Changes In Culture Conditions Research Articles

Comparative Transcriptomic Analysis on the Effect of Sesamol on the Two-Stages Fermentation of Aurantiochytrium sp. for Enhancing DHA Accumulation.

Aurantiochytrium is a well-known long-chain polyunsaturated fatty acids (PUFAs) producer, especially docosahexaenoic acid (DHA). In order to reduce the cost or improve the productivity of DHA, many researchers are focusing on exploring the high-yield strain, reducing production costs, changing culture conditions, and other measures. In this study, DHA production was improved by a two-stage fermentation. In the first stage, efficient and cheap soybean powder was used instead of conventional peptone, and the optimization of fermentation conditions (optimal fermentation conditions: temperature 28.7 °C, salinity 10.7‱, nitrogen source concentration 1.01 g/L, and two-nitrogen ratio of yeast extract to soybean powder 2:1) based on response surface methodology resulted in a 1.68-fold increase in biomass concentration. In the second stage, the addition of 2.5 mM sesamol increased the production of fatty acid and DHA by 93.49% and 98.22%, respectively, as compared to the optimal culture condition with unadded sesamol. Transcriptome analyses revealed that the addition of sesamol resulted in the upregulation of some genes related to fatty acid synthesis and antioxidant enzymes in Aurantiochytrium. This research provides a low-cost and effective culture method for the commercial production of DHA by Aurantiochytrium sp.

Amino Acid Uptake Limitations during Human Mesenchymal Stem Cell-Based Chondrogenesis.

A mino acids are the essential building blocks for collagen and proteoglycan, which are the main constituents for cartilage extracellular matrix (ECM). Synthesis of ECM proteins requires the uptake of various essential/nonessential amino acids. Analyzing amino acid metabolism during chondrogenesis can help to relate tissue quality to amino acid metabolism under different conditions. In our study, we studied amino acid uptake/secretion using human mesenchymal stem cell (hMSC)-based aggregate chondrogenesis in a serum-free induction medium with a defined chemical formulation. The initial glucose level and medium-change frequency were varied. Our results showed that essential amino acid uptake increased with time during hMSCs chondrogenesis for all initial glucose levels and medium-change frequencies. Essential amino acid uptake rates were initial glucose-level independent. The DNA-normalized glycosaminoglycans and hydroxyproline content of chondrogenic aggregates correlated with cumulative uptake of leucine, valine, and tryptophan regardless of initial glucose levels and medium-change frequencies. Collectively, our results show that amino acid uptake rates during in vitro chondrogenesis were insufficient to produce a tissue with an ECM content similar to that of human neonatal cartilage or adult cartilage. Furthermore, this deficiency was likely related to the downregulation of some key amino acid transporters in the cells. Such deficiency could be partially improved by increasing the amino acid availability in the chondrogenic medium by changing culture conditions.

Features of exotoxin production of vaccine strains of anthrax pathogen for use in the veterinary industry

Exotoxins that produce vaccine strains of the anthrax pathogen are the main source of immunogenicity of anti-selective vaccines used in veterinary medicine. The relevance of the study is due to the search for the most suitable vaccine strains of the anthrax pathogen to obtain high production of exotoxin as a factor of the effectiveness of drugs for the implementation of preventive and safety measures in the field of veterinary medicine. In this regard, the purpose of the study was to examine the productive properties of microbes of the Bacillus genus regarding the production of exotoxin under changes in cultivation conditions during incubation. Microbiological and biotechnological methods and comparative statistical analysis are used to examine vaccine strains of the anthrax pathogen. Strains are selected according to the intensity of growth on nutrient media. A biotechnological approach to obtaining a specific anthrax protein is used to analyse the production of exotoxin by vaccine strains of the anthrax pathogen. When cultured on identical nutrient media, the vaccine strains produce different amounts of exotoxin. Virulent (B. anthracis IBM-92 Z), vaccine (B. anthracis K-79 Z, B. anthracis Sterne 34F 2, B. anthracis 55, B. anthracis SB. anthracis Tsenkovsky II) strains, and anthrax cultures (B. cereus 8035, B. anthracoides 67, B. subtilis BKM 17) are examined. In the course of experimental work, it is determined that the production of exotoxin of various anthrax pathogen strains depends on the medium’s pH. It is established that with identical pH values of the medium and cultivation conditions, the highest production of exotoxin was shown by the vaccine strain B. anthracis K-79 Z. The titer of a specific anthrax protein was 1:64. Changes in the pH of the medium during the cultivation of strains affect the amount of exotoxin formation – the main factor in the formation of specific immunity against the anthrax pathogen. The results of the study can be applied by specialists of the veterinary service to select antigen producers in the development of new drugs against anthrax in animals based on exotoxins

