Chemical Optimization of Selective Pseudomonas aeruginosa LasB Elastase Inhibitors and Their Impact on LasB-Mediated Activation of IL-1β in Cellular and Animal Infection Models

LasB elastase is a broad-spectrum exoprotease and a key virulence factor of Pseudomonas aeruginosa, a major pathogen causing lung damage and inflammation in acute and chronic respiratory infections. Here, we describe the chemical optimization of specific LasB inhibitors with druglike properties and investigate their impact in cellular and animal models of P. aeruginosa infection. Competitive inhibition of LasB was demonstrated through structural and kinetic studies. In vitro LasB inhibition was confirmed with respect to several host target proteins, namely, elastin, IgG, and pro-IL-1 beta. Furthermore, inhibition of LasBmediated IL-1 beta activation was demonstrated in macrophage and mouse lung infection models. In mice, intravenous administration of inhibitors also resulted in reduced bacterial numbers at 24 h. These highly potent, selective, and soluble LasB inhibitors constitute valuable tools to study the proinflammatory impact of LasB in P. aeruginosa infections and, most importantly, show clear potential for the clinical development of a novel therapy for life-threatening respiratory infections caused by this opportunistic pathogen

Extending working life and the management of change. Is the workplace ready for the ageing worker?

Increasing longevity and the strain on state and occupational pensions have brought into question long-held assumptions about the age of retirement, and raised the prospect of a workplace populated by ageing workers. In the United Kingdom the default retirement age has gone, incremental increases in state pension age are being implemented and ageism has been added to workplace anti-discrimination laws. These changes are yet to bring about the anticipated transformation in workplace demographics, but it is coming, making it timely to ask if the workplace is ready for the ageing worker and how the extension of working life will be managed. We report findings from qualitative case studies of five large organisations located in the United Kingdom. Interviews and focus groups were conducted with employees, line managers, occupational health staff and human resources managers. Our findings reveal a high degree of uncertainty and ambivalence among workers and managers regarding the desirability and feasibility of extending working life; wide variations in how older workers are managed within workplaces; a gap between policies and practices; and evidence that while casualisation might be experienced negatively by younger workers, it may be viewed positively by financially secure older workers seeking flexibility. We conclude with a discussion of the challenges facing employers and policy makers in making the modern workplace fit for the ageing worker

Drosophila Duplication Hotspots Are Associated with Late-Replicating Regions of the Genome

Duplications play a significant role in both extremes of the phenotypic spectrum of newly arising mutations: they can have severe deleterious effects (e.g. duplications underlie a variety of diseases) but can also be highly advantageous. The phenotypic potential of newly arisen duplications has stimulated wide interest in both the mutational and selective processes shaping these variants in the genome. Here we take advantage of the Drosophila simulans–Drosophila melanogaster genetic system to further our understanding of both processes. Regarding mutational processes, the study of two closely related species allows investigation of the potential existence of shared duplication hotspots, and the similarities and differences between the two genomes can be used to dissect its underlying causes. Regarding selection, the difference in the effective population size between the two species can be leveraged to ask questions about the strength of selection acting on different classes of duplications. In this study, we conducted a survey of duplication polymorphisms in 14 different lines of D. simulans using tiling microarrays and combined it with an analogous survey for the D. melanogaster genome. By integrating the two datasets, we identified duplication hotspots conserved between the two species. However, unlike the duplication hotspots identified in mammalian genomes, Drosophila duplication hotspots are not associated with sequences of high sequence identity capable of mediating non-allelic homologous recombination. Instead, Drosophila duplication hotspots are associated with late-replicating regions of the genome, suggesting a link between DNA replication and duplication rates. We also found evidence supporting a higher effectiveness of selection on duplications in D. simulans than in D. melanogaster. This is also true for duplications segregating at high frequency, where we find evidence in D. simulans that a sizeable fraction of these mutations is being driven to fixation by positive selection

Synthesis of fragments of hexacyclinic acid

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Indian Hedgehog is essential for definitive Haematopoiesis in the fetal liver

