Distinguishing transgenic from non-transgenic Arabidopsis plants by 1H NMR-based metabolic fingerprinting

We have recently reported the construction of an nuclear magnetic resonance （NMR）-based metabonomics study platform, Automics. To examine the application of Automics in transgenic plants, we performed metabolic fingerprinting analysis, i.e., 1H NMR spectroscopy and multivariate analysis, on wild-type and transgenic Arabidopsis. We found that it was possible to distinguish wild-type from four transgenic plants by PLS-DA following application of orthogonal signal correction （OSC）. Scores plot following OSC clearly demonstrates significant variation between the transgenic and non-transgenic groups, suggesting that the metabolic changes among wild-type and transgenic lines are possibly associated with transgenic event, We also found that the major contributing metabolites were some specific amino acids （i.e., threonine and alanine）, which could correspond to the insertion of the selective marker BAR gene in the transgenic plants. Our data suggests that NMR-based metabonomics is an efficient method to distinguish fingerprinting difference between wild-type and transgenic plants, and can potentially be applied in the bio-safety assessment of transgenic plants.     

Intra- and inter-metabolite correlation spectroscopy of tomato metabolomics data obtained by liquid chromatography-mass spectrometry and nuclear magnetic resonance

Nuclear magnetic resonance (NMR) and liquid chromatography-mass spectrometry (LCMS) are frequently used as technological platforms for metabolomics applications. In this study, the metabolic profiles of ripe fruits from 50 different tomato cultivars, including beef, cherry and round types, were recorded by both ¹H NMR and accurate mass LC-quadrupole time-of-flight (QTOF) MS. Different analytical selectivities were found for these both profiling techniques. In fact, NMR and LCMS provided complementary data, as the metabolites detected belong to essentially different metabolic pathways. Yet, upon unsupervised multivariate analysis, both NMR and LCMS datasets revealed a clear segregation of, on the one hand, the cherry tomatoes and, on the other hand, the beef and round tomatoes. Intra-method (NMR–NMR, LCMS–LCMS) and inter-method (NMR–LCMS) correlation analyses were performed enabling the annotation of metabolites from highly correlating metabolite signals. Signals belonging to the same metabolite or to chemically related metabolites are among the highest correlations found. Inter-method correlation analysis produced highly informative and complementary information for the identification of metabolites, even in de case of low abundant NMR signals. The applied approach appears to be a promising strategy in extending the analytical capacities of these metabolomics techniques with regard to the discovery and identification of biomarkers and yet unknown metabolites. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Characterization of insoluble calcium alginates by solid-state NMR

Three series of 9 insoluble calcium alginate powders with different average calcium contents (1.5, 3.5 and 8%, w/w) are investigated by means of ¹³C solid-state NMR spectroscopy. The effect of the increased calcium content on the determination of the mannuronate (M) to guluronate (G) ratio from spectral deconvolution of the ¹³C CP/MAS spectra is discussed, and the variations observed are commented in function of possible structural modifications related to the interaction with the divalent cations. The possibility of using solid-state NMR spectroscopy for the quantification of the calcium content in unknown alginate samples is explored performing principal component analysis (PCA) of the spectra. The results obtained show that a clear separation of alginates with slightly different calcium content is possible. The proposed method relies on the sole use of the chemical shifts of the signals corresponding to pyranose carbons, suggesting that PCA of solid-state NMR data holds promises as a rapid and undestructive method for screening the calcium content of alginate-based materials with biomedical uses.     

Phenotypic biomonitoring using multivariate flow cytometric analysis of multi-stained microorganisms

