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NASA NTRS1988

Methanohalophilus zhilinae sp. nov., an alkaliphilic, halophilic, methylotrophic methanogen

Methanohalophilus zhilinae, a new alkaliphilic, halophilic, methylotrophic species of methanogenic bacteria, is described. Strain WeN5T (T = type strain) from Bosa Lake of the Wadi el Natrun in Egypt was designated the type strain and was further characterized. This strain was nonmotile, able to catabolize dimethylsulfide, and able to grow in medium with a methyl group-containing substrate (such as methanol or trimethylamine) as the sole organic compound added. Sulfide (21 mM) inhibited cultures growing on trimethylamine. The antibiotic susceptibility pattern of strain WeN5T was typical of the pattern for archaeobacteria, and the guanine-plus-cytosine content of the deoxyribonucleic acid was 38 mol%. Characterization of the 16S ribosomal ribonucleic acid sequence indicated that strain WeN5T is phylogenetically distinct from members of previously described genera other than Methanohalophilus and supported the partition of halophilic methanogens into their own genus.

Non-NASA Center↗

NASA NTRS2001

Microarray analysis of genes differentially expressed in HepG2 cells cultured in simulated microgravity: preliminary report

Developed at NASA, the rotary cell culture system (RCCS) allows the creation of unique microgravity environment of low shear force, high-mass transfer, and enables three-dimensional (3D) cell culture of dissimilar cell types. Recently we demonstrated that a simulated microgravity is conducive for maintaining long-term cultures of functional hepatocytes and promote 3D cell assembly. Using deoxyribonucleic acid (DNA) microarray technology, it is now possible to measure the levels of thousands of different messenger ribonucleic acids (mRNAs) in a single hybridization step. This technique is particularly powerful for comparing gene expression in the same tissue under different environmental conditions. The aim of this research was to analyze gene expression of hepatoblastoma cell line (HepG2) during early stage of 3D-cell assembly in simulated microgravity. For this, mRNA from HepG2 cultured in the RCCS was analyzed by deoxyribonucleic acid microarray. Analyses of HepG2 mRNA by using 6K glass DNA microarray revealed changes in expression of 95 genes (overexpression of 85 genes and downregulation of 10 genes). Our preliminary results indicated that simulated microgravity modifies the expression of several genes and that microarray technology may provide new understanding of the fundamental biological questions of how gravity affects the development and function of individual cells.

Non-NASA Center↗

NASA NTRS2004

Oligodeoxynucleotide Probes for Detecting Intact Cells

A rapid, sensitive test using chemiluminescent oligodeoxynucleotide probes has been developed for detecting, identifying, and enumerating intact cells. The test is intended especially for use in detecting and enumerating bacteria and yeasts in potable water. As in related tests that have been developed recently for similar purposes, the oligodeoxynucleotide probes used in this test are typically targeted at either singlecopy deoxyribonucleic acid (DNA) genes (such as virulence genes) or the multiple copies (10,000 to 50,000 copies per cell) of 16S ribosomal ribonucleic acids (rRNAs). Some of those tests involve radioisotope or fluorescent labeling of the probes for reporting hybridization of probes to target nucleic acids. Others of those tests involve labeling with enzymes plus the use of chemiluminescent or chromogenic substrates to report hybridization via color or the emission of light, respectively. The present test is of the last-mentioned type. The chemiluminescence in the present test can be detected easily with relatively simple instrumentation. In developing the present test, the hybridization approach was chosen because hybridization techniques are very specific. Hybridization detects stable, inheritable genetic targets within microorganisms. These targets are not dependent on products of gene expression that can vary with growth conditions or physiological states of organisms in test samples. Therefore, unique probes can be designed to detect and identify specific genera or species of bacteria or yeast (in terms of rRNA target sequences) or can be designed to detect and identify virulence genes (genomic target sequences). Because of the inherent specificity of this system, there are few problems of cross-reactivity. Hybridization tests are rapid, but hybridization tests now available commercially lack sensitivity; typically, between 10(exp 6) and 10(exp 7) cells of the target organism are needed to ensure a reliable test. Consequently, the numbers of target bacteria in samples are usually amplified by overnight pre-enrichment growth. These tests are usually performed in laboratories by skilled technicians. The present test was designed to overcome the shortcomings of the commercial hybridization tests. The figure summarizes the major steps of the test. It is important to emphasize that the hybridization process used in this test differs from that of other hybridization tests in that it does not extract target nucleic acids. This process is based on intact-cell hybridization (so-called in situ hybridization ), wherein the intact cells act as immobilizing agents. The cells are identified and enumerated by measuring the chemiluminescence emitted from alkaline phosphatase-probe (AP-probe) hybridization; the chemiluminescence is detected or measured by use of photographic film or a luminometer, respectively.

