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At least 1,369 records · Page 76

Microbial identification by immunohybridization assay of artificial RNA labels

Ribosomal RNA (rRNA) and engineered stable artificial RNAs (aRNAs) are frequently used to monitor bacteria in complex ecosystems. In this work, we describe a solid-phase immunocapture hybridization assay that can be used with low molecular weight RNA targets. A biotinylated DNA probe is efficiently hybridized in solution with the target RNA, and the DNA-RNA hybrids are captured on streptavidin-coated plates and quantified using a DNA-RNA heteroduplex-specific antibody conjugated to alkaline phosphatase. The assay was shown to be specific for both 5S rRNA and low molecular weight (LMW) artificial RNAs and highly sensitive, allowing detection of as little as 5.2 ng (0.15 pmol) in the case of 5S rRNA. Target RNAs were readily detected even in the presence of excess nontarget RNA. Detection using DNA probes as small as 17 bases targeting a repetitive artificial RNA sequence in an engineered RNA was more efficient than the detection of a unique sequence.

Non-NASA Center↗

Normal human mammary epithelial cells spontaneously escape senescence and acquire genomic changes

Senescence and genomic integrity are thought to be important barriers in the development of malignant lesions. Human fibroblasts undergo a limited number of cell divisions before entering an irreversible arrest, called senescence. Here we show that human mammary epithelial cells (HMECs) do not conform to this paradigm of senescence. In contrast to fibroblasts, HMECs exhibit an initial growth phase that is followed by a transient growth plateau (termed selection or M0; refs 3-5), from which proliferative cells emerge to undergo further population doublings (approximately 20-70), before entering a second growth plateau (previously termed senescence or M1; refs 4-6). We find that the first growth plateau exhibits characteristics of senescence but is not an insurmountable barrier to further growth. HMECs emerge from senescence, exhibit eroding telomeric sequences and ultimately enter telomere-based crisis to generate the types of chromosomal abnormalities seen in the earliest lesions of breast cancer. Growth past senescent barriers may be a pivotal event in the earliest steps of carcinogenesis, providing many genetic changes that predicate oncogenic evolution. The differences between epithelial cells and fibroblasts provide new insights into the mechanistic basis of neoplastic transformation.

NASA Discipline Radiation Health↗

Promoter-dependent and -independent activation of insulin-like growth factor binding protein-5 gene expression by prostaglandin E2 in primary rat osteoblasts

Insulin-like growth factor (IGF) action is mediated by high affinity cell surface IGF receptors and modulated by a family of secreted IGF binding proteins (IGFBPs). IGFBP-5, the most conserved of six IGFBPs characterized to date, uniquely potentiates the anabolic actions of IGF-I for skeletal cells. In osteoblasts, IGFBP-5 production is stimulated by prostaglandin E2 (PGE2), a local factor that mediates certain effects induced by parathyroid hormone, cytokines such as interleukin-1 and transforming growth factor-beta, and mechanical strain. In this study, we show that transcriptional and post-transcriptional events initiated by PGE2 collaborate to enhance IGFBP-5 gene expression in primary fetal rat osteoblast cultures. PGE2 treatment stimulated up to a 7-fold rise in steady-state levels of IGFBP-5 mRNA throughout 32 h of incubation. Analysis of nascent IGFBP-5 mRNA suggested that PGE2 had only a modest stimulatory effect on IGFBP-5 gene transcription, and transient transfection studies with IGFBP-5 promoter-reporter genes confirmed that PGE2 enhanced promoter activity by approximately 2-fold. Similar stimulatory effects were seen with forskolin. A DNA fragment with only 51 base pairs of the 5'-flanking sequence retained hormonal responsiveness, which may be mediated by a binding site for transcription factor AP-2 located at positions -44 to -36 in the proximal IGFBP-5 promoter. Incubation of osteoblasts with the mRNA transcriptional inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole demonstrated that PGE2 enhanced IGFBP-5 mRNA stability by 2-fold, increasing the t1/2 from 9 to 18 h. The effects of PGE2 on steady-state IGFBP-5 transcripts were abrogated by preincubating cells with cycloheximide, indicating that the effects of PGE2 on both gene transcription and mRNA stability required ongoing protein synthesis. Therefore, both promoter-dependent and -independent pathways converge to enhance IGFBP-5 gene expression in response to PGE2 in osteoblasts.

