[Determination of 24 elements migration in Yixing clay pottery by inductively coupled plasma mass spectrometry].

OBJECTIVE: To establish an analytical method for determining the migration of 24 elements in Yixing clay pottery in 4% acetic acid simulated solution by inductively coupled plasma mass spectrometry. METHODS: Four types of Yixing clay pottery, including Yixing clay teapot, Yixing clay kettle, Yixing clay pot, and Yixing clay electric stew pot, were immersed in 4% acetic acid as a food simulant for testing. The migration amount of 24 elements in the migration solution was determined using inductively coupled plasma mass spectrometry. RESULTS: Lithium, magnesium, aluminum, iron, and barium elements with a mass concentration of 1000 µg/L; Lead, cadmium, total arsenic, chromium, nickel, copper, vanadium, manganese, antimony, tin, zinc, cobalt, molybdenum, silver, beryllium, thallium, titanium, and strontium elements within 100 µg/L there was a linear relationship within, the r value was between 0.998 739 and 0.999 989. Total mercury at 5.0 µg/L, there was a linear relationship within, the r value of 0.995 056. The detection limit of the elements measured by this method was between 0.5 and 45.0 µg/L, the recovery rate was 80.6%-108.9%, and the relative standard deviation was 1.0%-4.8%(n=6). A total of 32 samples of four types of Yixing clay pottery sold on the market, including teapots, boiling kettles, casseroles, and electric stewing pots, were tested. It was found that the migration of 16 elements, including beryllium, titanium, chromium, nickel, cobalt, zinc, silver, cadmium, antimony, total mercury, thallium, tin, copper, total arsenic, molybdenum, and lead, were lower than the quantitative limit. The element with the highest migration volume teapot was aluminum, magnesium, and barium; The kettle was aluminum and magnesium; Casserole was aluminum, magnesium, and lithium; The electric stew pot was aluminum. CONCLUSION: This method is easy to operate and has high accuracy, providing an effective and feasible detection method for the determination and evaluation of element migration in Yixing clay pottery.

Enhanced stability and tunable photoluminescence in Mn2+-doped one-dimensional hybrid lead halide perovskites for high-performance white light emitting diodes.

Organic-inorganic hybrid low-dimensional lead halides have garnered significant interest in the realm of solid-state optical materials due to their unique properties and potential applications. In this study, we report the synthesis, characterization and application of Mn2+-doped one-dimensional (1D) [AEP]PbCl5·H2O hybrid lead halide perovskites with tunable photoluminescence properties. The Mn2+ doping leads to a redshift of the dominant emission wavelength from 463 nm to 630 nm, with the optimal doping concentration resulting in an enhanced photoluminescence quantum yield (PLQY) from less than 1% to 8.96%. The structural and optical stability of these doped perovskites have been thoroughly investigated revealing excellent performance under humid and high-temperature conditions. Perovskite-PVP composite films exhibit high crystallization and bright orange-red emission under UV excitation. Furthermore, we demonstrate the successful fabrication of a white LED device using the Mn2+-doped perovskite in combination with commercial green and blue phosphors. The fabricated LED exhibits a high color rendering index (CRI) of 87.2 and stable electroluminescence performance under various operating currents and extended operation times. Our findings highlight the potential of Mn2+-doped 1D hybrid lead halide perovskites as efficient and stable phosphors for high-performance white light emitting diodes and other optoelectronic applications.

Lignin decolorization in organic solvents and their application in natural sunscreen.

In order to improve the utilization of industrial lignin as an effective component for ultraviolet (UV) shielding, organic solvent (methanol, ethanol, and acetone) fractionation was applied to improve its UV absorption performance and reduce its apparent color. Physicochemical properties of lignin and lignin-based sunscreens, such as molar mass fraction, functional group content, color change and UV shielding properties, were characterized in detail by GPC, UV spectroscopy, 31P NMR and HSQC-NMR spectroscopy. The results showed that the color and UV-shielding properties of the soluble fraction were significantly superior to those of the original and insoluble fractions. Different lignin fractions were acted as the only active substance in the pure cream and its UV-shielding properties were compared. Among them, the composite sunscreen by adding 5 wt% acetone fractionated lignin had highest sun protection factor (SPF) value of 6.6, approximately 4.5 times higher than those sunscreens mixed with pristine lignin. Overall, this work offers the potential of industrial lignin in value-added applications such as UV protection and cosmetics.

