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Results for “cell metabolism”

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At least 19 records

Label-free nanoscopy of cell metabolism by ultrasensitive reweighted visible stimulated Raman scattering

Nanoscopic imaging of cell metabolism is hindered by the incompatibility of small metabolites with fluorescent dyes and the limited resolution of imaging mass spectrometry. We present ultrasensitive reweighted visible stimulated Raman scattering (URV-SRS), a label-free vibrational nanoscopy technique that enables multiplexed detection of metabolic nanostructures within cells. We developed an extensively chirped spectral focusing visible SRS microscope that achieves a detection limit of 4,000 molecules and introduced a self-supervised learning-based denoiser to robustly suppress non-independent SRS noise by over 7.2 dB. The instrumentation-based signal enhancement and computation-based noise suppression synergistically improved the detection sensitivity by 50 times over near-infrared SRS. Leveraging this enhanced sensitivity, we further pushed the resolution to nanoscopic levels by introducing Fourier reweighting to amplify sub-100 nm spatial frequencies previously overwhelmed by noise. Validated by Fourier ring correlation, URV-SRS achieves a lateral resolution of 86 nm in cellular imaging. Here, we applied URV-SRS to elucidate the reprogramming of metabolic nanostructures associated with virus replication in Vero E6 host cells and to compositionally delineate subcellular fatty acid synthesis in engineered Escherichia coli, demonstrating its capability towards nanoscopic spatial metabolomics.

59 BASIC BIOLOGICAL SCIENCES

Shear stress induced stimulation of mammalian cell metabolism

A flow apparatus was developed for the study of the metabolic response of anchorage dependent cells to a wide range of steady and pulsatile shear stresses under well controlled conditions. Human umbilical vein endothelial cell monolayers were subjected to steady shear stresses of up to 24 dynes/sq cm, and the production of prostacyclin was determined. The onset of flow led to a burst in prostacyclin production which decayed to a long term steady state rate (SSR). The SSR of cells exposed to flow was greater than the basal release level, and increased linearly with increasing shear stress. It is demonstrated that shear stresses in certain ranges may not be detrimental to mammalian cell metabolism. In fact, throughout the range of shear stresses studied, metabolite production is maximized by maximizing shear stress.

Mcintire, L. V.

Red cell metabolism studies on Skylab

Blood samples from Spacelab crewmembers were studied for possible environment effects on red cell components. Analysis involved peroxidation of red cell lipids, enzymes of red cell metabolism, and levels of 2,3-diphosphoglyceric acid and adenosine triphosphate. Results show that there is no evidence of lipid peroxidation, that biochemical effect known to be associated with irreversible red cell damage. Changes observed in glycolytic intermediates and enzymes cannot be directly implicated as indicating evidence of red cell damage.

Mengel, C. E.

Red cell metabolism studies on Skylab

On the basis of these background data, metabolic studies were performed on humans involved in space flight. These studies included the Skylab experiences. The primary purpose of the investigations was to study red cells for: (1) evidences of lipid peroxidation, or (2) changes at various points in the glycolytic pathway. The Skylab missions were an opportunity to study blood samples before, during, and after flight and to compare results with simultaneous controls. No direct evidence that lipid peroxidation had occurred in the red blood cells was apparent in the studies.

Mengel, C. E.

Red blood cell metabolism (M114), part D

Statistically significant differences were found between Skylab simulation crews and controls for glycolytic enzymes. The absence of simultaneous controls for the pre- and postchamber analyses leaves the significance of the findings in the crew during these periods indeterminate.

Mengel, C. E.

