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At least 163 records · Page 9

Regulation of Osteoblast Survival by the Extracellular Matrix and Gravity

Spaceflight adversely affects the skeleton, posing a substantial risk to astronaut's health during long duration missions. The reduced bone mass observed in growing animals following spaceflight is due at least in part to inadequate bone formation by osteoblasts. Thus, it is of central importance to identify basic cellular mechanisms underlying normal bone formation. The fundamental ideas underlying our research are that interactions between extracellular matrix proteins, integrin adhesion receptors, cytoplasmic signaling and cytoskeletal proteins are key ingredients for the proper functioning of osteoblasts, and that gravity impacts these interactions. As an in vitro model system we used primary fetal rat calvarial cells which faithfully recapitulate osteoblast differentiation characteristically observed in vivo. We showed that specific integrin receptors ((alpha)3(beta)1), ((alpha)5(beta)1), ((alpha)8(betal)1) and extracellular matrix proteins (fibronectin, laminin) were needed for the differentiation of immature osteoblasts. In the course of maturation, cultured osteoblasts switched from depending on fibronectin and laminin for differentiation to depending on these proteins for their very survival. Furthermore, we found that manipulating the gravity vector using ground-based models resulted in activation of key intracellular survival signals generated by integrin/extracellular matrix interactions. We are currently testing the in vivo relevance of some of these observations using targeted transgenic technology. In conclusion, mechanical factors including gravity may participate in regulating survival via cellular interactions with the extracellular matrix. This leads us to speculate that microgravity adversely affects the survival of osteoblasts and contributes to spaceflight-induced osteoporosis.

Globus. Ruth K.↗

NASA, Remote Sensing and Archaeology: An Example from Southeast Louisiana

NASA Stennis Space Center, located in Mississippi, USA, undertook an archaeological survey of the southeastern Louisiana marshes beginning in 2003. Progress on this activity was severely hampered by the 2005 hurricane season when both Katrina and Rita devastated the study area. In 2008, the NASA team reinitiated the analysis of the project data and that work continues today. The project was conducted initially in partnership with the U.S. Army, Corps of Engineers New Orleans District and Tulane University. NASA and its partners utilized a wide variety of satellite and airborne remote sensing instruments combined with field verification surveys to identify prehistoric archeological sites in the Southeastern Louisiana delta, both known and still undiscovered. The main approach was to carefully map known sites and use the spectral characteristics of these sites to locate high probability targets elsewhere in the region. The archaeological activities were conducted in support of Coast 2050 whose stated goals is to sustain and restore a coastal ecosystem that supports and protects the environment, economy and culture of southern Louisiana. As the Coast 2050 report states: [T]he rate of coastal land loss in Louisiana has reached catastrophic proportions. Within the last 50 years, land loss rates have exceeded 40 square miles per year, and in the 1990's the rate has been estimated to be between 25 and 35 square miles each year. This loss represents 80% of the coastal wetland loss in the entire continental United States.

Giardino, Marco J.↗

Characterization of Truncated Tumor-Associated NADH Oxidase (ttNOX)

Bacterial, plant and animal cells possess novel surface proteins that exhibit both NADH oxidation (NOX) or hydroquinone and protein disulfide-thiol interchange. These enzymatic activities alternate to yield oscillating patterns wjth period lengths of approximately 24 minutes. The catalytic period of NOX proteins are temperature compensated and gravity responsive. We report the cloning, expression and characterization of truncated tumor-associated NADH oxidase (ttNOX), in which the membrane spanning region has been deleted. The cDNA (originated from HeLa cells) was cloned into pET-34b and pET-14b (Novagen) vectors for E. coli expression. Optimized expression and purification protocols yielded greater than 300mg per liter of culture with greater than 95% purity. Circular dichroism data was collected from a 2.7mg/ml solution in a 0.1mm cuvette with variable scanning using an Olis RSM CD spectrophotometer. The ellipticity values were scanned from 190 to 260nm. The spectra recorded have characteristics for alpha proteins with band maxima at 216nm and a possible shoulder at 212nm at 12OC and 250 C. Protein crystal screens are in progress and, to date, only small crystals have been observed. The regular periodic oscillatory change in the ttNOX protein is indicative of a possible time-keeping functional role. A single protein possessing alternating catalytic activities, with a potential biological clock function, is unprecedented and structural determination is paramount to understanding this role.

