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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 73 records · Page 4
All sourcesNASA NTRSDOE OSTI
NASA NTRS1986

Immunological analyses of U.S. Space Shuttle crewmembers

Changes in the immunoresponsiveness of 'T' lymphocytes following space flight have been reported previously. Additional data collected before and after 11 Shuttle space flights show that absolute lymphocyte numbers, lymphocyte blastogenic capability, and eosinophil percent in the peripheral blood of crewmembers are generally depressed postflight. These responses resemble those associated with physical and emotional stress and may not be related to flight per se. Additional data from Space Shuttle flights 41B and 41D, involving 11 crewmembers, indicate a postflight decrease in cells reacting with 'B' lymphocyte and monocyte monoclonal antibody tags. Further, the loss of 'T' lymphocyte blast capability correlates with the decreased monocyte count (correlation coefficient = 0.697). This finding implies that the previously reported loss of blastogenic capability may be a function of decreased monocyte control, as noted in several nonspaceflight related studies.

Taylor, G. R.↗
NASA NTRS1999

The influence of SV40 immortalization of human fibroblasts on p53-dependent radiation responses

The simian virus 40 large tumor antigen (SV40 Tag) has been ascribed many functions critical to viral propagation, including binding to the mammalian tumor suppressor p53. Recent studies have demonstrated that SV40-transformed murine cells have functional p53. The status of p53 in SV40-immortalized human cells, however, has not been characterized. We have found that in response to ionizing radiation, p53-dependent p21 transactivation activity is present, albeit reduced, in SV40-immortalized cells and that this activity can be further reduced with either dominant negative p53 expression or higher SV40 Tag expression. Furthermore, overexpression of p53 in SV40-immortalized ataxia-telangiectasia (A-T) cells restores p53-dependent p21 induction to typical A-T levels. All SV40-immortalized cell lines exhibited an absence of G1 arrest. Moreover, all SV40-immortalized cell lines exhibited increased apoptosis relative to primary cells in response to ionizing radiation, suggesting that SV40 immortalization results in a unique phenotype with regard to DNA damage responses. Copyright 1999 Academic Press.

NASA Discipline Radiation Health↗
DOE OSTI2021

A Palette of Minimally Tagged Sucrose Analogues for Real-Time Raman Imaging of Intracellular Plant Metabolism

Sucrose is the main saccharide used for longdistance transport in plants and plays an essential role in energy metabolism; however, there are no analogues for real-time imaging in live cells. We have optimised a synthetic approach to prepare sucrose analogues including very small ( 50 Da or less) Raman tags in the fructose moiety. Spectroscopic analysis identified the alkyne-tagged compound 6 as a sucrose analogue recognised by endogenous transporters in live cells and with higher Raman intensity than other sucrose derivatives. Herein, we demonstrate the application of compound 6 as the first optical probe to visualise real-time uptake and intracellular localisation of sucrose in live plant cells using Raman microscopy.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

STORM Super-Resolution Visualization of Self-Assembled γPFD Chaperone Ultrastructures in Methanocaldococcus jannaschii

Gamma-prefoldin (γPFD), a unique chaperone found in the extremely thermophilic methanogen Methanocaldococcus jannaschii, self-assembles into filaments in vitro, which so far have been observed using transmission electron microscopy and cryo-electron microscopy. Utilizing three-dimensional stochastic optical reconstruction microscopy (3D-STORM), here we achieve ~20 nm resolution by precisely locating individual fluorescent molecules, hence resolving γPFD ultrastructure both in vitro and in vivo. Through CF647 NHS ester labeling, we first demonstrate the accurate visualization of filaments and bundles with purified γPFD. Next, by implementing immunofluorescence labeling after creating a 3xFLAG-tagged γPFD strain, we successfully visualize γPFD in M. jannaschii cells. Through 3D-STORM and two-color STORM imaging with DNA, we show the widespread distribution of filamentous γPFD structures within the cell. These findings provide valuable insights into the structure and localization of γPFD, opening up possibilities for studying intriguing nanoscale components not only in archaea but also in other microorganisms.

