Neurosciences research program bulletin volume 3, no. 3 - information storage and processing in biomolecular systems
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Preservation of Lipid Biomarkers Under Prolonged and Extreme Hyperaridity in Atacama Desert Soils
Molecular biomarkers are the most direct biosignatures of life on early Earth and a key target in the search for life on Mars. Lipid biomarkers are of particular interest given their ability to survive oxidative degradation and record microbial presence and activity of microorganisms that occurred billions of years ago (Eigenbrode, 2008). Environmental conditions that suspend biotic and abiotic degradative processes prior to lithification can lead to enhanced biomolecular preservation over geological time-scales. The hyperarid core of the Atacama Desert in northern Chile offers a unique environment to investigate lipid biomarker taphonomy under extreme and prolonged dryness. We investigated the accumulation and degree of preservation of lipid biomarkers in million-year-old hyperarid soils where primarily abiotic conditions influence their taphonomy. Soils were extracted and free and membrane bound lipids were analyzed across a vertical profile of 2.5 meters in the Yungay hyper-arid core of the Atacama Desert. Due to the extremely low inventory of biomass in Atacama soils, samples were collected by scientists wearing cleanroom suits to minimize anthropogenic contamination during sampling. Fatty acids were found to be well preserved in Yungay soils, and were most abundant in the clay-rich soils at approx.2 m depth (approx.750 ng of fatty acid methyl ester/g of soil). These buried clays layers were fluvially deposited approximately 2 million years ago, and have been excluded from exposure to rainwater and modern surficial processes since their emplacement (Ewing et al., 2008). Monocarboxylic fatty acid, monohydroxy fatty acid, glycerol tetraether, and n-alkane hydrocarbon content was found to change with depth. Lipid biomarker content in deeper soil layers is suggestive of soils having been formed at a time when environmental conditions were capable of supporting active microbial communities and plants. In short, total lipid extracts reveal a remarkable degree of lipid biomarker preservation even in the oldest soils analyzed (ca. 2 Myr) indicating that typical diagenetic processes of lipid destruction are arrested under extreme dryness. This result has implications for the search for molecular biomarkers on Mars, which could have experienced millions to billions of years of extreme hyperaridity.
Lipid Biomarkers↗
Excess of L-Alanine in Amino Acids Synthesized in a Plasma Torch Generated by a Hypervelocity Meteorite Impact Reproduced in the Laboratory
We present a laboratory reproduction of hypervelocity impacts of a carbon containing meteorite on a mineral substance representative of planetary surfaces. The physical conditions of the resulting impact plasma torch provide favorable conditions for abiogenic synthesis of protein amino acids: We identified glycine and alanine, and in smaller quantities serine, in the produced material. Moreover, we observe breaking of alanine mirror symmetry with L excess, which coincides with the bioorganic world. Therefore the selection of L-amino acids for the formation of proteins for living matter could have been the result from plasma processes occurring during the impact meteorites on the surface. This indicates that the plasma torch from meteorite impacts could play an important role in the formation of biomolecular homochirality. Thus, meteorite impacts possibly were the initial stage of this process and promoted conditions for the emergence of a living matter.
