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Function (Oxford, England) 2022Targeting chondrocyte dynamics is a strategy for slowing osteoarthritis progression during aging. We describe a stable-isotope method using in vivo deuterium oxide...
An In Vivo Stable Isotope Labeling Method to Investigate Individual Matrix Protein Synthesis, Ribosomal Biogenesis, and Cellular Proliferation in Murine Articular Cartilage.
Targeting chondrocyte dynamics is a strategy for slowing osteoarthritis progression during aging. We describe a stable-isotope method using in vivo deuterium oxide labeling and mass spectrometry to measure protein concentration, protein half-life, cell proliferation, and ribosomal biogenesis in a single sample of murine articular cartilage. We hypothesized that a 60-d labeling period would capture age-related declines in cartilage matrix protein content, protein synthesis rates, and cellular proliferation. Knee cartilage was harvested to the subchondral bone from 25- to 90-wk-old female C57BL/6J mice treated with deuterium oxide for 15, 30, 45, and 60 d. We measured protein concentration and half-lives using targeted high resolution accurate mass spectrometry and d2ome data processing software. Deuterium enrichment was quantified in isolated DNA and RNA to measure cell proliferation and ribosomal biogenesis, respectively. Most collagen isoforms were less abundant in aged animals, with negligible collagen synthesis at either age. In contrast, age altered the concentration and half-lives of many proteoglycans and other matrix proteins, including several with greater concentration and half-lives in older mice such as proteoglycan 4, clusterin, and fibronectin-1. Cellular proteins were less abundant in older animals, consistent with reduced cellularity. Nevertheless, deuterium was maximally incorporated into 60% of DNA and RNA by 15 d of labeling in both age groups, suggesting the presence of two large pools of either rapidly (<15 d) or slowly (>60 d) proliferating cells. Our findings indicate that age-associated changes in cartilage matrix protein content and synthesis occur without detectable changes in the relative number of proliferating cells.
Topics: Mice; Animals; Female; Cartilage, Articular; Matrilin Proteins; Isotope Labeling; Deuterium Oxide; Deuterium; Mice, Inbred C57BL; Collagen; Cell Proliferation; DNA; Protein Biosynthesis; RNA
PubMed: 35399495
DOI: 10.1093/function/zqac008 -
Journal of Biomedical Optics Nov 2023Changes in lipid, water, and collagen (LWC) content in tissue are associated with numerous medical abnormalities (cancer, atherosclerosis, and Alzheimer's disease)....
SIGNIFICANCE
Changes in lipid, water, and collagen (LWC) content in tissue are associated with numerous medical abnormalities (cancer, atherosclerosis, and Alzheimer's disease). Standard imaging modalities are limited in resolution, specificity, and/or penetration for quantifying these changes. Short-wave infrared (SWIR) photoacoustic imaging (PAI) has the potential to overcome these challenges by exploiting the unique optical absorption properties of .
AIM
This study's aim is to harness SWIR PAI for mapping LWC changes in tissue. The focus lies in devising a reflection-mode PAI technique that surmounts current limitations related to SWIR light delivery.
APPROACH
To enhance light delivery for reflection-mode SWIR PAI, we designed a deuterium oxide (, "heavy water") gelatin (HWG) interface for opto-acoustic coupling, intended to significantly improve light transmission above 1200 nm.
RESULTS
HWG permits light delivery up to 1850 nm, which was not possible with water-based coupling ( light delivery up to 1350 nm). PAI using the HWG interface and the Visualsonics Vevo LAZR-X reveals a signal increase up to 24 dB at 1720 nm in lipid-rich regions.
CONCLUSIONS
By overcoming barriers related to light penetration, the HWG coupling interface enables accurate quantification/monitoring of biomarkers like LWC using reflection-mode PAI. This technological stride offers potential for tracking changes in chronic diseases () and evaluating their responses to therapeutic interventions.
Topics: Deuterium Oxide; Photoacoustic Techniques; Diagnostic Imaging; Water; Lipids
PubMed: 38078156
DOI: 10.1117/1.JBO.28.11.116001 -
The Journal of General Physiology Jul 2007Determining the mechanisms of flux through protein channels requires a combination of structural data, permeability measurement, and molecular dynamics (MD) simulations....
