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Subtilisins from microbial sources, especially from the Bacillaceae family, are of particular interest for biotechnological applications and serve the currently growing enzyme market as efficient and novel biocatalysts. Biotechnological applications include use in detergents, cosmetics, leather processing, wastewater treatment and pharmaceuticals. To identify a possible candidate for the enzyme market, here we cloned the gene of the subtilisin SPFA from Fictibacillus arsenicus DSM 15822ᵀ (obtained through a data mining-based search) and expressed it in Bacillus subtilis DB104. After production and purification, the protease showed a molecular mass of 27.57 kDa and a pI of 5.8. SPFA displayed hydrolytic activity at a temperature optimum of 80 °C and a very broad pH optimum between 8.5 and 11.5, with high activity up to pH 12.5. SPFA displayed no NaCl dependence but a high NaCl tolerance, with decreasing activity up to concentrations of 5 m NaCl. The stability enhanced with increasing NaCl concentration. Based on its substrate preference for 10 synthetic peptide 4-nitroanilide substrates with three or four amino acids and its phylogenetic classification, SPFA can be assigned to the subgroup of true subtilisins. Moreover, SPFA exhibited high tolerance to 5% (w/v) SDS and 5% H₂O₂ (v/v). The biochemical properties of SPFA, especially its tolerance of remarkably high pH, SDS and H₂O₂, suggest it has potential for biotechnological applications.
The first and last mile of a railway journey, in both freight and transit applications, constitutes a high effort and is either non-productive (e.g. in the case of depot operations) or highly inefficient (e.g. in industrial railways). These parts are typically managed on-sight, i.e. with no signalling and train protection systems ensuring the freedom of movement. This is possible due to the rather short braking distances of individual vehicles and shunting consists. The present article analyses the braking behaviour of such shunting units. For this purpose, a dedicated model is developed. It is calibrated on published results of brake tests and validated against a high-definition model for low-speed applications. Based on this model, multiple simulations are executed to obtain a Monte Carlo simulation of the resulting braking distances. Based on the distribution properties and established safety levels, the risk of exceeding certain braking distances is evaluated and maximum braking distances are derived. Together with certain parameters of the system, these can serve in the design and safety assessment of driver assistance systems and automation of these processes.
This study evaluates neuromechanical control and muscle-tendon interaction during energy storage and dissipation tasks in hypergravity. During parabolic flights, while 17 subjects performed drop jumps (DJs) and drop landings (DLs), electromyography (EMG) of the lower limb muscles was combined with in vivo fascicle dynamics of the gastrocnemius medialis, two-dimensional (2D) kinematics, and kinetics to measure and analyze changes in energy management. Comparisons were made between movement modalities executed in hypergravity (1.8 G) and gravity on ground (1 G). In 1.8 G, ankle dorsiflexion, knee joint flexion, and vertical center of mass (COM) displacement are lower in DJs than in DLs; within each movement modality, joint flexion amplitudes and COM displacement demonstrate higher values in 1.8 G than in 1 G. Concomitantly, negative peak ankle joint power, vertical ground reaction forces, and leg stiffness are similar between both movement modalities (1.8 G). In DJs, EMG activity in 1.8 G is lower during the COM deceleration phase than in 1 G, thus impairing quasi-isometric fascicle behavior. In DLs, EMG activity before and during the COM deceleration phase is higher, and fascicles are stretched less in 1.8 G than in 1 G. Compared with the situation in 1 G, highly task-specific neuromuscular activity is diminished in 1.8 G, resulting in fascicle lengthening in both movement modalities. Specifically, in DJs, a high magnitude of neuromuscular activity is impaired, resulting in altered energy storage. In contrast, in DLs, linear stiffening of the system due to higher neuromuscular activity combined with lower fascicle stretch enhances the buffering function of the tendon, and thus the capacity to safely dissipate energy.
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.
Research on robotic lunar exploration has seen a broad revival, especially since the Google Lunar X-Prize increasingly brought private endeavors into play. This development is supported by national agencies with the aim of enabling long-term lunar infrastructure for in-situ operations and the establishment of a moon village. One challenge for effective exploration missions is developing a compact and lightweight robotic rover to reduce launch costs and open the possibility for secondary payload options. Existing micro rovers for exploration missions are clearly limited by their design for one day of sunlight and their low level of autonomy. For expanding the potential mission applications and range of use, an extension of lifetime could be reached by surviving the lunar night and providing a higher level of autonomy. To address this objective, the paper presents a system design concept for a lightweight micro rover with long-term mission duration capabilities, derived from a multi-day lunar mission scenario at equatorial regions. Technical solution approaches are described, analyzed, and evaluated, with emphasis put on the harmonization of hardware selection due to a strictly limited budget in dimensions and power.
