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At least 19 records

Pseudomonas aeruginosa gene PA4880 encodes a Dps-like protein with a Dps fold, bacterioferritin-type ferroxidase centers, and endonuclease activity

We report the biochemical, structural, and functional characterization of the protein coded by gene PA4880 in the P. aeruginosa PAO1 genome. The PA4880 gene had been annotated as coding a probable bacterioferritin. Our structural work shows that the product of gene PA4880 is a protein that adopts the Dps subunit fold, which oligomerizes into a 12-mer quaternary structure. Unlike Dps, however, the ferroxidase di-iron centers and iron coordinating ligands are buried within each subunit, in a manner identical to that observed in the ferroxidase center of P. aeruginosa bacterioferritin. Since these structural characteristics correspond to Dps-like proteins, we term the protein as P. aeruginosa Dps-like, or Pa DpsL. The ferroxidase centers in Pa DpsL catalyze the oxidation of Fe 2+ utilizing O 2 or H 2 O 2 as oxidant, and the resultant Fe 3+ is compartmentalized in the interior cavity. Interestingly, incubating Pa DpsL with plasmid DNA results in efficient nicking of the DNA and at higher concentrations of Pa DpsL the DNA is linearized and eventually degraded. The nickase and endonuclease activities suggest that Pa DpsL, in addition to participating in the defense of P. aeruginosa cells against iron-induced toxicity, may also participate in the innate immune mechanisms consisting of restriction endonucleases and cognate methyl transferases.

59 BASIC BIOLOGICAL SCIENCES↗

Pseudomonas aeruginosa Dps (PA0962) Functions in H 2 O 2 Mediated Oxidative Stress Defense and Exhibits In Vitro DNA Cleaving Activity

We report the structural, biochemical, and functional characterization of the product of gene PA0962 from Pseudomonas aeruginosa PAO1. The protein, termed Pa Dps, adopts the Dps subunit fold and oligomerizes into a nearly spherical 12-mer quaternary structure at pH 6.0 or in the presence of divalent cations at neutral pH and above. The 12-Mer Pa Dps contains two di-iron centers at the interface of each subunit dimer, coordinated by conserved His, Glu, and Asp residues. In vitro, the di-iron centers catalyze the oxidation of Fe 2+ utilizing H 2 O 2 (not O 2 ) as an oxidant, suggesting Pa Dps functions to aid P. aeruginosa to survive H 2 O 2 -mediated oxidative stress. In agreement, a P. aeruginosa Δdps mutant is significantly more susceptible to H 2 O 2 than the parent strain. The Pa Dps structure harbors a novel network of Tyr residues at the interface of each subunit dimer between the two di-iron centers, which captures radicals generated during Fe 2+ oxidation at the ferroxidase centers and forms di-tyrosine linkages, thus effectively trapping the radicals within the Dps shell. Surprisingly, incubating Pa Dps and DNA revealed unprecedented DNA cleaving activity that is independent of H 2 O 2 or O 2 but requires divalent cations and 12-mer Pa Dps.

59 BASIC BIOLOGICAL SCIENCES↗

Independent Orbiter Assessment (IOA): Analysis of the DPS subsystem

The results of the Independent Orbiter Assessment (IOA) of the Failure Modes and Effects Analysis/Critical Items List (FMEA/CIL) is presented. The IOA approach features a top-down analysis of the hardware to independently determine failure modes, criticality, and potential critical items. The independent analysis results corresponding to the Orbiter Data Processing System (DPS) hardware are documented. The DPS hardware is required for performing critical functions of data acquisition, data manipulation, data display, and data transfer throughout the Orbiter. Specifically, the DPS hardware consists of the following components: Multiplexer/Demultiplexer (MDM); General Purpose Computer (GPC); Multifunction CRT Display System (MCDS); Data Buses and Data Bus Couplers (DBC); Data Bus Isolation Amplifiers (DBIA); Mass Memory Unit (MMU); and Engine Interface Unit (EIU). The IOA analysis process utilized available DPS hardware drawings and schematics for defining hardware assemblies, components, and hardware items. Each level of hardware was evaluated and analyzed for possible failure modes and effects. Criticality was assigned based upon the severity of the effect for each failure mode. Due to the extensive redundancy built into the DPS the number of critical items are few. Those identified resulted from premature operation and erroneous output of the GPCs.