Karr, Laurel J.↗

Antiproliferative effect of elevated glucose in human microvascular endothelial cells

Diabetic microangiopathy has been implicated as a fundamental feature of the pathological complications of diabetes including retinopathy, neuropathy, and diabetic foot ulceration. However, previous studies devoted to examining the deleterious effects of elevated glucose on the endothelium have been performed largely in primary cultured cells of macrovessel origin. Difficulty in the harvesting and maintenance of microvascular endothelial cells in culture have hindered the study of this relevant population. Therefore, the objective of this study was to characterize the effect of elevated glucose on the proliferation and involved signaling pathways of an immortalized human dermal microvascular endothelial cell line (HMEC-1) that possess similar characteristics to their in vivo counterparts. Human dermal microvascular endothelial cells (HMEC-1) were grown in the presence of normal (5 mM) or high D-glucose (20 mM) for 14 days. The proliferative response of HMEC-1 was compared under these conditions as well as the cAMP and PKC pathways by in vitro assays. Elevated glucose significantly inhibited (P < 0.05) HMEC-1 proliferation after 7, 10, and 14 days. This effect was not mimicked by 20 mM mannitol. The antiproliferative effect was more pronounced with longer exposure (1-14 days) to elevated glucose and was irreversible 4 days after a 10-day exposure. The antiproliferative effect was partially reversed in the presence of a PKA inhibitor, Rp-cAMP (10-50 microM), and/or a PKC inhibitor, Calphostin C (10 nM). HMEC-1 exposed to elevated glucose (20 mM) for 14 days caused an increase in cyclic AMP accumulation, PKA, and PKC activity but was not associated with the activation of downstream events such as CRE and AP-1 binding activity. These data support the hypothesis that HMEC-1 is a suitable model to study the deleterious effects of elevated glucose on microvascular endothelial cells. Continued studies with HMEC-1 may prove advantageous in delineation of the molecular pathophysiology associated with diabetic microangiopathy.

NASA Discipline Cell Biology↗

Introduction to Computational Methods for Stability and Control (COMSAC)

This Symposium is intended to bring together the often distinct cultures of the Stability and Control (S&C) community and the Computational Fluid Dynamics (CFD) community. The COMSAC program is itself a new effort by NASA Langley to accelerate the application of high end CFD methodologies to the demanding job of predicting stability and control characteristics of aircraft. This talk is intended to set the stage for needing a program like COMSAC. It is not intended to give details of the program itself. The topics include: 1) S&C Challenges; 2) Aero prediction methodology; 3) CFD applications; 4) NASA COMSAC planning; 5) Objectives of symposium; and 6) Closing remarks.

Hall, Robert M.↗

Marine Algae Industrialization Consortium (MAGIC): Combining biofuel and high-value bioproducts to meet the RFS

