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At least 199 records · Page 11
All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

NEAR: Neural Embeddings for Amino acid Relationships

Protein language models (PLMs) have recently demonstrated potential to supplant classical protein database search methods based on sequence alignment, but are slower than common alignment-based tools and appear to be prone to a high rate of false labeling. Here, we present NEAR, a method based on neural representation learning that is designed to improve both speed and accuracy of search for likely homologs in a large protein sequence database. NEAR’s ResNet embedding model is trained using contrastive learning guided by trusted sequence alignments. It computes per-residue embeddings for target and query protein sequences, and identifies alignment candidates with a pipeline consisting of residue-level k-NN search and a simple neighbor aggregation scheme. Tests on a benchmark consisting of trusted remote homologs and randomly shuffled decoy sequences reveal that NEAR substantially improves accuracy relative to state-of-the-art PLMs, with lower memory requirements and faster embedding and search speed. While these results suggest that the NEAR model may be useful for standalone homology detection with increased sensitivity over standard alignment-based methods, in this manuscript we focus on a more straightforward analysis of the model’s value as a high-speed pre-filter for sensitive annotation. In that context, NEAR is at least 5x faster than the pre-filter currently used in the widely-used profile hidden Markov model (pHMM) search tool HMMER3, and also outperforms the pre-filter used in our fast pHMM tool, nail.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1999

Integrin-like proteins are localized to plasma membrane fractions, not plastids, in Arabidopsis

Integrins are a large family of integral membrane proteins that function in signal transduction in animal systems. These proteins are conserved in vertebrates, invertebrates, and fungi. Evidence from previous research suggests that integrin-like proteins may be present in plants as well, and that these proteins may function in signal transduction during gravitropism. In past studies, researchers have used monoclonal and polyclonal antibodies to localize beta 1 integrin-like proteins in plants. However, there is a disparity between data collected from these studies, especially since molecular weights obtained from these investigations range from 55-120 kDa for integrin-like proteins. To date, a complete investigation which employs all three basic immunolabeling procedures, immunoblotting, immunofluorescence microscopy, and immunogold labeling, in addition to extensive fractionation and exhaustive controls, has been lacking. In this paper, we demonstrate that use of a polyclonal antibody against the cytoplasmic domain of avian beta 1-integrin can produce potential artifacts in immunolocalization studies. However, these problems can be eliminated through use of starchless mutants or proper specimen preparation prior to electrophoresis. We also show that this antibody, when applied within the described parameters and with careful controls, identifies a large (100 kDa) integrin-like protein that is localized to plasma membrane fractions in Arabidopsis.

NASA Discipline Plant Biology↗
DOE OSTI2025

Imaging and spatially resolved mass spectrometry applications in nephrology

The application of spatially resolved mass spectrometry (MS) and MS imaging approaches for studying biomolecular processes in the kidney is rapidly growing. These powerful methods, which enable label-free and multiplexed detection of many molecular classes across omics domains (including metabolites, drugs, proteins and protein post-translational modifications), are beginning to reveal new molecular insights related to kidney health and disease. Further, the complexity of the kidney often necessitates multiple scales of analysis for interrogating biofluids, whole organs, functional tissue units, single cells and subcellular compartments. Various MS methods can generate omics data across these spatial domains and facilitate both basic science and pathological assessment of the kidney. Optimal processes related to sample preparation and handling for different MS applications are rapidly evolving. Emerging technology and methods, improvement of spatial resolution, broader molecular characterization, multimodal and multiomics approaches and the use of machine learning and artificial intelligence approaches promise to make these applications even more valuable in the field of nephology. Overall, spatially resolved MS and MS imaging methods have the potential to fill much of the omics gap in systems biology analysis of the kidney and provide functional outputs that cannot be obtained using genomics and transcriptomic methods.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Modification of cell wall polysaccharide guides cell division in Streptococcus mutans

