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Uranus Flagship-class Orbiter and Probe Using Aerocapture

Exploration of the Ice Giants, especially Uranus, via orbiter and atmospheric probes, is required to answer pressing science questions that have been raised in the latest National Academies of Sciences Planetary Decadal Survey. Since the Ice Giants are the farthest planets from Earth, traditional fully-propulsive orbit insertion missions have transit times to the planetary bodies bordering 13-15 years and require a large amount of propellant (wet mass percentages of around 60-70%) for the orbit insertion maneuver, leaving less mass for the scientific payload and a planetary probe. Aerocapture uses aerodynamic forces generated by flight within a planetary atmosphere to decelerate and achieve orbit insertion. Aerocapture has been considered for several past missions but it has not been demonstrated. However, recent developments in thermal protection systems (TPS), guidance and control (G&C), and interplanetary navigation capabilities show the potential for using rigid, heritage entry vehicle configurations already flown at other planetary bodies for Ice Giants aerocapture. Aerocapture can robustly deliver spacecraft to Ice Giant orbits, while substantially increasing on-orbit payload mass (more than 40%) that can be used for a robust atmospheric entry probe. Additionally, the aerocapture maneuver would reduce the interplanetary transit time by 2-5 years (15-30%) relative to fully-propulsive orbit insertion. Recent work has shown that a flagship-class mission can be conducted in a shorter time than fully-propulsive missions if using aerocapture. This paper will consider the merits of including aerocapture as the orbit-insertion mechanism for a Uranus mission. Specifically, the implications of aerocapture orbit insertion for in-situ atmospheric probes will be discussed. The Uranus Orbiter and Probe concept mission study [3] is considered as the potential payload. Results from a recent NASA Space Technology Mission Directorate (STMD)-funded activity that is designing an aerocapture mission for a Uranus orbiter will be presented.

Soumyo Dutta↗
NASA NTRS2024

Aerocapture: An Enabling Technology for Flagship-Class Uranus Orbiter and Probe Mission