The Marine Algae Industrialization Consortium (MAGIC) was formed to address pressing challenges in the commercialization of microalgae as a source of biofuel. The “Marine Algae Industrialization Consortium (MAGIC): Combining biofuel and high-value bioproducts to meet the RFS” project formally addressed two US Department of Energy Bioenergy Technologies Office (BETO) goals: (1) Model the sustainable supply of 1 million metric tonnes ash free dry weight (AFDW) cultivated algal biomass and (2) Demonstrate valuable co-products produced along with biofuel intermediates to increase value of algal biomass by 30%. To achieve these goals, the project demonstrated and validated high-value co-products to drive down the cost of biofuel by increasing the value of algae “co-products” towards increasing the selling price of total algae biomass as one of the key drivers of economics and adoption. This was accomplished through five core, interdependent tasks including: (1) strain selection to identify and deliver strains for mass culture, (2) mass culture using a hybrid cultivation system and following key operating parameters for downstream applications to provide algae feedstock, (3) recovery and conversion to evaluate two alternative methods to separate dry algae biomass into oil and residuals for downstream testing, (4) product assessment to determine biofuel, aquafeed or poultry feed product efficacy using algae biomass fractions as well as to provide critical performance data for valuation and (5) commercialization to use technoeconomic and life cycle assessments (TEA/LCA) as iterative design and assessment tools including consideration of target markets, competitors, and distribution channels to guide product assessment, development and valuation. A total of 46 peer-review publications, many open-access, provide detail of much of the work carried out and the results of the tasks. Additional reports and presentations provide other technical and public engagement material. At a high level, using a variety of approaches, more than 1000 marine microalgae strains were evaluated to ultimately identify the seven winners that were down-selected to be grown in mass culture. Strain selection demonstrated that there were no ‘super strains’ and that each candidate had strengths and limitations for specific products, growth conditions or operational considerations. Mass culture growth of these seven strains at >5000 L / 29 m 2 scale found that four them were suitable for product assessment. More than 250 kg of biomass was produced across hundreds of pond runs along with thousands of cultivation entries on the growth and biomass characteristics as well as environmental parameters. In the process, dozens of standard operating procedures were generated as was custom software to process and analyze cultivation data. Recovery and conversion of algae biomass demonstrated that a hexane solvent based extraction protocol was most effective at recovering oil (biocrude) from algae and four strains were processed to produce oil and lipid extracted algae (residuals) for downstream testing. Membrane-based oil separation was less successful, but may still be applicable to other commercial applications in the future. Product testing demonstrated that algae biocrude is of high quality and hydrotreating generated numerous fractions of high quality composition for fuel and lubricate based applications. Aquafeed studies performed at a variety of scales showed that both whole and defatted (lipid extracted algae) microalgae were suitable as a feed ingredient, but that the specifics of the fed animal and biochemical composition of the algae are critical factors when determining formulation. Similarly, poultry studies on whole and defatted microalgae generally showed positive outcomes on animal growth and health, with some microalgae providing enhanced nutritional composition of the animal product. Economic and life cycle assessments covered a wide range of possible commercialization and sustainability scenarios. Replacement value, improved product value added, consumer values marketing added valuation and improved animal health were considered as alternatives for microalgae valuation. Using the open pond system, algae productivity was identified as the key driver of commercialization economics, but combination of co-products (e.g. animal feed) with biofuel production substantially increased the total selling price of algae. Modeled microalgae selling price exceeded $\$$1500/tonne and could generate competitive biofuel selling prices below $\$$5 gallon gas equivalents using realistic algal productivities. Short (process scale) and longer (decadal trends) sustainability assessments show that marine microalgae can enhance the sustainability of energy production and lead to other realized benefits in water, fertilizer and land use for other sectors (e.g. agriculture). This project successfully demonstrated all of the components of an end-to-end process from mass microalgae cultivation and dewatering, to recovery and conversion of algae biomass components, to final product demonstration and process valuation; the combined results provide a framework for future commercialization of algae based biofuels.

09 BIOMASS FUELS↗

Antibacterial activity of iron oxide, iron nitride, and tobramycin conjugated nanoparticles against Pseudomonas aeruginosa biofilms

Novel methods are necessary to reduce morbidity and mortality of patients suffering from infections with Pseudomonas aeruginosa. Being the most common infectious species of the Pseudomonas genus, P. aeruginosa is the primary Gram-negative etiology responsible for nosocomial infections. Due to the ubiquity and high adaptability of this species, an effective universal treatment method for P. aeruginosa infection still eludes investigators, despite the extensive research in this area. Results We report bacterial inhibition by iron-oxide (nominally magnetite) nanoparticles (NPs) alone, having a mean hydrodynamic diameter of ~ 16 nm, as well as alginate-capped iron-oxide NPs. Alginate capping increased the average hydrodynamic diameter to ~ 230 nm. We also investigated alginate-capped iron-oxide NP-drug conjugates, with a practically unchanged hydrodynamic diameter of ~ 232 nm. Susceptibility and minimum inhibitory concentration (MIC) of the NPs, NP-tobramycin conjugates, and tobramycin alone were determined in the PAO1 bacterial colonies. Investigations into susceptibility using the disk diffusion method were done after 3 days of biofilm growth and after 60 days of growth. MIC of all compounds of interest was determined after 60-days of growth, to ensure thorough establishment of biofilm colonies. Conclusions Positive inhibition is reported for uncapped and alginate-capped iron-oxide NPs, and the corresponding MICs are presented. We report zero susceptibility to iron-oxide NPs capped with polyethylene glycol, suggesting that the capping agent plays a major role in enabling bactericidal ability in of the nanocomposite. Our findings suggest that the alginate-coated nanocomposites investigated in this study have the potential to overcome the bacterial biofilm barrier. Magnetic field application increases the action, likely via enhanced diffusion of the iron-oxide NPs and NP-drug conjugates through mucin and alginate barriers, which are characteristic of cystic-fibrosis respiratory infections. We demonstrate that iron-oxide NPs coated with alginate, as well as alginate-coated magnetite–tobramycin conjugates inhibit P. aeruginosa growth and biofilm formation in established colonies. We have also determined that susceptibility to tobramycin decreases for longer culture times. However, susceptibility to the iron-oxide NP compounds did not demonstrate any comparable decrease with increasing culture time. These findings imply that iron-oxide NPs are promising lower-cost alternatives to silver NPs in antibacterial coatings, solutions, and drugs, as well as other applications in which microbial abolition or infestation prevention is sought.

