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Oxidative modification of membrane lipids by hypochlorous acid could be an important element in the mechanism of membrane disruption by activated neutrophils. We have previously shown that hypochlorous acid reacts with unsaturated fatty acids of membrane phospholipids to give fatty acid chlorohydrins (Winterbourn et al. 1992. Arch. Biochem. Biophys. 296: 547-555). In the present study, we examined the reaction of cholesterol in bilayers with an inert phospholipid carrier. Product separation and identification was performed using gas chromatography-mass spectrometry after trimethylsilyl-derivatization. Unlike the reaction of hypochlorous acid with unsaturated fatty acids, no chlorohydrin derivatives were found with cholesterol. Instead, the main oxidation products were identified as the epimeric cholesterol 5,6-epoxides and 4-hydroxycholesterol, while several other hydroxy- and keto-derivatives were also found in smaller amounts. Analysis of the products obtained after reaction of vesicles composed of a mixture of several unsaturated phospholipid species plus cholesterol revealed that the individual fatty acids and cholesterol all exhibit comparable susceptibilities toward hypochlorous acid. Using myeloperoxidase to generate hypochlorous acid, basically the same products and product distribution were obtained. These studies show that unsaturated phospholipids and cholesterol can be profoundly modified by reaction with hypochlorous acid. This warrants further investigation to define the role of lipid modifications in neutrophil-mediated membrane disruption.

This study was designed to determine the effects of a variety of naturally occurring saturated fats on aortic lesion formation in C57BL/6J mice that are susceptible to diet-induced fatty streak lesions. Groups of female mice were randomly assigned to one of seven treatment groups and were fed diets containing 15% (w/w) hydrogenated coconut oil, hydrogenated soy oil, hydrogenated palm oil, cocoa butter, lard, tallow, or dairy butter, 1% cholesterol, and 0.5% cholic acid. Plasma lipid levels were measured to determine whether lesion formation was related to specific changes in these parameters. Lesions, which were observed in all groups of mice, ranged from 420 to 3220 microns2/aortic cross section. Lesion area was positively correlated to the percentage of saturated fatty acids contained in the fat sources and the ratio of combined VLDL plus LDL-cholesterol to HDL-cholesterol and inversely correlated to monounsaturated fatty acids content and to HDL-cholesterol levels. Results from this study demonstrate that inbred mice may provide a good model for dissecting the genetic basis for the differential atherogenic responses to diet-induction and for studying the effects of dietary factors on aortic lesion development.

Anemia in patients with sickle cell disease (SCD) increases 2,3-diphosphoglycerate (2,3-DPG), decreasing hemoglobin-oxygen (HbO 2 ) affinity to improve oxygen offloading and promote hemoglobin polymerization (sickling) of red blood cells (RBCs). We report the discovery of FT-4202, an investigational, selective pyruvate kinase type-R (PKR) activator with a multimodal mechanism of action and potential to increase ATP and decrease 2,3-DPG, resulting in increased HbO 2 affinity, decreased Hb polymerization, and improved RBC health. FT-4202 was identified via structure-enabled lead optimization medicinal chemistry using X-ray crystallography, molecular modeling, and thermal shift assays. FT-4202, an allosteric PKR activator, stabilizes the tetrameric enzyme and increases PKR activity in human and mouse RBCs in vitro. Seven-day oral administration of FT-4202 in Berkeley SCD mice reduced 2,3-DPG, increased HbO 2 affinity, and reduced RBC sickling versus control. There were no adverse in vitro safety findings. FT-4202 offers a therapeutic opportunity to modify the course of SCD., Competing Interests: Conflict of Interest Disclosure This work was funded by Forma Therapeutics, Inc., Watertown, Massachusetts, which was acquired by Novo Nordisk, Watertown, in October 2022. AE, DJR, EW, DRL, SF, PT, XZ, AT, CD, LY, DM, KF, MR, SMG, and GM were employees of Forma Therapeutics, Inc., at the time the work was conducted. FAK and SL were employees of the Children's Hospital Oakland Research Institute. SMG became an employee of Novo Nordisk (following their acquisition of Forma Therapeutics, Inc.) FAK and SL received institutional compensation for the assays performed in the study. AE, DJR, DRL, MR, and GM declare co-invention of multiple patent applications relating to this research project: 1) Applicant: Forma Therapeutics; Inventors: Anna Ericsson, Neal Green, Gary Gustafson, David R. Lancia, Jr., Gary Marshall, Lorna Mitchell, David Richard, Zhongguo Wang, Sanjeev Forsyth, Patrick Kelly, Madhu Mondal, Maria Ribadeneira, Patricia Schroeder; Application number: US-2020129485-A1; Status: Pending; Specific aspect of manuscript covered in patent application: Discovery and use of FT-4202 to treat sickle cell disease. 2) Applicant: Forma Therapeutics; Inventor(s): Anna Ericsson, Neal Green, Gary Gustafson, David R. Lancia, Jr., Gary Marshall, Lorna Mitchell, David Richard, Zhongguo Wang; Patent number: US 10,675,274 B2; Status: Awarded; Specific aspect of manuscript covered in patent application: Compound synthesis, biochemical, and cellular activities of compounds. 3) Applicant: Forma Therapeutics; Inventor(s): David R. Lancia, Gary Gustafson, Neal Green, Lorna Mitchell, Anna Ericsson, David Richard; Patent Number: US 11,001,588 B2; Status: Awarded; Specific aspect of manuscript covered in patent application: Compound synthesis and biochemical activities of compounds. 4) Applicant: Forma Therapeutics; Inventor(s): Anna Ericsson, Neal Green, Gary Gustafson, Bingsong Han, David R. Lancia, Jr., Lorna Mitchell, David Richard, Tatiana Shelekhin, Chase C. Smith, Zhongguo Wang, Xiaozhang Zheng; Patent number: US 11,014,927 B2; Status: Awarded; Specific aspect of manuscript covered in patent application: Compound synthesis, biochemical, and cellular activities of compounds. 5) Applicant: Forma Therapeutics; Inventor(s): Anna Ericsson, Neal Green, Gary Gustafson, David R. Lancia, Jr., Gary Marshall, Lorna Mitchell, David Richard, Zhongguo Wang; Patent number: US 11,071,725 B2; Status: Awarded; Specific aspect of manuscript covered in patent application: Compound synthesis, biochemical, and cellular activities of compounds. AE, EW, SF, PT, XZ, AT, CD, LY, DM, MR, and GM have no additional financial or non-financial competing interests to declare., (Copyright © 2024 International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.)

Abstract: Etavopivat is an investigational, once daily, oral, selective erythrocyte pyruvate kinase (PKR) activator. A multicenter, randomized, placebo-controlled, double-blind, 3-part, phase 1 study was conducted to characterize the safety and clinical activity of etavopivat. Thirty-six patients with sickle cell disease (SCD) were enrolled into 4 cohorts: 1 single-dose, 2 multiple ascending doses, and 1 open-label (OL). In the OL cohort, 15 patients (median age 33.0 years [range, 17-55]) received 400 mg etavopivat once daily for 12 weeks; 14 patients completed treatment. Consistent with the mechanism of PKR activation, increases in adenosine triphosphate and decreases in 2,3-diphosphoglycerate were observed and sustained over 12 weeks' treatment. This translated clinically to an increase in hemoglobin (Hb; mean maximal increase 1.6 g/dL [range, 0.8-2.8]), with >1 g/dL increase in 11 (73%) patients during treatment. In addition, the oxygen tension at which Hb is 50% saturated was reduced (P = .0007) with a concomitant shift in point of sickling (P = .0034) to lower oxygen tension in oxygen-gradient ektacytometry. Hemolysis markers (absolute reticulocyte count, indirect bilirubin, and lactate dehydrogenase) decreased from baseline, along with matrix metalloproteinase-9 and erythropoietin. In the OL cohort, adverse events (AEs) were mostly grade 1/2, consistent with underlying SCD; 5 patients had serious AEs. Vaso-occlusive pain episode was the most common treatment-emergent AE (n = 7) in the OL cohort. In this, to our knowledge, the first study of etavopivat in SCD, 400 mg once daily for 12 weeks was well tolerated, resulting in rapid and sustained increases in Hb, improved red blood cell physiology, and decreased hemolysis. This trial was registered at www.ClinicalTrials.gov as #NCT03815695., (© 2024 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.)

