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NASA NTRS1992

Endochondral bone formation in embryonic mouse pre-metatarsals

Long term exposure to a reduced gravitational environment has a deleterious effect on bone. The developmental events which occur prior to initial bone deposition will provide insight into the regulation of mature bone physiology. We have characterized a system in which the events preceding bone formation take place in an isolated in vitro organ culture environment. We show that cultured pre-metatarsal tissue parallels development of pre-metatarsal tissue in the embryo. Both undergo mesenchyme differentiation and morphogenesis to form a cartilage rod, which resembles the future bone, followed by terminal chondrocyte differentiation in a definite morphogenetic pattern. These sequential steps occur prior to osteoblast maturation and bone matrix deposition in the developing organism. Alkaline phosphatase (ALP) activity is a distinctive enzymatic marker for mineralizing tissues. We have measured this activity throughout pre-metatarsal development and show (a) where in the tissue it is predominantly found, and (b) that this is indeed the mineralizing isoform of the enzyme.

NASA Discipline Number 93-10↗

NASA NTRS1994

Gravity in mammalian organ development: differentiation of cultured lung and pancreas rudiments during spaceflight

Organ culture of embryonic mouse lung and pancreas rudiments has been used to investigate development and differentiation, and to assess the effects of microgravity on culture differentiation, during orbital spaceflight of the shuttle Endeavour (mission STS-54). Lung rudiments continue to grow and branch during spaceflight, an initial result that should allow future detailed study of lung morphogenesis in microgravity. Cultured embryonic pancreas undergoes characteristic exocrine acinar tissue and endocrine islet tissue differentiation during spaceflight, and in ground controls. The rudiments developing in the microgravity environment of spaceflight appear to grow larger than their ground counterparts, and they may have differentiated more rapidly than controls, as judged by exocrine zymogen granule presence.

STS-54 Shuttle Project↗

NASA NTRS1994

Neonatal rat heart cells cultured in simulated microgravity

In vitro characteristics of cardiac cells cultured in simulated microgravity are reported. Tissue culture methods performed at unit gravity constrain cells to propagate, differentiate, and interact in a two dimensional (2D) plane. Neonatal rat cardiac cells in 2D culture organize predominantly as bundles of cardiomyocytes with the intervening areas filled by non-myocyte cell types. Such cardiac cell cultures respond predictably to the addition of exogenous compounds, and in many ways they represent an excellent in vitro model system. The gravity-induced 2D organization of the cells, however, does not accurately reflect the distribution of cells in the intact tissue. We have begun characterizations of a three-dimensional (3D) culturing system designed to mimic microgravity. The NASA designed High-Aspect-Ratio-Vessel (HARV) bioreactors provide a low shear environment which allows cells to be cultured in static suspension. HARV-3D cultures were prepared on microcarrier beads and compared to control-2D cultures using a combination of microscopic and biochemical techniques. Both systems were uniformly inoculated and medium exchanged at standard intervals. Cells in control cultures adhered to the polystyrene surface of the tissue culture dishes and exhibited typical 2D organization. Cells in cultured in HARV's adhered to microcarrier beads, the beads aggregated into defined clusters containing 8 to 15 beads per cluster, and the clusters exhibited distinct 3D layers: myocytes and fibroblasts appeared attached to the surfaces of beads and were overlaid by an outer cell type. In addition, cultures prepared in HARV's using alternative support matrices also displayed morphological formations not seen in control cultures. Generally, the cells prepared in HARV and control cultures were similar, however, the dramatic alterations in 3D organization recommend the HARV as an ideal vessel for the generation of tissue-like organizations of cardiac cells in simulated microgravity.

Akins, Robert E.↗

NASA NTRS2003

Loss of T cell precursors after spaceflight and exposure to vector-averaged gravity

Using fetal thymus organ culture (FTOC), we examined the effects of spaceflight and vector-averaged gravity on T cell development. Under both conditions, the development of T cells was significantly attenuated. Exposure to spaceflight for 16 days resulted in a loss of precursors for CD4+, CD8+, and CD4+CD8+ T cells in a rat/mouse xenogeneic co-culture. A significant decrease in the same precursor cells, as well as a decrease in CD4-CD8- T cell precursors, was also observed in a murine C57BL/6 FTOC after rotation in a clinostat to produce a vector-averaged microgravity-like environment. The block in T cell development appeared to occur between the pre-T cell and CD4+CD8+ T cell stage. These data indicate that gravity plays a decisive role in the development of T cells.

Flight Experiment↗

NASA NTRS1969

Cyclic adenosine monophosphate - Stimulation of melatonin and serotonin synthesis in cultured rat pineals.

