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Results for “spatial sequencing”

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PubMed1993

Purification and sequence determination of guanylate kinase from pig brain.

A purification procedure for guanylate kinase from pig brain has been developed consisting of ammonium sulfate precipitation and heptane extraction of the crude extract, hydrophobic-interaction chromatography, affinity chromatography and chromatofocussing. From 1.75 kg pig brain, 1.2 mg enzyme was isolated with a yield of 18% and a purity of about 90%. For sequence determination, the protein was cleaved with trypsin, cyanogen bromide and endoproteinase Glu-C. Some of the isolated peptides were subcleaved with chymotrypsin, thermolysin or trifluoroacetic acid. The blocked N-terminus was analyzed by mass spectrometry and by amino acid analysis of a tryptic peptide, while the C-terminus was found in a tryptic and a chymotryptic peptide and confirmed by a carboxypeptidase Y digestion. The sequence contains 197 amino acids with a M(r) of 21,831, one tryptophan and one cysteine residue. It has been compared to those of the homologous enzymes of yeast and Escherichia coli, as well as to proteins from sequence data banks that show similarities. The sequence is discussed in the light of the known spatial structure of yeast guanylate kinase.

Amino Acid Sequence↗

PubMed1995

Analysis of DNA diversity by spatial autocorrelation.

Two statistics are proposed for summarizing spatial patterns of DNA diversity. These autocorrelation indices for DNA analysis, or AIDAs, can be applied to RFLP and sequence data; the resulting set of autocorrelation coefficients, or correlogram, measures whether, and to what extent, individual DNA sequences or haplotypes resemble the haplotypes sampled at arbitrarily chosen spatial distances. Analyses of computer-generated sets of data, and of RFLP data from two natural populations, show that AIDAs allow one to objectively and simply identify basic patterns in the spatial distribution of haplotypes. These statistics, therefore, seem to be a useful tool both to explore the genetic structure of a population and to suggest hypotheses on the evolutionary processes that shaped the observed patterns.

DNA↗

PubMed2003

Discriminant snakes for 3D reconstruction of anatomical organs.

In this work a new statistic deformable model for 3D segmentation of anatomical organs in medical images is proposed. A statistic discriminant snake performs a supervised learning of the object boundary in an image slice to segment the next slice of the image sequence. Each part of the object boundary is projected in a feature space generated by a bank of Gaussian filters. Then, clusters corresponding to different boundary pieces are constructed by means of linear discriminant analysis. Finally, a parametric classifier is generated from each contour in the image slice and embodied into the snake energy-minimization process to guide the snake deformation in the next image slice. The discriminant snake selects and classifies image features by the parametric classifier and deforms to minimize the dissimilarity between the learned and found image features. The new approach is of particular interest for segmenting 3D images with anisotropic spatial resolution, and for tracking temporal image sequences. In particular, several anatomical organs from different imaging modalities are segmented and the results compared to expert tracings.

Algorithms↗

PubMed2006

Phylogenetic mapping of recombination hotspots in human immunodeficiency virus via spatially smoothed change-point processes.

We present a Bayesian framework for inferring spatial preferences of recombination from multiple putative recombinant nucleotide sequences. Phylogenetic recombination detection has been an active area of research for the last 15 years. However, only recently attempts to summarize information from several instances of recombination have been made. We propose a hierarchical model that allows for simultaneous inference of recombination breakpoint locations and spatial variation in recombination frequency. The dual multiple change-point model for phylogenetic recombination detection resides at the lowest level of our hierarchy under the umbrella of a common prior on breakpoint locations. The hierarchical prior allows for information about spatial preferences of recombination to be shared among individual data sets. To overcome the sparseness of breakpoint data, dictated by the modest number of available recombinant sequences, we a priori impose a biologically relevant correlation structure on recombination location log odds via a Gaussian Markov random field hyperprior. To examine the capabilities of our model to recover spatial variation in recombination frequency, we simulate recombination from a predefined distribution of breakpoint locations. We then proceed with the analysis of 42 human immunodeficiency virus (HIV) intersubtype gag recombinants and identify a putative recombination hotspot.

Animals↗

PubMed2001

Contrast-enhanced thoracic 3D-MR angiography in infants and children.

