Beating Bias in the Directed Evolution of Proteins: Combining High‐Fidelity on‐Chip Solid‐Phase Gene Synthesis with Efficient Gene Assembly for Combinatorial Library Construction

Saturation mutagenesis (SM) constitutes a widely used technique in the directed evolution of selective enzymes as catalysts in organic chemistry and in the manipulation of metabolic paths and genomes, but the quality of the libraries is far from optimal due to the inherent amino acid bias. Herein, it is shown how this fundamental problem can be solved by applying high‐fidelity solid‐phase chemical gene synthesis on silicon chips followed by efficient gene assembly. Limonene epoxide hydrolase was chosen as the catalyst in the model desymmetrization of cyclohexene oxide with the stereoselective formation of (R,R)‐ and (S,S)‐cyclohexane‐1,2‐diol. A traditional combinatorial PCR‐based SM library, produced by simultaneous randomization at several residues by using a reduced amino acid alphabet, and the respective synthetic library were constructed and compared. Statistical analysis at the DNA level with massive sequencing demonstrates that, in the synthetic approach, 97 % of the theoretically possible DNA mutants are formed, whereas the traditional SM library contained only about 50 %. Screening at the protein level also showed the superiority of the synthetic library; many highly (R,R)‐ and (S,S)‐selective variants being discovered are not found in the traditional SM library. With the prices of synthetic genes decreasing, this approach may point the way to future directed evolution.     

Low‐temperature crystallization of La0.15Sr0.775TiO3 using ionic liquids

The n‐type thermoelectric oxide La_0.15Sr_0.775TiO₃ (LST) has been synthesized at 600°C using an ionic liquid method. The method uses the ionic liquid 1‐ethyl 3‐methylimidazolium acetate as the sole complexing agent: the lack of a second, carbon‐rich template decreases the quantity of reduced intermediate phases which form during heating. By suppressing these phases, greatly reduced temperatures can be used to crystallize the perovskite LST phase, on the nanoscale. These nanoparticles have the potential to be used to increase the figure of merit in n‐type thermoelectric oxide devices.     

Suppression of electroosmotic flow and prevention of wall adsorption in capillary zone electrophoresis using zwitterionic surfactants

Addition of zwitterionic surfactants such as dodecyldimethyl(3-sulfopropyl)ammonium hydroxide, hexadecyldimethyl(3-sulfopropyl)ammonium hydroxide, and coco (amidopropyl)hydroxyldimethylsulfobetaine (Rewoteric AM CAS U) to an electrophoretic buffer suppress the electroosmotic flow by 50-90%. Onset of suppression occurs at approximately the critical micelle concentration of the surfactant. CAS U effectively suppresses the electroosmotic flow over the pH range 3-12. Addition of 2 mM CAS U to the electrophoretic buffer prevents adsorption of cationic proteins lysozyme, α-chymotrypsinogen A, cytochrome c, and ribonuclease A. Migration time reproducibility for these proteins is ～1% RSD within 1 day and 2-5% from day to day. Efficiencies in excess of 750?000 plates/m and recoveries of >80% were observed for protein injections from distilled water. Alternatively if 2 mM CAS U is added to samples, recoveries were quantitative, although efficiencies decreased to 325?000-600?000 plates/m. The natural electroosmotic flow of the capillaries is regenerated simply by rinsing with sodium hydroxide.     

Multiple token‐based neighbor discovery for directional sensor networks

Directional sensor networks (DSNs) can significantly improve the performance of a network by employing energy efficient communication protocols. Neighbor discovery is a vital part of medium access control (MAC) and routing protocol, which influences the establishment of communication between neighboring nodes. Neighbor discovery is a challenging task in DSNs due to the limited coverage provided by directional antennas. Furthermore, in these networks, communication can only take place when the beams of the directional antennas are pointed toward each other. In this article, we propose a novel multiple token‐based neighbor discovery (MuND) protocol, in which multiple tokens are transmitted based on an area exploration algorithm. The performance of the protocol is evaluated using the Cooja simulator. The simulation results reveal that the proposed MuND protocol achieves lower neighbor discovery latency, with a 100% neighbor discovery ratio, and has a relatively low communication overhead and low energy consumption.     

Accurate micro-computed tomography imaging of pore spaces in collagen-based scaffold

In this work we have used X-ray micro-computed tomography (μCT) as a method to observe the morphology of 3D porous pure collagen and collagen-composite scaffolds useful in tissue engineering. Two aspects of visualizations were taken into consideration: improvement of the scan and investigation of its sensitivity to the scan parameters. Due to the low material density some parts of collagen scaffolds are invisible in a μCT scan. Therefore, here we present different contrast agents, which increase the contrast of the scanned biopolymeric sample for μCT visualization. The increase of contrast of collagenous scaffolds was performed with ceramic hydroxyapatite microparticles (HAp), silver ions (Ag⁺) and silver nanoparticles (Ag-NPs). Since a relatively small change in imaging parameters (e.g. in 3D volume rendering, threshold value and μCT acquisition conditions) leads to a completely different visualized pattern, we have optimized these parameters to obtain the most realistic picture for visual and qualitative evaluation of the biopolymeric scaffold. Moreover, scaffold images were stereoscopically visualized in order to better see the 3D biopolymer composite scaffold morphology. However, the optimized visualization has some discontinuities in zoomed view, which can be problematic for further analysis of interconnected pores by commonly used numerical methods. Therefore, we applied the locally adaptive method to solve discontinuities issue. The combination of contrast agent and imaging techniques presented in this paper help us to better understand the structure and morphology of the biopolymeric scaffold that is crucial in the design of new biomaterials useful in tissue engineering.     

Influence of stimulants on electrodermal studies in Fragile X syndrome

Attention Deficit Hyperactivity Disorder (ADHD) is seen in the majority of children with Fragile X Syndrome (FraX). Previous work has documented an enhanced sweat response to stimuli in children with FraX compared to controls utilizing electrodermal response (EDR) measures. The present study assesses the EDRs both on and off stimulants in 19 children with ADHD and FraX compared to 17 age- and IQ-matched control patients with ADHD and developmental delays. Although the baseline EDRs were comparable between FraX patients and controls, the patients with FraX had a significant decrease in EDR amplitude and number of peaks when treated with stimulants compared to controls. This suggests that patients with FraX are more responsive to the enhancement of inhibitory systems that occur with stimulant use for ADHD. The use of a quantifiable measure, such as EDR, is recommended in future studies of treatment efficacy.     

Research in progress

Environmental designers and researchers have taken advantage of visual surrogates for problem solving in a variety of physical settings and for theory building in the areas of environmental perception and behaviour. However, validity issues—external validity, ecological validity and incremental validity—have to be considered while selecting an environmental surrogate. In representing water features the dynamic qualities of water, sound and movement, have to be portrayed. A method is described for representing built waterscapes by videotape images, which takes into account these three validity concerns (although the validity of the surrogate is not tested). The surrogate attempts to recognize that human environmental perception is multi-modal and that humans are active participants throughout the environment. Such a surrogate seeks to more realistically reflect actual human environmental experience, but the effectiveness of the videos and their application to non-built settings requires further empirical support.