Activity-based profiling of retaining β-glucosidases: a comparative study

Activity-based protein profiling (ABPP) is a versatile strategy to report on enzyme activity in vitro, in situ, and in vivo. The development and use of ABPP tools and techniques has met with considerable success in monitoring physiological processes involving esterases and proteases. Activity-based profiling of glycosidases, on the other hand, has proven more difficult, and to date no broad-spectrum glycosidase activity-based probes (ABPs) have been reported. In a comparative study, we investigated both 2-deoxy-2-fluoroglycosides and cyclitol epoxides for their utility as a starting point towards retaining β-glucosidase ABP. We also investigated the merits of direct labeling and two-step bio-orthogonal labeling in reporting on glucosidase activity under various conditions. Our results demonstrate that 1) in general cyclitol epoxides are the superior glucosidase ABPs, 2) that direct labeling is the more efficient approach but it hinges on the ability of the glucosidase to be accommodated in the active site of the reporter (BODIPY) entity, and 3) that two-step bio-orthogonal labeling can be achieved on isolated enzymes but translating this protocol to cell extracts requires more investigation.     

Increasing robustness against changes in the interferent structure by incorporating prior information in the augmented classical least-squares framework

A class of multivariate calibration methods called augmented classical least squares (ACLS) has been proposed which combines an explicit linear additive model with the predictive power of inverse models, such as principal component regression (PCR) and partial least squares (PLS). Because of its use of the explicit linear additive model, ACLS provides an interesting framework to incorporate different sources of prior information, such as measured pure component spectra, in the model. In this study, the predictive power of ACLS models incorporating different amounts of prior information has been compared to that of PCR and PLS using two examples, a designed experiment and one with biological samples. In both cases, the ACLS models showed predictive power comparable to PLS under idealized validation conditions. When a different interferent structure was present in the validation samples, the predictive power of the inverse models (PCR and PLS) dramatically decreased, with an increase in root-mean-squared error of prediction by a factor of 3.5 for the first example and a factor of 2 in the second example. The incorporation of prior information in the ACLS framework was found to considerably reduce or even completely remove these dramatic effects, especially when the pure component contributions for the interferents were taken into account.     

The effect of shear deformations in floorbeams on the moment distribution in orthotropic plated bridge decks

Floorbeams of orthotropic plated bridge decks are generally elements with a low slenderness, especially in the case of railway bridges. When loaded, such elements generate not only flexural deformations but shear deformations as well. Depending on the configuration, these shear deformations can be considerably large and should certainly not be neglected. In a design according to the Pelikan-Esslinger method, this deformation is taken into account in the second stage of the calculation of the orthotropic deck. At this stage, the additional bending moments, shear forces and floorbeam reactions due to the floorbeam flexibility are evaluated. The deflection of a directly loaded floorbeam creates a distribution of the load to adjacent non-loaded floorbeams. In addition, the deflection will affect the longitudinal ribs, increasing the sagging moments at midspan and decreasing the bending moments at the supports of the ribs provided by the floorbeams. In this paper a method is developed to take the shear deformation in the floorbeam into account, thus correcting the Pelikan-Esslinger method. The correction is especially important since the floorbeam web is frequently further weakened by cutouts for the longitudinal ribs and in some cases additional cope holes. The floorbeam web resisting the shear deformation is further reduced by these openings, increasing the importance of shear deformations even for more slender floorbeams. The validity of the proposed method is checked by full finite element calculation using shell elements which inherently comprise shear deformation. The effect of the correction on the calculations in the second stage of the calculation are relatively small for the floorbeam reactions; on the contrary, it can be quite large for the additional bending moments in the longitudinal ribs. In that case, the effect is worth considering.     