George G Managadze↗
Investigating Biological Responses to Deep Space Radiation for Missions Beyond Low Earth Orbit (LEO) using Yeast
To enable long-term spaceflight missions and establish habitation on the Moon and Mars, we require a comprehensive understanding of the effects of chronic deep space radiation exposure on humans. BioSentinel is NASA’s first biological CubeSat to venture beyond Low Earth Orbit (LEO). It utilizes Saccharomyces cerevisiae (budding yeast) as a model organism to study biological responses to deep space radiation. Yeast share significant genetic homology with humans, including basic cellular metabolism and DNA repair mechanisms. In addition, unlike human cell cultures, yeast can survive the duration and constraints of a deep space mission. BioSentinel measures biological responses using an optical system and alamarBlue oxidation-reduction (redox) dye. Two strains of yeast are studied - a wild-type and a rad51 mutant strain that is deficient in DNA repair. Changes in metabolism and growth are monitored throughout the nominal 6-month mission. Preliminary tests indicate a significant change in the alamarBlue response to low-dose ionizing radiation (IR). Additionally, rad51 cells have shown an IR dose-dependent decrease in glucose uptake and accumulation of oxidized NADH (NAD+). These biomolecules are involved in reactions responsible for basic cell processes, including growth and development, signaling, and respiration. The current study expanded upon previous data by exposing yeast to deep space-relevant radiation. Glucose and NADH/NAD+ assays were conducted on yeast subjected to varying dosages of high-energy Fe-56 and simulated galactic cosmic rays (GCRs). The resulting data was analyzed using Excel and GraphPad Prism. A particular focus was to identify biomolecules resulting from aerobic respiration, which requires the presence of oxygen, or anaerobic processes. As long-term spaceflight missions draw near, it is increasingly important to characterize biological processes affected by the conditions of deep space. Studying biomolecular damage caused by deep space radiation may enable the development of engineering controls or biomedical therapeutics that mitigate health complications for future astronauts.
Kyra Keenan↗
Impact of Track Structure Effects on Shielding and Dosimetry
Galactic cosmic rays (GCR) consisting of nuclei of all the known elements with kinetic energies extending from tens to millions of MeV pose a significant health hazard to future deep space operations. Even half of the radiation exposures expected in ISS will result from GCR components. The biological actions of these radiations are known to depend on the details of the energy deposition (not just linear energy transfer, LET, but the lateral dispersion of energy deposition about the particle track). Energy deposits in tissues are dominated by the transfer of tens to hundreds of eV to the tissue's atomic electrons. In the case of low LET radiations, the collisions are separated by large dimensions compared to the size of important biomolecular structures. If such events are also separated in time, then the radiation adds little to the background of radicals occurring from ordinary metabolic processes and causes little or no biological injury. Hence, dose rate is a strong determinant of the action of low LET exposures. The GCR exposures are dominated by ions of high charge and energy (HZE) characterized by many collisions with atomic electrons over biomolecular dimensions, resulting in high radical- density events associated with a few isolated ion paths through the cell and minimal dose rate dependence at ordinary exposure levels. The HZE energy deposit declines quickly laterally and merges with the background radical density in the track periphery for which the exact lateral distribution of the energy deposit is the determinant of the biological injury. Although little data exists on human exposures from HZE radiations, limited studies in mice and mammalian cell cultures allow evaluation of the effects of track structure on shield attenuation properties and evaluation of implications for dosimetry. The most complete mammalian cell HZE exposure data sets have been modeled including the C3H10T1/2 survival and transformation data of Yang et al., the V79 survival and mutation data of various groups, and the Harderian gland tumor data of Alpen et al. Model results for the Harderian gland tumor data in comparison with data from Alpen et al. The Harderian target cell initiation cross section compares closely with the transformation cross section found for the C3H10T1/2 cell transformation data of Yang et al. The most notable feature of the cross sections are the multivalued cross sections for a given LET which implies the corresponding relative biological effectiveness (RBE) is dependent not only on the LET but also the ion type. This fact is at variance with the latest ICRP recommended quality factor which is a defined function of only the LET.
