Presynaptic inhibition upon CB1 or mGlu2/3 receptor activation requires ERK/MAPK phosphorylation of Munc18-1.

Presynaptic cannabinoid (CB1R) and metabotropic glutamate receptors (mGluR2/3) regulate synaptic strength by inhibiting secretion. Here, we reveal a presynaptic inhibitory pathway activated by extracellular signal-regulated kinase (ERK) that mediates CB1R- and mGluR2/3-induced secretion inhibition. This pathway is triggered by a variety of events, from foot shock-induced stress to intense neuronal activity, and induces phosphorylation of the presynaptic protein Munc18-1. Mimicking constitutive phosphorylation of Munc18-1 results in a drastic decrease in synaptic transmission. ERK-mediated phosphorylation of Munc18-1 ultimately leads to degradation by the ubiquitin-proteasome system. Conversely, preventing ERK-dependent Munc18-1 phosphorylation increases synaptic strength. CB1R- and mGluR2/3-induced synaptic inhibition and depolarization-induced suppression of excitation (DSE) are reduced upon ERK/MEK pathway inhibition and further reduced when ERK-dependent Munc18-1 phosphorylation is blocked. Thus, ERK-dependent Munc18-1 phosphorylation provides a major negative feedback loop to control synaptic strength upon activation of presynaptic receptors and during intense neuronal activity.

Competencies for rating perceived exertion in amateur soccer players with and without intellectual disabilities.

BACKGROUND: Perception of exertion is essential for self-regulation in sports. The ability to rate perceived exertion (RPE) is regarded as psychophysiological competence, although cognitive components of RPE are largely unknown. The present study tested the hypothesis that cognitive processing speed, perseveration and figural fluency correlate with RPE. METHODS: The present study tested relationships between the performance in neuropsychological tests and the competence for RPE assessed during soccer training in 30 adults with and 22 adults without intellectual disabilities. RESULTS: Mean correlation coefficients for RPE and heart rate differed significantly between participants with intellectual disabilities (r = .41) and participants without intellectual disabilities (r = .71). The variance of RPE could be partially explained by neuropsychological performance measures reflecting cognitive processing speed and perseveration and by age. CONCLUSIONS: The results point to an impaired perception of exertion in people with intellectual disabilities, which can be partially explained by individual neuropsychological competencies.

Prediction of radiation-induced toxicity by in vitro radiosensitivity of lymphocytes in prostate cancer patients.

AIM: To identify predictive assays for radiation-induced toxicity in prostate cancer patients. PATIENTS & METHODS: Patients have been surveyed prospectively before and up to 16 months after radiotherapy using a validated questionnaire. Subgroups of 25 patients with minor and larger score changes, respectively, were selected for γ-H2AX, G2 and Annexin V assays. RESULTS: A significantly higher spontaneous chromatid aberration yield (HR: 1.46 [95% CI: 1.02-2.09]; p = 0.04), higher levels of early apoptotic (HR: 1.12 [95% CI: 1.01-1.24]; p = 0.04) and late apoptotic and necrotic (HR: 1.10 [95% CI: 0.99-1.23]; p = 0.08) lymphocytes 24 h post-irradiation were found in patients with a bowel bother score decrease greater than 20 points more than 1 year after treatment. CONCLUSION: Chromatid aberration and apoptosis/necrosis assays appear to be suitable for the prediction of radiation-induced toxicity.

Programme Dementia Prevention (pdp): A Nationwide Program for Personalized Prevention in Luxembourg.

BACKGROUND: With continuously aging societies, an increase in the number of people with cognitive decline is to be expected. Aside from the development of causative treatments, the successful implementation of prevention strategies is of utmost importance to reduce the high societal burden caused by neurodegenerative diseases leading to dementia among which the most common cause is Alzheimer's disease. OBJECTIVE: The aim of the Luxembourgish "programme dementia prevention (pdp)" is to prevent or at least delay dementia in an at-risk population through personalized multi-domain lifestyle interventions. The current work aims to provide a detailed overview of the methodology and presents initial results regarding the cohort characteristics and the implementation process. METHODS: In the frame of the pdp, an extensive neuropsychological evaluation and risk factor assessment are conducted for each participant. Based on the results, individualized multi-domain lifestyle interventions are suggested. RESULTS: A total number of 450 participants (Mean age = 69.5 years; SD = 10.8) have been screened at different recruitment sites throughout the country, among whom 425 participants (94.4%) met the selection criteria. CONCLUSIONS: We provide evidence supporting the feasibility of implementing a nationwide dementia prevention program and achieving successful recruitment of the target population by establishing a network of different healthcare providers.

