Glucose Concentration In Medium Research Articles

Glucose-induced RYBP suppresses tumor cell aerobic glycolysis and migration

RYBP (Ring 1 and YY1 binding protein) has been frequently reported to play an important role during body development, stem cell differentiation, apoptosis and tumorigenesis, but whether RYBP carries out additional functions remains mysterious. Here, we demonstrated that RYBP protein levels elevate with increasing glucose concentration in cell culture medium in human tumorigenic cell lines, but an opposite trend was observed in non-tumorigenic cells. Mechanistic exploration disclosed that glucose inhibits polyubiquitination and proteasomal degradation, leading to RYBP stabilization in tumor cells. Further study showed that RYBP inhibits the glycolysis of tumor cells, as both extracellular acidification rate (ECAR) and lactate production increase when RYBP is knocked down, and decrease when RYBP is over-expressed, and this effect is unrelated to the glucose uptake ability of tumor cells. The functional study showed that RYBP is involved in the regulation of glucose on tumor cell migration. Compared to low glucose culture and their wildtypes, high glucose significantly enhanced tumor cell migration in RYBP knockdown or knockout tumor cells. Taken together, our current study uncovered a previously unknown function of RYBP in tumor metabolism, and this finding will enhance the exploration of the interplay between RYBP and nutrients during tumor cell metabolic reprogramming.

β cell acetate production and release are negligible

ABSTRACT Background Studies suggest that short chain fatty acids (SCFAs), which are primarily produced from fermentation of fiber, regulate insulin secretion through free fatty acid receptors 2 and 3 (FFA2 and FFA3). As these are G-protein coupled receptors (GPCRs), they have potential therapeutic value as targets for treating type 2 diabetes (T2D). The exact mechanism by which these receptors regulate insulin secretion and other aspects of pancreatic β cell function is unclear. It has been reported that glucose-dependent release of acetate from pancreatic β cells negatively regulates glucose stimulated insulin secretion. While these data raise the possibility of acetate’s potential autocrine action on these receptors, these findings have not been independently confirmed, and multiple concerns exist with this observation, particularly the lack of specificity and precision of the acetate detection methodology used. Methods Using Min6 cells and mouse islets, we assessed acetate and pyruvate production and secretion in response to different glucose concentrations, via liquid chromatography mass spectrometry. Results Using Min6 cells and mouse islets, we showed that both intracellular pyruvate and acetate increased with high glucose conditions; however, intracellular acetate level increased only slightly and exclusively in Min6 cells but not in the islets. Further, extracellular acetate levels were not affected by the concentration of glucose in the incubation medium of either Min6 cells or islets. Conclusions Our findings do not substantiate the glucose-dependent release of acetate from pancreatic β cells, and therefore, invalidate the possibility of an autocrine inhibitory effect on glucose stimulated insulin secretion.

PANC-1 Cell Line as an Experimental Model for Characterizing PIVKA-II Production, Distribution, and Molecular Mechanisms Leading to Protein Release in PDAC.

Pancreatic ductal adenocarcinoma (PDAC) represents a highly aggressive malignancy with a lack of reliable diagnostic biomarkers. Protein induced by vitamin K absence (PIVKA-II) is a protein increased in various cancers (particularly in hepatocellular carcinoma), and it has recently exhibited superior diagnostic performance in PDAC detection compared to other biomarkers. The aim of our research was to identify an in vitro model to study PIVKA-II production, distribution, and release in PDAC. We examined the presence of PIVKA-II protein in a panel of stabilized pancreatic cancer cell lines by Western blot analysis and indirect immunofluorescence (IFA). After quantitative evaluation of PIVKA-II in PaCa 44, H-Paf II, Capan-1, and PANC-1, we adopted the latter as a reference model. Subsequently, we analyzed the effect of glucose addiction on PIVKA-II production in a PANC-1 cell line in vitro; PIVKA-II production seems to be directly related to an increase in glucose concentration in the culture medium. Finally, we evaluated if PIVKA-II released in the presence of increasing doses of glucose is concomitant with the expression of two well-acknowledged epithelial-mesenchymal transition (EMT) markers (Vimentin and Snail). According to our experimental model, we can speculate that PIVKA-II release by PANC-1 cells is glucose-dependent and occurs jointly with EMT activation.

