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DOE OSTI2021

Liquid Chromatography Coupled to Refractive Index or Mass Spectrometric Detection for Metabolite Profiling in Lysate-based Cell-free Systems

Engineering cellular metabolism for targeted biosynthesis can require extensive design-build-test-learn (DBTL) cycles as the engineer works around the cell's survival requirements. Alternatively, carrying out DBTL cycles in cell-free environments can accelerate this process and alleviate concerns with host compatibility. A promising approach to cell-free metabolic engineering (CFME) leverages metabolically active crude cell extracts as platforms for biomanufacturing and for rapidly discovering and prototyping modified proteins and pathways. Realizing these capabilities and optimizing CFME performance requires methods to characterize the metabolome of lysate-based cell-free platforms. That is, analytical tools are necessary for monitoring improvements in targeted metabolite conversions and in elucidating alterations to metabolite flux when manipulating lysate metabolism. In this work, metabolite analyses using high-performance liquid chromatography (HPLC) coupled with either optical or mass spectrometric detection were applied to characterize metabolite production and flux in E. coli S30 lysates. Specifically, this report describes the preparation of samples from CFME lysates for HPLC analyses using refractive index detection (RID) to quantify the generation of central metabolic intermediates and by-products in the conversion of low-cost substrates (i.e., glucose) to various high-value products. The analysis of metabolite conversion in CFME reactions fed with 13 C-labeled glucose through reversed-phase liquid chromatography coupled to tandem mass spectrometry (MS/MS), a powerful tool for characterizing specific metabolite yields and lysate metabolic flux from starting materials, is also presented. Altogether, applying these analytical methods to CFME lysate metabolism enables the advancement of these systems as alternative platforms for executing faster or novel metabolic engineering tasks.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2016

Quasars as a tracer of large-scale structures in the distant universe

We study the dependence of the number density and properties of quasars on the background galaxy density using the currently largest spectroscopic data sets of quasars and galaxies. We construct a galaxy number density field smoothed over the variable smoothing scale of between approximately 10 and 20 h {sup −1} Mpc over the redshift range 0.46 < z < 0.59 using the Sloan Digital Sky Survey (SDSS) Data Release 12 (DR12) Constant MASS galaxies. The quasar sample is prepared from the SDSS-I/II DR7. We examine the correlation of incidence of quasars with the large-scale background density and the dependence of quasar properties such as bolometric luminosity, black hole mass, and Eddington ratio on the large-scale density. We find a monotonic correlation between the quasar number density and large-scale galaxy number density, which is fitted well with a power-law relation, n{sub Q}∝ρ{sub G}{sup 0.618}. We detect weak dependences of quasar properties on the large-scale density such as a positive correlation between black hole mass and density, and a negative correlation between luminosity and density. We discuss the possibility of using quasars as a tracer of large-scale structures at high redshifts, which may be useful for studies of the growth of structures in the high-redshift universe.

79 ASTRONOMY AND ASTROPHYSICS↗

DOE OSTI2018

Effect of relative humidity on the composition of secondary organic aerosol from the oxidation of toluene

The effect of relative humidity (RH) on the chemical composition of secondary organic aerosol (SOA) formed from low-NO x toluene oxidation in the absence of seed particles was investigated. SOA samples were prepared in an aerosol smog chamber at < 2 % RH and 75 % RH, collected on Teflon filters, and analyzed with nanospray desorption electrospray ionization high-resolution mass spectrometry (nano-DESI–HRMS). Measurements revealed a significant reduction in the fraction of oligomers present in the SOA generated at 75 % RH compared to SOA generated under dry conditions. In a separate set of experiments, the particle mass concentrations were measured with a scanning mobility particle sizer (SMPS) at RHs ranging from < 2 to 90 %. It was found that the particle mass loading decreased by nearly an order of magnitude when RH increased from < 2 to 75–90 % for low-NO x toluene SOA. The volatility distributions of the SOA compounds, estimated from the distribution of molecular formulas using the “molecular corridor” approach, confirmed that low-NO x toluene SOA became more volatile on average under high-RH conditions. In contrast, the effect of RH on SOA mass loading was found to be much smaller for high-NO x toluene SOA. The observed increase in the oligomer fraction and particle mass loading under dry conditions were attributed to the enhancement of condensation reactions, which produce water and oligomers from smaller compounds in low-NO x toluene SOA. The reduction in the fraction of oligomeric compounds under humid conditions is predicted to partly counteract the previously observed enhancement in the toluene SOA yield driven by the aerosol liquid water chemistry in deliquesced inorganic seed particles.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2024

