Creation of a TILLING population in barley after chemical mutagenesis with sodium azide and MNU

Since the development of the Targeting Induced Local Lesions in Genome (TILLING) strategy, it has been applied in both plants and animals in many studies. The creation of an appropriate population is the first and most crucial step of TILLING. The goal is to obtain a highly mutagenized population that allows many mutations in any gene of interest to be found. Therefore, an effective method of mutation induction should be developed. A high mutation density is associated with saving time, costs, and the labor required for the development of a TILLING platform. The proper handling of the mutated generations, the establishment of a seed bank, and the development of a DNA library are essential for creating a TILLING population. The database in which all of the data from the molecular and phenotypic analyses are collected is a very useful tool for maintaining such population. Once developed, a TILLING population can serve as a renewable resource of mutations for research that uses both forward and reverse genetic approaches. In this chapter, we describe the methods for the development and maintenance of a TILLING population in barley

Mutation in HvCBP20 (Cap binding protein 20) adapts barley to drought stress at phenotypic and transcriptomic levels

This work was supported by the European Regional Development Fund through the Innovative Economy for Poland 2007–2013, project WND-POIG.01.03.01-00-101/08 POLAPGEN-BD “Biotechnological tools for breeding cereals with increased resistance to drought,” task 22; National Science Centre, Poland, project SONATA 2015/19/D/NZ9/03573 “Translational genomics approach to identify the mechanisms of CBP20 signalosome in Arabidopsis and barley under drought stress.”CBP20 (Cap-Binding Protein 20) encodes a small subunit of the cap-binding complex (CBC), which is involved in the conserved cell processes related to RNA metabolism in plants and, simultaneously, engaged in the signaling network of drought response, which is dependent on ABA. Here, we report the enhanced tolerance to drought stress of barley mutant in the HvCBP20 gene manifested at the morphological, physiological, and transcriptomic levels. Physiological analyses revealed differences between the hvcbp20.ab mutant and its WT in response to a water deficiency. The mutant exhibited a higher relative water content (RWC), a lower stomatal conductance and changed epidermal pattern compared to the WT after drought stress. Transcriptome analysis using the Agilent Barley Microarray integrated with observed phenotypic traits allowed to conclude that the hvcbp20.ab mutant exhibited better fitness to stress conditions by its much more efficient and earlier activation of stress-preventing mechanisms. The network hubs involved in the adjustment of hvcbp20.ab mutant to the drought conditions were proposed. These results enabled to make a significant progress in understanding the role of CBP20 in the drought stress response.European Regional Development Fund; National Science Centre, Polan

Pseudanabaena galeata CCNP1313 : biological activity and peptides production

Even cyanobacteria from ecosystems of low biodiversity, such as the Baltic Sea, can constitute a rich source of bioactive metabolites. Potent toxins, enzyme inhibitors, and anticancer and antifungal agents were detected in both bloom-forming species and less commonly occurring cyanobacteria. In previous work on the Baltic Pseudanabaena galeata CCNP1313, the induction of apoptosis in the breast cancer cell line MCF-7 was documented. Here, the activity of the strain was further explored using human dermal fibroblasts, African green monkey kidney, cancer cell lines (T47D, HCT-8, and A549(ACE2/TMPRSS2)) and viruses (SARS-CoV-2, HCoV-OC43, and WNV). In the tests, extracts, chromatographic fractions, and the main components of the P. galeata CCNP1313 fractions were used. The LC-MS/MS analyses of the tested samples led to the detection of forty-five peptides. For fourteen of the new peptides, putative structures were proposed based on MS/MS spectra. Although the complex samples (i.e., extracts and chromatographic fractions) showed potent cytotoxic and antiviral activities, the effects of the isolated compounds were minor. The study confirmed the significance of P. galeata CCNP1313 as a source of metabolites with potent activity. It also illustrated the difficulties in assigning the observed biological effects to specific metabolites, especially when they are produced in minute amounts

HorTILLUS - a rich and renewable source of induced mutations for forward/reverse genetics and pre-breeding programs in barley (Hordeum vulgare L.)

TILLING (Targeting Induced Local Lesions IN Genomes) is a strategy used for functional analysis of genes that combines the classical mutagenesis and a rapid, high-throughput identification of mutations within a gene of interest. TILLING has been initially developed as a discovery platform for functional genomics, but soon it has become a valuable tool in development of desired alleles for crop breeding, alternative to transgenic approach. Here we present the HorTILLUS (Hordeum—TILLING—University of Silesia) population created for spring barley cultivar “Sebastian” after double-treatment of seeds with two chemical mutagens: sodium azide (NaN3) and N-methyl-N-nitrosourea (MNU). The population comprises more than 9,600 M2 plants from which DNA was isolated, seeds harvested, vacuum-packed, and deposited in seed bank. M3 progeny of 3,481 M2 individuals was grown in the field and phenotyped. The screening for mutations was performed for 32 genes related to different aspects of plant growth and development. For each gene fragment, 3,072–6,912 M2 plants were used for mutation identification using LI-COR sequencer. In total, 382 mutations were found in 182.2Mb screened. The average mutation density in the HorTILLUS, estimated as 1 mutation per 477 kb, is among the highest mutation densities reported for barley. The majority of mutations were G/C to A/T transitions, however about 8% transversions were also detected. Sixty-one percent of mutations found in coding regions were missense, 37.5% silent and 1.1% nonsense. In each gene, the missense mutations with a potential effect on protein function were identified. The HorTILLUS platformis the largest of the TILLING populations reported for barley and best characterized. The population proved to be a useful tool, both in functional genomic studies and in forward selection of barley mutants with required phenotypic changes. We are constantly renewing the HorTILLUS population, which makes it a permanent source of new mutations.We offer the usage of this valuable resource to the interested barley researchers on cooperative basis