Polarization of skylight in the O(2)A band: effects of aerosol properties

Motivated by several observations of the degree of linear polarization of skylight in the oxygen A (O(2)A) band that do not yet have a quantitative explanation, we analyze the influence of aerosol altitude, microphysics, and optical thickness on the degree of linear polarization of the zenith skylight in the spectral region of the O(2)A band, between 755 to 775 nm. It is shown that the degree of linear polarization inside the O(2)A band is particularly sensitive to aerosol altitude. The sensitivity is strongest for aerosols within the troposphere and depends also on their microphysical properties and optical thickness. The polarization of the O(2)A band can be larger than the polarization of the continuum, which typically occurs for strongly polarizing aerosols in an elevated layer, or smaller, which typically occurs for depolarizing aerosols or cirrus clouds in an elevated layer. We show that in the case of a single aerosol layer in the atmosphere a determination of the aerosol layer altitude may be obtained. Furthermore, we show limitations of the aerosol layer altitude determination in case of multiple aerosol layers. To perform these simulations we developed a fast method for multiple scattering radiative transfer calculations in gaseous absorption bands including polarization. The method is a combination of doubling-adding and k-binning methods. We present an error estimation of this method by comparing with accurate line-by-line radiative transfer simulations. For the Motivated by several observations of the degree of linear polarization of skylight in the oxygen A (O(2)A) band that do not yet have a quantitative explanation, we analyze the influence of aerosol altitude, microphysics, and optical thickness on the degree of linear polarization of the zenith skylight in the spectral region of the O(2)A band, between 755 to 775 nm. It is shown that the degree of linear polarization inside the O(2)A band is particularly sensitive to aerosol altitude. The sensitivity is strongest for aerosols within the troposphere and depends also on their microphysical properties and optical thickness. The polarization of the O(2)A band can be larger than the polarization of the continuum, which typically occurs for strongly polarizing aerosols in an elevated layer, or smaller, which typically occurs for depolarizing aerosols or cirrus clouds in an elevated layer. We show that in the case of a single aerosol layer in the atmosphere a determination of the aerosol layer altitude may be obtained. Furthermore, we show limitations of the aerosol layer altitude determination in case of multiple aerosol layers. To perform these simulations we developed a fast method for multiple scattering radiative transfer calculations in gaseous absorption bands including polarization. The method is a combination of doubling-adding and k-binning methods. We present an error estimation of this method by comparing with accurate line-by-line radiative transfer simulations. For the O(2)A band, the errors in the degree of linear polarization are less than 0.11% for transmitted light, and less than 0.31% for reflected light. band, the errors in the degree of linear polarization are less than 0.11% for transmitted light, and less than 0.31% for reflected light.     

Negligible depletion solid-phase microextraction with radiolabeled analytes to study free concentrations and protein binding: an example with [3H]estradiol

A new method is presented that enables sensitive measurement of free concentrations of radiolabeled ligands. Additionally, protein binding of radiochemicals in complex matrixes can be determined with this new technique that combines negligible depletion solid-phase microextraction (nd-SPME) with liquid scintillation counting (LSC) as detection. [3H]Estradiol was taken as an example compound. Possible matrix effects of protein on fiber uptake kinetics were studied. No matrix effect was found, either by fouling of the fiber, or by changed uptake kinetics. The validity of the method was shown in the determination of the affinity constant (Ka) of estradiol for human serum albumin (HSA). The Ka was estimated at 8.9 x 10(4) M(-1), which corresponds well with literature values. This study shows that nd-SPME is suitable to study the free concentration and protein binding of [3H]estradiol. The method described in this paper combines the advantages of nd-SPME with the advantages of radiolabeled analytes, creating a timesaving, simple, and sensitive analytical tool that will be particularly useful in complex matrixes containing many potential interferences for chromatographic methods.     

Microtubule gliding and cross-linked microtubule networks on micropillar interfaces

We combined biochemical and topographical patterning to achieve motor-driven microtubule gliding on top of microfabricated pillar arrays with limited and controllable surface interactions of gliding microtubules. Kinesins immobilized on pillar heads pushed microtubules from the top of one micropillar to the next bridging up to 20 mum deep gaps filled with buffer solution. Distances of more than 10 mum were by-passed, and microtubule buckling was occasionally observed. The velocity distributions of microtubules gliding on poly(dimethylsiloxane) (PDMS) pillars, on flat PDMS, and on glass were found to be different, most likely due to topological and/or chemical differences between the substrates. We also used pillar arrays to suspend cross-linked microtubule networks, whose structural characteristics were governed by the topographical characteristics of the pillar pattern. These experiments open new possibilities to study the dynamics and the self-organization of motor/microtubule networks in defined topologically structured environments.     

