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Biomedical subjects

C J Roberts

Publications and source records attributed to C J Roberts.

At least 19 recordsLinked to original sources
PubMed2007

Effect of exercise, training, circadian rhythm, age, and sex on insulin-like growth factor-1 in the horse.

Insulin-like growth factor-1 could be a useful marker in the horse for diagnostic, selection, or forensic purposes, provided its physiological regulation is well understood. The objective of this study was to investigate factors, such as acute exercise, fitness training, time of day, sex, and age, that may influence serum IGF-1 in normal, healthy horses. Throughout a 9-wk training program, 6 geldings maintained a mean (+/- SEM) IGF-1 concentration of 302 +/- 29 ng/mL. Moderate or high intensity exercise had no effect on IGF-1 concentrations, when pre- and postexercise values were compared. Over a 24-h period, there was some variation in IGF-1 concentrations but no clear diurnal rhythm. Concentrations of IGF-1 were measured in a large population of thoroughbred horses (1,880) on 3 continents. The population deviated slightly from a normal distribution (P < 0.001) because of large IGF-1 concentrations in 10 horses. The global mean IGF-1 concentration was 310 +/- 2.2 ng/mL, with a greater mean value (P < 0.001) in gonad-intact males (336 +/- 5.6 ng/mL) than in females (303 +/- 3.2 ng/mL) or geldings (302 +/- 3.2 ng/mL). However, the greatest IGF-1 concentrations observed for all stallions, mares, and geldings were 627, 676, and 709 ng/mL, respectively. In mares and geldings, IGF-1 concentrations showed a gradual decrease with advancing age (P < 0.001), but the effect was much less marked in stallions. This study confirms that IGF-1 concentrations are stable, compared with GH concentrations, in the horse and that a meaningful measure of IGF-1 status can be obtained from a daily serum sample.

Aging↗
PubMed2005

Structural study of DNA condensation induced by novel phosphorylcholine-based copolymers for gene delivery and relevance to DNA protection.

Poly[2-(dimethylamino)ethyl methacrylate-b-2-methacryloyloxyethyl phosphorylcholine] (DMA-MPC) is currently under investigation as a new vector candidate for gene therapy. The DMA block has been previously demonstrated to condense DNA effectively. The MPC block contains a phosphorylcholine (PC) headgroup, which can be found naturally in the outside of the cell membrane. This PC-based polymer is extremely hydrophilic and acts as a biocompatible steric stabilizer. In this study, we assess in detail the morphologies of DNA complexes obtained using the diblock copolymer series DMA(x)MPC30 (where the mean degree of polymerization of the MPC block was fixed at 30 and the DMA block length was systematically varied) using transmission electron microscopy (TEM) and liquid atomic force microscopy (AFM). Both techniques indicate more compact complex morphologies (more efficient condensation) as the length of the cationic DMA block increases. However, the detailed morphologies of the DMA(x)MPC30-DNA complexes observed by TEM in vacuo and by AFM in aqueous medium are different. This phenomena is believed to be related to the highly hydrophilic nature of the MPC block. TEM studies revealed that the morphology of the complexes changes from loosely condensed structures to highly condensed rods, toroids, and oval-shaped particles as the DMA moiety increases. In contrast, morphological changes from plectonemic loops to flower-like and rectangular block-like structures, with an increase in highly condensed central regions, are observed by in situ AFM studies. The relative population of each structure is clearly dependent on the polymer molecular composition. Enzymatic degradation assays revealed that only the DMA homopolymer provided effective DNA protection against DNase I degradation, while other highly condensed copolymer complexes, as judged from TEM and gel electrophoresis, only partially protected the DNA. However, AFM images indicated that the same highly condensed complexes have less condensed regions, which we believe to be the initiation sites for enzymatic attack. This indicates that the open structures observed by AFM of the DNA complexation by the DMA(x)MPC30 copolymer series are closer to in vivo morphology when compared to TEM.

Biopolymers↗
PubMed2004

Modified polyethylenimines as non-viral gene delivery systems for aerosol gene therapy: investigations of the complex structure and stability during air-jet and ultrasonic nebulization.