PURPOSE: To optimise breath-hold contrast-enhanced MR angiography (MRA) in infants and children with suspected congenital heart or thoracic vessel malformation. MATERIAL AND METHODS: Thirty-nine children (median age 1 year) were examined, using five different ultrafast MRA sequences with a TR between 3.2 and 5.0 ms and the contrast agent meglumine gadoterate. A test injection was used to determine contrast travel time. Different parameters for contrast injection were evaluated. Signal-to-noise ratio (SNR) measurements were performed and image quality and injection timing were evaluated. RESULTS: MRA was successful in all patients and image quality was considered very good in 52%. Adequate SNR was achieved with no significant differences between the MR sequences. SNR decreased only 25-30% between subsequent scans. The mean contrast dose was 0.23 mmol/kg. The mean scan time was 12.5 +/- 3.8 s; the shorter scan times made dynamic examinations possible with high temporal resolution. Highest spatial resolution was obtained with TR 4.6/5.0 sequences. CONCLUSION: A contrast dose of 0.2 mmol/kg b.w. is recommended with an injection rate of 0.5 to 1.2 ml/s, depending on patient size and scan time. The scan delay time should equal the contrast travel time for optimal vessel enhancement. In the future, contrast-enhanced MRA may be a potential alternative to angiocardiography in infants and children.

Adolescent↗

PubMed2007

3D radial projection technique with ultrashort echo times for sodium MRI: clinical applications in human brain and skeletal muscle.

(23)Na MRI has the potential to noninvasively detect sodium (Na) content changes in vivo. The goal of this study was to implement (23)Na MRI in a clinical setting for neurooncological and muscular imaging. Due to the biexponential T(2) decay of the tissue Na signal with a short component, which ranges between 0.5-8 ms, the measurement of total Na content requires imaging techniques with echo times (TEs) below 0.5 ms. A 3D radial pulse sequence with a TE of 0.2 ms at a spatial resolution of 4 x 4 x 4 mm(3) was developed that allows the acquisition and presentation of Na images on the scanner. This sequence was evaluated in patients with low- and high-grade gliomas, and higher (23)Na MR signals corresponding to an increased Na content were found in the tumor regions. The contrast-to-noise ratio (CNR) between tumor and white matter increased from 0.8 +/- 0.2 to 1.3 +/- 0.3 with tumor grade. In patients with an identified muscular (23)Na channelopathy (Paramyotonia congenita (PC)), induced muscle weakness led to a signal increase of approximately 18% in the (23)Na MR images, which was attributed to intracellular Na(+) accumulation in this region.

Adult↗

PubMed1995

Demonstration by pulsed neutron scattering that the arrangement of the Fab and Fc fragments in the overall structures of bovine IgG1 and IgG2 in solution is similar.

The bovine IgG1 and IgG2 isotypes exhibit large differences in effector functions. To examine the structural basis for this, the 12-domain structures of IgG1 and IgG2 were investigated by pulsed neutron scattering using a recently developed camera LOQ. This method reports on the average relative disposition in solution of the Fab and Fc fragments in IgG. The radii of gyration (RG) were found to be similar at 5.64 and 5.71 nm for IgG1 and IgG2 respectively in 100% 2H2O buffers. The two cross-sectional radii of gyration (RXS) were also similar at 2.38-2.41 and 0.98-1.02 nm. Similar values were obtained for porcine IgG. Both bovine IgG1 and IgG2 possess similar overall solution structures, despite sequence differences at the hinge region at the centre of their structures. An automated computer survey of possible IgG structures was developed, in which coordinates for the two Fab fragments were displaced in a two-dimensional plane relative to those of the Fc fragment in 0.25 nm steps. The scattering curves calculated from these structures were found to be sensitive to relative displacements of the three fragments, but not on their rotational orientation about their longest axes. Good agreement with the solution scattering data was obtained with a planar IgG model in which the C-terminus of the CH1 domain of Fab was 3.6 nm from the N-terminus of Fc in both IgG1 and IgG2, with a precision of 0.7 nm. Energy refinement showed that this spatial separation is compatible with the hinge sequences of bovine IgG1 and IgG2. The results show that multidomain protein structures can be modelled using LOQ data, and that a long hinge sequence does not necessarily reflect a large distance between Fab and Fc. The steric accessibility of Fc sites for interactions with cell-surface Fc receptors and C1q of complement is shown to be generally similar for IgG1 and IgG2, and the difference in effector function between IgG1 and IgG2 is probably based on deletions in the IgG2 hinge sequence.

Amino Acid Sequence↗

PubMed2004

Angiotensin II inhibitory peptide found in the receptor sequence using peptide array.