New antiferroelectric compound studied by complementary methods

New antiferroelectric compound has been studied by electrooptic methods. The electrooptic parameters such as spontaneous polarization, tilt angle and switching time were measured. Using texture observation and differential scanning calorimetric methods, the phase sequences at cooling and heating have been obtained

Comparative Analysis of the Wear of NC11LV and Hardox 600 Steel Used in Tools for Extrusion of Clay Strands in the Process of Producing Ceramic Roof Tiles

This article presents the results of a comparative analysis performed with respect to the wear of tools used for the extrusion of a clay strand (for ceramic roof tile) made from two materials: steel NC11LV and steel Hardox 600. The aim of the studies was to determine the causes and mechanisms of wear as well as to evaluate the possibility of choosing the optimal material, mostly in respect to its resistance to intensive wear as well as an increase in the operation time. The results of the conducted investigations included: an analysis of the technology, thermovision measurements of the forming process, a macroscopic analysis combined with 3D scanning of the worn tools, ball-on-disc tests of the sliding wear resistance and hardness measurements. The obtained results demonstrated that the tools made of steel NC11LV were much less worn than those made of steel Hardox 600, as the operation time for the NC11LV steel tools was almost three times longer. The results of the ball-on-disc tests showed a similar manner of wear for both materials (with the working temperature of about 50 °C). The higher durability of the tools made from steel NC11LV can be an effect of a slightly lower coefficient of friction in the initial period of operation as well as the presence of hard carbides, which means increased hardness and thus also higher wear resistance at working temperatures

Preliminary Studies of the Durability of Tools Used to Form Ceramic Tiles Made of Hardox 600 and NC11LV Steel

The study performs a comparative analysis of the wear of tools made of two wear-resistant materials: steel Hardox 600 and NC11LV, used in the process of forming a band for roofing tiles. The analyses were to allow the assessment of the possibility of replacing the standard material for tools in this process with a much less expensive tool steel for cold work after heat treatment (with a large number of carbides), as an alternative material dedicated to tools resistant to intense abrasive wear. The performed investigations included a macroscopic and geometrical analysis with the use of 3D scanning, microstructural analyses conducted by means of a light microscope, as well as an analysis of the topography of the working areas of the tools with the use of SEM, and microhardness tests. The obtained results demonstrate that the tools made of both materials were characterized with a similar level of wear, which, in the most critical area, reached over 4 mm, while the tools made of steel NC11LV worked over a much longer period of time without regeneration, equaling 912 h, and an insert made of steel Hardox 600 operated for 384 h. A higher tool life in the case of NC11LV steel may be the result of higher hardness and the presence of hard carbides

Electro-optic and dielectric properties of new binary ferroelectric and antiferroelectric liquid crystalline mixtures

Several new binary liquid crystalline mixtures have been designed and their properties were studied by complementary methods. It has been shown that even both pure components used for the mixture design possess the ferroelectric behaviour; the induced antiferroelectric smectic phase has been detected for one of the prepared mixtures. The phase diagram has been constructed and the existence of the antiferroelectric phase was confirmed by switching time and dielectric spectroscopy measurements. Some of the resulted mixtures possess very high values of the tilt angle that reaches close below 45° degrees at saturation. Values of spontaneous polarisation were found within 50–200 nC/cm2 in dependence of the mixture’s composition. Due to specific properties, the obtained mixtures might be interesting for further design of multicomponent mixtures and formulation of the advanced nanocomposite systems

Evaluation of the abilities of the staff to perform medical rescue procedures at the advanced level in the State Fire Service in Poland in connection with an update of the Principles of Medical Rescue Organisation in the National Emergency and Fire System

Introduction: Analysis of the medical education of State Fire Service (SFS) officers in terms of the possibility to perform medical emergency procedures at the advanced level in connection with an update of the Rules of Medical Rescue Organisation in National Emergency and Fire System (NEFS). Material and methods: An analysis was conducted of the nationwide staff-related data on the medical education of SFS officers, both in the day-based and shift-based system. The analysis also covered additional qualifications for the pursuit of the profession of a physician, nurse and medical emergency worker among the firefighters/medical emergency workers in Poland, satisfaction of the obligation of professional training and additional employment in healthcare facilities. Results: 2220 officers with medical education work in the organisational units of SFS. Nearly 40% of the officers with medical education in SFS additionally work in healthcare units and more than a half pursues the statutory professional training. To ensure one medical emergency worker per professional shift on the ready, every SFS EFU should have ca. 5.6 full-time positions for medical emergency workers — this is a coefficient including absences of leave, sickness absences and other variables. Therefore, the above calculations indicate that to ensure one medical emergency worker per professional shift in one SFS EFU (E ≈ 5.6), ca. 2817 officers with medical education should be employed. With the current staff level (SRM = 2220), ca. 597 officers should be employed to fill the demand (Mrt) if the advanced level is to be achieved Conclusions: The medical rescue in SFS as one of the disciplines of medicine requires continual development in terms of solutions for the system, equipment and first of all the staff. A higher number of medical emergency workers in SFS may have a positive effect on the quality of the medical procedures carried out during isolated emergency medical incidents. The organisational units of SFS in most voivodeships in Poland face a shortage of medical emergency workers on duty, which has a negative effect on the possibility to perform Medical Rescue Procedures at the advanced level