RESUMO
Inflammatory bowel disease (IBD) is associated with altered microbiota composition and metabolism, but it is unclear whether these changes precede inflammation or are the result of it since current studies have mainly focused on changes after the onset of disease. We previously showed differences in mucus gut microbiota composition preceded colitis-induced inflammation and stool microbial differences only became apparent at colitis onset. In the present study, we aimed to investigate whether microbial dysbiosis was associated with differences in both predicted microbial gene content and endogenous metabolite profiles. We examined the functional potential of mucus and stool microbial communities in the mdr1a -/- mouse model of colitis and littermate controls using PICRUSt on 16S rRNA sequencing data. Our findings indicate that despite changes in microbial composition, microbial functional pathways were stable before and during the development of mucosal inflammation. LC-MS-based metabolic phenotyping (metabotyping) in urine samples confirmed that metabolite profiles in mdr1a -/- mice were remarkably unaffected by development of intestinal inflammation and there were no differences in previously published metabolic markers of IBD. Metabolic profiles did, however, discriminate the colitis-prone mdr1a -/- genotype from controls. Our results indicate resilience of the metabolic network irrespective of inflammation. Importantly as metabolites differentiated genotype, genotype-differentiating metabolites could potentially predict IBD risk.
Assuntos
Colite/etiologia , Colite/metabolismo , Microbioma Gastrointestinal , Metaboloma , Metabolômica , Fenótipo , Animais , Cromatografia Líquida de Alta Pressão , Modelos Animais de Doenças , Suscetibilidade a Doenças , Genótipo , Doenças Inflamatórias Intestinais/etiologia , Doenças Inflamatórias Intestinais/metabolismo , Masculino , Espectrometria de Massas , Metabolômica/métodos , Metagenoma , Metagenômica , Camundongos , Camundongos Knockout , RNA Ribossômico 16S/genéticaRESUMO
MOTIVATION: In recent years, the gulf between the mass of accumulating-research data and the massive literature describing and analyzing those data has widened. The need for intelligent tools to bridge this gap, to rescue the knowledge being systematically isolated in literature and data silos, is now widely acknowledged. RESULTS: To this end, we have developed Utopia Documents, a novel PDF reader that semantically integrates visualization and data-analysis tools with published research articles. In a successful pilot with editors of the Biochemical Journal (BJ), the system has been used to transform static document features into objects that can be linked, annotated, visualized and analyzed interactively (http://www.biochemj.org/bj/424/3/). Utopia Documents is now used routinely by BJ editors to mark up article content prior to publication. Recent additions include integration of various text-mining and biodatabase plugins, demonstrating the system's ability to seamlessly integrate on-line content with PDF articles. AVAILABILITY: http://getutopia.com.
Assuntos
Serviços de Informação , Literatura , Publicações , Pesquisa , Software , Internet , Publicações Periódicas como Assunto , Publicações/classificação , EditoraçãoRESUMO
The development of aptamers on custom synthesized DNA microarrays, which has been demonstrated in recent publications, can facilitate detailed analyses of sequence and fitness relationships. Here we use the technique to observe the paths taken through sequence-fitness space by three different evolutionary regimes: asexual reproduction, recombination and model-based evolution. The different evolutionary runs are made on the same array chip in triplicate, each one starting from a small population initialized independently at random. When evolving to a common target protein, glucose-6-phosphate dehydrogenase (G6PD), these nine distinct evolutionary runs are observed to develop aptamers with high affinity and to converge on the same motif not present in any of the starting populations. Regime specific differences in the evolutions, such as speed of convergence, could also be observed.
