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PubMed2004

Resolution of 8-aminonaphthalene-1,3,6-trisulfonic acid-labeled glucose oligomers in polyacrylamide gel electrophoresis at low gel concentration.

A discontinuous Tris-Cl/acetate (OAc) buffer system, unprecedently containing OAc as the trailing constituent, and operative in polyacrylamide gel electrophoresis (PAGE) at low polyacrylamide concentration (T = 4.8%) is described in the paper. The characteristics of the electrophoretic system are illustrated by the resolution of fluorescent 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS)-labeled malto-oligosaccharides and dextran homopolymers. In this buffer system, the resolving phase is constituted by Tris-OAc behind a moving boundary formed between the leading chloride ion of Tris-HCl gel buffer and the trailing OAc ion provided by a catholyte of NH(4)OAc. In contrast with the results obtained with Tris-CI/glycinate buffer commonly used in electrophoresis, or with Tris-CI/borate, the best resolution of the glucose oligomers containing 1-4 glucose units in Tris-OAc, pH 8.8, ionic strength of 0.08, was obtained at 4.8% polyacrylamide concentration, using 0.5 M NH(4)OAc, pH 9.5 as the catholyte. Under those conditions, the ANTS-glucose oligomers were separated with mobilities decreasing from glucose to maltohexaose. The linear Ferguson plots (log relative mobility, R(f), vs.%T) of the glucose oligomers show that the surface net charge of those oligomers is inversely related to their sizes, given by the slopes, K(R), of the plots. The molecular weight of the oligomers is directly but nonlinearly related to K(R). The novel electrophoretic system illustrated here for separation of short ANTS-saccharides can be potentially applied to the resolution of other biomolecules such as rapidly migrating DNA, peptides or proteins.

Buffers↗

PubMed2004

Chiral capillary electrophoretic resolution of baclofen, gabaergic ligand, using highly sulfated cyclodextrins.

Using cyclodextrin-capillary zone electrophoresis (CD-CZE), baseline separation of baclofen, a potent GABA(B) agonist; was achieved. A method for the enantioresolution of this gamma-aminobutyric acid (GABA) and determination of enantiomeric purity was developed using CDs (highly sulfated-CD or highly S-CD) as chiral selectors and capillaries dynamically coated with polyethylene oxide (PEO). Operational parameters, such as the nature and concentration of the chiral selectors, buffer concentration, organic modifiers, and applied voltage, were investigated. The use of charged CDs provides a driving force in the opposite direction of the positively charged baclofen in the running buffer and enantiomeric resolution by inclusion of compounds in the CD cavity. Highly S-beta-CD was found to be the most effective complexing agent, allowing good enantiomeric resolution. The complete resolution was obtained using 25 mM phosphate buffer, pH 2.5, containing 3% w/v highly S-beta-CD at 25 degrees C with aN applied field of 0.40 kV/cm. The apparent association constants of the inclusion complexes were calculated. This optimized method was validated in terms of repeatability and limits of detection (0.13 microg x mL(-1)) and quantification. The migration order was determined.

Baclofen↗

PubMed2003

Retention modeling and resolution optimization for a group of N-phenylpyrazole derivatives in micellar electrokinetic chromatography using empirical and physicochemical models.

The optimization of the separation resolution for a group of N-phenylpyrazole derivatives in micellar electrokinetic chromatography (MEKC) as a function of the separation buffer composition (surfactant and organic modifier concentration) has been performed. In order to achieve our purpose, the first step has been the prediction of the migration times of the electroosmotic flow (t(0)) and micelles (t(m)), and the retention factors of solutes (k), as a function of surfactant (sodium dodecyl sulfate) and alcohol (n-propanol or n-butanol) concentrations, by means of empirical equations. Also, some physicochemical models have been applied to relate the retention factors to the surfactant and the organic modifier concentrations in order to optimize the separation resolution and to increase our knowledge of the separation process. Finally, a comparison of the resolution optimization through the use of the physicochemical and empirical models selected has been made in order to obtain the optimum separation buffer composition for the separation of a group of 17 N-phenylpyrazole derivatives as test solutes.

