Search

Cellular metabolism regulates oxidative and proteostatic stresses in hematopoietic stem cells (HSCs) during replication and aging. Metabolic adaptations drive the chemoresistance and immune evasion capacities of leukemic stem cells (LSCs) in disease initiation and recurrence. Metabolic adaptations and epigenetic mechanisms interact in LSCs. Metabolic changes impact both HSCs and LSCs through cell-intrinsic and -extrinsic mechanisms. Hematopoietic stem cells (HSCs) and leukemic stem cells (LSCs) are crucial for ensuring hematopoietic homeostasis and driving leukemia progression, respectively. Recent research has revealed that metabolic adaptations significantly regulate the function and survival of these stem cells. In this review, we provide an overview of how metabolic pathways regulate oxidative and proteostatic stresses in HSCs during homeostasis and aging. Furthermore, we highlight targetable metabolic pathways and explore their interactions with epigenetics and the microenvironment in addressing the chemoresistance and immune evasion capacities of LSCs. The metabolic differences between HSCs and LSCs have profound implications for therapeutic strategies. [ABSTRACT FROM AUTHOR]

CX-5461, a first-in-class compound, is widely recognized as a selective inhibitor of RNA polymerase I. Recently, it has been reported to possess novel immunosuppressive properties with significant therapeutic effects in transplantation immune rejection. However, the potential use of CX-5461 for Systemic Lupus Erythematosus (SLE) treatment remains unknown. In this study, we elucidated the mechanism underlying the therapeutic efficacy of CX-5461 in lupus. Our findings demonstrated that CX-5461 selectively targets B cells and effectively reduces the proportions of B cells, germinal center B cells, and plasma cells in MRL/MPJ-Faslpr and Resiquimod (R848)-induced lupus mice. Molecular studies revealed that CX-5461 modulates CD36-Acyl-CoA Synthetase Long Chain Family Member 4 (ACSL4)-mediated glycerolipid metabolism in B cells, triggering ferroptosis through the p53- Solute Carrier Family 7 Member 11 (SLC7A11)- Arachidonate 12-Lipoxygenase (ALOX12) pathway, thereby decreasing IgG and Anti-Double-Stranded Deoxyribonucleic Acid (dsDNA) antibody levels and attenuating lupus. Collectively, these results suggest that CX-5461 holds promise as an effective candidate for targeted therapy against lupus. [Display omitted] • CX-5461 significantly inhibited lupus manifestation of MRL/MPJ-Faslpr mice and Resiquimod-induced mice. • CX-5461 suppressed B cell subsets in two independent lupus animal models. • CX-5461 triggered B-cell ferroptosis in vitro and vivo. • CX-5461 modulates CD36 ACSL4 -mediated glycerolipid metabolism in B cells, triggering ferroptosis through the p53- SLC7A11- ALOX12 pathway. [ABSTRACT FROM AUTHOR]

Striated muscle cells, encompassing cardiac myocytes and skeletal muscle fibers, are fundamental to athletic performance, facilitating blood circulation and coordinated movement through contraction. Despite their distinct functional roles, these muscle types exhibit similarities in cytoarchitecture, protein expression, and excitation-contraction coupling. Both muscle types also undergo molecular remodeling in energy metabolism and cell size in response to acute and repeated exercise stimuli to enhance exercise performance. Reactive oxygen species (ROS) produced by NADPH oxidase (NOX) isoforms 2 and 4 have emerged as signaling molecules that regulate exercise adaptations. This review systematically compares NOX2 and NOX4 expression, regulation, and roles in cardiac and skeletal muscle responses across exercise modalities. We highlight the many gaps in our knowledge and opportunities to let future skeletal muscle research into NOX-dependent mechanisms be inspired by cardiac muscle studies and vice versa. Understanding these processes could enhance the development of exercise routines to optimize human performance and health strategies that capitalize on the advantages of physical activity. [Display omitted] • Striated muscle cells exhibit many redox-regulated similarities, including the expression of NOX2 and NOX4. • NOX2 and NOX4 are linked to responses to exercise in skeletal and cardiac muscles, influencing glucose metabolism and cell growth. • Further studies on NOX-dependent redox regulation of striated skeletal muscle may improve our understanding of the factors that limit human performance. [ABSTRACT FROM AUTHOR]

Calcific aortic valve disease (CAVD) is one of the most common forms of valvulopathy, with a 50 % elevated risk of a fatal cardiovascular event, and greater than 15,000 annual deaths in North America alone. The treatment standard is valve replacement as early diagnostic, mitigation, and drug strategies remain underdeveloped. The development of early diagnostic and therapeutic strategies requires the fabrication of effective in vitro valve mimetic models to elucidate early CAVD mechanisms. In this study, we developed a multilayered physiologically relevant 3D valve-on-chip (VOC) system that incorporated aortic valve mimetic extracellular matrix (ECM), porcine aortic valve interstitial cell (VIC) and endothelial cell (VEC) co-culture and dynamic mechanical stimuli. Collagen and glycosaminoglycan (GAG) based hydrogels were assembled in a bilayer to mimic healthy or diseased compositions of the native fibrosa and spongiosa. Multiphoton imaging and proteomic analysis of healthy and diseased VOCs were performed. Collagen-based bilayered hydrogel maintained the phenotype of the VICs. Proteins related to cellular processes like cell cycle progression, cholesterol biosynthesis, and protein homeostasis were found to be significantly altered and correlated with changes in cell metabolism in diseased VOCs. This study suggested that diseased VOCs may represent an early, adaptive disease initiation stage, which was corroborated by human aortic valve proteomic assessment. In this study, we developed a collagen-based bilayered hydrogel to mimic healthy or diseased compositions of the native fibrosa and spongiosa layers. When the gels were assembled in a VOC with VECs and VICs, the diseased VOCs revealed key insights about the CAVD initiation process. Calcific aortic valve disease (CAVD) elevates the risk of death due to cardiovascular pathophysiology by 50 %, however, prevention and mitigation strategies are lacking, clinically. Developing tools to assess early disease would significantly aid in the prevention of disease and in the development of therapeutics. Previously, studies have utilized collagen and glycosaminoglycan-based hydrogels for valve cell co-cultures, valve cell co-cultures in dynamic environments, and inorganic polymer-based multilayered hydrogels; however, these approaches have not been combined to make a physiologically relevant model for CAVD studies. We fabricated a bi-layered hydrogel that closely mimics the aortic valve and used it for valve cell co-culture in a dynamic platform to gain mechanistic insights into the CAVD initiation process using proteomic and multiphoton imaging assessment. [Display omitted] [ABSTRACT FROM AUTHOR]

Cellular organelles maintain areas of close apposition with other organelles at which the cytosolic gap in between them is reduced to a minimum. These membrane contact sites (MCS) are vital for organelle communication and are formed by molecular tethers that physically connect opposing membranes. Although many regulatory pathways are known to converge at MCS, a link between MCS and transcriptional regulation—the primary mechanism through which cells adapt their metabolism to environmental cues—remains largely elusive. In this study, we performed RNA-sequencing on Saccharomyces cerevisiae cells lacking tricalbin proteins (Tcb1, Tcb2, and Tcb3), a family of tethering proteins that connect the endoplasmic reticulum with the plasma membrane and Golgi, to investigate if gene expression is altered when MCS are disrupted. Our results indicate that in the tcb1Δ2Δ3Δ strain, pathways responsive to a high-glucose environment, including glycolysis, fermentation, amino acid synthesis, and low-affinity glucose uptake, are upregulated. Conversely, pathways crucial during glucose depletion, such as the tricarboxylic acid cycle, respiration, high-affinity glucose uptake, and amino acid uptake are downregulated. In addition, we demonstrate that the altered gene expression of tcb1Δ2Δ3Δ in glucose metabolism correlates with increased growth, glucose consumption, CO2 production, and ethanol generation. In conclusion, our findings reveal that tricalbin protein deletion induces a shift in gene expression patterns mimicking cellular responses to a high-glucose environment. This suggests that MCS play a role in sensing and signaling pathways that modulate gene transcription in response to glucose availability. [ABSTRACT FROM AUTHOR]

Propionic acid links the oxidation of branched-chain amino acids and odd-chain fatty acids to the TCA cycle. Gut microbes ferment complex fiber remnants, generating high concentrations of short chain fatty acids, acetate, propionate and butyrate, which are shared with the host as fuel sources. Analysis of vitamin B12-dependent propionate utilization in skin biopsy samples has been used to characterize and diagnose underlying inborn errors of cobalamin (or B12) metabolism. In these cells, the B12-dependent enzyme, methylmalonyl-CoA mutase (MMUT), plays a central role in funneling propionate to the TCA cycle intermediate, succinate. Our understanding of the fate of propionate in other cell types, specifically, the involvement of the β-oxidation-like and methylcitrate pathways, is limited. In this study, we have used [14C]-propionate tracing in combination with genetic ablation or inhibition of MMUT, to reveal the differential utilization of the B12-dependent and independent pathways for propionate metabolism in fibroblast versus colon cell lines. We demonstrate that itaconate can be used as a tool to investigate MMUT-dependent propionate metabolism in cultured cell lines. While MMUT gates the entry of propionate carbons into the TCA cycle in fibroblasts, colon-derived cell lines exhibit a quantitatively significant or exclusive reliance on the β-oxidation-like pathway. Lipidomics and metabolomics analyses reveal that propionate elicits pleiotropic changes, including an increase in odd-chain glycerophospholipids, and perturbations in the purine nucleotide cycle and arginine/nitric oxide metabolism. The metabolic rationale and the regulatory mechanisms underlying the differential reliance on propionate utilization pathways at a cellular, and possibly tissue level, warrant further elucidation. [ABSTRACT FROM AUTHOR]

