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PubMed2005

Accurate multiplex polony sequencing of an evolved bacterial genome.

We describe a DNA sequencing technology in which a commonly available, inexpensive epifluorescence microscope is converted to rapid nonelectrophoretic DNA sequencing automation. We apply this technology to resequence an evolved strain of Escherichia coli at less than one error per million consensus bases. A cell-free, mate-paired library provided single DNA molecules that were amplified in parallel to 1-micrometer beads by emulsion polymerase chain reaction. Millions of beads were immobilized in a polyacrylamide gel and subjected to automated cycles of sequencing by ligation and four-color imaging. Cost per base was roughly one-ninth as much as that of conventional sequencing. Our protocols were implemented with off-the-shelf instrumentation and reagents.

Acrylic Resins↗
PubMed1999

Identification of mycobacterial species by comparative sequence analysis of the RNA polymerase gene (rpoB).

For the differentiation and identification of mycobacterial species, the rpoB gene, encoding the beta subunit of RNA polymerase, was investigated. rpoB DNAs (342 bp) were amplified from 44 reference strains of mycobacteria and clinical isolates (107 strains) by PCR. The nucleotide sequences were directly determined (306 bp) and aligned by using the multiple alignment algorithm in the MegAlign package (DNASTAR) and the MEGA program. A phylogenetic tree was constructed by the neighbor-joining method. Comparative sequence analysis of rpoB DNAs provided the basis for species differentiation within the genus Mycobacterium. Slowly and rapidly growing groups of mycobacteria were clearly separated, and each mycobacterial species was differentiated as a distinct entity in the phylogenetic tree. Pathogenic Mycobacterium kansasii was easily differentiated from nonpathogenic M. gastri; this differentiation cannot be achieved by using 16S rRNA gene (rDNA) sequences. By being grouped into species-specific clusters with low-level sequence divergence among strains of the same species, all of the clinical isolates could be easily identified. These results suggest that comparative sequence analysis of amplified rpoB DNAs can be used efficiently to identify clinical isolates of mycobacteria in parallel with traditional culture methods and as a supplement to 16S rDNA gene analysis. Furthermore, in the case of M. tuberculosis, rifampin resistance can be simultaneously determined.

Amino Acid Sequence↗
PubMed2004

Inversion of thicknesses of multi-layered structures from eddy current testing measurements.

Luquire et al.'s impedance change model of a rectangular cross section probe coil above a structure with an arbitrary number of parallel layers was used to study the principle of measuring thicknesses of multi-layered structures in terms of eddy current testing voltage measurements. An experimental system for multi-layered thickness measurement was developed and several fitting models to formulate the relationships between detected impedance/voltage measurements and thickness are put forward using least square method. The determination of multi-layered thicknesses was investigated after inversing the voltage outputs of the detecting system. The best fitting and inversion models are presented.

Algorithms↗
PubMed2004

Generality of the structurally constrained protein evolution model: assessment on representatives of the four main fold classes.

The Structurally Constrained Protein Evolution (SCPE) model simulates protein evolution by introducing random mutations into the evolving sequences and selecting them against too much structural perturbation. Given a single protein structure, the SCPE model can be used to obtain a whole set of site-dependent amino acid substitution matrices. The set of SCPE substitution matrices for a given protein family can be seen as an independent-sites model of evolution for that family. Thus, these matrices can be compared with other substitution-matrix-based models of evolution. So far, SCPE has been tested only on left-handed parallel beta helix (LbetaH) proteins. Here, we address the question of generality by assessing the SCPE model on representatives of the four main classes of folds: alpha, beta, alpha+beta, and alpha/beta. We compare with other models using the likelihood ratio test with parametric bootstrapping. We show that SCPE performs better than the popular JTT model for all cases considered. Furthermore, by considering the relative contributions of mutation and selection, we found that the key to the success of the SCPE model is the selection step.

Algorithms↗
PubMed2004

Practical use of alpha blockade strategy in the management of hypoplastic left heart syndrome following stage one palliation with a Blalock-Taussig shunt.

