Effects of heat-treatment on the stability and composition of metabolomic extracts from the earthworm <Emphasis Type="Italic">Eisenia fetida</Emphasis>

Environmental metabolomics studies employing earthworms as sentinels for soil contamination are numerous, but the instability of the metabolite extracts from these organisms has been minimally addressed. This study evaluated the efficacy of adding a heat-treatment step in two commonly used extraction protocols (Bligh and Dyer and D₂O phosphate buffer) as a pre-analytical stabilization method. The resulting metabolic profiles of Eisenia fetida were assessed using principal component analysis and NMR spectral evaluations. The heated Bligh and Dyer extractions produced stabilized profiles with minimal variation of the extracted metabolomic profiles over time, providing a more suitable method for metabolomic analysis of earthworm extracts.     

Identifying biochemical phenotypic differences between cryptic species

Molecular genetic methods can distinguish divergent evolutionary lineages in what previously appeared to be single species, but it is not always clear what functional differences exist between such cryptic species. We used a metabolomic approach to profile biochemical phenotype (metabotype) differences between two putative cryptic species of the earthworm Lumbricus rubellus. There were no straightforward metabolite biomarkers of lineage, i.e. no metabolites that were always at higher concentration in one lineage. Multivariate methods, however, identified a small number of metabolites that together helped distinguish the lineages, including uncommon metabolites such as Nε-trimethyllysine, which is not usually found at high concentrations. This approach could be useful for characterizing functional trait differences, especially as it is applicable to essentially any species group, irrespective of its genome sequencing status.     

Concordance of N bio-indicators: Dandelion and earthworms in a rotational pasture

Earthworms are important soil metabionts indicative of N enrichment in pastures. A rotational pasture in central Nova Scotia was tested for earthworms using chemical extraction followed by excavation and hand sorting in 28 paired micro plots placed in areas with low versus high proportion of the N indicator plant species dandelion (Taraxacum officinale). Species richness was low with five earthworm species of the Lumbricidae recovered in the following order of abundance: Lumbricus rubellus, Lumbricus terrestris, Aporrectodea turgida, Aporrectodea tuberculata, and Aporrectodea trapezoides. All species occurred at high constancy except the rare A. trapezoides. The inventory revealed spatial differentiation of earthworm abundance and community structure at the field level. High proportion of dandelion reduced pasture sward biomass while abundance of L. rubellus and A. tuberculata significantly (p < 0.05) increased with a concomitant increase in epigeic earthworm dominance at the expense of the anecic L. terrestris. Thus, low cost and non-destructive floristic surveys of N indicators, such as dandelion, allow for concordant inferences about the environmental impact of intensive cow pasture on earthworms and ecosystem function. High earthworm counts may run contrary to the notion of ecological integrity depending on specific earthworm abundances. Reduced earthworm benefits due to any de-intensification of rotational pasture must be assessed against increased risks of N-leaching in intensive pastures with high proportion of dandelion.     

The biological interpretation of metabolomic data can be misled by the extraction method used

The field of metabolomics is getting more and more popular and a wide range of different sample preparation procedures are in use by different laboratories. Chemical extraction methods using one or more organic solvents as the extraction agent are the most commonly used approach to extract intracellular metabolites and generate metabolite profiles. Metabolite profiles are the scaffold supporting the biological interpretation in metabolomics. Therefore, we aimed to address the following fundamental question: can we obtain similar metabolomic results and, consequently, reach the same biological interpretation by using different protocols for extraction of intracellular metabolites? We have used four different methods for extraction of intracellular metabolites using four different microbial cell types (Gram negative bacterium, Gram positive bacterium, yeast, and a filamentous fungus). All the quenched samples were pooled together before extraction, and, therefore, they were identical. After extraction and GC?CMS analysis of metabolites, we did not only detect different numbers of compounds depending on the extraction method used and regardless of the cell type tested, but we also obtained distinct metabolite levels for the compounds commonly detected by all methods (P-value?<?0.001). These differences between methods resulted in contradictory biological interpretation regarding the activity of different metabolic pathways. Therefore, our results show that different solvent-based extraction methods can yield significantly different metabolite profiles, which impact substantially in the biological interpretation of metabolomics data. Thus, development of alternative extraction protocols and, most importantly, standardization of sample preparation methods for metabolomics should be seriously pursued by the scientific community.     

