RESUMO
Analytical errors caused by suboptimal performance of the chosen platform for a number of metabolites and instrumental drift are a major issue in large-scale metabolomics studies. Especially for MS-based methods, which are gaining common ground within metabolomics, it is difficult to control the analytical data quality without the availability of suitable labeled internal standards and calibration standards even within one laboratory. In this paper, we suggest a workflow for significant reduction of the analytical error using pooled calibration samples and multiple internal standard strategy. Between and within batch calibration techniques are applied and the analytical error is reduced significantly (increase of 25% of peaks with RSD lower than 20%) and does not hamper or interfere with statistical analysis of the final data.
Assuntos
Metabolômica , Algoritmos , Calibragem/normas , Espectrometria de Massas/métodos , Metabolômica/métodos , Metabolômica/normas , Fenótipo , Controle de Qualidade , Reprodutibilidade dos TestesRESUMO
Cesium chloride centrifugation of each of the DNAs extracted from eight strains of Crithidia revealed a main band at rho = 1.717 g/cm(3) and a satellite band varying from rho = 1.701 to 1.705 g/cm(3) for the different strains By electron microscopy each DNA was shown to include circular molecules, 0.69-0.80 micro in mean contour length, and large, topologically two-dimensional masses of DNA in which the molecules appeared in the form of rosettes. DNA isolated from kinetoplast fractions of Crithidia acanthocephali was shown to consist of light satellite DNA and to be mainly in the form of large masses, 0.8 micro (mol wt = 1.54 x 10(6) daltons) circular molecules, and a few long, linear molecules. The results of experiments involving ultracentrifugation, heating, and quenching, sonication, and endodeoxyribonuclease digestion, combined with electron microscopy, are consistent with the following hypothesis. The large DNA masses are associations of 0.8 micro circles which are mainly covalently closed. The circles are held together in groups (the rosettes) of up to 46 by the topological interlocking of each circle with many other circles in the group. A group of circles is attached to an adjacent group by one or more circles, each interlocking with many circles of both groups. Each of the associations comprises, on the average, about 27,000 circles (total mol wt approximately 41 x 10(9) daltons). A model is proposed for the in situ arrangement of the associations which takes into consideration their form and structure, and appearance in thin sections
Assuntos
DNA/análise , Eucariotos/citologia , Organoides , Acridinas/farmacologia , Centrifugação com Gradiente de Concentração , Densitometria , Etídio/farmacologia , Microscopia Eletrônica , Modelos Estruturais , Peso Molecular , Raios UltravioletaRESUMO
Cesium chloride centrifugation of DNA extracted from cells of blood strain Trypanosoma lewisi revealed a main band, rho = 1.707, a light satellite, rho = 1.699, and a heavy satellite, rho = 1.721. Culture strain T. lewisi DNA comprised only a main band, rho = 1.711, and a light satellite, rho = 1.699. DNA isolated from DNase-treated kinetoplast fractions of both the blood and culture strains consisted of only the light satellite DNA. Electron microscope examination of rotary shadowed preparations of lysates revealed that DNA from kinetoplast fractions was mainly in the form of single 0.4 micro circular molecules and large masses of 0.4 micro interlocked circles with which longer, often noncircular molecules were associated. The 0.4 micro circular molecules were mainly in the covalently closed form: they showed a high degree of resistance to thermal denaturation which was lost following sonication; and they banded at a greater density than linear DNA in cesium chloride-ethidium bromide gradients. Interpretation of the large masses of DNA as comprising interlocked covalently closed 0.4 micro circles was supported by the findings that they banded with single circular molecules in cesium chloride-ethidium bromide gradients, and following breakage of some circles by mild sonication, they disappeared and were replaced by molecules made up of low numbers of apparently interlocked 0.4 micro circles. When culture strain cells were grown in the presence of either ethidium bromide or acriflavin, there was a loss of stainable kinetoplast DNA in cytological preparations. There was a parallel loss of light satellite and of circular molecules from DNA extracted from these cells.
