Nuclear Magnetic Resonance Assays Research Articles

Searching for Frataxin Function: Exploring the Analogy with Nqo15, the Frataxin-like Protein of Respiratory Complex I from Thermus thermophilus.

Nqo15 is a subunit of respiratory complex I of the bacterium Thermus thermophilus, with strong structural similarity to human frataxin (FXN), a protein involved in the mitochondrial disease Friedreich's ataxia (FRDA). Recently, we showed that the expression of recombinant Nqo15 can ameliorate the respiratory phenotype of FRDA patients' cells, and this prompted us to further characterize both the Nqo15 solution's behavior and its potential functional overlap with FXN, using a combination of in silico and in vitro techniques. We studied the analogy of Nqo15 and FXN by performing extensive database searches based on sequence and structure. Nqo15's folding and flexibility were investigated by combining nuclear magnetic resonance (NMR), circular dichroism, and coarse-grained molecular dynamics simulations. Nqo15's iron-binding properties were studied using NMR, fluorescence, and specific assays and its desulfurase activation by biochemical assays. We found that the recombinant Nqo15 isolated from complex I is monomeric, stable, folded in solution, and highly dynamic. Nqo15 does not share the iron-binding properties of FXN or its desulfurase activation function.

Separation of avibactam by gemini quaternary ammonium salt: Extraction, precipitation, and application in large-scale production

β-lactamase inhibitors have important clinical significance to restore the antibacterial effect of antibiotics. Non-β-lactam β-lactamase inhibitor represented by avibactam contained a sulfonic acid group, which posed a considerable challenge to separation and purification. To separate avibactam, eight gemini quaternary ammonium salts were developed for extraction, and the extraction conditions were investigated and optimized. Notably, some gemini quaternary ammonium salts can directly precipitate avibactam in aqueous solution with the excellent yield and purity. The probable mechanism of precipitation was studied by critical micelle concentration, transmission electron microscopy, and nuclear magnetic resonance assays. The precipitation process of gemini quaternary ammonium salt N,N,N,N′,N′,N′-hexabutylbutanediammonium dibromide was successfully applied in separation and purification of a 2 kg avibactam production, showing unique efficient and green advantages. Our research offered an effective and scale-up approach to separation and purification of β-lactamase inhibitor containing sulfonic acid group.

A-207 Analytical Validation of Helena SPIFE Touch to Determine ApoB-Containing Lipoproteins Lp(a)-P, LDL-P and VLDL-P

Abstract Background Elevated Lp(a) is an independent risk factor for atherosclerotic (ASCVD) events and associates with aortic stenosis. Accurate immunochemical measurement of Lp(a) is complicated by heterogeneity of Lp(a) molecular size. The objective of this study was to validate the analytical performance of an electrophoretic assay for measurement of ApoB-containing lipoprotein particles including Lp(a)-P, LDL-P, and VLDL-P and to compare results to other standard clinical methods. Method Lp(a)-P, LDL-P, and VLDL-P were quantified by electrophoretic serum separation followed by immuno-staining with an ApoB antibody using the SPIFE Touch (Helena Laboratories, Beaumont, TX). Stability was evaluated by comparison to results at time zero (n = 10). Precision was assessed by 5 measurements twice daily for five days. Limit of quantitation (LoQ) was determined by 20 replicate measurements. Linearity was assessed by mixing high and low concentrations at various ratios. Accuracy was assessed by split sample comparison (n = 126) with Helena Laboratories. Lp(a)-P and LDL-P results were compared to nuclear magnetic resonance (NMR) using a Bruker Ascend 600 NMR (Bruker, Billerica, MA) with AXINON software (numares, AG, Regensberg, Germany) (n = 114). Lp(a)-P is included with LDL-P by NMR. Therefore, the SPIFE Lp(a)-P + LDL-P was compared to NMR LDL-P. SPIFE Lp(a)-P was compared directly to Roche cobas c501 Lp(a) immunoassay calibrated to molar units (Roche Diagnostics, Indianapolis, IN) (n = 126). Results The analytical performance of Lp(a)-P, LDL-P, and VLDL-P measured on the SPIFE Touch met all acceptance criteria. Bias to the reference method was <20% with R2 > 0.98. All analytes were stable 14 days refrigerated and 14 days frozen. Lipoprotein particle measurements on SPIFE Touch correlated to those obtained by both NMR and Roche assays. Conclusion The analytical performance of the SPIFE Touch for measuring the concentration of ApoB-containing lipoproteins is acceptable. Further studies are needed to determine clinical implications and utility compared to NMR and Roche assays.

