An engineered Tetrahymena tRNA(Gln) for in vivo incorporation of unnatural amino acids into proteins by nonsense suppression

A new tRNA, THG73, has been designed and evaluated as a vehicle for incorporating unnatural amino acids site-specifically into proteins expressed in vivo using the stop codon suppression technique. The construct is a modification of tRNAGln(CUA) from Tetrahymena thermophila, which naturally recognizes the stop codon UAG. Using electrophysiological studies of mutations at several sites of the nicotinic acetylcholine receptor, it is established that THG73 represents a major improvement over previous nonsense suppressors both in terms of efficiency and fidelity of unnatural amino acid incorporation. Compared with a previous tRNA used for in vivo suppression, THG73 is as much as 100-fold less likely to be acylated by endogenous synthetases of the Xenopus oocyte. This effectively eliminates a major concern of the in vivo suppression methodology, the undesirable incorporation of natural amino acids at the suppression site. In addition, THG73 is 4-10-fold more efficient at incorporating unnatural amino acids in the oocyte system. Taken together, these two advances should greatly expand the range of applicability of the in vivo nonsense suppression methodology

Diagnostic development for determining the joint temperature/soot statistics in hydrocarbon-fueled pool fires : LDRD final report.

A joint temperature/soot laser-based optical diagnostic was developed for the determination of the joint temperature/soot probability density function (PDF) for hydrocarbon-fueled meter-scale turbulent pool fires. This Laboratory Directed Research and Development (LDRD) effort was in support of the Advanced Simulation and Computing (ASC) program which seeks to produce computational models for the simulation of fire environments for risk assessment and analysis. The development of this laser-based optical diagnostic is motivated by the need for highly-resolved spatio-temporal information for which traditional diagnostic probes, such as thermocouples, are ill-suited. The in-flame gas temperature is determined from the shape of the nitrogen Coherent Anti-Stokes Raman Scattering (CARS) signature and the soot volume fraction is extracted from the intensity of the Laser-Induced Incandescence (LII) image of the CARS probed region. The current state of the diagnostic will be discussed including the uncertainty and physical limits of the measurements as well as the future applications of this probe

Dopamine transporter trafficking and Rit2 GTPase: Mechanism of action and in vivo impact

Following its evoked release, DA signaling is rapidly terminated by presynaptic reuptake, mediated by the cocaine-sensitive DAT. DAT surface availability is dynamically regulated by endocytic trafficking, and direct PKC activation acutely diminishes DAT surface expression by accelerating DAT internalization. Previous cell line studies demonstrated that PKC-stimulated DAT endocytosis requires both Ack1 inactivation, which releases a DAT-specific endocytic brake, and the neuronal GTPase, Rit2, which binds DAT. However, it is unknown whether Rit2 is required for PKC-stimulated DAT endocytosis in DAergic terminals, or whether there are region- and/or sex-dependent differences in PKC-stimulated DAT trafficking. Moreover, the mechanisms by which Rit2 controls PKC-stimulated DAT endocytosis are unknown. Here, we directly examined these important questions. Ex vivo studies revealed that PKC activation acutely decreased DAT surface expression selectively in ventral, but not dorsal, striatum. AAV-mediated, conditional Rit2 knockdown in DAergic neurons impacted baseline DAT surface:intracellular distribution in DAergic terminals from female ventral, but not dorsal, striatum. Further, Rit2 was required for PKC-stimulated DAT internalization in both male and female ventral striatum. FRET and surface pulldown studies in cell lines revealed that PKC activation drives DAT-Rit2 surface dissociation, and that the DAT N-terminus is required for both PKC-mediated DAT-Rit2 dissociation and DAT internalization. Finally, we found that Rit2 and Ack1 independently converge on DAT to facilitate PKC-stimulated DAT endocytosis. Together, our data provide greater insight into mechanisms that mediate PKC-regulated DAT internalization, and reveal unexpected region-specific differences in PKC-stimulated DAT trafficking in bona fide DAergic terminals

EUV mask reflectivity measurements with micron-scale spatial resolution

The effort to produce defect-free mask blanks for EUV lithography relies on increasing the detection sensitivity of advanced mask inspection tools, operating at several wavelengths. We describe the unique measurement capabilities of a prototype actinic (EUV wavelength) microscope that is capable of detecting small defects and reflectivity changes that occur on the scale of microns to nanometers. Types of defects: (a) Buried Substrate Defects: particles & pits (causes amplitude and/or phase variations); (b) Surface Contamination (reduces reflectivity and (possibly) contrast); (c) Damage from Inspection and Use (reduces the reflectivity of the multilayer coating). This paper presents an overview of several topics where scanning actinic inspection makes a unique contribution to EUVL research. We describe the role of actinic scanning inspection in four cases: defect repair studies; observations of laser damage; after scanning electron microscopy; and native and programmed defects

