Analysis of Mitragynine and Speciociliatine in Umbilical Cord by LC-MS-MS for Detecting Prenatal Exposure to Kratom.

Kratom is an herbal drug that is legal in the USA. While it is marketed as a safer alternative to opioids, it can cause opioid-like withdrawal symptoms when discontinued after regular use. Several case studies have shown that kratom exposure in utero can lead to symptoms in newborns consistent with neonatal abstinence syndrome. Here, we present a validated method for the detection of kratom in umbilical cord by liquid chromatography--tandem mass spectrometry. The umbilical cord is homogenized in solvent and kratom analytes are purified by solid phase extraction (strong cation exchange). Diastereomeric kratom alkaloids mitragynine (MG), speciociliatine (SC), speciogynine and mitraciliatine are separated by reverse phase chromatography on a phenyl-hexyl column. Applying this method to residual umbilical cords submitted to our laboratory for drug testing, 29 positive specimens exhibiting varied kratom analyte distributions were observed. MG and SC were the most abundant kratom analytes and were selected as biomarkers of kratom exposure. A cutoff concentration of 0.08 ng/g was established for both MG and SC.

Measurement of Urinary Free Cortisol and Cortisone by LC-MS/MS.

Monitoring urinary free cortisol (UFC) excretion helps assess adrenal function and is used to screen for endogenous Cushing's syndrome caused by an adrenal or pituitary tumor. While serum cortisol levels fluctuate in response to time of day, stress, and concentrations of cortisol-binding globulin (CBG), a 24-h urine collection measures the cortisol produced over the entire day and does not suffer from as much variability as a serum measurement.We describe here a method of measurement of urinary free cortisol (UFC) and cortisone using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Urine samples, combined with stable isotope-labeled internal standards, are extracted by liquid-liquid extraction using ethyl acetate and hexane. An API 5500 mass spectrometer operated in positive atmospheric pressure chemical ionization (APCI) mode is used for detection.

Quantitation of Fatty Acids in Serum/Plasma and Red Blood Cells by Gas Chromatography-Negative Chemical Ionization-Mass Spectrometry.

Quantitation of long-chain fatty acids in serum/plasma and red blood cells is a useful diagnostic tool in the evaluation of nutritional status and assessment of risk for essential fatty acid deficiency (EFAD). Serum/plasma has been the traditional sample type for this method, yet it requires prolonged fasting which is not compatible with some patient populations. More recently, red blood cells have become an important sample type due to less intraindividual variability and obviating the need for fasting. Here we present a method for the quantitation of 22 fatty acids in serum/plasma or red blood cells. Fatty acids are hydrolyzed and extracted from the biological matrix, followed by derivatization with pentafluorobenzyl bromide and subsequent analysis by gas chromatography-negative chemical ionization-mass spectrometry (GC-NCI-MS).

LC-MS/MS Method for Measurement of Thiopurine Nucleotides (TN) in Erythrocytes and Association of TN Concentrations With TPMT Enzyme Activity.

Monitoring concentrations of thiopurine metabolites is used clinically to prevent adverse effects in patients on thiopurine drug therapy. We developed a LC-MS/MS method for the quantification of 6-thioguanine (6-TG) and 6-methylmercaptopurine (6-MMP) in red blood cells (RBCs). This method utilizes an automated cell washer for RBC separation from whole blood samples and washing of the separated RBCs. The lower limit of quantification of the method was 0.2 µmol/L for 6-TG (∼50 pmol/8 × 108 RBC) and 4 µmol/L for 6-MMP (∼1,000 pmol/8 × 108 RBC). The total imprecision of the assay was <3.0%. The upper limit of linearity for 6-TG and 6-MMP was 7.5 µmol/L and 150 µmol/L, respectively. The stability of the thiopurine metabolites under pre- and post-analytically relevant conditions was also evaluated. A good agreement was observed between this method and validated LC-MS/MS methods from three laboratories, except for ∼40% low bias for 6-MMP observed in one of the methods. The assessment of the association between 6-TG and 6-MMP concentrations with thiopurine S-methyltransferase (TPMT) phenotype and genotype demonstrated a statistically significant difference in the thiopurine metabolite concentrations between the TPMT groups with normal and intermediate activity of 6-MMP (p < 0.0001), while the difference in 6-TG concentrations was statistically not significant (p = 0.096). Among the samples with normal TPMT activity, higher concentrations of 6-MMP (p = 0.015) were observed in pediatric samples than in the samples of adults. No statistically significant differences were observed in the distributions of 6-TG and 6-MMP concentrations among the evaluated genotypes.