Importance of Intracellular Energy Status on High-Hydrostatic-Pressure Inactivation of sake Yeast Saccharomyces cerevisiae.

The HHP inactivation behaviors of Niigata sake yeast Saccharomyces cerevisiae strain S9arg and its aerobic respiratory-deficient mutant strains were investigated after cultivating them in a YPD media containing 2% to 15% glucose, as well as in moromi mash, in a laboratory-scale sake brewing process. The piezotolerance of strain S9arg, shown after cultivation in a YPD medium containing 2% glucose, decreased to become piezosensitive with increasing glucose concentrations in YPD media. In contrast, the piezosensitivity of a mutant strain UV1, shown after cultivation in the YPD medium containing 2% glucose, decreased to become piezotolerant with increasing glucose concentrations in the YPD medium. The intracellular ATP concentrations were analyzed for an S. cerevisiae strain with intact aerobic respiratory ability, as well as for strain UV1. The higher concentration of ATP after cultivation suggested a higher energy status and may be closely related to higher piezotolerance for the yeast strains. The decreased piezotolerance of strain S9arg observed after a laboratory-scale sake brewing test may be due to a lower energy status resulting from a high glucose concentration in moromi mash during the early period of brewing, as well as a lower aeration efficiency during the brewing process, compared with cultivation in a YPD medium containing 2% glucose.

Novel Technologies for Butyric Acid Fermentation: Use of Cellulosic Biomass, Rapid Bioreactor, and Efficient Product Recovery

Butyric acid, a four-carbon fatty acid, is an important industrial chemical and feedstock. To produce this chemical, a control fermentation was run with a 126.5 g.L−1 glucose concentration in the feed medium. In this medium, the strain produced 44.8 g.L−1 total acid with a productivity of 0.23 g.L−1h−1 and a yield of 0.41 g.g−1. The strain (Clostridium tyrobutyricum ATCC 25755) was also able to utilize glucose and xylose simultaneously with similar fermentation performance. The culture was also used to produce butyric acid from wheat straw hydrolysate (WSH) employing a hot water pretreatment. In a batch system, the strain resulted in a productivity and yield of 0.27 g.L−1h−1 and 0.44 g.g−1, respectively, which was an improvement over the use of glucose or xylose alone or mixtures of both. To improve reactor productivity, a membrane cell recycle bioreactor was used which resulted in a productivity of 1.89 g.L−1h−1. This productivity was 822% of that achieved in the glucose or xylose batch fermentation. Furthermore, a butyric acid recovery method was developed using XAD-4 adsorbent resin. In this system, up to 206.1 g.L−1 of butyric acid was used in the feed and, as a result of the quick adsorption, the residual butyric acid concentration was 29.5 g.L−1. In this experiment, the rate of acid removal of 1059.4 g.L−1h−1 was achieved.

A fluorescent probe for selective detection of lysosomal β-hexosaminidase in live cells

