Malachite green derivatives for two-photon RNA detection

The design, preparation and characterisation of a library of malachite green (MG) derivatives for two-photon RNA labelling is described. Some of these MG derivatives exhibit an increased affinity for an MG-aptamer, as well as improved two-photon sensitivity when compared to the classical malachite green chloride. The underlying mechanisms and potential benefits for in vivo RNA visualisation are discussed.     

Enzymatic esterification of glycerol I. Lipase-catalyzed synthesis of regioisomerically pure 1,3-sn-diacylglycerols

Regioisomerically pure 1,3-sn-diacylglycerols are conveniently prepared in high yields (>80%) and in large quantities by enzymatic esterification of glycerol in the presence of various 1,3-selective lipases(Chromobacterium viscosum, Rhizopus delemar, Rhizomucor miehei) and a variety of different acyl donors like free fatty acids, fatty acid alkyl esters and vinyl esters. All reactions are carried out in aprotic organic solvents of low water content, namelyn-hexane, diethyl ether or tBuOMe. The creation of an artificial interphase between the solvent-immiscible hydrophilic glycerol and the hydrophobic reaction media by the adsorption of glycerol onto a solid support prior to use was essential for the success of these transformations. The effects of reaction conditions and the regioselectivities of the lipases on the product yields are described in detail.     

Aging Behavior of Propellants investigated by heat generation,stabilizer consumption,and molar mass degradation

Four double-base rocket propellants and a single-base gun propellant were tested to find out stabilizer consumption molar mass degradation, and heat generation between 50 °C and 90 °C and between 40 °C and 110 °C, respectively. Stabilizer depletion was measured by high performance liquid chromatography (HPLC), molar mass degradation by gel permeation chromatography (GPC), and heat generation by microcalorimetry. The conditioning times at different temperatures were determined with one of the rocket propellants, and the influence of the long-term storage on heat generation at 60 °C for 2191 dys was investigated. A correlated experiment between stabilizer consumption and heat generation showed that heat production does not increase substantially until the stabilizer has been consumed almost completely. The experimental data of molar mass degradation are described by a kinetic model based on statistical chain scission, and the stabilizer decrease by a first order reaction. For the gun propellant the Arrhenius plots of the reaction rate constants show two temperatures of teh stabilizer consumption, molar mass degradation and heat generation were determined.     

Processing of High-Density Submicrometer Al2O3 for New Applications

Sintered corundum components with submicrometer grain sizes exhibit properties which enable numerous new applications. Wet powder processing is developed to associate minimum grain sizes at highest densities with the lowest population of macrodefects. A closest ratio of powder particle size and sintered grain size is important for obtaining most fine-grained microstructures. This target was approached best by using powders with particle sizes in the range of 100–200 nm rather than with smaller nanoparticles.     

Alumina Grown during Deposition of Thermal Barrier Coatings on NiCrAlY

The microstructure of Al₂O₃ formed by oxidation of a model NiCrAlY alloy during electron-beam physical vapor deposition of ZrO₂–7.6 mol% YO_1.5 is examined and compared with that formed on the bare substrate. The growth rate, morphology, and chemical composition of the oxide vary among the different constituents of the alloy surface and are further influenced by the O₂ partial pressure and the physical presence of the thermal barrier coating (TBC) layer. These differences, however, are largely limited to the outer oxide layer. The interplay between the TBC and the growing oxide leads to the formation of a fine-grain Al₂O₃–ZrO₂"mixed zone" within the thermally grown oxide. A mechanism is outlined to explain this behavior, based on the dissolution of ZrO₂ in a transient Al₂O₃ structure growing by outward diffusion of Al, and its subsequent reprecipitation when the metastable phase transforms to the stable α-Al₂O₃ form.     

Miscibility and Crystallization Behaviors of Polyamide 6/Polytetrafluoroethylene Blends

Summary: The miscibility and crystallization behaviors of polyamide 6 (PA 6)/polytetrafluoroethylene (PTFE) blends, prepared via reactive extrusion, are systematically investigated by means of wide‐angle X‐ray diffraction (WAXD), differential scanning calorimetry (DSC), and dynamic mechanical thermal analysis (DMTA). WAXD measurements show that no co‐crystallization occurred between two components, while DSC and DMTA measurements suggest that a certain degree of miscibility between them might exist due to the formation of some copolymers during the reactive extrusion.

DMTA curves for the pure PA 6 sample and PA 6/PTFE blends with various compositions.     