Altering translation allows E. coli to overcome chemically stabilized G-quadruplexes

Genomic DNA from each sample was prepared using the Wizard Genomic DNA Purification Kit (Promega) and after, DNA was quantified using the QuantiFluor ONE dsDNA System (Promega). Genomic DNA underwent shearing to ~200 bp fragments via sonication and the gDNA fragments were prepared for sequencing using the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB). Bead-based size selection was used to select ~200 bp fragments and the fragments then underwent a splinkerette PCR using a Tn5-enriching forward primer and custom reverse primers for multiplexing. A final bead-based size selection was used to select for the correct length DNA. DNA was sequenced at the University of Michigan Advanced Genomics Core using Illumina sequencing with a custom read primer reading the last 10 nt of the transposon. PhiX174 DNA spike was added to the run to ensure sufficient sequence diversity on the flow cell. Then, a custom index read primer and standard Illumina primer were used to sequence the index reads and PhiX174, respectively.

Keck, James L.↗

DOE OSTI2023

Diazotroph diversity across a land-management gradient

Soil diazotrophs are ubiquitous bacteria and archaea responsible for converting atmospheric nitrogen into plant-available ammonium. This process, if properly harnessed, can ameliorate excessive fertilizer inputs in conventional agricultural systems. However, the effect of diazotroph biodiversity on free-living nitrogen fixation (FLNF) is poorly understood. Here, we characterized diazotroph biodiversity across a land-management intensity gradient, determining the interactive effects of land management and diazotroph biodiversity on their ecosystem functioning (FLNF). Subsequently, we evaluated the functional redundancy of diazotroph biodiversity on FLNF in-vitro by performing a biodiversity reduction assay with chloroform fumigation, incubating soils with chloroform for either 24 or 72 hours. We estimated diazotroph biodiversity for both in-situ and chloroform-fumigated samples by preparing and sequencing nifH gene libraries. The raw sequencing data of our nifH library is deposited here.

Kristy, Brandon↗

NASA NTRS1972

Infrared absorptivities of transition metals at room and liquid-helium temperatures.

Evaluation of experimental data concerning the normal spectral absorptivities of the transition metals, nickel, iron, platinum, and chromium, at both room and liquid-helium temperatures in the wavelength range from 2.5 to 50 microns. The absorptivities were derived from reflectivity measurements made relative to a room-temperature vapor-deposited gold reference mirror. The absorptivity of the gold reference mirror was measured calorimetrically, by use of infrared laser sources. Investigation of various methods of sample-surface preparation resulted in the choice of a vacuum-annealing process as the final stage. The experimental results are discussed on the basis of the anomalous-skin-effect theory modified for multiple conduction bands. As predicted, the results approach a single-band model toward the longer wavelengths. Agreement between theory and experiment is considerably improved by taking into account the modification of the relaxation time due to the photon-electron-phonon interaction proposed by Holstein (1954) and Gurzhi (1958); but, particularly at helium temperatures, the calculated curve is consistently below the experimental results.

Jones, M. C.↗

NASA NTRS1977

Superconductivity in sputtered CuMO6S8

Samples were prepared by melting the metals, followed by annealing to various temperatures. The result was a structurally weak material. Sputtered films on sapphire substrates were prepared and studied. The substrates give the films mechanical strength and permit easy attachment of electrical leads. Materials were characterized by X-ray diffraction, electron microscopy, electrical resistance vs. temperature, and critical current measurements. Some of the results on CuMo6S8 are presented.

Alterovitz, S.↗

NASA NTRS1976

X-ray topographic observations of magnetic domains in Czochralski-grown nickel single crystals in anomalous transmission geometry

The anomalous transmission effect has been observed in nickel single crystals grown by the Czochralski technique. Sample crystals prepared for X-ray topography are 1.5 to 2.5 cm in diameter and thicker than 0.4 mm; the product of the ordinary linear absorption coefficient and thickness ranges from 17 to 45 for Cu K-alpha radiation. Topographs taken with an asymmetric-crystal-topographic (ACT) camera have revealed extremely straight line images and rectangular arrangements of short line-segment images, along with the images of crystal imperfections. The line images change their arrangement upon application of magnetic fields. These images are attributed to magnetic domains in the interior of crystals. The formation of domain walls near the crystal surface is also observed and distinguished clearly from magnetic domains in the bulk with the use of the ACT camera.

