Biomedical subjects

Lukas M Wick

Publications and source records attributed to Lukas M Wick.

6 recordsLinked to original sources

PubMed2006

Influence of dangling ends and surface-proximal tails of targets on probe-target duplex formation in 16S rRNA gene-based diagnostic arrays.

Dangling ends and surface-proximal tails of gene targets influence probe-target duplex formation and affect the signal intensity of probes on diagnostic microarrays. This phenomenon was evaluated using an oligonucleotide microarray containing 18-mer probes corresponding to the 16S rRNA genes of 10 waterborne pathogens and a number of synthetic and PCR-amplified gene targets. Signal intensities for Klenow/random primer-labeled 16S rRNA gene targets were dissimilar from those for 45-mer synthetic targets for nearly 73% of the probes tested. Klenow/random primer-labeled targets resulted in an interaction with a complex mixture of 16S rRNA genes (used as the background) 3.7 times higher than the interaction of 45-mer targets with the same mixture. A 7-base-long dangling end sequence with perfect homology to another single-stranded background DNA sequence was sufficient to produce a cross-hybridization signal that was as strong as the signal obtained by the probe-target duplex itself. Gibbs free energy between the target and a well-defined background was found to be a better indicator of hybridization signal intensity than the sequence or length of the dangling end alone. The dangling end (Gibbs free energy of -7.6 kcal/mol) was found to be significantly more prone to target-background interaction than the surface-proximal tail (Gibbs free energy of -64.5 kcal/mol). This study underlines the need for careful target preparation and evaluation of signal intensities for diagnostic arrays using 16S rRNA and other gene targets due to the potential for target interaction with a complex background.

Bacteria↗

PubMed2006

On-chip non-equilibrium dissociation curves and dissociation rate constants as methods to assess specificity of oligonucleotide probes.

Nucleic acid hybridization serves as backbone for many high-throughput systems for detection, expression analysis, comparative genomics and re-sequencing. Specificity of hybridization between probes and intended targets is always critical. Approaches to ensure and evaluate specificity include use of mismatch probes, obtaining dissociation curves rather than single temperature hybridizations, and comparative hybridizations. In this study, we quantify effects of mismatch type and position on intensity of hybridization signals and provide a new approach based on dissociation rate constants to evaluate specificity of hybridized signals in complex target mixtures. Using an extensive set of 18mer oligonucleotide probes on an in situ synthesized biochip platform, we demonstrate that mismatches in the center of the probe are more discriminating than mismatches toward the extremities of the probe and mismatches toward the attached end are less discriminating than those toward the loose end. The observed destabilizing effect of a mismatch type agreed in general with predictions using the nearest neighbor model. Use of a new parameter, specific dissociation temperature (T(d-w), temperature of maximum specific dissociation rate constant), obtained from probe-target duplex dissociation profiles considerably improved the evaluation of specificity. These results have broad implications for hybridization data obtained from complex mixtures of nucleic acids.

Base Pair Mismatch↗

PubMed2005

Evolution of genomic content in the stepwise emergence of Escherichia coli O157:H7.

Genome comparisons have demonstrated that dramatic genetic change often underlies the emergence of new bacterial pathogens. Evolutionary analysis of Escherichia coli O157:H7, a pathogen that has emerged as a worldwide public health threat in the past two decades, has posited that this toxin-producing pathogen evolved in a series of steps from O55:H7, a recent ancestor of a nontoxigenic pathogenic clone associated with infantile diarrhea. We used comparative genomic hybridization with 50-mer oligonucleotide microarrays containing probes from both pathogenic and nonpathogenic genomes to infer when genes were acquired and lost. Many ancillary virulence genes identified in the O157 genome were already present in an O55:H7-like progenitor, with 27 of 33 genomic islands of >5 kb and specific for O157:H7 (O islands) that were acquired intact before the split from this immediate ancestor. Most (85%) of variably absent or present genes are part of prophages or phage-like elements. Divergence in gene content among these closely related strains was approximately 140 times greater than divergence at the nucleotide sequence level. A >100-kb region around the O-antigen gene cluster contained highly divergent sequences and also appears to be duplicated in its entirety in one lineage, suggesting that the whole region was cotransferred in the antigenic shift from O55 to O157. The beta-glucuronidase-positive O157 variants, although phylogenetically closest to the Sakai strain, were divergent for multiple adherence factors. These observations suggest that, in addition to gains and losses of phage elements, O157:H7 genomes are rapidly diverging and radiating into new niches as the pathogen disseminates.

