The Serotonin Transporter (SERT) regulates extracellular serotonin levels and is the target of most current drugs used to treat depression. The mechanisms by which inhibition of SERT activity ...influences behavior are poorly understood. To address this question in the model organism Drosophila melanogaster, we developed new loss of function mutations in Drosophila SERT (dSERT). Previous studies in both flies and mammals have implicated serotonin as an important neuromodulator of sleep, and our newly generated dSERT mutants show an increase in total sleep and altered sleep architecture that is mimicked by feeding the SSRI citalopram. Differences in daytime versus nighttime sleep architecture as well as genetic rescue experiments unexpectedly suggest that distinct serotonergic circuits may modulate daytime versus nighttime sleep. dSERT mutants also show defects in copulation and food intake, akin to the clinical side effects of SSRIs and consistent with the pleomorphic influence of serotonin on the behavior of D. melanogaster. Starvation did not overcome the sleep drive in the mutants and in male dSERT mutants, the drive to mate also failed to overcome sleep drive. dSERT may be used to further explore the mechanisms by which serotonin regulates sleep and its interplay with other complex behaviors.
The decision to move towards a mating partner or a food source is essential for life. The mechanisms underlying these behaviors are not well understood. Here, we investigated the role of octopamine - ...the invertebrate analogue of noradrenaline - in innate olfactory attraction to ethanol. We confirmed that preference is caused via an olfactory stimulus by dissecting the function of the olfactory co-receptor Orco (formally known as OR83b). Orco function is not required for ethanol recognition per se, however it plays a role in context dependent recognition of ethanol. Odor-evoked ethanol preference requires the function of Tbh (Tyramine β hydroxalyse), the rate-limiting enzyme of octopamine synthesis. In addition, neuronal activity in a subset of octopaminergic neurons is necessary for olfactory ethanol preference. Notably, a specific neuronal activation pattern of tyraminergic/octopaminergic neurons elicit preference and is therefore sufficient to induce preference. In contrast, dopamine dependent increase in locomotor activity is not sufficient for olfactory ethanol preference. Consistent with the role of noradrenaline in mammalian drug induced rewards, we provide evidence that in adult Drosophila the octopaminergic neurotransmitter functions as a reinforcer and that the molecular dissection of the innate attraction to ethanol uncovers the basic properties of a response selection system.
The hangover gene defines a cellular stress pathway that is required for rapid ethanol tolerance in Drosophila melanogaster. To understand how cellular stress changes neuronal function, we analyzed ...Hangover function on a cellular and neuronal level. We provide evidence that Hangover acts as a nuclear RNA binding protein and we identified the phosphodiesterase 4d ortholog dunce as a target RNA. We generated a transcript-specific dunce mutant that is impaired not only in ethanol tolerance but also in the cellular stress response. At the neuronal level, Dunce and Hangover are required in the same neuron pair to regulate experience-dependent motor output. Within these neurons, two cyclic AMP (cAMP)-dependent mechanisms balance the degree of tolerance. The balance is achieved by feedback regulation of Hangover and dunce transcript levels. This study provides insight into how nuclear Hangover/RNA signaling is linked to the cytoplasmic regulation of cAMP levels and results in neuronal adaptation and behavioral changes.
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•The nuclear stress protein Hangover binds RNA.•The Pde4d ortholog dunce is a target of Hangover signaling.•Both mediate experience-dependent behavioral changes in the same neurons.•Hangover links two cAMP-dependent mechanisms to regulate ethanol tolerance.
Cellular stressors like ethanol cause specific behavioral changes. Ruppert et al. show that the nuclear stress RNA-interacting protein Hangover is broadly expressed but only required in neurons mediating experience-dependent changes in behavior. At the cellular level, Hangover links nuclear signaling to cAMP regulation via the phosphodiesterase 4d ortholog Dunce.