Persisting ring chromosomes detected by mFISH in lymphocytes of a cancer patient-a case report.

We report the case of an 84 years old prostate cancer patient with severe side effects after radiotherapy in 2006. He was cytogenetically analysed in 2009 and in 2012 in a comparative study for individual radiosensitivity of prostate cancer patients. No other patient had clonal aberrations, but this patient showed ring chromosomes in the range of 21-25% of lymphocytes. He received 5 cycles of 5-fluorouracil/folic acid for chemotherapy of sigmoid colon carcinoma in 2003, three years before radiotherapy of prostate cancer. Blood samples were irradiated ex vivo with Cs-137 γ-rays (0.7Gy/min) in the G0-phase of the cell cycle. 100 FISH painted metaphases were analysed for the control and the irradiated samples each. Multicolour in situ hybridisation techniques like mFISH and mBand as well as MYC locus, telomere and centromere painting probes were used to characterise ring metaphases. Metaphase search and autocapture was performed with a Zeiss Axioplan 2 imaging microscope followed by scoring and image analysis using Metafer 4/ISIS software (MetaSystems). In 2009 chromosome 8 rings were found in about 25% of lymphocytes. Rings were stable over time and increased to about 30% until 2012. The ring chromosome 8 always lacked telomere signals and a small amount of rings displayed up to four centromere signals. In aberrant metaphases 8pter and 8qter were either translocated or deleted. Further analyses revealed that the breakpoint at the p arm is localised at 8p21.2-22. The breakpoint at the q arm turned out to be distal from the MYC locus at 8q23-24. We hypothesise that the ring chromosome 8 has been developed during the 5 FU/folic acid treatments in 2003. The long term persistence might be due to clonal expansion of a damaged but viable hematopoietic stem cell giving rise to cycling progenitor cells that permit cell survival and proliferation.

Increased diversity of Malassezia species on the skin of Parkinson's disease patients.

Background: Parkinson's disease (PD) is characterized by motor disorders and the composition of Lewy bodies (LBs) in the substantia nigra. Due to the lack of a definitive biomarker, the current treatments do not modify the progression of PD. Recently, researchers revealed lipid dysregulation and some potential volatile biomarkers of PD related to a unique odor from PD patients by metabolomics of sebum, which is supposed to cause a potential change for skin microflora. In this study, we identified the 4 Malassezia species in PD patients and compared them with healthy controls. Methods: We collected 95 sebum samples (47 PDs and 48 Controls) by cotton swabs and extracted the DNA. The identification of Malassezia species was performed by Nested PCR. Specific primers for each species were used to amplify corresponding yeasts in each sample. Results: M. restricta and M. globosa are the most common species for both groups. The prevalence of M. slooffiae and M. sympodialis were significantly higher in the PD group compared with controls (63.8% vs. 29.1 and 74.5% vs. 54.2% respectively), the binary logistic regression model further indicated that M. slooffiae (OR = 9.358, p < 0.001) was associated with PD. Moreover, the diversity of Malassezia species was significantly greater (3.5 vs. 2.9 species per individual, p = 0.002) in the PD group. Conclusion: Based on our results, we preliminarily observed a change in Malassezia species incidence and diversity on the skin of PD patients, which could be associated with lipid dysregulation; meanwhile, it might also be a noninvasive biomarker for PD.

Abnormal subpopulations of monocytes in the cerebrospinal fluid of patients with Parkinson's disease.

We performed immune cell profiling by multiparameter flow cytometry in cerebrospinal fluid (CSF) and peripheral blood (PB) of Parkinson's Disease (PD) patients (n = 9) and healthy controls (n = 8). Classical and non-classical monocytes were increased in the CSF of PD patients.

Co-digestion of solid waste: Towards a simple model to predict methane production.

