Showing papers by "Douglas B. Kell published in 2000"

Detection of the dipicolinic acid biomarker in Bacillus spores using Curie-point pyrolysis mass spectrometry and Fourier transform infrared spectroscopy.

Abstract: Thirty-six strains of aerobic endospore-forming bacteria confirmed by polyphasic taxonomic methods to belong to Bacillus amyloliquefaciens, Bacillus cereus, Bacillus licheniformis, Bacillus megaterium, Bacillus subtilis (including Bacillus niger and Bacillus globigii), Bacillus sphaericus, and Brevi laterosporus were grown axenically on nutrient agar, and vegetative and sporulated biomasses were analyzed by Curie-point pyrolysis mass spectrometry (PyMS) and diffuse reflectance−absorbance Fourier-transform infrared spectroscopy (FT-IR). Chemometric methods based on rule induction and genetic programming were used to determine the physiological state (vegetative cells or spores) correctly, and these methods produced mathematical rules which could be simply interpreted in biochemical terms. For PyMS it was found that m/z 105 was characteristic and is a pyridine ketonium ion (C6H3ON+) obtained from the pyrolysis of dipicolinic acid (pyridine-2,6-dicarboxylic acid; DPA), a substance found in spores but not in ...

The Kyoto encyclopedia of genes and genomes--KEGG.

Abstract: The aim of the present study was to explore the function and interaction of differentially expressed genes (DEGs) in pulmonary embolism (PE). The gene expression profile GSE13535, was downloaded from the Gene Expression Omnibus database. The DEGs 2 and 18 h post-PE initiation were identified using the affy package in R software. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of the DEGs were analyzed using Database for Annotation Visualization and Integrated Discovery (DAVID) online analytical tools. In addition, protein-protein interaction (PPI) networks of the DEGs were constructed using the Search Tool for the Retrieval of Interacting Genes/Proteins. The PPI network at 18 h was modularized using ClusterONE, and a functional enrichment analysis of the DEGs in the top three modules was performed with DAVID. Overall, 80 and 346 DEGs were identified 2 and 18 h after PE initiation, respectively. The KEGG pathways, including chemokine signaling and toll‐like receptor signaling, were shown to be significantly enriched. The five highest degree nodes in the PPI networks at 2 or 18 h were screened. The module analysis of the PPI network at 18 h revealed 11 hub nodes. A Gene Ontology terms analysis demonstrated that the DEGs in the top three modules were associated with the inflammatory, defense and immune responses. The results of the present study suggest that the DEGs identified, including chemokine‐related genes TFPI2 and TNF, may be potential target genes for the treatment of PE. The chemokine signaling pathway, inflammatory response and immune response were explored, and it may be suggested that these pathways have important roles in PE.

On-line, real time measurements of cellular biomass using dielectric spectroscopy

Abstract: Introduction All else being equal, the productivity of a biological process is determined by the quantity of biomass present. There is therefore a major requirement for the accurate measurement and control of the biomass within fermentors, at both laboratory and industrial scales. Presently the range of sensors available that can be used in situ and reliably for the monitoring and regulation of biotechnological processes in general is rather limited. These sensors normally rely upon physical (e.g. optical, mechanical and electrical) or chemical variables (e.g. pH and concentration) rather than biological ones per se (Sarra et al., 1996; Pons, 1991). However only physical methods allow the on-line, real-time estimation of biomass (Harris and Kell, 1985). As well as physical methods, any easily determinable chemical that is produced or consumed by cells at an essentially constant rate during cell growth may also be used to assess biomass, e.g. carbon dioxide evolution and oxygen consumption. In these indirect methods biomass is then calculated based upon mass balances, stoichiometric relationships or empirical constants. However, this type of approach has the great disadvantage that it does not generally discriminate between biomass and necromass (Kell et al., 1990). Even if biomass was easily measurable there is still the question of what is biologically relevant information for fermentation control and how can one define and quantify it (e.g. metabolism, viability, vitality, morphology) (Kell et al., 1987; Kell, 1987a;

Culturability of Mycobacterium tuberculosis cells isolated from murine macrophages: a bacterial growth factor promotes recovery

Abstract: 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.

Explanatory Analysis of the Metabolome Using Genetic Programming of Simple, Interpretable Rules

Abstract: Genetic programming, in conjunction with advanced analytical instruments, is a novel tool for the investigation of complex biological systems at the whole-tissue level. In this study, samples from tomato fruit grown hydroponically under both high- and low-salt conditions were analysed using Fourier-transform infrared spectroscopy (FTIR), with the aim of identifying spectral and biochemical features linked to salinity in the growth environment. FTIR spectra of whole tissue extracts are not amenable to direct visual analysis, so numerical modelling methods were used to generate models capable of classifying the samples based on their spectral characteristics. Genetic programming (GP) provided models with a better prediction accuracy to the conventional data modelling methods used, whilst being much easier to interpret in terms of the variables used. Examination of the GP-derived models showed that there were a small number of spectral regions that were consistently being used. In particular, the spectral region containing absorbances potentially due to a cyanide/nitrile functional group was identified as discriminatory. The explanatory power of the GP models enabled a chemical interpretation of the biochemical differences to be proposed. The combination of FTIR and GP is therefore a powerful and novel analytical tool that, in this study, improves our understanding of the biochemistry of salt tolerance in tomato plants.