Up to Date of Cyanobacterial Natural Products

More than 2000 compounds have been reported from cyanobacteria. The most successful example is dolastatin 10, of which a related compound monomethylauristatin E is used as antibody-drug conjugate (ADC) for Hodgkin lymphoma and systemic anaplastic large cell lymphoma. Recently genome-based analyses by Piel led to the discovery of novel compounds from cyanobacteria. W. H. Gerwick found a potential as anti-SARS-CoV-2 agent in gallinamide A, which was reported as a cathepsin L inhibitor. In our group columbamides were isolated from the marine cyanobacterium Moorena bouillonii. The geometry of the double bond was determined by the coupling constant obtained using non-decoupled heteronuclear single quantum coherence (HSQC). The configuration of chloromethine in a long-chain acyl moiety was determined by the Ohrui method at room temperature using a chiral HPLC column. Columbamide D showed biosurfactant activity. One strain many compounds (OSMAC) is a method to discover new compounds by changing culture conditions. Prior to our experiments, attempts to apply OSMAC in cyanobacteria resulted in the induction or up-regulation of only known compounds. The heat shock culture of the freshwater cyanobacterium Microcystis aeruginosa up-regulated a ribosomal peptide argicyclamide C. At the same time, we discovered bis-prenylated and monoprenylated argicyclamides A and B. More recently iron-limited culture produced hydroxylated argicyclamide A. OSMAC and genome-based screening could lead the discovery of unique biologically active compounds from cyanobacteria.

Identification of CNOT4 As a Positive Regulator of Erythroid Cell Growth Using a Genome Wide CRISPR Knock-out Screen

Red blood cells, also known as erythrocytes, are the principle means of oxygen delivery to the body, and inadequate or disordered erythrocyte production is a major cause of human disease. While the cell-extrinsic factors that control erythrocyte production have been well studied, our understanding of the cell-intrinsic regulation of this process is incomplete. To identify genes that regulate erythroid proliferation and/or maturation, we performed a genome-scale CRISPR knock-out screen in the immortalized human erythroid cell line, HUDEP-2. HUDEP-2 cells can be maintained at the pro-erythroblast stage and then sequentially differentiated into basophilic, polychromatic, and orthochromatic erythroblasts by changing culture conditions, which permits separate evaluation of the impact of genetic perturbations on pro-erythroblast viability and terminal erythroid differentiation capacity. To perform our screen, we used the commercially available GeCKO-v2 library, which includes 6 sgRNAs targeting virtually every protein-coding gene in the human genome. This library was packaged into lentiviral particles, each expressing Cas9, an sgRNA, and a puromycin resistance gene. HUDEP-2 cells were transduced with the pooled GeCKO-v2 library at low multiplicity of infection to ensure delivery of only one sgRNA per cell. Following puromycin selection, pro-erythroblasts were collected prior to differentiation and orthochromatic erythroblasts were sorted at day 12 of differentiation (selecting for cells that downregulated CD49d). By identifying sgRNAs that were under-represented in orthochromatic erythroblasts compared to the proerythroblasts, we identified several genes that when deleted lead to impaired erythroid differentiation. One of these genes was CNOT4, which encodes a RING E3 ligase member of the CCR4-NOT complex, a conserved transcriptional regulator. This gene has been previously reported to positively regulate JAK/STAT signaling, which is known to play a key role in erythroid growth and differentiation. Additionally, only 30 out of 1095 cell lines tested in the Depmap portal exhibit diminished growth following CNOT4 deletion, suggesting a rather specific impact on erythroid cells. To define the role of CNOT4 in erythropoiesis, we electroporated human CD34+ hematopoietic stem and progenitor cells (HSPCs) with a ribonucleoprotein (RNP) complex consisting of Cas9 protein and CNOT4-targeting sgRNA. Three unique CNOT4-targeting sgRNAs were tested. Following RNP electroporation, cells were differentiated using a four-phase erythroid differentiation culture protocol and assessed for growth, indel frequencies, and erythroid differentiation capacity. For all three sgRNAs tested, CNOT4 disruption resulted in 10-fold reduced proliferation over the course of differentiation compared to cells electroporated with non-targeting sgRNAs. Erythroid differentiation, assessed by flow cytometry using CD49d and CD233 expression, was identical in CNOT4 deleted and control cells. However, evaluation of cytospins revealed reduced cell size with diminished cytoplasm to nuclear ratio in CNOT4 deficient compared to control cells. Notably, the findings above were validated in erythroid cells differentiated from HSPCs harvested from two independent donors. Taken together, these findings suggest a novel role for CNOT4 and/or the CCR4-NOT complex in erythroid differentiation. Studies are ongoing to determine the mechanism by which CNOT4 disruption negatively impacts erythropoiesis and to examine if CNOT4 overexpression results in enhanced proliferation/differentiation of erythroid cells.