Assuntos
Aptâmeros de Nucleotídeos/química , Análise de Sequência com Séries de Oligonucleotídeos , Algoritmos , Aptâmeros de Nucleotídeos/genética , Simulação por Computador , Glucosefosfato Desidrogenase/química , Glucosefosfato Desidrogenase/metabolismo , Modelos GenéticosRESUMO
Being born small for gestational age (SGA) confers significantly increased risks of perinatal morbidity and mortality. Accumulating evidence suggests that an SGA fetus results from a poorly perfused and abnormally developed placenta. Some of the placental features seen in SGA, such as abnormal cell turnover and impaired nutrient transport, can be reproduced by culture of placental explants in hypoxic conditions. Metabolic footprinting offers a hypothesis-generating strategy to investigate factors absorbed by and released from this tissue in vitro. Previously, metabolic footprinting of the conditioned culture media has identified differences in placental explants cultured under normoxic and hypoxic conditions and between normal pregnancies and those complicated by pre-eclampsia. In this study we aimed to examine the differences in the metabolic footprint of placental villous explants cultured at different oxygen (O(2)) tensions between women who deliver an SGA baby (n = 9) and those from normal controls (n = 8). Placental villous explants from cases and controls were cultured for 96 h in 1% (hypoxic), 6% (normoxic) and 20% (hyperoxic) O(2). Metabolic footprints were analysed by Ultra Performance Liquid Chromatography coupled to an electrospray hybrid LTQ-Orbitrap Mass Spectrometry (UPLC-MS). 574 metabolite features showed significant difference between SGA and normal at one or more of the oxygen tensions. SGA explant media cultured under hypoxic conditions was observed, on a univariate level, to exhibit the same metabolic signature as controls cultured under normoxic conditions in 49% of the metabolites of interest, suggesting that SGA tissue is acclimatised to hypoxic conditions in vivo. No such behaviour was observed under hyperoxic culture conditions. Glycerophospholipid and tryptophan metabolism were highlighted as areas of particular interest.
Assuntos
Hipóxia/metabolismo , Recém-Nascido Pequeno para a Idade Gestacional/metabolismo , Oxigênio/metabolismo , Placenta/metabolismo , Feminino , Humanos , Recém-Nascido , Metabolômica/métodos , Oxigênio/administração & dosagem , Gravidez , Análise de Componente Principal , Espectrometria de Massas por Ionização por Electrospray , Técnicas de Cultura de TecidosRESUMO
Pre-eclampsia (PE) is a multi-system disorder thought to be mediated by circulating factors released from damaged placental villous trophoblast. There is extensive evidence of changes in the villous tissue in PE, some of which may be replicated by culturing villous tissue in hypoxic conditions. Metabolic footprinting offers a hypothesis-generating strategy to investigate factors released from this tissue in vitro. This study investigated differences in the factors released from villous trophoblast from uncomplicated pregnancies (n=6) and those with PE (n=6). In both cases, explanted placental villous fragments were cultured for 96 h in 1% O(2) (hypoxia) or 6% O(2) (placental normoxia). Metabolites consumed from and released into serum-conditioned culture medium were analysed by Ultra Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS). The relative concentration of 154 features of the metabolic footprint were observed to change in culture medium from uncomplicated pregnancies cultured in normoxic and hypoxic conditions (p<0.00005). 21 and 80 features were also different in culture medium from PE versus uncomplicated pregnancies cultured in hypoxic and normoxic conditions, respectively (p<0.00005). When comparing all 4 groups, 47 metabolic features showed a similar relative concentration in PE-derived media cultured in normoxic conditions to conditioned media from normal villous tissue cultured in hypoxic conditions. These data suggest that hypoxia may have a role in the placental pathogenesis of PE. Three areas of metabolism were highlighted for systems biology investigation; glutamate and glutamine, tryptophan metabolism and leukotriene or prostaglandin metabolism.
