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Biomedical subjects

Ronald W Davis

Publications and source records attributed to Ronald W Davis.

At least 55 records · Page 3Linked to original sources
PubMed2005

Single DNA molecule detection using nanopipettes and nanoparticles.

Single DNA molecules labeled with nanoparticles can be detected by blockades of ionic current as they are translocated through a nanopipette tip formed by a pulled glass capillary. The nanopipette detection technique can provide not only tools for detection and identification of single DNA and protein molecules but also deeper insight and understanding of stochastic interactions of various biomolecules with their environment.

Biosensing Techniques↗
PubMed2005

Highly multiplexed molecular inversion probe genotyping: over 10,000 targeted SNPs genotyped in a single tube assay.

Large-scale genetic studies are highly dependent on efficient and scalable multiplex SNP assays. In this study, we report the development of Molecular Inversion Probe technology with four-color, single array detection, applied to large-scale genotyping of up to 12,000 SNPs per reaction. While generating 38,429 SNP assays using this technology in a population of 30 trios from the Centre d'Etude Polymorphisme Humain family panel as part of the International HapMap project, we established SNP conversion rates of approximately 90% with concordance rates >99.6% and completeness levels >98% for assays multiplexed up to 12,000plex levels. Furthermore, these individual metrics can be "traded off" and, by sacrificing a small fraction of the conversion rate, the accuracy can be increased to very high levels. No loss of performance is seen when scaling from 6,000plex to 12,000plex assays, strongly validating the ability of the technology to suppress cross-reactivity at high multiplex levels. The results of this study demonstrate the suitability of this technology for comprehensive association studies that use targeted SNPs in indirect linkage disequilibrium studies or that directly screen for causative mutations.

Chromosome Inversion↗
PubMed2005

A novel method for STR-based DNA profiling using microarrays.

We describe a novel method for rapidly identifying and distinguishing between different DNA sequences using short tandem repeat (STR) analysis and DNA microarrays. The method can be used to deduce identity, length, and number of STRs of the target molecule. We refer to this technique as the "variable-length probe array" method for STR profiling (VLPA). The method involves hybridization of the unknown STR target sequence to a DNA microarray displaying complementary probes that vary in length to cover the range of possible STRs. A post-hybridization enzymatic digestion of the DNA hybrids is then used to selectively remove labeled single-stranded regions of DNA from the microarray surface. The number of repeats in the unknown target is then deduced based on the pattern of target DNA that remains hybridized to the array. This DNA profiling technique is useful for performing forensic analysis to uniquely identify individual humans or other species.

DNA↗
PubMed2004

A multi-enzyme model for Pyrosequencing.

Pyrosequencing is a DNA sequencing technique based on sequencing-by-synthesis enabling rapid real-time sequence determination. This technique employs four enzymatic reactions in a single tube to monitor DNA synthesis. Nucleotides are added iteratively to the reaction and in case of incorporation, pyrophosphate (PPi) is released. PPi triggers a series of reactions resulting in production of light, which is proportional to the amount of DNA and number of incorporated nucleotides. Generated light is detected and recorded by a detector system in the form of a peak signal, which reflects the activity of all four enzymes in the reaction. We have developed simulations to model the kinetics of the enzymes. These simulations provide a full model for the Pyrosequencing four-enzyme system, based on which the peak height and shape can be predicted depending on the concentrations of enzymes and substrates. Simulation results are shown to be compatible with experimental data. Based on these simulations, the rate-limiting steps in the chain can be determined, and K(M) and kcat of all four enzymes in Pyrosequencing can be calculated.

Apyrase↗
PubMed2004

A transcriptional profile of aging in the human kidney.

In this study, we found 985 genes that change expression in the cortex and the medulla of the kidney with age. Some of the genes whose transcripts increase in abundance with age are known to be specifically expressed in immune cells, suggesting that immune surveillance or inflammation increases with age. The age-regulated genes show a similar aging profile in the cortex and the medulla, suggesting a common underlying mechanism for aging. Expression profiles of these age-regulated genes mark not only age, but also the relative health and physiology of the kidney in older individuals. Finally, the set of aging-regulated kidney genes suggests specific mechanisms and pathways that may play a role in kidney degeneration with age.

Adult↗
PubMed2004

Whole blood and leukocyte RNA isolation for gene expression analyses.

