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Structure of Pseudoknot PK26 Shows 3D Domain Swapping in an RNA

3D domain swapping provides a facile pathway for the evolution of oligomeric proteins and allosteric mechanisms and a means for using monomer-oligomer equilibria to regulate biological activity. The term "3D domain swapping" describes the exchange of identical domains between two protein monomers to create an oligomer. 3D domain swapping has, so far, only been recognized in proteins. In this study, the structure of the pseudoknot PK26 is reported and it is a clear example of 3D domain swapping in RNA. PK26 was chosen for study because RNA pseudoknots are required structures in several biological processes and they arise frequently in in vitro selection experiments directed against protein targets. PK26 specifically inhibits HIV-1 reverse transcriptase with nanomolar affinity. We have now determined the 3.1 A resolution crystal structure of PK26 and find that it forms a 3D domain swapped dimer. PK26 shows extensive base pairing between and within strands. Formation of the dimer requires the linker region between the pseudoknot folds to adopt a unique conformation that allows a base within a helical stem to skip one base in the stacking register. Rearrangement of the linker would permit a monomeric pseudoknot to form. This structure shows how RNA can use 3D domain swapping to build large scale oligomers like the putative hexamer in the packaging RNA of bacteriophage Phi29.

Lietzke, Susan E↗

Urine Pretreatment Configuration and Test Results for Space Applications

Pretreatment of urine using Oxone and sulfuric acid is baselined in the International Space Station (ISS) waste water reclamation system to control odors, fix urea and control microbial growth. In addition, pretreatment is recommended for long term flight use of urine collection and two phase separation to reduce or eliminate fouling of the associated hardware and plumbing with urine precipitates. This is important for ISS application because the amount of maintenance time for cleaning and repairing hardware must be minimized. This paper describes the development of a chemical pretreatment system based on solid tablet shapes which are positioned in the urine collection hose and are dissolved by the intrained urine at the proper ratio of pretreatment to urine. Building upon the prior success of the developed and tested solid Oxone tablet a trade study was completed to confirm if a similar approach, or alternative, would be appropriate for the sulfuric acid injection method. In addition, a recommended handling and packaging approach of the solid tablets for long term, safe and convenient use on ISS was addressed. Consequently, the solid tablet concept with suitable packaging was identified as the Urine Pretreat / Prefilter Assembly (UPPA). Testing of the UPPA configuration confirmed the disolution rates and ratios required by ISS were achieved. This testing included laboratory controlled methods as well as a 'real world' test evaluation that occurred during the 150 day Stage 10 Water Recovery Test (WRT) conducted at NASA Marshall Space Flight Center (MSFC).

Howard, Stanley G.↗

SBIR Success Stories at NASA Glenn Research Center

This booklet of success stories summarizes the NASA Glenn Research Center's accomplishments and successes by the Small Business Innovation Research (SBIR) and Small Business Technology Transfer (STTR) programs. These success stories are the results of selecting projects that best support NASA missions and also have commercialization potential. Each success story describes the innovation accomplished, commercialization of the technology, and further applications and usages. The company name and the NASA contact person are identified to encourage further interest and communication to occur.

Kim, Walter S.↗

Ergonomic Evaluation of the Foot Restraint Equipment Device (FRED)

Within the scope of the Microgravity Workstation and Restraint Evaluation project, funded by the NASA Headquarters Life Sciences Division, evaluations were proposed to be conducted in ground, KC-135, and/or Shuttle environments to investigate the human factors engineering (HFE) issues concerning confined/unique workstations, including crew restraint requirements. As part of these evaluations, KC-135 flights were conducted to investigate user/ workstation/ restraint integration for microgravity use of the FRED with the RMS workstation. This evaluation was a pre-cursor to Detailed Supplementary Objective (DSO) - 904 on STS-88. On that mission, a small-statured astronaut will be using the FRED restraint while working at the Aft RMS workstation. The DSO will collect video for later posture analyses, as well as subjective data in the form of an electronic questionnaire. This report describes the current FRED KC-135 evaluations. The primary objectives were to evaluate the usability of the FRED and to verify the DSO in-flight setup. The restraint interface evaluation consisted of four basic areas of restraint use: 1) adjustability; 2) general usability and comfort; 3) usability at the RMS workstation; and 4) assembly and disassembly.

