166 research outputs found

    Engineered split in Pfu DNA polymerase fingers domain improves incorporation of nucleotide γ-phosphate derivative

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    Using compartmentalized self-replication (CSR), we evolved a version of Pyrococcus furiosus (Pfu) DNA polymerase that tolerates modification of the γ-phosphate of an incoming nucleotide. A Q484R mutation in α-helix P of the fingers domain, coupled with an unintended translational termination-reinitiation (split) near the finger tip, dramatically improve incorporation of a bulky γ-phosphate-O-linker-dabcyl substituent. Whether synthesized by coupled translation from a bicistronic (−1 frameshift) clone, or reconstituted from separately expressed and purified fragments, split Pfu mutant behaves identically to wild-type DNA polymerase with respect to chromatographic behavior, steady-state kinetic parameters (for dCTP), and PCR performance. Although naturally-occurring splits have been identified previously in the finger tip region of T4 gp43 variants, this is the first time a split (in combination with a point mutation) has been shown to broaden substrate utilization. Moreover, this latest example of a split hyperthermophilic archaeal DNA polymerase further illustrates the modular nature of the Family B DNA polymerase structure

    Evaluating the capability of regional-scale air quality models to cature the vertical distribution of pollutants

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    This study is conducted in the framework of the Air Quality Modelling Evaluation International Initiative (AQMEII) and aims at the operational evaluation of an ensemble of 12 regional-scale chemical transport models used to predict air quality over the North American (NA) and European (EU) continents for 2006. The modelled concentrations of ozone and CO, along with the meteorological fields of wind speed (WS) and direction (WD), temperature (T), and relative humidity (RH), are compared against high-quality in-flight measurements collected by instrumented commercial aircraft as part of the Measurements of OZone, water vapour, carbon monoxide and nitrogen oxides by Airbus In-service airCraft (MOZAIC) programme. The evaluation is carried out for five model domains positioned around four major airports in NA (Portland, Philadelphia, Atlanta, and Dallas) and one in Europe (Frankfurt), from the surface to 8.5 km. We compare mean vertical profiles of modelled and measured variables for all airports to compute error and variability statistics, perform analysis of altitudinal error correlation, and examine the seasonal error distribution for ozone, including an estimation of the bias introduced by the lateral boundary conditions (BCs). The results indicate that model performance is highly dependent on the variable, location, season, and height (e.g. surface, planetary boundary layer (PBL) or free troposphere) being analysed. While model performance for T is satisfactory at all sites (correlation coefficient in excess of 0.90 and fractional bias ≤ 0.01 K), WS is not replicated as well within the PBL (exhibiting a positive bias in the first 100 m and also underestimating observed variability), while above 1000 m, the model performance improves (correlation coefficient often above 0.9). The WD at NA airports is found to be biased in the PBL, primarily due to an overestimation of westerly winds. RH is modelled well within the PBL, but in the free troposphere large discrepancies among models are observed, especially in EU. CO mixing ratios show the largest range of modelled-to-observed standard deviations of all the examined species at all heights and for all airports. Correlation coefficients for CO are typically below 0.6 for all sites and heights, and large errors are present at all heights, particularly in the first 250 m. Model performance for ozone in the PBL is generally good, with both bias and error within 20%. Profiles of ozone mixing ratios depend strongly on surface processes, revealed by the sharp gradient in the first 2 km (10 to 20 ppb km−1). Modelled ozone in winter is biased low at all locations in the NA, primarily due to an underestimation of ozone from the BCs. Most of the model error in the PBL is due to surface processes (emissions, transport, photochemistry), while errors originating aloft appear to have relatively limited impact on model performance at the surface. Suggestions for future work include interpretation of the model-to-model variability and common sources of model bias, and linking CO and ozone bias to the bias in the meteorological fields. Based on the results from this study, we suggest possible in-depth, process-oriented and diagnostic investigations to be carried out next

    Climate Change and Developing-Country Cities: Implications For Environmental Health and Equity

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    Climate change is an emerging threat to global public health. It is also highly inequitable, as the greatest risks are to the poorest populations, who have contributed least to greenhouse gas (GHG) emissions. The rapid economic development and the concurrent urbanization of poorer countries mean that developing-country cities will be both vulnerable to health hazards from climate change and, simultaneously, an increasing contributor to the problem. We review the specific health vulnerabilities of urban populations in developing countries and highlight the range of large direct health effects of energy policies that are concentrated in urban areas. Common vulnerability factors include coastal location, exposure to the urban heat-island effect, high levels of outdoor and indoor air pollution, high population density, and poor sanitation. There are clear opportunities for simultaneously improving health and cutting GHG emissions most obviously through policies related to transport systems, urban planning, building regulations and household energy supply. These influence some of the largest current global health burdens, including approximately 800,000 annual deaths from ambient urban air pollution, 1.2 million from road-traffic accidents, 1.9 million from physical inactivity, and 1.5 million per year from indoor air pollution. GHG emissions and health protection in developing-country cities are likely to become increasingly prominent in policy development. There is a need for a more active input from the health sector to ensure that development and health policies contribute to a preventive approach to local and global environmental sustainability, urban population health, and health equity

    Comparison of exercise testing and CMR measured myocardial perfusion reserve for predicting outcome in asymptomatic aortic stenosis: the PRognostic Importance of MIcrovascular Dysfunction in Aortic Stenosis (PRIMID AS) study

