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Does Collection Time Bias the Ecology of Cleanroom Air Samples?

Microbial monitoring of astromaterials collections has taken on increased importance with the return of biologically sensitive samples from the asteroids Ryugu and Bennu and the initiation of the Mars Sample Return Program. Terrestrial bacteria and fungi can alter the mineralogy and organic composition of our collections causing irreversible contamination of pristine samples and increasing the risk of false positives for life detection measurements. NASA has conducted routine microbial monitoring of its existing collections since 20181. Initial monitoring focused on surface samples collected with foam swabs. Although, airborne microbiology is often decoupled from surface microbiology in the built environment2 culture-based air sampling techniques like impactors were not compliant with existing contamination control requirements. Bringing organic rich media, gelatin or liquids into curation cleanrooms presents an unacceptable risk to pristine samples. In 2022 NASA purchased a materials complaint air sampler and began collecting air samples from the cleanrooms in addition to surface samples3. The new instrument uses an electret filter to collect samples that are suitable for cultivating organisms or for direct DNA sequencing. Preliminary DNA sequencing results appeared to indicate that longer sampling times biased the microbial community in favor of hearty, spore-forming bacteria3. We present the results of a study comparing overnight sampling (17 hours) to short (1 hour) sampling of unoccupied curation cleanrooms. The results will help us optimize our monitoring protocols and develop a more detailed inventory of the ecology of astromaterials curation cleanrooms. Methods: We analyzed 72 paired air samples from six different cleanrooms including the meteorite processing lab (ISO 7 equivalent, 16 samples), the lunar lab (ISO 6 equivalent, 10 samples), the stardust lab (ISO 5 equivalent 14 samples), the OSIRIS-REx lab (ISO 5 equivalent, 12 samples), the Hayabusa2 lab (ISO 5 equivalent, 14 samples), and the Genesis lab (ISO 4 equivalent, 6 samples). All the samples were collected with an InnovaPrep Bobcat air sampler operating at a sampling rate of 200 L/min. The sampler operates for 5 minutes out of every 20 minute period. Half of the samples were collected by filtering 3,000L (15 min. of active sampling) of air across an electret filter for one hour. The rest of the samples were collected by filtering approximately 51,000 L air across the filter overnight (~17 hours, 255 min. of active sampling). Cells were eluted from the filter using 6-7 ml of pressurized 0.15% tween 20 in PBS (phosphate buffered saline). This liquid was used to cultivate bacteria according to previously published methods1,4,5 and for DNA extraction and next generation sequencing. DNA was extracted with a Qiagen MagAttract PowerMicrobiome kit6. To identify bacteria and archaea, the 16S rRNA gene was amplified using Earth Microbiome primers for the V4 region 7. The amplified DNA was sequenced on an Illumina MiSeq using a V3 reagent kit. The resulting sequences were processed using DADA2 and QIIME2 as implemented on the EDGE bioinformatics platform8–10. Results: Only two of the 72 samples had no amplifiable DNA. Amplified DNA concentrations ranged from 2.67 – 0.272 ng/µl. The median concentration of amplified DNA for the 1 hour samples was 0.770 ± 0.368 ng/µl. The median concentration of amplified DNA for the overnight samples was 0.877 ± 0.434 ng/µl. On average the overnight samples had slightly more sequences (58,960 vs. 59,456) and ASV’s (amplicon sequence variants) (60 vs 64.5) than the one hour samples, but these differences are not statistically significant. The most abundant ASV in every sample mapped to the genus Cupravidus. ASV’s mapping to the genuses Bacillus, Schlegelella, Thermus, and Staphylococcus were also common. Discussion and Future Work: Alpha diversity statistics like Shannon Entropy and Faith Phylogenetic Diversity are used to describe the diversity of organisms in a single sample. If a longer sampling time was biasing the data, we would expect to see a change in these diversity statistics vs. sample time. However, we did not observe this in our data. The median Shannon entropy was slightly higher for the overnight samples (3.773 vs 3.611) as was the Faith Phylogenetic Diversity (4.042 vs 3.596), but both values were within a standard deviation of each other for the two sampling times (Fig. 1). It is unlikely, that the longer sampling time is introducing bias into our data. We do observe a significant decrease in diversity when comparing the air samples by lab. The Genesis lab (ISO 4 equivalent) has a lower median number of ASV’s (45.5) than the other labs (62). Median values for Shannon Entropy (3.717 vs. 3.430) and Faith Phylogenetic Diversity (3.796 vs. 3.548) are also lower for Genesis, but those values are with one standard deviation of each other for the different sampling times. This is consistent with previous culture-based results suggesting that the environment in cleanrooms tends to select for a core group of organisms capable of surviving under dry, low nutrient, conditions. The presence of the ASV’s mapping to Cupravidus and Thermus in our sequencing blanks and controls suggests that several of the most common organisms in our samples represent contaminants from the reagents used to perform the DNA extractions and sequencing. Further work is needed to identify these contaminants, remove them from our data and recalculate the diversity statistics. This is a systematic error. Therefore, we do not expect removing the sequencing contaminants to change our conclusions. Longer air sample collection times appear to result in slightly higher diversity and do not bias the results towards “hardy” bacteria like spore-formers. Based on these preliminary results we conclude that sampling at least 3,000 liters of air is sufficient to capture the microbial diversity of cleanrooms, and that air samples can also be collected overnight without negatively impacting diversity. These results allow us to be flexible when designing microbial monitoring plans so that they do not interfere with routine lab activity. References: 1. Regberg, A. B. et al. 49th Lunar and Planetary Science Conference (2018). 2. The United States Pharmacopeial Convention. USP General Chapter <1116> (2013). 3. Regberg, A. B., et al. 54th Lunar and Planetary Science Conference (2023). 4. Regberg, A. B. et al. 53rd Lunar and Planetary Science Conference ( 2022). 5. Davis, R. E.,et al. 50th Lunar and Planetary Science Conference (2019). 6. Qiagen. MagAttract® PowerMicrobiome® DNA/RNA EP Kit Handbook. (2018). 7. Walters, W. et al. mSystems 1, (2015). 8. Callahan, B. J. et al. Nat. Methods 13, 581–583 (2016). 9. Hall, M. & Beiko, R. G. Microbiome Analysis: Methods and Protocols113–129 (Springer, 2018). 10. Philipson, C. et al. Bio-Protoc. 7, e2622 (2017).

