35 results on '"Stefanoni, D"'
Search Results
2. Myelomonocytic cells in giant cell arteritis activate trained immunity programs sustaining inflammation and cytokine production
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Eleonora Cantoni, Ivan Merelli, Davide Stefanoni, Alessandro Tomelleri, Corrado Campochiaro, Vito Giordano, Maddalena Panigada, Elena M Baldissera, Laura Merlo Pich, Valentina Natoli, Athanasios Ziogas, Jorge Domínguez-Andrés, Giacomo De Luca, Davide Mazza, Samuel Zambrano, Daniela Gnani, Marina Ferrarini, Elisabetta Ferrero, Alessandra Agresti, Barbara Vergani, Biagio Eugenio Leone, Simone Cenci, Angelo Ravelli, Marco Matucci-Cerinic, Angelo D’Alessandro, Leo A B Joosten, Lorenzo Dagna, Mihai G Netea, Raffaella Molteni, Giulio Cavalli, Cantoni, E, Merelli, I, Stefanoni, D, Tomelleri, A, Campochiaro, C, Giordano, V, Panigada, M, Baldissera, E, Merlo Pich, L, Natoli, V, Ziogas, A, Domínguez-Andrés, J, De Luca, G, Mazza, D, Zambrano, S, Gnani, D, Ferrarini, M, Ferrero, E, Agresti, A, Vergani, B, Leone, B, Cenci, S, Ravelli, A, Matucci-Cerinic, M, D'Alessandro, A, Joosten, L, Dagna, L, Netea, M, Molteni, R, and Cavalli, G
- Subjects
IL-6 ,trained immunity ,Rheumatology ,immunometabolism ,Pharmacology (medical) ,monocyte/macrophage ,epigenetic - Abstract
ObjectiveTrained immunity (TI) is a de facto memory program of innate immune cells, characterized by immunometabolic and epigenetic changes sustaining enhanced production of cytokines. TI evolved as a protective mechanism against infections; however, inappropriate activation can cause detrimental inflammation and might be implicated in the pathogenesis of chronic inflammatory diseases. In this study, we investigated the role of TI in the pathogenesis of giant cell arteritis (GCA), a large-vessel vasculitis characterized by aberrant macrophage activation and excess cytokine production.MethodsMonocytes from GCA patients and from age- and sex-matched healthy donors were subjected to polyfunctional studies, including cytokine production assays at baseline and following stimulation, intracellular metabolomics, chromatin immunoprecipitation-qPCR, and combined ATAC/RNA sequencing. Immunometabolic activation (i.e. glycolysis) was assessed in inflamed vessels of GCA patients with FDG-PET and immunohistochemistry (IHC), and the role of this pathway in sustaining cytokine production was confirmed with selective pharmacologic inhibition in GCA monocytes.ResultsGCA monocytes exhibited hallmark molecular features of TI. Specifically, these included enhanced IL-6 production upon stimulation, typical immunometabolic changes (e.g. increased glycolysis and glutaminolysis) and epigenetic changes promoting enhanced transcription of genes governing pro-inflammatory activation. Immunometabolic changes of TI (i.e. glycolysis) were a feature of myelomonocytic cells in GCA lesions and were required for enhanced cytokine production.ConclusionsMyelomonocytic cells in GCA activate TI programs sustaining enhanced inflammatory activation with excess cytokine production.
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- 2023
3. Inhibition of asparagine synthetase effectively retards polycystic kidney disease progression.
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Clerici S, Podrini C, Stefanoni D, Distefano G, Cassina L, Steidl ME, Tronci L, Canu T, Chiaravalli M, Spies D, Bell TA 3rd, Costa AS, Esposito A, D'Alessandro A, Frezza C, Bachi A, and Boletta A
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- Animals, Humans, Mice, Disease Progression, Kidney pathology, Kidney metabolism, Oligonucleotides, Antisense pharmacology, Oligonucleotides, Antisense therapeutic use, Aspartate-Ammonia Ligase metabolism, Aspartate-Ammonia Ligase genetics, Aspartate-Ammonia Ligase antagonists & inhibitors, Disease Models, Animal, Polycystic Kidney Diseases metabolism, Polycystic Kidney Diseases drug therapy, Polycystic Kidney Diseases pathology, Polycystic Kidney Diseases genetics
- Abstract
Polycystic kidney disease (PKD) is a genetic disorder characterized by bilateral cyst formation. We showed that PKD cells and kidneys display metabolic alterations, including the Warburg effect and glutaminolysis, sustained in vitro by the enzyme asparagine synthetase (ASNS). Here, we used antisense oligonucleotides (ASO) against Asns in orthologous and slowly progressive PKD murine models and show that treatment leads to a drastic reduction of total kidney volume (measured by MRI) and a prominent rescue of renal function in the mouse. Mechanistically, the upregulation of an ATF4-ASNS axis in PKD is driven by the amino acid response (AAR) branch of the integrated stress response (ISR). Metabolic profiling of PKD or control kidneys treated with Asns-ASO or Scr-ASO revealed major changes in the mutants, several of which are rescued by Asns silencing in vivo. Indeed, ASNS drives glutamine-dependent de novo pyrimidine synthesis and proliferation in cystic epithelia. Notably, while several metabolic pathways were completely corrected by Asns-ASO, glycolysis was only partially restored. Accordingly, combining the glycolytic inhibitor 2DG with Asns-ASO further improved efficacy. Our studies identify a new therapeutic target and novel metabolic vulnerabilities in PKD., (© 2024. The Author(s).)
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- 2024
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4. Myelomonocytic cells in giant cell arteritis activate trained immunity programs sustaining inflammation and cytokine production.
- Author
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Cantoni E, Merelli I, Stefanoni D, Tomelleri A, Campochiaro C, Giordano V, Panigada M, Baldissera EM, Merlo Pich L, Natoli V, Ziogas A, Domínguez-Andrés J, De Luca G, Mazza D, Zambrano S, Gnani D, Ferrarini M, Ferrero E, Agresti A, Vergani B, Leone BE, Cenci S, Ravelli A, Matucci-Cerinic M, D'Alessandro A, Joosten LAB, Dagna L, Netea MG, Molteni R, and Cavalli G
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- Humans, Monocytes metabolism, Trained Immunity, Inflammation, Cytokines, Giant Cell Arteritis pathology
- Abstract
Objective: Trained immunity (TI) is a de facto memory program of innate immune cells, characterized by immunometabolic and epigenetic changes sustaining enhanced production of cytokines. TI evolved as a protective mechanism against infections; however, inappropriate activation can cause detrimental inflammation and might be implicated in the pathogenesis of chronic inflammatory diseases. In this study, we investigated the role of TI in the pathogenesis of giant cell arteritis (GCA), a large-vessel vasculitis characterized by aberrant macrophage activation and excess cytokine production., Methods: Monocytes from GCA patients and from age- and sex-matched healthy donors were subjected to polyfunctional studies, including cytokine production assays at baseline and following stimulation, intracellular metabolomics, chromatin immunoprecipitation-qPCR, and combined ATAC/RNA sequencing. Immunometabolic activation (i.e. glycolysis) was assessed in inflamed vessels of GCA patients with FDG-PET and immunohistochemistry (IHC), and the role of this pathway in sustaining cytokine production was confirmed with selective pharmacologic inhibition in GCA monocytes., Results: GCA monocytes exhibited hallmark molecular features of TI. Specifically, these included enhanced IL-6 production upon stimulation, typical immunometabolic changes (e.g. increased glycolysis and glutaminolysis) and epigenetic changes promoting enhanced transcription of genes governing pro-inflammatory activation. Immunometabolic changes of TI (i.e. glycolysis) were a feature of myelomonocytic cells in GCA lesions and were required for enhanced cytokine production., Conclusions: Myelomonocytic cells in GCA activate TI programs sustaining enhanced inflammatory activation with excess cytokine production., (© The Author(s) 2023. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)
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- 2023
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5. Metabolic Signatures of Performance in Elite World Tour Professional Male Cyclists.
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Nemkov T, Cendali F, Stefanoni D, Martinez JL, Hansen KC, San-Millán I, and D'Alessandro A
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- Humans, Male, Bicycling physiology, Exercise physiology, Lactates, Succinates, Athletic Performance
- Abstract
Background and Objective: Metabolomics studies of recreational and elite athletes have been so far limited to venipuncture-dependent blood sample collection in the setting of controlled training and medical facilities. However, limited to no information is currently available to determine if findings in laboratory settings are translatable to a real-world scenario in elite competitions. The goal of this study was to define molecular signatures of exertion under controlled exercise conditions and use these signatures as a framework for assessing cycling performance in a World Tour competition., Methods: To characterize molecular profiles of exertion in elite athletes during cycling, we performed metabolomics analyses on blood isolated from 28 international-level, elite, World Tour professional male athletes from a Union Cycliste Internationale World Team taken before and after a graded exercise test to volitional exhaustion and before and after a long aerobic training session. Moreover, established signatures were then used to characterize the metabolic physiology of five of these cyclists who were selected to represent the same Union Cycliste Internationale World Team during a seven-stage elite World Tour race., Results: Using dried blood spot collection to circumvent logistical hurdles associated with field sampling, these studies defined metabolite signatures and fold change ranges of anaerobic or aerobic exertion in elite cyclists, respectively. Blood profiles of lactate, carboxylic acids, fatty acids, and acylcarnitines differed between exercise modes. The graded exercise test elicited significant two- to three-fold accumulations in lactate and succinate, in addition to significant elevations in free fatty acids and acylcarnitines. Conversely, the long aerobic training session elicited a larger magnitude of increase in fatty acids and acylcarnitines without appreciable increases in lactate or succinate. Comparable signatures were revealed after sprinting and climbing stages, respectively, in a World Tour race. In addition, signatures of elevated fatty acid oxidation capacity correlated with competitive performance., Conclusions: Collectively, these studies provide a unique view of alterations in the blood metabolome of elite athletes during competition and at the peak of their performance capabilities. Furthermore, they demonstrate the utility of dried blood sampling for omics analysis, thereby enabling molecular monitoring of athletic performance in the field during training and competition., (© 2023. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)
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- 2023
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6. Induction of Drug-Resistance and Production of a Culture Medium Able to Induce Drug-Resistance in Vinblastine Untreated Murine Myeloma Cells.
