Photoconvertible fluorescent protein EosFP: biophysical properties and cell biology applications

EosFP is a fluorescent protein from the coral Lobophyllia hemprichii that changes its fluorescence emission from green to red upon irradiation with near-UV light. Here we present the spectroscopic properties of wild-type EosFP and a variety of monomeric and dimeric mutants and provide a structural interpretation of its oligomerization and photoconversion, which is based on X-ray structure analysis of the green and red species that we reported recently. Because functional expression of the monomeric EosFP variant is limited to temperatures of 30 degrees C, we have developed a tandem dimer. This construct, in which two EosFP subunits are connected by a flexible 12 amino acid linker, expresses well after fusion with the androgen and endothelin A receptors at 37 degrees C. A variety of applications in cellular imaging, developmental biology and automated high-content screening applications are presented, which demonstrate that EosFP is a powerful tool for in vivo monitoring of cellular processes.     

Stick-slip of the three-phase line in measurements of dynamic contact angles

Contact angles of a series of n-alkanes (i.e., n-heptane to n-hexadecane) are studied on two functionalized maleimide copolymers (i.e., poly(ethene-alt-N-(4-(perfluoroheptylcarbonyl)aminobutyl)maleimide) (ETMF) and poly(octadecene-alt-N-(4-(perfluoroheptylcarbonyl)aminobutyl)maleimide) (ODMF)). On the homogeneous ETMF films, all liquids show a smooth motion of the three-phase line. In contrast, on ODMF surfaces that are found to consist of mainly fluorocarbons and small patches of hydrocarbons, short-chain n-alkanes show a stick-slip pattern. By increasing the chain length of the probe liquids, stick-slip is reduced significantly. The phenomenon is discussed in the framework of the Cassie equation. It is found that the upper limit of contact angles in the stick-slip pattern is given by the advancing angle that would be obtained on the pure fluorocarbon surface, whereas the lower limit of the stick-slip pattern is given by the Cassie angle.     

Interaction of the indoor air pollutant acetone with Degussa P25 TiO2 studied by chemical ionization mass spectrometry

Preventing a build-up of indoor pollutant concentrations has emerged as a major goal in environmental chemistry. Here, we have applied chemical ionization mass spectrometry to study the interaction of acetone, a common indoor air pollutant, with Degussa P25 TiO2, an inexpensive catalyst that is widely used for the degradation of volatile organic compounds into CO2 and water. To better understand the adsorption of acetone onto Degussa P25, the necessary first step for its degradation, the experiments were carried out at room temperature in the absence of UV light. This allowed for the deconvolution of the nonreactive and reactive thermal binding processes on Degussa P25 at acetone partial pressures (10(-7)-10(-4) Torr) commonly found in indoor environments. On average, 30% of the adsorbed acetone is bound irreversibly, resulting in a surface coverage of irreversibly bound acetone of approximately 1 x 10(12) molecules/cm2 at 3-4 x 10(-5) Torr. Equilibrium and dynamic experiments yield a sticking coefficient of approximately 1 x 10(-4) that is independent of the acetone partial pressures examined here. Equilibrium binding constants and free energies of adsorption are reported.     

Comparison of protein A affinity sorbents III. Life time study

Protein A affinity chromatography is a popular purification method for immunoglobulins applied at various scales, ranging from micro-tube up to 1000l column format. Three novel high capacity protein A affinity chromatography media have been subjected to a lifetime study using 50 consecutive purification cycles of a cell culture supernatant (CCS) containing a monoclonal antibody. Chromatographic conditions followed protocols used in industrial antibody processing, including stripping and cleaning-in-place of the resins. For all three media, no significant loss of purification performance (measured by sodium dodecylsulfate polyacrylamide gel electrophoresis and analytical size-exclusion chromatography (SEC)) could be observed over 50 cycles. Eluate samples were analyzed for leaked protein A and host cell protein (HCP) content. MabSelect SuRe, the first protein A affinity medium compatible with alkaline regeneration conditions, exhibited the lowest leakage levels, in the range of 1-3 ppm. For the media MabSelect Xtra and ProSep-vA Ultra, leakage levels were in the range of 30-40 ppm. Host cell protein content of eluates from MabSelect Xtra and SuRe were between 300 and 700 ppm, whereas for ProSep-vA Ultra 3000-4000 ppm was achieved.     