Determining the activity of lysosomal β-hexosaminidase in cells is of great importance for understanding the roles that these enzymes play in pathophysiological events. Herein, we designed the new fluorescent probe, βGalNAc-Rhod-CM(NEt2), which consisted of a βGalNAc-linked rhodol unit serving as a β-hexosaminidase reactive fluorogenic moiety and a N,N′-diethylaminocoumarin (CM(NEt2)) group acting as a fluorescence marker for determining the degree of cell permeabilization. Treatment of βGalNAc-Rhod-CM(NEt2) with β-hexosaminidase promoted generation of Rhod-CM(NEt2), thereby leading to an increase in the intensity of fluorescence of Rhod. However, this probe did not respond to the functionally related glycosidase, O-GlcNAcase. The detection limit of βGalNAc-Rhod-CM(NEt2) for β-hexosaminidase was determined to be 0.52 nM, indicating that it has high sensitivity for this enzyme. Furthermore, the probe functioned as an excellent fluorogenic substrate for β-hexosaminidase with kcat and Km values of 17 sec−1 and 22 μM, respectively. The results of cell studies using βGalNAc-Rhod-CM(NEt2) showed that levels of β-hexosaminidase activity in cells can be determined by measuring the intensity of fluorescence arising from Rhod and that the intensity of fluorescence of CM(NEt2) can be employed to determine the degree of cell permeabilization of the probe. Utilizing the new probe, we assessed β-hexosaminidase activities in several types of cells and evaluated the effect of glucose concentrations in culture media on the activity of this enzyme.

Influence of physiological glucose levels on SIM‐A9 microglial cell immune response

AbstractBackgroundMicroglia play an important role in the early onset of Alzheimer’s disease pathology1. They are responsible for maintaining brain homeostasis, where changes in energy metabolism alter microglial metabolic profile and function2. The majority of cell culture‐based studies have relied on growing microglia under optimal media conditions, not reflecting the brain’s physiological levels of key metabolites. This study aims to define a suitable experimental culture system that reflects the brain’s physiological glucose level, approximately 1mM3, so the effects of altered glucose concentration can be tested.MethodSIM‐A9 mouse immortalised microglial cells were initially grown in DMEM/F‐12 medium (17.5mM glucose) containing 10% FBS and 0.1% Penicillin/Streptomycin (Gibco, UK). After 20 hours cells were re‐seeded in Neurobasal medium containing 1x B‐27 supplement (Gibco, UK) and either 1mM or 5mM glucose and grown for 20 hours. Cell proliferation and viability was assessed over 40 hours using an Incucyte imaging system (Essen BioScience, UK) and Trypan Blue assay. Media glucose concentration, cellular glucose uptake (Promega, UK), microglia activation and metabolic marker expression using qRT‐PCR were measured following stimulation of cells with Lipopolysaccharide (LPS) (2.5ng/ml)ResultMaintaining SIM‐A9 cells in 1mM and 5mM glucose medium showed static proliferation but similar viability over 20 hours compared to growth in DMEM/F12. LPS treatment increased TNFa and IL1b expression in SIM‐A9 cells in all glucose conditions, indicating the cells’ competency to produce a pro‐inflammatory response. No change in pro‐inflammatory activation markers (TNFa, IL1b) was seen after maintenance in 1mM, but in 5mM glucose for 20 hours, compared to LPS treated cells. Media glucose concentration was 0.7mM over 20 hours in 1mM and 1mM in 5mM glucose media. Expression of glucose transporters Glut1 and Glut3 and glycolysis marker PFKFB3 under these glucose conditions was investigated.ConclusionUntil now microglial metabolic studies have not always reflected the physiological concentrations of glucose in the brain. The results show that culturing microglia in 1mM glucose medium does not induce pro‐inflammatory activation, while maintaining minimal cell death and unvaried confluency.

Optimization of L-asparaginase production from endophytic bacteria isolated from the mangrove Rhizophora mucronata