Dibutyryl cyclic adenosine monophosphate stimulation of melatonin and serotonin synthesis from C-14 labeled tryptophan by rat pineals in organ culture

Shein, H. M.↗

NASA NTRS2014

Interdisciplinary Interactions During R&D and Early Design of Large Engineered Systems

Designing Large-Scale Complex Engineered Systems (LaCES) such as aircraft and submarines requires the input of thousands of engineers and scientists whose work is proximate in neither time nor space. Comprehensive knowledge of the system is dispersed among specialists whose expertise is in typically one system component or discipline. This study examined the interactive work practices among such specialists seeking to improve engineering practice through a rigorous and theoretical understanding of current practice. This research explored current interdisciplinary practices and perspectives during R&D and early LaCES design and identified why these practices and perspectives prevail and persist. The research design consisted of a three-fold, integrative approach that combined an open-ended survey, semi-structured interviews, and ethnography. Significant empirical data from experienced engineers and scientists in a large engineering organization were obtained and integrated with theories from organization science and engineering. Qualitative analysis was used to obtain a holistic, contextualized understanding. The over-arching finding is that issues related to cognition, organization, and social interrelations mostly dominate interactions across disciplines. Engineering issues, such as the integration of hardware or physics-based models, are not as significant. For example, organization culture is an important underlying factor that guided researchers more toward individual sovereignty over cross-disciplinarity. The organization structure and the engineered system architecture also serve as constraints to the engineering work. Many differences in work practices were observed, including frequency and depth of interactions, definition or co-construction of requirements, clarity or creation of the system architecture, work group proximity, and cognitive challenges. Practitioners are often unaware of these differences resulting in confusion and incorrect assumptions regarding work expectations. Cognitively, the enactment and coconstruction of knowledge are the fundamental tasks of the interdisciplinary interactions. Distributed and collective cognition represent most of the efforts. Argument, ignorance, learning, and creativity are interrelated aspects of the interactions that cause discomfort but yield benefits such as problem mitigation, broader understanding, and improved system design and performance. The quality and quantity of social interrelations are central to all work across disciplines with reciprocity, respectful engagement, and heedful interrelations being significant to the effectiveness of the engineering and scientific work.

McGowan, Anna-Maria Rivas↗

NASA NTRS1993

Utilization of microgravity bioreactors for differentiation of mammalian skeletal tissue

Bioreactor cell and tissue culture vessels can be used to study bone development in a simulated microgravity environment. These vessels will also provide an advantageous, low maintenance culture system on space station Freedom. Although many types of cells and tissues can potentially utilize this system, our particular interest is in developing bone tissue. We have characterized an organ culture system utilizing embryonic mouse pre-metatarsal mesenchyme, documenting morphogenesis and differentiation as cartilage rods are formed, with subsequent terminal chondrocyte differentiation to hypertrophied cells. Further development to form bone tissue is achieved by supplementation of the culture medium. Research using pre-metatarsal tissue, combined with the bioreactor culture hardware, could give insight into the advantages and/or disadvantages of conditions experienced in microgravity. Studies such as these have the potential to enhance understanding of bone development and adult bone physiology, and may help define the processes of bone demineralization experienced in space and in pathological conditions here on earth.

Non-NASA Center↗

NASA NTRS1989

Metabolic alterations induced in cultured skeletal muscle by stretch-relaxation activity

Muscle cells differentiated in vitro are repetitively stretched and relaxed in order to determine the presence of short- and long-term alterations occurring in glucose uptake and lactate efflux that are similar to the metabolic alterations occurring in stimulated organ-cultured muscle and in vivo skeletal muscle during the active state. It is observed that whereas mechanical stimulation increases these metabolic parameters within 4-6 h of starting activity, unstimulated basal rates in control cultures also increase during this period of time, and by 8 h, their rates have reached or exceeded the rates in continuously stimulated cells. Measurements of these parameters in media of different compositions show that activity-induced long-term alterations in the parameters occur independently of growth factors in serium and embryo extracts.

