13C-Metabolic flux analysis detected a hyperoxemia-induced reduction of tricarboxylic acid cycle metabolism in granulocytes during two models of porcine acute subdural hematoma and hemorrhagic shock.

Introduction: Supplementation with increased inspired oxygen fractions has been suggested to alleviate the harmful effects of tissue hypoxia during hemorrhagic shock (HS) and traumatic brain injury. However, the utility of therapeutic hyperoxia in critical care is disputed to this day as controversial evidence is available regarding its efficacy. Furthermore, in contrast to its hypoxic counterpart, the effect of hyperoxia on the metabolism of circulating immune cells remains ambiguous. Both stimulating and detrimental effects are possible; the former by providing necessary oxygen supply, the latter by generation of excessive amounts of reactive oxygen species (ROS). To uncover the potential impact of increased oxygen fractions on circulating immune cells during intensive care, we have performed a 13C-metabolic flux analysis (MFA) on PBMCs and granulocytes isolated from two long-term, resuscitated models of combined acute subdural hematoma (ASDH) and HS in pigs with and without cardiovascular comorbidity. Methods: Swine underwent resuscitation after 2 h of ASDH and HS up to a maximum of 48 h after HS. Animals received normoxemia (PaO2 = 80 - 120 mmHg) or targeted hyperoxemia (PaO2 = 200 - 250 mmHg for 24 h after treatment initiation, thereafter PaO2 as in the control group). Blood was drawn at time points T1 = after instrumentation, T2 = 24 h post ASDH and HS, and T3 = 48 h post ASDH and HS. PBMCs and granulocytes were isolated from whole blood to perform electron spin resonance spectroscopy, high resolution respirometry and 13C-MFA. For the latter, we utilized a parallel tracer approach with 1,2-13C2 glucose, U-13C glucose, and U-13C glutamine, which covered essential pathways of glucose and glutamine metabolism and supplied redundant data for robust Bayesian estimation. Gas chromatography-mass spectrometry further provided multiple fragments of metabolites which yielded additional labeling information. We obtained precise estimations of the fluxes, their joint credibility intervals, and their relations, and characterized common metabolic patterns with principal component analysis (PCA). Results: 13C-MFA indicated a hyperoxia-mediated reduction in tricarboxylic acid (TCA) cycle activity in circulating granulocytes which encompassed fluxes of glutamine uptake, TCA cycle, and oxaloacetate/aspartate supply for biosynthetic processes. We further detected elevated superoxide levels in the swine strain characterized by a hypercholesterolemic phenotype. PCA revealed cell type-specific behavioral patterns of metabolic adaptation in response to ASDH and HS that acted irrespective of swine strains or treatment group. Conclusion: In a model of resuscitated porcine ASDH and HS, we saw that ventilation with increased inspiratory O2 concentrations (PaO2 = 200 - 250 mmHg for 24 h after treatment initiation) did not impact mitochondrial respiration of PBMCs or granulocytes. However, Bayesian 13C-MFA results indicated a reduction in TCA cycle activity in granulocytes compared to cells exposed to normoxemia in the same time period. This change in metabolism did not seem to affect granulocytes' ability to perform phagocytosis or produce superoxide radicals.

Protocol to profile the bioenergetics of organoids using Seahorse.

Addressing bioenergetics is key to evaluate the impact of metabolism on the regulation of biological processes and its alteration in disease. Organoids are in vitro grown self-organizing structures derived from healthy and diseased tissue that recapitulate with high fidelity the tissue of origin. Bioenergetics is commonly analyzed by Seahorse XF analysis. However, its application to organoid studies is technically challenging. Here, we share our in-house optimized protocols to examine organoid bioenergetics in response to drugs, gene knockdown, or to characterize the metabolism of specific cell types. For complete details on the use and execution of this protocol, please refer to Ludikhuize et al. (2020).

NetRed, an algorithm to reduce genome-scale metabolic networks and facilitate the analysis of flux predictions.

