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Angewandte Chemie (International Ed. in... Oct 2021The template-directed synthesis of RNA played an important role in the transition from prebiotic chemistry to the beginnings of RNA based life, but the mechanism of RNA...
The template-directed synthesis of RNA played an important role in the transition from prebiotic chemistry to the beginnings of RNA based life, but the mechanism of RNA copying chemistry is incompletely understood. We measured the kinetics of template copying with a set of primers with modified 3'-nucleotides and determined the crystal structures of these modified nucleotides in the context of a primer/template/substrate-analog complex. pH-rate profiles and solvent isotope effects show that deprotonation of the primer 3'-hydroxyl occurs prior to the rate limiting step, the attack of the alkoxide on the activated phosphate of the incoming nucleotide. The analogs with a E ribose conformation show the fastest formation of 3'-5' phosphodiester bonds. Among those derivatives, the reaction rate is strongly correlated with the electronegativity of the 2'-substituent. We interpret our results in terms of differences in steric bulk and charge distribution in the ground vs. transition states.
Topics: Arabinose; Crystallography, X-Ray; DNA Primers; Deuterium Oxide; Imidazoles; Kinetics; Nucleic Acid Conformation; Nucleotides; RNA; Structure-Activity Relationship; Templates, Genetic; Water
PubMed: 34428345
DOI: 10.1002/anie.202109714 -
Analytical Chemistry Jun 2023Hydrogen-deuterium exchange mass spectrometry (HDX/MS) is increasingly used to study the dynamics of protein conformation. Coupled to native MS, HDX can also...
Hydrogen-deuterium exchange mass spectrometry (HDX/MS) is increasingly used to study the dynamics of protein conformation. Coupled to native MS, HDX can also characterize the conformations of oligonucleotides and their binding to cations, small molecules, and proteins. Data processing and visualization of native HDX/MS of oligonucleotides requires dedicated software solutions. OligoR is a web-browser-based application that addresses the specific needs of DNA HDX/MS and native MS experiments from raw data in an open format to visualization and export of results. Whole experiments spanning many time points can be processed in minutes for several mass-separated species. To access valuable folding dynamics information, we have developed a simple and robust approach to deconvolute bimodal isotope distributions, even when they are highly overlapping. This approach is based on modeling physically possible isotope distributions determined from chemical formulae and could be extended to any type of analyte (proteins, peptides, sugars, and small molecules). All results are presented in interactive data tables, and publication-quality figures can be generated, customized, and exported.
Topics: Oligonucleotides; Deuterium Exchange Measurement; Hydrogen Deuterium Exchange-Mass Spectrometry; Proteins; Peptides; Protein Conformation
PubMed: 37310448
DOI: 10.1021/acs.analchem.3c01321 -
Scientific Reports Sep 2023In vivo deuterated water (HO) labeling leads to deuterium (H) incorporation into biomolecules of proliferating cells and provides the basis for its use in cell kinetics...
In vivo deuterated water (HO) labeling leads to deuterium (H) incorporation into biomolecules of proliferating cells and provides the basis for its use in cell kinetics research. We hypothesized that rapidly proliferating cancer cells would become preferentially labeled with H and, therefore, could be visualized by deuterium magnetic resonance imaging (dMRI) following a brief period of in vivo systemic HO administration. We initiated systemic HO administration in two xenograft mouse models harboring either human colorectal, HT-29, or pancreatic, MiaPaCa-2, tumors and HO level of ~ 8% in total body water (TBW). Three schemas of HO administration were tested: (1) starting at tumor seeding and continuing for 7 days of in vivo growth with imaging on day 7, (2) starting at tumor seeding and continuing for 14 days of in vivo growth with imaging on day 14, and (3) initiation of labeling following a week of in vivo tumor growth and continuing until imaging was performed on day 14. Deuterium chemical shift imaging of the tumor bearing limb and contralateral control was performed on either day 7 of 14 after tumor seeding, as described. After 14 days of in vivo tumor growth and 7 days of systemic labeling with HO, a clear deuterium contrast was demonstrated between the xenografts and normal tissue. Labeling in the second week after tumor implantation afforded the highest contrast between neoplastic and healthy tissue in both models. Systemic labeling with HO can be used to create imaging contrast between tumor and healthy issue, providing a non-radioactive method for in vivo cancer imaging.
