Results for “cell envelope” — Page 14

DOE OSTI2022

A neutralizing antibody target in early HIV-1 infection was recapitulated in rhesus macaques immunized with the transmitted/founder envelope sequence

Transmitted/founder (T/F) HIV-1 envelope proteins (Envs) from infected individuals that developed neutralization breadth are likely to possess inherent features desirable for vaccine immunogen design. To explore this premise, we conducted an immunization study in rhesus macaques (RM) using T/F Env sequences from two human subjects, one of whom developed potent and broad neutralizing antibodies (Z1800M) while the other developed little to no neutralizing antibody responses (R66M) during HIV-1 infection. Using a DNA/MVA/protein immunization protocol, 10 RM were immunized with each T/F Env. Within each T/F Env group, the protein boosts were administered as either monomeric gp120 or stabilized trimeric gp140 protein. All vaccination regimens elicited high titers of antigen-specific IgG, and two animals that received monomeric Z1800M Env gp120 developed autologous neutralizing activity. Using early Env escape variants isolated from subject Z1800M as guides, the serum neutralizing activity of the two immunized RM was found to be dependent on the gp120 V5 region. Interestingly, the exact same residues of V5 were also targeted by a neutralizing monoclonal antibody (nmAb) isolated from the subject Z1800M early in infection. Glycan profiling and computational modeling of the Z1800M Env gp120 immunogen provided further evidence that the V5 loop is exposed in this T/F Env and was a dominant feature that drove neutralizing antibody targeting during infection and immunization. An expanded B cell clonotype was isolated from one of the neutralization-positive RM and nmAbs corresponding to this group demonstrated V5-dependent neutralization similar to both the RM serum and the human Z1800M nmAb. The results demonstrate that neutralizing antibody responses elicited by the Z1800M T/F Env in RM converged with those in the HIV-1 infected human subject, illustrating the potential of using immunogens based on this or other T/F Envs with well-defined immunogenicity as a starting point to drive breadth.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS2008

Computational Analyses in Support of Sub-scale Diffuser Testing for the A-3 Facility: Unsteady Analyses and Risk Assessment - Part 2

Simulation technology can play an important role in rocket engine test facility design and development by assessing risks, providing analysis of dynamic pressure and thermal loads, identifying failure modes and predicting anomalous behavior of critical systems. This is especially true for facilities such as the proposed A-3 facility at NASA SSC because of a challenging operating envelope linked to variable throttle conditions at relatively low chamber pressures. Design Support of the feasibility of operating conditions and procedures is critical in such cases due to the possibility of startup/shutdown transients, moving shock structures, unsteady shock-boundary layer interactions and engine and diffuser unstart modes that can result in catastrophic failure. Analyses of such systems is difficult due to resolution requirements needed to accurately capture moving shock structures, shock-boundary layer interactions, two-phase flow regimes and engine unstart modes. In a companion paper, we will demonstrate with the use of CFD, steady analyses advanced capability to evaluate supersonic diffuser and steam ejector performance in the sub-scale A-3 facility. In this paper we will address transient issues with the operation of the facility especially at startup and shutdown, and assess risks related to afterburning due to the interaction of a fuel rich plume with oxygen that is a by-product of the steam ejectors. The primary areas that will be addressed in this paper are: (1) analyses of unstart modes due to flow transients especially during startup/ignition, (2) engine safety during the shutdown process (3) interaction of steam ejectors with the primary plume i.e. flow transients as well as probability of afterburning. In this abstract we discuss unsteady analyses of the engine shutdown process. However, the final paper will include analyses of a staged startup, drawdown of the engine test cell pressure, and risk assessment of potential afterburning in the facility. Unsteady simulations have been carried out to study the engine shutdown process in the facility and understand the physics behind the interactions between the steam ejectors, the test cell and the supersonic diffuser. As a first approximation, to understand the dominant unsteady mechanisms in the engine test cell and the supersonic diffuser, the turning duct in the facility was removed. As the engine loses power a rarefaction wave travels downstream that disrupts the shock cell structure in the supersonic diffuser. Flow from the test cell is seen to expand into the supersonic diffuser section and re-pressurizes the area around the nozzle along with a upstream traveling compression wave that emanates from near the first stage ejectors. Flow from the first stage ejector expands to the center of the duct and a new shock train is formed between the first and second stage ejectors. Both stage ejectors keep the facility pressurized and prevent any large amplitude pressure fluctuations from affecting the engine nozzle. The resultant pressure loads the nozzle experiences in the shutdown process are small.

