SC-PROV: A Provenance Vocabulary for Social Computation

The research described here is supported by the award made by the RCUK Digital Economy programme to the dot.rural Digital Economy Hub; award reference: EP/G066051/1.

Yeast microarrays for genome wide parallel genetic and gene expression analysis

We have developed high-density DNA microarrays of yeast ORFs. These microarrays can monitor hybridization to ORFs for applications such as quantitative differential gene expression analysis and screening for sequence polymorphisms. Automated scripts retrieved sequence information from public databases to locate predicted ORFs and select appropriate primers for amplification. The primers were used to amplify yeast ORFs in 96-well plates, and the resulting products were arrayed using an automated micro arraying device. Arrays containing up to 2,479 yeast ORFs were printed on a single slide. The hybridization of fluorescently labeled samples to the array were detected and quantitated with a laser confocal scanning microscope. Applications of the microarrays are shown for genetic and gene expression analysis at the whole genome level.

Patches of synchronized activity in the cerebellar cortex evoked by mossy-fiber stimulation: Questioning the role of parallel fibers

The discrepancy between the structural longitudinal organization of the parallel-fiber system in the cerebellar cortex and the functional mosaic-like organization of the cortex has provoked controversial theories about the flow of information in the cerebellum. We address this issue by characterizing the spatiotemporal organization of neuronal activity in the cerebellar cortex by using optical imaging of voltage-sensitive dyes in isolated guinea-pig cerebellum. Parallel-fiber stimulation evoked a narrow beam of activity, which propagated along the parallel fibers. Stimulation of the mossy fibers elicited a circular, nonpropagating patch of synchronized activity. These results strongly support the hypothesis that a beam of parallel fibers, activated by a focal group of granule cells, fails to activate the Purkinje cells along most of its length. It is thus the ascending axon of the granule cell, and not its parallel branches, that activates and defines the basic functional modules of the cerebellar cortex.

Propagating structure of Alzheimer’s β-amyloid(10–35) is parallel β-sheet with residues in exact register

The pathognomonic plaques of Alzheimer’s disease are composed primarily of the 39- to 43-aa β-amyloid (Aβ) peptide. Crosslinking of Aβ peptides by tissue transglutaminase (tTg) indicates that Gln15 of one peptide is proximate to Lys16 of another in aggregated Aβ. Here we report how the fibril structure is resolved by mapping interstrand distances in this core region of the Aβ peptide chain with solid-state NMR. Isotopic substitution provides the source points for measuring distances in aggregated Aβ. Peptides containing a single carbonyl 13C label at Gln15, Lys16, Leu17, or Val18 were synthesized and evaluated by NMR dipolar recoupling methods for the measurement of interpeptide distances to a resolution of 0.2 Å. Analysis of these data establish that this central core of Aβ consists of a parallel β-sheet structure in which identical residues on adjacent chains are aligned directly, i.e., in register. Our data, in conjunction with existing structural data, establish that the Aβ fibril is a hydrogen-bonded, parallel β-sheet defining the long axis of the Aβ fibril propagation.

Transformation by v-Src: Ras-MAPK and PI3K-mTOR Mediate Parallel Pathways

An increase in the level of active, GTP-bound Ras is not necessary for transformation of chicken embryo fibroblasts (CEF) by v-Src. This suggests that other Ras-independent pathways contribute to transformation by v-Src. To address the possibility that activation of phosphatidylinositol-3-kinase (PI3K) and the mammalian target of rapamycin (mTOR/FRAP), represents one of these pathways, we have examined the effect of simultaneous inhibition of the Ras-MAPK and PI3K-mTOR pathways on transformation of CEF by v-Src. Transformation was assessed by the standard parameters of morphological alteration, increased hexose uptake, loss of density inhibition, and anchorage-independent growth. Inhibition of the Ras-MAPK pathway by expression of the dominant-negative Ras mutant HRasN17 or by addition of the MAPK kinase (MEK) inhibitor PD98059 reduced several of these parameters but failed to block transformation. Similarly, inhibition of the PI3K-mTOR pathway by addition of the PI3K inhibitor 2-[4-morpholinyl]-8-phenyl-4H-1-benzopyran-4-one (LY294002) or the mTOR inhibitor rapamycin, although reducing several parameters of transformation, also failed to block transformation. However, simultaneous inhibition of signaling by the Ras-MAPK pathway and the PI3K-mTOR pathway essentially blocked transformation. These data indicate that transformation of CEF by v-Src is mediated by two parallel pathways, the Ras-MAPK pathway and the PI-3K-mTOR pathway, which both contribute to transformation. The possibility that simultaneous activation of other pathways is also required is not excluded.

