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Proteomics Nov 2023This pilot experiment examines if a loss in muscle proteostasis occurs in people with obesity and whether endurance exercise positively influences either the abundance...
This pilot experiment examines if a loss in muscle proteostasis occurs in people with obesity and whether endurance exercise positively influences either the abundance profile or turnover rate of proteins in this population. Men with (n = 3) or without (n = 4) obesity were recruited and underwent a 14-d measurement protocol of daily deuterium oxide (D O) consumption and serial biopsies of vastus lateralis muscle. Men with obesity then completed 10-weeks of high-intensity interval training (HIIT), encompassing 3 sessions per week of cycle ergometer exercise with 1 min intervals at 100% maximum aerobic power interspersed by 1 min recovery periods. The number of intervals per session progressed from 4 to 8, and during weeks 8-10 the 14-d measurement protocol was repeated. Proteomic analysis detected 352 differences (p < 0.05, false discovery rate < 5%) in protein abundance and 19 (p < 0.05) differences in protein turnover, including components of the ubiquitin-proteasome system. HIIT altered the abundance of 53 proteins and increased the turnover rate of 22 proteins (p < 0.05) and tended to benefit proteostasis by increasing muscle protein turnover rates. Obesity and insulin resistance are associated with compromised muscle proteostasis, which may be partially restored by endurance exercise.
PubMed: 37963832
DOI: 10.1002/pmic.202300395 -
BioRxiv : the Preprint Server For... Nov 2023Production of soluble proteins is essential for structure/function studies, however, this usually requires milligram amounts of protein, which can be difficult to obtain...
Production of soluble proteins is essential for structure/function studies, however, this usually requires milligram amounts of protein, which can be difficult to obtain with traditional expression systems. Recently, the Gram-negative bacterium appeared as a novel and alternative host platform for production of proteins in high yields. Here, we used a commercial strain derived from (Vmax X2) to produce soluble bacterial and fungal proteins in milligram scale, which we struggled to achieve in . These proteins include the cholera toxin (CT) and -acetyl glucosamine binding protein A (GbpA) from , the heat-labile enterotoxin (LT) from and the fungal nematotoxin CCTX2 from . CT, GbpA and LT are secreted by the Type II secretion system in their natural hosts. When these three proteins were produced in Vmax, they were also secreted, and could be recovered from the growth media. This simplified the downstream purification procedure and resulted in considerably higher protein yields compared to production in (6- to 26-fold increase). We also tested Vmax for protein deuteration using deuterated minimal media with deuterium oxide as solvent, and achieved a 3-fold increase in yield compared to the equivalent protocol in . This is good news since isotopic labeling is expensive and often ineffective, but represents a necessary prerequisite for some structural techniques. Thus, Vmax represents a promising host for production of challenging expression targets and for protein deuteration in amounts suitable for structural biology studies.
PubMed: 37961550
DOI: 10.1101/2023.11.03.565449 -
The Journal of Membrane Biology Dec 2023Vigna radiata H-translocating pyrophosphatases (VrH-PPases, EC 3.6.1.1) are present in various endomembranes of plants, bacteria, archaea, and certain protozoa. They...
Vigna radiata H-translocating pyrophosphatases (VrH-PPases, EC 3.6.1.1) are present in various endomembranes of plants, bacteria, archaea, and certain protozoa. They transport H into the lumen by hydrolyzing pyrophosphate, which is a by-product of many essential anabolic reactions. Although the crystal structure of H-PPases has been elucidated, the H translocation mechanism of H-PPases in the solution state remains unclear. In this study, we used hydrogen-deuterium exchange (HDX) coupled with mass spectrometry (MS) to investigate the dynamics of H-PPases between the previously proposed R state (resting state, Apo form), I state (intermediate state, bound to a substrate analog), and T state (transient state, bound to inorganic phosphate). When hydrogen was replaced by proteins in deuterium oxide solution, the backbone hydrogen atoms, which were exchanged with deuterium, were identified through MS. Accordingly, we used deuterium uptake to examine the structural dynamics and conformational changes of H-PPases in solution. In the highly conserved substrate binding and proton exit regions, HDX-MS revealed the existence of a compact conformation with deuterium exchange when H-PPases were bound with a substrate analog and product. Thus, a novel working model was developed to elucidate the in situ catalytic mechanism of pyrophosphate hydrolysis and proton transport. In this model, a proton is released in the I state, and the TM5 inner wall serves as a proton piston.