Flux balance analysis (FBA) of large, genome-scale stoichiometric models (GSMs) is a powerful and popular method to predict cell-wide metabolic activity. FBA typically generates a flux vector containing O(1,000) fluxes. The interpretation of such a flux vector is difficult, even for expert users, because of the large size and complex topology of the underlying metabolic network. This interpretation could be simplified by condensing the network to a reduced, yet fully representative version. Toward this goal we report NetRed, an algorithm that systematically reduces a stoichiometric matrix and a corresponding flux vector to a more easily interpretable form. The reduction offered by NetRed is transparent because it relies purely on matrix algebra and not on optimization. Uniquely, it involves zero information loss; therefore, the original unreduced network can be easily recovered from the reduced network. The inputs to NetRed are (i) a stoichiometric matrix, (ii) a flux vector with numerical flux values, and (iii) a list of "protected" metabolites recommended by the user to remain in the reduced network. NetRed outputs a reduced metabolic network containing a reduced number of metabolites, of which the protected metabolites are a subset. The algorithm also generates a corresponding reduced flux vector. Due to its simplified presentation and easier interpretability, the reduced network allows the user to quickly find fluxes through metabolites and reaction modes or pathways of interest. In this manuscript, we first demonstrate NetRed on a simple network consisting of glycolysis and the pentose phosphate pathway (PPP), wherein NetRed reduced the PPP to a single net reaction. We followed this with applications of NetRed to E. coli and yeast GSMs. NetRed reduced the size of an E. coli GSM by 20- to 30-fold and enabled a comprehensive comparison of aerobic and anaerobic metabolism. The application of NetRed to a yeast GSM allowed for easy mechanistic interpretation of a double-gene knockout that rerouted flux toward dihydroartemisinic acid. When applied to an E. coli strain engineered for enhanced valine production, NetRed allowed for a holistic interpretation of the metabolic rerouting resulting from multiple genetic interventions.

Vitamin E does not prevent Western diet-induced NASH progression and increases metabolic flux dysregulation in mice.

Fatty liver involves ectopic lipid accumulation and dysregulated hepatic oxidative metabolism, which can progress to a state of elevated inflammation and fibrosis referred to as nonalcoholic steatohepatitis (NASH). The factors that control progression from simple steatosis to NASH are not fully known. Here, we tested the hypothesis that dietary vitamin E (VitE) supplementation would prevent NASH progression and associated metabolic alterations induced by a Western diet (WD). Hyperphagic melanocortin-4 receptor-deficient (MC4R-/-) mice were fed chow, chow+VitE, WD, or WD+VitE starting at 8 or 20 weeks of age. All groups exhibited extensive hepatic steatosis by the end of the study (28 weeks of age). WD feeding exacerbated liver disease severity without inducing proportional changes in liver triglycerides. Eight weeks of WD accelerated liver pyruvate cycling, and 20 weeks of WD extensively upregulated liver glucose and oxidative metabolism assessed by 2H/13C flux analysis. VitE supplementation failed to reduce the histological features of NASH. Rather, WD+VitE increased the abundance and saturation of liver ceramides and accelerated metabolic flux dysregulation compared with 8 weeks of WD alone. In summary, VitE did not limit NASH pathogenesis in genetically obese mice, but instead increased some indicators of metabolic dysfunction.

Enhancing hydrogen-dependent growth of and carbon dioxide fixation by Clostridium ljungdahlii through nitrate supplementation.

Synthesis gas (syngas) fermentation via the Wood-Ljungdahl pathway is receiving growing attention as a possible platform for the fixation of CO2 and renewable production of fuels and chemicals. However, the pathway operates near the thermodynamic limit of life, resulting in minimal adenosine triphosphate (ATP) production and long doubling times. This calls into question the feasibility of producing high-energy compounds at industrially relevant levels. In this study, we investigated the possibility of co-utilizing nitrate as an inexpensive additional electron acceptor to enhance ATP production during H2 -dependent growth of Clostridium ljungdahlii, Moorella thermoacetica, and Acetobacterium woodii. In contrast to other acetogens tested, growth rate and final biomass titer were improved for C. ljungdahlii growing on a mixture of H2 and CO2 when supplemented with nitrate. Transcriptomic analysis, 13CO2 labeling, and an electron balance were used to understand how electron flux was partitioned between CO2 and nitrate. We further show that, with nitrate supplementation, the ATP/adenosine diphosphate (ADP) ratio and acetyl-CoA pools were increased by fivefold and threefold, respectively, suggesting that this strategy could be useful for the production of ATP-intensive heterologous products from acetyl-CoA. Finally, we propose a pathway for enhanced ATP production from nitrate and use this as a basis to calculate theoretical yields for a variety of products. This study demonstrates a viable strategy for the decoupling of ATP production from carbon dioxide fixation, which will serve to significantly improve the CO2 fixation rate and the production metrics of other chemicals from CO2 and H2 in this host.