Non-NASA Center↗

A Subsurface Soil Composition and Physical Properties Experiment to Address Mars Regolith Stratigraphy

Previous in-situ measurements of soil-like materials on the surface of Mars, in particular during the on-going Mars Exploration Rover missions, have shown complex relationships between composition, exposure to the surface environment, texture, and local rocks. In particular, a diversity in both compositional and physical properties could be established that is interpreted to be diagnostic of the complex geologic history of the martian surface layer. Physical and chemical properties vary laterally and vertically, providing insight into the composition of rocks from which soils derive, and environmental conditions that led to soil formation. They are central to understanding whether habitable environments existed on Mars in the distant past. An instrument the Mole for Soil Compositional Studies and Sampling (MOCSS) - is proposed to allow repeated access to subsurface regolith on Mars to depths of up to 1.5 meters for in-situ measurements of elemental composition and of physical and thermophysical properties, as well as for subsurface sample acquisition. MOCSS is based on the compact PLUTO (PLanetary Underground TOol) Mole system developed for the Beagle 2 lander and incorporates a small X-ray fluorescence spectrometer within the Mole which is a new development. Overall MOCSS mass is approximately 1.4 kilograms. Taken together, the MOCSS science data support to decipher the geologic history at the landing site as compositional and textural stratigraphy if they exist - can be detected at a number of places if the MOCSS were accommodated on a rover such as MSL. Based on uncovered stratigraphy, the regional sequence of depositional and erosional styles can be constrained which has an impact on understanding the ancient history of the Martian near-surface layer, considering estimates of Mars soil production rates of 0.5... 1.0 meters per billion years on the one hand and Mole subsurface access capability of approximately 1.5 meters. An overview of the MOCSS, XRS instrument accomodation and the impact that these instruments have on Mars science is discussed.

Richter, L.↗

SATS HVO Concept Validation Experiment

A human-in-the-loop simulation experiment was conducted at the NASA Langley Research Center s (LaRC) Air Traffic Operations Lab (ATOL) in an effort to comprehensively validate tools and procedures intended to enable the Small Aircraft Transportation System, Higher Volume Operations (SATS HVO) concept of operations. The SATS HVO procedures were developed to increase the rate of operations at non-towered, non-radar airports in near all-weather conditions. A key element of the design is the establishment of a volume of airspace around designated airports where pilots accept responsibility for self-separation. Flights operating at these airports, are given approach sequencing information computed by a ground based automated system. The SATS HVO validation experiment was conducted in the ATOL during the spring of 2004 in order to determine if a pilot can safely and proficiently fly an airplane while performing SATS HVO procedures. Comparative measures of flight path error, perceived workload and situation awareness were obtained for two types of scenarios. Baseline scenarios were representative of today s system utilizing procedure separation, where air traffic control grants one approach or departure clearance at a time. SATS HVO scenarios represented approaches and departure procedures as described in the SATS HVO concept of operations. Results from the experiment indicate that low time pilots were able to fly SATS HVO procedures and maintain self-separation as safely and proficiently as flying today's procedures.

Consiglio, Maria↗

Nova V2362 Cygni (Nova Cygni 2006): Spitzer, Swift, and Ground-Based Spectral Evolution

Nova V2362 Cygni has undergone a number of very unusual changes. Ground-based spectroscopy initially revealed a normal sequence of events: the object faded and its near-infrared emission lines gradually shifted to higher excitation conditions until about day 100 when the optical fading reversed and the object slowly brightened. This was accompanied by a rise in the Swift X-ray telescope flux and a sudden shift in excitation of the visible and IR spectrum back to low levels. The new lower excitation spectrum revealed broad line widths and many P-Cygni profiles, all indicative of the ejection of a second shell. Eventually, dust formed, the X-ray brightness -- apparently unaffected by dust formation -- peaked and then declined, and the object faded at all wavelengths. The Spitzer dust spectra revealed a number of solid-state emission features that, at this time, are not identified.

