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Copper-doped beryllium spheres are an attractive ablator for inertial confinement fusion experiments. Beryllium spheres are made by sputtering beryllium onto spherical plastic mandrels which must t...

Aniridia is a rare congenital syndrome that is associated with reduced visual acuity and progressive loss of vision. Aniridia patients may also develop systemic health issues associated with defects in the pancreas, digestive, and central nervous systems. The spectrum of symptoms associated with aniridia is due to haploinsufficiency of the paired box 6 gene (PAX6) and its role in the development and maintenance of the affected tissues. Here, we isolated pancreatic islets from mice heterozygous for Pax6 to test whether a Pax6-specific miRNA suppression (target protector) strategy can restore PAX6 protein levels. We show that miR-7 and miR-375 target specific sites within the Pax6 3′ UTR in a mouse pancreatic β-insulinoma cell line. Tough decoys (Tuds) against miR-7 and miR-375 increase expression of a mouse Pax6 3′ UTR luciferase reporter and increase PAX6 protein levels in these cells. Finally, we demonstrate that the shielding of the miR-7 binding site with a target protector restores PAX6 protein levels in the Pax6 heterozygous islets. The data presented here represent a proof of concept for RNA-based therapy for the progressive defects associated with aniridia and suggest the target protector approach may be a useful therapeutic strategy for other haploinsufficiency diseases., Graphical Abstract

Double-shell inertial confinement fusion targets represent a unique platform for achieving ignition. They consist of a low-Z outer ablator, a high-Z inner pusher layer, and a low-density foam layer sandwiched in between. There is the possibility that double-shell targets may achieve ignition at lower ion temperatures due to the containment of radiation and conduction losses as well as requiring smaller convergence ratios. We have explored using magnetron sputtering to make the inner high-Z pusher layers and have demonstrated a W-Cr bilayer inner-shell design. An Al-Be mixture was explored as one of the outer ablator materials. This material takes advantage of Al X-ray M-band absorption to reduce preheating and still retain Be high-ablation speeds. Typical commercial Al-Be materials suffer from phase separation. However, by using magnetron sputtering we have been able to demonstrate homogeneous Al-Be ablator coatings. The sputtered material forms with nanosized grains and has demonstrated excellent...

Dysregulated activity of the protease matriptase is a key contributor to aggressive tumor growth, cancer metastasis, and osteoarthritis. Methods for the detection and quantification of matriptase activity and inhibition would be useful tools. To address this need, we developed a matriptase-sensitive protein biosensor based on a dimerization-dependent red fluorescent protein (ddRFP) reporter system. In this platform, two adjoining protein domains, connected by a protease-labile linker, produce fluorescence when assembled and are nonfluorescent when the linker is cleaved by matriptase. A panel of ddRFP-based matriptase biosensor designs was created that contained different linker lengths between the protein domains. These constructs were characterized for linker-specific cleavage, matriptase activity, and matriptase selectivity; a biosensor containing a RSKLRVGGH linker (termed B4) was expressed at high yields and displayed both high catalytic efficiency and matriptase specificity. This biosensor detects matriptase inhibition by soluble and yeast cell surface expressed inhibitor domains with up to a 5-fold dynamic range and also detects matriptase activity expressed by human cancer cell lines. In addition to matriptase, we highlight a strategy that can be used to create effective biosensors for quantifying activity and inhibition of other proteases of interest. [Image: see text]

We describe a multi-purpose technology platform, termed μSCALE (Microcapillary Single Cell Analysis and Laser Extraction), that enables massively parallel, quantitative biochemical and biophysical measurements on millions of protein variants expressed from yeast or bacteria. μSCALE spatially segregates single cells within a microcapillary array, enabling repeated imaging, cell growth, and protein expression. We performed high-throughput analysis of cells and their protein products using a range of fluorescent assays, including binding affinity measurements and dynamic enzymatic assays. A precise laser-based extraction method allows rapid recovery of live clones and their genetic material from microcapillaries for further study. With μSCALE, we discovered a new antibody against a clinical cancer target, evolved an orange fluorescent protein biosensor, and engineered an enzyme with reduced sensitivity to its inhibitor. These three distinct protein analysis and engineering applications, each with unique assay requirements and different host organisms, highlight the flexibility and technical capabilities of our platform.

