Determination of four sequential stages during microautophagy in vitro.

Microautophagy is the transfer of cytosolic components into the lysosome by direct invagination of the lysosomal membrane and subsequent budding of vesicles into the lysosomal lumen. This process is topologically equivalent to membrane invagination during multivesicular body formation and to the budding of enveloped viruses. Vacuoles are lysosomal compartments of yeasts. Vacuolar membrane invagination can be reconstituted in vitro with purified yeast vacuoles, serving as a model system for budding of vesicles into the lumen of an organelle. Using this in vitro system, we defined different reaction states. We identified inhibitors of microautophagy in vitro and used them as tools for kinetic analysis. This allowed us to characterize four biochemically distinguishable steps of the reaction. We propose that these correspond to sequential stages of vacuole invagination and vesicle scission. Formation of vacuolar invaginations was slow and temperature-dependent, whereas the final scission of the vesicle from a preformed invagination was fast and proceeded even on ice. Our observations suggest that the formation of invaginations rather than the scission of vesicles is the rate-limiting step of the overall reaction.

Cost-Benefit Analysis of Sequential Warning Lights in Nighttime Work Zone Tapers, TPF-5(081), 2011

Improving safety at nighttime work zones is important because of the extra visibility concerns. The deployment of sequential lights is an innovative method for improving driver recognition of lane closures and work zone tapers. Sequential lights are wireless warning lights that flash in a sequence to clearly delineate the taper at work zones. The effectiveness of sequential lights was investigated using controlled field studies. Traffic parameters were collected at the same field site with and without the deployment of sequential lights. Three surrogate performance measures were used to determine the impact of sequential lights on safety. These measures were the speeds of approaching vehicles, the number of late taper merges and the locations where vehicles merged into open lane from the closed lane. In addition, an economic analysis was conducted to monetize the benefits and costs of deploying sequential lights at nighttime work zones. The results of this study indicates that sequential warning lights had a net positive effect in reducing the speeds of approaching vehicles, enhancing driver compliance, and preventing passenger cars, trucks and vehicles at rural work zones from late taper merges. Statistically significant decreases of 2.21 mph mean speed and 1 mph 85% speed resulted with sequential lights. The shift in the cumulative speed distributions to the left (i.e. speed decrease) was also found to be statistically significant using the Mann-Whitney and Kolmogorov-Smirnov tests. But a statistically significant increase of 0.91 mph in the speed standard deviation also resulted with sequential lights. With sequential lights, the percentage of vehicles that merged earlier increased from 53.49% to 65.36%. A benefit-cost ratio of around 5 or 10 resulted from this analysis of Missouri nighttime work zones and historical crash data. The two different benefitcost ratios reflect two different ways of computing labor costs.

Randomised trial of oral versus sequential intravenous/oral cephalosporins in children with pyelonephritis.

The hypothesis was tested that oral antibiotic treatment in children with acute pyelonephritis and scintigraphy-documented lesions is equally as efficacious as sequential intravenous/oral therapy with respect to the incidence of renal scarring. A randomised multi-centre trial was conducted in 365 children aged 6 months to 16 years with bacterial growth in cultures from urine collected by catheter. The children were assigned to receive either oral ceftibuten (9 mg/kg once daily) for 14 days or intravenous ceftriaxone (50 mg/kg once daily) for 3 days followed by oral ceftibuten for 11 days. Only patients with lesions detected on acute-phase dimercaptosuccinic acid (DMSA) scintigraphy underwent follow-up scintigraphy. Efficacy was evaluated by the rate of renal scarring after 6 months on follow-up scintigraphy. Of 219 children with lesions on acute-phase scintigraphy, 152 completed the study; 80 (72 females, median age 2.2 years) were given ceftibuten and 72 (62 females, median age 1.6 years) were given ceftriaxone/ceftibuten. Patients in the intravenous/oral group had significantly higher C-reactive protein (CRP) concentrations at baseline and larger lesion(s) on acute-phase scintigraphy. Follow-up scintigraphy showed renal scarring in 21/80 children treated with ceftibuten and 33/72 with ceftriaxone/ceftibuten (p = 0.01). However, after adjustment for the confounding variables (CRP and size of acute-phase lesion), no significant difference was observed for renal scarring between the two groups (p = 0.2). Renal scarring correlated with the extent of the acute-phase lesion (r = 0.60, p < 0.0001) and the grade of vesico-ureteric reflux (r = 0.31, p = 0.03), and was more frequent in refluxing renal units (p = 0.04). The majority of patients, i.e. 44 in the oral group and 47 in the intravenous/oral group, were managed as out-patients. Side effects were not observed. From this study, we can conclude that once-daily oral ceftibuten for 14 days yielded comparable results to sequential ceftriaxone/ceftibuten treatment in children aged 6 months to 16 years with DMSA-documented acute pyelonephritis and it allowed out-patient management in the majority of these children.