Propionate-induced changes in cardiac metabolism, notably CoA trapping, are not altered by l-carnitine.

High concentrations of propionate and its metabolites are found in several diseases that are often associated with the development of cardiac dysfunction, such as obesity, diabetes, propionic acidemia, and methylmalonic acidemia. In the present work, we employed a stable isotope-based metabolic flux approach to understand propionate-mediated perturbation of cardiac energy metabolism. Propionate led to accumulation of propionyl-CoA (increased by ~101-fold) and methylmalonyl-CoA (increased by 36-fold). This accumulation caused significant mitochondrial CoA trapping and inhibited fatty acid oxidation. The reduced energy contribution from fatty acid oxidation was associated with increased glucose oxidation. The enhanced anaplerosis of propionate and CoA trapping altered the pool sizes of tricarboxylic acid cycle (TCA) metabolites. In addition to being an anaplerotic substrate, the accumulation of proprionate-derived malate increased the recycling of malate to pyruvate and acetyl-CoA, which can enter the TCA for energy production. Supplementation of 3 mM l-carnitine did not relieve CoA trapping and did not reverse the propionate-mediated fuel switch. This is due to new findings that the heart appears to lack the specific enzyme catalyzing the conversion of short-chain (C3 and C4) dicarboxylyl-CoAs to dicarboxylylcarnitines. The discovery of this work warrants further investigation on the relevance of dicarboxylylcarnitines, especially C3 and C4 dicarboxylylcarnitines, in cardiac conditions such as heart failure.

13C-metabolic flux analysis of ethanol-assimilating Saccharomyces cerevisiae for S-adenosyl-L-methionine production.

BACKGROUND: Saccharomyces cerevisiae is a host for the industrial production of S-adenosyl-L-methionine (SAM), which has been widely used in pharmaceutical and nutritional supplement industries. It has been reported that the intracellular SAM content in S. cerevisiae can be improved by the addition of ethanol during cultivation. However, the metabolic state in ethanol-assimilating S. cerevisiae remains unclear. In this study, 13C-metabolic flux analysis (13C-MFA) was conducted to investigate the metabolic regulation responsible for the high SAM production from ethanol. RESULTS: The comparison between the metabolic flux distributions of central carbon metabolism showed that the metabolic flux levels of the tricarboxylic acid cycle and glyoxylate shunt in the ethanol culture were significantly higher than that of glucose. Estimates of the ATP balance from the 13C-MFA data suggested that larger amounts of excess ATP was produced from ethanol via increased oxidative phosphorylation. The finding was confirmed by the intracellular ATP level under ethanol-assimilating condition being similarly higher than glucose. CONCLUSIONS: These results suggest that the enhanced ATP regeneration due to ethanol assimilation was critical for the high SAM accumulation.

Nitrogen and phosphorus flux from the production of Nile tilapia through the application of environmental indicators / Fluxo de nitrogênio e fósforo na produção de tilápia-do-nilo (Oreochromis niloticus) a partir da aplicação de indicadores ambientais

