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PCR Based Microbial Monitor for Analysis of Recycled Water Aboard the ISSA: Issues and Prospects

The monitoring of spacecraft life support systems for the presence of health threatening microorganisms is paramount for crew well being and successful completion of missions. Development of technology to monitor spacecraft recycled water based on detection and identification of the genetic material of contaminating microorganisms and viruses would be a substantial improvement over current NASA plans to monitor recycled water samples that call for the use of conventional microbiology techniques which are slow, insensitive, and labor intensive. The union of the molecular biology techniques of DNA probe hybridization and polymerase chain reaction (PCR) offers a powerful method for the detection, identification, and quantification of microorganisms and viruses. This technology is theoretically capable of assaying samples in as little as two hours with specificity and sensitivity unmatched by any other method. A major advance in probe-hybridization/PCR has come about in a technology called TaqMan(TM), which was invented by Perkin Elmer. Instrumentation using TaqMan concepts is evolving towards devices that could meet NASA's needs of size, low power use, and simplicity of operation. The chemistry and molecular biology needed to utilize these probe-hybridization/PCR instruments must evolve in parallel with the hardware. The following issues of chemistry and biology must be addressed in developing a monitor: Early in the development of a PCR-based microbial monitor it will be necessary to decide how many and which organisms does the system need the capacity to detect. We propose a set of 17 different tests that would detect groups of bacteria and fungus, as well as specific eukaryotic parasites and viruses; In order to use the great sensitivity of PCR it will be necessary to concentrate water samples using filtration. If a lower limit of detection of 1 microorganism per 100 ml is required then the microbes in a 100 ml sample must be concentrated into a volume that can be added to a PCR assay; There are not likely to be contaminants in ISSA recycled water that would inhibit PCR resulting in false-negative results; The TaqMan PCR product detection system is the most promising method for developing a rapid, highly automated gene-based microbial monitoring system. The method is inherently quantitative. NASA and other government agencies have invested in other technologies that, although potentially could lead to revolutionary advances, are not likely to mature in the next 5 years into working systems; PCR-based methods cannot distinguish between DNA or RNA of a viable microorganism and that of a non-viable organism. This may or may not be an important issue with reclaimed water on the ISSA. The recycling system probably damages the capacity of the genetic material of any bacteria or viruses killed during processing to serve as a template in a PCR desinged to amplify a large segment of DNA (less than 650 base pairs). If necessary, vital dye staining could be used in addition to PCR, to enumerate the viable cells in a water sample; The quality control methods have been developed to insure that PCR's are working properly, and that reactions are not contaminated with PCR carryover products which could lead to the generation of false-positive results; and The sequences of the small rRNA subunit gene for a large number of microorganisms are known, and they consititue the best database for rational development of the oligonucleotide reagents that give PCR its great specificity. From those gene sequences, sets of oligonucleotide primers for PCR and Taqman detection that could be used in a NASA microbial monitor were constructed using computer based methods. In addition to space utilization, a microbial monitior will have tremendous terrestrial applications. Analysis of patient samples for microbial pathogens, testing industrial effluent for biofouling bacteria, and detection biological warfare agents on the battlefield are but a few of the diverse potential uses for this technology. Once fully developed, gene-based microbial monitors will become the fundamental tool in every lab that tests for microbial contaminants, and serve as a powerful weapon in mankind's war with the germ world.

Cassell, Gail H.↗

Magnetic Resonance–Guided High-Intensity Focused Ultrasound Hyperthermia for Recurrent Rectal Cancer: MR Thermometry Evaluation and Preclinical Validation

Purpose: To evaluate the feasibility of magnetic resonance–guided high-intensity focused ultrasound (MR-HIFU) mild hyperthermia in deep tissue targets for enhancing radiation therapy and chemotherapy in the context of recurrent rectal cancer. A preclinical study was performed to evaluate the safety and performance of MR-HIFU mild hyperthermia. A prospective imaging study was performed in volunteers with rectal cancer to evaluate MR thermometry quality near the rectum and accessibility of rectal tumors using MR-HIFU. Methods and Materials: Mild hyperthermia was performed in pig thigh (9 sonications, 6 pigs) using a clinical MR-HIFU system. Targets near the rectal wall and deep thigh were evaluated. Thermal maps obtained in 6 planes every 3.2 seconds were used to control sonications in 18-mm diameter treatment regions at temperatures of 42°C to 42.5°C for 10 to 60 minutes. Volunteer imaging-only studies to assess the quality of MR thermometry (without heating) were approved by the institutional research ethics board. Anatomic and MR thermometry images were acquired in consenting volunteers with rectal cancer. In 3 of 6 study participants, rectal filling with saline was used to reduce motion-related MR thermometry artifacts near the tumor. Results: In pigs, mean target temperature matched the desired hyperthermia temperature within 0.2°C; temporal standard deviation ≤0.5°C. With optimized control thresholds, no undesired tissue damage was observed. In human volunteers, MR temperature measurements had adequate precision and stability, especially when rectal filling was used to reduce bowel motion. Conclusions: In pigs, MR-HIFU can safely deliver mild hyperthermia (41°C-43°C) to a targeted volume for 30 minutes. In humans, careful patient selection and preparation will enable adequate targeting for recurrent rectal cancers and sufficient MR temperature mapping stability to control mild hyperthermia. These results enable human trials of MR-HIFU hyperthermia.