BACKGROUND: There is a direct relationship between bodyweight and risk of diabetes. Lorcaserin, a selective serotonin 2C receptor agonist that suppresses appetite, has been shown to facilitate sustained weight loss in obese or overweight patients. We aimed to evaluate the long-term effects of lorcaserin on diabetes prevention and remission. METHODS: In this randomised, double-blind, placebo-controlled trial done in eight countries, we recruited overweight or obese patients (body-mass index ≥27 kg/m2) with or at high risk for atherosclerotic vascular disease. Eligible patients were aged 40 years or older; patients at high risk for atherosclerotic vascular disease had to be aged 50 years or older with diabetes and at least one other risk factor. Patients were randomly assigned to receive either lorcaserin (10 mg twice daily) or matching placebo. Additionally, all patients had access to a standardised weight management programme based on lifestyle modification. The prespecified primary metabolic efficacy endpoint of time to incident diabetes was assessed in patients with prediabetes at baseline. The prespecified secondary outcomes for efficacy were incident diabetes in all patients without diabetes, achievement of normoglycaemia in patients with prediabetes, and change in glycated haemoglobin (HbA1c) in patients with diabetes. Hypoglycaemia was a prespecified safety outcome. Analysis was by intention to treat, using Cox proportional hazard models for time-to-event analyses. This trial is registered with ClinicalTrials.gov, number NCT02019264. FINDINGS: Between Feb 7, 2014, and Nov 20, 2015, 12 000 patients were randomly assigned to lorcaserin or placebo (6000 patients in each group) and followed up for a median of 3·3 years (IQR 3·0-3·5). At baseline, 6816 patients (56·8%) had diabetes, 3991 (33·3%) prediabetes, and 1193 (9·9%) normoglycaemia. At 1 year, patients treated with lorcaserin had a net weight loss beyond placebo of 2·6 kg (95% CI 2·3-2·9) for those with diabetes, 2·8 kg (2·5-3·2) for those with prediabetes, and 3·3 kg (2·6-4·0) for those with normoglycaemia (p

Research in the field of cut-off grade optimisation has shown a relationship between cut-off grade, project life and Net Present Value. Lane’s theory demonstrates that cut-off grades can be optimised in order to maximise project profitability. Although the theory forms the basis for many open pit mining projects, application of the theory in underground mining remains limited to-date. The main reason for this is the complex interaction between all processes in underground mine planning which makes it difficult to apply Lane’s mathematical optimisation approach. Recently a new Stope Optimiser product was released. The AMS Stope Optimiser automates the design of underground stopes at user defined cut-off grades and allows for rapid evaluation of mine designs at different cut-off grades. Using this software, an optimisation approach was developed and validated on an underground gold deposit in northern Sweden. Potential project NPV increased by approximately 30% when using this new approach. Spatial grade uncertainty in mineral resources was identified to be a major risk in underground stope design. The optimisation approach was further extended to account for grade risk using estimated and stochastic simulated resource models. The resulting optimisation process accounts for grade risk early in the design process and reduces the risk of a stope not meeting the cut-off grade with subsequent financial loss.

TWIK-1 two-pore domain K+ channels are highly expressed in mature hippocampal astrocytes. While the TWIK-1 activity is readily detectable on astrocyte membrane, the majority of channels are retained in the intracellular compartments, which raises an intriguing question of whether the membrane TWIK-1 channels could be dynamically regulated for functions yet unknown. Here, the regulation of TWIK-1 membrane expression by Gi/Go-coupled metabotropic glutamate receptor 3 (mGluR3) and its functional impact on ammonium uptake has been studied. Activation of mGluR3 induced a marked translocation of TWIK-1 channels from the cytoplasm to the membrane surface. Consistent with our early observation that membrane TWIK-1 behaves as nonselective monovalent cation channel, mGluR3-mediated TWIK-1 membrane expression was associated with a depolarizing membrane potential (V M). As TWIK-1 exhibits a discernibly high permeability to ammonium (NH4 +), a critical substrate in glutamate-glutamine cycle for neurotransmitter replenishment, regulation of NH4 + uptake capacity by TWIK-1 membrane expression was determined by response of astrocyte V M to bath application of 5 mM NH4Cl. Stimulation of mGluR3 potentiated NH4 +-induced V M depolarization by ∼30 % in wild type, but not in TWIK-1 knockout astrocytes. Furthermore, activation of mGluR3 mediated a coordinated translocation of TWIK-1 channels with recycling endosomes toward astrocyte membrane and the mGluR3-mediated potentiation of NH4 + uptake required a functional Rab-mediated trafficking pathway. Altogether, we demonstrate that the activation of mGluR3 up-regulates the membrane expression of TWIK-1 that in turn enhances NH4 + uptake in astrocytes, a mechanism potentially important for functional coupling of astrocyte glutamate-glutamine cycle with the replenishment of neurotransmitters in neurons.

ARS2 is a regulator of RNA polymerase II transcript processing through its role in the maturation of distinct nuclear cap-binding complex (CBC)-controlled RNA families. In this study, we examined ARS2 domain function in transcript processing. Structural modeling based on the plant ARS2 orthologue, SERRATE, revealed 2 previously uncharacterized domains in mammalian ARS2: an N-terminal domain of unknown function (DUF3546), which is also present in SERRATE, and an RNA recognition motif (RRM) that is present in metazoan ARS2 but not in plants. Both the DUF3546 and zinc finger domain (ZnF) were required for association with microRNA and replication-dependent histone mRNA. Mutations in the ZnF disrupted interaction with FLASH, a key component in histone pre-mRNA processing. Mutations targeting the Mid domain implicated it in DROSHA interaction and microRNA biogenesis. The unstructured C terminus was required for interaction with the CBC protein CBP20, while the RRM was required for cell cycle progression and for binding to FLASH. Together, our results support a bridging model in which ARS2 plays a central role in RNA recognition and processing through multiple protein and RNA interactions.

