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Journal of Magnetic Resonance Open Dec 2023In light of the growing interest deuterium metabolic imaging, hyperpolarized C, N, He, and Xe imaging, as well as P spectroscopy and imaging in large animals on...
In light of the growing interest deuterium metabolic imaging, hyperpolarized C, N, He, and Xe imaging, as well as P spectroscopy and imaging in large animals on clinical MR scanners, we demonstrate the use of a (radio)frequency converter system to allow X-nuclear MR spectroscopy (MRS) and MR imaging (MRI) on standard clinical MRI scanners without multinuclear capability. This is not only an economical alternative to the multinuclear system (MNS) provided by the scanner vendors, but also overcomes the frequency bandwidth problem of some vendor-provided MNSs that prohibit users from applications with X-nuclei of low magnetogyric ratio, such as deuterium (6.536 MHz/Tesla) and N (-4.316 MHz/Tesla). Here we illustrate the design of the frequency converter system and demonstrate its feasibility for P (17.235 MHz/Tesla), C (10.708 MHz/Tesla), and N MRS and MRI on a clinical MRI scanner without vendor-provided multinuclear hardware.
PubMed: 38046796
DOI: 10.1016/j.jmro.2023.100118 -
BioRxiv : the Preprint Server For... Feb 2024The G protein-coupled metabotropic glutamate receptors form homodimers and heterodimers with highly diverse responses to glutamate and varying physiological function....
The G protein-coupled metabotropic glutamate receptors form homodimers and heterodimers with highly diverse responses to glutamate and varying physiological function. The molecular basis for this diversity remains poorly delineated. We employ molecular dynamics, single-molecule spectroscopy, and hydrogen-deuterium exchange to dissect the pathway of activation triggered by glutamate. We find that activation entails multiple loosely coupled steps and identify a novel pre-active intermediate whose transition to the active state forms dimer interactions that set signaling efficacy. Such subunit interactions generate functional diversity that differs across homodimers and heterodimers. The agonist-bound receptor is remarkably dynamic, with low occupancy of G protein-coupling conformations, providing considerable headroom for modulation of the landscape by allosteric ligands. Sites of sequence diversity within the dimerization interface and diverse coupling between activation rearrangements may contribute to precise decoding of glutamate signals and transients over broad spatial and temporal scales.
PubMed: 38464305
DOI: 10.1101/2024.02.28.582567 -
Clinical Nutrition (Edinburgh, Scotland) Jan 2024Athletes vary in hydration status due to ongoing training regimes, diet demands, and extreme exertion. With water being one of the largest body composition compartments,...
BACKGROUND
Athletes vary in hydration status due to ongoing training regimes, diet demands, and extreme exertion. With water being one of the largest body composition compartments, its variation can cause misinterpretation of body composition assessments meant to monitor strength and training progress. In this study, we asked what accessible body composition approach could best quantify body composition in athletes with a variety of hydration levels.
METHODS
The Da Kine Study recruited collegiate and intramural athletes to undergo a variety of body composition assessments including air-displacement plethysmography (ADP), deuterium-oxide dilution (DO), dual-energy X-ray absorptiometry (DXA), underwater-weighing (UWW), 3D-optical (3DO) imaging, and bioelectrical impedance (BIA). Each of these methods generated 2- or 3-compartment body composition estimates of fat mass (FM) and fat-free mass (FFM) and was compared to equivalent measures of the criterion 6-compartment model (6CM) that accounts for variance in hydration. Body composition by each method was used to predict abdominal and thigh strength, assessed by isokinetic/isometric dynamometry.
RESULTS
In total, 70 (35 female) athletes with a mean age of 21.8 ± 4.2 years were recruited. Percent hydration (Body Water/FFM) had substantial variation in both males (63-73 %) and females (58-78 %). ADP and DXA FM and FF M had moderate to substantial agreement with the 6C model (Lin's Concordance Coefficient [CCC] = 0.90-0.95) whereas the other measures had lesser agreement (CCC <0.90) with one exception of 3DO FFM in females (CCC = 0.91). All measures of FFM produced excellent precision with %CV < 1.0 %. However, FM measures in general had worse precision (% CV < 2.0 %). Increasing quartiles (significant p < 0.001 trend) of 6CM FFM resulted in increasing strength measures in males and females. Moreover, the stronger the agreement between the alternative methods to the 6CM, the more robust their correlation with strength, irrespective of hydration status.
