Metabolic flux sampling predicts strain-dependent differences related to aroma production among commercial wine yeasts

Background Metabolomics coupled with genome-scale metabolic modeling approaches have been employed recently to quantitatively analyze the physiological states of various organisms, including Saccharomyces cerevisiae. Although yeast physiology in laboratory strains is well-studied, the metabolic states under industrially relevant scenarios such as winemaking are still not sufficiently understood, especially as there is considerable variation in metabolism between commercial strains. To study the potential causes of strain-dependent variation in the production of volatile compounds during enological conditions, random flux sampling and statistical methods were used, along with experimental extracellular metabolite flux data to characterize the differences in predicted intracellular metabolic states between strains. Results It was observed that four selected commercial wine yeast strains (Elixir, Opale, R2, and Uvaferm) produced variable amounts of key volatile organic compounds (VOCs). Principal component analysis was performed on extracellular metabolite data from the strains at three time points of cell cultivation (24, 58, and 144 h). Separation of the strains was observed at all three time points. Furthermore, Uvaferm at 24 h, for instance, was most associated with propanol and ethyl hexanoate. R2 was found to be associated with ethyl acetate and Opale could be associated with isobutanol while Elixir was most associated with phenylethanol and phenylethyl acetate. Constraint-based modeling (CBM) was employed using the latest genome-scale metabolic model of yeast (Yeast8) and random flux sampling was performed with experimentally derived fluxes at various stages of growth as constraints for the model. The flux sampling simulations allowed us to characterize intracellular metabolic flux states and illustrate the key parts of metabolism that likely determine the observed strain differences. Flux sampling determined that Uvaferm and Elixir are similar while R2 and Opale exhibited the highest degree of differences in the Ehrlich pathway and carbon metabolism, thereby causing strain-specific variation in VOC production. The model predictions also established the top 20 fluxes that relate to phenotypic strain variation (e.g. at 24 h). These fluxes indicated that Opale had a higher median flux for pyruvate decarboxylase reactions compared with the other strains. Conversely, R2 which was lower in all VOCs, had higher median fluxes going toward central metabolism. For Elixir and Uvaferm, the differences in metabolism were most evident in fluxes pertaining to transaminase and hexokinase associated reactions. The applied analysis of metabolic divergence unveiled strain-specific differences in yeast metabolism linked to fusel alcohol and ester production. Conclusions Overall, this approach proved useful in elucidating key reactions in amino acid, carbon, and glycerophospholipid metabolism which suggest genetic divergence in activity in metabolic subsystems among these wine strains related to the observed differences in VOC formation. The findings in this study could steer more focused research endeavors in developing or selecting optimal aroma-producing yeast stains for winemaking and other types of alcoholic fermentations.

Effects of the tumour microenvironment on protoporphyrin IX accumulation in glioblastoma

Aims Glioblastoma is the most common primary brain tumour and has a poor prognosis. Standard clinical intervention involves the resection of the tumour volume, chemotherapy and radiotherapy. However, achieving gross-total resection is challenging due to poorly defined boundaries as a result of tumour infiltration. Fluorescence-guided surgery (FGS) utilises an apparently selective accumulation of protoporphyrin IX (PPIX) that occurs in areas of glioblastoma after systemic administration of the metabolite 5-aminolevulinic acid (5-ALA). We have investigated the metabolic basis for the heterogeneity of the PPIX fluorescent signal, and its implications for glioma biology. Method Using glioblastoma cell lines and patient-derived primary cells, we have monitored the uptake of 5-ALA and conversion to the fluorescent molecule PPIX. Stable isotope tracing coupled with GCMS and LCMS was used to analyse intra- and extracellular metabolite levels arising from exogenous 5-ALA administration under both normoxic (21% O2) and hypoxic (1% O2) conditions. Results Uptake of exogenous 5-ALA from culture media and conversion to PPIX is observed in a time and dose-dependent manner in both normoxia and hypoxia. High levels of PPIX accumulation are associated with reduced cell proliferation despite the majority of the PPIX synthesised not being retained within the tumour cell, but exported into the medium. Under hypoxic conditions, reduced fluorescence is observed as a result of the decrease in oxygen availability likely affecting the oxygen-dependent enzymes. Stable isotope tracing experiments indicate an increase in the glutamine-derived succinate pool in response to exogenous 5-ALA, which is dependent on flux through the heme pathway. Conclusion Our data suggest that different microenvironments within the tumour alter the activity of the heme biosynthetic pathway, resulting in differential fluorescence in glioblastoma. It paves the way by which we could work to alter the glioblastoma microenvironment in order to further improve the use of FGS in guiding surgery across these devastating tumours.

Thermodynamic Genome-Scale Metabolic Modeling of Metallodrug Resistance in Colorectal Cancer

Background: Mass spectrometry-based metabolomics approaches provide an immense opportunity to enhance our understanding of the mechanisms that underpin the cellular reprogramming of cancers. Accurate comparative metabolic profiling of heterogeneous conditions, however, is still a challenge. Methods: Measuring both intracellular and extracellular metabolite concentrations, we constrain four instances of a thermodynamic genome-scale metabolic model of the HCT116 colorectal carcinoma cell line to compare the metabolic flux profiles of cells that are either sensitive or resistant to ruthenium- or platinum-based treatments with BOLD-100/KP1339 and oxaliplatin, respectively. Results: Normalizing according to growth rate and normalizing resistant cells according to their respective sensitive controls, we are able to dissect metabolic responses specific to the drug and to the resistance states. We find the normalization steps to be crucial in the interpretation of the metabolomics data and show that the metabolic reprogramming in resistant cells is limited to a select number of pathways. Conclusions: Here, we elucidate the key importance of normalization steps in the interpretation of metabolomics data, allowing us to uncover drug-specific metabolic reprogramming during acquired metal-drug resistance.