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

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

O-GlcNAc modification mediates aquaporin 3 to coordinate endometrial cell glycolysis and affects embryo implantation.

Introduction: O-linked ß-D-N-acetylglucosamine (O-GlcNAc) modification is a post-translational modification in which a single O-GlcNAc is added to serine or threonine residues in nuclear, cytoplasmic, and mitochondrial proteins, and is involved in a variety of physiological processes. Objectives: In the present study, the role of O-GlcNAcylation in embryo implantation was evaluated. Furthermore, whether O-GlcNAcylation is involved in orchestrating glucose metabolism to influence endometrial cell physiological functions was investigated. Methods: Different endometrial tissues were detected using immunohistochemistry. Pregnant mouse models were established to verify molecular expression. O-GlcNAc transferase and aquaporin 3 (AQP3) knockdown were used to detect embryo implantation efficiency in vitro and in vivo. Western blotting and immunofluorescence were used to detect protein expression and stability. Dual luciferase reporter assay and chromatin immunoprecipitation (ChIP) were used to verify the binding transcription factor. Glycolysis was detected using bioenergy analyzer, and metabolites were analyzed using isotope 13C-labeled LC-MS. Metabolic-related genes were determined using RNA sequencing. Results: Activation of endometrial hexosamine biosynthetic pathway (HBP) caused elevated O-GlcNAcylation during the window of implantation, affecting endometrial cell function and embryo implantation. Specifically, elevated O-GlcNAcylation increased glucose uptake via glucose transporter 1 (GLUT1) leading to glucose metabolic flow into the pentose phosphate pathways and HBP, which regulate the metabolic reprogramming of endometrial cells. Furthermore, O-GlcNAcylation mediated the intracellular transport of glycerol to support and compensate for glycolysis through regulation of AQP3. Unexpectedly, elevated AQP3 also increased glucose uptake via GLUT1. These processes maintained higher metabolic requirements for endometrial physiology. Furthermore, the transcription factor SP1 specifically bound to the AQP3 promoter region, and O-GlcNAcylation of SP1 increased its stability and transcriptional regulation of AQP3 which is associated with O-GlcNAcylation of SP1. Conclusion: Overall, O-GlcNAcylation regulated glucose metabolism in endometrial cells, and AQP3-mediated compensation provides new insights into the communication between glycolysis and O-GlcNAcylation.

[Application of different pretreatment methods in the determination of aluminum in reference substances].

OBJECTIVE: The pretreatment method was studied to solve the disagreement of aluminum in the reference substances between detection and the value of the certificate. METHODS: The pretreatment method of wet digestion with nitric acid, microwave digestion of hydrogen fluoride and dry ashing at different temperatures were used to determine aluminum in the reference substances by plasma emission spectrometry. RESULTS: Pretreatment method affected the test result of aluminum in reference substances directly. The result showed that wet digestion with nitric acid and dry ashing at 500-550 ℃ could not reach the value of the certificate of reference substances, meanwhile microwave digestion of hydrogen fluoride( HNO_3-HF-H_2O_2= 8 : 1 : 1, V/V/V) and dry ashing at more higher temperature( 800-950 ℃) could meet the testing requirements. CONCLUSION: The determination of aluminum in reference substances is recommended by hydrofluoric acid system digestion or higher temperature ashing method to ensure the accuracy of the test result.

Hypoxia modulates the purine salvage pathway and decreases red blood cell and supernatant levels of hypoxanthine during refrigerated storage.

