Development of nutrient medium for riboflavin biosynthesis by Eremothecium ashbyi ascomycetes

The object of the study is the riboflavin producer Eremothecium ashbyi Guilliermond 1935 VKPM F-340, the subject of the study is the regularities of riboflavin biosynthesis by the E. ashbyi F-340 strain under different cultivation conditions. Riboflavin is an important micronutrient that is a precursor of the coenzymes flavin mononucleotide and flavinadine dinucleotide, it is necessary for biochemical reactions in all living cells. Population growth and an increase in human needs for vitamin-fortified food and agricultural products is the reason for an increase in demand for riboflavin preparations. Considering this, it is important and economically beneficial to improve the technology for the production of vitamin B2. An important factor that affects the yield of the product is the nutrient medium. At present, the influence of agricultural waste on the biosynthesis of riboflavin is being actively studied in the world. However, not all of the studied types of raw materials are typical for the agriculture of this or that country. Therefore, in order to determine whether this direction of research is promising, it is important to check the effect on the biosynthetic activity of the riboflavin producer of the most common wastes of the domestic industry. In this work, this is done on the example of Ukraine. In the course of the study, microbiological (surface and deep cultivation of E. ashbyi F-340), physicochemical (determination of the amount of biomass by the gravimetric method, determination of the concentration of riboflavin by the spectrophotometric method) and mathematical methods were used. The proposed media with the addition of agricultural waste, providing a higher yield of riboflavin compared to conventional media. The influence of different types of agricultural waste on the biosynthesis of riboflavin by the producer E. ashbyi F-340 was evaluated. The efficiency of using sunflower cake as a component of the nutrient medium is shown. The optimal sources of carbon for the nutrient medium with oil cake have been determined, which increases the yield of riboflavin. Due to the large amount of sunflower cake obtained in Ukraine, its use for modifying the nutrient medium in order to increase the yield of riboflavin in the future will lead to a decrease in the cost of the target product due to the use of cheap and ecological raw materials.

Multiple Irradiation Affects Cellular and Extracellular Components of the Mouse Brain Tissue and Adhesion and Proliferation of Glioblastoma Cells in Experimental System In Vivo

Intensive adjuvant radiotherapy (RT) is a standard treatment for glioblastoma multiforme (GBM) patients; however, its effect on the normal brain tissue remains unclear. Here, we investigated the short-term effects of multiple irradiation on the cellular and extracellular glycosylated components of normal brain tissue and their functional significance. Triple irradiation (7 Gy*3 days) of C57Bl/6 mouse brain inhibited the viability, proliferation and biosynthetic activity of normal glial cells, resulting in a fast brain-zone-dependent deregulation of the expression of proteoglycans (PGs) (decorin, biglycan, versican, brevican and CD44). Complex time-point-specific (24–72 h) changes in decorin and brevican protein and chondroitin sulfate (CS) and heparan sulfate (HS) content suggested deterioration of the PGs glycosylation in irradiated brain tissue, while the transcriptional activity of HS-biosynthetic system remained unchanged. The primary glial cultures and organotypic slices from triple-irradiated brain tissue were more susceptible to GBM U87 cells’ adhesion and proliferation in co-culture systems in vitro and ex vivo. In summary, multiple irradiation affects glycosylated components of normal brain extracellular matrix (ECM) through inhibition of the functional activity of normal glial cells. The changed content and pattern of PGs and GAGs in irradiated brain tissues are accompanied by the increased adhesion and proliferation of GBM cells, suggesting a novel molecular mechanism of negative side-effects of anti-GBM radiotherapy.

Progesterone-dependent and -independent modulation of luminal epithelial transcription to support pregnancy in cattle

In cattle, starting 4-5 days after estrus, pre-implantation embryonic development occurs in the confinement of the uterine lumen. Cells in the endometrial epithelial layer control the molecular traffic to and from the lumen and, thereby determine luminal composition. Starting early post-estrus, endometrial function is regulated by sex-steroids, but the effects of progesterone on luminal cells transcription have not been measured in vivo. First objective was to determine the extent to which progesterone controls transcription in luminal epithelial cells 4 d (D4) after estrus. Second objective was to discover luminal transcripts that predict pregnancy outcomes, when the effect of progesterone is controlled. Endometrial luminal epithelial cells were collected from embryo transfer recipients on D4 using a cytological brush and their transcriptome determined by RNASeq. Pregnancy by embryo transfer was measured on D30 (25 pregnant and 18 non-pregnant). Progesterone concentration on D4 was associated positively (n= 182) and negatively (n= 58) with gene expression. Progesterone-modulated transcription indicated an increase in oxidative phosphorylation, biosynthetic activity and proliferation of epithelial cells. When these effects of progesterone were controlled, different genes affected positively (n= 22) and negatively (n= 292) odds of pregnancy. These set of genes indicated that a receptive uterine environment was characterized by the inhibition of phosphoinositide signaling and innate immune system responses. A panel of 25 genes predicted the pregnancy outcome with sensitivity and specificity ranging from 64-96% and 44-83%, respectively. In conclusion, in the early diestrus, both progesterone-dependent and -independent mechanisms regulate luminal epithelial transcription associated with pregnancy outcomes in cattle.