Assuntos
DNA/análise , Acridinas/farmacologia , Animais , Centrifugação com Gradiente de Concentração , Césio , Densitometria , DesoxirribonucleasesRESUMO
Messenger RNA levels change on a minutes scale due to both degradation and de novo transcription. Consequently, alterations in the transcript profiles that are not representative for the condition of interest are easily introduced during sample harvesting and work-up. In order to avoid these unwanted changes we have validated a -45 degrees C methanol-based quenching method for obtaining reliable and reproducible 'snapshot' samples of Lactobacillus plantarum cells for transcriptome analyses. Transcript profiles of cells harvested with the quenching method were compared with transcript profiles of cells that were harvested according to two different commonly applied protocols. Significant differences between the transcript profiles of cells harvested by the different methods from the same steady-state culture were observed. In total, 42 genes or operons were identified from which the transcript levels were altered when the cells were not immediately quenched upon harvesting. Among these, several have previously been associated with cold-shock response. Furthermore, the reproducibility of transcript profiles improved, as indicated by the fact that the variation in the data sets obtained from the quenched cells was smaller than in the data sets obtained from the cells that were harvested under non-quenched conditions.
Assuntos
Análise Serial de Proteínas/métodos , Lactobacillus/genética , Metanol , RNA Bacteriano/genética , RNA Mensageiro/genética , Reprodutibilidade dos Testes , Transcrição GênicaAssuntos
DNA/análise , Organoides/análise , Trypanosoma/análise , Animais , Densitometria , Histocitoquímica , Microscopia Eletrônica , Mitocôndrias , Mutação , RatosRESUMO
Genomics is based on the ability to determine the transcriptome, proteome, and metabolome of a cell. These technologies only have added value when they are integrated and based on robust and reproducible workflows. This chapter describes the experimental design, sampling, sample pretreatment, data evaluation, integration, and interpretation. The actual generation of the data is not covered in this chapter since it is highly depended on available equipment and infrastructure. The enormous amount of data generated by these technologies are integrated and interpreted inorder to generate leads for strain and process improvement. Biostatistics are becoming very important for the whole work flow therefore, some general recommendations how to set up experimental design and how to use biostatistics in enhancing the quality of the data and the selection of biological relevant leads for strain engineering and target identification are described.
Assuntos
Interpretação Estatística de Dados , Fungos/genética , Fungos/metabolismo , Perfilação da Expressão Gênica/métodos , Engenharia Metabólica , Metaboloma , Modelos Estatísticos , Proteoma/genética , Proteoma/metabolismo , Proteômica , RNA Fúngico/genética , RNA Fúngico/isolamento & purificação , RNA Fúngico/metabolismo , Biologia de SistemasRESUMO
Metabolomics involves the unbiased quantitative and qualitative analysis of the complete set of metabolites present in cells, body fluids and tissues (the metabolome). By analyzing differences between metabolomes using biostatistics (multivariate data analysis; pattern recognition), metabolites relevant to a specific phenotypic characteristic can be identified. However, the reliability of the analytical data is a prerequisite for correct biological interpretation in metabolomics analysis. In this review the challenges in quantitative metabolomics analysis with regards to analytical as well as data preprocessing steps are discussed. Recommendations are given on how to optimize and validate comprehensive silylation-based methods from sample extraction and derivatization up to data preprocessing and how to perform quality control during metabolomics studies. The current state of method validation and data preprocessing methods used in published literature are discussed and a perspective on the future research necessary to obtain accurate quantitative data from comprehensive GC-MS data is provided.
Assuntos
Replicação do DNA , Escherichia coli/metabolismo , Álcoois/farmacologia , Aminoácidos/metabolismo , Isótopos de Carbono , Centrifugação com Gradiente de Concentração , Cloranfenicol/farmacologia , Cromossomos Bacterianos , Biossíntese de Proteínas , Timina/metabolismo , Fatores de Tempo , TrítioRESUMO
A longitudinal experimental design in combination with metabolomics and multiway data analysis is a powerful approach in the identification of metabolites whose correlation with bioproduct formation shows a shift in time. In this paper, a strategy is presented for the analysis of longitudinal microbial metabolomics data, which was performed in order to identify metabolites that are likely inducers of phenylalanine production by Escherichia coli. The variation in phenylalanine production as a function of differences in metabolism induced by the different environmental conditions in time was described by a validated multiway statistical model. Notably, most of the metabolites showing the strongest relations with phenylalanine production seemed to hardly change in time. Apparently, potential bottlenecks in phenylalanine seem to hardly change in the course of a batch fermentation. The approach described in this study is not limited to longitudinal microbial studies but can also be applied to other (biological) studies in which similar longitudinal data need to be analyzed.