Lowery, H. J.↗

Battery Systems for X-38 Crew Return Vehicle (CRV) and Deorbit Propulsion Stage (DPS)

A 28V 32 Ah cell Li/MnO2 and a 28V NiMH battery systems for the Deorbit Propulsion Stage (DPS) and the X-38 Crew Return Vehicle (CRV) are developed in Friwo-Silforkraft, Germany with the following objectives and approach: Provide safe battery designs for lowest volume and cost, and within schedule; Take advantage of less complex requests for V201 vs OPS CRV to simplify design and reduce cost; Use only existing commercial cell designs as building blocks for larger battery; Derive battery designs from the ASTRO-SPAS design which is the largest lithium battery design with Shuttle flight experience; Place maximum amount of battery energy on DPS; DPS battery is non rechargeable; and CRV batteries are rechargeable. This paper contains the following sections: a brief introduction on CRV requirements, CRV advantages over Soyuz, and X-38 programs; Battery objectives and approach; Battery requirements and groundrules (performance, on-orbit operation, etc); Design trades, solutions, redundancy plan, and margins; Envelope, size, and mass; Interfaces (structural, electrical & thermal); and Deviation from OPS CRV.

Darcy, Eric↗

Dynamic Positioning System (DPS) Risk Analysis Using Probabilistic Risk Assessment (PRA)

The National Aeronautics and Space Administration (NASA) Safety & Mission Assurance (S&MA) directorate at the Johnson Space Center (JSC) has applied its knowledge and experience with Probabilistic Risk Assessment (PRA) to projects in industries ranging from spacecraft to nuclear power plants. PRA is a comprehensive and structured process for analyzing risk in complex engineered systems and/or processes. The PRA process enables the user to identify potential risk contributors such as, hardware and software failure, human error, and external events. Recent developments in the oil and gas industry have presented opportunities for NASA to lend their PRA expertise to both ongoing and developmental projects within the industry. This paper provides an overview of the PRA process and demonstrates how this process was applied in estimating the probability that a Mobile Offshore Drilling Unit (MODU) operating in the Gulf of Mexico and equipped with a generically configured Dynamic Positioning System (DPS) loses location and needs to initiate an emergency disconnect. The PRA described in this paper is intended to be generic such that the vessel meets the general requirements of an International Maritime Organization (IMO) Maritime Safety Committee (MSC)/Circ. 645 Class 3 dynamically positioned vessel. The results of this analysis are not intended to be applied to any specific drilling vessel, although provisions were made to allow the analysis to be configured to a specific vessel if required.

Thigpen, Eric B.↗

Dynamic Positioning System (DPS) Risk Analysis Using Probabilistic Risk Assessment (PRA)

The National Aeronautics and Space Administration (NASA) Safety & Mission Assurance (S&MA) directorate at the Johnson Space Center (JSC) has applied its knowledge and experience with Probabilistic Risk Assessment (PRA) to projects in industries ranging from spacecraft to nuclear power plants. PRA is a comprehensive and structured process for analyzing risk in complex engineered systems and/or processes. The PRA process enables the user to identify potential risk contributors such as, hardware and software failure, human error, and external events. Recent developments in the oil and gas industry have presented opportunities for NASA to lend their PRA expertise to both ongoing and developmental projects within the industry. This paper provides an overview of the PRA process and demonstrates how this process was applied in estimating the probability that a Mobile Offshore Drilling Unit (MODU) operating in the Gulf of Mexico and equipped with a generically configured Dynamic Positioning System (DPS) loses location and needs to initiate an emergency disconnect. The PRA described in this paper is intended to be generic such that the vessel meets the general requirements of an International Maritime Organization (IMO) Maritime Safety Committee (MSC)/Circ. 645 Class 3 dynamically positioned vessel. The results of this analysis are not intended to be applied to any specific drilling vessel, although provisions were made to allow the analysis to be configured to a specific vessel if required.

