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At least 433 records · Page 24

Microfabricated Genomic Analysis System

Genetic sequencing and many genetic tests and assays require electrophoretic separation of DNA. In this technique, DNA fragments are separated by size as they migrate through a sieving gel under the influence of an applied electric field. In order to conduct these analyses on-orbit, it is essential to acquire the capability to efficiently perform electrophoresis in a microgravity environment. Conventional bench top electrophoresis equipment is large and cumbersome and does not lead itself to on-orbit utilization. Much of the previous research regarding on-orbit electrophoresis involved altering conventional electrophoresis equipment for bioprocessing, purification, and/or separation technology applications. A new and more efficient approach to on-orbit electrophoresis is the use of a microfabricated electrophoresis platform. These platforms are much smaller, less expensive to produce and operate, use less power, require smaller sample sizes (nanoliters), and achieve separation in a much shorter distance (a few centimeters instead of 10 s or 100 s of centimeters.) In contrast to previous applications, this platform would be utilized as an analytical tool for life science/medical research, environmental monitoring, and medical diagnoses. Identification of infectious agents as well as radiation related damage are significant to NASA s efforts to maintain, study, and monitor crew health during and in support of near-Earth and interplanetary missions. The capability to perform genetic assays on-orbit is imperative to conduct relevant and insightful biological and medical research, as well as continuing NASA s search for life elsewhere. This technology would provide an essential analytical tool for research conducted in a microgravity environment (Shuttle, ISS, long duration/interplanetary missions.) In addition, this technology could serve as a critical and invaluable component of a biosentinel system to monitor space environment genotoxic insults to include radiation.

Gonda, Steve↗

Program for Editing Spacecraft Command Sequences

Sequence Translator, Editor, and Expander Resource (STEER) is a computer program that facilitates construction of sequences and blocks of sequences (hereafter denoted generally as sequence products) for commanding a spacecraft. STEER also provides mechanisms for translating among various sequence product types and quickly expanding activities of a given sequence in chronological order for review and analysis of the sequence. To date, construction of sequence products has generally been done by use of such clumsy mechanisms as text-editor programs, translating among sequence product types has been challenging, and expanding sequences to time-ordered lists has involved arduous processes of converting sequence products to "real" sequences and running them through Class-A software (defined, loosely, as flight and ground software critical to a spacecraft mission). Also, heretofore, generating sequence products in standard formats has been troublesome because precise formatting and syntax are required. STEER alleviates these issues by providing a graphical user interface containing intuitive fields in which the user can enter the necessary information. The STEER expansion function provides a "quick and dirty" means of seeing how a sequence and sequence block would expand into a chronological list, without need to use of Class-A software.

Gladden, Roy↗

Statistical properties of DNA sequences

We review evidence supporting the idea that the DNA sequence in genes containing non-coding regions is correlated, and that the correlation is remarkably long range--indeed, nucleotides thousands of base pairs distant are correlated. We do not find such a long-range correlation in the coding regions of the gene. We resolve the problem of the "non-stationarity" feature of the sequence of base pairs by applying a new algorithm called detrended fluctuation analysis (DFA). We address the claim of Voss that there is no difference in the statistical properties of coding and non-coding regions of DNA by systematically applying the DFA algorithm, as well as standard FFT analysis, to every DNA sequence (33301 coding and 29453 non-coding) in the entire GenBank database. Finally, we describe briefly some recent work showing that the non-coding sequences have certain statistical features in common with natural and artificial languages. Specifically, we adapt to DNA the Zipf approach to analyzing linguistic texts. These statistical properties of non-coding sequences support the possibility that non-coding regions of DNA may carry biological information.

Non-NASA Center↗

Homology and the optimization of DNA sequence data

Three methods of nucleotide character analysis are discussed. Their implications for molecular sequence homology and phylogenetic analysis are compared. The criterion of inter-data set congruence, both character based and topological, are applied to two data sets to elucidate and potentially discriminate among these parsimony-based ideas. c2001 The Willi Hennig Society.