Hypoxanthine catabolism in vivo is potentially dangerous as it fuels production of urate and, most importantly, hydrogen peroxide. However, it is unclear whether accumulation of intracellular and supernatant hypoxanthine in stored red blood cell units is clinically relevant for transfused recipients. Leukoreduced red blood cells from glucose-6-phosphate dehydrogenase-normal or -deficient human volunteers were stored in AS-3 under normoxic, hyperoxic, or hypoxic conditions (with oxygen saturation ranging from <3% to >95%). Red blood cells from healthy human volunteers were also collected at sea level or after 1-7 days at high altitude (>5000 m). Finally, C57BL/6J mouse red blood cells were incubated in vitro with 13C1-aspartate or 13C5-adenosine under normoxic or hypoxic conditions, with or without deoxycoformycin, a purine deaminase inhibitor. Metabolomics analyses were performed on human and mouse red blood cells stored for up to 42 or 14 days, respectively, and correlated with 24 h post-transfusion red blood cell recovery. Hypoxanthine increased in stored red blood cell units as a function of oxygen levels. Stored red blood cells from human glucose-6-phosphate dehydrogenase-deficient donors had higher levels of deaminated purines. Hypoxia in vitro and in vivo decreased purine oxidation and enhanced purine salvage reactions in human and mouse red blood cells, which was partly explained by decreased adenosine monophosphate deaminase activity. In addition, hypoxanthine levels negatively correlated with post-transfusion red blood cell recovery in mice and - preliminarily albeit significantly - in humans. In conclusion, hypoxanthine is an in vitro metabolic marker of the red blood cell storage lesion that negatively correlates with post-transfusion recovery in vivo Storage-dependent hypoxanthine accumulation is ameliorated by hypoxia-induced decreases in purine deamination reaction rates.

Metabolism of Citrate and Other Carboxylic Acids in Erythrocytes As a Function of Oxygen Saturation and Refrigerated Storage.

State-of-the-art proteomics technologies have recently helped to elucidate the unanticipated complexity of red blood cell metabolism. One recent example is citrate metabolism, which is catalyzed by cytosolic isoforms of Krebs cycle enzymes that are present and active in mature erythrocytes and was determined using quantitative metabolic flux analysis. In previous studies, we reported significant increases in glycolytic fluxes in red blood cells exposed to hypoxia in vitro or in vivo, an observation relevant to transfusion medicine owing to the potential benefits associated with hypoxic storage of packed red blood cells. Here, using a combination of steady state and quantitative tracing metabolomics experiments with 13C1,2,3-glucose, 13C6-citrate, 13C515N2-glutamine, and 13C1-aspartate via ultra-high performance liquid chromatography coupled on line with mass spectrometry, we observed that hypoxia in vivo and in vitro promotes consumption of citrate and other carboxylates. These metabolic reactions are theoretically explained by the activity of cytosolic malate dehydrogenase 1 and isocitrate dehydrogenase 1 (abundantly represented in the red blood cell proteome), though moonlighting functions of additional enzymes cannot be ruled out. These observations enhance understanding of red blood cell metabolic responses to hypoxia, which could be relevant to understand systemic physiological and pathological responses to high altitude, ischemia, hemorrhage, sepsis, pulmonary hypertension, or hemoglobinopathies. Results from this study will also inform the design and testing of novel additive solutions that optimize red blood cell storage under oxygen-controlled conditions.

Structural and Functional Insight of Sphingosine 1-Phosphate-Mediated Pathogenic Metabolic Reprogramming in Sickle Cell Disease.

Elevated sphingosine 1-phosphate (S1P) is detrimental in Sickle Cell Disease (SCD), but the mechanistic basis remains obscure. Here, we report that increased erythrocyte S1P binds to deoxygenated sickle Hb (deoxyHbS), facilitates deoxyHbS anchoring to the membrane, induces release of membrane-bound glycolytic enzymes and in turn switches glucose flux towards glycolysis relative to the pentose phosphate pathway (PPP). Suppressed PPP causes compromised glutathione homeostasis and increased oxidative stress, while enhanced glycolysis induces production of 2,3-bisphosphoglycerate (2,3-BPG) and thus increases deoxyHbS polymerization, sickling, hemolysis and disease progression. Functional studies revealed that S1P and 2,3-BPG work synergistically to decrease both HbA and HbS oxygen binding affinity. The crystal structure at 1.9 Å resolution deciphered that S1P binds to the surface of 2,3-BPG-deoxyHbA and causes additional conformation changes to the T-state Hb. Phosphate moiety of the surface bound S1P engages in a highly positive region close to α1-heme while its aliphatic chain snakes along a shallow cavity making hydrophobic interactions in the "switch region", as well as with α2-heme like a molecular "sticky tape" with the last 3-4 carbon atoms sticking out into bulk solvent. Altogether, our findings provide functional and structural bases underlying S1P-mediated pathogenic metabolic reprogramming in SCD and novel therapeutic avenues.