Alpha blockade with phenoxybenzamine has been used in the postoperative management after the Norwood operation. The principle of this approach is the increase in systemic cardiac output by maximal dilation of the systemic circulation. This effect results in a more stable parallel circulation through prevention of fluctuations in systemic vascular resistance in the early postoperative period. We have incorporated this approach in our postoperative management protocol and have observed a decrease in sudden cardiac collapse. This article describes the practical use of alpha blockade strategy, its clinical application, and the most common clinical scenarios observed during the initial hours following stage one palliation with a Blalock-Taussig shunt for hypoplastic left heart syndrome in our cardiac surgery unit.

Adrenergic alpha-Antagonists↗
PubMed1992

Limitations of Augustinsson plots.

The Augustinsson plot transforms the Michaelis-Menten equation by graphing velocity, v, as a function of velocity/substrate, v/s. The plot is linear with a negative slope for a single class of saturable sites and curvilinear for multiple classes of sites. Statistically, Augustinsson plots suffer from the limitation that there is no true independent variable. Experimental errors form a Gaussian distribution that rests neither parallel to the v axis nor v/s axis, but rather along a line emanating from the origin. Widening the range of s values produces increasingly large errors in the calculated Km and Vm values, especially when multiple sites are present. Exact data containing no error produces accurate constants using either the Michaelis-Menten or Augustinsson equation. With Km and Vm constants previously derived by fitting data with the statistically valid Michaelis-Menten equation, the Augustinsson plot may be used accurately to display multiple site data.

Algorithms↗
PubMed2003

Cholinergic reversal of isoflurane anesthesia in rats as measured by cross-approximate entropy of the electroencephalogram.

BACKGROUND: Pharmacologic modulation of the state of consciousness is of interest for clinical practice and for a better understanding of anesthetic mechanisms. The cholinergic activating system is an important regulator of the state of consciousness during general anesthesia. Entropy of the electroencephalogram has been proposed as a promising measure of anesthetic depth. The authors have shown that volatile anesthetics decrease cross-approximate entropy (C-ApEn) of the bihemispheric frontal electroencephalogram in rats. The effect of cholinergic agents on C-ApEn has not been examined. Here, the authors test the hypothesis that cholinergic activation reverses the effect of isoflurane anesthesia on C-ApEn. METHODS: An electroencephalogram in the 1- to 100-Hz range was recorded bipolarly, with epidural leads from the frontal cortex of both hemispheres, and used to calculate C-ApEn, which reflects statistical independence of bihemispheric electroencephalographic activity. Cholinesterase inhibitor, neostigmine (25 mug), or the muscarinic agonist oxotremorine (25 mug) were infused intracerebroventricularly while the rats were inhaling 1.0% (0.7 minimum alveolar concentration) isoflurane. In other animals, isoflurane was lowered to 0.4% (0.3 minimum alveolar concentration) to assess the electroencephalogram in a sedated, waking state. RESULTS: At 1.0% isoflurane, C-ApEn decreased by 54% compared with that at 0.4%, but the motor reflex response to tail pinch was still present. Cholinergic agents reversed the electroencephalogram-depressant effect of isoflurane, i.e., C-ApEn rose to the level measured at 0.4% isoflurane. The rise in C-ApEn was paralleled by the appearance of spontaneous limb and orofacial explorative movements, suggesting a return of consciousness. In contrast, cholinergic agents fully blocked the motor reflex to tail pinch. CONCLUSIONS: C-ApEn of the bihemispheric electroencephalogram correlates with the return of spontaneous motor signs but not with the nociceptive reflex. Cerebral cholinergic activation dissociates central and peripheral anesthetic effects. C-ApEn, a novel measure of interhemispheric electroencephalogram independence, is a promising correlate of depth of sedation and state of consciousness.

Algorithms↗
PubMed2004

The effect of unrealistic thyroid vertical position on thyroid dose in the MIRD phantom.