The analysis of gene expression data in clinical medicine has been plagued by the lack of a critical evaluation of accepted methodologies for the collection, processing, and labeling of RNA. In the present report, the reliability of two commonly used techniques to isolate RNA from whole blood or its leukocyte compartment was compared by examining their reproducibility, variance, and signal-to-noise ratios. Whole blood was obtained from healthy subjects and was either untreated or stimulated ex vivo with Staphylococcus enterotoxin B (SEB). Blood samples were also obtained from trauma patients but were not stimulated with SEB ex vivo. Total RNA was isolated from whole blood with the PAXgene proprietary blood collection system or from isolated leukocytes. Biotin-labeled cRNA was hybridized to Affymetrix GeneChips. The Pearson correlation coefficient for gene expression measurements in replicates from healthy subjects with both techniques was excellent, exceeding 0.985. Unsupervised analyses, including hierarchical cluster analysis, however, revealed that the RNA isolation method resulted in greater differences in gene expression than stimulation with SEB or among different trauma patients. The intraclass correlation, a measure of signal-to-noise ratio, of the difference between SEB-stimulated and unstimulated blood from healthy subjects was significantly higher in leukocyte-derived samples than in whole blood: 0.75 vs. 0.46 (P = 0.002). At the P < 0.001 level of significance, twice as many probe sets discriminated between SEB-stimulated and unstimulated blood with leukocyte isolation than with PAXgene. The findings suggest that the method of RNA isolation from whole blood is a critical variable in the design of clinical studies using microarray analyses.

Adolescent↗
PubMed2004

Characterization of synthetic DNA bar codes in Saccharomyces cerevisiae gene-deletion strains.

Incorporation of strain-specific synthetic DNA tags into yeast Saccharomyces cerevisiae gene-deletion strains has enabled identification of gene functions by massively parallel growth rate analysis. However, it is important to confirm the sequences of these tags, because mutations introduced during construction could lead to significant errors in hybridization performance. To validate this experimental system, we sequenced 11,812 synthetic 20-mer molecular bar codes and adjacent sequences (>1.8 megabases synthetic DNA) by pyrosequencing and Sanger methods. At least 31% of the genome-integrated 20-mer tags contain differences from those originally synthesized. However, these mutations result in anomalous hybridization in only a small subset of strains, and the sequence information enables redesign of hybridization probes for arrays. The robust performance of the yeast gene-deletion dual oligonucleotide bar-code design in array hybridization validates the use of molecular bar codes in living cells for tracking their growth phenotype.

DNA Primers↗
PubMed2004

Sister-chromatid cohesion mediated by the alternative RF-CCtf18/Dcc1/Ctf8, the helicase Chl1 and the polymerase-alpha-associated protein Ctf4 is essential for chromatid disjunction during meiosis II.

Cohesion between sister chromatids mediated by a multisubunit complex called cohesin is established during DNA replication and is essential for the orderly segregation of chromatids during anaphase. In budding yeast, a specialized replication factor C called RF-C(Ctf18/Dcc1/Ctf8) and the DNA-polymerase-alpha-associated protein Ctf4 are required to maintain sister-chromatid cohesion in cells arrested for long periods in mitosis. We show here that CTF8, CTF4 and a helicase encoded by CHL1 are required for efficient sister chromatid cohesion in unperturbed mitotic cells, and provide evidence that Chl1 functions during S-phase. We also show that, in contrast to mitosis, RF-C(Ctf18/Dcc1/Cft8), Ctf4 and Chl1 are essential for chromosome segregation during meiosis and for the viability of meiotic products. Our finding that cells deleted for CTF8, CTF4 or CHL1 undergo massive meiosis II non-disjunction suggests that the second meiotic division is particularly sensitive to cohesion defects. Using a functional as well as a cytological assay, we demonstrate that CTF8, CHL1 and CTF4 are essential for cohesion between sister centromeres during meiosis but dispensable for cohesin's association with centromeric DNA. Our finding that mutants in fission yeast ctf18 and dcc1 have similar defects suggests that the involvement of the alternative RF-C(Ctf18/Dcc1/Ctf8) complex in sister chromatid cohesion might be highly conserved.

Chromatids↗
PubMed2004

Integrative analysis of the mitochondrial proteome in yeast.