Chromosome Mapping↗

PubMed2004

Potential of DNA microarrays for developing parallel detection tools (PDTs) for microorganisms relevant to biodefense and related research needs.

Development of parallel detection tools using microarrays is critically reviewed in view of the need for screening multiple microorganisms in a single test. Potential research needs with respect to probe design and specificity, validation, sample concentration, selective target enrichment and amplification, and data analysis are discussed. Data illustrating selected probe design issues for detecting multiple targets in mixed microbial systems is presented. Challenges with respect to cost, time, and ease of use compared to other methods are also summarized.

Animals↗

PubMed2004

Molecular components of physiological stress responses in Escherichia coli.

In order to survive under and adapt to different conditions Escherichia coli has evolved elaborate systems that are able to sense and respond to environmental stimuli. Very often, different stresses act on a bacterium simultaneously and a variety of stresses have similar effects on cellular molecules and processes. Therefore, the various stress response systems have to interact (cross talk) with each other. A complex network of global regulatory systems with a multitude of molecular components ensures a coordinated and effective answer. Such regulatory components include DNA, mRNAs, sRNAs, proteins, such as DNA-and RNA binding proteins, alternative sigma factors and two-component systems, as well as small molecular weight molecules, as for example (p)ppGpp. These regulatory systems govern the expression of a plethora of further effectors that aim at maintaining stability of the cellular equilibrium under the various conditions. Using five of the most important stress response systems, we will discuss the roles and mechanisms of such regulatory and effector molecules in more detail. The heat shock response, controlled by the sigma factor sigma32, and the envelope stress response, controlled by the sigma factor sigmaE and the Cpx two-component system, both result in an increased expression of chaperones and proteases in response to misfolded proteins. The cold shock response governs expression of RNA chaperones and ribosomal factors, ensuring accurate translation at low temperatures. The general stress response depends on the sigma factor sigmaS, which controls the expression of more than 50 genes conferring resistance to many different stresses. The (p)ppGpp-dependent stringent response reduces the cellular protein synthesis capacity and controls further global responses upon nutritional downshift. A lot has been learned in recent years about the mechanisms of action of single components. However, the main challenge for the future is to achieve an understanding of the interactions of these components under different physiological conditions.

Adaptation, Physiological↗

PubMed2002

The apparent clock-like evolution of Escherichia coli in glucose-limited chemostats is reproducible at large but not at small population sizes and can be explained with Monod kinetics.

To follow and model evolution of a microbial population in the chemostat, parameters are needed that give an indication of the absolute extent of evolution at a high resolution of time. In this study the evolution of the maximum specific growth rate ( micro (max)) and the residual glucose concentration was followed for populations of Escherichia coli K-12 under glucose-limited conditions at dilution rates of 0.1 x h(-1), 0.3 x h(-1) and 0.53 x h(-1) during 500-700 h in continuous culture. Whereas micro (max) improved only during the initial 150 h, the residual glucose concentration decreased constantly during 500 h of cultivation and therefore served as a convenient parameter to monitor the evolution of a population at a high time resolution with respect to its affinity for the growth-limiting substrate. The evolution of residual glucose concentrations was reproducible in independent chemostats with a population size of 10(11) cells, whereas no reproducibility was found in chemostats containing 10(7) cells. A model based on Monod kinetics assuming successive take-overs of mutants with improved kinetic parameters (primarily K(s)) was able to simulate the experimentally observed evolution of residual glucose concentrations. Similar values for the increase in glucose affinity of mutant phenotypes (K(s(mutant)) approximately equal 0.6 x K(s(parent))) and similar mutation rates per cell per generation leading to these mutant phenotypes (1-5 x1 0(-7)) were estimated in silico for all dilution rates. The model predicts a maximum rate of evolution at a dilution rate slightly below micro (max)/2. With increasing and decreasing dilution rates the evolution slows down, which also explains why in special cases a selection-driven evolution can exhibit apparent clock-like behaviour. The glucose affinity for WT cells was dependent on the dilution rate with highest values at dilution rates around micro (max)/2. Below 0.3 x h(-1) poorer affinity was mainly due to the effects of rpoS.

Adaptation, Physiological↗