A yeast metabolite extraction protocol optimised for time-series analyses

There is an increasing call for the absolute quantification of time-resolved metabolite data. However, a number of technical issues exist, such as metabolites being modified/degraded either chemically or enzymatically during the extraction process. Additionally, capillary electrophoresis mass spectrometry (CE-MS) is incompatible with high salt concentrations often used in extraction protocols. In microbial systems, metabolite yield is influenced by the extraction protocol used and the cell disruption rate. Here we present a method that rapidly quenches metabolism using dry-ice ethanol bath and methanol N-ethylmaleimide solution (thus stabilising thiols), disrupts cells efficiently using bead-beating and avoids artefacts created by live-cell pelleting. Rapid sample processing minimised metabolite leaching. Cell weight, number and size distribution was used to calculate metabolites to an attomol/cell level. We apply this method to samples obtained from the respiratory oscillation that occurs when yeast are grown continuously.     

Rhabditis terricola: An opportunistic nematode parasite of earthworm cocoons

During a study on the breeding rate of the earthworms Lumbricus rubellus and Eisenia foetida, nematodes identified as Rhabditis terricola were found consistently in large numbers in earthworm cocoons that failed to hatch after an adequate incubation period. This nematode species, which was previously known as a saprophyte, was found to invade earthworm cocoons and reproduce within, causing extensive productivity losses in earthworm cultures. In this study, R. terricola was effectively eradicated from earthworm cultures by rinsing the earthworms in tap water and transferring them repeatedly to sterile bedding.     

Palatability of Selected Alpine Plant Litters for the Decomposer Lumbricus rubellus (Lumbricidae)

On alpine pastureland the decline in large-bodied earthworm numbers and biomass after abandonment of management might be the result of a shift from highly palatable grass litter to poorly digestible leaf litter of dwarf shrubs. To test this hypothesis, we analysed nitrogen, phosphorous and total phenolic contents of fresh and aged litter of eight commonly occuring alpine plant species and compared consumption rates of these food sources in a controlled feeding experiment with Lumbricus rubellus (Lumbricidae). Furthermore, we analysed the microbial community structure of aged litter materials to check for a relationship between the microbial characteristics of the different plant litter types and the food choice of earthworms. Plant litters differed significantly in their chemical composition, earthworms, however, showed no preference for any litter species, but generally rejected fresh litter material. Microbial community structures of the litter types were significantly different, but we could find no evidence for selective feeding of L. rubellus. We conclude that L. rubellus is a widespread, adaptable ubiquist, which is able to feed on a variety of food sources differing in quality and palatability, as long as they have been exposed to wheathering.     

A rapid, high resolution high performance liquid chromatography profiling procedure for plant and microbial aromatic secondary metabolites

High performance liquid chromatography protocols have been developed to allow the simultaneous analysis of a very wide range of soluble aromatic secondary metabolites in unfractionated biological extracts. The methods are simple, sensitive, and highly reproducible. They are applicable to a wide variety of natural product investigations in both plants and microorganisms. High resolution of metabolites is achieved in 25 minutes by chromatography on a reverse phase C18 column in a gradient of 0 to 55% acetonitrile in water at pH 3. For example, near-baseline resolution of over 20 phenylpropanoid metabolites and 18 naturally occurring metabolites of indole-3-acetic acid can be obtained. The methods can be applied directly to whole tissue extracts without prepurification or enrichment. Moreover, the simplicity and sensitivity of the protocols allow their application to a large number of very small tissue samples, such as those encountered in research on host-microbe interactions. Such profiles allow one to monitor simultaneously the various alternative metabolic fates of a complex array of molecules. Examination of the profiles over time thus provides one with a powerful tool to correlate many concurrent molecular events that may relate to a given biological phenomenon. The final protocol requires as little as 1 milligram of tissue, which is extracted directly in a microfuge tube in 80% ethanol. With a variable wavelength detector, as little as 100 femtomoles of a given metabolite can be analyzed. Examples of the application of the protocols to a number of plant and microbial secondary product investigations and to screening for flavonoid mutants of Arabidopsis thaliana (L.) Heynh. are given.     

