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At least 289 records · Page 16
All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

In vitro investigation of protein assembly by combined microscopy and infrared spectroscopy at the nanometer scale

The nanoscale structure and dynamics of proteins on surfaces has been extensively studied using various imaging techniques, such as transmission electron microscopy and atomic force microscopy (AFM) in liquid environments. These powerful imaging techniques, however, can potentially damage or perturb delicate biological material and do not provide chemical information, which prevents a fundamental understanding of the dynamic processes underlying their evolution under physiological conditions. Here, we use a platform developed in our laboratory that enables acquisition of infrared (IR) spectroscopy and AFM images of biological material in physiological liquids with nanometer resolution in a cell closed by atomically thin graphene membranes transparent to IR photons. In this work, we studied the self-assembly process of S-layer proteins at the graphene-aqueous solution interface. The graphene acts also as the membrane separating the solution containing the proteins and Ca 2+ ions from the AFM tip, thus eliminating sample damage and contamination effects. The formation of S-layer protein lattices and their structural evolution was monitored by AFM and by recording the amide I and II IR absorption bands, which reveal the noncovalent interaction between proteins and their response to the environment, including ionic strength and solvation. Our measurement platform opens unique opportunities to study biological material and soft materials in general.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Trajectories for the evolution of bacterial CO 2 -concentrating mechanisms

Cyanobacteria rely on CO 2 -concentrating mechanisms (CCMs) to grow in today’s atmosphere (0.04% CO 2 ). These complex physiological adaptations require ≈15 genes to produce two types of protein complexes: inorganic carbon (Ci) transporters and 100+ nm carboxysome compartments that encapsulate rubisco with a carbonic anhydrase (CA) enzyme. Mutations disrupting any of these genes prohibit growth in ambient air. If any plausible ancestral form—i.e., lacking a single gene—cannot grow, how did the CCM evolve? Here, we test the hypothesis that evolution of the bacterial CCM was “catalyzed” by historically high CO 2 levels that decreased over geologic time. Using an E. coli reconstitution of a bacterial CCM, we constructed strains lacking one or more CCM components and evaluated their growth across CO 2 concentrations. We expected these experiments to demonstrate the importance of the carboxysome. Instead, we found that partial CCMs expressing CA or Ci uptake genes grew better than controls in intermediate CO 2 levels (≈1%) and observed similar phenotypes in two autotrophic bacteria, Halothiobacillus neapolitanus and Cupriavidus necator . To understand how CA and Ci uptake improve growth, we model autotrophy as colimited by CO 2 and HCO 3 − , as both are required to produce biomass. Our experiments and model delineated a viable trajectory for CCM evolution where decreasing atmospheric CO 2 induces an HCO 3 − deficiency that is alleviated by acquisition of CA or Ci uptake, thereby enabling the emergence of a modern CCM. This work underscores the importance of considering physiology and environmental context when studying the evolution of biological complexity.

Flamholz, Avi I.↗
DOE OSTI2024

Myosin in autoinhibited off state(s), stabilized by mavacamten, can be recruited in response to inotropic interventions

Mavacamten is a FDA-approved small-molecule therapeutic designed to regulate cardiac function at the sarcomere level by selectively but reversibly inhibiting the enzymatic activity of myosin. It shifts myosin toward ordered off states close to the thick filament backbone. It remains elusive whether these myosin heads in the off state(s) can be recruited in response to physiological stimuli when required to boost cardiac output. We show that cardiac myosins stabilized in these off state(s) by mavacamten are recruitable by 1) Ca 2+ , 2) increased chronotropy [heart rate (HR)], 3) stretch, and 4) β-adrenergic (β-AR) stimulation, all known physiological inotropic interventions. At the molecular level, we show that Ca 2+ increases myosin ATPase activity by shifting mavacamten-stabilized myosin heads from the inactive super-relaxed state to the active disordered relaxed state. At the myofilament level, both Ca 2+ and passive lengthening can shift mavacamten-ordered off myosin heads from positions close to the thick filament backbone to disordered on states closer to the thin filaments. In isolated rat cardiomyocytes, increased stimulation rates enhanced shortening fraction in mavacamten-treated cells. This observation was confirmed in vivo in telemetered rats, where left-ventricular dP/dt max , an index of inotropy, increased with HR in mavacamten-treated animals. Finally, we show that β-AR stimulation in vivo increases left-ventricular function and stroke volume in the setting of mavacamten. Our data demonstrate that the mavacamten-promoted off states of myosin in the thick filament are at least partially activable, thus preserving cardiac reserve mechanisms.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Identification and characterization of a small-molecule metallophore involved in lanthanide metabolism