Periphytic microbial response to environmental phosphate bioavailability – relevance to P management in paddy fields

Periphyton occurs widely in shallow-water ecosystems such as paddy fields and plays critical parts in regulating local phosphorus cycling. As such, understanding the mechanisms of the biofilm’s response to environmental P variability may lead to better perceptions of P utilization and retention in rice farms. Present study aims at exploring the biological and biochemical processes underlying periphyton’s P buffering capability through examining changes in community structure, phosphorus uptake and storage, and molecular makeup of exometabolome at different levels of P availability. Under stressed (both excessive and scarce) phosphorus conditions, we found increased populations of the bacterial genus capable of transforming orthophosphate to polyphosphate, as well as mixotrophic algae who can survive through phagotrophy. These results were corroborated by observed polyphosphate buildup under low and high P treatment. Exometabolomic analyses further revealed that periphytic organisms may substitute S-containing lipids for phospholipids, use siderophores to dissolve iron (hydr)oxides to scavenge adsorbed P, and synthesize auxins to resist phosphorus starvation. These findings not only shed light on the mechanistic insights responsible for driving the periphytic P buffer but attest to the ecological roles of periphyton in aiding plants such as rice to overcome P limitations in natural environment. Importance The ability of periphyton to buffer environmental P in shallow aquatic ecosystems may be a natural lesson on P utilization and retention in paddy fields. This work revealed the routes and tools through which periphytic organisms adapt to and regulate ambient P fluctuation. The mechanistic understanding further implicates that the biofilm may serve rice plants to alleviate P stress. Additional results from extracellular metabolite analyses suggest the dissolved periphytic exometabolome can be a valuable nutrient source for soil microbes and plants to reduce biosynthetic costs. These discoveries have the potential to improve our understanding of biogeochemical cycling of phosphorus in general and to refine P management strategies for rice farm in particular.

Osteogenic Response of Human Mesenchymal Stem Cells Analysed Using Combined Intracellular and Extracellular Metabolomic Monitoring

Background/Aims: The skeleton is a metabolically active organ undergoing continuous remodelling initiated by mesenchymal progenitors present in bone and bone marrow. Under certain pathological conditions this remodelling balance shifts towards increased resorption resulting in weaker bone microarchitecture, and there is consequently a therapeutic need to identify pathways that could inversely enhance bone formation from stem cells. Metabolomics approaches recently applied to stem cell characterisation could help identify new biochemical markers involved in osteogenic differentiation. Methods: Combined intra- and extracellular metabolite profiling was performed by liquid chromatography-mass spectrometry (LC-MS) on human mesenchymal stem cells (MSCs) undergoing osteogenic differentiation in vitro. Using a combination of univariate and multivariate analyses, changes in metabolite and nutrient concentration were monitored in cultures under osteogenic treatment over 10 days. Results: A subset of differentially detected compounds was identified in differentiating cells, suggesting a direct link to metabolic processes involved in osteogenic response. Conclusion: These results highlight new metabolite candidates as potential biomarkers to monitor stem cell differentiation towards the bone lineage.

Thermodynamic genome-scale metabolic modeling of metallodrug resistance in colorectal cancer

Background: Mass spectrometry-based metabolomics approaches provide an immense opportunity to enhance our understanding of the mechanisms that underpin the cellular reprogramming of cancers. Accurate comparative metabolic profiling of heterogeneous conditions, however, is still a challenge. Methods: Measuring both intracellular and extracellular metabolite concentrations, we constrain four instances of a thermodynamic genome-scale metabolic model of the HCT116 colorectal carcinoma cell line to compare the metabolic flux profiles of cells that are either sensitive or resistant to ruthenium- or platinum-based treatments with BOLD-100/KP1339 and oxaliplatin, respectively. Results: Normalizing according to growth rate and normalizing resistant cells according to their respective sensitive controls, we are able to dissect metabolic responses specific to the drug and to the resistance states. We find the normalization steps to be crucial in the interpretation of the metabolomics data and show that the metabolic reprogramming in resistant cells is limited to a select number of pathways. Conclusions: Here we elucidate the key importance of normalization steps in the interpretation of metabolomics data, allowing us to uncover drug-specific metabolic reprogramming during acquired metal-drug resistance.

Deteksi Golongan Senyawa Ekstrak Kasar Metabolit Ekstrasel Mikroalga Laut Spirulina sp. Sebagai Agen Antioksidan

Microalgae excrete antioxidant compounds as a defense system to protect themselves from the danger of ultraviolet rays. These compounds also can be used as the organic materials of cosmetics or medicines. This study aimed to determine the antioxidant activity of marine microalgal extracellular metabolite extract of Spirulina sp.. Extracellular metabolites were extracted from the residual media filtrate from the harvesting. The qualitative antioxidant test’s results using the thin-layer chromatography technique and 2,2-diphenylpycrilhydrazil (DPPH) reagent showed antioxidant activity. Moreover, the component identification using ninhydrin and Dragendorff reagent in thin layer chromatography test showed alkaloid and peptide compounds. To support the results, the identification using infrared spectrum analysis showed the peaks at 1117 cm-1 (C-N and C-C stretching), 1458 cm-1 (C-H bending of methyl group), 1635 cm-1 (C=O stretching of amide group), and 3454 cm-1 (N-H stretching of amine and amide groups).