Quantitative 1 H Nuclear Magnetic Resonance Assay for the Rapid Detection of Pyrazinamide Resistance in Mycobacterium tuberculosis from Sputum Samples

Tuberculosis (TB), caused by Mycobacterium tuberculosis, is one of the 10 leading killer diseases in the world. At least one-quarter of the population has been infected, and there are 1.3 million deaths annually. The emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains challenges TB treatments. One of the drugs widely used in first- and second-line regimens is pyrazinamide (PZA). Statistically, 50% of MDR and 90% of XDR clinical strains are resistant to PZA, and recent studies have shown that its use in patients with PZA-resistant strains is associated with higher mortality rates. Therefore, the is an urgent need for the development of an accurate and efficient PZA susceptibility assay. PZA crosses the M. tuberculosis membrane and is hydrolyzed to its active form, pyrazinoic acid (POA), by a nicotinamidase encoded by the pncA gene. Up to 99% of clinical PZA-resistant strains have mutations in this gene, suggesting that this is the most likely mechanism of resistance. However, not all pncA mutations confer PZA resistance, only the ones that lead to limited POA production. Therefore, susceptibility to PZA may be addressed simply by its ability to form, or not, POA. Here, we present a nuclear magnetic resonance method to accurately quantify POA directly in the supernatant of sputum cultures collected from TB patients. The ability of the clinical sputum culture to hydrolyze PZA was determined, and the results were correlated with the results of other biochemical and molecular PZA drug susceptibility assays. The excellent sensitivity and specificity values attained suggest that this method could become the new gold standard for the determination of PZA susceptibility.

Identification of peptide binding sequence of TRIM25 on 14-3-3σ by bioinformatics and biophysical techniques

14-3-3σ protein is one of the seven isoforms from the highly conserved eukaryotic 14-3-3 protein family. Downregulation of 14-3-3σ expression has been observed in various tumors. TRIM25 is responsible for the proteolytic degradation of 14-3-3σ, in which abrogation of TRIM25 suppressed tumor growth through 14-3-3σ upregulation. However, to date, the exact 14-3-3σ interacting residues of TRIM25 have yet to be resolved. Thus, this study attempts to identify the peptide binding sequence of TRIM25 on 14-3-3σ via both bioinformatics and biophysical techniques. Multiple sequence alignment of the CC domain of TRIM25 revealed five potential peptide binding sequences (Peptide 1–5). Nuclear magnetic resonance (NMR) assay (1H CPMG) identified Peptide 1 as an important sequence for binding to 14-3-3σ. Competition NMR assay suggested that Peptide 1 binds to the amphipathic pocket of 14-3-3σ with an estimated KD of 116.4 µM by isothermal titration calorimetry. Further in silico docking and molecular dynamics simulations studies proposed that Peptide 1 is likely to interact with Lys49, Arg56, Arg129, and Tyr130 residues at the amphipathic pocket of 14-3-3σ. These results suggest that Peptide 1 may serve as a biological probe or a template to design inhibitors of TRIM25-14-3-3σ interaction as a potentially novel class of anticancer agents. Communicated by Ramaswamy H. Sarma

Vantera Mediated Quantification of Urine Citrate and Creatinine: A New Technology to Assess Risk of Nephrolithiasis.