Astrocytes are extensively coupled through gap junctions into a syncytium. However, the basic role of this major brain network remains largely unknown. Using electrophysiological and computational modeling methods, we demonstrate that the membrane potential (VM) of an individual astrocyte in a hippocampal syncytium, but not in a single, freshly isolated cell preparation, can be well-maintained at quasi-physiological levels when recorded with reduced or K(+) free pipette solutions that alter the K(+) equilibrium potential to non-physiological voltages. We show that an astrocyte's associated syncytium provides powerful electrical coupling, together with ionic coupling at a lesser extent, that equalizes the astrocyte's VM to levels comparable to its neighbors. Functionally, this minimizes VM depolarization attributable to elevated levels of local extracellular K(+) and thereby maintains a sustained driving force for highly efficient K(+) uptake. Thus, gap junction coupling functions to achieve isopotentiality in astrocytic networks, whereby a constant extracellular environment can be powerfully maintained for crucial functions of neural circuits.

We have developed a versatile new class of genetically encoded fluorescent biosensor based on reversible exchange of the heterodimeric partners of green and red dimerization-dependent fluorescent proteins. We demonstrate the use of this strategy to construct both intermolecular and intramolecular ratiometric biosensors for qualitative imaging of caspase activity, Ca(2+) concentration dynamics and other second-messenger signaling activities.

Amphibian biology is intricate, and there are many inter-related factors that need to be understood before establishing successful Conservation Breeding Programs (CBPs). Nutritional needs of amphibians are highly integrated with disease and their husbandry needs, and the diversity of developmental stages, natural habitats, and feeding strategies result in many different recommendations for proper care and feeding. This review identifies several areas where there is substantial room for improvement in maintaining healthy ex situ amphibian populations specifically in the areas of obtaining and utilizing natural history data for both amphibians and their dietary items, achieving more appropriate environmental parameters, understanding stress and hormone production, and promoting better physical and population health. Using a scientific or research framework to answer questions about disease, nutrition, husbandry, genetics, and endocrinology of ex situ amphibians will improve specialists' understanding of the needs of these species. In general, there is a lack of baseline data and comparative information for most basic aspects of amphibian biology as well as standardized laboratory approaches. Instituting a formalized research approach in multiple scientific disciplines will be beneficial not only to the management of current ex situ populations, but also in moving forward with future conservation and reintroduction projects. This overview of gaps in knowledge concerning ex situ amphibian care should serve as a foundation for much needed future research in these areas.

A novel series of benzyl substituted thieno[2,3-d]pyrimidines were identified as potent A2A receptor antagonists. Several five- and six-membered heterocyclic replacements for the optimized methylfuran were explored. Select compounds effectively reverse catalepsy in mice when dosed orally.

The discovery of naturally evolved fluorescent proteins and their subsequent tuning by protein engineering provided the basis for a large family of genetically encoded biosensors that report a variety of physicochemical processes occurring in living tissue. These optogenetic reporters are powerful tools for live-cell microscopy and quantitative analysis at the subcellular level. In this review, we present an overview of the transduction mechanisms that have been exploited for engineering these genetically encoded reporters. Finally, we discuss current and future efforts towards the combined use of various optogenetic actuators and reporters for simultaneously controlling and imaging the physiology of cells and tissues.

Protein toxin splicing mediated by split inteins can be used as a strategy for conditional cell ablation. The approach requires artificial fragmentation of a potent protein toxin and tethering each toxin fragment to a split intein fragment. The toxin-intein fragments are, in turn, fused to dimerization domains, such that addition of a dimerizing agent reconstitutes the split intein. These chimeric toxin-intein fusions remain nontoxic until the dimerizer is added, resulting in activation of intein splicing and ligation of toxin fragments to form an active toxin. Considerations for the engineering and implementation of conditional toxin splicing (CTS) systems include: choice of toxin split site, split site (extein) chemistry, and temperature sensitivity. The following method outlines design criteria and implementation notes for CTS using a previously engineered system for splicing a toxin called sarcin, as well as for developing alternative CTS systems.

Protein toxin splicing mediated by split inteins can be used as a strategy for conditional cell ablation. The approach requires artificial fragmentation of a potent protein toxin and tethering each toxin fragment to a split intein fragment. The toxin-intein fragments are, in turn, fused to dimerization domains, such that addition of a dimerizing agent reconstitutes the split intein. These chimeric toxin-intein fusions remain nontoxic until the dimerizer is added, resulting in activation of intein splicing and ligation of toxin fragments to form an active toxin. Considerations for the engineering and implementation of conditional toxin splicing (CTS) systems include: choice of toxin split site, split site (extein) chemistry, and temperature sensitivity. The following method outlines design criteria and implementation notes for CTS using a previously engineered system for splicing a toxin called sarcin, as well as for developing alternative CTS systems.

