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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 55 records · Page 3
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Advances in Cell Wall Matrix Research with a Focus on Mixed-Linkage Glucan

Mixed β(1,3;1,4)-linkage glucan (MLG) is commonly found in the monocot lineage, at particularly high levels in the Poaceae family, but also in the evolutionally distant genus, Equisetum. MLG has several properties that make it unique from other plant cell wall polysaccharides. It consists of β1,4-linked polymers of glucose interspersed with β1,3-linkages, but the presence of β1,3-linkages provides quite different physical properties compared to its closest form of the cell wall component, cellulose. The mechanisms of MLG biosynthesis have been investigated to understand whether single or multiple enzymes are required to build mixed linkages in the glucan chain. Currently, MLG synthesis by a single enzyme is supported by mutagenesis analyses of cellulose synthase-like F6, the major MLG synthase, but further investigation is needed to gather mechanistic insights. Because of transient accumulation of MLG in elongating cells and vegetative tissues, several hypotheses have been proposed to explain the role of MLG in the plant cell wall. Studies have been carried out to identify gene expression regulators during development and light cycles as well as enzymes involved in MLG organization in the cell wall. A role of MLG as a storage molecule in grains is evident, but the role of MLG in vegetative tissues is still not well understood. Characterization of a cell wall component is difficult due to the complex heterogeneity of the plant cell wall. Furthermore, as detailed in this review, recent exciting research has made significant impacts in the understanding of MLG biology in plants.

Transglucosylase/hydrolase (XTH)↗
DOE OSTI2023

Coordination and divergence in community assembly processes across co-occurring microbial groups separated by cell size

Setting the pace of life and constraining the role of members in food webs, body size can affect the structure and dynamics of communities across multiple scales of biological organization (e.g., from the individual to the ecosystem). However, its effects on shaping microbial communities, as well as underlying assembly processes, remain poorly known. Here, we analyzed microbial diversity in the largest urban lake in China and disentangled the ecological processes governing microbial eukaryotes and prokaryotes using 16S and 18S amplicon sequencing. We found that pico/nano-eukaryotes (0.22–20 μm) and micro-eukaryotes (20–200 μm) showed significant differences in terms of both community composition and assembly processes even though they were characterized by similar phylotype diversity. We also found scale dependencies whereby micro-eukaryotes were strongly governed by environmental selection at the local scale and dispersal limitation at the regional scale. Interestingly, it was the micro-eukaryotes, rather than the pico/nano-eukaryotes, that shared similar distribution and community assembly patterns with the prokaryotes. This indicated that assembly processes of eukaryotes may be coupled or decoupled from prokaryotes’ assembly processes based on eukaryote cell size. While the results support the important influence of cell size, there may be other factors leading to different levels of assembly process coupling across size classes. Additional studies are needed to quantitatively parse the influence of cell size versus other factors as drivers of coordinated and divergent community assembly processes across microbial groups. Regardless of the governing mechanisms, our results show that there are clear patterns in how assembly processes are coupled across sub-communities defined by cell size. These size-structured patterns could be used to help predict shifts in microbial food webs in response to future disturbance.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

