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David Carter

Publications and source records attributed to David Carter.

17 recordsLinked to original sources
PubMed2006

Vertebrate gene finding from multiple-species alignments using a two-level strategy.

BACKGROUND: One way in which the accuracy of gene structure prediction in vertebrate DNA sequences can be improved is by analyzing alignments with multiple related species, since functional regions of genes tend to be more conserved. RESULTS: We describe DOGFISH, a vertebrate gene finder consisting of a cleanly separated site classifier and structure predictor. The classifier scores potential splice sites and other features, using sequence alignments between multiple vertebrate species, while the structure predictor hypothesizes coding transcripts by combining these scores using a simple model of gene structure. This also identifies and assigns confidence scores to possible additional exons. Performance is assessed on the ENCODE regions. We predict transcripts and exons across the whole human genome, and identify over 10,000 high confidence new coding exons not in the Ensembl gene set. CONCLUSION: We present a practical multiple species gene prediction method. Accuracy improves as additional species, up to at least eight, are introduced. The novel predictions of the whole-genome scan should support efficient experimental verification.

Animals↗
PubMed2006

Cellular transcriptomics -- the next phase of endocrine expression profiling.

Transcriptome analysis, or global gene expression profiling, has become a commonly used and valuable tool in both basic and clinical endocrine research. Novel endocrine regulators have 'surfaced' and greater consideration is now given to understanding function at the level of gene networks. Recent developments have shown that the transcriptome is considerably larger and more divergently expressed than was previously thought. Endocrine cells express a great variety of coding and noncoding RNAs in a highly cell-specific manner. If further value is to be taken from this research area, then steps towards defined cellular transcriptomics must be taken. New sampling techniques that utilize novel genetic models are a key first step.

Animals↗
PubMed2006

Nanoscale glassification of gold substrates for surface plasmon resonance analysis of protein toxins with supported lipid membranes.

Surface plasmon resonance (SPR) spectroscopy, a powerful tool for biosensing and protein interaction analysis, is currently confined to gold substrates and the relevant surface chemistries involving dextran and functional thiols. Drawbacks of using self-assembled monolayers (SAMs) for SPR-related surface modification include limited stability, pinhole defects, bioincompatibility, and nonspecific protein adsorption. Here we report the development of stable nanometer-scale glass (silicate) layers on gold substrates for SPR analysis of protein toxins. The nanoscale silicate layers were built up with layer-by-layer deposition of poly(allylamine hydrochloride) and sodium silicate, followed by calcination at high temperature. The resulting silicate films have a thickness ranging from 2 to 15 nm and demonstrate outstanding stability in flow cell conditions. The use of these surfaces as a platform to construct supported bilayer membranes (SBMs) is demonstrated, and improved performance against protein adsorption on SBM-coated surfaces is quantified by SPR measurements. SBMs can be formed reproducibly on the silicate surface via vesicle fusion and quantitatively removed using injection of 5% Triton X-100 solution, generating a fresh surface for each test. Membrane properties such as lateral diffusion of the SBMs on the silicate films are characterized with photobleaching methods. Studies of protein binding with biotin/avidin and ganglioside/cholera toxin systems show detection limits lower than 1 microg/mL (i.e., nanomolar range), and the response reproducibility is better than 7% RSD. The method reported here allows many assay techniques developed for glass surfaces to be transferred to label-free SPR analysis without the need for adaptation of protocols and time-consuming synthetic development of thiol-based materials and opens new avenues for developing novel bioanalytical technologies for protein analysis.

Avidin↗
PubMed2006

Specialized transcription factories.

We have previously suggested a model for the eukaryotic genome based on the structure of the bacterial nucleoid where active RNA polymerases cluster to loop the intervening DNA. This organization of polymerases into clusters--which we call transcription 'factories'--has important consequences. For example, in the nucleus of a HeLa cell the concentration of soluble RNA polymerase II is approximately 1 mM, but the local concentration in a factory is 1000-fold higher. Because a promoter can diffuse approximately 100 nm in 15 s, one lying near a factory is likely to initiate; moreover, when released at termination, it will still lie near a factory, and the movement and modifications (e.g. acetylation) accompanying elongation will leave it in an 'open' conformation. Another promoter out in a long loop is less likely to initiate, because the promoter concentration falls off with the cube of the distance from the factory. Moreover, a long tether will buffer it from transcription-induced movement, making it prone to deacetylation, deposition of HP1 (heterochromatin protein 1), and incorporation into heterochromatin. The context around a promoter will then be self-sustaining: productive collisions of an active promoter with the factory will attract factors increasing the frequency of initiation, and the longer an inactive promoter remains inactive, the more it becomes embedded in heterochromatin. We review here the evidence that different factories may specialize in the transcription of different groups of genes.