Assuntos
Escherichia coli/metabolismo , Metabolômica/métodos , Algoritmos , Fermentação , Análise dos Mínimos Quadrados , Modelos Biológicos , Fenilalanina/metabolismo , Análise de Componente Principal , Análise de RegressãoRESUMO
Metabolomics is an emerging, powerful, functional genomics technology that involves the comparative non-targeted analysis of the complete set of metabolites in an organism. We have set-up a robust quantitative metabolomics platform that allows the analysis of 'snapshot' metabolomes. In this study, we have applied this platform for the comprehensive analysis of the metabolite composition of Pseudomonas putida S12 grown on four different carbon sources, i.e. fructose, glucose, gluconate and succinate. This paper focuses on the microbial aspects of analyzing comprehensive metabolomes, and demonstrates that metabolomes can be analyzed reliably. The technical (i.e. sample work-up and analytical) reproducibility was on average 10%, while the biological reproducibility was approximately 40%. Moreover, the energy charge values of the microbial samples generated were determined, and indicated that no biotic or abiotic changes had occurred during sample work-up and analysis. In general, the metabolites present and their concentrations were very similar after growth on the different carbon sources. However, specific metabolites showed large differences in concentration, especially the intermediates involved in the degradation of the carbon sources studied. Principal component discriminant analysis was applied to identify metabolites that are specific for, i.e. not necessarily the metabolites that show those largest differences in concentration, cells grown on either of these four carbon sources. For selected enzymatic reactions, i.e. the glucose-6-phosphate isomerase, triosephosphate isomerase and phosphoglyceromutase reactions, the apparent equilibrium constants (K(app)) were calculated. In several instances a carbon source-dependent deviation between the apparent equilibrium constant (K(app)) and the thermodynamic equilibrium constant (K(eq)) was observed, hinting towards a potential point of metabolic regulation or towards bottlenecks in biosynthesis routes. For glucose-6-phosphate isomerase and phosphoglyceromutase, the K(app) was larger than K(eq), and the results suggested that the specific enzymatic activities of these two enzymes were too low to reach the thermodynamic equilibrium in growing cells. In contrast, with triosephosphate isomerase the K(app) was smaller than K(eq), and the results suggested that this enzyme is kinetically controlled.
Assuntos
Carbono/metabolismo , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Genômica , Pseudomonas putida/genética , Pseudomonas putida/metabolismo , Metabolismo Energético , Metabolismo , Reprodutibilidade dos TestesRESUMO
Metabolite profiling in combination with multivariate statistics is a sophisticated method for quality assessment of natural products. For the development of a quality control strategy in Traditional Chinese Medicine (TCM), we have measured the metabolite fingerprints of Rehmannia glutinosa by GC-MS. Plants were grown under different climate and soil conditions in a phytotron and were processed by a variable number of repetitive steps to investigate the effects on both growth conditions and processing for material medica of R. glutinosa. The GC-MS data have been analyzed by principal component analysis (PCA) and the new approach of the ANOVA-simultaneous component analysis (ASCA) which can combine the information from a structured data design with multivariate analysis. The results clearly show the effect of the different factors and indicate directions for process improvement. When plants were grown under various temperatures, humidity and light intensities for a short period (3 weeks), no significant changes on studied metabolites were observed. However, significant changes were found between different processing cycles. The present data clearly indicate the importance of strictly controlling processing in R. glutinosa and illustrate the impact of growth conditions. This is the first report on the metabolite profile of R. glutinosa that provides a base for the establishment of a quality control strategy.
Assuntos
Medicamentos de Ervas Chinesas/química , Fitoterapia , Rehmannia/crescimento & desenvolvimento , Clima , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Análise de Componente Principal , Controle de Qualidade , SoloRESUMO
The value of the multivariate data analysis tools principal component analysis (PCA) and principal component discriminant analysis (PCDA) for prioritizing leads generated by microarrays was evaluated. To this end, Pseudomonas putida S12 was grown in independent triplicate fermentations on four different carbon sources, i.e. fructose, glucose, gluconate and succinate. RNA isolated from these samples was analysed in duplicate on an anonymous clone-based array to avoid bias during data analysis. The relevant transcripts were identified by analysing the loadings of the principal components (PC) and discriminants (D) in PCA and PCDA, respectively. Even more specifically, the relevant transcripts for a specific phenotype could also be ranked from the loadings under an angle (biplot) obtained after PCDA analysis. The leads identified in this way were compared with those identified using the commonly applied fold-difference and hierarchical clustering approaches. The different data analysis methods gave different results. The methods used were complementary and together resulted in a comprehensive picture of the processes important for the different carbon sources studied. For the more subtle, regulatory processes in a cell, the PCDA approach seemed to be the most effective. Except for glucose and gluconate dehydrogenase, all genes involved in the degradation of glucose, gluconate and fructose were identified. Moreover, the transcriptomics approach resulted in potential new insights into the physiology of the degradation of these carbon sources. Indications of iron limitation were observed with cells grown on glucose, gluconate or succinate but not with fructose-grown cells. Moreover, several cytochrome- or quinone-associated genes seemed to be specifically up- or downregulated, indicating that the composition of the electron-transport chain in P. putida S12 might change significantly in fructose-grown cells compared to glucose-, gluconate- or succinate-grown cells.