The NtrYX Two-Component System Regulates the Bacterial Cell Envelope

ABSTRACT Activity of the NtrYX two-component system has been associated with important processes in diverse bacteria, ranging from symbiosis to nitrogen and energy metabolism. In the facultative alphaproteobacterium Rhodobacter sphaeroides , loss of the two-component system NtrYX results in increased lipid production and sensitivity to some known cell envelope-active compounds. In this study, we show that NtrYX directly controls multiple properties of the cell envelope. We find that the response regulator NtrX binds upstream of cell envelope genes, including those involved in peptidoglycan biosynthesis and modification and in cell division. We show that loss of NtrYX impacts the cellular levels of peptidoglycan precursors and lipopolysaccharide and alters cell envelope structure, increasing cell length and the thickness of the periplasm. Cell envelope function is also disrupted in the absence of NtrYX, resulting in increased outer membrane permeability. Based on the properties of R. sphaeroides cells lacking NtrYX and the target genes under direct control of this two-component system, we propose that NtrYX plays a previously undescribed, and potentially conserved, role in the assembly, structure, and function of the cell envelope in a variety of bacteria. IMPORTANCE The bacterial cell envelope provides many important functions. It protects cells from harsh environments, serves as a selective permeability barrier, houses bioenergetic functions, defines sensitivity to antibacterial agents, and plays a crucial role in biofilm formation, symbiosis, and virulence. Despite the important roles of this cellular compartment, we lack a detailed understanding of the biosynthesis and remodeling of the cell envelope. Here, we report that the R. sphaeroides two-component signaling system NtrYX is a previously undescribed regulator of cell envelope processes, providing evidence that it is directly involved in controlling transcription of genes involved in cell envelope assembly, structure, and function in this and possibly other bacteria. Thus, our data report on a newly discovered process used by bacteria to assemble and remodel the cell envelope.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Identification of new marker genes from plant single‐cell RNA‐seq data using interpretable machine learning methods

Summary An essential step in the analysis of single‐cell RNA sequencing data is to classify cells into specific cell types using marker genes. In this study, we have developed a machine learning pipeline called single‐cell predictive marker (SPmarker) to identify novel cell‐type marker genes in the Arabidopsis root. Unlike traditional approaches, our method uses interpretable machine learning models to select marker genes. We have demonstrated that our method can: assign cell types based on cells that were labelled using published methods; project cell types identified by trajectory analysis from one data set to other data sets; and assign cell types based on internal GFP markers. Using SPmarker, we have identified hundreds of new marker genes that were not identified before. As compared to known marker genes, the new marker genes have more orthologous genes identifiable in the corresponding rice single‐cell clusters. The new root hair marker genes also include 172 genes with orthologs expressed in root hair cells in five non‐ Arabidopsis species, which expands the number of marker genes for this cell type by 35–154%. Our results represent a new approach to identifying cell‐type marker genes from scRNA‐seq data and pave the way for cross‐species mapping of scRNA‐seq data in plants.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2022

High-Throughput Microfluidics Platform for Intracellular Delivery and Sampling of Biomolecules from Live Cells

Nondestructive cell membrane permeabilization systems enable the intracellular delivery of exogenous biomolecules for cell engineering tasks as well as the temporal sampling of cytosolic contents from live cells for the analysis of dynamic processes. Here, in this paper, we report a microwell array format live-cell analysis device (LCAD) that can perform localized-electroporation induced membrane permeabilization, for cellular delivery or sampling, and directly interfaces with surface-based biosensors for analyzing the extracted contents. We demonstrate the capabilities of the LCAD via an automated high-throughput workflow for multimodal analysis of live-cell dynamics, consisting of quantitative measurements of enzyme activity using self-assembled monolayers for MALDI mass spectrometry (SAMDI) and deep-learning enhanced imaging and analysis. By combining a fabrication protocol that enables robust assembly and operation of multilayer devices with embedded gold electrodes and an automated imaging workflow, we successfully deliver functional molecules (plasmid and siRNA) into live cells at multiple time-points and track their effect on gene expression and cell morphology temporally. Furthermore, we report sampling performance enhancements, achieving saturation levels of protein tyrosine phosphatase activity measured from as few as 60 cells, and demonstrate control over the amount of sampled contents by optimization of electroporation parameters using a lumped model. Lastly, we investigate the implications of cell morphology on electroporation-induced sampling of fluorescent molecules using a deep-learning enhanced image analysis workflow.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

N6-methyladenine demethylase ALKBH1 inhibits the differentiation of skeletal muscle

