Fluorescence quenching-based assays for hydrolyzing enzymes. Application of time-resolved fluorometry in assays for caspase, helicase, and phosphatase

We have developed assay technologies to measure hydrolyzing enzymes based on homogeneous time-resolved fluorescence quenching (TruPoint). High sensitivity was obtained using fluorescent europium chelates as labels, internally quenched by suitable quenchers and released upon enzymatic reaction. This approach allows robust and sensitive monitoring of low enzyme activities. The assay technology and the choice of donor-acceptor pairs were evaluated in three different enzymatic assays, a protease related to apoptosis, helicase involved in DNA unwinding, and phosphatase having an important role in cellular signaling cascades. All the assays produced an increasing signal, were sensitive, and had a good dynamic range. There were significant differences in optimized quenchers for each of the assays depending on the size, flexibility, and rigidity of the substrates. Also, clear differences in the energy-transfer reactions, their requirements for spectral overlapping, ionic interactions, and energy-transfer distances were found. Each of the enzymatic assays was briefly tested in a high-throughput screening environment by analyzing signal dynamics and statistical relevance as Z' factors.     

Line‐Sweep Ambient Obscurance

Screen‐space ambient occlusion and obscurance have become established methods for rendering global illumination effects in real‐time applications. While they have seen a steady line of refinements, their computational complexity has remained largely unchanged and either undersampling artefacts or too high render times limit their scalability. In this paper we show how the fundamentally quadratic per‐pixel complexity of previous work can be reduced to a linear complexity. We solve obscurance in discrete azimuthal directions by performing line sweeps across the depth buffer in each direction. Our method builds upon the insight that scene points along each line can be incrementally inserted into a data structure such that querying for the largest occluder among the visited samples along the line can be achieved at an amortized constant cost. The obscurance radius therefore has no impact on the execution time and our method produces accurate results with smooth occlusion gradients in a few milliseconds per frame on commodity hardware.     

Closing the 1,3-propanediol route enhances hydrogen production from glycerol by Halanaerobium saccharolyticum subsp. saccharolyticum

The effect of vitamin B₁₂ on hydrogen (H₂) and 1,3-propanediol (1,3-PD) production in glycerol fermentation of Halanaerobium saccharolyticum subsp. saccharolyticum was studied in batch cultivations. In addition, the effect of vitamin B₁₂ on glucose fermentation of H. saccharolyticum subsp. saccharolyticum and on glycerol fermentation of H. saccharolyticum subsp. senegalensis, that is a non-1,3-PD producing bacterium, and of Clostridium butyricum, that is known for vitamin B₁₂-independent 1,3-PD production, were studied for comparison. 1,3-PD production of H. saccharolyticum subsp. saccharolyticum was observed to be vitamin B₁₂-dependent and the H₂ production in glycerol fermentation was remarkably enhanced when 1,3-PD production was blocked by the lack of vitamin B₁₂ supplementation. The highest H₂ yield obtained by H. saccharolyticum subsp. saccharolyticum was 2.16 mol/mol glycerol. No significant effects of vitamin B₁₂ on metabolite production in glucose fermentation of H. saccharolyticum subsp. saccharolyticum or in glycerol fermentation of H. saccharolyticum subsp. senegalensis or C. butyricum were observed.     

Short Vapour Residence Time Catalytic Pyrolysis of Spruce Sawdust in a Bubbling Fluidized-Bed Reactor with HZSM-5 Catalysts

Catalytic pyrolysis of spruce sawdust was carried out in a bubbling fluidized-bed reactor using HZSM-5 catalysts. The effects of space velocity, catalyst deactivation, catalyst acidity and catalyst regeneration were studied. The use of catalysts decreased the yield of organic liquids compared to non-catalytic yields while the yields of pyrolytic water and gases increased. Decreasing the space velocity enhanced these effects. The rate of catalyst deactivation depended on the acidity of the catalyst, with more acidic catalysts deactivating more rapidly. Using a catalyst with a Si/Al ratio of 140 resulted in the largest changes in bio-oil properties. Periodic regeneration of the catalyst in the fluidized-bed reactor was also demonstrated using varying regeneration times and temperatures. It was shown that compared to BFB reactors, CFB reactor types would offer better operating characteristics for commercial scale catalytic pyrolysis processes in regard to vapour residence times, and catalyst activity and regeneration.     

High-level parameterizable area estimation modeling for ASIC designs

Architectural design space exploration and early area budgeting for ASIC and IP block development require accurate high level gate count estimation methods without requiring the hardware being fully specified. The proposed method uses hierarchical and parameterizable models requiring minimal amount of information about the implementation technology to meet this goal. The modeling process flow is to: (1) create a block diagram of the design, (2) create a model for each block, and (3) sum up estimates of all sub-blocks by supplying the correct parameters to each sub-model. We discuss the model creation for a few parameterized library blocks as well as three communication blocks and a processor core from real IC projects ranging from 22 to 250 kgates. The average relative estimation error of the proposed method for the library blocks is 3.2% and for the real world examples 4.0%. The best application of this method is early in the design phase when different implementation architectures are compared.     

