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PubMed1982

Reproducibility of electron diffraction intensity data obtained from hydrated microcrystals of rat hemoglobin.

Analysis of electron diffraction patterns from rat hemoglobin taken at 200 kV on a wet stage yields intensity data to a resolution of 2-3 A which are as reproducible as those from typical X-ray diffraction. Some crystals were so similar that the differences in measured intensities were insignificant (R = 0.056), but in other cases real differences between crystals were observed (R = 0.33). Dynamic scattering was insignificant under our diffraction conditions; however, exposures to electron doses as low as 10(-2) e/A2 produced detectable changes in measured intensities. Limits to the reproducibility of the data are set by radiation damage and errors in microdensitometry.

Animals↗
PubMed2001

Cochlear implantation in healthy and otitis-prone children: a prospective study.

OBJECTIVE: To evaluate and compare the timing of surgery, intraoperative findings, and otitis media-related outcome of cochlear implantation in children who are otitis-prone with their counterparts who are not otitis-prone. STUDY DESIGN: Prospective. METHODS: Children referred for cochlear implantation were assigned to a non-otitis-prone group (group A: normal otoscopy on their first visit after referral) or an otitis-prone group (group B: current or a recent history of otitis media at referral). Group B patients were managed using a structured protocol aimed at preimplantation otitis media control. The study reviewed pre-, intra-, and postoperative data. RESULTS: Of the 18 children studied, 8 were assigned to group A (mean age at referral, 40.6 mo) and 10 to group B (mean age at referral, 31.6 mo). For otitis media control, all otitis-prone children underwent ventilating tube insertion (various numbers of procedures before implantation). Only one otitis-prone child required cortical mastoidectomy also. Time from referral to implantation was similar in the two groups (mean, 6.6 mo). High-resolution computed tomography data showed mastoid pneumatization to be significantly smaller in the otitis-prone group, but the facial recess was not smaller in this group. During implantation, 10 children had inflamed middle ear mucosa. Seven of these belonged to group B. All of these seven children had a round window niche obliterated by the inflamed mucosa, which had to be removed for round window membrane identification. After implantation, only one child had drainage through the ventilating tube for more than 1 week. Two children in group B developed otitis media (1 year postimplantation) that was overcome within 1 week. There were no otitis media-related complications. CONCLUSIONS: If a structured protocol is used for the control of otitis media before cochlear implantation, otitis media should not require a delay in implantation. In otitis media-prone children, the round window niche is often obscured by inflamed mucosa. Its removal is mandatory for identification of the round window membrane. After cochlear implantation, otitis media is not a frequent occurrence.

Acute Disease↗
PubMed2003

Chemometric approach for the resolution and quantification of unresolved peaks in gas chromatography--selected-ion mass spectrometry data.

A semiautomated and integrated chemometric approach is presented for the resolution and quantification of unresolved target-analyte signals in gas chromatography-selected-ion monitoring (GC-SIM) data collected using scanning mass spectrometers. The chemometric approach utilizes an unskewing algorithm and two multivariate chemometric methods known as rank alignment and the generalized rank annihilation method (GRAM). The unskewing algorithm corrects the retention-time differences within a single GC-SIM data matrix caused by using a scanning mass spectrometer. Rank alignment objectively corrects the run-to-run retention-time difference between a sample GC-SIM data matrix and a standard addition GC-SIM data matrix. GRAM analysis uses the sample and standard addition data matrices to mathematically resolve and quantify the target-analyte signal(s). The resolution and quantification of severely unresolved target-analyte signals are demonstrated using GC-SIM data obtained from conventional heart-cut two-dimensional gas chromatography with mass spectrometric detection. In addition, the GC-SIM data is used to demonstrate the result of chemometric analysis when the absence of a target-analyte signal is obscured by interference. Chemometric analysis is shown to unambiguously detect an analyte based on its resolved mass chromatograms in situations where the traditional approach of measuring peak height fails to positively detect it. The predicted analyte concentrations are within 8% of the reference concentrations.

Gas Chromatography-Mass Spectrometry↗
PubMed2005

Protein-ligand interaction probed by time-resolved crystallography.

