Bulk and spatially resolved extracellular metabolomics of free-living nitrogen fixation

Soil microorganisms drive ecosystem function, but challenges of scale between microbe and ecosystem hinder our ability to accurately quantify and predictively model the soil microbe-ecosystem function relationship. Quantifying this relationship necessitates studies that systematically characterize multi-omics of soil microorganisms and their activity across sampling scales from spatially resolved to bulk measures, and structural complexity, from liquid pure culture to in situ. To address this need, we cultured two diazotrophic bacteria in liquid and solid media, with and without nitrogen (N) to quantify differences in extracellular metabolites associated with nitrogen fixation under increasing environmental structural complexity. We also quantified extracellular metabolites across sampling scales including bulk sampling via GC-MS analysis and spatially resolved analysis via MALDI mass spectrometry imaging. We found extracellular production of inorganic and organic N during free-living nitrogen fixation activity, highlighting a key mechanism of terrestrial N contributions from this process. Additionally, our results emphasize the need to consider the structural complexity of the environment and spatial scale when quantifying microbial activity. We found differences in metabolite profiles between culture conditions, supporting previous work indicating environmental structure influences microbial function, and across scales, underscoring the need to quantify microbial scale conditions to accurately interpret microbial function.

Biochemistry Behind Protein Adaptations in Extremophiles

Extremophiles are the mortals that tolerate in the most limiting and aggravating conditions to life. Because of these fantastic ecological criticisms, extremophiles have substituted innumerable intriguing transformations to cell films, proteins, and extracellular metabolites. These stimulatingly regulated usual particles and frameworks as of now play parts in numerous biotechnological fields. Compounds from extremophilic microorganisms as a rule catalyse synthetic responses in non-standard conditions. Such conditions advance accumulation, precipitation, and denaturation, diminishing the movement of most non-extremophilic catalysts, regularly because of the shortfall of adequate hydration. Extremophilic catalysts can go after hydration by means of modifications particularly to their surface through more noteworthy surface charges and expanded sub-atomic movement. These assets have permitted few extremophilic compounds to work within the sight of non-fluid natural solvents, with potential for plan of valuable impetuses.

Partial Purification and Characterization of Bacteriocin-Like Inhibitory Substances Produced by Streptomyces sp. Isolated from the Gut of Chanos chanos

Bacteriocin-like inhibitory substances (BLIS) have sparked great interest because of their promising use in food as natural antimicrobial agents. In this work, six Streptomyces isolates obtained from the gut of Chanos chanos demonstrated their ability to produce extracellular metabolites with inhibitory activity against Salmonella enterica serovar Typhimurium, Escherichia coli, Listeria monocytogenes, and Staphylococcus aureus. Exposure of the extracellular metabolites to proteolytic enzymes (i.e., proteinase-K, trypsin, and pepsin) revealed high sensitivity and confirmed their proteinaceous nature. The metabolites were stable at high temperatures (up to 100°C for 30 min) and a wide range of pH (pH 2.0–7.0). Fractionation of the crude BLIS by filtration yielded three fractions based on molecular weight: <3 kDa, 3–10 kDa, and >10 kDa. Analysis of the antibacterial activity of these fractions showed increased specific activity, especially in the fraction with a molecular weight (MW) of <3 kDa, relative to the crude sample. The fraction with MW < 3 kDa had minimum inhibitory and bactericidal concentrations in ranges 0.04–0.62 mg·mL−1 and 0.08–1.25 mg·mL−1, respectively. This fraction also showed better temperature and pH stability compared with crude BLIS. Brine shrimp toxicity assay revealed that this fraction has moderate toxicity with a 50% lethal concentration of 226.975 μg·mL−1 (i.e., moderate toxicity) to Artemia salina. Identification of the peptide sequences of this fraction by liquid chromatography–tandem mass spectrometry yielded 130 proteins with retention times of 15.21–19.57 min. Eleven proteins with MWs of 1345.66–2908.35 Da and composed of less than 30 amino acid residues with high hydrophobicity (15.34–26.22 kcal·mol−1) appeared to be responsible for the antibacterial activity of the fraction. This study revealed the potential application of BLIS from Streptomyces, especially BLIS SCA-8, as antibacterial agents.

Metabolic and Transcriptional Changes across Osteogenic Differentiation of Mesenchymal Stromal Cells

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.

