The most prevalent genetic cause of ALS-FTD,C9orf72 synergizes the toxicity of ATXN2 intermediate polyglutamine repeats through the autophagy pathway

The most common genetic cause for amyotrophic lateral sclerosis and frontotemporal dementia (ALS-FTD) is repeat expansion of a hexanucleotide sequence (GGGGCC) within the C9orf72 genomic sequence. To elucidate the functional role of C9orf72 in disease pathogenesis, we identified certain molecular interactors of this factor. We determined that C9orf72 exists in a complex with SMCR8 and WDR41 and that this complex acts as a GDP/GTP exchange factor for RAB8 and RAB39, 2 RAB GTPases involved in macroautophagy/autophagy. Consequently, C9orf72 depletion in neuronal cultures leads to accumulation of unresolved aggregates of SQSTM1/p62 and phosphorylated TARDBP/TDP-43. However, C9orf72 reduction does not lead to major neuronal toxicity, suggesting that a second stress may be required to induce neuronal cell death. An intermediate size of polyglutamine repeats within ATXN2 is an important genetic modifier of ALS-FTD. We found that coexpression of intermediate polyglutamine repeats (30Q) of ATXN2 combined with C9orf72 depletion increases the aggregation of ATXN2 and neuronal toxicity. These results were confirmed in zebrafish embryos where partial C9orf72 knockdown along with intermediate (but not normal) repeat expansions in ATXN2 causes locomotion deficits and abnormal axonal projections from spinal motor neurons. These results demonstrate that C9orf72 plays an important role in the autophagy pathway while genetically interacting with another major genetic risk factor, ATXN2, to contribute to ALS-FTD pathogenesis.     

Sensitivity to reinforcement in successive and delayed discriminations

Pigeons' responses were recorded in successive 15-s subintervals of 60-s components of several multiple variable-interval schedules of food reinforcement. In the standard multiple schedule or successive discrimination, discriminative stimuli were present throughout each component. In the delayed discrimination or memory procedure, red or green stimuli were present in the first 15 s of components and were followed by a white stimulus for the remainder of both components. Ratios of responses in the first 15 s of the two components, where discriminative stimuli were present, were sensitive to ratios of reinforcers obtained in the two components, to the same extent in both multiple and memory procedures. In both procedures, sensitivity to reinforcement decreased systematically over component subintervals, but to a greater extent in the memory procedure where discriminative stimuli were absent. The reduction in sensitivity with time since presentation of prior discriminative stimuli in the memory procedure was therefore influenced by two main factors: delayed stimulus control by the discriminative stimuli presented earlier in the component, and a decrease in sensitivity to reinforcement with increasing time since component alternation.     

New Insights into Handling Missing Values in Environmental Epidemiological Studies

Missing data are unavoidable in environmental epidemiologic surveys. The aim of this study was to compare methods for handling large amounts of missing values: omission of missing values, single and multiple imputations (through linear regression or partial least squares regression), and a fully Bayesian approach. These methods were applied to the PARIS birth cohort, where indoor domestic pollutant measurements were performed in a random sample of babies'' dwellings. A simulation study was conducted to assess performances of different approaches with a high proportion of missing values (from 50% to 95%). Different simulation scenarios were carried out, controlling the true value of the association (odds ratio of 1.0, 1.2, and 1.4), and varying the health outcome prevalence. When a large amount of data is missing, omitting these missing data reduced statistical power and inflated standard errors, which affected the significance of the association. Single imputation underestimated the variability, and considerably increased risk of type I error. All approaches were conservative, except the Bayesian joint model. In the case of a common health outcome, the fully Bayesian approach is the most efficient approach (low root mean square error, reasonable type I error, and high statistical power). Nevertheless for a less prevalent event, the type I error is increased and the statistical power is reduced. The estimated posterior distribution of the OR is useful to refine the conclusion. Among the methods handling missing values, no approach is absolutely the best but when usual approaches (e.g. single imputation) are not sufficient, joint modelling approach of missing process and health association is more efficient when large amounts of data are missing.     

Optimization of factors influencing enzyme activity and product selectivity and the role of proton transfer in the catalytic mechanism of patchoulol synthase

The patchoulol synthase (PTS) from Pogostemon cablin is a versatile sesquiterpene synthase and produces more than 20 valuable sesquiterpenes by conversion of the natural substrate farnesyl pyrophosphate (FPP). PTS has the potential to be used as a biocatalyst for the production of valuable sesquiterpenes such as (−)-patchoulol. The objective of the present study is to develop an efficient biotransformation and to characterize the biocatalytic mechanism of the PTS in detail. For this purpose, soluble PTS was prepared using an optimized cultivation protocol and continuous downstream process with a purity of 98%. The PTS biotransformation was then optimized regarding buffer composition, pH-value, and temperature for biotransformation as well as functional and kinetic properties to improve productivity. For the bioconversion of FPP, the highest enzyme activity was reached with the 2-(N-morphlino)ethanesulfonic acid (MES) buffer containing 10% (v/v) glycerol and 10 mM MgCl₂ at pH 6.4 and 34°C. The PTS showed an unusual substrate inhibition for sesquiterpene synthases indicating an intermediate sesquiterpene formed in the active center. Deuteration experiments were used to gain further insights into the biocatalytic mechanism described in literature. Thus it could be shown that a second substrate binding site must be responsible for substrate inhibition and that further protonation and deprotonation steps are involved in the reaction mechanism.