Glucose Medium Research Articles

Cleavage and in vitro cultivation rates monitoring in culture media supplemented with energy sources, non-essential amino acids, and antioxidants in the buffalo embryos

The study was designed to monitor the cleavage rate (CR) and in-vitro cultivation rate (IVC) after addition of energy sources, non-essential amino acids, and antioxidants to the Synthetic oviductal fluid (SOF) and FertiCult. After in-vitro maturation and in-vitro fertilization, presumptive zygotes were cultured in one of two culture media: FertiCult media and SOF medium, supplemented with pyruvate, glucose, and sodium lactate as energy sources, as well as 10, 20, 250, 500, and 750 mg non-essential amino acids, and antioxidants. All stages of cleavage rate (CR), and in-vitro cultivation rate (IVC) of embryonic development including morula stage (MOR) and blastocyst (BLAS) have been assessed. The findings revealed that there were no significant differences in the CR between the control and other treated groups with sources of energy when added to SOF media (P > 0.05), while there were significant differences (P < 0.05) in the IVC of embryonic development between groups (The percentages of MOR stage in the control, pyruvate, glucose and mixture of source of energy (MIX) were at 50%, 62.5%, 60%, and 63.6%, respectively). The highest percentage of the BLAS was recorded after SOF supplementation with glucose (40%). Similarly, there were no significant differences (P > 0.05) in the CR between control and FertiCult supplemented with sources of energy, while the IVC stages increased significantly (P < 0.05) in the FertiCult media supplemented with glucose, pyruvate, sodium lactate, and MIX. The percentages of the MOR stage in the control, pyruvate, glucose and mix media were at 50%, 55.6%, 55.6%, 54.5%, 57.1% respectively. The lowest percentage of the BLAS was recorded after FertiCult supplementation with pyruvate (11.1%). Replenishing the SOF maturation media with 20 mg of non-essential amino acids significantly (P < 0.05) enhanced the MOR stage (100%). There was also an improvement in the development of BLAS stage, where it reached 31.2% and 47.4% in the SOF maturation media supplemented with 10, and 750 mg non-essential amino acids, respectively. There were no significant differences (P > 0.05) in neither CR nor IVC between control and FertiCult supplemented with antioxidants. There were significant differences (P < 0.05) in the MOR stages (control, 42.9% & treated, 57.9%) and BLAS stages (control, 21.4% & treated, 42.1%) in antioxidant supplemented SOF maturation media compared to control. In conclusion, supplementation of SOF cultivation medium with energy sources, 20 mg of non-essential amino acids and antioxidant addition may improve the cleavage rate (CR) and in vitro cultivation rate (IVC) of buffalos’ embryonic development.

Measuring Cell Dimensions in Fission Yeast Using Machine Learning.

In fission yeast (Schizosaccharomyces pombe), cell length is a crucial indicator of cell cycle progression. Microscopy screens that examine the effect of agents or genotypes suspected of altering genomic or metabolic stability and thus cell size are crucial for studying disruptions to cell cycle dynamics. This method is based on using an automated cell segmentation algorithm to measure S. pombe cells imaged by brightfield (BF) microscopy methods. PhotoPhenosizer (PP) is a machine learning-based tool designed for automated cell measuring and dimensional analysis of morphology frequency distributions. Integration of this method into large-scale pipelines for tracking cell dimension change streamlines morphological measurements, which facilitates the examination of cellular responses to genomic and metabolic stresses. In this protocol, we use PP to observe the effect of genomic instability on cell size dynamics over a 12-day chronological lifespan assay. Our results show that relative to wild-type cells, a replication stress mutant shows larger cells during chronological aging in excess glucose media. Our results are consistent with activation of checkpoints that regulate cell morphology in response to DNA damage. This method's application highlights the relevance of its incorporation in experimental routines that require large-scale image processing and its adoption by users with routine needs in S. pombe molecular research projects.

Quorum sensing in Vibrio controls carbon metabolism to optimize growth in changing environmental conditions.