A. B. Regberg↗
NASA NTRS1998

Formation of oligonucleotide-PNA-chimeras by template-directed ligation

DNA sequences have previously been reported to act as templates for the synthesis of PNA, and vice versa. A continuous evolutionary transition from an informational replicating system based on one polymer to a system based on the other would be facilitated if it were possible to form chimeras, that is molecules that contain monomers of both types. Here we show that ligation to form chimeras proceeds efficiently both on PNA and on DNA templates. The efficiency of ligation is primarily determined by the number of backbone bonds at the ligation site and the relative orientation of template and substrate strands. The most efficient reactions result in the formation of chimeras with ligation junctions resembling the structures of the backbones of PNA and DNA and with antiparallel alignment of both components of the chimera with the template, that is, ligations involving formation of 3'-phosphoramidate and 5'-ester bonds. However, double helices involving PNA are stable both with antiparallel and parallel orientation of the two strands. Ligation on PNA but not on DNA templates is, therefore, sometimes possible on templates with reversed orientation. The relevance of these findings to discussions of possible transitions between genetic systems is discussed.

NASA Discipline Exobiology↗
NASA NTRS1991

Representations of mechanical assembly sequences

Five types of representations for assembly sequences are reviewed: the directed graph of feasible assembly sequences, the AND/OR graph of feasible assembly sequences, the set of establishment conditions, and two types of sets of precedence relationships. (precedence relationships between the establishment of one connection between parts and the establishment of another connection, and precedence relationships between the establishment of one connection and states of the assembly process). The mappings of one representation into the others are established. The correctness and completeness of these representations are established. The results presented are needed in the proof of correctness and completeness of algorithms for the generation of mechanical assembly sequences.