Abstract. Nafisaturrahmah A, Susilowati A, Pangastuti A. 2023. Optimization of L-asparaginase production from endophytic bacteria isolated from the mangrove Rhizophora mucronata. Nusantara Bioscience 15: 279-287. L-asparaginase is an enzyme that hydrolyes L-asparaginase to L-aspartate and ammonia. L-asparaginase has the potential to treat acute lymphoblastic leukemia and other malignant cancers. So far, purified L-asparaginase from Escherichia coli and Erwinea chrysanthemi has been available and applied clinically in humans. However, this treatment has side effects such as allergy, cross-interaction, immune system stimulation, drug resistance, and nonspecific L-glutaminase activity. These side effects can be overcome by discovering new sources of L-asparaginase, which are serologically different but have similar therapeutic effects. This study aims to determine the optimal conditions of endophytic bacterial culture in producing L-asparaginase. Endophytic bacteria were screened using an M9 medium with asparagine as a substrate; the L-asparaginase-producing isolates showed pink zones around the colonies. Optimization of L-aparaginase production by endophytic bacteria is carried out by One Factor at A Time (OFAT). Optimization of enzyme production includes incubation time, temperature, pH, ammonium sulfate levels, and glucose concentration in the bacterial growth medium; determination of enzyme production by Nesslerization method. The results showed that 8 isolates could produce high L-asparaginase, 14 isolates had medium ability, 30 isolates had low ability, and 2 bacterial isolates did not produce L-asparaginase. Endophytic isolates were able to produce the highest L-Aparaginase under different optimal conditions. The optimal incubation time for endophytic isolates in this study was 60-84 hours, the optimal temperature was 37ºC, the optimal pH was 7, the nitrogen content was 0.25 mg/L, and the optimal glucose level was 3%.

Glucose restriction in Saccharomyces cerevisiae modulates the phosphorylation pattern of the 20S proteasome and increases its activity

Caloric restriction is known to extend the lifespan and/or improve diverse physiological parameters in a vast array of organisms. In the yeast Saccharomyces cerevisiae, caloric restriction is performed by reducing the glucose concentration in the culture medium, a condition previously associated with increased chronological lifespan and 20S proteasome activity in cell extracts, which was not due to increased proteasome amounts in restricted cells. Herein, we sought to investigate the mechanisms through which glucose restriction improved proteasome activity and whether these activity changes were associated with modifications in the particle conformation. We show that glucose restriction increases the ability of 20S proteasomes, isolated from Saccharomyces cerevisiae cells, to degrade model substrates and whole proteins. In addition, threonine 55 and/or serine 56 of the α5-subunit, were/was consistently found to be phosphorylated in proteasomes isolated from glucose restricted cells, which may be involved in the increased proteolysis capacity of proteasomes from restricted cells. We were not able to observe changes in the gate opening nor in the spatial conformation in 20S proteasome particles isolated from glucose restricted cells, suggesting that the changes in activity were not accompanied by large conformational alterations in the 20S proteasome but involved allosteric activation of proteasome catalytic site.

Evaluating the potential of semi-continuous itaconic acid fermentation by Aspergillus terreus: operational profile and experiences

Itaconic acid is an important bio-based chemical. The present study aims to evaluate the applicability of semi-continuous fermentation technique for itaconic acid production by Aspergillus terreus. The fermentation is planned to be connected with bipolar membrane electrodialysis unit for acid recovery. This process allows the reuse of residual glucose from the effluent. Our particular attention was focused on the effect of glucose concentration. Two different glucose supplementation strategies were tested: constant glucose concentration in the refilling medium and adjusted glucose concentration in order to maintain a continuously high – 120 g/L – glucose concentration in the fermentor. The itaconic acid titre, yield and productivity for the 24 h time periods between draining/refilling interventions were investigated. The constantly high glucose concentration in the fermentor resulted in doubled biomass formation. The average itaconic acid titre was 32.9 ± 2.7 g/L. The producing strain formed numerous spores during semi-continuous fermentation that germinated continuously. Yield and volumetric productivity showed a periodic pattern during the procedure.

EFFECT OF LACTATE DEHYDROGENASE INHIBITION BY OXAMATE ON LEWIS LUNG CARCINOMA CELLS WITH DIFFERENT METASTATIC POTENTIAL.