Bacteria sense population density via the cell-cell communication system called quorum sensing (QS). The evolution of QS and its maintenance or loss in mixed bacterial communities is highly relevant to understanding how cell-cell signaling impacts bacterial fitness and competition, particularly under varying environmental conditions such as nutrient availability. We uncovered a phenomenon in which Vibrio cells grown in minimal medium optimize expression of the methionine and tetrahydrofolate (THF) synthesis genes via QS. Strains that are genetically "locked" at high cell density grow slowly in minimal glucose media and suppressor mutants accumulate via inactivating-mutations in metF (methylenetetrahydrofolate reductase) and luxR (the master QS transcriptional regulator). In mixed cultures, QS mutant strains initially coexist with wild type, but as glucose is depleted, wild type outcompetes the QS mutants. Thus, QS regulation of methionine/THF synthesis is a fitness benefit that links nutrient availability and cell density, preventing accumulation of QS-defective mutants.

Carbon starvation drives biosynthesis of high mannose-composition polysaccharides in Ganoderma lucidum

Polysaccharides with high mannose composition exhibit favorable antitumor activity, but there is currently a lack of well-defined and practical approaches to efficiently manufacture this type of polysaccharide. This study explores the impact of carbon utilization on exopolysaccharide (EPS) production and monosaccharide composition in Ganoderma lucidum, aiming to promote the biosynthesis of polysaccharides with high mannose composition. Sucrose supply significantly increased the mannose proportion to 35.52 % in EPS via carbon starvation. To enhance polysaccharide production with high mannose composition, a joint control by carbon starvation and glucose feeding strategy was developed, achieving 0.42 g/L EPS and 18.67 % mannose with 5 g/L glucose supplementation on day 6 in an initial 10 g/L glucose medium. Using this approach, G. lucidum mycelia exhibited normal growth patterns similar to those in sucrose culture. Essentially, carbon starvation upregulated key genes in the GDP-mannose synthesis pathway, including phosphomannose isomerase (PMI), phosphomannose mutase (PMM), and GDP-mannose pyrophosphorylase (GMP), by over 2-fold, facilitating high mannose-composition polysaccharide synthesis in both sucrose and carbon starvation conditions. This work provides insights into the biosynthesis of structurally specific polysaccharides in G. lucidum and offers a foundation for developing polysaccharides with diverse applications in food, medicine, and other fields.

Emodin regulated lactate metabolism by inhibiting MCT1 to delay non-small cell lung cancer progression.

Lung cancer is one of the most common malignant tumors in the world, with high incidence rate and mortality. Monocarboxylate transporter (MCT) 1 has been found to be widely expressed in various tumors and plays a crucial role in regulating energy metabolism. Emodin, as an important traditional Chinese medicine in China, has been reported to inhibit the progression of lung cancer. However, its potential mechanism has not been fully elucidated. The effects of emodin and MCT1 inhibitor AZD3965 on the proliferation, migration, and invasion of lung cancer cells were detected using cell counting kit-8 (CCK-8) assay, wound-healing assay, and transwell small chamber assay. The content of glucose, lactate, and pyruvate in the cell culture medium was detected using a glucose, lactate, and pyruvate detection kit, and also detected protein expression using western blotting. In addition, to investigate the effects of emodin and AZD3965 on lung cancer in vivo, we constructed nude mice subcutaneous transplant tumor model by subcutaneous injection of lung cancer cells. The results showed that emodin and AZD3965 could inhibit the proliferation, migration, and invasion of lung cancer cells. At the same time, they could inhibit the expression of MCT1 in lung cancer cells and promote the release of lactate, but did not affect the content of glucose and pyruvate. In vivo experiments had shown that emodin and AZD3965 could effectively inhibit the growth of lung cancer and inhibit the expression of MCT1. All in all, our data suggested that emodin inhibited the proliferation, migration, and invasion of lung cancer cells, possibly by inhibiting MCT1, providing important theoretical basis for elucidating the mechanism of emodin in treating lung cancer.

