The suhB gene of Burkholderia cenocepacia is required for protein secretion, biofilm formation, motility and polymyxin B resistance

Burkholderia cenocepacia is a member of the Burkholderia cepacia complex (Bcc), a group of Gram-negative opportunistic pathogens that cause severe lung infections in patients with cystic fibrosis and display extreme intrinsic resistance to antibiotics including antimicrobial peptides. B. cenocepacia BCAL2157 encodes a protein homologous to SuhB, an inositol-1-monophosphatase from Escherichia coli, which was suggested to participate in posttranscriptional control of gene expression. In this work we show that a deletion of the suhB-like gene in B. cenocepacia (?suhBBc) was associated with pleiotropic phenotypes. The ?suhBBc mutant had a growth defect manifested by an almost 2-fold increase in the generation time relative to the parental strain. The mutant also had a general defect in protein secretion, motility and biofilm formation. Further analysis of the Type-2 and the Type-6 secretion systems activities revealed that these secretion systems were inactive in the ?suhBBc mutant. In addition, the mutant exhibited increased susceptibility to polymyxin B but not to aminoglycosides like gentamicin and kanamycin. Together, our results demonstrate that suhBBc deletion compromises general protein secretion including the activity of T2SS and T6SS, and affects polymyxin B resistance, motility, and biofilm formation. The pleiotropic effects observed upon suhBBc deletion demonstrate that suhBBc plays a critical role in the physiology of B. cenocepacia.

Computationally efficient sequential learning algorithms for direct link resource-allocating networks

Computionally efficient sequential learning algorithms are developed for direct-link resource-allocating networks (DRANs). These are achieved by decomposing existing recursive training algorithms on a layer by layer and neuron by neuron basis. This allows network weights to be updated in an efficient parallel manner and facilitates the implementation of minimal update extensions that yield a significant reduction in computation load per iteration compared to existing sequential learning methods employed in resource-allocation network (RAN) and minimal RAN (MRAN) approaches. The new algorithms, which also incorporate a pruning strategy to control network growth, are evaluated on three different system identification benchmark problems and shown to outperform existing methods both in terms of training error convergence and computational efficiency. (c) 2005 Elsevier B.V. All rights reserved.

On the solubilization of water with ethanol in hydrophobic hexafluorophosphate ionic liquids

The solubility of water in the hydrophobic 1-alkyl-3-methylimidazolium hexafluorophosphate (alkyl = butyl, hexyl, and octyl) ionic liquids, can be significantly increased in the presence of ethanol as a co-solute. 1-Hexyl-3-methylimidazolium hexafluorophosphate and 1-octyl-3-methylimidazolium hexafluorophosphate are completely miscible with ethanol, and immiscible with water, whereas 1-butyl-3-methylimidazolium hexafluorophosphate is totally miscible with aqueous ethanol only between 0.5-0.9 mole fraction ethanol at 25degreesC. At higher and lower mole fraction of ethanol, the aqueous and IL components are only partially miscible and a biphasic system is obtained upon mixing equal volumes of the IL and aqueous ethanol. The observation of a large range of total miscibility between water and the IL in the three-component system has important implications for purifications and separations from IL.

Temporal information and children's and adults' causal inferences

Three experiments examined whether children and adults would use temporal information as a cue to the causal structure of a three-variable system, and also whether their judgements about the effects of interventions on the system would be affected by the temporal properties of the event sequence. Participants were shown a system in which two events B and C occurred either simultaneously (synchronous condition) or in a temporal sequence (sequential condition) following an initial event A. The causal judgements of adults and 6-7-year-olds differed between the conditions, but this was not the case for 4-year-olds' judgements. However, unlike those of adults, 6-7-year-olds' intervention judgements were not affected by condition, and causal and intervention judgements were not reliably consistent in this age group. The findings support the claim that temporal information provides an important cue to causal structure, at least in older children. However, they raise important issues about the relationship between causal and intervention judgements.

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.