CONCLUSION
The criterion 6CM showed the best association to strength regardless of the hydration status of the athletes for both males and females. Simpler methods showed high precision for both FM and FFM and those with the strongest agreement to the 6CM had the highest strength associations.
SUMMARY BOX
This study compared various body composition analysis methods in 70 athletes with varying states of hydration to the criterion 6-compartment model and assessed their relationship to muscle strength. The results showed that accurate and precise estimates of body composition can be determined in athletes, and a more accurate body composition measurement produces better strength estimates. The best laboratory-based techniques were air displacement plethysmography and dual-energy x-ray absorptiometry, while the commercial methods had moderate-poor agreement. Prioritizing accurate body composition assessment ensures better strength estimates in athletes.
Topics: Male; Humans; Female; Adolescent; Young Adult; Adult; Body Composition; Body Water; Athletes; Absorptiometry, Photon; Electric Impedance; Muscle Strength; Reproducibility of Results
PubMed: 38104490
DOI: 10.1016/j.clnu.2023.11.040 -
Protein Science : a Publication of the... Nov 2023Ca /CaM-dependent protein kinase kinases 1 and 2 (CaMKK1 and CaMKK2) phosphorylate and enhance the catalytic activity of downstream kinases CaMKI, CaMKIV, and protein...
Ca /CaM-dependent protein kinase kinases 1 and 2 (CaMKK1 and CaMKK2) phosphorylate and enhance the catalytic activity of downstream kinases CaMKI, CaMKIV, and protein kinase B. Accordingly, CaMKK1 and CaMKK2 regulate key physiological and pathological processes, such as tumorigenesis, neuronal morphogenesis, synaptic plasticity, transcription factor activation, and cellular energy homeostasis, and promote cell survival. Both CaMKKs are partly inhibited by phosphorylation, which in turn triggers adaptor and scaffolding protein 14-3-3 binding. However, 14-3-3 binding only significantly affects CaMKK1 function. CaMKK2 activity remains almost unchanged after complex formation for reasons still unclear. Here, we aim at structurally characterizing CaMKK1:14-3-3 and CaMKK2:14-3-3 complexes by SAXS, H/D exchange coupled to MS, and fluorescence spectroscopy. The results revealed that complex formation suppresses the interaction of both phosphorylated CaMKKs with Ca /CaM and affects the structure of their kinase domains and autoinhibitory segments. But these effects are much stronger on CaMKK1 than on CaMKK2 because the CaMKK1:14-3-3γ complex has a more compact and rigid structure in which the active site of the kinase domain directly interacts with the last two C-terminal helices of the 14-3-3γ protein, thereby inhibiting CaMKK1. In contrast, the CaMKK2:14-3-3 complex has a looser and more flexible structure, so 14-3-3 binding only negligibly affects the catalytic activity of CaMKK2. Therefore, Ca /CaM binding suppression and the interaction of the kinase active site of CaMKK1 with the last two C-terminal helices of 14-3-3γ protein provide the structural basis for 14-3-3-mediated CaMKK1 inhibition.
Topics: 14-3-3 Proteins; Catalytic Domain; Scattering, Small Angle; X-Ray Diffraction; Phosphorylation; Calcium-Calmodulin-Dependent Protein Kinase Kinase
PubMed: 37817008
DOI: 10.1002/pro.4805 -
The Journal of Physical Chemistry. B Aug 2023Protein-DNA interactions play an important role in numerous biological functions within the living cell. In many of these interactions, the DNA helix is significantly...
