Preservation of Features of Anatomical Polymorphism of Deschampsia antarctica É. Desv. (Poaceae) During In Vitro Clonal Reproduction

We studied the anatomical features of the leaf structure of Deschampsia antarctica É. Desv. (Poaceae) obtained from seeds collected from different habitats of the maritime Antarctic. These plants have been maintained in culture in vitro for more than 7 years. The plants include stable chromosome forms comprising diploids (2 n = 26), a diploid plant with B-chromosomes (2 n = 26 + 0–3B), a triploid with rearranged chromosome morphology, and myxoploids with different ratios of diploid and nondiploid cells in the root meristem. The D. antarctica plants that were studied generally had a similar anatomical structure in culture in vitro and in nature. At the same time, plants of different cultivated genotypes also displayed their own leaf structure features. In particular, qualitative features included asymmetric four-ribs and five-rib leaves instead of typical three-rib leaves for some genotypes, some individuals characterized by the presence of unicellular nonglandular pointed trichomes on an adaxial side, differences in vascular bundle sheath, and other features. No clear dependence of the anatomical structural features on the chromosomal status of the studied genotypes was evident. At the same time, differences were evident in traits that included the number of stomata, thickness of the outer cell walls of the epidermis, bundles sheath state, number of leaf ribs, and the persistent presence of trichomes under prolonged in vitro cloning. The findings indicate that D. antarctica plants collected from different locations of the maritime Antarctic for long-term cultivation in vitro under standard cultivation conditions retain the anatomical characteristics of the wild plants. The polymorphism of in vitro-cultivated plants is not related to the polymorphism of their karyotype, but is apparently due to the heterogeneity of the original plants and possibly the epigenetic fixation of a number of anatomical features produced in the natural microhabitats of D. antarctica populations.

Tissue dermal equivalent – а cellular product based on human dermal keratinocytes and fibroblasts: the properties of equivalent components and perspectives of practical application

The nature and ways of isolation and cultivation in vitro of keratinocytes and fibroblasts, the main cellular components of skin to prepare a new biomedical product, tissue dermal equivalent were considered. The main attention was payed to optimization of upbuilding dermal cell biomass including selection of medium compositions and conditions of cultivation. The information was given on main parameters of cell cultures as proliferation activity, viability and phenotype of the cells. Genotoxicity of fibroblasts and biocompatibility of the cells with organic matrixes to find the optimal carrier for cellular elements of tissue dermal equivalent were studied. The composition, the process of preparation of tissue dermal equivalent and perspectives of its practical application were discussed.

sTNFRII-Fc modification protects human UC-MSCs against apoptosis/autophagy induced by TNF-α and enhances their efficacy in alleviating inflammatory arthritis

Background Tumor necrosis factor (TNF)-α inhibitors represented by Etanercept (a fusion protein containing soluble TNF receptor II (sTNFRII) and the Fc segment of human IgG1) play a pivotal role in Rheumatoid arthritis (RA) treatment. However, long-term use increases the risk of infection and tumors for their systemic inhibition of TNF-α, which disrupts the regular physiological function of this molecular. Mesenchymal stem cells (MSCs)-based delivery system provides new options for RA treatment with their “homing” and immune-regulation capacities, whereas inflammatory environment (especially TNF-α) is not conducive to MSCs' therapeutic effects by inducing apoptosis/autophagy. Here, we constructed a strain of sTNFRII-Fc-expressing MSCs (sTNFRII-MSC), aiming to offset the deficiency of those two interventions. Methods Constructed sTNFRII-Fc lentiviral vector was used to infect human umbilical cord-derived MSCs, and sTNFRII-MSC stable cell line was generated by monoclonal cultivation. In vitro and vivo characteristics of sTNFRII-MSC were assessed by coculture assay and an acute inflammatory model in NOD/SCID mice. The sTNFRII-MSC were transplanted into CIA model, pathological and immunological indicators were detected to evaluate the therapeutic effects of sTNFRII-MSC. The distribution of sTNFRII-MSC was determined by immunofluorescence assay. Apoptosis and autophagy were analyzed by flow cytometry, western blot and immunofluorescence. Results sTNFRII-Fc secreted by sTNFRII-MSC present biological activity both in vitro and vivo. sTNFRII-MSC transplantation effectively alleviates mice collagen-induced arthritis (CIA) via migrating to affected area, protecting articular cartilage destruction, modulating immune balance and sTNFRII-MSC showed prolonged internal retention via resisting apoptosis/autophagy induced by TNF-α. Conclusion sTNFRII-Fc modification protects MSCs against apoptosis/autophagy induced by TNF-α, in addition to releasing sTNFRII-Fc neutralizing TNF-α to block relevant immune-inflammation cascade, and thus exert better therapeutic effects in alleviating inflammatory arthritis.