Hatfaludy, Sophia↗

NASA NTRSDate not supplied

Unlocking the Microbiome of the International Space Station

With the start of human occupation more than 22 years ago, the microbiome of International Space Station (ISS) has been monitored to assess risk to both crew and craft. Historically, this monitoring has been achieved through onboard culture and ground-based analyses. Data spanning this timeframe are descriptive of a semi-closed, human occupied environment with associations to crew changes and process escapes within the environmental control and life support systems. While this approach has served to provide alerts to anomalies and overall confidence in the controls in place, the data are limited to the media type and growth conditions used. The bias toward the detection of culturable organisms has depicted an overall lack in biodiversity. As NASA leaves the ISS to focus on exploration, it is critical to fully understand its microbiome and its possible association to the noted positive influence on crew and vehicle health. The implementation of culture-independent, nanopore sequencing-based studies, both onboard the ISS and with returned ground samples, is revealing a more thorough depiction of the microbiome. As noted with pervious culture-based data, there is a common core microbiome across time and location, but key distinct areas of greater diversity exist. Through further investigation, these areas are emerging as unique ecological niches, potentially resulting in environmentally driven microbial selection. Moreover, the presence of some noted taxa within these unique locations has implications for crew health, planetary protection, and controls used in future spacecraft systems. The ability to perform in situ profiling of the ISS microbiome is transforming how NASA assesses risk and is a critical tool towards monitoring the establishment of the environmental microbiome in exploration spacecraft.

Sarah Stahl-Rommel↗

NASA NTRS2021

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↗

NASA NTRS2006

The Molecular Ecology of Guerrero Negro: Justifying the Need for Environmental Genomics

The record of life on the only planet where it is known to exist is contained in the biogeochemical processes that organisms catalyze for their survival, in the compounds that they produce, and in their phylogenetic (evolutionary) relationships to each other. We manipulated sulfate and nutrient concentrations in intact microbial mats over periods of time up to a year. The objectives of the manipulations were: 1) characterize the diversity of process-associated functional genes; 2) understand environmental conditions leading to shifts in microbial guilds; 3) monitor/identify competitive responses of organisms sharing a metabolic niche. Characterization of functional genes associated with carbon (mcrA), nitrogen (nifH, nirK) and sulfur (dsrkB) cycling performed to date provided insight into the diversity and metabolic potential of the system; however, we only identified broad scale correlations between gene abundances and changes in mat physiology. For instance, increases in methane production by mats subjected to lowered sulfate and salinity concentrations were correlated with an observed increase in abundance of hydrogenotroph-like mcrA genes. However, due to low sequence similarity to any cultured isolates, phylogenetic associations only allow order level taxonomic commentary, preventing any associations being made on the cellular level. In each of the genes characterized from these experiments, a significant portion of sequences recovered show minimal phylogenetic affiliation to cultured organisms, preventing any understanding of inter-community dynamics and the functional capacities of these unknown organisms. Environmental genomics may provide insight into mat systems by allowing the correlation of functional genes with phylogenetic markers.

Smith, Jason M.↗

NASA NTRSDate not supplied

Culture-Independent Microbial Air Profiling using a Spaceflight-Compatible Nanopore Sequencing Method

Microbial monitoring of spacecraft air is critical toward assessing the efficacy of microbial controls within the environmental control and life support systems to protect both the crew and the vehicle environment. Additionally, understanding of the atmospheric microbial profiles will be essential toward Mars-forward planetary protection planning. Currently, onboard the International Space Station (ISS), the air is monitored on a quarterly basis using an impaction air sampler. With this method, bacterial and fungal cells and spores are pulled onto plates containing growth medium. Following five days of ambient incubation onboard the ISS, the crew compares the growth to density charts and provides the approximations to the ground. Upon return of the culture plates to the Johnson Space Center’s Microbiology Laboratory, the isolates present are identified for crew health risk assessments. As NASA moves beyond the low-Earth orbit of ISS, sample return will be impractical. As such, a near real-time monitoring capability for the assessment of the spacecraft atmosphere is necessary. Significant strides have been made in recent years to utilize a molecular-based method for microbial profiling of ISS surfaces. The developed method is not dependent on microbial culture, thus removing the associated risk to the crew from high microbial levels, the bias toward detecting only the culturable organisms, and eliminates the need for sample return. The work described here details the evaluation of three different air sampling platforms whose product is amenable to downstream molecular processing and nanopore sequencing. The three samplers were compared in terms of mass and power requirements, ease-of -use, and the resulting data. For the two highest ranking samplers, a basic concept of operations was developed to transfer the sample into the already established preparation and sequencing process. Using these concepts of operations, an in-depth comparison of the molecular data generated was compared to the historical culture-based method. Data from both methods detailed similar microbial profiles, while the molecular method detailed microbial identifications that could not be identified through the culture-based method. The developed method will enable the generation of near real-time microbial profiles of the spacecraft atmosphere.

