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Molecular & Cellular Proteomics : MCP Aug 2023Proteomic studies in facioscapulohumeral muscular dystrophy (FSHD) could offer new insight into disease mechanisms underpinned by post-transcriptional processes. We used...
Proteomic studies in facioscapulohumeral muscular dystrophy (FSHD) could offer new insight into disease mechanisms underpinned by post-transcriptional processes. We used stable isotope (deuterium oxide; DO) labeling and peptide mass spectrometry to investigate the abundance and turnover rates of proteins in cultured muscle cells from two individuals affected by FSHD and their unaffected siblings (UASb). We measured the abundance of 4420 proteins and the turnover rate of 2324 proteins in each (n = 4) myoblast sample. FSHD myoblasts exhibited a greater abundance but slower turnover rate of subunits of mitochondrial respiratory complexes and mitochondrial ribosomal proteins, which may indicate an accumulation of "older" less viable mitochondrial proteins in myoblasts from individuals affected by FSHD. Treatment with a 2'-O-methoxyethyl modified antisense oligonucleotide targeting exon 3 of the double homeobox 4 (DUX4) transcript tended to reverse mitochondrial protein dysregulation in FSHD myoblasts, indicating the effect on mitochondrial proteins may be a DUX4-dependent mechanism. Our results highlight the importance of post-transcriptional processes and protein turnover in FSHD pathology and provide a resource for the FSHD research community to explore this burgeoning aspect of FSHD.
Topics: Humans; Muscular Dystrophy, Facioscapulohumeral; Proteome; Proteomics; Homeodomain Proteins; Myoblasts; Muscle, Skeletal
PubMed: 37353005
DOI: 10.1016/j.mcpro.2023.100605 -
Journal of Biomedical Optics Sep 2023The shortwave infrared (SWIR, to 2000 nm) holds promise for label-free measurements of water and lipid content in thick tissue, owed to the chromophore-specific...
SIGNIFICANCE
The shortwave infrared (SWIR, to 2000 nm) holds promise for label-free measurements of water and lipid content in thick tissue, owed to the chromophore-specific absorption features and low scattering in this range. water and lipid estimations have potential applications including the monitoring of hydration, volume status, edema, body composition, weight loss, and cancer. To the best of our knowledge, there are currently no point-of-care or wearable devices available that exploit the SWIR wavelength range, limiting clinical and at-home translation of this technology.
AIM
To design and fabricate a diffuse optical wearable SWIR probe for water and lipid quantification in tissue.
APPROACH
Simulations were first performed to confirm the theoretical advantage of SWIR wavelengths over near infrared (NIR). The probe was then fabricated, consisting of light emitting diodes at three wavelengths (980, 1200, 1300 nm) and four source-detector (S-D) separations (7, 10, 13, 16 mm). validation was then performed on emulsion phantoms containing varying concentrations of water, lipid, and deuterium oxide (). A deep neural network was developed as the inverse model for quantity estimation.
RESULTS
Simulations indicated that SWIR wavelengths could reduce theoretical water and lipid extraction errors from to when compared to NIR wavelengths. The SWIR probe had good signal-to-noise ratio ( up to 10 mm S-D) and low drift ( up to 10 mm S-D). Quantification error in emulsion phantoms was for water and for lipid. Water estimation during a dilution experiment had an error of .
CONCLUSIONS
This diffuse optical SWIR probe was able to quantify water and lipid contents with good accuracy, opening the door to human investigations.
Topics: Humans; Emulsions; Deep Learning; Water; Wearable Electronic Devices; Lipids
PubMed: 37313427
DOI: 10.1117/1.JBO.28.9.094808 -
Trials Jun 2023The decline in skeletal muscle mass experienced following a short-term period (days to weeks) of muscle disuse is mediated by impaired rates of muscle protein synthesis...
The effect of combined β-lactoglobulin supplementation and resistance exercise training prior to limb immobilisation on muscle protein synthesis rates in healthy young adults: study protocol for a randomised controlled trial.
BACKGROUND
The decline in skeletal muscle mass experienced following a short-term period (days to weeks) of muscle disuse is mediated by impaired rates of muscle protein synthesis (MPS). Previous RCTs of exercise or nutrition prehabilitation interventions designed to mitigate disuse-induced muscle atrophy have reported limited efficacy. Hence, the aim of this study is to investigate the impact of a complex prehabilitation intervention that combines β-lactoglobulin (a novel milk protein with a high leucine content) supplementation with resistance exercise training on disuse-induced changes in free-living integrated rates of MPS in healthy, young adults.