Modeling methane production is a key issue for solid waste co-digestion. Here, the effect of a step-wise increase in the organic loading rate (OLR) on reactor performance was investigated, and four new models were evaluated to predict methane yields using data acquired in batch mode. Four co-digestion experiments of mixtures of 2 solid substrates were conducted in semi-continuous mode. Experimental methane yields were always higher than the BMP values of mixtures calculated from the BMP of each substrate, highlighting the importance of endogenous production (methane produced from auto-degradation of microbial community and generated solids). The experimental methane productions under increasing OLRs corresponded well to the modeled data using the model with constant endogenous production and kinetics identified at 80% from total batch time. This model provides a simple and useful tool for technical design consultancies and plant operators to optimize the co-digestion and the choice of the OLRs.

The Luxembourg Parkinson's Study: A Comprehensive Approach for Stratification and Early Diagnosis.

While genetic advances have successfully defined part of the complexity in Parkinson's disease (PD), the clinical characterization of phenotypes remains challenging. Therapeutic trials and cohort studies typically include patients with earlier disease stages and exclude comorbidities, thus ignoring a substantial part of the real-world PD population. To account for these limitations, we implemented the Luxembourg PD study as a comprehensive clinical, molecular and device-based approach including patients with typical PD and atypical parkinsonism, irrespective of their disease stage, age, comorbidities, or linguistic background. To provide a large, longitudinally followed, and deeply phenotyped set of patients and controls for clinical and fundamental research on PD, we implemented an open-source digital platform that can be harmonized with international PD cohort studies. Our interests also reflect Luxembourg-specific areas of PD research, including vision, gait, and cognition. This effort is flanked by comprehensive biosampling efforts assuring high quality and sustained availability of body liquids and tissue biopsies. We provide evidence for the feasibility of such a cohort program with deep phenotyping and high quality biosampling on parkinsonism in an environment with structural specificities and alert the international research community to our willingness to collaborate with other centers. The combination of advanced clinical phenotyping approaches including device-based assessment will create a comprehensive assessment of the disease and its variants, its interaction with comorbidities and its progression. We envision the Luxembourg Parkinson's study as an important research platform for defining early diagnosis and progression markers that translate into stratified treatment approaches.

Induction of the chromosomal translocation t(14;18) by targeting the BCL-2 locus with specific binding I-125-labeled triplex-forming oligonucleotides.

Triplex-Forming oligonucleotides (TFO) bind sequence-specific to the DNA double helix in-vitro and in-vivo and are a promising tool to manipulate genes or gene regulatory elements. TFO as a carrier molecule for short-range particle emitter such as Auger-Electron-Emitters (AEE) bear the potential to introduce radiation-induced site-specific complex DNA lesions, which are known to induce chromosomal translocations. We studied gene expression, translocation frequency and protein expression in SCL-II cells after transfection with the AEE Iodine-125 (I-125) labeled TFO-BCL2 targeting the human BCL2 gene. The TFO-BCL2 binds to the BCL2 gene in close proximity to a known major-breakage-region (mbr). SCL-II cells were transfected with I-125 labeled TFO and stored for decay accumulation. Monitoring of BCL2 translocations was done with the Fluorescence-In-Situ-Hybridization (FISH) method. The utilized FISH probes were designed to detect a t(14;18) translocation of the BCL2 gene, which is a common translocation leading to an overexpression of BCL2 protein. Analysis of BCL2 gene expression levels was done via quantitative Real-Time PCR. Verification of gene expression on the protein level was analyzed by Western blotting. The relative gene expression of BCL2 in I-125-TFO-BCL2 transfected cells showed a significant up-regulation when compared to controls. Analysis of the BCL2 t(14;18) translocation frequency revealed a significant 1.8- to 2-fold increase when compared to control cells. This 2-fold increase was not reflected on the protein level. We conclude that I-125 decays within the BCL2 gene facilitate the t(14;18) chromosomal translocation in the SCL-II cells and that the increased frequency contributes to the observed overall enhanced BCL2 gene expression.

Increasing genomic instability during cancer therapy in a patient with Li-Fraumeni syndrome.

