NMR-based leaf metabolic profiling of V. planifolia and three endemic Vanilla species from the Peruvian Amazon.

The fruit of Vanilla planifolia is broadly preferred by the agroindustry and gourmet markets due to its refined flavor and aroma. Peruvian Vanilla has been proposed as a possible source for genetic improvement of existing Vanilla cultivars, but, little has been done to facilitate comprehensive studies of these and other Vanilla. Here, a nuclear magnetic resonance (NMR) metabolomic platform was developed to profile for the first time the leaves - organ known to accumulate vanillin putative precursors - of V. planifolia and those of Peruvian V. pompona, V. palmarum, and V. ribeiroi, with the aim to determine metabolic differences among them. Analysis of the NMR spectra allowed the identification of thirty-six metabolites, twenty-five of which were quantified. One-way ANOVA and post-hoc Tukey test revealed that these metabolites changed significantly among species, whilst multivariate-analyses allowed the identification of malic and homocitric acids, together with two vanillin precursors, as relevant metabolic markers for species differentiation.

Guidelines for correlation coefficient threshold settings in metabolite correlation networks exemplified on a potato association panel.

BACKGROUND: Correlation network analysis has become an integral tool to study metabolite datasets. Networks are constructed by omitting correlations between metabolites based on two thresholds-namely the r and the associated p-values. While p-value threshold settings follow the rules of multiple hypotheses testing correction, guidelines for r-value threshold settings have not been defined. RESULTS: Here, we introduce a method that allows determining the r-value threshold based on an iterative approach, where different networks are constructed and their network topology is monitored. Once the network topology changes significantly, the threshold is set to the corresponding correlation coefficient value. The approach was exemplified on: (i) a metabolite and morphological trait dataset from a potato association panel, which was grown under normal irrigation and water recovery conditions; and validated (ii) on a metabolite dataset of hearts of fed and fasted mice. For the potato normal irrigation correlation network a threshold of Pearson's |r|≥ 0.23 was suggested, while for the water recovery correlation network a threshold of Pearson's |r|≥ 0.41 was estimated. For both mice networks the threshold was calculated with Pearson's |r|≥ 0.84. CONCLUSIONS: Our analysis corrected the previously stated Pearson's correlation coefficient threshold from 0.4 to 0.41 in the water recovery network and from 0.4 to 0.23 for the normal irrigation network. Furthermore, the proposed method suggested a correlation threshold of 0.84 for both mice networks rather than a threshold of 0.7 as applied earlier. We demonstrate that the proposed approach is a valuable tool for constructing biological meaningful networks.

Assessment of the Presence of Lipophilic Phycotoxins in Scallops (Argopecten purpuratus) Farmed along Peruvian Coastal Waters.

ABSTRACT: Some harmful algal blooms produce lipophilic marine biotoxins (LMTs) such as okadaic acid (OA; and its analogs dinophysistoxins [DTXs]), yessotoxins (YTXs), pectenotoxins (PTXs), and azaspiracids (AZAs), all of which may accumulate in filter-feeding bivalve mollusks. European health regulations stipulate a limit of 160 µg/kg for OA or DTXs, PTXs, and AZAs and 3.75 mg/kg for YTXs. Argopecten purpuratus is a valuable commercial marine bivalve exploited in Peru. Despite its importance and the periodic reports of the presence of harmful algal blooms in Peruvian coastal waters, information regarding potential contamination of these scallops by LMTs is lacking. We evaluated LMTs in 115 samples of A. purpuratus collected between November 2013 and March 2015 from 18 production areas distributed along the Peruvian coast. The hepatopancreas, which accumulates most of the toxins in the scallop, was analyzed with liquid chromatography-tandem mass spectrometry to quantify OA in its free form, YTX, AZA-1, and PTX-2. Baseline separation was achieved in 19 min. Linearity (R2 > 0.997), precision (coefficient of variation < 15%), and limits of quantification (0.155 to 0.479 ng/mL) were satisfactory. YTX was found in 72 samples, and PTX-2 was found in 17 samples, but concentrations of both biotoxins were below the regulatory limits. Free OA and AZA-1 were not detected in the scallop samples. This atypical profile (i.e., presence of PTX-2 and absence of OA) may be linked to the presence of the dinoflagellate Dinophysis acuminata. The production of YTX could be associated with the phytoplankton Gonyaulax spinifera and Protoceratium reticulatum. This is the first systematic assessment of the four types of LMTs in shellfish from Peruvian coastal waters. The results suggest low prevalence of LMTs in Peruvian bay scallops but support continued surveillance and analysis of LMTs in Peru.

Morphological and metabolic profiling of a tropical-adapted potato association panel subjected to water recovery treatment reveals new insights into plant vigor.

Potato (Solanum tuberosum L.) is one of the world's most important crops, but it is facing major challenges due to climatic changes. To investigate the effects of intermittent drought on the natural variability of plant morphology and tuber metabolism in a novel potato association panel comprising 258 varieties we performed an augmented block design field study under normal irrigation and under water-deficit and recovery conditions in Ica, Peru. All potato genotypes were profiled for 45 morphological traits and 42 central metabolites via nuclear magnetic resonance. Statistical tests and norm of reaction analysis revealed that the observed variations were trait specific, that is, genotypic versus environmental. Principal component analysis showed a separation of samples as a result of conditional changes. To explore the relational ties between morphological traits and metabolites, correlation-based network analysis was employed, constructing one network for normal irrigation and one network for water-recovery samples. Community detection and difference network analysis highlighted the differences between the two networks, revealing a significant correlational link between fumarate and plant vigor. A genome-wide association study was performed for each metabolic trait. Eleven single nucleotide polymorphism (SNP) markers were associated with fumarate. Gene Ontology analysis of quantitative trait loci regions associated with fumarate revealed an enrichment of genes regulating metabolic processes. Three of the 11 SNPs were located within genes, coding for a protein of unknown function, a RING domain protein and a zinc finger protein ZAT2. Our findings have important implications for future potato breeding regimes, especially in countries suffering from climate change.