Polyelectrolyte complexes between DNA and polyethylenimine (PEI) are promising non-viral delivery systems for pulmonary inhalation gene therapy and thus require sufficient stability during nebulization. The structure and stability of four different PEI-DNA polyplexes, namely branched (bPEI), linear (linPEI), poly(ethylene glycol)-grafted PEI (PEGPEI), biodegradable (bioPEI) PEI with DNA, were investigated. Using atomic force microscopy, the morphology of DNA and polyplexes before and after both air-jet and ultrasonic nebulization was characterized. The influence of nebulization on physico-chemical properties, particle size and zeta potential, was studied. Efficient DNA condensation to spherical particles was achieved with bPEI (90 nm) and PEGPEI (110 nm). By contrast, incomplete DNA condensations, seen as flower structures, were observed with linPEI (110 nm) and bioPEI (105 nm). Air-jet nebulization altered the polyplex structure to a greater extent than ultrasonic nebulization and resulted mainly in smaller and non-spherical particles (30-200 nm). Ultrasonic nebulization did not change the spherical structure or particle size of the polyplexes. In particular, the shape and size of the PEGPEI polyplexes did not change. We conclude that ultrasonic nebulization is a milder aerosolization method for gene delivery systems based on PEI. Additionally, PEGPEI-DNA polyplexes seem to be more stable than their counterparts, which may be advantageous in pulmonary inhalation gene therapy.

Aerosols↗
PubMed2004

AFM studies of the crystallization and habit modification of an excipient material, adipic acid.

Atomic force microscopy (AFM) has been used to investigate the (1 0 0) face of crystalline adipic acid, both in air and liquid environments. In air, surface reorganization occurred during scanning of the AFM probe, which has been investigated using single point force-distance analysis under a controlled relative humidity (RH) environment. We suggest such reorganization can be attributed to the influence of a network of water molecules bound to the hydrophilic (1 0 0) surface permitting local AFM tip-enhanced dissolution and reorganization of the solute. In situ imaging was also carried out on the crystals, revealing etch-pit formation during dissolution, and rapid growth at higher levels of supersaturation (sigma), both of which are direct consequences of the hydrophilic nature of the (1 0 0) face. Also presented here are nanoscale observations of the effect of octanoic acid, a structurally-related habit modifier, on crystalline adipic acid. Using AFM, we have been able to show that the presence of octanoic acid at low concentration has little observable affect on the development of the (1 0 0) face; however, as this concentration is increased, there are clear changes in step morphology and growth mode on the (1 0 0) face of the crystal. At a concentration of 1.26 mmol dm(-3) (a concentration corresponding to a molar ratio of approximately 1:175 octanoic acid:adipic acid), growth on the (1 0 0) face is inhibited, with in situ AFM imaging indicating this is a direct consequence of octanoic acid binding to the surface, and pinning the monomolecular growth steps.

Adipates↗
PubMed2004

Using microscopic techniques to reveal the mechanism of anion exchange in crystalline co-ordination polymers.

Co-ordination polymers are currently attracting extensive interest due to their potential applications as supramolecular hosts, vessels, and frameworks for storage and separations. Many applications rely on the ion exchange capabilities of these compounds, and considerable debate surrounds the mechanism by which ion exchange occurs in co-ordination polymers. Here AFM and SEM were applied, for the first time, to investigate this class of materials. In situ AFM studies revealed the mechanism by which anion exchange and the subsequent structural transformations of the crystalline co-ordination polymers [[Ag(4,4'-bipy)]BF(4)](infinity) and [[Ag(4,4'-bipy)]NO(3)](infinity) occur. The process is initiated by the dissolution of the metastable crystalline polymer, followed by the subsequent crystallization of the new stable phase on the surface of the original crystal. The formation of deep clefts in the metastable polymer crystal during the transformation allows the solution to access the successive crystalline layers. Thus, the entire process can be viewed as a self-perpetuating cascade of dissolution and recrystallization throughout the macroscopic crystal. SEM data consolidate the findings of AFM. These techniques collectively illustrate that the anion exchange, and subsequent structural transformation, proceeds via a solvent-mediated mechanism, rather than a purely solid-state one.

Journal Article↗
PubMed2004

Direct atomic force microscopy observations of monovalent ion induced binding of DNA to mica.

Multivalent ions in solution are known to mediate attraction between two like-charged molecules. Such attraction has proved useful in atomic force microscopy (AFM) where DNA may be immobilized to a mica surface facilitating direct imaging in liquid. Theories of DNA immobilization suggest that either 'salt bridging' or fluctuation in the positions of counter ions about both the mica surface and DNA backbone secure DNA to the mica substrate. Whilst both theoretical and experimental evidence suggest that immobilization is possible in the presence of divalent ions, very few studies identify that such immobilization is possible with monovalent ions. Here we present direct AFM evidence of DNA immobilized to mica in the presence of only monovalent ions. Our data depict E. coli plasmid pBR322 adsorbed onto the negatively charged mica both after short (10 min) and long (24 h) incubation periods. These data suggest the need to re-explore current theories of like-charge attraction to include the possibility of monovalent interactions. We suggest that this DNA immobilization strategy may offer the potential to image natural processes with limited immobilization forces and hence enable maximum conformational freedom of the immobilized biomolecule.