Peptide array consisting of hundreds of peptides spatially addressed and synthesized on a cellulose membrane support was used to screen ligand-inhibitory peptides. As a model, angiotensin II (Ang II), a significant peptide related to the treatment of cardiovascular diseases, was chosen as the target ligand. Peptide arrays covering the Ang II receptor type 1 sequence were prepared, and peptide domains with high affinity to the Ang II fluorescein conjugate were investigated. The peptide (VVIVIY) within the first transmembrane region exhibited the highest affinity to Ang II. The synthesized soluble VVIVIY peptide had an 84% inhibitory effect on Ang II-induced aorta contraction. These results indicate that our screening strategy utilizing peptide array is an effective approach for the peptide drug development.

Amino Acid Sequence↗

PubMed1994

Vaccination of sheep against Fasciola hepatica with glutathione S-transferase. Identification and mapping of antibody epitopes on a three-dimensional model of the antigen.

The glutathione S-transferases (FhGST) of the liver fluke Fasciola hepatica have been identified as novel vaccine candidates that protect sheep against a fluke infection. With the use of overlapping peptides covering the predicted amino acid sequences of four FhGST cDNAs, we have defined the linear epitopes recognized by polyclonal antibody from sheep vaccinated with FhGST. Dominant and minor epitopes were found to be present on all four of the sequences although some epitopes were shown to be specific to particular FhGST. A high percentage of the FhGST peptides were found to be antigenic although considerable variability in response to the peptides was observed among the animals. This analysis was extended to the IgG1 and IgG2 response at the peptide level. Based on the recently solved crystal structure of the rat mu-class GST 3-3, a three-dimensional model of one of the FhGST sequences was generated that allowed the predicted spatial localization of defined epitopes. Most epitopes were localized on regions of high flexibility and accessibility. A comparison of epitopes on FhGST with the B cell epitopes on Sm28, a 28-kDa GST from Schistosoma mansoni, has found few similarities. There was no correlation between an antibody response to linear peptide epitopes and the level of protection induced in sheep by vaccination with FhGST.

Amino Acid Sequence↗

PubMedDate not supplied

[Analysis of exciton parameters in DNA. Exciton waves in DNA as one of the reasons of mutagenesis].

Formulae were obtained for the quantitative analysis of the following parameters of excitons in DNA: 1) the lifetime of electronic excitation; 2) the numbers of exciton runs along the DNA sequence; 3) the energy loss by an exciton for one run; 4) the maximum length of the DNA sequence capable of deactivating an exciton for one run. The maximum and minimum ranges for the constant of electronic excitation migration was determined to meet the requirement of inductive-resonance energy transfer for the case of strong interaction. The constant of exciton energy migration was shown to depend on the activation energy, which is equal to the energy of absorbed quantum. An analytical formula was derived to determine the number of quanta the DNA molecule is able to absorb, depending on its length, without nonlinear effects (without overlapping of spatial areas of electronic excitation). By this formula, DNA sequences consisting of only identical AT and GC nucleotide pairs and aggregate AT + GC (in the ratio 1:1) DNA sequences ranging from 1 up to 10(10) base pairs were analyzed. The results of the analysis suggest that the overlapping of spatial areas of electronic excitation induced by a single ultraviolet quantum occurs in short DNA sequences characteristic of prokaryotes. To achieve the same effects on long DNA sequences specific for eukaryotes, DNA must synchronously absorb a great number of ultraviolet quanta. Based on the above results, the following conclusions were made: 1) disturbances in the normal activity of DNA and RNA polymerases may be due to electromagnetic field, which is caused by the oscillatory relaxation of vibronic levels of nucleotides. The energy enters the vibronic levels of nucleotides from an exciton running along the DNA sequence; 2) the increase in the noncoding DNA sequences in eukaryotes due to evolution is a way of DNA protection from undesirable mutations; 3) prokaryotes must possess a greater potentiality and a higher rate of mutagenesis in comparison with eukaryotes, which is proved by their greater diversity in nature.

Algorithms↗

PubMed2003

A neural model of how the brain represents and compares multi-digit numbers: spatial and categorical processes.