Topics: Inorganic Pyrophosphatase; Vigna; Protons; Deuterium; Diphosphates; Deuterium Exchange Measurement; Hydrogen; Mass Spectrometry
PubMed: 37955797
DOI: 10.1007/s00232-023-00295-9 -
Analytical Chemistry Nov 2023In drug research and development, knowledge of the precise structure of an active ingredient is crucial. However, it is equally important to know the water content of...
In drug research and development, knowledge of the precise structure of an active ingredient is crucial. However, it is equally important to know the water content of the drug molecule, particularly the number of crystal waters present in its structure. Such knowledge ensures the avoidance of drug dosage and formulation errors since the number of water molecules affects the physicochemical and pharmaceutical properties of the molecule. Several methods have been used for crystal water measurements of organic compounds, of which thermogravimetry and crystallography may be the most common ones. To the best of our knowledge, solution-state NMR spectroscopy has not been used for crystal water determination in deuterium oxide. Quantitative NMR (qNMR) method will be presented in the paper with a comparison of single-crystal X-ray diffraction and thermogravimetric analysis results. The qNMR method for water content measurement is straightforward, reproducible, and accurate, including measurement of H NMR spectrum before and after the addition of the analyte compound, and the result can be calculated after integration of the reference compound, analyte, and HDO signals using the given equation. In practical terms, there is no need for weighing the samples under study, which makes it simple and is a clear advantage to the current determination methods. In addition, the crystal structures of two model bisphosphonates used herein are reported: that of monopotassium etidronate dihydrate and monosodium zoledronate trihydrate.
PubMed: 37923567
DOI: 10.1021/acs.analchem.3c03689 -
Frontiers in Nutrition 2023Neurologically impaired (NI) children are at risk of malnutrition, which consequently impacts their health and quality of life. Accurate nutrition assessment is an...
Measurement of body composition by deuterium oxide dilution technique and development of a predictive equation for body fat mass among severe neurologically impaired children.
INTRODUCTION
Neurologically impaired (NI) children are at risk of malnutrition, which consequently impacts their health and quality of life. Accurate nutrition assessment is an important step in guiding appropriate nutrition support. Conventional anthropometric measurements among NI children have some limitations. Determining body composition requires more complex equipment, which is not routinely performed. This study was conducted to evaluate the association between anthropometric parameters and body composition assessed using the deuterium dilution technique (DDT) in NI children.
METHODS
A cross-sectional study enrolled severe NI children aged 1-20 years who received home enteral nutrition for at least 3 months. Weight, length, and 4-site skinfold thickness were measured. Body composition was determined using DDT following the International Atomic Energy Agency (IAEA) protocol.
RESULTS
A total of 37 NI children (56.76% male, median age 7.2 years) were enrolled. The prevalence of underweight, stunting, and overweight were 22, 38, and 35%, respectively. Body composition analysis showed the mean (SD) of total body water (TBW) and fat mass (FM) were 10.52 (4.51) kg and 9.51 (6.04) kg, respectively. Multivariate GLM analysis showed that the factors associated with FM were age (β = 0.07 [0.05,0.08]; < 0.001), body mass index (BMI) (β = 0.82 [0.52, 1.12]; < 0.001), biceps skinfold thickness (BSF) (β = 0.49 [0.23,0.75]; = 0.001), and subscapular skinfold thickness (SSF) (β = -0.24 [-0.46,0.03]; = 0.030). A predictive equation for FM was constructed.
CONCLUSION
A high prevalence of malnutrition was found among severe NI children despite enteral nutrition support. Our findings showed that age, BMI, BSF, and SSF were associated with FM. The predictive equation of FM was proposed and needed to be further validated and applied to clinical practice.
