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International Journal of Molecular... Jul 2023The administration of low doses of DO to living organisms was used for decades for the investigation of metabolic pathways and for the measurement of the turnover rate...
The administration of low doses of DO to living organisms was used for decades for the investigation of metabolic pathways and for the measurement of the turnover rate for specific compounds. Usually, the investigation of the deuterium uptake in lipids is performed by measuring the deuteration level of the palmitic acid residue using GC-MS instruments, and to our knowledge, the application of the modern untargeted LC-MS/MS lipidomics approaches was only reported a few times. Here, we investigated the deuterium uptake for >500 lipids for 13 organs and body liquids of mice (brain, lung, heart, liver, kidney, spleen, plasma, urine, etc.) after 4 days of 100% DO administration. The maximum deuteration level was observed in the liver, plasma, and lung, while in the brain and heart, the deuteration level was lower. Using MS/MS, we demonstrated the incorporation of deuterium in palmitic and stearic fragments in lipids (PC, PE, TAG, PG, etc.) but not in the corresponding free forms. Our results were analyzed based on the metabolic pathways of lipids.
Topics: Mice; Animals; Deuterium; Chromatography, Liquid; Tandem Mass Spectrometry; Lipidomics; Palmitic Acid
PubMed: 37511483
DOI: 10.3390/ijms241411725 -
Analytical Chemistry Jun 2019The intracellular pathogen Salmonella enterica has evolved an array of traits for propagation and invasion of the intestinal layers. It remains largely elusive how...
The intracellular pathogen Salmonella enterica has evolved an array of traits for propagation and invasion of the intestinal layers. It remains largely elusive how Salmonella adjusts its metabolic states to survive inside immune host cells. In this study, single-cell Raman biotechnology combined with deuterium isotope probing (Raman-DIP) have been applied to reveal metabolic changes of the typhoidal Salmonella Typhi Ty2, the nontyphoidal Salmonella Typhimurium LT2, and a clinical isolate Typhimurium D23580. By initially labeling the Salmonella strains with deuterium, we employed reverse labeling to track their metabolic changes in the time-course infection of THP-1 cell line, human monocyte-derived dendritic cells (MoDCs) and macrophages (Mf). We found that, in comparison with a noninvasive serovar, the invasive Salmonella strains Ty2 and D23580 have downregulated metabolic activity inside human macrophages and dendritic cells and used lipids as alternative carbon source, perhaps a strategy to escape from the host immune response. Proteomic analysis using high sensitivity mass spectrometry validated the findings of Raman-DIP analysis.
Topics: Cell Line; Deuterium; Down-Regulation; Humans; Isotope Labeling; Macrophages; Metabolome; Principal Component Analysis; Salmonella typhi; Single-Cell Analysis; Spectrum Analysis, Raman
PubMed: 31117406
DOI: 10.1021/acs.analchem.9b01010 -
Journal of Chemical Theory and... Dec 2016We present a united-atom model (gb-fb15) for the molecular dynamics simulation of hydrated liquid-crystalline dipalmitoylphosphatidylcholine (DPPC) phospholipid...
We present a united-atom model (gb-fb15) for the molecular dynamics simulation of hydrated liquid-crystalline dipalmitoylphosphatidylcholine (DPPC) phospholipid bilayers. This model was constructed through the parameter-space minimization of a regularized least-squares objective function via the ForceBalance method. The objective function was computed using a training set of experimental bilayer area per lipid and deuterium order parameter. This model was validated by comparison to experimental volume per lipid, X-ray scattering form factor, thermal area expansivity, area compressibility modulus, and lipid lateral diffusion coefficient. These comparisons demonstrate that gb-fb15 is robust to temperature variation and an improvement over the original model for both the training and validation properties.
Topics: 1,2-Dipalmitoylphosphatidylcholine; Deuterium; Lipid Bilayers; Liquid Crystals; Molecular Dynamics Simulation; Temperature
PubMed: 27786477
DOI: 10.1021/acs.jctc.6b00801 -
MAbs 2021Human carbonic anhydrase (hCAIX), an extracellular enzyme that catalyzes the reversible hydration of CO, is often overexpressed in solid tumors. This enzyme is...