Possible effects of organelle charge and density on cell metabolism

A system of perception and transduction involving the gravity-induced asymmetric distribution of a plant growth hormone is studied. A theory is constructed which assumes that the perception of the gravitational stimulus involved a perturbation of the plant's bioelectric field and that the transduction of the stimulus involved voltage-gating of hormone movement from the plant's vascular tissue into the hormone responsive growing tissue. Particular attention is focused on the barriers to indole-3-acetic acid (IAA) transport from the seed to the mesocotyl cortex, the protoinhibition of IAA movement from the endosperm to the shoot, the effects of the gravitational stimulus on the movement of IAA from the kernel to the shoot, electrochemical gating as a target for the gravity stimulus, and the gravity sensing mechanism.

Bandurski, Robert S.

Multicellular magnetotactic bacteria are genetically heterogeneous consortia with metabolically differentiated cells

Consortia of multicellular magnetotactic bacteria (MMB) are currently the only known example of bacteria without a unicellular stage in their life cycle. Because of their recalcitrance to cultivation, most previous studies of MMB have been limited to microscopic observations. To study the biology of these unique organisms in more detail, we use multiple culture-independent approaches to analyze the genomics and physiology of MMB consortia at single-cell resolution. We separately sequenced the metagenomes of 22 individual MMB consortia, representing 8 new species, and quantified the genetic diversity within each MMB consortium. This revealed that, counter to conventional views, cells within MMB consortia are not clonal. Single consortia metagenomes were then used to reconstruct the species-specific metabolic potential and infer the physiological capabilities of MMB. To validate genomic predictions, we performed stable isotope probing (SIP) experiments and interrogated MMB consortia using fluorescence in situ hybridization (FISH) combined with nanoscale secondary ion mass spectrometry (NanoSIMS). By coupling FISH with bioorthogonal noncanonical amino acid tagging (BONCAT), we explored their in situ activity as well as variation of protein synthesis within cells. We demonstrate that MMB consortia are mixotrophic sulfate reducers and that they exhibit metabolic differentiation between individual cells, suggesting that MMB consortia are more complex than previously thought. These findings expand our understanding of MMB diversity, ecology, genomics, and physiology, as well as offer insights into the mechanisms underpinning the multicellular nature of their unique lifestyle.

59 BASIC BIOLOGICAL SCIENCES

Cellular changes in microgravity and the design of space radiation experiments

Cell metabolism, secretion and cell-cell interactions can be altered during space flight. Early radiobiology experiments have demonstrated synergistic effects of radiation and microgravity as indicated by increased mutagenesis, increased chromosome aberrations, inhibited development, and retarded growth. Microgravity-induced changes in immune cell functions include reduced blastogenesis and cell-mediated, delayed-type hypersensitivity responses, increased cytokine secretions, but inhibited cytotoxic effects an macrophage differentiation. These effects are important because of the high radiosensitivity of immune cells. It is difficult to compare ground studies with space radiation biology experiments because of the complexity of the space radiation environment, types of radiation damage and repair mechanisms. Altered intracellular functions and molecular mechanisms must be considered in the design and interpretation of space radiation experiments. Critical steps in radiocarcinogenesis could be affected. New cell systems and hardware are needed to determine the biological effectiveness of the low dose rate, isotropic, multispectral space radiation and the potential usefulness of radioprotectants during space flight.

Morrison, D. R.

Plant metabolism and cell wall formation in space (microgravity) and on Earth

Variations in cell wall chemistry provide vascular plants with the ability to withstand gravitational forces, as well as providing facile mechanisms for correctional responses to various gravitational stimuli, e.g., in reaction wood formation. A principal focus of our current research is to precisely and systematically dissect the essentially unknown mechanism(s) of vascular plant cell wall assembly, particularly with respect to formation of its phenolic constituents, i.e., lignins and suberins, and how gravity impacts upon these processes. Formation of these phenolic polymers is of particular interest, since it appears that elaboration of their biochemical pathways was essential for successful land adaptation. By extrapolation, we are also greatly intrigued as to how the microgravity environment impacts upon 'normal' cell wall assembly mechanisms/metabolism.

Lewis, Norman G.

