Search

The lymphatic fluid is the conduit by which part of the tissue "omics" is transported to the draining lymph node for immunosurveillance. Following cannulation of the pre-nodal cervical and mesenteric afferent lymphatics, herein we investigate the lymph proteomic composition, uncovering that its composition varies according to the tissue of origin. Tissue specificity is also reflected in the dendritic cell-major histocompatibility complex class II-eluted immunopeptidome harvested from the cervical and mesenteric nodes. Following inflammatory disruption of the gut barrier, the lymph antigenic and inflammatory loads are analyzed in both mice and subjects with inflammatory bowel diseases. Gastrointestinal tissue damage reflects the lymph inflammatory and damage-associated molecular pattern signatures, microbiome-derived by-products, and immunomodulatory molecules, including metabolites of the gut-brain axis, mapped in the afferent mesenteric lymph. Our data point to the relevance of the lymphatic fluid to probe the tissue-specific antigenic and inflammatory load transported to the draining lymph node for immunosurveillance., Competing Interests: Declaration of interests The authors declare that they do not have any conflict of interest., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Platelet (PLT) product transfusion is a life-saving therapy for actively bleeding patients. There is an urgent need to maintain PLT function and extend shelf life to improve outcomes in these patients. Cold-stored PLT (CS-PLT) maintain hemostatic potential better than room temperature-stored PLT (RT-PLT). However, whether function in long-term CS-PLT is maintained under physiological flow regimes and/or determined by cold-induced metabolic changes is unknown., Objectives: This study aimed to (i) compare the function of RT-PLT and CS-PLT under physiological flow conditions, (ii) determine whether CS-PLT maintain function after 3 weeks of storage, and (iii) identify metabolic pathways associated with the CS-PLT lesion., Methods: We performed phenotypic and functional assessments of RT- and CS-PLT (22 °C and 4 °C storage, respectively; N = 10 unique donors) at storage days 0, 5, and/or 21 via metabolomics, flow cytometry, aggregation, thrombin generation, viscoelastic testing, and a microfluidic assay to measure primary hemostatic function., Results: Day 21 4 °C PLT formed an occlusive thrombus under arterial shear at a similar rate to day 5 22 °C PLT. Day 21 4 °C PLTs had enhanced thrombin generation capacity compared with day 0 PLT and maintained functionality comparable to day RT-PLT across all assays performed. Key metrics from microfluidic assessment, flow cytometry, thrombin generation, and aggregation were associated with 4 °C storage, and metabolites involved in taurine and purine metabolism significantly correlated with these metrics. Taurine supplementation of PLT during storage improved hemostatic function under flow., Conclusion: CS-PLT stored for 3 weeks maintain hemostatic activity, and storage-induced phenotype and function are associated with taurine and purine metabolism., Competing Interests: Declaration of competing interests P.C.S. is a consultant for Cerus Corporation and Haima Therapeutics. All other authors have no competing interests to disclose., (Copyright © 2023 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

Background: Due to their complexity and to the presence of common clinical features, differentiation between asthma and chronic obstructive pulmonary disease (COPD) can be a challenging task, complicated in such cases also by asthma-COPD overlap syndrome. The distinct immune/inflammatory and structural substrates of COPD and asthma are responsible for significant differences in the responses to standard pharmacologic treatments. Therefore, an accurate diagnosis is of central relevance to assure the appropriate therapeutic intervention in order to achieve safe and effective patient care. Induced sputum (IS) accurately mirrors inflammation in the airways, providing a more direct picture of lung cell metabolism in comparison to those specimen that reflect analytes in the systemic circulation., Methods: An integrated untargeted metabolomics and lipidomics analysis was performed in IS of asthmatic (n = 15) and COPD (n = 22) patients based on Ultra-High-Pressure Liquid Chromatography-Mass Spectrometry (UHPLC-MS) and UHPLC-tandem MS (UHPLC-MS/MS). Partial Least Squares-Discriminant Analysis (PLS-DA) was applied to resulting dataset. The analysis of main enriched metabolic pathways and the association of the preliminary metabolites/lipids pattern identified to clinical parameters of asthma/COPD differentiation were explored. Multivariate ROC analysis was performed in order to determine the discriminatory power and the reliability of the putative biomarkers for diagnosis between COPD and asthma., Results: PLS-DA indicated a clear separation between COPD and asthmatic patients. Among the 15 selected candidate biomarkers based on Variable Importance in Projection scores, putrescine showed the highest score. A differential IS bio-signature of 22 metabolites and lipids was found, which showed statistically significant variations between asthma and COPD. Of these 22 compounds, 18 were decreased and 4 increased in COPD compared to asthmatic patients. The IS levels of Phosphatidylethanolamine (PE) (34:1), Phosphatidylglycerol (PG) (18:1;18:2) and spermine were significantly higher in asthmatic subjects compared to COPD., Conclusions: This is the first pilot study to analyse the IS metabolomics/lipidomics signatures relevant in discriminating asthma vs COPD. The role of polyamines, of 6-Hydroxykynurenic acid and of D-rhamnose as well as of other important players related to the alteration of glycerophospholipid, aminoacid/biotin and energy metabolism provided the construction of a diagnostic model that, if validated on a larger prospective cohort, might be used to rapidly and accurately discriminate asthma from COPD., (© 2024. The Author(s).)