Human carbonic anhydrase (hCAIX), an extracellular enzyme that catalyzes the reversible hydration of CO, is often overexpressed in solid tumors. This enzyme is instrumental in maintaining the survival of cancer cells in a hypoxic and acidic tumor microenvironment. Absent in most normal tissues, hCAIX is a promising therapeutic target for detection and treatment of solid tumors. Screening of a library of anti-hCAIX monoclonal antibodies (mAbs) previously identified three therapeutic candidates (mAb c2C7, m4A2 and m9B6) with distinct biophysical and functional characteristics. Selective binding to the catalytic domain was confirmed by yeast surface display and isothermal calorimetry, and deeper insight into the dynamic binding profiles of these mAbs upon binding were highlighted by bottom-up hydrogen-deuterium exchange mass spectrometry (HDX-MS). Here, a conformational and allosterically silent epitope was identified for the antibody-drug conjugate candidate c2C7. Unique binding profiles are described for both inhibitory antibodies, m4A2 and m9B6. M4A2 reduces the ability of the enzyme to hydrate CO by steric gating at the entrance of the catalytic cavity. Conversely, m9B6 disrupts the secondary structure that is necessary for substrate binding and hydration. The synergy of these two inhibitory mechanisms is demonstrated in activity assays and HDX-MS. Finally, the ability of m4A2 to modulate extracellular pH and intracellular metabolism is reported. By highlighting three unique modes by which hCAIX can be targeted, this study demonstrates both the utility of HDX-MS as an important tool in the characterization of anti-cancer biotherapeutics, and the underlying value of CAIX as a therapeutic target.
Topics: Antibodies, Monoclonal; Catalytic Domain; Deuterium; Deuterium Exchange Measurement; Epitope Mapping; Humans; Hydrogen Deuterium Exchange-Mass Spectrometry
PubMed: 34812124
DOI: 10.1080/19420862.2021.1997072 -
Journal of Applied Physiology... May 2020The use of deuterium oxide (DO) has greatly expanded the scope of what is possible for the measurement of protein synthesis. The greatest asset of DO labeling is that it...
The use of deuterium oxide (DO) has greatly expanded the scope of what is possible for the measurement of protein synthesis. The greatest asset of DO labeling is that it facilitates the measurement of synthesis rates over prolonged periods of time from single proteins through integrated tissue-based measurements. Because the ease of administration, the method is amenable for use in a variety of models and conditions. Although the method adheres to the same rules as other isotope methods, the flexibility can create conditions that are not the same as other approaches and thus requires careful execution to maintain validity and reliability. For this CORP article, we provide a history that gave rise to the method and discuss the advantages and disadvantages of the method, the critical assumptions, guidelines, and best practices based on instrumentation, models, and experimental design. The goal of this CORP article is to propagate additional use of DO in a manner that produces reliable and valid data.
Topics: Deuterium; Deuterium Oxide; Protein Biosynthesis; Reproducibility of Results; Water
PubMed: 32213116
DOI: 10.1152/japplphysiol.00855.2019 -
Proceedings of the National Academy of... Dec 2020The hydrogen isotope ratio of water cryogenically extracted from plant stem samples (δH) is routinely used to aid isotope applications that span hydrological,...
The hydrogen isotope ratio of water cryogenically extracted from plant stem samples (δH) is routinely used to aid isotope applications that span hydrological, ecological, and paleoclimatological research. However, an increasing number of studies have shown that a key assumption of these applications-that δH is equal to the δH of plant source water (δH)-is not necessarily met in plants from various habitats. To examine this assumption, we purposedly designed an experimental system to allow independent measurements of δH, δH, and δH of water transported in xylem conduits (δH) under controlled conditions. Our measurements performed on nine woody plant species from diverse habitats revealed a consistent and significant depletion in δH compared with both δH and δH Meanwhile, no significant discrepancy was observed between δH and δH in any of the plants investigated. These results cast significant doubt on the long-standing view that deuterium fractionation occurs during root water uptake and, alternatively, suggest that measurement bias inherent in the cryogenic extraction method is the root cause of δH depletion. We used a rehydration experiment to show that the stem water cryogenic extraction error could originate from a dynamic exchange between organically bound deuterium and liquid water during water extraction. In light of our finding, we suggest caution when partitioning plant water sources and reconstructing past climates using hydrogen isotopes, and carefully propose that the paradigm-shifting phenomenon of ecohydrological separation ("two water worlds") is underpinned by an extraction artifact.