Automated parallel DNA sequencing on multiple channel microchips

We report automated DNA sequencing in 16-channel microchips. A microchip prefilled with sieving matrix is aligned on a heating plate affixed to a movable platform. Samples are loaded into sample reservoirs by using an eight-tip pipetting device, and the chip is docked with an array of electrodes in the focal plane of a four-color scanning detection system. Under computer control, high voltage is applied to the appropriate reservoirs in a programmed sequence that injects and separates the DNA samples. An integrated four-color confocal fluorescent detector automatically scans all 16 channels. The system routinely yields more than 450 bases in 15 min in all 16 channels. In the best case using an automated base-calling program, 543 bases have been called at an accuracy of >99%. Separations, including automated chip loading and sample injection, normally are completed in less than 18 min. The advantages of DNA sequencing on capillary electrophoresis chips include uniform signal intensity and tolerance of high DNA template concentration. To understand the fundamentals of these unique features we developed a theoretical treatment of cross-channel chip injection that we call the differential concentration effect. We present experimental evidence consistent with the predictions of the theory.

Computation, prediction, and experimental tests of fitness for bacteriophage T7 mutants with permuted genomes

We created a simulation based on experimental data from bacteriophage T7 that computes the developmental cycle of the wild-type phage and also of mutants that have an altered genome order. We used the simulation to compute the fitness of more than 105 mutants. We tested these computations by constructing and experimentally characterizing T7 mutants in which we repositioned gene 1, coding for T7 RNA polymerase. Computed protein synthesis rates for ectopic gene 1 strains were in moderate agreement with observed rates. Computed phage-doubling rates were close to observations for two of four strains, but significantly overestimated those of the other two. Computations indicate that the genome organization of wild-type T7 is nearly optimal for growth: only 2.8% of random genome permutations were computed to grow faster, the highest 31% faster, than wild type. Specific discrepancies between computations and observations suggest that a better understanding of the translation efficiency of individual mRNAs and the functions of qualitatively “nonessential” genes will be needed to improve the T7 simulation. In silico representations of biological systems can serve to assess and advance our understanding of the underlying biology. Iteration between computation, prediction, and observation should increase the rate at which biological hypotheses are formulated and tested.

Estrogen-induced transcription of the progesterone receptor gene does not parallel estrogen receptor occupancy

The activation of the silent endogenous progesterone receptor (PR) gene by 17-β-estradiol (E2) in cells stably transfected with estrogen receptor (ER) was used as a model system to study the mechanism of E2-induced transcription. The time course of E2-induced PR transcription rate was determined by nuclear run-on assays. No marked effect on specific PR gene transcription rates was detected at 0 and 1 h of E2 treatment. After 3 h of E2 treatment, the PR mRNA synthesis rate increased 2.0- ± 0.2-fold and continued to increase to 3.5- ± 0.4-fold by 24 h as compared with 0 h. The transcription rate increase was followed by PR mRNA accumulation. No PR mRNA was detectable at 0, 1, and 3 h of E2 treatment. PR mRNA accumulation was detected at 6 h of E2 treatment and continued to accumulate until 18 h, the longest time point examined. Interestingly, this slow and gradual transcription rate increase of the endogenous PR gene did not parallel binding of E2 to ER, which was maximized within 30 min. Furthermore, the E2–ER level was down-regulated to 15% at 3 h as compared with 30 min of E2 treatment and remained low at 24 h of E2 exposure. These paradoxical observations indicate that E2-induced transcription activation is more complicated than just an association of the occupied ER with the transcription machinery.

Molecular computation: RNA solutions to chess problems

We have expanded the field of “DNA computers” to RNA and present a general approach for the solution of satisfiability problems. As an example, we consider a variant of the “Knight problem,” which asks generally what configurations of knights can one place on an n × n chess board such that no knight is attacking any other knight on the board. Using specific ribonuclease digestion to manipulate strands of a 10-bit binary RNA library, we developed a molecular algorithm and applied it to a 3 × 3 chessboard as a 9-bit instance of this problem. Here, the nine spaces on the board correspond to nine “bits” or placeholders in a combinatorial RNA library. We recovered a set of “winning” molecules that describe solutions to this problem.

Multiple quantum solid-state NMR indicates a parallel, not antiparallel, organization of β-sheets in Alzheimer's β-amyloid fibrils

Senile plaques associated with Alzheimer's disease contain deposits of fibrils formed by 39- to 43-residue β-amyloid peptides with possible neurotoxic effects. X-ray diffraction measurements on oriented fibril bundles have indicated an extended β-sheet structure for Alzheimer's β-amyloid fibrils and other amyloid fibrils, but the supramolecular organization of the β-sheets and other structural details are not well established because of the intrinsically noncrystalline, insoluble nature of amyloid fibrils. Here we report solid-state NMR measurements, using a multiple quantum (MQ) 13C NMR technique, that probe the β-sheet organization in fibrils formed by the full-length, 40-residue β-amyloid peptide (Aβ1–40). Although an antiparallel β-sheet organization often is assumed and is invoked in recent structural models for full-length β-amyloid fibrils, the MQNMR data indicate an in-register, parallel organization. This work provides site-specific, atomic-level structural constraints on full-length β-amyloid fibrils and applies MQNMR to a significant problem in structural biology.