Non-NASA Center↗

Geochemical and Microbial Dynamics of Hydrogen in a Methane Storage Reservoir

Hydrogen has been identified as a flexible energy carrier with zero or negative emission across multiple energy systems, and existing natural gas infrastructure could be leveraged if hydrogen gas (H2) was blended with methane (CH4). For example, subsurface methane storage reservoirs could be slightly modified to also store hydrogen if a methane/hydrogen blend were injected. However, the compatibility of methane storage reservoirs to include H2 injection has not been fully demonstrated, and this could lead to geochemical and microbiological reactions that alter the reservoir and stored gas content. It is essential that we understand the impact of H2 gas on the biogeochemistry of subsurface storage reservoirs before deploying large-scale H2-CH4 storage, We collected produced fluid from two separate methane storage reservoirs in the Southwestern US. First, we completed a baseline analysis of the biogeochemistry through qPCR, 16S rRNA sequencing, metagenomic sequencing, and geochemical analysis. Each reservoir was found to have unique geochemical conditions and a unique microbial community structure, with Site 1 having a higher TDS and an abundance of Shewanella and Site 2 having a lower TDS and high abundance of Eubacterium and Acetobacterium. Next, we ran a series of high pressure, high temperature reactors under hydrogen storage conditions with the biological sample from one of the storage reservoirs and a 20% H2-80% CH4 gas blend for up to 7 days. Our results show a decrease of hydrogen by 5% in reactors as early as 1-3 days. Previous hydrogen storage work has linked subsurface microorganisms with methanogenesis hydrogen sulfide production, acid production, and microbial corrosion. Our results show minimal change in the fluid chemistry, with the exception of a decrease in dissolved sulfate concentrations. Taxonomic sequencing demonstrated the presence of microorganisms capable of iron redox, acid generation, and hydrogen sulfide production throughout the reactors, suggesting microbial hydrogen consumption may occur through various metabolic pathways. This work demonstrates that site-specific geochemistry and microbiology may impact the efficiency of hydrogen storage in methane storage reservoirs.

environmental microbiology↗

High yield production of 3-hydroxypropionic acid using Issatchenkia orientalis

Biomanufacturing provides a more sustainable alternative to fossil-based chemical manufacturing. 3-Hydroxypropionic acid (3HP) is a top Department of Energy value-added chemical and precursor to bioplastics, yet cost-effective microbial production remains elusive. Here, we establish the acid-tolerant yeast Issatchenkia orientalis as a robust host for low-pH 3HP biosynthesis. Genome-scale modeling identifies the β-alanine pathway as optimal, offering the highest theoretical yield and lowest oxygen requirement. Thermodynamic analysis confirms its favorability under acidic conditions. Using sequence similarity network analysis, we discover highly active aspartate 1-decarboxylase (PAND), β-alanine-pyruvate aminotransferase (BAPAT), and 3HP dehydrogenase (YDFG), which significantly improve the pathway efficiency. Next, to further elevate the production, pathway optimization through multi-copy PAND integration, byproduct elimination (knockouts of pyruvate decarboxylase and glycerol-3-phosphate dehydrogenase), and reinforcement of aspartate flux by overexpression of pyruvate carboxylase and aspartate amino transferase improves the titer to 29 g/L in shake flasks. Fed-batch fermentation at pH 4 with low-cost corn steep liquor medium further increases the production to 92 g/L with 0.7 g/g yield and 0.55 g/L/h productivity. Techno-economic analysis indicates that such performance could potentially enable a financially viable process for sustainable acrylic acid production. This work establishes I. orientalis as a next-generation platform for cost-effective 3HP production and paves the way toward industrial commercialization.

Biotechnology↗

Considerations in STS payload environmental verification

Considerations regarding the Space Transportation System (STS) payload environmental verification are reviewed. It is noted that emphasis is placed on testing at the subassembly level and that the basic objective of structural dynamic payload verification is to ensure reliability in a cost-effective manner. Structural analyses consist of: (1) stress analysis for critical loading conditions, (2) model analysis for launch and orbital configurations, (3) flight loads analysis, (4) test simulation analysis to verify models, (5) kinematic analysis of deployment/retraction sequences, and (6) structural-thermal-optical program analysis. In addition to these approaches, payload verification programs are being developed in the thermal-vacuum area. These include the exposure to extreme temperatures, temperature cycling, thermal-balance testing and thermal-vacuum testing.