Whitmore, Mihriban↗

Microgravity Science Glovebox

The Microgravity Science Glovebox (MSG) is a versatile research facility designed to permit the flexibility of crew manipulated investigations on the International Space Station (ISS). The MSG configuration has been planned around the concept of an experimental workstation where a variety of experiments can be installed and operated in a fashion very similar to their operation in a ground-based laboratory. The approach has been to provide a large working volume with a significant set of power, data and imaging resources, all enclosed, but accessible by the crew through sealed glove ports. This arrangement allows the advantage of interactive experimentation without unduly compromising the experiment design with restrictions imposed by protective and containment challenges that normally arise in manned space-flight laboratories. In addition, the data and imaging resources allow cooperative monitoring of experiment progress between the crew and ground-based scientists. As ISS utilization evolves, the MSG is scheduled to become a major pathfinder for developing and exploiting the scientific advantages of truly enabling the coupling of experimentation in space with an evaluative response from the crew and investigators.

Roark, Walt↗

Communications System Architecture Development for Air Traffic Management and Aviation Weather Information Dissemination

This document is the NASA AATT Task Order 24 Final Report. NASA Research Task Order 24 calls for the development of eleven distinct task reports. Each task was a necessary exercise in the development of comprehensive communications systems architecture (CSA) for air traffic management and aviation weather information dissemination for 2015, the definition of the interim architecture for 2007, and the transition plan to achieve the desired End State. The eleven tasks are summarized along with the associated Task Order reference. The output of each task was an individual task report. The task reports that make up the main body of this document include Task 5, Task 6, Task 7, Task 8, Task 10, and Task 11. The other tasks provide the supporting detail used in the development of the architecture. These reports are included in the appendices. The detailed user needs, functional communications requirements and engineering requirements associated with Tasks 1, 2, and 3 have been put into a relational database and are provided electronically.

Gallagher, Seana↗

Alleviation of Facility/Engine Interactions in an Open-Jet Scramjet Test Facility

Results of a series of shakedown tests to eliminate facility/engine interactions in an open-jet scramjet test facility are presented. The tests were conducted with the NASA DFX (Dual-Fuel eXperimental scramjet) engine in the NASA Langley Combustion Heated Scramjet Test Facility (CHSTF) in support of the Hyper-X program, The majority of the tests were conducted at a total enthalpy and pressure corresponding to Mach 5 flight at a dynamic pressure of 734 psf. The DFX is the largest engine ever tested in the CHSTF. Blockage, in terms of the projected engine area relative to the nozzle exit area, is 81% with the engine forebody leading edge aligned with the upper edge of the facility nozzle such that it ingests the nozzle boundary layer. The blockage increases to 95% with the engine forebody leading edge positioned 2 in. down in the core flow. Previous engines successfully tested in the CHSTF have had blockages of no more than 51%. Oil flow studies along with facility and engine pressure measurements were used to define flow behavior. These results guided modifications to existing aeroappliances and the design of new aeroappliances. These changes allowed fueled tests to be conducted without facility interaction effects in the data with the engine forebody leading edge positioned to ingest the facility nozzle boundary layer. Interaction effects were also reduced for tests with the engine forebody leading edge positioned 2 in. into the core flow, however some interaction effects were still evident in the engine data. A new shroud and diffuser have been designed with the goal of allowing fueled tests to be conducted with the engine forebody leading edge positioned in the core without facility interaction effects in the data. Evaluation tests of the new shroud and diffuser will be conducted once ongoing fueled engine tests have been completed.