Background: The coagulopathy of traumatic brain injury (TBI) remains poorly understood. Contradictory descriptions highlight the distinction between systemic and local coagulation, with descriptions of systemic hypercoagulability despite intracranial hypocoagulopathy. This perplexing coagulation profile has been hypothesized to be due to tissue factor release. The objective of this study was to assess the coagulation profile of TBI patients undergoing neurosurgical procedures. We hypothesize that dura violation is associated with higher tissue factor and conversion to a hypercoagulable profile and unique metabolomic and proteomic phenotype., Methods: This is a prospective, observational cohort study of all adult TBI patients at an urban, Level I trauma center who underwent a neurosurgical procedure from 2019 to 2021. Whole blood samples were collected before and then 1 hour following dura violation. Citrated rapid and tissue plasminogen activator (tPA) thrombelastography (TEG) were performed, in addition to measurement of tissue factory activity, metabolomics, and proteomics., Results: Overall, 57 patients were included. The majority (61%) were male, the median age was 52 years, 70% presented after blunt trauma, and the median Glasgow Coma Score was 7. Compared with pre-dura violation, post-dura violation blood demonstrated systemic hypercoagulability, with a significant increase in clot strength (maximum amplitude of 74.4 mm vs. 63.5 mm; p < 0.0001) and a significant decrease in fibrinolysis (LY30 on tPAchallenged TEG of 1.4% vs. 2.6%; p = 0.04). There were no statistically significant differences in tissue factor. Metabolomics revealed notable increases in metabolites involved in late glycolysis, cysteine, and one-carbon metabolites, and metabolites involved in endothelial dysfunction/arginine metabolism/responses to hypoxia. Proteomics revealed notable increase in proteins related to platelet activation and fibrinolysis inhibition., Conclusion: A systemic hypercoagulability is observed in TBI patients, characterized by increased clot strength and decreased fibrinolysis and a unique metabolomic and proteomics phenotype independent of tissue factor levels., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Metabolomics studies in sickle cell disease (SCD) have been so far limited to tens of samples, owing to technical and experimental limitations. To overcome these limitations, we performed plasma metabolomics analyses on 596 samples from patients with SCD enrolled in the WALK-PHaSST study (clinicaltrials gov. Identifier: NCT00492531). Clinical covariates informed the biological interpretation of metabolomics data, including genotypes (hemoglobin [Hb] SS, hemoglobin SC), history of recent transfusion (HbA%), response to hydroxyurea treatment (fetal Hb%). We investigated metabolic correlates to the degree of intravascular hemolysis, cardiorenal function, as determined by tricuspid regurgitation velocity (TRV), estimated glomerular filtration rate (eGFR), and overall hazard ratio (unadjusted or adjusted by age). Recent transfusion events or hydroxyurea treatment were associated with elevation in plasma-free fatty acids and decreases in acyl-carnitines, urate, kynurenine, indoles, carboxylic acids, and glycine- or taurine-conjugated bile acids. High levels of these metabolites, along with low levels of plasma S1P and L-arginine were identified as top markers of hemolysis, cardiorenal function (TRV, eGFR), and overall hazard ratio. We thus uploaded all omics and clinical data on a novel online portal that we used to identify a potential mechanism of dysregulated red cell S1P synthesis and export as a contributor to the more severe clinical manifestations in patients with the SS genotype compared to SC. In conclusion, plasma metabolic signatures - including low S1P, arginine and elevated kynurenine, acyl-carnitines and bile acids - are associated with clinical manifestation and therapeutic efficacy in SCD patients, suggesting new avenues for metabolic interventions in this patient population.

