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Results for “cell tagging”

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 19 records
All sourcesNASA NTRSDOE OSTI
NASA NTRS1972

Pulmonary Edema and Plasma Volume Changes in Dysbarism

Two groups of anesthetized, fasted pigs were utilized. One group of 13 animals (8.5 to 16.6 kilograms) was exposed to a high-pressure environment, and the other group of eight animals (6.9 to 20.0 kilograms) constituted the control group. The experimental group was subjected to an atmosphere of 90 percent nitrogen and 10 percent oxygen at a pressure of 50 psig for 30 minutes and then decompressed at a rate 10 psi/min. Plasma volumes, using both iodine-125-tagged-albumin and chromium-51-tagged-cell dilution techniques, were measured before, immediately after, and at 30 and 60 minutes after decompression. Aortic and right-ventricular systolic pressures were also recorded. At 60 minutes after decompression, blood samples were taken, the animals were sacrificed, and the water content of the lungs, kidneys, livers, and spleens was estimated by measuring tissue wet weight and dry weight. Protein extravasation and tissue blood volumes were determined by measuring the iodine-125-tagged-albumin and chromium-51-tagged-cell spaces in homo-genates of the organs under investigation.

Joki, J. A.↗
NASA NTRS1988

Antibody enhancement of free-flow electrophoresis

Specific T cell clones and antibodies (ABs) were developed to study the efficiency of purifying closely associated T cells using Continuous Flow Electrophoresis System. Enhanced separation is accomplished by tagging cells first with ABs directed against the antigenic determinants on the cell surface and then with ABs against the Fc portion of the first AB. This second AB protrudes sufficiently beyond the cell membrane and glycocalyx to become the major overall cell surface potential determinant and thus causes a reduction of electrophoretic mobility. This project was divided into three phases. Phase one included development of specific T cell clones and separation of these specific clones. Phase two extends these principles to the separation of T cells from spleen cells and immunized lymph node cells. Phase three applies this double antibody technique to the separation of T cytotoxic cells from bone marrow.

Cohly, H. H. P.↗
DOE OSTI2024

Updraft Width Modulates Ambient Atmospheric Controls on Convective Cloud Depth

Abstract The depth of convective clouds affects vertical transport of atmospheric constituents, influencing downstream weather and climate. Atmospheric controls on the maximum depth reached by moist convection are investigated with radar‐tracked convective cells tagged with sounding‐derived atmospheric parameters from a field campaign in central Argentina. Regression analyses show that narrow (<12‐km diameter) and wide (>16‐km diameter) cell depths respond to disparate factors, where cell areas are defined using composite reflectivity signatures. Undiluted lifted parcel indices including convective available potential energy (CAPE) and level of neutral buoyancy (LNB) are top predictors of wide cell maximum depth while mid‐tropospheric relative humidity is the top predictor of narrow cell maximum depth. Because narrow cells are more numerous than wide cells, the overall outcome of the full cell population does not strongly correlate with CAPE and LNB conditions. Tracked cells and atmospheric conditions in a simulation with 3‐km grid spacing covering the field campaign produce similar results to those observed. Narrow cells that are relatively deep have a cooler and moister mid‐troposphere with weaker free tropospheric subsidence, while relatively deep wide cells have much warmer and moister lower tropospheric conditions. These atmospheric differences are present 1 hr before cell initiation at both a fixed observing site and variable cell initiation locations. Simulated narrow cell maximum equivalent potential temperature decreases with height at a rate similar to the ambient vertical gradient, causing these cells to fall short of their LNB and supporting the view that entrainment‐driven dilution is a dominant control on their depth.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2021

Laser particles with omnidirectional emission for cell tracking

The ability to track individual cells in space over time is crucial to analyzing heterogeneous cell populations. Recently, microlaser particles have emerged as unique optical probes for massively multiplexed single-cell tagging. However, the microlaser far-field emission is inherently direction-dependent, which causes strong intensity fluctuations when the orientation of the particle varies randomly inside cells. Here, we demonstrate a general solution based on the incorporation of nanoscale light scatterers into microlasers. Two schemes are developed by introducing either boundary defects or a scattering layer into microdisk lasers. The resulting laser output is omnidirectional, with the minimum-to-maximum ratio of the angle-dependent intensity improving from 0.007 (-24 dB) to > 0.23 (-6 dB). After transfer into live cells in vitro, the omnidirectional laser particles within moving cells could be tracked continuously with high signal-to-noise ratios for 2 h, while conventional microlasers exhibited frequent signal loss causing tracking failure.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Impact of moisture on microbial decomposition phenotypes and enzyme dynamics

