Suspension-cultured plant cells as a platform for obtaining recombinant proteins

Production of recombinant proteins in suspension cultures of genetically modified plant cells is a promising and rapidly developing area of plant biotechnology. In the present review article, advantages related to using plant systems for expression of recombinant proteins are considered. Here, the main focus is covering the literature on optimization of cultivation conditions of suspension-cultured plant cells to obtain a maximal yield of target proteins. In particular, certain examples of successful use of such cells to produce pharmaceuticals were described

Plant expression systems for production of recombinant pharmaceutically important proteins

The market of pharmaceutically valuable proteins is the fastest growing segment of the economy. Most biopharmaceuticals have been obtained in mammalian and microorganism cells, but both systems have a number of disadvantages. Plant cells combine the advantages of the eukaryotic system of protein production and the simplicity and cheapness of the bacterial, and the use of plants for the production of recombinant proteins is an economically important and promising direction. The advantage of plant systems is the lower cost of cell cultivation. They are free from unwanted components, such as bacterial endotoxins, hyperglycosylated proteins produced by yeast, animal and human pathogens in cell cultures of transgenic animals. In addition, plants are higher eukaryotes, and therefore fullvalue folding and the formation of multimeric protein complexes occur in their cells, as well as a significant portion of post­translational modifications similar to those in mammalian cells. The currently developed plant expression systems for recombinant proteins are extremely diverse and number more than 100 different technologies based on different plant species, gene transfer methods, expression strategies, methods for the subsequent extraction of the target protein, etc. This is nuclear and plastid transformation, transient and stable expression during transformation using agrobacterial transport, bombardment or electroporation, cultivation of whole terrestrial or aquatic plants, plant tissues or suspension cell cultures as expression systems. The review examines the current state of research in the use of plant expression systems for the production of recombinant proteins for pharmaceuticals. The emphasis was placed on the advantages of plant cell cultures in comparison with other expression systems. Specific examples discuss promising plant systems for the production of recombinant proteins, such as transplastomic plants, moss and aquatic plant cultures, as well as suspension cultures of cells of higher plants. The current state of the market for recombinant proteins obtained using plant expression systems is considered. The prospects of creating plant (“edible”) vaccines based on genetically modified plants are discussed

Cartoon Image of the Mother, Its Perception by Elementary School Students and Correction in the Process of Media Education

Received 11 June 2021. Accepted 8 September 2021. Published online 11 October 2021.This article presents a psychological study of the mother image projected in cartoons and its perception by elementary school students. The research provides evidence for the importance of an integral approach to the analysis of media texts addressed to children, as well as for the necessity of considering their narrative, verbal, and descriptive components. A psychological analysis was conducted on the material of three cartoons: “Chunya” (USSR), “Barboskiny” [The Barkers] (Russia), and “Peppa Pig” (UK). Hypotheses were formulated about the potential influence of the cartoons on the younger audience. 70 elementary school students (χ = 9.5 years old) took part in the study. The research was conducted using the method of semantic differential; the data obtained were processed using factor analysis. The results show that the categorization of images follows the factors of “education”, “love”, “patience”, and “respect”. Differences in the semantic assessment of the cartoons under study are presented. Children perceive the events taking place in a cartoon directly, without reflection. Artistic mediation—polysemy, metaphors, and the category of the comic—does not evoke an aesthetic reaction in children, as assumed by the authors. It was found that the semantic assessment of the word “mother” by elementary school students did not agree with the traditional cultural status of a mother. Thus, the respondents ranked such indicators as “understanding” and “prestige” at a low level. A developmental experiment was conducted to correct the mother image as perceived by elementary school students. During the experimental program (8 lessons in total), the schoolchildren watched and discussed the cartoons together with a psychologist. At the end of the experiment, the semantic assessment of the verbal stimulus “mom” by the respondents showed a statistically significantly increase (р ≤ 0.05) in the indicators of “understanding” and “prestige”

Design and assembly of plant-based COVID-19 candidate vaccines: reсent development and future prospects

An outbreak of a new variant of the coronavirus infection, known as COVID-19, occurred at the end of 2019 in China, in the city of Wuhan. It was caused by the SARS-CoV-2 virus. This variant of the virus is characterized by a high degree of variability and, as the current situation with its spread across different regions of the globe shows, it can lead to a progressive spread of infection among the human population and become the cause of a pandemic. The world scientific community is making tremendous efforts to develop means of protection, prevention and treatment of this disease based on modern advances in molecular biology, immunology and vaccinology. This review provides information on the current state of research in the field of vaccine development against COVID-19 with an emphasis on the role of plants in solving this complex problem. Although plants have long been used by mankind as sources of various medicinal substances, in a pandemic, plant expression systems become attractive as biofactories or bioreactors for the production of artificially created protein molecules that include protective antigens against viral infection. The design and creation of such artificial molecules underlies the development of recombinant subunit vaccines aimed at a rapid response against the spread of infections with a high degree of variability. The review presents the state of research covering a period of just over two years, i. e. since the emergence of the new outbreak of coronavirus infection. The authors tried to emphasize the importance of rapid response of research groups from various scientific fields towards the use of existing developments to create means of protection against various pathogens. With two plant expression systems – stable and transient – as examples, the development of work on the creation of recombinant subunit vaccines against COVID-19 in various laboratories and commercial companies is shown. The authors emphasize that plant expression systems have promise for the development of not only protective means under conditions of rapid response (subunit vaccines), but also therapeutic agents in the form of monoclonal antibodies against COVID-19 synthesized in plant cells