Indian hedgehog (Ihh) is a member of the hedgehog family of secreted proteins which play diverse roles in development. Members of this family have established roles in craniofacial development, long bone formation and spermatogenesis. Mammals have three hedgehog proteins which all act through the Patched receptor to activate smoothened and downstream zinc finger transcription factors of the Gli family. Ihh can induce differentiation of primitive red blood cells during primitive streak formation, and definitive red blood cells from CD34+ cord blood stem cells. Both Ihh and the Patched receptor are expressed in the fetal liver. In this report we show Ihh knockout mice display a partially penetrant defect in fetal liver haematopoiesis characterised by severe anaemia and apoptosis of the fetal liver erythroid compartment. Primitive haematopoiesis is normal, so mutant embryos survive until ~E14. Components of the hedgehog signalling pathway are expressed in stromal, stem cell and progenitor cell components of the haematopoietic system. Lastly, fetal liver progenitor cells (CFU-e, BFU-e, and myeloid CFUs) are normal in Ihh null mice, suggesting a critical requirement for Ihh in the fetal liver stem cell niche

A recessive screen for genes regulating hematopoietic stem cells

Identification of genes that regulate the development, self-renewal, and differentiation of stem cells is of vital importance for understanding normal organogenesis and cancer; such knowledge also underpins regenerative medicine. Here we demonstrate that chemical mutagenesis of mice combined with advances in hematopoietic stem cell reagents and genome resources can efficiently recover recessive mutations and identify genes essential for generation and proliferation of definitive hematopoietic stem cells and/or their progeny. We used high-throughput fluorescence-activated cell sorter to analyze 9 subsets of blood stem cells, progenitor cells, circulating red cells, and platelets in more than 1300 mouse embryos at embryonic day (E) 14.5. From 45 pedigrees, we recovered 6 strains with defects in definitive hematopoiesis. We demonstrate rapid identification of a novel mutation in the c-Myb transcription factor that results in thrombocythemia and myelofibrosis as proof of principal of the utility of our fluorescence-activated cell sorter-based screen. Such phenotype-driven approaches will provide new knowledge of the genes, protein interactions, and regulatory networks that underpin stem cell biology. (Blood. 2010;116(26):5849-5858

Genomic organization and regulation of murine alpha haemoglobin stabilizing protein by erythroid Kruppel-like factor

Alpha haemoglobin stabilising protein (AHSP) binds free alpha-globin chains and plays an important role in the protection of red cells, particularly during beta-thalassaemia. Murine ASHP was discovered as a GATA-1 target gene and human AHSP is directly regulated by GATA-1. More recently, AHSP was rediscovered as a highly erythroid Kruppel-like factor (EKLF) -dependent transcript. We have determined the organisation of the murine AHSP gene and compared it to orthologs. There are two CACC box elements in the proximal promoter. The proximal element is absolutely conserved, but does not bind EKLF as it is not a canonical binding site. In rodents, the distal element contains a 3 bp insertion that disrupts the typical EKLF binding consensus region. Nevertheless, EKLF binds this atypical site by gel mobility shift assay, specifically occupies the AHSP promoter in vivo in a chromatin immunoprecipitation assay, and transactivates AHSP through this CACC site in promoter-reporter assays. Our results suggest EKLF can occupy CACC elements in vivo that are not predictable from the consensus binding site inferred from structural studies. We also propose that absence of AHSP in EKLF-null red cells exacerbates the toxicity of free alpha-globin chains, which exist because of the defect in beta-globin gene activation

Optimization of Sphingosine-1-phosphate‑1 Receptor Agonists: Effects of Acidic, Basic, and Zwitterionic Chemotypes on Pharmacokinetic and Pharmacodynamic Profiles

The efficacy of the recently approved drug fingolimod (FTY720) in multiple sclerosis patients results from the action of its phosphate metabolite on sphingosine-1-phosphate S1P₁ receptors, while a variety of side effects have been ascribed to its S1P₃ receptor activity. Although S1P and phospho-fingolimod share the same structural elements of a zwitterionic headgroup and lipophilic tail, a variety of chemotypes have been found to show S1P₁ receptor agonism. Here we describe a study of the tolerance of the S1P₁ and S1P₃ receptors toward bicyclic heterocycles of systematically varied shape and connectivity incorporating acidic, basic, or zwitterionic headgroups. We compare their physicochemical properties, their performance in <i>in vitro</i> and <i>in vivo</i> pharmacokinetic models, and their efficacy in peripheral lymphocyte lowering. The campaign resulted in the identification of several potent S1P₁ receptor agonists with good selectivity vs S1P₃ receptors, efficacy at <1 mg/kg oral doses, and developability properties suitable for progression into preclinical development