Proteostasis and metabolic dysfunction in a distinct subset of storage-induced senescent erythrocytes targeted for clearance.

Although refrigerated storage slows the metabolism of volunteer donor RBCs, cellular aging still occurs throughout this in vitro process, which is essential in transfusion medicine. Storage-induced microerythrocytes (SMEs) are morphologically-altered senescent RBCs that accumulate during storage and which are cleared from circulation following transfusion. However, the molecular and cellular alterations that trigger clearance of this RBC subset remain to be identified. Using a staining protocol that sorts long-stored SMEs (i.e., CFSE high ) and morphologically-normal RBCs (CFSE low ), these in vitro aged cells were characterized. Metabolomics analysis identified depletion of energy, lipid-repair, and antioxidant metabolites in CFSE high RBCs. By redox proteomics, irreversible protein oxidation primarily affected CFSE high RBCs. By proteomics, 96 proteins, mostly in the proteostasis family, had relocated to CFSE high RBC membranes. CFSE high RBCs exhibited decreased proteasome activity and deformability; increased phosphatidylserine exposure, osmotic fragility, and endothelial cell adherence; and were cleared from the circulation during human spleen ex vivo perfusion. Conversely, molecular, cellular, and circulatory properties of long-stored CFSE low RBCs resembled those of short-stored RBCs. CFSE high RBCs are morphologically and metabolically altered, have irreversibly oxidized and membrane-relocated proteins, and exhibit decreased proteasome activity. In vitro aging during storage selectively alters metabolism and proteostasis in SMEs, targeting these senescent cells for clearance.

Blocking Tryptophan Catabolism Reduces Triple-Negative Breast Cancer Invasive Capacity.

SIGNIFICANCE: TDO2 is more highly expressed than the nonhomologous TRP-catabolizing enzyme IDO1 in TNBC. We find that TDO2 knockdown can lead to a compensatory increase in IDO1. Therefore, we tested a newly developed TDO2/IDO1 dual inhibitor and found that it decreases TRP catabolism, anchorage-independent survival, and invasive capacity.

Bone Density in Transgender Youth on Gender-Affirming Hormone Therapy.

Some transgender youth are treated with gonadotropin-releasing hormone agonists (GnRHa) followed by testosterone or estradiol, which may impact bone mineral density (BMD). This cross-sectional study of transgender youth (n = 56, aged 10.4-19.8 years, 53% assigned female at birth [AFAB]) utilized total body dual-energy x-ray absorptiometry to evaluate BMD Z-scores, and associations between GnRHa duration, body mass index (BMI), and BMD. Participants on GnRHa alone (n = 19, 14 assigned male at birth [AMAB], 5 AFAB) at the time of the study visit were 13.8 [12.8, 15.3] (median [IQR]) years old, had been on GnRHa for 10 [5.5, 19.5] months, and began GnRHa at age 12 [10.4, 12.6] years. Total body BMD Z-score for individuals on GnRHa monotherapy was -0.10 [-0.8, 0.4] (AFAB, female norms) and -0.65 [-1.4, 0.22] (AMAB, male norms). AFAB participants (n = 21) on testosterone were age 16.7 [15.9, 17.8] years, had been on testosterone for 11 [7.3, 14.5] months, and started testosterone at age 16 [14.8, 16.8] years; total body BMD Z-score -0.2 [-0.5, 0] (male norms) and 0.4 [-0.2, 0.7] (female norms). AMAB participants (n = 16) were age 16.2 [15.1, 17.4] years, had been on estradiol for 11 [5.6, 13.7] months, and started estradiol at age 16 [14.4, 16.7] years; total body BMD Z-score -0.4 [-1.1, 0.3] (male norms) and -0.2 [-0.7, 0.6] (female norms). BMD Z-score was negatively correlated with GnRHa duration (male norms: r = -0.5, P = .005; female norms: r = -0.4, P = .029) and positively correlated with BMI (male norms: r = 0.4, P = .003; female norms: r = 0.4, P = .004). In this cross-sectional cohort, total body BMD Z-scores were slightly below average, but lowest in the AMAB group on GnRHa monotherapy.

Centering equity in a longitudinal health systems science curricula.

