Modeling of RNA nanotubes using molecular dynamics simulation.

In this study, we construct novel RNA nanoclusters, RNA nanotubes made of several nanorings up to the size of 20 nm, utilizing the molecular dynamics simulation, and study their structural properties [i.e., the root mean square deviation, the radius of gyration and the radial distribution function (RDF)] in physiological solutions that can be used for drug delivery into the human body. The patterns of energy and temperature variations of the systems are also discussed. Furthermore, we study the concentration of ions around the tube as a function of time at a particular temperature. We have found that when the temperature increases, the number of ions increases within a certain distance of the tube. We report that the number of ions within this distance around the tubes decreases in quenched runs. This indicates that some ions evaporate with decrease in temperature, as has been observed in the case of the nanoring. RDF plots also demonstrate a similar trend with temperature, as was found in the case of RNA nanorings.

Ultraviolet B irradiation and activation of protein kinase D in primary mouse epidermal keratinocytes.

Our previous studies demonstrated that protein kinase D (PKD), a serine/threonine kinase implicated in various cell processes, is upregulated in basal cell carcinoma (BCC), supporting a possible tumorigenic role for PKD in skin. As the greatest risk factor for BCC is sun exposure, the ability of ultraviolet B (UVB) irradiation to activate PKD in primary mouse keratinocytes was investigated. Using western analysis with two autophosphorylation-specific antibodies, we show for the first time that UVB activated PKD in a time- and dose-dependent manner. UVB-induced PKD activation was verified using an in vitro kinase assay. Furthermore, activation was reduced by antioxidant pretreatment, suggesting a link with oxidative stress. UVB-induced PKD activation was mediated primarily by Src family tyrosine kinases rather than protein kinase C (PKC), and in fact, UVB did not alter PKC-mediated transphosphorylation. UVB induced apoptosis dose dependently, and this death could be prevented by overexpression of wild-type PKD, but not mutant PKD or the empty adenovirus. Indeed, a mutant that cannot be phosphorylated by Src kinases exacerbated UVB-elicited apoptosis. Thus, our data indicate that UVB irradiation of keratinocytes induces Src-mediated activation of PKD, which protects cells from UVB-stimulated apoptosis, providing a possible explanation for the observed upregulation of PKD in BCC.

Prediction of DNA single-strand conformation polymorphism: analysis by capillary electrophoresis and computerized DNA modeling.

We have analyzed previously three representative p53 single-point mutations by capillary-electrophoresis single-strand conformation polymorphism (CE-SSCP). In the current study, we compared our CE-SSCP results with the potential secondary structures predicted by an RNA/DNA-folding algorithm with DNA energy rules, used in conjunction with a computer analysis workbench called STRUCTURELAB. Each of these mutations produces measurable shifts in CE migration times relative to wild type. Using computerized folding analysis, each of the mutations was found to have a conformational difference relative to wild type, which accounts for the observed differences in CE migration. Additional properties exhibited in the CE electropherograms were also explained using the computerized analysis. These include the appearance of secondary peaks and the temperature dependence of the electrophoretic patterns. The results yield insight into the mechanism of SSCP and how the conditions of this measurement, especially temperature, may be optimized to improve the sensitivity of the SSCP method. The results may also impact other diagnostic methods, which would benefit by a better understanding of DNA single-strand conformation polymorphisms to optimize conditions for enzymatic cleavage and DNA hybridization reactions.

RNA folding pathway functional intermediates: their prediction and analysis.

The massively parallel genetic algorithm (GA) for RNA structure prediction uses the concepts of mutation, recombination, and survival of the fittest to evolve a population of thousands of possible RNA structures toward a solution structure. As described below, the properties of the algorithm are ideally suited to use in the prediction of possible folding pathways and functional intermediates of RNA molecules given their sequences. Utilizing Stem Trace, an interactive visualization tool for RNA structure comparison, analysis of not only the solution ensembles developed by the algorithm, but also the stages of development of each of these solutions, can give strong insight into these folding pathways. The GA allows the incorporation of information from biological experiments, making it possible to test the influence of particular interactions between structural elements on the dynamics of the folding pathway. These methods are used to reveal the folding pathways of the potato spindle tuber viroid (PSTVd) and the host killing mechanism of Escherichia coli plasmid R1, both of which are successfully explored through the combination of the GA and Stem Trace. We also present novel intermediate folds of each molecule, which appear to be phylogenetically supported, as determined by use of the methods described below.

The massively parallel genetic algorithm for RNA folding: MIMD implementation and population variation.