Introduction: The current decadal survey published by the National Academies of Sciences has informed National Aeronautics and Space Administration (NASA) to prioritize the study of the Ice Giants, especially Uranus. To gather the required data that addresses the science questions raised in this survey, a mission to Uranus with an orbiter and atmospheric probe must be designed. The Uranus Orbiter and Probe (UOP) study, which the survey identified as the flagship mission of this decade, proposes a 2031 launch to take advantage of a Jupiter fly-by and utilizes a fully propulsive orbit insertion design with an Earth-to-Uranus transit times ranging from 13 to 15 years. This fully propulsive orbit insertion at Uranus will be very fuel expensive (wet mass percentages of around 60-70\%) thereby leaving less mass for the scientific payload and additional planetary probes. In addition, scientists are more interested in visiting Uranus before 2049, when the Spring Equinox will occur, as it allows studying Uranus seasons not seen during Voyager 2's flyby in 1987. A NASA Flagship-Class mission would require at least 10 years of lead time prior to launch thereby making the 2031 launch to take advantage of the Jupiter fly-by extremely challenging. The consequence of missing the Jupiter fly-by and launching in the late 2030s is the challenge of a fully-propulsive mission like UOP to have a feasible alternative interplanetary trajectory that reaches Uranus before 2050. As an alternative, to address the shortcomings of the fully propulsive mission, one can design a mission to Uranus using aerocapture. What is aerocapture: Aerocapture is an atmospheric maneuver that uses aerodynamic forces generated by flight within a planetary atmosphere to decelerate and achieve orbit insertion. Using aerocapture allows one to provide the change in velocity (Delta V) needed to slow down from the approach hyperbolic trajectory to achieve the desired captured orbit around the target planet using aerodynamic forces of the vehicle (lift and drag). Using aerodynamic forces instead of fully-propulsive maneuvers results in significant savings in the fuel. Furthermore, aerocapture can also allow one to consider interplanetary trajectories with higher approach hyperbolic velocities, thus reducing the mission transit times. Aerocapture as an enabling technology: To use aerocapture as an enabling technology for the Uranus exploration mission, one would require an integrated system-level design, including a Thermal Protection System (TPS), hardware needed for aerodynamic modulation, and autonomous Guidance, Navigation, and Control (GNC) systems. Aerocapture has yet to be demonstrated, despite considering it for several past missions. Recent advancements in TPS and GNC capabilities show the potential for using rigid, heritage entry vehicle configurations already flown at other planetary bodies for Uranus aerocapture. Aerocapture can be a robust technology that can deliver spacecraft to Uranus science orbits while substantially increasing on-orbit payload mass (more than 40\%) that can enable robust atmospheric entry probes. Additionally, the aerocapture maneuver would reduce the interplanetary transit time by 2–5 years (15-30\%) relative to fully-propulsive orbit insertion. Recent work has shown that one can conduct a flagship-class mission in a shorter period than fully-propulsive missions if using aerocapture. What does aerocapture bring in for a Uranus Mission: Using aerocapture for a Uranus orbiter and atmospheric probe mission can provide one with considerable propellant savings. Spacecraft in interplanetary trajectories to Uranus typically need an Delta V in orders of kilometers per second to insert into science orbit. One would require thousands of kilograms of fuel to achieve such a Delta V using a traditional fully propulsive maneuver, leaving less mass for payload during the mission launch. Aerocapture can reduce the propulsion needs by dissipating energy in the sizable atmosphere of Uranus without a significant mass increase due to the need for an aeroshell. One can use the mass savings achieved using the aerocapture to reduce the launch vehicle requirements. In addition, one can also have additional science instruments on the orbiter or create a robust instrumentation suite on an atmospheric probe that can significantly increase the science outcome of the Uranus exploration mission. Furthermore, since aerocapture performance is relatively insensitive to increases in hyperbolic excess velocity, one can design the interplanetary trajectory to arrive at Uranus faster, reducing the interplanetary transit time and operations cost. All these savings, achieved using aerocapture, could help fit a larger class mission, such as the Uranus mission within a smaller capital, e.g., a Flagship-class orbiter mission in a New Frontiers class capital. Summary: This talk will provide an overview of how aerocapture can enable the Uranus exploration mission. Specifically, this talk will discuss the latest advancements made in the Uranus aerocapture study, such as investigating interplanetary trajectories with higher hyperbolic approach velocities and their implications on the aero heating and the TPS design, incorporating FNPAG (an advanced numerical-predictor guidance) and comparison of multiple navigation approaches. In addition, this talk will focus on mechanical design that can house more than one atmospheric probe and the six degrees of freedom simulation of aerocapture at Uranus. Findings from a recent NASA Space Technology Mission Directorate (STMD)-funded activity studying the aerocapture as an enabling technology for a Uranus orbiter will be presented. Using the science payload recommended by the Decadal Survey for Uranus exploration, this work shows many improvements over the baseline fully-propulsive mission. These improvements include a shorter cruise phase, flexibility in launch opportunities late into the 2030s while reaching Uranus before the 2050 equinox for the desired science opportunities, and lower propellant mass needs. This talk will highlight how aerocapture can be utilized for Uranus science orbit insertion using a lower-risk, heritage entry vehicle configuration used extensively as a Mars entry, descent, and landing vehicle. Furthermore, this talk will explore how the demonstration of aerocapture at Earth can benefit the aerocapture-enabled Uranus mission.

Aerocapture↗
DOE OSTI2019

Combinatorial design of chemical-dependent protein switches for controlling intracellular electron transfer

One concern with controlling electron flow in cells is the lack of biomolecules that directly couple the sensing of environmental conditions to electron transfer efficiency. To overcome this protein component limitation, we randomly inserted the ligand binding domain (LBD) from the human estrogen receptor (ER) into a thermostable 2Fe-2S ferredoxin (Fd) from Mastigocladus laminosus and used a bacterial selection to identify Fd-LBD fusion proteins that support electron transfer from a Fd-NADP reductase (FNR) to a Fd-dependent sulfite reductase (SIR). Mapping LBD insertion sites onto structure revealed that Fd tolerates domain insertion adjacent to or within the tetracysteine motif that coordinates the 2Fe-2S metallocluster. With both classes of the fusion proteins, cellular ET was enhanced by the ER antagonist 4-hydroxytamoxifen, albeit to different extents. In addition, one of Fds arising from ER-LBD insertion within the tetracysteine motif was found to acquire an oxygen-tolerant 2Fe-2S cluster, suggesting that ET is regulated through post-translational ligand binding. Furthermore, these findings show that domain insertion can be used to create protein electron carriers whose ET is regulated by environmental conditions. These metalloprotein switches are expected to be useful for achieving fast regulation of ET in engineered metabolic pathways and between electroactive bacteria and conductive materials.

