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Douglas R Smith

Publications and source records attributed to Douglas R Smith.

15 recordsLinked to original sources
PubMed2006

Improved repeat identification and masking in Dipterans.

Repetitive sequences are a major constituent of many eukaryote genomes and play roles in gene regulation, chromosome inheritance, nuclear architecture, and genome stability. The identification of repetitive elements has traditionally relied on in-depth, manual curation and computational determination of close relatives based on DNA identity. However, the rapid divergence of repetitive sequence has made identification of repeats by DNA identity difficult even in closely related species. Hence, the presence of unidentified repeats in genome sequences affects the quality of gene annotations and annotation-dependent analyses (e.g. microarray analyses). We have developed an enhanced repeat identification pipeline using two approaches. First, the de novo repeat finding program PILER-DF was used to identify interspersed repetitive elements in several recently finished Dipteran genomes. Repeats were classified, when possible, according to their similarity to known elements described in Repbase and GenBank, and also screened against annotated genes as one means of eliminating false positives. Second, we used a new program called RepeatRunner, which integrates results from both RepeatMasker nucleotide searches and protein searches using BLASTX. Using RepeatRunner with PILER-DF predictions, we masked repeats in thirteen Dipteran genomes and conclude that combining PILER-DF and RepeatRunner greatly enhances repeat identification in both well-characterized and un-annotated genomes.

Animals↗
PubMed2006

Integration of an on-line protein digestion microreactor to a nanoelectrospray emitter for peptide mapping.

A method for integrating nanoelectrospray mass spectrometry with a microreactor for on-line digestion and fast peptide mass mapping from dilute protein samples is presented. Fused silica capillaries (i.d. 50 microm, o.d. 360 microm) are employed as the digestion microreactor and the nanoelectrospray emitter by immobilizing trypsin onto the surface of the inner wall of the fused silica capillary tubing. The procedure is demonstrated using solutions of 1pmol/mul angiotensin II, cytochrome c, hemoglobin, and beta-casein. Because the inner walls of the capillaries are modified by covalent chemical bonds, the adsorption of peptides and proteins to the inner walls of the capillaries is suppressed. This procedure was performed with solutions as dilute as 1fmol/mul (1nM) cytochrome c. This method shows generation of tryptic peptides with sequence coverage up to 90% within minutes; trypsin autolysis products are not detected. In addition, the immobilized enzyme can be cleaned easily, enabling the microreactor to be reused for nanoelectrospray.

Amino Acid Sequence↗
PubMed2006

SB-399885 is a potent, selective 5-HT6 receptor antagonist with cognitive enhancing properties in aged rat water maze and novel object recognition models.

SB-399885 (N-[3,5-dichloro-2-(methoxy)phenyl]-4-(methoxy)-3-(1-piperazinyl)benzenesulfonamide) has high affinity for human recombinant and native 5-HT(6) receptors, with pK(i) values 9.11+/-0.03 and 9.02+/-0.05, respectively and is a potent competitive antagonist (pA(2) 7.85+/-0.04). It displays over 200-fold selectivity for the 5-HT(6) receptor over all other receptors, ion channels and enzymes tested to date. SB-399885 inhibited ex vivo [(125)I]SB-258585 (4-Iodo-N-[4-methoxy-3-(4-methyl-piperazin-1-yl)-phenyl]-benzenesulfonamide) binding with an ED(50) of 2.0+/-0.24 mg/kg p.o. in rats. It had a minimum effective dose of 1 mg/kg p.o. in a rat maximal electroshock seizure threshold test and a long duration of action, overall demonstrating an excellent pharmacokinetic-pharmacodynamic correlation. Repeated administration of this agent (10 mg/kg p.o., b.i.d. for 7 days) significantly reversed a scopolamine-induced deficit (0.5 mg/kg i.p.) in a rat novel object recognition paradigm. Moreover, in aged rats (22 months old) SB-399885 (10 mg/kg p.o., b.i.d. for 7 days) fully reversed the age-dependent deficit in water maze spatial learning compared to vehicle-treated age-matched controls and significantly improved recall of the task measured by increases in the searching of the target quadrant on post-training days 1, 3 and 7. In vivo microdialysis in the rat medial prefrontal cortex demonstrated that acute SB-399885 (10 mg/kg p.o.) significantly increased extracellular acetylcholine levels. These data demonstrate that SB-399885 is a potent, selective, brain penetrant, orally active 5-HT(6) receptor antagonist with cognitive enhancing properties that are likely to be mediated by enhancements of cholinergic function. These studies provide further support for the potential therapeutic utility of 5-HT(6) receptor antagonists in disorders characterised by cognitive deficits such as Alzheimer's disease and schizophrenia.

