MetaboMiner – semi-automated identification of metabolites from 2D NMR spectra of complex biofluids

<p>Abstract</p> <p>Background</p> <p>One-dimensional (1D) ¹H nuclear magnetic resonance (NMR) spectroscopy is widely used in metabolomic studies involving biofluids and tissue extracts. There are several software packages that support compound identification and quantification via 1D ¹H NMR by spectral fitting techniques. Because 1D ¹H NMR spectra are characterized by extensive peak overlap or spectral congestion, two-dimensional (2D) NMR, with its increased spectral resolution, could potentially improve and even automate compound identification or quantification. However, the lack of dedicated software for this purpose significantly restricts the application of 2D NMR methods to most metabolomic studies.</p> <p>Results</p> <p>We describe a standalone graphics software tool, called MetaboMiner, which can be used to automatically or semi-automatically identify metabolites in complex biofluids from 2D NMR spectra. MetaboMiner is able to handle both ¹H-¹H total correlation spectroscopy (TOCSY) and ¹H-¹³C heteronuclear single quantum correlation (HSQC) data. It identifies compounds by comparing 2D spectral patterns in the NMR spectrum of the biofluid mixture with specially constructed libraries containing reference spectra of ~500 pure compounds. Tests using a variety of synthetic and real spectra of compound mixtures showed that MetaboMiner is able to identify >80% of detectable metabolites from good quality NMR spectra.</p> <p>Conclusion</p> <p>MetaboMiner is a freely available, easy-to-use, NMR-based metabolomics tool that facilitates automatic peak processing, rapid compound identification, and facile spectrum annotation from either 2D TOCSY or HSQC spectra. Using comprehensive reference libraries coupled with robust algorithms for peak matching and compound identification, the program greatly simplifies the process of metabolite identification in complex 2D NMR spectra.</p

The compound (3-{5-[(2,5-dimethoxyphenyl)amino]-1,3,4-thiadiazolidin-2-yl}-5,8-methoxy-2H-chromen-2-one) inhibits the prion protein conversion from PrPC to PrPSc with lower IC50 in ScN2a cells

Published ArticlePrion diseases are fatal neurodegenerative disorders of the central nervous system characterized by the accumulation of a protease resistant form (PrPSc) of the cellular prion protein (PrPC) in the brain. Two types of cellular prion (PrPC) compounds have been identified that appear to affect prion conversion are known as Effective Binders (EBs) and Accelerators (ACCs). Effective binders shift the balance in favour of PrPC, whereas Accelerators favour the formation of PrPSc. Molecular docking indicates EBs and ACCs both bind to pocket-D of the SHaPrPC molecule. However, EBs and ACCs may have opposing effects on the stability of the salt bridge between Arg156 and Glu196/Glu200. Computational docking data indicate that the hydrophobic benzamide group of the EB, GFP23 and the 1-(3,3-dimethylcyclohexylidene)piperidinium group of the ACC, GFP22 play an important role in inhibition and conversion from SHaPrPC to SHaPrPSc, respectively. Experimentally, NMR confirmed the amide chemical shift perturbations observed upon the binding of GFP23 to pocket-D of SHaPrPC. Consistent with its role as an ACC, titration of GFP22 resulted in widespread chemical shift changes and signal intensity loss due to protein unfolding. Virtual screening of a ligand database using the molecular scaffold developed from the set of EBs identified six of our compounds (previously studied using fluorescence quenching) as being among the top 100 best binders. Among them, compounds 5 and 6 were found to be particularly potent in decreasing the accumulation SHaPrPSc in ScN2a cells with an IC50 of 35 mM and 20 mM

The compound (3-{5-[(2,5-dimethoxyphenyl)amino]-1,3,4-thiadiazolidin-2-yl}-5,8-methoxy-2H-chromen-2-one) inhibits the prion protein conversion from PrPC to PrPSc with lower IC50 in ScN2a cells