METHODS/DESIGN
To address this aim, we will recruit 24 healthy young (18-45 years) males and females to conduct a parallel, double-blind, 2-arm, randomised placebo-controlled trial. The intervention group will combine a 7-day structured resistance exercise training programme with thrice daily dietary supplementation with 23 g of β-lactoglobulin. The placebo group will combine the same training programme with an energy-matched carbohydrate (dextrose) control. The study protocol will last 16 days for each participant. Day 1 will be a familiarisation session and days 2-4 will be the baseline period. Days 5-11 represent the 'prehabilitation period' whereby participants will combine resistance training with their assigned dietary supplementation regimen. Days 12-16 represent the muscle disuse-induced 'immobilisation period' whereby participants will have a single leg immobilised in a brace and continue their assigned dietary supplementation regimen only (i.e. no resistance training). The primary endpoint of this study is the measurement of free-living integrated rates of MPS using deuterium oxide tracer methodology. Measurements of MPS will be calculated at baseline, over the 7-day prehabilitation period and over the 5-day immobilisation period separately. Secondary endpoints include measurements of muscle mass and strength that will be collected on days 4 (baseline), 11 (end of prehabilitation) and 16 (end of immobilisation).
DISCUSSION
This novel study will establish the impact of a bimodal prehabilitation strategy that combines ß-lactoglobulin supplementation and resistance exercise training in modulating MPS following a short-term period of muscle disuse. If successful, this complex intervention may be translated to clinical practice with application to patients scheduled to undergo, for example, hip or knee replacement surgery.
TRIAL REGISTRATION
NCT05496452. Registered on August 10, 2022.
PROTOCOL VERSION
16-12-2022/1.
Topics: Female; Male; Humans; Young Adult; Muscle Proteins; Resistance Training; Muscles; Lactoglobulins; Dietary Supplements; Randomized Controlled Trials as Topic
PubMed: 37312095
DOI: 10.1186/s13063-023-07329-6 -
Advanced Science (Weinheim,... Aug 2023Gas-solid reactions are important for many redox processes that underpin the energy and sustainability transition. The specific case of hydrogen-based iron oxide...
Gas-solid reactions are important for many redox processes that underpin the energy and sustainability transition. The specific case of hydrogen-based iron oxide reduction is the foundation to render the global steel industry fossil-free, an essential target as iron production is the largest single industrial emitter of carbon dioxide. This perception of gas-solid reactions has not only been limited by the availability of state-of-the-art techniques which can delve into the structure and chemistry of reacted solids, but one continues to miss an important reaction partner that defines the thermodynamics and kinetics of gas phase reactions: the gas molecules. In this investigation, cryogenic-atom probe tomography is used to study the quasi in situ evolution of iron oxide in the solid and gas phases of the direct reduction of iron oxide by deuterium gas at 700°C. So far several unknown atomic-scale characteristics are observed, including, D accumulation at the reaction interface; formation of a core (wüstite)-shell (iron) structure; inbound diffusion of D through the iron layer and partitioning of D among phases and defects; outbound diffusion of oxygen through the wüstite and/or through the iron to the next free available inner/outer surface; and the internal formation of heavy nano-water droplets at nano-pores.
PubMed: 37290039
DOI: 10.1002/advs.202300626 -
The Journal of Nutrition Jun 2023Muscle mass and strength decrease during short periods of immobilization and slowly recover during remobilization. Recent artificial intelligence applications have... (Randomized Controlled Trial)
Randomized Controlled Trial
Vicia faba Peptide Network Supplementation Does Not Differ From Milk Protein in Modulating Changes in Muscle Size During Short-Term Immobilization and Subsequent Remobilization, but Increases Muscle Protein Synthesis Rates During Remobilization in Healthy Young Men.
BACKGROUND
Muscle mass and strength decrease during short periods of immobilization and slowly recover during remobilization. Recent artificial intelligence applications have identified peptides that appear to possess anabolic properties in in vitro assays and murine models.
OBJECTIVES
This study aimed to compare the impact of Vicia faba peptide network compared with milk protein supplementation on muscle mass and strength loss during limb immobilization and regain during remobilization.
METHODS
Thirty young (24 ± 5 y) men were subjected to 7 d of one-legged knee immobilization followed by 14 d of ambulant recovery. Participants were randomly allocated to ingest either 10 g of the Vicia faba peptide network (NPN_1; n = 15) or an isonitrogenous control (milk protein concentrate; MPC; n = 15) twice daily throughout the study. Single-slice computed tomography scans were performed to assess quadriceps cross-sectional area (CSA). Deuterium oxide ingestion and muscle biopsy sampling were applied to measure myofibrillar protein synthesis rates.