Soil organic matter decomposition is a complex process reflecting microbial composition and environmental conditions. Moisture can modulate the connectivity and interactions of microbes. Due to heterogeneity, a deeper understanding of the influence of soil moisture on the dynamics of organic matter decomposition and resultant phenotypes remains a challenge. Soils from a long-term field experiment exposed to high and low moisture treatments were incubated in the laboratory to investigate organic matter decomposition using chitin as a model substrate. By combining enzymatic assays, biomass measurements, and microbial enrichment via activity-based probes, we determined the microbial functional response to chitin amendments and field moisture treatments at both the community and cell scales. Chitinolytic activities showed significant responses to the amendment of chitin, independent of differences in field moisture treatments. However, for other measurements of carbon metabolism and cellular functions, soils from high moisture field treatments had greater potential enzyme activity than soils from low moisture field treatments. A cell tagging approach was used to enrich and quantify bacterial taxa that are actively producing chitin-degrading enzymes. By integrating organism, community, and soil core measurements we show that (i) a small subset of taxa compose the majority (>50%) of chitinase production despite broad functional redundancy, (ii) the identity of key chitin degraders varies with moisture level, and (iii) extracellular enzymes that are not cell-associated account for most potential chitinase activity measured in field soil.

activity-based probes↗
DOE OSTI2020

Bump-and-Hole Engineering Identifies Specific Substrates of Glycosyltransferases in Living Cells

Studying posttranslational modifications classically relies on experimental strategies that oversimplify the complex biosynthetic machineries of living cells. Protein glycosylation contributes to essential biological processes, but correlating glycan structure, underlying protein, and disease-relevant biosynthetic regulation is currently elusive. Here, we engineer living cells to tag glycans with editable chemical functionalities while providing information on biosynthesis, physiological context, and glycan fine structure. We introduce a non-natural substrate biosynthetic pathway and use engineered glycosyltransferases to incorporate chemically tagged sugars into the cell surface glycome of the living cell. We apply the strategy to a particularly redundant yet disease-relevant human glycosyltransferase family, the polypeptide N-acetylgalactosaminyl transferases. This approach bestows a gain-of-chemical-functionality modification on cells, where the products of individual glycosyltransferases can be selectively characterized or manipulated to understand glycan contribution to major physiological processes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Nitrogen-dependent coordination of cell cycle, quiescence and TAG accumulation in Chlamydomonas

Abstract Microalgae hold great promises as sustainable cellular factories for the production of alternative fuels, feeds, and biopharmaceuticals for human health. While the biorefinery approach for fuels along with the coproduction of high-value compounds with industrial, therapeutic, or nutraceutical applications have the potential to make algal biofuels more economically viable, a number of challenges continue to hamper algal production systems at all levels. One such hurdle includes the metabolic trade-off often observed between the increased yields of desired products, such as triacylglycerols (TAG), and the growth of an organism. Initial genetic engineering strategies to improve lipid productivity in microalgae, which focused on overproducing the enzymes involved in fatty acid and TAG biosynthesis or inactivating competing carbon (C) metabolism, have seen some successes albeit at the cost of often greatly reduced biomass. Emergent approaches that aim at modifying the dynamics of entire metabolic pathways by engineering of pertinent transcription factors or signaling networks appear to have successfully achieved a balance between growth and neutral lipid accumulation. However, the biological knowledge of key signaling networks and molecular components linking these two processes is still incomplete in photosynthetic eukaryotes, making it difficult to optimize metabolic engineering strategies for microalgae. Here, we focus on nitrogen (N) starvation of the model green microalga, Chlamydomonas reinhardtii , to present the current understanding of the nutrient-dependent switch between proliferation and quiescence, and the drastic reprogramming of metabolism that results in the storage of C compounds following N starvation. We discuss the potential components mediating the transcriptional repression of cell cycle genes and the establishment of quiescence in Chlamydomonas, and highlight the importance of signaling pathways such as those governed by the target of rapamycin (TOR) and sucrose nonfermenting-related (SnRK) kinases in the coordination of metabolic status with cellular growth. A better understanding of how the cell division cycle is regulated in response to nutrient scarcity and of the signaling pathways linking cellular growth to energy and lipid homeostasis, is essential to improve the prospects of biofuels and biomass production in microalgae.

