Biomedical subjects

Nick Thomas

Publications and source records attributed to Nick Thomas.

9 recordsLinked to original sources

PubMed2007

Fluorescent proteins and engineered cell lines.

Green fluorescent protein and other fluorescent proteins provide powerful tools for high content analysis of cellular processes. Engineering fluorescent protein sensors for expression in cellular assays requires consideration of a wide range of design factors to produce fusion proteins capable of generating informative and biologically relevant data while meeting the rigorous demands of high content screening. The target protein, fluorescent protein, host cell line, construct components and orientation, expression level, and other factors all contribute to the performance of the sensor. This chapter reviews the process of selecting an optimum combination of these design elements for engineering cells for high content analysis.

Animals↗

PubMed2006

Guidelines for the diagnosis and management of individuals with neurofibromatosis 1.

Neurofibromatosis 1 (NF1) is a common neurocutaneous condition with an autosomal dominant pattern of inheritance. The complications are diverse and disease expression varies, even within families. Progress in molecular biology and neuroimaging and the development of mouse models have helped to elucidate the aetiology of NF1 and its clinical manifestations. Furthermore, these advances have raised the prospect of therapeutic intervention for this complex and distressing disease. Members of the United Kingdom Neurofibromatosis Association Clinical Advisory Board collaborated to produce a consensus statement on the current guidelines for diagnosis and management of NF1. The proposals are based on published clinical studies and on the pooled knowledge of experts in neurofibromatosis with experience of providing multidisciplinary clinical and molecular services for NF1 patients. The consensus statement discusses the diagnostic criteria, major differential diagnoses, clinical manifestations and the present strategies for monitoring and management of NF1 complications.

Child↗

PubMed2006

Dynamic green fluorescent protein sensors for high-content analysis of the cell cycle.

We have developed two dynamic sensors that report cell cycle position in living mammalian cells. The sensors use well-characterized components from proteins that are spatially and temporally regulated through the cell cycle. Coupling of these components to Enhanced Green Fluorescent Protein (EGFP) has been used to engineer fusion proteins that report G1/S and G2/M transitions during the cell cycle without perturbing cell cycle progression. Expression of these sensors in stable cell lines allows high content analysis of the effects of drugs and gene knockdown on the cell cycle using automated image analysis to determine cell cycle position and to abstract correlative data from multiplexed sensors and morphological analysis.

Animals↗

PubMed2006

Adenoviral sensors for high-content cellular analysis.

To maximize the potential of high-content cellular analysis for investigating complex cellular signaling pathways and processes, we have generated a library of adenoviral encoded cellular sensors based on protein translocation and reporter gene activation that enable a diverse set of assays to be applied to lead compound profiling in drug discovery and development. Adenoviral vector transduction is an efficient and technically simple system for expression of cellular sensors in diverse cell types, including primary cells. Adenoviral vector-mediated transient expression of cellular sensors, either as fluorescent protein fusions or live cell gene reporters, allows rapid assay development for profiling the activities of candidate drugs across multiple cellular systems selected for biological and physiological relevance to the target disease state.

Adenoviridae↗

PubMed2005

Characterization and gene expression profiling of a stable cell line expressing a cell cycle GFP sensor.

The use of stable cell lines expressing fusions with green fluorescent protein (GFP) has increased significantly in recent years. In this study we have used a range of complimentary analytical techniques to examine the characteristics of a cell line stably expressing a EGFP cell cycle sensor relative to parental U2OS cells. Analysis of cell cycle duration and cell cycle phase distribution by cell growth assays and flow cytometry revealed that the two cell lines had identical doubling times and cell cycle distributions. Measurement of EGFP fusion protein mRNA by quantitative RT-PCR indicated a EGFP sensor expression level equivalent to endogenous Cyclin B1 (7000 copies/cell in G2). Microarray analysis showed a 0.9% (>2 fold at p<0.001 across 20,000 genes) difference in global gene expression levels between parental and EGFP expressing U2OS cells, with no significant differences in expression of A, B, C, D, E, F, G, H, I, K, L, M or T type Cyclins between the two cell types. These results confirm that engineering a stable cell line for low expression of a EGFP cell cycle sensor is minimally perturbing to the cell cycle and cellular gene expression.

Cell Cycle↗

PubMed2003

Image-based screening of signal transduction assays.