62 RADIOLOGY AND NUCLEAR MEDICINE↗

Benefits of adaptive radiation therapy in lung cancer as a function of replanning frequency

Purpose: To quantify the potential benefit associated with daily replanning in lung cancer in terms of normal tissue dose sparing and to characterize the tradeoff between adaptive benefit and replanning frequency. Methods: A set of synthetic images and contours, derived from weekly active breathing control images of 12 patients who underwent radiation therapy treatment for nonsmall cell lung cancer, is generated for each fraction of treatment using principal component analysis in a way that preserves temporal anatomical trends (e.g., tumor regression). Daily synthetic images and contours are used to simulate four different treatment scenarios: (1) a “no-adapt” scenario that simulates delivery of an initial plan throughout treatment, (2) a “midadapt” scenario that implements a single replan for fraction 18, (3) a “weekly adapt” scenario that simulates weekly adaptations, and (4) a “full-adapt” scenario that simulates daily replanning. An initial intensity modulated radiation therapy plan is created for each patient and replanning is carried out in an automated fashion by reoptimizing beam apertures and weights. Dose is calculated on each image and accumulated to the first in the series using deformable mappings utilized in synthetic image creation for comparison between simulated treatments. Results: Target coverage was maintained and cord tolerance was not exceeded for any of the adaptive simulations. Average reductions in mean lung dose (MLD) and volume of lung receiving 20 Gy or more (V20{sub lung}) were 65 ± 49 cGy (p = 0.000 01) and 1.1% ± 1.2% (p = 0.0006), respectively, for all patients. The largest reduction in MLD for a single patient was 162 cGy, which allowed an isotoxic escalation of the target dose of 1668 cGy. Average reductions in cord max dose, mean esophageal dose (MED), dose received by 66% of the heart (D66{sub heart}), and dose received by 33% of the heart (D33{sub heart}), were 158 ± 280, 117 ± 121, 37 ± 77, and 99 ± 120 cGy, respectively. Average incremental reductions in MLD for the midadapt, weekly adapt, and full-adapt treatments were 38, 18, and 8 cGy, respectively. Incremental reductions in MED for the same treatments were 57, 37, and 23 cGy. Reductions in MLD and MED for the full-adapt treatment were correlated with the absolute decrease in the planning target volume (r = 0.34 and r = 0.26). Conclusions: Adaptive radiation therapy for lung cancer yields clinically relevant reductions in normal tissue doses for frequencies of adaptation ranging from a single replan up to daily replanning. Increased frequencies of adaptation result in additional benefit while magnitude of benefit decreases.

60 APPLIED LIFE SCIENCES↗

waveSZ: A Hardware-Algorithm Co-Design of Efficient Lossy Compression for Scientific Data

Error-bounded lossy compression is critical to the success of extreme-scale scientific research because of ever-increasing volumes of data produced by today's high-performance computing (HPC) applications. Not only can error-controlled lossy compressors significantly reduce the I/O and storage burden but they can retain high data fidelity for post analysis. Existing state-of-the-art lossy compressors, however, generally suffer from relatively low compression and decompression throughput (up to hundreds of megabytes per second on a single CPU core), which considerably restrict the adoption of lossy compression by many HPC applications especially those with a fairly high data production rate. In this paper, we propose a highly efficient lossy compression approach based on field programmable gate arrays (FPGAs) under the state-of-the-art lossy compression model SZ. Our contributions are fourfold. (1) We adopt a wavefront memory layout to alleviate the data dependency during the prediction for higher-dimensional predictors, such as the Lorenzo predictor. (2) We propose a co-design framework named WAVESZ based on the wavefront memory layout and the characteristics of SZ algorithm and carefully implement it by using high-level synthesis. (3) We propose a hardware-algorithm co-optimization method to improve the performance. (4) We evaluate our proposed WAVESZ on three real-world HPC simulation datasets from the Scientific Data Reduction Benchmarks and compare it with other state-of-the-art methods on both CPUs and FPGAs. Experiments show that our WAVESZ can improve SZ's compression throughput by 6.9x similar to 8.7x over the production version running on a state-of-the-art CPU and improve the compression ratio and throughput by 2.1x and 5.8x on average, respectively, respectively, compared with the state-of-the-art FPGA design.