A new method for monitoring phenotypic profiles of pure cultures and complex microbial communities was evaluated. The approach was to stain microorganisms with a battery of fluorescent dyes prior to flow cytometry analysis (FCM) and to analyse the data using multivariate methods, including principal component analysis and partial least squares. The FCM method was quantitatively evaluated using different mixtures of pure cultures as well as microbial communities. The results showed that the method could quantitatively and reproducibly resolve both populations and communities of microorganisms with 5% abundance in a diverse microbial background. The feasibility of monitoring complex microbial communities over time during the biodegradation of naphthalene using the FCM method was demonstrated. The biodegradation of naphthalene occurred to differing extents in microcosms representing three different types of aromatic-contaminated groundwater and a sample of bio-basin water. The FCM method distinguished each of these four microbial communities. The phenotypic profiles were compared with genotypic profiles generated by random-amplified polymorphic DNA analysis. The genotypic profiles of the microbial communities described only the microbial composition, and not their functional change, whereas the phenotypic profiles seemed to contain information on both the composition and the functional change of the microorganisms. Furthermore, event analysis of the FCM data showed that microbial communities with initially differing compositions could converge towards a similar composition if they had a capacity for high levels of degradation, whereas microbial communities with similar initial compositions could diverge if they differed in biodegrading ability.     

Evaluation of circulant partial diallel crosses in maize

Summary The present study was conducted in maize (Zea mays L.) on crosses among 20 diverse parents. The materials were evaluated in four different environments for eight characters. Combining ability analysis was carried out following diallel and partial diallel crosses. The number of crosses per parent (s) varied from 3 to 19 and the results were studied to identify the critical value of s that would provide an adequate information with minimum resources. The S₅ partial diallel was as good as the S₁₉ for the detection of differences among general combining ability (GCA) effects. Even the S₃ gave adequate information in the case of characters with high heritability. However, partial diallel analysis was less efficient in detecting the differences due to specific combining ability (SCA) effects. These results varied with environments, and characters with low heritability were more prone to misinterpretation. GCA effects showed fluctuations in partial diallel analysis which were more pronounced in S₅ and S₃, particularly for characters with low heritability. The average standard error of difference between GCA effects increased with a decrease in s, with a steep slope for s < 7. The partial diallel analysis was more efficient for the estimation of the variance component of GCA than for SCA, as the estimate of SCA was biased upwards. Estimates of broad sense heritability obtained from the partial diallels agreed with the full diallel analysis better than the narrow sense estimates. Smaller partial diallels gave erratic estimates of heritability, particularly for the characters with low heritability.     

Direct NMR analysis of cannabis water extracts and tinctures and semi-quantitative data on delta9-THC and delta9-THC-acid

Cannabis sativa L. is the source for a whole series of chemically diverse bioactive compounds that are currently under intensive pharmaceutical investigation. In this work, hot and cold water extracts as well as ethanol/water mixtures (tinctures) of cannabis were compared in order to better understand how these extracts differ in their overall composition. NMR analysis and in vitro cell assays of crude extracts and fractions were performed. Manufacturing procedures to produce natural remedies can strongly affect the final composition of the herbal medicines. Temperature and polarity of the solvents used for the extraction resulted to be two factors that affect the total amount of Delta(9)-THC in the extracts and its relative quantity with respect to Delta(9)-THC-acid and other metabolites. Diffusion-edited (1)H NMR (1D DOSY) and (1)H NMR with suppression of the ethanol and water signals were used. With this method it was possible, without any evaporation or separation step, to distinguish between tinctures from different cannabis cultivars. This approach is proposed as a direct analysis of plant tinctures.     

Relationship of restriction fragment length polymorphisms to single-cross hybrid performance of maize

Summary Isozymes and restriction fragment length polymorphisms (RFLPs) have been proposed for use in varietal identification and selection for agronomic traits. Although the use of isozymes for these purposes has been well documented, evaluation of the efficacy of RFLP technology as applied to crop improvement is far from complete. This investigation was conducted to study the relationship between RFLP-derived genotypes and heterotic patterns of a group of maize (Zea mays L.) inbred lines. A total of 22 inbreds was crossed to four testers (B73, B76, Mo17, and Va26) in combinations that minimized crossing within heterotic groups. Forty-seven single-cross progeny were subsequently evaluated for several agronomic traits (including grain yield and moisture, ear height, and root lodging) over 2–4 consecutive years at two to four Iowa locations in a randomized complete-block design. The inbred lines were subjected to RFLP analysis, which involved 47 genomic clones and the restriction enzymes EcoRI and HindIII. Hybrid RFLP patterns were predicted from their inbred parents. Modified Roger's distances were computed to estimate genetic distance among the inbred lines. Principal component analysis facilitated ascertainment of relative dispersion of the inbreds based on the frequency of variants at specific RFLP loci. Evident associations of variants with genes affecting agronomic traits were identified by principal component regression analysis, in which adjusted hybrid means were regressed on the matrix of hybrid variants frequencies. The hybrid means were adjusted by removing environmental effects, using residuals as dependent variables in the regression analysis. Results from this study suggest that RFLP analysis may be of value in allocating maize inbreds to heterotic groups, but no relationship between RFLP-based genetic distance and hybrid performance was apparent. Principal component regression identified variants potentially linked to genes that control specific agronomic traits.Joint contribution: USDA-ARS and Journal Paper No. J-13590 of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA 50011, USA. Projects No. 2818 and 2778     