The reliability and methodology of genome-scale metabolic models (GEMs) of Chinese hamster ovary (CHO) cells have advanced. CHO-GEMs have aided in cell line and process development, thus impacting on biomanufacturing efficiency. An integrative model structure can incorporate multiple layers and capture condition-specific cell regulation. Integration of CHO-GEMs with artificial intelligence (AI) and advanced algorithms will enable autonomous bioreactor management for digital biomanufacturing. Genome-scale metabolic models (GEMs) of Chinese hamster ovary (CHO) cells are valuable for gaining mechanistic understanding of mammalian cell metabolism and cultures. We provide a comprehensive overview of past and present developments of CHO-GEMs and in silico methods within the flux balance analysis (FBA) framework, focusing on their practical utility in rational cell line development and bioprocess improvements. There are many opportunities for further augmenting the model coverage and establishing integrative models that account for different cellular processes and data for future applications. With supportive collaborative efforts by the research community, we envisage that CHO-GEMs will be crucial for the increasingly digitized and dynamically controlled bioprocessing pipelines, especially because they can be successfully deployed in conjunction with artificial intelligence (AI) and systems engineering algorithms. [ABSTRACT FROM AUTHOR]

The skin is made up of different layers with various gradients, which maintain a complex microenvironment, particularly in terms of oxygen levels. However, all types of skin cells are cultured in conventional incubators that do not reproduce physiological oxygen levels. Instead, they are cultured at atmospheric oxygen levels, a condition that is far removed from physiology and may lead to the generation of free radicals known to induce skin ageing. This review aims to summarize the current literature on the effect of physiological oxygen levels on skin cells, highlight the shortcomings of current in vitro models, and demonstrate the importance of respecting skin oxygen levels. We begin by clarifying the terminology used about oxygen levels and describe the specific distribution of oxygen in the skin. We review and discuss how skin cells adapt their oxygen consumption and metabolism to oxygen levels environment, as well as the changes that are induced, particularly, their redox state, life cycle and functions. We examine the effects of oxygen on both simple culture models and more complex reconstructed skin models. Finally, we present the implications of oxygen modulation for a more therapeutic approach. [Display omitted] • Skin cells are physiologically in a microenvironment poor in oxygen called physioxia. • Skin physioxia is not taken into account in in vitro models. • Physioxia affects cutaneous cell metabolism. • Physioxia impacts skin cell proliferation, differentiation and senescence. • Physioxia modulates skin cell migration, pigmentation and angiogenesis. [ABSTRACT FROM AUTHOR]

The circadian clock and cancer cell metabolism mutually regulate each other through regulation of oncogenes or the tumor microenvironment. Dysregulation of the circadian clock broadly impacts cancer cell metabolism and survival. The core clock genes possess noncanonical and transcription activity-independent functions to reprogram cancer cell metabolism. The circadian clock and cell metabolism are both dysregulated in cancer cells through intrinsic cell-autonomous mechanisms and external influences from the tumor microenvironment. The intricate interplay between the circadian clock and cancer cell metabolism exerts control over various metabolic processes, including aerobic glycolysis, de novo nucleotide synthesis, glutamine and protein metabolism, lipid metabolism, mitochondrial metabolism, and redox homeostasis in cancer cells. Importantly, oncogenic signaling can confer a moonlighting function on core clock genes, effectively reshaping cellular metabolism to fuel cancer cell proliferation and drive tumor growth. These interwoven regulatory mechanisms constitute a distinctive feature of cancer cell metabolism. [ABSTRACT FROM AUTHOR]

Cellular stress response pathways (CSRPs) constantly monitor a changing microenvironment. Intrinsic and extrinsic stresses activate specific CSRPs that converge at the level of RNA metabolism, aiding cell survival. RNA G-quadruplexes (rG4s) are emerging stress-responsive cis -elements found in both the coding and noncoding transcriptomes. The dynamics of rG4s contribute to different aspects of RNA metabolism from transcription to decay. Cellular stress affects rG4s' dynamics and the functions of rG4-bearing transcripts. The rG4 proteome is highly enriched with RNA-binding proteins (RBPs) that contribute to cellular stress responses. Multiple RBPs bind rG4 transcripts and modulate their localization, stability, and translation in a stress-responsive manner. Interactions between rG4 transcripts and RBPs contribute to liquid–liquid phase separation (LLPS), the phenomenon that underlies the formation of membraneless compartments. Specifically, rG4s are implicated in the formation and dynamics of stress-responsive RNA granules, such as stress granules. Stress-induced rG4s are potential therapeutic targets for human disease. RNA G-quadruplexes (rG4s) are noncanonical secondary structures formed by guanine-rich sequences that are found in different regions of RNA molecules. These structures have been implicated in diverse biological processes, including translation, splicing, and RNA stability. Recent studies have suggested that rG4s play a role in the cellular response to stress. This review summarizes the current knowledge on rG4s under stress, focusing on their formation, regulation, and potential functions in stress response pathways. We discuss the molecular mechanisms that regulate the formation of rG4 under different stress conditions and the impact of these structures on RNA metabolism, gene expression, and cell survival. Finally, we highlight the potential therapeutic implications of targeting rG4s for the treatment of stress-related diseases through modulating cell survival. [ABSTRACT FROM AUTHOR]

Vγ9Vδ2 T cells are under investigation as alternative effector cells for adoptive cell therapy (ACT) in cancer. Despite promising in vitro results, anti-tumor efficacies in early clinical studies have been lower than expected, which could be ascribed to the complex interplay of tumor and immune cell metabolism competing for the same nutrients in the tumor microenvironment. To contribute to the scarce knowledge regarding gamma delta T-cell metabolism, we investigated the metabolic phenotype of 25-day-expanded Vγ9Vδ2 T cells and how it is intertwined with functionality. We found that Vγ9Vδ2 T cells displayed a quiescent metabolism, utilizing both glycolysis and oxidative phosphorylation (OXPHOS) for energy production, as measured in Seahorse assays. Upon T-cell receptor activation, both pathways were upregulated, and inhibition with metabolic inhibitors showed that Vγ9Vδ2 T cells were dependent on glycolysis and the pentose phosphate pathway for proliferation. The dependency on glucose for proliferation was confirmed in glucose-free conditions. Cytotoxicity against malignant melanoma was reduced by glycolysis inhibition but not OXPHOS inhibition. These findings lay the groundwork for further studies on manipulation of Vγ9Vδ2 T-cell metabolism for improved ACT outcome. [Display omitted] [ABSTRACT FROM AUTHOR]

Ischemic heart diseases and cardiomyopathies are characterized by hypoxia, energy starvation and mitochondrial dysfunction. HIF-1 acts as a cellular oxygen sensor, tuning the balance of metabolic and oxidative stress pathways to provide ATP and sustain cell survival. Acting on mitochondria, HIF-1 regulates different processes such as energy substrate utilization, oxidative phosphorylation and mitochondrial dynamics. In turn, mitochondrial homeostasis modifications impact HIF-1 activity. This underlies that HIF-1 and mitochondria are tightly interconnected to maintain cell homeostasis. Despite many evidences linking HIF-1 and mitochondria, the mechanistic insights are far from being understood, particularly in the context of cardiac diseases. Here, we explore the current understanding of how HIF-1, reactive oxygen species and cell metabolism are interconnected, with a specific focus on mitochondrial function and dynamics. We also discuss the divergent roles of HIF in acute and chronic cardiac diseases in order to highlight that HIF-1, mitochondria and oxidative stress interaction deserves to be deeply investigated. While the strategies aiming at stabilizing HIF-1 have provided beneficial effects in acute ischemic injury, some deleterious effects were observed during prolonged HIF-1 activation. Thus, deciphering the link between HIF-1 and mitochondria will help to optimize HIF-1 modulation and provide new therapeutic perspectives for the treatment of cardiovascular pathologies. [Display omitted] • Interplay between HIF-1 mitochondria and ROS stand at the forefront of cardiac diseases. • Depending on the hypoxic stress, HIF-1 deciphers a paradoxical response that deserves to be deeply investigated. • "HIF-1 and mitochondria" crosstalk exploration will open new avenues to understand cardiac diseases • Exploration of the "HIF-1-mitochondria" crosstalk will open new avenues to propose new therapeutic strategies. [ABSTRACT FROM AUTHOR]

As a main source of erythrocyte degradation products, the process of hemolysis is a key determinant in skewing proinflammatory signatures in myeloid cells. Hemolytic products such as heme and iron can elicit proinflammatory effects, activating endothelial tissues and innate immunity. Iron plays an important role in myeloid cell polarization by altering cellular signaling and metabolism, while also affecting chromatin modifications. The interplay between iron and myeloid cells can influence the progression of several diseases, including sickle cell disease, multiple sclerosis (MS), and cancers. Iron-loaded myeloid cells are spatially organized, often surrounding lesions in MS and tumor masses. Myeloid cell polarization is linked to the iron scavenging and storage functions of these cells in a tissue-dependent manner. New studies are advancing our knowledge of iron as a modulator of immune functions, cell metabolism, and myeloid cell polarization into proinflammatory or anti-inflammatory functions. Considering the significance of myeloid cells at steady state and under disease conditions, it is important to understand the different functions and effects of iron-loaded myeloid cells. Iron-associated myeloid cell functions can be disease-dependent and the cells may exhibit polarizing functions during inflammation. Myeloid cells that populate all human organs and blood are a versatile class of innate immune cells. They are crucial for sensing and regulating processes as diverse as tissue homeostasis and inflammation and are frequently characterized by their roles in either regulating or promoting inflammation. Recent studies in cultured cells and mouse models highlight the role of iron in skewing the functional properties of myeloid cells in tissue damage and repair. Here, we review certain emerging concepts on how iron influences and determines myeloid cell polarization in the context of its uptake, storage, and metabolism, including in conditions such as multiple sclerosis (MS), sickle cell disease, and tumors. [ABSTRACT FROM AUTHOR]

