Hereditary xerocytosis (HX, also known as Dehydrated Stomatocytosis) is an autosomal dominant hemolytic anemia characterized by dehydrated erythrocytes with increased cation leak and decreased osmotic fragility. HX is caused by heterozygous mutations in the PIEZO1 or KCNN4 genes. Most HX mutations are missense mutations with mutation-specific gain-of-function properties in heterologous expression assays and in affected erythrocytes. The nM potency KCa3.1 inhibitor, senicapoc, has been tested in humans with sickle cell disease and several lung diseases. Oral administration of senicapoc has achieved KCa3.1 target engagement across multiple animal species, in multiple mouse models of human disease, and in humans without significant drug toxicity. We hypothesized that patients with HX attributed to KCa3.1 gain-of-function would benefit from senicapoc treatment. We therefore undertook an exploratory clinical trial of senicapoc in four HX patients of a previously studied single family, each heterozygous for KCa3.1 variant V282M. Senicapoc treatment lowered serum bilirubin in all four subjects, but lowered reticulocyte count in only one subject. None of the four subjects met the endpoints for increased blood hemoglobin content, decreased serum LDH, decreased MCHC or improved serum haptoglobin, despite LC/MS-confirmed compliance. Indeed, revisiting electrophysiological analysis demonstrated greatly reduced senicapoc sensitivity of the KCa3.1 HX variant V282M. We conclude that senicapoc treatment did not improve anemia in four members of a single family with HX attributed to the heterozygous KCa3.1 variant V282M, consistent with reduced sensitivity of this channel pore mutant to inhibition by senicapoc. (ClinicalTrials.gov ID# NCT04372498).

The 1,4-dihydropyridines, drugs with well-established bioavailability and toxicity profiles, have proven efficacy in treating human hypertension, peripheral vascular disorders, and coronary artery disease. Every 1,4-dihydropyridine in clinical use blocks L-type voltage-gated calcium channels. We now report our development, using selective optimization of a side activity (SOSA), of a class of 1,4-dihydropyridines that selectively and potently inhibit the intermediate-conductance calcium-activated K+ channel KCa3.1, a validated therapeutic target for diseases affecting many organ systems. One of these 1,4-dihydropyridines, DHP-103, blocked KCa3.1 with an IC50 of 6 nM and exhibited exquisite selectivity over calcium channels and a panel of >100 additional molecular targets. Using high-resolution structure determination by cryogenic electron microscopy together with mutagenesis and electrophysiology, we delineated the drug binding pocket for DHP-103 within the water-filled central cavity of the KCa3.1 channel pore, where bound drug directly impedes ion permeation. DHP-103 inhibited gain-of-function mutant KCa3.1 channels that cause hereditary xerocytosis, suggesting its potential use as a therapeutic for this hemolytic anemia. In a rat model of acute ischemic stroke, the second leading cause of death worldwide, DHP-103 administered 12 h postischemic insult in proof-of-concept studies reduced infarct volume, improved balance beam performance (measure of proprioception) and decreased numbers of activated microglia in infarcted areas. KCa3.1-selective 1,4-dihydropyridines hold promise for the many diseases for which KCa3.1 has been experimentally confirmed as a therapeutic target.

The behaviour of a tungsten first wall is studied under the irradiation conditions predicted for the different operation scenarios of the European Laser fusion project HiPER, which is based on direct drive targets and an evacuated dry wall chamber. The scenarios correspond to different stages in the development of a nuclear fusion reactor, from proof of principle (bunch mode facility) to economic feasibility (pre-commercial power plant). This work constitutes a quantitative study to evaluate the first wall performance under realistic irradiation conditions in the different scenarios. We calculated the radiation fluxes assuming the geometrical configurations reported so far for HiPER. Then, we calculated the irradiation-induced first wall temperature evolution and the thermo-mechanical response of the material. The results indicate that the first wall will plastically deform up to a few microns underneath the surface. Continuous operation in power plant leads to fatigue failure with crack generation and growth. Finally, the crack propagation and the minimum W thickness required to fulfil the first wall protection role is studied. The response of tungsten as first wall material as well as its main limitations will be discussed for the HiPER scenarios.