09 BIOMASS FUELS↗
DOE OSTI2021

Raman-probes for monitoring metabolites and nutrient fate in Yarrowia lipolytica using deuterated glucose

The aim of this study was to explore the application of deuterium (D) labeled glucose-D 7 as Raman-tag in Yarrowia lipolytica to assess triacyl glycerides (TAG) biosynthesis. Cells were grown in N depleted media in: (i) glucose in H 2 O (control), (ii) glucose-D 7 in H 2 O and (iii) glucose-D 7 in D 2 O, and the resulting biomass and lipids quantified. Infrared and Raman spectroscopies showed the incorporation of D with the presence of a C-D stretching bands in the silent region (1800–2600 cm -1 ) for cells grown in glucose-D 7 in H 2 O and glucose-D 7 in D 2 O as compared to the control. Fatty acid methyl ester analysis of cells grown in the three media showed significant differences in lipid profiles. Cells grown on glucose-D 7 in H 2 O and glucose-D 7 in D 2 O showed unequivocally D incorporation in fatty acids and TAG by mass spectrometry. Higher D incorporation was observed when cells were grown in D 2 O than H 2 O. Deuterium was also incorporated into the cell wall polysaccharides.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Aggregation‐Induced Stimulated Emission of 100% Dye Microspheres

Dyes with aggregation-induced emission (AIE) properties have gained interest due to their bright luminescence in solid-state aggregates. While fluorescence from AIE dyes is widely exploited, relatively little is known about aggregation-induced stimulated emission. Here, stimulated emission of tetraphenylethene (TPE)-based organoboron AIE dyes, TPEQBN, in thin films and in microcavity lasers is investigated. Using femtosecond pump–probe spectroscopy, gain coefficients up to 230 cm -1 at 500 nm are measured. Using rate equations, concentration- and pump-dependent gain dynamics as well as laser build-up dynamics are analyzed. During laser oscillation, radiative stimulated emission allows high instantaneous quantum yield greater than 90% to be achieved. Solid-state microspheres made of 100% AIE dyes are fabricated via microfluidic emulsion and solvent evaporation method. Coupled with high gain and high refractive index of 1.76, microspheres as small as 2 µm in diameter show lasing by nanosecond pumping with a threshold of ≈10 pJ µm –2 . Polymer coated, but not bare, microspheres are internalized by live cells and generate narrowband cavity-mode emission from within the cytoplasm. This work shows the potential of AIE dyes as laser materials.

36 MATERIALS SCIENCE↗
DOE OSTI2021

Lasofoxifene as a potential treatment for therapy-resistant ER-positive metastatic breast cancer