Today, the ability for metabolic reprogramming is considered one of the distinguishing features of metastatically active tumor cells, a classic example of which is aerobic glycolysis. Despite a large number of studies in this direction, the question of the relationship between the intensity of aerobic glycolysis and the metastatic potential of tumor cells remains almost completely open. The work aimed to investigate the effect of the lactate dehydrogenase (LDH) inhibitor on the viability and several characteristics of Lewis lung carcinoma cells with different metastatic potential. High-metastatic (LLC) and low-metastatic (LLC/R9) variants of Lewis lung carcinoma cells were used. After 24 h of tumor cells incubation with or without 40 mM sodium oxamate, cell viability, the concentration of glucose and lactate in the incubation medium, distribution of cells by the cell cycle phases, and intracellular ROS production were estimated. It was revealed that regardless of the metastatic potential, LLC cells are heterogeneous in terms of both the involvement of aerobic glycolysis in their growth and survival processes and the sensitivity to the cytotoxic/cytostatic action of an LDH inhibitor. 35% of cells of either LLC variant form an oxamate-resistant subpopulation while 65% are oxamate-sensitive. The rate of glucose consumption of LLC/R9 cells in the absence of oxamate is almost twice higher compared to LLC and, as a result, the sensitivity of these cells to the cytotoxic/cytostatic effect of oxamate also is significantly higher (the IC50 for LLC/R9 cells is by 35.8% lower than that for LLC cells, p < 0.05). Approximately one-third of the cells of both LLC and LLC/R9 variants can survive and proliferate when aerobic glycolysis is completely inhibited by oxamate. This indicates metabolic reprogramming (either pre-existing or dynamically arising in response to inhibition of glycolysis) of this subpopulation of cells, within which not only the survival of cells but also their proliferative activity is most likely based on glutamine metabolism. Such metabolic heterogeneity of metastatically active cells indicates that inhibition of glycolysis as monotherapy is insufficient for effective antimetastatic therapy. Presumably, more effective would be to involve various inhibitors of metabolic processes that ensure the metabolic plasticity of metastatic cells.

MiR-143-3p/FNDC5 axis: a novel regulator of insulin sensitivity.

Insulin resistance is a key hallmark in type 2 diabetes. In recent decades, there have been numerous studies of the causes of insulin resistance. microRNAs (miRNAs) participate in the regulation of multiple aspects of energy metabolism and miR-143-3p has been shown to induce insulin resistance. We aimed to predict the downstream targets of miR-143-3p and found a miR-143-3p binding site on the 3'-untranslated region of FNDC5 (Fibronectin type III domain containing 5) mRNA. We first confirmed that FNDC5 mRNA is a target of miR-143-3p using a double luciferase experiment, then constructed a prokaryotic expression system for the mature form of FNDC5, irisin, and expressed and purified irisin protein. We transfected a miR-143-3p mimic into HepG2-NTCP (Na+-taurocholate cotransporting polypeptide) cells using an NTCP targeting vector, then 24 h later, the glucose concentration of the culture medium, western blot analysis was analyzed. We next co-incubated the cells transfected with the miR-143-3p mimic with irisin for 12 h following by the assay of glucose uptake and AKT phosphorylation. The glucose concentration of the culture medium was higher than that associated with control miRNA-transfected cells (p < 0.01). Western blot analysis showed that the miR-143-3p mimic significantly reduced the expression of FNDC5 (p < 0.05) and the phosphorylation of AKT (Protein kinase B) (p < 0.05), implying impaired insulin signaling. which increased the glucose uptake (p < 0.0001) and AKT phosphorylation in the cells (p < 0.05). We conclude that FNDC5 is a direct target of miR-143-3p and that miR-143-3p induces insulin resistance by reducing its expression.