Water diffusion in cytoplasmic streaming in Elodea internodal cells under the effect of antimitotic agents

The translational displacement of the cytoplasmic water in Elodea stem cells resulting from protein motor activity was measured using the NMR method. A 24-h treatment with vincristine results in a reduction of the translational displacement of the cytoplasmic water. With a constant cytoplasmic streaming velocity, the dynamics of the translational displacement of the cytoplasmic water under the effect of taxol are characterized by a continuous increase at a concentration of 0.05 mM, and reaching a plateau at a concentration of 0.5 mM.     

Discrimination of Ipomoea aquatica cultivars and bioactivity correlations using NMR-based metabolomics approach

Ipomoea aquatica Forsk is a green leafy vegetable that is a rich source of minerals, proteins, vitamins, amino acids, and secondary metabolites. Different types of I. aquatica cultivars are grown for consumption but little is known about the metabolites variation. Proton nuclear magnetic resonance (¹H NMR) spectroscopy combined with multivariate data analysis was applied for metabolic profiling of three I. aquatica cultivars including “broad leaf (K-25)”, “bamboo leaf (K-88)”, and “special pointed leaf (K-11)”. The orthogonal partial least squares discriminant analysis (OPLS-DA) indicated a clear separation among cultivars. The relative levels of various compounds, such as amino acids, organic acids, sugars, and phenolic compounds were specific to each cultivar. The K-11 cultivar was different from the other cultivars due to a high phenolic content. The content of sugars and some amino acids was higher in K-88 and K-25 possessed a higher content of organic acids. The in vitro study revealed that the I. aquatica cultivars exhibited potent antioxidant and α-glucosidase activities. The results of this study indicate that the K-11 cultivar was the most active due to the abundance of epicatechin, 4,5-dicaffeoylquinic, protocatechuic acid, and rutin.     

Solid state NMR studies on the structural and conformational properties of natural maize starches

Natural maize starches having a range of amylose contents have been characterised by CP/MAS NMR spectroscopy. Chemical shifts, relative resonance intensities, line-widths and spectral shapes were compared at different moisture contents. At 10% moisture content, these parameters showed few significant differences across a range of apparent amylose levels from 0 to 84%. After hydration of the granules to ≈30% moisture, it was found that the amylose content significantly affected the relative signal intensities and line-widths especially of C-1 and C-4 resonances. Narrower line-widths after hydration were attributed to (i) an increased degree of crystallinity, and (ii) disappearance of the signals of amorphous material which, on becoming more mobile, became invisible to the CP/MAS experiment. The enhanced resolution at higher moisture levels revealed signals which were assigned to the amylose–lipid complex, i.e. V-type amylose. The amount of V-amylose detected by NMR increased with both amylose content and lipid content of the granule. Prolonged treatment of the granules with iodine vapour significantly increased the amount of V-type amylose in the high amylose samples, but caused a decrease in their degree of crystallinity. Waxy-maize starch was barely affected by iodination. The results provide evidence that amylose tends to disrupt the structural order within amylopectin crystallities. This effect is enhanced by the formation of the amylose–iodine complex, indicating that V-amylose could be a major crystallite-disrupting agent in native starch granules.     

