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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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33 records · Page 2
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Incorporation and Assembly of a Light-Emitting Enzymatic Reaction into Model Protein Condensates

Eukaryotic cells partition enzymes and other cellular components into distinct subcellular compartments to generate specialized biochemical niches. A subclass of these compartments form in the absence of lipid membranes, via liquid-liquid phase separation of proteins to form biomolecular condensates or “membraneless organelles” such as nucleoli, stress granules, and P-bodies. Because of their ability to form compartments from simple starting materials, membraneless organelles are an attractive target for engineering new functionalities in both living cells and protocells. In this work, we demonstrate incorporation of novel enzymatic activity in protein coacervates with a light-generating enzyme, NanoLuc, to produce bioluminescence. Using condensates comprised of the disordered RGG domain of C. elegans LAF-1, we show functionalization of condensates with enzymatic activity in vitro and that localization to protein coacervates enhances the assembly and activity of split enzymes. To build condensates that function as light emitting reactors, we designed a NanoLuc enzyme flanked by RGG domains. Furthermore, the resulting condensates concentrated NanoLuc by 10-fold over bulk solution and display significantly increased net reaction rates. We further show that condensate viscosity impacts light emission due to diffusion-limited behavior. By splitting NanoLuc enzyme into its constituent components, we demonstrate that NanoLuc activity can be reconstituted via co-condensation. Further, we demonstrate control of the spatial localization of enzyme within condensates by targettng NanoLuc to the surface of in vitro condensates. Collectively, this work demonstrates that membraneless organelles can be endowed with localized enzymatic activity, and that this activity can be spatially and temporally controlled via enzyme reconstitution and design of protein surfactants.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

Regeneration of critical-sized defects, in a goat model, using a dextrin-based hydrogel associated with granular synthetic bone substitute

Abstract The development of injectable bone substitutes (IBS) have obtained great importance in the bone regeneration field, as a strategy to reach hardly accessible defects using minimally invasive techniques and able to fit to irregular topographies. In this scenario, the association of injectable hydrogels and bone graft granules is emerging as a well-established trend. Particularly, in situ forming hydrogels have arisen as a new IBS generation. An in situ forming and injectable dextrin-based hydrogel (HG) was developed, aiming to act as a carrier of granular bone substitutes and bioactive agents. In this work, the HG was associated to a granular bone substitute (Bonelike®) and implanted in goat critical-sized calvarial defects (14 mm) for 3, 6 and 12 weeks. The results showed that HG improved the handling properties of the Bonelike® granules and did not affect its osteoconductive features, neither impairing the bone regeneration process. Human multipotent mesenchymal stromal cells from the umbilical cord, extracellular matrix hydrolysates and the pro-angiogenic peptide LLKKK18 were also combined with the IBS. These bioactive agents did not enhance the new bone formation significantly under the conditions tested, according to micro-computed tomography and histological analysis.

Pereira, Isabel↗
DOE OSTI2025

Secretory stimuli distinctly regulate insulin secretory granule maturation through structural remodeling

Insulin secretory granule (ISG) maturation is a crucial aspect of insulin secretion and glucose homeostasis. The regulation of this maturation remains poorly understood, especially how secretory stimuli affect ISG maturity and subcellular localization. In this study, we used soft X-ray tomography (SXT) to quantitatively map ISG morphology, density, and location in single INS-1E and mouse pancreatic β cells under the effect of various secretory stimuli. We found that the activation of glucokinase (GK), gastric inhibitory polypeptide receptor (GIPR), glucagon-like peptide-1 receptor (GLP-1R), and G protein-coupled receptor 40 (GPR40) promotes ISG maturation. Each stimulus induces unique structural remodeling in ISGs, by altering size and density, depending on the specific signaling cascades activated. These distinct ISG subpopulations mobilize and redistribute in the cell, altering the overall cellular structural organization. Our results provide insight into how current diabetes and obesity therapies impact ISG maturation and may inform the development of future treatments that target maturation specifically.

insulin granule maturation↗
DOE OSTI2020

Productivity and bioproduct formation in phototropic knock/out mutants in micro algae