Chromatography, Micellar Electrokinetic Capillary↗

PubMed2003

Enhancement of selectivity and resolution in the enantioseparation of uncharged compounds using mixtures of oppositely charged cyclodextrins in capillary electrophoresis.

The enantiomeric separation of some nonsteroidal anti-inflammatory drugs (NSAIDs) was investigated in capillary electrophoresis (CE) using dual systems with mixtures of charged cyclodextrin (CD) derivatives. A significant enhancement of selectivity and resolution could be achieved in the enantioseparation of these analytes in their uncharged form by the simultaneous addition of two oppositely charged CD derivatives to the background electrolyte. The combination of the single-isomer cationic CD, permethyl-6-monoamino-6-monodeoxy-beta-CD (PMMAbetaCD) and the single-isomer polyanionic CD, heptakis-6-sulfato-beta-cyclodextrin (HSbetaCD) in a pH 2.5 phosphoric acid-triethanolamine buffer, was designed and employed for the enantioseparation of profens. The improvement in selectivity and resolution can be attributed to the fact that the two CDs, which lead to independent and enantioselective complexation with the analyte enantiomers, have not only opposite effects on the electrophoretic mobility of these compounds but also opposite affinity patterns towards the enantiomers of these compounds. Binding constants for these enantiomers with each CD were determined using linear regression approach, in order to be able to predict the effect of the concentrations of the two CDs on enantiomeric selectivity and resolution in such dual systems.

Anti-Inflammatory Agents, Non-Steroidal↗

PubMed2004

Peak compression and resolution for electrophoretic separations in diverging microchannels.

We report the results of experiments and simulations on electrokinetic flow in diverging microchannels (with cross-sectional area that increases with distance along the channel). Because of conservation of mass and charge, the velocity of an analyte in the channel decreases as the channel cross-section increases. Consequently, the leading edge of a band of sample moves more slowly than the trailing edge and the sample band is compressed. Sample peak widths, rather than increasing diffusively with time, can then be controlled by the geometry of the channel and can even be made to decrease with time. We consider the possibility of using this peak compression effect to improve the resolution of electrophoretic separations. Our results indicate that for typical separations that are dispersion limited, this peak compression effect is more than offset by the decreased distance between peaks, and the separation resolution in diverging channels is worse than that found for straight channels at the same applied voltage. For separations in very short channels or at very high field strengths, however, when the separation efficiency is injection limited, the peak compression effect is dominant and diverging channels can then be used to achieve improved separation resolution.

Electrophoresis, Microchip↗

PubMed2005

Rapid on-chip postcolumn labeling and high-resolution separations of DNA.

When performing genetic analysis on microfluidic systems, labeling the sample DNA for detection is a critical preparation step. Labeling procedures often involve fluorescently tagged primers and PCRs, which lengthen experimental run times and introduce higher levels of complexity, increasing the overall cost per analysis. Alternatively, on-chip labeling techniques based on intercalating dyes permit rapid labeling of DNA fragments. However, as noted in the literature, the stochastic nature of dye-DNA complex formation hinders the native electrophoretic migration of DNA fragments, degrading the separation resolution. In this study, we present a novel method of controllably labeling DNA fragments at the end of the electrophoretic separation channel in a glass microfluidic chip. Permitting the DNA to separate and labeling just before detection, achieves the rapid labeling associated with intercalators while maintaining the high resolution of native DNA separations. Our analyses are completed in minutes, rather than the hours typical of sample prelabeling. We demonstrate an electrophoretic microchip-based intercalator labeling technique that achieves higher resolution performance than reported in the literature to date.

DNA↗

PubMed2003

Improvement in variability of the horizontal meridian of the primary visual area following high-resolution spatial normalization.