Urine citrate is often used to identify patients at risk of recurrent nephrolithiasis or kidney stones. A high-throughput assay was developed to measure urine citrate and creatinine on the Vantera® Clinical Analyzer, a nuclear magnetic resonance (NMR) instrument designed for the clinical laboratory. Assay performance was evaluated and comparisons between the NMR and chemistry results were conducted. Linearity was demonstrated over a wide range of concentrations for citrate (6 and 2040 mg/L) and creatinine (2.8 and 1308 mg/dL). Intra-and inter-assay precision (%CV) ranged from 0.9 to 3.7% for citrate and 0.4 to 2.1% for creatinine. The correlation coefficients for the comparison between NMR and chemistry results were 0.98 (Y = 1.00X + 5.0) for citrate and 0.96 (Y = 0.968X + 0.97) for creatinine. The reference intervals for both analytes were confirmed. Ten endogenous and exogenous substances were tested and none were found to interfere with the assay results. In conclusion, the newly developed high-throughput NMR assay exhibited robust performance and generated results comparable to the currently utilized chemistry tests, thereby providing an alternative means to simultaneously quantify urine citrate and creatinine for clinical and research use. Furthermore, the NMR assay does not exhibit the same interference limitations as the chemistry tests and it enables multiplexing with other urine metabolite assays which saves time and costs.

Retrospective comparison of results for simultaneous orders for LDL particle count, apolipoprotein B, and LDL-C

BackgroundAnalysis of lipoprotein size and composition by nuclear magnetic resonance (NMR) has been advocated as a method for identifying individuals at high CVD risk. We compared risk stratification between NMR-based LDL particle number (LDL-PNUM), LDL-cholesterol (LDL-C), and apolipoprotein B (apoB). MethodsRetrospective data from patients with simultaneous orders for LDL-PNUM, LDL-C, and apoB were analyzed and included data from an NMR assay (Numares). Quantitative and qualitative analyses were performed. Additional lipid parameters were investigated for patients with discordant risk classifications in LDL-related measurements. The percent change of LDL-PNUM was compared to the percent change of LDL-C or apoB for patients with serial measurements. ResultsWe observed good quantitative and qualitative correlation when comparing LDL-PNUM to either LDL-C or apoB (Spearman’s ρ ≥ 0.83, percent agreements ≥ 85%). Among the patients with discordant risk stratification, most had increased LDL-PNUM and normal LDL-C and apoB. For patients with serial measurements, a strong correlation between the LDL-PNUM percent change and the LDL-C or apoB percent change was observed (Spearman’s ρ > 0.93). ConclusionFor many patients, risk stratification of LDL-PNUM is similar to apoB or LDL-C using cut-offs proposed by guidelines.

Octahedral ruthenium and magnesium naringenin 5-alkoxide complexes: NMR analysis of diastereoisomers and in-vivo antibacterial activity against Xylella fastidiosa

Although many copper-based antimicrobial compounds have been developed to control pathogenic bacteria and fungi in plants and applied for crop protection, there is evidence that several plant pathogens have developed resistance to copper-based antimicrobial compounds, including some Xanthomonas species. Xylella is a bacterial genus belonging to the Xanthomonas family; and X. fastidiosa, which is responsible for citrus variegated chlorosis (CVC) in sweet orange, may develop resistance to one or more copper-based antimicrobials. Because of the time required for the development and approval of new antimicrobials for commercial use, the discovery of novel bactericidal compounds is essential before the development of resistance to the antimicrobials currently in use becomes widespread. Here, we explored the antimicrobial potential of two newly synthesized antimicrobials complexes and one natural compound against X. fastidiosa. Several nuclear magnetic resonance (NMR) assays with high resolution and sensitivity were developed to identify new diastereoisomers in the context of octahedral ruthenium - [Ru(narin)(phen)2]PF6-and magnesium naringenin 5-alkoxide - [Mg(narin)(phen)2]OAc - complexes, obtained in the present work. The NMR assays proved to be powerful tools for the identification of isomers in metal complexes. Moreover, a protocol for the in-vivo determination of the effects of these complexes against X. fastidiosa was developed. The main trunks of X. fastidiosa infected plants were injected with the two complexes as well as with the limonoid azadirachtin using a syringe; the number of bacterial cells in the plants following treatment was estimated via real-time quantitative PCR (qPCR). Importantly, the administration of both complexes and of azadirachtin drastically reduced the number of X. fastidiosa cells in vivo.