Wilson, J. W.↗
Kinetics of DSB rejoining and formation of simple chromosome exchange aberrations
PURPOSE: To investigate the role of kinetics in the processing of DNA double strand breaks (DSB), and the formation of simple chromosome exchange aberrations following X-ray exposures to mammalian cells based on an enzymatic approach. METHODS: Using computer simulations based on a biochemical approach, rate-equations that describe the processing of DSB through the formation of a DNA-enzyme complex were formulated. A second model that allows for competition between two processing pathways was also formulated. The formation of simple exchange aberrations was modelled as misrepair during the recombination of single DSB with undamaged DNA. Non-linear coupled differential equations corresponding to biochemical pathways were solved numerically by fitting to experimental data. RESULTS: When mediated by a DSB repair enzyme complex, the processing of single DSB showed a complex behaviour that gives the appearance of fast and slow components of rejoining. This is due to the time-delay caused by the action time of enzymes in biomolecular reactions. It is shown that the kinetic- and dose-responses of simple chromosome exchange aberrations are well described by a recombination model of DSB interacting with undamaged DNA when aberration formation increases with linear dose-dependence. Competition between two or more recombination processes is shown to lead to the formation of simple exchange aberrations with a dose-dependence similar to that of a linear quadratic model. CONCLUSIONS: Using a minimal number of assumptions, the kinetics and dose response observed experimentally for DSB rejoining and the formation of simple chromosome exchange aberrations are shown to be consistent with kinetic models based on enzymatic reaction approaches. A non-linear dose response for simple exchange aberrations is possible in a model of recombination of DNA containing a DSB with undamaged DNA when two or more pathways compete for DSB repair.
DNA Damage↗
Cross-Disciplinarity in the Advance of Antarctic Ecosystem Research
The biodiversity, ecosystem services and climate variability of the Antarctic continent, and the Southern Ocean are major components of the whole Earth system. Antarctic ecosystems are driven more strongly by the physical environment than many other marine and terrestrial ecosystems. As a consequence, to understand ecological functioning, cross-disciplinary studies are especially important in Antarctic research. The conceptual study presented here is based on a workshop initiated by the Research Programme Antarctic Thresholds - Ecosystem Resilience and Adaption of the Scientific Committee on Antarctic Research, which focused on challenges in identifying and applying cross-disciplinary approaches in the Antarctic. Novel ideas, and first steps in their implementation, were clustered into eight themes, ranging from scale problems, risk maps, organism and ecosystem responses to multiple environmental changes, to evolutionary processes. Scaling models and data across different spatial and temporal scales were identified as an overarching challenge. Approaches to bridge gaps in the research programmes included multi-disciplinary monitoring, linking biomolecular findings and simulated physical environments, as well as integrative ecological modelling. New strategies in academic education are proposed. The results of advanced cross-disciplinary approaches can contribute significantly to our knowledge of ecosystem functioning, the consequences of climate change, and to global assessments that ultimately benefit humankind.
Scaling↗
DspaceOgre 3D Graphics Visualization Tool
This general-purpose 3D graphics visualization C++ tool is designed for visualization of simulation and analysis data for articulated mechanisms. Examples of such systems are vehicles, robotic arms, biomechanics models, and biomolecular structures. DspaceOgre builds upon the open-source Ogre3D graphics visualization library. It provides additional classes to support the management of complex scenes involving multiple viewpoints and different scene groups, and can be used as a remote graphics server. This software provides improved support for adding programs at the graphics processing unit (GPU) level for improved performance. It also improves upon the messaging interface it exposes for use as a visualization server.
Jain, Abhinandan↗
NASA Tech Briefs, February 2006
Topics discussed include: Nearly Direct Measurement of Relative Permittivity; DCS-Neural-Network Program for Aircraft Control and Testing; Dielectric Heaters for Testing Spacecraft Nuclear Reactors; Using Doppler Shifts of GPS Signals To Measure Angular Speed; Monitoring Temperatures of Tires Using Luminescent Materials; Highly Efficient Multilayer Thermoelectric Devices; Very High-Speed Digital Video Capability for In-Flight Use; MMIC DHBT Common-Base Amplifier for 172 GHz; Modular, Microprocessor-Controlled Flash Lighting System; Generic Environment for Simulating Launch Operations; Modular Aero-Propulsion System Simulation; X-Windows Socket Widget Class; Infrastructure for Rapid Development of Java GUI Programs; Processing Raman Spectra of High-Pressure Hydrogen Flames; X-Windows Information Sharing Protocol Widget Class; Simulating Humans as Integral Parts of Spacecraft Missions; Analyzing Power Supply and Demand on the ISS; Polyimides From a-BPDA and Aromatic Diamines; Making Plant-Support Structures From Waste Plant Fiber; Large Deployable Reflectarray Antenna; Periodically Discharging, Gas-Coalescing Filter; Ion Milling On Steps for Fabrication of Nanowires; Neuro-Prosthetic Implants With Adjustable Electrode Arrays; Microfluidic Devices for Studying Biomolecular Interactions; Studying Functions of All Yeast Genes Simultaneously; Polarization Phase-Compensating Coats for Metallic Mirrors; Tunable-Bandwidth Filter System; Methodology for Designing Fault-Protection Software; and Ground-Based Localization of Mars Rovers.