Compression Ratio↗

X-Ray Microanalysis and Electron Energy Loss Spectrometry in the Analytical Electron Microscope: Review and Future Directions

This paper reviews and discusses future directions in analytical electron microscopy for microchemical analysis using X-ray and Electron Energy Loss Spectroscopy (EELS). The technique of X-ray microanalysis, using the ratio method and k(sub AB) factors, is outlined. The X-ray absorption correction is the major barrier to the objective of obtaining I% accuracy and precision in analysis. Spatial resolution and Minimum Detectability Limits (MDL) are considered with present limitations of spatial resolution in the 2 to 3 microns range and of MDL in the 0.1 to 0.2 wt. % range when a Field Emission Gun (FEG) system is used. Future directions of X-ray analysis include improvement in X-ray spatial resolution to the I to 2 microns range and MDL as low as 0.01 wt. %. With these improvements the detection of single atoms in the analysis volume will be possible. Other future improvements include the use of clean room techniques for thin specimen preparation, quantification available at the I% accuracy and precision level with light element analysis quantification available at better than the 10% accuracy and precision level, the incorporation of a compact wavelength dispersive spectrometer to improve X-ray spectral resolution, light element analysis and MDL, and instrument improvements including source stability, on-line probe current measurements, stage stability, and computerized stage control. The paper reviews the EELS technique, recognizing that it has been slow to develop and still remains firmly in research laboratories rather than in applications laboratories. Consideration of microanalysis with core-loss edges is given along with a discussion of the limitations such as specimen thickness. Spatial resolution and MDL are considered, recognizing that single atom detection is already possible. Plasmon loss analysis is discussed as well as fine structure analysis. New techniques for energy-loss imaging are also summarized. Future directions in the EELS technique will be the development of new spectrometers and improvements in thin specimen preparation. The microanalysis technique needs to be simplified and software developed so that the EELS technique approaches the relative simplicity of the X-ray technique. Finally, one can expect major improvements in EELS imaging as data storage and processing improvements occur.

Goldstein, J. I.↗

SEM petrography of dispersed organic matter in black shales: A review

Organic matter (OM)-hosted pores are important constituents of the pore system of black shales and play a crucial role in determining their methane adsorption capacity and porosity. OM-hosted pores are generally observed and described with scanning electron microscope (SEM) on Ar ion-milled surfaces. However, SEM imaging is not able to reliably distinguish OM types and relate the observed pores to specific macerals. Partly because of this inability to relate organic pores to macerals, the evolution of organic porosity during thermal maturation remains poorly understood. In this paper, we review the petrographic characteristics of dispersed organic matter (DOM) in black shales under the SEM. Organic petrographic classification of DOM developed for reflected-light microscopy is so far the most practical method when describing DOM in black shales under the SEM because this classification has information on the origin of DOM. Therefore, correlative microscopy (combination of reflected-light and electron microscopy) is the most effective method to identify both OM types and OM-hosted pores. This method, however, is not readily available to most researchers. Although identifying OM on the basis of SEM observations is a challenging task, it is achievable provided there is a good understanding of the studied shales, especially their thermal maturity and original OM composition. Therefore, the overall objective of this paper is to review petrographic characteristics of DOM in black shales under the SEM to provide some guidelines for identifying DOM from SEM observations. We also review factors that control the formation and preservation of OM-hosted pores. OM-hosted pores consist of primary and secondary organic pores. Primary organic pores are pores inherited from the biological structure of the original OM. Secondary organic pores develop during hydrocarbon generation and expulsion from oil-prone OM and are hosted by solid bitumen or pyrobitumen. The development of secondary organic pores is controlled by thermal maturity and OM type, and their preservation is subject to thermal maturity, OM content, and mineralogical composition. The presented view of the evolution of micropore and mesopore characteristics of OM with thermal maturity is based on data from the literature. The specific surface area and pore volume of OM in black shales follow a parabolic pattern with increasing thermal maturity (quantified via vitrinite reflectance, R o ). The initial increase reflects development of OM-hosted pores, and the subsequent decrease is due to denser stacking of aromatic units in the macromolecular structure of OM, with maximum values (specific surface area ~ 300 m 2 /g and pore volume ~ 0.3 cm 3 /g) reached at R o values in the 2.5-3.5% range. The contribution of OM-hosted pores to the pore characteristics of black shales depends on OM content, OM type, and thermal maturity.