Abstract We aimed in this study utilize environmental indicators as a quantitative method to evaluate and discuss the nitrogen (TN) and phosphorus (TP) flux by a production stage grow-out (termination) of Nile tilapia (Oreochromis niloticus) in fishpond. The TN and TP load, the mass balance, the input of TN and TP via feed and the converted nutrients in fish biomass are the environmental indicators applied in this study. During the production cycle (128 days), the system exported 15,931 g TN and 4,189 g TP that were related to the amount of feed supplied (r Pearson = 0.8825 and r = 0.8523, respectively), corroborated by the feed conversion ratio (1.61:1). The indicators showed that 26% TN and 45% TP were reversed into fish biomass, 62% TN and 40% TP were retained in the fishpond, and 12% TN and 15% TP were exported via effluent. The largest contribution of nutrients generated by the system and exported via effluent was observed in phase III and IV. This result is supported by the feed conversion ratio 2.14 and 2.21:1 obtained at this phase, a fact explained by the amount of feed offered and the fish metabolism. Application of environmental indicators showed to be an efficient tool to quantify flux of TN and TP produced during the grow-out period of Nile tilapia and therefore, guide management practices more sustainable. Concerning the environmental sustainability of the activity the implementation of best management practices such as the better control of the feed amount offered would lead to a smaller loss of TN and TP to the water. Furthermore, the use of better quality feeds would allow greater nutrient assimilation efficiency.

Resumo Nós objetivamos neste estudo, utilizar indicadores ambientais como método quantitativo para avaliar e discutir sobre o fluxo de nitrogênio (TN) e fósforo (TP) na etapa final de crescimento (terminação) de tilápia-do-nilo (Oreochromis niloticus) em viveiro escavado. A carga de TN e TP, o balanço de massa, a entrada de nutrientes via ração e o TN e TP convertido em biomassa de peixe foram os indicadores ambientais utilizados neste estudo. Durante o ciclo produtivo (128 dias), o sistema exportou 15.931 g NT e 4.189 g PT os quais foram relacionadas às quantidades de alimento fornecido (r Pearson = 0,8825 e r = 0,8523, respectivamente), corroborada pela conversão alimentar (1,61:1). Os indicadores evidenciaram que 26% NT e 45% PT foram revertidos em biomassa de peixe, 62% NT e 40% PT ficaram retidos no viveiro e 12% NT e 15% PT foram exportados via efluente. O maior aporte de nutrientes gerado pelo sistema e exportado via efluente foi verificado nas fases III e IV. Este resultado é corroborado pelas taxas de conversão alimentar de 2,14 e 2,21:1 obtida nestas fases, fato explicado pela quantidade de ração ofertada e pelo metabolismo dos peixes. A aplicação dos indicadores ambientais mostrou ser uma ferramenta eficiente para quantificar o fluxo de TN e TP produzidos durante a etapa final de crescimento de tilápia-do-nilo e com isso orientar práticas de manejo mais sustentáveis. Com vistas à sustentabilidade ambiental da atividade, a implantação de boas práticas de manejo tais como o melhor controle da quantidade de alimento ofertado levaria a menor perda de NT e PT para a água. Além disso, o uso de rações de melhor qualidade permitiria maior eficiência de assimilação desses nutrientes.

Nitrogen and phosphorus flux from the production of Nile tilapia through the application of environmental indicators.

We aimed in this study utilize environmental indicators as a quantitative method to evaluate and discuss the nitrogen (TN) and phosphorus (TP) flux by a production stage grow-out (termination) of Nile tilapia (Oreochromis niloticus) in fishpond. The TN and TP load, the mass balance, the input of TN and TP via feed and the converted nutrients in fish biomass are the environmental indicators applied in this study. During the production cycle (128 days), the system exported 15,931 g TN and 4,189 g TP that were related to the amount of feed supplied (r Pearson = 0.8825 and r = 0.8523, respectively), corroborated by the feed conversion ratio (1.61:1). The indicators showed that 26% TN and 45% TP were reversed into fish biomass, 62% TN and 40% TP were retained in the fishpond, and 12% TN and 15% TP were exported via effluent. The largest contribution of nutrients generated by the system and exported via effluent was observed in phase III and IV. This result is supported by the feed conversion ratio 2.14 and 2.21:1 obtained at this phase, a fact explained by the amount of feed offered and the fish metabolism. Application of environmental indicators showed to be an efficient tool to quantify flux of TN and TP produced during the grow-out period of Nile tilapia and therefore, guide management practices more sustainable. Concerning the environmental sustainability of the activity the implementation of best management practices such as the better control of the feed amount offered would lead to a smaller loss of TN and TP to the water. Furthermore, the use of better quality feeds would allow greater nutrient assimilation efficiency.