The nuclear magnetic resonance metabolic profile: Impact of fasting status

IntroductionMeasurement of lipoprotein subclass concentration (-c), particle number (-p), and size (-s) by nuclear magnetic resonance (NMR) has gained traction in the clinical laboratory due to associations between smaller lipid particle sizes and atherogenic risk, especially for LDL-p. The standard protocols for lipoprotein measurements by NMR require fasting blood samples; however, patients may not fast properly before sample collection. The study objective was to evaluate the impact of fasting status on the NMR-based lipid profile and to identify key parameters differentiating between fasting and post-meal specimens. MethodsForty-eight self-reported healthy male and female participants were recruited. Blood was collected after a 12 h fast and 4 h after a high fat meal. Samples were analyzed using the AXINON LipoFIT by NMR assay. The measurements included triglyceride, total cholesterol, IDL-c, and LDL, HDL, VLDL concentration, particle number, and size, as well as glucose, and four amino acids (alanine, valine, leucine and isoleucine). ResultsAs expected, triglycerides increased after the meal (58%, p < 0.0001). Significant changes were also observed for VLDL, LDL, and HDL parameters, and the branched chain amino acids. The ratio of Valine*VLDL-c/LDL-c or Isoleucine*VLDL-c/LDL-c provided equally effective differentiation of fasting and post-meal samples. The ratio cutoffs (79.1 and 23.6 when calculated using valine and isoleucine, respectively) had sensitivities of 86% and specificities of 93–95%. ConclusionsThe clinical impact on NMR results from post-meal samples warrants further evaluation. Algorithms to differentiate fasting and post-meal specimens may be useful in identifying suboptimal specimens.

Discovery of Berberine that Targetedly Induces Autophagic Degradation of both BCR-ABL and BCR-ABL T315I through Recruiting LRSAM1 for Overcoming Imatinib Resistance.

Imatinib, the breakpoint cluster region protein (BCR)/Abelson murine leukemia viral oncogene homolog (ABL) inhibitor, is widely used to treat chronic myeloid leukemia (CML). However, imatinib resistance develops in many patients. Therefore, new drugs with improved therapeutic effects are urgently needed. Berberine (BBR) is a potent BCR-ABL inhibitor for imatinib-sensitive and -resistant CML. Protein structure analysis and virtual screening were used to identify BBR targets in CML. Molecular docking analysis, surface plasmon resonance imaging, nuclear magnetic resonance assays, and thermoshift assays were performed to confirm the BBR target. The change in BCR-ABL protein expression after BBR treatment was assessed by Western blotting. The effects of BBR were assessed in vitro in cell lines, in vivo in mice, and in human CML bone marrow cells as a potential strategy to overcome imatinib resistance. We discovered that BBR bound to the protein tyrosine kinase domain of BCR-ABL. BBR inhibited the activity of BCR-ABL and BCR-ABL with the T315I mutation, and it also degraded these proteins via the autophagic lysosome pathway by recruiting E3 ubiquitin-protein ligase LRSAM1. BBR inhibited the cell viability and colony formation of CML cells and prolonged survival in CML mouse models with imatinib sensitivity and resistance. The results show that BBR directly binds to and degrades BCR-ABL and BCR-ABL T315I via the autophagic lysosome pathway by recruiting LRSAM1. The use of BBR is a new strategy to improve the treatment of patients with CML with imatinib sensitivity or resistance.See related commentary by Elf, p. 3899.

Ligand-aided 1H Nuclear Magnetic Resonance Spectroscopy for Non-destructive Estimation of Sulfate Content in Sulfated Saccharides.