Topics: Cold Temperature; Deuterium; Groundwater; Hydrogen; Hydrology; Oxygen Isotopes; Plant Stems; Plants; Time Factors; Water
PubMed: 33318208
DOI: 10.1073/pnas.2014422117 -
Biophysical Journal May 2015In this report, we applied site-specifically deuterated N-stearoylsphingomyelins (SSMs) to raft-exhibiting ternary mixtures containing SSM,...
In this report, we applied site-specifically deuterated N-stearoylsphingomyelins (SSMs) to raft-exhibiting ternary mixtures containing SSM, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and cholesterol (Chol) and successfully acquired deuterium quadrupole coupling profiles of SSM from liquid-ordered (Lo) and liquid-disordered (Ld) domains. To our knowledge, this is the first report that shows detailed lipid chain dynamics separately and simultaneously obtained from coexisting Lo and Ld domains. We also found that the quadrupole profile of the Lo phase in the ternary system was almost identical to that in the SSM-Chol binary mixture, suggesting that the order profile of the binary system is essentially applicable to more complicated membrane systems in terms of the acyl chain order. We also demonstrated that (2)H NMR spectroscopy, in combination with organic synthesis of deuterated components, could be used to reveal the accurate mole fractions of each component distributed in the Lo and Ld domains. As compared with the reported tie-line analysis of phase diagrams, the merit of our (2)H NMR analysis is that the domain-specific compositional fractions are directly attainable without experimental complexity and ambiguity. The accurate compositional distributions as well as lipid order profiles in ternary mixtures are relevant to understanding the molecular mechanism of lipid raft formation.
Topics: Cholesterol; Deuterium; Magnetic Resonance Spectroscopy; Membrane Microdomains; Phosphatidylcholines; Sphingomyelins
PubMed: 25992728
DOI: 10.1016/j.bpj.2015.04.008 -
Journal of Applied Microbiology 2003The aim was to develop reliable and economical protocols for the production of fully deuteriated biomolecules by bacteria. This required the preparation of...
AIMS
The aim was to develop reliable and economical protocols for the production of fully deuteriated biomolecules by bacteria. This required the preparation of deuterium-tolerant bacterial strains and an understanding of the physiological mechanisms of acquisition of deuterium tolerance.
METHODS AND RESULTS
We report here improved methods for the cultivation of Escherichia coli on fully deuteriated minimal medium. A multi-stage adaptation protocol was developed; this included repeated plating and selection of colonies and resulted in highly deuterium-tolerant cell cultures. Three E. coli strains, JM109, MRE600 and MRE600Rif, were adapted to growth on deuteriated succinate medium. This is the first report of JM109 being adapted to deuteriated minimal media. The adapted strains showed good, consistent growth rates and were capable of being transformed with plasmids. Expression of heterologous proteins in these strains was reliable and yields were consistently high (100-200 mg l-1). We also show that all E. coli cells are inherently capable of growth on deuteriated media.
CONCLUSIONS
We have developed a new adaptation protocol that resulted in three highly deuterium-tolerant E. coli strains. Deuterium-adapted cultures produced good yields of a deuteriated recombinant protein. We suggest that E. coli cells are inherently capable of growth on deuteriated media, but that non-specific mutations enhance deuterium tolerance. Thus plating and selection of colonies leads to highly deuterium-tolerant strains.
SIGNIFICANCE AND IMPACT OF STUDY
An understanding of the mechanism of adaptation of E. coli to growth on deuteriated media allows strategies for the development of disabled deuterium-tolerant strains suitable for high-level production of deuteriated recombinant proteins and other biomolecules. This is of particular importance for nuclear magnetic resonance and neutron scattering studies of biomolecules.
Topics: Adaptation, Physiological; Bacteriological Techniques; Culture Media; Deuterium; Drug Tolerance; Electroporation; Escherichia coli; Peptide Elongation Factor Tu; Recombinant Proteins; Transformation, Genetic
PubMed: 12631193
DOI: 10.1046/j.1365-2672.2003.01866.x -
Biochemistry Jan 2021Water is key to protein structure and stability, yet the relationship between protein-water interactions and structure is poorly understood, in part because there are...