Structural study of copper(I)-bleomycin

Previous NMR studies on Cu(I)-bleomycin have suggested that this adduct has a geometry distinct from Fe(II)BLM. The coordination chemistry of this bleomycin derivative has been investigated through the extension of the NMR data reported previously, and the use of molecular dynamics calculations. The data collected from the NMR experiments support the coordination to the metal center of the primary and secondary amines in beta-aminoalanine and the pyrimidine ring. The detection in the NMR spectra of the signal derived from the amide hydrogen in beta-hydroxyhistidine indicates that this amide is protonated in Cu(I)-bleomycin, precluding participation of the pyrimidinyl carboxamide nitrogen in the coordination of Cu(I), as previously reported. Three-dimensional solution structures compatible with the NMR data have been assayed for Cu(I)-bleomycin for the first time by way of molecular dynamics calculations, and two models showing four and five coordination have been found to be those that better fit the experimental data. In both models the primary amine in beta-aminoalanine is coordinated such that it is located on the same side, with respect to the coordination cage, as the peptide linker fragment. This result seems important for the favored models to be compatible with either their possible oxidation to become one of the reported structures for Cu(II)BLM, or their transformation into Fe(II) adducts able to cause DNA damage.     

1H NMR and GC/MS metabolomics of earthworm responses to sub-lethal DDT and endosulfan exposure

The metabolic response of the earthworm Eisenia fetida to two pesticides, dichlorodiphenyltrichloroethane (DDT) and endosulfan, was characterized in contact tests using proton nuclear magnetic resonance (¹H NMR) and principal component analysis (PCA). PCA loading plots suggested that maltose, leucine and alanine were important metabolites contributing to the differences in dosed and control earthworms for both compounds at doses of 0.5, 1.0 and 2.0 μg/cm². Gas chromatography/mass spectrometry (GC/MS) was used to quantify the metabolites identified in E. fetida and determine if the changes in maltose, leucine and alanine following exposure to DDT and endosulfan (at 0.5 and 1.0 μg/cm²) were reproducible and greater than the natural variability. Quantification by GC/MS suggested that maltose was not a reliable biomarker since it both increased and decreased in earthworms exposed to DDT and increased by just 3% with exposure to endosulfan. Leucine was not stable with the GC/MS derivitization method used in this study and could not be confirmed as a reliable biomarker. However, alanine consistently increased for both DDT and endosulfan exposed E. fetida. Alanine showed considerable variability in control earthworms (±41.6%), yet the variability in alanine to glycine ratios was just ±10.5%. Increases in the alanine to glycine ratio were statistically significant at the P = 0.05 level for the 1.0 μg/cm² DDT dose and both the 0.5 and 1.0 μg/cm² endosulfan doses, suggesting that deviations from the normal homeostatic ratio of 1.5 for alanine to glycine is a potential biomarker of DDT and endosulfan exposure warranting further study. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Environmental Metabolomics Special Issue of Metabolomics.     

A computer-based protocol for semiautomated assignments and 3D structure determination of proteins

Summary A strategy is presented for the semiautomated assignment and 3D structure determination of proteins from heteronuclear multidimensional Nuclear Magnetic Resonance (NMR) data. This approach involves the computer-based assignment of the NMR signals, identification of distance restraints from nuclear Overhauser effects, and generation of 3D structures by using the NMR-derived restraints. The protocol is described in detail and illustrated on a resonance assignment and structure determination of the FK506 binding protein (FKBP, 107 amino acids) complexed to the immunosuppressant, ascomycin. The 3D structures produced from this automated protocol attained backbone and heavy atom rmsd of 1.17 and 1.69 Å, respectively. Although more highly resolved structures of the complex have been obtained by standard interpretation of NMR data (Meadows et al. (1993) Biochemistry, 32, 754–765), the structures generated with this automated protocol required minimal manual intervention during the spectral assignment and 3D structure calculations stages. Thus, the protocol may yield an approximate order of magnitude reduction in the time required for the generation of 3D structures of proteins from NMR data.     

