Joint transmutation of stable Cs and Sr isotopes in microbiological systems and prospects for accelerated deactivation of liquid radioactive waste

It was found during the research that in the experimental and control bioreactors, which at the beginning of the experiments contained only cesium and strontium, by the end of the experiments, yttrium and barium were found. These isotopes are formed as a result of low-energy nuclear reactions involving protons. In addition, in experimental bioreactors with an optimal composition, a two to threefold increase in the concentration of yttrium was recorded in comparison with the control non-optimal experiments. Accumulation of strontium and cesium in biomass was registered, which is explained by the process of biosorption. It is known that biosorption is the first step towards nuclear transformation (biotransmutation). At the same time, one of the main conditions for the nuclear transformation of biomass elements is its maximum efficient growth. An unexpected fact discovered during the experiment is that yttrium and barium were also found in the control bioreactor, where no biomass was added before the experiment, but only deionized water, glucose, and the initial stable cesium and strontium salts. It is important to note that these elements were not detected in the analysis of the initial salts, substrates, and deionized water. Most likely, the presence of yttrium and barium is due to inoculation of the control fluid of the bioreactor (where no biomass pellets were added) with microorganisms from the experimental bioreactors during their periodic opening for taking current pH samples and adding glucose. Also, the work recorded a decrease in the content of cesium and strontium in the liquid by 20% and 55%, respectively, which goes beyond the statistical error.

Gene introduction approaches in chloroplast transformation and its applications

Background Chloroplast is a type of plastid that is believed to be originated from ancestral cyanobacteria. Chloroplast besides being a major component for photosynthesis, also takes part in another major plant metabolism, making it one of the major components of plants. Main body Chloroplast transformation is an alternative and better genetic engineering approach compared to the nuclear transformation that has been widely applied in plant genetic engineering. Chloroplast transformation has exhibited various positive effects as compared to nuclear transformation. This is a more preferred technique by researchers. To carry out chloroplast transformation, the vector design must be performed, and a selectable marker needs to be incorporated before the chloroplast could uptake the construct. The common way of introducing a gene into the host, which is the chloroplast, involves the biolistic, PEG-mediated, carbon nanotubes carriers, UV-laser microbeam, and Agrobacterium-mediated transformation approaches. Apart from discussing the processes involved in introducing the gene into the chloroplast, this review also focuses on the various applications brought about by chloroplast transformation, particularly in the field of agriculture and environmental science. Conclusion Chloroplast transformation has shown a lot of advantages and proven to be a better alternative compared to nuclear genome transformation. Further studies must be conducted to uncover new knowledge regarding chloroplast transformation as well as to discover its additional applications in the fields of biotechnology.

Chlamydomonas reinhardtii nuclear transformation by electroporation. v2

This protocols describe the steps required for nuclear transformation of Chlamydomonas reinhardtii by electroporation. Here you can find a video following the protocol.

Agrobacterium tumefaciens-Mediated Nuclear Transformation of a Biotechnologically Important Microalga—Euglena gracilis

Euglena gracilis (E. gracilis) is an attractive organism due to its evolutionary history and substantial potential to produce biochemicals of commercial importance. This study describes the establishment of an optimized protocol for the genetic transformation of E. gracilis mediated by Agrobacterium (A. tumefaciens). E. gracilis was found to be highly sensitive to hygromycin and zeocin, thus offering a set of resistance marker genes for the selection of transformants. A. tumefaciens-mediated transformation (ATMT) yielded hygromycin-resistant cells. However, hygromycin-resistant cells hosting the gus gene (encoding β-glucuronidase (GUS)) were found to be GUS-negative, indicating that the gus gene had explicitly been silenced. To circumvent transgene silencing, GUS was expressed from the nuclear genome as transcriptional fusions with the hygromycin resistance gene (hptII) (encoding hygromycin phosphotransferase II) with the foot and mouth disease virus (FMDV)-derived 2A self-cleaving sequence placed between the coding sequences. ATMT of Euglena with the hptII-2A–gus gene yielded hygromycin-resistant, GUS-positive cells. The transformation was verified by PCR amplification of the T-DNA region genes, determination of GUS activity, and indirect immunofluorescence assays. Cocultivation factors optimization revealed that a higher number of transformants was obtained when A. tumefaciens LBA4404 (A600 = 1.0) and E. gracilis (A750 = 2.0) cultures were cocultured for 48 h at 19 °C in an organic medium (pH 6.5) containing 50 µM acetosyringone. Transformation efficiency of 8.26 ± 4.9% was achieved under the optimized cocultivation parameters. The molecular toolkits and method presented here can be used to bioengineer E. gracilis for producing high-value products and fundamental studies.