Sulfated saccharides exhibit diverse physiological activities, but a lack of any convenient assay hinders their evaluation. Herein, an assay for the analysis of sulfated saccharides is described using 1H nuclear magnetic resonance (NMR) spectroscopy by employing ligands that can form ionic complexes with the sulfate groups. Based on the change in the chemical shift (Δδ) of the ligands by sulfated mono- to tetrasaccharide, imidazole was found to be a good ligand, showing the maximum Δδ; neutral saccharides do not show any change in the δ value. A marked and constant downfield δ value observed was changed dramatically at a molar ratio of >1:1 (imidazole:sulfated saccharides), allowing a sulfate content estimation based on the concentration of imidazole at the Δδ inflection point. By the proposed ligand-aided 1H NMR assay, the sulfate content of natural sulfated polysaccharide, fucoidan, was non-destructively estimated to be 2.1 mmol/g-fucoidan.

SAR by (Protein-Observed) 19F NMR.

Inhibitor discovery for protein-protein interactions has proven difficult due to the large protein surface areas and dynamic interfaces involved. This is particularly the case when targeting transcription-factor-protein interactions. To address this challenge, structural biology approaches for ligand discovery using X-ray crystallography, mass spectrometry, and nuclear magnetic resonance (NMR) spectroscopy have had a significant impact on advancing small molecule inhibitors into the clinic, including the U.S. Food and Drug Administration approved drug, Venetoclax. Inspired by the protein-observed NMR approach using 1H-15N-HSQC NMR which detects chemical shift perturbations of 15N-labeled amides, we have applied a complementary protein-observed 19F NMR approach using 19F-labeled side-chains that are enriched at protein-protein-interaction interfaces. This protein-observed 19F NMR assay is abbreviated PrOF NMR to distinguish the experiment from the more commonly employed ligand-observed 19F NMR methods. In this Account, we describe our efforts using PrOF NMR as a ligand discovery tool, particularly for fragment-based ligand discovery (FBLD). We metabolically label the aromatic amino acids on proteins due to the enrichment of aromatic residues at protein interfaces. We choose the 19F nucleus due to its high signal sensitivity and the hyperresponsiveness of 19F to changes in chemical environment. Simultaneous labeling with two different types of fluorinated aromatic amino acids for PrOF NMR has also been achieved. We first describe the technical aspects of considering the application of PrOF NMR for characterizing native protein-protein interactions and for ligand screening. Several test cases are further described with a focus on a transcription factor coactivator interaction with the KIX domain of CBP/p300 and two epigenetic regulatory domains, the bromodomains of BRD4 and BPTF. Through these case studies, we highlight medicinal chemistry applications in FBLD, selectivity screens, structure-activity relationship (SAR) studies, and ligand deconstruction approaches. These studies have led to the discovery of some of the first inhibitors for BPTF and a novel inhibitor class for the N-terminal bromodomain of BRD4. The speed, ease of interpretation, and relatively low concentration of protein needed for NMR-based binding experiments affords a rapid, structural biology-based method to discover and characterize both native and new ligands for bromodomains, and it may find utility in the study of additional epigenetic proteins and transcription-factor-protein interactions.

Nuclear Magnetic Resonance (NMR)–Based Serum Metabolic Profiles: Impact of Fasting Status

