Metabolomics meets functional assays: coupling LC-MS and microfluidic cell-based receptor-ligand analyses.

INTRODUCTION: Metabolomics has become a valuable tool in many research areas. However, generating metabolomics-based biochemical profiles without any related bioactivity is only of indirect value in understanding a biological process. Therefore, metabolomics research could greatly benefit from tools that directly determine the bioactivity of the detected compounds. OBJECTIVE: We aimed to combine LC-MS metabolomics with a cell based receptor assay. This combination could increase the understanding of biological processes and may provide novel opportunities for functional metabolomics. METHODS: We developed a flow through biosensor with human cells expressing both the TRPV1, a calcium ion channel which responds to capsaicin, and the fluorescent intracellular calcium ion reporter, YC3.6. We have analysed three contrasting Capsicum varieties. Two were selected with contrasting degrees of spiciness for characterization by HPLC coupled to high mass resolution MS. Subsequently, the biosensor was then used to link individual pepper compounds with TRPV1 activity. RESULTS: Among the compounds in the crude pepper fruit extracts, we confirmed capsaicin and also identified both nordihydrocapsaicin and dihydrocapsaicin as true agonists of the TRPV1 receptor. Furthermore, the biosensor was able to detect receptor activity in extracts of both Capsicum fruits as well as a commercial product. Sensitivity of the biosensor to this commercial product was similar to the sensory threshold of a human sensory panel. CONCLUSION: Our results demonstrate that the TRPV1 biosensor is suitable for detecting bioactive metabolites. Novel opportunities may lie in the development of a continuous functional assay, where the biosensor is directly coupled to the LC-MS.

Multi-platform metabolomics analyses of a broad collection of fragrant and non-fragrant rice varieties reveals the high complexity of grain quality characteristics.

The quality of rice in terms not only of its nutritional value but also in terms of its aroma and flavour is becoming increasingly important in modern rice breeding where global targets are focused on both yield stability and grain quality. In the present paper we have exploited advanced, multi-platform metabolomics approaches to determine the biochemical differences in 31 rice varieties from a diverse range of genetic backgrounds and origin. All were grown under the specific local conditions for which they have been bred and all aspects of varietal identification and sample purity have been guaranteed by local experts from each country. Metabolomics analyses using 6 platforms have revealed the extent of biochemical differences (and similarities) between the chosen rice genotypes. Comparison of fragrant rice varieties showed a difference in the metabolic profiles of jasmine and basmati varieties. However with no consistent separation of the germplasm class. Storage of grains had a significant effect on the metabolome of both basmati and jasmine rice varieties but changes were different for the two rice types. This shows how metabolic changes may help prove a causal relationship with developing good quality in basmati rice or incurring quality loss in jasmine rice in aged grains. Such metabolomics approaches are leading to hypotheses on the potential links between grain quality attributes, biochemical composition and genotype in the context of breeding for improvement. With this knowledge we shall establish a stronger, evidence-based foundation upon which to build targeted strategies to support breeders in their quest for improved rice varieties.

Comprehensive metabolomics to evaluate the impact of industrial processing on the phytochemical composition of vegetable purees.

The effects of conventional industrial processing steps on global phytochemical composition of broccoli, tomato and carrot purees were investigated by using a range of complementary targeted and untargeted metabolomics approaches including LC-PDA for vitamins, (1)H NMR for polar metabolites, accurate mass LC-QTOF MS for semi-polar metabolites, LC-MRM for oxylipins, and headspace GC-MS for volatile compounds. An initial exploratory experiment indicated that the order of blending and thermal treatments had the highest impact on the phytochemicals in the purees. This blending-heating order effect was investigated in more depth by performing alternate blending-heating sequences in triplicate on the same batches of broccoli, tomato and carrot. For each vegetable and particularly in broccoli, a large proportion of the metabolites detected in the purees was significantly influenced by the blending-heating order, amongst which were potential health-related phytochemicals and flavour compounds like vitamins C and E, carotenoids, flavonoids, glucosinolates and oxylipins. Our metabolomics data indicates that during processing the activity of a series of endogenous plant enzymes, such as lipoxygenases, peroxidases and glycosidases, including myrosinase in broccoli, is key to the final metabolite composition and related quality of the purees.

MSClust: a tool for unsupervised mass spectra extraction of chromatography-mass spectrometry ion-wise aligned data.

Mass peak alignment (ion-wise alignment) has recently become a popular method for unsupervised data analysis in untargeted metabolic profiling. Here we present MSClust-a software tool for analysis GC-MS and LC-MS datasets derived from untargeted profiling. MSClust performs data reduction using unsupervised clustering and extraction of putative metabolite mass spectra from ion-wise chromatographic alignment data. The algorithm is based on the subtractive fuzzy clustering method that allows unsupervised determination of a number of metabolites in a data set and can deal with uncertain memberships of mass peaks in overlapping mass spectra. This approach is based purely on the actual information present in the data and does not require any prior metabolite knowledge. MSClust can be applied for both GC-MS and LC-MS alignment data sets. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11306-011-0368-2) contains supplementary material, which is available to authorized users.