Vertical external-cavity surface-emitting laser for dual-wavelength generation

We report an optically pumped vertical external-cavity surface-emitting laser that is designed for emission at two wavelengths simultaneously. Single transverse mode continuous-wave (CW) operation is demonstrated at the wavelengths of 984 and 1042 nm. The device produces a CW optical power of 140 mW for the 984-nm component and 115 mW for the 1042-nm component, exhibiting over 10% efficiency. At a high pump power, self-pulsation of the components appeared on top of the CW-mode components. The maximum total output of 1 W was obtained at the pump power of 9 W.     

Novel Online Diagnostic Analysis for In-Flight Particle Properties in Cold Spraying

In cold spraying, powder particles are accelerated by preheated supersonic gas stream to high velocities and sprayed on a substrate. The particle velocities depend on the equipment design and process parameters, e.g., on the type of the process gas and its pressure and temperature. These, in turn, affect the coating structure and the properties. The particle velocities in cold spraying are high, and the particle temperatures are low, which can, therefore, be a challenge for the diagnostic methods. A novel optical online diagnostic system, HiWatch HR, will open new possibilities for measuring particle in-flight properties in cold spray processes. The system employs an imaging measurement technique called S-PTV (sizing-particle tracking velocimetry), first introduced in this research. This technique enables an accurate particle size measurement also for small diameter particles with a large powder volume. The aim of this study was to evaluate the velocities of metallic particles sprayed with HPCS and LPCS systems and with varying process parameters. The measured in-flight particle properties were further linked to the resulting coating properties. Furthermore, the camera was able to provide information about variations during the spraying, e.g., fluctuating powder feeding, which is important from the process control and quality control point of view.     

Effect of hot drawing on properties of wet‐spun poly(L,D‐lactide) copolymer multifilament fibers

Polylactide stereocopolymer multifilament fibers were prepared by wet spinning and subsequent hot drawing. The stereocopolymers were poly‐(L,D ‐lactide) [P(L,D )LA], L/D ratio 96/4, and poly‐(L,DL ‐lactide) [P(L,DL )LA], L/DL ratio 70/30. They were dissolved in dichloromethane and coagulated in a spin bath containing ethanol. The hot‐drawing temperature was 65°C. The draw ratios (DR) were upto 4.5 to the P(L,D )LA 96/4 filaments and upto 3 to the P(L,DL )LA 70/30 filaments. Wet spinning decreased crystallinities of both copolymers. Hot drawing increased the crystallinity of the P(L,D )LA 96/4 filament but not to the level of the original copolymer, whereas the as‐spun and the hot‐drawn P(L,DL )LA 70/30 filaments were amorphous. The filament diameter, tenacity, Young's modulus, and elongation at break were dependent on the DR. The maximum tenacity (285 MPa) and Young's modulus (2.0 GPa) were achieved with the P(L,D )LA 96/4 filament at the DR of 4.5. Respectively, the maximum tenacity of the hot‐drawn P(L,DL )LA 70/30 filament was 175 MPa and Young's modulus 1.3 GPa at the DR of 3. Hot drawing slowed down in vitro degradation rate of both stereocopolymer filaments. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2010     

Chlorate Formation in Chlorine Dioxide Delignification—An Analysis via Elementary Kinetic Modeling

Abstract

Elementary kinetic modeling was used to study the mechanism of chlorate formation in chlorine dioxide delignification. Reaction conditions reflecting typical industrial processes (T = 50°C, pH 1.5–4) were examined. Fe mediated Cl(III) decomposition and a reaction between hypochlorous acid and chlorous acid (or their equilibrium counterparts) were found to be the major reaction routes responsible for chlorate formation at pH < 3. The latter route accounts for chlorate formation at pH ≥ 3. The rate of chlorous acid (HClO₂) self-decomposition was too slow either to compete against the other routes (pH < 3) or to yield notable amounts of chlorate within the given time frame (pH ≥ 3). The results suggest that chlorate formation could be suppressed, without adverse effects on chlorine dioxide regeneration, by aiming for end pH 3–3.5, ensuring a moderate chloride ion concentration and by favoring concentrated solutions/suspensions.     

Prospecting hydrogen production of Escherichia coli by metabolic network modeling

Genome-scale model was applied to analyze the anaerobic metabolism of Escherichia coli. Three different methods were used to find deletions affecting fermentative hydrogen production: flux balance analysis (FBA), algorithm for blocking competing pathways (ABCP), and manual selection. Based on these methods, 81 E. coli mutants possessing one gene deletion were selected and cultivated in batch experiments. Experimental results of H₂ and biomass production were compared against the results of FBA. Several gene deletions enhancing H₂ production were found. Correctness of gene essentiality predictions of FBA for the selected genes was 78% and 77% in glucose and galactose media, respectively. 33% of the mutations that were predicted by FBA to increase H₂ production had a positive effect in experiments. Batch cultivation is a simple and straightforward experimental way to screen improvements in H₂ production. However, the ability of FBA to predict the H₂ production rate cannot be evaluated by batch experiments. Metabolic network models provide a method for gaining broader understanding of the complicated metabolic system of a cell and can aid in prospecting suitable gene deletions for enhancing H₂ production.