Fast and Efficient Fragment-Based Lead Generation by Fully Automated Processing and Analysis of Ligand-Observed NMR Binding Data.

PubMed

Peng, Chen; Frommlet, Alexandra; Perez, Manuel; Cobas, Carlos; Blechschmidt, Anke; Dominguez, Santiago; Lingel, Andreas

2016-04-14

NMR binding assays are routinely applied in hit finding and validation during early stages of drug discovery, particularly for fragment-based lead generation. To this end, compound libraries are screened by ligand-observed NMR experiments such as STD, T1ρ, and CPMG to identify molecules interacting with a target. The analysis of a high number of complex spectra is performed largely manually and therefore represents a limiting step in hit generation campaigns. Here we report a novel integrated computational procedure that processes and analyzes ligand-observed proton and fluorine NMR binding data in a fully automated fashion. A performance evaluation comparing automated and manual analysis results on (19)F- and (1)H-detected data sets shows that the program delivers robust, high-confidence hit lists in a fraction of the time needed for manual analysis and greatly facilitates visual inspection of the associated NMR spectra. These features enable considerably higher throughput, the assessment of larger libraries, and shorter turn-around times.

Fragment-Linking Approach Using (19)F NMR Spectroscopy To Obtain Highly Potent and Selective Inhibitors of β-Secretase.

PubMed

Jordan, John B; Whittington, Douglas A; Bartberger, Michael D; Sickmier, E Allen; Chen, Kui; Cheng, Yuan; Judd, Ted

2016-04-28

Fragment-based drug discovery (FBDD) has become a widely used tool in small-molecule drug discovery efforts. One of the most commonly used biophysical methods in detecting weak binding of fragments is nuclear magnetic resonance (NMR) spectroscopy. In particular, FBDD performed with (19)F NMR-based methods has been shown to provide several advantages over (1)H NMR using traditional magnetization-transfer and/or two-dimensional methods. Here, we demonstrate the utility and power of (19)F-based fragment screening by detailing the identification of a second-site fragment through (19)F NMR screening that binds to a specific pocket of the aspartic acid protease, β-secretase (BACE-1). The identification of this second-site fragment allowed the undertaking of a fragment-linking approach, which ultimately yielded a molecule exhibiting a more than 360-fold increase in potency while maintaining reasonable ligand efficiency and gaining much improved selectivity over cathepsin-D (CatD). X-ray crystallographic studies of the molecules demonstrated that the linked fragments exhibited binding modes consistent with those predicted from the targeted screening approach, through-space NMR data, and molecular modeling.

NMR Fragment Screening Hit Induces Plasticity of BRD7/9 Bromodomains.

PubMed

Wang, Na; Li, Fudong; Bao, Hongyu; Li, Jie; Wu, Jihui; Ruan, Ke

2016-08-03

The complex biology associated with inhibition of bromodomain and extra-terminal (BET) domains by chemical probes has attracted increasing attention, and there is a need to identify non-BET bromodomain (BD) inhibitors. Several potent inhibitors of the BRD9 BD have recently been discovered, with anticancer and anti-inflammation activity. However, its paralogue, BRD7 BD, remains unexploited. Here, we identified new chemotypes targeting BRD7 BD by using NMR fragment-based screening. BRD7/9 BDs exhibit similar patterns of chemical-shift perturbation upon the titration of hit compound 1. The crystal structure revealed that 1 repels the Y222 group of BRD9 BD in a similar way to that for butyryllysine, but not acetyllysine and known inhibitors. Hit 1 induced less rearrangement of residue F161 of BRD9 BD than acetyllysine, butyryllysine, and crotonyllysine. Our study provides structural insight into a new generation of butyryllysine mimics for probing the function of BRD7/9 BD. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Recombinant Kinase Production and Fragment Screening by NMR Spectroscopy.

PubMed

Han, Byeonggu; Ahn, Hee-Chul

2016-01-01

During the past decade fragment-based drug discovery (FBDD) has rapidly evolved and several drugs or drug candidates developed by FBDD approach are clinically in use or in clinical trials. For example, vemurafenib, a V600E mutated BRAF inhibitor, was developed by utilizing FBDD approach and approved by FDA in 2011. In FBDD, screening of fragments is the starting step for identification of hits and lead generation. Fragment screening usually relies on biophysical techniques by which the protein-bound small molecules can be detected. NMR spectroscopy has been extensively used to study the molecular interaction between the protein and the ligand, and has many advantages in fragment screening over other biophysical techniques. This chapter describes the practical aspects of fragment screening by saturation transfer difference NMR.

Methylpyrrole inhibitors of BET bromodomains

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hasvold, Lisa A.; Sheppard, George S.; Wang, Le

2017-05-01

An NMR fragment screen for binders to the bromodomains of BRD4 identified 2-methyl-3-ketopyrroles 1 and 2. Elaboration of these fragments guided by structure-based design provided lead molecules with significant activity in a mouse tumor model. Further modifications to the methylpyrrole core provided compounds with improved properties and enhanced activity in a mouse model of multiple myeloma.

Identification of some novel pyrazolo[1,5-a]pyrimidine derivatives as InhA inhibitors through pharmacophore-based virtual screening and molecular docking.

PubMed

Modi, Palmi; Patel, Shivani; Chhabria, Mahesh T

2018-05-04

The InhA inhibitors play key role in mycolic acid synthesis by preventing the fatty acid biosynthesis pathway. In this present article, Pharmacophore modelling and molecular docking study followed by in silico virtual screening could be considered as effective strategy to identify newer enoyl-ACP reductase inhibitors. Pyrrolidine carboxamide derivatives were opted to generate pharmacophore models using HypoGen algorithm in Discovery studio 2.1. Further it was employed to screen Zinc and Minimaybridge databases to identify and design newer potent hit molecules. The retrieved newer hits were further evaluated for their drug likeliness and docked against enoyl acyl carrier protein reductase. Here, novel pyrazolo[1,5-a]pyrimidine analogues were designed and synthesized with good yields. Structural elucidation of synthesized final molecules was perform through IR, MASS, 1 H-NMR, 13 C-NMR spectroscopy and further tested for its in vitro anti-tubercular activity against H37Rv strain using Microplate Alamar blue assay (MABA) method. Most of the synthesized compounds displayed strong anti-tubercular activities. Further, these potent compounds were gauged for MDR-TB, XDR-TB and cytotoxic study.

Target immobilization as a strategy for NMR-based fragment screening: comparison of TINS, STD, and SPR for fragment hit identification.

PubMed

Kobayashi, Masakazu; Retra, Kim; Figaroa, Francis; Hollander, Johan G; Ab, Eiso; Heetebrij, Robert J; Irth, Hubertus; Siegal, Gregg

2010-09-01

Fragment-based drug discovery (FBDD) has become a widely accepted tool that is complementary to high-throughput screening (HTS) in developing small-molecule inhibitors of pharmaceutical targets. Because a fragment campaign can only be as successful as the hit matter found, it is critical that the first stage of the process be optimized. Here the authors compare the 3 most commonly used methods for hit discovery in FBDD: high concentration screening (HCS), solution ligand-observed nuclear magnetic resonance (NMR), and surface plasmon resonance (SPR). They selected the commonly used saturation transfer difference (STD) NMR spectroscopy and the proprietary target immobilized NMR screening (TINS) as representative of the array of possible NMR methods. Using a target typical of FBDD campaigns, the authors find that HCS and TINS are the most sensitive to weak interactions. They also find a good correlation between TINS and STD for tighter binding ligands, but the ability of STD to detect ligands with affinity weaker than 1 mM K(D) is limited. Similarly, they find that SPR detection is most suited to ligands that bind with K(D) better than 1 mM. However, the good correlation between SPR and potency in a bioassay makes this a good method for hit validation and characterization studies.

NMR-based platform for fragment-based lead discovery used in screening BRD4-targeted compounds

PubMed Central

Yu, Jun-lan; Chen, Tian-tian; Zhou, Chen; Lian, Fu-lin; Tang, Xu-long; Wen, Yi; Shen, Jing-kang; Xu, Ye-chun; Xiong, Bing; Zhang, Nai-xia

2016-01-01

Aim: Fragment-based lead discovery (FBLD) is a complementary approach in drug research and development. In this study, we established an NMR-based FBLD platform that was used to screen novel scaffolds targeting human bromodomain of BRD4, and investigated the binding interactions between hit compounds and the target protein. Methods: 1D NMR techniques were primarily used to generate the fragment library and to screen compounds. The inhibitory activity of hits on the first bromodomain of BRD4 [BRD4(I)] was examined using fluorescence anisotropy binding assay. 2D NMR and X-ray crystallography were applied to characterize the binding interactions between hit compounds and the target protein. Results: An NMR-based fragment library containing 539 compounds was established, which were clustered into 56 groups (8–10 compounds in each group). Eight hits with new scaffolds were found to inhibit BRD4(I). Four out of the 8 hits (compounds 1, 2, 8 and 9) had IC50 values of 100–260 μmol/L, demonstrating their potential for further BRD4-targeted hit-to-lead optimization. Analysis of the binding interactions revealed that compounds 1 and 2 shared a common quinazolin core structure and bound to BRD4(I) in a non-acetylated lysine mimetic mode. Conclusion: An NMR-based platform for FBLD was established and used in discovery of BRD4-targeted compounds. Four potential hit-to-lead optimization candidates have been found, two of them bound to BRD4(I) in a non-acetylated lysine mimetic mode, being selective BRD4(I) inhibitors. PMID:27238211

Metabolomics-Driven Discovery of a Prenylated Isatin Antibiotic Produced by Streptomyces Species MBT28.

PubMed

Wu, Changsheng; Du, Chao; Gubbens, Jacob; Choi, Young Hae; van Wezel, Gilles P

2015-10-23

Actinomycetes are a major source of antimicrobials, anticancer compounds, and other medically important products, and their genomes harbor extensive biosynthetic potential. Major challenges in the screening of these microorganisms are to activate the expression of cryptic biosynthetic gene clusters and the development of technologies for efficient dereplication of known molecules. Here we report the identification of a previously unidentified isatin-type antibiotic produced by Streptomyces sp. MBT28, following a strategy based on NMR-based metabolomics combined with the introduction of streptomycin resistance in the producer strain. NMR-guided isolation by tracking the target proton signal resulted in the characterization of 7-prenylisatin (1) with antimicrobial activity against Bacillus subtilis. The metabolite-guided genome mining of Streptomyces sp. MBT28 combined with proteomics identified a gene cluster with an indole prenyltransferase that catalyzes the conversion of tryptophan into 7-prenylisatin. This study underlines the applicability of NMR-based metabolomics in facilitating the discovery of novel antibiotics.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Taylor-Pashow, K.; Fondeur, F.; White, T.

Savannah River National Laboratory (SRNL) was tasked with identifying and developing at least one, but preferably two methods for quantifying the suppressor in the Next Generation Solvent (NGS) system. The suppressor is a guanidine derivative, N,N',N"-tris(3,7-dimethyloctyl)guanidine (TiDG). A list of 10 possible methods was generated, and screening experiments were performed for 8 of the 10 methods. After completion of the screening experiments, the non-aqueous acid-base titration was determined to be the most promising, and was selected for further development as the primary method. {sup 1}H NMR also showed promising results from the screening experiments, and this method was selected formore » further development as the secondary method. Other methods, including {sup 36}Cl radiocounting and ion chromatography, also showed promise; however, due to the similarity to the primary method (titration) and the inability to differentiate between TiDG and TOA (tri-n-ocytlamine) in the blended solvent, {sup 1}H NMR was selected over these methods. Analysis of radioactive samples obtained from real waste ESS (extraction, scrub, strip) testing using the titration method showed good results. Based on these results, the titration method was selected as the method of choice for TiDG measurement. {sup 1}H NMR has been selected as the secondary (back-up) method, and additional work is planned to further develop this method and to verify the method using radioactive samples. Procedures for analyzing radioactive samples of both pure NGS and blended solvent were developed and issued for the both methods.« less

Automatic 1H-NMR Screening of Fatty Acid Composition in Edible Oils

PubMed Central

Castejón, David; Fricke, Pascal; Cambero, María Isabel; Herrera, Antonio

2016-01-01

In this work, we introduce an NMR-based screening method for the fatty acid composition analysis of edible oils. We describe the evaluation and optimization needed for the automated analysis of vegetable oils by low-field NMR to obtain the fatty acid composition (FAC). To achieve this, two scripts, which automatically analyze and interpret the spectral data, were developed. The objective of this work was to drive forward the automated analysis of the FAC by NMR. Due to the fact that this protocol can be carried out at low field and that the complete process from sample preparation to printing the report only takes about 3 min, this approach is promising to become a fundamental technique for high-throughput screening. To demonstrate the applicability of this method, the fatty acid composition of extra virgin olive oils from various Spanish olive varieties (arbequina, cornicabra, hojiblanca, manzanilla, and picual) was determined by 1H-NMR spectroscopy according to this protocol. PMID:26891323

TINS, target immobilized NMR screening: an efficient and sensitive method for ligand discovery.

PubMed

Vanwetswinkel, Sophie; Heetebrij, Robert J; van Duynhoven, John; Hollander, Johan G; Filippov, Dmitri V; Hajduk, Philip J; Siegal, Gregg

2005-02-01

We propose a ligand screening method, called TINS (target immobilized NMR screening), which reduces the amount of target required for the fragment-based approach to drug discovery. Binding is detected by comparing 1D NMR spectra of compound mixtures in the presence of a target immobilized on a solid support to a control sample. The method has been validated by the detection of a variety of ligands for protein and nucleic acid targets (K(D) from 60 to 5000 muM). The ligand binding capacity of a protein was undiminished after 2000 different compounds had been applied, indicating the potential to apply the assay for screening typical fragment libraries. TINS can be used in competition mode, allowing rapid characterization of the ligand binding site. TINS may allow screening of targets that are difficult to produce or that are insoluble, such as membrane proteins.

Mass spectrometry for fragment screening.

PubMed

Chan, Daniel Shiu-Hin; Whitehouse, Andrew J; Coyne, Anthony G; Abell, Chris

2017-11-08

Fragment-based approaches in chemical biology and drug discovery have been widely adopted worldwide in both academia and industry. Fragment hits tend to interact weakly with their targets, necessitating the use of sensitive biophysical techniques to detect their binding. Common fragment screening techniques include differential scanning fluorimetry (DSF) and ligand-observed NMR. Validation and characterization of hits is usually performed using a combination of protein-observed NMR, isothermal titration calorimetry (ITC) and X-ray crystallography. In this context, MS is a relatively underutilized technique in fragment screening for drug discovery. MS-based techniques have the advantage of high sensitivity, low sample consumption and being label-free. This review highlights recent examples of the emerging use of MS-based techniques in fragment screening. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Application of pulsed field gradient NMR techniques for investigating binding of flavor compounds to macromolecules.

PubMed

Jung, Da-Mi; De Ropp, Jeffrey S; Ebeler, Susan E

2002-07-17

Two diffusion-based NMR techniques are presented and used to investigate the binding of selected flavor compounds to macromolecules. A pulsed field gradient NMR (PFG-NMR) method was applied to measure the apparent diffusion coefficients of four alkanone compounds as they associated with bovine serum albumin (BSA). The change in the apparent diffusion coefficient as a function of the BSA/alkanone ratio was fitted to yield binding constants (K(a)()) and binding stoichiometry (n) for each alkanone. The results showed that the apparent diffusion coefficients of alkanones increased with a decrease in the BSA/alkanone ratios, and the measured values of K(a)() and n were comparable with those obtained with other methods and depended on the alkanone structure. A diffusion-based nuclear Overhauser effect (called diffusion NOE pumping) method was also applied to screen mixtures of flavor compounds and identify those that have a binding affinity to complex macromolecules. Using this technique benzaldehyde and vanillin were observed to bind with bovine serum albumin, whereas 2-phenylethanol was identified as a nonbinding or weakly binding ligand with BSA. The diffusion NOE pumping method was also applied to a hydro alcoholic solution of cacao bean tannin extracts to which a mixture of ethylbenzoate, benzaldehyde, and 2-phenylethanol was added. The diffusion NOE pumping technique clearly indicated that ethylbenzoate had a stronger binding affinity to the polymeric (-)-epicatechin units of the cacao bean tannin extracts than the other two flavor compounds. The results successfully demonstrate the potential applications of diffusion-based NMR techniques for studying flavors and nonvolatile food matrix interactions.

Saturation-Transfer Difference (STD) NMR: A Simple and Fast Method for Ligand Screening and Characterization of Protein Binding

ERIC Educational Resources Information Center

Viegas, Aldino; Manso, Joao; Nobrega, Franklin L.; Cabrita, Eurico J.

2011-01-01

Saturation transfer difference (STD) NMR has emerged as one of the most popular ligand-based NMR techniques for the study of protein-ligand interactions. The success of this technique is a consequence of its robustness and the fact that it is focused on the signals of the ligand, without any need of processing NMR information about the receptor…

Enhanced detection of aldehydes in Extra-Virgin Olive Oil by means of band selective NMR spectroscopy

NASA Astrophysics Data System (ADS)

Dugo, Giacomo; Rotondo, Archimede; Mallamace, Domenico; Cicero, Nicola; Salvo, Andrea; Rotondo, Enrico; Corsaro, Carmelo

2015-02-01

High resolution Nuclear Magnetic Resonance (NMR) spectroscopy is a very powerful tool for comprehensive food analyses and especially for Extra-Virgin Olive Oils (EVOOs). We use the NMR technique to study the spectral region of aldehydes (8-10 ppm) for EVOOs coming from the south part of Italy. We perform novel experiments by using mono and bidimensional band selective spin-echo pulse sequences and identify four structural classes of aldehydes in EVOOs. For the first time such species are identified in EVOOs without any chemical treatment; only dilution with CDCl3 is employed. This would allow the discrimination of different EVOOs for the aldehydes content increasing the potentiality of the NMR technique in the screening of metabolites for geographical characterization of EVOOs.

Operation of a 500 MHz high temperature superconducting NMR: towards an NMR spectrometer operating beyond 1 GHz.

PubMed

Yanagisawa, Y; Nakagome, H; Tennmei, K; Hamada, M; Yoshikawa, M; Otsuka, A; Hosono, M; Kiyoshi, T; Takahashi, M; Yamazaki, T; Maeda, H

2010-04-01

We have begun a project to develop an NMR spectrometer that operates at frequencies beyond 1 GHz (magnetic field strength in excess of 23.5 T) using a high temperature superconductor (HTS) innermost coil. As the first step, we developed a 500 MHz NMR with a Bi-2223 HTS innermost coil, which was operated in external current mode. The temporal magnetic field change of the NMR magnet after the coil charge was dominated by (i) the field fluctuation due to a DC power supply and (ii) relaxation in the screening current in the HTS tape conductor; effect (i) was stabilized by the 2H field-frequency lock system, while effect (ii) decreased with time due to relaxation of the screening current induced in the HTS coil and reached 10(-8)(0.01 ppm)/h on the 20th day after the coil charge, which was as small as the persistent current mode of the NMR magnet. The 1D (1)H NMR spectra obtained by the 500 MHz LTS/HTS magnet were nearly equivalent to those obtained by the LTS NMR magnet. The 2D-NOESY, 3D-HNCO and 3D-HNCACB spectra were achieved for ubiquitin by the 500 MHz LTS/HTS magnet; their quality was closely equivalent to that achieved by a conventional LTS NMR. Based on the results of numerical simulation, the effects of screening current-induced magnetic field changes are predicted to be harmless for the 1.03 GHz NMR magnet system. 2010 Elsevier Inc. All rights reserved.

Molecular Docking and NMR Binding Studies to Identify Novel Inhibitors of Human Phosphomevalonate Kinase

PubMed Central

Boonsri, Pornthip; Neumann, Terrence S.; Olson, Andrew L.; Cai, Sheng; Herdendorf, Timothy J.; Miziorko, Henry M.; Hannongbua, Supa; Sem, Daniel S.

2012-01-01

Phosphomevalonate kinase (PMK) phosphorylates mevalonate-5-phosphate (M5P) in the mevalonate pathway, which is the sole source of isoprenoids and steroids in humans. We have identified new PMK inhibitors with virtual screening, using Autodock. Promising hits were verified and their affinity measured using NMR-based 1H-15N Heteronuclear Single Quantum Coherence (HSQC) chemical shift perturbation and fluorescence titrations. Chemical shift changes were monitored, plotted, and fitted to obtain dissociation constants (Kd). Tight binding compounds with Kd’s ranging from 6–60 µM were identified. These compounds tended to have significant polarity and negative charge, similar to the natural substrates (M5P and ATP). HSQC crosspeak changes suggest that binding induces a global conformational change, such as domain closure. Compounds identified in this study serve as chemical genetic probes of human PMK, to explore pharmacology of the mevalonate pathway, as well as starting points for further drug development. PMID:23146631

Metabolomic tools for secondary metabolite discovery from marine microbial symbionts.

PubMed

Macintyre, Lynsey; Zhang, Tong; Viegelmann, Christina; Martinez, Ignacio Juarez; Cheng, Cheng; Dowdells, Catherine; Abdelmohsen, Usama Ramadam; Gernert, Christine; Hentschel, Ute; Edrada-Ebel, RuAngelie

2014-06-05

Marine invertebrate-associated symbiotic bacteria produce a plethora of novel secondary metabolites which may be structurally unique with interesting pharmacological properties. Selection of strains usually relies on literature searching, genetic screening and bioactivity results, often without considering the chemical novelty and abundance of secondary metabolites being produced by the microorganism until the time-consuming bioassay-guided isolation stages. To fast track the selection process, metabolomic tools were used to aid strain selection by investigating differences in the chemical profiles of 77 bacterial extracts isolated from cold water marine invertebrates from Orkney, Scotland using liquid chromatography-high resolution mass spectrometry (LC-HRMS) and nuclear magnetic resonance (NMR) spectroscopy. Following mass spectrometric analysis and dereplication using an Excel macro developed in-house, principal component analysis (PCA) was employed to differentiate the bacterial strains based on their chemical profiles. NMR 1H and correlation spectroscopy (COSY) were also employed to obtain a chemical fingerprint of each bacterial strain and to confirm the presence of functional groups and spin systems. These results were then combined with taxonomic identification and bioassay screening data to identify three bacterial strains, namely Bacillus sp. 4117, Rhodococcus sp. ZS402 and Vibrio splendidus strain LGP32, to prioritize for scale-up based on their chemically interesting secondary metabolomes, established through dereplication and interesting bioactivities, determined from bioassay screening.

One Question, Multiple Answers: Biochemical and Biophysical Screening Methods Retrieve Deviating Fragment Hit Lists.

PubMed

Schiebel, Johannes; Radeva, Nedyalka; Köster, Helene; Metz, Alexander; Krotzky, Timo; Kuhnert, Maren; Diederich, Wibke E; Heine, Andreas; Neumann, Lars; Atmanene, Cedric; Roecklin, Dominique; Vivat-Hannah, Valérie; Renaud, Jean-Paul; Meinecke, Robert; Schlinck, Nina; Sitte, Astrid; Popp, Franziska; Zeeb, Markus; Klebe, Gerhard

2015-09-01

Fragment-based lead discovery is gaining momentum in drug development. Typically, a hierarchical cascade of several screening techniques is consulted to identify fragment hits which are then analyzed by crystallography. Because crystal structures with bound fragments are essential for the subsequent hit-to-lead-to-drug optimization, the screening process should distinguish reliably between binders and non-binders. We therefore investigated whether different screening methods would reveal similar collections of putative binders. First we used a biochemical assay to identify fragments that bind to endothiapepsin, a surrogate for disease-relevant aspartic proteases. In a comprehensive screening approach, we then evaluated our 361-entry library by using a reporter-displacement assay, saturation-transfer difference NMR, native mass spectrometry, thermophoresis, and a thermal shift assay. While the combined results of these screening methods retrieve 10 of the 11 crystal structures originally predicted by the biochemical assay, the mutual overlap of individual hit lists is surprisingly low, highlighting that each technique operates on different biophysical principles and conditions. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Characterization of Protein-Carbohydrate Interactions by NMR Spectroscopy.

PubMed

Grondin, Julie M; Langelaan, David N; Smith, Steven P

2017-01-01

Solution-state nuclear magnetic resonance (NMR) spectroscopy can be used to monitor protein-carbohydrate interactions. Two-dimensional 1 H- 15 N heteronuclear single quantum coherence (HSQC)-based techniques described in this chapter can be used quickly and effectively to screen a set of possible carbohydrate binding partners, to quantify the dissociation constant (K d ) of any identified interactions, and to map the carbohydrate binding site on the structure of the protein. Here, we describe the titration of a family 32 carbohydrate binding module from Clostridium perfringens (CpCBM32) with the monosaccharide N-acetylgalactosamine (GalNAc), in which we calculate the apparent dissociation of the interaction, and map the GalNAc binding site onto the structure of CpCBM32.

Combining automated peak tracking in SAR by NMR with structure-based backbone assignment from 15N-NOESY

PubMed Central

2012-01-01

Background Chemical shift mapping is an important technique in NMR-based drug screening for identifying the atoms of a target protein that potentially bind to a drug molecule upon the molecule's introduction in increasing concentrations. The goal is to obtain a mapping of peaks with known residue assignment from the reference spectrum of the unbound protein to peaks with unknown assignment in the target spectrum of the bound protein. Although a series of perturbed spectra help to trace a path from reference peaks to target peaks, a one-to-one mapping generally is not possible, especially for large proteins, due to errors, such as noise peaks, missing peaks, missing but then reappearing, overlapped, and new peaks not associated with any peaks in the reference. Due to these difficulties, the mapping is typically done manually or semi-automatically, which is not efficient for high-throughput drug screening. Results We present PeakWalker, a novel peak walking algorithm for fast-exchange systems that models the errors explicitly and performs many-to-one mapping. On the proteins: hBclXL, UbcH5B, and histone H1, it achieves an average accuracy of over 95% with less than 1.5 residues predicted per target peak. Given these mappings as input, we present PeakAssigner, a novel combined structure-based backbone resonance and NOE assignment algorithm that uses just 15N-NOESY, while avoiding TOCSY experiments and 13C-labeling, to resolve the ambiguities for a one-to-one mapping. On the three proteins, it achieves an average accuracy of 94% or better. Conclusions Our mathematical programming approach for modeling chemical shift mapping as a graph problem, while modeling the errors directly, is potentially a time- and cost-effective first step for high-throughput drug screening based on limited NMR data and homologous 3D structures. PMID:22536902

Advances in fragment-based drug discovery platforms.

PubMed

Orita, Masaya; Warizaya, Masaichi; Amano, Yasushi; Ohno, Kazuki; Niimi, Tatsuya

2009-11-01

Fragment-based drug discovery (FBDD) has been established as a powerful alternative and complement to traditional high-throughput screening techniques for identifying drug leads. At present, this technique is widely used among academic groups as well as small biotech and large pharmaceutical companies. In recent years, > 10 new compounds developed with FBDD have entered clinical development, and more and more attention in the drug discovery field is being focused on this technique. Under the FBDD approach, a fragment library of relatively small compounds (molecular mass = 100 - 300 Da) is screened by various methods and the identified fragment hits which normally weakly bind to the target are used as starting points to generate more potent drug leads. Because FBDD is still a relatively new drug discovery technology, further developments and optimizations in screening platforms and fragment exploitation can be expected. This review summarizes recent advances in FBDD platforms and discusses the factors important for the successful application of this technique. Under the FBDD approach, both identifying the starting fragment hit to be developed and generating the drug lead from that starting fragment hit are important. Integration of various techniques, such as computational technology, X-ray crystallography, NMR, surface plasmon resonance, isothermal titration calorimetry, mass spectrometry and high-concentration screening, must be applied in a situation-appropriate manner.

Utility of metabolic profiling of serum in the diagnosis of pregnancy complications.

PubMed

Powell, Katie L; Carrozzi, Anthony; Stephens, Alexandre S; Tasevski, Vitomir; Morris, Jonathan M; Ashton, Anthony W; Dona, Anthony C

2018-06-01

Currently there are no clinical screening tests available to identify pregnancies at risk of developing preeclampsia (PET) and/or intrauterine growth restriction (IUGR), both of which are associated with abnormal placentation. Metabolic profiling is now a stable analytical platform used in many laboratories and has successfully been used to identify biomarkers associated with various pathological states. We used nuclear magnetic resonance spectroscopy (NMR) to metabolically profile serum samples collected from 143 pregnant women at 26-41 weeks gestation with pregnancy outcomes of PET, IUGR, PET IUGR or small for gestational age (SGA) that were age-matched to normal pre/term pregnancies. Spectral analysis found no difference in the measured metabolites from normal term, pre-term and SGA samples, and of 25 identified metabolites, only glutamate was marginally different between groups. Of the identified metabolites, 3-methylhistidine, creatinine, acetyl groups and acetate, were determined to be independent predictors of PET and produced area under the curves (AUC) = 0.938 and 0.936 for the discovery and validation sets. Only 3-hydroxybutyrate was determined to be an independent predictor of IUGR, however the model had low predictive power (AUC = 0.623 and 0.581 for the discovery and validation sets). A sub-panel of metabolites had strong predictive power for identifying PET samples in a validation dataset, however prediction of IUGR was more difficult using the identified metabolites. NMR based metabolomics can identify metabolites strongly associated with disease and has the potential to be useful in developing early clinical screening tests for at risk pregnancies. Copyright © 2018 Elsevier Ltd. All rights reserved.

Preparation of Protein Samples for NMR Structure, Function, and Small Molecule Screening Studies

PubMed Central

Acton, Thomas B.; Xiao, Rong; Anderson, Stephen; Aramini, James; Buchwald, William A.; Ciccosanti, Colleen; Conover, Ken; Everett, John; Hamilton, Keith; Huang, Yuanpeng Janet; Janjua, Haleema; Kornhaber, Gregory; Lau, Jessica; Lee, Dong Yup; Liu, Gaohua; Maglaqui, Melissa; Ma, Lichung; Mao, Lei; Patel, Dayaban; Rossi, Paolo; Sahdev, Seema; Shastry, Ritu; Swapna, G.V.T.; Tang, Yeufeng; Tong, Saichiu; Wang, Dongyan; Wang, Huang; Zhao, Li; Montelione, Gaetano T.

2014-01-01

In this chapter, we concentrate on the production of high quality protein samples for NMR studies. In particular, we provide an in-depth description of recent advances in the production of NMR samples and their synergistic use with recent advancements in NMR hardware. We describe the protein production platform of the Northeast Structural Genomics Consortium, and outline our high-throughput strategies for producing high quality protein samples for nuclear magnetic resonance (NMR) studies. Our strategy is based on the cloning, expression and purification of 6X-His-tagged proteins using T7-based Escherichia coli systems and isotope enrichment in minimal media. We describe 96-well ligation-independent cloning and analytical expression systems, parallel preparative scale fermentation, and high-throughput purification protocols. The 6X-His affinity tag allows for a similar two-step purification procedure implemented in a parallel high-throughput fashion that routinely results in purity levels sufficient for NMR studies (> 97% homogeneity). Using this platform, the protein open reading frames of over 17,500 different targeted proteins (or domains) have been cloned as over 28,000 constructs. Nearly 5,000 of these proteins have been purified to homogeneity in tens of milligram quantities (see Summary Statistics, http://nesg.org/statistics.html), resulting in more than 950 new protein structures, including more than 400 NMR structures, deposited in the Protein Data Bank. The Northeast Structural Genomics Consortium pipeline has been effective in producing protein samples of both prokaryotic and eukaryotic origin. Although this paper describes our entire pipeline for producing isotope-enriched protein samples, it focuses on the major updates introduced during the last 5 years (Phase 2 of the National Institute of General Medical Sciences Protein Structure Initiative). Our advanced automated and/or parallel cloning, expression, purification, and biophysical screening technologies are suitable for implementation in a large individual laboratory or by a small group of collaborating investigators for structural biology, functional proteomics, ligand screening and structural genomics research. PMID:21371586

A general analytical platform and strategy in search for illegal drugs.

PubMed

Johansson, Monika; Fransson, Dick; Rundlöf, Torgny; Huynh, Ngoc-Hang; Arvidsson, Torbjörn

2014-11-01

An effective screening procedure to identify and quantify active pharmaceutical substances in suspected illegal medicinal products is described. The analytical platform, consisting of accurate mass determination with liquid chromatography time-of-flight mass spectrometry (LC-QTOF-MS) in combination with nuclear magnetic resonance (NMR) spectroscopy provides an excellent analytical tool to screen for unknowns in medicinal products, food supplements and herbal formulations. This analytical approach has been successfully applied to analyze thousands of samples. The general screening method usually starts with a methanol extraction of tablets/capsules followed by liquid chromatographic separation on a Halo Phenyl-Hexyl column (2.7μm; 100mm×2.1mm) using an acetonitrile/0.1% formic acid gradient as eluent. The accurate mass of peaks of interest was recorded and a search made against an in-house database containing approximately 4200 substances, mostly pharmaceutical compounds. The search could be general or tailored against different classes of compounds. Hits were confirmed by analyzing a reference substance and/or by NMR. Quantification was normally performed with quantitative NMR (qNMR) spectroscopy. Applications for weight-loss substances like sibutramine and orlistat, sexual potency enhancement (PDE-5 inhibitors), and analgesic drugs are presented in this study. We have also identified prostaglandin analogues in eyelash growth serum, exemplified by isopropyl cloprostenate and bimatoprost. For creams and ointments, matrix solid-phase dispersion (MSPD) was found to give a clean extracts with high recovery prior to LC-MS analyses. The structural elucidation of cetilistat, a new weight-loss substance recently found in illegal medicines purchased over the Internet, is also presented. Copyright © 2014 Elsevier B.V. All rights reserved.

A more detailed picture of the interactions between virtual screening-derived hits and the DNA G-quadruplex: NMR, molecular modelling and ITC studies.

PubMed

Trotta, Roberta; De Tito, Stefano; Lauri, Ilaria; La Pietra, Valeria; Marinelli, Luciana; Cosconati, Sandro; Martino, Luigi; Conte, Maria R; Mayol, Luciano; Novellino, Ettore; Randazzo, Antonio

2011-08-01

The growing amount of literature about G-quadruplex DNA clearly demonstrates that such a structure is no longer viewed as just a biophysical strangeness but it is instead being considered as an important target for the treatment of various human disorders such as cancers or venous thrombosis. In this scenario, with the aim of finding brand new molecular scaffolds able to interact with the groove of the DNA quadruplex [d(TGGGGT)](4), we recently performed a successful structure-based virtual screening (VS) campaign. As a result, six molecules were found to be somehow groove binders. Herein, we report the results of novel NMR titration experiments of these VS-derived ligands with modified quadruplexes, namely [d(TGG(Br)GGT)](4) and [d(TGGGG(Br)T)](4). The novel NMR spectroscopy experiments combined with molecular modelling studies, allow for a more detailed picture of the interaction between each binder and the quadruplex DNA. Noteworthy, isothermal titration calorimetry (ITC) measurements on the above-mentioned compounds revealed that 2, 4, and 6 besides their relatively small dimensions bind the DNA quadruplex [d(TGGGGT)](4) with higher affinity than distamycin A, to the best of our knowledge, the most potent groove binder identified thus far. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

Fragment-Based, Structure-Enabled Discovery of Novel Pyridones and Pyridone Macrocycles as Potent Bromodomain and Extra-Terminal Domain (BET) Family Bromodomain Inhibitors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Le; Pratt, John K.; Soltwedel, Todd

Members of the BET family of bromodomain containing proteins have been identified as potential targets for blocking proliferation in a variety of cancer cell lines. A two-dimensional NMR fragment screen for binders to the bromodomains of BRD4 identified a phenylpyridazinone fragment with a weak binding affinity (1, Ki = 160 μM). SAR investigation of fragment 1, aided by X-ray structure-based design, enabled the synthesis of potent pyridone and macrocyclic pyridone inhibitors exhibiting single digit nanomolar potency in both biochemical and cell based assays. Advanced analogs in these series exhibited high oral exposures in rodent PK studies and demonstrated significant tumormore » growth inhibition efficacy in mouse flank xenograft models.« less

The rise of fragment-based drug discovery.

PubMed

Murray, Christopher W; Rees, David C

2009-06-01

The search for new drugs is plagued by high attrition rates at all stages in research and development. Chemists have an opportunity to tackle this problem because attrition can be traced back, in part, to the quality of the chemical leads. Fragment-based drug discovery (FBDD) is a new approach, increasingly used in the pharmaceutical industry, for reducing attrition and providing leads for previously intractable biological targets. FBDD identifies low-molecular-weight ligands (∼150 Da) that bind to biologically important macromolecules. The three-dimensional experimental binding mode of these fragments is determined using X-ray crystallography or NMR spectroscopy, and is used to facilitate their optimization into potent molecules with drug-like properties. Compared with high-throughput-screening, the fragment approach requires fewer compounds to be screened, and, despite the lower initial potency of the screening hits, offers more efficient and fruitful optimization campaigns. Here, we review the rise of FBDD, including its application to discovering clinical candidates against targets for which other chemistry approaches have struggled.

Authentication of beef versus horse meat using 60 MHz 1H NMR spectroscopy

PubMed Central

Jakes, W.; Gerdova, A.; Defernez, M.; Watson, A.D.; McCallum, C.; Limer, E.; Colquhoun, I.J.; Williamson, D.C.; Kemsley, E.K.

2015-01-01

This work reports a candidate screening protocol to distinguish beef from horse meat based upon comparison of triglyceride signatures obtained by 60 MHz 1H NMR spectroscopy. Using a simple chloroform-based extraction, we obtained classic low-field triglyceride spectra from typically a 10 min acquisition time. Peak integration was sufficient to differentiate samples of fresh beef (76 extractions) and horse (62 extractions) using Naïve Bayes classification. Principal component analysis gave a two-dimensional “authentic” beef region (p = 0.001) against which further spectra could be compared. This model was challenged using a subset of 23 freeze–thawed training samples. The outcomes indicated that storing samples by freezing does not adversely affect the analysis. Of a further collection of extractions from previously unseen samples, 90/91 beef spectra were classified as authentic, and 16/16 horse spectra as non-authentic. We conclude that 60 MHz 1H NMR represents a feasible high-throughput approach for screening raw meat. PMID:25577043

Fragment-based discovery of a potent NAMPT inhibitor.

PubMed

Korepanova, Alla; Longenecker, Kenton L; Pratt, Steve D; Panchal, Sanjay C; Clark, Richard F; Lake, Marc; Gopalakrishnan, Sujatha M; Raich, Diana; Sun, Chaohong; Petros, Andrew M

2017-12-12

NAMPT expression is elevated in many cancers, making this protein a potential target for anticancer therapy. We have carried out both NMR based and TR-FRET based fragment screens against human NAMPT and identified six novel binders with a range of potencies. Co-crystal structures were obtained for two of the fragments bound to NAMPT while for the other four fragments force-field driven docking was employed to generate a bound pose. Based on structural insights arising from comparison of the bound fragment poses to that of bound FK866 we were able to synthetically elaborate one of the fragments into a potent NAMPT inhibitor. Copyright © 2017 Elsevier Ltd. All rights reserved.

Identification of a xanthine oxidase-inhibitory component from Sophora flavescens using NMR-based metabolomics.

PubMed

Suzuki, Ryuichiro; Hasuike, Yuka; Hirabayashi, Moeka; Fukuda, Tatsuo; Okada, Yoshihito; Shirataki, Yoshiaki

2013-10-01

We demonstrate that NMR-based metabolomics studies can be used to identify xanthine oxidase-inhibitory compounds in the diethyl ether soluble fraction prepared from a methanolic extract of Sophora flavescens. Loading plot analysis, accompanied by direct comparison of 1H NMR spectraexhibiting characteristic signals, identified compounds exhibiting inhibitory activity. NMR analysis indicated that these characteristic signals were attributed to flavanones such as sophoraflavanone G and kurarinone. Sophoraflavanone G showed inhibitory activity towards xanthine oxidase in an in vitro assay.

Potential antimicrobial agents from triazole-functionalized 2H-benzo[b][1,4]oxazin-3(4H)-ones.

PubMed

Bollu, Rajitha; Banu, Saleha; Bantu, Rajashaker; Reddy, A Gopi; Nagarapu, Lingaiah; Sirisha, K; Kumar, C Ganesh; Gunda, Shravan Kumar; Shaik, Kamal

2017-12-01

A series of substituted triazole functionalized 2H-benzo[b][1,4]oxazin-3(4H)-ones were synthesized by employing click chemistry and further characterized based on 1 H NMR, 13 C NMR, IR and mass spectral studies. All the synthesized derivatives were screened for their in vitro antimicrobial activities. Further, molecular docking studies were accomplished to explore the binding interactions between 1,2,3-triazol-4-yl-2H-benzo[b][1,4]oxazin-3(4H)-one and the active site of Staphylococcus aureus (CrtM) dehydrosqualene synthase (PDB ID: 2ZCS). These docking studies revealed that the synthesized derivatives showed high binding energies and strong H-bond interactions with the dehydrosqualene synthase validating the observed antimicrobial activity data. Based on antimicrobial activity and docking studies, the compounds 9c, 9d and 9e were identified as promising antimicrobial leads. Copyright © 2017 Elsevier Ltd. All rights reserved.

Fragment-based protein-protein interaction antagonists of a viral dimeric protease

PubMed Central

Gable, Jonathan E.; Lee, Gregory M.; Acker, Timothy M.; Hulce, Kaitlin R.; Gonzalez, Eric R.; Schweigler, Patrick; Melkko, Samu; Farady, Christopher J.; Craik, Charles S.

2016-01-01

Fragment-based drug discovery has shown promise as an approach for challenging targets such as protein-protein interfaces. We developed and applied an activity-based fragment screen against dimeric Kaposi’s sarcoma-associated herpesvirus protease (KSHV Pr) using an optimized fluorogenic substrate. Dose response determination was performed as a confirmation screen and NMR spectroscopy was used to map fragment inhibitor binding to KSHV Pr. Kinetic assays demonstrated that several initial hits also inhibit human cytomegalovirus protease (HCMV Pr). Binding of these hits to HCMV Pr was also confirmed via NMR spectroscopy. Despite the use of a target-agnostic fragment library, more than 80% of confirmed hits disrupted dimerization and bound to a previously reported pocket at the dimer interface of KSHV Pr, not to the active site. One class of fragments, an aminothiazole scaffold, was further explored using commercially available analogs. These compounds demonstrated greater than 100-fold improvement of inhibition. This study illustrates the power of fragment-based screening for these challenging enzymatic targets and provides an example of the potential druggability of pockets at protein-protein interfaces. PMID:26822284

A Fragment-Based Ligand Screen Against Part of a Large Protein Machine: The ND1 Domains of the AAA+ ATPase p97/VCP.

PubMed

Chimenti, Michael S; Bulfer, Stacie L; Neitz, R Jeffrey; Renslo, Adam R; Jacobson, Matthew P; James, Thomas L; Arkin, Michelle R; Kelly, Mark J S

2015-07-01

The ubiquitous AAA+ ATPase p97 functions as a dynamic molecular machine driving several cellular processes. It is essential in regulating protein homeostasis, and it represents a potential drug target for cancer, particularly when there is a greater reliance on the endoplasmic reticulum-associated protein degradation pathway and ubiquitin-proteasome pathway to degrade an overabundance of secreted proteins. Here, we report a case study for using fragment-based ligand design approaches against this large and dynamic hexamer, which has multiple potential binding sites for small molecules. A screen of a fragment library was conducted by surface plasmon resonance (SPR) and followed up by nuclear magnetic resonance (NMR), two complementary biophysical techniques. Virtual screening was also carried out to examine possible binding sites for the experimental hits and evaluate the potential utility of fragment docking for this target. Out of this effort, 13 fragments were discovered that showed reversible binding with affinities between 140 µM and 1 mM, binding stoichiometries of 1:1 or 2:1, and good ligand efficiencies. Structural data for fragment-protein interactions were obtained with residue-specific [U-(2)H] (13)CH3-methyl-labeling NMR strategies, and these data were compared to poses from docking. The combination of virtual screening, SPR, and NMR enabled us to find and validate a number of interesting fragment hits and allowed us to gain an understanding of the structural nature of fragment binding. © 2015 Society for Laboratory Automation and Screening.

Increasing Chemical Space Coverage by Combining Empirical and Computational Fragment Screens

PubMed Central

2015-01-01

Most libraries for fragment-based drug discovery are restricted to 1,000–10,000 compounds, but over 500,000 fragments are commercially available and potentially accessible by virtual screening. Whether this larger set would increase chemotype coverage, and whether a computational screen can pragmatically prioritize them, is debated. To investigate this question, a 1281-fragment library was screened by nuclear magnetic resonance (NMR) against AmpC β-lactamase, and hits were confirmed by surface plasmon resonance (SPR). Nine hits with novel chemotypes were confirmed biochemically with KI values from 0.2 to low mM. We also computationally docked 290,000 purchasable fragments with chemotypes unrepresented in the empirical library, finding 10 that had KI values from 0.03 to low mM. Though less novel than those discovered by NMR, the docking-derived fragments filled chemotype holes from the empirical library. Crystal structures of nine of the fragments in complex with AmpC β-lactamase revealed new binding sites and explained the relatively high affinity of the docking-derived fragments. The existence of chemotype holes is likely a general feature of fragment libraries, as calculation suggests that to represent the fragment substructures of even known biogenic molecules would demand a library of minimally over 32,000 fragments. Combining computational and empirical fragment screens enables the discovery of unexpected chemotypes, here by the NMR screen, while capturing chemotypes missing from the empirical library and tailored to the target, with little extra cost in resources. PMID:24807704

Micro-scale NMR Screening of New Detergents for Membrane Protein Structural Biology

PubMed Central

Zhang, Qinghai; Horst, Reto; Geralt, Michael; Ma, Xingquan; Hong, Wen-Xu; Finn, M. G.; Stevens, Raymond C.; Wüthrich, Kurt

2008-01-01

The rate limiting step in biophysical characterization of membrane proteins is often the availability of suitable amounts of protein material. It was therefore of interest to demonstrate that micro-coil nuclear magnetic resonance (NMR) technology can be used to screen microscale quantities of membrane proteins for proper folding in samples destined for structural studies. Micoscale NMR was then used to screen a series of newly designed zwitterionic phosphocholine detergents for their ability to reconstitute membrane proteins, using the previously well characterized β-barrel E.coli outer membrane protein OmpX as a test case. Fold screening was thus achieved with μg-amounts of uniformly 2H,15N-labeld OmpX and affordable amounts of the detergents, and prescreening with SDS-gel electrophoresis ensured efficient selection of the targets for NMR studies. A systematic approach to optimize the phosphocholine motif for membrane protein refolding led to the identification of two new detergents, 138-Fos and 179-Fos, that yield 2D [15N,1H]-TROSY correlation NMR spectra of natively folded reconstituted OmpX. PMID:18479092

Screening protein – Single stranded RNA complexes by NMR spectroscopy for structure determination☆

PubMed Central

Foot, Jaelle N.; Feracci, Mikael; Dominguez, Cyril

2014-01-01

In the past few years, RNA molecules have been revealed to be at the center of numerous biological processes. Long considered as passive molecules transferring genetic information from DNA to proteins, it is now well established that RNA molecules play important regulatory roles. Associated with that, the number of identified RNA binding proteins (RBPs) has increased considerably and mutations in RNA molecules or RBP have been shown to cause various diseases, such as cancers. It is therefore crucial to understand at the molecular level how these proteins specifically recognise their RNA targets in order to design new generation drug therapies targeting protein–RNA complexes. Nuclear magnetic resonance (NMR) is a particularly well-suited technique to study such protein–RNA complexes at the atomic level and can provide valuable information for new drug discovery programs. In this article, we describe the NMR strategy that we and other laboratories use for screening optimal conditions necessary for structural studies of protein-single stranded RNA complexes, using two proteins, Sam68 and T-STAR, as examples. PMID:24096002

Racial differences in the relationship between rate of nicotine metabolism and nicotine intake from cigarette smoking

PubMed Central

Ross, Kathryn C.; Gubner, Noah R.; Tyndale, Rachel F.; Hawk, Larry W.; Lerman, Caryn; George, Tony P.; Cinciripini, Paul; Schnoll, Robert A.; Benowitz, Neal L.

2016-01-01

Rate of nicotine metabolism has been identified as an important factor influencing nicotine intake and can be estimated using the nicotine metabolite ratio (NMR), a validated biomarker of CYP2A6 enzyme activity. Individuals who metabolize nicotine faster (higher NMR) may alter their smoking behavior to titrate their nicotine intake in order to maintain similar levels of nicotine in the body compared to slower nicotine metabolizers. There are known racial differences in the rate of nicotine metabolism with African Americans on average having a slower rate of nicotine metabolism compared to Whites. The goal of this study was to determine if there are racial differences in the relationship between rate of nicotine metabolism and measures of nicotine intake assessed using multiple biomarkers of nicotine and tobacco smoke exposure. Using secondary analyses of the screening data collected in a recently completed clinical trial, treatment-seeking African American and White daily smokers (10 or more cigarettes per day) were grouped into NMR quartiles so that the races could be compared at the same NMR, even though the distribution of NMR within race differed. The results indicated that rate of nicotine metabolism is a more important factor influencing nicotine intake in White smokers. Specifically, Whites were more likely to titrate their nicotine intake based on the rate at which they metabolize nicotine. However, this relationship was not found in African Americans. Overall there was a greater step down, linear type relationship between NMR groups and cotinine or cotinine/cigarette in African Americans, which is consistent with the idea that differences in blood cotinine levels between the African American NMR groups were primarily due to differences in CYP2A6 enzyme activity without titration of nicotine intake among faster nicotine metabolizers. PMID:27180107

Authentication of beef versus horse meat using 60 MHz 1H NMR spectroscopy.

PubMed

Jakes, W; Gerdova, A; Defernez, M; Watson, A D; McCallum, C; Limer, E; Colquhoun, I J; Williamson, D C; Kemsley, E K

2015-05-15

This work reports a candidate screening protocol to distinguish beef from horse meat based upon comparison of triglyceride signatures obtained by 60 MHz (1)H NMR spectroscopy. Using a simple chloroform-based extraction, we obtained classic low-field triglyceride spectra from typically a 10 min acquisition time. Peak integration was sufficient to differentiate samples of fresh beef (76 extractions) and horse (62 extractions) using Naïve Bayes classification. Principal component analysis gave a two-dimensional "authentic" beef region (p=0.001) against which further spectra could be compared. This model was challenged using a subset of 23 freeze-thawed training samples. The outcomes indicated that storing samples by freezing does not adversely affect the analysis. Of a further collection of extractions from previously unseen samples, 90/91 beef spectra were classified as authentic, and 16/16 horse spectra as non-authentic. We conclude that 60 MHz (1)H NMR represents a feasible high-throughput approach for screening raw meat. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

A Preliminary Investigation of NSCL/P Plasma and Urine in Guizhou Province in China Using NMR-Based Metabonomics.

PubMed

Lei, Huang Guang; Hong, Luo; Kun, Song Ju; Hai, Yin Xin; Dong, Wang Ya; Ke, Zhao; Ping, Xu; Hao, Chen

2013-09-01

Objective : To assess the feasibility of metabonomics in clinical studies. This is a pilot study introducing nuclear magnetic resonance (NMR)-based metabonomics to elucidate and compare the metabolism of patients with nonsyndromic cleft lip and/or palate (NSCL/P) and children without orofacial clefts. Methods : High-resolution (1)H NMR spectroscopy was performed on plasma and urine samples obtained from NSCL/P and healthy children. The (1)H NMR spectra were further analyzed with principal component analysis. Results : Compared to the control group, the level of low-molecular-weight metabolites in plasma such as asparagine was higher in NSCL/P patients, while arginine, lysine, acetate, lactate, proline, glutamine, pyruvate, creatinine, choline, and β-glucose were lower. The carnitine, citrate, and formate excretion in urine appeared to be higher in the healthy children, while the NSCL/P group excreted higher concentrations of aspartic acid and phenylalanine in urine. Conclusion : The present study clearly demonstrated the great potential of NMR-based metabonomics in elucidating NSCL/P plasma metabolism and the possible application of this technology in clinical diagnosis and screening.

Determination of Diethyl Phthalate and Polyhexamethylene Guanidine in Surrogate Alcohol from Russia

PubMed Central

Monakhova, Yulia B.; Kuballa, Thomas; Leitz, Jenny; Lachenmeier, Dirk W.

2011-01-01

Analytical methods based on spectroscopic techniques were developed and validated for the determination of diethyl phthalate (DEP) and polyhexamethylene guanidine (PHMG), which may occur in unrecorded alcohol. Analysis for PHMG was based on UV-VIS spectrophotometry after derivatization with Eosin Y and 1H NMR spectroscopy of the DMSO extract. Analysis of DEP was performed with direct UV-VIS and 1H NMR methods. Multivariate curve resolution and spectra computation methods were used to confirm the presence of PHMG and DEP in the investigated beverages. Of 22 analysed alcohol samples, two contained DEP or PHMG. 1H NMR analysis also revealed the presence of signals of hawthorn extract in three medicinal alcohols used as surrogate alcohol. The simple and cheap UV-VIS methods can be used for rapid screening of surrogate alcohol samples for impurities, while 1H NMR is recommended for specific confirmatory analysis if required. PMID:21647285

Determination of diethyl phthalate and polyhexamethylene guanidine in surrogate alcohol from Russia.

PubMed

Monakhova, Yulia B; Kuballa, Thomas; Leitz, Jenny; Lachenmeier, Dirk W

2011-01-01

Analytical methods based on spectroscopic techniques were developed and validated for the determination of diethyl phthalate (DEP) and polyhexamethylene guanidine (PHMG), which may occur in unrecorded alcohol. Analysis for PHMG was based on UV-VIS spectrophotometry after derivatization with Eosin Y and (1)H NMR spectroscopy of the DMSO extract. Analysis of DEP was performed with direct UV-VIS and (1)H NMR methods. Multivariate curve resolution and spectra computation methods were used to confirm the presence of PHMG and DEP in the investigated beverages. Of 22 analysed alcohol samples, two contained DEP or PHMG. (1)H NMR analysis also revealed the presence of signals of hawthorn extract in three medicinal alcohols used as surrogate alcohol. The simple and cheap UV-VIS methods can be used for rapid screening of surrogate alcohol samples for impurities, while (1)H NMR is recommended for specific confirmatory analysis if required.

Fragment-Based Protein-Protein Interaction Antagonists of a Viral Dimeric Protease.

PubMed

Gable, Jonathan E; Lee, Gregory M; Acker, Timothy M; Hulce, Kaitlin R; Gonzalez, Eric R; Schweigler, Patrick; Melkko, Samu; Farady, Christopher J; Craik, Charles S

2016-04-19

Fragment-based drug discovery has shown promise as an approach for challenging targets such as protein-protein interfaces. We developed and applied an activity-based fragment screen against dimeric Kaposi's sarcoma-associated herpesvirus protease (KSHV Pr) using an optimized fluorogenic substrate. Dose-response determination was performed as a confirmation screen, and NMR spectroscopy was used to map fragment inhibitor binding to KSHV Pr. Kinetic assays demonstrated that several initial hits also inhibit human cytomegalovirus protease (HCMV Pr). Binding of these hits to HCMV Pr was also confirmed by NMR spectroscopy. Despite the use of a target-agnostic fragment library, more than 80 % of confirmed hits disrupted dimerization and bound to a previously reported pocket at the dimer interface of KSHV Pr, not to the active site. One class of fragments, an aminothiazole scaffold, was further explored using commercially available analogues. These compounds demonstrated greater than 100-fold improvement of inhibition. This study illustrates the power of fragment-based screening for these challenging enzymatic targets and provides an example of the potential druggability of pockets at protein-protein interfaces. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

N-15 NMR Spectroscopy as a Method for Comparing the Rates of Imidization of Several Diamines

NASA Technical Reports Server (NTRS)

Johnson, J. Christopher; Kuczmarski, Maria A.

2006-01-01

The relative rates of the conversion of amide-acid to imide was measured for a series or aromatic diamines that have been identified as potential replacements for 4,4'-methylene dianiline (MDA) in high-temperature polyimides and polymer composites. These rates were compared with the N-15 NMR resonances of the unreacted amines. The initial rates of imidization track with the difference in chemical shift between the amine nitrogens in MDA and those in the subject diamines. This comparison demonstrated that N-15 NMR spectroscopy is appropriate for the rapid screening of candidate diamines to determine their reactivity relative to MDA, and can serve to provide guidance to the process of creating the time-temperature profiles used in processing these materials into polymer matrix composites.

Rapid screening, identification, and purification of neuraminidase inhibitors from Lithospermum erythrorhizon Sieb.et Zucc. by ultrafiltration with HPLC-ESI-TOF-MS combined with semipreparative HPLC.

PubMed

Zhang, Minmin; Zhao, Hengqiang; Zhao, Zhiguo; Yan, Huijiao; Lv, Ruimin; Cui, Li; Yuan, Jinpeng; Wang, Daijie; Geng, Yanling; Liu, Daicheng; Wang, Xiao

2016-06-01

We put forward an efficient strategy based on bioassay guidance for the rapid screening, identification, and purification of the neuraminidase inhibitors from traditional Chinese medicines, and apply to the discovery of anti-influenza components from Lithospermiun erythrorhizon Sieb.et Zucc. Ultrafiltration with high-performance liquid chromatography and electrospray ionization time-of-flight mass spectrometry was employed for the rapid screening and preliminarily identification of anti-influenza components from Zicao. Semipreparative high-performance liquid chromatography was used for the rapid separation and purification of the target compounds. NMR spectroscopy, mass spectrometry, and UV spectroscopy were used for further structural identification, and the activity of the compounds was verified by in vitro assay. Five compounds were found to have neuraminidase inhibitory activity by this method. Subsequently, the five compounds were separated by semipreparative high-performance liquid chromatography with the purity over 98% for all of them by high-performance liquid chromatography test. Combined with the NMR spectroscopy, mass spectrometry, and UV spectroscopy data, they were identified as alkannin, acetylalkannin, isobutyrylalkannin, β,β-dimethylacryloylalkannin and isovalerylalkannin. The in vitro assay showed that all five compounds had good neuraminidase inhibitory activities. These results suggested that the method is highly efficient, and it can provide platform and methodology supports for the rapid discovery of anti-influenza active ingredients from complex Chinese herbal medicines. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Molecular docking study, synthesis and biological evaluation of Mannich bases as Hsp90 inhibitors.

PubMed

Gupta, Sayan Dutta; Bommaka, Manish Kumar; Mazaira, Gisela I; Galigniana, Mario D; Subrahmanyam, Chavali Venkata Satya; Gowrishankar, Naryanasamy Lachmana; Raghavendra, Nulgumnalli Manjunathaiah

2015-09-01

The ubiquitously expressed heat shock protein 90 is an encouraging target for the development of novel anticancer agents. In a program directed towards uncovering novel chemical scaffolds against Hsp90, we performed molecular docking studies using Tripos-Sybyl drug designing software by including the required conserved water molecules. The results of the docking studies predicted Mannich bases derived from 2,4-dihydroxy acetophenone/5-chloro 2,4-dihydroxy acetophenone as potential Hsp90 inhibitors. Subsequently, a few of them were synthesized (1-6) and characterized by IR, (1)H NMR, (13)C NMR and mass spectral analysis. The synthesized Mannich compounds were evaluated for their potential to suppress Hsp90 ATPase activity by the colorimetric Malachite green assay. Subsequently, the molecules were screened for their antiproilferative effect against PC3 pancreatic carcinoma cells by adopting the 3-(4,5-dimethythiazol- 2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay method. The activity profile of the identified derivatives correlated well with their docking results. Copyright © 2015 Elsevier B.V. All rights reserved.

1H NMR based metabolic profiling of eleven Algerian aromatic plants and evaluation of their antioxidant and cytotoxic properties.

PubMed

Brahmi, Nabila; Scognamiglio, Monica; Pacifico, Severina; Mekhoukhe, Aida; Madani, Khodir; Fiorentino, Antonio; Monaco, Pietro

2015-10-01

Eleven Algerian medicinal and aromatic plants (Aloysia triphylla, Apium graveolens, Coriandrum sativum, Laurus nobilis, Lavandula officinalis, Marrubium vulgare, Mentha spicata, Inula viscosa, Petroselinum crispum, Salvia officinalis, and Thymus vulgaris) were selected and their hydroalcoholic extracts were screened for their antiradical and antioxidant properties in cell-free systems. In order to identify the main metabolites constituting the extracts, 1 H NMR-based metabolic profiling was applied. Data obtained emphasized the antiradical properties of T. vulgaris, M. spicata and L. nobilis extracts (RACI 1.37, 0.97 and 0.93, respectively), whereas parsley was the less active as antioxidant (RACI -1.26). When the cytotoxic effects of low and antioxidant doses of each extract were evaluated towards SK-N-BE(2)C neuronal and HepG2 hepatic cell lines, it was observed that all the extracts weakly affected the metabolic redox activity of the tested cell lines. Overall, data strongly plead in favor of the use of these plants as potential food additives in replacement of synthetic compounds. Copyright © 2015 Elsevier Ltd. All rights reserved.

NMR-based metabolomic urinalysis: a rapid screening test for urinary tract infection.

PubMed

Lam, Ching-Wan; Law, Chun-Yiu; To, Kelvin Kai-Wang; Cheung, Stanley Kwok-Kuen; Lee, Kim-Chung; Sze, Kong-Hung; Leung, Ka-Fai; Yuen, Kwok-Yung

2014-09-25

Urinary tract infection (UTI) is one of the most common bacterial infections in humans; however, there is no accurate and fast quantitative test to detect UTI. Dipstick urinalysis is semi-quantitative with a limited diagnostic accuracy, while urine culture is accurate but takes time. We described a quantitative biochemical method for the diagnosis of bacteriuria using a single marker. We compared the urine metabolomes from 88 patients with bacterial UTI and 61 controls using (1)H NMR spectroscopy followed by principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA). The biomarker identified was subsequently validated using independent samples. The urine acetic acid/creatinine (mmol/mmol) level was determined to be the most discriminatory marker for bacterial UTI with an area-under-receiver operating characteristic curve=0.97, sensitivity=91% and specificity=95% at the optimal cutoff 0.03 mmol/mmol. For validation, 60 samples were recruited prospectively. Using the optimal cutoff for acetic acid/creatinine, this method showed sensitivity=96%, specificity=94%, positive predictive value=92%, negative predictive value=97% and an overall accuracy=95%. The diagnostic performance was superior to dipstick urinalysis or microscopy. In addition, we also observed an increase of urinary trimethylamine (TMA) in patients with Escherichia coli-associated UTI. TMA is a mammalian-microbial co-metabolite and the high level of TMA generated is related to the bacterial enzyme, trimethylamine N-oxide (TMAO) reductase which reduces TMAO to TMA. Urine acetic acid is a neglected metabolite that can be used for rapid diagnosis of UTI and TMA can be used for etiologic diagnosis of UTI. With the introduction of NMR-based clinical analyzers to clinical laboratories, NMR-based urinalysis can be translated for clinical use. Copyright © 2014 Elsevier B.V. All rights reserved.

Microplate assay for screening the antibacterial activity of Schiff bases derived from substituted benzopyran-4-one.

PubMed

Amin, Rehab M; Abdel-Kader, Nora S; El-Ansary, Aida L

2012-09-01

Schiff bases (SB(1)-SB(3)) were synthesized from the condensation of 6-formyl-7-hydroxy-5-methoxy-2-methylbenzopyran-4-one with 2-aminopyridine (SB(1)), p-phenylenediamine (SB(2)) and o-phenylenediamine (SB(3)), while Schiff bases (SB(4)-SB(6)) were synthesized by condensation of 5,7-dihydroxy-6-formyl-2-methylbenzopyran-4-one with 2-aminopyridine (SB(4)), p-phenylenediamine (SB(5)) and o-phenylenediamine (SB(6)). Schiff bases were characterized using elemental analysis, IR, UV-Vis, (1)H NMR, (13)C NMR and mass spectroscopy. These compounds were screened for antibacterial activities by micro-plate assay technique. Escherichia coli and Staphylococcus capitis were exposed to different concentrations of the Schiff bases. Results showed that the antibacterial effect of these Schiff bases on Gram-negative bacteria were higher than that on Gram-positive bacteria moreover, the Schiff bases containing substituent OCH(3) on position five have higher antibacterial activity than that containing hydroxy group on the same position. Copyright © 2012 Elsevier B.V. All rights reserved.

Mixing and Matching Detergents for Membrane Protein NMR Structure Determination

DOE Office of Scientific and Technical Information (OSTI.GOV)

Columbus, Linda; Lipfert, Jan; Jambunathan, Kalyani

2009-10-21

One major obstacle to membrane protein structure determination is the selection of a detergent micelle that mimics the native lipid bilayer. Currently, detergents are selected by exhaustive screening because the effects of protein-detergent interactions on protein structure are poorly understood. In this study, the structure and dynamics of an integral membrane protein in different detergents is investigated by nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopy and small-angle X-ray scattering (SAXS). The results suggest that matching of the micelle dimensions to the protein's hydrophobic surface avoids exchange processes that reduce the completeness of the NMR observations. Based onmore » these dimensions, several mixed micelles were designed that improved the completeness of NMR observations. These findings provide a basis for the rational design of mixed micelles that may advance membrane protein structure determination by NMR.« less

Potential effect of diaper and cotton ball contamination on NMR- and LC/MS-based metabonomics studies of urine from newborn babies.

PubMed

Goodpaster, Aaron M; Ramadas, Eshwar H; Kennedy, Michael A

2011-02-01

Nuclear magnetic resonance (NMR) and liquid chromatography/mass spectrometry (LC/MS) based metabonomics screening of urine has great potential for discovery of biomarkers for diseases that afflict newborn and preterm infants. However, urine collection from newborn infants presents a potential confounding problem due to the possibility that contaminants might leach from materials used for urine collection and influence statistical analysis of metabonomics data. In this manuscript, we have analyzed diaper and cotton ball contamination using synthetic urine to assess its potential to influence the outcome of NMR- and LC/MS-based metabonomics studies of human infant urine. Eight diaper brands were examined using the "diaper plus cotton ball" technique. Data were analyzed using conventional principal components analysis, as well as a statistical significance algorithm developed for, and applied to, NMR data. Results showed most diaper brands had distinct contaminant profiles that could potentially influence NMR- and LC/MS-based metabonomics studies. On the basis of this study, it is recommended that diaper and cotton ball brands be characterized using metabonomics methodologies prior to initiating a metabonomics study to ensure that contaminant profiles are minimal or manageable and that the same diaper and cotton ball brands be used throughout a study to minimize variation.

A combination of 19F NMR and surface plasmon resonance for site-specific hit selection and validation of fragment molecules that bind to the ATP-binding site of a kinase.

PubMed

Nagatoishi, Satoru; Yamaguchi, Sou; Katoh, Etsuko; Kajita, Keita; Yokotagawa, Takane; Kanai, Satoru; Furuya, Toshio; Tsumoto, Kouhei

2018-05-01

19 F NMR has recently emerged as an efficient, sensitive tool for analyzing protein binding to small molecules, and surface plasmon resonance (SPR) is also a popular tool for this purpose. Herein a combination of 19 F NMR and SPR was used to find novel binders to the ATP-binding pocket of MAP kinase extracellular regulated kinase 2 (ERK2) by fragment screening with an original fluorinated-fragment library. The 19 F NMR screening yielded a high primary hit rate of binders to the ERK2 ATP-binding pocket compared with the rate for the SPR screening. Hit compounds were evaluated and categorized according to their ability to bind to different binding sites in the ATP-binding pocket. The binding manner was characterized by using isothermal titration calorimetry and docking simulation. Combining 19 F NMR with other biophysical methods allows the identification of multiple types of hit compounds, thereby increasing opportunities for drug design using preferred fragments. Copyright © 2018 Elsevier Ltd. All rights reserved.

Harnessing biosynthesis and quantitative NMR for late stage functionalization of lead molecules: Application to the M1 positive allosteric modulator (PAM) program.

PubMed

Brodney, Michael A; Sharma, Raman; Lazzaro, John T; Walker, Gregory S; Scott Obach, R

2018-06-15

A facile method for late stage diversification of lead molecules for the M1 PAM program using biosynthesis is described. Liver microsomes from several species are screened to identify a high turnover system. Subsequent incubations using less than 1 mg of substrate generate nanomole quantities of drug metabolites that are purified, characterized by microcryoprobe NMR spectroscopy, and quantified to known concentrations to enable rapid biology testing. The late-stage diversification of lead compounds provides rapid SAR feedback to the medicinal chemistry design cycle. Copyright © 2018 Elsevier Ltd. All rights reserved.

Characterization of the Raf kinase inhibitory protein (RKIP) binding pocket: NMR-based screening identifies small-molecule ligands.

PubMed

Shemon, Anne N; Heil, Gary L; Granovsky, Alexey E; Clark, Mathew M; McElheny, Dan; Chimon, Alexander; Rosner, Marsha R; Koide, Shohei

2010-05-05

Raf kinase inhibitory protein (RKIP), also known as phoshaptidylethanolamine binding protein (PEBP), has been shown to inhibit Raf and thereby negatively regulate growth factor signaling by the Raf/MAP kinase pathway. RKIP has also been shown to suppress metastasis. We have previously demonstrated that RKIP/Raf interaction is regulated by two mechanisms: phosphorylation of RKIP at Ser-153, and occupation of RKIP's conserved ligand binding domain with a phospholipid (2-dihexanoyl-sn-glycero-3-phosphoethanolamine; DHPE). In addition to phospholipids, other ligands have been reported to bind this domain; however their binding properties remain uncharacterized. In this study, we used high-resolution heteronuclear NMR spectroscopy to screen a chemical library and assay a number of potential RKIP ligands for binding to the protein. Surprisingly, many compounds previously postulated as RKIP ligands showed no detectable binding in near-physiological solution conditions even at millimolar concentrations. In contrast, we found three novel ligands for RKIP that specifically bind to the RKIP pocket. Interestingly, unlike the phospholipid, DHPE, these newly identified ligands did not affect RKIP binding to Raf-1 or RKIP phosphorylation. One out of the three ligands displayed off target biological effects, impairing EGF-induced MAPK and metabolic activity. This work defines the binding properties of RKIP ligands under near physiological conditions, establishing RKIP's affinity for hydrophobic ligands and the importance of bulky aliphatic chains for inhibiting its function. The common structural elements of these compounds defines a minimal requirement for RKIP binding and thus they can be used as lead compounds for future design of RKIP ligands with therapeutic potential.

Synthesis, spectroscopic characterization and antimicrobial studies of Co(II), Ni(II), Cu(II) and Zn(II) complexes with Schiff bases derived from 5-bromo-salicylaldehyde

NASA Astrophysics Data System (ADS)

Kursunlu, Ahmed Nuri; Guler, Ersin; Sevgi, Fatih; Ozkalp, Birol

2013-09-01

In this study, the new Schiff base ligands derived from condensation of amine and 5-bromo-salicylaldehyde were characterized. All compounds, the Schiff bases and the metal complexes, were characterized by elemental analyzes, FT-IR, 1H NMR, 13C NMR and magnetic susceptibility measurements. The synthesized ligands, along with their metal (II) complexes, were screened for their in vitro antibacterial activity against four Gram-negative (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Salmonella enteritidis) and four Gram-positive (Streptococcus pyogones, Bacillus cereus, Staphylococcus aureus and Methicillin-resistant S. aureus) bacterial strains by using disc diffusion and broth microdilution techniques.

Effective progression of nuclear magnetic resonance-detected fragment hits.

PubMed

Eaton, Hugh L; Wyss, Daniel F

2011-01-01

Fragment-based drug discovery (FBDD) has become increasingly popular over the last decade as an alternate lead generation tool to HTS approaches. Several compounds have now progressed into the clinic which originated from a fragment-based approach, demonstrating the utility of this emerging field. While fragment hit identification has become much more routine and may involve different screening approaches, the efficient progression of fragment hits into quality lead series may still present a major bottleneck for the broadly successful application of FBDD. In our laboratory, we have extensive experience in fragment-based NMR screening (SbN) and the subsequent iterative progression of fragment hits using structure-assisted chemistry. To maximize impact, we have applied this approach strategically to early- and high-priority targets, and those struggling for leads. Its application has yielded a clinical candidate for BACE1 and lead series in about one third of the SbN/FBDD projects. In this chapter, we will give an overview of our strategy and focus our discussion on NMR-based FBDD approaches. Copyright © 2011 Elsevier Inc. All rights reserved.

Spatial and Temporal Variations of a Screening Current Induced Magnetic Field in a Double-Pancake HTS Insert of an LTS/HTS NMR Magnet

PubMed Central

Ahn, Min Cheol; Yagai, Tsuyoshi; Hahn, Seungyong; Ando, Ryuya; Bascuñán, Juan; Iwasa, Yukikazu

2010-01-01

This paper presents experimental and simulation results of a screening current induced magnetic field (SCF) in a high temperature superconductor (HTS) insert that constitutes a low-/high-temperature superconductor (LTS/HTS) NMR magnet. In this experiment, the HTS insert, a stack of 50 double-pancake coils, each wound with Bi2223 tape, was operated at 77 K. A screening current was induced in the HTS insert by three magnetic field sources: 1) a self field from the HTS insert; 2) an external field from a 5-T background magnet; and 3) combinations of 1) and 2). For each field excitation, which induced an SCF, its axial field distribution and temporal variations were measured and compared with simulation results based on the critical state model. Agreement on field profile between experiment and simulation is satisfactory but more work is needed to make the simulation useful for designing shim coils that will cancel the SCF. PMID:20401187

Determinants of Ligand Subtype-Selectivity at α1A-Adrenoceptor Revealed Using Saturation Transfer Difference (STD) NMR.

PubMed

Yong, Kelvin J; Vaid, Tasneem M; Shilling, Patrick J; Wu, Feng-Jie; Williams, Lisa M; Deluigi, Mattia; Plückthun, Andreas; Bathgate, Ross A D; Gooley, Paul R; Scott, Daniel J

2018-04-20

α 1A - and α 1B -adrenoceptors (α 1A -AR and α 1B -AR) are closely related G protein-coupled receptors (GPCRs) that modulate the cardiovascular and nervous systems in response to binding epinephrine and norepinephrine. The GPCR gene superfamily is made up of numerous subfamilies that, like α 1A -AR and α 1B -AR, are activated by the same endogenous agonists but may modulate different physiological processes. A major challenge in GPCR research and drug discovery is determining how compounds interact with receptors at the molecular level, especially to assist in the optimization of drug leads. Nuclear magnetic resonance spectroscopy (NMR) can provide great insight into ligand-binding epitopes, modes, and kinetics. Ideally, ligand-based NMR methods require purified, well-behaved protein samples. The instability of GPCRs upon purification in detergents, however, makes the application of NMR to study ligand binding challenging. Here, stabilized α 1A -AR and α 1B -AR variants were engineered using Cellular High-throughput Encapsulation, Solubilization, and Screening (CHESS), allowing the analysis of ligand binding with Saturation Transfer Difference NMR (STD NMR). STD NMR was used to map the binding epitopes of epinephrine and A-61603 to both receptors, revealing the molecular determinants for the selectivity of A-61603 for α 1A -AR over α 1B -AR. The use of stabilized GPCRs for ligand-observed NMR experiments will lead to a deeper understanding of binding processes and assist structure-based drug design.

LC-NMR Technique in the Analysis of Phytosterols in Natural Extracts

PubMed Central

Horník, Štěpán; Sajfrtová, Marie; Sýkora, Jan; Březinová, Anna; Wimmer, Zdeněk

2013-01-01

The ability of LC-NMR to detect simultaneously free and conjugated phytosterols in natural extracts was tested. The advantages and disadvantages of a gradient HPLC-NMR method were compared to the fast composition screening using SEC-NMR method. Fractions of free and conjugated phytosterols were isolated and analyzed by isocratic HPLC-NMR methods. The results of qualitative and quantitative analyses were in a good agreement with the literature data. PMID:24455424

Fragment Screening and HIV Therapeutics

PubMed Central

Bauman, Joseph D.; Patel, Disha; Arnold, Eddy

2013-01-01

Fragment screening has proven to be a powerful alternative to traditional methods for drug discovery. Biophysical methods, such as X-ray crystallography, NMR spectroscopy, and surface plasmon resonance, are used to screen a diverse library of small molecule compounds. Although compounds identified via this approach have relatively weak affinity, they provide a good platform for lead development and are highly efficient binders with respect to their size. Fragment screening has been utilized for a wide-range of targets, including HIV-1 proteins. Here, we review the fragment screening studies targeting HIV-1 proteins using X-ray crystallography or surface plasmon resonance. These studies have successfully detected binding of novel fragments to either previously established or new sites on HIV-1 protease and reverse transcriptase. In addition, fragment screening against HIV-1 reverse transcriptase has been used as a tool to better understand the complex nature of ligand binding to a flexible target. PMID:21972022

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wenzel, Carsten; Riefke, Björn; Gründemann, Stephan

Cancer cells in poorly vascularized tumor regions need to adapt to an unfavorable metabolic microenvironment. As distance from supplying blood vessels increases, oxygen and nutrient concentrations decrease and cancer cells react by stopping cell cycle progression and becoming dormant. As cytostatic drugs mainly target proliferating cells, cancer cell dormancy is considered as a major resistance mechanism to this class of anti-cancer drugs. Therefore, substances that target cancer cells in poorly vascularized tumor regions have the potential to enhance cytostatic-based chemotherapy of solid tumors. With three-dimensional growth conditions, multicellular tumor spheroids (MCTS) reproduce several parameters of the tumor microenvironment, including oxygenmore » and nutrient gradients as well as the development of dormant tumor regions. We here report the setup of a 3D cell culture compatible high-content screening system and the identification of nine substances from two commercially available drug libraries that specifically target cells in inner MCTS core regions, while cells in outer MCTS regions or in 2D cell culture remain unaffected. We elucidated the mode of action of the identified compounds as inhibitors of the respiratory chain and show that induction of cell death in inner MCTS core regions critically depends on extracellular glucose concentrations. Finally, combinational treatment with cytostatics showed increased induction of cell death in MCTS. The data presented here shows for the first time a high-content based screening setup on 3D tumor spheroids for the identification of substances that specifically induce cell death in inner tumor spheroid core regions. This validates the approach to use 3D cell culture screening systems to identify substances that would not be detectable by 2D based screening in otherwise similar culture conditions. - Highlights: • Establishment of a novel method for 3D cell culture based high-content screening. • First reported high-content screen for the identification of compounds that target cells in dormant tumor spheroid regions. • Metabolite profiling by NMR spectroscopy on 3D tumor spheroids. • Identification of respiratory chain inhibitors to specifically induce cell death in inner tumor spheroid regions. • Respiratory chain inhibitors enhance cytostatic based therapy in vitro.« less

Coupling HPLC-SPE-NMR with a microplate-based high-resolution antioxidant assay for efficient analysis of antioxidants in food--validation and proof-of-concept study with caper buds.

PubMed

Wiese, Stefanie; Wubshet, Sileshi G; Nielsen, John; Staerk, Dan

2013-12-15

This work describes the coupling of a microplate-based antioxidant assay with a hyphenated system consisting of high-performance liquid chromatography-solid-phase extraction-nuclear magnetic resonance spectroscopy, i.e., HPLC-SPE-NMR/high-resolution antioxidant assay, for the analysis of complex food extracts. The applicability of the microplate-based antioxidant assay for high-resolution screening of common food phenolics as well as parameters related to their trapping efficiency, elution behavior, and recovery on/from SPE cartridges are described. It was found that the microplate-based high-resolution antioxidant assay is an attractive and easy implementable alternative to direct on-line screening methods. Furthermore, it was shown that Resin SH and Resin GP SPE material are superior to RP C18HD for trapping of phenolic compounds. Proof-of-concept study was performed with caper bud extract, revealing the most important antioxidants to be quercetin, kaempferol, rutin, kaempferol-3-O-β-rutinoside and N(1),N(5),N(10)-triphenylpropenoyl spermidine amides. Targeted isolation of the latter, and comprehensive NMR experiments showed them to be N(1),N(10)-di-(E)-caffeoyl-N(5)-p-(E)-coumaroyl spermidine, N(1)-(E)-caffeoyl-N(5),N(10)-di-p-(E)-coumaroyl spermidine, N(10)-(E)-caffeoyl-N(1),N(5)-di-p-(E)-coumaroyl spermidine, and N(1),N(5),N(10)-tri-p-(E)-coumaroyl spermidine amides. Copyright © 2013 Elsevier Ltd. All rights reserved.

Design, synthesis and antimalarial screening of some hybrid 4-aminoquinoline-triazine derivatives against pf-DHFR-TS.

PubMed

Sahu, Supriya; Ghosh, Surajit Kumar; Kalita, Junmoni; Dutta, Mayurakhi; Bhat, Hans Raj

2016-04-01

Existing antifolate antimalarial drugs have shown resistance due to the mutations at some amino acid positions of Plasmodium falciparum DHFR-TS. In the present study, to overcome this resistance, a new series of hybrid 4-aminoquinoline-triazine derivatives were designed and docked into the active site of Pf-DHFR-TS (PDB i.d. 1J3K) using validated CDOCKER protocol. Binding energy was calculated by applying CHARMm forcefield. Binding energy and the pattern of interaction of the docked compounds were analysed. Fifteen compounds were selected for synthesis based on their binding energy values and docking poses. Synthesized compounds were characterised by FTIR, (1)H NMR, (13)C NMR, mass spectroscopy and were screened for antimalarial activity against 3D7 strain of Plasmodium falciparum. Copyright © 2016 Elsevier Inc. All rights reserved.

Discovery and structure-activity relationship of oxalylarylaminobenzoic acids as inhibitors of protein tyrosine phosphatase 1B.