Keegan, W. B.↗

Rover Sequencing and Visualization Program

The Rover Sequencing and Visualization Program (RSVP) is the software tool for use in the Mars Exploration Rover (MER) mission for planning rover operations and generating command sequences for accomplishing those operations. RSVP combines three-dimensional (3D) visualization for immersive exploration of the operations area, stereoscopic image display for high-resolution examination of the downlinked imagery, and a sophisticated command-sequence editing tool for analysis and completion of the sequences. RSVP is linked with actual flight-code modules for operations rehearsal to provide feedback on the expected behavior of the rover prior to committing to a particular sequence. Playback tools allow for review of both rehearsed rover behavior and downlinked results of actual rover operations. These can be displayed simultaneously for comparison of rehearsed and actual activities for verification. The primary inputs to RSVP are downlink data products from the Operations Storage Server (OSS) and activity plans generated by the science team. The activity plans are high-level goals for the next day s activities. The downlink data products include imagery, terrain models, and telemetered engineering data on rover activities and state. The Rover Sequence Editor (RoSE) component of RSVP performs activity expansion to command sequences, command creation and editing with setting of command parameters, and viewing and management of rover resources. The HyperDrive component of RSVP performs 2D and 3D visualization of the rover s environment, graphical and animated review of rover-predicted and telemetered state, and creation and editing of command sequences related to mobility and Instrument Deployment Device (IDD) operations. Additionally, RoSE and HyperDrive together evaluate command sequences for potential violations of flight and safety rules. The products of RSVP include command sequences for uplink that are stored in the Distributed Object Manager (DOM) and predicted rover state histories stored in the OSS for comparison and validation of downlinked telemetry. The majority of components comprising RSVP utilize the MER command and activity dictionaries to automatically customize the system for MER activities. Thus, RSVP, being highly data driven, may be tailored to other missions with minimal effort. In addition, RSVP uses a distributed, message-passing architecture to allow multitasking, and collaborative visualization and sequence development by scattered team members.

Cooper, Brian↗

Update on Rover Sequencing and Visualization Program

The Rover Sequencing and Visualization Program (RSVP) has been updated. RSVP was reported in Rover Sequencing and Visualization Program (NPO-30845), NASA Tech Briefs, Vol. 29, No. 4 (April 2005), page 38. To recapitulate: The Rover Sequencing and Visualization Program (RSVP) is the software tool to be used in the Mars Exploration Rover (MER) mission for planning rover operations and generating command sequences for accomplishing those operations. RSVP combines three-dimensional (3D) visualization for immersive exploration of the operations area, stereoscopic image display for high-resolution examination of the downlinked imagery, and a sophisticated command-sequence editing tool for analysis and completion of the sequences. RSVP is linked with actual flight code modules for operations rehearsal to provide feedback on the expected behavior of the rover prior to committing to a particular sequence. Playback tools allow for review of both rehearsed rover behavior and downlinked results of actual rover operations. These can be displayed simultaneously for comparison of rehearsed and actual activities for verification. The primary inputs to RSVP are downlink data products from the Operations Storage Server (OSS) and activity plans generated by the science team. The activity plans are high-level goals for the next day s activities. The downlink data products include imagery, terrain models, and telemetered engineering data on rover activities and state. The Rover Sequence Editor (RoSE) component of RSVP performs activity expansion to command sequences, command creation and editing with setting of command parameters, and viewing and management of rover resources. The HyperDrive component of RSVP performs 2D and 3D visualization of the rover s environment, graphical and animated review of rover predicted and telemetered state, and creation and editing of command sequences related to mobility and Instrument Deployment Device (robotic arm) operations. Additionally, RoSE and HyperDrive together evaluate command sequences for potential violations of flight and safety rules. The products of RSVP include command sequences for uplink that are stored in the Distributed Object Manager (DOM) and predicted rover state histories stored in the OSS for comparison and validation of downlinked telemetry. The majority of components comprising RSVP utilize the MER command and activity dictionaries to automatically customize the system for MER activities.

Cooper, Brian↗

Assembly of catalytic complexes from randomized oligonucleotides

The early evolution of life relied on catalytic RNAs (ribozymes) for central functions. To test whether early catalysts could have assembled from multiple short nucleic acid fragments in random sequence environments, we performed an in vitro selection from a short RNA library in the presence of 256 different DNA 20-nucleotide oligomers. High-throughput sequencing and biochemical analysis showed that most of the selected 1331 RNA sequences required at least one DNA for activity. Representatives for four of six RNA clusters that depended on DNA cofactors were active even when the 256 DNAs were replaced by completely random DNA 20-nucleotide oligomers. The formation of these catalytic complexes and the recruitment of oligonucleotide cofactors from completely random libraries demonstrate an important principle for the emergence of the earliest oligonucleotide catalysts.