Ahuja, Vineet↗

DOE OSTI2021

Potential Activities and Long Lifetimes of Organic Carbon-Degrading Extracellular Enzymes in Deep Subsurface Sediments of the Baltic Sea

Heterotrophic microorganisms in marine sediments produce extracellular enzymes to hydrolyze organic macromolecules, so their products can be transported inside the cell and used for energy and growth. Therefore, extracellular enzymes may mediate the fate of organic carbon in sediments. The Baltic Sea Basin is a primarily depositional environment with high potential for organic matter preservation. The potential activities of multiple organic carbon-degrading enzymes were measured in samples obtained by the International Ocean Discovery Program Expedition 347 from the Little Belt Strait, Denmark, core M0059C. Potential maximum hydrolysis rates (V max ) were measured at depths down to 77.9mbsf for the following enzymes: alkaline phosphatase, β- d -xylosidase, β- d -cellobiohydrolase, N-acetyl-β- d -glucosaminidase, β-glucosidase, α-glucosidase, leucyl aminopeptidase, arginyl aminopeptidase, prolyl aminopeptidase, gingipain, and clostripain. Extracellular peptidase activities were detectable at depths shallower than 54.95mbsf, and alkaline phosphatase activity was detectable throughout the core, albeit against a relatively high activity in autoclaved sediments. β-glucosidase activities were detected above 30mbsf; however, activities of other glycosyl hydrolases (β-xylosidase, β-cellobiohydrolase, N-acetyl-β-glucosaminidase, and α-glucosidase) were generally indistinguishable from zero at all depths. These extracellular enzymes appear to be extremely stable: Among all enzymes, a median of 51.3% of enzyme activity was retained after autoclaving for an hour. We show that enzyme turnover times scale with the inverse of community metabolic rates, such that enzyme lifetimes in subsurface sediments, in which metabolic rates are very slow, are likely to be extraordinarily long. A back-of-the-envelope calculation suggests enzyme lifetimes are, at minimum, on the order of 230days, and may be substantially longer. These results lend empirical support to the hypothesis that a population of subsurface microbes persist by using extracellular enzymes to slowly metabolize old, highly degraded organic carbon.

54 ENVIRONMENTAL SCIENCES↗

NASA NTRS2003

Mixing and Transport in the Solar Nebula

Boss & Vanhala (2000, 2001) prepared reviews of triggered collapse and injection models, using Prudence Foster's finite differences code at very high spatial resolution (440 x 1440 cells) to demonstrate the convergence of the R-T fingers in triggered injection models. A two dimensional hydrodynamical calculation with unprecedentedly high spatial resolution (960 x 2880 zones, or almost 3 million grid points) demonstrated that it suitable shock front can both trigger the collapse of an otherwise stable presolar cloud, and inject shock front particles into the collapsing cloud through the formation of what become Rayleigh-Taylor fingers of compressed fluid layers falling into the gravitational potential well of the growing protostar. These calculations suggest that heterogeneity derived from these R-T fingers will persist down to the scale of their injection onto the surface of the solar nebula. Haghighipour developed a numerical code capable of calculating the orbital evolution of dust grains of varied sizes in a gaseous nebula, subject to Epstein and Stokes drag as well as the self-gravity of the disk. In collaboration with the PI and George W. Wetherill, Haghighipour has been involved in development of a new idea on the possibility of rapid formation of ice giant planets via the disk instability mechanism. Haghighipour studied the stability of a five-body system consisting of the Sun and four protoplanets by numerically integrating their equations of motions. Using Levison and Duncan s SWIFT integrator, Haghighipour showed that, depending on the orbital parameters of the bodies, such a system can be stable for 0.1-10 Myr. Time periods of 1 Myr or more are long enough to be consistent with the time scale proposed for the formation of giant planets by the disk instability mechanism and the photoevaporation of the gaseous envelopes of the outermost protoplanets by a nearby OB star, resulting in the formation of ice giant planets. The PI has used his three dimensional models of marginally gravitationally unstable disks to study the preservation of isotopic heterogeneity in evolving protoplanetary disks. Such heterogeneity might arise from the infall onto the disk s surface of solids processed in the X-wind region of the disk, or derived from stellar nucleosynthesis and injected by R-T fingers. The technique used consists of solving a color equation, identical to the gas continuity equation, which follows the time evolution in three space dimensions of an arbitrarily placed initial color field, i.e., a dye inserted the disk. The models show that significant concentrations of color could persist for time periods of about a thousand years or more, even in the most dynamically active region of such a disk. Such a time period might be long enough for solids to coagulate and grow to significant sizes while retaining the isotopic signature of their birth region in the nebula.