Characterization of the AtsR Hybrid Sensor Kinase Phosphorelay Pathway and Identification of Its Response Regulator in Burkholderia cenocepacia

AtsR is a membrane-bound hybrid sensor kinase of Burkholderia cenocepacia that negatively regulates quorum sensing and virulence factors such as biofilm production, type 6-secretion and protease secretion. Here, we elucidate the mechanism of AtsR phosphorelay by site-directed mutagenesis of predicted histidine and aspartic acid phosphoacceptor residues. We demonstrate by in vitro phosphorylation that histidine-245 and aspartic acid-536 are conserved sites of phosphorylation in AtsR, and we also identify the cytosolic response regulator AtsT (BCAM0381) as a key component of the AtsR phosphorelay pathway. Monitoring the function of AtsR and its derivatives in vivo by measuring extracellular protease activity and swarming motility confirmed the in vitro phosphorylation results. Together, we find that the AtsR receiver domain plays a fine-tuning role in determining the levels of phosphotransfer from its sensor kinase domain to the AtsT response regulator.

A statistical analysis of person–to–vehicle communications channels in an urban environment at 5.8 GHz

In recent years, the embracement of smart devices carried or worn by people have transformed how society interact with one another. This trend has also been observed in the advancement of vehicular networks. Here, developments in wireless technologies for vehicle-to-vehicle (V2V) and vehicle-to-roadside (V2R) communications are leading to a new generation of vehicular networks. A natural extension of both types of networks will be their eventual wireless integration. Both people and vehicles will undoubtedly form integral parts of future mobile networks of people and things. Central to this will be the person-to-vehicle (P2V) communications channel. As the P2V channel will be subject to different signal propagation characteristics than either type of communication system considered in isolation, it is imperative the characteristics of the wireless channel must first be fully understood. To the best of the author's knowledge, this is a topic which has not yet been addressed in the open literature. In this paper we will present our most recent research on the statistical characterization of the 5.8 GHz person-to-vehicle channel in an urban environment.

Observational Constraints on the Progenitors of Core-Collapse Supernovae: The Case for Missing High-Mass Stars

Over the last 15 years, the supernova community has endeavoured to directly identify progenitor stars for core-collapse supernovae discovered in nearby galaxies. These precursors are often visible as resolved stars in high-resolution images from space-and ground-based telescopes. The discovery rate of progenitor stars is limited by the local supernova rate and the availability and depth of archive images of galaxies, with 18 detections of precursor objects and 27 upper limits. This review compiles these results (from 1999 to 2013) in a distance-limited sample and discusses the implications of the findings. The vast majority of the detections of progenitor stars are of type II-P, II-L, or IIb with one type Ib progenitor system detected and many more upper limits for progenitors of Ibc supernovae (14 in all). The data for these 45 supernovae progenitors illustrate a remarkable deficit of high-luminosity stars above an apparent limit of log L/L-circle dot similar or equal to 5.1 dex. For a typical Salpeter initial mass function, one would expect to have found 13 high-luminosity and high-mass progenitors by now. There is, possibly, only one object in this time-and volume-limited sample that is unambiguously high-mass (the progenitor of SN2009ip) although the nature of that supernovae is still debated. The possible biases due to the influence of circumstellar dust, the luminosity analysis, and sample selection methods are reviewed. It does not appear likely that these can explain the missing high-mass progenitor stars. This review concludes that the community's work to date shows that the observed populations of supernovae in the local Universe are not, on the whole, produced by high-mass (M greater than or similar to 18 M-circle dot) stars. Theoretical explosions of model stars also predict that black hole formation and failed supernovae tend to occur above an initial mass of M similar or equal to 18 M-circle dot. The models also suggest there is no simple single mass division for neutron star or black-hole formation and that there are islands of explodability for stars in the 8-120 M-circle dot range. The observational constraints are quite consistent with the bulk of stars above M similar or equal to 18 M-circle dot collapsing to form black holes with no visible supernovae.

