Nuclear Pyruvate Dehydrogenase Complex Regulates Histone Acetylation and Transcriptional Regulation in the Ethylene Response.

Ethylene plays its essential roles in plant development, growth, and defense responses by controlling the transcriptional reprograming, in which EIN2-C-directed regulation of histone acetylation is the first key-step for chromatin to perceive ethylene signaling. But how the nuclear acetyl coenzyme A (acetyl CoA) is produced to ensure the ethylene-mediated histone acetylation is unknown. Here we report that ethylene triggers the accumulation of the pyruvate dehydrogenase complex (PDC) in the nucleus to synthesize nuclear acetyl CoA to regulate ethylene response. PDC is identified as an EIN2-C nuclear partner, and ethylene triggers its nuclear accumulation. Mutations in PDC lead to an ethylene-hyposensitivity that results from the reduction of histone acetylation and transcription activation. Enzymatically active nuclear PDC synthesize nuclear acetyl CoA for EIN2-C-directed histone acetylation and transcription regulation. These findings uncover a mechanism by which PDC-EIN2 converges the mitochondrial enzyme mediated nuclear acetyl CoA synthesis with epigenetic and transcriptional regulation for plant hormone response.

Quantification of aminobutyric acids and their clinical applications as biomarkers for osteoporosis.

Osteoporosis is a highly prevalent chronic aging-related disease that frequently is only detected after fracture. We hypothesized that aminobutyric acids could serve as biomarkers for osteoporosis. We developed a quick, accurate, and sensitive screening method for aminobutyric acid isomers and enantiomers yielding correlations with bone mineral density (BMD) and osteoporotic fracture. In serum, γ-aminobutyric acid (GABA) and (R)-3-aminoisobutyric acid (D-BAIBA) have positive associations with physical activity in young lean women. D-BAIBA positively associated with hip BMD in older individuals without osteoporosis/osteopenia. Lower levels of GABA were observed in 60-80 year old women with osteoporotic fractures. Single nucleotide polymorphisms in seven genes related to these metabolites associated with BMD and osteoporosis. In peripheral blood monocytes, dihydropyrimidine dehydrogenase, an enzyme essential to D-BAIBA generation, exhibited positive association with physical activity and hip BMD. Along with their signaling roles, BAIBA and GABA might serve as biomarkers for diagnosis and treatments of osteoporosis.

Targeted quantification of lipid mediators in skeletal muscles using restricted access media-based trap-and-elute liquid chromatography-mass spectrometry.

Lipid mediators (LMs) are a class of bioactive metabolites of the essential polyunsaturated fatty acids (PUFA), which are involved in many physiological processes. Their quantification in biological samples is critical for understanding their functions in lifestyle and chronic diseases, such as diabetes, as well allergies, cancers, and in aging processes. We developed a rapid, and sensitive LC-MS/MS method to quantify the concentrations of 14 lipid mediators of interest in mouse skeletal muscle tissue without time-consuming liquid-liquid or solid-phase extractions. A restricted-access media (RAM) based trap was used prior to LC-MS as cleanup process to prevent the analytical column from clogging and deterioration. The system enabled automatic removal of residual proteins and other biological interferences presented in the tissue extracts; the target analytes were retained in the trap and then eluted to an analytical column for separation. Matrix evaluation tests demonstrated that the use of the combined RAM trap and chromatographic separation efficiently eliminated the biological or chemical matrix interferences typically encountered in bioanalytical analysis. Using 14 LM standards and 12 corresponding deuterated compounds as internal standards, the five-point calibration curves, established over the concentration range of 0.031-320 ng mL-1, demonstrated good linearity of r2 > 0.9903 (0.9903-0.9983). The lower detection limits obtained were 0.016, 0.031, 0.062, and 0.31 ng mL-1 (0.5, 1, 2, and 10 pg on column), respectively, depending on the specific compounds. Good accuracy (87.1-114.5%) and precision (<13.4%) of the method were observed for low, medium, and high concentration quality control samples. The method was applied to measure the amount of 14 target LMs in mouse skeletal muscle tissues. All 14 analytes in this study were successfully detected and quantified in the gastrocnemius muscle samples, which provided crucial information for both age and gender-related aspects of LMs signaling in skeletal muscles previously unknown. This method could be applied to advance the understanding of skeletal muscle pathophysiology to study the role of LMs in health and disease. Furthermore, we will expand the application of this methodology to humans and other tissues/matrices in the near future.