Abstract Introduction Measurement of lipoprotein subclass concentration (-c), particle number (-p), and size (-s) by nuclear magnetic resonance (NMR) has gained traction recently in the clinical laboratory due to associations between smaller lipid particle sizes and atherogenic risk, especially for LDL-p. Since patients may not comply with the clinician’s recommendation of fasting before sample collection, our objective was to evaluate the impact of fasting status on NMR-based lipid and amino acid determinations. Methods Fifty self-reported healthy participants were recruited using an internal review board-approved protocol: males (n = 25; 41.3 ± 10.5 years; BMI 27.7 ± 4.6 kg/m2) and females (n = 25; 35.6 ± 9.1 years; BMI 25.3 ± 5.4 kg/m2). Blood was collected after overnight fast and 4 hours after a high-fat meal. Samples were analyzed using the AXINON lipoFIT by NMR assay (Numares) with an Avance III NMR spectrometer and Ascend 600 MHz magnet (Bruker). This assay included measurements of triglycerides, total cholesterol, IDL-c, and LDL, HDL, VLDL concentrations; particle numbers; and sizes. Additionally, NMR-based glucose, alanine, valine, leucine, and isoleucine concentrations were acquired. Conventional lipid parameters were obtained using cobas 8000 chemistry system (Roche). Fasting and postprandial results were compared for significant differences. Results As expected, mean triglycerides increased after the meal (53%, P &lt; .0001). Increases were also observed for VLDL-c (22%, P &lt; .0001) and large VLDL-p (56%, P &lt; .0001), as well as LDL-s, HDL-s, and VLDL-s (all &lt;3.5%, P &lt; .005). In contrast, LDL-p, small LDL-p, large LDL-p, HDL-p, small HDL-p, glucose, total cholesterol, and LDL, HDL, and IDL cholesterols demonstrated at most an 11% decrease (all P &lt; .002). Valine, leucine, and isoleucine also increased after the meal (31%-46%, P &lt; .0001 for all). Differences in individual lipids or amino acids were not sufficient to distinguish between fasting vs postprandial states, although incorporation of valine into a ratio with VLDL-c and LDL cholesterol (Valine*VLDL-c/LDL cholesterol) provided adequate differentiation (ROC analysis: AUC 0.91 [SD 0.03, P &lt; .0001]). A cutoff of 75 for the ratio had a sensitivity of 88.9% (95% CI, 76.0%-96.3%) and specificity of 85.4% (95% CI, 72.2%-93.9%). Interference with NMR results occurred for three participants’ postprandial samples. These individuals had significantly higher fasting triglycerides, LDL-p, small LDL-p, large VLDL-p, VLDL-s, VLDL-c, IDL-c and lower LDL-s, and HDL-s compared to other participants. Conclusions The clinical impact on results from postprandial samples warrants further evaluation prior to accepting nonfasting samples for NMR analysis. Considering a subpar compliance with fasting, algorithms to differentiate fasting and postprandial specimens may be useful in identifying suboptimal specimens.

Evaluating the Effects of Fibrinogen αC Mutations on the Ability of Factor XIII to Crosslink the Reactive αC Glutamines (Q237, Q328, Q366).

Fibrinogen (Fbg) levels and extent of fibrin polymerization have been associated with various pathological conditions such as cardiovascular disease, arteriosclerosis, and coagulation disorders. Activated factor XIII (FXIIIa) introduces γ-glutamyl-ε-lysinyl isopeptide bonds between reactive glutamines and lysines in the fibrin network to form a blood clot resistant to fibrinolysis. FXIIIa crosslinks the γ-chains and at multiple sites in the αC region of Fbg. Fbg αC contains a FXIII binding site involving αC (389-402) that is located near three glutamines whose reactivities rank Q237 >> Q366 ≈ Q328. Mass spectrometry and two-dimensional heteronuclear single-quantum correlation nuclear magnetic resonance assays were used to probe the anchoring role that αC E396 may play in controlling FXIII function and characterize the effects of Q237 on the reactivities of Q328 and Q366. Studies with αC (233-425) revealed that the E396A mutation does not prevent the transglutaminase function of FXIII A2 or A2B2. Other residues must play a compensatory role in targeting FXIII to αC. Unlike full Fbg, Fbg αC (233-425) did not promote thrombin cleavage of FXIII, an event contributing to activation. With the αC (233-425) E396A mutant, Q237 exhibited slower reactivities compared with αC wild-type (WT) consistent with difficulties in directing this N-terminal segment toward an anchored FXIII interacting at a weaker binding region. Q328 and Q366 became less reactive when Q237 was replaced with inactive N237. Q237 crosslinking is proposed to promote targeting of Q328 and Q366 to the FXIII active site. FXIII thus uses Fbg αC anchoring sites and distinct Q environments to regulate substrate specificity.