Protein lipoylation is critical for the optimal function of several key enzymes in mitochondria; thus, regulating lipoylation greatly impacts mitochondrial function and cell metabolism. As a novel cell death pathway, cuproptosis involves two essential mechanisms: protein lipoylation and copper overload. Recent studies indicate that the ferredoxin 1 (FDX1)/ lipoic acid synthase (LIAS) axis links lipoylation and excessive copper ions and plays a key role in cuproptosis. New techniques suggest there are more potential lipoylated proteins. Given the pivotal role of mitochondria in cell metabolism, targeting protein lipoylation could be a promising strategy for disease therapy. Protein lipoylation, a crucial post-translational modification (PTM), plays a pivotal role in mitochondrial function and emerges as a key player in cell death through cuproptosis. This novel copper-driven cell death pathway is activated by excessive copper ions binding to lipoylated mitochondrial proteins, disrupting energy production and causing lethal protein aggregation and cell death. The intricate relationship among protein lipoylation, cellular energy metabolism, and cuproptosis offers a promising avenue for regulating essential cellular functions. This review focuses on the mechanisms of lipoylation and its significant impact on cell metabolism and cuproptosis, emphasizing the key genes involved and their implications for human diseases. It offers valuable insights into targeting dysregulated cellular metabolism for therapeutic purposes. [ABSTRACT FROM AUTHOR]

Many biopharmaceutical manufacturers and researchers seek a comprehensive understanding and real-time monitoring of cell growth and metabolism to enhance the cell culture process. However, the biological complexity of cells and the intricacies of cell culture process have impeded the development of analytical and monitoring techniques. This study aims to present a complete picture of cell culture processes using in-line Raman spectroscopy. Initially, we proposed an efficient method for discriminating and removing anomalous spectra. Subsequently, Raman-based models for 27 components including amino acid, organic acid, lipid, organic alkali, alkaloid, alcohol, saccharide, biomass, physicochemical parameter, ion, and protein titer were established. The Q2 obtained by these models was greater than 0.8, and the relative percent difference (RPD) from external validation was greater than 2.0 except for glucose. Additionally, the proposed control charts effectively monitored normal batches and abnormal conditions such as bacterial contamination and insufficient feeding. In cases of unexplained culture abnormalities, the proposed workflow offered explanations. [Display omitted] • Monitoring and analysis of CHO cell culture processes using Raman spectroscopy. • Raman-based models of 27 different components in bioreactors were established. • Process trajectory of normal cultivation was described. • Abnormal cultivation was identified and explained. [ABSTRACT FROM AUTHOR]

• Cells increase traction forces and energy usage with substrate stiffness. • Cells on stiffer substrates convert energy into traction forces more efficiently. • Cell behaviors are regulated by both energy production and utilization efficiency. To form blood vessels, endothelial cells rearrange their cytoskeleton, generate traction stresses, migrate, and proliferate, all of which require energy. Despite these energetic costs, stiffening of the extracellular matrix promotes tumor angiogenesis and increases cell contractility. However, the interplay between extracellular matrix, cell contractility, and cellular energetics remains mechanistically unclear. Here, we utilized polyacrylamide substrates with various stiffnesses, a real-time biosensor of ATP, and traction force microscopy to show that endothelial cells exhibit increasing traction forces and energy usage trend as substrate stiffness increases. Inhibition of cytoskeleton reorganization via ROCK inhibition resulted in decreased cellular energy efficiency, and an opposite trend was found when cells were treated with manganese to promote integrin affinity. Altogether, our data reveal a link between matrix stiffness, cell contractility, and cell energetics, suggesting that endothelial cells on stiffer substrates can better convert intracellular energy into cellular traction forces. Given the critical role of cellular metabolism in cell function, our study also suggests that not only energy production but also the efficiency of its use plays a vital role in regulating cell behaviors and may help explain how increased matrix stiffness promotes angiogenesis. [ABSTRACT FROM AUTHOR]

Pyridoxal kinase (PdxK) is a vitamin B 6 salvage pathway enzyme which produces pyridoxal phosphate. We have investigated the impact of PdxK deletion in Leishmania donovani on parasite survivability, infectivity and cellular metabolism. Ld PdxK mutants were generated by gene replacement strategy. All mutants showed significant reduction in growth in comparison to wild type. For PdxK mediated biochemical perturbations, only heterozygous mutants and complementation mutants were used as the growth of null mutants were compromised. Heterozygous mutant showed reduction in vitro infectivity and higher cytosolic and mitochondrial ROS levels. Glutathione levels decreased significantly in heterozygous mutant indicating its involvement in cellular oxidative metabolism. Pyridoxal kinase gene deletion resulted in reduced ATP levels in parasites and arrest at G 0 /G 1 phase of cell cycle. All these perturbations were rescued by PdxK gene complementation. This is the first report to confirm that Ld PdxK plays an indispensable role in cell survival, pathogenicity, redox metabolism and cell cycle progression of L. donovani parasites. These results provide substantial evidence supporting PdxK as a therapeutic target for the development of specific antileishmanial drug candidates. [Display omitted] • Leishmania donovani pdxk alleles were deleted from parasite genome. • The growth of null mutants were compromised pointing towards the indispensable nature of pyridoxal kinase. • Deletion of pdxk alleles led to increased ROS generation and decreased GSH levels. • pdxk deletion led to G o /G 1 phase cell cycle arrest. [ABSTRACT FROM AUTHOR]

With investigators looking to expand engineered T cell therapies such as CAR-T to new tumor targets and patient populations, a variety of cell manufacturing platforms have been developed to scale manufacturing capacity using closed and/or automated systems. Such platforms are particularly useful for solid tumor targets, which typically require higher CAR-T cell doses. Although T cell phenotype and function are key attributes that often correlate with therapeutic efficacy, how manufacturing platforms influence the final CAR-T cell product is currently unknown. We compared 4 commonly used T cell manufacturing platforms (CliniMACS Prodigy, Xuri W25 rocking platform, G-Rex gas-permeable bioreactor, static bag culture) using identical media, stimulation, culture length, and donor starting material. Selected CD4+CD8+ cells were transduced with lentiviral vector incorporating a CAR targeting FGFR4, a promising target for pediatric sarcoma. We observed significant differences in overall expansion over the 14-day culture; bag cultures had the highest capacity for expansion while the Prodigy had the lowest (481-fold versus 84-fold, respectively). Strikingly, we also observed considerable differences in the phenotype of the final product, with the Prodigy significantly enriched for CCR7+CD45RA+ naïve/stem central memory (T n/scm)-like cells at 46% compared to bag and G-Rex with 16% and 13%, respectively. Gene expression analysis also showed that Prodigy CAR-Ts are more naïve, less cytotoxic and less exhausted than bag, G-Rex, and Xuri CAR-Ts, and pointed to differences in cell metabolism that were confirmed via metabolic assays. We hypothesized that dissolved oxygen level, which decreased substantially during the final 3 days of the Prodigy culture, may contribute to the observed differences in T cell phenotype. By culturing bag and G-Rex cultures in 1% O 2 from day 5 onward, we could generate >60% T n/scm -like cells, with longer time in hypoxia correlating with a higher percentage of T n/scm -like cells. Intriguingly, our results suggest that oxygenation is responsible, at least in part, for observed differences in T cell phenotype among bioreactors and suggest hypoxic culture as a potential strategy prevent T cell differentiation during expansion. Ultimately, our study demonstrates that selection of bioreactor system may have profound effects not only on the capacity for expansion, but also on the differentiation state of the resulting CAR-T cells. [ABSTRACT FROM AUTHOR]

Itaconate (ITA) research began as an industrial application as a precursor for polymer synthesis and has advanced significantly since the discovery of the Irg1 gene in 1995. A deep exploration of ITA as an immunomodulatory metabolite has provided a profound understanding of the functional significance and mechanistic basis of metabolic reprogramming in the context of inflammatory responses and cancer development. ITA rapidly accumulates to high levels in myeloid cells during both infectious and sterile inflammatory conditions. This metabolite acts as a signaling molecule to modulate immune response by binding, covalently and noncovalently, to multiple proteins and regulating the oxidative response, cell metabolism, epigenetic modification, and gene expression intrinsically, and activating G protein-coupled receptor (GPCR) signaling via paracrine signaling. Genetic and preclinical studies in animal models support the therapeutic potential for treating a wide range of inflammatory diseases and cancer. Developing novel therapies based on ITA and targeting aconitate decarboxylase (ACOD1) holds exciting potential for treating many diseases that currently lack effective therapy. Immune cells undergo rapid and extensive metabolic changes during inflammation. In addition to contributing to energetic and biosynthetic demands, metabolites can also function as signaling molecules. Itaconate (ITA) rapidly accumulates to high levels in myeloid cells under infectious and sterile inflammatory conditions. This metabolite binds to and regulates the function of diverse proteins intracellularly to influence metabolism, oxidative response, epigenetic modification, and gene expression and to signal extracellularly through binding the G protein-coupled receptor (GPCR). Administration of ITA protects against inflammatory diseases and blockade of ITA production enhances antitumor immunity in preclinical models. In this article, we review ITA metabolism and its regulation, discuss its target proteins and mechanisms, and conjecture a rationale for developing ITA-based therapeutics to treat inflammatory diseases and cancer. [ABSTRACT FROM AUTHOR]