PubMed

Liu, Gang; Szczepankiewicz, Bruce G; Pei, Zhonghua; Janowick, David A; Xin, Zhili; Hajduk, Philip J; Abad-Zapatero, Cele; Liang, Heng; Hutchins, Charles W; Fesik, Stephen W; Ballaron, Steve J; Stashko, Mike A; Lubben, Tom; Mika, Amanda K; Zinker, Bradley A; Trevillyan, James M; Jirousek, Michael R

2003-05-22

Protein Tyrosine phosphatase 1B (PTP1B) has been implicated as a key negative regulator of both insulin and leptin signaling pathways. Using an NMR-based screening approach with 15N- and 13C-labeled PTP1B, we have identified 2,3-dimethylphenyloxalylaminobenzoic acid (1) as a general, reversible, and competitive PTPase inhibitor. Structure-based approach guided by X-ray crystallography facilitated the development of 1 into a novel series of potent and selective PTP1B inhibitors occupying both the catalytic site and a portion of the noncatalytic, second phosphotyrosine binding site. Interestingly, oral biovailability has been observed in rats for some compounds. Furthermore, we demonstrated in vivo plasma glucose lowering effects with compound 12d in ob/ob mice.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Smith, R.E.

This report describes the analysis of carbonxyl-terminated butadiene (CTB), carboxyl-terminated butadiene/acrylonitrile (CTBN), and a CTBN adduct prepared by reaction with Epon 828. Data from gel permeation chromatography, nuclear magnetic resonance spectroscopy, high performance liquid chromatography, and ion chromatography are presented and discussed. Quantitative methods based on carbon-13 and proton NMR for analyzing CTBN are described. Proton NMR was found to be useful in identifying lots that have an abnormal amount of CTBN protons. One such lot exhibited a phase separation of a polybutadiene impurity. Carbon-13 NMR was found to be capable of determining nitrile content directly. Carbon-13 NMR had amore » relative standard deviation of 8.3% and a proton NMR of 3.9%. Proton NMR was found to be useful in identifying lots that have 5% more CTBN protons than other lots. 3 refs., 11 figs., 4 tabs.« less

A pilot study of NMR-based sensory prediction of roasted coffee bean extracts.

PubMed

Wei, Feifei; Furihata, Kazuo; Miyakawa, Takuya; Tanokura, Masaru

2014-01-01

Nuclear magnetic resonance (NMR) spectroscopy can be considered a kind of "magnetic tongue" for the characterisation and prediction of the tastes of foods, since it provides a wealth of information in a nondestructive and nontargeted manner. In the present study, the chemical substances in roasted coffee bean extracts that could distinguish and predict the different sensations of coffee taste were identified by the combination of NMR-based metabolomics and human sensory test and the application of the multivariate projection method of orthogonal projection to latent structures (OPLS). In addition, the tastes of commercial coffee beans were successfully predicted based on their NMR metabolite profiles using our OPLS model, suggesting that NMR-based metabolomics accompanied with multiple statistical models is convenient, fast and accurate for the sensory evaluation of coffee. Copyright © 2013 Elsevier Ltd. All rights reserved.

Characterization of the Raf Kinase Inhibitory Protein (RKIP) Binding Pocket: NMR-Based Screening Identifies Small-Molecule Ligands

PubMed Central

Granovsky, Alexey E.; Clark, Mathew M.; McElheny, Dan; Chimon, Alexander; Rosner, Marsha R.; Koide, Shohei

2010-01-01

Background Raf kinase inhibitory protein (RKIP), also known as phoshaptidylethanolamine binding protein (PEBP), has been shown to inhibit Raf and thereby negatively regulate growth factor signaling by the Raf/MAP kinase pathway. RKIP has also been shown to suppress metastasis. We have previously demonstrated that RKIP/Raf interaction is regulated by two mechanisms: phosphorylation of RKIP at Ser-153, and occupation of RKIP's conserved ligand binding domain with a phospholipid (2-dihexanoyl-sn-glycero-3-phosphoethanolamine; DHPE). In addition to phospholipids, other ligands have been reported to bind this domain; however their binding properties remain uncharacterized. Methods/Findings In this study, we used high-resolution heteronuclear NMR spectroscopy to screen a chemical library and assay a number of potential RKIP ligands for binding to the protein. Surprisingly, many compounds previously postulated as RKIP ligands showed no detectable binding in near-physiological solution conditions even at millimolar concentrations. In contrast, we found three novel ligands for RKIP that specifically bind to the RKIP pocket. Interestingly, unlike the phospholipid, DHPE, these newly identified ligands did not affect RKIP binding to Raf-1 or RKIP phosphorylation. One out of the three ligands displayed off target biological effects, impairing EGF-induced MAPK and metabolic activity. Conclusions/Significance This work defines the binding properties of RKIP ligands under near physiological conditions, establishing RKIP's affinity for hydrophobic ligands and the importance of bulky aliphatic chains for inhibiting its function. The common structural elements of these compounds defines a minimal requirement for RKIP binding and thus they can be used as lead compounds for future design of RKIP ligands with therapeutic potential. PMID:20463977

Structure Elucidation of Unknown Metabolites in Metabolomics by Combined NMR and MS/MS Prediction

DOE PAGES

Boiteau, Rene M.; Hoyt, David W.; Nicora, Carrie D.; ...

2018-01-17

Here, we introduce a cheminformatics approach that combines highly selective and orthogonal structure elucidation parameters; accurate mass, MS/MS (MS 2), and NMR in a single analysis platform to accurately identify unknown metabolites in untargeted studies. The approach starts with an unknown LC-MS feature, and then combines the experimental MS/MS and NMR information of the unknown to effectively filter the false positive candidate structures based on their predicted MS/MS and NMR spectra. We demonstrate the approach on a model mixture and then we identify an uncatalogued secondary metabolite in Arabidopsis thaliana. The NMR/MS 2 approach is well suited for discovery ofmore » new metabolites in plant extracts, microbes, soils, dissolved organic matter, food extracts, biofuels, and biomedical samples, facilitating the identification of metabolites that are not present in experimental NMR and MS metabolomics databases.« less

Structure Elucidation of Unknown Metabolites in Metabolomics by Combined NMR and MS/MS Prediction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Boiteau, Rene M.; Hoyt, David W.; Nicora, Carrie D.

Here, we introduce a cheminformatics approach that combines highly selective and orthogonal structure elucidation parameters; accurate mass, MS/MS (MS 2), and NMR in a single analysis platform to accurately identify unknown metabolites in untargeted studies. The approach starts with an unknown LC-MS feature, and then combines the experimental MS/MS and NMR information of the unknown to effectively filter the false positive candidate structures based on their predicted MS/MS and NMR spectra. We demonstrate the approach on a model mixture and then we identify an uncatalogued secondary metabolite in Arabidopsis thaliana. The NMR/MS 2 approach is well suited for discovery ofmore » new metabolites in plant extracts, microbes, soils, dissolved organic matter, food extracts, biofuels, and biomedical samples, facilitating the identification of metabolites that are not present in experimental NMR and MS metabolomics databases.« less

Structure Elucidation of Unknown Metabolites in Metabolomics by Combined NMR and MS/MS Prediction

PubMed Central

Hoyt, David W.; Nicora, Carrie D.; Kinmonth-Schultz, Hannah A.; Ward, Joy K.

2018-01-01

We introduce a cheminformatics approach that combines highly selective and orthogonal structure elucidation parameters; accurate mass, MS/MS (MS2), and NMR into a single analysis platform to accurately identify unknown metabolites in untargeted studies. The approach starts with an unknown LC-MS feature, and then combines the experimental MS/MS and NMR information of the unknown to effectively filter out the false positive candidate structures based on their predicted MS/MS and NMR spectra. We demonstrate the approach on a model mixture, and then we identify an uncatalogued secondary metabolite in Arabidopsis thaliana. The NMR/MS2 approach is well suited to the discovery of new metabolites in plant extracts, microbes, soils, dissolved organic matter, food extracts, biofuels, and biomedical samples, facilitating the identification of metabolites that are not present in experimental NMR and MS metabolomics databases. PMID:29342073

NMR-Spectroscopy for Nontargeted Screening and Simultaneous Quantification of Health-Relevant Compounds in Foods: The Example of Melamine

PubMed Central

2009-01-01

The recent melamine crisis in China has pointed out a serious deficiency in current food control systems, namely, they specifically focus on selected known compounds. This targeted approach allowed the presence of melamine in milk products to be overlooked for a considerable time. To avoid such crises in the future, we propose that nontargeted screening methods need to be developed and applied. To this end, NMR has an extraordinary potential that just started to be recognized and exploited. Our research shows that, from the very same set of spectra, 1H NMR at 400 MHz can distinguish between melamine-contaminated and melamine-free infant formulas and can provide quantitative information by integration of individual lines after identification. For contaminated Chinese infant formulas or candy, identical results were obtained when comparing NMR with SPE-LC/MS/MS. NMR was found to be suitable for routine nontargeted and targeted analyses of foods, and its use will significantly increase food safety. PMID:20349917

Synthesis, characterization, antioxidant and brine shrimp cytotoxic activity of novel 3-benzothioyl-1-(3-hydroxy-3-phenyl -3-propyl)-1-methylthiourea.

PubMed

Shoaib, Mohammad; Ullah, Abid; Shah, Syed Wadood Ali; Tahir, Muhammad Nawaz

2017-07-01

In the present research work novel ephedrine based thiourea derivative, 3-benzothioyl-1-(3-hydroxy-3-phenyl -3-propyl)-1-methylthiourea 4is synthesized and then characterized elemental analyzed via various techniques i.e., Proton NMR, carbon13 NMR and fatherly confirmed via X-ray crystallography. Compound 4 was then screened for their possible antioxidant and cytotoxic potentials. Benzoyl chloride was treated with an equimolar potassium thiocyanate in acetone to achieve benzoyl isothiocyantes. It was then treated with an equimolar (1R, 2S)-(-)-Ephedrine to obtain the 3-benzothioyl-1-(3-hydroxy-3-phenyl-3-propyl)-1-methyl thiourea4. It was then screened for antioxidant and cytotoxic potentials. The compound 4 showed excellent antioxidant activity almost comparable to ascorbic acid (standard) and have significant cytotoxic activity with LC 50 value 05±0.58 ppm.

Accessibility of selenomethionine proteins by total chemical synthesis: structural studies of human herpesvirus-8 MIP-II.

PubMed

Shao, W; Fernandez, E; Wilken, J; Thompson, D A; Siani, M A; West, J; Lolis, E; Schweitzer, B I

1998-12-11

The determination of high resolution three-dimensional structures by X-ray crystallography or nuclear magnetic resonance (NMR) is a time-consuming process. Here we describe an approach to circumvent the cloning and expression of a recombinant protein as well as screening for heavy atom derivatives. The selenomethionine-modified chemokine macrophage inflammatory protein-II (MIP-II) from human herpesvirus-8 has been produced by total chemical synthesis, crystallized, and characterized by NMR. The protein has a secondary structure typical of other chemokines and forms a monomer in solution. These results indicate that total chemical synthesis can be used to accelerate the determination of three-dimensional structures of new proteins identified in genome programs.

Integrating computational and chemical biology tools in the discovery of antiangiogenic small molecule ligands of FGF2 derived from endogenous inhibitors

PubMed Central

Foglieni, Chiara; Pagano, Katiuscia; Lessi, Marco; Bugatti, Antonella; Moroni, Elisabetta; Pinessi, Denise; Resovi, Andrea; Ribatti, Domenico; Bertini, Sabrina; Ragona, Laura; Bellina, Fabio; Rusnati, Marco; Colombo, Giorgio; Taraboletti, Giulia

2016-01-01

The FGFs/FGFRs system is a recognized actionable target for therapeutic approaches aimed at inhibiting tumor growth, angiogenesis, metastasis, and resistance to therapy. We previously identified a non-peptidic compound (SM27) that retains the structural and functional properties of the FGF2-binding sequence of thrombospondin-1 (TSP-1), a major endogenous inhibitor of angiogenesis. Here we identified new small molecule inhibitors of FGF2 based on the initial lead. A similarity-based screening of small molecule libraries, followed by docking calculations and experimental studies, allowed selecting 7 bi-naphthalenic compounds that bound FGF2 inhibiting its binding to both heparan sulfate proteoglycans and FGFR-1. The compounds inhibit FGF2 activity in in vitro and ex vivo models of angiogenesis, with improved potency over SM27. Comparative analysis of the selected hits, complemented by NMR and biochemical analysis of 4 newly synthesized functionalized phenylamino-substituted naphthalenes, allowed identifying the minimal stereochemical requirements to improve the design of naphthalene sulfonates as FGF2 inhibitors. PMID:27000667

A LEAN approach toward automated analysis and data processing of polymers using proton NMR spectroscopy.

PubMed

de Brouwer, Hans; Stegeman, Gerrit

2011-02-01

To maximize utilization of expensive laboratory instruments and to make most effective use of skilled human resources, the entire chain of data processing, calculation, and reporting that is needed to transform raw NMR data into meaningful results was automated. The LEAN process improvement tools were used to identify non-value-added steps in the existing process. These steps were eliminated using an in-house developed software package, which allowed us to meet the key requirement of improving quality and reliability compared with the existing process while freeing up valuable human resources and increasing productivity. Reliability and quality were improved by the consistent data treatment as performed by the software and the uniform administration of results. Automating a single NMR spectrophotometer led to a reduction in operator time of 35%, doubling of the annual sample throughput from 1400 to 2800, and reducing the turn around time from 6 days to less than 2. Copyright © 2011 Society for Laboratory Automation and Screening. Published by Elsevier Inc. All rights reserved.

Fast and global authenticity screening of honey using ¹H-NMR profiling.

PubMed

Spiteri, Marc; Jamin, Eric; Thomas, Freddy; Rebours, Agathe; Lees, Michèle; Rogers, Karyne M; Rutledge, Douglas N

2015-12-15

An innovative analytical approach was developed to tackle the most common adulterations and quality deviations in honey. Using proton-NMR profiling coupled to suitable quantification procedures and statistical models, analytical criteria were defined to check the authenticity of both mono- and multi-floral honey. The reference data set used was a worldwide collection of more than 800 honeys, covering most of the economically significant botanical and geographical origins. Typical plant nectar markers can be used to check monofloral honey labeling. Spectral patterns and natural variability were established for multifloral honeys, and marker signals for sugar syrups were identified by statistical comparison with a commercial dataset of ca. 200 honeys. Although the results are qualitative, spiking experiments have confirmed the ability of the method to detect sugar addition down to 10% levels in favorable cases. Within the same NMR experiments, quantification of glucose, fructose, sucrose and 5-HMF (regulated parameters) was performed. Finally markers showing the onset of fermentation are described. Copyright © 2014 Elsevier Ltd. All rights reserved.

TLC screening for antioxidant activity of extracts from fifteen bamboo species and identification of antioxidant flavone glycosides from leaves of Bambusa. textilis McClure.

PubMed

Wang, Jin; Yue, Yong-De; Tang, Feng; Sun, Jia

2012-10-19

Interest in the antioxidant activity of bamboo leaves is growing. To discover new sources of natural antioxidants, a TLC bioautography method combined with a new image processing method was developed to evaluate the antioxidant activity of leaf extracts from 15 different species of bamboo. The results showed that the methanolic extract of Bambusa. textilis McClure possessed the highest antioxidant activity among the selected bamboo species. To rapidly identify the antioxidant compounds, the crude extract of B. textilis McClure was analysed by HPLC-UV, and HPLC-micro-fractionation of the extract was carried out. Based on TLC bioautography-guided fractionation, three antioxidant fractions were isolated from B. textilis McClure by preparative chromatography. These three antioxidant compounds were identified as isoorientin 4''-O β-D-xylopyranoside, isoorientin 2''-O-α-L-rhamnoside and isoorientin according to their UV, MS, and NMR data. The proposed TLC screening method could therefore be an easy way to evaluate the antioxidant activity of bamboo leaves, and the results achieved should prove very helpful for promoting their utilization, as B. textilis McClure can be considered a promising plant source of natural antioxidants.

Synthesis, antioxidant and analgesic activities of Schiff bases of 4-amino-1,2,4-triazole derivatives containing a pyrazole moiety.

PubMed

Karrouchi, K; Chemlal, L; Taoufik, J; Cherrah, Y; Radi, S; El Abbes Faouzi, M; Ansar, M

2016-11-01

A series of Schiff bases of 4-amino-1,2,4-triazole derivatives containing pyrazole (5a-h) were synthesized from condensation of 4-amino-5-(5-methyl-1H-pyrazol-3-yl)-4H-1,2,4-triazole-3-thiol (3) derivative with various aromatic aldehydes (4a-h). The structures of the synthesized compounds were elucidated by IR, 1 H NMR, 13 C NMR, and mass spectrometry. All the synthesized compounds (5a-h) were screened for their in vivo analgesic and in vitro antioxidant activities revealing significant analgesic and antioxidant properties. Copyright © 2016 Académie Nationale de Pharmacie. Published by Elsevier Masson SAS. All rights reserved.

Synthesis, biological activity and molecular modeling study of new Schiff bases incorporated with indole moiety.

PubMed

Halawa, Ahmed H; El-Gilil, Shimaa Mohamed Abd; Bedair, Ahmed H; Shaaban, Mohamed; Frese, Marcel; Sewald, Norbert; Eliwa, Essam M; El-Agrody, Ahmed M

2017-10-26

A new series of heterocyclic Schiff bases 2-9 containing indole moiety were synthesized by facile and efficient condensation of indole-3/2/5-carboxaldehyde (1a/1b/1c) with different aromatic and heterocyclic primary amines using conventional and/or microwave irradiation methods. The structures of the obtained compounds were assigned by sophisticated spectroscopic and spectrometric techniques (1D-NMR, 2D-NMR and MS). The synthesized compounds were screened for their cytotoxicity and antibacterial activities. In vitro cytotoxicity screening revealed that compound 5 exhibited moderate activity against KB-3-1 cell line (IC50=57.7 μM) while 5-indolylimino derivative 7 indicated close to the activity (IC50=19.6 μM) in comparison with the positive control (+)-Griseofulvin (IC50=19.2 μM), while the tested compounds 5, 6b, 7 and 9 revealed good or moderate antibacterial activity. In addition, molecular docking study of Schiff bases 2-9 was performed by Molecular Operating Environment (MOE 2014.09) program on the matrix metalloproteinase-8 (MMP-8) (Protein Data Bank (PDB) ID: 1MNC) in an attempt to explore their mode of action as anticancer drugs.

Structure-Based Virtual Ligand Screening on the XRCC4/DNA Ligase IV Interface

NASA Astrophysics Data System (ADS)

Menchon, Grégory; Bombarde, Oriane; Trivedi, Mansi; Négrel, Aurélie; Inard, Cyril; Giudetti, Brigitte; Baltas, Michel; Milon, Alain; Modesti, Mauro; Czaplicki, Georges; Calsou, Patrick

2016-03-01

The association of DNA Ligase IV (Lig4) with XRCC4 is essential for repair of DNA double-strand breaks (DSBs) by Non-homologous end-joining (NHEJ) in humans. DSBs cytotoxicity is largely exploited in anticancer therapy. Thus, NHEJ is an attractive target for strategies aimed at increasing the sensitivity of tumors to clastogenic anticancer treatments. However the high affinity of the XRCC4/Lig4 interaction and the extended protein-protein interface make drug screening on this target particularly challenging. Here, we conducted a pioneering study aimed at interfering with XRCC4/Lig4 assembly. By Molecular Dynamics simulation using the crystal structure of the complex, we first delineated the Lig4 clamp domain as a limited suitable target. Then, we performed in silico screening of ~95,000 filtered molecules on this Lig4 subdomain. Hits were evaluated by Differential Scanning Fluorimetry, Saturation Transfer Difference - NMR spectroscopy and interaction assays with purified recombinant proteins. In this way we identified the first molecule able to prevent Lig4 binding to XRCC4 in vitro. This compound has a unique tripartite interaction with the Lig4 clamp domain that suggests a starting chemotype for rational design of analogous molecules with improved affinity.

Development of novel HER2 inhibitors against gastric cancer derived from flavonoid source of Syzygium alternifolium through molecular dynamics and pharmacophore-based screening

PubMed Central

Babu, Tirumalasetty Muni Chandra; Rammohan, Aluru; Baki, Vijaya Bhaskar; Devi, Savita; Gunasekar, Duvvuru; Rajendra, Wudayagiri

2016-01-01

Continuous usage of synthetic chemotherapeutic drugs causes adverse effects, which prompted for the development of alternative therapeutics for gastric cancer from natural source. This study was carried out with a specific aim to screen gastroprotective compounds from the fruits of Syzygium alternifolium (Myrtaceae). Three flavonoids, namely, 1) 5-hydroxy-7,4′-dimethoxy-6,8-di-C-methylflavone, 2) kaempferol-3-O-β-d-glucopyranoside, and 3) kaempferol-3-O-α-l-rhamnopyranoside were isolated from the above medicinal plant by employing silica gel column chromatography and are characterized by NMR techniques. Antigastric cancer activity of these flavonoids was examined on AGS cell lines followed by cell cycle progression assay. In addition, pharmacophore-based screening and molecular dynamics of protein–ligand complex were carried out to identify potent scaffolds. The results showed that compounds 2 and 3 exhibited significant cytotoxic effect, whereas compound 1 showed moderate effect on AGS cells by inhibiting G2/M phase of cell cycle. Molecular docking analysis revealed that compound 2 has higher binding energies on human growth factor receptor-2 (HER2). The constructed pharmacophore models reveal that the compounds have more number of H-bond Acc/Don features which contribute to the inhibition of HER2 activity. By selecting these features, 34 hits were retrieved using the query compound 2. Molecular dynamic simulations (MDS) of protein–ligand complexes demonstrated conspicuous inhibition of HER2 as evidenced by dynamic trajectory analysis. Based on these results, the compound ZINC67903192 was identified as promising HER2 inhibitor against gastric cancer. The present work provides a basis for the discovery a new class of scaffolds from natural products for gastric carcinoma. PMID:27853354

Development of novel HER2 inhibitors against gastric cancer derived from flavonoid source of Syzygium alternifolium through molecular dynamics and pharmacophore-based screening.

PubMed

Babu, Tirumalasetty Muni Chandra; Rammohan, Aluru; Baki, Vijaya Bhaskar; Devi, Savita; Gunasekar, Duvvuru; Rajendra, Wudayagiri

2016-01-01

Continuous usage of synthetic chemotherapeutic drugs causes adverse effects, which prompted for the development of alternative therapeutics for gastric cancer from natural source. This study was carried out with a specific aim to screen gastroprotective compounds from the fruits of Syzygium alternifolium (Myrtaceae). Three flavonoids, namely, 1) 5-hydroxy-7,4'-dimethoxy-6,8-di-C-methylflavone, 2) kaempferol-3-O- β -d-glucopyranoside, and 3) kaempferol-3-O- α -l-rhamnopyranoside were isolated from the above medicinal plant by employing silica gel column chromatography and are characterized by NMR techniques. Antigastric cancer activity of these flavonoids was examined on AGS cell lines followed by cell cycle progression assay. In addition, pharmacophore-based screening and molecular dynamics of protein-ligand complex were carried out to identify potent scaffolds. The results showed that compounds 2 and 3 exhibited significant cytotoxic effect, whereas compound 1 showed moderate effect on AGS cells by inhibiting G2/M phase of cell cycle. Molecular docking analysis revealed that compound 2 has higher binding energies on human growth factor receptor-2 (HER2). The constructed pharmacophore models reveal that the compounds have more number of H-bond Acc/Don features which contribute to the inhibition of HER2 activity. By selecting these features, 34 hits were retrieved using the query compound 2. Molecular dynamic simulations (MDS) of protein-ligand complexes demonstrated conspicuous inhibition of HER2 as evidenced by dynamic trajectory analysis. Based on these results, the compound ZINC67903192 was identified as promising HER2 inhibitor against gastric cancer. The present work provides a basis for the discovery a new class of scaffolds from natural products for gastric carcinoma.

Microwave assisted synthesis and anti-lipase activity of some new fluorine-containing benzimidazoles.

PubMed

Menteşe, E; Yilmaz, F; Ülker, S; Kahveci, B

2015-01-01

In this study, a new series of fluorine containing benzimidazoles (4a-l) and bisbenzimidazoles (6a-c, 8) were synthesized by the reaction of o-phenylenediamines with iminoester hydrochlorides (3a-l, 7) in methanol under microwave irradiation. The structures of these newly synthesized compounds were identified by IR, (1)H-NMR, (13)C-NMR, mass spectroscopy and elemental analysis data. The synthesized compounds were screened for their pancreatic lipase activities. Our results indicate that the compounds 6a, 6b and 6c can serve as an anti-lipase agent. The compounds 6b and 6c inhibited pancreatic lipase activity by 84.03% and 97.49% at a concentration of 3 µg/mL, respectively. © Georg Thieme Verlag KG Stuttgart · New York.

NMR spectroscopy of filtered serum of prostate cancer: A new frontier in metabolomics.

PubMed

Kumar, Deepak; Gupta, Ashish; Mandhani, Anil; Sankhwar, Satya Narain

2016-09-01

To address the shortcomings of digital rectal examinations (DRE), serum prostate-specific antigen (PSA), and trans-rectal ultrasound (TRUS) for precise determination of prostate cancer (PC) and differentiation from benign prostatic hyperplasia (BPH), we applied (1) H-nuclear magnetic resonance (NMR) spectroscopy as a surrogate tactic for probing and prediction of PC and BPH. The study comprises 210 filtered sera from suspected PC, BPH, and a healthy subjects' cohort (HC). The filtered serum approach delineates to identify and quantify 52 metabolites using (1) H NMR spectroscopy. All subjects had undergone clinical evaluations (DRE, PSA, and TRUS) followed by biopsy for Gleason score, if needed. NMR-measured metabolites and clinical evaluation data were examined separately using linear multivariate discriminant function analysis (DFA) to probe the signature descriptors for each cohort. DFA indicated that glycine, sarcosine, alanine, creatine, xanthine, and hypoxanthine were able to determine abnormal prostate (BPH + PC). DFA-based classification presented high precision (86.2% by NMR and 68.1% by clinical laboratory method) in discriminating HC from BPH + PC. DFA reveals that alanine, sarcosine, creatinine, glycine, and citrate were able to discriminate PC from BPH. DFA-based categorization exhibited high accuracy (88.3% by NMR and 75.2% by clinical laboratory method) to differentiate PC from BPH. (1) H NMR-based metabolic profiling of filtered-serum sample appears to be assuring, swift, and least-invasive for probing and prediction of PC and BPH with its signature metabolic profile. This novel technique is not only on a par with histopathological evaluation of PC determination but is also comparable to liquid chromatography-based mass spectrometry to identify the metabolites. Prostate 76:1106-1119, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Solution state nuclear magnetic resonance spectroscopy for biological metabolism and pathway intermediate analysis.

PubMed

Nealon, Gareth L; Howard, Mark J

2016-12-15

Using nuclear magnetic resonance (NMR) spectroscopy in the study of metabolism has been immensely popular in medical- and health-related research but has yet to be widely applied to more fundamental biological problems. This review provides some NMR background relevant to metabolism, describes why 1 H NMR spectra are complex as well as introducing relevant terminology and definitions. The applications and practical considerations of NMR metabolic profiling and 13 C NMR-based flux analyses are discussed together with the elegant 'enzyme trap' approach for identifying novel metabolic pathway intermediates. The importance of sample preparation and data analysis are also described and explained with reference to data precision and multivariate analysis to introduce researchers unfamiliar with NMR and metabolism to consider this technique for their research interests. Finally, a brief glance into the future suggests NMR-based metabolism has room to expand in the 21st century through new isotope labels, and NMR technologies and methodologies. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

Classification using NMR-based metabolomics of Sophora flavescens grown in Japan and China.

PubMed

Suzuki, Ryuichiro; Ikeda, Yuriko; Yamamoto, Akari; Saima, Toyoe; Fujita, Tatsuya; Fukuda, Tatsuo; Fukuda, Eriko; Baba, Masaki; Okada, Yoshihito; Shirataki, Yoshiaki

2012-11-01

We demonstrate that NMR-based metabolomics can be used to identify the country of growth (Japan or China) of Sophora flavescens plants. Principle Component Analysis (PCA) conducted on extracts of S. flavescens grown in China provided data distinct from that of extracts of plants grown in Japan. Loading plot analysis showed signals characteristic of Japanese S. flavescens. NMR analyses showed these signals to be due to kurarinol (1) and kushenol H (2). These compounds were confirmed by HPLC analysis to be distinctive markers for Japanese S. flavescens.

High-Resolution Magic Angle Spinning Nuclear Magnetic Resonance of Intact Zebrafish Embryos Detects Metabolic Changes Following Exposure to Teratogenic Polymethoxyalkenes from Algae

PubMed Central

Roy, Upasana; Jaja-Chimedza, Asha; Sanchez, Kristel; Matysik, Joerg

2016-01-01

Abstract Techniques based on nuclear magnetic resonance (NMR) for imaging and chemical analyses of in vivo, or otherwise intact, biological systems are rapidly emerging and finding diverse applications within a wide range of fields. Very recently, several NMR-based techniques have been developed for the zebrafish as a model animal system. In the current study, the novel application of high-resolution magic angle spinning (HR-MAS) NMR is presented as a means of metabolic profiling of intact zebrafish embryos. Toward investigating the utility of HR-MAS NMR as a toxicological tool, these studies specifically examined metabolic changes of embryos exposed to polymethoxy-1-alkenes (PMAs)—a recently identified family of teratogenic compounds from freshwater algae—as emerging environmental contaminants. One-dimensional and two-dimensional HR-MAS NMR analyses were able to effectively identify and quantify diverse metabolites in early-stage (≤36 h postfertilization) embryos. Subsequent comparison of the metabolic profiles between PMA-exposed and control embryos identified several statistically significant metabolic changes associated with subacute exposure to the teratogen, including (1) elevated inositol as a recognized component of signaling pathways involved in embryo development; (2) increases in several metabolites, including inositol, phosphoryl choline, fatty acids, and cholesterol, which are associated with lipid composition of cell membranes; (3) concomitant increase in glucose and decrease in lactate; and (4) decreases in several biochemically related metabolites associated with central nervous system development and function, including γ-aminobutyric acid, glycine, glutamate, and glutamine. A potentially unifying model/hypothesis of PMA teratogenicity based on the data is presented. These findings, taken together, demonstrate that HR-MAS NMR is a promising tool for metabolic profiling in the zebrafish embryo, including toxicological applications. PMID:27348393

Fragment growing and linking lead to novel nanomolar lactate dehydrogenase inhibitors.

PubMed

Kohlmann, Anna; Zech, Stephan G; Li, Feng; Zhou, Tianjun; Squillace, Rachel M; Commodore, Lois; Greenfield, Matthew T; Lu, Xiaohui; Miller, David P; Huang, Wei-Sheng; Qi, Jiwei; Thomas, R Mathew; Wang, Yihan; Zhang, Sen; Dodd, Rory; Liu, Shuangying; Xu, Rongsong; Xu, Yongjin; Miret, Juan J; Rivera, Victor; Clackson, Tim; Shakespeare, William C; Zhu, Xiaotian; Dalgarno, David C

2013-02-14

Lactate dehydrogenase A (LDH-A) catalyzes the interconversion of lactate and pyruvate in the glycolysis pathway. Cancer cells rely heavily on glycolysis instead of oxidative phosphorylation to generate ATP, a phenomenon known as the Warburg effect. The inhibition of LDH-A by small molecules is therefore of interest for potential cancer treatments. We describe the identification and optimization of LDH-A inhibitors by fragment-based drug discovery. We applied ligand based NMR screening to identify low affinity fragments binding to LDH-A. The dissociation constants (K(d)) and enzyme inhibition (IC(50)) of fragment hits were measured by surface plasmon resonance (SPR) and enzyme assays, respectively. The binding modes of selected fragments were investigated by X-ray crystallography. Fragment growing and linking, followed by chemical optimization, resulted in nanomolar LDH-A inhibitors that demonstrated stoichiometric binding to LDH-A. Selected molecules inhibited lactate production in cells, suggesting target-specific inhibition in cancer cell lines.

Benzophenone based fluorophore for selective detection of Sn2+ ion: Experimental and theoretical study.

PubMed

Jadhav, Amol G; Shinde, Suvidha S; Lanke, Sandip K; Sekar, Nagaiyan

2017-03-05

Synthesis of novel benzophenone-based chemosensor is presented for the selective sensing of Sn 2+ ion. Screening of competitive metal ions was performed by competitive experiments. The specific cation recognition ability of chemosensor towards Sn 2+ was investigated by experimental (UV-visible, fluorescence spectroscopy, 1 H NMR, 13 C NMR, FTIR and HRMS) methods and further supported by Density Functional Theory study. The stoichiometric binding ratio and binding constant (K a ) for complex is found to be 1:1 and 1.50×10 4 , respectively. The detection limit of Sn 2+ towards chemosensor was found to be 0.3898ppb. Specific selectivity and superiority of chemosensor over another recently reported chemosensor is presented. Copyright © 2016 Elsevier B.V. All rights reserved.

Experimental and analytical variation in human urine in 1H NMR spectroscopy-based metabolic phenotyping studies.

PubMed

Maher, Anthony D; Zirah, Séverine F M; Holmes, Elaine; Nicholson, Jeremy K

2007-07-15

1H NMR spectroscopy potentially provides a robust approach for high-throughput metabolic screening of biofluids such as urine and plasma, but sample handling and preparation need careful optimization to ensure that spectra accurately report biological status or disease state. We have investigated the effects of storage temperature and time on the 1H NMR spectral profiles of human urine from two participants, collected three times a day on four different days. These were analyzed using modern chemometric methods. Analytical and preparation variation (tested between -40 degrees C and room temperature) and time of storage (to 24 h) were found to be much less influential than biological variation in sample classification. Statistical total correlation spectroscopy and discriminant function methods were used to identify the specific metabolites that were hypervariable due to preparation and biology. Significant intraindividual variation in metabolite profiles were observed even for urine collected on the same day and after at least 6 h fasting. The effect of long-term storage at different temperatures was also investigated, showing urine is stable if frozen for at least 3 months and that storage at room temperature for long periods (1-3 months) results in a metabolic profile explained by bacterial activity. Presampling (e.g., previous day) intake of food and medicine can also strongly influence the urinary metabolic profiles indicating that collective detailed participant historical meta data are important for interpretation of metabolic phenotypes and for avoiding false biomarker discovery.

Ebselen, a Small-Molecule Capsid Inhibitor of HIV-1 Replication.

PubMed

Thenin-Houssier, Suzie; de Vera, Ian Mitchelle S; Pedro-Rosa, Laura; Brady, Angela; Richard, Audrey; Konnick, Briana; Opp, Silvana; Buffone, Cindy; Fuhrmann, Jakob; Kota, Smitha; Billack, Blase; Pietka-Ottlik, Magdalena; Tellinghuisen, Timothy; Choe, Hyeryun; Spicer, Timothy; Scampavia, Louis; Diaz-Griffero, Felipe; Kojetin, Douglas J; Valente, Susana T

2016-04-01

The human immunodeficiency virus type 1 (HIV-1) capsid plays crucial roles in HIV-1 replication and thus represents an excellent drug target. We developed a high-throughput screening method based on a time-resolved fluorescence resonance energy transfer (HTS-TR-FRET) assay, using the C-terminal domain (CTD) of HIV-1 capsid to identify inhibitors of capsid dimerization. This assay was used to screen a library of pharmacologically active compounds, composed of 1,280in vivo-active drugs, and identified ebselen [2-phenyl-1,2-benzisoselenazol-3(2H)-one], an organoselenium compound, as an inhibitor of HIV-1 capsid CTD dimerization. Nuclear magnetic resonance (NMR) spectroscopic analysis confirmed the direct interaction of ebselen with the HIV-1 capsid CTD and dimer dissociation when ebselen is in 2-fold molar excess. Electrospray ionization mass spectrometry revealed that ebselen covalently binds the HIV-1 capsid CTD, likely via a selenylsulfide linkage with Cys198 and Cys218. This compound presents anti-HIV activity in single and multiple rounds of infection in permissive cell lines as well as in primary peripheral blood mononuclear cells. Ebselen inhibits early viral postentry events of the HIV-1 life cycle by impairing the incoming capsid uncoating process. This compound also blocks infection of other retroviruses, such as Moloney murine leukemia virus and simian immunodeficiency virus, but displays no inhibitory activity against hepatitis C and influenza viruses. This study reports the use of TR-FRET screening to successfully identify a novel capsid inhibitor, ebselen, validating HIV-1 capsid as a promising target for drug development. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Genome-Wide Association of the Laboratory-Based Nicotine Metabolite Ratio in Three Ancestries.

PubMed

Baurley, James W; Edlund, Christopher K; Pardamean, Carissa I; Conti, David V; Krasnow, Ruth; Javitz, Harold S; Hops, Hyman; Swan, Gary E; Benowitz, Neal L; Bergen, Andrew W

2016-09-01

Metabolic enzyme variation and other patient and environmental characteristics influence smoking behaviors, treatment success, and risk of related disease. Population-specific variation in metabolic genes contributes to challenges in developing and optimizing pharmacogenetic interventions. We applied a custom genome-wide genotyping array for addiction research (Smokescreen), to three laboratory-based studies of nicotine metabolism with oral or venous administration of labeled nicotine and cotinine, to model nicotine metabolism in multiple populations. The trans-3'-hydroxycotinine/cotinine ratio, the nicotine metabolite ratio (NMR), was the nicotine metabolism measure analyzed. Three hundred twelve individuals of self-identified European, African, and Asian American ancestry were genotyped and included in ancestry-specific genome-wide association scans (GWAS) and a meta-GWAS analysis of the NMR. We modeled natural-log transformed NMR with covariates: principal components of genetic ancestry, age, sex, body mass index, and smoking status. African and Asian American NMRs were statistically significantly (P values ≤ 5E-5) lower than European American NMRs. Meta-GWAS analysis identified 36 genome-wide significant variants over a 43 kilobase pair region at CYP2A6 with minimum P = 2.46E-18 at rs12459249, proximal to CYP2A6. Additional minima were located in intron 4 (rs56113850, P = 6.61E-18) and in the CYP2A6-CYP2A7 intergenic region (rs34226463, P = 1.45E-12). Most (34/36) genome-wide significant variants suggested reduced CYP2A6 activity; functional mechanisms were identified and tested in knowledge-bases. Conditional analysis resulted in intergenic variants of possible interest (P values < 5E-5). This meta-GWAS of the NMR identifies CYP2A6 variants, replicates the top-ranked single nucleotide polymorphism from a recent Finnish meta-GWAS of the NMR, identifies functional mechanisms, and provides pan-continental population biomarkers for nicotine metabolism. This multiple ancestry meta-GWAS of the laboratory study-based NMR provides novel evidence and replication for genome-wide association of CYP2A6 single nucleotide and insertion-deletion polymorphisms. We identify three regions of genome-wide significance: proximal, intronic, and distal to CYP2A6. We replicate the top-ranking single nucleotide polymorphism from a recent GWAS of the NMR in Finnish smokers, identify a functional mechanism for this intronic variant from in silico analyses of RNA-seq data that is consistent with CYP2A6 expression measured in postmortem lung and liver, and provide additional support for the intergenic region between CYP2A6 and CYP2A7. © The Author 2016. Published by Oxford University Press on behalf of the Society for Research on Nicotine and Tobacco.

Genome-Wide Association of the Laboratory-Based Nicotine Metabolite Ratio in Three Ancestries

PubMed Central

Baurley, James W.; Edlund, Christopher K.; Pardamean, Carissa I.; Conti, David V.; Krasnow, Ruth; Javitz, Harold S.; Hops, Hyman; Swan, Gary E.; Benowitz, Neal L.

2016-01-01

Introduction: Metabolic enzyme variation and other patient and environmental characteristics influence smoking behaviors, treatment success, and risk of related disease. Population-specific variation in metabolic genes contributes to challenges in developing and optimizing pharmacogenetic interventions. We applied a custom genome-wide genotyping array for addiction research (Smokescreen), to three laboratory-based studies of nicotine metabolism with oral or venous administration of labeled nicotine and cotinine, to model nicotine metabolism in multiple populations. The trans-3′-hydroxycotinine/cotinine ratio, the nicotine metabolite ratio (NMR), was the nicotine metabolism measure analyzed. Methods: Three hundred twelve individuals of self-identified European, African, and Asian American ancestry were genotyped and included in ancestry-specific genome-wide association scans (GWAS) and a meta-GWAS analysis of the NMR. We modeled natural-log transformed NMR with covariates: principal components of genetic ancestry, age, sex, body mass index, and smoking status. Results: African and Asian American NMRs were statistically significantly (P values ≤ 5E-5) lower than European American NMRs. Meta-GWAS analysis identified 36 genome-wide significant variants over a 43 kilobase pair region at CYP2A6 with minimum P = 2.46E-18 at rs12459249, proximal to CYP2A6. Additional minima were located in intron 4 (rs56113850, P = 6.61E-18) and in the CYP2A6-CYP2A7 intergenic region (rs34226463, P = 1.45E-12). Most (34/36) genome-wide significant variants suggested reduced CYP2A6 activity; functional mechanisms were identified and tested in knowledge-bases. Conditional analysis resulted in intergenic variants of possible interest (P values < 5E-5). Conclusions: This meta-GWAS of the NMR identifies CYP2A6 variants, replicates the top-ranked single nucleotide polymorphism from a recent Finnish meta-GWAS of the NMR, identifies functional mechanisms, and provides pan-continental population biomarkers for nicotine metabolism. Implications: This multiple ancestry meta-GWAS of the laboratory study-based NMR provides novel evidence and replication for genome-wide association of CYP2A6 single nucleotide and insertion–deletion polymorphisms. We identify three regions of genome-wide significance: proximal, intronic, and distal to CYP2A6. We replicate the top-ranking single nucleotide polymorphism from a recent GWAS of the NMR in Finnish smokers, identify a functional mechanism for this intronic variant from in silico analyses of RNA-seq data that is consistent with CYP2A6 expression measured in postmortem lung and liver, and provide additional support for the intergenic region between CYP2A6 and CYP2A7. PMID:27113016

High-Throughput Screening of Australian Marine Organism Extracts for Bioactive Molecules Affecting the Cellular Storage of Neutral Lipids

PubMed Central

Rae, James; Fontaine, Frank; Salim, Angela A.; Lo, Harriet P.; Capon, Robert J.; Parton, Robert G.; Martin, Sally

2011-01-01

Mammalian cells store excess fatty acids as neutral lipids in specialised organelles called lipid droplets (LDs). Using a simple cell-based assay and open-source software we established a high throughput screen for LD formation in A431 cells in order to identify small bioactive molecules affecting lipid storage. Screening an n-butanol extract library from Australian marine organisms we identified 114 extracts that produced either an increase or a decrease in LD formation in fatty acid-treated A431 cells with varying degrees of cytotoxicity. We selected for further analysis a non-cytotoxic extract derived from the genus Spongia (Heterofibria). Solvent partitioning, HPLC fractionation and spectroscopic analysis (NMR, MS) identified a family of related molecules within this extract with unique structural features, a subset of which reduced LD formation. We selected one of these molecules, heterofibrin A1, for more detailed cellular analysis. Inhibition of LD biogenesis by heterofibrin A1 was observed in both A431 cells and AML12 hepatocytes. The activity of heterofibrin A1 was dose dependent with 20 µM inhibiting LD formation and triglyceride accumulation by ∼50% in the presence of 50 µM oleic acid. Using a fluorescent fatty acid analogue we found that heterofibrin A1 significantly reduces the intracellular accumulation of fatty acids and results in the formation of distinct fatty acid metabolites in both cultured cells and in embryos of the zebrafish Danio rerio. In summary we have shown using readily accessible software and a relatively simple assay system that we can identify and isolate bioactive molecules from marine extracts, which affect the formation of LDs and the metabolism of fatty acids both in vitro and in vivo. PMID:21857959

Detergent/Nanodisc Screening for High-Resolution NMR Studies of an Integral Membrane Protein Containing a Cytoplasmic Domain

PubMed Central

Maslennikov, Innokentiy; Choe, Senyon; Riek, Roland

2013-01-01

Because membrane proteins need to be extracted from their natural environment and reconstituted in artificial milieus for the 3D structure determination by X-ray crystallography or NMR, the search for membrane mimetic that conserve the native structure and functional activities remains challenging. We demonstrate here a detergent/nanodisc screening study by NMR of the bacterial α-helical membrane protein YgaP containing a cytoplasmic rhodanese domain. The analysis of 2D [15N,1H]-TROSY spectra shows that only a careful usage of low amounts of mixed detergents did not perturb the cytoplasmic domain while solubilizing in parallel the transmembrane segments with good spectral quality. In contrast, the incorporation of YgaP into nanodiscs appeared to be straightforward and yielded a surprisingly high quality [15N,1H]-TROSY spectrum opening an avenue for the structural studies of a helical membrane protein in a bilayer system by solution state NMR. PMID:23349867

Rapid Identification of Synthetic Cannabinoids in Herbal Incenses with DART-MS and NMR.

PubMed

Marino, Michael A; Voyer, Brandy; Cody, Robert B; Dane, A John; Veltri, Mercurio; Huang, Ling

2016-01-01

The usage of herbal incenses containing synthetic cannabinoids has caused an increase in medical incidents and triggered legislations to ban these products throughout the world. Law enforcement agencies are experiencing sample backlogs due to the variety of the products and the addition of new and still-legal compounds. In our study, proton nuclear magnetic resonance (NMR) spectroscopy was employed to promptly screen the synthetic cannabinoids after their rapid, direct detection on the herbs and in the powders by direct analysis in real time mass spectrometry (DART-MS). A simple sample preparation protocol was employed on 50 mg of herbal sample matrices for quick NMR detection. Ten synthetic cannabinoids were discovered in fifteen herbal incenses. The combined DART-MS and NMR methods can be used to quickly screen synthetic cannabinoids in powder and herbal samples, serving as a complementary approach to conventional GC-MS or LC-MS methods. © 2015 American Academy of Forensic Sciences.

Screening for Triterpenoid Saponins in Plants Using Hyphenated Analytical Platforms.

PubMed

Khakimov, Bekzod; Tseng, Li Hong; Godejohann, Markus; Bak, Søren; Engelsen, Søren Balling

2016-11-24

Recently the number of studies investigating triterpenoid saponins has drastically increased due to their diverse and potentially attractive biological activities. Currently the literature contains chemical structures of few hundreds of triterpenoid saponins of plant and animal origin. Triterpenoid saponins consist of a triterpene aglycone with one or more sugar moieties attached to it. However, due to similar physico-chemical properties, isolation and identification of a large diversity of triterpenoid saponins remain challenging. This study demonstrates a methodology to screen saponins using hyphenated analytical platforms, GC-MS, LC-MS/MS, and LC-SPE-NMR/MS, in the example of two different phenotypes of the model plant Barbarea vulgaris (winter cress), glabrous (G) and pubescent (P) type that are known to differ by their insect resistance. The proposed methodology allows for detailed comparison of saponin profiles from intact plant extracts as well as saponin aglycone profiles from hydrolysed samples. Continuously measured 1D proton NMR data during LC separation along with mass spectrometry data revealed significant differences, including contents of saponins, types of aglycones and numbers of sugar moieties attached to the aglycone. A total of 49 peaks were tentatively identified as saponins from both plants; they are derived from eight types of aglycones and with 2-5 sugar moieties. Identification of two previously known insect-deterrent saponins, hederagenin cellobioside and oleanolic acid cellobioside, demonstrated the applicability of the methodology for relatively rapid screening of bioactive compounds.

Combined Application of UHPLC-QTOF/MS, HPLC-ELSD and 1 H-NMR Spectroscopy for Quality Assessment of DA-9801, A Standardised Dioscorea Extract.

PubMed

Kang, Kyo Bin; Ryu, Jayoung; Cho, Youngwoong; Choi, Sang-Zin; Son, Miwon; Sung, Sang Hyun

2017-05-01

DA-9801, a standardised 50% aqueous ethanolic extract of a mixture of Dioscorea japonica and D. nipponica, is a botanical drug candidate for the treatment of diabetic neuropathy, which finished its US phase II clinical trials recently. An advanced quality control method is needed for further development of DA-9801, considering its high contents of both primary and secondary metabolites. Development of a quality assessment strategy for DA-9801, based on the combination of UHPLC-QTOF/MS, HPLC-ELSD, and 1 H-NMR spectroscopy. The method was developed and tested with 15 batch products of DA-9801. The steroidal saponins of DA-9801 were tentatively identified by UHPLC-QTOF/MS and were quantified with the validated HPLC-ELSD method. Primary metabolites of DA-9801 were identified and profiled using 1 H-NMR spectrometry. The batch-to-batch equivalence of DA-9801 was tested with the 1 H-NMR spectra using spectral binning, correlation analysis, and principal component analysis. Six major saponins of DA-9801 were tentatively identified by UHPLC-QTOF/MS. Among them, protodioscin and dioscin were quantified by the validated HPLC-ELSD method. Twenty-six metabolites were identified in 1 H-NMR spectra. The similarity between DA-9801 batches could be evaluated with the NMR spectra of DA-9801. The 1 H-NMR method also revealed that two Dioscorea species contributed distinct amino acids to the contents of DA-9801. This study validates the effectiveness of UHPLC-QTOF/MS, HPLC-ELSD, and 1 H NMR-combined method for quality control of DA-9801 and its crude materials. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Ultra-low field nuclear magnetic resonance and magnetic resonance imaging to discriminate and identify materials

DOEpatents

Kraus, Robert H.; Matlashov, Andrei N.; Espy, Michelle A.; Volegov, Petr L.

2010-03-30

An ultra-low magnetic field NMR system can non-invasively examine containers. Database matching techniques can then identify hazardous materials within the containers. Ultra-low field NMR systems are ideal for this purpose because they do not require large powerful magnets and because they can examine materials enclosed in conductive shells such as lead shells. The NMR examination technique can be combined with ultra-low field NMR imaging, where an NMR image is obtained and analyzed to identify target volumes. Spatial sensitivity encoding can also be used to identify target volumes. After the target volumes are identified the NMR measurement technique can be used to identify their contents.

Application of Fragment Based Drug Discovery to Membrane Proteins: Biophysical Identification of Ligands of the Integral Membrane Enzyme DsbB

PubMed Central

Früh, Virginie; Zhou, Yunpeng; Chen, Dan; Loch, Caroline; Eiso, AB; Grinkova, Yelena N.; Verheij, Herman; Sligar, Stephen G; Bushweller, John H.; Siegal, Gregg

2014-01-01

Summary Membrane proteins are important pharmaceutical targets, but they pose significant challenges for fragment based drug discovery approaches. Here we present the first successful use of biophysical methods to screen for fragment ligands to an integral membrane protein. The E. coli inner membrane protein DsbB was solubilized in detergent micelles and lipid bilayer nanodiscs. The solubilized protein was immobilized with retention of functionality and used to screen 1,071 drug fragments for binding using Target Immobilized NMR Screening. Biochemical and biophysical validation of the 8 most potent hits revealed an IC50 range of 7 to 200 μM. The ability to insert a broad array of membrane proteins into nanodiscs, combined with the efficiency of TINS, demonstrates the feasibility of finding fragments targeting membrane proteins. PMID:20797617

A Small Molecule Causes a Population Shift in the Conformational Landscape of an Intrinsically Disordered Protein.

PubMed

Ban, David; Iconaru, Luigi I; Ramanathan, Arvind; Zuo, Jian; Kriwacki, Richard W

2017-10-04

Intrinsically disordered proteins (IDPs) have roles in myriad biological processes and numerous human diseases. However, kinetic and amplitude information regarding their ground-state conformational fluctuations has remained elusive. We demonstrate using nuclear magnetic resonance (NMR)-based relaxation dispersion that the D2 domain of p27 Kip1 , a prototypical IDP, samples multiple discrete, rapidly exchanging conformational states. By combining NMR with mutagenesis and small-angle X-ray scattering (SAXS), we show that these states involve aromatic residue clustering through long-range hydrophobic interactions. Theoretical studies have proposed that small molecules bind promiscuously to IDPs, causing expansion of their conformational landscapes. However, on the basis of previous NMR-based screening results, we show here that compound binding only shifts the populations of states that existed within the ground state of apo p27-D2 without changing the barriers between states. Our results provide atomic resolution insight into how a small molecule binds an IDP and emphasize the need to examine motions on the low microsecond time scale when probing these types of interactions.

Accurate Identification of Unknown and Known Metabolic Mixture Components by Combining 3D NMR with Fourier Transform Ion Cyclotron Resonance Tandem Mass Spectrometry.

PubMed

Wang, Cheng; He, Lidong; Li, Da-Wei; Bruschweiler-Li, Lei; Marshall, Alan G; Brüschweiler, Rafael

2017-10-06

Metabolite identification in metabolomics samples is a key step that critically impacts downstream analysis. We recently introduced the SUMMIT NMR/mass spectrometry (MS) hybrid approach for the identification of the molecular structure of unknown metabolites based on the combination of NMR, MS, and combinatorial cheminformatics. Here, we demonstrate the feasibility of the approach for an untargeted analysis of both a model mixture and E. coli cell lysate based on 2D/3D NMR experiments in combination with Fourier transform ion cyclotron resonance MS and MS/MS data. For 19 of the 25 model metabolites, SUMMIT yielded complete structures that matched those in the mixture independent of database information. Of those, seven top-ranked structures matched those in the mixture, and four of those were further validated by positive ion MS/MS. For five metabolites, not part of the 19 metabolites, correct molecular structural motifs could be identified. For E. coli, SUMMIT MS/NMR identified 20 previously known metabolites with three or more 1 H spins independent of database information. Moreover, for 15 unknown metabolites, molecular structural fragments were determined consistent with their spin systems and chemical shifts. By providing structural information for entire metabolites or molecular fragments, SUMMIT MS/NMR greatly assists the targeted or untargeted analysis of complex mixtures of unknown compounds.

Towards Using NMR to Screen for Spoiled Tomatoes Stored in 1,000 L, Aseptically Sealed, Metal-Lined Totes

PubMed Central

Pinter, Michael D.; Harter, Tod; McCarthy, Michael J.; Augustine, Matthew P.

2014-01-01

Nuclear magnetic resonance (NMR) spectroscopy is used to track factory relevant tomato paste spoilage. It was found that spoilage in tomato paste test samples leads to longer spin lattice relaxation times T1 using a conventional low magnetic field NMR system. The increase in T1 value for contaminated samples over a five day room temperature exposure period prompted the work to be extended to the study of industry standard, 1,000 L, non-ferrous, metal-lined totes. NMR signals and T1 values were recovered from a large format container with a single-sided NMR sensor. The results of this work suggest that a handheld NMR device can be used to study tomato paste spoilage in factory process environments. PMID:24594611

Towards using NMR to screen for spoiled tomatoes stored in 1,000 L, aseptically sealed, metal-lined totes.

PubMed

Pinter, Michael D; Harter, Tod; McCarthy, Michael J; Augustine, Matthew P

2014-03-03

Nuclear magnetic resonance (NMR) spectroscopy is used to track factory relevant tomato paste spoilage. It was found that spoilage in tomato paste test samples leads to longer spin lattice relaxation times T1 using a conventional low magnetic field NMR system. The increase in T1 value for contaminated samples over a five day room temperature exposure period prompted the work to be extended to the study of industry standard, 1,000 L, non-ferrous, metal-lined totes. NMR signals and T1 values were recovered from a large format container with a single-sided NMR sensor. The results of this work suggest that a handheld NMR device can be used to study tomato paste spoilage in factory process environments.

Carbonic anhydrase inhibition of Schiff base derivative of imino-methyl-naphthalen-2-ol: Synthesis, structure elucidation, molecular docking, dynamic simulation and density functional theory calculations

NASA Astrophysics Data System (ADS)

Abbas, Saghir; Nasir, Hafiza Huma; Zaib, Sumera; Ali, Saqib; Mahmood, Tariq; Ayub, Khurshid; Tahir, Muhammad Nawaz; Iqbal, Jamshed

2018-03-01

In the present study, we have designed and synthesized a Schiff base derivative 3 and characterized by FT-IR, 1H and 13C NMR spectroscopy. Single crystal X-ray diffraction and NMR studies were also performed. The synthetic compound was screened for its inhibitory potential against carbonic anhydrase II. The experimental results were validated by molecular docking and dynamic simulations of compound 3 in the active pocket of enzyme. Important binding interactions with the key residues in the active site of the carbonic anhydrase enzyme were revealed. Moreover, supramolecular assembly of the title compound was analyzed by density functional theory (DFT) calculations. These studies rendered a more clear understanding for the demonstration of novel molecular mechanism involved in CA II inhibition by the synthesized compound.

The application of NMR-based milk metabolite analysis in milk authenticity identification.

PubMed

Li, Qiangqiang; Yu, Zunbo; Zhu, Dan; Meng, Xianghe; Pang, Xiumei; Liu, Yue; Frew, Russell; Chen, He; Chen, Gang

2017-07-01

Milk is an important food component in the human diet and is a target for fraud, including many unsafe practices. For example, the unscrupulous adulteration of soymilk into bovine and goat milk or of bovine milk into goat milk in order to gain profit without declaration is a health risk, as the adulterant source and sanitary history are unknown. A robust and fit-for-purpose technique is required to enforce market surveillance and hence protect consumer health. Nuclear magnetic resonance (NMR) is a powerful technique for characterization of food products based on measuring the profile of metabolites. In this study, 1D NMR in conjunction with multivariate chemometrics as well as 2D NMR was applied to differentiate milk types and to identify milk adulteration. Ten metabolites were found which differed among milk types, hence providing characteristic markers for identifying the milk. These metabolites were used to establish mathematical models for milk type differentiation. The limit of quantification (LOQ) of adulteration was 2% (v/v) for soymilk in bovine milk, 2% (v/v) for soymilk in goat milk and 5% (v/v) for bovine milk in goat milk, with relative standard deviation (RSD) less than 10%, which can meet the needs of daily inspection. The NMR method described here is effective for milk authenticity identification, and the study demonstrates that the NMR-based milk metabolite analysis approach provides a means of detecting adulteration at expected levels and can be used for dairy quality monitoring. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

Standardizing the experimental conditions for using urine in NMR-based metabolomic studies with a particular focus on diagnostic studies: a review.

PubMed

Emwas, Abdul-Hamid; Luchinat, Claudio; Turano, Paola; Tenori, Leonardo; Roy, Raja; Salek, Reza M; Ryan, Danielle; Merzaban, Jasmeen S; Kaddurah-Daouk, Rima; Zeri, Ana Carolina; Nagana Gowda, G A; Raftery, Daniel; Wang, Yulan; Brennan, Lorraine; Wishart, David S

The metabolic composition of human biofluids can provide important diagnostic and prognostic information. Among the biofluids most commonly analyzed in metabolomic studies, urine appears to be particularly useful. It is abundant, readily available, easily stored and can be collected by simple, noninvasive techniques. Moreover, given its chemical complexity, urine is particularly rich in potential disease biomarkers. This makes it an ideal biofluid for detecting or monitoring disease processes. Among the metabolomic tools available for urine analysis, NMR spectroscopy has proven to be particularly well-suited, because the technique is highly reproducible and requires minimal sample handling. As it permits the identification and quantification of a wide range of compounds, independent of their chemical properties, NMR spectroscopy has been frequently used to detect or discover disease fingerprints and biomarkers in urine. Although protocols for NMR data acquisition and processing have been standardized, no consensus on protocols for urine sample selection, collection, storage and preparation in NMR-based metabolomic studies have been developed. This lack of consensus may be leading to spurious biomarkers being reported and may account for a general lack of reproducibility between laboratories. Here, we review a large number of published studies on NMR-based urine metabolic profiling with the aim of identifying key variables that may affect the results of metabolomics studies. From this survey, we identify a number of issues that require either standardization or careful accounting in experimental design and provide some recommendations for urine collection, sample preparation and data acquisition.

A Discovery-Based Hydrochlorination of Carvone Utilizing a Guided-Inquiry Approach to Determine the Product Structure from [superscript 13]C NMR Spectra

ERIC Educational Resources Information Center

Pelter, Michael W.; Walker, Natalie M.

2012-01-01

This experiment describes a discovery-based method for the regio- and stereoselective hydrochlorination of carvone, appropriate for a 3-h second-semester organic chemistry laboratory. The product is identified through interpretation of the [superscript 13]C NMR and DEPT spectra are obtained on an Anasazi EFT-60 at 15 MHz as neat samples. A…

Establish an automated flow injection ESI-MS method for the screening of fragment based libraries: Application to Hsp90.

PubMed

Riccardi Sirtori, Federico; Caronni, Dannica; Colombo, Maristella; Dalvit, Claudio; Paolucci, Mauro; Regazzoni, Luca; Visco, Carlo; Fogliatto, Gianpaolo

2015-08-30

ESI-MS is a well established technique for the study of biopolymers (nucleic acids, proteins) and their non covalent adducts, due to its capacity to detect ligand-target complexes in the gas phase and allows inference of ligand-target binding in solution. In this article we used this approach to investigate the interaction of ligands to the Heat Shock Protein 90 (Hsp90). This enzyme is a molecular chaperone involved in the folding and maturation of several proteins which has been subjected in the last years to intensive drug discovery efforts due to its key role in cancer. In particular, reference compounds, with a broad range of dissociation constants from 40pM to 100μM, were tested to assess the reliability of ESI-MS for the study of protein-ligand complexes. A good agreement was found between the values measured with a fluorescence polarization displacement assay and those determined by mass spectrometry. After this validation step we describe the setup of a medium throughput screening method, based on ESI-MS, suitable to explore interactions of therapeutic relevance biopolymers with chemical libraries. Our approach is based on an automated flow injection ESI-MS method (AFI-MS) and has been applied to screen the Nerviano Medical Sciences proprietary fragment library of about 2000 fragments against Hsp90. In order to discard false positive hits and to discriminate those of them interacting with the N-terminal ATP binding site, competition experiments were performed using a reference inhibitor. Gratifyingly, this group of hits matches with the ligands previously identified by NMR FAXS techniques and confirmed by X-ray co-crystallization experiments. These results support the use of AFI-MS for the screening of medium size libraries, including libraries of small molecules with low affinity typically used in fragment based drug discovery. AFI-MS is a valid alternative to other techniques with the additional opportunities to identify compounds interacting with unpredicted or allosteric sites, without the need of any binding probes. Copyright © 2015 Elsevier B.V. All rights reserved.

Surface and interfacial properties of soy-based polysoaps

USDA-ARS?s Scientific Manuscript database

Soybean oil (SO) was polymerized by the reaction of its double bonds in the presence of a catalyst. The resulting polymer (PSO) was positively identified using a combination of FTIR, 1H NMR, 13C NMR, and GPC methods. PSO was hydrolyzed into polysoaps with Na+, K+ or TEA+ (triethanol amine) counter i...

Combining NMR and X-ray crystallography in fragment-based drug discovery: discovery of highly potent and selective BACE-1 inhibitors.

PubMed

Wyss, Daniel F; Wang, Yu-Sen; Eaton, Hugh L; Strickland, Corey; Voigt, Johannes H; Zhu, Zhaoning; Stamford, Andrew W

2012-01-01

Fragment-based drug discovery (FBDD) has become increasingly popular over the last decade. We review here how we have used highly structure-driven fragment-based approaches to complement more traditional lead discovery to tackle high priority targets and those struggling for leads. Combining biomolecular nuclear magnetic resonance (NMR), X-ray crystallography, and molecular modeling with structure-assisted chemistry and innovative biology as an integrated approach for FBDD can solve very difficult problems, as illustrated in this chapter. Here, a successful FBDD campaign is described that has allowed the development of a clinical candidate for BACE-1, a challenging CNS drug target. Crucial to this achievement were the initial identification of a ligand-efficient isothiourea fragment through target-based NMR screening and the determination of its X-ray crystal structure in complex with BACE-1, which revealed an extensive H-bond network with the two active site aspartate residues. This detailed 3D structural information then enabled the design and validation of novel, chemically stable and accessible heterocyclic acylguanidines as aspartic acid protease inhibitor cores. Structure-assisted fragment hit-to-lead optimization yielded iminoheterocyclic BACE-1 inhibitors that possess desirable molecular properties as potential therapeutic agents to test the amyloid hypothesis of Alzheimer's disease in a clinical setting.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Longenecker, Kenton L.; Stamper, Geoffrey F.; Hajduk, Philip J.

In a broad genomics analysis to find novel protein targets for antibiotic discovery, MurF was identified as an essential gene product for Streptococcus pneumonia that catalyzes a critical reaction in the biosynthesis of the peptidoglycan in the formation of the cell wall. Lacking close relatives in mammalian biology, MurF presents attractive characteristics as a potential drug target. Initial screening of the Abbott small-molecule compound collection identified several compounds for further validation as pharmaceutical leads. Here we report the integrated efforts of NMR and X-ray crystallography, which reveal the multidomain structure of a MurF-inhibitor complex in a compact conformation that differsmore » dramatically from related structures. The lead molecule is bound in the substrate-binding region and induces domain closure, suggestive of the domain arrangement for the as yet unobserved transition state conformation for MurF enzymes. The results form a basis for directed optimization of the compound lead by structure-based design to explore the suitability of MurF as a pharmaceutical target.« less

NMR Spectroscopy Identifies Metabolites Translocated from Powdery Mildew Resistant Rootstocks to Susceptible Watermelon Scions.

PubMed

Mahmud, Iqbal; Kousik, Chandrasekar; Hassell, Richard; Chowdhury, Kamal; Boroujerdi, Arezue F

2015-09-16

Powdery mildew (PM) disease causes significant loss in watermelon. Due to the unavailability of a commercial watermelon variety that is resistant to PM, grafting susceptible cultivars on wild resistant rootstocks is being explored as a short-term management strategy to combat this disease. Nuclear magnetic resonance-based metabolic profiles of susceptible and resistant rootstocks of watermelon and their corresponding susceptible scions (Mickey Lee) were compared to screen for potential metabolites related to PM resistance using multivariate principal component analysis. Significant score plot differences between the susceptible and resistant groups were revealed through Mahalanobis distance analysis. Significantly different spectral buckets and their corresponding metabolites (including choline, fumarate, 5-hydroxyindole-3-acetate, and melatonin) have been identified quantitatively using multivariate loading plots and verified by volcano plot analyses. The data suggest that these metabolites were translocated from the powdery mildew resistant rootstocks to their corresponding powdery mildew susceptible scions and can be related to PM disease resistance.

Spectroscopic and Statistical Techniques for Information Recovery in Metabonomics and Metabolomics

NASA Astrophysics Data System (ADS)

Lindon, John C.; Nicholson, Jeremy K.

2008-07-01

Methods for generating and interpreting metabolic profiles based on nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry (MS), and chemometric analysis methods are summarized and the relative strengths and weaknesses of NMR and chromatography-coupled MS approaches are discussed. Given that all data sets measured to date only probe subsets of complex metabolic profiles, we describe recent developments for enhanced information recovery from the resulting complex data sets, including integration of NMR- and MS-based metabonomic results and combination of metabonomic data with data from proteomics, transcriptomics, and genomics. We summarize the breadth of applications, highlight some current activities, discuss the issues relating to metabonomics, and identify future trends.

Spectroscopic and statistical techniques for information recovery in metabonomics and metabolomics.

PubMed

Lindon, John C; Nicholson, Jeremy K

2008-01-01

Methods for generating and interpreting metabolic profiles based on nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry (MS), and chemometric analysis methods are summarized and the relative strengths and weaknesses of NMR and chromatography-coupled MS approaches are discussed. Given that all data sets measured to date only probe subsets of complex metabolic profiles, we describe recent developments for enhanced information recovery from the resulting complex data sets, including integration of NMR- and MS-based metabonomic results and combination of metabonomic data with data from proteomics, transcriptomics, and genomics. We summarize the breadth of applications, highlight some current activities, discuss the issues relating to metabonomics, and identify future trends.

Design, synthesis, structural characterization by IR, (1) H, (13) C, (15) N, 2D-NMR, X-ray diffraction and evaluation of a new class of phenylaminoacetic acid benzylidene hydrazines as pfENR inhibitors.

PubMed

Samal, Ramanuj P; Khedkar, Vijay M; Pissurlenkar, Raghuvir R S; Bwalya, Angela Gono; Tasdemir, Deniz; Joshi, Ramesh A; Rajamohanan, P R; Puranik, Vedavati G; Coutinho, Evans C

2013-06-01

Recent studies have revealed that plasmodial enoyl-ACP reductase (pfENR, FabI), one of the crucial enzymes in the plasmodial type II fatty acid synthesis II (FAS II) pathway, is a promising target for liver stage malaria infections. Hence, pfENR inhibitors have the potential to be used as causal malarial prophylactic agents. In this study, we report the design, synthesis, structural characterization and evaluation of a new class of pfENR inhibitors. The search for inhibitors began with a virtual screen of the iResearch database by molecular docking. Hits obtained from the virtual screen were ranked according to their Glide score. One hit was selected as a lead and modified to improve its binding to pfENR; from this, a series of phenylamino acetic acid benzylidene hydrazides were designed and synthesized. These molecules were thoroughly characterized by IR, (1) H, (13) C, (15) N, 2D-NMR (COSY, NOESY, (1) H-(13) C, (1) H-(15) N HSQC and HMBC), and X-ray diffraction. NMR studies revealed the existence of conformational/configurational isomers around the amide and imine functionalities. The major species in DMSO solution is the E, E form, which is in dynamic equilibrium with the Z, E isomer. In the solid state, the molecule has a completely extended conformation and forms helical structures that are stabilized by strong hydrogen bond interactions, forming a helical structure stabilized by N-H…O interactions, a feature unique to this class of compounds. Furthermore, detailed investigation of the NMR spectra indicated the presence of a minor impurity in most compounds. The structure of this impurity was deduced as an imidazoline-4-one derivative based on (1) H-(13) C and (1) H-(15) H HMBC spectra and was confirmed from the NOESY spectra. The molecules were screened for in vitro activity against recombinant pfENR enzyme by a spectrophotometric assay. Four molecules, viz. 17, 7, 10, and 12 were found to be active at 7, 8, 10, and 12 μm concentration, respectively, showing promising pfENR inhibitory potential. A classification model was derived based on a binary QSAR approach termed recursive partitioning (RP) to highlight structural characteristics that could be tuned to improve activity. © 2013 John Wiley & Sons A/S.

1H and 31P benchtop NMR of liquids and solids used in and/or produced during the manufacture of methamphetamine by the HI reduction of pseudoephedrine/ephedrine.

PubMed

Bogun, Ben; Moore, Sarah

2017-09-01

In this study, the use of benchtop NMR spectroscopy in the analysis of solids and liquids used and/or produced during the HI reduction of pseudoephedrine was evaluated. The study focused on identifying organic precursors and phosphorus containing compounds used in and/or produced during the manufacturing process. Samples taken from clandestine laboratories, where this synthesis process was suspected of occurring, were also analysed and evaluated. Benchtop NMR was able to distinguish between ephedrine, pseudoephedrine and methamphetamine as the free base and hydrochloride salt. This technique was also effective at identifying and distinguishing between phosphorus containing compounds used and/or produced during the manufacture of methamphetamine. Benchtop NMR was also determined to be effective at analysing samples from suspected clandestine laboratories. Copyright © 2017 Elsevier B.V. All rights reserved.

Synthesis, characterization and antibacterial studies of 2-chloro-5-fluoro-N-[dibenzyl carbamothioyl] benzamide thiourea

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sapari, Suhaila; Yamin, Bohari M.; Hasbullah, Aishah

Synthesis, characterization and antibacterial studies of 2-chloro-5-fluoro-N-[dibenzyl carbamothioyl] benzamide thiourea has been reported. The compound characterized by using elementary analysis CHNS, IR, {sup 1}H NMR and {sup 13}C NMR spectroscopies. The compounds have been screened for their antibacterial studies.

Residue-level global and local ensemble-ensemble comparisons of protein domains.

PubMed

Clark, Sarah A; Tronrud, Dale E; Karplus, P Andrew

2015-09-01

Many methods of protein structure generation such as NMR-based solution structure determination and template-based modeling do not produce a single model, but an ensemble of models consistent with the available information. Current strategies for comparing ensembles lose information because they use only a single representative structure. Here, we describe the ENSEMBLATOR and its novel strategy to directly compare two ensembles containing the same atoms to identify significant global and local backbone differences between them on per-atom and per-residue levels, respectively. The ENSEMBLATOR has four components: eePREP (ee for ensemble-ensemble), which selects atoms common to all models; eeCORE, which identifies atoms belonging to a cutoff-distance dependent common core; eeGLOBAL, which globally superimposes all models using the defined core atoms and calculates for each atom the two intraensemble variations, the interensemble variation, and the closest approach of members of the two ensembles; and eeLOCAL, which performs a local overlay of each dipeptide and, using a novel measure of local backbone similarity, reports the same four variations as eeGLOBAL. The combination of eeGLOBAL and eeLOCAL analyses identifies the most significant differences between ensembles. We illustrate the ENSEMBLATOR's capabilities by showing how using it to analyze NMR ensembles and to compare NMR ensembles with crystal structures provides novel insights compared to published studies. One of these studies leads us to suggest that a "consistency check" of NMR-derived ensembles may be a useful analysis step for NMR-based structure determinations in general. The ENSEMBLATOR 1.0 is available as a first generation tool to carry out ensemble-ensemble comparisons. © 2015 The Protein Society.

Residue-level global and local ensemble-ensemble comparisons of protein domains

PubMed Central

Clark, Sarah A; Tronrud, Dale E; Andrew Karplus, P

2015-01-01

Many methods of protein structure generation such as NMR-based solution structure determination and template-based modeling do not produce a single model, but an ensemble of models consistent with the available information. Current strategies for comparing ensembles lose information because they use only a single representative structure. Here, we describe the ENSEMBLATOR and its novel strategy to directly compare two ensembles containing the same atoms to identify significant global and local backbone differences between them on per-atom and per-residue levels, respectively. The ENSEMBLATOR has four components: eePREP (ee for ensemble-ensemble), which selects atoms common to all models; eeCORE, which identifies atoms belonging to a cutoff-distance dependent common core; eeGLOBAL, which globally superimposes all models using the defined core atoms and calculates for each atom the two intraensemble variations, the interensemble variation, and the closest approach of members of the two ensembles; and eeLOCAL, which performs a local overlay of each dipeptide and, using a novel measure of local backbone similarity, reports the same four variations as eeGLOBAL. The combination of eeGLOBAL and eeLOCAL analyses identifies the most significant differences between ensembles. We illustrate the ENSEMBLATOR's capabilities by showing how using it to analyze NMR ensembles and to compare NMR ensembles with crystal structures provides novel insights compared to published studies. One of these studies leads us to suggest that a “consistency check” of NMR-derived ensembles may be a useful analysis step for NMR-based structure determinations in general. The ENSEMBLATOR 1.0 is available as a first generation tool to carry out ensemble-ensemble comparisons. PMID:26032515

Recommendations and Standardization of Biomarker Quantification Using NMR-Based Metabolomics with Particular Focus on Urinary Analysis.

PubMed

Emwas, Abdul-Hamid; Roy, Raja; McKay, Ryan T; Ryan, Danielle; Brennan, Lorraine; Tenori, Leonardo; Luchinat, Claudio; Gao, Xin; Zeri, Ana Carolina; Gowda, G A Nagana; Raftery, Daniel; Steinbeck, Christoph; Salek, Reza M; Wishart, David S

2016-02-05

NMR-based metabolomics has shown considerable promise in disease diagnosis and biomarker discovery because it allows one to nondestructively identify and quantify large numbers of novel metabolite biomarkers in both biofluids and tissues. Precise metabolite quantification is a prerequisite to move any chemical biomarker or biomarker panel from the lab to the clinic. Among the biofluids commonly used for disease diagnosis and prognosis, urine has several advantages. It is abundant, sterile, and easily obtained, needs little sample preparation, and does not require invasive medical procedures for collection. Furthermore, urine captures and concentrates many "unwanted" or "undesirable" compounds throughout the body, providing a rich source of potentially useful disease biomarkers; however, incredible variation in urine chemical concentrations makes analysis of urine and identification of useful urinary biomarkers by NMR challenging. We discuss a number of the most significant issues regarding NMR-based urinary metabolomics with specific emphasis on metabolite quantification for disease biomarker applications and propose data collection and instrumental recommendations regarding NMR pulse sequences, acceptable acquisition parameter ranges, relaxation effects on quantitation, proper handling of instrumental differences, sample preparation, and biomarker assessment.

Classification of Coffee Beans by GC-C-IRMS, GC-MS, and (1)H-NMR.

PubMed

Arana, Victoria Andrea; Medina, Jessica; Esseiva, Pierre; Pazos, Diego; Wist, Julien

2016-01-01

In a previous work using (1)H-NMR we reported encouraging steps towards the construction of a robust expert system for the discrimination of coffees from Colombia versus nearby countries (Brazil and Peru), to assist the recent protected geographical indication granted to Colombian coffee in 2007. This system relies on fingerprints acquired on a 400 MHz magnet and is thus well suited for small scale random screening of samples obtained at resellers or coffee shops. However, this approach cannot easily be implemented at harbour's installations, due to the elevated operational costs of cryogenic magnets. This limitation implies shipping the samples to the NMR laboratory, making the overall approach slower and thereby more expensive and less attractive for large scale screening at harbours. In this work, we report on our attempt to obtain comparable classification results using alternative techniques that have been reported promising as an alternative to NMR: GC-MS and GC-C-IRMS. Although statistically significant information could be obtained by all three methods, the results show that the quality of the classifiers depends mainly on the number of variables included in the analysis; hence NMR provides an advantage since more molecules are detected to obtain a model with better predictions.

Classification of Coffee Beans by GC-C-IRMS, GC-MS, and 1H-NMR

PubMed Central

Arana, Victoria Andrea; Esseiva, Pierre; Pazos, Diego

2016-01-01

In a previous work using 1H-NMR we reported encouraging steps towards the construction of a robust expert system for the discrimination of coffees from Colombia versus nearby countries (Brazil and Peru), to assist the recent protected geographical indication granted to Colombian coffee in 2007. This system relies on fingerprints acquired on a 400 MHz magnet and is thus well suited for small scale random screening of samples obtained at resellers or coffee shops. However, this approach cannot easily be implemented at harbour's installations, due to the elevated operational costs of cryogenic magnets. This limitation implies shipping the samples to the NMR laboratory, making the overall approach slower and thereby more expensive and less attractive for large scale screening at harbours. In this work, we report on our attempt to obtain comparable classification results using alternative techniques that have been reported promising as an alternative to NMR: GC-MS and GC-C-IRMS. Although statistically significant information could be obtained by all three methods, the results show that the quality of the classifiers depends mainly on the number of variables included in the analysis; hence NMR provides an advantage since more molecules are detected to obtain a model with better predictions. PMID:27516919

Advanced solid-state NMR spectroscopy of natural organic matter.

PubMed

Mao, Jingdong; Cao, Xiaoyan; Olk, Dan C; Chu, Wenying; Schmidt-Rohr, Klaus

2017-05-01

Solid-state NMR is essential for the characterization of natural organic matter (NOM) and is gaining importance in geosciences and environmental sciences. This review is intended to highlight advanced solid-state NMR techniques, especially a systematic approach to NOM characterization, and their applications to the study of NOM. We discuss some basics of how to acquire high-quality and quantitative solid-state 13 C NMR spectra, and address some common technical mistakes that lead to unreliable spectra of NOM. The identification of specific functional groups in NOM, primarily based on 13 C spectral-editing techniques, is described and the theoretical background of some recently-developed spectral-editing techniques is provided. Applications of solid-state NMR to investigating nitrogen (N) in NOM are described, focusing on limitations of the widely used 15 N CP/MAS experiment and the potential of improved advanced NMR techniques for characterizing N forms in NOM. Then techniques used for identifying proximities, heterogeneities and domains are reviewed, and some examples provided. In addition, NMR techniques for studying segmental dynamics in NOM are reviewed. We also briefly discuss applications of solid-state NMR to NOM from various sources, including soil organic matter, aquatic organic matter, organic matter in atmospheric particulate matter, carbonaceous meteoritic organic matter, and fossil fuels. Finally, examples of NMR-based structural models and an outlook are provided. Copyright © 2016 Elsevier B.V. All rights reserved.

Carbon nuclear magnetic resonance spectroscopic fingerprinting of commercial gasoline: pattern-recognition analyses for screening quality control purposes.

PubMed

Flumignan, Danilo Luiz; Boralle, Nivaldo; Oliveira, José Eduardo de

2010-06-30

In this work, the combination of carbon nuclear magnetic resonance ((13)C NMR) fingerprinting with pattern-recognition analyses provides an original and alternative approach to screening commercial gasoline quality. Soft Independent Modelling of Class Analogy (SIMCA) was performed on spectroscopic fingerprints to classify representative commercial gasoline samples, which were selected by Hierarchical Cluster Analyses (HCA) over several months in retails services of gas stations, into previously quality-defined classes. Following optimized (13)C NMR-SIMCA algorithm, sensitivity values were obtained in the training set (99.0%), with leave-one-out cross-validation, and external prediction set (92.0%). Governmental laboratories could employ this method as a rapid screening analysis to discourage adulteration practices. Copyright 2010 Elsevier B.V. All rights reserved.