Major Histocompatibility Complex (MHC) molecules have been proposed to play a role in Sickle Cell Disease (SCD) pathophysiology. Endothelial cells express MHC molecules following exposure to cytokines. SCD is characterized, in part, by vascular endothelial cell activation, increased oxidative stress, sickle cell adhesion, and excess levels of endothelin-1 (ET-1) contributing to vaso-occlusive crises. ET-1 activates endothelial cells, induces oxidative stress and inflammation, and alters erythrocyte volume homeostasis. However, the role of ET-1 on MHC regulation in SCD is unclear. We first studied two sickle transgenic knockout mouse models of moderate to severe disease phenotype, βS-Antilles and Berkeley (BERK) mice. We observed significant increases in H2-Aa mRNA levels in spleens, lungs, and kidneys from transgenic sickle mice when compared to transgenic knockout mice expressing human hemoglobin A (HbA). Mice treated for 14 days with ET-1 receptor antagonists significantly reduced H2-Aa mRNA levels. We characterized the effect of ET-1 on MHC class II expression in the human endothelial cell line EA.hy926. We observed dose-dependent increases in the expression of MHC class II (HLA-DRA) and MHC transcription factor (CIITA) that were significantly blocked by treatment with BQ788, a selective blocker of ET-1 type B receptors. Chromatin immunoprecipitation studies in EA.hy926 cells showed that ET-1 increased Histone H3 acetylation of the HLA-DRA promoter, an event blocked by BQ788 treatment. These results implicate ET-1 as a novel regulator of MHC class II molecules and suggest that ET-1 receptor blockade represents a promising therapeutic approach to regulate both immune and vascular responses in SCD.

Increased endothelin-1 (ET-1) levels in patients with sickle cell disease (SCD) and transgenic mouse models of SCD contribute to disordered hematological, vascular, and inflammatory responses. Mineralocorticoid receptor (MR) activation by aldosterone, a critical component of the Renin-Angiotensin- Aldosterone- System, modulates inflammation and vascular reactivity, partly through increased ET-1 expression. However, the role of MR in SCD remains unclear. We hypothesized that MR blockade in transgenic SCD mice would reduce ET-1 levels, improve hematological parameters, and reduce inflammation. Berkeley SCD (BERK) mice, a model of severe SCD, were randomized to either sickle standard chow or chow containing the MR antagonist (MRA), eplerenone (156 mg/Kg), for 14 days. We found that MRA treatment reduced ET-1 plasma levels (p =.04), improved red cell density gradient profile (D-50; p <.002), and increased mean corpuscular volume in both erythrocytes (p <.02) and reticulocytes (p <.024). MRA treatment also reduced the activity of the erythroid intermediate-conductance Ca2+-activated K+ channel -K(Ca)3.1 (Gardos channel, KCNN4), reduced cardiac levels of mRNAs encoding ET-1, Tumor Necrosis Factor Receptor-1, and protein disulfide isomerase (PDI) (p <.01), and decreased plasma PDI and myeloperoxidase activity. Aldosterone (10(-8) M for 24 h in vitro) also increased PDI mRNA levels (p <.01) and activity (p <.003) in EA.hy926 human endothelial cells, in a manner blocked by pre-incubation with the MRA canrenoic acid (1 mu M; p <.001). Our results suggest a novel role for MR activation in SCD that may exacerbate SCD pathophysiology and clinical complications.

SLC26A4/Pendrin is the major electroneutral Cl- /HCO3- exchanger of the apical membrane of the Type B intercalated cell (IC) of the connecting segment (CNT) and cortical collecting duct (CCD). Pendrin mediates both base secretion in response to systemic base load and Cl- reabsorption in response to systemic volume depletion, manifested as decreased nephron salt and water delivery to the distal nephron. Pendrin-mediated Cl- /HCO3- exchange in the apical membrane is upregulated through stimulation of the β-IC apical membrane G protein-coupled receptor, 2-oxoglutarate receptor 1 (OXGR1/GPR99), by its ligand α-ketoglutarate (αKG). αKG is both filtered by the glomerulus and lumenally secreted by proximal tubule apical membrane organic anion transporters (OATs). OXGR1-mediated regulation of Pendrin by αKG has been documented in transgenic mice and in isolated perfused CCD. However, aspects of the OXGR1 signaling pathway have remained little investigated since its original discovery in lymphocytes. Moreover, no ex vivo cellular system has been reported in which to study the OXGR1 signaling pathway of Type B-IC, a cell type refractory to survival in culture in its differentiated state. As Xenopus oocytes express robust heterologous Pendrin activity, we investigated OXGR1 regulation of Pendrin in oocytes. Despite functional expression of OXGR1 in oocytes, co-expression of Pendrin and OXGR1 failed to exhibit αKG-sensitive stimulation of Pendrin-mediated Cl- /anion exchange under a wide range of conditions. We conclude that Xenopus oocytes lack one or more essential molecular components or physical conditions required for OXGR1 to regulate Pendrin activity.