Many bacteria secrete metallophores, low-molecular-weight organic compounds that bind ions with high selectivity and affinity, in order to access essential metals from the environment. Previous work has elucidated the structures and biosynthetic machinery of metallophores specific for iron, zinc, nickel, molybdenum, and copper. No physiologically relevant lanthanide-binding metallophore has been discovered despite the knowledge that lanthanide metals (Ln) have been revealed to be essential cofactors for certain alcohol dehydrogenases across a diverse range of phyla. Here, we report the biosynthetic machinery, the structure, and the physiological relevance of a lanthanophore, methylolanthanin. The structure of methylolanthanin exhibits a unique 4-hydroxybenzoate moiety which has not previously been described in other metallophores. We find that production of methylolanthanin is required for normal levels of Ln accumulation in the methylotrophic bacterium Methylobacterium extorquens AM1, while overexpression of the molecule greatly increases bioaccumulation and adsorption. Our results provide a clearer understanding of how Ln-utilizing bacteria sense, scavenge, and store Ln; essential processes in the environment where Ln are poorly bioavailable. More broadly, the identification of this lanthanophore opens doors for study of how biosynthetic gene clusters are repurposed for additional functions and the complex relationship between metal homeostasis and fitness.

Science & Technology - Other Topics↗
DOE OSTI2025

Cholesterol-dependent enzyme activity of human TSPO1

The amino acid sequence of the tryptophan-rich sensory proteins (TSPO) is substantially conserved throughout all kingdoms of life. Human mitochondrial TSPO1 (HsTSPO1) binds to porphyrins and steroids, although its interactions with these molecules remains unknown.HsTSPO1 is associated with numerous physiological and pathological disorders, but the underlying molecular mechanisms are unknown. Here, we disclose the finding of human mitochondrial TSPO as a cholesterol-dependent protoporphyrin IX oxygenase. The results of our biochemical characterization are consistent with structural data and evolutionary analysis. The dependence ofHsTSPO1 activity on cholesterol may be the result of the coevolution of this membrane protein with the membrane system. Our study provides a molecular foundation for comprehending the various roles played by mitochondrial TSPO in normal physiological and pathological situations.

Science & Technology - Other Topics↗
DOE OSTI2025

Cyclization of archaeal membrane lipids impacts membrane protein activity and archaellum formation

Enhancement of the cyclization of membrane lipids GDGTs (glycerol dialkyl glycerol tetraethers) is a critical strategy for archaea to adapt to various environmental stresses. However, the physiological function of membrane lipid cyclization remains unclear. Here, we reported that the GDGT ring synthases mutant, deficient in GDGT cyclization, inhibited archaellum formation and reduced cell motility in thermoacidophilic crenarchaeon Sulfolobus acidocaldarius . This inhibition was caused by decreased transcription of the archaellum operon, likely due to cleavage of the C-terminal domains in transmembrane proteins ArnRs, the transcription factors that regulate archaellum operon expression. The transcriptomic and proteomic analysis showed deficiency of GDGT cyclization broadly impacted the expression of membrane associate proteins, including respiratory chain proteins, and decreased cellular ATP concentration. Moreover, phylogenetic analysis demonstrated that the correlation between GDGT cyclization and archaellum formation is widespread among (hyper)thermophilic archaea, and this was further verified in the euryarchaeon Thermococcus kodakarensis. Our findings suggested that archaea modify their membrane lipids to profoundly alter cellular appendages and cell physiology to adapt to environmental fluctuations.