BACKGROUND: Li-Fraumeni syndrome (LFS) is a cancer predisposition disorder characterized by germline mutations of the p53 tumor-suppressor gene. In response to DNA damage, p53 stimulates protective cellular processes including cell-cycle arrest and apoptosis to prevent aberrant cell proliferation. Current cancer therapies involve agents that damage DNA, which also affect non-cancerous hematopoietic stem/progenitor cells. Here, we report on a child with LFS who developed genomic instability during craniospinal irradiation for metastatic choroid plexus carcinoma (CPC). CASE PRESENTATION: This previously healthy 4-year-old boy presented with parieto-temporal brain tumor, diagnosed as CPC grade-3. Screening for cancer-predisposing syndrome revealed heterozygous p53 germline mutation, leading to LFS diagnosis. After tumour resection and systemic chemotherapy, entire craniospinal axis was irradiated due to leptomeningeal seeding, resulting in disease stabilization for nearly 12 months. Blood lymphocytes of LFS patient (p53-deficient) and age-matched tumor-children (p53-proficient) were collected before, during and after craniospinal irradiation and compared with asymptomatic carriers for identical p53 mutation, not exposed to DNA-damaging treatment. In p53-deficient lymphocytes of LFS patient radiation-induced DNA damage failed to induce cell-cycle arrest or apoptosis. Although DNA repair capacity was not impaired, p53-deficient blood lymphocytes of LFS patient showed significant accumulation of 53BP1-foci during and even several months after irradiation, reflecting persistent DNA damage. Electron microscopy revealed DNA abnormalities ranging from simple unrepaired lesions to chromosomal abnormalities. Metaphase spreads of p53-deficient lymphocytes explored by mFISH revealed high amounts of complex chromosomal aberrations after craniospinal irradiation. CONCLUSIONS: Tumor suppressor p53 plays a central role in maintaining genomic stability by promoting cell-cycle checkpoints and apoptosis. Here, we demonstrate that a patient with LFS receiving craniospinal irradiation including large volumes of bone marrow developed progressive genomic instability of the hematopoietic system. During DNA-damaging radiotherapy, genome-stabilizing mechanisms in proliferating stem/progenitor cells are perturbed by p53 deficiency, increasing the risk of cancer initiation and progression.

Chromosome aberrations induced by the Auger electron emitter (125)I.

DNA-associated Auger electron emitters (AEE) cause cellular damage leading to high-LET type cell survival curves indicating an enhanced relative biological effectiveness. Double strand breaks (DSBs) induced by Iodine-125-deoxyuridine ((125)I-UdR) decays are claimed to be very complex. To elucidate the assumed genotoxic potential of (125)I-UdR, chromatid aberrations were analysed in exposed human peripheral blood lymphocytes (PBL). PBL were stimulated with medium containing phytohaemagglutinin (PHA). After 24h, cultures were labelled with (125)I-UdR for 18h (activity concentration 1-45 kBq) during the S-phase. Following standard cytogenetic procedure, at least 100 metaphases were analysed microscopically for each activity concentration. Cell death was measured by apoptosis assay using flow cytometry. Radiation doses were determined by using point kernel calculations. After 18h labelling with (125)I-UdR the cell cycle distribution is severely disturbed. About 40% of PBL are fully labelled and 20% show a moderate labelling of (125)I-UdR, whereas 40% of cells remain un-labelled. The dose-response relationship fits to a polynomial curve in the low dose range, whereas a linear fit supplies a better estimation in the high dose range. Even the lowest dose of 0.2Gy leads to a 13-fold increase of aberrations compared to the controls. On average every fifth (125)I-decay produces a single chromatid aberration in PBL. Additionally, a dose-dependent increase of cell death is observed. (125)I-UdR has a very strong genotoxic capacity in human PBL, even at 0.2Gy. Efficiently labelled cells displaying a prolonged cell cycle compared to moderately labelled cells and cell death contribute substantially to the desynchronisation of the cell cycle. Our data, showing for the first time, that one (125)I-decay induces ∼ 0.2 chromatid aberrations, are in very good accordance to DSB data, stating that ∼0.26 DSB are induced per decay, indicating that it takes on average 250 decays to induce one chromosome aberration (CA). [Corrected]

Degradation efficiency of agricultural biogas plants--a full-scale study.