Macrophage polarization towards the M1 phenotype under bacterial product-related exposure (LPS) requires a rapid change in gene expression patterns and cytokine production along with a metabolic rewiring. Metabolic pathways and redox reactions are such tightly connected, giving rise to an area of research referred to as immunometabolism. A role in this context has been paid to the master redox-sensitive regulator Nuclear factor erythroid 2-related factor 2 (Nrf2) and to the 5'-ectonucleotidase CD73, a marker related to macrophage metabolism rearrangement under pro-inflammatory conditions. In this light, a cell model of LPS-stimulated macrophages has been established and nine 4,7-dihydro-4-ethylpyrazolo[ l ,5- a ]pyrimidin-7-ones with a potential anti-inflammatory effect have been administered. Our data highlight that two selected compounds (namely, 5 and 8) inhibit the LPS-induced Nrf2 nuclear translocation and ameliorate the activity rate of the antioxidant enzyme catalase. Additionally, the pyridine-containing compound (8) promotes the shift from the pro-inflammatory immunophenotype M1 to the pro-resolving M2 one, by downregulating CD80 and iNOS and by enhancing CD163 and TGFβ1 expression. Most importantly, CD73 is modulated by these compounds as well as the lactate production. Our data demonstrate that pyrazolo[ l ,5- a ]pyrimidine derivatives are effective as anti-inflammatory compounds. Furthermore, these pyrazolo[ l ,5- a ]pyrimidines exert their action via CD73-related signaling and modulation of cell metabolism of activated macrophages. • 4,7-Dihydro-4-ethylpyrazolo[ l ,5- a ]pyrimidin-7-ones displayed potential anti-inflammatory effect. • Two compounds modulated the LPS-induced Nrf2 nuclear translocation. • CD73, a protein related to macrophage metabolism under pro-inflammatory conditions, is modulated. • The expression of markers associated with the shift from M1 to M2 immunophenotype in macrophages was studied. [ABSTRACT FROM AUTHOR]

As part of the classical renin-angiotensin system, the peptidase angiotensin-converting enzyme (ACE) makes angiotensin II which has myriad effects on systemic cardiovascular function, inflammation, and cellular proliferation. Less well known is that macrophages and neutrophils make ACE in response to immune activation which has marked effects on myeloid cell function independent of angiotensin II. Here, we discuss both classical (angiotensin) and nonclassical functions of ACE and highlight mice called ACE 10/10 in which genetic manipulation increases ACE expression by macrophages and makes these mice much more resistant to models of tumors, infection, atherosclerosis, and Alzheimer’s disease. In another model called NeuACE mice, neutrophils make increased ACE and these mice are much more resistant to infection. In contrast, ACE inhibitors reduce neutrophil killing of bacteria in mice and humans. Increased expression of ACE induces a marked increase in macrophage oxidative metabolism, particularly mitochondrial oxidation of lipids, secondary to increased peroxisome proliferator-activated receptor α expression, and results in increased myeloid cell ATP. ACE present in sperm has a similar metabolic effect, and the lack of ACE activity in these cells reduces both sperm motility and fertilization capacity. These nonclassical effects of ACE are not due to the actions of angiotensin II but to an unknown molecule, probably a peptide, that triggers a profound change in myeloid cell metabolism and function. Purifying and characterizing this peptide could offer a new treatment for several diseases and prove potentially lucrative. [ABSTRACT FROM AUTHOR]

There is a paucity of experimental and mathematical models that take the extracellular and cellular components of the tumour microenvironment into account. Basic models only integrate cancer cells in isolation of the stromal and matrix surrounding and are flawed due to the lack of cancer-supporting cells and factors of the extracellular matrix, which are both essential for cell–cell and cell–matrix interactions and to produce metabolites that fuel cancer cells. The tumour microenvironment has a poor oxygen diffusion, low nutrient levels, and high interstitial pressure and, as a consequence, to exist in this nutrient-limiting conditions, cancer cells reprogram their metabolism, uptake of glucose, and mitochondrial pathways. We summarise approaches to model the metabolic crosstalk within the tumour microenvironment from a biological, engineering, and mathematical perspective, by discussing the perspectives and limitations. Biomaterial-based platforms and tumour-engineered systems are better approaches to model the tumour metabolism. Tumours are heterogeneous tissues containing diverse populations of cells and an abundant extracellular matrix (ECM). This tumour microenvironment prompts cancer cells to adapt their metabolism to survive and grow. Besides epigenetic factors, the metabolism of cancer cells is shaped by crosstalk with stromal cells and extracellular components. To date, most experimental models neglect the complexity of the tumour microenvironment and its relevance in regulating the dynamics of the metabolism in cancer. We discuss emerging strategies to model cellular and extracellular aspects of cancer metabolism. We highlight cancer models based on bioengineering, animal, and mathematical approaches to recreate cell–cell and cell–matrix interactions and patient-specific metabolism. Combining these approaches will improve our understanding of cancer metabolism and support the development of metabolism-targeting therapies. [ABSTRACT FROM AUTHOR]

Acute lung injury (ALI) represents a critical respiratory condition typified by rapid-onset lung inflammation, contributing to elevated morbidity and mortality rates. Central to ALI pathogenesis lies macrophage dysfunction, characterized by an overabundance of pro-inflammatory cytokines and a shift in metabolic activity towards glycolysis. This study emphasizes the crucial function of glucose metabolism in immune cell function under inflammatory conditions and identifies hexokinase 2 (HK2) as a key regulator of macrophage metabolism and inflammation. Given the limitations of HK2 inhibitors, we propose the CRISPR/Cas9 system for precise HK2 downregulation. We developed an aerosolized core-shell liposomal nanoplatform (CSNs) complexed with CaP for efficient drug loading, targeting lung macrophages. Various CSNs were synthesized to encapsulate an mRNA based CRISPR/Cas9 system (mCas9/gHK2), and their gene editing efficiency and HK2 knockout were examined at both gene and protein levels in vitro and in vivo. The CSN-mCas9/gHK2 treatment demonstrated a significant reduction in glycolysis and inflammation in macrophages. In an LPS-induced ALI mouse model, inhaled CSN-mCas9/gHK2 mitigated the proinflammatory tumor microenvironment and reprogrammed glucose metabolism in the lung, suggesting a promising strategy for ALI prevention and treatment. This study highlights the potential of combining CRISPR/Cas9 gene editing with inhalation delivery systems for effective, localized pulmonary disease treatment, underscoring the importance of targeted gene modulation and metabolic reprogramming in managing ALI. This study investigates an inhalable CRISPR/Cas9 gene editing system targeting pulmonary macrophages, with the aim of modulating glucose metabolism to alleviate Acute Lung Injury (ALI). The research highlights the role of immune cell metabolism in inflammation, as evidenced by changes in macrophage glucose metabolism and a notable reduction in pulmonary edema and inflammation. Additionally, observed alterations in macrophage polarization and cytokine levels in bronchoalveolar lavage fluid suggest potential therapeutic implications. These findings not only offer insights into possible ALI treatments but also contribute to the understanding of immune cell metabolism in inflammatory diseases, which could be relevant for various inflammatory and metabolic disorders. [Display omitted] [ABSTRACT FROM AUTHOR]

Modulating the metabolism of cancer cells, immune cells, or both is a promising strategy to potentiate cancer immunotherapy in the nutrient-competitive tumor microenvironment. Glutamine has emerged as an ideal target as cancer cells highly rely on glutamine for replenishing the tricarboxylic acid cycle in the process of aerobic glycolysis. However, non-specific glutamine restriction may induce adverse effects in unconcerned tissues and therefore glutamine inhibitors have achieved limited success in the clinic so far. Here we report the synthesis and evaluation of a redox-responsive prodrug of 6-Diazo-5-oxo-L-norleucine (redox-DON) for tumor-targeted glutamine inhibition. When applied to treat mice bearing subcutaneous CT26 mouse colon carcinoma, redox-DON exhibited equivalent antitumor efficacy but a greatly improved safety profile, particularly, in spleen and gastrointestinal tract, as compared to the state-of-the-art DON prodrug, JHU083. Furthermore, redox-DON synergized with checkpoint blockade antibodies leading to durable cures in tumor-bearing mice. Our results suggest that redox-DON is a safe and effective therapeutic for tumor-targeted glutamine inhibition showing promise for enhanced metabolic modulatory immunotherapy. The approach of reversible chemical modification may be generalized to other metabolic modulatory drugs that suffer from overt toxicity. [Display omitted] [ABSTRACT FROM AUTHOR]

Heme is an iron-containing prosthetic group necessary for the function of several proteins termed "hemoproteins." Erythrocytes contain most of the body's heme in the form of hemoglobin and contain high concentrations of free heme. In nonerythroid cells, where cytosolic heme concentrations are 2 to 3 orders of magnitude lower, heme plays an essential and often overlooked role in a variety of cellular processes. Indeed, hemoproteins are found in almost every subcellular compartment and are integral in cellular operations such as oxidative phosphorylation, amino acid metabolism, xenobiotic metabolism, and transcriptional regulation. Growing evidence reveals the participation of heme in dynamic processes such as circadian rhythms, NO signaling, and the modulation of enzyme activity. This dynamic view of heme biology uncovers exciting possibilities as to how hemoproteins may participate in a range of physiologic systems. Here, we discuss how heme is regulated at the level of its synthesis, availability, redox state, transport, and degradation and highlight the implications for cellular function and whole organism physiology. [ABSTRACT FROM AUTHOR]

