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Typical hemolytic uremic syndrome (HUS) can occur as a severe systemic complication of infections with Shiga toxin (Stx)-producing Escherichia coli. Its pathology can be induced by Stx types, resulting in toxin-mediated damage to renal barriers, inflammation, and the development of acute kidney injury (AKI). Two sphingosine kinase (SphK) isozymes, SphK1 and SphK2, have been shown to be involved in barrier maintenance and renal inflammatory diseases. Therefore, we sought to determine their role in the pathogenesis of HUS. Experimental HUS was induced by the repeated administration of Stx2 in wild-type (WT) and SphK1 (SphK1-/-) or SphK2 (SphK2-/-) null mutant mice. Disease severity was evaluated by assessing clinical symptoms, renal injury and dysfunction, inflammatory status and sphingolipid levels on day 5 of HUS development. Renal inflammation and injury were found to be attenuated in the SphK2-/- mice, but exacerbated in the SphK1-/- mice compared to the WT mice. The divergent outcome appeared to be associated with oppositely altered sphingolipid levels. This study represents the first description of the distinct roles of SphK1-/- and SphK2-/- in the pathogenesis of HUS. The identification of sphingolipid metabolism as a potential target for HUS therapy represents a significant advance in the field of HUS research.
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Lesión Renal Aguda , Síndrome Hemolítico-Urémico , Ratones Noqueados , Fosfotransferasas (Aceptor de Grupo Alcohol) , Animales , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Lesión Renal Aguda/metabolismo , Lesión Renal Aguda/patología , Lesión Renal Aguda/etiología , Lesión Renal Aguda/genética , Ratones , Síndrome Hemolítico-Urémico/patología , Síndrome Hemolítico-Urémico/genética , Modelos Animales de Enfermedad , Esfingolípidos/metabolismo , Riñón/patología , Riñón/metabolismo , Ratones Endogámicos C57BL , Toxina Shiga II , Eliminación de Gen , MasculinoRESUMEN
Semiconductor quantum dots are promising candidates for the generation of nonclassical light. Coupling a quantum dot to a device capable of providing polarization-selective enhancement of optical transitions is highly beneficial for advanced functionalities, such as efficient resonant driving schemes or applications based on optical cyclicity. Here, we demonstrate broadband polarization-selective enhancement by coupling a quantum dot emitting in the telecom O-band to an elliptical bullseye resonator. We report bright single-photon emission with a degree of linear polarization of 96%, Purcell factor of 3.9 ± 0.6, and count rates up to 3 MHz. Furthermore, we present a measurement of two-photon interference without any external polarization filtering. Finally, we demonstrate compatibility with compact Stirling cryocoolers by operating the device at temperatures up to 40 K. These results represent an important step toward practical integration of optimal quantum dot photon sources in deployment-ready setups.
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Antiplatelet medication is standard of care in acute myocardial infarction (AMI). However, it may have obscured beneficial properties of the activated platelet secretome. We identify platelets as major source of a sphingosine-1-phosphate (S1P) burst during AMI, and find its magnitude to favorably associate with cardiovascular mortality and infarct size in STEMI patients over 12 months. Experimentally, administration of supernatant from activated platelets reduces infarct size in murine AMI, which is blunted in platelets deficient for S1P export (Mfsd2b) or production (Sphk1) and in mice deficient for cardiomyocyte S1P receptor 1 (S1P1). Our study reveals an exploitable therapeutic window in antiplatelet therapy in AMI as the GPIIb/IIIa antagonist tirofiban preserves S1P release and cardioprotection, whereas the P2Y12 antagonist cangrelor does not. Here, we report that platelet-mediated intrinsic cardioprotection is an exciting therapeutic paradigm reaching beyond AMI, the benefits of which may need to be considered in all antiplatelet therapies.