Recombinant Expression of Thrombolytic Agent Reteplase in Marine Microalga Tetraselmis subcordiformis (Chlorodendrales, Chlorophyta)

Tetraselmis subcordiformis, a unicellular marine green alga, is used widely in aquaculture as an initial feeding for fish, bivalve mollusks, penaeid shrimp larvae, and rotifers because of its rich content of amino acids and fatty acids. A stable nuclear transformation system using the herbicide phosphinothricin (PPT) as a selective reagent was established previously. In this research, the recombinant expression in T. subcordiformis was investigated by particle bombardment with the rt-PA gene that encodes the recombinant human tissue-type plasminogen activator (Reteplase), which is a thrombolytic agent for acute myocardial infarction treatment. Transgenic algal strains were selected by their resistance to PPT, and expression of rt-PA was validated by PCR, Southern blotting, and Western blotting, and bioactivity of rt-PA was confirmed by the fibrin agarose plate assay for bioactivity. The results showed that rt-PA was integrated into the genome of T. subcordiformis, and the expression product was bioactive, indicating proper post-transcriptional modification of rt-PA in T. subcordiformis. This report contributes to efforts that take advantage of marine microalgae as cell factories to prepare recombinant drugs and in establishing a characteristic pathway of oral administration in aquaculture.

Efficient Transient Expression of Recombinant Proteins Using DNA Viral Vectors in Freshwater Microalgal Species

The increase in the world population, the advent of new infections and health issues, and the scarcity of natural biological products have spotlighted the importance of recombinant protein technology and its large-scale production in a cost-effective manner. Microalgae have become a significant promising platform with the potential to meet the increasing demand for recombinant proteins and other biologicals. Microalgae are safe organisms that can grow rapidly and are easily cultivated with basic nutrient requirements. Although continuous efforts have led to considerable progress in the algae genetic engineering field, there are still many hurdles to overcome before these microorganisms emerge as a mature expression system. Hence, there is a need to develop efficient expression approaches to exploit microalgae for the production of recombinant proteins at convenient yields. This study aimed to test the ability of the DNA geminiviral vector with Rep-mediated replication to transiently express recombinant proteins in the freshwater microalgal species Chlamydomonas reinhardtii and Chlorella vulgaris using Agrobacterium-mediated transformation. The SARS-CoV-2 receptor binding domain (RBD) and basic fibroblast growth factor (bFGF) are representative antigen proteins and growth factor proteins, respectively, that were subcloned in a geminiviral vector and were used for nuclear transformation to transiently express these proteins in C. reinhardtii and C. vulgaris. The results showed that the geminiviral vector allowed the expression of both recombinant proteins in both algal species, with yields at 48 h posttransformation of up to 1.14 μg/g RBD and 1.61 ng/g FGF in C. vulgaris and 1.61 μg/g RBD and 1.025 ng/g FGF in C. reinhardtii. Thus, this study provides a proof of concept for the use of DNA viral vectors for the simple, rapid, and efficient production of recombinant proteins that repress the difficulties faced in the genetic transformation of these unicellular green microalgae. This concept opens an avenue to explore and optimize green microalgae as an ideal economically valuable platform for the production of therapeutic and industrially relevant recombinant proteins in shorter time periods with significant yields.