Protein-DNA interactions play an important role in numerous biological functions within the living cell. In many of these interactions, the DNA helix is significantly distorted upon protein-DNA complex formation. The I restriction-modification system is one such system, where the methylation target is flipped out of the helix when bound to the methyltransferase. However, the base flipping mechanism is not well understood. The dynamics of the binding site of the I methyltransferase and endonuclease (underlined) within the DNA oligomer [d(GATATATC)] are studied using deuterium solid-state NMR (SSNMR). SSNMR spectra obtained from DNAs deuterated on the base of nucleotides within and flanking the [5'-GCGC-3'] sequence indicate that all of these positions are structurally flexible. Previously, conformational flexibility within the phosphodiester backbone and furanose ring within the target sequence has been observed and hypothesized to play a role in the distortion mechanism. However, whether that distortion was occurring through an active or passive mechanism remained unclear. These NMR data demonstrate that although the [5'-GCGC-3'] sequence is dynamic, the target cytosine is not passively flipping out of the double-helix on the millisecond-picosecond time scale. Additionally, although previous studies have shown that both the furanose ring and phosphodiester backbone experience a change in dynamics upon methylation, which may play a role in recognition and cleavage by the endonuclease, our observations here indicate that methylation has no effect on the dynamics of the base itself.
Topics: Protein Binding; Binding Sites; DNA; Magnetic Resonance Spectroscopy; Protein Domains; DNA Methylation; Nucleic Acid Conformation
PubMed: 37561575
DOI: 10.1021/acs.jpcb.3c03687 -
STAR Protocols Dec 2023In biological systems, protein function depends on spatial and temporal changes known as protein dynamics, which can be probed by amide hydrogen/deuterium (H/D)...
In biological systems, protein function depends on spatial and temporal changes known as protein dynamics, which can be probed by amide hydrogen/deuterium (H/D) exchange. Here, we present a protocol for determining protein dynamics by Fourier-transform infrared (FT-IR) spectroscopy. We describe steps for protein sample preparation and FT-IR spectra collection. We then detail procedures for spectra analysis. Applications include the effects of protein mutation or protein and metal ion or ligand interactions on the protein H/D exchange rate. For complete details on the use and execution of this protocol, please refer to Yu et al. (2013)..
Topics: Spectroscopy, Fourier Transform Infrared; Proteins
PubMed: 38043057
DOI: 10.1016/j.xpro.2023.102587 -
Molecules (Basel, Switzerland) Jan 2024Radical reactions in water or aqueous media are important for organic synthesis, realizing high-yielding processes under non-toxic and environmentally friendly... (Review)
Review
Radical reactions in water or aqueous media are important for organic synthesis, realizing high-yielding processes under non-toxic and environmentally friendly conditions. This overview includes (i) a general introduction to organic chemistry in water and aqueous media, (ii) synthetic approaches in, on, and with water as well as in heterogeneous phases, (iii) reactions of carbon-centered radicals with water (or deuterium oxide) activated through coordination with various Lewis acids, (iv) photocatalysis in water and aqueous media, and (v) synthetic applications bioinspired by naturally occurring processes. A wide range of chemical processes and synthetic strategies under different experimental conditions have been reviewed that lead to important functional group translocation and transformation reactions, leading to the preparation of complex molecules. These results reveal how water as a solvent/medium/reagent in radical chemistry has matured over the last two decades, with further discoveries anticipated in the near future.
PubMed: 38338314
DOI: 10.3390/molecules29030569 -
Frontiers in Nutrition 2023To date, body composition assessments in Hispanics, computed via bioimpedance devices, have primarily focused on body fat percent, fat mass, and fat-free mass instead of...
BACKGROUND
To date, body composition assessments in Hispanics, computed via bioimpedance devices, have primarily focused on body fat percent, fat mass, and fat-free mass instead of total body water (TBW). Additionally, virtually no information is available on which type of bioimpedance device is preferred for TBW assessments in Hispanic populations.
PURPOSE
The purpose of this study was to validate two bioimpedance devices for the estimate of TBW in Hispanics adults when using a criterion deuterium oxide (DO) technique.