Establishing age-stratified red blood cell fatty acid reference ranges using model-based clustering and iterative application of the harris-boyd method.

BACKGROUND: The current assessment of nutritional status and diagnosis of essential fatty acids deficiency (EFAD) utilizes the analysis of long-chain fatty acids (LCFAs) in serum or plasma; however, these concentrations do not represent habitual LCFA intake. LCFAs in red blood cells (RBCs) are less prone to intra-individual variability and exclude the need for fasting, which is unrealistic in pediatric populations. Our study objective was to characterize the RBC LCFA profiles in pediatric and adult reference populations and establish age-specific reference intervals (RIs). METHODS: Twenty-one LCFAs in RBCs were measured in 523 pediatric and adult controls by gas chromatography-mass spectrometry. Model-based clustering was used to identify possible age subgroups. After removing outliers by the Tukey method, initial age subgroups were then compared using the Harris-Boyd method in an iterative manner. RIs (95%), with confidence intervals (90%), in the final age groups were established using parametric or non-parametric statistics. RESULTS: Our data showed heterogeneous changes in the concentrations of most LCFAs and the EFAD biomarkers (mead acid, Triene/Tetraene ratio) during infancy. Model-based clustering identified six initial age subgroups per fatty acid, on average. Our application of the iterative Harris-Boyd method decreased the average number of age groups to three per fatty acid, with 13 total unique age cut-offs. Finally, using these age groups, we established age-specific RIs for 21 fatty acids, six group totals, and the Triene/Tetraene ratio. CONCLUSION: Our study revealed significant age-dependent changes in RBC fatty acid profiles warranting separate pediatric and adults RIs. Model-based clustering and the iterative application of the Harris-Boyd method were successfully used to establish RBC fatty acid RIs for an objective assessment of long-term nutritional status in pediatric and adult populations.

Preserving Vascular Integrity Protects Mice against Multidrug-Resistant Gram-Negative Bacterial Infection.

The rise in multidrug-resistant (MDR) organisms portends a serious global threat to the health care system with nearly untreatable infectious diseases, including pneumonia and its often fatal sequelae, acute respiratory distress syndrome (ARDS) and sepsis. Gram-negative bacteria (GNB), including Acinetobacter baumannii, Pseudomonas aeruginosa, and carbapenemase-producing Klebsiella pneumoniae (CPKP), are among the World Health Organization's and National Institutes of Health's high-priority MDR pathogens for targeted development of new therapies. Here, we show that stabilizing the host's vasculature by genetic deletion or pharmacological inhibition of the small GTPase ADP-ribosylation factor 6 (ARF6) increases survival rates of mice infected with A. baumannii, P. aeruginosa, and CPKP. We show that the pharmacological inhibition of ARF6-GTP phenocopies endothelium-specific Arf6 disruption in enhancing the survival of mice with A. baumannii pneumonia, suggesting that inhibition is on target. Finally, we show that the mechanism of protection elicited by these small-molecule inhibitors acts by the restoration of vascular integrity disrupted by GNB lipopolysaccharide (LPS) activation of the TLR4/MyD88/ARNO/ARF6 pathway. By targeting the host's vasculature with small-molecule inhibitors of ARF6 activation, we circumvent microbial drug resistance and provide a potential alternative/adjunctive treatment for emerging and reemerging pathogens.

Coordination of the filament stabilizing versus destabilizing activities of cofilin through its secondary binding site on actin.

