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Sensitive and rapid detection of cholera toxin subunit B using magnetic frequency mixing detection
(2019)
Cholera is a life-threatening disease caused by the cholera toxin (CT) as produced by some Vibrio cholerae serogroups. In this research we present a method which directly detects the toxin’s B subunit (CTB) in drinking water. For this purpose we performed a magnetic sandwich immunoassay inside a 3D immunofiltration column. We used two different commercially available antibodies to capture CTB and for binding to superparamagnetic beads. ELISA experiments were performed to select the antibody combination. The beads act as labels for the magnetic frequency mixing detection technique. We show that the limit of detection depends on the type of magnetic beads. A nonlinear Hill curve was fitted to the calibration measurements by means of a custom-written python software. We achieved a sensitive and rapid detection of CTB within a broad concentration range from 0.2 ng/ml to more
than 700 ng/ml.
Herein, fibroin, polylactide (PLA), and carbon are investigated for their suitability as biocompatible and biodegradable materials for amperometric biosensors. For this purpose, screen-printed carbon electrodes on the biodegradable substrates fibroin and PLA are modified with a glucose oxidase membrane and then encapsulated with the biocompatible material Ecoflex. The influence of different curing parameters of the carbon electrodes on the resulting biosensor characteristics is studied. The morphology of the electrodes is investigated by scanning electron microscopy, and the biosensor performance is examined by amperometric measurements of glucose (0.5–10 mM) in phosphate buffer solution, pH 7.4, at an applied potential of 1.2 V versus a Ag/AgCl reference electrode. Instead of Ecoflex, fibroin, PLA, and wound adhesive are tested as alternative encapsulation compounds: a series of swelling tests with different fibroin compositions, PLA, and Ecoflex has been performed before characterizing the most promising candidates by chronoamperometry. Therefore, the carbon electrodes are completely covered with the particular encapsulation material. Chronoamperometric measurements with H2O2 concentrations between 0.5 and 10 mM enable studying the leakage current behavior.
It has been shown that muscle fascicle curvature increases with increasing contraction level and decreasing muscle–tendon complex length. The analyses were done with limited examination windows concerning contraction level, muscle–tendon complex length, and/or intramuscular position of ultrasound imaging. With this study we aimed to investigate the correlation between fascicle arching and contraction, muscle–tendon complex length and their associated architectural parameters in gastrocnemius muscles to develop hypotheses concerning the fundamental mechanism of fascicle curving. Twelve participants were tested in five different positions (90°/105°*, 90°/90°*, 135°/90°*, 170°/90°*, and 170°/75°*; *knee/ankle angle). They performed isometric contractions at four different contraction levels (5%, 25%, 50%, and 75% of maximum voluntary contraction) in each position. Panoramic ultrasound images of gastrocnemius muscles were collected at rest and during constant contraction. Aponeuroses and fascicles were tracked in all ultrasound images and the parameters fascicle curvature, muscle–tendon complex strain, contraction level, pennation angle, fascicle length, fascicle strain, intramuscular position, sex and age group were analyzed by linear mixed effect models. Mean fascicle curvature of the medial gastrocnemius increased with contraction level (+5 m−1 from 0% to 100%; p = 0.006). Muscle–tendon complex length had no significant impact on mean fascicle curvature. Mean pennation angle (2.2 m−1 per 10°; p < 0.001), inverse mean fascicle length (20 m−1 per cm−1; p = 0.003), and mean fascicle strain (−0.07 m−1 per +10%; p = 0.004) correlated with mean fascicle curvature. Evidence has also been found for intermuscular, intramuscular, and sex-specific intramuscular differences of fascicle curving. Pennation angle and the inverse fascicle length show the highest predictive capacities for fascicle curving. Due to the strong correlations between pennation angle and fascicle curvature and the intramuscular pattern of curving we suggest for future studies to examine correlations between fascicle curvature and intramuscular fluid pressure.
Low emission zones and truck bans, the rising price of diesel and increases in road tolls: all of these factors are putting serious pressure on the transport industry. Commercial vehicle manufacturers and their suppliers are in the process of identifying new solutions to these challenges as part of their efforts to meet the EEV (enhanced environmentally friendly vehicle) limits, which are currently the most robust European exhaust and emissions standards for trucks and buses.