Phototropin is a blue light receptor, which mediates a variety of blue-light elicited physiological processes in plants and algae. In higher plants these processes include phototropism, chloroplast movement and stomatal opening. In the green alga Chlamydomonas reinhardtii, phototropin plays a vital role in progression of the sexual life cycle and in the control of the eye spot size and light sensitivity. Phototropin is also involved in blue-light mediated changes in the synthesis of chlorophylls, carotenoids, chlorophyll binding proteins. We compared the transcriptome of phototropin knock out (PHOT KO) mutant and wild-type parent to analyze differences in gene expression in high light grown cultures (500 μmol photons m⁻²s⁻¹). Our results indicate the up-regulation of genes involved in photosynthetic electron transport chain, carbon fixation pathway, starch, lipid, and cell cycle control genes. With respect to photosynthetic electron transport genes, genes encoding proteins of the cytochrome b6f and ATP synthase complex were up regulated potentially facilitating proton-coupled electron transfer. In addition genes involved in limiting steps in the Calvin cycle Ribulose-1,5-biphosphate carboxylase/oxygenase (RuBisCO), Sidoheptulose 1,7 biophosphatase (SBPase), Glyceraldehyde-3-phosphate dehydrogenase (3PGDH) and that mediate cell-cycle control (CDK) were also up regulated along with starch synthase and fatty acid biosynthesis genes involved in starch and lipid synthesis. In addition, transmission electron micrographs show increased accumulation of starch granules in PHOT mutant compared to wild type, which is consistent with the higher expression of starch synthase genes. Collectively, the altered patterns of gene expression in the PHOT mutants were associated with a two-fold increase in growth and biomass accumulation compared to wild type when grown in environmental photobioreactors (Phenometrics) that simulate a pond environment. In conclusion, our studies suggest that phototropin may be a master gene regulator that suppresses rapid cell growth and promotes gametogenesis and sexual recombination in wild type strains.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

HDX–MS finds that partial unfolding with sequential domain activation controls condensation of a cellular stress marker

Eukaryotic cells form condensates to sense and adapt to their environment [S. F. Banani, H. O. Lee, A. A. Hyman, M. K. Rosen,Nat. Rev. Mol. Cell Biol.18, 285–298 (2017), H. Yoo, C. Triandafillou, D. A. Drummond,J. Biol. Chem.294, 7151–7159 (2019)]. Poly(A)-binding protein (Pab1), a canonical stress granule marker, condenses upon heat shock or starvation, promoting adaptation [J. A. Ribacket al.,Cell168, 1028–1040.e19 (2017)]. The molecular basis of condensation has remained elusive due to a dearth of techniques to probe structure directly in condensates. We apply hydrogen–deuterium exchange/mass spectrometry to investigate the mechanism of Pab1’s condensation. Pab1’s four RNA recognition motifs (RRMs) undergo different levels of partial unfolding upon condensation, and the changes are similar for thermal and pH stresses. Although structural heterogeneity is observed, the ability of MS to describe populations allows us to identify which regions contribute to the condensate’s interaction network. Our data yield a picture of Pab1’s stress-triggered condensation, which we term sequential activation (Fig. 1A), wherein each RRM becomes activated at a temperature where it partially unfolds and associates with other likewise activated RRMs to form the condensate. Subsequent association is dictated more by the underlying free energy surface than specific interactions, an effect we refer to as thermodynamic specificity. Our study represents an advance for elucidating the interactions that drive condensation. Furthermore, our findings demonstrate how condensation can use thermodynamic specificity to perform an acute response to multiple stresses, a potentially general mechanism for stress-responsive proteins.

Science & Technology - Other Topics↗
DOE OSTI2021

Influence of Uranium Concentration and pH on U-Phosphate Biomineralization by Caulobacter OR37

Uranium contamination of soils and groundwater in the United States represents a significant health risk and will require multiple remediation approaches. Microbial phosphatase activity coupled to the addition of an organic P source has recently been studied as a remediation strategy that provides an extended release of inorganic P (Pi) into U-contaminated sites, resulting in the precipitation of meta-autunite minerals. Previous laboratory- and field-based biomineralization studies have investigated environments with relatively high U concentrations (>20 μM). However, most contaminated sites have much lower U concentrations (<2 μM). The Environmental Protection Agency (EPA) limit for U in drinking water is 0.126 μM. Reaching this regulatory limit becomes challenging as U concentrations approach autunite solubility. Furthermore, we studied the precipitation of U(VI)-phosphate minerals by an environmental isolate of Caulobacter sp. (strain OR37) from an Oak Ridge, Tennessee, U-contaminated site. Abiotic U(VI) solubility experiments reveal that U(VI)-phosphate minerals do not form in the presence of excess Pi (500 μM) when U(VI) concentrations are <1 μM and pH is <5. When OR37 cells are reacted under the same conditions with Pi or glycerol-2-phosphate, U(VI)-phosphate mineral formation was observed, along with the formation of intracellular polyphosphate granules. These results show that bacteria provide supersaturated microenvironments needed for U(VI)-phosphate mineralization while hydrolyzing organic P sources. This provides a pathway to lower U concentrations to below EPA limits for drinking water.

