Complete absence of GLUT1 does not impair human terminal erythroid differentiation.

The Glucose transporter 1 (GLUT1) is one of the most abundant proteins within the erythrocyte membrane and is required for glucose and dehydroascorbic acid (Vitamin C precursor) transport. It is widely recognized as a key protein for red cell structure, function, and metabolism. Previous reports highlighted the importance of GLUT1 activity within these uniquely glycolysis-dependent cells, in particular for increasing antioxidant capacity needed to avoid irreversible damage from oxidative stress in humans. However, studies of glucose transporter roles in erythroid cells are complicated by species-specific differences between humans and mice. Here, using CRISPR-mediated gene editing of immortalized erythroblasts and adult CD34+ hematopoietic progenitor cells, we generate committed human erythroid cells completely deficient in expression of GLUT1. We show that absence of GLUT1 does not impede human erythroblast proliferation, differentiation, or enucleation. This work demonstrates for the first-time generation of enucleated human reticulocytes lacking GLUT1. The GLUT1-deficient reticulocytes possess no tangible alterations to membrane composition or deformability in reticulocytes. Metabolomic analyses of GLUT1-deficient reticulocytes reveal hallmarks of reduced glucose import, downregulated metabolic processes and upregulated AMPK-signalling, alongside alterations in antioxidant metabolism, resulting in increased osmotic fragility and metabolic shifts indicative of higher oxidant stress. Despite detectable metabolic changes in GLUT1 deficient reticulocytes, the absence of developmental phenotype, detectable proteomic compensation or impaired deformability comprehensively alters our understanding of the role of GLUT1 in red blood cell structure, function and metabolism. It also provides cell biological evidence supporting clinical consensus that reduced GLUT1 expression does not cause anaemia in GLUT1 deficiency syndrome.

Metabolic rewiring induced by ranolazine improves melanoma responses to targeted therapy and immunotherapy.

Resistance of melanoma to targeted therapy and immunotherapy is linked to metabolic rewiring. Here, we show that increased fatty acid oxidation (FAO) during prolonged BRAF inhibitor (BRAFi) treatment contributes to acquired therapy resistance in mice. Targeting FAO using the US Food and Drug Administration-approved and European Medicines Agency-approved anti-anginal drug ranolazine (RANO) delays tumour recurrence with acquired BRAFi resistance. Single-cell RNA-sequencing analysis reveals that RANO diminishes the abundance of the therapy-resistant NGFRhi neural crest stem cell subpopulation. Moreover, by rewiring the methionine salvage pathway, RANO enhances melanoma immunogenicity through increased antigen presentation and interferon signalling. Combination of RANO with anti-PD-L1 antibodies strongly improves survival by increasing antitumour immune responses. Altogether, we show that RANO increases the efficacy of targeted melanoma therapy through its effects on FAO and the methionine salvage pathway. Importantly, our study suggests that RANO could sensitize BRAFi-resistant tumours to immunotherapy. Since RANO has very mild side-effects, it might constitute a therapeutic option to improve the two main strategies currently used to treat metastatic melanoma.

Proteomic analysis of the supernatant of red blood cell units: the effects of storage and leucoreduction.

BACKGROUND: Red blood cell (RBC) transfusion is a life-saving intervention for critically ill patients; however, it has been linked to increased morbidity and mortality. We hypothesize that a number of important proteins accumulate during routine storage of RBCs, which may explain some of the adverse effects seen in transfused patients. STUDY DESIGN: Five RBC units were drawn and divided (half prestorage leucoreduced (LR-RBC) and half left as an unmodified control (RBC). The supernatant was separated on days 1 and 42 of storage and proteomic analyses completed with in-gel tryptic digestion and nano-liquid chromatography tandem mass spectrometry. RESULTS: In RBC supernatants, 401 proteins were identified: 203 increased with storage, 114 decreased, and 84 were unchanged. In LR-RBC supernatant, 231 proteins were identified: 84 increased with storage, 30 decreased, and 117 were unchanged. Prestorage leucoreduction removed many platelet- and leucocyte-derived structural proteins; however, a number of intracellular proteins accumulated including peroxiredoxins (Prdx) 6 and latexin. The increases were confirmed by immunoblotting, including the T-phosphorylation of Prdx-6, indicating that it may be functioning as an active phospholipase. Active matrix metalloproteinase-9 also increased with a coinciding decrease in the metalloproteinase inhibitor 1 and cystatin C. CONCLUSION: We conclude that a number of proteins increase with RBC storage, which is partially ameliorated with leucoreduction, and transfusion of stored RBCs may introduce mediators that result in adverse events in the transfused host.

Expression of SDF-1-CXCR4 axis and an anti-remodelling effectiveness of foetal-liver stem cell transplantation in the infarcted rat heart.

SDF-1, a chemokine secreted by injured tissues, may be instrumental in chemoattracting CXCR4(+) stem cells (SCs) for repair of infarcted myocardium. We hypothesize that the myocardial SDF-1 expression determines also the engraftment and beneficial effects of SCs transplanted into the infarcted heart. Myocardial infarction (MI) was induced in rats by coronary artery ligation. The animals were either sacrificed at 2, 7, 16, 21 or 28 days after MI or were re-operated at 2, 7 or 14 days after MI to receive SCs transplantation, and were sacrificed 14 days later. SCs transplantation consisted of 3 x 15 microl injections of SCs isolated from foetal rat liver (FLSCs) into the myocardium bordering the infarction zone (5 x 10(6) cells/heart, labelled with PKH2 Green Fluorescent Cell Linker, approximately 20% CXCR4(+)). In the MI border zone, SDF-1 and CXCR4 immunostaining was transiently increased after MI, picking at 2 days and down regulating to the sham level by 21 days after MI. Simultaneously, an increased incorporation of CXCR4(+) and CD133(+) cells into capillaries was evident. AMD1300, a blocker of CXCR4, prevented the post-MI expression of CXCR4. In the MI border zone, the cardiomyocyte cross-sectional diameter increased and capillary/cardiomyocyte ratio decreased systematically during the 28 post-MI days, while an interstitial collagen accumulation demonstrated transient increase. FLSCs did not survive in the non-infarcted hearts. In infarcted hearts, FLSCs survived best when they were injected at 2 days after MI. The survival was negligible again when the injection was performed at 14 days after MI. FLSCs transplanted at 2 days after MI caused a further rise in SDF-1, CXCR4, and CD133 expression, compared with the untreated infarcted hearts. Only FLSCs transplanted at 2 days, but not later, attenuated cardiomyocyte hypertrophy and increased capillary/cardiomyocyte ratio in the MI border zone. These results suggest that myocardial signalling for homing of the endogenous and the exogenous SCs is transiently activated early after MI, that SDF-1 is instrumental in this process, and that there is only a narrow time-window after MI when SCs transplantation results in their efficient myocardial engraftment and beneficial anti-remodelling effect.