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The adaptor molecule stimulator of IFN genes (STING) is central to production of type I IFNs in response to infection with DNA viruses and to presence of host DNA in the cytosol. Excessive release of type I IFNs through STING-dependent mechanisms has emerged as a central driver of several interferonopathies, including systemic lupus erythematosus (SLE), Aicardi–Goutières syndrome (AGS), and stimulator of IFN genes-associated vasculopathy with onset in infancy (SAVI). The involvement of STING in these diseases points to an unmet need for the development of agents that inhibit STING signaling. Here, we report that endogenously formed nitro-fatty acids can covalently modify STING by nitro-alkylation. These nitro-alkylations inhibit STING palmitoylation, STING signaling, and subsequently, the release of type I IFN in both human and murine cells. Furthermore, treatment with nitro-fatty acids was sufficient to inhibit production of type I IFN in fibroblasts derived from SAVI patients with a gain-of-function mutation in STING. In conclusion, we have identified nitro-fatty acids as endogenously formed inhibitors of STING signaling and propose for these lipids to be considered in the treatment of STING-dependent inflammatory diseases.
Background: In endothelial cells, activation of the AMP-activated protein kinase (AMPK) has been linked with anti-inflammatory actions but the events downstream of kinase activation are not well understood. Here, we addressed the effects of AMPK activation/deletion on the activation of NFKappaB and determined whether the AMPK could contribute to the anti-inflammatory actions of nitric oxide (NO). Methodology/Principal Findings: Overexpression of a dominant negative AMPKalpha2 mutant in tumor necrosis factor-alpha-stimulated human endothelial cells resulted in increased NFKappaB activity, E-selectin expression and monocyte adhesion. In endothelial cells from AMPKalpha2-/- mice the interleukin (IL)-1beta induced expression of E-selectin was significantly increased. DETA-NO activated the AMPK and attenuated NFKappaB activation/E-selectin expression, effects not observed in human endothelial cells in the presence of the dominant negative AMPK, or in endothelial cells from AMPKalpha2-/- mice. Mechanistically, overexpression of constitutively active AMPK decreased the phosphorylation of IKappaB and p65, indicating a link between AMPK and the IKappaB kinase (IKK). Indeed, IKK (more specifically residues Ser177 and Ser181) was found to be a direct substrate of AMPKalpha2 in vitro. The hyper-phosphorylation of the IKK, which is known to result in its inhibition, was also apparent in endothelial cells from AMPKalpha2+/+ versus AMPKalpha2-/- mice. Conclusions: These results demonstrate that the IKK is a direct substrate of AMPKalpha2 and that its phosphorylation on Ser177 and Ser181 results in the inhibition of the kinase and decreased NFKappaB activation. Moreover, as NO potently activates AMPK in endothelial cells, a portion of the anti-inflammatory effects of NO are mediated by AMPK.
Increased oxidative stress and reduced nitric oxide (NO) bioavailability play a causal role in endothelial cell dysfunction occurring in the vasculature of diabetic patients. In this review, we summarized the molecular mechanisms underpinning diabetic endothelial and vascular dysfunction. In particular, we focused our attention on the complex interplay existing among NO, reactive oxygen species (ROS), and one crucial regulator of intracellular ROS production, p66Shc protein.
In cultured human endothelial cells, physiological levels of NO prevent apoptosis and interfere with the activation of the caspase cascade. In vitro data have demonstrated that NO inhibits the activity of caspase-3 by S-nitrosation of the enzyme. Here we present evidence for the in vivo occurrence and functional relevance of this novel antiapoptotic mechanism. To demonstrate that the cysteine residue Cys-163 of caspase-3 is S-nitrosated, cells were transfected with the Myc-tagged p17 subunit of caspase-3. After incubation of the transfected cells with different NO donors, Myc-tagged p17 was immunoprecipitated with anti-Myc antibody. S-Nitrosothiol was detected in the immunoprecipitate by electron spin resonance spectroscopy after liberation and spin trapping of NO by N-methyl-D-glucamine-dithiocarbamate-iron complex. Transfection of cells with a p17 mutant, where the essential Cys-163 was mutated into alanine, completely prevented S-nitrosation of the enzyme. As a functional correlate, in human umbilical vein endothelial cells the NO donors sodium nitroprusside or PAPA NONOate (50 microM) significantly reduced the increase in caspase-3-like activity induced by overexpressing caspase-3 by 75 and 70%, respectively. When human umbilical vein endothelial cells were cotransfected with beta-galactosidase, morphological analysis of stained cells revealed that cell death induction by overexpression of caspase-3 was completely suppressed in the presence of sodium nitroprusside, PAPA NONOate, or S-nitroso-L-cysteine (50 microM). Thus, NO supplied by exogenous NO donors serves in vivo as an antiapoptotic regulator of caspase activity via S-nitrosation of the Cys-163 residue of caspase-3.
