Functional Validation of cas9/GuideRNA Constructs for Site-Directed Mutagenesis of Triticale ABA8′OH1 loci

Cas endonuclease-mediated genome editing provides a long-awaited molecular biological approach to the modification of predefined genomic target sequences in living organisms. Although cas9/guide (g)RNA constructs are straightforward to assemble and can be customized to target virtually any site in the plant genome, the implementation of this technology can be cumbersome, especially in species like triticale that are difficult to transform, for which only limited genome information is available and/or which carry comparatively large genomes. To cope with these challenges, we have pre-validated cas9/gRNA constructs (1) by frameshift restitution of a reporter gene co-introduced by ballistic DNA transfer to barley epidermis cells, and (2) via transfection in triticale protoplasts followed by either a T7E1-based cleavage assay or by deep-sequencing of target-specific PCR amplicons. For exemplification, we addressed the triticale ABA 8′-HYDROXYLASE 1 gene, one of the putative determinants of pre-harvest sprouting of grains. We further show that in-del induction frequency in triticale can be increased by TREX2 nuclease activity, which holds true for both well- and poorly performing gRNAs. The presented results constitute a sound basis for the targeted induction of heritable modifications in triticale genes.     

New Halogenated Water‐Soluble Chlorin and Bacteriochlorin as Photostable PDT Sensitizers: Synthesis,Spectroscopy, Photophysics,and in vitro Photosensitizing Efficacy

Chlorin and bacteriochlorin derivatives of 5,10,15,20‐tetrakis(2‐chloro‐5‐sulfophenyl)porphyrin have intense absorptions in the phototherapeutic window, high water solubility, high photostability, low fluorescence quantum yield, long triplet lifetimes, and high singlet oxygen quantum yields. Biological studies revealed their negligible dark cytotoxicity, yet significant photodynamic effect against A549 (human lung adenocarcinoma), MCF7 (human breast carcinoma) and SK‐MEL‐188 (human melanoma) cell lines upon red light irradiation (cutoff λ<600 nm) at low light doses. Time‐dependent cellular accumulation of the chlorinated sulfonated chlorin reached a plateau at 2 h, as previously observed for the related porphyrin. However, the optimal incubation time for the bacteriochlorin derivative was significantly longer (12 h). The spectroscopic, photophysical, and biological properties of the compounds are discussed in relevance to their PDT activity, leading to the conclusion that the bacteriochlorin derivative is a promising candidate for future in vivo experiments.     

A stress analysis of a shaft with a press-fitted hub subjected to cyclic axial loading

This paper presents a calculation of the stress distribution in a shaft with a press-fitted hub subjected to axial fretting fatigue. Both normal contact stresses and frictional shear stresses at the shaft-hub interface are included in the model. The solution of Airy's function is obtained by means of Fourier integrals. The results are presented for different combinations of hub length, shaft radius and non-slip area and for various values of the coefficient of friction. Special consideration was given to the axial component of the stress, σ_z, as this is the most important stress component in the initiation and propagation of fatigue cracks. Finally, the paper deduces the implications arising from the stress analysis on the fretting fatigue of the junction studied.     

Modelling of composite crystallization

Metal matrix composites(MMCs)have received much attention due to their promising advanced mechanical properties.The aim of this work is to create a micro-macro model of composite crystallization.The developed model is coupled with the process of heat flow in the macroscopic scale,resulting from the heat emission during the nucleation and the growth of grains.Taking into account both of these phenomena,the proposed model is distinguished by a good reflection of reality.Moreover,the presented model assumes that the function of grain density depends on the maximal supercooling and the mass volume of the reinforcement phase particles.The knowledge of the equations,describing the function of grain density depending on the degree of supercooling,is necessary in the,more and more often used,numerical modelling of the casting structure.     

Analysis of Microencapsulation Ability of Essential Oils during Spray Drying

Results of preliminary investigations of the preservation of elemi (E) and peppermint (M) oils in a maltodextrin microcapsule during spray drying of emulsion in a laboratory dryer are discussed. The ratio of maltodextrin to water in the emulsion was 30:70, and the content of each oil 10, 20, and 30 wt·% in relation to maltodextrin. Product quality and results of the process were estimated on the basis of microscopic features of the powder (the shape and size of microparticles) and its density, moisture content and water vapor sorptivity. The efficiency of encapsulation was estimated by specifying the content of essential oil in the product. The compositions of initial oils and the oils contained in the emulsion, inside and on the capsule surface were compared. The positive results of this study justify the necessity of further research and provide a perspective for developing novel technologies.     

Comparative performance of membrane filters vs. high-surface area filtration cartridges for the determination of element concentrations in freshwater systems

Interdisciplinary studies on trace element cycles in aquatic environments may require the simultaneous use of small- and large-scale filtration devices to collect all the necessary sample aliquots for the different scientific objectives (e.g. trace element and organic carbon analysis, chronic toxicity testing, and characterization of colloidal matter). This study compares the performance of membrane filters (MF, Millipore-approx. surface area 0.002 m2) and two large-surface area filtration cartridges (Calyx, MSI and Polypro, CUNO approx. surface area 1 m2) in three freshwater systems with contrasting characteristics. Membrane filters and filtration cartridges fractionate water samples in a comparable way except for some elements (Al, Mn, Pb, and Ti) and when dealing with high-turbidity, organic-rich matrices. Estimation of the actual filters' pore size using the single particle counting technique gives cut-offs of 0.2-0.3, 0.7 and 2 microm for membrane filters, Polypro cartridge and Calyx cartridge respectively, explaining many of the differences observed between membrane and cartridge filters. Direct involvement of filter manufacturers to determine actual filter cut-offs during environmental applications would be highly beneficial to increase the comparability of the measurements of filterable element concentrations. Better harmonization of filtration procedures (filter type, actual filter cut-off, in situ vs. ex situ filtration) among laboratories in different countries is also necessary in order to improve consistency of environmental databases.     

Modeling and physical interpretation of photocatalytic oxidation efficiency in indoor air applications

Volatile organic compounds (VOCs) are the major pollutants in indoor air, which significantly impact indoor air quality (IAQ). As a promising technique to remove VOCs, photocatalytic oxidation (PCO) takes the advantages of oxidation of a large range of VOCs with low energy consumption. In this study, the mass transports and reaction mechanism involved in the PCO process have been studied. In addition, the kinetic models of PCO on the different conditions of elementary reactions have been critically reviewed. Moreover, the factors that may affect the efficiency of PCO were interpreted based on the established fundamental mechanism of PCO. Some recommendations were made for future work to improve the efficiency of PCO system for building applications.     

Structural Systems Composed of Concentric Hoops and Designed for Lightweight Domes of Large Spans

The paper presents a selected group of tension-strut structural systems designed for the construction of lightweight dome covers of large spans, which can be comparatively easy to assembly and have rises of which can be relatively small. This will allow significant decrease costs of erection and maintenance of objects covered by these roof structures. The proposed systems have been obtained from the results of suitable transformations of a chosen type of double-layer space frame and an appropriate arrangement of tetrahedron modules in the space of each of the newly designed type of the structural system. All these systems are built by means of concentric hoops having their own integral spatial stiffness obtained after an appropriate pre-stressing. Particular hoops can be mounted on the ground level and then one by one will be hoisted to the designed positions where they will be connected by means of special sets of the tension members. Due to these structural features, the assembly process of each system should be relatively simple, fast and not expensive. The whole tension-strut structure has to be connected to the compression perimeter ring and suitably pre-stressed. There are presented visualizations of the proposed systems prepared on the basis of the appropriate numerical models especially defined for each particular structure.