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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 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

A red-emitting carborhodamine for monitoring and measuring membrane potential

Biological membrane potentials, or voltages, are a central facet of cellular life. Optical methods to visualize cellular membrane voltages with fluorescent indicators are an attractive complement to traditional electrode-based approaches, since imaging methods can be high throughput, less invasive, and provide more spatial resolution than electrodes. Recently developed fluorescent indicators for voltage largely report changes in membrane voltage by monitoring voltage-dependent fluctuations in fluorescence intensity. However, it would be useful to be able to not only monitor changes but also measure values of membrane potentials. This study discloses a fluorescent indicator which can address both. We describe the synthesis of a sulfonated tetramethyl carborhodamine fluorophore. When this carborhodamine is conjugated with an electron-rich, methoxy (-OMe) containing phenylenevinylene molecular wire, the resulting molecule, CRhOMe, is a voltage-sensitive fluorophore with red/far-red fluorescence. Using CRhOMe, changes in cellular membrane potential can be read out using fluorescence intensity or lifetime. In fluorescence intensity mode, CRhOMe tracks fast-spiking neuronal action potentials (APs) with greater signal-to-noise than state-of-the-art BeRST 1 (another voltage-sensitive fluorophore). CRhOMe can also measure values of membrane potential. The fluorescence lifetime of CRhOMe follows a single exponential decay, substantially improving the quantification of membrane potential values using fluorescence lifetime imaging microscopy (FLIM). The combination of red-shifted excitation and emission, mono-exponential decay, and high voltage sensitivity enable fast FLIM recording of APs in cardiomyocytes. The ability to both monitor and measure membrane potentials with red light using CRhOMe makes it an important approach for studying biological voltages.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Wireless Bioelectronic Modulation of Membrane Potential in Glioblastoma Using Carbon Nanotube Porins

Disruption of membrane potential (V mem ) can activate pathways associated with cancer proliferation. Manipulating ion channels may therefore present an effective strategy for treating cancers that fail to respond to conventional therapies. One approach to target these channels is to manipulate the membrane charge, which involves the use of wireless bipolar electrodes such as carbon nanotube porins (CNTPs) inserted into cell membranes to modulate membrane charge and ionic flux. By utilizing membrane dyes, we observed alterations in V mem induced by CNTPs and externally applied voltages. Analyses of cellular behaviors and processes indicated that V mem is more receptive to stimuli in invasive cancers, while it leads to increased metabolism in less invasive cancers, with notable changes in the cell cycle occurring at approximately 48 h post-treatment in Glioblastoma (GB) cell lines. This work shows that CNTPs, in combination and with externally applied voltages, can modulate V mem and alter cancer cell processes, supporting their potential as a therapeutic.

bioelectricity↗
DOE OSTI2021

Point-localized, site-specific membrane potential optical recording by single fluorescent nanodiscs

Nanodisc technology was implemented as a platform for voltage nanosensors. A fluorescence (Förster) resonance energy transfer (FRET)-based voltage-sensing scheme employing fluorescent nanodiscs and the hydrophobic ion dipicrylamine was developed and utilized to optically record membrane potentials on the single-nanodisc level. Ensemble and single-nanosensor recordings were demonstrated for HEK293 cells and primary cortical neuron cells. Conjugation of nanodiscs to anti-GABA A antibodies allowed for site-specific membrane potential measurements from postsynaptic sites.

77 NANOSCIENCE AND NANOTECHNOLOGY↗
DOE OSTI2022

Membrane potential sensing: Material design and method development for single particle optical electrophysiology

