Ubiquitin-mediated proteolysis in Xenopus extract

The small protein modifier, ubiquitin, can be covalently attached to proteins in the process of ubiquitylation, resulting in a variety of functional outcomes. In particular, the most commonly-associated and well-studied fate for proteins modified with ubiquitin is their ultimate destruction: degradation by the 26S proteasome via the ubiquitin-proteasome system, or digestion in lysosomes by proteolytic enzymes. From the earliest days of ubiquitylation research, a reliable and versatile “cell-in-a-test-tube” system has been employed in the form of cytoplasmic extracts from the eggs and embryos of the frog Xenopus laevis. Biochemical studies of ubiquitin and protein degradation using this system have led to significant advances particularly in the study of ubiquitin-mediated proteolysis, while the versatility of Xenopus as a developmental model has allowed investigation of the in vivo consequences of ubiquitylation. Here we describe the use and history of Xenopus extract in the study of ubiquitin-mediated protein degradation, and highlight the versatility of this system that has been exploited to uncover mechanisms and consequences of ubiquitylation and proteolysis.Research in AP’s laboratory is supported by Medical Research Council grants MR/K018329/1 and MR/L021129/1, and core support from the Wellcome Trust and MRC to the Wellcome Trust – Medical Research Council Cambridge Stem Cell Institute. GM was supported by Michael Levin at Tufts University through grants from the G. Harold and Leila Y. Mathers Charitable Foundation and the Physical Science Oncology Center supported by Award Number U54CA143876 from the National Cancer Institute

Facilitative parenting and children's social, emotional and behavioural adjustment

Facilitative parenting (FP) supports the development of children’s social and emotional competence and effective peer relationships. Previous research has shown that FP discriminates between children bullied by peers from children who are not bullied, according to reports of teachers. This study investigates the association between FP and children’s social, emotional and behavioral problems, over and above the association with dysfunctional parenting (DP). 215 parents of children aged 5–11 years completed questionnaires about parenting and child behavior, and children and teachers completed measures of child bullying victimization. As predicted, FP accounted for variance in teacher reports of children’s bullying victimization as well as parent reports of children’s social and emotional problems and prosocial behavior better than that accounted for by DP. However for children’s reports of peer victimization the whole-scale DP was a better predictor than FP. Contrary to predictions, FP accounted for variance in conduct problems and hyperactivity better than DP. When analyses were replicated substituting subscales of dysfunctional and FP, a sub-set of FP subscales including Warmth, Supports Friendships, Not Conflicting, Child Communicates and Coaches were correlated with low levels of problems on a broad range of children’s adjustment problems. Parent–child conflict accounted for unique variance in children’s peer victimization (teacher report), peer problems, depression, emotional problems, conduct problems and hyperactivity. The potential relevance of FP as a protective factor for children against a wide range of adjustment problems is discussed

Assessing the order of magnitude of outcomes in single-arm cohorts through systematic comparison with corresponding cohorts: An example from the AMOS study

<p>Abstract</p> <p>Background</p> <p>When a therapy has been evaluated in the first clinical study, the outcome is often compared descriptively to outcomes in corresponding cohorts receiving other treatments. Such comparisons are often limited to selected studies, and often mix different outcomes and follow-up periods. Here we give an example of a systematic comparison to all cohorts with identical outcomes and follow-up periods.</p> <p>Methods</p> <p>The therapy to be compared (anthroposophic medicine, a complementary therapy system) had been evaluated in one single-arm cohort study: the Anthroposophic Medicine Outcomes Study (AMOS). The five largest AMOS diagnosis groups (A-cohorts: asthma, depression, low back pain, migraine, neck pain) were compared to all retrievable corresponding cohorts (C-cohorts) receiving other therapies with identical outcomes (SF-36 scales or summary measures) and identical follow-up periods (3, 6 or 12 months). Between-group differences (pre-post difference in an A-cohort minus pre-post difference in the respective C-cohort) were divided with the standard deviation (SD) of the baseline score of the A-cohort.</p> <p>Results</p> <p>A-cohorts (5 cohorts with 392 patients) were similar to C-cohorts (84 cohorts with 16,167 patients) regarding age, disease duration, baseline affection and follow-up rates. A-cohorts had ≥ 0.50 SD larger improvements than C-cohorts in 13.5% (70/517) of comparisons; improvements of the same order of magnitude (small or minimal differences: -0.49 to 0.49 SD) were found in 80.1% of comparisons; and C-cohorts had ≥ 0.50 SD larger improvements than A-cohorts in 6.4% of comparisons. Analyses stratified by diagnosis had similar results. Sensitivity analyses, restricting the comparisons to C-cohorts with similar study design (observational studies), setting (primary care) or interventions (drugs, physical therapies, mixed), or restricting comparisons to SF-36 scales with small baseline differences between A- and C-cohorts (-0.49 to 0.49 SD) also had similar results.</p> <p>Conclusion</p> <p>In this descriptive analysis, anthroposophic therapy was associated with SF-36 improvements largely of the same order of magnitude as improvements following other treatments. Although these non-concurrent comparisons cannot assess comparative effectiveness, they suggest that improvements in health status following anthroposophic therapy can be clinically meaningful. The analysis also demonstrates the value of a systematic approach when comparing a therapy cohort to corresponding therapy cohorts.</p

