Journal articles on the topic 'Single cell metabolic flux'
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Wagner, Allon, Chao Wang, David DeTomaso, Arman Koul, Aviv Regev, Vijay K. Kuchroo, and Nir Yosef. "Cell-specific metabolic models reveal novel metabolic regulators of Th17 pathogenicity: from single-cell RNA-Seq to actionable metabolic targets." Journal of Immunology 200, no. 1_Supplement (May 1, 2018): 163.21. http://dx.doi.org/10.4049/jimmunol.200.supp.163.21.
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Abstract Cellular metabolism is a powerful regulator of immune response. In order to unbiasedly search for novel metabolic regulators of Th17 development and function, we have developed COMPASS, a computational algorithm to characterize the metabolic landscape of single cells based on single-cell RNA-Seq profiles and flux balance analysis. We used COMPASS to characterize the metabolic heterogeneity in Th17 cells, whose pathogenic state triggers auto-immunity, yet whose non-pathogenic form promotes tissue homeostasis and barrier functions. COMPASS recovered known metabolic switches and predicted that the polyamine pathway should be a novel, powerful regulator of Th17 pathogenicity. We validated the pathway’s effect through an array of transcriptome, LC/MS metabolome, and functional assays. Deletion of polyamine enzymes in T cells resulted in altered metabolic space, T cell functions and, most importantly, aggravated symptoms in EAE, a murine model of multiple sclerosis. While our study is concerned with Th17 cells, COMPASS is generally applicable, and can be used to unbiasedly characterize the metabolic states of any cell population based on its single-cell transcriptome profiles. Furthermore, COMPASS predicts metabolic switches between cell states that present testable, mechanistic hypotheses.
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Land, S. C., D. M. Porterfield, R. H. Sanger, and P. J. Smith. "The self-referencing oxygen-selective microelectrode: detection of transmembrane oxygen flux from single cells." Journal of Experimental Biology 202, no. 2 (January 15, 1999): 211–18. http://dx.doi.org/10.1242/jeb.202.2.211.
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A self-referencing, polarographic, oxygen-selective microelectrode was developed for measuring oxygen fluxes from single cells. This technique is based on the translational movement of the microelectrode at a known frequency through an oxygen gradient, between known points. The differential current of the electrode was converted into a directional measurement of flux using the Fick equation. Operational characteristics of the technique were determined using artificial gradients. Calculated oxygen flux values matched theoretical values derived from static measurements. A test preparation, an isolated neuron, yielded an oxygen flux of 11.46+/−1.43 pmol cm-2 s-1 (mean +/− s.e.m.), a value in agreement with those available in the literature for single cells. Microinjection of metabolic substrates or a metabolic uncoupler increased oxygen flux, whereas microinjection of KCN decreased oxygen flux. In the filamentous alga Spirogyra greveilina, the probe could easily differentiate a 16.6% difference in oxygen flux with respect to the position of the spiral chloroplast (13.3+/−0.4 pmol cm-2 s-1 at the chloroplast and 11.4+/−0.4 pmol cm-2 s-1 between chloroplasts), despite the fact that these positions averaged only 10.6+/−1.8 microm apart (means +/− s.e.m.). A light response experiment showed real-time changes in measured oxygen flux correlated with changes in lighting. Taken together, these results show that the self-referencing oxygen microelectrode technique can be used to detect local oxygen fluxes with a high level of sensitivity and spatial resolution in real time. The oxygen fluxes detected reliably correlated with the metabolic state of the cell.
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Lee, Geonhui, Thomas Ruan, Claudia Wong, Kofi Deh, Alli Abolarin, Alexander Correa, Kayvan R. Keshari, and Sangmoo Jeong. "Micro-Slab Coil Design for Hyperpolarized Metabolic Flux Analysis in Multiple Samples." Bioengineering 10, no. 1 (December 21, 2022): 14. http://dx.doi.org/10.3390/bioengineering10010014.
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Abnormal metabolism is a hallmark of cancer cells. Accumulating evidence suggests that metabolic changes are likely to occur before other cellular responses in cancer cells upon drug treatment. Therefore, the metabolic activity or flux in cancer cells could be a potent biomarker for cancer detection and treatment monitoring. Magnetic resonance (MR)-based sensing technologies have been developed with hyperpolarized molecules for real-time flux analysis, but they still suffer from low sensitivity and throughput. To address this limitation, we have developed an innovative miniaturized MR coil, termed micro-slab MR coil, for simultaneous analysis of metabolic flux in multiple samples. Combining this approach with hyperpolarized probes, we were able to quantify the pyruvate-to-lactate flux in two different leukemic cell lines in a non-destructive manner, simultaneously. Further, we were able to rapidly assess flux changes with drug treatment in a single hyperpolarization experiment. This new multi-sample system has the potential to transform our ability to assess metabolic dynamics at scale.
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Wagner, Allon, Chao Wang, David DeTomaso, Julian Avila Pacheco, Sarah Zaghouani, Johannes Fessler, Elliot Akama-Garren, et al. "In Silico Modeling of Metabolic State in Single Th17 Cells Reveals Novel Regulators of Inflammation and Autoimmunity." Journal of Immunology 204, no. 1_Supplement (May 1, 2020): 150.22. http://dx.doi.org/10.4049/jimmunol.204.supp.150.22.
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Abstract Cellular metabolism is a major regulator of immune response, but it is difficult to study the metabolic status of an individual immune cell using current technologies. Here, we present Compass, an algorithm to characterize the metabolic landscape of single cells in silico based on single-cell RNA-Seq and flux balance analysis. We used Compass to study the landscape of metabolic heterogeneity in Th17 cells and search for novel metabolic regulators of their inflammatory function. In central carbon metabolism, Compass predicted a metabolic switch between glycolysis and fatty acid oxidation that mirrors the Th17 vs. Treg phenotype, which we validated through transcriptomic, metabolic and functional assays. The TCA cycle was predicted to break at two points, both of which have been independently identified by other groups in M1 macrophage polarization. Surprisingly, and contrary to common immunometabolic understanding, Compass predicted that glycolysis too was divided into functional modules, and that one of them supported an anti-inflammatory phenotype. We validate the paradoxical prediction and demonstrate that inhibition of this module promotes a pro-inflammatory transcriptional program in Th17 cells, resulting in neuroinflammation in an adoptive transfer model of autoimmune disease. In conclusion, Compass is a widely applicable algorithm to characterize metabolic states at single cell resolution. It allows associating cellular metabolic states with effector functions and detection of metabolic targets that regulate effector phenotypes. We expect it to become a widely used tool with the increasing availability of single-cell RNA-Seq data, spurred by efforts such as the human cell atlas.
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Dai, David, Nicholas Horvath, and Jeffrey Varner. "Dynamic Sequence Specific Constraint-Based Modeling of Cell-Free Protein Synthesis." Processes 6, no. 8 (August 17, 2018): 132. http://dx.doi.org/10.3390/pr6080132.
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Cell-free protein expression has emerged as an important approach in systems and synthetic biology, and a promising technology for personalized point of care medicine. Cell-free systems derived from crude whole cell extracts have shown remarkable utility as a protein synthesis technology. However, if cell-free platforms for on-demand biomanufacturing are to become a reality, the performance limits of these systems must be defined and optimized. Toward this goal, we modeled E. coli cell-free protein expression using a sequence specific dynamic constraint-based approach in which metabolite measurements were directly incorporated into the flux estimation problem. A cell-free metabolic network was constructed by removing growth associated reactions from the iAF1260 reconstruction of K-12 MG1655 E. coli. Sequence specific descriptions of transcription and translation processes were then added to this metabolic network to describe protein production. A linear programming problem was then solved over short time intervals to estimate metabolic fluxes through the augmented cell-free network, subject to material balances, time rate of change and metabolite measurement constraints. The approach captured the biphasic cell-free production of a model protein, chloramphenicol acetyltransferase. Flux variability analysis suggested that cell-free metabolism was potentially robust; for example, the rate of protein production could be met by flux through the glycolytic, pentose phosphate, or the Entner-Doudoroff pathways. Variation of the metabolite constraints revealed central carbon metabolites, specifically upper glycolysis, tricarboxylic acid (TCA) cycle, and pentose phosphate, to be the most effective at training a predictive model, while energy and amino acid measurements were less effective. Irrespective of the measurement set, the metabolic fluxes (for the most part) remained unidentifiable. These findings suggested dynamic constraint-based modeling could aid in the design of cell-free protein expression experiments for metabolite prediction, but the flux estimation problem remains challenging. Furthermore, while we modeled the cell-free production of only a single protein in this study, the sequence specific dynamic constraint-based modeling approach presented here could be extended to multi-protein synthetic circuits, RNA circuits or even small molecule production.
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Yasukawa, Tomoyuki, Andrew Glidle, Jonathan M. Cooper, and Tomokazu Matsue. "Electroanalysis of Metabolic Flux from Single Cells in Simple Picoliter-Volume Microsystems." Analytical Chemistry 74, no. 19 (October 2002): 5001–8. http://dx.doi.org/10.1021/ac025836u.
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Güell, Oriol, Francesco Alessandro Massucci, Francesc Font-Clos, Francesc Sagués, and M. Ángeles Serrano. "Mapping high-growth phenotypes in the flux space of microbial metabolism." Journal of The Royal Society Interface 12, no. 110 (September 2015): 20150543. http://dx.doi.org/10.1098/rsif.2015.0543.
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Experimental and empirical observations on cell metabolism cannot be understood as a whole without their integration into a consistent systematic framework. However, the characterization of metabolic flux phenotypes is typically reduced to the study of a single optimal state, such as maximum biomass yield that is by far the most common assumption. Here, we confront optimal growth solutions to the whole set of feasible flux phenotypes (FFPs), which provides a benchmark to assess the likelihood of optimal and high-growth states and their agreement with experimental results. In addition, FFP maps are able to uncover metabolic behaviours, such as aerobic fermentation accompanying exponential growth on sugars at nutrient excess conditions, that are unreachable using standard models based on optimality principles. The information content of the full FFP space provides us with a map to explore and evaluate metabolic behaviour and capabilities, and so it opens new avenues for biotechnological and biomedical applications.
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Sengupta, Debanti, Amy Mongersun, Tae Jin Kim, Kellen Mongersun, Rie von Eyben, Paul Abbyad, and Guillem Pratx. "Multiplexed Single-Cell Measurements of FDG Uptake and Lactate Release Using Droplet Microfluidics." Technology in Cancer Research & Treatment 18 (January 1, 2019): 153303381984106. http://dx.doi.org/10.1177/1533033819841066.
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Introduction: Glucose utilization and lactate release are 2 important indicators of cancer metabolism. Most tumors consume glucose and release lactate at a higher rate than normal tissues due to enhanced aerobic glycolysis. However, these 2 indicators of metabolism have not previously been studied on a single-cell level, in the same cell. Objective: To develop and characterize a novel droplet microfluidic device for multiplexed measurements of glucose uptake (via its analog 18F-fluorodeoxyglucose) and lactate release, in single live cells encapsulated in an array of water-in-oil droplets. Results: Surprisingly, 18F-fluorodeoxyglucose uptake and lactate release were only marginally correlated at the single-cell level, even when assayed in a standard cell line (MDA-MB-231). While 18F-fluorodeoxyglucose-avid cells released substantial amounts of lactate, the reverse was not true, and many cells released high amounts of lactate without taking up 18F-fluorodeoxyglucose. Discussion: These results confirm that cancer cells rely on multiple metabolic pathways in addition to aerobic glycolysis and that the use of these pathways is highly heterogeneous, even under controlled culture conditions. Clinically, the large cell-to-cell variability suggests that positron emission tomography measurements of 18F-fluorodeoxyglucose uptake represent metabolic flux only in an aggregate sense, not for individual cancer cells within the tumor.
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Chung, J., R. Clifford, G. Sriram, and C. Keefer. "68 Flux analysis of aerobic glycolysis in bovine blastocysts and CT1 cells." Reproduction, Fertility and Development 31, no. 1 (2019): 159. http://dx.doi.org/10.1071/rdv31n1ab68.
