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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.
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).
In proton therapy, the dose from secondary neutrons to the patient can contribute to side effects and the creation of secondary cancer. A simple and fast detection system to distinguish between dose from protons and neutrons both in pretreatment verification as well as potentially in vivo monitoring is needed to minimize dose from secondary neutrons. Two 3 mm long, 1 mm diameter organic scintillators were tested for candidacy to be used in a proton–neutron discrimination detector. The SCSF-3HF (1500) scintillating fibre (Kuraray Co. Chiyoda-ku, Tokyo, Japan) and EJ-260 plastic scintillator (Eljen Technology, Sweetwater, TX, USA) were irradiated at the TRIUMF Neutron Facility and the Proton Therapy Research Centre. In the proton beam, we compared the raw Bragg peak and spread-out Bragg peak response to the industry standard Markus chamber detector. Both scintillator sensors exhibited quenching at high LET in the Bragg peak, presenting a peak-to-entrance ratio of 2.59 for the EJ-260 and 2.63 for the SCSF-3HF fibre, compared to 3.70 for the Markus chamber. The SCSF-3HF sensor demonstrated 1.3 times the sensitivity to protons and 3 times the sensitivity to neutrons as compared to the EJ-260 sensor. Combined with our equations relating neutron and proton contributions to dose during proton irradiations, and the application of Birks’ quenching correction, these fibres provide valid candidates for inexpensive and replicable proton-neutron discrimination detectors
Optical Fibers as Dosimeter Detectors for Mixed Proton/Neutron Fields - A Biological Dosimeter
(2023)
In recent years, proton therapy has gained importance as a cancer treatment modality due to its conformality with the tumor and the sparing of healthy tissue. However, in the interaction of the protons with the beam line elements and patient tissues, potentially harmful secondary neutrons are always generated. To ensure that this neutron dose is as low as possible, treatment plans could be created to also account for and minimize the neutron dose. To monitor such a treatment plan, a compact, easy to use, and inexpensive dosimeter must be developed that not only measures the physical dose, but which can also distinguish between proton and neutron contributions. To that end, plastic optical fibers with scintillation materials (Gd₂O₂S:Tb, Gd₂O₂S:Eu, and YVO₄:Eu) were irradiated with protons and neutrons. It was confirmed that sensors with different scintillation materials have different sensitivities to protons and neutrons. A combination of these three scintillators can be used to build a detector array to create a biological dosimeter.
Seismic excited liquid filled tanks are subjected to extreme loading due to hydrodynamic pressures, which can lead to nonlinear stability failure of the thinwalled cylindrical tanks, as it is known from past earthquakes. A significant reduction of the seismically induced loads can be obtained by the application of base isolation systems, which have to be designed carefully with respect to the modified hydrodynamic behaviour of the tank in interaction with the liquid. For this reason a highly sophisticated fluid-structure interaction model has to be applied for a realistic simulation of the overall dynamic system. In the following, such a model is presented and compared with the results of simplified mathematical models for rigidly supported tanks. Finally, it is examined to what extent a simple mechanical model can represent the behaviour of a base isolated tank in case of seismic excitation
In order for traditional masonry to stay a competitive building material in seismically active regions there is an urgent demand for modern, deformation-based verification procedures which exploit the nonlinear load bearing reserves. The Capacity Spectrum Method (CSM) is a widely accepted design approach in the field of reinforced concrete and steel construction. It compares the seismic action with the load-bearing capacity of the building considering nonlinear material behavior with its post-peak capacity. The bearing capacity of the building is calculated iteratively using single wall capacity curves. This paper presents a new approach for the bilinear approximation of single wall capacity curves in the style of EC6/EC8 respectively FEMA 306/FEMA 356 based on recent shear wall test results of the European Collective-Research Project “ESECMaSE”. The application of the CSM to masonry structures by using bilinear approximations of capacity curves as input is demonstrated on the example of a typical German residential home.