Excessive accumulation of the extracellular matrix is a hallmark of many inflammatory and fibrotic diseases, including those of the kidney. This study addresses the question whether NO, in addition to inhibiting the expression of MMP-9, a prominent metalloprotease expressed by mesangial cells, additionally modulates expression of its endogenous inhibitor TIMP-1. We demonstrate that exogenous NO has no modulatory effect on the extracellular TIMP-1 content but strongly amplifies the early increase in cytokine-induced TIMP-1 mRNA and protein levels. We examined whether transforming growth factor beta (TGFbeta), a potent profibrotic cytokine, is involved in the regulation of NO-dependent TIMP-1 expression. Experiments utilizing a pan-specific neutralizing TGFbeta antibody demonstrate that the NO-induced amplification of TIMP-1 is mediated by extracellular TGFbeta. Mechanistically, NO causes a rapid increase in Smad-2 phosphorylation, which is abrogated by the addition of neutralizing TGFbeta antisera. Similarly, the NO-dependent increase in Smad-2 phosphorylation is prevented in the presence of an inhibitor of TGFbeta-RI kinase, indicating that the NO-dependent activation of Smad-2 occurs via the TGFbeta-type I receptor. Furthermore, activation of the Smad signaling cascade by NO is corroborated by the NO-dependent increase in nuclear Smad-4 level and is paralleled by increased DNA binding of Smad-2/3 containing complexes to a TIMP-1-specific Smad-binding element (SBE). Reporter gene assays revealed that NO activates a 0.6-kb TIMP-1 gene promoter fragment as well as a TGFbeta-inducible and SBE-driven control promoter. Chromatin immunoprecipitation analysis also demonstrated DNA binding activity of Smad-3 and Smad-4 proteins to the TIMP-1-specific SBE. Finally, by enzyme-linked immunosorbent assay, we demonstrated that NO causes a rapid increase in TGFbeta(1) levels in cell supernatants. Together, these experiments demonstrate that NO by induction of the Smad signaling pathway modulates TIMP-1 expression.
Ceramide levels are strongly increased by stimulation of renal mesangial cells with nitric oxide (NO). This effect was shown previously to be due to a dual action of NO, comprising an activation of sphingomyelinases and an inhibition of ceramidase activity. In this study we show that the NO-triggered inhibition of neutral ceramidase activity is paralleled by a down-regulation at the protein level. A complete loss of neutral ceramidase protein is obtained after 24 h of stimulation. Whereas the selective proteasome inhibitor lactacystin blocked NO-evoked ceramidase degradation, several caspase inhibitors were ineffective. Moreover, the NO-induced degradation is reversed by the protein kinase C (PKC) activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), and also by the physiological PKC activators platelet-derived growth factor-BB (PDGF), angiotensin II and ATP, resulting in a normalization of neutral ceramidase protein as well as activity. In vivophosphorylation studies using 32Pi-labeled mesangial cells revealed that TPA, PDGF, angiotensin II, and ATP trigger an increased phosphorylation of the neutral ceramidase, which is blocked by the broad spectrum PKC inhibitor Ro-31 8220 but not by CGP 41251, which has a preferential action on Ca2+-dependent isoforms, thus suggesting the involvement of a Ca2+-independent PKC isoform. In vitro phosphorylation assays using recombinant PKC isoenzymes and neutral ceramidase immunoprecipitated from unstimulated mesangial cells show that particularly the PKC-δ isoform and to a lesser extent the PKC-α isoform are efficient in directly phosphorylating neutral ceramidase. In summary, our data show that NO is able to induce degradation of neutral ceramidase, thereby promoting accumulation of ceramide in the cell. This effect is reversed by PKC activation, most probably by the PKC-δ isoenzyme, which can directly phosphorylate and thereby prevent neutral ceramidase degradation. These novel regulatory interactions will provide therapeutically valuable information to target neutral ceramidase stability and subsequent ceramide accumulation.