Rosson, Reinhardt A.↗

NASA NTRSDate not supplied

Rapid Nucleic Acid Isolation Method and Fluid Handling Devices Overview

Sample preparation is a common bottleneck in genetic analysis. Tools that reduce time and effort are of value in a laboratory setting. There is a need for a genetic analysis/detection system that is not only mobile, ruggedized, easy-to-use but also provides an enclosed fluid handling that diminishes the risks of Ribonucleic acid (RNA) contamination and degradation during processing. NASA has developed a novel assay methodology and suite of devices to isolate nucleic acids and prepare samples for real-time (quantitative) polymerase chain reaction (PCR) analysis that solves the problem of contamination and degradation because of the advanced microfluidics which provides better fluid handling. The assay employs an aqueous-based non-alcohol method that yields robust RNA quality. The suite of ready-to-use devices provide pre-loaded reagents in liquid and lyophilized formats to enable rapid, cost effective, and portable manual operation in the laboratory or remote field environments.

Travis Boone↗

DOE OSTI2024

Supramolecular assembly of polycation/mRNA nanoparticles and in vivo monocyte programming

Size-dependent phagocytosis is a well-characterized phenomenon in monocytes and macrophages. However, this size effect for preferential gene delivery to these important cell targets has not been fully exploited because commonly adopted stabilization methods for electrostatically complexed nucleic acid nanoparticles, such as PEGylation and charge repulsion, typically arrest the vehicle size below 200 nm. Here, we bridge the technical gap in scalable synthesis of larger submicron gene delivery vehicles by electrostatic self-assembly of charged nanoparticles, facilitated by a polymer structurally designed to modulate internanoparticle Coulombic and van der Waals forces. Specifically, our strategy permits controlled assembly of small poly(β-amino ester)/messenger ribonucleic acid (mRNA) nanoparticles into particles with a size that is kinetically tunable between 200 and 1,000 nm with high colloidal stability in physiological media. We found that assembled particles with an average size of 400 nm safely and most efficiently transfect monocytes following intravenous administration and mediate their differentiation into macrophages in the periphery. When a CpG adjuvant is co-loaded into the particles with an antigen mRNA, the monocytes differentiate into inflammatory dendritic cells and prime adaptive anticancer immunity in the tumor-draining lymph node. This platform technology offers a unique ligand-independent, particle-size-mediated strategy for preferential mRNA delivery and enables therapeutic paradigmsviamonocyte programming.

