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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 1,171 records · Page 65
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

An integrated in vivo/in vitro framework to enhance cell-free biosynthesis with metabolically rewired yeast extracts

Cell-free systems using crude cell extracts present appealing opportunities for designing biosynthetic pathways and enabling sustainable chemical synthesis. However, the lack of tools to effectively manipulate the underlying host metabolism in vitro limits the potential of these systems. Here, we create an integrated framework to address this gap that leverages cell extracts from host strains genetically rewired by multiplexed CRISPR-dCas9 modulation and other metabolic engineering techniques. As a model, we explore conversion of glucose to 2,3-butanediol in extracts from flux-enhanced Saccharomyces cerevisiae strains. We show that cellular flux rewiring in several strains of S. cerevisiae combined with systematic optimization of the cell-free reaction environment significantly increases 2,3-butanediol titers and volumetric productivities, reaching productivities greater than 0.9 g/L-h. We then show the generalizability of the framework by improving cell-free itaconic acid and glycerol biosynthesis. Our coupled in vivo/in vitro metabolic engineering approach opens opportunities for synthetic biology prototyping efforts and cell-free biomanufacturing.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Cycling ferulate in monocot cell walls

Cell walls in grasses contain arabinoxylan polysaccharides decorated with ferulate groups but the mechanism by which the ferulate is attached to arabinoxylans has long remained unknown. Here, a new study shows that ferulate is transferred to arabinoxylan from a 3,6-di-O-feruloyl sucrose intermediate formed in a sucrose ferulate cycle.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Facile One-Pot Nanoproteomics for Label-Free Proteome Profiling of 50–1000 Mammalian Cells

Recent advances in sample preparation enable label-free MS-based proteome profiling of small numbers of mammalian cells. However, specific devices are often required to downscale sample processing volume from the standard 50-200 µL to sub-µL for effective nanoproteomics, which greatly impedes the implementation of current nanoproteomics methods by broad proteomics research community. Here we report a facile one-pot nanoproteomics method termed SOPs-MS (Surfactant-assisted One-Pot sample processing at the standard volume coupled with MS) for convenient proteome profiling of 50-1000 mammalian cells. Building upon our recent development of SOP-MS for label-free single-cell proteomics at low µL volume (Commun Bio 2021, 4, 265), we have systematically evaluated its processing volume at 10-200 µL using 100 human cells for robust reproducible nanoproteomic analysis. The processing volume of 50 µL which is in the range of volume for standard proteomics sample preparation, has been selected for easy sample handling with benchtop micropipette. Using the commonly accessible LC-MS platform, SOPs-MS allows for reliable label-free quantification of ~1200-2700 protein groups from 50-1000 MCF10A cells. When applied to small subpopulations of mouse colon crypt cells, SOPs-MS can reveal distinct protein signatures between any two subpopulation cells with identification of ~1500-2500 protein groups for each subpopulation. SOPs-MS may pave the way for routine deep proteome profiling of small numbers of cells as well as low-input samples.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Structural delineation and phase-dependent activation of the costimulatory CD27:CD70 complex

CD27 is a tumor necrosis factor (TNF) receptor, which stimulates lymphocytes and promotes their differentiation upon activation by TNF ligand CD70. Activation of the CD27 receptor provides a costimulatory signal to promote T cell, B cell, and NK cell activity to facilitate antitumor and anti-infection immunity. Aberrant increased and focused expression of CD70 on many tumor cells renders CD70 an attractive therapeutic target for direct tumor killing. However, despite their use as drug targets to treat cancers, the molecular basis and atomic details of CD27 and CD70 interaction remain elusive. Here we report the crystal structure of human CD27 in complex with human CD70. Analysis of our structure shows that CD70 adopts a classical TNF ligand homotrimeric assembly to engage CD27 receptors in a 3:3 stoichiometry. By combining structural and rational mutagenesis data with reported disease-correlated mutations, we identified the key amino acid residues of CD27 and CD70 that control this interaction. We also report increased potency for plate-bound CD70 constructs compared with solution-phase ligand in a functional activity to stimulate T-cells in vitro. These findings offer new mechanistic insight into this critical costimulatory interaction.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2021

Upregulation of a novel LncRNA AC104958.2 stabilized by PCBP2 promotes proliferation and microvascular invasion in hepatocellular carcinoma