Exposure to both oxidative and shear stress, a condition that the red blood cell (RBC) continuously experiences in the circulation in vivo can be mimicked in a Couette type viscometer and monitored by ektacytometry. RBCs maintain their deformation and orientation under shear stress and oxidative stress until a threshold is reached at which these conditions appear to overwhelm the elaborate and complex pathways that maintain a proper redox environment in the cell. Oxidative stress under shear alters the ability of the cell to deform, changes cell morphology, its orientation in the shear stress field, and appears to alter intracellular and membrane characteristics. The application of the RoxyScan technology allows the comparison of oxidant effects and the role of antioxidant systems. This provides the opportunity to study the ability of RBC to deal with oxidative stress in various conditions, including RBC disorders such as sickle cell disease (SCD)., (© 2024. The Author(s).)

Peroxisomes and the endoplasmic reticulum (ER) are intimately linked subcellular organelles, physically connected at membrane contact sites. While collaborating in lipid metabolism, for example, of very long-chain fatty acids (VLCFAs) and plasmalogens, the ER also plays a role in peroxisome biogenesis. Recent work identified tethering complexes on the ER and peroxisome membranes that connect the organelles. These include membrane contacts formed via interactions between the ER protein VAPB (vesicle-associated membrane protein-associated protein B) and the peroxisomal proteins ACBD4 and ACBD5 (acyl-coenzyme A-binding domain protein). Loss of ACBD5 has been shown to cause a significant reduction in peroxisome-ER contacts and accumulation of VLCFAs. However, the role of ACBD4 and the relative contribution these two proteins make to contact site formation and recruitment of VLCFAs to peroxisomes remain unclear. Here, we address these questions using a combination of molecular cell biology, biochemical, and lipidomics analyses following loss of ACBD4 or ACBD5 in HEK293 cells. We show that the tethering function of ACBD5 is not absolutely required for efficient peroxisomal β-oxidation of VLCFAs. We demonstrate that loss of ACBD4 does not reduce peroxisome-ER connections or result in the accumulation of VLCFAs. Instead, the loss of ACBD4 resulted in an increase in the rate of β-oxidation of VLCFAs. Finally, we observe an interaction between ACBD5 and ACBD4, independent of VAPB binding. Overall, our findings suggest that ACBD5 may act as a primary tether and VLCFA recruitment factor, whereas ACBD4 may have regulatory functions in peroxisomal lipid metabolism at the peroxisome-ER interface., Competing Interests: Conflict of interest All authors declare that they have no conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Individuals with sickle cell anemia (SCA) exhibit decreased exercise capacity. Anemia limits oxygen-carrying capacity and affects cardiopulmonary fitness. The drug voxelotor raises hemoglobin in SCA. We hypothesized that voxelotor improves exercise capacity in youths with SCA., Methods: In a single-center, open-label, single-arm, longitudinal interventional pilot study (NCT04581356), SCA patients aged 12 and older, stably maintained on hydroxyurea, were treated with 1500 mg voxelotor daily, and performed cardiopulmonary exercise testing before (CPET#1) and after voxelotor (CPET#2). A modified Bruce Protocol was performed on a motorized treadmill, and breath-by-breath gas exchange data were collected. Peak oxygen consumption (peak VO 2 ), anaerobic threshold, O 2 pulse, VE/VCO 2 slope, and time exercised were compared for each participant. The primary endpoint was change in peak VO 2 . Hematologic parameters were measured before each CPET. Patient Global Impression of Change (PGIC) and Clinician Global Impression of Change (CGIC) surveys were collected., Results: Ten hemoglobin SS patients aged 12-24 completed the study. All demonstrated expected hemoglobin rise, with average +1.6 g/dL (p = .003) and P 50 left shift of average -11 mmHg (p < .0001) with decreased oxygen off-loading at low pO 2 . The change in % predicted peak VO 2 from CPET#1 to CPET#2 ranged from -12.8% to +11.3%, with significant improvement of more than 5% in one subject, more than 5% decrease in five subjects, and insignificant change of less than 5% in four subjects. All 10 CGIC and seven of 10 PGIC responses were positive., Conclusion: In a plot study of 10 youths with SCA, voxelotor treatment did not improve peak VO 2 in 9 out of 10 patients., (© 2023 The Authors. Pediatric Blood & Cancer published by Wiley Periodicals LLC.)

Intravascular hemolysis results in the release of cell-free hemoglobin and heme in plasma. In sickle cell disease, the fragility of the sickle red blood cell leads to chronic hemolysis, which can contribute to oxidative damage and activation of inflammatory pathways. The scavenger proteins haptoglobin and hemopexin provide pathways to remove hemoglobin and heme, respectively, from the circulation. Heme also intercalates in membranes of blood cells and endothelial cells in the vasculature and associates with other plasma components such as albumin and lipoproteins. Hemopexin has a much higher affinity and can strip heme from the other pools and detoxify plasma from cell-free circulatory heme. However, due to chronic hemolysis, hemopexin is depleted in individuals with sickle cell disease. Thus, cell-free unbound heme is expected to accumulate in plasma. We developed a methodology for the accurate quantification of the fraction of heme, which is pathologically relevant in sickle cell disease, that does not appear to be sequestered to a plasma compartment. Our data show significant variation in the concentration of unbound heme, and rather unexpectedly, the size of the unbound fraction does not correlate to the degree of hemolysis, as measured by the concentration of bound heme. Very high heme concentrations (>150 µM) were obtained in some plasma with unbound concentrations that were several fold lower than in plasma with much lower hemolysis (<50 µM). These findings underscore the long-term effects of chronic hemolysis on the blood components and of the disruption of the essential equilibrium between release of hemoproteins/heme in the circulation and adaptative response of the scavenging/removal mechanisms. Understanding the clinical implications of this loss of response may provide insights into diagnostic and therapeutic targets in patients with sickle cell disease.