Male infertility occurs approximately in about 50% of all infertility cases and represents a serious concern worldwide. Traditional semen analysis alone is insufficient to diagnose male infertility. Over the past two decades, advances in omics technologies have led to the widespread application of metabolomics profiling as a valuable diagnostic tool for various diseases and disorders. Seminal plasma represents a rich and easily accessible source of metabolites surrounding spermatozoa, a milieu that provides several indispensable nutrients to sustain sperm motility and fertilization. Changes of metabolic profiles in seminal plasma reflect male reproductive tract disorders. Here, we performed seminal plasma metabolomics and lipidomics profiling to identify a new pattern of biomarkers of male infertility. Seminal plasma samples from unfertile subjects ( n = 31) and fertile controls ( n = 19) were analyzed using an untargeted metabolomics/lipidomics integrated approach, based on Ultra-High-Pressure Liquid Chromatography-tandem Mass Spectrometry. Partial Least Squares-Discriminant Analysis showed a distinct separation between healthy fertile men and infertile subjects. Among the 15 selected candidate biomarkers based on Variable Importance in Projection scores, phosphatidylethanolamine (PE) (18:1; 18:1) resulted with the highest score. In total, 40 molecular species showed statistically significant variations between fertile and infertile men. Heat-map and volcano plot analysis indicated that acylcarnitines, phosphatidylserine (PS) (40:2) and lactate were decreased, while PE (18:1; 18:1), Phosphatidic acid (PA) (O-19:2; 18:1), Lysophosphatidylethanolamine (LPE) (O-16:1) and Phosphatidylcholine (PC) (O-16:2; 18:1)-CH3 were increased in the infertile group. The present study is the first one to analyze the metabolomics/lipidomics dysregulation in seminal plasma between fertile and infertile individuals regardless of sub-infertility condition. Association of several metabolites/lipids dysregulation with male infertility reinforced data of previous studies performed with different approaches. In particular, we confirmed significantly decreased levels of PS and carnitines in infertile patients as well as the positive correlation with sperm motility and morphology. If validated on a larger prospective cohort, the metabolite biomarkers of infertility in seminal plasma we identified in the present study might inform novel strategies for diagnosis and interventions to overcome male infertility., Competing Interests: Author AF was employed by Urogyn Biotech S. R. L. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Correnti, Preianò, Fregola, Gamboni, Stephenson, Savino, D’Alessandro and Terracciano.)

Sickle cell disease and β-thalassemia represent hemoglobinopathies arising from dysfunctional or underproduced β-globin chains, respectively. In both diseases, red blood cell injury and anemia are the impetus for end organ injury. Because persistent erythrophagocytosis is a hallmark of these genetic maladies, it is critical to understand how macrophage phenotype polarizations in tissue compartments can inform on disease progression. Murine models of sickle cell disease and β-thalassemia allow for a basic understanding of the mechanisms and provide for translation to human disease. A multi-omics approach to understanding the macrophage metabolism and protein changes in two murine models of β-globinopathy was performed on peripheral blood mononuclear cells as well as spleen and liver macrophages isolated from Berkley sickle cell disease (Berk-ss) and heterozygous B1/B2 globin gene deletion (Hbb th3/+ ) mice. The results from these experiments revealed that the metabolome and proteome of macrophages are polarized to a distinct phenotype in Berk-ss and Hbb th3/+ compared with each other and their common-background mice (C57BL6/J). Further, spleen and liver macrophages revealed distinct disease-specific phenotypes, suggesting that macrophages become differentially polarized and reprogrammed within tissue compartments. We conclude that tissue recruitment, polarization, and metabolic and proteomic reprogramming of macrophages in Berk-ss and Hbb th3/+ mice may be relevant to disease progression in other tissue.

Outcomes for patients with acute myeloid leukemia (AML) remain poor due to the inability of current therapeutic regimens to fully eradicate disease-initiating leukemia stem cells (LSC). Previous studies have demonstrated that oxidative phosphorylation (OXPHOS) is an essential process that is targetable in LSC. Sirtuin 3 (SIRT3), a mitochondrial deacetylase with a multi-faceted role in metabolic regulation, has been shown to regulate OXPHOS in cancer models; however, it has not yet been studied in the context of LSC. Thus, we sought to identify if SIRT3 is important for LSC function. Using RNAi and a SIRT3 inhibitor (YC8-02), we demonstrate that SIRT3 is a critical target for the survival of primary human LSC but is not essential for normal human hematopoietic stem and progenitor cell function. In order to elucidate the molecular mechanisms by which SIRT3 is essential in LSC we combined transcriptomic, proteomic, and lipidomic approaches, showing that SIRT3 is important for LSC function through the regulation of fatty acid oxidation (FAO) which is required to support OXPHOS and ATP production in human LSC. Further, we discovered two approaches to further sensitize LSC to SIRT3 inhibition. First, we found that LSC tolerate the toxic effects of fatty acid accumulation induced by SIRT3 inhibition by upregulating cholesterol esterification. Disruption of cholesterol homeostasis sensitizes LSC to YC8-02 and potentiates LSC death. Second, SIRT3 inhibition sensitizes LSC to the BCL-2 inhibitor venetoclax. Together, these findings establish SIRT3 as a regulator of lipid metabolism and potential therapeutic target in primitive AML cells.