Discovery of Potent Myeloid Cell Leukemia 1 (Mcl-1) Inhibitors Using Fragment-Based Methods and Structure-Based Design

DOE Office of Scientific and Technical Information (OSTI.GOV)

Friberg, Anders; Vigil, Dominico; Zhao, Bin

2012-12-17

Myeloid cell leukemia 1 (Mcl-1), a member of the Bcl-2 family of proteins, is overexpressed and amplified in various cancers and promotes the aberrant survival of tumor cells that otherwise would undergo apoptosis. Here we describe the discovery of potent and selective Mcl-1 inhibitors using fragment-based methods and structure-based design. NMR-based screening of a large fragment library identified two chemically distinct hit series that bind to different sites on Mcl-1. Members of the two fragment classes were merged together to produce lead compounds that bind to Mcl-1 with a dissociation constant of <100 nM with selectivity for Mcl-1 over Bcl-xLmore » and Bcl-2. Structures of merged compounds when complexed to Mcl-1 were obtained by X-ray crystallography and provide detailed information about the molecular recognition of small-molecule ligands binding Mcl-1. The compounds represent starting points for the discovery of clinically useful Mcl-1 inhibitors for the treatment of a wide variety of cancers.« less

Synthesis, characterization, antimicrobial screening and in silico studies of Schiff bases derived from trans-para-methoxycinnamaldehyde

NASA Astrophysics Data System (ADS)

Obasi, N. L.; Kaior, G. U.; Ibezim, A.; Ochonogor, Alfred E.; Rhyman, Lydia; Uahengo, Veikko; Lutter, Michael; Jurkschat, Klaus; Ramasami, Ponnadurai

2017-12-01

Two Schiff bases namely N,N‧-Bis-[3-(4-metoxy-phenyl)-allylidene]ethane-1,2-diamine (TPMC/EDA) and [3-(4-methoxy-phenyl)-allylidene]-phenyl-amine (TPMC/AN) were synthesized. They were characterized using elemental microanalysis, IR, NMR, UV and mass spectroscopies. Single crystals of TPMC/AN were also analyzed by X-ray diffraction and the compound was examined using B3LYP/6-311++G(d,p) method. A Monoclinic crystal system and space groups of P21/c were obtained for the crystal. Docking calculations on the compounds showed they interacted with fungal N-myristoyltransferase and bacteria DNA gyrase at 2.62-2.95 and 190.26-98.99 μM ranges. The predicted docked poses identified unique binding modes of the compounds and vital intermolecular interactions. The anti-microbial screening of the compounds were carried out against Escherichia coli, Bacillus subtillis, Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans and Aspergillus niger using agar well diffusion method. The standard drugs used were the anti-bacterial ciprofloxacin and the anti-fungal fluconazole. The compounds showed activity against all the microorganisms comparable to the used standard drugs. TPMC/EDA was more active than the standard fungal drug in the screening against the fungi strain, Aspergillus niger. It showed the MIC and IZD of 1.3 mg/ml and 9.0 mm respectively. These suggest that the compounds are potential bactericidal and fungicidal candidates.

Targeting SDF-1/CXCL12 with a ligand that prevents activation of CXCR4 through structure based drug design

PubMed Central

Veldkamp, Christopher T.; Ziarek, Joshua J.; Peterson, Francis C.; Chen, Yu; Volkman, Brian F.

2010-01-01

CXCL12 is an attractive target for clinical therapy because of its involvement in autoimmune diseases, cancer growth, metastasis, and neovascularization. Tyrosine sulfation at three positions in the CXCR4 N-terminus is crucial for specific, high-affinity CXCL12 binding. An NMR structure of the complex between the CXCL12 dimer and a sulfotyrosine-containing CXCR4 fragment enabled high-throughput in silico screening for inhibitors of the chemokine-receptor interface. A total of 1.4 million compounds from the ZINC database were docked into a cleft on the CXCL12 surface normally occupied by sulfotyrosine 21 (sY21), and five were selected for experimental screening. NMR titrations with CXCL12 revealed that four compounds occupy the sY21 site, one of which binds with a Kd of 64 µM. This compound selectively inhibits SDF1-induced CXCR4 signaling in THP1 cells. Our results suggest that sulfotyrosine recognition sites can be targeted for the development of novel chemokine inhibitors. PMID:20459090

Nonlinear Behavior of a Shim Coil in an LTS/HTS NMR Magnet With an HTS Insert Comprising Double-Pancake HTS-Tape Coils

PubMed Central

Hahn, Seung-yong; Ahn, Min Cheol; Bascuñán, Juan; Yao, Weijun; Iwasa, Yukikazu

2010-01-01

This paper reports results, experimental and analytical, of the nonlinear behavior of a shim coil in the presence of an HTS coil assembled with double-pancake (DP) HTS-tape coils. The experimental results are from: 1) operation of a 700 MHz LTS/HTS NMR magnet (LH700) consisting of a 600 MHz LTS NMR magnet (L600) equipped with superconducting shim coils and a 100 MHz DP-assembled HTS insert (H100) and; 2) an experiment with a room-temperature (RT) Z1 shim coil coupled to a 50 MHz DP-as-sembled HTS insert (H50). A field mapping theory with a harmonic analysis is applied to interpret both results. Based on experimental results and analyses, we conclude that the screening-current-induced field (SCF) generated by a DP-assembled HTS insert is primarily responsible for the nonlinear behavior, including strength degradation, of a shim coil. PMID:20383282

SpecPad: device-independent NMR data visualization and processing based on the novel DART programming language and Html5 Web technology.

PubMed

Guigas, Bruno

2017-09-01

SpecPad is a new device-independent software program for the visualization and processing of one-dimensional and two-dimensional nuclear magnetic resonance (NMR) time domain (FID) and frequency domain (spectrum) data. It is the result of a project to investigate whether the novel programming language DART, in combination with Html5 Web technology, forms a suitable base to write an NMR data evaluation software which runs on modern computing devices such as Android, iOS, and Windows tablets as well as on Windows, Linux, and Mac OS X desktop PCs and notebooks. Another topic of interest is whether this technique also effectively supports the required sophisticated graphical and computational algorithms. SpecPad is device-independent because DART's compiled executable code is JavaScript and can, therefore, be run by the browsers of PCs and tablets. Because of Html5 browser cache technology, SpecPad may be operated off-line. Network access is only required during data import or export, e.g. via a Cloud service, or for software updates. A professional and easy to use graphical user interface consistent across all hardware platforms supports touch screen features on mobile devices for zooming and panning and for NMR-related interactive operations such as phasing, integration, peak picking, or atom assignment. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

Evaluation of short-chain-length polyhydroxyalkanoate accumulation in Bacillus aryabhattai.

PubMed

Balakrishna Pillai, Aneesh; Jaya Kumar, Arjun; Thulasi, Kavitha; Kumarapillai, Harikrishnan

This study was focused on the polyhydroxybutyrate (PHB) accumulation property of Bacillus aryabhattai isolated from environment. Twenty-four polyhydroxyalkanoate (PHA) producers were screened out from sixty-two environmental bacterial isolates based on Sudan Black B colony staining. Based on their PHA accumulation property, six promising isolates were further screened out. The most productive isolate PHB10 was identified as B. aryabhattai PHB10. The polymer production maxima were 3.264g/L, 2.181g/L, 1.47g/L, 1.742g/L and 1.786g/L in glucose, fructose, maltose, starch and glycerol respectively. The bacterial culture reached its stationary and declining phases at 18h and 21h respectively and indicated growth-associated PHB production. Nuclear Magnetic Resonance (NMR) spectra confirmed the material as PHB. The material has thermal stability between 30 and 140°C, melting point at 170°C and maximum thermal degradation at 287°C. The molecular weight and poly dispersion index of the polymer were found as 199.7kDa and 2.67 respectively. The bacterium B. aryabhattai accumulating PHB up to 75% of cell dry mass utilizing various carbon sources is a potential candidate for large scale production of bacterial polyhydroxybutyrate. Copyright © 2017 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

Screening of immunomodulatory components in Yu-ping-feng-san using splenocyte binding and HPLC.

PubMed

Hong, Min; Wang, Xin-zhi; Wang, Liang; Hua, Yong-qing; Wen, Hong-mei; Duan, Jin-ao

2011-01-05

Yu-ping-feng-san (YPFS) is a widely used immunomodulatory herbal medication used in traditional Chinese medicine, but the active molecules remain obscure. To screen for bioactive components we combined splenocyte binding with high performance liquid chromatography (SB-HPLC). After enrichment by splenocyte binding, two YPFS components (C1 and C2) were analyzed by HPLC. Compound C2 was identified as linoleic acid (LA) based on UV absorption and mass spectrometry. Silica gel chromatography was used to purify compound C1 from Radix Saposhnikoviae, a major constituent of YPFS. This allowed identification of the molecule as panaxynol (PAN) based on EI-MS and NMR spectrometry. Bioassay in vitro demonstrated that PAN significantly inhibited splenocyte proliferation induced by concanavalin A (ConA) in a concentration-dependent manner, whereas LA had no significant effect on splenocyte proliferation. In vivo, PAN was found to attenuate allergic contact dermatitis in a mouse model of delayed-type hypersensitivity (DTH), a pharmacological activity not previously reported for this molecule. It is suggested that PAN contributes to the anti-DTH effects of YPFS. SB-HPLC provides a rapid and efficient method for the identification of potential immunomodulatory components in traditional Chinese medicines. Copyright © 2010 Elsevier B.V. All rights reserved.

H-1 Nuclear Magnetic Resonance Metabolomics Analysis Identifies Novel Urinary Biomarkers for Lung Function

DOE Office of Scientific and Technical Information (OSTI.GOV)

MCClay, Joseph L.; Adkins, Daniel E.; Isern, Nancy G.

2010-06-04

Chronic obstructive pulmonary disease (COPD), characterized by chronic airflow limitation, is a serious and growing public health concern. The major environmental risk factor for COPD is tobacco smoking, but the biological mechanisms underlying COPD are not well understood. In this study, we used proton nuclear magnetic resonance (1H-NMR) spectroscopy to identify and quantify metabolites associated with lung function in COPD. Plasma and urine were collected from 197 adults with COPD and from 195 adults without COPD. Samples were assayed using a 600 MHz NMR spectrometer, and the resulting spectra were analyzed against quantitative spirometric measures of lung function. After correctingmore » for false discoveries and adjusting for covariates (sex, age, smoking) several spectral regions in urine were found to be significantly associated with baseline lung function. These regions correspond to the metabolites trigonelline, hippurate and formate. Concentrations of each metabolite, standardized to urinary creatinine, were associated with baseline lung function (minimum p-value = 0.0002 for trigonelline). No significant associations were found with plasma metabolites. Two of the three urinary metabolites positively associated with baseline lung function, i.e. hippurate and formate, are often related to gut microflora. This suggests that the microbiome composition is variable between individuals with different lung function. Alternatively, the nature and origins of all three associated metabolites may reflect lifestyle differences affecting overall health. Our results will require replication and validation, but demonstrate the utility of NMR metabolomics as a screening tool for identifying novel biomarkers of lung disease or disease risk.« less

NMR data-driven structure determination using NMR-I-TASSER in the CASD-NMR experiment

PubMed Central

Jang, Richard; Wang, Yan

2015-01-01

NMR-I-TASSER, an adaption of the I-TASSER algorithm combining NMR data for protein structure determination, recently joined the second round of the CASD-NMR experiment. Unlike many molecular dynamics-based methods, NMR-I-TASSER takes a molecular replacement-like approach to the problem by first threading the target through the PDB to identify structural templates which are then used for iterative NOE assignments and fragment structure assembly refinements. The employment of multiple templates allows NMR-I-TASSER to sample different topologies while convergence to a single structure is not required. Retroactive and blind tests of the CASD-NMR targets from Rounds 1 and 2 demonstrate that even without using NOE peak lists I-TASSER can generate correct structure topology with 15 of 20 targets having a TM-score above 0.5. With the addition of NOE-based distance restraints, NMR-I-TASSER significantly improved the I-TASSER models with all models having the TM-score above 0.5. The average RMSD was reduced from 5.29 to 2.14 Å in Round 1 and 3.18 to 1.71 Å in Round 2. There is no obvious difference in the modeling results with using raw and refined peak lists, indicating robustness of the pipeline to the NOE assignment errors. Overall, despite the low-resolution modeling the current NMR-I-TASSER pipeline provides a coarse-grained structure folding approach complementary to traditional molecular dynamics simulations, which can produce fast near-native frameworks for atomic-level structural refinement. PMID:25737244

Probing hydrogen bonding in cocrystals and amorphous dispersions using (14)N-(1)H HMQC solid-state NMR.

PubMed

Tatton, Andrew S; Pham, Tran N; Vogt, Frederick G; Iuga, Dinu; Edwards, Andrew J; Brown, Steven P

2013-03-04

Cocrystals and amorphous solid dispersions have generated interest in the pharmaceutical industry as an alternative to more established solid delivery forms. The identification of intermolecular hydrogen bonding interactions in a nicotinamide palmitic acid cocrystal and a 50% w/w acetaminophen-polyvinylpyrrolidone solid dispersion are reported using advanced solid-state magic-angle spinning (MAS) NMR methods. The application of a novel (14)N-(1)H HMQC experiment, where coherence transfer is achieved via through-space couplings, is shown to identify specific hydrogen bonding motifs. Additionally, (1)H isotropic chemical shifts and (14)N electric field gradient (EFG) parameters, both accessible from (14)N-(1)H HMQC experiments, are shown to be sensitive to changes in hydrogen bonding geometry. Numerous indicators of molecular association are accessible from this experiment, including NH cross-peaks occurring from intermolecular hydrogen bonds and changes in proton chemical shifts or electric field gradient parameters. First-principles calculations using the GIPAW approach that yield accurate estimates of isotropic chemical shifts, and EFG parameters were used to assist in assignment. It is envisaged that (14)N-(1)H HMQC solid state NMR experiments could become a valuable screening technique of solid delivery forms in the pharmaceutical industry.

Fermentation, Isolation, Structure, and antidiabetic activity of NFAT-133 produced by Streptomyces strain PM0324667

PubMed Central

2011-01-01

Type-2 diabetes is mediated by defects in either insulin secretion or insulin action. In an effort to identify extracts that may stimulate glucose uptake, similar to insulin, a high throughput-screening assay for measuring glucose uptake in skeletal muscle cells was established. During the screening studies to discover novel antidiabetic compounds from microbial resources a Streptomyces strain PM0324667 (MTCC 5543, the Strain accession number at Institute of Microbial Technology, Chandigarh, India), an isolate from arid soil was identified which expressed a secondary metabolite that induced glucose uptake in L6 skeletal muscle cells. By employing bioactivity guided fractionation techniques, a tri-substituted simple aromatic compound with anti-diabetic potential was isolated. It was characterized based on MS and 2D NMR spectral data and identified as NFAT-133 which is a known immunosuppressive agent that inhibits NFAT-dependent transcription in vitro. Our investigations revealed the antidiabetic potential of NFAT-133. The compound induced glucose uptake in differentiated L6 myotubes with an EC50 of 6.3 ± 1.8 μM without activating the peroxisome proliferator-activated receptor-γ. Further, NFAT-133 was also efficacious in vivo in diabetic animals and reduced systemic glucose levels. Thus it is a potential lead compound which can be considered for development as a therapeutic for the treatment of type-2 diabetes. We have reported herewith the isolation of the producer microbe, fermentation, purification, in vitro, and in vivo antidiabetic activity of the compound. PMID:22104600

Monoterpene Unknowns Identified Using IR, [to the first power]H-NMR, [to the thirteenth power]C-NMR, DEPT, COSY, and HETCOR

ERIC Educational Resources Information Center

Alty, Lisa T.

2005-01-01

A study identifies a compound from a set of monoterpenes using infrared (IR) and one-dimensional (1D) nuclear magnetic resonance (NMR) techniques. After identifying the unknown, each carbon and proton signal can be interpreted and assigned to the structure using the information in the two-dimensional (2D) NMR spectra, correlation spectroscopy…

Hyperpolarization of “Neat” Liquids by NMR Signal Amplification by Reversible Exchange

PubMed Central

2016-01-01

We report NMR Signal Amplification by Reversible Exchange (SABRE) hyperpolarization of the rare isotopes in “neat” liquids, each composed only of an otherwise pure target compound with isotopic natural abundance (n.a.) and millimolar concentrations of dissolved catalyst. Pyridine (Py) or Py derivatives are studied at 0.4% isotopic natural abundance 15N, deuterated, 15N enriched, and in various combinations using the SABRE-SHEATH variant (microTesla magnetic fields to permit direct 15N polarization from parahydrogen via reversible binding and exchange with an Ir catalyst). We find that the dilute n.a. 15N spin bath in Py still channels spin order from parahydrogen to dilute 15N spins, without polarization losses due to the presence of 14N or 2H. We demonstrate P15N ≈ 1% (a gain of 2900 fold relative to thermal polarization at 9.4 T) at high substrate concentrations. This fundamental finding has a significant practical benefit for screening potentially hyperpolarizable contrast agents without labeling. The capability of screening at n.a. level of 15N is demonstrated on examples of mono- and dimethyl-substituted Py (picolines and lutidines previously identified as promising pH sensors), showing that the presence of a methyl group in the ortho position significantly decreases SABRE hyperpolarization. PMID:26029349

STD-NMR experiments identify a structural motif with novel second-site activity against West Nile virus NS2B-NS3 protease.

PubMed

Schöne, Tobias; Grimm, Lena Lisbeth; Sakai, Naoki; Zhang, Linlin; Hilgenfeld, Rolf; Peters, Thomas

2017-10-01

West Nile virus (WNV) belongs to the genus Flavivirus of the family Flaviviridae. This mosquito-borne virus that is highly pathogenic to humans has been evolving into a global threat during the past two decades. Despite many efforts, neither antiviral drugs nor vaccines are available. The viral protease NS2B-NS3 pro is essential for viral replication, and therefore it is considered a prime drug target. However, success in the development of specific NS2B-NS3 pro inhibitors had been moderate so far. In the search for new structural motifs with binding affinity for NS2B-NS3 pro , we have screened a fragment library, the Maybridge Ro5 library, employing saturation transfer difference (STD) NMR experiments as readout. About 30% of 429 fragments showed binding to NS2B-NS3 pro . Subsequent STD-NMR competition experiments using the known active site fragment A as reporter ligand yielded 14 competitively binding fragments, and 22 fragments not competing with A. In a fluorophore-based protease assay, all of these fragments showed inhibition in the micromolar range. Interestingly, 10 of these 22 fragments showed a notable increase of STD intensities in the presence of compound A suggesting cooperative binding. The most promising non-competitive inhibitors 1 and 2 (IC 50 ∼ 500 μM) share a structural motif that may guide the development of novel second-site (potentially allosteric) inhibitors of NS2B-NS3 pro . To identify the matching protein binding site, chemical shift perturbation studies employing 1 H, 15 N-TROSY-HSQC experiments with uniformly 2 H, 15 N-labeled protease were performed in the presence of 1, and in the concomitant absence or presence of A. The data suggest that 1 interacts with Met 52* of NS2B, identifying a secondary site adjacent to the binding site of A. Therefore, our study paves the way for the synthesis of novel bidentate NS2B-NS3 pro inhibitors. Copyright © 2017 Elsevier B.V. All rights reserved.

Identification of polymethoxylated flavones from green tangerine peel (Pericarpium Citri Reticulatae Viride) by chromatographic and spectroscopic techniques.

PubMed

Wang, Dandan; Wang, Jian; Huang, Xuehui; Tu, Ying; Ni, Kunyi

2007-05-09

Polymethoxylated flavones (PMFs) were extracted from Pericarpium Citri Reticulatae Viride using a procedure that obtained a consistent mixture of PMFs both in identity and proportion. The mixture consisted of isosinensetin (0.2%) (1), sinensetin (1.7%) (4), tetramethyl-o-isoscutellarein (0.3%) (5), nobiletin (40.5%) (6), tetramethyl-o-scutellarein (1.2%) (7), tangeretin (45.6%) (10), 5-demethylnobiletin (8.7%) (12), 5-demethyl tangeretin (0.8%) (14) and other flavonoids including heptamethoxyflavone (1.0%) (9), among which, compounds 1, 4, 5, 7 and 9 were identified based on their UV spectra, MS data and elution order described in the literature while compounds 6, 10, 12 and 14 were isolated and identified by UV, IR, MS, (1)H NMR, (13)C NMR and 2D NMR spectral studies. In addition, compound 14 was isolated and identified for the first time from Citrus.

Stereoselective synthesis, spectral and antimicrobial studies of some cyanoacetyl hydrazones of 3-alkyl-2,6-diarylpiperidin-4-ones

NASA Astrophysics Data System (ADS)

Velayutham Pillai, M.; Rajeswari, K.; Vidhyasagar, T.

2014-11-01

A series of novel cyanoacetyl hydrazones of 3-alkyl-2,6-diarylpiperidin-4-ones were synthesized stereoselectively and characterized by IR, Mass, 1H NMR, 13C NMR, 1H-1H COSY and 1H-13C COSY spectra. The stereochemistry of the synthesized compounds was established using NMR spectra. Antimicrobial screening of the synthesized compounds revealed their antibacterial and antifungal potencies. Growth inhibition of Enterobacter Aerogenes by compound 15 was found to be superior to the standard drug.

Inhibition of CDC25B Phosphatase Through Disruption of Protein–Protein Interaction

DOE PAGES

Lund, George; Dudkin, Sergii; Borkin, Dmitry; ...

2014-11-25

CDC25 phosphatases are key cell cycle regulators and represent very attractive but challenging targets for anticancer drug discovery. Here in this paper, we explored whether fragment-based screening represents a valid approach to identify inhibitors of CDC25B. This resulted in identification of 2-fluoro-4-hydroxybenzonitrile, which directly binds to the catalytic domain of CDC25B. Interestingly, NMR data and the crystal structure demonstrate that this compound binds to the pocket distant from the active site and adjacent to the protein–protein interaction interface with CDK2/Cyclin A substrate. Furthermore, we developed a more potent analogue that disrupts CDC25B interaction with CDK2/Cyclin A and inhibits dephosphorylation ofmore » CDK2. Based on these studies, we provide a proof of concept that targeting CDC25 phosphatases by inhibiting their protein–protein interactions with CDK2/Cyclin A substrate represents a novel, viable opportunity to target this important class of enzymes.« less

Expedited Selection of NMR Chiral Solvating Agents for Determination of Enantiopurity

PubMed Central

2016-01-01

The use of NMR chiral solvating agents (CSAs) for the analysis of enantiopurity has been known for decades, but has been supplanted in recent years by chromatographic enantioseparation technology. While chromatographic methods for the analysis of enantiopurity are now commonplace and easy to implement, there are still individual compounds and entire classes of analytes where enantioseparation can prove extremely difficult, notably, compounds that are chiral by virtue of very subtle differences such as isotopic substitution or small differences in alkyl chain length. NMR analysis using CSAs can often be useful for such problems, but the traditional approach to selection of an appropriate CSA and the development of an NMR-based analysis method often involves a trial-and-error approach that can be relatively slow and tedious. In this study we describe a high-throughput experimentation approach to the selection of NMR CSAs that employs automation-enabled screening of prepared libraries of CSAs in a systematic fashion. This approach affords excellent results for a standard set of enantioenriched compounds, providing a valuable comparative data set for the effectiveness of CSAs for different classes of compounds. In addition, the technique has been successfully applied to challenging pharmaceutical development problems that are not amenable to chromatographic solutions. Overall, this methodology provides a rapid and powerful approach for investigating enantiopurity that compliments and augments conventional chromatographic approaches. PMID:27280168

Chemical Composition and Seasonality of Aromatic Mediterranean Plant Species by NMR-Based Metabolomics

PubMed Central

Scognamiglio, Monica; D'Abrosca, Brigida; Esposito, Assunta; Fiorentino, Antonio

2015-01-01

An NMR-based metabolomic approach has been applied to analyse seven aromatic Mediterranean plant species used in traditional cuisine. Based on the ethnobotanical use of these plants, the approach has been employed in order to study the metabolic changes during different seasons. Primary and secondary metabolites have been detected and quantified. Flavonoids (apigenin, quercetin, and kaempferol derivatives) and phenylpropanoid derivatives (e.g., chlorogenic and rosmarinic acid) are the main identified polyphenols. The richness in these metabolites could explain the biological properties ascribed to these plant species. PMID:25785229

Structure-based manual screening and automatic networking for systematically exploring sansanmycin analogues using high performance liquid chromatography tandem mass spectroscopy.

PubMed

Jiang, Zhi-Bo; Ren, Wei-Cong; Shi, Yuan-Yuan; Li, Xing-Xing; Lei, Xuan; Fan, Jia-Hui; Zhang, Cong; Gu, Ren-Jie; Wang, Li-Fei; Xie, Yun-Ying; Hong, Bin

2018-05-18

Sansanmycins (SS), one of several known uridyl peptide antibiotics (UPAs) possessing a unique chemical scaffold, showed a good inhibitory effect on the highly refractory pathogens Pseudomonas aeruginosa and Mycobacterium tuberculosis, especially on the multi-drug resistant M. tuberculosis. This study employed high performance liquid chromatography-mass spectrometry detector (HPLC-MSD) ion trap and LTQ orbitrap tandem mass spectrometry (MS/MS) to explore sansanmycin analogues manually and automatically by re-analysis of the Streptomyces sp. SS fermentation broth. The structure-based manual screening method, based on analysis of the fragmentation pathway of known UPAs and on comparisons of the MS/MS spectra with that of sansanmycin A (SS-A), resulted in identifying twenty sansanmycin analogues, including twelve new structures (1-12). Furthermore, to deeply explore sansanmycin analogues, we utilized a GNPS based molecular networking workflow to re-analyze the HPLC-MS/MS data automatically. As a result, eight more new sansanmycins (13-20) were discovered. Compound 1 was discovered to lose two amino acids of residue 1 (AA 1 ) and (2S, 3S)-N 3 -methyl-2,3-diamino butyric acid (DABA) from the N-terminus, and compounds 6, 11 and 12 were found to contain a 2',3'-dehydrated 4',5'-enamine-3'-deoxyuridyl moiety, which have not been reported before. Interestingly, three trace components with novel 5,6-dihydro-5'-aminouridyl group (16-18) were detected for the first time in the sansanmycin-producing strain. Their structures were primarily determined by detail analysis of the data from MS/MS. Compounds 8 and 10 were further confirmed by nuclear magnetic resonance (NMR) data, which proved the efficiency and accuracy of the method of HPLC-MS/MS for exploration of novel UPAs. Comparing to manual screening, the networking method can provide systematic visualization results. Manual screening and networking method may complement with each other to facilitate the mining of novel UPAs. Copyright © 2018 Elsevier B.V. All rights reserved.

Los Alamos Team Demonstrates Bottle Scanner Technology

DOE Office of Scientific and Technical Information (OSTI.GOV)

Espy, Michelle; Schultz, Larry

2014-05-06

Los Alamos scientists are demonstrating a Nuclear Magnetic Resonance Imaging (NMR) technology that may provide a breakthrough for screening liquids at airport security. By adding low-power X-ray data to the NMR mix, scientists believe they have unlocked a new detection technology. Funded in part by the Department of Homeland Security's Science and Technology Directorate, the new technology is called MagRay.

Los Alamos Team Demonstrates Bottle Scanner Technology

ScienceCinema

Espy, Michelle; Schultz, Larry

2018-02-13

Los Alamos scientists are demonstrating a Nuclear Magnetic Resonance Imaging (NMR) technology that may provide a breakthrough for screening liquids at airport security. By adding low-power X-ray data to the NMR mix, scientists believe they have unlocked a new detection technology. Funded in part by the Department of Homeland Security's Science and Technology Directorate, the new technology is called MagRay.

Metabolic discrimination of sea buckthorn from different Hippophaë species by 1H NMR based metabolomics.

PubMed

Liu, Yue; Fan, Gang; Zhang, Jing; Zhang, Yi; Li, Jingjian; Xiong, Chao; Zhang, Qi; Li, Xiaodong; Lai, Xianrong

2017-05-08

Sea buckthorn (Hippophaë; Elaeagnaceae) berries are widely consumed in traditional folk medicines, nutraceuticals, and as a source of food. The growing demand of sea buckthorn berries and morphological similarity of Hippophaë species leads to confusions, which might cause misidentification of plants used in natural products. Detailed information and comparison of the complete set of metabolites of different Hippophaë species are critical for their objective identification and quality control. Herein, the variation among seven species and seven subspecies of Hippophaë was studied using proton nuclear magnetic resonance ( 1 H NMR) metabolomics combined with multivariate data analysis, and the important metabolites were quantified by quantitative 1 H NMR (qNMR) method. The results showed that different Hippophaë species can be clearly discriminated and the important interspecific discriminators, including organic acids, L-quebrachitol, and carbohydrates were identified. Statistical differences were found among most of the Hippophaë species and subspecies at the content levels of the aforementioned interspecific discriminators via qNMR and one-way analysis of variance (ANOVA) test. These findings demonstrated that 1 H NMR-based metabolomics is an applicable and effective approach for simultaneous metabolic profiling, species differentiation and quality assessment.

Recommendations and Standardization of Biomarker Quantification Using NMR-Based Metabolomics with Particular Focus on Urinary Analysis

PubMed Central

2016-01-01

NMR-based metabolomics has shown considerable promise in disease diagnosis and biomarker discovery because it allows one to nondestructively identify and quantify large numbers of novel metabolite biomarkers in both biofluids and tissues. Precise metabolite quantification is a prerequisite to move any chemical biomarker or biomarker panel from the lab to the clinic. Among the biofluids commonly used for disease diagnosis and prognosis, urine has several advantages. It is abundant, sterile, and easily obtained, needs little sample preparation, and does not require invasive medical procedures for collection. Furthermore, urine captures and concentrates many “unwanted” or “undesirable” compounds throughout the body, providing a rich source of potentially useful disease biomarkers; however, incredible variation in urine chemical concentrations makes analysis of urine and identification of useful urinary biomarkers by NMR challenging. We discuss a number of the most significant issues regarding NMR-based urinary metabolomics with specific emphasis on metabolite quantification for disease biomarker applications and propose data collection and instrumental recommendations regarding NMR pulse sequences, acceptable acquisition parameter ranges, relaxation effects on quantitation, proper handling of instrumental differences, sample preparation, and biomarker assessment. PMID:26745651

Metabolism of 7-nitrogenz[a]anthracene by intestinal microflora

DOE Office of Scientific and Technical Information (OSTI.GOV)

Morehead, M.C.; Franklin, W.; Fu, P.P.

1994-12-31

Pure cultures of anaerobic intestinal bacteria and mixed fecal microflora from human, rat, mouse, and pig were screened for the ability to metabolize 7-nitrobenz[a]anthracene. Based on analysis by high-performance liquid chromatography (HPLC) and by ultraviolent (UV), mass, and nuclear magnetic resonance (NMR) spectral techniques, the compounds were identified as 7-aminobenz[a]anthracene and benz[a]anthracene 7,12-dione. Identification of 7-NH{sub 2}BA as a metabolite of 7-NO{sub 2}BA indicates that the anaerobic intestinal bacteria are capable of reducing 7-NO{sub 2}BA to potentially bioactive intermediates. The reductive capacities of the mixed intestinal microflora were generally greater than those of pure cultures. Thus, metabolism of 7-NO{sub 2}BAmore » in the intestinal tract may be underestimated if pure cultures are used as the sole method for evaluating the potential hazard.« less

1H NMR-Based Metabolomic Analysis of Sub-Lethal Perfluorooctane Sulfonate Exposure to the Earthworm, Eisenia fetida, in Soil

PubMed Central

Lankadurai, Brian P.; Furdui, Vasile I.; Reiner, Eric J.; Simpson, André J.; Simpson, Myrna J.

2013-01-01

1H NMR-based metabolomics was used to measure the response of Eisenia fetida earthworms after exposure to sub-lethal concentrations of perfluorooctane sulfonate (PFOS) in soil. Earthworms were exposed to a range of PFOS concentrations (five, 10, 25, 50, 100 or 150 mg/kg) for two, seven and fourteen days. Earthworm tissues were extracted and analyzed by 1H NMR. Multivariate statistical analysis of the metabolic response of E. fetida to PFOS exposure identified time-dependent responses that were comprised of two separate modes of action: a non-polar narcosis type mechanism after two days of exposure and increased fatty acid oxidation after seven and fourteen days of exposure. Univariate statistical analysis revealed that 2-hexyl-5-ethyl-3-furansulfonate (HEFS), betaine, leucine, arginine, glutamate, maltose and ATP are potential indicators of PFOS exposure, as the concentrations of these metabolites fluctuated significantly. Overall, NMR-based metabolomic analysis suggests elevated fatty acid oxidation, disruption in energy metabolism and biological membrane structure and a possible interruption of ATP synthesis. These conclusions obtained from analysis of the metabolic profile in response to sub-lethal PFOS exposure indicates that NMR-based metabolomics is an excellent discovery tool when the mode of action (MOA) of contaminants is not clearly defined. PMID:24958147

[Screening and optimization of cholesterol conversion strain].

PubMed

Fan, Dan; Xiong, Bingjian; Pang, Cuiping; Zhu, Xiangdong

2014-10-04

Bacterial strain SE-1 capable of transforming cholesterol was isolated from soil and characterized. The transformation products were identified. Fermentation conditions were optimized for conversion. Cholesterol was used as sole carbon source to isolate strain SE-1. Morphology, physiological and biochemical characteristics of strain SE-1 were studied. 16S rRNA gene was sequenced and subjected to phylogenetic analysis. Fermentation supernatants were extracted with chloroform, the transformation products were analyzed by silica gel thin layer chromatography and Sephadex LH20. Their structures were identified by 1H-NMR and 13C-NMR. Fermentation medium including carbon and nitrogen, methods of adding substrates and fermentation conditions for Strain SE-1 were optimized. Strain SE-1 was a Gram-negative bacterium, exhibiting the highest homologs to Burkholderia cepacia based on the physiological analysis. The sequence analysis of 16S rRNA gene of SE-1 strain and comparison with related Burkholderia show that SE-1 strain was very close to B. cepacia (Genbank No. U96927). The similarity was 99%. The result of silica gel thin layer chromatography shows that strain SE-1 transformed cholesterol to two products, 7beta-hydroxycholesterol and the minor product was 7-oxocholesterol. The optimum culture conditions were: molasses 5%, (NH4 )2SO4 0.3%, 4% of inoculation, pH 7.5 and 36 degrees C. Under the optimum culture condition, the conversion rate reached 34.4% when concentration of cholesterol-Tween 80 was 1 g/L. Cholesterol 7beta-hydroxylation conversion rate under optimal conditions was improved by 20.8%. Strain SE-1 isolated from soil is capable of converting cholesterol at lab-scale.

Identification and characterization of a novel high affinity metal-binding site in the hammerhead ribozyme.

PubMed Central

Hansen, M R; Simorre, J P; Hanson, P; Mokler, V; Bellon, L; Beigelman, L; Pardi, A

1999-01-01

A novel metal-binding site has been identified in the hammerhead ribozyme by 31P NMR. The metal-binding site is associated with the A13 phosphate in the catalytic core of the hammerhead ribozyme and is distinct from any previously identified metal-binding sites. 31P NMR spectroscopy was used to measure the metal-binding affinity for this site and leads to an apparent dissociation constant of 250-570 microM at 25 degrees C for binding of a single Mg2+ ion. The NMR data also show evidence of a structural change at this site upon metal binding and these results are compared with previous data on metal-induced structural changes in the core of the hammerhead ribozyme. These NMR data were combined with the X-ray structure of the hammerhead ribozyme (Pley HW, Flaherty KM, McKay DB. 1994. Nature 372:68-74) to model RNA ligands involved in binding the metal at this A13 site. In this model, the A13 metal-binding site is structurally similar to the previously identified A(g) metal-binding site and illustrates the symmetrical nature of the tandem G x A base pairs in domain 2 of the hammerhead ribozyme. These results demonstrate that 31P NMR represents an important method for both identification and characterization of metal-binding sites in nucleic acids. PMID:10445883

Chemical Screens Against A Reconstituted Multi-Protein Complex: Myricetin Blocks DnaJ Regulation of DnaK through an Allosteric Mechanism

PubMed Central

Chang, Lyra; Miyata, Yoshinari; Ung, Peter M. U.; Bertelsen, Eric B.; McQuade, Thomas J.; Carlson, Heather A.; Zuiderweg, Erik R. P.; Gestwicki, Jason E.

2011-01-01

SUMMARY DnaK is a molecular chaperone responsible for multiple aspects of proteostasis. The intrinsically slow ATPase activity of DnaK is stimulated by its co-chaperone, DnaJ, and these proteins often work in concert. To identify inhibitors, we screened plant-derived extracts against a re-constituted mixture of DnaK and DnaJ. This approach resulted in the identification of flavonoids, including myricetin, which inhibited activity by up to 75%. Interestingly, myricetin prevented DnaJ-mediated stimulation of ATPase activity, with minimal impact on either DnaK’s intrinsic turnover rate or its stimulation by another co-chaperone, GrpE. Using NMR, we found that myricetin binds DnaK at an unanticipated site between the IB and IIB subdomains and that it allosterically blocked binding of DnaJ. Together, these results highlight a “gray box” screening approach, which approximates a limited amount of the complexity expected in physiological, multi-protein systems. PMID:21338918

PAINS in the Assay: Chemical Mechanisms of Assay Interference and Promiscuous Enzymatic Inhibition Observed during a Sulfhydryl-Scavenging HTS

PubMed Central

2015-01-01

Significant resources in early drug discovery are spent unknowingly pursuing artifacts and promiscuous bioactive compounds, while understanding the chemical basis for these adverse behaviors often goes unexplored in pursuit of lead compounds. Nearly all the hits from our recent sulfhydryl-scavenging high-throughput screen (HTS) targeting the histone acetyltransferase Rtt109 were such compounds. Herein, we characterize the chemical basis for assay interference and promiscuous enzymatic inhibition for several prominent chemotypes identified by this HTS, including some pan-assay interference compounds (PAINS). Protein mass spectrometry and ALARM NMR confirmed these compounds react covalently with cysteines on multiple proteins. Unfortunately, compounds containing these chemotypes have been published as screening actives in reputable journals and even touted as chemical probes or preclinical candidates. Our detailed characterization and identification of such thiol-reactive chemotypes should accelerate triage of nuisance compounds, guide screening library design, and prevent follow-up on undesirable chemical matter. PMID:25634295

Use of NMR and NMR Prediction Software to Identify Components in Red Bull Energy Drinks

ERIC Educational Resources Information Center

Simpson, Andre J.; Shirzadi, Azadeh; Burrow, Timothy E.; Dicks, Andrew P.; Lefebvre, Brent; Corrin, Tricia

2009-01-01

A laboratory experiment designed as part of an upper-level undergraduate analytical chemistry course is described. Students investigate two popular soft drinks (Red Bull Energy Drink and sugar-free Red Bull Energy Drink) by NMR spectroscopy. With assistance of modern NMR prediction software they identify and quantify major components in each…

Development of peptoid-based ligands for the removal of cadmium from biological media

DOE PAGES

Knight, Abigail S.; Zhou, Effie Y.; Francis, Matthew B.

2015-05-14

Cadmium poisoning poses a serious health concern due to cadmium's increasing industrial use, yet there is currently no recommended treatment. The selective coordination of cadmium in a biological environment—i.e. in the presence of serum ions, small molecules, and proteins—is a difficult task. To address this challenge, a combinatorial library of peptoid-based ligands has been evaluated to identify structures that selectively bind to cadmium in human serum with minimal chelation of essential metal ions. Eighteen unique ligands were identified in this screening procedure, and the binding affinity of each was measured using metal titrations monitored by UV-vis spectroscopy. To evaluate themore » significance of each chelating moiety, sequence rearrangements and substitutions were examined. Analysis of a metal–ligand complex by NMR spectroscopy highlighted the importance of particular residues. Depletion experiments were performed in serum mimetics and human serum with exogenously added cadmium. These depletion experiments were used to compare and demonstrate the ability of these peptoids to remove cadmium from blood-like mixtures. In one of these depletion experiments, the peptoid sequence was able to deplete the cadmium to a level comparable to the reported acute toxicity limit. Evaluation of the metal selectivity in buffered solution and in human serum was performed to verify minimal off-target binding. These studies highlight a screening platform for the identification of metal–ligands that are capable of binding in a complex environment. They additionally demonstrate the potential utility of biologically-compatible ligands for the treatment of heavy metal poisoning.« less

Development of peptoid-based ligands for the removal of cadmium from biological media

DOE Office of Scientific and Technical Information (OSTI.GOV)

Knight, Abigail S.; Zhou, Effie Y.; Francis, Matthew B.

Cadmium poisoning poses a serious health concern due to cadmium's increasing industrial use, yet there is currently no recommended treatment. The selective coordination of cadmium in a biological environment—i.e. in the presence of serum ions, small molecules, and proteins—is a difficult task. To address this challenge, a combinatorial library of peptoid-based ligands has been evaluated to identify structures that selectively bind to cadmium in human serum with minimal chelation of essential metal ions. Eighteen unique ligands were identified in this screening procedure, and the binding affinity of each was measured using metal titrations monitored by UV-vis spectroscopy. To evaluate themore » significance of each chelating moiety, sequence rearrangements and substitutions were examined. Analysis of a metal–ligand complex by NMR spectroscopy highlighted the importance of particular residues. Depletion experiments were performed in serum mimetics and human serum with exogenously added cadmium. These depletion experiments were used to compare and demonstrate the ability of these peptoids to remove cadmium from blood-like mixtures. In one of these depletion experiments, the peptoid sequence was able to deplete the cadmium to a level comparable to the reported acute toxicity limit. Evaluation of the metal selectivity in buffered solution and in human serum was performed to verify minimal off-target binding. These studies highlight a screening platform for the identification of metal–ligands that are capable of binding in a complex environment. They additionally demonstrate the potential utility of biologically-compatible ligands for the treatment of heavy metal poisoning.« less

Nuclear magnetic resonance (NMR)-based metabolomics for cancer research.

PubMed

Ranjan, Renuka; Sinha, Neeraj

2018-05-07

Nuclear magnetic resonance (NMR) has emerged as an effective tool in various spheres of biomedical research, amongst which metabolomics is an important method for the study of various types of disease. Metabolomics has proved its stronghold in cancer research by the development of different NMR methods over time for the study of metabolites, thus identifying key players in the aetiology of cancer. A plethora of one-dimensional and two-dimensional NMR experiments (in solids, semi-solids and solution phases) are utilized to obtain metabolic profiles of biofluids, cell extracts and tissue biopsy samples, which can further be subjected to statistical analysis. Any alteration in the assigned metabolite peaks gives an indication of changes in metabolic pathways. These defined changes demonstrate the utility of NMR in the early diagnosis of cancer and provide further measures to combat malignancy and its progression. This review provides a snapshot of the trending NMR techniques and the statistical analysis involved in the metabolomics of diseases, with emphasis on advances in NMR methodology developed for cancer research. Copyright © 2018 John Wiley & Sons, Ltd.

High resolution NMR measurements using a 400MHz NMR with an (RE)Ba2Cu3O7-x high-temperature superconducting inner coil: Towards a compact super-high-field NMR.

PubMed

Piao, R; Iguchi, S; Hamada, M; Matsumoto, S; Suematsu, H; Saito, A T; Li, J; Nakagome, H; Takao, T; Takahashi, M; Maeda, H; Yanagisawa, Y

2016-02-01

Use of high-temperature superconducting (HTS) inner coils in combination with conventional low-temperature superconducting (LTS) outer coils for an NMR magnet, i.e. a LTS/HTS NMR magnet, is a suitable option to realize a high-resolution NMR spectrometer with operating frequency >1GHz. From the standpoint of creating a compact magnet, (RE: Rare earth) Ba2Cu3O7-x (REBCO) HTS inner coils which can tolerate a strong hoop stress caused by a Lorentz force are preferred. However, in our previous work on a first-generation 400MHz LTS/REBCO NMR magnet, the NMR resolution and sensitivity were about ten times worse than that of a conventional LTS NMR magnet. The result was caused by a large field inhomogeneity in the REBCO coil itself and the shielding effect of a screening current induced in that coil. In the present paper, we describe the operation of a modified 400MHz LTS/REBCO NMR magnet with an advanced field compensation technology using a combination of novel ferromagnetic shimming and an appropriate procedure for NMR spectrum line shape optimization. We succeeded in obtaining a good NMR line shape and 2D NOESY spectrum for a lysozyme aqueous sample. We believe that this technology is indispensable for the realization of a compact super-high-field high-resolution NMR. Copyright © 2016 Elsevier Inc. All rights reserved.

Mixtures of the 1-ethyl-3-methylimidazolium acetate ionic liquid with different inorganic salts: insights into their interactions.

PubMed

Oliveira, Filipe S; Cabrita, Eurico J; Todorovic, Smilja; Bernardes, Carlos E S; Lopes, José N Canongia; Hodgson, Jennifer L; MacFarlane, Douglas R; Rebelo, Luís P N; Marrucho, Isabel M

2016-01-28

In this work, we explore the interactions between the ionic liquid 1-ethyl-3-methylimidazolim acetate and different inorganic salts belonging to two different cation families, those based on ammonium and others based on sodium. NMR and Raman spectroscopy are used to screen for changes in the molecular environment of the ions in the ionic liquid + inorganic salt mixtures as compared to pure ionic liquid. The ion self-diffusion coefficients are determined from NMR data, allowing the discussion of the ionicity values of the ionic liquid + inorganic salt mixtures calculated using different methods. Our data reveal that preferential interactions are established between the ionic liquid and ammonium-based salts, as opposed to sodium-based salts. Computational calculations show the formation of aggregates between the ionic liquid and the inorganic salt, which is consistent with the spectroscopic data, and indicate that the acetate anion of the ionic liquid establishes preferential interactions with the ammonium cation of the inorganic salts, leaving the imidazolium cation less engaged in the media.

Operation of a 400MHz NMR magnet using a (RE:Rare Earth)Ba2Cu3O7-x high-temperature superconducting coil: Towards an ultra-compact super-high field NMR spectrometer operated beyond 1GHz.

PubMed

Yanagisawa, Y; Piao, R; Iguchi, S; Nakagome, H; Takao, T; Kominato, K; Hamada, M; Matsumoto, S; Suematsu, H; Jin, X; Takahashi, M; Yamazaki, T; Maeda, H

2014-12-01

High-temperature superconductors (HTS) are the key technology to achieve super-high magnetic field nuclear magnetic resonance (NMR) spectrometers with an operating frequency far beyond 1GHz (23.5T). (RE)Ba 2 Cu 3 O 7- x (REBCO, RE: rare earth) conductors have an advantage over Bi 2 Sr 2 Ca 2 Cu 3 O 10- x (Bi-2223) and Bi 2 Sr 2 CaCu 2 O 8- x (Bi-2212) conductors in that they have very high tensile strengths and tolerate strong electromagnetic hoop stress, thereby having the potential to act as an ultra-compact super-high field NMR magnet. As a first step, we developed the world's first NMR magnet comprising an inner REBCO coil and outer low-temperature superconducting (LTS) coils. The magnet was successfully charged without degradation and mainly operated at 400MHz (9.39T). Technical problems for the NMR magnet due to screening current in the REBCO coil were clarified and solved as follows: (i) A remarkable temporal drift of the central magnetic field was suppressed by a current sweep reversal method utilizing ∼10% of the peak current. (ii) A Z2 field error harmonic of the main coil cannot be compensated by an outer correction coil and therefore an additional ferromagnetic shim was used. (iii) Large tesseral harmonics emerged that could not be corrected by cryoshim coils. Due to those harmonics, the resolution and sensitivity of NMR spectra are ten-fold lower than those for a conventional LTS NMR magnet. As a result, a HSQC spectrum could be achieved for a protein sample, while a NOESY spectrum could not be obtained. An ultra-compact 1.2GHz NMR magnet could be realized if we effectively take advantage of REBCO conductors, although this will require further research to suppress the effect of the screening current. Copyright © 2014 Elsevier Inc. All rights reserved.

Ammonificins C and D, Hydroxyethylamine Chromene Derivatives from a Cultured Marine Hydrothermal Vent Bacterium, Thermovibrio ammonificans

PubMed Central

Andrianasolo, Eric H.; Haramaty, Liti; Rosario-Passapera, Richard; Vetriani, Costantino; Falkowski, Paul; White, Eileen; Lutz, Richard

2012-01-01

Chemical and biological investigation of the cultured marine hydrothermal vent bacterium, Thermovibrio ammonifican led to the isolation of two hydroxyethylamine chromene derivatives, ammonificins C and D. Their structures were elucidated using combination of NMR and mass spectrometry. Absolute stereochemistry was ascertained by comparison of experimental and calculated CD spectra. Biological evaluation and assessment were determined using the patented ApopScreen cell-based screen for apoptosis-induction. Ammonificins C and D induce apoptosis in micromolar concentrations. To our knowledge, this finding is the first report of chemical compounds that induce apoptosis from the cultured deep-sea marine organism, hydrothermal vent bacterium, Thermovibrio ammonificans. PMID:23170085

Fragment screening for drug leads by weak affinity chromatography (WAC-MS).

PubMed

Ohlson, Sten; Duong-Thi, Minh-Dao

2018-02-23

Fragment-based drug discovery is an important tool for design of small molecule hit-to-lead compounds against various biological targets. Several approved drugs have been derived from an initial fragment screen and many such candidates are in various stages of clinical trials. Finding fragment hits, that are suitable for optimisation by medicinal chemists, is still a challenge as the binding between the small fragment and its target is weak in the range of mM to µM of K d and irrelevant non-specific interactions are abundant in this area of transient interactions. Fortunately, there are methods that can study weak interactions quite efficiently of which NMR, surface plasmon resonance (SPR) and X-ray crystallography are the most prominent. Now, a new technology based on zonal affinity chromatography, weak affinity chromatography (WAC), has been introduced which has remedied many of the problems with other technologies. By combining WAC with mass spectrometry (WAC-MS), it is a powerful tool to identify binders quantitatively in terms of affinity and kinetics either from fragment libraries or from complex mixtures of biological extracts. As WAC-MS can be multiplexed by analysing mixtures of fragments (20-100 fragments) in one sample, this approach yields high throughput, where a whole library of e.g. >2000 fragments can be analysed quantitatively within a day. WAC-MS is easy to perform, where the robustness and quality of HPLC is fully utilized. This review will highlight the rationale behind the application of WAC-MS for fragment screening in drug discovery. Copyright © 2018 Elsevier Inc. All rights reserved.

Recent developments and applications of saturation transfer difference nuclear magnetic resonance (STD NMR) spectroscopy.

PubMed

Wagstaff, Jane L; Taylor, Samantha L; Howard, Mark J

2013-04-05

This review aims to illustrate that STD NMR is not simply a method for drug screening and discovery, but has qualitative and quantitative applications that can answer fundamental and applied biological and biomedical questions involving molecular interactions between ligands and proteins. We begin with a basic introduction to the technique of STD NMR and report on recent advances and biological applications of STD including studies to follow the interactions of non-steroidal anti-inflammatories, minimum binding requirements for virus infection and understating inhibition of amyloid fibre formation. We expand on this introduction by reporting recent STD NMR studies of live-cell receptor systems, new methodologies using scanning STD, magic-angle spinning STD and approaches to use STD NMR in a quantitative fashion for dissociation constants and group epitope mapping (GEM) determination. We finish by outlining new approaches that have potential to influence future applications of the technique; NMR isotope-editing, heteronuclear multidimensional STD and (19)F STD methods that are becoming more amenable due to the latest NMR equipment technologies.

Proton magnetic resonance imaging with para-hydrogen induced polarization.

PubMed

Dechent, Jan F; Buljubasich, Lisandro; Schreiber, Laura M; Spiess, Hans W; Münnemann, Kerstin

2012-02-21

A major challenge in imaging is the detection of small amounts of molecules of interest. In the case of magnetic resonance imaging (MRI) their signals are typically concealed by the large background signal of e.g. the body. This problem can be tackled by hyperpolarization which increases the NMR signals up to several orders of magnitude. However, this strategy is limited for (1)H, the most widely used nucleus in NMR and MRI, because the enormous number of protons in the body screens the small amount of hyperpolarized ones. Here, we describe a method giving rise to high (1)H MRI contrast for hyperpolarized molecules against a large background signal. The contrast is based on the J-coupling induced rephasing of the NMR signal of molecules hyperpolarized via PHIP and it can easily be implemented in common pulse sequences. We discuss several scenarios with different or equal dephasing times T(2)* for the hyperpolarized and thermally polarized compounds and verify our approach by experiments. This method may open up unprecedented opportunities to use the standard MRI nucleus (1)H for e.g. metabolic imaging in the future.

Counterfeit Adderall Containing Aceclofenac from Internet Pharmacies.

PubMed

Gaudiano, Maria Cristina; Borioni, Anna; Antoniella, Eleonora; Valvo, Luisa

2016-07-01

A nontargeted approach based on liquid chromatography equipped with a quadrupole time-of-flight mass detector (LC-MS Q-TOF) joined to nuclear magnetic resonance (NMR) analysis allowed rapid identification and quantification of the anti-inflammatory drug aceclofenac in illegal Adderall tablets. The largest chromatographic peak had m/z = 354.030 and m/z = 376.012 matching, respectively, the ionic structures (M + H) + and (M + Na) + of a molecule M. The accurate mass data generated the molecular formula C 16 H 13 Cl 2 NO 4 . A screening of the pharmaceutical active substances having that molecular formula together with the MS/MS fragmentation pattern suggested aceclofenac. Aceclofenac structure was unambiguously confirmed by 1 H and 13 C NMR experiments. The aceclofenac content was 90 mg/tablet (RSD 2%) as detected by quantitative NMR. Information on the identity and content of illegal drugs is required for legal purposes; it supports in evaluating the effective impact on users safety, and it is useful for control laboratories using a targeted approach in their analytical activities. © 2016 American Academy of Forensic Sciences.

Fluid-Rock Characterization and Interactions in NMR Well Logging

DOE Office of Scientific and Technical Information (OSTI.GOV)

George J. Hirasaki; Kishore K. Mohanty

2005-09-05

The objective of this report is to characterize the fluid properties and fluid-rock interactions that are needed for formation evaluation by NMR well logging. The advances made in the understanding of NMR fluid properties are summarized in a chapter written for an AAPG book on NMR well logging. This includes live oils, viscous oils, natural gas mixtures, and the relation between relaxation time and diffusivity. Oil based drilling fluids can have an adverse effect on NMR well logging if it alters the wettability of the formation. The effect of various surfactants on wettability and surface relaxivity are evaluated for silicamore » sand. The relation between the relaxation time and diffusivity distinguishes the response of brine, oil, and gas in a NMR well log. A new NMR pulse sequence in the presence of a field gradient and a new inversion technique enables the T{sub 2} and diffusivity distributions to be displayed as a two-dimensional map. The objectives of pore morphology and rock characterization are to identify vug connectivity by using X-ray CT scan, and to improve NMR permeability correlation. Improved estimation of permeability from NMR response is possible by using estimated tortuosity as a parameter to interpolate between two existing permeability models.« less

Identification of Imitation Cheese and Imitation Ice Cream Based on Vegetable Fat Using NMR Spectroscopy and Chemometrics

PubMed Central

Monakhova, Yulia B.; Godelmann, Rolf; Andlauer, Claudia; Kuballa, Thomas; Lachenmeier, Dirk W.

2013-01-01

Vegetable oils and fats may be used as cheap substitutes for milk fat to manufacture imitation cheese or imitation ice cream. In this study, 400 MHz nuclear magnetic resonance (NMR) spectroscopy of the fat fraction of the products was used in the context of food surveillance to validate the labeling of milk-based products. For sample preparation, the fat was extracted using an automated Weibull-Stoldt methodology. Using principal component analysis (PCA), imitation products can be easily detected. In both cheese and ice cream, a differentiation according to the type of raw material (milk fat and vegetable fat) was possible. The loadings plot shows that imitation products were distinguishable by differences in their fatty acid ratios. Furthermore, a differentiation of several types of cheese (Edamer, Gouda, Emmentaler, and Feta) was possible. Quantitative data regarding the composition of the investigated products can also be predicted from the same spectra using partial least squares (PLS) regression. The models obtained for 13 compounds in cheese (R 2 0.75–0.95) and 17 compounds in ice cream (R 2 0.83–0.99) (e.g., fatty acids and esters) were suitable for a screening analysis. NMR spectroscopy was judged as suitable for the routine analysis of dairy products based on milk or on vegetable fat substitutes. PMID:26904597

School-Based Screening to Identify At-Risk Students Not Already Known to School Professionals: The Columbia Suicide Screen

PubMed Central

Wilcox, Holly C.; Schonfeld, Irvin Sam; Davies, Mark; Hicks, Roger C.; Turner, J. Blake; Shaffer, David

2009-01-01

Objectives. We sought to determine the degree of overlap between students identified through school-based suicide screening and those thought to be at risk by school administrative and clinical professionals. Methods. Students from 7 high schools in the New York metropolitan area completed the Columbia Suicide Screen; 489 of the 1729 students screened had positive results. The clinical status of 641 students (73% of those who had screened positive and 23% of those who had screened negative) was assessed with modules from the Diagnostic Interview Schedule for Children. School professionals nominated by their principal and unaware of students' screening and diagnostic status were asked to indicate whether they were concerned about the emotional well-being of each participating student. Results. Approximately 34% of students with significant mental health problems were identified only through screening, 13.0% were identified only by school professionals, 34.9% were identified both through screening and by school professionals, and 18.3% were identified neither through screening nor by school professionals. The corresponding percentages among students without mental health problems were 9.1%, 24.0%, 5.5%, and 61.3%. Conclusions. School-based screening can identify suicidal and emotionally troubled students not recognized by school professionals. PMID:19059865

Developing advanced X-ray scattering methods combined with crystallography and computation.

PubMed

Perry, J Jefferson P; Tainer, John A

2013-03-01

The extensive use of small angle X-ray scattering (SAXS) over the last few years is rapidly providing new insights into protein interactions, complex formation and conformational states in solution. This SAXS methodology allows for detailed biophysical quantification of samples of interest. Initial analyses provide a judgment of sample quality, revealing the potential presence of aggregation, the overall extent of folding or disorder, the radius of gyration, maximum particle dimensions and oligomerization state. Structural characterizations include ab initio approaches from SAXS data alone, and when combined with previously determined crystal/NMR, atomistic modeling can further enhance structural solutions and assess validity. This combination can provide definitions of architectures, spatial organizations of protein domains within a complex, including those not determined by crystallography or NMR, as well as defining key conformational states of a protein interaction. SAXS is not generally constrained by macromolecule size, and the rapid collection of data in a 96-well plate format provides methods to screen sample conditions. This includes screening for co-factors, substrates, differing protein or nucleotide partners or small molecule inhibitors, to more fully characterize the variations within assembly states and key conformational changes. Such analyses may be useful for screening constructs and conditions to determine those most likely to promote crystal growth of a complex under study. Moreover, these high throughput structural determinations can be leveraged to define how polymorphisms affect assembly formations and activities. This is in addition to potentially providing architectural characterizations of complexes and interactions for systems biology-based research, and distinctions in assemblies and interactions in comparative genomics. Thus, SAXS combined with crystallography/NMR and computation provides a unique set of tools that should be considered as being part of one's repertoire of biophysical analyses, when conducting characterizations of protein and other macromolecular interactions. Copyright © 2013 Elsevier Inc. All rights reserved.

Enriching the biological space of natural products and charting drug metabolites, through real time biotransformation monitoring: The NMR tube bioreactor.

PubMed

Chatzikonstantinou, Alexandra V; Chatziathanasiadou, Maria V; Ravera, Enrico; Fragai, Marco; Parigi, Giacomo; Gerothanassis, Ioannis P; Luchinat, Claudio; Stamatis, Haralambos; Tzakos, Andreas G

2018-01-01

Natural products offer a wide range of biological activities, but they are not easily integrated in the drug discovery pipeline, because of their inherent scaffold intricacy and the associated complexity in their synthetic chemistry. Enzymes may be used to perform regioselective and stereoselective incorporation of functional groups in the natural product core, avoiding harsh reaction conditions, several protection/deprotection and purification steps. Herein, we developed a three step protocol carried out inside an NMR-tube. 1st-step: STD-NMR was used to predict the: i) capacity of natural products as enzyme substrates and ii) possible regioselectivity of the biotransformations. 2nd-step: The real-time formation of multiple-biotransformation products in the NMR-tube bioreactor was monitored in-situ. 3rd-step: STD-NMR was applied in the mixture of the biotransformed products to screen ligands for protein targets. Herein, we developed a simple and time-effective process, the "NMR-tube bioreactor", that is able to: (i) predict which component of a mixture of natural products can be enzymatically transformed, (ii) monitor in situ the transformation efficacy and regioselectivity in crude extracts and multiple substrate biotransformations without fractionation and (iii) simultaneously screen for interactions of the biotransformation products with pharmaceutical protein targets. We have developed a green, time-, and cost-effective process that provide a simple route from natural products to lead compounds for drug discovery. This process can speed up the most crucial steps in the early drug discovery process, and reduce the chemical manipulations usually involved in the pipeline, improving the environmental compatibility. Copyright © 2017 Elsevier B.V. All rights reserved.

Fragment-Based Optimization of Small Molecule CXCL12 Inhibitors for Antagonizing the CXCL12/CXCR4 Interaction

PubMed Central

Ziarek, Joshua J.; Liu, Yan; Smith, Emmanuel; Zhang, Guolin; Peterson, Francis C.; Chen, Jun; Yu, Yongping; Chen, Yu; Volkman, Brian F.; Li, Rongshi

2013-01-01

The chemokine CXCL12 and its G protein-coupled receptor (GPCR) CXCR4 are high-priority clinical targets because of their involvement in metastatic cancers (also implicated in autoimmune disease and cardiovascular disease). Because chemokines interact with two distinct sites to bind and activate their receptors, both the GPCRs and chemokines are potential targets for small molecule inhibition. A number of chemokines have been validated as targets for drug development, but virtually all drug discovery efforts focus on the GPCRs. However, all CXCR4 receptor antagonists with the exception of MSX-122 have failed in clinical trials due to unmanageable toxicities, emphasizing the need for alternative strategies to interfere with CXCL12/CXCR4-guided metastatic homing. Although targeting the relatively featureless surface of CXCL12 was presumed to be challenging, focusing efforts at the sulfotyrosine (sY) binding pockets proved successful for procuring initial hits. Using a hybrid structure-based in silico/NMR screening strategy, we recently identified a ligand that occludes the receptor recognition site. From this initial hit, we designed a small fragment library containing only nine tetrazole derivatives using a fragment-based and bioisostere approach to target the sY binding sites of CXCL12. Compound binding modes and affinities were studied by 2D NMR spectroscopy, X-ray crystallography, molecular docking and cell-based functional assays. Our results demonstrate that the sY binding sites are conducive to the development of high affinity inhibitors with better ligand efficiency (LE) than typical protein-protein interaction inhibitors (LE ≤ 0.24). Our novel tetrazole-based fragment 18 was identified to bind the sY21 site with a Kd of 24 μM (LE = 0.30). Optimization of 18 yielded compound 25 which specifically inhibits CXCL12-induced migration with an improvement in potency over the initial hit 9. The fragment from this library that exhibited the highest affinity and ligand efficiency (11: Kd = 13 μM, LE = 0.33) may serve as a starting point for development of inhibitors targeting the sY12 site. PMID:23368099

[Chemical-genetics based screening for furanonaphthoquinone producing endophytic actinomycetes from seeds of Trewia nudiflora].

PubMed

Li, Fang; Kang, Qianjin; Yao, Xiaoling; Li, Yanyan; Wei, Maolong; Cao, Yong; Lin, Shuangjun; Bai, Linquan; Ma, Wei; Deng, Zixin

2012-04-04

The seeds of Trewia nudiflora containing maytansine (an anticancer agent), was investigated to explore the endophytic actinomycetes diversity and screen for naphthoquinones producing strain. The seeds of Trewia nudiflora were sliced and plated on different selective media after surface sterilization. Clones that looked like actinomycetes were selected, and classified according to the 16S rRNA sequences. Isolated strains were screened for furanonaphthoquinone biosynthesis gene by PCR, and tested for antibacterial and antifungal activity using Staphyloccocusaureus, Pseudomon-asaeruginosa, Bacillus subtilis, Rhizoctoniasolani and Gibberellasaubinetii. LC-MS and NMR were used to determine the structure of candidate compounds. More than 100 endophytic bacteria were isolated. Among them 66 were streptomycetes. FNQ6 (polyketide synthase Type III) and FNQ21 (carboxymuconate cycloisomerase) were only detected in Streptomyces sp. HTZ 27. We got 5 mg pure furanonaphthoquinone (FNQI) from 1 liter Streptomyces sp. HTZ 27 agar fermentation medium. The use of chemical-genetics method increased the efficiency of screening for target compound producing bacteria.

A rapid NMR-based method for discrimination of strain-specific cell wall teichoic acid structures reveals a third backbone type in Lactobacillus plantarum.

PubMed

Tomita, Satoru; Tanaka, Naoto; Okada, Sanae

2017-03-01

The lactic acid bacterium Lactobacillus plantarum is capable of producing strain-specific structures of cell wall teichoic acid (WTA), an anionic polysaccharide found in the Gram-positive bacterial cell wall. In this study, we established a rapid, NMR-based procedure to discriminate WTA structures in this species, and applied it to 94 strains of L. plantarum. Six previously reported glycerol- and ribitol-containing WTA subtypes were successfully identified from 78 strains, suggesting that these were the dominant structures. However, the level of structural variety differed markedly among bacterial sources, possibly reflecting differences in strain-level microbial diversity. WTAs from eight strains were not identified based on NMR spectra and were classified into three groups. Structural analysis of a partial degradation product of an unidentified WTA produced by strain TUA 1496L revealed that the WTA was 1-O-β-d-glucosylglycerol. Two-dimensional NMR analysis of the polymer structure showed phosphodiester bonds between C-3 and C-6 of the glycerol and glucose residues, suggesting a polymer structure of 3,6΄-linked poly(1-O-β-d-glucosyl-sn-glycerol phosphate). This is the third WTA backbone structure in L. plantarum, following 3,6΄-linked poly(1-O-α-d-glucosyl-sn-glycerol phosphate) and 1,5-linked poly(ribitol phosphate). © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Insecticidal peptides from the theraposid spider Brachypelma albiceps: an NMR-based model of Ba2.

PubMed

Corzo, Gerardo; Bernard, Cedric; Clement, Herlinda; Villegas, Elba; Bosmans, Frank; Tytgat, Jan; Possani, Lourival D; Darbon, Herve; Alagón, Alejandro

2009-08-01

Soluble venom and purified fractions of the theraposid spider Brachypelma albiceps were screened for insecticidal peptides based on toxicity to crickets. Two insecticidal peptides, named Ba1 and Ba2, were obtained after the soluble venom was separated by high performance liquid chromatography and cation exchange chromatography. The two insecticidal peptides contain 39 amino acid residues and three disulfide bonds, and based on their amino acid sequence, they are highly identical to the insecticidal peptides from the theraposid spiders Aphonopelma sp. from the USA and Haplopelma huwenum from China indicating a relationship among these genera. Although Ba1 and Ba2 were not able to modify currents in insect and vertebrate cloned voltage-gated sodium ion channels, they have noteworthy insecticidal activities compared to classical arachnid insecticidal toxins indicating that they might target unknown receptors in insect species. The most abundant insecticidal peptide Ba2 was submitted to NMR spectroscopy to determine its 3-D structure; a remarkable characteristic of Ba2 is a cluster of basic residues, which might be important for receptor recognition.

DNA cleavage, antibacterial, antifungal and anthelmintic studies of Co(II), Ni(II) and Cu(II) complexes of coumarin Schiff bases: Synthesis and spectral approach

NASA Astrophysics Data System (ADS)

Patil, Sangamesh A.; Prabhakara, Chetan T.; Halasangi, Bhimashankar M.; Toragalmath, Shivakumar S.; Badami, Prema S.

2015-02-01

The metal complexes of Co(II), Ni(II) and Cu(II) have been synthesized from 6-formyl-7,8-dihydroxy-4-methylcoumarin with o-toluidine/3-aminobenzotrifluoride. The synthesized Schiff bases and their metal complexes were structurally characterized based on IR, 1H NMR, 13C NMR, UV-visible, ESR, magnetic, thermal, fluorescence, mass and ESI-MS studies. The molar conductance values indicate that complexes are non-electrolytic in nature. Elemental analysis reveals ML2·2H2O [M = Co(II), Ni(II) and Cu(II)] stoichiometry, where 'L' stands for a singly deprotonated ligand. The presence of co-ordinated water molecules were confirmed by thermal studies. The spectroscopic studies suggest the octahedral geometry. Redox behavior of the complexes were confirmed by cyclic voltammetry. All the synthesized compounds were screened for their antibacterial (Escherichia coli, Pseudomonas auregenosa, klebsiella, Proteus, Staphylococcus aureus and salmonella) antifungal (Candida, Aspergillus niger and Rhizopus), anthelmintic (Pheretima posthuma) and DNA cleavage (Calf Thymus DNA) activity.

DNA cleavage, antibacterial, antifungal and anthelmintic studies of Co(II), Ni(II) and Cu(II) complexes of coumarin Schiff bases: synthesis and spectral approach.

PubMed

Patil, Sangamesh A; Prabhakara, Chetan T; Halasangi, Bhimashankar M; Toragalmath, Shivakumar S; Badami, Prema S

2015-02-25

The metal complexes of Co(II), Ni(II) and Cu(II) have been synthesized from 6-formyl-7,8-dihydroxy-4-methylcoumarin with o-toluidine/3-aminobenzotrifluoride. The synthesized Schiff bases and their metal complexes were structurally characterized based on IR, (1)H NMR, (13)C NMR, UV-visible, ESR, magnetic, thermal, fluorescence, mass and ESI-MS studies. The molar conductance values indicate that complexes are non-electrolytic in nature. Elemental analysis reveals ML2·2H2O [M = Co(II), Ni(II) and Cu(II)] stoichiometry, where 'L' stands for a singly deprotonated ligand. The presence of co-ordinated water molecules were confirmed by thermal studies. The spectroscopic studies suggest the octahedral geometry. Redox behavior of the complexes were confirmed by cyclic voltammetry. All the synthesized compounds were screened for their antibacterial (Escherichia coli, Pseudomonas auregenosa, klebsiella, Proteus, Staphylococcus aureus and salmonella) antifungal (Candida, Aspergillus niger and Rhizopus), anthelmintic (Pheretima posthuma) and DNA cleavage (Calf Thymus DNA) activity. Copyright © 2014 Elsevier B.V. All rights reserved.

Integration of 1H NMR and UPLC-Q-TOF/MS for a Comprehensive Urinary Metabonomics Study on a Rat Model of Depression Induced by Chronic Unpredictable Mild Stress

PubMed Central

Jia, Hong-mei; Feng, Yu-fei; Liu, Yue-tao; Chang, Xing; Chen, Lin; Zhang, Hong-wu; Ding, Gang; Zou, Zhong-mei

2013-01-01

Depression is a type of complex psychiatric disorder with long-term, recurrent bouts, and its etiology remains largely unknown. Here, an integrated approach utilizing 1H NMR and UPLC-Q-TOF/MS together was firstly used for a comprehensive urinary metabonomics study on chronic unpredictable mild stress (CUMS) treated rats. More than twenty-nine metabolic pathways were disturbed after CUMS treatment and thirty-six potential biomarkers were identified by using two complementary analytical technologies. Among the identified biomarkers, nineteen (10, 11, 16, 17, 21–25, and 27–36) were firstly reported as potential biomarkers of CUMS-induced depression. Obviously, this paper presented a comprehensive map of the metabolic pathways perturbed by CUMS and expanded on the multitude of potential biomarkers that have been previously reported in the CUMS model. Four metabolic pathways, including valine, leucine and isoleucine biosynthesis; phenylalanine, tyrosine and tryptophan biosynthesis; tryptophan metabolism; synthesis and degradation of ketone bodies had the deepest influence in the pathophysiologic process of depression. Fifteen potential biomarkers (1–2, 4–6, 15, 18, 20–23, 27, 32, 35–36) involved in the above four metabolic pathways might become the screening criteria in clinical diagnosis and predict the development of depression. Moreover, the results of Western blot analysis of aromatic L-amino acid decarboxylase (DDC) and indoleamine 2, 3-dioxygenase (IDO) in the hippocampus of CUMS-treated rats indicated that depletion of 5-HT and tryptophan, production of 5-MT and altered expression of DDC and IDO together played a key role in the initiation and progression of depression. In addition, none of the potential biomarkers were detected by NMR and LC-MS simultaneously which indicated the complementary of the two kinds of detection technologies. Therefore, the integration of 1H NMR and UPLC-Q-TOF/MS in metabonomics study provided an approach to identify the comprehensive potential depression-related biomarkers and helpful in further understanding the underlying molecular mechanisms of depression through the disturbance of metabolic pathways. PMID:23696839

Application of NMR Methods to Identify Detection Reagents for Use in the Development of Robust Nanosensors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cosman, M; Krishnan, V V; Balhorn, R

2004-04-29

Nuclear Magnetic Resonance (NMR) spectroscopy is a powerful technique for studying bi-molecular interactions at the atomic scale. Our NMR lab is involved in the identification of small molecules, or ligands that bind to target protein receptors, such as tetanus (TeNT) and botulinum (BoNT) neurotoxins, anthrax proteins and HLA-DR10 receptors on non-Hodgkin's lymphoma cancer cells. Once low affinity binders are identified, they can be linked together to produce multidentate synthetic high affinity ligands (SHALs) that have very high specificity for their target protein receptors. An important nanotechnology application for SHALs is their use in the development of robust chemical sensors ormore » biochips for the detection of pathogen proteins in environmental samples or body fluids. Here, we describe a recently developed NMR competition assay based on transferred nuclear Overhauser effect spectroscopy (trNOESY) that enables the identification of sets of ligands that bind to the same site, or a different site, on the surface of TeNT fragment C (TetC) than a known ''marker'' ligand, doxorubicin. Using this assay, we can identify the optimal pairs of ligands to be linked together for creating detection reagents, as well as estimate the relative binding constants for ligands competing for the same site.« less

Rapid Identification of Candida Species by Using Nuclear Magnetic Resonance Spectroscopy and a Statistical Classification Strategy

PubMed Central

Himmelreich, Uwe; Somorjai, Ray L.; Dolenko, Brion; Lee, Ok Cha; Daniel, Heide-Marie; Murray, Ronan; Mountford, Carolyn E.; Sorrell, Tania C.

2003-01-01

Nuclear magnetic resonance (NMR) spectra were acquired from suspensions of clinically important yeast species of the genus Candida to characterize the relationship between metabolite profiles and species identification. Major metabolites were identified by using two-dimensional correlation NMR spectroscopy. One-dimensional proton NMR spectra were analyzed by using a staged statistical classification strategy. Analysis of NMR spectra from 442 isolates of Candida albicans, C. glabrata, C. krusei, C. parapsilosis, and C. tropicalis resulted in rapid, accurate identification when compared with conventional and DNA-based identification. Spectral regions used for the classification of the five yeast species revealed species-specific differences in relative amounts of lipids, trehalose, polyols, and other metabolites. Isolates of C. parapsilosis and C. glabrata with unusual PCR fingerprinting patterns also generated atypical NMR spectra, suggesting the possibility of intraspecies discontinuity. We conclude that NMR spectroscopy combined with a statistical classification strategy is a rapid, nondestructive, and potentially valuable method for identification and chemotaxonomic characterization that may be broadly applicable to fungi and other microorganisms. PMID:12902244

Design and Development of ChemInfoCloud: An Integrated Cloud Enabled Platform for Virtual Screening.

PubMed

Karthikeyan, Muthukumarasamy; Pandit, Deepak; Bhavasar, Arvind; Vyas, Renu

2015-01-01

The power of cloud computing and distributed computing has been harnessed to handle vast and heterogeneous data required to be processed in any virtual screening protocol. A cloud computing platorm ChemInfoCloud was built and integrated with several chemoinformatics and bioinformatics tools. The robust engine performs the core chemoinformatics tasks of lead generation, lead optimisation and property prediction in a fast and efficient manner. It has also been provided with some of the bioinformatics functionalities including sequence alignment, active site pose prediction and protein ligand docking. Text mining, NMR chemical shift (1H, 13C) prediction and reaction fingerprint generation modules for efficient lead discovery are also implemented in this platform. We have developed an integrated problem solving cloud environment for virtual screening studies that also provides workflow management, better usability and interaction with end users using container based virtualization, OpenVz.

Design of a multi-purpose fragment screening library using molecular complexity and orthogonal diversity metrics

NASA Astrophysics Data System (ADS)

Lau, Wan F.; Withka, Jane M.; Hepworth, David; Magee, Thomas V.; Du, Yuhua J.; Bakken, Gregory A.; Miller, Michael D.; Hendsch, Zachary S.; Thanabal, Venkataraman; Kolodziej, Steve A.; Xing, Li; Hu, Qiyue; Narasimhan, Lakshmi S.; Love, Robert; Charlton, Maura E.; Hughes, Samantha; van Hoorn, Willem P.; Mills, James E.

2011-07-01

Fragment Based Drug Discovery (FBDD) continues to advance as an efficient and alternative screening paradigm for the identification and optimization of novel chemical matter. To enable FBDD across a wide range of pharmaceutical targets, a fragment screening library is required to be chemically diverse and synthetically expandable to enable critical decision making for chemical follow-up and assessing new target druggability. In this manuscript, the Pfizer fragment library design strategy which utilized multiple and orthogonal metrics to incorporate structure, pharmacophore and pharmacological space diversity is described. Appropriate measures of molecular complexity were also employed to maximize the probability of detection of fragment hits using a variety of biophysical and biochemical screening methods. In addition, structural integrity, purity, solubility, fragment and analog availability as well as cost were important considerations in the selection process. Preliminary analysis of primary screening results for 13 targets using NMR Saturation Transfer Difference (STD) indicates the identification of uM-mM hits and the uniqueness of hits at weak binding affinities for these targets.

Design of a multi-purpose fragment screening library using molecular complexity and orthogonal diversity metrics.

PubMed

Lau, Wan F; Withka, Jane M; Hepworth, David; Magee, Thomas V; Du, Yuhua J; Bakken, Gregory A; Miller, Michael D; Hendsch, Zachary S; Thanabal, Venkataraman; Kolodziej, Steve A; Xing, Li; Hu, Qiyue; Narasimhan, Lakshmi S; Love, Robert; Charlton, Maura E; Hughes, Samantha; van Hoorn, Willem P; Mills, James E

2011-07-01

Fragment Based Drug Discovery (FBDD) continues to advance as an efficient and alternative screening paradigm for the identification and optimization of novel chemical matter. To enable FBDD across a wide range of pharmaceutical targets, a fragment screening library is required to be chemically diverse and synthetically expandable to enable critical decision making for chemical follow-up and assessing new target druggability. In this manuscript, the Pfizer fragment library design strategy which utilized multiple and orthogonal metrics to incorporate structure, pharmacophore and pharmacological space diversity is described. Appropriate measures of molecular complexity were also employed to maximize the probability of detection of fragment hits using a variety of biophysical and biochemical screening methods. In addition, structural integrity, purity, solubility, fragment and analog availability as well as cost were important considerations in the selection process. Preliminary analysis of primary screening results for 13 targets using NMR Saturation Transfer Difference (STD) indicates the identification of uM-mM hits and the uniqueness of hits at weak binding affinities for these targets.

One-pot microwave assisted synthesis under green chemistry conditions, antioxidant screening, and cytotoxicity assessments of benzimidazole Schiff bases and pyrimido[1,2-a]benzimidazol-3(4H)-ones.

PubMed

Neochoritis, Constantinos G; Zarganes-Tzitzikas, Tryfon; Tsoleridis, Constantinos A; Stephanidou-Stephanatou, Julia; Kontogiorgis, Christos A; Hadjipavlou-Litina, Dimitra J; Choli-Papadopoulou, Theodora

2011-01-01

The synthesis of a number of benzimidazole Schiff bases 3 and 3-oxo-pyrimido[1,2-a]benzimidazoles 4 in excellent yields by a one-step sequence from the reaction of 2-aminobenzimidazole under green chemistry conditions is described. Structural assignments of the new compounds as well as complete assignment of (1)H and (13)C NMR signals have been unambiguously achieved based on the analysis of their (1)H and (13)C NMR (1D and 2D), IR, MS and elemental analysis data. To the synthesized Schiff bases the E-configuration was assigned on the basis of comparison of experimental and calculated (DFT) (13)C NMR chemical shifts. Compounds 3 and 4 were evaluated as inhibitors of lipoxygenase (LOX) and of lipid peroxidation (LPO). All the tested derivatives showed inhibition of lipid peroxidation, whereas most of them were found to have higher activation than the reference compound trolox; The Schiff bases 3e, 3h, and 3i, and the pyrimidobenzimidazoles 4a, 4e and 4f were found to be the most potent. The most potent LOX inhibitor within the subset of Schiff bases was found compound 3i, followed by 3f, whereas compounds 4a and 4g were found the most potent of the 3-oxo-pyrimido[1,2-a]benzimidazole group. Moreover, some cytotoxicity assessments were undertaken, whereupon it was found that Schiff base 3i and pyrimidobenzimidazoles 4e and 4f did not exhibit cytotoxicity at similar concentrations resembling thus the inhibitory activity of lipid peroxidation. The most cytotoxic Schiff base and pyrimidobenzimidazole were found to be 3d and 4c, respectively. Copyright © 2010 Elsevier Masson SAS. All rights reserved.