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Embryo quality and maternal recognition are crucial for successful initiation of bovine pregnancy. Previous studies have proposed that better quality embryos use aerobic glycolysis to meet a high demand for biomass components. While hexoses are the principal carbon sources that provide energy to glycolysis, little is known about partitioning of hexoses into metabolic pathways or alteration of partitioning when different hexoses are simultaneously available. Specific metabolic utilisation of 13C-labelled substrates can be quantified by gas chromatography-mass spectrometry, an excellent noninvasive approach for studying cellular metabolism. To assess hexose flux through central metabolism, bovine blastocysts and CT1 cells (a bovine trophectoderm cell line) were cultured in SOF-based media supplemented with combinations of 50% uniformly labelled (U) and 50% naturally abundant (NA) glucose (Glc) or fructose (Fru) (U−13C Glc+NA Glc, U−13C Fru+NA Fru, U−13C Glc+NA Fru, and U−13C Fru+NA Glc), such that total hexose concentration was 1.5mM. Metabolites in spent media from 24-h cultures of single or 5 blastocysts (40-μL drops; 5% CO2, 5% O2, 90% N2) and 1-, 2-, 3-, 6-, 8-, and 24-h incubations of CT1 cells (150 μL; ~3×104 cells per well; 5% CO2, 95% air) were extracted with a MeOH-CHCl3 reagent, derivatized, and analysed by gas chromatography-mass spectrometry. Measurement of mass isotopomer distributions of metabolites, chiefly pyruvate, lactate, and amino acids, followed by correction for natural abundances and metabolic modelling, revealed several insights. For instance, five Day 7 or Day 8 blastocysts (Day 0=fertilization) supplied with U−13C Glc+NA Fru displayed 13C enrichments of 80.3%±1.4% for pyruvate and 71.6%±2.8% for lactate, whereas when supplied with U−13C Fru+NA Glc, they displayed lower 13C enrichments of 5.7%±2.4% for pyruvate and 2.8%±0.4% lactate (mean±standard deviation, n=3 to 4). Metabolic modelling revealed that when Glc and Fru are simultaneously available, the blastocysts used 2.5±0.2 moles of Fru per 100 moles of Glc used. Furthermore, 13C enrichment of pyruvate was 42.0±0.6% when U−13C Glc+NA Glc was supplied and 37.8±2.7% when U−13C Fru+NA Fru was supplied. Lactate enrichments followed a similar trend. This indicates that, individually, Glc and Fru were utilised majorly through aerobic glycolysis with some involvement of the pentose phosphate pathway. Alanine was negligibly labelled in all of the experiments, suggesting either a low TCA flux or that alanine is diluted by extra- or intracellular amino or fatty acids. Single blastocysts and CT1 cells showed a similar labelling pattern when hexoses were available. Following Glc depletion at 8h in CT1 cultures, the 13C enrichments of alanine and citrate in the media increased, suggesting a sharp alteration of metabolic state. These findings demonstrate that metabolic flux can be comprehensively analysed for single bovine blastocysts and CT1 cell metabolism models that of the blastocyst. This project was supported by Agriculture and Food Research Initiative Competitive Grant no. 2015-67015-23237 from the USDA National Institute of Food and Agriculture.
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De Martino, A., D. Granata, E. Marinari, C. Martelli, and V. Van Kerrebroeck. "Optimal Fluxes, Reaction Replaceability, and Response to Enzymopathies in the Human Red Blood Cell." Journal of Biomedicine and Biotechnology 2010 (2010): 1–10. http://dx.doi.org/10.1155/2010/415148.
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Characterizing the capabilities, key dependencies, and response to perturbations of genome-scale metabolic networks is a basic problem with important applications. A key question concerns the identification of the potentially most harmful reaction knockouts. The integration of combinatorial methods with sampling techniques to explore the space of viable flux states may provide crucial insights on this issue. We assess the replaceability of every metabolic conversion in the human red blood cell by enumerating the alternative paths from substrate to product, obtaining a complete map of he potential damage of single enzymopathies. Sampling the space of optimal steady state fluxes in the healthy and in the mutated cell reveals both correlations and complementarity between topologic and dynamical aspects.
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Zhang, Zhichao, Qing Zhang, Shaohua Guan, and Hualin Shi. "Quantitative Connection between Cell Size and Growth Rate by Phospholipid Metabolism." Cells 9, no. 2 (February 8, 2020): 391. http://dx.doi.org/10.3390/cells9020391.
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The processes involved in cell growth are extremely complicated even for a single cell organism such as Escherichia coli, while the relationship between growth rate and cell size is simple. We aimed to reveal the systematic link between them from the aspect of the genome-scale metabolic network. Since the growth rate reflects metabolic rates of bacteria and the cell size relates to phospholipid synthesis, a part of bacterial metabolic networks, we calculated the cell length from the cardiolipin synthesis rate, where the cardiolipin synthesis reaction is able to represent the phospholipid metabolism of Escherichia coli in the exponential growth phase. Combined with the flux balance analysis, it enables us to predict cell length and to examine the quantitative relationship between cell length and growth rate. By simulating bacteria growing in various nutrient media with the flux balance analysis and calculating the corresponding cell length, we found that the increase of the synthesis rate of phospholipid, the cell width, and the protein fraction in membranes caused the increase of cell length with growth rate. Different tendencies of phospholipid synthesis rate changing with growth rate result in different relationships between cell length and growth rate. The effects of gene deletions on cell size and growth rate are also examined. Knocking out the genes, such as
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tktA,
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yqaB,
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pgm, and
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cysQ, affects growth rate largely while affecting cell length slightly. Results of this method are in good agreement with experiments.
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Sarkar, Debolina, Marine Landa, Anindita Bandyopadhyay, Himadri B. Pakrasi, Jonathan P. Zehr, and Costas D. Maranas. "Elucidation of trophic interactions in an unusual single-cell nitrogen-fixing symbiosis using metabolic modeling." PLOS Computational Biology 17, no. 5 (May 7, 2021): e1008983. http://dx.doi.org/10.1371/journal.pcbi.1008983.
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Marine nitrogen-fixing microorganisms are an important source of fixed nitrogen in oceanic ecosystems. The colonial cyanobacterium Trichodesmium and diatom symbionts were thought to be the primary contributors to oceanic N2 fixation until the discovery of the unusual uncultivated symbiotic cyanobacterium UCYN-A (Candidatus Atelocyanobacterium thalassa). UCYN-A has atypical metabolic characteristics lacking the oxygen-evolving photosystem II, the tricarboxylic acid cycle, the carbon-fixation enzyme RuBisCo and de novo biosynthetic pathways for a number of amino acids and nucleotides. Therefore, it is obligately symbiotic with its single-celled haptophyte algal host. UCYN-A receives fixed carbon from its host and returns fixed nitrogen, but further insights into this symbiosis are precluded by both UCYN-A and its host being uncultured. In order to investigate how this syntrophy is coordinated, we reconstructed bottom-up genome-scale metabolic models of UCYN-A and its algal partner to explore possible trophic scenarios, focusing on nitrogen fixation and biomass synthesis. Since both partners are uncultivated and only the genome sequence of UCYN-A is available, we used the phylogenetically related Chrysochromulina tobin as a proxy for the host. Through the use of flux balance analysis (FBA), we determined the minimal set of metabolites and biochemical functions that must be shared between the two organisms to ensure viability and growth. We quantitatively investigated the metabolic characteristics that facilitate daytime N2 fixation in UCYN-A and possible oxygen-scavenging mechanisms needed to create an anaerobic environment to allow nitrogenase to function. This is the first application of an FBA framework to examine the tight metabolic coupling between uncultivated microbes in marine symbiotic communities and provides a roadmap for future efforts focusing on such specialized systems.
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Cook, Daniel J., John Whitman, Nicole Liadis, and John Cole. "Abstract P1-05-07: Spatially-resolved single-cell tumor heterogeneity captured by TumorScope biophysical modeling software using MR Imaging." Cancer Research 82, no. 4_Supplement (February 15, 2022): P1–05–07—P1–05–07. http://dx.doi.org/10.1158/1538-7445.sabcs21-p1-05-07.
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Abstract Background: Dysregulated cellular metabolism is a hallmark of breast cancer, and targeting it has promising implications for improving care and patient outcomes. Specifically, heterogeneity in tumor metabolism is thought to play a role in determining chemotherapy response, the development of resistance, and promoting metastastasis. Despite this, metabolic tumor heterogeneity for individual breast cancer patients has not been characterized completely. Methods: In this study, we used state-of-the-art techniques to characterize metabolic heterogeneity within individual patient tumors by integrating single cell RNA-seq data with genome-scale metabolic modeling. Using SimBioSys’ TumorScope - a commercially available biophysical modeling platform, we compared intra-tumoral metabolic heterogeneity from experimental single cell RNA-seq data to simulated intra tumoral heterogeneity. Results: Using single cell RNA-seq data, we found that intra-tumoral gradients in nutrient availability are widely present within patient tumors (for a single luminal A patient, glucose import flux ranged from 0.19 - 1.25 g/gDW/day, while glutathione import ranged from 0.004 - 0.054 g/gDW/day). We also found that these gradients lead to cellular growth rate gradients within individual tumors (for our representative patient, median SGR = 0.62 %/day +/- 0.33 %/day stdev). Using TumorScope, we found this same gradient behavior within patient tumors. Selecting a similarly growing luminal A patient from our TumorScope simulations resulted in gradients in glucose import (range = 0.17 - 1.26 g/gDW/day), glutathione import (range = 0.024 - 0.058 g/gDW/day), and tumor SGR (median = 0.40 %/day, stdev = 0.42 %/day), which closely match metabolism from single cells (comparing maximum-scaled SGR distributions between single cells and TumorScope yielded a p-value = 0.10). We next examined which nutrients govern heterogeneity in tumor SGR. We found that glucose availability with the tumor microenvironment is more limiting to cell growth than oxygen availability, and this result was consistent between metabolic profiles from both single cell RNA-seq data and TumorScope simulations. TumorScope’s spatially resolved simulations offered the additional insight that gradients in nutrient availability are caused by heterogeneity in the distribution of macro- and micro-vasculature and the composition of the tumor microenvironment. We then used data reduction techniques to compare populations of single cells with differing metabolic phenotypes to identify molecular behavior at the single cells in higher molecular resolution. We found that single cells collected from the clinic co-cluster with single cells from TumorScope simulations, suggesting that a significant amount of intra-tumoral metabolic heterogeneity observed in patients is captured by TumorScope simulations. Conclusion: Accessing tumor heterogeneity has traditionally required specialized equipment, analytic expertise, and invasive procedures, largely limiting its study to large, academic hospitals. Currently, metabolic heterogeneity is only understood in 2D and for few markers (using pathology slides) or in relatively few cells with little or no spatial resolution (for single cell RNA-seq). TumorScope provides a novel approach to simulate metabolic heterogeneity at the single cell scale in 3D across a whole tumor. TumorScope democratizes the study of tumor heterogeneity by making it accessible to clinicians and researchers from MRI data alone. TumorScope has the capability to capture tumor metabolic heterogeneity at a higher scale than previously achievable which will allow for a dramatic increase in our understanding of tumor biology and ultimately improve clinical decision making. Citation Format: Daniel J Cook, John Whitman, Nicole Liadis, John Cole. Spatially-resolved single-cell tumor heterogeneity captured by TumorScope biophysical modeling software using MR Imaging [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-05-07.
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Lavoie, Michel, Blanche Saint-Béat, Jan Strauss, Sébastien Guérin, Antoine Allard, Simon V. Hardy, Angela Falciatore, and Johann Lavaud. "Genome-Scale Metabolic Reconstruction and in Silico Perturbation Analysis of the Polar Diatom Fragilariopsis cylindrus Predicts High Metabolic Robustness." Biology 9, no. 2 (February 17, 2020): 30. http://dx.doi.org/10.3390/biology9020030.
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Diatoms are major primary producers in polar environments where they can actively grow under extremely variable conditions. Integrative modeling using a genome-scale model (GSM) is a powerful approach to decipher the complex interactions between components of diatom metabolism and can provide insights into metabolic mechanisms underlying their evolutionary success in polar ecosystems. We developed the first GSM for a polar diatom, Fragilariopsis cylindrus, which enabled us to study its metabolic robustness using sensitivity analysis. We find that the predicted growth rate was robust to changes in all model parameters (i.e., cell biochemical composition) except the carbon uptake rate. Constraints on total cellular carbon buffer the effect of changes in the input parameters on reaction fluxes and growth rate. We also show that single reaction deletion of 20% to 32% of active (nonzero flux) reactions and single gene deletion of 44% to 55% of genes associated with active reactions affected the growth rate, as well as the production fluxes of total protein, lipid, carbohydrate, DNA, RNA, and pigments by less than 1%, which was due to the activation of compensatory reactions (e.g., analogous enzymes and alternative pathways) with more highly connected metabolites involved in the reactions that were robust to deletion. Interestingly, including highly divergent alleles unique for F. cylindrus increased its metabolic robustness to cellular perturbations even more. Overall, our results underscore the high robustness of metabolism in F. cylindrus, a feature that likely helps to maintain cell homeostasis under polar conditions.
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Keuls, Rachel A., Karin Kojima, Brittney Lozzi, John W. Steele, Qiuying Chen, Steven S. Gross, Richard H. Finnell, and Ronald J. Parchem. "MiR-302 Regulates Glycolysis to Control Cell-Cycle during Neural Tube Closure." International Journal of Molecular Sciences 21, no. 20 (October 13, 2020): 7534. http://dx.doi.org/10.3390/ijms21207534.