Automated procedure for candidate compound selection in GC-MS metabolomics based on prediction of Kovats retention index.

MOTIVATION: Matching both the retention index (RI) and the mass spectrum of an unknown compound against a mass spectral reference library provides strong evidence for a correct identification of that compound. Data on retention indices are, however, available for only a small fraction of the compounds in such libraries. We propose a quantitative structure-RI model that enables the ranking and filtering of putative identifications of compounds for which the predicted RI falls outside a predefined window. RESULTS: We constructed multiple linear regression and support vector regression (SVR) models using a set of descriptors obtained with a genetic algorithm as variable selection method. The SVR model is a significant improvement over previous models built for structurally diverse compounds as it covers a large range (360-4100) of RI values and gives better prediction of isomer compounds. The hit list reduction varied from 41% to 60% and depended on the size of the original hit list. Large hit lists were reduced to a greater extend compared with small hit lists. AVAILABILITY: http://appliedbioinformatics.wur.nl/GC-MS. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Inconsistencies between human genetic cytolocations and those derived using genomic sequence.

One result of the publishing of the human genome sequence is the ability to define objects through their position on the consensus sequence. While this has simplified the process of creating order maps for genes on a chromosome, it has created discrepancies between the published cytolocations of human genes, as presented through genetic references, and those locations derived computationally from the genomic sequence. For the 6,830 records with HUGO gene symbols shared between the online version of Mendelian Inheritance in Man and Ensembl, 18% of the records have a discrepancy of at least one cytogenetic band between the datasets. Discordance between data sets at this frequency would have a significant impact on the utility of datasets created by the amalgamation of numerous biological databases.

Improving the specificity of 16S rDNA-based polymerase chain reaction for detecting Borrelia burgdorferi sensu lato-causative agents of human Lyme disease.

AIMS: 16S rDNA sequences of Borrelia burgdorferi sensu lato were aligned with the 16S rDNA sequences of Borrelia hermsii, Borrelia turicatae, and Borrelia lonestari in order to identify primers that might be used to more specifically identify agents of human Lyme disease in ticks in human skin samples. METHODS AND RESULTS: Standard polymerase chain reaction (PCR), using an oligonucleotide sequence, designated TEC1, was shown, in combination with a previously developed primer (LD2) to amplify strains of B. burgdorferi sensu stricto, Borrelia afzelii, and Borrelia garinii, but not the non-Lyme causing B. hermsii or B. turicatae. This primer pair, designated Bbsl, was successfully used to amplify B. burgdorferi sensu lato from skin biopsies of patients with Lyme disease symptoms as well as from Ixodes scapularis, Amblyomma americanum and Dermacentor variabilis ticks. CONCLUSIONS: The primer set Bbsl allows for the rapid detection and differentiation of B. burgdorferi sensu lato from non-Lyme disease-causing Borrelia species in ticks and human tissues. SIGNIFICANCE AND IMPACT OF THE STUDY: The PCR primer set, Bbsl, will greatly facilitate detection of the causative agents of Lyme disease in infected ticks and human skin samples assisting in epidemiological studies, and potentially allowing for a more rapid diagnosis of the disease in patients.

Ground based studies of gene expression in Arabidopsis exposed to gravity stresses.

As a link in the preparation of the MULTIGEN experiment, which will take place on the International Space Station, ground based studies of the gene expression in Arabidopsis thaliana were performed. Microarray technology was used to screen Arabidopsis seedlings exposed to simulated hypogravity on a Random Positioning Machine and a 1 x g control sample. This screening showed differential expression in 177 out of approximately 8000 genes. Some of these genes can be grouped into functional categories, e.g. general metabolism, biogenesis of cellular components, cellular transport and transport facilitation, and cell rescue and defense response. However, about 50% of the genes encode proteins with unknown function. Based on the above results a new "in-house" cDNA microarray was constructed. Some of the selected genes on this microarray (e.g. Xyloglucan endotransglycosylase, At2g18800) showed differential expression both in Arabidopsis exposed to hypergravity and simulated hypogravity by use of a centrifuge and a Random Positioning Machine.

Gene expression during anthesis and senescence in Iris flowers.