MAP kinase-dependent phosphorylation processes have been shown to interfere with the degradation of the antiapoptotic protein Bcl-2. The cytosolic MAP kinase phosphatase MAP kinase phosphatase-3 (MKP-3) induces apoptosis of endothelial cells in response to tumor necrosis factor alpha (TNFalpha) via dephosphorylation of the MAP kinase ERK1/2, leading to Bcl-2 proteolysis. Here we report that the endothelial cell survival factor nitric oxide (NO) down-regulated MKP-3 by destabilization of MKP-3 mRNA. This effect of NO was paralleled by a decrease in MKP-3 protein levels. Moreover, ERK1/2 was found to be protected against TNFalpha-induced dephosphorylation by coincubation of endothelial cells with the NO donor. Subsequently, both the decrease in Bcl-2 protein levels and the mitochondrial release of cytochrome c in response to TNFalpha were largely prevented by exogenous NO. In cells overexpressing MKP-3, no differences in phosphatase activity in the presence or absence of NO were found, excluding potential posttranslational modifications of MKP-3 protein by NO. These data demonstrate that upstream of the S-nitrosylation of caspase-3, NO exerts additional antiapoptotic effects in endothelial cells, which rely on the down-regulation of MKP-3 mRNA.
Nitric oxide donors induce stress signaling via ceramide formation in rat renal mesangial cells
(1999)
Exogenous NO is able to trigger apoptosis of renal mesangial cells, and thus may contribute to acute lytic phases as well as to resolution of glomerulonephritis. However, the mechanism involved in these events is still unclear. We report here that chronic exposure of renal mesangial cells for 24 h to compounds releasing NO, including spermine-NO, (Z)-1-{N-methyl-N-[6-(N-methylammoniohexyl)amino]}diazen-1-ium-1,2-diolate (MAHMA-NO), S-nitrosoglutathione (GS-NO), and S-nitroso-N-acetyl-d,l-penicillamine (SNAP) results in a potent and dose-dependent increase in the lipid signaling molecule ceramide. Time courses reveal that significant effects occur after 2–4 h of stimulation with NO donors and reach maximal levels after 24 h of stimulation. No acute (within minutes) ceramide production can be detected. When cells were stimulated with NO donors in the presence of phorbol ester, a direct activator of protein kinase C, both ceramide production and DNA fragmentation are completely abolished. Furthermore, addition of exogenous ceramide partially reversed the inhibitory effect of phorbol ester on apoptosis, thus suggesting a negative regulation of protein kinase C on ceramide formation and apoptosis. In contrast to exogenous NO, tumor necrosis factor (TNF)-α stimulates a very rapid and transient increase in ceramide levels within minutes but fails to induce the late-phase ceramide formation. Moreover, TNF fails to induce apoptosis in mesangial cells. Interestingly, NO and TNFα cause a chronic activation of acidic and neutral sphingomyelinases, the ceramide-generating enzymes, whereas acidic and neutral ceramidases, the ceramide-metabolizing enzymes, are inhibited by NO, but potently stimulated by TNFα. Furthermore, in the presence of an acidic ceramidase inhibitor,N-oleoylethanolamine, TNFα leads to a sustained accumulation of ceramide and in parallel induces DNA fragmentation. In summary, our data demonstrate that exogenous NO causes a chronic up-regulation of ceramide levels in mesangial cells by activating sphingomyelinases and concomitantly inhibiting ceramidases, and that particularly the late-phase of ceramide generation may be responsible for the further processing of a proapoptotic signal.
Upregulations of neuronal nitric oxide synthase (nNOS) in the rodent brain have been associated with neuronal aging. To address underlying mechanisms we generated SH-SY5Y neuronal cells constitutively expressing nNOS at a level similar to mouse brain (nNOS+ versus MOCK). Initial experiments revealed S-nitrosylations (SNO) of key players of protein homeostasis: heat shock cognate HSC70/HSPA8 within its nucleotide-binding site, and UBE2D ubiquitin conjugating enzymes at the catalytic site cysteine. HSPA8 is involved in protein folding, organelle import/export and chaperone-mediated LAMP2a-dependent autophagy (CMA). A set of deep redox and full proteome analyses, plus analysis of autophagy, CMA and ubiquitination with rapamycin and starvation as stimuli confirmed the initial observations and revealed a substantial increase of SNO modifications in nNOS+ cells, in particular targeting protein networks involved in protein catabolism, ubiquitination, carbohydrate metabolism and cell cycle control. Importantly, NO-independent reversible oxidations similarly occurred in both cell lines. Functionally, nNOS caused an accumulation of proteins, including CMA substrates and loss of LAMP2a. UBE2D activity and proteasome activity were impaired, resulting in dysregulations of cell cycle checkpoint proteins. The observed changes of protein degradation pathways caused an expansion of the cytoplasm, large lysosomes, slowing of the cell cycle and suppression of proliferation suggesting a switch of the phenotype towards aging, supported by downregulations of neuronal progenitor markers but increase of senescence-associated proteins. Hence, upregulation of nNOS in neuronal cells imposes aging by SNOing of key players of ubiquitination, chaperones and of substrate proteins leading to interference with crucial steps of protein homeostasis.