NAMPT-mediated NAD+ biosynthesis suppresses activation of hepatic stellate cells and protects against CCl4-induced liver fibrosis in mice

Background: Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the rate-limiting step in the salvage pathway of mammalian nicotinamide adenine dinucleotide (NAD+) biosynthesis. Through its NAD+-biosynthetic activity, NAMPT is able to regulate the development of hepatic steatosis and inflammation induced by diet or alcohol. However, the roles NAMPT plays in the development of liver fibrosis remain obscure. Purpose: To investigate the roles of NAMPT-mediated NAD+ biosynthesis in hepatic stellate cell (HSC) activation and liver fibrosis. Research Design: Realtime RT-PCR and western blot analyses were performed to analyze the expression of profibrogenic genes. Sirius red staining was conducted to examine the fibrosis in liver. Mouse liver fibrosis was induced by intraperitoneal injection of carbon tetrachloride (CCl4) 2 times a week for 6 weeks. Adenovirus-mediated NAMPT overexpression or nicotinamide mononucleotide (NMN) administration was carried out to study the effects of elevation of NAD+ levels on protecting CCl4-induced liver fibrosis in mice. LX2 cells or primary HSCs were used to study the role of NAMPT overexpression or NMN treatment in reducing profibrogenic gene expression in vitro. ResultsCCl4 administration suppresses NAMPT expression in liver and reduces hepatic NAD+ content. Tgfβ1 treatment decreases intracellular NAD+ levels and NAMPT expression in LX2 cells. Adenovirus-mediated NAMPT overexpression augments liver NAD+ levels, inhibits HSC activation and alleviates CCl4-induced liver fibrosis in mice. Administration of NMN also suppresses HSC activation and protects against CCl4-induced liver fibrosis in mice. Conclusions: NAMPT-mediated NAD+ biosynthesis inhibits HSC activation and protects against CCl4-induced liver fibrosis.

Rapid sanitary and technological control of culturing the producers of amylolytic enzymes.

It has been established that the biotechnological process of culturing bacillary microbial producers of amylolytic enzymes can be express-controlled by determining their ATP bioluminescence. The advantages of the method have been shown. The analytical review of producers of hydrolytic enzymes has made it clear how practical it is to use bacillary microorganisms for targeted secretion of amylolytic enzymes in biotechnological production. After monitoring bacillary microorganisms, it has been found advisable to choose Bacillus subtilis ATCC 6633 as the working culture due to its time of production of amylolytic enzymes and its biosynthetic activity. Reasons have been given for using the rapid ATP control method, which is based on the principle of bioluminescence. Different growth media have been compared and evaluated in order to intensify the quantitative biosynthetic activity of the microbial culture in the technological process of culturing bacillary microorganisms. The experiments have proved that growth media can be modified by introducing a number of carbohydrate–protein substrates as inducers of amylolytic complex gene expression. The latter manifests itself in the amylolytic activity accelerated by 12–24 hours, and causes an increase in the number of microorganisms (1.87–3.99 times as many as in the reference culture). Two methods of control (rapid bioluminescent and classic microbiological) have been used for correlative determination of the quantitative growth of Bacillus subtilis cells. Mathematical straight-line correlations have been obtained in a semilogarithmic system for the number of cells of the bacillary producer of the amylolytic enzyme complex. These correlations allow carrying out rapid control in a production environment. Along with the traditional rapid sanitary control in biotechnological production, which includes controlling the contamination of the equipment, personnel’s hands, and water, it has been suggested to perform proprietary technological express control of amylolytic enzyme biosynthesis using the culture Bacillus subtilis ATCC 6633

Proteomic signatures of human visceral and subcutaneous adipocytes

Objective Adipose tissue distribution is a key factor influencing metabolic health and risk in obesity-associated comorbidities. Here we aim to compare the proteomic profiles of mature adipocytes from different depots. Methods Abdominal subcutaneous (SA) and omental visceral adipocytes (VA) were isolated from paired AT biopsies obtained during bariatric surgery of 19 severely obese women (BMI > 30 kg/m 2) and analysed using state-of-the-art mass spectrometry. Differential expression analysis and weighted gene co-expression network analysis (WGCNA) were performed to investigate proteome signature properties and to examine a possible association of the protein expression with the clinical data. Results We identified 3,686 protein groups and found 1,140 differentially expressed proteins (adj. p-value < 0.05), of which 576 proteins were upregulated in SA and 564 in VA samples. We provide a global protein profile of abdominal SA and omental VA, present the most differentially expressed pathways and processes distinguishing SA from VA, and correlate them with clinical and body composition data. We show that SA are significantly more active in processes linked to vesicular transport and secretion, and to increased lipid metabolism activity. Conversely, the expression of proteins involved in the mitochondrial energy metabolism and translational or biosynthetic activity is higher in VA. Conclusion Our analysis represents a valuable resource of protein expression profiles in abdominal SA and omental VA, highlighting key differences in their role in obesity.