Highlights: • AC104958.2 is a novel lncRNA identified to be upregulated in HCC tissues and associated with MVI. • Knockout of AC104958.2 inhibited cell proliferation, invasion, migration and EMT. • AC104958.2 interacted with PCBP2. • PCBP2 was upregulated in HCC and enhanced the RNA stability of AC104958.2. Long non-coding RNAs (lncRNAs) were reported to be involved in tumorigenesis and progression of hepatocellular carcinoma (HCC). Microvascular invasion (MVI) is an independent predictor for early recurrence and overall survival in postoperative patients with HCC. However, the mechanisms how lncRNAs affect HCC and MVI remain elusive. By RNA sequencing (RNA-seq) in a series of 65 HCC samples and 30 paired adjacent non-tumor liver tissue, we identified a novel lncRNA AC104958.2 that was significantly upregulated in HCC tissues and associated with MVI. Overexpression of AC104958.2 obviously elevated cell viability, metastasis, invasion and epithelial-mesenchymal transition (EMT), while knockout of AC104958.2 mediated by CRISPR/Cas9 technique showed the opposite effects. In addition, the interaction between AC104958.2 and Poly (rC) binding protein 2 (PCBP2) was identified by RNA pull down and mass spectrometry (MS), which was further validated by RNA immunoprecipitation (RIP). PCBP2 was also upregulated in HCC and associated with MVI. High expression of both AC104958.2 and PCBP2 was correlated with tumor size, TNM stage and MVI in HCC. Overexpression of PCBP2 greatly increased the cell viability, metastasis, invasion and EMT. Moreover, actinomycin D assay showed that overexpression of PCBP2 enhanced the RNA stability of AC104958.2. In conclusion, our study showed that a novel lncRNA AC104958.2 exerted oncogenic roles in HCC and might be a promising biomarker and therapeutic target.

60 APPLIED LIFE SCIENCES↗
NASA NTRS1992

The Acid Growth Theory of auxin-induced cell elongation is alive and well

Plant cells elongate irreversibly only when load-bearing bonds in the walls are cleaved. Auxin causes the elongation of stem and coleoptile cells by promoting wall loosening via cleavage of these bonds. This process may be coupled with the intercalation of new cell wall polymers. Because the primary site of auxin action appears to be the plasma membrane or some intracellular site, and wall loosening is extracellular, there must be communication between the protoplast and the wall. Some "wall-loosening factor" must be exported from auxin-impacted cells, which sets into motion the wall loosening events. About 20 years ago, it was suggested that the wall-loosening factor is hydrogen ions. This idea and subsequent supporting data gave rise to the Acid Growth Theory, which states that when exposed to auxin, susceptible cells excrete protons into the wall (apoplast) at an enhanced rate, resulting in a decrease in apoplastic pH. The lowered wall pH then activates wall-loosening processes, the precise nature of which is unknown. Because exogenous acid causes a transient (1-4 h) increase in growth rate, auxin must also mediate events in addition to wall acidification for growth to continue for an extended period of time. These events may include osmoregulation, cell wall synthesis, and maintenance of the capacity of walls to undergo acid-induced wall loosening. At present, we do not know if these phenomena are tightly coupled to wall acidification or if they are the products of multiple independent signal transduction pathways.

NASA Discipline Number 40-50↗
DOE OSTI2022

Identification and demonstration of roGFP2 as an environmental sensor for cryogenic correlative light and electron microscopy

Cryogenic correlative light and electron microscopy (cryo-CLEM) seeks to leverage orthogonal information present in two powerful imaging modalities. While recent advances in cryogenic electron microscopy (cryo-EM) allow for the visualization and identification of structures within cells at the nanometer scale, information regarding the cellular environment, such as pH, membrane potential, ionic strength, etc., which influences the observed structures remains absent. Fluorescence microscopy can potentially be used to reveal this information when specific labels, known as fluorescent biosensors, are used, but there has been minimal use of such biosensors in cryo-CLEM to date. Here we demonstrate the applicability of one such biosensor, the fluorescent protein roGFP2, for cryo-CLEM experiments. At room temperature, the ratio of roGFP2 emission brightness when excited at 425 nm or 488 nm is known to report on the local redox potential. When samples containing roGFP2 are rapidly cooled to 77 K in a manner compatible with cryo-EM, the ratio of excitation peaks remains a faithful indicator of the redox potential at the time of freezing. Using purified protein in different oxidizing/reducing environments, we generate a calibration curve which can be used to analyze in situ measurements. As a proof-of-principle demonstration, we investigate the oxidation/reduction state within vitrified Caulobacter crescentus cells. The polar organizing protein Z (PopZ) localizes to the polar regions of C. crescentus where it is known to form a distinct microdomain. Finally, by expressing an inducible roGFP2-PopZ fusion we visualize individual microdomains in the context of their redox environment.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Molecular insights into the complex mechanics of plant epidermal cell walls