Assuntos
Hipóxia/metabolismo , Metabolômica , Placenta/metabolismo , Pré-Eclâmpsia/metabolismo , Cromatografia Líquida , Meios de Cultivo Condicionados , Feminino , Humanos , Espectrometria de Massas , Gravidez/metabolismoRESUMO
The chemical identification of mass spectrometric signals in metabolomic applications is important to provide conversion of analytical data to biological knowledge about metabolic pathways. The complexity of electrospray mass spectrometric data acquired from a range of samples (serum, urine, yeast intracellular extracts, yeast metabolic footprints, placental tissue metabolic footprints) has been investigated and has defined the frequency of different ion types routinely detected. Although some ion types were expected (protonated and deprotonated peaks, isotope peaks, multiply charged peaks) others were not expected (sodium formate adduct ions). In parallel, the Manchester Metabolomics Database (MMD) has been constructed with data from genome scale metabolic reconstructions, HMDB, KEGG, Lipid Maps, BioCyc and DrugBank to provide knowledge on 42,687 endogenous and exogenous metabolite species. The combination of accurate mass data for a large collection of metabolites, theoretical isotope abundance data and knowledge of the different ion types detected provided a greater number of electrospray mass spectrometric signals which were putatively identified and with greater confidence in the samples studied. To provide definitive identification metabolite-specific mass spectral libraries for UPLC-MS and GC-MS have been constructed for 1,065 commercially available authentic standards. The MMD data are available at http://dbkgroup.org/MMD/.
Assuntos
Bases de Dados Factuais , Espectrometria de Massas , Metabolômica/métodos , Cromatografia Líquida de Alta Pressão , Testes de Química Clínica , Feminino , Humanos , Internet , Masculino , Saccharomyces cerevisiae/metabolismoRESUMO
Pre-eclampsia (PE) is a multi-system disorder of pregnancy hypothesised to arise from circulating factors derived from an unhealthy placenta. Some changes in placental phenotype seen in PE can be reproduced by culture in altered oxygen (O2) tension. Currently, these circulating factors are unidentified, partly due to the complexity of maternal plasma. Investigation of factors released from placental tissue provides a potential method to identify bioactive compounds. Experimental strategies to study compounds present in a biological system have expanded greatly in recent years. Metabolomics can detect and identify endogenous and secreted metabolites. We aimed to determine whether metabolites could be identified in placental cultures with acceptable experimental variability and to determine whether altered O2 tension affects the composition of the placental metabolome. In this study we used gas-chromatography-mass spectroscopy to determine the presence of metabolites in conditioned culture medium (CCM) and tissue lysates of placental villous explants cultured in 1, 6 and 20% atmospheric O2 for 96h. This experimental strategy had an intra-assay variation of 6.1-11.6%. Intra and inter-placental variability were 15.7-35.8% and 44.8-46.2% respectively. Metabolic differences were identified between samples cultured in 1, 6 and 20% O2 in both CCM and tissue lysate. Differentially expressed metabolites included: 2-deoxyribose, threitol or erythritol and hexadecanoic acid. We conclude that metabolomic strategies offer a novel approach to investigate placental function. When conducted under carefully controlled conditions, with appropriate statistical analysis, metabolic differences can be identified in placental explants in response to altered O2 tension. Metabolomics could be used to identify changes in conditions associated with placental pathology.