Cofilin is a ubiquitous modulator of actin cytoskeleton dynamics that can both stabilize and destabilize actin filaments depending on its concentration and/or the presence of regulatory co-factors. Three charge-reversal mutants of yeast cofilin, located in cofilin's filament-specific secondary binding site, were characterized in order to understand why disruption of this site leads to enhanced filament disassembly. Crystal structures of the mutants showed that the mutations specifically affect the secondary actin-binding interface, leaving the primary binding site unaltered. The mutant cofilins show enhanced activity compared to wild-type cofilin in severing and disassembling actin filaments. Electron microscopy and image analysis revealed long actin filaments in the presence of wild-type cofilin, while the mutants induced many short filaments, consistent with enhanced severing. Real-time fluorescence microscopy of labeled actin filaments confirmed that the mutants, unlike wild-type cofilin, were functioning as constitutively active severing proteins. In cells, the mutant cofilins delayed endocytosis, which depends on rapid actin turnover. We conclude that mutating cofilin's secondary actin-binding site increases cofilin's ability to sever and de-polymerize actin filaments. We hypothesize that activators of cofilin severing, like Aip1p, may act by disrupting the interface between cofilin's secondary actin-binding site and the actin filament.

Structural biology of the proteasome.

The proteasome refers to a collection of complexes centered on the 20S proteasome core particle (20S CP), a complex of 28 subunits that houses proteolytic sites in its hollow interior. Proteasomes are found in eukaryotes, archaea, and some eubacteria, and their activity is critical for many cellular pathways. Important recent advances include inhibitor binding studies and the structure of the immunoproteasome, whose specificity is altered by the incorporation of inducible catalytic subunits. The inherent repression of the 20S CP is relieved by the ATP-independent activators 11S and Blm10/PA200, whose structures reveal principles of proteasome mechanism. The structure of the ATP-dependent 19S regulatory particle, which mediates degradation of polyubiquitylated proteins, is being revealed by a combination of crystal or NMR structures of individual subunits and electron microscopy reconstruction of the intact complex. Other recent structural advances inform us about mechanisms of assembly and the role of conformational changes in the functional cycle.

Structure of a proteasome Pba1-Pba2 complex: implications for proteasome assembly, activation, and biological function.

The 20S proteasome is an essential, 28-subunit protease that sequesters proteolytic sites within a central chamber, thereby repressing substrate degradation until proteasome activators open the entrance/exit gate. Two established activators, Blm10 and PAN/19S, induce gate opening by binding to the pockets between proteasome α-subunits using C-terminal HbYX (hydrophobic-tyrosine-any residue) motifs. Equivalent HbYX motifs have been identified in Pba1 and Pba2, which function in proteasome assembly. Here, we demonstrate that Pba1-Pba2 proteins form a stable heterodimer that utilizes its HbYX motifs to bind mature 20S proteasomes in vitro and that the Pba1-Pba2 HbYX motifs are important for a physiological function of proteasomes, the maintenance of mitochondrial function. Other factors that contribute to proteasome assembly or function also act in the maintenance of mitochondrial function and display complex genetic interactions with one another, possibly revealing an unexpected pathway of mitochondrial regulation involving the Pba1-Pba2 proteasome interaction. Our determination of a proteasome Pba1-Pba2 crystal structure reveals a Pba1 HbYX interaction that is superimposable with those of known activators, a Pba2 HbYX interaction that is different from those reported previously, and a gate structure that is disrupted but not sufficiently open to allow entry of even small peptides. These findings extend understanding of proteasome interactions with HbYX motifs and suggest multiple roles for Pba1-Pba2 interactions throughout proteasome assembly and function.

Interaction of the Thermoplasma acidophilum A1A0-ATP synthase peripheral stalk with the catalytic domain.

The peripheral stalk of the archaeal ATP synthase (A1A0)-ATP synthase is formed by the heterodimeric EH complex and is part of the stator domain, which counteracts the torque of rotational catalysis. Here we used nuclear magnetic resonance spectroscopy to probe the interaction of the C-terminal domain of the EH heterodimer (E(CT1)H(CT)) with the N-terminal 23 residues of the B subunit (B(NT)). The data show a specific interaction of B(NT) peptide with 26 residues of the E(CT1)H(CT) domain, thereby providing a molecular picture of how the peripheral stalk is anchored to the A3B3 catalytic domain in A1A0.