Coupling Radiotracer Experiments with Chemical Fractionation for the Estimation of Respiratory Fluxes.

Carbohydrates catabolized via respiratory processes are not only used for energy production but also for biosynthesis of cellular components including soluble molecules (sugars, amino acids, organic acids, and their derivatives) and insoluble macromolecules (proteins, starch, and cell wall). Radiotracer experiments using 14C-labeled glucose provide a global picture of the fate of respired carbon in the metabolic network. This method is based on a chemical fractionation of biomolecules in 14C-glucose fed plant materials and the subsequent determination of radioactivity in each fraction. Metabolic flux into each fraction can be estimated from the specific activity of the hexose phosphate pool. Here, we describe the procedure for glucose metabolism in potato tuber but similar protocols can be adopted for various plant organs and substrates.

A dynamic metabolic flux analysis of ABE (acetone-butanol-ethanol) fermentation by Clostridium acetobutylicum ATCC 824, with riboflavin as a by-product.

The present study reveals that supplementing sodium acetate (NaAc) strongly stimulates riboflavin production in acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum ATCC 824 with xylose as carbon source. Riboflavin production increased from undetectable concentrations to ∼0.2 g L-1 (0.53 mM) when supplementing 60 mM NaAc. Of interest, solvents production and biomass yield were also promoted with fivefold acetone, 2.6-fold butanol, and 2.4-fold biomass adding NaAc. A kinetic metabolic model, developed to simulate ABE biosystem, with riboflavin production, revealed from a dynamic metabolic flux analysis (dMFA) simultaneous increase of riboflavin (ribA) and GTP (precursor of riboflavin) (PurM) synthesis flux rates under NaAc supplementation. The model includes 23 fluxes, 24 metabolites, and 72 kinetic parameters. It also suggested that NaAc condition has first stimulated the accumulation of intracellular metabolite intermediates during the acidogenic phase, which have then fed the solventogenic phase leading to increased ABE production. In addition, NaAc resulted in higher intracellular levels of NADH during the whole culture. Moreover, lower GTP-to-adenosine phosphates (ATP, ADP, AMP) ratio under NaAc supplemented condition suggests that GTP may have a minor role in the cell energetic metabolism compared to its contribution to riboflavin synthesis.

Targeting Metabolic Reprogramming by Influenza Infection for Therapeutic Intervention.

Influenza is a worldwide health and financial burden posing a significant risk to the immune-compromised, obese, diabetic, elderly, and pediatric populations. We identified increases in glucose metabolism in the lungs of pediatric patients infected with respiratory pathogens. Using quantitative mass spectrometry, we found metabolic changes occurring after influenza infection in primary human respiratory cells and validated infection-associated increases in c-Myc, glycolysis, and glutaminolysis. We confirmed these findings with a metabolic drug screen that identified the PI3K/mTOR inhibitor BEZ235 as a regulator of infectious virus production. BEZ235 treatment ablated the transient induction of c-Myc, restored PI3K/mTOR pathway homeostasis measured by 4E-BP1 and p85 phosphorylation, and reversed infection-induced changes in metabolism. Importantly, BEZ235 reduced infectious progeny but had no effect on the early stages of viral replication. BEZ235 significantly increased survival in mice, while reducing viral titer. We show metabolic reprogramming of host cells by influenza virus exposes targets for therapeutic intervention.

Bioenergetics-based modeling of Plasmodium falciparum metabolism reveals its essential genes, nutritional requirements, and thermodynamic bottlenecks.

