Biomedical subjects

Paul Rigby

Publications and source records attributed to Paul Rigby.

8 recordsLinked to original sources

PubMed2006

Determination of the orientational distribution and orientation factor for transfer between membrane-bound fluorophores using a confocal microscope.

Orientational fluorophores have been a useful tool in physical chemistry, biochemistry, and more recently structural biology due to the polarized nature of the light they emit and that fact that energy can be transferred between them. We present a practical scheme in which measurements of the intensity of emitted fluorescence can be used to determine limits on the mean and distribution of orientation of the absorption transition moment of membrane-bound fluorophores. We demonstrate how information about the orientation of fluorophores can be used to calculate the orientation factor kappa(2) required for use in FRET spectroscopy. We illustrate the method using images of AlexaFluor probes bound to MscL mechanosensitive transmembrane channel proteins in spherical liposomes.

Algorithms↗

PubMed2006

Antigen from the anterior chamber of the eye travels in a soluble form to secondary lymphoid organs via lymphatic and vascular routes.

PURPOSE: To determine the afferent pathways linking the anterior chamber (AC) of the eye to the secondary lymphoid organs. METHODS: Single intracameral, subconjunctival, or intravenous injections and topical application on the conjunctiva of 3 muL (30 mug) of cascade-blue-labeled Dextran (CB-Dx) were performed in Lewis rats. In addition, bilateral intracameral injections (CB-Dx into the right AC and FITC-Dx into the left AC) or a combination of intracameral (CB-Dx) and intravenous (FITC-Dx) injections were performed. Distribution of antigen-positive cells and free antigen in frozen sections of lymphoid organs from animals killed at 24 hours after these various types of injection was analyzed by fluorescence and confocal microscopy. RESULTS: After intracameral and subconjunctival injections, antigen reaches the ipsilateral lymph node of the head and neck predominantly via the conjunctival lymphatics. Intraocular antigen entering the venous circulation reaches the spleen and mesenteric lymph nodes, but also a small proportion enters the lymph nodes of the head and neck. After bilateral intracameral injections, individual cells bearing both fluorescent antigens were identified in lymphoid organs draining the eye. Similarly, double antigen-positive marginal zone macrophages were observed after simultaneous intracameral injection of CB-Dx and intravenous injection of FITC-Dx. CONCLUSIONS: These data demonstrate that in the first 24 hours after injection of antigen into the anterior chamber of the eye, antigen reaches the lymphoid organs mainly in a soluble form via both the blood and lymph.

Administration, Topical↗

PubMed2005

A flexible approach to the calculation of resonance energy transfer efficiency between multiple donors and acceptors in complex geometries.

Resonance energy transfer provides a practical way to measure distances in the range of 10-100 A between sites in biological molecules. Although the relationship between the efficiency of energy transfer and the distance between sites is well described for a single pair of fluorophores, the situation is more difficult when more than two fluorophores are present. Using a Monte Carlo calculation scheme, we demonstrate how resonance energy transfer can be used to measure distances between fluorophores in complex geometries. We demonstrate the versatility of the approach by calculating the efficiency of energy transfer for individual fluorophores randomly distributed in two and three dimensions, for linked pairs of donors and acceptors and pentameric structures of five linked fluorophores. This approach can be used to relate the efficiency of energy transfer to the distances between fluorophores, R0, molecular concentrations, laser power, and donor/acceptor ratios in ensembles of molecules or when many fluorophores are attached to a single molecule such as in multimeric proteins.

Algorithms↗

PubMed2005

Conformational changes involved in MscL channel gating measured using FRET spectroscopy.

We demonstrate that fluorescence resonance energy transfer spectroscopy is a powerful tool for in situ structural analysis of multimeric membrane proteins by measuring the conformational changes involved in gating the mechanosensitive ion channel of large conductance. Ensemble analysis is used to analyze the intensity of light emitted by AlexaFluor-labeled cysteine mutants reconstituted into artificial liposomes before and after acceptor photobleaching. The diameter of the protein is found to increase by 16 A upon channel activation.

Escherichia coli Proteins↗

PubMed2005

Visualisation of the mechanosensitive channel of large conductance in bacteria using confocal microscopy.

The mechanosensitive channel of large conductance (MscL) plays an important role in the survival of bacterial cells to hypo-osmotic shock. This channel has been extensively studied and its sequence, structure and electrophysiological characteristics are well known. Here we present a method to visualise MscL in living bacteria using confocal microscopy. By creating a gene fusion between mscl and the gene encoding the green fluorescent protein (GFP) we were able to express the fusion protein MscL-GFP in bacteria. We show that MscL-GFP is present in the cytoplasmic membrane and forms functional channels. These channels have the same characteristics as wild-type MscL, except that they require more pressure to open. This method could prove an interesting, non-invasive, tool to study the localisation and the regulation of expression of MscL in bacteria.

