No description available.

No description available.

No description available.

A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) are a family of proteins that are closely related to the matrix metalloproteinases (MMPs). It has been suggested that the proteins have a critical role in the breakdown of articular cartilage during osteoarthritis (OA). More recently it has been suggested that their actions could potentially regulate atherosclerotic plaque stability. Atherosclerosis is a chronic, inflammatory disorder characterised by lipid and cholesterol accumulation and the development of fibrotic plaques within the walls of large and medium arteries. The stability of the plaques is very important because clinical symptoms are only presented after rupture of the unstable plaques, leading to thrombosis and ischemia. During the current study, immunohistochemical analysis confirmed that ADAMTS-­1, -­4 and -­5 were being expressed within human carotid atherosclerotic lesions; macrophages were identified as major contributors to their expressions. Following on from this THP-­1 macrophages were stimulated with transforming growth factor-­β (TGF-­β), interferon-­γ (IFN-­γ), TNF-­like protein 1A (TL1A), interleukin (IL)-­17A and IL-­33. The regulation of ADAMTS-­1, -­4 and -­5 expressions were analysed using quantitative polymerase chain reactions (QPCR) and western blots. It was shown that TGF-­β increased the expressions of ADAMTS-­1 and -­5 and decreased the expression of ADAMTS-­4. IL-­33 decreased the expressions of ADAMTS-­1, -­4 and -­5 and IFN-­γ also decreased the expression of ADAMTS-­1. TL1A and IL-­17A stimulation of macrophages had no regulatory actions over ADAMTS-­1, -­4 or -­5 expressions. Looking at evidence from previous studies, TL1A and IL-­17A were identified as agents that could potentially act in synergy to amplify pro­inflammatory cytokine responses. To investigate this further, THP-­1 macrophages were stimulated with TL1A and IL-­17A, TL1A and IFN-­γ and also IL-­17A combined with IFN-­γ. TL1A and IL-­17A were shown to act in synergy to increase the expressions of ADAMTS-­1, -­4 and -­5 in macrophages. The regulation of ADAMTS-­1, -­4 and -­5 expressions in macrophages by IL-­33 was studied further. The mechanism of signal transduction was studied using RNA interference (RNAi) targeting extracellular signal-­‐regulated kinases (ERK)-­1, ERK-­2, p38, c-­Jun N-­terminal kinases(JNK)-­1/2, c-­Jun, phosphoinositide 3-­kinase(PI3K)-­γ, PI3K-­δ, p50, p65 and Janus kinase(JAK)-­1/2. It was determined that the attenuation of ADAMTS-­1, -­4 and -­5 expressions occurred through transcriptional regulation that was dependent on the ST2 receptor. ERK-­1, ERK-­2, JNK-­1/2, c-­Jun, PI3K-­γ and PI3K-­δ were also involved in the signal transduction of the response. The cellular roles of ADAMTS activity within atherosclerotic disease progression remain poorly understood. During the current study adenoviral vectors were created that delivered shRNA-­targeting ADAMTS-­1, -­4 and -­5. The adenoviral vectors were utilised in studies designed to investigate the roles of ADAMTS-­1, -­4 and -­5 during macrophage migration and foam cell formation. The studies showed that knockdown of ADAMTS-­1, -­4 and -­5 had no effect on macrophage migration or foam cell formation. More research is required into the cellular roles that ADAMTS proteases play during atherosclerotic disease progression. The field of research is now growing and could potentially provide some exciting opportunities for novel therapeutics of the future.

Recent studies have indicated the presence of a high degree of cryptic genetic diversity in some clitellate sentinel species. One of these species, the earthworm Lumbricus rubellus, has been recently found to comprise two divergent clades in the UK, and are possibly cryptic species. L. rubellus is commonly used in ecotoxicological assays, where undetected differences in contaminant responses between cryptic lineages may lead to confusing or misleading results. Furthermore, given the key role that earthworm species play in the soil ecosystem, a better understanding of cryptic diversity is necessary to investigate whether divergent lineages play different roles within their ecosystems. In this study, the phylogenomics of the acid-tolerant, cosmopolitan, epigeic species Lumbricus rubellus was investigated, with regard to demography during the glacial stages of the Pleistocene and the recent post-glacial colonization of North Europe using mitochondrial DNA markers, next-generation sequencing and environmental niche modelling tools. The niche suitability of L.rubellus during the last 120.000 years was inferred, allowing hypotheses on survival and recolonisation to be constructed. Phylogenetic, population structure and coalescent-based analyses resulted in the discovery of 11 deep divergent lineages (with levels of divergence up to 18% for mitochondrial markers), which most likely survived in refugia during Pleistocene glaciations. Signatures of expansions point to a possible recolonisation of central Europe during the last Glaciation, survival of one of the clades in a northern cryptic glacial refugium and a consequent recolonisation of northern Europe during the last 10,000 years. Genetic evidence and divergence time ultimately suggest that L. rubellus is a cryptic species complex, which clades diverged as far as ~5MY ago. The entire mitochondrial genome of the species complex is described here for the first time, and a survey of the deep phylogenetic signal over the mitochondrial genomes of eight selected individuals was carried out, supporting and deepening the phylogeny constructed using only two mitochondrial genes. Finally, whole genome analysis of genetic divergence supported the hypothesis of cryptic divergence for the two most divergent lineages selected