Published ArticlePrion diseases are fatal neurodegenerative disorders of the central nervous system characterized by the accumulation of a protease resistant form (PrPSc) of the cellular prion protein (PrPC) in the brain. Two types of cellular prion (PrPC) compounds have been identified that appear to affect prion conversion are known as Effective Binders (EBs) and Accelerators (ACCs). Effective binders shift the balance in favour of PrPC, whereas Accelerators favour the formation of PrPSc. Molecular docking indicates EBs and ACCs both bind to pocket-D of the SHaPrPC molecule. However, EBs and ACCs may have opposing effects on the stability of the salt bridge between Arg156 and Glu196/Glu200. Computational docking data indicate that the hydrophobic benzamide group of the EB, GFP23 and the 1-(3,3-dimethylcyclohexylidene)piperidinium group of the ACC, GFP22 play an important role in inhibition and conversion from SHaPrPC to SHaPrPSc, respectively. Experimentally, NMR confirmed the amide chemical shift perturbations observed upon the binding of GFP23 to pocket-D of SHaPrPC. Consistent with its role as an ACC, titration of GFP22 resulted in widespread chemical shift changes and signal intensity loss due to protein unfolding. Virtual screening of a ligand database using the molecular scaffold developed from the set of EBs identified six of our compounds (previously studied using fluorescence quenching) as being among the top 100 best binders. Among them, compounds 5 and 6 were found to be particularly potent in decreasing the accumulation SHaPrPSc in ScN2a cells with an IC50 of 35 mM and 20 mM

Analisis Penerapan Sistem Akuntansi Penjualan Kredit Dan Penerimaan Kas Dalam Mendukung Pengendalian Intern Perusahaan (Studi Kasus PT. Smart Tbk Refinery Surabaya)

System of selling credit accounting and system of cash receiving from account receivable is the source of life to achieving company goals. This research on the system of credit sales and cash receipts to support the company internal control. This research was conducted at PT. SMART Tbk Refinery Surabaya. PT. SMART Tbk Refinery Surabaya only selling cooking oil in the form of branded product and trading product on credit. PT. SMART Tbk Refinery Surabaya still has any weakness on system of selling credit accounting and system of cash receiving from account receivable, some of the sales transaction activity that occurred less supportive of the company\u27s internal control. This study aims to provide information to companies about the advantages and weakness of credit sales accounting system and cash receipts that have been applied by the company

The Human Urine Metabolome

Urine has long been a “favored” biofluid among metabolomics researchers. It is sterile, easy-to-obtain in large volumes, largely free from interfering proteins or lipids and chemically complex. However, this chemical complexity has also made urine a particularly difficult substrate to fully understand. As a biological waste material, urine typically contains metabolic breakdown products from a wide range of foods, drinks, drugs, environmental contaminants, endogenous waste metabolites and bacterial by-products. Many of these compounds are poorly characterized and poorly understood. In an effort to improve our understanding of this biofluid we have undertaken a comprehensive, quantitative, metabolome-wide characterization of human urine. This involved both computer-aided literature mining and comprehensive, quantitative experimental assessment/validation. The experimental portion employed NMR spectroscopy, gas chromatography mass spectrometry (GC-MS), direct flow injection mass spectrometry (DFI/LC-MS/MS), inductively coupled plasma mass spectrometry (ICP-MS) and high performance liquid chromatography (HPLC) experiments performed on multiple human urine samples. This multi-platform metabolomic analysis allowed us to identify 445 and quantify 378 unique urine metabolites or metabolite species. The different analytical platforms were able to identify (quantify) a total of: 209 (209) by NMR, 179 (85) by GC-MS, 127 (127) by DFI/LC-MS/MS, 40 (40) by ICP-MS and 10 (10) by HPLC. Our use of multiple metabolomics platforms and technologies allowed us to identify several previously unknown urine metabolites and to substantially enhance the level of metabolome coverage. It also allowed us to critically assess the relative strengths and weaknesses of different platforms or technologies. The literature review led to the identification and annotation of another 2206 urinary compounds and was used to help guide the subsequent experimental studies. An online database containing the complete set of 2651 confirmed human urine metabolite species, their structures (3079 in total), concentrations, related literature references and links to their known disease associations are freely available at http://www.urinemetabolome.ca

Microbiome and metabolome modifying effects of several cardiovascular disease interventions in apo-E-/- mice