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Plaquetas , Infarto del Miocardio , Humanos , Ratones , Animales , Infarto del Miocardio/tratamiento farmacológico , Esfingosina , Lisofosfolípidos/uso terapéutico , Miocitos CardíacosRESUMEN
AIMS: Therapeutic options targeting post-ischaemic cardiac remodelling are sparse. The bioactive sphingolipid sphingosine-1-phosphate (S1P) reduces ischaemia/reperfusion injury. However, its impact on post-ischaemic remodelling independently of its infarct size (IS)-reducing effect is yet unknown and was addressed in this study. METHODS AND RESULTS: Acute myocardial infarction (AMI) in mice was induced by permanent ligation of the left anterior descending artery (LAD). C57Bl6 were treated with the S1P lyase inhibitor 4-deoxypyridoxine (DOP) starting 7 days prior to AMI to increase endogenous S1P concentrations. Cardiac function and myocardial healing were assessed by cardiovascular magnetic resonance imaging (cMRI), murine echocardiography, histomorphology, and gene expression analysis. DOP effects were investigated in cardiomyocyte-specific S1P receptor 1 deficient (S1PR1 Cardio Cre+) and Cre- control mice and S1P concentrations measured by LC-MS/MS. IS and cardiac function did not differ between control and DOP-treated groups on day one after LAD-ligation despite fourfold increase in plasma S1P. In contrast, cardiac function was clearly improved and myocardial scar size reduced, respectively, on Day 21 in DOP-treated mice. The latter also exhibited smaller cardiomyocyte size and reduced embryonic gene expression. The benefit of DOP treatment was abolished in S1PR1 Cardio Cre+. CONCLUSIONS: S1P improves cardiac function and myocardial healing post AMI independently of initial infarct size and accomplishes this via the cardiomyocyte S1PR1. Hence, in addition to its beneficial effects on I/R injury, S1PR1 may be a promising target in post-infarction myocardial remodelling as adjunctive therapy to revascularization as well as in patients not eligible for standard interventional procedures.
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Infarto del Miocardio , Receptores de Lisoesfingolípidos , Ratones , Animales , Receptores de Esfingosina-1-Fosfato/uso terapéutico , Cromatografía Liquida , Receptores de Lisoesfingolípidos/genética , Receptores de Lisoesfingolípidos/metabolismo , Receptores de Lisoesfingolípidos/uso terapéutico , Espectrometría de Masas en Tándem , Infarto del Miocardio/tratamiento farmacológicoRESUMEN
The yeast-to-hypha transition is a key virulence attribute of the opportunistic human fungal pathogen Candida albicans, since it is closely tied to infection-associated processes such as tissue invasion and escape from phagocytes. While the nature of hypha-associated gene expression required for fungal virulence has been thoroughly investigated, potential morphotype-dependent activity of metabolic pathways remained unclear. Here, we combined global transcriptome and metabolome analyses for the wild-type SC5314 and the hypha-defective hgc1Δ and cph1Δefg1Δ strains under three hypha-inducing (human serum, N-acetylglucosamine, and alkaline pH) and two yeast-promoting conditions to identify metabolic adaptions that accompany the filamentation process. We identified morphotype-related activities of distinct pathways and a metabolic core signature of 26 metabolites with consistent depletion or enrichment during the yeast-to-hypha transition. Most strikingly, we found a hypha-associated activation of de novo sphingolipid biosynthesis, indicating a connection of this pathway and filamentous growth. Consequently, pharmacological inhibition of this partially fungus-specific pathway resulted in strongly impaired filamentation, verifying the necessity of de novo sphingolipid biosynthesis for proper hypha formation. IMPORTANCE The reversible switch of Candida albicans between unicellular yeast and multicellular hyphal growth is accompanied by a well-studied hypha-associated gene expression, encoding virulence factors like adhesins, toxins, or nutrient scavengers. The investigation of this gene expression consequently led to fundamental insights into the pathogenesis of this fungus. In this study, we applied this concept to hypha-associated metabolic adaptations and identified morphotype-dependent activities of distinct pathways and a stimulus-independent metabolic signature of hyphae. Most strikingly, we found the induction of de novo sphingolipid biosynthesis as hypha associated and essential for the filamentation of C. albicans. These findings verified the presence of morphotype-specific metabolic traits in the fungus, which appear connected to the fungal virulence. Furthermore, the here-provided comprehensive description of the fungal metabolome will help to foster future research and lead to a better understanding of fungal physiology.