Topics: Humans; Animals; Mice; Heterografts; Deuterium; Magnetic Resonance Imaging; Transplantation, Heterologous; Administration, Cutaneous; Disease Models, Animal; Neoplasm Seeding
PubMed: 37679461
DOI: 10.1038/s41598-023-41163-9 -
Angewandte Chemie (International Ed. in... Nov 2023Deuterium labelling of organic compounds is an important process in chemistry. We report the first example of photocatalytic dehalogenative deuteration of both...
Deuterium labelling of organic compounds is an important process in chemistry. We report the first example of photocatalytic dehalogenative deuteration of both arylhalides and alkylhalides (40 substrates) over a metal-organic framework, MFM-300(Cr), using CD CN as the deuterium source at room temperature. MFM-300(Cr) catalyses high deuterium incorporation and shows excellent tolerance to various functional groups. Synchrotron X-ray powder diffraction reveals the activation of halogenated substrates via confined binding within MFM-300(Cr). In situ electron paramagnetic resonance spectroscopy confirms the formation of carbon-based radicals as intermediates and reveals the reaction pathway. This protocol removes the use of precious-metal catalysts from state-of-the-art processes based upon direct hydrogen isotope exchange and shows high photocatalytic stability, thus enabling multiple catalytic cycles.
PubMed: 37783657
DOI: 10.1002/anie.202306267 -
Nutrients Mar 2022The evaluation of body composition (BC) is relevant in the evaluation of children's health-disease states. Different methods and devices are used to estimate BC. The...
Body Composition Assessment in Mexican Children and Adolescents. Part 1: Comparisons between Skinfold-Thickness, Dual X-ray Absorptiometry, Air-Displacement Plethysmography, Deuterium Oxide Dilution, and Magnetic Resonance Imaging with the 4-C Model.
The evaluation of body composition (BC) is relevant in the evaluation of children's health-disease states. Different methods and devices are used to estimate BC. The availability of methods and the clinical condition of the patient usually defines the ideal approach to be used. In this cross-sectional study, we evaluate the accuracy of different methods to estimate BC in Mexican children and adolescents, using the 4-C model as the reference. In a sample of 288 Mexican children and adolescents, 4-C body composition assessment, skinfold-thickness (SF), dual-energy X-ray absorptiometry (DXA), air displacement plethysmography (ADP), and deuterium dilution (DO) were performed, along with MRI in a subsample (52 participants). The analysis of validity was performed by correlation analysis, linear regression, and the Bland-Altman method. All methods analyzed showed strong correlations for FM with 4-C values and between each other; however, DXA and MRI overestimated FM, whereas skinfolds and ADP under-estimated FM. Conclusion: The clinical assessment of BC by means of SF, ADP, DXA, MRI and DO correlated well with the 4-C model and between them, providing evidence of their clinical validity and utility. The results from different methods are not interchangeable. Preference between methods may depend on their availability and the specific clinical setting.
Topics: Absorptiometry, Photon; Adolescent; Body Composition; Child; Cross-Sectional Studies; Deuterium Oxide; Humans; Magnetic Resonance Imaging; Plethysmography
PubMed: 35268047
DOI: 10.3390/nu14051073 -
Frontiers in Immunology 2022Although computational structure prediction has had great successes in recent years, it regularly fails to predict the interactions of large protein complexes with... (Review)
Review
Although computational structure prediction has had great successes in recent years, it regularly fails to predict the interactions of large protein complexes with residue-level accuracy, or even the correct orientation of the protein partners. The performance of computational docking can be notably enhanced by incorporating experimental data from structural biology techniques. A rapid method to probe protein-protein interactions is hydrogen-deuterium exchange mass spectrometry (HDX-MS). HDX-MS has been increasingly used for epitope-mapping of antibodies (Abs) to their respective antigens (Ags) in the past few years. In this paper, we review the current state of HDX-MS in studying protein interactions, specifically Ab-Ag interactions, and how it has been used to inform computational structure prediction calculations. Particularly, we address the limitations of HDX-MS in epitope mapping and techniques and protocols applied to overcome these barriers. Furthermore, we explore computational methods that leverage HDX-MS to aid structure prediction, including the computational simulation of HDX-MS data and the combination of HDX-MS and protein docking. We point out challenges in interpreting and incorporating HDX-MS data into Ab-Ag complex docking and highlight the opportunities they provide to build towards a more optimized hybrid method, allowing for more reliable, high throughput epitope identification.