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Masci VL, Stefanoni D, D'Alessandro A, Zambelli M, Modesti L, Pollini D, Ovidi E, and Tiezzi A
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- Mice, Humans, Animals, Drug Resistance, Neoplasm, Tumor Cells, Cultured, Neoplasm Recurrence, Local, Vinblastine pharmacology, Multiple Myeloma
- Abstract
Cancer therapies use different compounds of synthetic and natural origin. However, despite some positive results, relapses are common, as standard chemotherapy regimens are not fully capable of completely eradicating cancer stem cells. While vinblastine is a common chemotherapeutic agent in the treatment of blood cancers, the development of vinblastine resistance is often observed. Here, we performed cell biology and metabolomics studies to investigate the mechanisms of vinblastine resistance in P3X63Ag8.653 murine myeloma cells. Treatment with low doses of vinblastine in cell media led to the selection of vinblastine-resistant cells and the acquisition of such resistance in previously untreated, murine myeloma cells in culture. To determine the mechanistic basis of this observation, we performed metabolomic analyses of resistant cells and resistant drug-induced cells in a steady state, or incubation with stable isotope-labeled tracers, namely,
13 C15 N-amino acids. Taken together, these results indicate that altered amino acid uptake and metabolism could contribute to the acquisition of vinblastine resistance in blood cancer cells. These results will be useful for further research on human cell models.- Published
- 2023
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7. Warburg-like metabolic transformation underlies neuronal degeneration in sporadic Alzheimer's disease.
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Traxler L, Herdy JR, Stefanoni D, Eichhorner S, Pelucchi S, Szücs A, Santagostino A, Kim Y, Agarwal RK, Schlachetzki JCM, Glass CK, Lagerwall J, Galasko D, Gage FH, D'Alessandro A, and Mertens J
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- Glycolysis, Humans, Protein Isoforms genetics, Protein Isoforms metabolism, Pyruvate Kinase genetics, Pyruvate Kinase metabolism, Alzheimer Disease, Neoplasms pathology
- Abstract
The drivers of sporadic Alzheimer's disease (AD) remain incompletely understood. Utilizing directly converted induced neurons (iNs) from AD-patient-derived fibroblasts, we identified a metabolic switch to aerobic glycolysis in AD iNs. Pathological isoform switching of the glycolytic enzyme pyruvate kinase M (PKM) toward the cancer-associated PKM2 isoform conferred metabolic and transcriptional changes in AD iNs. These alterations occurred via PKM2's lack of metabolic activity and via nuclear translocation and association with STAT3 and HIF1α to promote neuronal fate loss and vulnerability. Chemical modulation of PKM2 prevented nuclear translocation, restored a mature neuronal metabolism, reversed AD-specific gene expression changes, and re-activated neuronal resilience against cell death., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)
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- 2022
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8. BRAF Modulates Lipid Use and Accumulation.
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Turner JA, Paton EL, Van Gulick R, Stefanoni D, Cendali F, Reisz J, Tobin RP, McCarter M, D'Alessandro A, Torres RM, Robinson WA, Couts KL, and Schlaepfer IR
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There is increasing evidence that oxidative metabolism and fatty acids play an important role in BRAF-driven tumorigenesis, yet the effect of BRAF mutation and expression on metabolism is poorly understood. We examined how BRAF mutation and expression modulates metabolite abundance. Using the non-transformed NIH3T3 cell line, we generated cells that stably overexpressed BRAF V600E or BRAF WT. We found that cells expressing BRAF V600E were enriched with immunomodulatory lipids. Further, we found a unique transcriptional signature that was exclusive to BRAF V600E expression. We also report that BRAF V600E mutation promoted accumulation of long chain polyunsaturated fatty acids (PUFAs) and rewired metabolic flux for non-Warburg behavior. This cancer promoting mutation further induced the formation of tunneling nanotube (TNT)-like protrusions in NIH3T3 cells that preferentially accumulated lipid droplets. In the plasma of melanoma patients harboring the BRAF V600E mutation, levels of lysophosphatidic acid, sphingomyelin, and long chain fatty acids were significantly increased in the cohort of patients that did not respond to BRAF inhibitor therapy. Our findings show BRAF V600 status plays an important role in regulating immunomodulatory lipid profiles and lipid trafficking, which may inform future therapy across cancers.
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- 2022
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9. Immunometabolic activation of macrophages leads to cytokine production in the pathogenesis of KRAS-mutated histiocytosis.
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Ferrero E, Villa A, Stefanoni D, Nemkov T, D'Alessandro A, Tengesdal I, Belloni D, Molteni R, Vergani B, De Luca G, Grassini G, Cangi MG, Dagna L, Doglioni C, Cavalli G, and Ferrarini M
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- Cytokines metabolism, Gene Expression, Humans, Macrophages metabolism, Mutation, Histiocytosis genetics, Histiocytosis pathology, Proto-Oncogene Proteins p21(ras) genetics
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- 2022
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10. Beta thalassemia minor is a beneficial determinant of red blood cell storage lesion.
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Tzounakas VL, Anastasiadi AT, Stefanoni D, Cendali F, Bertolone L, Gamboni F, Dzieciatkowska M, Rousakis P, Vergaki A, Soulakis V, Tsitsilonis OE, Stamoulis K, Papassideri IS, Kriebardis AG, D'Alessandro A, and Antonelou MH
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- Blood Preservation, Erythrocyte Transfusion, Erythrocytes metabolism, Hemolysis, Humans, beta-Thalassemia genetics, beta-Thalassemia metabolism
- Abstract
Blood donor genetics and lifestyle affect the quality of red blood cell (RBC) storage. Heterozygotes for beta thalassemia (bThal+) constitute a non-negligible proportion of blood donors in the Mediterranean and other geographical areas. The unique hematological profile of bThal+ could affect the capacity of enduring storage stress, however, the storability of bThal+ RBC is largely unknown. In this study, RBC from 18 bThal+ donors were stored in the cold and profiled for primary (hemolysis) and secondary (phosphatidylserine exposure, potassium leakage, oxidative stress) quality measures, and metabolomics, versus sex- and age-matched controls. The bThal+ units exhibited better levels of storage hemolysis and susceptibility to lysis following osmotic, oxidative and mechanical insults. Moreover, bThal+ RBC had a lower percentage of surface removal signaling, reactive oxygen species and oxidative defects to membrane components at late stages of storage. Lower potassium accumulation and higher uratedependent antioxidant capacity were noted in the bThal+ supernatant. Full metabolomics analyses revealed alterations in purine and arginine pathways at baseline, along with activation of the pentose phosphate pathway and glycolysis upstream to pyruvate kinase in bThal+ RBC. Upon storage, substantial changes were observed in arginine, purine and vitamin B6 metabolism, as well as in the hexosamine pathway. A high degree of glutamate generation in bThal+ RBC was accompanied by low levels of purine oxidation products (IMP, hypoxanthine, allantoin). The bThal mutations impact the metabolism and the susceptibility to hemolysis of stored RBC, suggesting good post-transfusion recovery. However, hemoglobin increment and other clinical outcomes of bThal+ RBC transfusion deserve elucidation by future studies.
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- 2022
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11. Metabolic alterations mediated by STAT3 promotes drug persistence in CML.