Acetylcholine↗
PubMed2006

Dredging of drainage ditches increases short-term transport of soluble phosphorus.

Managed drainage ditches are common in the midwestern United States. These ditches are designed to remove water from fields as quickly as possible, and sediment buildup necessitates dredging, to ensure adequate water removal. This laboratory study was conducted to determine the impact of ditch dredging on soluble phosphorus (P) transport. Ditch sediments were collected from a drainage ditch in northeastern Indiana immediately before and after dredging. The sediments were placed in a stream simulator, and stream water was loaded with 0.55 mM P for 5 d (adsorption experiment). Water was then removed, and "clean" water (no P added) was used for a desorption experiment, lasting 1 d. During the adsorption experiment, pre-dredged sediments were able to remove P from the water column quicker, and P concentrations 120 h after introduction of high P water were lower for the pre-dredged sediments (0.075 mM P) than the dredged sediments (0.111 mM P). During the desorption experiment, P was released to the water column slower in the pre-dredged treatment than the dredged treatment (instantaneous flux at t = 0 was 0.205 microM P h(-1) for pre-dredged and 0.488 microM P h(-1) for dredged). This occurred despite higher Mehlich 3-extractable P in the pre-dredged sediments than the dredged sediments. Equilibrium phosphorus concentrations (EPCo) were lower in the pre-dredged sediments during both adsorption and desorption experiments. Transport of soluble P immediately after dredging will likely increase in drainage ditches; however, dredging is a necessary management tool to ensure adequate discharge of water from surrounding fields.

Adsorption↗
PubMed2006

Analytical performance characteristics of nanoelectrospray emitters as a function of conductive coating.

As miniaturization of electrospray continues to become more prevalent in the mass spectrometry arsenal, numerous types of conductive coatings have been developed with miniaturized electrospray emitters. Different conductive coatings have different properties that may lead to differences in analytical performance. This paper investigates and compares the analytical properties of a series of applied conductive coatings for low-flow electrospray ionization developed in this laboratory vs. commercially-available types. Evaporated graphite is thoroughly compared with commercially available polyaniline (PANI) coated emitters and metal coated emitters. Each set of emitters was investigated to determine various performance characteristics, including susceptibility to electrical discharge in both positive and negative ionization modes, as well as emitter reproducibility and generation of a standard curve to determine each emitter coating's limit of detection and limit of quantitation. Furthermore, evaporated graphite and polyaniline coated fused silica capillaries were investigated to determine which coating is more stable over long-term analyses and during electrical discharge.

Boron↗
PubMed2005

Genome sequence, comparative analysis and haplotype structure of the domestic dog.

Here we report a high-quality draft genome sequence of the domestic dog (Canis familiaris), together with a dense map of single nucleotide polymorphisms (SNPs) across breeds. The dog is of particular interest because it provides important evolutionary information and because existing breeds show great phenotypic diversity for morphological, physiological and behavioural traits. We use sequence comparison with the primate and rodent lineages to shed light on the structure and evolution of genomes and genes. Notably, the majority of the most highly conserved non-coding sequences in mammalian genomes are clustered near a small subset of genes with important roles in development. Analysis of SNPs reveals long-range haplotypes across the entire dog genome, and defines the nature of genetic diversity within and across breeds. The current SNP map now makes it possible for genome-wide association studies to identify genes responsible for diseases and traits, with important consequences for human and companion animal health.