Bacteria↗
PubMed2005

Stable and fluid ethylphosphocholine membranes in a poly(dimethylsiloxane) microsensor for toxin detection in flooded waters.

Highly stable and fluid supported bilayer membranes were fabricated by fusion of positively charged ethylphosphocholine (DOPC+) vesicles into poly(dimethylsiloxane) (PDMS) microchannels for immunosensing of cholera toxin (CT) in flooded waters. Compared to phosphatidylcholine (PC) layers in the microchannels, DOPC+ membranes show exceptionally strong resistance to air-dry damage, as demonstrated by fluorescence recovery after photobleaching (FRAP) measurements and protein adsorption studies. In FRAP experiments, the mobile fraction of PC membranes was found to decrease by 10% upon drying/rehydration and the lateral diffusion coefficient decreased from 2.2 to 1.6 microm(2)/s, whereas the mobile fraction and diffusion coefficient for DOPC+ membranes remain virtually unchanged during this process. Characterization by confocal microscopy reveals that only 1% of the DOPC+ membrane in the microchannels was removed by the drying/rehydration process, as compared to 11% for PC. Protein adsorption trends indicate that the charge of DOPC+ membranes allows for tuning of solution conditions to enable the desired protein-membrane interaction to predominate at the interface. A flow-based immunoassay for bacterial toxin was developed with 5% GM1/DOPC+ membranes in PDMS channels, and a detection limit of 250 amol for CT was obtained from the calibration curves. The assay was successfully applied to detection of CT spiked in water samples from the Santa Ana River, with nearly identical response and sensitivity.

Biosensing Techniques↗
PubMed2005

Developmental regulation of the beta-globin gene locus.

The beta-globin genes have become a classical model for studying regulation of gene expression. Wide-ranging studies have revealed multiple levels of epigenetic regulation that coordinately ensure a highly specialised, tissue- and stage-specific gene transcription pattern. Key players include cis-acting elements involved in establishing and maintaining specific chromatin conformations and histone modification patterns, elements engaged in the transcription process through long-range regulatory interactions, transacting general and tissue-specific factors. On a larger scale, molecular events occurring at the locus level take place in the context of a highly dynamic nucleus as part of the cellular epigenetic programme.

Animals↗
PubMed2005

A TASK3 channel (KCNK9) mutation in a genetic model of absence epilepsy.

Childhood absence epilepsy is an idiopathic, generalized, nonconvulsive epilepsy with a multifactorial genetic etiology. The KCNK9 gene coding for the TASK3 (Twik-like acid-sensitive K</U)+) channel is present on chromosome 8 at position 8q24, a locus that has shown positive linkage to the human absence epilepsy phenotype. Sequencing of the KCNK9 gene in the genetic absence epilepsy rats from Strasbourg (GAERS), a well established genetic model of this disease, reveals an additional alanine residue in a polyalanine tract within the C-terminal intracellular domain. This additional alanine is absent in the inbred nonepileptic control (NEC) strain, Wistar, and Wistar albino Glaxo strain bred in Rijswijk, another inbred rat model of absence epilepsy. Expression of the mutant channel in CHO cells produces a K+ current that is blocked by acidic pH and millimolar concentrations of barium or ruthenium red and is not different from the wild-type channel. In brain slices, thalamic neurons display a prominent pH-sensitive tonic K+ current, but no difference was observed between GAERS and NEC or Wistar rats. Ruthenium red had no effect in cortical, reticular thalamic, or sensory thalamic neurons in either GAERS or NEC, indicating that the TASK3 homodimer is not present in these structures. Twik-like acid-sensitive K+(TASK3) channels, therefore, are probably associated with TASK1 to form ruthenium red-insensitive heterodimers in these neurons. Finally, no difference was found between GAERS and NEC rats in the modulation of the leak K+ current following activation of muscarinic receptors. These studies describe the first mutation found in a genetic model of absence epilepsy. Although our experiments showed no difference in the leak K+ current between GAERS and NEC rats, further work is needed to ascertain whether this mutation contributes to the generation of absence seizures, possibly by mechanisms related to the expansion of the polyalanine run.