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Neural tube closure is a critical early step in central nervous system development that requires precise control of metabolism to ensure proper cellular proliferation and differentiation. Dysregulation of glucose metabolism during pregnancy has been associated with neural tube closure defects (NTDs) in humans suggesting that the developing neuroepithelium is particularly sensitive to metabolic changes. However, it remains unclear how metabolic pathways are regulated during neurulation. Here, we used single-cell mRNA-sequencing to analyze expression of genes involved in metabolism of carbon, fats, vitamins, and antioxidants during neurulation in mice and identify a coupling of glycolysis and cellular proliferation to ensure proper neural tube closure. Using loss of miR-302 as a genetic model of cranial NTD, we identify misregulated metabolic pathways and find a significant upregulation of glycolysis genes in embryos with NTD. These findings were validated using mass spectrometry-based metabolite profiling, which identified increased glycolytic and decreased lipid metabolites, consistent with a rewiring of central carbon traffic following loss of miR-302. Predicted miR-302 targets Pfkp, Pfkfb3, and Hk1 are significantly upregulated upon NTD resulting in increased glycolytic flux, a shortened cell cycle, and increased proliferation. Our findings establish a critical role for miR-302 in coordinating the metabolic landscape of neural tube closure.
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Fan, Yang-Yi, Laurie A. Davidson, Evelyn S. Callaway, Gus A. Wright, Stephen Safe, and Robert S. Chapkin. "A bioassay to measure energy metabolism in mouse colonic crypts, organoids, and sorted stem cells." American Journal of Physiology-Gastrointestinal and Liver Physiology 309, no. 1 (July 1, 2015): G1—G9. http://dx.doi.org/10.1152/ajpgi.00052.2015.
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Evidence suggests that targeting cancer cell energy metabolism might be an effective therapeutic approach for selective ablation of malignancies. Using a Seahorse Extracellular Flux Analyzer, we have demonstrated that select environmental agents can alter colonic mitochondrial function by increasing respiration-induced proton leak, thereby inducing apoptosis, a marker of colon cancer risk. To further probe bioenergetics in primary intestinal cells, we developed methodology that can be modified and adapted to measure the bioenergetic profiles of colonic crypts, the basic functional unit of the colon, and colonic organoids, an ex vivo 3D culture of colonic crypts. Furthermore, in combination with the MoFlo Astrios High-Speed Cell Sorter, we were able to measure the bioenergetic profiles of colonic adult stem and daughter cells from Lgr5-EGFP-IRES-creERT2 transgenic mice. We examined the effects of 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD), a full arylhydrocarbon receptor agonist, known to affect gastrointestinal function and cancer risk, on the bioenergetic profiles of intestinal epithelial cells. Mouse colonic crypts, organoids, or sorted single cells were seeded onto Matrigel-precoated Seahorse XF24 microplates for extracellular flux analysis. Temporal analyses revealed distinct energy metabolic profiles in crypts and organoids challenged with TCDD. Furthermore, sorted Lgr5+ stem cells exhibited a Warburg-like metabolic profile. This is noteworthy because perturbations in stem cell dynamics are generally believed to represent the earliest step toward colon tumorigenesis. We propose that our innovative methodology may facilitate future in vivo/ex vivo metabolic studies using environmental agents affecting colonocyte energy metabolism.
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Agius, Loranne, Josep Centelles, and Marta Cascante. "Multiple glucose 6-phosphate pools or channelling of flux in diverse pathways?" Biochemical Society Transactions 30, no. 2 (April 1, 2002): 38–43. http://dx.doi.org/10.1042/bst0300038.
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Glucose 6-phosphate is an intermediate of pathways of glucose utilization and production as well as a regulator of enzyme activity and gene expression. Studies on the latter functions are in part based on measurement of the glucose 6-phosphate content in a whole-cell extract. Several studies have suggested that there are multiple subcellular pools of glucose 6-phosphate. It is proposed that this data can be interpreted in terms of channelling of metabolic intermediates through multiple pathways of glucose metabolism with leakage of glucose 6-phosphate from the channels into a single free pool. It is also proposed that measurement of total tissue content of glucose 6-phosphate approximates the free pool.
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Covert, Markus W., Nan Xiao, Tiffany J. Chen, and Jonathan R. Karr. "Integrating metabolic, transcriptional regulatory and signal transduction models in Escherichia coli." Bioinformatics 24, no. 18 (July 10, 2008): 2044–50. http://dx.doi.org/10.1093/bioinformatics/btn352.
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AbstractMotivation: The effort to build a whole-cell model requires the development of new modeling approaches, and in particular, the integration of models for different types of processes, each of which may be best described using different representation. Flux-balance analysis (FBA) has been useful for large-scale analysis of metabolic networks, and methods have been developed to incorporate transcriptional regulation (regulatory FBA, or rFBA). Of current interest is the integration of these approaches with detailed models based on ordinary differential equations (ODEs).Results: We developed an approach to modeling the dynamic behavior of metabolic, regulatory and signaling networks by combining FBA with regulatory Boolean logic, and ordinary differential equations. We use this approach (called integrated FBA, or iFBA) to create an integrated model of Escherichia coli which combines a flux-balance-based, central carbon metabolic and transcriptional regulatory model with an ODE-based, detailed model of carbohydrate uptake control. We compare the predicted Escherichia coli wild-type and single gene perturbation phenotypes for diauxic growth on glucose/lactose and glucose/glucose-6-phosphate with that of the individual models. We find that iFBA encapsulates the dynamics of three internal metabolites and three transporters inadequately predicted by rFBA. Furthermore, we find that iFBA predicts different and more accurate phenotypes than the ODE model for 85 of 334 single gene perturbation simulations, as well for the wild-type simulations. We conclude that iFBA is a significant improvement over the individual rFBA and ODE modeling paradigms.Availability: All MATLAB files used in this study are available at http://www.simtk.org/home/ifba/.Contact: covert@stanford.eduSupplementary information: Supplementary data are available at Bioinformatics online.
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Pareek, Vidhi, Hua Tian, Nicholas Winograd, and Stephen J. Benkovic. "Metabolomics and mass spectrometry imaging reveal channeled de novo purine synthesis in cells." Science 368, no. 6488 (April 16, 2020): 283–90. http://dx.doi.org/10.1126/science.aaz6465.
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Metabolons, multiprotein complexes consisting of sequential enzymes of a metabolic pathway, are proposed to be biosynthetic “hotspots” within the cell. However, experimental demonstration of their presence and functions has remained challenging. We used metabolomics and in situ three-dimensional submicrometer chemical imaging of single cells by gas cluster ion beam secondary ion mass spectrometry (GCIB-SIMS) to directly visualize de novo purine biosynthesis by a multienzyme complex, the purinosome. We found that purinosomes comprise nine enzymes that act synergistically, channeling the pathway intermediates to synthesize purine nucleotides, increasing the pathway flux, and influencing the adenosine monophosphate/guanosine monophosphate ratio. Our work also highlights the application of high-resolution GCIB-SIMS for multiplexed biomolecular analysis at the level of single cells.
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Banerjee, Arindam, Charles N. Birts, Matthew Darley, Rachel Parker, Alex H. Mirnezami, Jonathan West, Ramsey I. Cutress, Stephen A. Beers, Matthew J. J. Rose-Zerilli, and Jeremy P. Blaydes. "Stem cell-like breast cancer cells with acquired resistance to metformin are sensitive to inhibitors of NADH-dependent CtBP dimerization." Carcinogenesis 40, no. 7 (January 22, 2019): 871–82. http://dx.doi.org/10.1093/carcin/bgy174.
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AbstractAltered flux through major metabolic pathways is a hallmark of cancer cells and provides opportunities for therapy. Stem cell-like cancer (SCLC) cells can cause metastasis and therapy resistance. They possess metabolic plasticity, theoretically enabling resistance to therapies targeting a specific metabolic state. The C-terminal binding protein (CtBP) transcriptional regulators are potential therapeutic targets in highly glycolytic cancer cells, as they are activated by the glycolytic coenzyme nicotinamide adenine dinucleotide (NADH). However, SCLC cells commonly exist in an oxidative state with low rates of glycolysis. Metformin inhibits complex I of the mitochondrial electron transport chain; it can kill oxidative SCLC cells and has anti-cancer activity in patients. SCLC cells can acquire resistance to metformin through increased glycolysis. Given the potential for long-term metformin therapy, we have studied acquired metformin resistance in cells from the claudin-low subtype of breast cancer. Cells cultured for 8 weeks in sub-IC50 metformin concentration proliferated comparably to untreated cells and exhibited higher rates of glucose uptake. SCLC cells were enriched in metformin-adapted cultures. These SCLC cells acquired sensitivity to multiple methods of inhibition of CtBP function, including a cyclic peptide inhibitor of NADH-induced CtBP dimerization. Single-cell mRNA sequencing identified a reprogramming of epithelial–mesenchymal and stem cell gene expression in the metformin-adapted SCLC cells. These SCLC cells demonstrated an acquired dependency on one of these genes, Tenascin C. Thus, in addition to acquisition of sensitivity to glycolysis-targeting therapeutic strategies, the reprograming of gene expression in the metformin-adapted SCLC cells renders them sensitive to potential therapeutic approaches not directly linked to cell metabolism.
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Heng, Jacob S., Amir Rattner, Genevieve L. Stein-O’Brien, Briana L. Winer, Bryan W. Jones, Hilary J. Vernon, Loyal A. Goff, and Jeremy Nathans. "Hypoxia tolerance in the Norrin-deficient retina and the chronically hypoxic brain studied at single-cell resolution." Proceedings of the National Academy of Sciences 116, no. 18 (April 15, 2019): 9103–14. http://dx.doi.org/10.1073/pnas.1821122116.
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The mammalian CNS is capable of tolerating chronic hypoxia, but cell type-specific responses to this stress have not been systematically characterized. In the Norrin KO (NdpKO) mouse, a model of familial exudative vitreoretinopathy (FEVR), developmental hypovascularization of the retina produces chronic hypoxia of inner nuclear-layer (INL) neurons and Muller glia. We used single-cell RNA sequencing, untargeted metabolomics, and metabolite labeling from 13C-glucose to compare WT and NdpKO retinas. In NdpKO retinas, we observe gene expression responses consistent with hypoxia in Muller glia and retinal neurons, and we find a metabolic shift that combines reduced flux through the TCA cycle with increased synthesis of serine, glycine, and glutathione. We also used single-cell RNA sequencing to compare the responses of individual cell types in NdpKO retinas with those in the hypoxic cerebral cortex of mice that were housed for 1 week in a reduced oxygen environment (7.5% oxygen). In the hypoxic cerebral cortex, glial transcriptome responses most closely resemble the response of Muller glia in the NdpKO retina. In both retina and brain, vascular endothelial cells activate a previously dormant tip cell gene expression program, which likely underlies the adaptive neoangiogenic response to chronic hypoxia. These analyses of retina and brain transcriptomes at single-cell resolution reveal both shared and cell type-specific changes in gene expression in response to chronic hypoxia, implying both shared and distinct cell type-specific physiologic responses.
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Delgoffe, Greg M. "Abstract IA09: Tumor cell oxidative metabolism as a barrier to antitumor immunity in melanoma." Cancer Research 80, no. 19_Supplement (October 1, 2020): IA09. http://dx.doi.org/10.1158/1538-7445.mel2019-ia09.
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Abstract The treatment of melanoma has been fundamentally changed by immunotherapies like blockade of coinhibitory “checkpoint” molecules like CTLA-4 and PD-1. However, despite these remarkable successes, even within T cell-inflamed, high-mutational-burden tumors, response rates remain low, likely due to additional immune inhibitory mechanisms present in the tumor microenvironment. It is now generally appreciated that the tumor microenvironment is characterized by a distinct metabolic landscape, having low oxygen tension, depleted levels of essential nutrients, and buildup of toxic byproducts. We sought to determine whether this metabolic landscape was variable from patient to patient and how this landscape affected antitumor immunity and thus response to immunotherapy. Using extracellular metabolic flux analysis, we profiled the metabolic derangement of patient-derived cell lines as well as direct ex vivo patient tumor samples, revealing striking heterogeneity not only in the degree of metabolic derangement but also in the type of metabolism a tumor cell was dependent on. Analysis of the tumor infiltrate revealed that a tumor’s preference for oxidative metabolism was associated with immune dysfunction, suggesting that the ability to generate hypoxic regions in vivo is a key aspect of immune resistance. We confirmed this finding in vivo using a novel model of melanoma in which individual metabolic pathways can be selectively targeted in a single-cell derived line: loss of oxidative metabolism in tumor cells sensitizes animals to PD-1 blockade immunotherapy, while loss of glycolytic metabolism has no appreciable effect on antitumor immunity. Analysis of melanoma patient biopsy samples prior to initiation of immunotherapy revealed that increased tumor oxidative metabolism and intratumoral hypoxia was associated with poorer responses to checkpoint blockade. Our data highlight the use of tumor microenvironment hypoxia as a potential predictor of immunotherapy resistance and shed light on the use of modulators of tumor cell oxidation as a means to improve responses to checkpoint blockade. Citation Format: Greg M. Delgoffe. Tumor cell oxidative metabolism as a barrier to antitumor immunity in melanoma [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr IA09.