We review the development of “single” nanoparticle-based inorganic and organic voltage sensors, which can eventually become a viable tool for “non-genetic optogenetics.” The voltage sensing is accomplished with optical imaging at the fast temporal response and high spatial resolutions in a large field of view. Inorganic voltage nanosensors utilize the Quantum Confined Stark Effect (QCSE) to sense local electric fields. Engineered nanoparticles achieve substantial single-particle voltage sensitivity (~2% Δλ spectral Stark shift up to ~30% ΔF/F per 160 mV) at room temperature due to enhanced charge separation. A dedicated home-built fluorescence microscope records spectrally resolved images to measure the QCSE induced spectral shift at the single-particle level. Biomaterial based surface ligands are designed and developed based on theoretical simulations. The hybrid nanobiomaterials satisfy anisotropic facet-selective coating, enabling effective compartmentalization beyond non-specific staining. Self-spiking- and patched-HEK293 cells and cortical neurons, when stained with hybrid nanobiomaterials, show clear photoluminescence intensity changes in response to membrane potential (MP) changes. Organic voltage nanosensors based on polystyrene beads and nanodisk technology utilize Fluorescence (Förster) Resonance Energy Transfer (FRET) to sense local electric fields. Voltage sensing FRET pairs achieve voltage sensitivity up to ~35% ΔF/F per 120 mV in cultures. Non-invasive MP recording from individual targeted sites (synapses and spines) with nanodisks has been realized. However, both of these QCSE- and FRET-based voltage nanosensors yet need to reach the milestone of recording individual action potentials from individual targeted sites.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Electrically controlling and optically observing the membrane potential of supported lipid bilayers

Supported lipid bilayers are a well-developed model system for the study of membranes and their associated proteins, such as membrane channels, enzymes, and receptors. These versatile model membranes can be made from various components, ranging from simple synthetic phospholipids to complex mixtures of constituents, mimicking the cell membrane with its relevant physiochemical and molecular phenomena. In addition, the high stability of supported lipid bilayers allows for their study via a wide array of experimental probes. In this work, we describe a platform for supported lipid bilayers that is accessible both electrically and optically, and demonstrate direct optical observation of the transmembrane potential of supported lipid bilayers. We show that the polarization of the supported membrane can be electrically controlled and optically probed using voltage-sensitive dyes. Membrane polarization dynamics is understood through electrochemical impedance spectroscopy and the analysis of an equivalent electrical circuit model. In addition, we describe the effect of the conducting electrode layer on the fluorescence of the optical probe through metal-induced energy transfer, and show that while this energy transfer has an adverse effect on the voltage sensitivity of the fluorescent probe, its strong distance dependency allows for axial localization of fluorescent emitters with ultrahigh accuracy. We conclude with a discussion on possible applications of this platform for the study of voltage-dependent membrane proteins and other processes in membrane biology and surface science.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Bacterial Homologs of Progestin and AdipoQ Receptors (PAQRs) Affect Membrane Energetics Homeostasis but Not Fluidity

Membrane potential homeostasis is essential for cell survival. Defects in membrane potential lead to pleiotropic phenotypes, consistent with the central role of membrane energetics in cell physiology. Homologs of the progestin and AdipoQ receptors (PAQRs) are conserved in multiple phyla of Bacteria and Eukarya. In eukaryotes, PAQRs are proposed to modulate membrane fluidity and fatty acid (FA) metabolism. The role of bacterial homologs has not been elucidated. Here, we use Escherichia coli and Bacillus subtilis to show that bacterial PAQR homologs, which we name “TrhA,” have a role in membrane energetics homeostasis. Using transcriptional fusions, we show that E. coli TrhA (encoded by yqfA) is part of the unsaturated fatty acid biosynthesis regulon. Fatty acid analyses and physiological assays show that a lack of TrhA in both E. coli and B. subtilis (encoded by yplQ) provokes subtle but consistent changes in membrane fatty acid profiles that do not translate to control of membrane fluidity. Instead, membrane proteomics in E. coli suggested a disrupted energy metabolism and dysregulated membrane energetics in the mutant, though it grew similarly to its parent. These changes translated into a disturbed membrane potential in the mutant relative to its parent under various growth conditions. Similar dysregulation of membrane energetics was observed in a different E. coli strain and in the distantly related B. subtilis. Together, our findings are consistent with a role for TrhA in membrane energetics homeostasis, through a mechanism that remains to be elucidated.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Stress-regulated Arabidopsis GAT2 is a low affinity γ-aminobutyric acid transporter

