Tautomerization and Isomerization in Quantitative NMR: A Case Study with 4-Deoxynivalenol (DON).

The regulated mycotoxin 4-deoxynivalenol (DON) has a heterocyclic structure that is readily amenable to tautomerization and conformational isomerization in solution. An analysis of DON in solution by NMR revealed the presence of hemiacetal tautomer(s) and putative conformational isomers, which maintain the intact enone functional group. The extent and type of tautomerization/isomerization vary according to the NMR solvent used and produce different signal patterns in the NMR spectra. Thus, the same proton produces multiple signals depending on which isomer/tautomer it belongs to. To maintain the accuracy of quantitative NMR (qNMR) measurements, it was essential to conclusively identify all signals belonging to the same proton to avoid underestimating its integral value. A strategy to overcome the complications of DON tautomerization and isomerization in solution during qNMR is reported. Of all proton atoms on the DON carbo-skeleton, H-10 produced clearly defined signals centered at 6.6 ppm for suspected conformational isomers and at 5.5 ppm for hemiacetal tautomers. To determine the purity of DON by quantitative proton NMR, the collective integrals of all isomeric and tautomeric signals belonging to H-10 provided the most accurate value. The purity of DON obtained with this protocol is highly accurate and suitable for the value assignment of certified reference materials (CRMs).

Improved Isolation Procedures for Okadaic Acid Group Toxins from Shellfish (Mytilus edulis) and Microalgae (Prorocentrum lima).

Okadaic acid (OA) group toxins may accumulate in shellfish and can result in diarrhetic shellfish poisoning when consumed by humans, and are therefore regulated. Purified toxins are required for the production of certified reference materials used to accurately quantitate toxin levels in shellfish and water samples, and for other research purposes. An improved procedure was developed for the isolation of dinophysistoxin-2 (DTX2) from shellfish (M. edulis), reducing the number of purification steps from eight to five, thereby increasing recoveries to ~68%, compared to ~40% in a previously reported method, and a purity of >95%. Cell densities and toxin production were monitored in cultures of Prorocentrum lima, that produced OA, DTX1, and their esters, over ~1.5 years with maximum cell densities of ~70,000 cells mL-1 observed. Toxin accumulation progressively increased over the study period, to ~0.7 and 2.1 mg L-1 of OA and DTX1 (including their esters), respectively, providing information on appropriate harvesting times. A procedure for the purification of OA and DTX1 from the harvested biomass was developed employing four purification steps, with recoveries of ~76% and purities of >95% being achieved. Purities were confirmed by LC-HRMS, LC-UV, and NMR spectroscopy. Additional stability observations led to a better understanding of the chemistry of these toxins.

Structure Elucidation and Relative Toxicity of (24R)-24-Hydroxyyessotoxin from a Namibian Isolate of Gonyaulax spinifera.

Liquid chromatography-high-resolution mass spectrometry (LC-HRMS) analysis of a Namibian strain of Gonyaulax spinifera showed the presence of a number of yessotoxins (YTXs). Principal among these were YTX (1), homoYTX (2), and a tentative hydroxylated analogue that did not correspond to any previously confirmed YTX structures. Culturing the G. spinifera strain afforded sufficient biomass for purification of the new analogue through a series of solvent partitioning and chromatographic steps, yielding ∼0.9 mg as a solid. NMR spectroscopy, ion-trap mass spectrometry, and HRMS identified the new analogue as 24-hydroxyYTX (7). Purified 24-hydroxyYTX was quantitated by NMR, and its relative toxicity evaluated using two embryonic zebrafish toxicity assays. 24-HydroxyYTX demonstrated reduced toxicity compared to YTX.

Identification of Host Proteins Predictive of Early Stage Mycobacterium tuberculosis Infection.

The objective of this study was to identify blood-based protein biomarkers of early stage Mycobacterium tuberculosis (Mtb) infection. We utilized plasma and serum specimens from TB patients and their contacts (age≥12) enrolled in a household contact study in Uganda. In the discovery phase cross-sectional samples from 104 HIV-uninfected persons classified as either active TB, latent Mtb infection (LTBI), tuberculin skin test (TST) converters, or persistent TST-negative were analyzed. Two hundred eighty-nine statistically significant (false discovery rate corrected p<0.05) differentially expressed proteins were identified across all comparisons. Proteins associated with cellular immunity and lipid metabolism were induced early after Mtb infection. One hundred and fifty-nine proteins were selected for a targeted mass spectrometry assay. A set of longitudinal samples from 52 TST-negative subjects who converted to TST-positive or remained TST-negative were analyzed, and multivariate logistic regression was used to identify unique protein panels able to predict TST conversion with cross-validated AUC>0.85. Panel performance was confirmed with an independent validation set of longitudinal samples from 16 subjects. These candidate protein biomarkers may allow for the identification of recently Mtb infected individuals at highest risk for developing active TB and most likely to benefit from preventive therapy.

Host Protein Biomarkers Identify Active Tuberculosis in HIV Uninfected and Co-infected Individuals.