Estrogen receptor (ER) signaling is modulated by systemic metabolism in ER+ breast tumors. Endocrine therapies can alter cellular metabolism in ER+ breast tumors. Gut microbiota-derived factors change ER activity and endocrine therapy responsiveness. Targeting of metabolic pathways using dietary or pharmacological approaches can be used in the clinic to improve endocrine therapy efficacy. Estrogen receptor-positive (ER+) breast tumors have a better overall prognosis than ER− tumors; however, there is a sustained risk of recurrence. Mounting evidence indicates that genetic and epigenetic changes associated with resistance impact critical signaling pathways governing cell metabolism. This review delves into recent literature concerning the metabolic pathways regulated in ER+ breast tumors by the availability of nutrients and endocrine therapies and summarizes research on how changes in systemic and gut microbial metabolism can affect ER activity and responsiveness to endocrine therapy. As targeting of metabolic pathways using dietary or pharmacological approaches enters the clinic, we provide an overview of the supporting literature and suggest future directions. [ABSTRACT FROM AUTHOR]

Cisplatin (cis -Dichlorodiamineplatinum[II], CDDP) is generally accepted as a platinum-based alkylating agent type of the DNA-damaging anticancer drug, which is widely administrated in clinical treatment of many solid tumors. The pharmacological effect of CDDP is mainly achieved by replacing the chloride ion (Cl − ) in its structure with H 2 O to form active substances with the strong electrophilic properties and then react with any nucleophilic molecules, primarily leading to genomic DNA damage and subsequent cell death. In this process, those target genes driven by the consensus electrophilic and/or antioxidant response elements (EpREs/AREs) in their promoter regions are also activated or repressed by CDDP. Thereby, we here examined the expression profiling of such genes regulated by two principal antioxidant transcription factors Nrf1 and Nrf2 (both encoded by Nfe2l1 and Nfe2l2, respectively) in diverse cellular signaling responses to this intervention. The results demonstrated distinct cellular metabolisms, molecular pathways and signaling response mechanisms by which Nrf1 and Nrf2 as the drug targets differentially contribute to the anticancer efficacy of CDDP on hepatoma cells and xenograft tumor mice. Interestingly, the role of Nrf1, rather than Nrf2, is required for the anticancer effect of CDDP, to suppress malignant behavior of HepG2 cells by differentially monitoring multi-hierarchical signaling to gene regulatory networks. To our surprise, it was found there exists a closer relationship of Nrf1α than Nrf2 with DNA repair, but the hyperactive Nrf2 in Nrf1α –∕– cells manifests a strong correlation with its resistance to CDDP, albeit their mechanistic details remain elusive. [Display omitted] • Differential activation of Nrf1 and Nrf2 in distinct cellular responses to cisplatin. • Distinct mechanisms whereby Nrf1 and Nrf2 contribute to the efficacy of cisplatin. • Substantial repression of WT rather than Nrf1α –∕– tumor by cisplatin in model mice. • Significant chemoresistance of Nrf1α –∕– rather than Nrf2 –∕– cell lines to cisplatin. • A rather requirement of Nrf1 than Nrf2 for transcription of H2AX in the DNA repair. [ABSTRACT FROM AUTHOR]

Schizosaccharomyces pombe Php4 is the regulatory subunit of the CCAAT-binding complexes and plays an important role in the regulation of iron homeostasis and iron-dependent metabolism. Here, we show that Php4 undergoes ubiquitindependent degradation in the late logarithmic and stationary phases. The degradation and ubiquitination of Php4 could be attenuated by deletion of hul6, a gene encoding a putative HECT-type E3 ubiquitin ligase. The expression levels of Hul6 and Php4 are oppositely regulated during cell growth. Hul6 interacts with the C-terminal region of Php4. Two lysine residues (K217 and K274) located in the C-terminal region of Php4 are required for its polyubiquitination. Increasing the levels of Php4 by deletion of hul6 or overexpression of php4 decreased expression of Php4 target proteins involved in iron-dependent metabolic pathways such as the tricarboxylic cycle and mitochondrial oxidative phosphorylation, thus causing increased sensitivity to high-iron and reductions in succinate dehydrogenase and mitochondrial complex II activities. Hul6 is located primarily in the mitochondrial outer membrane and most likely targets cytosolic Php4 for ubiquitination and degradation. Taken together, our data suggest that Hul6 regulates irondependent metabolism through degradation of Php4 under normal growth conditions. Our results also suggest that Hul6 promotes iron-dependent metabolism to help the cell to adapt to a nutrient-starved growth phase. [ABSTRACT FROM AUTHOR]

Atherosclerosis is the main cause of a series of fatal cardiovascular diseases, characterized by pathological accumulation of apoptotic cells and lipids. Pro-phagocytic antibody-based or pro-autophagy gene-based therapies are currently being explored to stimulate the phagocytic clearance of apoptotic cells and lipid metabolism; however, monotherapies are only moderately effective or require high doses with unacceptable side effects. Herein, we engineered a specific nano-bioconjugate loaded with antisense oligonucleotides (ASOs) of mammalian target of rapamycin (mTOR) and modified with anti-signal-regulated protein-α antibody (aSIRPα) for macrophage-mediated atherosclerosis therapy. The specific nano-bioconjugate utilized acid-responsive calcium phosphate (CaP) as a carrier to load mTOR ASOs, coated with lipid on the surface of CaP nanoparticles (ASOs@CaP), and subsequently modified with aSIRPα. The resulting nano-bioconjugates could accumulate within atherosclerotic plaques, target to macrophages and reactivate lesional phagocytosis through blocking the CD47-SIRPα signaling axis. In addition, efficient delivery of mTOR ASOs inhibited mTOR expression, which significantly restored impaired autophagy. The combined action of mTOR ASOs and aSIRPα reduced apoptotic cells and lipids accumulation. This nanotherapy significantly reduced plaque burden and inhibited progression of atherosclerotic lesions. These results show the potential of specific nano-bioconjugates for the prevention of atherosclerotic cardiovascular disease. Atherosclerosis is the main cause of a series of fatal cardiovascular diseases. Pro-phagocytic antibody-based or pro-autophagy gene-based therapies are currently being explored to stimulate the phagocytic clearance of apoptotic cells and lipid metabolism; however, monotherapies are only moderately effective or require high doses with unacceptable side effects. Herein, we engineered a specific nano-bioconjugate loaded with antisense oligonucleotides (ASOs) of mammalian target of rapamycin (mTOR) and modified with anti-signal-regulated protein-α antibody (aSIRPα) for macrophage-mediated atherosclerosis therapy. Our study demonstrated that the combined action of mTOR ASOs and aSIRPα reduced apoptotic cells and lipids accumulation. This nanotherapy significantly reduced plaque burden and inhibited progression of atherosclerotic lesions. These results show the potential of specific nano-bioconjugates for the prevention of atherosclerotic cardiovascular disease. [Display omitted] [ABSTRACT FROM AUTHOR]

There is dynamic and bidirectional regulation of cell metabolism and cell cycle progression that is incompletely understood. Changes to cellular metabolism, including redox state, can directly affect the activity of cell cycle regulators and signaling pathways to impact cell cycle control. Mitochondrial metabolism changes dramatically during the cell cycle and most acutely during mitosis. Cells adjust cell cycle duration and growth rate to couple cell size to cell cycle progression and maintain uniform cell size across generations. Disruption of protein synthesis or DNA replication can drive cells to exit the cell cycle and become quiescent. The relationship between metabolism and cell cycle progression is complex and bidirectional. Cells must rewire metabolism to meet changing biosynthetic demands across cell cycle phases. In turn, metabolism can influence cell cycle progression through direct regulation of cell cycle proteins, through nutrient-sensing signaling pathways, and through its impact on cell growth, which is linked to cell division. Furthermore, metabolism is a key player in mediating quiescence–proliferation transitions in physiologically important cell types, such as stem cells. How metabolism impacts cell cycle progression, exit, and re-entry, as well as how these processes impact metabolism, is not fully understood. Recent advances uncovering mechanistic links between cell cycle regulators and metabolic processes demonstrate a complex relationship between metabolism and cell cycle control, with many questions remaining. [ABSTRACT FROM AUTHOR]

Sirtuin 3 (SIRT3) shows distinct roles in reprogramming cell metabolism and shaping tumor phenotypes, mainly by deacetylating diverse substrates. The context-specific functions of SIRT3 may both promote and suppress tumorigenesis. Recent advances in SIRT3-targeted drug discovery, including inhibitors and activators, hold significant promise for cancer therapeutics. A deep understanding of the mechanisms of SIRT3-targeted small molecules could prompt the development of SIRT3-targeted therapy. Sirtuin 3 (SIRT3), an NAD+-dependent deacetylase, plays a key role in the modulation of metabolic reprogramming and regulation of cell death, as well as in shaping tumor phenotypes. Owing to its critical role in determining tumor-type specificity or the direction of tumor evolution, the development of small-molecule modulators of SIRT3, including inhibitors and activators, is of significant interest. In this review, we discuss recent studies on the oncogenic or tumor-suppressive functions of SIRT3, evaluate advances in SIRT3-targeted drug discovery, and present potential avenues for the design of small-molecule modulators of SIRT3 for cancer therapy. [ABSTRACT FROM AUTHOR]