Albertson, Cindy W.↗

Crystallization of bFGF-DNA Aptamer Complexes Using a Sparse Matrix Designed for Protein-Nucleic Acid Complexes

The Sparse Matrix approach for obtaining lead crystallization conditions has proven to be very fruitful for the crystallization of proteins and nucleic acids. Here we report a Sparse Matrix developed specifically for the crystallization of protein-DNA complexes. This method is rapid and economical, typically requiring 2.5 mg of complex to test 48 conditions. The method was originally developed to crystallize basic fibroblast growth factor (bFGF) complexed with DNA sequences identified through in vitro selection, or SELEX, methods. Two DNA aptamers that bind with approximately nanomolar affinity and inhibit the angiogenic properties of bFGF were selected for co-crystallization. The Sparse Matrix produced lead crystallization conditions for both bFGF-DNA complexes.

Cannone, Jaime J.↗

First Protein Crystallization Experiments on The International Space Station: Sweet Success in Space With Thaumatin

We determined the room temperature 1.2 A structure of thaumatin using a crystal grown in the first protein crystallization experiment conducted aboard the International Space Station (ISS). The crystals were grown in the Enhanced Gaseous Nitrogen Dewar (EGN) developed by Alexander McPherson and co-workers. EGN transports frozen solutions contained in tygon tubing in a liquid nitrogen Dewar to ISS where the tubes then thaw. Batch, free interface diffusion (FID), or vapor diffusion crystallization occurs after thawing. EGN was flown to the ISS on STS-106 on September 8, 2000. This was a "risk mitigation" flight that tested EGN performance and the process of conducting experiments on ISS. We focused on how to map a hanging drop crystallization recipe to the EGN FID method. Thaumatin was chosen as the test system. Three series of crystallization recipes were set-up. Each series tested different volume ratios of protein-rich solution to precipitant-rich solution. The series differed from each other by fixing either the protein concentration or the amount of protein in the solutions. Upon return of the samples to Earth on October 24 by STS-92, bubbles that spanned the diameter of the tubing were observed in all tubes. Such bubbles interrupt liquid-liquid diffusion and force vapor diffusion equilibration to occur instead. Nonetheless, crystals grew in 9 of 30 tubes. Many large crystals were grown, the largest being 2.0 x 1.1 x 1.0 cubic mm. The largest crystal was used to collect data at room temperature on beamline 7-1 of the Stanford Synchrotron Radiation Source to a maximum resolution of 1.2 A. The structure was refined anisotropically using SHELX with a data to parameter ratio of 4.5 to give an R(sub factor) of 15.8% (R(sub free) = 18.2%) for ail reflections without generated hydrogens. This refinement is proceeding. Comparisons of this 1.2 A microgravity structure to previous reports of the thaumatin structure at 1.75 A and to ground control crystals will be presented.

Kundrot, Craig E.↗

Thaumatin Crystallization Aboard the International Space Station Using Liquid-Liquid Diffusion in the Enhanced Gaseous Nitrogen Dewar (EGN)

This paper reports results from the first biological crystal growth experiment on the International Space Station (ISS). Crystals of thaumatin were grown using liquid-liquid diffusion in Tygon tubing transported in the Enhanced Gaseous Nitrogen Dewar (EGN). Different Volume ratios and concentrations of protein and precipitant were used to test different adaptations of the vapor diffusion crystallization recipe to the liquid-liquid diffusion method. The EGN warmed up from -196 C to 0 C in about four days, about the same time it took to warm from 0 C to 20 C. The temperature within the EGN was 20 - 24 C for the majority of the experiment. Air gaps that blocked liquid-liquid diffusion formed in the tubes. Nonetheless, crystals were grown. Synchrotron diffraction data collected from the best space grown crystal extended to 1.28 Angstroms, comparable to previous studies of space-grown thaumatin crystals. The resolution of the best ground control crystal was only 1.47 Angstroms. It is not clear if the difference in diffraction limit is due to factors other than crystal size. Improvements in temperature control and the elimination of air gaps are needed, but the results show that EGN on the ISS can be used to produce space grown crystals that diffract to high resolution.