Time-resolved (TR) crystallography is a unique method for determining the structures of intermediates in biomolecular reactions. The technique reached its mature stage with the development of the powerful third-generation synchrotron X-ray sources, and the advances in data processing and analysis of time-resolved Laue crystallographic data. A time resolution of 100 ps has been achieved and relatively small structural changes can be detected even from only partial reaction initiation. The remaining challenge facing the application of this technique to a broad range of biological systems is to find an efficient and rapid, system-specific method for the reaction initiation in the crystal. Other frontiers for the technique involve the continued improvement in time resolution and further advances in methods for determining intermediate structures and reaction mechanisms. The time-resolved technique, combined with trapping methods and computational approaches, holds the promise for a complete structure-based description of biomolecular reactions.

Bacterial Proteins↗
PubMed1990

Predictability of SNR and reader preference in clinical MR imaging.

Fifty-four independent scans were performed in two volunteers covering one anatomic region in each (the brain and knee) with the purpose of ascertaining the agreement between predicted and measured signal-to-noise ratios (SNR). Systematically varied parameters were number of excitations (NEX), field of view (FOV), section thickness (dz), and the number of phase-encoding steps (Ny). Correlation coefficients of measured versus predicted SNR were 0.82 and 0.86, respectively, in the anatomies studied. Significantly improved correlations were found for data subpopulations in which NEX was held constant. To assess the criteria guiding reader preference, a blinded study was performed in which radiologists were asked to rate images from least to most desirable. In order to quantitatively determine the criteria for reader preference, plots of mean rating versus SNR, voxel volume, and an image quality index [IQI = SNR/(voxel volume)] were performed. The latter was found to be a better predictor of reader preference than either SNR or spatial resolution alone. The data suggests T1-weighted scan protocols yielding SNR of approximately 20 are preferable with any excess SNR being traded for smaller voxel size or shorter scan times.

Brain↗
PubMed2000

Identification of uncommon plant metabolites based on calculation of elemental compositions using gas chromatography and quadrupole mass spectrometry.

Unknown compounds in polar fractions of Arabidopsis thaliana crude leaf extracts were identified on the basis of calculations of elemental compositions obtained from gas chromatography/low-resolution quadrupole mass spectrometric data. Plant metabolites were methoximated and silylated prior to analysis. All known peaks were used as internal references to construct polynomial recalibration curves of from raw mass spectrometric data. Mass accuracies of 0.005 +/- 0.003 amu and isotope ratio errors of 0.5 +/- 0.3% (A + 1/A), respectively, 0.3 +/- 0.2% (A + 2/A), could be achieved. Both masses and isotope ratios were combined when the elemental compositions of unknown peaks were calculated. After calculation, compound identities were elucidated by searching metabolic databases, interpreting spectra, and, finally, by comparison with reference compounds. Sum formulas of more than 70 peaks were determined throughout single GC/MS chromatograms. Exact masses were confirmed by high-resolution mass spectrometric data. More than 15 uncommon plant metabolites were identified, some of which are novel in Arabidopsis, such as tartronate semialdehyde, citramalic acid, allothreonine, or glycolic amide.

Amino Acids↗
PubMed2000

Crystal structure of yeast initiation factor 4A, a DEAD-box RNA helicase.

The eukaryotic translation initiation factor 4A (eIF4A) is a member of the DEA(D/H)-box RNA helicase family, a diverse group of proteins that couples an ATPase activity to RNA binding and unwinding. Previous work has provided the structure of the amino-terminal, ATP-binding domain of eIF4A. Extending those results, we have solved the structure of the carboxyl-terminal domain of eIF4A with data to 1.75 A resolution; it has a parallel alpha-beta topology that superimposes, with minor variations, on the structures and conserved motifs of the equivalent domain in other, distantly related helicases. Using data to 2.8 A resolution and molecular replacement with the refined model of the carboxyl-terminal domain, we have completed the structure of full-length eIF4A; it is a "dumbbell" structure consisting of two compact domains connected by an extended linker. By using the structures of other helicases as a template, compact structures can be modeled for eIF4A that suggest (i) helicase motif IV binds RNA; (ii) Arg-298, which is conserved in the DEA(D/H)-box RNA helicase family but is absent from many other helicases, also binds RNA; and (iii) motifs V and VI "link" the carboxyl-terminal domain to the amino-terminal domain through interactions with ATP and the DEA(D/H) motif, providing a mechanism for coupling ATP binding and hydrolysis with conformational changes that modulate RNA binding.