Synergistic ecoclimate teleconnections from forest loss in different regions structure global ecological responses

ABSTRACT: Forest loss in hotspots around the world impacts not only local climate where loss occurs, but also influences climate and vegetation in remote parts of the globe through ecoclimate teleconnections. The magnitude and mechanism of remote impacts likely depends on the location and distribution of forest loss hotspots, but the nature of these dependencies has not been investigated. We use global climate model simulations to estimate the distribution of ecologically-relevant climate changes resulting from forest loss in two hotspot regions: western North America (wNA), which is experiencing accelerated dieoff, and the Amazon basin, which is subject to high rates of deforestation. The remote climatic and ecological net effects of simultaneous forest loss in both regions differed from the combined effects of loss from the two regions simulated separately, as evident in three impacted areas. Eastern South American Gross Primary Productivity (GPP) increased due to changes in seasonal rainfall associated with Amazon forest loss and changes in temperature related to wNA forest loss. Eurasia’s GPP declined with wNA forest loss due to cooling temperatures increasing soil ice volume. Southeastern North American productivity increased with simultaneous forest loss, but declined with only wNA forest loss due to changes in VPD. Our results illustrate the need for a new generation of local-to-global scale analyses to identify potential ecoclimate teleconnections, their underlying mechanisms, and most importantly, their synergistic interactions, to predict the responses to increasing forest loss under future land use change and climate change

Inside the guts of wood-eating catfishes: can they digest wood?

To better understand the structure and function of the gastrointestinal (GI) tracts of wood-eating catfishes, the gross morphology, length, and microvilli surface area (MVSA) of the intestines of wild-caught Panaque nocturnus, P. cf. nigrolineatus “Marañon”, and Hypostomus pyrineusi were measured, and contrasted against these same metrics of a closely related detritivore, Pterygoplichthys disjunctivus. All four species had anatomically unspecialized intestines with no kinks, valves, or ceca of any kind. The wood-eating catfishes had body size-corrected intestinal lengths that were 35% shorter than the detritivore. The MVSA of all four species decreased distally in the intestine, indicating that nutrient absorption preferentially takes place in the proximal and mid-intestine, consistent with digestive enzyme activity and luminal carbohydrate profiles for these same species. Wild-caught Pt. disjunctivus, and P. nigrolineatus obtained via the aquarium trade, poorly digested wood cellulose (<33% digestibility) in laboratory feeding trials, lost weight when consuming wood, and passed stained wood through their digestive tracts in less than 4 h. Furthermore, no selective retention of small particles was observed in either species in any region of the gut. Collectively, these results corroborate digestive enzyme activity profiles and gastrointestinal fermentation levels in the fishes’ GI tracts, suggesting that the wood-eating catfishes are not true xylivores such as beavers and termites, but rather, are detritivores like so many other fishes from the family Loricariidae

Application of microarray technology in pulmonary diseases

Microarrays are a powerful tool that have multiple applications both in clinical and cell biology arenas of common lung diseases. To exemplify how this tool can be useful, in this review, we will provide an overview of the application of microarray technology in research relevant to common lung diseases and present some of the future perspectives