Aluminum Silicates↗
PubMed2003

Electrostatic interactions observed when imaging proteins with the atomic force microscope.

The atomic force microscope (AFM) is now an established and valuable tool for the study of biological macromolecules in aqueous environments. In this paper we form a patterned boundary via the microcontact printing of individually isolated proteins, covalently attached to a solid support. We use this boundary to investigate electrostatic interactions that can occur between an AFM tip and a protein surface during imaging in solution. The observed height variations of the protein film are found to be a combination of not only structural considerations and thickness of the protein film, but also the repulsive contribution from electrostatic interactions between the AFM tip and the sample. These variations in measured heights of the protein surface can be described by Derjaguin, Landau, Verway, Overbeek (DLVO) theory. Our experimental results show that height measurements can be manipulated either negatively or positively by adjusting the pH and concentration of the electrolyte buffer that is utilised.

Algorithms↗
PubMed2003

Strabismus following endoscopic orbital decompression for thyroid eye disease.

Endoscopic orbital decompression may be used to treat disfiguring proptosis or sight threatening optic nerve compression in patients with thyroid eye disease. Strabismus is common in thyroid eye disease and frequently follows decompression surgery. We retrospectively reviewed patients undergoing endoscopic decompression for thyroid eye disease, by a single surgeon, from 1994 to 2000. Twenty-three patients (21 female, 2 male) were identified with a mean age of 47.5 years. At presentation, 21 patients had proptosis, 8 optic nerve compression (2 without proptosis) and 11 strabismus (9 complained of diplopia) with a mean BSV score of 24.5 before decompression. Forty orbits were decompressed with a mean decrease in proptosis of 3.3 mm. Following decompression, the mean BSV score was 25, and 17 patients had manifest strabismus in primary gaze (3 at near only) of whom 10 had pre-existing strabismus. Five patients had new diplopia (22%). Eleven patients ultimately required strabismus surgery of whom 8 had manifest strabismus before decompression. Following strabismus surgery, the mean BSV score was 37. The final BSV score for those not requiring strabismus surgery was 29. Mean follow-up was 28 months. Endoscopic orbital decompression can effectively treat disfiguring proptosis. Diplopia is a common complication, but pre-existing diplopia may improve.

Adult↗
PubMed2002

A study of single drug particle adhesion interactions using atomic force microscopy.

This paper aims to use Atomic Force Microscopy (AFM) to characterise the interaction forces between micronized salbutamol particles, an active ingredient frequently used in metered dose inhalers, and also to glass, lactose and a fluoropolymer. The methodology used involves challenging a salbutamol functionalized AFM tip to the surfaces of interest and measuring the force experienced by the cantilever as a function of tip-sample separation. Analysis of this force-distance data allows quantification of the particle-substrate adhesion. This study yields a ranking of adhesion as glass>lactose>salbutamol>polytetrafluoroethylene (PTFE). An increase in the interaction force between the salbutamol particle and PTFE on repeated contact due to tribocharging is also observed.

Adhesiveness↗
PubMed2002

Interactions of 3T3 fibroblasts and endothelial cells with defined pore features.

The colonization of biodegradable polymer scaffolds with cell populations has been established as the foundation for the engineering of a number of tissues, including cartilage, liver, and bone. Within these scaffolds, the cells encounter a porous environment in which they must migrate across the convoluted polymer surface to generate a homogenous cell distribution. Predicting the interactions between cells and pores is important if scaffold characteristics are to be optimized. Therefore, we investigated the behavior of two model cell types over a range of defined pore features. These pore features range from 5 to 90 microm in diameter and have been fabricated by photolithographic techniques. Quantitatively, the behavior of the cells is dependent on three factors: 1) percentage cell coverage of the surface; 2) pore size; and 3) cell type. Fibroblast cells displayed a co-operative pattern of cell spreading in which pores with diameters greater than the cell dimensions were bridged by groups of cells using their neighbors as supports. Endothelial cells were unable to use neighbors as support structures and failed to bridge pores greater than the cell diameter.

3T3 Cells↗
PubMed2002

Contact lenses in the management of high anisometropic amblyopia.