We investigated the decrease in intersubject functional variability in the horizontal meridian (HM) of the primary visual area (V1) before and after individual anatomical variability was significantly reduced using a high-resolution spatial normalization (HRSN) method. The analyzed dataset consisted of 10 normal, right-handed volunteers who had undergone both an O-15 PET study, which localized retinotopic visual area (V1), and a high-resolution anatomical MRI. Individual occipital lobes were manually segmented from anatomical images and transformed into a common space using an in-house high-resolution regional spatial normalization method called OSN. Individual anatomical and functional variability was quantified before and after HRSN processing. The reduction of individual anatomical variability was judged by the reduction in gray matter (GM) mismatch and by the improvement in overlap frequency between individual calcarine sulci. The reduction in intersubject functional variability of HM was determined by measurements of the overlap frequency between individual HM areas and by improvement in intersubject Z-score maps. The HRSN processing significantly reduced the individual anatomical variability: GM mismatch was reduced by a factor of two and the mean calcarine sulcus overlap frequency was improved from 37 to 68%. The reduction in functional variability was more subtle. However, both HM mean overlap (increased from 18 to 28%) and the average Z-score (increased from 2.2 to 2.55) were significantly improved. Although, functional registration was significantly improved by matching sulci, there was still residual variability. This is believed to be the variability of individual areas within the calcarine sulcus, and cannot be resolved by sulcal match. Thus, the proposed methodology provides an efficient, unbiased, and automated way to study structure-functional relationship in human brain.

Brain↗

PubMed2004

Protocol for volumetric segmentation of medial temporal structures using high-resolution 3-D magnetic resonance imaging.

Quantitative analysis of brain structures in normal subjects and in different neurological conditions can be carried out in vivo through magnetic resonance imaging (MRI) volumetric studies. The use of high-resolution MRI combined with image post-processing that allows simultaneous multiplanar view may facilitate volumetric segmentation of temporal lobe structures. We define a protocol for volumetric studies of medial temporal lobe structures using high-resolution MR images and we studied 30 healthy subjects (19 women; mean age, 33 years; age range, 21-55 years). Images underwent field non-homogeneity correction and linear stereotaxic transformation into a standard space. Structures of interest comprised temporopolar, entorhinal, perirhinal, parahippocampal cortices, hippocampus, and the amygdala. Segmentation was carried out with multiplanar assessment. There was no statistically significant left/right-sided asymmetry concerning any structure analyzed. Neither gender nor age influenced the volumes obtained. The coefficient of repeatability showed no significant difference of intra- and interobserver measurements. Imaging post-processing and simultaneous multiplanar view of high-resolution MRI facilitates volumetric assessment of the medial portion of the temporal lobe with strict adherence to anatomic landmarks. This protocol shows no significant inter- and intraobserver variations and thus is reliable for longitudinal studies.

Adult↗

PubMed2005

Visualization of thalamic nuclei on high resolution, multi-averaged T1 and T2 maps acquired at 1.5 T.

The ability to differentiate noninvasively between the primary nuclear divisions of the thalamus has immediate clinical applicability for surgical planning and guidance of functional stereotactic procedures. Comparison of prior qualitative magnetic resonance imaging (MRI) studies carried out at field strengths of 1.5 and 4 Tesla have revealed contrast within the thalamus that varies with field strength, suggesting possible differences in the inherent T1 and T2 relaxation times of the constituent nuclei. We investigate this hypothesis through acquisition of high-resolution, multi-averaged deep-brain T1 and T2 maps of a healthy volunteer. Fourteen nuclei were identified using their center-of-mass coordinates (in Talairach space) and average T1 and T2 values obtained from regions of interest placed within each. Results from this analysis revealed significant differences in T1 and T2 between the nuclei with a T1 range from 700 to 1,400 ms and a T2 range from 89 to 122 ms, allowing visual discrimination between the major nuclei groups. Furthermore, the high-resolution images showed distinct borders of T1 and T2 hypointensity surrounding each nucleus, revealing structure not reported previously. These results confirm our hypothesis and demonstrate the potential high-resolution quantitative imaging for nucleus visualization and surgical planning.