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    Aims To assess cardiovascular magnetic resonance (CMR) measured myocardial perfusion reserve (MPR) and exercise testing in asymptomatic patients with moderate-severe AS. Methods and results Multi-centre, prospective, observational study, with blinded analysis of CMR data. Patients underwent adenosine stress CMR, symptom-limited exercise testing (ETT) and echocardiography and were followed up for 12–30 months. The primary outcome was a composite of: typical AS symptoms necessitating referral for AVR, cardiovascular death and major adverse cardiovascular events. 174 patients were recruited: mean age 66.2 ± 13.34 years, 76% male, peak velocity 3.86 ± 0.56 m/s and aortic valve area index 0.57 ± 0.14 cm2/m2. A primary outcome occurred in 47 (27%) patients over a median follow-up of 374 (IQR 351–498) days. The mean MPR in those with and without a primary outcome was 2.06 ± 0.65 and 2.34 ± 0.70 (P = 0.022), while the incidence of a symptom-limited ETT was 45.7% and 27.0% (P = 0.020), respectively. MPR showed moderate association with outcome area under curve (AUC) = 0.61 (0.52–0.71, P = 0.020), as did exercise testing (AUC = 0.59 (0.51–0.68, P = 0.027), with no significant difference between the two. Conclusions MPR was associated with symptom-onset in initially asymptomatic patients with AS, but with moderate accuracy and was not superior to symptom-limited exercise testing. ClinicalTrials.gov (NCT01658345)

    Age and skill bias of trade liberalisation? : heterogeneous employment effects of EU Eastern Enlargement

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    This study analyses the 2004 Eastern Enlargement to the European Union to obtain evidence on the employment effects of an increase in trade liberalisation. The Enlargement is thought to generate a trade-induced demand shock with no (or only limited) supply effects. Besides the variation over time induced by the Enlargement, identification of the effects is based on a Melitz (2003) type productivity term to differentiate firms by the extent of exposure to the demand shock. The idea is that the effects of the demand shock should be driven by differences in firm-level productivity from the period before the new member countries actually entered the EU. German linked employer-employee data allow to observe the relation of initial establishment productivity with employment changes over a long panel from 1995 to 2009. The estimates show that the Enlargement had a negative effect on establishment-level employment growth, which is driven by increased worker separations and increased job destruction. Besides the overall employment effect, the study focuses on effect heterogeneity across age and skill groups of the workforce. These estimates point to a skill bias in the effect of the Enlargement that disadvantages low- and medium-skilled workers in terms of higher worker separation and job destruction. In addition, lowskilled workers suffer fewer accessions by firms, where against medium-skilled workers enjoy increased accessions and creation of new jobs. Besides this indication for a skill bias, there are no clear indications that point to an age bias in the employment effect of the Eastern Enlargement

    Seasonal ozone vertical profiles over North America using the AQMEII3 group of air quality models: model inter-comparison and stratospheric intrusions

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    This study evaluates simulated vertical ozone profiles produced in the framework of the third phase of the Air Quality Model Evaluation International Initiative (AQMEII3) against ozonesonde observations in North America for the year 2010. Four research groups from the United States (US) and Europe have provided modeled ozone vertical profiles to conduct this analysis. Because some of the modeling systems differ in their meteorological drivers, wind speed and temperature are also included in the analysis. In addition to the seasonal ozone profile evaluation for 2010, we also analyze chemically inert tracers designed to track the influence of lateral boundary conditions on simulated ozone profiles within the modeling domain. Finally, cases of stratospheric ozone intrusions during May–June 2010 are investigated by analyzing ozonesonde measurements and the corresponding model simulations at Intercontinental Chemical Transport Experiment Ozonesonde Network Study (IONS) experiment sites in the western United States. The evaluation of the seasonal ozone profiles reveals that, at a majority of the stations, ozone mixing ratios are underestimated in the 1–6&thinsp;km range. The seasonal change noted in the errors follows the one seen in the variance of ozone mixing ratios, with the majority of the models exhibiting less variability than the observations. The analysis of chemically inert tracers highlights the importance of lateral boundary conditions up to 250&thinsp;hPa for the lower-tropospheric ozone mixing ratios (0–2&thinsp;km). Finally, for the stratospheric intrusions, the models are generally able to reproduce the location and timing of most intrusions but underestimate the magnitude of the maximum mixing ratios in the 2–6&thinsp;km range and overestimate ozone up to the first kilometer possibly due to marine air influences that are not accurately described by the models. The choice of meteorological driver appears to be a greater predictor of model skill in this altitude range than the choice of air quality model.</p

    Thermostable DNA Polymerase from a Viral Metagenome Is a Potent RT-PCR Enzyme

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    Viral metagenomic libraries are a promising but previously untapped source of new reagent enzymes. Deep sequencing and functional screening of viral metagenomic DNA from a near-boiling thermal pool identified clones expressing thermostable DNA polymerase (Pol) activity. Among these, 3173 Pol demonstrated both high thermostability and innate reverse transcriptase (RT) activity. We describe the biochemistry of 3173 Pol and report its use in single-enzyme reverse transcription PCR (RT-PCR). Wild-type 3173 Pol contains a proofreading 3′-5′ exonuclease domain that confers high fidelity in PCR. An easier-to-use exonuclease-deficient derivative was incorporated into a PyroScript RT-PCR master mix and compared to one-enzyme (Tth) and two-enzyme (MMLV RT/Taq) RT-PCR systems for quantitative detection of MS2 RNA, influenza A RNA, and mRNA targets. Specificity and sensitivity of 3173 Pol-based RT-PCR were higher than Tth Pol and comparable to three common two-enzyme systems. The performance and simplified set-up make this enzyme a potential alternative for research and molecular diagnostics
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