Background: Endocrine therapy remains the mainstay of treatment for estrogen receptor-positive (ER+) breast cancer. Constitutively active mutations in the ligand binding domain of ERα render tumors resistant to endocrine agents. Breast cancers with the two most common ERα mutations, Y537S and D538G, have low sensitivity to fulvestrant inhibition, a typical second-line endocrine therapy. Lasofoxifene is a selective estrogen receptor modulator with benefits on bone health and breast cancer prevention potential. This study investigated the anti-tumor activity of lasofoxifene in breast cancer xenografts expressing Y537S and D538G ERα mutants. The combination of lasofoxifene with palbociclib, a CDK4/6 inhibitor, was also evaluated. Methods: Luciferase-GFP tagged MCF7 cells bearing wild-type, Y537S, or D538G ERα were injected into the mammary ducts of NSG mice (MIND model), which were subsequently treated with lasofoxifene or fulvestrant as single agents or in combination with palbociclib. Tumor growth and metastasis were monitored with in vivo and ex vivo luminescence imaging, terminal tumor weight measurements, and histological analysis. Results: As a monotherapy, lasofoxifene was more effective than fulvestrant at inhibiting primary tumor growth and reducing metastases. Adding palbociclib improved the effectiveness of both lasofoxifene and fulvestrant for tumor suppression and metastasis prevention at four distal sites (lung, liver, bone, and brain), with the combination of lasofoxifene/palbociclib being generally more potent than that of fulvestrant/palbociclib. X-ray crystallography of the ERα ligand binding domain (LBD) shows that lasofoxifene stabilizes an antagonist conformation of both wild-type and Y537S LBD. The ability of lasofoxifene to promote an antagonist conformation of Y537S, combined with its long half-life and bioavailability, likely contributes to the observed potent inhibition of primary tumor growth and metastasis of MCF7 Y537S cells. Conclusions: We report for the first time the anti-tumor activity of lasofoxifene in mouse models of endocrine therapy-resistant breast cancer. The results demonstrate the potential of using lasofoxifene as an effective therapy for women with advanced or metastatic ER+ breast cancers expressing the most common constitutively active ERα mutations.

60 APPLIED LIFE SCIENCES↗
NASA NTRS1990

Microspheres

Vital information on a person's physical condition can be obtained by identifying and counting the population of T-cells and B-cells, lymphocytes of the same shape and size that help the immune system protect the body from the invasion of disease. The late Dr. Alan Rembaum developed a method for identifying the cells. The method involved tagging the T-cells and B-cells with microspheres of different fluorescent color. Microspheres, which have fluorescent dye embedded in them, are chemically treated so that they can link with antibodies. With the help of a complex antibody/antigen reaction, the microspheres bind themselves to specific 'targets,' in this case the T-cells or B-cells. Each group of cells can then be analyzed by a photoelectronic instrument at different wavelengths emitted by the fluorescent dyes. Same concept was applied to the separation of cancer cells from normal cells. Microspheres were also used to conduct many other research projects. Under a patent license Magsphere, Inc. is producing a wide spectrum of microspheres on a large scale and selling them worldwide for various applications.

Source record↗
DOE OSTI2022

Controlling Protein Enrichment in Lipid Sponge Phase Droplets using SNAP–Tag Bioconjugation

All cells use organized lipid compartments to facilitate specific biological functions. Membrane-bound organelles create defined spatial environments that favor unique chemical reactions while isolating incompatible biological processes. Despite the fundamental role of cellular organelles, there is a scarcity of methods for preparing functional artificial lipid-based compartments. In this work, we demonstrate a robust bioconjugation system for sequestering proteins into zwitterionic lipid sponge phase droplets. Incorporation of benzylguanine (BG)-modified phospholipids that form stable covalent linkages with an O 6 -methylguanine DNA methyltransferase (SNAP-tag) fusion protein enables programmable control of protein capture. We show that this methodology can be used to anchor hydrophilic proteins at the lipid-aqueous interface, concentrating them within an accessible but protected chemical environment. SNAP-tag technology enables the integration of proteins that regulate complex biological functions in lipid sponge phase droplets, and should facilitate the development of advanced lipid-based artificial organelles.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1986

Real-time garbage collection for list processing

In a list processing system, small reference counters are maintained in conjunction with memory cells for the purpose of identifying memory cells that become available for re-use. The counters are updated as references to the cells are created and destroyed, and when a counter of a cell is decremented to logical zero the cell is immediately returned to a list of free cells. In those cases where a counter must be incremented beyond the maximum value that can be represented in a small counter, the cell is restructured so that the additional reference count can be represented. The restructuring involves allocating an additional cell, distributing counter, tag, and pointer information among the two cells, and linking both cells appropriately into the existing list structure.

Shuler, R. L., Jr.↗
NASA NTRS1990

Real-time garbage collection for list processing using restructured cells for increased reference counter size

In a list processing system, small reference counters are maintained in conjunction with memory cells for the purpose of identifying memory cells that become available for re-use. The counters are updated as references to the cells are created and destroyed, and when a counter of a cell is decremented to logical zero the cell is immediately returned to a list of free cells. In those cases where a counter must be incremented beyond the maximum value that can be represented in a small counter, the cell is restructured so that the additional reference count can be represented. The restructuring involves allocating an additional cell, distributing counter, tag, and pointer information among the two cells, and linking both cells appropriately into the existing list structure.