Lesch-Nyhan syndrome (LN) is an is an X-linked recessive inborn error of metabolism that arises from a deficiency of purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT). The disease manifests severely, causing intellectual deficits and other neural abnormalities, hypercoagulability, uncontrolled self-injury, and gout. While allopurinol is used to alleviate gout, other symptoms are less understood, impeding treatment. Herein, we present a high-throughput multi-omics analysis of red blood cells (RBCs) from three pediatric siblings carrying a novel S162N HPRT1 mutation. RBCs from both parents-the mother, a heterozygous carrier, and the father, a clinically healthy control-were also analyzed. Global metabolite analysis of LN RBCs shows accumulation of glycolytic intermediates upstream of pyruvate kinase, unsaturated fatty acids, and long chain acylcarnitines. Similarly, highly unsaturated phosphatidylcholines are also elevated in LN RBCs, while free choline is decreased. Intracellular iron, zinc, selenium, and potassium are also decreased in LN RBCs. Global proteomics documented changes in RBC membrane proteins, hemoglobin, redox homeostasis proteins, and the enrichment of coagulation proteins. These changes were accompanied by elevation in protein glutamine deamidation and methylation in the LN children and carrier mother. Treatment with allopurinol incompletely reversed the observed phenotypes in the two older siblings currently on this treatment. This unique data set provides novel opportunities for investigations aimed at potential therapies for LN-associated sequelae.

Despite a wealth of exploratory plasma metabolomics studies in sickle cell disease (SCD), no study to date has evaluate a large and well phenotyped cohort to compare the primary erythrocyte metabolome of hemoglobin SS, SC and transfused AA red blood cells (RBCs) in vivo. The current study evaluates the RBC metabolome of 587 subjects with sickle cell sickle cell disease (SCD) from the WALK-PHaSST clinical cohort. The set includes hemoglobin SS, hemoglobin SC SCD patients, with variable levels of HbA related to RBC transfusion events. Here we explore the modulating effects of genotype, age, sex, severity of hemolysis, and transfusion therapy on sickle RBC metabolism. Results show that RBCs from patients with Hb SS genotypes-compared to AA RBCs from recent transfusion events or SC RBCs-are characterized by significant alterations of RBC acylcarnitines, pyruvate, sphingosine 1-phosphate, creatinine, kynurenine and urate metabolism. Surprisingly, the RBC metabolism of SC RBCs is dramatically different from SS, with all glycolytic intermediates significantly elevated in SS RBCs, with the exception of pyruvate. This result suggests a metabolic blockade at the ATP-generating phosphoenolpyruvate to pyruvate step of glycolysis, which is catalyzed by redox-sensitive pyruvate kinase. Metabolomics, clinical and hematological data were collated in a novel online portal. In conclusion, we identified metabolic signatures of HbS RBCs that correlate with the degree of steady state hemolytic anemia, cardiovascular and renal dysfunction and mortality., (© 2023 Wiley Periodicals LLC.)

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., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Background: The Red blood cell (RBC) storage lesion results in decreased circulation and function of transfused RBCs. Elevated oxidant stress and impaired energy metabolism are a hallmark of the storage lesion in both human and murine RBCs. Although human studies don't suffer concerns that findings may not translate, they do suffer from genetic and environmental variability amongst subjects. Murine models can control for genetics, environment, and much interventional experimentation can be carried out in mice that is neither technically feasible nor ethical in humans. However, murine models are only useful to the extent that they have similar biology to humans. Hypoxic storage has been shown to mitigate the storage lesion in human RBCs, but has not been investigated in mice., Materials and Methods: RBCs from a C57BL6/J mouse strain were stored under normoxic (untreated) or hypoxic conditions (SO2 ~ 26%) for 1h, 7 and 12 days. Samples were tested for metabolomics at steady state, tracing experiments with 1,2,3- 13 C 3 -glucose, proteomics and end of storage post transfusion recovery., Results: Hypoxic storage improved post-transfusion recovery and energy metabolism, including increased steady state and 13 C 3 -labeled metabolites from glycolysis, high energy purines (adenosine triphosphate) and 2,3-diphospholgycerate. Hypoxic storage promoted glutaminolysis, increased glutathione pools, and was accompanied by elevation in the levels of free fatty acids and acyl-carnitines., Discussion: This study isolates hypoxia, as a single independent variable, and shows similar effects as seen in human studies. These findings also demonstrate the translatability of murine models for hypoxic RBC storage and provide a pre-clinical platform for ongoing study.