UNLABELLED: Poster at College Congress, Birmingham 2001. Oral presentation at European Strabismus Association, Florence 2001. PURPOSE: Anisometropia of more than one dioptre during the sensitive visual period may cause amblyopia. Its management requires refractive correction, and occlusion. Compliance with treatment is critical if visual improvement is to obtained. High anisometropia, poor initial acuity and mixed strabismic/anisometropia amblyopia are predictive factors for a poor outcome. We evaluated contact lens use in the management of high anisometropic amblyopia. METHODS: Retrospective analysis of anisometropic amblyopia managed in a paediatric contact lens clinic (July 1996-July 2000), after standard amblyopia therapy of spectacles and occlusion therapy had been tried. Presenting age, acuity and refraction, duration of lens wear and occlusion, and final visual outcomes were noted. RESULTS: Seven children (four male, three female) presented at age 3.5-6 years (mean 4.5). Six had myopic anisometropia 6.0-18.4 dioptres (mean 10.4 dioptres) and one 6.75 dioptres hypermetropic anisometropia. The initial corrected acuities of the amblyopic eyes were 6/18 to 1/60. Five patients used contact lenses with a range from 5 months to 4 years. Final acuities were 6/12-1/60. Two myopes with 6 dioptres anisometropia improved three to four Snellen lines, one with 8.8 dioptres improved one line. Three with >10 dioptres anisometropia did not improve. The hypermetropic patient improved part of one Snellen line. CONCLUSIONS: High anisometropic amblyopia is challenging to treat. In our study contact lenses improved visual acuity in myopic anisometropia of up to 9 dioptres.

Amblyopia↗
PubMed2001

Role of scaffolds in MAP kinase pathway specificity revealed by custom design of pathway-dedicated signaling proteins.

BACKGROUND: Signal transduction pathways with shared components must be insulated from each other to avoid the inappropriate activation of multiple pathways by a single stimulus. Scaffold proteins are thought to contribute to this specificity by binding select substrates. RESULTS: We have studied the ability of scaffold proteins to influence signaling by the yeast kinase Ste11, a MAPKKK molecule that participates in three distinct MAP kinase pathways: mating, filamentation, and HOG. We used protein fusions to force Ste11 to associate preferentially with a subset of its possible binding partners in vivo, including Ste5, Ste7, and Pbs2. Signaling became confined to a particular pathway when Ste11 was covalently attached to these scaffolds or substrates. This pathway bias was conferred upon both stimulus-activated and constitutively active forms of Ste11. We also used membrane-targeted derivatives of the mating pathway scaffold, Ste5, to show that stimulus-independent signaling initiated by this scaffold remained pathway specific. Finally, we demonstrate that loss of pathway insulation has a negative physiological consequence, as nonspecific activation of both the HOG and mating pathways interfered with proper execution of the mating pathway. CONCLUSIONS: The signaling properties of these kinase fusions support a model in which scaffold proteins dictate substrate choice and promote pathway specificity by presenting preferred substrates in high local concentration. Furthermore, insulation is inherent to scaffold-mediated signaling and does not require that signaling be initiated by pathway-specific stimuli or activator proteins. Our results give insight into the mechanisms and physiological importance of pathway insulation and provide a foundation for the design of customized signaling proteins.

MAP Kinase Kinase Kinases↗
PubMed2001

Dissolution behavior of porcine somatotropin with simultaneous gel formation and lysine Schiff-base hydrolysis.

The primary goal of this work was to develop a reliable in vitro dissolution model to evaluate the effects of Schiff-base hydrolysis and gel formation on the dissolution kinetics of pellets of porcine somatotropin (pST) and pST conjugated with ortho-vanillin (ov-pST) via Schiff-base formation, in an effort to develop an extended-release pST implant. Experimentally, dissolution was investigated as a function of ov concentration in pH 7.4, phosphate buffered saline under sink conditions where steady-state (constant flux) dissolution is typically observed. However, the resulting dissolution profiles displayed variable release rates due to gel formation at the solid-liquid interface, which impeded pST release. Chemical modification of pST with ov reduced gel formation and also resulted in much lower release rates. A mathematical model was developed that quantitatively accounts for changes in dissolution rates due to transient gel formation via irreversible aggregation, along with the effects of reversible Schiff-base hydrolysis involving ov, and predicts dissolution rates in the presence of ov decrease due to the much lower solubility of ov-pST. Our results indicate that although ov-pST is less prone to aggregate, pST/ov-pST equilibration is rapid compared to dissolution and therefore aggregation remains the limiting factor, and ultimately precludes this approach as a viable extended-release delivery system.

Animals↗
PubMed2001

Force-induced melting of a short DNA double helix.

