Extending the chemistry that supports genetic information transfer in vivo: phosphorothioate DNA, phosphorothioate RNA, 2'-O-methyl RNA, and methylphosphonate DNA.

DNA and RNA are the polynucleotides known to carry genetic information in life. Chemical variants of DNA and RNA backbones have been used in structure-function and biosynthesis studies in vitro, and in antisense pharmacology, where their properties of nuclease resistance and enhanced cellular uptake are important. This study addressed the question of whether the base(s) attached to artificial backbones encodes genetic information that can be transferred in vivo. Oligonucleotides containing chemical variants of DNA or RNA were used as primers for site-specific mutagenesis of bacteriophage f1. Progeny phage were scored both genetically and physically for the inheritance of information originally encoded by bases attached to the nonstandard backbones. Four artificial backbone chemistries were tested: phosphorothioate DNA, phosphorothioate RNA, 2'-O-methyl RNA and methylphosphonate DNA. All four were found capable of faithful information transfer from their attached bases when one or three artificial positions were flanked by normal DNA. Among oligonucleotides composed entirely of nonstandard backbones, only phosphorothioate DNA supported genetic information transfer in vivo.

Truncated WT1 mutants alter the subnuclear localization of the wild-type protein.

WT1 encodes a zinc-finger protein, expressed as distinct isoforms, that is inactivated in a subset of Wilms tumors. Both constitutional and somatic mutations disrupting the DNA-binding domain of WT1 result in a potentially dominant-negative phenotype. In generating inducible cell lines expressing wild-type isoforms of WT1 and WT1 mutants, we observed dramatic differences in the subnuclear localization of the induced proteins. The WT1 isoform that binds with high affinity to a defined DNA target, WT1(-KTS), was diffusely localized throughout the nucleus. In contrast, expression of an alternative splicing variant with reduced DNA binding affinity, WT1 (+KTS), or WT1 mutants with a disrupted zinc-finger domain resulted in a speckled pattern of expression within the nucleus. Although similar in appearance, the localization of WT1 variants to subnuclear clusters was clearly distinct from that of the essential splicing factor SC35, suggesting that WT1 is not directly involved in pre-mRNA splicing. Localization to subnuclear clusters required the N terminus of WT1, and coexpression of a truncated WT1 mutant and wild-type WT1(-KTS) resulted in their physical association, the redistribution of WT1(-KTS) from a diffuse to a speckled pattern, and the inhibition of its transactivational activity. These observations suggest that different WT1 isoforms and WT1 mutants have distinct subnuclear compartments. Dominant-negative WT1 proteins physically associate with wild-type WT1 in vivo and may result in its sequestration within subnuclear structures.

Antisense DNA downregulation of the ERBB2 oncogene measured by a flow cytometric assay.

A causal role has been inferred for ERBB2 overexpression in the etiology of breast cancer and other epithelial malignancies. The development of therapeutics that inhibit this tyrosine kinase cell surface receptor remains a high priority. This report describes the specific downregulation of ERBB2 protein and mRNA in the breast cancer cell line SK-BR-3 by using antisense DNA phosphorothioates. An approach was developed to examine antisense effects which allows simultaneous measurements of antisense dose and gene specific regulation on a per cell basis. A fluorescein isothiocyanate end-labeled tracer oligonucleotide was codelivered with antisense DNA followed by immunofluorescent staining for ERBB2 protein expression. Two-color flow cytometry measured the amount of both intracellular oligonucleotide and ERBB2 protein. In addition, populations of cells that received various doses of nucleic acids were physically separated and studied. In any given transfection, a 100-fold variation in oligonucleotide dosage was found. ERBB2 protein expression was reduced greater than 50%, but only in cells within a relatively narrow uptake range. Steady-state ERBB2 mRNA levels were selectively diminished, indicating a specific antisense effect. Cells receiving the optimal antisense dose were sorted and analyzed for cell cycle changes. After 2 days of ERBB2 suppression, breast cancer cells showed an accumulation in the G1 phase of the cell cycle.

The product of the ataxia-telangiectasia group D complementing gene, ATDC, interacts with a protein kinase C substrate and inhibitor.

