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All sourcesNASA NTRSDOE OSTI
NASA NTRS1997

Activation and proliferation of lymphocytes and other mammalian cells in microgravity

The experimental findings reviewed in this chapter support the following conclusions: Proliferation. Human T-lymphocytes, associated with monocytes as accessory cells, show dramatic changes in the centrifuge, in the clinostat and in space. In free-floating cells the mitogenic response is depressed by 90% in microgravity, whereas in cells attached to a substratum activation is enhanced by 100% compared to 1-G ground and inflight controls. The duration of phase G1 of the mitotic cycle of HeLa cells is reduced in hypergravity, resulting in an increased proliferation rate. Other systems like Friend cells and WI38 human embryonic lung cells do not show significant changes. Genetic expression and signal transduction. T-lymphocytes and monocytes show important changes in the expression of cytokines like interleukin-1, interleukin-2, interferon-gamma and tumor necrosis factor. The data from space experiments in Spacelab, Space Shuttle mid-deck, and Biokosmos have helped to clarify certain aspects of the mechanism of T-cell activation. Epidermoid A431 cells show changes in the genetic expression of the proto-oncogenes c-fos and c-jun in the clinostat and in sounding rockets. Membrane function, in particular the binding of ligates as first messengers of a signal, is not changed in most of the cell systems in microgravity. Morphology and Mortility. Free cells, lymphocytes in particular, are able to move and form aggregates in microgravity, indicating that cell-cell contacts and cell communications do take place in microgravity. Dramatic morphological and ultrastructural changes are not detected in cells cultured in microgravity. Important experiments with single mammalian cells, including immune cells, were carried out recently in three Spacelab flights, (SL-J, D-2, and IML-2 in 1992, 1993, and 1994, respectively). The results of the D-2 mission have been published in ref. 75; those of the IML-2 mission in ref. 76. Finally, many cell biology experiments in space have suffered in the past from a lack of adequate controls (like 1-G centrifuges) and of proper experimental conditions (like well-controlled temperature). In this respect the availability of Biorack, outfitted with proper incubators with 1-G control centrifuge as well as a glovebox with a microscope, is a great advantage. It is also desirable that cell biology experiments in space are accompanied or even preceded by a program of ground-based investigations in the fast rotating clinostat and in the centrifuge, and that preparatory experiments be done in parabolic flights and sounding rockets, whenever possible. Proper publication of the results of space experiments is another important need. A great number of data have been published in proceedings and reports that are not available to the broad scientific community. To guarantee the credibility and the international recognition of space biology it is important that the results be published in international, peer reviewed journals.

manned↗
NASA NTRS1995

Structurally complex and highly active RNA ligases derived from random RNA sequences

Seven families of RNA ligases, previously isolated from random RNA sequences, fall into three classes on the basis of secondary structure and regiospecificity of ligation. Two of the three classes of ribozymes have been engineered to act as true enzymes, catalyzing the multiple-turnover transformation of substrates into products. The most complex of these ribozymes has a minimal catalytic domain of 93 nucleotides. An optimized version of this ribozyme has a kcat exceeding one per second, a value far greater than that of most natural RNA catalysts and approaching that of comparable protein enzymes. The fact that such a large and complex ligase emerged from a very limited sampling of sequence space implies the existence of a large number of distinct RNA structures of equivalent complexity and activity.

Non-NASA Center↗
NASA NTRS1993

Modeling study on the cleavage step of the self-splicing reaction in group I introns

A three-dimensional model of the Tetrahymena thermophila group I intron is used to further explore the catalytic mechanism of the transphosphorylation reaction of the cleavage step. Based on the coordinates of the catalytic core model proposed by Michel and Westhof (Michel, F., Westhof, E. J. Mol. Biol. 216, 585-610 (1990)), we first converted their ligation step model into a model of the cleavage step by the substitution of several bases and the removal of helix P9. Next, an attempt to place a trigonal bipyramidal transition state model in the active site revealed that this modified model for the cleavage step could not accommodate the transition state due to insufficient space. A lowering of P1 helix relative to surrounding helices provided the additional space required. Simultaneously, it provided a better starting geometry to model the molecular contacts proposed by Pyle et al. (Pyle, A. M., Murphy, F. L., Cech, T. R. Nature 358, 123-128. (1992)), based on mutational studies involving the J8/7 segment. Two hydrated Mg2+ complexes were placed in the active site of the ribozyme model, using the crystal structure of the functionally similar Klenow fragment (Beese, L.S., Steitz, T.A. EMBO J. 10, 25-33 (1991)) as a guide. The presence of two metal ions in the active site of the intron differs from previous models, which incorporate one metal ion in the catalytic site to fulfill the postulated roles of Mg2+ in catalysis. The reaction profile is simulated based on a trigonal bipyramidal transition state, and the role of the hydrated Mg2+ complexes in catalysis is further explored using molecular orbital calculations.

