Biological and genetic determinants of glycolysis: Phosphofructokinase isoforms boost energy status of stored red blood cells and transfusion outcomes.

Mature red blood cells (RBCs) lack mitochondria and thus exclusively rely on glycolysis to generate adenosine triphosphate (ATP) during aging in vivo or storage in blood banks. Here, we leveraged 13,029 volunteers from the Recipient Epidemiology and Donor Evaluation Study to identify associations between end-of-storage levels of glycolytic metabolites and donor age, sex, and ancestry-specific genetic polymorphisms in regions encoding phosphofructokinase 1, platelet (detected in mature RBCs); hexokinase 1 (HK1); and ADP-ribosyl cyclase 1 and 2 (CD38/BST1). Gene-metabolite associations were validated in fresh and stored RBCs from 525 Diversity Outbred mice and via multi-omics characterization of 1,929 samples from 643 human RBC units during storage. ATP and hypoxanthine (HYPX) levels-and the genetic traits linked to them-were associated with hemolysis in vitro and in vivo, both in healthy autologous transfusion recipients and in 5,816 critically ill patients receiving heterologous transfusions, suggesting their potential as markers to improve transfusion outcomes.

Ferroptosis regulates hemolysis in stored murine and human red blood cells.

Red blood cell (RBC) metabolism regulates hemolysis during aging in vivo and in the blood bank. Here, we leveraged a diversity outbred mouse population to map the genetic drivers of fresh/stored RBC metabolism and extravascular hemolysis upon storage and transfusion in 350 mice. We identify the ferrireductase Steap3 as a critical regulator of a ferroptosis-like process of lipid peroxidation. Steap3 polymorphisms were associated with RBC iron content, in vitro hemolysis, and in vivo extravascular hemolysis both in mice and 13,091 blood donors from the Recipient Epidemiology and Donor evaluation Study. Using metabolite Quantitative Trait Loci analyses, we identified a network of gene products (FADS1/2, EPHX2 and LPCAT3) - enriched in donors of African descent - associated with oxylipin metabolism in stored human RBCs and related to Steap3 or its transcriptional regulator, the tumor protein TP53. Genetic variants were associated with lower in vivo hemolysis in thousands of single-unit transfusion recipients. Highlights: Steap3 regulates lipid peroxidation and extravascular hemolysis in 350 diversity outbred miceSteap3 SNPs are linked to RBC iron, hemolysis, vesiculation in 13,091 blood donorsmQTL analyses of oxylipins identified ferroptosis-related gene products FADS1/2, EPHX2, LPCAT3Ferroptosis markers are linked to hemoglobin increments in transfusion recipients.

A Multiomics Assessment of Preoperative Exercise in Pancreatic Cancer Survivors Receiving Neoadjuvant Therapy: A Case Series.

To molecularly characterize the impact of exercise on mitigating neoadjuvant treatment (NAT)-induced physical decline in pancreatic ductal adenocarcinoma (PDAC) patients, a multi-omics approach was employed for the analysis of plasma samples before and after a personalized exercise intervention. Consisting of personalized aerobic and resistance exercises, this intervention was associated with significant molecular changes that correlated with improvements in lean mass, appendicular skeletal muscle index (ASMI), and performance in the 400-m walk test (MWT) and sit-to-stand test. These alterations indicated exercise-induced modulation of inflammation and mitochondrial function markers. This case study provides proof-of-principal application for multiomics-based assessments of supervised exercise, thereby supporting this intervention as a feasible and beneficial intervention for PDAC patients to potentially enhance treatment response and patient quality of life. The molecular changes observed here underscore the importance of physical activity in cancer treatment protocols, advocating for the development of accessible multiomics-guided exercise programs for cancer patients.

Inflammation promotes aging-associated oncogenesis in the lung.