Biotechnology & Applied Microbiology↗

Genomic Insights into Moderately Thermophilic Methanotrophs of the Genus Methylocaldum

Considering the increasing interest in understanding the biotic component of methane removal from our atmosphere, it becomes essential to study the physiological characteristics and genomic potential of methanotroph isolates, especially their traits allowing them to adapt to elevated growth temperatures. The genetic signatures of Methylocaldum species have been detected in many terrestrial and aquatic ecosystems. A small set of representatives of this genus has been isolated and maintained in culture. The genus is commonly described as moderately thermophilic, with the growth optimum reaching 50 °C for some strains. Here, we present a comparative analysis of genomes of three Methylocaldum strains—two terrestrial M. szegediense strains (O-12 and Norfolk) and one marine strain, Methylocaldum marinum (S8). The examination of the core genome inventory of this genus uncovers significant redundancy in primary metabolic pathways, including the machinery for methane oxidation (numerous copies of pmo genes) and methanol oxidation (duplications of mxaF, xoxF1-5 genes), three pathways for one-carbon (C1) assimilation, and two methods of carbon storage (glycogen and polyhydroxyalkanoates). We also investigate the genetics of melanin production pathways as a key feature of the genus.

59 BASIC BIOLOGICAL SCIENCES↗

Tribal Revegetation Project Final Project Report: 92-Acre Area, Area 5 Radioactive Waste Management Complex, Nevada National Security Site, Nevada