Background/aims: Cells adapt to chronic extracellular hypotonicity by altering metabolism. Corresponding effects of sustained hypotonic exposure at the whole-person level remain to be confirmed and characterized in clinical and population-based studies. This analysis aimed to 1) describe changes in urine and serum metabolomic profiles associated with four weeks of sustained > +1 L/d drinking water in healthy, normal weight, young men, 2) identify metabolic pathways potentially impacted by chronic hypotonicity, and 3) explore if effects of chronic hypotonicity differ by type of specimen and/or acute hydration condition., Materials: Untargeted metabolomic assays were completed for specimen stored from Week 1 and Week 6 of the Adapt Study for four men (20-25 years) who changed hydration classification during that period. Each week, first-morning urine was collected after overnight food and water restriction, and urine (t+60 min) and serum (t+90 min) were collected after a 750 mL bolus of drinking water. Metaboanalyst 5.0 was used to compare metabolomic profiles., Results: In association with four weeks of > + 1 L/d drinking water, urine osmolality decreased below 800 mOsm/kg H 2 O and saliva osmolality decreased below 100 mOsm/kg H 2 O. Between Week 1 and Week 6, 325 of 562 metabolic features in serum changed by 2-fold or more relative to creatinine. Based on hypergeometric test p-value <0.05 or Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway impact factor >0.2, the sustained > + 1 L/d of drinking water was associated with concurrent changes in carbohydrate, protein, lipid, and micronutrient metabolism, a metabolomic pattern of carbohydrate oxidation via the tricarboxylic acid (TCA) cycle, instead of glycolysis to lactate, and a reduction of chronic disease risk factors in Week 6. Similar metabolic pathways appeared potentially impacted in urine, but the directions of impact differed by specimen type., Conclusion: In healthy, normal weight, young men with initial total water intake below 2 L/d, sustained > + 1 L/d drinking water was associated with profound changes in serum and urine metabolomic profile, which suggested normalization of an aestivation-like metabolic pattern and a switch away from a Warburg-like pattern. Further research is warranted to pursue whole-body effects of chronic hypotonicity that reflect cell-level effects and potential beneficial effects of drinking water on chronic disease risk.

Anemia is a continuing global public health concern and a priority for international action. The prevalence of anemia is estimated from the hemoglobin (Hb) levels within target populations, yet the procedures for measuring Hb are not standardized and different approaches may result in discrepancies. Several analytical variables have been proposed to influence Hb measurements, but it is difficult to understand the impact on specific variables from large population or field studies. Therefore, we designed a highly controlled protocol that minimized most technical parameters to specifically investigate the impact of blood draw site and analytic device on Hb measurements. A diverse cohort of sixty healthy adults each provided a sequential capillary and venous blood sample that were measured for Hb using an automated hematology analyzer (ADVIA-2120) and two point-of-care devices (HemoCue 201+ and HemoCue 301). Comparing blood draw sites, the mean Hb content was 0.32-0.47 g/dL (2-4%) higher in capillary compared to venous blood from the same donors. Comparing different Hb measuring instruments, the mean Hb content was 0.19-0.46 g/dL (1-4%) higher measured with HemoCue devices compared to ADVIA-2120 in both capillary and venous blood from the same donors. The maximum variance in measurement was also higher with HemoCue devices using blood from venous (5-6% CV) and capillary (21-25% CV) sites compared to ADVIA-2120 (0.6-2% CV). Other variables including blood collection tube manufacturer did not affect mean Hb content. These results demonstrate that even when most technical variables are minimized, the blood draw site and the analytical device can have a small but statistically significant effect on the mean and dispersion of Hb measurements. Even in this study, the few participants identified as mildly anemic using venous blood measured by ADVIA-2120 would not have been classified as anemic using their capillary blood samples or point-of-care analyzers. Thus, caution is warranted when comparing Hb values between studies having differences in blood draw site and Hb measuring device. Future anemia testing should maintain consistency in these analytical variables., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2022 Killilea et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The transfer of acyl chains to proteins and lipids from acyl-CoA donor molecules is achieved by the actions of diverse enzymes and proteins, including the acyl-CoA binding domain-containing protein ACBD6. N-myristoyl-transferase (NMT) enzymes catalyze the covalent attachment of a 14-carbon acyl chain from the relatively rare myristoyl-CoA to the N-terminal glycine residue of myr-proteins. The interaction of the ankyrin-repeat domain of ACBD6 with NMT produces an active enzymatic complex for the use of myristoyl-CoA protected from competitive inhibition by acyl donor competitors. The absence of the ACBD6/NMT complex in ACBD6.KO cells increased the sensitivity of the cells to competitors and significantly reduced myristoylation of proteins. Protein palmitoylation was not altered in those cells. The specific defect in myristoyl-transferase activity of the ACBD6.KO cells provided further evidence of the essential functional role of the interaction of ACBD6 with the NMT enzymes. Acyl-CoAs bound to the acyl-CoA binding domain of ACBD6 are acyl donors for the lysophospholipid acyl-transferase enzymes (LPLAT), which acylate single acyl-chain lipids, such as the bioactive molecules LPA and LPC. Whereas the formation of acyl-CoAs was not altered in ACBD6.KO cells, lipid acylation processes were significantly reduced. The defect in PC formation from LPC by the LPCAT enzymes resulted in reduced lipid droplets content. The diversity of the processes affected by ACBD6 highlight its dual function as a carrier and a regulator of acyl-CoA dependent reactions. The unique role of ACBD6 represents an essential common feature of (acyl-CoA)-dependent modification pathways controlling the lipid and protein composition of human cell membranes.

Objectives: L-Glutamine was FDA-approved for sickle cell disease (SCD) in 2017, yet the mechanism(s)-of-action are poorly understood. This study investigates the potential activation of autophagy as a previously unexplored mechanism-of-benefit., Design: Prospective, open-label, 8-week, phase-2 trial of oral L-glutamine (10 g TID) in patients with SCD at risk for pulmonary hypertension identified by Doppler-echocardiography by an elevated tricuspid-regurgitant-jet-velocity (TRV)≥ 2.5 m/s. Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples taken from SCD patients at baseline, two, four, six and eight weeks of glutamine therapy, and from controls at baseline; BAX (pro-apoptotic marker) and LC3-II/LC3-I (autophagy marker) were measured via western blot analysis to assess apoptosis and autophagy respectively., Setting: Comprehensive SCD Center in Oakland, California., Results: Patients with SCD (n = 8) had a mean age of 44 ± 16, 50% were male; 63% Hb-SS, and mean TRV= 3.1 ± 0.7 m/s. Controls' mean age (n = 5) was 32 ± 12% and 57% were male; all were Hb-AA with a mean TRV= 1.8 ± 0.6. At baseline, SCD-PBMCs had 2-times higher levels of BAX and LC3-I versus controls (both p = 0.03). Levels of BAX expression increased by 300% after 8-weeks of glutamine supplementation (p = 0.005); LC3-I protein levels decreased while LC3-II levels increased by 70%, giving a significant increase in the LC3-II/LC3-I ratio (p = 0.02)., Conclusion: PBMCs from glutamine-supplemented SCD patients have upregulated apoptotic and autophagy proteins. The parallel increase in BAX and the LC3-II / LC3-I ratio with glutamine supplementation suggest a possible role of autophagic cell death. The increase in apoptotic markers provide insight into a possible mechanism used by peripheral PBMCs during glutamine supplementation in patients with SCD., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The cytokine storm (CS) in hyperinflammation is characterized by high levels of cytokines, extreme activation of innate as well as adaptive immune cells and initiation of apoptosis. High levels of apoptotic cells overwhelm the proper recognition and removal system of these cells. Phosphatidylserine on the apoptotic cell surface, which normally provides a recognition signal for removal, becomes a target for hemostatic proteins and secretory phospholipase A2. The dysregulation of these normal pathways in hemostasis and the inflammasome result in a prothrombotic state, cellular death, and end-organ damage. In this review, we provide the argument that this imbalance in recognition and removal is a common denominator regardless of the inflammatory trigger. The complex reaction of the immune defense system in hyperinflammation leads to self-inflicted damage. This common endpoint may provide additional options to monitor the progression of the inflammatory syndrome, predict severity, and may add to possible treatment strategies.