Catechol siderophores framed on 2,3-dihydroxybenzoyl-L-serine from Streptomyces varsoviensis.

Enterobactin is an archetypical catecholate siderophore that plays a key role in the acquisition of ferric iron by microorganisms. Catechol moieties have been shown to be promising siderophore cores. Variants of the conserved 2,3-dihydroxybenzoate (DHB) moiety with structural modifications expand the bioactivity. Streptomyces are characterized by metabolites with diverse structures. The genomic sequence of Streptomyces varsoviensis indicated that it possessed a biosynthetic gene cluster for DHB containing siderophores and metabolic profiling revealed metabolites correlated with catechol-type natural products. Here, we report the discovery of a series of catecholate siderophores produced by S. varsoviensis and a scale-up fermentation was performed to purify these compounds for structural elucidation. A biosynthetic route for the catecholate siderophores is also proposed. These new structural features enrich the structural diversity of the enterobactin family compounds. One of the new linear enterobactin congeners shows moderate activity against a food-borne pathogen Listeria monocytogenes. This work demonstrated that changing culture conditions is still a promising approach to explore unexplored chemical diversity. The availability of the biosynthetic machinery will enrich the genetic toolbox of catechol siderophores and facilitate such engineering efforts.

A Study of the Metabolic Profiles of Penicillium dimorphosporum KMM 4689 Which Led to Its Re-Identification as Penicillium hispanicum

Changes in cultivation conditions, in particular salinity and temperature, affect the production of secondary fungal metabolites. In this work, the extracts of fungus previously described as Penicillium dimorphosporum cultivated in various salinity and temperature conditions were investigated using HPLC UV/MS techniques, and their DPPH radical scavenging and cytotoxicity activities against human prostate cancer PC-3 cells and rat cardiomyocytes H9c2 were tested. In total, 25 compounds, including 13 desoxyisoaustamide-related alkaloids and eight anthraquinones, were identified in the studied extracts and their relative amounts were estimated. The production of known neuroprotective alkaloids 5, 6 and other brevianamide alkaloids was increased in hypersaline and high-temperature conditions, and this may be an adaptation to extreme conditions. On the other hand, hyposalinity stress may induce the synthesis of unidentified antioxidants with low cytotoxicity that could be very interesting for future investigation. The study of secondary metabolites of the strain KMM 4689 showed that although brevianamide-related alkaloids and anthraquinone pigments are widely distributed in various fungi, these metabolites have not been described for P. dimorphosporum and related species. For this reason, the strain KMM 4689 was re-sequenced using the β-tubulin gene and ITS regions as molecular markers and further identified as P. hispanicum.

Mitochondrial plasticity supports proliferative outgrowth and invasion of ovarian cancer spheroids during adhesion.

