Characterization of Fluorescent Proteins with Intramolecular Photostabilization**

Genetically encodable fluorescent proteins have revolutionized biological imaging in vivo and in vitro. Despite their importance, their photophysical properties, i. e., brightness, count-rate and photostability, are relatively poor compared to synthetic organic fluorophores or quantum dots. Intramolecular photostabilizers were recently rediscovered as an effective approach to improve photophysical properties of organic fluorophores. Here, direct conjugation of triplet-state quenchers or redox-active substances creates high local concentrations of photostabilizer around the fluorophore. In this paper, we screen for effects of covalently linked photostabilizers on fluorescent proteins. We produced a double cysteine mutant (A206C/L221C) of α-GFP for attachment of photostabilizer-maleimides on the β-barrel near the chromophore. Whereas labelling with photostabilizers such as trolox, a nitrophenyl group, and cyclooctatetraene, which are often used for organic fluorophores, had no effect on α-GFP-photostability, a substantial increase of photostability was found upon conjugation to azobenzene. Although the mechanism of the photostabilizing effects remains to be elucidated, we speculate that the higher triplet-energy of azobenzene might be crucial for triplet-quenching of fluorophores in the blue spectral range. Our study paves the way for the development of fluorescent proteins with photostabilizers in the protein barrel by methods such as unnatural amino acid incorporation.     

Process Design for the Production of Prebiotic Oligosaccharides in an Enzyme Membrane Bioreactor: Interaction between Enzymatic Reaction and Membrane Filtration

Appropriately designed and operated enzyme membrane bioreactors (EMBRs) can fully utilize the activity of free, soluble enzymes while maximizing enzyme reusability and simultaneously removing the enzyme-free product in situ. A filtration model system was applied to determine critical EMBR design factors suitable for continuous enzymatic production of prebiotic fructo-oligosaccharides. The filtration flux and separation efficiency were investigated in terms of the membrane cutoff rating. In addition, the influence of operational conditions on enzyme activity was also determined.     

Weight-scheduling for linear time-variant model predictive wind turbine control toward field testing

Forschung im Ingenieurwesen - Modern multi-megawatt wind turbines require powerful control algorithms which consider several control objectives at the same time and respect process constraints....     

Attenuation of gossypol cytotoxicity by cyclic AMP in a rat liver cell line

We showed previously that supplementation for 30 d with 800 IU (727 mg) vitamin E/d did not adversely affect healthy elderly persons. We have now assessed the effects of 4 mo of supplementation with 60, 200, or 800 IU (55, 182, or 727 mg) all-rac-alpha-tocopherol/d on general health, nutrient status, liver enzyme function, thyroid hormone concentrations, creatinine concentrations, serum autoantibodies, killing of Candida albicans by neutrophils, and bleeding time in 88 healthy subjects aged >65 y participating in a double-blind, placebo-controlled trial. No side effects were reported by the subjects. Vitamin E supplementation had no effect on body weight, plasma total proteins, albumin, glucose, plasma lipids or the lipoprotein profile, total bilirubin, alkaline phosphatase, serum aspartate aminotransferase, serum alanine aminotransferase, lactate dehydrogenase, serum urea nitrogen, total red blood cells, white blood cells or white blood cell differential counts, platelet number, bleeding time, hemoglobin, hematocrit, thyroid hormones, or urinary or serum creatinine concentrations. Values from all supplemented groups were within normal ranges for older adults and were not significantly different from values in the placebo group. Vitamin E supplementation had no significant effects on plasma concentrations of other antioxidant vitamins and minerals, glutathione peroxidase, superoxide dismutase, or total homocysteine. There was no significant effect of vitamin E on serum nonspecific immunoglobulin concentrations or anti-DNA and anti-thyroglobulin antibodies. The cytotoxic ability of neutrophils against Candida albicans was not compromised. Thus, 4 mo of supplementation with 60-800 IU vitamin E/d had no adverse effects. These results are relevant for determining risk-to-benefit ratios for vitamin E supplementation.     

Endometrial connexin expression in the mare and pig: evidence for the suppression of cell-cell communication in uterine luminal epithelium

AIMS: To study the dose-response effects of intravenous terbutaline on the cardiovascular and respiratory autonomic nervous regulation. METHODS: The study followed a randomized, placebo-controlled crossover design in six healthy adult volunteers. The terbutaline dose ranged from 10 to 30 microg min(-1) We continuously measured electrocardiogram, finger systolic arterial pressure (SAP) and flow-volume spirometry in supine and upright positions at baseline and during 3 h drug infusion. The periodic variability components of R-R intervals (time between successive heart beats) and SAP in relation to respiration were assessed using spectral analysis techniques. The regularity of the time series was assessed by approximate entropy (ApEn) and the convolutedness by fractal dimension (FD). RESULTS: Terbutaline dose-dependently decreased total variability of R-R intervals, low frequency (LF) variability of R-R intervals (10 s waves), high frequency (HF) variability of R-R intervals (respiratory variability), total variability of SAP, HF variability of SAP, baroreflex sensitivity, plasma potassium concentration, approximate entropy of R-R interval and of SAP as well as fractal dimension of R-R interval. Terbutaline dose-dependently increased heart rate, LF/HF ratios of R-R intervals and of SAP, LF variability of SAP, minute ventilation and plasma terbutaline concentration. CONCLUSIONS: Terbutaline infusion decreases parasympathetic cardiovascular reactivity, baroreflex sensitivity, dimensionality of heart rate and plasma potassium concentration; it increases sympathetic dominance in cardiovascular autonomic balance, minute ventilation, and the regularity of heart rate and blood pressure time series.     