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Choi, Soohee, Suree Kim, Jiyoung Park, Seung Eun Lee, Chaewon Kim, and Dongmin Kang. "Diclofenac: A Nonsteroidal Anti-Inflammatory Drug Inducing Cancer Cell Death by Inhibiting Microtubule Polymerization and Autophagy Flux." Antioxidants 11, no. 5 (May 20, 2022): 1009. http://dx.doi.org/10.3390/antiox11051009.
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Diclofenac, a nonsteroidal anti-inflammatory drug (NSAID) used to treat inflammatory diseases induces cellular toxicity by increasing the production of reactive oxygen species (ROS) and impairing autophagic flux. In this study, we investigated whether diclofenac induces cancer cell death and the mechanism by which diclofenac causes cell death. We observed that diclofenac induces mitotic arrest with a half-maximal effective concentration of 170 μM and cell death with a half-maximal lethal dose of 200 µM during 18-h incubation in HeLa cells. Cellular microtubule imaging and in vitro tubulin polymerization assays demonstrated that treatment with diclofenac elicits microtubule destabilization. Autophagy relies on microtubule-mediated transport and the fusion of autophagic vesicles. We observed that diclofenac inhibits both phagophore movement, an early step of autophagy, and the fusion of autophagosomes and lysosomes, a late step of autophagy. Diclofenac also induces the fragmentation of mitochondria and the Golgi during cell death. We found that diclofenac induces cell death further in combination with 5-fuorouracil, a DNA replication inhibitor than in single treatment in cancer cells. Pancreatic cancer cells, which have high basal autophagy, are particularly sensitive to cell death by diclofenac. Our study suggests that microtubule destabilization by diclofenac induces cancer cell death via compromised spindle assembly checkpoints and increased ROS through impaired autophagy flux. Diclofenac may be a candidate therapeutic drug in certain type of cancers by inhibiting microtubule-mediated cellular events in combination with clinically utilized nucleoside metabolic inhibitors, including 5-fluorouracil, to block cancer cell proliferation.
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Zhang, Yiru, Trang Nguyen, Junfei Zhao, Enyuan Shang, Consuelo Torrini, Peter D. Canoll, Georg Karpel-Massler, and Markus Siegelin. "CBMT-15. MET INHIBITION DRIVES PGC1A DEPENDENT METABOLIC REPROGRAMMING AND ELICITS UNIQUE METABOLIC VULNERABILITIES IN GLIOBLASTOMA." Neuro-Oncology 21, Supplement_6 (November 2019): vi36. http://dx.doi.org/10.1093/neuonc/noz175.137.
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Abstract The receptor kinase, c-MET, has emerged as a target for glioblastoma therapy. However, treatment resistance evolves inevitably. By performing a global metabolite screen with metabolite set enrichment coupled with transcriptome and gene set enrichment analysis and proteomic screening, we have identified substantial reprogramming of tumor metabolism, involving oxidative phosphorylation and fatty acid oxidation (FAO) with a substantial accumulation of acyl-carnitines accompanied by an increase of PGC1a in response to genetic (shRNA and CRISPR/Cas9) and pharmacological (crizotinib) inhibition of c-MET. Extracellular flux and carbon tracing analyses (U-13C-Glucose and U-13C-Glutamine) demonstrated enhanced oxidative metabolism, which was driven by FAO and supported by increased anaplerosis of glucose carbons. These findings were observed in concert with increased number and fusion of mitochondria and production of reactive oxygen species (ROS). Genetic interference with PGC1a rescued this oxidative phenotype driven by c-MET inhibition. Silencing and chromatin immunoprecipitation experiments demonstrated that CREB regulates the expression of PGC1a in the context of c-MET inhibition. Interference with both oxidative phosphorylation (metformin, oligomycin) and beta-oxidation of fatty acids (etomoxir) enhanced the anti-tumor efficacy of c-MET inhibition. Moreover, based on a high-throughput drug screen, we show that gamitrinib along with c-MET inhibition results in synergistic cell death. Finally, utilizing patient-derived xenograft models, we provide evidence that the combination treatments (crizotinib+etomoxir and crizotinib+gamitrinib) were significantly more efficacious than single treatment without induction of toxicity. Collectively, we have unraveled the mechanistic underpinnings of c-MET inhibitor treatment and identified novel combination therapies that may enhance the therapeutic efficacy of c-MET inhibition.
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Oshima, Nobu, Yuki Aisu, Shigeo Hisamori, Shigeru Tsunoda, Hiroki Hashida, Kenji Uryuhara, Hiroyuki Kobayashi, et al. "Abstract 6222: Comprehensive monitoring of pyruvate metabolism in cancer cells and tumors reveals vulnerability to metabolic inhibition therapy with small molecules." Cancer Research 82, no. 12_Supplement (June 15, 2022): 6222. http://dx.doi.org/10.1158/1538-7445.am2022-6222.
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Abstract Introduction: Pyruvate(Pyr) metabolism is a keystone in cancer metabolism, as well as normal cells, and has two major metabolic fluxes; Glycolysis and Oxidative Phosphorylation (OXPHOS). Especially in cancer cells, increased Pyr metabolism meets the demand of higher energy consumption during rapid cell growing state. Therefore, monitoring tumor Pyr metabolic flux in vitro and in vivo should be necessary to develop a therapeutic strategy using small molecules which can inhibit the fluxes.Aim: We performed in vitro metabolic analysis on pancreatic cancer cells (MiaPaCa2) and applied a novel in vivo imaging technology “hyperpolarized 13C-pyruvate magnetic resonance spectroscopic imaging (HP-MRSI)” to dynamic monitoring of the impact of Glycolysis and OXPHOS inhibitors on the MiaPaCa2 xenografts. Methods: We used a novel LDH inhibitor “NCI-006” for Glycolysis inhibition and Mitochondrial Complex 1(MC1) Inhibitor "Metformin(Met)", which is already clinically available, and “IACS-010759(IACS)” for OXPHOS inhibition, in vitro and in vivo. Extracellular flux (EXF) analysis and HP-MRSI were performed before and after administration of each or both to assess inhibitor impact on metabolic flux in vitro and in vivo, respectively. Results: HP-MRSI confirmed that LDH activity in the tumor was suppressed by NCI-006 (83.3±4.4% decrease) and accelerated by Met (69.9±6.6% increase) and IACS (88.6±25.1% increase). We confirmed these in vivo observations are fully consistent with the effect of those inhibitors in vitro on the energy profile of MiaPaCa2, and also the close correlation of these data with the results of the ex vivo LDH activity assay, suggesting HP-MRSI can reliably monitor in vivo on target effects of the inhibitors without need for tissue sampling. In addition, combined treatment with the NCI-006 and MC1 inhibitor significantly suppressed in vitro cell growth and tumor growth in an efficacy study of MiaPaCa2 xenografts, compared to each single administration. Apoptosis assay showed the combined treatment induced apoptosis in MIA Paca2 cells. Conclusion: Using EXF analyzer and HP-MRSI revealed that Glycolysis inhibition redirects tumor Pyr toward OXPHOS and also MC1 inhibition redirects tumor Pyr toward Glycolysis. Combination therapy suppresses metabolic plasticity, causing metabolic quiescence in vitro and tumor growth inhibition in vivo. HP-MRSI is thought to be useful for pancreatic cancer patients to establish the current treatment model because of no need to obtain clinical samples, such as patients-derived cancer cells or spheroid from the patients. The current proof of concept can be of great value in developing new therapeutic strategies using metabolic inhibitors to treat cancers. Citation Format: Nobu Oshima, Yuki Aisu, Shigeo Hisamori, Shigeru Tsunoda, Hiroki Hashida, Kenji Uryuhara, Hiroyuki Kobayashi, Masato Kondo, koji Kitamura, Satoshi Kaihara, Kazutaka Obama, Krishna Murali, Len Neckers. Comprehensive monitoring of pyruvate metabolism in cancer cells and tumors reveals vulnerability to metabolic inhibition therapy with small molecules [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6222.
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Land, S. C., R. H. Sanger, and P. J. S. Smith. "O2 availability modulates transmembrane Ca2+ flux via second-messenger pathways in anoxia-tolerant hepatocytes." Journal of Applied Physiology 82, no. 3 (March 1, 1997): 776–83. http://dx.doi.org/10.1152/jappl.1997.82.3.776.
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Land, S. C., R. H. Sanger, and P. J. S. Smith.O2 availability modulates transmembrane Ca2+ flux via second-messenger pathways in anoxia-tolerant hepatocytes. J. Appl. Physiol. 82(3): 776–783, 1997.—Transmembrane Ca2+-flux was studied from single isolated turtle hepatocytes by using a noninvasive Ca2+-selective self-referencing microelectrode. Cells in Ca2+-reduced culture medium demonstrated a vanadate-and lanthanum-inhibitable Ca2+-efflux of 4 × 10−17 mol Ca2+ ⋅ μm−2 ⋅ s−1continuously over 170 h. This flux diminished with 50 nM phorbol 12-myristate 13-acetate, a protein kinase C (PKC) activator, and was reinstated on PKC deactivation with sphingosine. Progressive hypoxia resulted in a reversible suppression of Ca2+ efflux to 90% of normoxic controls with an apparent Michaelis constant for oxygen of 145 μM. PKC activation was critical in this suppression, as anaerobic administration of sphingosine caused a Ca2+ influx and cell rupture. Hypoxia was also associated with an altered pattern of adenosine-mediated control over Ca2+ efflux. Adenosine (100 μM) elevated Ca2+ efflux twofold in normoxia, but neither adenosine nor the A1-purinoreceptor antagonist 8-phenyltheophylline altered the observed anaerobic suppression. Aerobic administration of 2–10 mM KCN failed to reproduce the anaerobic suppression; however, in conjunction with 10 mM iodoacetate, complete metabolic blockade caused a Ca2+ influx and cell rupture. These observations suggest modulatory control by oxygen over transmembrane Ca2+ efflux involving second-messenger systems in the hypoxic transition.
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Gwon, Hyeokjo, Kitae Park, Soon-Chun Chung, Ryoung-Hee Kim, Jin Kyu Kang, Sang Min Ji, Nag-Jong Kim, et al. "A safe and sustainable bacterial cellulose nanofiber separator for lithium rechargeable batteries." Proceedings of the National Academy of Sciences 116, no. 39 (September 9, 2019): 19288–93. http://dx.doi.org/10.1073/pnas.1905527116.
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Bacterial cellulose nanofiber (BCNF) with high thermal stability produced by an ecofriendly process has emerged as a promising solution to realize safe and sustainable materials in the large-scale battery. However, an understanding of the actual thermal behavior of the BCNF in the full-cell battery has been lacking, and the yield is still limited for commercialization. Here, we report the entire process of BCNF production and battery manufacture. We systematically constructed a strain with the highest yield (31.5%) by increasing metabolic flux and improved safety by introducing a Lewis base to overcome thermochemical degradation in the battery. This report will open ways of exploiting the BCNF as a “single-layer” separator, a good alternative to the existing chemical-derived one, and thus can greatly contribute to solving the environmental and safety issues.
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Mun, Edward C., Kevin J. Tally, and Jeffrey B. Matthews. "Characterization and regulation of adenosine transport in T84 intestinal epithelial cells." American Journal of Physiology-Gastrointestinal and Liver Physiology 274, no. 2 (February 1, 1998): G261—G269. http://dx.doi.org/10.1152/ajpgi.1998.274.2.g261.
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Adenosine release from mucosal sources during inflammation and ischemia activates intestinal epithelial Cl−secretion. Previous data suggest that A2b receptor-mediated Cl− secretory responses may be dampened by epithelial cell nucleoside scavenging. The present study utilizes isotopic flux analysis and nucleoside analog binding assays to directly characterize the nucleoside transport system of cultured T84 human intestinal epithelial cells and to explore whether adenosine transport is regulated by secretory agonists, metabolic inhibition, or phorbol ester. Uptake of adenosine across the apical membrane displayed characteristics of simple diffusion. Kinetic analysis of basolateral uptake revealed a Na+-independent, nitrobenzylthioinosine (NBTI)-sensitive facilitated-diffusion system with low affinity but high capacity for adenosine. NBTI binding studies indicated a single population of high-affinity binding sites basolaterally. Neither forskolin, 5′-( N-ethylcarboxamido)-adenosine, nor metabolic inhibition significantly altered adenosine transport. However, phorbol 12-myristate 13-acetate significantly reduced both adenosine transport and the number of specific NBTI binding sites, suggesting that transporter number may be decreased through activation of protein kinase C. This basolateral facilitated adenosine transporter may serve a conventional function in nucleoside salvage and a novel function as a regulator of adenosine-dependent Cl− secretory responses and hence diarrheal disorders.