α-MnO2 with a cryptomelane structure for the non-enzymatic glucose electrooxidation in a neutral medium

A high demand for non-enzymatic glucose sensors and continuous glucose monitoring systems encourages the development of stable and active catalysts for the enzymeless anodic oxidation of glucose in the neutral medium. In regard to this reaction, we analyze the prospects of alpha manganese dioxide with a cryptomelane structure synthesized via an aqueous chemical route. To provide various specific surface area, the hydrated α-MnO2 oxide is annealed in a wide temperature range from 60 to 700 °C. Furthermore, we investigate the influence of the oxide annealing temperature on the amount of crystal water, chemical composition, morphology, electrochemical recharging behavior, electrocatalytic activity and stability in the glucose oxidation carried out in an isotonic NaCl-based phosphate-buffered saline (pH 7.40). To solidly determine potentials of the irreversible cathodic dissolution of α-MnO2 as well as O2/Cl2 evolution on α-MnO2, a rotating ring-disk electrode is applied. For the most active α-MnO2 sample with the 100 μg cm−2geo loading the glucose oxidation current of ca. 40 μA cm−2geo after 1 h at +0.8 V (SCE) for 7 mM of glucose is achieved. The linear concentration dependence in a physiological glucose range (1–30 mM) with the sensitivity of ca. 17.8 μA mM−0.5 cm−2geo at 25 °C is observed. Finally, glucose selectivity of α-MnO2 in relation to fructose, galactose, xylose, ribose, urea, lactic acid, acetone, ascorbic acid and paracetamol is revealed.

Identification of a cellular role of hemolysin co-regulatory protein (Hcp) in Vibrio alginolyticus modulating substrate metabolism and biofilm formation by cAMP-CRP

Cyclic AMP (cAMP) and cAMP receptor protein (CRP) system controls catabolic enzyme expression based on metabolite concentrations in bacteria. Hemolysin co-regulatory protein (Hcp) is well known as a molecular chaperone for virulence factor secretion of the type VI secretion system (T6SS). However, the intracellular role of Hcp involving in bacterial physiological processes remains unknown. To clarify that, we constructed a single hcp mutant strain and analyzed their effects on the physiological processes of Vibrio alginolyticus. The omics results revealed the extensive involvement of Hcp in the catabolic metabolism in bacteria. Simultaneously, Hcp1 and Hcp2 played opposing regulatory roles on the bacterial growth, biofilm formation, and intracellular cAMP-CRP levels during cultivation in a glucose medium. Furthermore, the interacting protein screening and co-immunoprecipitation (Co-IP) assays confirmed that the glucose-specific phosphoenolpyruvate (PEP)-phosphotransferase system (PTS) enzyme IIA component (EIIAglc) was a key interacting partner with Hcp proteins as well as class I adenylyl cyclase (AC-I) in Vibrio alginolyticus. These results indicated that, to achieve cellular homeostasis, Hcp1 and Hcp2 might exert antagonistic and synergistic effects, respectively, on the interaction between EIIAglc and AC thus cooperatively regulating intracellular cAMP-CRP production.

Copper nanoparticles biosynthesis by Priestia megaterium and its application as antibacterial and antitumor agents

The growth of material science and technology places high importance on creating better processes for synthesizing copper nanoparticles. Thus, an easy, ecological, and benign process for producing copper nanoparticles (CuNPs) has been developed using Priestia sp. bacteria utilizing a variety of low-cost agro-industrial wastes and byproducts. The biosynthesis of CuNPs was conducted using glucose medium and copper ions salt solution, then it was replaced by utilizing low-cost agro-industrial wastes. UV–visible spectroscopy, dynamic light scattering (DLS), X-ray diffraction (XRD), High-resolution transmission electron microscope (HR-TEM), Attenuated Total Reflectance and Fourier transform infrared (ATR-FTIR), and zeta potential were used to characterize the biosynthesized CuNPs. The cytotoxicity of CuNPs using Vero -CCL-81 cell lines, and antibacterial and antitumor properties using human colon epithelial colorectal adenocarcinoma Caco-2-HTB-37 cell lines were assessed. The UV–visible and DLS studies revealed CuNPs formation, with a maximum concentration of 6.19 ppm after 48 h, as indicated by a 0.58 Surface plasmon resonance (SPR) within 450 nm and 57.73 nm particle size. The 16S rRNA gene analysis revealed that Priestia sp. isolate is closely related to Priestia megaterium and has been deposited in the NCBI GenBank with accession number AMD 2024. The biosynthesis with various agro-industrial wastes indicated blackstrap sugar cane molasses being the most effective for reducing CuNPs size to 3.12 nm owing to various reducing and stabilizing active compounds. The CuNPs were free of contaminants, with a sphere-shaped structure and a cytotoxicity assessment with an IC50 of 367.27 μg/mL. The antibacterial activity exhibited by the most susceptible bacteria were Bacillus cereus ATCC 11788 and Staphylococcus aureus ATCC 6538 with inhibition zones of 26.0 mm and 28.0 mm, respectively. The antitumor effect showed an IC50 dose of 175.36 μg/mL. Based on the findings, the current work sought to lower product costs and provide a practical solution to the environmental contamination issues brought on by the buildup of agricultural wastes. In addition, the obtained CuNPs could be applied in many fields such as pharmaceuticals, water purification, and agricultural applications as future aspects.