The Effect of Magnetite Nanoparticles on the Growth and Development of Nicotiana Tabacum Plants in Vivo and in Vitro Culture

Background. Nanomaterials are easily modified and have unique characteristics associated with a large reactive surface Due to these properties, nanomaterials are used in various branches of sciences and technology, such as pharmaceuticals, biotechnology, chemical technology, etc. Recently, the effect of magnetite nanoparticles on the morphological properties of plants has been actively studied for their further use as nanoadditives to increase yields and improve the properties of agricultural plants. Tobacco (Nicotiana tabacum) is a model object of plant biotechnology, it is used to study the effect of various factors on dicotyledonous plants, so it was chosen to study the effect of magnetite on the growth, development, and mass accumulation by plants. Objective. We are aimed to study the effect of magnetite nanoparticles on the growth and development of Nicotiana tabacum in vivo and in vitro. Methods. The ability of tobacco to produce biogenic magnetic nanoparticles by searching for mammal proteins homologues in theNicotiana tabacum proteome using the Blast NCBI program was studied using comparative genomics methods. The plants were divided into groups (control, magnetite nanoparticle concentration 0.1 mg/cm3, magnetite nanoparticle concentration 1 mg/cm3) for both in vivo and in vitro experiments. Analysis of plant parameters was performed every 14 days to study the dynamics of the effects of magnetite nanoparticles. Results. It was determined that magnetite nanoparticles at a concentration of 0.1 mg/cm3 in culture in vitro and in vivo significantly affect the growth of the root system and sprouts of Nicotiana tabacum. On the 56th day of plant cultivation in vitro on a salivary medium supplemented with magnetite nanoparticles at a concentration of 0.1 mg/cm3, an increase in the shoot length by 13.3%, root length by 31.7%, and the mass of absolutely dry substances by 18.75% was observed compared to the control. Treatment of magnetite nanoparticles with a suspension at a concentration of 0.1 mg/cm3 led to more pronounced results when growing tobacco in vivo. So, on the56th day, the root length increased by 23.3%, the length of the shoot – by 19.2%, and the mass of absolutely dry substances – by2 times, the first leaves appeared 2 days earlier compared to the control. The addition of magnetite nanoparticles to the substrate on which the plants were grown in vivo at a concentration of 1 mg/cm3 inhibits the growth of tobacco. Conclusions. Studies have shown the expediency of using magnetic nanoparticles at a concentration of 0.1 mg/cm3 as nanofertilizers in tobacco cultivation.

Dynamics of growth parameters of Gentiana lutea L. in vitro plants under different lighting conditions