Novel antimalarial therapies are urgently needed for the fight against drug-resistant parasites. The metabolism of malaria parasites in infected cells is an attractive source of drug targets but is rather complex. Computational methods can handle this complexity and allow integrative analyses of cell metabolism. In this study, we present a genome-scale metabolic model (iPfa) of the deadliest malaria parasite, Plasmodium falciparum, and its thermodynamics-based flux analysis (TFA). Using previous absolute concentration data of the intraerythrocytic parasite, we applied TFA to iPfa and predicted up to 63 essential genes and 26 essential pairs of genes. Of the 63 genes, 35 have been experimentally validated and reported in the literature, and 28 have not been experimentally tested and include previously hypothesized or novel predictions of essential metabolic capabilities. Without metabolomics data, four of the genes would have been incorrectly predicted to be non-essential. TFA also indicated that substrate channeling should exist in two metabolic pathways to ensure the thermodynamic feasibility of the flux. Finally, analysis of the metabolic capabilities of P. falciparum led to the identification of both the minimal nutritional requirements and the genes that can become indispensable upon substrate inaccessibility. This model provides novel insight into the metabolic needs and capabilities of the malaria parasite and highlights metabolites and pathways that should be measured and characterized to identify potential thermodynamic bottlenecks and substrate channeling. The hypotheses presented seek to guide experimental studies to facilitate a better understanding of the parasite metabolism and the identification of targets for more efficient intervention.

A bioenergetics assay for studying the effects of environmental stressors on mitochondrial function in vivo in zebrafish larvae.

Mitochondria, an integral component of cellular energy metabolism and other key functions, are extremely vulnerable to damage by environmental stressors. Although methods to measure mitochondrial function in vitro exist, sensitive, medium- to high-throughput assays that assess respiration within physiologically-relevant whole organisms are needed to identify drugs and/or chemicals that disrupt mitochondrial function, particularly at sensitive early developmental stages. Consequently, we have developed and optimized an assay to measure mitochondrial bioenergetics in zebrafish larvae using the XFe24 Extracellular Flux Analyzer. To prevent larval movement from confounding oxygen consumption measurements, we relied on MS-222-based anesthetization. We obtained stable measurement values in the absence of effects on average oxygen consumption rate and subsequently optimized the use of pharmacological agents for metabolic partitioning. To confirm assay reproducibility we demonstrated that triclosan, a positive control, significantly decreased spare respiratory capacity. We then exposed zebrafish from 5 hours post-fertilization (hpf) to 6days post-fertilization (dpf) to three polycyclic aromatic hydrocarbons (PAHs) - benzo(a)pyrene (BaP), phenanthrene (Phe), and fluoranthene (FL) - and measured various fundamental parameters of mitochondrial respiratory chain function, including maximal respiration, spare respiratory capacity, mitochondrial and non-mitochondrial respiration. Exposure to all three PAHs decreased spare respiratory capacity and maximal respiration. Additionally, Phe exposure increased non-mitochondrial respiration and FL exposure decreased mitochondrial respiration and increased non-mitochondrial respiration. Overall, this whole organism-based assay provides a platform for examining mitochondrial dysfunction in vivo at critical developmental stages. It has important implications in biomedical sciences, toxicology and ecophysiology, particularly to examine the effects of environmental chemicals and/or drugs on mitochondrial bioenergetics.

Assessment of metabolism-dependent drug efficacy and toxicity on a multilayer organs-on-a-chip.

Pharmaceutical development is greatly hindered by the poor predictive power of existing in vitro models for drug efficacy and toxicity testing. In this work, we present a new and multilayer organs-on-a-chip device that allows for the assessment of drug metabolism, and its resultant drug efficacy and cytotoxicity in different organ-specific cells simultaneously. Four cell lines representing the liver, tumor (breast cancer and lung cancer), and normal tissue (gastric cells) were cultured in the compartmentalized micro-chambers of the multilayer microdevice. We adopted the prodrug capecitabine (CAP) as a model drug. The intermediate metabolites 5'-deoxy-5-fluorocytidine (DFUR) of CAP that were metabolized from liver and its active metabolite 5-fluorouracil (5-FU) from the targeted cancer cells and normal tissue cells were identified using mass spectrometry. CAP exhibited strong cytoxicity on breast cancer and lung cancer cells, but not in normal gastric cells. Moreover, the drug-induced cytotoxicity on cells varied in various target tissues, suggesting the metabolism-dependent drug efficacy in different tissues as exisits in vivo. This in vitro model can not only allow for characterizing the dynamic metabolism of anti-cancer drugs in different tissues simultaneously, but also facilitate the assessment of drug bioactivity on various target tissues in a simple way, indicating the utility of this organs-on-chip for applications in pharmacodynamics/pharmacokinetics studies, drug efficacy and toxicity testing.