The effect of fluid mechanical stress on cellular arachidonic acid metabolism

The effect of sublytic levels of mechanical perturations of cells on cell metabolism were investigated by analyzing the products of arachidonic acid (used as a marker metabolite) in blood platelets, polymorphonuclear leucocytes, and cultured umbilical-vein endothelial cells after the suspensions of these cells were subjected to a shear stress in a modified viscometer. It is shown that the sublytic levels of mechanical stress stimulated the arachidonic acid metabolism in all these cell types. Possible biological implications of this stress-metabolism coupling are discussed.

Mcintire, L. V.

Spaceflight effects on cultured embryonic chick bone cells

A model calcifying system of primary osteoblast cell cultures derived from normal embryonic chicken calvaria has been flown aboard the shuttle, Endeavour, during the National Aeronautics and Space Administration (NASA) mission STS-59 (April 9-20, 1994) to characterize unloading and other spaceflight effects on the bone cells. Aliquots of cells (approximately 7 x 10(6)) grown in Dulbecco's modified Eagle's medium (DMEM) + 10% fetal bovine serum (FBS) were mixed with microcarrier beads, inoculated into cartridge culture units of artificial hollow fiber capillaries, and carried on the shuttle. To promote cell differentiation, cartridge media were supplemented with 12.5 microg/ml ascorbate and 10 mM beta-glycerophosphate for varying time periods before and during flight. Four cartridges contained cells from 17-day-old embryos grown for 5 days in the presence of ascorbate prior to launch (defined as flight cells committed to the osteoblastic lineage) and four cartridges supported cells from 14-day-old embryos grown for 10 days with ascorbate before launch (uncommitted flight cells). Eight cartridges prepared in the same manner were maintained under normal gravity throughout the flight (control cells) and four additional identical cartridges under normal gravity were terminated on the day of launch (basal cells). From shuttle launch to landing, all cartridges were contained in closed hardware units maintaining 5% CO2, 37 degrees C, and media delivery at a rate of approximately 1.5 ml/6 h. During day 3 and day 5 of flight, duplicate aliquots of conditioned media and accumulated cell products were collected in both the flight and the control hardware units. At the mission end, comparisons among flight, basal, and control samples were made in cell metabolism, gene expression for type I collagen and osteocalcin, and ultrastructure. Both committed and uncommitted flight cells were metabolically active, as measured by glucose uptake and lactate production, at approximately the same statistical levels as control counterparts. Flight cells elaborated a less extensive extracellular matrix, evidenced by a reduced collagen gene expression and collagen protein appearance compared with controls. Osteocalcin was expressed by all cells, a result indicating progressive differentiation of both flight and control osteoblasts, but its message levels also were reduced in flight cells compared with ground samples. This finding suggested that osteoblasts subjected to flight followed a slower progression toward a differentiated function. The summary of data indicates that spaceflight, including microgravity exposure, demonstrably affects bone cells by down-regulating type I collagen and osteocalcin gene expression and thereby inhibiting expression of the osteogenic phenotype notably by committed osteoblasts. The information is important for insight into the response of bone cells to changes of gravity and of force in general.

Non-NASA Center

Kinetic Tetrazolium Microtiter Assay

Kinetic tetrazolium microtiter assay (KTMA) involves use of tetrazolium salts and Triton X-100 (or equivalent), nontoxic, in vitro color developer solubilizing colored metabolite formazan without injuring or killing metabolizing cells. Provides for continuous measurement of metabolism and makes possible to determine rate of action of antimicrobial agent in real time as well as determines effective inhibitory concentrations. Used to monitor growth after addition of stimulatory compounds. Provides for kinetic determination of efficacy of biocide, greatly increasing reliability and precision of results. Also used to determine relative effectiveness of antimicrobial agent as function of time. Capability of generating results on day of test extremely important in treatment of water and waste, disinfection of hospital rooms, and in pharmaceutical, agricultural, and food-processing industries. Assay also used in many aspects of cell biology.