Boss, Alan P.↗

DOE OSTI2025

Physiological roles of an Acinetobacter -specific σ factor

ABSTRACT The Gram-negative pathogen Acinetobacter baumannii is considered an “urgent threat” to human health due to its propensity to become antibiotic resistant. Understanding the distinct regulatory paradigms used by A. baumannii to mitigate cellular stresses may uncover new therapeutic targets. Many γ-proteobacteria use the extracytoplasmic function (ECF) σ factor, RpoE, to invoke envelope homeostasis networks in response to stress. Acinetobacter species contain the poorly characterized ECF “SigAb”; however, it is unclear if SigAb has the same physiological role as RpoE. Here, we show that SigAb is a metal stress-responsive ECF that appears unique to Acinetobacter species and distinct from RpoE-like ECFs. We combine promoter mutagenesis, motif scanning, and chromatin immunoprecipitation-sequencing (ChIP-seq) to define the direct SigAb regulon, which consists of genes encoding SigAb itself, the stringent response mediator, RelA, and the uncharacterized small RNA, “SabS.” However, RNA-seq of strains overexpressing SigAb revealed a large, indirect regulon containing hundreds of genes. Metal resistance genes are key elements of the indirect regulon, as CRISPRi knockdown of sigAb or sabS resulted in increased copper sensitivity and excess copper-induced SigAb-dependent transcription. Furthermore, we found that two uncharacterized genes in the sigAb operon, “ aabA ” and “ aabB ,” have anti-SigAb activity. Finally, employing a targeted Tn-seq approach that uses CRISPR-associated transposons, we show that sigAb , aabA , and aabB are important for fitness even during optimal growth conditions. Our work reveals new physiological roles for SigAb and SabS, provides a novel approach for assessing gene fitness, and highlights the distinct regulatory architecture of A. baumannii . IMPORTANCE Acinetobacter baumannii is a hospital-acquired pathogen, and many strains are resistant to multiple antibiotics. Understanding how A. baumannii senses and responds to stress may uncover novel routes to treat infections. Here, we examine how the Acinetobacter -specific transcription factor, SigAb, mitigates stress. We find that SigAb directly regulates only a small number of genes, but indirectly controls hundreds of genes that have substantial impacts on cell physiology. We show that SigAb is required for maximal growth, even during optimal conditions, and is acutely required during growth in the presence of elevated copper. Given that copper toxicity plays roles in pathogenesis and on copper-containing surfaces in hospitals, we speculate that SigAb function may be important in clinically relevant contexts.