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

Soft X-ray Tomography Reveals HSV-1-Induced Remodeling of Human B Cells

Upon infection, viruses hijack the cell machinery and remodel host cell structures to utilize them for viral proliferation. Since viruses are about a thousand times smaller than their host cells, imaging virus-host interactions at high spatial resolution is like looking for a needle in a haystack. Scouting gross cellular changes with fluorescent microscopy is only possible for well-established viruses, where fluorescent tagging is developed. Soft X-ray tomography (SXT) offers 3D imaging of entire cells without the need for chemical fixation or labeling. Here, we use full-rotation SXT to visualize entire human B cells infected by the herpes simplex virus 1 (HSV-1). We have mapped the temporospatial remodeling of cells during the infection and observed changes in cellular structures, such as the presence of cytoplasmic stress granules and multivesicular structures, formation of nuclear virus-induced dense bodies, and aggregates of capsids. Our results demonstrate the power of SXT imaging for scouting virus-induced changes in infected cells and understanding the orchestration of virus-host remodeling quantitatively.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1991

An area model for on-chip memories and its application

An area model suitable for comparing data buffers of different organizations and arbitrary sizes is described. The area model considers the supplied bandwidth of a memory cell and includes such buffer overhead as control logic, driver logic, and tag storage. The model gave less than 10 percent error when verified against real caches and register files. It is shown that, comparing caches and register files in terms of area for the same storage capacity, caches generally occupy more area per bit than register files for small caches because the overhead dominates the cache area at these sizes. For larger caches, the smaller storage cells in the cache provide a smaller total cache area per bit than the register set. Studying cache performance (traffic ratio) as a function of area, it is shown that, for small caches, direct-mapped caches perform significantly better than four-way set-associative caches and, for caches of medium areas, both direct-mapped and set-associative caches perform better than fully associative caches.

Mulder, Johannes M.↗
DOE OSTI2021

Spatially Resolved Mass Spectrometry at the Single Cell: Recent Innovations in Proteomics and Metabolomics

Biological systems are composed of heterogeneous populations of cells that intercommunicate to form a functional living tissue. Biological function varies greatly across populations of cells, as each single cell has a unique transcriptome, proteome, and metabolome that translates to functional differences within single species and across kingdoms. Over the past decade, substantial advancements in our ability to characterize omic profiles on a single cell level have occurred, including in multiple spectroscopic and mass spectrometry (MS)-based techniques. Of these technologies, spatially resolved mass spectrometry approaches, including mass spectrometry imaging (MSI), have shown the most progress for single cell proteomics and metabolomics. For example, reporter-based methods using heavy metal tags have allowed for targeted MS investigation of the proteome at the subcellular level, and development of technologies such as laser ablation electrospray ionization mass spectrometry (LAESI-MS) now mean that dynamic metabolomics can be performed in situ. In this Perspective, we showcase advancements in single cell spatial metabolomics and proteomics over the past decade and highlight important aspects related to high-throughput screening, data analysis, and more which are vital to the success of achieving proteomic and metabolomic profiling at the single cell scale. Finally, using this broad literature summary, we provide a perspective on how the next decade may unfold in the area of single cell MS-based proteomics and metabolomics.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRSDate not supplied

Considering Cell Proliferation to Optimize Detection of Radiation-induced 53BP1+ Foci in 15 Mouse Strains ex vivo

Due to high metabolic activity, proliferating cells continuously generate free radicals, which induce DNA double strand breaks (DSB). Fluorescently tagged nuclear foci of DNA repair protein 53 binding protein-1(53BP1) are used as a standard metric for measuring DSB formation at baseline and in response to environmental insults such as radiation. Here we demonstrate that the background level of spontaneous 53BP1+ foci formation can be modeled mathematically as a function of cell confluence, which is a metric of proliferation rate. This model was validated using spontaneous 53BP1+ foci data from 68 cultures of primary skin fibroblasts derived from 15 different strains of mice, showing a ~10 fold decrease from low to full confluence that is independent of mouse strain. We developed an online open access tool to correct for the impact of cell confluence on the detection of radiation-induced 53BP1+ foci (RIF). This tool provides guidelines for the number of cells required to reach statistical significance for the detection of excess foci induced by low doses of ionizing radiation as a function of confluence and time post-irradiation. We hope that this quantification tool will help the radiation biology community in the design of future experiments that utilize 53BP1+ foci-based quantification of radiation responses in vitro. Our “tool for enhanced results of RIF in cells” (terRIFic) can be found at: https://radbiolab.shinyapps.io/terrific/