Single-scan 2D NMR: An Emerging Tool in Analytical Spectroscopy

PubMed Central

Giraudeau, Patrick; Frydman, Lucio

2016-01-01

Two-dimensional Nuclear Magnetic Resonance (2D NMR) spectroscopy is widely used in chemical and biochemical analyses. Multidimensional NMR is also witnessing an increased use in quantitative and metabolic screening applications. Conventional 2D NMR experiments, however, are affected by inherently long acquisition durations, arising from their need to sample the frequencies involved along their indirect domains in an incremented, scan-by-scan nature. A decade ago a so-called “ultrafast” (UF) approach was proposed, capable to deliver arbitrary 2D NMR spectra involving any kind of homo- or hetero-nuclear correlations, in a single scan. During the intervening years the performance of this sub-second 2D NMR methodology has been greatly improved, and UF 2D NMR is rapidly becoming a powerful analytical tool witnessing an expanded scope of applications. The present reviews summarizes the principles and the main developments which have contributed to the success of this approach, and focuses on applications which have been recently demonstrated in various areas of analytical chemistry –from the real time monitoring of chemical and biochemical processes, to extensions in hyphenated techniques and in quantitative applications. PMID:25014342

Synthesis of novel ferrocenyl Mannich bases and their antibacterial activities

NASA Astrophysics Data System (ADS)

Liu, Yuting; Xin, Hong; Yin, Jingyi; Yin, Dawei; Yang, Zijiang; Li, Jie

2018-04-01

In this work, a series of Mannich bases bearing ferrocenyl groups were synthesized via Mannich reactions. The effects of different conditions on the reaction were explored, and the newly synthesized compounds were well characterized by NMR and FT-IR. All compounds have been screened for in vitro antibacterial and antifungal, and the compounds 1-ferrocenyl-3-phenyl-3-(m-nitrophenylamino)-1-acetone (2f) and 1-ferrocenyl-3-phenyl-3-(p-nitrophenylamino)-1-acetone (2g) were shown to be significant activity against all the tested bacterial strains, such as Staphylococcus aureus, Streptococcus, Actinomycete, Escherichia coli, Saccharomyces cerevisiae.

Identification of Multiple Druggable Secondary Sites by Fragment Screening against DC-SIGN.

PubMed

Aretz, Jonas; Baukmann, Hannes; Shanina, Elena; Hanske, Jonas; Wawrzinek, Robert; Zapol'skii, Viktor A; Seeberger, Peter H; Kaufmann, Dieter E; Rademacher, Christoph

2017-06-12

DC-SIGN is a cell-surface receptor for several pathogenic threats, such as HIV, Ebola virus, or Mycobacterium tuberculosis. Multiple attempts to develop inhibitors of the underlying carbohydrate-protein interactions have been undertaken in the past fifteen years. Still, drug-like DC-SIGN ligands are sparse, which is most likely due to its hydrophilic, solvent-exposed carbohydrate-binding site. Herein, we report on a parallel fragment screening against DC-SIGN applying SPR and a reporter displacement assay, which complements previous screenings using 19 F NMR spectroscopy and chemical fragment microarrays. Hit validation by SPR and 1 H- 15 N HSQC NMR spectroscopy revealed that although no fragment bound in the primary carbohydrate site, five secondary sites are available to harbor drug-like molecules. Building on key interactions of the reported fragment hits, these pockets will be targeted in future approaches to accelerate the development of DC-SIGN inhibitors. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Unambiguous metabolite identification in high-throughput metabolomics by hybrid 1D 1 H NMR/ESI MS 1 approach: Hybrid 1D 1 H NMR/ESI MS 1 metabolomics method

DOE Office of Scientific and Technical Information (OSTI.GOV)

Walker, Lawrence R.; Hoyt, David W.; Walker, S. Michael

We present a novel approach to improve accuracy of metabolite identification by combining direct infusion ESI MS1 with 1D 1H NMR spectroscopy. The new approach first applies standard 1D 1H NMR metabolite identification protocol by matching the chemical shift, J-coupling and intensity information of experimental NMR signals against the NMR signals of standard metabolites in metabolomics library. This generates a list of candidate metabolites. The list contains false positive and ambiguous identifications. Next, we constrained the list with the chemical formulas derived from high-resolution direct infusion ESI MS1 spectrum of the same sample. Detection of the signals of a metabolitemore » both in NMR and MS significantly improves the confidence of identification and eliminates false positive identification. 1D 1H NMR and direct infusion ESI MS1 spectra of a sample can be acquired in parallel in several minutes. This is highly beneficial for rapid and accurate screening of hundreds of samples in high-throughput metabolomics studies. In order to make this approach practical, we developed a software tool, which is integrated to Chenomx NMR Suite. The approach is demonstrated on a model mixture, tomato and Arabidopsis thaliana metabolite extracts, and human urine.« less

Advantages of Molecular Weight Identification during Native MS Screening.

PubMed

Khan, Ahad; Bresnick, Anne; Cahill, Sean; Girvin, Mark; Almo, Steve; Quinn, Ronald

2018-05-09

Native mass spectrometry detection of ligand-protein complexes allowed rapid detection of natural product binders of apo and calcium-bound S100A4 (a member of the metal binding protein S100 family), T cell/transmembrane, immunoglobulin (Ig), and mucin protein 3, and T cell immunoreceptor with Ig and ITIM (immunoreceptor tyrosine-based inhibitory motif) domains precursor protein from extracts and fractions. Based on molecular weight common hits were detected binding to all four proteins. Seven common hits were identified as apigenin 6- C - β - D -glucoside 8- C - α - L -arabinoside, sweroside, 4',5-dihydroxy-7-methoxyflavanone-6- C -rutinoside, loganin acid, 6- C -glucosylnaringenin, biochanin A 7- O -rutinoside and quercetin 3- O -rutinoside. Mass guided isolation and NMR identification of hits confirmed the mass accuracy of the ligand in the ligand-protein MS complexes. Thus, molecular weight ID from ligand-protein complexes by electrospray ionization Fourier transform mass spectrometry allowed rapid dereplication. Native mass spectrometry using electrospray ionization Fourier transform mass spectrometry is a tool for dereplication and metabolomics analysis. Georg Thieme Verlag KG Stuttgart · New York.

Insight into small molecule binding to the neonatal Fc receptor by X-ray crystallography and 100 kHz magic-angle-spinning NMR

PubMed Central

Macpherson, Alex; Smith-Penzel, Susanne; Basse, Nicolas; Lecomte, Fabien; Deboves, Hervé; Taylor, Richard D.; Norman, Tim; Porter, John; Waters, Lorna C.; Westwood, Marta; Cossins, Ben; Cain, Katharine; White, James; Griffin, Robert; Prosser, Christine; Kelm, Sebastian; Sullivan, Amy H.; Fox, David; Carr, Mark D.; Henry, Alistair; Taylor, Richard; Meier, Beat H.; Oschkinat, Hartmut; Lawson, Alastair D.

2018-01-01

Aiming at the design of an allosteric modulator of the neonatal Fc receptor (FcRn)–Immunoglobulin G (IgG) interaction, we developed a new methodology including NMR fragment screening, X-ray crystallography, and magic-angle-spinning (MAS) NMR at 100 kHz after sedimentation, exploiting very fast spinning of the nondeuterated soluble 42 kDa receptor construct to obtain resolved proton-detected 2D and 3D NMR spectra. FcRn plays a crucial role in regulation of IgG and serum albumin catabolism. It is a clinically validated drug target for the treatment of autoimmune diseases caused by pathogenic antibodies via the inhibition of its interaction with IgG. We herein present the discovery of a small molecule that binds into a conserved cavity of the heterodimeric, extracellular domain composed of an α-chain and β2-microglobulin (β2m) (FcRnECD, 373 residues). X-ray crystallography was used alongside NMR at 100 kHz MAS with sedimented soluble protein to explore possibilities for refining the compound as an allosteric modulator. Proton-detected MAS NMR experiments on fully protonated [13C,15N]-labeled FcRnECD yielded ligand-induced chemical-shift perturbations (CSPs) for residues in the binding pocket and allosteric changes close to the interface of the two receptor heterodimers present in the asymmetric unit as well as potentially in the albumin interaction site. X-ray structures with and without ligand suggest the need for an optimized ligand to displace the α-chain with respect to β2m, both of which participate in the FcRnECD–IgG interaction site. Our investigation establishes a method to characterize structurally small molecule binding to nondeuterated large proteins by NMR, even in their glycosylated form, which may prove highly valuable for structure-based drug discovery campaigns. PMID:29782488

Insight into small molecule binding to the neonatal Fc receptor by X-ray crystallography and 100 kHz magic-angle-spinning NMR.

PubMed

Stöppler, Daniel; Macpherson, Alex; Smith-Penzel, Susanne; Basse, Nicolas; Lecomte, Fabien; Deboves, Hervé; Taylor, Richard D; Norman, Tim; Porter, John; Waters, Lorna C; Westwood, Marta; Cossins, Ben; Cain, Katharine; White, James; Griffin, Robert; Prosser, Christine; Kelm, Sebastian; Sullivan, Amy H; Fox, David; Carr, Mark D; Henry, Alistair; Taylor, Richard; Meier, Beat H; Oschkinat, Hartmut; Lawson, Alastair D

2018-05-01

Aiming at the design of an allosteric modulator of the neonatal Fc receptor (FcRn)-Immunoglobulin G (IgG) interaction, we developed a new methodology including NMR fragment screening, X-ray crystallography, and magic-angle-spinning (MAS) NMR at 100 kHz after sedimentation, exploiting very fast spinning of the nondeuterated soluble 42 kDa receptor construct to obtain resolved proton-detected 2D and 3D NMR spectra. FcRn plays a crucial role in regulation of IgG and serum albumin catabolism. It is a clinically validated drug target for the treatment of autoimmune diseases caused by pathogenic antibodies via the inhibition of its interaction with IgG. We herein present the discovery of a small molecule that binds into a conserved cavity of the heterodimeric, extracellular domain composed of an α-chain and β2-microglobulin (β2m) (FcRnECD, 373 residues). X-ray crystallography was used alongside NMR at 100 kHz MAS with sedimented soluble protein to explore possibilities for refining the compound as an allosteric modulator. Proton-detected MAS NMR experiments on fully protonated [13C,15N]-labeled FcRnECD yielded ligand-induced chemical-shift perturbations (CSPs) for residues in the binding pocket and allosteric changes close to the interface of the two receptor heterodimers present in the asymmetric unit as well as potentially in the albumin interaction site. X-ray structures with and without ligand suggest the need for an optimized ligand to displace the α-chain with respect to β2m, both of which participate in the FcRnECD-IgG interaction site. Our investigation establishes a method to characterize structurally small molecule binding to nondeuterated large proteins by NMR, even in their glycosylated form, which may prove highly valuable for structure-based drug discovery campaigns.

Design of compound libraries for fragment screening

NASA Astrophysics Data System (ADS)

Blomberg, Niklas; Cosgrove, David A.; Kenny, Peter W.; Kolmodin, Karin

2009-08-01

Approaches to the design of libraries for fragment screening are illustrated with reference to a 20 k generic fragment screening library and a 1.2 k generic NMR screening library. Tools and methods for library design that have been developed within AstraZeneca are described, including Foyfi fingerprints and the Flush program for neighborhood characterization. It will be shown how Flush and the BigPicker, which selects maximally diverse sets of compounds, are used to apply the Core and Layer method for library design. Approaches to partitioning libraries into cocktails are also described.

Synthesis, characterization, antimicrobial, DNA-cleavage and antioxidant activities of 3-((5-chloro-2-phenyl-1H-indol-3-ylimino)methyl)quinoline-2(1H)-thione and its metal complexes

NASA Astrophysics Data System (ADS)

Vivekanand, B.; Mahendra Raj, K.; Mruthyunjayaswamy, B. H. M.

2015-01-01

Schiff base 3-((5-chloro-2-phenyl-1H-indol-3-ylimino)methyl)quinoline-2(1H)-thione and its Cu(II), Co(II), Ni(II), Zn(II) and Fe(III), complexes have been synthesized and characterized by elemental analysis, UV-Visible, IR, 1H NMR, 13C NMR and mass spectra, molar conductance, magnetic susceptibility, ESR and TGA data. The ligand and its metal complexes have been screened for their antibacterial activity against Staphylococcus aureus and Pseudomonas aeruginosa, antifungal activity against Aspergillus niger and Aspergillus flavus in minimum inhibition concentration (MIC) by cup plate method respectively, antioxidant activity using 1,1-diphenyl-2-picryl hydrazyl (DPPH), which was compared with that of standard drugs vitamin-C and vitamin-E and DNA cleavage activity using calf-thymus DNA.

A Systematic Evaluation of Field-Based Screening Methods for the Assessment of Anterior Cruciate Ligament (ACL) Injury Risk.

PubMed

Fox, Aaron S; Bonacci, Jason; McLean, Scott G; Spittle, Michael; Saunders, Natalie

2016-05-01

Laboratory-based measures provide an accurate method to identify risk factors for anterior cruciate ligament (ACL) injury; however, these methods are generally prohibitive to the wider community. Screening methods that can be completed in a field or clinical setting may be more applicable for wider community use. Examination of field-based screening methods for ACL injury risk can aid in identifying the most applicable method(s) for use in these settings. The objective of this systematic review was to evaluate and compare field-based screening methods for ACL injury risk to determine their efficacy of use in wider community settings. An electronic database search was conducted on the SPORTDiscus™, MEDLINE, AMED and CINAHL databases (January 1990-July 2015) using a combination of relevant keywords. A secondary search of the same databases, using relevant keywords from identified screening methods, was also undertaken. Studies identified as potentially relevant were independently examined by two reviewers for inclusion. Where consensus could not be reached, a third reviewer was consulted. Original research articles that examined screening methods for ACL injury risk that could be undertaken outside of a laboratory setting were included for review. Two reviewers independently assessed the quality of included studies. Included studies were categorized according to the screening method they examined. A description of each screening method, and data pertaining to the ability to prospectively identify ACL injuries, validity and reliability, recommendations for identifying 'at-risk' athletes, equipment and training required to complete screening, time taken to screen athletes, and applicability of the screening method across sports and athletes were extracted from relevant studies. Of 1077 citations from the initial search, a total of 25 articles were identified as potentially relevant, with 12 meeting all inclusion/exclusion criteria. From the secondary search, eight further studies met all criteria, resulting in 20 studies being included for review. Five ACL-screening methods-the Landing Error Scoring System (LESS), Clinic-Based Algorithm, Observational Screening of Dynamic Knee Valgus (OSDKV), 2D-Cam Method, and Tuck Jump Assessment-were identified. There was limited evidence supporting the use of field-based screening methods in predicting ACL injuries across a range of populations. Differences relating to the equipment and time required to complete screening methods were identified. Only screening methods for ACL injury risk were included for review. Field-based screening methods developed for lower-limb injury risk in general may also incorporate, and be useful in, screening for ACL injury risk. Limited studies were available relating to the OSDKV and 2D-Cam Method. The LESS showed predictive validity in identifying ACL injuries, however only in a youth athlete population. The LESS also appears practical for community-wide use due to the minimal equipment and set-up/analysis time required. The Clinic-Based Algorithm may have predictive value for ACL injury risk as it identifies athletes who exhibit high frontal plane knee loads during a landing task, but requires extensive additional equipment and time, which may limit its application to wider community settings.

A new cinnamic acid glycoside from roots of Heracleum dissectum.

PubMed

Wang, Zhi-Gang; Mi, Jie; Wang, Xin-Rui; Huo, Ya-Yu; Peng, Ya-Jie; Zhang, Hai-Min; Gao, Yang; Zhang, Hai-Long

2018-01-01

From the roots of Heracleum dissectum Lebb., one new cinnamic acid glycoside derivative named dissectumoside (1), together with eight known compounds including three phenolics, three phenolic glycosides and two phenylpropanoic glycoside were isolated using various chromatographic methods. Among them compound 2-9 was isolated from the plant for the first time. Their structures were elucidated and identified on the basis of their physicochemical properties and by extensive analyses of NMR spectroscopy and high-resolution mass spectrometry. The results of triglyceride accumulation screening in 3T3-L1 cells showed that compounds 1, 5 and 9 exhibited significantly accelerating activities of adipogenesis in adipocytes.

Synthesis of a novel BODIPY library and its application in the discovery of a fructose sensor.

PubMed

Zhai, Duanting; Lee, Sung-Chan; Vendrell, Marc; Leong, Lai Peng; Chang, Young-Tae

2012-02-13

We prepared a new library of 160 compounds by conjugation of a BODIPY core to a collection of aldehydes. This library was screened against 52 biologically relevant analytes and we identified one fluorescent sensor of fructose (Fructose Orange). Fructose Orange showed a 24-fold fluorescence increase upon recognition of fructose and an outstanding selectivity among 24 different saccharides. NMR studies confirmed that five different binding interactions were formed between the sensor and fructose. Furthermore, Fructose Orange was applied to the quantification of fructose in soft drinks, being the most selective fluorescent sensor for fructose reported to date.

Relativistic Zeroth-Order Regular Approximation Combined with Nonhybrid and Hybrid Density Functional Theory: Performance for NMR Indirect Nuclear Spin-Spin Coupling in Heavy Metal Compounds.

PubMed

Moncho, Salvador; Autschbach, Jochen

2010-01-12

A benchmark study for relativistic density functional calculations of NMR spin-spin coupling constants has been performed. The test set contained 47 complexes with heavy metal atoms (W, Pt, Hg, Tl, Pb) with a total of 88 coupling constants involving one or two heavy metal atoms. One-, two-, three-, and four-bond spin-spin couplings have been computed at different levels of theory (nonhybrid vs hybrid DFT, scalar vs two-component relativistic). The computational model was based on geometries fully optimized at the BP/TZP scalar relativistic zeroth-order regular approximation (ZORA) and the conductor-like screening model (COSMO) to include solvent effects. The NMR computations also employed the continuum solvent model. Computations in the gas phase were performed in order to assess the importance of the solvation model. The relative median deviations between various computational models and experiment were found to range between 13% and 21%, with the highest-level computational model (hybrid density functional computations including scalar plus spin-orbit relativistic effects, the COSMO solvent model, and a Gaussian finite-nucleus model) performing best.

Application of ¹H NMR for the characterisation and authentication of ''Tonda Gentile Trilobata" hazelnuts from Piedmont (Italy).

PubMed

Caligiani, Augusta; Coisson, Jean Daniel; Travaglia, Fabiano; Acquotti, Domenico; Palla, Gerardo; Palla, Luigi; Arlorio, Marco

2014-04-01

The Italian hazelnut (Corylus avellana L.) cultivar "Tonda Gentile Trilobata" (TGT) is covered by protected geographical indication "Nocciola Piemonte" and is well-known as the best-suited hazelnut for the industrial transformation into roasted kernel. The hazelnut cultivar identification is primarily based on morphological characteristics, so there is the need for more objective analytical methods for high quality hazelnut authentication. This study reports the (1)H NMR fingerprinting of raw and roasted hazelnut, with the aim of obtaining hazelnut classification based on their spectroscopic pattern. (1)H NMR analyses were carried out on polar extracts of TGT and other cultivars: the data were analysed with multivariate statistical methods. Results showed that (1)H NMR combined with chemometrics is useful to characterise the hazelnuts as a function of the cultivars, both on raw and roasted form. The classification models allowed identifying molecular markers useful to distinguish TGT from other types, among these trigonelline, amino acids and an unidentified orto-disubstituted aromatic compound. Copyright © 2013 Elsevier Ltd. All rights reserved.

Urine metabonomic profiling of a female adolescent with PIT-1 mutation before and during growth hormone therapy: insights into the metabolic effects of growth hormone.

PubMed

Abd Rahman, Shaffinaz; Schirra, Horst Joachim; Lichanska, Agnieszka M; Huynh, Tony; Leong, Gary M

2013-01-01

Growth hormone (GH) is a protein hormone with important roles in growth and metabolism. The objective of this study was to investigate the metabolism of a human subject with severe GH deficiency (GHD) due to a PIT-1 gene mutation and the metabolic effects of GH therapy using Nuclear Magnetic Resonance (NMR)-based metabonomics. NMR-based metabonomics is a platform that allows the metabolic profile of biological fluids such as urine to be recorded, and any alterations in the profile modulated by GH can potentially be detected. Urine samples were collected from a female subject with severe GHD before, during and after GH therapy, and from healthy age- and sex-matched controls and analysed with NMR-based metabonomics. The samples were collected at a hospital and the study was performed at a research facility. We studied a 17 year old female adolescent with severe GHD secondary to PIT-1 gene mutation who had reached final adult height and who had ceased GH therapy for over 3 years. The subject was subsequently followed for 5 years with and without GH therapy. Twelve healthy age-matched female subjects acted as control subjects. The GH-deficient subject re-commenced GH therapy at a dose of 1 mg/day to normalise serum IGF-1 levels. Urine metabolic profiles were recorded using NMR spectroscopy and analysed with multivariate statistics to distinguish the profiles at different time points and identify significant metabolites affected by GH therapy. NMR-based metabonomics revealed that the metabolic profile of the GH-deficient subject altered with GH therapy and that her profile was different from healthy controls before, and during withdrawal of GH therapy. This study illustrates the potential use of NMR-based metabonomics for monitoring the effects of GH therapy on metabolism by profiling the urine of GH-deficient subjects. Further controlled studies in larger numbers of GH-deficient subjects are required to determine the clinical benefits of NMR-based metabonomics in subjects receiving GH therapy. Copyright © 2012 Elsevier Ltd. All rights reserved.

Isolation, Purification and Characterization of Vinblastine and Vincristine from Endophytic Fungus Fusarium oxysporum Isolated from Catharanthus roseus

PubMed Central

Kumar, Ashutosh; Patil, Deepak; Rajamohanan, Pattuparambil Ramanpillai; Ahmad, Absar

2013-01-01

Endophytic fungi reside in a symbiotic fashion inside their host plants, mimic their chemistry and interestingly, produce the same natural products as their hosts and are thus being screened for the production of valuable compounds like taxol, camptothecin, podophyllotoxin, etc. Vinblastine and vincristine are excellent anti-cancer drugs but their current production using plants is non-abundant and expensive. In order to make these drugs readily available to the patients at affordable prices, we isolated the endophytic fungi from Catharanthus roseus plant and found a fungus AA-CRL-6 which produces vinblastine and vincristine in appreciable amounts. These drugs were purified by TLC and HPLC and characterized using UV-Vis spectroscopy, ESI-MS, MS/MS and 1H NMR. One liter of culture filtrate yielded 76 µg and 67 µg of vinblastine and vincristine respectively. This endophytic fungal strain was identified as Fusarium oxysporum based upon its cultural and morphological characteristics and internal transcribed spacer (ITS) sequence analysis. PMID:24066024

A comprehensive NMR methodology to assess the composition of biobased and biodegradable polymers in contact with food.

PubMed

Gratia, Audrey; Merlet, Denis; Ducruet, Violette; Lyathaud, Cédric

2015-01-01

A nuclear magnetic resonance (NMR) methodology was assessed regarding the identification and quantification of additives in three types of polylactide (PLA) intended as food contact materials. Additives were identified using the LNE/NMR database which clusters NMR datasets on more than 130 substances authorized by European Regulation No. 10/2011. Of the 12 additives spiked in the three types of PLA pellets, 10 were rapidly identified by the database and correlated with spectral comparison. The levels of the 12 additives were estimated using quantitative NMR combined with graphical computation. A comparison with chromatographic methods tended to prove the sensitivity of NMR by demonstrating an analytical difference of less than 15%. Our results therefore demonstrated the efficiency of the proposed NMR methodology for rapid assessment of the composition of PLA. Copyright © 2014 Elsevier B.V. All rights reserved.

Rapid NMR method for the quantification of organic compounds in thin stillage.

PubMed

Ratanapariyanuch, Kornsulee; Shen, Jianheng; Jia, Yunhua; Tyler, Robert T; Shim, Youn Young; Reaney, Martin J T

2011-10-12

Thin stillage contains organic and inorganic compounds, some of which may be valuable fermentation coproducts. This study describes a thorough analysis of the major solutes present in thin stillage as revealed by NMR and HPLC. The concentration of charged and neutral organic compounds in thin stillage was determined by excitation sculpting NMR methods (double pulse field gradient spin echo). Compounds identified by NMR included isopropanol, ethanol, lactic acid, 1,3-propanediol, acetic acid, succinic acid, glycerophosphorylcholine, betaine, glycerol, and 2-phenylethanol. The concentrations of lactic and acetic acid determined with NMR were comparable to those determined using HPLC. HPLC and NMR were complementary, as more compounds were identified using both methods. NMR analysis revealed that stillage contained the nitrogenous organic compounds betaine and glycerophosphorylcholine, which contributed as much as 24% of the nitrogen present in the stillage. These compounds were not observed by HPLC analysis.

A solid-phase extraction procedure coupled to 1H NMR, with chemometric analysis, to seek reliable markers of the botanical origin of honey.

PubMed

Beretta, Giangiacomo; Caneva, Enrico; Regazzoni, Luca; Bakhtyari, Nazanin Golbamaki; Maffei Facino, Roberto

2008-07-14

The aim of this work was to establish an analytical method for identifying the botanical origin of honey, as an alternative to conventional melissopalynological, organoleptic and instrumental methods (gas-chromatography coupled to mass spectrometry (GC-MS), high-performance liquid chromatography HPLC). The procedure is based on the (1)H nuclear magnetic resonance (NMR) profile coupled, when necessary, with electrospray ionisation-mass spectrometry (ESI-MS) and two-dimensional NMR analyses of solid-phase extraction (SPE)-purified honey samples, followed by chemometric analyses. Extracts of 44 commercial Italian honeys from 20 different botanical sources were analyzed. Honeydew, chestnut and linden honeys showed constant, specific, well-resolved resonances, suitable for use as markers of origin. Honeydew honey contained the typical resonances of an aliphatic component, very likely deriving from the plant phloem sap or excreted into it by sap-sucking aphids. Chestnut honey contained the typical signals of kynurenic acid and some structurally related metabolite. In linden honey the (1)H NMR profile gave strong signals attributable to the mono-terpene derivative cyclohexa-1,3-diene-1-carboxylic acid (CDCA) and to its 1-O-beta-gentiobiosyl ester (CDCA-GBE). These markers were not detectable in the other honeys, except for the less common nectar honey from rosa mosqueta. We compared and analyzed the data by multivariate techniques. Principal component analysis found different clusters of honeys based on the presence of these specific markers. The results, although obviously only preliminary, suggest that the (1)H NMR profile (with HPLC-MS analysis when necessary) can be used as a reference framework for identifying the botanical origin of honey.

1H NMR based pharmacometabolomics analysis of urine identifies metabolic phenotype of clopidogrel high on treatment platelets reactivity in coronary artery disease patients.

PubMed

Amin, Arwa M; Sheau Chin, Lim; Teh, Chin-Hoe; Mostafa, Hamza; Mohamed Noor, Dzul Azri; Sk Abdul Kader, Muhamad Ali; Kah Hay, Yuen; Ibrahim, Baharudin

2017-11-30

Clopidogrel high on treatment platelets reactivity (HTPR) has burdened achieving optimum therapeutic outcome. Although there are known genetic and non-genetic factors associated with clopidogrel HTPR, which explain in part clopidogrel HTPR, yet, great portion remains unknown, often hindering personalizing antiplatelet therapy. Nuclear magnetic resonance ( 1 H NMR) pharmacometabolomics analysis is useful technique to phenotype drug response. We investigated using 1 H NMR analysis to phenotype clopidogrel HTPR in urine. Urine samples were collected from 71 coronary artery disease (CAD) patients who were planned for interventional angiographic procedure prior to taking 600mg clopidogrel loading dose (LD) and 6h post LD. Patients' platelets function testing was assessed with the VerifyNow ® P2Y12 assay at 6h after LD. Urine samples were analysed using 1 H NMR. Multivariate statistical analysis was used to identify metabolites associated with clopidogrel HTPR. In pre-dose samples, 16 metabolites were associated with clopidogrel HTPR. However, 18 metabolites were associated with clopidogrel HTPR in post-dose samples. The pathway analysis of the identified biomarkers reflected that multifactorial conditions are associated with clopidogrel HTPR. It also revealed the implicated role of gut microbiota in clopidogrel HTPR. Pharmacometabolomics not only discovered novel biomarkers of clopidogrel HTPR but also revealed implicated pathways and conditions. Copyright © 2017 Elsevier B.V. All rights reserved.

Contributions of computational chemistry and biophysical techniques to fragment-based drug discovery.

PubMed

Gozalbes, Rafael; Carbajo, Rodrigo J; Pineda-Lucena, Antonio

2010-01-01

In the last decade, fragment-based drug discovery (FBDD) has evolved from a novel approach in the search of new hits to a valuable alternative to the high-throughput screening (HTS) campaigns of many pharmaceutical companies. The increasing relevance of FBDD in the drug discovery universe has been concomitant with an implementation of the biophysical techniques used for the detection of weak inhibitors, e.g. NMR, X-ray crystallography or surface plasmon resonance (SPR). At the same time, computational approaches have also been progressively incorporated into the FBDD process and nowadays several computational tools are available. These stretch from the filtering of huge chemical databases in order to build fragment-focused libraries comprising compounds with adequate physicochemical properties, to more evolved models based on different in silico methods such as docking, pharmacophore modelling, QSAR and virtual screening. In this paper we will review the parallel evolution and complementarities of biophysical techniques and computational methods, providing some representative examples of drug discovery success stories by using FBDD.

Three-dimensional structure and ligand interactions of the low molecular weight protein tyrosine phosphatase from Campylobacter jejuni.

PubMed

Tolkatchev, Dmitri; Shaykhutdinov, Rustem; Xu, Ping; Plamondon, Josée; Watson, David C; Young, N Martin; Ni, Feng

2006-10-01

A putative low molecular weight protein tyrosine phosphatase (LMW-PTP) was identified in the genome sequence of the bacterial pathogen, Campylobacter jejuni. This novel gene, cj1258, has sequence homology with a distinctive class of phosphatases widely distributed among prokaryotes and eukaryotes. We report here the solution structure of Cj1258 established by high-resolution NMR spectroscopy using NOE-derived distance restraints, hydrogen bond data, and torsion angle restraints. The three-dimensional structure consists of a central four-stranded parallel beta-sheet flanked by five alpha-helices, revealing an overall structural topology similar to those of the eukaryotic LMW-PTPs, such as human HCPTP-A, bovine BPTP, and Saccharomyces cerevisiae LTP1, and to those of the bacterial LMW-PTPs MPtpA from Mycobacterium tuberculosis and YwlE from Bacillus subtilis. The active site of the enzyme is flexible in solution and readily adapts to the binding of ligands, such as the phosphate ion. An NMR-based screen was carried out against a number of potential inhibitors and activators, including phosphonomethylphenylalanine, derivatives of the cinnamic acid, 2-hydroxy-5-nitrobenzaldehyde, cinnamaldehyde, adenine, and hypoxanthine. Despite its bacterial origin, both the three-dimensional structure and ligand-binding properties of Cj1258 suggest that this novel phosphatase may have functional roles close to those of eukaryotic and mammalian tyrosine phosphatases. The three-dimensional structure along with mapping of small-molecule binding will be discussed in the context of developing high-affinity inhibitors of this novel LMW-PTP.

Automatic analysis of quantitative NMR data of pharmaceutical compound libraries.

PubMed

Liu, Xuejun; Kolpak, Michael X; Wu, Jiejun; Leo, Gregory C

2012-08-07

In drug discovery, chemical library compounds are usually dissolved in DMSO at a certain concentration and then distributed to biologists for target screening. Quantitative (1)H NMR (qNMR) is the preferred method for the determination of the actual concentrations of compounds because the relative single proton peak areas of two chemical species represent the relative molar concentrations of the two compounds, that is, the compound of interest and a calibrant. Thus, an analyte concentration can be determined using a calibration compound at a known concentration. One particularly time-consuming step in the qNMR analysis of compound libraries is the manual integration of peaks. In this report is presented an automated method for performing this task without prior knowledge of compound structures and by using an external calibration spectrum. The script for automated integration is fast and adaptable to large-scale data sets, eliminating the need for manual integration in ~80% of the cases.

Neonatal, infant and under-five mortalities in Nigeria: An examination of trends and drivers (2003-2013).

PubMed

Morakinyo, Oyewale Mayowa; Fagbamigbe, Adeniyi Francis

2017-01-01

Neonatal (NMR), infant (IMR) and under-five (U5M) mortality rates remain high in Nigeria. Evidence-based knowledge of trends and drivers of child mortality will aid proper interventions needed to combat the menace. Therefore, this study assessed the trends and drivers of NMR, IMR, and U5M over a decade in Nigeria. A nationally representative data from three consecutive Nigeria Demographic and Household Surveys (NDHS) was used. A total of 66,158 live births within the five years preceding the 2003 (6029), 2008 (28647) and 2013 (31482) NDHS were included in the analyses. NMR was computed using proportions while IMR and U5 were computed using life table techniques embedded in Stata version 12. Probit regression model and its associated marginal effects were used to identify the predisposing factors to NMR, IMR, and U5M. The NMR, IMR, and U5M per 1000 live births in 2003, 2008 and 2013 were 52, 41, 39; 100, 75, 69; and 201, 157, 128 respectively. The NMR, IMR, and U5M were consistently lower among children whose mothers were younger, living in rural areas and from richer households. Generally, the probability of neonate death in 2003, 2008 and 2013 were 0.049, 0.039 and 0.038 respectively, the probability of infant death was 0.093, 0.071 and 0.064 while the probability of under-five death was 0.140, 0.112 and 0.092 for the respective survey years. While adjusting for other variables, the likelihood of infant and under-five deaths was significantly reduced across the survey years. Maternal age, mothers' education, place of residence, child's sex, birth interval, weight at birth, skill of birth attendant, delivery by caesarean operation or not significantly influenced NMR, IMR, and U5M. The NMR, IMR, and U5M in Nigeria reduced over the studied period. Multi-sectoral interventions targeted towards the identified drivers should be instituted to improve child survival.

A 1H-NMR based metabolomics study of the intervention effect of mangiferin on hyperlipidemia hamsters induced by a high-fat diet.

PubMed

Guo, Fuchuan; Zi, Tianqi; Liu, Liyan; Feng, Rennan; Sun, Changhao

2017-07-19

It has been demonstrated that mangiferin can ameliorate hypertriglyceridemia by modulating the expression levels of genes involved in lipid metabolism in animal experiments, but its effects on the serum metabolic fingerprinting of hyperlipidemia animal models have not been reported. Thus, a NMR-based metabolomics approach was conducted to explore the effects of mangiferin on hyperlipidemia hamsters and to gain a better understanding of the involved metabolic pathways. Hamsters fed with a high-fat diet were orally administered with mangiferin 150 mg per kg BW once a day for 8 weeks. Serum samples were analysed by 1 H NMR, and multivariate statistical analysis was applied to the data to identify potential biomarkers. In total, 20 discriminating metabolites were identified. It turned out that mangiferin administration can partly reverse the metabolism disorders induced by a high-fat diet and exerted a good anti-hypertriglyceridemia effect. Mangiferin ameliorated hyperlipidemia by intervening in some major metabolic pathways, involving glycolysis, the TCA cycle, synthesis of ketone bodies, and BCAAs as well as choline and lipid metabolism. These findings provided new essential information on the effects of mangiferin and demonstrated the great potential of this nutrimetabolomics approach.

Functional gene-based discovery of phenazines from the actinobacteria associated with marine sponges in the South China Sea.

PubMed

Karuppiah, Valliappan; Li, Yingxin; Sun, Wei; Feng, Guofang; Li, Zhiyong

2015-07-01

Phenazines represent a large group of nitrogen-containing heterocyclic compounds produced by the diverse group of bacteria including actinobacteria. In this study, a total of 197 actinobacterial strains were isolated from seven different marine sponge species in the South China Sea using five different culture media. Eighty-seven morphologically different actinobacterial strains were selected and grouped into 13 genera, including Actinoalloteichus, Kocuria, Micrococcus, Micromonospora, Mycobacterium, Nocardiopsis, Prauserella, Rhodococcus, Saccharopolyspora, Salinispora, Serinicoccus, and Streptomyces by the phylogenetic analysis of 16S rRNA gene. Based on the screening of phzE genes, ten strains, including five Streptomyces, two Nocardiopsis, one Salinispora, one Micrococcus, and one Serinicoccus were found to be potential for phenazine production. The level of phzE gene expression was highly expressed in Nocardiopsis sp. 13-33-15, 13-12-13, and Serinicoccus sp. 13-12-4 on the fifth day of fermentation. Finally, 1,6-dihydroxy phenazine (1) from Nocardiopsis sp. 13-33-15 and 13-12-13, and 1,6-dimethoxy phenazine (2) from Nocardiopsis sp. 13-33-15 were isolated and identified successfully based on ESI-MS and NMR analysis. The compounds 1 and 2 showed antibacterial activity against Bacillus mycoides SJ14, Staphylococcus aureus SJ51, Escherichia coli SJ42, and Micrococcus luteus SJ47. This study suggests that the integrated approach of gene screening and chemical analysis is an effective strategy to find the target compounds and lays the basis for the production of phenazine from the sponge-associated actinobacteria.

Synthesis and characterization of crystalline structures based on phenylboronate ligands bound to alkaline earth cations.

PubMed

Reinholdt, Marc; Croissant, Jonas; Di Carlo, Lidia; Granier, Dominique; Gaveau, Philippe; Bégu, Sylvie; Devoisselle, Jean-Marie; Mutin, P Hubert; Smith, Mark E; Bonhomme, Christian; Gervais, Christel; van der Lee, Arie; Laurencin, Danielle

2011-08-15

We describe the preparation of the first crystalline compounds based on arylboronate ligands PhB(OH)(3)(-) coordinated to metal cations: [Ca(PhB(OH)(3))(2)], [Sr(PhB(OH)(3))(2)]·H(2)O, and [Ba(PhB(OH)(3))(2)]. The calcium and strontium structures were solved using powder and single-crystal X-ray diffraction, respectively. In both cases, the structures are composed of chains of cations connected through phenylboronate ligands, which interact one with each other to form a 2D lamellar structure. The temperature and pH conditions necessary for the formation of phase-pure compounds were investigated: changes in temperature were found to mainly affect the morphology of the crystallites, whereas strong variations in pH were found to affect the formation of pure phases. All three compounds were characterized using a wide range of analytical techniques (TGA, IR, Raman, XRD, and high resolution (1)H, (11)B, and (13)C solid-state NMR), and the different coordination modes of phenylboronate ligands were analyzed. Two different kinds of hydroxyl groups were identified in the structures: those involved in hydrogen bonds, and those that are effectively "free" and not involved in hydrogen bonds of any significant strength. To position precisely the OH protons within the structures, an NMR-crystallography approach was used: the comparison of experimental and calculated NMR parameters (determined using the Gauge Including Projector Augmented Wave method, GIPAW) allowed the most accurate positions to be identified. In the case of the calcium compound, it was found that it is the (43)Ca NMR data that are critical to help identify the best model of the structure. © 2011 American Chemical Society

Novel menadione hybrids: Synthesis, anticancer activity, and cell-based studies.

PubMed

Prasad, Chakka Vara; Nayak, Vadithe Lakshma; Ramakrishna, Sistla; Mallavadhani, Uppuluri Venkata

2018-01-01

A series of novel menadione-based triazole hybrids were designed and synthesized by employing copper-catalyzed azide-alkyne cycloaddition (CuAAC). All the synthesized hybrids were characterized by their spectral data ( 1 H NMR, 13 C NMR, IR, and HRMS). The synthesized compounds were evaluated for their anticancer activity against five selected cancer cell lines including lung (A549), prostate (DU-145), cervical (Hela), breast (MCF-7), and mouse melanoma (B-16) using MTT assay. The screening results showed that majority of the synthesized compounds displayed significant anticancer activity. Among the tested compounds, the triazoles 5 and 6 exhibited potent activity against all cell lines. In particular, compound 6 showed higher potency than the standard tamoxifen and parent menadione against MCF-7 cell line. Flow cytometric analysis revealed that compound 6 arrested cell cycle at G0/G1 phase and induced apoptotic cell death which was further confirmed by Hoechst staining, measurement of mitochondrial membrane potential (ΔΨm) and Annexin-V-FITC assay. Thus, compound 6 can be considered as lead molecule for further development as potent anticancer therapeutic agent. © 2017 John Wiley & Sons A/S.

Metabolite profiling of Clinacanthus nutans leaves extracts obtained from different drying methods by 1H NMR-based metabolomics

NASA Astrophysics Data System (ADS)

Hashim, Noor Haslinda Noor; Latip, Jalifah; Khatib, Alfi

2016-11-01

The metabolites of Clinacanthus nutans leaves extracts and their dependence on drying process were systematically characterized using 1H nuclear magnetic resonance spectroscopy (NMR) multivariate data analysis. Principal component analysis (PCA) and partial least square-discriminant analysis (PLS-DA) were able to distinguish the leaves extracts obtained from different drying methods. The identified metabolites were carbohydrates, amino acid, flavonoids and sulfur glucoside compounds. The major metabolites responsible for the separation in PLS-DA loading plots were lupeol, cycloclinacosides, betulin, cerebrosides and choline. The results showed that the combination of 1H NMR spectroscopy and multivariate data analyses could act as an efficient technique to understand the C. nutans composition and its variation.

Fragment-based hit discovery and structure-based optimization of aminotriazoloquinazolines as novel Hsp90 inhibitors.

PubMed

Casale, Elena; Amboldi, Nadia; Brasca, Maria Gabriella; Caronni, Dannica; Colombo, Nicoletta; Dalvit, Claudio; Felder, Eduard R; Fogliatto, Gianpaolo; Galvani, Arturo; Isacchi, Antonella; Polucci, Paolo; Riceputi, Laura; Sola, Francesco; Visco, Carlo; Zuccotto, Fabio; Casuscelli, Francesco

2014-08-01

In the last decade the heat shock protein 90 (Hsp90) has emerged as a major therapeutic target and many efforts have been dedicated to the discovery of Hsp90 inhibitors as new potent anticancer agents. Here we report the identification of a novel class of Hsp90 inhibitors by means of a biophysical FAXS-NMR based screening of a library of fragments. The use of X-ray structure information combined with modeling studies enabled the fragment evolution of the initial triazoloquinazoline hit to a class of compounds with nanomolar potency and drug-like properties suited for further lead optimization. Copyright © 2014 Elsevier Ltd. All rights reserved.

NMR-based urine analysis in rats: prediction of proximal tubule kidney toxicity and phospholipidosis.

PubMed

Lienemann, Kai; Plötz, Thomas; Pestel, Sabine

2008-01-01

The aim of safety pharmacology is early detection of compound-induced side-effects. NMR-based urine analysis followed by multivariate data analysis (metabonomics) identifies efficiently differences between toxic and non-toxic compounds; but in most cases multiple administrations of the test compound are necessary. We tested the feasibility of detecting proximal tubule kidney toxicity and phospholipidosis with metabonomics techniques after single compound administration as an early safety pharmacology approach. Rats were treated orally, intravenously, inhalatively or intraperitoneally with different test compounds. Urine was collected at 0-8 h and 8-24 h after compound administration, and (1)H NMR-patterns were recorded from the samples. Variation of post-processing and feature extraction methods led to different views on the data. Support Vector Machines were trained on these different data sets and then aggregated as experts in an Ensemble. Finally, validity was monitored with a cross-validation study using a training, validation, and test data set. Proximal tubule kidney toxicity could be predicted with reasonable total classification accuracy (85%), specificity (88%) and sensitivity (78%). In comparison to alternative histological studies, results were obtained quicker, compound need was reduced, and very importantly fewer animals were needed. In contrast, the induction of phospholipidosis by the test compounds could not be predicted using NMR-based urine analysis or the previously published biomarker PAG. NMR-based urine analysis was shown to effectively predict proximal tubule kidney toxicity after single compound administration in rats. Thus, this experimental design allows early detection of toxicity risks with relatively low amounts of compound in a reasonably short period of time.

Synthesis and spectral characterization of Zn(II) microsphere series for antimicrobial application

NASA Astrophysics Data System (ADS)

Singh, Ajay K.; Pandey, Sarvesh K.; Pandey, O. P.; Sengupta, S. K.

2014-09-01

Microsphere series have been synthesized by reacting zinc(II) acetate dihydrate with Schiff bases derived from 2-hydrazino-5-[substituted phenyl]-1,3,4-thiadiazole/oxadiazole/triazole with salicylaldehyde. Elemental analysis suggests that the complexes have 1:2 and 1:1 stoichiometry of the type [Zn(L)2(H2O)2] and [Zn(L‧)(H2O)2]; LH = Schiff bases derived from 2-hydrazino-5-[substituted phenyl]-1,3,4-thia/oxadiazole with salicylaldehyde; L‧H2 = Schiff bases derived from 3-(substituted phenyl)-4-amino-5-hydrazino-1,2,4-triazole and salicylaldehyde and were characterized by elemental analyses, IR, 1H NMR and 13C NMR spectral data. Scanning electron microscopy (SEM) showed that synthesized materials have microsphere like structure and there EDX analysis comparably matches with elemental analysis. For the antimicrobial application Schiff bases and their zinc(II) complexes were screened for four bacteria e.g. Bacillus subtilis, Pseudomonas aeruginosa, Salmonella typhi, Streptococcus pyogenes and four fungi e.g. Cyrtomium falcatum, Aspergillus niger, Fusarium oxysporium and Curvularia pallescence by the reported method. Schiff base and Zn(II) compounds showed significant antimicrobial activities. However, activities increase upon chelation. Thermal analysis (TGA) data of compound (10) showed its stability up to 300 °C.

NMR-based metabonomics and correlation analysis reveal potential biomarkers associated with chronic atrophic gastritis.

PubMed

Cui, Jiajia; Liu, Yuetao; Hu, Yinghuan; Tong, Jiayu; Li, Aiping; Qu, Tingli; Qin, Xuemei; Du, Guanhua

2017-01-05

Chronic atrophic gastritis (CAG) is one of the most important pre-cancerous states with a high prevalence. Exploring of the underlying mechanism and potential biomarkers is of significant importance for CAG. In the present work, 1 H NMR-based metabonomics with correlative analysis was performed to analyze the metabolic features of CAG. 19 plasma metabolites and 18 urine metabolites were enrolled to construct the circulatory and excretory metabolome of CAG, which was in response to alterations of energy metabolism, inflammation, immune dysfunction, as well as oxidative stress. 7 plasma biomarkers and 7 urine biomarkers were screened to elucidate the pathogenesis of CAG based on the further correlation analysis with biochemical indexes. Finally, 3 plasma biomarkers (arginine, succinate and 3-hydroxybutyrate) and 2 urine biomarkers (α-ketoglutarate and valine) highlighted the potential to indicate risks of CAG in virtue of correlation with pepsin activity and ROC analysis. Here, our results paved a way for elucidating the underlying mechanisms in the development of CAG, and provided new avenues for the diagnosis of CAG and presented potential drug targets for treatment of CAG. Copyright © 2016 Elsevier B.V. All rights reserved.

Fe (III), Co(II), Ni(II), Cu(II) and Zn(II) complexes of schiff bases based-on glycine and phenylalanine: Synthesis, magnetic/thermal properties and antimicrobial activity

NASA Astrophysics Data System (ADS)

Sevgi, Fatih; Bagkesici, Ugur; Kursunlu, Ahmed Nuri; Guler, Ersin

2018-02-01

Zinc (II), copper (II), nickel (II), cobalt (II) and iron (III) complexes of Schiff bases (LG, LP) derived from 2-hydroxynaphthaldehyde with glycine and phenylalanine were reported and characterized by 1H NMR, 13C NMR, elemental analyses, melting point, FT-IR, magnetic susceptibility and thermal analyses (TGA). TGA data show that iron and cobalt include to the coordinated water and metal:ligand ratio is 1:2 while the complex stoichiometry for Ni (II), Cu (II) and Zn (II) complexes is 1:1. As expected, Ni (II) and Zn (II) complexes are diamagnetic; Cu (II), Co (II) and Fe (III) complexes are paramagnetic character due to a strong ligand of LG and LP. The LG, LP and their metal complexes were screened for their antimicrobial activities against five Gram-positive (Staphylococcus aureus, Methicillin resistant Staphylococcus aureus (MRSA), Bacillus cereus, Streptococcus mutans and Enterococcus faecalis) and three Gram-negative (Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa) and one fungi (Candida albicans) by using broth microdilution techniques. The activity data show that ligands and their metal complexes exhibited moderate to good activity against Gram-positive bacteria and fungi.

Modelling the acid/base 1H NMR chemical shift limits of metabolites in human urine.

PubMed

Tredwell, Gregory D; Bundy, Jacob G; De Iorio, Maria; Ebbels, Timothy M D

2016-01-01

Despite the use of buffering agents the 1 H NMR spectra of biofluid samples in metabolic profiling investigations typically suffer from extensive peak frequency shifting between spectra. These chemical shift changes are mainly due to differences in pH and divalent metal ion concentrations between the samples. This frequency shifting results in a correspondence problem: it can be hard to register the same peak as belonging to the same molecule across multiple samples. The problem is especially acute for urine, which can have a wide range of ionic concentrations between different samples. To investigate the acid, base and metal ion dependent 1 H NMR chemical shift variations and limits of the main metabolites in a complex biological mixture. Urine samples from five different individuals were collected and pooled, and pre-treated with Chelex-100 ion exchange resin. Urine samples were either treated with either HCl or NaOH, or were supplemented with various concentrations of CaCl 2 , MgCl 2 , NaCl or KCl, and their 1 H NMR spectra were acquired. Nonlinear fitting was used to derive acid dissociation constants and acid and base chemical shift limits for peaks from 33 identified metabolites. Peak pH titration curves for a further 65 unidentified peaks were also obtained for future reference. Furthermore, the peak variations induced by the main metal ions present in urine, Na + , K + , Ca 2+ and Mg 2+ , were also measured. These data will be a valuable resource for 1 H NMR metabolite profiling experiments and for the development of automated metabolite alignment and identification algorithms for 1 H NMR spectra.

Investigating Protein-Ligand Interactions by Solution Nuclear Magnetic Resonance Spectroscopy.

PubMed

Becker, Walter; Bhattiprolu, Krishna Chaitanya; Gubensäk, Nina; Zangger, Klaus

2018-04-17

Protein-ligand interactions are of fundamental importance in almost all processes in living organisms. The ligands comprise small molecules, drugs or biological macromolecules and their interaction strength varies over several orders of magnitude. Solution NMR spectroscopy offers a large repertoire of techniques to study such complexes. Here, we give an overview of the different NMR approaches available. The information they provide ranges from the simple information about the presence of binding or epitope mapping to the complete 3 D structure of the complex. NMR spectroscopy is particularly useful for the study of weak interactions and for the screening of binding ligands with atomic resolution. © 2018 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

Molecular evolution of the hyaluronan synthase 2 gene in mammals: implications for adaptations to the subterranean niche and cancer resistance

PubMed Central

Faulkes, Christopher G.; Davies, Kalina T. J.; Rossiter, Stephen J.; Bennett, Nigel C.

2015-01-01

The naked mole-rat (NMR) Heterocephalus glaber is a unique and fascinating mammal exhibiting many unusual adaptations to a subterranean lifestyle. The recent discovery of their resistance to cancer and exceptional longevity has opened up new and important avenues of research. Part of this resistance to cancer has been attributed to the fact that NMRs produce a modified form of hyaluronan—a key constituent of the extracellular matrix—that is thought to confer increased elasticity of the skin as an adaptation for living in narrow tunnels. This so-called high molecular mass hyaluronan (HMM-HA) stems from two apparently unique substitutions in the hyaluronan synthase 2 enzyme (HAS2). To test whether other subterranean mammals with similar selection pressures also show molecular adaptation in their HAS2 gene, we sequenced the HAS2 gene for 11 subterranean mammals and closely related species, and combined these with data from 57 other mammals. Comparative screening revealed that one of the two putatively important HAS2 substitutions in the NMR predicted to have a significant effect on hyaluronan synthase function was uniquely shared by all African mole-rats. Interestingly, we also identified multiple other amino acid substitutions in key domains of the HAS2 molecule, although the biological consequences of these for hyaluronan synthesis remain to be determined. Despite these results, we found evidence of strong purifying selection acting on the HAS2 gene across all mammals, and the NMR remains unique in its particular HAS2 sequence. Our results indicate that more work is needed to determine whether the apparent cancer resistance seen in NMR is shared by other members of the African mole-rat clade. PMID:25948568

Multi-platform metabolomics assays for human lung lavage fluids in an air pollution exposure study.

PubMed

Surowiec, Izabella; Karimpour, Masoumeh; Gouveia-Figueira, Sandra; Wu, Junfang; Unosson, Jon; Bosson, Jenny A; Blomberg, Anders; Pourazar, Jamshid; Sandström, Thomas; Behndig, Annelie F; Trygg, Johan; Nording, Malin L

2016-07-01

Metabolomics protocols are used to comprehensively characterize the metabolite content of biological samples by exploiting cutting-edge analytical platforms, such as gas chromatography (GC) or liquid chromatography (LC) coupled to mass spectrometry (MS) assays, as well as nuclear magnetic resonance (NMR) assays. We have developed novel sample preparation procedures combined with GC-MS, LC-MS, and NMR metabolomics profiling for analyzing bronchial wash (BW) and bronchoalveolar lavage (BAL) fluid from 15 healthy volunteers following exposure to biodiesel exhaust and filtered air. Our aim was to investigate the responsiveness of metabolite profiles in the human lung to air pollution exposure derived from combustion of biofuels, such as rapeseed methyl ester biodiesel, which are increasingly being promoted as alternatives to conventional fossil fuels. Our multi-platform approach enabled us to detect the greatest number of unique metabolites yet reported in BW and BAL fluid (82 in total). All of the metabolomics assays indicated that the metabolite profiles of the BW and BAL fluids differed appreciably, with 46 metabolites showing significantly different levels in the corresponding lung compartments. Furthermore, the GC-MS assay revealed an effect of biodiesel exhaust exposure on the levels of 1-monostearylglycerol, sucrose, inosine, nonanoic acid, and ethanolamine (in BAL) and pentadecanoic acid (in BW), whereas the LC-MS assay indicated a shift in the levels of niacinamide (in BAL). The NMR assay only identified lactic acid (in BW) as being responsive to biodiesel exhaust exposure. Our findings demonstrate that the proposed multi-platform approach is useful for wide metabolomics screening of BW and BAL fluids and can facilitate elucidation of metabolites responsive to biodiesel exhaust exposure. Graphical Abstract Graphical abstract illustrating the study workflow. NMR Nuclear Magnetic Resonance, LC-TOFMS Liquid chromatography-Time Of Flight Mass Spectrometry, GC Gas Chromatography-Mass spectrometry.

Flow-through lipid nanotube arrays for structure-function studies of membrane proteins by solid-state NMR spectroscopy.

PubMed

Chekmenev, Eduard Y; Gor'kov, Peter L; Cross, Timothy A; Alaouie, Ali M; Smirnov, Alex I

2006-10-15

A novel method for studying membrane proteins in a native lipid bilayer environment by solid-state NMR spectroscopy is described and tested. Anodic aluminum oxide (AAO) substrates with flow-through 175 nm wide and 60-mum-long nanopores were employed to form macroscopically aligned peptide-containing lipid bilayers that are fluid and highly hydrated. We demonstrate that the surfaces of both leaflets of such bilayers are fully accessible to aqueous solutes. Thus, high hydration levels as well as pH and desirable ion and/or drug concentrations could be easily maintained and modified as desired in a series of experiments with the same sample. The method allows for membrane protein NMR experiments in a broad pH range that could be extended to as low as 1 and as high as 12 units for a period of up to a few hours and temperatures as high as 70 degrees C without losing the lipid alignment or bilayers from the nanopores. We demonstrate the utility of this method by a solid-state 19.6 T (17)O NMR study of reversible binding effects of mono- and divalent ions on the chemical shift properties of the Leu(10) carbonyl oxygen of transmembrane pore-forming peptide gramicidin A (gA). We further compare the (17)O shifts induced by binding metal ions to the binding of protons in the pH range from 1 to 12 and find a significant difference. This unexpected result points to a difference in mechanisms for ion and proton conduction by the gA pore. We believe that a large number of solid-state NMR-based studies, including structure-function, drug screening, proton exchange, pH, and other titration experiments, will benefit significantly from the method described here.

Influence of Freezing and Storage Procedure on Human Urine Samples in NMR-Based Metabolomics

PubMed Central

Rist, Manuela J.; Muhle-Goll, Claudia; Görling, Benjamin; Bub, Achim; Heissler, Stefan; Watzl, Bernhard; Luy, Burkhard

2013-01-01

It is consensus in the metabolomics community that standardized protocols should be followed for sample handling, storage and analysis, as it is of utmost importance to maintain constant measurement conditions to identify subtle biological differences. The aim of this work, therefore, was to systematically investigate the influence of freezing procedures and storage temperatures and their effect on NMR spectra as a potentially disturbing aspect for NMR-based metabolomics studies. Urine samples were collected from two healthy volunteers, centrifuged and divided into aliquots. Urine aliquots were frozen either at −20 °C, on dry ice, at −80 °C or in liquid nitrogen and then stored at −20 °C, −80 °C or in liquid nitrogen vapor phase for 1–5 weeks before NMR analysis. Results show spectral changes depending on the freezing procedure, with samples frozen on dry ice showing the largest deviations. The effect was found to be based on pH differences, which were caused by variations in CO2 concentrations introduced by the freezing procedure. Thus, we recommend that urine samples should be frozen at −20 °C and transferred to lower storage temperatures within one week and that freezing procedures should be part of the publication protocol. PMID:24957990

Influence of Freezing and Storage Procedure on Human Urine Samples in NMR-Based Metabolomics.

PubMed

Rist, Manuela J; Muhle-Goll, Claudia; Görling, Benjamin; Bub, Achim; Heissler, Stefan; Watzl, Bernhard; Luy, Burkhard

2013-04-09

It is consensus in the metabolomics community that standardized protocols should be followed for sample handling, storage and analysis, as it is of utmost importance to maintain constant measurement conditions to identify subtle biological differences. The aim of this work, therefore, was to systematically investigate the influence of freezing procedures and storage temperatures and their effect on NMR spectra as a potentially disturbing aspect for NMR-based metabolomics studies. Urine samples were collected from two healthy volunteers, centrifuged and divided into aliquots. Urine aliquots were frozen either at -20 °C, on dry ice, at -80 °C or in liquid nitrogen and then stored at -20 °C, -80 °C or in liquid nitrogen vapor phase for 1-5 weeks before NMR analysis. Results show spectral changes depending on the freezing procedure, with samples frozen on dry ice showing the largest deviations. The effect was found to be based on pH differences, which were caused by variations in CO2 concentrations introduced by the freezing procedure. Thus, we recommend that urine samples should be frozen at -20 °C and transferred to lower storage temperatures within one week and that freezing procedures should be part of the publication protocol.

Bone volume-to-total volume ratio measured in trabecular bone by single-sided NMR devices.

PubMed

Brizi, Leonardo; Barbieri, Marco; Baruffaldi, Fabio; Bortolotti, Villiam; Fersini, Chiara; Liu, Huabing; Nogueira d'Eurydice, Marcel; Obruchkov, Sergei; Zong, Fangrong; Galvosas, Petrik; Fantazzini, Paola

2018-01-01

Reduced bone strength is associated with a loss of bone mass, usually evaluated by dual-energy X-ray absorptiometry, although it is known that the bone microstructure also affects the bone strength. Here, a method is proposed to measure (in laboratory) the bone volume-to-total volume ratio by single-sided NMR scanners, which is related to the microstructure of the trabecular bone. Three single-sided scanners were used on animal bone samples. These low-field, mobile, low-cost devices are able to detect the NMR signal, regardless of the sample sizes, without the use of ionizing radiations, with the further advantage of signal localization offered by their intrinsic magnetic field gradients. The performance of the different single-sided scanners have been discussed. The results have been compared with bone volume-to-total volume ratio by micro CT and MRI, obtaining consistent values. Our results demonstrate the feasibility of the method for laboratory analyses, which are useful for measurements like porosity on bone specimens. This can be considered as the first step to develop an NMR method based on the use of a mobile single-sided device, for the diagnosis of osteoporosis, through the acquisition of the signal from the appendicular skeleton, allowing for low-cost, wide screening campaigns. Magn Reson Med 79:501-510, 2018. © 2017 International Society for Magnetic Resonance in Medicine. © 2017 International Society for Magnetic Resonance in Medicine.

The multiple roles of computational chemistry in fragment-based drug design

NASA Astrophysics Data System (ADS)

Law, Richard; Barker, Oliver; Barker, John J.; Hesterkamp, Thomas; Godemann, Robert; Andersen, Ole; Fryatt, Tara; Courtney, Steve; Hallett, Dave; Whittaker, Mark

2009-08-01

Fragment-based drug discovery (FBDD) represents a change in strategy from the screening of molecules with higher molecular weights and physical properties more akin to fully drug-like compounds, to the screening of smaller, less complex molecules. This is because it has been recognised that fragment hit molecules can be efficiently grown and optimised into leads, particularly after the binding mode to the target protein has been first determined by 3D structural elucidation, e.g. by NMR or X-ray crystallography. Several studies have shown that medicinal chemistry optimisation of an already drug-like hit or lead compound can result in a final compound with too high molecular weight and lipophilicity. The evolution of a lower molecular weight fragment hit therefore represents an attractive alternative approach to optimisation as it allows better control of compound properties. Computational chemistry can play an important role both prior to a fragment screen, in producing a target focussed fragment library, and post-screening in the evolution of a drug-like molecule from a fragment hit, both with and without the available fragment-target co-complex structure. We will review many of the current developments in the area and illustrate with some recent examples from successful FBDD discovery projects that we have conducted.

Integration of Microfractionation, qNMR and Zebrafish Screening for the In Vivo Bioassay-Guided Isolation and Quantitative Bioactivity Analysis of Natural Products

PubMed Central

Maes, Jan; Siverio-Mota, Dany; Marcourt, Laurence; Munck, Sebastian; Kamuhabwa, Appolinary R.; Moshi, Mainen J.; Esguerra, Camila V.; de Witte, Peter A. M.; Crawford, Alexander D.; Wolfender, Jean-Luc

2013-01-01

Natural products (NPs) are an attractive source of chemical diversity for small-molecule drug discovery. Several challenges nevertheless persist with respect to NP discovery, including the time and effort required for bioassay-guided isolation of bioactive NPs, and the limited biomedical relevance to date of in vitro bioassays used in this context. With regard to bioassays, zebrafish have recently emerged as an effective model system for chemical biology, allowing in vivo high-content screens that are compatible with microgram amounts of compound. For the deconvolution of the complex extracts into their individual constituents, recent progress has been achieved on several fronts as analytical techniques now enable the rapid microfractionation of extracts, and microflow NMR methods have developed to the point of allowing the identification of microgram amounts of NPs. Here we combine advanced analytical methods with high-content screening in zebrafish to create an integrated platform for microgram-scale, in vivo NP discovery. We use this platform for the bioassay-guided fractionation of an East African medicinal plant, Rhynchosia viscosa, resulting in the identification of both known and novel isoflavone derivatives with anti-angiogenic and anti-inflammatory activity. Quantitative microflow NMR is used both to determine the structure of bioactive compounds and to quantify them for direct dose-response experiments at the microgram scale. The key advantages of this approach are (1) the microgram scale at which both biological and analytical experiments can be performed, (2) the speed and the rationality of the bioassay-guided fractionation – generic for NP extracts of diverse origin – that requires only limited sample-specific optimization and (3) the use of microflow NMR for quantification, enabling the identification and dose-response experiments with only tens of micrograms of each compound. This study demonstrates that a complete in vivo bioassay-guided fractionation can be performed with only 20 mg of NP extract within a few days. PMID:23700445

Regioselective reaction: synthesis, characterization and pharmacological activity of some new Mannich and Schiff bases containing sydnone.

PubMed

Nithinchandra; Kalluraya, B; Aamir, S; Shabaraya, A R

2012-08-01

A novel series of 1-substituted aminomethyl-3-[1-(4-isobutylphenyl)ethyl]-4-(3-aryl-4-sydnonylidene) amino-1,2,4-triazol-5-thiones (9), was prepared from the 3-[1-(4-isobutylphenyl)ethyl]-4-(3-aryl-4-sydnonylidene) amino 5-mercapto-1,2,4-triazoles (8) by aminomethylation with formaldehyde and secondary amine. The structures of Schiff bases (8) and Mannich bases (9) were characterized on the basis of IR, NMR, mass spectra1 data and elemental analysis. The newly synthesized compounds were screened for their anti-inflammatory and analgesic activities. Mannich bases (9) carrying piperidine and morpholine residues showed promising anti-inflammatory and analgesic activity. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

Can NMR solve some significant challenges in metabolomics?

PubMed Central

Gowda, G.A. Nagana; Raftery, Daniel

2015-01-01

The field of metabolomics continues to witness rapid growth driven by fundamental studies, methods development, and applications in a number of disciplines that include biomedical science, plant and nutrition sciences, drug development, energy and environmental sciences, toxicology, etc. NMR spectroscopy is one of the two most widely used analytical platforms in the metabolomics field, along with mass spectrometry (MS). NMR's excellent reproducibility and quantitative accuracy, its ability to identify structures of unknown metabolites, its capacity to generate metabolite profiles using intact biospecimens with no need for separation, and its capabilities for tracing metabolic pathways using isotope labeled substrates offer unique strengths for metabolomics applications. However, NMR's limited sensitivity and resolution continue to pose a major challenge and have restricted both the number and the quantitative accuracy of metabolites analyzed by NMR. Further, the analysis of highly complex biological samples has increased the demand for new methods with improved detection, better unknown identification, and more accurate quantitation of larger numbers of metabolites. Recent efforts have contributed significant improvements in these areas, and have thereby enhanced the pool of routinely quantifiable metabolites. Additionally, efforts focused on combining NMR and MS promise opportunities to exploit the combined strength of the two analytical platforms for direct comparison of the metabolite data, unknown identification and reliable biomarker discovery that continue to challenge the metabolomics field. This article presents our perspectives on the emerging trends in NMR-based metabolomics and NMR's continuing role in the field with an emphasis on recent and ongoing research from our laboratory. PMID:26476597

Can NMR solve some significant challenges in metabolomics?

NASA Astrophysics Data System (ADS)

Nagana Gowda, G. A.; Raftery, Daniel

2015-11-01

The field of metabolomics continues to witness rapid growth driven by fundamental studies, methods development, and applications in a number of disciplines that include biomedical science, plant and nutrition sciences, drug development, energy and environmental sciences, toxicology, etc. NMR spectroscopy is one of the two most widely used analytical platforms in the metabolomics field, along with mass spectrometry (MS). NMR's excellent reproducibility and quantitative accuracy, its ability to identify structures of unknown metabolites, its capacity to generate metabolite profiles using intact bio-specimens with no need for separation, and its capabilities for tracing metabolic pathways using isotope labeled substrates offer unique strengths for metabolomics applications. However, NMR's limited sensitivity and resolution continue to pose a major challenge and have restricted both the number and the quantitative accuracy of metabolites analyzed by NMR. Further, the analysis of highly complex biological samples has increased the demand for new methods with improved detection, better unknown identification, and more accurate quantitation of larger numbers of metabolites. Recent efforts have contributed significant improvements in these areas, and have thereby enhanced the pool of routinely quantifiable metabolites. Additionally, efforts focused on combining NMR and MS promise opportunities to exploit the combined strength of the two analytical platforms for direct comparison of the metabolite data, unknown identification and reliable biomarker discovery that continue to challenge the metabolomics field. This article presents our perspectives on the emerging trends in NMR-based metabolomics and NMR's continuing role in the field with an emphasis on recent and ongoing research from our laboratory.

1H-NMR and UPLC-MS metabolomics: Functional tools for exploring chemotypic variation in Sceletium tortuosum from two provinces in South Africa.

PubMed

Zhao, Jianping; Khan, Ikhlas A; Combrinck, Sandra; Sandasi, Maxleene; Chen, Weiyang; Viljoen, Alvaro M

2018-05-17

Sceletium tortuosum (Aizoaceae) is widely recognised for the treatment of stress, anxiety and depression, as well as for obsessive compulsive disorders. A comprehensive intraspecies chemotypic variation study, using samples harvested from two distinct regions of South Africa, was done using both proton nuclear magnetic resonance ( 1 H-NMR) spectroscopy of methanol extracts (N = 145) and ultra performance liquid chromatography-mass spectrometry (UPLC-MS) of acid/base extracts (N = 289). Chemometric analysis of the 1 H-NMR data indicated two main clusters that were region-specific (Northern Cape and Western Cape provinces). Two dimensional (2D) NMR was used to identify analytes that contributed to the clustering as revealed by the S-plot. The sceletium alkaloids, pinitol and two alkylamines, herein reported for the first time from S. tortuosum, were identified as markers that distinguished the two groups. Relative quantification of the marker analytes conducted by qNMR indicated that samples from the Northern Cape generally contained higher concentrations of all the markers than samples from the Western Cape. Quantitative analysis of the four mesembrine alkaloids using a validated UPLC-photo diode array (PDA) detection method confirmed the results of qNMR with regard to the total alkaloid concentrations. Samples from the Northern Cape Province were found to contain, on average, very high total alkaloids, ranging from 4938.0 to 9376.8 mg/kg dry w. Regarding the Western Cape samples, the total yields of the four mesembrine alkaloids were substantially lower (averages 16.4-4143.2 mg/kg). Hierarchical cluster analysis of the UPLC-MS data, based on the alkaloid chemistry, revealed three branches, with one branch comprising samples primarily from the Northern Cape that seemed somewhat chemically conserved, while the other two branches represented mainly samples from the Western Cape. The construction of an orthogonal projections to latent structures-discriminant analysis model and subsequent loadings plot, allowed alkaloid markers to be identified for each cluster. The diverse sceletium alkaloid chemistry of samples from the three clusters may facilitate the recognition of alkaloid profiles, rather than individual compounds, that exert targeted effects on various brain receptors involved in stress, anxiety or depression. Copyright © 2018 Elsevier Ltd. All rights reserved.

Sterols from Sargassum oligocystum, a brown algae from the Persian Gulf, and their bioactivity.

PubMed

Permeh, Parisa; Saeidnia, Soodabeh; Mashinchian-Moradi, Ali; Gohari, Ahmad R

2012-01-01

Sargassum oligocystum (Heterokontophyta) is one of the most abundant algae distributed in the Persian Gulf. In this study, the cytotoxic effects of this algae on brine shrimp larvae were evaluated and the main sterols of the algae identified. Separation and purification of the compounds was carried out using silica gel column chromatography and HPLC to obtain eight pure compounds, 1-8. Structural elucidation of the constituents was based on the data obtained from (1)H-NMR, (13)C-NMR, HSQC, HMBC, DEPT and EI-MS. The compounds separated from S. oligocystum were identified as 22-dehydrocholesterol (1), cholesterol (2), fucosterol (3), 29-hydroperoxystigmasta-5,24(28)-dien-3β-ol (4), 24-hydroperoxy-24-vinylcholesterol (5), a mixture of 24(S)-hydroxy-24-vinylcholesterol (6) and 24(R)-hydroxy-24-vinylcholesterol (7), and ostreasterol (8) based on their spectral data and from comparison with those previously reported in the literature.

Phytochemical investigation and hair growth studies on the rhizomes of Nardostachys jatamansi DC

PubMed Central

Gottumukkala, Venkateswara Rao; Annamalai, Tiruganasambandham; Mukhopadhyay, Triptikumar

2011-01-01

Nardostachys jatamansi DC rhizomes were subjected to extraction, fractionation, and isolation of terpenoid compounds. Three terpenoid compounds were isolated which are nardal, jatamansic acid, and nardin. These compounds were identified based on physical and spectral data (UV, IR,1H and13C NMR, 2D NMR, Mass) and comparison with authentic compounds. The crude extract, fractions, and two of the isolated compounds were tested for their hair growth activity. The hair growth studies showed good activities for the extract, fraction, and the isolated compounds. PMID:21716625

Applications of SHAPES screening in drug discovery.

PubMed

Lepre, Christopher A; Peng, Jeffrey; Fejzo, Jasna; Abdul-Manan, Norzehan; Pocas, Jennifer; Jacobs, Marc; Xie, Xiaoling; Moore, Jonathan M

2002-12-01

The SHAPES strategy combines nuclear magnetic resonance (NMR) screening of a library of small drug-like molecules with a variety of complementary methods, such as virtual screening, high throughput enzymatic assays, combinatorial chemistry, X-ray crystallography, and molecular modeling, in a directed search for new medicinal chemistry leads. In the past few years, the SHAPES strategy has found widespread utility in pharmaceutical research. To illustrate a variety of different implementations of the method, we will focus in this review on recent applications of the SHAPES strategy in several drug discovery programs at Vertex Pharmaceuticals.

Comprehensive non-targeted analysis of contaminated groundwater of a former ammunition destruction site using 1H-NMR and HPLC-SPE-NMR/TOF-MS.

PubMed

Godejohann, Markus; Heintz, Lea; Daolio, Cristina; Berset, Jean-Daniel; Muff, Daniel

2009-09-15

The aim of the present study was to explore the capabilities of the combination of 1H NMR (proton nuclear magnetic resonance) mixture analysis and HPLC-SPE-NMR/TOF-MS (high-performance liquid chromatography coupled to solid-phase extraction and nuclear magnetic resonance and time-of-flight mass spectrometry) for the characterization of xenobiotic contaminants in groundwater samples. As an example, solid-phase extracts of two groundwater samples taken from a former ammunition destruction site in Switzerland were investigated. 1H NMR spectra of postcolumn SPE enriched compounds, together with accurate mass measurements, allowed the structural elucidation of unknowns. This untargeted approach allowed us to identify expected residues of explosives such as 2,4,6-trinitrotoluene (2,4,6-TNT), Hexogen (RDX) and Octogen (HMX), degradation products of TNT (1,3,5-trinitrobenzene (1,3,5-TNB), 2-amino-4,6-dinitrotoluene (2-A-4,6-DNT), 3,5-dinitrophenol (3,5-DNP), 3,5-dinitroaniline (3,5-DNA), 2,6-dinitroanthranite, and 2-Hydroxy-4,6-dinitrobenzonitrile), benzoic acid, Bisphenol A (a known endocrine disruptor compound), and some toxicologically relevant additives for propelling charges: Centralite I (1,3-diethyl-1,3-diphenylurea), DPU (N,N-diphenylurethane), N,N-diphenylcarbamate (Acardite II), and N-methyl-N-phenylurethane. To our knowledge, this is the first report of the presence of these additives in environmental samples. Extraction recoveries for Centralite I and DPU have been determined. Contaminants identified by our techniques were quantified based on HPLC-UV (HPLC-ultraviolet detection) and 1H NMR mixture analysis. The concentrations of the contaminants ranged between 0.1 and 48 microg/L assuming 100% recovery for the SPE step.

An overview of methods using (13)C for improved compound identification in metabolomics and natural products.

PubMed

Clendinen, Chaevien S; Stupp, Gregory S; Ajredini, Ramadan; Lee-McMullen, Brittany; Beecher, Chris; Edison, Arthur S

2015-01-01

Compound identification is a major bottleneck in metabolomics studies. In nuclear magnetic resonance (NMR) investigations, resonance overlap often hinders unambiguous database matching or de novo compound identification. In liquid chromatography-mass spectrometry (LC-MS), discriminating between biological signals and background artifacts and reliable determination of molecular formulae are not always straightforward. We have designed and implemented several NMR and LC-MS approaches that utilize (13)C, either enriched or at natural abundance, in metabolomics applications. For LC-MS applications, we describe a technique called isotopic ratio outlier analysis (IROA), which utilizes samples that are isotopically labeled with 5% (test) and 95% (control) (13)C. This labeling strategy leads to characteristic isotopic patterns that allow the differentiation of biological signals from artifacts and yield the exact number of carbons, significantly reducing possible molecular formulae. The relative abundance between the test and control samples for every IROA feature can be determined simply by integrating the peaks that arise from the 5 and 95% channels. For NMR applications, we describe two (13)C-based approaches. For samples at natural abundance, we have developed a workflow to obtain (13)C-(13)C and (13)C-(1)H statistical correlations using 1D (13)C and (1)H NMR spectra. For samples that can be isotopically labeled, we describe another NMR approach to obtain direct (13)C-(13)C spectroscopic correlations. These methods both provide extensive information about the carbon framework of compounds in the mixture for either database matching or de novo compound identification. We also discuss strategies in which (13)C NMR can be used to identify unknown compounds from IROA experiments. By combining technologies with the same samples, we can identify important biomarkers and corresponding metabolites of interest.

Structural analysis and biomedical applications of dextran produced by a new isolate Pediococcus pentosaceus screened from biodiversity hot spot Assam.

PubMed

Patel, Seema; Kasoju, Naresh; Bora, Utpal; Goyal, Arun

2010-09-01

Dextran produced by a natural isolate of Pediococcus pentosaceus, screened from Assam, in the Northeastern region of India, was estimated, purified, structure characterised and functionality analysed. The dextran concentration in the cell free supernatant of the isolate P. pentosaceus was 10.2mg/ml. FT-IR analysis revealed the hydroxyl and carboxyl functional groups present in the dextran. (1)H NMR and (13)C NMR spectral data revealed that the dextran has a linear backbone of alpha-(1-->6) linked D-glucose residues. The decrease in viscosity of dextran solution with the increase in shear rate, threw light on its typical non-Newtonian pseudoplastic behaviour. The cytotoxicity tests on human cervical cancer (HeLa) cell line was studied which showed the dextran is non-toxic and biocompatible, rendering it safe for drug delivery, tissue engineering and various other biomedical applications. (c) 2010 Elsevier Ltd. All rights reserved.

Polymeric material prepared from Schiff base based on O-carboxymethyl chitosan and its Cu(II) and Pd(II) complexes

NASA Astrophysics Data System (ADS)

Baran, Talat; Menteş, Ayfer

2016-07-01

In this study, a new eco-friendly Schiff base based on O-carboxymethyl chitosan ([OCMCS-7a]) and its copper(II) and palladium(II) complexes were synthesized. Characterizations of [OCMCS-7a] and its metal complexes were conducted using FTIR, 1H NMR, 13C NMR, TG/DTG, XRD, SEM-EDAX, ICP, UV-VIS, GC-MS, elemental analysis, magnetic moment and molar conductivity measurements. The degree of substitution (DS) of [OCMCS-7a] was determined by elemental analysis to be 0.44. It was shown by the solubility test that [OCMCS-7a] was completely soluble in water. Surface images of chitosan, [OCMCS-7a] and its Cu(II) and Pd(II) complexes were investigated using the SEM-EDAX technique. Their thermal behaviors and crystallinities of the synthesized complexes were determined by TG/DTG and X-ray powder diffraction techniques, respectively. The metal contents of the obtained complexes were determined using an ICP-OES instrument. From the analyses, it was noted that the thermal stabilities and crystallinities of [OCMCS-7a] and its complexes decreased compared to chitosan. As a consequence of surface screening, it was also noted that the surface structure of the chitosan was smoother than that of the obtained compounds.

WaVPeak: picking NMR peaks through wavelet-based smoothing and volume-based filtering.

PubMed

Liu, Zhi; Abbas, Ahmed; Jing, Bing-Yi; Gao, Xin

2012-04-01

Nuclear magnetic resonance (NMR) has been widely used as a powerful tool to determine the 3D structures of proteins in vivo. However, the post-spectra processing stage of NMR structure determination usually involves a tremendous amount of time and expert knowledge, which includes peak picking, chemical shift assignment and structure calculation steps. Detecting accurate peaks from the NMR spectra is a prerequisite for all following steps, and thus remains a key problem in automatic NMR structure determination. We introduce WaVPeak, a fully automatic peak detection method. WaVPeak first smoothes the given NMR spectrum by wavelets. The peaks are then identified as the local maxima. The false positive peaks are filtered out efficiently by considering the volume of the peaks. WaVPeak has two major advantages over the state-of-the-art peak-picking methods. First, through wavelet-based smoothing, WaVPeak does not eliminate any data point in the spectra. Therefore, WaVPeak is able to detect weak peaks that are embedded in the noise level. NMR spectroscopists need the most help isolating these weak peaks. Second, WaVPeak estimates the volume of the peaks to filter the false positives. This is more reliable than intensity-based filters that are widely used in existing methods. We evaluate the performance of WaVPeak on the benchmark set proposed by PICKY (Alipanahi et al., 2009), one of the most accurate methods in the literature. The dataset comprises 32 2D and 3D spectra from eight different proteins. Experimental results demonstrate that WaVPeak achieves an average of 96%, 91%, 88%, 76% and 85% recall on (15)N-HSQC, HNCO, HNCA, HNCACB and CBCA(CO)NH, respectively. When the same number of peaks are considered, WaVPeak significantly outperforms PICKY. WaVPeak is an open source program. The source code and two test spectra of WaVPeak are available at http://faculty.kaust.edu.sa/sites/xingao/Pages/Publications.aspx. The online server is under construction. statliuzhi@xmu.edu.cn; ahmed.abbas@kaust.edu.sa; majing@ust.hk; xin.gao@kaust.edu.sa.