WHAT WAS THE EDUCATIONAL CHALLENGE?: Health inequity impedes care at every level of the health care system. Despite this, health equity is not the foundation for most health systems science (HSS) curricula. WHAT WAS THE SOLUTION?: We reframed our HSS curricula to focus on health equity. HOW WAS THE SOLUTION IMPLEMENTED?: We integrated equity concepts into all HSS content areas. First-year content emphasizes structural competency and is delivered through didactics, discussions, interprofessional education, panels, and service-learning requirements. Second-year content applies HSS principles in the clinical space through direct patient care and assignments. Third- and fourth-year content focuses on HSS advocacy and leadership. WHAT LESSONS WERE LEARNED THAT ARE RELEVANT TO A WIDER GLOBAL AUDIENCE?: It is crucial to center health equity in medical curricula to improve patient outcomes. Proper faculty development, non-judgmental discussions, and integration with clinical and medical sciences are critical to successful implementation. WHAT ARE THE NEXT STEPS?: We will address feedback, emphasize relevance to patients and populations, and refine outcome measures.

Light-Driven Metabolic Pathways in Non-Photosynthetic Biohybrid Bacteria.

Biomanufacturing via microorganisms relies on carbon substrates for molecular feedstocks and a source of energy to carry out enzymatic reactions. This creates metabolic bottlenecks and lowers the efficiency for substrate conversion. Nanoparticle biohybridization with proteins and whole cell surfaces can bypass the need for redox cofactor regeneration for improved secondary metabolite production in a non-specific manner. Here we propose using nanobiohybrid organisms (Nanorgs), intracellular protein-nanoparticle hybrids formed through the spontaneous coupling of core-shell quantum dots (QDs) with histidine-tagged enzymes in non-photosynthetic bacteria, for light-mediated control of bacterial metabolism. This proved to eliminate metabolic constrictions and replace glucose with light as the source of energy in Escherichia coli, with an increase in growth by 1.7-fold in 75 % reduced nutrient media. Metabolomic tracking through carbon isotope labeling confirmed flux shunting through targeted pathways, with accumulation of metabolites downstream of respective targets. Finally, application of Nanorgs with the Ehrlich pathway improved isobutanol titers/yield by 3.9-fold in 75 % less sugar from E. coli strains with no genetic alterations. These results demonstrate the promise of Nanorgs for metabolic engineering and low-cost biomanufacturing.

Comparing Antepartum and Postpartum Opioid-Related Maternal Deaths in the State of Michigan From 2007 to 2015.

Objective Opioid use disorder (OUD) continues to be a leading cause of maternal death in the United States. The impact of OUD on pregnancy has dramatically grown in recent years, with OUD-related maternal deaths between 2007 and 2016 nearly doubling. However, the characteristics of pregnancy-associated-not-related (PANR) deaths from opioid overdose are not well understood. Specifically, the timing of OUD-related maternal deaths relative to the partum periods has not been fully described. In this study, we aimed to better characterize high-risk time periods for people with OUD, with the goal of elucidating factors that may contribute to opioid-related PANR deaths. Methods In this retrospective cohort study, we analyzed the Michigan Department of Health and Human Services Maternal Mortality Surveillance Program database from 2007 to 2015 to investigate the temporal trends in opioid-related PANR deaths. Results There was an over fourfold increase in opioid-related PANR from 2007 to 2015 and a maternal mortality ratio of 23.0 per 100,000 births attributable to opioid-related PANR deaths. Ante- and postpartum opioid-related PANR deaths shared similar demographic distribution, were associated with polysubstance use, and had low rates of medication-assisted treatment (MAT). Most opioid-related PANR deaths occurred at a steady rate during the postpartum period. Only 3.6% of people who died in the postpartum period were uninsured, compared to 42.1% of people who died in the antepartum period. Conclusion Though ante and postpartum deaths share many characteristics, our study revealed key distinctions that can help better inform the care of pregnant patients with OUD.

Retention of functional mitochondria in mature red blood cells from patients with sickle cell disease.