Radiation, DNA damage, confluence↗
DOE OSTI2023

A previously uncharacterized divisome-associated lipoprotein, DalA, is needed for normal cell division in Rhodobacterales

ABSTRACT The bacterial cell envelope is a key subcellular compartment with important roles in antibiotic resistance, nutrient acquisition, and cell morphology. We seek to gain a better understanding of proteins that contribute to the function of the cell envelope in Alphaproteobacteria . Using Rhodobacter sphaeroides , we show that a previously uncharacterized protein, RSP_1200, is an outer membrane (OM) lipoprotein that non-covalently binds peptidoglycan (PG). Using a fluorescently tagged version of this protein, we find that RSP_1200 undergoes a dynamic repositioning during the cell cycle and is enriched at the septum during cell division. We show that the position of RSP_1200 mirrors the location of FtsZ rings, leading us to propose that RSP_1200 is a newly identified component of the R. sphaeroides ’ divisome. Additional support for this hypothesis includes the co-precipitation of RSP_1200 with FtsZ, the Pal protein, and several predicted PG L,D-transpeptidases. We also find that a ∆ RSP_1200 mutation leads to defects in cell division, sensitivity to PG-active antibiotics, and results in the formation of OM protrusions at the septum during cell division. Based on these results, we propose to name RSP_1200 DalA (for division-associated lipoprotein A) and postulate that DalA serves as a scaffold to position or modulate the activity of PG transpeptidases that are needed to form envelope invaginations during cell division. We find that DalA homologs are present in members of the Rhodobacterales order within Alphaproteobacteria . Therefore, we propose that further analysis of this and related proteins will increase our understanding of the macromolecular machinery and proteins that participate in cell division in Gram-negative bacteria. IMPORTANCE Multi-protein complexes of the bacterial cell envelope orchestrate key processes like growth, division, biofilm formation, antimicrobial resistance, and production of valuable compounds. The subunits of these protein complexes are well studied in some bacteria, and differences in their composition and function are linked to variations in cell envelope composition, shape, and proliferation. However, some envelope protein complex subunits have no known homologs across the bacterial phylogeny. We find that Rhodobacter sphaeroides RSP_1200 is a newly identified lipoprotein (DalA) and that loss of this protein causes defects in cell division and changes the sensitivity to compounds, affecting cell envelope synthesis and function. We find that DalA forms a complex with proteins needed for cell division, binds the cell envelope polymer peptidoglycan, and colocalizes with enzymes involved in the assembly of this macromolecule. The analysis of DalA provides new information on the cell division machinery in this and possibly other Alphaproteobacteria.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Inactivation of Fluorescent Lipid Bilayers by Irradiation With 300 keV Electrons Using Liquid Cell Transmission Electron Microscopy

Liquid cell transmission electron microscopy allows for imaging of samples in a fully hydrated state at high resolution and has the potential for visualizing static or dynamic biological structures. However, the ionizing nature of the electron beam makes it difficult to discern real physiological dynamics from radiation induced artifacts within liquid cell samples. Electron flux thresholds for achieving high resolution structures from biological samples frozen in ice have been described extensively by the cryo-electron microscopy field, while electron flux thresholds which do not result in a functional change for biological samples within the hydrated environment of a transmission electron microscope liquid cell is less clear. Establishing these functional thresholds for biologically relevant samples is important for accurate interpretation of results from liquid cell experiments. Here we demonstrate the electron damage threshold of fluorescently tagged lipid bilayers by quantifying the change in fluorescence before and after electron exposure. We observe the reduction of fluorescent signal in bilayers by 25% after only 0.0005 e − /Å 2 and a reduction of over 90% after 0.01 e − /Å 2 . These results indicate that the loss of function occurs at irradiation thresholds far below a typical single high resolution (scanning) transmission electron microscopy image and orders of magnitude below fluxes used for preserving structural features with cryo-electron microscopy.

