Differential elemental uptake in three pseudo-metallophyte C<Subscript>4</Subscript> grasses in situ in the eastern USA

Background and aims

Elemental uptake in serpentine floras in eastern North America is largely unknown. The objective of this study was to determine major and trace element concentrations in soil and leaves of three native pseudo-metallophyte C₄ grasses in situ at five sites with three very different soil types, including three serpentine sites, in eastern USA.

Methods

Pseudo-total and extractible concentrations of 15 elements were measured and correlated from the soils and leaves of three species at the five sites.

Results

Element concentrations in soils of pseudo-metallophytes varied up to five orders of magnitude. Soils from metalliferous sites exhibited higher concentrations of their characteristic elements than non-metalliferous. In metallicolous populations, elemental concentrations depended on the element. Concentrations of major elements (Ca, Mg, K) in leaves were lower than typical toxicity thresholds, whereas concentrations of Zn were higher.

Conclusions

In grasses, species can maintain relatively low metal concentrations in their leaves even when soil concentrations are richer. However, in highly Zn-contaminated soil, we found evidence of a threshold concentration above which Zn uptake increases drastically. Finally, absence of main characteristics of serpentine soil at one site indicated the importance of soil survey and restoration to maintain serpentinophytes communities and avoid soil encroachment.

     

Untargeted and stable isotope-assisted metabolomic analysis of MDA-MB-231 cells under hypoxia

Introduction

Hypoxia commonly occurs in cancers and is highly related with the occurrence, development and metastasis of cancer. Treatment of triple negative breast cancer remains challenge. Knowledge about the metabolic status of triple negative breast cancer cell lines in hypoxia is valuable for the understanding of molecular mechanisms of this tumor subtype to develop effective therapeutics.

Objectives

Comprehensively characterize the metabolic profiles of triple negative breast cancer cell line MDA-MB-231 in normoxia and hypoxia and the pathways involved in metabolic changes in hypoxia.

Methods

Differences in metabolic profiles affected pathways of MDA-MB-231 cells in normoxia and hypoxia were characterized using GC–MS based untargeted and stable isotope assisted metabolomic techniques.

Results

Thirty-three metabolites were significantly changed in hypoxia and nine pathways were involved. Hypoxia increased glycolysis, inhibited TCA cycle, pentose phosphate pathway and pyruvate carboxylation, while increased glutaminolysis in MDA-MB-231 cells.

Conclusion

The current results provide metabolic differences of MDA-MB-231 cells in normoxia and hypoxia conditions as well as the involved metabolic pathways, demonstrating the power of combined use of untargeted and stable isotope-assisted metabolomic methods in comprehensive metabolomic analysis.

     

The bacterial community associated with rose-scented geranium (<Emphasis Type="Italic">Pelargonium graveolens</Emphasis>) leaves responds to anthracnose symptoms

Background

The fungus Colletotrichum is a plant pathogen that causes the anthracnose disease, resulting in huge losses in various crops including the rose-scented geranium (Pelargonium graveolens). Although the bacterial community associated with plants has an important role in the establishment of plant diseases, little is known about what happens in P. graveolens.

Aims

To increase the knowledge about the bacterial community associated with P. graveolens and its relationship with anthracnose disease symptoms.

Methods

Quantitative PCR and high-throughput sequencing were combined to determine the presence of the fungus Colletotrichum and to reveal the bacterial communities associated with different plant parts – root, stem and leaf – and in the rhizosphere and bulk soil, and also to determine the respective bacterial communities associated with P. graveolens leaves symptomatic and asymptomatic for anthracnose disease.

Results

The fungus Colletotrichum was detected in all plant parts and in the surrounding soil. Bacterial communities varied spatially in plants, and the disease symptoms also influenced the composition of the bacterial community. Abundances of operational taxonomic units (OTUs) assigned to the phylum Actinobacteria and to the genus Streptococcus were greatly increased in asymptomatic leaves.

Conclusions

The bacterial community associated to geranium leaves responds to anthracnose symptoms.

     

Tartrate inhibition of prostatic acid phosphatase improves seminal fluid metabolite stability

Introduction

Human seminal fluid (hSF) has been suggested as a biofluid suitable to characterise male reproductive organ pathology with metabolomics. However, various enzymatic processes, including phosphorylcholine hydrolysis mediated by prostatic acid phosphatase (PAP), cause unwanted metabolite variation that may complicate metabolomic analysis of fresh hSF samples.

Objectives

To investigate the effects of PAP inhibition with tartrate.