Highlights: • The DNA N6-mA modification increased during skeletal muscle development, accompanied by a strong decrease of ALKBH1. • ALKBH1 enhanced proliferation and inhibited differentiation of C2C12 cells. • ALKBH1 impaired multiple genes and signaling pathways to promote proliferation and inhibit differentiation of C2C12 cells. DNA N{sup 6}-methyladenine (N6-mA) was recently recognized as a new epigenetic modification in mammalian genome, and ALKBH1 was discovered as its demethylase. Knock-out mice studies revealed that ALKBH1 was indispensable for normal embryonic development. However, the function of ALKBH1 in myogenesis is largely unknown. In this study, we found that N6-mA showed a steady increase, going along with a strong decrease of ALKBH1 during skeletal muscle development. Our results also showed that ALKBH1 enhanced proliferation and inhibited differentiation of C2C12 cells. Genome-wide transcriptome analysis and reporter assays further revealed that ALKBH1 accomplished the differentiation inhibiting function by regulating a core set of genes and multiple signaling pathways, including increasing chemokine (C-X-C motif) ligand 14 (CXCL14) and activating ERK signaling. Taken together, our results demonstrated that ALKBH1 is critical for the myogenic differentiation of C2C12 cells, and suggested that N6-mA might be a new epigenetic mechanism for the regulation of myogenesis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Dynamics and growth rate implications of ribosomes and mRNAs interaction in E. coli

Understanding how cells grow and adapt under various nutrient conditions is pivotal in the study of biological stoichiometry. Recent studies provide empirical evidence that cells use multiple strategies to maintain an optimal protein production rate under different nutrient conditions. Mathematical models can provide a solid theoretical foundation that can explain experimental observations and generate testable hypotheses to further our understanding of the growth process. In this study, we generalize a modeling framework that centers on the translation process and study its asymptotic behaviors to validate algebraic manipulations involving the steady states. Using experimental results on the growth of E. coli under C-, N-, and P-limited environments, we simulate the expected quantitative measurements to show the feasibility of using the model to explain empirical evidence. Our results support the findings that cells employ multiple strategies to maintain a similar protein production rate across different nutrient limitations. Moreover, we find that the previous study underestimates the significance of certain biological rates, such as the binding rate of ribosomes to mRNA and the transition rate between different ribosomal stages. Furthermore, our simulation shows that the strategies used by cells under C- and P-limitations result in a faster overall growth dynamics than under N-limitation. In conclusion, the general modeling framework provides a valuable platform to study cell growth under different nutrient supply conditions, which also allows straightforward extensions to the coupling of transcription, translation, and energetics to deepen our understanding of the growth process.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Single-nuclei transcriptome analysis of the shoot apex vascular system differentiation in Populus

ABSTRACT Differentiation of stem cells in the plant apex gives rise to aerial tissues and organs. Presently, we lack a lineage map of the shoot apex cells in woody perennials – a crucial gap considering their role in determining primary and secondary growth. Here, we used single-nuclei RNA-sequencing to determine cell type-specific transcriptomes of the Populus vegetative shoot apex. We identified highly heterogeneous cell populations clustered into seven broad groups represented by 18 transcriptionally distinct cell clusters. Next, we established the developmental trajectories of the epidermis, leaf mesophyll and vascular tissue. Motivated by the high similarities between Populus and Arabidopsis cell population in the vegetative apex, we applied a pipeline for interspecific single-cell gene expression data integration. We contrasted the developmental trajectories of primary phloem and xylem formation in both species, establishing the first comparison of vascular development between a model annual herbaceous and a woody perennial plant species. Our results offer a valuable resource for investigating the principles underlying cell division and differentiation conserved between herbaceous and perennial species while also allowing us to examine species-specific differences at single-cell resolution.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Optical tweezer measurements of asymptotic nonlinearities in complex fluids