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Bayley, Nicholas, Christopher Tse, Henan Zhu, Lisa Ta, Lynn Baufeld, Laura Gosa, Weihong Yan, et al. "EPCO-10. INTRATUMORAL HETEROGENEITY OF ENVIRONMENT-INDUCED EXPRESSION PROGRAMS IN GLIOMA PATIENTS AND DERIVED MODEL SYSTEMS." Neuro-Oncology 23, Supplement_6 (November 2, 2021): vi3. http://dx.doi.org/10.1093/neuonc/noab196.009.
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Abstract Bulk tumor and single-cell RNA sequencing have revealed the remarkable molecular heterogeneity and plasticity of gliomas. This has led to the creation of tumor subtypes and cellular states describing inter- and intra-tumoral heterogeneity, respectively. While there has been great interest in creating and revising new classifications, the biological reasons for this degree of heterogeneity and the selective pressures driving it remain to be fully described. The brain tumor microenvironment (TME) is a complex, regionally heterogeneous ecosystem of communicating normal and malignant cell types and scavenge-able nutrients and metabolites. We hypothesized that distinct cellular interactions and metabolic flux in the TME may drive the inter- and intra-tumoral heterogeneity of gliomas. To identify tumorigenic programs impacted by environmental context we performed bulk RNA sequencing of over 35 triplets of patient glioma samples and their matched derivative models established in direct orthotopic mouse xenografts (DPDOX) and conventional gliomasphere cultures (GS). This analysis revealed environment-specific programs including in vivo immune and neuroglial signaling, in vitro lipid metabolism, and cell migration altered in model systems. These environmental programs are enriched in specific tumor subtypes and neuroglial signaling programs lost in vitro are dynamically upregulated upon re-transplantation in vivo. To further investigate associations between tumor cellular state and environment-driven programs we performed single-cell RNA sequencing of 3 patient and model system triplets. By annotating with brain cell atlases from previous single-cell characterizations, 6 major clusters and over 20 sub-clusters of tumor cell states and hybrid states were identified. Overlaying results from bulk sequencing revealed cell state-specific expression of environment-induced programs. Further, these cellular state “niches” diverge in model environments suggesting that environmental factors modulate not only the composition of cellular states but also their biological roles within gliomas.
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Rashidi, Aida, Alex Cordero, Brandyn Castro, David Hou, Mark Dapash, Peng Zhang, Yu Han, et al. "TAMI-25. UPREGULATION OF CREATINE METABOLISM BY MYELOID CELLS RESULTS IN GLIOBLASTOMA PROGRESSION." Neuro-Oncology 23, Supplement_6 (November 2, 2021): vi203. http://dx.doi.org/10.1093/neuonc/noab196.809.
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Abstract Malignant brain tumors are uniquely immunosuppressive, with a predominant infiltration of immunosuppressive tumor-associated myeloid cells (TAMCs) and a deficit in T-cells unrivaled to any other tumor. This unique tumor microenvironment (TME) promotes resistance to both conventional and immune therapies for this disease. The underlying mechanisms by which TAMCs promote glioblastoma (GBM) progression are not fully understood. We found that TAMCs specifically upregulate de-novo creatine metabolism within GBM using unbiased genetic and metabolic screening. This metabolic phenotype was confirmed in human GBM patients by comparing peripheral versus tumor-infiltrating myeloid cells. Examination of de-novo creatine generation using Carbon13 arginine flux revealed that TAMCs, but not tumor-infiltrating CD8+ T-cells, can produce creatine. Furthermore, we demonstrate that TAMCs actively secrete de-novo generated creatine into cell cultures. Examination of the single-cell microenvironment of GBM revealed that malignant cells preferentially express the creatine transporter, indicating that TAMC-derived creatine is taken up by GBM. Notably, SLC6A8 is directly upregulated in the context of hypoxia and suggests that creatine uptake is a mechanism to promote survival under hypoxic stress. Indeed, exogenous creatine supplementation promoted both the migration and survival of multiple glioblastoma cell lines in-vitro. Utilizing an established inhibitor of creatine metabolism, β-Guanidinopropionic acid (β -GPA), we found that β -GPA blocks both the migration and survival of glioma cells under hypoxic stress. Lastly, β -GPA also inhibited creatine secretion by TAMCs, showing that creatine blockade can also influence TAMC metabolic phenotype. In the future, we will examine the importance of creatine metabolism on both immune suppression and tumor progression in-vivo. This work provides novel insights into the role of creatine metabolism in GBM and identifies a unique therapeutic avenue for this devastating disease.
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Wang, Jenny, and Jonathan Zippin. "2173." Journal of Clinical and Translational Science 1, S1 (September 2017): 2. http://dx.doi.org/10.1017/cts.2017.25.
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OBJECTIVES/SPECIFIC AIMS: The soluble adenylyl cyclase (sAC) is a noncanonical source of cAMP in mammalian cells. sAC is an ATP/bicarbonate ion sensor, whose activity responds to intracellular signals such as pH changes and metabolism. Unlike the more traditionally studied transmembrane adenylyl cyclase, sAC is not tethered to the cell membrane and instead is found in subcellular microdomains like the mitochondria and nucleus. In particular, sAC localization in the mitochondria has been implicated in oxidative phosphorylation and mitochondrial metabolism. Specific changes in sAC microdomain localization have diagnostic utility in a wide variety of cancers, namely melanoma. We have recently found that loss of sAC leads to tumorigenesis and a Warburg/cancer-like metabolic phenotype, consisting of an activated flux through glycolysis, increased lactate production, and dependence on glucose metabolism. In addition, computational analysis of the metabolomics profile of sAC null cells suggests an increased flux through serine synthetic pathways. We hypothesized that specific sAC microdomains are responsible for this cancer-like metabolic state. METHODS/STUDY POPULATION: We have established oncogenic SV40 large T antigen and HPV16-E6 expressing mouse embryonic fibroblasts lacking sAC expression (SV40 KO and E6 KO, respectively). Using these parental lines, we reintroduced sAC by targeting the protein to specific microdomains. sAC was either driven into the mitochondria (mito-sAC) or was driven into all possible microdomains (WT sAC). Single clones were generated and sAC expression was confirmed by Western analysis. Stable cell lines were evaluated for mitochondrial metabolism, glucose sensitivity, and serine sensitivity. RESULTS/ANTICIPATED RESULTS: We found that reintroduction of WT sAC into sAC null cells rescued sensitivity to glycolytic inhibition compared with control cells (p<0.01). The effect was not dependent on the method of immortalization as it was seen in both SV40 and E6 KO cell lines. sAC activity was not directly proportional to expression suggesting that additional regulatory pathways exist. Interestingly, targeted delivery of sAC to the mitochondria was not as effective in rescuing glucose sensitivity as untargeted delivery of sAC into all possible microdomains. Therefore, even though mitochondrial sAC is known to influence metabolism, our data suggests that the nonmitochondrial isoform is most important for cancer cell metabolism. Although metabolomics analysis suggested that serine synthetic pathways are activated in sAC null cells, there is no evidence to suggest that serine is required for sAC null cell growth. Neither inhibition of serine synthesis nor serine starvation differentially affected the growth of sAC null cells compared with WT sAC. DISCUSSION/SIGNIFICANCE OF IMPACT: These data suggest that the Warburg metabolic phenotype in sAC null cells is regulated by specific sAC microdomains. By targeting sAC to specific microdomains, we can further distinguish the role of sAC localization in cellular metabolism. Cancer cells have been shown to exhibit altered metabolic circuitry of pathways like glycolysis, which allow them to adapt to increased metabolic demands of cellular proliferation and waning environmental resources. Beyond helping us improve the use of sAC immunolocalization as a cancer diagnostic, a better understanding of sAC microdomains in transformed cells will help us understand how this signaling pathway is important in cancer. Pharmacologic manipulation of sAC signaling may represent a new cancer therapeutic strategy.
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Goretzki, Alexandra, Yen-Ju Lin, Jennifer Zimmermann, Hannah Rainer, Ann-Christine Junker, Sonja Wolfheimer, Stefan Vieths, Stephan Scheurer, and Stefan Schülke. "Role of Glycolysis and Fatty Acid Synthesis in the Activation and T Cell-Modulating Potential of Dendritic Cells Stimulated with a TLR5-Ligand Allergen Fusion Protein." International Journal of Molecular Sciences 23, no. 20 (October 21, 2022): 12695. http://dx.doi.org/10.3390/ijms232012695.
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Trained immune responses, based on metabolic and epigenetic changes in innate immune cells, are de facto innate immune memory and, therefore, are of great interest in vaccine development. In previous studies, the recombinant fusion protein rFlaA:Betv1, combining the adjuvant and toll-like receptor (TLR)5-ligand flagellin (FlaA) and the major birch pollen allergen Bet v 1 into a single molecule, significantly suppressed allergic sensitization in vivo while also changing the metabolism of myeloid dendritic cells (mDCs). Within this study, the immune–metabolic effects of rFlaA:Betv1 during mDC activation were elucidated. In line with results for other well-characterized TLR-ligands, rFlaA:Betv1 increased glycolysis while suppressing oxidative phosphorylation to different extents, making rFlaA:Betv1 a suitable model to study the immune–metabolic effects of TLR-adjuvanted vaccines. In vitro pretreatment of mDCs with cerulenin (inhibitor of fatty acid biosynthesis) led to a decrease in both rFlaA:Betv1-induced anti-inflammatory cytokine Interleukin (IL) 10 and T helper cell type (TH) 1-related cytokine IL-12p70, while the pro-inflammatory cytokine IL 1β was unaffected. Interestingly, pretreatment with the glutaminase inhibitor BPTES resulted in an increase in IL-1β, but decreased IL-12p70 secretion while leaving IL-10 unchanged. Inhibition of the glycolytic enzyme hexokinase-2 by 2-deoxyglucose led to a decrease in all investigated cytokines (IL-10, IL-12p70, and IL-1β). Inhibitors of mitochondrial respiration had no effect on rFlaA:Betv1-induced IL-10 level, but either enhanced the secretion of IL-1β (oligomycin) or decreased IL-12p70 (antimycin A). In extracellular flux measurements, mDCs showed a strongly enhanced glycolysis after rFlaA:Betv1 stimulation, which was slightly increased after respiratory shutdown using antimycin A. rFlaA:Betv1-stimulated mDCs secreted directly antimicrobial substances in a mTOR- and fatty acid metabolism-dependent manner. In co-cultures of rFlaA:Betv1-stimulated mDCs with CD4+ T cells, the suppression of Bet v 1-specific TH2 responses was shown to depend on fatty acid synthesis. The effector function of rFlaA:Betv1-activated mDCs mainly relies on glycolysis, with fatty acid synthesis also significantly contributing to rFlaA:Betv1-mediated cytokine secretion, the production of antimicrobial molecules, and the modulation of T cell responses.
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Wagner, Tristan, Gwénaëlle André-Leroux, Valérie Hindie, Nathalie Barilone, María-Natalia Lisa, Sylviane Hoos, Bertrand Raynal, et al. "Structural insights into the functional versatility of an FHA domain protein in mycobacterial signaling." Science Signaling 12, no. 580 (May 7, 2019): eaav9504. http://dx.doi.org/10.1126/scisignal.aav9504.
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Forkhead-associated (FHA) domains are modules that bind to phosphothreonine (pThr) residues in signaling cascades. The FHA-containing mycobacterial protein GarA is a central element of a phosphorylation-dependent signaling pathway that redirects metabolic flux in response to amino acid starvation or cell growth requirements. GarA acts as a phosphorylation-dependent ON/OFF molecular switch. In its nonphosphorylated ON state, the GarA FHA domain engages in phosphorylation-independent interactions with various metabolic enzymes that orchestrate nitrogen flow, such as 2-oxoglutarate decarboxylase (KGD). However, phosphorylation at the GarA N-terminal region by the protein kinase PknB or PknG triggers autoinhibition through the intramolecular association of the N-terminal domain with the FHA domain, thus blocking all downstream interactions. To investigate these different FHA binding modes, we solved the crystal structures of the mycobacterial upstream (phosphorylation-dependent) complex PknB-GarA and the downstream (phosphorylation-independent) complex GarA-KGD. Our results show that the phosphorylated activation loop of PknB serves as a docking site to recruit GarA through canonical FHA-pThr interactions. However, the same GarA FHA–binding pocket targets an allosteric site on nonphosphorylated KGD, where a key element of recognition is a phosphomimetic aspartate. Further enzymatic and mutagenesis studies revealed that GarA acted as a dynamic allosteric inhibitor of KGD by preventing crucial motions in KGD that are necessary for catalysis. Our results provide evidence for physiological phosphomimetics, supporting numerous mutagenesis studies using such approaches, and illustrate how evolution can shape a single FHA-binding pocket to specifically interact with multiple phosphorylated and nonphosphorylated protein partners.