Toward Arabidopsis thaliana hydrophilic metabolome: assessment of extraction methods and quantitative 1H NMR

Our goal was to establish the hydrophilic metabolome of heterotrophic Arabidopsis thaliana cells grown in suspension, a cellular model of plant sink tissues. Water‐soluble metabolites were extracted using four protocols: perchloric acid, boiling ethanol, methanol and methanol/chloroform (M/Chl). They were detected and quantified using ¹H nuclear magnetic resonance (NMR) spectroscopy at 400 MHz. Extraction yields and reproducibility of the extraction methods were investigated. The effects of cell harvest protocol, cell grinding and lyophilization and storage conditions on the measured metabolic profiles were also studied. These quantitative studies demonstrated for the first time that the four extraction protocols commonly used do lead to quite similar molecular compositions as analyzed by ¹H NMR. The M/Chl method proved effective and reliable to prepare series of physiologically significant extracts from plant cells for ¹H NMR analysis. Reproducibility of the detected metabolome was assessed over long periods of time by analyzing a large number of separate extracts prepared from independent cultures. Larger variations in the NMR metabolite profiles could be correlated to changes in physiological parameters of the culture medium. Quantitative resolved ¹H NMR of cell extracts proved to be robust and reliable for routine metabolite profiling of plant cell cultures.     

Exhaustive extraction of cyclopeptides from Amanita phalloides: Guidelines for working with complex mixtures of secondary metabolites

1. Understanding plant‐insect interactions is an active area of research in both ecology and evolution. Much attention has been focused on the impact of secondary metabolites in the host plant or fungi on these interactions. Plants and fungi contain a variety of biologically active compounds, and the secondary metabolite profile can vary significantly between individual samples. However, many experiments characterize the biological effects of only a single secondary metabolite or a subset of these compounds.
2. Here, we develop an exhaustive extraction protocol using an accelerated solvent extraction protocol to recover the complete suite of cyclopeptides and other secondary metabolites found in Amanita phalloides (death cap mushrooms) and compare its efficacy to the “Classic” extraction method used in earlier works.
3. We demonstrate that our extraction protocol recovers the full suite of cyclopeptides and other secondary metabolites in A. phalloides unlike the “Classic” method that favors polar cyclopeptides.
4. Based on these findings, we provide recommendations for how to optimize protocols to ensure exhaustive extracts and also the best practices when using natural extracts in ecological experiments.

     

Comparative analysis of fecal DNA extraction methods with phylogenetic microarray: Effective recovery of bacterial and archaeal DNA using mechanical cell lysis

Several different protocols are used for fecal DNA extraction, which is an integral step in all phylogenetic and metagenomic approaches to characterize the highly diverse intestinal ecosystem. We compared four widely used methods, and found their DNA yields to vary up to 35-fold. Bacterial, archaeal and human DNA was quantified by real-time PCR, and a compositional analysis of different extracts was carried out using the Human Intestinal Tract Chip, a 16S rRNA gene-based phylogenetic microarray. The overall microbiota composition was highly similar between the methods in contrast to the profound differences between the subjects (Pearson correlations > 0.899 and 0.735, respectively). A detailed comparative analysis of mechanical and enzymatic methods showed that despite their overall similarity, the mechanical cell disruption by repeated bead beating showed the highest bacterial diversity and resulted in significantly improved DNA extraction efficiency of archaea and some bacteria, including Clostridium cluster IV. By applying the mechanical disruption method a high prevalence (67%) of methanogenic archaea was detected in healthy subjects (n = 24), exceeding the typical values reported previously. The assessment of performance differences between different methodologies serves as a concrete step towards the comparison and reliable meta-analysis of the results obtained in different laboratories.     

High-throughput extraction and quantification method for targeted metabolomics in murine tissues

Introduction

Global metabolomics analyses using body fluids provide valuable results for the understanding and prediction of diseases. However, the mechanism of a disease is often tissue-based and it is advantageous to analyze metabolomic changes directly in the tissue. Metabolomics from tissue samples faces many challenges like tissue collection, homogenization, and metabolite extraction.