Provider: - Institution: - Data provided by Europeana Collections- Köln, Universität zu Köln, Diss., 2013- All metadata published by Europeana are available free of restriction under the Creative ...Commons CC0 1.0 Universal Public Domain Dedication. However, Europeana requests that you actively acknowledge and give attribution to all metadata sources including Europeana
Increased ZAP-70 expression predicts poor prognosis in chronic lymphocytic leukemia (CLL). Current methods for accurately measuring ZAP-70 expression are problematic, preventing widespread ...application of these tests in clinical decision making. We therefore used comprehensive DNA methylation profiling of the ZAP-70 regulatory region to identify sites important for transcriptional control.
High-resolution quantitative DNA methylation analysis of the entire ZAP-70 gene regulatory regions was conducted on 247 samples from patients with CLL from four independent clinical studies.
Through this comprehensive analysis, we identified a small area in the 5' regulatory region of ZAP-70 that showed large variability in methylation in CLL samples but was universally methylated in normal B cells. High correlation with mRNA and protein expression, as well as activity in promoter reporter assays, revealed that within this differentially methylated region, a single CpG dinucleotide and neighboring nucleotides are particularly important in ZAP-70 transcriptional regulation. Furthermore, by using clustering approaches, we identified a prognostic role for this site in four independent data sets of patients with CLL using time to treatment, progression-free survival, and overall survival as clinical end points.
Comprehensive quantitative DNA methylation analysis of the ZAP-70 gene in CLL identified important regions responsible for transcriptional regulation. In addition, loss of methylation at a specific single CpG dinucleotide in the ZAP-70 5' regulatory sequence is a highly predictive and reproducible biomarker of poor prognosis in this disease. This work demonstrates the feasibility of using quantitative specific ZAP-70 methylation analysis as a relevant clinically applicable prognostic test in CLL.
Bleomycin-induced lung injury leads to surfactant dysfunction and permanent loss of alveoli due to a remodeling process called collapse induration. Collapse induration also occurs in acute ...interstitial lung disease and idiopathic pulmonary fibrosis in humans. We hypothesized that surfactant dysfunction aggravates lung injury and early remodeling resulting in collapse induration within 7 days after lung injury. Rats received bleomycin to induce lung injury and either repetitive surfactant replacement therapy (SRT: 100 mg Curosurf/kg BW = surf group) or saline (0.9% NaCl = saline group). After 3 (D3) or 7 (D7) days, invasive pulmonary function tests were performed to determine tissue elastance (H) and static compliance (Cst). Bronchoalveolar lavage (BAL) was taken for surfactant function, inflammatory markers, and protein measurements. Lungs were fixed by vascular perfusion for design-based stereology and electron microscopic analyses. SRT significantly improved minimum surface tension of alveolar surfactant as well as H and Cst at D3 and D7. At D3 decreased inflammatory markers including neutrophilic granulocytes, IL-1β, and IL-6 correlated with reduced BAL-protein levels after SRT. Numbers of open alveoli were significantly increased at D3 and D7 in SRT groups whereas at D7 there was also a significant reduction in septal wall thickness and parenchymal tissue volume. Septal wall thickness and numbers of open alveoli highly correlated with improved lung mechanics after SRT. In conclusion, reduction in surface tension was effective to stabilize alveoli linked with an attenuation of parameters of acute lung injury at D3 and collapse induration at D7. Hence, SRT modifies disease progression to collapse induration.
La maladie de Ménière (MM) est une pathologie vestibulaire chronique invalidante. Le traitement d’un syndrome d’apnées du sommeil concomitant permettrait de diminuer le handicap et d’améliorer ...l’atteinte auditive.
Caractériser le sommeil, la vigilance diurne (somnolence, fatigue) et rechercher des troubles du sommeil (insomnie, syndrome d’apnées hypopnées obstructives du sommeil) dans une cohorte de patients suivis pour une MM.