SIMSAFADIM-CLASTIC: A new approach to mathematical 3D forward simulation modelling for terrigenous and carbonate marine sedimentation

Most sedimentary modelling programs developed in recent years focus on either terrigenous or carbonate marine sedimentation. Nevertheless, only a few programs have attempted to consider mixed terrigenous-carbonate sedimentation, and most of these are two-dimensional, which is a major restriction since geological processes take place in 3D. This paper presents the basic concepts of a new 3D mathematical forward simulation model for clastic sediments, which was developed from SIMSAFADIM, a previous 3D carbonate sedimentation model. The new extended model, SIMSAFADIM-CLASTIC, simulates processes of autochthonous marine carbonate production and accumulation, together with clastic transport and sedimentation in three dimensions of both carbonate and terrigenous sediments. Other models and modelling strategies may also provide realistic and efficient tools for prediction of stratigraphic architecture and facies distribution of sedimentary deposits. However, SIMSAFADIM-CLASTIC becomes an innovative model that attempts to simulate different sediment types using a process-based approach, therefore being a useful tool for 3D prediction of stratigraphic architecture and facies distribution in sedimentary basins. This model is applied to the neogene Vallès-Penedès half-graben (western Mediterranean, NE Spain) to show the capacity of the program when applied to a realistic geologic situation involving interactions between terrigenous clastics and carbonate sediments.

MMPBSA decomposition of the binding energy throughout a molecular dynamics simulation of Amyloid-Beta (Aß10-35) aggregation

Recent experiments with amyloid-beta (Aß) peptides indicate that the formation of toxic oligomers may be an important contribution to the onset of Alzheimer's disease. The toxicity of Aß oligomers depend on their structure, which is governed by assembly dynamics. However, a detailed knowledge of the structure of at the atomic level has not been achieved yet due to limitations of current experimental techniques. In this study, replica exchange molecular dynamics simulations are used to identify the expected diversity of dimer conformations of Aß10-35 monomers. The most representative dimer conformation has been used to track the dimer formation process between both monomers. The process has been characterized by means of the evolution of the decomposition of the binding free energy, which provides an energetic profile of the interaction. Dimers undergo a process of reorganization driven basically by inter-chain hydrophobic and hydrophilic interactions and also solvation/desolvation processes.

Sequential therapist interventions and the therapeutic alliance: a pilot study

(from the journal abstract) Scientific interest for the concept of alliance has been maintained and stimulated by repeated findings that a strong alliance is associated with facilitative treatment process and favourable treatment outcome. However, because the alliance is not in itself a therapeutic technique, these findings were unsuccessful in bringing about significant improvements in clinical practice. An essential issue in modern psychotherapeutic research concerns the relation between common factors which are known to explain great variance in empirical results and the specific therapeutic techniques which are the primary basis of clinical training and practice. This pilot study explored sequences in therapist interventions over four sessions of brief psychodynamic investigation. It aims at determining if patterns of interventions can be found during brief psychodynamic investigation and if these patterns can be associated with differences in the therapeutic alliance. Therapist interventions where coded using the Psychodynamic Intervention Rating Scale (PIRS) which enables the classification of each therapist utterance into one of 9 categories of interpretive interventions (defence interpretation, transference interpretation), supportive interventions (question, clarification, association, reflection, supportive strategy) or interventions about the therapeutic frame (work-enhancing statement, contractual arrangement). Data analysis was done using lag sequential analysis, a statistical procedure which identifies contingent relationships in time among a large number of behaviours. The sample includes N = 20 therapist-patient dyads assigned to three groups with: (1) a high and stable alliance profile, (2) a low and stable alliance profile and (3) an improving alliance profile. Results suggest that therapists most often have one single intention when interacting with patients. Large sequences of questions, associations and clarifications were found, which indicate that if a therapist asks a question, clarifies or associates, there is a significant probability that he will continue doing so. A single theme sequence involving frame interventions was also observed. These sequences were found in all three alliance groups. One exception was found for mixed sequences of interpretations and supportive interventions. The simultaneous use of these two interventions was associated with a high or an improving alliance over the course of treatment, but not with a low and stable alliance where only single theme sequences of interpretations were found. In other words, in this last group, therapists were either supportive or interpretative, whereas with high or improving alliance, interpretations were always given along with supportive interventions. This finding provides evidence that examining therapist interpretation individually can only yield incomplete findings. How interpretations were given is important for alliance building. It also suggests that therapists should carefully dose their interpretations and be supportive when necessary in order to build a strong therapeutic alliance. And from a research point of view, to study technical interventions, we must look into dynamic variables such as dosage, the supportive quality of an intervention, and timing. (PsycINFO Database Record (c) 2005 APA, all rights reserved)