Coronavirus disease 2019 (COVID-19) is caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and can affect multiple organs, among which is the circulatory system. Inflammation and mortality risk markers were previously detected in COVID-19 plasma and red blood cells (RBCs) metabolic and proteomic profiles. Additionally, biophysical properties, such as deformability, were found to be changed during the infection. Based on such data, we aim to better characterize RBC functions in COVID-19. We evaluate the flow properties of RBCs in severe COVID-19 patients admitted to the intensive care unit by using microfluidic techniques and automated methods, including artificial neural networks, for an unbiased RBC analysis. We find strong flow and RBC shape impairment in COVID-19 samples and demonstrate that such changes are reversible upon suspension of COVID-19 RBCs in healthy plasma. Vice versa, healthy RBCs resemble COVID-19 RBCs when suspended in COVID-19 plasma. Proteomics and metabolomics analyses allow us to detect the effect of plasma exchanges on both plasma and RBCs and demonstrate a new role of RBCs in maintaining plasma equilibria at the expense of their flow properties. Our findings provide a framework for further investigations of clinical relevance for therapies against COVID-19 and possibly other infectious diseases., Competing Interests: SR, GS, LK, SQ Though unrelated to the contents of this manuscript, the authors declare that SMR, GS, LK, and SQ are co-founders of Cysmic GmbH, ML Affiliated with Cysmic GmbH; the author has no financial interests to declare, FG, MD, PM, KZ, Av, CW No competing interests declared, AD Though unrelated to the contents of this manuscript, AD is a founder of Omix Technologies Inc and Altis Biosciences LLC, and a scientific advisory board member for Hemanext Inc and Forma Therapeutics, (© 2022, Recktenwald, Simionato et al.)

Background: Platelets are part of innate immunity and comprise the cellular portion of hemostasis. Platelets express sex hormone receptors on their plasma membrane and sex hormones can alter their function in vitro. Little is known about how age and sex may affect platelet biology; thus, we hypothesized that platelets from males and females have different metabolomic profiles, which may be altered by age and in vitro treatment with sex hormones., Methods: Day 1 apheresis platelets were drawn from five 18-53-year-old, premenopausal younger females (YF), five ≥54-year-old, postmenopausal, older females (OF), five 18-44-year-old younger males (YM), and four ≥45-year-old older males (OM). Platelets were normalized to a standard concentration and metabolomics analyses were completed. Unsupervised statistical analyses and hierarchical clustering with principal component analyses were completed., Results: Platelets from OM had (1) elevated mono-, di- and tri-carboxylates, (2) increased levels of free fatty acids, acyl-carnitines, and free amino acids, and (3) increased purine breakdown and deamination products. In vitro incubation with sex hormones only affected platelets from OM donors with trends towards increased ATP and other high-energy purines and decreases in L-proline and other amino acids., Conclusion: Platelets from OM's versus YF, OF, and YM have a different metabolome implying increased energy metabolism, more free fatty acids, acylcarnitines, and amino acids, and increased breakdown of purines and deamination products. However, only platelets from OM were affected by sex hormones in vitro. Platelets from OM are metabolically distinct, which may impart functional differences when transfused., (© 2022 AABB.)

Objectives: Identify the metabolites that are increased in the plasma of severely injured patients that developed ARDS versus severely injured patients that did not, and assay if these increased metabolites prime pulmonary sequestration of neutrophils (PMNs) and induce pulmonary sequestration in an animal model of ARDS. We hypothesize that metabolic derangement due to advanced shock in critically injured patients leads to the PMNs, which serves as the first event in the ARDS. Summary of Background Data: Intracellular metabolites accumulate in the plasma of severely injured patients., Methods: Untargeted metabolomics profiling of 67 critically injured patients was completed to establish a metabolic signature associated with ARDS development. Metabolites that significantly increased were assayed for PMN priming activity in vitro. The metabolites that primed PMNs were tested in a 2-event animal model of ARDS to identify a molecular link between circulating metabolites and clinical risk for ARDS., Results: After controlling for confounders, 4 metabolites significantly increased: creatine, dehydroascorbate, fumarate, and succinate in trauma patients who developed ARDS ( P < 0.05). Succinate alone primed the PMN oxidase in vitro at physiologically relevant levels. Intravenous succinate-induced PMN sequestration in the lung, a first event, and followed by intravenous lipopolysaccharide, a second event, resulted in ARDS in vivo requiring PMNs. SUCNR1 inhibition abrogated PMN priming, PMN sequestration, and ARDS. Conclusion: Significant increases in plasma succinate post-injury may serve as the first event in ARDS. Targeted inhibition of the SUCNR1 may decrease ARDS development from other disease states to prevent ARDS globally., Competing Interests: The authors declare no conflict of interest., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

Biological therapeutics represent an increasing and critical component of newly approved drugs; however, the inability to deliver biologics intracellularly in a controlled manner remains a major limitation. We have developed a semi-synthetic, tunable phage-like particle (PLP) platform derived from bacteriophage λ. The shell surface can be decorated with small-molecule, biological and synthetic moieties, alone or in combination and in defined ratios. Here, we demonstrate that the platform can be used to deliver biological macromolecules intracellularly and in a controlled manner. Ubiquitin-specific protease 7 (USP7) is a deubiquitinating enzyme that has been widely recognized as an ideal target for the treatment of a variety of cancers. Recently, UbV.7.2, a novel biologic derived from the ubiquitin scaffold, was developed for inhibition of USP7, but issues remain in achieving efficient and controlled intracellular delivery of the biologic. We have shown that decoration of PLPs with trastuzumab (Trz), a HER2-targeted therapeutic used in the treatment of various cancers, results in specific targeting and uptake of Trz-PLPs into HER2-overexpressing breast cancer cells. By simultaneously decorating PLPs with Trz and UbV.7.2, we now show that these particles are also internalized by HER2-positive cells, thus providing a means for intracellular delivery of the biologic in a controlled fashion. Internalized particles retain USP7 inhibition activity of UbV.7.2 and alter the metabolic and proteomic landscapes of these cells. This study demonstrates that the λ "designer nanoparticles" represent a powerful system for the intracellular delivery of biologics in a defined dose.