Red blood cell ß-adrenergic receptors contribute to diet-induced energy expenditure by increasing O2 supply.

Diet-induced obesity (DIO) represents the major cause for the current obesity epidemic, but the mechanism underlying DIO is unclear. ß-Adrenergic receptors (ß-ARs) play a major role in sympathetic nervous system-mediated (SNS-mediated) diet-induced energy expenditure (EE). Rbc express abundant ß-ARs; however, a potential role for rbc in DIO remains untested. Here, we demonstrated that high-fat, high-caloric diet (HFD) feeding increased both EE and blood O2 content, and the HFD-induced increases in blood O2 level and in body weight gain were negatively correlated. Deficiency of ß-ARs in rbc reduced glycolysis and ATP levels, diminished HFD-induced increases in both blood O2 content and EE, and resulted in DIO. Importantly, specific activation of cAMP signaling in rbc promoted HFD-induced EE and reduced HFD-induced tissue hypoxia independent of obesity. Both HFD and pharmacological activation cAMP signaling in rbc led to increased glycolysis and ATP levels. These results identify a previously unknown role for rbc ß-ARs in mediating the SNS action on HFD-induced EE by increasing O2 supply, and they demonstrate that HFD-induced EE is limited by blood O2 availability and can be augenmented by increased O2 supply.

Erythrocyte purinergic signaling components underlie hypoxia adaptation.

Erythrocytes are vital to human adaptation under hypoxic conditions because of their abundance in number and irreplaceable function of delivering oxygen (O2). However, although multiple large-scale altitude studies investigating the overall coordination of the human body for hypoxia adaptation have been conducted, detailed research with a focus on erythrocytes was missing due to lack of proper techniques. The recently maturing metabolomics profiling technology appears to be the answer to this limitation. Metabolomics profiling provides unbiased high-throughput screening data that reveal the overall metabolic status of erythrocytes. Recent studies have exploited this new technology and provided novel insight into erythrocyte physiology and pathology. In particular, a series of studies focusing on erythrocyte purinergic signaling have reported that adenosine signaling, coupled with 5' AMP-activated protein kinase (AMPK) and the production of erythrocyte-enriched bioactive signaling lipid sphingosine 1-phosphate, regulate erythrocyte glucose metabolism for more O2 delivery. Moreover, an adenosine-dependent "erythrocyte hypoxic memory" was discovered that provides an explanation for fast acclimation upon re-ascent. These findings not only shed new light on our understanding of erythrocyte function and hypoxia adaptation, but also offer a myriad of novel therapeutic possibilities to counteract various hypoxic conditions.

Elevated Adenosine Induces Placental DNA Hypomethylation Independent of A2B Receptor Signaling in Preeclampsia.

Preeclampsia is a prevalent pregnancy hypertensive disease with both maternal and fetal morbidity and mortality. Emerging evidence indicates that global placental DNA hypomethylation is observed in patients with preeclampsia and is linked to altered gene expression and disease development. However, the molecular basis underlying placental epigenetic changes in preeclampsia remains unclear. Using 2 independent experimental models of preeclampsia, adenosine deaminase-deficient mice and a pathogenic autoantibody-induced mouse model of preeclampsia, we demonstrate that elevated placental adenosine not only induces hallmark features of preeclampsia but also causes placental DNA hypomethylation. The use of genetic approaches to express an adenosine deaminase minigene specifically in placentas, or adenosine deaminase enzyme replacement therapy, restored placental adenosine to normal levels, attenuated preeclampsia features, and abolished placental DNA hypomethylation in adenosine deaminase-deficient mice. Genetic deletion of CD73 (an ectonucleotidase that converts AMP to adenosine) prevented the elevation of placental adenosine in the autoantibody-induced preeclampsia mouse model and ameliorated preeclampsia features and placental DNA hypomethylation. Immunohistochemical studies revealed that elevated placental adenosine-mediated DNA hypomethylation predominantly occurs in spongiotrophoblasts and labyrinthine trophoblasts and that this effect is independent of A2B adenosine receptor activation in both preeclampsia models. Extending our mouse findings to humans, we used cultured human trophoblasts to demonstrate that adenosine functions intracellularly and induces DNA hypomethylation without A2B adenosine receptor activation. Altogether, both mouse and human studies reveal novel mechanisms underlying placental DNA hypomethylation and potential therapeutic approaches for preeclampsia.