PubMed: 37920288
DOI: 10.3389/fnut.2023.1162956 -
International Journal of Molecular... Oct 2023Mass spectrometry has been an essential technique for the investigation of the metabolic pathways of living organisms since its appearance at the beginning of the 20th...
Mass spectrometry has been an essential technique for the investigation of the metabolic pathways of living organisms since its appearance at the beginning of the 20th century. Due to its capability to resolve isotopically labeled species, it can be applied together with stable isotope tracers to reveal the transformation of particular biologically relevant molecules. However, low-resolution techniques, which were used for decades, had limited capabilities for untargeted metabolomics, especially when a large number of compounds are labelled simultaneously. Such untargeted studies may provide new information about metabolism and can be performed with high-resolution mass spectrometry. Here, we demonstrate the capabilities of high-resolution mass spectrometry to obtain insights on the metabolism of a model plant, , germinated in DO and HO-enriched media. In particular, we demonstrated that in vivo labeling with heavy water helps to identify if a compound is being synthesized at a particular stage of germination or if it originates from seed content, and tandem mass spectrometry allows us to highlight the substructures with incorporated isotope labels. Additionally, we found in vivo labeling useful to distinguish between isomeric compounds with identical fragmentation patterns due to the differences in their formation rates that can be compared by the extent of heavy atom incorporation.
Topics: Deuterium Oxide; Isotope Labeling; Metabolomics; Tandem Mass Spectrometry; Plants; Isotopes
PubMed: 37895078
DOI: 10.3390/ijms242015396 -
GeroScience Apr 2024Age-associated declines in aerobic capacity promote the development of various metabolic diseases. In rats selectively bred for high/low intrinsic aerobic capacity,...
Age-associated declines in aerobic capacity promote the development of various metabolic diseases. In rats selectively bred for high/low intrinsic aerobic capacity, greater aerobic capacity reduces susceptibility to metabolic disease while increasing longevity. However, little remains known how intrinsic aerobic capacity protects against metabolic disease, particularly with aging. Here, we tested the effects of aging and intrinsic aerobic capacity on systemic energy expenditure, metabolic flexibility and mitochondrial protein synthesis rates using 24-month-old low-capacity (LCR) or high-capacity runner (HCR) rats. Rats were fed low-fat diet (LFD) or high-fat diet (HFD) for eight weeks, with energy expenditure (EE) and metabolic flexibility assessed utilizing indirect calorimetry during a 48 h fast/re-feeding metabolic challenge. Deuterium oxide (D2O) labeling was used to assess mitochondrial protein fraction synthesis rates (FSR) over a 7-day period. HCR rats possessed greater EE during the metabolic challenge. Interestingly, HFD induced changes in respiratory exchange ratio (RER) in male and female rats, while HCR female rat RER was largely unaffected by diet. In addition, analysis of protein FSR in skeletal muscle, brain, and liver mitochondria showed tissue-specific adaptations between HCR and LCR rats. While brain and liver protein FSR were altered by aerobic capacity and diet, these effects were less apparent in skeletal muscle. Overall, we provide evidence that greater aerobic capacity promotes elevated EE in an aged state, while also regulating metabolic flexibility in a sex-dependent manner. Modulation of mitochondrial protein FSR by aerobic capacity is tissue-specific with aging, likely due to differential energetic requirements by each tissue.
Topics: Rats; Male; Female; Animals; Energy Metabolism; Liver; Diet, High-Fat; Metabolic Diseases; Mitochondrial Proteins
PubMed: 37880490
DOI: 10.1007/s11357-023-00985-1 -
Magnetic Resonance in Medicine Feb 2024Tail-vein catheterization and subsequent in-magnet infusion is a common route of administration of deuterium ( H)-labeled substrates in small-animal deuterium (D) MR...
PURPOSE
Tail-vein catheterization and subsequent in-magnet infusion is a common route of administration of deuterium ( H)-labeled substrates in small-animal deuterium (D) MR studies. With mice, because of the tail vein's small diameter, this procedure is challenging. It requires considerable personnel training and practice, is prone to failure, and may preclude serial studies. Motivated by the need for an alternative, the time courses for common small-molecule deuterated substrates and downstream metabolites in brain following subcutaneous infusion were determined in mice and are presented herein.