ВЛИЯНИЕ РАЗЛИЧНЫХ КОНЦЕНТРАЦИЙ ГЛЮКОЗЫ В ПИТАТЕЛЬНОЙ СРЕДЕ НА МОРФОГЕНЕЗ РАСТЕНИЙ ВИНОГРАДА IN VITRO

To study the organic nutrition of grape plants in in vitro culture, we conducted research on the effect of glucose introduced into the nutrient medium on the morphogenesis of test-tube grape plants. The experiments were carried out on two model varieties (Sibirkovyi and Krasnostop Karpi) selected by us from the laboratory collection of in vitro healthy grape plants. These varieties, in addition to differences in genotypes, most noticeably differ in the features of development in in vitro culture, which we noted earlier when they were cultivated and deposited in the collection. The Sibirkovyi variety is one of the fastest growing, and Krasnostop Karpi is one of the slowest developing varieties in in vitro culture. In the experiment, glucose concentrations from 0.0 (sucrose control 10.0 g/l) to 40.0 g/l were used. The varietal response of the model varieties in the experiment with different concentrations of glucose in the nutrient medium was different. The variety Sibirkovyi was already one and a half times ahead of the variety Krasnostop Karpi in development in the first terms of accounting. At the same time, for the Sibirkovyi variety, the optimal range of concentrations for the development and preservation of microplants according to most indicators was the range from 5 to 20 g/l, and for the Krasnostop Karpi variety, the optimal range was narrower – 0-20 g/l. At high glucose concentrations of 30,0-40,0 g/l, the plants of both varieties experienced stress, which was manifested in the intensive release of phenols into the nutrient medium (this was most clearly manifested in the Sibirkovyi variety), and often the plants did not reach the top of the cultivation vessel during ontogenesis. besides, they showed earlier aging processes. At the same time, at high concentrations of glucose, a slowdown in growth processes and its ripening of shoots in individual microplants was noted.

Antimicrobial susceptibility testing using infrared attenuated total reflection (IR-ATR) spectroscopy to monitor metabolic activity

Fast and accurate detection of antimicrobial resistance in pathogens remains a challenge, and with the increase in antimicrobial resistance due to mis- and overuse of antibiotics, it has become an urgent public health problem. We demonstrate how infrared attenuated total reflection (IR-ATR) can be used as a simple method for assessment of bacterial susceptibility to antibiotics. This is achieved by monitoring the metabolic activities of bacterial cells via nutrient consumption and using this as an indicator of bacterial viability. Principal component analysis of the obtained spectra provides a tool for fast and simple discrimination of antimicrobial resistance in the acquired data. We demonstrate this concept using four bacterial strains and four different antibiotics, showing that the change in glucose concentration in the growth medium after 2 h, as monitored by IR-ATR, can be used as a spectroscopic diagnostic technique, to reduce detection time and to improve quality in the assessment of antimicrobial resistance in pathogens.

Glucose Starvation Stimulates the Promoting Strength of a Novel Evolved Suc2 Promoter.

Promoters are essential for designing Saccharomyces cerevisiae cell factories. Identifying novel promoters tuned to produce specific metabolites under increasingly diverse industrial stresses is required to improve the economic feasibility of whole fermentation processes. In this study, a positively evolved Suc2 promoter (SUC 2p) with promoter activity stronger than that of the wild-type Suc2 promoter (SUC 2wtp) was obtained. Quantitative real-time PCR, fluorescence analysis, Western blotting, and a β-galactosidase activity assay revealed that SUC 2p is a medium-strength promoter compared with eight reported promoters at a medium glucose concentration (2% (w/v)). Different glucose concentrations modulated the strength of SUC 2p. Low glucose concentrations (0.05 and 0.5% (w/v)) enhanced the promoter strength of SUC 2p dramatically, with promoter activity higher than that of reported strong promoters. Glucose starvation resulted in the formation of a new Msn2/4 binding site on SUC 2p. Our work should facilitate the development of promoters with novel fine-tuning properties and the construction of S. cerevisiae cell factories suitable for the industrial production of essential chemicals under glucose-deprived conditions.