Ultra-Clean Pure Shift 1H-NMR applied to metabolomics profiling.

Even though Pure Shift NMR methods have conveniently been used in the assessment of crowded spectra, they are not commonly applied to the analysis of metabolomics data. This paper exploits the recently published SAPPHIRE-PSYCHE methodology in the context of plant metabolome. We compare single pulse, PSYCHE, and SAPPHIRE-PSYCHE spectra obtained from aqueous extracts of Physalis peruviana fruits. STOCSY analysis with simplified SAPPHIRE-PSYCHE spectra of six types of Cape gooseberry was carried out and the results attained compared with classical STOCSY data. PLS coefficients analysis combined with 1D-STOCSY was performed in an effort to simplify biomarker identification. Several of the most compromised proton NMR signals associated with critical constituents of the plant mixture, such as amino acids, organic acids, and sugars, were more cleanly depicted and their inter and intra correlation better reveled by the Pure Shift methods. The simplified data allowed the identification of glutamic acid, a metabolite not observed in previous studies of Cape gooseberry due to heavy overlap of its NMR signals. Overall, the results attained indicated that Ultra-Clean Pure Shift spectra increase the performance of metabolomics data analysis such as STOCSY and multivariate coefficients analysis, and therefore represent a feasible and convenient additional tool available to metabolomics.

Backbone chemical shift assignment of macrophage infectivity potentiator virulence factor of Trypanosoma cruzi.

Chagas disease is a trypanosomiasis disease inflicted by Trypanosoma cruzi parasite. In Latin America, at least 10 million people are infected and annually, 10,000 casualties are deplored. Macrophage infectivity potentiator protein is one of the major virulence factors secreted by T. cruzi (TcMIP) in order to infect its host but little is known about its mechanism of action. Studies confer TcMIP an important role in the extracellular matrix transmigration and basal lamina penetration. Here, we report the backbone 1H, 13C, and 15N resonance assignment of TcMIP and the comparison of the secondary structure obtained against reported X-ray crystallography data.

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

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.

Validated HPLC-Diode Array Detector Method for Simultaneous Evaluation of Six Quality Markers in Coffee.

Nicotinic acid, N-methylpyridinium ion, and trigonelline are well studied nutritional biomarkers present in coffee, and they are indicators of thermal decomposition during roasting. However, no method is yet available for their simultaneous determination. This paper describes a rapid and validated HPLC-diode array detector method for the simultaneous quantitation of caffeine, trigonelline, nicotinic acid, N-methylpyridinium ion, 5-caffeoylquinic acid, and 5-hydroxymethyl furfural that is applicable to three coffee matrixes: green, roasted, and instant. Baseline separation among all compounds was achieved in 30 min using a phenyl-hexyl RP column (250×4.6 mm, 5 µm particle size), 0.3% aqueous formic buffer (pH 2.4)-methanol mobile phase at a flow rate of 1 mL/min, and a column temperature at 30°C. The method showed good linear correlation (r2>0.9985), precision (less than 3.9%), sensitivity (LOD=0.023-0.237 µg/mL; LOQ=0.069-0.711 µg/mL), and recovery (84-102%) for all compounds. This simplified method is amenable for a more complete routine evaluation of coffee in industry.

Exploration of Vanilla pompona from the Peruvian Amazon as a potential source of vanilla essence: quantification of phenolics by HPLC-DAD.

This study provides the first chemical investigation of wild-harvested fruits of Vanilla pompona ssp. grandiflora (Lindl.) Soto-Arenas developed in their natural habitat in the Peruvian Amazon. Flowers were hand-pollinated and the resulting fruits were analysed at different developmental stages using an HPLC-DAD method validated for the quantification of glucovanillin and seven other compounds. The method showed satisfactory linearity (r(2)>0.9969), precision (coefficient of variation <2%), recoveries (70-100%), limit of detection (0.008-0.212 µg/ml), and limit of quantification (0.027-0.707 µg/ml). The evaluation of crude and enzyme-hydrolyzed Soxhlet-extracted samples confirmed the leading role of glucosides in fruit development. LC-ESI-MS studies corroborated the identities of four glucosides and seven aglycones, among them vanillin (5.7/100 g), 4-hydroxybenzyl alcohol (3.6/100 g), and anisyl alcohol (7.1/100 g) were found in high concentrations. The attractive flavor/aroma profile exhibited by wild V. pompona fruits supports studies focused on the development of this species as a specialty crop.

The use of natural product scaffolds as leads in the search for trypanothione reductase inhibitors.

Twenty-three heterocyclic compounds were evaluated for their potential as trypanothione reductase inhibitors. As a result, the harmaline, 10-thiaisoalloxazine, and aspidospermine frameworks were identified as the basis of inhibitors of Trypanosoma cruzi trypanothione reductase. Two new compounds showed moderately strong, linear competitive inhibition, namely N,N-dimethyl-N-[3-(7-methoxy-1-methyl-3,4-dihydro-9H-beta-carbolin-9-yl)propyl]amine (15) and 1,3-bis[3-(dimethylamino)propyl]-1,5-dihydro-2H-pyrimido[4,5-b][1,4]benzothiazine-2,4(3H)-dione (21), with K(i) values of 35.1+/-3.5microM and 26.9+/-1.9microM, respectively. Aspidospermine (25) inhibited T. cruzi TryR with a K(i) of 64.6+/-6.2microM. None of the compounds inhibited glutathione reductase. Their toxicity toward promastigotes of Leishmania amazonensis was assessed.