Intracellular survival of Burkholderia cepacia complex in phagocytic cells

Burkholderia cepacia complex (Bcc) species are a group of Gram-negative opportunistic pathogens that infect the airways of cystic fibrosis patients, and occasionally they infect other immunocompromised patients. Bcc bacteria display high-level multidrug resistance, and chronically persist in the infected host while eliciting robust inflammatory responses. Studies using macrophages, neutrophils and dendritic cells, combined with advances to genetically manipulate these bacteria have increased our understanding of the molecular mechanisms of virulence in these pathogens and the molecular details of cell-host responses triggering inflammation. This article discusses our current view of the intracellular survival of B. cenocepacia within macrophages.

Preparing to prescribe: How do clerkship students learn in the midst of complexity?

Prescribing tasks, which involve pharmacological knowledge, clinical decision-making and practical skill, take place within unpredictable social environments and involve interactions within and between endlessly changing health care teams. Despite this, curriculum designers commonly assume them to be simple to learn and perform. This research used mixed methods to explore how undergraduate medical students learn to prescribe in the 'real world'. It was informed by cognitive psychology, sociocultural theory, and systems thinking. We found that learning to prescribe occurs as a dynamic series of socially negotiated interactions within and between individuals, communities and environments. As well as a thematic analysis, we developed a framework of three conceptual spaces in which learning opportunities for prescribing occur. This illustrates a complex systems view of prescribing education and defines three major system components: the "social space", where the environmental conditions influence or bring about a learning experience; the "process space", describing what happens during the learning experience; and the intra-personal "cognitive space", where the learner may develop aspects of prescribing expertise. This conceptualisation broadens the scope of inquiry of prescribing education research by highlighting the complex interplay between individual and social dimensions of learning. This perspective is also likely to be relevant to students' learning of other clinical competencies.

Modelling the effectiveness of grass buffer strips in managing muddy floods under a changing climate

Muddy floods occur when rainfall generates runoff on agricultural land, detaching and transporting sediment into the surrounding natural and built environment. In the Belgian Loess Belt, muddy floods occur regularly and lead to considerable economic costs associated with damage to property and infrastructure. Mitigation measures designed to manage the problem have been tested in a pilot area within Flanders and were found to be cost-effective within three years. This study assesses whether these mitigation measures will remain effective under a changing climate. To test this, the Water Erosion Prediction Project (WEPP) model was used to examine muddy flooding diagnostics (precipitation, runoff, soil loss and sediment yield) for a case study hillslope in Flanders where grass buffer strips are currently used as a mitigation measure. The model was run for present day conditions and then under 33 future site-specific climate scenarios. These future scenarios were generated from three earth system models driven by four representative concentration pathways and downscaled using quantile mapping and the weather generator CLIGEN. Results reveal that under the majority of future scenarios, muddy flooding diagnostics are projected to increase, mostly as a consequence of large scale precipitation events rather than mean changes. The magnitude of muddy flood events for a given return period is also generally projected to increase. These findings indicate that present day mitigation measures may have a reduced capacity to manage muddy flooding given the changes imposed by a warming climate with an enhanced hydrological cycle. Revisions to the design of existing mitigation measures within existing policy frameworks are considered the most effective way to account for the impacts of climate change in future mitigation planning. 

Irish Credit Unions: Differential Regulation based on Business Model Complexity

This study examines the business model complexity of Irish credit unions using a latent class approach to measure structural performance over the period 2002 to 2013. The latent class approach allows the endogenous identification of a multi-class framework for business models based on credit union specific characteristics. The analysis finds a three class system to be appropriate with the multi-class model dependent on three financial viability characteristics. This finding is consistent with the deliberations of the Irish Commission on Credit Unions (2012) which identified complexity and diversity in the business models of Irish credit unions and recommended that such complexity and diversity could not be accommodated within a one size fits all regulatory framework. The analysis also highlights that two of the classes are subject to diseconomies of scale. This may suggest credit unions would benefit from a reduction in scale or perhaps that there is an imbalance in the present change process. Finally, relative performance differences are identified for each class in terms of technical efficiency. This suggests that there is an opportunity for credit unions to improve their performance by using within-class best practice or alternatively by switching to another class.