We investigated changes in gene expression in Iris hollandica flowers by microarray technology. Flag tepals were sampled daily, from three days prior to flower opening to the onset of visible senescence symptoms. Gene expression profiles were compared with biochemical data including lipid and protein degradation and DNA coiling, and with morphological data. Plasmodesmata of mesophyll cells closed about two days before flower opening, while in the epidermis they closed concomitant with opening. Similarly, the onset of visible senescence in the epidermis cells occurred about two days later than in the mesophyll. About 1400 PCR-amplified clones, derived from a subtractive cDNA library enriched for tepal-specific genes, were spotted and about 240 clones, including 200 that were expressed most differentially, were sequenced. The expression patterns showed three main clusters. One exhibited high expression during tepal growth (cluster A). These genes were putatively associated with pigmentation, cell wall synthesis and metabolism of lipids and proteins. The second cluster (B) was highly expressed during flower opening. The third cluster (C) related to the final stages of senescence, with genes putatively involved in signal transduction, and the remobilization of phospholipids, proteins, and cell wall compounds. Throughout the sampling period, numerous plant defence genes were highly expressed. We identified an ion channel protein putatively involved in senescence, and some putative regulators of transcription and translation, including a MADS-domain factor.

The Neurospora crassa genome: cosmid libraries sorted by chromosome.

A Neurospora crassa cosmid library of 12,000 clones (at least nine genome equivalents) has been created using an improved cosmid vector pLorist6Xh, which contains a bacteriophage lambda origin of replication for low-copy-number replication in bacteria and the hygromycin phosphotransferase marker for direct selection in fungi. The electrophoretic karyotype of the seven chromosomes comprising the 42.9-Mb N. crassa genome was resolved using two translocation strains. Using gel-purified chromosomal DNAs as probes against the new cosmid library and the commonly used medium-copy-number pMOcosX N. crassa cosmid library in two independent screenings, the cosmids were assigned to chromosomes. Assignments of cosmids to linkage groups on the basis of the genetic map vs. the electrophoretic karyotype are 93 +/- 3% concordant. The size of each chromosome-specific subcollection of cosmids was found to be linearly proportional to the size of the particular chromosome. Sequencing of an entire cosmid containing the qa gene cluster indicated a gene density of 1 gene per 4 kbp; by extrapolation, 11,000 genes would be expected to be present in the N. crassa genome. By hybridizing 79 nonoverlapping cosmids with an average insert size of 34 kbp against cDNA arrays, the density of previously characterized expressed sequence tags (ESTs) was found to be slightly <1 per cosmid (i.e., 1 per 40 kbp), and most cosmids, on average, contained an identified N. crassa gene sequence as a starting point for gene identification.

Plant cell culture initiation. Practical tips.

The use of cultured plant cells in either organized or unorganized form has increased vey considerably in the last 10-15 yr. Many new technologies have been developed and applications in both fundamental and applied research have led to the development of some powerful tools for improving our knowledge of botanical systems and for gaining external influence over some of the key processes involved in inter- and intracellular organization. This is particularly the case when cell culture techniques are combined with those for the genetic modification of plant cells. Being able to regenerate whole plants that have gained or lost the expression of one or more specific genes has revolutionized the way in which we approach scientific questions and has opened up many additional possibilities for the molecular dissection of plants. The success or fall of all plant cell culture technologies lies with culture initiation. The choice of plant material, its physiologival state and cultivation history, the media used, and their means of preparation are just some of the factors that can greatly influence whether the desired end result will be achieved. In this article are described some of the practical aspects involved in successful plant cell culture initiation and the choices that have to be made. Attention is given to some of the pitfalls that can occur and how to avoid them. A good start is half the work.

Chrysomya rufifacies (Macquart) (Diptera: Calliphoridae) established in the vicinity of Knoxville, Tennessee, USA.

The hairy maggot blow fly, Chrsomya rufifacies (Macquart) (Diptera: Calliphoridae) was collected in large numbers as both adults and immatures in the Knoxville, Tennessee, area during 1998 and is likely established there. The distribution of this species in the Old World, isothermal data, and its collection from mid-Michigan during 1998 suggest that it will eventually occupy most of the U.S. The forensic importance of C. rufifacies makes it probable that it will factor into an increasing number of medicolegal cases, but the expanding distribution of this species decreases its utility as a geographic indicator when postmortem movement of decedents is suspected.

Pattern of degeneration of the spiral ganglion cell and its processes in the C57BL/6J mouse.

Although degeneration of spiral ganglion cells has been described as a histopathologic correlate of hearing loss both in animals and humans, the pattern and sequence of this degeneration remain controversial. Degeneration of hair cells and of spiral ganglion cells and their dendritic processes was evaluated in the C57BL/6J mouse, in which there is a genetically determined progressive sensorineural loss starting in the high frequencies that is similar to the pattern commonly seen in the human. Auditory function was evaluated by brainstem evoked responses, and degeneration of hair cells, ganglion cells and their dendrites was evaluated histologically at 3, 8, 12 and 18 months of age. Progressive loss of auditory sensitivity was correlated with the loss of outer and inner hair cells and spiral ganglion cells and their dendritic processes. In addition, dendritic counts were consistently lower at a distal location in the osseous spiral lamina (i.e. near the organ of Corti) than at a proximal location (i.e. near the spiral ganglion), and the difference between the number of distal dendrites and the number of proximal dendrites tended to be greater with advancing age. These observations suggest an age-related progressive retrograde degeneration of spiral ganglion cells. Thus, in degenerating cochleas, some remaining spiral ganglion cells may have no distal dendritic processes near the organ of Corti. This may have implications for successful stimulation of the cochlear neuron in cochlear implantation.