Sickle cell disease (SCD) is an inherited blood disorder characterized by sickled red blood cells (RBCs), which are more sensitive to haemolysis and can contribute to disease pathophysiology. Although treatment of SCD can include RBC transfusion, patients with SCD have high rates of alloimmunization. We hypothesized that RBCs from patients with SCD have functionally active mitochondria and can elicit a type 1 interferon response. We evaluated blood samples from more than 100 patients with SCD and found elevated frequencies of mitochondria in reticulocytes and mature RBCs, as compared to healthy blood donors. The presence of mitochondria in mature RBCs was confirmed by flow cytometry, electron microscopy, and proteomic analysis. The mitochondria in mature RBCs were metabolically competent, as determined by enzymatic activities and elevated levels of mitochondria-derived metabolites. Metabolically-active mitochondria in RBCs may increase oxidative stress, which could facilitate and/or exacerbate SCD complications. Coculture of mitochondria-positive RBCs with neutrophils induced production of type 1 interferons, which are known to increase RBC alloimmunization rates. These data demonstrate that mitochondria retained in mature RBCs are functional and can elicit immune responses, suggesting that inappropriate retention of mitochondria in RBCs may play an underappreciated role in SCD complications and be an RBC alloimmunization risk factor.

p53-driven lipidome influences non-cell-autonomous lysophospholipids in pancreatic cancer.

Adaptation of the lipid metabolism participates  in cancer pathogenesis, facilitating energy storage and influencing cell fate and control of molecular signalling. The tumour suppressor protein p53 is a molecular hub of cell metabolism, supporting antioxidant capabilities and counteracting oncogene-induced metabolic switch. Despite extensive work has described the p53-dependent metabolic pathways, a global profiling of p53 lipidome is still missing. By high-throughput untargeted lipidomic analysis of pancreatic ductal adenocarcinoma (PDAC) cells, we profile the p53-dependent lipidome, revealing intracellular and secreted lysophospholipids as one of the most affected class. Lysophospholipids are hydrolysed forms of phospholipids that results from phospholipase activity, which can function as signalling molecules, exerting non-cell-autonomous effects and instructing cancer microenvironment and immunity. Here, we reveal that p53 depletion reduces abundance of intracellular lysophosphatidyl-choline, -ethanolamine and -serine and their secretion in the extracellular environment. By integrating this with genomic and transcriptomic studies from in vitro models and human PDAC patients, we identified potential clinically relevant candidate p53-dependent phospholipases. In particular PLD3, PLCB4 and PLCD4 expression is regulated by p53 and chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) indicates a direct transcriptional control on their chromatin accessible genomic loci. Consistently, PLD3, PLCB4 and PLCD4 expression correlates with p53 mutational status in PDAC patients, and these genes display prognostic significance. Overall, our data provide insights into lipidome rewiring driven by p53 loss and identify alterations of lysophospholipids as a potential molecular mechanism for p53-mediated non-cell-autonomous molecular signalling that instructs cancer microenvironment and immunity during PDAC pathogenesis.

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.

Red Blood Cell Metabolism in Pyruvate Kinase Deficient Patients.

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.

Complement-containing small extracellular vesicles from adventitial fibroblasts induce proinflammatory and metabolic reprogramming in macrophages.

Pulmonary hypertension (PH) is a severe cardiopulmonary disease characterized by complement-dependent, fibroblast-induced perivascular accumulation and proinflammatory activation of macrophages. We hypothesized that, in PH, nanoscale-sized small extracellular vesicles (sEVs), released by perivascular/adventitial fibroblasts, are critical mediators of complement-dependent proinflammatory activation of macrophages. Pulmonary adventitial fibroblasts were isolated from calves with severe PH (PH-Fibs) and age-matched controls (CO-Fibs). PH-Fibs exhibited increased secretion of sEVs, compared with CO-Fibs, and sEV biological activity was tested on mouse and bovine bone marrow-derived macrophages (BMDMs) and showed similar responses. Compared with sEVs derived from CO-Fibs, sEVs derived from PH-Fibs (PH-Fib-sEVs) induced augmented expression of proinflammatory cytokines/chemokines and metabolic genes in BMDMs. Pharmacological blockade of exosome release from PH-Fibs resulted in significant attenuation of proinflammatory activation of BMDMs. "Bottom-up" proteomic analyses revealed significant enrichment of complement and coagulation cascades in PH-Fib-sEVs, including augmented expression of the complement component C3. We therefore examined whether the PH-Fib-sEV-mediated proinflammatory activation of BMDMs was complement C3 dependent. Treatment of PH-Fibs with siC3-RNA significantly attenuated the capacity of PH-Fib-sEVs for proinflammatory activation of BMDMs. PH-Fib-sEVs mediated proglycolytic alterations and complement-dependent activation of macrophages toward a proinflammatory phenotype, as confirmed by metabolomic studies. Thus, fibroblast-released sEVs served as critical mediators of complement-induced perivascular/microenvironmental inflammation in PH.

Impact of taurine on red blood cell metabolism and implications for blood storage.

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.