Background: Lung cancer is the leading cause of cancer death in the world. While cigarette smoking is the major preventable factor for cancers in general and lung cancer in particular, old age is also a major risk factor. Aging-related chronic, low-level inflammation, termed inflammaging, has been widely documented; however, it remains unclear how inflammaging contributes to increased lung cancer incidence. Aim: To establish connections between aging-associated changes in the lungs and cancer risk. Methods: We analyzed public databases of gene expression for normal and cancerous human lungs and used mouse models to understand which changes were dependent on inflammation, as well as to assess the impact on oncogenesis. Results: Analyses of GTEx and TCGA databases comparing gene expression profiles from normal lungs, lung adenocarcinoma, lung squamous cell carcinoma of subjects across age groups revealed upregulated pathways such as inflammatory response, TNFA signaling via NFκB, and interferon-gamma response. Similar pathways were identified comparing the gene expression profiles of young and old mouse lungs. Transgenic expression of alpha 1 antitrypsin (AAT) partially reverses increases in markers of aging-associated inflammation and immune deregulation. Using an orthotopic model of lung cancer using cells derived from EML4-ALK fusion-induced adenomas, we demonstrated an increased tumor outgrowth in lungs of old mice while NLRP3 knockout in old mice decreased tumor volumes, suggesting that inflammation contributes to increased lung cancer development in aging organisms. Conclusions: These studies reveal how expression of an anti-inflammatory mediator (AAT) can reduce some but not all aging-associated changes in mRNA and protein expression in the lungs. We further show that aging is associated with increased tumor outgrowth in the lungs, which may relate to an increased inflammatory microenvironment.

Biological and Genetic Determinants of Glycolysis: Phosphofructokinase Isoforms Boost Energy Status of Stored Red Blood Cells and Transfusion Outcomes.

Mature red blood cells (RBCs) lack mitochondria, and thus exclusively rely on glycolysis to generate adenosine triphosphate (ATP) during aging in vivo or storage in the blood bank. Here we leveraged 13,029 volunteers from the Recipient Epidemiology and Donor Evaluation Study to identify an association between end-of-storage levels of glycolytic metabolites and donor age, sex, and ancestry-specific genetic polymorphisms in regions encoding phosphofructokinase 1, platelet (detected in mature RBCs), hexokinase 1, ADP-ribosyl cyclase 1 and 2 (CD38/BST1). Gene-metabolite associations were validated in fresh and stored RBCs from 525 Diversity Outbred mice, and via multi-omics characterization of 1,929 samples from 643 human RBC units during storage. ATP and hypoxanthine levels - and the genetic traits linked to them - were associated with hemolysis in vitro and in vivo, both in healthy autologous transfusion recipients and in 5,816 critically ill patients receiving heterologous transfusions, suggesting their potential as markers to improve transfusion outcomes. Highlights: Blood donor age and sex affect glycolysis in stored RBCs from 13,029 volunteers;Ancestry, genetic polymorphisms in PFKP, HK1, CD38/BST1 influence RBC glycolysis;Modeled PFKP effects relate to preventing loss of the total AXP pool in stored RBCs;ATP and hypoxanthine are biomarkers of hemolysis in vitro and in vivo.

Dasatinib and Trametinib Promote Anti-Tumor Metabolic Activity.

Thyroid cancer is the most common endocrine neoplasm, and despite its overall high survival rate, patients with metastatic disease or tumors that resist radioactive iodine experience a significantly worse prognosis. Helping these patients requires a better understanding of how therapeutics alter cellular function. Here, we describe the change in metabolite profiles after treating thyroid cancer cells with the kinase inhibitors dasatinib and trametinib. We reveal alterations to glycolysis, the TCA cycle, and amino acid levels. We also highlight how these drugs promote short-term accumulation of the tumor-suppressive metabolite 2-oxoglutarate, and demonstrate that it reduces the viability of thyroid cancer cells in vitro. These results show that kinase inhibition profoundly alters the metabolome of cancer cells and highlight the need to better understand how therapeutics reprogram metabolic processes, and ultimately, cancer cell behavior.

Vitamin B5 and succinyl-CoA improve ineffective erythropoiesis in SF3B1-mutated myelodysplasia.