Determining the mechanisms of flux through protein channels requires a combination of structural data, permeability measurement, and molecular dynamics (MD) simulations. To further clarify the mechanism of flux through aquaporin 1 (AQP1), osmotic p(f) (cm(3)/s/pore) and diffusion p(d) (cm(3)/s/pore) permeability coefficients per pore of H(2)O and D(2)O in AQP1 were calculated using MD simulations. We then compared the simulation results with experimental measurements of the osmotic AQP1 permeabilities of H(2)O and D(2)O. In this manner we evaluated the ability of MD simulations to predict actual flux results. For the MD simulations, the force field parameters of the D(2)O model were reparameterized from the TIP3P water model to reproduce the experimentally observed difference in the bulk self diffusion constants of H(2)O vs. D(2)O. Two MD systems (one for each solvent) were constructed, each containing explicit palmitoyl-oleoyl-phosphatidyl-ethanolamine (POPE) phospholipid molecules, solvent, and AQP1. It was found that the calculated value of p(f) for D(2)O is approximately 15% smaller than for H(2)O. Bovine AQP1 was reconstituted into palmitoyl-oleoyl-phosphatidylcholine (POPC) liposomes, and it was found that the measured macroscopic osmotic permeability coefficient P(f) (cm/s) of D(2)O is approximately 21% lower than for H(2)O. The combined computational and experimental results suggest that deuterium oxide permeability through AQP1 is similar to that of water. The slightly lower observed osmotic permeability of D(2)O compared to H(2)O in AQP1 is most likely due to the lower self diffusion constant of D(2)O.
Topics: Animals; Aquaporin 1; Cattle; Cell Membrane Permeability; Computer Simulation; Deuterium Oxide; Models, Chemical; Models, Molecular; Protein Conformation; Reproducibility of Results; Water
PubMed: 17591989
DOI: 10.1085/jgp.200709810 -
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 -
Protein Science : a Publication of the... Sep 2018The stability of a protein is vital for its biological function, and proper folding is partially driven by intermolecular interactions between protein and water. In many...
The stability of a protein is vital for its biological function, and proper folding is partially driven by intermolecular interactions between protein and water. In many studies, H O is replaced by D O because H O interferes with the protein signal. Even this small perturbation, however, affects protein stability. Studies in isotopic waters also might provide insight into the role of solvation and hydrogen bonding in protein folding. Here, we report a complete thermodynamic analysis of the reversible, two-state, thermal unfolding of the metastable, 7-kDa N-terminal src-homology 3 domain of the Drosophila signal transduction protein drk in H O and D O using one-dimensional F NMR spectroscopy. The stabilizing effect of D O compared with H O is enthalpic and has a small to insignificant effect on the temperature of maximum stability, the entropy, and the heat capacity of unfolding. We also provide a concise summary of the literature about the effects of D O on protein stability and integrate our results into this body of data.
Topics: Animals; Deuterium Oxide; Drosophila; Drosophila Proteins; Protein Stability; Thermodynamics; src Homology Domains
PubMed: 30052291
DOI: 10.1002/pro.3477 -
BMB Reports Jan 2008The present study was aimed to elucidate the mechanism of stabilization of tubulin by deuterium oxide (D(2)O). Rate of decrease of tryptophan fluorescence during aging...
The present study was aimed to elucidate the mechanism of stabilization of tubulin by deuterium oxide (D(2)O). Rate of decrease of tryptophan fluorescence during aging of tubulin at 4 degrees C and 37 degrees C was significantly lower in D(2)O than in H(2)O. Circular dichroism spectra of tubulin after incubation at 4 degrees C, suggested that complete stabilization of the secondary structure in D(2)O during the first 24 hours of incubation. The number of available cysteine measured by DTNB reaction was decreased to a lesser extent in D(2)O than in H(2)O. During the increase in temperature of tubulin, the rate of decrease of fluorescence at 335 nm and change of CD value at 222 nm was lesser in D(2)O. Differential Scanning calorimetric experiments showed that the T(m) values for tubulin unfolding in D(2)O were 58.6 degrees C and 62.17 degrees C, and in H(2)O those values were 55.4 degrees C and 59.35 degrees C.