WaVPeak: picking NMR peaks through wavelet-based smoothing and volume-based filtering

PubMed Central

Liu, Zhi; Abbas, Ahmed; Jing, Bing-Yi; Gao, Xin

2012-01-01

Motivation: Nuclear magnetic resonance (NMR) has been widely used as a powerful tool to determine the 3D structures of proteins in vivo. However, the post-spectra processing stage of NMR structure determination usually involves a tremendous amount of time and expert knowledge, which includes peak picking, chemical shift assignment and structure calculation steps. Detecting accurate peaks from the NMR spectra is a prerequisite for all following steps, and thus remains a key problem in automatic NMR structure determination. Results: We introduce WaVPeak, a fully automatic peak detection method. WaVPeak first smoothes the given NMR spectrum by wavelets. The peaks are then identified as the local maxima. The false positive peaks are filtered out efficiently by considering the volume of the peaks. WaVPeak has two major advantages over the state-of-the-art peak-picking methods. First, through wavelet-based smoothing, WaVPeak does not eliminate any data point in the spectra. Therefore, WaVPeak is able to detect weak peaks that are embedded in the noise level. NMR spectroscopists need the most help isolating these weak peaks. Second, WaVPeak estimates the volume of the peaks to filter the false positives. This is more reliable than intensity-based filters that are widely used in existing methods. We evaluate the performance of WaVPeak on the benchmark set proposed by PICKY (Alipanahi et al., 2009), one of the most accurate methods in the literature. The dataset comprises 32 2D and 3D spectra from eight different proteins. Experimental results demonstrate that WaVPeak achieves an average of 96%, 91%, 88%, 76% and 85% recall on 15N-HSQC, HNCO, HNCA, HNCACB and CBCA(CO)NH, respectively. When the same number of peaks are considered, WaVPeak significantly outperforms PICKY. Availability: WaVPeak is an open source program. The source code and two test spectra of WaVPeak are available at http://faculty.kaust.edu.sa/sites/xingao/Pages/Publications.aspx. The online server is under construction. Contact: statliuzhi@xmu.edu.cn; ahmed.abbas@kaust.edu.sa; majing@ust.hk; xin.gao@kaust.edu.sa PMID:22328784

31P NMR Characterization of Tricin and Its Structurally Similar Flavonoids

DOE PAGES

Li, Mi; Pu, Yunqiao; Tschaplinski, Timothy J.; ...

2017-04-24

Tricin, a flavonoid metabolite, has been recently identified as a component of lignin in select monocot plants. This finding has initiated consideration on updating the lignin biosynthesis pathway. Here, we report a rapid method of determination of tricin in corn stover lignin, based on 31P nuclear magnetic resonance (NMR) spectroscopy by phosphitylating with 2-chloro-4,4,5,5-tetramethyl-1,3,2-dioxaphospholane (TMDP). Nine other flavonoids, with similar structure to tricin, have also been examined using the current method. The application of 31P NMR enables rapid identification of tricin-like flavonoids in the heterogeneous lignin polymer. The well resolved spectroscopic peaks from these derivatized flavonoids and lignin functional groupsmore » provide important information for the determination of flavonoids individually or their association with lignin.« less

31P NMR Characterization of Tricin and Its Structurally Similar Flavonoids

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Mi; Pu, Yunqiao; Tschaplinski, Timothy J.

Tricin, a flavonoid metabolite, has been recently identified as a component of lignin in select monocot plants. This finding has initiated consideration on updating the lignin biosynthesis pathway. Here, we report a rapid method of determination of tricin in corn stover lignin, based on 31P nuclear magnetic resonance (NMR) spectroscopy by phosphitylating with 2-chloro-4,4,5,5-tetramethyl-1,3,2-dioxaphospholane (TMDP). Nine other flavonoids, with similar structure to tricin, have also been examined using the current method. The application of 31P NMR enables rapid identification of tricin-like flavonoids in the heterogeneous lignin polymer. The well resolved spectroscopic peaks from these derivatized flavonoids and lignin functional groupsmore » provide important information for the determination of flavonoids individually or their association with lignin.« less

Monitoring the Electrochemical Processes in the Lithium–Air Battery by Solid State NMR Spectroscopy

PubMed Central

2013-01-01

A multi-nuclear solid-state NMR approach is employed to investigate the lithium–air battery, to monitor the evolution of the electrochemical products formed during cycling, and to gain insight into processes affecting capacity fading. While lithium peroxide is identified by 17O solid state NMR (ssNMR) as the predominant product in the first discharge in 1,2-dimethoxyethane (DME) based electrolytes, it reacts with the carbon cathode surface to form carbonate during the charging process. 13C ssNMR provides evidence for carbonate formation on the surface of the carbon cathode, the carbonate being removed at high charging voltages in the first cycle, but accumulating in later cycles. Small amounts of lithium hydroxide and formate are also detected in discharged cathodes and while the hydroxide formation is reversible, the formate persists and accumulates in the cathode upon further cycling. The results indicate that the rechargeability of the battery is limited by both the electrolyte and the carbon cathode stability. The utility of ssNMR spectroscopy in directly detecting product formation and decomposition within the battery is demonstrated, a necessary step in the assessment of new electrolytes, catalysts, and cathode materials for the development of a viable lithium–oxygen battery. PMID:24489976

High-throughput screening for bioactive components from traditional Chinese medicine.

PubMed

Zhu, Yanhui; Zhang, Zhiyun; Zhang, Meng; Mais, Dale E; Wang, Ming-Wei

2010-12-01

Throughout the centuries, traditional Chinese medicine has been a rich resource in the development of new drugs. Modern drug discovery, which relies increasingly on automated high throughput screening and quick hit-to-lead development, however, is confronted with the challenges of the chemical complexity associated with natural products. New technologies for biological screening as well as library building are in great demand in order to meet the requirements. Here we review the developments in these techniques under the perspective of their applicability in natural product drug discovery. Methods in library building, component characterizing, biological evaluation, and other screening methods including NMR and X-ray diffraction are discussed.

An Eco-Friendly Ultrasound-Assisted Synthesis of Novel Fluorinated Pyridinium Salts-Based Hydrazones and Antimicrobial and Antitumor Screening

PubMed Central

Rezki, Nadjet; Al-Sodies, Salsabeel A.; Aouad, Mohamed R.; Bardaweel, Sanaa; Messali, Mouslim; El Ashry, El Sayed H.

2016-01-01

The present work reports an efficient synthesis of fluorinated pyridinium salts-based hydrazones under both conventional and eco-friendly ultrasound procedures. The synthetic approach first involves the preparation of halogenated pyridinium salts through the condensation of isonicotinic acid hydrazide (1) with p-fluorobenzaldehyde (2) followed by the nucleophilic alkylation of the resulting N-(4-fluorobenzylidene)isonicotinohydrazide (3) with a different alkyl iodide. The iodide counteranion of 5–10 was subjected to an anion exchange metathesis reaction in the presence of an excess of the appropriate metal salts to afford a new series of fluorinated pyridinium salts tethering a hydrazone linkage 11–40. Ultrasound irradiation led to higher yields in considerably less time than the conventional methods. The newly synthesized ILs were well-characterized with FT-IR, 1H NMR, 13C NMR, 11B, 19F, 31P and mass spectral analyses. The ILs were also screened for their antimicrobial and antitumor activities. Within the series, the salts tethering fluorinated counter anions 11–13, 21–23, 31–33 and 36–38 were found to be more potent against all bacterial and fungal strains at MIC 4–8 µg/mL. The in vitro antiproliferative activity was also investigated against four tumor cell lines (human ductal breast epithelial tumor T47D, human breast adenocarcinoma MCF-7, human epithelial carcinoma HeLa and human epithelial colorectal adenocarcinoma Caco-2) using the MTT assay, which revealed that promising antitumor activity was exhibited by compounds 5, 12 and 14. PMID:27213367

Structure of dimethylphenyl betaine hydrochloride studied by X-ray diffraction, DFT calculation, NMR and FTIR spectra

NASA Astrophysics Data System (ADS)

Szafran, M.; Katrusiak, A.; Dega-Szafran, Z.; Kowalczyk, I.

2013-01-01

The structure of dimethylphenyl betaine hydrochloride (1) has been studied by X-ray diffraction, DFT calculations, NMR and FTIR spectra. The crystals are monoclinic, space group P21/c. In the crystal, the Cl- anion is connected with protonated betaine through the O-H⋯Cl- hydrogen bond of 2.943(2) Å. The structures in the gas phase (2) and water solution (3) have been optimized by the B3LYP/6-311++G(d,p) approach and the geometrical results have been compared with the X-ray data of 1. The FTIR spectrum of the solid compound is consistent with the X-ray results. The probable assignments of the anharmonic experimental vibrational frequencies of the investigated chloride (1) based on the calculated harmonic frequencies in water solution (3) are proposed. The correlations between the experimental 1H and 13C NMR chemical shifts (δexp) of 1 in D2O and the magnetic isotropic shielding constants (σcalc) calculated by the GIAO/B3LYP/6-311G++(d,p) approach, using the screening solvation model (COSMO), δexp = a + b σcalc, for optimized molecule 3 in water solution are linear and correctly reproduce the experimental chemical shifts.

Chemical characterization of a prominent phosphomonoester resonance from mammalian brain. 31P and 1H NMR analysis at 4.7 and 14.1 tesla

NASA Astrophysics Data System (ADS)

Pettegrew, J. W.; Kopp, S. J.; Dadok, J.; Minshew, N. J.; Feliksik, J. M.; Glonek, T.; Cohen, M. M.

A prominent 31P NMR resonance at 3.84 ppm in mammalian brain has been identified as ethanolamine phosphate. The identification was based on 1H and 31P NMR findings (including pH titrations) at 4.7 and 14.1 T, as well as thin-layer chromatography studies. We previously incorrectly assigned the 3.84 ppm resonance to ribose-5-phosphate. The incorrect assignment occurred because the two compounds have very similar 31P chemical shifts, and because we did not carefully consider the effects of counter ions and ionic strengths when interpreting the 31P chemical shifts. In separate preliminary studies we have demonstrated ethanolamine phosphate to be high in immature developing brain and in the degenerating brain of Alzheimer's and Huntington's disease patients. Ethanolamine phosphate may therefore serve as a sensitive marker of membrane phospholipid turnover for both in vitro and in vivo31P NMR studies.

Taxol-producing [corrected] fungal endophyte, Pestalotiopsis species isolated from Taxus cuspidata.

PubMed

Kumaran, Rangarajulu Senthil; Kim, Hyung Joo; Hur, Byung-Ki

2010-11-01

The endophytic fungi, Pestalotiopsis versicolor and Pestalotiopsis neglecta, were isolated from the healthy leaves and bark of the Japanese Yew tree, Taxus cuspidata. The fungal species were identified by their characteristic culture morphology and molecular analysis. For the first time, the test fungi were screened for the production of taxol in modified liquid medium. The presence of taxol was confirmed by HPLC, (1)H NMR, and LC-MS methods of analysis. The maximum amount of taxol production in P. versicolor was recorded as 478 μg/l. The production rate was increased to 9560-fold than that found in the culture broth of earlier reported fungus, Taxomyces andreanae. The extracted fungal taxol showed a strong cytotoxic activity in the in vitro culture of tested human cancer cells by apoptotic assay indicating that the increase in taxol concentration induces increased cell death. A PCR-based screening for ts, a unique gene in the formation of the taxane skeleton, confirmed the molecular blueprint for taxol biosynthesis. The results designate that the fungal endophyte, P. versicolor, is an excellent candidate for an alternate source of taxol supply and can also serve as a potential species for genetic engineering to enhance the production of taxol to a higher level. Copyright © 2010 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Screening Mixtures of Small Molecules for Binding to Multiple Sites on the Surface Tetanus Toxin C Fragment by Bioaffinity NMR

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cosman, M; Zeller, L; Lightstone, F C

2002-01-01

The clostridial neurotoxins include the closely related tetanus (TeNT) and botulinum (BoNT) toxins. Botulinum toxin is used to treat severe muscle disorders and as a cosmetic wrinkle reducer. Large quantities of botulinum toxin have also been produced by terrorists for use as a biological weapon. Because there are no known antidotes for these toxins, they thus pose a potential threat to human health whether by an accidental overdose or by a hostile deployment. Thus, the discovery of high specificity and affinity compounds that can inhibit their binding to neural cells can be used as antidotes or in the design ofmore » chemical detectors. Using the crystal structure of the C fragment of the tetanus toxin (TetC), which is the cell recognition and cell surface binding domain, and the computational program DOCK, sets of small molecules have been predicted to bind to two different sites located on the surface of this protein. While Site-1 is common to the TeNT and BoNTs, Site-2 is unique to TeNT. Pairs of these molecules from each site can then be linked together synthetically to thereby increase the specificity and affinity for this toxin. Electrospray ionization mass spectroscopy was used to experimentally screen each compound for binding. Mixtures containing binders were further screened for activity under biologically relevant conditions using nuclear magnetic resonance (NMR) methods. The screening of mixtures of compounds offers increased efficiency and throughput as compared to testing single compounds and can also evaluate how possible structural changes induced by the binding of one ligand can influence the binding of the second ligand. In addition, competitive binding experiments with mixtures containing ligands predicted to bind the same site could identify the best binder for that site. NMR transfer nuclear Overhauser effect (trNOE) confirm that TetC binds doxorubicin but that this molecule is displaced by N-acetylneuraminic acid (sialic acid) in a mixture that also contains 3-sialyllactose (another predicted site 1 binder) and bisbenzimide 33342 (non-binder). A series of five predicted Site-2 binders were then screened sequentially in the presence of the Site-1 binder doxorubicin. These experiments showed that the compounds lavendustin A and naphthofluorescein-di-({beta}-D-galactopyranoside) binds along with doxorubicin to TetC. Further experiments indicate that doxorubicin and lavendustin are potential candidates to use in preparing a bidendate inhibitor specific for TetC. The simultaneous binding of two different predicted Site-2 ligands to TetC suggests that they may bind multiple sites. Another possibility is that the conformations of the binding sites are dynamic and can bind multiple diverse ligands at a single site depending on the pre-existing conformation of the protein, especially when doxorubicin is already bound.« less

Development of a method to screen and isolate potential xanthine oxidase inhibitors from Panax japlcus var via ultrafiltration liquid chromatography combined with counter-current chromatography.

PubMed

Li, Sainan; Tang, Ying; Liu, Chunming; Li, Jing; Guo, Liping; Zhang, Yuchi

2015-03-01

Panax japlcus var is a typical Chinese herb with a large number of saponins existing in all parts of it. The common methods of screening and isolating saponins are mostly labor-intensive and time-consuming. In this study, a new assay based on ultrafiltration-liquid chromatography-mass spectrometry (UF-LC-MS) was developed for the rapid screening and identifying of the ligands for xanthine oxidase from the extract of P. japlcus. Six saponins were identified as xanthine oxidase inhibitors from the extract. Subsequently, the specific binding ligands, namely, 24 (R)-majoroside R1, chikusetsusaponin IVa, oleanolic acid-28-O-β-D-glucopyranoside, notoginsenoside Fe, ginsenoside Rb2 and ginsenoside Rd (the purities of them were 95.74%, 96.12%, 93.19%, 94.83%, 95.07% and 94.62%, respectively) were separated by high-speed counter-current chromatography (HSCCC). The component ratio of the solvent system of HSCCC was calculated with the help of a multiexponential function model was optimized. The partition coefficient (K) values of the target compounds and resolutions of peaks were employed as the research indicators, and exponential function and binomial formulas were used to optimize the solvent system and flow rate of the mobile phases in a two-stage separation. An optimized two-phase solvent system composed of ethyl acetate, isopropanol, 0.1% aqueous formic acid (1.9:1.0:1.3, v/v/v, for the first-stage) and that composed of methylene chloride, acetonitrile, isopropanol, 0.1% aqueous formic acid (5.6:1.0:2.4:5.2, v/v/v/v, for the second-stage) were used to isolate the six compounds from P. japlcus. The targeted compounds isolated, collected and purified by HSCCC were analyzed by high performance liquid chromatography (UPLC), and the chemical structures of all the six compounds were identified by UV, MS and NMR. The results demonstrate that UF-LC-MS combined with HSCCC might provide not only a powerful tool for screening and isolating xanthine oxidase inhibitors in complex samples but also a useful platform for discovering bioactive compounds for the prevention and treatment of gout. Copyright © 2014 Elsevier B.V. All rights reserved.

New cholinesterase inhibitors for Alzheimer's disease: Structure Activity Studies (SARs) and molecular docking of isoquinolone and azepanone derivatives.

PubMed

Bacalhau, Patrícia; San Juan, Amor A; Marques, Carolina S; Peixoto, Daniela; Goth, Albertino; Guarda, Cátia; Silva, Mara; Arantes, Sílvia; Caldeira, A Teresa; Martins, Rosário; Burke, Anthony J

2016-08-01

A library of isoquinolinone and azepanone derivatives were screened for both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activity. The strategy adopted included (a) in vitro biological assays, against eel AChE (EeAChE) and equine serum BuChE (EqBuChE) in order to determine the compounds IC50 and their dose-response activity, consolidated by (b) molecular docking studies to evaluate the docking poses and interatomic interactions in the case of the hit compounds, validated by STD-NMR studies. Compound (1f) was identified as one of these hits with an IC50 of 89.5μM for EeAChE and 153.8μM for EqBuChE, (2a) was identified as a second hit with an IC50 of 108.4μM (EeAChE) and 277.8μM (EqBuChE). In order to gain insights into the binding mode and principle active site interactions of these molecules, (R)-(1f) along with 3 other analogues (also as the R-enantiomer) were docked into both RhAChE and hBuChE models. Galantamine was used as the benchmark. The docking study was validated by performing an STD-NMR study of (1f) with EeAChE using galantamine as the benchmark. Copyright © 2016 Elsevier Inc. All rights reserved.

Composition and Quantitation of Microalgal Lipids by ERETIC 1H NMR Method

PubMed Central

Nuzzo, Genoveffa; Gallo, Carmela; d’Ippolito, Giuliana; Cutignano, Adele; Sardo, Angela; Fontana, Angelo

2013-01-01

Accurate characterization of biomass constituents is a crucial aspect of research in the biotechnological application of natural products. Here we report an efficient, fast and reproducible method for the identification and quantitation of fatty acids and complex lipids (triacylglycerols, glycolipids, phospholipids) in microalgae under investigation for the development of functional health products (probiotics, food ingredients, drugs, etc.) or third generation biofuels. The procedure consists of extraction of the biological matrix by modified Folch method and direct analysis of the resulting material by proton nuclear magnetic resonance (1H NMR). The protocol uses a reference electronic signal as external standard (ERETIC method) and allows assessment of total lipid content, saturation degree and class distribution in both high throughput screening of algal collection and metabolic analysis during genetic or culturing studies. As proof of concept, the methodology was applied to the analysis of three microalgal species (Thalassiosira weissflogii, Cyclotella cryptica and Nannochloropsis salina) which drastically differ for the qualitative and quantitative composition of their fatty acid-based lipids. PMID:24084790

Screening molecular associations with lipid membranes using natural abundance 13C cross-polarization magic-angle spinning NMR and principal component analysis.

PubMed

Middleton, David A; Hughes, Eleri; Madine, Jillian

2004-08-11

We describe an NMR approach for detecting the interactions between phospholipid membranes and proteins, peptides, or small molecules. First, 1H-13C dipolar coupling profiles are obtained from hydrated lipid samples at natural isotope abundance using cross-polarization magic-angle spinning NMR methods. Principal component analysis of dipolar coupling profiles for synthetic lipid membranes in the presence of a range of biologically active additives reveals clusters that relate to different modes of interaction of the additives with the lipid bilayer. Finally, by representing profiles from multiple samples in the form of contour plots, it is possible to reveal statistically significant changes in dipolar couplings, which reflect perturbations in the lipid molecules at the membrane surface or within the hydrophobic interior.

Examining national and district-level trends in neonatal health in Peru through an equity lens: a success story driven by political will and societal advocacy.

PubMed

Huicho, Luis; Huayanay-Espinoza, Carlos A; Herrera-Perez, Eder; Niño de Guzman, Jessica; Rivera-Ch, Maria; Restrepo-Méndez, Maria Clara; Barros, Aluisio J D

2016-09-12

Peru has impressively reduced its neonatal mortality rate (NMR). We aimed, for the period 2000-2013, to: (a) describe national and district NMR variations over time; (b) assess NMR trends by wealth quintile and place of residence; (c) describe evolution of mortality causes; (d) assess completeness of registered mortality; (e) assess coverage and equity of NMR-related interventions; and (f) explore underlying driving factors. We compared national NMR time trends from different sources. To describe NMR trends by wealth quintiles, place of residence and districts, we pooled data on births and deaths by calendar year for neonates born to women interviewed in multiple surveys. We disaggregated coverage of NMR-related interventions by wealth quintiles and place of residence. To identify success factors, we ran regression analyses and combined desk reviews with qualitative interviews and group discussions. NMR fell by 51 % from 2000 to 2013, second only to Brazil in Latin America. Reduction was higher in rural and poorest segments (52 and 58 %). District NMR change varied by source. Regarding cause-specific NMRs, prematurity decreased from 7.0 to 3.2 per 1,000 live births, intra-partum related events from 2.9 to 1.2, congenital abnormalities from 2.4 to 1.8, sepsis from 1.9 to 0.8, pneumonia from 0.9 to 0.4, and other conditions from 1.2 to 0.7. Under-registration of neonatal deaths decreased recently, more in districts with higher development index and lower rural population. Coverage of family planning, antenatal care and skilled birth attendance increased more in rural areas and in the poorest quintile. Regressions did not show consistent associations between mortality and predictors. During the study period social determinants improved substantially, and dramatic out-of-health-sector and health-sector changes occurred. Rural areas and the poorest quintile experienced greater NMR reduction. This progress was driven, within a context of economic growth and poverty reduction, by a combination of strong societal advocacy and political will, which translated into pro-poor implementation of evidence-based interventions with a rights-based approach. Although progress in Peru for reducing NMR has been remarkable, future challenges include closing remaining gaps for urban and rural populations and improving newborn health with qualified staff and intermediate- and intensive-level health facilities.

CSI 3.0: a web server for identifying secondary and super-secondary structure in proteins using NMR chemical shifts

PubMed Central

Hafsa, Noor E.; Arndt, David; Wishart, David S.

2015-01-01

The Chemical Shift Index or CSI 3.0 (http://csi3.wishartlab.com) is a web server designed to accurately identify the location of secondary and super-secondary structures in protein chains using only nuclear magnetic resonance (NMR) backbone chemical shifts and their corresponding protein sequence data. Unlike earlier versions of CSI, which only identified three types of secondary structure (helix, β-strand and coil), CSI 3.0 now identifies total of 11 types of secondary and super-secondary structures, including helices, β-strands, coil regions, five common β-turns (type I, II, I′, II′ and VIII), β hairpins as well as interior and edge β-strands. CSI 3.0 accepts experimental NMR chemical shift data in multiple formats (NMR Star 2.1, NMR Star 3.1 and SHIFTY) and generates colorful CSI plots (bar graphs) and secondary/super-secondary structure assignments. The output can be readily used as constraints for structure determination and refinement or the images may be used for presentations and publications. CSI 3.0 uses a pipeline of several well-tested, previously published programs to identify the secondary and super-secondary structures in protein chains. Comparisons with secondary and super-secondary structure assignments made via standard coordinate analysis programs such as DSSP, STRIDE and VADAR on high-resolution protein structures solved by X-ray and NMR show >90% agreement between those made with CSI 3.0. PMID:25979265

Synthesis, spectroscopic characterization, DFT optimization and biological activities of Schiff bases and their metal (II) complexes

NASA Astrophysics Data System (ADS)

Rauf, Abdur; Shah, Afzal; Munawar, Khurram Shahzad; Khan, Abdul Aziz; Abbasi, Rashda; Yameen, Muhammad Arfat; Khan, Asad Muhammad; Khan, Abdur Rahman; Qureshi, Irfan Zia; Kraatz, Heinz-Bernhard; Zia-ur-Rehman

2017-10-01

A Novel Schiff base, 3-(((4-chlorophenyl)imino)methyl)benzene-1,2-diol (HL1) was successfully synthesized along with a structurally similar Schiff base 3-(((4-bromophenyl)imino)methyl)benzene-1,2-diol (HL2). Both the Schiff bases were used to synthesize their zinc (II) and cobalt (II) complexes. These compounds were characterized by FTIR, 1H NMR, 13C NMR and elemental analysis. Metal complexes were confirmed by TGA. Crystals of Schiff bases were also characterized by X-ray analysis and experimental parameters were found in line with the theoretical parameters. Quantum mechanical approach was also used to fine useful structural parameters and to ensure the geometry of metal complexes. The photometric behaviors of all the synthesized compounds were investigated in a wide pH range using BR buffers. The appearance of isosbestic points indicated the existence of Schiff bases in more than one isomeric form. Moreover, these compounds were screened for enzyme inhibition; antibacterial, cytotoxic and in vivo antidiabetic activities and compounds were found active against one or other activity. Results indicate that ZnL22 is a good inhibitor of alkaline phosphatase enzyme and possess highest potential against diabetes, blood cholesterol level and cancer cells. This effort just provides preliminary data for some biological properties. Further investigations are required to precisely determine mechanistic pathways of their use towards drug development.

NMR-based metabolomics reveals the metabolite profiles of Vibrio parahaemolyticus under ferric iron stimulation.

PubMed

Zhou, Jun; Lu, Chenyang; Zhang, Dijun; Ma, Chennv; Su, Xiurong

2017-08-01

Vibrio parahaemolyticus is a halophilic bacterium endemic to coastal areas, and its pathogenicity has caused widespread seafood poisoning. In our previous research, the protein expression of V. parahaemolyticus in Fe 3+ medium was determined using isobaric tags for relative and absolute quantitation (iTRAQ). Here, nuclear magnetic resonance (NMR) was used to detect changes in the V. parahaemolyticus metabolome. NMR spectra were obtained using methanol-water extracts of intracellular metabolites from V. parahaemolyticus under various culture conditions, and 62 metabolites were identified, including serine, arginine, alanine, ornithine, tryptophan, glutamine, malate, NAD + , NADP + , oxypurinol, xanthosine, dCTP, uracil, thymine, hypoxanthine, and betaine. Among these, 21 metabolites were up-regulated after the stimulation of the cells by ferric iron, and 9 metabolites were down-regulated. These metabolites are involved in amino acid and protein synthesis, energy metabolism, DNA and RNA synthesis and osmolality. Based on these results, we conclude that Fe 3+ influences the metabolite profiles of V. parahaemolyticus.

NMR-based metabolic study of fruits of Physalis peruviana L. grown in eight different Peruvian ecosystems.

PubMed

Maruenda, Helena; Cabrera, Rodrigo; Cañari-Chumpitaz, Cristhian; Lopez, Juan M; Toubiana, David

2018-10-01

The berry of Physalis peruviana L. (Solanaceae) represents an important socio-economical commodity for Latin America. The absence of a clear phenotype renders it difficult to trace its place of origin. In this study, Cape gooseberries from eight different regions within the Peruvian Andes were profiled for their metabolism implementing a NMR platform. Twenty-four compounds could be unequivocally identified and sixteen quantified. One-way ANOVA and post-hoc Tukey test revealed that all of the quantified metabolites changed significantly among regions: Bambamarca I showed the most accumulated significant differences. The coefficient of variation demonstrated high phenotypic plasticity for amino acids, while sugars displayed low phenotypic plasticity. Correlation analysis highlighted the closely coordinated behavior of the amino acid profile. Finally, PLS-DA revealed a clear separation among the regions based on their metabolic profiles, accentuating the discriminatory capacity of NMR in establishing significant phytochemical differences between producing regions of the fruit of P. peruviana L. Copyright © 2018 Elsevier Ltd. All rights reserved.

Fragment-based discovery of potent inhibitors of the anti-apoptotic MCL-1 protein.

PubMed

Petros, Andrew M; Swann, Steven L; Song, Danying; Swinger, Kerren; Park, Chang; Zhang, Haichao; Wendt, Michael D; Kunzer, Aaron R; Souers, Andrew J; Sun, Chaohong

2014-03-15

Apoptosis is regulated by the BCL-2 family of proteins, which is comprised of both pro-death and pro-survival members. Evasion of apoptosis is a hallmark of malignant cells. One way in which cancer cells achieve this evasion is thru overexpression of the pro-survival members of the BCL-2 family. Overexpression of MCL-1, a pro-survival protein, has been shown to be a resistance factor for Navitoclax, a potent inhibitor of BCL-2 and BCL-XL. Here we describe the use of fragment screening methods and structural biology to drive the discovery of novel MCL-1 inhibitors from two distinct structural classes. Specifically, cores derived from a biphenyl sulfonamide and salicylic acid were uncovered in an NMR-based fragment screen and elaborated using high throughput analog synthesis. This culminated in the discovery of selective and potent inhibitors of MCL-1 that may serve as promising leads for medicinal chemistry optimization efforts. Copyright © 2014 Elsevier Ltd. All rights reserved.

Studies on silicon NMR characterization and kinetic modeling of the structural evolution of siloxane-based materials and their applications in drug delivery and adsorption

NASA Astrophysics Data System (ADS)

Ambati, Jyothirmai

This dissertation presents studies of the synthetic processes and applications of siloxane-based materials. Kinetic investigations of bridged organoalkoxysilanes that are precursors to organic-inorganic hybrid polysilsesquioxanes are a primary focus. Quick gelation despite extensive cyclization is found during the polymerization of bridged silane precursors except for silanes with certain short bridges. This work is an attempt to characterize and understand some of the distinct features of bridged silanes using experimental characterization, kinetic modeling and simulation. In addition to this, the dissertation shows how the properties of siloxane-materials can be engineered for drug delivery and adsorption. The phase behavior of polymerizing mixtures is first investigated to identify the solutions that favor kinetic characterization. Microphase separation is found to cause gradual loss of NMR signal for certain initial compositions. Distortionless Enhancement by Polarization Transfer 29Si NMR is employed to identify the products of polymerization of some short-bridged silanes under no signal loss conditions. This technique requires knowing indirect 29Si-1H scalar coupling constants which sometimes cannot be measured due to second-order effects. However, the B3LYP density functional method with 6-31G basis set is found to predict accurate 29Si- 1H coupling constants of organoalkoxysilanes and siloxanes. The scalar coupling constants thus estimated are employed to resolve non-trivial coupled NMR spectra and quantitative kinetic modeling is performed using the DEPT Si NMR transients. In order to investigate the role of the organic bridging group, the structural evolution of bridged and non-bridged silanes are compared using Monte Carlo simulations. Kinetic and simulation models suggest that cyclization plays a key role right from the onset of polymerization for bridged silanes even more than in non-bridged silanes. The simulations indicate that the carbosiloxane rings formed from short-bridged precursors slow down but do not prevent gelation. The tuning of siloxane-based materials for adsorption technologies are also discussed here. In the first example, antioxidant enzyme loading is investigated as a means to reduce oxidative stress generated by silica nanoparticle drug carriers. Materials are engineered for promising enzyme loading and protection from proteolysis. Second, the potential of copper sulfate impregnation to enhance adsorption of ammonia by silica is explored by molecular simulation. KEYWORDS: Sol-gel Polymerization, Kinetic Investigation, Si NMR, Bridged Silanes, DFT Calculations.

The health indicators associated with screen-based sedentary behavior among adolescent girls: a systematic review.

PubMed

Costigan, Sarah A; Barnett, Lisa; Plotnikoff, Ronald C; Lubans, David R

2013-04-01

Evidence suggests sitting time is independently associated with a range of health issues in adults, yet the relationship between sedentary behavior and health indicators in young people is less clear. Age-related increases in sedentary behavior are well-documented; the behavioral patterns of adolescent girls are of particular concern. More than one third of adolescent girls' sedentary behavior time is accumulated through use of recreational screen-based behaviors. The objective of this review was to investigate the association between recreational screen-based sedentary behavior and the physical, behavioral, and psychosocial health indicators for adolescent girls. A secondary objective was to identify studies that have adjusted sedentary behavior indicators for physical activity. A structured electronic search of all publication years (through December 2011) was conducted to identify studies in: CINAHL, Communications and Mass Media Complete, ERIC, MEDLINE with Full Text, PsycINFO, and SPORTDiscus with Full Text. Included publications were observational and interventional studies involving adolescent girls (12-18 years) that examined associations between screen-based, sedentary behavior and health indicators (physical, psychosocial, and/or behavioral). The search identified 33 studies that evaluated health indicators of screen-based sedentary behaviors among adolescent girls. Strong evidence for a positive association between screen-based sedentary behavior and weight status was found. A positive association was observed between screen-time and sleep problems, musculoskeletal pain and depression. Negative associations were identified between screen time and physical activity/fitness, screen time and psychological well-being, and screen time and social support. The relationship between screen-based sedentary behavior and diet quality was inconclusive. Less than half of the studies adjusted sedentary behavior indicators for physical activity. Screen-based sedentary behavior is associated with a range of adverse health consequences, but additional longitudinal studies are needed to better understand the health impacts. In addition, screen-time guidelines for youth should be regularly revised and updated to reflect rapid technological changes. Copyright © 2013 Society for Adolescent Health and Medicine. Published by Elsevier Inc. All rights reserved.

In situ {sup 13}C MAS NMR study of n-hexane conversion on Pt and Pd supported on basic materials

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ivanova, I.I.; Pasau-Claerbout, A.; Seivert, M.

n-Hexane conversion was studied in situ on Pt and Pd supported on aluminum-stabilized magnesium oxide and Pt on Zeolite KL catalysts (Pt/Mg(Al)O, Pd/Mg(Al)O and Pt/KL) by means of {sup 13}C MAS NMR spectroscopy. n-Hexane 1-{sup 13}C was used as a labelled reactant. Forty NMR lines corresponding to 14 different products were resolved and identified. The NMR line assignments were confirmed by adsorption of model compounds. The NMR results were further quantified and compared with continuous flow microreactor tests. Four parallel reaction pathways were identified under flow conditions: isomerization, cracking, dehydrocyclization, and dehydrogenation. Aromatization occurs via two reaction routes: (1) n-hexanemore » dehydrogenation towards hexadienes and hexatrienes, followed by dehydrogenation of a cyclic intermediate. The former reaction pathway is prevented under NMR batch conditions. High pressures induced in the NMR cells at high reaction temperatures (573, 653 K) shift the reaction equilibrium towards hydrogenation. NMR experiments showed that on Pt catalysts aromatization occurs via a cyclohexane intermediate, whereas on Pd it takes place via methylcyclopentane ring enlargement. 54 refs., 15 figs., 3 tabs.« less

A new method of evaluating tight gas sands pore structure from nuclear magnetic resonance (NMR) logs

NASA Astrophysics Data System (ADS)

Xiao, Liang; Mao, Zhi-qiang; Xie, Xiu-hong

2016-04-01

Tight gas sands always display such characteristics of ultra-low porosity, permeability, high irreducible water, low resistivity contrast, complicated pore structure and strong heterogeneity, these make that the conventional methods are invalid. Many effective gas bearing formations are considered as dry zones or water saturated layers, and cannot be identified and exploited. To improve tight gas sands evaluation, the best method is quantitative characterizing rock pore structure. The mercury injection capillary pressure (MICP) curves are advantageous in predicting formation pore structure. However, the MICP experimental measurements are limited due to the environment and economy factors, this leads formation pore structure cannot be consecutively evaluated. Nuclear magnetic resonance (NMR) logs are considered to be promising in evaluating rock pore structure. Generally, to consecutively quantitatively evaluate tight gas sands pore structure, the best method is constructing pseudo Pc curves from NMR logs. In this paper, based on the analysis of lab experimental results for 20 core samples, which were drilled from tight gas sandstone reservoirs of Sichuan basin, and simultaneously applied for lab MICP and NMR measurements, the relationships of piecewise power function between nuclear magnetic resonance (NMR) transverse relaxation T2 time and pore-throat radius Rc are established. A novel method, which is used to transform NMR reverse cumulative curve as pseudo capillary pressure (Pc) curve is proposed, and the corresponding model is established based on formation classification. By using this model, formation pseudo Pc curves can be consecutively synthesized. The pore throat radius distribution, and pore structure evaluation parameters, such as the average pore throat radius (Rm), the threshold pressure (Pd), the maximum pore throat radius (Rmax) and so on, can also be precisely extracted. After this method is extended into field applications, several tight gas sandstone reservoirs are processed, and the predicted results are compared with core derived results. Good consistency between evaluated results with core derived results illustrates the dependability of the proposed method. Comparing with the previous methods, this presented model is much more theoretical, and the applicability is much improved. Combining with the evaluated results, our target tight gas sands are well evaluated, and many potential gas-bearing layers are effectively identified.

Trace level detection of compounds related to the chemical weapons convention by 1H-detected 13C NMR spectroscopy executed with a sensitivity-enhanced, cryogenic probehead.

PubMed

Cullinan, David B; Hondrogiannis, George; Henderson, Terry J

2008-04-15

Two-dimensional 1H-13C HSQC (heteronuclear single quantum correlation) and fast-HMQC (heteronuclear multiple quantum correlation) pulse sequences were implemented using a sensitivity-enhanced, cryogenic probehead for detecting compounds relevant to the Chemical Weapons Convention present in complex mixtures. The resulting methods demonstrated exceptional sensitivity for detecting the analytes at trace level concentrations. 1H-13C correlations of target analytes at < or = 25 microg/mL were easily detected in a sample where the 1H solvent signal was approximately 58,000-fold more intense than the analyte 1H signals. The problem of overlapping signals typically observed in conventional 1H spectroscopy was essentially eliminated, while 1H and 13C chemical shift information could be derived quickly and simultaneously from the resulting spectra. The fast-HMQC pulse sequences generated magnitude mode spectra suitable for detailed analysis in approximately 4.5 h and can be used in experiments to efficiently screen a large number of samples. The HSQC pulse sequences, on the other hand, required roughly twice the data acquisition time to produce suitable spectra. These spectra, however, were phase-sensitive, contained considerably more resolution in both dimensions, and proved to be superior for detecting analyte 1H-13C correlations. Furthermore, a HSQC spectrum collected with a multiplicity-edited pulse sequence provided additional structural information valuable for identifying target analytes. The HSQC pulse sequences are ideal for collecting high-quality data sets with overnight acquisitions and logically follow the use of fast-HMQC pulse sequences to rapidly screen samples for potential target analytes. Use of the pulse sequences considerably improves the performance of NMR spectroscopy as a complimentary technique for the screening, identification, and validation of chemical warfare agents and other small-molecule analytes present in complex mixtures and environmental samples.

Neonatal, infant and under-five mortalities in Nigeria: An examination of trends and drivers (2003-2013)

PubMed Central

Fagbamigbe, Adeniyi Francis

2017-01-01

Neonatal (NMR), infant (IMR) and under-five (U5M) mortality rates remain high in Nigeria. Evidence-based knowledge of trends and drivers of child mortality will aid proper interventions needed to combat the menace. Therefore, this study assessed the trends and drivers of NMR, IMR, and U5M over a decade in Nigeria. A nationally representative data from three consecutive Nigeria Demographic and Household Surveys (NDHS) was used. A total of 66,158 live births within the five years preceding the 2003 (6029), 2008 (28647) and 2013 (31482) NDHS were included in the analyses. NMR was computed using proportions while IMR and U5 were computed using life table techniques embedded in Stata version 12. Probit regression model and its associated marginal effects were used to identify the predisposing factors to NMR, IMR, and U5M. The NMR, IMR, and U5M per 1000 live births in 2003, 2008 and 2013 were 52, 41, 39; 100, 75, 69; and 201, 157, 128 respectively. The NMR, IMR, and U5M were consistently lower among children whose mothers were younger, living in rural areas and from richer households. Generally, the probability of neonate death in 2003, 2008 and 2013 were 0.049, 0.039 and 0.038 respectively, the probability of infant death was 0.093, 0.071 and 0.064 while the probability of under-five death was 0.140, 0.112 and 0.092 for the respective survey years. While adjusting for other variables, the likelihood of infant and under-five deaths was significantly reduced across the survey years. Maternal age, mothers’ education, place of residence, child’s sex, birth interval, weight at birth, skill of birth attendant, delivery by caesarean operation or not significantly influenced NMR, IMR, and U5M. The NMR, IMR, and U5M in Nigeria reduced over the studied period. Multi-sectoral interventions targeted towards the identified drivers should be instituted to improve child survival. PMID:28793340

Which BRCA genetic testing programs are ready for implementation in health care? A systematic review of economic evaluations.

PubMed

D'Andrea, Elvira; Marzuillo, Carolina; De Vito, Corrado; Di Marco, Marco; Pitini, Erica; Vacchio, Maria Rosaria; Villari, Paolo

2016-12-01

There is considerable evidence regarding the efficacy and effectiveness of BRCA genetic testing programs, but whether they represent good use of financial resources is not clear. Therefore, we aimed to identify the main health-care programs for BRCA testing and to evaluate their cost-effectiveness. We performed a systematic review of full economic evaluations of health-care programs involving BRCA testing. Nine economic evaluations were included, and four main categories of BRCA testing programs were identified: (i) population-based genetic screening of individuals without cancer, either comprehensive or targeted based on ancestry; (ii) family history (FH)-based genetic screening, i.e., testing individuals without cancer but with FH suggestive of BRCA mutation; (iii) familial mutation (FM)-based genetic screening, i.e., testing individuals without cancer but with known familial BRCA mutation; and (iv) cancer-based genetic screening, i.e., testing individuals with BRCA-related cancers. Currently BRCA1/2 population-based screening represents good value for the money among Ashkenazi Jews only. FH-based screening is potentially very cost-effective, although further studies that include costs of identifying high-risk women are needed. There is no evidence of cost-effectiveness for BRCA screening of all newly diagnosed cases of breast/ovarian cancers followed by cascade testing of relatives, but programs that include tools for identifying affected women at higher risk for inherited forms are promising. Cost-effectiveness is highly sensitive to the cost of BRCA1/2 testing.Genet Med 18 12, 1171-1180.

Genome-wide association study of a nicotine metabolism biomarker in African American smokers: impact of chromosome 19 genetic influences.

PubMed

Chenoweth, Meghan J; Ware, Jennifer J; Zhu, Andy Z X; Cole, Christopher B; Cox, Lisa Sanderson; Nollen, Nikki; Ahluwalia, Jasjit S; Benowitz, Neal L; Schnoll, Robert A; Hawk, Larry W; Cinciripini, Paul M; George, Tony P; Lerman, Caryn; Knight, Joanne; Tyndale, Rachel F

2018-03-01

The activity of CYP2A6, the major nicotine-inactivating enzyme, is measurable in smokers using the nicotine metabolite ratio (NMR; 3'hydroxycotinine/cotinine). Due to its role in nicotine clearance, the NMR is associated with smoking behaviours and response to pharmacotherapies. The NMR is highly heritable (~80%), and on average lower in African Americans (AA) versus whites. We previously identified several reduce and loss-of-function CYP2A6 variants common in individuals of African descent. Our current aim was to identify novel genetic influences on the NMR in AA smokers using genome-wide approaches. Genome-wide association study (GWAS). Multiple sites within Canada and the United States. AA smokers from two clinical trials: Pharmacogenetics of Nicotine Addiction Treatment (PNAT)-2 (NCT01314001; n = 504) and Kick-it-at-Swope (KIS)-3 (NCT00666978; n = 450). Genome-wide SNP genotyping, the NMR (phenotype) and population substructure and NMR covariates. Meta-analysis revealed three independent chromosome 19 signals (rs12459249, rs111645190 and rs185430475) associated with the NMR. The top overall hit, rs12459249 (P = 1.47e-39; beta = 0.59 per C (versus T) allele, SE = 0.045), located ~9.5 kb 3' of CYP2A6, remained genome-wide significant after controlling for the common (~10% in AA) non-functional CYP2A6*17 allele. In contrast, rs111645190 and rs185430475 were not genome-wide significant when controlling for CYP2A6*17. In total, 96 signals associated with the NMR were identified; many were not found in prior NMR GWASs in individuals of European descent. The top hits were also associated with the NMR in a third cohort of AA (KIS2; n = 480). None of the hits were in UGT or OCT2 genes. Three independent chromosome 19 signals account for ~20% of the variability in the nicotine metabolite ratio in African American smokers. The hits identified may contribute to inter-ethnic variability in nicotine metabolism, smoking behaviours and tobacco-related disease risk. © 2017 Society for the Study of Addiction.

Can NMR solve some significant challenges in metabolomics?

PubMed

Nagana Gowda, G A; Raftery, Daniel

2015-11-01

The field of metabolomics continues to witness rapid growth driven by fundamental studies, methods development, and applications in a number of disciplines that include biomedical science, plant and nutrition sciences, drug development, energy and environmental sciences, toxicology, etc. NMR spectroscopy is one of the two most widely used analytical platforms in the metabolomics field, along with mass spectrometry (MS). NMR's excellent reproducibility and quantitative accuracy, its ability to identify structures of unknown metabolites, its capacity to generate metabolite profiles using intact bio-specimens with no need for separation, and its capabilities for tracing metabolic pathways using isotope labeled substrates offer unique strengths for metabolomics applications. However, NMR's limited sensitivity and resolution continue to pose a major challenge and have restricted both the number and the quantitative accuracy of metabolites analyzed by NMR. Further, the analysis of highly complex biological samples has increased the demand for new methods with improved detection, better unknown identification, and more accurate quantitation of larger numbers of metabolites. Recent efforts have contributed significant improvements in these areas, and have thereby enhanced the pool of routinely quantifiable metabolites. Additionally, efforts focused on combining NMR and MS promise opportunities to exploit the combined strength of the two analytical platforms for direct comparison of the metabolite data, unknown identification and reliable biomarker discovery that continue to challenge the metabolomics field. This article presents our perspectives on the emerging trends in NMR-based metabolomics and NMR's continuing role in the field with an emphasis on recent and ongoing research from our laboratory. Copyright © 2015 Elsevier Inc. All rights reserved.

Tumour xenograft detection through quantitative analysis of the metabolic profile of urine in mice

NASA Astrophysics Data System (ADS)

Moroz, Jennifer; Turner, Joan; Slupsky, Carolyn; Fallone, Gino; Syme, Alasdair

2011-02-01

The metabolic content of urine from NIH III nude mice (n = 22) was analysed before and after inoculation with human glioblastoma multiforme (GBM) cancer cells. An age- and gender-matched control population (n = 14) was also studied to identify non-tumour-related changes. Urine samples were collected daily for 6 weeks, beginning 1 week before cell injection. Metabolite concentrations were obtained via targeted profiling with Chenomx Suite 5.1, based on nuclear magnetic resonance (NMR) spectra acquired on an Oxford 800 MHz cold probe NMR spectrometer. The Wilcoxon rank sum test was used to evaluate the significance of the change in metabolite concentration between the two time points. Both the metabolite concentrations and the ratios of pairs of metabolites were studied. The complicated inter-relationships between metabolites were assessed through partial least-squares discriminant analysis (PLS-DA). Receiver operating characteristic (ROC) curves were generated for all variables and the area under the curve (AUC) calculated. The data indicate that the number of statistically significant changes in metabolite concentrations was more pronounced in the tumour-bearing population than in the control animals. This was also true of the ratios of pairs of metabolites. ROC analysis suggests that the ratios were better able to differentiate between the pre- and post-injection samples compared to the metabolite concentrations. PLS-DA models produced good separation between the populations and had the best AUC results (all models exceeded 0.937). These results demonstrate that metabolomics may be used as a screening tool for GBM cells grown in xenograft models in mice.

Insights into (S)-rivastigmine inhibition of butyrylcholinesterase (BuChE): Molecular docking and saturation transfer difference NMR (STD-NMR).

PubMed

Bacalhau, Patrícia; San Juan, Amor A; Goth, Albertino; Caldeira, A Teresa; Martins, Rosário; Burke, Anthony J

2016-08-01

Rivastigmine is a very important drug prescribed for the treatment of Alzheimer's disease (AD) symptoms. It is a dual inhibitor, in that it inhibits both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). For our screening program on the discovery of new rivastigmine analogue hits for human butyrylcholinesterase (hBuChE) inhibition, we investigated the interaction of this inhibitor with BuChE using the complimentary approach of the biophysical method, saturation transfer difference (STD)-NMR and molecular docking. This allowed us to obtain essential information on the key binding interactions between the inhibitor and the enzyme to be used for screening of hit compounds. The main conclusions obtained from this integrated study was that the most dominant interactions were (a) H-bonding between the carbamate carbonyl of the inhibitor and the NH group of the imidazole unit of H434, (b) stacking of the aromatic unit of the inhibitor and the W82 aromatic unit in the choline binding pocket via π-π interactions and (c) possible CH/π interactions between the benzylic methyl group and the N-methyl groups of the inhibitor and W82 of the enzyme. Copyright © 2016 Elsevier Inc. All rights reserved.

Creating Novel Activated Factor XI Inhibitors through Fragment Based Lead Generation and Structure Aided Drug Design

PubMed Central

Fjellström, Ola; Akkaya, Sibel; Beisel, Hans-Georg; Eriksson, Per-Olof; Erixon, Karl; Gustafsson, David; Jurva, Ulrik; Kang, Daiwu; Karis, David; Knecht, Wolfgang; Nerme, Viveca; Nilsson, Ingemar; Olsson, Thomas; Redzic, Alma; Roth, Robert; Sandmark, Jenny; Tigerström, Anna; Öster, Linda

2015-01-01

Activated factor XI (FXIa) inhibitors are anticipated to combine anticoagulant and profibrinolytic effects with a low bleeding risk. This motivated a structure aided fragment based lead generation campaign to create novel FXIa inhibitor leads. A virtual screen, based on docking experiments, was performed to generate a FXIa targeted fragment library for an NMR screen that resulted in the identification of fragments binding in the FXIa S1 binding pocket. The neutral 6-chloro-3,4-dihydro-1H-quinolin-2-one and the weakly basic quinolin-2-amine structures are novel FXIa P1 fragments. The expansion of these fragments towards the FXIa prime side binding sites was aided by solving the X-ray structures of reported FXIa inhibitors that we found to bind in the S1-S1’-S2’ FXIa binding pockets. Combining the X-ray structure information from the identified S1 binding 6-chloro-3,4-dihydro-1H-quinolin-2-one fragment and the S1-S1’-S2’ binding reference compounds enabled structure guided linking and expansion work to achieve one of the most potent and selective FXIa inhibitors reported to date, compound 13, with a FXIa IC50 of 1.0 nM. The hydrophilicity and large polar surface area of the potent S1-S1’-S2’ binding FXIa inhibitors compromised permeability. Initial work to expand the 6-chloro-3,4-dihydro-1H-quinolin-2-one fragment towards the prime side to yield molecules with less hydrophilicity shows promise to afford potent, selective and orally bioavailable compounds. PMID:25629509

Targeting lysine specific demethylase 4A (KDM4A) tandem TUDOR domain - A fragment based approach.

PubMed

Upadhyay, Anup K; Judge, Russell A; Li, Leiming; Pithawalla, Ron; Simanis, Justin; Bodelle, Pierre M; Marin, Violeta L; Henry, Rodger F; Petros, Andrew M; Sun, Chaohong

2018-06-01

The tandem TUDOR domains present in the non-catalytic C-terminal half of the KDM4A, 4B and 4C enzymes play important roles in regulating their chromatin localizations and substrate specificities. They achieve this regulatory role by binding to different tri-methylated lysine residues on histone H3 (H3-K4me3, H3-K23me3) and histone H4 (H4-K20me3) depending upon the specific chromatin environment. In this work, we have used a 2D-NMR based fragment screening approach to identify a novel fragment (1a), which binds to the KDM4A-TUDOR domain and shows modest competition with H3-K4me3 binding in biochemical as well as in vitro cell based assays. A co-crystal structure of KDM4A TUDOR domain in complex with 1a shows that the fragment binds stereo-specifically to the methyl lysine binding pocket forming a network of strong hydrogen bonds and hydrophobic interactions. We anticipate that the fragment 1a can be further developed into a novel allosteric inhibitor of the KDM4 family of enzymes through targeting their C-terminal tandem TUDOR domain. Copyright © 2018 Elsevier Ltd. All rights reserved.

Catching TFSI: A Computational-Experimental Approach to β-Cyclodextrin-Based Host-Guest Systems as electrolytes for Li-Ion Batteries.

PubMed

Jeschke, Steffen; Jankowski, Piotr; Best, Adam S; Johansson, Patrik

2018-03-12

Cyclodextrins (CDs) are pyranoside-based macromolecules with a hydrophobic cavity to encapsulate small molecules. They are used as molecular vehicles, for instance in pharmaceutical drug delivery or as solubility enhancer of monomers for their polymerization in aqueous solution. In this context, it was discovered about 10 years ago that the bis(trifluoromethylsulonyl)imide (TFSI) anion forms host-guest complexes with βCD in aqueous media. This sparked interest in using the TFSI anion in lithium-based battery electrolytes open for its encapsulation by βCD as an attractive approach to increase the contribution of the cation to the total ion conductivity. By using semi-empirical quantum mechanical (SQM) methods and the conductor-like screening model for a real solvent (COSMO-RS), a randomly methylated βCD (RMβCD) is here identified as a suitable host for TFSI when using organic solvents often used in battery technology. By combining molecular dynamics (MD) simulations with different NMR and FTIR experiments, the formation of the corresponding RMβCD-TFSI complex was investigated. Finally, the effects of the addition RMβCD to a set of electrolytes on the ion conductivity are measured and explained using three distinct scenarios. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Structure-Function Study of Tertiary Amines as Switchable Polarity Solvents

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aaron D. Wilson; Frederick F. Stewart

2014-02-01

A series of tertiary amines have been screened for their function as switchable polarity solvents (SPS). The relative ratios of tertiary amine and carbonate species as well as maximum possible concentration were determined through quantitative 1H and 13C NMR spectroscopy. The viscosities of the polar SPS solutions were measured and ranged from near water in dilute systems through to gel formation at high concentrations. The van't Hoff indices for SPS solutions were measured through freezing point depression studies as a proxy for osmotic pressures. A new form of SPS with an amine : carbonate ratio significantly greater than unity hasmore » been identified. Tertiary amines that function as SPS at ambient pressures appear to be limited to molecules with fewer than 12 carbons. The N,N-dimethyl-n-alkylamine structure has been identified as important to the function of an SPS.« less

Alkaloids from roots of Stephania rotunda and their cholinesterase inhibitory activity.

PubMed

Hung, Tran Manh; Dang, Nguyen Hai; Kim, Jin Cheol; Jang, Han-Su; Ryoo, Sung-Woo; Lee, Jeong Hyung; Choi, Jae Sue; Bae, Kihwan; Min, Byung Sun

2010-10-01

In the course of screening plants used in folk medicine as memory enhancers, a 70% ethanolic extract of Stephania rotunda roots showed significant AChE inhibitory activity. Repeated column chromatography led to the isolation of a new protoberberine alkaloid, which we named stepharotudine (1), and seven known compounds (2-8). The chemical structures of the isolated compounds were elucidated based on extensive 1D and 2D NMR spectroscopic data. Compounds 1-8 were investigated in vitro for their anticholinesterase activity using a rat cortex AChE enzyme. © Georg Thieme Verlag KG Stuttgart · New York.

Discovering [superscript 13]C NMR, [superscript 1]H NMR, and IR Spectroscopy in the General Chemistry Laboratory through a Sequence of Guided-Inquiry Exercises

ERIC Educational Resources Information Center

Iler, H. Darrell; Justice, David; Brauer, Shari; Landis, Amanda

2012-01-01

This sequence of three guided-inquiry labs is designed for a second-semester general chemistry course and challenges students to discover basic theoretical principles associated with [superscript 13]C NMR, [superscript 1]H NMR, and IR spectroscopy. Students learn to identify and explain basic concepts of magnetic resonance and vibrational…

Synthesis, spectral and antimicrobial activity of Zn(II) complexes with Schiff bases derived from 2-hydrazino-5-[substituted phenyl]-1,3,4-thiadiazole and benzaldehyde/2-hydroxyacetophenone/indoline-2,3-dione

NASA Astrophysics Data System (ADS)

Singh, Ajay K.; Pandey, O. P.; Sengupta, S. K.

2013-09-01

Zn(II) complexes have been synthesized by reacting zinc acetate with Schiff bases derived from 2-hydrazino-5-[substituted phenyl]-1,3,4-thiadiazole and 2-hydroxyacetophenone/benzaldehyde/indoline-2,3-dione. All these complexes are soluble in DMF and DMSO; low molar conductance values indicate that they are non electrolytes. Elemental analyses suggest that the complexes have 1:2 metal to ligands stoichiometry of the types [ZnL2(H2O)2](L = monoanionic Schiff bases derived from 2-hydrazino-5-[substituted phenyl]-1,3,4-thiadiazole and 2-hydroxyacetophenone/indoline-2,3-dione) [ZnL2‧(OOCCH3)2(H2O)2](L‧ = neutral Schiff bases derived from 2-hydrazino-5-[substituted phenyl]-1,3,4-thiadiazole and benzaldehyde), and they were characterized by IR, 1H NMR, and 13C NMR. Particle sizes of synthesized compounds were measured with dynamic light scattering (DLS) analyser which indicates that particle diameter are of the range ca. 100-200 nm. All these Schiff bases and their complexes have also been screened for their antibacterial (Bacillus subtilis (B. subtilis), Escherichia coli (E. coli) and antifungal activities (Colletotrichum falcatum (C. falcatum), Aspergillus niger (A. niger), Fusarium oxysporium (F. oxysporium) Curvularia pallescence (C. pallescence). The antimicrobial activities have shown that upon complexation the activity increases.

Nutrition screening tools: an analysis of the evidence.

PubMed

Skipper, Annalynn; Ferguson, Maree; Thompson, Kyle; Castellanos, Victoria H; Porcari, Judy

2012-05-01

In response to questions about tools for nutrition screening, an evidence analysis project was developed to identify the most valid and reliable nutrition screening tools for use in acute care and hospital-based ambulatory care settings. An oversight group defined nutrition screening and literature search criteria. A trained analyst conducted structured searches of the literature for studies of nutrition screening tools according to predetermined criteria. Eleven nutrition screening tools designed to detect undernutrition in patients in acute care and hospital-based ambulatory care were identified. Trained analysts evaluated articles for quality using criteria specified by the American Dietetic Association's Evidence Analysis Library. Members of the oversight group assigned quality grades to the tools based on the quality of the supporting evidence, including reliability and validity data. One tool, the NRS-2002, received a grade I, and 4 tools-the Simple Two-Part Tool, the Mini-Nutritional Assessment-Short Form (MNA-SF), the Malnutrition Screening Tool (MST), and Malnutrition Universal Screening Tool (MUST)-received a grade II. The MST was the only tool shown to be both valid and reliable for identifying undernutrition in the settings studied. Thus, validated nutrition screening tools that are simple and easy to use are available for application in acute care and hospital-based ambulatory care settings.

Refinement of NMR structures using implicit solvent and advanced sampling techniques.

PubMed

Chen, Jianhan; Im, Wonpil; Brooks, Charles L

2004-12-15

NMR biomolecular structure calculations exploit simulated annealing methods for conformational sampling and require a relatively high level of redundancy in the experimental restraints to determine quality three-dimensional structures. Recent advances in generalized Born (GB) implicit solvent models should make it possible to combine information from both experimental measurements and accurate empirical force fields to improve the quality of NMR-derived structures. In this paper, we study the influence of implicit solvent on the refinement of protein NMR structures and identify an optimal protocol of utilizing these improved force fields. To do so, we carry out structure refinement experiments for model proteins with published NMR structures using full NMR restraints and subsets of them. We also investigate the application of advanced sampling techniques to NMR structure refinement. Similar to the observations of Xia et al. (J.Biomol. NMR 2002, 22, 317-331), we find that the impact of implicit solvent is rather small when there is a sufficient number of experimental restraints (such as in the final stage of NMR structure determination), whether implicit solvent is used throughout the calculation or only in the final refinement step. The application of advanced sampling techniques also seems to have minimal impact in this case. However, when the experimental data are limited, we demonstrate that refinement with implicit solvent can substantially improve the quality of the structures. In particular, when combined with an advanced sampling technique, the replica exchange (REX) method, near-native structures can be rapidly moved toward the native basin. The REX method provides both enhanced sampling and automatic selection of the most native-like (lowest energy) structures. An optimal protocol based on our studies first generates an ensemble of initial structures that maximally satisfy the available experimental data with conventional NMR software using a simplified force field and then refines these structures with implicit solvent using the REX method. We systematically examine the reliability and efficacy of this protocol using four proteins of various sizes ranging from the 56-residue B1 domain of Streptococcal protein G to the 370-residue Maltose-binding protein. Significant improvement in the structures was observed in all cases when refinement was based on low-redundancy restraint data. The proposed protocol is anticipated to be particularly useful in early stages of NMR structure determination where a reliable estimate of the native fold from limited data can significantly expedite the overall process. This refinement procedure is also expected to be useful when redundant experimental data are not readily available, such as for large multidomain biomolecules and in solid-state NMR structure determination.

Synthesis, in vitro anticancer and antimycobacterial evaluation of new 5-(2,5-dimethoxyphenyl)-1,3,4-thiadiazole-2-amino derivatives.

PubMed

Polkam, Naveen; Rayam, Parsharamulu; Anireddy, Jaya Shree; Yennam, Satyanarayana; Anantaraju, Hasitha Shilpa; Dharmarajan, Sriram; Perumal, Yogeeswari; Kotapalli, Sudha Sravanti; Ummanni, Ramesh; Balasubramanian, Sridhar

2015-04-01

A series of 2,5-disubstituted-1,3,4-thiadiazole derivatives 5a-5l, 7a-7e and 9 have been synthesised and screened for in vitro antimycobacterial activity against Mycobacterium smegmatis MC-155. In addition these compounds have also been screened for cytotoxic activity against cancer cell lines HT-29, MDA-MB-231 by MTT colorimetric assay. The compounds are well characterized by spectral analysis viz. (1)H NMR, (13)C NMR, FT-IR, mass and HRMS. Screening results indicate that compounds 5g, 7a possess good antitubercular activity with MIC value 65.74 and 40.86, respectively, compounds 5g, 7a, 7b, 7d, 7e and 9 displayed promising cytotoxic activity against the cell lines tested. 5g and 7a stand out to be potent antimycobacterial and anticancer agents among the tested series. Further the title compounds were also tested on human normal cells HEK293T and are found to be safer with lesser cytotoxicity. It is interesting to observe that compound 5g has come out to be safer, potent anticancer and antimycobacterial agent. Copyright © 2015 Elsevier Ltd. All rights reserved.

DNA Binding, Cleavage and Antibacterial Activity of Mononuclear Cu(II), Ni(II) and Co(II) Complexes Derived from Novel Benzothiazole Schiff Bases.

PubMed

Vamsikrishna, Narendrula; Kumar, Marri Pradeep; Tejaswi, Somapangu; Rambabu, Aveli; Shivaraj

2016-07-01

A series of novel bivalent metal complexes M(L1)2 and M(L2)2 where M = Cu(II), Ni(II), Co(II) and L1 = 2-((benzo [d] thiazol-6-ylimino)methyl)-4-bromophenol [BTEMBP], L2 = 1-((benzo [d] thiazol-6-ylimino)methyl) naphthalen-2-ol [BTEMNAPP] were synthesized. All the compounds have been characterized by elemental analysis, SEM, Mass, (1)H NMR, (13)C NMR, UV-Vis, IR, ESR, spectral data and magnetic susceptibility measurements. Based on the analytical and spectral data four-coordinated square planar geometry is assigned to all the complexes. DNA binding properties of these complexes have been investigated by electronic absorption spectroscopy, fluorescence and viscosity measurements. It is observed that these binary complexes strongly bind to calf thymus DNA by an intercalation mode. DNA cleavage efficacy of these complexes was tested in presence of H2O2 and UV light by gel electrophoresis and found that all the complexes showed better nuclease activity. Finally the compounds were screened for antibacterial activity against few pathogens and found that the complexes have potent biocidal activity than their free ligands.

Synthesis, structure-activity relationship and molecular docking of cyclohexenone based analogous as potent non-nucleoside reverse-transcriptase inhibitors

NASA Astrophysics Data System (ADS)

Nazar, Muhammad Faizan; Abdullah, Muhammad Imran; Badshah, Amir; Mahmood, Asif; Rana, Usman Ali; Khan, Salah Ud-Din

2015-04-01

The chalcones core in compounds is advantageously chosen effective synthons, which offer exciting perspectives in biological and pharmacological research. The present study reports the successful development of eight new cyclohexenone based anti-reverse transcriptase analogous using rational drug design synthesis principles. These new cyclohexenone derivatives (CDs) were synthesized by following a convenient route of Robinson annulation, and the molecular structure of these CDs were later confirmed by various analytical techniques such as 1H NMR, 13C NMR, FT-IR, UV-Vis spectroscopy and mass spectrometry. All the synthesized compounds were screened theoretically and experimentally against reverse transcriptase (RT) and found potentially active reverse transcriptase (RT) inhibitors. Of the compounds studied, the compound 2FC4 showed high interaction with RT at non-nucleoside binding site, contributing high free binding energy (ΔG -8.01 Kcal) and IC50 (0.207 μg/ml), respectively. Further results revealed that the compounds bearing more halogen groups, with additional hydrophobic character, offered superior anti-reverse transcriptase activity as compared to rest of compounds. It is anticipate that the present study would be very useful for the selection of potential reverse transcriptase inhibitors featuring inclusive pharmacological profiles.

CSI 3.0: a web server for identifying secondary and super-secondary structure in proteins using NMR chemical shifts.

PubMed

Hafsa, Noor E; Arndt, David; Wishart, David S

2015-07-01

The Chemical Shift Index or CSI 3.0 (http://csi3.wishartlab.com) is a web server designed to accurately identify the location of secondary and super-secondary structures in protein chains using only nuclear magnetic resonance (NMR) backbone chemical shifts and their corresponding protein sequence data. Unlike earlier versions of CSI, which only identified three types of secondary structure (helix, β-strand and coil), CSI 3.0 now identifies total of 11 types of secondary and super-secondary structures, including helices, β-strands, coil regions, five common β-turns (type I, II, I', II' and VIII), β hairpins as well as interior and edge β-strands. CSI 3.0 accepts experimental NMR chemical shift data in multiple formats (NMR Star 2.1, NMR Star 3.1 and SHIFTY) and generates colorful CSI plots (bar graphs) and secondary/super-secondary structure assignments. The output can be readily used as constraints for structure determination and refinement or the images may be used for presentations and publications. CSI 3.0 uses a pipeline of several well-tested, previously published programs to identify the secondary and super-secondary structures in protein chains. Comparisons with secondary and super-secondary structure assignments made via standard coordinate analysis programs such as DSSP, STRIDE and VADAR on high-resolution protein structures solved by X-ray and NMR show >90% agreement between those made with CSI 3.0. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Discovery of Small Molecules that Inhibit the Disordered Protein, p27Kip1

PubMed Central

Iconaru, Luigi I.; Ban, David; Bharatham, Kavitha; Ramanathan, Arvind; Zhang, Weixing; Shelat, Anang A.; Zuo, Jian; Kriwacki, Richard W.

2015-01-01

Disordered proteins are highly prevalent in biological systems, they control myriad signaling and regulatory processes, and their levels and/or cellular localization are often altered in human disease. In contrast to folded proteins, disordered proteins, due to conformational heterogeneity and dynamics, are not considered viable drug targets. We challenged this paradigm by identifying through NMR-based screening small molecules that bound specifically, albeit weakly, to the disordered cell cycle regulator, p27Kip1 (p27). Two groups of molecules bound to sites created by transient clusters of aromatic residues within p27. Conserved chemical features within these two groups of small molecules exhibited complementarity to their binding sites within p27, establishing structure-activity relationships for small molecule:disordered protein interactions. Finally, one compound counteracted the Cdk2/cyclin A inhibitory function of p27 in vitro, providing proof-of-principle that small molecules can inhibit the function of a disordered protein (p27) through sequestration in a conformation incapable of folding and binding to a natural regulatory target (Cdk2/cyclin A). PMID:26507530

Discovery of Small Molecules that Inhibit the Disordered Protein, p27 Kip1

DOE PAGES

Iconaru, Luigi I.; Ban, David; Bharatham, Kavitha; ...

2015-10-28

In disordered proteins we see that they are highly prevalent in biological systems. They control myriad signaling and regulatory processes, and their levels and/or cellular localization are often altered in human disease. In contrast to folded proteins, disordered proteins, due to conformational heterogeneity and dynamics, are not considered viable drug targets. We challenged this paradigm by identifying through NMR-based screening small molecules that bound specifically, albeit weakly, to the disordered cell cycle regulator, p27 Kip1 (p27). Moreover, two groups of molecules bound to sites created by transient clusters of aromatic residues within p27. Conserved chemical features within these two groupsmore » of small molecules exhibited complementarity to their binding sites within p27, establishing structure-activity relationships for small molecule: disordered protein interactions. Finally, one compound counteracted the Cdk2/cyclin A inhibitory function of p27 in vitro, providing proof-of- principle that small molecules can inhibit the function of a disordered protein (p27) through sequestration in a conformation incapable of folding and binding to a natural regulatory target (Cdk2/cyclin A).« less

The polar warhead of a TRIM24 bromodomain inhibitor rearranges a water-mediated interaction network.

PubMed

Liu, Jiuyang; Li, Fudong; Bao, Hongyu; Jiang, Yiyang; Zhang, Shuya; Ma, Rongsheng; Gao, Jia; Wu, Jihui; Ruan, Ke

2017-04-01

Tripartite motif-containing protein 24 (TRIM24) is closely correlated with multiple cancers, and a recent study demonstrated that the bromodomain of TRIM24 is essential for the proliferation of lethal castration-resistant prostate cancer. Here, we identify three new inhibitors of the TRIM24 bromodomain using NMR fragment-based screening. The crystal structures of two new inhibitors in complex with the TRIM24 bromodomain reveal that the water-bridged interaction network is conserved in the same fashion as those for known benzoimidazolone inhibitors. Interestingly, the polar substitution on the warhead of one new inhibitor pulls the whole ligand approximately 2 Å into the inner side pocket of the TRIM24 bromodomain, and thus exhibits a binding mode significantly different from other known bromodomain ligands. This mode provides a useful handle for further hit-to-lead evolution toward novel inhibitors of the TRIM24 bromodomain. Structural data are available in the PDB under the accession numbers 5H1T, 5H1U, and 5H1V. © 2017 Federation of European Biochemical Societies.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Iconaru, Luigi I.; Ban, David; Bharatham, Kavitha

In disordered proteins we see that they are highly prevalent in biological systems. They control myriad signaling and regulatory processes, and their levels and/or cellular localization are often altered in human disease. In contrast to folded proteins, disordered proteins, due to conformational heterogeneity and dynamics, are not considered viable drug targets. We challenged this paradigm by identifying through NMR-based screening small molecules that bound specifically, albeit weakly, to the disordered cell cycle regulator, p27 Kip1 (p27). Moreover, two groups of molecules bound to sites created by transient clusters of aromatic residues within p27. Conserved chemical features within these two groupsmore » of small molecules exhibited complementarity to their binding sites within p27, establishing structure-activity relationships for small molecule: disordered protein interactions. Finally, one compound counteracted the Cdk2/cyclin A inhibitory function of p27 in vitro, providing proof-of- principle that small molecules can inhibit the function of a disordered protein (p27) through sequestration in a conformation incapable of folding and binding to a natural regulatory target (Cdk2/cyclin A).« less

Isolation of 4,5-O-Dicaffeoylquinic Acid as a Pigmentation Inhibitor Occurring in Artemisia capillaris Thunberg and Its Validation In Vivo.

PubMed

Tabassum, Nadia; Lee, Ji-Hyung; Yim, Soon-Ho; Batkhuu, Galzad Javzan; Jung, Da-Woon; Williams, Darren R

2016-01-01

There is a continual need to develop novel and effective melanogenesis inhibitors for the prevention of hyperpigmentation disorders. The plant Artemisia capillaris Thunberg (Oriental Wormwood) was screened for antipigmentation activity using murine cultured cells (B16-F10 malignant melanocytes). Activity-based fractionation using HPLC and NMR analyses identified the compound 4,5-O-dicaffeoylquinic acid as an active component in this plant. 4,5-O-Dicaffeoylquinic acid significantly reduced melanin synthesis and tyrosinase activity in a dose-dependent manner in the melanocytes. In addition, 4,5-O-dicaffeoylquinic acid treatment reduced the expression of tyrosinase-related protein-1. Significantly, we could validate the antipigmentation activity of this compound in vivo, using a zebrafish model. Moreover, 4,5-O-dicaffeoylquinic acid did not show toxicity in this animal model. Our discovery of 4,5-O-dicaffeoylquinic acid as an inhibitor of pigmentation that is active in vivo shows that this compound can be developed as an active component for formulations to treat pigmentation disorders.

An update on the use of C. elegans for preclinical drug discovery: screening and identifying anti-infective drugs.

PubMed

Kim, Wooseong; Hendricks, Gabriel Lambert; Lee, Kiho; Mylonakis, Eleftherios

2017-06-01

The emergence of antibiotic-resistant and -tolerant bacteria is a major threat to human health. Although efforts for drug discovery are ongoing, conventional bacteria-centered screening strategies have thus far failed to yield new classes of effective antibiotics. Therefore, new paradigms for discovering novel antibiotics are of critical importance. Caenorhabditis elegans, a model organism used for in vivo, offers a promising solution for identification of anti-infective compounds. Areas covered: This review examines the advantages of C. elegans-based high-throughput screening over conventional, bacteria-centered in vitro screens. It discusses major anti-infective compounds identified from large-scale C. elegans-based screens and presents the first clinically-approved drugs, then known bioactive compounds, and finally novel small molecules. Expert opinion: There are clear advantages of using a C. elegans-infection based screening method. A C. elegans-based screen produces an enriched pool of non-toxic, efficacious, potential anti-infectives, covering: conventional antimicrobial agents, immunomodulators, and anti-virulence agents. Although C. elegans-based screens do not denote the mode of action of hit compounds, this can be elucidated in secondary studies by comparing the results to target-based screens, or conducting subsequent target-based screens, including the genetic knock-down of host or bacterial genes.

Utility of magnetic resonance imaging and nuclear magnetic resonance-based metabolomics for quantification of inflammatory lung injury

PubMed Central

Serkova, Natalie J.; Van Rheen, Zachary; Tobias, Meghan; Pitzer, Joshua E.; Wilkinson, J. Erby; Stringer, Kathleen A.

2008-01-01

Magnetic resonance imaging (MRI) and metabolic nuclear magnetic resonance (NMR) spectroscopy are clinically available but have had little application in the quantification of experimental lung injury. There is a growing and unfulfilled need for predictive animal models that can improve our understanding of disease pathogenesis and therapeutic intervention. Integration of MRI and NMR could extend the application of experimental data into the clinical setting. This study investigated the ability of MRI and metabolic NMR to detect and quantify inflammation-mediated lung injury. Pulmonary inflammation was induced in male B6C3F1 mice by intratracheal administration of IL-1β and TNF-α under isoflurane anesthesia. Mice underwent MRI at 2, 4, 6, and 24 h after dosing. At 6 and 24 h lungs were harvested for metabolic NMR analysis. Data acquired from IL-1β+TNF-α-treated animals were compared with saline-treated control mice. The hyperintense-to-total lung volume (HTLV) ratio derived from MRI was higher in IL-1β+TNF-α-treated mice compared with control at 2, 4, and 6 h but returned to control levels by 24 h. The ability of MRI to detect pulmonary inflammation was confirmed by the association between HTLV ratio and histological and pathological end points. Principal component analysis of NMR-detectable metabolites also showed a temporal pattern for which energy metabolism-based biomarkers were identified. These data demonstrate that both MRI and metabolic NMR have utility in the detection and quantification of inflammation-mediated lung injury. Integration of these clinically available techniques into experimental models of lung injury could improve the translation of basic science knowledge and information to the clinic. PMID:18441091

Characterization and antioxidative activities of rare C(50) carotenoids-sarcinaxanthin, sarcinaxanthin monoglucoside, and sarcinaxanthin diglucoside-obtained from Micrococcus yunnanensis.

PubMed

Osawa, Ayako; Ishii, Yoko; Sasamura, Nao; Morita, Marie; Kasai, Hiroaki; Maoka, Takashi; Shindo, Kazutoshi

2010-01-01

While screening for antioxidative carotenoids from marine bacteria, we isolated and identified sarcinaxanthin and its glucosylated compounds (sarcinaxanthin monoglucoside and sarcinaxanthin diglucoside) from a moderately halophilic bacterium-Micrococcus yunnanensis strain AOY-1. In the singlet oxygen ((¹O₂) quenching model, the IC(50) values of the antioxidative activities of these carotenoids were as follows: sarcinaxanthin , 57 µM; sarcinaxanthin monoglucoside, 54 µM; and sarcinaxanthin diglucoside, 74 µM. In addition, the complete proton nuclear magnetic resonance (¹H NMR) assignments of sarcinaxanthin monoglucoside pentaacetate and sarcinaxanthin diglucoside octaacetate, and fast atom bombardment mass spectrometry/mass spectrometry (FAB-MS/MS) analyses of sarcinaxanthin and sarcinaxanthin monoglucoside are reported for the first time.

Synthesis of soybean oil-based polymeric surfactants in supercritical carbon dioxide and investigation of their surface properties

USDA-ARS?s Scientific Manuscript database

This paper reports the preparation of polymeric surfactants (HPSO) via a two-step synthetic procedure: polymerization of soybean oil (PSO) in supercritical carbon dioxide and followed by hydrolysis of PSO with a base. HPSO was characterized and identified by using a combination of FTIR, 1H NMR, 13C...

Child and teacher acceptability of school-based echocardiographic screening for rheumatic heart disease in Uganda.

PubMed

Ploutz, Michelle; Aliku, Twalib; Bradley-Hewitt, Tyler; Dantin, Andrea; Lemley, Bethan; Gillespie, Catherine W; Lwabi, Peter; Sable, Craig; Beaton, Andrea

2017-01-01

Introduction Rheumatic heart disease causes substantial morbidity in children in low-income countries. School-based echocardiographic screening has been suggested as a means to identify children with latent disease; however, little is known about the experience of children and teachers participating in screenings. The aim of our study was to assess students' and teachers' experience of school-based echocardiographic screening and identify areas for improvement. Materials and methods A school-based echocardiographic screening programme was conducted in five schools in Northern Uganda in 2013. After 8 months, an age- and gender-stratified population that included 5% of the participating students and teachers completed a questionnaire via an in-person interview. Responses were reviewed by question and coded to identify key themes. A total of 255 students (mean 10.7 years; 48% male) and 35 teachers participated in our study. In total, 95% of the students and 100% of the teachers were happy to have participated in the screening; however, students reported feeling scared (35%) and nervous (48%) during the screening process. Programmatic strengths included the following: knowing one's health status, opportunity to receive treatment, and staff interactions. Although 43% of the patients did not suggest a change with open-ended questioning, concerns regarding privacy, fear of the screening process, and a desire to include others in the community were noted. Discussion School-based echocardiographic rheumatic heart disease screening was well received by students and teachers. Future programmes would likely benefit from improved pre-screening education regarding the screening process and diagnosis of rheumatic heart disease. Furthermore, education of teachers and students could improve screening perception and establish realistic expectations regarding the scope of screening.

Strain Selection for Generation of O-Antigen-Based Glycoconjugate Vaccines against Invasive Nontyphoidal Salmonella Disease

PubMed Central

Saul, Allan; MacLennan, Calman A.; Micoli, Francesca; Rondini, Simona

2015-01-01

Nontyphoidal Salmonellae, principally S. Typhimurium and S. Enteritidis, are a major cause of invasive bloodstream infections in sub-Saharan Africa with no vaccine currently available. Conjugation of lipopolysaccharide O-antigen to a carrier protein constitutes a promising vaccination strategy. Here we describe a rational process to select the most appropriate isolates of Salmonella as source of O-antigen for developing a bivalent glycoconjugate vaccine. We screened a library of 30 S. Typhimurium and 21 S. Enteritidis in order to identify the most suitable strains for large scale O-antigen production and generation of conjugate vaccines. Initial screening was based on growth characteristics, safety profile of the isolates, O-antigen production, and O-antigen characteristics in terms of molecular size, O-acetylation and glucosylation level and position, as determined by phenol sulfuric assay, NMR, HPLC-SEC and HPAEC-PAD. Three animal isolates for each serovar were identified and used to synthesize candidate glycoconjugate vaccines, using CRM197 as carrier protein. The immunogenicity of these conjugates and the functional activity of the induced antibodies was investigated by ELISA, serum bactericidal assay and flow cytometry. S. Typhimurium O-antigen showed high structural diversity, including O-acetylation of rhamnose in a Malawian invasive strain generating a specific immunodominant epitope. S. Typhimurium conjugates provoked an anti-O-antigen response primarily against the O:5 determinant. O-antigen from S. Enteritidis was structurally more homogeneous than from S. Typhimurium, and no idiosyncratic antibody responses were detected for the S. Enteritidis conjugates. Of the three initially selected isolates, two S. Typhimurium (1418 and 2189) and two S. Enteritidis (502 and 618) strains generated glycoconjugates able to induce high specific antibody levels with high breadth of serovar-specific strain coverage, and were selected for use in vaccine production. The strain selection approach described is potentially applicable to the development of glycoconjugate vaccines against other bacterial pathogens. PMID:26445460

Regioselective reaction: synthesis and pharmacological study of Mannich bases containing ibuprofen moiety.

PubMed

Sujith, K V; Rao, Jyothi N; Shetty, Prashanth; Kalluraya, Balakrishna

2009-09-01

A series of 4-[(4-aryl)methylidene]amino-2-(substituted-4-ylmethyl)-5-{1-[4-(2-methylpropyl)phenyl]ethyl}-2,4-dihydro-3H-1,2,4-triazole-3-thione (6) were synthesized from an arylpropionic acid namely, ibuprofen by a three-component Mannich reaction. Aminomethylation of 4-[(4-aryl)methylidene]amino-5-{1-[4-(2-methylpropyl)phenyl] ethyl}-4H-1,2,4-triazole-3-thiol (5) with formaldehyde and a secondary amine furnished this novel series of Mannich bases (6). Both Schiff bases (5) and Mannich bases (6) were well characterized on the basis of IR, NMR, mass spectral data and elemental analysis. They were screened for their anti-inflammatory, analgesic, antibacterial and antifungal activities. Some of the Mannich bases (6) carrying morpholino and N-methylpiperazino residues were found to be promising anti-inflammatory and analgesic agents.