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KAVANAGH, Kathryn L., Mario KLIMACEK, Bernd NIDETZKY, and David K. WILSON. "Structure of xylose reductase bound to NAD+ and the basis for single and dual co-substrate specificity in family 2 aldo-keto reductases." Biochemical Journal 373, no. 2 (July 15, 2003): 319–26. http://dx.doi.org/10.1042/bj20030286.
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Xylose reductase (XR; AKR2B5) is an unusual member of aldo-keto reductase superfamily, because it is one of the few able to efficiently utilize both NADPH and NADH as co-substrates in converting xylose into xylitol. In order to better understand the basis for this dual specificity, we have determined the crystal structure of XR from the yeast Candida tenuis in complex with NAD+ to 1.80 Å resolution (where 1 Å=0.1 nm) with a crystallographic R-factor of 18.3%. A comparison of the NAD+- and the previously determined NADP+-bound forms of XR reveals that XR has the ability to change the conformation of two loops. To accommodate both the presence and absence of the 2′-phosphate, the enzyme is able to adopt different conformations for several different side chains on these loops, including Asn276, which makes alternative hydrogen-bonding interactions with the adenosine ribose. Also critical is the presence of Glu227 on a short rigid helix, which makes hydrogen bonds to both the 2′- and 3′-hydroxy groups of the adenosine ribose. In addition to changes in hydrogen-bonding of the adenosine, the ribose unmistakably adopts a 3′-endo conformation rather than the 2′-endo conformation seen in the NADP+-bound form. These results underscore the importance of tight adenosine binding for efficient use of either NADH or NADPH as a co-substrate in aldo-keto reductases. The dual specificity found in XR is also an important consideration in designing a high-flux xylose metabolic pathway, which may be improved with an enzyme specific for NADH.
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Leyn, Semen A., Xiaoqing Li, Qingxiang Zheng, Pavel S. Novichkov, Samantha Reed, Margaret F. Romine, James K. Fredrickson, Chen Yang, Andrei L. Osterman, and Dmitry A. Rodionov. "Control of Proteobacterial Central Carbon Metabolism by the HexR Transcriptional Regulator." Journal of Biological Chemistry 286, no. 41 (August 17, 2011): 35782–94. http://dx.doi.org/10.1074/jbc.m111.267963.
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Bacteria exploit multiple mechanisms for controlling central carbon metabolism (CCM). Thus, a bioinformatic analysis together with some experimental data implicated the HexR transcriptional factor as a global CCM regulator in some lineages of Gammaproteobacteria operating as a functional replacement of the Cra regulator characteristic of Enterobacteriales. In this study, we combined a large scale comparative genomic reconstruction of HexR-controlled regulons in 87 species of Proteobacteria with the detailed experimental analysis of the HexR regulatory network in the Shewanella oneidensis model system. Although nearly all of the HexR-controlled genes are associated with CCM, remarkable variations were revealed in the scale (from 1 to 2 target operons in Enterobacteriales up to 20 operons in Aeromonadales) and gene content of HexR regulons between 11 compared lineages. A predicted 17-bp pseudo-palindrome with a consensus tTGTAATwwwATTACa was confirmed as a HexR-binding motif for 15 target operons (comprising 30 genes) by in vitro binding assays. The negative effect of the key CCM intermediate, 2-keto-3-deoxy-6-phosphogluconate, on the DNA-regulator complex formation was verified. A dual mode of HexR action on various target promoters, repression of genes involved in catabolic pathways and activation of gluconeogenic genes, was for the first time predicted by the bioinformatic analysis and experimentally verified by changed gene expression pattern in S. oneidensis ΔhexR mutant. Phenotypic profiling revealed the inability of this mutant to grow on lactate or pyruvate as a single carbon source. A comparative metabolic flux analysis of wild-type and mutant strains of S. oneidensis using [13C]lactate labeling and GC-MS analysis confirmed the hypothesized HexR role as a master regulator of gluconeogenic flux from pyruvate via the transcriptional activation of phosphoenolpyruvate synthase (PpsA).
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Rashidi Othman, Norazian Mohd. Hassan, Ainaa Eliah Abu Bakar, Nur Hidayah Noh, Nurrulhidayah Ahmad Fadzillah, and Noraini Mahmad. "Manipulation of Environmental Stress Towards Lutein Production in Chlorella fusca Cell Culture." Journal of Pharmacy and Nutrition Sciences 9, no. 5 (January 5, 2019): 251–57. http://dx.doi.org/10.29169/1927-5951.2019.09.05.3.
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All carotenoids originate from a single, common precursor, phytoene. The colour of carotenoids is determinedby desaturation, isomerization, cyclization, hydroxylation and epoxidation of the 40-carbon phytoene. The conjugated double-bond structure and nature of end ring groups confer on the carotenoids properties such as colour and antioxidant activity. Algae may become major sources of carotenoids but the extent of environmental stress and genetic influences on algae carotenoid biosynthesis are poorly understood. Carotenoid biosynthesis can be influenced by many aspects and is liable to geometric isomerization with the existence of oxygen, light and heat which affect the colour degradation and oxidation. Therefore, in this study carotenoid biogenesis is investigated in cell culture of Chlorella fusca as a potential model system for rapid initiation, and extraction of carotenoids by providing stringent control of genetic, developmental and environmental factors. The value of this experimental system for investigating key factors controlling the carotenoid accumulation is then tested by assessing the effects of environmental variables, such as drought stress, light intensity, nutrient strength and media formulation on carotenoid accumulation. Our findings revealed that the conversion of violaxanthin to lutein is due to irradiance stress condition, nutrient strength as well as drought stress. As a result, manipulation of environmental variables will up-regulate lutein concentration. This reaction will restrict the supply of precursors for ABA biosynthesis and the algae cell culture responds by increasing carotenogenic metabolic flux to compensate for this restriction. In conclusion, selecting the appropriate algae species for the appropriate environmental conditions is not only important for yield production, but also for nutritional value quality of carotenoid.
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Forte, Dorian, Roberto Maria Pellegrino, Francesco Fabbri, Ivan Vannini, Samantha Bruno, Giulia Corradi, Rafael J. Argüello, et al. "Circulating Extracellular Vesicles from Acute Myeloid Leukemia Patients Drive Distinct Metabolic Profile of Leukemic Cells and Reveal Crucial Lipidomic Biomarkers." Blood 138, Supplement 1 (November 5, 2021): 3471. http://dx.doi.org/10.1182/blood-2021-150339.
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Abstract Background. Extracellular vesicles (EVs) are submicron vesicles released from various cell types including blood cells with pleiotropic effects on cell signalling and metabolism. EV cargos are enriched in nucleic acids, proteins, and lipids that can be delivered to target cells to influence surrounding microenvironment. Thus, EVs represent a powerful tool for liquid biopsy in hematological malignancies including acute myeloid leukemia (AML). AML is an aggressive disease with high relapse rate and less invasive tools are urgently needed to investigate disease (metabolic) dynamics. Accumulating evidence has reported a key role for EVs in shaping the AML bone marrow niche. However, at present, the metabolic function and the lipidomic signature driven by circulating EVs have yet to fully emerge. Methods. Peripheral blood (PB) and bone marrow (BM) were collected from AML patients at diagnosis (n=40) and PB from age/sex-matched healthy donors (HD, n=20). EVs were purified from platelet-poor plasma by size exclusion chromatography and quantified using the NanoSight technology. Immunomagnetically isolated CD34+ cells from umbilical cord blood (CB) or AML patients were characterized by analyzing the hematopoietic stem/progenitor cell (HSPC)-specific cluster of differentiation marker expression, redox metabolic profiling (using CellROX, glutathione detection reagent and MitoTracker) after 24 hours co-culture with EVs. Quantitative lipidomic profiling of circulating EVs was performed by Liquid Chromatography coupled with High-Resolution Mass Spectrometry (LC/HRMS). Seahorse extracellular flux analyses were performed in leukemia cell lines (including KG-1, KASUMI-1, MOLM-13, THP-1 and OCI-AML3). To functionally define the metabolic reprogramming of leukemic cellular components within their microenvironment, leukemic stem cell subsets were assessed by flow cytometry-based SCENITH (Single Cell ENergetic metabolism by profilIng Translation inHibition) method in both whole blood and BM samples (n=4). Results. In our work, plasma-derived EVs from AML patients showed a significant increase in the size and protein amounts compared to HD counterparts. To explore the metabolic perturbation triggered by EVs, we developed a co-culture system with circulating EVs from either HD or AML patients with CB or AML CD34+. We found a reduction in the frequency of AML CD34+ with high ROS levels in the presence of AML EVs without affecting the ROS levels in normal CB CD34+. In parallel, AML EVs increased the frequency of AML CD34+ with both high mitochondrial activity and glutathione, a key antioxidant molecule involved in many metabolic pathways. Similar metabolic profiles were also confirmed in human leukemic cell lines tested. Specifically, Seahorse flux analysis revealed that EVs induced a cell energy phenotype consistent with quiescent and chemoresistant state in human leukemic cell lines, showing a more glycolytic state in MOLM-13. Interestingly, both CD34+ and CD34+/CD38- leukemic fractions from whole blood and BM of the same AML patients were analyzed by SCENITH after co-cultures with HD/AML EVs. Remarkably, PB CD34+/CD38- leukemic fractions were more dependent on mitochondrial activity in the presence of AML EVs, suggesting a metabolic shift triggered by leukemic EV that apparently occur in the leukemic fractions out of the BM niche. In addition, to give insights into lipidomic signatures of EVs as disease biomarkers, we detected a total of 25 (out of 200) independent lipid species significantly different between AML-derived EVs and HD (n=20, respectively). We reported the abundance of both glycerolipid and fatty acids species in AML EVs. Also, through a multivariate statistical analysis of EV lipidomic profile, we revealed that AML EVs were depleted in sphingomyelin classes, a class of lipids that are interconnected to HSC metabolism. Finally, according to the 2017 ELN risk stratification system, we observed the depletion in important modulators of EV release and formation as ether-linked phosphatidylethanolamine and phosphatidylethanolamine species in adverse-risk AML patients. Conclusion. Overall, our study provides the basis for further investigations on the metabolic alterations trigger by EVs within the BM microenvironment and suggests prognostic biomarkers for leukemic patients that might reveal novel metabolic vulnerabilities in AML scenario. Disclosures Cavo: Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: TRAVEL, ACCOMMODATIONS, EXPENSES, Speakers Bureau; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel Accommodations, Speakers Bureau; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Adaptive Biotechnologies: Consultancy, Honoraria; GlaxoSmithKline: Consultancy, Honoraria; Bristol-Myers Squib: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Curti: Jazz Pharma: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees.
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Czechowski, Tomasz, Tony R. Larson, Theresa M. Catania, David Harvey, Geoffrey D. Brown, and Ian A. Graham. "Artemisia annua mutant impaired in artemisinin synthesis demonstrates importance of nonenzymatic conversion in terpenoid metabolism." Proceedings of the National Academy of Sciences 113, no. 52 (December 7, 2016): 15150–55. http://dx.doi.org/10.1073/pnas.1611567113.
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Artemisinin, a sesquiterpene lactone produced by Artemisia annua glandular secretory trichomes, is the active ingredient in the most effective treatment for malaria currently available. We identified a mutation that disrupts the amorpha-4,11-diene C-12 oxidase (CYP71AV1) enzyme, responsible for a series of oxidation reactions in the artemisinin biosynthetic pathway. Detailed metabolic studies of cyp71av1-1 revealed that the consequence of blocking the artemisinin biosynthetic pathway is the redirection of sesquiterpene metabolism to a sesquiterpene epoxide, which we designate arteannuin X. This sesquiterpene approaches half the concentration observed for artemisinin in wild-type plants, demonstrating high-flux plasticity in A. annua glandular trichomes and their potential as factories for the production of novel alternate sesquiterpenes at commercially viable levels. Detailed metabolite profiling of leaf maturation time-series and precursor-feeding experiments revealed that nonenzymatic conversion steps are central to both artemisinin and arteannuin X biosynthesis. In particular, feeding studies using 13C-labeled dihydroartemisinic acid (DHAA) provided strong evidence that the final steps in the synthesis of artemisinin are nonenzymatic in vivo. Our findings also suggest that the specialized subapical cavity of glandular secretory trichomes functions as a location for both the chemical conversion and the storage of phytotoxic compounds, including artemisinin. We conclude that metabolic engineering to produce high yields of novel secondary compounds such as sesquiterpenes is feasible in complex glandular trichomes. Such systems offer advantages over single-cell microbial hosts for production of toxic natural products.