GENE TRANSFER IN TOBACCO MITOCHONDRIA IN VITRO AND IN VIVO

Earlier, we had showed that isolated mitochondria from different organisms can import DNA. Exploiting this mechanism, we assessed the possibility of genes transfer in tobacco mitochondria in vitro and in vivo. Whereas homologous recombination is a rare occasion in higher plant nuclei, recombination between the large direct repeats in plant mitochondrial genome generates its multipartite structure. Following transfection of isolated organelles with constructs composed of a partial gfp gene flanked by mitochondrial DNA fragments, we showed the homologous recombination of imported DNA with the resident DNA and the integration of the reporter gene. The recombination yielded an insertion of a continuous exogenous DNA fragment including the gfp sequence and at least the 0.5 kb of the flanking sequence on each side. Using of transfection constructs carrying multiple sequences homologous to mitochondrial DNA could be suitable for insertion of a target gene into any region of the mitochondrial genome, which turns this approach to be of a general and methodical importance. Usually mitochondrial reactive oxygen species (ROS) level is under strict control of the antioxidant system including the Mn-containing superoxide dismutase (MnSOD). MnSOD is presented in multiple forms encoded by several genes in plants. Possibly, this enzyme, beside its catalytic function, fulfills as well some unknown biochemical functions. Thus, one of maize SOD enzymes (SOD3.4) could bind with mitochondrial DNA. Another SOD form (SOD3.1) is located in close proximity to mitochondrial respiratory complexes, where ROS are generated. To study possible physiological functions of this enzyme, we cloned the maize SOD3.1 gene. Compared to the SOD3.4, this enzyme didn't demonstrate DNA-binding activity. At the same time, SOD3.1 didn't show non-specific DNA-hydrolyzing activity as Cu/ZnSOD does. It means that this enzyme might have some DNA protective function. We made NtPcob-sod3.1-IGR-cob* genetic construct with integrative properties. It contains the selective gene and the gene of interest under control of the 5'-regulatory regions of Arabidopsis orf262 gene and the tobacco cob gene. We used modified variant of the tobacco apocytochrome b gene as a gene for selection with the nucleotide substitution G128T (G43V) which results in antimycin A resistance. The maize sod3.1 gene was used as a gene of interest. The construct was delivered into tobacco callus cells and leaf disks by biolistic method. The callus lines demonstrating the high growth rates in the presence of antimycin A in comparison with the non-transformed control lines were selected. PCR analysis of transformed callus lines revealed the presence of heterologous maize sod3.1 sequence and the integration of the construct elements in tobacco mitochondrial genome

Тривожність та спортивна мотивація гімнасток у тренувальному процесі та у змагальний період (порівняльний аналіз).

The paper presents the results of testing the level of gymnasts' anxiety and motivational condition in the training process and in the competition period, the comparative analysis is provided. The authors introduce a new indicator for research practice – the desired level of anxiety and motivational condition of the sportsman in the competitive period, modifying Spielberger's and Sopov's scale. Basing on the zoning methodology, the authors draw conclusions about the features of the psychological condition of the gymnasts, using the correlation between the level of anxiety and motivational condition of young sportsmenПредставлены результаты тестирования уровня тревожности и мотивационного состояния гимнасток в тренировочном процессе и в соревновательном периоде, проведен сравнительный анализ. Авторы вводят в исследовательскую практику новый показатель желаемого уровня тревожности и мотивационного состояния спортсмена в соревновательный период, модифицируя шкалы Ч. Спилбергера и В. Сопова. Опираясь на методику «зонирования», авторы делают выводы об особенностях психического состояния гимнасток, используя взаимосвязь между уровнем тревожности и мотивационным состоянием юных спортсменок.Представлено результати тестування рівня тривожності та мотиваційного стану гімнасток у тренувальному процесі та у змагальному періоді, проведено порівняльний аналіз. Автори вводять у дослідницьку практику новий показник бажаного рівня тривожності та мотиваційного стану спортсмена у змагальний період, модифікуючи шкали Ч. Спілбергера і В. Сопова. Спираючись на методику «зонування», автори роблять висновки що до особливостей психічного стану гімнасток, використовуючи взаємозв'язок між рівнем тривожності і мотиваційним станом юних спортсмено

Candidate vaccine construction against tick-born encephalitis based on hybrid recombinant flagG-protE-protein