Pierson, Duane L.

Portable Immune-Assessment System

Portable immune-assessment system developed for use in rapidly identifying infections or contaminated environment. System combines few specific fluorescent reagents for identifying immune-cell dysfunction, toxic substances, buildup of microbial antigens or microbial growth, and potential identification of pathogenic microorganisms using fluorescent microplate reader linked to laptop computer. By using few specific dyes for cell metabolism, DNA/RNA conjugation, specific enzyme activity, or cell constituents, one makes immediate, onsite determination of person's health or of contamination of environment.

Pierson, Duane L.

PET-FBA: A lightweight enzyme allocation and thermodynamics-constrained flux analysis approach to explore Escherichia coli metabolic adaptation to intracellular acidification

Escherichia coli employs diverse strategies to adapt to acidic environments that disrupt enzyme activity and the thermodynamic feasibility of essential reactions. To understand the impact of pH stress on cell metabolism, we present the PET-FBA (pH-, Enzyme protein allocation-, and Thermodynamics-constrained Flux Balance Analysis) framework. PET-FBA extends genome-scale modeling by integrating enzyme protein costs and reaction Gibbs free energy changes. Additionally, by incorporating pH-dependent enzyme kinetics in response to intracellular acidification, this framework enables the simulation of E. coli's metabolic adjustments across varying external pH levels. The model's accuracy is validated by comparing in silico growth simulations with experimental measurements under both anaerobic and aerobic conditions, as well as in silico gene knockouts of essential genes. By explicitly incorporating pH effects, our model accurately replicates the metabolic shift towards lactate production as the primary fermentation product at low pH in anaerobic conditions. This shift is only predicted when enzyme kinetics are dynamically adjusted as a function of pH. Further analysis revealed that this shift can be attributed to the reduced protein efficiency of the acetyl-CoA branch compared to lactate dehydrogenase under acidic stress, which then becomes crucial for maintaining NAD regeneration and cell growth at low pH. Furthermore, we identified strategies for enhancing cell growth under acidic anaerobic conditions by improving the enzyme activity of lactate dehydrogenase and pyruvate formate lyase, which increases NAD production efficiency and reduces enzyme protein allocation costs. Designed as a lightweight yet versatile framework, PET-FBA enables efficient genome-scale metabolic analysis. Using E. coli as a model system, our framework provides a systematic approach to understanding metabolic responses to environmental stress, pinpointing key metabolic bottlenecks, and identifying potential targets for strain optimization.

42 ENGINEERING

Advancing Torpor Inducing Transfer Habitats for Human Stasis to Mars: II - Phase

SpaceWorks Enterprises, Inc. (SEI) has conducted an evaluation of an advanced habitat system designed to transportcrews between the Earth and Mars. This new and innovative habitat concept is capable of placing crew members ininactive, torpor states during transit phases of a deep space mission. This substantially reduces the mass and size ofthe habitat, which ultimately leads to significant reductions in the overall architecture size.Our approach for achieving this is based on extending the current and evolving medical practice of TherapeuticHypothermia (TH) - a proven and effective treatment for various traumatic injuries. TH is a medical treatment thatlowers a patient's body temperature by just 5 to 10 degrees Fahrenheit causing human metabolic rate to decreasesignificantly and the body to enter an unconscious state. This method avoids the intractable challenges often associatedwith cell metabolic cessation through cryogenic freezing and other highly speculative approaches.The initial results obtained from the research and analysis conducted in the Phase I effort warranted further study ofthis concept and technology. The specific objectives of the continued work include:1. Addressing critical medical aspects and risks for inducing torpor via Therapeutic Hypothermia and theapproach for providing nutrition and hydration for the crew during torpor. 2. Focusing on mitigation aspects and technology potential for solving key human spaceflight challenges. 3. Addressing critical engineering aspects of the design that may impact the initial performance and cost resultsobtained in Phase I. 4. Examining the broader extensibility and enabling capabilities of this concept through applicability toadditional exploration missions beyond Mars. 5. Establishing a technology development roadmap, addressing both medical and engineering aspects, thatindicate a logical and scientifically achievable path forward for maturation of this technology.