Topics: Animals; Brain Chemistry; Calorimetry, Differential Scanning; Circular Dichroism; Deuterium Oxide; Fluorescence; Goats; Molecular Conformation; Protein Folding; Protein Structure, Secondary; Temperature; Tubulin
PubMed: 18304452
DOI: 10.5483/bmbrep.2008.41.1.062 -
Scientific Reports Jun 2020Increased glucose uptake and aerobic glycolysis are striking features of many cancers. These features have led to many techniques for screening and diagnosis, but many... (Comparative Study)
Comparative Study
Increased glucose uptake and aerobic glycolysis are striking features of many cancers. These features have led to many techniques for screening and diagnosis, but many are expensive, less feasible or have harmful side-effects. Here, we report a sensitive H/H NMR method to measure the kinetics of lactate isotopomer and HDO production using a deuterated tracer. To test this hypothesis, HUH-7 hepatocellular carcinoma and AML12 normal hepatocytes were incubated with [H]glucose. H/H NMR data were recorded for cell media as a function of incubation time. The efflux rate of lactate-CH, lactate-CHD and lactate-CHD was calculated as 0.0033, 0.0071, and 0.0.012 µmol/10cells/min respectively. Differential production of lactate isotopomers was due to deuterium loss during glycolysis. Glucose uptake and HDO production by HUH-7 cells showed a strong correlation, indicating that monitoring the HDO production could be a diagnostic feature in cancers. Deuterium mass balance of [H]glucose uptake to H-lactate and HDO production is quantitatively matched, suggesting increasing HDO signal could be used to diagnose Warburg (cancer) metabolism. Measuring the kinetics of lactate isotopomer and HDO production by H and H MR respectively are highly sensitive. Increased T of H-lactate isotopomers indicates inversion/saturation recovery methods may be a simple means of generating metabolism-based contrast.
Topics: Aerobiosis; Carcinoma, Hepatocellular; Cell Line, Tumor; Culture Media; Deuterium Oxide; Early Detection of Cancer; Glucose; Glycolysis; Humans; Lactic Acid; Liver Neoplasms; Proton Magnetic Resonance Spectroscopy
PubMed: 32483190
DOI: 10.1038/s41598-020-65839-8 -
Chemical & Pharmaceutical Bulletin 2020The high-order functions of molecular capture and chiral recognition of tea gallated catechins (-)-epigallocatechin-3-O-gallate (EGCg) in water were investigated. A... (Review)
Review
The high-order functions of molecular capture and chiral recognition of tea gallated catechins (-)-epigallocatechin-3-O-gallate (EGCg) in water were investigated. A solution of equimolar amounts of a variety of heterocyclic compounds and EGCg in water afforded adhesive precipitates containing the heterocyclic compounds and EGCg at a molar ratio of 1 : 1, based on the integrated value of NMR proton signals. The molecular capture abilities of a variety of heterocyclic compounds using EGCg from the aqueous solutions were evaluated with the ratios of the heterocyclic compounds included in the precipitates of EGCg complex to the total heterocyclic compounds used. In the H-NMR spectrum of a solution containing cyclo(L-Pro-Gly), cyclo(D-Pro-Gly), and EGCg in a DO solution, a difference in the chemical shift of the H-NMR signal for some protons of the Pro residue was observed. Judging from the crystal structures of the 2 : 2 EGCg complexes of cyclo(L-Pro-Gly), cyclo(D-Pro-Gly), the difference in the chemical shift derived mainly from a magnetic anisotropic shielding effect by the ring current from the B ring of EGCg.In the H-NMR spectrum of a solution containing the pharmaceuticals racemic (R,S)-propranolol, (R,S)-diprophylline, (R,S)-proxyphylline and EGCg in DO, splitting of the H-NMR signals of the pharmaceuticals was observed. It was suggested that the pharmaceuticals formed diastereomers of EGCg complexes, as a result chirality of the pharmaceuticals was recognized by EGCg in the DO solution.