03 NATURAL GAS↗

MRI-Guided High–Dose-Rate Intracavitary Brachytherapy for Treatment of Cervical Cancer: The University of Pittsburgh Experience

Purpose: Image-based brachytherapy is increasingly used for gynecologic malignancies. We report early outcomes of magnetic resonance imaging (MRI)-guided brachytherapy. Methods and Materials: Consecutive patient cases with FIGO stage IB1 to IVA cervical cancer treated at a single institution were retrospectively reviewed. All patients received concurrent cisplatin with external beam radiation therapy along with interdigitated high–dose-rate intracavitary brachytherapy. Computed tomography or MRI was completed after each application, the latter acquired for at least 1 fraction. High-risk clinical target volume (HRCTV) and organs at risk were identified by Groupe Européen de Curiethérapie and European SocieTy for Radiotherapy and Oncology guidelines. Doses were converted to equivalent 2-Gy doses (EQD{sub 2}) with planned HRCTV doses of 75 to 85 Gy. Results: From 2007 to 2013, 128 patients, median 52 years of age, were treated. Predominant characteristics included stage IIB disease (58.6%) with a median tumor size of 5 cm, squamous histology (82.8%), and no radiographic nodal involvement (53.1%). Most patients (67.2%) received intensity modulated radiation therapy (IMRT) at a median dose of 45 Gy, followed by a median brachytherapy dose of 27.5 Gy (range, 25-30 Gy) in 5 fractions. At a median follow up of 24.4 months (range, 2.1-77.2 months), estimated 2-year local control, disease-free survival, and cancer-specific survival rates were 91.6%, 81.8%, and 87.6%, respectively. Predictors of local failure included adenocarcinoma histology (P<.01) and clinical response at 3 months (P<.01). Among the adenocarcinoma subset, receiving HRCTV D{sub 90} EQD{sub 2} ≥84 Gy was associated with improved local control (2-year local control rate 100% vs 54.5%, P=.03). Grade 3 or greater gastrointestinal or genitourinary late toxicity occurred at a 2-year actuarial rate of 0.9%. Conclusions: This study constitutes one of the largest reported series of MRI-guided brachytherapy in North America, demonstrating excellent local control with acceptable morbidity. Dose escalation may be warranted when feasible for adenocarcinomas to offset the risk of local failure.

62 RADIOLOGY AND NUCLEAR MEDICINE↗

Supranutrition of microalgal docosahexaenoic acid and calcidiol improved growth performance, tissue lipid profiles, and tibia characteristics of broiler chickens

Abstract Background Docosahexaenoic acid (DHA) and calcidiol could be enriched in chicken for improving public nutrition and health. It remains unclear if supranutritional levels of DHA and calcidiol impair growth performance or metabolism of broiler chickens. This study was to determine singular and combined effects of high levels of supplemental DHA-rich microalgal biomass or oil and calcidiol on growth performance, concentrations of triglycerides, cholesterol, and nonesterfied fatty acids in plasma, liver, breast, and thigh, and biophysical properties of tibia. Methods In Exp. 1, 144 day-old Cornish chicks were divided into 4 groups (6 cages/treatment, 6 birds/cage), and were fed a corn-soybean meal basal diet (BD), BD + 10,000 IU calcidiol/kg (BD + Cal), BD + 1% DHA-rich Aurantiochytrium (1.2 g DHA/kg; BD + DHA), and BD + Cal + DHA for 6 weeks. In Exp. 2, 180 day-old chicks were divided into 5 groups, and were fed: BD, BD + DHA (0.33% to 0.66% oil, 1.5 to 3.0 g DHA/kg), BD + DHA + EPA (1.9% to 3.8% eicosapentaenoic acid-rich Nannochloropsis sp. CO18, 0.3 to 0.6 g EPA/kg), BD + DHA + calcidiol (6000 to 12,000 IU/kg diet), and BD + DHA + EPA + Cal for 6 weeks. Results Birds fed BD + Cal diet in Exp. 1 and BD + DHA + EPA diet in Exp. 2 had higher ( P < 0.05) body weight gain (10%–11%) and gain:feed ratio (7%), and lower ( P < 0.05) total cholesterol and triglyceride concentrations in plasma (18%–54%), liver (8%–26%), breast (19%–26%), and thigh (10%–19%), respectively, over the controls. The two diets also improved ( P < 0.05) tibial breaking strength (8%–24%), total bone volume (2%–13%), and (or) bone mineral density (3%–19%) of chickens. Conclusion Supranutrition of dietary calcidiol and DHA alone or together did not produce adverse effects, but led to moderate improvements of growth performance, lipid profiles of plasma and muscle, and bone properties of broiler chickens.