Methods

Using NMR spectroscopy, the kinetics of phosphorylcholine to choline hydrolysis was characterized in hSF samples from three subjects at different temperatures and tartrate concentrations. Principal components analysis was used to characterise the effects of tartrate and temperature on personal differences in metabolite profiles. Potential effects of tartrate on RNA quantification were also determined.

Results

Metabolite profiles and the kinetics of phosphorylcholine degradation are reproducible in independent samples from three ostensibly normal subjects. Increasing concentrations of tartrate and refrigerated sample storage (279 K) resulted in greatly reduced reaction rates as judged by apparent rate constants. Multivariate statistical analysis showed that personal differences in metabolite profiles are not overshadowed by tartrate addition, which stabilises phosphorylcholine and choline concentrations. The tartrate signal also served as an internal concentration standard in the samples, allowing the determination of absolute metabolite concentrations in hSF. Furthermore, the presence of tartrate did not affect RNA expression analysis by qPCR.

Conclusion

Based on these results we recommend as standard protocol for the collection of hSF samples, that 10 mM tartrate are added immediately to samples, followed by sample storage/handling at 277 K until clinical processing within 6 h to remove/inactivate enzymes and isolate metabolite supernatant and other cellular fractions.

     

Soil microalgae modulate grain arsenic accumulation by reducing dimethylarsinic acid and enhancing nutrient uptake in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Background and aims

Microalgae are ubiquitous in paddy soils. However, their roles in arsenic (As) accumulation and transport in rice plants remains unknown.

Methods

Two green algae and five cyanobacteria were used in pot experiments under continuously flooded conditions to ascertain whether a microalgal inoculation could influence rice growth and rice grain As accumulation in plants grown in As-contaminated soils.

Results

The microalgal inoculation greatly enhanced nutrient uptake and rice growth. The presence of representative microalga Anabaena azotica did not significantly differ the grain inorganic As concentrations but remarkably decreased the rice root and grain DMA concentrations. The translocation of As from roots to grains was also markedly decreased by rice inoculated with A. azotica. This subsequently led to a decrease in the total As concentration in rice grains.

Conclusions

The results of the study indicate that the microalgal inoculation had a strong influence on soil pH, soil As speciation, and soil nutrient bioavailability, which significantly affected the rice growth, nutrient uptake, and As accumulation and translocation in rice plants. The results suggest that algae inoculation can be an effective strategy for improving nutrient uptake and reducing As translocation from roots to grains by rice grown in As-contaminated paddy soils.

     

Uncovering the metabolic response of abalone (<Emphasis Type="Italic">Haliotis midae)</Emphasis> to environmental hypoxia through metabolomics

Introduction

Oxygen is essential for metabolic processes and in the absence thereof alternative metabolic pathways are required for energy production, as seen in marine invertebrates like abalone. Even though hypoxia has been responsible for significant losses to the aquaculture industry, the overall metabolic adaptations of abalone in response to environmental hypoxia are as yet, not fully elucidated.

Objective

To use a multiplatform metabolomics approach to characterize the metabolic changes associated with energy production in abalone (Haliotis midae) when exposed to environmental hypoxia.

Methods

Metabolomics analysis of abalone adductor and foot muscle, left and right gill, hemolymph, and epipodial tissue samples were conducted using a multiplatform approach, which included untargeted NMR spectroscopy, untargeted and targeted LC–MS spectrometry, and untargeted and semi-targeted GC-MS spectrometric analyses.

Results

Increased levels of anaerobic end-products specific to marine animals were found which include alanopine, strombine, tauropine and octopine. These were accompanied by elevated lactate, succinate and arginine, of which the latter is a product of phosphoarginine breakdown in abalone. Primarily amino acid metabolism was affected, with carbohydrate and lipid metabolism assisting with anaerobic energy production to a lesser extent. Different tissues showed varied metabolic responses to hypoxia, with the largest metabolic changes in the adductor muscle.

Conclusions

From this investigation, it becomes evident that abalone have well-developed (yet understudied) metabolic mechanisms for surviving hypoxic periods. Furthermore, metabolomics serves as a powerful tool for investigating the altered metabolic processes in abalone.

     

Identification of a urine metabolite constellation characteristic for kidney allograft rejection

Introduction

Allograft rejection is still an important complication after kidney transplantation. Currently, monitoring of these patients mostly relies on the measurement of serum creatinine and clinical evaluation. The gold standard for diagnosing allograft rejection, i.e. performing a renal biopsy is invasive and expensive. So far no adequate biomarkers are available for routine use.

Objectives

We aimed to develop a urine metabolite constellation that is characteristic for acute renal allograft rejection.

Methods

NMR-Spectroscopy was applied to a training cohort of transplant recipients with and without acute rejection.