38 RADIATION CHEMISTRY, RADIOCHEMISTRY, AND NUCLEA↗
DOE OSTI2024

Quantitative Analysis of Rhodobacter sphaeroides Storage Organelles via Cryo-Electron Tomography and Light Microscopy

Bacterial cytoplasmic organelles are diverse and serve many varied purposes. Here, we employed Rhodobacter sphaeroides to investigate the accumulation of carbon and inorganic phosphate in the storage organelles, polyhydroxybutyrate (PHB) and polyphosphate (PP), respectively. Using cryo-electron tomography (cryo-ET), these organelles were observed to increase in size and abundance when growth was arrested by chloramphenicol treatment. The accumulation of PHB and PP was quantified from three-dimensional (3D) segmentations in cryo-tomograms and the analysis of these 3D models. The quantification of PHB using both segmentation analysis and liquid chromatography and mass spectrometry (LCMS) each demonstrated an over 10- to 20-fold accumulation of PHB. The cytoplasmic location of PHB in cells was assessed with fluorescence light microscopy using a PhaP-mNeonGreen fusion-protein construct. The subcellular location and enumeration of these organelles were correlated by comparing the cryo-ET and fluorescence microscopy data. A potential link between PHB and PP localization and possible explanations for co-localization are discussed. Finally, the study of PHB and PP granules, and their accumulation, is discussed in the context of advancing fundamental knowledge about bacterial stress response, the study of renewable sources of bioplastics, and highly energetic compounds.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Autotrophic biofilms sustained by deeply sourced groundwater host diverse bacteria implicated in sulfur and hydrogen metabolism

Abstract Background Biofilms in sulfide-rich springs present intricate microbial communities that play pivotal roles in biogeochemical cycling. We studied chemoautotrophically based biofilms that host diverse CPR bacteria and grow in sulfide-rich springs to investigate microbial controls on biogeochemical cycling. Results Sulfide springs biofilms were investigated using bulk geochemical analysis, genome-resolved metagenomics, and scanning transmission X-ray microscopy (STXM) at room temperature and 87 K. Chemolithotrophic sulfur-oxidizing bacteria, including Thiothrix and Beggiatoa , dominate the biofilms, which also contain CPR Gracilibacteria, Absconditabacteria, Saccharibacteria, Peregrinibacteria, Berkelbacteria, Microgenomates, and Parcubacteria. STXM imaging revealed ultra-small cells near the surfaces of filamentous bacteria that may be CPR bacterial episymbionts. STXM and NEXAFS spectroscopy at carbon K and sulfur L 2,3 edges show that filamentous bacteria contain protein-encapsulated spherical elemental sulfur granules, indicating that they are sulfur oxidizers, likely Thiothrix . Berkelbacteria and Moranbacteria in the same biofilm sample are predicted to have a novel electron bifurcating group 3b [NiFe]-hydrogenase, putatively a sulfhydrogenase, potentially linked to sulfur metabolism via redox cofactors. This complex could potentially contribute to symbioses, for example, with sulfur-oxidizing bacteria such as Thiothrix that is based on cryptic sulfur cycling. One Doudnabacteria genome encodes adjacent sulfur dioxygenase and rhodanese genes that may convert thiosulfate to sulfite. We find similar conserved genomic architecture associated with CPR bacteria from other sulfur-rich subsurface ecosystems. Conclusions Our combined metagenomic, geochemical, spectromicroscopic, and structural bioinformatics analyses of biofilms growing in sulfide-rich springs revealed consortia that contain CPR bacteria and sulfur-oxidizing Proteobacteria, including Thiothrix , and bacteria from a new family within Beggiatoales. We infer roles for CPR bacteria in sulfur and hydrogen cycling.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Metal Ion Fluxes Controlling Amphibian Fertilization

Mammalian oocytes undergo major changes in zinc content and localization to be fertilized, the most striking being the rapid exocytosis of over 10 billion zinc ions in what are known as zinc sparks. Here, we report that fertilization of amphibian Xenopus laevis eggs also initiates a zinc spark that progresses across the cell surface in coordination with dynamic calcium waves. This zinc exocytosis is accompanied by a newly recognized loss of intracellular manganese. Synchrotron-based X-ray fluorescence and analytical electron microscopy reveal that zinc and manganese are sequestered in a system of cortical granules that are abundant at the animal pole. Through electron-nuclear double-resonance studies, we rule out Mn2+ complexation with phosphate or nitrogenous ligands in intact eggs, but the data are consistent with a carboxylate coordination environment. Our observations suggest that zinc and manganese fluxes are a conserved feature of fertilization in vertebrates and that they function as part of a physiological block to polyspermy.