36 MATERIALS SCIENCE↗
NASA NTRS1993

Phenotypic characterization of xpr, a global regulator of extracellular virulence factors in Staphylococcus aureus

We recently described a Tn551 insertion in the chromosome of Staphylococcus aureus S6C that resulted in drastically reduced expression of extracellular lipase (M. S. Smeltzer, S. R. Gill, and J. J. Iandolo, J. Bacteriol. 174:4000-4006, 1992). The insertion was localized to a chromosomal site (designated omega 1058) distinct from the lipase structural gene (geh) and the accessory gene regulator (agr), both of which were structurally intact in the lipase-negative (Lip-) mutants. In this report, we describe a phenotypic comparison between strains S6C, a hyperproducer of enterotoxin B; KSI9051, a derivative of S6C carrying the Tn551 insertion at omega 1058; ISP546, an 8325-4 strain that carries a Tn551 insertion in the agr locus; and ISP479C, the parent strain of ISP546 cured of the Tn551 delivery plasmid pI258repA36. Compared with their respective parent strains, ISP546 and KSI9051 produced greatly reduced amounts of lipase, alpha-toxin, delta-toxin, protease, and nuclease. KSI9051 also produced reduced amounts of staphylococcal enterotoxin B. Coagulase production was increased in ISP546 but not in KSI9051. Using a mouse model, we also demonstrated that ISP546 and KSI9051 were far less virulent than ISP479C and S6C. We have designated the genetic element defined by the Tn551 insertion at omega 1058 xpr to denote its role as a regulator of extracellular protein synthesis. We conclude that xpr and agr are similar and possibly interactive regulatory genes that play an important role in pathogenesis of staphylococcal disease.

NASA Discipline Cell Biology↗
DOE OSTI2016

Comparison of Subdivided Versus Full-Length Fuel Pin Burnup in the ATF-2 Safety Analysis

The Accident Tolerant Fuels (ATF) campaign, under the Fuel Cycle Research and Development (FCRD) Advanced Fuels program, was developed after the Fukushima Daiichi Nuclear Power Plant disaster in 2011. Under ATF, fuel and cladding concepts are to be developed and tested that may more readily withstand an accident similar to the one that occurred at Fukushima Daiichi. ATF is a combination of industry, universities, and national laboratories in an effort to develop advanced accident tolerant cladding and fuel concepts. The end goal of the ATF program is to insert a lead test rod (LTR) or lead test assembly (LTA) into an operating commercial power light water reactor by 2022. In order to meet this 2022 insertion goal, ATF is split into three separate phases: ATF-1, ATF-2, and ATF-3. The first phase of the ATF program, ATF-1, is the concept feasibility study and early irradiation testing. Feasibility, fabrication, and out-of-pile testing are happening at universities and laboratories all over the United States. Irradiation testing of these concepts is happening at the Advanced Test Reactor (ATR) at the Idaho National Laboratory (INL). Early irradiation testing involves testing clad and/or fuel in drop-in capsules in the ATR. Currently, these capsules are in the relatively low-flux positions in the ATR, but some newer concepts and concepts that require higher burnup will be moved to the higher-flux positions later on during irradiation. Currently, Oak Ridge National Laboratory (ORNL), Westinghouse Electric Company, Areva, and General Electric (GE) have inserted capsules for ATF-1, although more concepts are planned. The second phase of ATF, ATF-2, is the focus of this paper. ATF-2 is the loop testing of fuel pins with new cladding and fuel concepts to simulate pressurized water reactor (PWR) and boiling water reactor (BWR) environments. ATF-2 will concurrently be happening at the ATR and at the Halden Reactor in Norway. The ATF-2 irradiation at the ATR will only simulate a PWR environment. ATF-2 will be inserted into the ATR pressurized water loop, Loop 2A, in the Center Flux Trap (CFT), where chemistry, temperature, and pressure can all be controlled separately from the primary coolant system and other loops in ATR. Loop 2A will be borated, pressurized to 2200 psi nominal, and have an average water temperature of 300 deg. C. A borated loop with fueled rodlets is extremely rare for Nuclear Energy experiments in the ATR, so ATF-2 will demonstrate new and unique capabilities for INL. ATF-3 is the testing of fresh and irradiated ATF concepts at the Transient Reactor Test (TREAT) Facility. The TREAT Reactor is planned for restart, re-establishing U.S. transient testing capabilities after nearly 20 years of non-operations. TREAT will test ATF concepts under transient conditions, such as a reactivity insertion accident. In addition to fresh fuel pins, TREAT will use from ATF2 and do transient testing on those irradiated pins. The information TREAT will provide to the vendors to support fuel licensing will be valuable, showing that these concepts do enhance the accident tolerance of the fuel in commercial reactors. (author)