The impacts of interferon (IFN) signaling on COVID-19 pathology are multiple, with both protective and harmful effects being documented. We report here a multiomics investigation of systemic IFN signaling in hospitalized COVID-19 patients, defining the multiomics biosignatures associated with varying levels of 12 different type I, II, and III IFNs. The antiviral transcriptional response in circulating immune cells is strongly associated with a specific subset of IFNs, most prominently IFNA2 and IFNG. In contrast, proteomics signatures indicative of endothelial damage and platelet activation associate with high levels of IFNB1 and IFNA6. Seroconversion and time since hospitalization associate with a significant decrease in a specific subset of IFNs. Additionally, differential IFN subtype production is linked to distinct constellations of circulating myeloid and lymphoid immune cell types. Each IFN has a unique metabolic signature, with IFNG being the most associated with activation of the kynurenine pathway. IFNs also show differential relationships with clinical markers of poor prognosis and disease severity. For example, whereas IFNG has the strongest association with C-reactive protein and other immune markers of poor prognosis, IFNB1 associates with increased neutrophil to lymphocyte ratio, a marker of late severe disease. Altogether, these results reveal specialized IFN action in COVID-19, with potential diagnostic and therapeutic implications., Competing Interests: Competing interest statement: J.M.E. serves on the COVID Development Advisory Board for Eli Lilly., (Copyright © 2022 the Author(s). Published by PNAS.)

Acute myeloid leukemia (AML) is characterized by the presence of leukemia stem cells (LSCs), and failure to fully eradicate this population contributes to disease persistence/relapse. Prior studies have characterized metabolic vulnerabilities of LSCs, which demonstrate preferential reliance on oxidative phosphorylation (OXPHOS) for energy metabolism and survival. In the present study, using both genetic and pharmacologic strategies in primary human AML specimens, we show that signal transducer and activator of transcription 3 (STAT3) mediates OXPHOS in LSCs. STAT3 regulates AML-specific expression of MYC, which in turn controls transcription of the neutral amino acid transporter gene SLC1A5. We show that genetic inhibition of MYC or SLC1A5 acts to phenocopy the impairment of OXPHOS observed with STAT3 inhibition, thereby establishing this axis as a regulatory mechanism linking STAT3 to energy metabolism. Inhibition of SLC1A5 reduces intracellular levels of glutamine, glutathione, and multiple tricarboxylic acid (TCA) cycle metabolites, leading to reduced TCA cycle activity and inhibition of OXPHOS. Based on these findings, we used a novel small molecule STAT3 inhibitor, which binds STAT3 and disrupts STAT3-DNA, to evaluate the biological role of STAT3. We show that STAT3 inhibition selectively leads to cell death in AML stem and progenitor cells derived from newly diagnosed patients and patients who have experienced relapse while sparing normal hematopoietic cells. Together, these findings establish a STAT3-mediated mechanism that controls energy metabolism and survival in primitive AML cells., (© 2022 by The American Society of Hematology.)

Aging and obesity independently contribute toward an endothelial dysfunction that results in an imbalanced VWF to ADAMTS13 ratio. In addition, plasma thrombin and plasmin generation are elevated and reduced, respectively, with increasing age and also with increasing body mass index (BMI). The severity risk of Corona Virus Disease 2019 (COVID-19) increases in adults older than 65 and in individuals with certain pre-existing health conditions, including obesity (>30 kg/m 2 ). The present cross-sectional study focused on an analysis of the VWF/ADAMTS13 axis, including measurements of von Willebrand factor (VWF) antigen (VWF:AG), VWF collagen binding activity (VWF:CBA), Factor VIII antigen, ADAMTS13 antigen, and ADAMTS13 activity, in addition to thrombin and plasmin generation potential, in a demographically diverse population of COVID-19 negative (-) ( n = 288) and COVID-19 positive (+) ( n = 543) patient plasmas collected at the time of hospital presentation. Data were analyzed as a whole, and then after dividing patients by age (<65 and ≥65) and independently by BMI [<18.5, 18.5-24.9, 25-29.9, >30 (kg/m 2 )]. These analyses suggest that VWF parameters (i.e., the VWF/ADAMTS13 activity ratio) and thrombin and plasmin generation differed in COVID-19 (+), as compared to COVID-19 (-) patient plasma. Further, age (≥65) more than BMI contributed to aberrant plasma indicators of endothelial coagulopathy. Based on these findings, evaluating both the VWF/ADAMTS13 axis, along with thrombin and plasmin generation, could provide insight into the extent of endothelial dysfunction as well as the plasmatic imbalance in coagulation and fibrinolysis potential, particularly for at-risk patient populations., Competing Interests: IA is a consultant for Pharmacosmos Therapeutics. DR receives consulting fees from Portola Pharmaceuticals. AD'A and TN are founders of Omix Technologies Inc and Altis Biosciences LLC. AD'A and SLS are consultants for Hemanext Inc. SLS is also a consultant for Tioma, Inc. and TCIP, Inc., and the Executive Director of the Worldwide Initiative for Rh Disease Eradication WIRhE. AD'A is a consultant for FORMA LLC. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Thangaraju, Katneni, Akpan, Tanaka, Thomas, Setua, Reisz, Cendali, Gamboni, Nemkov, Kahn, Wei, Valk, Hudson, Roh, Moriconi, Zimring, D'Alessandro, Spitalnik, Francis and Buehler.)

