Burkholderia cenocepacia-induced delay of acidification and phagolysosomal fusion in cystic fibrosis transmembrane conductance regulator (CFTR)-defective macrophages

The Burkholderia cepacia complex (Bcc) is a group of opportunistic bacteria chronically infecting the airways of patients with cystic fibrosis (CF). Several laboratories have shown that Bcc members, in particular B. cenocepacia, survive within a membrane-bound vacuole inside phagocytic and epithelial cells. We have previously demonstrated that intracellular B. cenocepacia causes a delay in phagosomal maturation, as revealed by impaired acidification and slow accumulation of the late phagolysosomal marker LAMP-1. In this study, we demonstrate that uninfected cystic fibrosis transmembrane conductance regulator (CFTR)-defective macrophages or normal macrophages treated with a CFTR-specific drug inhibitor display normal acidification. However, after ingestion of B. cenocepacia, acidification and phagolysosomal fusion of the bacteria-containing vacuoles occur in a lower percentage of CFTR-negative macrophages than CFTR-positive cells, suggesting that loss of CFTR function contributes to enhance bacterial intracellular survival. The CFTR-associated phagosomal maturation defect was absent in macrophages exposed to heat-inactivated B. cenocepacia and macrophages infected with a non-CF pathogen such as Salmonella enterica, an intracellular pathogen that once internalized rapidly traffics to acidic compartments that acquire lysosomal markers. These results suggest that not only a defective CFTR but also viable B. cenocepacia are required for the altered trafficking phenotype. We conclude that CFTR may play a role in the mechanism of clearance of the intracellular infection, as we have shown before that B. cenocepacia cells localized to the lysosome lose cell envelope integrity. Therefore, the prolonged maturation arrest of the vacuoles containing B. cenocepacia within cftr(-/-) macrophages could be a contributing factor in the persistence of the bacteria within CF patients.

Burkholderia cenocepacia requires RpoE for growth under stress conditions and delay of phagolysosomal fusion in macrophages

Burkholderia cenocepacia is an opportunistic pathogen causing serious infections in patients with cystic fibrosis. The widespread distribution of this bacterium in the environment suggests that it must adapt to stress to be able to survive. We identified in B. cenocepacia K56-2 a gene predicted to encode RpoE, the extra-cytoplasmic stress response regulator. The rpoE gene is the first gene of a predicted operon encoding proteins homologous to RseA, RseB, MucD and a protein of unknown function. The genomic organization and the co-transcription of these genes were confirmed by PCR and RT-PCR. The mucD and rpoE genes were mutated, giving rise to B. cenocepacia RSF24 and RSF25, respectively. While mutant RSF24 did not demonstrate any growth defects under the conditions tested, RSF25 was compromised for growth under temperature (44 degrees C) and osmotic stress (426 mM NaCl). Expression of RpoE in trans could complement the osmotic growth defect but exacerbated temperature sensitivity in both RSF25 and wild-type K56-2. Inactivation of rpoE altered the bacterial cell surface, as indicated by increased binding of the fluorescent dye calcofluor white and by an altered outer-membrane protein profile. These cell surface changes were restored by complementation with a plasmid encoding rpoE. Macrophage infections in which bacterial colocalization with fluorescent dextran was examined demonstrated that the rpoE mutant could not delay the fusion of B. cenocepacia-containing vacuoles with lysosomes, in contrast to the parental strain K56-2. These data show that B. cenocepacia rpoE is required for bacterial growth under certain stress conditions and for the ability of intracellular bacteria to delay phagolysosomal fusion in macrophages.

Intracellular survival of Burkholderia cenocepacia in macrophages is associated with a delay in the maturation of bacteria-containing vacuoles

Strains of the Burkholderia cepacia complex (Bcc) are opportunistic bacteria that can cause life-threatening infections in patients with cystic fibrosis and chronic granulomatous disease. Previous work has shown that Bcc isolates can persist in membrane-bound vacuoles within amoeba and macrophages without bacterial replication, but the detailed mechanism of bacterial persistence is unknown. In this study, we have investigated the survival of the Burkholderia cenocepacia strain J2315 within RAW264.7 murine macrophages. Strain J2315 is a prototypic isolate of the widespread and transmissible ET12 clone. Unlike heat-inactivated bacteria, which reach lysosomes shortly after internalization, vacuoles containing live B. cenocepacia J2315 accumulate the late endosome/lysosome marker LAMP-1 and start fusing with lysosomal compartments only after 6 h post internalization. Using fluorescent fluid-phase probes, we also demonstrated that B. cenocepacia-containing vacuoles continued to interact with newly formed endosomes, and maintained a luminal pH of 6.4 +/- 0.12. In contrast, vacuoles containing heat-inactivated bacteria had an average pH of 4.8 +/- 0.03 and rapidly merged with lysosomes. Additional experiments using concanamycin A, a specific inhibitor of the vacuolar H+-ATPase, revealed that vacuoles containing live bacteria did not exclude the H+-ATPase. This mode of bacterial survival did not require type III secretion, as no differences were found between wild type and a type III secretion mutant strain. Collectively, our results suggest that intracellular B. cenocepacia cause a delay in the maturation of the phagosome, which may contribute to facilitate bacterial escape from the microbicidal activities of the host cell.

How (In)Consistent is the Court of Justice in the Application of the Market Access Test?”