NASA Discipline Exobiology↗
NASA NTRS1988

Serotonin, atherosclerosis, and collateral vessel spasm

Studies on animal models demonstrate that platelet products contribute to vascular spasm in ischemic syndromes and that this is reversible with administration of ketanserin and thromboxane synthesis inhibitors. Laboratory animals (dogs, rabbits, and rats) that had femoral artery ligations exhibited supersensitivity to serotonin within days in their collateral blood vessels. This supersensitivity lasted at least 6 months. The response to serotonin was reversed by ketanserin, but not by 5HT-1 antagonists. Supersensitivity does not extend to norepinephrine, and alpha blockers do not influence the response to serotonin. It appears that platelet activation by endothelial injury contributes to ischemia through blood vessel occlusion and vascular spasm. When platelet activation occurs in vivo, blood vessel occlusion and vascular spasm are reversible in part by using ketanserin or agents that block thromboxane synthesis or its action. Combining both classes of agents reverses spasm completely. These findings support existing evidence that platelet products contribute to vascular disease, and provide an approach to improved management with currently available pharmacologic agents.

Review, Tutorial↗
NASA NTRS2012

Processes that Drove the Transition from Chemistry to Biology: Concepts and Evidence

Two properties are particularly germane to the transition from chemistry to biology. One is the emergence of complex molecules (polymers) capable of performing non-trivial functions, such as catalysis, energy transduction or transport across cell walls. The other is the ability of several functions to work in concert to provide reproductive advantage to systems hosting these functions. Biological systems exhibit these properties at remarkable levels of efficiency and accuracy in a way that appears effortless. However, dissection of these properties reveals great complexities that are involved. This opens a question: how a simple, ancestral system could have acquired the required properties? Other questions follow. What are the chances that a functional polymer emerges at random? What is the minimum structural complexity of a polymer to carry out a function at a reasonable level of efficiency? Can we identify concrete, protobiologically plausible mechanisms that yield advantageous coupling between different functions? These and similar questions are at the core of the main topic of this session: how soulless chemistry became life? Clearly, we do not have complete answers to any of these questions. However, in recent years a number of new and sometimes unexpected clues have been brought to light. Of particular interest are proteins because they are the main functional polymers in contemporary cells. The emergence of protein functions is a puzzle. It is widely accepted that a well ]defined, compact structure (fold) is a prerequisite for function. It is equally widely accepted that compact folds are rare among random amino acid polymers. Then, how did protein functionality start? According to one hypothesis well folded were preceded by their poorly folded, yet still functional ancestors. Only recently, however, experimental evidence supporting this hypothesis has been presented. In particular, a small enzyme capable of ligating two RNA fragments with the rate of 106 above background was evolved in vitro. This enzyme does not look like any contemporary protein. It is very flexible and its structure is kept together just by a single salt bridge between a charged residue and a coordinating zinc. A similar picture emerges from studies of simple transmembrane channels that mimic those in ancestral cells. Again, they are extremely flexible and do not form a conventional pore. Yet, they efficiently mediate ion transport. Studies on simple proteins that are on-going in several laboratories hold promise of revealing crucial links between chemical and biological catalysis and other ubiquitous cell functions. Interaction between composition, growth and division of protobiologically relevant vesicles and metabolic reactions that they encapsulate is an example of coupling between simple functions that promotes reproduction and evolution. Recent studies have demonstrated possible mechanisms by which vesicles might have evolved their composition from fatty acids to phospholipids, thus facilitating a number of new cellular functions. Conversely, it has been also demonstrated that an encapsulated metabolism might drive vesicle division. These are, again, examples of processes that might have driven the transition from chemistry to biology.