Amino Acid Sequence↗
PubMed2003

An algorithm for three-way data analysis that alternatively minimizes coupled vector (COV) resolution error and PARAFAC error.

A novel algorithm, alternatively minimizing coupled vector (COV) resolution error and PARAFAC error algorithm, is proposed in this paper. This algorithm can overcome the problem of slow convergence and is insensitive to the estimation of component number, such problems are unavoidable while using the traditional parallel factors analysis (PARAFAC) algorithm. In other words, this algorithm is capable of improving the computing speed and providing accurate resolutions provided that the number of factors used in the computation is no less than that of the actual underlying ones. The characteristic performances were demonstrated with a novel fluorescence data array.

Journal Article↗
PubMed1998

A model of the quaternary structure of the Escherichia coli F1 ATPase from X-ray solution scattering and evidence for structural changes in the delta subunit during ATP hydrolysis.

The shape and subunit arrangement of the Escherichia coli F1 ATPase (ECF1 ATPase) was investigated by synchrotron radiation x-ray solution scattering. The radius of gyration and the maximum dimension of the enzyme complex are 4.61 +/- 0.03 nm and 15.5 +/- 0.05 nm, respectively. The shape of the complex was determined ab initio from the scattering data at a resolution of 3 nm, which allowed unequivocal identification of the volume occupied by the alpha3beta3 subassembly and further positioning of the atomic models of the smaller subunits. The delta subunit was positioned near the bottom of the alpha3beta3 hexamer in a location consistent with a beta-delta disulfide formation in the mutant ECF1 ATPase, betaY331W:betaY381C:epsilonS108C, when MgADP is bound to the enzyme. The position and orientation of the epsilon subunit were found by interactively fitting the solution scattering data to maintain connection of the two-helix hairpin with the alpha3beta3 complex and binding of the beta-sandwich domain to the gamma subunit. Nucleotide-dependent changes of the delta subunit were investigated by stopped-flow fluorescence technique at 12 degrees C using N-[4-[7-(dimethylamino)-4-methyl]coumarin-3-yl]maleimide (CM) as a label. Fluorescence quenching monitored after addition of MgATP was rapid [k = 6.6 s-1] and then remained constant. Binding of MgADP and the noncleavable nucleotide analog AMP . PNP caused an initial fluorescent quenching followed by a slower decay back to the original level. This suggests that the delta subunit undergoes conformational changes and/or rearrangements in the ECF1 ATPase during ATP hydrolysis.

Adenosine Diphosphate↗
PubMed2005

A comparison of two commercial treatment-planning systems to IMRT.

This study compared the clinical functionality of BrainSCAN (BrainLAB) and Helios (Eclipse, Varian) for intensity-modulated radiation therapy (IMRT) treatment planning with the aim of identifying practical and technical issues. The study considered implementation and commissioning, dose optimization, and plan assessment. Both systems were commissioned for the same 6 MV photon beam equipped with a high-resolution multileaf collimator (Varian Millennium 120 leaf). The software was applied to three test plans having identical imaging and contour data. Analysis considered 3D axial dose distributions, dose-volume histograms, and monitor unit calculations. Each system requires somewhat different input data to characterize the beam prior to use, so the same data cannot be used for commissioning. In addition, whereas measured beam data was entered directly into Helios with minimal data processing, the BrainSCAN system required configured beam data to be sent to BrainLAB before clinical use. One key difference with respect to system commissioning was that BrainSCAN required high resolution data, which necessitated the use of detectors with small active volumes. This difference was found to impact on the ability of the systems to accurately calculate dose for highly modulated fields, with BrainSCAN being more successful than Helios. In terms of functionality, the BrainSCAN system uses a dynamically penalized likelihood inverse planning algorithm and calculates four plans at once with various relative weighting of the planning target and organ-at-risk volumes. Helios uses a gradient algorithm that allows the user to make changes to some of the input parameters during optimization. An analysis of the dosimetry output shows that, although the systems are different in many respects, they are each capable of producing substantially equivalent dose plans in terms of target coverage and normal tissue sparing.