Brain Mapping↗

PubMed2006

Retrospective motion correction protocol for high-resolution anatomical MRI.

Modern computational brain morphology methods require that anatomical images be acquired at high resolution and with a high signal-to-noise ratio. This often translates into long acquisition times (>20 minutes) and images susceptible to head motion. In this study we tested retrospective motion correction (RMC), common for functional MRI (fMRI) and PET image motion correction, as a means to improve the quality of high-resolution 3-D anatomical MR images. RMC methods are known to be effective for correcting interscan motion; therefore, a single high-resolution 3-D MRI brain study was divided into six shorter acquisition segments to help shift intrascan motion into interscan motion. To help reduce intrascan head motion, each segment image was reviewed for motion artifacts and repeated if necessary. Interscan motion correction was done by spatially registering images to the third image and forming a single average motion-corrected image. RMC was tested on 35 subjects who were considered at high risk for head motion. Our results show that RMC provided better contrast-to-noise ratio and boundary detail when compared to nonmotion-corrected averaged images.

Adolescent↗

PubMed2007

Comparison of different cortical connectivity estimators for high-resolution EEG recordings.

The aim of this work is to characterize quantitatively the performance of a body of techniques in the frequency domain for the estimation of cortical connectivity from high-resolution EEG recordings in different operative conditions commonly encountered in practice. Connectivity pattern estimators investigated are the Directed Transfer Function (DTF), its modification known as direct DTF (dDTF) and the Partial Directed Coherence (PDC). Predefined patterns of cortical connectivity were simulated and then retrieved by the application of the DTF, dDTF, and PDC methods. Signal-to-noise ratio (SNR) and length (LENGTH) of EEG epochs were studied as factors affecting the reconstruction of the imposed connectivity patterns. Reconstruction quality and error rate in estimated connectivity patterns were evaluated by means of some indexes of quality for the reconstructed connectivity pattern. The error functions were statistically analyzed with analysis of variance (ANOVA). The whole methodology was then applied to high-resolution EEG data recorded during the well-known Stroop paradigm. Simulations indicated that all three methods correctly estimated the simulated connectivity patterns under reasonable conditions. However, performance of the methods differed somewhat as a function of SNR and LENGTH factors. The methods were generally equivalent when applied to the Stroop data. In general, the amount of available EEG affected the accuracy of connectivity pattern estimations. Analysis of 27 s of nonconsecutive recordings with an SNR of 3 or more ensured that the connectivity pattern could be accurately recovered with an error below 7% for the PDC and 5% for the DTF. In conclusion, functional connectivity patterns of cortical activity can be effectively estimated under general conditions met in most EEG recordings by combining high-resolution EEG techniques, linear inverse estimation of the cortical activity, and frequency domain multivariate methods such as PDC, DTF, and dDTF.

Analysis of Variance↗

PubMedDate not supplied

High resolution CT scan of temporal bone fractures: association of facial nerve paralysis with temporal bone fractures.

This radiologic study analyzed high resolution computed tomographic (CT) scans of 22 patients with temporal bone fractures. There were 19 males and three females. Fifteen of 22 had clinical evidence of facial nerve injury ranging from mild paresis to complete paralysis. The high resolution CT scan analysis identified a characteristic fracture of the temporal bone in every patient with facial nerve injury. A high percentage of these fractures (68%) could be classified as mixed and did not fall into a longitudinal or transverse fracture category. The characteristic fracture extends from the petrotympanic fissure at the glenoid fossa to the anterior inferior aspect of the medial bony external auditory canal. It resumes at the superior aspect of the external auditory canal (scutum) extending laterally along the external canal wall. If the vector force of the fracture is projected medially, it will cross the facial nerve in its horizontal portion. Often, the evaluation of trauma patients with routine CT scans for central nervous system (CNS) (brain) evaluation is inadequate for evaluation of temporal bone fractures. A high resolution CT scan should be performed when clinical criteria warrant its use. It is recognized that the incidence of facial nerve injury may be higher in this select population.

