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Journal of Biomolecular NMR Jun 2024Deuterium (H) spin relaxation of CHD methyl groups has been widely applied to investigate picosecond-to-nanosecond conformational dynamics in proteins by solution-state...
Deuterium (H) spin relaxation of CHD methyl groups has been widely applied to investigate picosecond-to-nanosecond conformational dynamics in proteins by solution-state NMR spectroscopy. The B dependence of the H spin relaxation rates is represented by a linear relationship between the spectral density function at three discrete frequencies J(0), J(ω) and J(2ω). In this study, the linear relation between H relaxation rates at B fields separated by a factor of two and the interpolation of rates at intermediate frequencies are combined for a more robust approach for spectral density mapping. The general usefulness of the approach is demonstrated on a fractionally deuterated (55%) and alternate C-C labeled sample of E. coli RNase H. Deuterium relaxation rate constants (R, R, R, R) were measured for 57 well-resolved CHD moieties in RNase H at H frequencies of 475 MHz, 500 MHz, 900 MHz, and 950 MHz. The spectral density mapping of the 475/950 MHz data combination was performed independently and jointly to validate the expected relationship between data recorded at B fields separated by a factor of two. The final analysis was performed by jointly analyzing 475/950 MHz rates with 700 MHz rates interpolated from 500/900 MHz data to yield six J(ω) values for each methyl peak. The J(ω) profile for each peak was fit to the original (τ, S, τ) or extended model-free function (τ, S, S, τ, τ) to obtain optimized dynamic parameters.
PubMed: 38856928
DOI: 10.1007/s10858-024-00443-w -
The Journal of Organic Chemistry Jun 2024Aromatic sulfones are the prevailing scaffolds in pharmaceutical and material sciences. However, compared to their widespread application, the selective deuterium...
Aromatic sulfones are the prevailing scaffolds in pharmaceutical and material sciences. However, compared to their widespread application, the selective deuterium labeling of these structures is restricted due to their electron-deficient properties. This study presents two comprehensive strategies for the deuteration of aromatic sulfones. The base-promoted deuteration uses DMSO- as the deuterium source, resulting in a rapid H/D exchange within 2 h. Meanwhile, a silver-catalyzed protocol offers a much milder option by using economical DO to furnish the labeled sulfones.
PubMed: 38856238
DOI: 10.1021/acs.joc.4c00352 -
International Journal of Mass... May 2024Capillary vibrating sharp-edge spray ionization (cVSSI) combined with hydrogen/deuterium exchange-mass spectrometry (HDX-MS) has been utilized to characterize different...
Capillary vibrating sharp-edge spray ionization (cVSSI) combined with hydrogen/deuterium exchange-mass spectrometry (HDX-MS) has been utilized to characterize different solution-phase DNA conformers including DNA G-quadruplex topologies as well as triplex DNA and duplex DNA. In general, G-quadruplex DNA shows a wide range of protection of hydrogens extending from ~12% to ~21% deuterium incorporation. Additionally, the DNA sequences selected to represent parallel, antiparallel, and hybrid G-quadruplex topologies exhibit slight differences in deuterium uptake levels which appear to loosely relate to overall conformer stability. Notably, the exchange level for one of the hybrid sequence sub topologies of G-quadruplex DNA (24 TTG) is significantly different (compared with the others studied here) despite the DNA sequences being highly comparable. For the quadruplex-forming sequences, correlation analysis suggests protection of base hydrogens involved in tetrad hydrogen bonding. For duplex DNA ~19% deuterium incorporation is observed while only ~16% is observed for triplex DNA. This increased protection of hydrogens may be due to the added backbone scaffolding and Hoogsteen base pairing of the latter species. These experiments lay the groundwork for future studies aimed at determining the structural source of this protection as well as the applicability of the approach for ascertaining different oligonucleotide folds, co-existing conformations, and/or overall conformer flexibility.