Nitric oxide causes ADP-ribosylation and inhibition of glyceraldehyde-3-phosphate dehydrogenase
(1992)
Nitric oxide and nitric oxide-generating agents like 3-morpholinosydnonimine (SIN-1) stimulate the mono-ADP-ribosylation of a cytosolic, 39-kDa protein in various tissues. This protein was purified from human platelet cytosol by conventional and fast protein liquid chromatography techniques. N-terminal sequence analysis identified the isolated protein as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Nitric oxide stimulates the auto-ADP-ribosylation of GAPDH in a time and concentration-dependent manner with maximal effects after about 60 min. Associated with ADP-ribosylation is a loss of enzymatic activity. NAD(+)-free enzyme is not inhibited by SIN-1, indicating the absolute requirement of NAD+ as the substrate of the ADP-ribosylation reaction. Inhibition of the glycolytic enzyme GAPDH may be relevant as a cytotoxic effect of NO complementary to its inhibitory actions on iron-sulfur enzymes like aconitase and electron transport proteins of the respiratory chain.
Aim: To assess outcomes in patients with advanced adenocarcinoma non-small-cell lung cancer who received nintedanib plus docetaxel after progression on prior chemotherapy followed by immune checkpoint inhibitor (ICI) therapy. Patients & methods: VARGADO is a prospective, noninterventional study. We describe initial data from a cohort of 22 patients who received nintedanib plus docetaxel after chemotherapy and ICI therapy. Results: Median progression-free survival with nintedanib plus docetaxel was 5.5 months (95% CI: 1.9–8.7 months). The objective response rate was 7/12 (58%) and the disease control rate was 10/12 (83%). Data for overall survival rate 12 months after the start of treatment (primary end point) are not yet mature and are not reported. Of 22 patients, 73% experienced drug-related adverse events; adverse events led to treatment discontinuation in 32% of patients. Conclusion: These data highlight the potential clinical benefit of nintedanib plus docetaxel in patients who failed prior ICI therapy.
Trial registration number: NCT02392455
Background: Nerve injury induced protein 1 (Ninjurin 1 (Ninj1)) was first identified in Schwann cells and neurons contributing to cell adhesion and nerve regeneration. Recently, the role of Ninj1 has been linked to inflammatory processes in the central nervous system where functional repression reduced leukocyte infiltration and clinical disease activity during experimental autoimmune encephalomyelitis in mice [1]. But Ninj1 is also expressed outside the nervous system in various organs such as the liver and kidney as well as on leukocytes [2,3]. Therefore, we hypothesized that Ninj1 contributes to inflammation in general; that is, also outside the nervous system, with special interest in the pathogenesis of sepsis.
Methods: Ninj1 was repressed by transfecting HMEC-1 cells, a human dermal microvascular endothelial cell line with siRNA targeting Ninj1 (siNinj1) or a negative control (siC). Subsequently, cells were stimulated with 100 ng/ml LPS (TLR4 agonist), 3 μg/ml LTA (TLR2 agonist) or 100 n/ml poly(I:C) (TLR3 agonist) for 3 hours. The inflammatory response was analyzed by real-time PCR. In addition, transmigration of neutrophils across a HMEC-1 monolayer was measured using transwell plates (pore size 3 μm).
Results: Repression of Ninj1 by siRNA reduced Ninj1 mRNA expression in HMEC about 90% (Figure 1A). Reduced Ninj1 expression decreased neutrophil migration to 62.5% (Figure 1B) and TLR signaling. In detail, knockdown of Ninj1 significantly reduced TLR-2 and TLR-4 triggered expression of ICAM-1 and IL-6 (Figure 1C,D) while poly(I:C)-induced expression was only slightly reduced. To analyze a more specific TLR-3 target, we measured IP-10 mRNA expression, which was also significantly reduced in siNinj1-transfected cells (Figure 1E).
Conclusion: Our in vitro data strongly indicated that Ninj1 is involved in regulation of TLR signaling and therewith contributes to inflammation. In vivo experiments will clarify its impact on systemic inflammation.