Anatomically, the thyroid gland is placed in the lower neck, extending from the level of the fifth cervical vertebra down to the first thoracic vertebra. However, the thyroid of the Medical Internal Radiation Dose (MIRD) phantom, which has been widely used for dosimetric calculation, is located right above the top of the torso and is completely included in the neck region. To investigate the effect of the unrealistic position of the thyroid in the MIRD phantom on dose calculation, thyroid absorbed doses at various vertical positions were calculated for the idealized external broad parallel photon beam from anterior-posterior, posterior-anterior, right lateral (RLAT), and left lateral (LLAT) direction using the Monte Carlo method. The thyroid absorbed dose decreased by as much as about 60% for 0.05 MeV photon in both RLAT and LLAT irradiations when the thyroid was relocated to realistic position (inserted into the torso). The effective dose also decreased by 10%, consequently. The thyroid dose of the widely accepted stylized model, the MIRD phantom, is overestimated in RLAT and LLAT irradiation geometries.

Algorithms↗
PubMedDate not supplied

Optimization of the dose delivery in a few field techniques using radiobiological objective functions.

A method for finding optimal primary fluence profiles for multiple field external beam radiation therapy techniques has been developed using a radiobiologically based objective function that quantifies the probability of achieving complication-free tumor control, P+. The objective function P+ has the valuable property of giving the highest possible dose to the tumor without causing severe damage to normal tissues at risk. This radiobiologically based objective function selects suitable dose levels but also takes into account the dose homogeneity in the target volume to the extent that it is not causing an excessive risk of local recurrence or damage to surrounding normal tissues. The biological parameters used can either be patient specific, as determined by a predictive assay on biopsy specimens, or taken from a library of radiobiological parameter values characteristic for different tissue types of a reference patient. In its present form the method can be used to determine the optimum incident photon fluence profiles for each beam. The method has been used to investigate for a given target volume a large number of combinations of beam entry directions to find the best beam orientations with respect to the probability of achieving complication-free tumor control. It is demonstrated that when nonuniform dose delivery is available it is unsuitable to combine parallel opposed beams in two-beam techniques and to a lesser extent also to use perpendicular beams. In two-beam techniques the best angle between the beams is generally in the 100 degrees-120 degrees range. The major symmetry characteristics of the P+ phase space for two-beam techniques are also identified. The method can easily be extended from two to three dimensions and noncoplanar geometry, but it is presented here in its two-dimensional form for clarity and speed of calculation.

Algorithms↗
PubMed2003

Pumping of drugs by P-glycoprotein: a two-step process?

The apparent inhibition constant, Kapp, for the blockade of P-glycoprotein (P-gp) by four drugs, verapamil, cyclosporin A, XR9576 (tariquidar), and vinblastine, was measured by studying their ability to inhibit daunorubicin and calcein-AM efflux from four strains of Ehrlich cells with different levels of drug resistance and P-gp content. For daunorubicin as a transport substrate, Kapp was independent of [P-gp] for verapamil but increased strictly linearly with [P-gp] for vinblastine, cyclosporin A, and XR9576. A theoretical analysis of the kinetics of drug pumping and its reversal shows that Kapp for inhibition should increase linearly with the amount of pumps present in the membrane for a reverser that inhibits pumping from the cytoplasmic face. In contrast, if the reverser acts by blocking transport from the outer face, i.e., preemptively, Kapp should be independent of the number of pumps present. The experimental data suggest that verapamil blocks pumping at the extracellular face of the membrane, whereas the other three blockers act on pumping from the cytoplasmic phase. The maximum degree of inhibition was the same for all four blockers; thus, they do not act in parallel but rather, in serial, i.e., a drug that is pumped from the cytoplasmic phase has to pass the preemptive route upon leaving the cell. Our results are consistent with the Sauna-Ambudkar two-step model for pumping by P-gp. We suggest that the vinblastine/cyclosporin A/XR9576-binding site accepts daunorubicin at the cytoplasmic face and transfers it to the verapamil-binding site, from where daunorubicin is emptied at the extracellular surface.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2006

Towards computing with proteins.

Can proteins be used as computational devices to address difficult computational problems? In recent years there has been much interest in biological computing, that is, building a general purpose computer from biological molecules. Most of the current efforts are based on DNA because of its ability to self-hybridize. The exquisite selectivity and specificity of complex protein-based networks motivated us to suggest that similar principles can be used to devise biological systems that will be able to directly implement any logical circuit as a parallel asynchronous computation. Such devices, powered by ATP molecules, would be able to perform, for medical applications, digital computation with natural interface to biological input conditions. We discuss how to design protein molecules that would serve as the basic computational element by functioning as a NAND logical gate, utilizing DNA tags for recognition, and phosphorylation and exonuclease reactions for information processing. A solution of these elements could carry out effective computation. Finally, the model and its robustness to errors were tested in a computer simulation.