More Efficient Breakdown of PET Plastic by Modifying the PETase Enzyme

Overproduction and improper disposal methods have caused the world's oceans and ecosystems to be polluted with plastic waste. Polyethylene terephthalate (PET) is a thermoplastic polyester resin widely used in plastic products. PET plastic contains a high ratio of aromatic components that are poorly reactive and difficult to degrade, so PET remains in the environment for an extensive time. In 2016, the novel bacterium Ideonella sakaiensis was discovered to be capable of using PET as a carbon source for growth. The enzyme PETase was isolated from the bacterium and found to break down PET plastic at a slow rate. Our goal is to modify the PETase enzyme to hydrolyze PET plastic with a higher efficiency. The over‐expression and purification of the PETase protein has been optimized. High performance liquid chromatography and nuclear magnetic resonance assays are being used to measure the rate and efficiency of PET breakdown. PETase with various amino acid substitutions has been generated, with the goal of improving enzyme activity under commercially relevant conditions. An optimized PETase enzyme capable of degrading these plastics efficiently on a large scale could be the first step to repairing our polluted ecosystems.Support or Funding InformationThis work was supported in part by the FUTURE program at the University of Iowa Carver College of Medicine.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Benchtop low-field 1H Nuclear Magnetic Resonance for detecting falsified medicines

Falsified medicines represent a serious threat to public health. Among the different measures to effectively combat this scourge, analytical methods play a key role in their detection and removal from the market before they reach patients. The present study evaluates for the first time the potential of a benchtop low-field (LF) Nuclear Magnetic Resonance (NMR) spectrometer for uncovering drug falsification by focusing on the analysis of fifteen erectile dysfunction and nine antimalarial medicines, the most commonly reported falsified medicines in developed and developing countries respectively. After a simple and rapid sample preparation and ≈ 5 min of spectrum recording, LF 1H NMR allows to conclude on the quality of the medicine: presence or absence of the expected active pharmaceutical ingredient (API), presence of unexpected API, absence of any API. Some 2D experiments are also described but although conclusive they are hampered by the duration of the experiments. The LF 1H NMR assay, based on the internal standard method, is validated by the determination of its accuracy, repeatability, limits of detection (LOD) and quantification (LOQ), and by comparison of the data obtained on some medicines after 45 min of spectrum recording to those measured with high-field 1H NMR. Because of its saving capabilities (cost, space, user experience), LF 1H NMR spectroscopy might become a routine screening tool in laboratories in charge of detecting falsified medicines.

Phlebotomy tube interference with nuclear magnetic resonance (NMR) lipoprotein subclass analysis

BackgroundLipoprotein subclass analysis by nuclear magnetic resonance (NMR) can be used in risk assessment of atherosclerotic cardiovascular disease (ASCVD). There is little information in the literature regarding phlebotomy tube interferences with NMR testing. MethodsPooled human serum was exposed to phlebotomy tubes manufactured by Becton Dickinson (BD), Greiner Bio-One, or Sarstedt. Serum was analyzed on the Axinon lipoFIT by NMR assay and by conventional lipid assays performed on a Roche Cobas 8000 system. The effect of incomplete fill volume was also assessed. ResultsAnalytical interference in NMR lipoprotein subclass results was observed across many different tube types. The 5 mL Greiner Bio-One Z Serum Sep Clot Activator tube correlated the best with non-gel containing serum tubes from BD and Greiner Bio-One. BD Serum Separator Tubes (SSTs) displayed strong interferences across several NMR analytes that were enhanced with decreased tube fill volumes. Interferences were also observed with different sizes of Greiner Bio-One Z Serum Sep Clot Activator tubes. Interference was generally not observed with conventional lipid testing, although minor interference was found for some tubes with lipoprotein(a) [Lp(a)]. ConclusionsNMR lipoprotein subclass analysis should be standardized by both tube type and tube size to prevent risk of analytical interference.

The Molecular Mechanism of Intermolecular Signal Transduction in Cystathionine‐β‐Synthase (CBS)