A massively parallel Genetic Algorithm (GA) has been applied to RNA sequence folding on three different computer architectures. The GA, an evolution-like algorithm that is applied to a large population of RNA structures based on a pool of helical stems derived from an RNA sequence, evolves this population in parallel. The algorithm was originally designed and developed for a 16384 processor SIMD (Single Instruction Multiple Data) MasPar MP-2. More recently it has been adapted to a 64 processor MIMD (Multiple Instruction Multiple Data) SGI ORIGIN 2000, and a 512 processor MIMD CRAY T3E. The MIMD version of the algorithm raises issues concerning RNA structure data-layout and processor communication. In addition, the effects of population variation on the predicted results are discussed. Also presented are the scaling properties of the algorithm from the perspective of the number of physical processors utilized and the number of virtual processors (RNA structures) operated upon.

New techniques for DNA sequence classification.

DNA sequence classification is the activity of determining whether or not an unlabeled sequence S belongs to an existing class C. This paper proposes two new techniques for DNA sequence classification. The first technique works by comparing the unlabeled sequence S with a group of active motifs discovered from the elements of C and by distinction with elements outside of C. The second technique generates and matches gapped fingerprints of S with elements of C. Experimental results obtained by running these algorithms on long and well conserved Alu sequences demonstrate the good performance of the presented methods compared with FASTA. When applied to less conserved and relatively short functional sites such as splice-junctions, a variation of the second technique combining fingerprinting with consensus sequence analysis gives better results than the current classifiers employing text compression and machine learning algorithms.

A Boltzmann filter improves the prediction of RNA folding pathways in a massively parallel genetic algorithm.

RNA folding using the massively parallel genetic algorithm (GA) has been enhanced by the addition of a Boltzmann filter. The filter uses the Boltzmann probability distribution in conjunction with Metropolis' relaxation algorithm. The combination of these two concepts within the GA's massively parallel computational environment helps guide the genetic algorithm to more accurately reflect RNA folding pathways and thus final solution structures. Helical regions (base-paired stems) now form in the structures based upon the stochastic properties of the thermodynamic parameters that have been determined from experiments. Thus, structural changes occur based upon the relative energetic impact that the change causes rather than just geometric conflicts alone. As a result, when comparing the predictions to phylogenetically determined structures, over multiple runs, fewer false-positive stems (predicted incorrectly) and more true-positive stems (predicted correctly) are generated, and the total number of predicted stems representing a solution is diminished. In addition, the significance (rate of occurrence) of the true-positive stems is increased. Thus, the predicted results more accurately reflect phylogenetically determined structures.

Predicting RNA H-type pseudoknots with the massively parallel genetic algorithm.

MOTIVATION: Using the genetic algorithm (GA) for RNA folding on a massively parallel supercomputer, MasPar MP-2 with 16,384 processors, we successfully predicted the existence of H-type pseudoknots in several sequences. RESULTS: The GA is applied to folding the tRNA-like 3' end of turnip yellow mosaic virus (TYMV) RNA sequence with 82 nucleotides, the 3' UTRs of satellite tobacco necrosis virus (STNV)-2 RNA sequence with 619 nucleotides and STNV-I RNA sequence with 622 nucleotides, and the bacteriophage T2, T4 and T6 gene 32 mRNA sequences with 946, 1340 and 946 nucleotides, respectively. The GA's results match the phylogenetically supported tertiary structures of these sequences.

Secondary structure computer prediction of the poliovirus 5' non-coding region is improved by a genetic algorithm.

Comparison of the secondary structure of the 5' non-coding region of poliovirus 3 RNA derived from the genetic algorithm with the model of Skinner et al. (J. Mol. Biol., 207, 379-392, 1989) demonstrates many of the confirmed structural elements. The genetic algorithm (Shapiro and Navetta, J. Supercomput., 8, 195-201, 1994) generates a population of all possible stems, then mixes, combines, and recombines these stems in multiple iterations on a massively parallel computer, ultimately selecting a most fit structure based on its energy. The secondary structure of the region containing the determinants of neurovirulence was better predicted using the genetic algorithm, whereas the dynamic programming algorithm (Zuker, Science, 244, 48-52, 1989) required phylogenetic comparative sequence analysis to arrive at the correct conclusion. In addition, artificial mutations were introduced throughout this region of the genome and although rearrangements in structure may occur, many structures persisted, suggesting that the given structures thus selected may have evolved to withstand isolated mutations. The genetic algorithm-derived structure for the 5' non-coding region compares favorably with the biological data and functions previously described, and contains all of the 'persistent' structures, suggesting also that the persistence factor may be an aid to validating structures.