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.

Background: Pyruvate kinase deficiency (PKD) is the most frequent congenital enzymatic defect of glycolysis, and one of the most common causes of hereditary non spherocytic hemolytic anemia. Therapeutic interventions are limited, in part because of the incomplete understanding of the molecular mechanisms that compensate for the metabolic defect. Methods: Mass spectrometry-based metabolomics analyses were performed on red blood cells (RBCs) from healthy controls ( n =10) and PKD patients ( n =5). Results: In PKD patients, decreases in late glycolysis were accompanied by accumulation of pentose phosphate pathway (PPP) metabolites, as a function of oxidant stress to purines (increased breakdown and deamination). Markers of oxidant stress included increased levels of sulfur-containing compounds (methionine and taurine), polyamines (spermidine and spermine). Markers of hypoxia such as succinate, sphingosine 1-phosphate (S1P), and hypoxanthine were all elevated in PKD subjects. Membrane lipid oxidation and remodeling was observed in RBCs from PKD patients, as determined by increases in the levels of free (poly-/highly-unsaturated) fatty acids and acyl-carnitines. Conclusion: In conclusion, in the present study, we provide the first overview of RBC metabolism in patients with PKD. Though limited in scope, the study addresses the need for basic science to investigate pathologies targeting underrepresented minorities (Amish population in this study), with the ultimate goal to target treatments to health disparities., Competing Interests: AD’A is a founder of Omix Technologies Inc. and Altis Biosciences LLC. AD’A is a consultant for Rubius Therapeutics and an advisory board member for Hemanext Inc. and FORMA Therapeutics Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Roy, Cendali, Ooyama, Gamboni, Morton and D’Alessandro.)

The Corona Virus Disease 2019 (COVID-19) pandemic represents an ongoing worldwide challenge. The present large study sought to understand independent and overlapping metabolic features of samples from acutely ill patients (n = 831) that tested positive (n = 543) or negative (n = 288) for COVID-19. High-throughput metabolomics analyses were complemented with antigen and enzymatic activity assays on plasma from acutely ill patients collected while in the emergency department, at admission, or during hospitalization. Lipidomics analyses were also performed on COVID-19-positive or -negative subjects with the lowest and highest body mass index (n = 60/group). Significant changes in amino acid and fatty acid/acylcarnitine metabolism emerged as highly relevant markers of disease severity, progression, and prognosis as a function of biological and clinical variables in these patients. Further, machine learning models were trained by entering all metabolomics and clinical data from half of the COVID-19 patient cohort and then tested on the other half, yielding ~78% prediction accuracy. Finally, the extensive amount of information accumulated in this large, prospective, observational study provides a foundation for mechanistic follow-up studies and data sharing opportunities, which will advance our understanding of the characteristics of the plasma metabolism in COVID-19 and other acute critical illnesses.