Thigpen, Eric B.↗

Probabilistic Risk Analysis (PRA) of a Mobile Offshore Drilling Unit (MODU) Dynamic Positioning System (DPS)

Probabilistic Risk Assessment (PRA) has been utilized by NASA in a variety of space oriented projects. It has served as one of the primary risk identification and ranking tools. Recent developments in the oil and gas industry have presented opportunities for NASA to lend their PRA expertise to both ongoing and developmental projects within the industry. As a result, NASA has entered into an agreement with Anadarko Petroleum Company (APC) to collaboratively develop PRAs for different aspects of the subsea drilling and completion process of well development. This paper documents how PRA was applied to estimate the probability that a Mobile Offshore Drilling Unit (MODU) equipped with a generically configured Dynamic Positioning System (DPS) loses location and needs to initiate an emergency disconnect. Since this project was in essence a pilot project, the PRA described in this paper is intended to be generic such that the vessel meets the general requirements of an International Maritime Organization (IMO) Maritime Safety Committee (MSC)/Circ. 645 Class 3 dynamically positioned vessel. The results of this analysis are not intended to be applied to any specific drilling vessel, although provisions were made to allow the analysis to be configured to a specific vessel if required.

Thigpen, Eric B.↗

A study of discrete control signal fault conditions in the shuttle DPS

An analysis of the effects of discrete failures on the data processing subsystem is presented. A functional description of each discrete together with a list of software modules that use this discrete are included. A qualitative description of the consequences that may ensue due to discrete failures is given followed by a probabilistic reliability analysis of the data processing subsystem. Based on the investigation conducted, recommendations were made to improve the reliability of the subsystem.

Reddi, S. S.↗

Determination of the Interfacial Energy between Graphene Nanoplatelets and Deuterated or Hydrogenated Polystyrene

The tracer diffusion coefficients of various graphene nanoplatelet-polystyrene (GNP-PS) systems were determined by using dynamic secondary ion mass spectrometry, which provided insights into the interactions within the polymer nanocomposite system. Bilayer films, where one contained trace amounts of deuterated polystyrene (dPS), were produced, and the interdiffusion was measured by detecting the position of the dPS interface. GNPs were placed in both or only one of the two layers. The diffusion coefficient was decreased when GNPs were present in both layers as compared to the samples without GNP, indicating an attractive interaction between GNP and polystyrene. Surprisingly, the coefficient increased relative to the control when GNPs were present only in the PS containing layer, whereas it decreased most strongly when GNP and dPS were present only in the same layer, indicating that an additional preference existed for GNPs and dPS. A significant difference in interfacial energies was measured using contact angle goniometry of PS or dPS droplets on flat GNP layers, confirming the preferential interaction. DFT calculations were used to calculate the interfacial interaction between GNP and dPS or PS, and they showed an attractive potential which was maximal when the PS rings were conformal with the GNP structure. Furthermore, the differential energy between PS and dPS at the GNP interface was in excellent agreement with the measured value. In contrast, contact angle goniometry indicated that the interaction between GNP and poly(methyl methacrylate) (PMMA) was unfavorable, and no difference in interfacial energy between dPMMA and PMMA was observed. Therefore, these techniques can provide a means for determining the relative affinity between GNP and different polymer hosts, which can be an important consideration in the mechanics and adhesion properties of the compounds.

36 MATERIALS SCIENCE↗

NMR Spectroscopy for Protein Higher Order Structure Similarity Assessment in Formulated Drug Products

Peptide and protein drug molecules fold into higher order structures (HOS) in formulation and these folded structures are often critical for drug efficacy and safety. Generic or biosimilar drug products (DPs) need to show similar HOS to the reference product. The solution NMR spectroscopy is a non-invasive, chemically and structurally specific analytical method that is ideal for characterizing protein therapeutics in formulation. However, only limited NMR studies have been performed directly on marketed DPs and questions remain on how to quantitively define similarity. Here, NMR spectra were collected on marketed peptide and protein DPs, including calcitonin-salmon, liraglutide, teriparatide, exenatide, insulin glargine and rituximab. The 1D 1 H spectral pattern readily revealed protein HOS heterogeneity, exchange and oligomerization in the different formulations. Principal component analysis (PCA) applied to two rituximab DPs showed consistent results with the previously demonstrated similarity metrics of Mahalanobis distance (D M ) of 3.3. The 2D 1 H- 13 C HSQC spectral comparison of insulin glargine DPs provided similarity metrics for chemical shift difference (Δδ) and methyl peak profile, i.e., 4 ppb for 1 H, 15 ppb for 13 C and 98% peaks with equivalent peak height. Finally, 2D 1 H- 15 N sofast HMQC was demonstrated as a sensitive method for comparison of small protein HOS. The application of NMR procedures and chemometric analysis on therapeutic proteins offer quantitative similarity assessments of DPs with practically achievable similarity metrics.