Bacterial Physiological Phenomena↗

PubMed2005

Enteral nutritional support in prevention and treatment of pressure ulcers: a systematic review and meta-analysis.

BACKGROUND: There have been few systematic reviews and no meta-analyses of the clinical benefits of nutritional support in patients with, or at risk of developing, pressure ulcers. Therefore, this systematic review and meta-analysis was undertaken to address the impact of enteral nutritional support on pressure ulcer incidence and healing and a range of other clinically relevant outcome measures in this group. METHODS: Fifteen studies (including eight randomised controlled trials (RCTs)) of oral nutritional supplements (ONS) or enteral tube feeding (ETF), identified using electronic databases (including Pub Med and Cochrane) and bibliography searches, were included in the systematic review. Outcomes including pressure ulcer incidence, pressure ulcer healing, quality of life, complications, mortality, anthropometry and dietary intake were recorded, with the aim of comparing nutritional support versus routine care (e.g. usual diet and pressure ulcer care) and nutritional formulas of different composition. Of these 15 studies, 5 RCTs comparing ONS (4 RCTs) and ETF (1 RCT) with routine care could be included in a meta-analysis of pressure ulcer incidence. RESULTS: Meta-analysis showed that ONS (250-500 kcal, 2-26 weeks) were associated with a significantly lower incidence of pressure ulcer development in at-risk patients compared to routine care (odds ratio 0.75, 95% CI 0.62-0.89, 4 RCTs, n=1224, elderly, post-surgical, chronically hospitalised patients). Similar results were obtained when a combined meta-analysis of ONS (4 RCT) and ETF (1 RCT) trials was performed (OR 0.74, 95% CI 0.62-0.88, 5 RCTs, n=1325). Individual studies showed a trend towards improved healing of existing pressure ulcers with disease-specific (including high protein) versus standard formulas, although robust RCTs are required to confirm this. Although some studies indicate that total nutritional intake is improved, data on other outcome measures (quality of life) are lacking. CONCLUSIONS: This systematic review shows enteral nutritional support, particularly high protein ONS, can significantly reduce the risk of developing pressure ulcers (by 25%). Although studies suggest ONS and ETF may improve healing of PU, further research to confirm this trend is required.

Aged↗

PubMed2003

Macrophage recognition and phagocytosis of apoptotic fibroblasts is critically dependent on fibroblast-derived thrombospondin 1 and CD36.

The induction of fibroblast apoptosis and their clearance by phagocytes is essential for normal wound healing and prevention of scarring. However, little is known about the clearance of apoptotic fibroblasts and whether apoptotic cells are active participants in the recruitment and activation of phagocytes. In this study, we provide the first evidence that apoptotic fibroblasts actively release increased amounts of thrombospondin (TSP1) to actively recruit macrophages. Expression of TSP1 and its receptor CD36 was increased on the surface of apoptotic fibroblasts. By chemical cross-linking and immunoprecipitation we show that TSP1 and CD36 were directly associated. This was confirmed by confocal microscopy. Blockade of either CD36 or TSP1 on apoptotic fibroblasts inhibited phagocytosis. Blockade of alpha v beta 3 integrins as well as CD36 and TSP1 on macrophages inhibited phagocytosis. In contrast, phosphatidylserine or lectins were not involved. These findings suggest that apoptotic fibroblasts release TSP1 as a signal to recruit macrophages while the up-regulated expression of the CD36/TSP1 complex on their cell surface may form a ligand bridging the fibroblast to a complex consisting of alpha v beta 3/CD36/TSP1 on macrophages. These results establish fundamental mechanisms for the clearance of apoptotic fibroblasts and may provide insights into the processes involved in normal wound repair.

Antigens, CD↗

PubMed2002

Airway nerves: detection and visualisation.

The importance of understanding the roles of nerves in regulating lung function cannot be overestimated if we are to successfully address the therapeutic management of respiratory diseases such as asthma and chronic obstructive pulmonary disease. Critical to this understanding is a more complete appreciation of airway innervation patterns, densities and functions. Accordingly, there is increasing demand for cost-effective techniques that enable the detection and visualisation of airway nerves. Immunofluorescence approaches, including confocal microscopy, are increasingly popular methods in pursuit of this important information, although the selection of a technique should be guided primarily by the type and quality of the information required from the study. Importantly, quantification of tissue nerve density is now feasible, adding a new dimension to the assessment of the significance of innervation patterns.

Animals↗