Metabolic differences in MSTN and FGF5 dual-gene edited sheep muscle cells during myogenesis.

Dynamic metabolic reprogramming occurs at different stages of myogenesis and contributes to the fate determination of skeletal muscle satellite cells (MuSCs). Accumulating evidence suggests that mutations in myostatin (MSTN) have a vital role in regulating muscle energy metabolism. Here, we explored the metabolic reprogramming in MuSCs and myotube cells in MSTN and FGF5 dual-gene edited sheep models prepared previously, and also focused on the metabolic alterations during myogenic differentiation of MuSCs. Our study revealed that the pathways of nucleotide metabolism, pantothenate and CoA biosynthesis were weakened, while the unsaturated fatty acids biosynthesis were strengthened during myogenic differentiation of sheep MuSCs. The MSTN and FGF5 dual-gene editing mainly inhibited nucleotide metabolism and biosynthesis of unsaturated fatty acids in sheep MuSCs, reduced the number of lipid droplets in per satellite cell, and promoted the pentose phosphate pathway, and the interconversion of pentose and glucuronate. The MSTN and FGF5 dual-gene editing also resulted in the inhibition of nucleotide metabolism and TCA cycle pathway in differentiated myotube cells. The differential metabolites we identified can be characterized as biomarkers of different cellular states, and providing a new reference for MSTN and FGF5 dual-gene editing in regulation of muscle development. It may also provide a reference for the development of muscle regeneration drugs targeting biomarkers.

Quantitative comparison of bile acid glucuronides sub-metabolome between intrahepatic cholestasis and healthy pregnant women.

Because of the pathological indication and the physiological functions, bile acids (BAs) have occupied the research hotspot in recent decades. Although extensive efforts have been paid onto BAs sub-metabolome characterization, as the subfamily, BA glucuronides (gluA-BAs) profile is seldom concerned. Here, we made efforts to develop a LC-MS/MS program enabling quantitative gluA-BAs sub-metabolome characterization and to explore the differential species in serum between intrahepatic cholestasis of pregnancy (ICP) patients and healthy subjects. To gain as many authentic gluA-BAs as possible, liver microsomes from humans, rats, and mice were deployed to conjugate glucuronyl group to authentic BAs through in vitro incubation. Eighty gluA-BAs were captured and subsequently served as authentic compounds to correlate MS/MS spectral behaviors to structural features using squared energy-resolved MS program. Optimal collision energy (OCE) of [M-H]->[M-H-176.1]- was jointly administrated by [M-H]- mass and glucuronidation site, and identical exciting energies corresponding to 50% survival rate of 1st-generation fragment ion (EE50) were observed merely when the aglycone of a gluA-BA was consistent with the suspected structure. Through integrating high-resolution m/z, OCE, and EE50 information to identify gluA-BAs in a BAs pool, 97 ones were found and identified, and further, quantitative program was built for all annotated gluA-BAs by assigning OCEs to [M-H]->[M-H-176.1]- ion transitions. Quantitative gluA-BAs sub-metabolome of ICP was different from that of the healthy group. More GCDCA-3-G, GDCA-3-G, TCDCA-7-G, TDCA-3-G, and T-ß-MCA-3-G were distributed in the ICP group. Above all, this study not only offered a promising analytical tool for in-depth gluA-BAs sub-metabolome characterization, but also clarified gluA-BAs allowing the differentiation of ICP and healthy subjects.

Novel Insights into the Adverse Health Effects of per- and Polyfluoroalkyl Substances on the Kidney via Human Urine Metabolomics.

Per- and polyfluoroalkyl substances (PFAS) receive significant research attention due to their potential adverse effects on human health. Evidence shows that the kidney is one of the target organs of PFAS. In occupational exposure scenarios, high PFAS concentrations may adversely affect kidney metabolism, but whether this effect is reflected in the small metabolic molecules contained in urine remains unknown. In this study, 72 matched serum and urine samples from occupational workers of a fluorochemical manufactory as well as 153 urine samples from local residents were collected, and 23 PFAS levels were quantified. The concentrations of Σ23PFAS in the serum and urine samples of workers were 5.43 ± 1.02 µg/mL and 201 ± 46.9 ng/mL, respectively, while the Σ23PFAS concentration in the urine of the residents was 6.18 ± 0.76 ng/mL. For workers, high levels of urinary PFAS were strongly correlated with levels in serum (r = 0.57-0.93), indicating that urinary PFAS can be a good indicator for serum PFAS levels. Further, a urine nontargeted metabolomics study was conducted. The results of association models, including Bayesian kernel machine regression, demonstrated positive correlations between urinary PFAS levels and key small kidney molecules. A total of eight potential biomarkers associated with PFAS exposure were identified, and all of them showed significant positive correlations with markers of kidney function. These findings provide the first evidence that urine can serve as a matrix to indicate the adverse health effects of high levels of exposure to PFAS on the kidneys.

BART: A transferable liquid chromatography retention time library for bile acids.

Identification of unknown bile acids, especially the distinguishment between isomers, requires retention times of a large number of reference standards, which are often not commercially available. Meanwhile, published retention information cannot be directly transferred across labs due to the differences between liquid chromatography (LC) systems, such as different extra column volume and dwell volume. To improve this situation, a transferrable retention time library for bile acids named BART was developed. BART was composed of isocratic retention models of 272 bile acids and a software tool to predict their gradient retention times on various LC systems. The isocratic retention times of bile acids were acquired on a Waters BEH C18 column with mobile phases of acidic ammonium acetate buffer and acetonitrile, and fit to the quadratic solvent strength model (QSSM). Segmented linear gradient retention times were calculated with holdup time (t0), dwell time (tD) and actual gradient profile corrected using 21 bile acid calibration standards. In addition to the reference system where the isocratic retention times were acquired, this approach has been validated on four other LC-MS systems in four labs with two gradient methods. Average root mean square errors (RMSE) between predicted and experimental retention times were 0.052 and 0.054 min for the two gradients tested, which were 9-fold more accurate than referring to a static retention time library. The library is freely available at https://bafinder.github.io/.