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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.

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.

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.

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.

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.

Thick (>150 μm) beryllium coatings are studied as an ablator material of interest for fusion fuel capsules for the National Ignition Facility (NIF). As an added complication, the coatings are deposited on mm-scale spherical substrates, as opposed to flats. DC magnetron sputtering is used because of the relative controllability of the processing temperature and energy of the deposits. We used ultra small angle x-ray spectroscopy (USAXS) to characterize the void fraction and distribution along the spherical surface. We investigated the void structure using a combination focused ion beam (FIB) and scanning electron microscope (SEM), along with transmission electron microscopy (TEM). Our results show a few volume percent of voids and a typical void diameter of less than two hundred nanometers. Understanding how the stresses in the deposited material develop with thickness is important so that we can minimize film cracking and delamination. To that end, an in-situ multiple optical beam stress sensor (MOSS) was used to measure the stress behavior of thick Beryllium coatings on flat substrates as the material was being deposited. We will show how the film stress saturates with thickness and changes with pressure.

Multilayer (ML) structures composed of Mo-Be, Ru-Be and Rh-Be with bilayer periods of - 6 nm have been grown using dc magnetron sputter deposition. The ML microstructure has been characterized using x-ray diffraction and high-resolution transmission electron microscopy, and the normal incidence reflectivity has been measured at soft x-ray wavelengths.

The effects on sleep of four β-Blockers, atenolol, propranolol, metoprolol and pindolol, were studied in a placebo-controlled trial. Drugs were administered in random order to 10 female volunteers who acted as their own controls. Subjects were tested five times, each test period lasting 10 nights (2 baseline, 2 low dose, 4 high dose, and 2 withdrawal). A questionnaire concerning subjective appreciation of the quality of the previous night's sleep was completed each morning. Night recordings of muscle tension, eye movement, heart rate and electroencephalogram were recorded on paper and magnetic tape. Analysis of the subjective questionnaires showed that recollection of dreaming and awakening in the night was increased by the three lipophilic drugs, propranolol, metoprolol, and pindolol. These results confirm reports in the literature but are contrary to those expected from considering the effects of noradrenaline on sleep. Analysis of physiological records confirmed subjects' reports that waking was increased by the lipophilic drugs. Dreaming (rapid eye movement sleep, REM) was reduced, as predicted from knowledge of the effect of noradrenaline on sleep. Increased awakening leads to an increase in remembered dreaming which explains the otherwise paradoxical results. Although atenolol had no effect on subjective measures of sleep this hydrophilic drug also reduced REM frequency, suggesting that either it has some central effect, or that REM reduction is due to a peripheral ‘shielding’ effect.

THE report of a postulated lactose intolerance in Asians1 said of food aid programmes: “It could be that aid in this form with the subsequent induction of diarrhoea is not the most efficient method of helping a malnourished community”. Subsequent criticism of this comment2 questioned the validity of extending the results of a laboratory experiment, in which the administration of lactose induced gastric disorders among adult Asians, to food aid programmes because “To be meaningful in terms of the place of milk and milk products in aid schemes … the emphasis should be on the reactions of children rather than of adults, for most food aid schemes are directed towards improving the health of children”.