PubMed: 38854816
DOI: 10.1016/j.ijms.2024.117231 -
ACS Omega Jun 2024The mode of action of antibiotics can be broadly classified as bacteriostatic and bactericidal. The bacteriostatic mode leads to the arrested growth of the cells, while...
The mode of action of antibiotics can be broadly classified as bacteriostatic and bactericidal. The bacteriostatic mode leads to the arrested growth of the cells, while the bacteriocidal mode causes cell death. In this work, we report the applicability of deuterium stable isotope probing (DSIP) in combination with Raman spectroscopy (Raman DSIP) for discriminating the mode of action of antibiotics at the community level. a well-known model microbe, was used as an organism for the study. We optimized the concentration of deuterium oxide required for metabolic activity monitoring without compromising the microbial growth. Our findings suggest that changes in the intensity of the C-D band in the high-wavenumber region could serve as a quantifiable marker for determining the antibiotic mode of action. This can be used for early identification of the antibiotic's mode of action. Our results explore the new perspective that supports the utility of deuterium-based vibrational tags in the field of clinical spectroscopy. Understanding the antibiotic's mode of action on bacterial cells in a short and objective manner can significantly enhance the clinical management abilities of infectious diseases and may also help in personalized antimicrobial therapy.
PubMed: 38854576
DOI: 10.1021/acsomega.4c01666 -
Chemosphere Jun 2024Antimicrobial resistance (AMR) in oceans poses a significant threat to human health through the seafood supply chain. Ammonia-oxidizing archaea (AOA) are important...
Antimicrobial resistance (AMR) in oceans poses a significant threat to human health through the seafood supply chain. Ammonia-oxidizing archaea (AOA) are important marine microorganisms and play a key role in the biogeochemical nitrogen cycle around the world. However, the AMR of marine AOA to aquicultural antibiotics is poorly explored. Here, Raman-deuterium isotope probing (Raman-DIP), a single-cell tool, was developed to reveal the AMR of a typical marine species of AOA, Nitrosopumilus maritimus (designated SCM1), against six antibiotics, including erythromycin, tetracycline, novobiocin, neomycin, bacitracin, and vancomycin. The DO concentration (30% v/v) and culture period (9 days) were optimized for the precise detection of metabolic activity in SCM1 cells through Raman-DIP. The relative metabolic activity of SCM1 upon exposure to antibiotics was semi-quantitatively calculated based on single-cell Raman spectra. SCM1 exhibited high resistance to erythromycin, tetracycline, novobiocin, neomycin, and vancomycin, with minimum inhibitory concentration (MIC) values between 100 and 400 mg/L while SCM1 is very sensitive to bacitracin (MIC: 0.8 mg/L). Notably, SCM1 cells were completely inactive under the metabolic activity minimum inhibitory concentration conditions (MA-MIC: 1.6∼800 mg/L) for the six antibiotics. Further genomic analysis revealed the antibiotic resistance genes (ARGs) of SCM1 including 14 types categorized into 33 subtypes. This work increases our knowledge of the AMR of marine AOA by linking the resistant phenome to the genome, contributing to the risk assessment of AMR in the underexplored ocean environment. As antibiotic resistance in marine microorganisms is significantly affected by the concentration of antibiotics in coastal environments, we encourage more studies concentrating on both the phenotypic and genotypic antibiotic resistance of marine archaea. This may facilitate a comprehensive evaluation of the capacity of marine microorganisms to spread AMR and the implementation of suitable control measures to protect environmental safety and human health.
PubMed: 38852635
DOI: 10.1016/j.chemosphere.2024.142500 -
Journal of Colloid and Interface Science May 2024The solubilization of sodium diclofenac (Na-DFC) in a glycerol monooleate-based emulsion triggers series of structural changes. Incorporation of Na-DFC, leads to...