Source record↗
Radiation Risk Assessment of the Individual Astronaut: A Complement to Radiation Interests at the NIH
Predicting human risks following exposure to space radiation is uncertain in part because of unpredictable distribution of high-LET and low-dose-derived damage amongst cells in tissues, unknown synergistic effects of microgravity upon gene- and protein-expression, and inadequately modeled processing of radiation-induced damage within cells to produce rare and late-appearing malignant cancers. Furthermore, estimation of risks of radiogenic outcome within small numbers of astronauts is not possible using classic epidemiologic study. It therefore seems useful to develop strategies of risk-assessment based upon large datasets acquired from correlated biological models useful for resolving radiogenic risk-assessment for irradiated individuals. In this regard, it is suggested that sensitive cellular biodosimeters that simultaneously report 1) the quantity of absorbed dose after exposure to ionizing radiation, 2) the quality of radiation delivering that dose, and 3) the biomolecular risk of malignant transformation be developed in order to resolve these NASA-specific challenges. Multiparametric cellular biodosimeters could be developed using analyses of gene-expression and protein-expression whereby large datasets of cellular response to radiation-induced damage are analyzed for markers predictive for acute response as well as cancer-risk. A new paradigm is accordingly addressed wherein genomic and proteomic datasets are registered and interrogated in order to provide statistically significant dose-dependent risk estimation in individual astronauts. This evaluation of the individual for assessment of radiogenic outcomes connects to NIH program in that such a paradigm also supports assignment of a given patient to a specific therapy, the diagnosis of response of that patient to therapy, and the prediction of risks accumulated by that patient during therapy - such as risks incurred by scatter and neutrons produced during high-energy Intensity-Modulated Radiation Therapy. Value of assessment of radiogenic outcome for individuals exposed to radiation is suggested to be common to both NASA and NIH.
Richmond, Robert C.↗
The Icebreaker Life Mission to Mars: A Search for Biomolecular Evidence for Life
The search for evidence of life on Mars is the primary motivation for the exploration of that planet. The results from previous missions, and the Phoenix mission in particular, indicate that the ice-cemented ground in the north polar plains is likely to be the most recently habitable place that is currently known on Mars. The near-surface ice likely provided adequate water activity during periods of high obliquity, ~ 5 Myr ago. Carbon dioxide and nitrogen is present in the atmosphere, and nitrates may be present in the soil. Perchlorate in the soil together with iron in basaltic rock provides a possible energy source for life. Furthermore, the presence of organics must once again be considered, as the results of the Viking GCMS are now suspect given the discovery of the thermally reactive perchlorate. Ground-ice may provide a way to preserve organic molecules for extended periods of time, especially organic biomarkers. The Mars Icebreaker Life mission focuses on the following science goals: 1. Search for specific biomolecules that would be conclusive evidence of life. 2. A general search for organic molecules in the ground ice. 3. Determine the processes of ground ice formation and the role of liquid water. 4. Understand the mechanical properties of the Mars polar ice-cemented soil. 5. Assess the recent habitability of the environment with respect to required elements to support life, energy sources, and possible toxic elements. And 6. Compare the elemental composition of the northern plains with mid-latitude sites. The Icebreaker Life payload has been designed around the Phoenix spacecraft and is targeted to a site near the Phoenix landing site. However, the Icebreaker payload could be supported on other Mars landing systems. Preliminary studies of the SpaceX Dragon lander show that it could support the Icebreaker payload for a landing either at the Phoenix site or at mid-latitudes. Duplicate samples could be cached as a target for possible return by a Mars Sample Return mission. If the samples were shown to contain organic biomarkers interest in returning them to Earth would be high.