Essential roles of Lon protease in the morpho-physiological traits of the rice pathogen Burkholderia glumae

The highly conserved ATP-dependent Lon protease plays important roles in diverse biological processes. The lon gene is usually nonessential for viability; however, lon mutants of several bacterial species, although viable, exhibit cellular defects. Here, we show that a lack of Lon protease causes pleiotropic effects in the rice pathogen Burkholderia glumae. The null mutation of lon produced three colony types, big (BLONB), normal (BLONN), and small (BLONS), in Luria–Bertani (LB) medium. Colonies of the BLONB and BLONN types were re-segregated upon subculture, while those of the BLONS type were too small to manipulate. The BLONN type was chosen for further studies, as only this type was fully genetically complemented. BLONN-type cells did not reach the maximum growth capacity, and their population decreased drastically after the stationary phase in LB medium. BLONN-type cells were defective in the biosynthesis of quorum sensing (QS) signals and exhibited reduced oxalate biosynthetic activity, causing environmental alkaline toxicity and population collapse. Addition of excessive N-octanoyl-homoserine lactone (C8-HSL) to BLONN-type cell cultures did not fully restore oxalate biosynthesis, suggesting that the decrease in oxalate biosynthesis in BLONN-type cells was not due to insufficient C8-HSL. Co-expression of lon and tofR in Escherichia coli suggested that Lon negatively affects the TofR level in a C8-HSL-dependent manner. Lon protease interacted with the oxalate biosynthetic enzymes, ObcA and ObcB, indicating potential roles for the oxalate biosynthetic activity. These results suggest that Lon protease influences colony morphology, growth, QS system, and oxalate biosynthesis in B. glumae.

Hydrolytic activity of the microbial consortium of secondary raw materials of food production

В результате проведенных исследований выявлен базальный уровень микробных гидролаз для перевода структурных биополимеров пшеничных отрубей, рапсового и соевого жмыха в формы, доступные для усвоения и проявления биосинтетической активности выявленного консорциума микроорганизмов. As a result of the conducted studies, the basal level of microbial hydrolases for the conversion of structural biopolymers of wheat bran, rapeseed and soy cake into forms available for assimilation and manifestation of the biosynthetic activity of the identified consortium of microorganisms was revealed.

Ultrastructural Alterations in Cells of Sunflower Linear Glandular Trichomes during Maturation

Sunflower and related taxa are known to possess a characteristic type of multicellular uniseriate trichome which produces sesquiterpenes and flavonoids of yet unknown function for this plant. Contrary to the metabolic profile, the cytological development and ultrastructural rearrangements during the biosynthetic activity of the trichome have not been studied in detail so far. Light, fluorescence and transmission electron microscopy were employed to investigate the functional structure of different trichome cells and their subcellular compartmentation in the pre-secretory, secretory and post-secretory phase. It was shown that the trichome was composed of four cell types, forming the trichome basis with a basal and a stalk cell, a variable number (mostly from five to eight) of barrel-shaped glandular cells and the tip consisting of a dome-shaped apical cell. Metabolic activity started at the trichome tip sometimes accompanied by the formation of small subcuticular cavities at the apical cell. Subsequently, metabolic activity progressed downwards in the upper glandular cells. Cells involved in the secretory process showed disintegration of the subcellular compartments and lost vitality in parallel to deposition of fluorescent and brownish metabolites. The subcuticular cavities usually collapsed in the early secretory stage, whereas the colored depositions remained in cells of senescent hairs.

Temporal compartmentalization of viral infection in bacterial cells

Virus infection causes major rearrangements in the subcellular architecture of eukaryotes, but its impact in prokaryotic cells was much less characterized. Here, we show that infection of the bacterium Bacillus subtilis by bacteriophage SPP1 leads to a hijacking of host replication proteins to assemble hybrid viral–bacterial replisomes for SPP1 genome replication. Their biosynthetic activity doubles the cell total DNA content within 15 min. Replisomes operate at several independent locations within a single viral DNA focus positioned asymmetrically in the cell. This large nucleoprotein complex is a self-contained compartment whose boundaries are delimited neither by a membrane nor by a protein cage. Later during infection, SPP1 procapsids localize at the periphery of the viral DNA compartment for genome packaging. The resulting DNA-filled capsids do not remain associated to the DNA transactions compartment. They bind to phage tails to build infectious particles that are stored in warehouse compartments spatially independent from the viral DNA. Free SPP1 structural proteins are recruited to the dynamic phage-induced compartments following an order that recapitulates the viral particle assembly pathway. These findings show that bacteriophages restructure the crowded host cytoplasm to confine at different cellular locations the sequential processes that are essential for their multiplication.