Plant growth regulators have a vital role in plant growth and development. The cellular response to these regulators depends on the presence and the action of specific receptors. The plant growth regulators and their receptors act together in complexes which determine the final effects of the plant growth regulators. In the research reported here, emphasis has been given to the regulation of the activity of the receptors themselves. The regulation of the N-l-naphthylphthalamic acid (N~A) receptor through phosphorylation and dephosphorylation and the regulation of the auxin binding protein (ABP) through gene manipulation have been investigated. NPA, an auxin transport inhibitor, was found to bind specifically to a crude membrane preparation from sugar beet seedling leaf cell suspension cultures. The in vitro binding was optimal at pH 4.5 and 4?C. Binding parameters for NP A binding were determined by Scatchard analysis. The dissociation constant (Kd) and binding protein concentration were found to be 1.71 x 10-7 mol dm-3 and 220 pmoles g-I membrane protein respectively. It was found that the amount of specific 3H-NPA binding was significantly increased by adding Mg2+ A TP to the binding assay solution; treatment of membrane preparations with acid phosphatase, prior to the NP A binding assay, resulted in lower specific binding. A TP activation and phosphatase inactivation were culture stage dependent. Although a considerable effect could be detected when using cells from day 8 (representing the linear phase), the same treatment did not alter the binding if cells from day I (representing lag phase) or day 14 (representing the stationary phase) were used. These observations have strongly highlighted the possible involvement of a phosphorylation and dephosphorylation mechanism in vivo in the regulation of the activity of the NP A receptor. High phosphatase activity was found in the supernatant, but not in the membrane pellet, after 50,000 g centrifugation. The presence of a membrane-bound auxin receptor, ABP, was demonstrated by Scatchard analysis in sugar beet seedlings. The Kd value and the receptor concentration were found to be 2.15 x 10-6 mol dm-3 and 68 pmoles g-I membrane protein. The protein could be solubilised either with the detergent Triton X-I 00 or by acetone-washing, with a recovery of about 40%. An acetone-solubilised ABP preparation could be partially purified by DEAE-Sephacel ion exchange chromatography, NAA-linked AH-Sepharose 4B affmity chromatography or Sephadex G-200 gel filtration. The recovery after any of these chromatographic treatments was very low so that successive chromatography for further purification was unsuccessful. The low level of detectable binding after purification resulted mainly from the low abundance of ABP in the plant material. Non-radioactive labelling and detection techniques were used to show that an ABP-probe hybridized to sugar beet genomic DNA during dot blotting. The present study has indicated that receptor activity could be regulated by a phosphorylation and dephosphorylation mechanism in plants. The investigation has also suggested that the effect of plant growth regulators on plant development could be regulated through the manipulation of the expression of their receptor genes.

Cancer cachexia is characterised by selective depletion of skeletal muscle protein reserves. The ubiquitin-proteasome proteolytic pathway has been shown to be responsible for muscle wasting in a range of cachectic conditions including cancer cachexia. To establish the importance of this pathway in muscle wasting during cancer (and sepsis), a quantitative competitive RT-PCR (QcRT-PCR) method was developed to measure the mRNA levels of the proteasome sub units C2a and C5ß and the ubiquitin-conjugating enzyme E214k. Western blotting was also used to measure the 20S proteasome and E214k protein expression. In vivo studies in mice bearing a cachexia inducing murine colon adenocarcinoma (MAC16) demonstrated the effect of progressive weight loss on the mRNA and protein expression for 20S proteasome subunits, as well as the ubiquitin-conjugating enzyme, E214k, in gastrocnemius and pectoral muscles. QcRT-PCR measurements showed a good correlation between expression of the proteasome subunits (C2 and CS) and the E214k enzyme mRNA and weight loss in gastrocnemius muscle, where expression increased with increasing weight loss followed by a decrease in expression at higher weight losses (25-27%). Similar results were obtained in pectoral muscles, but with the expression being several fold lower in comparison to that in gastrocnemius muscle, reflecting the different degrees of protein degradation in the two muscles during the process of cancer cachexia. Western blot analysis of 20S and E214k protein expression followed a similar pattern with respect to weight loss as that found with mRNA. In addition, mRNA and protein expression of the 20S proteasome subunits and E214k enzyme was measured in biopsies from cachectic cancer patients, which also showed a good correlation between weight loss and proteasome expression, demonstrating a progressive increase in expression of the proteasome subunits and E214k mRNA and protein in cachectic patients with progressively increasing weight loss. The effect of the cachexia-inducing tumour product PIF (proteolysis inducing factor) and 15-hydroxyeicosatetraenoic acid (15-HETE), the arachidoinic acid metabolite (thought to be the intracellular transducer of PIF action) has also been determined. Using a surrogate model system for skeletal muscle, C2C12 myotubes in vitro, it was shown that both PIF and 15-HETE increased proteasome subunit expression (C2a and C5ß) as well as the E214k enzyme. This increase gene expression was attenuated by preincubation with EPA or the 15-lipoxygenase inhibitor CV-6504; immunoblotting also confirmed these findings. Similarly, in sepsis-induced cachexia in NMRI mice there was increased mRNA and protein expression of the 20S proteasome subunits and the E214k enzyme, which was inhibited by EPA treatment. These results suggest that 15-HETE is the intracellular mediator for PIF induced protein degradation in skeletal muscle, and that elevated muscle catabolism is accomplished through upregulation of the ubiquitin-proteasome-proteolytic pathway. Furthermore, both EPA and CV -6504 have shown anti-cachectic properties, which could be used in the future for the treatment of cancer cachexia and other similar catabolic conditions.