Fatty acid and metabolomic profiling approaches differentiate heterotrophic and mixotrophic culture conditions in a microalgal food supplement 'Euglena'.

BACKGROUND: Microalgae have been recognized as a good food source of natural biologically active ingredients. Among them, the green microalga Euglena is a very promising food and nutritional supplements, providing high value-added poly-unsaturated fatty acids, paramylon and proteins. Different culture conditions could affect the chemical composition and food quality of microalgal cells. However, little information is available for distinguishing the different cellular changes especially the active ingredients including poly-saturated fatty acids and other metabolites under different culture conditions, such as light and dark. RESULTS: In this study, together with fatty acid profiling, we applied a gas chromatography-mass spectrometry (GC-MS)-based metabolomics to differentiate hetrotrophic and mixotrophic culture conditions. CONCLUSIONS: This study suggests metabolomics can shed light on understanding metabolomic changes under different culture conditions and provides a theoretical basis for industrial applications of microalgae, as food with better high-quality active ingredients.

Extraction, purification, methylation and GC-MS analysis of short-chain carboxylic acids for metabolic flux analysis.

Dynamic metabolic flux analysis requires efficient and effective methods for extraction, purification and analysis of a plethora of naturally-occurring compounds. One area of metabolism that would be highly informative to study using metabolic flux analysis is the tricarboxylic acid (TCA) cycle, which consists of short-chain carboxylic acids. Here, we describe a newly-developed method for extraction, purification, derivatization and analysis of short-chain carboxylic acids involved in the TCA cycle. The method consists of snap-freezing the plant material, followed by maceration and a 12-15h extraction at -80 °C. The extracts are then subject to reduction (to stabilize ß-keto acids), purified by strong anion exchange solid phase extraction and methylated with methanolic HCl. This method could also be readily adapted to quantify many other short-chain carboxylic acids.

Diurnal Regulation of Cellular Processes in the Cyanobacterium Synechocystis sp. Strain PCC 6803: Insights from Transcriptomic, Fluxomic, and Physiological Analyses.

UNLABELLED: Synechocystis sp. strain PCC 6803 is the most widely studied model cyanobacterium, with a well-developed omics level knowledgebase. Like the lifestyles of other cyanobacteria, that of Synechocystis PCC 6803 is tuned to diurnal changes in light intensity. In this study, we analyzed the expression patterns of all of the genes of this cyanobacterium over two consecutive diurnal periods. Using stringent criteria, we determined that the transcript levels of nearly 40% of the genes in Synechocystis PCC 6803 show robust diurnal oscillating behavior, with a majority of the transcripts being upregulated during the early light period. Such transcripts corresponded to a wide array of cellular processes, such as light harvesting, photosynthetic light and dark reactions, and central carbon metabolism. In contrast, transcripts of membrane transporters for transition metals involved in the photosynthetic electron transport chain (e.g., iron, manganese, and copper) were significantly upregulated during the late dark period. Thus, the pattern of global gene expression led to the development of two distinct transcriptional networks of coregulated oscillatory genes. These networks help describe how Synechocystis PCC 6803 regulates its metabolism toward the end of the dark period in anticipation of efficient photosynthesis during the early light period. Furthermore, in silico flux prediction of important cellular processes and experimental measurements of cellular ATP, NADP(H), and glycogen levels showed how this diurnal behavior influences its metabolic characteristics. In particular, NADPH/NADP(+) showed a strong correlation with the majority of the genes whose expression peaks in the light. We conclude that this ratio is a key endogenous determinant of the diurnal behavior of this cyanobacterium. IMPORTANCE: Cyanobacteria are photosynthetic microbes that use energy from sunlight and CO2 as feedstock. Certain cyanobacterial strains are amenable to facile genetic manipulation, thus enabling synthetic biology and metabolic engineering applications. Such strains are being developed as a chassis for the sustainable production of food, feed, and fuel. To this end, a holistic knowledge of cyanobacterial physiology and its correlation with gene expression patterns under the diurnal cycle is warranted. In this report, a genomewide transcriptional analysis of Synechocystis PCC 6803, the most widely studied model cyanobacterium, sheds light on the global coordination of cellular processes during diurnal periods. Furthermore, we found that, in addition to light, the redox level of NADP(H) is an important endogenous regulator of diurnal entrainment of Synechocystis PCC 6803.

