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Results for “spatial sequencing”

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PubMed1993

Structure of the thiamine- and flavin-dependent enzyme pyruvate oxidase.

Pyruvate oxidase from Lactobacillus plantarum is a tetrameric enzyme that decarboxylates pyruvate, producing hydrogen peroxide and the energy-storage metabolite acetylphosphate. Structure determination at 2.1 angstroms showed that the cofactors thiamine pyrophosphate (TPP) and flavin adenine dinucleotide (FAD) are bound at the carboxyl termini of six-stranded parallel beta sheets. The pyrophosphate moiety of TPP is bound to a metal ion and to a beta alpha alpha beta unit corresponding to an established sequence fingerprint. The spatial arrangement of TPP and FAD suggests that the oxidation of the oxyethyl intermediate does not occur by hydride displacement but rather by a two-step transfer of two electrons.

Amino Acid Sequence↗

PubMed2005

Vestige: maximum likelihood phylogenetic footprinting.

BACKGROUND: Phylogenetic footprinting is the identification of functional regions of DNA by their evolutionary conservation. This is achieved by comparing orthologous regions from multiple species and identifying the DNA regions that have diverged less than neutral DNA. Vestige is a phylogenetic footprinting package built on the PyEvolve toolkit that uses probabilistic molecular evolutionary modelling to represent aspects of sequence evolution, including the conventional divergence measure employed by other footprinting approaches. In addition to measuring the divergence, Vestige allows the expansion of the definition of a phylogenetic footprint to include variation in the distribution of any molecular evolutionary processes. This is achieved by displaying the distribution of model parameters that represent partitions of molecular evolutionary substitutions. Examination of the spatial incidence of these effects across regions of the genome can identify DNA segments that differ in the nature of the evolutionary process. RESULTS: Vestige was applied to a reference dataset of the SCL locus from four species and provided clear identification of the known conserved regions in this dataset. To demonstrate the flexibility to use diverse models of molecular evolution and dissect the nature of the evolutionary process Vestige was used to footprint the Ka/Ks ratio in primate BRCA1 with a codon model of evolution. Two regions of putative adaptive evolution were identified illustrating the ability of Vestige to represent the spatial distribution of distinct molecular evolutionary processes. CONCLUSION: Vestige provides a flexible, open platform for phylogenetic footprinting. Underpinned by the PyEvolve toolkit, Vestige provides a framework for visualising the signatures of evolutionary processes across the genome of numerous organisms simultaneously. By exploiting the maximum-likelihood statistical framework, the complex interplay between mutational processes, DNA repair and selection can be evaluated both spatially (along a sequence alignment) and temporally (for each branch of the tree) providing visual indicators to the attributes and functions of DNA sequences.

Algorithms↗

PubMed1995

The sigma 54-dependent promoter Ps of the TOL plasmid of Pseudomonas putida requires HU for transcriptional activation in vivo by XylR.

In the presence of toluene and xylenes, the sigma 54-dependent Ps promoter of the TOL (toluene biodegradation) plasmid pWW0 of Pseudomonas putida is activated at a distance by the XylR protein, of the NtrC family of transcriptional regulators. Since contacts between XylR bound to upstream activating sites and the RNA polymerase require the looping out of the intervening DNA segment, the intrinsic curvature, the bendability of the corresponding sequence, and the spatial effects of protein-induced DNA bending have an influence on promoter activity. Unlike other sigma 54-dependent promoters, Ps does not require the structural aid of the integration host factor to assemble a specific promoter geometry required for transcriptional initiation. In vivo analysis of transcriptional activity in various genetic backgrounds suggests, instead, that the looping out of intervening DNA sequences in Ps would result from the exacerbation of a preexisting static bend within the region, assisted by the histone-like protein HU.

Bacterial Proteins↗

PubMed2004

Regulation of transcription of the steroidogenic acute regulatory protein (StAR) gene: temporal and spatial changes in transcription factor binding and histone modification.

We examined the binding of transcription factors and histone modifications associated with induction of expression of the steroidogenic acute regulatory protein (StAR) gene in MA-10 cells using a quantitative chromatin immunoprecipitation (ChIP) assay. GATA-4, SF-1/Ad4BP, and cyclic AMP response element binding protein binding protein (CBP) bind rapidly to the StAR proximal promoter, but in different patterns following 8-Br-cAMP stimulation. Concomitantly, histone modifications occur in a spatial and temporal sequence including increased association of acetylated histone H3 with the proximal promoter region, increased association of dimethylated lysine 4 histone H3 with exonic sequences, a modification that marks actively transcribed regions, and reduced association of a marker linked to gene silencing (lysine 9 dimethylated histone H3). Our findings demonstrate that transcription factors and coactivators are rapidly associated with the StAR proximal promoter, that the patterns of binding differ which has implications for postulated direct interactions among these factors, and that multiple histone modifications are demonstrable in a spatially- and temporally-specific pattern along the StAR gene. These observations suggest that a combinatorial code of transcription factors including reciprocal changes in histone modifications associated with active transcription and gene silencing control StAR gene expression.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1986

Multivariate assessment of locomotor behavior: pharmacological and behavioral analyses.