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Patel SB, Nemkov T, Stefanoni D, Benavides GA, Bassal MA, Crown BL, Matkins VR, Camacho V, Kuznetsova V, Hoang AT, Tenen DE, Wolock SL, Park J, Ying L, Yue Z, Chen JY, Yang H, Tenen DG, Ferrell PB, Lu R, Darley-Usmar V, D'Alessandro A, Bhatia R, and Welner RS
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- Animals, Apoptosis, Female, Glycolysis, Leukemia, Myelogenous, Chronic, BCR-ABL Positive genetics, Leukemia, Myelogenous, Chronic, BCR-ABL Positive metabolism, Leukemia, Myelogenous, Chronic, BCR-ABL Positive pathology, Male, Mice, Neoplastic Stem Cells metabolism, Neoplastic Stem Cells pathology, Protein Kinase Inhibitors pharmacology, STAT3 Transcription Factor genetics, Drug Resistance, Neoplasm, Leukemia, Myelogenous, Chronic, BCR-ABL Positive drug therapy, Metabolome, Neoplastic Stem Cells drug effects, STAT3 Transcription Factor metabolism, Small Molecule Libraries pharmacology, Transcriptome
- Abstract
Leukemic stem cells (LSCs) can acquire non-mutational resistance following drug treatment leading to therapeutic failure and relapse. However, oncogene-independent mechanisms of drug persistence in LSCs are incompletely understood, which is the primary focus of this study. We integrated proteomics, transcriptomics, and metabolomics to determine the contribution of STAT3 in promoting metabolic changes in tyrosine kinase inhibitor (TKI) persistent chronic myeloid leukemia (CML) cells. Proteomic and transcriptional differences in TKI persistent CML cells revealed BCR-ABL-independent STAT3 activation in these cells. While knockout of STAT3 inhibited the CML cells from developing drug-persistence, inhibition of STAT3 using a small molecule inhibitor sensitized the persistent CML cells to TKI treatment. Interestingly, given the role of phosphorylated STAT3 as a transcription factor, it localized uniquely to genes regulating metabolic pathways in the TKI-persistent CML stem and progenitor cells. Subsequently, we observed that STAT3 dysregulated mitochondrial metabolism forcing the TKI-persistent CML cells to depend on glycolysis, unlike TKI-sensitive CML cells, which are more reliant on oxidative phosphorylation. Finally, targeting pyruvate kinase M2, a rate-limiting glycolytic enzyme, specifically eradicated the TKI-persistent CML cells. By exploring the role of STAT3 in altering metabolism, we provide critical insight into identifying potential therapeutic targets for eliminating TKI-persistent LSCs., (© 2021. The Author(s).)
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- 2021
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12. Oncogene-induced maladaptive activation of trained immunity in the pathogenesis and treatment of Erdheim-Chester disease.
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Molteni R, Biavasco R, Stefanoni D, Nemkov T, Domínguez-Andrés J, Arts RJ, Merelli I, Mazza D, Zambrano S, Panigada M, Cantoni E, Tengesdal IW, Maksud P, Piras F, Cesana D, Cassina L, Distefano G, Loffreda A, Gnani D, De Luca G, Tomelleri A, Campochiaro C, Joosten LAB, Dinarello CA, Kajaste-Rudnitski A, Haroche J, Cardaci S, Cenci S, Dagna L, Doglioni C, Ferrarini M, Ferrero E, Boletta A, D'Alessandro A, Montini E, Netea MG, and Cavalli G
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- Cells, Cultured, Epigenesis, Genetic, Erdheim-Chester Disease immunology, Erdheim-Chester Disease pathology, Humans, Immunity, Inflammation immunology, Inflammation pathology, Macrophages immunology, Macrophages metabolism, Macrophages pathology, Oncogenes, Point Mutation, Proto-Oncogene Proteins B-raf immunology, Erdheim-Chester Disease genetics, Inflammation genetics, Proto-Oncogene Proteins B-raf genetics
- Abstract
Trained immunity (TI) is a proinflammatory program induced in monocyte/macrophages upon sensing of specific pathogens and is characterized by immunometabolic and epigenetic changes that enhance cytokine production. Maladaptive activation of TI (ie, in the absence of infection) may result in detrimental inflammation and development of disease; however, the exact role and extent of inappropriate activation of TI in the pathogenesis of human diseases is undetermined. In this study, we uncovered the oncogene-induced, maladaptive induction of TI in the pathogenesis of a human inflammatory myeloid neoplasm (Erdheim-Chester disease, [ECD]), characterized by the BRAFV600E oncogenic mutation in monocyte/macrophages and excess cytokine production. Mechanistically, myeloid cells expressing BRAFV600E exhibit all molecular features of TI: activation of the AKT/mammalian target of rapamycin signaling axis; increased glycolysis, glutaminolysis, and cholesterol synthesis; epigenetic changes on promoters of genes encoding cytokines; and enhanced cytokine production leading to hyperinflammatory responses. In patients with ECD, effective therapeutic strategies combat this maladaptive TI phenotype; in addition, pharmacologic inhibition of immunometabolic changes underlying TI (ie, glycolysis) effectively dampens cytokine production by myeloid cells. This study revealed the deleterious potential of inappropriate activation of TI in the pathogenesis of human inflammatory myeloid neoplasms and the opportunity for inhibition of TI in conditions characterized by maladaptive myeloid-driven inflammation., (© 2021 by The American Society of Hematology.)
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- 2021
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13. Metabolism navigates neural cell fate in development, aging and neurodegeneration.
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Traxler L, Lagerwall J, Eichhorner S, Stefanoni D, D'Alessandro A, and Mertens J
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- Brain metabolism, Energy Metabolism, Humans, Neurons metabolism, Aging metabolism, Alzheimer Disease metabolism
- Abstract
An uninterrupted energy supply is critical for the optimal functioning of all our organs, and in this regard the human brain is particularly energy dependent. The study of energy metabolic pathways is a major focus within neuroscience research, which is supported by genetic defects in the oxidative phosphorylation mechanism often contributing towards neurodevelopmental disorders and changes in glucose metabolism presenting as a hallmark feature in age-dependent neurodegenerative disorders. However, as recent studies have illuminated roles of cellular metabolism that span far beyond mere energetics, it would be valuable to first comprehend the physiological involvement of metabolic pathways in neural cell fate and function, and to subsequently reconstruct their impact on diseases of the brain. In this Review, we first discuss recent evidence that implies metabolism as a master regulator of cell identity during neural development. Additionally, we examine the cell type-dependent metabolic states present in the adult brain. As metabolic states have been studied extensively as crucial regulators of malignant transformation in cancer, we reveal how knowledge gained from the field of cancer has aided our understanding in how metabolism likewise controls neural fate determination and stability by directly wiring into the cellular epigenetic landscape. We further summarize research pertaining to the interplay between metabolic alterations and neurodevelopmental and psychiatric disorders, and expose how an improved understanding of metabolic cell fate control might assist in the development of new concepts to combat age-dependent neurodegenerative diseases, particularly Alzheimer's disease., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)
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- 2021
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14. In Vitro Characterization and Metabolomic Analysis of Cold-Stored Platelets.
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Zhao HW, Serrano K, Stefanoni D, D'Alessandro A, and Devine DV
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- Cold Temperature, Metabolomics, Platelet Aggregation, Temperature, Blood Platelets, Blood Preservation
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Platelet concentrates are currently stored at room temperature (RP) under constant agitation for up to 5-7 days depending on national regulations. However, platelet quality deteriorates during storage and room-temperature storage also increases the risk of bacterial growth. Previous studies have shown that cold-stored platelets (CPs) have higher hemostatic functions and can be stored for up to 3 weeks. While these studies have compared the metabolic phenotypes of CPs and RPs, they have neither compared the impact of storage temperature and cold agitation (CPAs) on platelet function nor identified metabolic correlates to such parameters. In vitro analysis showed that CPAs and CPs had reduced count, faster CD62P expression, and increased lactadherin binding. Furthermore, CPAs and CPs had higher maximal aggregation and a reduced aggregation lag phase compared to RPs. Metabolomic analysis revealed that CPAs and CPs exhibited lower oxidative stress shown by preserved glutathione and pentose phosphate pools. CPAs and CPs also had reduced markers of beta-oxidation and amino acid catabolism, demonstrating reduced needs for energy. Agitation did not significantly impact in vitro function or metabolomic parameters of cold-stored platelets. Correlation of in vitro and metabolomic results highlighted important metabolites that may contribute to stored platelet functions. Raw data are publicly available through Metabolomics Workbench with the study identifier ST001644.
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- 2021
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15. The impact of donor sex and age on stored platelet metabolism and post-transfusion recovery.
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D'Alessandro A, Stefanoni D, Slichter SJ, Fu X, and Zimring JC
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- Adult, Blood Platelets cytology, Blood Preservation, Female, Humans, Male, Middle Aged, Platelet Function Tests, Young Adult, Blood Donors, Blood Platelets metabolism, Platelet Transfusion
- Abstract
Background: The impact of donor biology on blood component storability is increasingly appreciated as a determinant of the storage lesion and post-transfusion performances. Platelet metabolism is affected by age and it is critical to platelet responses to activating stimuli in an age-dependent manner. Sex has been previously highlighted as a contributing factor to the platelet proteomics lesion. However, little is known about the impact of donor sex and age on stored platelet metabolism and post-transfusion capacity to circulate., Materials and Methods: Apheresis platelets were donated via apheresis by 21 healthy volunteers (12 males and 9 females; ages 20 to 59). Metabolomics analyses were performed at day 0 and after 5 days of storage at 22+2 °C, along with autologous post-transfusion recovery and survival studies with
51 Cr and111 In., Results: Sex and age significantly impacted platelet metabolism at baseline and upon storage. Platelets from older, male donors were characterised by higher levels of Krebs cycle metabolites, pentose phosphate pathway intermediates and byproducts, deaminated purines and long chain fatty acids. These metabolites ranked amongst the top significant correlates to post-transfusion recoveries. Glutathione homeostasis and sphingosine 1-phosphate were the top positive correlates to long term survival, which was lower in platelets from older, male donors - without reaching statistical significance., Discussion: In this study we report that donor sex and age have a significant impact on platelet metabolism. Novel metabolic correlates to platelet post-transfusion performances (24 h recovery and long-term survival) were identified through high-resolution, stable isotope-labeled internal standard-assisted metabolomics approach.- Published
- 2021
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16. The anti-inflammatory cytokine interleukin-37 is an inhibitor of trained immunity.