Mast cell regranulation involves a metabolic switch promoted by the interaction between mTORC1 and a glucose-6-phosphate transporter

Mast cells (MCs) are highly granulated tissue resident hematopoietic cells and because of their capacity to degranulate and release many proinflammatory mediators, they are major effectors of chronic inflammatory disorders including asthma and urticaria. As MCs have the unique capacity to reform their granules following degranulation in vitro, their potential to undergo multiple cycles of degranulation and regranulation in vivo has been linked to their pathogenesis. However, it is not known what factors regulate MC regranulation let alone if MC regranulation occurs in vivo. Here, we report that IgE-sensitized mice can undergo multiple bouts of regranulation, following repeated anaphylactic reactions. mTORC1, a critical nutrient sensor that activates protein and lipid synthesis, was found necessary for MC regranulation. mTORC1 activity in MCs was regulated by a glucose-6-phosphate transporter, Slc37a2, which was found to be necessary for increased glucose-6-phosphate and ATP levels during regranulation, two upstream signals of mTOR. Slc37a2 is highly expressed at the cell periphery early during regranulation where it appears to colocalize with mTORC1. Additionally, this transporter was found to concentrate extracellular metabolites within endosomes which are trafficked directly into nascent granules. Thus, the metabolic switch associated with MC regranulation is mediated by the interactions of a cellular metabolic sensor and a transporter of extracellular metabolites into MC granules.

Metabolomics Comparison of Drug-Resistant and Drug-Susceptible Pseudomonas aeruginosa Strain (Intra- and Extracellular Analysis)

Pseudomonas aeruginosa is a common human pathogen belonging to the ESKAPE group. The multidrug resistance of bacteria is a considerable problem in treating patients and may lead to increased morbidity and mortality rate. The natural resistance in these organisms is caused by the production of specific enzymes and biofilm formation, while acquired resistance is multifactorial. Precise recognition of potential antibiotic resistance on different molecular levels is essential. Metabolomics tools may aid in the observation of the flux of low molecular weight compounds in biochemical pathways yielding additional information about drug-resistant bacteria. In this study, the metabolisms of two P. aeruginosa strains were compared—antibiotic susceptible vs. resistant. Analysis was performed on both intra- and extracellular metabolites. The 1H NMR method was used together with multivariate and univariate data analysis, additionally analysis of the metabolic pathways with the FELLA package was performed. The results revealed the differences in P. aeruginosa metabolism of drug-resistant and drug-susceptible strains and provided direct molecular information about P. aeruginosa response for different types of antibiotics. The most significant differences were found in the turnover of amino acids. This study can be a valuable source of information to complement research on drug resistance in P. aeruginosa.

Optimisation of Culture media through Response surface methodology to improve Antioxidant activity of E. coli

Antioxidants play a significant role in oxidative stress management and health protection. One variable at a time response surface methodology (RSM) was used to formulate different composition of media to increase the antioxidant property of selected microorganism (Escherichia coli MTCC no. 40). 1, 1-diphenyl-2-picrylhydrazyl (DPPH) assays was used to measure antioxidant activity of selected microorganism. The reduction potentiality of DPPH radical was determined by the DPPH% antioxidant or scavenging activity of the solution. In the present study, we have measured antioxidant activity of both intracellular and extracellular metabolites in dry and wet extracellular metabolites in supernatant respectively. In DPPH assay, extracellular metabolites showed better antioxidant potential in comparison with the reference compound. The comparison was based on antioxidant activity in different responses (number 1-16) for both dry and wet biomass. According to the results maximum DPPH % antioxidant or scavenging activity was showed by response 9 (wet biomass) and response 6 (dry biomass) that is 24.8447 and 35.0142 respectively in comparison to standard (4.4636). Hence, response surface methodology is used in bioprocess technology to optimize the medium components which is the primary step involved to enhance the antioxidant activity of particular microorganism.

The Extracellular Metabolome Stratifies Low and High Risk Potentially Premalignant Oral Keratinocytes and Identifies Citrate as a Potential Non-Invasive Marker of Tumour Progression

Premalignant oral lesions (PPOLs) which bypass senescence (IPPOL) have a much greater probability of progressing to malignancy, but pre-cancerous fields also contain mortal PPOL keratinocytes (MPPOL) that possess tumour-promoting properties. To identify metabolites that could potentially separate IPPOL, MPPOL and normal oral keratinocytes non-invasively in vivo, we conducted an unbiased screen of their conditioned medium. MPPOL keratinocytes showed elevated levels of branch-chain amino acid, lipid, prostaglandin, and glutathione metabolites, some of which could potentially be converted into volatile compounds by oral bacteria and detected in breath analysis. Extracellular metabolites were generally depleted in IPPOL, and only six were elevated, but some metabolites distinguishing IPPOL from MPPOL have been associated with progression to oral squamous cell carcinoma (OSCC) in vivo. One of the metabolites elevated in IPPOL relative to the other groups, citrate, was confirmed by targeted metabolomics and, interestingly, has been implicated in cancer growth and metastasis. Although our investigation is preliminary, some of the metabolites described here are detectable in the saliva of oral cancer patients, albeit at a more advanced stage, and could eventually help detect oral cancer development earlier.