Medium and process optimizations for Euglena gracilis with high biomass production enriched with protein

Axenic Euglena cultures were grown under different LED conditions, medium compositions, and durations of cultivation to identify the factors involved in tuning Euglena biomass quality, especially protein and paramylon contents. Compositional analysis suggests that paramylon and protein concentrations in biomass can be tuned to different extents depending on medium and process conditions, including medium compositions, glucose concentrations, and illuminations of various LED spectra. Through the optimization of these parameters, the biomass productivities under specified conditions reached ~50–68 g/L. The protein-rich (>50 % dry basis) Euglena cultures can be produced with higher biomass productivities (38 g/L) under mixotrophic conditions with the presence of white and red LED lights and the modified medium composition. The cell morphology studies of Euglena cultures under various cultivation conditions indicate the correlation of slender cell-shape to the cell feature of high-protein contents in the biomass. The fresh cellular volumes and dry weights of paramylon-rich round-shaped cells were also estimated to be 5–9 times higher than those of protein-rich slender-shaped cells. The present study demonstrates an effective method to produce high-protein Euglena biomass with higher productivity in a shorter time.

Submerged fermentation using Aspergillus tubingensis as an efficient strategy to obtain antioxidant extracts from black cricket proteins

In recent years, there has been growing interest in edible insects as a novel food source due to their high nutritional value and more sustainable cultivation system compared to traditional animal sources. Edible insects are rich in proteins, which can be used to produce bioactive peptides through enzymatic hydrolysis or fermentation. The main objective of this study was to evaluate the production of an antioxidant extract using proteins extracted from the black cricket (Gryllus assimilis) as a substrate for the submerged fermentative process conducted by the filamentous fungus Aspergillus tubingensis. A response surface methodology (CCRD) was employed to investigate the effect of glucose and black cricket protein concentrations in the culture medium on the release of the peptides. The results demonstrated that the maximum values for DPPH (290.79 µmol TE g–1), ABTS (862.82 µmol TE g–1), and FRAP (1020.11 µmol TE g–1) were obtained at 72 or 96 h of fermentation using lower concentrations of both protein and glucose. These findings confirm that black cricket proteins are suitable for producing an antioxidant extract via microbial fermentation.

Biochemical Properties of Oregano b-Glucosidase Supports Origanum Onites L. as a Feed Additive

Background: Feed additives are used for different purposes and include different substances. Recently, the feed additive potential of aromatic plants has been frequently investigated. β-glucosidases are used as feed additive enzymes to increase nutritional value. This study was carried out to determine the biochemical properties of Oregano onites L. β-glucosidase in terms of expected biochemical properties from feed additive enzymes. Methods: Oregano β-glucosidase was purified by hydrophobic interaction chromatography. The purified enzyme was checked the purity on SDS-PAGE. The biochemical properties (optimal pH and temperature, thermal stability, glucose and alcohol tolerance) of the purified enzyme were determined using para-Nitrophenyl-β-D-glucopyranoside as a substrate. Results: Oregano β-glucosidase was purified 23.15-fold with a yield of 8.2%. The purified enzyme was visualised as a single band at 65.7 kDa on SDS-PAGE. The enzyme had maximum activity at pH 4.0 and 45 °C and retained over 50% activity at pH 4-7. The enzyme maintained up to 50% of its activity after 60 minutes of incubation at 400C, 500C, and 60°C. The effect of glucose on the purified enzyme was determined by finding the relative activity at different glucose concentrations. When the glucose concentration in the reaction medium was 0.6 M, the relative activity was 52%, and the enzyme showed a high tolerance to glucose. The enzyme was also found to be tolerant to alcohol. The enzyme's high activity at acidic pH, its ability to maintain its activity for a long time, and its tolerance to glucose and alcohol make the enzyme valuable. β-glucosidase of Origanum onites L. used as a feed additive was purified for the first time and some biochemical properties were determined. It showed it’s biochemical properties similar to exogenous enzymes recommended as feed additives. Therefore, the results of this study provide information supporting the use of oregano as a feed additive.