Nikotinabhänigkeit und -abstinenz sind mit erniedrigtem bzw. erhöhtem Körpergewicht assoziiert. Dem Nucleus arcuatus kommt eine Schlüsselrolle bei der Körpergewichtsregulation zu, und es wurde bereits gezeigt, dass eine inadäquate Genexpression in diesem hypothalamischen Kerngebiet zu Adipositas führen kann. Obwohl bekannt ist, dass Nikotin die Genexpression im Hypothalamus beeinflussen kann, sind die genauen Mechanismen, die zu den anorektischen Effekten führen, noch weitgehend unbekannt. Diese Studie wurde durchgeführt, um eine Methode zu etablieren, mit der umfassende und informative Genexpressionsprofile im Nucleus arcuatus - unter anderem - in Abhänigkeit von Nikotinwirkungen erstellt werden können. Hierzu wurden Microarrays mit einer limitierten Zahl an Genen hergestellt, für die teilweise bereits Informationen zu Nikotin-abhängiger Gegenregulation verfügbar waren. Fünfundzwanzig Fischer-Ratten wurden miniosmotische Pumpen implantiert, die entweder Nikotin (9mg/kg Körpergewicht/ Tag) oder physiologische Kochsalzlösung freisetzten. Nach einer Woche wurde ein Teil der Tiere getötet, während bei den übrigen Tieren die Effekte einer dreitägigen Nikotinabstinenz untersucht wurden. Die Nikotinbehandlung war mit einer reduzierten Gewichtszunahme sowie einer erhöhten Rektaltemperatur (p=0,001) assoziiert. Die Nikotinabstinenz führte dagegen zu einer gesteigerten Gewichtszunahme (p=0,012), einer erniedrigten Rektaltemperatur sowie einer höheren Serumglukosekonzentration (p=0,028). Für die Serumkonzentrationen von Insulin, Leptin, Cholesterin oder Triglyceriden konnten keine signifikanten Unterschiede aufgezeigt werden, was möglicherweise aber auf die relativ geringe Anzahl an Versuchstieren zurückzuführen ist. Mit den Microarray-Studien ließen sich die vorbeschriebenen Daten für die Nikotin-abhängige Regulation der Genexpression von NPY sowie teilweise auch von POMC bestätigen. Darüberhinaus konnten neue Informationen im Hinblick auf die Nikotinabhängige Regulation der Genexpression von AGRP, LepRb, Insulinrezeptor, MC3R, NPY-Y5-Rezeptor, Prohormone-Convertase-1, POMC und SOCS-3 gewonnen werden. Zusätzlich zu den neuen Daten stellen wir somit einen methodischen Ansatz vor, mit dem Expressionsprofile für viele hundert hypothalamischen Genen in Abhängigkeit von Manipulationen des Körpergewichts erstellt werden können, um so dann tiefgreifende Einsichten in die neuronalen molekularen Mechanismen der Körpergewichtskontrolle nehmen zu können.
Einleitung: Die chronische Hepatitis C gehört zu den häufigen Ursachen einer Leberzirrhose. Durch die Entwicklung von direkt antiviralen Medikamenten (direct acting antiviral agent, DAA) können Heilungsraten von über 90% bei chronischer Hepatitis C erreicht werden. Der Einfluss des Therapieerfolgs auf Fibroseregression und portale Hypertension ist insbesondere für Patienten mit Leberzirrhose bisher nicht ausreichend geklärt. Elastographische Messungen von Leber und Milz können als Surrogatmarker zur nicht-invasiven Evaluation von Fibroseregression und Rückgang von portaler Hypertension dienen. Ziel der vorliegenden Arbeit war die Evaluation der Dynamik von Leberfibrose und portaler Hypertension mittels transienter Elastographie der Leber (L-TE) und Acoustic Radiation Force Impulse Elastographie von Leber (L-ARFI) und Milz (M-ARFI) bei Patienten mit DAA-induziertem anhaltendem virologischen Ansprechen (sustained virological response, SVR) und Hepatitis C Virus (HCV) assoziierter Leberzirrhose.
Patienten und Methoden: In dieser prospektiven, monozentrischen Studie wurden Daten von 56 Patienten mit chronischer Hepatitis C und assoziierter Leberzirrhose analysiert, die eine SVR nach 12-24 Wochen antiviraler Hepatitis C Therapie mit DAA's erreichten. Dabei wurden zu vier Zeitpunkten (Therapiebeginn [BL], Therapieende [EOT], 24 Wochen nach Therapieende [FU24] und 48 Wochen nach Therapieende [FU48]) Messungen vorgenommen. Zusätzlich wurden Leberzirrhose assoziierte Scores und Komplikationen sowie Laborparameter erhoben.
Ergebnisse: Die elastographischen Messungen der Leber zeigten signifikante Verbesserungen im Studienverlauf, am stärksten während der antiviralen Therapie. Im L-TE zeigten sich Verbesserungen zwischen BL [Median(Min-Max), 32,45(9,1-75)kPa] und FU48[Median (Min-Max), 17,1 (3,7-59,3) kPa] (p<0,0001) und im L-ARFI zwischen BL [Median (Min-Max), 2,7 (1,2-4,1) m/s] und FU24 [Median (Min-Max), 2,5 (1,2-3,9)m/s (p=0,011), wobei signifikante Verbesserungen (≥30%) bei 35/49 (71%) Patienten im L-TE und bei 6/55 (11%) im L-ARFI auftraten. Die M-ARFI Messungen als Korrelat der portalen Hypertension, zeigten kaum Veränderungen. Die medianen Werte blieben zwischen BL [Median (Min-Max), 3,4 (1,9-4,3) m/s] und FU48 [Median (Min-Max), 3,4(2-4,4) m/s] konstant (p=0,9). Nur 2/54 (4%) der Patentien zeigten signifikante Verbesserungen. In der univariaten Analyse zeigten sich 65 als positive Einflussfaktoren der L-TE- und L-ARFI-Verbesserung ein MELD Score bis 10 sowie ein BMI bis 30 kg/m².