Lynch, David K.↗

Enhancing Cassini Operations & Science Planning Tools

The Cassini team uses a variety of software utilities as they manage and coordinate their mission to Saturn. Most of these tools have been unchanged for many years, and although stability is a virtue for long-lived space missions, there are some less-fragile tools that could greatly benefit from modern improvements. This report shall describe three such upgrades, including their architectural differences and their overall impact. Emphasis is placed on the motivation and rationale behind architectural choices rather than the final product, so as to illuminate the lessons learned and discoveries made.These three enhancements included developing a strategy for migrating Science Planning utilities to a new execution model, rewriting the team's internal portal for ease of use and maintenance, and developing a web-based agenda application for tracking the sequence of files being transmitted to the Cassini spacecraft. Of this set, the first two have been fully completed, while the agenda application is currently in the early prototype stage.

science planning↗

Numerical classification of coding sequences

DNA sequences coding for protein may be represented by counts of nucleotides or codons. A complete reading frame may be abbreviated by its base count, e.g. A76C158G121T74, or with the corresponding codon table, e.g. (AAA)0(AAC)1(AAG)9 ... (TTT)0. We propose that these numerical designations be used to augment current methods of sequence annotation. Because base counts and codon tables do not require revision as knowledge of function evolves, they are well-suited to act as cross-references, for example to identify redundant GenBank entries. These descriptors may be compared, in place of DNA sequences, to extract homologous genes from large databases. This approach permits rapid searching with good selectivity.

Non-NASA Center↗

FOAM (Functional Ontology Assignments for Metagenomes): A Hidden Markov Model (HMM) database with environmental focus

A new functional gene database, FOAM (Functional Ontology Assignments for Metagenomes), was developed to screen environmental metagenomic sequence datasets. FOAM provides a new functional ontology dedicated to classify gene functions relevant to environmental microorganisms based on Hidden Markov Models (HMMs). Sets of aligned protein sequences (i.e. ‘profiles’) were tailored to a large group of target KEGG Orthologs (KOs) from which HMMs were trained. The alignments were checked and curated to make them specific to the targeted KO. Within this process, sequence profiles were enriched with the most abundant sequences available to maximize the yield of accurate classifier models. An associated functional ontology was built to describe the functional groups and hierarchy. FOAM allows the user to select the target search space before HMM-based comparison steps and to easily organize the results into different functional categories and subcategories. FOAM is publicly available at http://portal.nersc.gov/project/m1317/FOAM/.

54 ENVIRONMENTAL SCIENCES↗

ROTATION PERIODS OF 34,030 KEPLER MAIN-SEQUENCE STARS: THE FULL AUTOCORRELATION SAMPLE

We analyzed three years of data from the Kepler space mission to derive rotation periods of main-sequence stars below 6500 K. Our automated autocorrelation-based method detected rotation periods between 0.2 and 70 days for 34,030 (25.6%) of the 133,030 main-sequence Kepler targets (excluding known eclipsing binaries and Kepler Objects of Interest), making this the largest sample of stellar rotation periods to date. In this paper we consider the detailed features of the now well-populated period-temperature distribution and demonstrate that the period bimodality, first seen by McQuillan et al. in the M-dwarf sample, persists to higher masses, becoming less visible above 0.6 M {sub ☉}. We show that these results are globally consistent with the existing ground-based rotation-period data and find that the upper envelope of the period distribution is broadly consistent with a gyrochronological age of 4.5 Gyr, based on the isochrones of Barnes, Mamajek, and Hillenbrand and Meibom et al. We also performed a detailed comparison of our results to those of Reinhold et al. and Nielsen et al., who measured rotation periods of field stars observed by Kepler. We examined the amplitude of periodic variability for the stars with detection rotation periods, and found a typical range between ∼950 ppm (5th percentile) and ∼22,700 ppm (95th percentile), with a median of ∼5600 ppm. We found typically higher amplitudes for shorter periods and lower effective temperatures, with an excess of low-amplitude stars above ∼5400 K.

