L-Glutamic Acid Assay Kit

Mesenchymal stromal cells (MSCs) are multipotent post-natal stem cells with applications in tissue engineering and regenerative medicine. MSCs can differentiate into osteoblasts, chondrocytes, or adipocytes, with functional differences in cells during osteogenesis accompanied by metabolic changes. The temporal dynamics of these metabolic shifts have not yet been fully characterized and are suspected to be important for therapeutic applications such as osteogenesis optimization. Here, our goal was to characterize the metabolic shifts that occur during osteogenesis. We profiled five key extracellular metabolites longitudinally (glucose, lactate, glutamine, glutamate, and ammonia) from MSCs from four donors to classify osteogenic differentiation into three metabolic stages, defined by changes in the uptake and secretion rates of the metabolites in cell culture media. We used a combination of untargeted metabolomic analysis, targeted analysis of ¹³C-glucose labelled intracellular data, and RNA-sequencing data to reconstruct a gene regulatory network and further characterize cellular metabolism. The metabolic stages identified in this proof-of-concept study provide a framework for more detailed investigations aimed at identifying biomarkers of osteogenic differentiation and small molecule interventions to optimize MSC differentiation for clinical applications.

Jeotgal containing abundant free amino acids plays an important role in the unique savory taste (umami) and flavor in kimchi. However, it is also responsible for the unpleasant fishy smell and high salt content of kimchi. Therefore, the present study aimed to identify alternative jeotgal sources and investigate the fermentation properties of jeotgal alternatives added to kimchi. The tomato hot-water extract (TH2) and dry-aged beef hot-water extract (DBH) were selected as jeotgal alternatives for kimchi preparation based on their glutamic acid contents. Characteristics of kimchi with TH2 alone (JA1) and TH2 and DBH in combination (1:1, JA2) were compared with kimchi prepared using commercially available anchovy fish sauce (CON). The pH of JA1 and JA2 was slightly decreased during fermentation, whereas the salinity was significantly lower than CON (p < 0.05). Notably, the most effective factor of the savory taste of kimchi, glutamic acid contents of JA1 and JA2 were significantly higher than that of CON (P < 0.05). In conclusion, JA1 showed slower fermentation with lower salinity and higher glutamic acid content than CON. Overall, this study showed that JA1 derived from TH2 could improve the taste and quality of kimchi by increasing glutamic acid content and decreasing the unpleasant flavor.

Multi-marker tests hold promise for identifying symptomatic women at risk of imminent preterm delivery (PTD, <37 week’s gestation). This study sought to determine the relationship of inflammatory mediators and metabolites in cervicovaginal fluid (CVF) with spontaneous PTD (sPTD) and delivery within 14 days of presentation with symptoms of preterm labour (PTL). CVF samples from 94 (preterm = 19, term = 75) singleton women with symptoms of PTL studied between 19⁺⁰-36⁺⁶ weeks’ gestation were analysed for cytokines/chemokines by multiplexed bead-based immunoassay, while metabolites were quantified by enzyme-based spectrophotometry in a subset of 61 women (preterm = 16, term = 45). Prevalence of targeted vaginal bacterial species was determined for 70 women (preterm = 14, term = 66) by PCR. Overall, 10 women delivered within 14 days of sampling. Predictive capacities of individual biomarkers and cytokine-metabolite combinations for sPTD and delivery within 14 days of sampling were analysed by logistic regression models and area under the receiver operating characteristic curve. Fusobacterium sp., Mubiluncus mulieris and Mycoplasma hominis were detected in more preterm-delivered than term women (P<0.0001), while, M. curtisii was found in more term-delivered than preterm women (P<0.0001). RANTES (0.91, 0.65-1.0), IL-6 (0.79, 0.67-0.88), and Acetate/Glutamate ratio (0.74, 0.61-0.85) were associated with delivery within 14 days of sampling (AUC, 95% CI). There were significant correlations between cytokines and metabolites, and several cytokine-metabolite combinations were associated with sPTD or delivery within 14 days of sampling (e.g. L/D-lactate ratio+Acetate/Glutamate ratio+IL-6: 0.84, 0.67-0.94). Symptomatic women destined to deliver preterm and within 14 days of sampling express significantly higher pro-inflammatory mediators at mid to late gestation. In this cohort, IL-6, Acetate/Glutamate ratio and RANTES were associated with delivery within 14 days of sampling, consistent with their roles in modulating infection-inflammation-associated preterm labour in women presenting with symptoms of preterm birth. Replication of these observations in larger cohorts of women could show potential clinical utility.

Ectoine, a cyclic tetrahydropyrimidine (2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid) is a compatible solute, serves as a protective compound in many halophilic eubacterial cells under stress. In this study, the ectoine biosynthesis genes (ectA, B and C) from the genomic DNA of a deep sea eubacteria, Bacillus clausii NIOT-DSB04 was PCR amplified, cloned into the expression vector pQE30 with a 6 × histidine tag and expressed in M15 cells. The lysates of induced cells with diaminobutyric acid aminotransferase and ectoine synthase disclosed two clear expressed bands with molecular masses of 46 kDa and 15 kDa as estimated by SDS-PAGE. The recombinant ectoine synthase activity of the expressed cells was at higher level than that of uninduced cells. In silico sequence and phylogenetic analysis of nucleotides and amino acids revealed that the ectA, B and C sequences of Bacillus clausii NIOT-DSB04 were conserved in many eubacteria.