Moser, Trevor↗
DOE OSTI2021

Arabidopsis thaliana EARLY RESPONSIVE TO DEHYDRATION 7 Localizes to Lipid Droplets via Its Senescence Domain

Lipid droplets (LDs) are neutral-lipid-containing organelles found in all kingdoms of life and are coated with proteins that carry out a vast array of functions. Compared to mammals and yeast, relatively few LD proteins have been identified in plants, particularly those associated with LDs in vegetative (non-seed) cell types. Thus, to better understand the cellular roles of LDs in plants, a more comprehensive inventory and characterization of LD proteins is required. Here, we performed a proteomics analysis of LDs isolated from drought-stressed Arabidopsis leaves and identified EARLY RESPONSIVE TO DEHYDRATION 7 (ERD7) as a putative LD protein. mCherry-tagged ERD7 localized to both LDs and the cytosol when ectopically expressed in plant cells, and the protein’s C-terminal senescence domain (SD) was both necessary and sufficient for LD targeting. Phylogenetic analysis revealed that ERD7 belongs to a six-member family in Arabidopsis that, along with homologs in other plant species, is separated into two distinct subfamilies. Notably, the SDs of proteins from each subfamily conferred targeting to either LDs or mitochondria. Further, the SD from the ERD7 homolog in humans, spartin, localized to LDs in plant cells, similar to its localization in mammals; although, in mammalian cells, spartin also conditionally localizes to other subcellular compartments, including mitochondria. Disruption of ERD7 gene expression in Arabidopsis revealed no obvious changes in LD numbers or morphology under normal growth conditions, although this does not preclude a role for ERD7 in stress-induced LD dynamics. Consistent with this possibility, a yeast two-hybrid screen using ERD7 as bait identified numerous proteins involved in stress responses, including some that have been identified in other LD proteomes. Collectively, these observations provide new insight to ERD7 and the SD-containing family of proteins in plants and suggest that ERD7 may be involved in functional aspects of plant stress response that also include localization to the LD surface.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2021

Evaluation of Differential Peptide Loading on Tandem Mass Tag-Based Proteomic and Phosphoproteomic Data Quality

Global and phosphoproteome profiling has demonstrated great utility for the analysis of clinical specimens. One major barrier to the broad clinical application of proteomic profiling is the large amount of biological material required, particularly for phosphoproteomics—currently on the order of 25 mg wet tissue weight, depending on tissue type. For hematopoietic cancers such as acute myeloid leukemia (AML), the sample requirement is in excess of 10 million (1E7) peripheral blood mononuclear cells (PBMCs). Throughout the course of a prospective study, this requirement will certainly exceed what is obtainable from many of the individual patients/timepoints. For this reason, we were interested in examining the impact of differential peptide loading across multiplex channels on proteomic data quality. Methods: To achieve this, we tested a range of channel loading amounts (20, 40, 100, 200, and 400 µg of tryptic peptides, or approximately the material obtainable from 5E5, 1E6, 2.5E6, 5E6, and 1E7 AML patient cells) to assess proteome coverage, quantification reproducibility and accuracy in experiments utilizing isobaric tandem mass tag (TMT) labeling. As expected, we found that fewer missing values are observed in TMT channels with higher peptide loading amounts compared to those with lower loading. Moreover, channels with lower loading amounts have greater quantitative variability than channels with higher loading amounts. Statistical analysis of the differences in means among the five loading groups showed that the 20 µg loading group was significantly different from the 400 µg loading group. However, no significant differences were detected among the 40, 100, 200 and 400 µg loading groups. Conclusions: These assessment data demonstrate the practical limits of loading differential quantities of peptides across channels in TMT multiplexes, and provide a basis for designing the optimal clinical proteomics study when specimen quantities are limited.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Structural basis for ring-opening fluorescence by the RhoBAST RNA aptamer

