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  1. High-throughput cell-free screening of eukaryotic membrane protein expression in lipidic mimetics

    Bruni, Renato ; Laguerre, Aisha ; Kaminska, Anna‐Maria ; ... - Protein Science
    Membrane proteins play essential roles in cellular function and metabolism. Nonetheless, biophysical and structural studies of membrane proteins are impeded by the difficulty of their expression in and purification from heterologous cell-based systems. As an alternative to these cell-based systems, cell-free protein synthesis has proven to be an exquisite method for screening membrane protein targets in a variety of lipidic mimetics. Here we report a high-throughput screening workflow and applied it to screen 61eukaryotic membrane protein targets. For each target, we tested its expression in lipidic mimetics: two detergents, two liposomes, and two nanodiscs. We show that 35 membrane proteinsmore » (57%) can be expressed in a soluble fraction in at least one of the mimetics with the two detergents performing significantly better than nanodiscs and liposomes, in that order. Using the established cell-free workflow, we studied the production and biophysical assays for mitochondrial pyruvate carrier (MPC) complexes. Our studies show that the complexes produced in cell-free are functional competent in complex formation and substrate binding. Our results highlight the utility of using cell-free systems for screening and production of eukaryotic membrane proteins.« less

  2. Patterned anodes with sub-millimeter spatial resolution for large-area MCP-based photodetector systems

    Park, Jinseo ; Wu, Fangjian ; Angelico, Evan ; ... - Nuclear Instruments and Methods in Physics Research. Section A, Accelerators, Spectrometers, Detectors and Associated Equipment
    Micro-channel-plate-based photo-detectors are unique in being capable of covering very large areas such as those required in elementary particle and nuclear physics, while providing sub-millimeter space resolution, time resolutions of less than 10 picoseconds for charged particles, and time resolutions of 30 psec–50 psec for single photons. In such systems the electronic channel count is a major cost driver. Incorporating a capacitively-coupled anode allows the use of external pickup electrodes with patterns of individual channels optimized for occupancy, rate, and time/space resolution. The signal pickup antenna can be economically implemented as a printed circuit card with a 2-dimensional array ofmore » pads for high-occupancy/high-rate applications such as in particle colliders and medical imaging, or a 1-dimensional array of strips for a lower channel count in low-occupancy/low-rate applications such as large neutrino detectors. In this paper we present pad patterns that enhance signal-sharing between pads to lower the channel count per unit area in large-area systems by factors up to 4, while maintaining spatial resolutions of approximately 100 to 200 μ m for charged particles and 400 to 1000 μ m for single photons. Patterns that use multiple signal layers in the signal-pickup board can lower the channel count even further, moving the scaling behavior in the number of pads versus total area from quadratic to linear.« less

  3. Impacts of glass composition, pH, and temperature on glass forward dissolution rate

    Vienna, John D. ; Neeway, James J. ; Ryan, Joseph V. ; ... - npj Materials Degradation
    Nuclear waste glasses dissolve at the forward dissolution rate (rf) in very dilute aqueous solutions, which can isolate the impact of the glass composition from solution feedback and alteration product effects. While it has long been known that pH and temperature (T) strongly impact rf, the impacts of glass composition have remained uncertain. In this work, rf data from 19 nuclear waste glasses were used with the aim of identifying the effect of glass composition on rf. The rf values were modeled as: rf= k010–ηpHexp(-Ea/RT), with k0, η, Ea, and R, respectively, being the intrinsic rate constant, pH coefficient, apparentmore » activation energy, and gas constant. However, no predictive correlation could be established between the individual model parameters (log[k0], η, and Ea) and glass composition for the glasses considered in this study, an outcome that was attributed to the strong positive correlation between the log[$$k_0$$] and Ea parameters. Therefore, a model was fitted directly to the combined rf from all 19 glasses. This approach showed that 90% of the variation in rf data could be accounted for solely by T and pH effects. Therefore, any composition effects must be relatively small. After normalizing for differences in pH and T, the only notable differences in rf between the glasses were found to correlate with variations in the fraction of glass forming tetrahedra contributed by tetrahedral boron, $$f$$([4]B), with an abrupt threshold at a high value of $$f$$([4]B) (~0.22), where higher rf are predicted with no discernable composition effects below the threshold.« less

  4. Pore-scale water dynamics during drying and the impacts of structure and surface wettability

    Cruz, Brian C. ; Furrer, Jessica M. ; Guo, Yi-Syuan ; ... - Water Resources Research
    Plants and microbes secrete mucilage into soil during dry conditions, which can alter soil structure and increase contact angle. Structured soils exhibit a broad pore size distribution with many small and many large pores, and strong capillary forces in narrow pores can retain moisture in soil aggregates. Meanwhile, contact angle determines the water repellency of soils, which can result in suppressed evaporation rates. Although they are often studied independently, both structure and contact angle influence water movement, distribution, and retention in soils. Here drying experiments were conducted using soil micromodels patterned to emulate different aggregation states of a sandy loammore » soil. Micromodels were treated to exhibit contact angles representative of those in bulk soil (8.4° ± 1.9°) and the rhizosphere (65° ± 9.2°). Drying was simulated using a lattice Boltzmann single-component, multiphase model. In our experiments, micromodels with higher contact angle surfaces took 4 times longer to completely dry versus micromodels with lower contact angle surfaces. Microstructure influenced drying rate as a function of saturation and controlled the spatial distribution of moisture within micromodels. In conclusion, lattice Boltzmann simulations accurately predicted pore-scale moisture retention patterns within micromodels with different structures and contact angles.« less
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