Human cystathionine‐β‐synthase (hCBS) is a pyridoxal phosphate‐dependent (PLP) enzyme that contains heme and catalyzes the β‐replacement reactions of serine (L‐Ser) by homocysteine (L‐Hcy) to produce cystathionine. Mutation in this key mammalian enzyme causes hyperhomocysteinemia, which leads to aggressive arterial diseases, a clinical phenotype observed in patients. Although the role of PLP is similar to other PLP dependent enzymes that catalyze the exchange of the hydroxyl group of serine to thiolate in homocysteine, little is known about the role that heme plays in regulating the catalytic process. My project seeks to develop a continuous, kinetic assay to measure CBS activity. Two approaches are being explored: 1) A coupled kinetic assay, in which the cystathionine produced by CBS is used by Cystathionine‐β‐lyase (hCBL) to generate pyruvate, and pyruvate production is measured phototrically; 2) A novel nuclear magnetic resonance assay, in which we will monitor the direct production of cystathionine over time. These methods will be compared to the standard, but discontinuous, radiometric assay. These assays will be used to understand the effect of small ligand binding to the heme and point mutations at the heme cofactor in relation to the enzyme activity. The long‐term goal of this work is to use time‐resolved absorption and magnetic circular dichroism spectroscopy to understand how small ligand binding to the heme induces conformational changes at the PLP active site in CBS and to measure activity of CBS with various mutated active site residues.Support or Funding InformationArnold and Mabel Beckman Foundation; NIH MARC: T34‐GM008574This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Discovery of alkyl bis(oxy)dibenzimidamide derivatives as novel protein arginine methyltransferase 1 (PRMT1) inhibitors.

Protein arginine methylation, a post-translational modification critical for a variety of biological processes, is catalyzed by protein arginine N-methyltransferases (PRMTs). In particular, PRMT1 is responsible for over 85% of the arginine methylation in mammalian cells. Dysregulation of PRMT1 is involved in diverse pathological diseases including cancers. However, most current PRMT1 inhibitors are lack of specificity, efficacy, and bioavailability. Herein, a series of alkyl bis(oxy)dibenzimidamide derivatives were identified as selective PRMT1 inhibitors. Among them, the most potent compound corresponds to hexamidine (IC50 =5.9±1.7μm), which is an antimicrobial agent. The binding between hexamidine and PRMT1 was further validated by thermal shift assays and nuclear magnetic resonance (NMR) experiments. Molecular docking and NMR assays indicated that hexamidine occupied the substrate binding pocket. Furthermore, hexamidine effectively blocked cell proliferation in cancer cell lines related to PRMT1 overexpression. Taken together, this study has provided a druggable scaffold targeting PRMT1 as well as a new way to repurpose old drugs which is a complementary tool for the discovery of new lead compounds.

NMR quantification of trimethylamine-N-oxide in human serum and plasma in the clinical laboratory setting

Background and objectivesTrimethylamine-N-oxide (TMAO) produced by gut microbiota metabolism of dietary choline and carnitine has been shown to be associated with increased risk of cardiovascular disease (CVD) and to provide incremental clinical prognostic utility beyond traditional risk factors for assessing a patient's CVD risk. The aim of this study was to develop an automated nuclear magnetic resonance (NMR) spectroscopy assay for quantification of TMAO concentration in serum and plasma using a high-throughput NMR clinical analyzer. MethodsKey steps in assay development included: (i) shifting the TMAO analyte peak to a less crowded region of the spectrum with a pH buffer/reagent, (ii) attenuating the broad protein background signal in the spectrum and (iii) using a non-negative least squares algorithm for peak deconvolution. Assay performance was evaluated according to Clinical and Laboratory Standards Institute guidelines. A method comparison study was performed to compare TMAO concentrations quantified by NMR and mass spectrometry (MS). ResultsThe within-run and within-lab imprecision ranged from 4.3 to 14.5%. Under the acquisition method employed, the NMR assay had a limit of blank, detection and quantitation of 1.6, 3.0 and 3.3μM, respectively. Linearity was demonstrated within the reportable range of 3.3 to 3000μM. TMAO measurements using the NMR assay, which involves minimal sample preparation, compared well with values obtained with the MS-based assay (R2=0.98). ConclusionsThe NMR based assay provides a simple and accurate measurement of circulating TMAO levels amenable to the high-throughput demands of the clinical chemistry laboratory. Moreover, assay performance enables the levels of TMAO to be quantified in serum or plasma at clinically actionable concentrations for the assessment of cardiovascular disease risks and individualized dietary monitoring.