79 ASTRONOMY AND ASTROPHYSICS↗

LINEBACkER: Bio-inspired Data Reduction Toward Real Time Network Traffic Analysis

Abstract—One essential component of resilient cyber applications is the ability to detect adversaries and protect systems with the same flexibility adversaries will use to achieve their goals. Current detection techniques do not enable this degree of flexibility because most existing applications are built using exact or regular-expression matching to libraries of rule sets. Further, network traffic defies traditional cyber security approaches that focus on limiting access based on the use of passwords and examination of lists of installed or downloaded programs. These approaches do not readily apply to network traffic occurring beyond the access control point, and when the data in question are combined control and payload data of ever increasing speed and volume. Manual analysis of network traffic is not normally possible because of the magnitude of the data that is being exchanged and the length of time that this analysis takes. At the same time, using an exact matching scheme to identify malicious traffic in real time often fails because the lists against which such searches must operate grow too large. In this work, we introduce an alternative method for cyber network detection based on similarity-measuring algorithms for gene sequence analysis. These methods are ideal because they were designed to identify similar but nonidentical sequences. We demonstrate that our method is generally applicable to the problem of network traffic analysis by illustrating its use in two different areas both based on different attributes of network traffic. Our approach provides a logical framework for organizing large collections of network data, prioritizing traffic of interest to human analysts, and makes it possible to discover traffic signatures without the bias introduced by expert-directed signature generation. Pattern recognition on reduced representations of network traffic offers a fast, efficient, and more robust way to detect anomalies.

Teuton, Jeremy R.↗

Energetics of the lattice: packing elements in crystals of four-stranded intercalated cytosine-rich DNA molecules

Condensation of single molecules from solution into crystals represents a transition between distinct energetic states. In solution, the atomic interactions within the molecule dominate. In the crystalline state, however, a set of additional interactions are formed between molecules in close contact in the lattice--these are the packing interactions. The crystal structures of d(CCCT), d(TAACCC), d(CCCAAT), and d(AACCCC) have in common a four-stranded intercalated cytosine segment, built by stacked layers of cytosine.cytosine+ (C.C+) base pairs coming from two parallel duplexes that intercalate into each other with opposite polarity. The intercalated cytosine segments in these structures are similar in their geometry, even though the sequences crystallized in different space groups. In the crystals, adenine and thymine residues of the sequences are used to build the three-dimensional crystal lattice by elaborately interacting with symmetry-related molecules. The packing elements observed provide novel insight about the copious ways in which nucleic acid molecules can interact with each other--for example, when folded in more complicated higher order structures, such as mRNA and chromatin.

Non-NASA Center↗

Real-Time Culture-Independent Microbial Profiling Onboard the International Space Station using Nanopore Sequencing