Ataxia-telangiectasia (AT) is an autosomal recessive human genetic disease characterized by immunological, neurological, and developmental defects and an increased risk of cancer. Cells from individuals with AT show sensitivity to ionizing radiation, elevated recombination, cell cycle abnormalities, and aberrant cytoskeletal organization. The molecular basis of the defect is unknown. A candidate AT gene (ATDC) was isolated on the basis of its ability to complement the ionizing radiation sensitivity of AT group D fibroblasts. Whether ATDC is mutated in any AT patients is not known. We have found that the ATDC protein physically interacts with the intermediate-filament protein vimentin, which is a protein kinase C substrate and colocalizing protein, and with an inhibitor of protein kinase C, hPKCI-1. Indirect immunofluorescence analysis of cultured cells transfected with a plasmid encoding an epitope-tagged ATDC protein localizes the protein to vimentin filaments. We suggest that the ATDC and hPKCI-1 proteins may be components of a signal transduction pathway that is induced by ionizing radiation and mediated by protein kinase C.

Transforming growth factor beta induces the cyclin-dependent kinase inhibitor p21 through a p53-independent mechanism.

The transforming growth factor beta s (TGF-beta s) are a group of multifunctional growth factors which inhibit cell cycle progression in many cell types. The TGF-beta-induced cell cycle arrest has been partially attributed to the regulatory effects of TGF-beta on both the levels and the activities of the G1 cyclins and their kinase partners. The activities of these kinases are negatively regulated by a number of small proteins, p21 (WAF1, Cip1), p27Kip1, p16, and p15INK4B, that physically associate with cyclins, cyclin-dependent kinases, or cyclin-Cdk complexes. p21 has been previously shown to be transcriptionally induced by DNA damage through p53 as a mediator. We demonstrate that TGF-beta also causes a rapid transcriptional induction of p21, suggesting that p21 can respond to both intracellular and extracellular signals for cell cycle arrest. In contrast to DNA damage, however, induction of p21 by TGF-beta is not dependent on wild-type p53. The cell line studied in these experiments, HaCaT, contains two mutant alleles of p53, which are unable to activate transcription from the p21 promoter when overexpressed. In addition, TGF-beta and p53 act through distinct elements in the p21 promoter. Taken together, these findings suggest that TGF-beta can induce p21 through a p53-independent pathway. Previous findings have implicated p27Kip1 and p15INK2B as effectors mediating the TGF-beta growth inhibitory effect. These results demonstrate that a single extracellular antiproliferative signal, TGF-beta, can act through multiple signaling pathways to elicit a growth arrest response.

The Impact of Transit Oriented Development on the Travel Behaviors of Workers in Denver, Colorado

To combat unsustainable transportation systems characterized by reliance on petroleum, polluting emissions, traffic congestion and suburban sprawl, planners encourage mixed use, densely populated areas that provide individuals with opportunities to live, work, eat and shop without necessarily having to drive private automobiles to accommodate their needs. Despite these attempts, the frequency and duration of automobile trips has consistently increased in the United States throughout past decades. While many studies have focused on how residential proximity to transit influences travel behavior, the effect of workplace location has largely been ignored. This paper asks, does working near a TOD influence the travel behaviors of workers differently than workers living near a TOD? We examine the non-work travel behaviors of workers based upon their commuting mode and proximity to TODs. The data came from a 2009 travel behavior survey by the Denver Regional Council of Governments, which contains 8,000 households, 16,000 individuals, and nearly 80,000 trips. We measure sustainable travel behaviors as reduced mileage, reduced number of trips, and increased use of non-automobile transportation. The results of this study indicate that closer proximity of both households and workplaces to TODs decrease levels of car commuting and that non-car commuting leads to more sustainable personal travel behaviors characterized by more trips made with alternative modes.

Models and methods for the direct simulation of rough and micropatterned surfaces

Friction in hydrodynamic bearings are a major source of losses in car engines ([69]). The extreme loading conditions in those bearings lead to contact between the matching surfaces. In such conditions not only the overall geometry of the bearing is relevant, but also the small-scale topography of the surface determines the bearing performance. The possibility of shaping the surface of lubricated bearings down to the micrometer ([57]) opened the question of whether friction can be reduced by mean of micro-textures, with mixed results. This work focuses in the development of efficient numerical methods to solve thin film (lubrication) problems down to the roughness scale of measured surfaces. Due to the high velocities and the convergent-divergent geometries of hydrodynamic bearings, cavitation takes place. To treat cavitation in the lubrication problem the Elrod- Adams model is used, a mass-conserving model which has proven in careful numerical ([12]) and experimental ([119]) tests to be essential to obtain physically meaningful results. Another relevant aspect of the modeling is that the bearing inertial effects are considered, which is necessary to correctly simulate moving textures. As an application, the effects of micro-texturing the moving surface of the bearing were studied. Realistic values are assumed for the physical parameters defining the problems. Extensive fundamental studies were carried out in the hydrodynamic lubrication regime. Mesh-converged simulations considering the topography of real measured surfaces were also run, and the validity of the lubrication approximation was assessed for such rough surfaces.