Diskussion und Schlussfolgerung: In Zusammenschau der Ergebnisse kann bei einem Teil der Patienten von einer Fibroseregression und dem Rückgang der portalen Hypertension nach DAA-basierter, interferon-freier antiviraler Therapie ausgegangen werden. Die mittels L-ARFI und L-TE gemessene Verbesserung der Lebersteifigkeit unter antiviraler Therapie scheint auf einer Verbesserung von Nekroinflammation und Leberfibrose zu basieren. Bei Nichtbeachtung des Einflusses der Nekroinflammation auf nicht-invasive Messungen der Lebersteifigkeit kann es zur Überschätzung der Fibroseregression nach SVR kommen. Da es sich bei der Zirrhose- bzw. Fibroseregression anscheinend um einen langsamen Prozess handelt, sind ausreichend lange Nachbeobachtungszeiten in entsprechenden Studien zu fordern. Für die mit einer Leberzirrhose assoziierte portale Hypertension gelten ähnliche Überlegungen.
Zusammenfassung der 4-Zeilen vs. 16-Zeilen MDCT Untersuchung der Bypässe Der Zweck dieser Studie war es die Bildqualität von aortokoronaren Bypassdarstellungen in 4- und 16-Zeilen Multidetektor-CT mit verschiedenen Bildreformationen zu untersuchen. Materialien und Methoden: 116 Patienten unterzogen sich einer CTUntersuchung nach einer Bypassoperation. Gruppe A (n=58) wurde mit einem 4-Zeiler untersucht; Gruppe B (n=58) wurde mit einem 16-Zeiler untersucht. Verschiedene Bypasstypen wie die LITA zur LAD und venöse Gefäße zur RCA und zur RCX wurden mit in die Studie eingeschlossen. Eine Fünf-Punkte-Lickert-Skala wurde zur Beurteilung der Bildqualität benutzt. Jeder Bypass wurde mitverschiedenen Bildreformationen betrachtet: MIP thin, MPR, VRT. Besonderes Augenmerk wurde auf die Darstellbarkeit der distalen Anastomose gelegt. Die Korrelation zwischen den Untersuchern wurde festgestellt. Resultate: Von 289 untersuchten Bypassgefäßen wurden 279 (96,54%) als funktionsfähig und 10 (3,46%) als nicht funktionsfähig klassifiziert. Bis auf die distalen Anastomosen zeigte der 16-Zeiler signifikant bessere Resultate in allen Segmenten der Bypasses. Vergleiche der Reformationen innerhalb der Gruppen A und B zeigten das mit der MIP thin (p<0,05) und der VRT (p<0,05) bessere Bildqualitäten möglich sind als im Vergleich mit MPR. Schlussfolgerung: Signifikant bessere Darstellbarkeit aller Bypasstypen ist mit dem 16-Zeiler im Vergleich zum 4-Zeiler möglich. Bei der distalen Anastomose scheint kein Unterschied bei der Darstellbarkeit zwischen den beiden 4- bzw. 16- Zeiler Technologien zu bestehen. Zusammenfassung der 4-Zeilen vs. 16-Zeilen MDCT proximale Aortenkonnektoren Untersuchung Der Zweck dieser Studie war es die Bildqualität der proximalen Anastomose von aortokoronaren Bypässen zu untersuchen die mit Hilfe von Nitinolkonnektoren an der Aorta befestigt wurden. Materialien und Methoden: 33 Patienten wurden sofort nach der Bypassoperation mit dem CT untersucht. Die selben Patienten wurden am fünften postoperativen Tag (4Zeilen CT/Gruppe A) und ein Jahr später nochmals (16-Zeiler CT/Gruppe B) untersucht. 23 ACVB auf RCX Bypässe und 27 ACVB auf RCA Bypässe wurden in die Studie eingeschlossen. Jede Anastomose wurde mit verschiedenen Reformationen (MIP,MPR und VRT) ausgewertet. Die Korrelation zwischen den Untersuchern wurde festgestellt. Resultate: Fünf Tage nach der Operation wurden 48 von 50 Bypässe als durchgängig und funktionsfähig klassifiziert. Ein Jahr später sind es noch 42 von 50 Bypässe. In beiden Gruppen konnte der Aortenkonnektor in guter Qualität dargestellt werden: 1.82±1.10 (Gruppe A) und 1.93±1.22 (Gruppe B) für ACVB auf RCA Bypässe, die selben Ergebnisse für ACVB auf RCX Bypässe (MPR). Die Bildqualität auf der Aortenkonnektorseite zeigte keinen signifikanten Unterschied zwischen jeglichen Reformationen beider Gruppen). Innerhalb der Gruppen zeigte die MPR eine bessere Darstellbarkeit als die MIP und VRT (P>0.05). Schlussfolgerung: In der Anwesenheit von metallischen Implantaten, hier im Falle der Nitinolkonnektoren, bietet das CT vertrauenwürdige Daten zur Untersuchung der proximalen Anastomose. Das 16-Zeilen CT bringt keine signifikante Verbesserung der Bildqualität. MPR zeigte die beste Darstellbarkeit innerhalb beider Gruppen.