Topics: Catechin; Deuterium Oxide; Drug Development; Hydrophobic and Hydrophilic Interactions; Molecular Structure; Stereoisomerism
PubMed: 33268646
DOI: 10.1248/cpb.c20-00197 -
International Breastfeeding Journal Jun 2020Evidence of interventions that are effective in improving exclusive breastfeeding (EBF) practices is needed to help countries revise their strategies. To assess whether... (Comparative Study)
Comparative Study
Participation in the "nutrition at the Centre" project through women's group improved exclusive breastfeeding practices, as measured by the deuterium oxide dose-to-mother technique.
BACKGROUND
Evidence of interventions that are effective in improving exclusive breastfeeding (EBF) practices is needed to help countries revise their strategies. To assess whether mothers who had participated in the Nutrition at the Centre (N@C) project effectively demonstrated better EBF practices than did those who did not participate, we documented the processes of this nutritional intervention in Benin.
METHODS
This study was a cross-sectional design comparing the intervention group, namely, the Village Saving and Loan Association (VSLA-N@C), to the control group. The N@C project was an educational intervention based on behavioural and social changes related to nutrition. Through VSLA groups installed in communities, mothers were connected to the project; had weekly discussions around the process, benefits and challenges linked to EBF, and advocated during Breastfeeding Week celebrations. The study participants were mothers with children aged 4-5.5 months from the VSLA-N@C group (n = 53) and mothers (n = 50) from non-intervention areas who served as controls. With the deuterium oxide dose-to-mother technique, we quantified human milk intake (HMI) and non-milk oral intake (NMOI) and compared both groups using Student's t-test. A child is considered to be exclusively breastfed if the NMOI is less than 86.6 g/day. Multivariate regression logistics adjusted for VSLA membership, mothers' body mass index, and children's age, weight-for-age and weight-for-length, thus enabling us to measure differences in EBF rates.
RESULTS
Children of mothers from the VSLA-N@C group consumed significantly more human milk than those of mothers in the control group (900.2 ± 152.5 g/day vs 842.2 ± 188.6 g/day, P = 0.044). Children in the VSLA-N@C group had significantly less non-milk oral intake than did those in the control group (difference: 148.2 g/day, P = 0.000). Therefore, the EBF rate was significantly higher in the VSLA group (38% vs 8%, P < 0.0001), and mothers in VSLAs were 14 times more likely to practise EBF than were those in the control group (adjusted odds ratio [AOR] = 13.9, 95% CI 1.9-116.5, P = 0.015).
CONCLUSION
The EBF rate was significantly higher in the group of mothers who participated in the VSLA-N@C project than in those who did not receive the intervention. The N@C model could be promoted as a strategy for increasing EBF practices in poor and rural contexts, where it is possible to organize mothers into VSLA groups to discuss the process, benefits and challenges of EBF.
Topics: Adolescent; Adult; Benin; Breast Feeding; Child, Preschool; Cross-Sectional Studies; Deuterium Oxide; Dose-Response Relationship, Drug; Female; Health Promotion; Humans; Infant; Infant Food; Male; Milk, Human; Mothers; Young Adult
PubMed: 32590987
DOI: 10.1186/s13006-020-00302-y -
Proteomics. Clinical Applications Aug 2014Monitoring protein dynamics, compared to measuring static protein expression profiles taken with snapshot evaluations, have recently been the focus of proteomics studies...
Monitoring protein dynamics, compared to measuring static protein expression profiles taken with snapshot evaluations, have recently been the focus of proteomics studies examining tissue or blood samples where time course changes occur. Using deuterium oxide ((2) H2 O) to label amino acids is a useful method to monitor protein turnover rates. The synthesis rate for individual proteins is calculated from the rate of (2) H incorporation into specific proteins analyzed by high resolution MS. In this issue, Wang and colleagues measured the plasma protein turnover dynamics in healthy humans by in vivo (2) H2 O labeling [Wang, D. et al., Proteomics Clin. Appl. 2014, 8, 610-619]. The authors developed and validated a safe and accessible (2) H2 O administration protocol to record the turnover dynamics of 542 plasma proteins using MS. Their study demonstrates a promising new way to evaluate plasma protein dynamics in clinical trials where such knowledge could help for prognosis and evaluating treatment efficacy.
Topics: Animals; Blood Proteins; Deuterium Oxide; Humans; Isotope Labeling; Proteomics; Rats; Safety
PubMed: 25044642
DOI: 10.1002/prca.201400066