Results

We obtained a metabolite constellation of four metabolites that shows promising performance to detect renal allograft rejection in the cohorts used (AUC of 0.72 and 0.74, respectively).

Conclusion

A metabolite constellation was defined with the potential for further development of an in-vitro diagnostic test that can support physicians in their clinical assessment of a kidney transplant patient.

     

rDolphin: a GUI R package for proficient automatic profiling of 1D <Superscript>1</Superscript>H-NMR spectra of study datasets

Introduction

Adoption of automatic profiling tools for ¹H-NMR-based metabolomic studies still lags behind other approaches in the absence of the flexibility and interactivity necessary to adapt to the properties of study data sets of complex matrices.

Objectives

To provide an open source tool that fully integrates these needs and enables the reproducibility of the profiling process.

Methods

rDolphin incorporates novel techniques to optimize exploratory analysis, metabolite identification, and validation of profiling output quality.

Results

The information and quality achieved in two public datasets of complex matrices are maximized.

Conclusion

rDolphin is an open-source R package (http://github.com/danielcanueto/rDolphin) able to provide the best balance between accuracy, reproducibility and ease of use.

     

Short-term partitioning of Cd recently taken up between sunflowers organs (<Emphasis Type="Italic">Helianthus annuus</Emphasis>) at flowering and grain filling stages: effect of plant transpiration and allometry

Aims

This work concentrated on understanding the allocation of Cd recently taken up between the organs of sunflower at early and middle reproductive growth stages. The roles of transpiration and allometry were investigated.

Methods

Sunflowers were grown hydroponically in greenhouse, being exposed to low concentrations of Cd (pCd²⁺ = 11.03). At flower bud and grain filling stages, plants were exposed for three days to ¹¹¹Cd and at the same time, subjected or not to fans to increase the transpiration. The partitioning of ¹¹¹Cd between plant organs measured by high resolution ICP-MS was then modelled.

Results

Although the use of fans increased the plant water uptake and transpiration by about 20%, there were no significant effects on the partitioning of recent Cd. Most of the recent Cd was recovered in roots (60%) and only 2.8% were found in seeds (0.8% for the husk and 2.0% for the almonds). The sequestration of recent Cd in a plant organ was successfully explained by its biomass and except for leaves, by the biomass of other organs acting as competitive sinks.

Conclusions

This work proposes a modelling approach for the partitioning of the labelled Cd between plant organs in sunflower.

     

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

Introduction

Despite the use of buffering agents the ¹H 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.

Objectives

To investigate the acid, base and metal ion dependent ¹H NMR chemical shift variations and limits of the main metabolites in a complex biological mixture.

Methods

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 CaCl₂, MgCl₂, NaCl or KCl, and their ¹H NMR spectra were acquired.

Results

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⁺, Ca²⁺ and Mg²⁺, were also measured.

Conclusion

These data will be a valuable resource for ¹H NMR metabolite profiling experiments and for the development of automated metabolite alignment and identification algorithms for ¹H NMR spectra.

     

Metabolomic signatures of increases in temperature and ocean acidification from the reef-building coral, <Emphasis Type="Italic">Pocillopora damicornis</Emphasis>

Introduction

As a changing climate threatens the persistence of terrestrial and marine ecosystems by altering community composition and function, differential performance of taxa highlights the need for predictive metrics and mechanistic understanding of the factors underlying positive performance in the face of environmental disturbances. Biochemical reactions within cells provide a snapshot of molecular regulation and flexibility during exposure to environmental stressors. However, because the organism is the unit of selection there is a need for the integration of metabolite data with organism physiology to understand mechanisms responsible for individual success under a changing climate.

Objectives

Our study aims to characterize the molecular response of reef corals to simulated global climate change stressors. Furthermore, we seek to relate changes in the molecular physiology to observations in overall colony response.

Methods

To this end, we applied a non-targeted metabolomic approach to describe lipid and primary metabolite composition after exposure of the reef-building coral Pocillopora damicornis to ambient and elevated experimental climate change conditions. We compared these metabolite data to organism physiology, specifically the key processes of photosynthesis, respiration, and calcification.

Results

Corals significantly altered their lipid and primary metabolite profiles in response to experimental treatments. Primary metabolite profiles predicted organisms’ net photosynthesis, but not calcification or respiration measures. Despite challenges in metabolome annotation, our data indicated corals alter carbohydrate composition, cell structural lipids, and signaling compounds in response to elevated treatment conditions.

Conclusions

The integration of metabolite and physiological data highlights the predictive power of metabolomics in defining organism performance and provides biomarkers for future studies. Here, we present a multivariate biomarker approach to assess climate change impacts and advance our mechanistic understanding of stress response in this keystone species.