In ovoid-shaped, Gram-positive bacteria, MapZ guides FtsZ-ring positioning at cell equators. The cell wall of the ovococcus Streptococcus mutans contains peptidoglycan decorated with serotype c carbohydrates (SCCs). In the present study, we identify the major cell separation autolysin AtlA as an SCC-binding protein. AtlA binding to SCC is attenuated by the glycerol phosphate (GroP) modification. Using fluorescently labeled AtlA constructs, we mapped SCC distribution on the streptococcal surface, revealing enrichment of GroP-deficient immature SCCs at the cell poles and equators. The immature SCCs co-localize with MapZ at the equatorial rings throughout the cell cycle. In GroP-deficient mutants, AtlA is mislocalized, resulting in dysregulated cellular autolysis. These mutants display morphological abnormalities associated with MapZ mislocalization, leading to FtsZ-ring misplacement. Altogether, our data support a model in which maturation of a cell wall polysaccharide provides the molecular cues for the recruitment of cell division machinery, ensuring proper daughter cell separation and FtsZ-ring positioning.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
NASA NTRS1978

Design of polymeric immunomicrospheres for cell labelling and cell separation

Synthesis of several classes of hydrophylic microspheres applied to cell labeling and cell separation is described. Five classes of cross-linked microspheres with functional groups such as carboxyl, hydroxyl, amide and/or pyridine groups were synthesized. These functional groups were used to bind covalently antibodies and other proteins to the surface of the microspheres. To optimize the derivatisation technique, polyglutaraldehyde immunomicrospheres were prepared and utilized. Specific populations of human and murine lymphocytes were labelled with microspheres synthesized by the emulsion of the ionizing radiation technique. The labelling of the cells by means of microspheres containing an iron core produced successful separation of B from T lymphocytes by means of a magnetic field.

Rembaum, A.↗
DOE OSTI2021

Immunotargeting of Nanocrystals by SpyCatcher Conjugation of Engineered Antibodies

Inorganic nanocrystals such as quantum dots (QDs) and upconverting nanoparticles (UCNPs) are uniquely suited for quantitative live-cell imaging and are typically functionalized with ligands to study specific receptors or cellular targets. Antibodies (Ab) are among the most useful targeting reagents owing to their high affinities and specificities, but common nanocrystal labeling methods may orient Ab incorrectly, be reversible or denaturing, or lead to Ab-NP complexes too large for some applications. Here, we report that SpyCatcher proteins, which bind and spontaneously form covalent isopeptide bonds with cognate SpyTag peptides, can conjugate engineered Ab to nanoparticle surfaces with control over stability, orientation, and stoichiometry. Compact SpyCatcher-functionalized QDs and UCNPs may be labeled with short-chain variable fragment Ab (scFv) engineered to bind urokinase-type plasminogen activator receptors (uPAR) that are overexpressed in many human cancers. Confocal imaging of anti-uPAR scFv-QD conjugates shows the antibody mediates specific binding and internalization by breast cancer cells expressing uPAR. Time-lapse imaging of photostable scFv-UCNP conjugates shows that Ab binding causes uPAR internalization with a ~20 min half-life on the cell surface, and uPAR is internalized to endolysosomal compartments distinct from general membrane stains and without significant recycling to the cell surface. The controlled and stable conjugation of engineered Ab to NPs enables targeting of diverse receptors for live-cell study of their distribution, trafficking, and physiology.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

First comprehensive identification of cardiac proteins with putative increased O-GlcNAc levels during pressure overload hypertrophy