Mitochondrial toxicity of selected natural compounds: in vitro assessment and in silico molecular docking and dynamics simulation

Prangos uechtritzii Boiss & Hausskn stands out for its rich bioactive constituents including prantschimgin (PRA), imperatorin (IMP), suberosin (SUB), adicardin (ADI), and oxypeucedanin hydrate (OPH) in the Apiaceae family. Although these molecules contribute to several biological activities, their mitochondrial toxicity were not illuminated in depth with the appropriate in vitro and in silico models. Cell viability studies investigated the cytotoxic activities of molecules in HepG2 cells by replacing glucose with galactose due to Warburg effects. Mitochondrial toxicity (mitotoxicity) parameters such as cellular adenosine triphosphate (ATP) and mitochondrial membrane potential (MMP) levels were assessed with cytotoxic concentrations of selected molecules. Molecular docking and dynamics studies were also conducted against mitochondrial electron transport chain (ETC) complexes (I-V) with selected compounds. In vitro results showed that PRA, SUB, and IMP reduced cell viability more in galactose media compared to high glucose media in a dose-dependent manner. PRA, IMP, and SUB decreased ATP levels and MMP, especially in the galactose medium. The in silico study revealed that PRA, IMP, and SUB might bind to complexes I-V at different levels. The docking study demonstrated that PRA had the highest binding potential with the complexes, higher than the standard ligands in some cases. The molecular dynamics (MD) simulation study showed that PRA formed stable complexes with complexes II, III, and IV. In addition, PRA was anticipated to remain inside the binding site of complex II most stably during the 230 ns simulation period. Our study suggests that PRA, IMP, and SUB exhibit mitotoxicity.

Alpha(α)-mangostin (Xanthone of Garcinia mangostana L.): Augmenting Macrophages Activity for an Effective Diabetic Wound Healing

Diabetic wounds are particularly difficult to treat medically because they heal at a slower pace than regular wounds. Macrophages are essential in all stages of normal wound healing, including inflammation, proliferation and rem odelling. When wound healing is affected, macrophages can reduce the level of growth factor and increase the level of interleukin-6 (IL-6), disrupt the balance between tissue inhibitors of metalloproteinase (TIMPs) and matrix metalloproteinase (MMPs), which can slow down healing. Alpha(α)-mangostin, a natural xanthone derived from the pericarp of the mangosteen, has gained considerable attention due to its anti-inflammatory properties, suggesting its potential to promote wound healing. However, its exact role in healing diabetic foot ulcers, common in diabetes, remains unclear. Hence, this study aims to explore how α-mangostin might affect diabetic wound healing by evaluating its impact on PDGF, CTGF, BFGF, VEGF, TGF-β, MMP-9, TIMP-2 and IL-6 secretion in macrophage cells. Human monocytic macrophages (THP-1) were incubated with a 35 mM glucose solution for 72 h to create a glucose-enriched medium. The cells were then incubated with α-mangostin (0.15, 2.5 and 5 µg/mL) together with 35 mM glucose. Carboxymethyl cellulose (CMC) served as positive controls; glucose-enriched media and media-alone served as negative controls. Protein expression was measured using ELISA. α-mangostin (2.5 µg/mL) increased the levels of PDGF and VEGF and decreased the level of MMP-9 compared to glucose controls. There was no significant difference in other growth factors, TIMP-2 and IL-6 protein levels across any of the treatment groups compared to glucose controls. In conclusion, α-mangostin particularly at 2.5 µg/mL demonstrated a significant increase in PDGF and VEGF levels while simultaneously reducing MMP-9 in macrophage cells under glucose-induced conditions. These findings suggest that α-mangostin holds the potential for enhancing the healing of chronic wounds in diabetic conditions. HIGHLIGHTS Treatment of macrophages (THP-1 cells) with 2.5 µg/mL Alpha (α)-mangostin in a high glucose medium led to a significant increase in PDGF and VEGF secretion compared to glucose control. Treatment of the cells with 2.5 µg/mL Alpha (α)-mangostin in a high glucose medium led to a significant reduction of MMP-9 secretion compared to glucose control. There is no significant effects of Alpha (α)-mangostin on IL-6 and TIMP secretion compared to controls This study suggests that Alpha (α)-mangostin may be beneficial in promoting diabetic wound healing via stimulation of PDGF and VEGF secretion and reducing MMP-9 levels. GRAPHICAL ABSTRACT