COVID-19 pathology involves dysregulation of diverse molecular, cellular, and physiological processes. To expedite integrated and collaborative COVID-19 research, we completed multi-omics analysis of hospitalized COVID-19 patients, including matched analysis of the whole-blood transcriptome, plasma proteomics with two complementary platforms, cytokine profiling, plasma and red blood cell metabolomics, deep immune cell phenotyping by mass cytometry, and clinical data annotation. We refer to this multidimensional dataset as the COVIDome. We then created the COVIDome Explorer, an online researcher portal where the data can be analyzed and visualized in real time. We illustrate herein the use of the COVIDome dataset through a multi-omics analysis of biosignatures associated with C-reactive protein (CRP), an established marker of poor prognosis in COVID-19, revealing associations between CRP levels and damage-associated molecular patterns, depletion of protective serpins, and mitochondrial metabolism dysregulation. We expect that the COVIDome Explorer will rapidly accelerate data sharing, hypothesis testing, and discoveries worldwide., Competing Interests: Declaration of interests J.M.E. serves in the COVID Development Advisory Board for Elly Lilly and has provided consulting services to Gilead Sciences Inc. J.M.E. also serves on the Cell Reports Advisory Board. The remaining authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Increases in the red blood cell (RBC) degree of fatty acid desaturation are reported in response to exercise, aging, or diseases associated with systemic oxidant stress. However, no studies have focused on the presence and activity of fatty acid desaturases (FADS) in the mature RBC., Study Design and Methods: Steady state metabolomics and isotope-labeled tracing experiments, immunofluorescence approaches, and pharmacological interventions were used to determine the degree of fatty acid unsaturation, FADS activity as a function of storage, oxidant stress, and G6PD deficiency in human and mouse RBCs., Results: In 250 blood units from the REDS III RBC Omics recalled donor population, we report a storage-dependent accumulation of free mono-, poly-(PUFAs), and highly unsaturated fatty acids (HUFAs), which occur at a faster rate than saturated fatty acid accumulation. Through a combination of immunofluorescence, pharmacological inhibition, tracing experiments with stable isotope-labeled fatty acids, and oxidant challenge with hydrogen peroxide, we demonstrate the presence and redox-sensitive activity of FADS2, FADS1, and FADS5 in the mature RBC. Increases in PUFAs and HUFAs in human and mouse RBCs correlate negatively with storage hemolysis and positively with posttransfusion recovery. Inhibition of these enzymes decreases accumulation of free PUFAs and HUFAs in stored RBCs, concomitant to increases in pyruvate/lactate ratios. Alterations of this ratio in G6PD deficient patients or units supplemented with pyruvate-rich rejuvenation solutions corresponded to decreased PUFA and HUFA accumulation., Conclusion: Fatty acid desaturases are present and active in mature RBCs. Their activity is sensitive to oxidant stress, storage duration, and alterations of the pyruvate/lactate ratio., (© 2021 AABB.)

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.)

We previously demonstrated that leukemia stem cells (LSCs) in de novo acute myeloid leukemia (AML) patients are selectively reliant on amino acid metabolism and that treatment with the combination of venetoclax and azacitidine (ven/aza) inhibits amino acid metabolism, leading to cell death. In contrast, ven/aza fails to eradicate LSCs in relapsed/refractory (R/R) patients, suggesting altered metabolic properties. Detailed metabolomic analysis revealed elevated nicotinamide metabolism in relapsed LSCs, which activates both amino acid metabolism and fatty acid oxidation to drive OXPHOS, thereby providing a means for LSCs to circumvent the cytotoxic effects of ven/aza therapy. Genetic and pharmacological inhibition of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in nicotinamide metabolism, demonstrated selective eradication of R/R LSCs while sparing normal hematopoietic stem/progenitor cells. Altogether, these findings demonstrate that elevated nicotinamide metabolism is both the mechanistic basis for ven/aza resistance and a metabolic vulnerability of R/R LSCs., Competing Interests: Declaration of Interests D.A.P. receives research funding from Abbvie and has served as a consultant for Abbvie. C.L.J. is currently employed at the Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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.)

Background: Red blood cell exchange (RCE) transfusions are a mainstay in the treatment of sickle cell anemia (SCA), and allow a temporary restoration of physiological parameters with respect to erythrocyte oxygen carrying capacity and systems metabolism. Recently, we noted that 1) RCE significantly impacts recipients' metabolism in SCA; 2) fresh and end-of-storage red blood cell (RBC) units differently impact systems of metabolism in healthy autologous recipients; and 3) phosphate/inosine/pyruvate/adenine (PIPA) solution reverses the metabolic age of stored RBCs. Therefore, we hypothesized that RCE with PIPA-treated RBC units could further increase the metabolic benefits of RCE in SCA patients., Study Design and Methods: Circulating plasma and erythrocytes were collected from patients with SCA before and after RCE, with either conventional or PIPA-treated RBC units, prior to metabolomics analyses., Results: Consistent with prior work, RCE significantly decreased circulating levels of markers of systemic hypoxemia (lactate, succinate) and decreased plasma levels of acyl-carnitines and amino acids. However, PIPA-treated exchanges were superior to untreated RCEs, with a higher energy state and an increased capacity to activate the pentose phosphate pathway and glutamine metabolism. In addition, RBCs and plasma from recipients of PIPA-treated RBC units resulted in significantly decreased levels of post-transfusion plasticizers, though at the expense of higher circulating levels of oxidized purines (hypoxanthine, xanthine, and the antioxidant urate)., Conclusion: Transfusion of PIPA-treated RBCs further increases the metabolic benefits of RCE to patients with SCA, significantly reducing the levels of post-transfusion plasticizers., (© 2019 AABB.)