The degradation efficiency of 21 full-scale agricultural CSTR biogas plants was investigated. The residual methane potential of the digestion stages was determined in batch digestion tests (20.0 and 37.0 °C). The results of this study showed that the residual methane yield is significantly correlated to the HRT (r=-0.73). An almost complete degradation of the input substrates was achieved due to a HRT of more than 100 days (0.097±0.017 Nm(3)/kg VS). The feedstock characteristics have the largest impact to the degradation time. It was found that standard values of the methane yield are a helpful tool for evaluating the degradation efficiency. Adapting the HRT to the input materials is the key factor for an efficient degradation in biogas plants. No influence of digester series configuration to the VS degradation was found. The mean VS degradation rate in the total reactor systems was 78±7%.

CONSORT item reporting quality in the top ten ranked journals of critical care medicine in 2011: a retrospective analysis.

INTRODUCTION: Reporting randomised controlled trials is a key element in order to disseminate research findings. The CONSORT statement was introduced to improve the reporting quality. We assessed the adherence to the CONSORT statement of randomised controlled trials published 2011 in the top ten ranked journals of critical care medicine (ISI Web of Knowledge 2011, Thomson Reuters, London UK). METHODS: Design. We performed a retrospective cross sectional data analysis. Setting. This study was executed at the University Hospital of RWTH, Aachen. Participants. We selected the following top ten listed journals according to ISI Web of Knowledge (Thomson Reuters, London, UK) critical care medicine ranking in the year 2011: American Journal of Respiratory and Critical Care Medicine, Critical Care Medicine, Intensive Care Medicine, CHEST, Critical Care, Journal of Neurotrauma, Resuscitation, Pediatric Critical Care Medicine, Shock and Minerva Anestesiologica. Main outcome measures. We screened the online table of contents of each included journal, to identify the randomised controlled trials. The adherence to the items of the CONSORT Checklist in each trial was evaluated. Additionally we correlated the citation frequency of the articles and the impact factor of the respective journal with the amount of reported items per trial. RESULTS: We analysed 119 randomised controlled trials and found, 15 years after the implementation of the CONSORT statement, that a median of 61,1% of the checklist-items were reported. Only 55.5% of the articles were identified as randomised trials in their titles. The citation frequency of the trials correlated significantly (rs = 0,433; p<0,001 and r = 0,331; p<0,001) to the CONSORT statement adherence. The impact factor showed also a significant correlation to the CONSORT adherence (r = 0,386; p<0,001). CONCLUSION: The reporting quality of randomised controlled trials in the field of critical care medicine remains poor and needs considerable improvement.

Chromosomal radiosensitivity analyzed by FISH in lymphocytes of prostate cancer patients and healthy donors.

It is known that about 5-10% of cancer patients show severe clinical side effects during and after radiotherapy due to enhanced sensitivity to ionizing radiation. Identification of those radiosensitive individuals by a reliable in vitro assay before onset of treatment would have a great impact on successful radiotherapy. We compared the radiosensitivity of the chromosomes 2, 11 and 17 in prostate cancer patients with and without severe side effects after radiotherapy and in age-matched healthy donors. Each cohort consisted of at least 10 donors. Peripheral blood lymphocytes were irradiated ex vivo with 0.5, 1 und 2 Gy ((137)Cs γ rays). We investigated the radiosensitivity of the chromosomes 2, 11 and 17 by scoring of 100 FISH painted metaphases for each dose point and donor group. Statistical analyses were performed by nonparametric tests as Mann-Whitney test and Kruskal-Wallis ANOVA, paired Wilcoxon rank test, χ(2) goodness-of-fit test and Spearman rank-order correlation at a significance level of P < 0.05. Analysis of the overall aberration yield revealed no significant differences between any donor groups. The translocation frequencies of the chromosomes 2, 11 and 17 coincided with their relative size. Thus, none of the chromosomes analyzed were more or less radiosensitive with respect to the genomic translocation frequency. Additionally, neither of the chromosomes showed enhanced or diminished radiosensitivity in one of the donor groups. Furthermore, variance analyses revealed that the distribution pattern of the aberrations per donor did not differ in each donor group even after exposure to 2 Gy. Prostate cancer patients with and without side effects cannot be distinguished from healthy donors based on aberration yield after irradiation with γ rays.

Liprin-α2 promotes the presynaptic recruitment and turnover of RIM1/CASK to facilitate synaptic transmission.