Topics: Antigen-Antibody Complex; Deuterium; Deuterium Exchange Measurement; Epitopes; Hydrogen Deuterium Exchange-Mass Spectrometry; Mass Spectrometry; Proteins
PubMed: 35720345
DOI: 10.3389/fimmu.2022.859964 -
The Journal of Nutrition May 2023Eating disorders (EDs) compromise individuals' nutritional status, affecting, among other organs and systems, bone health.
Body Composition Assessment in Adult Females with Anorexia Nervosa and Bulimia Nervosa-A Cross-Sectional Study Comparing Dual-Energy X-Ray Absorptiometry Scan and Isotopic Dilution of Deuterium.
BACKGROUND
Eating disorders (EDs) compromise individuals' nutritional status, affecting, among other organs and systems, bone health.
OBJECTIVES
This study aimed to assess and compare bone mineral density (BMD) from DXA scan and deuterium (DO) dilution of adult females with anorexia nervosa (AN) and bulimia nervosa (BN).
METHODS
This was a cross-sectional study with 53 female participants (18-49 y) with a diagnosis of AN (n = 25) or BN (n = 28). DXA scan was performed to assess BMD, FM, and FFM, and DO dilution was used to assess total body water (TBW), FM, and FFM. Interviews/questionnaires were used to assess symptoms, illness trajectory, and physical activity. t-test, chi-square test, Pearson's linear correlation, linear regressions, and Bland-Altman analyses were performed, with a significance level of 5%.
RESULTS
TBW below the recommended level for adult females (≥ 45%) was more frequent in BN (60%) compared with AN (21%; P = 0.013). FM index (FMI) (soft tissue only) (t-test P = 0.06), and FFM index (FFMI) (t-test P = 0.08) agreed between DXA scan and DO dilution. Only FFMI did not show systematic bias of proportion (β: -0.2, P = 0.177). The diagnosis of BN, binge-eating episodes, and physical activity in AN were associated with the differences in the methods' results. FMI was positively associated with BMD in AN, and both FMI and FFMI were positively associated with BMD in BN.
CONCLUSIONS
In adult females with EDs, DXA scan and DO dilution achieved agreement for FMI and FFMI. Changes in FM and FFM are important in understanding the mechanisms behind bone loss in EDs. Protocols for body composition assessment in EDs can help to minimize the effect of the ED diagnosis, ED behaviors (that is, excessive exercise and purging behaviors), and weight on the accuracy of measurements.
Topics: Humans; Adult; Female; Absorptiometry, Photon; Deuterium; Cross-Sectional Studies; Anorexia Nervosa; Bulimia Nervosa; Body Composition
PubMed: 36934952
DOI: 10.1016/j.tjnut.2023.03.019 -
Methods in Enzymology 2022Hydrogen deuterium exchange coupled to mass spectrometry (HDX-MS) is a valuable technique to investigate the dynamics of protein systems. The approach compares the...
Hydrogen deuterium exchange coupled to mass spectrometry (HDX-MS) is a valuable technique to investigate the dynamics of protein systems. The approach compares the deuterium uptake of protein backbone amides under multiple conditions to characterize protein conformation and interaction. HDX-MS is versatile and can be applied to diverse ligands, however, challenges remain when it comes to exploring complexes containing nucleic acids. In this chapter, we present procedures for the optimization and application of HDX-MS to studying RNA-binding proteins and use the RNA helicase Mtr4 as a demonstrative example. We highlight considerations in designing on-exchange, bottom-up, comparative studies on proteins with RNA. Our protocol details preliminary testing and optimization of experimental parameters. Difficulties arising from the inclusion of RNA, such as signal repression and sample carryover, are addressed. We discuss how chromatography parameters can be adjusted depending on the issues presented by the RNA, emphasizing reproducible peptide recovery in the absence and presence of RNA. Methods for visualization of HDX data integrated with statistical analysis are also reviewed with examples. These protocols can be applied to future studies of various RNA-protein complexes.