Étude monocentrique évaluant chez 26 patients avec MM avérée la sévérité de la maladie (anamnèse, explorations fonctionnelles des vertiges et de l’audition, score de handicap fonctionnel des vertiges AAO-HNS), les comorbidités (cardiovasculaires, anxio-dépressives, migraine), les troubles du sommeil cliniquement (classification ICSD-III, questionnaire de sommeil de Pittsburgh PSQI, échelles de somnolence d’Epworth ESS, de fatigue Pichot PSS) et par polygraphique ventilatoire ou polysomnographie (index d’apnées-hypopnées).
Un total de 58 % des patients se disaient insatisfaits de leur sommeil, mais 76 % avaient un score pathologique>5 au PSQI. Un total de 73 % avaient une insomnie (58 %) et/ou un SAOS. Un total de 42 % avaient un SAOS au moins modéré dont 38 % avec indication à une pression positive continue. Les plus invalidés par la MM étaient insomniaques et fatigués, mais pas somnolents. Une moins bonne audition et la nécessité d’une thérapeutique plus intensive étaient observées en cas d’un SAOS modéré ou sévère.
Des interactions bidirectionnelles entre oreille interne et sommeil ont été décrites. Les substrats physiopathologiques impliqués restent peu connus. Une exploration du sommeil semble pertinente en cas de suspicion de troubles du sommeil ou devant une intensification thérapeutique. Une amélioration du sommeil pourrait permettre une meilleure qualité de vie et avoir un impact positif sur la MM.
Les troubles du sommeil sont fréquents et probablement sous-diagnostiqués chez les patients atteints de MM. Des études supplémentaires sont nécessaires pour mieux appréhender les associations entre sommeil et MM.
ZAP-70 methylation 223 nucleotides downstream of transcription start (CpG+223) predicts outcome in chronic lymphocytic leukemia (CLL), but its impact relative to CD38 and ZAP-70 expression or ...immunoglobulin heavy chain variable region (IGHV) status is uncertain. Additionally, standardizing ZAP-70 expression analysis has been unsuccessful. CpG+223 methylation was quantitatively determined in 295 untreated CLL cases using MassARRAY. Impact on clinical outcome vs CD38 and ZAP-70 expression and IGHV status was evaluated. Cases with low methylation (<20%) had significantly shortened time to first treatment (TT) and overall survival (OS) (P < .0001). For TT, low methylation defined a large subset of ZAP-70 protein-negative cases with significantly shortened TT (median, 8.0 vs 3.9 years for high vs low methylation; hazard ratio HR = 0.43; 95% confidence interval CI, 0.25-0.74). Conversely, 16 ZAP-70 protein-positive cases with high methylation had poor outcome (median, 1.1 vs 2.3 years for high vs low methylation; HR = 1.62; 95% CI, 0.87-3.03). For OS, ZAP-70 methylation was the strongest risk factor; CD38 and ZAP-70 expression or IGHV status did not significantly improve OS prediction. A pyrosequencing assay was established that reproduced the MassARRAY data (κ coefficient > 0.90). Thus, ZAP-70 CpG+223 methylation represents a superior biomarker for TT and OS that can be feasibly measured, supporting its use in risk-stratifying CLL.
•Methylation analysis at ZAP-70 CpG+223 in CLL provides superior prognostic information vs IGHV status or CD38 or ZAP-70 expression.•A pyrosequencing method for the feasible assessment of CpG+223 methylation in CLL samples is provided.
Abstract 3865
ZAP-70 expression is a prognostic marker differentiating chronic lymphocytic leukemia (CLL) patients with indolent disease from those rapidly progressing after diagnosis and requiring ...treatment. We previously demonstrated in four independent clinical cohorts that DNA methylation at a single CpG dinucleotide in the ZAP-70 promoter region impacts on ZAP-70 transcriptional regulation and is prognostic in CLL (Claus et al. J Clin Oncol. 2012;30(20):2483–91). Here, we confirm and extend our previous findings by investigating the significance of ZAP-70 methylation relative to established prognostic biomarkers in CLL: IGHV mutation status or expression of ZAP-70 protein or CD38.