Gender inequalities in occupational health related to the unequal distribution of working and employment conditions: a systematic review

Introduction: Gender inequalities exist in work life, but little is known about their presence in relation to factors examined in occupation health settings. The aim of this study was to identify and summarize the working and employment conditions described as determinants of gender inequalities in occupational health in studies related to occupational health published between 1999 and 2010. Methods: A systematic literature review was undertaken of studies available in MEDLINE, EMBASE, Sociological Abstracts, LILACS, EconLit and CINAHL between 1999 and 2010. Epidemiologic studies were selected by applying a set of inclusion criteria to the title, abstract, and complete text. The quality of the studies was also assessed. Selected studies were qualitatively analysed, resulting in a compilation of all differences between women and men in the prevalence of exposure to working and employment conditions and work-related health problems as outcomes. Results: Most of the 30 studies included were conducted in Europe (n=19) and had a cross-sectional design (n=24). The most common topic analysed was related to the exposure to work-related psychosocial hazards (n=8). Employed women had more job insecurity, lower control, worse contractual working conditions and poorer self-perceived physical and mental health than men did. Conversely, employed men had a higher degree of physically demanding work, lower support, higher levels of effort-reward imbalance, higher job status, were more exposed to noise and worked longer hours than women did. Conclusions: This systematic review has identified a set of working and employment conditions as determinants of gender inequalities in occupational health from the occupational health literature. These results may be useful to policy makers seeking to reduce gender inequalities in occupational health, and to researchers wishing to analyse these determinants in greater depth.

A massive association around the obscured open cluster RSGC3

Context. Four clusters of red supergiants have been discovered in a region of the Milky Way close to base of the Scutum-Crux Arm and the tip of the Long Bar. Population synthesis models indicate that they must be very massive to harbour so many supergiants. If the clusters are physically connected, this Scutum Complex would be the largest and most massive star-forming region ever identified in the Milky Way. Aims. The spatial extent of one of these clusters, RSGC3, has not been investigated. In this paper we explore the possibility that a population of red supergiants could be located in its vicinity. Methods. We utilised 2MASS JHKS photometry to identify candidate obscured luminous red stars in the vicinity of RSGC3. We observed a sample of candidates with the TWIN spectrograph on the 3.5-m telescope at Calar Alto, obtaining intermediate-resolution spectroscopy in the 8000−9000 Å range. We re-evaluated a number of classification criteria proposed in the literature for this spectral range and found that we could use our spectra to derive spectral types and luminosity classes. Results. We measured the radial velocity of five members of RSGC3, finding velocities similar to the average for members of Stephenson 2. Among the candidates observed outside the cluster, our spectra revealed eight M-type supergiants at distances <18′ from the centre of RSGC3, distributed in two clumps. The southern clump is most likely another cluster of red supergiants, with reddening and age identical to RSGC3. From 2MASS photometry, we identified four likely supergiant members of the cluster in addition to the five spectroscopically observed. The northern clump may be a small cluster with similar parameters. Photometric analysis of the area around RSGC3 suggests the presence of a large (>30) population of red supergiants with similar colours. Conclusions. Our data suggest that the massive cluster RSGC3 is surrounded by an extended association, which may be very massive ( ≳ 105 M⊙). We also show that supergiants in the Scutum Complex may be characterised via a combination of 2MASS photometry and intermediate-to-high-resolution spectroscopy in the Z band.

Massive open star clusters using the VVV survey III. A young massive cluster at the far edge of the Galactic bar

Context. Young massive clusters are key to map the Milky Way’s structure, and near-infrared large area sky surveys have contributed strongly to the discovery of new obscured massive stellar clusters. Aims. We present the third article in a series of papers focused on young and massive clusters discovered in the VVV survey. This article is dedicated to the physical characterization of VVV CL086, using part of its OB-stellar population. Methods. We physically characterized the cluster using JHKS near-infrared photometry from ESO public survey VVV images, using the VVV-SkZ pipeline, and near-infrared K-band spectroscopy, following the methodology presented in the first article of the series. Results. Individual distances for two observed stars indicate that the cluster is located at the far edge of the Galactic bar. These stars, which are probable cluster members from the statistically field-star decontaminated CMD, have spectral types between O9 and B0 V. According to our analysis, this young cluster (1.0 Myr < age < 5.0 Myr) is located at a distance of 11+5-6 kpc, and we estimate a lower limit for the cluster total mass of (2.8+1.6-1.4) · 103 M⊙. It is likely that the cluster contains even earlier and more massive stars.