The presynaptic active zone mediates synaptic vesicle exocytosis, and modulation of its molecular composition is important for many types of synaptic plasticity. Here, we identify synaptic scaffold protein liprin-α2 as a key organizer in this process. We show that liprin-α2 levels were regulated by synaptic activity and the ubiquitin-proteasome system. Furthermore, liprin-α2 organized presynaptic ultrastructure and controlled synaptic output by regulating synaptic vesicle pool size. The presence of liprin-α2 at presynaptic sites did not depend on other active zone scaffolding proteins but was critical for recruitment of several components of the release machinery, including RIM1 and CASK. Fluorescence recovery after photobleaching showed that depletion of liprin-α2 resulted in reduced turnover of RIM1 and CASK at presynaptic terminals, suggesting that liprin-α2 promotes dynamic scaffolding for molecular complexes that facilitate synaptic vesicle release. Therefore, liprin-α2 plays an important role in maintaining active zone dynamics to modulate synaptic efficacy in response to changes in network activity.

Dendritic position is a major determinant of presynaptic strength.

Different regulatory principles influence synaptic coupling between neurons, including positional principles. In dendrites of pyramidal neurons, postsynaptic sensitivity depends on synapse location, with distal synapses having the highest gain. In this paper, we investigate whether similar rules exist for presynaptic terminals in mixed networks of pyramidal and dentate gyrus (DG) neurons. Unexpectedly, distal synapses had the lowest staining intensities for vesicular proteins vGlut, vGAT, Synaptotagmin, and VAMP and for many nonvesicular proteins, including Bassoon, Munc18, and Syntaxin. Concomitantly, distal synapses displayed less vesicle release upon stimulation. This dependence of presynaptic strength on dendritic position persisted after chronically blocking action potential firing and postsynaptic receptors but was markedly reduced on DG dendrites compared with pyramidal dendrites. These data reveal a novel rule, independent of neuronal activity, which regulates presynaptic strength according to dendritic position, with the strongest terminals closest to the soma. This gradient is opposite to postsynaptic gradients observed in pyramidal dendrites, and different cell types apply this rule to a different extent.

In vivo versus in vitro individual radiosensitivity analysed in healthy donors and in prostate cancer patients with and without severe side effects after radiotherapy.

BACKGROUND: A high cellular radiosensitivity may be connected with a risk for development of severe side effects after radiotherapy and indicate cancer susceptibility. Hence, a fast and robust in vitro test is desirable to identify radiosensitive individuals. MATERIALS AND METHODS: The study included 25 prostate cancer patients with severe side effects (S) and 25 patients without severe side effects (0) after radiotherapy as well as 23 male healthy age-matched donors. Blood samples were exposed to 0.5 Gy or 1 Gy of γ-rays. The initial level of double-strand breaks (dsb) and repair kinetics measured by phosphorylation of histone H2A (γ-H2AX-assay), apoptosis (Annexin V-assay) and the induction of chromatid aberrations after irradiation in the G2-phase of the cell cycle (G2-assay) were analysed. RESULTS: A significant higher chromatid aberration yield was found in lymphocytes from prostate cancer patients when compared to healthy donors. We found no significant differences between patients S and patients 0. CONCLUSIONS: There is no obvious correlation between clinical and cellular radiosensitivity in lymphocytes of prostate cancer patients when all chosen in vitro assays are considered. Although 25% of the patients showed both severe side effects and increased radiation-induced chromosomal sensitivity, predictive value of G2-assay is doubtful.

Munc13 controls the location and efficiency of dense-core vesicle release in neurons.

Neuronal dense-core vesicles (DCVs) contain diverse cargo crucial for brain development and function, but the mechanisms that control their release are largely unknown. We quantified activity-dependent DCV release in hippocampal neurons at single vesicle resolution. DCVs fused preferentially at synaptic terminals. DCVs also fused at extrasynaptic sites but only after prolonged stimulation. In munc13-1/2-null mutant neurons, synaptic DCV release was reduced but not abolished, and synaptic preference was lost. The remaining fusion required prolonged stimulation, similar to extrasynaptic fusion in wild-type neurons. Conversely, Munc13-1 overexpression (M13OE) promoted extrasynaptic DCV release, also without prolonged stimulation. Thus, Munc13-1/2 facilitate DCV fusion but, unlike for synaptic vesicles, are not essential for DCV release, and M13OE is sufficient to produce efficient DCV release extrasynaptically.