Adenosine Triphosphate↗
PubMed2006

Time delay and partial coherence analyses to identify cortical connectivities.

Recently it has been demonstrated by Albo that partial coherence analysis is sensitive to signal to noise ratio (SNR) and that it will always identify the signal with the highest SNR among the three signals as the main (driving) influence. We propose to use time delay analysis in parallel to partial coherence analysis to identify the connectivities between the multivariate time series. Both are applied to a theoretical model (used by Albo) to analyse the connections introduced in the model. Time delay analysis identifies the connections correctly. We also apply these analyses to the electroencephalogram (EEG) and electromyogram of essential tremor patients and EEG of normal subjects while bimanually tapping their index fingers. Biologically plausible cortico-muscular and cortico-cortical connections are identified by these methods.

Algorithms↗
PubMed1995

Computing with dynamic attractors in neural networks.

In this paper we report on some new architectures for neural computation, motivated in part by biological considerations. One of our goals is to demonstrate that it is just as easy for a neural net to compute with arbitrary attractors--oscillatory or chaotic--as with the more usual asymptotically stable fixed points. The advantages (if any) of such architectures are currently being investigated; but it seems reasonable that the much richer dynamics of recurrent networks, so obvious in recordings of brain activity, must be useful for something. On the other hand, the constraints of computing with biological wet-ware may make chaotic dynamics unavoidable in complex nervous systems. We hypothesize also that the as yet unrivaled capabilities of the human brain derive from an ability to integrate both analog intuitive pattern recognition operations, and digital symbolic logical operations at the ground level of its hardware. To investigate these possibilities, we have constructed a parallel distributed processing architecture inspired by the structure and dynamics of cerebral cortex. The construction assumes that cortex is a set of coupled associative memories with dynamic attractors. It is guided also by a particular concept of the physical structure required of macroscopic computational systems in general for reliable computation in the presence of noise. Our challenge is to accomplish real tasks that brains can do, using ordinary differential equations, in networks that are as faithful as possible to the known anatomy and dynamics of cortex.

Algorithms↗
PubMed2003

[Adaptation of control mechanisms involved in upright undisturbed stance maintenance during prolonged darkness].

AIMS OF THE STUDY: To assess to which extent the non visual somato-sensorial information may, through a recalibration process, induce a reorganisation by the central nervous system to control undisturbed upright stance. MATERIALS AND METHODS: Ten healthy adults were placed in complete darkness for a 24 min period. Their postural performance was recorded through a force platform on which they were required to stand still at regular intervals. Centre of Pressure (CP) displacements, recorded from the platform, were modelled as fractional brownian motion. Through this analysis, one may objectively assess from which distance and for how long the corrective process is initiated with the aim of slowing and retrace its steps. In addition, the degree to which the CP trajectories are successively controlled was determined. RESULTS: Once in complete darkness, an increase of the mean time intervals (Delta(t)) before the corrective process intervenes was observed, the effect being mostly significant for the mediolateral direction. In parallel, the mean distances covered at this Delta(t) were slightly affected for both mediolateral and anteroposterior directions. Lastly, the degree to which the CP trajectories are controlled tended to decrease. CONCLUSION: These data suggest a reorganisation of the control mechanisms called into play for maintaining an undisturbed upright stance, thus implying participation of the central nervous system. This short-term adaptation is discussed on the basis of our knowledge of long term adaptations previously observed in blind individuals, and also in a rehabilitation perspective.

Adaptation, Physiological↗
PubMed1998

[Prevention and treatment of headache due to intracranial hypotension].

