Phenotyping and genotyping of platelet defects in patient populations enriched in bleeding

Inherited platelet disorders vary in their associated bleeding risk. Individuals in families with known platelet gene mutations often have variable bleeding, suggesting that bleeding risk is multifactorial. Inherited platelet disorders are difficult to diagnose due to the absence of a gold standard laboratory technique and their variable bleeding phenotype, which often only manifests after haemostatic challenges. The work in this thesis furthers the previous studies in the genotyping and phenotyping of platelets project by significantly increasing the number of participants, allowing further characterisation of inherited platelet disorders in those with a normal platelet count. A bleeding assessment tool score was also recorded in all newly recruited adults. Two specific groups of patients are also studied: Those with unexplained menorrhagia, in whom the hypothesis was that some have an undiagnosed platelet defect. Those with inherited thrombocytopenia, in whom I sought to develop an assay to assess platelet function, as bleeding risk can not be predicted by platelet count alone, suggesting that qualitative defects may contribute. The genetic basis of platelet defects was investigated in many of the patients, leading to identification of mutations in genes known to be critical in platelet biology, and identification of several possible novel variants

Directed evolution of anti-HER2 DARPins by SNAP display reveals stability/function trade-offs in the selection process.

In vitro display technologies have proved to be powerful tools for obtaining high-affinity protein binders. We recently described SNAP display, an entirely in vitro DNA display system that uses the SNAP-tag to link protein with its encoding DNA in water-in-oil emulsions. Here, we apply SNAP display for the affinity maturation of a designed ankyrin repeat proteins (DARPin) that binds to the extracellular domain of HER2 previously isolated by ribosome display. After four SNAP display selection cycles, proteins that bound specifically to HER2 in vitro, with dissociation constants in the low- to sub-nanomolar range, were isolated. In vitro affinities of the panel of evolved DARPins directly correlated with the fluorescence intensities of evolved DARPins bound to HER2 on a breast cancer cell line. A stability trade-off is observed as the most improved DARPins have decreased thermostability, when compared with the parent DARPin used as a starting point for affinity maturation. Dissection of the framework mutations of the highest affinity variant, DARPin F1, shows that functionally destabilising and compensatory mutations accumulated throughout the four rounds of evolution.G.H. was supported by a CASE studentship from the Engineering and Physical Sciences Research Council and MedImmune and the Marie-Curie Research Training Network ENEFP. F.H. is a starting investigator of the European Research Council. Funding to pay the Open Access publication charges for this article was provided by the Engineering and Physical Sciences Research Council.This is the final version of the article. It first appeared from Oxford University Press via http://dx.doi.org/10.1093/protein/gzv02

Assessment of the anthelmintic effect of natural plant cysteine proteinases against the gastrointestinal nematode, Heligmosomoides polygyrus, in vitro

We examined the mechanism of action and compared the anthelmintic efficacy of cysteine proteinases from papaya, pineapple, fig, kiwi fruit and Egyptian milkweed in vitro using the rodent gastrointestinal nematode Heligmosomoides polygyrus. Within a 2 h incubation period, all the cysteine proteinases, with the exception of the kiwi fruit extract, caused marked damage to the cuticle of H. polygyrus adult male and female worms, reflected in the loss of surface cuticular layers. Efficacy was comparable for both sexes of worms, was dependent on the presence of cysteine and was completely inhibited by the cysteine proteinase inhibitor, E-64. LD50 values indicated that the purified proteinases were more efficacious than the proteinases in the crude latex, with purified ficin, papain, chymopapain, Egyptian milkweed latex extract and pineapple fruit extract, containing fruit bromelain, having the most potent effect. The mechanism of action of these plant enzymes (i.e. an attack on the protective cuticle of the worm) suggests that resistance would be slow to develop in the field. The efficacy and mode of action make plant cysteine proteinases potential candidates for a novel class of anthelmintics urgently required for the treatment of humans and domestic livestock

In vitro anthelmintic effects of cysteine proteinases from plants against intestinal helminths of rodents

Infections with gastrointestinal (GI) nematodes are amongst the most prevalent worldwide, especially in tropical climates. Control of these infections is primarily through treatment with anthelmintic drugs, but the rapid development of resistance to all the currently available classes of anthelmintic means that alternative treatments are urgently required. Cysteine proteinases from plants such as papaya, pineapple and fig are known to be substantially effective against three rodent GI nematodes, Heligmosomoides polygyrus, Trichuris muris and Protospirura muricola, both in vitro and in vivo. Here, based on in vitro motility assays and scanning electron microscopy, we extend these earlier reports, demonstrating the potency of this anthelmintic effect of plant cysteine proteinases against two GI helminths from different taxonomic groups – the canine hookworm, Ancylostoma ceylanicum, and the rodent cestode, Rodentolepis microstoma. In the case of hookworms, a mechanism of action targeting the surface layers of the cuticle indistinguishable from that reported earlier appears to be involved, and in the case of cestodes, the surface of the tegumental layers was also the principal location of damage. Hence, plant cysteine proteinases have a broad spectrum of activity against intestinal helminths (both nematodes and cestodes), a quality that reinforces their suitability for development as a muchneeded novel treatment against GI helminths of humans and livestock