11 NUCLEAR FUEL CYCLE AND FUEL MATERIALS↗
DOE OSTI2023

A cellular selection identifies elongated flavodoxins that support electron transfer to sulfite reductase

Flavodoxins (Flds) mediate the flux of electrons between oxidoreductases in diverse metabolic pathways. To investigate whether Flds can support electron transfer to a sulfite reductase (SIR) that evolved to couple with a ferredoxin, we evaluated the ability of Flds to transfer electrons from a ferredoxin-NADP reductase (FNR) to a ferredoxin-dependent SIR using growth complementation of an Escherichia coli strain with a sulfur metabolism defect. We show that Flds from cyanobacteria complement this growth defect when coexpressed with an FNR and an SIR that evolved to couple with a plant ferredoxin. When we evaluated the effect of peptide insertion on Fld-mediated electron transfer, we observed a sensitivity to insertions within regions predicted to be proximal to the cofactor and partner binding sites, while a high insertion tolerance was detected within loops distal from the cofactor and within regions of helices and sheets that are proximal to those loops. Bioinformatic analysis showed that natural Fld sequence variability predicts a large fraction of the motifs that tolerate insertion of the octapeptide SGRPGSLS. In conclusion, these results represent the first evidence that Flds can support electron transfer to assimilatory SIRs, and they suggest that the pattern of insertion tolerance is influenced by interactions with oxidoreductase partners.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2016

CIRSE Standards of Practice Guidelines on Gastrostomy

PurposeSurgical Gastrostomy has been around since the 19th century but in 1980 the first successful percutaneous endoscopic gastrostomy was reported. A year later the first successful percutaneous gastrostomy was performed using fluoroscopic guidance. The technique for percutaneous insertion and the equipment used has been refined since then and it is now considered the gold standard for gastrostomy insertion. Here we present guidelines for image-guided enteral feeding tubes in adults.Material and MethodWe performed a review and analysis of the scientific literature, other national and international guidelines and expert opinion.ResultsStudies have shown fluoroscopic techniques have consistently higher success rates with lower rates of major complications than endoscopic techniques. However, the Achilles' heel of many fluoroscopic techniques is the requirement for smaller gastrostomy tube sizes resulting in them being more prone to blockages and thus requiring further intervention.ConclusionRadiological feeding tube insertion is a safe and effective procedure. Success rates are higher, and complication rates lower than PEG or surgical gastrostomy tube placement and innovative techniques for gastric and jejunal access mean that there are very few cases in which RIG is not possible. The principal weakness of radiologically inserted gastrostomies is the limitiation on tube size which leads to a higher rate of tube blockage. Per-oral image-guided gastrostomies have to an extent addressed this but have not been popularised. Currently many centres still consider endoscopic gastrostomies as the first line unless patients are too unwell to undergo this procedure or previous attempts have failed, in which case radioloically inserted gastrostomies are the technique of choice.