Patients with myelodysplastic syndrome and ring sideroblasts (MDS-RS) present with symptomatic anemia due to ineffective erythropoiesis that impedes their quality of life and increases morbidity. More than 80% of patients with MDS-RS harbor splicing factor 3B subunit 1 (SF3B1) mutations, the founder aberration driving MDS-RS disease. Here, we report how mis-splicing of coenzyme A synthase (COASY), induced by mutations in SF3B1, affects heme biosynthesis and erythropoiesis. Our data revealed that COASY was up-regulated during normal erythroid differentiation, and its silencing prevented the formation of erythroid colonies, impeded erythroid differentiation, and precluded heme accumulation. In patients with MDS-RS, loss of protein due to COASY mis-splicing led to depletion of both CoA and succinyl-CoA. Supplementation with COASY substrate (vitamin B5) rescued CoA and succinyl-CoA concentrations in SF3B1mut cells and mended erythropoiesis differentiation defects in MDS-RS primary patient cells. Our findings reveal a key role of the COASY pathway in erythroid maturation and identify upstream and downstream metabolites of COASY as a potential treatment for anemia in patients with MDS-RS.

pH regulates hematopoietic stem cell potential via polyamines.

Upon ex vivo culture, hematopoietic stem cells (HSCs) quickly lose potential and differentiate into progenitors. The identification of culture conditions that maintain the potential of HSCs ex vivo is therefore of high clinical interest. Here, we demonstrate that the potential of murine and human HSCs is maintained when cultivated for 2 days ex vivo at a pH of 6.9, in contrast to cultivation at the commonly used pH of 7.4. When cultivated at a pH of 6.9, HSCs remain smaller, less metabolically active, less proliferative and show enhanced reconstitution ability upon transplantation compared to HSC cultivated at pH 7.4. HSCs kept at pH 6.9 show an attenuated polyamine pathway. Pharmacological inhibition of the polyamine pathway in HSCs cultivated at pH 7.4 with DFMO mimics phenotypes and potential of HSCs cultivated at pH 6.9. Ex vivo exposure to a pH of 6.9 is therefore a positive regulator of HSC function by reducing polyamines. These findings might improve HSC short-term cultivation protocols for transplantation and gene therapy interventions.

Sphingosine 1-phosphate has a negative effect on RBC storage quality.

Blood storage promotes the rapid depletion of red blood cell (RBC) high-energy adenosine triphosphate (ATP) and 2,3-diphosphoglycerate (DPG), which are critical regulators of erythrocyte physiology and function, as well as oxygen kinetics and posttransfusion survival. Sphingosine-1-phosphate (S1P) promotes fluxes through glycolysis. We hypothesized that S1P supplementation to stored RBC units would improve energy metabolism and posttransfusion recovery. We quantified S1P in 1929 samples (n = 643, storage days 10, 23, and 42) from the REDS RBC Omics study. We then supplemented human and murine RBCs from good storer (C57BL6/J) and poor storer strains (FVB) with S1P (1, 5, and 10 µM) before measurements of metabolism and posttransfusion recovery. Similar experiments were repeated for mice with genetic ablation of the S1P biosynthetic pathway (sphingosine kinase 1 [Sphk1] knockout [KO]). Sample analyses included metabolomics at steady state, tracing experiments with 1,2,3-13C3-glucose, proteomics, and analysis of end-of-storage posttransfusion recovery, under normoxic and hypoxic storage conditions. Storage promoted decreases in S1P levels, which were the highest in units donated by female or older donors. Supplementation of S1P to human and murine RBCs boosted the steady-state levels of glycolytic metabolites and glycolytic fluxes, ie the generation of ATP and DPG, at the expense of the pentose phosphate pathway. Lower posttransfusion recovery was observed upon S1P supplementation. All these phenomena were reversed in Sphk1 KO mice or with hypoxic storage. S1P is a positive regulator of energy metabolism and a negative regulator of antioxidant metabolism in stored RBCs, resulting in lower posttransfusion recoveries in murine models.

ZOOMICS : comparative metabolomics of red blood cells from dogs, cows, horses and donkeys during refrigerated storage for up to 42 days.