RESULTS
Leg immobilization decreased quadriceps CSA (primary outcome) from 81.9 ± 10.6 to 76.5 ± 9.2 cm and from 74.8 ± 10.6 to 71.5 ± 9.8 cm in the NPN_1 and MPC groups, respectively (P < 0.001). Remobilization partially recovered quadriceps CSA (77.3 ± 9.3 and 72.6 ± 10.0 cm, respectively; P = 0.009), with no differences between the groups (P > 0.05). During immobilization, myofibrillar protein synthesis rates (secondary outcome) were lower in the immobilized leg (1.07% ± 0.24% and 1.10% ± 0.24%/d, respectively) than in the non-immobilized leg (1.55% ± 0.27% and 1.52% ± 0.20%/d, respectively; P < 0.001), with no differences between the groups (P > 0.05). During remobilization, myofibrillar protein synthesis rates in the immobilized leg were greater with NPN_1 than those with MPC (1.53% ± 0.38% vs. 1.23% ± 0.36%/d, respectively; P = 0.027).
CONCLUSION
NPN_1 supplementation does not differ from milk protein in modulating the loss of muscle size during short-term immobilization and the regain during remobilization in young men. NPN_1 supplementation does not differ from milk protein supplementation in modulating the myofibrillar protein synthesis rates during immobilization but further increases myofibrillar protein synthesis rates during remobilization.
Topics: Male; Humans; Animals; Mice; Vicia faba; Muscle Proteins; Muscular Atrophy; Milk Proteins; Artificial Intelligence; Muscle Strength; Immobilization; Quadriceps Muscle; Dietary Supplements; Peptides; Muscle, Skeletal
PubMed: 37277162
DOI: 10.1016/j.tjnut.2023.01.014 -
Journal of Visualized Experiments : JoVE May 2023Fatty acid synthesis is a complex and highly energy demanding metabolic pathway with important functional roles in the control of whole-body metabolic homeostasis and...
Fatty acid synthesis is a complex and highly energy demanding metabolic pathway with important functional roles in the control of whole-body metabolic homeostasis and other physiological and pathological processes. Contrary to other key metabolic pathways, such as glucose disposal, fatty acid synthesis is not routinely functionally assessed, leading to incomplete interpretations of metabolic status. In addition, there is a lack of publicly available detailed protocols suitable for newcomers in the field. Here, we describe an inexpensive quantitative method utilizing deuterium oxide and gas chromatography mass spectrometry (GCMS) for the analysis of total fatty acid de novo synthesis in brown adipose tissue in vivo. This method measures the synthesis of the products of fatty acid synthase independently of a carbon source, and it is potentially useful for virtually any tissue, in any mouse model, and under any external perturbation. Details on the sample preparation for GCMS and downstream calculations are provided. We focus on the analysis of brown fat due to its high levels of de novo fatty acid synthesis and critical roles in maintaining metabolic homeostasis.
Topics: Mice; Animals; Adipose Tissue, Brown; Deuterium Oxide; Fatty Acids; Lipogenesis; Glucose; Adipose Tissue
PubMed: 37246886
DOI: 10.3791/64219 -
Journal of Visualized Experiments : JoVE May 2023Essential aromatic amino acids (AAAs) are building blocks for synthesizing new biomasses in cells and sustaining normal biological functions. For example, an abundant...
Essential aromatic amino acids (AAAs) are building blocks for synthesizing new biomasses in cells and sustaining normal biological functions. For example, an abundant supply of AAAs is important for cancer cells to maintain their rapid growth and division. With this, there is a rising demand for a highly specific, noninvasive imaging approach with minimal sample preparation to directly visualize how cells harness AAAs for their metabolism in situ. Here, we develop an optical imaging platform that combines deuterium oxide (D2O) probing with stimulated Raman scattering (DO-SRS) and integrates DO-SRS with two-photon excitation fluorescence (2PEF) into a single microscope to directly visualize the metabolic activities of HeLa cells under AAA regulation. Collectively, the DO-SRS platform provides high spatial resolution and specificity of newly synthesized proteins and lipids in single HeLa cell units. In addition, the 2PEF modality can detect autofluorescence signals of nicotinamide adenine dinucleotide (NADH) and Flavin in a label-free manner. The imaging system described here is compatible with both in vitro and in vivo models, which is flexible for various experiments. The general workflow of this protocol includes cell culture, culture media preparation, cell synchronization, cell fixation, and sample imaging with DO-SRS and 2PEF modalities.
Topics: Humans; HeLa Cells; Amino Acids, Aromatic; Microscopy; Proteins; Amino Acids
PubMed: 37246865
DOI: 10.3791/65121 -
Translational Vision Science &... May 2023The purpose of this study was to determine the effects of the Photoactivated Chromophore for Keratitis Corneal Cross-Linking (PACK-CXL) protocol modifications on corneal...
