Directed evolution of a small-molecule-triggered intein with improved splicing properties in mammalian cells

Laboratory-created small-molecule-dependent inteins enable protein structure and function to be controlled posttranslationally in living cells. Previously we evolved inteins that splice efficiently in Saccharomyces cerevisiae only in the presence of the cell-permeable small molecule 4-hydroxytamoxifen (4-HT). In mammalian cells, however, these inteins exhibited lower splicing efficiencies and slower splicing in the presence of 4-HT, as well as higher background splicing in the absence of 4-HT. Here we further evolved ligand-dependent inteins in yeast at 30°C and 37°C. The resulting second-generation evolved inteins exhibit substantially improved splicing yields and kinetics. The improvements carried over to mammalian cells, in which the newly evolved inteins spliced with substantially greater (～2- to 8-fold) efficiency while maintaining low background splicing levels. These second-generation inteins augment the promise of ligand-dependent protein splicing for probing protein function in mammalian cells.     

Synthesis, characterization, and photophysical study of fluorescent N-substituted benzo[ghi]perylene "swallow tail" monoimides

A set of N-substituted benzoperylene monoimide (BPI) fluorophores was synthesized and characterized structurally and photophysically. Condensation of benzo[ghi]perylene-1,2-dicarboxylic anhydride in the presence of "swallow tail" alkyl amines produced fluorophores that are soluble in a range of organic solvents, highly absorbing in the near-UV (ε(334) = 79,000 M(-1) cm(-1)), and fluorescent in the visible range. Photophysical behavior of the compounds was studied with steady-state and time-correlated single photon counting. The synthesized BPIs exhibit positive solvachromatic emission (λ(em) (hexane) = 469 nm; λ(em) (ethanol) = 550 nm) as a function of solvent polarity with little change in their excited-state lifetime (9.6-6.5 ns) and fluorescence quantum yield (0.27-0.44) over the polarity range studied. Solvachromatic shifts were analyzed using the Lippert-Mataga approach. In nonpolar hydrocarbon solvents evidence of dual emission from closely spaced (562 cm(-1)) S(1) and S(2) excited states is observed. Preliminary peak assignments for the anomalous S(2) emission are made.     

Energy Dispersive X-Ray Spectrometry by Microcalorimetry for the SEM

 Analytical X-ray spectrometry for electron beam instruments has advanced significantly with the development of the microcalorimeter energy dispersive X-ray spectrometer (μcal EDS). The μcal EDS operates by measuring the temperature rise when a single photon is absorbed in a metal target. A cryoelectronic circuit with electrothermal feedback and a superconducting transition edge sensor serves as the thermometer. Spectral resolution approaching 4.5 eV for high energy photons (6000 eV) and 2 eV for low energy photons below 2000 eV has been demonstrated in energy dispersive operation across a photon energy range from 250 eV to 8 keV. Spectra of a variety of materials demonstrate the power of the μcal EDS to solve practical problems while operating on a scanning electron microscope platform.     

Beam wave propagation within the second Rytov perturbation approximation

The applicability of the classical Rytov method in statistical wave propagation problems is reconsidered and expanded by demanding results that are of second order in the permittivity fluctuations, rather than limiting them to just the first Rytov perturbation approximation, as is traditionally done. It is shown that one must augment the well-known second order statistics (e.g., log-amplitude variance), as calculated from the first Rytov approximation, with first-order statistics (e.g., the average log-amplitude), as calculated from the second Rytov approximation. Thus, a complete solution is derived for the second Rytov approximation for general beam wave propagation through turbulent media, the permittivity fluctuations of which are described by the Kolmogorov-Obukhov spectrum. This then allows a complete and consistent treatment that yields the fact that the average log-amplitude is, in the general beam wave case, not equal to the additive inverse of the log-amplitude variance. This gives results from the Rytov method that are then in exact agreement with the corresponding limiting case of strong fluctuation theory, as well as a simplified analytical expression for beam wave broadening, and the correct theoretical explanation of the well-known applicability limit for the Rytov method.