Protein posttranslational modifications (PTMs) by O-GlcNAc globally rise during pressure-overload hypertrophy (POH). However, a major knowledge gap exists on the specific proteins undergoing changes in O-GlcNAc levels during POH primarily because this PTM is low abundance and easily lost during standard mass spectrometry (MS) conditions used for protein identification. Methodologies have emerged to enrich samples for O-GlcNAcylated proteins prior to MS analysis. Accordingly, our goal was to identify the specific proteins undergoing changes in O-GlcNAc levels during POH. We used C57/Bl6 mice subjected to Sham or transverse aortic constriction (TAC) to create POH. From the hearts, we labelled the O-GlcNAc moiety with tetramethylrhodamine azide (TAMRA) before sample enrichment by TAMRA immunoprecipitation (IP). We used LC-MS/MS to identify and quantify the captured putative O-GlcNAcylated proteins. We identified a total of 700 putative O-GlcNAcylated proteins in Sham and POH. Two hundred thirty-three of these proteins had significantly increased enrichment in POH over Sham suggesting higher O-GlcNAc levels whereas no proteins were significantly decreased by POH. We examined two MS identified metabolic enzymes, CPT1B and the PDH complex, to validate by immunoprecipitation. We corroborated increased O-GlcNAc levels during POH for CPT1B and the PDH complex. Enzyme activity assays suggests higher O-GlcNAcylation increases CPT1 activity and decreases PDH activity during POH. In summary, we generated the first comprehensive list of proteins with putative changes in O-GlcNAc levels during POH. Our results demonstrate the large number of potential proteins and cellular processes affected by O-GlcNAc and serve as a guide for testing specific O-GlcNAc-regulated mechanisms during POH.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Enhancement of anticancer drug sensitivity in multidrug resistance cells overexpressing ATP-binding cassette (ABC) transporter ABCC10 by CP55, a synthetic derivative of 5-cyano-6-phenylpyrimidin

Highlights: • Multidrug resistance (MDR) in cancer is one of the major obstacles in chemotherapy. • ABCC10 mediates resistance to wide spectrum of chemotherapeutic agents, but only limited number of studies are available. • Overexpression of ABCC10 has been linked to various types of cancers including colorectal, gastric and lung cancer. • CP55 is a potent ABCC10 inhibitor which enhances the anticancer drug sensitivity in ABCC10-overexpression MDR cells. ATP-binding cassette (ABC) transporter C10 (ABCC10), also named multidrug resistance protein 7 (MRP7), is a member of ABC transporter superfamily and has been revealed to transport a wide range of chemotherapeutic agents including taxanes, epothilone B, Vinca alkaloids, and anthracyclines. In our previous study, a 5-cyano-6-phenylpyrimidin derivative CP55 was synthesized and found significantly reversal effect of multidrug resistance (MDR) mediated by ABCB1. In this study, we found CP55 also efficiently reversed MDR mediated by ABCC10. Our in vitro study showed that co-treatment with CP55 significantly increased the efficacy of ABCC10-substrate anticancer drugs in MDR cells overexpressing ABCC10. Furthermore, we showed that treatment with CP55 increased the intracellular accumulation of [{sup 3}H]-labeled anticancer drugs and in-turn decreasing drug efflux by inhibiting the transport activity, without altering ABCC10 protein ex-pression level or cellular localization. Potential CP55-ABCC10 interactions were predicted via docking analysis using human ABCC10 homology model and obtained high docking score. Therefore, CP55 represents a promising therapeutic agent in the combinational treatment of chemo-resistant cancer related to ABCC10.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

D-Band EPR and ENDOR Spectroscopy of 15 N-Labeled Photosystem I

For billions of years, nature has optimized the photosynthetic machinery that converts light energy into chemical energy. Key primary reactions of photosynthesis occur in large membrane protein-cofactor complexes. The light-induced sequential electron transfer reactions occur through a chain of donor/acceptor cofactors embedded in the protein matrix resulting in a long-lived transmembrane charge-separated state. EPR is the method of choice to study electron transfer and the interaction of protein environment with redox-active cofactors. However, the spectra of organic cofactor radicals typically are not fully resolved and severely overlap at conventional X-band EPR. Even at Q-band EPR, this overlap is present and often a serious problem. As a result, there is a large variation of the reported EPR data and limited understanding of electronic structures of several redox-active cofactors. These serious problems can often be overcome by the excellent spectral resolution provided by high-frequency EPR (HF EPR). In this work, we study the electronic structure of the primary electron donor P 700 and the secondary electron acceptor A 1 of Photosystem I (PSI) using 130 GHz (D-band) EPR and Electron-Nuclear-Double-Resonance (ENDOR) spectroscopy. PSI was isotopically labeled with 15 N (I = $\frac{1/2}$) to avoid quadrupolar interactions in the most abundant nitrogen isotope 14 N (I = 1) and simplify the ENDOR spectra. ENDOR spectroscopy is central for determining the hyperfine coupling of nitrogen atoms of the two chlorophyll molecules comprising oxidized P 700 and the involvement of protein nitrogen atoms with reduced A 1 . While HF ENDOR of A 1 - allows identification of two nitrogen atoms, HF ENDOR of P 700 + still does not permit unique assignment of the recorded hyperfine couplings.