Linear Chain Method for Numerical Modelling of Burnup Systems

The theoretical aspects of the linear chain method for the numerical modelling of nuclear transmutation systems, and particularly regarding the transmutation trajectory analysis (TTA), are presented. The theoretical background of the TTA method, as an advanced version of the linear chain method, with the detailed description of the applied mathematical set-up and graphical visualisation of transformation chains, is shown. As the TTA method was initially developed at the AGH University of Science and Technology almost 25 years ago, several numerical implementations were introduced worldwide, yet the mathematical improvements or alternative forms of solutions and numerical algorithms were reported since then. The method was also implemented and tested by different research groups, also in confrontation with alternative approaches to the nuclear transformation problem known as the matrix method. The aim of the paper is to present the background of the developed method and its advantages, clarify misunderstandings in the method perception and suggest unexplored options in numerical algorithm implementation.

Chromochloris zofingiensis (Chlorophyceae) Divides by Consecutive Multiple Fission Cell-Cycle under Batch and Continuous Cultivation

Several green algae can divide by multiple fission and spontaneously synchronize their cell cycle with the available light regime. The yields that can be obtained from a microalgal culture are directly affected by cell cycle events. Chromochloris zofingiensis is considered as one of the most promising microalgae for biotechnological applications due to its fast growth and the flexible trophic capabilities. It is intensively investigated in the context of bio-commodities production (carotenoids, storage lipids); however, the pattern of cell-cycle events under common cultivation strategies was not yet characterized for C. zofingiensis. In this study, we have employed fluorescence microscopy to characterize the basic cell-cycle dynamics under batch and continuous modes of phototrophic C. zofingiensis cultivation. Staining with SYBR green—applied in DMSO solution—enabled, for the first time, the clear and simple visualization of polynuclear stages in this microalga. Accordingly, we concluded that C. zofingiensis divides by a consecutive pattern of multiple fission, whereby it spontaneously synchronizes growth and cell division according to the available illumination regime. In high-light continuous culture or low-light batch culture, C. zofingiensis cell-cycle was completed within several light-dark (L/D) cycles (14 h/10 h); however, cell divisions were synchronized with the dark periods only in the high-light continuous culture. In both modes of cultivation, daughter cell release was mainly facilitated by division of 8 and 16-polynuclear cells. The results of this study are of both fundamental and applied science significance and are also important for the development of an efficient nuclear transformation system for C. zofingiensis.

The effect of implantation materials based on equine and bovine xenogenic bone extracellular matrix on the formation of extracellular neutrophilic traps (experimental study)

Purpose To study the effect of osteoplastic materials based on the extracellular xenomatrix of bovine and equine bone tissue on the formation of neutrophil extracellular traps (NETs) in the peripheral blood of rabbits in the early post-operative period after implantation. Materials and methods The study was carried out on 18 male rabbits of the Soviet Chinchilla breed, aged from 8 months to 1.2 years, weighing from 3.0 to 4.5 kg. A perforated bone defect of a cylindrical shape measuring 2 x 6 mm in the distal metaphysis of the right and left femurs was modeled in the animals. The rabbits were divided into three groups, six animals each. In group I, the bone defect was left unfilled; in group II, the defect was filled with a bovine bone tissue xenomatrix, and an equine bone tissue xenomatrix was implanted in group III animals. The implantation material had the appearance of a yellowish crumb with a particle size of 0.5– 1 mm. Blood smears stained according to Romanovsky-Giemsa were used for counting extracellular neutrophil traps (NETs). The percentage of neutrophils that passed the stages of nuclear transformation and emitted free chromatin into the extracellular space in the form of network-like structures was calculated. Results On days 3–7 of the experiment, the number of NETs increased in the early stages of NETosis in all groups. There were no significant differences between the groups. In group I, on days 7 and 14, the number of early forms of NETs (stages 1a and 1b) returned to the values of the preoperative period. In groups II and III, normalization of NETs (stage 1a) did not occur, and the content of NETs (stage 1b) returned to the initial level only by day 30 of the experiment. On days 3, 7, 14, the number of mature NETs increased in all groups. The highest values were noted in group II, where the bovine xenogeneic matrix was implanted. Conclusion Implantation materials based on the extracellular matrix of equine and bovine xenogeneic bone stimulate excessive formation of early NETs on days 14–30 of the experimental period in response to xenotransplantation. Xenomaterials of bovine bone tissue, in comparison with xenomaterials of equine bone tissue, induce a more pronounced inflammatory reaction in the nearest time after defect filling, which is manifested by higher production of mature NETs on days 3–14 of the experiment.