Aim. Study of the dynamics Gentiana lutea L. plant growth processes in vitro depending on the light regime changes of their cultivation in order to develop a scheme to increase their adaptive potential. Methods. Methods of plant cultivation in vitro, biometric method, as well as ANOVA variance analysis and middle group analysis in pairs using the Tukey test (Tukey test) were used. Results. It is shown that the cultivation of G. lutea plants in vitro using 25 W/m2 light flux intensity in the region of photosynthetically active radiation and the ratio of blue (Eb): green (Eg): red (Er) ranges = 41.8%: 42.7 %: 15.5% triggers non-specific photomorphogenesis reactions for intact plants, which lead to poor root system development, stem elongation, formation of small leaves with a thin leaf blade, overall low productivity and low adaptation potential of G. lutea plants to ex vitro and in situ conditions. Increasing the light flux intensity to 44 W/m2 and increasing the red wave proportion up to 20.3% allows not only to improve the bioproductivity of G. lutea plants which are cultivated in vitro, but also to increase the coefficient of microclonal reproduction without the additional use of exogenous growth regulators. The greatest growth of the aboveground and underground parts, increase in effective leaf surface are observed in vitro plants during cultivation at 135 W/m2 light flux intensity and spectral composition Eb: Eg: Er = 29.5%: 32.5%: 38.0%. Conclusions. It is possible to improve plant bioproductivity by changing the light conditions of plant cultivation in vitro, and, accordingly, to increase the adaptive potential to ex vitro and in situ conditions. Keywords: Gentiana lutea L., in vitro plants, light flux intensity, spectral composition, growth parameters.

sTNFRII-Fc modification protects human UC-MSCs against apoptosis/autophagy induced by TNF-α and enhances their efficacy in alleviating inflammatory arthritis

Background Tumor necrosis factor (TNF)-α inhibitors represented by etanercept (a fusion protein containing soluble TNF receptor Ⅱ (sTNFRⅡ) and the Fc segment of human IgG1) play a pivotal role in Rheumatoid arthritis (RA) treatment. However, long-term use increases the risk of infection and tumors for their systemic inhibition of TNF-α, which disrupts the regular physiological function of this molecular. Mesenchymal stem cells (MSCs)-based delivery system provides new options for RA treatment with their “homing” and immune-regulation capacities, whereas inflammatory environment (especially TNF-α) is not conducive to MSCs' therapeutic effects by inducing apoptosis/autophagy. Here, we constructed a strain of sTNFRⅡ-Fc-expressing MSCs (sTNFRⅡ-MSC), aiming to offset the deficiency of those two interventions. Methods Constructed sTNFRII-Fc lentiviral vector was used to infect human umbilical cord-derived MSCs, and sTNFRII-MSC stable cell line was generated by monoclonal cultivation. In vitro and vivo characteristics of sTNFRII-MSC were assessed by coculture assay and an acute inflammatory model in NOD/SCID mice. The sTNFRII-MSC were transplanted into CIA model, pathological and immunological indicators were detected to evaluate the therapeutic effects of sTNFRII-MSC. The distribution of sTNFRII-MSC was determined by immunofluorescence assay. Apoptosis and autophagy was analyzed by flow cytometry, western blot and immunofluorescence. Results sTNFRⅡ-Fc secreted by sTNFRⅡ-MSC present biological activity both in vitro and vivo. sTNFRⅡ-MSC transplantation effectively alleviates mice collagen-induced arthritis (CIA) via migrating to affected area, protecting articular cartilage destruction, modulating immune balance and sTNFRⅡ-MSC showed prolonged internal retention via resisting apoptosis/autophagy induced by TNF-α. Conclusion sTNFRⅡ-Fc modification protects MSCs against apoptosis/autophagy induced by TNF-α, in addition to releasing sTNFRⅡ-Fc neutralizing TNF-α to block relevant immune-inflammation cascade, and thus exert better therapeutic effects in alleviating inflammatory arthritis.