The importance of both setting and intensity of physical activity in relation to non-clinical anxiety and depression

Physical activity is associated with good physical and mental health. Current recommendations suggest that people should achieve 30 minutes of moderate-intensity activity most days of the week to gain health benefits. This activity may be accumulated in leisure time, in active commuting, at work or in the home. Here we look at the cross-sectional relationship between physical activity and mental health as measured by the HADS anxiety and depression scores in a sample of 1,742 participants from a Scottish general population survey. The participants were men and women in three age cohorts aged around 24, 44 and 64 years who, in 1995, were interviewed face to face and also self-completed the HADS depression and anxiety scale. Respondents reported their levels of physical activity at work, in the home and in leisure time; the intensities of activity were also determined. Physical activity was related to depression scores but not to anxiety scores. There was no relationship between work physical activity and depression score. Among women, depression score increased with each additional episode of vigorous home activity. In both sexes, depression score decreased with each additional episode of vigorous leisure activity, but among men the decrease in depression score with moderate leisure activity was reversed if a lot of moderate activity was undertaken. We have found a variable relationship between depression scores and various settings for physical activity. Researchers, policymakers and practitioners who are interested in the relationship between physical activity and mental health should take into account the setting for activity as well as frequency, duration and intensity of activity

Food insecurity, school absenteeism and educational attainment of adolescents in Jimma Zone Southwest Ethiopia: a longitudinal study

<p>Abstract</p> <p>Background</p> <p>Food insecurity not only affects physical growth and health of children but also their intellectual development, school attendance and academic performance. However, most evidences are based on studies in high income countries. Although food insecurity is common in Ethiopia, to what extent it affects school attendance and educational attainment of adolescents is not explored. We hypothesized that food insecure adolescents would be more likely to be absent from school and have lower grades attained after 1 year compared to their food secure peers.</p> <p>Methods</p> <p>We used data from 2009 adolescents in the age group of 13-17 years from two consecutive surveys of a five year longitudinal family study in Southwest Ethiopia. A stratified random sampling was used to select participants. Regression analyses were used to compare school absenteeism and the highest grade attained after 1 year of follow-up in food secure and insecure adolescents. The analysis was adjusted for demographic factors, reported illness and workload.</p> <p>Results</p> <p>Significantly more (33.0%) food insecure adolescents were absent from school compared with their food secure peers (17.8%, P < 0.001). Multivariable logistic regression analyses showed that after adjusting for gender, place of residence and gender of the household head, adolescent food insecurity [OR 1.77 (1.34-2.33)], severe household food insecurity [OR 1.62 (1.27-2.06)], illness during the past one month before the survey [OR 2.26 (1.68-3.06)], the highest grade aspired to be completed by the adolescent [OR 0.92 (0.88-0.96)], and the number of days that the adolescent had to work per week [OR 1.16 (1.07-1.26)] were independent predictors of school absenteeism. Similarly after controlling for household income and gender of the household head, adolescent food insecurity(P < 0.001), severe household food insecurity(P < 0.001), illness during the last month(P < 0.001) and rural residence(P < 0.001) were inversely associated with highest grade attained, while age of the adolescent(P < 0.001), the highest grade intended to be completed(P < 0.001) and residence in semi urban area(P < 0.001) were positively associated with the highest grade attained.</p> <p>Conclusions</p> <p>Adolescent and household food insecurity are positively associated with school absenteeism and a lower educational attainment. Programs aiming to achieve universal access to primary education in food insecure environments should integrate interventions to ensure food security of adolescents.</p

Soot formation, transport, and radiation in unsteady diffusion flames : LDRD final report.