59 BASIC BIOLOGICAL SCIENCES↗

Lattice Dynamics of Sb 2 Se 3 from Inelastic Neutron and X-Ray Scattering

The lattice dynamics of orthorhombic Sb 2 Se 3 is studied by a combination of inelastic neutron and 121 Sb nuclear inelastic scattering giving access to the total and Sb partial density of phonon states (DPS). The Se partial DPS is determined from the difference between the total and Sb partial DPS. The total DPS is determined at 39, 150, and 300 K, and an analysis of the temperature-induced mode shifts in combination with low-temperature powder diffraction data is provided. Using an earlier reported theoretical approach, the corresponding total and partial DPS of Sb 2 Se 3 are calculated by first-principles calculations. Herein, a detailed analysis of the Grüneisen parameter, element-specific and bulk Debye temperatures, and the mean force constants as derived from the experimental data and discrete Fourier transform calculations is provided. In general, the calculations underestimate the strength of the covalent Sb-Se bonds.

72 PHYSICS OF ELEMENTARY PARTICLES AND FIELDS↗

Failure Mode Identification of Insulin Drug Products – Impact of Relevant Stress Conditions on the Quality of the Drug

Fast-acting insulin drug products (DPs) are carried and administered by diabetic patients to maintain their blood glucose level throughout the day, exposing the DPs to stress conditions. Apidra, Novolog, and Humalog insulin DPs were tested under various stress conditions. Dynamic light scattering (DLS), and size exclusion chromatography (SEC) were used to monitor the stability and aggregation. Thermal stress alone did not influence the stability. However, 24 hr exposure to vigorous mechanical stress shifted the DLS size peaks of Novolog and Humalog from 5 ± 1 nm to > 50.9 ± 25.6 nm, and the SEC native protein peak areas decreased 52% for Novolog and 18.4% for Humalog. Combined stress accelerated protein aggregation more drastically. Novolog and Humalog size shifted (>75 nm) after 3 hr and the peak area decreased > 97.9% after 6 hr exposure, indicating that high temperature accelerated the aggregation triggered by agitation. Soluble aggregates were captured by DLS early on compared to SEC. Apidra was comparably stable indicating DP formulation plays a critical role in stability. Here this study provides a greater understanding of potential failure modes patients and care givers may encounter while handling insulin DPs.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Long-term Dynamical Stability in the Outer Solar System. I. The Regular and Chaotic Evolution of the 34 Largest Trans-Neptunian Objects

We carried out an extensive analysis of the stability of the outer solar system, making use of the frequency analysis technique over short-term integrations of nearly 100,000 test particles, as well as a statistical analysis of 200 1 Gyr long numerical simulations, which consider the mutual perturbations of the giant planets and the 34 largest trans-Neptunian objects (we have called all 34 objects “dwarf planets,” DPs, even if probably only the largest of them are true DPs). From the frequency analysis, we produced statistical diffusion maps for a wide region of the a–e phase-space plane; we also present the average diffusion time for orbits as a function of perihelion. We later turned our attention to the 34 DPs, making an individualized analysis for each of them and producing a first approximation of their future stability. From the 200 distinct realizations of the orbital evolution of the 34 DPs, we classified the sample into three categories, including 17 stable, 11 unstable, and 6 resonant objects; we also found that, statistically, two objects from the sample will leave the trans-Neptunian region within the next gigayear, most likely being ejected from the solar system, but with a nonnegligible probability of going inside the orbit of Neptune, either to collide with a giant planet or even falling to the inner solar system, where our simulations are no longer able to resolve their continuous evolution.

79 ASTRONOMY AND ASTROPHYSICS↗