Objectives

We aimed to establish a metabolite extraction protocol optimized for tissue metabolite quantification by the targeted metabolomics AbsoluteIDQ? p180 Kit (Biocrates). The extraction method should be non-selective, applicable to different kinds and amounts of tissues, monophasic, reproducible, and amenable to high throughput.

Methods

We quantified metabolites in samples of eleven murine tissues after extraction with three solvents (methanol, phosphate buffer, ethanol/phosphate buffer mixture) in two tissue to solvent ratios and analyzed the extraction yield, ionization efficiency, and reproducibility.

Results

We found methanol and ethanol/phosphate buffer to be superior to phosphate buffer in regard to extraction yield, reproducibility, and ionization efficiency for all metabolites measured. Phosphate buffer, however, outperformed both organic solvents for amino acids and biogenic amines but yielded unsatisfactory results for lipids. The observed matrix effects of tissue extracts were smaller or in a similar range compared to those of human plasma.

Conclusion

We provide for each murine tissue type an optimized high-throughput metabolite extraction protocol, which yields the best results for extraction, reproducibility, and quantification of metabolites in the p180 kit. Although the performance of the extraction protocol was monitored by the p180 kit, the protocol can be applicable to other targeted metabolomics assays.

     

Parallelized small-scale production of uniformly C-labeled cell extract for quantitative metabolome analysis

The need for quantitative intracellular metabolome information is central to modern applied biotechnology and systems biology. In most cases, sample preparation and metabolite analysis result in degradation of metabolites and signal suppression due to metabolite instability and matrix effects during LC–MS analysis. Therefore the application of uniformly (U) ¹³C-labeled cell extract as an internal standard has gained interest in recent years. In this study a multiple-step protocol has been developed for efficient preparation of U-¹³C-labeled Escherichia coli cell extracts in stirred-tank bioreactors on a milliliter scale with a minimal supply of costly ¹³C-labeled substrate. Significant reduction of fermentation medium salt concentration in the U-¹³C-labeled cell extract was achieved to reduce ion-suppression effects during mass-spectrometric analysis. Additionally, variation of reaction conditions in parallel-operated stirred-tank bioreactors on a milliliter scale enables the simultaneous preparation of U-¹³C-labeled cell extracts with varying metabolite concentrations, which is shown by an example of the labeled phosphoenolpyruvate level in E. coli.     

Comparison of destructive and nondestructive DNA extraction methods for the metabarcoding of arthropod bulk samples

DNA metabarcoding is routinely used for biodiversity assessment, in particular targeting highly diverse groups for which limited taxonomic expertise is available. Various protocols are currently in use, although standardization is key to its application in large-scale monitoring. DNA metabarcoding of arthropod bulk samples can be conducted either destructively from sample tissue, or nondestructively from sample fixative or lysis buffer. Nondestructive methods are highly desirable for the preservation of sample integrity but have yet to be experimentally evaluated in detail. Here, we compare diversity estimates from 14 size-sorted Malaise trap samples processed consecutively with three nondestructive approaches (one using fixative ethanol and two using lysis buffers) and one destructive approach (using homogenized tissue). Extraction from commercial lysis buffer yielded comparable species richness and high overlap in species composition to the ground tissue extracts. A significantly divergent community was detected from preservative ethanol-based DNA extraction. No consistent trend in species richness was found with increasing incubation time in lysis buffer. These results indicate that nondestructive DNA extraction from incubation in lysis buffer could provide a comparable alternative to destructive approaches with the added advantage of preserving the specimens for postmetabarcoding taxonomic work but at a higher cost per sample.     