• The non-toxigenic M. wesenbergii FACHB-908 was more sensitive to C 14 -benzalkonium chloride (BAC-14) than the two toxigenic M. aeruginosa. • Photosynthetic and metabolic responses showed higher sensitivity to BAC-14 than growth inhibition. • BAC-14 specific responses in glycerophospholipid metabolism, synchronized by dysregulation of energy metabolism and amino acid biosynthesis. • BAC-14 induced elevation of extracellular microcystin contents in the absence of growth inhibition of the two toxigenic Microcystis. Quaternary ammonium compounds (QACs) are commonly used in a variety of consumer and commercial products, typically as a component of disinfectants. During the COVID-19 pandemic, QACs became one of the primary agents utilized to inactivate the SARS-CoV-2 virus on surfaces. However, the ecotoxicological effects of QACs upon aquatic organisms have not been fully assessed. In this study, we examined the effects of a widely used QAC (benzalkonium chloride-C 14 , BAC-14) on two toxigenic Microcystis strains and one non-toxigenic freshwater Microcystis strain and carried out an analysis focused on primary, adaptive and compensatory stress responses at apical (growth and photosynthesis) and metabolic levels. This analysis revealed that the two toxic Microcystis strains were more tolerant than the non-toxic strain, with 96 hr-EC 50 values of 0.70, 0.76, and 0.38 mg/L BAC-14 for toxigenic M. aeruginosa FACHB-905, toxigenic M. aeruginosa FACHB-469, and non-toxigenic M. wesenbergii FACHB-908, respectively. The photosynthetic activities of the Microcystis , assessed via F v /F m values, were significantly suppressed under 0.4 mg/L BAC-14. Furthermore, this analysis revealed that BAC-14 altered 14, 12, and 8 metabolic pathways in M. aeruginosa FACHB-905, M. aeruginosa FACHB-469, and M. wesenbergii FACHB-908, respectively. It is noteworthy that BAC-14 enhanced the level of extracellular microcystin production in the toxigenic Microcystis strains , although cell growth was not significantly affected. Collectively, these data show that BAC-14 disrupted the physiological and metabolic status of Microcystis cells and stimulated the production and release of microcystin, which could result in damage to aquatic systems. [Display omitted] [ABSTRACT FROM AUTHOR]

Cellular therapies are currently employed to treat a variety of disease processes. For T cell-based therapies, success often relies on the metabolic fitness of the T cell product, where cells with enhanced metabolic capacity demonstrate improved in vivo efficacy. AMP-activated protein kinase (AMPK) is a cellular energy sensor which combines environmental signals with cellular energy status to enforce efficient and flexible metabolic programming. We hypothesized that increasing AMPK activity in human T cells would augment their oxidative capacity, creating an ideal product for adoptive cellular therapies. Lentiviral transduction of the regulatory AMPKγ2 subunit stably enhanced intrinsic AMPK signaling and promoted mitochondrial respiration with increased basal oxygen consumption rates, higher maximal oxygen consumption rate, and augmented spare respiratory capacity. These changes were accompanied by increased proliferation and inflammatory cytokine production, particularly within restricted glucose environments. Introduction of AMPKγ2 into bulk CD4 T cells decreased RNA expression of canonical Th2 genes, including the cytokines interleukin (IL)-4 and IL-5, while introduction of AMPKγ2 into individual Th subsets universally favored proinflammatory cytokine production and a downregulation of IL-4 production in Th2 cells. When AMPKγ2 was overexpressed in regulatory T cells, both in vitro proliferation and suppressive capacity increased. Together, these data suggest that augmenting intrinsic AMPK signaling via overexpression of AMPKγ2 can improve the expansion and functional potential of human T cells for use in a variety of adoptive cellular therapies. [ABSTRACT FROM AUTHOR]

One-carbon metabolism is a central metabolic pathway critical for the biosynthesis of several amino acids, methyl group donors, and nucleotides. The pathway mostly relies on the transfer of a carbon unit from the amino acid serine, through the cofactor folate (in its several forms), and to the ultimate carbon acceptors that include nucleotides and methyl groups used for methylation of proteins, RNA, and DNA. Nucleotides are required for DNA replication, DNA repair, gene expression, and protein translation, through ribosomal RNA. Therefore, the one-carbon metabolism pathway is essential for cell growth and function in all cells, but is specifically important for rapidly proliferating cells. The regulation of onecarbon metabolism is a critical aspect of the normal and pathological function of the pathway, such as in cancer, where hijacking these regulatory mechanisms feeds an increased need for nucleotides. One-carbon metabolism is regulated at several levels: via gene expression, posttranslational modification, subcellular compartmentalization, allosteric inhibition, and feedback regulation. In this review, we aim to inform the readers of relevant one-carbon metabolism regulation mechanisms and to bring forward the need to further study this aspect of one-carbon metabolism. The review aims to integrate two major aspects of cancer metabolism--signaling downstream of nutrient sensing and one-carbon metabolism, because while each of these is critical for the proliferation of cancerous cells, their integration is critical for comprehensive understating of cellular metabolism in transformed cells and can lead to clinically relevant insights. [ABSTRACT FROM AUTHOR]

Branched chain α-ketoacid dehydrogenase complex (BCKDC) is the rate-limiting enzyme in branched chain amino acid (BCAA) catabolism, a metabolic pathway with great importance for human health. BCKDC belongs to the mitochondrial α-ketoacid dehydrogenase complex family, which also includes pyruvate dehydrogenase complex and oxoglutarate dehydrogenase complex. Here, we revealed that BCKDC can be substantially inhibited by reactive nitrogen species (RNS) via a mechanism similar to what we recently discovered with pyruvate dehydrogenase complex and oxoglutarate dehydrogenase complex—RNS can cause inactivating covalent modifications of the lipoic arm on its E2 subunit. In addition, we showed that such reaction between RNS and the lipoic arm of the E2 subunit can further promote inhibition of the E3 subunits of α-ketoacid dehydrogenase complexes. We examined the impacts of this RNS-mediated BCKDC inhibition in muscle cells, an important site of BCAA metabolism, and demonstrated that the nitric oxide production induced by cytokine stimulation leads to a strong inhibition of BCKDC activity and BCAA oxidation in myotubes and myoblasts. More broadly, nitric oxide production reduced the level of functional lipoic arms across the multiple α-ketoacid dehydrogenases and led to intracellular accumulation of their substrates (α-ketoacids), decrease of their products (acyl-CoAs), and a lower cellular energy charge. In sum, this work revealed a new mechanism for BCKDC regulation, demonstrated that RNS can generally inhibit all α-ketoacid dehydrogenases, which has broad physiological implications across multiple cell types, and elucidated the mechanistic connection between RNS-driven inhibitory modifications on the E2 and E3 subunits of αketoacid dehydrogenases. [ABSTRACT FROM AUTHOR]

The pharmacological behavior of natural killer (NK) cells is deeply affected by their metabolic activity, and their effector functions are a response to nutrient availability, glycolytic activity, and lipid metabolism in tumors. Disruptions in these processes lead to bioenergetic dysfunction via immunometabolic reprogramming. We discuss cutting-edge concepts in NK cell metabolism, in particular the notion that metabolic substrate flexibility enables NK cells to adapt to different nutrient sources, and their reprogrammed metabolism affects their pharmacology, trafficking, and activation. We focus on the relevance of the metabolic processes and oncometabolites that drive alterations in immunological signaling on NK cells, their growing relevance to immunotherapy, and how they can be pharmacologically targeted to regulate NK cells toward restored metabolic fitness. The adaptation of natural killer (NK) cells to conditions in the microenvironment of tumors is deeply affected by their metabolic activity, itself a result of nutrient availability and the metabolism of the cancer cells themselves. Elevated rates of glycolysis and lipid metabolism in cancers not only lead to the accumulation of immunosuppressive byproducts but also contribute to an environment of elevated concentrations of extracellular metabolites. This results in altered NK cell bioenergetics through changes in transcriptional and translational profiles, ultimately affecting their pharmacology and impairing NK cell responses. However, understanding the metabolic processes that drive alterations in immunological signaling on NK cells remains both difficult and vastly underexplored. We discuss the varied and complex drivers of NK cell metabolism in homeostasis and the tumor microenvironment (TME), challenges associated with their targetability, and unexplored therapeutic opportunities. [ABSTRACT FROM AUTHOR]

Cellular metabolism is an important feature of spermatozoa that deserves more insights to be fully understood, in particular in porcine semen physiology. The present study aims to characterize the balance between glycolytic and oxidative metabolism in boar sperm cells. Agilent Seahorse technology was used to assess both oxygen consumption rate (OCR), as an oxidative metabolism index, and extracellular acidification rate (ECAR), as a glycolytic index. Different metabolic parameters were studied on freshly ejaculated sperm cells (identified as day zero sample, d0) and after one day of storage at 17 °C in Androhep extender (d1). Mitochondrial ATP production rate (MitoATP) was higher than the glycolytic ATP production rate (glycoATP) at both d0 and d1 while at d1 the amount of ATP production decreased, in particular, due to OXPHOS reduction. Conversely, glycoATP was not significantly different between d0 and d1. Interestingly, OCR profile showed no different bioenergetic parameters (i.e. ATP turnover, basal or maximal respiration, and spare respiration) between d0 and d1, thus indicating that sperm cell metabolism was reversibly decreased by preservation conditions. Other metabolic parameters showed the same trend, irrespective of the storage time: under stressed conditions (oligomycin plus FCCP), spermatozoa showed an increase in mitochondrial respiration while the metabolic potential of glycolysis did not undergo variations when compared to baseline metabolism. The rate of oxidation of fuel substrates – glucose, fatty acids, and glutamine – showed that sperm reliance on glucose oxidation to maintain baseline respiration was higher than fatty acids or glutamine. Interestingly spermatozoa demonstrated to have a low "capacity" parameter, which indicates that they cannot use only a single fuel substrate to produce energy. This feature of sperm metabolism to be unable to increase oxidation of a particular fuel to compensate for inhibition of alternative fuel pathway(s) was demonstrated by the negative value of "flexibility". Our results showed that ATP production in boar sperm cells relied on mitochondrial oxidative metabolism in freshly ejaculated cells, while, under liquid storage conditions, their oxidative metabolism decreased while the glycolysis remained constant. These results open new fields of research in the preservation techniques of boar sperm cells. • Oxidative cell metabolism of boar spermatozoa was storage time dependent. • The glycolysis activity was irrespective of the storage time. • In freshly ejaculated sperm cells, ATP was predominantly synthesized in mitochondria. • In sperm cells, the primary substrate for ATP production is likely glucose rather than glutamine or fatty acids. [ABSTRACT FROM AUTHOR]

