SCHEMA Recombination of a Fungal Cellulase Uncovers a Single Mutation That Contributes Markedly to Stability

A quantitative linear model accurately (R^2 = 0.88) describes the thermostabilities of 54 characterized members of a family of fungal cellobiohydrolase class II (CBH II) cellulase chimeras made by SCHEMA recombination of three fungal enzymes, demonstrating that the contributions of SCHEMA sequence blocks to stability are predominantly additive. Thirty-one of 31 predicted thermostable CBH II chimeras have thermal inactivation temperatures higher than the most thermostable parent CBH II, from Humicola insolens, and the model predicts that hundreds more CBH II chimeras share this superior thermostability. Eight of eight thermostable chimeras assayed hydrolyze the solid cellulosic substrate Avicel at temperatures at least 5 °C above the most stable parent, and seven of these showed superior activity in 16-h Avicel hydrolysis assays. The sequence-stability model identified a single block of sequence that adds 8.5 °C to chimera thermostability. Mutating individual residues in this block identified the C313S substitution as responsible for the entire thermostabilizing effect. Introducing this mutation into the two recombination parent CBH IIs not featuring it (Hypocrea jecorina and H. insolens) decreased inactivation, increased maximum Avicel hydrolysis temperature, and improved long time hydrolysis performance. This mutation also stabilized and improved Avicel hydrolysis by Phanerochaete chrysosporium CBH II, which is only 55–56% identical to recombination parent CBH IIs. Furthermore, the C313S mutation increased total H. jecorina CBH II activity secreted by the Saccharomyces cerevisiae expression host more than 10-fold. Our results show that SCHEMA structure-guided recombination enables quantitative prediction of cellulase chimera thermostability and efficient identification of stabilizing mutations

Diversification of importin-α isoforms in cellular trafficking and disease states.

The human genome encodes seven isoforms of importin α which are grouped into three subfamilies known as α1, α2 and α3. All isoforms share a fundamentally conserved architecture that consists of an N-terminal, autoinhibitory, importin-β-binding (IBB) domain and a C-terminal Arm (Armadillo)-core that associates with nuclear localization signal (NLS) cargoes. Despite striking similarity in amino acid sequence and 3D structure, importin-α isoforms display remarkable substrate specificity in vivo. In the present review, we look at key differences among importin-α isoforms and provide a comprehensive inventory of known viral and cellular cargoes that have been shown to associate preferentially with specific isoforms. We illustrate how the diversification of the adaptor importin α into seven isoforms expands the dynamic range and regulatory control of nucleocytoplasmic transport, offering unexpected opportunities for pharmacological intervention. The emerging view of importin α is that of a key signalling molecule, with isoforms that confer preferential nuclear entry and spatiotemporal specificity on viral and cellular cargoes directly linked to human diseases

Asthma education for school staff.