Algorithms↗
PubMed1998

Submicrometre resolution phase-contrast radiography with the beam from an X-ray waveguide.

Experimental data with unprecedented submicrometre resolution obtained in a phase-contrast radiography experiment in a magnifying configuration are presented. The term 'phase contrast' here indicates that the phase retardation of coherent light in matter was utilized as the contrast mechanism. The coherent and divergent beam exiting an X-ray waveguide was used in a lensless configuration to magnify spatial variations in optical path length up to several hundred times. The defocused image of a nylon fibre was measured with a resolution of 0.14 micro m at the object. Sufficient contrast was found for exposure times of 0.1 s, i.e. in the regime for real-time studies.

Journal Article↗
PubMed1996

Molecular evolution of ependymin and the phylogenetic resolution of early divergences among euteleost fishes.

The rate and pattern of DNA evolution of ependymin, a single-copy gene coding for a highly expressed glycoprotein in the brain matrix of teleost fishes, is characterized and its phylogenetic utility for fish systematics is assessed. DNA sequences were determined from catfish, electric fish, and characiforms and compared with published ependymin sequences from cyprinids, salmon, pike, and herring. Among these groups, ependymin amino acid sequences were highly divergent (up to 60% sequence difference), but had surprisingly similar hydropathy profiles and invariant glycosylation sites, suggesting that functional properties of the proteins are conserved. Comparison of base composition at third codon positions and introns revealed AT-rich introns and GC-rich third codon positions, suggesting that the biased codon usage observed might not be due to mutational bias. Phylogenetic information content of third codon positions was surprisingly high and sufficient to recover the most basal nodes of the tree, in spite of the observation that pairwise distances (at third codon positions) were well above the presumed saturation level. This finding can be explained by the high proportion of phylogenetically informative nonsynonymous changes at third codon positions among these highly divergent proteins. Ependymin DNA sequences have established the first molecular evidence for the monophyly of a group containing salmonids and esociforms. In addition, ependymin suggests a sister group relationship of electric fish (Gymnotiformes) and Characiformes, constituting a significant departure from currently accepted classifications. However, relationships among characiform lineages were not completely resolved by ependymin sequences in spite of seemingly appropriate levels of variation among taxa and considerably low levels of homoplasy in the data (consistency index = 0.7). If the diversification of Characiformes took place in an "explosive" manner, over a relatively short period of time this pattern should also be observed using other phylogenetic markers. Poor conservation of ependymin's primary structure hinders the design of efficient primers for PCR that could be used in wide-ranging fish systematic studies. However, alternative methods like PCR amplification from cDNA used here should provide promising comparative sequence data for the resolution of phylogenetic relationships among other basal lineages of teleost fishes.

Amino Acid Sequence↗
PubMed1998

Manipulation and molecular resolution of a phosphatidylcholine-supported planar bilayer by atomic force microscopy.

The morphology of supported planar bilayers has been investigated below phase transition temperature by atomic force microscopy in contact and tapping mode. The bilayers were formed by the vesicle-spreading technique. In contact mode at low scanning forces of about 1 nN true molecular resolution could be achieved for supported phosphatidylcholine bilayers. The resolution was confirmed by experiments that captured the location, average area of individual lipid headgroups and the manipulation of the bilayer surface. Repeated scanning in contact mode shifted the random topology of the surface consecutively to a striped pattern. Height profiles of defect-containing bilayers were analyzed. The shape of the defects became smooth by repeated scanning. The height profiles allowed the estimation of the indentation of the tip into the surface-adsorbed membrane. In tapping mode a disordered pattern of headgroups became visible. Our morphological data at molecular resolution suggest that the native arrangement of the choline head-groups is disordered, free of large packing defects and becomes ordered in Schallamach waves by scanning in contact mode.