62 RADIOLOGY AND NUCLEAR MEDICINE↗
DOE OSTI2018

The importance of the membrane interface as the reference state for membrane protein stability

The insertion of nascent polypeptide chains into lipid bilayer membranes and the stability of membrane proteins crucially depend on the equilibrium partitioning of polypeptides. For this, the transfer of full sequences of amino-acid residues into the bilayer, rather than individual amino acids, must be understood. Earlier studies have revealed that the most likely reference state for partitioning very hydrophobic sequences is the membrane interface. We have used μs-scale simulations to calculate the interface-to-transmembrane partitioning free energies ΔG S→TM for two hydrophobic carrier sequences in order to estimate the insertion free energy for all 20 amino acid residues when bonded to the center of a partitioning hydrophobic peptide. Our findings reflect that prior single-residue scales likely overestimate the partitioning free energies of polypeptides. The correlation of ΔG S→TM with experimental full-peptide translocon insertion data is high, implying an important role for the membrane interface in translocon-based insertion. The choice of carrier sequence greatly modulates the contribution of each single-residue mutation to the overall partitioning free energy. Our results demonstrate the importance of quantifying the observed full-peptide partitioning equilibrium, which is between membrane interface and transmembrane inserted, rather than combining individual water-to-membrane amino acid transfer free energies.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Noble-Metal Substitution in Hemoproteins: An Emerging Strategy for Abiological Catalysis

Enzymes have evolved to catalyze a range of biochemical transformations with high efficiencies and unparalleled selectivities, including stereoselectivities, regioselectivities, chemoselectivities, and substrate selectivities, while typically operating under mild aqueous conditions. These properties have motivated extensive research to identify or create enzymes with reactivity that complements or even surpasses the reactivity of small-molecule catalysts for chemical reactions. One of the limitations preventing the wider use of enzymes in chemical synthesis, however, is the narrow range of bond constructions catalyzed by native enzymes. One strategy to overcome this limitation is to create artificial metalloenzymes (ArMs) that combine the molecular recognition of nature with the reactivity discovered by chemists. This Account describes a new approach for generating ArMs by the formal replacement of the natural iron found in the porphyrin IX (PIX) of hemoproteins with noble metals. Analytical techniques coupled with studies of chemical reactivity have demonstrated that expression of apomyoglobins and apocytochrome P450s (for which "apo-" denotes the cofactor-free protein) followed by reconstitution with metal-PIX cofactors in vitro creates proteins with little perturbation of the native structure, suggesting that the cofactors likely reside within the native active site. By means of this metal substitution strategy, a large number of ArMs have been constructed that contain varying metalloporphyrins and mutations of the protein. The studies discussed in this Account encompass the use of ArMs containing noble metals to catalyze a range of abiological transformations with high chemoselectivity, enantioselectivity, diastereoselectivity, and regioselectivity. These transformations include intramolecular and intermolecular insertion of carbenes into C-H, N-H, and S-H bonds, cyclopropanation of vinylarenes and of internal and nonconjugated alkenes, and intramolecular insertions of nitrenes into C-H bonds. The rates of intramolecular insertions into C-H bonds catalyzed by thermophilic P450 enzymes reconstituted with an Ir(Me)-PIX cofactor are now comparable to the rates of reactions catalyzed by native enzymes and, to date, 1000 times greater than those of any previously reported ArM. This reactivity also encompasses the selective intermolecular insertion of the carbene from ethyl diazoacetate into C-H bonds over dimerization of the carbene to form alkenes, a class of carbene insertion or selectivity not reported to occur with small-molecule catalysts. These combined results highlight the potential of well-designed ArMs to catalyze abiological transformations that have been challenging to achieve with any type of catalyst. The metal substitution strategy described herein should complement the reactivity of native enzymes and expand the scope of enzyme-catalyzed reactions.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2021

