In situ analysis of osmolyte mechanisms of proteome thermal stabilization.

Organisms use organic molecules called osmolytes to adapt to environmental conditions. In vitro studies indicate that osmolytes thermally stabilize proteins, but mechanisms are controversial, and systematic studies within the cellular milieu are lacking. We analyzed Escherichia coli and human protein thermal stabilization by osmolytes in situ and across the proteome. Using structural proteomics, we probed osmolyte effects on protein thermal stability, structure and aggregation, revealing common mechanisms but also osmolyte- and protein-specific effects. All tested osmolytes (trimethylamine N-oxide, betaine, glycerol, proline, trehalose and glucose) stabilized many proteins, predominantly via a preferential exclusion mechanism, and caused an upward shift in temperatures at which most proteins aggregated. Thermal profiling of the human proteome provided evidence for intrinsic disorder in situ but also identified potential structure in predicted disordered regions. Our analysis provides mechanistic insight into osmolyte function within a complex biological matrix and sheds light on the in situ prevalence of intrinsically disordered regions.

ATP-independent substrate recruitment to proteasomal degradation in mycobacteria.

Mycobacteria and other actinobacteria possess proteasomal degradation pathways in addition to the common bacterial compartmentalizing protease systems. Proteasomal degradation plays a crucial role in the survival of these bacteria in adverse environments. The mycobacterial proteasome interacts with several ring-shaped activators, including the bacterial proteasome activator (Bpa), which enables energy-independent degradation of heat shock repressor HspR. However, the mechanism of substrate selection and processing by the Bpa-proteasome complex remains unclear. In this study, we present evidence that disorder in substrates is required but not sufficient for recruitment to Bpa-mediated proteasomal degradation. We demonstrate that Bpa binds to the folded N-terminal helix-turn-helix domain of HspR, whereas the unstructured C-terminal tail of the substrate acts as a sequence-specific threading handle to promote efficient proteasomal degradation. In addition, we establish that the heat shock chaperone DnaK, which interacts with and co-regulates HspR, stabilizes HspR against Bpa-mediated proteasomal degradation. By phenotypical characterization of Mycobacterium smegmatis parent and bpa deletion mutant strains, we show that Bpa-dependent proteasomal degradation supports the survival of the bacterium under stress conditions by degrading HspR that regulates vital chaperones.

Hypothesis testing in multivariate normal models with block circular covariance structures.

In this article, we address the problem of simultaneous testing hypothesis about mean and covariance matrix for repeated measures data when both the mean vector and covariance matrix are patterned. In particular, tests about the mean vector under block circular and doubly exchangeable covariance structures have been considered. The null distributions are established for the corresponding likelihood ratio test statistics, and expressions for the exact or near-exact probability density and cumulative distribution functions are obtained. The application of the results is illustrated by both a simulation study and a real-life data example.

Survival in Hostile Conditions: Pupylation and the Proteasome in Actinobacterial Stress Response Pathways.

Bacteria employ a multitude of strategies to cope with the challenges they face in their natural surroundings, be it as pathogens, commensals or free-living species in rapidly changing environments like soil. Mycobacteria and other Actinobacteria acquired proteasomal genes and evolved a post-translational, ubiquitin-like modification pathway called pupylation to support their survival under rapidly changing conditions and under stress. The proteasomal 20S core particle (20S CP) interacts with ring-shaped activators like the hexameric ATPase Mpa that recruits pupylated substrates. The proteasomal subunits, Mpa and pupylation enzymes are encoded in the so-called Pup-proteasome system (PPS) gene locus. Genes in this locus become vital for bacteria to survive during periods of stress. In the successful human pathogen Mycobacterium tuberculosis, the 20S CP is essential for survival in host macrophages. Other members of the PPS and proteasomal interactors are crucial for cellular homeostasis, for example during the DNA damage response, iron and copper regulation, and heat shock. The multiple pathways that the proteasome is involved in during different stress responses suggest that the PPS plays a vital role in bacterial protein quality control and adaptation to diverse challenging environments.

The Sävsjö-school-project: a cluster-randomized trial aimed at improving the literacy of beginners-achievements, mental health, school satisfaction and reading capacity at the end of grade three using an alternative school curriculum.

BACKGROUND: A curriculum was planned using modern concepts based on the "old" principles to test if such an educational intervention provided pupils with good mental health and a solid basis for good reading and writing skills, as well as generated a positive attitude to learn. These "old" principles were based on previous knowledge derived from school psychiatry (which in Sweden was a branch of child and adolescent psychiatry 1915-1970), educational psychology and the educational approach from the differentiating Swedish School system of 1946-1970 (itself based on the principles of curative education "Heilpädagogie", which was later renamed mental health care). METHODS: All six available schools in the small Swedish city of Sävsjö participated in the study. In these six schools there were eight preschool classes that included every 6-year old child living in the city. In total there were 184 families with 186 children (including 2 pairs of twins) who belonged to these preschool classes and were invited to take part in the study. One family moved just before school-start and 8 decided not to participate, thus 177 children (84 boys and 93 girls, aged 5.6-6.6 years) entered the study. The preschool classes were randomized into an experimental group with four preschool classes and a comparison group with four preschool classes. The experimental group followed a teaching program from the start of the preschool year until the end of grade 3 that was tailored to each student's individual capacity based on the concepts of school maturation and curative education used in the Swedish schools during the period 1946-1970. The comparison group followed today's average Swedish school curriculum. The project was planned as an intervention study covering the preschool year and the first 3 years of elementary school, which was to form a basis for a follow-up when the pupils had left senior high, the 12th year in Swedish public school. The outcome and the achievements were measured at end of grade 3 using standardized tests on reading, writing and mathematical skills. Behavior was assessed at school start and at end of grade 3 using the Child Behavior Check List (CBCL-scales) in addition to a questionnaire on Attention Deficit Hyperactivity Disorder (AD/HD) with criteria from DSM-IV. The children made a self-evaluation of their attitude towards learning. RESULTS: At the end of school year 3, the children in the experimental group had an improved reading capacity (p = 0.002, effect size(es) = 4.35) and reading comprehension (p = 0.03, es = 0.04). They evaluated their own reading (p = 0.02, es = 0.23), writing (p = 0.007, es = 0.35) and mathematical skills (p = 0.003, es = 0.48) as going "very well" when compared to comparison group. Differences regarding intelligence quotas between the groups at the start of school had disappeared by the end of grade 3. No differences referring to CBCL were found at end of grade 3. One child in the comparison group fulfilled criteria for AD/HD, according to parents and teachers. CONCLUSIONS: The alternative curriculum covering the preschool year through the first 3 years of elementary school based on the old principles from curative education ("Heilpädagogie"), educational psychology and school psychiatry gave the children in the experimental group a better reading capacity and reading comprehension.Trial registration The study started in 1998. The data were collected longitudinally and prospectively but have not been analyzed until now, with the children having left senior high. A retrospective registration in the ISRCTN is pending.

Influence diagnostics for count data under AB-BA crossover trials.

This paper aims to develop diagnostic measures to assess the influence of data perturbations on estimates in AB-BA crossover studies with a Poisson distributed response. Generalised mixed linear models with normally distributed random effects are utilised. We show that in this special case, the model can be decomposed into two independent sub-models which allow to derive closed-form expressions to evaluate the changes in the maximum likelihood estimates under several perturbation schemes. The performance of the new influence measures is illustrated by simulation studies and the analysis of a real dataset.