This article presents micro-medium-amplitude oscillatory shear (μMAOS), a method to measure the frequency-dependent micromechanical properties of soft materials in the asymptotically nonlinear regime using optical tweezers. For this work, we have developed a theoretical framework to extract these nonlinear mechanical properties of the material from experimental measurements and also proposed a physical interpretation of the third-order nonlinearities measured in single-tone oscillatory tests. We validate the method using a well-characterized surfactant solution of wormlike micelles, and subsequently employ this technique to demonstrate that the cytoplasm of a living cell undergoes strain softening and shear thinning when locally subjected to weakly nonlinear oscillatory deformations.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

BefA, a microbiota-secreted membrane disrupter, disseminates to the pancreas and increases β cell mass

Microbiome dysbiosis is a feature of diabetes, but how microbial products influence insulin production is poorly understood. Here, we report the mechanism of BefA, a microbiome-derived protein that increases proliferation of insulin-producing β cells during development in gnotobiotic zebrafish and mice. BefA disseminates systemically by multiple anatomic routes to act directly on pancreatic islets. We detail BefA’s atomic structure, containing a lipid-binding SYLF domain, and demonstrate that it permeabilizes synthetic liposomes and bacterial membranes. A BefA mutant impaired in membrane disruption fails to expand β cells, whereas the pore-forming host defense protein, Reg3, stimulates β cell proliferation. Our work demonstrates that membrane permeabilization by microbiome-derived and host defense proteins is necessary and sufficient for β cell expansion during pancreas development, potentially connecting microbiome composition with diabetes risk.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Determining protein polarization proteome-wide using physical dissection of individual Stentor coeruleus cells

Cellular components are non-randomly arranged with respect to the shape and polarity of the whole cell. Patterning within cells can extend down to the level of individual proteins and mRNA. But how much of the proteome is actually localized with respect to cell polarity axes? Proteomics combined with cellular fractionation has shown that most proteins localize to one or more organelles but does not tell us how many proteins have a polarized localization with respect to the large-scale polarity axes of the intact cell. Genome-wide localization studies in yeast found that only a few percent of proteins have a localized position relative to the cell polarity axis defined by sites of polarized cell growth. Here, we describe an approach for analyzing protein distribution within a cell with a visibly obvious global patterning - the giant ciliate Stentor coeruleus. Ciliates, including Stentor, have highly polarized cell shapes with visible surface patterning. A Stentor cell is roughly 2mmlong, allowing a ‘‘proteomic dissection’’ in which microsurgery is used to separate cellular fragments along the anterior-posterior axis, followed by comparative proteomic analysis. In our analysis, 25% of the proteome, including signaling proteins, centrin/SFI proteins, and GAS2 orthologs, shows a polarized location along the cell’s anterior-posterior axis. We conclude that a large proportion of all proteins are polarized with respect to global cell polarity axes and that proteomic dissection provides a simple and effective approach for spatial proteomics.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

LxCxD motif of the APC/C coactivator subunit FZR1 is critical for interaction with the retinoblastoma protein

Highlights: • Several oncogenic viral proteins bind to tumor suppressor pRB via LxCxE motif. • LxCxE present in many cellular proteins that interact with pRB. • APC/C interacts with pRB via this motif in its FZR1 subunit. • FZR1 LxCxE-like LxCxD critical for FZR1-pRB direct binding and SKP2 ubiquitylation. • Mutation of FZR1 LxCxD alters cell cycle. Retinoblastoma protein (pRB) regulates cell cycle by utilizing different regions of its pocket domain for interacting with E2F family of transcription factors and with cellular and viral proteins containing an LxCxE motif. An LxCxE-like motif, LxCxD, is present in FZR1, an adaptor protein of the multi-subunit E3 ligase complex anaphase-promoting complex/cyclosome (APC/C). The APC/C{sup FZR1} complex regulates the timely degradation of multiple cell cycle proteins for mitotic exit and maintains G1 state. We report that FZR1 interacts with pRB via its LxCxD motif. By using point mutations, we found that the cysteine residue in the FZR1 LxCxD motif is critical for direct interaction with pRb. The direct binding of the LxCxD motif of FZR1 to the pRB LxCxE binding pocket is confirmed by using human papillomavirus protein E7 as a competitor, both in vitro and in vivo. While mutation of the cysteine residue significantly disrupts FZR1 interaction with pRB, this motif does not affect FZR1 and core APC/C association. Expression of the FZR1 point mutant results in accumulation of S-phase kinase-associated protein 2 (SKP2) and Polo-like kinase 1 (PLK1), while p27{sup Kip1} and p21{sup Cip1} proteins are downregulated, indicating a G1 cell cycle defect. Consistently, cells containing point mutant FZR1 enter the S phase prematurely. Together our results suggest that the LxCxD motif of FZR1 is a critical determinant for the interaction between FZR1 and pRB and is important for G1 restriction.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