METHODS
Three H-labeled substrates-[6,6- H ]glucose, [ H ]acetate, and [3,4,4,4- H ]beta-hydroxybutyrate-and H O were administered to mice in-magnet via subcutaneous catheter. Brain time courses of the substrates and downstream metabolites (and semi-heavy water) were determined via single-voxel DMRS.
RESULTS
Subcutaneous catheter placement and substrate administration was readily accomplished with limited personnel training. Substrates reached pseudo-steady state in brain within ∼30-40 min of bolus infusion. Time constants characterizing the appearance in brain of deuterated substrates or semi-heavy water following H O administration were similar (∼15 min).
CONCLUSION
Administration of deuterated substrates via subcutaneous catheter for in vivo DMRS experiments with mice is robust, requires limited personnel training, and enables substantial dosing. It is suitable for metabolic studies where pseudo-steady state substrate administration/accumulation is sufficient. It is particularly advantageous for serial longitudinal studies over an extended period because it avoids inevitable damage to the tail vein following multiple catheterizations.
Topics: Mice; Animals; Deuterium Oxide; Deuterium; Tail; Brain
PubMed: 37849055
DOI: 10.1002/mrm.29888 -
Chemistry, An Asian Journal Dec 2023A novel synthetic approach to 2,3,6-trisubstituted pyridines, their 4,5-dideuterated derivatives, 4,5- C - and doubly-labeled D - C -pyridines has been developed using...
A novel synthetic approach to 2,3,6-trisubstituted pyridines, their 4,5-dideuterated derivatives, 4,5- C - and doubly-labeled D - C -pyridines has been developed using catalyst-free [4+2] cycloaddition of 1,2,4-triazines and in situ generated acetylene or labeled acetylene. Calcium carbide and water or deuterium oxide were used for the in situ generation of acetylene and dideuteroacetylene. Calcium carbide- C in the mixture with water or deuterium oxide was applied as C -acetylene and D - C -acetylene source.
PubMed: 37843978
DOI: 10.1002/asia.202300781 -
International Journal of Molecular... Sep 2023In the heavy petroleum industry, the development of efficient demulsifiers for the effective breaking of interfacially active asphaltenes (IAA)-stabilized water-in-heavy...
In the heavy petroleum industry, the development of efficient demulsifiers for the effective breaking of interfacially active asphaltenes (IAA)-stabilized water-in-heavy oil (W/HO) emulsions is a highly attractive but challenging goal. Herein, a novel nitrogen and oxygen containing demulsifier (JXGZ) with strong hydrogen bonding has been successfully synthesized through combining esterification, polymerization and amidation. Bottle tests indicated that JXGZ is effectual in quickly demulsifying the IAA-stabilized W/HO emulsions; complete dehydration (100%) to the emulsions could be achieved in 4 min at 55 °C using 400 ppm of JXGZ. In addition, the effects of demulsifier concentration, temperature and time on the demulsification performance of JXGZ are systematically analyzed. Demulsification mechanisms reveal that the excellent demulsification performance of JXGZ is attributed to the strong hydrogen bonding between JXGZ and water molecules (dual swords synergistic effect under hydrogen bond reconstruction). The interaction of the "dual swords synergistic effect" generated by two types of hydrogen bonds can quickly break the non-covalent interaction force (π-π stacking, Van der Waals force, hydrogen bonds) of IAA at the heavy oil-water interface, quickly promote the aggregation and coalescence of water molecules and finally achieve the demulsification of W/HO emulsions. These findings indicate that the JXGZ demulsifier shows engineering application prospects in the demulsification of heavy oil-water emulsions, and this work provides the key information for developing more efficient chemical demulsifiers suitable for large-scale industrial applications.
Topics: Emulsions; Hydrogen Bonding; Water; Petroleum; Deuterium Oxide
PubMed: 37834251
DOI: 10.3390/ijms241914805