%. The linearity studies gave correlation coefficients between 0.9957 and 0.9993 for RNPAs against concentration. Detection limits were between 3 and 6 fmol or approximately 2 pg of each amino acid. Basic amino acids were separated in a MECC system using sodium cholate. Procedures and problems using Dns derivatisation for amino acids analysed by the MECC methods are described. Finally, examples of analyses of hydrolysates of real complex samples show, that this method can be applied to determine the amino acid composition of proteins in feedstuffs and skin.

Amino Acid Sequence↗