Computational analysis of cell walls Layers of intertwined fibers make up plant cell walls. The various types of fibers respond differently to deformation. Cellulose microfibrils, for example, can stretch or curve, changing their end-to-end length, and can also slide past each other, reorient relative directions, and bundle with neighboring microfibrils. Zhang et al. developed a computational model based on observations of onion skin epidermis that describes how these complex changes in space govern cell wall mechanics. The results inform ways to engineer multifunctional fibrous materials. Science , this issue p. 706

Science & Technology - Other Topics↗
DOE OSTI2020

Vip1 is a kinase and pyrophosphatase switch that regulates inositol diphosphate signaling

Inositol diphosphates (PP-IPs), also known as inositol pyrophosphates, are high-energy cellular signaling codes involved in nutrient and regulatory responses. We report that the evolutionarily conserved gene product, Vip1, possesses autonomous kinase and pyrophosphatase domains capable of synthesis and destruction of D-1 PP-IPs. Our studies provide atomic-resolution structures of the PP-IP products and unequivocally define that the Vip1 gene product is a highly selective 1-kinase and 1-pyrophosphatase enzyme whose activities arise through distinct active sites. Kinetic analyses of kinase and pyrophosphatase parameters are consistent with Vip1 evolving to modulate levels of 1-IP 7 and 1,5-IP 8 . Individual perturbations in kinase and pyrophosphatase activities in cells result in differential effects on vacuolar morphology and osmotic responses. Analogous to the dual-functional key energy metabolism regulator, phosphofructokinase 2, Vip1 is a kinase and pyrophosphatase switch whose 1-PP-IP products play an important role in a cellular adaptation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Mechanical Regulation of Photosynthesis in Cyanobacteria

Photosynthetic organisms regulate their responses to many diverse stimuli in an effort to balance light harvesting with utilizable light energy for carbon fixation and growth (source–sink regulation). This balance is critical to prevent the formation of reactive oxygen species that can lead to cell death. However, investigating the molecular mechanisms that underlie the regulation of photosynthesis in cyanobacteria using ensemble-based measurements remains a challenge due to population heterogeneity. Here, to address this problem, we used long-term quantitative time-lapse fluorescence microscopy, transmission electron microscopy, mathematical modelling and genetic manipulation to visualize and analyse the growth and subcellular dynamics of individual wild-type and mutant cyanobacterial cells over multiple generations. We reveal that mechanical confinement of actively growing Synechococcus sp. PCC 7002 cells leads to the physical disassociation of phycobilisomes and energetic decoupling from the photosynthetic reaction centres. We suggest that the mechanical regulation of photosynthesis is a critical failsafe that prevents cell expansion when light and nutrients are plentiful, but when space is limiting. These results imply that cyanobacteria must convert a fraction of the available light energy into mechanical energy to overcome frictional forces in the environment, providing insight into the regulation of photosynthesis and how microorganisms navigate their physical environment.

BASIC BIOLOGICAL SCIENCES,BIOMASS FUELS↗
DOE OSTI2026

Multiphasic droplet microfluidics platform for controlled bacteria and mammalian cell co-culture

Microfluidics has revolutionized high-throughput miniaturized biological assays. However, co-culture of mammalian cells and bacteria remains challenging in microfluidic systems due to incompatible growth requirements, limited spatial control, and the requirement for a mammalian cell adhesion matrix. Here, we present a microfluidic platform that generates multiphasic droplets which encapsulate mammalian and bacterial cells, enabling their direct and indirect co-culture. By combining photopolymerizable hydrogels with polymer phase separation, we generate core–shell droplets composed of a liquid and a hydrogel compartment. The hydrogel compartment supports mammalian cell adhesion and culture, while the liquid compartment sustains bacterial growth. We demonstrate two droplet architectures that allow physical bacteria–mammalian cell contacts or enforce complete physical separation, representing direct and indirect co-culture. Our multiphasic droplets are stable, customizable, able to sustain co-culture for over 24 hours, and compatible with fluorescence-based cell sorting technologies. Overall, our multiphasic droplet microfluidic platform provides a scalable and versatile tool for high-throughput co-culture and screening of host–microbe interactions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Distinguishing Mesoscale Polar Order (Unidirectional vs Bidirectional) of Cellulose Microfibrils in Plant Cell Walls Using Sum Frequency Generation Spectroscopy