AbstractBackgroundDisordered endothelial cell activation plays an important role in the pathophysiology of atherosclerosis, cancer, sepsis, viral infections, and inflammatory responses. There is interest in developing novel therapeutics to regulate endothelial cell function in atherothrombotic, metabolic, vascular, and hematological diseases. Extracts from leaves of theSyzygium jambos(L.) Alston (S. jambos) trees have been proposed to treat cardiovascular diseases and diabetes through unclear mechanisms. We investigated the effects of theS. jambosextract on biomarkers of endothelial dysfunction and immune responses in the human endothelial cell line, EA.hy926.MethodsLeaves ofS. jamboswere collected, concocted and lyophilized. To study the effects ofS. jamboson endothelial cell activation, we used the human endothelial cell line. IL-6 levels were measured using qPCR and ELISA. PDI activity was measured using Insulin Turbidity and Di-E-GSSG assays. CM-H2DCFDA was used to study ROS levels. Migration assay was used to studyS. jamboseffect on ex vivo human polymorphonuclear and human mononuclear cells.ResultsOur results show that incubation of EA.hy926 cells with ET-1 led to a 6.5 ± 1.6 fold increase in IL-6 expression by qPCR, an event that was blocked byS. jambos. Also, we observed that ET-1 increased extracellular protein disulfide isomerase (PDI) activity that was likewise dose-dependently blocked byS. jambos(IC50 = 14 μg/mL). Consistent with these observations, ET-1 stimulatedex vivohuman polymorphonuclear and mononuclear cell migration that also was dose-dependently blocked byS. jambos. In addition, ET-1 stimulation led to significant increases in ROS production that were sensitive toS. jambos.ConclusionOur results suggest that theS. jambosextract represents a novel cardiovascular protective pharmacological approach to regulate endothelial cell activation, IL-6 expression, and immune-cell responses.

Paracrine ATP release by erythrocytes has been shown to regulate endothelial cell function via purinergic signaling, and this erythoid-endothelial signaling network is pathologically dysregulated in sickle cell disease. We tested the role of extracellular ATP-mediated purinergic signaling in the activation of Psickle, the mechanosensitive Ca2+-permeable cation channel of human sickle erythrocytes (SS RBC). Psickle activation increases intracellular [Ca2+] to stimulate activity of the RBC Gardos channel, KCNN4/KCa3.1, leading to cell shrinkage and accelerated deoxygenation-activated sickling.We found that hypoxic activation of Psickle recorded by cell-attached patch clamp in SS RBC is inhibited by extracellular apyrase, which hydrolyzes extracellular ATP. Hypoxic activation of Psickle was also inhibited by the pannexin-1 inhibitor, probenecid, and by the P2 antagonist, suramin. A Psickle-like activity was also activated in normoxic SS RBC (but not in control red cells) by bath pH 6.0. Acid-activated Psickle-like activity was similarly blocked by apyrase, probenecid, and suramin, as well as by the Psickle inhibitor, Grammastola spatulata mechanotoxin-4 (GsMTx-4).In vitro-differentiated cultured human sickle reticulocytes (SS cRBC), but not control cultured reticulocytes, also exhibited hypoxia-activated Psickle activity that was abrogated by GsMTx-4. Psickle-like activity in SS cRBC was similarly elicited by normoxic exposure to acid pH, and this acid-stimulated activity was nearly completely blocked by apyrase, probenecid, and suramin, as well as by GsMTx-4.Thus, hypoxia-activated and normoxic acid-activated cation channel activities are expressed in both SS RBC and SS cRBC, and both types of activation appear to be mediated or greatly amplified by autocrine or paracrine purinergic signaling.