For the past two decades, microbial monitoring of the International Space Station (ISS) has relied on culture-dependent methods that require return to Earth for analysis. This has a number of limitations, with the most significant being bias towards the detection of culturable organisms and the inherent delay between sample collection and ground-based analysis. In recent years, portable and easy-to-use molecular-based tools, such as Oxford Nanopore Technologies’ MinION™ sequencer and miniPCR bio’s miniPCR™ thermal cycler, have been validated onboard the ISS. Here, we report on the development, validation, and implementation of a swab-to-sequencer method that provides a culture-independent solution to real-time microbial profiling onboard the ISS. Method development focused on analysis of swabs collected in a low-biomass environment with limited facility resources and stringent controls on allowed processes and reagents. ISS-optimized procedures included enzymatic DNA extraction from a swab tip, bead-based purifications, altered buffers, and the use of miniPCR and the MinION. Validation was conducted through extensive ground-based assessments comparing current standard culture-dependent and newly developed culture-independent methods. Similar microbial distributions were observed between the two methods; however, as expected, the culture-independent data revealed microbial profiles with greater diversity. Protocol optimization and verification was established during NASA Extreme Environment Mission Operations (NEEMO) analog missions 21 and 22, respectively. Unique microbial profiles obtained from analog testing validated the swab-to-sequencer method in an extreme environment. Finally, four independent swab-to-sequencer experiments were conducted onboard the ISS by two crewmembers. Microorganisms identified from ISS swabs were consistent with historical culture-based data, and primarily consisted of commonly observed human-associated microbes. This simplified method has been streamlined for high ease-of-use for a non-trained crew to complete in an extreme environment, thereby enabling environmental and human health diagnostics in real-time as future missions take us beyond low-Earth orbit.

microbiology↗

Laboratory characterization of silicon avalanche photodiodes (APD) for pulse position modulation (PPM) detection

Two commercially available large area silicon avalanche photodiodes (APD) were characterized in the laboratory. Specifically, the response of the APD’s to a sequence of 8-bit pulse position modulated (256-PPM) laser pulses, with and without background noise, was recorded and stored for post analysis. Empirical probability distribution functions (pdf) were constructed from the signal and noise slot data and compared to pdf’s predicted by an analytical model based on Webb/Gaussian statistics. The limited pulse sequence was also used to generate bit-error rate (BER) versus signal photons per pulse values, albeit with large error bars. These BER measurements were also compared to results predicted using the Gaussian and Webb/Gaussian models for APD channel statistics. While the measurements qualitatively reflect features predicted by theory, significant quantitative deviations are displayed between the measurements and theory. The source of these discrepancies is not currently well understood, but it is surmised that inaccurate knowledge of detector parameters such as gain and noise equivalent temperature models may explain the discrepancies.

Biswas, A.↗

Evolution of I-SceI Homing Endonucleases with Increased DNA Recognition Site Specificity

Elucidating how homing endonucleases undergo changes in recognition site specificity will facilitate efforts to engineer proteins for gene therapy applications. I-SceI is a monomeric homing endonuclease that recognizes and cleaves within an 18-bp target. It tolerates limited degeneracy in its target sequence, including substitution of a C:G{sub +4} base pair for the wild-type A:T{sub +4} base pair. Libraries encoding randomized amino acids at I-SceI residue positions that contact or are proximal to A:T{sub +4} were used in conjunction with a bacterial one-hybrid system to select I-SceI derivatives that bind to recognition sites containing either the A:T{sub +4} or the C:G{sub +4} base pairs. As expected, isolates encoding wild-type residues at the randomized positions were selected using either target sequence. All I-SceI proteins isolated using the C:G{sub +4} recognition site included small side-chain substitutions at G100 and either contained (K86R/G100T, K86R/G100S and K86R/G100C) or lacked (G100A, G100T) a K86R substitution. Interestingly, the binding affinities of the selected variants for the wild-type A:T{sub +4} target are 4- to 11-fold lower than that of wild-type I-SceI, whereas those for the C:G{sub +4} target are similar. The increased specificity of the mutant proteins is also evident in binding experiments in vivo. These differences in binding affinities account for the observed -36-fold difference in target preference between the K86R/G100T and wild-type proteins in DNA cleavage assays. An X-ray crystal structure of the K86R/G100T mutant protein bound to a DNA duplex containing the C:G{sub +4} substitution suggests how sequence specificity of a homing enzyme can increase. This biochemical and structural analysis defines one pathway by which site specificity is augmented for a homing endonuclease.