Imaging techniques have played a vital role in signal transduction research over several decades. Recently, industrialized macro- and micro-imaging systems have found application in drug discovery laboratories, where they increase the throughput and efficiency of drug screening. Macro-imagers are used for primary screening, where they favor compound conservation (through assay miniaturization), and achieve unprecedented rates of throughput. Micro-imaging systems achieve relatively high throughput, at the same time providing sub-cellular resolution with fixed or living cells. These micro-imaging analyses were previously conducted at very low throughput and, typically, were the sole domain of the academic researcher. Although both macro and micro forms of image-based screening remain technologies in development, they have already made substantial contributions to screening programs and will continue to do so.

Fluorescence Resonance Energy Transfer↗

PubMed2002

Genotype-phenotype correlations in X-linked myotubular myopathy.

X-linked myotubular myopathy is a severe congenital myopathy that presents in the neonatal period with profound hypotonia and an inability to establish spontaneous respiration. Usually death occurs in infancy from respiratory failure. However, there is phenotypic variability; a number of affected boys have achieved respiratory independence and become ambulatory. Disease-causing mutations have been identified throughout the MTM1 gene on Xq28. MTM1 encodes the protein myotubularin, which is expressed ubiquitously. The main objectives of this study were to establish whether the nature or site of the mutation in the MTM1 gene could predict severity of the disease and to investigate whether early intensive clinical intervention facilitated survival until spontaneous improvement occurred. An association was demonstrated between the presence of a non-truncating mutation of the MTM1 gene and the mild phenotype. However, many non-truncating mutations were also seen in association with the severe phenotype and these were not confined to recognized functional domains of the protein. This suggests that the use of mutation analysis to predict prognosis in the early period following diagnosis is limited. Unexpectedly, over 50 patients surviving for more than 1 year were identified in this study. Further information obtained on 40 of these cases revealed that 50% were receiving 24-h ventilatory support, while 27% were ventilated at night only. The high survival rate for this disorder therefore reflects intensive medical intervention without which the majority of these boys would not survive.

Chi-Square Distribution↗

PubMed2002

The evolution of the vertebrate beta-globin gene promoter.

Complexity analysis is capable of highlighting those gross evolutionary changes in gene promoter regions (loosely termed "promoter shuffling") that are undetectable by conventional DNA sequence alignment. Complexity analysis was therefore used here to identify the modular components (blocks) of the orthologous beta-globin gene promoter sequences of 22 vertebrate species, from zebrafish to humans. Considerable variation between the beta-globin gene promoters was apparent in terms of block presence/absence, copy number, and relative location. Some sequence blocks appear to be ubiquitous, whereas others are restricted to a specific taxon. Block similarities were also evident between the promoters of the paralogous human beta-like globin genes. It may be inferred that a wide variety of different mutational mechanisms have operated upon the beta-globin gene promoter over evolutionary time. Because these include gross changes such as deletion, duplication, amplification, elongation, contraction, and fusion, as well as the steady accumulation of single base-pair substitutions, it is clear that some redefinition of the term "promoter shuffling" is required. This notwithstanding, and as previously described for the vertebrate growth hormone gene promoter, the modular structure of the beta-globin promoter region and those of its paralogous counterparts have continually been rearranged into new combinations through the alteration, or shuffling, of preexisting blocks. Some of these changes may have had no influence on promoter function, but others could have altered either the level of gene expression or the responsiveness of the promoter to external stimuli. The comparative study of vertebrate beta-globin gene promoter regions described here confirms the generality of the phenomenon of sequence block shuffling and thus supports the view that it could have played an important role in the evolution of differential gene expression.

Amino Acid Sequence↗

PubMedDate not supplied

Lighting the circle of life: fluorescent sensors for covert surveillance of the cell cycle.

The cell cycle is the collective mechanism through which all of us develop, exist and in many cases, when it goes wrong, die. Despite enormous progress in unravelling the complexity of the cell cycle through intensive study over the past 100 years, development of new tools to analyse the process and associated cellular events has not kept pace. All standard cell cycle analysis methods preclude real time dynamic analysis of the cell cycle in live cells at single cell resolution. To address the needs of cell cycle investigations across a range of analysis platforms we are currently developing a range of cell cycle phase markers based on GFP expression controlled by well characterised cell cycle components to allow covert surveillance of the cell cycle in living cells.

Biomarkers↗