Objectives: Validation of the measurement of erythrocyte deformability as a useful prognostic, rheological biomarker for patients with sickle cell disease (SCD)., Methods: The degree of reduced deformability was based on the value of the maximum elongation index (EI max ) of the deformability curve of an osmotic gradient ektacytometer. The performance of this technique was analytically and clinically validated by analysing 200 normal subjects and 100 patients with well-documented thalassemia's and Hb variants in relation to their clinical condition., Results: In this study, we show that EI max is a reproducible parameter with a small inter-individual coefficient of (Biological) variation (CV)=1.6% and a small intra-individual CV=3.5%. We demonstrate that loss of deformability correlates with the clinical condition and the various mutations underlying sickle cell disease and thalassemia. For SCD patients, a strongly reduced EI max with a cut-off =0.360 is a signal for future vaso-occlusive (VOC) events requiring hospitalisation with a specificity=85%, sensitivity=80%, PPV=81% and NPV=84% based on a ROC curve (AUC=0.89)., Conclusion: This study validated the clinical utility of EI max as a prognostic marker for future clinical problems in individual high-risk SCD patients. In addition, EI max may help to achieve an adequate personal transfusion policy for an optimal blood flow in anaemic patients with SCD., (© 2022 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Objectives: L-Glutamine is FDA-approved for sickle cell disease (SCD), yet the mechanism(s)-of-action are poorly understood. We performed a pharmacokinetics (pK) study to determine the metabolic fate of glutamine supplementation on plasma and erythrocyte amino acids in patients with SCD., Design: A pK study was performed where patients with SCD fasting for > 8 h received oral L-glutamine (10 g). Blood was analyzed at baseline, 30/60/90 min/2/3/4/8 hrs. A standardized diet was administered to all participants at 3 established time-points (after 2/5/7hrs). A subset of patients also had pK studies performed without glutamine supplementation to follow normal diurnal fluctuations in amino acids., Setting: Comprehensive SCD Center in Oakland, California RESULTS: Five patients with SCD were included, three of whom performed pK studies both with and without glutamine supplementation. Average age was 50.6 ± 5.6 years, 60% were female, 40% SS, 60% SC. Plasma glutamine levels increased significantly after oral glutamine supplementation, compared to minimal fluctuations with diet. Plasma glutamine concentration peaked within 30-min of ingestion (p = 0.01) before decreasing to a plateau by 2-h that remained higher than baseline by 8 h. Oral glutamine also increased plasma arginine concentration, which peaked by 4-h (p = 0.03) and remained elevated through 8-h. Erythrocyte glutamine levels began to increase by 8-h, while erythrocyte arginine concentration peaked at 4-h., Conclusions: Oral glutamine supplementation acutely improved glutamine and arginine bioavailability in both plasma and erythrocytes. This is the first study to demonstrate that glutamine therapy increases arginine bioavailability and may provide insight into shared mechanisms-of-action between these conditionally-essential amino acids., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Secretory phospholipase 2 (sPLA2) acts as a mediator between proximal and distal events of the inflammatory cascade. Its role in SARS-CoV-2 infection is unknown, but could contribute to COVID-19 inflammasome activation and cellular damage. We present the first report of plasma sPLA2 levels in adults and children with COVID-19 compared with controls. Currently asymptomatic adults with a history of recent COVID-19 infection (≥4 weeks before) identified by SARS-CoV-2 IgG antibodies had sPLA2 levels similar to those who were seronegative (9 ± 6 vs.17 ± 28 ng/mL, P  = 0.26). In contrast, children hospitalized with severe COVID-19 had significantly elevated sPLA2 compared with those with mild or asymptomatic SARS-CoV-2 infection (269 ± 137 vs. 2 ± 3 ng/mL, P  = 0.01). Among children hospitalized with multisystem inflammatory syndrome in children (MIS-C), all had severe disease requiring pediatric intensive care unit (PICU) admission. sPLA2 levels were significantly higher in those with acute illness <10 days versus convalescent disease ≥10 days (540 ± 510 vs. 2 ± 1, P  = 0.04). Thus, sPLA2 levels correlated with COVID-19 severity and acute MIS-C in children, implicating a role in inflammasome activation and disease pathogenesis. sPLA2 may be a useful biomarker to stratify risk and guide patient management for children with acute COVID-19 and MIS-C. Therapeutic compounds targeting sPLA2 and inflammasome activation warrant consideration.

Alleviating anaemia in patients with sickle cell disease (SCD) is crucial in managing acute complications, mitigating end-organ damage and preventing early mortality. Some disease-modifying and curative therapies have increased haemoglobin (Hb) levels to exceed 100 g/l, a threshold above which complications from red blood cell (RBC) transfusions have occurred, raising concern about whole-blood viscosity-related complications with these therapies. Here we discuss the rationale behind this limit, the effect of viscosity on blood flow and the applicability of this Hb threshold to therapies for SCD beyond RBC transfusions., (© 2021 The Authors. British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.)

The replication of Plasmodium falciparum parasites within red blood cells (RBCs) causes severe disease in humans, especially in Africa. Deleterious alleles like hemoglobin S are well-known to confer strong resistance to malaria, but the effects of common RBC variation are largely undetermined. Here, we collected fresh blood samples from 121 healthy donors, most with African ancestry, and performed exome sequencing, detailed RBC phenotyping, and parasite fitness assays. Over one-third of healthy donors unknowingly carried alleles for G6PD deficiency or hemoglobinopathies, which were associated with characteristic RBC phenotypes. Among non-carriers alone, variation in RBC hydration, membrane deformability, and volume was strongly associated with P. falciparum growth rate. Common genetic variants in PIEZO1 , SPTA1/SPTB , and several P. falciparum invasion receptors were also associated with parasite growth rate. Interestingly, we observed little or negative evidence for divergent selection on non-pathogenic RBC variation between Africans and Europeans. These findings suggest a model in which globally widespread variation in a moderate number of genes and phenotypes modulates P. falciparum fitness in RBCs., Competing Interests: EE, FK, CL, DP, EE No competing interests declared, (© 2021, Ebel et al.)

The etiology of necrotizing enterocolitis (NEC) is not known. Alterations in gut microbiome, mucosal barrier function, immune cell activation, and blood flow are characterized events in its development, with stress as a contributing factor. The hormone corticotropin-releasing factor (CRF) is a key mediator of stress responses and influences these aforementioned processes. CRF signaling is modulated by NEC's main risk factors of prematurity and formula feeding. Using an established neonatal rat model of NEC, we tested hypotheses that: (i) increased CRF levels-as seen during stress-promote NEC in formula-fed (FF) newborn rats, and (ii) antagonism of CRF action ameliorates NEC. Newborn pups were formula-fed to initiate gut inflammation and randomized to: no stress, no stress with subcutaneous CRF administration, stress (acute hypoxia followed by cold exposure-NEC model), or stress after pretreatment with the CRF peptide antagonist Astressin. Dam-fed unstressed and stressed littermates served as controls. NEC incidence and severity in the terminal ileum were determined using a histologic scoring system. Changes in CRF, CRF receptor (CRFRs), and toll-like receptor 4 (TLR4) expression levels were determined by immunofluorescence and immunoblotting, respectively. Stress exposure in FF neonates resulted in 40.0% NEC incidence, whereas exogenous CRF administration resulted in 51.7% NEC incidence compared to 8.7% in FF non-stressed neonates (p<0.001). Astressin prevented development of NEC in FF-stressed neonates (7.7% vs. 40.0%; p = 0.003). CRF and CRFR immunoreactivity increased in the ileum of neonates with NEC compared to dam-fed controls or FF unstressed pups. Immunoblotting confirmed increased TLR4 protein levels in FF stressed (NEC model) animals vs. controls, and Astressin treatment restored TLR4 to control levels. Peripheral CRF may serve as specific pharmacologic target for the prevention and treatment of NEC., Competing Interests: No competing interests exist.