Improving accuracy in detecting acoustic onsets.

In current cognitive psychology, naming latencies are commonly measured by electronic voice keys that detect when sound exceeds a certain amplitude threshold. However, recent research (e.g., K. Rastle & M. H. Davis, 2002) has shown that these devices are particularly inaccurate in precisely detecting acoustic onsets. In this article, the authors discuss the various problems and solutions that have been put forward with respect to this issue and show that classical voice keys may trigger several tens of milliseconds later than acoustic onset. The authors argue that a solution to this problem may come from voice keys that use a combination of analogue and digital noise (nonspeech sound) detection. It is shown that the acoustic onsets detected by such a device are only a few milliseconds delayed and correlate highly (up to .99) with reaction time values obtained by visual waveform inspection. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Ester-functionalized poly(3-alkylthiophene) copolymers: Synthesis,physicochemical characterization and performance in bulk heterojunction organic solar cells

The introduction of functional moieties in the donor polymer (side chains) offers a potential pathway toward selective modification of the nanomorphology of conjugated polymer:fullerene active layer blends applied in bulk heterojunction organic photovoltaics, pursuing morphology control and solar cell stability. For this purpose, two types of poly(3-alkylthiophene) random copolymers, incorporating different amounts (10/30/50%) of ester-functionalized side chains, were efficiently synthesized using the Rieke method. The solar cell performance of the functionalized copolymers was evaluated and compared to the pristine P3HT:PCBM system. It was observed that the physicochemical and opto-electronic characteristics of the polythiophene donor material can be modified to a certain extent via copolymerization without (too much) jeopardizing the OPV efficiency, as far as the functionalized side chains are introduced in a moderate ratio (<30%) and that preference is given to side chains with a small molar volume. A range of complementary techniques – UV–Vis spectroscopy, (modulated temperature) differential scanning calorimetry, transmission electron microscopy and X-ray diffraction analysis – indicated that variations in polymer crystallinity, while maintaining a high level of regioregularity, are probably the main factor responsible for the observed differences.     

Quantifying Biomediated Ground Improvement by Ureolysis: Large-Scale Biogrout Experiment

Biogrouting is a biological ground improvement method, in which microorganisms are used to induce carbonate precipitation in the subsurface in order to increase the strength and stiffness of granular soils. In this paper the results of a large-scale experiment (100?m3) are presented, in which the feasibility of biogrouting as a ground improvement method is investigated using techniques and equipment similar to those used in potential applications. In situ geophysical measurements were used to monitor the biogrouting process during treatment and indicated that the stiffness had increased significantly after one day of treatment. The results of unconfined compressive strength tests on samples which were excavated after treatment were used to assess the distribution of mechanical properties throughout the cemented sand body, which correlated quite well with the results of the in situ geophysical measurements. The stiffness increase could be quantified as a function of the injected volume of grouting agents and the distance from the injection points. These results will serve as an important benchmark for future applications of biomediated ground improvement.     

Hybrid cascode feedforward compensation for nano-scale low-power ultra-area-efficient three-stage amplifiers

A modified frequency compensation technique is proposed for low-power area-efficient three-stage amplifiers driving medium to large capacitive loads. Coined hybrid cascode feedforward compensation (HCFC), the total compensation capacitor is divided and shared between two internal high-speed feedback loops instead of only one loop as is common in prior art. Detailed analysis of this technique shows significant improvement in terms of bandwidth and stability. This is verified for a 1.2-V amplifier driving a 500-pF capacitive load in 90-nm CMOS technology, where HCFC reduces the total capacitor size and improves the gain-bandwidth by at least 30% and 40% respectively, compared to the prevailing schemes.