Stochastic modelling and control of antibiotic subtilin production.

A stochastic hybrid model for the production of the antibiotic subtilin by the Bacillus subtilis is investigated. This model consists of 5 variables with four possible discrete dynamical states and this high dimensionality represents a bottleneck for using statistical tools that require to solve the corresponding Fokker-Planck problem. For this reason, a suitable reduced model with 3 variables and two dynamical states is proposed. The corresponding Fokker-Planck hyperbolic system is used to validate the evolution statistics and to construct a robust feedback control strategy to increase subtilin production. Results of numerical experiments are presented that show the effectiveness of the proposed control scheme.

Canonical and noncanonical equilibrium distribution.

We address the problem of the dynamical foundation of noncanonical equilibrium. We consider, as a source of divergence from ordinary statistical mechanics, the breakdown of the condition of time scale separation between microscopic and macroscopic dynamics. We show that this breakdown has the effect of producing a significant deviation from the canonical prescription. We also show that, while the canonical equilibrium can be reached with no apparent dependence on dynamics, the specific form of noncanonical equilibrium is, in fact, determined by dynamics. We consider the special case where the thermal reservoir driving the system of interest to equilibrium is a generator of intermittent fluctuations. We assess the form of the noncanonical equilibrium reached by the system in this case. Using both theoretical and numerical arguments we demonstrate that Lévy statistics are the best description of the dynamics and that the Lévy distribution is the correct basin of attraction. We also show that the correct path to noncanonical equilibrium by means of strictly thermodynamic arguments has not yet been found, and that further research has to be done to establish a connection between dynamics and thermodynamics.

Spike-driven synaptic plasticity: theory, simulation, VLSI implementation.

We present a model for spike-driven dynamics of a plastic synapse, suited for aVLSI implementation. The synaptic device behaves as a capacitor on short timescales and preserves the memory of two stable states (efficacies) on long timescales. The transitions (LTP/LTD) are stochastic because both the number and the distribution of neural spikes in any finite (stimulation) interval fluctuate, even at fixed pre- and postsynaptic spike rates. The dynamics of the single synapse is studied analytically by extending the solution to a classic problem in queuing theory (Takacs process). The model of the synapse is implemented in aVLSI and consists of only 18 transistors. It is also directly simulated. The simulations indicate that LTP/LTD probabilities versus rates are robust to fluctuations of the electronic parameters in a wide range of rates. The solutions for these probabilities are in very good agreement with both the simulations and measurements. Moreover, the probabilities are readily manipulable by variations of the chip's parameters, even in ranges where they are very small. The tests of the electronic device cover the range from spontaneous activity (3-4 Hz) to stimulus-driven rates (50 Hz). Low transition probabilities can be maintained in all ranges, even though the intrinsic time constants of the device are short (approximately 100 ms). Synaptic transitions are triggered by elevated presynaptic rates: for low presynaptic rates, there are essentially no transitions. The synaptic device can preserve its memory for years in the absence of stimulation. Stochasticity of learning is a result of the variability of interspike intervals; noise is a feature of the distributed dynamics of the network. The fact that the synapse is binary on long timescales solves the stability problem of synaptic efficacies in the absence of stimulation. Yet stochastic learning theory ensures that it does not affect the collective behavior of the network, if the transition probabilities are low and LTP is balanced against LTD.

Fluctuation-dissipation process without a time scale

We study the influence of a dissipation process on diffusion dynamics triggered by fluctuations with long-range correlations. We make the assumption that the perturbation process involved is of the same kind as those recently studied numerically and theoretically, with a good agreement between theory and numerical treatment. As a result of this assumption the equilibrium distribution departs from the ordinary canonical distribution. The distribution tails are truncated, the distribution border is signaled by sharp peaks, and, in the weak dissipation limit, the central distribution body becomes identical to a truncated Levy distribution.

Characterization of six murine monoclonal antibodies specific for toxin B of Clostridium difficile.

Six murine hybridoma cell lines producing monoclonal antibodies (MAbs) specific for Toxin B of Clostridium difficile have been generated from toxin-immunized female RBF/DnJ mice. All six antibodies were reactive in Western blots with a > 200-kD protein in the supernatants of the toxigenic strain 10463 and were unreactive with similarly prepared material from the nontoxigenic strain 2037. Polyclonal antisera from rabbits immunized with Toxin B reacted on Western blots primarily with Toxin B, a 40-kD and a 55-kD band with a minor set of triplet bands at approximately 100 kD. None of the MAbs reacted in a direct EIA with purified Toxin A from C. difficile but two MAbs reacted weakly with a trypsin-sensitive band (> 200 kD) in Western blots of C. sordellii. Polyclonal antisera developed against Toxin B reacted strongly with supernatants from C. sordellii, C. bifermentans, and the nontoxigenic strain 2037. Toxin B-specific antisera was unreactive with supernatants from C. perfringens or purified Toxin A from C. difficile in direct EIA. Toxin B-specific MAbs linked to an affinity column were able to deplete bacterial supernatant of cytotoxigenic activity.

p-maleimidophenyl isocyanate: a novel heterobifunctional linker for hydroxyl to thiol coupling.

p-Maleimidophenyl isocyanate (PMPI, 1) is a heterobifunctional cross-linking agent useful for thiol to hydroxyl coupling. Several maleimide-activated compounds were prepared and characterized and then shown to be reactive with thiol-containing proteins. Examples include activation of vitamin B12, digoxigenin, digitoxigenin, estradiol, progesterone, and some serine-containing peptides.

Fluorometric enzyme immunoassay for measurement of infectious feline leukemia virus and its neutralization.

A fluorometric enzyme immunoassay using an alkaline phosphatase-conjugated monoclonal antibody was developed to quantitate feline leukemia virus (FeLV) infection. Monoclonal antibodies, directed against the FeLV structural protein p27, were conjugated with alkaline phosphatase using a modified maleimide method. The enzyme immunoassay requires only 4 days to reproducibly measure FeLV production instead of the 12 days required for the commonly used transformation assay using C81 cells. A linear correlation was found between the virion associated reverse transcriptase activity and the amount of intracellular p27 as determined by the fluorometric enzyme immunoassay. An immunocytochemical assay using the same conjugated monoclonal with a different substrate gave visible plaques in infected cell monolayers and was therefore used to titrate FeLV in plaque-forming units. The results obtained by all the procedures followed single hit kinetics for FeLV infection. The fluorometric enzyme immunoassay was adapted to measure FeLV neutralizing antibodies, allowing a sensitive and accurate determination of neutralizing titers.

Efficient purification of mouse monoclonal antibodies from ascites fluid by medium-performance anion exchange chromatography.

Medium-performance anion-exchange chromatography was applied to the purification of murine IgG class monoclonal antibodies from ascites fluid. The separations were performed under mild conditions at pH 8 using relatively low sodium-chloride concentrations. Recoveries for monoclonal antibodies of subclasses IgG1, IgG2a, and IgG2b were about 90%. The IgG preparations were free of other ascites fluid proteins.