Bacon, Emily E. (ORCID:0000000180907689)↗

NASA NTRS2008

Computational Analyses in Support of Sub-scale Diffuser Testing for the A-3 Facility: Steady Predictions - Part 1

Simulation technology can play an important role in rocket engine test facility design and development by assessing risks, providing analysis of dynamic pressure and thermal loads, identifying failure modes and predicting anomalous behavior of critical systems. Advanced numerical tools assume greater significance in supporting testing and design of high altitude testing facilities and plume induced testing environments of high thrust engines because of the greater inter-dependence and synergy in the functioning of the different sub-systems. This is especially true for facilities such as the proposed A-3 facility at NASA SSC because of a challenging operating envelope linked to variable throttle conditions at relatively low chamber pressures. Facility designs in this case will require a complex network of diffuser ducts, steam ejector trains, fast operating valves, cooling water systems and flow diverters that need to be characterized for steady state performance. In this paper, we will demonstrate with the use of CFD analyses s advanced capability to evaluate supersonic diffuser and steam ejector performance in a sub-scale A-3 facility at NASA Stennis Space Center (SSC) where extensive testing was performed. Furthermore, the focus in this paper relates to modeling of critical sub-systems and components used in facilities such as the A-3 facility. The work here will address deficiencies in empirical models and current CFD analyses that are used for design of supersonic diffusers/turning vanes/ejectors as well as analyses for confined plumes and venting processes. The primary areas that will be addressed are: (1) supersonic diffuser performance including analyses of thermal loads (2) accurate shock capturing in the diffuser duct; (3) effect of turning duct on the performance of the facility (4) prediction of mass flow rates and performance classification for steam ejectors (5) comparisons with test data from sub-scale diffuser testing and assessment of confidence levels in CFD based flowpath modeling of the facility. The analyses tools used here expand on the multi-element unstructured CFD which has been tailored and validated for impingement dynamics of dry plumes, complex valve/feed systems, and high pressure propellant delivery systems used in engine and component test stands at NASA SSC. The analyses performed in the evaluation of the sub-scale diffuser facility explored several important factors that influence modeling and understanding of facility operation such as (a) importance of modeling the facility with Real Gas approximation, (b) approximating the cluster of steam ejector nozzles as a single annular nozzle, (c) existence of mixed subsonic/supersonic flow downstream of the turning duct, and (d) inadequacy of two-equation turbulence models in predicting the correct pressurization in the turning duct and expansion of the second stage steam ejectors. The procedure used for modeling the facility was as follows: (i) The engine, test cell and first stage ejectors were simulated with an axisymmetric approximation (ii) the turning duct, second stage ejectors and the piping downstream of the second stage ejectors were analyzed with a three-dimensional simulation utilizing a half-plane symmetry approximation. The solution i.e. primitive variables such as pressure, velocity components, temperature and turbulence quantities were passed from the first computational domain and specified as a supersonic boundary condition for the second simulation. (iii) The third domain comprised of the exit diffuser and the region in the vicinity of the facility (primary included to get the correct shock structure at the exit of the facility and entrainment characteristics). The first set of simulations comprising the engine, test cell and first stage ejectors was carried out both as a turbulent real gas calculation as well as a turbulent perfect gas calculation. A comparison for the two cases (Real Turbulent and Perfect gas turbulent) of the Ma Number distribution and temperature distributions are shown in Figures 1 and 2 respectively. The Mach Number distribution shows small yet distinct differences between the two cases such as locations of shocks/shock reflections and a slightly different impingement point on the wall of the diffuser from the expansion at the exit of the nozzle. Similarly the temperature distribution indicates different flow recirculation patterns in the test cell. Both cases capture all the essential flow phenomena such as the shock-boundary layer interaction, plume expansion, expansion of the first stage ejectors, mixing between the engine plume and the first stage ejector flow and pressurization due to the first stage ejectors. The final paper will discuss thermal loads on the walls of the diffuser and cooling mechanisms investigated.