Dimerization-dependent fluorescent proteins (ddFP) are a recently introduced class of genetically encoded reporters that can be used for the detection of protein interactions in live cells. The progenitor of this class of tools was a red fluorescent ddFP (ddRFP) derived from a homodimeric variant of Discosoma red fluorescent protein. Here, we describe the engineering and application of an expanded palette of ddFPs, which includes green (ddGFP) and yellow (ddYFP) variants. These optimized variants offer several advantages relative to ddRFP including increased in vitro contrast and brightness for ddGFP and increased brightness and a lowered pK a for ddYFP. We demonstrate that both variants are useful as biosensors for protease activity in live cells. Using the ddGFP tool, we generated a highly effective indicator of endomembrane proximity that can be used to image the mitochondria-associated membrane (MAM) interface of endoplasmic reticulum (ER) and mitochondria.

The design and characterization of two, dual adenosine A2A/A1 receptor antagonists in several animal models of Parkinson's disease is described. Compound 1 was previously reported as a potential treatment for Parkinson's disease. Further characterization of 1 revealed that it was metabolized to reactive intermediates that caused the genotoxicity of 1 in the Ames and mouse lymphoma L51784 assays. The identification of the metabolites enabled the preparation of two optimized compounds 13 and 14 that were devoid of the metabolic liabilities associated with 1. Compounds 13 and 14 are potent dual A2A/A1 receptor antagonists that have excellent activity, after oral administration, across a number of animal models of Parkinson's disease including mouse and rat models of haloperidol-induced catalepsy, mouse and rat models of reserpine-induced akinesia, and the rat 6-hydroxydopamine (6-OHDA) lesion model of drug-induced rotation.

Thick (>150 μm) beryllium coatings are studied as an ablator material of interest for fusion fuel capsules for the National Ignition Facility. DC magnetron sputtering is used because of the relative controllability of the processing temperature and energy of the deposits. However, coatings produced by DC magnetron sputtering leak the fuel gas D2. By using ion-assisted DC magnetron, sputtered coatings can be made that are leak-tight. Transmission electron microscopy (TEM) studies revealed microstructural changes that lead to leak-tight coating. Ultrasmall angle x-ray spectroscopy is used to characterize the void distribution and volume along the spherical surface along with a combination of focused ion beam, scanning electron microscope, and TEM. An in situ multibeam optical stress sensor was used to measure the stress behavior of thick beryllium coatings on flat substrates as the material was being deposited.

Sputter coating of beryllium on spherical mandrels has been used at Lawrence Livermore National Laboratory and at General Atomics to produce graded, copper doped beryllium shells. While these coatings have consistent microstructure and acceptable void content, different coaters produced different results with respect to argon implantation. Each individual system met the requirements for argon implantation, but the deviation from one system to another and from run to run exceeded the variability requirements as specified by the National Ignition Facility target design requirements. We redesigned the fixturing within one system to improve reproducibility. Then, we reconfigured the coaters so that the vertical and lateral alignments of the shells under the gun varied

Two reactive metabolites were identified in vivo for the dual A2A/A1 receptor antagonist 1. Two strategies were implemented to successfully mitigate the metabolic liabilities associated with 1. Optimization of the arylindenopyrimidines led to a number of amide, ether, and amino analogs having comparable in vitro and in vivo activity.

Tidal freshwater sections of the Cooper River Estuary (South Carolina) include extensive wetlands, which were formerly impounded for rice culture during the 1,700s and 1,800s. Most of these former rice fields are now open to tidal exchange and have developed into productive wetlands that vary in bottom topography, tidal hydrography and vegetation dominants. The purpose of this project was to quantify nitrogen (N) transport via tidal exchange between the main estuarine channel and representative wetland types and to relate exchange patterns to the succession of vegetation dominants. We examined N concentration and mass exchange at the main tidal inlets for the three representative wetland types (submerged aquatic vegetation [SAV], floating leaf vegetation, and intertidal emergent marsh) over 18-21 tidal cycles (July 1998–August 2000). Nitrate + nitrite concentrations were significantly lower during ebb flow at all study sites, suggesting potential patterns of uptake by all wetland types. The magnitude of nitrate decline during ebb flow was negatively correlated with oxygen concentration, reflecting the potential importance of denitrification and nitrate reduction within hypoxic wetland waters and sediments. The net tidal exchange of nitrate + nitrite was particularly consistent for the intertidal emergent marsh, where flow-weighted ebb concentrations were usually 18–40% lower than during flood tides. Seasonal patterns for the emergent marsh indicated higher rates of nitrate + nitrite uptake during the spring and summer (> 400 μmol N m-2 tide-1) with an annual mean uptake of 248 ± 162 μmol m–2 tide–1. The emergent marsh also removed ammonium through most of the year (207 ± 109 μmol m–2 tide–1), and exported dissolved organic nitrogen (DON) in the fall (1,690 ± 793 μmol m–2 tide–1), suggesting an approximate annual balance between the dissolved inorganic N uptake and DON export. The other wetland types (SAV and floating leaf vegetation) were less consistent in magnitude and direction of N exchange. Since the emergent marsh site had the highest bottom elevation and the highest relative cover of intertidal habitat, these results suggest that the nature of N exchange between the estuarine waters and bordering wetlands is affected by wetland morphometry, tidal hydrography, and corresponding vegetation dominants. With the recent diversion of river discharge, water levels in the upper Cooper estuary have dropped more than 10 cm, leading to a succession of wetland communities from subtidal habitats toward more intertidal habitats. Results of this study suggest that current trends of wetland succession in the upper Cooper River may result in higher rates of system-wide inorganic N removal and DON inputs by the growing distributions of intertidal emergent marshes.