Germany is a frontrunner in setting frameworks for the transition to a low-carbon system. The mobility sector plays a significant role in this shift, affecting different people and groups on multiple levels. Without acceptance from these stakeholders, emission targets are out of reach. This research analyzes how the heterogeneous preferences of various stakeholders align with the transformation of the mobility sector, looking at the extent to which the German transformation paths are supported and where stakeholders are located.
Under the research objective of comparing stakeholders' preferences to identify which car segments require additional support for a successful climate transition, a status quo of stakeholders and car performance criteria is the foundation for the analysis. Stakeholders' hidden preferences hinder the derivation of criteria weightings from stakeholders; therefore, a ranking from observed preferences is used. This study's inverse multi-criteria decision analysis means that weightings can be predicted and used together with a recalibrated performance matrix to explore future preferences toward car segments.
Results show that stakeholders prefer medium-sized cars, with the trend pointing towards the increased potential for alternative propulsion technologies and electrified vehicles. These insights can guide the improved targeting of policy supporting the energy and mobility transformation. Additionally, the method proposed in this work can fully handle subjective approaches while incorporating a priori information. A software implementation of the proposed method completes this work and is made publicly available.
The aim of the present study was the characterisation of three true subtilisins and one phylogenetically intermediate subtilisin from halotolerant and halophilic microorganisms. Considering the currently growing enzyme market for efficient and novel biocatalysts, data mining is a promising source for novel, as yet uncharacterised enzymes, especially from halophilic or halotolerant Bacillaceae, which offer great potential to meet industrial needs. Both halophilic bacteria Pontibacillus marinus DSM 16465ᵀ and Alkalibacillus haloalkaliphilus DSM 5271ᵀ and both halotolerant bacteria Metabacillus indicus DSM 16189 and Litchfieldia alkalitelluris DSM 16976ᵀ served as a source for the four new subtilisins SPPM, SPAH, SPMI and SPLA. The protease genes were cloned and expressed in Bacillus subtilis DB104. Purification to apparent homogeneity was achieved by ethanol precipitation, desalting and ion-exchange chromatography. Enzyme activity could be observed between pH 5.0–12.0 with an optimum for SPPM, SPMI and SPLA around pH 9.0 and for SPAH at pH 10.0. The optimal temperature for SPMI and SPLA was 70 °C and for SPPM and SPAH 55 °C and 50 °C, respectively. All proteases showed high stability towards 5% (w/v) SDS and were active even at NaCl concentrations of 5 M. The four proteases demonstrate potential for future biotechnological applications.
The aerodynamic performance of propellers strongly depends on their geometry and, consequently, on aeroelastic deformations. Knowledge of the extent of the impact is crucial for overall aircraft performance. An integrated simulation environment for steady aeroelastic propeller simulations is presented. The simulation environment is applied to determine the impact of elastic deformations on the aerodynamic propeller performance. The aerodynamic module includes a blade element momentum approach to calculate aerodynamic loads. The structural module is based on finite beam elements, according to Timoshenko theory, including moderate deflections. Several fixed-pitch propellers with thin-walled cross sections made of both isotropic and non-isotropic materials are investigated. The essential parameters are varied: diameter, disc loading, sweep, material, rotational, and flight velocity. The relative change of thrust between rigid and elastic blades quantifies the impact of propeller elasticity. Swept propellers of large diameters or low disc loadings can decrease the thrust significantly. High flight velocities and low material stiffness amplify this tendency. Performance calculations without consideration of propeller elasticity can lead to decreased efficiency. To avoid cost- and time-intense redesigns, propeller elasticity should be considered for swept planforms and low disc loadings.
Several species of (poly)saccharides and organic acids can be found often simultaneously in various biological matrices, e.g., fruits, plant materials, and biological fluids. The analysis of such matrices sometimes represents a challenging task. Using Aloe vera (A. vera) plant materials as an example, the performance of several spectro-scopic methods (80 MHz benchtop NMR, NIR, ATR-FTIR and UV–vis) for the simultaneous analysis of quality parameters of this plant material was compared. The determined parameters include (poly)saccharides such as aloverose, fructose and glucose as well as organic acids (malic, lactic, citric, isocitric, acetic, fumaric, benzoic and sorbic acids). 500 MHz NMR and high-performance liquid chromatography (HPLC) were used as the reference methods.