Cellulose in plant cell walls are synthesized as crystalline microfibrils with diameters of 3–4 nm and lengths of around 1–10 μm. These microfibrils are known to be the backbone of cell walls, and their multiscale three-dimensional organization plays a critical role in cell wall functions including plant growth and recalcitrance to degradation. The mesoscale organization of microfibrils over a 1–100 nm range in cell walls is challenging to resolve because most characterization techniques investigating this length scale suffer from low spatial resolution, sample preparation artifacts, or inaccessibility of specific cell types. Here, we report a sum frequency generation (SFG) study determining the mesoscale polarity of cellulose microfibrils in intact plant cell walls. SFG is a nonlinear optical spectroscopy technique sensitive to the molecular-to-mesoscale order of noncentrosymmetric domains in amorphous matrices. However, the quantitative theoretical model to unravel the effect of polarity in packing of noncentrosymmetric domains on SFG spectral features has remained unresolved. In this work, we show how the phase synchronization principle of the SFG process is used to predict the relative intensities of vibrational modes with different polar angles from the noncentrosymmetric domain axis. Applying this model calculation for the first time and employing SFG microscopy, we found that cellulose microfibrils in certain xylem cell walls are deposited unidirectionally (or biased in one direction) instead of the bidirectional polarity which was believed to be dominant in plant cell walls from volume-averaged characterizations of macroscopic samples. Here, with this advancement in SFG analysis, one can now determine the relative polarity of noncentrosymmetric domains such as crystalline biopolymers interspersed in amorphous polymer matrices, which will open opportunities to study new questions that have not been conceived in the past.

36 MATERIALS SCIENCE↗
DOE OSTI2020

A pipeline for targeted metagenomics of environmental bacteria

Background:Metagenomics and single cell genomics provide a window into the genetic repertoire of yet uncultivated microorganisms, but both methods are usually taxonomically untargeted. The combination of fluorescence in situ hybridization (FISH) and fluorescence activated cell sorting (FACS) has the potential to enrich taxonomically well-defined clades for genomic analyses. Methods:Cells hybridized with a taxon-specific FISH probe are enriched based on their fluorescence signal via flow cytometric cell sorting. A recently developed FISH procedure, the hybridization chain reaction (HCR)-FISH, provides the high signal intensities required for flow cytometric sorting while maintaining the integrity of the cellular DNA for subsequent genome sequencing. Sorted cells are subjected to shotgun sequencing, resulting in targeted metagenomes of low diversity. Results: Pure cultures of different taxonomic groups were used to (1) adapt and optimize the HCR-FISH protocol and (2) assess the effects of various cell fixation methods on both the signal intensity for cell sorting and the quality of subsequent genome amplification and sequencing. Best results were obtained for ethanol-fixed cells in terms of both HCR-FISH signal intensity and genome assembly quality. Our newly developed pipeline was successfully applied to a marine plankton sample from the North Sea yielding good quality metagenome assembled genomes from a yet uncultivated flavobacterial clade. Conclusions: With the developed pipeline, targeted metagenomes at various taxonomic levels can be efficiently retrieved from environmental samples. The resulting metagenome assembled genomes allow for the description of yet uncharacterized microbial clades.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Pectic-AGP is a major form of Arabidopsis AGPs

As a key component in cell walls of numerous organisms ranging from green algae to higher plants, AGPs play principal roles in many biological processes such as cell-cell adhesion and regulating Ca 2+ signaling pathway as a Ca 2+ -capacitor. Consistently, AGP structures vary from species to species and from tissue to tissue. To understand the functions of AGPs, it is vital to know their structural differences relative to their location in the plant. Thus, AGPs were purified from different Arabidopsis tissues. Analyses of these AGPs demonstrated that the AGPs comprised covalently linked pectin and AGP, referred to as pectic-AGPs. Importantly, these pectic-AGPs were glycosylated with a remarkable variety of polysaccharides including homogalacturonan, rhamnogalacturonan-I, and type II arabinogalactan at different ratios and lengths. Furthermore, this result not only suggests that pectic-AGP is a major form of Arabidopsis AGPs, but also supports AGPs serve as crosslinkers covalently connecting pectins with structures tailored for tissue-specific functions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