A Behavioral Pattern Monitor (BPM) is described which is designed to assess the spatial and temporal sequences of the locomotor movements, investigatory holepokes, and rearings of rats. The system records these behavioral responses with 0.1 sec resolution in time and 1.5 inch resolution in space, and permanently stores all the resulting data. The sequences of these responses may then be displayed on a video terminal or on paper and are also available for the calculation of a variety of descriptive statistics. Studies are described in which rats were tested repeatedly without any pharmacological treatments or in single test sessions following the administration of saline or one of five stimulant drugs. A variety of descriptive measures of the temporal or spatial patterning of the animals' behavior are described and applied to the data resulting from the studies of the various stimulants. It is concluded that the combination of these measures enables distinctions to be made among these drugs which cannot be made on the basis of measures of the amount of locomotor activity.

Amphetamine↗

PubMed2004

High-resolution, multicontrast three-dimensional-MRI characterizes atherosclerotic plaque composition in ApoE-/- mice ex vivo.

PURPOSE: To systematically investigate intrinsic MR contrast mechanisms that would facilitate plaque characterization and quantification in the aortic root and brachiocephalic artery of ApoE-/- mice ex vivo. MATERIALS AND METHODS: To establish unambiguous MR parameters for routinely analyzing atherosclerotic plaque ex vivo at 11.7 T, relaxation times of plaque components were quantitatively assessed. Magnetization transfer and lipid-proton three-dimensional MR imaging was investigated for visualization of collagen- and lipid-rich plaque regions, respectively. A three-dimensional multiecho sequence with a spatial resolution of 47 x 47 x 63 microm was implemented providing a variable degree of T2-weighting. RESULTS: Relaxation time measurements showed clear tissue heterogeneity between atherosclerotic plaque components in the T2-values, but similar T1-values at 11.7 T (T1/T2 mean +/- SD; cellular plaque component: 1.2 +/- 0.3 seconds/26.3 +/- 0.4 msec; fibrofatty plaque component: 1.1 +/- 0.2 seconds/13.7 +/- 2.0 msec). The three-dimensional multiecho sequence allowed the calculation of the intrinsic proton density and T2-maps. The sum of the multiecho data provided strong T2-weighting that facilitated quantification of various components of atherosclerotic plaque in the mouse aortic root and correlated well with histology (P < 0.0001). CONCLUSION: High-resolution MRI allows for accurate classification and quantification of atherosclerotic plaque components in the aortic root of mice.

Animals↗

PubMed2004

Functional sodium magnetic resonance imaging of the intact rat kidney.

BACKGROUND: Renal fluid homeostasis depends to a large extent on the sodium concentration gradient along the corticomedullary axis. The spatial distribution and extent of this gradient were previously determined by invasive methods, which yielded a range of results. We demonstrate here the capacity of sodium magnetic resonance imaging (MRI) to quantify non-invasively renal sodium distribution in the intact kidney. METHODS: Sodium MRI was applied to study normal, diuretic, and obstructed rat kidneys in vivo. The images were recorded at 4.7 Tesla using a 3-dimensional gradient echo sequence, with high spatial and temporal resolution. The tissue sodium concentration (TSC) was obtained by taking into account the measured nuclear relaxation rates and MRI visibility relative to a reference saline solution. RESULTS: The corticomedullary sodium gradient increased linearly from the cortex to the inner medulla by approximately 31 mmol/L/mm, from a TSC of approximately 60 mmol/L to approximately 360 mmol/L. Furosemide induced a 50% reduction in the inner-medulla sodium and a 25% increase in the cortical sodium. The kinetics of these changes was related to the specific site and mechanism of the loop diuretic. Distinct profiles of the sodium gradient were observed in acute obstructed kidneys, as well as spontaneously obstructed kidneys. The changes in the sodium gradient correlated with the extent of damage and the residual function of the kidneys. CONCLUSION: Quantitative assessment of the renal corticomedullary sodium gradient by high resolution sodium MRI may help verify new aspects of the kidney concentrating mechanism and serve as a non-invasive diagnostic method of renal function.