Ovarian cancer cells aggregate during or after exfoliation from the primary tumor to form threedimensional spheroids. Spheroid formation provides a survival advantage during peritoneal dissemination in nutrient and oxygen-depleted conditions which is accompanied by a suppressed metabolic phenotype and fragmented mitochondria. Upon arrival to their metastatic sites, spheroids adhere to peritoneal organs and transition to a more epithelial phenotype to support outgrowth and invasion. In this study, we investigated the plasticity of mitochondrial morphology, dynamics, and function upon adhesion. Using our slow-developing (MOSE-L) and fast-developing (MOSE-LTICv) ovarian cancer models, we mimicked adhesion and reoxygenation conditions by plating the spheroids onto tissue culture dishes and changing culture conditions from hypoxia and low glucose to normoxia with high glucose levels after adhesion. We used Western Blot, microscopy and Seahorse analyses to determine the plasticity of mitochondrial morphology and functions upon adhesion, and the impact on proliferation and invasion capacities. Independent of culture conditions, all spheroids adhered to and began to grow onto the culture plates. While the bulk of the spheroid was unresponsive, the mitochondrial morphology in the outgrowing cells was indistinguishable from cells growing in monolayers, indicating that mitochondrial fragmentation in spheroids was indeed reversible. This was accompanied by an increase in regulators of mitobiogenesis, PGC1a, mitochondrial mass, and respiration. Reoxygenation increased migration and invasion in both cell types but only the MOSE-L responded with increased proliferation to reoxygenation. The highly aggressive phenotype of the MOSE-LTICv was characterized by a relative independence of oxygen and the preservation of higher levels of proliferation, migration and invasion even in limiting culture conditions but a higher reliance on mitophagy. Further, the outgrowth in these aggressive cells relies mostly on proliferation while the MOSE-L cells both utilize proliferation and migration to achieve outgrowth. Suppression of proliferation with cycloheximide impeded aggregation, reduced outgrowth and invasion via repression of MMP2 expression and the flattening of the spheroids. Our studies indicate that the fragmentation of the mitochondria is reversible upon adhesion. The identification of regulatory signaling molecules and pathways of these key phenotypic alterations that occur during primary adhesion and invasion is critical for the identification of druggable targets for therapeutic intervention to prevent aggressive metastatic disease.

Hybrid Modeling of CHO Cell Cultivation in Monoclonal Antibody Production with an Impurity Generation Module

Representative cultivation models are needed for designing efficient monoclonal antibody (mAb) production processes. Simple Monod-type kinetic models could fail to capture changes at different phases and conditions. Current models rarely account for process-related impurities, which hinders optimizing integrated processes. A three-module hybrid approach is thus introduced here. Module 1 is a kinetic metabolism model until cell death based on Monod-type equations. Module 2 is a data-driven module for updating parameters based on system dynamics and changes in cultivation conditions. Module 3 proposes a kinetic model for the generation of host cell proteins and DNA. The model is applied to newly established high-performance CHO cell lines and is validated with experiments at different operating conditions. The adopted modular approach helps identify weak model links, which can be corrected via the data-driven module. The proposed model combines accuracy and simplicity for design and control applications of cultivation processes considering quality aspects.

Adaptivity of sweet pepper varieties cultivated in the Astrakhan region

In agricultural production, it is important not only to achieve a high productivity, but also to ensure its stability. To obtain high and stable yields, it is necessary to have information on the adaptability of crops varieties and hybrids to a specific cultivation zone. The article analyzes the results of the field experiment on the variety testing of sweet pepper. To assess the variety for adaptability to natural conditions and cultivation technology in the Astrakhan region, such parameters as plasticity and stability of the variety were used. To characterize the ecological plasticity, the regression coefficient bi was determined, which characterizes response of the cultivar to changes in cultivation conditions. To characterize the parameter of stability, the standard deviation from the regression line 2d. was calculated. All calculations were carried out according to S.A. Eberhart, W.A. Russell as presented by V.Z. Pakudin.

Influence of Amino Acid Feeding on Production of Calcimycin and Analogs in Streptomyces chartreusis.