Pohorille, Andrew↗
NASA NTRS2014

Proteins with Novel Structure, Function and Dynamics

Recently, a small enzyme that ligates two RNA fragments with the rate of 10(exp 6) above background was evolved in vitro (Seelig and Szostak, Nature 448:828‐831, 2007). This enzyme does not resemble any contemporary protein (Chao et al., Nature Chem. Biol. 9:81‐83, 2013). It consists of a dynamic, catalytic loop, a small, rigid core containing two zinc ions coordinated by neighboring amino acids, and two highly flexible tails that might be unimportant for protein function. In contrast to other proteins, this enzyme does not contain ordered secondary structure elements, such as alpha‐helix or beta‐sheet. The loop is kept together by just two interactions of a charged residue and a histidine with a zinc ion, which they coordinate on the opposite side of the loop. Such structure appears to be very fragile. Surprisingly, computer simulations indicate otherwise. As the coordinating, charged residue is mutated to alanine, another, nearby charged residue takes its place, thus keeping the structure nearly intact. If this residue is also substituted by alanine a salt bridge involving two other, charged residues on the opposite sides of the loop keeps the loop in place. These adjustments are facilitated by high flexibility of the protein. Computational predictions have been confirmed experimentally, as both mutants retain full activity and overall structure. These results challenge our notions about what is required for protein activity and about the relationship between protein dynamics, stability and robustness. We hypothesize that small, highly dynamic proteins could be both active and fault tolerant in ways that many other proteins are not, i.e. they can adjust to retain their structure and activity even if subjected to mutations in structurally critical regions. This opens the doors for designing proteins with novel functions, structures and dynamics that have not been yet considered.

Proteins↗
NASA NTRS2015

Life in the Clouds of Venus? An Experimental Synthetic Biology Approach

The surface of Venus constitutes the most hellish and biologically inhospitable planetary surface in our solar system, boasting a pH of 0, blistering winds that can melt lead, and pressures of 60 atm. However, during the earlier years of the solar system, without the runaway greenhouse effect that has plagued the planet, Venus potentially housed oceans and perhaps even life. There is a possibility that microbes could have retreated into hospitable niches in the atmosphere, as suggested by Carl Sagan as early as 1967 [1]. For example, 50 km above the raging hell of the Venusian surface, exists a relatively temperate environment that might serve as reservoir for life. This astrobiology project seeks to explore life at the extremes and to theorize whether microbial communities could not only survive but also reproduce in the Venusian atmosphere. Specifically, we ask: are aerosols viable microbial environments? But before we can test for life in the clouds, we have to develop a proper reporter to visualize cell growth in situ. For this purpose, we aimed to develop cell-growth dependent reporters to serve as remote biosensors for cell growth. We developed two using the polA promoter, a DNA-replication dependent promoter, and nrd operon promoter, a cell-cycle dependent promoter. Using these cell-growth reporters, the next step is to aerosolize microbes expressing these reporters in a suspension chamber adapted from a Millikan Drop Apparatus to assay reproduction in an aerosolized environment. Better yet is to test the reproduction of microbes in a microgravity regime such as on ISS.Approach: We engineered two cell-cycle dependent genetic reporters. One was the polA promoter which codes for DNA Polymerase I, a gene active in DNA replication [2]. The other was the nrdP. The activation of ribonucleotide reductase reduces ribonucleotides into deoxyribonucleotides and is involved in the bacterial cell cycle [3]. This promoter began activation during the initiation of DNA replication and is cell-cycle dependent [4]. These promoters were fused to a GFP reporter, transformed into E. coli. The constructs were deposited in the iGEM registry as K847210: Escherichia coli DNA-replication dependent polA promoter K847211: Escherichia coli cell-division dependent nrd promoter Results: Our constructs displayed fluorescence when transformed into NEB-5alpha competent cells. While nrdP-E0840 displayed sufficient fluorescence as verified by fluorescent microscopy, the original polAP-E0840 construct (which uses mut3b GFP) exhibited low expression; while fluorescence was visible under the microscope, the signal was too weak for the camera to recognize. The polA promoter was therefore digest-ed with EcoRI and SpeI then ligated into plasmid pNCS containing a RBS, Clover, and a terminator. Clover is a highly engineered green fluorescent protein that exhibits extreme brightness [5] Fluoresence time course data demonsrated that the genes were induced in a cell cycle dependant manner [6]. Our assays via microscopy and the bulk assay shows that our promoters are functional as cell cycle reporters.Conclusions: The application of such tools are widespread and not limited to astrobiology; nrdP could be used to determine doubling times empirically and could possibly extrapolate DNA content from intensity of signals expressed by polAP. However, we are pri-marily interested in its use in astrobiology.