Assuntos
Biomarcadores/metabolismo , Vilosidades Coriônicas/efeitos dos fármacos , Vilosidades Coriônicas/metabolismo , Redes e Vias Metabólicas/efeitos dos fármacos , Oxigênio/farmacologia , Biomarcadores/análise , Caproatos/metabolismo , Técnicas de Cultura de Células , Desoxirribose/metabolismo , Relação Dose-Resposta a Droga , Feminino , Humanos , Redes e Vias Metabólicas/fisiologia , Técnicas de Cultura de Órgãos , Oxirredução/efeitos dos fármacos , Gravidez , Álcoois Açúcares/metabolismoRESUMO
In answering the question 'Systems Biology--will it work?' (which it self-evidently has already), it is appropriate to highlight advances in philosophy, in new technique development and in novel findings. In terms of philosophy, we see that systems biology involves an iterative interplay between linked activities--instance, between theory and experiment, between induction and deduction and between measurements of parameters and variables--with more emphasis than has perhaps been common now being focused on the first in each of these pairs. In technique development, we highlight closed loop machine learning and its use in the optimization of scientific instrumentation, and the ability to effect high-quality and quasi-continuous optical images of cells. This leads to many important and novel findings. In the first case, these may involve new biomarkers for disease, whereas in the second case, we have determined that many biological signals may be frequency-rather than amplitude-encoded. This leads to a very different view of how signalling 'works' (equations such as that of Michaelis and Menten which use only amplitudes, i.e. concentrations, are inadequate descriptors), lays emphasis on the signal processing network elements that lie 'downstream' of what are traditionally considered the signals, and allows one simply to understand how cross-talk may be avoided between pathways which nevertheless use common signalling elements. The language of cells is much richer than we had supposed, and we are now well placed to decode it.
Assuntos
Fenômenos Fisiológicos Celulares , Simulação por Computador , Metabolismo , Modelos Biológicos , Biologia de Sistemas , Genômica , Transdução de SinaisRESUMO
AIMS: To determine the utility of vacuum-packed polythene bags as a convenient, flexible and cost-effective alternative to fixed volume glass vessels for lab-scale silage studies. METHODS AND RESULTS: Using perennial ryegrass or red clover forage, similar fermentations (as assessed by pH measurement) occurred in glass tube and vacuum-packed silos over a 35-day period. As vacuum-packing devices allow modification of initial packing density, the effect of four different settings (initial packing densities of 0.397, 0.435, 0.492 and 0.534 g cm(-3)) on the silage fermentation over 16 days was examined. Significant differences in pH decline and lactate accumulation were observed at different vacuum settings. Gas accumulation was apparent within all bags and changes in bag volume with time was observed to vary according to initial packing density. CONCLUSIONS: Vacuum-packed silos do provide a realistic model system for lab-scale silage fermentations. SIGNIFICANCE AND IMPACT OF THE STUDY: Use of vacuum-packed silos holds potential for lab-scale evaluations of silage fermentations, allowing higher throughput of samples, more consistent packing as well as the possibility of investigating the effects of different initial packing densities and use of different wrapping materials.
Assuntos
Ração Animal , Microbiologia de Alimentos , Silagem , Monitoramento Ambiental/instrumentação , Monitoramento Ambiental/métodos , Fermentação , Modelos Biológicos , VácuoRESUMO
In previous work, we studied the behaviour of a model of part of the NF-kappaB signalling pathway. The model displayed oscillations that varied both in number, amplitude and frequency when its parameters were varied. Sensitivity analysis showed that just nine of the 64 reaction parameters were mainly responsible for the control of the oscillations when these parameters were varied individually. However, the control of the properties of any complex system is distributed, and, as many of these reactions are highly non-linear, we expect that their interactions will be too. Pairwise modulation of these nine parameters gives a search space some 50 times smaller (81 against 4096) than that required for the pairwise modulation of all 64 reactions, and this permitted their study (which would otherwise have been effectively intractable). Strikingly synergistic effects were observed, in which the effect of one of the parameters was strongly (and even qualitatively) dependent on the values of another parameter. Regions of parameter space could be found in which the amplitude, but not the frequency (timing), of oscillations varied, and vice versa. Such modelling will permit the design and performance of experiments aimed at disentangling the role of the dynamics of oscillations, rather than simply their amplitude, in determining cell fate. Overall, the analyses reveal a level of complexity in these dynamic models that is not apparent from study of their individual parameters alone and point to the value of manipulating multiple elements of complex networks to achieve desired physiological effects.