Biomarkers for active tuberculosis (TB) are urgently needed to improve rapid TB diagnosis. The objective of this study was to identify serum protein expression changes associated with TB but not latent Mycobacterium tuberculosis infection (LTBI), uninfected states, or respiratory diseases other than TB (ORD). Serum samples from 209 HIV uninfected (HIV(-)) and co-infected (HIV(+)) individuals were studied. In the discovery phase samples were analyzed via liquid chromatography and mass spectrometry, and in the verification phase biologically independent samples were analyzed via a multiplex multiple reaction monitoring mass spectrometry (MRM-MS) assay. Compared to LTBI and ORD, host proteins were significantly differentially expressed in TB, and involved in the immune response, tissue repair, and lipid metabolism. Biomarker panels whose composition differed according to HIV status, and consisted of 8 host proteins in HIV(-) individuals (CD14, SEPP1, SELL, TNXB, LUM, PEPD, QSOX1, COMP, APOC1), or 10 host proteins in HIV(+) individuals (CD14, SEPP1, PGLYRP2, PFN1, VASN, CPN2, TAGLN2, IGFBP6), respectively, distinguished TB from ORD with excellent accuracy (AUC = 0.96 for HIV(-) TB, 0.95 for HIV(+) TB). These results warrant validation in larger studies but provide promise that host protein biomarkers could be the basis for a rapid, blood-based test for TB.

N-Heterocycles from chromium aminocarbenes.

The initial [2 + 2]-cycloadduct between a chromium aminocarbene and a tethered alkene undergoes a ß-hydrogen elimination very efficiently when triphenylphosphine is added as a ligand. The reaction gives cyclic enamines or homoenamines depending on the substitution on the alkene.

Elevated plasma gas6 levels are associated with venous thromboembolic disease.

Growth arrest-specific 6 (gas6), a novel vitamin K-dependent protein, has been demonstrated to have a role in thrombus stabilization as gas6 null mice are resistant to lethal venous and arterial thrombosis. However, the association between gas6 and venous thromboembolism has not been elucidated in humans. The present study aims to assess the role of gas6 in human venous thromboembolic (VTE) disease. Using a highly specific ELISA method, we measured plasma levels of gas6 in plasma samples obtained from 279 patients with VTE and 79 healthy volunteers. Medication history, comorbid conditions and VTE characteristics were documented. Mean gas6 levels were higher in patients with VTE as compared to healthy volunteers, being 46 ±11 ng/ml and 35 ±6.4 ng/ml respectively (P < 0.001). Odds ratios (OR) for VTE given elevated (≥90th percentile of healthy volunteers) gas6 levels were estimated in regression models in the whole study population. After adjustment for age, sex, medications and comorbidity, subjects with elevated gas6 had an increased risk of VTE (OR of 16.3 (95% CI 5.8-45.7, P < 0.001) compared to those with lower levels of gas6. This association remains significant even among patients with a comparable age distribution. Among patients with VTE, mean gas levels showed a trend of higher levels in those with more extensive thrombi. There was no correlation between elevated gas6 levels and recurrent VTE. In conclusion, we demonstrate an association between VTE and elevated gas6 levels consistent with in vivo murine models of thrombosis. This constitutes a potential novel mechanism for thrombosis in humans and may aid in the understanding of the pathophysiology of VTE.

Gas6-mediated signaling is dependent on the engagement of its gamma-carboxyglutamic acid domain with phosphatidylserine.

Gas6 is a vitamin K-dependent protein containing gamma-carboxyglutamic acid (Gla) at its N-terminus and a receptor binding domain at its C-terminus. Gas6-Axl binding is necessary but not sufficient to support endothelial cell survival as decarboxylated gas6 inhibits the pro-survival function of gas6 by binding and inhibiting Axl, even though decarboxylated gas6 cannot support endothelial cell survival itself. It is hypothesized that interactions between the Gla domain of gas6 and phosphatidylserine (PS), though not required for gas6 binding to Axl, are necessary for gas6-Axl function. In support of this hypothesis are results showing that (1) two specific inhibitors of Gla-PS interactions, namely soluble PS and Annexin V, abrogate gas6-mediated endothelial cell survival and (2) Soluble PS inhibits Akt activation, a downstream intracellular event triggered by gas6-Axl binding. In conclusion, we propose a heretofore unknown function of Gla, where Gla-PS binding on the N-terminus of gas6 is necessary for a gas6 function mediated through its binding to Axl via its C-terminus.

The Gla domain of factor IXa binds to factor VIIIa in the tenase complex.

During blood coagulation factor IXa binds to factor VIIIa on phospholipid membranes to form an enzymatic complex, the tenase complex. To test whether there is a protein-protein contact site between the gamma-carboxyglutamic acid (Gla) domain of factor IXa and factor VIIIa, we demonstrated that an antibody to the Gla domain of factor IXa inhibited factor VIIIa-dependent factor IXa activity, suggesting an interaction of the factor IXa Gla domain with factor VIIIa. To study this interaction, we synthesized three analogs of the factor IXa Gla domain (FIX1-47) with Phe-9, Phe-25, or Val-46 replaced, respectively, with benzoylphenylalanine (BPA), a photoactivatable cross-linking reagent. These factor IX Gla domain analogs maintain native tertiary structure, as demonstrated by calcium-induced fluorescence quenching and phospholipid binding studies. In the absence of phospholipid membranes, FIX1-47 was able to inhibit factor IXa activity. This inhibition is dependent on the presence of factor VIIIa, suggesting a contact site between the factor IXa Gla domain and factor VIIIa. To demonstrate a direct interaction we did cross-linking experiments with FIX1-479BPA, FIX1-4725BPA, and FIX1-4746BPA. Covalent cross-linking to factor VIIIa was observed primarily with FIX1-4725BPA and to a much lesser degree with FIX1-4746BPA. Immunoprecipitation experiments with an antibody to the C2 domain of factor VIIIa indicate that the factor IX Gla domain cross-links to the A3-C1-C2 domain of factor VIIIa. These results suggest that the factor IXa Gla domain contacts factor VIIIa in the tenase complex through a contact site that includes phenylalanine 25 and perhaps valine 46.