Identification of novel drug scaffolds for inhibition of SARS-CoV 3-Chymotrypsin-like protease using virtual and high-throughput screenings.

PubMed

Lee, Hyun; Mittal, Anuradha; Patel, Kavankumar; Gatuz, Joseph L; Truong, Lena; Torres, Jaime; Mulhearn, Debbie C; Johnson, Michael E

2014-01-01

We have used a combination of virtual screening (VS) and high-throughput screening (HTS) techniques to identify novel, non-peptidic small molecule inhibitors against human SARS-CoV 3CLpro. A structure-based VS approach integrating docking and pharmacophore based methods was employed to computationally screen 621,000 compounds from the ZINC library. The screening protocol was validated using known 3CLpro inhibitors and was optimized for speed, improved selectivity, and for accommodating receptor flexibility. Subsequently, a fluorescence-based enzymatic HTS assay was developed and optimized to experimentally screen approximately 41,000 compounds from four structurally diverse libraries chosen mainly based on the VS results. False positives from initial HTS hits were eliminated by a secondary orthogonal binding analysis using surface plasmon resonance (SPR). The campaign identified a reversible small molecule inhibitor exhibiting mixed-type inhibition with a K(i) value of 11.1 μM. Together, these results validate our protocols as suitable approaches to screen virtual and chemical libraries, and the newly identified compound reported in our study represents a promising structural scaffold to pursue for further SARS-CoV 3CLpro inhibitor development. Copyright © 2013. Published by Elsevier Ltd.

Ultra-low field nuclear magnetic resonance and magnetic resonance imaging to discriminate and identify materials

DOEpatents

Matlashov, Andrei Nikolaevich; Urbaitis, Algis V.; Savukov, Igor Mykhaylovich; Espy, Michelle A.; Volegov, Petr Lvovich; Kraus, Jr., Robert Henry

2013-03-05

Method comprising obtaining an NMR measurement from a sample wherein an ultra-low field NMR system probes the sample and produces the NMR measurement and wherein a sampling temperature, prepolarizing field, and measurement field are known; detecting the NMR measurement by means of inductive coils; analyzing the NMR measurement to obtain at least one measurement feature wherein the measurement feature comprises T1, T2, T1.rho., or the frequency dependence thereof; and, searching for the at least one measurement feature within a database comprising NMR reference data for at least one material to determine if the sample comprises a material of interest.

Simple 1H NMR spectroscopic method for assay of salts of the contrast agent diatrizoate in commercial solutions.

PubMed

Hanna, G M; Lau-Cam, C A

1996-01-01

A simple, accurate, and specific 1H NMR spectroscopic method was developed for the assay of diatrizoate meglumine or the combination diatrizoate meglumine and diatrizoate sodium in commercial solutions for injection. A mixture of injectable solution and sodium acetate, the internal standard, was diluted with D2O and the 1H NMR spectrum of the solution was obtained. Two approaches were used to calculate the drug content, based on the integral values for the -N-CO-CH3 protons of diatrizoic acid at 2.23 ppm, and -N-CH3 protons of meglumine at 2.73 ppm, and the CH3-CO-protons of sodium acetate at 1.9 ppm. Recoveries (mean +/- standard deviation) of diatrizoic acid and meglumine from 10 synthetic mixtures of various amounts of these compounds with a fixed amount of internal standard were 100.3 +/- 0.55% and 100.1 +/- 0.98%, respectively. In addition to providing a direct means of simultaneously assaying diatrizoic acid and meglumine, the proposed NMR method can also be used to identify diatrizoate meglumine and each of its molecular components.

Patient-completed or symptom-based screening tools for endometriosis: a scoping review.

PubMed

Surrey, Eric; Carter, Cathryn M; Soliman, Ahmed M; Khan, Shahnaz; DiBenedetti, Dana B; Snabes, Michael C

2017-08-01

The objective of this review was to evaluate existing patient-completed screening questionnaires and/or symptom-based predictive models with respect to their potential for use as screening tools for endometriosis in adult women. Validated instruments were of particular interest. We conducted structured searches of PubMed and targeted searches of the gray literature to identify studies reporting on screening instruments used in endometriosis. Studies were screened according to inclusion and exclusion criteria that followed the PICOS (population, intervention, comparison, outcomes, study design) framework. A total of 16 studies were identified, of which 10 described measures for endometriosis in general, 2 described measures for endometriosis at specific sites, and 4 described measures for deep-infiltrating endometriosis. Only 1 study evaluated a questionnaire that was solely patient-completed. Most measures required physician, imaging, or laboratory assessments in addition to patient-completed questionnaires, and several measures relied on complex scoring. Validation for use as a screening tool in adult women with potential endometriosis was lacking in all studies, as most studies focused on diagnosis versus screening. This literature review did not identify any fully validated, symptom-based, patient-reported questionnaires for endometriosis screening in adult women.

Characterization and Comparison of Fast Pyrolysis Bio-oils from Pinewood, Rapeseed Cake, and Wheat Straw Using 13C NMR and Comprehensive GC × GC.

PubMed

Negahdar, Leila; Gonzalez-Quiroga, Arturo; Otyuskaya, Daria; Toraman, Hilal E; Liu, Li; Jastrzebski, Johann T B H; Van Geem, Kevin M; Marin, Guy B; Thybaut, Joris W; Weckhuysen, Bert M

2016-09-06

Fast pyrolysis bio-oils are feasible energy carriers and a potential source of chemicals. Detailed characterization of bio-oils is essential to further develop its potential use. In this study, quantitative 13 C nuclear magnetic resonance ( 13 C NMR) combined with comprehensive two-dimensional gas chromatography (GC × GC) was used to characterize fast pyrolysis bio-oils originated from pinewood, wheat straw, and rapeseed cake. The combination of both techniques provided new information on the chemical composition of bio-oils for further upgrading. 13 C NMR analysis indicated that pinewood-based bio-oil contained mostly methoxy/hydroxyl (≈30%) and carbohydrate (≈27%) carbons; wheat straw bio-oil showed to have high amount of alkyl (≈35%) and aromatic (≈30%) carbons, while rapeseed cake-based bio-oil had great portions of alkyl carbons (≈82%). More than 200 compounds were identified and quantified using GC × GC coupled to a flame ionization detector (FID) and a time of flight mass spectrometer (TOF-MS). Nonaromatics were the most abundant and comprised about 50% of the total mass of compounds identified and quantified via GC × GC. In addition, this analytical approach allowed the quantification of high value-added phenolic compounds, as well as of low molecular weight carboxylic acids and aldehydes, which exacerbate the unstable and corrosive character of the bio-oil.

Application of metabonomics in a compound ranking study in early drug development revealing drug-induced excretion of choline into urine.

PubMed

Dieterle, Frank; Schlotterbeck, Götz; Ross, Alfred; Niederhauser, Urs; Senn, Hans

2006-09-01

Selecting drug candidates based on toxicity is an important step in early drug development. In this case study, it is shown how metabonomics is applied to a ranking study, in which drug candidates with equal pharmacological activities are selected based on least toxic side effects. The metabonomic analyses were carried out on an animal study that followed an established protocol for pilot toxicology/ranking studies in rats, however, not specifically modified for a metabonomic assessment. It is shown how conditions not specificially adopted for metabonomics investigations can significantly influence the metabolic profiles recorded by NMR. Furthermore, it is shown how the multivariate analysis of the NMR spectra identified an extreme excretion of an endogenous metabolite into urine induced by two out of the five drug candidates. The subsequent structure elucidation by two-dimensional NMR experiments and a subsequent validation by spiking experiments identified the metabolite as choline. The discussion of the mechanistic background for the excretion of choline, which is usually well-conserved in the body, results in two hypotheses of either a massive degradation of cell membranes or an inhibition of the choline oxidation. Although the validation of these hypotheses needs a follow-up study, the finding of a increased excretion of the important metabolite choline warrants exclusion of these two compounds as viable drug candidates from a metabonomics point of view.

Magnetic resonance imaging of granular materials

NASA Astrophysics Data System (ADS)

Stannarius, Ralf

2017-05-01

Magnetic Resonance Imaging (MRI) has become one of the most important tools to screen humans in medicine; virtually every modern hospital is equipped with a Nuclear Magnetic Resonance (NMR) tomograph. The potential of NMR in 3D imaging tasks is by far greater, but there is only "a handful" of MRI studies of particulate matter. The method is expensive, time-consuming, and requires a deep understanding of pulse sequences, signal acquisition, and processing. We give a short introduction into the physical principles of this imaging technique, describe its advantages and limitations for the screening of granular matter, and present a number of examples of different application purposes, from the exploration of granular packing, via the detection of flow and particle diffusion, to real dynamic measurements. Probably, X-ray computed tomography is preferable in most applications, but fast imaging of single slices with modern MRI techniques is unmatched, and the additional opportunity to retrieve spatially resolved flow and diffusion profiles without particle tracking is a unique feature.

Solution-state 2D NMR of ball-milled plant cell wall gels in DMSO-d6/pyridine-d5†

PubMed Central

Ralph, John

2014-01-01

NMR fingerprinting of the components of finely divided plant cell walls swelled in DMSO has been recently described. Cell wall gels, produced directly in the NMR tube with perdeutero-dimethylsulfoxide, allowed the acquisition of well resolved/dispersed 2D 13C–1H correlated solution-state NMR spectra of the entire array of wall polymers, without the need for component fractionation. That is, without actual solubilization, and without apparent structural modification beyond that inflicted by the ball milling and ultrasonication steps, satisfactorily interpretable spectra can be acquired that reveal compositional and structural details regarding the polysaccharide and lignin components in the wall. Here, the profiling method has been improved by using a mixture of perdeuterated DMSO and pyridine (4:1, v/v). Adding pyridine provided not only easier sample handling because of the better mobility compared to the DMSO-d6-only system but also considerably elevated intensities and improved resolution of the NMR spectra due to the enhanced swelling of the cell walls. This modification therefore provides a more rapid method for comparative structural evaluation of plant cell walls than is currently available. We examined loblolly pine (Pinus taeda, a gymnosperm), aspen (Populus tremuloides, an angiosperm), kenaf (Hibiscus cannabinus, an herbaceous plant), and corn (Zea mays L., a grass, i.e., from the Poaceae family). In principle, lignin composition (notably, the syringyl : guaiacyl : p-hydroxyphenyl ratio) can be quantified without the need for lignin isolation. Correlations for p-coumarate units in the corn sample are readily seen, and a variety of the ferulate correlations are also well resolved; ferulates are important components responsible for cell wall cross-linking in grasses. Polysaccharide anomeric correlations were tentatively assigned for each plant sample based on standard samples and various literature data. With the new potential for chemometric analysis using the 2D NMR fingerprint, this gel-state method may provide the basis for an attractive approach to providing a secondary screen for selecting biomass lines and for optimizing biomass processing and conversion efficiencies. PMID:20090974

Computational Diffusion Magnetic Resonance Imaging Based on Time-Dependent Bloch NMR Flow Equation and Bessel Functions.

PubMed

Awojoyogbe, Bamidele O; Dada, Michael O; Onwu, Samuel O; Ige, Taofeeq A; Akinwande, Ninuola I

2016-04-01

Magnetic resonance imaging (MRI) uses a powerful magnetic field along with radio waves and a computer to produce highly detailed "slice-by-slice" pictures of virtually all internal structures of matter. The results enable physicians to examine parts of the body in minute detail and identify diseases in ways that are not possible with other techniques. For example, MRI is one of the few imaging tools that can see through bones, making it an excellent tool for examining the brain and other soft tissues. Pulsed-field gradient experiments provide a straightforward means of obtaining information on the translational motion of nuclear spins. However, the interpretation of the data is complicated by the effects of restricting geometries as in the case of most cancerous tissues and the mathematical concept required to account for this becomes very difficult. Most diffusion magnetic resonance techniques are based on the Stejskal-Tanner formulation usually derived from the Bloch-Torrey partial differential equation by including additional terms to accommodate the diffusion effect. Despite the early success of this technique, it has been shown that it has important limitations, the most of which occurs when there is orientation heterogeneity of the fibers in the voxel of interest (VOI). Overcoming this difficulty requires the specification of diffusion coefficients as function of spatial coordinate(s) and such a phenomenon is an indication of non-uniform compartmental conditions which can be analyzed accurately by solving the time-dependent Bloch NMR flow equation analytically. In this study, a mathematical formulation of magnetic resonance flow sequence in restricted geometry is developed based on a general second order partial differential equation derived directly from the fundamental Bloch NMR flow equations. The NMR signal is obtained completely in terms of NMR experimental parameters. The process is described based on Bessel functions and properties that can make it possible to distinguish cancerous cells from normal cells. A typical example of liver distinguished from gray matter, white matter and kidney is demonstrated. Bessel functions and properties are specifically needed to show the direct effect of the instantaneous velocity on the NMR signal originating from normal and abnormal tissues.

Composition of Indigo naturalis.

PubMed

Plitzko, Inken; Mohn, Tobias; Sedlacek, Natalie; Hamburger, Matthias

2009-06-01

A proposal for a European Pharmacopoeia monograph concerning Indigo naturalis has recently been published, whereby the indigo (1) and indirubin (2) content should be determined by HPLC-UV. This method was tested, but problems were seen with the dosage of indigo due to poor solubility. A quantitative assay for indigo based on (1)H-NMR was developed as an alternative. The HPLC and qNMR assays were compared with eight Indigo naturalis samples. The HPLC assay consistently gave much lower indigo concentrations because solubility was the limiting factor in sample preparation. In one sample, sucrose was identified by (1)H-NMR as an organic additive. Simple wet chemistry assays for undeclared additives such as sugars and starch were tested with artificially spiked Indigo naturalis samples to establish their limits of detection, and sulfate ash determinations were carried out in view of a better assessment of Indigo naturalis in a future European monograph.

Toward a structure determination method for biomineral-associated protein using combined solid- state NMR and computational structure prediction.

PubMed

Masica, David L; Ash, Jason T; Ndao, Moise; Drobny, Gary P; Gray, Jeffrey J

2010-12-08

Protein-biomineral interactions are paramount to materials production in biology, including the mineral phase of hard tissue. Unfortunately, the structure of biomineral-associated proteins cannot be determined by X-ray crystallography or solution nuclear magnetic resonance (NMR). Here we report a method for determining the structure of biomineral-associated proteins. The method combines solid-state NMR (ssNMR) and ssNMR-biased computational structure prediction. In addition, the algorithm is able to identify lattice geometries most compatible with ssNMR constraints, representing a quantitative, novel method for investigating crystal-face binding specificity. We use this method to determine most of the structure of human salivary statherin interacting with the mineral phase of tooth enamel. Computation and experiment converge on an ensemble of related structures and identify preferential binding at three crystal surfaces. The work represents a significant advance toward determining structure of biomineral-adsorbed protein using experimentally biased structure prediction. This method is generally applicable to proteins that can be chemically synthesized. Copyright © 2010 Elsevier Ltd. All rights reserved.

Toxicity Assessment of 17 alpha-ethinylestradiol by Cell-Culture Based NMR Metabolomics

EPA Science Inventory

A zebrafish liver cell line (ZFL) established from adult zebrafish has been used in a variety of biological research, including toxicology, pharmacology, developmental biology and molecular genetics. The goal of this study is to develop an in vitro approach to identify the respo...

Analysis of GAA/TTC DNA triplexes using nuclear magnetic resonance and electrospray ionization mass spectrometry.

PubMed

Mariappan, S V Santhana; Cheng, Xun; van Breemen, Richard B; Silks, Louis A; Gupta, Goutam

2004-11-15

The formation of a GAA/TTC DNA triplex has been implicated in Friedreich's ataxia. The destabilization of GAA/TTC DNA triplexes either by pH or by binding to appropriate ligands was analyzed by nuclear magnetic resonance (NMR) and positive-ion electrospray mass spectrometry. The triplexes and duplexes were identified by changes in the NMR chemical shifts of H8, H1, H4, 15N7, and 15N4. The lowest pH at which the duplex is detectable depends upon the overall stability and the relative number of Hoogsteen C composite function G to T composite function A basepairs. A melting pH (pHm) of 7.6 was observed for the destabilization of the (GAA)2T4(TTC)2T4(CTT)2 triplex to the corresponding Watson-Crick duplex and the T4(CTT)2 overhang. The mass spectrometric analyses of (TTC)6.(GAA)6 composite function(TTC)6 triplex detected ions due to both triplex and single-stranded oligonucleotides under acidic conditions. The triplex ions disappeared completely at alkaline pH. Duplex and single strands were detectable only at neutral and alkaline pH values. Mass spectrometric analyses also showed that minor groove-binding ligands berenil, netropsin, and distamycin and the intercalating ligand acridine orange destabilize the (TTC)6.(GAA)6 composite function (TTC)6 triplex. These NMR and mass spectrometric methods may function as screening assays for the discovery of agents that destabilize GAA/TTC triplexes and as general methods for the characterization of structure, dynamics, and stability of DNA and DNA-ligand complexes.

Synthesis, spectroscopy, X-ray crystallography, DFT calculations, DNA binding and molecular docking of a propargyl arms containing Schiff base.

PubMed

Balakrishnan, C; Subha, L; Neelakantan, M A; Mariappan, S S

2015-11-05

A propargyl arms containing Schiff base (L) was synthesized by the condensation of 1-[2-hydroxy-4-(prop-2-yn-1-yloxy)phenyl]ethanone with trans-1,2-diaminocyclohexane. The structure of L was characterized by IR, (1)H NMR, (13)C NMR and UV-Vis spectroscopy and by single crystal X-ray diffraction analysis. The UV-Visible spectral behavior of L in different solvents exhibits positive solvatochromism. Density functional calculation of the L in gas phase was performed by using DFT (B3LYP) method with 6-31G basis set. The computed vibrational frequencies and NMR signals of L were compared with the experimental data. Tautomeric stability study inferred that the enolimine is more stable than the ketoamine form. The charge delocalization has been analyzed using natural bond orbital (NBO) analysis. Electronic absorption and emission spectral studies were used to study the binding of L with CT-DNA. The molecular docking was done to identify the interaction of L with A-DNA and B-DNA. Copyright © 2015 Elsevier B.V. All rights reserved.

NMR Structure of Francisella tularensis Virulence Determinant Reveals Structural Homology to Bet v1 Allergen Proteins.

PubMed

Zook, James; Mo, Gina; Sisco, Nicholas J; Craciunescu, Felicia M; Hansen, Debra T; Baravati, Bobby; Cherry, Brian R; Sykes, Kathryn; Wachter, Rebekka; Van Horn, Wade D; Fromme, Petra

2015-06-02

Tularemia is a potentially fatal bacterial infection caused by Francisella tularensis, and is endemic to North America and many parts of northern Europe and Asia. The outer membrane lipoprotein, Flpp3, has been identified as a virulence determinant as well as a potential subunit template for vaccine development. Here we present the first structure for the soluble domain of Flpp3 from the highly infectious Type A SCHU S4 strain, derived through high-resolution solution nuclear magnetic resonance (NMR) spectroscopy; the first structure of a lipoprotein from the genus Francisella. The Flpp3 structure demonstrates a globular protein with an electrostatically polarized surface containing an internal cavity-a putative binding site based on the structurally homologous Bet v1 protein family of allergens. NMR-based relaxation studies suggest loop regions that potentially modulate access to the internal cavity. The Flpp3 structure may add to the understanding of F. tularensis virulence and contribute to the development of effective vaccines. Copyright © 2015 Elsevier Ltd. All rights reserved.

Breast cancer screening in an era of personalized regimens: a conceptual model and National Cancer Institute initiative for risk-based and preference-based approaches at a population level.

PubMed

Onega, Tracy; Beaber, Elisabeth F; Sprague, Brian L; Barlow, William E; Haas, Jennifer S; Tosteson, Anna N A; D Schnall, Mitchell; Armstrong, Katrina; Schapira, Marilyn M; Geller, Berta; Weaver, Donald L; Conant, Emily F

2014-10-01

Breast cancer screening holds a prominent place in public health, health care delivery, policy, and women's health care decisions. Several factors are driving shifts in how population-based breast cancer screening is approached, including advanced imaging technologies, health system performance measures, health care reform, concern for "overdiagnosis," and improved understanding of risk. Maximizing benefits while minimizing the harms of screening requires moving from a "1-size-fits-all" guideline paradigm to more personalized strategies. A refined conceptual model for breast cancer screening is needed to align women's risks and preferences with screening regimens. A conceptual model of personalized breast cancer screening is presented herein that emphasizes key domains and transitions throughout the screening process, as well as multilevel perspectives. The key domains of screening awareness, detection, diagnosis, and treatment and survivorship are conceptualized to function at the level of the patient, provider, facility, health care system, and population/policy arena. Personalized breast cancer screening can be assessed across these domains with both process and outcome measures. Identifying, evaluating, and monitoring process measures in screening is a focus of a National Cancer Institute initiative entitled PROSPR (Population-based Research Optimizing Screening through Personalized Regimens), which will provide generalizable evidence for a risk-based model of breast cancer screening, The model presented builds on prior breast cancer screening models and may serve to identify new measures to optimize benefits-to-harms tradeoffs in population-based screening, which is a timely goal in the era of health care reform. © 2014 American Cancer Society.

NMR spectroscopic and analytical ultracentrifuge analysis of membrane protein detergent complexes.

PubMed

Maslennikov, Innokentiy; Kefala, Georgia; Johnson, Casey; Riek, Roland; Choe, Senyon; Kwiatkowski, Witek

2007-11-08

Structural studies of integral membrane proteins (IMPs) are hampered by inherent difficulties in their heterologous expression and in the purification of solubilized protein-detergent complexes (PDCs). The choice and concentrations of detergents used in an IMP preparation play a critical role in protein homogeneity and are thus important for successful crystallization. Seeking an effective and standardized means applicable to genomic approaches for the characterization of PDCs, we chose 1D-NMR spectroscopic analysis to monitor the detergent content throughout their purification: protein extraction, detergent exchange, and sample concentration. We demonstrate that a single NMR measurement combined with a SDS-PAGE of a detergent extracted sample provides a useful gauge of the detergent's extraction potential for a given protein. Furthermore, careful monitoring of the detergent content during the process of IMP production allows for a high level of reproducibility. We also show that in many cases a simple sedimentation velocity measurement provides sufficient data to estimate both the oligomeric state and the detergent-to-protein ratio in PDCs, as well as to evaluate the homogeneity of the samples prior to crystallization screening. The techniques presented here facilitate the screening and selection of the extraction detergent, as well as help to maintain reproducibility in the detergent exchange and PDC concentration procedures. Such reproducibility is particularly important for the optimization of initial crystallization conditions, for which multiple purifications are routinely required.

NMR spectroscopic and analytical ultracentrifuge analysis of membrane protein detergent complexes

PubMed Central

Maslennikov, Innokentiy; Kefala, Georgia; Johnson, Casey; Riek, Roland; Choe, Senyon; Kwiatkowski, Witek

2007-01-01

Background Structural studies of integral membrane proteins (IMPs) are hampered by inherent difficulties in their heterologous expression and in the purification of solubilized protein-detergent complexes (PDCs). The choice and concentrations of detergents used in an IMP preparation play a critical role in protein homogeneity and are thus important for successful crystallization. Results Seeking an effective and standardized means applicable to genomic approaches for the characterization of PDCs, we chose 1D-NMR spectroscopic analysis to monitor the detergent content throughout their purification: protein extraction, detergent exchange, and sample concentration. We demonstrate that a single NMR measurement combined with a SDS-PAGE of a detergent extracted sample provides a useful gauge of the detergent's extraction potential for a given protein. Furthermore, careful monitoring of the detergent content during the process of IMP production allows for a high level of reproducibility. We also show that in many cases a simple sedimentation velocity measurement provides sufficient data to estimate both the oligomeric state and the detergent-to-protein ratio in PDCs, as well as to evaluate the homogeneity of the samples prior to crystallization screening. Conclusion The techniques presented here facilitate the screening and selection of the extraction detergent, as well as help to maintain reproducibility in the detergent exchange and PDC concentration procedures. Such reproducibility is particularly important for the optimization of initial crystallization conditions, for which multiple purifications are routinely required. PMID:17988403

Immobilized magnetic beads-based multi-target affinity selection coupled with HPLC-MS for screening active compounds from traditional Chinese medicine and natural products.

PubMed

Chen, Yaqi; Chen, Zhui; Wang, Yi

2015-01-01

Screening and identifying active compounds from traditional Chinese medicine (TCM) and other natural products plays an important role in drug discovery. Here, we describe a magnetic beads-based multi-target affinity selection-mass spectrometry approach for screening bioactive compounds from natural products. Key steps and parameters including activation of magnetic beads, enzyme/protein immobilization, characterization of functional magnetic beads, screening and identifying active compounds from a complex mixture by LC/MS, are illustrated. The proposed approach is rapid and efficient in screening and identification of bioactive compounds from complex natural products.

Mechanistic insight into formation and changes of nanoparticles in MgF2 sols evidenced by liquid and solid state NMR.

PubMed

Karg, M; Scholz, G; König, R; Kemnitz, E

2012-02-28

The fluorolytic sol-gel reaction of magnesium methoxide with HF in methanol was studied by (19)F, (1)H and (13)C liquid and solid state NMR. In (19)F NMR five different species were identified, three of which belong to magnesium fluoride nanoparticles, i.e. NMR gave access to local structures of solid particles in suspensions. The long-term evolution of (19)F signals was followed and along with (19)F MAS NMR experiments of sols rotating at 13 kHz mechanistic insights into the ageing processes were obtained.

A systematic review on US-based community health navigator (CHN) interventions for cancer screening promotion--comparing community- versus clinic-based navigator models.

PubMed

Hou, Su-I; Roberson, Kiersten

2015-03-01

This study synthesized lessons learned from US-based community and clinic health navigator (CHN) interventions on cancer screening promotion to identify characteristics of models and approaches for addressing cancer disparities. The combination terms "cancer screening" and "community health workers or navigators" or "patient navigators" were used in searching Medline, CINAHL, and PsycInfo. A total of 27 articles published during January 2005∼April 2014 were included. Two CHN models were identified: community-based (15 studies) and clinic/hospital-based (12 studies). While both models used the term "navigators," most community-based programs referred them as community health workers/navigators/advisors, whereas clinic-based programs often called them patient navigators. Most community-based CHN interventions targeted specific racial/ethnic minority or rural groups, while clinic-based programs mostly targeted urban low income or mixed ethnic groups. Most community-based CHN programs outreached members from community networks, while clinic-based programs commonly worked with pre-identified in-service clients. Overall, regardless model type, CHNs had similar roles and responsibilities, and interventions demonstrated effective outcomes. Our review identified characteristics of CHN interventions with attention to different settings. Lessons learned have implication on the dissemination and implementation of CHN interventions for cancer screening promotion across setting and target groups.

Two-dimensional NMR spectroscopy links structural moieties of soil organic matter to the temperature sensitivity of its decomposition

NASA Astrophysics Data System (ADS)

Soucemarianadin, Laure; Erhagen, Björn; Öquist, Mats; Nilsson, Mats; Schleucher, Jürgen

2015-04-01

Soil organic matter (SOM) represents a huge carbon pool, specifically in boreal ecosystems. Warming-induced release of large amounts of CO2 from the soil carbon pool might become a significant exacerbating feedback to global warming, if decomposition rates of boreal soils were more sensitive to increased temperatures. Despite a large number of studies dedicated to the topic, it has proven difficult to elucidate how the organo-chemical composition of SOM influences its decomposition, or its quality as a substrate for microbial metabolism. A great part of this challenge results from our inability to achieve a detailed characterization of the complex composition of SOM on the level of molecular structural moieties. 13C nuclear magnetic resonance (NMR) spectroscopy is a common tool to characterize SOM. However, SOM is a very complex mixture and the chemical shift regions distinguished in the 13C NMR spectra often represent many different molecular fragments. For example, in the carbohydrates region, signals of all monosaccharides present in many different polymers overlap. This overlap thwarts attempts to identify molecular moieties, resulting in insufficient information to characterize SOM composition. We applied two-dimensional (2D) NMR to characterize SOM with highly increased resolution. We directly dissolved finely ground litters and forest floors'fibric and humic horizons'of both coniferous and deciduous boreal forests in dimethyl sulfoxide and analyzed the resulting solution with a 2D 1H-13C NMR experiment. In the 2D planes of these spectra, signals of CH groups can be resolved based on their 13C and 1H chemical shifts, hence the resolving power and information content of these NMR spectra is hugely increased. The 2D spectra indeed resolved overlaps observed in 1D 13C spectra, so that hundreds of distinct CH groups could be observed and many molecular fragments could be identified. For instance, in the aromatics region, signals from individual lignin units could be recognized. It was hence possible to follow the fate of specific structural moieties in soils. We observed differences between litter and soil samples, and were able to relate them to the decomposition of identifiable moieties. Using multivariate data analysis, we aimed at linking the detailed chemical fingerprints of SOM to turnover rates in a soil incubation experiment. With the multivariate models, we were able to relate signal patterns in the 2D spectra and intensities of identifiable molecular moieties to variability in the temperature response of organic matter decomposition, as assessed by Q10. In conclusion, the characterization of SOM composition at the molecular level by solution-state 2D NMR spectroscopy is highly promising; it offers unprecedented possibilities to link SOM molecular composition to ecosystem processes, and their responses to environmental changes.

Ab initio NMR parameters of BrCH3 and ICH3 with relativistic and vibrational corrections

NASA Astrophysics Data System (ADS)

Uhlíková, Tereza; Urban, Štěpán

2018-05-01

This study is focused on two effects identified when NMR parameters are calculated based on first principles. These effects are 1. vibrational correction of properties when using ab initio optimized equilibrium geometry; 2. relativistic effects and limits of using the Flygare equation. These effects have been investigated and determined for nuclear spin-rotation constants and nuclear magnetic shieldings for the CH3Br and CH3I molecules. The most significant result is the difference between chemical shieldings determined based on the ab initio relativistic four-component Dirac-Coulomb Hamiltonian and chemical shieldings calculated using experimental values and the Flygare equation. This difference is approximately 320 ppm and 1290 ppm for 79Br and 127I in the CH3X molecule, respectively.

Analysis of the defect clusters in congruent lithium tantalate

NASA Astrophysics Data System (ADS)

Vyalikh, Anastasia; Zschornak, Matthias; Köhler, Thomas; Nentwich, Melanie; Weigel, Tina; Hanzig, Juliane; Zaripov, Ruslan; Vavilova, Evgenia; Gemming, Sibylle; Brendler, Erica; Meyer, Dirk C.

2018-01-01

A wide range of technological applications of lithium tantalate (LT) is closely related to the defect chemistry. In literature, several intrinsic defect models have been proposed. Here, using a combinational approach based on DFT and solid-state NMR, we demonstrate that distribution of electric field gradients (EFGs) can be employed as a fingerprint of a specific defect configuration. Analyzing the distribution of 7Li EFGs, the FT-IR and electron spin resonance (ESR) spectra, and the 7Li spin-lattice relaxation behavior, we have found that the congruent LT samples provided by two manufacturers show rather different defect concentrations and distributions although both were grown by the Czochralski method. After thermal treatment hydrogen out-diffusion and homogeneous distribution of other defects have been observed by ESR, NMR, and FT-IR. The defect structure in one of two congruent LT crystals after annealing has been identified and proved by defect formation energy considerations, whereas the more complex defect configuration, including the presence of extrinsic defects, has been suggested for the other LT sample. The approach of searching the EFG fingerprints from DFT calculations in NMR spectra can be applied for identifying the defect clusters in other complex oxides.

Profiling the metabolome changes caused by cranberry procyanidins in plasma of female rats using (1) H NMR and UHPLC-Q-Orbitrap-HRMS global metabolomics approaches.

PubMed

Liu, Haiyan; Garrett, Timothy J; Tayyari, Fariba; Gu, Liwei

2015-11-01

The objective was to investigate the metabolome changes in female rats gavaged with partially purified cranberry procyanidins (PPCP) using (1) H NMR and UHPLC-Q-Orbitrap-HRMS metabolomics approaches, and to identify the contributing metabolites. Twenty-four female Sprague-Dawley rats were randomly separated into two groups and administered PPCP or partially purified apple procyanidins (PPAP) for three times using a 250 mg extracts/kg body weight dose. Plasma was collected 6 h after the last gavage and analyzed using (1) H NMR and UHPLC-Q-Orbitrap-HRMS. No metabolome difference was observed using (1) H NMR metabolomics approach. However, LC-HRMS metabolomics data show that metabolome in the plasma of female rats administered PPCP differed from those gavaged with PPAP. Eleven metabolites were tentatively identified from a total of 36 discriminant metabolic features based on accurate masses and/or product ion spectra. PPCP caused a greater increase of exogenous metabolites including p-hydroxybenzoic acid, phenol, phenol-sulphate, catechol sulphate, 3, 4-dihydroxyphenylvaleric acid, and 4'-O-methyl-(-)-epicatechin-3'-O-beta-glucuronide in rat plasma. Furthermore, the plasma level of O-methyl-(-)-epicatechin-O-glucuronide, 4-hydroxy-5-(hydroxyphenyl)-valeric acid-O-sulphate, 5-(hydroxyphenyl)-ϒ-valerolactone-O-sulphate, 4-hydroxydiphenylamine, and peonidin-3-O-hexose were higher in female rats administered with PPAP. The metabolome changes caused by cranberry procyanidins were revealed using an UHPLC-Q-Orbitrap-HRMS global metabolomics approach. Exogenous and microbial metabolites were the major identified discriminate biomarkers. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Screening and structural elucidation of the zwitterionic cocrystal o-picolinic acid with p-nitro aniline

NASA Astrophysics Data System (ADS)

Mekala, R.; Jagdish, P.; Mathammal, R.; Sangeetha, K.

2017-04-01

The cocrystal was screened by solvent drop grinding method and the crystals were grown by slow evaporation method at ambient conditions. The cocrystal formation of o-picolinic acid with p-nitro aniline was initially analysed by powder X-ray diffraction. Further the structural properties of the grown crystal were confirmed by the single X-ray diffraction which indicates that the cocrystal were connected by the strong N+sbnd H-⋯O hydrogen bond interaction. The cell parameters of the grown crystal were a = 14.2144(5) Å, b = 5.7558(2) Å, c = 16.0539(6) Å. The functional groups were identified using Fourier transform infrared and Raman spectral analysis. The excitation and emission state of the sample was analysed by the UV-Visible and Fluorescence studies. The red emission was observed from the Fluorescence studies. NMR studies revealed the chemical shift of the cocrystal. Thermal stability and its melting behaviour were studied by TGA and DSC analytical techniques. Electrical behaviour was studied using the dielectric studies. The intermolecular charge transfer within the molecule were analysed using HOMO- LUMO plots.

Consistency and sources of divergence in recommendations on screening with questionnaires for presently experienced health problems or symptoms: a comparison of recommendations from the Canadian Task Force on Preventive Health Care, UK National Screening Committee, and US Preventive Services Task Force.

PubMed

Thombs, Brett D; Saadat, Nazanin; Riehm, Kira E; Karter, Justin Michael; Vaswani, Akansha; Andrews, Bonnie K; Simons, Peter; Cosgrove, Lisa

2017-08-09

Recently, health screening recommendations have gone beyond screening for early-stage, asymptomatic disease to include "screening" for presently experienced health problems and symptoms using self-report questionnaires. We examined recommendations from three major national guideline organizations to determine the consistency of recommendations, identify sources of divergent recommendations, and determine if guideline organizations have identified any direct randomized controlled trial (RCT) evidence for the effectiveness of questionnaire-based screening. We reviewed recommendation statements listed by the Canadian Task Force on Preventive Health Care (CTFPHC), the United Kingdom National Screening Committee (UKNSC), and the United States Preventive Services Task Force (USPSTF) as of 5 September 2016. Eligible recommendations focused on using self-report questionnaires to identify patients with presently experienced health problems or symptoms. Within each recommendation and accompanying evidence review we identified screening RCTs. We identified 22 separate recommendations on questionnaire-based screening, including three CTFPHC recommendations against screening, eight UKNSC recommendations against screening, four USPSTF recommendations in favor of screening (alcohol misuse, adolescent depression, adult depression, intimate partner violence), and seven USPSTF recommendations that did not recommend for or against screening. In the four cases where the USPSTF recommended screening, either the CTFPHC, the UKNSC, or both recommended against. When recommendations diverged, the USPSTF expressed confidence in benefits based on indirect evidence, evaluated potential harms as minimal, and did not consider cost or resource use. CTFPHC and UKNSC recommendations against screening, on the other hand, focused on the lack of direct evidence of benefit and raised concerns about harms to patients and resource use. Of six RCTs that directly evaluated screening interventions, five did not report any statistically significant primary or secondary health outcomes in favor of screening, and one trial reported equivocal results. Only the USPSTF has made any recommendations for screening with questionnaires for presently experienced problems or symptoms. The CTFPHC and UKNSC recommended against screening in all of their recommendations. Differences in recommendations appear to reflect differences in willingness to assume benefit from indirect evidence and different approaches to assessing possible harms and resource consumption. There were no examples in any recommendations of RCTs with direct evidence of improved health outcomes.

Thalassosamide, a Siderophore Discovered from the Marine-Derived Bacterium Thalassospira profundimaris.

PubMed

Zhang, Fan; Barns, Kenneth; Hoffmann, F Michael; Braun, Doug R; Andes, David R; Bugni, Tim S

2017-09-22

Here we describe the rapid identification and prioritization of novel active marine natural products using an improved dereplication strategy. During the course of our screening of marine natural product libraries, a new cyclic trihydroxamate compound, thalassosamide, was discovered from the α-proteobacterium Thalassospira profundimaris. Its structure was determined by 2D NMR and MS/MS experiments, and the absolute configuration of the lysine-derived units was established by Marfey's analysis, whereas that of C-9, 9', and 9″ was determined via the circular dichroism data of the [Rh 2 (OCOCF 3 ) 4 ] complex and DFT NMR calculations. Thalassosamide showed moderate in vivo efficacy against Pseudomonas aeruginosa.

The conformation of cyclo(-D-Pro-Ala4-) as a model for cyclic pentapeptides of the DL4 type.

PubMed

Heller, Markus; Sukopp, Martin; Tsomaia, Natia; John, Michael; Mierke, Dale F; Reif, Bernd; Kessler, Horst

2006-10-25

The conformation of the cyclic pentapeptide cyclo(-D-Pro-Ala(4)-) in solution and in the solid state was reinvestigated using modern NMR techniques. To allow unequivocal characterization of hydrogen bonds, relaxation behavior, and intramolecular distances, differently labeled isotopomers were synthesized. The NMR results, supported by extensive MD simulations, demonstrate unambiguously that the preferred conformation previously described by us, but recently questioned, is indeed correct. The validation of the conformational preferences of this cyclic peptide is important given that this system is a template for several bioactive compounds and for controlled "spatial screening" for the search of bioactive conformations.

Rapid Determination of Major Compounds in the Ethanol Extract of Geopropolis from Malaysian Stingless Bees, Heterotrigona itama, by UHPLC-Q-TOF/MS and NMR.

PubMed

Zhao, Lingling; Yu, Mengjiao; Sun, Minghui; Xue, Xiaofeng; Wang, Tongtong; Cao, Wei; Sun, Liping

2017-11-10

A reliable, rapid analytical method was established for the characterization of constituents of the ethanol extract of geopropolis (EEGP) produced by Malaysian stingless bees- Heterotrigona itama -by combining ultra-high-performance liquid chromatography with quadruple time-of-flight mass spectrometry (UHPLC-Q-TOF/MS). Based on known standards, the online METLIN database, and published literature, 28 compounds were confirmed. Phenolic acids, flavones, triterpenes and phytosterol were identified or tentatively identified using characteristic diagnostic fragment ions. The results indicated that terpenoids were the main components of EEGP, accompanied by low levels of phenolic acids, flavonoids, and phytosterol. Two major components were further purified by preparative high-performance liquid chromatography (PHPLC) and identified by nuclear magnetic resonance (NMR) as 24( E )-cycloart-24-ene-26-ol-3-one and 20-hydroxy-24-dammaren-3-one. These two triterpenes, confirmed in this geopropolis for the first time, are potential chemical markers for the identification of geopropolis from Malaysian stingless bees, H. itama .

Database-Guided Discovery of Potent Peptides to Combat HIV-1 or Superbugs

PubMed Central

Wang, Guangshun

2013-01-01

Antimicrobial peptides (AMPs), small host defense proteins, are indispensable for the protection of multicellular organisms such as plants and animals from infection. The number of AMPs discovered per year increased steadily since the 1980s. Over 2,000 natural AMPs from bacteria, protozoa, fungi, plants, and animals have been registered into the antimicrobial peptide database (APD). The majority of these AMPs (>86%) possess 11–50 amino acids with a net charge from 0 to +7 and hydrophobic percentages between 31–70%. This article summarizes peptide discovery on the basis of the APD. The major methods are the linguistic model, database screening, de novo design, and template-based design. Using these methods, we identified various potent peptides against human immunodeficiency virus type 1 (HIV-1) or methicillin-resistant Staphylococcus aureus (MRSA). While the stepwise designed anti-HIV peptide is disulfide-linked and rich in arginines, the ab initio designed anti-MRSA peptide is linear and rich in leucines. Thus, there are different requirements for antiviral and antibacterial peptides, which could kill pathogens via different molecular targets. The biased amino acid composition in the database-designed peptides, or natural peptides such as θ-defensins, requires the use of the improved two-dimensional NMR method for structural determination to avoid the publication of misleading structure and dynamics. In the case of human cathelicidin LL-37, structural determination requires 3D NMR techniques. The high-quality structure of LL-37 provides a solid basis for understanding its interactions with membranes of bacteria and other pathogens. In conclusion, the APD database is a comprehensive platform for storing, classifying, searching, predicting, and designing potent peptides against pathogenic bacteria, viruses, fungi, parasites, and cancer cells. PMID:24276259

Exploration of multiple Sortase A protein conformations in virtual screening

NASA Astrophysics Data System (ADS)

Gao, Chunxia; Uzelac, Ivana; Gottfries, Johan; Eriksson, Leif A.

2016-02-01

Methicillin resistant Staphylococcus aureus (MRSA) has become a major health concern which has brought about an urgent need for new therapeutic agents. As the S. aureus Sortase A (SrtA) enzyme contributes to the adherence of the bacteria to the host cells, inhibition thereof by small molecules could be employed as potential antivirulence agents, also towards resistant strains. Albeit several virtual docking SrtA campaigns have been reported, no strongly inhibitatory non-covalent binders have as yet emerged therefrom. In order to better understand the binding modes of small molecules, and the effect of different receptor structures employed in the screening, we herein report on an exploratory study employing 10 known binders and 500 decoys on 100 SrtA structures generated from regular or steered molecular dynamics simulations on four different SrtA crystal/NMR structures. The results suggest a correlation between the protein structural flexibility and the virtual screening performance, and confirm the noted immobilization of the β6/β7 loop upon substrate binding. The NMR structures reported appear to perform slightly better than the Xray-crystal structures, but the binding modes fluctuate tremendously, and it might be suspected that the catalytic site is not necessarily the preferred site of binding for some of the reported active compounds.

Exploration of multiple Sortase A protein conformations in virtual screening

PubMed Central

Gao, Chunxia; Uzelac, Ivana; Gottfries, Johan; Eriksson, Leif A.

2016-01-01

Methicillin resistant Staphylococcus aureus (MRSA) has become a major health concern which has brought about an urgent need for new therapeutic agents. As the S. aureus Sortase A (SrtA) enzyme contributes to the adherence of the bacteria to the host cells, inhibition thereof by small molecules could be employed as potential antivirulence agents, also towards resistant strains. Albeit several virtual docking SrtA campaigns have been reported, no strongly inhibitatory non-covalent binders have as yet emerged therefrom. In order to better understand the binding modes of small molecules, and the effect of different receptor structures employed in the screening, we herein report on an exploratory study employing 10 known binders and 500 decoys on 100 SrtA structures generated from regular or steered molecular dynamics simulations on four different SrtA crystal/NMR structures. The results suggest a correlation between the protein structural flexibility and the virtual screening performance, and confirm the noted immobilization of the β6/β7 loop upon substrate binding. The NMR structures reported appear to perform slightly better than the Xray-crystal structures, but the binding modes fluctuate tremendously, and it might be suspected that the catalytic site is not necessarily the preferred site of binding for some of the reported active compounds. PMID:26846342

Thyroxine-Based Screening for Congenital Hypothyroidism in Neonates with Down Syndrome.

PubMed

Erlichman, Ira; Mimouni, Francis B; Erlichman, Matityahu; Schimmel, Michael S

2016-06-01

To ascertain whether thyroxine (T4)-based screening programs for congenital hypothyroidism (initial measurement of total T4 [tT4] followed by thyroid stimulating hormone [TSH] measurement in patients with tT4 <10th percentile) identifies congenital hypothyroidism in all neonates with Down syndrome. Retrospective cohort study of 159 neonates with Down syndrome, born during the period 1998-2007 were included. Screening test results were compared with those of the general population. All primary care physicians of these infants were contacted and infants' thyroid status verified. tT4 concentrations in children with Down syndrome were significantly lower, and TSH higher than those in the general population; tT4 concentrations did not correlate with screening TSH concentrations. Twenty children with Down syndrome were treated with L-thyroxin within the first month of life although only 10 babies had been identified by the routine screening test. T4-based screening does not identify many cases of congenital hypothyroidism in neonates with Down syndrome. We recommend that neonates with Down syndrome be screened by simultaneous measurements of both tT4 and TSH. Copyright © 2016 Elsevier Inc. All rights reserved.

Synthesis, spectral and antimicrobial activity of Zn(II) complexes with Schiff bases derived from 2-hydrazino-5-[substituted phenyl]-1,3,4-thiadiazole and benzaldehyde/2-hydroxyacetophenone/indoline-2,3-dione.

PubMed

Singh, Ajay K; Pandey, O P; Sengupta, S K

2013-09-01

Zn(II) complexes have been synthesized by reacting zinc acetate with Schiff bases derived from 2-hydrazino-5-[substituted phenyl]-1,3,4-thiadiazole and 2-hydroxyacetophenone/benzaldehyde/indoline-2,3-dione. All these complexes are soluble in DMF and DMSO; low molar conductance values indicate that they are non electrolytes. Elemental analyses suggest that the complexes have 1:2 metal to ligands stoichiometry of the types [ZnL2(H2O)2](L=monoanionic Schiff bases derived from 2-hydrazino-5-[substituted phenyl]-1,3,4-thiadiazole and 2-hydroxyacetophenone/indoline-2,3-dione) [ZnL2(')(OOCCH3)2(H2O)2](L'=neutral Schiff bases derived from 2-hydrazino-5-[substituted phenyl]-1,3,4-thiadiazole and benzaldehyde), and they were characterized by IR, (1)H NMR, and (13)C NMR. Particle sizes of synthesized compounds were measured with dynamic light scattering (DLS) analyser which indicates that particle diameter are of the range ca. 100-200nm. All these Schiff bases and their complexes have also been screened for their antibacterial (Bacillus subtilis (B. subtilis), Escherichia coli (E. coli) and antifungal activities (Colletotrichum falcatum (C. falcatum), Aspergillus niger (A. niger), Fusarium oxysporium (F. oxysporium) Curvularia pallescence (C. pallescence). The antimicrobial activities have shown that upon complexation the activity increases. Copyright © 2013 Elsevier B.V. All rights reserved.

Spectroscopic characterization of metal complexes of novel Schiff base. Synthesis, thermal and biological activity studies

NASA Astrophysics Data System (ADS)

Omar, M. M.; Mohamed, Gehad G.; Ibrahim, Amr A.

2009-07-01

Novel Schiff base (HL) ligand is prepared via condensation of 4-aminoantipyrine and 2-aminobenzoic acid. The ligand is characterized based on elemental analysis, mass, IR and 1H NMR spectra. Metal complexes are reported and characterized based on elemental analyses, IR, 1H NMR, solid reflectance, magnetic moment, molar conductance and thermal analyses (TGA, DrTGA and DTA). The molar conductance data reveal that all the metal chelates are non-electrolytes. IR spectra show that HL is coordinated to the metal ions in a uninegatively tridentate manner with NNO donor sites of the azomethine N, amino N and deprotonated caroxylic-O. From the magnetic and solid reflectance spectra, it is found that the geometrical structures of these complexes are octahedral. The thermal behaviour of these chelates shows that the hydrated complexes losses water molecules of hydration in the first step followed immediately by decomposition of the anions and ligand molecules in the subsequent steps. The activation thermodynamic parameters, such as, E*, ΔH*, ΔS* and ΔG* are calculated from the DrTG curves using Coats-Redfern method. The synthesized ligands, in comparison to their metal complexes also were screened for their antibacterial activity against bacterial species, Escherichia Coli, Pseudomonas aeruginosa, Staphylococcus Pyogones and Fungi (Candida). The activity data show that the metal complexes to be more potent/antibacterial than the parent Shciff base ligand against one or more bacterial species.

Use of diffusion-ordered NMR spectroscopy and HPLC-UV-SPE-NMR to identify undeclared synthetic drugs in medicines illegally sold as phytotherapies.

PubMed

Silva, Lorena M A; Filho, Elenilson G A; Thomasi, Sérgio S; Silva, Bianca F; Ferreira, Antonio G; Venâncio, Tiago

2013-09-01

The informal (and/or illegal) e-commerce of pharmaceutical formulations causes problems that governmental health agencies find hard to control, one of which concerns formulas sold as natural products. The purpose of this work was to explore the advantages and limitations of DOSY and HPLC-UV-SPE-NMR. These techniques were used to identify the components of a formula illegally marketed in Brazil as an herbal medicine possessing anti-inflammatory and analgesic properties. DOSY was able to detect the major components present at higher concentrations. Complete characterization was achieved using HPLC-UV-SPE-NMR, and 1D and 2D NMR analyses enabled the identification of known synthetic drugs. These were ranitidine and a mixture of orphenadrine citrate, piroxicam, and dexamethasone, which are co-formulated in a remedy called Rheumazim that is used to relieve severe pain, but it is prohibited in Brazil because of a lack of sufficient pharmacokinetic and pharmacodynamic information. Copyright © 2013 John Wiley & Sons, Ltd.

A new glycation product ‘norpronyl-lysine,’ and direct characterization of cross linking and other glycation adducts: NMR of model compounds and collagen

PubMed Central

Bullock, Peter T. B.; Reid, David G.; Ying Chow, W.; Lau, Wendy P. W.; Duer, Melinda J.

2014-01-01

NMR is ideal for characterizing non-enzymatic protein glycation, including AGEs (advanced glycation endproducts) underlying tissue pathologies in diabetes and ageing. Ribose, R5P (ribose-5-phosphate) and ADPR (ADP-ribose), could be significant and underinvestigated biological glycating agents especially in chronic inflammation. Using [U-13C]ribose we have identified a novel glycoxidation adduct, 5-deoxy-5-desmethylpronyl-lysine, ‘norpronyl-lysine’, as well as numerous free ketones, acids and amino group reaction products. Glycation by R5P and ADPR proceeds rapidly with R5P generating a brown precipitate with PLL (poly-L-lysine) within hours. ssNMR (solid-state NMR) 13C–13C COSY identifies several crosslinking adducts such as the newly identified norpronyl-lysine, in situ, from the glycating reaction of 13C5-ribose with collagen. The same adducts are also identifiable after reaction of collagen with R5P. We also demonstrate for the first time bio-amine (spermidine, N-acetyl lysine, PLL) catalysed ribose 2-epimerization to arabinose at physiological pH. This work raises the prospect of advancing understanding of the mechanisms and consequences of glycation in actual tissues, in vitro or even ex vivo, using NMR isotope-labelled glycating agents, without analyses requiring chemical or enzymatic degradations, or prior assumptions about glycation products. PMID:27919030

Selective excitation enables assignment of proton resonances and (1)H-(1)H distance measurement in ultrafast magic angle spinning solid state NMR spectroscopy.

PubMed

Zhang, Rongchun; Ramamoorthy, Ayyalusamy

2015-07-21

Remarkable developments in ultrafast magic angle spinning (MAS) solid-state NMR spectroscopy enabled proton-based high-resolution multidimensional experiments on solids. To fully utilize the benefits rendered by proton-based ultrafast MAS experiments, assignment of (1)H resonances becomes absolutely necessary. Herein, we propose an approach to identify different proton peaks by using dipolar-coupled heteronuclei such as (13)C or (15)N. In this method, after the initial preparation of proton magnetization and cross-polarization to (13)C nuclei, transverse magnetization of desired (13)C nuclei is selectively prepared by using DANTE (Delays Alternating with Nutations for Tailored Excitation) sequence and then, it is transferred to bonded protons with a short-contact-time cross polarization. Our experimental results demonstrate that protons bonded to specific (13)C atoms can be identified and overlapping proton peaks can also be assigned. In contrast to the regular 2D HETCOR experiment, only a few 1D experiments are required for the complete assignment of peaks in the proton spectrum. Furthermore, the finite-pulse radio frequency driven recoupling sequence could be incorporated right after the selection of specific proton signals to monitor the intensity buildup for other proton signals. This enables the extraction of (1)H-(1)H distances between different pairs of protons. Therefore, we believe that the proposed method will greatly aid in fast assignment of peaks in proton spectra and will be useful in the development of proton-based multi-dimensional solid-state NMR experiments to study atomic-level resolution structure and dynamics of solids.

Phylogenetically Novel LuxI/LuxR-Type Quorum Sensing Systems Isolated Using a Metagenomic Approach

PubMed Central

Nasuno, Eri; Fujita, Masaki J.; Nakatsu, Cindy H.; Kamagata, Yoichi; Hanada, Satoshi

2012-01-01

A great deal of research has been done to understand bacterial cell-to-cell signaling systems, but there is still a large gap in our current knowledge because the majority of microorganisms in natural environments do not have cultivated representatives. Metagenomics is one approach to identify novel quorum sensing (QS) systems from uncultured bacteria in environmental samples. In this study, fosmid metagenomic libraries were constructed from a forest soil and an activated sludge from a coke plant, and the target genes were detected using a green fluorescent protein (GFP)-based Escherichia coli biosensor strain whose fluorescence was screened by spectrophotometry. DNA sequence analysis revealed two pairs of new LuxI family N-acyl-l-homoserine lactone (AHL) synthases and LuxR family transcriptional regulators (clones N16 and N52, designated AubI/AubR and AusI/AusR, respectively). AubI and AusI each produced an identical AHL, N-dodecanoyl-l-homoserine lactone (C12-HSL), as determined by nuclear magnetic resonance (NMR) and mass spectrometry. Phylogenetic analysis based on amino acid sequences suggested that AusI/AusR was from an uncultured member of the Betaproteobacteria and AubI/AubR was very deeply branched from previously described LuxI/LuxR homologues in isolates of the Proteobacteria. The phylogenetic position of AubI/AubR indicates that they represent a QS system not acquired recently from the Proteobacteria by horizontal gene transfer but share a more ancient ancestry. We demonstrated that metagenomic screening is useful to provide further insight into the phylogenetic diversity of bacterial QS systems by describing two new LuxI/LuxR-type QS systems from uncultured bacteria. PMID:22983963

Community-based screening for obstetric fistula in Nigeria: a novel approach

PubMed Central

2014-01-01

Background Obstetric fistula continues to have devastating effects on the physical, social, and economic lives of thousands of women in many low-resource settings. Governments require credible estimates of the backlog of existing cases requiring care to effectively plan for the treatment of fistula cases. Our study aims to quantify the backlog of obstetric fistula cases within two states via community-based screenings and to assess the questions in the Demographic Health Survey (DHS) fistula module. Methods The screening sites, all lower level health facilities, were selected based on their geographic coverage, prior relationships with the communities and availability of fistula surgery facilities in the state. This cross-sectional study included women who presented for fistula screenings at study facilities based on their perceived fistula-like symptoms. Research assistants administered the pre-screening questionnaire. Nurse-midwives then conducted a medical exam. Univariate and bivariate analyses are presented. Results A total of 268 women attended the screenings. Based on the pre-screening interview, the backlog of fistula cases reported was 75 (28% of women screened). The backlog identified after the medical exam was 26 fistula cases (29.5% of women screened) in Kebbi State sites and 12 cases in Cross River State sites (6.7%). Verification assessment showed that the DHS questionnaire had 92% sensitivity, 83% specificity with 47% positive predictive value and 98% negative predictive value for identifying women afflicted by fistula among women who came for the screenings. Conclusions This methodology, involving effective, locally appropriate messaging and community outreach followed up with medical examination by nurse-midwives at lower level facilities, is challenging, but represents a promising approach to identify the backlog of women needing surgery and to link them with surgical facilities. PMID:24456506

How to identify students for school-based depression intervention: can school record review be substituted for universal depression screening?

PubMed

Kuo, Elena S; Vander Stoep, Ann; Herting, Jerald R; Grupp, Katherine; McCauley, Elizabeth

2013-02-01

Early identification and intervention are critical for reducing the adverse effects of depression on academic and occupational performance. Cost-effective approaches are needed for identifying adolescents at high depression risk. This study evaluated the utility of school record review versus universal school-based depression screening for determining eligibility for an indicated depression intervention program implemented in the middle school setting. Algorithms derived from grades, attendance, suspensions, and basic demographic information were evaluated with regard to their ability to predict students' depression screening scores. The school information-based algorithms proved poor proxies for individual students' depression screening results. However, school records showed promise for identifying low, medium, and high-yield subgroups on the basis of which efficient screening targeting decisions could be made. Study results will help to guide school nurses who coordinate indicated depression intervention programs in school settings as they evaluate options of approaches for determining which students are eligible for participation. © 2012 Wiley Periodicals, Inc.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Xiangmei; Mort, Andrew

A comore » mmercial apple pectin was sequentially digested with the cloned enzymes endopolygalacturonase, galactanase, arabinofuranosidase, xylogalacturonase, and rhamnogalacturonan hydrolase. The rhamnogalacturonan hydrolase-generated oligosaccharides were separated by ultrafiltration, anion exchange, and size-exclusion chromatography. Fractions from the ion exchange chromatography were pooled, lyophilized, and screened by MALDI-TOF MS. An oligosaccharide (RGP14P3) was identified and its structure, α -D-Gal p A- ( 1 → 2 ) - α -L-Rha p - ( 1 → 4 ) - α -D-Gal p A- ( 1 → 2 ) - α -L-Rha p - ( 1 → 4 ) - α -D-Gal p A, determined by 1D and 2D NMR spectrometry. This oligosaccharide probably represents a direct connection between homogalacturonan and rhamnogalacturonan in pectin. Alternatively, it could indicate that the nonreducing end of rhamnogalacturonan starts with a galacturonic acid residue.« less

nmrML: A Community Supported Open Data Standard for the Description, Storage, and Exchange of NMR Data.

PubMed

Schober, Daniel; Jacob, Daniel; Wilson, Michael; Cruz, Joseph A; Marcu, Ana; Grant, Jason R; Moing, Annick; Deborde, Catherine; de Figueiredo, Luis F; Haug, Kenneth; Rocca-Serra, Philippe; Easton, John; Ebbels, Timothy M D; Hao, Jie; Ludwig, Christian; Günther, Ulrich L; Rosato, Antonio; Klein, Matthias S; Lewis, Ian A; Luchinat, Claudio; Jones, Andrew R; Grauslys, Arturas; Larralde, Martin; Yokochi, Masashi; Kobayashi, Naohiro; Porzel, Andrea; Griffin, Julian L; Viant, Mark R; Wishart, David S; Steinbeck, Christoph; Salek, Reza M; Neumann, Steffen

2018-01-02

NMR is a widely used analytical technique with a growing number of repositories available. As a result, demands for a vendor-agnostic, open data format for long-term archiving of NMR data have emerged with the aim to ease and encourage sharing, comparison, and reuse of NMR data. Here we present nmrML, an open XML-based exchange and storage format for NMR spectral data. The nmrML format is intended to be fully compatible with existing NMR data for chemical, biochemical, and metabolomics experiments. nmrML can capture raw NMR data, spectral data acquisition parameters, and where available spectral metadata, such as chemical structures associated with spectral assignments. The nmrML format is compatible with pure-compound NMR data for reference spectral libraries as well as NMR data from complex biomixtures, i.e., metabolomics experiments. To facilitate format conversions, we provide nmrML converters for Bruker, JEOL and Agilent/Varian vendor formats. In addition, easy-to-use Web-based spectral viewing, processing, and spectral assignment tools that read and write nmrML have been developed. Software libraries and Web services for data validation are available for tool developers and end-users. The nmrML format has already been adopted for capturing and disseminating NMR data for small molecules by several open source data processing tools and metabolomics reference spectral libraries, e.g., serving as storage format for the MetaboLights data repository. The nmrML open access data standard has been endorsed by the Metabolomics Standards Initiative (MSI), and we here encourage user participation and feedback to increase usability and make it a successful standard.

Exposure-based screening for Nipah virus encephalitis, Bangladesh.

PubMed

Sazzad, Hossain M S; Luby, Stephen P; Ströher, Ute; Daszak, Peter; Sultana, Sharmin; Afroj, Sayma; Rahman, Mahmudur; Gurley, Emily S

2015-02-01

We measured the performance of exposure screening questions to identify Nipah virus encephalitis in hospitalized encephalitis patients during the 2012-13 Nipah virus season in Bangladesh. The sensitivity (93%), specificity (82%), positive predictive value (37%), and negative predictive value (99%) results suggested that screening questions could more quickly identify persons with Nipah virus encephalitis.

Breast Cancer Screening in an Era of Personalized Regimens

PubMed Central

Onega, Tracy; Beaber, Elisabeth F.; Sprague, Brian L.; Barlow, William E.; Haas, Jennifer S.; Tosteson, Anna N.A.; Schnall, Mitchell D.; Armstrong, Katrina; Schapira, Marilyn M.; Geller, Berta; Weaver, Donald L.; Conant, Emily F.

2014-01-01

Breast cancer screening holds a prominent place in public health, health care delivery, policy, and women’s health care decisions. Several factors are driving shifts in how population-based breast cancer screening is approached, including advanced imaging technologies, health system performance measures, health care reform, concern for “overdiagnosis,” and improved understanding of risk. Maximizing benefits while minimizing the harms of screening requires moving from a “1-size-fits-all” guideline paradigm to more personalized strategies. A refined conceptual model for breast cancer screening is needed to align women’s risks and preferences with screening regimens. A conceptual model of personalized breast cancer screening is presented herein that emphasizes key domains and transitions throughout the screening process, as well as multilevel perspectives. The key domains of screening awareness, detection, diagnosis, and treatment and survivorship are conceptualized to function at the level of the patient, provider, facility, health care system, and population/policy arena. Personalized breast cancer screening can be assessed across these domains with both process and outcome measures. Identifying, evaluating, and monitoring process measures in screening is a focus of a National Cancer Institute initiative entitled PROSPR (Population-based Research Optimizing Screening through Personalized Regimens), which will provide generalizable evidence for a risk-based model of breast cancer screening, The model presented builds on prior breast cancer screening models and may serve to identify new measures to optimize benefits-to-harms tradeoffs in population-based screening, which is a timely goal in the era of health care reform. PMID:24830599

Fragment-Based Drug Discovery Using NMR Spectroscopy

PubMed Central

Harner, Mary J.; Frank, Andreas O.; Fesik, Stephen W.

2013-01-01

Nuclear magnetic resonance (NMR) spectroscopy has evolved into a powerful tool for fragment-based drug discovery over the last two decades. While NMR has been traditionally used to elucidate the three-dimensional structures and dynamics of biomacromolecules and their interactions, it can also be a very valuable tool for the reliable identification of small molecules that bind to proteins and for hit-to-lead optimization. Here, we describe the use of NMR spectroscopy as a method for fragment-based drug discovery and how to most effectively utilize this approach for discovering novel therapeutics based on our experience. PMID:23686385

Newborn screening education on the internet: a content analysis of North American newborn screening program websites.

PubMed

Araia, Makda H; Potter, Beth K

2011-09-01

The Internet is a potentially important medium for communication about public health programs including newborn screening. This study explores whether the information available on official newborn screening program websites is consistent with existing guidelines regarding educational content for parents. We conducted a systematic search of the public websites of newborn screening programs in the US and Canada, identifying web pages and downloadable brochures that contained educational information. Two researchers independently reviewed all documents to determine the extent to which they included 14 key recommended educational messages. We identified 85 documents containing educational information on 46 US and 6 Canadian newborn screening program websites. The documents contained from 1 to 14 of the recommended messages. The majority of identified materials emphasized the importance and benefits of screening. The differences between US and Canadian materials were related to the importance of parental involvement in follow-up and issues of consent and storage of blood spots. Our findings are consistent with studies of non-web-based newborn screening education materials. The results emphasize the need for further evaluation of newborn screening education, including internet-based resources, particularly in terms of the impact of particular messages on parental attitudes and behaviors.

Anti-inflammatory and enzyme inhibitory activities of a crude extract and a pterocarpan isolated from the aerial parts of Vitex agnus-castus.

PubMed

Ahmad, Bashir; Azam, Sadiq; Bashir, Shumaila; Khan, Ibrar; Adhikari, Achyut; Choudhary, Muhammad Iqbal

2010-11-01

A new compound, 6a,11a-dihydro-6H-[1] benzofuro [3,2-c][1,3]dioxolo[4,5-g]chromen-9-ol was isolated from the ethyl acetate fraction of Vitex agnus-castus. The structure of this compound was identified with the help of spectroscopic techniques ((13)C NMR, (1)H NMR, HMBC, HMQC, NOESY and COSY). The compound showed low urease- (32.0%) and chymotrypsin- (31.4%) inhibitory activity, and moderate (41.3%) anti-inflammatory activity. The crude extract and various fractions obtained from the aerial parts of the plant were also screened for possible in vitro hemagglutination, antibacterial and phytotoxic activities. No hemagglutination activity against human erythrocytes was observed in crude extracts and fractions of V. agnus-castus. The fractions and crude methanolic extract showed moderate and low antibacterial activity. Exceptions were the CHCl(3) fraction, which showed significant antibacterial activity against Klebsiella pneumonia (81% with MIC(50)=2.19 mg/mL), the n-hexane fraction, which exhibited no activity against Salmonella typhi, and the CHCl(3) and aqueous fractions, which showed no activity against Bacillus pumalis. Moderate phytotoxic activity (62.5%) was observed by n-hexane fraction of V. agnus-castus against Lemna minor L at 1000 μg/mL.

NMR study of complexes between low molecular mass inhibitors and the West Nile virus NS2B-NS3 protease.

PubMed

Su, Xun-Cheng; Ozawa, Kiyoshi; Yagi, Hiromasa; Lim, Siew P; Wen, Daying; Ekonomiuk, Dariusz; Huang, Danzhi; Keller, Thomas H; Sonntag, Sebastian; Caflisch, Amedeo; Vasudevan, Subhash G; Otting, Gottfried

2009-08-01

The two-component NS2B-NS3 protease of West Nile virus is essential for its replication and presents an attractive target for drug development. Here, we describe protocols for the high-yield expression of stable isotope-labelled samples in vivo and in vitro. We also describe the use of NMR spectroscopy to determine the binding mode of new low molecular mass inhibitors of the West Nile virus NS2B-NS3 protease which were discovered using high-throughput in vitro screening. Binding to the substrate-binding sites S1 and S3 is confirmed by intermolecular NOEs and comparison with the binding mode of a previously identified low molecular mass inhibitor. Our results show that all these inhibitors act by occupying the substrate-binding site of the protease rather than by an allosteric mechanism. In addition, the NS2B polypeptide chain was found to be positioned near the substrate-binding site, as observed previously in crystal structures of the protease in complex with peptide inhibitors or bovine pancreatic trypsin inhibitor. This indicates that the new low molecular mass compounds, although inhibiting the protease, also promote the proteolytically active conformation of NS2B, which is very different from the crystal structure of the protein without inhibitor.

A personal computer-based nuclear magnetic resonance spectrometer

NASA Astrophysics Data System (ADS)

Job, Constantin; Pearson, Robert M.; Brown, Michael F.

1994-11-01

Nuclear magnetic resonance (NMR) spectroscopy using personal computer-based hardware has the potential of enabling the application of NMR methods to fields where conventional state of the art equipment is either impractical or too costly. With such a strategy for data acquisition and processing, disciplines including civil engineering, agriculture, geology, archaeology, and others have the possibility of utilizing magnetic resonance techniques within the laboratory or conducting applications directly in the field. Another aspect is the possibility of utilizing existing NMR magnets which may be in good condition but unused because of outdated or nonrepairable electronics. Moreover, NMR applications based on personal computer technology may open up teaching possibilities at the college or even secondary school level. The goal of developing such a personal computer (PC)-based NMR standard is facilitated by existing technologies including logic cell arrays, direct digital frequency synthesis, use of PC-based electrical engineering software tools to fabricate electronic circuits, and the use of permanent magnets based on neodymium-iron-boron alloy. Utilizing such an approach, we have been able to place essentially an entire NMR spectrometer console on two printed circuit boards, with the exception of the receiver and radio frequency power amplifier. Future upgrades to include the deuterium lock and the decoupler unit are readily envisioned. The continued development of such PC-based NMR spectrometers is expected to benefit from the fast growing, practical, and low cost personal computer market.

Synthesis and antimicrobial activities of new higher amino acid Schiff base derivatives of 6-aminopenicillanic acid and 7-aminocephalosporanic acid

NASA Astrophysics Data System (ADS)

Özdemir (nee Güngör), Özlem; Gürkan, Perihan; Özçelik, Berrin; Oyardı, Özlem

2016-02-01

Novel β-lactam derivatives (1c-3c) (1d-3d) were produced by using 6-aminopenicillanic acid (6-APA), 7-aminocephalosporanic acid (7-ACA) and the higher amino acid Schiff bases. The synthesized compounds were characterized by elemental analysis, IR, 1H/13C NMR and UV-vis spectra. Antibacterial activities of all the higher amino acid Schiff bases (1a-3a) (1b-3b) and β-lactam derivatives were screened against three gram negative bacteria (Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Acinetobacter baumannii RSKK 02026), three gram positive bacteria (Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 07005, Bacillus subtilis ATCC 6633) and their drug-resistant isolates by using broth microdilution method. Two fungi (Candida albicans and Candida krusei) were used for antifungal activity.

(1)H-NMR-based metabolomic analysis of the effect of moderate wine consumption on subjects with cardiovascular risk factors.

PubMed

Vázquez-Fresno, Rosa; Llorach, Rafael; Alcaro, Francesca; Rodríguez, Miguel Ángel; Vinaixa, Maria; Chiva-Blanch, Gemma; Estruch, Ramon; Correig, Xavier; Andrés-Lacueva, Cristina

2012-08-01

Moderate wine consumption is associated with health-promoting activities. An H-NMR-based metabolomic approach was used to identify urinary metabolomic differences of moderate wine intake in the setting of a prospective, randomized, crossover, and controlled trial. Sixty-one male volunteers with high cardiovascular risk factors followed three dietary interventions (28 days): dealcoholized red wine (RWD) (272mL/day, polyphenol control), alcoholized red wine (RWA) (272mL/day) and gin (GIN) (100mL/day, alcohol control). After each period, 24-h urine samples were collected and analyzed by (1) H-NMR. According to the results of a one-way ANOVA, significant markers were grouped in four categories: alcohol-related markers (ethanol); gin-related markers; wine-related markers; and gut microbiota markers (hippurate and 4-hydroxphenylacetic acid). Wine metabolites were classified into two groups; first, metabolites of food metabolome: tartrate (RWA and RWD), ethanol, and mannitol (RWA); and second, biomarkers that relates to endogenous modifications after wine consumption, comprising branched-chain amino acid (BCAA) metabolite (3-methyl-oxovalerate). Additionally, a possible interaction between alcohol and gut-related biomarkers has been identified. To our knowledge, this is the first time that this approach has been applied in a nutritional intervention with red wine. The results show the capacity of this approach to obtain a comprehensive metabolome picture including food metabolome and endogenous biomarkers of moderate wine intake. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Characterization and Comparison of Fast Pyrolysis Bio-oils from Pinewood, Rapeseed Cake, and Wheat Straw Using 13C NMR and Comprehensive GC × GC

PubMed Central

2016-01-01

Fast pyrolysis bio-oils are feasible energy carriers and a potential source of chemicals. Detailed characterization of bio-oils is essential to further develop its potential use. In this study, quantitative 13C nuclear magnetic resonance (13C NMR) combined with comprehensive two-dimensional gas chromatography (GC × GC) was used to characterize fast pyrolysis bio-oils originated from pinewood, wheat straw, and rapeseed cake. The combination of both techniques provided new information on the chemical composition of bio-oils for further upgrading. 13C NMR analysis indicated that pinewood-based bio-oil contained mostly methoxy/hydroxyl (≈30%) and carbohydrate (≈27%) carbons; wheat straw bio-oil showed to have high amount of alkyl (≈35%) and aromatic (≈30%) carbons, while rapeseed cake-based bio-oil had great portions of alkyl carbons (≈82%). More than 200 compounds were identified and quantified using GC × GC coupled to a flame ionization detector (FID) and a time of flight mass spectrometer (TOF-MS). Nonaromatics were the most abundant and comprised about 50% of the total mass of compounds identified and quantified via GC × GC. In addition, this analytical approach allowed the quantification of high value-added phenolic compounds, as well as of low molecular weight carboxylic acids and aldehydes, which exacerbate the unstable and corrosive character of the bio-oil. PMID:27668136

Reliable resonance assignments of selected residues of proteins with known structure based on empirical NMR chemical shift prediction

NASA Astrophysics Data System (ADS)

Li, Da-Wei; Meng, Dan; Brüschweiler, Rafael

2015-05-01

A robust NMR resonance assignment method is introduced for proteins whose 3D structure has previously been determined by X-ray crystallography. The goal of the method is to obtain a subset of correct assignments from a parsimonious set of 3D NMR experiments of 15N, 13C labeled proteins. Chemical shifts of sequential residue pairs are predicted from static protein structures using PPM_One, which are then compared with the corresponding experimental shifts. Globally optimized weighted matching identifies the assignments that are robust with respect to small changes in NMR cross-peak positions. The method, termed PASSPORT, is demonstrated for 4 proteins with 100-250 amino acids using 3D NHCA and a 3D CBCA(CO)NH experiments as input producing correct assignments with high reliability for 22% of the residues. The method, which works best for Gly, Ala, Ser, and Thr residues, provides assignments that serve as anchor points for additional assignments by both manual and semi-automated methods or they can be directly used for further studies, e.g. on ligand binding, protein dynamics, or post-translational modification, such as phosphorylation.

Reliable Resonance Assignments of Selected Residues of Proteins with Known Structure Based on Empirical NMR Chemical Shift Prediction

PubMed Central

Li, Da-Wei; Meng, Dan; Brüschweiler, Rafael

2015-01-01

A robust NMR resonance assignment method is introduced for proteins whose 3D structure has previously been determined by X-ray crystallography. The goal of the method is to obtain a subset of correct assignments from a parsimonious set of 3D NMR experiments of 15N, 13C labeled proteins. Chemical shifts of sequential residue pairs are predicted from static protein structures using PPM_One, which are then compared with the corresponding experimental shifts. Globally optimized weighted matching identifies the assignments that are robust with respect to small changes in NMR cross-peak positions. The method, termed PASSPORT, is demonstrated for 4 proteins with 100 – 250 amino acids using 3D NHCA and a 3D CBCA(CO)NH experiments as input producing correct assignments with high reliability for 22% of the residues. The method, which works best for Gly, Ala, Ser, and Thr residues, provides assignments that serve as anchor points for additional assignments by both manual and semi-automated methods or they can be directly used for further studies, e.g. on ligand binding, protein dynamics, or post-translational modification, such as phosphorylation. PMID:25863893

Conformational heterogeneity in the Hsp70 chaperone-substrate ensemble identified from analysis of NMR-detected titration data.

PubMed

Sekhar, Ashok; Nagesh, Jayashree; Rosenzweig, Rina; Kay, Lewis E

2017-11-01

The Hsp70 chaperone system plays a critical role in cellular homeostasis by binding to client protein molecules. We have recently shown by methyl-TROSY NMR methods that the Escherichia coli Hsp70, DnaK, can form multiple bound complexes with a small client protein, hTRF1. In an effort to characterize the interactions further we report here the results of an NMR-based titration study of hTRF1 and DnaK, where both molecular components are monitored simultaneously, leading to a binding model. A central finding is the formation of a previously undetected 3:1 hTRF1-DnaK complex, suggesting that under heat shock conditions, DnaK might be able to protect cytosolic proteins whose net concentrations would exceed that of the chaperone. Moreover, these results provide new insight into the heterogeneous ensemble of complexes formed by DnaK chaperones and further emphasize the unique role of NMR spectroscopy in obtaining information about individual events in a complex binding scheme by exploiting a large number of probes that report uniquely on distinct binding processes. © 2017 The Protein Society.

Unraveling the active hypoglycemic agent trigonelline in Balanites aegyptiaca date fruit using metabolite fingerprinting by NMR.

PubMed

Farag, Mohamed A; Porzel, Andrea; Wessjohann, Ludger A

2015-11-10

Trigonelline (3-carboxy-1-methyl pyridinium) was identified as a relevant bioactivity and taste imparting component in Balanites aegyptiaca fruit, using (1)H NMR of crude extracts without any fractionation or isolation step. The structural integrity of trigonelline was established within the extract matrix via(1)H NMR, (1)H-(1)H COSY, HMQC and HMBC and by comparison with authentic standard. A quantitative (1)H NMR method (qHNMR) was used to determine trigonelline concentrations in the peel and pulp of B. aegyptiaca fruit of 8 and 13mgg(-1), respectively. Trigonelline so far has not been reported from B. aegyptiaca or its genus as it easily escapes LC-MS based detection. Its discovery provides novel insight into the balanite fruits antidiabetic properties as the compound is known for a pronounced hypoglycemic effect. In addition, it is likely to impart the perceptible bitter taste portion to balanites sweet bitter taste. UPLC-MS of the crude extract additionally revealed the fruit flavonoid pattern showing quercetin/isorhamnetin flavonol conjugates in addition to epicatechin, the latter being present at much lower levels. Copyright © 2015 Elsevier B.V. All rights reserved.

Automatic NMR-Based Identification of Chemical Reaction Types in Mixtures of Co-Occurring Reactions

PubMed Central

Latino, Diogo A. R. S.; Aires-de-Sousa, João

2014-01-01

The combination of chemoinformatics approaches with NMR techniques and the increasing availability of data allow the resolution of problems far beyond the original application of NMR in structure elucidation/verification. The diversity of applications can range from process monitoring, metabolic profiling, authentication of products, to quality control. An application related to the automatic analysis of complex mixtures concerns mixtures of chemical reactions. We encoded mixtures of chemical reactions with the difference between the 1H NMR spectra of the products and the reactants. All the signals arising from all the reactants of the co-occurring reactions were taken together (a simulated spectrum of the mixture of reactants) and the same was done for products. The difference spectrum is taken as the representation of the mixture of chemical reactions. A data set of 181 chemical reactions was used, each reaction manually assigned to one of 6 types. From this dataset, we simulated mixtures where two reactions of different types would occur simultaneously. Automatic learning methods were trained to classify the reactions occurring in a mixture from the 1H NMR-based descriptor of the mixture. Unsupervised learning methods (self-organizing maps) produced a reasonable clustering of the mixtures by reaction type, and allowed the correct classification of 80% and 63% of the mixtures in two independent test sets of different similarity to the training set. With random forests (RF), the percentage of correct classifications was increased to 99% and 80% for the same test sets. The RF probability associated to the predictions yielded a robust indication of their reliability. This study demonstrates the possibility of applying machine learning methods to automatically identify types of co-occurring chemical reactions from NMR data. Using no explicit structural information about the reactions participants, reaction elucidation is performed without structure elucidation of the molecules in the mixtures. PMID:24551112

Automatic NMR-based identification of chemical reaction types in mixtures of co-occurring reactions.

PubMed

Latino, Diogo A R S; Aires-de-Sousa, João

2014-01-01

The combination of chemoinformatics approaches with NMR techniques and the increasing availability of data allow the resolution of problems far beyond the original application of NMR in structure elucidation/verification. The diversity of applications can range from process monitoring, metabolic profiling, authentication of products, to quality control. An application related to the automatic analysis of complex mixtures concerns mixtures of chemical reactions. We encoded mixtures of chemical reactions with the difference between the (1)H NMR spectra of the products and the reactants. All the signals arising from all the reactants of the co-occurring reactions were taken together (a simulated spectrum of the mixture of reactants) and the same was done for products. The difference spectrum is taken as the representation of the mixture of chemical reactions. A data set of 181 chemical reactions was used, each reaction manually assigned to one of 6 types. From this dataset, we simulated mixtures where two reactions of different types would occur simultaneously. Automatic learning methods were trained to classify the reactions occurring in a mixture from the (1)H NMR-based descriptor of the mixture. Unsupervised learning methods (self-organizing maps) produced a reasonable clustering of the mixtures by reaction type, and allowed the correct classification of 80% and 63% of the mixtures in two independent test sets of different similarity to the training set. With random forests (RF), the percentage of correct classifications was increased to 99% and 80% for the same test sets. The RF probability associated to the predictions yielded a robust indication of their reliability. This study demonstrates the possibility of applying machine learning methods to automatically identify types of co-occurring chemical reactions from NMR data. Using no explicit structural information about the reactions participants, reaction elucidation is performed without structure elucidation of the molecules in the mixtures.