Aufgrund der leichten Handhabung und des Nachweises einer Mortalitätssenkung gilt der Nachweis von okkultem Blut (FOBT) im Stuhl derzeit als das am weitesten verbreitete Screeningverfahren für das kolorektale Karzinom. Als nachteilig erweisen sich allerdings eine unzureichende Sensitivität, insbesondere beim Nachweis früher Stadien und eine nach wie vor geringe Akzeptanz in der Bevölkerung. Vorläufige Daten zum Nachweis von Calprotectin oder der Tumor-M2-PK im Stuhl ließen bessere Screeningeigenschaften erwarten. Aber auch hierschränkt die geringe Sensitivität für frühe Vorstufen und unzureichende Spezifität mit zu erwartenden hohen Folgekosten die Tauglichkeit der Tests deutlich ein. Die kürzlich entwickelten immunologischen FOBTs (I-FOBT)erweisen sich als spezifischer und sensitiver. Sie beruhen auf dem Nachweis von humanem Hämoglobin mittels spezifischer Antikörper und sind somit unabhängig von diätetischen oder medikamentösen Faktoren, was zu einer deutlich besseren Akzeptanz führt. Sie gelten derzeit als kosteneffektivste Verfahren unter den nichtinvasiven Screeningmaßnahmen. Der Nachweis von Tumor-DNA im Stuhl eröffnet eine neue Ära zum frühzeitigen Nachweis kolorektaler Karzinome. Erste kleinere Studien weisen auf eine sehr gute Sensitivität dieser Verfahren hin. Sie lagen für kolorektale Karzinome zwischen 62–91% und für Adenome zwischen 26–73% bei mit 93–100% sehr guter Spezifität. Als nachteilig im Ver-gleich zu den derzeit verfügbaren Screeningtests erweisen sich allerdings die vergleichsweise hohen Kosten.
NF-κB is involved in immune responses, inflammation, oncogenesis, cell proliferation and apoptosis. Even though NF-κB can be activated by DNA damage via Ataxia telangiectasia-mutated (ATM) signalling, little was known about an involvement in DNA repair. In this work, we dissected distinct DNA double-strand break (DSB) repair mechanisms revealing a stimulatory role of NF-κB in homologous recombination (HR). This effect was independent of chromatin context, cell cycle distribution or cross-talk with p53. It was not mediated by the transcriptional NF-κB targets Bcl2, BAX or Ku70, known for their dual roles in apoptosis and DSB repair. A contribution by Bcl-xL was abrogated when caspases were inhibited. Notably, HR induction by NF-κB required the targets ATM and BRCA2. Additionally, we provide evidence that NF-κB interacts with CtIP-BRCA1 complexes and promotes BRCA1 stabilization, and thereby contributes to HR induction. Immunofluorescence analysis revealed accelerated formation of replication protein A (RPA) and Rad51 foci upon NF-κB activation indicating HR stimulation through DSB resection by the interacting CtIP-BRCA1 complex and Rad51 filament formation. Taken together, these results define multiple NF-κB-dependent mechanisms regulating HR induction, and thereby providing a novel intriguing explanation for both NF-κB-mediated resistance to chemo- and radiotherapies as well as for the sensitization by pharmaceutical intervention of NF-κB activation
Lysine-specific demethylase 1 (LSD1), a histone lysine demethylase with the main specificity for H3K4me2, has been shown to be overexpressed in rhabdomyosarcoma (RMS) tumor samples. However, its role in RMS biology is not yet well understood. Here, we identified a new role of LSD1 in regulating adhesion of RMS cells. Genetic knockdown of LSD1 profoundly suppressed clonogenic growth in a panel of RMS cell lines, whereas LSD1 proved to be largely dispensable for regulating cell death and short-term survival. Combined RNA and ChIP-sequencing performed to analyze RNA expression and histone methylation at promoter regions revealed a gene set enrichment for adhesion-associated terms upon LSD1 knockdown. Consistently, LSD1 knockdown significantly reduced adhesion to untreated surfaces. Importantly, precoating of the plates with the adhesives collagen I or fibronectin rescued this reduced adhesion of LSD1 knockdown cells back to levels of control cells. Using KEGG pathway analysis, we identified 17 differentially expressed genes (DEGs) in LSD1 knockdown cells related to adhesion processes, which were validated by qRT-PCR. Combining RNA and ChIP-sequencing results revealed that, within this set of genes, SPP1, C3AR1, ITGA10 and SERPINE1 also exhibited increased H3K4me2 levels at their promoter regions in LSD1 knockdown compared to control cells. Indeed, LSD1 ChIP experiments confirmed enrichment of LSD1 at their promoter regions, suggesting a direct transcriptional regulation by LSD1. By identifying a new role of LSD1 in the modulation of cell adhesion and clonogenic growth of RMS cells, these findings highlight the importance of LSD1 in RMS.