Both animals and humans represent and compare numerical quantities, but only humans have evolved multi-digit place-value number systems. This article develops a Spatial Number Network, or SpaN, model to explain how these shared numerical capabilities are computed using a spatial representation of number quantities in the Where cortical processing stream, notably the inferior parietal cortex. Multi-digit numerical representations that obey a place-value principle are proposed to arise through learned interactions between categorical language representations in the What cortical processing stream and the Where spatial representation. Learned semantic categories that symbolize separate digits, as well as place markers like 'ty,' 'hundred,' and 'thousand,' are associated through learning with the corresponding spatial locations of the Where representation. Such What-to-Where auditory-to-visual learning generates place-value numbers as an emergent property, and may be compared with other examples of multi-modal cross-modality learning, including synesthesia. The model quantitatively simulates error rates in quantification and numerical comparison tasks, and reaction times for number priming and numerical assessment and comparison tasks. In the Where cortical process, transient responses to inputs are integrated before they activate an ordered spatial map that selectively responds to the number of events in a sequence and exhibits Weber law properties. Numerical comparison arises from activity pattern changes across the spatial map that define a 'directional comparison wave.' Variants of these model mechanisms have elsewhere been used to explain data about other Where stream phenomena, such as motion perception, spatial attention, and target tracking. The model is compared with other models of numerical representation.

Animals↗

PubMed2006

Four-dimensional ultrafast electron microscopy of phase transitions.

Reported here is direct imaging (and diffraction) by using 4D ultrafast electron microscopy (UEM) with combined spatial and temporal resolutions. In the first phase of UEM, it was possible to obtain snapshot images by using timed, single-electron packets; each packet is free of space-charge effects. Here, we demonstrate the ability to obtain sequences of snapshots ("movies") with atomic-scale spatial resolution and ultrashort temporal resolution. Specifically, it is shown that ultrafast metal-insulator phase transitions can be studied with these achieved spatial and temporal resolutions. The diffraction (atomic scale) and images (nanometer scale) we obtained manifest the structural phase transition with its characteristic hysteresis, and the time scale involved (100 fs) is now studied by directly monitoring coordinates of the atoms themselves.

Crystallization↗

PubMed2006

Micro-RNA-like effects of complete intronic sequences.

MicroRNAs (miRNAs) have been suggested as suppressors of numerous target genes in human cells. In this report, we present gene chip array data indicating that in the absence of miRNA sequences, complete human introns are similarly capable of coordinating expression of large numbers of gene products at spatially diverse sites within the genome. The expression of selected intronic sequences (6a, 14b and 23) derived from the cystic fibrosis transmembrane conductance regulator (CFTR) gene caused extensive and specific transcriptional changes in epithelial cells (HeLa) that do not normally express this gene product. Each intron initiated a distinctive pattern of gene transcription. Affected genes such as FOXF1, sucrase-isomaltase, collagen, interferon, complement and thrombospondin 1 have previously been linked to CFTR function or are known to contribute to the related processes of epithelial differentiation and repair. A possible regulatory function of this nature has not been demonstrated previously for non-coding sequences within eukaryotic DNA. The results are consistent with the observation that splicesomal introns are found only in eukaryotic organisms and that the number of introns increases with phylogenetic complexity.

Cell Differentiation↗

PubMed1994

Genetic and biochemical analysis of IHF/HU hybrid proteins.

Integration host factor (IHF) is a small heterodimeric DNA-binding protein of E coli composed of two subunits, alpha and beta, encoded by the himA and hip genes, respectively. IHF binds to DNA at a consensus sequence and bends DNA. HU protein, encoded by the hupA and hupB genes, is similar to IHF except that it does not bind to a specific DNA sequence. To investigate the protein determinants for IHF specificity we exchanged progressively longer segments from the C-terminus of Hip with those of HupA, and followed the activity in vivo and in vitro of four such IHF/HU hybrids. Replacement of 11 residues from the C-terminal alpha helix of Hip by the complementary eight residues of HupA (hybrid 1), had only minor effects on the DNA binding activity of the protein. As progressively longer segments of Hip were replaced by HupA, a precipitous decrease in IHF activity was observed. The hybrid with the longest substitution, hybrid 4, was totally inactive in vivo and could not be purified. None of the hybrid proteins could complement HU activity. Comparing the activities of hybrid 1, hybrid 2 and IHF point mutants, led us to conclude that the structural integrity of the C-terminal alpha helix and its spatial position, but not its amino acid sequence, are important for DNA binding specificity. We favor the hypothesis that alpha helices 3 of both IHF subunits interact with the body of IHF so as to anchor the arms. This interaction stabilizes the arms to permit DNA binding specificity. Thus the C-termini of IHF influence, in an indirect way, the recognition of specific sites on DNA.