In this study yeast mitochondria were used as a model system to apply, evaluate, and integrate different genomic approaches to define the proteins of an organelle. Liquid chromatography mass spectrometry applied to purified mitochondria identified 546 proteins. By expression analysis and comparison to other proteome studies, we demonstrate that the proteomic approach identifies primarily highly abundant proteins. By expanding our evaluation to other types of genomic approaches, including systematic deletion phenotype screening, expression profiling, subcellular localization studies, protein interaction analyses, and computational predictions, we show that an integration of approaches moves beyond the limitations of any single approach. We report the success of each approach by benchmarking it against a reference set of known mitochondrial proteins, and predict approximately 700 proteins associated with the mitochondrial organelle from the integration of 22 datasets. We show that a combination of complementary approaches like deletion phenotype screening and mass spectrometry can identify over 75% of the known mitochondrial proteome. These findings have implications for choosing optimal genome-wide approaches for the study of other cellular systems, including organelles and pathways in various species. Furthermore, our systematic identification of genes involved in mitochondrial function and biogenesis in yeast expands the candidate genes available for mapping Mendelian and complex mitochondrial disorders in humans.

Chemical Fractionation↗
PubMed2004

Genomic and proteomic determinants of outcome in patients undergoing thoracoabdominal aortic aneurysm repair.

Thoracoabdominal aortic aneurysm repair, with its requisite intraoperative mesenteric ischemia-reperfusion, often results in the development of systemic inflammatory response syndrome, multiorgan dysfunction syndrome (MODS), and death. In the present study, an adverse clinical outcome following thoracoabdominal aortic aneurysm repair was identified by blood leukocyte genomic and plasma proteomic responses. Time-dependent changes in the expression of 146 genes from blood leukocytes were observed (p < 0.001). Expression of 138 genes (p < 0.001) and the concentration of seven plasma proteins discriminated between patients who developed MODS and those who did not, and many of these differences were evident even before surgery. These findings suggest that changes in blood leukocyte gene expression and plasma protein concentrations can illuminate pathophysiological processes that are subsequently associated with the clinical sequelae of systemic inflammatory response syndrome and MODS. These changes in gene expression and plasma protein concentrations are often observed before surgery, consistent with either a genetic predisposition or pre-existing inflammatory state.

Aged↗
PubMed2004

The diploid genome sequence of Candida albicans.

We present the diploid genome sequence of the fungal pathogen Candida albicans. Because C. albicans has no known haploid or homozygous form, sequencing was performed as a whole-genome shotgun of the heterozygous diploid genome in strain SC5314, a clinical isolate that is the parent of strains widely used for molecular analysis. We developed computational methods to assemble a diploid genome sequence in good agreement with available physical mapping data. We provide a whole-genome description of heterozygosity in the organism. Comparative genomic analyses provide important clues about the evolution of the species and its mechanisms of pathogenesis.

Candida albicans↗
PubMed2004

Ultra-high-efficiency strong cation exchange LC/RPLC/MS/MS for high dynamic range characterization of the human plasma proteome.

High-efficiency nanoscale reversed-phase liquid chromatography (chromatographic peak capacities of approximately 1000: Shen, Y.; Zhao, R.; Berger, S. J.; Anderson, G. A.; Rodriguez, N.; Smith, R. D. Anal. Chem. 2002, 74, 4235. Shen, Y.; Moore, R. J.; Zhao, R.; Blonder, J.; Auberry, D. L.; Masselon, C.; Pasa-Tolic, L.; Hixson, K. K.; Auberry, K. J.; Smith, R. D. Anal. Chem. 2003, 75, 3596.) and strong cation exchange LC was used to obtain ultra-high-efficiency separations (combined chromatographic peak capacities of >10(4)) in conjunction with tandem mass spectrometry (MS/MS) for characterization of the human plasma proteome. Using conservative SEQUEST peptide identification criteria (i.e., without considering chymotryptic or elastic peptides) and peptide LC normalized elution time constraints, the separation quality enabled the identification of proteins over a dynamic range of greater than 8 orders of magnitude in relative abundance using ion trap MS/MS instrumentation. Between 800 and 1682 human proteins were identified, depending on the criteria used for identification, from a total of 365 microg of human plasma. The analyses identified relatively low-level (approximately pg/mL) proteins (e.g., cytokines) coexisting with high-abundance proteins (e.g., mg/mL-level serum albumin).