Fires pose the dominant risk to the safety and security of nuclear weapons, nuclear transport containers, and DOE and DoD facilities. The thermal hazard from these fires primarily results from radiant emission from high-temperature flame soot. Therefore, it is necessary to understand the local transport and chemical phenomena that determine the distributions of soot concentration, optical properties, and temperature in order to develop and validate constitutive models for large-scale, high-fidelity fire simulations. This report summarizes the findings of a Laboratory Directed Research and Development (LDRD) project devoted to obtaining the critical experimental information needed to develop such constitutive models. A combination of laser diagnostics and extractive measurement techniques have been employed in both steady and pulsed laminar diffusion flames of methane, ethylene, and JP-8 surrogate burning in air. For methane and ethylene, both slot and coannular flame geometries were investigated, as well as normal and inverse diffusion flame geometries. For the JP-8 surrogate, coannular normal diffusion flames were investigated. Soot concentrations, polycyclic aromatic hydrocarbon (PAH) laser-induced fluorescence (LIF) signals, hydroxyl radical (OH) LIF, acetylene and water vapor concentrations, soot zone temperatures, and the velocity field were all successfully measured in both steady and unsteady versions of these various flames. In addition, measurements were made of the soot microstructure, soot dimensionless extinction coefficient (&amp;), and the local radiant heat flux. Taken together, these measurements comprise a unique, extensive database for future development and validation of models of soot formation, transport, and radiation

Nicotinic acetylcholine receptor subunit variants are associated with blood pressure; findings in the Old Order Amish and replication in the Framingham Heart Study

<p>Abstract</p> <p>Background</p> <p>Systemic blood pressure, influenced by both genetic and environmental factors, is regulated via sympathetic nerve activity. We assessed the role of genetic variation in three subunits of the neuromuscular nicotinic acetylcholine receptor positioned on chromosome 2q, a region showing replicated evidence of linkage to blood pressure.</p> <p>Methods</p> <p>We sequenced <it>CHRNA1</it>, <it>CHRND </it>and <it>CHRNG </it>in 24 Amish subjects from the Amish Family Diabetes Study (AFDS) and identified 20 variants. We then performed association analysis of non-redundant variants (n = 12) in the complete AFDS cohort of 1,189 individuals, and followed by genotyping blood pressure-associated variants (n = 5) in a replication sample of 1,759 individuals from the Framingham Heart Study (FHS).</p> <p>Results</p> <p>The minor allele of a synonymous coding SNP, rs2099489 in <it>CHRNG</it>, was associated with higher systolic blood pressure in both the Amish (p = 0.0009) and FHS populations (p = 0.009) (minor allele frequency = 0.20 in both populations).</p> <p>Conclusion</p> <p><it>CHRNG </it>is currently thought to be expressed only during fetal development. These findings support the Barker hypothesis, that fetal genotype and intra-uterine environment influence susceptibility to chronic diseases later in life. Additional studies of this variant in other populations, as well as the effect of this variant on acetylcholine receptor expression and function, are needed to further elucidate its potential role in the regulation of blood pressure. This study suggests for the first time in humans, a possible role for genetic variation in the neuromuscular nicotinic acetylcholine receptor, particularly the gamma subunit, in systolic blood pressure regulation.</p

Renal artery sympathetic denervation:observations from the UK experience

Background: Renal denervation (RDN) may lower blood pressure (BP); however, it is unclear whether medication changes may be confounding results. Furthermore, limited data exist on pattern of ambulatory blood pressure (ABP) response—particularly in those prescribed aldosterone antagonists at the time of RDN. Methods: We examined all patients treated with RDN for treatment-resistant hypertension in 18 UK centres. Results: Results from 253 patients treated with five technologies are shown. Pre-procedural mean office BP (OBP) was 185/102 mmHg (SD 26/19; n = 253) and mean daytime ABP was 170/98 mmHg (SD 22/16; n = 186). Median number of antihypertensive drugs was 5.0: 96 % ACEi/ARB; 86 % thiazide/loop diuretic and 55 % aldosterone antagonist. OBP, available in 90 % at 11 months follow-up, was 163/93 mmHg (reduction of 22/9 mmHg). ABP, available in 70 % at 8.5 months follow-up, was 158/91 mmHg (fall of 12/7 mmHg). Mean drug changes post RDN were: 0.36 drugs added, 0.91 withdrawn. Dose changes appeared neutral. Quartile analysis by starting ABP showed mean reductions in systolic ABP after RDN of: 0.4; 6.5; 14.5 and 22.1 mmHg, respectively (p &lt; 0.001 for trend). Use of aldosterone antagonist did not predict response (p &lt; 0.2). Conclusion: In 253 patients treated with RDN, office BP fell by 22/9 mmHg. Ambulatory BP fell by 12/7 mmHg, though little response was seen in the lowermost quartile of starting blood pressure. Fall in BP was not explained by medication changes and aldosterone antagonist use did not affect response