Lipid Bilayers↗
PubMed1991

Isolation, crystallization, crystal structure analysis and refinement of constitutive C-phycocyanin from the chromatically adapting cyanobacterium Fremyella diplosiphon at 1.66 A resolution.

Constitutive phycocyanin from cyanobacterium Fremyella diplosiphon (Calothrix sp. PCC 7601) grown in green light, has been isolated and crystallized. The crystals belong to the space group R3 with cell constants a = b = 180.26 A, c = 61.24 A, alpha = beta = 90 degrees, gamma = 120 degrees. The crystal structure has been determined by Patterson search techniques using the molecular model of C-phycocyanin from the cyanobacterium Agmenellum quadruplicatum. The asymmetric unit of the crystal cell consists of two (alpha beta)-monomers related by a local dyad. Three asymmetric units are arranged around a crystallographic triad and form an (alpha beta)6-hexamer, the functional unit in the native antenna rod. The initial structure has been refined in a cyclic manner by energy-restrained crystallographic refinement and modelling until the conventional crystallographic R-factor converged at 18.1% with data to a resolution of 1.66 A. The molecular structure resembles closely the C-phycocyanins of Mastigocladus laminosus and A. quadruplicatum. The conformation and configuration of the alpha-84 and beta-84 chromophores is very similar to the corresponding chromophores in the trimeric C-phycocyanin of M. laminosus, whereas the beta-155 chromophore differs in configuration with C(4)-Z, C(10)-Z and C(15)-Z compared to C(4)-Z, C(10)-Z, C(15)-Z,E. The stereochemistry of the beta-155 chiral centres is C(2)-RC(3)-R and C(31)-S, respectively, whereas alpha-84 and beta-84 have C(2)-RC(3)-R and C(31)-R. The amino acid sequences of constitutive and inducible phycocyanin differ mainly in residues located on the surface of the beta-subunits that mediate the inter-hexameric contacts.

Amino Acid Sequence↗
PubMed2005

Analytical evidence of amorphous microdomains within nitridosilicate and nitridoaluminosilicate single crystals.

Single crystals of new nitridosilicates and nitridoaluminosilicates with excellent R values in X-ray investigations were analysed quantitatively using 30 to 60 microm single-spot LA-ICP-MS. Significant discrepancies between expected and measured chemical composition could not be explained by the crystallographic data. High spatial resolution analysis using electron probe microanalysis (EPMA, 10 microm) leads to the discovery of inhomogeneities in the crystalline material. The application of standard single-spot LA-ICP-MS with a spatial resolution of 30 to 60 microm is not suitable for the analysis of these crystals as the existing inhomogeneities dominate and alter the determined concentrations. However, owing to the better detection capabilities, a scanning LA-ICP-MS procedure enables a more representative analysis of single crystals of Ca(5)Si(2)Al(2)N(8) than single-spot LA-ICP-MS as a result of a larger sampling volume. It is highly likely that these impurities consist of amorphous, vitreous phases as powder diffraction X-ray data indicates the existence of a significant fraction of an X-ray amorphous material besides crystalline silicates. These microdomains contain less aluminium, silicon and calcium or are nearly free of aluminium, which explains the detected discrepancies in the chemical composition.

Journal Article↗
PubMed2006

Factors affecting flow karyotype resolution.