Nuwu (Southern Paiute), Newe (Western Shoshone), and Nuumu (Owens Valley Paiute) are linguistically related, Numic-speaking peoples who are part of the broader Uto-Aztecan language group. Numic peoples view the land as a holistic, living, sentient being with feelings and purpose. The land is personified with human characteristics and it needs to be experienced to be understood through “learning by doing.” Numic peoples do not support ground disturbing activities within their ancestral lands, including activities tied to the storage of low-level radioactive waste or classified materials on the NNSS, which they view as culturally inappropriate. These deep-rooted ancestral connections are the impetus for reinforcing Numic responsibility for healing disturbed areas by integrating respect and patience with consistent Tribal interaction. Tribal Ecological Knowledge (TEK) is the science of describing Tribal approaches for understanding natural resources. Numic TEK is embedded in traditional teachings learned incrementally over time though experience and it evolves through lessons learned and responses to environmental changes over millennia. Therefore, TEK can broaden and enhance Western scientific knowledge associated with revegetation, especially in highly disturbed areas. The project blended TEK with Western scientific ecological methods to create a vegetative cover within test plots on the 92-Acre Area located at the Radioactive Waste Management Complex (RWMC) located in Area 5 on the Nevada National Security Site (NNSS). The vegetated test plots were systematically created for the Department of Energy (DOE) in tandem with the existing Federal Facilities Agreement and Consent Order (FFACO) with the Nevada Department of Environmental Protection (NDEP). Three previous contractor-lead attempts at revegetation, one targeting full cover revegetation and two targeting test plot revegetation, did not achieve the anticipated results at this location. When presented to the 16 American Indian Tribal nations and affiliated groups with cultural and historical ties to the NNSS, the group appointed a Tribal Revegetation Committee (TRC) that included six expert Tribal knowledge holders to collaborate with an ethnoecologist/cultural anthropologist and two biologists. Project design, planning, seed and outplant selection, spiritual land preparation, and methodology were guided by the TRC and an ethnoecologist/cultural anthropologist from Portland State University (PSU) and biologists from Desert Research Institute (DRI). Using TEK, the TRC recommended a specific seed mixture that contained nine native plant species and three species of outplants. The revegetation effort included preparing and planting thirty 10 m × 10 m (32.8 ft × 32.8 ft) seeded plots, twelve of which also included outplants; and eight 10 m × 100 m (32.8 ft × 328 ft) plots that only received outplants, all atop a waste cell cover. The TRC and the project team creatively adapted TEK with Western scientific methods so that the planned revegetation efforts could occur within the safety and security parameters of the RWMC. Test plots were subjected to one of five soil treatments with varying combinations of straw or mulch applications, soil amendment, and/or outplant planting and one of two watering treatments (watered or unwatered). Planting was divided into two events: one in the fall season and another during the subsequent spring season based on TEK and a corresponding recommendation from the TRC. Monitoring and spiritual management was conducted by the TRC to evaluate plant progress on a monthly basis (in conjunction with the biologist and anthropologist) during each respective growing season for a period of three years after planting. This approach allowed Tribal members the opportunity to conduct traditional blessings and other culturally appropriate activities to restore balance to the land in accordance with Tribal protocols. Following TEK-guided methods, successful plant establishment from seed stock and outplants was observed in many plots. Overall, plots planted in the spring, as recommended by the TRC, showed higher rates of outplant survival and native seedling emergence than those planted in the fall. This finding is significant because it is contrary to the original guidance and previous approaches provided for planting in this region. The TRC believes the frequent co-occurrence of native seedlings near surviving outplants indicates an important symbiotic relationship understood by Tribal communities and overlooked by others. Watered outplants displayed much higher survivability than unwatered plants, even after watering was reduced after the plants were established. Soil amendments and mulch created higher densities of native plants from seed. Many native seedlings showed significant delays in germination, which is considered a normal adaptation to desert climates. Some native plants did not germinate until the third growing year, whereas others germinated during the first growing year, which demonstrates the complexity of the desert environment. Evidence of native insects, reptiles, mammals, and birds, as well as native plants that were not part of the planted species, were noted and considered culturally significant. Despite the presence of non-native plants, native outplants continued to thrive and the incidence of native plant germination from seed increased over time. These successful revegetation results where previous efforts were unsuccessful reinforce the importance of integrating regionally appropriate, TEK-guided methodology with Western science to achieve positive results and the necessity of integrating Tribal involvement in all stages of the revegetation effort. Expanded approaches coupled with Tribal knowledge and tools from Western science addressed a complex problem tied to revegetating atop a low-level radioactive waste cell. The level of Tribal participation serves as a progressive model for building collaborative relationships and addressing ecological challenges on the NNSS.

54 ENVIRONMENTAL SCIENCES↗

Activation of polycystin-1 signaling by binding of stalk-derived peptide agonists

Polycystin-1 (PC1) is the protein product of thePKD1gene whose mutation causes autosomal dominant Polycystic Kidney Disease (ADPKD). PC1 is an atypical G protein-coupled receptor (GPCR) with an autocatalytic GAIN domain that cleaves PC1 into extracellular N-terminal and membrane-embedded C-terminal (CTF) fragments. Recently, activation of PC1 CTF signaling was shown to be regulated by a stalk tethered agonist (TA), resembling the mechanism observed for adhesion GPCRs. Here, synthetic peptides of the first 9- (p9), 17- (p17), and 21-residues (p21) of the PC1 stalk TA were shown to re-activate signaling by a stalkless CTF mutant in human cell culture assays. Novel Peptide Gaussian accelerated molecular dynamics (Pep-GaMD) simulations elucidated binding conformations of p9, p17, and p21 and revealed multiple specific binding regions to the stalkless CTF. Peptide agonists binding to the TOP domain of PC1 induced close TOP-putative pore loop interactions, a characteristic feature of stalk TA-mediated PC1 CTF activation. Additional sequence coevolution analyses showed the peptide binding regions were consistent with covarying residue pairs identified between the TOP domain and the stalk TA. These insights into the structural dynamic mechanism of PC1 activation by TA peptide agonists provide an in-depth understanding that will facilitate the development of therapeutics targeting PC1 for ADPKD treatment.