Life detection↗
Archean geochemistry of formaldehyde and cyanide and the oligomerization of cyanohydrin
The sources and speciation of reduced carbon and nitrogen inferred for the early Archean are reviewed in terms of current observations and models, and known chemical reactions. Within this framework hydrogen cyanide and cyanide ion in significant concentration would have been eliminated by reaction with excess formaldehyde to form cyanohydrin (glycolonitrile), and with ferrous ion to formferrocyanide. Natural reactions of these molecules would under such conditions deserve special consideration in modeling of primordial organochemical processes. As a step in this direction, transformation reactions have been investigated involving glycolonitrile in the presence of water. We find that glycolonitrile, formed from formaldehyde and hydrogen cyanide or cyanide ion, spontaneously cyclodimerizes to 4-amino-2-hydroxymethyloxazole. The crystalline dimer is the major product at low temperatue (approximately 0 C); the yield diminishes with increasing temperature at the expense of polymerization and hydrolysis products. Hydrolysis of glycolamide and of oxazole yields a number of simpler organic molecules, including ammonia and glycolamide. The spontaneous polymerization of glycolonitrile and its dimer gives rise to soluble, cationic oligomers of as yet unknown structure, and, unless arrested, to a viscous liquid, insoluble in water. A loss of cyanide by reaction with formaldehyde, inferred for the early terrestrial hydrosphere and cryosphere would present a dilemma for hypotheses invoking cyanide and related compounds as concentrated reactants capable of forming biomolecular precursor species. Attempts to escape from its horns may take advantage of the efficient concentration and separation of cyanide as solid ferriferrocyanide, and most directly of reactions of glycolonitrile and its derivatives.
Arrhenius, T.↗
Radiation Effect on Human Tissue
Predicting the occurrence of human cancer following exposure of an epidemiologic population to any agent causing genetic damage is a difficult task. To an approximation, this is because the uncertainty of uniform exposure to the damaging agent, and the uncertainty of uniform processing of that damage within a complex set of biological variables, degrade the confidence of predicting the delayed expression of cancer as a relatively rare event within clinically normal individuals. This situation begs the need for alternate controlled experimental models that are predictive for the development of human cancer following exposures to agents causing genetic damage. Such models historically have not been of substantial proven value. It is more recently encouraging, however, that developments in molecular and cell biology have led to an expanded knowledge of human carcinogenesis, and of molecular markers associated with that process. It is therefore appropriate to consider new laboratory models developed to accomodate that expanded knowledge in order to assess the cancer risks associated with exposures to genotoxic agents. When ionizing radiation of space is the genotoxic agent, then a series of additional considerations for human cancer risk assessment must also be applied. These include the dose of radiation absorbed by tissue at different locations in the body, the quality of the absorbed radiation, the rate at which absorbed dose accumulates in tissue, the way in which absorbed dose is measured and calculated, and the alterations in incident radiation caused by shielding materials. It is clear that human cancer risk assessment for damage caused by ionizing radiation is a multidisciplinary responsibility, and that within this responsibility no single discipline can hold disproportionate sway if a risk assessment model of radiation-induced human cancer is to be developed that has proven value. Biomolecular and cellular markers from the work reported here are considered for use in assessing human cancer risk related to exposure to space radiation. This potential use must be integrated within the specified multidisciplinary context in order to create a new tool of molecular epidemiology that can hopefully then realistically assess this cancer risk.