Science & Technology - Other Topics↗

NASA NTRS2009

Discrimination of Spore-Forming Bacilli Using spoIVA

A method of discriminating between spore-forming and non-spore-forming bacteria is based on a combination of simultaneous sporulation-specific and non-sporulation-specific quantitative polymerase chain reactions (Q-PCRs). The method was invented partly in response to the observation that for the purposes of preventing or reducing biological contamination affecting many human endeavors, ultimately, only the spore-forming portions of bacterial populations are the ones that are problematic (or, at least, more problematic than are the non-spore-forming portions). In some environments, spore-forming bacteria constitute small fractions of the total bacterial populations. The use of sporulation-specific primers in Q-PCR affords the ability to assess the spore-forming fraction of a bacterial population present in an environment of interest. This assessment can provide a more thorough and accurate understanding of the bacterial contamination in the environment, thereby making it possible to focus contamination- testing, contamination-prevention, sterilization, and decontamination resources more economically and efficiently. The method includes the use of sporulation-specific primers in the form of designed, optimized deoxyribonucleic acid (DNA) oligonucleotides specific for the bacterial spoIVA gene (see table). [In "spoIVA," "IV" signifies Roman numeral four and the entire quoted name refers to gene A for the fourth stage of sporulation.] These primers are mixed into a PCR cocktail with a given sample of bacterial cells. A control PCR cocktail into which are mixed universal 16S rRNA primers is also prepared. ["16S rRNA" denotes a ribosomal ribonucleic acid (rRNA) sequence that is common to all organisms.] Following several cycles of heating and cooling according to the PCR protocol to amplify amounts of DNA molecules, the amplification products can be analyzed to determine the types of bacterial cells present within the samples. If the amplification product is strong, relative to the product of a control PCR sequence, then it is concluded that the bacterial population in the sample consists predominantly of spore-forming cells. If the amplification product is weak or nonexistent, then it is concluded that the bacterial population in the sample consists predominantly or entirely of non-spore-forming cells.

Venkateswaran, Kasthuri↗

NASA NTRS1971

Evolution of proteins.

The amino acid sequences of proteins from living organisms are dealt with. The structure of proteins is first discussed; the variation in this structure from one biological group to another is illustrated by the first halves of the sequences of cytochrome c, and a phylogenetic tree is derived from the cytochrome c data. The relative geological times associated with the events of this tree are discussed. Errors which occur in the duplication of cells during the evolutionary process are examined. Particular attention is given to evolution of mutant proteins, globins, ferredoxin, and transfer ribonucleic acids (tRNA's). Finally, a general outline of biological evolution is presented.

Dayhoff, M. O.↗

DOE OSTI2024

Adjusting the Energy Profile for CH–O Interactions Leads to Improved Stability of RNA Stem-Loop Structures in MD Simulations

The role of ribonucleic acid (RNA) in biology continues to grow, but insight into important aspects of RNA behavior is lacking, such as dynamic structural ensembles in different environments, how flexibility is coupled to function, and how function might be modulated by small molecule binding. In the case of proteins, much progress in these areas has been made by complementing experiments with atomistic simulations, but RNA simulation methods and force fields are less mature. It remains challenging to generate stable RNA simulations, even for small systems where well-defined, thermostable structures have been established by experiments. Further many different aspects of RNA energetics have been adjusted in force fields, seeking improvements that are transferable across a variety of RNA structural motifs. In this work, the role of weak CH···O interactions is explored, which are ubiquitous in RNA structure but have received less attention in RNA force field development. By comparing data extracted from high-resolution RNA crystal structures to energy profiles from quantum mechanics and force field calculations, it is shown that CH···O interactions are overly repulsive in the widely used Amber RNA force fields. A simple, targeted adjustment of CH···O repulsion that leaves the remainder of the force field unchanged was developed. Then, the standard and modified force fields were tested using molecular dynamics (MD) simulations with explicit water and salt, amassing over 300 μs of data for multiple RNA systems containing important features such as the presence of loops, base stacking interactions as well as canonical and noncanonical base pairing. In this work and others, standard force fields lead to reproducible unfolding of the NMR-based structures. Including a targeted CH···O adjustment in an otherwise identical protocol dramatically improves the outcome, leading to stable simulations for all RNA systems tested.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2020

High Mass Analysis with a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer: From Inorganic Salt Clusters to Antibody Conjugates and Beyond