The present work describes the construction of the gene encoding the recombinant protein flagG­protE, its synthesis, purification and study. The recombinant flagG­protE protein is a promising molecule for developing a candidate recombinant vaccine against tickborne encephalitis by the ability to bind to monoclonal antibodies (MCA) against native protein E of tick­borne encephalitis virus. The antigenic determinants of two recombinant proteins were studied: protE and flagG­protE using a panel of 8 MCA. The recombinant protein protE comprises the tick­borne encephalitis virus envelope protein and the flagGprotE recombinant protein has an additional flagG domain encoding flagellin G of Salmonella typhi. It was found that the MCA tested revealed epitopes on the recombinant protein protE. This indicates that the investigated recombinant protein has an antigenic structure similar to the antigenic structure of the native tick­borne encephalitis virus protein E. In the study of the recombinant protein flagG­protE by the ability to bind a panel of 8 MCA, only five of them react with epitopes of the tested protein. MCA 4F6, 7F10, and 6B9 did not recognize the corresponding epitope in the recombinant flagG­protE protein, while in the recombinant protein protE, these epitopes were detected successfully. Our data indicate that the antigenic structure of recombinant protE­protein can be changed under the influence of the flagellin domain, which in turn can lead to the unavailability of some antigenic determinants. This fact must be taken into account when constructing recombinant molecules with antigenic properties. Nevertheless, the fundamentally important regions in the region of the fusion peptide and III domain are antigenically present on the surface of the recombinant protein. This should ensure the formation of neutralizing antibodies, and the presence of a complete amino acid sequence of protein E in the recombinant protein induces the formation of a T­cell immune response. The emergence of a new generation of vaccines against tick­borne encephalitis with a higher level of safety and immunogenicity will improve the vaccine prevention of the population from tick­borne encephalitis

The role of copper in bifacial CdTe based solar cells

We present an innovative back contact for CdTe solar cell by the application of a transparent conducting oxide, typically ITO, as a back electrical contact on CdTe/CdS photovoltaic devices that acts as a free-Cu stable back contact and at the same time allows to realize bifacial CdTe solar cells, which can be illuminated from either or both sides. The controlled insertion of a very limited amount of copper into the ITO back contact permits to have reproducible devices with high efficiencies still keeping the bifacial configuration. Thin CdTe layer solar cells with ITO back contact have been realized with efficiencies exceeding 10%, the reduced thickness of CdTe allows to have a better performance on the back-side illumination and reduces the amount of CdTe material

The multi-stripe travelling salesman problem

In the classical Travelling Salesman Problem (TSP), the objective function sums the costs for travelling from one city to the next city along the tour. In the q-stripe TSP with q ≥ 1, the objective function sums the costs for travelling from one city to each of the next q cities along the tour. The resulting q-stripe TSP generalizes the TSP and forms a special case of the quadratic assignment problem. We analyze the computational complexity of the q-stripe TSP for various classes of specially structured distance matrices. We derive NP-hardness results as well as polyomially solvable cases. One of our main results generalizes a well-known theorem of Kalmanson from the classical TSP to the q-stripe TSP

Novel Interactome of \u3cem\u3eSaccharomyces cerevisiae\u3c/em\u3e Myosin Type II Identified by a Modified Integrated Membrane Yeast Two-Hybrid (iMYTH) Screen

Nonmuscle myosin type II (Myo1p) is required for cytokinesis in the budding yeast Saccharomyces cerevisiae. Loss of Myo1p activity has been associated with growth abnormalities and enhanced sensitivity to osmotic stress, making it an appealing antifungal therapeutic target. The Myo1p tail-only domain was previously reported to have functional activity equivalent to the full-length Myo1p whereas the head-only domain did not. Since Myo1p tail-only constructs are biologically active, the tail domain must have additional functions beyond its previously described role in myosin dimerization or trimerization. The identification of new Myo1p-interacting proteins may shed light on the other functions of the Myo1p tail domain. To identify novel Myo1p-interacting proteins, and determine if Myo1p can serve as a scaffold to recruit proteins to the bud neck during cytokinesis, we used the integrated split-ubiquitin membrane yeast two-hybrid (iMYTH) system. Myo1p was iMYTH-tagged at its C-terminus, and screened against both cDNA and genomic prey libraries to identify interacting proteins. Control experiments showed that the Myo1p-bait construct was appropriately expressed, and that the protein colocalized to the yeast bud neck. Thirty novel Myo1p-interacting proteins were identified by iMYTH. Eight proteins were confirmed by coprecipitation (Ape2, Bzz1, Fba1, Pdi1, Rpl5, Tah11, and Trx2) or mass spectrometry (AP-MS) (Abp1). The novel Myo1p-interacting proteins identified come from a range of different processes, including cellular organization and protein synthesis. Actin assembly/disassembly factors such as the SH3 domain protein Bzz1 and the actin-binding protein Abp1 represent likely Myo1p interactions during cytokinesis