Yang, Wei (ORCID:0000000262755981)↗
DOE OSTI2021

Dependence of thick filament structure in relaxed mammalian skeletal muscle on temperature and interfilament spacing

Contraction of skeletal muscle is regulated by structural changes in both actin-containing thin filaments and myosin-containing thick filaments, but myosin-based regulation is unlikely to be preserved after thick filament isolation, and its structural basis remains poorly characterized. Here, we describe the periodic features of the thick filament structure in situ by high-resolution small-angle x-ray diffraction and interference. We used both relaxed demembranated fibers and resting intact muscle preparations to assess whether thick filament regulation is preserved in demembranated fibers, which have been widely used for previous studies. We show that the thick filaments in both preparations exhibit two closely spaced axial periodicities, 43.1 nm and 45.5 nm, at near-physiological temperature. The shorter periodicity matches that of the myosin helix, and x-ray interference between the two arrays of myosin in the bipolar filament shows that all zones of the filament follow this periodicity. The 45.5-nm repeat has no helical component and originates from myosin layers closer to the filament midpoint associated with the titin super-repeat in that region. Cooling relaxed or resting muscle, which partially mimics the effects of calcium activation on thick filament structure, disrupts the helical order of the myosin motors, and they move out from the filament backbone. Compression of the filament lattice of demembranated fibers by 5% Dextran, which restores interfilament spacing to that in intact muscle, stabilizes the higher-temperature structure. The axial periodicity of the filament backbone increases on cooling, but in lattice-compressed fibers the periodicity of the myosin heads does not follow the extension of the backbone. Thick filament structure in lattice-compressed demembranated fibers at near-physiological temperature is similar to that in intact resting muscle, suggesting that the native structure of the thick filament is largely preserved after demembranation in these conditions, although not in the conditions used for most previous studies with this preparation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

The structural and functional integrities of porcine myocardium are mostly preserved by cryopreservation

Structural and functional studies of heart muscle are important to gain insights into the physiological bases of cardiac muscle contraction and the pathological bases of heart disease. While fresh muscle tissue works best for these kinds of studies, this is not always practical to obtain, especially for heart tissue from large animal models and humans. Conversely, tissue banks of frozen human hearts are available and could be a tremendous resource for translational research. It is not well understood, however, how liquid nitrogen freezing and cryostorage may impact the structural integrity of myocardium from large mammals. In this study, we directly compared the structural and functional integrity of never-frozen to previously frozen porcine myocardium to investigate the consequences of freezing and cryostorage. X-ray diffraction measurements from hydrated tissue under near-physiological conditions and electron microscope images from chemically fixed porcine myocardium showed that prior freezing has only minor effects on structural integrity of the muscle. Furthermore, mechanical studies similarly showed no significant differences in contractile capabilities of porcine myocardium with and without freezing and cryostorage. These results demonstrate that liquid nitrogen preservation is a practical approach for structural and functional studies of myocardium.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Chronically underestimated: a reassessment of US heat waves using the extended heat index

Abstract The heat index, or apparent temperature, was never defined for extreme heat and humidity, leading to the widespread adoption of a polynomial extrapolation designed by the United States National Weather Service. Recently, however, the heat index has been extended to all combinations of temperature and humidity, presenting an opportunity to reassess past heat waves. Here, three-hourly temperature and humidity are used to evaluate the extended heat index over the contiguous United States during the years 1984–2020. It is found that the 99.9th percentile of the daily maximum heat index is highest over the Midwest. Identifying and ranking heat waves by the spatially integrated exceedance of that percentile, the Midwest once again stands out as home to the most extreme heat waves, including the top-ranked July 2011 and July 1995 heat waves. The extended heat index can also be used to evaluate the physiological stress induced by heat and humidity. It is found that the most extreme Midwest heat waves tax the cardiovascular system with a skin blood flow that is elevated severalfold, approaching the physiological limit. These effects are not captured by the National Weather Service’s polynomial extrapolation, which also underestimates the heat index by as much as 10 ∘ C (20 ∘ F) during severe heat waves.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2023

Thermal acclimation of plant photosynthesis and autotrophic respiration in a northern peatland