Water is key to protein structure and stability, yet the relationship between protein-water interactions and structure is poorly understood, in part because there are few techniques that permit the study of dehydrated protein structure at high resolution. Here, we describe liquid-observed vapor exchange (LOVE) NMR, a solution NMR-based method that provides residue-level information about the structure of dehydrated proteins. Using the model protein GB1, we show that LOVE NMR measurements reflect the fraction of the dried protein population trapped in a conformation where a given residue is protected from exchange with DO vapor. Comparisons to solution hydrogen-deuterium exchange data affirm that the dried protein structure is strongly influenced by local solution stability and that the mechanism of dehydration protection exerted by the widely used protectant trehalose differs from its mechanism of stabilization in solution. Our results highlight the need for refined models of cosolute-mediated dehydration protection and demonstrate the ability of LOVE NMR to inform such models.
Topics: Amino Acids; Bacterial Proteins; Deuterium; Deuterium Exchange Measurement; Freeze Drying; Hydrogen; Magnetic Resonance Spectroscopy; Protein Conformation; Streptococcaceae
PubMed: 33400518
DOI: 10.1021/acs.biochem.0c00863 -
Anesthesiology Oct 1998Desflurane, enflurane and isoflurane can be degraded to carbon monoxide (CO) by carbon dioxide absorbents, whereas sevoflurane and halothane form negligible amounts of...
BACKGROUND
Desflurane, enflurane and isoflurane can be degraded to carbon monoxide (CO) by carbon dioxide absorbents, whereas sevoflurane and halothane form negligible amounts of CO. Carbon monoxide formation is greater with drier absorbent, and with barium hydroxide, than with soda lime. The mechanism, role of absorbent composition and water, and anesthetic structures determining CO formation are unknown. This investigation examined sequential steps in anesthetic degradation to CO.
METHODS
Carbon monoxide formation from anesthetics and desiccated barium hydroxide lime or soda lime was determined at equimole and equiMAC concentrations. Carbon monoxide formation from deuterium-substituted anesthetics was also quantified. Proton abstraction from anesthetics by strong base was determined by deuterium isotope exchange. A reactive chemical intermediate was trapped and identified by gas chromatography-mass spectrometry. The source of the oxygen in CO was identified by 18O incorporation.
RESULTS
Desflurane,enflurane,andisoflurane(difluoromethylethyl ethers), but not sevoflurane (monofluoromethyl ether), methoxyflurane (methy-ethyl ether), or halothane (alkane) were degraded to CO. The amount of CO formed was desflurane > or = enflurane > isoflurane at equiMAC and enflurane > desflurane > isoflurane at equimole concentrations. Proton abstraction from the difluoromethoxy carbon was greater with potassium than with sodium hydroxide, but unmeasurable with barium hydroxide. Carbon monoxide formation was correlated (r = 0.95-1.00) with difluoromethoxy (enflurane > desflurane > isoflurane > or = methoxyflurane = sevoflurane = 0) but not ethyl carbon proton abstraction. Deuterium substitution on enflurane and desflurane diminished CO formation. Chemical trapping showed formation of a difluorocarbene intermediate from enflurane and desflurane. Incorporation of H2(18)O in barium hydroxide lime resulted in C18O formation from unlabeled enflurane and desflurane.
CONCLUSIONS
A difluoromethoxy group is a structural requirement for haloether degradation to CO. Results are consistent with initial base-catalyzed difluoromethoxy proton abstraction (potassium > sodium hydroxide, thus greater CO formation with barium hydroxide lime vs. soda lime) forming a carbanion (reprotonated by water to regenerate the anesthetic, hence requirements for relatively dry absorbent), carbanion decomposition to a difluorocarbene, and subsequent difluorocarbene reaction to form CO.
Topics: Absorption; Anesthetics, Inhalation; Barium Compounds; Carbon Monoxide; Deuterium; Kinetics; Mass Spectrometry; Oxygen Isotopes; Protons
PubMed: 9778011
DOI: 10.1097/00000542-199810000-00018