Acute Disease↗

PubMed1997

[T1-weighted excretory MR urography using GD-DTPA after low-dose diuretic administration].

PURPOSE: Assessment of the morphology of the urinary tract by gadolinium (Gd)-enhanced T1-weighted MR-urography (MRU) and precontrast diuretic injection. METHODS: MRU was performed in 5 patients using Gd-DTPA-enhanced coronal T1-weighted 3D-gradient-echo sequences with different spatial resolution. Prior to contrast injection, 10 mg of furosemide were administered intravenously. RESULTS: Secondary to the precontrast furosemide injection, the increase of the renal excretion rate allows complete visualisation of the entire collecting system within 10 minutes following Gd injection. Maximum intensity projections provide a three-dimensional overview of the ureters separate from the bladder. To achieve detailed anatomic imaging of the calices, both optimal distension and high resolution sequences are prerequisites which cannot be realized by means of survey urograms only. CONCLUSIONS: Contrast-enhanced excretory MRU performed after injection of a diuretic offers a rapid approach to visualising the morphology of the nondilated urinary tract. Imaging of caliceal details must be done with high-resolution 3D-gradient-echo sequences.

Adult↗

PubMed2002

The 1.62 A structure of Thermoascus aurantiacus endoglucanase: completing the structural picture of subfamilies in glycoside hydrolase family 5.

The crystal structure of Thermoascus aurantiacus endoglucanase (Cel5A), a family 5 glycoside hydrolase, has been determined to 1.62 A resolution by multiple isomorphous replacement with anomalous scattering. It is the first report of a structure in the subfamily to which Cel5A belongs. Cel5A consists solely of a catalytic module with compact eight-fold beta/alpha barrel architecture. The length of the tryptophan-rich substrate binding groove suggests the presence of substrate binding subsites -4 to +3. Structural comparison shows that two glycines are completely conserved in the family, in addition to the two catalytic glutamates and six other conserved residues previously identified. Gly 44 in particular is part of a type IV C-terminal helix capping motif, whose disruption is likely to affect the position of an essential conserved arginine. One aromatic residue (Trp 170 in Cel5A), not conserved in term of sequence, is nonetheless spatially conserved in the substrate binding groove. Its role might be to force the bend that occurs in the polysaccharide chain on binding, thus favoring substrate distortion at subsite -1.

Ascomycota↗

PubMed2002

Assessing the impact of comparative genomic sequence data on the functional annotation of the Drosophila genome.

BACKGROUND: It is widely accepted that comparative sequence data can aid the functional annotation of genome sequences; however, the most informative species and features of genome evolution for comparison remain to be determined. RESULTS: We analyzed conservation in eight genomic regions (apterous, even-skipped, fushi tarazu, twist, and Rhodopsins 1, 2, 3 and 4) from four Drosophila species (D. erecta, D. pseudoobscura, D. willistoni, and D. littoralis) covering more than 500 kb of the D. melanogaster genome. All D. melanogaster genes (and 78-82% of coding exons) identified in divergent species such as D. pseudoobscura show evidence of functional constraint. Addition of a third species can reveal functional constraint in otherwise non-significant pairwise exon comparisons. Microsynteny is largely conserved, with rearrangement breakpoints, novel transposable element insertions, and gene transpositions occurring in similar numbers. Rates of amino-acid substitution are higher in uncharacterized genes relative to genes that have previously been studied. Conserved non-coding sequences (CNCSs) tend to be spatially clustered with conserved spacing between CNCSs, and clusters of CNCSs can be used to predict enhancer sequences. CONCLUSIONS: Our results provide the basis for choosing species whose genome sequences would be most useful in aiding the functional annotation of coding and cis-regulatory sequences in Drosophila. Furthermore, this work shows how decoding the spatial organization of conserved sequences, such as the clustering of CNCSs, can complement efforts to annotate eukaryotic genomes on the basis of sequence conservation alone.

Animals↗

PubMed2001

Some measures of verbal and spatial working memory in eight- and nine-year-old hearing-impaired children with cochlear implants.