Amino Acid Sequence↗
PubMed2004

Active genes dynamically colocalize to shared sites of ongoing transcription.

The intranuclear position of many genes has been correlated with their activity state, suggesting that migration to functional subcompartments may influence gene expression. Indeed, nascent RNA production and RNA polymerase II seem to be localized into discrete foci or 'transcription factories'. Current estimates from cultured cells indicate that multiple genes could occupy the same factory, although this has not yet been observed. Here we show that, during transcription in vivo, distal genes colocalize to the same transcription factory at high frequencies. Active genes are dynamically organized into shared nuclear subcompartments, and movement into or out of these factories results in activation or abatement of transcription. Thus, rather than recruiting and assembling transcription complexes, active genes migrate to preassembled transcription sites.

Animals↗
PubMed2004

On-Pump Beating Heart Surgery Offers an Alternative for Unstable Patients Undergoing Coronary Artery Bypass Grafting.

Abstract Background: Cardiac surgery has expanded the available approaches to aortocoronary artery bypass grafting to include approaches from minimally invasive surgery to full sternotomy. The heart can be arrested, left beating, or assisted with a right ventricular assist device or cardiopulmonary bypass pump. We have examined the 4 surgical modes that we use routinely in our large multisurgeon practice to determine our selection biases and the outcomes of the different techniques. Methods: Of the 4733 coronary artery bypass grafting (CABG) patients we studied from January 2000 through December 2002, 2332 (49.3%) operations were done on-pump on the arrested heart, 1908 (40.3%) were performed off-pump, 364 (7.7%) were performed on-pump on the beating heart, and 129 (2.7%) were performed with right heart assist. The preoperative risk factors, operative variables, and postoperative outcomes of the groups were analyzed. Results: Patients selected for on-pump beating heart procedures tended to be sicker with the highest predicted risk of death. We also selected patients who were in cardiogenic shock, in resuscitation, in emergent or salvage status, on dialysis, and with preoperative intra-aortic balloon pump (IABP) use for on-pump beating heart procedures at higher than expected rates. Patients with renal failure with or without dialysis, and those having a history of cerebrovascular accident tended not to be chosen for on-pump arrested heart procedures. Off-pump beating heart procedures were avoided for patients with cardiogenic shock or resuscitation, in emergent or salvage status, and with preoperative IABP use. The mortality rate in these patients was slightly worse in the on-pump beating heart group (4.4%) than in the on-pump arrested heart (3.5%) and off-pump (2.3%) groups (analysis of variance [ANOVA], P =.04). Atrial fibrillation occurred more frequently in both the on-pump beating heart (20.1%) and on-pump arrested heart (23.8%) groups (ANOVA, P <.001). The on-pump groups had higher rates of blood product use and reoperation for bleeding and a prolonged ventilation rate, compared with the other procedures. On-pump patients had a statistically longer length of stay than either off-pump or right heart-assisted patients ( P <.05) and required longer times on the ventilator and in the intensive care unit. Conclusions: Normothermic cardiopulmonary bypass with a beating heart is safe and efficacious and may be the method of choice for patients in cardiogenic shock, requiring resuscitation, or with previous CABG surgery, recent myocardial infarction, a low ejection fraction, or unstable arrhythmias.

Journal Article↗
PubMed2003

The VTI family of SNARE proteins is necessary for plant viability and mediates different protein transport pathways.

The Arabidopsis genome contains a family of v-SNAREs: VTI11, VTI12, and VTI13. Only VTI11 and VTI12 are expressed at appreciable levels. Although these two proteins are 60% identical, they complement different transport pathways when expressed in the yeast vti1 mutant. VTI11 was identified recently as the mutated gene in the shoot gravitropic mutant zig. Here, we show that the vti11 zig mutant has defects in vascular patterning and auxin transport. An Arabidopsis T-DNA insertion mutant, vti12, had a normal phenotype under nutrient-rich growth conditions. However, under nutrient-poor conditions, vti12 showed an accelerated senescence phenotype, suggesting that VTI12 may play a role in the plant autophagy pathway. VTI11 and VTI12 also were able to substitute for each other in their respective SNARE complexes, and a double-mutant cross between zig and vti12 was embryo lethal. These results suggest that some VTI1 protein was necessary for plant viability and that the two proteins were partially functionally redundant.