BACKGROUND: The use of omics technologies in human transfusion medicine has improved our understanding of the red blood cell (RBC) storage lesion(s). Despite significant progress towards understanding the storage lesion(s) of human RBCs, a comparison of basal and post-storage RBC metabolism across multiple species using omics technologies has not yet been reported, and is the focus of this study. MATERIALS AND METHODS: Blood was collected in a standard bag system (CPD-SAG-Mannitol) from dogs (n=8), horses, bovines, and donkeys (n=6). All bags were stored at 4°C for up to 42 days (i.e., the end of the shelf life in Italian veterinary clinics) and sampled weekly for metabolomics analyses. In addition, data comparisons to our ongoing Zoomics project are included to compare this study's results with those of non-human primates and humans. RESULTS: Significant interspecies differences in RBC metabolism were observed at baseline, at the time of donation, with bovine showing significantly higher levels of metabolites in the tryptophan/kynurenine pathway; dogs showing elevated levels of high-energy compounds (especially adenosine triphosphate and S-adenosyl-methionine) and equine (donkey and horse) RBCs showing almost overlapping phenotypes, with the highest levels of free branched chain amino acids, glycolytic metabolites (including 2,3-diphosphoglycerate), higher total glutathione pools, and elevated metabolites of the folate pathway compared to the other species. Strikingly, previously described metabolic markers of the storage lesion(s) in humans followed similar trends across all species, though the rate of accumulation/depletion of metabolites in energy and redox metabolism varied by species, with equine blood showing the lowest degree of storage lesion(s). DISCUSSION: These results interrogate RBC metabolism across a range of mammalian species and improve our understanding of both human and veterinary blood storage and transfusion.

Coagulation potential and the integrated omics of extracellular vesicles from COVID-19 positive patient plasma.

Extracellular vesicles (EVs) participate in cell-to-cell communication and contribute toward homeostasis under physiological conditions. But EVs can also contribute toward a wide array of pathophysiology like cancer, sepsis, sickle cell disease, and thrombotic disorders. COVID-19 infected patients are at an increased risk of aberrant coagulation, consistent with elevated circulating levels of ultra-high molecular weight VWF multimers, D-dimer and procoagulant EVs. The role of EVs in COVID-19 related hemostasis may depend on cells of origin, vesicular cargo and size, however this is not well defined. We hypothesized that the procoagulant potential of EV isolates from COVID-19 (+) patient plasmas could be defined by thrombin generation assays. Here we isolated small EVs (SEVs) and large EVs (LEVs) from hospitalized COVID-19 (+) patient (n = 21) and healthy donor (n = 20) plasmas. EVs were characterized by flow cytometry, Transmission electron microscopy, nanoparticle tracking analysis, plasma thrombin generation and a multi-omics approach to define coagulation potential. These data were consistent with differences in EV metabolite, lipid, and protein content when compared to healthy donor plasma isolated SEVs and LEVs. Taken together, the effect of EVs on plasma procoagulant potential as defined by thrombin generation and supported by multi-omics is enhanced in COVID-19. Further, we observe that this effect is driven both by EV size and phosphatidyl serine.

Omics Markers of Red Blood Cell Transfusion in Trauma.

Red blood cell (RBC) transfusion is a life-saving intervention for millions of trauma patients every year worldwide. While hemoglobin thresholds are clinically driving the need for RBC transfusion, limited information is available with respect to transfusion efficacy at the molecular level in clinically relevant cohorts. Here, we combined plasma metabolomic and proteomic measurements in longitudinal samples (n = 118; up to 13 time points; total samples: 690) from trauma patients enrolled in the control of major bleeding after trauma (COMBAT) study. Samples were collected in the emergency department and at continuous intervals up to 168 h (seven days) post-hospitalization. Statistical analyses were performed to determine omics correlate to transfusions of one, two, three, five, or more packed RBC units. While confounded by the concomitant transfusion of other blood components and other iatrogenic interventions (e.g., surgery), here we report that transfusion of one or more packed RBCs­mostly occurring within the first 4 h from hospitalization in this cohort­results in the increase in circulating levels of additive solution components (e.g., mannitol, phosphate) and decreases in the levels of circulating markers of hypoxia, such as lactate, carboxylic acids (e.g., succinate), sphingosine 1-phosphate, polyamines (especially spermidine), and hypoxanthine metabolites with potential roles in thromboinflammatory modulation after trauma. These correlations were the strongest in patients with the highest new injury severity scores (NISS > 25) and lowest base excess (BE < −10), and the effect observed was proportional to the number of units transfused. We thus show that transfusion of packed RBCs transiently increases the circulating levels of plasticizers­likely leaching from the blood units during refrigerated storage in the blood bank. Changes in the levels of arginine metabolites (especially citrulline to ornithine ratios) are indicative of an effect of transfusion on nitric oxide metabolism, which could potentially contribute to endothelial regulation. RBC transfusion was associated with changes in the circulating levels of coagulation factors, fibrinogen chains, and RBC-proteins. Changes in lysophospholipids and acyl-carnitines were observed upon transfusion, suggestive of an effect on the circulating lipidome­though cell-extrinsic/intrinsic effects and/or the contribution of other blood components cannot be disentangled. By showing a significant decrease in circulating markers of hypoxia, this study provides the first multi-omics characterization of RBC transfusion efficacy in a clinically relevant cohort of trauma patients.