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Cavalli G, Tengesdal IW, Gresnigt M, Nemkov T, Arts RJW, Domínguez-Andrés J, Molteni R, Stefanoni D, Cantoni E, Cassina L, Giugliano S, Schraa K, Mills TS, Pietras EM, Eisenmensser EZ, Dagna L, Boletta A, D'Alessandro A, Joosten LAB, Netea MG, and Dinarello CA
- Subjects
- Animals, Candidiasis genetics, Candidiasis immunology, Candidiasis microbiology, Epigenesis, Genetic drug effects, Glycolysis drug effects, Glycolysis genetics, Host-Pathogen Interactions drug effects, Host-Pathogen Interactions immunology, Humans, Male, Mice, Inbred C57BL, Neutrophils drug effects, Neutrophils metabolism, Mice, Anti-Inflammatory Agents pharmacology, Immunity, Innate drug effects, Interleukin-1 pharmacology
- Abstract
Trained immunity (TI) is a de facto innate immune memory program induced in monocytes/macrophages by exposure to pathogens or vaccines, which evolved as protection against infections. TI is characterized by immunometabolic changes and histone post-translational modifications, which enhance production of pro-inflammatory cytokines. As aberrant activation of TI is implicated in inflammatory diseases, tight regulation is critical; however, the mechanisms responsible for this modulation remain elusive. Interleukin-37 (IL-37) is an anti-inflammatory cytokine that curbs inflammation and modulates metabolic pathways. In this study, we show that administration of recombinant IL-37 abrogates the protective effects of TI in vivo, as revealed by reduced host pro-inflammatory responses and survival to disseminated candidiasis. Mechanistically, IL-37 reverses the immunometabolic changes and histone post-translational modifications characteristic of TI in monocytes, thus suppressing cytokine production in response to infection. IL-37 thereby emerges as an inhibitor of TI and as a potential therapeutic target in immune-mediated pathologies., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2021
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17. Targeting tumor-derived NLRP3 reduces melanoma progression by limiting MDSCs expansion.
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Tengesdal IW, Menon DR, Osborne DG, Neff CP, Powers NE, Gamboni F, Mauro AG, D'Alessandro A, Stefanoni D, Henen MA, Mills TS, De Graaf DM, Azam T, Vogeli B, Palmer BE, Pietras EM, DeGregori J, Tan AC, Joosten LAB, Fujita M, Dinarello CA, and Marchetti C
- Subjects
- Animals, CD8-Positive T-Lymphocytes immunology, Humans, Interleukin-1beta genetics, Interleukin-1beta immunology, Melanoma, Experimental genetics, Melanoma, Experimental pathology, Mice, Mice, Knockout, NLR Family, Pyrin Domain-Containing 3 Protein genetics, Neoplasm Proteins genetics, Signal Transduction genetics, Signal Transduction immunology, T-Lymphocytes, Regulatory immunology, Melanoma, Experimental immunology, Myeloid-Derived Suppressor Cells immunology, NLR Family, Pyrin Domain-Containing 3 Protein immunology, Neoplasm Proteins immunology
- Abstract
Interleukin-1β (IL-1β)-mediated inflammation suppresses antitumor immunity, leading to the generation of a tumor-permissive environment, tumor growth, and progression. Here, we demonstrate that nucleotide-binding domain, leucine-rich containing family, pyrin domain-containing-3 (NLRP3) inflammasome activation in melanoma is linked to IL-1β production, inflammation, and immunosuppression. Analysis of cancer genome datasets (TCGA and GTEx) revealed greater NLRP3 and IL-1β expression in cutaneous melanoma samples ( n = 469) compared to normal skin ( n = 324), with a highly significant correlation between NLRP3 and IL-1β ( P < 0.0001). We show the formation of the NLRP3 inflammasome in biopsies of metastatic melanoma using fluorescent resonance energy transfer analysis for NLRP3 and apoptosis-associated speck-like protein containing a CARD. In vivo, tumor-associated NLRP3/IL-1 signaling induced expansion of myeloid-derived suppressor cells (MDSCs), leading to reduced natural killer and CD8
+ T cell activity concomitant with an increased presence of regulatory T (Treg) cells in the primary tumors. Either genetic or pharmacological inhibition of tumor-derived NLRP3 by dapansutrile (OLT1177) was sufficient to reduce MDSCs expansion and to enhance antitumor immunity, resulting in reduced tumor growth. Additionally, we observed that the combination of NLRP3 inhibition and anti-PD-1 treatment significantly increased the antitumor efficacy of the monotherapy by limiting MDSC-mediated T cell suppression and tumor progression. These data show that NLRP3 activation in melanoma cells is a protumor mechanism, which induces MDSCs expansion and immune evasion. We conclude that inhibition of NLRP3 can augment the efficacy of anti-PD-1 therapy., Competing Interests: Competing interest statement: L.A.B.J. serves on Olatec’s Scientific Advisory Board and receives compensation. C.A.D. serves as Chairman of Olatec’s Scientific Advisory Board, is co-Chief Scientific Officer, receives compensation, and has equity in Olatec. C.M. serves as Director for Olatec’s Innovative Science Program and has equity in Olatec., (Copyright © 2021 the Author(s). Published by PNAS.)- Published
- 2021
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18. Blood donor exposome and impact of common drugs on red blood cell metabolism.
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Nemkov T, Stefanoni D, Bordbar A, Issaian A, Palsson BO, Dumont LJ, Hay A, Song A, Xia Y, Redzic JS, Eisenmesser EZ, Zimring JC, Kleinman S, Hansen KC, Busch MP, and D'Alessandro A
- Subjects
- Adolescent, Adult, Aged, Animals, Energy Metabolism drug effects, Erythrocyte Transfusion, Female, Glycolysis drug effects, Healthy Volunteers, Hemoglobins metabolism, High-Throughput Screening Assays, Humans, In Vitro Techniques, Machine Learning, Male, Metabolomics, Mice, Mice, Inbred C57BL, Mice, Knockout, Middle Aged, Models, Biological, Oxidation-Reduction drug effects, Phosphotransferases (Alcohol Group Acceptor) deficiency, Phosphotransferases (Alcohol Group Acceptor) genetics, Ranitidine pharmacology, Young Adult, Blood Donors, Erythrocytes drug effects, Erythrocytes metabolism, Exposome, Nonprescription Drugs adverse effects, Nonprescription Drugs pharmacokinetics, Prescription Drugs adverse effects, Prescription Drugs pharmacokinetics
- Abstract
Computational models based on recent maps of the RBC proteome suggest that mature erythrocytes may harbor targets for common drugs. This prediction is relevant to RBC storage in the blood bank, in which the impact of small molecule drugs or other xenometabolites deriving from dietary, iatrogenic, or environmental exposures ("exposome") may alter erythrocyte energy and redox metabolism and, in so doing, affect red cell storage quality and posttransfusion efficacy. To test this prediction, here we provide a comprehensive characterization of the blood donor exposome, including the detection of common prescription and over-the-counter drugs in blood units donated by 250 healthy volunteers in the Recipient Epidemiology and Donor Evaluation Study III Red Blood Cell-Omics (REDS-III RBC-Omics) Study. Based on high-throughput drug screenings of 1366 FDA-approved drugs, we report that approximately 65% of the tested drugs had an impact on erythrocyte metabolism. Machine learning models built using metabolites as predictors were able to accurately predict drugs for several drug classes/targets (bisphosphonates, anticholinergics, calcium channel blockers, adrenergics, proton pump inhibitors, antimetabolites, selective serotonin reuptake inhibitors, and mTOR), suggesting that these drugs have a direct, conserved, and substantial impact on erythrocyte metabolism. As a proof of principle, here we show that the antacid ranitidine - though rarely detected in the blood donor population - has a strong effect on RBC markers of storage quality in vitro. We thus show that supplementation of blood units stored in bags with ranitidine could - through mechanisms involving sphingosine 1-phosphate-dependent modulation of erythrocyte glycolysis and/or direct binding to hemoglobin - improve erythrocyte metabolism and storage quality.
- Published
- 2021
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19. Acute Cycling Exercise Induces Changes in Red Blood Cell Deformability and Membrane Lipid Remodeling.
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Nemkov T, Skinner SC, Nader E, Stefanoni D, Robert M, Cendali F, Stauffer E, Cibiel A, Boisson C, Connes P, and D'Alessandro A
- Subjects
- Adult, Erythrocyte Count, Erythrocyte Deformability genetics, Humans, Lipidomics, Male, Metabolomics, Oxygen Consumption, Physical Exertion physiology, Erythrocytes metabolism, Exercise physiology, Membrane Lipids blood, Physical Exertion genetics
- Abstract
Here we describe the effects of a controlled, 30 min, high-intensity cycling test on blood rheology and the metabolic profiles of red blood cells (RBCs) and plasma from well-trained males. RBCs demonstrated decreased deformability and trended toward increased generation of microparticles after the test. Meanwhile, metabolomics and lipidomics highlighted oxidative stress and activation of membrane lipid remodeling mechanisms in order to cope with altered properties of circulation resulting from physical exertion during the cycling test. Of note, intermediates from coenzyme A (CoA) synthesis for conjugation to fatty acyl chains, in parallel with reversible conversion of carnitine and acylcarnitines, emerged as metabolites that significantly correlate with RBC deformability and the generation of microparticles during exercise. Taken together, we propose that RBC membrane remodeling and repair plays an active role in the physiologic response to exercise by altering RBC properties.