Purinergic control of red blood cell metabolism: novel strategies to improve red cell storage quality.

Transfusion of stored blood is regarded as one of the great advances in modern medicine. However, during storage in the blood bank, red blood cells (RBCs) undergo a series of biochemical and biomechanical changes that affect cell morphology and physiology and potentially impair transfusion safety and efficacy. Despite reassuring evidence from clinical trials, it is universally accepted that the storage lesion(s) results in the altered physiology of long-stored RBCs and helps explain the rapid clearance of up to one-fourth of long-stored RBCs from the recipient's bloodstream at 24 hours after administration. These considerations explain the importance of understanding and mitigating the storage lesion. With the emergence of new technologies that have enabled large-scale and in-depth screening of the RBC metabolome and proteome, recent studies have provided novel insights into the molecule-level metabolic changes underpinning the accumulation of storage lesions to RBCs in the blood bank and alternative storage strategies to mitigate such lesion(s). These approaches borrow from recent insights on the biochemistry of RBC adaptation to high altitude hypoxia. We recently conducted investigations in genetically modified mice and revealed novel insights into the role of adenosine signalling in response to hypoxia as a previously unrecognised cascade regulating RBC glucose metabolism and increasing O2 release, while decreasing inflammation and tissue injuries in animal models. Here, we will discuss the molecular mechanisms underlying the role of purinergic molecules, including adenosine and adenosine triphosphate in manipulating RBCs and blood vessels in response to hypoxia. We will also speculate about new therapeutic possibilities to improve the quality of stored RBCs and the prognosis after transfusion.

Iron overload correlates with serum liver fibrotic markers and liver dysfunction: Potential new methods to predict iron overload-related liver fibrosis in thalassemia patients.

BACKGROUND: Early detection of liver fibrosis in thalassemia patients and rapid initiation of treatment to interfere with its progression are extremely important. OBJECTIVE: This study aimed to find a sensitive, easy-to-detect and noninvasive method other than liver biopsy for early detection of liver fibrosis in thalassemia patients. METHODS: A total of 244 Chinese Thalassemia patients with non-transfusion-dependent thalassemia (NTDT, n = 105) or thalassemia major (TM, n = 139) and 120 healthy individuals were recruited into the present study, and blood collagen type IV (C IV), precollagen type III (PIIINPC) and hyaluronic acid (HA), aspartate aminotransferase (AST), alanine aminotransferase (ALT) and ferritin were measured. Liver iron concentration was determined by MRI. The correlation of serum markers with liver iron load and liver function was evaluated. RESULTS: Serum C IV, PIIINPC and HA were significantly elevated in Chinese patients with NTDT and further elevated in TM patients. Moreover, C IV, PIIINPC and HA were also positively correlated to serum ferritin and liver iron concentration and further elevated during the progression to multi-organ damage in NTDT patients. Finally, serum ferritin and liver iron concentration were significantly correlated with liver dysfunction determined by AST and ALT. CONCLUSION: Taken together, our results indicate that monitoring serum C IV, PIIINPC and HA is a potentially sensitive method to predict the risks for iron overload-related liver fibrosis in Chinese thalassemia patients.

Erythrocytes retain hypoxic adenosine response for faster acclimatization upon re-ascent.