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Candida albicans , Hifa , Humanos , Candida albicans/genética , Hifa/genética , Proteínas Fúngicas/genética , Factores de Virulencia/metabolismo , Esfingolípidos/metabolismoRESUMEN
The development of efficient sources of single photons and entangled photon pairs emitting in the low-loss wavelength region around 1550 nm is crucial for long-distance quantum communication. Moreover, direct fiber coupling and electrical carrier injection are highly desirable for deployment in compact and user-friendly systems integrated with the existing fiber infrastructure. Here we present a detailed design study of circular Bragg gratings fabricated in InP slabs and operating in the telecom C-band. These devices enable the simultaneous enhancement of the X and XX spectral lines, with collection efficiency in numerical aperture 0.65 close to 90% for the wavelength range 1520 - 1580 nm and Purcell factor up to 15. We also investigate the coupling into a single mode fiber, which exceeds 70% in UHNA4. Finally, we propose a modified device design directly compatible with electrical carrier injection, reporting Purcell factors up to 20 and collection efficiency in numerical aperture 0.65 close to 70% for the whole telecom C-band.
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Cholesterol is highly abundant within all human body cells and modulates critical cellular functions related to cellular plasticity, metabolism, and survival. The cholesterol-binding toxin pneumolysin represents an essential virulence factor of Streptococcus pneumoniae in establishing pneumonia and other pneumococcal infections. Thus, cholesterol scavenging of pneumolysin is a promising strategy to reduce S. pneumoniae induced lung damage. There may also be a second cholesterol-dependent mechanism whereby pneumococcal infection and the presence of pneumolysin increase hepatic sterol biosynthesis. Here we investigated a library of polymer particles varying in size and composition that allow for the cellular delivery of cholesterol and their effects on cell survival mechanisms following pneumolysin exposure. Intracellular delivery of cholesterol by nanocarriers composed of Eudragit E100-PLGA rescued pneumolysin-induced alterations of lipid homeostasis and enhanced cell survival irrespective of neutralization of pneumolysin.
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Doxorubicin (Dox) is a chemotherapeutic agent with cardiotoxicity associated with profibrotic effects. Dox increases ceramide levels with pro-inflammatory effects, cell death, and fibrosis. The purpose of our study was to identify the underlying ceramide signaling pathways. We aimed to characterize the downstream effects on cell survival, metabolism, and fibrosis. Human fibroblasts (hFSF) were treated with 0.7 µM of Dox or transgenically overexpressed ceramide synthase 2 (FLAG-CerS2). Furthermore, cells were pre-treated with MitoTempo (MT) (2 h, 20 µM) or Fumonisin B1 (FuB) (4 h, 100 µM). Protein expression was measured by Western blot or immunofluorescence (IF). Ceramide levels were determined with mass spectroscopy (MS). Visualizations were conducted using laser scanning microscopy (LSM) or electron microscopy. Mitochondrial activity was measured using seahorse analysis. Dox and CerS2 overexpression increased CerS2 protein expression. Coherently, ceramides were elevated with the highest peak for C24:0. Ceramide- induced mitochondrial ROS production was reduced with MT or FuB preincubation. Mitochondrial homeostasis was reduced and accompanied by reduced ATP production. Our data show that the increase in pro-inflammatory ceramides is an essential contributor to Dox side-effects. The accumulation of ceramides resulted in a lipotoxic shift and subsequently mitochondrial structural and functional damage, which was partially reversible following inhibition of ceramide synthesis.
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Ceramidas/metabolismo , Doxorrubicina/efectos adversos , Prepucio/patología , Proteínas de la Membrana/genética , Esfingosina N-Aciltransferasa/genética , Proteínas Supresoras de Tumor/genética , Adenosina Trifosfato/metabolismo , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibrosis , Prepucio/citología , Prepucio/efectos de los fármacos , Humanos , Masculino , Espectrometría de Masas , Proteínas de la Membrana/metabolismo , Mitocondrias Cardíacas/efectos de los fármacos , Mitocondrias Cardíacas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Esfingosina N-Aciltransferasa/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Regulación hacia ArribaRESUMEN
Jaundice, the clinical hallmark of infection-associated liver dysfunction, reflects altered membrane organization of the canalicular pole of hepatocytes and portends poor outcomes. Mice lacking phosphoinositide 3-kinase-γ (PI3Kγ) are protected against membrane disintegration and hepatic excretory dysfunction. However, they exhibit a severe immune defect that hinders neutrophil recruitment to sites of infection. To exploit the therapeutic potential of PI3Kγ inhibition in sepsis, a targeted approach to deliver drugs to hepatic parenchymal cells without compromising other cells, in particular immune cells, seems warranted. Here, we demonstrate that nanocarriers functionalized through DY-635, a fluorescent polymethine dye, and a ligand of organic anion transporters can selectively deliver therapeutics to hepatic parenchymal cells. Applying this strategy to a murine model of sepsis, we observed the PI3Kγ-dependent restoration of biliary canalicular architecture, maintained excretory liver function, and improved survival without impairing host defense mechanisms. This strategy carries the potential to expand targeted nanomedicines to disease entities with systemic inflammation and concomitantly impaired barrier functionality.