60 APPLIED LIFE SCIENCES↗

Analysis of Gas Membrane Ultra-High Purification of Small Quantities of Mono-Isotopic Silane

A small quantity of high-value, crude, mono-isotopic silane is a prospective gas for a small-scale, high-recovery, ultra-high membrane purification process. This is an unusual application of gas membrane separation for which we provide a comprehensive analysis of a simple purification model. The goal is to develop direct analytic expressions for estimating the feasibility and efficiency of the method, and guide process design; this is only possible for binary mixtures of silane in the dilute limit which is a somewhat realistic case. Among the common impurities in crude silane, methane poses a special membrane separation challenge since it is chemically similar to silane. Other potential problematic surprises are: ethylene, diborane and ethane (in this order). Nevertheless, we demonstrate, theoretically, that a carefully designed membrane system may be able to purify mono-isotopic, crude silane to electronics-grade level in a reasonable amount of time and expenses. We advocate a combination of membrane materials that preferentially reject heavy impurities based on mobility selectivity, and light impurities based on solubility selectivity. We provide estimates for the purification of significant contaminants of interest. To improve the separation selectivity, it is advantageous to use a permeate chamber under vacuum, however this also requires greater control of in-leakage of impurities in the system. In this study, we suggest cellulose acetate and polydimethylsiloxane as examples of membrane materials on the basis of limited permeability data found in the open literature. We provide estimates on the membrane area needed and priming volume of the cell enclosure for fabrication purposes when using the suggested membrane materials. These estimates are largely theoretical in view of the absence of reliable experimental data for the permeability of silane. Last but not least, future extension of this work to the non-dilute limit may apply to the recovery of silane from rejected streams of natural silicon semi-conductor processes.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

From tides to seasons: How cyclic tidal drivers and plant physiology interact to affect carbon cycling at the terrestrial-estuarine boundary (Final technical report)

Coastal ecosystems are among the most biologically and biogeochemically active and diverse systems on Earth. Because they act as important linkages between terrestrial ecosystems and the open ocean, their incorporation in Earth system models (ESMs) is critical to predict coastal and global responses to environmental changes. However, they vary greatly in the magnitude of tides and the volume and timing of freshwater input from land, making it challenging to model the major biogeochemical reactions that control productivity and greenhouse gas emissions across coastal terrestrial aquatic interfaces (TAIs). Our overall objective was to improve mechanistic process understanding and modeling of tidal wetland hydro-biogeochemistry in coastal TAIs. We established a new flux tower site (Ameriflux US-PLo) in the oligohaline part of the Parker River to continuously monitor ecosystem-scale carbon fluxes under temporally varying salinity conditions. The site is co-located with long-term monitoring plots of the Plum Island Ecosystems LTER project. We installed wells and redox sensors in the marsh interior and creek bank, established biomass monitoring plots and deployed novel optode sensors in both locations. We used this data to parameterize plant-mediated transport in PFLOTRAN and tested the impact of soil heterogeneity on porewater constituents and gas fluxes. We collected observations of root oxygen release with a novel planar optode system in the field. Flux data collected during the measurement period encompasses a large variation in salinity ranging from drought to record precipitation years. We developed a method to extract functional relationships from the flux data using artificial neural networks, identifying salinity thresholds for CH 4 fluxes. Finally, we are using the coupled ELM-PFLOTRAN model to test the impact of antecedent hydrological conditions on the salinity-CH 4 flux relationship. This grant contributed to the professional development of one postdoc, three research assistants and one graduate student. The sensor data has been shared with external collaborators.

54 ENVIRONMENTAL SCIENCES↗

Comparison of wind-farm control strategies under realistic offshore wind conditions: wake quantities of interest