The Eph receptors and their ligands, the ephrins, are thought to act at points of close cell-cell contact to elicit bi-directional signaling in receptor and ligand expressing cells. However, when cultured in vitro, some A-type ephrins are released from the cell surface and it is unclear if these soluble ephrins participate in Eph receptor activation. We show that soluble ephrin A5 is subject to oligomerization. Ephrins A1 and A5 are substrates for a cross-linking enzyme, tissue transglutaminase, which mediates the formation of oligomeric ephrin. Transglutaminase-cross-linked ephrin binds to A-type Eph receptors, stimulates Eph kinase activity, and promotes invasion and migration of HeLa cells. Transglutaminase-mediated oligomerization of soluble ephrin potentially represents a novel mechanism of forward signaling through Eph receptors and may extend the influence of A-type ephrins beyond cell contact mediated signaling.

Sputtered beryllium and copper-doped beryllium coatings as thick as 170μm have been deposited on spherical substrates to produce hollow shells that are required as targets for inertial fusion experiments. Be coatings by magnetron sputtering achieved ∼95±2% bulk density consistently up to 170μm thick. Coatings on the spherical substrates exhibit the typical columnar structure throughout the entire thickness. Transmission electron microscopy indicates the presence of submicron, nearly spherical voids mainly aggregated along the columnar structure and grain boundaries, as well as some smaller intragranular elongated voids. Holes have been drilled in beryllium shells produced in this manner to allow filling with deuterium (the fusion fuel). Gas retention of these shells has been examined using mass spectrometry. It appears that a fraction of the pores in the coatings are interconnected, which leads to leakage of the hollow shells. Boron-doped Be layers near the eutectic phase, at a concentration of ∼11at.%, h...

Various morphologies have been observed in sputter-deposited Be ablator capsules, including nodular growth, cone growth and twisted grain growth. By devising an agitation method that includes both bouncing and rolling the spherical mandrels during deposition, and by reducing the coating rate, consistent columnar grain structure has now been obtained up to 170 mm. Low mode deformation of the shells is observed on thin CH mandrels, but is suppressed if stiffer mandrels are used. Ablator density measured by weighing and x-ray radiography is 93%–95% of bulk density of Be. Transmission electron microscopy shows 100.200 nm size voids in the film and striations inside the grains. Be shells produced with rolling agitation have met most of the NIF specifications. Some of the few remaining issues will be discussed.

Ablative targets for the National Ignition Campaign (NIC) have been fabricated by sputter coating spherical mandrels made of glow discharge polymer (GDP) with graded copper doped beryllium (Be) layers. The inner mandrel must be completely removed to meet specific ignition design requirements. The process of removing the mandrel requires elevated temperature in the presence of oxygen. However, elevating the temperature in air also oxidizes the Be and can cause blistering on the inner surface of the Be shell. This paper will discuss a refined technique, which removes the GDP mandrel without compromising the integrity of the inner Be surface. The oxygen gradient that develops during the mandrel removal and the impact of its presence will also be discussed.

The choice of the mandrel material has a significant bearing on the properties of the sputter-coated beryllium shell needed for NIF targets. Here, we present our work on screening four mandrel materials, their impact on the Be shell and issues related to their subsequent removal. Beryllium shells sputter deposited on hollow glow discharge polymer or GDP spheres met most of the target specifications. However, they had greater opacity due to partial oxidation of the Be during the GDP burnout step. Poly (α-methyl styrene), silicon and nickel beads were explored as alternative mandrels but were plagued with problems such as cracking of the Be shell or incomplete removal. The most promising approach was a two-step coating process mediated by a thin 6μm Be mandrel that is made using GDP.

Graded copper-doped Be shells have been fabricated by sputter coating on spherical mandrels. While such coatings have consistent microstructure and acceptable void content and size, we have found that they suffer from sufficient interconnected porosity leading to relatively rapid gas leakage. In this paper, we present an extensive study of D 2 leakage out of Be shells made by sputter coating. The leakage appears to follow molecular flow dynamics as determined by examining the temperature dependence of the flow. Furthermore, the time dependence of the leakage suggests that the flow channels are nanometerish in diameter, propagating through the thickness of the coating, possibly brought about by residual stress in the coatings. We have investigated the D 2 leakage time constant as a function of a large number of coating parameters, including the effect of introducing boron-doped layers. Addition of thin 0.25 μm amorphous boron-doped layers near the inside surface has been most effective in producing shells with long time constants (greater than 7 days to immeasurable) with yield of greater than 50%. There is still substantial scatter in the data, even within a given coating batch, suggesting a possible stochastic cracking process driven by residual stress in the coating.