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Hepatopatías , Sepsis , Animales , Ratones , Infiltración Neutrófila , Fosfatidilinositol 3-Quinasas , Inhibidores de las Quinasa Fosfoinosítidos-3 , Sepsis/tratamiento farmacológicoRESUMEN
Oversupply of fatty acids (FAs) to cardiomyocytes (CMs) is associated with increased ceramide content and elevated the risk of lipotoxic cardiomyopathy. Here we investigate the role of ceramide accumulation on mitochondrial function and mitophagy in cardiac lipotoxicity using CMs derived from human induced pluripotent stem cell (hiPSC). Mature CMs derived from hiPSC exposed to the diabetic-like environment or transfected with plasmids overexpressing serine-palmitoyltransferase long chain base subunit 1 (SPTLC1), a subunit of the serine-palmitoyltransferase (SPT) complex, resulted in increased intracellular ceramide levels. Accumulation of ceramides impaired insulin-dependent phosphorylation of Akt through activating protein phosphatase 2A (PP2A) and disturbed gene and protein levels of key metabolic enzymes including GLUT4, AMPK, PGC-1α, PPARα, CD36, PDK4, and PPARγ compared to controls. Analysis of CMs oxidative metabolism using a Seahorse analyzer showed a significant reduction in ATP synthesis-related O2 consumption, mitochondrial ß-oxidation and respiratory capacity, indicating an impaired mitochondrial function under diabetic-like conditions or SPTLC1-overexpression. Further, ceramide accumulation increased mitochondrial fission regulators such as dynamin-related protein 1 (DRP1) and mitochondrial fission factor (MFF) as well as auto/mitophagic proteins LC3B and PINK-1 compared to control. Incubation of CMs with the specific SPT inhibitor (myriocin) showed a significant increase in mitochondrial fusion regulators the mitofusin 2 (MFN2) and optic atrophy 1 (OPA1) as well as p-Akt, PGC-1 α, GLUT-4, and ATP production. In addition, a significant decrease in auto/mitophagy and apoptosis was found in CMs treated with myriocin. Our results suggest that ceramide accumulation has important implications in driving insulin resistance, oxidative stress, increased auto/mitophagy, and mitochondrial dysfunction in the setting of lipotoxic cardiomyopathy. Therefore, modulation of the de novo ceramide synthesis pathway may serve as a novel therapeutic target to treat metabolic cardiomyopathy.