OBJECTIVE: The purpose of this study was to examine working memory for sequences of auditory and visual stimuli in prelingually deafened pediatric cochlear implant users with at least 4 yr of device experience. DESIGN: Two groups of 8- and 9-yr-old children, 45 normal-hearing and 45 hearing-impaired users of cochlear implants, completed a novel working memory task requiring memory for sequences of either visual-spatial cues or visual-spatial cues paired with auditory signals. In each sequence, colored response buttons were illuminated either with or without simultaneous auditory presentation of verbal labels (color-names or digit-names). The child was required to reproduce each sequence by pressing the appropriate buttons on the response box. Sequence length was varied and a measure of memory span corresponding to the longest list length correctly reproduced under each set of presentation conditions was recorded. Additional children completed a modified task that eliminated the visual-spatial light cues but that still required reproduction of auditory color-name sequences using the same response box. Data from 37 pediatric cochlear implant users were collected using this modified task. RESULTS: The cochlear implant group obtained shorter span scores on average than the normal-hearing group, regardless of presentation format. The normal-hearing children also demonstrated a larger "redundancy gain" than children in the cochlear implant group-that is, the normal-hearing group displayed better memory for auditory-plus-lights sequences than for the lights-only sequences. Although the children with cochlear implants did not use the auditory signals as effectively as normal-hearing children when visual-spatial cues were also available, their performance on the modified memory task using only auditory cues showed that some of the children were capable of encoding auditory-only sequences at a level comparable with normal-hearing children. CONCLUSIONS: The finding of smaller redundancy gains from the addition of auditory cues to visual-spatial sequences in the cochlear implant group as compared with the normal-hearing group demonstrates differences in encoding or rehearsal strategies between these two groups of children. Differences in memory span between the two groups even on a visual-spatial memory task suggests that atypical working memory development irrespective of input modality may be present in this clinical population.

Audiometry, Pure-Tone↗

PubMed2026

A High-Resolution Stereo-Seq Spatial Transcriptomic Resource for Adult Holstein Cattle Liver.

The bovine liver is a highly compartmentalized organ that plays essential roles in continuous gluconeogenesis and nitrogen recycling; however, its spatial molecular architecture has remained largely uncharacterized due to the limitations of traditional bulk and single-cell approaches. To address this gap, Spatial Enhanced Resolution Omics-sequencing (Stereo-seq) was utilized to generate a subcellular-resolution (500 nm) transcriptomic map of an adult Holstein cattle liver, and a refined reference-guided workflow was implemented to overcome standard annotation limitations in livestock. Raw sequencing data were processed using the Stereo-seq Analysis Workflow and analyzed with Stereopy, Seurat, SingleR, and reference-guided workflows. Spatial aggregation was evaluated at Bin20, Bin50, Bin100, Bin150, and Bin200. Increasing bin size increased molecular identifier counts and detected-gene complexity while progressively reducing spatial granularity. Bin50, corresponding to 50 × 50 DNA nanoballs and an approximate nominal footprint of 25 × 25 µm, was therefore selected as a practical intermediate aggregation level for the primary analyses. Quality-control assessment, Leiden clustering, UMAP visualization, reference-based cell-type annotation, cluster-marker analysis, and spatial mapping of canonical hepatic genes demonstrated preservation of biologically interpretable liver transcriptional organization. Raw sequencing data processed spatial matrices, annotated objects, and analysis code are publicly available to support reanalysis and computational benchmarking. In summary, we present a Stereo-seq spatial transcriptomic resource generated from liver tissue of an adult Holstein cow. This initial resource provides a valuable foundation for future studies of bovine liver biology, comparative genomics, and the spatial basis of livestock health and production traits.

Animals↗

PubMed2006

Determination of network of residues that regulate allostery in protein families using sequence analysis.

Allosteric interactions between residues that are spatially apart and well separated in sequence are important in the function of multimeric proteins as well as single-domain proteins. This observation suggests that, among the residues that are involved in long-range communications, mutation at one site should affect interactions at a distant site. By adopting a sequence-based approach, we present an automated approach that uses a generalization of the familiar sequence entropy in conjunction with a coupled two-way clustering algorithm, to predict the network of interactions that trigger allosteric interactions in proteins. We use the method to identify the subset of dynamically important residues in three families, namely, the small PDZ family, G protein-coupled receptors (GPCR), and the Lectins, which are cell-adhesion receptors that mediate the tethering and rolling of leukocytes on inflamed endothelium. For the PDZ and GPCR families, our procedure predicts, in agreement with previous studies, a network containing a small number of residues that are involved in their function. Application to the Lectin family reveals a network of residues interspersed throughout the C-terminal end of the structure that are responsible for binding to ligands. Based on our results and previous studies, we propose that functional robustness requires that only a small subset of distantly connected residues be involved in transmitting allosteric signals in proteins.

Algorithms↗

PubMed1994

Noninvasive measurement of renal hemodynamic functions using gadolinium enhanced magnetic resonance imaging.