BACKGROUND: Teachers and school staff should be competent in managing asthma in schools. Demonstrated low levels of asthma knowledge mean that staff may not know how best to protect a child with asthma in their care, or may fail to take appropriate action in the event of a serious attack. Education about asthma could help to improve this knowledge and lead to better asthma outcomes for children. OBJECTIVES: To assess the effectiveness and safety of asthma education programmes for school staff, and to identify content and attributes underpinning them. SEARCH METHODS: We conducted the most recent searches on 29 November 2016. SELECTION CRITERIA: We included randomised controlled trials comparing an intervention to educate school staff about asthma versus a control group. We included studies reported as full text, those published as abstract only and unpublished data. DATA COLLECTION AND ANALYSIS: At least two review authors screened the searches, extracted outcome data and intervention characteristics from included studies and assessed risk of bias. Primary outcomes for the quantitative synthesis were emergency department (ED) or hospital visits, mortality and asthma control; we graded the main results and presented evidence in a 'Summary of findings' table. We planned a qualitative synthesis of intervention characteristics, but study authors were unable to provide the necessary information.We analysed dichotomous data as odds ratios, and continuous data as mean differences or standardised mean differences, all with a random-effects model. We assessed clinical, methodological and statistical heterogeneity when performing meta-analyses, and we narratively described skewed data. MAIN RESULTS: Five cluster-RCTs of 111 schools met the review eligibility criteria. Investigators measured outcomes in participating staff and often in children or parents, most often at between 1 and 12 months.All interventions were educational programmes but duration, content and delivery varied; some involved elements of training for pupils or primary care providers. We noted risk of selection, performance, detection and attrition biases, although to a differing extent across studies and outcomes.Quanitative and qualitative analyses were limited. Only one study reported visits to the ED or hospital and provided data that were too skewed for analysis. No studies reported any deaths or adverse events. Studies did not report asthma control consistently, but results showed no difference between groups on the paediatric asthma quality of life questionnaire (mean difference (MD) 0.14, 95% confidence interval (CI) -0.03 to 0.31; 1005 participants; we downgraded the quality of evidence to low for risk of bias and indirectness). Data for symptom days, night-time awakenings, restricted activities of daily living and school absences were skewed or could not be analysed; some mean scores were better in the trained group, but most differences between groups were small and did not persist to 24 months.Schools that received asthma education were more adherent to asthma policies, and staff were better prepared; more schools that had received staff asthma training had written asthma policies compared with control schools, more intervention schools showed improvement in measures taken to prevent or manage exercise-induced asthma attacks and more staff at intervention schools reported that they felt able to administer salbutamol via a spacer. However, the quality of the evidence was low; results show imbalances at baseline, and confidence in the evidence was limited by risk of bias and imprecision. Staff knowledge was higher in groups that had received asthma education, although results were inconsistent and difficult to interpret owing to differences between scales (low quality).Available information about the interventions was insufficient for review authors to conduct a meaningful qualitative synthesis of the content that led to a successful intervention, or of the resources required to replicate results accurately. AUTHORS' CONCLUSIONS: Asthma education for school staff increases asthma knowledge and preparedness, but studies vary and all available evidence is of low quality. Studies have not yet captured whether this improvement in knowledge has led to appreciable benefits over the short term or the longer term for the safety and health of children with asthma in school. Randomised evidence does not contribute to our knowledge of content or attributes of interventions that lead to the best outcomes, or of resources required for successful implementation.Complete reporting of the content and resources of educational interventions is essential for assessment of their effectiveness and feasibility for implementation. This applies to both randomised and non-randomised studies, although the latter may be better placed to observe important clinical outcomes such as exacerbations and mortality in the longer term

A Single cis Element Maintains Repression of the Key Developmental Regulator Gata2

In development, lineage-restricted transcription factors simultaneously promote differentiation while repressing alternative fates. Molecular dissection of this process has been challenging as transcription factor loci are regulated by many trans-acting factors functioning through dispersed cis elements. It is not understood whether these elements function collectively to confer transcriptional regulation, or individually to control specific aspects of activation or repression, such as initiation versus maintenance. Here, we have analyzed cis element regulation of the critical hematopoietic factor Gata2, which is expressed in early precursors and repressed as GATA-1 levels rise during terminal differentiation. We engineered mice lacking a single cis element −1.8 kb upstream of the Gata2 transcriptional start site. Although Gata2 is normally repressed in late-stage erythroblasts, the −1.8 kb mutation unexpectedly resulted in reactivated Gata2 transcription, blocked differentiation, and an aberrant lineage-specific gene expression pattern. Our findings demonstrate that the −1.8 kb site selectively maintains repression, confers a specific histone modification pattern and expels RNA Polymerase II from the locus. These studies reveal how an individual cis element establishes a normal developmental program via regulating specific steps in the mechanism by which a critical transcription factor is repressed

Constraints on the χ_(c1) versus χ_(c2) polarizations in proton-proton collisions at √s = 8 TeV

The polarizations of promptly produced χ_(c1) and χ_(c2) mesons are studied using data collected by the CMS experiment at the LHC, in proton-proton collisions at √s=8  TeV. The χ_c states are reconstructed via their radiative decays χ_c → J/ψγ, with the photons being measured through conversions to e⁺e⁻, which allows the two states to be well resolved. The polarizations are measured in the helicity frame, through the analysis of the χ_(c2) to χ_(c1) yield ratio as a function of the polar or azimuthal angle of the positive muon emitted in the J/ψ → μ⁺μ⁻ decay, in three bins of J/ψ transverse momentum. While no differences are seen between the two states in terms of azimuthal decay angle distributions, they are observed to have significantly different polar anisotropies. The measurement favors a scenario where at least one of the two states is strongly polarized along the helicity quantization axis, in agreement with nonrelativistic quantum chromodynamics predictions. This is the first measurement of significantly polarized quarkonia produced at high transverse momentum