BACKGROUND: One of the major factors which influences the chromosome purity achievable particularly during high speed sorting is the analytical resolution of individual chromosome peaks in the flow karyotype, as well as the amount of debris and fragmented chromosomes. We have investigated the factors involved in the preparation of chromosome suspensions that influence karyotype resolution. METHODS: Chromosomes were isolated from various human and animal cell types using a series of polyamine buffer isolation protocols modified with respect to pH, salt concentration, and chromosome staining time. Each preparation was analyzed on a MoFlo sorter (DAKO) configured for high speed sorting and the resolution of the flow karyotypes compared. RESULTS: High resolution flow cytometric data was obtained with chromosomes optimally isolated using hypotonic solution buffered at pH 8.0 and polyamine isolation buffer (with NaCl excluded) between pH 7.50 and 8.0. Extending staining time to more than 8 h with chromosome suspensions isolated from cell lines subjected to sufficient metaphase arrest times gave the best result with the lowest percentage of debris generated, tighter chromosome peaks with overall lower coefficients of variation, and a 1- to 5-fold increase in the yield of isolated chromosomes. CONCLUSIONS: Optimization of buffer pH and the length of staining improved karyotype resolution particularly for larger chromosomes and reduced the presence of chromosome fragments (debris). However, the most interesting and surprising finding was that the exclusion of NaCl in PAB buffer improved the yield and resolution of larger chromosomes.

Animals↗
PubMed1990

Higher plant origins and the phylogeny of green algae.

5S rRNA sequences from six additional green algae lend strong molecular support for the major outlines of higher plant and green algae phylogeny that have been proposed under varying naming conventions by several authors. In particular, the molecular evidence now available unequivocally supports the existence of at least two well-separated divisions of the Chlorobionta: the Chlorophyta and the Streptophyta (i.e., charophytes) (according to the nomenclature of Bremer). The chlamydomonad 5S rRNAs are, however, sufficiently distinct from both clusters that it may ultimately prove preferable to establish a third taxon for them. In support of these conclusions 5S rRNA sequence data now exist for members of four diverse classes of chlorophytes. These sequences all exhibit considerably more phylogenetic affinity to one another than any of them show toward members of the other cluster, the Streptophyta, or the two Chlamydomonas strains. Among the Charophyceae, new 5S rRNA sequences are provided herein for three genera, Spirogyra, Klebsormidium, and Coleochaete. All of these sequences and the previously published Nitella sequence show greater resemblance among themselves and to the higher plants than they do to any of the other green algae examined to date. These results demonstrate that an appropriately named taxon that includes these green algae and the higher plants is strongly justified. The 5S rRNA data lack the resolution needed, however, to unequivocally determine which of several subdivisions of the charophytes is the sister group of the land plants. The evolutionary diversity of Chlamydomonas relative to the other green algae was recognized in earlier 5S rRNA studies but was unanticipated by ultrastructural work.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed2007

Accuracy of high-resolution peripheral quantitative computed tomography for measurement of bone quality.

The introduction of three-dimensional high-resolution peripheral in vivo quantitative computed tomography (HR-pQCT) (XtremeCT, Scanco Medical, Switzerland; voxel size 82 microm) provides a new approach to monitor micro-architectural bone changes longitudinally. The accuracy of HR-pQCT for three important determinants of bone quality, including bone mineral density (BMD), architectural measurements and bone mechanics, was determined through a comparison with micro-computed tomography (microCT) and dual energy X-ray absorptiometry (DXA). Forty measurements from 10 cadaver radii with low bone mass were scanned using the three modalities, and image registration was used for 3D data to ensure identical regions were analyzed. The areal BMD of DXA correlated well with volumetric BMD by HR-pQCT despite differences in dimensionality (R(2) = 0.69), and the correlation improved when non-dimensional bone mineral content was assessed (R(2) = 0.80). Morphological parameters measured by HR-pQCT in a standard patient analysis, including bone volume ratio, trabecular number, derived trabecular thickness, derived trabecular separation, and cortical thickness correlated well with muCT measures (R(2) = 0.59-0.96). Additionally, some non-metric parameters such as connectivity density (R(2) = 0.90) performed well. The mechanical stiffness assessed by finite element analysis of HR-pQCT images was generally higher than for microCT data due to resolution differences, and correlated well at the continuum level (R(2) = 0.73). The validation here of HR-pQCT against gold-standards microCT and DXA provides insight into the accuracy of the system, and suggests that in addition to the standard patient protocol, additional indices of bone quality including connectivity density and mechanical stiffness may be appropriate to include as part of a standard patient analysis for clinical monitoring of bone quality.

Absorptiometry, Photon↗