Richmond, Robert C.↗
Biodosimetry as a New Paradigm for Determination of Radiation Risks and Risk-Mitigation in Astronauts Exposed to Space Radiation
Predicting risk of cancer in astronauts exposed to space radiation is challenging partly because uncertainties of absorption of dose and the processing of dose-related damage at the cellular level degrade the confidence of predicting the expression of cancer. Cellular biodosimeters that simultaneously report: 1) the quantity of absorbed dose after exposure to ionizing radiation, 2) the quality of radiation delivering that dose, and 3) the macromolecular profiles related to malignant transformation in cells absorbing that dose would therefore be useful. An approach to such a multiparametric biodosimeter will be reported, This is the demonstration of two dose-responsive field-effects of enhanced protein-expression. In one case, expression of keratin 18 (K18) in cultures of human mammary epithelial cells (HMEC) irradiated with cesium-137 gamma-rays is enhanced following exposure of log phase cells to relatively low doses of 30 to 90 cGy. K18 has been reported by a marker for tumor staging and for apoptosis. In the second case, expression of connexin 43 (Cx43) is increased in irradiated stationary phase cultures of HMEC, indicating enhanced formation of gap junctions. Gap junctions have been reported to be involved in bystander effects following irradiation. It is a biodosimeter for assessing radiogenic damage. It is suggested further that such biomolecular dosimetry may introduce a new paradigm for assessing cancer risk and risk-mitigation in individuals, a requirement for managing radiation health in astronauts during extended missions in space. This new paradigm is built upon the statistical power provided by the use of functional genomics and proteomics represented in combined gene- and protein-expression assays.
Richmond, Robert↗
Rational Design of Nanoplasmonic Array Geometries for Biosensing
Background: Molecular diagnostics provide early and accurate diagnosis, which is essential for the prevention and treatment of infectious as well as chronic diseases. These tests are designed to detect disease-specific bioanalytes such as nucleic acid (DNA or RNA) or protein (antigens, antibodies) biomarkers. In the context of infectious disease diagnosis, nucleic acid-based detection methods are known to provide more specific and sensitive results. Here, the presence of a unique sequence belonging to the pathogenic genomic material is targeted to identify species, organism, genera and/or antimicrobial resistant gene markers. The majority of the common nucleic acid based diagnostic techniques require amplification (polymerase chain reaction, isothermal amplification etc.) of the pathogenic genetic material prior to detection impacting diagnostic speed, complexity, and cost thereby limiting ease of use. Thus, the development of simplified nucleic acid-based diagnostics that can be even used in resource-poor settings may hugely benefit patients across the globe. Nanopath is a molecular diagnostics company utilizing a solid-state nanosensor to enable sequence-specific detection of target nucleic acids without the need of amplification. These nanostructures enable ultra-sensitive biomarker detection using geometric, feature-dependent properties highly dependent on the local dielectric environment, allowing them to be sensitive to low concentration binding events. This paper describes an application of this approach to provide highly relevant clinical information within a single doctor’s office visit. Intro: The Nanopath team is in collaboration with NASA (National Aeronautics and Space Administration) and NIST (National Institute of Standards and Technology) to push the bounds of the fundamental physics associated with their biosensing platform. The ability of metals to support electromagnetic surface waves gives rise to surface plasmons when optically illuminated. This property, and its strong sensitivity to changes in the local refractive index, allows for the use of metal nanoparticles as ultra-sensitive transducers. In prior work by members of this team, ensembles of randomly oriented nanoparticles (i.e., colloidal nanorods dispersed on chip) were employed for sequence-specific nucleic acid sensing (1-3). While these particle sensors have the advantage of rapid fabrication, they suffer from low sensitivity and quality factor due to the random particle dispersity. In contrast, in this study we employ ordered array nanoparticle ensembles which can be used to improve sensor sensitivity and figure-of-merit. Study Methods Overview: In this talk, we detail the results of sensing experiments and computational simulations to outline a rational design of the structure