The solubilization of sodium diclofenac (Na-DFC) in a glycerol monooleate-based emulsion triggers series of structural changes. Incorporation of Na-DFC, leads to formation of a reverse hexagonal mesophase between 2 and 5 wt% Na-DFC. Between 6 and 9 wt% Na-DFC, the hexagonal symmetry gradually transitions to a disordered lamellar mesophase. These structural shifts impact the system's storage modulus, structuring enthalpy, and structural diffusivity. Despite these transitions, the driving force for Na-DFC release remains consistent, leading to hypothesize that the interfacial structure remains unchanged during Na-DFC release. The nano-structural modifications imposed by the Na-DFC load and release were assessed by small-angle X-ray diffraction (SAXD), spin-probe electron paramagnetic resonance (EPR), and nuclear quadrupole resonance (NQR). The selective solubilization of Na-DFC was demonstrated by SAXD peak fittings, revealing an increase of hexagonally oriented rods at the expense of non-oriented micelles, rather than gradual micellar elongation. Computation of the EPR spectra also showcased the selective solubilization of Na-DFC at an enhanced free energy interface (γ), evidenced by step-wise variations in polarity, microviscosity, and order parameters. Additionally, NQR analysis highlighted a higher anisotropy for sodium compared to deuterium, linking the selective solubilization of Na-DFC to heterogeneous structural transformations. These findings underscore the heterogeneous nature of solubilization-release processes, driven by locally increased micellar free energy. Consequently, the loaded Na-DFC interfaces maintain a constant γ, ensuring a consistent release driving force despite the structural transitions affecting the matrix. The ability to selectively solubilize guest molecules may herald a new era in the utilization of selective molecular interfacial loading.
PubMed: 38852356
DOI: 10.1016/j.jcis.2024.05.096 -
The Journal of Physical Chemistry. B Jun 2024The reaction of benzylsuccinate synthase, the radical-based addition of toluene to a fumarate cosubstrate, is initiated by hydrogen transfer from a conserved cysteine to...
The reaction of benzylsuccinate synthase, the radical-based addition of toluene to a fumarate cosubstrate, is initiated by hydrogen transfer from a conserved cysteine to the nearby glycyl radical in the active center of the enzyme. In this study, we analyze this step by comprehensive computer modeling, predicting (i) the influence of bound substrates or products, (ii) the energy profiles of forward- and backward hydrogen-transfer reactions, (iii) their kinetic constants and potential mechanisms, (iv) enantiospecificity differences, and (v) kinetic isotope effects. Moreover, we support several of the computational predictions experimentally, providing evidence for the predicted H/D-exchange reactions into the product and at the glycyl radical site. Our data indicate that the hydrogen transfer reactions between the active site glycyl and cysteine are principally reversible, but their rates differ strongly depending on their stereochemical orientation, transfer of protium or deuterium, and the presence or absence of substrates or products in the active site. This is particularly evident for the isotope exchange of the remaining protium atom of the glycyl radical to deuterium, which appears dependent on substrate or product binding, explaining why the exchange is observed in some, but not all, glycyl-radical enzymes.
Topics: Biocatalysis; Kinetics; Carbon-Sulfur Lyases; Catalytic Domain; Models, Molecular; Cysteine; Hydrogen; Free Radicals; Carbon-Carbon Lyases
PubMed: 38848492
DOI: 10.1021/acs.jpcb.4c01237 -
Inorganic Chemistry Jun 2024A series of ruthenium complexes () bearing new NNN-pincer ligands were synthesized in 58-78% yields. All of the complexes are air and moisture stable and were...
A series of ruthenium complexes () bearing new NNN-pincer ligands were synthesized in 58-78% yields. All of the complexes are air and moisture stable and were characterized by IR, NMR, and high-resolution mass spectra (HRMS). In addition, the structures of were confirmed by X-ray crystallographic analysis. These Ru(II) complexes exhibited high catalytic efficiency and broad functional group tolerance in the N-methylation reaction of amines using CHOH as both the C1 source and solvent. Experimental results indicated that the electronic effect of the substituents on the ligands considerably affects the catalytic reactivity of the complexes in which bearing an electron-donating OMe group showed the highest activity. Deuterium labeling and control experiments suggested that the dehydrogenation of methanol to generate ruthenium hydride species was the rate-determining step in the reaction. Furthermore, this protocol also provided a ready approach to versatile trideuterated -methylamines under mild conditions using CDOD as a deuterated methylating agent.