Experimental Validation of an &lt;i&gt;In Vitro&lt;/i&gt; Method for Assessing Insulin-Dependent Glucose Uptake

Russian and international regulatory documents require that analytical procedures for establishing bioequivalence and comparability of quality attributes of biotechnological (biological) products, carrying out batch release of medicinal products, and conducting other equally important in vitro studies must be validated. These in vitro studies include molecule–receptor binding and product bioactivity assays. However, at present, there is no single approach to validation of in vitro bioanalytical methods not involving the determination of active ingredient concentrations in biological fluids. The aim of the work was to validate a procedure for assessing insulin-dependent glucose uptake and demonstrate the suitability of GOD-PAP GLUCOSE kits for glucose determination in culture media. Materials and methods: The study used RinFast® insulin aspart by Geropharm, Russia; a placebo for insulin aspart; the L6J1 rat myogenic cell line; and a GOD-PAP GLUCOSE kit for quantitative determination of glucose. The study was carried out on differentiated cells cultured for 7 days. To encourage L6J1 differentiation, the authors used DMEM with 4.5 g/L glucose and 2% horse serum. The statistical analysis of results was performed using Prism 9. Results: The study demonstrated the analytical procedure’s specificity, as the concentration of residual glucose in the culture medium observed with the placebo was 4 times higher than that with the maximum concentration of the medicinal product. The determination of precision showed the repeatability of 4–9% and the intralaboratory precision of 11–16%. The coefficient of variation for robustness amounted to 14% in 4 independent experiments comprising a total of 9 analytical runs. The authors compared insulin products (the insulin aspart and a genetically engineered human insulin), and the half-maximal inhibitory concentration (IC50) values differed by 1.5 times. For the GOD-PAP GLUCOSE kit, the linear regression coefficient of determination was 0.9983, the sensitivity amounted to 1 mmol/ L, and the accuracy ranged between 95% and 107%. Conclusions: the procedure using L6J1 rat myoblasts as a test system may be considered specific, highly precise, and robust in assessing insulin-dependent glucose uptake and suitable for detecting biological activity of insulin preparations in vitro.

Air–liquid interface cultures trigger a metabolic shift in intestinal epithelial cells (IPEC-1)

An improved oxygen availability in air–liquid interface (ALI) cultures of enterocytes of the small intestine seems to be primarily responsible for morphological, metabolic, and functional changes. Intestinal porcine epithelial cells 1 (IPEC-1) are less investigated and are rarely used as model for intestinal barrier but showed a profound change of cell shape during ALI cultivation. We aim to answer the following question: Are the observed morphological effects accompanied by changes in metabolic function? A microarray analysis of submerged culture (SMC) and ALI cultures identified 830 significantly regulated genes. Subsequent functional clustering revealed alterations in 31 pathways, with the highest number of regulated genes in metabolic pathways, carbon metabolism, glycolysis, and hypoxia-inducible factor (HIF) signaling. Furthermore, HIF-1α as a mediator of a metabolic switch between glycolysis and oxidative phosphorylation showed a trend of increased mRNA levels in ALI in contrast to a reduced nuclear HIF-1α content in the nucleus. Candidate genes of oxidative phosphorylation such as a mitochondrial marker exhibited enhanced mRNA levels, which was confirmed by western blot analysis. Cytochrome C oxidase (COX) subunit 5B protein was decreased in ALI, although mRNA level was increased. The oxidation of ferrocytochrome C to ferricytochrome C was used for detection of cytochrome C oxidase activity of isolated mitochondria and resulted in a trend of higher activity in ALI. Furthermore, quantification of glucose and lactate concentrations in cell culture medium revealed significantly reduced glucose levels and decreased lactate production in ALI. To evaluate energy metabolism, we measured cellular adenosine triphosphate (ATP) aggregation in homogenized cell suspensions showing similar levels. However, application of the uncoupling agent FCCP reduced ATP levels in ALI but not in SMC. In contrast, blocking with 2-desoxy-d-glucose (2DG) significantly reduced ATP content in ALI and SMC. These results indicate a metabolic shift in IPEC-1 cultured under ALI conditions enhancing oxidative phosphorylation and suppressing glycolysis.