The effect of smoking on visceral fat accumulation in Czech men and women

Many studies indicate that smoking is one of risk factors influencing the accumulation of visceral fat (VFA). The mechanism by which smoking contributes to the accumulation of visceral fat is not yet fully understood, but it is assumed that smoking increases the level of plasma cortisol, causes imbalance between male and female sex hormones in women, and decreases testosterone levels in men (Chiolero et al., 2008). The objection of this study was to evaluate the effect of smoking on the accumulation of VFA in the population of the Czech Republic. The research included 1,412 individuals of both genders divided into categories of smokers vs nonsmokers and physically active vs. physically inactive. VFA was measured by the device InBody 720 and information about physical activity and smoking was collected via a questionnaire. We supposed that smokers would have a higher percentage of VFA than non-smokers. The results showed that smokers had a higher amount of VFA, but the difference was not statistically significant. In contrast, statistically significant differences were found in the waist-to-hip ratio (WHR) and total % body fat. Physically active individuals had a much more favourable results than physically inactive individuals. Remarkably, the amount of VFA, WHR and % body fat tended to be smaller in smoking men than in non-smoking men, but higher in smoking women than in non-smoking women.

Measuring the stellar wind parameters in IGR J17544-2619 and Vela X-1 constrains the accretion physics in supergiant fast X-ray transient and classical supergiant X-ray binaries

Context. Classical supergiant X-ray binaries (SGXBs) and supergiant fast X-ray transients (SFXTs) are two types of high-mass X-ray binaries (HMXBs) that present similar donors but, at the same time, show very different behavior in the X-rays. The reason for this dichotomy of wind-fed HMXBs is still a matter of debate. Among the several explanations that have been proposed, some of them invoke specific stellar wind properties of the donor stars. Only dedicated empiric analysis of the donors’ stellar wind can provide the required information to accomplish an adequate test of these theories. However, such analyses are scarce. Aims. To close this gap, we perform a comparative analysis of the optical companion in two important systems: IGR J17544-2619 (SFXT) and Vela X-1 (SGXB). We analyze the spectra of each star in detail and derive their stellar and wind properties. As a next step, we compare the wind parameters, giving us an excellent chance of recognizing key differences between donor winds in SFXTs and SGXBs. Methods. We use archival infrared, optical and ultraviolet observations, and analyze them with the non-local thermodynamic equilibrium (NLTE) Potsdam Wolf-Rayet model atmosphere code. We derive the physical properties of the stars and their stellar winds, accounting for the influence of X-rays on the stellar winds. Results. We find that the stellar parameters derived from the analysis generally agree well with the spectral types of the two donors: O9I (IGR J17544-2619) and B0.5Iae (Vela X-1). The distance to the sources have been revised and also agree well with the estimations already available in the literature. In IGR J17544-2619 we are able to narrow the uncertainty to d = 3.0 ± 0.2 kpc. From the stellar radius of the donor and its X-ray behavior, the eccentricity of IGR J17544-2619 is constrained to e< 0.25. The derived chemical abundances point to certain mixing during the lifetime of the donors. An important difference between the stellar winds of the two stars is their terminal velocities (ν∞ = 1500 km s-1 in IGR J17544-2619 and ν∞ = 700 km s-1 in Vela X-1), which have important consequences on the X-ray luminosity of these sources. Conclusions. The donors of IGR J17544-2619 and Vela X-1 have similar spectral types as well as similar parameters that physically characterize them and their spectra. In addition, the orbital parameters of the systems are similar too, with a nearly circular orbit and short orbital period. However, they show moderate differences in their stellar wind velocity and the spin period of their neutron star which has a strong impact on the X-ray luminosity of the sources. This specific combination of wind speed and pulsar spin favors an accretion regime with a persistently high luminosity in Vela X-1, while it favors an inhibiting accretion mechanism in IGR J17544-2619. Our study demonstrates that the relative wind velocity is critical in class determination for the HMXBs hosting a supergiant donor, given that it may shift the accretion mechanism from direct accretion to propeller regimes when combined with other parameters.

