Stereoselective Amine-omics Using Heavy Atom Isotope Labeled l- and d-Marfey's Reagents.

Biological amines and amino acids play essential roles in many biochemical processes. The chemical complexity of biological samples is challenging, and the selective identification and quantification of amines and amino acid stereoisomers would be very useful for amine-focused "amino-omics" studies. Many amines and amino acids are chiral, and their stereoisomers cannot be resolved on achiral media without chiral derivatization.

Deconvolution of multichannel LC-MS/MS chromatograms of glucosamine-phosphates: Evidence of a GlmS regulatory difference between and .

Resolving isomeric analytes is challenging given their physical similarity - making chromatographic resolution difficult, and their identical masses - making simple mass resolution impossible. MS/MS data provides a means to resolve isomeric analytes if their MS/MS intensity profiles are sufficiently different. Glucosamine-6-phosphate (GlcN-6P) and glucosamine-1-phosphate (GlcN-1P) are early bacterial cell wall intermediates.

Screening the NCI diversity set V for anti-MRSA activity: cefoxitin synergy and LC-MS/MS confirmation of folate/thymidine biosynthesis inhibition.

New antibacterial agents are urgently needed to counter increasingly resistant bacteria. One approach to this problem is library screening for new antibacterial agents. Library screening efforts can be improved by increasing the information content of the screening effort.

Electrosynthesis and Microanalysis in Thin Layer: An Electrochemical Pipette for Rapid Electrolysis and Mechanistic Study of Electrochemical Reactions.

Electrochemistry represents unique approaches for the promotion and mechanistic study of chemical reactions and has garnered increasing attention in different areas of chemistry. This expansion necessitates the enhancement of the traditional electrochemical cells that are intrinsically constrained by mass transport limitations. Herein, we present an approach for designing an electrochemical cell by limiting the reaction chamber to a thin layer of solution, comparable to the thickness of the diffusion layer.

Synergistic Combinations of FDA-Approved Drugs with Ceftobiprole against Methicillin-Resistant Staphylococcus aureus.

New strategies are urgently needed to address the public health threat of antimicrobial resistance. Synergistic agent combinations provide one possible pathway toward addressing this need and are also of fundamental mechanistic interest. Effective methods for comprehensively identifying synergistic agent combinations are required for such efforts.

Library Screening for Synergistic Combinations of FDA-Approved Drugs and Metabolites with Vancomycin against VanA-Type Vancomycin-Resistant Enterococcus faecium.

Antimicrobial resistance is a major public health threat, and there is an urgent need for new strategies to address this issue. In a recent study, a library screening strategy was developed in which an FDA-approved drug library was screened against methicillin-resistant Staphylococcus aureus (MRSA) in both its original (unmetabolized [UM]) and its human liver microsome metabolized (postmetabolized [PM]) forms and in the absence and presence of a resistant-to antibiotic. This allows the identification of agents with active metabolites and agents that can act synergistically with the resistant-to antibiotic.

Effect of Vancomycin on Cytoplasmic Peptidoglycan Intermediates and Operon mRNA Levels in VanA-Type Vancomycin-Resistant Enterococcus faecium.

Resistance in VanA-type vancomycin-resistant Enterococcus faecium (VREfm) is due to an inducible gene cassette encoding seven proteins (). This provides for an alternative peptidoglycan (PG) biosynthesis pathway whereby D-Ala-D-Ala is replaced by D-Ala-d-lactate (Lac), to which vancomycin cannot bind effectively. This study aimed to quantify cytoplasmic levels of normal and alternative pathway PG intermediates in VanA-type VREfm by liquid chromatography-tandem mass spectrometry before and after vancomycin exposure and to correlate these changes with changes in operon mRNA levels measured by real-time quantitative PCR (RT-qPCR).

Dimensionally Enhanced Antibacterial Library Screening.

The emergence and spread of antimicrobial resistance is a major public health threat, and there is an urgent need to develop new strategies to address the issue. In this study, the possibility of enhancing a whole cell based antibacterial library screen by increasing the dimensionality of the screening effort is explored using methicillin-resistant (MRSA) as the target organism. One dimension involved generating and screening a human liver microsome metabolized FDA approved drug library.

LC-MS/MS-Based Separation and Quantification of Marfey's Reagent Derivatized Proteinogenic Amino Acid DL-Stereoisomers.

D-Amino acids are important biological molecules. Improved analytical methods for their resolution and quantification remain of keen interest. In this study, we investigated the use of Marfey's reagent (chiral) derivatization coupled with LC-MS/MS-based separation and detection of the resulting diastereomers for quantification of the 19 common L- and D-amino acids and glycine.

Efficient Purification and LC-MS/MS-based Assay Development for Ten-Eleven Translocation-2 5-Methylcytosine Dioxygenase.

The epigenetic transcription regulation mediated by 5-methylcytosine (5mC) has played a critical role in eukaryotic development. Demethylation of these epigenetic marks is accomplished by sequential oxidation by ten-eleven translocation dioxygenases (TET1-3), followed by the thymine-DNA glycosylase-dependent base excision repair. Inactivation of the TET2 gene due to genetic mutations or by other epigenetic mechanisms is associated with a poor prognosis in patients with diverse cancers, especially hematopoietic malignancies.

Derivation and numerical profile analysis of a hierarchically formulated microscopic model of hemoglobin oxygen binding.