A technique for the assessment of single kidney hemodynamic functions utilizing a novel MR pulse sequence in conjunction with MR contrast material administration is described. Renal extraction fraction (EF) is derived by measuring the concentration of the incoming contrast agent in the renal artery and the outgoing concentration in the renal vein. The glomerular filtration rate (GFR) can then be determined by the product of EF and renal plasma flow. A modified inversion recovery MR pulse sequence is used to measure the T1 of moving blood. This pulse sequence uses a spatially nonselective inversion pulse. A series of small flip angle detection pulses are then used to monitor the recovery of longitudinal spin magnetization in an image plane intersecting the renal vessels. The recovery rate is measured in each vessel and the T1 of blood determined. These T1 measurements are then used to determine the ratio of contrast concentration in the renal arteries and veins. Blood flow measurements can be obtained simultaneously with T1 measurements by inserting flow-encoding magnetic field gradients into the pulse sequence. Preliminary results in human volunteers suggest the feasibility of noninvasively determining hemodynamic functions with magnetic resonance.

Algorithms↗

PubMed1999

Characterization and comparison of protein structures. Part I-characterization.

The quantitative criteria characterizing the regularity of Calpha-backbones in the protein structures are presented. A technique is based on the Fourier remapping of the Cartesian coordinates for the Calpha-chain. The Fourier spectra identify the hidden periodicities and symmetries in protein structures, while the integral regularity is assessed via the spectral structural entropies. The formal unification of digitizing and the similarities in statistics for the random counterparts allow study of the direct correlations between the distribution of physico-chemical characteristics along the amino acid sequence and the spatial conformation of the polypeptide chain. The significant correlations are found for both hydrophobicity and side-chain volumes, though, as expected, the effects for hydrophobicity turn out essentially stronger. A scheme is illustrated by the set of 120 protein structures comprising the representatives from the main superfamilies and superfolds.

Amino Acid Sequence↗

PubMed1993

Topology of P-glycoprotein as determined by epitope mapping of MRK-16 monoclonal antibody.

There is growing evidence for the direct role of P-glycoprotein mediating multidrug resistance in tumor cells. P-glycoprotein is thought to function as an energy-dependent drug efflux pump. The monoclonal antibody MRK-16 binds to an external domain of P-glycoprotein and partially inhibits drug efflux in multidrug-resistant cells. As an approach toward elucidating the mechanism by which MRK-16 affects drug transport, we undertook the definition of the precise binding site of this antibody. In this study we have mapped the epitope of MRK-16 monoclonal antibody to a resolution of a single amino acid using a series of overlapping synthetic peptides. We demonstrate that MRK-16 recognizes only the class I isoform (MDR1) of human P-glycoprotein and that its epitope encompasses at least two (first and fourth) of the six predicted extracellular peptide loops. These results suggest that the epitope of MRK-16 is discontinuous and that the sequences involved which are separated by about 625 amino acids in the linear sequence must be spatially situated in close proximity in the native protein. Based on these results, we present a model for transmembrane alpha-helical packing of P-glycoprotein in the lipid bilayer. This may have implications for understanding the function of P-glycoprotein in drug transport.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2005

Spatio-temporal free-form registration of cardiac MR image sequences.

In this paper we present two registration algorithms for the spatio-temporal alignment of cardiac MR image sequences. Both algorithms have the ability to correct spatial misalignment between the images sequences caused by global and local shape differences. In addition, they have the ability to correct temporal misalignment caused by differences in the length of the cardiac cycles and by differences in the dynamic properties of the hearts. The algorithms use a 4D deformable transformation model which is separated into spatial and temporal components. The first registration algorithm optimizes the spatial and temporal transformation models simultaneously, while the second registration algorithm optimizes the temporal transformation component before optimizing the spatial component. For the evaluation of the spatio-temporal registration methods we have acquired 15 MR image sequences from healthy volunteers. The registration methods were quantitatively evaluated by measuring the overlap and surface distance of anatomical regions and qualitatively by visual inspection. The results demonstrate that a significant improvement in the alignment of the image sequences is achieved by the use of the deformable spatio-temporal transformation model. We demonstrate the use of the method for the construction of a probabilistic MR cardiac atlas representing the anatomy and function of a healthy heart.

Algorithms↗

PubMed1990

Disintegrins: a family of integrin inhibitory proteins from viper venoms.

Disintegrins represent a new class of low molecular weight, RGD-containing, cysteine-rich peptides isolated from the venom of various snakes. They interact with the beta 1 and beta 3 families of integrins and their potency is at least 500-2000 times higher than short RGDX peptides. Analysis of the amino acid sequences of 14 different disintegrins suggests that the RGD sequence, in the spatial configuration determined by the appropriate pairing of the cysteine residues, functions as a cell recognition site. However, certain nonconserved amino acids appear to modify the activity of disintegrins, their specificity for various receptors, and their ability to compete specifically with various ligands.

Amino Acid Sequence↗