Background: There is strong evidence indicating that gut microbiota have the potential to modify, or be modified by the drugs and nutritional interventions that we rely upon. This study aims to characterize the compositional and functional effects of several nutritional, neutraceutical, and pharmaceutical cardiovascular disease interventions on the gut microbiome, through metagenomic and metabolomic approaches. Apolipoprotein-E-deficient mice were fed for 24 weeks either high-fat/cholesterol diet alone (control, HFC) or high-fat/cholesterol in conjunction with one of three dietary interventions, as follows: plant sterol ester (PSE), oat β-glucan (OBG) and bile salt hydrolase-active Lactobacillus reuteri APC 2587 (BSH), or the drug atorvastatin (STAT). The gut microbiome composition was then investigated, in addition to the host fecal and serum metabolome. Results: We observed major shifts in the composition of the gut microbiome of PSE mice, while OBG and BSH mice displayed more modest fluctuations, and STAT showed relatively few alterations. Interestingly, these compositional effects imparted by PSE were coupled with an increase in acetate and reduction in isovalerate (p < 0.05), while OBG promoted n-butyrate synthesis (p < 0.01). In addition, PSE significantly dampened the microbial production of the proatherogenic precursor compound, trimethylamine (p < 0.05), attenuated cholesterol accumulation, and nearly abolished atherogenesis in the model (p < 0.05). However, PSE supplementation produced the heaviest mice with the greatest degree of adiposity (p < 0.05). Finally, PSE, OBG, and STAT all appeared to have considerable impact on the host serum metabolome, including alterations in several acylcarnitines previously associated with a state of metabolic dysfunction (p < 0.05). Conclusions: We observed functional alterations in microbial and host-derived metabolites, which may have important implications for systemic metabolic health, suggesting that cardiovascular disease interventions may have a significant impact on the microbiome composition and functionality. This study indicates that the gut microbiome-modifying effects of novel therapeutics should be considered, in addition to the direct host effects

Molecular docking of thiamine reveals similarity in binding properties between the prion protein and other thiamine-binding proteins

Prion-induced diseases are a global health concern. The lack of effective therapy and 100 % mortality rates for such diseases have made the prion protein an important target for drug discovery. Previous NMR experimental work revealed that thiamine and its derivatives bind the prion protein in a pocket near the N-terminal loop of helix 1, and conserved intermolecular interactions were noted between thiamine and other thiamine-binding proteins. Furthermore, water-mediated interactions were observed in all of the X-ray crystallographic structures of thiamine-binding proteins, but were not observed in the thiamine-prion NMR study. To better understand the potential role of water in thiamine-prion binding, a docking study was employed using structural X-ray solvent. Before energy minimization, docked thiamine assumed a "V" shape similar to some of the known thiamine-dependent proteins. Following minimization with NMR-derived restraints, the "F" conformation was observed. Our findings confirmed that water is involved in ligand stabilization and phosphate group interaction. The resulting refined structure of thiamine bound to the prion protein allowed the 4-aminopyrimidine ring of thiamine to \u3c0-stack with Tyr150, and facilitated hydrogen bonding between Asp147 and the amino group of 4-aminopyrimidine. Investigation of the \u3c0-stacking interaction through mutation of the tyrosine residue further revealed its importance in ligand placement. The resulting refined structure is in good agreement with previous experimental restraints, and is consistent with the pharmacophore model of thiamine-binding proteins. \ua9 2013 Springer-Verlag Berlin Heidelberg.Peer reviewed: YesNRC publication: Ye

Detailed biophysical characterization of the acid-induced PrPc to PrP\u3b2 conversion process

Prions are believed to spontaneously convert from a native, monomeric highly helical form (called PrP c) to a largely \u3b2-sheet-rich, multimeric and insoluble aggregate (called PrP sc). Because of its large size and insolubility, biophysical characterization of PrP sc has been difficult, and there are several contradictory or incomplete models of the PrP sc structure. A \u3b2-sheet-rich, soluble intermediate, called PrP \u3b2, exhibits many of the same features as PrP sc and can be generated using a combination of low pH and/or mild denaturing conditions. Studies of the PrP c to PrP \u3b2 conversion process and of PrP \u3b2 folding intermediates may provide insights into the structure of PrP sc. Using a truncated, recombinant version of Syrian hamster PrP \u3b2 (shPrP(90-232)), we used NMR spectroscopy, in combination with other biophysical techniques (circular dichroism, dynamic light scattering, electron microscopy, fluorescence spectroscopy, mass spectrometry, and proteinase K digestion), to characterize the pH-driven PrP c to PrP \u3b2 conversion process in detail. Our results show that below pH 2.8 the protein oligomerizes and conversion to the \u3b2-rich structure is initiated. At pH 1.7 and above, the oligomeric protein can recover its native monomeric state through dialysis to pH 5.2. However, when conversion is completed at pH 1.0, the large oligomer "locks down" irreversibly into a stable, \u3b2-rich form. At pH values above 3.0, the protein is amenable to NMR investigation. Chemical shift perturbations, NOE, amide line width, and T 2 measurements implicate the putative "amylome motif" region, "NNQNNF" as the region most involved in the initial helix-to-\u3b2 conversion phase. We also found that acid-induced PrP \u3b2 oligomers could be converted to fibrils without the use of chaotropic denaturants. The latter finding represents one of the first examples wherein physiologically accessible conditions (i.e., only low pH) were used to achieve PrP conversion and fibril formation. \ua9 2010 American Chemical Society.Peer reviewed: YesNRC publication: Ye