Background: Transient receptor potential cation channel subfamily V member 1 (TRPV1) are sensitive to heat, capsaicin, pungent chemicals and other noxious stimuli. They play important roles in the pain pathway where in concert with proinflammatory factors such as leukotrienes they mediate sensitization and hyperalgesia. TRPV1 is the target of several novel analgesics drugs under development and therefore, TRPV1 genetic variants might represent promising candidates for pharmacogenetic modulators of drug effects.
Methods: A next-generation sequencing (NGS) panel was created for the human TRPV1 gene and in addition, for the leukotriene receptors BLT1 and BLT2 recently described to modulate TRPV1 mediated sensitisation processes rendering the coding genes LTB4R and LTB4R2 important co-players in pharmacogenetic approaches involving TRPV1. The NGS workflow was based on a custom AmpliSeq™ panel and designed for sequencing of human genes on an Ion PGM™ Sequencer. A cohort of 80 healthy subjects of Western European descent was screened to evaluate and validate the detection of exomic sequences of the coding genes with 25 base pair exon padding.
Results: The amplicons covered approximately 97% of the target sequence. A median of 2.81 x 10 6 reads per run was obtained. This identified approximately 140 chromosome loci where nucleotides deviated from the reference sequence GRCh37 hg19 comprising the three genes TRPV1, LTB4R and LTB4R2. Correspondence between NGS and Sanger derived nucleotide sequences was 100%.
Conclusions: Results suggested that the NGS approach based on AmpliSeq™ libraries and Ion Personal Genome Machine (PGM) sequencing is a highly efficient mutation detection method. It is suitable for large-scale sequencing of TRPV1 and functionally related genes. The method adds a large amount of genetic information as a basis for complete analysis of TRPV1 ion channel genetics and its functional consequences.
Background: The opioid system is involved in the control of pain, reward, addictive behaviors and vegetative effects. Opioids exert their pharmacological actions through the agonistic binding at opioid receptors and variation in the coding genes has been found to modulate opioid receptor expression or signaling. However, a limited selection of functional opioid receptor variants is perceived as insufficient in providing a genetic diagnosis of clinical phenotypes and therefore, unrestricted access to opioid receptor genetics is required.
Methods: Next-generation sequencing (NGS) workflow was based on a custom AmpliSeq™ panel and designed for sequencing of human genes related to the opioid receptor group (OPRM1, OPRD1, OPRK1, SIGMA1, OPRL1) on an Ion PGM™ Sequencer. A cohort of 79 previously studied chronic pain patients was screened to evaluate and validate the detection of exomic sequences of the coding genes with 25 base pair exon padding. In-silico analysis was performed using SNP and Variation Suite® software.
Results: The amplicons covered approximately 90% of the target sequence. A median of 2.54 × 106 reads per run was obtained generating a total of 35,447 nucleotide reads from each DNA sample. This identified approximately 100 chromosome loci where nucleotides deviated from the reference sequence GRCh37 hg19, including functional variants such as the OPRM1 rs1799971 SNP (118 A > G) as the most scientifically regarded variant or rs563649 SNP coding for μ-opioid receptor splice variants. Correspondence between NGS and Sanger derived nucleotide sequences was 100%.
Conclusion: Results suggested that the NGS approach based on AmpliSeq™ libraries and Ion PGM sequencing is a highly efficient mutation detection method. It is suitable for large-scale sequencing of opioid receptor genes. The method includes the variants studied so far for functional associations and adds a large amount of genetic information as a basis for complete analysis of human opioid receptor genetics and its functional consequences.