John A. Rose

Rapid modulation of long-term depression and spinogenesis via synaptic estrogen receptors in hippocampal principal neurons

Rapid modulation of hippocampal synaptic plasticity by estrogen has long been a hot topic, but analysis of molecular mechanisms via synaptic estrogen receptors has been seriously difficult. Here, two types of independent synaptic plasticity, long‐term depression (LTD) and spinogenesis, were investigated, in response to 17β‐estradiol and agonists of estrogen receptors using hippocampal slices from adult male rats. Multi‐electrode investigations demonstrated that estradiol rapidly enhanced LTD not only in CA1 but also in CA3 and dentate gyrus. Dendritic spine morphology analysis demonstrated that the density of thin type spines was selectively increased in CA1 pyramidal neurons within 2 h after application of 1 nm estradiol. This enhancement of spinogenesis was completely suppressed by mitogen‐activated protein (MAP) kinase inhibitor. Only the estrogen receptor (ER) alpha agonist, (propyl‐pyrazole‐trinyl)tris‐phenol (PPT), induced the same enhancing effect as estradiol on both LTD and spinogenesis in the CA1. The ERbeta agonist, (4‐hydroxyphenyl)‐propionitrile (DPN), suppressed LTD and did not affect spinogenesis. Because the mode of synaptic modulations by estradiol was mostly the same as that by the ERalpha agonist, a search was made for synaptic ERalpha using purified RC‐19 antibody qualified using ERalpha knockout (KO) mice. Localization of ERalpha in spines of principal glutamatergic neurons was demonstrated using immunogold electron microscopy and immunohistochemistry. ERalpha was also located in nuclei, cytoplasm and presynapses.

A DNA based implementation of an evolutionary search for good encodings for DNA computation

Computation based on manipulation of DNA molecules has the potential to solve problems with massive parallelism. DNA computation, however, is implemented with chemical reactions between the nucleotide bases, and therefore, the results can be error-prone. Application of DNA based computation to traditional computing paradigms requires error-free computation, which the DNA chemistry is unable to support. Careful encoding of the nucleotide sequences can alleviate the production of errors, but these good encodings are difficult to find. In this paper, an algorithm for evolutionary computation with DNA is sketched. Evolutionary computation does not require error-free DNA chemistry, and in fact, takes advantage of errors to produce change and variation in the population. An application of the DNA based evolution program to a search for good DNA encodings is sketched.

A Software Tool for Generating Non-crosshybridizing Libraries of DNA Oligonucleotides

Under an all or nothing hybridization model, the problem of finding a library of non-crosshybridizing DNA oligonucleotides is shown to be equivalent to finding an independent set of vertices in a graph. Individual oligonucleotides or Watson-Crick pairs are represented as vertices. Indicating a hybridization, an edge is placed between vertices (oligonu-cleotides or pairs) if the minimum free energy of hybridization, according to the nearest-neighbor model of duplex thermal stability, is less than some threshold value. Using this equivalence, an algorithm is implemented to find maximal libraries. Sequence designs were generated for a test of a modified PCR protocol. The results indicated that the designed structures formed as planned, and that there was little to no secondary structure present in the single-strands. In addition, simulations to find libraries of 10-mers and 20-mers were done, and the base composition of the non-crosshybridizing libraries was found to be 2/3 A-T and 1/3 G-C under high salt conditions, and closer to uniform for lower salt concentrations.

Enhancement of nitric oxide production by association of nitric oxide synthase with N-methyl-D-aspartate receptors via postsynaptic density 95 in genetically engineered Chinese hamster ovary cells: real-time fluorescence imaging using nitric oxide sensitive dye.

The current quantitative study demonstrates that the recruitment of neuronal nitric oxide synthase (nNOS) beneath N‐methyl‐d‐aspartate (NMDA) receptors, via postsynaptic density 95 (PSD‐95) proteins significantly enhances nitric oxide (NO) production. Real‐time single‐cell fluorescence imaging was applied to measure both NO production and Ca2+ influx in Chinese hamster ovary (CHO) cells expressing recombinant NMDA receptors (NMDA‐R), nNOS, and PSD‐95. We examined the relationship between the rate of NO production and Ca2+ influx via NMDA receptors using the NO‐reactive fluorescent dye, diaminofluorescein‐FM (DAF‐FM) and the Ca2+‐sensitive yellow cameleon 3.1 (YC3.1), conjugated with PSD‐95 (PSD‐95‐YC3.1). The presence of PSD‐95 enhanced the rate of NO production by 2.3‐fold upon stimulation with 100 µm NMDA in CHO1(+) cells (expressing NMDA‐R, nNOS and PSD‐95) when compared with CHO1(–) cells (expressing NMDA‐R and nNOS lacking PSD‐95). The presence of nNOS inhibitor or NMDA‐R blocker almost completely suppressed this NMDA‐stimulated NO production. The Ca2+ concentration beneath the NMDA‐R, [Ca2+]NR, was determined to be 5.4 µm by stimulating CHO2 cells (expressing NMDA‐R and PSD‐95‐YC3.1) with 100 μm NMDA. By completely permealizing CHO1 cells with ionomycin, a general relationship curve of the rate of NO production versus the Ca2+ concentration around nNOS, [Ca2+]NOS, was obtained over the wide range of [Ca2+]NOS. This sigmoidal curve had an EC50 of approximately 1.2 µm of [Ca2+]NOS, implying that [Ca2+]NR = 5.4 µm can activate nNOS effectively.