Streptomyces chartreusis NRRL 3882 produces the polyether ionophore calcimycin and a variety of analogs, which originate from the same biosynthetic gene cluster. The role of calcimycin and its analogs for the producer is unknown, but calcimycin has strong antibacterial activity. Feeding experiments were performed in chemically defined medium systematically supplemented with proteinogenic amino acids to analyze their individual effects on calcimycin synthesis. In the culture supernatants, in addition to known calcimycin analogs, eight so far unknown analogs were detected using LC-MS/MS. Under most conditions cezomycin was the compound produced in highest amounts. The highest production of calcimycin was detected upon feeding with glutamine. Supplementation of the medium with glutamic acid resulted in a decrease in calcimycin production, and supplementation of other amino acids such as tryptophan, lysine, and valine resulted in the decrease in the synthesis of calcimycin and of the known intermediates of the biosynthetic pathway. We demonstrated that the production of calcimycin and its analogs is strongly dependent on amino acid supply. Utilization of amino acids as precursors and as nitrogen sources seem to critically influence calcimycin synthesis. Even amino acids not serving as direct precursors resulted in a different product profile regarding the stoichiometry of calcimycin analogs. Only slight changes in cultivation conditions can lead to major changes in the metabolic output, which highlights the hidden potential of biosynthetic gene clusters. We emphasize the need to further study the extent of this potential to understand the ecological role of metabolite diversity originating from single biosynthetic gene clusters.

Development of a base editor for protein evolution via in situ mutation in vivo.

Protein evolution has significantly enhanced the development of life science. However, it is difficult to achieve in vitro evolution of some special proteins because of difficulties with heterologous expression, purification, and function detection. To achieve protein evolution via in situ mutation in vivo, we developed a base editor by fusing nCas with a cytidine deaminase in Bacillus subtilis through genome integration. The base editor introduced a cytidine-to-thymidine mutation of approximately 100% across a 5 nt editable window, which was much higher than those of other base editors. The editable window was expanded to 8 nt by extending the length of sgRNA, and conversion efficiency could be regulated by changing culture conditions, which was suitable for constructing a mutant protein library efficiently in vivo. As proof-of-concept, the Sec-translocase complex and bacitracin-resistance-related protein BceB were successfully evolved in vivo using the base editor. A Sec mutant with 3.6-fold translocation efficiency and the BceB mutants with different sensitivity to bacitracin were obtained. As the construction of the base editor does not rely on any additional or host-dependent factors, such base editors (BEs) may be readily constructed and applicable to a wide range of bacteria for protein evolution via in situ mutation.

Strategies for enhancing terpenoids accumulation in microalgae.

Terpenoids represent one of the largest class of chemicals in nature, which play important roles in food and pharmaceutical fields due to diverse biological and pharmacological activities. Microorganisms are recognized as a promising source of terpenoids due to its short growth cycle and sustainability. Importantly, microalgae can fix inorganic carbon through photosynthesis for the growth of themselves and the biosynthesis of various terpenoids. Moreover, microalgae possess effective biosynthesis pathways of terpenoids, both the eukaryotic mevalonic acid (MVA) pathway and the prokaryotic methyl-D-erythritol 4-phosphate (MEP) pathway. In recent years, various genetic engineering strategies have been applied to increase target terpenoid yields, including overexpression of the rate-limited enzymes and inhibition of the competing pathways. However, since gene-editing tools are only built in some model microalgae, fermentation strategies that are easier to be operated have been widely successful in promoting the production of terpenoids, such as changing culture conditions and addition of chemical additives. In addition, an economical and effective downstream process is also an important consideration for the industrial production of terpenoids, and the solvent extraction and the supercritical fluid extraction method are the most commonly used strategies, especially in the industrial production of β-carotene and astaxanthin from microalgae. In this review, recent advancements and novel strategies used for terpenoid production are concluded and discussed, and new insights to move the field forward are proposed. KEY POINTS: • The MEP pathway is more stoichiometrically efficient than the MVA pathway. • Advanced genetic engineering and fermentation strategies can increase terpene yield. • SFE has a higher recovery of carotenoids than solvent extraction.