     

Quantitative analysis of tetrahydrofolate metabolites from <Emphasis Type="Italic">clostridium autoethanogenum</Emphasis>

Introduction

Quantification of tetrahydrofolates (THFs), important metabolites in the Wood–Ljungdahl pathway (WLP) of acetogens, is challenging given their sensitivity to oxygen.

Objective

To develop a simple anaerobic protocol to enable reliable THFs quantification from bioreactors.

Methods

Anaerobic cultures were mixed with anaerobic acetonitrile for extraction. Targeted LC–MS/MS was used for quantification.

Results

Tetrahydrofolates can only be quantified if sampled anaerobically. THF levels showed a strong correlation to acetyl-CoA, the end product of the WLP.

Conclusion

Our method is useful for relative quantification of THFs across different growth conditions. Absolute quantification of THFs requires the use of labelled standards.

     

Automated metabolite identification from biological fluid <Superscript>1</Superscript>H NMR spectra

Introduction

Metabolite identification in biological samples using Nuclear Magnetic Resonance (NMR) spectra is a challenging task due to the complexity of the biological matrices.

Objectives

This paper introduces a new, automated computational scheme for the identification of metabolites in 1D ¹H NMR spectra based on the Human Metabolome Database.

Methods

The methodological scheme comprises of the sequential application of preprocessing, data reduction, metabolite screening and combination selection.

Results

The proposed scheme has been tested on the 1D ¹H NMR spectra of: (a) an amino acid mixture, (b) a serum sample spiked with the amino acid mixture, (c) 20 blood serum, (d) 20 human amniotic fluid samples, (e) 160 serum samples from publicly available database. The methodological scheme was compared against widely used software tools, exhibiting good performance in terms of correct assignment of the metabolites.

Conclusions

This new robust scheme accomplishes to automatically identify peak resonances in ¹H-NMR spectra with high accuracy and less human intervention with a wide range of applications in metabolic profiling.

     

Extraction of natural products from bark of <Emphasis Type="Italic">Betula pendula</Emphasis> using ionic liquids

Introduction

Aqueous–methanol mixtures have successfully been applied to extract a broad range of metabolites from plant tissue. However, a certain amount of material remains insoluble.

Objectives

To enlarge the metabolic compendium, two ionic liquids were selected to extract the methanol insoluble part of trunk from Betula pendula.

Methods

The extracted compounds were analyzed by LC/MS and GC/MS.

Results

The results show that 1-butyl-3-methylimidazolium acetate (IL-Ac) predominantly resulted in fatty acids, whereas 1-ethyl-3-methylimidazolium tosylate (IL-Tos) mostly yielded phenolic structures. Interestingly, bark yielded more ionic liquid soluble metabolites compared to interior wood.

Conclusion

From this one can conclude that the application of ionic liquids may expand the metabolic snapshot.

     

Metabolic risks at birth of neonates exposed in utero to HIV-antiretroviral therapy relative to unexposed neonates: an NMR metabolomics study of cord blood

Introduction

Antiretroviral therapy (ART) for HIV-infected pregnant women is highly effective in preventing mother-to-child transmission (PMTCT) of the virus, but deleterious metabolic and mitochondrial observations in infants born to HIV-infected women treated with ART during pregnancy are periodically reported.

Objectives

This study addresses the concern of HIV-ART-induced metabolic perturbations through a metabolomics study of cord blood collected during transitional neonatal hypoglycaemia following birth from newborns either exposed or unexposed to fetal HIV-ART.

Methods

Proton magnetic resonance spectra from cord blood of 11 in utero HIV-ART-exposed and 14 unexposed newborns, as well as serum from 8 control infants, generated 114 spectral bins which were used to identify significant metabolites by means of univariate and multivariate statistical analyses.

Results

The metabolite profiles differed significantly between that from the unexposed newborns and that from infants—interpreted to characterize the state of transitional neonatal hypoglycaemia (low glucose and high lactic acid and ketone bodies). Quantitative analysis of potential ATP generation showed no meaningful difference in the global metabolite profiles of HIV-ART-exposed and unexposed neonates, but Volcano plot analysis, affirmed by odds ratios, indicated that exposure to HIV-ART affected the plasma 3-hydroxybutyric acid and hypoxanthine concentrations.

Conclusions

The metabolite profile for transitional neonatal hypoglycaemia indicated that HIV-ART did not compromise the exposed neonates to the energy stress of allostasis experienced at birth. Increased hypoxanthine and 3-hydroxybutyric acid indicates metabolic stress at birth in some of the newborns exposed to HIV-ART and raises a concern about unrecognized prolonged allostasis with potential neurological consequences for these infants.