Wind-farm control strategies aim to increase the efficiency, and therefore lower the levelized cost of energy, of wind farms. This is done by using turbine settings such as the yaw angle, blade pitch angles, or generator torque to manipulate the wake that negatively affects downstream turbines in the farm. Two inherently different wind-farm control methods have been identified in the literature: wake steering (WS) and active wake mixing (AWM). As one of two companion papers focused on understanding practical aspects of these two wind-farm control strategies using large-eddy simulation (LES), we below analyze the wake quantities of interest for a single wind turbine performing WS and AWM, while the companion article (Frederik et al., 2025) focuses on turbine quantities of interest including power and structural loads for the same computational setup and also includes two-turbine arrays with full and partial wake overlap. The simulations, which are based in the LES solver AMR-Wind, are tailored to have inflow conditions representative of measurements from a site off the East Coast of the US, including with strong veer and low turbulence. The turbine, which is modeled in OpenFAST and coupled to the LES, is the IEA 15 MW, an open-source offshore design. After presenting an overview of the wake recovery for the different wake-control cases, the analysis probes the fluid-dynamic causes for the different performance of the arrays reported in the companion article by examining control volumes around the wakes and the budget of the mean-flow kinetic energy (MKE) within these volumes. In the high veer environment considered, the MKE recovery is dominated by mean convection, and this is shown to especially benefit the WS strategy when a neighboring turbine is directly downstream: there is ≈65 % more available power for a downstream turbine than in the baseline case, and this power is gained primarily through mean convection on the left-tip and top-tip faces of the control volume. However, the case with imperfect knowledge of the exact wind direction favors the pulse-type AWM strategy, largely because of ≈9 % increased turbulent entrainment from aloft versus the baseline that could be related to an apparent resistance to skewing in the pulsed wake. The general reduced effectiveness of helix-type and other individual-pitch-based AWM strategies for inflow with high veer and low turbulence as reported in the companion paper is due, in part, to low magnitudes of phase-averaged turbulent entrainment. Two main findings of this study are thus that veer has a significant impact on the effectiveness of different wake-control strategies and that pulse-type AWM may be a useful strategy when the objective is power maximization in realistic, offshore flow environments with imperfect knowledge of the exact wake overlap position on the downstream turbine.

17 WIND ENERGY↗

Heat transfer computer programs

On May 7, 1964, at the invitation of Dr. Raymond L. Bisplinghoff, Associate Administrator for Advanced Research and Technology, a confer­ence was held in NASA Headquarters, on the subject "Heat Transfer Computer Programs." The conference was one of several held in Headquarters during the first half of 1964 for review and planning of NASA's research program on space vehicle thermal radiation and temperature control. The conference brought about a useful interchange of information between the NASA Centers, including JPL, on computer programs which had been developed for the thermal design of spacecraft. The conferees concluded that in addition to our annual conference in this area, a useful con­tinuing exchange of information could be through the establishment of a quarterly newsletter issued from Headquarters describing, current efforts. Such a newsletter, on heat transfer. computer programs, has been established and it is hope that the publication of the present proceedings will be a further milestone in advancing the technology of using electronic computers as a tool in the thermal design of space vehicles. The present volume includes all of the formal presentations made by staff members of the various NASA Centers. Their cooperation, which led to a highly successful conference, is greatly appreciated.

SATELLITE↗

Down-regulation of Transducin-Like Enhancer of Split protein 4 in hepatocellular carcinoma promotes cell proliferation and epithelial-Mesenchymal-Transition

Background: Transducin-Like Enhancer of Split protein 4 (TLE4) has been reported to be involved in some subsets of acute myeloid leukemia and colorectal cancer. In the present study, we aimed to explore the role of TLE4 in tumorigenesis and cancer progression in hepatocellular carcinoma (HCC). Methods: The expression pattern of TLE4 in HCC was determined by Western-blot and qRT-PCR, gain-of-function and loss-of-function was used to explore the biological role of TLE4 in HCC cells. A xenograft model was established to confirm its effects on proliferation. Results: The protein expression levels of TLE4 were significantly down-regulated in HCC tissues compared to matched adjacent normal liver tissues. In vitro, down-regulation of TLE4 in Huh7 or SMMC-7721 promoted cell proliferation and ectopical expression of TLE4 in Hep3B or Bel-7404 suppressed cell proliferation. In addition, the cell colony formation ability was enhanced after down-regulation of TLE4 expression in Huh-7 but suppressed after over-expression in Hep3B. Furthermore, down-regulation of TLE4 increased the cell invasion ability, as well as increased the expression level of Vimentin and decreased that of E-cadherin, indicating a phenotype of epithelial-mesenchymal transition (EMT) in HCC cells. On the contrary, ectopical expression of TLE4 in HCC cells decreased the cell invasion ability and inhibited EMT. In vivo, compared to control group, xenograft tumor volumes were significantly decreased in TLE4 overexpression group. Conclusions: These results demonstrated that TLE4 might play important regulatory roles in cellular proliferation and EMT process in HCC. - Highlights: • TLE4 is significantly down-regulated in HCC samples. • Down regulated of TLE4 in HCC cells promotes cell proliferation. • Down regulated of TLE4 in HCC cells promotes epithelial-to-mesenchymal transition.