The dynamic behaviour of DNA is of fundamental importance to many cellular processes. One principal characteristic, central to transcription and replication, is the ability of the duplex to "melt". It has recently been shown that dynamic force spectroscopy provides information about the energetics of biomolecular dissociation. We have employed this technique to investigate the unbinding of single dodecanucleotide molecules. To separate the duplex to single-stranded DNA, forces ranging from 17 to 40 pN were required over a range of loading rates. Interpretation of the dependence of melting force on loading rate revealed that the energy barrier to rupture is between 9 and 13 kcal mol-1 in height and situated 0.58 nm from an intermediate structural state. Thermal melting studies show that, prior to dissociation, the oligonucleotide underwent a transition which required between 7 and 11 kcal mol-1 in energy. Through combined dynamic force spectroscopy and thermal melting studies we show the derivation of an energy landscape to dissociate a 12-mer duplex. Until very recently, this type of information was only accessible by computational analysis. Additionally, the force spectroscopy data allow an estimation of the kinetics of duplex formation and melting.

Base Sequence↗
PubMed2001

Synthesis and characterisation of a degradable poly(lactic acid)-poly(ethylene glycol) copolymer with biotinylated end groups.

Poly(lactic acid)-poly(ethylene glycol)-biotin (PLA-PEG-biotin) is a degradable polymer with protein resistant properties that can undergo rapid surface engineering in aqueous media to create biomimetic surfaces. Surface engineering of this polymer is dependent on biomolecular interactions between the biotin end group and the protein avidin. Given the vigorous conditions of synthesis, it is essential that the manufacture of the polymer does not alter the biotin structure or its molecular recognition. Equally, it is important that the incorporation of biotin does not adversely affect the physicochemical properties of the polymer. (1)H NMR provides evidence of biotin attachment and structural integrity. (1)H NMR, gel permeation chromatography (GPC), and differential scanning calorimetry (DSC) analysis shows there is no significant effect on bulk properties induced by the biotin end group. Surface plasmon resonance (SPR) and fluorescent spectroscopy studies using the 2-(4'-hydroxyazobenzene) benzoic acid (HABA)/avidin complex show that the biotin moieties binding capabilities are not impaired by the synthesis.

Avidin↗
PubMed2001

An in situ dissolution study of aspirin crystal planes (100) and (001) by atomic force microscopy.

PURPOSE: To observe in situ and on individual aspirin crystal faces the comparative rates and processes of dissolution of the dominant faces. METHODS: The kinetics of the dissolution rate of two aspirin crystal planes (001) and (100) under 0.05M HCl are studied in situ at room temperature using Atomic Force Microscopy. The dissolution process of each crystal plane was followed by observed changes in topographic features. RESULTS: The results revealed that crystal plane (001) dissolves by receding step edges, and has a dissolution rate of 0.45 nm s(-1). Conversely. plane (100) displays crystal terrace sinking at an average rate of 2.93 nm s(-1). Calculated intrinsic dissolution values (g s(-1) cm(-2)) for planes (001) and (100) are 1.37 x 10(-7) gs(-1) cm(-2) and 8.36 x 10(-7) gs(-1) cm(-2), respectively. CONCLUSIONS: These values indicate that the rate of flux of material from plane (100) is approximately six times greater than that from plane (001), under 0.05M HCl. Interpretation of the data, based upon intrinsic dissolution rates and dissolution rate velocities, correlate with reported variations in the dissolution behavior of commercial aspirin products. These observations illustrate the suitability of the technique for characterizing the dissolution behavior of crystalline drugs.

Aspirin↗
PubMed2001

Molecular patterning on carbon based surfaces through photobiotin activation.

We have demonstrated the site-specific adhesion of photobiotin as a method of producing protein micropatterns. These patterns were created by the selective UV irradiation of a thin film of deposited photobiotin. The UV activated areas of photobiotin were then developed using fluorescently labelled avidin. The size of pattern produced is an order of magnitude smaller than those previously reported by this method. The patterns were characterised, using atomic force microscopy (AFM) to determine their microstructure. It was found that the AFM could discriminate between the areas of protein immobilised to the surface through the activated photobiotin, and the bare substrate surface where the inactivated photobiotin had been removed during the washing process. The potential of these patterns as sensing surfaces is demonstrated through the creation of a spatially patterned immunosensing surface. In this case, a biotinylated antibody was bound to the surface and the pattern developed using a second antibody specific to the immobilised biotinylated antibody. This technique could thus provide a simple and efficient method of producing high density immunoassay systems.

Journal Article↗