GRIM-19 inhibits proliferation and induces apoptosis in a p53-dependent manner in colorectal cancer cells through the SIRT7/PCAF/MDM2 axis

Colorectal cancer (CRC) is the leading deadly cancer worldwide. Gene associated with retinoid-IFN-induced mortality-19 (GRIM-19), a novel tumor suppressor, has been reported to be expressed at low levels in human CRC. However, the role of GRIM-19 in CRC progression and the corresponding detailed mechanisms are unclear. The results of this study indicated that GRIM-19 expression is related to CRC progression. Overexpression of GRIM-19 was found to inhibit CRC cell proliferation and induce apoptosis in vitro and in vivo. Our results demonstrated that GRIM-19 suppresses CRC through posttranslational regulation of p53, in which SIRT7 is activated by GRIM-19 and triggers PCAF-mediated MDM2 ubiquitination, eventually stabilizing the p53 protein. We also observed that GRIM-19 enhances the effect of oxaliplatin against CRC. In conclusion, GRIM-19 plays an important role in CRC development and is a potential biomarker and therapeutic target for clinical treatment of CRC.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

Redox Stability Controls the Cellular Uptake and Activity of Ruthenium‐Based Inhibitors of the Mitochondrial Calcium Uniporter (MCU)

Abstract The mitochondrial calcium uniporter (MCU) is the ion channel that mediates Ca 2+ uptake in mitochondria. Inhibitors of the MCU are valuable as potential therapeutic agents and tools to study mitochondrial Ca 2+ . The best‐known inhibitor of the MCU is the ruthenium compound Ru360. Although this compound is effective in permeabilized cells, it does not work in intact biological systems. We have recently reported the synthesis and characterization of Ru265, a complex that selectively inhibits the MCU in intact cells. Here, the physical and biological properties of Ru265 and Ru360 are described in detail. Using atomic absorption spectroscopy and X‐ray fluorescence imaging, we show that Ru265 is transported by organic cation transporter 3 (OCT3) and taken up more effectively than Ru360. As an explanation for the poor cell uptake of Ru360, we show that Ru360 is deactivated by biological reductants. These data highlight how structural modifications in metal complexes can have profound effects on their biological activities.

Woods, Joshua J.↗
NASA NTRS2003

Tensegrity II. How structural networks influence cellular information processing networks

The major challenge in biology today is biocomplexity: the need to explain how cell and tissue behaviors emerge from collective interactions within complex molecular networks. Part I of this two-part article, described a mechanical model of cell structure based on tensegrity architecture that explains how the mechanical behavior of the cell emerges from physical interactions among the different molecular filament systems that form the cytoskeleton. Recent work shows that the cytoskeleton also orients much of the cell's metabolic and signal transduction machinery and that mechanical distortion of cells and the cytoskeleton through cell surface integrin receptors can profoundly affect cell behavior. In particular, gradual variations in this single physical control parameter (cell shape distortion) can switch cells between distinct gene programs (e.g. growth, differentiation and apoptosis), and this process can be viewed as a biological phase transition. Part II of this article covers how combined use of tensegrity and solid-state mechanochemistry by cells may mediate mechanotransduction and facilitate integration of chemical and physical signals that are responsible for control of cell behavior. In addition, it examines how cell structural networks affect gene and protein signaling networks to produce characteristic phenotypes and cell fate transitions during tissue development.

Review↗
DOE OSTI2020

Biomolecular complex viewed by dynamic nuclear polarization solid-state NMR spectroscopy

Solid-state nuclear magnetic resonance (ssNMR) is an indispensable tool for elucidating the structure and dynamics of insoluble and non-crystalline biomolecules. The recent advances in the sensitivity-enhancing technique magic-angle spinning dynamic nuclear polarization (MAS-DNP) have substantially expanded the territory of ssNMR investigations and enabled the detection of polymer interfaces in a cellular environment. This article highlights the emerging MAS-DNP approaches and their applications to the analysis of biomolecular composites and intact cells to determine the folding pathway and ligand binding of proteins, the structural polymorphism of low-populated biopolymers, as well as the physical interactions between carbohydrates, proteins, and lignin. Furthermore, these structural features provide an atomic-level understanding of many cellular processes, promoting the development of better biomaterials and inhibitors. It is anticipated that the capabilities of MAS-DNP in biomolecular and biomaterial research will be further enlarged by the rapid development of instrumentation and methodology.

36 MATERIALS SCIENCE↗