Life Sciences & Biomedicine - Other Topics↗

Microbial reduction of manganese oxides - Interactions with iron and sulfur

Alteromonas putrefaciens (strain MR-1) is capable of rapid Mn(IV) reduction under conditions of neutral pH and temperatures characteristic of the Oneida Lake, New York, sediments from which it was isolated. MR-1 also reduces Fe(3+) to Fe(2+), and disproportionates thiosulfate to sulfide and sulfite; independently, the Fe(2+) and sulfide act as rapid reductants of Mn. The addition of Fe(3+) or thiosulfate to cultures of MR-1 in the presence of oxidized Mn increases the rate and the extent of Mn reduction relative to that observed in the absence of Fe(3+) or thiosulfate. Furthermore, when Fe(3+) and Mn oxides are present conjointly, Fe(2+) does not appear until the reduction of the oxidized Mn is complete. These results demonstrate that the observed rates of Fe(2+) and sulfide production may underestimate the total rates of Fe and sulfate reduction in those environments containing oxidized Mn. These results also demonstrate the potential impact that a single microbe can exert on sediment geochemistry, and provide the basis for preliminary models of the complexity of microbial and geochemical interactions that occur.

Myers, Charles R.↗

The spread of technological innovations: effects of psychology, culture and policy interventions

Technological innovations drive the evolution of human societies. The success of innovations depends not only on their actual benefits but also on how potential adopters perceive them and how their beliefs are affected by their social and cultural environment. To deepen our understanding of socio-psychological processes affecting the new technology spread, we model the joint dynamics of three interlinked processes: individual learning and mastering the new technology, changes in individual attitudes towards it, and changes in individual adoption decisions. We assume that the new technology can potentially lead to a higher benefit but achieving it requires learning. We posit that individual decision-making process as well as their attitudes are affected by cognitive dissonance and conformity with peers and an external authority. Individuals vary in different psychological characteristics and in their attitudes. We investigate both transient dynamics and long-term equilibria observed in our model. We show that early adopters are usually individuals who are characterized by low cognitive dissonance and low conformity with peers but are sensitive to the effort of an external authority promoting the innovation. We examine the effectiveness of five different intervention strategies aiming to promote the diffusion of a new technology: training individuals, providing subsidies for early adopters, increasing the visibility of peer actions, simplifying the exchange of opinions between people, and increasing the effort of an external authority. We also discuss the effects of culture on the spread of innovations. Finally, we demonstrate that neglecting the cognitive forces and the dynamic nature of individual attitudes can lead to wrong conclusions about adoption of innovations. Our results can be useful in developing more efficient policies aiming to promote the spread of new technologies in different societies, cultures and countries.

99 GENERAL AND MISCELLANEOUS↗

Anaerobic biosynthesis of unsaturated fatty acids in the cyanobacterium, Oscillatoria limnetica

The mechanism for synthesis of monounsaturated fatty acids under aerobic and anaerobic conditions was studied in the facultative anaerobic cyanobacterium, Oscillatoria limnetica. The hexadecenoic acid (C16:1) of aerobically grown O. limnetica was shown to contain both the delta 7 (79%) and delta 9 (21%) isomers, while the octadecenoic (C18:1) acid was entirely the delta 9 acid. Incorporation of [2-14C] acetate into the fatty acids under aerobic conditions resulted in synthesis of the delta 7 and delta 9 C16:1 and the delta 9 C18:1. Synthesis of unsaturated fatty acids in the presence of DCMU required sulfide. Anaerobic incubations in the presence of DCMU and sulfide (less than 0.003% atmospheric oxygen) resulted in a two-fold increase in monounsaturated fatty acids of both delta 7 and delta 9 C16:1 and delta 9 and delta 11 C18:1. The synthesis of these is characteristic of a bacterial-type, anaerobic pathway.