Abstract The four-carbon non-proteinogenic amino acid γ-aminobutyric acid (GABA) accumulates to high levels in plants in response to various abiotic and biotic stress stimuli, and plays a role in C:N balance, signaling, and as a transport regulator. Expression in Xenopus oocytes and voltage-clamping allowed the characterization of Arabidopsis GAT2 (At5g41800) as a low affinity GABA transporter with a K0.5GABA ~8 mM. l-Alanine and butylamine represented additional substrates. GABA-induced currents were strongly dependent on the membrane potential, reaching the highest affinity and highest transport rates at strongly negative membrane potentials. Mutation of Ser17, previously reported to be phosphorylated in planta, did not result in altered affinity. In a short-term stress experiment, AtGAT2 mRNA levels were up-regulated at low water potential and under osmotic stress (polyethylene glycol and mannitol). Furthermore, AtGAT2 promoter activity was detected in vascular tissues, maturating pollen, and the phloem unloading region of young seeds. Even though this suggested a role for AtGAT2 in long-distance transport and loading of sink organs, under the conditions tested neither AtGAT2-overexpressing plants, atgat2 or atgat1 T-DNA insertion lines, nor atgat1 atgat2 doubleknockout mutants differed from wild-type plants in growth on GABA, amino acid levels, or resistance to salt and osmotic stress.

Plant Sciences↗
DOE OSTI2023

The Genetics of Aerotolerant Growth in Zymomonas mobilis

Reduced genome bacteria are genetically simplified systems that facilitate biological study and industrial use. The free-living Alphaproteobacterium, Zymomonas mobilis, has a naturally reduced genome containing fewer than 2000 protein coding genes. Despite its small genome, Z. mobilis thrives in diverse conditions including the presence or absence of atmospheric oxygen. However, insufficient characterization of essential and conditionally essential genes has limited broader adoption of Z. mobilis as a model Alphaproteobacterium. Here, we use genome-scale CRISPRi-seq to systematically identify and characterize Z. mobilis genes that are conditionally essential for aerotolerant or anaerobic growth, or are generally essential across both conditions. Comparative genomics revealed that the essentiality of most "generally essential" genes was shared between Z. mobilis and other Alphaproteobacteria, validating Z. mobilis as reduced genome model. Among conditionally essential genes, we found that the DNA repair gene, recJ, was critical only for aerobic growth but reduced the mutation rate under both conditions. Further, we show that genes encoding the F1FO ATP synthase and Rnf respiratory complex are required for anaerobic growth of Z. mobilis. Combining CRISPRi partial knockdowns with metabolomics and membrane potential measurements, we determined that the ATP synthase generates membrane potential that is consumed by Rnf to power downstream processes. Rnf knockdown strains accumulated isoprenoid biosynthesis intermediates, suggesting a key role for Rnf in powering essential biosynthetic reactions. Our work establishes Z. mobilis as a streamlined model for alphaproteobacterial genetics, has broad implications in bacterial energy coupling, and informs Z. mobilis genome manipulation for optimized production of valuable isoprenoid-based bioproducts

Alphaproteobacteria↗
DOE OSTI2023

The genetics of aerotolerant growth in an alphaproteobacterium with a naturally reduced genome