59 BASIC BIOLOGICAL SCIENCES↗

Sequence polymorphism in an insect RNA virus field population: A snapshot from a single point in space and time reveals stochastic differences among and within individual hosts

Population structure of Homalodisca coagulata Virus-1 (HoCV-1) among and within field-collected insects sampled from a single point in space and time was examined. Polymorphism in complete consensus sequences among single-insect isolates was dominated by synonymous substitutions. The mutant spectrum of the C2 helicase region within each single-insect isolate was unique and dominated by nonsynonymous singletons. Bootstrapping was used to correct the within-isolate nonsynonymous:synonymous arithmetic ratio (N:S) for RT-PCR error, yielding an N:S value ~one log-unit greater than that of consensus sequences. Probability of all possible single-base substitutions for the C2 region predicted N:S values within 95% confidence limits of the corrected within-isolate N:S when the only constraint imposed was viral polymerase error bias for transitions over transversions. These results indicate that bottlenecks coupled with strong negative/purifying selection drive consensus sequences toward neutral sequence space, and that most polymorphism within single-insect isolates is composed of newly-minted mutations sampled prior to selection. -- Highlights: •Sampling protocol minimized differential selection/history among isolates. •Polymorphism among consensus sequences dominated by negative/purifying selection. •Within-isolate N:S ratio corrected for RT-PCR error by bootstrapping. •Within-isolate mutant spectrum dominated by new mutations yet to undergo selection.

60 APPLIED LIFE SCIENCES↗

PWR ENDF/B-VII cross-section libraries for ORIGEN-ARP

New pressurized water reactor (PWR) cross-section libraries were generated for use with the ORIGEN-ARP depletion sequence in the SCALE nuclear analysis code system. These libraries are based on ENDF/B-VII nuclear data and were generated using the two-dimensional depletion sequence, TRITON/NEWT, in SCALE 6.1. The libraries contain multiple burnup-dependent cross-sections for seven PWR fuel designs, with enrichments ranging from 1.5 to 6 wt% {sup 235}U. The burnup range has been extended from the 72 GWd/MTU used in previous versions of the libraries to 90 GWd/MTU. Validation of the libraries using radiochemical assay measurements and decay heat measurements for PWR spent fuel showed good agreement between calculated and experimental data. Verification against detailed TRITON simulations for the considered assembly designs showed that depletion calculations performed in ORIGEN-ARP with the pre-generated libraries provide similar results as obtained with direct TRITON depletion, while greatly reducing the computation time. (authors)

21 SPECIFIC NUCLEAR REACTORS AND ASSOCIATED PLANTS↗

Leveraging Existing Mission Tools in a Re-Usable, Component-Based Software Environment

Emerging methods in component-based software development offer significant advantages but may seem incompatible with existing mission operations applications. In this paper we relate our positive experiences integrating existing mission applications into component-based tools we are delivering to three missions. In most operations environments, a number of software applications have been integrated together to form the mission operations software. In contrast, with component-based software development chunks of related functionality and data structures, referred to as components, can be individually delivered, integrated and re-used. With the advent of powerful tools for managing component-based development, complex software systems can potentially see significant benefits in ease of integration, testability and reusability from these techniques. These benefits motivate us to ask how component-based development techniques can be relevant in a mission operations environment, where there is significant investment in software tools that are not component-based and may not be written in languages for which component-based tools even exist. Trusted and complex software tools for sequencing, validation, navigation, and other vital functions cannot simply be re-written or abandoned in order to gain the advantages offered by emerging component-based software techniques. Thus some middle ground must be found. We have faced exactly this issue, and have found several solutions. Ensemble is an open platform for development, integration, and deployment of mission operations software that we are developing. Ensemble itself is an extension of an open source, component-based software development platform called Eclipse. Due to the advantages of component-based development, we have been able to vary rapidly develop mission operations tools for three surface missions by mixing and matching from a common set of mission operation components. We have also had to determine how to integrate existing mission applications for sequence development, sequence validation, and high level activity planning, and other functions into a component-based environment. For each of these, we used a somewhat different technique based upon the structure and usage of the existing application.

Greene, Kevin↗