Analysis of proteins and complexes under native mass spectrometric (MS) and solution conditions was typically performed using time-of-flight (ToF) analyzers, due to their routine high m/z transmission and detection capabilities. However, over recent years, the ability of Orbitrap-based mass spectrometers to transmit and detect a range of high molecular weight species is well documented. Herein, we describe how a 15 Tesla Fourier transform ion cyclotron resonance mass spectrometer (15 T FT-ICR MS) is more than capable of analyzing a wide range of ions in the high m/z scale (>5000), in both positive and negative instrument polarities, ranging from the inorganic cesium iodide salt clusters; a humanized IgG1k monoclonal antibody (mAb; 148.2 kDa); an IgG1-mertansine drug conjugate (148.5 kDa, drug-to-antibody ratio; DAR 2.26); an IgG1-siRNA conjugate (159.1 kDa; ribonucleic acid to antibody ratio; RAR 1); the membrane protein aquaporin-Z (97.2 kDa) liberated from a C8E4 detergent micelle; the empty MSP1D1-nanodisc (142.5 kDa) and the tetradecameric chaperone protein complex GroEL (806.2 kDa; GroEL dimer at 1.6 MDa). We also investigate different regions of the FT-ICR MS that impact ion transmission and desolvation. Finally, we demonstrate how the transmission of these species and resultant spectra are highly consistent with those previously generated on both quadrupole-ToF (Q-ToF) and Orbitrap instrumentation. This article serves as an impactful example of how FT-ICR mass analyzers are competitive to Q-ToFs and Orbitraps for high mass detection at high m/z.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2025

Viroid-like “obelisk” agents are widespread in the ocean and exceed the abundance of RNA viruses in the prokaryotic fraction

Abstract “Obelisks” are recently discovered ribonucleic acid (RNA) viroid-like elements present in diverse environments with no phylogenetic similarity to any known biological agent. obelisks were first identified in the human gut and in a commensal bacterium acting as a replicative host. They have a circular ∼1 kb RNA genome, rod-like secondary structures, and the encoding of a protein superfamily called “Oblins”. We performed a large-scale search of obelisks in the ocean using the Pebblescout program and the transcriptomic Sequence Archive Read databases, revealing the biogeography and abundance of these viroid-like RNA elements. We detected 55 obelisk genomes resulting in 35 marine clusters at the species level. These obelisks were detected in the prokaryotic fraction and to a lesser extent in the eukaryotic fraction, and distributed across all the oceans from surface to mesopelagic including the Arctic, and even in the coldest seawater of Earth beneath the Antarctic Ross Ice Shelf. The obelisk hallmark protein Oblin-1 confirmed by 3D models was found in various marine samples. Some of the detected marine obelisks harbor hammerhead self-cleaving ribozymes in both polarities. In the prokaryotic, but not the eukaryotic, fraction of the Tara Ocean dataset, relative abundance of obelisks calculated by transcriptomic fragment recruitment indicated that they are abundant in marine samples, reaching or even exceeding the relative abundance of the previously discovered uncultured RNA viruses. In conclusion, obelisks are abundant and widespread viroid-like elements that should be included in ocean biogeochemical models.

Environmental Sciences & Ecology↗

DOE OSTI2025

Structural basis for aminoacylation of cellular modified tRNALys3 by human lysyl-tRNA synthetase