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Liu, Yuxuan, Lucille Stuani, Dorra Jedoui, Milton Merchant, Astraea Jager, Jolanda Sarno, Charles G. Mullighan, Felix J. Hartmann, Sean Bendall, and Kara L. Davis. "Inhibition of Pre-BCR Signaling Mediates a Metabolic Switch in B-Cell Progenitor Acute Lymphoblastic Leukemia." Blood 138, Supplement 1 (November 5, 2021): 615. http://dx.doi.org/10.1182/blood-2021-145956.
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Abstract Despite improvements in overall survival for children with B-cell progenitor acute lymphoblastic leukemia (BCP-ALL), it remains the second-leading cause of cancer related death in children with approximately 200 deaths per year in the U.S. Thus, there remains a critical need for a definitive cure to prevent relapse for patients with BCP ALL. The accumulation of BCP ALL blasts results from the disruption of normal developmental checkpoints. One of these checkpoints, as pro-B cells transition to become pre-B cells, involves surface expression of the precursor-B-cell receptor (pre-BCR). Prior work has categorized BCP ALL into pre-BCR positive and pre-BCR negative subtypes based on the protein expression of Ig light chain and active signaling of SRC family kinases, SYK, BTK. Combining single cell analysis and machine learning, we previously identified pre-B cells with activation of pre-BCR signaling, namely CREB, 4EBP1, rpS6 and SYK, that are present at diagnosis and highly predictive of relapse. We call these relapse predictive cells. Relapse predictive cells were enriched in relapse samples, demonstrating their persistence from diagnosis to relapse and making them an actionable target to prevent relapse altogether. To better understand relapse predictive cells, we enriched pre-B cells from patients with known relapse status and performed whole transcriptome sequencing. Relapse predictive cells demonstrated significant upregulation of genes in the oxidative phosphorylation (OXPHOS), glycolysis, and reactive oxygen species (ROS) pathways compared to pre-B-like leukemia cells from patients who will not go on to relapse. Analysis of public genome-wide CRISPR screen datasets in 2 pre-BCR+ and 4 pre-BCR- cell lines found 69 essential genes uniquely present in pre-BCR+ cell lines, related to mitochondria translation, OXPHOS and TCA cycle pathway. We performed CRISPR knock down of proximal pre-BCR related tyrosine kinase SYK in pre-BCR+ (Nalm6, Kasumi-2) and pre-BCR- (697, REH, SUPB15) cell lines to understand how activated pre-BCR impacts cellular metabolism in pre-BCR+ and pre-BCR- cells. CyTOF analysis of pre-BCR signaling demonstrated effective inhibition of downstream pre-BCR pathway members in the KD cells (pSYK, pBLNK, pBTK). RNA sequencing demonstrated upregulation of mitochondrial translation and OXPHOS pathways with downregulation of hypoxia pathways in pre-BCR+ but not pre-BCR- SYK KD cells. Functional extracellular flux experiments by Seahorse confirmed pre-BCR+ SYK KD cells to have higher basal oxygen consumption rate (OCR) and lower extracellular acidification rate (ECAR) compared to wild-type pre-BCR+ cells, indicating a switch from highly glycolytic to aerobic metabolism. To determine the interplay between pre-BCR signaling and cellular metabolism at the single cell level, we performed CYTOF with a panel examining pre-BCR pathway members, developmental phenotype and metabolism in these cell lines as well as matched diagnosis-relapse patient-derived xenografts. These results indicate, in line with the RNA sequencing and Seahorse data, that inhibiting pre-BCR signaling is accompanied by inhibition of glycolysis with lower protein expression of glycolytic related enzymes HIF1A, GLUT1, PFKFB4, GAPDH, ENO1 and LDHA. Further, we observed in cells completely deficient in the ability to initiate pre-BCR signal (SYK knock out), activated p4EBP1 indicating signaling feedback from the PI3K-AKT pathway and a metabolic adaption indicating utilization of energy sources other than glucose in cells surviving SYK loss. Finally, to determine the impact of loss of pre-BCR signaling on proliferation, in vitro competition assays demonstrated SYK KD cells to be less proliferative in all the cell lines except pre-BCR- cell line 697. In vivo, SYK KO demonstrated significantly slower engraftment (median %hCD45: 84% vs 54%, P=0.009) in NSG mice and significantly longer survival time than the mice xenografted with wild-type cells (median survival 28 vs 39 days, P=0.0004). Together, our data indicate that individual BCP ALL cells with active pre-BCR signaling are associated with relapse and that these cells have a unique metabolic state that relies on active glycolysis and metabolic flexibility supporting proliferation in vitro as well as engraftment and aggressivity in vivo. Further metabolomics experiments and characterization of primary patient samples are underway. Disclosures Mullighan: Pfizer: Research Funding; Illumina: Membership on an entity's Board of Directors or advisory committees; AbbVie: Research Funding; Amgen: Current equity holder in publicly-traded company. Davis: Novartis Pharmaceuticals: Honoraria; Jazz Pharmaceuticals: Research Funding.
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Sanchez, Anthony M. J., Henri Bernardi, Guillaume Py, and Robin B. Candau. "Autophagy is essential to support skeletal muscle plasticity in response to endurance exercise." American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 307, no. 8 (October 15, 2014): R956—R969. http://dx.doi.org/10.1152/ajpregu.00187.2014.
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Physical exercise is a stress that can substantially modulate cellular signaling mechanisms to promote morphological and metabolic adaptations. Skeletal muscle protein and organelle turnover is dependent on two major cellular pathways: Forkhead box class O proteins (FOXO) transcription factors that regulate two main proteolytic systems, the ubiquitin-proteasome, and the autophagy-lysosome systems, including mitochondrial autophagy, and the MTORC1 signaling associated with protein translation and autophagy inhibition. In recent years, it has been well documented that both acute and chronic endurance exercise can affect the autophagy pathway. Importantly, substantial efforts have been made to better understand discrepancies in the literature on its modulation during exercise. A single bout of endurance exercise increases autophagic flux when the duration is long enough, and this response is dependent on nutritional status, since autophagic flux markers and mRNA coding for actors involved in mitophagy are more abundant in the fasted state. In contrast, strength and resistance exercises preferentially raise ubiquitin-proteasome system activity and involve several protein synthesis factors, such as the recently characterized DAGK for mechanistic target of rapamycin activation. In this review, we discuss recent progress on the impact of acute and chronic exercise on cell component turnover systems, with particular focus on autophagy, which until now has been relatively overlooked in skeletal muscle. We especially highlight the most recent studies on the factors that can impact its modulation, including the mode of exercise and the nutritional status, and also discuss the current limitations in the literature to encourage further works on this topic.
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Heart, Emma, and Peter J. S. Smith. "Rhythm of the β-cell oscillator is not governed by a single regulator: multiple systems contribute to oscillatory behavior." American Journal of Physiology-Endocrinology and Metabolism 292, no. 5 (May 2007): E1295—E1300. http://dx.doi.org/10.1152/ajpendo.00648.2006.
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Pulsatile insulin output, paralleled by oscillations in intracellular Ca2+, reflect oscillating metabolism within β-cells in response to secretory fuels. Here we question whether oscillatory periodicity is conserved or varied from stimulation to stimulation, whether glycolysis is essential for the manifestation of an oscillatory response, and if an environment of nutrient oversupply affects oscillatory regularity. We have determined that a β-cell oscillatory Ca2+ pattern is independent of the type of applied secretory fuel (glucose, methyl-pyruvate, or α-ketoisocaproate). In addition, single cells respond with the same pattern when repeatedly stimulated, regardless of the type of stimulatory fuel. Presence of substimulatory glucose is not necessary to obtain an oscillatory responses to methyl-pyruvate or α-ketoisocaproate. Glucose-6-phosphate, as a measure of glycolytic flux, is not detectable under these conditions. These data suggest that multiple systems, rather than a single enzyme component, can contribute to the β-cell oscillatory behavior. Prolonged exposure to high levels of palmitate impaired oscillatory regularity in the individual β-cells. This supports the hypothesis that a high-fat environment might contribute to loss of regular oscillatory pattern in diabetic subjects, acting, at least in part, at the level of the single β-cell.
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Issitt, Theo, Sean T. Sweeney, William J. Brackenbury, and Kelly R. Redeker. "Sampling and Analysis of Low-Molecular-Weight Volatile Metabolites in Cellular Headspace and Mouse Breath." Metabolites 12, no. 7 (June 27, 2022): 599. http://dx.doi.org/10.3390/metabo12070599.
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Volatile compounds, abundant in breath, can be used to accurately diagnose and monitor a range of medical conditions. This offers a noninvasive, low-cost approach with screening applications; however, the uptake of this diagnostic approach has been limited by conflicting published outcomes. Most published reports rely on large scale screening of the public, at single time points and without reference to ambient air. Here, we present a novel approach to volatile sampling from cellular headspace and mouse breath that incorporates multi-time-point analysis and ambient air subtraction revealing compound flux as an effective proxy of active metabolism. This approach to investigating breath volatiles offers a new avenue for disease biomarker discovery and diagnosis. Using gas chromatography mass spectrometry (GC/MS), we focus on low molecular weight, metabolic substrate/by-product compounds and demonstrate that this noninvasive technique is sensitive (reproducible at ~1 µg cellular protein, or ~500,000 cells) and capable of precisely determining cell type, status and treatment. Isolated cellular models represent components of larger mammalian systems, and we show that stress- and pathology-indicative compounds are detectable in mice, supporting further investigation using this methodology as a tool to identify volatile targets in human patients.
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Klein, Vivien Jessica, Marta Irla, Marina Gil López, Trygve Brautaset, and Luciana Fernandes Brito. "Unravelling Formaldehyde Metabolism in Bacteria: Road towards Synthetic Methylotrophy." Microorganisms 10, no. 2 (January 20, 2022): 220. http://dx.doi.org/10.3390/microorganisms10020220.
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Formaldehyde metabolism is prevalent in all organisms, where the accumulation of formaldehyde can be prevented through the activity of dissimilation pathways. Furthermore, formaldehyde assimilatory pathways play a fundamental role in many methylotrophs, which are microorganisms able to build biomass and obtain energy from single- and multicarbon compounds with no carbon–carbon bonds. Here, we describe how formaldehyde is formed in the environment, the mechanisms of its toxicity to the cells, and the cell’s strategies to circumvent it. While their importance is unquestionable for cell survival in formaldehyde rich environments, we present examples of how the modification of native formaldehyde dissimilation pathways in nonmethylotrophic bacteria can be applied to redirect carbon flux toward heterologous, synthetic formaldehyde assimilation pathways introduced into their metabolism. Attempts to engineer methylotrophy into nonmethylotrophic hosts have gained interest in the past decade, with only limited successes leading to the creation of autonomous synthetic methylotrophy. Here, we discuss how native formaldehyde assimilation pathways can additionally be employed as a premise to achieving synthetic methylotrophy. Lastly, we discuss how emerging knowledge on regulation of formaldehyde metabolism can contribute to creating synthetic regulatory circuits applied in metabolic engineering strategies.
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Schwartz, Laurent, Luc Benichou, Jules Schwartz, Maxime Pontié, and Marc Henry. "Is the Second Law of Thermodynamics Able to Classify Drugs?" Substantia 6, no. 1 (March 7, 2022): 37–47. http://dx.doi.org/10.36253/substantia-1364.
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Specialization characterizes pharmacology, with the consequence of classifying the various treatments into unrelated categories. Treating a specific disease usually requires the design of a specific drug. The second law of thermodynamics is the driving force both for chemical reactions and for life. It applies to diseases and treatment. In most common diseases, there is a metabolic shift toward anabolism and anaerobic glycolysis, resulting in the release of entropy in the form of biomass. In accordance with the second principle of thermodynamics, treatment should aim at decreasing the entropy flux, which stays inside the body in the form of biomass. Most treatments aim at increasing the amount of entropy that is released by the cell in the form of thermal photons. As clinically different diseases often requires similar drugs, this calls for reinforcement in a quest for a single unified framework. For example, treatment of aggressive autoimmune diseases requires the same cytotoxic chemotherapy than for cancer. This strongly suggests that despite their apparent disparity, there is an underlying unity in the diseases and the treatments. The shift toward increased entropy release in the form of heat offers sound guidelines for the repurposing of drugs.
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Rosenberg, Evgenia, Valeria Voevoda, Hila Magen, Olga Ostrovsky, Avichai Shimoni, Amnon Peled, Arnon Nagler, and Katia Beider. "Venetoclax Reverses Metabolic Reprogramming Induced By S1P Modulator FTY720, Suppresses Oxidative Phosphorylation and Synergistically Targets Multiple Myeloma." Blood 138, Supplement 1 (November 5, 2021): 1195. http://dx.doi.org/10.1182/blood-2021-149372.