Photobiostimulation of Chlorella sorokiniana using light emitting diodes (LEDs) for increasing lipid and biodiesel production

At present, the major body of research is focused on weaning the world from fossil fuels. The problem is that the world is running out of fossil fuel. Therefore, an alternative source must be identified. The biofuels are promising alternatives. In the case of petrodiesel, a promising alternative is biodiesel production from algae. The ability of microalgae to generate large quantities of lipids with a fast growth rate made them superior biodiesel producers. Using light-emitting diodes (LEDs) as an energy source in microalgal cultivation was recently increased owing to its large spectrum, endurance, and low-energy utilization. Changes in cultivation conditions, limited capabilities of harvesting light, and self-shading of microalgae were the most important problems. Therefore, the photobiostimulation of algae using LEDs radiation led to an increase in algal growth rate which results in increased lipid production. This research investigated the influence of monochromatic LEDs on the growth of Chlorella sorokiniana microalga. At the first phase, microalgae growth and algal biomass significantly increased under red LEDs [2.3 g/L], blue LEDs [1.8 g/L], green LEDs [0.7 g/L], and white LEDs (0.6) g/L as a control, respectively. At the second phase, microalgal growth and algal biomass significantly increased under red LEDs [2.9 g/L], blue LEDs 2.3 g/L, and white LEDs (1.5) g/L as a control, respectively. The percentage of extracted oil (%) or the yield of extracted oil of microalgae was 10.38 % (white LEDs), 16.94 % (blue LEDs), and 15.55 % (red LEDs) respectively. It was concluded that the photobiostimulation of algae using LEDs led to the enhanced weight of algal biomass, therefore increased of lipids and biodiesel production. The red LEDs were the best one in terms of increasing the weight of algal biomass. The blue LEDs were the best one in terms of increasing the percentage of extracted oil. However, the green LEDs were not effective.

Physiological responses and productivity of the seaweed Ulva ohnoi (Chlorophyta) under changing cultivation conditions in pilot large land-based ponds

Land based intensive cultivation systems have been proposed as an ideal option for the commercial production of high value products from seaweeds. However, many cultures on Ulva and other seaweeds are based on relatively small-scale facilities. The high variability of culture conditions can strongly affect the physiological performance of seaweeds, but few studies examine their phenotypic plasticity by integrating critical biological descriptors, e.g. photobiology, oxidative stress, nutrient acquisition. The purpose of this study was to determine the physiological plasticity and growth of Ulva ohnoi during its cultivation in land-based 40 m3 ponds. Through an entire cultivation cycle (four-weeks), photosynthesis, respiration, pigments, antioxidant capacity and nutrient content were measured. Light, temperature, pH, and dissolved inorganic nitrogen (DIN) were simultaneously monitored in seawater. Additionally, the N-uptake kinetics of U. ohnoi were examined in the laboratory in order to explain the efficiency of the seaweed biomass for DIN-incorporation in the ponds after fertilization. Generally, the gradual increase in seaweed density throughout the cultivation period was directly associated to a drop in light availability and dissolved inorganic carbon (i.e. higher pH) within the ponds. These changes in cultivation conditions were related to a reduction of photosynthetic capacities, nutrient content and growth of U.ohnoi. N-uptake kinetics of U. ohnoi and the behavior of DIN within the ponds after fertilization, indicated that U. ohnoi was able to incorporate ammonium more efficiently than nitrate, and the presence of the former likely inhibits nitrate acquisition. The understanding of the capacity of U. ohnoi to acclimate to the extreme changing culture conditions, could be applied to improve its productivity and chemical composition.

Implementation of the productivity potential of forage crops in the west of the Briansk region

In the non-black soil zone of Russia on sod-podzolic and floodplain soils, the adaptive properties of forage crops of the bluegrass family were assessed in terms of ecological stability and plasticity and the effect of mineral fertilizers on them according to the yield criterion. The analysis of changes in the yield of forage crops was carried out from 2009 to 2015. It was characterized by different weather conditions. As a result of the research, it was found that the environmental index varied over the years from –6.36 to 6.08. The highest adaptation coefficient was observed in Sudanese grass (1.37) and millet (1.33). The smallest yield variability was found in the cultivation of awnless brome (10.8%), millet (13.2%) and annual ryegrass (14.6%). The highest stress resistance was found in one-year ryegrass (–6.00) awnless brome (–6.20) and oats (–6.50) – the main forage crops in the study area. The minimum value of the yield range in contrasting conditions was set in millet (20.4) and awnless brome (22.0). The most stable crops in obtaining the harvest were two-source reed, annual ryegrass, Scleranthus, meadow fescue and Sudanese grass. Sudanese grass, millet and oats were the most responsive to changes in cultivation conditions. The most intense (bi = 2.40, and Sd 2 = 0.39) fodder crop in the western Briansk region is Sudan grass, while millet and oats are highly responsive in combination with low yield stability. The awnless brome reacts poorly to the improvement of external conditions, but has a fairly high yield stability. The use of mineral fertilizers increases the environmental index, adaptation, variability of the yield of forage crops, increases the gap between the maximum and minimum yield, and also enhances the stability and reduces the responsiveness of crops to environmental changes.