How the owl resolves auditory coding ambiguity

The barn owl (Tyto alba) uses interaural time difference (ITD) cues to localize sounds in the horizontal plane. Low-order binaural auditory neurons with sharp frequency tuning act as narrow-band coincidence detectors; such neurons respond equally well to sounds with a particular ITD and its phase equivalents and are said to be phase ambiguous. Higher-order neurons with broad frequency tuning are unambiguously selective for single ITDs in response to broad-band sounds and show little or no response to phase equivalents. Selectivity for single ITDs is thought to arise from the convergence of parallel, narrow-band frequency channels that originate in the cochlea. ITD tuning to variable bandwidth stimuli was measured in higher-order neurons of the owl’s inferior colliculus to examine the rules that govern the relationship between frequency channel convergence and the resolution of phase ambiguity. Ambiguity decreased as stimulus bandwidth increased, reaching a minimum at 2–3 kHz. Two independent mechanisms appear to contribute to the elimination of ambiguity: one suppressive and one facilitative. The integration of information carried by parallel, distributed processing channels is a common theme of sensory processing that spans both modality and species boundaries. The principles underlying the resolution of phase ambiguity and frequency channel convergence in the owl may have implications for other sensory systems, such as electrolocation in electric fish and the computation of binocular disparity in the avian and mammalian visual systems.

A model of long-term memory storage in the cerebellar cortex: A possible role for plasticity at parallel fiber synapses onto stellate/basket interneurons

By evoking changes in climbing fiber activity, movement errors are thought to modify synapses from parallel fibers onto Purkinje cells (pf*Pkj) so as to improve subsequent motor performance. Theoretical arguments suggest there is an intrinsic tradeoff, however, between motor adaptation and long-term storage. Assuming a baseline rate of motor errors is always present, then repeated performance of any learned movement will generate a series of climbing fiber-mediated corrections. By reshuffling the synaptic weights responsible for any given movement, such corrections will degrade the memories for other learned movements stored in overlapping sets of synapses. The present paper shows that long-term storage can be accomplished by a second site of plasticity at synapses from parallel fibers onto stellate/basket interneurons (pf*St/Bk). Plasticity at pf*St/Bk synapses can be insulated from ongoing fluctuations in climbing fiber activity by assuming that changes in pf*St/Bk synapses occur only after changes in pf*Pkj synapses have built up to a threshold level. Although climbing fiber-dependent plasticity at pf*Pkj synapses allows for the exploration of novel motor strategies in response to changing environmental conditions, plasticity at pf*St/Bk synapses transfers successful strategies to stable long-term storage. To quantify this hypothesis, both sites of plasticity are incorporated into a dynamical model of the cerebellar cortex and its interactions with the inferior olive. When used to simulate idealized motor conditioning trials, the model predicts that plasticity develops first at pf*Pkj synapses, but with additional training is transferred to pf*St/Bk synapses for long-term storage.

Protein-free parallel triple-stranded DNA complex formation

A 14 nt DNA sequence 5′-AGAATGTGGCAAAG-3′ from the zinc finger repeat of the human KRAB zinc finger protein gene ZNF91 bearing the intercalator 2-methoxy,6-chloro,9-amino acridine (Acr) attached to the sugar–phosphate backbone in various positions has been shown to form a specific triple helix (triplex) with a 16 bp hairpin (intramolecular) or a two-stranded (intermolecular) duplex having the identical sequence in the same (parallel) orientation. Intramolecular targets with the identical sequence in the antiparallel orientation and a non-specific target sequence were tested as controls. Apparent binding constants for formation of the triplex were determined by quantitating electrophoretic band shifts. Binding of the single-stranded oligonucleotide probe sequence to the target led to an increase in the fluorescence anisotropy of acridine. The parallel orientation of the two identical sequence segments was confirmed by measurement of fluorescence resonance energy transfer between the acridine on the 5′-end of the probe strand as donor and BODIPY-Texas Red on the 3′-amino group of either strand of the target duplex as acceptor. There was full protection from OsO4-bipyridine modification of thymines in the probe strand of the triplex, in accordance with the presumed triplex formation, which excluded displacement of the homologous duplex strand by the probe–intercalator conjugate. The implications of these results for the existence of protein-independent parallel triplexes are discussed.

Using three-dimensional microfluidic networks for solving computationally hard problems

This paper describes the design of a parallel algorithm that uses moving fluids in a three-dimensional microfluidic system to solve a nondeterministically polynomial complete problem (the maximal clique problem) in polynomial time. This algorithm relies on (i) parallel fabrication of the microfluidic system, (ii) parallel searching of all potential solutions by using fluid flow, and (iii) parallel optical readout of all solutions. This algorithm was implemented to solve the maximal clique problem for a simple graph with six vertices. The successful implementation of this algorithm to compute solutions for small-size graphs with fluids in microchannels is not useful, per se, but does suggest broader application for microfluidics in computation and control.