This paper reports on the steps involved in the development of a novel electrochemical immunoassay for secretory immunoglobulin A (sIgA) determinations in human sweat. This novel immunoassay involves an initial step whereby sweat sIgA is adsorbed onto a polyvinylidene fluoride (PVDF) membrane (sweat patch). Following a wash step, a solution containing bovine serum albumin (BSA) is used to block any sites at which non-specific binding might occur. After a second wash step, the PVDF sweat patch is transferred to a second tube to which is added diluent and anti-sIgA antibody conjugated to horseradish peroxidase (HRP). Following an incubation step, an aliquot of supernatant containing unbound antibody is transferred to a well in a 96 well plate format which had been previously coated with sIgA. After a second incubation step and wash step, 3,3′,5,5′-tetramethylbenzidine (TMB) is added to the same well and left to undergo enzymatic oxidation. Finally a screen-printed carbon electrode (SPCE) is inserted into the well and any oxidised TMB is detected by chronoamperometry using an applied potential of +50 mV. The resulting reduction current is then referred to a calibration plot to deduce the unknown sIgA concentration. The optimisation of the steps involved in this assay is described in detail. Some preliminary data is presented on sweat sIgA levels collected from human volunteers who had undergone a controlled exercise regime on a bicycle (Ergociser). To our knowledge this is the first report where a sweat patch and a SPCE have been successfully incorporated into an immunoassay for sIgA.

The synthesis and in vivo SAR of 2-(2,2,2)-trifluoroethyl-benzimidazoles are described. Prostate antagonism and/or levator ani agonism can be modulated by varying the substitution at the 2-position of 5,6-dichloro-benzimidazoles. Potent androgen agonists on the muscle were discovered that strongly bind to the androgen receptor (2-17 nM) and show potent in vivo efficacy (0.03-0.11 mg/day). True SARMs showing both prostate antagonism and levator ani agonism were revealed.

The synthesis and in vivo SAR of 5,6-dichloro-benzimidazole derivatives as novel selective androgen receptor antagonists are described. During screening of 2-alkyl benzimidazoles, it was found that a trifluoromethyl group greatly enhances antagonist activity in the prostate. Benzimidazole 1 is a potent AR antagonist in the rat prostate (ID50 = 0.15 mg/day).

Mature astrocytes exhibit a linear current-to-voltage K+ membrane conductance (passive conductance) and an extremely low membrane resistance ( Rm) in situ. The combination of these electrophysiological characteristics establishes a highly negative and stable membrane potential that is essential for basic functions, such as K+ spatial buffering and neurotransmitter uptake. However, astrocytes are coupled extensively in situ. It remains to be determined whether the observed passive behavior and low Rm are attributable to the intrinsic properties of membrane ion channels or to gap junction coupling in functionally mature astrocytes. In the present study, freshly dissociated hippocampal tissues were used as a new model to examine this basic question in young adult animals. The morphologically intact single astrocytes could be reliably dissociated from animals postnatal day 21 and older. At this animal age, dissociated single astrocytes exhibit passive conductance and resting membrane potential similar to those exhibited by astrocytes in situ. To precisely measure the Rm from single astrocytes, dual-patch single-astrocyte recording was performed. We show that dissociated single astrocytes exhibit a low Rm similarly to syncytial coupled astrocytes. Functionally, the symmetric expression of high-K+ conductance enabled rapid change in the intracellular K+ concentrations in response to changing K+ drive force. Altogether, we demonstrate that freshly dissociated tissue preparation is a highly useful model for study of the functional expression and regulation of ion channels, receptors, and transporters in astrocytes and that passive behavior and low Rm are the intrinsic properties of mature astrocytes.

The sputtering of beryllium (Be) has been used at LLNL for nearly 30 years in the fabrication of laser targets. Several years ago the prospect of using sputtering to fabricate spherical Be capsules for National Ignition Facility (NIF) targets began to be explored and a basic strategy was developed that involved sputtering down onto plastic mandrels bouncing in a pan. While this appears to be very straightforward in principle, in practice sputtering has been used almost exclusively to make thin films (< 1 micron) on flat substrates. Thick films pose a significant challenge for sputtering while materials on spherical substrates are essentially unexplored. More recently, based on computational results, the point design for the first NIF ignition target capsule was specified as a Be capsule with Cu-doped layers of specific thickness, each layer with a different concentration of copper. While the work described here was motivated by the need to make the layered capsules, the primary progress on Be capsules has been the development of a more complete metallurgical understanding of the materials that are fabricated and the beginning of the exploration of the relationship between the sputter processing and microstructure of these spherical samples. At least two barriers tomore » growth to full thickness (i.e. 170 microns) have been identified and efforts to overcome these barriers are underway.« less