Evaluation of an on-demand, ex vivo bedside blood gas monitor on pulmonary artery blood gas determinations.

Critically ill patients often have cardiopulmonary perturbations that require rapid and frequent assessment for optimal care, including cardiac output determinations, measurement of cardiac filling pressures, and arterial and mixed venous blood gas determinations. We evaluated the performance of a rapid, on-demand bedside blood gas monitor to determine arterial and mixed venous blood gas values. The blood gas monitor uses fluorescent optode technology to directly measure Po2, Pco2, and pH. This measurement is accomplished by aspirating blood from the artery or vein into a sampling chamber where it interfaces with the fluorescent optode. After approximately 90 s of equilibration, the blood gas values are reported. Since the blood is drawn into the sampling chamber, it can be returned to the patient, thus eliminating the need for phlebotomy. We studied 15 critically ill patients requiring systemic and pulmonary arterial catheterization. Conventional blood gas analysis was performed simultaneously. The results obtained from the blood gas monitor were compared with those obtained via traditional blood gas analysis using Bland-Altman plots and examination of bias and precision. The results were well within the expected clinical variance. During the study period, there was no interference with patient care or adverse events related to the use of the monitoring system. In conclusion, the blood gas monitor can provide rapid, accurate determinations of arterial and mixed venous blood gases allowing optimal therapeutic interventions in critically ill patients.

STRUCTURELAB: a heterogeneous bioinformatics system for RNA structure analysis.

STRUCTURELAB is a computational system that has been developed to permit the use of a broad array of approaches for the analysis of the structure of RNA. The goal of the development is to provide a large set of tools that can be well integrated with experimental biology to aid in the process of the determination of the underlying structure of RNA sequences. The approach taken views the structure determination problem as one of dealing with a database of many computationally generated structures and provides the capability to analyze this data set from different perspectives. Many algorithms are integrated into one system that also utilizes a heterogeneous computing approach permitting the use of several computer architectures to help solve the posed problems. These different computational platforms make it relatively easy to incorporate currently existing programs as well as newly developed algorithms and to best match these algorithms to the appropriate hardware. The system has been written in Common Lisp running on SUN or SGI Unix workstations, and it utilizes a network of participating machines defined in reconfigurable tables. A window-based interface makes this heterogeneous environment as transparent to the user as possible.

Microbial contamination of blood conservation devices during routine use in the critical care setting: results of a prospective, randomized trial.

OBJECTIVES: To compare microbial contamination of two different blood conservation devices; to determine if there was an association between contamination of the blood conservation devices and clinical infections; to determine if there was a significant user preference for either of the two devices. DESIGN: Prospective, randomized trial. SETTING: Medical, neurosurgical, and spinal cord intensive care units of an urban, university hospital. PATIENTS: Forty patients who required clinically indicated intrafierial catheters placed at new sites. INTERVENTIONS: The two most widely available blood conservation devices at the time of the study (Venous Arterial blood Management Protection system [VAMP], Baxter Edwards Critical-Care, Irvine, CA; and Safe Draw, Ohmeda, Madison, WI) were chosen for comparison. After the normal 48 to 72 hrs of device use, the blood conservation systems were removed and semi-quantitative and quantitative cultures were taken from comparable sites of the two devices. Positive cultures from the patients were recorded and correlated with cultures obtained from the devices. In order to assess preference for either device, a survey tool was administered to the nursing staff who participated in the study. MEASUREMENTS AND MAIN RESULTS: Quantitative cultures from all sites cultured in both groups demonstrated mean colony counts of < 10(3) colony-forming units (cfu)/mL. There were no statistically significant differences in the colony counts at any of the sites compared between the two groups. There were no statistically significant relationships between positive cultures and patient age, gender, duration of device utilization, frequency of device entry, or the intensive care unit in which the study was conducted. In no circumstance did positive cultures from any of the blood conservation devices correlate with positive culture results from any sites of clinical infection. The clinical survey demonstrated a statistically significant preference for the VAMP system, which persisted despite increased experience with the Safe Draw system. CONCLUSIONS: The levels of microbial contamination noted in these devices were not consistent with clinical infection (defined as 10(3) cfu/mL on quantitative cultures). There was no significant difference in degree or pattern of contamination between the two devices. When utilized and changed according to the Centers for Disease Control guidelines, blood conservation devices are not harbors of infection in the critical care setting. Blood conservation devices can be used as part of a comprehensive blood conservation program in the critical care setting without undue concern for exacerbating infectious processes.