Allgood, Daniel C.↗

NASA NTRS2010

Computational Analyses in Support of Sub-scale Diffuser Testing for the A-3 Facility: Steady Predictions - Part 1

Simulation technology can play an important role in rocket engine test facility design and development by assessing risks, providing analysis of dynamic pressure and thermal loads, identifying failure modes and predicting anomalous behavior of critical systems. Advanced numerical tools assume greater significance in supporting testing and design of high altitude testing facilities and plume induced testing environments of high thrust engines because of the greater inter-dependence and synergy in the functioning of the different sub-systems. This is especially true for facilities such as the proposed A-3 facility at NASA SSC because of a challenging operating envelope linked to variable throttle conditions at relatively low chamber pressures. Facility designs in this case will require a complex network of diffuser ducts, steam ejector trains, fast operating valves, cooling water systems and flow diverters that need to be characterized for steady state performance. In this paper, we will demonstrate with the use of CFD analyses s advanced capability to evaluate supersonic diffuser and steam ejector performance in a sub-scale A-3 facility at NASA Stennis Space Center (SSC) where extensive testing was performed. Furthermore, the focus in this paper relates to modeling of critical sub-systems and components used in facilities such as the A-3 facility. The work here will address deficiencies in empirical models and current CFD analyses that are used for design of supersonic diffusers/turning vanes/ejectors as well as analyses for confined plumes and venting processes. The primary areas that will be addressed are: (1) supersonic diffuser performance including analyses of thermal loads (2) accurate shock capturing in the diffuser duct; (3) effect of turning duct on the performance of the facility (4) prediction of mass flow rates and performance classification for steam ejectors (5) comparisons with test data from sub-scale diffuser testing and assessment of confidence levels in CFD based flowpath modeling of the facility. The analyses tools used here expand on the multi-element unstructured CFD which has been tailored and validated for impingement dynamics of dry plumes, complex valve/feed systems, and high pressure propellant delivery systems used in engine and component test stands at NASA SSC. The analyses performed in the evaluation of the sub-scale diffuser facility explored several important factors that influence modeling and understanding of facility operation such as (a) importance of modeling the facility with Real Gas approximation, (b) approximating the cluster of steam ejector nozzles as a single annular nozzle, (c) existence of mixed subsonic/supersonic flow downstream of the turning duct, and (d) inadequacy of two-equation turbulence models in predicting the correct pressurization in the turning duct and expansion of the second stage steam ejectors. The procedure used for modeling the facility was as follows: (i) The engine, test cell and first stage ejectors were simulated with an axisymmetric approximation (ii) the turning duct, second stage ejectors and the piping downstream of the second stage ejectors were analyzed with a three-dimensional simulation utilizing a half-plane symmetry approximation. The solution i.e. primitive variables such as pressure, velocity components, temperature and turbulence quantities were passed from the first computational domain and specified as a supersonic boundary condition for the second simulation. (iii) The third domain comprised of the exit diffuser and the region in the vicinity of the facility (primary included to get the correct shock structure at the exit of the facility and entrainment characteristics). The first set of simulations comprising the engine, test cell and first stage ejectors was carried out both as a turbulent real gas calculation as well as a turbulent perfect gas calculation. A comparison for the two cases (Real Turbulent and Perfect gas turbulent) of the Ma Number distribution and temperature distributions are shown in Figures 1 and 2 respectively.

Allgood, Daniel C.↗

DOE OSTI2022

Silver Nanowire-Indium Zinc Oxide Composite Flexible Transparent Conducting Electrodes Made by Spin- coating and Photonic Curing