Transposon sequencing analysis of Bradyrhizobium diazoefficiens 110 spc4

Bradyrhizobium diazoefficiens USDA110 is one of the most effective nitrogen-fixing symbionts of soybeans. Here we carried out a large-scale transposon insertion sequencing (Tn-seq) analysis of strain Bd110spc4, which is derived from USDA110, with the goal of increasing available resources for identifying genes crucial for the survival of this plant symbiont under diverse conditions. We prepared two transposon (Tn) insertion libraries of Bd110spc4 with 155,042 unique Tn insertions when the libraries were combined, which is an average of one insertion every 58.7 bp of the reference USDA110 genome. Application of bioinformatic filtering steps to remove genes too small to be expected to have Tn insertions, resulted in a list of genes that were classified as putatively essential. Comparison of this gene set with genes putatively essential for the growth of the closely related alpha-proteobacterium, Rhodopseudomonas palustris, revealed a small set of five genes that may be collectively essential for closely related members of the family Bradyrhizobiaceae. This group includes bacteria with diverse lifestyles ranging from plant symbionts to animal-associated species to free-living species.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

Comparison of the polymorphs of VOPO 4 as multi-electron cathodes for rechargeable alkali-ion batteries

Multi-electron polyanion cathodes offer the potential for achieving both high voltage and high capacity in rechargeable alkali-ion batteries. Among the few materials known to exhibit multi-electron cycling, the polymorphs of VOPO 4 , which operate on the V 3+ –V 4+ –V 5+ redox couples, are particularly promising due to the high gravimetric capacities that have been achieved and the high voltage of the V 4+/5+ couple. In this work, we performed a systematic first principles investigation, supported by careful electrochemical characterization and published experimental data, of the relative thermodynamic stability, voltage, band gap, and diffusion kinetics for alkali intercalation into the β, ε and αI polymorphs of VOPO 4 . We find that all VOPO4 polymorphs remain reasonably stable with the insertion of one alkali ion per V, but are significantly destabilized with the insertion of two alkali ions per V. The voltages for Na insertion are ~0.33–0.69 V lower than those for Li insertion. We find that the αI polymorph is predicted to have higher Li + migration barriers and larger band gaps than the β and ε polymorphs, which account for the relatively worse electrochemical cycling performance observed. On the other hand, only the αI polymorph exhibits reasonably low barriers for Na + migration compared to the β and ε polymorphs, which are consistent with observed electrochemical performances reported thus far in the literature. We also show that differences in the voltage, kinetics and rate capability of these different polymorphs for Li and Na insertion can be traced back to their fundamentally different VO 6 /VO 5 -PO 4 frameworks.

Lin, Yuh-Chieh↗
DOE OSTI2014

Near-surface depletion of antimony during the growth of GaAsSb and GaAs/GaAsSb nanowires

The near-surface reduction of the Sb mole fraction during the growth of GaAsSb nanowires (NWs) and GaAs NWs with GaAsSb inserts has been studied using quantitative high-angle annular dark field scanning transmission electron microscopy (STEM). A model for diffusion of Sb in the hexagonal NWs was developed and employed in combination with the quantitative STEM analysis. GaAsSb NWs grown by Ga-assisted molecular beam epitaxy (MBE) and GaAs/GaAsSb NWs grown by Ga- and Au-assisted MBE were investigated. At the high temperatures employed in the NW growth, As-Sb exchange at and outward diffusion of Sb towards the surface take place, resulting in reduction of the Sb concentration at and near the surface in the GaAsSb NWs and the GaAsSb inserts. In GaAsSb NWs, an increasing near-surface depletion of Sb was observed towards the bottom of the NW due to longer exposure to the As beam flux. In GaAsSb inserts, an increasing change in the Sb concentration profile was observed with increasing post-insert axial GaAs growth time, resulting from a combined effect of radial GaAs overgrowth and diffusion of Sb. The effect of growth temperature on the diffusion of Sb in the GaAsSb inserts was identified. The consequences of these findings for growth optimization and the optoelectronic properties of GaAsSb are discussed.

77 NANOSCIENCE AND NANOTECHNOLOGY↗
DOE OSTI2016

Electronic resonant tunneling on graphene superlattice heterostructures with a tunable graphene layer

We have theoretically investigated the electronic resonant tunneling effect in graphene superlattice heterostructures, where a tunable graphene layer is inserted between two different superlattices. It is found that a complete tunneling state appears inside the enlarged forbidden gap of the heterostructure by changing the thickness of the inserted graphene layer and the transmittance of the tunneling state depends on the thickness of the inserted layer. Furthermore, the frequency of the tunneling state changes with the thickness of the inserted graphene layer but it always located in the little overlapped forbidden gap of two graphene superlattices. Therefore, both a perfect tunneling state and an ultrawide forbidden gap are realized in such heterostrutures. Since maximum probability densities of the perfect tunneling state are highly localized near the interface between the inserted graphene layer and one graphene superlattice, it can be named as an interface-like state. Such structures are important to fabricate high-Q narrowband electron wave filters.