Members of the large multigene family of acyl-CoA binding domain containing proteins (ACBDs) share a conserved motif required for binding of Coenzyme A esterified fatty acids of various chain length. These proteins are present in the three kingdoms of life, and despite their predicted roles in cellular lipid metabolism, knowledge about the precise functions of many ACBD proteins remains scarce. Interestingly, several ACBD proteins are now suggested to function at organelle contact sites, and are recognized as host interaction proteins for different pathogens including viruses and bacteria. Here, we present a thorough phylogenetic analysis of the ACBD family and discuss their structure and evolution. We summarize recent findings on the various functions of animal and fungal ACBDs with particular focus on peroxisomes, the role of ACBD proteins at organelle membranes, and their increasing recognition as targets for pathogens., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Glycine N-myristoylation is an essential acylation modification modulating the functions, stability, and membrane association of diverse cytosolic proteins in human cells. Myristoyl-CoA is the 14-carbon acyl donor of the acyltransferase reaction. Acyl-CoAs of a chain length compatible with the binding site of the N-myristoyltransferase enzymes (NMT) are competitive inhibitors, and the mechanism protecting these enzymes from unwanted acyl-CoA species requires the acyl-CoA binding protein ACBD6. The acyl-CoA binding domain (ACB) and the ankyrin-repeat motifs (ANK) of ACBD6 can perform their functions independently. Interaction of ANK with human NMT2 was necessary and sufficient to provide protection. Fusion of the ANK module to the acyl-CoA binding protein ACBD1 was sufficient to confer the NMT-stimulatory property of ACBD6 to the chimera. The ACB domain is dispensable and sequestration of the competitor was not the basis for NMT2 protection. Acyl-CoAs bound to ACB modulate the function of the ANK module and act as positive effector of the allosteric activation of the enzyme. The functional relevance of homozygous mutations in ACBD6 gene, which have not been associated with a disease so far, is presented. Skin-derived fibroblasts of two unrelated individuals with neurodevelopmental disorder and carrying loss of function mutations in the ACBD6 gene were deficient in protein N-myristoylation. These cells were sensitive to substrate analog competing for myristoyl-CoA binding to NMT. These findings account for the requirement of an ANK-containing acyl-CoA binding protein in the cellular mechanism protecting the NMT enzymes and establish that in human cells, ACBD6 supports the N-myristoylation of proteins., Competing Interests: The authors have declared that no competing interests exist.

Red blood cells (RBCs) from sickle cell patients exposed to a low oxygen tension reveal highly heterogeneous cell morphologies due to the polymerization of sickle hemoglobin (HbS). We show that angle-resolved light scattering approach with the use of image-based flow cytometry provides reliable quantitative data to define the change in morphology of large populations of RBCs from sickle cell patients when the cells are exposed for different times to low oxygen. We characterize the RBC morphological profile by means of a set of morphological and physical parameters, which includes cell shape, size, and orientation. These parameters define the cell as discocyte, sickle, elongated, as well as irregularly or abnormal RBC shaped cells, including echinocytes, holly-leaf, and granular structures. In contrast to microscopy, quick assessment of large numbers of cells provides statistically relevant information of the dynamic process of RBC sickling in time. The use of this approach facilitates the understanding of the processes that define the propensity of sickle blood samples to change their shape, and the ensuing vaso-occlusive events in the circulation of the patients. Moreover, it assists in the evaluation of treatments that include the use of anti-sickling agents, gene therapy-based hemoglobin modifications, as well as other approaches to improve the quality of life of sickle cell patients. © 2019 International Society for Advancement of Cytometry., (© 2019 International Society for Advancement of Cytometry.)

Members of the human acyl-CoA binding domain-containing (ACBD) family regulate processes as diverse as viral replication, stem-cell self-renewal, organelle organization, and protein acylation. These functions are defined by nonconserved motifs present downstream of the ACBD. The human ankyrin-repeat-containing ACBD6 protein supports the reaction catalyzed by the human and Plasmodium N -myristoyltransferase (NMT) enzymes. Likewise, the newly identified Plasmodium ACBD6 homologue regulates the activity of the NMT enzymes. The relatively low abundance of myristoyl-CoA in the cell limits myristoylation. Binding of myristoyl-CoA to NMT is competed by more abundant acyl-CoA species such as palmitoyl-CoA. ACBD6 also protects the Plasmodium NMT enzyme from lauryl-CoA and forces the utilization of the myristoyl-CoA substrate. The phosphorylation of two serine residues of the acyl-CoA binding domain of human ACBD6 improves ligand binding capacity, prevents competition by unbound acyl-CoAs, and further enhances the activity of NMT. Thus, ACBD6 proteins promote N -myristoylation in mammalian cells and in one of their intracellular parasites under unfavorable substrate-limiting conditions., (Copyright © 2019 Soupene and Kuypers.)

De novo lipid synthesis and scavenging of fatty acids (FA) are processes essential for the formation of the membrane of the human pathogen Chlamydia trachomatis (C.t.). Host FA are assimilated via esterification by the bacterial acyl-acyl carrier protein (ACP) synthase AasC but inhibitors of the host acyl-CoA synthetase enymes ACSL also impaired growth of C.t. in human cells. In E. coli, activity of AasC was sensitive to triacsin C and rosiglitazone G. The absence of a triacsin C-insensitive pathway and the increased inhibition by rosiglitazone G confirmed the sensitivity of the bacterial acyl-ACP synthase to these drugs in infected human cells. We found no evidence that the human ACSL enzymes are required for lipid formation by C.t. The broad substrate specificity of acyltransferase CT775 provides C.t. with the capacity to incorporate straight-chain and bacterial specific branched-chain fatty acids. CT775 accepts both acyl-ACP and acyl-CoA as acyl donors and, 1- or 2-acyl isomers of lysophosphoplipids as acyl acceptors. The enzyme responsible for remodeling of human phosphatidylserine to bacterial phosphatidylethanolamine was identified as CT699. These findings provide evidence that the pathogen has the ability to extend the lipid diversity of its membrane.

In sickle cell disease (SCD), alterations of cholesterol metabolism is in part related to abnormal levels and activity of plasma proteins such as lecithin cholesterol acyltransferase (LCAT), and apolipoprotein A-I (ApoA-I). In addition, the size distribution of ApoA-I high density lipoproteins (HDL) differs from normal blood. The ratio of the amount of HDL 2 particle relative to the smaller higher density pre-β HDL (HDL 3 ) particle was shifted toward HDL 2 . This lipoprotein imbalance is exacerbated during acute vaso-occlusive episodes (VOE) as the relative levels of HDL 3 decrease. HDL 3 deficiency in SCD plasma was found to relate to a slower ApoA-I exchange rate, which suggests an impaired ABCA1-mediated cholesterol efflux in SCD. HDL 2 isolated from SCD plasma displayed an antioxidant capacity normally associated with HDL 3 , providing evidence for a change in function of HDL 2 in SCD as compared to HDL 2 in normal plasma. Although SCD plasma is depleted in HDL 3 , this altered capacity of HDL 2 could account for the lack of difference in pro-inflammatory HDL levels in SCD as compared to normal. Exposure of human umbilical vein endothelial cells to HDL 2 isolated from SCD plasma resulted in higher mRNA levels of the acute phase protein long pentraxin 3 (PTX3) as compared to incubation with HDL 2 from control plasma. Addition of the heme-scavenger hemopexin protein prevented increased expression of PTX3 in sickle HDL 2 -treated cells. These findings suggest that ApoA-I lipoprotein composition and functions are altered in SCD plasma, and that whole blood transfusion may be considered as a blood replacement therapy in SCD. Impact statement Our study adds to the growing evidence that the dysfunctional red blood cell (RBC) in sickle cell disease (SCD) affects the plasma environment, which contributes significantly in the vasculopathy that defines the disease. Remodeling of anti-inflammatory high density lipoprotein (HDL) to pro-inflammatory entities can occur during the acute phase response. SCD plasma is depleted of the pre-β particle (HDL 3 ), which is essential for stimulation of reverse cholesterol from macrophages, and the function of the larger HDL 2 particle is altered. These dysfunctions are exacerbated during vaso-occlusive episodes. Interaction of lipoproteins with endothelium increases formation of inflammatory mediators, a process counteracted by the heme-scavenger hemopexin. This links hemolysis to lipoprotein-mediated inflammation in SCD, and hemopexin treatment could be considered. The use of RBC concentrates in transfusion therapy of SCD patients underestimates the importance of the dysfunctional plasma compartment, and transfusion of whole blood or plasma may be warranted.