60 APPLIED LIFE SCIENCES↗

Stereotactic Ablative Radiation Therapy for Subcentimeter Lung Tumors: Clinical, Dosimetric, and Image Guidance Considerations

Purpose: Use of stereotactic ablative radiation therapy (SABR) for subcentimeter lung tumors is controversial. We report our outcomes for tumors with diameter ≤1 cm and their visibility on cone beam computed tomography (CBCT) scans and retrospectively evaluate the planned dose using a deterministic dose calculation algorithm (Acuros XB [AXB]). Methods and Materials: We identified subcentimeter tumors from our institutional SABR database. Tumor size was remeasured on an artifact-free phase of the planning 4-dimensional (4D)-CT. Clinical plan doses were generated using either a pencil beam convolution or an anisotropic analytic algorithm (AAA). All AAA plans were recalculated using AXB, and differences among D95 and mean dose for internal target volume (ITV) and planning target volume (PTV) on the average intensity CT dataset, as well as for gross tumor volume (GTV) on the end respiratory phases were reported. For all AAA patients, CBCT scans acquired during each treatment fraction were evaluated for target visibility. Progression-free and overall survival rates were calculated using the Kaplan-Meier method. Results: Thirty-five patients with 37 subcentimeter tumors were eligible for analysis. For the 22 AAA plans recalculated using AXB, Mean D95 ± SD values were 2.2 ± 4.4% (ITV) and 2.5 ± 4.8% (PTV) lower using AXB; whereas mean doses were 2.9 ± 4.9% (ITV) and 3.7 ± 5.1% (PTV) lower. Calculated AXB doses were significantly lower in one patient (difference in mean ITV and PTV doses, as well as in mean ITV and PTV D95 ranged from 22%-24%). However, the end respiratory phase GTV received at least 95% of the prescription dose. Review of 92 CBCT scans from all AAA patients revealed that the tumor was visualized in 82 images, and its position could be inferred in other images. The 2-year local progression-free survival was 100%. Conclusions: Patients with subcentimeter lung tumors are good candidates for SABR, given the dosimetry, ability to localize tumors with image guidance, and excellent local control.

62 RADIOLOGY AND NUCLEAR MEDICINE↗

Proteomic Retrieval from Nucleic Acid Depleted Space-Flown Human Cells

Compared to experiments utilizing humans in microgravity, cell-based approaches to questions about subsystems of the human system afford multiple advantages, such as crew safety and the ability to achieve statistical significance. To maximize the science return from flight samples, an optimized method was developed to recover protein from samples depleted of nucleic acid. This technique allows multiple analyses on a single cellular sample and when applied to future cellular investigations could accelerate solutions to significant biomedical barriers to human space exploration. Cell cultures grown in American Fluoroseal bags were treated with an RNA stabilizing agent (RNAlater - Ambion), which enabled both RNA and immunoreactive protein analyses. RNA was purified using an RNAqueous(registered TradeMark) kit (Ambion) and the remaining RNA free supernatant was precipitated with 5% trichloroacetic acid. The precipitate was dissolved in SDS running buffer and tested for protein content using a bicinchoninic acid assay (1) (Sigma). Equal loads of protein were placed on SDS-PAGE gels and either stained with CyproOrange (Amersham) or transferred using Western Blotting techniques (2,3,4). Protein recovered from RNAlater-treated cells and stained with protein stain, was measured using Imagequant volume measurements for rectangles of equal size. BSA treated in this way gave quantitative data over the protein range used (Fig 1). Human renal cortical epithelial (HRCE) cells (5,6,7) grown onboard the International Space Station (ISS) during Increment 3 and in ground control cultures exhibited similar immunoreactivity profiles for antibodies to the Vitamin D receptor (VDR) (Fig 2), the beta isoform of protein kinase C (PKC ) (Fig 3), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Fig 4). Parallel immunohistochemical studies on formalin-fixed flight and ground control cultures also showed positive immunostaining for VDR and other biomarkers (Fig 5). These results are consistent with data from additional antigenic recovery experiments performed on human Mullerian tumor cells cultured in microgravity (8).

Hammond, D. K.↗

SU-F-P-64: The Impact of Plan Complexity Parameters On the Plan Quality and Deliverability of Volumetric Modulated Arc Therapy with Canonical Correlation Analysis