An example of lipophiloselectivity: the preferred oxidation, in water, of hydrophobic 2-alkanols catalyzed by a cross-linked polyethyleneimine-polyoxometalate catalyst assembly

A cross-linked polyethyleneimine polymer containing the [ZnWZn2(H2O)2(ZnW9O34)2]12- polyoxometalate was prepared from branched polyethyleneimine (Mw = 600), the polyoxometalate, and a n-octylamine-epichlorohydrin cross-linking reagent. This catalytic assembly was active for the selective oxidation of 2-alkanols to 2-alkanones with aqueous H2O2 with reactions presumably occurring at a hydrophobic domain. Most importantly, the catalyst showed distinctive lipophiloselectivity, that is selectivity as a function of the lipophilic nature of a reaction substrate. The lipophiloselectivity was proportional to the relative partition coefficient (1-octanol/water) of the substrates.     

Recent progress in axisymmetric drop shape analysis (ADSA)

Axisymmetric Drop Shape Analysis (ADSA) is a powerful technique for the measurement of interfacial properties from the shape of drops/bubbles. It relies on the best fit between theoretical curves with known surface tension values and an experimental profile. Despite the general success of ADSA, inconsistent results are obtained for nearly spherical drop shapes. Since the source of this and possible other limitations are unknown, the entire ADSA technique including hardware and software is systematically scrutinized. The hardware consists of electronics, and optical and mechanical components that generate a digital image of a drop. Since the quality of images has a considerable impact on the surface tension measurements, general guidelines for the use of hardware components are developed to enhance the quality of the image. The scrutiny of the software of ADSA is significantly more involved. The software consists of image analysis and numerical schemes. One of the key elements is the modularization of the software, since a generic software package is not suitable for all experimental situations. As a result, a more versatile image analysis module is introduced. In this context a variety of state-of-the-art edge detection techniques are studied, and a more robust technique is adopted. The two existing ADSA numerical schemes are also compared systematically, and the more efficient one is implemented. It is shown that even this superior numerical scheme has convergence problems for nearly spherical drops. This difficulty is due to numerical truncation and accumulation of round-off errors, which are the ultimate limitation of all numerical schemes. This intrinsic limitation becomes more pronounced as drops become closer to spherical in shape, but there were no objective criteria available to define "close to spherical drops". Therefore, a quantitative criterion called shape parameter is introduced to identify the range of applicability of ADSA. The improved version of ADSA not only determines the interfacial properties more accurately but, through the shape parameter, also provides an a priori knowledge of the accuracy of the results.     

Surface imprinting strategies for the detection of trypsin

Self-organized receptor layers are synthesized by molecular imprinting methods directly on pre-coated 10 MHz quartz-crystal microbalances (QCMs). The surface-imprinting is performed by three methods using amorphous, crystalline and solubilized trypsin, respectively, as templates. These attempts allowed us to compare imprinting results obtained with templating proteins in the dry state as well as in aqueous solution. All methods are generally applicable for surface imprinting of thin films. The biomimetic sensor layers allow selective enzyme enrichment on the imprinted electrode with detection limits as low as 100 ng ml(-1) and response times of a few minutes. Solution-based polymer imprinting with native trypsin as template resulted in the highest specific enzyme recognition, which even allowed us to distinguish denatured trypsin from the native form.     

Palladium mediated activation of C-F bonds in 2,4,6-trifluoropyrimidine: Synthesis and structure of palladium pyrimidyloxy and pyrimidinone derivatives

[PdMe₂(dcpm)] (1) reacts with 2,4,6-trifluoropyrimidine in the presence of water to give the palladium derivative [PdMe{4-C₄N₂F₂H(O)}(dcpm)] (2). When additional triethylamine is present complex [PdMe(2-OC₄N₂F₂H)(dcpm)] (4) in addition to 2 is formed. Compound 2 converts slowly into the binuclear complex [Pd{4-C₄N₂F₂H(O)}(μ-dcpm)]₂ (5). The molecular structure of 5 was determined by X-ray crystallography. The palladium-palladium distance is 2.5898(3) Å.     

Two-dimensional spectral shearing interferometry for few-cycle pulse characterization

We present a new method for measuring the spectral phase of ultrashort pulses that utilizes spectral shearing interferometry with zero delay. Unlike conventional spectral phase interferometry for direct electric-field reconstruction, which encodes phase as a sensitively calibrated fringe in the spectral domain, two-dimensional spectral shearing interferometry robustly encodes phase along a second dimension. This greatly reduces demands on the spectrometer and allows for complex phase spectra to be measured over extremely large bandwidths, potentially exceeding 1.5 octaves.