Assuntos
Relógios Biológicos/fisiologia , Fenômenos Fisiológicos Celulares , Modelos Biológicos , NF-kappa B/metabolismo , Transdução de Sinais/fisiologia , Animais , Simulação por Computador , Retroalimentação/fisiologia , Regulação da Expressão Gênica/fisiologia , HumanosRESUMO
Signaling by the transcription factor nuclear factor kappa B (NF-kappaB) involves its release from inhibitor kappa B (IkappaB) in the cytosol, followed by translocation into the nucleus. NF-kappaB regulation of IkappaBalpha transcription represents a delayed negative feedback loop that drives oscillations in NF-kappaB translocation. Single-cell time-lapse imaging and computational modeling of NF-kappaB (RelA) localization showed asynchronous oscillations following cell stimulation that decreased in frequency with increased IkappaBalpha transcription. Transcription of target genes depended on oscillation persistence, involving cycles of RelA phosphorylation and dephosphorylation. The functional consequences of NF-kappaB signaling may thus depend on number, period, and amplitude of oscillations.
Assuntos
Regulação da Expressão Gênica , NF-kappa B/metabolismo , Transdução de Sinais , Transporte Ativo do Núcleo Celular , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Simulação por Computador , Citoplasma/metabolismo , Etoposídeo/farmacologia , Retroalimentação Fisiológica , Células HeLa , Humanos , Proteínas I-kappa B/genética , Proteínas I-kappa B/metabolismo , Modelos Biológicos , Inibidor de NF-kappaB alfa , Fosforilação , Proteínas Recombinantes de Fusão/metabolismo , Fator de Transcrição RelA , Transcrição Gênica , Transfecção , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Analysis of cellular signalling interactions is expected to create an enormous informatics challenge, perhaps even greater than that of analysing the genome. A key step in the evolution towards a more quantitative understanding of signalling is to specify explicitly the kinetics of all chemical reaction steps in a pathway. We have reconstructed a model of the nuclear factor, kappaB (NF-kappaB) signalling pathway, containing 64 parameters and 26 variables, including steps in which the activation of the NF-kappaB transcription factor is intimately associated with the phosphorylation and ubiquitination of its inhibitor kappaB by a membrane-associated kinase, and its translocation from the cytoplasm to the nucleus. We apply sensitivity analysis to the model. This identifies those parameters in this (IkappaB)/NF-kappaB signalling system (containing only induced IkappaBalpha isoform) that most affect the oscillatory concentration of nuclear NF-kappaB (in terms of both period and amplitude). The intention is to provide guidance on which proteins are likely to be most significant as drug targets or should be exploited for further, more detailed experiments. The sensitivity coefficients were found to be strongly dependent upon the magnitude of the parameter change studied, indicating the highly non-linear nature of the system. Of the 64 parameters in the model, only eight to nine exerted a major control on nuclear NF-kappaB oscillations, and each of these involved as reaction participants either the IkappaB kinase (IKK) or IkappaBalpha, directly. This means that the dominant dynamics of the pathway can be reflected, in addition to that of nuclear NF-kappaB itself, by just two of the other pathway variables. This is conveniently observed in a phase-plane plot.
Assuntos
Relógios Biológicos/fisiologia , Quinase I-kappa B/metabolismo , Modelos Biológicos , NF-kappa B/metabolismo , Transdução de Sinais/fisiologia , Animais , Simulação por Computador , Retroalimentação/fisiologia , Humanos , Sensibilidade e EspecificidadeRESUMO
The newly sequenced genome of Streptomyces coelicolor is estimated to encode 7825 theoretical proteins. We have mapped approximately 10% of the theoretical proteome experimentally using two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. Products from 770 different genes were identified, and the types of proteins represented are discussed in terms of their annotated functional classes. An average of 1.2 proteins per gene was observed, indicating extensive post-translational regulation. Examples of modification by N-acetylation, adenylylation and proteolytic processing were characterized using mass spectrometry. Proteins from both primary and certain secondary metabolic pathways are strongly represented on the map, and a number of these enzymes were identified at more than one two-dimensional gel location. Post-translational modification mechanisms may therefore play a significant role in the regulation of these pathways. Unexpectedly, one of the enzymes for synthesis of the actinorhodin polyketide antibiotic appears to be located outside the cytoplasmic compartment, within the cell wall matrix. Of 20 gene clusters encoding enzymes characteristic of secondary metabolism, eight are represented on the proteome map, including three that specify the production of novel metabolites. This information will be valuable in the characterization of the new metabolites.