Homem De Mello, Luiz S.↗
NASA NTRS2003

Iterative pass optimization of sequence data

The problem of determining the minimum-cost hypothetical ancestral sequences for a given cladogram is known to be NP-complete. This "tree alignment" problem has motivated the considerable effort placed in multiple sequence alignment procedures. Wheeler in 1996 proposed a heuristic method, direct optimization, to calculate cladogram costs without the intervention of multiple sequence alignment. This method, though more efficient in time and more effective in cladogram length than many alignment-based procedures, greedily optimizes nodes based on descendent information only. In their proposal of an exact multiple alignment solution, Sankoff et al. in 1976 described a heuristic procedure--the iterative improvement method--to create alignments at internal nodes by solving a series of median problems. The combination of a three-sequence direct optimization with iterative improvement and a branch-length-based cladogram cost procedure, provides an algorithm that frequently results in superior (i.e., lower) cladogram costs. This iterative pass optimization is both computation and memory intensive, but economies can be made to reduce this burden. An example in arthropod systematics is discussed. c2003 The Willi Hennig Society. Published by Elsevier Science (USA). All rights reserved.

NASA Discipline Evolutionary Biology↗
NASA NTRS1995

Linear Prediction Of Stationary Sequences Of Vectors

Report discusses direct approach to linear prediction of stationary sequence of vectors. Derives set of equations constituting minimal-order linear predictor. Using this approach, it proposes two specific computationally efficient schemes to predict vectors in sequence.

Baram, Yoram↗
NASA NTRS1992

How should Fitts' Law be applied to human-computer interaction?

The paper challenges the notion that any Fitts' Law model can be applied generally to human-computer interaction, and proposes instead that applying Fitts' Law requires knowledge of the users' sequence of movements, direction of movement, and typical movement amplitudes as well as target sizes. Two experiments examined a text selection task with sequences of controlled movements (point-click and point-drag). For the point-click sequence, a Fitts' Law model that used the diagonal across the text object in the direction of pointing (rather than the horizontal extent of the text object) as the target size provided the best fit for the pointing time data, whereas for the point-drag sequence, a Fitts' Law model that used the vertical size of the text object as the target size gave the best fit. Dragging times were fitted well by Fitts' Law models that used either the vertical or horizontal size of the terminal character in the text object. Additional results of note were that pointing in the point-click sequence was consistently faster than in the point-drag sequence, and that pointing in either sequence was consistently faster than dragging. The discussion centres around the need to define task characteristics before applying Fitts' Law to an interface design or analysis, analyses of pointing and of dragging, and implications for interface design.

NASA Program Space Human Factors↗
NASA NTRS1989

Design of laminated plates for maximum buckling load

The buckling load of laminated plates having midplane symmetry is maximized for a given total thickness. The thicknesses of the layers are taken as the design variables. Buckling analysis is carried out using the finite element method. The optimality equations are solved by a homotopy method which permits tracing optima as a function of total thickness. It is shown that for any design with a given stacking sequence of ply orientations, there exists a design associated with any other stacking sequence which possesses the same bending stiffness matrix and same total thickness. Hence, from the optimum design for a given stacking sequence, one can directly determine the optimum design for any rearrangement of the ply orientations, and the optimum buckling load is independent of the stacking sequence.

Shin, Yung S.↗
NASA NTRS2020

Solar System Escape Trajectories Using Outer Planetary Gravity Assists

A broad search of outer planet gravity assist sequences reveals flyby conditions that are naturally amenable for solar system escape. The optimal flyby conditions depend on the arrival velocity at the final body and provide the maximum possible escape speed for purely ballistic sequences. Trajectories in the 2030-2060 time frame are categorized by their encounter year with an emphasis on solar system escape speed and direction. All resulting sequences require a Jupiter gravity assist, and a considerable increase in escape speed is found in trajectories that utilize an additional Saturn, Uranus, or Neptune flyby. However, these solutions are limited by their availability and range of right ascensions. All cataloged trajectories are able to escape the solar system at least 3 au/year, and the highest energy sequences are in excess of 5.5 au/year. The search space is then used to find outer planet gravity assist trajectories to Kuiper Belt Objects (KBOs), and several cases are optimized.