- Published
- 2021
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20. The NLRP3 inflammasome inhibitor OLT1177 rescues cognitive impairment in a mouse model of Alzheimer's disease.
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Lonnemann N, Hosseini S, Marchetti C, Skouras DB, Stefanoni D, D'Alessandro A, Dinarello CA, and Korte M
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- Administration, Oral, Alzheimer Disease drug therapy, Alzheimer Disease genetics, Alzheimer Disease immunology, Amyloid beta-Protein Precursor genetics, Animals, Behavior Observation Techniques, Behavior, Animal drug effects, Cerebral Cortex drug effects, Cerebral Cortex immunology, Cerebral Cortex pathology, Cognitive Dysfunction immunology, Cognitive Dysfunction pathology, Disease Models, Animal, Humans, Inflammasomes immunology, Male, Mice, Mice, Transgenic, Microglia drug effects, Microglia immunology, Microglia pathology, NLR Family, Pyrin Domain-Containing 3 Protein immunology, Neuronal Plasticity drug effects, Neuronal Plasticity immunology, Nitriles therapeutic use, Presenilin-1 genetics, Spatial Memory drug effects, Alzheimer Disease complications, Cognitive Dysfunction drug therapy, Inflammasomes antagonists & inhibitors, NLR Family, Pyrin Domain-Containing 3 Protein antagonists & inhibitors, Nitriles pharmacology
- Abstract
Numerous studies demonstrate that neuroinflammation is a key player in the progression of Alzheimer's disease (AD). Interleukin (IL)-1β is a main inducer of inflammation and therefore a prime target for therapeutic options. The inactive IL-1β precursor requires processing by the the nucleotide-binding oligomerization domain-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome into a mature and active form. Studies have shown that IL-1β is up-regulated in brains of patients with AD, and that genetic inactivation of the NLRP3 inflammasome improves behavioral tests and synaptic plasticity phenotypes in a murine model of the disease. In the present study, we analyzed the effect of pharmacological inhibition of the NLRP3 inflammasome using dapansutrile (OLT1177), an oral NLRP3-specific inhibitor that is safe in humans. Six-month-old WT and APP/PS1 mice were fed with standard mouse chow or OLT1177-enriched chow for 3 mo. The Morris water maze test revealed an impaired learning and memory ability of 9-mo-old APP/PS1 mice ( P = 0.001), which was completely rescued by OLT1177 fed to mice ( P = 0.008 to untreated APP/PS1). Furthermore, our findings revealed that 3 mo of OLT1177 diet can rescue synaptic plasticity in this mouse model of AD ( P = 0.007 to untreated APP/PS1). In addition, microglia were less activated ( P = 0.07) and the number of plaques was reduced in the cortex ( P = 0.03) following NLRP3 inhibition with OLT1177 administration. We also observed an OLT1177 dose-dependent normalization of plasma metabolic markers of AD to those of WT mice. This study suggests the therapeutic potential of treating neuroinflammation with an oral inhibitor of the NLRP3 inflammasome., Competing Interests: Competing interest statement: C.M. serves as Director for Olatec’s Innovative Science Program and has equity in Olatec; D.B.S. serves as Chairman and Chief Executive Officer of Olatec; C.A.D. serves as Chairman of Olatec’s Scientific Advisory Board, is co-Chief Scientific Officer, and has equity in Olatec., (Copyright © 2020 the Author(s). Published by PNAS.)
- Published
- 2020
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21. Nitrogen recycling buffers against ammonia toxicity from skeletal muscle breakdown in hibernating arctic ground squirrels.
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Rice SA, Ten Have GAM, Reisz JA, Gehrke S, Stefanoni D, Frare C, Barati Z, Coker RH, D'Alessandro A, Deutz NEP, and Drew KL
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- Amino Acids metabolism, Animals, Arctic Regions, Arousal, Kidney metabolism, Myofibrils metabolism, Torpor physiology, Urea metabolism, gamma-Glutamyl Hydrolase metabolism, Ammonia toxicity, Hibernation physiology, Muscle, Skeletal physiology, Nitrogen metabolism, Sciuridae physiology
- Abstract
Hibernation is a state of extraordinary metabolic plasticity. The pathways of amino acid metabolism as they relate to nitrogen homeostasis in hibernating mammals in vivo are unknown. Here we show, using pulse isotopic tracing, evidence of increased myofibrillar (skeletal muscle) protein breakdown and suppressed whole-body production of metabolites in vivo throughout deep torpor. As whole-body production of metabolites is suppressed, amino acids with nitrogenous side chains accumulate during torpor, while urea cycle intermediates do not. Using
15 N stable isotope methodology in arctic ground squirrels (Urocitellus parryii), we provide evidence that free nitrogen is buffered and recycled into essential amino acids, non-essential amino acids and the gamma-glutamyl system during the inter-bout arousal period of hibernation. In the absence of nutrient intake or physical activity, our data illustrate the orchestration of metabolic pathways that sustain the provision of essential and non-essential amino acids and prevent ammonia toxicity during hibernation.- Published
- 2020
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22. Evidence of Structural Protein Damage and Membrane Lipid Remodeling in Red Blood Cells from COVID-19 Patients.
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Thomas T, Stefanoni D, Dzieciatkowska M, Issaian A, Nemkov T, Hill RC, Francis RO, Hudson KE, Buehler PW, Zimring JC, Hod EA, Hansen KC, Spitalnik SL, and D'Alessandro A
- Subjects
- Betacoronavirus, COVID-19, Humans, Lipidomics, Membrane Proteins analysis, Membrane Proteins chemistry, Membrane Proteins metabolism, Metabolome physiology, Models, Molecular, Proteome analysis, Proteome chemistry, Proteome metabolism, SARS-CoV-2, Coronavirus Infections blood, Coronavirus Infections pathology, Coronavirus Infections physiopathology, Erythrocytes chemistry, Erythrocytes cytology, Erythrocytes pathology, Membrane Lipids analysis, Membrane Lipids chemistry, Membrane Lipids metabolism, Pandemics, Pneumonia, Viral blood, Pneumonia, Viral pathology, Pneumonia, Viral physiopathology
- Abstract
The SARS-CoV-2 beta coronavirus is the etiological driver of COVID-19 disease, which is primarily characterized by shortness of breath, persistent dry cough, and fever. Because they transport oxygen, red blood cells (RBCs) may play a role in the severity of hypoxemia in COVID-19 patients. The present study combines state-of-the-art metabolomics, proteomics, and lipidomics approaches to investigate the impact of COVID-19 on RBCs from 23 healthy subjects and 29 molecularly diagnosed COVID-19 patients. RBCs from COVID-19 patients had increased levels of glycolytic intermediates, accompanied by oxidation and fragmentation of ankyrin, spectrin beta, and the N-terminal cytosolic domain of band 3 (AE1). Significantly altered lipid metabolism was also observed, in particular, short- and medium-chain saturated fatty acids, acyl-carnitines, and sphingolipids. Nonetheless, there were no alterations of clinical hematological parameters, such as RBC count, hematocrit, or mean corpuscular hemoglobin concentration, with only minor increases in mean corpuscular volume. Taken together, these results suggest a significant impact of SARS-CoV-2 infection on RBC structural membrane homeostasis at the protein and lipid levels. Increases in RBC glycolytic metabolites are consistent with a theoretically improved capacity of hemoglobin to off-load oxygen as a function of allosteric modulation by high-energy phosphate compounds, perhaps to counteract COVID-19-induced hypoxia. Conversely, because the N-terminus of AE1 stabilizes deoxyhemoglobin and finely tunes oxygen off-loading and metabolic rewiring toward the hexose monophosphate shunt, RBCs from COVID-19 patients may be less capable of responding to environmental variations in hemoglobin oxygen saturation/oxidant stress when traveling from the lungs to peripheral capillaries and vice versa.
- Published
- 2020
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23. ZOOMICS: Comparative Metabolomics of Red Blood Cells From Old World Monkeys and Humans.