Abstract Tagging RNAs with fluorogenic aptamers has enabled imaging of transcripts in living cells, revealing novel aspects of RNA metabolism and dynamics. While a diverse set of fluorogenic aptamers has been developed, a new generation of aptamers uses the ring-opening of spirocyclic rhodamine dyes to achieve robust performance in live mammalian cells. These fluorophores have two chemical states: a cell-permeable spirocyclic state and a fluorescent zwitterionic state. The SpyRho555 dye is mostly non-fluorescent in solution and becomes fluorescent in complex with the RhoBAST aptamer. To understand the basis for RhoBAST-SpyRho555 fluorogenicity, we have determined crystal structures of RhoBAST in complex with 5-carboxytetramethylrhodamine and a SpyRho555 analogue, MaP555. RhoBAST is organized by a perfect four-way junction that positions two loops to form the dye-binding pocket. The core of the ligand resides between a tri-adenine floor and a single guanine base, largely driven by π-stacking interactions. Importantly, the unpaired guanine interacts with the 3-position group of MaP555 to stabilize the open conformation, supported by mutagenesis data, and may play an active role in promoting the open conformation of the dye. This work has implications for the development of new fluorogenic aptamers with improved properties using structure-guided design approaches.

Biochemistry & Molecular Biology↗
DOE OSTI2023

Investigating and Optimizing the Lysate-Based Expression of Nonribosomal Peptide Synthetases Using a Reporter System

Lysate-based cell-free expression (CFE) systems are accessible platforms for expressing proteins that are difficult to synthesize in vivo, such as nonribosomal peptide synthetases (NRPSs). NRPSs are large (>100 kDa), modular enzyme complexes that synthesize bioactive peptide natural products. This synthetic process is analogous to transcription/translation (TX/TL) in lysates, resulting in potential resource competition between NRPS expression and NRPS activity in cell-free environments. Moreover, CFE conditions depend on the size and structure of the protein. Here, a reporter system for rapidly investigating and optimizing reaction environments for NRPS CFE is described. This strategy is demonstrated in E. coli lysate reactions using blue pigment synthetase A (BpsA), a model NRPS, carrying a C-terminal tetracysteine (TC) tag which forms a fluorescent complex with the biarsenical dye, FlAsH. A colorimetric assay was adapted for lysate reactions to detect the blue pigment product, indigoidine, of cell-free expressed BpsA-TC, confirming that the tagged enzyme is catalytically active. An optimized protocol for end point TC/FlAsH complex measurements in reactions enables quick comparisons of full-length BpsA-TC expressed under different reaction conditions, defining unique requirements for NRPS expression that are related to the protein’s catalytic activity and size. Importantly, these protein-dependent CFE conditions enable higher indigoidine titer and improve the expression of other monomodular NRPSs. Notably, these conditions differ from those used for the expression of superfolder GFP (sfGFP), a common reporter for optimizing lysate-based CFE systems, indicating the necessity for tailored reporters to optimize expression for specific enzyme classes. In conclusion, the reporter system is anticipated to advance lysate-based CFE systems for complex enzyme synthesis, enabling natural product discovery.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Large fluxes of fatty acids from membranes to triacylglycerol and back during N-deprivation and recovery in Chlamydomonas

Abstract Microalgae accumulate triacylglycerol (TAG) during nutrient deprivation and break it down after nutrient resupply, and these processes involve dramatic shifts in cellular carbon allocation. Due to the importance of algae in the global carbon cycle, and the potential of algal lipids as feedstock for chemical and fuel production, these processes are of both ecophysiological and biotechnological importance. However, the metabolism of TAG is not well understood, particularly the contributions of fatty acids (FAs) from different membrane lipids to TAG accumulation and the fate of TAG FAs during degradation. Here, we used isotopic labeling time course experiments on Chlamydomonas reinhardtii to track FA synthesis and transfer between lipid pools during nitrogen (N)-deprivation and resupply. When cells were labeled before N-deprivation, total levels of label in cellular FAs were unchanged during subsequent N-deprivation and later resupply, despite large fluxes into and out of TAG and membrane lipid pools. Detailed analyses of FA levels and labeling revealed that about one-third of acyl chains accumulating in TAG during N-deprivation derive from preexisting membrane lipids, and in total, at least 45% of TAG FAs passed through membrane lipids at one point. Notably, most acyl chains in membrane lipids during recovery after N-resupply come from TAG. Fluxes of polyunsaturated FAs from plastidic membranes into TAG during N-deprivation were particularly noteworthy. These findings demonstrate a high degree of integration of TAG and membrane lipid metabolism and highlight a role for TAG in storage and supply of membrane lipid components.