Escherichia↗
NASA NTRS2015

CRISPR/Cas9-Assisted Transformation-Efficient Reaction (CRATER) for Near-Perfect Selective Transformation

The CRISPR (Clustered, Regularly Interspaced, Short Palindromic Repeats)/Cas9 system has revolutionized genome editing by providing unprecedented DNA-targeting specificity. Here we demonstrate that this system can be also applied in vitro to fundamental cloning steps to facilitate efficient plasmid selection for transformation and selective gene insertion into plasmid vectors by cleaving unwanted plasmid byproducts with a single-guide RNA (sgRNA)-Cas9 nuclease complex. Using fluorescent and chromogenic proteins as reporters, we demonstrate that CRISPR/Cas9 cleavage excludes multiple plasmids as well as unwanted ligation byproducts resulting in an unprecedented increase in the transformation success rate from approximately 20% to nearly 100%. Thus, this CRISPR/Cas9-Assisted Transformation-Efficient Reaction (CRATER) protocol is a novel, inexpensive, and convenient application to conventional molecular cloning to achieve near-perfect selective transformation.

molecular cloning↗
NASA NTRS2018

Recombinant Spidroins Fully Replicate Primary Mechanical Properties of Natural Spider Silk

Dragline spider silk is among the strongest and toughest bio-based materials, capable of outperforming most synthetic polymers and even some metal alloys.1,2,3,4 These properties have gained spider silk a growing list of potential applications that, coupled with the impracticalities of spider farming, have driven a decades-long effort to produce recombinant spider silk proteins (spidroins) in engineered heterologous hosts.2 However, these efforts have so far been unable to yield synthetic silk fibers with mechanical properties equivalent to natural spider silk, largely due to an inability to stably produce highly repetitive, high molecular weight (MW) spidroins in heterologous hosts.1,5 Here we address these issues by combining synthetic biology techniques with split intein (SI)- mediated ligation for the bioproduction of spidroins with unprecedented MW (556 kDa), containing 192 repeat motifs of the Nephila clavipes MaSp1 dragline spidroin. Fibers spun from these synthetic spidroins display ultimate tensile strength (σ), modulus (E), extensibility (ε), and toughness (UT) of 1.03 +/- 0.11 GPa, 13.7 +/- 3.0 GPa, 18 +/- 6%, and 114 +/- 51 MJ/m3, respectively-equivalent to the performance of natural N. clavipes dragline silk.6 This work demonstrates for the first time that microbially produced synthetic silk fibers can match the performance of natural silk fibers by all common metrics (σ, E, ε, UT), providing a more dependable source of high-strength fibers to replace natural spider silks for mechanically demanding applications. Furthermore, our biosynthetic platform can be potentially expanded for the assembly and production of other protein-based materials with high MW and repetitive sequences that have so far been impossible to synthesize by genetic means alone.

spider silk↗
NASA NTRS2017

Spectrum

Spectrum is a multispectral fluorescence imager designed for capturing in vivo genetic expression in a variety of biological organisms, providing a capability that does not currently exist on the International Space Station (ISS). Researching organisms that have been transformed with in vivo reporter genes ligated with fluorescent proteins allows the scientific community to further understand the fundamental biological responses of these organisms when subjected to space environments. Model organisms that may utilize multispectral imaging on the ISS include unicellular organisms (e.g. Saccharomyces cerevisiae), plants (e.g. Arabidopsis thaliana), and invertebrates (e.g. Caenorhabditis elegans).

Levine, Howard G.↗
NASA NTRS2017

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↗
DOE OSTI2020

Electrochemical Deposition of Aluminum-Based Coatings From Aqueous Systems

The electroplating of aluminum has represented a challenge for the coatings industry for decades. The reactivity of aluminum metal leads to a very negative reduction potential. This leads to abundant gas evolution instead of aluminum reduction in water. Current aluminum deposition technology overcomes this limitation by excluding oxygen and water from the process with rigorously dry solvent and airtight plating tanks. These adaptations yield metallic aluminum depositions with significant impurities affecting its properties. In addition, this approach requires a great capital investment and cost of process maintenance. An alternative approach to Al electrodeposition has been developed based on the the use of organic ligands to stabilize the Al3+ ion in water-based solutions. By using a ligation approach, we can create aluminum species that remain stable in water while lowering the reduction potential of the aluminum complex by 0.5V. This improvement in reduction potential gives the possibility for the deposition of aluminum without significant competition from hydrogen evolution. With this platform we can deposit highly adherent aluminum-based layers on metal surfaces. The aluminum/aluminum oxide layers appear as thin, coherent and highly adherent coatings. This aluminum/alumina layer can then be chemically treated to achieve desirable surface properties which were previously inaccessible. For example, the coating can be made hydrophobic, highly adherent to different polymer coatings or even dyed using common anodizing dyes.