     

Iridoid and phenylethanoid/phenylpropanoid metabolite profiles of <Emphasis Type="Italic">Scrophularia</Emphasis> and <Emphasis Type="Italic">Verbascum</Emphasis> species used medicinally in North America

Introduction

Botanicals containing iridoid and phenylethanoid/phenylpropanoid glycosides are used worldwide for the treatment of inflammatory musculoskeletal conditions that are primary causes of human years lived with disability, such as arthritis and lower back pain.

Objectives

We report the analysis of candidate anti-inflammatory metabolites of several endemic Scrophularia species and Verbascum thapsus used medicinally by peoples of North America.

Methods

Leaves, stems, and roots were analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) and partial least squares-discriminant analysis (PLS-DA) was performed in MetaboAnalyst 3.0 after processing the datasets in Progenesis QI.

Results

Comparison of the datasets revealed significant and differential accumulation of iridoid and phenylethanoid/phenylpropanoid glycosides in the tissues of the endemic Scrophularia species and Verbascum thapsus.

Conclusions

Our investigation identified several species of pharmacological interest as good sources for harpagoside and other important anti-inflammatory metabolites.

     

High abundance of non-mycorrhizal plant species in severely phosphorus-impoverished Brazilian <Emphasis Type="Italic">campos rupestres</Emphasis>

Background and aims

We sought to describe the species and functional composition of Brazilian campos rupestres plant communities on severely nutrient-impoverished white sands, to test hypotheses relating plant communities and physiological adaptations to infertile soils. Based on recently-published information on a south-western Australian dune chronosequence, we hypothesised that campos rupestres plant communities would similarly contain a relatively large proportion of non-mycorrhizal species, because of the phosphorus-(P) impoverished nature of the soils. We also sought to test the hypothesis that many of these non-mycorrhizal species have high leaf manganese (Mn) concentrations as a consequence of carboxylate exudation to mobilise soil P.

Methods

We conducted flora surveys and quantified mycorrhizal status and foliar Mn concentrations in field sites with strongly-weathered sandy soils. Rhizosphere carboxylates were collected from glasshouse-grown plants to assess a potential correlation of carboxylates and leaf Mn concentrations.

Results

Soils were depleted of all major plant nutrients. Non-mycorrhizal plants were abundant in most field sites (mean relative cover = 48%). Vellozia species were dominant aboveground; belowground, roots were colonised more by dark septate endophytic fungi than by mycorrhizal fungi. From the field sites, foliar Mn concentrations in non-mycorrhizal species increased with decreasing soil P concentrations, but only when soil Mn concentrations were above a minimum threshold (exchangeable [Mn] above detection limit). Across all species, however, there was no relationship of foliar Mn concentrations with soil P concentrations.

Conclusions

Our hypothesis that white-sand campos rupestres communities contain a relatively large proportion of non-mycorrhizal plants was supported. Comparison with similar ecosystems in south-western Australia suggests that plant communities on severely P-impoverished sandy soils, despite differing evolutionary histories and little overlap in plant families, follow convergent evolutionary paths towards increasing abundance of non-mycorrhizal species.

     

Removing the bottlenecks of cell culture metabolomics: fast normalization procedure,correlation of metabolites to cell number,and impact of the cell harvesting method

Introduction

Although cultured cells are nowadays regularly analyzed by metabolomics technologies, some issues in study setup and data processing are still not resolved to complete satisfaction: a suitable harvesting method for adherent cells, a fast and robust method for data normalization, and the proof that metabolite levels can be normalized to cell number.

Objectives

We intended to develop a fast method for normalization of cell culture metabolomics samples, to analyze how metabolite levels correlate with cell numbers, and to elucidate the impact of the kind of harvesting on measured metabolite profiles.

Methods

We cultured four different human cell lines and used them to develop a fluorescence-based method for DNA quantification. Further, we assessed the correlation between metabolite levels and cell numbers and focused on the impact of the harvesting method (scraping or trypsinization) on the metabolite profile.

Results

We developed a fast, sensitive and robust fluorescence-based method for DNA quantification showing excellent linear correlation between fluorescence intensities and cell numbers for all cell lines. Furthermore, 82–97 % of the measured intracellular metabolites displayed linear correlation between metabolite concentrations and cell numbers. We observed differences in amino acids, biogenic amines, and lipid levels between trypsinized and scraped cells.

Conclusion

We offer a fast, robust, and validated normalization method for cell culture metabolomics samples and demonstrate the eligibility of the normalization of metabolomics data to the cell number. We show a cell line and metabolite-specific impact of the harvesting method on metabolite concentrations.