Transport and Use of a Centaur Second Stage in Space

As nations continue to explore space, the desire to reduce costs will continue to grow. As a method of cost reduction, transporting and/or use of launch system components as integral components of missions may become more commonplace in the future. There have been numerous scenarios written for using launch vehicle components (primarily space shuttle used external tanks) as part of flight missions or future habitats. Future studies for possible uses of launch vehicle upper stages might include asteroid diverter using gravity orbital perturbation, orbiting station component, raw material at an outpost, and kinetic impactor. The LCROSS (Lunar CRater Observation and Sensing Satellite) mission was conceived as a low-cost means of determining whether water exists at the polar regions of the moon. Manifested as a secondary payload with the LRO (Lunar Reconnaissance Orbiter) spacecraft aboard an Atlas V launch vehicle, LCROSS guided its spent Centaur Earth Departure Upper Stage (EDUS) into the lunar crater Cabeu's, as a kinetic impactor. This paper describes some of the challenges that the LCROSS project encountered in planning, designing, launching with and carrying the Centaur upper stage to the moon

Microgravity validation of a novel system for RNA isolation and multiplex quantitative real time PCR analysis of gene expression on the International Space Station

<div><p>The International Space Station (ISS) National Laboratory is dedicated to studying the effects of space on life and physical systems, and to developing new science and technologies for space exploration. A key aspect of achieving these goals is to operate the ISS National Lab more like an Earth-based laboratory, conducting complex end-to-end experimentation, not limited to simple microgravity exposure. Towards that end NASA developed a novel suite of molecular biology laboratory tools, reagents, and methods, named WetLab-2, uniquely designed to operate in microgravity, and to process biological samples for real-time gene expression analysis on-orbit. This includes a novel fluidic RNA Sample Preparation Module and fluid transfer devices, all-in-one lyophilized PCR assays, centrifuge, and a real-time PCR thermal cycler. Here we describe the results from the WetLab-2 validation experiments conducted in microgravity during ISS increment 47/SPX-8. Specifically, quantitative PCR was performed on a concentration series of DNA calibration standards, and Reverse Transcriptase-quantitative PCR was conducted on RNA extracted and purified on-orbit from frozen <i>Escherichia coli</i> and mouse liver tissue. Cycle threshold (Ct) values and PCR efficiencies obtained on-orbit from DNA standards were similar to Earth (1 g) controls. Also, on-orbit multiplex analysis of gene expression from bacterial cells and mammalian tissue RNA samples was successfully conducted in about 3 h, with data transmitted within 2 h of experiment completion. Thermal cycling in microgravity resulted in the trapping of gas bubbles inside septa cap assay tubes, causing small but measurable increases in Ct curve noise and variability. Bubble formation was successfully suppressed in a rapid follow-up on-orbit experiment using standard caps to pressurize PCR tubes and reduce gas release during heating cycles. The WetLab-2 facility now provides a novel operational on-orbit research capability for molecular biology and demonstrates the feasibility of more complex wet bench experiments in the ISS National Lab environment.</p></div

Validation of lyophilized assays.

<p>Validation of lyophilized assays.</p

Operations for the WetLab-2 system.

<p>Isolation and purification of RNA from biological tissues on–orbit starts with introduction of cells or tissues for lysis and homogenization into the Sample Preparation Module (SPM), followed by RNA binding to an affinity column, washing, and elution from the module. A Pipette Loader (PL) tool is provided for bubble free fluid transfer to a repeater pipette. This is then used to dispense accurate volumes of purified RNA into a centrifuge rotor/rack of lyophilized reagent tubes with enzymes and regents for reverse transcription and Taqman RT-qPCR. Data is available on-orbit within 3h of initiating the experiment, and transmitted by ISS to NASA Marshall Space Flight Center for emailing to investigators within 2 h of clean-up. A more detailed description of the process can be found in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183480#pone.0183480.s001" target="_blank">S1 Text</a>.</p

qPCR validation in microgravity.

<p>(A) qPCR assays targeting the <i>E</i>. <i>coli</i> 16S ribosomal gene were validated on the ground using seven tenfold dilutions of template to measure sensitivity and efficiency (inset). (B) Amplification curve from qPCR run conducted on the ISS with photo of a representative tube pre- and post- run in microgravity (inset). (C-F) qPCR amplification curves from the microgravity (C & D) and 1 g control (E & F) runs. All curves use the default SmartCycler values with the exception that the boxcar correction is set to 3. Efficiency graphs are shown on the insets. (G & H) Ct values for low (0.01 ng/test), mid (1 ng/test), and high (100 ng/test) assays during both experimental runs are shown using scatter plots with jitter. μg = microgravity.</p

<i>E</i>. <i>coli</i> and mouse liver assay validation using 100 ng control RNA.

<p><i>E</i>. <i>coli</i> and mouse liver assay validation using 100 ng control RNA.</p

RNA isolation and RT-qPCR in microgravity.

<p>(A) Photo of SPM. (B) Typical RNA quality from SPM with <i>E</i>. <i>coli</i> (left panel) and mouse liver (right panel), control Qiagen (left lane) SPM (right lane). Center panel shows RNA quality from the 1 g control (left lane) and the returned microgravity sample from ISS (right lane). (C-E) Scatter plots with jitter of the microgravity and 1 g control <i>E</i>. <i>coli</i> singleplex (C), duplex (D) and triplex (E) reactions. One outlier is indicated by the open marker in C. One of the microgravity triplex tubes did not give a dnaK-FAM signal (E). (F-H) Scatter plots with jitter of the microgravity and 1 g control mouse liver singleplex (F), duplex (G) and triplex (H) reactions. One outlier from the microgravity triplex fn1 plot is indicated by the open marker and no gapdh-FAM signal was seen in the microgravity triplex reactions (H).</p

<i>E</i>. <i>coli</i> and mouse liver primers and probes used in this study.

<p><i>E</i>. <i>coli</i> and mouse liver primers and probes used in this study.</p

HEX amplification curves and photos from microgravity reactions comparing different caps.

<p>(A) HEX amplification curves when our modified septa caps are used. (B) HEX amplification curves from tubes sealed with the standard commercial SmartTube Caps. Post run photos of representative tubes are shown above.</p