Chemical induction of neurogenic properties in mammalian Müller glia.

Müller glia (MG), cells that maintain homeostasis in the retina, are dormant stem cells that can regenerate neurons upon injury. However, the regenerative property of MG, which is reproducibly displayed in the lower vertebrates, is not readily observed in the mammals even upon forced expression of regulatory genes or exposure to growth factors. Here, we demonstrate a reproducible unmasking of the neurogenic properties of enriched rodent MG by serial exposure to different combinations of small molecules. The enriched MG, in response to changing culture conditions, silenced glia-specific genes and acquired transcriptional signature of neurons, accompanied by upregulation of genes known to regulate neuronal potential of MG. The MG-derived neurons expressed immunoreactivities corresponding to neuronal proteins and displayed electrophysiological features of immature neurons. Our study presents a proof of principle of pharmacological activation of neurogenic properties of mammalian MG, which may be utilized for therapeutic regeneration.

Assortment of black currant cultivars for the Southern Urals

The aim of the study was to assess black currant cultivars for yield, environmental plasticity and stability in Chelyabinsk Province. We identified adaptable black currant cvs. ‘Seyanets Druzhnoy’ (4.43 t/ha; AQ = 1.54), ‘Polar’ (3.59 t/ha; 1.38), ‘Venera’ (4.48 t/ha; 1.35), ‘Zhemchuzhina’ (4.49 t/ha; 1.34), ‘Sudarushka’ (4.43 t/ha; 1.30), ‘Bolero’ (3.42 t/ha; 1.20), ‘Gera’ (3.97 t/ha; 1.16), ‘Mortti’ (3.46 t/ha; 1.06), ‘Podarok Ilyinoy’ (3.89 t/ha; 1.03), ‘Selva’ (3.21 t/ha; 1.02) and ‘Mayak’ (3.54 t/ha; 1.01). The most interesting are intensive-type cultivars, capable of significantly increasing their productivity with the improved growing conditions. Intensive-type cultivars developed in Chelyabinsk are cvs. ‘Podarok Ilyinoy’, ‘Sudarushka’, ‘Zhemchuzhina’, ‘Gera’, ‘Mayak’ and ‘Venera’ (bi = 1.70, 1.68, 1.52, 1.46, 1.40, and 1.28, respectively).The locally developed cv. ‘Seyanets Druzhnoy’, environmentally plastic but insufficiently stable (bi = 1.17; Si2 = 6.1), exceeded in its average yield the environmentally plastic and stable cvs. ‘Selva’ (bi = 0.77; Si2 = 0.7) and ‘Mortti’ (bi = 0.93; Si2 = 1.1). Cv. ‘Bolero’ developed by the Vavilov Institute (VIR) and the Swedish cv. ‘Polar’ are neutral, i.e., they weakly respond to changes in cultivation conditions. High yields were recorded for cvs. ‘Zhemchuzhina, ‘Venera’, ‘Sudarushka’, ‘Seyanets Druzhnoy’, ‘Gera’, ‘Podarok Ilyinoy’, ‘Polar’, ‘Mayak’, ‘Mortti’ and ‘Bolero’.The yield of the following cultivars was at the average level for the experiment: ‘Rusalka’ (bi = 1.50), ‘Orloviya’ (bi = 1.56), ‘Pigmey’ (bi = 1.30), plus the yield (3.36 to 3.25 t/ha) of environmentally stable cvs. ‘Legenda’ (bi = 1.24; Si2 = 0.3), ‘Kama’ (bi = 1.00; Si2 = 0.8), and ‘Dochka’ (bi = 1.15; Si2 = 0.5). Cvs. ‘Zem Zarin’ (1.79 t/ha), ‘Chernecha’ (1.82 t/ha), ‘Zorya Galitskaya’‚ (2.18 t/ha), ‘Sibilla’ (2.37 t/ha), ‘Krasa Lvova’ (2.52 t/ha) and ‘Shakhalevskaya’ (2.84 t/ha) demonstrated yield levels significantly lower than the average.