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Células Madre Pluripotentes Inducidas , Mitofagia , Ceramidas/metabolismo , Humanos , Miocitos Cardíacos/metabolismo , Estrés OxidativoRESUMEN
BACKGROUND: Angiotensin converting enzyme inhibitors (ACEI) and angiotensin II receptor blockers (ARB) are important in the prevention of cardiovascular disease. The "ARB-MI paradox" implies that no risk reduction of myocardial infarction (MI) was found in ARB-treated patients despite target blood pressure control. Sphingosine-1-phosphate (S1P) is a cardioprotective sphingolipid which is released by platelets during activation. In this study we aimed to investigate differences of S1P homeostasis mediated by bradykinin and sphingosine kinases during ACEI/ARB treatment. METHODS: In this hypothesis generating pilot study, we investigated S1P plasma concentrations in 34 patients before and 3 months after ARB/ACEI medication. S1P levels were measured via liquid chromatography-tandem mass spectrometry. Bradykinin levels were measured by an enzyme-linked immunosorbent assay. RESULTS: Patient characteristics were not different between the ACEI and ARB group. Baseline S1P plasma concentrations were similar before ARB and ACEI treatment (7.4 SD 1.9 pmol vs. 7.8 SD 2.7 pmol, p = 0.54). After 3 months, S1P plasma levels were significantly higher in ACEI (9.3 SD 2.2 pmol) as compared to ARB treated patients (7.4 SD 2.4 pmol, p = 0.001). Pearson correlation showed no significant association between bradykinin and S1P levels before (r = -0.219; 95% CI [-0.54-0.15]; p = 0.245) or after three months of treatment with ACEI or ARB (r = -0.015; 95% CI [-0.48-0.45]; p = 0.95). CONCLUSIONS: S1P plasma concentrations are higher in ACE treated patients as compared to ARB treatment. This leads to the hypothesis, that differences in S1P metabolism might partially explain the ARB-MI paradox. This needs to be tested in clinical trials.
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Antagonistas de Receptores de Angiotensina , Infarto del Miocardio , Inhibidores de la Enzima Convertidora de Angiotensina , Humanos , Lisofosfolípidos , Proyectos Piloto , Esfingosina/análogos & derivadosRESUMEN
Sphingosine 1-phosphate (S1P) is a bioactive phospholipid and ligand for five G protein-coupled cell-surface receptors designated S1PR1-5. The determination of low levels of S1P remains a challenge and usually requires sophisticated analytical instrumentation and methodology. This report describes a technique using the linear ion trap mode of a basic QTrap triple-quadrupole mass spectrometer. S1P was extracted from acidified biological samples using a modified Folch extraction procedure. After the addition of C17-sphingosine as an internal standard, a step gradient LC method was used to separate the analytes on a reversed-phase C18 MultoHigh analytical column. After the internal standard C17-sphingosine was detected by multiple reaction monitoring (MRM), the detection mode was switched to enhanced product ion (EPI) mode for the detection of S1P. The mode was switched back to MRM again for the detection of other analytes. Using this QTrap method, we reached a limit of detection of 1 nM and a limit of quantification of 3 nM for S1P, which was up to 30 times more sensitive than the MRM mode with the same instrument. Intra-day precision ranged between -3.8 and 6.3%, and inter-day precision was between -13.8 and 3.3%, depending on the spiked S1P concentration.
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Cromatografía Líquida de Alta Presión/métodos , Lisofosfolípidos/análisis , Esfingosina/análogos & derivados , Espectrometría de Masas en Tándem/métodos , Animales , Límite de Detección , Modelos Lineales , Hígado/química , Ratones , Ratones Endogámicos C57BL , Miocardio/química , Reproducibilidad de los Resultados , Esfingosina/análisisRESUMEN
Sphingosine 1-phosphate (S1P) is an important immune modulator responsible for physiological cellular responses like lymphocyte development and function, positioning and emigration of T and B cells and cytokine secretion. Recent reports indicate that S1P does not only regulate immunity, but can also protect the function of organs by inducing disease tolerance. S1P also influences the replication of certain pathogens, and sphingolipids are also involved in pathogen recognition and killing. Certain carrier molecules for S1P like serum albumin and high density lipoproteins contribute to the regulation of S1P effects. They are able to associate with S1P and modulate its signaling properties. Similar to S1P, both carrier molecules are also decreased in sepsis patients and likely contribute to sepsis pathology and severity. In this review, we will introduce the concept of disease tolerance and the involvement of S1P. We will also discuss the contribution of S1P and its precursor sphingosine to host defense mechanisms against pathogens. Finally, we will summarize current data demonstrating the influence of carrier molecules for differential S1P signaling. The presented data may lead to new strategies for the prevention and containment of sepsis.