Purpose: To evaluate the impact of complexity indices on the plan quality and deliverability of volumetric modulated arc therapy (VMAT), and to determine the most significant parameters in the generation of an ideal VMAT plan. Methods: A multi-dimensional exploratory statistical method, canonical correlation analysis (CCA) was adopted to study the correlations between VMAT parameters of complexity, quality and deliverability, as well as their contribution weights with 32 two-arc VMAT nasopharyngeal cancer (NPC) patients and 31 one-arc VMAT prostate cancer patients. Results: The MU per arc (MU/Arc) and MU per control point (MU/CP) of NPC were 337.8±25.2 and 3.7±0.3, respectively, which were significantly lower than those of prostate cancer patients (MU/Arc : 506.9±95.4, MU/CP : 5.6±1.1). The plan complexity indices indicated that two-arc VMAT plans were more complex than one-arc VMAT plans. Plan quality comparison confirmed that one-arc VMAT plans had a high quality than two-arc VMAT plans. CCA results implied that plan complexity parameters were highly correlated with plan quality with the first two canonical correlations of 0.96, 0.88 (both p<0.001) and significantly correlated with deliverability with the first canonical correlation of 0.79 (p<0.001), plan quality and deliverability was also correlated with the first canonical correlation of 0.71 (p=0.02). Complexity parameters of MU/CP, segment area (SA) per CP, percent of MU/CP less 3 and planning target volume (PTV) were weighted heavily in correlation with plan quality and deliveability . Similar results obtained from individual NPC and prostate CCA analysis. Conclusion: Relationship between complexity, quality, and deliverability parameters were investigated with CCA. MU, SA related parameters and PTV volume were found to have strong effect on the plan quality and deliverability. The presented correlation among different quantified parameters could be used to improve the plan quality and the efficiency of the radiotherapy process when creating a complex VMAT plan.

60 APPLIED LIFE SCIENCES↗

Arc-Fault Primer: Numerical, Analytical, and Experimental Characteristics of Initiation and Sustainment of Arc Plasmas (DRAFT)

While arc-faults are rare in electrical installations, many documented events have led to fires that resulted in significant damage to energy-generation, commercial and residential systems, as well as surrounding structures, in both the United States and abroad. Arc-plasma discharges arise over time due to a variety of reliability issues related to cable material degradation, electrical and mechanical stresses or acute conductive wiring dislocations. These may lead to discontinuity between energized conductors, facilitating arcing events and fires. Arc-flash events rapidly release significant energy in a localized volume, where the electric arc experiences a reduction in resistance. This facilitates a reduction in electrical resistance as the arc temperature and pressure can increase rapidly. Strong pressure waves, electromagnetic interference (EMI), and intense light from an arc pose a threat to electrical worker safety and system equipment. This arc-fault primer provides basic fundamental insight into arc-fault plasma discharges, and an overview of direct current (DC) and alternating current (AC) arc-fault phenomena. This primer also covers pressure waves and EMI arc-fault hazard analyses related to incident energy prediction and potential damage analysis. Mitigation strategies are also discussed related to engineering design and employment of protective devices including arc-fault circuit interrupters (AFCIs). Best practices related to worker safety are also covered, especially as they pertain to electrical codes and standards, particularly Institute of Electrical and Electronics Engineers (IEEE) 1584 and National Fire Protection Agency (NFPA) 70E. Throughout the primer various modelling and test capabilities at Sandia National Laboratories are also covered, especially as they relate to novel methods of arc-fault/arc-flash characterization and mitigation approaches. Herein, this work describes methods for producing and characterizing controlled, sustained arcs at atmospheric pressures as well as methods for mitigation with novel materials.

42 ENGINEERING↗

Evolving Design Criteria for Very Large Aperture Space-Based Telescopes and Their Influence on the Need for Integrated Tools in the Optimization Process

NASA's Advanced Mirror Technology Development (AMTD) program has been developing the means to design and build the future generations of space based telescopes. With the nearing completion of the James Webb Space Telescope (JWST), the astrophysics community is already starting to define the requirements for follow on observatories. The restrictions of available launch vehicles and the possibilities of planned future vehicles have fueled the competition between monolithic primaries (with better optical quality) and segmented primaries (with larger apertures, but with diffraction, costs and figure control issues). Regardless of the current shroud sizes and lift capacities, these competing architectures share the need for rapid design tools. As part of the AMTD program a number of tools have been developed and tested to speed up the design process. Starting with the Arnold Mirror Modeler (which creates Finite Element Models (FEM) for structural analysis) and now also feeds these models into thermal stability analyses. They share common file formats and interchangeable results. During the development of the program, numerous trade studies were created for 4 meter and 8 meter monolithic primaries, complete with support systems. Evaluation of these results has led to a better understanding of how the specification drives the results. This paper will show some of the early trade studies for typical specification requirements such as lowest mirror bending frequency and suspension system lowest frequency. The results use representative allowable stress values for each mirror substrate material and construction method and generic material properties. These studies lead to some interesting relationships between feasible designs and the realities of actually trying to build these mirrors. Much of the traditional specifications were developed for much smaller systems, where the mass and volume of the primary where a small portion of the overall satellite. JWST shows us that as the aperture grows, the primary takes up the majority of the mass and volume and the established rules need to be adjusted. For example, a small change in lowest frequency requirement can change the cost by millions of dollars.

Space-based telescopes↗