Lam, Try↗
NASA NTRS1994

Aircraft stress sequence development: A complex engineering process made simple

Development of stress sequences for critical aircraft structure requires flight measured usage data, known aircraft loads, and established relationships between aircraft flight loads and structural stresses. Resulting cycle-by-cycle stress sequences can be directly usable for crack growth analysis and coupon spectra tests. Often, an expert in loads and spectra development manipulates the usage data into a typical sequence of representative flight conditions for which loads and stresses are calculated. For a fighter/trainer type aircraft, this effort is repeated many times for each of the fatigue critical locations (FCL) resulting in expenditure of numerous engineering hours. The Aircraft Stress Sequence Computer Program (ACSTRSEQ), developed by Southwest Research Institute under contract to San Antonio Air Logistics Center, presents a unique approach for making complex technical computations in a simple, easy to use method. The program is written in Microsoft Visual Basic for the Microsoft Windows environment.

Schrader, K. H.↗
NASA NTRS1995

Sequences of correlated hard X-ray and type III bursts during solar flares

Acceleration and injection of electron beams in solar flares can be traced from radio type III (or type U) bursts and correlated hard X-ray pulses with similar timescales and nonthermal spectra. We perform a systematic survey of such correlated radio and hard X-ray (HXR) pulses with timescales of less than or approximately 2 s in flares simultaneously observed with the radio spectrometer Ikarus and the Hard X-Ray Burst Spectrometer (HXRBS) on solar maximum mission (SMM). We applied an epoch-folding technique to enhance correlated time patterns in burst sequences at the two wavelengths. We present the results from the strongest (10) flares with a HXRBS count rate greater than or = 3000 counts/s, which have a satisfactory signal-to-noise ratio for subsecond pulses. The major findings of this study are presented. These observations strongly suggest that particle acceleration in solar flares occurs in a pulsed mode where electron beams are simultaneously injected in upward and downward directions. Since the sequences of correlated HXR and radio bursts show identical durations and intervals at the two wavelengths, they are believed to reflect most directly the temporal dynamics of the underlying common accelerator. As a consequence, thick-target models should be reconsidered under the aspect of electron injection with pulse durations of 0.2-2.0 s and duty cycles of approximately = 50%.

Aschwanden, Markus J.↗
NASA NTRS2010

Enhanced Modeling of First-Order Plant Equations of Motion for Aeroelastic and Aeroservoelastic Applications

A methodology is described for generating first-order plant equations of motion for aeroelastic and aeroservoelastic applications. The description begins with the process of generating data files representing specialized mode-shapes, such as rigid-body and control surface modes, using both PATRAN and NASTRAN analysis. NASTRAN executes the 146 solution sequence using numerous Direct Matrix Abstraction Program (DMAP) calls to import the mode-shape files and to perform the aeroelastic response analysis. The aeroelastic response analysis calculates and extracts structural frequencies, generalized masses, frequency-dependent generalized aerodynamic force (GAF) coefficients, sensor deflections and load coefficients data as text-formatted data files. The data files are then re-sequenced and re-formatted using a custom written FORTRAN program. The text-formatted data files are stored and coefficients for s-plane equations are fitted to the frequency-dependent GAF coefficients using two Interactions of Structures, Aerodynamics and Controls (ISAC) programs. With tabular files from stored data created by ISAC, MATLAB generates the first-order aeroservoelastic plant equations of motion. These equations include control-surface actuator, turbulence, sensor and load modeling. Altitude varying root-locus plot and PSD plot results for a model of the F-18 aircraft are presented to demonstrate the capability.

Pototzky, Anthony S.↗
NASA NTRS1993

Mutations in the putative calcium-binding domain of polyomavirus VP1 affect capsid assembly