Enhancement of Bioactive Compounds and Survival of Lactobacillus acidophilus Grown in the Omega-6, -7 Riched Cyanobacteria Spirulina platensis.

Lactobacillus acidophilus is a probiotic commonly used in aquaculture to enhance the growth and immune system of aquatic species through the synthesis of various enzymes, and antimicrobial compounds like lactic acid. Traditional method of growing L. acidophilus involes using the De Man-Rogosa-Sharpe (MRS) medium. However, L. acidophilus belongs to a non-spore forming group, which make it vulnerable to stress conditions, especially during the usage process. Therefore, the present study aimed to improve the survival rate, antibacterial activity, and enrich the polyunsaturated fatty acids (PUFAs) content of L. acidophilus LB when cultured in an algae-supplemented medium, thus increasing its benefits in aquaculture applications. Using different algae biomass species as an alternative to MRS medium for the growth of L. acidophilus LB, the results showed that Spirulina platensis promoted the highest density of L. acidophilus LB. When grown in (S. platensis + glucose) medium, L. acidophilus LB produced the highest lactic acid concentration of 18.24 ± 2.43mg/mL and survived in extreme conditions such as 4% NaCl, pH 1.0-2.0, and 50ºC, and inhibited 99.82 ± 0.24% of Vibrio parahaemolyticus population after 2days of treatment. Additionally, it was observed that the PUFAs content, specifically omega-6, and -7, also increased in the fermentation mixture as compared to the control sample. These findings highlighted the potential of utilizing the cyanobacteria S. platensis as an alternative, eco-friendly growth substance for L. acidophilus LB to enhance its bioactivity and viability under extreme conditions.

Determination of beneficial effects of cuminaldehyde on hyperglycemiaassociated kidney malfunctions.

Type 1 diabetes mellitus is defined by the autoimmune destruction of pancreatic β cells, with diabetic nephropathy being a significant consequence. Recently, cuminaldehyde has been shown protective ability against various pathophysiology. However, its nephroprotective and anti-diabetic potential has not yet been fully understood. We, therefore, conducted the present study to evaluate the anti-hyperglycemic potential of cuminaldehyde in NRK52E cells without (control) or with high glucose medium to emulate hyperglycemic conditions. Cuminaldehyde pre-treatment at an optimal concentration of 175μM prior to high glucose addition restricted excessive reactive oxygen species (ROS) production and maintained cellular morphology to almost normal. The inhibitor study using N-acetyl-l-cysteine confirmed that blocking of ROS assists NRK52E cells in evading apoptosis. In addition, hyperglycemia was induced in 6-week-old Swiss albino mice in this investigation through the intraperitoneal injection of streptozotocin (150mgkg-1 body weight). Hyperglycemia increased the kidney-to-body weight ratio, lowered serum insulin levels, and led to significant renal tissue damage compared to control mice. Moreover, hyperglycemia disturbs cellular redox equilibrium by decreasing antioxidant enzyme functions and promoting inflammatory cytokines in kidney tissue. Administering cuminaldehyde at a dosage of 10mgkg-1 body weight for 5weeks daily after the onset of diabetes effectively ameliorated the aforementioned anomalies and reversed kidney damage by regulating inflammation-induced cell death. Overall, the research demonstrated that cuminaldehyde has hypoglycemic, antioxidant, anti-inflammatory, and anti-apoptotic properties. We believe that after conducting extensive research, this unique molecule can be used in clinical trials against diabetic nephropathy in future.