Kundrot, Craig↗

Successes of Small Business Innovation Research at NASA Glenn Research Center

This booklet of success stories highlights the NASA Glenn Research Center's accomplishments and successes by the Small Business Innovation Research (SBIR) and Small Business Technology Transfer (STTR) Programs. These success stories are the results of selecting projects that support NASA missions and also have high commercialization potential. Each success story describes the innovation accomplished, commercialization of the technology, and further applications and usages. This booklet emphasizes the integration and incorporation of technologies into NASA missions and other government projects. The company name and the NASA contact person are identified to encourage further usage and application of the SBIR developed technologies and also to promote further commercialization of these products.

Kim, Walter S.↗

Equilibrium Kinetics Studies and Crystallization Aboard the International Space Station (ISS) Using the Protein Crystallization Apparatus for Microgravity (PCAM)

Protein Crystallization Apparatus in Microgravity (PCAM) trays have been used in Shuttle missions to crystallize proteins in a microgravity environment. The crystallization experiments are 'sitting drops' similar to that in Cryschem trays, but the reservoir solution is soaked in a wick. From early 2001, crystallization experiments are conducted on the International Space Station using mission durations of months rather than two weeks on previous shuttle missions. Experiments were set up in April 2001 on Flight 6A to characterize the time crystallization experiments will take to reach equilibrium in a microgravity environment using salts, polyethylene glycols and an organic solvent as precipitants. The experiments were set up to gather data for a series of days of activation with different droplet volumes and precipitants. The experimental set up on ISS and results of this study will be presented. These results will help future users of PCAM to choose precipitants to optimize crystallization conditions for their target macromolecules for a particular mission with known mission duration. Changes in crystal morphology and size between the ground and space grown crystals of a protein and a protein -DNA complex flown on the same mission will also be presented.

Achari, Aniruddha↗

The Indirect Effect of UV: Some Good News for Microbes?

Ultraviolet (UV) radiation is of great concern because its biological effects are predominantly harmful. UV damage may be direct or indirect, the latter mediated through the photochemical production of reactive oxygen species such as hydrogen peroxide. We measured the effect of H2O2 on various microbes both in the lab and in nature. At our study site in Yellowstone National Park, there is a UV-induced diurnal fluctuation of H2O2 extending up to one micron. Levels of DNA synthesis resulting from exposure to H2O2 were measured in several algal mats. Within naturally-occurring concentrations of H2O2, DNA synthesis increased. Laboratory studies showed that similar concentrations of H2O2 induce mitosis. We hypothesize that the low levels of H2O2 encountered in nature are inducing mitotic division. At higher levels of H2O2 a second peak in DNA synthesis was found which we interpret to represent DNA damage repair. These experiments suggest that in nature, the low levels of H2O2 produced may have a mitogenic rather than damaging effect. Assuming early levels of UV radiation were substantially higher at the time protists evolved, differential mitogenic effects could have influenced protistan evolution. With H2O2 likely to be present on such bodies as Mars and Europa, the response of organisms will be concentration-dependent and not linear.

Rothschild, Lynn J.↗

Vapor Compression Distillation Flight Experiment

One of the major requirements associated with operating the International Space Station is the transportation -- space shuttle and Russian Progress spacecraft launches - necessary to re-supply station crews with food and water. The Vapor Compression Distillation (VCD) Flight Experiment, managed by NASA's Marshall Space Flight Center in Huntsville, Ala., is a full-scale demonstration of technology being developed to recycle crewmember urine and wastewater aboard the International Space Station and thereby reduce the amount of water that must be re-supplied. Based on results of the VCD Flight Experiment, an operational urine processor will be installed in Node 3 of the space station in 2005.

