Probing the Conformation States of Neurotensin Receptor 1 Variants by NMR Site-Directed Methyl Labeling

G protein-coupled receptors (GPCRs) are key players in mediating signal transduction across the cell membrane. However, due to their intrinsic instability, many GPCRs are not suitable for structural investigations. Various approaches have been developed in recent years to remedy this situation, ranging from the use of more native membrane mimetics to protein-stabilization methods. The latter approach typically results in GPCRs that contain various numbers of mutations. However, probing the functionality of such variants by in vitro and in vivo assays is often time consuming. In addition, to validate the suitability of such GPCRs for structural investigations, an assessment of their conformation state is required. NMR spectroscopy has been proven to be suitable to probe the conformation state of GPCRs in solution. Here, by using chemical labeling with an isotope-labeled methyl probe, we show that the activity and the conformation state of stabilized neurotensin receptor 1 variants obtained from directed evolution can be efficiently assayed in 2D NMR experiments. This strategy enables the quantification of the active and inactive conformation states and the derivation of an estimation of the basal as well as agonist-induced activity of the receptor. Furthermore, this assay can be used as a readout when re-introducing agonist-dependent signaling into a highly stabilized, and thus rigidified, receptor by mutagenesis. This approach will be useful in cases where low production yields do not permit the addition of labeled compounds to the growth medium and where 1D NMR spectra of selectively ¹⁹F-labeled receptors are not sufficient to resolve signal overlap for a more detailed analysis.     

Mechanical Properties and Oxidation Behavior of Al2O3–AlN–TiN Composites

The study examines the effect which the composition of hot-pressed electroconductive ceramics has on their structure, mechanical properties, and oxidation behavior, for ceramics of the type AIN–Al₂O₃–42 wt% TiN, differing in the AIN/Al₂O₃ ratio. The results are physico-mechanical property data, including density, hardness, strength, fracture toughness, and wear resistance. A correlation was found between the wear resistance and fracture toughness. The analysis of oxidation products revealed the formation of α-Al₂O₃ and rutile in the temperature range from 600° to 1100°C and aluminum titanate above 1200°C. The spallation of the oxide layer caused low oxidation resistance of Al₂O₃-rich composites above 1250°C. The oxidation of composites was compared with the oxidation of pure TiN. The relationship is discussed between material properties, composition, phases, and processing parameters.     

Lanthanide-binding tags as versatile protein coexpression probes

Comprehensive proteomic analyses require new methodologies to accelerate the correlation of gene sequence with protein function. Key tools for such efforts include biophysical probes that integrate into the covalent architecture of proteins. Lanthanide-binding tags (LBTs) are expressible, multitasking fusion partners that are optimized to bind lanthanide ions and have several desirable attributes, which include long-lived luminescence, excellent X-ray scattering power for phase determination, and magnetic properties to facilitate NMR spectroscopic structure elucidation. Herein, we present peptide sequences with a 40-fold higher affinity for Tb(3+) ions and significantly brighter luminescence intensity compared with existing peptides. Incorporation of an LBT onto ubiquitin as a prototype fusion protein allows the use of powerful protein-visualization techniques, which include rapid luminescence detection of LBT-tagged proteins in SDS-PAGE gels, as well as determination of protein concentrations in complex mixtures. The LBT strategy is a new alternative for expressing fluorescent fusion proteins by routine molecular biological techniques.     

COMPREHENSIVE STUDY OF THE GAS HOLD UP AND BUBBLE SIZE DISTRIBUTION IN HIGHLY VISCOUS LIQUIDS II. CMC SOLUTIONS

Following properties of short bubble columns employing CMC solutions (1.0, 1.4 and 2.0%) and perforated plates (0.5, 1.0 and 3.0 mm hole diameters) were determined: relative mean gas hold up, E_G, bubble size distribution, Sauter bubble diameter, d_s, and the specific geometrical bubble surface areas, a' and a' due to the “intermediate to large bubbles.” The a' values were compared with the corresponding volumetric mass transfer coefficients, k_La's and the mass transfer coefficients, k_L, were estimated. The properties of these systems were investigated as function of the superficial gas velocity, W_SG, CMC concentration and aerator type.     