Peatlands contain one-third of global soil carbon (C), but the responses of peatland ecosystems to long-term warming are not well understood. Here, we pursue an emergent understanding of warming effects on ecosystem C fluxes at peatlands by constraining a process-oriented model, the terrestrial ECOsystem model, with observational data from a long-term warming experiment at the Spruce and Peatland Responses Under Changing Environments site. Model-based assessments show that ecosystem-level photosynthesis and autotrophic respiration exhibited significant thermal acclimation, with temperature sensitivities being linearly decreased with warming. Using the thermal-acclimated parameter values, simulated gross primary production, net primary production, and plant autotrophic respiration (R a ), were all lower than those simulated with non-thermal acclimated parameter values. In contrast, ecosystem respiration simulated with thermal acclimated parameter values was higher than that simulated with non-thermal acclimated parameter values. Net ecosystem CO 2 exchange was much higher after constraining model parameters with observational data from the warming treatments, releasing C at a rate of 28.3 g C m -2 yr -1 °C -1 . Our data-model integration study suggests that peatlands are likely to release more C than previously estimated. Earth system models may overestimate C uptake by peatlands under warming if physiological thermal acclimation of plants is not incorporated. Thus, it is critical to consider the long-term physiological thermal acclimation of plants in the models to better predict global C dynamics under future climate and their feedback to climate change.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2020

Non-coding RNAs: update on mechanisms and therapeutic targets from the ESC Working Groups of Myocardial Function and Cellular Biology of the Heart

Abstract Vast parts of mammalian genomes are actively transcribed, predominantly giving rise to non-coding RNA (ncRNA) transcripts including microRNAs, long ncRNAs, and circular RNAs among others. Contrary to previous opinions that most of these RNAs are non-functional molecules, they are now recognized as critical regulators of many physiological and pathological processes including those of the cardiovascular system. The discovery of functional ncRNAs has opened up new research avenues aiming at understanding ncRNA-related disease mechanisms as well as exploiting them as novel therapeutics in cardiovascular therapy. In this review, we give an update on the current progress in ncRNA research, particularly focusing on cardiovascular physiological and disease processes, which are under current investigation at the ESC Working Groups of Myocardial Function and Cellular Biology of the Heart. This includes a range of topics such as extracellular vesicle-mediated communication, neurohormonal regulation, inflammation, cardiac remodelling, cardio-oncology as well as cardiac development and regeneration, collectively highlighting the wide-spread involvement and importance of ncRNAs in the cardiovascular system.

Bär, Christian↗
DOE OSTI2022

Bacterial exometabolites influence Chlamydomonas cell cycle and double algal productivity

Algal-bacterial interactions provide clues to algal physiology, but mutualistic interactions are complicated by dynamic exchange. In this study, we characterized the response of Chlamydomonas reinhardtii to the presence of a putative alga-benefitting commensal bacterium ( Arthrobacter strain ‘P2b’). Co-cultivation promoted chlorophyll content, biomass, average cell size, and number of dividing cells, relative to axenic cultures. Addition of bacterial spent medium (whole, size-fractionated and heat-treated) had similar effects, indicating P2b does not require algal interaction to promote growth. Nutrients and pH were excluded as putative effectors, collectively indicating a commensal interaction mediated by Arthrobacter -released small exometabolite(s). Proteogenomic comparison revealed similar response to co-cultivation and spent media, including differential cell cycle regulation, extensive downregulation of flagellar genes and histones, carbonic anhydrase and RubisCO downregulation, upregulation of some chlorophyll, amino acid and carbohydrate biosynthesis genes, and changes to redox and Fe homeostasis. Further, Arthrobacter protein expression indicated some highly expressed putative secondary metabolites. Together, these results revealed that low molecular weight bacterial metabolites can elicit major physiological changes in algal cell cycle regulation, perhaps through a more productive G1 phase, that lead to substantial increases in photosynthetically-produced biomass. This work illustrates that model commensal interactions can be used to shed light on algal response to stimulating bacteria.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Integrating Intermediate Traits in Phylogenetic Genotype-to-Phenotype Studies