15N↗
DOE OSTI2023

Enhancing Nanoparticle Detection in Interferometric Scattering (iSCAT) Microscopy Using a Mask R-CNN

Interferometric scattering microscopy (iSCAT) is a label-free optical microscopy technique that enables imaging of individual nano-objects such as nanoparticles, viruses, and proteins. Essential to this technique is the suppression of background scattering and identification of signals from nano-objects. In the presence of substrates with high roughness, scattering heterogeneities in the background, when coupled with tiny stage movements, cause features in the background to be manifested in background-suppressed iSCAT images. Traditional computer vision algorithms detect these background features as particles, limiting the accuracy of object detection in iSCAT experiments. Here, in this paper, we present a pathway to improve particle detection in such situations using supervised machine learning via a mask region-based convolutional neural network (mask R-CNN). Using a model iSCAT experiment of 19.2 nm gold nanoparticles adsorbing to a rough layer-by-layer polyelectrolyte film, we develop a method to generate labeled datasets using experimental background images and simulated particle signals and train the mask R-CNN using limited computational resources via transfer learning. We then compare the performance of the mask R-CNN trained with and without inclusion of experimental backgrounds in the dataset against that of a traditional computer vision object detection algorithm, Haar-like feature detection, by analyzing data from the model experiment. Results demonstrate that including representative backgrounds in training datasets improved the mask R-CNN in differentiating between background and particle signals and elevated performance by markedly reducing false positives. The methodology for creating a labeled dataset with representative experimental backgrounds and simulated signals facilitates the application of machine learning in iSCAT experiments with strong background scattering and thus provides a useful workflow for future researchers to improve their image processing capabilities.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
NASA NTRS2000

CaMKIIalpha 3' untranslated region-directed mRNA translocation in living neurons: visualization by GFP linkage

The CaMKIIalpha mRNA extends into distal hippocampal dendrites, and the 3' untranslated region (3'UTR) is sufficient to mediate this localization. We labeled the 3'UTR of the CaMKIIalpha mRNA in hippocampal cultures by using a green fluorescent protein (GFP)/MS2 bacteriophage tagging system. The CaMKIIalpha 3'UTR formed discrete granules throughout the dendrites of transfected cells. The identity of the fluorescent granules was verified by in situ hybridization. Over 30 min time periods these granules redistributed without a net increase in granule number; with depolarization there is a tendency toward increased numbers of granules in the dendrites. These observations suggest that finer time resolution of granule motility might reveal changes in the motility characteristics of granules after depolarization. So that motile granules could be tracked, shorter periods of observation were required. The movements of motile granules can be categorized as oscillatory, unidirectional anterograde, or unidirectional retrograde. Colocalization of CaMKIIalpha 3'UTR granules and synapses suggested that oscillatory movements allowed the granules to sample several local synapses. Neuronal depolarization increased the number of granules in the anterograde motile pool. Based on the time frame over which the granule number increased, the translocation of granules may serve to prepare the dendrite for mounting an adequate local translation response to future stimuli. Although the resident pool of granules can respond to signals that induce local translation, the number of granules in a dendrite might reflect its activation history.