Chromatographic separation of racemic praziquantel and its residual determination in perch by LC-MS/MS.

An enantioselective depletion study of praziquantel (PZQ) in perch muscle was done using a simple, sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. Two cyclic polypeptides and two cyclic saccharide chromatographic columns were evaluated. The new hydroxypropyl ß-cyclodextrin (HP-RSP) superficially porous particle (SPP) column produced the optimum separation and was selected for the study. Deuterium labeled PZQ-d11 was used as the internal standard. The method was linear over concentration ranges of 5.00-5.00×102µgL-1 (r2≥0.99) for (-)-R-PZQ and (+)-S-PZQ. The average recoveries of R-PZQ and S-PZQ at three spiked levels of 5.00, 50.00 and 5.00×102µgkg-1 ranged from 86.1% to 98.2%, and the intra-day and inter-day relative standard deviations were less than 5%. The decision limit (CCα) and detection capability (CCß) of R-PZQ and S-PZQ in perch muscle matrices were all 1.0µgkg-1 and 5.0µgkg-1, respectively. The method was successfully applied in monitoring the depletion of praziquantel enantiomers in perch muscle following oral administration. The elimination rate of R-PZQ and S-PZQ in perch muscle tissue is equivalent.

Methyl labeling and TROSY NMR spectroscopy of proteins expressed in the eukaryote Pichia pastoris.

(13)C Methyl TROSY NMR spectroscopy has emerged as a powerful method for studying the dynamics of large systems such as macromolecular assemblies and membrane proteins. Specific (13)C labeling of aliphatic methyl groups and perdeuteration has been limited primarily to proteins expressed in E. coli, preventing studies of many eukaryotic proteins of physiological and biomedical significance. We demonstrate the feasibility of efficient (13)C isoleucine δ1-methyl labeling in a deuterated background in an established eukaryotic expression host, Pichia pastoris, and show that this method can be used to label the eukaryotic protein actin, which cannot be expressed in bacteria. This approach will enable NMR studies of previously intractable targets.

Bulk derivatization and cation exchange restricted access media-based trap-and-elute liquid chromatography-mass spectrometry method for determination of trace estrogens in serum.

Estrone (E1), estradiols (α/ß-E2), and estriol (E3) are four major metabolically active estrogens exerting strong biological activities at very low circulating concentrations. This paper reports a sensitive and efficient method with automated, on-line clean-up and detection to determine trace estrogens in a small volume of serum samples using liquid chromatography-electrospray ionization-tandem mass spectrometry directly, without off-line liquid-liquid or solid-phase extraction pretreatments. Serum aliquots (charcoal stripped fetal bovine serum, 100 µL) were spiked with four estrogen standards and their corresponding isotope-labeled internal standards, then bulk derivatized with 2-fluoro-1-methyl-pyridium p-toluenesulfonate (2-FMP) to establish the calibration curves and perform method validation. Calibration was established in the concentration ranges of 5-1000 pg mL(-1), and demonstrated good linearity of R(2) from 0.9944 to 0.9997 for the four derivatized estrogens. The lower detection limits obtained were 3-7 pg mL(-1). Good accuracy and precision in the range of 86-112% and 2.3-11.9%, respectively, were observed for the quality control (QC) samples at low, medium, and high concentration levels. The stability tests showed that the derivatized serum samples were stable 8h after derivatization at room temperature and at least to 48 h if stored at -20 °C. The method was applied to measure trace estrogens in real human and bovine serum samples, and three of four estrogen compounds studied were observed and quantified.

Corticosteroid decomposition via a mixed anhydride.

Oxidation of dexamethasone in an aqueous suspension by air during prolonged storage produces the 17alpha-formyloxy-17beta-carboxylic acid 4. A pathway to 4 is proposed that involves Baeyer-Villiger-type oxidation of keto aldehyde 1 to mixed anhydride 5, followed by intramolecular formyl transfer. Synthetically, acid 3 was reacted with N,N'-carbonyldiimidazole followed by triethylammonium formate in order to generate the transient anhydride 5 en route to an authentic sample of 4.