UV–vis data can be used only for identification of added preservatives (benzoic and sorbic acids) and drying agent (maltodextrin) and semiquantitative analysis of malic acid. NIR and MIR spectroscopies combined with multivariate regression can deliver more informative overview of A. vera extracts being able to additionally quantify glucose, aloverose, citric, isocitric, malic, lactic acids and fructose. Low-field NMR measurements can be used for the quantification of aloverose, glucose, malic, lactic, acetic, and benzoic acids. The benchtop NMR method was successfully validated in terms of robustness, stability, precision, reproducibility and limit of detection (LOD) and quantification (LOQ), respectively. All spectroscopic techniques are useful for the screening of (poly)saccharides and organic acids in plant extracts and should be applied according to its availability as well as information and confidence required for the specific analytical goal. Benchtop NMR spectroscopy seems to be the most feasible solution for quality control of A. vera products.
Background
Aminoacylases are highly promising enzymes for the green synthesis of acyl-amino acids, potentially replacing the environmentally harmful Schotten-Baumann reaction. Long-chain acyl-amino acids can serve as strong surfactants and emulsifiers, with application in cosmetic industries. Heterologous expression of these enzymes, however, is often hampered, limiting their use in industrial processes.
Results
We identified a novel mycobacterial aminoacylase gene from Mycolicibacterium smegmatis MKD 8, cloned and expressed it in Escherichia coli and Vibrio natriegens using the T7 overexpression system. The recombinant enzyme was prone to aggregate as inclusion bodies, and while V. natriegens Vmax™ could produce soluble aminoacylase upon induction with isopropyl β-d-1-thiogalactopyranoside (IPTG), E. coli BL21 (DE3) needed autoinduction with lactose to produce soluble recombinant protein. We successfully conducted a chaperone co-expression study in both organisms to further enhance aminoacylase production and found that overexpression of chaperones GroEL/S enhanced aminoacylase activity in the cell-free extract 1.8-fold in V. natriegens and E. coli. Eventually, E. coli ArcticExpress™ (DE3), which co-expresses cold-adapted chaperonins Cpn60/10 from Oleispira antarctica, cultivated at 12 °C, rendered the most suitable expression system for this aminoacylase and exhibited twice the aminoacylase activity in the cell-free extract compared to E. coli BL21 (DE3) with GroEL/S co-expression at 20 °C. The purified aminoacylase was characterized based on hydrolytic activities, being most stable and active at pH 7.0, with a maximum activity at 70 °C, and stability at 40 °C and pH 7.0 for 5 days. The aminoacylase strongly prefers short-chain acyl-amino acids with smaller, hydrophobic amino acid residues. Several long-chain amino acids were fairly accepted in hydrolysis as well, especially N-lauroyl-L-methionine. To initially evaluate the relevance of this aminoacylase for the synthesis of N-acyl-amino acids, we demonstrated that lauroyl-methionine can be synthesized from lauric acid and methionine in an aqueous system.
Conclusion
Our results suggest that the recombinant enzyme is well suited for synthesis reactions and will thus be further investigated.
The eVTOL industry is a rapidly growing mass market expected to start in 2024. eVTOL compete, caused by their predicted missions, with ground-based transportation modes, including mainly passenger cars. Therefore, the automotive and classical aircraft design process is reviewed and compared to highlight advantages for eVTOL development. A special focus is on ergonomic comfort and safety. The need for further investigation of eVTOL’s crashworthiness is outlined by, first, specifying the relevance of passive safety via accident statistics and customer perception analysis; second, comparing the current state of regulation and certification; and third, discussing the advantages of integral safety and applying the automotive safety approach for eVTOL development. Integral safety links active and passive safety, while the automotive safety approach means implementing standardized mandatory full-vehicle crash tests for future eVTOL. Subsequently, possible crash impact conditions are analyzed, and three full-vehicle crash load cases are presented.