Predictors of outcome after 6 and 12 months following anthroposophic therapy for adult outpatients with chronic disease: a secondary analysis from a prospective observational study

<p>Abstract</p> <p>Background</p> <p>Anthroposophic medicine is a physician-provided complementary therapy system involving counselling, artistic and physical therapies, and special medications. The purpose of this analysis was to identify predictors of symptom improvement in patients receiving anthroposophic treatment for chronic diseases.</p> <p>Methods</p> <p>913 adult outpatients from Germany participated in a prospective cohort study. Patients were starting anthroposophic treatment for mental (30.4% of patients, n = 278/913), musculoskeletal (20.2%), neurological (7.6%), genitourinary (7.4%) or respiratory disorders (7.2%) or other chronic indications. Stepwise multiple linear regression analysis was performed with the improvement of Symptom Score (patients' assessment, 0: not present, 10: worst possible) after 6 and 12 months as dependent variables. 61 independent variables pertaining to socio-demographics, life style, disease status, co-morbidity, health status (SF-36), depression, and therapy factors were analysed.</p> <p>Results</p> <p>Compared to baseline, Symptom Score improved by average 2.53 points (95% confidence interval 2.39-2.68, p < 0.001) after six months and by 2.49 points (2.32-2.65, p < 0.001) after 12 months. The strongest predictor for improvement after six months was baseline Symptom Score, which alone accounted for 25% of the variance (total model 32%). Improvement after six months was also positively predicted by better physical function, better general health, shorter disease duration, higher education level, a diagnosis of respiratory disorders, and by a higher therapy goal documented by the physician at baseline; and negatively predicted by the number of physiotherapy sessions in the pre-study year and by a diagnosis of genitourinary disorders. Seven of these nine variables (not the two diagnoses) also predicted improvement after 12 months. When repeating the 0-6 month analysis on two random subsamples of the original sample, three variables (baseline Symptom Score, physical function, general health) remained significant predictors in both analyses, and three further variables (education level, respiratory disorders, therapy goal) were significant in one analysis.</p> <p>Conclusion</p> <p>In adult outpatients receiving anthroposophic treatment for chronic diseases, symptom improvement after 6 and 12 months was predicted by baseline symptoms, health status, disease duration, education, and therapy goal. Other variables were not associated with the outcome. This secondary predictor analysis of data from a pre-post study does not allow for causal conclusions; the results are hypothesis generating and need verification in subsequent studies.</p

Role of Acetyl-Phosphate in Activation of the Rrp2-RpoN-RpoS Pathway in Borrelia burgdorferi

Borrelia burgdorferi, the Lyme disease spirochete, dramatically alters its transcriptome and proteome as it cycles between the arthropod vector and mammalian host. During this enzootic cycle, a novel regulatory network, the Rrp2-RpoN-RpoS pathway (also known as the σ54–σS sigma factor cascade), plays a central role in modulating the differential expression of more than 10% of all B. burgdorferi genes, including the major virulence genes ospA and ospC. However, the mechanism(s) by which the upstream activator and response regulator Rrp2 is activated remains unclear. Here, we show that none of the histidine kinases present in the B. burgdorferi genome are required for the activation of Rrp2. Instead, we present biochemical and genetic evidence that supports the hypothesis that activation of the Rrp2-RpoN-RpoS pathway occurs via the small, high-energy, phosphoryl-donor acetyl phosphate (acetyl∼P), the intermediate of the Ack-Pta (acetate kinase-phosphate acetyltransferase) pathway that converts acetate to acetyl-CoA. Supplementation of the growth medium with acetate induced activation of the Rrp2-RpoN-RpoS pathway in a dose-dependent manner. Conversely, the overexpression of Pta virtually abolished acetate-induced activation of this pathway, suggesting that acetate works through acetyl∼P. Overexpression of Pta also greatly inhibited temperature and cell density-induced activation of RpoS and OspC, suggesting that these environmental cues affect the Rrp2-RpoN-RpoS pathway by influencing acetyl∼P. Finally, overexpression of Pta partially reduced infectivity of B. burgdorferi in mice. Taken together, these findings suggest that acetyl∼P is one of the key activating molecule for the activation of the Rrp2-RpoN-RpoS pathway and support the emerging concept that acetyl∼P can serve as a global signal in bacterial pathogenesis