An effective protocol to solve the problem in genomic DNA isolation of tung tree

Tung tree (Vernicia fordii) is a native and oil-producing woody plant in China. The oil is industrially important and promising biodiesel raw material. However, until recently the lack of effective protocols for the extraction of genomic DNA had made DNA-based molecular studies of tung tree difficult. Here, four conventional protocols and one novel protocol were compared for their capacity in isolating DNA from tung tree leaves of different age. Our results showed that all the four conventional protocols could isolate DNA from old leaves, two from matured leaves, but none from young leaves. However, the detectable DNA samples contained many contaminations, leading to overestimation of DNA concentration measured by ultraviolet spectrophotometer, also interfering with the downstream PCR reaction. The novel protocol could produce high-yield and good-quality DNA from tung tree leaves regardless of leaf age. Its key steps were that a single leaf tissue sample could be recycled for DNA extraction for up to four times, and correspondingly four DNA precipitations (termed as the 1st, 2nd, 3rd and 4th DNA sample, respectively) were conducted. All the four DNA samples of a single tissue were good template for PCR reaction. The novel protocol is an effective method for genomic DNA isolation of tung tree.     

A new device for the freeze-clamping of tissue samples

Measurement of metabolite concentrations in tissue samples involves the following procedures: Removal of the sample from the animal, temporary arrest of metabolism, extraction (including weighing, homogenization, final fixation, and neutralization) and assay. Rapid temporary fixation following the sampling of tissue is essential to prevent autolytic changes in metabolite concentrations (1,2). The freeze-clamping technique described by Wollenberger et al. (3) meets this requirement as long as the final thickness of the freeze-clamped sample is sufficiently small. For brain tissue the limit seems to be about 2 mm (4).In our laboratory we have made extensive use of the freeze-clamping tongs of Wollenberger et al., especially for small tissue samples freeze-clamped in situ. However, when in situ clamping can not be used when more than 2–3 g of tissue must be sampled, the freeze-clamping press described below has proven very useful.     

Complexity and pitfalls of mass spectrometry-based targeted metabolomics in brain research

Current quantitative metabolomic research in brain tissue is challenged by several analytical issues. To compare data of metabolite pattern, ratios of individual metabolite concentrations and composed classifiers characterizing a distinct state, standardized workup conditions, and extraction medium are crucial. Differences in physicochemical properties of individual compounds and compound classes such as polarity determine extraction yields and, thus, ratios of compounds with varying properties. Also, variations in suppressive effects related to coextracted matrix components affect standards or references and their concentration-dependent responses.The selection of a common tissue extraction protocol is an ill-posed problem because it can be regarded as a multiple objective decision depending on factors such as sample handling practicability, measurement precision, control of matrix effects, and relevance of the chemical assay. This study systematically evaluates the impact of extraction solvents and the impact of the complex brain tissue on measured metabolite levels, taking into account ionization efficiency as well as challenges encountered in the trace-level quantification of the analytes in brain matrices. In comparison with previous studies that relied on nontargeted platforms, consequently emphasizing the global behavior of the metabolomic fingerprint, here we focus on several series of metabolites spanning over extensive polarity, concentration, and molecular mass ranges.     

Two strings to the systems biology bow: co-extracting the metabolome and proteome of yeast

Experimental samples are valuable and can represent a significant investment in time and resources. It is highly desirable at times to obtain as much information as possible from a single sample. This is especially relevant for systems biology approaches in which several ‘omics platforms are studied simultaneously. Unfortunately, each platform has a particular extraction methodology which increases sample number and sample volume requirements when multiple ‘omics are analyzed. We evaluated the integration of a yeast extraction method; specifically we explored whether fractions from a single metabolite extraction could be apportioned to multiple downstream ‘omics analytical platforms. In addition, we examined how variations to a chloroform/methanol yeast metabolite extraction regime influence metabolite recoveries. We show that protein suitable for proteomic analysis can be recovered from a metabolite extraction and that recovery of lipids, while reproducible, are not wholly quantitative. Higher quenching solution temperatures (?30 °C) can be used without significant leakage of intracellular metabolites when lower fermentation temperatures (20 °C) are employed. However, extended residence time in quenching solution, in combination with vigorous washing of quenched cell pellets, leads to extensive leakage of intracellular metabolites. Finally, there is minimal difference in metabolite amounts obtained when metabolite extractions are performed at 4 °C compared to extractions at ?20 °C. The evaluated extraction method delivers material suitable for metabolomic and proteomic analyses from the same sample preparation.