Biomedical subjects

Michael K Winson

Publications and source records attributed to Michael K Winson.

3 recordsLinked to original sources

PubMed2003

Using flow cytometry to quantify microbial heterogeneity.

Flow cytometry is a powerful technique for the study of single cells, and thus it is of particular utility in the study of heterogeneity in microbial populations. This review seeks to highlight the role of flow cytometric analyses in studies of microbial heterogeneity, drawing wherever possible on recently published research articles. Whilst microbial heterogeneity is well documented in both natural and laboratory environments, the underlying causes are less well understood. Possible sources for the heterogeneity that is observed in microbial systems are discussed, together with the flow cytometric tools that aid its study. The role of flow cytometry in molecular biology is discussed with reference to gene reporter systems, which enable heterogeneity of gene expression to be monitored. With the recent sequencing of a variety of microbial genomes, it is anticipated that flow cytometry will have an increasing role to play in studying the effects of gene expression and mutation on heterogeneity, and in resolving the interactions of genetics and physiology.

Bacteria↗

PubMed2002

Monitoring of complex industrial bioprocesses for metabolite concentrations using modern spectroscopies and machine learning: application to gibberellic acid production.

Two rapid vibrational spectroscopic approaches (diffuse reflectance-absorbance Fourier transform infrared [FT-IR] and dispersive Raman spectroscopy), and one mass spectrometric method based on in vacuo Curie-point pyrolysis (PyMS), were investigated in this study. A diverse range of unprocessed, industrial fed-batch fermentation broths containing the fungus Gibberella fujikuroi producing the natural product gibberellic acid, were analyzed directly without a priori chromatographic separation. Partial least squares regression (PLSR) and artificial neural networks (ANNs) were applied to all of the information-rich spectra obtained by each of the methods to obtain quantitative information on the gibberellic acid titer. These estimates were of good precision, and the typical root-mean-square error for predictions of concentrations in an independent test set was <10% over a very wide titer range from 0 to 4925 ppm. However, although PLSR and ANNs are very powerful techniques they are often described as "black box" methods because the information they use to construct the calibration model is largely inaccessible. Therefore, a variety of novel evolutionary computation-based methods, including genetic algorithms and genetic programming, were used to produce models that allowed the determination of those input variables that contributed most to the models formed, and to observe that these models were predominantly based on the concentration of gibberellic acid itself. This is the first time that these three modern analytical spectroscopies, in combination with advanced chemometric data analysis, have been compared for their ability to analyze a real commercial bioprocess. The results demonstrate unequivocally that all methods provide very rapid and accurate estimates of the progress of industrial fermentations, and indicate that, of the three methods studied, Raman spectroscopy is the ideal bioprocess monitoring method because it can be adapted for on-line analysis.

Algorithms↗

PubMed1997

Quorum sensing and Chromobacterium violaceum: exploitation of violacein production and inhibition for the detection of N-acylhomoserine lactones.

Quorum sensing relies upon the interaction of a diffusible signal molecule with a transcriptional activator protein to couple gene expression with cell population density. In Gram-negative bacteria, such signal molecules are usually N-acylhomoserine lactones (AHLs) which differ in the structure of their N-acyl side chains. Chromobacterium violaceum, a Gram-negative bacterium commonly found in soil and water, produces the characteristic purple pigment violacein. Previously the authors described a violacein-negative, mini-Tn5 mutant of C. violaceum (CV026) in which pigment production can be restored by incubation with supernatants from the wild-type strain. To develop this mutant as a general biosensor for AHLs, the natural C. violaceum AHL molecule was first chemically characterized. By using solvent extraction, HPLC and mass spectrometry, a single AHL, N-hexanoyl-L-homoserine lactone (HHL), was identified in wild-type C. violaceum culture supernatants which was absent from CV026. Since the production of violacein constitutes a simple assay for the detection of AHLs, we explored the ability of CV026 to respond to a series of synthetic AHL and N-acylhomocysteine thiolactone (AHT) analogues. In CV026, violacein is inducible by all the AHL and AHT compounds evaluated with N-acyl side chains from C4 to C8 in length, with varying degrees of sensitivity. Although AHL compounds with N-acyl side chains from C10 to C14 are unable to induce violacein production, if an activating AHL (e.g. HHL) is incorporated into the agar, these long-chain AHLs can be detected by their ability to inhibit violacein production. The versatility of CV026 in facilitating detection of AHL mixtures extracted from culture supernatants and separated by thin-layer chromatography is also demonstrated. These simple bioassays employing CV026 thus greatly extend the ability to detect a wide spectrum of AHL signal molecules.

Chromobacterium↗