Kuriyama, M.↗

NASA NTRS1975

Origin of 78235, a lunar norite cumulate

A chemical and petrographic study is reported of the phases from the rock 78235 which was returned on the Apollo 17 mission. Petrographic analysis of the thin sections from the bounder confirm its cumulate origin. In order to develop further the crystallization history for 78235, its subsequent shock history, and its relationship to other lunar crustal rocks, orthopyroxene, plagioclase, glass, and whole-rock samples were prepared and analyzed for major, minor, and trace elements. It is speculated that an early fractional crystallization event producing a layer of orthopyroxene-plagioclase cumulate with varying amounts of trapped liquid took place within 20 km of the surface of the moon.

Winzer, S. R.↗

NASA NTRS1980

Space fabrication demonstration system

The development effort on the composite beam cap fabricator was completed within cost and close to abbreviated goals. The design and analysis of flight weight primary and secondary beam builder structures proceeded satisfactorily but remains curtailed until further funding is made available to complete the work. The induction fastening effort remains within cost and schedule constraints. Tests of the LARC prototype induction welder is continuing in an instrumented test stand comprised of a Dumore drill press (air over oil feed for variable applied loads) and a dynamometer to measure actual welding loads. Continued testing shows that the interface screening must be well impregnated with resin to ensure proper flow when bonding graphite-acrylic lap shear samples. Specimens prepared from 0.030 inch thick graphite-polyethersulfone are also available for future induction fastening evaluation.

Source record↗

NASA NTRS1983

Chemical and isotopic study of extraterrestrial particles from the ocean floor

An Rb-Sr isotopic analysis of deep-sea spherules (DSS) from the Pacific, vesicular iridium-rich glassy objects (VIRGO) from an Antarctic-Ocean diatomaceous-ooze core, and equigranular objects (EGO) associated with or included in VIRGO, is reported. The techniques of sample collection, preparation, chemical analysis, and isotopic analysis (by mass spectrometry) are described in detail, and the results are presented in tables. The Sr-87/Sr-86 determination has a precision of 1-2 percent in microsamples of 2-8 x 10 to the 11th Sr atoms. The DSS are found to have Sr concentrations and Sr-87/Sr-86 (0.730-0.757) typical of chondritic meteorites. Extreme Rb depletion is attributed to volatization during meteoroid atmospheric heating. The VIRGO, while having major-element compositions like that of meteorites and high Ir levels suggesting extraterrestrial origin, have Sr-87/Sr-86 = 0.703-0.705; and the associated EGO has Sr-87/Sr-86 = 0.701 + or - 0.001, typical of MORB or basaltic achondrites. Hence their extraterrestrial origin is not proved, and some lithic component other than the EGO must be the source of the high Ir concentration.

Papanastassiou, D. A.↗

NASA NTRS1984

Chromium silicide formation by ion mixing

The formation of CrSi2 by ion mixing was studied as a function of temperature, silicide thickness and irradiated interface. Samples were prepared by annealing evaporated couples of Cr on Si and Si on Cr at 450 C for short times to form Si/CrSi2/Cr sandwiches. Xenon beams with energies up to 300 keV and fluences up to 8 x 10 to the 15th per sq cm were used for mixing at temperatures between 20 and 300 C. Penetrating only the Cr/CrSi2 interface at temperatures above 150 C induces further growth of the silicide as a uniform stoichiometric layer. The growth rate does not depend on the thickness of the initially formed silicide at least up to a thickness of 150 nm. The amount of growth depends linearly on the density of energy deposited at the interface. The growth is temperature dependent with an apparent activation energy of 0.2 eV. Irradiating only through the Si/CrSi2 interface does not induce silicide growth. It is concluded that the formation of CrSi2 by ion beam mixing is an interface-limited process and that the limiting reaction occurs at the Cr/CrSi2 interface.

Shreter, U.↗

NASA NTRS1984

Reflectance of metallic indium for solar energy applications

An investigation has been conducted in order to compile quantitative data on the reflective properties of metallic indium. The fabricated samples were of sufficiently high quality that differences from similar second-surface silvered mirrors were not apparent to the human eye. Three second-surface mirror samples were prepared by means of vacuum deposition techniques, yielding indium thicknesses of approximately 1000 A. Both hemispherical and specular measurements were made. It is concluded that metallic indium possesses a sufficiently high specular reflectance to be potentially useful in many solar energy applications.