Clinical and immunohistochemical assessment of vulval intraepithelial neoplasia following photodynamic therapy using a novel bioadhesive patch-type system loaded with 5-aminolevulinic acid

Background: The work in this study appraised photodynamic treatment (PDT) as a treatment method for vulval intraepithelial neoplasia (VIN) using a novel bioadhesive patch to deliver aminolevulinic acid. An analysis of changes in expression of apoptotic and cell cycle proteins (p53, p21, Mdm2, Blc-2, Bax, Ki-67) in response to PDT was evaluated. Methods: PDT was performed using non-laser light, either as a one or two-cycle treatment, with clinical and pathological assessment following after 6 weeks. Twenty-three patients with 25 VIN lesions underwent 49 cycles of PDT Patches were designed to conform to uneven vulval skin and contained 38 mg cm(-2) aminolevulinic acid. Assessment was carried out at 6 weeks post-treatment. Patient-based treatment assessment, along with clinical and pathological changes, were monitored. Immunohistochemical staining was used to elucidate a possible biomolecular basis for induced cellular changes. Results: Most patients (52%) reported a symptomatic response, with normal pathology restored in 38% of lesions. The patch was easy to apply and remove, causing minimal discomfort. Fluorescence inspection confirmed protoporphyrin accumulation. Pain during implementation of PDT was problematic, necessitating some form of local analgesia. Changes in expression of cell cycle and apoptotic-related proteins suggested involvement of apoptotic pathways. Down regulation of p21 and inverse changes in Bcl-2 and Bax were key findings. Conclusion: Treatment of VIN lesions using a novel bioadhesive patch induced changes in cell cycle and apoptotic proteins in response to PDT with possible utilisation of apoptotic pathways. The efficacy of PDT in treating VIN could be improved by a better understanding of these apoptotic mechanisms, the influence of factors, such as HPV status, and of the need for effective pain management.

Modelling the flow of congestive heart failure patients through a hospital system

The number of hospital admissions in England due to heart failure is projected to increase by over 50% during the next 25 years. This will incur greater pressures on hospital managers to allocate resources in an effective manner. A reliable indicator for measuring the quantity of resources consumed by hospital patients is their length of stay (LOS) in care. This paper proposes modelling the length of time heart failure patients spend in hospital using a special type of Markov model, where the flow of patients through hospital can be thought of as consisting of three stages of care—short-, medium- and longer-term care. If it is assumed that new admissions into the ward are replacements for discharges, such a model may be used to investigate the case-mix of patients in hospital and the expected patient turnover during some specified period of time. An example is illustrated by considering hospital admissions to a Belfast hospital in Northern Ireland, between 2000 and 2004.

Amphibian skin peptides and their corresponding cDNAs from single lyophilized secretion samples: identification of novel brevinins from three species of Chinese frogs

Brevinins are peptides of 24 amino acid residues, originally isolated from the skin of the Oriental frog, Rana brevipoda porsa, by nature of their microbicidal activity against a wide range of Gram-positive and Gram-negative bacteria and against strains of pathogenic fungi. cDNA libraries were constructed from lyophilized skin secretion of three, unstudied species of Chinese frog, Odorrana schmackeri, Odorrana versabilis and Pelophylax plancyi fukienensis, using our recently developed technique. In this report, we describe the “shotgun” cloning of novel brevinins by means of 3'-RACE, using a “universal” degenerate primer directed towards a highly conserved nucleic acid sequence domain within the 5'-untranslated region of previously characterized frog skin peptide cDNAs. Novel brevinins, deduced from cloned cDNA open-reading frames, were subsequently identified as mature peptides in the same samples of respective species skin secretions. Bioinformatic analysis of both prepro-brevinin nucleic acid sequences and translated open-reading frame amino acid sequences revealed a highly conserved signal peptide domain and a hypervariable anti-microbial peptide-encoding domain. The experimental approach described here can thus rapidly provide robust structural data on skin anti-microbial peptides without harming the donor amphibians.