IRS4 promotes the progression of non-small cell lung cancer and confers resistance to EGFR-TKI through the activation of PI3K/Akt and Ras-MAPK pathways

IRS4 is a member of the insulin receptor substrate (IRS) protein family. It acts as a cytoplasmic adaptor protein, integrating and transmitting signals from receptor protein tyrosine kinases to the intracellular environment. IRS4 can induce mammary tumorigenesis and is usually overexpressed in non-small cell lung cancer (NSCLC). However, little is known about the role of IRS4 in the development and progression of lung cancer. In this study, we show that IRS4 knockout suppresses the proliferation, colony formation, migration, and invasion of A549 lung cancer cells, as well as tumor growth in a nude mouse xenograft model. In contrast, stable expression of IRS4 showed the opposite effects. As expected, IRS4 was found to activate the PI3K/Akt and Ras-MAPK pathways, and we also showed that IRS4 depletion significantly enhanced the sensitivity of EGFR tyrosine kinase inhibitor (EGFR-TKI)-resistant cells to gefitinib. Taken together, these results show that IRS4 promotes NSCLC progression and may represent a potential therapeutic target for EGFR-TKI-resistant NSCLC.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Lost in transduction: Critical considerations when using viral vectors

The application of retroviral vectors in the laboratory requires considerations that often go overlooked but are often easy to circumvent. Here, we discuss the relationship between the observed transduction efficiency of a cell population and per-cell viral insertions—and describe how differential cell-type susceptibilities can confound results. We consider the math underlying this problem and review an alternative approach to the commonly used “multiplicity of infection” (MOI) method of titering and using viral vectors in the biomedical research laboratory.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Adherens junctions are involved in polarized contractile ring formation in dividing epithelial cells of Xenopus laevis embryos

Highlights: • The actomyosin contractile ring formation is polarized in epithelial cells. • Activation of the contractile ring is polarized in epithelial cells. • Adherens junctions are involved in the kinetics of the contractile ring formation. • Adherens junctions and the contractile ring are linked from the onset of cytokinesis. • High temporal resolution microscopy of epithelial cells in the Xenopus embryos. Cells dividing in the plane of epithelial tissues proceed by polarized constriction of the actomyosin contractile ring, leading to asymmetric ingression of the plasma mem brane. Asymmetric cytokinesis results in the apical positioning of the actomyosin contractile ring and ultimately of the midbody. Studies have indicated that the contractile ring is associated with adherens junctions, whose role is to maintain epithelial tissue cohesion. However, it is yet unknown when the contractile ring becomes associated with adherens junctions in epithelial cells. Here, we examined contractile ring formation and activation in the epithelium of Xenopus embryos and explored the implication of adherens junctions in the contractile ring formation. We show that accumulation of proteins involved in contractile ring formation and activation is polarized, starting at apical cell-cell contacts at the presumptive division site and spreading within seconds towards the cell basal side. We also show that adherens junctions are involved in the kinetics of contractile ring formation. Our study reveals that the link between the adherens junctions and the contractile ring is established from the onset of cytokinesis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