NASA Discipline Neuroscience↗
DOE OSTI2023

Dual-Responsive Turn-On T 1 Imaging-Guided Mild Photothermia for Precise Apoptotic Cancer Therapy

Apoptosis has gained increasing attention in cancer therapy as an intrinsic signaling pathway, which leads to minimal leakage of waste products from a dying cell to neighboring normal cells. Among various stimuli to trigger apoptosis, mild hyperthermia is attractive but confronts limitations of non-specific heating and acquired resistance from elevated expression of heat shock proteins. Here, a dual-stimulation activated turn-on T 1 imaging-based nanoparticulate system (DAS) is developed for mild photothermia (≈43 °C)-mediated precise apoptotic cancer therapy. In the DAS, a superparamagnetic quencher (ferroferric oxide nanoparticles, Fe 3 O 4 NPs) and a paramagnetic enhancer (Gd-DOTA complexes) are connected via the N6-methyladenine (m 6 A)-caged, Zn 2+ -dependent DNAzyme molecular device. The substrate strand of the DNAzyme contains one segment of Gd-DOTA complex-labeled sequence and another one of HSP70 antisense oligonucleotide. When the DAS is taken up by cancer cells, overexpressed fat mass and obesity-associated protein (FTO) specifically demethylates the m 6</-sup>A group, thereby activating DNAzymes to cleave the substrate strand and simultaneously releasing Gd-DOTA complex-labeled oligonucleotides. The restored T 1 signal from the liberated Gd-DOTA complexes lights up the tumor to guide the location and time of deploying 808 nm laser irradiation. Afterward, locally generated mild photothermia works in concert with HSP70 antisense oligonucleotides to promote apoptosis of tumor cells. This highly integrated design provides an alternative strategy for mild hyperthermia-mediated precise apoptotic cancer therapy.

70 PLASMA PHYSICS AND FUSION TECHNOLOGY↗
DOE OSTI2025

A plant-specific cytochrome b 5 –like protein is essential for phytosterol biosynthesis

Sterols are essential isoprenoid derivatives that contribute to membrane structure and function. In plants, they also serve as precursors to phytohormones and specialized metabolites important for development, defense, and health. Although the sterol biosynthetic pathway is considered well-characterized, we report the discovery of a plant-specific cytochrome b 5 –like protein, CB5LP, as a critical component of phytosterol biosynthesis. Loss of CB5LP in Arabidopsis causes embryonic defects, seedling lethality, and accumulation of 14α-methyl-sterols, with reduced levels of sitosterol and stigmasterol—indicating a defect in sterol 14α-demethylation. TurboID-based proximity labeling and in vitro assays show that CB5LP physically and functionally interacts with CYP51, a cytochrome P450 enzyme catalyzing this demethylation step. Unlike canonical cytochrome b 5 proteins, CB5LP has a reversed topology and is exclusive to plants, acting as an evolutionarily distinct electron donor. This discovery reveals an uncharacterized redox partnership essential for sterol biosynthesis and highlights a promising target for the development of selective herbicide.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Bimetallic Metal–Organic Framework Fe/Co-MIL-88(NH 2 ) Exhibiting High Peroxidase-like Activity and Its Application in Detection of Extracellular Vesicles

Metal–organic frameworks (MOFs) have many attractive features, including tunable composition, rigid structure, controllable pore size, and large specific surface area, and thus are highly applicable in molecular analysis. Depending on the MOF structure, a high number of un-saturated metal sites can be exposed to catalyze chemical reactions. In the present work, we report that by using both Co(II) and Fe(III) to prepare the MIL-88(NH 2 ) MOF, we can produce the bimetallic MOF that can catalyze the conversion of 3,3', 5,5"-tetramethylbenzidine (TMB) to a color product through reaction with H 2 O 2 at a higher reaction rate than the monometallic Fe-MIL-88(NH 2 ). The Michaelis constants (K m ) of the catalytic reaction for TMB and H 2 O 2 are 3-5 times smaller, and the catalytic constants (k cat ) are 5-10 times higher than those of the horse-radish peroxidase (HRP), supporting ultrahigh peroxidase-like activity. These values are also much more superior to those of the HRP-mimicking MOFs reported previously. Interestingly, the bimetallic MOF can be coupled with glucose oxidase (GOx) to trigger the cascade enzymatic reaction for highly sensitive detection of extracellular vesicles (EVs), a family of important biomarkers. Through conjugation to the aptamer that recognizes the marker protein on EV surface, the MOF can help isolate the EVs from biological matrices, which are subsequently labeled by GOx via antibody recognition. The cascade enzymatic reaction between MOF and GOx enables detection of EVs at a concentration as low as 7.8 × 10 4 particles/ml. Further, the assay can be applied to monitor EV secretion by cultured cells, and also can successfully detect the different EV quantities in the sera samples collected from cancer patients and healthy controls. Overall, we prove that the bimetallic Fe/Co-MIL-88(NH 2 ) MOF, with its high peroxidase-activity and high biocompatibility, is a valuable tool deployable in clinical assays to facility disease diagnosis and prognosis.

