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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.
New European Union (EU) regulations for UAS operations require an operational risk analysis, which includes an estimation of the potential danger of the UAS crashing. A key parameter for the potential ground risk is the kinetic impact energy of the UAS. The kinetic energy depends on the impact velocity of the UAS and, therefore, on the aerodynamic drag and the weight during free fall. Hence, estimating the impact energy of a UAS requires an accurate drag estimation of the UAS in that state. The paper at hand presents the aerodynamic drag estimation of small-scale multirotor UAS. Multirotor UAS of various sizes and configurations were analysed with a fully unsteady Reynolds-averaged Navier–Stokes approach. These simulations included different velocities and various fuselage pitch angles of the UAS. The results were compared against force measurements performed in a subsonic wind tunnel and provided good consistency. Furthermore, the influence of the UAS`s fuselage pitch angle as well as the influence of fixed and free spinning propellers on the aerodynamic drag was analysed. Free spinning propellers may increase the drag by up to 110%, depending on the fuselage pitch angle. Increasing the fuselage pitch angle of the UAS lowers the drag by 40% up to 85%, depending on the UAS. The data presented in this paper allow for increased accuracy of ground risk assessments.
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.
Amino acid-based surfactants are valuable compounds for cosmetic formulations. The chemical synthesis of acyl-amino acids is conventionally performed by the Schotten-Baumann reaction using fatty acyl chlorides, but aminoacylases have also been investigated for use in biocatalytic synthesis with free fatty acids. Aminoacylases and their properties are diverse; they belong to different peptidase families and show differences in substrate specificity and biocatalytic potential. Bacterial aminoacylases capable of synthesis have been isolated from Burkholderia, Mycolicibacterium, and Streptomyces. Although several proteases and peptidases from S. griseus have been described, no aminoacylases from this species have been identified yet. In this study, we investigated two novel enzymes produced by S. griseus DSM 40236ᵀ . We identified and cloned the respective genes and recombinantly expressed an α-aminoacylase (EC 3.5.1.14), designated SgAA, and an ε-lysine acylase (EC 3.5.1.17), designated SgELA, in S. lividans TK23. The purified aminoacylase SgAA was biochemically characterized, focusing on its hydrolytic activity to determine temperature- and pH optima and stabilities. The aminoacylase could hydrolyze various acetyl-amino acids at the Nα -position with a broad specificity regarding the sidechain. Substrates with longer acyl chains, like lauroyl-amino acids, were hydrolyzed to a lesser extent. Purified aminoacylase SgELA specific for the hydrolysis of Nε -acetyl-L-lysine was unstable and lost its enzymatic activity upon storage for a longer period but could initially be characterized. The pH optimum of SgELA was pH 8.0. While synthesis of acyl-amino acids was not observed with SgELA, SgAA catalyzed the synthesis of lauroyl-methionine.
Aspergillus oryzae is an industrially relevant organism for the secretory production of heterologous enzymes, especially amylases. The activities of potential heterologous amylases, however, cannot be quantified directly from the supernatant due to the high background activity of native α-amylase. This activity is caused by the gene products of amyA, amyB, and amyC. In this study, an in vitro CRISPR/Cas9 system was established in A. oryzae to delete these genes simultaneously. First, pyrG of A. oryzae NSAR1 was mutated by exploiting NHEJ to generate a counter-selection marker. Next, all amylase genes were deleted simultaneously by co-transforming a repair template carrying pyrG of Aspergillus nidulans and flanking sequences of amylase gene loci. The rate of obtained triple knock-outs was 47%. We showed that triple knockouts do not retain any amylase activity in the supernatant. The established in vitro CRISPR/Cas9 system was used to achieve sequence-specific knock-in of target genes. The system was intended to incorporate a single copy of the gene of interest into the desired host for the development of screening methods. Therefore, an integration cassette for the heterologous Fpi amylase was designed to specifically target the amyB locus. The site-specific integration rate of the plasmid was 78%, with exceptional additional integrations. Integration frequency was assessed via qPCR and directly correlated with heterologous amylase activity. Hence, we could compare the efficiency between two different signal peptides. In summary, we present a strategy to exploit CRISPR/Cas9 for gene mutation, multiplex knock-out, and the targeted knock-in of an expression cassette in A. oryzae. Our system provides straightforward strain engineering and paves the way for development of fungal screening systems.
Die Bereitstellung von nachhaltig erzeugtem Wasserstoff als Energieträger und Rohstoff ist eine wichtige Schlüsseltechnologie sowohl als Ersatz für fossile Energieträger, aber auch als Produkt im Zusammenhang mit Kreislaufprozessen. In der Abwasserbehandlung bestehen verschiedene Möglichkeiten Wasserstoff herzustellen. Mehrere Wege, mögliche Synergien, aber auch deren Nachteile werden vorgestellt.
A method for detecting and approximating fault lines or surfaces, respectively, or decision curves in two and three dimensions with guaranteed accuracy is presented. Reformulated as a classification problem, our method starts from a set of scattered points along with the corresponding classification algorithm to construct a representation of a decision curve by points with prescribed maximal distance to the true decision curve. Hereby, our algorithm ensures that the representing point set covers the decision curve in its entire extent and features local refinement based on the geometric properties of the decision curve. We demonstrate applications of our method to problems related to the detection of faults, to multi-criteria decision aid and, in combination with Kirsch’s factorization method, to solving an inverse acoustic scattering problem. In all applications we considered in this work, our method requires significantly less pointwise classifications than previously employed algorithms.
Im Verfahren gegen die Österreichische Post AG (Rs. C-300/21) befasste sich der EuGH erstmals mit dem in Art. 82 DS-GVO geregelten datenschutzrechtlichen Schadensersatzanspruch. Mit den Klarstellungen des EuGH verschieben sich die Probleme nun stärker zu den „klassischen“ Fragen des Schadensersatzrechts im Zivilprozess. Relevant sind dabei vor allem Aspekte der Darlegungs- und Beweislast und deren Besonderheiten mit Blick auf den Ersatz immaterieller Schäden. Der Beitrag fokussiert sich auf die Voraussetzungen und den dabei zu führenden Tatsachenbeweis bei der Klage des Betroffenen gegen den Verantwortlichen auf Ersatz immaterieller Schäden.
Kartellrecht vs. Datenschutzrecht: Rechtsgrundlagen für die Datenverarbeitung in sozialen Netzwerken
(2023)
Umsatzbasierte Bußgelder – wie sonst nur aus dem Kartellrecht bekannt – waren einer der Gründe, warum die Datenschutz-Grundverordnung (DSGVO) vor ihrem Inkrafttreten für erhebliches Aufsehen sorgte. Die vielfach relevanteren Schadensersatzansprüche, die, wie bei „Dieselgate“, aufgrund der Vielzahl von betroffenen Personen und der aus Sicht von Rechtsdienstleistern bestehenden Skalierbarkeit mit weitaus höheren Einbußen für Unternehmen einhergehen können, blieben zunächst unbeachtet. Inzwischen ist der Schadensersatzanspruch gem. Art. 82 DSGVO die Vorschrift, die die meisten Vorlagen zum Europäischen Gerichtshof (EuGH) der letzten Jahre hervorgerufen hat. Am 4.5.2023 hat nun der EuGH (Urteil v. 4.5.2023 - Rs. C-300/21, NWB GAAAJ-41389) in einem Grundsatzurteil über zentrale Fragen rund um den Ersatz immaterieller Schäden als Folge von Datenschutzverstößen entschieden.