77 NANOSCIENCE AND NANOTECHNOLOGY↗
DOE OSTI2024

Engineered mRNA–ribosome fusions for facile biosynthesis of selenoproteins

Ribosomes are often used in synthetic biology as a tool to produce desired proteins with enhanced properties or entirely new functions. However, repurposing ribosomes for producing designer proteins is challenging due to the limited number of engineering solutions available to alter the natural activity of these enzymes. In this study, we advance ribosome engineering by describing a novel strategy based on functional fusions of ribosomal RNA (rRNA) with messenger RNA (mRNA). Specifically, we create an mRNA–ribosome fusion called RiboU, where the 16S rRNA is covalently attached to selenocysteine insertion sequence (SECIS), a regulatory RNA element found in mRNAs encoding selenoproteins. When SECIS sequences are present in natural mRNAs, they instruct ribosomes to decode UGA codons as selenocysteine (Sec, U) codons instead of interpreting them as stop codons. This enables ribosomes to insert Sec into the growing polypeptide chain at the appropriate site. Our work demonstrates that the SECIS sequence maintains its functionality even when inserted into the ribosome structure. As a result, the engineered ribosomes RiboU interpret UAG codons as Sec codons, allowing easy and site-specific insertion of Sec in a protein of interest with no further modification to the natural machinery of protein synthesis. To validate this approach, we use RiboU ribosomes to produce three functional target selenoproteins in Escherichia coli by site-specifically inserting Sec into the proteins’ active sites. Overall, our work demonstrates the feasibility of creating functional mRNA–rRNA fusions as a strategy for ribosome engineering, providing a novel tool for producing Sec-containing proteins in live bacterial cells.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2014

SU-E-T-535: Preliminary 2D and 3D Gamma Calculation Comparison Using PRESAGE

Purpose: To compare gamma calculations from 2D and 3D dosimetry measurements for phantom quality assurance. Methods: An IROC Houston (RPC) head and neck phantom was irradiated with a 9 beam IMRT plan using two inserts: a TLD and film insert and a PRESAGE insert. Both inserts were irradiated 3 times. The film and PRESAGE doses were scaled to the TLD dose and 2D gamma calculations were made in the axial and sagittal planes bisecting the primary target. 3D gamma measurements were taken within the PRESAGE dosimeter volume. Gamma constraints of 3%/3mm distance to agreement (DTA), 5%/3mm DTA and 7%/4mm DTA were used in the study. The 3 irradiations for each insert were averaged together for comparison. Results: Film measurements for the 2D gamma showed 85% pixels passing at 3%/3mm in both planes. The 5%/3 mm constraint had 93% and 90% passing in the two planes. The 7%/4mm restraint resulted in 99% passing in both planes. The PRESAGE 2D gamma passed 66% and 61% of pixels in the both planes at 3%/3mm. At 5%/3mm 86% and 82% passed. For 7%/4mm, 94% of pixels passed in both planes. The 3D gamma resulted in a pass rate of 90% at 3%/3mm, 95% at 5%/3mm, and 99% at 7%/4mm. Conclusion: 2D gamma pass rates using film showed a higher pass rate than PRESAGE using the same criteria in the same planes. This may be due to poor 3D registration with the treatment plan compared to the 2D film registration system. The 3D gamma results had a higher pass rate (> 90% pass rate) possibly because it sampled many more pixels in noncritical volumes thus diluting the percent of pixels passing. 3D restraints should be more restrictive to be comparable to 2D results. Funding from NIH grant 5R01CA100835.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2014