NASA Discipline Exobiology↗

Dry Acid Deposition and Accumulation on the Surface of Mars and in the Atacama Desert, Chile

It has been discovered recently that soils from certain regions of the Chilean Atacama Desert have some characteristics that are similar to the surface materials tested by the Viking Landers. Navarro-Gonzalez et al. demonstrated that the quantity and diversity of heterotrophic bacteria increase as a function of local water availability in the Atacama, and that for some soil samples collected in the driest regions, no culturable bacteria could be isolated. Additionally, Navarro-Gonzalez et al. reported that pyrolysis-GCMS analysis of soils collected from these regions revealed extremely low levels of organic matter. Although the mechanism resulting in the low level of organics in these regions was not established by Navarro-Gonzalez, the condition of organic-depleted, near-sterile soil offers an interesting Earth analog of the martian surface material, as the Viking Gas Exchange (GEx) experiment and Labeled Release (LR) experiment were unable to demonstrate the presence of culturable bacteria, and the Viking pyrolysis- GCMS was unable to detect organic compounds.

Quinn, R. C.↗

Substrate flexibility regulates growth and apoptosis of normal but not transformed cells

One of the hallmarks of oncogenic transformation is anchorage-independent growth (27). Here we demonstrate that responses to substrate rigidity play a major role in distinguishing the growth behavior of normal cells from that of transformed cells. We cultured normal or H-ras-transformed NIH 3T3 cells on flexible collagen-coated polyacrylamide substrates with similar chemical properties but different rigidity. Compared with cells cultured on stiff substrates, nontransformed cells on flexible substrates showed a decrease in the rate of DNA synthesis and an increase in the rate of apoptosis. These responses on flexible substrates are coupled to decreases in cell spreading area and traction forces. In contrast, transformed cells maintained their growth and apoptotic characteristics regardless of substrate flexibility. The responses in cell spreading area and traction forces to substrate flexibility were similarly diminished. Our results suggest that normal cells are capable of probing substrate rigidity and that proper mechanical feedback is required for regulating cell shape, cell growth, and survival. The loss of this response can explain the unregulated growth of transformed cells.

Non-NASA Center↗

Growth and photomorphogenesis of pepper plants under red light-emitting diodes with supplemental blue or far-red lighting

Light-emitting diodes (LEDs) are a potential irradiation source for intensive plant culture systems and photobiological research. They have small size, low mass, a long functional life, and narrow spectral output. In this study, we measured the growth and dry matter partitioning of 'Hungarian Wax' pepper (Capsicum annuum L.) plants grown under red LEDs compared with similar plants grown under red LEDs with supplemental blue or far-red radiation or under broad spectrum metal halide (MH) lamps. Additionally, we describe the thermal and spectral characteristics of these sources. The LEDs used in this study had a narrow bandwidth at half peak height (25 nm) and a focused maximum spectral output at 660 nm for the red and 735 nm for the far-red. Near infrared radiation (800 to 3000 nm) was below detection and thermal infrared radiation (3000 to 50,000 nm) was lower in the LEDs compared to the MH source. Although the red to far-red ratio varied considerably, the calculated phytochrome photostationary state (phi) was only slightly different between the radiation sources. Plant biomass was reduced when peppers were grown under red LEDs in the absence of blue wavelengths compared to plants grown under supplemental blue fluorescent lamps or MH lamps. The addition of far-red radiation resulted in taller plants with greater stem mass than red LEDs alone. There were fewer leaves under red or red plus far-red radiation than with lamps producing blue wavelengths. These results indicate that red LEDs may be suitable, in proper combination with other wavelengths of light, for the culture of plants in tightly controlled environments such as space-based plant culture systems.

NASA Discipline Life Support Systems↗

Temporal Galactose‐Manganese Feeding in Fed‐Batch and Perfusion Bioreactors Modulates UDP‐Galactose Pools for Enhanced mAb Glycosylation Homogeneity