A Stingless Bee (Melipona seminigra) Marks Food Sources with a Pheromone from Its Claw Retractor Tendons

By depositing scent marks on flowers, bees reduce both the search time and the time spent with the handling of nonrewarding flowers. They thereby improve the efficiency of foraging. Whereas in honey bees the source of these scent marks is unknown, it is assumed to be the tarsal glands in bumble bees. According to histological studies, however, the tarsal glands lack any openings to the outside. Foragers of the stingless bee Melipona seminigra have previously been shown to deposit an attractant pheromone at sugar solution feeders, which is secreted at the tips of their tarsi. Here we show that the claw retractor tendons have specialized glandular epithelia within the femur and tibia of all legs that produce this pheromone. The secretion accumulates within the hollow tendon, which also serves as the duct to the outside, and is released from an opening at the base of the unguitractor plate. In choice experiments, M. seminigra was attracted by feeders baited with pentane extracts of the claw retractor tendons in the same way as it was attracted by feeders previously scent marked by foragers. Our results resolve the seeming contradiction between the importance of foot print secretions and the lack of openings of the tarsal glands.     

Synthesis of new boron-rich building blocks for boron neutron capture therapy or energy-filtering transmission electron microscopy

The synthesis of a new ortho-carborane derivative, tetracarboranylketone 4, is reported here. Ketone 4 was prepared from a tetraalkynylated ketone by the addition of decaborane. The keto group was then easily modified to yield the glycosides 17alpha and 18beta, which contain glucose or galactose, respectively, and the nucleotide 13b. In addition to ketone 4, which is acyclic, cyclic ketone 8 was also synthesised. X-ray diffraction analysis of compound 4 indicated the presence of two toluene guest molecules per molecule of the host compound. Furthermore, compound 4 displays a rather low cytotoxicity. These novel products can be used as building blocks to create a new class of biomolecules containing high-density carborane clusters. Such molecules may constitute powerful tools for applications like Boron Neutron Capture Therapy or Energy-Filtering Transmission Electron Microscopy.     

mRNA openers and closers: modulating AU-rich element-controlled mRNA stability by a molecular switch in mRNA secondary structure

Approximately 3 000 genes are regulated in a time-, tissue-, and stimulus-dependent manner by degradation or stabilization of their mRNAs. The process is mediated by interaction of AU-rich elements (AREs) in the mRNA's 3'-untranslated regions with trans-acting factors. AU-rich element-controlled genes of fundamentally different functional relevance depend for their activation on one positive regulator, HuR. Here we present a methodology to exploit this central regulatory process for specific manipulation of AU-rich element-controlled gene expression at the mRNA level. With a combination of single-molecule spectroscopy, computational biology, and molecular and cellular biochemistry, we show that mRNA recognition by HuR is dependent on the presentation of the sequence motif NNUUNNUUU in single-stranded conformation. The presentation of the HuR binding site in the mRNA secondary structure appears to act analogously to a regulatory on/off switch that specifically controls HuR access to mRNAs in cis. Based on this finding we present a methodology for manipulating ARE mRNA levels by actuating this conformational switch specifically in a target mRNA. Computationally designed oligonucleotides (openers) enhance the NNUUNNUUU accessibility by rearranging the mRNA conformation. Thereby they increase in vitro and endogenous HuR-mRNA complex formation which leads to specific mRNA stabilization (as demonstrated for TNFalpha and IL-2, respectively). Induced HuR binding both inside and outside the AU-rich element promotes functional IL-2 mRNA stabilization. This opener-induced mRNA stabilization mimics the endogenous IL-2 response to CD28 stimulation in human primary T-cells. We therefore propose that controlled modulation of the AU-rich element conformation by mRNA openers or closers allows message stabilization or destabilization in cis to be specifically triggered. The described methodology might provide a means for studying distinct pathways in a complex cellular network at the node of mRNA stability control. It allows ARE gene expression to be potentially silenced or boosted. This will be of particular value for drug-target validation, allowing the diseased phenotype to ameliorate or deteriorate. Finally, the mRNA openers provide a rational starting point for target-specific mRNA stability assays to screen for low-molecular-weight compounds acting as inhibitors or activators of an mRNA structure rearrangement.     

Solid‐Phase Gene Synthesis for Mutant Library Construction: The Future of Directed Evolution?

Directed evolution of stereo‐ and regioselective enzymes as catalysts in organic chemistry and biotechnology constitutes a complementary alternative to selective transition‐metal catalysts and organocatalysts. Saturation mutagenesis at sites lining the binding pocket has emerged as a key method in this endeavor, but it suffers from amino acid bias, which reduces the quality of the library at the DNA level and, thus, at the protein level. Chemical solid‐phase gene synthesis for library construction offers a solution to this fundamental problem, and the Sloning and Twist platforms are two possible options. This concept article analyzes these approaches and compares them to traditional PCR‐based saturation mutagenesis; the superior commercial Twist technique shows almost no bias.