PubMed: 38848310
DOI: 10.1021/acs.inorgchem.4c01561 -
Heliyon Jun 2024Viral double-stranded RNA (dsRNA) is sensed by toll-like receptor 3 (TLR3) and retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), including melanoma...
Viral double-stranded RNA (dsRNA) is sensed by toll-like receptor 3 (TLR3) and retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), including melanoma differentiation-associated gene 5 (MDA5). MDA5 recognizes the genome of dsRNA viruses and replication intermediates of single-stranded RNA viruses. MDA5 also plays an important role in the development of autoimmune diseases, such as Aicardi-Goutieres syndrome and type I diabetes. Patients with dermatomyositis with serum MDA5 autoantibodies (anti-CADM-140) are known to have a high risk of developing rapidly progressive interstitial lung disease and poor prognosis. However, there have been no reports on the soluble form of MDA5 in human serum. In the present study, we generated in-house monoclonal antibodies (mAbs) against human MDA5. We then performed immunohistochemical analysis and sensitive sandwich immunoassays to detect the MDA5 protein using two different mAbs (clones H27 and H46). As per the immunohistochemical analysis, the MDA5 protein was moderately expressed in the alveolar epithelia of normal lungs and was strongly expressed in the cytoplasm of lymphoid cells in the tonsils and acinar cells of the pancreas. Interestingly, soluble MDA5 protein was detectable in the serum, but not in the urine, of healthy donors. Soluble MDA5 protein was also detectable in the serum of patients with dermatomyositis. Immunoblot analysis showed that human cells expressed a 120 kDa MDA5 protein, while the 60 kDa MDA5 protein increased in the supernatant of peripheral mononuclear cells within 15 min after MDA5 agonist/double-strand RNA stimulation. Hydrogen deuterium exchange mass spectrometry revealed that an anti-MDA5 mAb (clone H46) bound to the epitope (415QILENSLLNL424) derived from the helicase domain of MDA5. These results indicate that a soluble MDA5 protein containing the helicase domain of MDA5 could be rapidly released from the cytoplasm of tissues after RNA stimulation.
PubMed: 38845920
DOI: 10.1016/j.heliyon.2024.e31727 -
Food Chemistry Jun 2024Pineapple aroma is one of the most important sensory quality traits that influences consumer purchasing patterns. Reported in this paper is a high throughput method to...
Pineapple aroma is one of the most important sensory quality traits that influences consumer purchasing patterns. Reported in this paper is a high throughput method to quantify in a single analysis the key volatile organic compounds that contribute to the aroma of pineapple cultivars grown in Australia. The method constituted stable isotope dilution analysis in conjunction with headspace solid-phase microextraction coupled with gas-chromatography mass spectrometry. Deuterium labelled analogues of the target analytes purchased commercially were used as internal standards. Twenty-six volatile organic compounds were targeted for quantification and the resulting calibration functions of the matrix -matched validated method had determination coefficients (R) ranging from 0.9772 to 0.9999. The method was applied to identify the key aroma volatile compounds produced by popular pineapple cultivars such as 'Aus Carnival', 'Aus Festival', 'Aus Jubilee', 'Aus Smooth (Smooth Cayenne)' and 'Aussie Gold (73-50)', grown in Queensland, Australia. Pineapple cultivars varied in its content and composition of free volatile components, which were predominantly comprised of esters, followed by terpenes, alcohols, aldehydes, and ketones.
PubMed: 38843713
DOI: 10.1016/j.foodchem.2024.139956