Adolescent↗

PubMedDate not supplied

Complete resolution of inflammatory activity following corticosteroid treatment of HBsAg-negative chronic active hepatitis.

To assess the frequency and significance of complete resolution of inflammatory activity following corticosteroid therapy, 115 patients with severe HBsAg-negative chronic active hepatitis were followed regularly for 84 +/- 5 months. Of 83 patients eligible to revert to normal liver tissue, 18 did so after 56 +/- 8 months. Five of the 18 relapsed after treatment withdrawal. Only patients who improved spontaneously after cessation of treatment from histologic features of chronic persistent hepatitis to normal invariably sustained the improvement. Of 32 patients with cirrhosis at presentation, 17 reverted to inactive cirrhosis after 48 +/- 10 months, but only 3 remained inactive after discontinuation of treatment. Mortality was similar in patients with and without reversion to normal tissue (0 vs. 14%, p greater than 0.2), but the frequency of relapse was less after complete resolution (28 vs. 76%, p less than 0.001). Reversion to inactive cirrhosis did not improve survival or reduce relapse frequency after remission and treatment withdrawal. Findings prior to therapy did not predict outcome. We conclude that complete resolution of inflammatory activity is possible, but that it occurs slowly, infrequently and unpredictably after therapy. In patients without cirrhosis, reversion to normal liver tissue decreases the likelihood of relapse and the requirement for retreatment. In patients with cirrhosis at presentation, elimination of inflammatory activity is rarely sustained and does not improve prognosis after remission and treatment withdrawal.

Adolescent↗

PubMed1996

Comparison of high-resolution endoluminal sonography to video endoscopy in the detection and evaluation of esophageal varices.

High-resolution endoluminal sonography (HRES) was used to image and measure esophageal varices in control subjects and patients with portal hypertension and compared with endoscopic findings. Nine control patients and 68 patients with known cirrhosis or noncirrhotic portal hypertension underwent videotaped HRES and videotaped esophagoscopy (EGD). Two blinded investigators reviewed the videotapes to determine the presence and size of the largest esophageal varix in each patient. The largest varix by HRES was measured with the esophagus at rest at a point where the varix appeared most circular. The largest varix seen on EGD was graded on a 5-point scale. All nine of the control patients were correctly identified by both EGD and HRES as grade I (no varices). Eight of the 10 patients with no varices seen on EGD had varices identified by HRES. The interobserver correlation for HRES was r = .88 and for EGD was r = .79. The correlation between EGD and HRES was r = .50. High resolution endoluminal sonography allows quantitative measurement of variceal size, is a more sensitive and reproducible imaging modality than esophagoscopy for the detection and sizing of esophageal varices. There is poor correlation between monographic measurement and endoscopic grading of esophageal varices. Assessment of esophageal variceal presence and the measurement of variceal size by high resolution endoluminal sonography is an accurate, reproducible method of determining the size of esophageal varices.

Adult↗

PubMed2005

BAC-based PCR fragment microarray: high-resolution detection of chromosomal deletion and duplication breakpoints.

The introduction of molecular techniques in conjunction with classical cytogenetic methods has in recent years greatly improved the diagnostic potential for chromosomal abnormalities. In particular, microarray-comparative genomic hybridization (CGH) based on the use of BAC clones promises a sensitive strategy for the detection of DNA copy-number changes on a genomewide scale, offering a resolution as high as >30,000 "bands" (as defined by the number of BACs within the currently highest-density BAC array) [Ishkanian et al., 2004]. We have tested the possibility of further increasing this resolution using PCR fragments generated from individual BAC clones. Using this approach, we have efficiently defined the proximal and distal breakpoints in two cytogenetic cases, one duplication and one deletion, to within 5-20 kb. The results support the potential use of BAC-based PCR fragments to further improve the resolution of the microarray-CGH strategy by an order of magnitude.

Chromosome Aberrations↗

PubMed2006

High resolution analysis of non-small cell lung cancer cell lines by whole genome tiling path array CGH.