METHODS
One-hundred thirty individuals (males: = 70; females: = 60) of Hispanic descent had TBW estimated via DO, single-frequency bioimpedance analysis ([SF-BIA] Quantum V, RJL Systems) and bioimpedance spectroscopy ([BIS] SFB7 Impedimed).
RESULTS
The mean values for SF-BIA were significantly lower than DO when evaluating the entire sample (37.4 L and 38.2 L, respectively; < 0.05). In contrast, TBW values were not statistically significant when comparing DO against BIS (38.4 L, > 0.05). Bland-Altman analysis indicated no proportional bias when evaluating the entire sample for SF-BIA or BIS. The standard error of estimate and total error values were ≤ 2.3 L and Lin's concordance correlation coefficient were ≥ 0.96 for all comparisons.
CONCLUSION
The SF-BIA and BIS devices evaluated in the current study hold promise for accurate estimation of TBW in Hispanic adults. While both methods demonstrated relatively low errors relative to the DO criterion, BIS exhibited a more consistent performance, particularly at the group level. These findings provide essential information for researchers and clinical nutrition practitioners assessing TBW in Hispanic adults.
PubMed: 37693242
DOI: 10.3389/fnut.2023.1221774 -
ELife Dec 2023BRAF is a key member in the MAPK signaling pathway essential for cell growth, proliferation, and differentiation. Mutant BRAF is often the underlying cause of various...
BRAF is a key member in the MAPK signaling pathway essential for cell growth, proliferation, and differentiation. Mutant BRAF is often the underlying cause of various types of cancer and mutant RAS, the upstream regulator of BRAF, is a driver of up to one-third of all cancers. BRAF interacts with RAS and undergoes a conformational change from an inactive, autoinhibited monomer to an active dimer, which propagates downstream signaling. Because of BRAF's complex regulation mechanism, the exact order and magnitude of its activation steps have yet to be confirmed experimentally. By studying the inter- and intramolecular interactions of BRAF, we unveil the domain-specific and isoform-specific details of BRAF regulation through pulldown assays, open surface plasmon resonance (OpenSPR), and hydrogen-deuterium exchange mass spectrometry (HDX-MS). We demonstrate that the BRAF specific region (BSR) and cysteine rich domain (CRD) play a crucial role in regulating the activation of BRAF in a RAS isoform-specific manner. Moreover, we quantified the binding affinities between BRAF N-terminal and kinase domains (KD) to reveal their individual roles in autoinhibition. Our findings also indicate that oncogenic BRAF-KD mutant has a lower affinity for the N-terminal domains, implicating that pathogenic BRAF acts through decreased propensity for autoinhibition. Collectively, our study provides valuable insight into the activation mechanism of BRAF kinase to guide the development of new therapeutic strategies for cancer treatment.
Topics: Proto-Oncogene Proteins B-raf; Biological Assay; Cell Cycle; Cell Differentiation; Protein Isoforms
PubMed: 38150000
DOI: 10.7554/eLife.88836 -
Journal of Proteome Research Sep 2023Structural proteomics techniques are useful for the determination of protein interaction interfaces. Each technique provides orthogonal structural information on the...
Structural proteomics techniques are useful for the determination of protein interaction interfaces. Each technique provides orthogonal structural information on the structure and the location of protein interaction sites. Here, we have characterized a monoclonal antibody epitope for a protein antigen by a combination of differential photoreactive surface modification (SM), cross-linking (CL), differential hydrogen-deuterium exchange (HDX), and epitope extraction/excision. We found that experimental data from different approaches agree with each other in determining the epitope of the monoclonal antibody on the protein antigens using the HIV-1 p24-mAb E complex as an illustrative example. A combination of these multiple structural proteomics approaches results in a detailed picture of the interaction of the proteins and increases confidence in the determination of the final structure of the protein interaction interface. Data are available via ProteomeXchange with identifier PXD040902.
Topics: Epitopes; Antibodies, Monoclonal; Epitope Mapping; Proteomics; Mass Spectrometry
PubMed: 37526474
DOI: 10.1021/acs.jproteome.3c00159