Red cell volume is a major determinant of HbS concentration in sickle cell disease. Cellular deoxy-HbS concentration determines the delay time, the interval between HbS deoxygenation and deoxy-HbS polymerization. Major membrane transporter protein determinants of sickle red cell volume include the SLC12/KCC K-Cl cotransporters KCC3/SLC12A6 and KCC1/SLC12A4, and the KCNN4/KCa3.1 Ca2+-activated K+ channel (Gardos channel). Among standard inhibitors of KCC-mediated K-Cl cotransport, only [(dihydroindenyl)oxy]acetic acid (DIOA) has been reported to lack inhibitory activity against the related bumetanide-sensitive erythroid Na-K-2Cl cotransporter NKCC1/SLC12A2. DIOA has been often used to inhibit K-Cl cotransport when studying the expression and regulation of other K+ transporters and K+ channels. We report here that DIOA at concentrations routinely used to inhibit K-Cl cotransport can also abrogate activity of the KCNN4/KCa3.1 Gardos channel in human and mouse red cells and in human sickle red cells. DIOA inhibition of A23187-stimulated erythroid K+ uptake (Gardos channel activity) was chloride-independent and persisted in mouse red cells genetically devoid of the principal K-Cl cotransporters KCC3 and KCC1. DIOA also inhibited YODA1-stimulated, chloride-independent erythroid K+ uptake. In contrast, DIOA exhibited no inhibitory effect on K+ influx into A23187-treated red cells of Kcnn4-/- mice. DIOA inhibition of human KCa3.1 was validated (IC50 42 µM) by whole cell patch clamp in HEK-293 cells. RosettaLigand docking experiments identified a potential binding site for DIOA in the fenestration region of human KCa3.1. We conclude that DIOA at concentrations routinely used to inhibit K-Cl cotransport can also block the KCNN4/KCa3.1 Gardos channel in normal and sickle red cells.

Investigation of erythrocytes from spontaneous or engineered germ-line mutant mice has been instrumental in characterizing the physiological functions of components of the red cell cytoskeleton and membrane. However, the red blood cell expresses some proteins whose germline loss-of-function is embryonic-lethal, perinatal-lethal, or confers reduced post-weaning viability. Promoter regions of erythroid-specific genes have been used to engineer erythroid-specific expression of Cre recombinase. Through breeding with mice carrying appropriately spaced insertions of loxP sequences, generation of erythroid-specific knockouts has been carried out for signaling enzymes, transcription factors, peptide hormones, and single transmembrane span signaling receptors. We report here the use of Cre recombinase expression driven by the erythropoietin receptor (EpoR) promoter to generate EpoR-Cre;Kcc3(f/f) mice, designed to express erythroid-specific knockout of the KCC3 K-Cl cotransporter encoded by Kcc3/Slc12A6. We confirm KCC3 as the predominant K-Cl cotransporter of adult mouse red cells in mice with better viability than previously exhibited by Kcc3(-/-) germline knockouts. We demonstrate roughly proportionate preservation of K-Cl stimulation by hypotonicity, staurosporine, and urea in the context of reduced, but not abrogated, K-Cl function in EpoR-Cre;Kcc3(f/f) mice. We also report functional evidence suggesting incomplete recombinase-mediated excision of the Kcc3 gene in adult erythroid tissues.

Painful crises in sickle cell disease (SCD) are associated with increased plasma cytokines levels, including endothelin-1 (ET-1). Reduced red cell magnesium content, mediated in part by increased Na+/Mg2+ exchanger (NME) activity, contributes to erythrocyte K+ loss, dehydration and sickling in SCD. However, the relationship between ET-1 and the NME in SCD has remained unexamined. We observed increased NME activity in sickle red cells incubated in the presence of 500 nM ET-1. Deoxygenation of sickle red cells, in contrast, led to decreased red cell NME activity and cellular dehydration that was reversed by the NME inhibitor, imipramine. Increased NME activity in sickle red cells was significantly blocked by pre-incubation with 100 nM BQ788, a selective blocker of ET-1 type B receptors. These results suggest an important role for ET-1 and for cellular magnesium homeostasis in SCD. Consistent with these results, we observed increased NME activity in sickle red cells of three mouse models of sickle cell disease greater than that in red cells of C57BL/J6 mice. In vivo treatment of BERK sickle transgenic mice with ET-1 receptor antagonists reduced red cell NME activity. Our results suggest that ET-1 receptor blockade may be a promising therapeutic approach to control erythrocyte volume and magnesium homeostasis in SCD and may thus attenuate or retard the associated chronic inflammatory and vascular complications of SCD.