Assuntos
Genes Bacterianos , Pseudomonas putida/genética , Metabolismo dos Carboidratos , Meios de Cultura , Análise Multivariada , Análise de Sequência com Séries de Oligonucleotídeos , Pseudomonas putida/química , Pseudomonas putida/enzimologiaRESUMO
Microbial production strains are currently improved using a combination of random and targeted approaches. In the case of a targeted approach, potential bottlenecks, feed-back inhibition, and side-routes are removed, and other processes of interest are targeted by overexpressing or knocking-out the gene(s) of interest. To date, the selection of these targets has been based at its best on expert knowledge, but to a large extent also on 'educated guesses' and 'gut feeling'. Therefore, time and thus money is wasted on targets that later prove to be irrelevant or only result in a very minor improvement. Moreover, in current approaches, biological processes that are not known to be involved in the formation of a specific product are overlooked and it is impossible to rank the relative importance of the different targets postulated. Metabolomics, a technology that involves the non-targeted, holistic analysis of the changes in the complete set of metabolites in the cell in response to environmental or cellular changes, in combination with multivariate data analysis (MVDA) tools like principal component discriminant analysis and partial least squares, allow the replacement of current empirical approaches by a scientific approach towards the selection and ranking of targets. In this review, we describe the technological challenges in setting up the novel metabolomics technology and the principle of MVDA algorithms in analyzing biomolecular data sets. In addition to strain improvement, the combined metabolomics and MVDA approach can also be applied to growth medium optimization, predicting the effect of quality differences of different batches of complex media on productivity, the identification of bioactives in complex mixtures, the characterization of mutant strains, the exploration of the production potential of strains, the assignment of functions to orphan genes, the identification of metabolite-dependent regulatory interactions, and many more microbiological issues.
Assuntos
Biologia Computacional , Genômica , Microbiologia Industrial/métodos , Proteômica , Bactérias/genética , Bactérias/metabolismo , Metabolismo Energético/genéticaRESUMO
A general method is presented for combining mass spectrometry-based metabolomics data. Such data are becoming more and more abundant, and proper tools for fusing these types of data sets are needed. Fusion of metabolomics data leads to a comprehensive view on the metabolome of an organism or biological system. The ideas presented draw upon established techniques in data analysis. Hence, they are also widely applicable to other types of X-omics data provided there is a proper pretreatment of the data. These issues are discussed using a real-life metabolomics data set from a microbial fermentation process.
Assuntos
Bases de Dados como Assunto , Escherichia coli/metabolismo , Processamento de Imagem Assistida por Computador , Espectrometria de Massas/métodosRESUMO
Temperature-sensitive simian virus (SV 40)-transformed 3T3 cells (tsSV3T3), which express the transformed phenotype when growing at 32 C but not at 39 C, were used to study changes in growth behavior during shift-up or shift-down experiments. In cultures of tsSV3T3 cells which had reached or were beyond monolayer density at 32 C, DNA synthesis reached very low levels within 24 to 48 h after shift-up. When cells which had been allowed to grow to high densities at 32 C were shifted to 39 C, not only cell growth stopped, but within two to three days the cultures shed a large number of cells into the medium. These cells were nonviable, and shedding stopped only when the number of cells attached had been reduced to that characteristic of the saturation density at 39 C. The remaining attached cells were viable and after the shift to 32 C were again able to grow from the monolayer to high cell densities. This behavior has been compared with that of normal 3T3 and wild-type SV3T3 cells under different conditions. We have also isolated new tsSV3T3 lines, using cells which had been infected with non-mutagenized wild-type SV40. This further demonstrates that the temperature sensitivity of these lines is due to a cellular rather than a viral mutation.