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Abstract Patients with multiple myeloma (MM) invariably relapse with chemotherapy-resistant disease, underscoring the need for new therapeutic modalities that bypass these resistance mechanisms. FTY720, also known as fingolimod, is an S1P modulator approved by the FDA to treat the relapsing form of multiple sclerosis. Previously we reported that FTY720 exhibits potent anti-myeloma effect in vitro and in vivo in disseminated xenograft model of MM (Beider et al., Clin Cancer Res 2017). Cytotoxic activity of FTY720 was associated with down-regulation of anti-apoptotic protein MCL-1 while not affecting BCL-2 levels. It is therefore conceivable that BCL-2 inhibition using BH3-mimetic venetoclax may improve responses to FTY720. Incubation of human MM cell lines (n=8) and primary MM cells (n=3) with venetoclax and FTY720 combination synergistically potentiated cell death (CI<0.02), regardless of the MM cells t (11; 14) status. The robust apoptosis induced by venetoclax /FTY720 treatment was accompanied by cytochrome C release, activation of caspase-3 and extensive DNA damage, demonstrated by increased TUNEL staining and elevated levels of phosphorylated histone H2AX, respectively . These effects were associated with down-regulation of BCL-2 protein, stabilization of pro-apoptotic Bak protein, loss of mitochondrial membrane potential, ER stress induction, and inhibition of the AKT/mTOR signaling pathway. Furthermore, the venetoclax /FTY720 combination markedly induced mitochondrial calcium flux and mitochondrial ROS generation. Corresponding with mitochondrial destabilization, venetoclax/FTY720 combination promoted the release of apoptosis-inducing factor (AIF) from the mitochondria to the cytosol and subsequently increased AIF nuclear localization, suggesting its functional role in the execution phase of the apoptosis in response to the dual treatment. AIF is a mitochondrial oxidoreductase that contributes to cell death programs and participates in the assembly of the respiratory chain. Of note, single-agent treatment with FTY720 profoundly up-regulated mitochondrial AIF levels. Given the regulative role of AIF in mitochondrial bioenergetics, we could suggest that increased mitochondrial levels of AIF upon FTY720 exposure may support adaptive responses and promote MM survival upon mitochondrial stress. We thus investigated a possible effect of venetoclax and FTY720 separately or in combination on the metabolic activity of MM cells, observing distinct metabolic profiles of single versus combined exposures. FTY720 significantly suppressed glycolysis, down-regulating the transcript levels of the glycolytic enzymes HK2, PDK1, and LDHA. Glycolytic suppression may result in upregulation of mitochondrial content, which maintains cell survival. In accordance, increased mitochondrial activity was detected in FTY720-treated MM cells, detected by high uptake of MitoSpy Red, a dye that stains mitochondria in a membrane potential-dependent manner. To determine if the changes in the mitochondrial content also altered mitochondrial function, bioenergetic analysis was undertaken. FTY720-treated MM cells demonstrated increased levels of NDUFB8 and UQCRC2 (subunits of mitochondrial respiratory complexes I and III, respectively). Furthermore, FTY720 exposure up-regulated ATP production, suggesting an increase in tumor-protective oxidative phosphorylation (OXPHOS). In agreement, inhibition of mitochondrial electron transport chain using rotenone sensitized MM cells to FTY720, synergistically promoting cell death. Notably, co-treatment with venetoclax effectively reversed the metabolic changes mediated by FTY720, reducing mitochondrial mass, suppressing mitochondrial activity and strongly down-regulating the pathways related to OXPHOS. Furthermore, venetoclax/FTY720 combination significantly reduced glutathione (GSH) levels, therefore suppressing antioxidative cell responses. To conclude, we unveil venetoclax role in the metabolic regulation in MM cells. Venetoclax reverses metabolic reprogramming induced by FTY720, suppresses mitochondrial respiration, induces vigorous mitochondrial damage and preferentially targets MM cells in combination with FTY720. These findings may provide the scientific basis for a novel combinatorial anti-myeloma therapy. Figure 1 Figure 1. Disclosures Peled: Biokine Therapeutics Ltd: Current Employment; Gamida Cell: Research Funding.
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Mertz, Joseph L., Shisheng Sun, Bojiao Yin, Yingwei Hu, Rahul Bhattacharya, Michael J. Bettenbaugh, Kevin J. Yarema, and Hui Zhang. "Comparison of Three Glycoproteomic Methods for the Analysis of the Secretome of CHO Cells Treated with 1,3,4-O-Bu3ManNAc." Bioengineering 7, no. 4 (November 10, 2020): 144. http://dx.doi.org/10.3390/bioengineering7040144.
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Comprehensive analysis of the glycoproteome is critical due to the importance of glycosylation to many aspects of protein function. The tremendous complexity of this post-translational modification, however, makes it difficult to adequately characterize the glycoproteome using any single method. To overcome this pitfall, in this report we compared three glycoproteomic analysis methods; first the recently developed N-linked glycans and glycosite-containing peptides (NGAG) chemoenzymatic method, second, solid-phase extraction of N-linked glycoproteins (SPEG), and third, hydrophilic interaction liquid chromatography (HILIC) by characterizing N-linked glycosites in the secretome of Chinese hamster ovary (CHO) cells. Interestingly, the glycosites identified by SPEG and HILIC overlapped considerably whereas NGAG identified many glycosites not observed in the other two methods. Further, utilizing enhanced intact glycopeptide identification afforded by the NGAG workflow, we found that the sugar analog 1,3,4-O-Bu3ManNAc, a “high flux” metabolic precursor for sialic acid biosynthesis, increased sialylation of secreted proteins including recombinant human erythropoietin (rhEPO).
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Stern, D., J. Brett, K. Harris, and P. Nawroth. "Participation of endothelial cells in the protein C-protein S anticoagulant pathway: the synthesis and release of protein S." Journal of Cell Biology 102, no. 5 (May 1, 1986): 1971–78. http://dx.doi.org/10.1083/jcb.102.5.1971.
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The protein C-protein S anticoagulant pathway is closely linked to the endothelium. In this paper the synthesis and release of the vitamin K-dependent coagulation factor protein S is demonstrated. Western blotting, after SDS PAGE of Triton X-100 extracts of bovine aortic endothelial cells grown in serum-free medium, demonstrated the presence of protein S. A single major band was observed at Mr approximately 75,000, closely migrating with protein S purified from plasma absent from cells treated with cycloheximide. Metabolic labeling of endothelial cells with [35S]methionine confirmed de novo synthesis of protein S. Using a radioimmunoassay, endothelium was found to release 180 fmol/10(5) cells per 24 h and contain 44 fmol/10(5) cells of protein S antigen. Protein S released from endothelium was functionally active and could promote activated protein C-mediated factor Va inactivation on the endothelial cell surface. Warfarin decreased secretion of protein S antigen by greater than 90% and increased intracellular accumulation by almost twofold. Morphological studies demonstrated intracellular protein S was in the Golgi complex, concentrated at the trans face, rough endoplasmic reticulum, lysosomes, and in vesicles at the periphery. In contrast, protein S was not found in vascular fibroblasts or smooth muscle cells. A pool of intracellular protein S could be released rapidly by the calcium ionophore A23187 (5 microM). This effect was dependent on the presence of calcium in the culture medium and could be blocked by LaCl3, which suggests that cytosolic calcium flux may be responsible for protein S release. These results demonstrate that endothelial cells, but not the subendothelial cells of the vessel wall, can synthesize and release protein S, which indicates a new mechanism by which the inner lining of the vessel wall can contribute to the prevention of thrombotic events.
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George, Sajan, and Heidi Abrahamse. "Redox Potential of Antioxidants in Cancer Progression and Prevention." Antioxidants 9, no. 11 (November 20, 2020): 1156. http://dx.doi.org/10.3390/antiox9111156.
Full textAbstract:
The benevolent and detrimental effects of antioxidants are much debated in clinical trials and cancer research. Several antioxidant enzymes and molecules are overexpressed in oxidative stress conditions that can damage cellular proteins, lipids, and DNA. Natural antioxidants remove excess free radical intermediates by reducing hydrogen donors or quenching singlet oxygen and delaying oxidative reactions in actively growing cancer cells. These reducing agents have the potential to hinder cancer progression only when administered at the right proportions along with chemo-/radiotherapies. Antioxidants and enzymes affect signal transduction and energy metabolism pathways for the maintenance of cellular redox status. A decline in antioxidant capacity arising from genetic mutations may increase the mitochondrial flux of free radicals resulting in misfiring of cellular signalling pathways. Often, a metabolic reprogramming arising from these mutations in metabolic enzymes leads to the overproduction of so called ’oncometabolites’ in a state of ‘pseudohypoxia’. This can inactivate several of the intracellular molecules involved in epigenetic and redox regulations, thereby increasing oxidative stress giving rise to growth advantages for cancerous cells. Undeniably, these are cell-type and Reactive Oxygen Species (ROS) specific, which is manifested as changes in the enzyme activation, differences in gene expression, cellular functions as well as cell death mechanisms. Photodynamic therapy (PDT) using light-activated photosensitizing molecules that can regulate cellular redox balance in accordance with the changes in endogenous ROS production is a solution for many of these challenges in cancer therapy.
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Sengupta, Annesha, Prem Pritam, Damini Jaiswal, Anindita Bandyopadhyay, Himadri B. Pakrasi, and Pramod P. Wangikar. "Photosynthetic Co-production of Succinate and Ethylene in a Fast-Growing Cyanobacterium, Synechococcus elongatus PCC 11801." Metabolites 10, no. 6 (June 16, 2020): 250. http://dx.doi.org/10.3390/metabo10060250.
Full textAbstract:
Cyanobacteria are emerging as hosts for photoautotrophic production of chemicals. Recent studies have attempted to stretch the limits of photosynthetic production, typically focusing on one product at a time, possibly to minimise the additional burden of product separation. Here, we explore the simultaneous production of two products that can be easily separated: ethylene, a gaseous product, and succinate, an organic acid that accumulates in the culture medium. This was achieved by expressing a single copy of the ethylene forming enzyme (efe) under the control of PcpcB, the inducer-free super-strong promoter of phycocyanin β subunit. We chose the recently reported, fast-growing and robust cyanobacterium, Synechococcus elongatus PCC 11801, as the host strain. A stable recombinant strain was constructed using CRISPR-Cpf1 in a first report of markerless genome editing of this cyanobacterium. Under photoautotrophic conditions, the recombinant strain shows specific productivities of 338.26 and 1044.18 μmole/g dry cell weight/h for ethylene and succinate, respectively. These results compare favourably with the reported productivities for individual products in cyanobacteria that are highly engineered. Metabolome profiling and 13C labelling studies indicate carbon flux redistribution and suggest avenues for further improvement. Our results show that S. elongatus PCC 11801 is a promising candidate for metabolic engineering.
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Krömer, Jens Olaf, Elmar Heinzle, Hartwig Schröder, and Christoph Wittmann. "Accumulation of Homolanthionine and Activation of a Novel Pathway for Isoleucine Biosynthesis in Corynebacterium glutamicum McbR Deletion Strains." Journal of Bacteriology 188, no. 2 (January 15, 2006): 609–18. http://dx.doi.org/10.1128/jb.188.2.609-618.2006.
Full textAbstract:
ABSTRACT In the present work, the metabolic consequences of the deletion of the methionine and cysteine biosynthesis repressor protein (McbR) in Corynebacterium glutamicum, which releases almost all enzymes of methionine biosynthesis and sulfate assimilation from transcriptional regulation (D. A. Rey, A. Pühler, and J. Kalinowski, J. Biotechnol. 103:51-65, 2003), were studied. C. glutamicum ATCC 13032 ΔmcbR showed no overproduction of methionine. Metabolome analysis revealed drastic accumulation of a single metabolite, which was not present in the wild type. It was identified by isotopic labeling studies and gas chromatography/mass spectrometry as l-homolanthionine {S-[(3S)-3-amino-3-carboxypropyl]-l-homocysteine}. The accumulation of homolanthionine to an intracellular concentration of 130 mM in the ΔmcbR strain was accompanied by an elevated intracellular homocysteine level. It was shown that cystathionine-γ-synthase (MetB) produced homolanthionine as a side reaction. MetB showed higher substrate affinity for cysteine (Km = 260 μM) than for homocysteine (Km = 540 μM). The cell is able to cleave homolanthionine at low rates via cystathionine-β-lyase (MetC). This cleavage opens a novel threonine-independent pathway for isoleucine biosynthesis via 2-oxobutanoate formed by MetC. In fact, the deletion mutant exhibited an increased intracellular isoleucine level. Metabolic flux analysis of C. glutamicum ΔmcbR revealed that only 24% of the O-acetylhomoserine at the entry of the methionine pathway is utilized for methionine biosynthesis; the dominating fraction is either stored as homolanthionine or redirected towards the formation of isoleucine. Deletion of metB completely prevents homolanthionine accumulation, which is regarded as an important step in the development of C. glutamicum strains for biotechnological methionine production.