Hyperglycemia is associated with decreased Mg2+ content in red blood cells (RBC), but mechanisms remain unclear. We characterized the regulation of Mg2+ efflux by glucose in ex vivo human RBC. We observed that hemoglobin A1C (HbA1C) values correlated with Na+-dependent Mg2+ efflux (Na+/Mg2+ exchange) and inversely correlated with cellular Mg content. Treatment of cells with 50 mM D-glucose, but not with sorbitol, lowered total cellular Mg (2.2 ± 0.1 to 2.0 ± 0.1 mM, p < 0.01) and enhanced Na+/Mg2+ exchange activity [0.60 ± 0.09 to 1.12 ± 0.09 mmol/1013 cell × h (flux units, FU), p < 0.05]. In contrast, incubation with selective Src family kinase inhibitors PP2 or SU6656 reduced glucose-stimulated exchange activation (p < 0.01). Na+/Mg2+ exchange activity was also higher in RBC from individuals with type 2 diabetes (T2D, 1.19 ± 0.13 FU) than from non-diabetic individuals (0.58 ± 0.05 FU, p < 0.01). Increased Na+/Mg2+ exchange activity in RBC from T2D subjects was associated with lower intracellular Mg content. Similarly increased exchange activity was evident in RBC from the diabetic db/db mouse model as compared to its non-diabetic control (p < 0.03). Extracellular exposure of intact RBC from T2D subjects to recombinant peptidyl-N-glycosidase F (PNGase F) reduced Na+/Mg2+ exchange activity from 0.98 ± 0.14 to 0.59 ± 0.13 FU (p < 0.05) and increased baseline intracellular Mg content (1.8 ± 0.1 mM) to normal values (2.1 ± 0.1 mM, p < 0.05). These data suggest that the reduced RBC Mg content of T2D RBC reflects enhanced RBC Na+/Mg2+ exchange subject to regulation by Src family kinases and by the N-glycosylation state of one or more membrane proteins. The data extend our understanding of dysregulated RBC Mg2+ homeostasis in T2D.

The molecular identity of Psickle, the deoxygenation-activated cation conductance of the human sickle erythrocyte, remains unknown. We observed in human sickle red cells that inhibitors of TRPA1 and TRPV1 inhibited Psickle, whereas a TRPV1 agonist activated a Psickle-like cation current. These observations prompted us to test the roles of TRPV1 and TRPA1 in Psickle in red cells of the SAD mouse model of sickle cell disease. We generated SAD mice genetically deficient in either TRPV1 or TRPA1. SAD;Trpv1-/- and SAD;Trpa1-/- mice were indistinguishable in appearance, hematological indices, and osmotic fragility from SAD mice. We found that deoxygenation-activated cation currents remained robust in SAD;Trpa1-/- and SAD;Trpv1-/- mice. In addition, 45Ca2+ influx into SAD mouse red cells during prolonged deoxygenation was not reduced in red cells from SAD;Trpa1-/- and SAD;Trpv1-/- mice. We conclude that the nonspecific cation channels TRPA1 and TRPV1 are not required for deoxygenation to stimulate Psickle-like activity in red cells of the SAD mouse model of sickle cell disease. (159).

Not available.

The Josephson effect results from the coupling of two superconductors across a spacer such as an insulator, a normal metal or a ferromagnet to yield a phase coherent quantum state. However, in junctions with ferromagnetic spacers, very long-range Josephson effects have remained elusive. Here we demonstrate extremely long-range (micrometric) high-temperature (tens of kelvins) Josephson coupling across the half-metallic manganite La0.7Sr0.3MnO3 combined with the superconducting cuprate YBa2Cu3O7. These planar junctions, in addition to large critical currents, display the hallmarks of Josephson physics, such as critical current oscillations driven by magnetic flux quantization and quantum phase locking effects under microwave excitation (Shapiro steps). The latter display an anomalous doubling of the Josephson frequency predicted by several theories. In addition to its fundamental interest, the marriage between high-temperature, dissipationless quantum coherent transport and full spin polarization brings opportunities for the practical realization of superconducting spintronics, and opens new perspectives for quantum computing.

The anomalous Hall effect (AHE) is an intriguing transport phenomenon occurring typically in ferromagnets as a consequence of broken time reversal symmetry and spin-orbit interaction. It can be caused by two microscopically distinct mechanisms, namely, by skew or side-jump scattering due to chiral features of the disorder scattering, or by an intrinsic contribution directly linked to the topological properties of the Bloch states. Here we show that the AHE can be artificially engineered in materials in which it is originally absent by combining the effects of symmetry breaking, spin orbit interaction and proximity-induced magnetism. In particular, we find a strikingly large AHE that emerges at the interface between a ferromagnetic manganite (La0.7Sr0.3MnO3) and a semimetallic iridate (SrIrO3). It is intrinsic and originates in the proximity-induced magnetism present in the narrow bands of strong spin-orbit coupling material SrIrO3, which yields values of anomalous Hall conductivity and Hall angle as high as those observed in bulk transition-metal ferromagnets. These results demonstrate the interplay between correlated electron physics and topological phenomena at interfaces between 3d ferromagnets and strong spin-orbit coupling 5d oxides and trace an exciting path towards future topological spintronics at oxide interfaces.