In sickle cell disease (SCD) cholesterol metabolism appears dysfunctional as evidenced by abnormal plasma cholesterol content in a subpopulation of SCD patients. Specific activity of the high density lipoprotein (HDL)-bound lecithin cholesterol acyltransferase (LCAT) enzyme, which catalyzes esterification of cholesterol, and generates lysoPC (LPC) was significantly lower in sickle plasma compared to normal. Inhibitory amounts of LPC were present in sickle plasma, and the red blood cell (RBC) lysophosphatidylcholine acyltransferase (LPCAT), essential for the removal of LPC, displayed a broad range of activity. The functionality of sickle HDL appeared to be altered as evidenced by a decreased HDL-Apolipoprotein A-I exchange in sickle plasma as compared to control. Increased levels of oxidized proteins including ApoA-I were detected in sickle plasma. In vitro incubation of sickle plasma with washed erythrocytes affected the ApoA-I-exchange supporting the view that the RBC blood compartment can affect cholesterol metabolism in plasma. HDL functionality appeared to decrease during acute vaso-occlusive episodes in sickle patients and was associated with an increase of secretory PLA 2 , a marker for increased inflammation. Simvastatin treatment to improve the anti-inflammatory function of HDL did not ameliorate HDL-ApoA-I exchange in sickle patients. Thus, the cumulative effect of an inflammatory and highly oxidative environment in sickle blood contributes to a decrease in cholesterol esterification and HDL function, related to hypocholesterolemia in SCD., (© 2016 by the Society for Experimental Biology and Medicine.)

This study reveals a novel interaction between deoxyhemoglobin, nitrite and the non-toxic compound, RRx-001, to generate supraphysiologic levels of nitric oxide (NO) in blood. We characterize the nitrite reductase activity of deoxyhemoglobin, which in the presence of bound RRx-001 reduces nitrite at a much faster rate, leading to markedly increased NO generation. These data expand on the paradigm that hemoglobin generates NO via nitrite reduction during hypoxia and ischemia when nitric oxide synthase (NOS) function is limited. Here, we demonstrate that RRx-001 greatly enhances NO generation from nitrite reduction. RRx-001 is thus the first example of a functional superagonist for nitrite reductase. We hypothesize that physiologically this reaction releases the potentially cytotoxic effector NO selectively in hypoxic tumor regions. It may be that a binary NO-H2O2 trigger is indirectly responsible for the observed tumoricidal activity of RRx-001 since NO is known to inhibit mitochondrial respiration.

The covalent attachment of a 14-carbon aliphatic tail on a glycine residue of nascent translated peptide chains is catalyzed in human cells by two N-myristoyltransferase (NMT) enzymes using the rare myristoyl-CoA (C(14)-CoA) molecule as fatty acid donor. Although, NMT enzymes can only transfer a myristate group, they lack specificity for C(14)-CoA and can also bind the far more abundant palmitoyl-CoA (C(16)-CoA) molecule. We determined that the acyl-CoA binding protein, acyl-CoA binding domain (ACBD)6, stimulated the NMT reaction of NMT2. This stimulatory effect required interaction between ACBD6 and NMT2, and was enhanced by binding of ACBD6 to its ligand, C(18:2)-CoA. ACBD6 also interacted with the second human NMT enzyme, NMT1. The presence of ACBD6 prevented competition of the NMT reaction by C(16)-CoA. Mutants of ACBD6 that were either deficient in ligand binding to the N-terminal ACBD or unable to interact with NMT2 did not stimulate activity of NMT2, nor could they protect the enzyme from utilizing the competitor C(16)-CoA. These results indicate that ACBD6 can locally sequester C(16)-CoA and prevent its access to the enzyme binding site via interaction with NMT2. Thus, the ligand binding properties of the NMT/ACBD6 complex can explain how the NMT reaction can proceed in the presence of the very abundant competitive substrate, C(16)-CoA., (Copyright © 2016 by the American Society for Biochemistry and Molecular Biology, Inc.)

We present a method for sub-population analysis of deformability distribution using single-cell microchamber array (SiCMA) technology. It is a unique method allowing the correlation of overall cellular characteristics with surface and cytosolic characteristics to define the distribution of individual cellular characteristics in heterogeneous cell populations. As a proof of principle, reticulocytes, the immature sub-population of red blood cells (RBC), were recognized from RBC population by a surface marker and different characteristics on deformability between these populations were characterized. The proposed technology can be used in a variety of applications that would benefit from the ability to measure the distribution of cellular characteristics in complex populations, especially important to define hematologic disorders.

The binding determinants of the human acyl-CoA binding domain-containing protein (ACBD) 6 and its function in lipid renewal of membranes were investigated. ACBD6 binds acyl-CoAs of a chain length of 6 to 20 carbons. The stoichiometry of the association could not be fitted to a 1-to-1 model. Saturation of ACBD6 by C16:0-CoA required higher concentration than less abundant acyl-CoAs. In contrast to ACBD1 and ACBD3, ligand binding did not result in the dimerization of ACBD6. The presence of fatty acids affected the binding of C18:1-CoA to ACBD6, dependent on the length, the degree of unsaturation, and the stereoisomeric conformation of their aliphatic chain. ACBD1 and ACBD6 negatively affected the formation of phosphatidylcholine (PC) and phosphatidylethanolamine in the red blood cell membrane. The acylation rate of lysophosphatidylcholine into PC catalyzed by the red cell lysophosphatidylcholine-acyltransferase 1 protein was limited by the transfer of the acyl-CoA substrate from ACBD6 to the acyltransferase enzyme. These findings provide evidence that the binding properties of ACBD6 are adapted to prevent its constant saturation by the very abundant C16:0-CoA and protect membrane systems from the detergent nature of free acyl-CoAs by controlling their release to acyl-CoA-utilizing enzymes., (Copyright © 2015 by the American Society for Biochemistry and Molecular Biology, Inc.)