of these plasmonic nanoparticle arrays for biomolecular sensing. Through simulation and experiment, we iteratively tailor nanostructure dimension to provide high quality signal and large resonance shifts upon modeled nucleic acid binding. In particular, full-wave electromagnetic simulations were conducted using Lumerical photonic simulation software in which periodic boundary conditions were applied in the x- and y- dimensions for each of the nanoplasmonic sensor geometries. To simulate the resonance response to changes in the bulk solution in contact with the sensor surface, the refractive index of the surrounding media was changed appropriately. Nucleic acid hybridization events were modeled using either using spherical structures approximating the relevant radius of genomic material as estimated by polymer models, or as conformal layers with the known refractive indices for nucleic acids. On the basis of initial simulations, nanosensors were fabricated using traditional electron-beam lithography protocols at NIST. To evaluate consensus between simulations and experiments, bulk sensing experiments were carried out in which the resonance peaks were obtained by submerging the sensors in refractive index standards. Key nanosensor characteristics including resonance peak locations, resonance peak shifts as a function of refractive index, and figure of merit (FOM) of extinction curves were examined between the experimental and simulation results prior to proceeding with simulations on additional geometries and more complex solution conditions, and further device fabrication. This iterative process is repeated toward a rational design of nanoplasmonic array geometries for biosensing optimizing response for targeted disease detection. In summary, this study puts forth a methodology for rational design and characterization of regularly spaced nanoparticle arrays for optics-based biosensing. The results of this study will allow for more informed design of nanostructure geometries towards sequence-specific nucleic acid detection. These improved designs have the potential to improve clinical sensitivity and limit-of-detection across disease indication.
sensor↗
Novel End-to-End Molecular Biology Approach for Direct Nanopore 1D cDNA Sequencing of Reverse Transcribed mRNAs Purified from Cell Cultures by the NASA ISS WetLab2 SPM
Continued space bioscience research onboard the International Space Station (ISS) and future long-duration flight missions to the Moon or Mars will require the ability to conduct on-orbit molecular analysis of biological samples independently from Earth. In the last year two new molecular analytic technologies have been installed and the technologies demonstrated onboard the ISS: The Sample Prep Module (SPM) WetLab-2 (WL2) qRT-PCR toolbox and the Oxford Nanopore MinIon Biomolecule Sequencer. Here we describe protocol development and integration into existing ISS technology for end-to-end on-orbit biological sample processing and molecular analysis with real time results generated utilizing only field offline analytic software. For this experiment we isolated primary cells from bone marrow flushes of wild type B6129SF2 mice (Jackson Labs) long bones. The cell isolate was then processed using the SPM to produce total 147nanograms of RNA. The total RNA was purified to only messenger RNA (mRNA) and transferred to Smartcycler Thermocycle ISS kit consumable tube using Eppendorf gel loading pipette tips for further processing. Complementary first strand cDNA was synthesized using OLIGO dT priming followed by addition of SuperScript II Reverse Transcriptase and thermal cycling as per manufacturers instruction. All thermal cycling was conducted using the ISS WetLab-2 Cephid Smarcycler real time thermal cycler. Our protocol takes advantage of mRNAs native poly(A) tail, synthesized in vivo to protect the mRNA from degradation by endonucleases, to eliminate end-prep for adapter ligation. The adapted library is purified using MyOne C1 Streptavidin beads before elution in buffer. The pre-sequencing library is diluted in the loading buffer and injected into the MinIon sample port, drawn into the nanopore window by capillary action, and sequenced using the MinKnown software with local basecalling. The sequencing read produced 34.5 million events and local basecalling produced 117,301 successful reads. NCBI Blast of the data for the mouse genome resulted in 2,462 successful nucleotide collection matches (gene sequences) exceeding 70 homology. These results demonstrate the viability of this novel flight ready end-to-end sample analytic methodology and provide a real time homolog for flight experimentation utilizing supply kits and technologies that have already been demonstrated on ISS.
MinIon↗