Amino Acid Sequence↗
PubMed2004

Chemogenomic profiling: identifying the functional interactions of small molecules in yeast.

We demonstrate the efficacy of a genome-wide protocol in yeast that allows the identification of those gene products that functionally interact with small molecules and result in the inhibition of cellular proliferation. Here we present results from screening 10 diverse compounds in 80 genome-wide experiments against the complete collection of heterozygous yeast deletion strains. These compounds include anticancer and antifungal agents, statins, alverine citrate, and dyclonine. In several cases, we identified previously known interactions; furthermore, in each case, our analysis revealed novel cellular interactions, even when the relationship between a compound and its cellular target had been well established. In addition, we identified a chemical core structure shared among three therapeutically distinct compounds that inhibit the ERG24 heterozygous deletion strain, demonstrating that cells may respond similarly to compounds of related structure. The ability to identify on-and-off target effects in vivo is fundamental to understanding the cellular response to small-molecule perturbants.

Antifungal Agents↗
PubMed2004

A genome-wide study of gene activity reveals developmental signaling pathways in the preimplantation mouse embryo.

The preimplantation development of the mammalian embryo encompasses a series of critical events: the transition from oocyte to embryo, the first cell divisions, the establishment of cellular contacts, the first lineage differentiation-all the first subtle steps toward a future body plan. Here, we use microarrays to explore gene activity during preimplantation development. We reveal robust and dynamic patterns of stage-specific gene activity that fall into two major phases, one up to the 2-cell stage (oocyte-to-embryo transition) and one after the 4-cell stage (cellular differentiation). The mouse oocyte and early embryo express components of multiple signaling pathways including those downstream of Wnt, BMP, and Notch, indicating that conserved regulators of cell fate and pattern formation are likely to function at the earliest embryonic stages. Overall, these data provide a detailed temporal profile of gene expression that reveals the richness of signaling processes in early mammalian development.

Animals↗
PubMed2004

SNP discovery in pooled samples with mismatch repair detection.

A targeted discovery effort is required to identify low frequency single nucleotide polymorphisms (SNPs) in human coding and regulatory regions. We here describe combining mismatch repair detection (MRD) with dideoxy terminator sequencing to detect SNPs in pooled DNA samples. MRD enriches for variant alleles in the pooled sample, and sequencing determines the nature of the variants. By using a genomic DNA pool as a template, approximately 100 fragments were amplified and subsequently combined and subjected en masse to the MRD procedure. The variant-enriched pool from this one MRD reaction is enriched for the population variants of all the tested fragments. Each fragment was amplified from the variant-enriched pool and sequenced, allowing the discovery of alleles with frequencies as low as 1% in the initial population. Our results support that MRD-based SNP discovery can be used for large-scale discovery of SNPs at low frequencies in a population.

Animals↗
PubMed2004

Regulatory networks affected by iron availability in Candida albicans.

Iron, an essential element for almost every organism, serves as a regulatory signal for the expression of virulence determinants in many prokaryotic and eukaryotic pathogens. Using a custom Affymetrix GeneChip representing the entire Candida albicans genome, we examined the changes in genome-wide gene expression in this opportunistic pathogen as a function of alterations in environmental concentrations of iron. A total of 526 open reading frame (ORF) transcripts are more highly expressed when the levels of available iron are low, while 626 ORF transcripts are more highly expressed in high-iron conditions. The transcripts dominantly affected by iron concentration range from those associated with cell-surface properties to others which affect mitochondrial function, iron transport and virulence-related secreted hydrolases. Moreover gene expression as assayed in DNA microarrays confirms and extends reports of alterations in cell-surface antigens and drug sensitivity correlated with iron availability. To understand how these genes and pathways might be regulated, we isolated a gene designated SFU1 that encodes a homologue of the Ustilago maydis URBS1, a transcriptional repressor of siderophore uptake/biosynthesis. Comparisons between wild-type and SFU1-null mutant strains revealed 139 potential target genes of Sfu1p; many of which are iron-responsive. Together, these results not only expand our understanding of global iron regulation in C. albicans, but also provide insights into the potential role of iron availability in C. albicans virulence.

Amino Acid Sequence↗