Towards understanding the influence of porosity on mechanical and fracture behaviour of quasi-brittle materials:experiments and modelling

In this work, porosity-property relationships of quasi-brittle materials are explored through a combined experimental and numerical approach. In the experimental part, hemihyrate gypsum plaster powder (CaSO 4 ⋅1/2H 2 O CaSO4⋅1/2H2O) and expanded spherical polystyrene beads (1.5–2.0 mm dia.) have been mixed to form a model material with controlled additions of porosity. The expanded polystyrene beads represent pores within the bulk due to their light weight and low strength compared with plaster. Varying the addition of infill allows the production of a material with different percentages of porosity: 0, 10, 20, 30 and 31 vol%. The size and location of these pores have been characterised by 3D X-ray computed tomography. Beams of the size of 20×20×150 20×20×150 mm were cast and loaded under four-point bending to obtain the mechanical characteristics of each porosity level. The elastic modulus and flexural strength are found to decrease with increased porosity. Fractography studies have been undertaken to identify the role of the pores on the fracture path. Based on the known porosity, a 3D model of each microstructure has been built and the deformation and fracture was computed using a lattice-based multi-scale finite element model. This model predicted similar trends as the experimental results and was able to quantify the fractured sites. The results from this model material experimental data and the lattice model predictions are discussed with respect to the role of porosity on the deformation and fracture of quasi-brittle materials

Towards understanding the influence of porosity on mechanical and fracture behaviour of quasi-brittle materials:experiments and modelling

In this work, porosity-property relationships of quasi-brittle materials are explored through a combined experimental and numerical approach. In the experimental part, hemihyrate gypsum plaster powder (CaSO 4 ⋅1/2H 2 O CaSO4⋅1/2H2O) and expanded spherical polystyrene beads (1.5–2.0 mm dia.) have been mixed to form a model material with controlled additions of porosity. The expanded polystyrene beads represent pores within the bulk due to their light weight and low strength compared with plaster. Varying the addition of infill allows the production of a material with different percentages of porosity: 0, 10, 20, 30 and 31 vol%. The size and location of these pores have been characterised by 3D X-ray computed tomography. Beams of the size of 20×20×150 20×20×150 mm were cast and loaded under four-point bending to obtain the mechanical characteristics of each porosity level. The elastic modulus and flexural strength are found to decrease with increased porosity. Fractography studies have been undertaken to identify the role of the pores on the fracture path. Based on the known porosity, a 3D model of each microstructure has been built and the deformation and fracture was computed using a lattice-based multi-scale finite element model. This model predicted similar trends as the experimental results and was able to quantify the fractured sites. The results from this model material experimental data and the lattice model predictions are discussed with respect to the role of porosity on the deformation and fracture of quasi-brittle materials

Mild bleeding disorders::what every clinician should know

Patients with mild bleeding disorders are under-recognized and frequently present to general physicians. The underlying reasons for bleeding are multifactorial. There is little evidence to guide diagnostic and management decision making in patients with mild bleeding disorders. This article outlines different types of mild bleeding disorders, with a particular focus on bleeding associated with low levels of von Willebrand factor and mild platelet defects. It gives practical, evidence-based advice on the investigation and management of patients with a suspected or known mild bleeding disorder, considering the scenarios of an acute bleed, stable outpatient, peri-surgical management and thrombosis. Patients with a mild bleeding disorder have variable bleeding because of the interplay of genetic and environmental factors. The clinical history remains of utmost importance in their general management. Liaison with a specialist centre, multidisciplinary assessment and a careful judgement of the balance of risk in each individual circumstance is required to safely manage these patients. </jats:p

CpG island methylation phenotype (CIMP) in oral cancer: associated with a marked inflammatory response and less aggressive tumour biology.

Studies in several tumour sites highlight the significance of the CpG island methylation phenotype (CIMP), with distinct features of histology, biological aggression and outcome. We utilise pyrosequencing techniques of quantitative methylation analysis to investigate the presence of CIMP in oral squamous cell carcinoma (OSCC) for the first time, and evaluate its correlation with allelic imbalance, pathology and clinical behaviour. Tumour tissue, control tissue and PBLs were obtained from 74 patients with oral squamous cell carcinoma. Pyrosequencing was used to analyse methylation patterns in 75-200 bp regions of the CpG rich gene promoters of 10 genes with a broad range of cellular functions. Allelic imbalance was investigated using a multiplexed panel of 11 microsatellite markers. Corresponding variables, histopathological staging and grading were correlated with these genetic and epigenetic aberrations. A cluster of tumours with a greater degree of promoter methylation than would be predicted by chance alone (P=0.001) were designated CIMP+ve. This group had less aggressive tumour biology in terms of tumour thickness (p=0.015) and nodal metastasis (P=0.012), this being apparently independent of tumour diameter. Further, it seems that these CIMP+ve tumours excited a greater host inflammatory response (P=0.019). The exact mechanisms underlying CIMP remain obscure but the association with a greater inflammatory host response supports existing theories relating these features in other tumour sites. As CIMP has significant associations with other well documented prognostic indicators, it may prove beneficial to include methylation analyses in molecular risk modelling of tumours