High level fructan accumulation in a transgenic sugar beet.

We have transformed sugar beet into a crop that produces fructans. The gene encoding 1-sucrose:sucrose fructosyl transferase (1-SST), which was isolated from Helianthus tuberosus, was introduced into sugar beet. In H. tuberosus, 1-SST mediates the first steps in fructan synthesis through the conversion of sucrose (GF) into low molecular weight fructans GF2, GF3, and GF4. In the taproot of sugar beet transformed with the 1-sst gene, the stored sucrose is almost totally converted into low molecular weight fructans. In contrast, 1-sst expression in the leaves resulted in only low levels of fructans. Despite the storage carbohydrate having been altered, the expression of the 1-sst gene did not have any visible effect on phenotype and did not affect the growth rate of the taproot as observed under greenhouse conditions.

The number of primary auditory afferents in the rat.

Published estimates of the number of primary auditory afferents in the rat differ by as much as 30%. We undertook to determine if the widely varying estimates were related to methodological differences, especially the difference between counting cells in Rosenthal's canal and fibers in the cochlear nerve. Type I ganglion cells and myelinated cochlear nerve fibers in the same ears were counted in Long-Evans and Sprague-Dawley strains. Type II spiral ganglion cells were also counted. In each strain the numbers of myelinated fibers and type I ganglion cells were essentially the same. Means for the Long-Evans were 18,036 fibers and 17,749 cells. Means for Sprague-Dawleys were higher: 19,444 fibers and 19,229 cells. The mean number of type II ganglion cells was also greater in Sprague-Dawley than in Long-Evans rats: 1,388 and 1,170, respectively. Cell and fiber counts from the two ears of the same animal differed on average by only 1%. The number of auditory afferents did not change with age over the range (2-10 months) studied here. Several methodological differences have probably contributed to the varying estimates of type I primary auditory afferents, but the discrepancies are not inherent in counts of fibers and spiral ganglion cells.

Stomatal Guard Cells Are Totipotent.

It has been successfully demonstrated, using epidermis explants of sugar beet (Beta vulgaris L.), that stomatal guard cells retain full totipotent capacity. Despite having one of the highest degrees of morphological adaptation and a unique physiological specialization, it is possible to induce a re-expression of full (embryogenic) genetic potential in these cells in situ by reversing their highly differentiated nature to produce regenerated plants via a callus stage. The importance of these findings both to stomatal research and to our understanding of cytodifferentiation in plants is discussed.

A high efficiency technique for the generation of transgenic sugar beets from stomatal guard cells.

An optimized protocol has been developed for the efficient and rapid genetic modification of sugar beet (Beta vulgaris L.). A polyethylene glycol-mediated DNA transformation technique could be applied to protoplast populations enriched specifically for a single totipotent cell type derived from stomatal guard cells, to achieve high transformation frequencies. Bialaphos resistance, conferred by the pat gene, produced a highly efficient selection system. The majority of plants were obtained within 8 to 9 weeks and were appropriate for plant breeding purposes. All were resistant to glufosinate-ammonium-based herbicides. Detailed genomic characterization has verified transgene integration, and progeny analysis showed Mendelian inheritance.

Computer-Assisted Identification of Protoplasts Responsible for Rare Division Events Reveals Guard-Cell Totipotency.

With the use of a computer-controlled microscope system to assist in the positioning and rapid relocation of large numbers of cultured cells, we were able to identify those protoplasts with the capacity to divide within a highly recalcitrant culture in which only a tiny fraction of the total population proceeds to produce viable microcalli. In the cultures used, comprising Beta vulgaris L. (sugar beet) leaf protoplasts, it was confirmed that these cells can be recognized solely on the basis of morphological characters. Therefore, a direct link exists between competence for cell division in vitro and cell type. Divergent callus morphologies and totipotent potential could also be ascribed to distinct protoplast types and hence to cells with a specific origin. The progenitors of the totipotent protoplasts in these cultures have been confirmed as being stomatal guard cells. Consequently, in plants even the most highly adapted living cells clearly retain and can reactivate all of the functional genetic information necessary to recreate the whole organism; an extreme degree of cytodifferentiation is, therefore, no hindrance to expressing totipotent potential. In addition to the considerable practical value of these findings, their implications concerning our understanding of both the control of gene expression and plant cell differentiation and its reversibility are of fundamental significance.