Background: A future bioeconomy relies on the development of technologies to convert waste into valuable compounds. We present here an attempt to design a biotechnological cascade for the conversion of vegetable waste into acetoin and electrical energy. Results: A vegetable waste dark fermentation effluent containing mainly acetate, butyrate and propionate was oxidized in a bioelectrochemical system. The achieved average current at a constant anode potential of 0 mV against standard hydrogen electrode was 177.5  ± 52.5 &microA/cm². During this step, acetate and butyrate were removed from the effluent while propionate was the major remaining component of the total organic carbon content comprising on average 75.6%. The key players with regard to carbon oxidation and electrode reduction were revealed using amplicon sequencing and metatranscriptomic analysis. Using nanofiltration, it was possible to concentrate the propionate in the effluent. The effluent was revealed to be a suitable medium for biotechnological production strains. As a proof of principle, the propionate in the effluent of the bioelectrochemical system was converted into the platform chemical acetoin with a carbon recovery of 86%. Conclusions: To the best of our knowledge this is the first report on a full biotechnological production chain leading from vegetable waste to the production of a single valuable platform chemical that integrates carbon elimination steps leading to the production of the valuable side product electrical energy.

Aims: The Lactobacillus casei group represents a widely explored group of lactic acid bacteria, characterized by a high level of biodiversity. In this study, the genetic and phenotypic diversity of a collection of more than 300 isolates of the Lact. casei group and their potential to produce volatile metabolites important for flavour development in dairy products, was examined. Methods and Results: Following confirmation of species by 16S rRNA PCR, the diversity of the isolates was determined by pulsed-field gel electrophoresis. The activities of enzymes involved in the proteolytic cascade were assessed and significant differences among the strains were observed. Ten strains were chosen based on the results of their enzymes activities and they were analysed for their ability to produce volatiles in media with increased concentrations of a representative aromatic, branched chain and sulphur amino acid. Volatiles were assessed using gas chromatography coupled with mass spectrometry. Strain-dependent differences in the range and type of volatiles produced were evident. Conclusions: Strains of the Lact. casei group are characterized by genetic and metabolic diversity which supports variability in volatile production. Significance and Impact of the Study: This study provides a screening approach for the knowledge-based selection of strains potentially enabling flavour diversification in fermented dairy products.

Itaconic acid, an unsaturated C5-dicarboxylic acid, is a biobased building block for the polymer industry. The purpose of this study was to establish proof of principle for an anaerobic fermentation process for the production of itaconic acid by modification of the mixed acid fermentation pathway of E. coli. E. coli BW25113 (DE3) and the phosphate acetyltransferase (pta) and lactate dehydrogenase (ldhA) deficient strain E. coli BW25113 (DE3) Δpta-ΔldhA were used to study anaerobic itaconate production in E. coli. Heterologous expression of the gene encoding cis-aconitate decarboxylase (cadA) from A. terreus in E. coli BW25113 (DE3) did not result in itaconate production under anaerobic conditions, but 0.08 mM of itaconate was formed when the genes encoding citrate synthase (gltA) and aconitase (acnA) from Corynebacterium glutamicum were also expressed. The same amount was produced when cadA was expressed in E. coli BW25113 (DE3) Δpta-ΔldhA. The titre increased 8 times to 0.66 mM (1.2 % Cmol) when E. coli BW25113 (DE3) Δpta-ΔldhA also expressed gltA and acnA. In addition, this strain produced 8.5 mM (13 % Cmol) of glutamate. The use of a nitrogen-limited growth medium reduced the accumulation of glutamate by nearly 50 % compared to the normal medium, and also resulted in a more than 3-fold increase of the itaconate titre to 2.9 mM. These results demonstrated that E. coli has potential to produce itaconate and glutamate under anaerobic conditions, closing the redox balance by co-production of succinate or ethanol with H₂ and CO₂.

Novel methods are needed for the development of nanodispersed drug formulations to enhance bioavailability of many hydrophobic pharmaceuticals. The poorly water-soluble quinine is a well-known anti-malaria drug which can be used as a promising model compound for the development of novel nanodispersed formulations. In addition to hydrophobic drug's own affecting properties, surfactants play an important role for the enhancement of their low bioavailability by preparing stable dispersions. Amphiphilic compounds can efficiently be used to stabilize colloidal fragments by preventing the precipitation or crystallization of poorly water-soluble active ingredients during fabrication. A novel biopolymer derivative based on the biotechnologically produced γ-polyglutamic acid (γ-PGA) from Bacillus licheniformis cultivation was developed for encapsulation of the active ingredient. High-molecular γ-PGA is an anionic polyelectrolyte that was optimized and modified with hydrophobic L-phenylalanine ethyl ester (L-PAE) to form an amphiphilic comb polymer P(γ-GA-r-L-PAE) with surfactive properties. The approach of the nanodispersion polymer concentration, molecular weight and grafting degree enables the efficient stabilization of the poorly water-soluble model drug. The research presented in this report indicates the potential benefits of hydrophobically modified γ-PGA and suggests its potential role in forming stable dispersions for future pharmaceutical applications.