Assuntos
Processamento de Proteína Pós-Traducional , Proteoma/metabolismo , Streptomyces/metabolismo , Acetilação , Proteínas de Bactérias/análise , Proteínas de Bactérias/metabolismo , Bases de Dados de Proteínas , Eletroforese em Gel Bidimensional , Internet , Proteoma/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Streptomyces/genéticaRESUMO
After growth of Rhodococcus rhodochrous in Sauton's medium, and further incubation for about 60 h in stationary phase, there was a transient (up to 5 log) decrease in the c.f.u. count, whereas the total count remained similar to its initial value. At the point of minimal viability, the most probable number (MPN) count was 10 times greater than the c.f.u. count. This difference was further magnified by 3-4 logs (giving values close to the total count) by incorporating supernatant taken from growing cultures. A small protein similar to Rpf (resuscitation-promoting factor of Micrococcus luteus) appeared to be responsible for some of the activity in the culture supernatant. The formation of "non-culturable" cells of the "Academia" strain of Mycobacterium tuberculosis was similarly observed following growth in Sauton's medium containing Tween 80 in sealed culture vessels, and further incubation for an extended stationary phase. This resulted in the formation, 4-5 months post-inoculation, of a homogeneous population of ostensibly "non-culturable" cells (zero c.f.u.). Remarkably, the MPN count for these cultures was 10(5) organisms ml(-1), and this value was further increased by one log using supernatant from an actively growing culture. Populations of "non-culturable" cells of Mycobacterium tuberculosis were also obtained by the filtration of "clumpy" cultures, which were grown in the absence of Tween 80. These small cells could only be grown in liquid medium (MPN) and their viability was enhanced by the addition of culture supernatant or Rpf. The "non-culturable" cells that accumulated during prolonged stationary phase in both the R. rhodochrous and the Mycobacterium tuberculosis cultures were small ovoid and coccoid forms with an intact permeability barrier, but with undetectable respiratory activity. The authors consider these non-culturable bacteria to be dormant. The observed activity of culture supernatants and Rpf with "non-culturable" bacterial suspensions invites the speculation that one, or more, of the cognate Mycobacterium tuberculosis Rpf-like molecule(s) could be involved in mechanisms of latency and reactivation of tuberculosis in vivo.
Assuntos
Proteínas de Bactérias , Interfase , Mycobacterium tuberculosis/citologia , Mycobacterium tuberculosis/crescimento & desenvolvimento , Rhodococcus/citologia , Rhodococcus/crescimento & desenvolvimento , Divisão Celular/efeitos dos fármacos , Contagem de Colônia Microbiana , Meios de Cultivo Condicionados/farmacologia , Citocinas/imunologia , Citocinas/metabolismo , Temperatura Alta , Interfase/efeitos dos fármacos , Micrococcus luteus , Microscopia Eletrônica de Varredura , Modelos Biológicos , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/ultraestrutura , Oxirredução , Rhodococcus/metabolismo , Rhodococcus/ultraestruturaRESUMO
Direct injection electrospray ionization mass spectrometry (ESI-MS) without prior analyte separation was investigated for the analysis of whole cell suspensions of bacteria. Thirty-six strains of aerobic endospore-forming bacteria, consisting of six Bacillus species and one Brevibacillus species, were studied
Assuntos
Bactérias Aeróbias/química , Esporos Bacterianos/química , Bacillus/química , Espectrometria de Massas por Ionização por Electrospray , UltracentrifugaçãoRESUMO
SUMMARY: We describe a program for the construction of spatially distributed metabolic models, which may then be simulated using the metabolic simulator GEPASI: This is useful for the modelling of heterogeneous systems whether as liquid cultures or as spatially organised systems with specified interconnections.