Solution 1H NMR study of the accommodation of the side chain of n-butyl-etiohemin-I incorporated into the active site of cyano-metmyoglobin

In order to identify the most readily deformable portion of the heme pocket in myoglobin, equine myoglobin was reconstituted with a meso-n-butyl substituent on centrosymmetric etiohemin-I. Solution ¹H NMR data for the low-spin iron(III) cyanide complex of oxidized myoglobin that include 2D nuclear Overhauser enhancement spectroscopy contacts, paramagnetic relaxation, and dipolar shifts resulting from magnetic anisotropy show that the heme binds uniquely to the iron in a manner that arranges the methyl and ethyl substituents on a given pyrrole in a clockwise manner when viewed from the proximal side, and with the n-butyl group seated at the canonical -meso position of native protohemin-IX. The butyl group is oriented sharply toward the proximal side and its protein contacts demonstrate that it is oriented largely into the xenon hole in myoglobin. The location of the n-butyl group on the proximal side near the vacancies places it within the region found to be most flexible in molecular dynamics simulation. A small, counterclockwise rotation of the pyrrole N–Fe–N vector of n-butyl-etiohemin-I relative to that for native protohemin, indicated by both the prosthetic group methyl contact shift pattern and the prosthetic group contacts to heme pocket residues, is proposed to allow the xenon hole to accommodate better the n-butyl group. In contrast to previous work, which showed that a bulky polar substituent on etiohemin-I preferentially seats at the canonical -meso position, the nonpolar n-butyl group selects the -meso position.Electronic Supplementary Material Supplementary material is available for this article at .     

Metabolomic analysis of biofluids from rats treated with Aconitum alkaloids using nuclear magnetic resonance and gas chromatography/time-of-flight mass spectrometry

The Aconitum alkaloids aconitine, mesaconitine, and hypaconitine are the main toxic components in a commonly used traditional Chinese herbal medicine Fu Zi. To provide guidelines for the safe use of this medicine, metabolic changes in Wistar rats caused by these compounds were investigated by means of integrated analysis of two metabonomic approaches: ¹H nuclear magnetic resonance (NMR) and gas chromatography/time-of-flight mass spectrometry (GC/TOF–MS). Rats were given a single dose of aconitine, mesaconitine, hypaconitine, or vehicle. The largest metabolic changes were observed 6 h after treatment. Every group receiving a dose had higher urine concentrations of glucose, acetate, dimethylglycine, succinate, and alanine and had lower concentrations of creatinine, citrate, 2-oxoglutarate, N-acetylated metabolites, and trimethylamine-N-oxide (TMAO) than did the control group. These results may reflect the perturbation of renal tubular function within the first 24 h after treatment. The results also revealed a larger perturbation of metabolic profiles in the aconitine group than in the mesaconitine and hypaconitine groups, illustrating how these alkaloids exhibit different toxicities. An analysis of plasma samples collected 7 days postdose showed that there were higher levels of lactate, alanine, and lipids along with lower levels of glucose, β-hydroxybutyrate, and creatine in the plasma of the aconitine and mesaconitine groups than there were in the control and hypaconitine groups. The GC/TOF–MS data from the plasma samples showed that the number of metabolites, with significant changes or with a tendency to change, in the aconitine and mesaconitine groups were dissimilar, suggesting a possible difference in the acute toxicity mechanisms of these alkaloids.     

A numerical phytosociological study of the summits of the majella massive (Italy)

Summary A phytosociological study of Majella's summits by the use of multivariate methods has allowed to discover the following new associations:Gnaphalio-Plantaginetum atratae on humocarbonatic soils in snowy depressions,Leontopodio-Elynetum on rendzina in wind exposed and stony places,Saxifrago-Papaveretum julici andCrepidi-Leontodontetum montani on protorendzina with gravel.Gnaphalio-Plantaginetum atratae andLeontopodio-Elynetum might be classified inElyno-Seslerietea, although the former presents character species ofSalicetea herbaceae; Saxifrago-Papaveretum julici andCrepidi-Leontodontetum montani are classified inThlaspidion stylosi (foeder. nova) ofThlaspidietea.Comparisons between some clustering methods and between ordination methods based on the mathematics of principal component analysis are discussed. The efficiency of sum of squares agglomeration when the plant communities vary in a continuum and the importance of a linearizing transformation in principal component analysis are stressed.The research was supported by a grant from the Italian CNR to Prof. D. Lausi. We wish to thank also Prof. L. Orlóci, Dr. E. van der Maarel and Prof. S. Pignatti for reading and discussing the text, and Mr. A. Zampar, S. Sedmak and T. Ubaldini for technical assistance.