Realizing high-throughput, low-cost perovskite solar cell (PSC) manufacturing is highly sought-after in photovoltaic (PV) research in recent years. To fully achieve roll-to-roll (R2R) manufacturing of PSCs, it is important to consider the flexible transparent electrode (TE). PET/ITO is a commonly used substrate for making flexible PSCs. When optimizing transparent conducting materials, there is a tradeoff between sheet resistance (Rsh) and optical transparency. Because commercial PET/ITO substrates are made with slow (~1 m/min) vacuum deposition processes, they tend to be expensive. Therefore, it would be advantageous to develop a high-throughput, R2R compatible, solution-deposition approach for fabricating the TE on PET substrates. While various solution-deposition processes, such as blade coating or slot-die coating, can achieve the desired web speed of > 10 m/min, there is still a need to improve the post-deposition annealing step. One promising post-deposition processing technique is intense-pulsed-light processing, also known as photonic curing. Photonic curing delivers short (0.01 – 100 ms) pulses of broadband (200 – 1500 nm) light from a xenon flash lamp to the samples. Any materials in the sample stack that absorb light will convert the impinging light pulse into heat within the sample, which drives changes in the sample (calcination, phase change, crystallization, etc.). Photonic curing has three main advantages over thermal annealing: 1. Faster processing speed (milliseconds or seconds). 2. Compatibility with plastic substrates. 3. Smaller physical footprint and less wasted energy. Since the light pulses are on for a short time, the intensity can be high while the total energy delivered to the sample is low, minimizing damages to the plastic substrates. In this work, a hybrid TE material is fabricated on PET substrates using photonic curing. The hybrid TE material contains a layer of silver nanowires (AgNWs) and a layer of metal-oxide (InOx, ITO, IZO, etc.). The AgNWs increase the light absorbed by the film during the photonic curing process, which leads to higher processing temperatures, possibly improving the conversion of the metal-oxide layer. The AgNWs also enhance the electrical conductivity of the final TE layer after photonic curing. A AgNW and metal-oxide bilayer is formed by spin coating each solution onto the PET substrate sequentially followed by a single photonic curing process. We use average optical transmittance (Tavg) from 400 to 700 nm and average Rsh to evaluate the TE performance. The following photonic curing parameters are varied to optimize Tavg (maximize) and Rsh (minimize): Pulse voltage, pulse envelope, number of micro-pulses, duty cycle, number of pulses, and pulse repetition rate. Preliminarily, we also observe a significant impact on the TE properties by the volume of AgNW deposited during the spin coating deposition step. Using dispense volumes of 80 µL and 20 µL, we achieve samples with Tavg = 73%, Rsh = 19 Ω/sq, and roughness = 9 nm, and Tavg = 83%, Rsh = 58 Ω/sq, and roughness = 5.6 nm, respectively, after photonic curing.

14 SOLAR ENERGY↗

DOE OSTI2021

Engineering well-expressed, V2-immunofocusing HIV-1 envelope glycoprotein membrane trimers for use in heterologous prime-boost vaccine regimens

HIV-1 vaccine immunofocusing strategies may be able to induce broadly-reactive neutralizing antibodies (NAbs). Here, we engineered a panel of diverse, membrane-resident native HIV-1 trimers vulnerable to two broad targets—the V2 apex and fusion peptide (FP). Selection criteria included i) high expression and ii) infectious function, so that trimer neutralization sensitivity can be profiled in pseudovirus (PV) assays. Initially, we boosted the expression of 17 candidate trimers by truncating gp41 and introducing a gp120-gp41 SOS disulfide to prevent gp120 shedding. "Repairs" were made to fill glycan holes and eliminate other strain-specific aberrations. A new neutralization assay allowed PV infection when our standard assay was insufficient. Trimers with exposed V3 loops, a target of non-NAbs, were discarded. To try to increase V2-sensitivity, we removed clashing glycans and modified the C-strand. Notably, a D167N mutation improved V2-sensitivity in several cases. Glycopeptide analysis of JR-FL trimers revealed near complete sequon occupation and that filling the N197 glycan hole was well-tolerated. In contrast, sequon optimization and inserting/removing glycans at other positions frequently had global "ripple" effects on glycan maturation and sequon occupation throughout the gp120 outer domain and gp41. V2 MAb CH01 selectively bound to trimers with small high mannose glycans near the base of the V1 loop, thereby avoiding clashes. Knocking in a rare N49 glycan was found to perturb gp41 glycans, increasing FP NAb sensitivity—and sometimes improving expression. Finally, a biophysical analysis of VLPs revealed that i) ~25% of particles bear Env spikes, ii) spontaneous particle budding is high and only increases 4-fold upon Gag transfection, and iii) Env+ particles express ~30–40 spikes. Taken together, we identified 7 diverse trimers with a range of sensitivities to two targets to allow rigorous testing of immunofocusing vaccine concepts.

60 APPLIED LIFE SCIENCES↗