36 MATERIALS SCIENCE↗
NASA NTRS2000

Plant nuclei can contain extensive grooves and invaginations

Plant cells can exhibit highly complex nuclear organization. Through dye-labeling experiments in untransformed onion epidermal and tobacco culture cells and through the expression of green fluorescent protein targeted to either the nucleus or the lumen of the endoplasmic reticulum/nuclear envelope in these cells, we have visualized deep grooves and invaginations into the large nuclei of these cells. In onion, these structures, which are similar to invaginations seen in some animal cells, form tubular or planelike infoldings of the nuclear envelope. Both grooves and invaginations are stable structures, and both have cytoplasmic cores containing actin bundles that can support cytoplasmic streaming. In dividing tobacco cells, invaginations seem to form during cell division, possibly from strands of the endoplasmic reticulum trapped in the reforming nucleus. The substantial increase in nuclear surface area resulting from these grooves and invaginations, their apparent preference for association with nucleoli, and the presence in them of actin bundles that support vesicle motility suggest that the structures might function both in mRNA export from the nucleus and in protein import from the cytoplasm to the nucleus.

Non-NASA Center↗
DOE OSTI2022

Probe for selectively characterizing enzymes involved in xenobiotic metabolism and method of making and using the same

Activity-based probes that can be used to selectively identify and characterize enzymes that are involved in different phases of xenobiotic metabolism in a host and its microbiota population(s) are described. The activity-based probes described specifically label only their target active enzymes involved in xenobiotic metabolism and therefore provide a measurement of true protein functional activity rather than transcript or protein abundance. The activity-based probes also provide multimodal profiling of these active enzymes. Methods for preparing the activity based probes and exemplary methods for their use also are disclosed.

Wright, Aaron T.↗
DOE OSTI2024

Probe for selectively characterizing enzymes involved in xenobiotic metabolism and method of making and using the same

Activity-based probes that can be used to selectively identify and characterize enzymes that are involved in different phases of xenobiotic metabolism in a host and its microbiota population(s) are described. The activity-based probes described specifically label only their target active enzymes involved in xenobiotic metabolism and therefore provide a measurement of true protein functional activity rather than transcript or protein abundance. The activity-based probes also provide multimodal profiling of these active enzymes. Methods for preparing the activity based probes and exemplary methods for their use also are disclosed.

Wright, Aaron T.↗
DOE OSTI2023

TopPICR: A Companion R Package for Top-Down Proteomics Data Analysis

Top-down proteomics is the analysis of proteins in their intact form without proteolysis, thus preserving valuable information about post-translational modifications, isoforms, and proteolytic processing. However, it is still a developing field due to limitations in the instrumentation, difficulties with interpretation of complex mass spectra, and a lack of well-established quantification approaches. TopPIC is one of the popular tools for proteoform identification. Here we extended its capabilities into label-free proteoform quantification by developing a companion R package (TopPICR). Key steps in the TopPICR pipeline include filtering identifications, inferring a minimal set of protein accessions explaining the observed sequences, aligning retention times, recalibrating measured masses, clustering features across datasets, and finally compiling feature intensities using the match-between-runs approach. The output of the pipeline is an MSnSet object which makes downstream data analysis seamlessly compatible with packages from the Bioconductor project. It also provides the capability for visualizing proteoforms within the context of the parent protein sequence. The functionality of TopPICR is demonstrated on top-down LC-MS/MS datasets of 10 human-in-mouse xenografts of luminal and basal breast tumor samples.

59 BASIC BIOLOGICAL SCIENCES↗