Human and Bacterial Toll-Interleukin Receptor Domains Exhibit Distinct Dynamic Features and Functions.

Toll-interleukin receptor (TIR) domains have emerged as critical players involved in innate immune signaling in humans but are also expressed as potential virulence factors within multiple pathogenic bacteria. However, there has been a shortage of structural studies aimed at elucidating atomic resolution details with respect to their interactions, potentially owing to their dynamic nature. Here, we used a combination of biophysical and biochemical studies to reveal the dynamic behavior and functional interactions of a panel of both bacterial TIR-containing proteins and mammalian receptor TIR domains. Regarding dynamics, all three bacterial TIR domains studied here exhibited an inherent exchange that led to severe resonance line-broadening, revealing their intrinsic dynamic nature on the intermediate NMR timescale. In contrast, the three mammalian TIR domains studied here exhibited a range in terms of their dynamic exchange that spans multiple timescales. Functionally, only the bacterial TIR domains were catalytic towards the cleavage of NAD+, despite the conservation of the catalytic nucleophile on human TIR domains. Our development of NMR-based catalytic assays allowed us to further identify differences in product formation for gram-positive versus gram-negative bacterial TIR domains. Differences in oligomeric interactions were also revealed, whereby bacterial TIR domains self-associated solely through their attached coil-coil domains, in contrast to the mammalian TIR domains that formed homodimers and heterodimers through reactive cysteines. Finally, we provide the first atomic-resolution studies of a bacterial coil-coil domain and provide the first atomic model of the TIR domain from a human anti-inflammatory IL-1R8 protein that undergoes a slow inherent exchange.

Multiomic Analysis Reveals Disruption of Cholesterol Homeostasis by Cannabidiol in Human Cell Lines.

The nonpsychoactive cannabinoid, cannabidiol (CBD), is Food and Dug Administration approved for treatment of two drug-resistant epileptic disorders and is seeing increased use among the general public, yet the mechanisms that underlie its therapeutic effects and side-effect profiles remain unclear. Here, we report a systems-level analysis of CBD action in human cell lines using temporal multiomic profiling. FRET-based biosensor screening revealed that CBD elicits a sharp rise in cytosolic calcium, and activation of AMP-activated protein kinase in human keratinocyte and neuroblastoma cell lines. CBD treatment leads to alterations in the abundance of metabolites, mRNA transcripts, and proteins associated with activation of cholesterol biosynthesis, transport, and storage. We found that CBD rapidly incorporates into cellular membranes, alters cholesterol accessibility, and disrupts cholesterol-dependent membrane properties. Sustained treatment with high concentrations of CBD induces apoptosis in a dose-dependent manner. CBD-induced apoptosis is rescued by inhibition of cholesterol synthesis and potentiated by compounds that disrupt cholesterol trafficking and storage. Our data point to a pharmacological interaction of CBD with cholesterol homeostasis pathways, with potential implications in its therapeutic use.

Deciphering Metabolic Adaptability of Leukemic Stem Cells.

Therapeutic targeting of leukemic stem cells is widely studied to control leukemia. An emerging approach gaining popularity is altering metabolism as a potential therapeutic opportunity. Studies have been carried out on hematopoietic and leukemic stem cells to identify vulnerable pathways without impacting the non-transformed, healthy counterparts. While many metabolic studies have been conducted using stem cells, most have been carried out in vitro or on a larger population of progenitor cells due to challenges imposed by the low frequency of stem cells found in vivo. This creates artifacts in the studies carried out, making it difficult to interpret and correlate the findings to stem cells directly. This review discusses the metabolic difference seen between hematopoietic stem cells and leukemic stem cells across different leukemic models. Moreover, we also shed light on the advancements of metabolic techniques and current limitations and areas for additional research of the field to study stem cell metabolism.

Unchecked oxidative stress in skeletal muscle prevents outgrowth of disseminated tumour cells.