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Bertolone L, Shin HK, Stefanoni D, Baek JH, Gao Y, Morrison EJ, Nemkov T, Thomas T, Francis RO, Hod EA, Zimring JC, Yoshida T, Karafin M, Schwartz J, Hudson KE, Spitalnik SL, Buehler PW, and D'Alessandro A
- Abstract
As part of the ZOOMICS project, we set out to investigate common and diverging metabolic traits in the blood metabolome across various species by taking advantage of recent developments in high-throughput metabolomics. Here we provide the first comparative metabolomics analysis of fresh and stored human ( n = 21, 10 males, 11 females), olive baboon ( n = 20), and rhesus macaque ( n = 20) red blood cells at baseline and upon 42 days of storage under blood bank conditions. The results indicated similarities and differences across species, which ultimately resulted in a differential propensity to undergo morphological alterations and lyse as a function of the duration of refrigerated storage. Focusing on purine oxidation, carboxylic acid, fatty acid, and arginine metabolism further highlighted species-specific metabolic wiring. For example, through a combination of steady state measurements and
13 C6 15 N4 -arginine tracing experiments, we report an increase in arginine catabolism into ornithine in humans, suggestive of species-specific arginase 1 activity and nitric oxide synthesis-an observation that may impact the translatability of cardiovascular disease studies carried out in non-human primates (NHPs). Finally, we correlated metabolic measurements to storage-induced morphological alterations via scanning electron microscopy and hemolysis, which were significantly lower in human red cells compared to both NHPs., (Copyright © 2020 Bertolone, Shin, Stefanoni, Baek, Gao, Morrison, Nemkov, Thomas, Francis, Hod, Zimring, Yoshida, Karafin, Schwartz, Hudson, Spitalnik, Buehler and D’Alessandro.)- Published
- 2020
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24. Red blood cell metabolism in Rhesus macaques and humans: comparative biology of blood storage.
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Stefanoni D, Shin HKH, Baek JH, Champagne DP, Nemkov T, Thomas T, Francis RO, Zimring JC, Yoshida T, Reisz JA, Spitalnik SL, Buehler PW, and D'Alessandro A
- Subjects
- Animals, Blood Banks, Erythrocyte Transfusion, Female, Humans, Macaca mulatta, Male, Blood Preservation, Erythrocytes
- Abstract
Macaques are emerging as a critical animal model in transfusion medicine, because of their evolutionary similarity to humans and perceived utility in discovery and translational science. However, little is known about the metabolism of Rhesus macaque red blood cells (RBC) and how this compares to human RBC metabolism under standard blood banking conditions. Metabolomic and lipidomic analyses, and tracing experiments with [1,2,3-
13 C3 ]glucose, were performed using fresh and stored RBC (sampled weekly until storage day 42) obtained from Rhesus macaques (n=20) and healthy human volunteers (n=21). These results were further validated with targeted quantification against stable isotope-labeled internal standards. Metabolomic analyses demonstrated inter-species differences in RBC metabolism independent of refrigerated storage. Although similar trends were observed throughout storage for several metabolic pathways, species- and sex-specific differences were also observed. The most notable differences were in glutathione and sulfur metabolites, purine and lipid oxidation metabolites, acylcarnitines, fatty acyl composition of several classes of lipids (including phosphatidylserines), glyoxylate pathway intermediates, and arginine and carboxylic acid metabolites. Species-specific dietary and environmental compounds were also detected. Overall, the results suggest an increased basal and refrigerator-storage-induced propensity for oxidant stress and lipid remodeling in Rhesus macaque RBC cells, as compared to human red cells. The overlap between Rhesus macaque and human RBC metabolic phenotypes suggests the potential utility of a translational model for simple RBC transfusions, although inter-species storage-dependent differences need to be considered when modeling complex disease states, such as transfusion in trauma/hemorrhagic shock models., (Copyright© 2020 Ferrata Storti Foundation.)- Published
- 2020
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25. COVID-19 infection alters kynurenine and fatty acid metabolism, correlating with IL-6 levels and renal status.
- Author
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Thomas T, Stefanoni D, Reisz JA, Nemkov T, Bertolone L, Francis RO, Hudson KE, Zimring JC, Hansen KC, Hod EA, Spitalnik SL, and D'Alessandro A
- Subjects
- Adult, Aged, Amino Acids metabolism, Betacoronavirus, Blood Glucose metabolism, COVID-19, Case-Control Studies, Creatine metabolism, Creatinine metabolism, Cystine, Fatty Acids, Nonesterified metabolism, Female, Humans, Male, Metabolome, Metabolomics, Methionine analogs & derivatives, Middle Aged, Pandemics, Polyamines metabolism, Proteolysis, SARS-CoV-2, Tryptophan metabolism, Coronavirus Infections metabolism, Fatty Acids metabolism, Interleukin-6 metabolism, Kynurenine metabolism, Oxidative Stress, Pneumonia, Viral metabolism, Renal Insufficiency metabolism
- Abstract
BACKGROUNDReprogramming of host metabolism supports viral pathogenesis by fueling viral proliferation, by providing, for example, free amino acids and fatty acids as building blocks.METHODSTo investigate metabolic effects of SARS-CoV-2 infection, we evaluated serum metabolites of patients with COVID-19 (n = 33; diagnosed by nucleic acid testing), as compared with COVID-19-negative controls (n = 16).RESULTSTargeted and untargeted metabolomics analyses identified altered tryptophan metabolism into the kynurenine pathway, which regulates inflammation and immunity. Indeed, these changes in tryptophan metabolism correlated with interleukin-6 (IL-6) levels. Widespread dysregulation of nitrogen metabolism was also seen in infected patients, with altered levels of most amino acids, along with increased markers of oxidant stress (e.g., methionine sulfoxide, cystine), proteolysis, and renal dysfunction (e.g., creatine, creatinine, polyamines). Increased circulating levels of glucose and free fatty acids were also observed, consistent with altered carbon homeostasis. Interestingly, metabolite levels in these pathways correlated with clinical laboratory markers of inflammation (i.e., IL-6 and C-reactive protein) and renal function (i.e., blood urea nitrogen).CONCLUSIONIn conclusion, this initial observational study identified amino acid and fatty acid metabolism as correlates of COVID-19, providing mechanistic insights, potential markers of clinical severity, and potential therapeutic targets.FUNDINGBoettcher Foundation Webb-Waring Biomedical Research Award; National Institute of General and Medical Sciences, NIH; and National Heart, Lung, and Blood Institute, NIH.
- Published
- 2020
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26. Evidence for structural protein damage and membrane lipid remodeling in red blood cells from COVID-19 patients.
- Author
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Thomas T, Stefanoni D, Dzieciatkowska M, Issaian A, Nemkov T, Hill RC, Francis RO, Hudson KE, Buehler PW, Zimring JC, Hod EA, Hansen KC, Spitalnik SL, and D'Alessandro A
- Abstract
The SARS-CoV-2 beta coronavirus is the etiological driver of COVID-19 disease, which is primarily characterized by shortness of breath, persistent dry cough, and fever. Because they transport oxygen, red blood cells (RBCs) may play a role in the severity of hypoxemia in COVID-19 patients. The present study combines state-of-the-art metabolomics, proteomics, and lipidomics approaches to investigate the impact of COVID-19 on RBCs from 23 healthy subjects and 29 molecularly-diagnosed COVID-19 patients. RBCs from COVID-19 patients had increased levels of glycolytic intermediates, accompanied by oxidation and fragmentation of ankyrin, spectrin beta, and the N-terminal cytosolic domain of band 3 (AE1). Significantly altered lipid metabolism was also observed, especially short and medium chain saturated fatty acids, acyl-carnitines, and sphingolipids. Nonetheless, there were no alterations of clinical hematological parameters, such as RBC count, hematocrit, and mean corpuscular hemoglobin concentration, with only minor increases in mean corpuscular volume. Taken together, these results suggest a significant impact of SARS-CoV-2 infection on RBC structural membrane homeostasis at the protein and lipid levels. Increases in RBC glycolytic metabolites are consistent with a theoretically improved capacity of hemoglobin to off-load oxygen as a function of allosteric modulation by high-energy phosphate compounds, perhaps to counteract COVID-19-induced hypoxia. Conversely, because the N-terminus of AE1 stabilizes deoxyhemoglobin and finely tunes oxygen off-loading, RBCs from COVID-19 patients may be incapable of responding to environmental variations in hemoglobin oxygen saturation when traveling from the lungs to peripheral capillaries and, as such, may have a compromised capacity to transport and deliver oxygen.
- Published
- 2020
- Full Text
- View/download PDF
27. Metabolomics of Endurance Capacity in World Tour Professional Cyclists.
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San-Millán I, Stefanoni D, Martinez JL, Hansen KC, D'Alessandro A, and Nemkov T
- Abstract
The study of elite athletes provides a unique opportunity to define the upper limits of human physiology and performance. Across a variety of sports, these individuals have trained to optimize the physiological parameters of their bodies in order to compete on the world stage. To characterize endurance capacity, techniques such as heart rate monitoring, indirect calorimetry, and whole blood lactate measurement have provided insight into oxygen utilization, and substrate utilization and preference, as well as total metabolic capacity. However, while these techniques enable the measurement of individual, representative variables critical for sports performance, they lack the molecular resolution that is needed to understand which metabolic adaptations are necessary to influence these metrics. Recent advancements in mass spectrometry-based analytical approaches have enabled the measurement of hundreds to thousands of metabolites in a single analysis. Here we employed targeted and untargeted metabolomics approaches to investigate whole blood responses to exercise in elite World Tour (including Tour de France) professional cyclists before and after a graded maximal physiological test. As cyclists within this group demonstrated varying blood lactate accumulation as a function of power output, which is an indicator of performance, we compared metabolic profiles with respect to lactate production to identify adaptations associated with physiological performance. We report that numerous metabolic adaptations occur within this physically elite population ( n = 21 males, 28.2 ± 4.7 years old) in association with the rate of lactate accumulation during cycling. Correlation of metabolite values with lactate accumulation has revealed metabolic adaptations that occur in conjunction with improved endurance capacity. In this population, cycling induced increases in tricarboxylic acid (TCA) cycle metabolites and Coenzyme A precursors. These responses occurred proportionally to lactate accumulation, suggesting a link between enhanced mitochondrial networks and the ability to sustain higher workloads. In association with lactate accumulation, altered levels of amino acids before and after exercise point to adaptations that confer unique substrate preference for energy production or to promote more rapid recovery. Cyclists with slower lactate accumulation also have higher levels of basal oxidative stress markers, suggesting long term physiological adaptations in these individuals that support their premier competitive status in worldwide competitions., (Copyright © 2020 San-Millán, Stefanoni, Martinez, Hansen, D’Alessandro and Nemkov.)