Statistical thermodynamic analysis and designof DNA-based computers

A principal research area in biomolecular computing is the development of analytical methods for evaluating computational fidelity and efficiency. In this work, the equilibrium theory of the DNA helix-coil transition is reviewed and expanded, as applied to the analysis and design of oligonucleotide-based computers. After a review of the equilibrium apparatus for modeling the helix-coil transition for single dsDNA species, application to complex hybridizing systems is discussed, via decomposition into component equilibria, which are presumed to proceed independently. The alternative approach, which involves estimation of a mean error probability per hybridized structure, or computational incoherence, ε is then presented, along with a discussion of a special-case exact solution (directed dimer formation), and an approximate general solution, applicable to conditions of uniform fractional-saturation. In order to clarify the opposing nature of the predictions of these two models, simulations are presented for the uniform saturation solution for ε, as applied to a small Tag–Antitag (TAT) system, along with the behavior expected via isolated melting curves. By a comparison with the predictions of a recent, TAT-specific solution for ε, the views provided by these generalized approximate models are shown to define the opposing limits of a more general error-response.

DNA computing: a review

DNA computing holds out the promise of important and significant connections between computers and living systems, as well as promising massively parallel computations. Before these promises are fulfilled, however, important challenges related to errors and practicality have to be addressed. On the other hand, new directions toward a synthesis of molecular evolution and DNA computing might circumvent the problems that have hindered development, so far.

A DNA based artificial immune system for self-nonself discrimination

Artificial immune systems attempt to distinguish self from nonself through string matching operations. A detector set of strings is selected by eliminating random strings that match the self strings. DNA based computers have been proposed to solve complex problems that defy solution on conventional computers. They are based on (hydrogen bonding based) matchings (called hybridizations) between Watson-Crick complementary pairs, Adenine-Thymine or Cytosine-Guanine. Therefore, a single strand (an oligonucleotide) will bind with other oligonucleotides that match most closely its sequence under the operation of Watson-Crick complementation. In this paper, an algorithm for implementing an artificial immune system for self-nonself discrimination based on DNA is described. This procedure takes advantage of the inherent pattern matching capability of DNA hybridization reactions and the notion of similarity naturally found in DNA hybridization.

A new readout approach in DNA computing based on real-time PCR with taqman probes

A new readout approach for the Hamiltonian Path Problem (HPP) in DNA computing based on the real-time polymerase chain reaction (PCR) is investigated. Several types of fluorescent probes and detection mechanisms are currently employed in real-time PCR, including SYBR Green, molecular beacons, and hybridization probes. In this study, real-time amplification performed using the TaqMan probes is adopted, as the TaqMan detection mechanism can be exploited for the design and development of the proposed readout approach. Double-stranded DNA molecules of length 120 base-pairs are selected as the input molecules, which represent the solving path for an HPP instance. These input molecules are prepared via the self-assembly of 20-mer and 30-mer single-stranded DNAs, by parallel overlap assembly. The proposed readout approach consists of two steps: real-time amplification in vitro using TaqMan-based real-time PCR, followed by information processing in silico to assess the results of real-time amplification, which in turn, enables extraction of the Hamiltonian path. The performance of the proposed approach is compared with that of conventional graduated PCR. Experimental results establish the superior performance of the proposed approach, relative to graduated PCR, in terms of implementation time.

The Fidelity of the Tag-Antitag System

In the universal DNA chip method, target RNAs are mapped onto a set of DNA tags. Parallel hybridization of these tags with an indexed, complementary antitag array then provides an estimate of the relative RNA concentrations in the original solution. Although both error estimation and error reduction are important to process application, a physical model of hybridization fidelity for the TAT system has yet to be proposed. In this work, an equilibrium chemistry model of TAT hybridation is used to estimate the error probability per hybridized tag (?). The temperature dependence of ? is then discussed in detail, and compared with the predictions of the stringency picture. In combination with a modified statistical zipper model of duplex formation, implemented by the Mjolnir software package, ? is applied to investigate the error behavior of small to moderate sized TAT sets. In the first simulation, the fidelities of (1) 105 random encodings, (2) a recently reported Hamming encoding, and (3) an ?-based, evolved encoding of a 32-strand, length- 16 TAT system are estimated, and discussed in detail. In the second simulation, the scaling behavior of the mean error rate of random TAT encodings is investigated. Results are used to discuss the ability of a random strategy to generate high fidelity TAT sets, as a function of set size and encoding length.

PNA-mediated Whiplash PCR

In Whiplash PCR( WPCR), autonomous molecular computation is achieved by the recursive, self-directed polymerase extension of a mixture of DNA hairpins. A barrier confronting efficient implementation, however, is a systematic tendency for encoded molecules towards backhybridization, a simple form of self-inhibition. In order to examine this effect, the length distribution of extended strands over the course of the reaction is examined by modeling the process of recursive extension as a Markov chain. The extension efficiency per polymerase encounter of WPCR is then discussed within the framework of a statistical thermodynamic model. The efficiency predicted by this model is consistent with the premature halting of computation reported in a recent in vitro WPCR implementation. The predicted scaling behavior also indicates that completion times are long enough to render WPCR-based massive parallelism infeasible. A modified architecture, PNA-mediated WPCR (PWPCR) is then proposed in which the formation of backhybridized structures is inhibited by targeted PNA2/DNA triplex formation. The efficiency of PWPCR is discussed, using a modified form of the model developed for WPCR. Application of PWPCR is predicted to result in an increase in computational efficiency sufficient to allow the implementation of autonomous molecular computation on a massive scale.