This paper investigates the effect of personal-tax progressivity on management performance and agency costs by examining measures of corporate operating efficiency. We study a sample of US-based manufacturing and service firms and variations in a cross-state tax policy. Using matched-pair testing and regression analysis, we find evidence consistent with the hypothesis that increased personal-tax progressivity negatively impacts management productivity and is manifested in reduced firm efficiency. We control for several other factors that the literature suggests is relevant to firm operating efficiency and find that our results are robust

The present prospective study was designed to determine the prevalence of pleural effusion at approximately 28 days after cardiac surgery and their subsequent course. This consecutive case study included 389 patients; 312 had only coronary artery bypass graft surgery (CABG) surgery, 37 had both valve and CABG surgery, and 40 had only valve surgery. Chest radiographs were obtained approximately 28 days postoperatively. Patients were subsequently contacted by telephone 3, 6, and 12 months postoperatively and questioned about the presence of fluid in their chest and related symptoms. The prevalence of pleural effusions in the patients undergoing only CABG surgery (63%) or CABG surgery plus valve surgery (62%) was significantly (p = 0.05) higher than that in the patients undergoing valve surgery only (45%). The prevalence of effusions occupying more than 25% of the hemithorax was 9.7%. The primary symptom associated with these larger effusions was dyspnea. Chest pain and fever were uncommon. Over the 12-month follow-up, the effusions tended to resolve. In conclusion, the prevalence of pleural effusions occupying more than 25% of the hemithorax is approximately 10%, 28 days postoperatively. These larger pleural effusions produce dyspnea but not chest pain or fever, and most of the effusions disappear gradually over the subsequent months.

When used with 1.06-mm beryllium (Be) capsules on the National Ignition Facility, gold hohlraums with the inner diameter of 5.75 mm and helium gas fill density of 1.6 mg/cm3 exhibit significant drive degradation due to laser energy backscatter (of order 14%–17%) and “missing” X-ray drive energy (about 32% during the main pulse). Also, hard to simulate cross-beam energy transfer (CBET) must be used to control the implosion symmetry. Larger, 6.72-mm hohlraums with fill densities ≤0.6 mg/cm3 generally offer improved drive efficiency, reduced hot-electron preheat, and better control of the implosion symmetry without CBET. Recently, we carried out an exploratory campaign to evaluate performance of 1.06-mm Be capsules in such hohlraums and determine optimal hohlraum parameters. Specifically, we performed a hohlraum fill-density scan with a three-shock, 9.5-ns laser pulse and found that an appropriate axial laser repointing and azimuthal outer-quad splitting resulted in significantly improved hohlraum energetics...

We present a photonic integrated circuit (PIC) composed of two strongly coupled distributed Bragg reflector (DBR) lasers. This PIC utilizes the dynamics of mutual injection locking to increase the relaxation resonance frequency from 3 GHz to beyond 30 GHz. Mutual injection-locking and external injection-locking operation are compared.

Protein splicing technology harnesses the ability of inteins to ligate protein fragments, forming a mature protein. This report describes our effort to engineer rapamycin-dependent protein splicing of a ribotoxin, called α-sarcin. Engineering this system required the investigation of important splicing parameters, including extein context and splicing temperature. We show α-sarcin splicing is dependent on rapamycin, is inducible with rapid kinetics, and triggers apoptosis in HeLa cells. These findings establish a proof-of-concept for a conditional cell ablation strategy.

We are studying the feasibility of using boron doping to refine the grain structure of sputter-deposited Be for NIF ignition capsule ablators. The goal is to improve the surface finish and homogene...

Boron carbide (B4C) is examined as a potential fuel container and ablator for implosion capsules on the National Ignition Facility (NIF). A capsule of pure B4C encasing a layer of solid DT implodes stably and ignites with anticipated NIF x-ray drives, producing 18 MJ of energy. Thin films of B4C were found to be resistant to oxidation and modestly transmitting in the infrared (IR), possibly enabling IR fuel characterization and enhancement for thin permeation barriers but not for full-thickness capsules. Polystyrene mandrels 0.5 mm in diameter were successfully coated with 0.15–2.0 µm of B4C. Thicknesses estimated from optical density agreed well with those measured by scanning electron microscopy (SEM). The B4C microstructure was columnar but finer than for Be made at the same conditions. B4C is a very strong material, with a fiber tensile strength capable of holding NIF fill pressures at room temperature, but it is also very brittle, and microscopic flaws or grain structure may limit the noncryo...