36 MATERIALS SCIENCE↗
DOE OSTI2020

ELECTROCHEMICAL DEPOSITION OF ALUMINUM-BASED COATINGS FROM AQUEOUS SYSTEMS

The electroplating of aluminum has represented a challenge for the coatings industry for decades. The reactivity of aluminum metal leads to a very negative reduction potential. This leads to abundant gas evolution instead of aluminum reduction in water. Current aluminum deposition technology overcomes this limitation by excluding oxygen and water from the process with rigorously dry solvent and airtight plating tanks. These adaptations yield metallic aluminum depositions with significant impurities affecting its properties. In addition, this approach requires a great capital investment and cost of process maintenance. An alternative approach to Al electrodeposition has been developed based on the the use of organic ligands to stabilize the Al3+ ion in water-based solutions. By using a ligation approach, we can create aluminum species that remain stable in water while lowering the reduction potential of the aluminum complex by 0.5V. This improvement in reduction potential gives the possibility for the deposition of aluminum without significant competition from hydrogen evolution. With this platform we can deposit highly adherent aluminum-based layers on metal surfaces. The aluminum/aluminum oxide layers appear as thin, coherent and highly adherent coatings. This aluminum/alumina layer can then be chemically treated to achieve desirable surface properties which were previously inaccessible. For example, the coating can be made hydrophobic, highly adherent to different polymer coatings or even dyed using common anodizing dyes.

36 MATERIALS SCIENCE↗
DOE OSTI2020

Rhodol fluorophores for near-infrared imaging

The present disclosure encompasses embodiments of a novel near-infrared-emitting molecular fluorophore and probes incorporating said fluorophore advantageous for in vitro and in vivo research studies. The fluorophore is robust, photostable, and possesses functionalities for easy bioorthogonal conjugation (e.g., click chemistry, hydrazone formation, Diels Alder, Staudinger ligation, etc.). It is biocompatible and emits at 711 nm in aqueous conditions. These fluorophores may be used to fluorescently tag biological molecules or structures of interest, or used as optical reporters (i.e., activatable molecular probes, fluorescent dyes) for specific biomarkers/analytes as they can be switched from “off” to “on.” This fluorophore is useful for cellular assays and preclinical small animal imaging as the near-infrared emission is highly penetrating, and the photophysical properties are outstanding. As such, the properties of this class of fluorophores could easily be translated for use in clinical applications.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

Synthetic apolipoproteins, and related compositions methods and systems for nanolipoprotein particles formation

Synthetic apolipoproteins based on native/naturally occurring homolog proteins can be prepared using solid-phase peptide synthesis approaches combined with native chemical ligation methods to create analogs of full length apolipoproteins. The chemical synthesis is expected to allow introduction of non-natural amino acids, e.g., α,α′-dialkyl amino acids, with a periodicity that encourages both helix formation and amphipathicity. Such apolipoprotein analogs are expected to encourage, in some embodiments, facile and more complete NLP formation, enabling consideration of full spectrum of nanoparticle-based biotechnology applications ranging from therapeutic sequestration and delivery to energy/biofuel production to biopolymer production.

Source record↗
DOE OSTI2023

Cloning and expression vectors and systems

Vectors for cloning, maintaining and expressing a wide range of coding sequences in inducible T7 expression systems in Escherichia coli expression hosts are disclosed herein. Target genes that can be stably maintained and expressed include those that specify proteins that are highly toxic to the host cell. Different configurations of vectors and expression hosts provide different rates of transcription and translation of target genes and therefore different rates of accumulation of target proteins. Methods for cloning by asymmetric ligation and co-expression of more than one target protein in a single vector are also disclosed, as are variants of BL21(DE3) having lower basal transcription by T7 RNA polymerase.

Studier, F. William↗
DOE OSTI2023

Methods for making polypeptides including d-amino acids

A method of making a polypeptide including one or more D-amino acids is provided. The method includes combining a ribosome with protein translation factors including (1) a template encoding the polypeptide, wherein the template encoding the polypeptide includes one or more codons which have been recoded to accept a tRNA attached to a D-amino acid, (2) a plurality of L-amino acids and a plurality of corresponding tRNAs, (3) a plurality of D-amino acids and their corresponding aminoacyl tRNA synthetase or a plurality of tRNAs ligated with a D-amino acid, and (4) elongation factor P in a concentration of 2 to 20 micromolar, wherein translation of the template encoding the polypeptide occurs to produce the polypeptide including one or more D-amino acids.

Church, George M.↗