Gamma-amino butyric acid (GABA) is a nonprotein amino acid found in a wide range of organisms including plants. Several studies have shown that GABA plays different roles in plant metabolism including carbon–nitrogen metabolism, energy balance, signaling and development. It has been suggested that the occurrence of GABA and the enzymes related to GABA biosynthesis in prokaryotes and eukaryotes may be important in evolution and diversification. However, studies of GABA biosynthesis and GABA levels in an evolutionary context are restricted to sequenced plant genomes. In this study we aimed to compare the activities of GDH and GAD enzymes and total nitrogen, and the contents of total soluble protein, succinate, glutamate, proline and GABA in plants from different phylogenetic levels including Ulva lactuca, Pseudevernia furfuracea, Nephrolepsis exaltata, Ginkgo biloba, Pinus pinea, Magnolia grandiflora, Nymphaea alba, Urtica dioica, Portulaca oleraceae, Malva sylvestris, Rosa canina, Lavandula stoechas, Washingtonia filifera, Avena barbata and Iris kaempferi. The activities of GAD and GDH enzymes differed according to the species and were not always parallel to GABA levels. The discrepancy in the contents of succinate and GABA between higher and primitive plants was also prominent. Glutamate levels were high with a few exceptions and proline contents were at similar low values as compared to other amino acids. Our results support the hypothesis that the GABA shunt plays a key role in carbon and nitrogen partitioning via linking amino acid metabolism and the tricarboxylic acid cycle which is essential for higher plant species.

Temperatures higher than the optimum negatively affects plant growth and development. Tolerance to high temperature is a complex process that involves several pathways. Understanding this process, especially in crops such as rice, is essential to prepare for predicted climate changes due to global warming. Here, we show that OsMYB55 is induced by high temperature and overexpression of OsMYB55 resulted in improved plant growth under high temperature and decreased the negative effect of high temperature on grain yield. Transcriptome analysis revealed an increase in expression of several genes involved in amino acids metabolism. We demonstrate that OsMYB55 binds to the promoter regions of target genes and directly activates expression of some of those genes including glutamine synthetase (OsGS1;2) glutamine amidotransferase (GAT1) and glutamate decarboxylase 3 (GAD3). OsMYB55 overexpression resulted in an increase in total amino acid content and of the individual amino acids produced by the activation of the above mentioned genes and known for their roles in stress tolerance, namely L-glutamic acid, GABA and arginine especially under high temperature condition. In conclusion, overexpression of OsMYB55 improves rice plant tolerance to high temperature, and this high tolerance is associated with enhanced amino acid metabolism through transcription activation.

The astrocytic glutamate transporters GLAST/EAAT1 and GLT-1/EAAT2 are crucial for the removal of glutamate from the synaptic cleft and are essential for maintaining a low concentration of extracellular glutamate in the brain. Enhanced transporter expression is neuroprotective. In the present study, we tested the neuropotective effects of maslinic acid, a natural product from the Olea europaea plant, on cultures of primary neurons from the cerebral cortex. Studies showed that astrocyte-conditioned medium from maslinic acid-treated astrocytes dose-dependently promoted neuron survival during glutamate toxicity by enhancing extracellular glutamate clearance. Real-time PCR and western blot analysis revealed that maslinic acid pre-treatment significantly increased the expression of GLAST and GLT-1 at the protein and mRNA levels. In addition, this neuroprotection was abolished by the glutamate transporter inhibitor, L-Threohydroxy aspartate (THA), in a co-culture of astrocytes and neurons. These findings suggest that maslinic acid regulates the extracellular glutamate concentration by increasing the expression of astrocytic glutamate transporters, which may, in turn, provide neuroprotection.

A selective method for the determination of L-glutamate in foodstuffs has been developed. It was based on the competition established between the analyte and the dye Coomassie brilliant blue G (CBBG) to interact with the surfactant didodecyldimethylammonium bromide (DDABr). The measurement parameter was the amount of DDABr required to reach a given dye-to-surfactant binding degree. It was obtained by photometric titration on the basis of the changes observed in the spectral characteristics of the dye when CBBG–DDABr aggregates were formed. The calibration graph obtained was linear in the L-glutamate concentration interval 0.2–5 mM (detection limit 0.05 mM). The high selectivity of the proposed method (other amino acids and food additives did not interfere at the concentrations present in foodstuffs) permitted the direct analysis of food samples after dissolution of the analyte in hot water. The accuracy of the surfactant to the dye binding degree method was demonstrated by determining L-glutamate in different kinds of foodstuffs (liquid and dried soups, seasonings, pasta sauces and dried mushroom creams) and comparing the results obtained with those provided by the commercial Boehringer Mannheim essay.