Invited panel speaker at a Jean Monnet Chair funded research workshop organised by the Europa Institute, School of Law, University of Edinburgh (9 December 2011), http://www.pol.ed.ac.uk/research_themes/index/jean_monnet_centre_of_excellence/principles_of_market_access_workshop

'Just be Brave' - The Experiences of Young Witnesses in Criminal Proceedings in Northern Ireland

The last three decades have witnessed considerable interest in the position of children and young people acting as witnesses in criminal cases and on how best to facilitate them to give their best evidence and minimise the trauma involved. This paper presents the findings of a small-scale study in Northern Ireland examining the experiences of young witnesses pre-trial, during the trial and post-trial. Interviews were carried out with 37 young witnesses and 33 parents, and a questionnaire was completed by 16 volunteers and practitioners working in a local young witness support scheme. The findings indicate that the prospect and actuality of giving evidence in a criminal trial are anxiety-provoking and stressful for the majority of young witnesses. Particular issues identified are delay, both in terms of cases coming to court and in waiting times at court, the availability of pre-trial preparation and support, facilities at court buildings and the treatment of young people during cross-examination by defence lawyers. The paper concludes that there is a continuing need to strive for improvement, and that this necessarily involves reviewing the experiences of young witnesses and seeking their views on measures designed to enable them to give their best evidence. Copyright © 2013 John Wiley & Sons, Ltd.

Human Rights and the United Kingdom Supreme Court

After setting the scene by explaining the constraints which are placed on the Justices of the UK Supreme Court, this book considers how human rights are conceptualized by the Court in general and how in particular the procedural questions thrown up by the Human Rights Act have been dealt with so far. It then examines on a right-by-right basis the Justices' position on all the European Convention rights and some additional international human rights standards incorporated into UK law.

Optimization of Composite Structures to Delay Mode Jump Instabilities

Numerous studies have shown that postbuckling stiffened panels may undergo abrupt changes in buckled mode
shape when loaded in uniaxial compression. This phenomenon is often referred to as a mode jump or secondary
instability. The resulting sudden release of stored energy may initiate damage in vulnerable regions within a
structure, for example, at the skin-stiffener interface of a stiffened composite panel. Current design practice is to
remove a mode jump by increasing the skin thickness of the postbuckling region. A layup optimization methodology,
based on a genetic algorithm, is presented, which delays the onset of secondary instabilities in a composite structure
while maintaining a constant weight and subject to a number of design constraints. A finite element model was
developed of a stiffened panel’s skin bay, which exhibited secondary instabilities. An automated numerical routine
extracted information directly from the finite element displacement results to detect the onset of initial buckling and
secondary instabilities. This routine was linked to the genetic algorithm to find a revised layup for the skin bay, within
appropriate design constraints, to delay the onset of secondary instabilities. The layup optimization methodology,
resulted in a panel that had a higher buckling load, prebuckling stiffness, and secondary instability load than the
baseline design.

Lay-up optimization of composite plates to delay mode-jump instabilities

Experimental and numerical studies have shown that the occurrence of abrupt secondary instabilities, or mode-jumps, in a postbuckling stiffened composite panel may initiate structural failure. This study presents an optimisation methodology, using a genetic algorithm and finite element analysis for the lay-up optimisation of postbuckling composite plates to delay the onset of mode-jump instabilities. A simple and novel approach for detecting modejumps is proposed, based on the RMS value of out-of-plane pseudo-velocities at a number of locations distributed over the postbuckling structure

Availability of stage at diagnosis, cancer treatment delay and compliance with cancer guidelines as cancer registry indicators for cancer care in Europe:Results of EUROCHIP-3 survey

EUROCHIP (European Cancer Health Indicators Project) focuses on understanding inequalities in the cancer burden, care and survival by the indicators "stage at diagnosis," "cancer treatment delay" and "compliance with cancer guidelines" as the most important indicators. Our study aims at providing insight in whether cancer registries collect well-defined variables to determine these indicators in a comparative way. Eighty-six general European population-based cancer registries (PBCR) from 32 countries responded to the questionnaire, which was developed by EUROCHIP in collaboration with ENCR (European Network of Cancer Registries) and EUROCOURSE. Only 15% of all the PBCR in EU had all three indicators available. The indicator "stage at diagnosis" was gathered for at least one cancer site by 81% (using TNM in 39%). Variables for the indicator "cancer treatment delay" were collected by 37%. Availability of type of treatment (30%), surgery date (36%), starting date of radiotherapy (26%) and starting date of chemotherapy (23%) resulted in 15% of the PBCRs to be able to gather the indicator "compliance to guidelines". Lack of data source access and qualified staff were the major reasons for not collecting all the variables. In conclusion, based on self-reporting, a few of the participating PBCRs had data available which could be used for clinical audits, evaluation of cancer care projects, survival and for monitoring national cancer control strategies. Extra efforts should be made to improve this very efficient tool to compare cancer burden and the effects of the national cancer plans over Europe and to learn from each other. © 2012 UICC.

Delay times and rates for Type Ia supernovae and thermonuclear explosions from double-detonation sub-Chandrasekhar mass models

We present theoretical delay times and rates of thermonuclear explosions that are thought to produce Type Ia supernovae (SNe Ia), including the double-detonation sub-Chandrasekhar mass model, using the population synthesis binary evolution code startrack. If detonations of sub-Chandrasekhar mass carbon-oxygen white dwarfs following a detonation in an accumulated layer of helium on the white dwarf's surface ('double-detonation' models) are able to produce thermonuclear explosions which are characteristically similar to those of SNe Ia, then these sub-Chandrasekhar mass explosions may account for at least some substantial fraction of the observed SN Ia rate. Regardless of whether all double-detonations look like 'normal' SNe Ia, in any case the explosions are expected to be bright and thus potentially detectable. Additionally, we find that the delay time distribution of double-detonation sub-Chandrasekhar mass SNe Ia can be divided into two distinct formation channels: the 'prompt' helium-star channel with delay times