Animals↗
PubMed2005

Serendipitous discovery of Wolbachia genomes in multiple Drosophila species.

BACKGROUND: The Trace Archive is a repository for the raw, unanalyzed data generated by large-scale genome sequencing projects. The existence of this data offers scientists the possibility of discovering additional genomic sequences beyond those originally sequenced. In particular, if the source DNA for a sequencing project came from a species that was colonized by another organism, then the project may yield substantial amounts of genomic DNA, including near-complete genomes, from the symbiotic or parasitic organism. RESULTS: By searching the publicly available repository of DNA sequencing trace data, we discovered three new species of the bacterial endosymbiont Wolbachia pipientis in three different species of fruit fly: Drosophila ananassae, D. simulans, and D. mojavensis. We extracted all sequences with partial matches to a previously sequenced Wolbachia strain and assembled those sequences using customized software. For one of the three new species, the data recovered were sufficient to produce an assembly that covers more than 95% of the genome; for a second species the data produce the equivalent of a 'light shotgun' sampling of the genome, covering an estimated 75-80% of the genome; and for the third species the data cover approximately 6-7% of the genome. CONCLUSIONS: The results of this study reveal an unexpected benefit of depositing raw data in a central genome sequence repository: new species can be discovered within this data. The differences between these three new Wolbachia genomes and the previously sequenced strain revealed numerous rearrangements and insertions within each lineage and hundreds of novel genes. The three new genomes, with annotation, have been deposited in GenBank.

Animals↗
PubMed2004

Protein interaction mapping on a functional shotgun sequence of Rickettsia sibirica.

Protein interaction maps can reveal novel pathways and functional complexes, allowing 'guilt by association' annotation of uncharacterized proteins. To address the need for large-scale protein interaction analyses, a bacterial two-hybrid system was coupled with a whole genome shotgun sequencing approach for microbial genome analysis. We report the first large-scale proteomics study using this system, integrating de novo genome sequencing with functional interaction mapping and annotation in a high-throughput format. We apply the approach by shotgun sequencing and annotating the genome of Rickettsia sibirica strain 246, an obligate intracellular human pathogen among the Spotted Fever Group rickettsiae. The bacteria invade endothelial cells and cause lysis after large amounts of progeny have accumulated. Little is known about specific Rickettsial virulence factors and their mode of pathogenicity. Analysis of the combined genomic sequence and protein-protein interaction data for a set of virulence related Type IV secretion system (T4SS) proteins revealed over 250 interactions and will provide insight into the mechanism of Rickettsial pathogenicity.

Bacterial Proteins↗
PubMed2003

Graphite-coated nanoelectrospray emitter for mass spectrometry.

A new, more rapid method for coating nanoelectrospray emitters with graphite is to use a vacuum deposition chamber and a graphite carbon electrode. This method allows for mass production of nanoelectrospray emitters in a short period of time. The emitters are laser-pulled borosilicate glass micropipets and have tapers of around 4 microm i.d. The conductive coating applied to the emitter is only 20-30 nm thick, allowing for optical transparency with the borosilicate emitters. The conductive coating is stable for a number of hours at the high voltages used for nanoelectrospray ionization and is durable in both positive and negative ion modes-even during electrical discharge. This stability will make it possible to couple these emitters with online separations such as capillary liquid chromatography or capillary electrophoresis.

Coated Materials, Biocompatible↗
PubMed2003

Genome visualization made fast and simple.

SUMMARY: The easiest way to gain a quick overall understanding of genomic data is with a visual display that allows the user to view information about an entire genome or chromosome at once. We have constructed GenomePlot, a Perl/Tk script with a series of modules that allow us to display many types of information simultaneously. Menu options allow the user to zoom to the desired view of the data and to print or save the image to a file to produce publication quality illustrations. AVAILABILITY: The program is available on request. A license is required for commercial use.