Seeler, John F.↗
DOE OSTI2021

Cellular metabolic stress responses via organelles

Metabolite fluctuations following nutrient metabolism or environmental stresses impact various intracellular signaling networks and stress responses to maintain cellular and organismal homeostasis. It has been shown that subcellular organelles, such as the endoplasmic reticulum, the Golgi apparatus, lysosomes and mitochondria serve as crucial hubs linking alterations in metabolite levels to cellular responses. This role is coordinated by molecular machineries that are associated with the lipid membranes of organelles, which sense the fluctuations in specific metabolites and activate the appropriate signaling and effector molecules. Moreover, recent studies have demonstrated that membraneless organelles, such as the nucleolus and stress granules, are involved in the metabolic stress response. Metabolite-induced post-translational modifications appear to play an important role in this process. Here, we review the molecular mechanisms of metabolite sensing and metabolite-mediated stress responses through membrane-bound and membraneless organelles in mammalian cells.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2025

Developing affordable and efficient heating devices for enhanced live cell imaging in confocal microscopy

Temperature control is crucial for live cell imaging, particularly in studies involving plant responses to high ambient temperatures and thermal stress. This study presents the design, development, and testing of two cost-effective heating devices tailored for confocal microscopy applications: an aluminum heat plate and a wireless mini-heater. The aluminum heat plate, engineered to integrate seamlessly with the standard 160 mm × 110 mm microscope stage, supports temperatures up to 36°C, suitable for studies in the range of non-stressful warm temperatures (e.g., 25-27°C forArabidopsis thaliana) and moderate heat stress (e.g., 30-36°C forA. thaliana). We also developed a wireless mini-heater that offers rapid, precise heating directly at the sample slide, with a temperature increase rate over 30 times faster than the heat plate. The wireless heater effectively maintained target temperatures up to 50°C, ideal for investigating severe heat stress and heat shock responses in plants. Both devices performed well in controlled studies, including the real-time analysis of heat shock protein accumulation and stress granule formation inA. thaliana. Our designs are effective and affordable, with total construction costs lower than $300. This accessibility makes them particularly valuable for small laboratories with limited funding. Future improvements could include enhanced heat uniformity, humidity control to mitigate evaporation, and more robust thermal management to minimize focus drift during extended imaging sessions. These modifications would further solidify the utility of our heating devices in live cell imaging, offering researchers reliable, budget-friendly tools for exploring plant thermal biology.

Plant Sciences↗
DOE OSTI2023

CryoET reveals organelle phenotypes in huntington disease patient iPSC-derived and mouse primary neurons

Huntington’s disease (HD) is caused by an expanded CAG repeat in the huntingtin gene, yielding a Huntingtin protein with an expanded polyglutamine tract. While experiments with patient-derived induced pluripotent stem cells (iPSCs) can help understand disease, defining pathological biomarkers remains challenging. Here, we used cryogenic electron tomography to visualize neurites in HD patient iPSC-derived neurons with varying CAG repeats, and primary cortical neurons from BACHD, deltaN17-BACHD, and wild-type mice. In HD models, we discovered sheet aggregates in double membrane-bound organelles, and mitochondria with distorted cristae and enlarged granules, likely mitochondrial RNA granules. We used artificial intelligence to quantify mitochondrial granules, and proteomics experiments reveal differential protein content in isolated HD mitochondria. Knockdown of Protein Inhibitor of Activated STAT1 ameliorated aberrant phenotypes in iPSC- and BACHD neurons. We show that integrated ultrastructural and proteomic approaches may uncover early HD phenotypes to accelerate diagnostics and the development of targeted therapeutics for HD.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Functional Insights of Salinity Stress-Related Pathways in Metagenome-Resolved Methanothrix Genomes