Metabolic flux profiling of MDCK cells during growth and canine adenovirus vector production.

Canine adenovirus vector type 2 (CAV2) represents an alternative to human adenovirus vectors for certain gene therapy applications, particularly neurodegenerative diseases. However, more efficient production processes, assisted by a greater understanding of the effect of infection on producer cells, are required. Combining [1,2-(13)C]glucose and [U-(13)C]glutamine, we apply for the first time (13)C-Metabolic flux analysis ((13)C-MFA) to study E1-transformed Madin-Darby Canine Kidney (MDCK) cells metabolism during growth and CAV2 production. MDCK cells displayed a marked glycolytic and ammoniagenic metabolism, and (13)C data revealed a large fraction of glutamine-derived labelling in TCA cycle intermediates, emphasizing the role of glutamine anaplerosis. (13)C-MFA demonstrated the importance of pyruvate cycling in balancing glycolytic and TCA cycle activities, as well as occurrence of reductive alphaketoglutarate (AKG) carboxylation. By turn, CAV2 infection significantly upregulated fluxes through most central metabolism, including glycolysis, pentose-phosphate pathway, glutamine anaplerosis and, more prominently, reductive AKG carboxylation and cytosolic acetyl-coenzyme A formation, suggestive of increased lipogenesis. Based on these results, we suggest culture supplementation strategies to stimulate nucleic acid and lipid biosynthesis for improved canine adenoviral vector production.

Anacardic Acid, Salicylic Acid, and Oleic Acid Differentially Alter Cellular Bioenergetic Function in Breast Cancer Cells.

Anacardic acid is a dietary and medicinal phytochemical that inhibits breast cancer cell proliferation and uncouples oxidative phosphorylation (OXPHOS) in isolated rat liver mitochondria. Since mitochondrial-targeted anticancer therapy (mitocans) may be useful in breast cancer, we examined the effect of anacardic acid on cellular bioenergetics and OXPHOS pathway proteins in breast cancer cells modeling progression to endocrine-independence: MCF-7 estrogen receptor α (ERα)+ endocrine-sensitive; LCC9 and LY2 ERα+, endocrine-resistant, and MDA-MB-231 triple negative breast cancer (TNBC) cells. At concentrations similar to cell proliferation IC50 s, anacardic acid reduced ATP-linked oxygen consumption rate (OCR), mitochondrial reserve capacity, and coupling efficiency while increasing proton leak, reflecting mitochondrial toxicity which was greater in MCF-7 compared to endocrine-resistant and TNBC cells. These results suggest tolerance in endocrine-resistant and TNBC cells to mitochondrial stress induced by anacardic acid. Since anacardic acid is an alkylated 2-hydroxybenzoic acid, the effects of salicylic acid (SA, 2-hydroxybenzoic acid moiety) and oleic acid (OA, monounsaturated alkyl moiety) were tested. SA inhibited whereas OA stimulated cell viability. In contrast to stimulation of basal OCR by anacardic acid (uncoupling effect), neither SA nor OA altered basal OCR- except OA inhibited basal and ATP-linked OCR, and increased ECAR, in MDA-MB-231 cells. Changes in OXPHOS proteins correlated with changes in OCR. Overall, neither the 2-hydroxybenzoic acid moiety nor the monounsaturated alky moiety of anacardic acid is solely responsible for the observed mitochondria-targeted anticancer activity in breast cancer cells and hence both moieties are required in the same molecule for the observed effects. J. Cell. Biochem. 117: 2521-2532, 2016. © 2016 Wiley Periodicals, Inc.