A major goal of research in evolution and genetics is linking genotype to phenotype. This work could be direct, such as determining the genetic basis of a phenotype by leveraging genetic variation or divergence in a developmental, physiological, or behavioral trait. The work could also involve studying the evolutionary phenomena (e.g., reproductive isolation, adaptation, sexual dimorphism, behavior) that reveal an indirect link between genotype and a trait of interest. When the phenotype diverges across evolutionarily distinct lineages, this genotype-to-phenotype problem can be addressed using phylogenetic genotype-to-phenotype (PhyloG2P) mapping, which uses genetic signatures and convergent phenotypes on a phylogeny to infer the genetic bases of traits. The PhyloG2P approach has proven powerful in revealing key genetic changes associated with diverse traits, including the mammalian transition to marine environments and transitions between major mechanisms of photosynthesis. However, there are several intermediate traits layered in between genotype and the phenotype of interest, including but not limited to transcriptional profiles, chromatin states, protein abundances, structures, modifications, metabolites, and physiological parameters. Each intermediate trait is interesting and informative in its own right, but synthesis across data types has great promise for providing a deep, integrated, and predictive understanding of how genotypes drive phenotypic differences and convergence. We argue that an expanded PhyloG2P framework (the PhyloG2P matrix) that explicitly considers intermediate traits, and imputes those that are prohibitive to obtain, will allow a better mechanistic understanding of any trait of interest. Furthermore, this approach provides a proxy for functional validation and mechanistic understanding in organisms where laboratory manipulation is impractical.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Gut microbiota carbon and sulfur metabolisms support Salmonella infections

Abstract Salmonella enterica serovar Typhimurium is a pervasive enteric pathogen and ongoing global threat to public health. Ecological studies in the Salmonella impacted gut remain underrepresented in the literature, discounting microbiome mediated interactions that may inform Salmonella physiology during colonization and infection. To understand the microbial ecology of Salmonella remodeling of the gut microbiome, we performed multi-omics on fecal microbial communities from untreated and Salmonella-infected mice. Reconstructed genomes recruited metatranscriptomic and metabolomic data providing a strain-resolved view of the expressed metabolisms of the microbiome during Salmonella infection. These data informed possible Salmonella interactions with members of the gut microbiome that were previously uncharacterized. Salmonella-induced inflammation significantly reduced the diversity of genomes that recruited transcripts in the gut microbiome, yet increased transcript mapping was observed for seven members, among which Luxibacter and Ligilactobacillus transcript read recruitment was most prevalent. Metatranscriptomic insights from Salmonella and other persistent taxa in the inflamed microbiome further expounded the necessity for oxidative tolerance mechanisms to endure the host inflammatory responses to infection. In the inflamed gut lactate was a key metabolite, with microbiota production and consumption reported amongst members with detected transcript recruitment. We also showed that organic sulfur sources could be converted by gut microbiota to yield inorganic sulfur pools that become oxidized in the inflamed gut, resulting in thiosulfate and tetrathionate that support Salmonella respiration. This research advances physiological microbiome insights beyond prior amplicon-based approaches, with the transcriptionally active organismal and metabolic pathways outlined here offering intriguing intervention targets in the Salmonella-infected intestine.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Detection of the Metabolic Response to Drought Stress Using Hyperspectral Reflectance

Drought is the most important limitation on crop yield. Understanding and detecting drought stress in crops is vital for improving water use efficiency through effective breeding and management. Leaf reflectance spectroscopy offers a rapid, non-destructive alternative to traditional techniques for measuring plant traits involved in a drought response. We measured drought stress in six glasshouse-grown agronomic species using physiological, biochemical and spectral data. In contrast to physiological traits, leaf metabolite concentrations revealed drought stress before it was visible to the naked eye. We used full-spectrum leaf reflectance data to predict metabolite concentrations using partial least-squares regression, with validation R2 values of 0.49 to 0.87. We show for the first time that spectroscopy may be used for the quantitative estimation of proline and abscisic acid, demonstrating the first use of hyperspectral data to detect a phytohormone. We used linear discriminant analysis and partial least squares discriminant analysis to differentiate between droughted and watered plants based on measured traits (accuracy: 71%) and raw spectral data (66%). Finally, we validated our glasshouse-developed models in an independent field trial. We demonstrate that spectroscopy can detect drought stress via underlying biochemical changes, before visual differences occur, representing a powerful advance for measuring limitations on yield.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2026