Hutchens, Cindy F.↗

Science Issues Associated with the Use of a Microfluidic Chip Designed Specifically for Protein Crystallization

The Iterative Biological Crystallization team in partnership with Caliper Technologies has produced a prototype microfluidic chip for batch crystallization that has been designed and tested. The chip is designed for the mixing and dispensing of up to five solutions with possible variation of the recipe being delivered to two growth wells. Developments that have led to the successful on-chip crystallization of a few model proteins have required investigative insight into many different areas, including fluid mixing dynamics, surface treatments, quantification and fidelity of reagent delivery. This presentation will encompass the ongoing studies and data accumulated toward these efforts.

Holmes, Anna M.↗

Development Status of the International Space Station Urine Processor Assembly

NASA, Marshall Space Flight Center (MSFC) is developing a Urine Processor Assembly (UPA) for the International Space Station (ISS). The UPA uses Vapor Compression Distillation (VCD) technology to reclaim water from pre-treated urine. This water is further processed by the Water Processor Assembly (WPA) to potable quality standards for use on the ISS. NASA has developed this technology over the last 25-30 years. Over this history, many technical issues were solved with thousands of hours of ground testing that demonstrate the ability of the UPA technology to reclaim water from urine. In recent years, NASA MSFC has been responsible for taking the UPA technology to "flight design" maturity. This paper will give a brief overview of the UPA design and a status of the major design and development efforts completed recently to mature the UPA to a flight level.

Holder, Donald W.↗

Determination of the Three-Dimensional Morphology of ALH84001 and Biogenic MV-1 Magnetite: Comparison of Results from Electron Tomography and Classical Transmission Electron Microscopy

Dated at approximately 3.9 billion years of age, carbonate disks, found within fractures of the host rock of Martian meteorite ALH84001, have been interpreted as secondary minerals that formed at low temperature in an aqueous medium. Heterogeneously distributed within these disks are magnetite nanocrystals that are of Martian origin. Approximately one quarter of these magnetites have morphological and chemical similarities to magnetite particles produced by magnetotactic bacteria strain MV-1, which are ubiquitous in aquatic habitats on Earth. Moreover, these types of magnetite particles are not known or expected to be produced by abiotic means either through geological processes or synthetically in the laboratory. The remaining three quarters of the ALH84001 magnetites are likely products of multiple processes including, but not limited to, precipitation from a hydrothermal fluid, thermal decomposition of the carbonate matrix in which they are embedded, and extracellular formation by dissimilatory Fe-reducing bacteria. We have proposed that the origins of magnetites in ALH84001 can be best explained as the products of multiple processes, one of which is biological. Recently the three-dimensional (3-D) external morphology of the purported biogenic fraction of the ALH84001 magnetites has been the subject of considerable debate. We report here the 3-D geometry of biogenic magnetite crystals extracted from MV-1 and of those extracted from ALH84001 carbonate disks using a combination of high resolution classical and tomographic transmission electron microscopy (TEM). We focus on answering the following questions: (1) which technique provides adequate information to deduce the 3-D external crystal morphology?; and, (2) what is the precise 3-D geometry of the ALH84001 and MV-1 magnetites?

Thomas-Keprta, Kathie L.↗

Results of the Vapor Compression Distillation Flight Experiment (VCD-FE)

Vapor Compression Distillation (VCD) is the chosen technology for urine processing aboard the International Space Station (ISS). Key aspects of the VCD design have been verified and significant improvements made throughout the ground;based development history. However, an important element lacking from previous subsystem development efforts was flight-testing. Consequently, the demonstration and validation of the VCD technology and the investigation of subsystem performance in micro-gravity were the primary goals of the VCD-FE. The Vapor Compression Distillation Flight Experiment (VCD-E) was a flight experiment aboard the Space Shuttle Columbia during the STS-107 mission. The VCD-FE was a full-scale developmental version of the Space Station Urine Processor Assembly (UPA) and was designed to test some of the potential micro-gravity issues with the design. This paper summarizes the experiment results.

Hutchens, Cindy↗