Arabidopsis↗
PubMed2002

Long-range chromatin regulatory interactions in vivo.

Communication between distal chromosomal elements is essential for control of many nuclear processes. For example, genes in higher eukaryotes often require distant enhancer sequences for high-level expression. The mechanisms proposed for long-range enhancer action fall into two basic categories. Non-contact models propose that enhancers act at a distance to create a favorable environment for gene transcription, or act as entry sites or nucleation points for factors that ultimately communicate with the gene. Contact models propose that communication occurs through direct interaction between the distant enhancer and the gene by various mechanisms that 'loop out' the intervening sequences. Although much attention has focused on contact models, the existence and nature of long-range interactions is still controversial and speculative, as there is no direct evidence that distant sequences physically interact in vivo. Here, we report the development of a widely applicable in situ technique to tag and recover chromatin in the immediate vicinity of an actively transcribed gene. We show that the classical enhancer element, HS2 of the prototypical locus control region (LCR) of the beta-globin gene cluster, is in close physical proximity to an actively transcribed HBB (beta-globin) gene located over 50 kb away in vivo, suggesting a direct regulatory interaction. The results give unprecedented insight into the in vivo structure of the LCR-gene interface and provide the first direct evidence of long-range enhancer communication.

Animals↗
PubMed2002

A first-generation linkage disequilibrium map of human chromosome 22.

DNA sequence variants in specific genes or regions of the human genome are responsible for a variety of phenotypes such as disease risk or variable drug response. These variants can be investigated directly, or through their non-random associations with neighbouring markers (called linkage disequilibrium (LD)). Here we report measurement of LD along the complete sequence of human chromosome 22. Duplicate genotyping and analysis of 1,504 markers in Centre d'Etude du Polymorphisme Humain (CEPH) reference families at a median spacing of 15 kilobases (kb) reveals a highly variable pattern of LD along the chromosome, in which extensive regions of nearly complete LD up to 804 kb in length are interspersed with regions of little or no detectable LD. The LD patterns are replicated in a panel of unrelated UK Caucasians. There is a strong correlation between high LD and low recombination frequency in the extant genetic map, suggesting that historical and contemporary recombination rates are similar. This study demonstrates the feasibility of developing genome-wide maps of LD.

Chromosome Mapping↗
PubMed2002

Comparison of gas chromatography and liquid chromatography mass spectrometric measurements for high accuracy analysis of cholesterol in human serum by isotope dilution mass spectrometry.

Cholesterol measurements are of vital clinical importance and reliable reference materials are essential for method validation. Gas chromatography with mass spectrometry (GC/MS) is usually used for the high accuracy analysis of cholesterol by isotope dilution. A certified reference material for cholesterol content in human serum was analysed by isotope dilution utilising GC/MS and liquid chromatography mass spectrometry (LC/MS). The use of LC/MS avoided the need for a derivatisation step. Both LC/MS and GC/MS produced results on the measurement of cholesterol that agreed within 0.5% of the certified value. Moreover, the precision obtained for ratio measurement using both techniques are comparable and lead to relative expanded standard uncertainties (with a coverage factor of 2) varying between 0.2 and 0.5%.

Cholesterol↗
PubMedDate not supplied

A cost-effectiveness analysis of eletriptan 40 and 80 mg versus sumatriptan 50 and 100 mg in the acute treatment of migraine.

OBJECTIVES: This article explores the application of cost-effectiveness analysis in a comparison of eletriptan and sumatriptan in the acute treatment of migraine. METHODS: The study employs data from a randomized, double-blind, placebo-controlled clinical trial comparison of oral eletriptan (40 and 80 mg) and oral sumatriptan (50 and 100 mg). Analyses were undertaken using two composite measures of treatment outcome constructed to reflect the requirements of patients more comprehensively than the conventional efficacy indicator of headache response at 2 hours. On the cost side of the equation, reflecting the health-care system perspective of the analysis, drug costs for initial dosing, second dosing for nonresponse, and recurrence and rescue medication were taken into account. RESULTS: The analysis found that eletriptan treatment resulted in lower costs per successfully treated attack than those of sumatriptan under both outcome criteria. CONCLUSION: Further refinement of outcomes measurement in migraine would be valuable and eletriptan has a potentially important role to play in the cost-effective management of the disorder.

Acute Disease↗