Synergistic regulation of chassis cell growth and screening of promoters, signal peptides and fusion protein linkers for enhanced recombinant protein expression in Bacillus subtilis

Growth-advantageous microbial chassis cells are beneficial for shortening fermentation period and boosting biomolecule productivity. This study focused on enhancing recombinant proteins synthesis efficiency in Bacillus subtilis by CRISPRi-mediated metabolism regulation for improved cell growth and screening expression elements. Specifically, by repressing odhA gene expression to reallocate cellular resource and overexpressing atpC, atpD and atpG genes to reprogram energy metabolism, the growth-advantageous chassis cell with high specific growth rate of 0.63 h−1 and biomass yield of 0.41 g DCW/g glucose in minimum medium was developed, representing 61.54 % and 46.43 % increasements compared to B. subtilis 168. Subsequently, using screened optimal P566 promoter and (EAAAK)3 protein linker, secretory bovine alpha-lactalbumin (α-LA) titer reached 1.02 mg/L. Finally, to test protein synthesis capability of cells, intracellular GFP, secretory α-LA and α-amylase were expressed with P566 promoter, representing 43.76 %, 75.49 % and 82.98 % increasements. The growth-advantageous B. subtilis chassis cells exhibit their potential to boost bioproduction productivity.

Prebiotic activity of enzymatically modified pea peel dietary fiber: An in vitro study

The study examined the prebiotic index of enzymatically modified insoluble dietary fiber from pea peel by using three probiotic bacterias; Lactobacillus rhamnosus ATCC 7469, Lactobacillus sakei ATCC 15521, Lactobacillus plantarum ATCC 8014 and one pathogenic bacteria; Escherichia coli ATCC 4157. A positive value for prebiotic potential was determined which was greater than inulin, unmodified dietary fiber, and standard glucose. The 30 h of cultivation, the cell density obtained from Lactobacillus rhamnosus ATCC 7469 culture conducted in MRS was 7.45 ± 0.08 log CFU/mL in glucose medium and 7.37 ± 0.07 log CFU/mL in dietary fiber medium. Growth kinetics analysis showed a positive effect for Lactobacillus spp. in DF medium. DF could boost cell biomass and relative growth rate of probiotic bacteria. Thus, the microbiome of the colon facilitates dietary fiber fermentation by bacteria shown in in vitro simulated gastrointestinal model.

Metabolism of primary high-grade serous ovarian carcinoma (HGSOC) cells under limited glutamine or glucose availability

BackgroundHigh-grade serous ovarian carcinoma (HGSOC) is the most common and aggressive subtype of epithelial ovarian carcinoma. It is primarily diagnosed at stage III or IV when the 5-year survival rate ranges between 20% and 40%. Here, we aimed to validate the hypothesis, based on HGSOC cell lines, that proposed the existence of two distinct groups of HGSOC cells with high and low oxidative phosphorylation (OXPHOS) metabolism, respectively, which are associated with their responses to glucose and glutamine withdrawal.MethodsWe isolated and cultivated primary cancer cell cultures from HGSOC and nontransformed ovarian fibroblasts from the surrounding ovarium of 45 HGSOC patients. We tested the metabolic flexibility of the primary cells, particularly in response to glucose and glutamine depletion, analyzed and modulated endoplasmic reticulum stress, and searched for indices of the existence of previously reported groups of HGSOC cells with high and low OXPHOS metabolism.ResultsThe primary HGSOC cells did not form two groups with high and low OXPHOS that responded differently to glucose and glutamine availabilities in the cell culture medium. Instead, they exhibited a continuum of OXPHOS phenotypes. In most tumor cell isolates, the responses to glucose or glutamine withdrawal were mild and surprisingly correlated with those of nontransformed ovarian fibroblasts from the same patients. The growth of tumor-derived cells in the absence of glucose was positively correlated with the lipid trafficking regulator FABP4 and was negatively correlated with the expression levels of HK2 and HK1. The correlations between the expression of electron transport chain (ETC) proteins and the oxygen consumption rates or extracellular acidification rates were weak. ER stress markers were strongly expressed in all the analyzed tumors. ER stress was further potentiated by tunicamycin but not by the recently proposed ER stress inducers based on copper(II)-phenanthroline complexes. ER stress modulation increased autophagy in tumor cell isolates but not in nontransformed ovarian fibroblasts.ConclusionsAnalysis of the metabolism of primary HGSOC cells rejects the previously proposed hypothesis that there are distinct groups of HGSOC cells with high and low OXPHOS metabolism that respond differently to glutamine or glucose withdrawal and are characterized by ETC protein levels.