- Published
- 2020
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28. Impact of taurine on red blood cell metabolism and implications for blood storage.
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Bertolone L, Roy MK, Hay AM, Morrison EJ, Stefanoni D, Fu X, Kanias T, Kleinman S, Dumont LJ, Stone M, Nemkov T, Busch MP, Zimring JC, and D'Alessandro A
- Subjects
- Animals, Humans, Metabolomics, Mice, Taurine pharmacology, Blood Donors, Blood Preservation, Erythrocytes metabolism, Oxidative Stress drug effects, Taurine pharmacokinetics
- Abstract
Background: Taurine is an antioxidant that is abundant in some common energy drinks. Here we hypothesized that the antioxidant activity of taurine in red blood cells (RBCs) could be leveraged to counteract storage-induced oxidant stress., Study Design and Methods: Metabolomics analyses were performed on plasma and RBCs from healthy volunteers (n = 4) at baseline and after consumption of a whole can of a common, taurine-rich (1000 mg/serving) energy drink. Reductionistic studies were also performed by incubating human RBCs with taurine ex vivo (unlabeled or
13 C15 N-labeled) at increasing doses (0, 100, 500, and 1000 μmol/L) at 37°C for up to 16 hours, with and without oxidant stress challenge with hydrogen peroxide (0.1% or 0.5%). Finally, we stored human and murine RBCs under blood bank conditions in additives supplemented with 500 μmol/L taurine, before metabolomics and posttransfusion recovery studies., Results: Consumption of energy drinks increased plasma and RBC levels of taurine, which was paralleled by increases in glycolysis and glutathione (GSH) metabolism in the RBC. These observations were recapitulated ex vivo after incubation with taurine and hydrogen peroxide. Taurine levels in the RBCs from the REDS-III RBC-Omics donor biobank were directly proportional to the total levels of GSH and glutathionylated metabolites and inversely correlated to oxidative hemolysis measurements. Storage of human RBCs in the presence of taurine improved energy and redox markers of storage quality and increased posttransfusion recoveries in FVB mice., Conclusion: Taurine modulates RBC antioxidant metabolism in vivo and ex vivo, an observation of potential relevance to transfusion medicine., (© 2020 AABB.)- Published
- 2020
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29. Nicotine exposure increases markers of oxidant stress in stored red blood cells from healthy donor volunteers.
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Stefanoni D, Fu X, Reisz JA, Kanias T, Nemkov T, Page GP, Dumont L, Roubinian N, Stone M, Kleinman S, Busch M, Zimring JC, and D'Alessandro A
- Subjects
- Erythrocytes pathology, Female, Humans, Male, Blood Donors, Blood Preservation, Cigarette Smoking adverse effects, Cigarette Smoking blood, Cigarette Smoking pathology, Erythrocytes metabolism, Nicotine adverse effects, Oxidative Stress
- Abstract
Background: Cigarette smoking is a frequent habit across blood donors (approx. 13% of the donor population), that could compound biologic factors and exacerbate oxidant stress to stored red blood cells (RBCs)., Study Design and Methods: As part of the REDS-III RBC-Omics (Recipient Epidemiology Donor Evaluation Study III Red Blood Cell-Omics) study, a total of 599 samples were sterilely drawn from RBC units stored under blood bank conditions at Storage Days 10, 23, and 42 days, before testing for hemolysis parameters and metabolomics. Quantitative measurements of nicotine and its metabolites cotinine and cotinine oxide were performed against deuterium-labeled internal standards., Results: Donors whose blood cotinine levels exceeded 10 ng/mL (14% of the tested donors) were characterized by higher levels of early glycolytic intermediates, pentose phosphate pathway metabolites, and pyruvate-to-lactate ratios, all markers of increased basal oxidant stress. Consistently, increased glutathionylation of oxidized triose sugars and lipid aldehydes was observed in RBCs donated by nicotine-exposed donors, which were also characterized by increased fatty acid desaturation, purine salvage, and methionine oxidation and consumption via pathways involved in oxidative stress-triggered protein damage-repair mechanisms., Conclusion: RBCs from donors with high levels of nicotine exposure are characterized by increases in basal oxidant stress and decreases in osmotic hemolysis. These findings indicate the need for future clinical studies aimed at addressing the impact of smoking and other sources of nicotine (e.g., nicotine patches, snuff, vaping, secondhand tobacco smoke) on RBC storage quality and transfusion efficacy., (© 2020 AABB.)
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- 2020
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30. Metabolic phenotypes of standard and cold-stored platelets.
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D'Alessandro A, Thomas KA, Stefanoni D, Gamboni F, Shea SM, Reisz JA, and Spinella PC
- Subjects
- Arginine metabolism, Blood Platelets cytology, Chromatography, High Pressure Liquid, Citric Acid Cycle, Cold Temperature, Humans, Mass Spectrometry, Plateletpheresis, Blood Platelets metabolism, Metabolome, Metabolomics methods
- Abstract
Background: Conventional platelet (PLT) storage at room temperature under continuous agitation results in a limited shelf life (5 days) and an increased risk of bacterial contamination. However, both of these aspects can be ameliorated by cold storage. Preliminary work has suggested that PLTs can be cold stored for up to 3 weeks, while preserving their metabolic activity longer than in PLTs stored at room temperature. As such, in the present study, we hypothesized that the metabolic phenotypes of PLTs stored at 4°C for 3 weeks could be comparable to that of room temperature-stored PLTs at 22°C for 5 days., Study Design and Methods: Metabolomics analyses were performed on nine apheresis PLT concentrates stored either at room temperature (22°C) for 5 days or refrigerated conditions (4°C) for up to 3 weeks., Results: Refrigeration did not impact the rate of decline in glutamine or the intracellular levels of Krebs cycle metabolites upstream to fumarate and malate. It did, however, decrease oxidant stress (to glutathione and purines) and slowed down the activation of the pentose phosphate pathway, glycolysis, and fatty acid metabolism (acyl-carnitines)., Conclusion: The overall metabolic phenotypes of 4°C PLTs at Storage Day 10 are comparable to PLTs stored at 22°C at the end of their 5-day shelf life, while additional changes in glycolysis, purine, and fatty acid metabolism are noted by Day 21., (© 2019 AABB.)
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- 2020
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31. COVID-19 infection results in alterations of the kynurenine pathway and fatty acid metabolism that correlate with IL-6 levels and renal status.
- Author
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Thomas T, Stefanoni D, Reisz JA, Nemkov T, Bertolone L, Francis RO, Hudson KE, Zimring JC, Hansen KC, Hod EA, Spitalnik SL, and D'Alessandro A
- Abstract
Previous studies suggest a role for systemic reprogramming of host metabolism during viral pathogenesis to fuel rapidly expanding viral proliferation, for example by providing free amino acids and fatty acids as building blocks. In addition, general alterations in metabolism can provide key understanding of pathogenesis. However, little is known about the specific metabolic effects of SARS-COV-2 infection. The present study evaluated the serum metabolism of COVID-19 patients (n=33), identified by a positive nucleic acid test of a nasopharyngeal swab, as compared to COVID-19-negative control patients (n=16). Targeted and untargeted metabolomics analyses specifically identified alterations in the metabolism of tryptophan into the kynurenine pathway, which is well-known to be involved in regulating inflammation and immunity. Indeed, the observed changes in tryptophan metabolism correlated with serum interleukin-6 (IL-6) levels. Metabolomics analysis also confirmed widespread dysregulation of nitrogen metabolism in infected patients, with decreased circulating levels of most amino acids, except for tryptophan metabolites in the kynurenine pathway, and increased markers of oxidant stress (e.g., methionine sulfoxide, cystine), proteolysis, and kidney dysfunction (e.g., creatine, creatinine, polyamines). Increased circulating levels of glucose and free fatty acids were also observed, consistent with altered carbon homeostasis in COVID-19 patients. Metabolite levels in these pathways correlated with clinical laboratory markers of inflammation and disease severity (i.e., IL-6 and C-reactive protein) and renal function (i.e., blood urea nitrogen). In conclusion, this initial observational study of the metabolic consequences of COVID-19 infection in a clinical cohort identified amino acid metabolism (especially kynurenine and cysteine/taurine) and fatty acid metabolism as correlates of COVID-19, providing mechanistic insights, potential markers of clinical severity, and potential therapeutic targets.