Topics: Deuterium; Deuterium Exchange Measurement; Hydrogen; Hydrogen Deuterium Exchange-Mass Spectrometry; Mass Spectrometry; Proteins; RNA
PubMed: 35965017
DOI: 10.1016/bs.mie.2022.04.002 -
Advances and prospects in deuterium metabolic imaging (DMI): a systematic review of in vivo studies.European Radiology Experimental Jun 2024Deuterium metabolic imaging (DMI) has emerged as a promising non-invasive technique for studying metabolism in vivo. This review aims to summarize the current... (Review)
Review
BACKGROUND
Deuterium metabolic imaging (DMI) has emerged as a promising non-invasive technique for studying metabolism in vivo. This review aims to summarize the current developments and discuss the futures in DMI technique in vivo.
METHODS
A systematic literature review was conducted based on the PRISMA 2020 statement by two authors. Specific technical details and potential applications of DMI in vivo were summarized, including strategies of deuterated metabolites detection, deuterium-labeled tracers and corresponding metabolic pathways in vivo, potential clinical applications, routes of tracer administration, quantitative evaluations of metabolisms, and spatial resolution.
RESULTS
Of the 2,248 articles initially retrieved, 34 were finally included, highlighting 2 strategies for detecting deuterated metabolites: direct and indirect DMI. Various deuterated tracers (e.g., [6,6'-H2]glucose, [2,2,2'-H3]acetate) were utilized in DMI to detect and quantify different metabolic pathways such as glycolysis, tricarboxylic acid cycle, and fatty acid oxidation. The quantifications (e.g., lactate level, lactate/glutamine and glutamate ratio) hold promise for diagnosing malignancies and assessing early anti-tumor treatment responses. Tracers can be administered orally, intravenously, or intraperitoneally, either through bolus administration or continuous infusion. For metabolic quantification, both serial time point methods (including kinetic analysis and calculation of area under the curves) and single time point quantifications are viable. However, insufficient spatial resolution remains a major challenge in DMI (e.g., 3.3-mL spatial resolution with 10-min acquisition at 3 T).
CONCLUSIONS
Enhancing spatial resolution can facilitate the clinical translation of DMI. Furthermore, optimizing tracer synthesis, administration protocols, and quantification methodologies will further enhance their clinical applicability.
RELEVANCE STATEMENT
Deuterium metabolic imaging, a promising non-invasive technique, is systematically discussed in this review for its current progression, limitations, and future directions in studying in vivo energetic metabolism, displaying a relevant clinical potential.
KEY POINTS
• Deuterium metabolic imaging (DMI) shows promise for studying in vivo energetic metabolism. • This review explores DMI's current state, limits, and future research directions comprehensively. • The clinical translation of DMI is mainly impeded by limitations in spatial resolution.
Topics: Humans; Deuterium; Animals
PubMed: 38825658
DOI: 10.1186/s41747-024-00464-y -
Communications Biology Jun 2022Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a technique to explore differential protein structure by examining the rate of deuterium incorporation for...
Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a technique to explore differential protein structure by examining the rate of deuterium incorporation for specific peptides. This rate will be altered upon structural perturbation and detecting significant changes to this rate requires a statistical test. To determine rates of incorporation, HDX-MS measurements are frequently made over a time course. However, current statistical testing procedures ignore the correlations in the temporal dimension of the data. Using tools from functional data analysis, we develop a testing procedure that explicitly incorporates a model of hydrogen deuterium exchange. To further improve statistical power, we develop an empirical Bayes version of our method, allowing us to borrow information across peptides and stabilise variance estimates for low sample sizes. Our approach has increased power, reduces false positives and improves interpretation over linear model-based approaches. Due to the improved flexibility of our method, we can apply it to a multi-antibody epitope-mapping experiment where current approaches are inapplicable due insufficient flexibility. Hence, our approach allows HDX-MS to be applied in more experimental scenarios and reduces the burden on experimentalists to produce excessive replicates. Our approach is implemented in the R-package "hdxstats": https://github.com/ococrook/hdxstats .
Topics: Bayes Theorem; Deuterium; Deuterium Exchange Measurement; Hydrogen Deuterium Exchange-Mass Spectrometry; Mass Spectrometry; Peptides
PubMed: 35705679
DOI: 10.1038/s42003-022-03517-3