Cas3-Mediated Genome Reduction: Demonstration in Cupriavidus Necator H16 Improves Growth on Heterotrophic and Autotrophic Carbon Sources

Genome reduction is widely used to improve microbial bioprocessing hosts by reducing the burden of inessential physiology. Rationally identifying genomic regions that are dispensable or even detrimental to bioprocessing is challenged by our inability to map genome sequence to function across complex regulation and physiology. Thus, there is a need for tools that rapidly generate reduced genome strains with improved performance in process-relevant conditions. Here, we report a Cascade-Cas3-enabled method called TRIM3 that generates large deletions by targeting a randomly integrated transposon, enabling facile generation of a genome-reduced mutant library. Mutants with improved performance were isolated following growth-coupled selection and analyzed by long-read DNA sequencing to identify deletions in their genomes. We deploy this system iteratively in the industrial host Cupriavidus necator H16 on fructose and on formate. After two rounds of TRIM3, we isolate a strain containing a total reduction of 1.4 Mb (18.4% of the genome) that grows 25% faster in a bioreactor on fructose and a strain with a total reduction of 0.5 Mb (7.3% of the genome) that grows 14% faster on formate. This work demonstrates a method for random, iterative, growth-selectable genome reduction that represents a new avenue for large-scale genome modifications and the development of improved bioprocessing hosts.

09 BIOMASS FUELS↗
DOE OSTI2021

Return of the Lemnaceae: duckweed as a model plant system in the genomics and postgenomics era

Abstract The aquatic Lemnaceae family, commonly called duckweed, comprises some of the smallest and fastest growing angiosperms known on Earth. Their tiny size, rapid growth by clonal propagation, and facile uptake of labeled compounds from the media were attractive features that made them a well-known model for plant biology from 1950 to 1990. Interest in duckweed has steadily regained momentum over the past decade, driven in part by the growing need to identify alternative plants from traditional agricultural crops that can help tackle urgent societal challenges, such as climate change and rapid population expansion. Propelled by rapid advances in genomic technologies, recent studies with duckweed again highlight the potential of these small plants to enable discoveries in diverse fields from ecology to chronobiology. Building on established community resources, duckweed is reemerging as a platform to study plant processes at the systems level and to translate knowledge gained for field deployment to address some of society’s pressing needs. This review details the anatomy, development, physiology, and molecular characteristics of the Lemnaceae to introduce them to the broader plant research community. We highlight recent research enabled by Lemnaceae to demonstrate how these plants can be used for quantitative studies of complex processes and for revealing potentially novel strategies in plant defense and genome maintenance.

Biochemistry & Molecular Biology↗
DOE OSTI2022

Time for a drought experiment: Do you know your plants’ water status?

Abstract Drought stress is an increasing concern because of climate change and increasing demands on water for agriculture. There are still many unknowns about how plants sense and respond to water limitation, including which genes and cellular mechanisms are impactful for ecology and crop improvement in drought-prone environments. A better understanding of plant drought resistance will require integration of several research disciplines. A common set of parameters to describe plant water status and quantify drought severity can enhance data interpretation and research integration across the research disciplines involved in understanding drought resistance and would be especially useful in integrating the flood of genomic data being generated in drought studies. Water potential (ψw) is a physical measure of the free energy status of water that, along with related physiological measurements, allows unambiguous description of plant water status that can apply across various soil types and environmental conditions. ψw and related physiological parameters can be measured with relatively modest investment in equipment and effort. Thus, we propose that increased use of ψw as a fundamental descriptor of plant water status can enhance the insight gained from many drought-related experiments and facilitate data integration and sharing across laboratories and research disciplines.

Juenger, Thomas E. (ORCID:0000000195509288)↗