Brandon J Dunbar↗

NASA NTRSDate not supplied

Culture-Independent Microbial Air Profiling using a Spaceflight-Compatible Nanopore Sequencing Method

Microbial monitoring of spacecraft air is critical toward assessing the efficacy of microbial controls within the environmental control and life support systems to protect the crew and vehicle environment. Currently, onboard the International Space Station (ISS), the air is monitored every quarter using an impaction air sampler. With this method, microbial cells and spores are pulled onto plates containing a growth medium. Following onboard incubation, the crew reports approximate microbial levels to the ground, but sample return is required for identification. Upon return of the plates, the isolates present are identified for crew health risk assessments. As NASA moves beyond low-Earth orbit, sample return will be impractical, and a near real-time monitoring capability is essential. Significant strides have been made in recent years to utilize a molecular-based method for microbial profiling of ISS surfaces. The developed method is independent of microbial culture, thus removing the bias toward detecting only culturable organisms, eliminating the need for sample return, and reducing the risk to crew health from exposure to high microbial levels. The work described here details the evaluation of three different air sampling platforms whose product is amenable to downstream molecular processing. The three samplers were compared in terms of mass and power requirements, ease of use, and the resulting data. For the two highest-ranking samplers, a basic concept of operations was developed to transfer the sample into the already established preparation and sequencing process. Using these concepts of operations, an in-depth comparison of the molecular data generated was compared to the historical culture-based method. Data from both methods detailed similar microbial profiles, while the molecular method detailed microbial identifications that were lacking from the culture data. The developed method will enable the generation of near real-time microbial profiles of the spacecraft atmosphere.

Brandon Dunbar↗

NASA NTRSDate not supplied

Culture-Independent Fungal Profiling for the International Space Station using Nanopore Sequencing: Method Development

Microbial monitoring of the International Space Station (ISS) environment is a crew health requirement that encompasses both bacterial and fungal identification. To achieve this currently, culture-based methods are used for sample collection, and these samples must be returned to the laboratory for analysis. The use of culture and the need for sample return to Earth results in a bias toward culturable organisms and causes a significant delay between sample collection and delivery of final data (weeks to months), respectively. Recently, advancements in molecular technology have aided a broad range of applications, including medical, industrial, and basic sciences. Additionally, increases in portability and ease-of-use of molecular platforms have provided point-of-use capabilities demonstrated by the miniPCR thermal cycler (miniPCR bio) and the MinION sequencer (Oxford Nanopore Technologies). Together, these devices have been applied to, and validated for, the identification of bacteria onboard the ISS. Building on this work, we have developed a spaceflight-compatible fungal workflow. Molecular-based fungal analysis is complicated by low biomass, difficult-to-lyse spores, debate regarding the region for taxonomic assignment, and the lack of bioinformatic pipelines and reference databases. To overcome these difficulties, primers yielding an ~ 2 Kb amplicon were validated against a wide range of ISS fungal isolates. The current spaceflight library preparation was substantially optimized, a bioinformatic pipeline was created, and refinements to the UNITE database were implemented. To compare this optimized method to the current culture-based standard, 30 sample sets (60 total swabs, two swabs held in tandem) were evaluated. Parallel fungal profiles were obtained between the two methods, with the culture-independent method revealing increased diversity. The addition of this method to the already established bacterial process fulfills the crew health identification requirement. Moreover, the implementation of this method onboard ISS will enhance our understanding of its unique fungal microbiome.

Hang N. Nguyen↗

NASA NTRS2001

Composition of Hydrothermal Vent Microbial Communities as Revealed by Analyses of Signature Lipids, Stable Carbon Isotopes and Aquificales Cultures