Stasis

Torpor Inducing Transfer Habitat for Human Stasis to Mars

SpaceWorks Enterprises, Inc. has performed an initial evaluation of an advanced habitat system designed to transport crews between the Earth and Mars. This new and innovative habitat design is capable of placing the crew in an inactive, torpor state for the duration of the in-space mission segments. This substantially reduces the mass and size of the habitat, which ultimately leads to significant reductions in the overall architecture size.Our approach for achieving this is based on extending the current and evolving medical practice of Therapeutic Hypothermia (TH) – a proven and effective treatment for various traumatic injuries. TH is a medical treatment that lowers a patient's body temperature by just 5 to 10 degrees Fahrenheit causing their metabolism to reduce significantly and the body to enter an unconscious state. This method avoids the intractable challenges often associated with cell metabolic cessation through cryogenic freezing and other highly speculative approaches.TH is a proven treatment for traumatic injuries; however it has not been applied for non-critical care purposes due to current lack of purpose (i.e. no practical need). The opportunity exists to use TH in this capacity to enable and enhance our human spaceflight capability. With this concept, we have the potential to simultaneously solve multiple exploration challenges.

Bradford, J.

Multimembrane Bioreactor

Set of hydrophilic and hydrophobic membranes in bioreactor allows product of reaction to be separated, while nutrients fed to reacting cells and byproducts removed from them. Separation process requires no externally supplied energy; free energy of reaction sufficient. Membranes greatly increase productivity of metabolizing cells by continuously removing product and byproducts, which might otherwise inhibit reaction, and by continuously adding oxygen and organic nutrients.

Cho, Toohyon

EvoNet: A phylogenomic and systems biology approach to identify genes underlying plant survival in marginal, low‐N soils

The DOE‐BER “EvoNet” project investigates the genetic and molecular basis of plant resilience in extreme environments. We do this by identifying key genes that enable “extreme survivor” species to thrive in the nitrogen-poor soils of Chile’s hyper-arid Atacama Desert. Our collections focus on 32 Atacama extremophile species, including seven grass species with potential biofuel applications. To identify genes-of-importance to survival we compared genomic and transcriptomic profiles of extremophile species that thrive in the Atacama to those of closely related “sister” species from nitrogen-rich arid and mesic regions of California. Deep RNA sequencing and de novo transcriptome assembly across these triplet species sets supported a phylogenomic framework for identifying positively selected genes associated with adaptive divergence. Our integrative analysis combined ecological and environmental data, metagenomics, evolutionary and systems biology, and metabolomics. This enabled us to create an unprecedented framework for systematically understanding how non-model plants have adapted to survive in extreme conditions. Our resulting database of positively selected ortholog groups in the extremophile plants offers promising targets for engineering crop and biofuel species with enhanced resilience to drought and extreme weather. Additionally, our newest dataset explores and exploits a complementary metabolomic approach. This new aspect provides innovative strategies to manipulate plant cell metabolism, further supporting efforts to improve agricultural productivity in the face of extreme climates. Importantly, our combined evolutionary- and metabolomic-based strategies focused on convergent patterns of adaptation, providing a genetic and metabolomic toolkit for improving crop and biofuel resilience across diverse plant species. Finally, our novel exploration of ecological and evolutionary dynamics delivered to the community a phylogenomic computational pipeline called “PhyloGeneious.” Our continued adaptations of this pipeline are publicly available to expedite evolutionary genomic research for future scientific discoveries. In total, our DOE-BER has provided genomic, metabolomic, and computational strategies to understand how extremophile plants provide evolutionary and physiological targets for improving agricultural and biofuel production.

59 BASIC BIOLOGICAL SCIENCES