Recently, methanogenic archaea belonging to the genus Methanothrix were reported to have a fundamental role in maintaining stable ecosystem functioning in anaerobic bioreactors under different configurations/conditions. In this study, we reconstructed three Methanothrix metagenome-assembled genomes (MAGs) from granular sludge collected from saline upflow anaerobic sludge blanket (UASB) reactors, where Methanothrix harundinacea was previously implicated with the formation of compact and stable granules under elevated salinity levels (up to 20 g/L Na + ). Genome annotation and pathway analysis of the Methanothrix MAGs revealed a genetic repertoire supporting their growth under high salinity. Specifically, the most dominant Methanothrix (MAG_279), classified as a subspecies of Methanothrix_A harundinacea_D, had the potential to augment its salinity resistance through the production of different glycoconjugates via the N-glycosylation process, and via the production of compatible solutes as N ε -acetyl-β-lysine and ectoine. The stabilization and reinforcement of the cell membrane via the production of isoprenoids was identified as an additional stress-related pathway in this microorganism. The improved understanding of the salinity stress-related mechanisms of M. harundinacea highlights its ecological niche in extreme conditions, opening new perspectives for high-efficiency methanisation of organic waste at high salinities, as well as the possible persistence of this methanogen in highly-saline natural anaerobic environments.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

VAMPs sensitive to tetanus toxin are required for cortical granule exocytosis in mouse oocytes

Highlights: • Cortical granule exocytosis is sensitive to tetanus toxin in mouse oocytes. • VAMP1 and VAMP3 proteins participate in cortical granule exocytosis. • This work confirms that cortical granule exocytosis is mediated by SNARE complex. Fusion of cortical granules with oocyte plasma membrane is one of the most significant secretory events to prevent polyspermy during oocyte activation. Cortical granule exocytosis (CGE) is distinct from most other exocytosis because cortical granules are not renewed after secretion. However, it is thought to be mediated by SNARE complex, which mediates membrane fusion in other exocytoses. SNAREs proteins are divided into Q (glutamine)- and R (arginine)-SNAREs. Q-SNAREs include Syntaxins and SNAP25 family, and R-SNAREs include VAMPs family. In mouse oocytes, Syntaxin4 and SNAP23 have been involved in CGE; nevertheless, it is unknown if VAMP is required. Here, we demonstrated by RT-PCR and immunoblotting that VAMP1 and VAMP3 are expressed in mouse oocyte, and they localized in the cortical region of this cell. Using a functional assay to quantify CGE, we showed that tetanus toxin –which specifically cleavages VAMP1, VAMP2 or VAMP3– inhibited CGE suggesting that at least one VAMP was necessary. Function blocking assays demonstrated that only the microinjection of anti-VAMP1 or anti-VAMP3 antibodies abolished CGE in activated oocytes. These findings demonstrate that R-SNAREs sensitive to tetanus toxin, VAMP1 and VAMP3 –but not VAMP2-, are required for CGE and demonstrate that CGE is mediated by the SNARE complex.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Sas20 is a highly flexible starch-binding protein in the Ruminococcus bromii cell-surface amylosome

Ruminococcus bromii is a keystone species in the human gut that has the rare ability to degrade dietary resistant starch (RS). This bacterium secretes a suite of starch-active proteins that work together within larger complexes called amylosomes that allow R. bromii to bind and degrade RS. Starch adherence system protein 20 (Sas20) is one of the more abundant proteins assembled within amylosomes, but little could be predicted about its molecular features based on amino acid sequence. Here, we performed a structure–function analysis of Sas20 and determined that it features two discrete starch-binding domains separated by a flexible linker. We show that Sas20 domain 1 contains an N-terminal β-sandwich followed by a cluster of α-helices, and the nonreducing end of maltooligosaccharides can be captured between these structural features. Furthermore, the crystal structure of a close homolog of Sas20 domain 2 revealed a unique bilobed starch-binding groove that targets the helical α1,4-linked glycan chains found in amorphous regions of amylopectin and crystalline regions of amylose. Affinity PAGE and isothermal titration calorimetry demonstrated that both domains bind maltoheptaose and soluble starch with relatively high affinity (K d ≤ 20 μM) but exhibit limited or no binding to cyclodextrins. Finally, small-angle X-ray scattering analysis of the individual and combined domains support that these structures are highly flexible, which may allow the protein to adopt conformations that enhance its starch-targeting efficiency. Taken together, we conclude that Sas20 binds distinct features within the starch granule, facilitating the ability of R. bromii to hydrolyze dietary RS.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