Pulmonary hypertension (PH) commonly develops in thalassaemia syndromes, but is poorly characterized. The goal of this study was to provide a comprehensive description of the cardiopulmonary and biological profile of patients with thalassaemia at risk for PH. A case-control study of thalassaemia patients at high versus low PH-risk was performed. A single cross-sectional measurement for variables reflecting cardiopulmonary status and biological pathophysiology were obtained, including Doppler-echocardiography, 6-min-walk-test, Borg Dyspnoea Score, New York Heart Association functional class, cardiac magnetic resonance imaging (MRI), chest-computerized tomography, pulmonary function testing and laboratory analyses targeting mechanisms of coagulation, inflammation, haemolysis, adhesion and the arginine-nitric oxide pathway. Twenty-seven thalassaemia patients were evaluated, 14 with an elevated tricuspid-regurgitant-jet-velocity (TRV) ≥ 2·5 m/s. Patients with increased TRV had a higher frequency of splenectomy, and significantly larger right atrial size, left atrial volume and left septal-wall thickness on echocardiography and/or MRI, with elevated biomarkers of abnormal coagulation, lactate dehydrogenase (LDH) levels and arginase concentration, and lower arginine-bioavailability compared to low-risk patients. Arginase concentration correlated significantly to several echocardiography/MRI parameters of cardiovascular function in addition to global-arginine-bioavailability and biomarkers of haemolytic rate, including LDH, haemoglobin and bilirubin. Thalassaemia patients with a TRV ≥ 2·5 m/s have additional echocardiography and cardiac-MRI parameters suggestive of right and left-sided cardiac dysfunction. In addition, low arginine bioavailability may contribute to cardiopulmonary dysfunction in β-thalassaemia., (© 2015 John Wiley & Sons Ltd.)

Background: The survival of malaria parasites, under substantial haem-induced oxidative stress in the red blood cells (RBCs) is dependent on the pentose phosphate pathway (PPP). The PPP is the only source of NADPH in the RBC, essential for the production of reduced glutathione (GSH) and for protection from oxidative stress. Glucose-6-phosphate dehydrogenase (G6PD) deficiency, therefore, increases the vulnerability of erythrocytes to oxidative stress. In Plasmodium, G6PD is combined with the second enzyme of the PPP to create a unique bifunctional enzyme, named glucose-6-phosphate dehydrogenase-6-phosphogluconolactonase (G6PD-6PGL). RRx-001 is a novel, systemically non-toxic, epigenetic anticancer agent currently in Phase 2 clinical development for multiple tumour types, with activity mediated through increased nitric oxide (NO) production and PPP inhibition. The inhibition of G6PD and NO overproduction induced by RRx-001 suggested its application in cerebral malaria (CM)., Methods: Plasmodium berghei ANKA (PbA) infection in C57BL/6 mice is an experimental model of cerebral malaria (ECM) with several similar pathological features to human CM. This study uses intravital microscopy methods with a closed cranial window model to quantify cerebral haemodynamic changes and leukocyte adhesion to endothelial cells in ECM., Results: RRx-001 had both single agent anti-parasitic activity and significantly increased the efficacy of artemether. In addition, RRx-001 preserved cerebral perfusion and reduced inflammation alone or combined with artemether. RRx-001's effects were associated with inhibition of PPP (G6PD and G6PD-6PGL) and by improvements in microcirculatory flow, which may be related to the NO donating properties of RRx-001., Conclusion: The results indicate that RRx-001 could be used to potentiate the anti-malarial action of artemisinin, particularly on resistant strains, and to prevent infection.

The bacterial human pathogen Chlamydia trachomatis invades cells as an infectious elementary body (EB). The EB is internalized into a vacuole that is hidden from the host defense mechanism, and is modified to sustain the development of the replicative reticulate body (RB). Inside this parasitophorous compartment, called the inclusion, the pathogen survives supported by an active exchange of nutrients and proteins with the host cell. We show that host lipids are scavenged and modified into bacterial-specific lipids by the action of a shared human-bacterial acylation mechanism. The bacterial acylating enzymes for the essential lipids 1-acyl-sn-glycerol 3-phosphate and 1-acyl-sn-phosphatidylcholine were identified as CT453 and CT775, respectively. Bacterial CT775 was found to be associated with lipid droplets (LDs). During the development of C. trachomatis, the human acyl-CoA carrier hACBD6 was recruited to cytosolic LDs and translocated into the inclusion. hACBD6 protein modulated the activity of CT775 in an acyl-CoA dependent fashion and sustained the activity of the bacterial acyltransferase by buffering the concentration of acyl-CoAs. We propose that disruption of the binding activity of the acyl-CoA carrier might represent a new drug-target to prevent growth of C. trachomatis., (© 2015 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Aims: Glucose 6-phosphate dehydrogenase (G6PD) is essential for maintenance of nicotinamide dinucleotide hydrogen phosphate (NADPH) levels and redox homeostasis. A number of drugs, such as antimalarial drugs, act to induce reactive oxygen species and hemolytic crisis in G6PD-deficient patients. We used diamide (DIA) to mimic drug-induced oxidative stress and studied how these drugs affect cellular metabolism using a metabolomic approach., Results: There are a few differences in metabolome between red blood cells (RBCs) from normal and G6PD-deficient individuals. DIA causes modest changes in normal RBC metabolism. In contrast, there are significant changes in various biochemical pathways, namely glutathione (GSH) metabolism, purine metabolism, and glycolysis, in G6PD-deficient cells. GSH depletion is concomitant with a shift in energy metabolism. Adenosine monophosphate (AMP) and adenosine diphosphate (ADP) accumulation activates AMP protein kinase (AMPK) and increases entry of glucose into glycolysis. However, inhibition of pyruvate kinase (PK) reduces the efficacy of energy production. Metabolic changes and protein oxidation occurs to a greater extent in G6PD-deficient RBCs than in normal cells, leading to severe irreversible loss of deformability of the former., Innovation and Conclusion: Normal and G6PD-deficient RBCs differ in their responses to oxidants. Normal cells have adequate NADPH regeneration for maintenance of GSH pool. In contrast, G6PD-deficient cells are unable to regenerate enough NADPH under a stressful situation, and switch to biosynthetic pathway for GSH supply. Rapid GSH exhaustion causes energy crisis and futile AMPK activation. Our findings suggest that drug-induced oxidative stress differentially affects metabolism and metabolite signaling in normal and G6PD-deficient cells. It also provides an insight into the pathophysiology of acute hemolytic anemia in G6PD-deficient patients.

Nitric oxide (NO) is a key regulator of vascular tone. Endothelial nitric oxide synthase (eNOS) is responsible for NO generation under normoxic conditions. Under hypoxia however, eNOS is inactive and red blood cells (RBC) provide an alternative NO generation pathway from nitrite to regulate hypoxic vasodilation. While nitrite reductase activity of hemoglobin is well acknowledged, little is known about generation of NO by intact RBC with physiological hemoglobin concentrations. We aimed to develop and apply a new approach to provide insights in the ability of RBC to convert nitrite into NO under hypoxic conditions. We established a novel experimental setup to evaluate nitrite uptake and the release of NO from RBC into the gas-phase under different conditions. NO measurements were similar to well-established clinical measurements of exhaled NO. Nitrite uptake was rapid, and after an initial lag phase NO release from RBC was constant in time under hypoxic conditions. The presence of oxygen greatly reduced NO release, whereas inhibition of eNOS and xanthine oxidoreductase (XOR) did not affect NO release. A decreased pH increased NO release under hypoxic conditions. Hypothermia lowered NO release, while hyperthermia increased NO release. Whereas fetal hemoglobin did not alter NO release compared to adult hemoglobin, sickle RBC showed an increased ability to release NO. Under all conditions nitrite uptake by RBC was similar. This study shows that nitrite uptake into RBC is rapid and release of NO into the gas-phase continues for prolonged periods of time under hypoxic conditions. Changes in the RBC environment such as pH, temperature or hemoglobin type, affect NO release.