Faster acclimatization to high altitude upon re-ascent is seen in humans; however, the molecular basis for this enhanced adaptive response is unknown. We report that in healthy lowlanders, plasma adenosine levels are rapidly induced by initial ascent to high altitude and achieved even higher levels upon re-ascent, a feature that is positively associated with quicker acclimatization. Erythrocyte equilibrative nucleoside transporter 1 (eENT1) levels are reduced in humans at high altitude and in mice under hypoxia. eENT1 deletion allows rapid accumulation of plasma adenosine to counteract hypoxic tissue damage in mice. Adenosine signalling via erythrocyte ADORA2B induces PKA phosphorylation, ubiquitination and proteasomal degradation of eENT1. Reduced eENT1 resulting from initial hypoxia is maintained upon re-ascent in humans or re-exposure to hypoxia in mice and accounts for erythrocyte hypoxic memory and faster acclimatization. Our findings suggest that targeting identified purinergic-signalling network would enhance the hypoxia adenosine response to counteract hypoxia-induced maladaptation.

AltitudeOmics: Red Blood Cell Metabolic Adaptation to High Altitude Hypoxia.

Red blood cells (RBCs) are key players in systemic oxygen transport. RBCs respond to in vitro hypoxia through the so-called oxygen-dependent metabolic regulation, which involves the competitive binding of deoxyhemoglobin and glycolytic enzymes to the N-terminal cytosolic domain of band 3. This mechanism promotes the accumulation of 2,3-DPG, stabilizing the deoxygenated state of hemoglobin, and cytosol acidification, triggering oxygen off-loading through the Bohr effect. Despite in vitro studies, in vivo adaptations to hypoxia have not yet been completely elucidated. Within the framework of the AltitudeOmics study, erythrocytes were collected from 21 healthy volunteers at sea level, after exposure to high altitude (5260 m) for 1, 7, and 16 days, and following reascent after 7 days at 1525 m. UHPLC-MS metabolomics results were correlated to physiological and athletic performance parameters. Immediate metabolic adaptations were noted as early as a few hours from ascending to >5000 m, and maintained for 16 days at high altitude. Consistent with the mechanisms elucidated in vitro, hypoxia promoted glycolysis and deregulated the pentose phosphate pathway, as well purine catabolism, glutathione homeostasis, arginine/nitric oxide, and sulfur/H2S metabolism. Metabolic adaptations were preserved 1 week after descent, consistently with improved physical performances in comparison to the first ascendance, suggesting a mechanism of metabolic memory.

Beneficial Role of Erythrocyte Adenosine A2B Receptor-Mediated AMP-Activated Protein Kinase Activation in High-Altitude Hypoxia.

BACKGROUND: High altitude is a challenging condition caused by insufficient oxygen supply. Inability to adjust to hypoxia may lead to pulmonary edema, stroke, cardiovascular dysfunction, and even death. Thus, understanding the molecular basis of adaptation to high altitude may reveal novel therapeutics to counteract the detrimental consequences of hypoxia. METHODS: Using high-throughput, unbiased metabolomic profiling, we report that the metabolic pathway responsible for production of erythrocyte 2,3-bisphosphoglycerate (2,3-BPG), a negative allosteric regulator of hemoglobin-O2 binding affinity, was significantly induced in 21 healthy humans within 2 hours of arrival at 5260 m and further increased after 16 days at 5260 m. RESULTS: This finding led us to discover that plasma adenosine concentrations and soluble CD73 activity rapidly increased at high altitude and were associated with elevated erythrocyte 2,3-BPG levels and O2 releasing capacity. Mouse genetic studies demonstrated that elevated CD73 contributed to hypoxia-induced adenosine accumulation and that elevated adenosine-mediated erythrocyte A2B adenosine receptor activation was beneficial by inducing 2,3-BPG production and triggering O2 release to prevent multiple tissue hypoxia, inflammation, and pulmonary vascular leakage. Mechanistically, we demonstrated that erythrocyte AMP-activated protein kinase was activated in humans at high altitude and that AMP-activated protein kinase is a key protein functioning downstream of the A2B adenosine receptor, phosphorylating and activating BPG mutase and thus inducing 2,3-BPG production and O2 release from erythrocytes. Significantly, preclinical studies demonstrated that activation of AMP-activated protein kinase enhanced BPG mutase activation, 2,3-BPG production, and O2 release capacity in CD73-deficient mice, in erythrocyte-specific A2B adenosine receptor knockouts, and in wild-type mice and in turn reduced tissue hypoxia and inflammation. CONCLUSIONS: Together, human and mouse studies reveal novel mechanisms of hypoxia adaptation and potential therapeutic approaches for counteracting hypoxia-induced tissue damage.