Efficient Improvement of Silage Additives by Using Genetic Algorithms

Abstract: The enormous variety of substances which may be added to forage in order to manipulate and improve the ensilage process presents an empirical, combinatorial optimization problem of great complexity. To investigate the utility of genetic algorithms for designing effective silage additive combinations, a series of small-scale proof of principle silage experiments were performed with fresh ryegrass. Having established that significant biochemical changes occur over an ensilage period as short as 2 days, we performed a series of experiments in which we used 50 silage additive combinations (prepared by using eight bacterial and other additives, each of which was added at six different levels, including zero [i.e., no additive]). The decrease in pH, the increase in lactate concentration, and the free amino acid concentration were measured after 2 days and used to calculate a “fitness” value that indicated the quality of the silage (compared to a control silage made without additives). This analysis also included a “cost” element to account for different total additive levels. In the initial experiment additive levels were selected randomly, but subsequently a genetic algorithm program was used to suggest new additive combinations based on the fitness values determined in the preceding experiments. The result was very efficient selection for silages in which large decreases in pH and high levels of lactate occurred along with low levels of free amino acids. During the series of five experiments, each of which comprised 50 treatments, there was a steady increase in the amount of lactate that accumulated; the best treatment combination was that used in the last experiment, which produced 4.6 times more lactate than the untreated silage. The additive combinations that were found to yield the highest fitness values in the final (fifth) experiment were assessed to determine a range of biochemical and microbiological quality parameters during full-term silage fermentation. We found that these combinations compared favorably both with uninoculated silage and with a commercial silage additive. The evolutionary computing methods described here are a convenient and efficient approach for designing silage additives.

Snapshots of Systems

Abstract: Following its original formulation in 1973 (Heinrich & Rapoport 1973, 1974; Kacser & Burns 1973) as a means of understanding the contribution of the individual steps of a biochemical pathway to the values of flux and metabolite concentrations observed, some 13 years were to pass before we first surveyed (Kell & Westerhoff, 1986a b) how the formalism, tools and terms of metabolic control analysis might usefully be applied to such systems in a biotechnological context. Since another such period has now elapsed, it is timely to take stock of progress, to recognize that the take-up of these methods among biotechnologists has been less than widespread, and (as requested by the Editors) to give a personal and critical review of successes, failures, problems and prospects for the use of metabolic control analysis in biotechnology. In what follows, it is taken that the reader has a good working knowledge of the essential principles of metabolic control analysis, as summarized for instance in Kell & Westerhoff (1986a), Kell et al. (1989), Cornish-Bowden & Cardenas (1990), Fell (1992, 1997), and Heinrich & Schuster (1996.); similar information is available on the Internet at http://gepasi.dbs.aber.ac.uk/metab/mca_home.htm and in links therefrom. In addition, we shall concentrate on unicellular systems, implicitly those most commonly exploited to make products of biotechnological interest.

Genomic computing: explanatory modelling for functional genomics

Abstract: Many newly discovered genes are of unknown function. DNA microarrays are a method for determining the expression levels of all genes in an organism for which a complete genome sequence is available. By comparing the expression changes under different conditions it should be possible to assign functions to these genes. However, many hundreds of thousands of data points may be produced over a series of experiments. Genetic programming provided simple explanatory rules for gene function from such datasets, where previous approaches had not succeeded.

A Portable Flow Cytometer for the Detection and Identification of Microorganisms

Abstract: Flow cytometry can be used for the detection and identification of microorganism [1]. However traditional instrumentation is bulky and complicated to operate and align. These factors combine to make flow cytometry less than ideal for the field detection of biological agents.

Dormancy in Non-Sporulating Bacteria: Its Significance for Environmental Monitoring

Abstract: In natural ecosystems, the total cell count obtained microscopically typically exceeds the viable count on non-selective media by orders of magnitude. The question therefore arises as to whether the “invisible”, apparently nonculturable cells are dead, are killed by our isolation media, or are merely in a dormant state from which we might in principle be able to resuscitate them if only we knew how. In particular the suggested “viable-but-nonculturable” (VBNC) bacteria have been invoked to explain phenomena as divergent as the epidemiology of some infections and the persistence of genetically marked organisms in the environment (e.g. the failure to isolate Vibrio cholerae and Campylobacter jejuni from clearly implicated sources or reservoirs of infection could be accounted for on the basis of their being present in a VBNC or dormant state). Application of flow cytometry may be a useful tool to visualize bacteria without their growing and to discriminate between dead and dormant bacteria. We found that dormant Micrococcus luteus can be physically separated from dead cells by flow cytometry sorter after cell staining with rhodamine 123. Resuscitation of dormant M.luteus cells in liquid medium does not proceed in the absence of a culture supernatant from batch-grown cells. This suggests that viable cells can excrete a pheromone-like substance necessary for the resuscitation of dormant cells.

The use of nonlinear dielectric spectroscopy to monitor the bioelectromagnetic effects of a weak pulsed magnetic field in real time.

Abstract: Nonlinear dielectric spectroscopy (NLDS) was used to detect interaction of a pulsed magnetic field (PMF) with membrane protein dynamics in aggregating Dictyostelium discoideum amoebae. In the experiments reported here, a strong nonlinear dielectric response of Dictyostelium discoideum cells is shown, and a distinctive nonlinear dielectric response of cells previously exposed to PMF is shown. The method of NLDS is shown to be capable of monitoring and charting the dynamic frequency response of the cell to an electromagnetic field. Bioelectromagnetics 21:25‐33, 2000. fl 2000 Wiley-Liss, Inc.

Intelligent Systems for the Characterization of Microorganisms from Hyperspectral Data

Abstract: Three rapid spectroscopic approaches for whole-organism fingerprinting, viz. pyrolysis mass spectrometry (PyMS), Fourier transform infra-red spectroscopy (FT-IR) and dispersive Raman microscopy, were used to analyze a group of 59 clinical bacterial isolates associated with urinary tract infection.