Analysis of the Effects of Low-Dose Radiation on Human Mesenchymal Stem Cells

Purpose: The aim of the study was to study the effect of low X-ray doses on human mesenchymal stem cells (MSCs) in long-term cultivation in vitro. Material and methods: MSCs of the mucosal gum tissue of human were used. Cells were irradiated using an RUST-M1 X-ray unit (Russia) at doses of 50, 80, 100, 250 and 1000 mGy (dose rate40 mGy/min) and then cultivated according to standard methods. Immunological characteristics and viability of MSCs were evaluated on a FACSCanto II flow cytometer (Becton Dickinson CA, USA) for early and late passengers. Proliferative activity (PA) was evaluated using an xCelligence real-time cell analyzer (ACEA Biosciencs, Inc.). Results: It was shown that the proliferative activity (PA) of MSCs of the mucosal gum tissue which were irradiated at dose 50 mGy is comparable with the control group in long-term cultivation while doses of 100 and 250 mGy showed a decrease of PA. Also non-irradiated MSCs showed a significant decrease of the PA during cultivation in a conditioned medium from cells that received dose of 1000 mGy and an increase of PA during cultivation in a conditioned medium of cells that received doses of 50, 100 and 250 mGy. The cells were previously irradiated at dose 250 mGy showed adaptive response during cultivation in conditioned medium from cells that received dose of 1000 mGy. Сonclusion: The assessment of the effects of low radiation doses was focused on the bystander effect in the presented study. It was noted after adding conditioned media from irradiated cells to previously irradiated and non-irradiated MSCs. The bystander effects for low and high doses are different and their biological meaning requires further study. The phenomenon of adaptive response was shown after addition conditioned media from cells irradiated at dose 1000 mGy to pre-irradiated MSCs received a dose of 250 mGy. The obtained result leads to the conclusion that the effects of low doses can be positive. Thus, the results of study mainly correspond to the threshold nonlinear concept, according to which the effect is not proportional to the received radiation dose.

DEVELOPMENT OF PROTOTYPES OF THERAPEUTIC AND PROPHYLACTIC ANTIOXIDANT AGENTS BASED ON NATURAL BIOLOGICALLY ACTIVE COMPOUNDS OF TROPICOGENIC REPRESENTATIVES OF ORCHIDACEAE JUSS.

Introduction. Plant extracts and bioactive substances (BAS) of natural origin possess a high biological activity and a low toxicity, therefore they are widely used in cosmetic, pharmaceutical, and food industries. Problem Statement. Nowadays, about 50% of all medical drugs, dietary supplements, and herbal medicines is produced from natural sources, so the search for plants with a high content of BAS, the development of biotechnologies for BAS production, and the design of composites providing the most effective use of BAS are of great importance. Purpose. The purpose is to study tropical orchid collection for identification of plants with a high BAS content; to develop a procedure for growing promising species in vitro; to design drug prototypes on the basis of plants extracts, nanosized silica, and gelatin biopolymer. Materials and Methods. The extracts prepared from orchids leaves, silica, and gelatin have been used as auxiliary components for composites synthesis. Spectrophotometric, chromatographic, quantum-chemical, anatomo-morphological, physiological-biochemical, and biotechnological methods have been used in the research. Results. The species of orchids, which contain a significant amount of BAS with high antioxidant properties have been identified, the main components of orchids extracts have been distinguished. Sterile cultures of selected species have been obtained; protocols for their cultivation in vitro have been developed. Two types of bioactive composites have been prepared; the interaction of extracts components with silica surface and gelatin molecules has been studied. It has been shown that the materials composed of silica and orchid extracts possess a high storage stability and a prolonged desorption of several BAS, while the materials based on extracts, silica, and gelatin ensure gradual release of all the extracts components. Conclusions. The orchids are valuable sources of natural antioxidants. The designed composites are promising in terms of producing drug formulations for prolonged release of antioxidants.

Bioactivity Comparison of Electrospun PCL Mats and Liver Extracellular Matrix as Scaffolds for HepG2 Cells

Cells grown on bioactive matrices have immensely advanced many aspects of biomedical research related to drug delivery and tissue engineering. Our main objective was to perform simple evaluation of the structural and biotic qualities of cell scaffolds made of affordable biomaterials for liver cell line (HepG2) cultivation in vitro. In this work the electrospun matrix made of synthetic polyester poly(ε-caprolactone) (PCL) was compared with the natural protein-based extracellular matrix isolated from porcine liver (ECM). Mechanical and structural analysis showed that ECM was about 12 times less resistant to tensile stress while it had significantly larger pore size and twice smaller water contact angle than PCL. Bioactivity assessment included comparison of cell growth and transfection efficiency on cell-seeded scaffolds. Despite the differences in composition and structure between the two respective matrices, the rate of cell spreading and the percentage of transfected cells on both scaffolds were fairly comparable. These results suggest that in an attempt to produce simple, cell carrying structures that adequately simulate the natural scaffold, one can rely on PCL electrospun mats.