Different pathways lead from the simple point mutation in hemoglobin to the membrane changes that characterize the altered interaction of the sickle red blood cell with its environment, including endothelial cells, white blood cells, and platelets. Polymerization and oxidation-induced damage to both lipid and protein components of the red cell membrane, as well as the generation of bioreactive membrane material (microparticles), has a profound effect on all tissues and organs, and defines the vasculopathy of the patient with sickle cell disease., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Biomarkers of chronic cell hydration status are needed to determine whether chronic hyperosmotic stress increases chronic disease risk in population-representative samples. In vitro, cells adapt to chronic hyperosmotic stress by upregulating protein breakdown to counter the osmotic gradient with higher intracellular amino acid concentrations. If cells are subsequently exposed to hypo-osmotic conditions, the adaptation results in excess cell swelling and/or efflux of free amino acids. This study explored whether increased red blood cell (RBC) swelling and/or plasma or urine amino acid concentrations after hypo-osmotic challenge might be informative about relative chronic hyperosmotic stress in free-living men. Five healthy men (20-25 years) with baseline total water intake below 2 L/day participated in an 8-week clinical study: four 2-week periods in a U-shaped A-B-C-A design. Intake of drinking water was increased by +0.8 ± 0.3 L/day in period 2, and +1.5 ± 0.3 L/day in period 3, and returned to baseline intake (0.4 ± 0.2 L/day) in period 4. Each week, fasting blood and urine were collected after a 750 mL bolus of drinking water, following overnight water restriction. The periods of higher water intake were associated with significant decreases in RBC deformability (index of cell swelling), plasma histidine, urine arginine, and urine glutamic acid. After 4 weeks of higher water intake, four out of five participants had ½ maximal RBC deformability below 400 mmol/kg; plasma histidine below 100 μmol/L; and/or undetectable urine arginine and urine glutamic acid concentrations. Work is warranted to pursue RBC deformability and amino acid concentrations after hypo-osmotic challenge as possible biomarkers of chronic cell hydration.

Pulmonary hypertension is a common but often overlooked complication associated with thalassemia syndromes. There are limited data on the safety and efficacy of selective pulmonary vasodilators in this at-risk population. We, therefore, designed a 12-week, open-label, phase 1/2, pilot-scale, proof-of-principle trial of sildenafil therapy in 10 patients with β-thalassemia and at increased risk of pulmonary hypertension based on an elevated tricuspid regurgitant jet velocity >2.5 m/s on Doppler-echocardiography. Variables compared at baseline and after 12 weeks of sildenafil treatment included Doppler-echocardiographic parameters, 6-minute walked distance, Borg Dyspnea Score, New York Heart Association functional class, pulmonary function, and laboratory parameters. Treatment with sildenafil resulted in a significant decrease in tricuspid regurgitant jet velocity by 13.3% (3.0±0.7 versus 2.6±0.5 m/s, P=0.04), improved left ventricular end systolic/diastolic volume, and a trend towards a improved New York Heart Association functional class. No significant change in 6-minute walked distance was noted. Sildenafil was well tolerated, although minor expected adverse events were commonly reported. The total dose of sildenafil (mg) was strongly correlated with percent change in nitric oxide metabolite concentration in the plasma (ρ=0.80, P=0.01). There were also significant increases in plasma and erythrocyte arginine concentrations. Our study suggests that sildenafil is safe and may improve pulmonary hemodynamics in patients at risk of pulmonary hypertension; however, it was not demonstrated to improve the distance walked in 6 minutes. Clinical trials are needed to identify the best treatment strategy for pulmonary hypertension in patients with β-thalassemia. (clinicaltrials.gov identifier: NCT00872170).

Painful episodes of vaso-occlusion are the leading cause of hospitalizations and emergency department visits in sickle cell disease, and are associated with increased mortality. Low nitric oxide bioavailability contributes to vasculopathy in sickle cell disease. Since arginine is the obligate substrate for nitric oxide production, and an acute deficiency is associated with pain, we hypothesized that arginine may be a beneficial treatment for pain related to sickle cell disease. Thirty-eight children with sickle cell disease hospitalized for 56 episodes of pain were randomized into this double-blinded placebo-controlled trial. Patients received L-arginine (100 mg/kg tid) or placebo for 5 days or until discharge. A significant reduction in total parenteral opioid use by 54% (1.9 ± 2.0 mg/kg versus 4.1 ± 4.1 mg/kg, P=0.02) and lower pain scores at discharge (1.9 ± 2.4 versus 3.9 ± 2.9, P=0.01) were observed in the treatment arm compared to the placebo one. There was no significant difference in hospital length of stay (4.1 ± 01.8 versus 4.8 ± 2.5 days, P=0.34), although a trend favored the arginine arm, and total opioid use was strongly correlated with the duration of the admission (r=0.86, P<0.0001). No drug-related adverse events were observed. Arginine therapy represents a novel intervention for painful vaso-occlusive episodes. A reduction of narcotic use by >50% is remarkable. Arginine is a safe and inexpensive intervention with narcotic-sparing effects that may be a beneficial adjunct to standard therapy for sickle cell-related pain in children. A large multi-center trial is warranted in order to confirm these observations.

Background: Unsaturated fatty acids are susceptible to oxidation and damaged chains are removed from glycerophospholipids by phospholipase A(2). De-acylated lipids are then re-acylated by lysophospholipid acyltransferase enzymes such as LPCAT1 which catalyses the formation of phosphatidylcholine (PC) from lysoPC and long-chain acyl-CoA., Results: Activity of LPCAT1 is inhibited by Ca(2+), and a Ca(2+)-binding motif of the EF-hand type, EFh-1, was identified in the carboxyl-terminal domain of the protein. The residues Asp-392 and Glu-403 define the loop of the hairpin structure formed by EFh-1. Substitution of D(392) and E(403) to alanine rendered an enzyme insensitive to Ca(2+), which established that Ca(2+) binding to that region negatively regulates the activity of the acyltransferase amino-terminal domain. Residue Cys-211 of the conserved motif III is not essential for catalysis and not sufficient for sensitivity to treatment by sulfhydryl-modifier agents. Among the several active cysteine-substitution mutants of LPCAT1 generated, we identified one to be resistant to treatment by sulfhydryl-alkylating and sulfhydryl-oxidizer agents., Conclusion: Mutant forms of LPCAT1 that are not inhibited by Ca(2+) and sulfhydryl-alkylating and -oxidizing agents will provide a better understanding of the physiological function of a mechanism that places the formation of PC, and the disposal of the bioactive species lysoPC, under the control of the redox status and Ca(2+) concentration of the cell.

We describe human chorionic mesenchymal stem cell (hCMSC) lines obtained from the chorion of human term placenta with high therapeutic potential in human organ pathology. hCMSCs propagated for more than 100 doublings without a decrease in telomere length and with no telomerase activity. Cells were highly positive for the embryonic stem cell markers OCT-4, NANOG, SSEA-3, and TRA-1-60. In vitro, cells could be differentiated into neuron-like cells (ectoderm), adipocytes, osteoblasts, endothelial-like cells (mesoderm), and hepatocytes (endoderm)-derivatives of all three germ layers. hCMSCs effectively facilitated repair of injured epithelium as demonstrated in an ex vivo-perfused human lung preparation injured by Escherichia coli endotoxin and in in vitro human lung epithelial cultures. We conclude that the chorion of human term placenta is an abundant source of multipotent stem cells that are promising candidates for cell-based therapies.