Influence of slab deflection on the out-of-plane capacity of unreinforced masonry partition walls
(2023)
Severe damage of non-structural elements is noticed in previous earthquakes, causing high economic losses and posing a life threat for the people. Masonry partition walls are one of the most commonly used non-structural elements. Therefore, their behaviour under earthquake loading in out-of-plane (OOP) direction is investigated by several researches in the past years. However, none of the existing experimental campaigns or analytical approaches consider the influence of prior slab deflection on OOP response of partition walls. Moreover, none of the existing construction techniques for the connection of partition walls with surrounding reinforced concrete (RC) is investigated for the combined slab deflection and OOP loading. However, the inevitable time-dependent behaviour of RC slabs leads to high values of final slab deflections which can further influence boundary conditions of partition walls. Therefore, a comprehensive study on the influence of slab deflection on the OOP capacity of masonry partitions is conducted. In the first step, experimental tests are carried out. Results of experimental tests are further used for the calibration of the numerical model employed for a parametric study. Based on the results, behaviour under combined loading for different construction techniques is explained. The results show that slab deflection leads either to severe damage or to a high reduction of OOP capacity. Existing practical solutions do not account for these effects. In this contribution, recommendations to overcome the problems of combined slab deflection and OOP loading on masonry partition walls are given. Possible interaction of in-plane (IP) loading, with the combined slab deflection and OOP loading on partition walls, is not investigated in this study.
Recent earthquakes as the 2012 Emilia earthquake sequence showed that recently built unreinforced masonry (URM) buildings behaved much better than expected and sustained, despite the maximum PGA values ranged between 0.20–0.30 g, either minor damage or structural damage that is deemed repairable. Especially low-rise residential and commercial masonry buildings with a code-conforming seismic design and detailing behaved in general very well without substantial damages. The low damage grades of modern masonry buildings that was observed during this earthquake series highlighted again that codified design procedures based on linear analysis can be rather conservative. Although advances in simulation tools make nonlinear calculation methods more readily accessible to designers, linear analyses will still be the standard design method for years to come. The present paper aims to improve the linear seismic design method by providing a proper definition of the q-factor of URM buildings. These q-factors are derived for low-rise URM buildings with rigid diaphragms which represent recent construction practise in low to moderate seismic areas of Italy and Germany. The behaviour factor components for deformation and energy dissipation capacity and for overstrength due to the redistribution of forces are derived by means of pushover analyses. Furthermore, considerations on the behaviour factor component due to other sources of overstrength in masonry buildings are presented. As a result of the investigations, rationally based values of the behaviour factor q to be used in linear analyses in the range of 2.0–3.0 are proposed.
Because of simple construction process, high energy efficiency, significant fire resistance and excellent sound isolation, masonry infilled reinforced concrete (RC) frame structures are very popular in most of the countries in the world, as well as in seismic active areas. However, many RC frame structures with masonry infills were seriously damaged during earthquake events, as the traditional infills are generally constructed with direct contact to the RC frame which brings undesirable infill/frame interaction. This interaction leads to the activation of the equivalent diagonal strut in the infill panel, due to the RC frame deformation, and combined with seismically induced loads perpendicular to the infill panel often causes total collapses of the masonry infills and heavy damages to the RC frames. This fact was the motivation for developing different approaches for improving the behaviour of masonry infills, where infill isolation (decoupling) from the frame has been more intensively studied in the last decade. In-plane isolation of the infill wall reduces infill activation, but causes the need for additional measures to restrain out-of-plane movements. This can be provided by installing steel anchors, as proposed by some researchers. Within the framework of European research project INSYSME (Innovative Systems for Earthquake Resistant Masonry Enclosures in Reinforced Concrete Buildings) the system based on a use of elastomers for in-plane decoupling and steel anchors for out-of-plane restrain was tested. This constructive solution was tested and deeply investigated during the experimental campaign where traditional and decoupled masonry infilled RC frames with anchors were subjected to separate and combined in-plane and out-of-plane loading. Based on a detailed evaluation and comparison of the test results, the performance and effectiveness of the developed system are illustrated.