Deammonification for nitrogen removal in municipal wastewater in temperate and cold climate zones is currently limited to the side stream of municipal wastewater treatment plants (MWWTP). This study developed a conceptual model of a mainstream deammonification plant, designed for 30,000 P.E., considering possible solutions corresponding to the challenging mainstream conditions in Germany. In addition, the energy-saving potential, nitrogen elimination performance and construction-related costs of mainstream deammonification were compared to a conventional plant model, having a single-stage activated sludge process with upstream denitrification. The results revealed that an additional treatment step by combining chemical precipitation and ultra-fine screening is advantageous prior the mainstream deammonification. Hereby chemical oxygen demand (COD) can be reduced by 80% so that the COD:N ratio can be reduced from 12 to 2.5. Laboratory experiments testing mainstream conditions of temperature (8–20°C), pH (6–9) and COD:N ratio (1–6) showed an achievable volumetric nitrogen removal rate (VNRR) of at least 50 gN/(m3∙d) for various deammonifying sludges from side stream deammonification systems in the state of North Rhine-Westphalia, Germany, where m3 denotes reactor volume. Assuming a retained Norganic content of 0.0035 kgNorg./(P.E.∙d) from the daily loads of N at carbon removal stage and a VNRR of 50 gN/(m3∙d) under mainstream conditions, a resident-specific reactor volume of 0.115 m3/(P.E.) is required for mainstream deammonification. This is in the same order of magnitude as the conventional activated sludge process, i.e., 0.173 m3/(P.E.) for an MWWTP of size class of 4. The conventional plant model yielded a total specific electricity demand of 35 kWh/(P.E.∙a) for the operation of the whole MWWTP and an energy recovery potential of 15.8 kWh/(P.E.∙a) through anaerobic digestion. In contrast, the developed mainstream deammonification model plant would require only a 21.5 kWh/(P.E.∙a) energy demand and result in 24 kWh/(P.E.∙a) energy recovery potential, enabling the mainstream deammonification model plant to be self-sufficient. The retrofitting costs for the implementation of mainstream deammonification in existing conventional MWWTPs are nearly negligible as the existing units like activated sludge reactors, aerators and monitoring technology are reusable. However, the mainstream deammonification must meet the performance requirement of VNRR of about 50 gN/(m3∙d) in this case.
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.
Background
Hip fractures are a common and costly health problem, resulting in significant morbidity and mortality, as well as high costs for healthcare systems, especially for the elderly. Implementing surgical preventive strategies has the potential to improve the quality of life and reduce the burden on healthcare resources, particularly in the long term. However, there are currently limited guidelines for standardizing hip fracture prophylaxis practices.
Methods
This study used a cost-effectiveness analysis with a finite-state Markov model and cohort simulation to evaluate the primary and secondary surgical prevention of hip fractures in the elderly. Patients aged 60 to 90 years were simulated in two different models (A and B) to assess prevention at different levels. Model A assumed prophylaxis was performed during the fracture operation on the contralateral side, while Model B included individuals with high fracture risk factors. Costs were obtained from the Centers for Medicare & Medicaid Services, and transition probabilities and health state utilities were derived from available literature. The baseline assumption was a 10% reduction in fracture risk after prophylaxis. A sensitivity analysis was also conducted to assess the reliability and variability of the results.
Results
With a 10% fracture risk reduction, model A costs between $8,850 and $46,940 per quality-adjusted life-year ($/QALY). Additionally, it proved most cost-effective in the age range between 61 and 81 years. The sensitivity analysis established that a reduction of ≥ 2.8% is needed for prophylaxis to be definitely cost-effective. The cost-effectiveness at the secondary prevention level was most sensitive to the cost of the contralateral side’s prophylaxis, the patient’s age, and fracture treatment cost. For high-risk patients with no fracture history, the cost-effectiveness of a preventive strategy depends on their risk profile. In the baseline analysis, the incremental cost-effectiveness ratio at the primary prevention level varied between $11,000/QALY and $74,000/QALY, which is below the defined willingness to pay threshold.
Conclusion
Due to the high cost of hip fracture treatment and its increased morbidity, surgical prophylaxis strategies have demonstrated that they can significantly relieve the healthcare system. Various key assumptions facilitated the modeling, allowing for adequate room for uncertainty. Further research is needed to evaluate health-state-associated risks.
The Cramér-von-Mises distance is applied to the distribution of the excess over a confidence level. Asymptotics of related statistics are investigated, and it is seen that the obtained limit distributions differ from the classical ones. For that reason, quantiles of the new limit distributions are given and new bootstrap techniques for approximation purposes are introduced and justified. The results motivate new one-sample goodness-of-fit tests for the distribution of the excess over a confidence level and a new confidence interval for the related fitting error. Simulation studies investigate size and power of the tests as well as coverage probabilities of the confidence interval in the finite sample case. A practice-oriented application of the Cramér-von-Mises tests is the determination of an appropriate confidence level for the fitting approach. The adoption of the idea to the well-known problem of threshold detection in the context of peaks over threshold modelling is sketched and illustrated by data examples.
Ambitious climate targets affect the competitiveness of industries in the international market. To prevent such industries from moving to other countries in the wake of increased climate protection efforts, cost adjustments may become necessary. Their design requires knowledge of country-specific production costs. Here, we present country-specific cost figures for different production routes of steel, paying particular attention to transportation costs. The data can be used in floor price models aiming to assess the competitiveness of different steel production routes in different countries (Rübbelke, 2022).