Large Scale Bacterial Colony Screening of Diversified FRET Biosensors

Biosensors based on Forster Resonance Energy Transfer (FRET) between fluorescent protein mutants have started to revolutionize physiology and biochemistry. However, many types of FRET biosensors show relatively small FRET changes, making measurements with these probes challenging when used under sub-optimal experimental conditions. Thus, a major effort in the field currently lies in designing new optimization strategies for these types of sensors. Here we describe procedures for optimizing FRET changes by large scale screening of mutant biosensor libraries in bacterial colonies. We describe optimization of biosensor expression, permeabilization of bacteria, software tools for analysis, and screening conditions. The procedures reported here may help in improving FRET changes in multiple suitable classes of biosensors

High-Throughput Platform for Optoacoustic Probing of Genetically Encoded Calcium Ion Indicators

Functional optoacoustic (OA) imaging assisted with genetically encoded calcium ion indicators (GECIs) holds promise for imaging large-scale neuronal activity at depths and spatiotemporal resolutions not attainable with existing optical microscopic techniques. However, currently available GECIs optimized for fluorescence (FL) imaging lack sufficient contrast for OA imaging and respond at wavelengths having limited penetration into the mammalian brain. Here we present an imaging platform capable of rapid assessment and cross-validation between OA and FL responses of sensor proteins expressed in Escherichia coli colonies. The screening system features optimized pulsed light excitation combined with ultrasensitive ultrasound detection to mitigate photobleaching while further allowing the dynamic characterization of calcium ion responses with millisecond precision. Targeted probing of up to six individual colonies per second in both calcium-loaded and calcium-unloaded states was possible with the system. The new platform greatly facilitates optimization of absorption-based labels, thus setting the stage for directed evolution of OA GECIs

Biologically active Phytophthora mating hormone prepared by catalytic asymmetric total synthesis

A Phytophthora mating hormone with an array of 1,5-stereogenic centers has been synthesized by using our recently developed methodology of catalytic enantioselective conjugate addition of Grignard reagents. We applied this methodology in a diastereo- and enantioselective iterative route and obtained two of the 16 possible stereoisomers of Phytophthora hormone α1. These synthetic stereoisomers induced the formation of sexual spores (oospores) in A2 mating type strains of three heterothallic Phytophthora species, P. infestans, P. capsici, and P. nicotianae but not in A1 mating type strains. The response was concentration-dependent, and the oospores were viable. These results demonstrate that the biological activity of the synthetic hormone resembles that of the natural hormone α1. Mating hormones are essential components in the sexual life cycle of a variety of organisms. For plant pathogens like Phytophthora, sexual reproduction is important as a source of genetic variation. Moreover, the thick-walled oospores are the most durable propagules that can survive harsh environmental conditions. Sexual reproduction can thus greatly affect disease epidemics. The availability of synthetic compounds mimicking the activity of Phytophthora mating hormone will be instrumental for further unravelling sexual reproduction in this important group of plant pathogens.

Crystal Plasticity and Fresh Lobster

Mechanics of few crystals Mechanics of many crystals 3D electron microscopy Chitin-composite

Tests of cosmic ray radiography for power industry applications

In this report, we assess muon multiple scattering tomography as a non-destructive inspection technique in several typical areas of interest to the nuclear power industry, including monitoring concrete degradation, gate valve conditions, and pipe wall thickness. This work is motivated by the need for radiographic methods that do not require the licensing, training, and safety controls of x-rays, and by the need to be able to penetrate considerable overburden to examine internal details of components that are otherwise inaccessible, with minimum impact on industrial operations. In some scenarios, we find that muon tomography may be an attractive alternative to more typical measurements.Comment: LA-UR-15-2212

The Microtubule-Targeting Agent Pretubulysin Impairs the Inflammatory Response in Endothelial Cells by a JNK-Dependent Deregulation of the Histone Acetyltransferase Brd4

The anti-inflammatory effects of depolymerizing microtubule-targeting agents on leukocytes are known for a long time, but the potential involvement of the vascular endothelium and the underlying mechanistic basis is still largely unclear. Using the recently synthesized depolymerizing microtubule-targeting agent pretubulysin, we investigated the antiinflammatory potential of pretubulysin and other microtubule-targeting agents with respect to the TNF-induced leukocyte adhesion cascade in endothelial cells, to improve our understanding of the underlying biomolecular background. We found that treatment with pretubulysin reduces inflammation in vivo and in vitro via inhibition of the TNF-induced adhesion of leukocytes to the vascular endothelium by down-regulation of the pro-inflammatory cell adhesion molecules ICAM-1 and VCAM-1 in a JNK-dependent manner. The underlying mechanism includes JNK-induced deregulation and degradation of the histone acetyltransferase Bromodomaincontaining protein 4. This study shows that depolymerizing microtubule-targeting agents, in addition to their established effects on leukocytes, also significantly decrease the inflammatory activation of vascular endothelial cells. These effects are not based on altered pro-inflammatory signaling cascades, but require deregulation of the capability of cells to enter constructive transcription for some genes, setting a baseline for further research on the prominent antiinflammatory effects of depolymerizing microtubule-targeting agents

Cable-driven parallel robot for curtain wall module installation

A cable-driven parallel robot (CDPR) was developed for the installation of curtain wall modules (CWM). The research addressed the question of whether the CDPR was capable installing CWMs with sufficient accuracy while being competitive compared to conventional manual methods. In order to develop and test such a system, a conceptual framework that consisted of three sub-systems was defined. The tests, carried out in two close-to-real demonstration buildings, revealed an absolute accuracy of the CWM installation of 4 to 23 mm. The working time for installing a CWM was reduced to 0.51 h. The results also show that the system is competitive for a workspace greater than 96 m2 compared to conventional manual methods. However, improvements such as reducing the hours for setting up the CDPR on the one hand and achieving a faster and more robust MEE on the other hand will be still necessary in the future.This project has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No. 73251

Biotechnological production of γ-decalactone, a peach like aroma, by Yarrowia lipolytica

The request for new flavourings increases every year. Consumer perception that everything natural is better is causing an increase demand for natural aroma additives. Biotechnology has become a way to get natural products. γ-Decalactone is a peach-like aroma widely used in dairy products, beverages and others food industries. In more recent years, more and more studies and industrial processes were endorsed to cost-effect this compound production. One of the best-known methods to produce -decalactone is from ricinoleic acid catalyzed by Yarrowia lipolytica, a generally regarded as safe status yeast. As yet, several factors affecting -decalactone production remain to be fully understood and optimized. In this review, we focus on the aromatic compound -decalactone and its production by Y. lipolytica. The metabolic pathway of lactone production and degradation are addressed. Critical analysis of novel strategies of bioprocess engineering, metabolic and genetic engineering and other strategies for the enhancement of the aroma productivity are presented.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684)