Xu Han↗

Deep mutational analysis reveals functional trade-offs in the sequences of EGFR autophosphorylation sites

Upon activation, the epidermal growth factor receptor (EGFR) phosphorylates tyrosine residues in its cytoplasmic tail, which triggers the binding of Src homology 2 (SH2) and phosphotyrosine-binding (PTB) domains and initiates downstream signaling. The sequences flanking the tyrosine residues (referred to as “phosphosites”) must be compatible with phosphorylation by the EGFR kinase domain and the recruitment of adapter proteins, while minimizing phosphorylation that would reduce the fidelity of signal transmission. To understand how phosphosite sequences encode these functions within a small set of residues, we carried out high-throughput mutational analysis of three phosphosite sequences in the EGFR tail. We used bacterial surface display of peptides coupled with deep sequencing to monitor phosphorylation efficiency and the binding of the SH2 and PTB domains of the adapter proteins Grb2 and Shc1, respectively. We found that the sequences of phosphosites in the EGFR tail are restricted to a subset of the range of sequences that can be phosphorylated efficiently by EGFR. Although efficient phosphorylation by EGFR can occur with either acidic or large hydrophobic residues at the -1 position with respect to the tyrosine, hydrophobic residues are generally excluded from this position in tail sequences. The mutational data suggest that this restriction results in weaker binding to adapter proteins but also disfavors phosphorylation by the cytoplasmic tyrosine kinases c-Src and c-Abl. Our results show how EGFR-family phosphosites achieve a trade-off between minimizing off-pathway phosphorylation and maintaining the ability to recruit the diverse complement of effectors required for downstream pathway activation.

Science & Technology - Other Topics↗

Knowledge-based decision support for Space Station assembly sequence planning

A complete Personal Analysis Assistant (PAA) for Space Station Freedom (SSF) assembly sequence planning consists of three software components: the system infrastructure, intra-flight value added, and inter-flight value added. The system infrastructure is the substrate on which software elements providing inter-flight and intra-flight value-added functionality are built. It provides the capability for building representations of assembly sequence plans and specification of constraints and analysis options. Intra-flight value-added provides functionality that will, given the manifest for each flight, define cargo elements, place them in the National Space Transportation System (NSTS) cargo bay, compute performance measure values, and identify violated constraints. Inter-flight value-added provides functionality that will, given major milestone dates and capability requirements, determine the number and dates of required flights and develop a manifest for each flight. The current project is Phase 1 of a projected two phase program and delivers the system infrastructure. Intra- and inter-flight value-added were to be developed in Phase 2, which has not been funded. Based on experience derived from hundreds of projects conducted over the past seven years, ISX developed an Intelligent Systems Engineering (ISE) methodology that combines the methods of systems engineering and knowledge engineering to meet the special systems development requirements posed by intelligent systems, systems that blend artificial intelligence and other advanced technologies with more conventional computing technologies. The ISE methodology defines a phased program process that begins with an application assessment designed to provide a preliminary determination of the relative technical risks and payoffs associated with a potential application, and then moves through requirements analysis, system design, and development.

Source record↗

Nanopore Sequencing in Space: The Advancement of In Situ Microbiome Analysis for the International Space Station and Beyond

Introduction: Routine assessment of the International Space Station (ISS) microbiome has been performed through in-flight culture and Earth-based analysis. Through utilization of the ISS, significant progress toward in situ microbial identification has been achieved. In 2016, the molecular space age began when DNA-based, Earth-prepared samples were amplified within miniPCR (Genes in Space-1), and subsequent samples were sequenced with the MinION (Biomolecule Sequencer). The following year, these platforms were used collectively to yield the first off-Earth identification of unknown bacteria collected and cultured onboard the ISS (Genes in Space-3). Further advancement occurred in 2018 when a culture-independent, direct swab-to-sequencer method revealed a more thorough depiction of the ISS surface microbiome (BEST). As NASA looks towards Artemis and extended exploration missions, it is critical to continue to harness the ISS to expand in situ nanopore sequencing-based analysis in support of microbial-related crew health, planetary protection, and space research initiatives.

Christian G. Mena↗

Analysis of xylem formation in pine by cDNA sequencing

Secondary xylem (wood) formation is likely to involve some genes expressed rarely or not at all in herbaceous plants. Moreover, environmental and developmental stimuli influence secondary xylem differentiation, producing morphological and chemical changes in wood. To increase our understanding of xylem formation, and to provide material for comparative analysis of gymnosperm and angiosperm sequences, ESTs were obtained from immature xylem of loblolly pine (Pinus taeda L.). A total of 1,097 single-pass sequences were obtained from 5' ends of cDNAs made from gravistimulated tissue from bent trees. Cluster analysis detected 107 groups of similar sequences, ranging in size from 2 to 20 sequences. A total of 361 sequences fell into these groups, whereas 736 sequences were unique. About 55% of the pine EST sequences show similarity to previously described sequences in public databases. About 10% of the recognized genes encode factors involved in cell wall formation. Sequences similar to cell wall proteins, most known lignin biosynthetic enzymes, and several enzymes of carbohydrate metabolism were found. A number of putative regulatory proteins also are represented. Expression patterns of several of these genes were studied in various tissues and organs of pine. Sequencing novel genes expressed during xylem formation will provide a powerful means of identifying mechanisms controlling this important differentiation pathway.