Chemoresistance constitute a major obstacle in cancer treatment, leading to limited options and decreased patient survival. Recent studies have revealed a novel mechanism of chemoresistance acquisition: the transfer of information via exosomes, small vesicles secreted by various cells. Exosomes play a crucial role in intercellular communication by carrying proteins, nucleic acids, and metabolites, influencing cancer cell behavior and response to treatment. One crucial mechanism of resistance is cancer metabolic reprogramming, which involves alterations in the cellular metabolic pathways to support the survival and proliferation of drug-resistant cancer cells. This metabolic reprogramming often includes increased glycolysis, providing cancer cells with the necessary energy and building blocks to evade the effects of chemotherapy. Notably, exosomes have been found to transport glycolytic enzymes, as identified in proteomic profiling, leading to the reprogramming of metabolic pathways, facilitating altered glucose metabolism and increased lactate production. As a result, they profoundly impact the tumor microenvironment, promoting tumor progression, survival, immune evasion, and drug resistance.Understanding the complexities of such exosome-mediated cell-to-cell communication might open new therapeutic avenues and facilitate biomarker development in managing cancers characterized by aggressive glycolytic features. Moreover, given the intricate nature of metabolic abnormalities combining future exosome-based-targeted therapies with existing treatments like chemotherapy, immunotherapy, and targeted therapies holds promise for achieving synergistic effects to overcome resistance and improve cancer treatment outcomes. [Display omitted] • Chemoresistance is a significant challenge for cancer patients' survival. • One crucial mechanism of chemoresistance involves cancer metabolic reprogramming. • Exosomes are emerging as key players in cell-communication which can profoundly influence the tumor and tumor microenvironment, promoting cancer metabolic reprogramming and chemoresistance, thus exacerbating the challenges of cancer treatment. • Understanding the complexities of exosome-mediated cell-to-cell communication may open new therapeutic avenues and facilitate biomarker development for managing cancers with aggressive glycolytic features. [ABSTRACT FROM AUTHOR]

Amelogenins are clinically used in periodontal regeneration as main components of root surface modifying agents, even without specifically preventing the premature colonization of the healing tissue defect by means of a physical barrier membrane. The objective of this study was to investigate the effects of human amelogenin on the proliferation, migration, and morphology of Immortalized Human Oral Keratinocytes (iHOKs). Immortalized Human Oral Keratinocytes were expanded in Keratinocyte Growth Medium-2 (KGM-2). Full-length recombinant amelogenin protein was diluted in KGM-2 in five concentrations (10 ng/ml, 100 ng/ml, 1.000 ng/ml, 5.000 ng/ml and 10.000 ng/ml). iHOKs were cultured in medium supplemented with the amelogenin dilutions. Samples without amelogenin served as control. Cell metabolism and cell proliferation together with cell migration were evaluated at day 7, 14, 21. At day 7, iHOKs treated with 10,000 ng/ml showed a significant decrease in keratinocytes´ proliferation. The metabolic activity at this timepoint was significantly lower for concentrations ≥ 1000 ng/ml. At days 14 and 21, both the addition of 5000 ng/ml and even more 10,000 ng/ml amelogenin reduced significantly the proliferation of keratinocytes. The effects on the metabolic activity for these timepoints were visible already with 100 ng/ml. Treatment of iHOKs with amelogenin of ≥ 1000 ng/ml led to inhibitory effects on cell migration already after 24 h. The full-length recombinant amelogenin has a significant biological impact on iHOKs. The increasing dose dependent inhibitory effects of amelogenin shown on iHOKs might explain the disruption of the apical migration of the junctional epithelium during regenerative healing. Amelogenin, presents time- and dose-dependent inhibitory effects on the growth of keratinocytes, which might explain the biological rationale behind its application in periodontal regeneration. [ABSTRACT FROM AUTHOR]

Here we contrast several carcinogenesis models. The somatic-mutation-theory posits mutations as main causes of malignancy. However, inconsistencies led to alternative explanations. For example, the tissue-organization-field-theory considers disrupted tissue-architecture as main cause. Both models can be reconciled using systems-biology-approaches, according to which tumors hover in states of self-organized criticality between order and chaos, are emergent results of multiple deviations and are subject to general laws of nature: inevitable variation(mutation) explainable by increased entropy(second-law-of-thermodynamics) or indeterminate decoherence upon measurement of superposed quantum systems(quantum mechanics), followed by Darwinian-selection. Genomic expression is regulated by epigenetics. Both systems cooperate. So cancer is neither just a mutational nor an epigenetic problem. Rather, epigenetics links environmental cues to endogenous genetics engendering a regulatory machinery that encompasses specific cancer-metabolic-networks. Interestingly, mutations occur at all levels of this machinery (oncogenes/tumor-suppressors, epigenetic-modifiers, structure-genes, metabolic-genes). Therefore, in most cases, DNA mutations may be the initial and crucial cancer-promoting triggers. [ABSTRACT FROM AUTHOR]

Although targeting the tumor metabolism is performed in cooperation with immunotherapy in the era of precision oncology, ignorance of immune cells' metabolism has resulted in unstable antitumor responses. Tumor-infiltrating regulatory T cells (TI-Tregs) are unique, overcoming the hypoxic, acidic, and nutrient-deficient tumor microenvironments (TMEs) and maintaining immunosuppressive functions. However, secondary autoimmunity caused by systemic Treg depletion remains the 'Sword of Damocles' for current Treg-targeted therapies. In this opinion piece, we propose that metabolically reprogrammed TI-Tregs might represent an obstacle to cancer therapies. Indeed, metabolism-based Treg-targeted therapy might provide higher selectivity for clearing TI-Tregs than traditional kinase/checkpoint inhibitors and chemokine/chemokine receptor blockade; it might also restore the efficacy of targeting the tumor metabolism and eliminate certain metabolic barriers to immunotherapy. Metabolic restrictions in the tumor microenvironment (TME) can force immune cells to develop a tolerant phenotype for metabolic adaptation, ultimately impairing the antitumor effectiveness of immune cells and immunotherapies. Drugs that target tumor metabolism have slowly progressed because of possible nonspecific targeting. Regulatory T cells (Tregs) are unique in terms of their immune and metabolic features. They limit the intensity of the immune response and can promote tumors' immune escape. They can use lactic acid as a fuel and shift to more efficient oxidative phosphorylation to avoid metabolic restrictions in the TME, reinforcing their function. Future efforts to improve the antitumor immune response might overcome the harmful effects of metabolic restrictions and tumor-infiltrating Tregs (TI-Tregs) and their interactions with the TME. Because tumor cells share some metabolic profiles with TI-Tregs, targeting the metabolism of TI-Tregs might have immune and metabolic benefits. Recent trials have noted that the tumor metabolism can hamper the efficacy of immunotherapy; however, the indiscriminate targeting of the tumor metabolism fails to achieve the desired outcomes because of the disturbance of metabolically reprogrammed immune cells, especially immunosuppressive regulatory T cells (Tregs). Tumor-infiltrating Tregs are restricted to a greater immunosuppressive state by tumor metabolites such as lactate, fatty acids (FAs), and kynurenine, which are intolerable to other immune cells. Therefore, targeting the metabolism of tumor-infiltrating Tregs has immune and metabolic significance, and might represent a candidate approach to overcome metabolic obstacles during immunotherapy. [ABSTRACT FROM AUTHOR]

Vaccination aims to generate a protective and persisting antibody response. Indeed, humoral vaccine-mediated protection depends on the quality and quantity of the produced antigen-specific antibodies for its initial magnitude and the persistence of the plasma cells for its duration. Therefore, understanding the mechanisms behind the generation, selection and maintenance of long-lived plasma cells secreting protective antibodies is of fundamental importance for understanding long-term immunity, vaccine responses, therapeutical approaches for autoimmune disease and multiple myeloma. Recent studies have observed correlations between the generation, function and lifespan of plasma cells and their metabolism, with metabolism being both a main driver and primary consequence of changes in cellular behavior. This review introduces how metabolic programs influence and drive immune cell functions in general and plasma cell differentiation and longevity more specifically, summarizing the current knowledge on metabolic pathways and their influences on cellular fate. In addition, available technologies to profile metabolism and their limitations are discussed, leading to the unique and open technological challenges for further advancement of this research field. [Display omitted] [ABSTRACT FROM AUTHOR]

p62 has been shown to be involved in many signaling pathways related to cancer cell metabolism and plays a crucial role in the regulation of cancer metabolism by participating in the activation or inhibition of these signaling pathways. p62 could regulate host metabolism through affecting the functions of tumor-infiltrating immune cells. Therapeutic intervention in cancers through the targeting of p62 is a promising cancer treatment strategy. Small-molecule compounds targeting the different domains of p62 could potentially be developed for cancer therapy. p62 is an important multifunctional adaptor protein participating in autophagy and many other activities. Many studies have revealed that p62 is highly expressed in multiple cancers and decreasing its level can effectively lower the proliferation ability of cancer cells. Moreover, much research has highlighted the significant role of the regulation of cancer cell metabolism in helping to treat tumors. Recent reports demonstrate that p62 could regulate cancer cell metabolism through various mechanisms. However, the relationship between p62 and cancer cell metabolism as well as the related mechanisms has not been fully elucidated. In this review, we describe glucose, glutamine, and fatty acid metabolism in tumor cells and some signaling pathways that can regulate cancer metabolism and are mediated by p62. [ABSTRACT FROM AUTHOR]