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Tolerancia Inmunológica , Lisofosfolípidos/inmunología , Sepsis/inmunología , Transducción de Señal/inmunología , Esfingosina/análogos & derivados , Animales , Humanos , Esfingosina/inmunologíaRESUMEN
Quantum networks are essential for realising distributed quantum computation and quantum communication. Entangled photons are a key resource, with applications such as quantum key distribution, quantum relays, and quantum repeaters. All components integrated in a quantum network must be synchronised and therefore comply with a certain clock frequency. In quantum key distribution, the most mature technology, clock rates have reached and exceeded 1GHz. Here we show the first electrically pulsed sub-Poissonian entangled photon source compatible with existing fiber networks operating at this clock rate. The entangled LED is based on InAs/InP quantum dots emitting in the main telecom window, with a multi-photon probability of less than 10% per emission cycle and a maximum entanglement fidelity of 89%. We use this device to demonstrate GHz clocked distribution of entangled qubits over an installed fiber network between two points 4.6km apart.
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An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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A hallmark of neurodegeneration is defective protein quality control. The E3 ligase Listerin (LTN1/Ltn1) acts in a specialized protein quality control pathway-Ribosome-associated Quality Control (RQC)-by mediating proteolytic targeting of incomplete polypeptides produced by ribosome stalling, and Ltn1 mutation leads to neurodegeneration in mice. Whether neurodegeneration results from defective RQC and whether defective RQC contributes to human disease have remained unknown. Here we show that three independently-generated mouse models with mutations in a different component of the RQC complex, NEMF/Rqc2, develop progressive motor neuron degeneration. Equivalent mutations in yeast Rqc2 selectively interfere with its ability to modify aberrant translation products with C-terminal tails which assist with RQC-mediated protein degradation, suggesting a pathomechanism. Finally, we identify NEMF mutations expected to interfere with function in patients from seven families presenting juvenile neuromuscular disease. These uncover NEMF's role in translational homeostasis in the nervous system and implicate RQC dysfunction in causing neurodegeneration.
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Enfermedades Neuromusculares/metabolismo , Ribosomas/metabolismo , Secuencia de Aminoácidos , Animales , Femenino , Humanos , Masculino , Ratones , Ratones Noqueados , Mutación , Enfermedades Neuromusculares/genética , Enfermedades Neuromusculares/patología , Proteolisis , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Ribosomas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alineación de SecuenciaRESUMEN
The breakdown of the endothelial cell (EC) barrier contributes significantly to sepsis mortality. Sphingosine 1-phosphate (S1P) is one of the most effective EC barrier-stabilizing signaling molecules. Stabilization is mainly transduced via the S1P receptor type 1 (S1PR1). Here, we demonstrate that S1P was autonomously produced by ECs. S1P secretion was significantly higher in primary human umbilical vein endothelial cells (HUVEC) compared to the endothelial cell line EA.hy926. Constitutive barrier stability of HUVEC, but not EA.hy926, was significantly compromised by the S1PR1 antagonist W146 and by the anti-S1P antibody Sphingomab. HUVEC and EA.hy926 differed in the expression of the S1P-transporter Spns2, which allowed HUVEC, but not EA.hy926, to secrete S1P into the extracellular space. Spns2 deficient mice showed increased serum albumin leakage in bronchoalveolar lavage fluid (BALF). Lung ECs isolated from Spns2 deficient mice revealed increased leakage of fluorescein isothiocyanate (FITC) labeled dextran and decreased resistance in electric cell-substrate impedance sensing (ECIS) measurements. Spns2 was down-regulated in HUVEC after stimulation with pro-inflammatory cytokines and lipopolysaccharides (LPS), which contributed to destabilization of the EC barrier. Our work suggests a new mechanism for barrier integrity maintenance. Secretion of S1P by EC via Spns2 contributed to constitutive EC barrier maintenance, which was disrupted under inflammatory conditions via the down-regulation of the S1P-transporter Spns2.