Bouquet, F. L.↗

NASA NTRS1983

High temperature deformation of hot-pressed polycrystalline orthoenstatite

Artificial hot pressed polycrystalline samples were prepared from purified powder of Bamble, Norway, orthoenstatite, (Mg0.86Fe0.14)SiO3. The uniaxial creep behavior of the polycrystalline orthoenstatite was studied over stress ranges of 10-180 MPa and temperatures of 1500-1700 K (0.82-0.93 T sub m) under two different oxygen fugacities, namely equilibrium (Mo-MoO2 buffer) and a reducing (graphite heating element) atmosphere, respectively. An intergranular glassy phase of different compositions with a cavitational creep deformation were observed. In the Mo-MoO2 buffer atmosphere with PO2 approx. 10 to the minus 11 power - 10 to the minus 13 power atmospheres, the results of an analytical electron microscopy analysis indicate that the glassy phases are richer in Ca and Al due to the residual impurities after hot pressing. In the reducing atmosphere with an oxygen fugacity of PO2 approx. 10 to the minus 3 power - 10 to the minus 25 power atmospheres, the results of analytical electron microscopy analysis indicate that the glassy phase is almost pure silica with the presence of free iron precipitate on grain facets and at triple junctions due to the reduction of bulk materials.

Dehghan-Banadaki, A.↗

NASA NTRS1987

Superconductivity at 52.5 K in the lanthanum-barium-copper-oxide system

The electrical properties of the (La/0/9/Ba/0.1/)CuO/4-y/ system are examined under ambient and hydrostatic pressures. The resistance, ac magnetic susceptibility, and superconductivity onset, midpoint, and intercept temperatures are measured. It is observed that at ambient pressure the resistance decreases with temperature decreases, and the ac susceptibility shows diamagnetic shifts starting at about 32 K. Under hydrostatic pressure a superconducting transition with an onset temperature of 52.5 K is observed, and the resistance increases at lower temperatures. The data reveal that the electrical properties of the La-Ba-Cu-O system are dependent on samples and preparation conditions. Various causes for the high temperature superconductivity of the system are proposed.

Chu, C. W.↗

NASA NTRS1990

Electronic state and superconductivity of YBa2Cu3-xO7-y (M=Al,Zn and Sn) systems

A series of YBa2Cu(3-x)MxO(7-y) (M=Al,Zn and Sn) single phase samples were prepared, and the measurements of the crystal structure, oxygen content, electric resistivity, thermoelectric power, Mossbauer spectrum, XPS and superconductivity were performed. The experimental results of X ray powder diffraction, Mossbauer spectrum and oxygen content show that the Zn(2+) and the Al(3+) occupy the Cu(2) site in Cu-O planes and the Cu(1) site in Cu-O chains respectively, but the Sn(4+) occupies both the Cu(1) sites. As regards the properties in superconducting state, both the Zn(2+) and the Al(3+) depress T(sub c) strongly, but the Sn(4+) does not. As for the electronic transport properties in normal state, the system doped by Al(3+) displays a rapid increase of resistivity and some electron localization-like effects, and the thermoelectric power enhances obviously; the series contained Zn(2+) almost shows no changes of electric resistivity but the sign of the thermoelectric power is reversed. Other results are given and briefly discussed.

Zhao, Y.↗

NASA NTRS1990

Constitutive parameter de-embedding using inhomogeneously-filled rectangular waveguides with longitudinal section modes

Constitutive parameter extraction from S parameter data using a rectangular waveguide whose cross section is partially filled with a material sample as opposed to being completely filled was examined. One reason for studying a partially filled geometry is to analyze the effect of air gaps between the sample and fixture for the extraction of constitutive parameters. Air gaps can occur in high temperature parameter measurements when the sample was prepared at room temperature. Single port and two port measurement approaches to parameter extraction are also discussed.

Park, A.↗

NASA NTRS1991

Developing and utilizing an Euler computational method for predicting the airframe/propulsion effects for an aft-mounted turboprop transport. Volume 2: User guide

This manual explains how to use an Euler based computational method for predicting the airframe/propulsion integration effects for an aft-mounted turboprop transport. The propeller power effects are simulated by the actuator disk concept. This method consists of global flow field analysis and the embedded flow solution for predicting the detailed flow characteristics in the local vicinity of an aft-mounted propfan engine. The computational procedure includes the use of several computer programs performing four main functions: grid generation, Euler solution, grid embedding, and streamline tracing. This user's guide provides information for these programs, including input data preparations with sample input decks, output descriptions, and sample Unix scripts for program execution in the UNICOS environment.

Chen, H. C.↗

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