2,3-Butanediol plus acetoin obtention by Enterobacter aerogenes ATCC 13048: inhibition by target products and cells reuse during fed-batch cultivation

2,3-Butanediol (2,3-BD) is a highly valued building block, and optimizing its production by fermentation, particularly with crude glycerol, is crucial. Enterobacter aerogenes is a key microorganism for this process; however, there are limited studies addressing the inhibition effects of products and by-products on 2,3-BD production. This study investigates these inhibition effects to maximize 2,3-BD production. Final concentrations of 2,3-BD plus acetoin reached 89.3, 92.7, and 71.1 g.L−1 with productivities of 1.22, 1.69, and 0.99 g.L−1.h−1 in pure glycerol, glucose, and crude glycerol media, respectively. Acetic acid was the main by-product, with concentrations ranging from 10 to 15 g.L−1. The reinoculation of E. aerogenes cells highlighted the strong effect of 2,3-BD and acetic acid on microbial growth and metabolism, with the cultivation environment exerting selective pressure. Notably, cells reuse enhanced performance in crude glycerol media, achieving a specific productivity in relation to biomass (YP/X) of 9.18 g.g−1; about 25% higher than in fed-batch without cells reuse. By combining results from two fed-batch cycles, the total final concentration of 2,3-BD plus acetoin reached 99.4 g.L−1, alongside a 33% reduction in total acetic acid production with reused cells.

Hyaluronic Acid Influences Amino Acid Metabolism via Differential L‐Type Amino Acid Transporter 1 Expression in the U87‐Malignant Glioma Cell Line

The glioblastoma (GBM) tumor microenvironment is heterogeneous, complex, and being increasingly understood as a significant contributor to tumor progression. In brain tumors, the extracellular matrix contains a large concentration of hyaluronic acid (HA) that makes it important to study its role in cancer progression. In particular, abnormal accumulation of HA is observed in gliomas and is often associated with poor prognosis. In addition, HA is a polymer and its molecular weight (MW) distribution may influence tumor cell activity. Herein, the influence of the MW of HA on tumor cell metabolism is evaluated. A 2D cell culture approach is used to expose the U87‐MG (medium glucose [MG]) cell line to different HA MWs (10, 60, and 500 kDa) and glucose concentrations (0, 5.5, and 25 mm). Notably, it is found that HA influences GBM amino acid metabolism via reduction in LAT1 transporter protein expression. Also an influence on mitochondrial respiration levels and a difference in the accumulation of some key products of cell metabolic activity (lactic acid, glutamic acid, and succinic acid) are reported. Overall, in these results, it is indicated that HA MW can influence GBM metabolic state, with implications for cell invasion and tumor progression.

Development of a programmable automated cell culture system to study the lung pathophysiology of Cystic Fibrosis-related diabetes

The study of many diseases is limited by the in vitro systems available. Cystic Fibrosis-Related Diabetes (CFRD), the main co-morbidity of Cystic Fibrosis (CF), is a perfect example. Cells in vivo experience glucose fluctuations after meals. In contrast, cells cultured in vitro are initially exposed to high glucose media. Glucose gets progressively depleted until the next media change days later, which is not physiologically relevant and could negatively impact the results of research studies. To better study the mechanisms driving CFRD pathophysiology, we developed a programmable and automated cell culture system (PACCS) capable of mimicking acute hyperglycemic episodes experienced by CFRD patients after meals. We adapted a commercially available perfusion system and performed 3D modeling to develop this system. Results show that PACCS can be successfully used to culture airway epithelial cells, both immortalized and primary cells. Further, CF cells responded differently to meal-like conditioning when compared to controls, suggesting impaired adaptative responses in CF cells. Overall, PACCS will allow us to better study CFRD pathophysiology, and it could be used for a wide range of other applications.