Reduced genome bacteria are genetically simplified systems that facilitate biological study and industrial use. The free-living alphaproteobacterium Zymomonas mobilis has a naturally reduced genome containing fewer than 2,000 protein-coding genes. Despite its small genome, Z. mobilis thrives in diverse conditions including the presence or absence of atmospheric oxygen. However, insufficient characterization of essential and conditionally essential genes has limited broader adoption of Z. mobilis as a model alphaproteobacterium. Here, we use genome-scale CRISPRi-seq (clustered regularly interspaced short palindromic repeats interference sequencing) to systematically identify and characterize Z. mobilis genes that are conditionally essential for aerotolerant or anaerobic growth or are generally essential across both conditions. Comparative genomics revealed that the essentiality of most “generally essential” genes was shared between Z. mobilis and other Alphaproteobacteria, validating Z. mobilis as a reduced genome model. Among conditionally essential genes, we found that the DNA repair gene, recJ, was critical only for aerobic growth but reduced the mutation rate under both conditions. Further, we show that genes encoding the F1FO ATP synthase and Rhodobacter nitrogen fixation (Rnf) respiratory complex are required for the anaerobic growth of Z. mobilis. Combining CRISPRi partial knockdowns with metabolomics and membrane potential measurements, we determined that the ATP synthase generates membrane potential that is consumed by Rnf to power downstream processes. Rnf knockdown strains accumulated isoprenoid biosynthesis intermediates, suggesting a key role for Rnf in powering essential biosynthetic reactions. Our work establishes Z. mobilis as a streamlined model for alphaproteobacterial genetics, has broad implications in bacterial energy coupling, and informs Z. mobilis genome manipulation for optimized production of valuable isoprenoid-based bioproducts.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Polyunsaturated Fatty Acids Mend Macrophage Transcriptome, Glycome, and Phenotype in the Patients with Neurodegenerative Diseases, Including Alzheimer’s Disease

Macrophages of healthy subjects have a pro-resolution phenotype, upload amyloid-β (Aβ) into endosomes, and degrade Aβ, whereas macrophages of patients with Alzheimer’s disease (AD) generally have a pro-inflammatory phenotype and lack energy for brain clearance of Aβ. To clarify the pathogenesis of sporadic AD and therapeutic effects of polyunsaturated fatty acids (PUFA) with vitamins B and D and antioxidants on monocyte/macrophage (MM) migration in the AD brain, MM transcripts in energy and Aβ degradation, MM glycome, and macrophage clearance of Aβ. We followed for 31.3 months (mean) ten PUFA-supplemented neurodegenerative patients: 3 with subjective cognitive impairment (SCI), 2 with mild cognitive impairment (MCI), 3 MCI/vascular cognitive impairment, 2 with dementia with Lewy bodies, and 7 non-supplemented caregivers. We examined: monocyte migration in the brain and a blood-brain barrier model by immunochemistry and electron microscopy; macrophage transcriptome by RNAseq; macrophage glycome by N-glycan profiling and LTQ-Orbitrap mass spectrometry; and macrophage phenotype and phagocytosis by immunofluorescence. MM invade Aβ plaques, upload but do not degrade Aβ, and release Aβ into vessels, which develop cerebrovascular amyloid angiopathy (CAA); PUFA upregulate energy and Aβ degradation enzyme transcripts in macrophages; PUFA enhance sialylated N-glycans in macrophages; PUFA reduce oxidative stress and increase pro-resolution MM phenotype, mitochondrial membrane potential, and Aβ phagocytosis (p < 0.001). Macrophages of SCI, MCI, and AD patients have interrelated defects in the transcriptome, glycome, Aβ phagocytosis, and Aβ degradation. PUFA mend macrophage transcriptome, enrich glycome, enhance Aβ clearance, and benefit the cognition of early-stage AD patients.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2025

Characterizing and modeling the mechanical behavior of an anion exchange membrane for carbon capture applications

A new direct air capture (DAC) technology uses a moisture swing (MS) process with anion exchange membranes, potentially offering a more energy-efficient way to remove CO₂ from the air. In this MS process, the membrane absorbs CO₂ as it dries and releases it when water is added. Understanding the mechanical behavior of these membranes is essential for improving the design and efficiency of DAC systems and prolonging sorbent lifetime. This study tested one anion exchange membrane, Fumasep’s FAA-3, under mechanical loading and various temperature and humidity conditions to measure its swelling, stiffness, strength, plastic deformation, and stress relaxation. Experimental results were used to identify a mechanical model for FAA-3 that can be used to predict the material’s nonlinear viscous behavior under various loads and environments. Unlike prior studies that assumed linear elastic behavior, this work incorporates humidity-dependent swelling, thermal expansion, and nonlinear viscoelasticity of FAA-3 in both the experiments and the model.