Shuler, Jr., Robert L.↗
NASA NTRS2002

Overexpression of neurofilament H disrupts normal cell structure and function

Studying exogenously expressed tagged proteins in live cells has become a standard technique for evaluating protein distribution and function. Typically, expression levels of experimentally introduced proteins are not regulated, and high levels are often preferred to facilitate detection. However, overexpression of many proteins leads to mislocalization and pathologies. Therefore, for normative studies, moderate levels of expression may be more suitable. To understand better the dynamics of intermediate filament formation, transport, and stability in a healthy, living cell, we inserted neurofilament heavy chain (NFH)-green fluorescent protein (GFP) fusion constructs in adenoviral vectors with tetracycline (tet)-regulated promoters. This system allows for turning on or off the synthesis of NFH-GFP at a selected time, for a defined period, in a dose-dependent manner. We used this inducible system for live cell imaging of changes in filament structure and cell shape, motility, and transport associated with increasing NFH-GFP expression. Cells with low to intermediate levels of NFH-GFP were structurally and functionally similar to neighboring, nonexpressing cells. In contrast, overexpression led to pathological alterations in both filament organization and cell function. Copyright 2002 Wiley-Liss, Inc.

Non-NASA Center↗
NASA NTRS1998

Transposon tagging and the study of root development in Arabidopsis

The maize Ac-Ds transposable element family has been used as the basis of transposon mutagenesis systems that function in a variety of plants, including Arabidopsis. We have developed modified transposons and methods which simplify the detection, cloning and analysis of insertion mutations. We have identified and are analyzing two plant lines in which genes expressed either in the root cap cells or in the quiescent cells, cortex/endodermal initial cells and columella cells of the root cap have been tagged with a transposon carrying a reporter gene. A gene expressed in root cap cells tagged with an enhancer-trap Ds was isolated and its corresponding EST cDNA was identified. Nucleotide and deduced amino acid sequences of the gene show no significant similarity to other genes in the database. Genetic ablation experiments have been done by fusing a root cap-specific promoter to the diphtheria toxin A-chain gene and introducing the fusion construct into Arabidopsis plants. We find that in addition to eliminating gravitropism, root cap ablation inhibits elongation of roots by lowering root meristematic activities.

NASA Discipline Plant Biology↗
DOE OSTI2025

Identification and Characterization of Lipid Droplet-Associated Protein (LDAP) Isoforms from Tung Tree (Vernicia fordii)

Lipid droplets (LDs) are cytoplasmic organelles responsible primarily for the storage of neutral lipids, such as triacyclglycerols (TAGs). Derived from the endoplasmic reticulum bilayer, LDs are composed of a hydrophobic lipid core encased by a phospholipid monolayer and surface-associated proteins. To date, only a relatively few LD ‘coat’ proteins in plants have been identified and characterized, most of which come from studies of the model plant Arabidopsis thaliana. To expand our knowledge of the plant LD proteome, the LD-associated protein (LDAP) family from the tung tree (Vernicia fordii), whose seeds are rich in a commercially valuable TAG containing the conjugated fatty acid α-eleostearic acid (C18:3Δ9cis,11trans,13trans [α-ESA]), was identified and characterized. Based on the tung tree transcriptome, three LDAP isoforms (VfLDAP1-3) were elucidated and the encoded proteins distinctly clustered into three clades along with their respective isoforms from other angiosperm species. Ectopic expression of the VfLDAPs in Nicotiana benthamiana leaves revealed that they localized specifically to LDs and influenced LD numbers and sizes, as well as increasing TAG content and altering TAG fatty acid composition. Interestingly, in a partially reconstructed TAG-ESA biosynthetic pathway, the co-expression of VfLDAP3 and, to a lesser degree, VfLDAP2, significantly increased the content of α-ESA stored within the LDs. These results suggest that the VfLDAPs can influence the steady-state content and composition of TAG in plant cells and that certain LDAP isoforms may have evolved to more efficiently package TAGs into LDs containing unusual fatty acids, such as α-ESA.

Plant Sciences↗