ABSTRACT Monoclonal antibodies (mAbs) represent a majority of biotherapeutics in the market today. These glycoproteins undergo posttranslational modifications, such as N‐linked glycosylation, that influence the structural & functional characteristics of the antibody. Glycosylation is a heterogenous posttranslational modification that may influence therapeutic glycoprotein stability and clinical efficacy, which is why it is often considered a critical quality attribute (CQA) of the mAb product. While much is known about the glycosylation pathways of Chinese Hamster Ovary (CHO) cells and how cell culture chemical modifiers may influence the N‐glycosylation profile of the final product, this knowledge is often based on the final cumulative glycan profile at the end of the batch process. Building a temporal understanding of N‐glycosylation and how mAb glycoform composition responds to real‐time changes in the biomanufacturing process will help build integrated process models that may allow for glycosylation control to produce a more homogenous product. Here, we look at the effect of specific nutrient feed media additives (e.g., galactose, manganese) and feeding times on the N‐glycosylation pathway to modulate N‐glycosylation of a Herceptin biosimilar mAb (i.e., Trastuzumab). We deploy the N‐GLYcanyzer process analytical technology (PAT) to monitor glycoforms in near real‐time for bench‐scale bioprocesses operated in both fed‐batch and perfusion modes to build an understanding of how temporal changes in mAb N‐glycosylation are dependent on specific media additives. We find that Trastuzumab terminal galactosylation is sensitive to media feeding times and intracellular nucleotide sugar pools. Temporal analysis reveals an increased desirable production of single and double galactose‐occupied glycoforms over time under glucose‐starved fed‐batch cultures. Comparable galactosylation profiles were also observed between fed‐batch (nutrient‐limited) and perfusion (non‐nutrient‐limited) bioprocess conditions. In summary, our results demonstrate the utility of real‐time monitoring of mAb glycoforms and feeding critical cell culture nutrients under fed‐batch and perfusion bioprocessing conditions to produce higher‐quality biologics.

Biotechnology & Applied Microbiology↗

A Comprehensive Characterization of Microorganisms and Allergens in Spacecraft Environment

The determination of risk from infectious disease during long-duration missions is composed of several factors including (1) the host#s susceptibility, (2) the host#s exposure to the infectious disease agent, and (3) the concentration of the infectious agent, and (4) the characteristics of the infectious agent. While stringent steps are taken to minimize the transfer of potential pathogens to spacecraft, several medically significant organisms have been isolated from both the Mir and International Space Station (ISS). Historically, the method for isolation and identification of microorganisms from spacecraft environmental samples depended upon their growth on culture media. Unfortunately, only a fraction of the organisms may grow on a culture medium, potentially omitting those microorganisms whose nutritional and physical requirements for growth are not met. Thus, several pathogens may not have been detected, such as Legionella pneumophila, the etiological agent of Legionnaire#s disease. We hypothesize that environmental analysis using non-culture-based technologies will reveal microorganisms, allergens, and microbial toxins not previously reported in spacecraft, allowing for a more complete health assessment. The development of techniques for this flight experiment, operationally named SWAB, has already provided advances in NASA laboratory processes and beneficial information toward human health risk assessment. The first accomplishment of the SWAB experiment was the incorporation of 16S ribosomal DNA sequencing for the identification of bacteria. The use of this molecular technique has increased bacterial speciation of environmental isolates from previous flights three fold compared to conventional methodology. This increased efficiency in bacterial speciation provides a better understanding of the microbial ecology and the potential risk to the crew. Additional SWAB studies focused on the use of molecular-based DNA fingerprinting using repetitive sequencebased polymerase chain reaction (rep-PCR). This technology has allowed contamination tracking of microorganisms between crewmembers and their environment. This study not only demonstrated that ISS has a greater diversity of organisms than originally expected, but also provided insight into possible routes of infection to the crew. Additional ground-based studies used rep-PCR and protein based assays to determine the potential of methicillin resistant Staphylococcus aureus (MRSA) aboard ISS. MRSA has become increasingly common on Earth and pose a treatment problem for infections during flight. While no MRSA have been isolated from ISS to date, the mecA gene product that is responsible for methicillin resistance was isolated in other Staphylococcus species aboard ISS suggesting a potential of MRSA through gene transfer. Using improved sample collection technologies, flight sampling for SWAB was initiated in August 2006 and should continue through spring of 2007. The focus of these flight samples is the collection of DNA for evaluation by Denaturing Gradient Gel Electrophoresis (DGGE). Unlike other techniques, DGGE does not depend on any microbial growth on culture media allowing a more comprehensive assessment of the spacecraft interior. This study should provide insight into the true microbial ecology that is experienced by the crew during flight. This information will lead toward an accurate microbial risk assessment to help set flight requirements to protect the safety, health, and performance of the crew.

Ott, C. M.↗