Multisite topographic microfluorometry of intracellular and exogenous fluorochromes.

Abstract. Microspectro fluorometry combined with microinjection of metabolites allows the monitoring of pathways regulating the energy metabolism, biosynthetic or metabolizing activity of the intact living cell. The topographic scan of ? 100–150 adjacent regions within a cell or the spectral scan of fluorescence from a single cell region is completed within 60 ms. The in situ activity of intracellular enzymes and substrate concentration vs rate relationships are derived from the kinetic analysis of transients (e.g. NAD(P) ? NAD(P)H) elicited by sequential injections with increasing doses of substrate (e.g. glucose-6-P, 6-phosphogluconate). A method of approximation is used to predict the non-linear behavior of whole biochemical systems (e.g. the sum of reactions involved in NAD(P) reduction-reoxidation transients). Approximation to power law kinetics is validated by prediction of system behavior over a 10–100 fold variation in the input concentration of substrate. The NAD-dependent glycolytic chain (faster rise kinetics) and NADPH-dependent shunt pathway (slower rise kinetics) are triggered optionally, by enhanced demand for degradation, synthesis or metabo-lization of exogenous compounds (hydrocarbons, aflatoxins). Compartmentation of NAD(P) pathways is suggested by differences in the reoxidation of cytoplasmic vs nuclear NAD(P)H. The intercellular transfer of molecules can be evaluated by microinjection of fluorescent tracers. The difference spectra ΔI_F due to microinjection of substrate are resolved in terms of bound vs free NAD(P)H (with differentiation of other endogenous and exogenous fluorochromes). On the basis of the above, a tentative scheme of intra- and intercellular dynamics can be developed.     

Self-sustained oscillations in a closed side branch system

Self-sustained oscillations of the flow in a closed side branch system due to a coupling of vortex shedding with acoustical resonances are considered. The configuration consists of two closed side branches of same length placed opposite to each other along a main pipe. This is called a cross-junction. Numerical simulations, based on the Euler equations for two-dimensional inviscid and compressible flows, are performed. As the radiation into the main pipe is negligible at the resonance frequency, this acoustically closed system is a good test-case of such Euler numerical calculations. The numerical results are compared to acoustical measurements and flow visualization obtained in a previous study. Depending on the flow conditions, the predicted pulsation amplitudes are about 30-40% higher than the measured amplitudes. This is partially due to the absence of visco-thermal dissipation in the numerical model but also to the effect of wall vibrations in experiments. A simple analytical model is proposed for the prediction of the pulsation amplitudes. This model is based on Nelson's representation of the shear layer as a row of discrete vortices convected at constant velocity from the upstream edge towards the downstream edge. When the downstream edge is sharp, this results in a spurious interaction between the singularity of the vortices and of the edge flow. This artefact is partially compensated by suppressing the singularity of the acoustical flow at the edge, or when a junction with rounded edges, as found in engineering practice, is considered. In spite of its crudeness, the analytical model provides a fair prediction (within 30%) which makes it useful for engineering applications.     

Speeding up chemical database searches using a proximity filter based on the logical exclusive or

In many large chemoinformatics database systems, molecules are represented by long binary fingerprint vectors whose components record the presence or absence in the molecular graphs of particular functional groups or combinatorial features, such as labeled paths or labeled trees. To speed up database searches, we propose to store with each fingerprint a small header vector containing primarily the result of applying the logical exclusive OR (XOR) operator to the fingerprint vector after modulo wrapping to a smaller number of bits, such as 128 bits. From the XOR headers of two molecules, tight bounds on the intersection and union of their fingerprint vectors can be rapidly obtained, yielding tight bounds on derived similarity measures, such as the Tanimoto measure. During a database search, every time these bounds are unfavorable, the corresponding molecule can be rapidly discarded with no need for further inspection. We derive probabilistic models that allow us to estimate precisely the behavior of the XOR headers and the level of pruning under different conditions in terms of similarity threshold and fingerprint density. These theoretical results are corroborated by experimental results on a large set of molecules. For a Tanimoto threshold of 0.5 (respectively 0.9), this approach requires searching less than 50% (respectively 10%) of the database, leading to typical search speedups of 2 to 3 times over the previous state-of-the-art.