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Células Endoteliales/metabolismo , Inflamación/metabolismo , Lisofosfolípidos/metabolismo , Esfingosina/análogos & derivados , Animales , Línea Celular Tumoral , Células Cultivadas , Células Endoteliales/patología , Células Endoteliales de la Vena Umbilical Humana , Humanos , Inflamación/patología , Proteínas de la Membrana/metabolismo , Ratones , Ratas , Transducción de Señal , Esfingosina/metabolismo , Receptores de Esfingosina-1-Fosfato/metabolismoRESUMEN
Introduction To correctly interpret plasma- or serum-sphingosine-1-phosphate (S1P) concentrations measured in clinical studies it is critical to understand all major determinants in healthy controls. Methods Serum- and plasma-S1P from 174 healthy blood donors was measured by liquid chromatography-tandem mass spectrometry and correlated to clinical laboratory data. Selected plasma samples, 10 with high and 10 with low S1P concentrations, were fractionated into very low-density lipoprotein (VLDL)-, low density lipoprotein (LDL)-, high density lipoprotein (HDL)-, and lipoprotein-free fractions. S1P was then measured in each fraction to determine its distribution. Results The mean S1P concentration in serum (1.04 ± 0.24 nmol/mL) was found 39% higher compared with plasma (0.75 ± 0.16 nmol/mL) and overall was not different between men and women. Only when stratified for age and gender, older women were found to exhibit higher circulatory S1P levels than men. In plasma, S1P levels correlate to red blood cell (RBC) counts but not to platelet counts. Conversely, serum-S1P correlates to platelet counts but not to RBC counts. In addition, eosinophil counts are strongly associated with serum-S1P concentrations. Both serum- and plasma-S1P correlate to total cholesterol but not to HDL-C. The distribution of S1P between VLDL-, LDL-, HDL-, and lipoprotein-free fractions is independent of total plasma-S1P concentrations. S1P concentrations in HDL but not in LDL are highly variable. Conclusion These data indicate S1P concentrations in plasma and serum to be differentially associated with cell counts and S1P carrier proteins. Besides platelets, eosinophil counts are identified as a novel determinant for serum-S1P concentrations further suggesting a role for S1P in eosinophil pathologies.
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In ribosome-associated quality control (RQC), Rqc2/NEMF closely supports the E3 ligase Ltn1/listerin in promoting ubiquitylation and degradation of aberrant nascent-chains obstructing large (60S) ribosomal subunits-products of ribosome stalling during translation. However, while Ltn1 is eukaryote-specific, Rqc2 homologs are also found in bacteria and archaea; whether prokaryotic Rqc2 has an RQC-related function has remained unknown. Here, we show that, as in eukaryotes, a bacterial Rqc2 homolog (RqcH) recognizes obstructed 50S subunits and promotes nascent-chain proteolysis. Unexpectedly, RqcH marks nascent-chains for degradation in a direct manner, by appending C-terminal poly-alanine tails that act as degrons recognized by the ClpXP protease. Furthermore, RqcH acts redundantly with tmRNA/ssrA and protects cells against translational and environmental stresses. Our results uncover a proteolytic-tagging mechanism with implications toward the function of related modifications in eukaryotes and suggest that RQC was already active in the last universal common ancestor (LUCA) to help cope with incomplete translation.
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Alanina/metabolismo , Bacillus subtilis/metabolismo , Células Procariotas/metabolismo , Proteolisis , Subunidades Ribosómicas Grandes Bacterianas/metabolismo , Células Eucariotas/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , ARN de Transferencia/metabolismo , Proteínas de Unión al ARN/metabolismo , Subunidades Ribosómicas Grandes de Eucariotas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
Alkbh1 is a mammalian homolog of the Escherichia coli DNA repair enzyme AlkB, an Fe(II) and 2-oxoglutarate dependent dioxygenase that removes alkyl lesions from DNA bases. The human homolog ALKBH1 has been associated with six different enzymatic activities including DNA, mRNA, or tRNA hydroxylation, cleavage at abasic (AP) sites in DNA, as well as demethylation of histones. The reported cellular roles of this protein reflect the diverse enzymatic activities and include direct DNA repair, tRNA modification, and histone modification. We demonstrate that ALKBH1 produced in mammalian cells (ALKBH1293) is similar to the protein produced in bacteria (ALKBH1Ec) with regard to its m6A demethylase and AP lyase activities. In addition, we find that ALKBH1293 forms a covalent adduct with the 5' product of the lyase product in a manner analogous to ALKBH1Ec. Localization and subcellular fractionation studies with the endogenous protein in two human cell strains confirm that ALKBH1 is primarily in the mitochondria. Two strains of CRISPR/Cas9-created ALKBH1-deficient HEK293 cells showed increases in mtDNA copy number and mitochondrial dysfunction as revealed by growth measurements and citrate synthase activity assays.