Direct air capture↗
DOE OSTI2023

Amphiphilic Co-Solvents Modulate the Structure of Membrane Domains

Biofuels are an increasing part of the sustainable energy picture. This makes it a societal and economic imperative to optimize biofuel production. Mitigating the toxic effects of amphiphilic co-solvents is one way to improve the efficiency of biofuel production. Amphiphiles partition into cellular membranes, leading to membrane thinning, destabilization, loss of membrane potential, and, ultimately, cell death. However, this picture of solvent toxicity misses the disruptive impact of co-solvents on lateral membrane organization, which is increasingly recognized as critical for membrane protein sorting and oligomerization. The alteration or disruption of membrane domains has deleterious effects on cellular processes. Here, in this work, we pursue the hypothesis that membrane lateral organization is disrupted by the presence of co-solvents at concentrations lower than those which lead to full membrane destabilization. The disruption occurs due to an increasing interfacial tension between the co-existing phases, resulting in conformational changes to minimize the interfacial length-to-area ratio. This represents an unrecognized mode of solvent-induced stress and a new target for interventions to improve fermentation yields.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

Protective Effect of Phloretin against Hydrogen Peroxide-Induced Oxidative Damage by Enhancing Autophagic Flux in DF-1 Cells

Phloretin (PHL) is a dihydrochalcone flavonoid isolated from the peel and root bark of apples, strawberries, and other plants with antioxidative characteristic. In this study, we aimed to investigate the protective effect and the potential mechanism of PHL on hydrogen peroxide (H2O2)-induced oxidative damage in DF-1 cells. The results showed that PHL exhibited no cytotoxic effect on DF-1 cells at concentration below 20 μM. PHL markedly increased H2O2-reduced cell viability, decreased H2O2-induced apoptosis, as evidenced by reduced apoptosis rate, the upregulation of gene and protein level of Bcl-2, and the downregulation of gene and protein level of Bax and Cleaved caspase3. In addition, PHL reduced H2O2-induced reactive oxygen species (ROS) production and restored antioxidant enzymes activities as well as mitochondrial membrane potential in a dose-dependent manner. Moreover, PHL prior to H2O2 further increased LC3-II level, promoted p62 turnover and improved lysosomal function. Importantly, autophagy inhibitor chloroquine (CQ) reversed the protective effect of PHL, and increased H2O2-induced apoptosis. Furthermore, PHL inhibited the phosphorylation levels of ERK, p38, and JNK. Collectively, these results indicate that PHL could attenuate H2O2-induced oxidative injury and apoptosis by maintaining lysosomal function and promoting autophagic flux, and MAPKs pathway may be involved in this process. Our study provides evidence that PHL could as a new strategy to against oxidative damage in poultry industry.

Song, Dan↗
DOE OSTI2021

Phagosomal chloride dynamics in the alveolar macrophage

Acidification in intracellular organelles is tightly linked to the influx of Cl – counteracting proton translocation by the electrogenic V-ATPase. We quantified the dynamics of Cl – transfer accompanying cargo incorporation into single phagosomes in alveolar macrophages (AMs). Phagosomal Cl – concentration and acidification magnitude were followed in real time with maximal acidification achieved at levels of approximately 200 mM. Live cell confocal microscopy verified that phagosomal Cl – influx utilized predominantly the Cl – channel CFTR. Relative levels of elemental chlorine (Cl) in hard X-ray fluorescence microprobe (XFM) analysis within single phagosomes validated the increase in Cl – content. XFM revealed the complex interplay between elemental K content inside the phagosome and changes in Cl – during phagosomal particle uptake. Cl – -dependent changes in phagosomal membrane potential were obtained using second harmonic generation (SHG) microscopy. These studies provide a mechanistic insight for screening studies in drug development targeting pulmonary inflammatory disease.

59 BASIC BIOLOGICAL SCIENCES↗
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↗