CTCF regulates hepatitis B virus cccDNA chromatin topology

Hepatitis B Virus (HBV) is a small DNA virus that replicates via an episomal covalently closed circular DNA (cccDNA) that serves as the transcriptional template for viral mRNAs. The host protein, CCCTC-binding factor (CTCF), is a key regulator of cellular transcription by maintaining epigenetic boundaries, nucleosome phasing, stabilisation of long-range chromatin loops and directing alternative exon splicing. We previously reported that CTCF binds two conserved motifs within Enhancer I of the HBV genome and represses viral transcripts, however, the underlying mechanisms were not identified. We show that CTCF depletion in cells harbouring cccDNA-like HBV molecules and in de novo infected cells resulted in an increase in spliced transcripts, which was most notable in the abundant SP1 spliced transcript. In contrast, depletion of CTCF in cell lines with integrated HBV DNA had no effect on the abundance of viral transcripts and in line with this observation there was limited evidence for CTCF binding to viral integrants, suggesting that CTCF-regulation of HBV transcription is specific to episomal cccDNA. Analysis of HBV chromatin topology by Assay for Transposase Accessibility/sequencing (ATAC-Seq) revealed an accessible region spanning Enhancers I and II and the basal core promoter (BCP). Mutating the CTCF binding sites within Enhancer I resulted in a dramatic rearrangement of chromatin accessibility where the open chromatin region was no longer detected, indicating loss of the phased nucleosome up- and down-stream of the HBV enhancer/BCP. These data demonstrate that CTCF functions to regulate HBV chromatin conformation and nucleosomal positioning in episomal maintained cccDNA, which has important consequences for HBV transcription regulation

CTCF regulates hepatitis B virus cccDNA chromatin topology

Hepatitis B Virus (HBV) is a small DNA virus that replicates via an episomal covalently closed circular DNA (cccDNA) that serves as the transcriptional template for viral mRNAs. The host protein, CCCTC-binding factor (CTCF), is a key regulator of cellular transcription by maintaining epigenetic boundaries, nucleosome phasing, stabilisation of long-range chromatin loops and directing alternative exon splicing. We previously reported that CTCF binds two conserved motifs within Enhancer I of the HBV genome and represses viral transcription, however, the underlying mechanisms were not identified. We show that CTCF depletion in cells harbouring cccDNA-like HBV molecules and in de novo infected cells resulted in an increase in spliced transcripts, which was most notable in the abundant SP1 spliced transcript. In contrast, depletion of CTCF in cell lines with integrated HBV DNA had no effect on the abundance of viral transcripts and in line with this observation there was limited evidence for CTCF binding to viral integrants, suggesting that CTCF-regulation of HBV transcription is specific to episomal cccDNA. Analysis of HBV chromatin topology by Assay for Transposase Accessible Chromatin Sequencing (ATAC-Seq) revealed an accessible region spanning Enhancers I and II and the basal core promoter (BCP). Mutating the CTCF binding sites within Enhancer I resulted in a dramatic rearrangement of chromatin accessibility where the open chromatin region was no longer detected, indicating loss of the phased nucleosome up- and down-stream of the HBV enhancer/BCP. These data demonstrate that CTCF functions to regulate HBV chromatin conformation and nucleosomal positioning in episomal maintained cccDNA, which has important consequences for HBV transcription regulation

Optimizing decomposition of software architecture for local recovery

Cataloged from PDF version of article.The increasing size and complexity of software systems has led to an amplified number of potential failures and as such makes it harder to ensure software reliability. Since it is usually hard to prevent all the failures, fault tolerance techniques have become more important. An essential element of fault tolerance is the recovery from failures. Local recovery is an effective approach whereby only the erroneous parts of the system are recovered while the other parts remain available. For achieving local recovery, the architecture needs to be decomposed into separate units that can be recovered in isolation. Usually, there are many different alternative ways to decompose the system into recoverable units. It appears that each of these decomposition alternatives performs differently with respect to availability and performance metrics. We propose a systematic approach dedicated to optimizing the decomposition of software architecture for local recovery. The approach provides systematic guidelines to depict the design space of the possible decomposition alternatives, to reduce the design space with respect to domain and stakeholder constraints and to balance the feasible alternatives with respect to availability and performance. The approach is supported by an integrated set of tools and illustrated for the open-source MPlayer software

Two-scale EHL: three-dimensional topography in tilted-pad bearings

Derived from the Heterogeneous Multiscale Methods (HMM), a two-scale method is developed for the analysis of Elastohydrodynamic Lubrication (EHL) and micro-EHL in tilted-pad bearings with three-dimensional topography. A relationship linking the pressure gradient to mass flow rate is derived and represented in the bearing domain through homogenisation of near-periodic simulations describing the Fluid Structure Interaction (FSI) of topographical features. For the parameters investigated the influence of compressibility and piezoviscosity was found to be more significant than that of non-Newtonian (shear-thinning) behaviour on textured bearing performance. As the size of topography increased two-scale solutions demonstrated that at constant load the coefficient of friction increased and the minimum film thickness decreased over a range of pad lengths and tilt angles

Fifth European Dirofilaria and Angiostrongylus Days (FiEDAD) 2016

Peer reviewe

Safety Case Driven Development for Medical Devices

Medical devices are safety-critical systems that must comply with standards during their development process because of their intrinsic potential of producing harms. Although the existing trend of an increasing complexity of medical hardware and software components, very little has been done in order to apply more mature safety practices already present on other industrial scenarios. This paper proposes a methodology to enhance the Model-Based System Engineering (MBSE) state-of-art practices from the safety perspective, encouraging the use of safety cases and providing guidance on how to show the correspondent traceability for the development artifacts. We illustrate our methodology and its usage in the context of an industrial Automated External Defibrillator (AED). We suggest that medical device industry could learn from other domains and adapt its development to take into account the hazards and risks along the development, providing more sophisticated justification, as, for example, the impact of design decisions