Sludge Pre-Treatment through Ozone Application: Alternative Sludge Reuse Possibilities for Recirculating Aquaculture System Optimization

Recirculating Aquaculture Systems (RAS) reduce water consumption by efficient filtration to maintain appropriate levels of accumulating compounds and sludge. Sludge is mechanically separated by drum filters and disposed of to the detriment of overall system water budgets. Dissolved nitrogen compounds are reduced via nitrification–denitrification filters, requiring commercial external carbon sources. The reuse of sludge after ozone pre-treatment may represent the next step in RAS optimization. The present study analyzes the content of sludge from RAS and tests ozonation as a pre-treatment for recycling as carbon source. The dissociative effect of ozone and the physicochemical changes due to ozonation lead to a significant increase in soluble carbon availability. Predominantly long-chain fatty acid (FA) (saturated and unsaturated) with 16 and 18 carbon atoms independently of the treatment were found in the profiles. Saturated FA concentrations in solution increased after 20, 40, and 60 min ozonation. The solid content of the sludge was practically unaffected by ozonation in terms of FA profile: only saturated FA slightly increases after 40 min treatment. The implications of these findings for denitrifying bacteria are discussed.

Abbreviations: Recirculating Aquaculture Systems (RAS); Advanced Oxidation Processes (AOPs)     

Engineering microfluidic papers: effect of fiber source and paper sheet properties on capillary-driven fluid flow

In the present study, we introduce a novel approach to control and modulate fluid transport inside microfluidic papers using lab-engineered paper sheets. Lab-sheets consisting of different fiber sources (eucalyptus sulfate and cotton linters pulp) and varying porosities were designed and further modified with small millimeter-scaled channels using hydrophobic barriers consisting of fiber-attached, hydrophobic polymers. The capillary-driven transport of an aqueous solution was monitored visually, and the influence of parameters such as fiber source, paper grammage, and channel width on the flow rates through the channel was investigated. The experimental results were compared with those obtained with commercially available filter papers. Our findings suggest that accurate control of fluid transport processes with standard filter papers is complex. Additionally, if the channel width is smaller than the mean fiber length, flow rates become dependent on the geometric parameters of the channel because of the formation of dead-end pores at the hydrophobic barriers. Finally, control of the paper sheets porosity, by varying the fiber density of the lab-made paper, affords the fabrication of chemically identical sheets whereby capillary flow is largely influenced and can be modulated accordingly by simple papermaking processes.     

The longitudinal impact of enterprise system users’ pre-adoption expectations and organizational support on post-adoption proficient usage

Although enterprise systems (ES) are ubiquitous, many firms report less than stellar payoffs from these costly investments, with underutilization often attributed to failures in the implementation process. Unfortunately, research has not provided sufficient insights into these failures, in part because it has focused on actual usage, as opposed to proficient usage, as the benchmark for successful implementation. Moreover, research has not generally examined how the adoption process unfolds over time, thus overlooking potential underlying mechanisms that may help explain how adopters achieve proficiency. To begin addressing these shortcomings, we study how adopters’ pre-adoption expectations, enacted over time, can influence their post-adoption proficiency, by shaping how and why they spend time using the system during the adoption period. We analyzed time-lagged survey data from 153 financial analysts, required to adopt new ES-based software, at a multinational bank. We found that adopters who hold pre-adoption expectations reflecting greater internal and external motives to adopt the system as well as systematically integrate it into their work routines are more apt to use the system in ways that enhance their cumulative knowledge of it, and subsequently develop higher levels of proficiency post-adoption. Moreover, greater organizational support enhanced the impact of adopters’ expectations on proficiency, except when their actual use is low in which case organizational support had an adverse effect.