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- 2020
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32. Hypoxic storage of red blood cells improves metabolism and post-transfusion recovery.
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DʼAlessandro A, Yoshida T, Nestheide S, Nemkov T, Stocker S, Stefanoni D, Mohmoud F, Rugg N, Dunham A, and Cancelas JA
- Subjects
- Adult, Blood Donors, Blood Preservation standards, Blood Transfusion standards, Cross-Sectional Studies, Female, Healthy Volunteers, Hemolysis, Humans, Male, Recovery of Function, Transplantation, Autologous, Blood Preservation methods, Erythrocytes metabolism, Hypoxia
- Abstract
Background: Blood transfusion is a lifesaving intervention for millions of recipients worldwide every year. Storing blood makes this possible but also promotes a series of alterations to the metabolism of the stored erythrocyte. It is unclear whether the metabolic storage lesion is correlated with clinically relevant outcomes and whether strategies aimed at improving the metabolic quality of stored units, such as hypoxic storage, ultimately improve performance in the transfused recipient., Study Design and Methods: Twelve healthy donor volunteers were recruited in a two-arm cross-sectional study, in which each subject donated 2 units to be stored under standard (normoxic) or hypoxic conditions (Hemanext technology). End-of-storage measurements of hemolysis and autologous posttransfusion recovery (PTR) were correlated to metabolomics measurements at Days 0, 21, and 42., Results: Hypoxic red blood cells (RBCs) showed superior PTR and comparable hemolysis to donor-paired standard units. Hypoxic storage improved energy and redox metabolism (glycolysis and 2,3-diphosphoglycerate), improved glutathione and methionine homeostasis, decreased purine oxidation and membrane lipid remodeling (free fatty acid levels, unsaturation and hydroxylation, acyl-carnitines). Intra- and extracellular metabolites in these pathways (including some dietary purines) showed significant correlations with PTR and hemolysis, though the degree of correlation was influenced by sulfur dioxide (SO
2 ) levels., Conclusion: Hypoxic storage improves energy and redox metabolism of stored RBCs, which results in improved posttransfusion recoveries in healthy autologous recipients-a Food and Drug Administration gold standard of stored blood quality. In addition, we identified candidate metabolic predictors of PTR for RBCs stored under standard and hypoxic conditions., (© 2020 AABB.)- Published
- 2020
- Full Text
- View/download PDF
33. Gene-Diet Interactions: Dietary Rescue of Metabolic Effects in spen -Depleted Drosophila melanogaster .
- Author
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Gillette CM, Hazegh KE, Nemkov T, Stefanoni D, D'Alessandro A, Taliaferro JM, and Reis T
- Subjects
- Animals, Dietary Carbohydrates metabolism, Drosophila Proteins deficiency, Drosophila melanogaster, Fat Body metabolism, Glycolysis, Larva growth & development, Larva metabolism, Lipid Metabolism, Diet, Drosophila Proteins genetics, Gene-Environment Interaction, Homeodomain Proteins genetics, Metabolome, RNA-Binding Proteins genetics
- Abstract
Obesity and its comorbidities are a growing health epidemic. Interactions between genetic background, the environment, and behavior ( i.e. , diet) greatly influence organismal energy balance. Previously, we described obesogenic mutations in the gene Split ends (Spen) in Drosophila melanogaster , and roles for Spen in fat storage and metabolic state. Lipid catabolism is impaired in Spen -deficient fat storage cells, accompanied by a compensatory increase in glycolytic flux and protein catabolism. Here, we investigate gene-diet interactions to determine if diets supplemented with specific macronutrients can rescue metabolic dysfunction in Spen-depleted animals. We show that a high-yeast diet partially rescues adiposity and developmental defects. High sugar partially improves developmental timing as well as longevity of mated females. Gene-diet interactions were heavily influenced by developmental-stage-specific organismal needs: extra yeast provides benefits early in development (larval stages) but becomes detrimental in adulthood. High sugar confers benefits to Spen-depleted animals at both larval and adult stages, with the caveat of increased adiposity. A high-fat diet is detrimental according to all tested criteria, regardless of genotype. Whereas Spen depletion influenced phenotypic responses to supplemented diets, diet was the dominant factor in directing the whole-organism steady-state metabolome. Obesity is a complex disease of genetic, environmental, and behavioral inputs. Our results show that diet customization can ameliorate metabolic dysfunction underpinned by a genetic factor., (Copyright © 2020 by the Genetics Society of America.)
- Published
- 2020
- Full Text
- View/download PDF
34. Red Blood Cell Metabolic Responses to Torpor and Arousal in the Hibernator Arctic Ground Squirrel.
- Author
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Gehrke S, Rice S, Stefanoni D, Wilkerson RB, Nemkov T, Reisz JA, Hansen KC, Lucas A, Cabrales P, Drew K, and D'Alessandro A
- Subjects
- Animals, Hibernation physiology, Sulfur metabolism, Tryptophan metabolism, Arousal physiology, Erythrocytes metabolism, Erythrocytes physiology, Metabolome physiology, Sciuridae blood, Sciuridae metabolism, Sciuridae physiology, Torpor physiology
- Abstract
Arctic ground squirrels provide a unique model to investigate metabolic responses to hibernation in mammals. During winter months these rodents are exposed to severe hypothermia, prolonged fasting, and hypoxemia. In the light of their role in oxygen transport/off-loading and owing to the absence of nuclei and organelles (and thus de novo protein synthesis capacity), mature red blood cells have evolved metabolic programs to counteract physiological or pathological hypoxemia. However, red blood cell metabolism in hibernation has not yet been investigated. Here we employed targeted and untargeted metabolomics approaches to investigate erythrocyte metabolism during entrance to torpor to arousal, with a high resolution of the intermediate time points. We report that torpor and arousal promote metabolism through glycolysis and pentose phosphate pathway, respectively, consistent with previous models of oxygen-dependent metabolic modulation in mature erythrocytes. Erythrocytes from hibernating squirrels showed up to 100-fold lower levels of biomarkers of reperfusion injury, such as the pro-inflammatory dicarboxylate succinate. Altered tryptophan metabolism during torpor was here correlated to the accumulation of potentially neurotoxic catabolites kynurenine, quinolinate, and picolinate. Arousal was accompanied by alterations of sulfur metabolism, including sudden spikes in a metabolite putatively identified as thiorphan (level 1 confidence)-a potent inhibitor of several metalloproteases that play a crucial role in nociception and inflammatory complication to reperfusion secondary to ischemia or hemorrhage. Preliminary studies in rats showed that intravenous injection of thiorphan prior to resuscitation mitigates metabolic and cytokine markers of reperfusion injury, etiological contributors to inflammatory complications after shock.
- Published
- 2019
- Full Text
- View/download PDF
35. Methylation of protein aspartates and deamidated asparagines as a function of blood bank storage and oxidative stress in human red blood cells.
- Author
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Reisz JA, Nemkov T, Dzieciatkowska M, Culp-Hill R, Stefanoni D, Hill RC, Yoshida T, Dunham A, Kanias T, Dumont LJ, Busch M, Eisenmesser EZ, Zimring JC, Hansen KC, and D'Alessandro A
- Subjects
- Erythrocytes cytology, Humans, Methylation, Proteomics, Time Factors, Asparagine metabolism, Aspartic Acid metabolism, Blood Banks, Blood Preservation, Erythrocytes metabolism, Oxidative Stress, Protein Processing, Post-Translational
- Abstract
Background: Being devoid of de novo protein synthesis capacity, red blood cells (RBCs) have evolved to recycle oxidatively damaged proteins via mechanisms that involve methylation of dehydrated and deamidated aspartate and asparagine residues. Here we hypothesize that such mechanisms are relevant to routine storage in the blood bank., Study Design and Methods: Within the framework of the REDS-III RBC-Omics (Recipient Epidemiology Donor Evaluation Study III Red Blood Cell-Omics) study, packed RBC units (n = 599) were stored under blood bank conditions for 10, 23, and 42 days and profiled for oxidative hemolysis and time-dependent metabolic dysregulation of the trans-sulfuration pathway., Results: In these units, methionine consumption positively correlated with storage age and oxidative hemolysis. Mechanistic studies show that this phenomenon is favored by oxidative stress or hyperoxic storage (sulfur dioxide >95%), and prevented by hypoxia or methyltransferase inhibition. Through a combination of proteomics approaches and
13 C-methionine tracing, we observed oxidation-induced increases in both Asn deamidation to Asp and formation of methyl-Asp on key structural proteins and enzymes, including Band 3, hemoglobin, ankyrin, 4.1, spectrin beta, aldolase, glyceraldehyde 3-phosphate dehydrogenase, biphosphoglycerate mutase, lactate dehydrogenase and catalase. Methylated regions tended to map proximal to the active site (e.g., N316 of glyceraldehyde 3-phosphate dehydrogenase) and/or residues interacting with the N-terminal cytosolic domain of Band 3., Conclusion: While methylation of basic amino acid residues serves as an epigenetic modification in nucleated cells, protein methylation at carboxylate side chains and deamidated asparagines is a nonepigenetic posttranslational sensor of oxidative stress and refrigerated storage in anucleated human RBCs., (© 2018 AABB.)- Published
- 2018
- Full Text
- View/download PDF
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