Abstract The average eukaryotic transfer ribonucleic acid (tRNA) contains 13 post-transcriptional modifications; however, their functional impact is largely unknown. Our understanding of the complex tRNA aminoacylation machinery in metazoans also remains limited. Herein, using a series of high-resolution cryo-electron microscopy (cryo-EM) structures, we provide the mechanistic basis for recognition and aminoacylation of fully modified cellular tRNALys3 by human lysyl-tRNA synthetase (h-LysRS). The tRNALys3 anticodon loop modifications S34 (mcm5s2U) and R37 (ms2t6A) play an integral role in recognition by h-LysRS. Modifications in the T-, variable-, and D-loops of tRNALys3 are critical for ordering the metazoan-specific N-terminal domain of LysRS. The two catalytic steps of tRNALys3 aminoacylation are structurally ordered; docking of the 3′-CCA end in the active site cannot proceed until the lysyl–adenylate intermediate is formed and the pyrophosphate byproduct is released. Association of the h-LysRS–tRNALys3 complex with a multi-tRNA synthetase complex-derived peptide shifts the equilibrium toward the 3′-CCA end “docked” conformation and allosterically increases h-LysRS catalytic efficiency. The insights presented here have broad implications for understanding the role of tRNA modifications in protein synthesis, the human aminoacylation machinery, and the growing catalog of metabolic and neurological diseases linked to it.

Devarkar, Swapnil C. (ORCID:000000029271243X)↗

DOE OSTI2020

Towards oilcane: Engineering hyperaccumulation of triacylglycerol into sugarcane stems

Abstract Metabolic engineering to divert carbon flux from sucrose to oil in high biomass crop like sugarcane is an emerging strategy to boost lipid yields per hectare for biodiesel production. Sugarcane stems comprise more than 70% of the crops' biomass and can accumulate sucrose in excess of 20% of their extracted juice. The energy content of oils in the form of triacylglycerol (TAG) is more than twofold that of carbohydrates. Here, we report a step change in TAG accumulation in sugarcane stem tissues achieving an average of 4.3% of their dry weight (DW) in replicated greenhouse experiments by multigene engineering. The metabolic engineering included constitutive co‐expression of wrinkled1; diacylglycerol acyltransferase1‐2; cysteine‐oleosin; and ribonucleic acid interference‐suppression of sugar‐dependent1. The TAG content in leaf tissue was also elevated by more than 400‐fold compared to non‐engineered sugarcane to an average of 8.0% of the DW and the amount of total fatty acids reached about 13% of the DW. With increasing TAG accumulation an increase of 18:1 unsaturated fatty acids was observed at the expense of 16:0 and 18:0 saturated fatty acids. Total biomass accumulation, soluble lignin, Brix and juice content were significantly reduced in the TAG hyperaccumulating sugarcane lines. Overcoming this yield drag by engineering lipid accumulation into late stem development will be critical to exceed lipid yields of current oilseed crops.

09 BIOMASS FUELS↗

DOE OSTI2021

Nanoparticle Mediated Delivery of Therapeutic mRNA for Protection Against Lung Damage

Medical countermeasures (MCMs) based on messenger ribonucleic acid (mRNA) are promising due to their programmability, targeting precision and specificity, predictable physicochemical properties, and amenability to scalable manufacture. However, safe and effective delivery vehicles are needed, especially for targeting the lung. We developed a generalized approach to nanoparticle-mediated mRNA delivery to lung, and used it to evaluate candidate therapies. In initial studies, reporter mRNA was delivered using lipid-coated mesoporous silica nanoparticles (LC-MSNs) and lipid nanoparticles (LNPs), the latter with greater consistency. Then, mRNA encoding known protein therapies were delivered using LNPs. These formulations showed some toxicity in mice with lung damage, but those with IL-1RA, sACE2-Ig, and ANGPT1 mRNA were modestly therapeutic on balance. Our work advances the state of the art for mRNA delivery to lung, and provides a foundation for evaluating and characterizing mRNA-based lung therapies, including three that appear to be exceptionally promising.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Using CRISPR/Cas13a technologies to detect miRNA [DOE SULI Internship Report]