Chromosomal regions harboring tumor suppressors and oncogenes are often deleted or amplified. Array comparative genomic hybridization detects segmental DNA copy number alterations in tumor DNA relative to a normal control. The recent development of a bacterial artificial chromosome array, which spans the human genome in a tiling path manner with >32,000 clones, has facilitated whole genome profiling at an unprecedented resolution. Using this technology, we comprehensively describe and compare the genomes of 28 commonly used non-small cell lung carcinoma (NSCLC) cell models, derived from 18 adenocarcinomas (AC), 9 squamous cell carcinomas and 1 large cell carcinoma. Analysis at such resolution not only provided a detailed genomic alteration template for each of these model cell lines, but revealed novel regions of frequent duplication and deletion. Significantly, a detailed analysis of chromosome 7 identified 6 distinct regions of alterations across this chromosome, implicating the presence of multiple novel oncogene loci on this chromosome. As well, a comparison between the squamous and AC cells revealed alterations common to both subtypes, such as the loss of 3p and gain of 5p, in addition to multiple hotspots more frequently associated with only 1 subtype. Interestingly, chromosome 3q, which is known to be amplified in both subtypes, showed 2 distinct regions of alteration, 1 frequently altered in squamous and 1 more frequently altered in AC. In summary, our data demonstrate the unique information generated by high resolution analysis of NSCLC genomes and uncover the presence of genetic alterations prevalent in the different NSCLC subtypes.

Carcinoma, Non-Small-Cell Lung↗

PubMed1985

Three-dimensional structure of the complex of actin and DNase I at 4.5 A resolution.

The shape of an actin subunit has been derived from an improved 6 A map of the complex of rabbit skeletal muscle actin and bovine pancreatic DNase I obtained by X-ray crystallographic methods. The three-dimensional structure of DNase I determined independently at 2.5 A resolution was compared with the DNase I electron density in the actin:DNase map. The two structures are very similar at 6 A resolution thus leading to an unambiguous identification of actin as well as DNase I electron density. Furthermore the correct hand of the actin structure is determined from the DNase I atomic structure. The resolution of the actin structure was extended to 4.5 A by using a single heavy-atom derivative and the knowledge of the atomic coordinates of DNase I. The dimensions of an actin subunit are 67 A X 40 A X 37 A. It consists of a small and a large domain, the small domain containing the N terminus. Actin is an alpha,beta-protein with a beta-pleated sheet in each domain. These sheets are surrounded by several alpha-helices, comprising at least 40% of the structure. The phosphate peak of the adenine nucleotide is located between the two domains. The complex of actin and DNase I as found in solution (i.e., the actin:DNase I contacts which do not depend on crystal packing) was deduced from a comparison of monoclinic with orthorhombic crystals. Residues 44-46, 51, 52, 60-62 of DNase I are close to a loop region in the small domain of actin. At a distance of approximately 15 A there is a second contact in the large domain in which Glu13 of DNase I is involved. A possible binding region for myosin is discussed.

Actins↗

PubMed1985

G.T wobble base-pairing in Z-DNA at 1.0 A atomic resolution: the crystal structure of d(CGCGTG).

The DNA oligomer d(CGCGTG) crystallizes as a Z-DNA double helix containing two guanine-thymine base pair mismatches of the wobble type. The crystal diffracts to 1 A resolution and the structure has been solved and refined. At this resolution, a large amount of information is revealed about the organization of the water molecules in the lattice generally and more specifically around the wobble base pairs. By comparing this structure with the analogous high resolution structure of d(CGCGCG) we can visualize the structural changes as well as the reorganization of the solvent molecules associated with wobble base pairing. There is only a small distortion of the Z-DNA backbone resulting from introduction of the GT mismatched base pairs. The water molecules cluster around the wobble base pair taking up all of the hydrogen bonding capabilities of the bases due to wobble pairing. These bridging water molecules serve to stabilize the base-base interaction and, thus, may be generally important for base mispairing either in DNA or in RNA molecules.

Base Sequence↗