A computational design method for condensation of binary vapours of immiscible liquidsEine Berechnungsmethode für die Kondensation eines binären Dampfgemisches aus unmischbaren Flüssigkeiten

Physically possible modes of condensation of binary vapours of immiscible liquids have been analysed. A mathematical model has been developed which enables the changes of the parameters of the vapour, interface and condensate as functions of the degree of condensation to be calculated. Calculations have been carried out for two systems and for various inlet vapour compositions and cooling medium temperatures. It has been found that, for typical industrial conditions, the condensation process does not differ much from the local total condensation. Accordingly, a simplified method of predicting the condenser area may be used in that case.     

Dynamic response of a single catalyst pellet

It has been shown previously that pulse-response experiments on single catalyst pellets can provide a simple, rapid method for determining effective diffusivities and adsorption equilibrium constants. Assumptions were made about boundary conditions in the earlier work in order to simplify the mathematical treatment. In this paper a more rigorous development is presented in order to evaluate the magnitude of likely errors in the simplified approach.By using large flow rates, small chamber volumes, and for catalyst pellets of moderate or low porosity, the error in using the approximate equations should be small (less than 10%). For short diffusion paths (short pellets) of highly porous catalysts, the error can be large.     

Interrogation of “surface,” “skin,” and “core” orientation in thermotropic liquid‐crystalline copolyester moldings by near‐edge X‐ray absorption fine structure and wide‐angle X‐ray scattering

Injection molding thermotropic liquid‐crystalline polymers (TLCPs) usually results in the fabrication of molded articles that possess complex states of orientation that vary greatly as a function of thickness. “Skin‐core” morphologies are often observed in TLCP moldings. Given that both “core” and “skin” orientation states may often differ both in magnitude and direction, deconvolution of these complex orientation states requires a method to separately characterize molecular orientation in the surface region. A combination of two‐dimensional wide‐angle X‐ray scattering (WAXS) in transmission and near‐edge X‐ray absorption fine structure (NEXAFS) spectroscopy is used to probe the molecular orientation in injection molded plaques fabricated from a 4,4′‐dihydroxy‐α‐methylstilbene (DHαMS)‐based thermotropic liquid crystalline copolyester. Partial electron yield (PEY) mode NEXAFS is a noninvasive ex situ characterization tool with exquisite surface sensitivity that samples to a depth of 2 nm. The effects of plaque geometry and injection molding processing conditions on surface orientation in the regions on‐ and off‐ axis to the centerline of injection molded plaques are presented and discussed. Quantitative comparisons are made between orientation parameters obtained by NEXAFS and those from 2D WAXS in transmission, which are dominated by the microstructure in the skin and core regions. Some qualitative comparisons are also made with 2D WAXS results from the literature. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci 2007     

Measurement of microscopic displacements in graphite/epoxy composite materials using SEM-generated fiducial markers

Graphite fiber-reinforced resin composite materials are widely used in commercial applications where high strength and low weight are critical factors. In order to predict the fracture toughness of a composite material from the constitutive properties of the resin and fibers, experimental methods for the analysis of microscopic displacements and strain fields that develop at the fracture crack tip within the composite material are required. Information derived from measurement of displacements, and calculation of strain fields can then be used to test micromechanical models of matrix-dominated fracture. A method was developed in which it is possible to conduct near real-time fracture analysis of epoxy-based composite materials, and to subsequently obtain micrometer-scale measurements of displacements in the region of the crack tip. A map matrix was generated on the surface of test specimens in an SEM equipped with a tensile stage, along with an x-ray spectroscopy and image analysis system. A 40 × 40 point digital map was introduced onto the surface of the specimen using the digital x-ray mapping function of the x-ray analysis system which produced a surface matrix with point spacing of 10 μm. The quality of maps varies with test specimens and therefore it is necessary to optimize microscope operation parameters for each resin tested. Reproducible results were obtained with both neat resins and graphite/epoxy composites. In situ analysis of a region of a propagating crack tip grown using the tensile stage reveals a deformation zone ahead of the crack tip and images of the stages of microcracking were captured by the image analyzer for subsequent measurement of displacement. Direct measurement of crack tip displacements from SEM electron beam-induced reference matrices provide an important tool in characterizing the fracture behavior of both neat resin and composite materials.