Mitochondrial dynamics and metabolism govern embryonic and adult neural stem cell (NSC) behavior. Quiescent adult NSCs have active mitochondria and rely on mitochondrial metabolism to sustain their cell state. Single-cell RNA sequencing reveals dynamic expression of mitochondrial metabolism genes in NSCs, but does not always provide an accurate picture of the metabolic state. Novel technologies exploiting metabolic parameters allow for separation of distinct NSC states in real time. Metabolism has emerged as a key regulator of stem cell behavior. Mitochondria are crucial metabolic organelles that are important for differentiated cells, yet considered less so for stem cells. However, recent studies have shown that mitochondria influence stem cell maintenance and fate decisions, inviting a revised look at this topic. In this review, we cover the current literature addressing the role of mitochondrial metabolism in mouse and human neural stem cells (NSCs) in the embryonic and adult brain. We summarize how mitochondria are implicated in fate regulation and how substrate oxidation affects NSC quiescence. We further explore single-cell RNA sequencing (scRNA-seq) data for metabolic signatures of adult NSCs, highlight emerging technologies reporting on metabolic signatures, and discuss mitochondrial metabolism in other stem cells. [ABSTRACT FROM AUTHOR]

As an important substrate of tumor glucose metabolism, glucose plays an important role in tumor metabolic reprogramming. Radiotherapy as a common means of tumor treatment, can directly kill cancer cells and reshaping the tumor immune microenvironment along with tumor metabolic reprogramming. However, the role of glucose in radiotherapy-mediated tumor immune microenvironment (TIME) reshaping and tumor metabolic reprogramming is still lacking. Therefore, our project aims to explore whether glucose supplementation can reshape tumor metabolism and enhance the efficacy of radiotherapy. The clinical data of patients were investigated to explore the correlation between blood glucose level and prognosis. To simulate changes in patient blood glucose levels, we established subcutaneous tumor models using mouse tumor cell lines such as MC38, CT26, B16F10, and CMT167, which were subjected to radiotherapy of one fraction of 15 Gy with or without daily subcutaneous administration of glucose or saline to explore the effects of supplementing glucose on hypofractionated radiotherapy. Flow cytometry was subsequently used to assess TIME. Real-time qPCR and O2K was used to monitor changes of glucose metabolism in tumor cells. Relevant mechanisms were validated through RNA sequencing, flow cytometry, in vitro co-culture, O2K, real-time qPCR, and western blotting. Through clinical data, it was found that within normal blood glucose levels, the prognosis of small cell lung cancer patients is significantly positively correlated with blood glucose levels. Various mouse subcutaneous tumor models have confirmed that supplementing glucose can enhance the efficacy of radiotherapy. Further analysis of flow cytometry confirmed increased infiltration and enhanced cytotoxic function of CD8+ T cells, thereby improving the TIME post-radiotherapy. Cell proliferation experiments demonstrated that supplementing glucose minimally impacted on radiotherapy-induced tumor cell proliferation and apoptosis. Furthermore, RNA sequencing and in vitro experiments confirmed that addition of glucose significantly enhanced activation of the IFN-I pathway after radiotherapy, which is depend on the increased levels of mitochondrial oxidative phosphorylation. While in the presence of oxidative phosphorylation inhibitors, boosted IFN-I pathway after radiotherapy by glucose addition disappeared. Further in vitro co-culture experiments showed that tumor cells cultured in high glucose levels post-radiotherapy promoted secretion of Cxcl10 of bone marrow dendritic cells. Our study found that glucose supplementation can enhance the efficacy of hypofractionated radiotherapy, which is partly attributed to activation of tumor oxidative phosphorylation and IFN-I pathway after radiotherapy, thus improving the tumor immune microenvironment. [ABSTRACT FROM AUTHOR]

Cellular crosstalk plays a vital role in maintaining pancreas homeostasis. Recently, in Cell Metabolism , Basile et al. demonstrated that aberrant upregulation of acinar-cell-specific pancreatic elastase CELA3B within endocrine islets reduces β-cell viability in type 2 diabetes (T2D). This identifies detrimental acinar–β-cell crosstalk as a novel pathogenic mechanism in diabetes. [ABSTRACT FROM AUTHOR]

Whereas it is known that p53 broadly regulates cell metabolism, the specific activities that mediate this regulation remain partially understood. Here, we identified carnitine ooctanoyltransferase (CROT) as a p53 transactivation target that is upregulated by cellular stresses in a p53-dependent manner. CROT is a peroxisomal enzyme catalyzing very long-chain fatty acids conversion to medium chain fatty acids that can be absorbed by mitochondria during β-oxidation. p53 induces CROT transcription through binding to consensus response elements in the 50-UTR of CROT mRNA. Overexpression of WT but not enzymatically inactive mutant CROT promotes mitochondrial oxidative respiration, while downregulation of CROT inhibits mitochondrial oxidative respiration. Nutrient depletion induces p53-dependent CROT expression that facilitates cell growth and survival; in contrast, cells deficient in CROT have blunted cell growth and reduced survival during nutrient depletion. Together, these data are consistent with a model where p53-regulated CROT expression allows cells to be more efficiently utilizing stored very long-chain fatty acids to survive nutrient depletion stresses. [ABSTRACT FROM AUTHOR]

[Display omitted] • Recent understanding of Bacillus nitrogen metabolism was systematically summarized. • The potential as well as practical applications of nitrogen metabolism regulation in synthetic biology were discussed. • The future research directions of nitrogen metabolism and synthetic biology based on nitrogen metabolism were prospected. • More attention needs to be paid to the role of nitrogen metabolism in improving cell factory performance. Nitrogen sources play an essential role in maintaining the physiological and biochemical activity of bacteria. Nitrogen metabolism, which is the core of microorganism metabolism, makes bacteria able to autonomously respond to different external nitrogen environments by exercising complex internal regulatory networks to help them stay in an ideal state. Although various studies have been put forth to better understand this regulation in Bacillus , and many valuable viewpoints have been obtained, these views need to be presented systematically and their possible applications need to be specified. The intention is to provide a deep and comprehensive understanding of nitrogen metabolism in Bacillus , an important industrial microorganism, and thereby apply this regulatory logic to synthetic biology to improve biosynthesis competitiveness. In addition, the potential researches in the future are also discussed. Understanding the meticulous regulation process of nitrogen metabolism in Bacillus not only could facilitate research on metabolic engineering but also could provide constructive insights and inspiration for studies of other microorganisms. [ABSTRACT FROM AUTHOR]

Development of prodrugs is a useful strategy to overcome some disadvantages of candidate drugs. Recently, we established a systematic approach to selecting appropriate prodrugs, and validated the utility of this approach using oseltamivir analogues. In this study, the utility of the approach was further examined using candesartan cilexetil and 20 kinds of its analogues having various types of side chain as model compounds. Log D values of analogues (2.5 to 4.7) were higher than that of candesartan (1.0), their active metabolite, and the results were reasonable for the purpose of improving permeability of candesartan. The analogues tended to be more soluble in artificial intestinal fluids than in artificial gastric fluid, owing to their acidic physicochemical characteristics. Their membrane permeabilities were not correlated with log D values, which can be attributed to the metabolism in Caco-2 cells used in this system. In human hepatocytes and enterocytes, 11 out of the 20 analogues were immediately hydrolyzed to candesartan, and species differences were observed in the hydrolysis efficiency. This study confirmed the utility of the systematic approach for selection of appropriate prodrugs that could be proceeded to in vivo pharmacokinetics study, with selection of suitable experimental animals. [ABSTRACT FROM AUTHOR]

Isotope-assisted metabolic flux analysis (iMFA) is a mathematical technique that estimates intracellular metabolic fluxes for complex biological systems. iMFA software uses experimental data (extracellular fluxes, isotope labeling patterns) and a curated network model as inputs and produces a quantitative metabolic flux map as the output. iMFA determines a set of flux values that produce the best match between the simulated and experimental mass distribution vectors using an iterative optimization process. iMFA is essential for the metabolic analysis of complex systems and enables the discovery of new metabolic pathways. Advances in iMFA are needed to enable analysis of dynamic metabolic states, multicellular co-cultures, and genome-scale networks. These new tools will significantly advance our understanding of metabolic complexity. Isotope-assisted metabolic flux analysis (iMFA) is a powerful method to mathematically determine the metabolic fluxome from experimental isotope labeling data and a metabolic network model. While iMFA was originally developed for industrial biotechnological applications, it is increasingly used to analyze eukaryotic cell metabolism in physiological and pathological states. In this review, we explain how iMFA estimates the intracellular fluxome, including data and network model (inputs), the optimization-based data fitting (process), and the flux map (output). We then describe how iMFA enables analysis of metabolic complexities and discovery of metabolic pathways. Our goal is to expand the use of iMFA in metabolism research, which is essential to maximizing the impact of metabolic experiments and continuing to advance iMFA and biocomputational techniques. [ABSTRACT FROM AUTHOR]