Micro ribonucleic acids (miRNA) give our immune systems the ability to recognize viruses and other pathogens by their complementary single-stranded RNA (ssRNA) produced in the reproduction of the pathogen in our cells. When miRNA of a specific sequence is detected in a cell sample, it can be assumed that the immune system is activated and attempting to track down the infection. This pathway can be utilized to diagnose infection from a pathogen before the individual even develops symptoms, aiding in early disease detection and proper treatment. One of the ways that we can detect miRNA is through an assay of clustered regularly interspaced short palindromic repeats or “CRISPR” and the bacterial protein Cas13a. This report details discoveries made while attempting to optimize this assay for miRNA detection. After looking at several different factors within the assay, it was determined that some factors, such as reporter type and metallic ion concentration, are more impactful on the overall assay sensitivity than other factors, such as the overall concentration of Cas13a, CRISPR RNA (crRNA), or ssRNA reporter. It was also discovered that different sequences with different lengths require renewed optimization efforts, as each target has a unique binding affinity determined by the sequence length and composition. This information is crucial in the development of point of care molecular detection devices as they become sensitive enough to identify pathogens before they spread.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2026

Diffusion and Solvation Dynamics of Ions in Water: Beyond the Brownian Approximation

The coupled dynamics of ions and water molecules in their first hydration shell impact a variety of processes including ion diffusion, selective ion transport in water-filled nanopores, and the kinetics of ion-pairing, ion adsorption, and metal-ligand binding reactions. In this work, we study these coupled dynamics for alkali metals (Li, Na, K, Rb, Cs), alkaline Earth metals (Mg, Ca, Sr, Ba), and chloride through the lens of their dependence on ion isotopic mass. Results are validated against previous measurements of the isotopic mass-dependence of ion diffusion coefficients in water and previous ab initio calculations of ion high-frequency dynamics in water. We find that the vibrational power spectra of ions in water consistently exhibit either two or three peaks, i.e., ions have several rattling frequencies within their solvations shells as previously reported for a subset of the species examined here. These frequencies have different sensititivies to isotopic mass that may serve as signatures of ion solvation processes (such as the tendency of ions to orient their first-shell water molecules) and that also may relate to Hofmeister-like effects including the relative affinity of different metals for ribonucleic acid (RNA).

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

NASA NTRS1972

Informational biopolymer structure in early living forms.

Some studies devoted to the search in various organisms for 'relics' of the biochemical nature of ancient organisms, preserved by the conservative nature of the evolutionary process in all living species, are reviewed. Investigations of five families of informational molecules constituting such 'relics' in very diverse organisms are reported. They include: cytochrome c, ferredoxin, trypsin, transfer ribonucleic acid (RNA), and 5S ribosomal RNA. It is shown that, even from these few informational molecules, some interesting inferences about early living organisms can be drawn.

Dayhoff, M. O.↗

NASA NTRS1980

Topochemical differences in the amount of RNA in the motoneurons of the spinal chord in hypoxia and hypokinesia

Reactions to hypoxia and hypoknesia were compared by measuring charges in the amount of ribonucleic acid (RNA) in the cytoplasm of neurons of the intumescentia cervicalis and lumbalis. Animals were subjected to hypoxia, hypokinesia and both combined and a control group to neither. A total of two groups of motoneurons were compared, one innervating the respiratory musculature, the other the musculature of the lower extremities, so that hypoxic hypoxia would probably affect the first group primarily and hypokinesia the second. Results indicate that neither affect the amount of RNA in the neurons of the first group but a significant increase is noted in neurons of the second group. Other significant results are reported.

Brumberg, V. A.↗

NASA NTRS1980

Effect of restricted mobility on RNA content and nucleotide composition and on protein content in motoneurons of spinal cord anterior horns

An investigation into the effect of hypokinesia on the ribonucleic acid (RNA) content, the nucleotide composition, and dynamics of protein content in the motoneuron of the rat spinal cord anterior horns is described. Methodology and findings are presented. The study results showed that the nucleotide composition of the total cellular RNA at all the studied periods of hypokinesia remained unchanged and is characteristic for the cytoplasmic, high polymer ribosomal RNA. This means that with a change in the functional state of the neuron the newly formed RNA of the nerve cell has the same composition of bases as the original RNA that belongs to the ribosomal type.

Gorbunova, A. V.↗