Non-NASA Center↗

4-Hydroxy-tetrahydrodipicolinate reductase from Neisseria gonorrhoeae – structure and interactions with coenzymes and substrate analog

Highlights: • N. gonorrhoeae is an obligate human pathogen which is a leading cause of communicable diseases. • 4-Hydroxy-tetrahydrodipicolinate reductase from N. gonorrhoeae (NgDapB) is a new drug target, and its structure was determined. • ITC we have demonstrated and characterized binding of NADH, NADPH and an inhibitor (2,6-PDC) to NgDapB. • In the case of NgDapB, the ITC results suggest random sequential mechanism for substrate binding. • A large scale analysis of DapBs' sequences revealed that DapBs can be segregated into three distinct groups. Neisseria gonorrhoeae, an obligate human pathogen, is a leading cause of communicable diseases globally. Due to rapid development of drug resistance, the rate of successfully curing gonococcal infections is rapidly decreasing. Hence, research is being directed toward finding alternative drugs or drug targets to help eradicate these infections. 4-Hydroxy-tetrahydrodipicolinate reductase (DapB), an important enzyme in the meso-diaminopimelate pathway, is a promising target for the development of new antibiotics. This manuscript describes the first structure of DapB from N. gonorrhoeae determined at 1.85 Å. This enzyme uses NAD(P)H as cofactor. Details of the interactions of the enzyme with its cofactors and a substrate analog/inhibitor are discussed. A large scale bioinformatics analysis of DapBs' sequences is also described.

60 APPLIED LIFE SCIENCES↗

The state of algal genome quality and diversity

The genomic era of biology has created unprecedented opportunity to study how life works, including understanding evolutionary principles, bioprospecting for novel antibiotics, and genetic manipulation of bioeconomically-relevant species. While genome sequencing and genomic analysis was previously restricted to large-scale projects and consortia, sequencing has become democratized. However, as genomics has become commonplace, the cataloging of sequenced organisms has become challenging, and standardized practices for sequencing, assembly, and annotation have not been adopted. This is equally true in research fields such as algal biology, despite the growing importance of algae in a bio-based economy. Here, in this study, we provide a comprehensive review of the state of eukaryotic algal genomics, explore the quality of algal genome assemblies, and identify the biases and gaps in the current species distribution to inform the development of future genome projects. Overall, we find a trend of declining quality of genomic resources, including a reduction in assembly quality, gene annotation quality, and genome completeness. Potential solutions to improve genome quality include widespread utilization of long read and scaffolding technologies, implementation of standards for assembly quality, evidence-based gene annotation and requisite publication, and support of continued development of gene prediction and genome assessment software.

59 BASIC BIOLOGICAL SCIENCES↗

A role for cysteine 3635 of RYR1 in redox modulation and calmodulin binding

Oxidation of the skeletal muscle Ca(2+) release channel (RYR1) increases its activity, produces intersubunit disulfide bonds, and blocks its interaction with calmodulin. Conversely, bound calmodulin protects RYR1 from the effects of oxidants (Zhang, J.-Z., Wu, Y., Williams, B. Y., Rodney, G., Mandel, F., Strasburg, G. M., and Hamilton, S. L. (1999) Am. J. Physiol. 276, Cell Physiol. C46-C53). In addition, calmodulin protects RYR1 from trypsin cleavage at amino acids 3630 and 3637 (Moore, C. P., Rodney, G., Zhang, J.-Z., Santacruz-Toloza, L., Strasburg, G. M., and Hamilton, S. L. (1999) Biochemistry 38, 8532-8537). The sequence between these two tryptic sites is AVVACFR. Alkylation of RYR1 with N-ethylmaleimide (NEM) blocks both (35)S-apocalmodulin binding and oxidation-induced intersubunit cross-linking. In the current work, we demonstrate that both cysteines needed for the oxidation-induced intersubunit cross-link are protected from alkylation with N-ethylmaleimide by bound calmodulin. We also show, using N-terminal amino acid sequencing together with analysis of the distribution of [(3)H]NEM labeling with each sequencing cycle, that cysteine 3635 of RYR1 is rapidly labeled by NEM and that this labeling is blocked by bound calmodulin. We propose that cysteine 3635 is located at an intersubunit contact site that is close to or within a calmodulin binding site. These findings suggest that calmodulin and oxidation modulate RYR1 activity by regulating intersubunit interactions in a mutually exclusive manner and that these interactions involve cysteine 3635.

Non-NASA Center↗