Domain architecture of the stator complex of the A1A0-ATP synthase from Thermoplasma acidophilum.

A key structural element in the ion translocating F-, A-, and V-ATPases is the peripheral stalk, an assembly of two polypeptides that provides a structural link between the ATPase and ion channel domains. Previously, we have characterized the peripheral stalk forming subunits E and H of the A-ATPase from Thermoplasma acidophilum and demonstrated that the two polypeptides interact to form a stable heterodimer with 1:1 stoichiometry (Kish-Trier, E., Briere, L. K., Dunn, S. D., and Wilkens, S. (2008) J. Mol. Biol. 375, 673-685). To define the domain architecture of the A-ATPase peripheral stalk, we have now generated truncated versions of the E and H subunits and analyzed their ability to bind each other. The data show that the N termini of the subunits form an alpha-helical coiled-coil, approximately 80 residues in length, whereas the C-terminal residues interact to form a globular domain containingalpha- and beta-structure. We find that the isolated C-terminal domain of the E subunit exists as a dimer in solution, consistent with a recent crystal structure of the related Pyrococcus horikoshii A-ATPase E subunit (Lokanath, N. K., Matsuura, Y., Kuroishi, C., Takahashi, N., and Kunishima, N. (2007) J. Mol. Biol. 366, 933-944). However, upon the addition of a peptide comprising the C-terminal 21 residues of the H subunit (or full-length H subunit), dimeric E subunit C-terminal domain dissociates to form a 1:1 heterodimer. NMR spectroscopy was used to show that H subunit C-terminal peptide binds to E subunit C-terminal domain via the terminal alpha-helices, with little involvement of the beta-sheet region. Based on these data, we propose a structural model of the A-ATPase peripheral stalk.

Structure of the yeast vacuolar ATPase.

The subunit architecture of the yeast vacuolar ATPase (V-ATPase) was analyzed by single particle transmission electron microscopy and electrospray ionization (ESI) tandem mass spectrometry. A three-dimensional model of the intact V-ATPase was calculated from two-dimensional projections of the complex at a resolution of 25 angstroms. Images of yeast V-ATPase decorated with monoclonal antibodies against subunits A, E, and G position subunit A within the pseudo-hexagonal arrangement in the V1, the N terminus of subunit G in the V1-V0 interface, and the C terminus of subunit E at the top of the V1 domain. ESI tandem mass spectrometry of yeast V1-ATPase showed that subunits E and G are most easily lost in collision-induced dissociation, consistent with a peripheral location of the subunits. An atomic model of the yeast V-ATPase was generated by fitting of the available x-ray crystal structures into the electron microscopy-derived electron density map. The resulting atomic model of the yeast vacuolar ATPase serves as a framework to help understand the role the peripheral stalk subunits are playing in the regulation of the ATP hydrolysis driven proton pumping activity of the vacuolar ATPase.

The stator complex of the A1A0-ATP synthase--structural characterization of the E and H subunits.

Archaeal ATP synthase (A-ATPase) is the functional homolog to the ATP synthase found in bacteria, mitochondria and chloroplasts, but the enzyme is structurally more related to the proton-pumping vacuolar ATPase found in the endomembrane system of eukaryotes. We have cloned, overexpressed and characterized the stator-forming subunits E and H of the A-ATPase from the thermoacidophilic Archaeon, Thermoplasma acidophilum. Size exclusion chromatography, CD, matrix-assisted laser desorption ionization time-of-flight mass spectrometry and NMR spectroscopic experiments indicate that both polypeptides have a tendency to form dimers and higher oligomers in solution. However, when expressed together or reconstituted, the two individual polypeptides interact with high affinity to form a stable heterodimer. Analyses by gel filtration chromatography and analytical ultracentrifugation show the heterodimer to have an elongated shape, and the preparation to be monodisperse. Thermal denaturation analyses by CD and differential scanning calorimetry revealed the more cooperative unfolding transitions of the heterodimer in comparison to those of the individual polypeptides. The data are consistent with the EH heterodimer forming the peripheral stalk(s) in the A-ATPase in a fashion analogous to that of the related vacuolar ATPase.