Plant Sciences↗
DOE OSTI2023

Cellulose synthase-like D movement in the plasma membrane requires enzymatic activity

Cellulose Synthase-Like D (CSLD) proteins, important for tip growth and cell division, are known to generate β-1,4-glucan. However, whether they are propelled in the membrane as the glucan chains they produce assemble into microfibrils is unknown. To address this, we endogenously tagged all eight CSLDs in Physcomitrium patens and discovered that they all localize to the apex of tip-growing cells and to the cell plate during cytokinesis. Actin is required to target CSLD to cell tips concomitant with cell expansion, but not to cell plates, which depend on actin and CSLD for structural support. Like Cellulose Synthase (CESA), CSLD requires catalytic activity to move in the plasma membrane. We discovered that CSLD moves significantly faster, with shorter duration and less linear trajectories than CESA. In contrast to CESA, CSLD movement was insensitive to the cellulose synthesis inhibitor isoxaben, suggesting that CSLD and CESA function within different complexes possibly producing structurally distinct cellulose microfibrils.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Multicellular magnetotactic bacteria are genetically heterogeneous consortia with metabolically differentiated cells

Consortia of multicellular magnetotactic bacteria (MMB) are currently the only known example of bacteria without a unicellular stage in their life cycle. Because of their recalcitrance to cultivation, most previous studies of MMB have been limited to microscopic observations. To study the biology of these unique organisms in more detail, we use multiple culture-independent approaches to analyze the genomics and physiology of MMB consortia at single-cell resolution. We separately sequenced the metagenomes of 22 individual MMB consortia, representing 8 new species, and quantified the genetic diversity within each MMB consortium. This revealed that, counter to conventional views, cells within MMB consortia are not clonal. Single consortia metagenomes were then used to reconstruct the species-specific metabolic potential and infer the physiological capabilities of MMB. To validate genomic predictions, we performed stable isotope probing (SIP) experiments and interrogated MMB consortia using fluorescence in situ hybridization (FISH) combined with nanoscale secondary ion mass spectrometry (NanoSIMS). By coupling FISH with bioorthogonal noncanonical amino acid tagging (BONCAT), we explored their in situ activity as well as variation of protein synthesis within cells. We demonstrate that MMB consortia are mixotrophic sulfate reducers and that they exhibit metabolic differentiation between individual cells, suggesting that MMB consortia are more complex than previously thought. These findings expand our understanding of MMB diversity, ecology, genomics, and physiology, as well as offer insights into the mechanisms underpinning the multicellular nature of their unique lifestyle.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

The development and use of metal-based probes for X-ray fluorescence microscopy

Abstract X-ray fluorescence microscopy (XFM) has become a widely used technique for imaging the concentration and distribution of metal ions in cells and tissues. Recent advances in synchrotron sources, optics, and detectors have improved the spatial resolution of the technique to <10 nm with attogram detection sensitivity. However, to make XFM most beneficial for bioimaging—especially at the nanoscale—the metal ion distribution must be visualized within the subcellular context of the cell. Over the years, a number of approaches have been taken to develop X-ray-sensitive tags that permit the visualization of specific organelles or proteins using XFM. In this review, we examine the types of X-ray fluorophore used, including nanomaterials and metal ions, and the approaches used to incorporate the metal into their target binding site via antibodies, genetically encoded metal-binding peptides, affinity labeling, or cell-specific peptides. We evaluate their advantages and disadvantages, review the scientific findings, and discuss the needs for future development.

36 MATERIALS SCIENCE↗