Amino Acid Sequence↗

PubMed1995

A velocity k-space analysis of flow effects in echo-planar and spiral imaging.

A velocity k-space formalism facilitates the analysis of flow effects for imaging sequences involving time-varying gradients such as echo-planar and spiral. For each sequence, the velocity k-space trajectory can be represented by kv(kr); that is, its velocity-frequency (kv) position as a function of spatial-frequency (kr) position. In an echo-planar sequence, kv is discontinuous and asymmetric. However, in a spiral sequence, kv is smoothly varying, circularly symmetric, and small near the kr origin. To compare the effects of these trajectory differences, simulated images were generated by computing the k-space values for an in-plane vessel with parabolic flow. Whereas the resulting echo-planar images demonstrate distortions and ghosting that depend on the vessel orientation, the spiral images exhibit minimal artifacts.

Artifacts↗

PubMed1990

Solution structure of phage lambda half-operator DNA by use of NMR, restrained molecular dynamics, and NOE-based refinement.

The structure of a DNA decamer comprising the left half of the OR3 operator from bacteriophage lambda is determined in solution by using nuclear magnetic resonance spectroscopy and restrained molecular mechanics calculations. Nuclear magnetic resonance assignments for nonexchangeable protons are obtained by two-dimensional correlated and nuclear Overhauser effect (NOE) spectroscopies. Exchangeable proton resonances are assigned by one-dimensional NOE experiments. Coupling constant measurements from one- and two-dimensional experiments are used to determine approximate dihedral angles within the deoxyribose ring. Distances between protons are estimated by extrapolating distances derived from the time-dependent NOE intensities to initial mixing times. The sets of dihedral angles and distances form a basis for structure determination by restrained molecular dynamics. Separate runs start from classical A and from B DNA and converge to essentially identical structures (atomic root mean square difference of 0.8 A). The structures are improved by NOE-based refinement in which observed NOE intensities are compared to those calculated by using a full matrix analysis procedure. Final NOE residual (R) factors were less than 0.19. The resultant structures are generally B type in character, but display local sequence-dependent variations in dihedral angles and in the spatial arrangement of adjacent base pairs. Although the entire structure exhibits a small bend, the central core of the half-operator, which comprises the sequence-specific recognition site for cro repressor, is straight.

Bacteriophage lambda↗

PubMed1987

Separate regulatory elements are responsible for the complex pattern of tissue-specific and developmental transcription of the yellow locus in Drosophila melanogaster.

DNA sequences involved in the control of the developmental and spatial expression of the yellow locus of Drosophila were identified by phenotypic analysis of germline transformants carrying various in vitro modified yellow genes. Two regions, located between -2873 bp and -1868 bp and between -1868 bp and -700 bp, act as tissue-specific enhancers which respectively regulate yellow transcription in the wings and body of the adult flies. Sequences situated closer to the mRNA cap site, between -225 bp and -91 bp from the start of transcription, are responsible for yellow expression in the denticle belts and mouth parts during larval development. Finally, coloration of the adult bristles is regulated by sequences located in the intron of the yellow gene. These results indicate the existence of several separate DNA elements responsible for the different patterns of temporal and spatial expression of the yellow locus.

Animals↗

PubMedDate not supplied

Theoretical and experimental evaluation of phase-dispersion effects caused by brain motion in diffusion and perfusion MR imaging.

We investigated intravoxel phase dispersion caused by pulsatile brain motion in diffusion spin-echo pulse sequences. Mathematical models were used to describe the spatial and temporal velocity distributions of human brain motion. The spatial distribution of brain-tissue velocity introduces a phase spread over one voxel, leading to signal loss. This signal loss was estimated theoretically, and effects on observed diffusion coefficient and perfused capillary fraction were assessed. When parameters from a diffusion pulse sequence without motion compensation were used, and ECG triggering with inappropriate delay times was assumed, the maximal signal loss caused by brain-motion-induced phase dispersion was predicted to be 21%. This corresponds to a 95% overestimation of the diffusion coefficient, and the perfusion-fraction error was small. Corresponding calculations for motion-compensated pulse sequences predicted a 1% to 1.5% signal loss due to undesired phase dispersion, whereas experimental results indicated a signal loss related to brain motion of 4%.

Adult↗