Optical channelizing filters with narrow linewidth are of interest for optical processing of microwave signals. Fabrication tolerances make it difficult to place exactly the optical resonance frequency within the microwave spectrum as is required for many applications. Therefore, efficient tuning of the filter resonance is essential. In this paper we present a tunable ring resonator filter with an integrated semiconductor optical amplifier (SOA) fabricated on an InP based photonic integrated circuit (PIC) platform. The ring resonance is tuned over 37 GHz with just 0.2 mA of current injection into a passive phase section. The use of current injection is often more efficient than thermal tuning using heaters making them useful for low-power applications. The single active ring resonator has an electrical FWHM of 1.5 GHz and shows greater than 16 dB of extinction between on and off resonance. The effects of SOA internal ring gain and induced passive loss on extinction and linewidth will be shown. Agreement between experimentally demonstrated devices and simulations are shown. The integration of the active and passive regions is done using quantum well intermixing and the resonators utilize buried heterostructure waveguides. The fabrication process of these filters is compatible with the monolithic integration of DBR lasers and high speed modulators enabling single chip highly functional PICs for the channelizing of RF signals.

The first indirect drive implosion experiments using Beryllium (Be) capsules at the National Ignition Facility confirm the superior ablation properties and elucidate possible Be-ablator issues such as hohlraum filling by ablator material. Since the 1990s, Be has been the preferred Inertial Confinement Fusion (ICF) ablator because of its higher mass ablation rate compared to that of carbon-based ablators. This enables ICF target designs with higher implosion velocities at lower radiation temperatures and improved hydrodynamic stability through greater ablative stabilization. Recent experiments to demonstrate the viability of Be ablator target designs measured the backscattered laser energy, capsule implosion velocity, core implosion shape from self-emission, and in-flight capsule shape from backlit imaging. The laser backscatter is similar to that from comparable plastic (CH) targets under the same hohlraum conditions. Implosion velocity measurements from backlit streaked radiography show that laser energy coupling to the hohlraum wall is comparable to plastic ablators. The measured implosion shape indicates no significant reduction of laser energy from the inner laser cone beams reaching the hohlraum wall as compared with plastic and high-density carbon ablators. These results indicate that the high mass ablation rate for beryllium capsules does not significantly alter hohlraum energetics. In addition, these data, together with data for low fill-density hohlraum performance, indicate that laser power multipliers, required to reconcile simulations with experimental observations, are likely due to our limited understanding of the hohlraum rather than the capsule physics since similar multipliers are needed for both Be and CH capsules as seen in experiments.

Study Objective To describe the prevalence of prescribing against-label statin-fibrate combination therapy. Design Retrospective cohort study. Data Source Medstat MarketScan Commercial Claims and Encounter database. Patients Adults (aged 18–89 yrs) who were prescribed statin-fibrate combination therapy between January 1, 2003, and June 30, 2009, had pharmacy claims demonstrating two or more concurrently filled prescriptions for a statin and a fibrate, and had continuous insurance enrollment for at least 12 months. Measurements and Main Results Claims data were used to identify patients with dyslipidemia based on International Classification of Diseases, Ninth Revision, Clinical Modification diagnosis codes. National Drug Codes were used to describe concurrent statin-fibrate combination therapy. The primary outcome was recent use of against-label statin-fibrate combination therapy, defined as use during the last 18 months (January 1, 2008–June 30, 2009) of the study period. Patients were stratified according to statin and dosage to identify against-label combination use (e.g., simvastatin > 10 mg/day with gemfibrozil). Within the recent-use period, 131,394 patients were prescribed concurrent statin-fibrate combination therapy; of these patients, 13,420 (10.2%) had against-label therapy. Simvastatin-gemfibrozil accounted for 8978 (66.9%) of all against-label combinations. Of all 9877 simvastatin-gemfibrozil combinations prescribed in the recent-use period (both on-label and against-label use), 8978 (90.9%) were against label. The secondary outcome was prevalence of against-label statin-fibrate combination therapy on an annual basis: 15.5% in 2003, 18.7% in 2004, 9.1% in 2005, 8.3% in 2006, 9.2% in 2007, and 9.8% in 2008. Conclusion Against-label statin-fibrate combination therapy continues to be prescribed despite established United States Food and Drug Administration (FDA) dose restrictions. Nearly every time the simvastatin-gemfibrozil combination was prescribed, it was against label because simvastatin exceeded the maximum dose restriction. Updated simvastatin labeling in June 2011 includes additional maximum dose restrictions and new contraindications, which include gemfibrozil. Different approaches in clinical practice are needed to ensure adherence with the revised FDA labeling.

SummaryThe expanding repertoire of genetically encoded biosensors constructed from variants of Aequorea victoria green fluorescent protein (GFP) enable the imaging of a variety of intracellular biochemical processes. To facilitate the imaging of multiple biosensors in a single cell, we undertook the development of a dimerization-dependent red fluorescent protein (ddRFP) that provides an alternative strategy for biosensor construction. An extensive process of rational engineering and directed protein evolution led to the discovery of a ddRFP with a Kd of 33 μM and a 10-fold increase in fluorescence upon heterodimer formation. We demonstrate that the dimerization-dependent fluorescence of ddRFP can be used for detection of a protein-protein interaction in vitro, imaging of the reversible Ca2+-dependent association of calmodulin and M13 in live cells, and imaging of caspase-3 activity during apoptosis.