ЖИРНОКИСЛОТНЫЙ СОСТАВ ОЗИМОГО РЫЖИКА В ЗАВИСИМОСТИ ОТ РЕГИОНА ВОЗДЕЛЫВАНИЯ

Camelina sylvestris (L.) Crantz. is a valuable oilseed known due to the vegetable oil used in human nutrition and industrial production. The current research is aimed to assess the content of fatty acids in Camelina sylvestris oilseeds depending on the hydrothermal cultivation conditions. Seeds of winter camelina variety ‘Penzyak’ grown in the Penza region and steppe Crimea in 2017–2019 served as the object of the research. In 2017, arid conditions were recorded in the Crimea, Selyaninov Hydrothermal Coefficient (HTC) = 0.61. In the Penza region, on the contrary, the level of moisture in 2017 was moderate, HTC = 1.10. Weather conditions in 2019 were characterized as arid in both regions; HTC was 0.63 and 0.60. In 2018, in the Crimea and Penza, HTC was 0.23 and 0.45, respectively. The soils of the experimental plots – chernozems leached (Penza) and southern low-humic (Crimea). Fatty acid methyl esters were isolated and analyzed according to GOST R 51 486–99. The concentration of oleic and linoleic acids increased in seeds grown in the Crimea and amounted to 15.50–18.56 % and 18.08–19.58 %. In the colder region (Penza), the content of these acids decreased to 11.90–14.82 and 16.12–17.10 %, respectively. The highest content of linolenic acid was in oilseeds from the Penza region and amounted to 36.80–38.50 %, which was 4.94–6.40 % higher than the content of linolenic acid in oilseeds from the Crimea. The content of erucic acid in the Camelina sylvestris (L.) Crantz. from both regions was 2.81–2.94 %. The coefficient of variation of this acid was 1.59 %. Linolenic and oleic acids are the most susceptible to changes in cultivation conditions, coefficient of variation – 10.08 and 16.32 %, respectively. The total content of polyunsaturated fatty acids was high and averaged 52.4–54.6 %. The highest concentration was noted in oilseeds from Penza, which exceeded that of from the Crimea by 2.2 %. The content of ω-9 fatty acids ranged from 33.7 % (Penza) to 35.9 % (Crimea) and was characterized by a low coefficient of variation – 4.14 %. The range of variation of ω-3 and ω-6 acid, on average, was 6.32–7.81 %. Their content was 31.5 and 20.9 % in oilseeds from the Crimea and 35.5 and 19.1 % in oilseeds from Penza. The ratio between omega-6 and omega-3 acids is 1.8:1.

A framework for the computational prediction and analysis of non-coding RNAs in microbial environmental populations and their experimental validation

Small regulatory RNAs and antisense RNAs play important roles in the regulation of gene expression in bacteria but are underexplored, especially in natural populations. While environmentally relevant microbes often are not amenable to genetic manipulation or cannot be cultivated in the laboratory, extensive metagenomic and metatranscriptomic datasets for these organisms might be available. Hence, dedicated workflows for specific analyses are needed to fully benefit from this information. Here, we identified abundant sRNAs from oceanic environmental populations of the ecologically important primary producer Prochlorococcus starting from a metatranscriptomic differential RNA-Seq (mdRNA-Seq) dataset. We tracked their homologs in laboratory isolates, and we provide a framework for their further detailed characterization. Several of the experimentally validated sRNAs responded to ecologically relevant changes in cultivation conditions. The expression of the here newly discovered sRNA Yfr28 was highly stimulated in low-nitrogen conditions. Its predicted top targets include mRNAs encoding cell division proteins, a sigma factor, and several enzymes and transporters, suggesting a pivotal role of Yfr28 in the coordination of primary metabolism and cell division. A cis-encoded antisense RNA was identified as a possible positive regulator of atpF encoding subunit b’ of the ATP synthase complex. The presented workflow will also be useful for other environmentally relevant microorganisms for which experimental validation abilities are frequently limiting although there is wealth of sequence information available.