PURPOSE
The purpose of this study was to determine the effects of the Photoactivated Chromophore for Keratitis Corneal Cross-Linking (PACK-CXL) protocol modifications on corneal resistance to enzymatic digestion and treatment depth.
METHODS
Eight hundred one ex vivo porcine eyes were randomly divided into groups of 12 to 86 corneas, treated with various epi-off PACK-CXL modifications, including acceleration (30 > 2 minutes, 5.4 J/cm2), increased fluence (5.4 > 32.4 J/cm2), deuterium oxide (D2O) supplementation, different carrier types (dextran versus hydroxypropyl methylcellulose [HPMC]), increased riboflavin concentration (0.1 > 0.4%), and riboflavin replenishment during irradiation (yes/no). Control group eyes did not receive PACK-CXL. A pepsin digestion assay was used to determine corneal resistance to enzymatic digestion. A phalloidin fluorescent imaging assay was used to determine the PACK-CXL treatment effect depth. Differences between groups were evaluated using a linear model and a derivative method, respectively.
RESULTS
PACK-CXL significantly increased corneal resistance to enzymatic digestion compared to no treatment (P < 0.03). When compared to a 10 minute, 5.4 J/cm2 PACK-CXL protocol, fluences of 16.2 J/cm2 and higher increased corneal resistance to enzymatic digestion by 1.5- to 2-fold (P < 0.001). Other protocol modifications did not significantly change corneal resistance. A 16.2 J/cm2 fluence also increased collagen compaction in the anterior stroma, whereas omitting riboflavin replenishment during irradiation increased PACK-CXL treatment depth.
CONCLUSIONS
Increasing fluence will likely optimize PACK-CXL treatment effectiveness. Treatment acceleration reduces treatment duration without compromising effectiveness.
TRANSLATIONAL RELEVANCE
The generated data help to optimize clinical PACK-CXL settings and direct future research efforts.
Topics: Swine; Animals; Photosensitizing Agents; Corneal Cross-Linking; Cornea; Riboflavin; Keratitis; Digestion; Cross-Linking Reagents
PubMed: 37191620
DOI: 10.1167/tvst.12.5.18 -
Food Chemistry: X Jun 2023DO-assisted moisture analysis of edible oils was investigated. The acetonitrile extract of the oil samples was split into two parts. The spectrum of one part was taken...
DO-assisted moisture analysis of edible oils was investigated. The acetonitrile extract of the oil samples was split into two parts. The spectrum of one part was taken as is, another was recorded after addition of excess DO. Changes in spectral absorption of the H-O-H bending band (1600-1660 cm) was used to calculate moisture in oil samples. To effectively depleting absorption of water in the acetonitrile extract, a 30-fold excess of DO is required. The typical OH-containing constituents in oil did not show significant interference on the H/D exchange. Validation experiments by using five oils with five levels of moisture spiked (50-1000 μg/g) suggested that the prediction tracked the spiked amounts well. The results of variance analysis indicate that there is no difference in terms of analytical methods and oil types used ( < 0.001). The DO method developed is generally applicable to the accurate analysis of moisture at trace levels (<100 μg/g) in edible oils.
PubMed: 37179978
DOI: 10.1016/j.fochx.2023.100679 -
Molecules (Basel, Switzerland) May 2023An atmospheric pressure glow discharge ionisation source was constructed and utilized to study the dopamine (DA) oxidation process coupling with mass spectrometry....
An atmospheric pressure glow discharge ionisation source was constructed and utilized to study the dopamine (DA) oxidation process coupling with mass spectrometry. During the DA oxidation process catalysed by polyphenol oxidase (PPO), six cationic intermediates were directly detected by the atmospheric pressure glow discharge mass spectrometry (APGD-MS). Combined with tandem mass spectrometry, the structures of the dopamine semiquinone radical (DASQ) and leukodopaminochrome radical (LDAC) intermediates and structures of the isomers of dopaminochrome (DAC) and 5,6-dihydroxyindole (DHI) were further characterised with the introduction of 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO) and deuterium oxide (DO) to APGD-MS. Meanwhile, UV-Vis studies confirmed the important role of PPO in catalyzing the DA oxidation reaction. Based on APGD-MS studies, a possible mechanism could be proposed for DA oxidation catalysed by PPO. Furthermore, APGD-MS could provide possibilities for the effective detection and characterisation of short-lived intermediates, even in complicated systems.
PubMed: 37175253
DOI: 10.3390/molecules28093844