INTRODUCTION: In spite of the large number of authors who have studied headache following lumbar puncture and spontaneous intra-cranial hypotension, many aspects remain little known, and there is still no completely satisfactory non-invasive treatment. DEVELOPMENT: In one group of patients there is an increased risk of post lumbar-puncture headache: adults between 20 and 50 years old, of low body weight and a previous history of chronic bilateral headache. In these patients all possible prophylactic measures should be taken whilst the lumbar puncture is being performed, so as to avoid the onset of headache. Use of modern atraumatic needles of small diameter and with a lateral opening have been shown by many studies to be satisfactory. There are also other manoevres which help to reduce the frequency of post lumbar-puncture headache, such as the insertion and withdrawal of the needle with the bevel parallel to the fibres of the dura mater and reinsertion of the stylet before withdrawing the needle. Once the patient has developed post lumbar-puncture headache, or spontaneous intracranial hypotension, initially treatment with cerebral vasoconstrictors such as oral caffeine may be used. After a period of time (2-4 weeks) which makes spontaneous regression of the headache unlikely, the treatment of choice involves epidural patches. CONCLUSION: Well-designed studies are necessary to demonstrate the efficacy of oral caffeine (and other drugs) in post lumbar-puncture headache.

Algorithms↗
PubMed2006

Parallel processing of large datasets from NanoLC-FTICR-MS measurements.

A new approach for automatic parallel processing of large mass spectral datasets in a distributed computing environment is demonstrated to significantly decrease the total processing time. The implementation of this novel approach is described and evaluated for large nanoLC-FTICR-MS datasets. The speed benefits are determined by the network speed and file transfer protocols only and allow almost real-time analysis of complex data (e.g., a 3-gigabyte raw dataset is fully processed within 5 min). Key advantages of this approach are not limited to the improved analysis speed, but also include the improved flexibility, reproducibility, and the possibility to share and reuse the pre- and postprocessing strategies. The storage of all raw data combined with the massively parallel processing approach described here allows the scientist to reprocess data with a different set of parameters (e.g., apodization, calibration, noise reduction), as is recommended by the proteomics community. This approach of parallel processing was developed in the Virtual Laboratory for e-Science (VL-e), a science portal that aims at allowing access to users outside the computer research community. As such, this strategy can be applied to all types of serially acquired large mass spectral datasets such as LC-MS, LC-MS/MS, and high-resolution imaging MS results.

Algorithms↗
PubMed2003

Analysis of frequency components of cortical potentials evoked by progressive misalignment of Kanizsa squares.

Cortical gamma oscillations (20-100 Hz) are thought to play an important role in encoding visual perception. If so they should emerge at about threshold. In the present investigation we examined the latter proposal. Visual responses were recorded in occipital, temporal and parietal areas (stimulus duration 512 ms). Oscillation strength and frequency were derived from FFT analysis and wavelet transforms. The specific goals of the present study are: 1: To examine the parallel between gamma oscillations and the psychometric threshold of perception of Kanizsa square (KS). The latter is gradually altered by a progressive misalignment of lower inducers (pacmen). Results show that the perception of the KS is altered by lateral displacements of the lower inducers as small as 0.1 to 0.2 degrees. In parallel, high frequency components of cortical responses gain in strength with misalignments. 2: Gamma oscillations emerge at or about the psychometric threshold. In addition, our data analysis demonstrates that gamma oscillations appear in short bursts (approx. 50 ms) in the time window between 200 and 500 ms after stimulus onset. Furthermore, controls indicated that these oscillations are of the induced-gamma type. Thus, our experiments suggest that gamma oscillations are associated with image structures and may be induced by local properties of the target.

Adult↗
PubMed2006

Time-dependent hierarchical regulation analysis: deciphering cellular adaptation.

Cells adapt to changes in their environment by the concerted action of many different regulatory mechanisms. Examples of such mechanisms are feedback inhibition by intermediates of metabolism, covalent modification of enzymes and changes in the abundance of mRNAs and proteins. These mechanisms act in parallel at different levels in the cellular hierarchy while regulating a single process. Existing hierarchical regulation analysis determines the relative importance of these mechanisms when the cell regulates a transition from one steady-state to another. Here, the analysis is extended to the regulation of time-dependent phenomena, for which two methods are introduced and illustrated with a kinetic model incorporating transcription and translation of metabolic enzymes.

Adaptation, Physiological↗

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