DNA methylation analysis of the ZAP-70 core promoter region was conducted in mononuclear cells of 295 untreated CLL patients, previously reported by the CRC (Rassenti et al. Blood 2008;112:1923–30) using MALDI-TOF mass spectrometry (MassARRAY, Sequenom). The 295 patients had a median age of 55 years (range 26–82), 69% were male, and 46% were Rai stage 0, 47% stage I/II, and 7% stage III/IV at diagnosis. ZAP-70 protein and CD38 expression were measured by four-color flow cytometry and IGHV mutational status was assessed, as previously described.
By hierarchical clustering, the previously identified single CpG dinucleotide (“CpG unit 1”, position +223 relative to the transcription start) separated patients with high methylation levels across the entire region from those with lower methylation levels at this specific site. With a median follow-up of 3.9 years in 50 patients who had not started treatment and 5.6 years among those still alive (n=196), patients with lower methylation levels at CpG unit 1 had shortened time to first treatment (TTT) and overall survival (OS) (both p<0.0001), confirming the prognostic relevance of this site. In our previous study, an optimal cut-off for CpG unit 1 methylation across four data sets was between 10% and 20%. In this cohort, the optimal cut-off across the two endpoints was between 15% and 25%. A natural gap in CpG unit 1 methylation levels occurred between 20% and 25%, adding potential biological significance, as lower values may represent patient cell samples in which both alleles are unmethylated. Applying a 20% cut-off, TTT and OS were significantly different for those with lower and higher methylation levels (both p<0.0001). The 5-year TTT estimates were 22% (95 % CI: 0.16–0.28) and 64% (95% CI: 0.52–0.74), respectively, for patients with low versus high methylation levels; 5-year OS estimates were 80% (95% CI: 0.73–0.85) and 90% (95% CI: 0.81–0.95). Low ZAP-70 methylation was significantly associated with CLL-cell expression of ZAP-70, CD38, or unmutated IGHV status (each p<0.0001). In a model of TTT, expression of ZAP-70 modified the strong effect of ZAP-70 methylation. In patients with CLL cells lacking ZAP-70 expression, high methylation was protective (hazard ratio HR=0.37, 95% CI: 0.23–0.58). In contrast, methylation had no significant effect in ZAP-70+ CLL (HR=1.47, 95% CI: 0.81–2.68); TTT was short regardless of methylation levels (Figure 1). IGHV mutation status and expression of CD38 did not provide significant additional prognostic information. The effect modification described for TTT was not observed for OS (p=0.57). Here, ZAP-70 methylation was the strongest risk factor (hazard ratio=0.32, 95% CI: 0.19–0.54; p<0.0001), and expression of ZAP-70, CD38 or unmutated IGHV did not significantly improve the ability to explain differences in OS.
Hence, the prognostic value of ZAP-70 CpG unit 1 methylation was confirmed in a large patient cohort with extensive follow-up. We defined a clinically applicable threshold for ZAP-70 methylation assessment and determined its relative value to current prognostic markers in CLL. ZAP-70 methylation predicted longer TTT among patients with CLL cells lacking ZAP-70 expression, but not among those with ZAP-70+ CLL cells, although this could reflect sampling bias due to the small number of patients (n=16) in our cohort with both high methylation and expression of ZAP-70. Lastly, methylation was the strongest risk factor for OS, and neither IGHV mutation status nor expression of ZAP-70 or CD38 significantly improved the ability to predict prognosis. In conclusion, ZAP-70 DNA methylation represents a clinically valuable biomarker for predicting TTT and OS in previously untreated patients with CLL.
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Gribben:Gilead: Honoraria; Mundipharma: Honoraria; GSK: Honoraria; Pharmacyclics: Honoraria; Roche: Honoraria; Celgene: Honoraria. Kay:Celgene: Research Funding.
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