Introducing Undergraduate Students to Metabolomics Using a NMR-Based Analysis of Coffee Beans

ERIC Educational Resources Information Center

Sandusky, Peter Olaf

2017-01-01

Metabolomics applies multivariate statistical analysis to sets of high-resolution spectra taken over a population of biologically derived samples. The objective is to distinguish subpopulations within the overall sample population, and possibly also to identify biomarkers. While metabolomics has become part of the standard analytical toolbox in…

Development of a Brief Pre-Implementation Screening Tool to Identify Teachers Who Are at Risk for Not Implementing Intervention Curriculum and High-Implementing Teachers

ERIC Educational Resources Information Center

Wang, Bo; Stanton, Bonita; Lunn, Sonja; Patel, Pooja; Koci, Veronica; Deveaux, Lynette

2017-01-01

Few questionnaires have been developed to screen for potentially poor implementers of school-based interventions. This study combines teacher characteristics, perceptions, and teaching/training experiences to develop a short screening tool that can identify potential "low-performing" or "high-performing" teachers…

Identification of sex-specific urinary biomarkers for major depressive disorder by combined application of NMR- and GC-MS-based metabonomics.

PubMed

Zheng, P; Chen, J-J; Zhou, C-J; Zeng, L; Li, K-W; Sun, L; Liu, M-L; Zhu, D; Liang, Z-H; Xie, P

2016-11-15

Women are more vulnerable to major depressive disorder (MDD) than men. However, molecular biomarkers of sex differences are limited. Here we combined gas chromatography-mass spectrometry (GC-MS)- and nuclear magnetic resonance (NMR)-based metabonomics to investigate sex differences of urinary metabolite markers in MDD, and further explore their potential of diagnosing MDD. Consequently, the metabolite signatures of women and men MDD subjects were significantly different from of that in their respective healthy controls (HCs). Twenty seven women and 36 men related differentially expressed metabolites were identified in MDD. Fourteen metabolites were changed in both women and men MDD subjects. Significantly, the women-specific (m-Hydroxyphenylacetate, malonate, glycolate, hypoxanthine, isobutyrate and azelaic acid) and men-specific (tyrosine, N-acetyl-d-glucosamine, N-methylnicotinamide, indoxyl sulfate, citrate and succinate) marker panels were further identified, which could differentiate men and women MDD patients from their respective HCs with higher accuracy than previously reported sex-nonspecific marker panels. Our findings demonstrate that men and women MDD patients have distinct metabonomic signatures and sex-specific biomarkers have promising values in diagnosing MDD.

Identification of low-molecular-weight compounds inhibiting growth of corynebacteria: potential lead compounds for antibiotics.

PubMed

Stark, Jaime L; Copeland, Jennifer C; Eletsky, Alexander; Somerville, Greg A; Szyperski, Thomas; Powers, Robert

2014-02-01

The bacterial genus Corynebacteria contains several pathogenic species that cause diseases such as diphtheria in humans and "cheesy gland" in goats and sheep. Thus, identifying new therapeutic targets to treat Corynebacteria infections is both medically and economically important. CG2496, a functionally uncharacterized protein from Corynebacterium glutamicum, was evaluated using an NMR ligand-affinity screen. A total of 11 compounds from a library of 460 biologically active compounds were shown to selectively bind CG2496 in a highly conserved region of the protein. The best binder was identified to be methiothepin (KD =54 ± 19 µM), an FDA-approved serotonin receptor antagonist. Methiothepin was also shown to inhibit the growth of C. glutamicum, but not bacteria that lack CG2496 homologs. Our results suggest that CG2496 is a novel therapeutic target and methiothepin is a potential lead compound or structural scaffold for developing new antibiotics specifically targeting Corynebacteria. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

BXR, an embryonic orphan nuclear receptor activated by a novel class of endogenous benzoate metabolites

PubMed Central

Blumberg, Bruce; Kang, Heonjoong; Bolado, Jack; Chen, Hongwu; Craig, A. Grey; Moreno, Tanya A.; Umesono, Kazuhiko; Perlmann, Thomas; De Robertis, Eddy M.; Evans, Ronald M.

1998-01-01

Nuclear receptors are ligand-modulated transcription factors that respond to steroids, retinoids, and thyroid hormones to control development and body physiology. Orphan nuclear receptors, which lack identified ligands, provide a unique, and largely untapped, resource to discover new principles of physiologic homeostasis. We describe the isolation and characterization of the vertebrate orphan receptor, BXR, which heterodimerizes with RXR and binds high-affinity DNA sites composed of a variant thyroid hormone response element. A bioactivity-guided screen of embryonic extracts revealed that BXR is activatable by low-molecular-weight molecules with spectral patterns distinct from known nuclear receptor ligands. Mass spectrometry and 1H NMR analysis identified alkyl esters of amino and hydroxy benzoic acids as potent, stereoselective activators. In vitro cofactor association studies, along with competable binding of radiolabeled compounds, establish these molecules as bona fide ligands. Benzoates comprise a new molecular class of nuclear receptor ligand and their activity suggests that BXR may control a previously unsuspected vertebrate signaling pathway. PMID:9573044

1H-NMR, 1H-NMR T2-edited, and 2D-NMR in bipolar disorder metabolic profiling.

PubMed

Sethi, Sumit; Pedrini, Mariana; Rizzo, Lucas B; Zeni-Graiff, Maiara; Mas, Caroline Dal; Cassinelli, Ana Cláudia; Noto, Mariane N; Asevedo, Elson; Cordeiro, Quirino; Pontes, João G M; Brasil, Antonio J M; Lacerda, Acioly; Hayashi, Mirian A F; Poppi, Ronei; Tasic, Ljubica; Brietzke, Elisa

2017-12-01

The objective of this study was to identify molecular alterations in the human blood serum related to bipolar disorder, using nuclear magnetic resonance (NMR) spectroscopy and chemometrics. Metabolomic profiling, employing 1 H-NMR, 1 H-NMR T 2 -edited, and 2D-NMR spectroscopy and chemometrics of human blood serum samples from patients with bipolar disorder (n = 26) compared with healthy volunteers (n = 50) was performed. The investigated groups presented distinct metabolic profiles, in which the main differential metabolites found in the serum sample of bipolar disorder patients compared with those from controls were lipids, lipid metabolism-related molecules (choline, myo-inositol), and some amino acids (N-acetyl-L-phenyl alanine, N-acetyl-L-aspartyl-L-glutamic acid, L-glutamine). In addition, amygdalin, α-ketoglutaric acid, and lipoamide, among other compounds, were also present or were significantly altered in the serum of bipolar disorder patients. The data presented herein suggest that some of these metabolites differentially distributed between the groups studied may be directly related to the bipolar disorder pathophysiology. The strategy employed here showed significant potential for exploring pathophysiological features and molecular pathways involved in bipolar disorder. Thus, our findings may contribute to pave the way for future studies aiming at identifying important potential biomarkers for bipolar disorder diagnosis or progression follow-up.

17alpha/H/ hopane identified in oil shale of the Green River formation /Eocene/ by carbon-13 NMR.

NASA Technical Reports Server (NTRS)

Balogh, B.; Wilson, D. M.; Christiansen, P.; Burlingame, A. L.

1973-01-01

During an investigation of C-13 NMR shifts and the structural correspondence of pentacyclic triterpenes a C-13 NMR study was conducted on one of the most abundant components of the hexane soluble fraction of oil shale bitumen of the Green River formation. A rigorous proof was derived exclusively from C-13 NMR data for the structure of the important triterpenoid fossil molecule. It was established that the structure of the isolated triterpane was 17alpha(H) hopane.

Prostate-Specific Antigen (PSA)–Based Population Screening for Prostate Cancer: An Economic Analysis

PubMed Central

Tawfik, A

2015-01-01

Background The prostate-specific antigen (PSA) blood test has become widely used in Canada to test for prostate cancer (PC), the most common cancer among Canadian men. Data suggest that population-based PSA screening may not improve overall survival. Objectives This analysis aimed to review existing economic evaluations of population-based PSA screening, determine current spending on opportunistic PSA screening in Ontario, and estimate the cost of introducing a population-based PSA screening program in the province. Methods A systematic literature search was performed to identify economic evaluations of population-based PSA screening strategies published from 1998 to 2013. Studies were assessed for their methodological quality and applicability to the Ontario setting. An original cost analysis was also performed, using data from Ontario administrative sources and from the published literature. One-year costs were estimated for 4 strategies: no screening, current (opportunistic) screening of men aged 40 years and older, current (opportunistic) screening of men aged 50 to 74 years, and population-based screening of men aged 50 to 74 years. The analysis was conducted from the payer perspective. Results The literature review demonstrated that, overall, population-based PSA screening is costly and cost-ineffective but may be cost-effective in specific populations. Only 1 Canadian study, published 15 years ago, was identified. Approximately $119.2 million is being spent annually on PSA screening of men aged 40 years and older in Ontario, including close to $22 million to screen men younger than 50 and older than 74 years of age (i.e., outside the target age range for a population-based program). A population-based screening program in Ontario would cost approximately $149.4 million in the first year. Limitations Estimates were based on the synthesis of data from a variety of sources, requiring several assumptions and causing uncertainty in the results. For example, where Ontario-specific data were unavailable, data from the United States were used. Conclusions PSA screening is associated with significant costs to the health care system when the cost of the PSA test itself is considered in addition to the costs of diagnosis, staging, and treatment of screen-detected PCs. PMID:26366237

Identifying Nursing Interventions in a Cancer Screening Program Using Nursing Interventions Classification Taxonomy.

PubMed

Benito, Llucia; Lluch, María Teresa; Falcó, Anna Marta; García, Montse; Puig, Montse

2017-04-01

This study aimed to investigate which Nursing Interventions Classification (NIC) labels correspond to specific nursing interventions provided during cancer screening to establish a nursing documentation system. This descriptive study was conducted to identify and classify the interventions that cancer screening nurses perform based on an initial list. The initial list was grouped into 15 interventions that corresponded to four domains and eight classes. The study found expert consensus regarding the duties of cancer screening nurses and identified 15 interventions that should be implemented in clinical practice for cancer screening care, according to the NIC taxonomy. This study is the first step in developing indicators to assess nursing performance in cancer screening, and it helps to establish the core competency requirements for cancer screening nurses. © 2015 NANDA International, Inc.

Sequence Effect on the Formation of DNA Minidumbbells.

PubMed

Liu, Yuan; Lam, Sik Lok

2017-11-16

The DNA minidumbbell (MDB) is a recently identified non-B structure. The reported MDBs contain two TTTA, CCTG, or CTTG type II loops. At present, the knowledge and understanding of the sequence criteria for MDB formation are still limited. In this study, we performed a systematic high-resolution nuclear magnetic resonance (NMR) and native gel study to investigate the effect of sequence variations in tandem repeats on the formation of MDBs. Our NMR results reveal the importance of hydrogen bonds, base-base stacking, and hydrophobic interactions from each of the participating residues. We conclude that in the MDBs formed by tandem repeats, C-G loop-closing base pairs are more stabilizing than T-A loop-closing base pairs, and thymine residues in both the second and third loop positions are more stabilizing than cytosine residues. The results from this study enrich our knowledge on the sequence criteria for the formation of MDBs, paving a path for better exploring their potential roles in biological systems and DNA nanotechnology.

A new chitosan Schiff base supported Pd(II) complex for microwave-assisted synthesis of biaryls compounds

NASA Astrophysics Data System (ADS)

Baran, Talat

2017-08-01

In this study, a new heterogeneous palladium (II) catalyst that contains O-carboxymethyl chitosan Schiff base has been designed for Suzuki coupling reactions. The chemical structures of the synthesized catalyst were characterized with the FTIR, TG/DTG, ICP-OES, SEM/EDAX, 1H NMR, 13C NMR, GC/MS, XRD, and magnetic moment techniques. The reusability and catalytic behavior of heterogeneous catalyst was tested towards Suzuki reactions. As a result of the tests, excellent selectivity was obtained, and by-products of homo coupling were not seen in the spectra. The biaryls products were identified on a GC/MS. In addition, it was determined in the reusability tests that the catalysts could be used several times (seven runs). More importantly, with very low catalyst loading (6 × 10-3 mol %) in very short reaction time (5 min), chitosan Schiff base supported Pd(II) complex gave high TON and TOF values. These findings showed that Schiff base supported Pd(II) catalyst is suitable for Suzuki cross coupling reactions.

CHARACTERIZATION OF METABOLITES IN SMALL FISH BIOFLUIDS AND TISSUES BY NMR SPECTROSCOPY

EPA Science Inventory

Nuclear magnetic resonance (NMR) spectroscopy has been utilized for assessing ecotoxicity in small fish models by means of metabolomics. Two fundamental challenges of NMR-based metabolomics are the detection limit and characterization of metabolites (or NMR resonance assignments...

Mass Screening: An Aid to Competency Based Program Development.

ERIC Educational Resources Information Center

Siehl, Peterann M.; Studer, Jeannine

Adolescent suicide is the second leading cause of death in the adolescent population and is on the rise. This study used a mass screening concept as a pre-test identifier of at risk clients for suicide ideation and depressions; development of a competency-based prevention group treatment program, and the post-testing of the identified at-risk…

A modified reverse one-hybrid screen identifies transcriptional activation in Phyochrome-Interacting Factor 3

USDA-ARS?s Scientific Manuscript database

Transcriptional activation domains (TAD) are difficult to predict and identify, since they are not conserved and have little consensus. Here, we describe a yeast-based screening method that is able to identify individual amino acid residues involved in transcriptional activation in a high throughput...

[Studies on chemical constituents from roots of Mirabilis jalapa].

PubMed

Lai, Guo-Fang; Luo, Shi-De; Cao, Jian-Xin; Wang, Yi-Fen

2008-01-01

To investigate the anti-HIV constituents from the root of Mirabilis jalapa. The compounds were isolated by column chromatography on silica gel, Sephadex LH - 20, MCI-gel CHP-20P and RP-18. The structure were identified by means of NMR and MS analyses (1H-NMR, 13C-NMR, MS). Eleven compounds were isolated and identified as astragaloside II (1), astragaloside II (2), astragaloside IV (3), astragaloside VI (4), flazin (5), 4'-hydroxy-2, 3-dihydroflavone 7-beta-D-glucopyranoside (6), gingerglycolipid A (7), 3, 4-dihydroxybenzaldehyd (8), p-hydroxybenzaldehyde (9), beta-sitosterol (10) and daucosterol (11). Compounds 1-9 were obtained from this genus for the first time.

Identifying at-risk children at school entry: the usefulness of multibehavioral problem profiles.

PubMed

Flanagan, Kelly S; Bierman, Karen L; Kam, Chi-Ming

2003-09-01

Found that 1st-grade teacher ratings of aggressive, hyperactive-inattentive, and low levels of prosocial behaviors made unique contributions to the prediction of school outcomes (measured 2 years later) for 755 children. Person-oriented analyses compared the predictive utility of 5 screening strategies based on child problem profiles to identify children at risk for school problems. A broad screening strategy, in which children with elevations in any 1 of the 3 behavior problem dimensions were identified as "at-risk," showed lower specificity but superior sensitivity, odds ratios, and overall accuracy in the prediction of school outcomes than the other screening strategies that were more narrowly focused or were based on a total problem score. Results are discussed in terms of implications for the screening and design of preventive interventions.

Development of a Clinical Forecasting Model to Predict Comorbid Depression Among Diabetes Patients and an Application in Depression Screening Policy Making.

PubMed

Jin, Haomiao; Wu, Shinyi; Di Capua, Paul

2015-09-03

Depression is a common but often undiagnosed comorbid condition of people with diabetes. Mass screening can detect undiagnosed depression but may require significant resources and time. The objectives of this study were 1) to develop a clinical forecasting model that predicts comorbid depression among patients with diabetes and 2) to evaluate a model-based screening policy that saves resources and time by screening only patients considered as depressed by the clinical forecasting model. We trained and validated 4 machine learning models by using data from 2 safety-net clinical trials; we chose the one with the best overall predictive ability as the ultimate model. We compared model-based policy with alternative policies, including mass screening and partial screening, on the basis of depression history or diabetes severity. Logistic regression had the best overall predictive ability of the 4 models evaluated and was chosen as the ultimate forecasting model. Compared with mass screening, the model-based policy can save approximately 50% to 60% of provider resources and time but will miss identifying about 30% of patients with depression. Partial-screening policy based on depression history alone found only a low rate of depression. Two other heuristic-based partial screening policies identified depression at rates similar to those of the model-based policy but cost more in resources and time. The depression prediction model developed in this study has compelling predictive ability. By adopting the model-based depression screening policy, health care providers can use their resources and time better and increase their efficiency in managing their patients with depression.

Optimal healthcare decision making under multiple mathematical models: application in prostate cancer screening.

PubMed

Bertsimas, Dimitris; Silberholz, John; Trikalinos, Thomas

2018-03-01

Important decisions related to human health, such as screening strategies for cancer, need to be made without a satisfactory understanding of the underlying biological and other processes. Rather, they are often informed by mathematical models that approximate reality. Often multiple models have been made to study the same phenomenon, which may lead to conflicting decisions. It is natural to seek a decision making process that identifies decisions that all models find to be effective, and we propose such a framework in this work. We apply the framework in prostate cancer screening to identify prostate-specific antigen (PSA)-based strategies that perform well under all considered models. We use heuristic search to identify strategies that trade off between optimizing the average across all models' assessments and being "conservative" by optimizing the most pessimistic model assessment. We identified three recently published mathematical models that can estimate quality-adjusted life expectancy (QALE) of PSA-based screening strategies and identified 64 strategies that trade off between maximizing the average and the most pessimistic model assessments. All prescribe PSA thresholds that increase with age, and 57 involve biennial screening. Strategies with higher assessments with the pessimistic model start screening later, stop screening earlier, and use higher PSA thresholds at earlier ages. The 64 strategies outperform 22 previously published expert-generated strategies. The 41 most "conservative" ones remained better than no screening with all models in extensive sensitivity analyses. We augment current comparative modeling approaches by identifying strategies that perform well under all models, for various degrees of decision makers' conservativeness.

Early Prediction of Lupus Nephritis Using Advanced Proteomics

DTIC Science & Technology

2010-06-01

SELDI-TOF-MS. Additional proteomic profiling studies using NMR- and MS-based metabonomics have been completed, and LC/MS based protein profiling using...Flight mass spectrometry (SELDI-TOF-MS). Changes in proteomic profiles will be confirmed and enhanced using NMR- and MS-based metabonomics , by Dr...performed using NMR- and MS-based metabonomics at Miami University, in the laboratory of Dr. Michael Kennedy. Initial spectra and profiles obtained show

Model for the interpretation of nuclear magnetic resonance relaxometry of hydrated porous silicate materials

NASA Astrophysics Data System (ADS)

Faux, D. A.; Cachia, S.-H. P.; McDonald, P. J.; Bhatt, J. S.; Howlett, N. C.; Churakov, S. V.

2015-03-01

Nuclear magnetic resonance (NMR) relaxation experimentation is an effective technique for probing the dynamics of proton spins in porous media, but interpretation requires the application of appropriate spin-diffusion models. Molecular dynamics (MD) simulations of porous silicate-based systems containing a quasi-two-dimensional water-filled pore are presented. The MD simulations suggest that the residency time of the water on the pore surface is in the range 0.03-12 ns, typically 2-5 orders of magnitude less than values determined from fits to experimental NMR measurements using the established surface-layer (SL) diffusion models of Korb and co-workers [Phys. Rev. E 56, 1934 (1997), 10.1103/PhysRevE.56.1934]. Instead, MD identifies four distinct water layers in a tobermorite-based pore containing surface Ca2 + ions. Three highly structured water layers exist within 1 nm of the surface and the central region of the pore contains a homogeneous region of bulklike water. These regions are referred to as layer 1 and 2 (L1, L2), transition layer (TL), and bulk (B), respectively. Guided by the MD simulations, a two-layer (2L) spin-diffusion NMR relaxation model is proposed comprising two two-dimensional layers of slow- and fast-moving water associated with L2 and layers TL+B, respectively. The 2L model provides an improved fit to NMR relaxation times obtained from cementitious material compared to the SL model, yields diffusion correlation times in the range 18-75 ns and 28-40 ps in good agreement with MD, and resolves the surface residency time discrepancy. The 2L model, coupled with NMR relaxation experimentation, provides a simple yet powerful method of characterizing the dynamical properties of proton-bearing porous silicate-based systems such as porous glasses, cementitious materials, and oil-bearing rocks.

Pharmacological and Toxicological Screening of Novel Benzimidazole-Morpholine Derivatives as Dual-Acting Inhibitors.

PubMed

Can, Nafiz Öncü; Çevik, Ulviye Acar; Sağlık, Begüm Nurpelin; Özkay, Yusuf; Atlı, Özlem; Baysal, Merve; Özkay, Ümide Demir; Can, Özgür Devrim

2017-08-19

The aim of this study was to investigate acetylcholinesterase (AChE), monoamine oxidase A (MAO-A), monoamine oxidase B (MAO-B), cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) enzyme inhibitory, and antimicrobial activities of a new series of 2-(4-substituted phenyl)-1-[2-(morpholin-4-yl)ethyl]-1 H -benzimidazole derivatives, for their possible use as multi-action therapeutic agents. Target compounds ( n = 15) were synthesized under microwave irradiation conditions in two steps, and their structures were elucidated by FT-IR, ¹H-NMR, 13 C-NMR and high resolution mass spectroscopic analyses. Pharmacological screening studies revealed that two of the compounds ( 2b and 2j ) have inhibitory potential on both COX-1 and COX-2 enzymes. In addition, cytotoxic and genotoxic properties of the compounds 2b , 2j and 2m were investigated via the well-known MTT and Ames tests, which revealed that the mentioned compounds are non-cytotoxic and non-genotoxic. As a concise conclusion, two novel compounds were characterized as potential candidates for treatment of frequently encountered inflammatory diseases.

Synthesis, antimicrobial, anti-biofilm evaluation, and molecular modelling study of new chalcone linked amines derivatives.

PubMed

El-Messery, Shahenda M; Habib, El-Sayed E; Al-Rashood, Sarah T A; Hassan, Ghada S

2018-12-01

A series of amide chalcones conjugated with different secondary amines were synthesised and characterised by different spectroscopic techniques 1 H NMR, 13 C NMR, and ESI-MS. They were screened for in vitro antibacterial activity. Compounds 36, 37, 38, 42, and 44 are the most active among the synthesised series exhibiting MIC value of 2.0-10.0 µg/ml against different bacterial strains. Compound 36 was equipotent to the standard drug Ampicillin displaying MBC value of 2.0 µg/ml against the bacterial strain Staphylococcus aureus. The products were screened for anti-biofilm activity. Compounds 36, 37, and 38 exhibited promising anti-biofilm activity with IC 50 value ranges from 2.4 to 8.6 µg. Molecular modelling was performed suggesting parameters of signalling anti-biofilm mechanism. AspB327 HisB340 (arene-arene interaction) and IleB328 amino acid residues seemed of higher importance to inhibit c-di-GMP. Hydrophobicity may be crucial for activity. ADME calculations suggested that compounds 36, 37, and 38 could be used as good orally absorbed anti-biofilm agents.

School-Based Screening for Suicide Risk: Balancing Costs and Benefits

PubMed Central

Wilcox, Holly; Huo, Yanling; Turner, J. Blake; Fisher, Prudence; Shaffer, David

2010-01-01

Objectives. We examined the effects of a scoring algorithm change on the burden and sensitivity of a screen for adolescent suicide risk. Methods. The Columbia Suicide Screen was used to screen 641 high school students for high suicide risk (recent ideation or lifetime attempt and depression, or anxiety, or substance use), determined by subsequent blind assessment with the Diagnostic Interview Schedule for Children. We compared the accuracy of different screen algorithms in identifying high-risk cases. Results. A screen algorithm comprising recent ideation or lifetime attempt or depression, anxiety, or substance-use problems set at moderate-severity level classed 35% of students as positive and identified 96% of high-risk students. Increasing the algorithm's threshold reduced the proportion identified to 24% and identified 92% of high-risk cases. Asking only about recent suicidal ideation or lifetime suicide attempt identified 17% of the students and 89% of high-risk cases. The proportion of nonsuicidal diagnosis–bearing students found with the 3 algorithms was 62%, 34%, and 12%, respectively. Conclusions. The Columbia Suicide Screen threshold can be altered to reduce the screen-positive population, saving costs and time while identifying almost all students at high risk for suicide. PMID:20634467

Study of water dynamics in the soaking, steaming, and solid-state fermentation of glutinous rice by LF-NMR: a novel monitoring approach.

PubMed

Li, Teng; Tu, Chuanhai; Rui, Xin; Gao, Yangwen; Li, Wei; Wang, Kun; Xiao, Yu; Dong, Mingsheng

2015-04-01

Solid-state fermentation (SSF) of starchy grain is a traditional technique for food and alcoholic beverage production in East Asia. In the present study, low-field nuclear magnetic resonance (LF-NMR) was introduced for the elucidation of water dynamics and microstructure alternations during the soaking, steaming, and SSF of glutinous rice as a rapid real-time monitoring method. Three different proton fractions with different mobilities were identified based on the degree of interaction between biopolymers and water. Soaking and steaming significantly changed the proton distribution of the sample. The different phases of SSF were reflected by the T2 parameters. In addition, the variations in the T2 parameters were explained by the microstructure changes of rice induced by SSF. The fermentation time and T2 parameters were sigmoidally correlated. Thus, LF-NMR may be an effective real-time monitoring method for SSF in starch systems.

1H NMR and PCA-based analysis revealed variety dependent changes in phenolic contents of apple fruit after drying.

PubMed

Francini, Alessandra; Romeo, Stefania; Cifelli, Mario; Gori, Daniele; Domenici, Valentina; Sebastiani, Luca

2017-04-15

Dry and fresh apples have been studied monitoring their polyphenolic profiles through 1 H NMR, antioxidant capacity and total polyphenol content. Six ancient and underutilized apple varieties (Mantovana, Mora, Nesta, Cipolla, Ruggina, Sassola) and a commercial one (Golden Delicious) were dried with an air-drying system at 45°C for 19h. Although some of their polyphenol constituents were lost during drying, the antioxidant capacity of some apple varieties remained higher compared to Golden Delicious. This result is very important for ancient and underutilized varieties that are not consumed on large scale as fresh product since they have low attractiveness, due to their ugly appearance. Combining quantitative NMR spectroscopy with principal component analysis we have identified and quantified several polyphenols (such as catechin, epicathechin, and chlorogenic acid) that are important to establish the nutraceutical value of the different investigated apple varieties. Copyright © 2016 Elsevier Ltd. All rights reserved.

Identifying Novel Molecular Structures for Advanced Melanoma by Ligand-Based Virtual Screening

PubMed Central

Wang, Zhao; Lu, Yan; Seibel, William; Miller, Duane D.; Li, Wei

2009-01-01

We recently discovered a new class of thiazole analogs that are highly potent against melanoma cells. To expand the structure-activity relationship study and to explore potential new molecular scaffolds, we performed extensive ligand-based virtual screening against a compound library containing 342,910 small molecules. Two different approaches of virtual screening were carried out using the structure of our lead molecule: 1) connectivity-based search using Scitegic Pipeline Pilot from Accelerys and 2) molecular shape similarity search using Schrodinger software. Using a testing compound library, both approaches can rank similar compounds very high and rank dissimilar compounds very low, thus validating our screening methods. Structures identified from these searches were analyzed, and selected compounds were tested in vitro to assess their activity against melanoma cancer cell lines. Several molecules showed good anticancer activity. While none of the identified compounds showed better activity than our lead compound, they provided important insight into structural modifications for our lead compound and also provided novel platforms on which we can optimize new classes of anticancer compounds. One of the newly synthesized analogs based on this virtual screening has improved potency and selectivity against melanoma. PMID:19445498

Computational Selection of Inhibitors of A-beta Aggregation and Neuronal Toxicity

PubMed Central

Chen, Deliang; Martin, Zane S.; Soto, Claudio; Schein, Catherine H.

2009-01-01

Alzheimer’s Disease (AD) is characterized by the cerebral accumulation of misfolded and aggregated amyloid-β protein (Aβ). Disease symptoms can be alleviated, in vitro and in vivo, by “β-sheet breaker” pentapeptides that reduce plaque volume. However the peptide nature of these compounds, made them biologically unstable and unable to penetrate membranes with high efficiency. The main goal of this study was to use computational methods to identify small molecule mimetics with better drug-like properties. For this purpose, the docked conformations of the active peptides were used to identify compounds with similar activities. A series of related β-sheet breaker peptides were docked to solid state NMR structures of a fibrillar form of Aβ. The lowest energy conformations of the active peptides were used to design three dimensional (3D)-pharmacophores, suitable for screening the NCI database with Unity. Small molecular weight compounds with physicochemical features in a conformation similar to the active peptides were selected, ranked by docking solubility parameters. Of 16 diverse compounds selected for experimental screening, 2 prevented and reversed Aβ aggregation at 2–3 μM concentration, as measured by Thioflavin T (ThT) fluorescence and ELISA assays. They also prevented the toxic effects of aggregated Aβ on neuroblastoma cells. Their low molecular weight and aqueous solubility makes them promising lead compounds for treating AD. PMID:19540126

CYP109E1 is a novel versatile statin and terpene oxidase from Bacillus megaterium.

PubMed

Putkaradze, Natalia; Litzenburger, Martin; Abdulmughni, Ammar; Milhim, Mohammed; Brill, Elisa; Hannemann, Frank; Bernhardt, Rita

2017-12-01

CYP109E1 is a cytochrome P450 monooxygenase from Bacillus megaterium with a hydroxylation activity for testosterone and vitamin D3. This study reports the screening of a focused library of statins, terpene-derived and steroidal compounds to explore the substrate spectrum of this enzyme. Catalytic activity of CYP109E1 towards the statin drug-precursor compactin and the prodrugs lovastatin and simvastatin as well as biotechnologically relevant terpene compounds including ionones, nootkatone, isolongifolen-9-one, damascones, and β-damascenone was found in vitro. The novel substrates induced a type I spin-shift upon binding to P450 and thus permitted to determine dissociation constants. For the identification of conversion products by NMR spectroscopy, a B. megaterium whole-cell system was applied. NMR analysis revealed for the first time the ability of CYP109E1 to catalyze an industrially highly important reaction, the production of pravastatin from compactin, as well as regioselective oxidations generating drug metabolites (6'β-hydroxy-lovastatin, 3'α-hydroxy-simvastatin, and 4″-hydroxy-simvastatin) and valuable terpene derivatives (3-hydroxy-α-ionone, 4-hydroxy-β-ionone, 11,12-epoxy-nootkatone, 4(R)-hydroxy-isolongifolen-9-one, 3-hydroxy-α-damascone, 4-hydroxy-β-damascone, and 3,4-epoxy-β-damascone). Besides that, a novel compound, 2-hydroxy-β-damascenone, produced by CYP109E1 was identified. Docking calculations using the crystal structure of CYP109E1 rationalized the experimentally observed regioselective hydroxylation and identified important amino acid residues for statin and terpene binding.

Identification of anti-HIV active dicaffeoylquinic- and tricaffeoylquinic acids in Helichrysum populifolium by NMR-based metabolomic guided fractionation.

PubMed

Heyman, Heino Martin; Senejoux, François; Seibert, Isabell; Klimkait, Thomas; Maharaj, Vinesh Jaichand; Meyer, Jacobus Johannes Marion

2015-06-01

South Africa being home to more than 35% of the world's Helichrysum species (c.a. 244) of which many are used in traditional medicine, is seen potentially as a significant resource in the search of new anti-HIV chemical entities. It was established that five of the 30 Helichrysum species selected for this study had significant anti-HIV activity ranging between 12 and 21 μg/mL (IC50) by using an in-house developed DeCIPhR method on a full virus model. Subsequent toxicity tests also revealed little or no toxicity for these active extracts. With the use of NMR-based metabolomics, the search for common chemical characteristics within the plant extract was conducted, which resulted in specific chemical shift areas identified that could be linked to the anti-HIV activity of the extracts. The NMR chemical shifts associated with the activity were identified to be 2.56-3.08 ppm, 5.24-6.28 ppm, 6.44-7.04 ppm and 7.24-8.04 ppm. This activity profile was then used to guide the fractionation process by narrowing down and focusing the fractionation and purification processes to speed up the putative identification of five compounds with anti-HIV activity in the most active species, Helichrysum populifolium. The anti-HIV compounds identified for the first time from H. populifolium were three dicaffeoylquinic acid derivatives, i.e. 3,4-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid and 4,5-dicaffeoylquinic acid as well as two tricaffeoylquinic acid derivatives i.e. 1,3,5-tricaffeoylquinic acid and either 5-malonyl-1,3,4-tricaffeoylquinic or 3-malonyl-1,4,5-tricaffeoylquinic acid, with the latter being identified for the first time in the genus. Copyright © 2015 Elsevier B.V. All rights reserved.

Analytical challenges in drug counterfeiting and falsification-The NMR approach.

PubMed

Holzgrabe, Ulrike; Malet-Martino, Myriam

2011-06-25

Counterfeiting of products is a global problem. As long as clothes, clocks, leather wear, etc. are faked there is no danger, but when it comes to drugs, counterfeiting can be life-threatening. In the last years sub-standard active pharmaceutical ingredients (APIs) were found more often even though the use of the quality-ensuring methods of international pharmacopoeias should have detected additional impurities and the low content of the API. Methods orthogonal to the separating methods used in the pharmacopoeias are necessary to find counterfeits. Beside Raman and NIR spectroscopies as well as powder X-ray analysis, NMR spectroscopy being a primary ratio method of measurement is highly suitable to identify and quantify a drug and its related substances as well as to recognize a drug of sub-standard quality. DOSY experiments are suitable to identify the ingredients of formulations and therefore to identify wrong and/or additional ingredients. This review gives an overview of the application of quantitative NMR spectroscopy and DOSY NMR in anticounterfeiting. Copyright © 2010 Elsevier B.V. All rights reserved.

Identification of Chlorogenic Acid as a Resistance Factor for Thrips in Chrysanthemum[C][OA

PubMed Central

Leiss, Kirsten A.; Maltese, Federica; Choi, Young Hae; Verpoorte, Robert; Klinkhamer, Peter G.L.

2009-01-01

Western flower thrips (Frankliniella occidentalis) has become a key insect pest of agricultural and horticultural crops worldwide. Little is known about host plant resistance to thrips. In this study, we investigated thrips resistance in chrysanthemum (Dendranthema grandiflora). We identified thrips-resistant chrysanthemums applying bioassays. Subsequently, nuclear magnetic resonance (NMR)-based metabolomics was applied to compare the metabolome of thrips-resistant and -susceptible chrysanthemums. NMR facilitates wide-range coverage of the metabolome. We show that thrips-resistant and -susceptible chrysanthemums can be discriminated on basis of their metabolomic profiles. Thrips-resistant chrysanthemums contained higher amounts of the phenylpropanoids chlorogenic acid and feruloyl quinic acid. Both phenylpropanoids are known for their inhibitory effect on herbivores as well as pathogens. Thus, chlorogenic and feruloyl quinic acid are the compounds of choice to improve host plants resistance to thrips in ornamentals and crops. The effect of chlorogenic acid on thrips was further studied in bioassays with artificial diets. These experiments confirmed the negative effects on thrips. Our results prove NMR to be an important tool to identify different metabolites involved in herbivore resistance. It constitutes a significant advance in the study of plant-insect relationships, providing key information on the implementation of herbivore resistance breeding strategies in plants. PMID:19448039

Comparison of traditional trigger tool to data warehouse based screening for identifying hospital adverse events.

PubMed

O'Leary, Kevin J; Devisetty, Vikram K; Patel, Amitkumar R; Malkenson, David; Sama, Pradeep; Thompson, William K; Landler, Matthew P; Barnard, Cynthia; Williams, Mark V

2013-02-01

Research supports medical record review using screening triggers as the optimal method to detect hospital adverse events (AE), yet the method is labour-intensive. This study compared a traditional trigger tool with an enterprise data warehouse (EDW) based screening method to detect AEs. We created 51 automated queries based on 33 traditional triggers from prior research, and then applied them to 250 randomly selected medical patients hospitalised between 1 September 2009 and 31 August 2010. Two physicians each abstracted records from half the patients using a traditional trigger tool and then performed targeted abstractions for patients with positive EDW queries in the complementary half of the sample. A third physician confirmed presence of AEs and assessed preventability and severity. Traditional trigger tool and EDW based screening identified 54 (22%) and 53 (21%) patients with one or more AE. Overall, 140 (56%) patients had one or more positive EDW screens (total 366 positive screens). Of the 137 AEs detected by at least one method, 86 (63%) were detected by a traditional trigger tool, 97 (71%) by EDW based screening and 46 (34%) by both methods. Of the 11 total preventable AEs, 6 (55%) were detected by traditional trigger tool, 7 (64%) by EDW based screening and 2 (18%) by both methods. Of the 43 total serious AEs, 28 (65%) were detected by traditional trigger tool, 29 (67%) by EDW based screening and 14 (33%) by both. We found relatively poor agreement between traditional trigger tool and EDW based screening with only approximately a third of all AEs detected by both methods. A combination of complementary methods is the optimal approach to detecting AEs among hospitalised patients.

Deformation and Failure of Protein Materials in Physiologically Extreme Conditions and Disease

DTIC Science & Technology

2009-03-01

resonance (NMR) spectroscopy and X- ray crystallography have advanced our ability to identify 3D protein structures57. Site-specific studies using NMR, a... ray crystallography, providing structural and temporal information about mechanisms of deformation and assembly (for example in intermediate...tens of thousands of 3D atomistic protein structures, identifying the structure of numerous proteins from varying species sources60. X- ray

Two High Throughput Screen Assays for Measurement of TNF-α in THP-1 Cells

PubMed Central

Leister, Kristin P; Huang, Ruili; Goodwin, Bonnie L; Chen, Andrew; Austin, Christopher P; Xia, Menghang

2011-01-01

Tumor Necrosis Factor-α (TNF-α), a secreted cytokine, plays an important role in inflammatory diseases and immune disorders, and is a potential target for drug development. The traditional assays for detecting TNF-α, enzyme linked immunosorbent assay (ELISA) and radioimmunoassay, are not suitable for the large size compound screens. Both assays suffer from a complicated protocol, multiple plate wash steps and/or excessive radioactive waste. A simple and quick measurement of TNF-α production in a cell based assay is needed for high throughput screening to identify the lead compounds from the compound library. We have developed and optimized two homogeneous TNF-α assays using the HTRF (homogeneous time resolved fluorescence) and AlphaLISA assay formats. We have validated the HTRF based TNF-α assay in a 1536-well plate format by screening a library of 1280 pharmacologically active compounds. The active compounds identified from the screen were confirmed in the AlphaLISA TNF-α assay using a bead-based technology. These compounds were also confirmed in a traditional ELISA assay. From this study, several beta adrenergic agonists have been identified as TNF-α inhibitors. We also identified several novel inhibitors of TNF-α, such as BTO-1, CCG-2046, ellipticine, and PD 169316. The results demonstrated that both homogeneous TNF-α assays are robust and suitable for high throughput screening. PMID:21643507

Selective excitation enables assignment of proton resonances and {sup 1}H-{sup 1}H distance measurement in ultrafast magic angle spinning solid state NMR spectroscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Rongchun; Ramamoorthy, Ayyalusamy, E-mail: ramamoor@umich.edu

2015-07-21

Remarkable developments in ultrafast magic angle spinning (MAS) solid-state NMR spectroscopy enabled proton-based high-resolution multidimensional experiments on solids. To fully utilize the benefits rendered by proton-based ultrafast MAS experiments, assignment of {sup 1}H resonances becomes absolutely necessary. Herein, we propose an approach to identify different proton peaks by using dipolar-coupled heteronuclei such as {sup 13}C or {sup 15}N. In this method, after the initial preparation of proton magnetization and cross-polarization to {sup 13}C nuclei, transverse magnetization of desired {sup 13}C nuclei is selectively prepared by using DANTE (Delays Alternating with Nutations for Tailored Excitation) sequence and then, it is transferredmore » to bonded protons with a short-contact-time cross polarization. Our experimental results demonstrate that protons bonded to specific {sup 13}C atoms can be identified and overlapping proton peaks can also be assigned. In contrast to the regular 2D HETCOR experiment, only a few 1D experiments are required for the complete assignment of peaks in the proton spectrum. Furthermore, the finite-pulse radio frequency driven recoupling sequence could be incorporated right after the selection of specific proton signals to monitor the intensity buildup for other proton signals. This enables the extraction of {sup 1}H-{sup 1}H distances between different pairs of protons. Therefore, we believe that the proposed method will greatly aid in fast assignment of peaks in proton spectra and will be useful in the development of proton-based multi-dimensional solid-state NMR experiments to study atomic-level resolution structure and dynamics of solids.« less

Targeted isolation and identification of bioactive compounds lowering cholesterol in the crude extracts of crabapples using UPLC-DAD-MS-SPE/NMR based on pharmacology-guided PLS-DA.

PubMed

Wen, Chao; Wang, Dongshan; Li, Xing; Huang, Tao; Huang, Cheng; Hu, Kaifeng

2018-02-20

The anti-hyperlipidemic effects of crude crabapple extracts derived from Malus 'Red jade', Malus hupehensis (Pamp.) Rehd. and Malus prunifolia (Willd.) Borkh. were evaluated on high-fat diet induced obese (HF DIO) mice. The results revealed that some of these extracts could lower serum cholesterol levels in HF DIO mice. The same extracts were also parallelly analyzed by LC-MS in both positive and negative ionization modes. Based on the pharmacological results, 22 LC-MS variables were identified to be correlated with the anti-hyperlipidemic effects using partial least square discriminant analysis (PLS-DA) and independent samples t-test. Further, under the guidance of the bioactivity-correlated LC-MS signals, 10 compounds were targetedly isolated and enriched using UPLC-DAD-MS-SPE and identified/elucidated by NMR together with MS/MS as citric acid(1), p-coumaric acid(2), hyperoside(3), myricetin(4), naringenin(5), quercetin(6), kaempferol(7), gentiopicroside(8), ursolic acid(9) and 8-epiloganic acid(10). Among these 10 compounds, 6 compounds, hyperoside(3), myricetin(4), naringenin(5), quercetin(6), kaempferol(7) and ursolic acid(9), were individually studied and reported to indeed have effects on lowering the serum lipid levels. These results demonstrated the efficiency of this strategy for drug discovery. In contrast to traditional routes to discover bioactive compounds in the plant extracts, targeted isolation and identification of bioactive compounds in the crude plant extracts using UPLC-DAD-MS-SPE/NMR based on pharmacology-guided PLS-DA of LC-MS data brings forward a new efficient dereplicated approach to natural products research for drug discovery. Copyright © 2017 Elsevier B.V. All rights reserved.

Histology Verification Demonstrates That Biospectroscopy Analysis of Cervical Cytology Identifies Underlying Disease More Accurately than Conventional Screening: Removing the Confounder of Discordance

PubMed Central

Gajjar, Ketan; Ahmadzai, Abdullah A.; Valasoulis, George; Trevisan, Júlio; Founta, Christina; Nasioutziki, Maria; Loufopoulos, Aristotelis; Kyrgiou, Maria; Stasinou, Sofia Melina; Karakitsos, Petros; Paraskevaidis, Evangelos; Da Gama-Rose, Bianca; Martin-Hirsch, Pierre L.; Martin, Francis L.

2014-01-01

Background Subjective visual assessment of cervical cytology is flawed, and this can manifest itself by inter- and intra-observer variability resulting ultimately in the degree of discordance in the grading categorisation of samples in screening vs. representative histology. Biospectroscopy methods have been suggested as sensor-based tools that can deliver objective assessments of cytology. However, studies to date have been apparently flawed by a corresponding lack of diagnostic efficiency when samples have previously been classed using cytology screening. This raises the question as to whether categorisation of cervical cytology based on imperfect conventional screening reduces the diagnostic accuracy of biospectroscopy approaches; are these latter methods more accurate and diagnose underlying disease? The purpose of this study was to compare the objective accuracy of infrared (IR) spectroscopy of cervical cytology samples using conventional cytology vs. histology-based categorisation. Methods Within a typical clinical setting, a total of n = 322 liquid-based cytology samples were collected immediately before biopsy. Of these, it was possible to acquire subsequent histology for n = 154. Cytology samples were categorised according to conventional screening methods and subsequently interrogated employing attenuated total reflection Fourier-transform IR (ATR-FTIR) spectroscopy. IR spectra were pre-processed and analysed using linear discriminant analysis. Dunn’s test was applied to identify the differences in spectra. Within the diagnostic categories, histology allowed us to determine the comparative efficiency of conventional screening vs. biospectroscopy to correctly identify either true atypia or underlying disease. Results Conventional cytology-based screening results in poor sensitivity and specificity. IR spectra derived from cervical cytology do not appear to discriminate in a diagnostic fashion when categories were based on conventional screening. Scores plots of IR spectra exhibit marked crossover of spectral points between different cytological categories. Although, significant differences between spectral bands in different categories are noted, crossover samples point to the potential for poor specificity and hampers the development of biospectroscopy as a diagnostic tool. However, when histology-based categories are used to conduct analyses, the scores plot of IR spectra exhibit markedly better segregation. Conclusions Histology demonstrates that ATR-FTIR spectroscopy of liquid-based cytology identifies the presence of underlying atypia or disease missed in conventional cytology screening. This study points to an urgent need for a future biospectroscopy study where categories are based on such histology. It will allow for the validation of this approach as a screening tool. PMID:24404130

Large scale meta-analysis of fragment-based screening campaigns: privileged fragments and complementary technologies.

PubMed

Kutchukian, Peter S; Wassermann, Anne Mai; Lindvall, Mika K; Wright, S Kirk; Ottl, Johannes; Jacob, Jaison; Scheufler, Clemens; Marzinzik, Andreas; Brooijmans, Natasja; Glick, Meir

2015-06-01

A first step in fragment-based drug discovery (FBDD) often entails a fragment-based screen (FBS) to identify fragment "hits." However, the integration of conflicting results from orthogonal screens remains a challenge. Here we present a meta-analysis of 35 fragment-based campaigns at Novartis, which employed a generic 1400-fragment library against diverse target families using various biophysical and biochemical techniques. By statistically interrogating the multidimensional FBS data, we sought to investigate three questions: (1) What makes a fragment amenable for FBS? (2) How do hits from different fragment screening technologies and target classes compare with each other? (3) What is the best way to pair FBS assay technologies? In doing so, we identified substructures that were privileged for specific target classes, as well as fragments that were privileged for authentic activity against many targets. We also revealed some of the discrepancies between technologies. Finally, we uncovered a simple rule of thumb in screening strategy: when choosing two technologies for a campaign, pairing a biochemical and biophysical screen tends to yield the greatest coverage of authentic hits. © 2014 Society for Laboratory Automation and Screening.

An efficient algorithm for automatic phase correction of NMR spectra based on entropy minimization

NASA Astrophysics Data System (ADS)

Chen, Li; Weng, Zhiqiang; Goh, LaiYoong; Garland, Marc

2002-09-01

A new algorithm for automatic phase correction of NMR spectra based on entropy minimization is proposed. The optimal zero-order and first-order phase corrections for a NMR spectrum are determined by minimizing entropy. The objective function is constructed using a Shannon-type information entropy measure. Entropy is defined as the normalized derivative of the NMR spectral data. The algorithm has been successfully applied to experimental 1H NMR spectra. The results of automatic phase correction are found to be comparable to, or perhaps better than, manual phase correction. The advantages of this automatic phase correction algorithm include its simple mathematical basis and the straightforward, reproducible, and efficient optimization procedure. The algorithm is implemented in the Matlab program ACME—Automated phase Correction based on Minimization of Entropy.

The clinical implementation of primary HPV screening.

PubMed

Mariani, Luciano; Igidbashian, Sarah; Sandri, Maria Teresa; Vici, Patrizia; Landoni, Fabio

2017-03-01

To evaluate, from a gynecology perspective, the transition from cytology-based HPV screening to primary HPV screening. Studies examining switching from cytology-based screening to primary HPV-DNA testing with triaging of patients with positive test results were retrieved and reviewed, with a particular focus on screening in an Italian setting. The increased complexity of patient-management decisions when implementing HPV-based screening was a critical issue discussed in the literature. The change in strategy represents a paradigm shift in moving from a medical perspective of identifying the disease in individual patients, to a public-healthcare perspective of excluding HPV from the healthy population and identifying a small sub-group of individuals at increased risk of HPV. With knowledge about HPV screening evolving rapidly, new programs and related algorithms need to be sufficiently flexible to be adjusted according to ongoing research and the validation of new assays. The establishment of a national working group (including epidemiologists, gynecologists, pathologists, and healthcare providers) will be necessary to properly implement and govern this important technical and cultural transition. © 2016 International Federation of Gynecology and Obstetrics.

Vivaldi: visualization and validation of biomacromolecular NMR structures from the PDB.

PubMed

Hendrickx, Pieter M S; Gutmanas, Aleksandras; Kleywegt, Gerard J

2013-04-01

We describe Vivaldi (VIsualization and VALidation DIsplay; http://pdbe.org/vivaldi), a web-based service for the analysis, visualization, and validation of NMR structures in the Protein Data Bank (PDB). Vivaldi provides access to model coordinates and several types of experimental NMR data using interactive visualization tools, augmented with structural annotations and model-validation information. The service presents information about the modeled NMR ensemble, validation of experimental chemical shifts, residual dipolar couplings, distance and dihedral angle constraints, as well as validation scores based on empirical knowledge and databases. Vivaldi was designed for both expert NMR spectroscopists and casual non-expert users who wish to obtain a better grasp of the information content and quality of NMR structures in the public archive. Copyright © 2013 Wiley Periodicals, Inc.

NMR-based fecal metabolomics fingerprinting as predictors of earlier diagnosis in patients with colorectal cancer.

PubMed

Lin, Yan; Ma, Changchun; Liu, Chengkang; Wang, Zhening; Yang, Jurong; Liu, Xinmu; Shen, Zhiwei; Wu, Renhua

2016-05-17

Colorectal cancer (CRC) is a growing cause of mortality in developing countries, warranting investigation into its earlier detection for optimal disease management. A metabolomics based approach provides potential for noninvasive identification of biomarkers of colorectal carcinogenesis, as well as dissection of molecular pathways of pathophysiological conditions. Here, proton nuclear magnetic resonance spectroscopy (1HNMR) -based metabolomic approach was used to profile fecal metabolites of 68 CRC patients (stage I/II=20; stage III=25 and stage IV=23) and 32 healthy controls (HC). Pattern recognition through principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA) was applied on 1H-NMR processed data for dimension reduction. OPLS-DA revealed that each stage of CRC could be clearly distinguished from HC based on their metabolomic profiles. Successive analyses identified distinct disturbances to fecal metabolites of CRC patients at various stages, compared with those in cancer free controls, including reduced levels of acetate, butyrate, propionate, glucose, glutamine, and elevated quantities of succinate, proline, alanine, dimethylglycine, valine, glutamate, leucine, isoleucine and lactate. These altered fecal metabolites potentially involved in the disruption of normal bacterial ecology, malabsorption of nutrients, increased glycolysis and glutaminolysis. Our findings revealed that the fecal metabolic profiles of healthy controls can be distinguished from CRC patients, even in the early stage (stage I/II), highlighting the potential utility of NMR-based fecal metabolomics fingerprinting as predictors of earlier diagnosis in CRC patients.

NMR-based fecal metabolomics fingerprinting as predictors of earlier diagnosis in patients with colorectal cancer

PubMed Central

Lin, Yan; Ma, Changchun; Liu, Chengkang; Wang, Zhening; Yang, Jurong; Liu, Xinmu; Shen, Zhiwei; Wu, Renhua

2016-01-01

Colorectal cancer (CRC) is a growing cause of mortality in developing countries, warranting investigation into its earlier detection for optimal disease management. A metabolomics based approach provides potential for noninvasive identification of biomarkers of colorectal carcinogenesis, as well as dissection of molecular pathways of pathophysiological conditions. Here, proton nuclear magnetic resonance spectroscopy (1HNMR) -based metabolomic approach was used to profile fecal metabolites of 68 CRC patients (stage I/II=20; stage III=25 and stage IV=23) and 32 healthy controls (HC). Pattern recognition through principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA) was applied on 1H-NMR processed data for dimension reduction. OPLS-DA revealed that each stage of CRC could be clearly distinguished from HC based on their metabolomic profiles. Successive analyses identified distinct disturbances to fecal metabolites of CRC patients at various stages, compared with those in cancer free controls, including reduced levels of acetate, butyrate, propionate, glucose, glutamine, and elevated quantities of succinate, proline, alanine, dimethylglycine, valine, glutamate, leucine, isoleucine and lactate. These altered fecal metabolites potentially involved in the disruption of normal bacterial ecology, malabsorption of nutrients, increased glycolysis and glutaminolysis. Our findings revealed that the fecal metabolic profiles of healthy controls can be distinguished from CRC patients, even in the early stage (stage I/II), highlighting the potential utility of NMR-based fecal metabolomics fingerprinting as predictors of earlier diagnosis in CRC patients. PMID:27107423

Screening and identification of steroidal saponins from Anemarrhena asphodeloides employing UPLC tandem triple quadrupole linear ion trap mass spectrometry.

PubMed

Xia, Yong-Gang; Guo, Xin-Dong; Liang, Jun; Yang, Bing-You; Kuang, Hai-Xue

2017-09-01

This study presents a practical and valid strategy for the screening and structural characterization of Anemarrhena asphodeloides Bge steroidal saponins (SSs) using ultra-high performance liquid chromatography coupled with triple quadrupole linear ion trap mass spectrometry. The whole analytical protocols integrate four-step procedures in the positive mode: (1) rational deduction of mass fragmentation pathways of A. asphodeloides SSs; (2) untargeted screening of potential A. asphodeloides SSs by multiple-ion monitoring-information-dependent-acquiring-enhanced product ion (MIM-IDA-EPI) scan through reverse phase liquid chromatography; (3) comprehensive construction of an ammoniated precursor ion database by combining untargeted MIM-IDA-EPI scans and data literature; and (4) structural interpretation of targeted A. asphodeloides SSs using MIM-IDA-EPI and multiple reaction monitoring (MRM)-IDA-EPI with an energy-resolved technique. The protocols were used to analyze SSs in A. asphodeloides; of the 87 detected SSs that were unambiguously characterized or tentatively identified, 19 compounds were the first to be reported from A. asphodeloides and 13 ones were characterized as potential new compounds. Accuracy of the analytical procedure was demonstrated by structural identification of three SSs by NMR spectroscopy. The proposed schemes hold an excellent promise in the structural prediction and interpretation of complex SSs from plant medicines by mass spectrometry. Copyright © 2017 Elsevier Inc. All rights reserved.

Structure of a Rhamnogalacturonan Fragment from Apple Pectin: Implications for Pectin Architecture

DOE PAGES

Wu, Xiangmei; Mort, Andrew

2014-01-01

A comore » mmercial apple pectin was sequentially digested with the cloned enzymes endopolygalacturonase, galactanase, arabinofuranosidase, xylogalacturonase, and rhamnogalacturonan hydrolase. The rhamnogalacturonan hydrolase-generated oligosaccharides were separated by ultrafiltration, anion exchange, and size-exclusion chromatography. Fractions from the ion exchange chromatography were pooled, lyophilized, and screened by MALDI-TOF MS. An oligosaccharide (RGP14P3) was identified and its structure, α -D-Gal p A- ( 1 → 2 ) - α -L-Rha p - ( 1 → 4 ) - α -D-Gal p A- ( 1 → 2 ) - α -L-Rha p - ( 1 → 4 ) - α -D-Gal p A, determined by 1D and 2D NMR spectrometry. This oligosaccharide probably represents a direct connection between homogalacturonan and rhamnogalacturonan in pectin. Alternatively, it could indicate that the nonreducing end of rhamnogalacturonan starts with a galacturonic acid residue.« less

Metabolic changes associated with papillary thyroid carcinoma: A nuclear magnetic resonance-based metabolomics study.

PubMed

Li, Yanyun; Chen, Minjian; Liu, Cuiping; Xia, Yankai; Xu, Bo; Hu, Yanhui; Chen, Ting; Shen, Meiping; Tang, Wei

2018-05-01

Papillary thyroid carcinoma (PTC) is the most common thyroid cancer. Nuclear magnetic resonance (NMR)‑based metabolomic technique is the gold standard in metabolite structural elucidation, and can provide different coverage of information compared with other metabolomic techniques. Here, we firstly conducted NMR based metabolomics study regarding detailed metabolic changes especially metabolic pathway changes related to PTC pathogenesis. 1H NMR-based metabolomic technique was adopted in conju-nction with multivariate analysis to analyze matched tumor and normal thyroid tissues obtained from 16 patients. The results were further annotated with Kyoto Encyclopedia of Genes and Genomes (KEGG), and Human Metabolome Database, and then were analyzed using modules of pathway analysis and enrichment analysis of MetaboAnalyst 3.0. Based on the analytical techniques, we established the models of principal component analysis (PCA), partial least squares-discriminant analysis (PLS-DA), and orthogonal partial least-squares discriminant analysis (OPLS‑DA) which could discriminate PTC from normal thyroid tissue, and found 15 robust differentiated metabolites from two OPLS-DA models. We identified 8 KEGG pathways and 3 pathways of small molecular pathway database which were significantly related to PTC by using pathway analysis and enrichment analysis, respectively, through which we identified metabolisms related to PTC including branched chain amino acid metabolism (leucine and valine), other amino acid metabolism (glycine and taurine), glycolysis (lactate), tricarboxylic acid cycle (citrate), choline metabolism (choline, ethanolamine and glycerolphosphocholine) and lipid metabolism (very-low‑density lipoprotein and low-density lipoprotein). In conclusion, the PTC was characterized with increased glycolysis and inhibited tricarboxylic acid cycle, increased oncogenic amino acids as well as abnormal choline and lipid metabolism. The findings in this study provide new insights into detailed metabolic changes of PTC, and hold great potential in the treatment of PTC.

DNA incision evaluation, binding investigation and biocidal screening of Cu(II), Ni(II) and Co(II) complexes with isoxazole Schiff bases.

PubMed

Ganji, Nirmala; Chityala, Vijay Kumar; Marri, Pradeep Kumar; Aveli, Rambabu; Narendrula, Vamsikrishna; Daravath, Sreenu; Shivaraj

2017-10-01

Two new series of binary metal complexes [M(L 1 ) 2 ] and [M(L 2 ) 2 ] where, M=Cu(II), Ni(II) & Co(II) and L 1 =4-((3,4-dimethylisoxazol-5-ylimino)methyl)benzene-1,3-diol; L 2 =2-((3,4-dimethylisoxazol-5-ylimino)methyl)-5-methoxyphenol were synthesized and characterized by elemental analysis, 1 H NMR, 13 C NMR, FT-IR, ESI mass, UV-Visible, magnetic moment, ESR, SEM and powder XRD studies. Based on these results, a square planar geometry is assigned for all the metal complexes where the Schiff base acts as uninegatively charged bidentate chelating agent via the hydroxyl oxygen and azomethine nitrogen atoms. DNA binding studies of all the complexes with calf thymus DNA have been comprehensively investigated using electronic absorption spectroscopy, fluorescence quenching and viscosity studies. The oxidative and photo cleavage affinity of metal complexes towards supercoiled pBR322 DNA has been ascertained by agarose gel electrophoresis assay. From the results, it is observed that all the metal complexes bind effectively to CT-DNA via an intercalative mode of binding and also cleave pBR322 DNA in a promising manner. Further the Cu(II) complexes have shown better binding and cleavage properties towards DNA. The antimicrobial activities of the Schiff bases and their metal complexes were studied on bacterial and fungal strains and the results denoted that the complexes are more potent than their Schiff base ligands. Copyright © 2017 Elsevier B.V. All rights reserved.

Unifying bacteria from decaying wood with various ubiquitous Gibbsiella species as G. acetica sp. nov. based on nucleotide sequence similarities and their acetic acid secretion.

PubMed

Geider, Klaus; Gernold, Marina; Jock, Susanne; Wensing, Annette; Völksch, Beate; Gross, Jürgen; Spiteller, Dieter

2015-12-01

Bacteria were isolated from necrotic apple and pear tree tissue and from dead wood in Germany and Austria as well as from pear tree exudate in China. They were selected for growth at 37 °C, screened for levan production and then characterized as Gram-negative, facultatively anaerobic rods. Nucleotide sequences from 16S rRNA genes, the housekeeping genes dnaJ, gyrB, recA and rpoB alignments, BLAST searches and phenotypic data confirmed by MALDI-TOF analysis showed that these bacteria belong to the genus Gibbsiella and resembled strains isolated from diseased oaks in Britain and Spain. Gibbsiella-specific PCR primers were designed from the proline isomerase and the levansucrase genes. Acid secretion was investigated by screening for halo formation on calcium carbonate agar and the compound identified by NMR as acetic acid. Its production by Gibbsiella spp. strains was also determined in culture supernatants by GC/MS analysis after derivatization with pentafluorobenzyl bromide. Some strains were differentiated by the PFGE patterns of SpeI digests and by sequence analyses of the lsc and the ppiD genes, and the Chinese Gibbsiella strain was most divergent. The newly investigated bacteria as well as Gibbsiella querinecans, Gibbsiella dentisursi and Gibbsiella papilionis, isolated in Britain, Spain, Korea and Japan, are taxonomically related Enterobacteriaceae, tolerate and secrete acetic acid. We therefore propose to unify them in the species Gibbsiella acetica sp. nov. Copyright © 2015. Published by Elsevier GmbH.

Online structural elucidation of alkaloids and other constituents in crude extracts and cultured cells of Nandina domestica by combination of LC-MS/MS, LC-NMR, and LC-CD analyses.

PubMed

Iwasa, Kinuko; Takahashi, Teturo; Nishiyama, Yumi; Moriyasu, Masataka; Sugiura, Makiko; Takeuchi, Atsuko; Tode, Chisato; Tokuda, Harukuni; Takeda, Kazuyoshi

2008-08-01

The combination of NMR, MS, and CD data permitted the structural elucidation including the absolute configuration of the known alkaloids and unknown components in the extract matrix solution of Nandina domestica without isolation and sample purification prior to the coupling experiments. Unstable natural stereoisomers were identified by LC-NMR and LC-MS. Five known alkaloids, (S)-isoboldine, (S)-domesticine, (S)-nantenine, sinoacutine, and menispermine, were identified from N. domestica. O-Methylpallidine and (E, E)-, (E, Z)-, and (Z, Z)-terrestribisamide were also characterized for the first time from this plant. Known jatrorrhizine, palmatine, and berberine and unknown (R)-carnegine and (E, E)-, (E, Z)-, and (Z, Z)-terrestribisamide were identified in the callus of N. domestica.

Novel long noncoding RNA NMR promotes tumor progression via NSUN2 and BPTF in esophageal squamous cell carcinoma.

PubMed

Li, Yuan; Li, Jiagen; Luo, Mei; Zhou, Chengcheng; Shi, Xuejiao; Yang, Wenhui; Lu, Zhiliang; Chen, Zhaoli; Sun, Nan; He, Jie

2018-05-12

Long noncoding RNAs (lncRNA) have been implicated in cancer but most of them remain largely unstudied. Here, we identified a novel NSUN2 methylated lncRNA (NMR), which was significantly upregulated in esophageal squamous cell carcinoma (ESCC), functioned as a key regulator of ESCC tumor metastasis and drug resistance. Upregulation of NMR correlated with tumor metastasis and indicated poor overall survival in ESCC patients. Functionally, NMR could promote tumor cell migration and invasion, inhibit cisplatin-induced apoptosis and increase drug resistance in ESCC cells. Mechanistically, transcription of NMR could be upregulated by NF-κB activation after IL-1β and TNF-α treatment. NMR was methylated by NSUN2 and might competitively inhibit methylation of potential mRNAs. NMR could directly bind to chromatin regulator BPTF, and potentially promote MMP3 and MMP10 expression by ERK1/2 pathway through recruiting BPTF to chromatin. Taken together, NMR functions as an oncogenic gene and may serve as new biomarker and therapeutic target in ESCC. Copyright © 2018 Elsevier B.V. All rights reserved.

A novel alkaloid isolated from Crotalaria paulina and identified by NMR and DFT calculations

NASA Astrophysics Data System (ADS)

Oliveira, Ramon Prata; Demuner, Antonio Jacinto; Alvarenga, Elson Santiago; Barbosa, Luiz Claudio Almeida; de Melo Silva, Thiago

2018-01-01

Pyrrolizidine alkaloids (PAs) are secondary metabolites found in Crotalaria genus and are known to have several biological activities. A novel macrocycle bislactone alkaloid, coined ethylcrotaline, was isolated and purified from the aerial parts of Crotalaria paulina. The novel macrocycle was identified with the aid of high resolution mass spectrometry and advanced nuclear magnetic resonance techniques. The relative stereochemistry of the alkaloid was defined by comparing the calculated quantum mechanical hydrogen and carbon chemical shifts of eight candidate structures with the experimental NMR data. The best fit between the eight candidate structures and the experimental NMR chemical shifts was defined by the DP4 statistical analyses and the Mean Absolute Error (MAE) calculations.

Multivariate Analysis of Two-Dimensional 1H, 13C Methyl NMR Spectra of Monoclonal Antibody Therapeutics To Facilitate Assessment of Higher Order Structure.

PubMed

Arbogast, Luke W; Delaglio, Frank; Schiel, John E; Marino, John P

2017-11-07

Two-dimensional (2D) 1 H- 13 C methyl NMR provides a powerful tool to probe the higher order structure (HOS) of monoclonal antibodies (mAbs), since spectra can readily be acquired on intact mAbs at natural isotopic abundance, and small changes in chemical environment and structure give rise to observable changes in corresponding spectra, which can be interpreted at atomic resolution. This makes it possible to apply 2D NMR spectral fingerprinting approaches directly to drug products in order to systematically characterize structure and excipient effects. Systematic collections of NMR spectra are often analyzed in terms of the changes in specifically identified peak positions, as well as changes in peak height and line widths. A complementary approach is to apply principal component analysis (PCA) directly to the matrix of spectral data, correlating spectra according to similarities and differences in their overall shapes, rather than according to parameters of individually identified peaks. This is particularly well-suited for spectra of mAbs, where some of the individual peaks might not be well resolved. Here we demonstrate the performance of the PCA method for discriminating structural variation among systematic sets of 2D NMR fingerprint spectra using the NISTmAb and illustrate how spectral variability identified by PCA may be correlated to structure.

A Policy Impact Analysis of the Mandatory NCAA Sickle Cell Trait Screening Program

PubMed Central

Tarini, Beth A; Brooks, Margaret Alison; Bundy, David G

2012-01-01

Objective To estimate the impact of the mandatory National Collegiate Athletic Association (NCAA) sickle cell trait (SCT) screening policy on the identification of sickle cell carriers and prevention of sudden death. Data Source We used NCAA reports, population-based SCT prevalence estimates, and published risks for exercise-related sudden death attributable to SCT. Study Design We estimated the number of sickle cell carriers identified and the number of potentially preventable sudden deaths with mandatory SCT screening of NCAA Division I athletes. We calculated the number of student-athletes with SCT using a conditional probability based upon SCT prevalence data and self-identified race/ethnicity status. We estimated sudden deaths over 10 years based on published attributable risk of exercise-related sudden death due to SCT. Principal Findings We estimate that over 2,000 NCAA Division I student-athletes with SCT will be identified under this screening policy and that, without intervention, about seven NCAA Division I student-athletes would die suddenly as a complication of SCT over a 10-year period. Conclusion Universal sickle cell screening of NCAA Division I student-athletes will identify a substantial number of sickle cell carriers. A successful intervention could prevent about seven deaths over a decade. PMID:22150647

Consumer interest in community pharmacy HIV screening services.

PubMed

Darin, Kristin M; Scarsi, Kimberly K; Klepser, Donald G; Klepser, Stephanie A; Reeves, Andrew; Young, Maria; Klepser, Michael E

2015-01-01

To evaluate consumers' interest in pharmacist-provided human immunodeficiency virus (HIV) screening and to evaluate potential barriers and facilitators to HIV screening in the community pharmacy setting. Cross-sectional survey of adult patients who presented to one of five community (chain and independent) pharmacies from November 2010 to August 2011. Based on 380 usable surveys, 135 (35.8%) participants were interested in pharmacy-based HIV screening. Independent predictors of interest in HIV screening identified in multivariate analysis (reference groups: ages 30 to 49 years old and white, non-Hispanic race) included younger age (18 to 29 years old) (odds ratio [OR], 2.48; 95% confidence interval [CI], 1.31 to 4.71); black, non-Hispanic race (OR, 2.37; CI, 1.40 to 4.03); and other race (OR, 4.58; CI, 1.63 to 12.87). Lack of perceived risk for HIV was the most commonly cited barrier to HIV screening; and free, rapid, or confidential HIV testing were identified as potential facilitators. Interest in pharmacy-based HIV screening was high among participants representing age and race groups disproportionately affected by HIV. Expansion of HIV screening efforts to community pharmacies warrants further consideration.

Use of a web-based survey to facilitate shared decision making for patients eligible for cancer screening.

PubMed

Brackett, Charles D; Kearing, Stephen

2015-04-01

Our aim was to facilitate shared decision making (SDM) during preventive visits by utilizing a web-based survey system to offer colorectal cancer (CRC) and prostate cancer screening decision aids (DAs) to appropriately identified patients prior to the visit. Patients completed a web-based questionnaire before their preventive medicine appointment. Age- and gender-appropriate patients completed additional questions to determine eligibility for CRC or prostate-specific antigen (PSA) screening. Eligible patients were offered a choice of video or print DA, and completed questions assessing their knowledge, values, and preferences regarding the screening decision. Responses were summarized and fed forward to clinician and patient reports. Overall, 11,493 CRC and 4,384 PSA questionnaires were completed. Patient responses were used to identify those eligible for cancer-screening DAs: 2,187 (19 %) for CRC and 2,962 (68 %) for PSA; 15 % of eligible patients requested a DA. Many patients declined a DA because they indicated they "already know enough to make their decision" (34 % for CRC, 46 % for PSA). A web-based questionnaire provides an efficient means to identify patients eligible for cancer screening decisions and to offer them DAs before an appointment. Pre-visit use of DAs along with reports giving feedback to patients and clinicians provides an opportunity for SDM to occur at the visit.

NMR/MS Translator for the Enhanced Simultaneous Analysis of Metabolomics Mixtures by NMR Spectroscopy and Mass Spectrometry: Application to Human Urine.

PubMed

Bingol, Kerem; Brüschweiler, Rafael

2015-06-05

A novel metabolite identification strategy is presented for the combined NMR/MS analysis of complex metabolite mixtures. The approach first identifies metabolite candidates from 1D or 2D NMR spectra by NMR database query, which is followed by the determination of the masses (m/z) of their possible ions, adducts, fragments, and characteristic isotope distributions. The expected m/z ratios are then compared with the MS(1) spectrum for the direct assignment of those signals of the mass spectrum that contain information about the same metabolites as the NMR spectra. In this way, the mass spectrum can be assigned with very high confidence, and it provides at the same time validation of the NMR-derived metabolites. The method was first demonstrated on a model mixture, and it was then applied to human urine collected from a pool of healthy individuals. A number of metabolites could be detected that had not been reported previously, further extending the list of known urine metabolites. The new analysis approach, which is termed NMR/MS Translator, is fully automated and takes only a few seconds on a computer workstation. NMR/MS Translator synergistically uses the power of NMR and MS, enhancing the accuracy and efficiency of the identification of those metabolites compiled in databases.

Barriers to Breast and Cervical Cancer Screening in Singapore: a Mixed Methods Analysis.

PubMed

Malhotra, Chetna; Bilger, Marcel; Liu, Joy; Finkelstein, Eric

2016-01-01

In order to increase breast and cervical cancer screening uptake in Singapore, women's perceived barriers to screening need to be identified and overcome. Using data from both focus groups and surveys, we aimed to assess perceived barriers and motivations for breast and cervical cancer screening. We conducted 8 focus groups with 64 women, using thematic analysis to identify overarching themes related to women's attitudes towards screening. Based on recurring themes from focus groups, several hypotheses regarding potential barriers and motivations to screen were generated and tested through a national survey of 801 women aged 25-64. Focus group participants had misconceptions related to screening, believing that the procedures were painful. Cost was an issue, as well as efficacy and fatalism. By identifying barriers to and motivators for screening through a mixed-method design that has both nuance and external validity, this study offers valuable suggestions to policymakers to improve breast and cervical cancer screening uptake in Singapore.

School-Based BMI and Body Composition Screening and Parent Notification in California: Methods and Messages

ERIC Educational Resources Information Center

Madsen, Kristine A.; Linchey, Jennifer

2012-01-01

Background: School-based body mass index (BMI) or body composition screening is increasing, but little is known about the process of parent notification. Since 2001, California has required annual screening of body composition via the FITNESSGRAM, with optional notification. This study sought to identify the prevalence of parental notification…

Cell and small animal models for phenotypic drug discovery.

PubMed

Szabo, Mihaly; Svensson Akusjärvi, Sara; Saxena, Ankur; Liu, Jianping; Chandrasekar, Gayathri; Kitambi, Satish S

2017-01-01

The phenotype-based drug discovery (PDD) approach is re-emerging as an alternative platform for drug discovery. This review provides an overview of the various model systems and technical advances in imaging and image analyses that strengthen the PDD platform. In PDD screens, compounds of therapeutic value are identified based on the phenotypic perturbations produced irrespective of target(s) or mechanism of action. In this article, examples of phenotypic changes that can be detected and quantified with relative ease in a cell-based setup are discussed. In addition, a higher order of PDD screening setup using small animal models is also explored. As PDD screens integrate physiology and multiple signaling mechanisms during the screening process, the identified hits have higher biomedical applicability. Taken together, this review highlights the advantages gained by adopting a PDD approach in drug discovery. Such a PDD platform can complement target-based systems that are currently in practice to accelerate drug discovery.

Service Use by At-Risk Youth after School-Based Suicide Screening

PubMed Central

2009-01-01

Objective We sought to examine follow-up service use by students identified at risk for suicidal behavior in a school-based screening program, and assess barriers to seeking services as perceived by youth and parents. Method We conducted a longitudinal study of 317 at-risk youth identified by a school-based suicide screening in six high schools in New York State. The at-risk teenagers and their parents were interviewed approximately two years after the initial screen to assess service use during the intervening period and identify barriers that may have interfered with seeking treatment. Results At the time of the screen, 72% of the at-risk students were not receiving any type of mental health service. Of these students, 51% were deemed in need of services and subsequently referred by us to a mental health professional. Nearly 70% followed through with the screening’s referral recommendations. Youth and their parents reported perceptions about mental health problems, specifically relating to the need for treatment, as the primary reasons for not seeking service. Conclusions Screening appears to be effective in enhancing the likelihood that students at risk for suicidal behavior will get into treatment. Well developed and systematic planning is needed to ensure that screening and referral services are coordinated so as to facilitate access for youth into timely treatment. PMID:19858758

A pre-classification strategy based on UPLC-Triple-TOF/MS for metabolic screening and identification of Radix glehniae in rats.

PubMed

Wang, Shuang; Qi, Pengcheng; Zhou, Na; Zhao, Minmin; Ding, Weijing; Li, Song; Liu, Minyan; Wang, Qiao; Jin, Shumin

2016-10-01

Traditional Chinese Medicines (TCMs) have gained increasing popularity in modern society. However, the profiles of TCMs in vivo are still unclear owing to their complexity and low level in vivo. In this study, UPLC-Triple-TOF techniques were employed for data acquiring, and a novel pre-classification strategy was developed to rapidly and systematically screen and identify the absorbed constituents and metabolites of TCMs in vivo using Radix glehniae as the research object. In this strategy, pre-classification for absorbed constituents was first performed according to the similarity of their structures. Then representative constituents were elected from every class and analyzed separately to screen non-target absorbed constituents and metabolites in biosamples. This pre-classification strategy is basing on target (known) constituents to screen non-target (unknown) constituents from the massive data acquired by mass spectrometry. Finally, the screened candidate compounds were interpreted and identified based on a predicted metabolic pathway, well - studied fragmentation rules, a predicted metabolic pathway, polarity and retention time of the compounds, and some related literature. With this method, a total of 111 absorbed constituents and metabolites of Radix glehniae in rats' urine, plasma, and bile samples were screened and identified or tentatively characterized successfully. This strategy provides an idea for the screening and identification of the metabolites of other TCMs.

Synthesis and characterization of tin(II) complexes of fluorinated Schiff bases derived from amino acids.

PubMed

Singh, Har Lal

2010-07-01

New tin(II) complexes of general formula Sn(L)(2) (L=monoanion of 3-methyl-4-fluoro-acetophenone phenylalanine L(1)H, 3-methyl-4-fluoro-acetophenone alanine L(2)H, 3-methyl-4-fluoro acetophenone tryptophan L(3)H, 3-methyl-4-fluoro-acetophenone valine L(4)H, 3-methyl-4-fluoro-acetophenone isoleucine L(5)H and 3-methyl-4-fluoro-acetophenone glycine L(6)H) have been prepared. It is characterized by elemental analyses, molar conductance measurements and molecular weight determinations. Bonding of these complexes is discussed in terms of their UV-visible, infrared, and nuclear magnetic resonance ((1)H, (13)C, (19)F and (119)Sn NMR) spectral studies. The ligands act as bidentate towards metal ions, via the azomethine nitrogen and deprotonated oxygen of the respective amino acid. Elemental analyses and NMR spectral data of the ligands with their tin(II) complexes agree with their proposed square pyramidal structures. A few representative ligands and their tin complexes have been screened for their antibacterial activities and found to be quite active in this respect. Copyright 2010 Elsevier B.V. All rights reserved.

Ultrasound-assisted synthesis, anticonvulsant activity, and docking study of indole-appended thiazolidin-4-ones.

PubMed

Nikalje, Anna Pratima G; Shaikh, Sameer I; Mulay, Abhineet; Khan, Firoz A K; Sangshetti, Jaiprakash N; Shaikh, Shoaib

2014-10-01

Two series of novel indolyl thiazolidin-4-one derivatives 4a-j and 5a-j were obtained by an ecofriendly synthetic protocol by treating a mixture of Schiff's bases (0.01 mol) with thioglycolic acid or thiolactic acid (0.01 mol) and anhydrous zinc chloride in catalytic amount in DMF as solvent under ultrasound irradiation, using an ultrasound synthesizer with a synthetic solid probe. The structures of the synthesized compounds were confirmed by IR, (1) H NMR, (13) C NMR, MS, and elemental analysis. The anticonvulsant activity and neurotoxicity of the newly synthesized compounds were established by MES and sc-PTZ model and by rotarod test, respectively, in vivo using mouse models. The actophotometer was used for the screening of behavioral activity. The compounds exhibited promising anticonvulsant activity; especially, the compounds showed maximum protection in the MES model at a dose of 100 mg/kg. Further, docking studies of the synthesized compounds were performed against the sodium channel receptor and showed good binding interactions with the receptor. A computational study was carried out to highlight the pharmacophore distance mapping, log p determination, and pharmacokinetic parameters. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

LC-UV-solid-phase extraction-NMR-MS combined with a cryogenic flow probe and its application to the identification of compounds present in Greek oregano.

PubMed

Exarchou, Vassiliki; Godejohann, Markus; van Beek, Teris A; Gerothanassis, Ioannis P; Vervoort, Jacques

2003-11-15

Structure elucidation of natural products usually relies on a combination of NMR spectroscopy with mass spectrometry whereby NMR trails MS in terms of the minimum sample amount required. In the present study, the usefulness of on-line solid-phase extraction (SPE) in LC-NMR for peak storage after the LC separation prior to NMR analysis is demonstrated. The SPE unit allows the use of normal protonated solvents for the LC separation and fully deuterated solvents for flushing the trapped compounds to the NMR probe. Thus, solvent suppression is no longer necessary. Multiple trapping of the same analyte from repeated LC injections was utilized to solve the problem of low concentration and to obtain 2D heteronuclear NMR spectra. In addition, a combination of the SPE unit with a recently developed cryoflow NMR probe and an MS was evaluated. This on-line LC-UV-SPE-NMR-MS system was used for the automated analysis of a Greek oregano extract. Combining the data provided by the UV, MS, and NMR spectra, the flavonoids taxifolin, aromadendrin, eriodictyol, naringenin, and apigenin, the phenolic acid rosmarinic acid, and the monoterpene carvacrol were identified. This automated technique is very useful for natural product analysis, and the large sensitivity improvement leads to significantly reduced NMR acquisition times.

[Study on Chemical Constituents of Peanut Hull].

PubMed

Zuo, Ai-xue; Sun, Yun; Qian, Shao-xiang; Rao, Gao-xiong

2015-02-01

To investigate the chemical constituents of peanut hull. Several chromatography methods such as silica gel and Sephadex LH-20 combined with recrystallization were applied to isolate the compounds. Based on spectrum technologies (MS,1H-NMR and 13C-NMR) and physico-chemical methods, structures of isolated compounds were identified. Twelve compounds were isolated and elucidated as luteolin (1), diosmetin (2), 5,7,3',4'-tetrahydroxy-8-prenyflavone (3),5,7,3'-trihydroxy-4'- methoxy-8-prenylflavone(4), eriodicrtyol (5), racemoflavone (6), hydnocarpin (7), 5,7-dihydroxy chromone (8), 5-hydroxy-chromone- 7-O-β-D-glucoside (9), ferulic acid (10), β-sitosterol (11) and daucosterol(12). Except compounds 1, 5 and 8, all compounds are obtained from peanut hull for the first time.

Nuclear magnetic resonance (NMR) study of the effect of cisplatin on the metabolic profile of MG-63 osteosarcoma cells.