Hyperglycemic environments directly compromise intestinal epithelial barrier function in an organoid model and hyaluronan (∼35 kDa) protects via a layilin dependent mechanism

BackgroundMetabolic syndrome and diabetes in obese individuals are strong risk factors for development of inflammatory bowel disease (IBD) and colorectal cancer. The pathogenic mechanisms of low-grade metabolic inflammation, including chronic hyperglycemic stress, in disrupting gut homeostasis are poorly understood. In this study, we sought to understand the impact of a hyperglycemic environment on intestinal barrier integrity and the protective effects of small molecular weight (35 kDa) hyaluronan on epithelial barrier function. MethodsIntestinal organoids derived from mouse colon were grown in normal glucose media (5 mM) or high glucose media (25 mM) to study the impact of hyperglycemic stress on the intestinal barrier. Additionally, organoids were pretreated with 35 kDa hyaluronan (HA35) to investigate the effect of hyaluronan on epithelial barrier under high glucose stress. Immunoblotting as well as confocal imaging was used to understand changes in barrier proteins, quantitative as well as spatial distribution, respectively. Alterations in barrier function were measured using trans-epithelial electrical resistance and fluorescein isothiocyanate flux assays. Untargeted proteomics analysis was performed to elucidate mechanisms by which HA35 exerts a protective effect on the barrier. Intestinal organoids derived from receptor knockout mice specific to various HA receptors were utilized to understand the role of HA receptors in barrier protection under high glucose conditions. ResultsWe found that high glucose stress decreased the protein expression as well as spatial distribution of two key barrier proteins, zona occludens-1 (ZO-1) and occludin. HA35 prevented the degradation or loss of ZO-1 and maintained the spatial distribution of both ZO-1 and occludin under hyperglycemic stress. Functionally, we also observed a protective effect of HA35 on the epithelial barrier under high glucose conditions. We found that HA receptor, layilin, was involved in preventing barrier protein loss (ZO-1) as well as maintaining spatial distribution of ZO-1 and occludin. Additionally, proteomics analysis showed that cell death and survival was the primary pathway upregulated in organoids treated with HA35 under high glucose stress. We found that XIAP associated factor 1 (Xaf1) was modulated by HA35 thereby regulating apoptotic cell death in the intestinal organoid system. Finally, we observed that spatial organization of both focal adhesion kinase (FAK) as well as F-actin was mediated by HA35 via layilin. ConclusionOur results highlight the impact of hyperglycemic stress on the intestinal barrier function. This is of clinical relevance, as impaired barrier function has been observed in individuals with metabolic syndrome. Additionally, we demonstrate barrier protective effects of HA35 through its receptor layilin and modulation of cellular apoptosis under high glucose stress.

Cell Geometry and Membrane Protein Crowding Constrain Growth Rate, Overflow Metabolism, Respiration, and Maintenance Energy.

A metabolic theory is presented for predicting maximum growth rate, overflow metabolism, respiration efficiency, and maintenance energy flux based on the intersection of cell geometry, membrane protein crowding, and metabolism. The importance of cytosolic macromolecular crowding on phenotype has been established in the literature but the importance of surface area has been largely overlooked due to incomplete knowledge of membrane properties. We demonstrate that the capacity of the membrane to host proteins increases with growth rate offsetting decreases in surface area-to-volume ratios (SA:V). This increase in membrane protein is hypothesized to be essential to competitive Escherichia coli phenotypes. The presented membrane-centric theory uses biophysical properties and metabolic systems analysis to successfully predict the phenotypes of E. coli K-12 strains, MG1655 and NCM3722, which are genetically similar but have SA:V ratios that differ up to 30%, maximum growth rates on glucose media that differ by 40%, and overflow phenotypes that start at growth rates that differ by 80%. These analyses did not consider cytosolic macromolecular crowding, highlighting the distinct properties of the presented theory. Cell geometry and membrane protein crowding are significant biophysical constraints on phenotype and provide a theoretical framework for improved understanding and control of cell biology.