Calcium ions appear to play a major role in maintaining the structural integrity of the polyomavirus and are likely involved in the processes of viral uncoating and assembly. Previous studies demonstrated that a VP1 fragment extending from Pro-232 to Asp-364 has calcium-binding capabilities. This fragment contains an amino acid stretch from Asp-266 to Glu-277 which is quite similar in sequence to the amino acids that make up the calcium-binding EF hand structures found in many proteins. To assess the contribution of this domain to polyomavirus structural integrity, the effects of mutations in this region were examined by transfecting mutated viral DNA into susceptible cells. Immunofluorescence studies indicated that although viral protein synthesis occurred normally, infective viral progeny were not produced in cells transfected with polyomavirus genomes encoding either a VP1 molecule lacking amino acids Thr-262 through Gly-276 or a VP1 molecule containing a mutation of Asp-266 to Ala. VP1 molecules containing the deletion mutation were unable to bind 45Ca in an in vitro assay. Upon expression in Escherichia coli and purification by immunoaffinity chromatography, wild-type VP1 was isolated as pentameric, capsomere-like structures which could be induced to form capsid-like structures upon addition of CaCl2, consistent with previous studies. However, although VP1 containing the point mutation was isolated as pentamers which were indistinguishable from wild-type VP1 pentamers, addition of CaCl2 did not result in their assembly into capsid-like structures. Immunogold labeling and electron microscopy studies of transfected mammalian cells provided in vivo evidence that a mutation in this region affects the process of viral assembly.

Non-NASA Center↗
NASA NTRS2017

Exploring Connectivity in Sequence Space of Functional RNA

Emergence of replicable genetic molecules was one of the marking points in the origin of life, evolution of which can be conceptualized as a walk through the space of all possible sequences. A theoretical concept of fitness landscape helps to understand evolutionary processes through assigning a value of fitness to each genotype. Then, evolution of a phenotype is viewed as a series of consecutive, single-point mutations. Natural selection biases evolution toward peaks of high fitness and away from valleys of low fitness. whereas neutral drift occurs in the sequence space without direction as mutations are introduced at random. Large networks of neutral or near-neutral mutations on a fitness landscape, especially for sufficiently long genomes, are possible or even inevitable. Their detection in experiments, however, has been elusive. Although a few near-neutral evolutionary pathways have been found, recent experimental evidence indicates landscapes consist of largely isolated islands. The generality of these results, however, is not clear, as the genome length or the fraction of functional molecules in the genotypic space might have been insufficient for the emergence of large, neutral networks. Thorough investigation on the structure of the fitness landscape is essential to understand the mechanisms of evolution of early genomes. RNA molecules are commonly assumed to play the pivotal role in the origin of genetic systems. They are widely believed to be early, if not the earliest, genetic and catalytic molecules, with abundant biochemical activities as aptamers and ribozymes, i.e. RNA molecules capable, respectively, to bind small molecules or catalyze chemical reactions. Here, we present results of our recent studies on the structure of the sequence space of RNA ligase ribozymes selected through in vitro evolution. Several hundred thousands of sequences active to a different degree were obtained by way of deep sequencing. Analysis of these sequences revealed several large clusters defined such that every sequence in a cluster can be reached from any other sequence in the same cluster through a series of single point mutations. Sequences in a single cluster appear to adopt more than one secondary structure. The mechanism of refolding within a single cluster was examined. To shed light on possible evolutionary paths in the space of ribozymes, the connectivity between clusters was investigated. The effect of length of RNA molecules on the structure of the fitness landscape and possible evolutionary paths was examined by way of comparing functional sequences of 20 and 80 nucleobases in length. It was found that sequences of different lengths shared secondary structure motifs that were presumed responsible for catalytic activity, with increasing complexity and global structural rearrangements emerging in longer molecules.

Wei, Chenyu↗
NASA NTRS1992

Planning Assembly Of Large Truss Structures In Outer Space

Report dicusses developmental algorithm used in systematic planning of sequences of operations in which large truss structures assembled in outer space. Assembly sequence represented by directed graph called "assembly graph", in which each arc represents joining of two parts or subassemblies. Algorithm generates assembly graph, working backward from state of complete assembly to initial state, in which all parts disassembled. Working backward more efficient than working forward because it avoids intermediate dead ends.