To address complex thermodynamic systems with multiple interacting events, we have developed the concept of hierarchical thermodynamic interactions. In this study, this concept is extended to protein-ligand systems with similar but not identical protein subunits, and applied to the analysis of previously published NMR and UV-vis monitored hemoglobin oxygen binding data. Non-linear regression provided estimated errors for statistically significant parameters, but not for null (zero) valued parameters.

β-aminoisobutyric Acid, l-BAIBA, Is a Muscle-Derived Osteocyte Survival Factor.

Exercise has beneficial effects on metabolism and on tissues. The exercise-induced muscle factor β-aminoisobutyric acid (BAIBA) plays a critical role in the browning of white fat and in insulin resistance. Here we show another function for BAIBA, that of a bone-protective factor that prevents osteocyte cell death induced by reactive oxygen species (ROS).

Antibiotic Effects on Methicillin-Resistant Staphylococcus aureus Cytoplasmic Peptidoglycan Intermediate Levels and Evidence for Potential Metabolite Level Regulatory Loops.

Cytoplasmic peptidoglycan (PG) precursor levels were determined in methicillin-resistant (MRSA) after exposure to several cell wall-targeting antibiotics. Three experiments were performed: (i) exposure to 4× MIC levels (acute); (ii) exposure to sub-MIC levels (subacute); (iii) a time course experiment of the effect of vancomycin. In acute exposure experiments, fosfomycin increased UDP-GlcNAc, as expected, and resulted in substantially lower levels of total UDP-linked metabolite accumulation relative to other pathway inhibitors, indicating reduced entry into this pathway.

Convenient expression, purification and quantitative liquid chromatography-tandem mass spectrometry-based analysis of TET2 5-methylcytosine demethylase.

5-Methylcytosine within CpG islands in DNA plays a crucial role in epigenetic transcriptional regulation during metazoan development. Recently, it has been established that the Ten-Eleven Translocation (TET) family, Fe(II)- and 2-oxoglutarate (2OG/αKG)-dependent oxygenases initiate 5-methylcytosine demethylation by iterative oxidation reactions. Mutations in the TET2 gene are frequently detected in patients with myeloid malignancies.

Gaussian and linear deconvolution of LC-MS/MS chromatograms of the eight aminobutyric acid isomers.

Isomeric molecules present a challenge for analytical resolution and quantification, even with MS-based detection. The eight aminobutyric acid (ABA) isomers are of interest for their various biological activities, particularly γ-aminobutyric acid (GABA) and the d- and l-isomers of β-aminoisobutyric acid (β-AIBA; BAIBA). This study aimed to investigate LC-MS/MS-based resolution of these ABA isomers as their Marfey's (Mar) reagent derivatives.

Cytoplasmic peptidoglycan intermediate levels in Staphylococcus aureus.

Intracellular cytoplasmic peptidoglycan (PG) intermediate levels were determined in Staphylococcus aureus during log-phase growth in enriched media. Levels of UDP-linked intermediates were quantitatively determined using ion pairing LC-MS/MS in negative mode, and amine intermediates were quantitatively determined stereospecifically as their Marfey's reagent derivatives in positive mode. Levels of UDP-linked intermediates in S.

Crocetinic acid inhibits hedgehog signaling to inhibit pancreatic cancer stem cells.

Pancreatic cancer is the fourth leading cause of cancer deaths in the US and no significant treatment is currently available. Here, we describe the effect of crocetinic acid, which we purified from commercial saffron compound crocetin using high performance liquid chromatography. Crocetinic acid inhibits proliferation of pancreatic cancer cell lines in a dose- and time-dependent manner.

Ion-pairing liquid chromatography-tandem mass spectrometry-based quantification of uridine diphosphate-linked intermediates in the Staphylococcus aureus cell wall biosynthesis pathway.

Bacterial cell wall biosynthesis is the target of several antibiotics and is of interest as a target for new inhibitor development. The cytoplasmic steps of this pathway involve a series of uridine diphosphate (UDP)-linked peptidoglycan intermediates. Quantification of these intermediates is essential for studies of current agents targeting this pathway and for the development of new agents targeting this pathway.

A liquid chromatography-tandem mass spectrometry assay for d-Ala-d-Lac: a key intermediate for vancomycin resistance in vancomycin-resistant enterococci.

Vancomycin exerts its antibacterial activity by binding to d-Ala-d-Ala in bacterial cell wall precursors. Vancomycin resistance in vancomycin-resistant enterococci (VRE) is due to an alternative cell wall biosynthesis pathway in which d-Ala-d-Ala is replaced, most commonly by d-Ala-d-Lac. In this study, we extend our recently developed Marfey's derivatization-based liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for l-Ala, d-Ala, and d-Ala-d-Ala to d-Ala-d-Lac and apply it to the quantitation of these metabolites in VRE.

Jatropha curcas: from biodiesel generation to medicinal applications.

Jatropha curcas (JC) is a multipurpose perennial plant that belongs to the Euphorbiaceae family and is native to arid and semiarid tropical regions worldwide. It has many attributes and considerable potential for renewable energy, fish and livestock feeding. Despite its rich application as a renewable source and for animal feeding, JC has barely been explored for its medicinal potential.

A liquid chromatography-tandem mass spectrometry assay for detection and quantitation of the dipeptide Gly-Gln in rat brain.

The enzymatic cleavage products of β-endorphin (β-endorphin1-27 and Gly-Gln) reduce voluntary alcohol consumption in alcohol-preferring (P) rats. Gly-Gln also inhibits the reward-benefiting effects of morphine and nicotine. It would be useful for the investigation of these effects to have an analytical method suitable for Gly-Gln detection and quantitation.