Sphingosine-1-phosphate promotes erythrocyte glycolysis and oxygen release for adaptation to high-altitude hypoxia.

Sphingosine-1-phosphate (S1P) is a bioactive signalling lipid highly enriched in mature erythrocytes, with unknown functions pertaining to erythrocyte physiology. Here by employing nonbiased high-throughput metabolomic profiling, we show that erythrocyte S1P levels rapidly increase in 21 healthy lowland volunteers at 5,260 m altitude on day 1 and continue increasing to 16 days with concurrently elevated erythrocyte sphingonisne kinase 1 (Sphk1) activity and haemoglobin (Hb) oxygen (O2) release capacity. Mouse genetic studies show that elevated erythrocyte Sphk1-induced S1P protects against tissue hypoxia by inducing O2 release. Mechanistically, we show that intracellular S1P promotes deoxygenated Hb anchoring to the membrane, enhances the release of membrane-bound glycolytic enzymes to the cytosol, induces glycolysis and thus the production of 2,3-bisphosphoglycerate (2,3-BPG), an erythrocyte-specific glycolytic intermediate, which facilitates O2 release. Altogether, we reveal S1P as an intracellular hypoxia-responsive biolipid promoting erythrocyte glycolysis, O2 delivery and thus new therapeutic opportunities to counteract tissue hypoxia.

Hypoxia-mediated impaired erythrocyte Lands' Cycle is pathogenic for sickle cell disease.

Although Lands' cycle was discovered in 1958, its function and cellular regulation in membrane homeostasis under physiological and pathological conditions remain largely unknown. Nonbiased high throughput metabolomic profiling revealed that Lands' cycle was impaired leading to significantly elevated erythrocyte membrane lysophosphatidylcholine (LysoPC) content and circulating and erythrocyte arachidonic acid (AA) in mice with sickle cell disease (SCD), a prevalent hemolytic genetic disorder. Correcting imbalanced Lands' cycle by knockdown of phospholipase 2 (cPLA2) or overexpression of lysophosphatidycholine acyltransferase 1 (LPCAT1), two key enzymes of Lands' cycle in hematopoietic stem cells, reduced elevated erythrocyte membrane LysoPC content and circulating AA levels and attenuated sickling, inflammation and tissue damage in SCD chimeras. Human translational studies validated SCD mouse findings and further demonstrated that imbalanced Lands' cycle induced LysoPC production directly promotes sickling in cultured mouse and human SCD erythrocytes. Mechanistically, we revealed that hypoxia-mediated ERK activation underlies imbalanced Lands' cycle by preferentially inducing the activity of PLA2 but not LPCAT in human and mouse SCD erythrocytes. Overall, our studies have identified a pathological role of imbalanced Lands' cycle in SCD erythrocytes, novel molecular basis regulating Lands' cycle and therapeutic opportunities for the disease.

Sustained Elevated Adenosine via ADORA2B Promotes Chronic Pain through Neuro-immune Interaction.

The molecular mechanisms of chronic pain are poorly understood and effective mechanism-based treatments are lacking. Here, we report that mice lacking adenosine deaminase (ADA), an enzyme necessary for the breakdown of adenosine, displayed unexpected chronic mechanical and thermal hypersensitivity due to sustained elevated circulating adenosine. Extending from Ada(-/-) mice, we further discovered that prolonged elevated adenosine contributed to chronic pain behaviors in two additional independent animal models: sickle cell disease mice, a model of severe pain with limited treatment, and complete Freund's adjuvant paw-injected mice, a well-accepted inflammatory model of chronic pain. Mechanistically, we revealed that activation of adenosine A2B receptors on myeloid cells caused nociceptor hyperexcitability and promoted chronic pain via soluble IL-6 receptor trans-signaling, and our findings determined that prolonged accumulated circulating adenosine contributes to chronic pain by promoting immune-neuronal interaction and revealed multiple therapeutic targets.