Extremely thermophilic microbial communities associated with the siliceous vent walls and outflow channel of Octopus Spring, Yellowstone National Park, have been examined for lipid biomarkers and carbon isotopic signatures. These data were compared with that obtained from representatives of three Aquificales genera. Thermocrinis ruber. "Thermocrinis sp. HI", Hydrogenobacter thermophilus TK-6, Aquifex pyrophilus and Aquifex aeolicus all contained phospholipids composed not only of the usual ester-linked fatty acids, but also ether-linked alkyls. The fatty acids of all cultured organisms were dominated by a very distinct pattern of n-C-20:1 and cy-C-21 compounds. The alkyl glycerol ethers were present primarily as CIS() monoethers with the expection of the Aquifex spp. in which dialkyl glycerol ethers with a boarder carbon-number distribution were also present. These Aquificales biomarker lipids were the major constituents in the lipid extracts of the Octopus Spring microbial samples. Two natural samples, a microbial biofilm growing in association with deposition of amorphous silica on the vent walls at 92 C, and the well-known 'pink-streamers community' (PSC), siliceous filaments of a microbial consortia growing in the upper outflow channel at 87 C were analyzed. Both the biofilm and PSC samples contained mono and dialkyl glycerol ethers with a prevalence of C-18 and C-20 alkyls. Phospholipid fatty acids were comprised of both the characteristic Aquificales n-C-20:1 and cy-C-21, and in addition, a series of iso-branched fatty acids from i-C-15:0 to i-C-21:0, With i-C-17:0 dominant in the PSC and i-C-19:0 in the biofilm, suggesting the presence of two major bacterial groups. Bacteriohopanepolyols were absent and the minute quantities of archaeol detected showed that Archaea were only minor constituents. Carbon isotopic compositions of the PSC yielded information about community structure and likely physiology. Biomass was C-13-depleted (10.9%) relative to available CO2 from the source water inorganic carbon pool with lipids further depleted by 6.3% relative to biomass The C-20-21 Aquificales fatty acids of the PSC were somewhat heavier than the iso-branched fatty acids. The carbon isotopic signatures of lipid biomarkers were also explored using a pure culture, T ruber, previously isolated from the PSC. Cells grown on C02 with O2 and both H2 and thiosulfate as electron donors were only slightly depleted (3.3%) relative to the C-source while cells grown on formate with O2 showed a major discrimination (19.7%), possibly the result of a metabolic branch point involving the assimilation of C-formate to biomass and the dissimilation to CO2 associated with energy production. T. ruber lipids were slightly heavier than biomass (+1.3%) whether cells were grown using CO2 or formate. Fatty acids from CO2 grown T. ruber cells were a so slightly heavier (average +2.1%) than biomass. The relatively depleted PSC C-20-21 fatty acids suggest that any associated Thermocrinis biomass would also be similarly depleted and much too light to be explained by growth on CO2. The C-fractionations determined with the pure culture suggest that growth of Thermocrinis in the PSC is more likely to occur on formate, presumably generated by geothermal activity. This study points to the value of the analysis of the structural and isotopic composition of lipid blomarkers both in pure culture studies, and in establishing community structure and physiology, as a complement to genomic profiles of microbial diversity. This is especially so when the members of the microbial community are novel and difficult to cultivate in the laboratory.

Jahnke, Linda L.↗

NASA NTRS2001

Composition of Hydrothermal Vent Microbial Communities as Revealed by Analyses of Signature Lipids, Stable Carbon Isotopes and Aquificales Cultures

Jahnke, Linda L.↗

NASA NTRS1991

Biofilms and the survival of opportunistic pathogens in recycled water

Microorganisms are likely to develop an organic film on pipes, water reservoirs and filters used for waste water reclamation during extended missions in space. These biofilms can serve to protect and concentrate potentially pathogenic microorganisms. Our investigation has emphasized the survival strategy of opportunistic pathogenic bacteria in distilled water. Pseudomonas aeruginosa and Staphylococcus aureus were used as test organisms. Cultures were incubated at 10 degrees, 25 degrees, and 37 degrees C. No viable Staphylococcus cells were detected after the first week of incubation. P. aeruginosa, however, survived in distilled water up to 5 months at all three temperatures tested. The starved cells were able to form a biofilm layer on stainless steel. The cells exhibited a negative surface charge. The charge may be involved in the adhesion of this bacterium to metal substrata. We are currently investigating the importance of adhesion in the survival of this and other potential human pathogens found in water recycling systems.

NASA Discipline Life Support Systems↗

NASA NTRS1997

Evidence for the involvement of carbonic anhydrase and urease in calcium carbonate formation in the gravity-sensing organ of Aplysia californica

To better understand the mechanisms that could modulate the formation of otoconia, calcium carbonate granules in the inner ear of vertebrate species, we examined statoconia formation in the gravity-sensing organ, the statocyst, of the gastropod mollusk Aplysia californica using an in vitro organ culture model. We determined the type of calcium carbonate present in the statoconia and investigated the role of carbonic anhydrase (CA) and urease in regulating statocyst pH as well as the role of protein synthesis and urease in statoconia production and homeostasis in vitro. The type of mineral present in statoconia was found to be aragonitic calcium carbonate. When the CA inhibitor, acetazolamide (AZ), was added to cultures of statocysts, the pH initially (30 min) increased and then decreased. The urease inhibitor, acetohydroxamic acid (AHA), decreased statocyst pH. Simultaneous addition of AZ and AHA caused a decrease in pH. Inhibition of urease activity also reduced total statoconia number, but had no effect on statoconia volume. Inhibition of protein synthesis reduced statoconia production and increased statoconia volume. In a previous study, inhibition of CA was shown to decrease statoconia production. Taken together, these data show that urease and CA play a role in regulating statocyst pH and the formation and maintenance of statoconia. CA produces carbonate ion for calcium carbonate formation and urease neutralizes the acid formed due to CA action, by production of ammonia.

Non-NASA Center↗