Greece and the European Agenda: EU Enlargement and European Monetary Union. Program for the Study of Germany & Europe Working Paper 8.2

In the decade to come, the European Union will embark on two new projects, each destined to transform it in fundamental ways: (i) Eastern enlargement, and (ii) economic and monetary union. Neither of these projects will affect all members equally or in the same way. But Greece will, for two reasons, be affected in a manner qualitatively different to all other member states. First, Greece is the only country physically affected by the Luxembourg Summit's decision to begin accession negotiations with some, but not all, Central and Eastern European applicant countries: as a result of this decision, she will continue, for at least another eight to ten years, to be the only member country not to share a common border with another member state, with all the consequent implications in economic and geostrategic tenns. Second, when the European Council meets in early May to select those member states that are deemed to have met the convergence criteria, it will find that Greece is the only member state falling short of those criteria. This development may create additional difficulties for her economy during the transitional period of derogation. It will also pose new risks to Greece, insofar as she will be absent during the initial-and crucial-years of establishing a common monetary policy.

The EU regime on biofuels in transport: Still in search of sustainability. Egmont Paper No. 68, July 2014

Like other regions of the world, the EU is developing biofuels in the transport sector to reduce oil consumption and mitigate climate change. To promote them, it has adopted favourable legislation since the 2000s. In 2009 it even decided to oblige each Member State to ensure that by 2020 the share of energy coming from renewable sources reached at least 10% of their final consumption of energy in the transport sector. Biofuels are considered the main instrument to reach that percentage since the development of other alternatives (such as hydrogen and electricity) will take much longer than expected. Meanwhile, these various legislative initiatives have driven the production and consumption of biofuels in the EU. Biofuels accounted for 4.7% of EU transport fuel consumption in 2011. They have also led to trade and investment in biofuels on a global scale. This large-scale expansion of biofuels has, however, revealed numerous negative impacts. These stem from the fact that first-generation biofuels (i.e., those produced from food crops), of which the most important types are biodiesel and bioethanol, are used almost exclusively to meet the EU’s renewable 10% target in transport. Their negative impacts are: socioeconomic (food price rises), legal (land-grabbing), environmental (for instance, water stress and water pollution; soil erosion; reduction of biodiversity), climatic (direct and indirect land-use effects resulting in more greenhouse gas emissions) and public finance issues (subsidies and tax relief). The extent of such negative impacts depends on how biofuel feedstocks are produced and processed, the scale of production, and in particular, how they influence direct land use change (DLUC) and indirect land use change (ILUC) and the international trade. These negative impacts have thus provoked mounting debates in recent years, with a particular focus on ILUC. They have forced the EU to re-examine how it deals with biofuels and submit amendments to update its legislation. So far, the EU legislation foresees that only sustainable biofuels (produced in the EU or imported) can be used to meet the 10% target and receive public support; and to that end, mandatory sustainability criteria have been defined. Yet they have a huge flaw. Their measurement of greenhouse gas savings from biofuels does not take into account greenhouse gas emissions resulting from ILUC, which represent a major problem. The Energy Council of June 2014 agreed to set a limit on the extent to which firstgeneration biofuels can count towards the 10% target. But this limit appears to be less stringent than the ones made previously by the European Commission and the European Parliament. It also agreed to introduce incentives for the use of advanced (second- and third-generation) biofuels which would be allowed to count double towards the 10% target. But this again appears extremely modest by comparison with what was previously proposed. Finally, the approach chosen to take into account the greenhouse gas emissions due to ILUC appears more than cautious. The Energy Council agreed that the European Commission will carry out a reporting of ILUC emissions by using provisional estimated factors. A review clause will permit the later adjustment of these ILUC factors. With such legislative orientations made by the Energy Council, one cannot consider yet that there is a major shift in the EU biofuels policy. Bolder changes would have probably meant risking the collapse of the high-emission conventional biodiesel industry which currently makes up the majority of Europe’s biofuel production. The interests of EU farmers would have also been affected. There is nevertheless a tension between these legislative orientations and the new Commission’s proposals beyond 2020. In any case, many uncertainties remain on this issue. As long as solutions have not been found to minimize the important collateral damages provoked by the first generation biofuels, more scientific studies and caution are needed. Meanwhile, it would be wise to improve alternative paths towards a sustainable transport sector, i.e., stringent emission and energy standards for all vehicles, better public transport systems, automobiles that run on renewable energy other than biofuels, or other alternatives beyond the present imagination.