Assuntos
Transformação Celular Neoplásica , Células Cultivadas/metabolismo , Vírus 40 dos Símios/crescimento & desenvolvimento , Temperatura , Animais , Autorradiografia , Isótopos de Carbono , Contagem de Células , Linhagem Celular , Células Cultivadas/citologia , Células Cultivadas/microbiologia , DNA de Neoplasias/biossíntese , Fibroblastos , Camundongos , Mitose , Mutação , Timidina/metabolismo , TrítioRESUMO
A procedure has been devised to isolate 3T3 mouse fibroblasts transformed by simian virus 40 (SV40) that express their transformed phenotype at low (32 degrees C) but not at high (39 degrees C) temperature. Three parameters typical of malignant growth in vitro: (a) high saturation density in culture, (b) ability to form colonies on monolayers of normal 3T3 cells, and (c) lack of contact inhibition of DNA synthesis, are temperature sensitive. These phenotypic changes are fully reversible. The serum requirement for growth appears to be largely unchanged by temperature. These cells seem to owe their behavior to a cellular, rather than to a viral, alteration since after fusion of the temperature-sensitive transformed cells with permissive monkey cells, a procedure that leads to rescue (i.e., multiplication of the virus), wild-type SV40 virus is produced.
Assuntos
Transformação Celular Neoplásica , Fibroblastos/metabolismo , Vírus 40 dos Símios/patogenicidade , Animais , Antígenos , Linhagem Celular , Cromossomos , Meios de Cultura , DNA Viral/biossíntese , Imunofluorescência , Haplorrinos , Rim , Camundongos , Mutação , Nitrosoguanidinas/farmacologia , Vírus 40 dos Símios/efeitos dos fármacos , Temperatura , Replicação Viral , gama-GlobulinasRESUMO
A binding assay that shows consistent differences in the amounts of tritium-labeled concanavalin A that bind to normal and virally transformed cells was used to study the kinetic changes of cell surface in SV40-transformed 3T3 cells that express the transformed phenotype in a temperature-sensitive manner (tsSV3T3 cells). The increase in concanavalin A binding, which paralleled the appearance of the characteristics of transformed cells, was dependent on the synthesis of cellular DNA. In agreement with the results of binding studies, exponentially growing tsSV3T3 cells agglutinated at much lower lectin titers (concanavalin A as well as wheat-germ agglutinin) at 32 degrees than at 39 degrees .
Assuntos
Sítios de Ligação , Transformação Celular Neoplásica , Lectinas , Vírus 40 dos Símios , Temperatura , Aglutinação , Animais , Células Cultivadas , Concanavalina A , DNA/biossíntese , DNA de Neoplasias/biossíntese , Glucosamina , Haptenos , Hidroxiureia/farmacologia , Cinética , Camundongos , Mutação , Fenótipo , TrítioRESUMO
Osteoarthritis (OA), one of the most common diseases among the elderly, is characterized by the progressive destruction of joint tissues. Its etiology is largely unclear and no effective disease-modifying treatment is currently available. Metabolic fingerprinting provides a novel tool for the identification of biomarkers. A metabolic fingerprint consists of a typical combination of metabolites in a biological fluid and is identified by a combination of (1)H NMR spectroscopy and multivariate data analysis (MVDA). The current feasibility study was aimed at identifying a metabolic fingerprint for OA and applying this in a nutritional intervention study. Urine samples were collected from osteoarthritic male Hartley guinea pigs (n = 44) at 10 and 12 mo of age, treated from 4 mo onward with variable vitamin C doses (2.5-3, 30 and 150 mg/d) and from healthy male Strain 13 guinea pigs (n = 8) at 12 mo of age, treated with 30 mg vitamin C/d. NMR measurements were performed on all urine samples. Subsequently, MVDA was carried out on the data obtained using NMR. An NMR fingerprint was identified that reflected the osteoarthritic changes in guinea pigs. The metabolites that comprised the fingerprint indicate that energy and purine metabolism are of major importance in OA. Metabolic fingerprinting also allowed detection of differences in OA-specific metabolites induced by different dietary vitamin C intakes. This study demonstrates the feasibility of metabolic fingerprinting to identify disease-specific profiles of urinary metabolites. NMR fingerprinting is a promising means of identifying new disease markers and of gaining fresh insights into the pathophysiology of disease.