Assuntos
Simulação por Computador , Metabolismo/fisiologia , Modelos Biológicos , SoftwareRESUMO
A large proportion of the 6,000 genes present in the genome of Saccharomyces cerevisiae, and of those sequenced in other organisms, encode proteins of unknown function. Many of these genes are "silent, " that is, they show no overt phenotype, in terms of growth rate or other fluxes, when they are deleted from the genome. We demonstrate how the intracellular concentrations of metabolites can reveal phenotypes for proteins active in metabolic regulation. Quantification of the change of several metabolite concentrations relative to the concentration change of one selected metabolite can reveal the site of action, in the metabolic network, of a silent gene. In the same way, comprehensive analyses of metabolite concentrations in mutants, providing "metabolic snapshots," can reveal functions when snapshots from strains deleted for unstudied genes are compared to those deleted for known genes. This approach to functional analysis, using comparative metabolomics, we call FANCY-an abbreviation for functional analysis by co-responses in yeast.
Assuntos
Metabolismo Energético/genética , Genoma Fúngico , Genômica/métodos , Mutação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Nucleotídeos de Adenina/metabolismo , Análise por Conglomerados , Genótipo , Hexosefosfatos/metabolismo , Fenótipo , Piruvatos/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimentoRESUMO
Most image-based analyses, using absorbance or fluorescence of the spatial distribution of identifiable structures in complex biological systems, use only a very small number of dimensions of possible spectral data for the generation and interpretation of the image. We here extend the concepts of hyperspectral imaging, being developed in remote sensing, into analytical biotechnology. The massive volume of information contained in hyperspectral spectroscopic images requires multivariate analysis in order to extract the chemical and spatial information contained within the data. We here describe the use of multivariate statistical methods to map and quantify common protein staining fluorophores (SYPRO Red, Orange and Tangerine) in electrophoretic gels. Specifically, we find (a) that the 'background' underpinning limits of detection is due more to proteins that have not migrated properly than to impurities or to ineffective destaining, (b) the detailed mechanisms of staining of SYPRO red and orange are apparently not identical, and in particular (c) that these methods can provide two orders of magnitude improvement in the detection limit per pixel, to levels well below the limit observable optically.
Assuntos
Biotecnologia/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Microscopia de Fluorescência/métodos , Corantes Fluorescentes/farmacologia , Análise MultivariadaRESUMO
Very little is known about the culturability and viability of mycobacteria following their phagocytosis by macrophages. We therefore studied populations of the avirulent 'Academia' strain of Mycobacterium tuberculosis isolated from murine peritoneal macrophage lysates several days post-infection in vivo. The resulting bacterial suspensions contained a range of morphological types including rods, ovoid forms and coccoid forms. Bacterial viability measured using the MPN method (dilution to extinction in liquid medium) was often much higher than that measured by CFU (plating on solid medium). Viability in the MPN assay was further enhanced when the Micrococcus luteus protein, Rpf, was incorporated into the liquid culture medium at picomolar concentrations. Rpf is an example of a family of autocrine growth factors found throughout the high G+C cohort of Gram-positive bacteria including M. tuberculosis. M. tuberculosis cells obtained from macrophages had altered surface properties, as compared with bacteria grown in vitro. This was indicated by loss of the ability to adsorb bacteriophage DS6A, a reduced tendency to form clumps, acquisition of ethidium bromide stainability following heat treatment, and loss of Rpf-mediated resuscitation following freezing and thawing. These results indicate that a proportion of 'unculturable' M. tuberculosis cells obtained from macrophages is either injured or dormant and that these cells may be recovered or resuscitated using Rpf in liquid medium.