Algorithms↗
PubMed2003

Exo-proofreading, a versatile SNP scoring technology.

We report the validation of a new assay for typing single nucleotide polymorphisms (SNPs) that takes advantage of the 3'-to-5' exonuclease proofreading activity of many DNA polymerases. The assay uses one or more primers labeled on the 3' nucleotide base, and can be implemented in a variety of formats including a one-step PCR reaction that allows SNP typing directly from genomic DNA samples. The detection of genotypes can be accomplished by means of fluorescence detection on assays that have been purified to remove excess primer, or by means of fluorescence polarization without any additional cleanup. We also demonstrate that the Exo-Proofreading SNP assay can be used on pooled samples to obtain allele frequency data.

Alleles↗
PubMed2002

Pharmacological profile of SB-357134: a potent, selective, brain penetrant, and orally active 5-HT(6) receptor antagonist.

N-(2,5-Dibromo-3-fluorophenyl)-4-methoxy-3-piperazin-1-ylbenzenesulfonamide (SB-357134) potently inhibited [125I]SB-258585 and [3H]LSD binding in a HeLa cell line expressing human 5-HT(6) receptors (pK(i)=8.6 and 8.54, respectively). Furthermore, SB-357134 inhibited [125I]SB-258585 binding in human caudate--putamen and in rat and pig striatum membranes (pK(i)=8.82, 8.44, and 8.61, respectively). SB-357134 displayed over 200-fold selectivity for the 5-HT(6) receptor versus 72 other receptors and enzymes. 5-HT-stimulated cyclic AMP (cAMP) accumulation in human 5-HT(6) receptors was competitively antagonised by SB-357134 (pA(2)=7.63). SB-357134 inhibited ex vivo [125I]SB-258585 binding in the rat with an ED(50) of 4.9 +/- 1.3 mg/kg po, 4 h postdose. In the rat maximal electroshock seizure threshold (MEST) test, SB-357134 produced a potent and dose-dependent increase in seizure threshold, with a minimum effective dose of 0.1 mg/kg po. At 10 mg/kg po, maximum activity occurred between 4 and 6 h postdose. Good exposure was observed with SB-357134 at 10 mg/kg po, reaching maximal blood and brain concentrations of 4.3 +/- 0.2 and 1.3 +/- 0.06 microM, respectively, 1 h postdose. In addition, SB-357134 (10 mg/kg po) enhanced memory and learning following chronic administration (twice a day for 7 days) in the rat water maze. Overall, these studies demonstrate that SB-357134 is a potent, selective, brain penetrant, and orally active 5-HT(6) receptor antagonist.

Algorithms↗
PubMedDate not supplied

Decreasing phosphorus runoff losses from land-applied poultry litter with dietary modifications and alum addition.

Phosphorus (P) losses from pastures fertilized with poultry litter contribute to the degradation of surface water quality in the United States. Dietary modification and manure amendments may reduce potential P runoff losses from pastures. In the current study, broilers were fed a normal diet, phytase diet, high available phosphorus (HAP) corn diet, or HAP corn + phytase diet. Litter treatments were untreated control and alum added at 10% by weight between flocks. Phytase and HAP corn diets reduced litter dissolved P content in poultry litter by 10 and 35%, respectively, compared with the normal diet (789 mg P kg(-1)). Alum treatment of poultry litter reduced the amount of dissolved P by 47%, while a 74% reduction was noted after alum treatment of litter from the HAP corn + phytase diet. The P concentrations in runoff water were highest from plots receiving poultry litter from the normal diet, whereas plots receiving poultry litter from phytase and HAP corn diets had reduced P concentrations. The addition of alum to the various poultry litters reduced P runoff by 52 to 69%; the greatest reduction occurred when alum was used in conjunction with HAP corn and phytase. This study demonstrates the potential added benefits of using dietary modification in conjunction with manure amendments in poultry operations. Integrators and producers should consider the use of phytase, HAP corn, and alum to reduce potential P losses associated with poultry litter application to pastures.

Alum Compounds↗