SU-E-T-609: Evaluation of Transit Dosimetry Software Using Heterogeneous Phantom

Purpose: The purpose of this work was to evaluate limits and capabilities in the transit dosimetry software for use with the TomoTherapy system by irradiating a heterogeneous phantom. Methods: Helical TomoTherapy plan was created using CIRS phantom (model 062M) with nine various tissue equivalent inserts (lung inhale 0.2 g/cm{sup 3}, lung exhale 0.5 g/cm{sup 3}, adipose 0.96 g/cm{sup 3}, breast 0.99 g/cm{sup 3}, water 1.01 g/cm{sup 3}, muscle 1.06 g/cm{sup 3}, liver 1.07 g/cm{sup 3}, trabecular bone 1.16 g/cm{sup 3}, and dense bone 1.53 g/cm{sup 3}). Targets were contoured within every insert. The phantom was scanned with a 50 cm field of view and 3 mm slice width. Images were imported into the TomoTherapy TPS. A plan was generated to deliver 20 Gy to every insert (2 Gy per fraction) with a jaw width of 2.5 cm, a pitch of 0.430 and an actual modulation factor of 2.621. After the radiation delivery the planning CT, the RT structure, the RT plan, and the RT dose (DICOM format) as well as the exit detector sinogram were imported into the Dosimetry Check software (Math Resolutions, LLC). The 3D delivered doses were reconstructed from the exit detector data by correcting for phantom and couch attenuation. The resulting dose distribution were compared with the TPS planned dose using gamma index. Results: Using the clinical gamma criteria, 3% and 3 mm, all tissue equivalent inserts had a passing percentage of 100% except for 0.2 g/cm{sup 3} and 0.5 g/cm{sup 3} density inserts (gamma value of 81.67% and 99.18% respectively). Conclusion: The evaluated transit dosimetry software provides an independent verification of helical TomoTherapy plans giving additional confidence in the treatment delivery, however, an overestimation of the reconstructed dose in low density materials has been revealed. Implementation of Monte Carlo algorithm for exit dose reconstruction should improve dosimetric accuracy in heterogeneous patient tissues. Agreement with Math Resolutions.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

A generalized reconstruction framework for unconventional PET systems

Purpose: Quantitative estimation of the radionuclide activity concentration in positron emission tomography (PET) requires precise modeling of PET physics. The authors are focused on designing unconventional PET geometries for specific applications. This work reports the creation of a generalized reconstruction framework, capable of reconstructing tomographic PET data for systems that use right cuboidal detector elements positioned at arbitrary geometry using a regular Cartesian grid of image voxels. Methods: The authors report on a variety of design choices and optimization for the creation of the generalized framework. The image reconstruction algorithm is maximum likelihood-expectation–maximization. System geometry can be specified using a simple script. Given the geometry, a symmetry seeking algorithm finds existing symmetry in the geometry with respect to the image grid to improve the memory usage/speed. Normalization is approached from a geometry independent perspective. The system matrix is computed using the Siddon’s algorithm and subcrystal approach. The program is parallelized through open multiprocessing and message passing interface libraries. A wide variety of systems can be modeled using the framework. This is made possible by modeling the underlying physics and data correction, while generalizing the geometry dependent features. Results: Application of the framework for three novel PET systems, each designed for a specific application, is presented to demonstrate the robustness of the framework in modeling PET systems of unconventional geometry. Three PET systems of unconventional geometry are studied. (1) Virtual-pinhole half-ring insert integrated into Biograph-40: although the insert device improves image quality over conventional whole-body scanner, the image quality varies depending on the position of the insert and the object. (2) Virtual-pinhole flat-panel insert integrated into Biograph-40: preliminary results from an investigation into a modular flat-panel insert are presented. (3) Plant PET system: a reconfigurable PET system for imaging plants, with resolution of greater than 3.3 mm, is shown. Using the automated symmetry seeking algorithm, the authors achieved a compression ratio of the storage and memory requirement by a factor of approximately 50 for the half-ring and flat-panel systems. For plant PET system, the compression ratio is approximately five. The ratio depends on the level of symmetry that exists in different geometries. Conclusions: This work brings the field closer to arbitrary geometry reconstruction. A generalized reconstruction framework can be used to validate multiple hypotheses and the effort required to investigate each system is reduced. Memory usage/speed can be improved with certain optimizations.

60 APPLIED LIFE SCIENCES↗