Skeletal muscle has long been recognized as an inhospitable site for disseminated tumour cells (DTCs). Yet its antimetastatic nature has eluded a thorough mechanistic examination. Here, we show that DTCs traffic to and persist within skeletal muscle in mice and in humans, which raises the question of how this tissue suppresses colonization. Results from mouse and organotypic culture models along with metabolomic profiling suggested that skeletal muscle imposes a sustained oxidative stress on DTCs that impairs their proliferation. Functional studies demonstrated that disrupting reduction-oxidation homeostasis via chemogenetic induction of reactive oxygen species slowed proliferation in a more fertile organ: the lung. Conversely, enhancement of the antioxidant potential of tumour cells through ectopic expression of catalase in the tumour or host mitochondria allowed robust colonization of skeletal muscle. These findings reveal a profound metabolic bottleneck imposed on DTCs and sustained by skeletal muscle. A thorough understanding of this biology could reveal previously undocumented DTC vulnerabilities that can be exploited to prevent metastasis in other more susceptible tissues.

Inductively-Coupled Plasma Mass Spectrometry-Novel Insights From an Old Technology Into Stressed Red Blood Cell Physiology.

BACKGROUND: Ion and metal homeostasis are critical to red blood cell physiology and Inductively Coupled Plasma (ICP) is a decades old approach to pursue elemental analysis. Recent evolution of ICP has resulted in its coupling to mass spectrometry (MS) instead of atomic absorption/emission. METHODS: Here we performed Inductively-coupled plasma mass spectrometry (ICP-MS) measurements of intra- and extra-cellular Na, K, Ca, Mg, Fe, and Cu in red blood cells undergoing ionic, heat, or starvation stress. Results were correlated with Ca measurements from other common platforms (e.g., fluorescence-based approaches) and extensive measurements of red blood cell metabolism. RESULTS: All stresses induced significant intra- and extracellular alterations of all measured elements. In particular, ionomycin treatment or hypertonic stress significantly impacted intracellular sodium and extracellular potassium and magnesium levels. Iron efflux was observed as a function of temperatures, with ionic and heat stress at 40°C causing the maximum decrease in intracellular iron pools and increases in the supernatants. Strong positive correlation was observed between calcium measurements via ICP-MS and fluorescence-based approaches. Correlation analyses with metabolomics data showed a strong positive association between extracellular calcium and intracellular sodium or magnesium levels and intracellular glycolysis. Extracellular potassium or iron were positively correlated with free fatty acids (especially mono-, poly-, and highly-unsaturated or odd-chain fatty acid products of lipid peroxidation). Intracellular iron was instead positively correlated with saturated fatty acids (palmitate, stearate) and negatively with methionine metabolism (methionine, S-adenosylmethionine), phosphatidylserine exposure and glycolysis. CONCLUSION: In the era of omics approaches, ICP-MS affords a comprehensive characterization of intracellular elements that provide direct insights on red blood cell physiology and represent meaningful covariates for data generated via other omics platforms such as metabolomics.

Irradiation Causes Alterations of Polyamine, Purine, and Sulfur Metabolism in Red Blood Cells and Multiple Organs.

Investigating the metabolic effects of radiation is critical to understand the impact of radiotherapy, space travel, and exposure to environmental radiation. In patients undergoing hemopoietic stem cell transplantation, iron overload is a common risk factor for poor outcomes. However, no studies have interrogated the multiorgan effects of these treatments concurrently. Herein, we use a model that recapitulates transfusional iron overload, a condition often observed in chronically transfused patients. We applied an omics approach to investigate the impact of both the iron load and irradiation on the host metabolome. The results revealed dose-dependent effects of irradiation in the red blood cells, plasma, spleen, and liver energy and redox metabolism. Increases in polyamines and purine salvage metabolites were observed in organs with high oxygen consumption including the heart, kidneys, and brain. Irradiation also impacted the metabolism of the duodenum, colon, and stool, suggesting a potential effect on the microbiome. Iron infusion affected the response to radiation in the organs and blood, especially in erythrocyte polyamines and spleen antioxidant metabolism, and affected glucose, methionine, and glutathione systems and tryptophan metabolism in the liver, stool, and the brain. Together, the results suggest that radiation impacts metabolism on a multiorgan level with a significant interaction of the host iron status.