De Mello, Luiz S. Homem↗
NASA NTRS1994

Sub-ply level scaling approach investigated for graphite-epoxy composite beam columns

Scale model graphite-epoxy composite specimens were fabricated using the 'sub-ply level' approach and tested as beam-columns under an eccentric axial load to determine the effect of specimen size on flexural response and failure. In the current research project, although the fiber diameters are not scaled, the thickness of the pre-preg material itself has been scaled by adjusting the number of fibers through the thickness of a single ply. Three different grades of graphite-epoxy composite material (AS4/3502) were obtained from Hercules, Inc., in which the number of fibers through the thickness of a single ply was reduced (Grade 190 with 12 to 16 fibers, Grade 95 with 6 to 8 fibers, and Grade 48 with 3 to 4 fibers). Thus, using the sub-ply level approach, a baseline eight ply quasi-isotropic laminate could be fabricated using either the Grade 48 or Grade 95 material and the corresponding full-scale laminate would be constructed from Grade 95 or standard Grade 190 material, respectively. Note that in the sub-ply level approach, the number of ply interfaces is constant for the baseline and full-scale laminates. This is not true for the ply level and sublaminate level scaled specimens. The three grades of graphite-epoxy composite material were used to fabricate scale model beam-column specimens with in-plane dimensions of 0.5*n x 5.75*n, where n=1,2,4 corresponsing to 1/4, 1/2, and full-scale factors. Angle ply, cross ply, and quasi-isotropic laminate stacking sequences were chosen for the investigation and the test matrices for each laminate type are given. Specimens in each laminate family with the same in-plane dimensions but different thicknesses were tested to isolate the influence of the thickness dimension on the flexural response and failure. Also, specific lay-ups were chosen with blocked plies and dispersed plies for each laminate type. Specimens were subjected to an eccentric axial load until failure. The load offset was introduced through a set of hinges which were attached to the platens of a standard load test machine. Three sets of geometrically scaled hinges were used to ensure that scaled loading conditions were applied. This loading condition was chosen because it promotes large flexural deformations and specimens fail at the center of the beam, away from the grip supports. Five channels of data including applied vertical load, end shortening displacement, strain from gages applied back-to-back at the midspan of the beam, and rotation of the hinge from a bubble inclinometer were recorded for each specimen. The beam-column test configuration was used previously to study size effects in ply level scaled composite specimens of the same material system, sizes, and stacking sequences. Thus, a direct comparison between the two scaling approaches is possible. Ply level scaled beam-columns with angle ply, cross ply, and quasi-isotropic lay-ups exhibited no size dependencies in the flexural response, but significant size effects in strength. The reduction in strength with increasing specimen size was not predicted successfully by analysis techniques. It is anticipated that results from this investigation will lead to a better understanding of the strength scale effect in composite structures.

Jackson, Karen E.↗
NASA NTRS1996

Whither microbiology? Phylogenetic trees

The direct cloning and sequencing of genes from uncultured microorganisms in a hot spring suggests that the diversity of life on Earth may be far in excess of that estimated from culturable species.

NASA Discipline Exobiology↗
NASA NTRS2018

The Turbolift: Linear Sled Hybrid Artificial Gravity Concept

Future crewed space exploration missions into deep space will require enhanced countermeasure technologies to ensure astronaut health. One such hazard is extended exposure to reduced gravity levels (i.e., microgravity, lunar gravity, or Martian gravity). Reduced gravity negatively impacts many physiological systems, leading to hydrostatic intolerance, musculoskeletal atrophy, sensorimotor impairment, bone demineralization, cardiovascular deconditioning, and visual alterations. Various countermeasures have been employed for mitigating these effects, such as exercise, pharmaceuticals, diet, and fluid loading. However, these approaches treat individual symptoms, such that each physiological system is addressed with typically one countermeasure. An alternative to this approach is artificial gravity (AG), which promises to be a holistic, comprehensive countermeasure. The traditional approach to creating AG is through centrifugation. However, centrifugation is not a "pure" form of AG and typically includes the drawbacks of Coriolis forces, gravity gradients, and vestibular cross-coupled illusions.As an alternative, we have proposed a Linear Sled Hybrid (LSH) AG system to mitigate astronauts' physiological deconditioning. This system functions by applying pure linear acceleration to produce footward loading. There is a half rotation (180_) to reorient the rider between acceleration and deceleration phases, such that the loading remains footward, as when standing on Earth. The rotation also provides some footward acceleration to the lower body through centripetal acceleration; hence the "hybrid" aspect of the design. At the end of the deceleration, the rider than accelerates back in the opposite direction and the sequence repeats.This proposed system could be integrated with future crewed space vehicles in a variety of manners.

Gruber, Jason↗