Logistics of defense: The contribution of endomembranes to plant innate immunity

Phytopathogens cause plant diseases that threaten food security. Unlike mammals, plants lack an adaptive immune system and rely on their innate immune system to recognize and respond to pathogens. Plant response to a pathogen attack requires precise coordination of intracellular traffic and signaling. Spatial and/or temporal defects in coordinating signals and cargo can lead to detrimental effects on cell development. The role of intracellular traffic comes into a critical focus when the cell sustains biotic stress. In this review, we discuss the current understanding of the post-immune activation logistics of plant defense. Specifically, we focus on packaging and shipping of defense-related cargo, rerouting of intracellular traffic, the players enabling defense-related traffic, and pathogen-mediated subversion of these pathways. We highlight the roles of the cytoskeleton, cytoskeleton–organelle bridging proteins, and secretory vesicles in maintaining pathways of exocytic defense, acting as sentinels during pathogen attack, and the necessary elements for building the cell wall as a barrier to pathogens. We also identify points of convergence between mammalian and plant trafficking pathways during defense and highlight plant unique responses to illustrate evolutionary adaptations that plants have undergone to resist biotic stress.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Intermittent supplementation with fisetin improves arterial function in old mice by decreasing cellular senescence

Cellular senescence and the senescence-associated secretory phenotype (SASP) contribute to age-related arterial dysfunction, in part, by promoting oxidative stress and inflammation, which reduce the bioavailability of the vasodilatory molecule nitric oxide (NO). In the present study, we assessed the efficacy of fisetin, a natural compound, as a senolytic to reduce vascular cell senescence and SASP factors and improve arterial function in old mice. We found that fisetin decreased cellular senescence in human endothelial cell culture. In old mice, vascular cell senescence and SASP-related inflammation were lower 1 week after the final dose of oral intermittent (1 week on—2 weeks off—1 weeks on dosing) fisetin supplementation. Old fisetin-supplemented mice had higher endothelial function. Leveraging old p16-3MR mice, a transgenic model allowing genetic clearance of p16 INK4A -positive senescent cells, we found that ex vivo removal of senescent cells from arteries isolated from vehicle- but not fisetin-treated mice increased endothelium-dependent dilation, demonstrating that fisetin improved endothelial function through senolysis. Enhanced endothelial function with fisetin was mediated by increased NO bioavailability and reduced cellular- and mitochondrial-related oxidative stress. Arterial stiffness was lower in fisetin-treated mice. Ex vivo genetic senolysis in aorta rings from p16-3MR mice did not further reduce mechanical wall stiffness in fisetin-treated mice, demonstrating lower arterial stiffness after fisetin was due to senolysis. Lower arterial stiffness with fisetin was accompanied by favorable arterial wall remodeling. The findings from this study identify fisetin as promising therapy for clinical translation to target excess cell senescence to treat age-related arterial dysfunction.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Catalytically inactive, purified RNase H1: A specific and sensitive probe for RNA–DNA hybrid imaging

R-loops are three-stranded nucleic acid structures with both physiological and pathological roles in cells. R-loop imaging generally relies on detection of the RNA–DNA hybrid component of these structures using the S9.6 antibody. We show that the use of this antibody for imaging can be problematic because it readily binds to double-stranded RNA (dsRNA) in vitro and in vivo, giving rise to nonspecific signal. In contrast, purified, catalytically inactive human RNase H1 tagged with GFP (GFP-dRNH1) is a more specific reagent for imaging RNA–DNA hybrids. GFP-dRNH1 binds strongly to RNA–DNA hybrids but not to dsRNA oligonucleotides in fixed human cells and is not susceptible to binding endogenous RNA. Furthermore, we demonstrate that purified GFP-dRNH1 can be applied to fixed cells to detect hybrids after their induction, thereby bypassing the need for cell line engineering. GFP-dRNH1 therefore promises to be a versatile tool for imaging and quantifying RNA–DNA hybrids under a wide range of conditions.

59 BASIC BIOLOGICAL SCIENCES↗