Microbiological research has uncovered the basis of fermentation, infectious disease, vaccination and antibiotics. Now, a technological revolution leveraging DNA, the code of life, has allowed us to unravel cellular and evolutionary processes in exquisite detail. Today our need for new innovation is still great. The modern world is a challenging environment: over-population, climate change and highly mobile populations create a high risk of pandemic disease especially from viruses and many bacteria are now resistant to our life saving antibiotic drugs due to overuse. In hospitals, the spread of pathogens can be rapid and life threatening. Whole-genome sequencing has the power to identify the source of infections and determine whether clusters of cases belong to an outbreak. Portable, real-time nanopore sequencing enables sequencing to be performed near the patient, even in resource-limited settings. Integrating with existing datasets allows digital surveillance able to detect outbreaks earlier while they can still be contained. Early demonstrations of the power of whole-genome sequencing for outbreak surveillance have made it an area of intense interest and further development in laboratory methods and infrastructure will make it an important tool that can be deployed in response to future outbreaks.

Biosynthetic steps in the mupirocin (pseudomonic acids) biosynthetic pathway of Pseudomonas fluorescens NCIMB 10586 have already been deduced. Putative functions of most of the genes of \(mup\) cluster have been assigned although exact sequence of steps in the pathway and their timings are not yet known. Thiomarinols are another group of anti-bacterials produced by Pseudoalteromonas sp. SANK 73390. Very little is known about the biosynthesis of thiomarinols that share striking structural similarity with pseudomonic acids in their polyketide and fatty acid moieties. This similarity is reflected at genetic level as significant similarity in amino acid identity between the products of at least 27 ORFs in these biosynthetic clusters. This project aimed to learn more of biosynthetic steps in the biosynthesis of mupirocin and thiomarinol antibiotics by testing for functional cross-complementation between pair of genes or a group of genes whose products show significant homology. Surprisingly, only two genes \(tmlJ\) and \(tmlS\) out of nine studied showed complementation in the \(mup\) system. Findings suggested protein-protein interactions limited interchangeability of equivalent functions between two biosynthetic systems. It was shown by expressing related genes as groups for complementation in the \(mup\) system that it was possible to confirm specificities of such interactions.

DNA associates with proteins to form chromatin which is essential for the compaction of the DNA into the cell nucleus and is highly dynamic in order to allow the different biological processes of the DNA to occur. Chromatin compaction is achieved at different hierarchical levels: the 10nm fibre (DNA associates to nucleosomes formed by different histones), the Higher Order Chromatin fibre and the 300 nm chromosome structures. This study has shown that both H1 and H4 histones play a crucial role in preserving meiotic as well as mitotic chromosome structure and functional genome integrity in Arabidopsis. The role of the different linker histone H1 isoforms as well as the core histone H4 in Arabidopsis thaliana was investigated using T-DNA and RNAi mutant lines which showed different meiotic defects. Chromosomal breaks as well as non-homologous connections in the h4RNAi were linked to 45S/5S rDNA disorganisation, suggesting that H4 preserves chromosome integrity at these rDNA regions. Ath1.1 mutant presented univalents and reduced chiasma frequency at metaphase I, linked to a severe defect in ASY1 localisation on the meiotic chromosome axes. Thus, indicating that histone H1.1 is vital for proper chromatin axis organization that permit normal loading of recombination machinery proteins in Arabidopsis.