PubMed

Duarte, Iola F; Lamego, Ines; Marques, Joana; Marques, M Paula M; Blaise, Benjamin J; Gil, Ana M

2010-11-05

In the present study, (1)H HRMAS NMR spectroscopy was used to assess the changes in the intracellular metabolic profile of MG-63 human osteosarcoma (OS) cells induced by the chemotherapy agent cisplatin (CDDP) at different times of exposure. Multivariate analysis was applied to the cells spectra, enabling consistent variation patterns to be detected and drug-specific metabolic effects to be identified. Statistical recoupling of variables (SRV) analysis and spectral integration enabled the most relevant spectral changes to be evaluated, revealing significant time-dependent alterations in lipids, choline-containing compounds, some amino acids, polyalcohols, and nitrogenated bases. The metabolic relevance of these compounds in the response of MG-63 cells to CDDP treatment is discussed.

Synthesis, spectroscopic characterization, biological screenings, DNA binding study and POM analyses of transition metal carboxylates.

PubMed

Uddin, Noor; Sirajuddin, Muhammad; Uddin, Nizam; Tariq, Muhammad; Ullah, Hameed; Ali, Saqib; Tirmizi, Syed Ahmed; Khan, Abdur Rehman

2015-04-05

This article contains the synthesis of a novel carboxylic acid derivative, its transition metal complexes and evaluation of biological applications. Six carboxylate complexes of transition metals, Zn(II) and Hg(II), have been successfully synthesized and characterized by FT-IR and NMR (1H, 13C). The ligand, HL, (4-[(2,6-Diethylphenyl)amino]-4-oxobutanoic acid) was also characterized by single crystal X-ray analysis. The complexation occurs via oxygen atoms of the carboxylate moiety. FT-IR date show the bidentate nature of the carboxylate moiety of the ligand as the Δν value in all complexes is less than that of the free ligand. The ligand and its complexes were screened for antifungal and antileishmanial activities. The results showed that the ligand and its complexes are active with few exceptions. UV-visible spectroscopy and viscometry results reveal that the ligand and its complexes interact with the DNA via intercalative mode of interaction. A new and efficient strategy to identify the pharmacophores and anti-pharmacophores sites in carboxylate derivatives for the antibacterial/antifungal activity using Petra, Osiris and Molinspiration (POM) analyses was also carried out. Copyright © 2014 Elsevier B.V. All rights reserved.

Synthesis, spectroscopic characterization, biological screenings, DNA binding study and POM analyses of transition metal carboxylates

NASA Astrophysics Data System (ADS)

Uddin, Noor; Sirajuddin, Muhammad; Uddin, Nizam; Tariq, Muhammad; Ullah, Hameed; Ali, Saqib; Tirmizi, Syed Ahmed; Khan, Abdur Rehman

2015-04-01

This article contains the synthesis of a novel carboxylic acid derivative, its transition metal complexes and evaluation of biological applications. Six carboxylate complexes of transition metals, Zn(II) and Hg(II), have been successfully synthesized and characterized by FT-IR and NMR (1H, 13C). The ligand, HL, (4-[(2,6-Diethylphenyl)amino]-4-oxobutanoic acid) was also characterized by single crystal X-ray analysis. The complexation occurs via oxygen atoms of the carboxylate moiety. FT-IR date show the bidentate nature of the carboxylate moiety of the ligand as the Δν value in all complexes is less than that of the free ligand. The ligand and its complexes were screened for antifungal and antileishmanial activities. The results showed that the ligand and its complexes are active with few exceptions. UV-visible spectroscopy and viscometry results reveal that the ligand and its complexes interact with the DNA via intercalative mode of interaction. A new and efficient strategy to identify the pharmacophores and anti-pharmacophores sites in carboxylate derivatives for the antibacterial/antifungal activity using Petra, Osiris and Molinspiration (POM) analyses was also carried out.

Synthesis, characterization and biological screening of sulfonamides derived form 2-phenylethylamine.

PubMed

Rehman, Aziz-ur; Afroz, Sumbel; Abbasi, Muhammad Athar; Tanveer, Wajeeha; Khan, Khalid Mohammed; Ashraf, Muhammad; Ahmad, Irshad; Afzal, Iftikhar; Ambreen, Nida

2012-10-01

In the present study, a series of N-substituted derivatives of 2-phenylethylamine has been synthesized. The reaction of 2-phenylethylamine (1) with benzene sulfonyl chloride (2) yielded N-(2-phenylethyl) benzenesulfonamide (3), which further on treatment with alkyl/acyl halides (4a-i) in the presence of sodium hydride furnished into N-substituted sulfonamides (5a-i). These derivatives were characterized by IR, (1)H-NMR and EI-MS and then screened against acetyl cholinesterase (AChE), butyryl cholinesterase (BChE) and lipoxygenase enzyme (LOX) and were found to be potent inhibitors of butyryl cholinesterase only.

Structure elucidation of the designer drug N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)-1-(5-fluoropentyl)-3-(4-fluorophenyl)-pyrazole-5-carboxamide and the relevance of predicted (13) C NMR shifts - a case study.

PubMed

Girreser, Ulrich; Rösner, Peter; Vasilev, Andrej

2016-07-01

The detailed structure elucidation process of the new cannabimimetic designer drug, N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)-1-(5-fluoropentyl)-3-(4-fluorophenyl)-pyrazole-5-carboxamide, with a highly substituted pyrazole skeleton, using nuclear magnetic resonance (NMR) spectroscopic and mass spectrometric (MS) techniques is described. After a first analysis of the NMR spectra and comparison with 48 possible pyrazole and imidazole structures, a subset of six positional isomeric pyrazoles and six imidazoles remained conceivable. Four substituents of the heterocyclic skeleton were identified: a proton bound to a pyrazole ring carbon atom; a 5-fluoropentyl group; a 4-fluorophenyl substituent; and a carbamoyl group, which is N-substituted with a methyl residue carrying a tert.-butyl and a carbamoyl substituent. The 5-fluoropentyl residue is situated at the nitrogen ring atom. Additional NMR experiments like the (1) H,(13) C HMBC were performed, but due to the small number of signals based on long-range couplings, the comparison of predicted and observed (13) C chemical shifts became necessary. The open access Internet shift prediction programs NMRDB, NMRSHIFTDB2, and CSEARCH were employed for the prediction of (13) C shift values which allowed an efficient and unambiguous structure determination. For the identified N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)-1-(5-fluoropentyl)-3-(4-fluorophenyl)-pyrazole-5-carboxamide, the best agreement between predicted (13) C shifts and the observed chemical shifts and long-range couplings for the pyrazole ring carbon atoms, with a standard error of about 2 ppm, was found with each of the predictions. For the comparison of measured and predicted chemical shifts model compounds with simple substituents proved helpful. The identified compound is a homologue of AZ-037 which is offered by Internet suppliers. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

Triprotic site-specific acid-base equilibria and related properties of fluoroquinolone antibacterials.

PubMed

Rusu, Aura; Tóth, Gergő; Szőcs, Levente; Kökösi, József; Kraszni, Márta; Gyéresi, Árpád; Noszál, Béla

2012-07-01

The complete macro- and microequilibrium analyses of six fluoroquinolone drugs - ciprofloxacin, enrofloxacin, norfloxacin, pefloxacin, ofloxacin and moxifloxacin - are presented. Previous controversial literature data are straightened up, the protonation centers are unambiguously identified, and the protonation macro- and microconstant values are reported. The macroconstants were determined by (1)H NMR-pH titrations while the microconstants were determined by a multi-modal spectroscopic-deductive methodology, in which methyl ester derivatives were synthesized and their NMR-pH titration data contributed to the evaluation of all the microconstants. The full (1)H, (13)C and (15)N NMR assignments, NMR-pH profiles, macro- and microprotonation schemes and species-specific diagrams are included. Our studies show that the fluoroquinolones have three protonation centers: the carboxylate group, the N-1' and N-4' piperazine nitrogens and concentration of the uncharged microspecies is way below the values published earlier. The results could be well interpreted in terms of structural properties. The protonation macro- and microconstant values allow the pre-planned method development in techniques such as capillary zone electrophoresis and also, the interpretation of fluoroquinolone mechanism of biological action, including the pharmacokinetic properties, and antibacterial activities that are all heavily influenced by the states of protonation. Copyright © 2012 Elsevier B.V. All rights reserved.

An unusual class of anthracyclines potentiate Gram-positive antibiotics in intrinsically resistant Gram-negative bacteria

PubMed Central

Cox, Georgina; Koteva, Kalinka; Wright, Gerard D.

2014-01-01

Objectives An orthogonal approach taken towards novel antibacterial drug discovery involves the identification of small molecules that potentiate or enhance the activity of existing antibacterial agents. This study aimed to identify natural-product rifampicin adjuvants in the intrinsically resistant organism Escherichia coli. Methods E. coli BW25113 was screened against 1120 actinomycete fermentation extracts in the presence of subinhibitory (2 mg/L) concentrations of rifampicin. The active molecule exhibiting the greatest rifampicin potentiation was isolated using activity-guided methods and identified using mass and NMR spectroscopy. Susceptibility testing and biochemical assays were used to determine the mechanism of antibiotic potentiation. Results The anthracycline Antibiotic 301A1 was isolated from the fermentation broth of a strain of Streptomyces (WAC450); the molecule was shown to be highly synergistic with rifampicin (fractional inhibitory concentration index = 0.156) and moderately synergistic with linezolid (FIC index = 0.25) in both E. coli and Acinetobacter baumannii. Activity was associated with inhibition of efflux and the synergistic phenotype was lost when tested against E. coli harbouring mutations within the rpoB gene. Structure–activity relationship studies revealed that other anthracyclines do not synergize with rifampicin and removal of the sugar moiety of Antibiotic 301A1 abolishes activity. Conclusions Screening only a subsection of our natural product library identified a small-molecule antibiotic adjuvant capable of sensitizing Gram-negative bacteria to antibiotics to which they are ordinarily intrinsically resistant. This result demonstrates the great potential of this approach in expanding antibiotic effectiveness in the face of the growing challenge of resistance in Gram-negatives. PMID:24627312

Questions About Cervical and Breast Cancer Screening Knowledge, Practice, and Outcomes: A Review of Demographic and Health Surveys.

PubMed

Viens, Laura; Perin, Doug; Senkomago, Virginia; Neri, Antonio; Saraiya, Mona

2017-05-01

United Nation's Sustainable Development Goals and the World Health Organization's Global Monitoring Framework support a strong global commitment to reducing the high burden of cervical and breast cancers among low- and middle-income countries. Strategies include vaccination, screening, and early diagnosis. Population-based surveys, such as those conducted by the Demographic and Health Surveys (DHS) Program, can collect the information needed to guide cancer control efforts in a standardized comparable manner. We identified and evaluated the breadth of breast and cervical cancer screening information that was collected by the DHS from 1984 through 2015. Then, we determined if these surveys currently provide the specific and measurable data about both the quantity and quality of cancer screening needed to guide national efforts to reduce the overall effects of cervical and breast cancers. We searched the DHS website to identify surveys conducted between the start of the DHS Program in 1984 and November 2015 that included questions about breast and cervical cancer screening. The relevant questions were extracted from the questionnaire, translated into English, and grouped by themes. Of the 90 countries where DHS surveys have been implemented, cervical cancer screening questions were included in 22 countries (24.4%) and breast cancer screening questions in 18 countries (20.0%). The common themes identified were disease knowledge, screening knowledge, screening practice, and screening outcomes. Most countries with survey questionnaires available for review addressed at least one aspect of screening practice (88.9% of cervical and 87.5% of breast), although few countries queried knowledge and outcomes. Questions that assess varied aspects of breast and cervical cancer screening have been incorporated into relatively few DHS surveys. The themes identified could guide the design of a standard set of questions for use in future population-based surveys and enable evaluation beyond the existence of screening, which would include assessment of the quality and impact of cervical and breast cancer screening.

Consolidated principles for screening based on a systematic review and consensus process.

PubMed

Dobrow, Mark J; Hagens, Victoria; Chafe, Roger; Sullivan, Terrence; Rabeneck, Linda

2018-04-09

In 1968, Wilson and Jungner published 10 principles of screening that often represent the de facto starting point for screening decisions today; 50 years on, are these principles still the right ones? Our objectives were to review published work that presents principles for population-based screening decisions since Wilson and Jungner's seminal publication, and to conduct a Delphi consensus process to assess the review results. We conducted a systematic review and modified Delphi consensus process. We searched multiple databases for articles published in English in 1968 or later that were intended to guide population-based screening decisions, described development and modification of principles, and presented principles as a set or list. Identified sets were compared for basic characteristics (e.g., number, categorization), a citation analysis was conducted, and principles were iteratively synthesized and consolidated into categories to assess evolution. Participants in the consensus process assessed the level of agreement with the importance and interpretability of the consolidated screening principles. We identified 41 sets and 367 unique principles. Each unique principle was coded to 12 consolidated decision principles that were further categorized as disease/condition, test/intervention or program/system principles. Program or system issues were the focus of 3 of Wilson and Jungner's 10 principles, but comprised almost half of all unique principles identified in the review. The 12 consolidated principles were assessed through 2 rounds of the consensus process, leading to specific refinements to improve their relevance and interpretability. No gaps or missing principles were identified. Wilson and Jungner's principles are remarkably enduring, but increasingly reflect a truncated version of contemporary thinking on screening that does not fully capture subsequent focus on program or system principles. Ultimately, this review and consensus process provides a comprehensive and iterative modernization of guidance to inform population-based screening decisions. © 2018 Joule Inc. or its licensors.

Consolidated principles for screening based on a systematic review and consensus process

PubMed Central

Hagens, Victoria; Chafe, Roger; Sullivan, Terrence; Rabeneck, Linda

2018-01-01

BACKGROUND: In 1968, Wilson and Jungner published 10 principles of screening that often represent the de facto starting point for screening decisions today; 50 years on, are these principles still the right ones? Our objectives were to review published work that presents principles for population-based screening decisions since Wilson and Jungner’s seminal publication, and to conduct a Delphi consensus process to assess the review results. METHODS: We conducted a systematic review and modified Delphi consensus process. We searched multiple databases for articles published in English in 1968 or later that were intended to guide population-based screening decisions, described development and modification of principles, and presented principles as a set or list. Identified sets were compared for basic characteristics (e.g., number, categorization), a citation analysis was conducted, and principles were iteratively synthesized and consolidated into categories to assess evolution. Participants in the consensus process assessed the level of agreement with the importance and interpretability of the consolidated screening principles. RESULTS: We identified 41 sets and 367 unique principles. Each unique principle was coded to 12 consolidated decision principles that were further categorized as disease/condition, test/intervention or program/system principles. Program or system issues were the focus of 3 of Wilson and Jungner’s 10 principles, but comprised almost half of all unique principles identified in the review. The 12 consolidated principles were assessed through 2 rounds of the consensus process, leading to specific refinements to improve their relevance and interpretability. No gaps or missing principles were identified. INTERPRETATION: Wilson and Jungner’s principles are remarkably enduring, but increasingly reflect a truncated version of contemporary thinking on screening that does not fully capture subsequent focus on program or system principles. Ultimately, this review and consensus process provides a comprehensive and iterative modernization of guidance to inform population-based screening decisions. PMID:29632037

Identifying metabolites related to nitrogen mineralisation using 1H NMR spectroscopy

NASA Astrophysics Data System (ADS)

. T McDonald, Noeleen; Graham, Stewart; Watson, Catherine; Gordon, Alan; Lalor, Stan; Laughlin, Ronnie; Elliott, Chris; . P Wall, David

2015-04-01

Exploring new analysis techniques to enhance our knowledge of the various metabolites within our soil systems is imperative. Principally, this knowledge would allow us to link key metabolites with functional influences on critical nutrient processes, such as the nitrogen (N) mineralisation in soils. Currently there are few studies that utilize proton nuclear magnetic resonance spectroscopy (1H NMR) to characterize multiple metabolites within a soil sample. The aim of this research study was to examine the effectiveness of 1H NMR for isolating multiple metabolites that are related to the mineralizable N (MN) capacity across a range of 35 Irish grassland soils. Soils were measured for MN using the standard seven day anaerobic incubation (AI-7). Additionally, soils were also analysed for a range of physio-chemical properties [e.g. total N, total C, mineral N, texture and soil organic matter (SOM)]. Proton NMR analysis was carried on these soils by extracting with 40% methanol:water, lyophilizing and reconstituting in deuterium oxide and recording the NMR spectra on a 400MHz Bruker AVANCE III spectrometer. Once the NMR data were spectrally processed and analysed using multivariate statistical analysis, seven metabolites were identified as having significant relationships with MN (glucose, trimethylamine, glutamic acid, serine, aspartic acid, 4-aminohippuirc acid and citric acid). Following quantification, glucose was shown to explain the largest percentage variability in MN (72%). These outcomes suggest that sources of labile carbon are essential in regulating N mineralisation and the capacity of plant available N derived from SOM-N pools in these soils. Although, smaller in concentration, the amino acids; 4-aminohippuirc acid, glutamic acid and serine also significantly (P<0.05) explained 43%, 27% and 19% of the variability in MN, respectively. This novel study highlights the effectiveness of using 1H NMR as a practical approach to profile multiple metabolites in soils simultaneously, and increasing the potential to identify those related to various soil processes.

Micro-scale NMR Experiments for Monitoring the Optimization of Membrane Protein Solutions for Structural Biology.

PubMed

Horst, Reto; Wüthrich, Kurt

2015-07-20

Reconstitution of integral membrane proteins (IMP) in aqueous solutions of detergent micelles has been extensively used in structural biology, using either X-ray crystallography or NMR in solution. Further progress could be achieved by establishing a rational basis for the selection of detergent and buffer conditions, since the stringent bottleneck that slows down the structural biology of IMPs is the preparation of diffracting crystals or concentrated solutions of stable isotope labeled IMPs. Here, we describe procedures to monitor the quality of aqueous solutions of [ 2 H, 15 N]-labeled IMPs reconstituted in detergent micelles. This approach has been developed for studies of β-barrel IMPs, where it was successfully applied for numerous NMR structure determinations, and it has also been adapted for use with α-helical IMPs, in particular GPCRs, in guiding crystallization trials and optimizing samples for NMR studies (Horst et al ., 2013). 2D [ 15 N, 1 H]-correlation maps are used as "fingerprints" to assess the foldedness of the IMP in solution. For promising samples, these "inexpensive" data are then supplemented with measurements of the translational and rotational diffusion coefficients, which give information on the shape and size of the IMP/detergent mixed micelles. Using microcoil equipment for these NMR experiments enables data collection with only micrograms of protein and detergent. This makes serial screens of variable solution conditions viable, enabling the optimization of parameters such as the detergent concentration, sample temperature, pH and the composition of the buffer.

Synthesis, characterization, antimicrobial screening and computational studies of 4-[3-(4-methoxy-phenyl)-allylideneamino]-1,5-dimethyl-2-phenyl-1,2-dihydro-pyrazol-3-one

NASA Astrophysics Data System (ADS)

Obasi, L. N.; Kaior, G. U.; Rhyman, L.; Alswaidan, Ibrahim A.; Fun, Hoong-Kun; Ramasami, P.

2016-09-01

The Schiff base, 4-[3-(4-methoxy-phenyl)-allylideneamino]-1,5-dimethyl-2-phenyl-1,2-dihydro-pyrazol-3-one (TPMC/AAP) was synthesized by the condensation of 4-aminoantipyrine (4-amino-1,5-dimethyl-2-phenylpyrazole-3-one) and trans-para-methoxycinnamaldehyde (trans-3,4-methoxyphenyl-2-propenal) in dry methanol at 75 °C. The compound was characterized using elemental microanalysis, IR, NMR, UV spectroscopies and single-crystal X-ray crystallography. The X-ray structure determination shows that the Schiff base, (TPMC/AAP) is orthorhombic with the Pbca space group. The anti-microbial screening of the compound was carried out with Escherichia coli, Bacillus subtillis, Staphylococcus aureus, Pseudemonas aeruginosa, Candida albicans and Aspergillus niger using agar well diffusion method. The Schiff base possesses significant antimicrobial activity. The minimum inhibitory concentration (MIC) of the compound was also determined and the activity was compared with that of conventional drugs ciprofloxacin and ketoconazole. The compound (TPMC/AAP) showed varying activity against the cultured bacteria and fungi used. To complement the experimental data, density functional theory (DFT) was used to have deeper understanding into the molecular parameters and infrared spectra of the compound.

Spectroscopic studies, antimicrobial activities and crystal structures of N-(2-hydroxy-3-methoxybenzalidene)1-aminonaphthalene

NASA Astrophysics Data System (ADS)

Ünver, Hüseyin; Yıldız, Mustafa; Dülger, Başaran; Özgen, Özen; Kendi, Engin; Durlu, Tahsin Nuri

2005-03-01

Schiff base N-(2-hydroxy-3-methoxybenzalidene)1-aminonaphthalene has been synthesized from the reaction of 2-hydroxy-3-methoxybenzaldehyde with 1-aminonaphthalene. The compound were characterized by elemental analysis, FT-IR, 1H NMR, 13C NMR and UV-visible techniques. The UV-visible spectra of the Schiff base were studied in polar and nonpolar solvents in acidic and basic media. The structure of the compound has been examined cyrstallographically. There are two independent molecules in the asymmetric unit. It crystallizes in the monoclinic space group P21/c, with unit cell parameters: a=14, 602(2), b=5,800(1), c=16, 899(1) Å, V=1394.4(2) Å 3, Dx=1.321 g cm -3 and Z=4. The crystal structure was solved by direct methods and refined by full-matrix least squares to a find R=0.041 of for 1179 observed reflections. The title compound's antimicrobial activities also have been studied. The antimicrobial activities of the ligand has been screened in vitro against the organisms Escherichia coli ATCC 11230, Staphylococcus aureus ATCC 6538, Klebsiella pneumoniae UC57, Micrococcus luteus La 2971, Proteus vulgaris ATCC 8427, Pseudomonas aeruginosa ATCC 27853, Mycobacterium smegmatis CCM 2067, Bacillus cereus ATCC 7064 and Listeria monocytogenes ATCC 15313, the yeast cultures Candida albicans ATCC 10231, Kluyveromyces fragilis NRRL 2415, Rhodotorula rubra DSM 70403, Debaryomyces hansenii DSM 70238 and Hanseniaspora guilliermondii DSM 3432.

Combined CRISPRi/a-Based Chemical Genetic Screens Reveal that Rigosertib Is a Microtubule-Destabilizing Agent. | Office of Cancer Genomics

Cancer.gov

Chemical libraries paired with phenotypic screens can now readily identify compounds with therapeutic potential. A central limitation to exploiting these compounds, however, has been in identifying their relevant cellular targets. Here, we present a two-tiered CRISPR-mediated chemical-genetic strategy for target identification: combined genome-wide knockdown and overexpression screening as well as focused, comparative chemical-genetic profiling.

HPLC-Based Activity Profiling: Discovery of Piperine as a Positive GABAA Receptor Modulator Targeting a Benzodiazepine-Independent Binding Site

PubMed Central

Zaugg, Janine; Baburin, Igor; Strommer, Barbara; Kim, Hyun-Jung; Hering, Steffen; Hamburger, Matthias

2011-01-01

A plant extract library was screened for GABAA receptor activity making use of a two-microelectrode voltage clamp assay on Xenopus laevis oocytes. An ethyl acetate extract of black pepper fruits [Piper nigrum L. (Piperaceae) 100 μg/mL] potentiated GABA-induced chloride currents through GABAA receptors (composed of α1, β2, and γ2S subunits) by 169.1 ± 2.4%. With the aid of an HPLC-based activity profiling approach, piperine (5) was identified as the main active compound, together with 12 structurally related less active or inactive piperamides (1–4, 6–13). Identification was achieved by on-line high-resolution mass spectrometry and off-line microprobe 1D and 2D NMR spectroscopy, using only milligram amounts of extract. Compound 5 induced a maximum potentiation of the chloride currents by 301.9 ± 26.5% with an EC50 of 52.4 ± 9.4 μM. A comparison of the modulatory activity of 5 and other naturally occurring piperamides enabled insights into structural features critical for GABAA receptor modulation. The stimulation of chloride currents through GABAA receptors by compound 5 was not antagonized by flumazenil (10 μM). These data show that piperine (5) represents a new scaffold of positive allosteric GABAA receptor modulators targeting a benzodiazepine-independent binding site. PMID:20085307

A (13)C NMR analysis of the effects of electron radiation on graphite/polyetherimide composites

NASA Technical Reports Server (NTRS)

Ferguson, Milton W.

1989-01-01

Initial investigations have been made into the use of high resolution nuclear magnetic resonance (NMR) for the characterization of radiation effects in graphite and Kevlar fibers, polymers, and the fiber/matrix interface in graphite/polyetherimide composites. Sample preparation techniques were refined. Essential equipment has been procured. A new NMR probe was constructed to increase the proton signal-to-noise ratio. Problem areas have been identified and plans developed to resolve them.

[Two new glycosides from Erigeron breviscapus (Vant.) Hand.-Mazz].

PubMed

Zhang, W D; Chen, W S; Wang, Y H; Liu, W Y; Kong, D Y; Li, H T

2001-10-01

To study the chemical constituents from the upground part of Erigeron breviscapus. The compounds were separated and purified by column chromatography with silica gel, and identified by IR, MS, NMR and 2D-NMR. Two new compounds were isolated and identified as 5,4'-dihydroxy flavonod-7-O-beta-D-pyranglycuronate buthyl ester(VI) and 3,5-dimethoxy benzene carbonic acid-4-O-beta-D-pyranglucose(VII). Compounds VI and VII were new compounds.

School scoliosis screening programme-a systematic review.

PubMed

Sabirin, J; Bakri, R; Buang, S N; Abdullah, A T; Shapie, A

2010-12-01

A systematic review on the effectiveness and cost-effectiveness of school scoliosis screening programme was carried out. A total of 248 relevant titles were identified, 117 abstracts were screened and 28 articles were included in the results. There was fair level of evidence to suggest that school scoliosis screening programme is safe, contributed to early detection and reduction of surgery. There was also evidence to suggest that school-based scoliosis screening programme is cost-effective. Based on the above review, screening for scoliosis among school children is recommended only for high risk group such as girls at twelve years of age.

Production of Two Novel Methoxy-Isoflavones from Biotransformation of 8-Hydroxydaidzein by Recombinant Escherichia coli Expressing O-Methyltransferase SpOMT2884 from Streptomyces peucetius

PubMed Central

Chiang, Chien-Min; Ding, Hsiou-Yu; Tsai, Ya-Ting; Chang, Te-Sheng

2015-01-01

Biotransformation of 8-hydroxydaidzein by recombinant Escherichia coli expressing O-methyltransferase (OMT) SpOMT2884 from Streptomyces peucetius was investigated. Two metabolites were isolated and identified as 7,4′-dihydroxy-8-methoxy-isoflavone (1) and 8,4′-dihydroxy-7-methoxy-isoflavone (2), based on mass, 1H-nuclear magnetic resonance (NMR) and 13C-NMR spectrophotometric analysis. The maximum production yields of compound (1) and (2) in a 5-L fermenter were 9.3 mg/L and 6.0 mg/L, respectively. The two methoxy-isoflavones showed dose-dependent inhibitory effects on melanogenesis in cultured B16 melanoma cells under non-toxic conditions. Among the effects, compound (1) decreased melanogenesis to 63.5% of the control at 25 μM. This is the first report on the 8-O-methylation activity of OMT toward isoflavones. In addition, the present study also first identified compound (1) with potent melanogenesis inhibitory activity. PMID:26610478

Performance of the WeNMR CS-Rosetta3 web server in CASD-NMR.

PubMed

van der Schot, Gijs; Bonvin, Alexandre M J J

2015-08-01

We present here the performance of the WeNMR CS-Rosetta3 web server in CASD-NMR, the critical assessment of automated structure determination by NMR. The CS-Rosetta server uses only chemical shifts for structure prediction, in combination, when available, with a post-scoring procedure based on unassigned NOE lists (Huang et al. in J Am Chem Soc 127:1665-1674, 2005b, doi: 10.1021/ja047109h). We compare the original submissions using a previous version of the server based on Rosetta version 2.6 with recalculated targets using the new R3FP fragment picker for fragment selection and implementing a new annotation of prediction reliability (van der Schot et al. in J Biomol NMR 57:27-35, 2013, doi: 10.1007/s10858-013-9762-6), both implemented in the CS-Rosetta3 WeNMR server. In this second round of CASD-NMR, the WeNMR CS-Rosetta server has demonstrated a much better performance than in the first round since only converged targets were submitted. Further, recalculation of all CASD-NMR targets using the new version of the server demonstrates that our new annotation of prediction quality is giving reliable results. Predictions annotated as weak are often found to provide useful models, but only for a fraction of the sequence, and should therefore only be used with caution.

Streamlined structure elucidation of an unknown compound in a pigment formulation.

PubMed

Yüce, Imanuel; Morlock, Gertrud E

2016-10-21

A fast and reliable quality control is important for ink manufacturers to ensure a constant production grade of mixtures and chemical formulations, and unknown components attract their attention. Structure elucidating techniques seem time-consuming in combination with column-based methods, but especially the low solubility of pigment formulations is challenging the analysis. In contrast, layer chromatography is more tolerant with regard to pigment particles. One PLC plate for NMR and FTIR analyses and one HPTLC plate for recording of high resolution mass spectra, MS/MS spectra and for gathering information on polarity and spectral properties were needed to characterize a structure, exemplarily shown for an unknown component in pigment Red 57:1 to be 3-hydroxy-2-naphtoic acid. A preparative layer chromatography (PLC) workflow was developed that used an Automated Multiple Development 2 (AMD 2) system. The 0.5-mm PLC plate could still be operated in the AMD 2 system and allowed a smooth switch from the analytical to the preparative gradient separation. Through automated gradient development and the resulting focusing of bands, the sharpness of the PLC bands was improved. For NMR, the necessary high load of the target compound on the PLC plate was achieved via a selective solvent extraction that discriminated the polar sample matrix and thus increased the application volume of the extract that could maximally be applied without overloading. By doing so, the yield for NMR analysis was improved by a factor of 9. The effectivity gain through a simple, but thoroughly chosen extraction solvent is often overlooked, and for educational purpose, it was clearly illustrated and demonstrated by an extended solvent screening. Thus, PLC using an automated gradient development after a selective extraction was proven to be a new powerful combination for structural elucidation by NMR. Copyright © 2016 Elsevier B.V. All rights reserved.

Investigating Sodium Storage Mechanisms in Tin Anodes: A Combined Pair Distribution Function Analysis, Density Functional Theory, and Solid-State NMR Approach.

PubMed

Stratford, Joshua M; Mayo, Martin; Allan, Phoebe K; Pecher, Oliver; Borkiewicz, Olaf J; Wiaderek, Kamila M; Chapman, Karena W; Pickard, Chris J; Morris, Andrew J; Grey, Clare P

2017-05-31

The alloying mechanism of high-capacity tin anodes for sodium-ion batteries is investigated using a combined theoretical and experimental approach. Ab initio random structure searching (AIRSS) and high-throughput screening using a species-swap method provide insights into a range of possible sodium-tin structures. These structures are linked to experiments using both average and local structure probes in the form of operando pair distribution function analysis, X-ray diffraction, and 23 Na solid-state nuclear magnetic resonance (ssNMR), along with ex situ 119 Sn ssNMR. Through this approach, we propose structures for the previously unidentified crystalline and amorphous intermediates. The first electrochemical process of sodium insertion into tin results in the conversion of crystalline tin into a layered structure consisting of mixed Na/Sn occupancy sites intercalated between planar hexagonal layers of Sn atoms (approximate stoichiometry NaSn 3 ). Following this, NaSn 2 , which is predicted to be thermodynamically stable by AIRSS, forms; this contains hexagonal layers closely related to NaSn 3 , but has no tin atoms between the layers. NaSn 2 is broken down into an amorphous phase of approximate composition Na 1.2 Sn. Reverse Monte Carlo refinements of an ab initio molecular dynamics model of this phase show that the predominant tin connectivity is chains. Further reaction with sodium results in the formation of structures containing Sn-Sn dumbbells, which interconvert through a solid-solution mechanism. These structures are based upon Na 5-x Sn 2 , with increasing occupancy of one of its sodium sites commensurate with the amount of sodium added. ssNMR results indicate that the final product, Na 15 Sn 4 , can store additional sodium atoms as an off-stoichiometry compound (Na 15+x Sn 4 ) in a manner similar to Li 15 Si 4 .

Investigating Sodium Storage Mechanisms in Tin Anodes: A Combined Pair Distribution Function Analysis, Density Functional Theory, and Solid-State NMR Approach

DOE PAGES

Stratford, Joshua M.; Mayo, Martin; Allan, Phoebe K.; ...

2017-05-04

Here, the alloying mechanism of high-capacity tin anodes for sodium-ion batteries is investigated using a combined theoretical and experimental approach. Ab initio random structure searching (AIRSS) and high-throughput screening using a species-swap method provide insights into a range of possible sodium–tin structures. These structures are linked to experiments using both average and local structure probes in the form of operando pair distribution function analysis, X-ray diffraction, and 23Na solid-state nuclear magnetic resonance (ssNMR), along with ex situ 119Sn ssNMR. Through this approach, we propose structures for the previously unidentified crystalline and amorphous intermediates. The first electrochemical process of sodium insertionmore » into tin results in the conversion of crystalline tin into a layered structure consisting of mixed Na/Sn occupancy sites intercalated between planar hexagonal layers of Sn atoms (approximate stoichiometry NaSn 3). Following this, NaSn 2, which is predicted to be thermodynamically stable by AIRSS, forms; this contains hexagonal layers closely related to NaSn 3, but has no tin atoms between the layers. NaSn 2 is broken down into an amorphous phase of approximate composition Na 1.2Sn. Reverse Monte Carlo refinements of an ab initio molecular dynamics model of this phase show that the predominant tin connectivity is chains. Further reaction with sodium results in the formation of structures containing Sn–Sn dumbbells, which interconvert through a solid-solution mechanism. These structures are based upon Na 5–xSn 2, with increasing occupancy of one of its sodium sites commensurate with the amount of sodium added. ssNMR results indicate that the final product, Na 15Sn 4, can store additional sodium atoms as an off-stoichiometry compound (Na 15+xSn 4) in a manner similar to Li 15Si 4.« less

Investigating Sodium Storage Mechanisms in Tin Anodes: A Combined Pair Distribution Function Analysis, Density Functional Theory, and Solid-State NMR Approach

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stratford, Joshua M.; Mayo, Martin; Allan, Phoebe K.

Here, the alloying mechanism of high-capacity tin anodes for sodium-ion batteries is investigated using a combined theoretical and experimental approach. Ab initio random structure searching (AIRSS) and high-throughput screening using a species-swap method provide insights into a range of possible sodium–tin structures. These structures are linked to experiments using both average and local structure probes in the form of operando pair distribution function analysis, X-ray diffraction, and 23Na solid-state nuclear magnetic resonance (ssNMR), along with ex situ 119Sn ssNMR. Through this approach, we propose structures for the previously unidentified crystalline and amorphous intermediates. The first electrochemical process of sodium insertionmore » into tin results in the conversion of crystalline tin into a layered structure consisting of mixed Na/Sn occupancy sites intercalated between planar hexagonal layers of Sn atoms (approximate stoichiometry NaSn 3). Following this, NaSn 2, which is predicted to be thermodynamically stable by AIRSS, forms; this contains hexagonal layers closely related to NaSn 3, but has no tin atoms between the layers. NaSn 2 is broken down into an amorphous phase of approximate composition Na 1.2Sn. Reverse Monte Carlo refinements of an ab initio molecular dynamics model of this phase show that the predominant tin connectivity is chains. Further reaction with sodium results in the formation of structures containing Sn–Sn dumbbells, which interconvert through a solid-solution mechanism. These structures are based upon Na 5–xSn 2, with increasing occupancy of one of its sodium sites commensurate with the amount of sodium added. ssNMR results indicate that the final product, Na 15Sn 4, can store additional sodium atoms as an off-stoichiometry compound (Na 15+xSn 4) in a manner similar to Li 15Si 4.« less

Screening and identification of radical scavengers from Neo-Taraxacum siphonanthum by online rapid screening method and nuclear magnetic resonance experiments.

PubMed

Shi, Shu Yun; Zhang, Yu Ping; Zhou, Hong Hao; Huang, Ke Long; Jiang, Xin Yu

2010-01-01

An online rapid screening method, the high-performance liquid chromatography (HPLC)-diode array detector (DAD)-radical scavenging detection (RSD)-electrospray ionization (ESI)-mass spectroscopy (MS)/MS system, was developed for the screening and identification of radical scavengers from Neo-Taraxacum siphonanthum, a new species found in China in 1989. For further characterization, the target compounds were isolated by silica column chromatography, preparative high-performance liquid chromatography (HPLC), HSCCC, and Sephadex LH-20 column chromatography and elucidated on the basis of ultraviolet (UV), ESI-MS/MS, and nuclear magnetic resonance (NMR) spectroscopy, as well as the chemical analysis. Eighteen antioxidative polyphenols (5 caffeic acid derivatives and 13 flavonoid derivatives) were characterized from Neo-T. siphonanthum. The distribution of all compounds was discussed in a chemosystematic context, which suggested that the genera Neo-Taraxacum and Taraxacum might relate chemosystematically.

[Determination of the Plant Origin of Licorice Oil Extract, a Natural Food Additive, by Principal Component Analysis Based on Chemical Components].

PubMed

Tada, Atsuko; Ishizuki, Kyoko; Sugimoto, Naoki; Yoshimatsu, Kayo; Kawahara, Nobuo; Suematsu, Takako; Arifuku, Kazunori; Fukai, Toshio; Tamura, Yukiyoshi; Ohtsuki, Takashi; Tahara, Maiko; Yamazaki, Takeshi; Akiyama, Hiroshi

2015-01-01

"Licorice oil extract" (LOE) (antioxidant agent) is described in the notice of Japanese food additive regulations as a material obtained from the roots and/or rhizomes of Glycyrrhiza uralensis, G. inflata or G. glabra. In this study, we aimed to identify the original Glycyrrhiza species of eight food additive products using LC/MS. Glabridin, a characteristic compound in G. glabra, was specifically detected in seven products, and licochalcone A, a characteristic compound in G. inflata, was detected in one product. In addition, Principal Component Analysis (PCA) (a kind of multivariate analysis) using the data of LC/MS or (1)H-NMR analysis was performed. The data of thirty-one samples, including LOE products used as food additives, ethanol extracts of various Glycyrrhiza species and commercially available Glycyrrhiza species-derived products were assessed. Based on the PCA results, the majority of LOE products was confirmed to be derived from G. glabra. This study suggests that PCA using (1)H-NMR analysis data is a simple and useful method to identify the plant species of origin of natural food additive products.

Novel mixed ligand complexes of bioactive Schiff base (E)-4-(phenyl (phenylimino) methyl) benzene-1,3-diol and 2-aminophenol/2-aminobenzoic acid: Synthesis, spectral characterization, antimicrobial and nuclease studies

NASA Astrophysics Data System (ADS)

Subbaraj, P.; Ramu, A.; Raman, N.; Dharmaraja, J.

2014-01-01

A novel bidentate Schiff base ligand has been synthesized using 2,4-dihydroxybenzophenone and aniline. Its mixed ligand complexes of MAB type [M = Mn(II), Co(II), Ni(II), Cu(II) and Zn(II); HA = Schiff base and B = 2-aminophenol/2-aminobenzoic acid] have been synthesized and characterized on the basis of spectral data UV-Vis, IR, 1H NMR, FAB-Mass, EPR, SEM and magnetic studies. All the complexes were soluble in DMF and DMSO. Elemental analysis and molar conductance values indicate that the complexes are non-electrolytes. HA binds with M(II) ions through azomethine and deprotonated phenolic group and B binds through the primary amine group and deprotonated phenolic/carboxylic groups. Using FAB-Mass the cleavage pattern of the ligand (HA) has been established. All the complexes adopt octahedral geometry around the metal ions. It has been confirmed with the help of UV-Vis, IR, 1H NMR and FAB-Mass spectral data. DNA binding activities of the complexes 1d and 2d are studied by UV-Vis spectroscopy and cleavage studies of Schiff base ligand and its complexes 1d and 2d have been by agarose gel electrophoresis method. In vitro biological activities of the free ligand (HA) and their metal complexes (1a-1e and 2a-2e) were screened against few bacteria, Escherichia coli, Staphylococcus saphyphiticus, Staphylococcus aureus, Pseudomonas aeruginosa and fungi Aspergillus niger, Enterobacter species, Candida albicans by well diffusion technique.

Chemical shift-based identification of monosaccharide spin-systems with NMR spectroscopy to complement untargeted glycomics.

PubMed

Klukowski, Piotr; Schubert, Mario

2018-06-15

A better understanding of oligosaccharides and their wide-ranging functions in almost every aspect of biology and medicine promises to uncover hidden layers of biology and will support the development of better therapies. Elucidating the chemical structure of an unknown oligosaccharide is still a challenge. Efficient tools are required for non-targeted glycomics. Chemical shifts are a rich source of information about the topology and configuration of biomolecules, whose potential is however not fully explored for oligosaccharides. We hypothesize that the chemical shifts of each monosaccharide are unique for each saccharide type with a certain linkage pattern, so that correlated data measured by NMR spectroscopy can be used to identify the chemical nature of a carbohydrate. We present here an efficient search algorithm, GlycoNMRSearch, that matches either a subset or the entire set of chemical shifts of an unidentified monosaccharide spin system to all spin systems in an NMR database. The search output is much more precise than earlier search functions and highly similar matches suggest the chemical structure of the spin system within the oligosaccharide. Thus searching for connected chemical shift correlations within all electronically available NMR data of oligosaccharides is a very efficient way of identifying the chemical structure of unknown oligosaccharides. With an improved database in the future, GlycoNMRSearch will be even more efficient deducing chemical structures of oligosaccharides and there is a high chance that it becomes an indispensable technique for glycomics. The search algorithm presented here, together with a graphical user interface, is available at http://glyconmrsearch.santos.pwr.edu.pl. Supplementary data are available at Bioinformatics online.

The Development of Metabolomic Sampling Procedures for Pichia pastoris, and Baseline Metabolome Data

PubMed Central

Tredwell, Gregory D.; Edwards-Jones, Bryn; Leak, David J.; Bundy, Jacob G.

2011-01-01

Metabolic profiling is increasingly being used to investigate a diverse range of biological questions. Due to the rapid turnover of intracellular metabolites it is important to have reliable, reproducible techniques for sampling and sample treatment. Through the use of non-targeted analytical techniques such as NMR and GC-MS we have performed a comprehensive quantitative investigation of sampling techniques for Pichia pastoris. It was clear that quenching metabolism using solutions based on the standard cold methanol protocol caused some metabolite losses from P. pastoris cells. However, these were at a low level, with the NMR results indicating metabolite increases in the quenching solution below 5% of their intracellular level for 75% of metabolites identified; while the GC-MS results suggest a slightly higher level with increases below 15% of their intracellular values. There were subtle differences between the four quenching solutions investigated but broadly, they all gave similar results. Total culture extraction of cells + broth using high cell density cultures typical of P. pastoris fermentations, was an efficient sampling technique for NMR analysis and provided a gold standard of intracellular metabolite levels; however, salts in the media affected the GC-MS analysis. Furthermore, there was no benefit in including an additional washing step in the quenching process, as the results were essentially identical to those obtained just by a single centrifugation step. We have identified the major high-concentration metabolites found in both the extra- and intracellular locations of P. pastoris cultures by NMR spectroscopy and GC-MS. This has provided us with a baseline metabolome for P. pastoris for future studies. The P. pastoris metabolome is significantly different from that of Saccharomyces cerevisiae, with the most notable difference being the production of high concentrations of arabitol by P. pastoris. PMID:21283710

Phage display selection of peptides that target calcium-binding proteins.

PubMed

Vetter, Stefan W

2013-01-01

Phage display allows to rapidly identify peptide sequences with binding affinity towards target proteins, for example, calcium-binding proteins (CBPs). Phage technology allows screening of 10(9) or more independent peptide sequences and can identify CBP binding peptides within 2 weeks. Adjusting of screening conditions allows selecting CBPs binding peptides that are either calcium-dependent or independent. Obtained peptide sequences can be used to identify CBP target proteins based on sequence homology or to quickly obtain peptide-based CBP inhibitors to modulate CBP-target interactions. The protocol described here uses a commercially available phage display library, in which random 12-mer peptides are displayed on filamentous M13 phages. The library was screened against the calcium-binding protein S100B.

Protective Factors Based Model for Screening for Posttraumatic Distress in Adolescents

ERIC Educational Resources Information Center

Pat-Horenczyk, Ruth; Kenan, Avraham Max; Achituv, Michal; Bachar, Eytan

2014-01-01

Background: There is growing application of school-based screening to identify post-traumatic distress in students following exposure to trauma. The consensus method is based on self-report questionnaires that assess posttraumatic symptoms, functional impairment, depression or anxiety. Objective: The current research explored the possibility of…

Molecular testing for Lynch syndrome in people with colorectal cancer: systematic reviews and economic evaluation.

PubMed

Snowsill, Tristan; Coelho, Helen; Huxley, Nicola; Jones-Hughes, Tracey; Briscoe, Simon; Frayling, Ian M; Hyde, Chris

2017-09-01

Inherited mutations in deoxyribonucleic acid (DNA) mismatch repair (MMR) genes lead to an increased risk of colorectal cancer (CRC), gynaecological cancers and other cancers, known as Lynch syndrome (LS). Risk-reducing interventions can be offered to individuals with known LS-causing mutations. The mutations can be identified by comprehensive testing of the MMR genes, but this would be prohibitively expensive in the general population. Tumour-based tests - microsatellite instability (MSI) and MMR immunohistochemistry (IHC) - are used in CRC patients to identify individuals at high risk of LS for genetic testing. MLH1 (MutL homologue 1) promoter methylation and BRAF V600E testing can be conducted on tumour material to rule out certain sporadic cancers. To investigate whether testing for LS in CRC patients using MSI or IHC (with or without MLH1 promoter methylation testing and BRAF V600E testing) is clinically effective (in terms of identifying Lynch syndrome and improving outcomes for patients) and represents a cost-effective use of NHS resources. Systematic reviews were conducted of the published literature on diagnostic test accuracy studies of MSI and/or IHC testing for LS, end-to-end studies of screening for LS in CRC patients and economic evaluations of screening for LS in CRC patients. A model-based economic evaluation was conducted to extrapolate long-term outcomes from the results of the diagnostic test accuracy review. The model was extended from a model previously developed by the authors. Ten studies were identified that evaluated the diagnostic test accuracy of MSI and/or IHC testing for identifying LS in CRC patients. For MSI testing, sensitivity ranged from 66.7% to 100.0% and specificity ranged from 61.1% to 92.5%. For IHC, sensitivity ranged from 80.8% to 100.0% and specificity ranged from 80.5% to 91.9%. When tumours showing low levels of MSI were treated as a positive result, the sensitivity of MSI testing increased but specificity fell. No end-to-end studies of screening for LS in CRC patients were identified. Nine economic evaluations of screening for LS in CRC were identified. None of the included studies fully matched the decision problem and hence a new economic evaluation was required. The base-case results in the economic evaluation suggest that screening for LS in CRC patients using IHC, BRAF V600E and MLH1 promoter methylation testing would be cost-effective at a threshold of £20,000 per quality-adjusted life-year (QALY). The incremental cost-effectiveness ratio for this strategy was £11,008 per QALY compared with no screening. Screening without tumour tests is not predicted to be cost-effective. Most of the diagnostic test accuracy studies identified were rated as having a risk of bias or were conducted in unrepresentative samples. There was no direct evidence that screening improves long-term outcomes. No probabilistic sensitivity analysis was conducted. Systematic review evidence suggests that MSI- and IHC-based testing can be used to identify LS in CRC patients, although there was heterogeneity in the methods used in the studies identified and the results of the studies. There was no high-quality empirical evidence that screening improves long-term outcomes and so an evidence linkage approach using modelling was necessary. Key determinants of whether or not screening is cost-effective are the accuracy of tumour-based tests, CRC risk without surveillance, the number of relatives identified for cascade testing, colonoscopic surveillance effectiveness and the acceptance of genetic testing. Future work should investigate screening for more causes of hereditary CRC and screening for LS in endometrial cancer patients. This study is registered as PROSPERO CRD42016033879. The National Institute for Health Research Health Technology Assessment programme.

A microscopy-based screen employing multiplex genome sequencing identifies cargo-specific requirements for dynein velocity

PubMed Central

Tan, Kaeling; Roberts, Anthony J.; Chonofsky, Mark; Egan, Martin J.; Reck-Peterson, Samara L.

2014-01-01

The timely delivery of membranous organelles and macromolecules to specific locations within the majority of eukaryotic cells depends on microtubule-based transport. Here we describe a screening method to identify mutations that have a critical effect on intracellular transport and its regulation using mutagenesis, multicolor-fluorescence microscopy, and multiplex genome sequencing. This screen exploits the filamentous fungus Aspergillus nidulans, which has many of the advantages of yeast molecular genetics but uses long-range microtubule-based transport in a manner more similar to metazoan cells. Using this method, we identified seven mutants that represent novel alleles of components of the intracellular transport machinery: specifically, kinesin-1, cytoplasmic dynein, and the dynein regulators Lis1 and dynactin. The two dynein mutations identified in our screen map to dynein's AAA+ catalytic core. Single-molecule studies reveal that both mutations reduce dynein's velocity in vitro. In vivo these mutants severely impair the distribution and velocity of endosomes, a known dynein cargo. In contrast, another dynein cargo, the nucleus, is positioned normally in these mutants. These results reveal that different dynein functions have distinct stringencies for motor performance. PMID:24403603

Demetalation of Fe, Mn, and Cu chelates and complexes: application to the NMR analysis of micronutrient fertilizers.

PubMed

López-Rayo, Sandra; Lucena, Juan J; Laghi, Luca; Cremonini, Mauro A

2011-12-28

The application of nuclear magnetic resonance (NMR) for the quality control of fertilizers based on Fe(3+), Mn(2+), and Cu(2+) chelates and complexes is precluded by the strong paramagnetism of metals. Recently, a method based on the use of ferrocyanide has been described to remove iron from commercial iron chelates based on the o,o-EDDHA [ethylenediamine-N,N'bis(2-hydroxyphenylacetic)acid] chelating agent for their analysis and quantification by NMR. The present work extended that procedure to other paramagnetic ions, manganese and copper, and other chelating, EDTA (ethylenediaminetetraacetic acid), IDHA [N-(1,2-dicarboxyethyl)-d,l-aspartic acid], and complexing agents, gluconate and heptagluconate. Results showed that the removal of the paramagnetic ions was complete, allowing us to obtain (1)H NMR spectra characterized by narrow peaks. The quantification of the ligands by NMR and high-performance liquid chromatography showed that their complete recovery was granted. The NMR analysis enabled detection and quantification of unknown impurities without the need of pure compounds as internal standards.

NMR-Based Serum Metabolomics Discriminates Takayasu Arteritis from Healthy Individuals: A Proof-of-Principle Study.

PubMed

Guleria, Anupam; Misra, Durga Prasanna; Rawat, Atul; Dubey, Durgesh; Khetrapal, Chunni Lal; Bacon, Paul; Misra, Ramnath; Kumar, Dinesh

2015-08-07

Takayasu arteritis (TA) is a debilitating, systemic disease that involves the aorta and large arteries in a chronic inflammatory process that leads to vessel stenosis. Initially, the disease remains clinically silent (or remains undetected) until the patients present with vascular occlusion. Therefore, new methods for appropriate and timely diagnosis of TA cases are needed to start proper therapy on time and also to monitor the patient's response to the given treatment. In this context, NMR-based serum metabolomic profiling has been explored in this proof-of-principle study for the first time to determine characteristic metabolites that could be potentially helpful for diagnosis and prognosis of TA. Serum metabolic profiling of TA patients (n = 29) and healthy controls (n = 30) was performed using 1D (1)H NMR spectroscopy, and possible biomarker metabolites were identified. Using projection to least-squares discriminant analysis, we could distinguish TA patients from healthy controls. Compared to healthy controls, TA patients had (a) increased serum levels of choline metabolites, LDL cholesterol, N-acetyl glycoproteins (NAGs), and glucose and (b) decreased serum levels of lactate, lipids, HDL cholesterol, and glucogenic amino acids. The results of this study are preliminary and need to be confirmed in a prospective study.

Evidence for altered metabolic pathways during environmental stress: (1)H-NMR spectroscopy based metabolomics and clinical studies on subjects of sea-voyage and Antarctic-stay.

PubMed

Yadav, Anand Prakash; Chaturvedi, Shubhra; Mishra, Kamla Prasad; Pal, Sunil; Ganju, Lilly; Singh, Shashi Bala

2014-08-01

The Antarctic context is an analogue of space travel, with close similarity in ambience of extreme climate, isolation, constrained living spaces, disrupted sleep cycles, and environmental stress. The present study examined the impact of the harsh habitat of Antarctica on human physiology and its metabolic pathways, by analyzing human serum samples, using (1)H-NMR spectroscopy for identification of metabolites; and quantifying other physiological and clinical parameters for correlation between expression data and metabolite data. Sera from seven adult males (of median age 36years) who participated in this study, from the 28th Indian Expeditionary group to the Antarctica station Maitri, were collected in chronological sequence. These included: i) baseline control; ii) during ship journey; iii) at Antarctica, in the months of March, May, August and November; to enable study of temporal evolution of monitored physiological states. 29 metabolites in serum were identified from the 400MHz (1)H-NMR spectra. Out of these, 19 metabolites showed significant variations in levels, during the ship journey and the stay at Maitri, compared to the base-line levels. Further biochemical analysis also supported these results, indicating that the ship journey, and the long-term Antarctic exposure, affected kidney and liver functioning. Our metabolite data highlights for the first time the effect of environmental stress on the patho-physiology of the human system. Multivariate analysis tools were employed for this metabonomics study, using (1)H-NMR spectroscopy. Copyright © 2014. Published by Elsevier Inc.

A flavone derivative from Sesbania sesban leaves and its cytotoxicity against murine leukemia P-388 cells

NASA Astrophysics Data System (ADS)

Dianhar, Hanhan; Syah, Yana Maolana; Mujahidin, Didin; Hakim, Euis Holisotan; Juliawaty, Lia Dewi

2014-03-01

Sesbania sesban, locally named as Jayanti, is one of Indonesia plants belonging to Fabaceae family. This species is traditionally used by Indonesian people to cure digestive disorders, fever, or headache. Jayanti can grow well in tropical to subtropical region, such as in Asia and Africa. Based on literature, qualitative analysis of the methanol extract of leaves of S. sesban showed that it contained flavonoids, alkaloids, saponins and glycosides. In addition, the activity assay of extracts of different tissues of this species showed antitumor, antimalarial, and antidiabetic activityies (leaves and seed extracts), antioxidants (flower extract), and analgesic (wood extract). Though the extracts of S. sesban parts showed interesting activities, chemical study of those extracts have not been widely reported. Therefore, the objective of this research was to isolate the secondary metabolites from methanol extract of leaves of S. sesban and to determine their cytotoxicity against murine leukemia P-388 cells. One compound has been obtained and identified as 3-hydroxy-4',7-dimethoxyflavone (1), a new isolated compound from nature. This compound was obtained through separation of methanol extract using various chromatographic techniques, such as vacuum liquid chromatography and radial chromatography. The structure elucidation of isolated compound was based on 1D NMR (1H-NMR and 13C-NMR) and 2D NMR (HMBC). The cytotoxicity of methanol extract and compound 1 against murine leukemia P-388 cells examined through MTT assay showed IC50 value of 60.04 μg/mL and 5.40 μg/mL, respectively.

A flavone derivative from Sesbania sesban leaves and its cytotoxicity against murine leukemia P-388 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dianhar, Hanhan, E-mail: liadewi@chem.itb.ac.id; Syah, Yana Maolana, E-mail: liadewi@chem.itb.ac.id; Mujahidin, Didin, E-mail: liadewi@chem.itb.ac.id

2014-03-24

Sesbania sesban, locally named as Jayanti, is one of Indonesia plants belonging to Fabaceae family. This species is traditionally used by Indonesian people to cure digestive disorders, fever, or headache. Jayanti can grow well in tropical to subtropical region, such as in Asia and Africa. Based on literature, qualitative analysis of the methanol extract of leaves of S. sesban showed that it contained flavonoids, alkaloids, saponins and glycosides. In addition, the activity assay of extracts of different tissues of this species showed antitumor, antimalarial, and antidiabetic activityies (leaves and seed extracts), antioxidants (flower extract), and analgesic (wood extract). Though themore » extracts of S. sesban parts showed interesting activities, chemical study of those extracts have not been widely reported. Therefore, the objective of this research was to isolate the secondary metabolites from methanol extract of leaves of S. sesban and to determine their cytotoxicity against murine leukemia P-388 cells. One compound has been obtained and identified as 3-hydroxy-4',7-dimethoxyflavone (1), a new isolated compound from nature. This compound was obtained through separation of methanol extract using various chromatographic techniques, such as vacuum liquid chromatography and radial chromatography. The structure elucidation of isolated compound was based on 1D NMR ({sup 1}H-NMR and {sup 13}C-NMR) and 2D NMR (HMBC). The cytotoxicity of methanol extract and compound 1 against murine leukemia P-388 cells examined through MTT assay showed IC{sub 50} value of 60.04 μg/mL and 5.40 μg/mL, respectively.« less

Squid detected NMR and MRI at ultralow fields

DOEpatents

Clarke, John [Berkeley, CA; McDermott, Robert [Louisville, CO; Pines, Alexander [Berkeley, CA; Trabesinger, Andreas Heinz [CH-8006 Zurich, CH

2007-05-15

Nuclear magnetic resonance (NMR) signals are detected in microtesla fields. Prepolarization in millitesla fields is followed by detection with an untuned dc superconducting quantum interference device (SQUID) magnetometer. Because the sensitivity of the SQUID is frequency independent, both signal-to-noise ratio (SNR) and spectral resolution are enhanced by detecting the NMR signal in extremely low magnetic fields, where the NMR lines become very narrow even for grossly inhomogeneous measurement fields. MRI in ultralow magnetic field is based on the NMR at ultralow fields. Gradient magnetic fields are applied, and images are constructed from the detected NMR signals.

Squid detected NMR and MRI at ultralow fields

DOEpatents

Clarke, John; McDermott, Robert; Pines, Alexander; Trabesinger, Andreas Heinz

2006-05-30

Nuclear magnetic resonance (NMR) signals are detected in microtesla fields. Prepolarization in millitesla fields is followed by detection with an untuned dc superconducting quantum interference device (SQUID) magnetometer. Because the sensitivity of the SQUID is frequency independent, both signal-to-noise ratio (SNR) and spectral resolution are enhanced by detecting the NMR signal in extremely low magnetic fields, where the NMR lines become very narrow even for grossly inhomogeneous measurement fields. MRI in ultralow magnetic field is based on the NMR at ultralow fields. Gradient magnetic fields are applied, and images are constructed from the detected NMR signals.

Squid detected NMR and MRI at ultralow fields

DOEpatents

Clarke, John [Berkeley, CA; Pines, Alexander [Berkeley, CA; McDermott, Robert F [Monona, WI; Trabesinger, Andreas H [London, GB

2008-12-16

Nuclear magnetic resonance (NMR) signals are detected in microtesla fields. Prepolarization in millitesla fields is followed by detection with an untuned dc superconducting quantum interference device (SQUID) magnetometer. Because the sensitivity of the SQUID is frequency independent, both signal-to-noise ratio (SNR) and spectral resolution are enhanced by detecting the NMR signal in extremely low magnetic fields, where the NMR lines become very narrow even for grossly inhomogeneous measurement fields. MRI in ultralow magnetic field is based on the NMR at ultralow fields. Gradient magnetic fields are applied, and images are constructed from the detected NMR signals.

SQUID detected NMR and MRI at ultralow fields

DOEpatents

Clarke, John; McDermott, Robert; Pines, Alexander; Trabesinger, Andreas Heinz

2006-10-03

Nuclear magnetic resonance (NMR) signals are detected in microtesla fields. Prepolarization in millitesla fields is followed by detection with an untuned dc superconducting quantum interference device (SQUID) magnetometer. Because the sensitivity of the SQUID is frequency independent, both signal-to-noise ratio (SNR) and spectral resolution are enhanced by detecting the NMR signal in extremely low magnetic fields, where the NMR lines become very narrow even for grossly inhomogeneous measurement fields. MRI in ultralow magnetic field is based on the NMR at ultralow fields. Gradient magnetic fields are applied, and images are constructed from the detected NMR signals.

Metabolic Characterization of Peripheral Host Responses to Drainage-Resistant Klebsiella pneumoniae Liver Abscesses by Serum 1H-NMR Spectroscopy.

PubMed

Chang, Zhihui; Wang, Hairui; Li, Beibei; Liu, Zhaoyu; Zheng, Jiahe

2018-01-01

Purpose: To explore the metabolic characterization of host responses to drainage-resistant Klebsiella pneumoniae liver abscesses (DRKPLAs) with serum 1H-nuclear magnetic resonance (NMR) spectroscopy. Materials and Methods: The hospital records of all patients with a diagnosis of a liver abscess between June 2015 and December 2016 were retrieved from an electronic hospital database. Eighty-six patients with Klebsiella pneumoniae ( K. pneumoniae ) liver abscesses who underwent percutaneous drainage were identified. Twenty patients with confirmed DRKPLAs were studied. Moreover, we identified 20 consecutive patients with drainage-sensitive Klebsiella pneumoniae liver abscesses (DSKPLAs) as controls. Serum samples from the two groups were analyzed with 1H NMR spectroscopy. Partial least squares discriminant analysis (PLS-DA) was used to perform 1H NMR metabolic profiling. Metabolites were identified using the Human Metabolome Database, and pathway analysis was performed with MetaboAnalyst 3.0. Results: The PLS-DA test was able to discriminate between the two groups. Five key metabolites that contributed to their discrimination were identified. Glucose, lactate, and 3-hydroxybutyrate were found to be upregulated in DRKPLAs, whereas glutamine and alanine were downregulated compared with the DSKPLAs. Pathway analysis indicated that amino acid metabolisms were significantly different between the DRKPLAs and the DSKPLAs. The D-glutamine and D-glutamate metabolisms exhibited the greatest influences. Conclusions: The five key metabolites identified in our study may be potential targets for guiding novel therapeutics of DRKPLAs and are worthy of additional investigation.

1H NMR Metabolomics Study of Spleen from C57BL/6 Mice Exposed to Gamma Radiation

PubMed Central

Xiao, X; Hu, M; Liu, M; Hu, JZ

2016-01-01

Due to the potential risk of accidental exposure to gamma radiation, it’s critical to identify the biomarkers of radiation exposed creatures. In the present study, NMR based metabolomics combined with multivariate data analysis to evaluate the metabolites changed in the C57BL/6 mouse spleen after 4 days whole body exposure to 3.0 Gy and 7.8 Gy gamma radiations. Principal component analysis (PCA) and orthogonal projection to latent structures analysis (OPLS) are employed for classification and identification potential biomarkers associated with gamma irradiation. Two different strategies for NMR spectral data reduction (i.e., spectral binning and spectral deconvolution) are combined with normalize to constant sum and unit weight before multivariate data analysis, respectively. The combination of spectral deconvolution and normalization to unit weight is the best way for identifying discriminatory metabolites between the irradiation and control groups. Normalized to the constant sum may achieve some pseudo biomarkers. PCA and OPLS results shown that the exposed groups can be well separated from the control group. Leucine, 2-aminobutyrate, valine, lactate, arginine, glutathione, 2-oxoglutarate, creatine, tyrosine, phenylalanine, π-methylhistidine, taurine, myoinositol, glycerol and uracil are significantly elevated while ADP is decreased significantly. These significantly changed metabolites are associated with multiple metabolic pathways and may be potential biomarkers in the spleen exposed to gamma irradiation. PMID:27019763

β-Helical architecture of cytoskeletal bactofilin filaments revealed by solid-state NMR

PubMed Central

Vasa, Suresh; Lin, Lin; Shi, Chaowei; Habenstein, Birgit; Riedel, Dietmar; Kühn, Juliane; Thanbichler, Martin; Lange, Adam

2015-01-01

Bactofilins are a widespread class of bacterial filament-forming proteins, which serve as cytoskeletal scaffolds in various cellular pathways. They are characterized by a conserved architecture, featuring a central conserved domain (DUF583) that is flanked by variable terminal regions. Here, we present a detailed investigation of bactofilin filaments from Caulobacter crescentus by high-resolution solid-state NMR spectroscopy. De novo sequential resonance assignments were obtained for residues Ala39 to Phe137, spanning the conserved DUF583 domain. Analysis of the secondary chemical shifts shows that this core region adopts predominantly β-sheet secondary structure. Mutational studies of conserved hydrophobic residues located in the identified β-strand segments suggest that bactofilin folding and polymerization is mediated by an extensive and redundant network of hydrophobic interactions, consistent with the high intrinsic stability of bactofilin polymers. Transmission electron microscopy revealed a propensity of bactofilin to form filament bundles as well as sheet-like, 2D crystalline assemblies, which may represent the supramolecular arrangement of bactofilin in the native context. Based on the diffraction pattern of these 2D crystalline assemblies, scanning transmission electron microscopy measurements of the mass per length of BacA filaments, and the distribution of β-strand segments identified by solid-state NMR, we propose that the DUF583 domain adopts a β-helical architecture, in which 18 β-strand segments are arranged in six consecutive windings of a β-helix. PMID:25550503

β-Helical architecture of cytoskeletal bactofilin filaments revealed by solid-state NMR.

PubMed

Vasa, Suresh; Lin, Lin; Shi, Chaowei; Habenstein, Birgit; Riedel, Dietmar; Kühn, Juliane; Thanbichler, Martin; Lange, Adam

2015-01-13

Bactofilins are a widespread class of bacterial filament-forming proteins, which serve as cytoskeletal scaffolds in various cellular pathways. They are characterized by a conserved architecture, featuring a central conserved domain (DUF583) that is flanked by variable terminal regions. Here, we present a detailed investigation of bactofilin filaments from Caulobacter crescentus by high-resolution solid-state NMR spectroscopy. De novo sequential resonance assignments were obtained for residues Ala39 to Phe137, spanning the conserved DUF583 domain. Analysis of the secondary chemical shifts shows that this core region adopts predominantly β-sheet secondary structure. Mutational studies of conserved hydrophobic residues located in the identified β-strand segments suggest that bactofilin folding and polymerization is mediated by an extensive and redundant network of hydrophobic interactions, consistent with the high intrinsic stability of bactofilin polymers. Transmission electron microscopy revealed a propensity of bactofilin to form filament bundles as well as sheet-like, 2D crystalline assemblies, which may represent the supramolecular arrangement of bactofilin in the native context. Based on the diffraction pattern of these 2D crystalline assemblies, scanning transmission electron microscopy measurements of the mass per length of BacA filaments, and the distribution of β-strand segments identified by solid-state NMR, we propose that the DUF583 domain adopts a β-helical architecture, in which 18 β-strand segments are arranged in six consecutive windings of a β-helix.

1H NMR metabolomics study of spleen from C57BL/6 mice exposed to gamma radiation

DOE PAGES

Xiao, Xiongjie; Hu, M.; Liu, M.; ...

2016-01-27

Due to the potential risk of accidental exposure to gamma radiation, it’s critical to identify the biomarkers of radiation exposed creatures. In the present study, NMR based metabolomics combined with multivariate data analysis to evaluate the metabolites changed in the C57BL/6 mouse spleen after 4 days whole body exposure to 3.0 Gy and 7.8 Gy gamma radiations. Principal component analysis (PCA) and orthogonal projection to latent structures analysis (OPLS) are employed for classification and identification potential biomarkers associated with gamma irradiation. Two different strategies for NMR spectral data reduction (i.e., spectral binning and spectral deconvolution) are combined with normalize tomore » constant sum and unit weight before multivariate data analysis, respectively. The combination of spectral deconvolution and normalization to unit weight is the best way for identifying discriminatory metabolites between the irradiation and control groups. Normalized to the constant sum may achieve some pseudo biomarkers. PCA and OPLS results shown that the exposed groups can be well separated from the control group. Leucine, 2-aminobutyrate, valine, lactate, arginine, glutathione, 2-oxoglutarate, creatine, tyrosine, phenylalanine, π-methylhistidine, taurine, myoinositol, glycerol and uracil are significantly elevated while ADP is decreased significantly. As a result, these significantly changed metabolites are associated with multiple metabolic pathways and may be potential biomarkers in the spleen exposed to gamma irradiation.« less

Bayesian reconstruction of projection reconstruction NMR (PR-NMR).

PubMed

Yoon, Ji Won

2014-11-01

Projection reconstruction nuclear magnetic resonance (PR-NMR) is a technique for generating multidimensional NMR spectra. A small number of projections from lower-dimensional NMR spectra are used to reconstruct the multidimensional NMR spectra. In our previous work, it was shown that multidimensional NMR spectra are efficiently reconstructed using peak-by-peak based reversible jump Markov chain Monte Carlo (RJMCMC) algorithm. We propose an extended and generalized RJMCMC algorithm replacing a simple linear model with a linear mixed model to reconstruct close NMR spectra into true spectra. This statistical method generates samples in a Bayesian scheme. Our proposed algorithm is tested on a set of six projections derived from the three-dimensional 700 MHz HNCO spectrum of a protein HasA. Copyright © 2014 Elsevier Ltd. All rights reserved.

Differential Epitope Mapping by STD NMR Spectroscopy To Reveal the Nature of Protein-Ligand Contacts.

PubMed

Monaco, Serena; Tailford, Louise E; Juge, Nathalie; Angulo, Jesus

2017-11-27

Saturation transfer difference (STD) NMR spectroscopy is extensively used to obtain epitope maps of ligands binding to protein receptors, thereby revealing structural details of the interaction, which is key to direct lead optimization efforts in drug discovery. However, it does not give information about the nature of the amino acids surrounding the ligand in the binding pocket. Herein, we report the development of the novel method differential epitope mapping by STD NMR (DEEP-STD NMR) for identifying the type of protein residues contacting the ligand. The method produces differential epitope maps through 1) differential frequency STD NMR and/or 2) differential solvent (D 2 O/H 2 O) STD NMR experiments. The two approaches provide different complementary information on the binding pocket. We demonstrate that DEEP-STD NMR can be used to readily obtain pharmacophore information on the protein. Furthermore, if the 3D structure of the protein is known, this information also helps in orienting the ligand in the binding pocket. © 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

In silico activity profiling reveals the mechanism of action of antimalarials discovered in a high-throughput screen

PubMed Central

Plouffe, David; Brinker, Achim; McNamara, Case; Henson, Kerstin; Kato, Nobutaka; Kuhen, Kelli; Nagle, Advait; Adrián, Francisco; Matzen, Jason T.; Anderson, Paul; Nam, Tae-gyu; Gray, Nathanael S.; Chatterjee, Arnab; Janes, Jeff; Yan, S. Frank; Trager, Richard; Caldwell, Jeremy S.; Schultz, Peter G.; Zhou, Yingyao; Winzeler, Elizabeth A.

2008-01-01

The growing resistance to current first-line antimalarial drugs represents a major health challenge. To facilitate the discovery of new antimalarials, we have implemented an efficient and robust high-throughput cell-based screen (1,536-well format) based on proliferation of Plasmodium falciparum (Pf) in erythrocytes. From a screen of ≈1.7 million compounds, we identified a diverse collection of ≈6,000 small molecules comprised of >530 distinct scaffolds, all of which show potent antimalarial activity (<1.25 μM). Most known antimalarials were identified in this screen, thus validating our approach. In addition, we identified many novel chemical scaffolds, which likely act through both known and novel pathways. We further show that in some cases the mechanism of action of these antimalarials can be determined by in silico compound activity profiling. This method uses large datasets from unrelated cellular and biochemical screens and the guilt-by-association principle to predict which cellular pathway and/or protein target is being inhibited by select compounds. In addition, the screening method has the potential to provide the malaria community with many new starting points for the development of biological probes and drugs with novel antiparasitic activities. PMID:18579783

Isolation, structure elucidation and antibacterial activity of methyl-4,8-dimethylundecanate from the marine actinobacterium Streptomyces albogriseolus ECR64.

PubMed

Thirumurugan, Durairaj; Vijayakumar, Ramasamy; Vadivalagan, Chithravel; Karthika, Pushparaj; Alam Khan, Md Khurshid

2018-05-25

Around 120 actinobacterial colonies were isolated from various regions of marine East coast region of Tamil Nadu, India. Among them, 33 were morphologically distinct and they were preliminarily screened for their antibacterial activity against Pseudomonas fluorescens, Vibrio cholerae, V. parahaemolyticus, V. alginolyticus, and Aeromonas hydrophila by cross-streak plate technique. Among the isolated, the isolate ECR64 exhibited maximum zone of inhibition against fish pathogenic bacteria. The crude bioactive compounds were extracted from the isolate ECR64 using different organic solvents which exhibited maximum antibacterial activity. Separation and purification of the bioactive compounds were made by column chromatography which yielded 27 fractions and were re-chromatographed to obtain the active compound. Ultra violet (UV), Fourier transform infrared (FT-IR) and nuclear magnetic resonance (NMR) spectral studies were used to predict the structure of the active compound which was identified as methyl-4,8-dimethylundecanate. The potential isolate ECR64 was identified as Streptomyces albogriseolus by phylogenetic, phenotypic and genotypic (16S rRNA gene sequence) analyses. The identified compound methyl-4,8-dimethylundecanate can be used as potential and alternative drug in disease management of aquaculture. Copyright © 2018 Elsevier Ltd. All rights reserved.

15N photo-CIDNP MAS NMR analysis of reaction centers of Chloracidobacterium thermophilum.

PubMed

Zill, Jeremias C; He, Zhihui; Tank, Marcus; Ferlez, Bryan H; Canniffe, Daniel P; Lahav, Yigal; Bellstedt, Peter; Alia, A; Schapiro, Igor; Golbeck, John H; Bryant, Donald A; Matysik, Jörg

2018-03-30

Photochemically induced dynamic nuclear polarization (photo-CIDNP) has been observed in the homodimeric, type-1 photochemical reaction centers (RCs) of the acidobacterium, Chloracidobacterium (Cab.) thermophilum, by 15 N magic-angle spinning (MAS) solid-state NMR under continuous white-light illumination. Three light-induced emissive (negative) signals are detected. In the RCs of Cab. thermophilum, three types of (bacterio)chlorophylls have previously been identified: bacteriochlorophyll a (BChl a), chlorophyll a (Chl a), and Zn-bacteriochlorophyll a' (Zn-BChl a') (Tsukatani et al. in J Biol Chem 287:5720-5732, 2012). Based upon experimental and quantum chemical 15 N NMR data, we assign the observed signals to a Chl a cofactor. We exclude Zn-BChl because of its measured spectroscopic properties. We conclude that Chl a is the primary electron acceptor, which implies that the primary donor is most likely Zn-BChl a'. Chl a and 8 1 -OH Chl a have been shown to be the primary electron acceptors in green sulfur bacteria and heliobacteria, respectively, and thus a Chl a molecule serves this role in all known homodimeric type-1 RCs.

GC-MS, LC-MS(n), LC-high resolution-MS(n), and NMR studies on the metabolism and toxicological detection of mesembrine and mesembrenone, the main alkaloids of the legal high "Kanna" isolated from Sceletium tortuosum.

PubMed

Meyer, Golo M J; Wink, Carina S D; Zapp, Josef; Maurer, Hans H

2015-01-01

Mesembrine and mesembrenone are the main alkaloids of Sceletium tortuosum, a plant species that was used for sedation and analgesia by the KhoiSan, previously known as Hottentots, a tribe in South Africa. After fermentation, the obtained preparation called "Kanna" or "Kougoed" was used by chewing, smoking, or sniffing. Today, Kanna gains popularity by drug users as legal high. For monitoring such consumption, metabolism studies are mandatory because the metabolites are mostly the analytical targets, especially in urine. Therefore, the metabolism of both alkaloids was investigated in rat urine and pooled human liver preparations after several sample work-up procedures. As both alkaloids were not commercially available, they were isolated from plant material by Soxhlet extraction, and their identity confirmed by NMR. The metabolites were identified using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography coupled to linear ion trap high resolution mass spectrometry (LC-HR-MS(n)). Both alkaloids were O- and N-demethylated, dihydrated, and/or hydroxylated at different positions. The phenolic metabolites were partly excreted as glucuronides and/or sulfates. Most of the phase I metabolites identified in rat urine could be detected also in the human liver preparations. After a common user's low dose application of mesembrine, mainly the O- and N demethyl-dihydro, hydroxy, and bis-demethyl-dihydro metabolites, and in case of mesembrenone only the N-demethyl and the N-demethyl-dihydro metabolite could be detected in rat urine using the authors' standard urine screening approaches (SUSA) by GC-MS or LC-MS(n). Thus, it should be possible to monitor a consumption of mesembrine and/or mesembrenone assuming similar pharmacokinetics in humans.

Chemical behavior of methylpyranomalvidin-3-O-glucoside in aqueous solution studied by NMR and UV-visible spectroscopy.

PubMed

Oliveira, Joana; Petrov, Vesselin; Parola, A Jorge; Pina, Fernando; Azevedo, Joana; Teixeira, Natércia; Brás, Natércia F; Fernandes, Pedro A; Mateus, Nuno; Ramos, Maria João; de Freitas, Victor

2011-02-17

In the present work, the proton-transfer reactions of the methylpyranomalvidin-3-O-glucoside pigment in water with different pH values was studied by NMR and UV-visible spectroscopies. The results showed four equilibrium forms: the methylpyranomalvidin-3-O-glucoside cation, the neutral quinoidal base, the respective anionic quinoidal base, and a dianionic base unprotonated at the methyl group. According to the NMR data, it seems that for methylpyranomalvidin-3-O-glucoside besides the acid-base equilibrium between the pyranoflavylium cation and the neutral quinoidal base, a new species is formed at pD 4.88-6.10. This is corroborated by the appearance of a new set of signals in the NMR spectrum that may be assigned to the formation of hemiketal/cis-chalcone species to a small extent. The two ionization constants (pK(a1) and pK(a2)) obtained by both methods (NMR and UV-visible) for methylpyranomalvidin-3-O-glucoside are in agreement (pK(a1) = 5.17 ± 0.03; pK(a2) = 8.85 ± 0.08; and pK(a1) = 4.57 ± 0.07; pK(a2) = 8.23 ± 0.04 obtained by NMR and UV-visible spectroscopies, respectively). Moreover, the fully dianionic unprotonated form (at the methyl group) of the methylpyranomalvidin-3-O-glucoside is converted slowly into a new structure that displays a yellow color at basic pH. On the basis of the results obtained through LC-MS and NMR, the proposed structure was found to correspond to the flavonol syringetin-3-glucoside.

FRET and BRET-based biosensors in live cell compound screens.

PubMed

Robinson, Katie Herbst; Yang, Jessica R; Zhang, Jin

2014-01-01

Live cell compound screening with genetically encoded fluorescence or bioluminescence-based biosensors offers a potentially powerful approach to identify novel regulators of a signaling event of interest. In particular, compound screening in living cells has the added benefit that the entire signaling network remains intact, and thus the screen is not just against a single molecule of interest but against any molecule within the signaling network that may modulate the distinct signaling event reported by the biosensor in use. Furthermore, only molecules that are cell permeable or act at cell surface receptors will be identified as "hits," thus reducing further optimization of the compound in terms of cell penetration. Here we discuss a detailed protocol for using genetically encoded biosensors in living cells in a 96-well format for the execution of high throughput compound screens and the identification of small molecules which modulate a signaling event of interest.

NMR Crystallography of Enzyme Active Sites: Probing Chemically-Detailed, Three-Dimensional Structure in Tryptophan Synthase

PubMed Central

Dunn, Michael F.

2013-01-01

Conspectus NMR crystallography – the synergistic combination of X-ray diffraction, solid-state NMR spectroscopy, and computational chemistry – offers unprecedented insight into three-dimensional, chemically-detailed structure. From its initial role in refining diffraction data of organic and inorganic solids, NMR crystallography is now being developed for application to active sites in biomolecules, where it reveals chemically-rich detail concerning the interactions between enzyme site residues and the reacting substrate that is not achievable when X-ray, NMR, or computational methodologies are applied in isolation. For example, typical X-ray crystal structures (1.5 to 2.5 Å resolution) of enzyme-bound intermediates identify possible hydrogen-bonding interactions between site residues and substrate, but do not directly identify the protonation state of either. Solid-state NMR can provide chemical shifts for selected atoms of enzyme-substrate complexes, but without a larger structural framework in which to interpret them, only empirical correlations with local chemical structure are possible. Ab initio calculations and molecular mechanics can build models for enzymatic processes, but rely on chemical details that must be specified. Together, however, X-ray diffraction, solid-state NMR spectroscopy, and computational chemistry can provide consistent and testable models for structure and function of enzyme active sites: X-ray crystallography provides a coarse framework upon which models of the active site can be developed using computational chemistry; these models can be distinguished by comparison of their calculated NMR chemical shifts with the results of solid-state NMR spectroscopy experiments. Conceptually, each technique is a puzzle piece offering a generous view of the big picture. Only when correctly pieced together, however, can they reveal the big picture at highest resolution. In this Account, we detail our first steps in the development of NMR crystallography for application to enzyme catalysis. We begin with a brief introduction to NMR crystallography and then define the process that we have employed to probe the active site in the β-subunit of tryptophan synthase with unprecedented atomic-level resolution. This approach has resulted in a novel structural hypothesis for the protonation state of the quinonoid intermediate in tryptophan synthase and its surprising role in directing the next step in the catalysis of L-Trp formation. PMID:23537227