Paul Dickinson

Viral mediated redirection of NEMO/IKKγ to autophagosomes curtails the inflammatory cascade.

The early host response to viral infections involves transient activation of pattern recognition receptors leading to an induction of inflammatory cytokines such as interleukin-1β (IL-1β) and tumor necrosis factor α (TNFα). Subsequent activation of cytokine receptors in an autocrine and paracrine manner results in an inflammatory cascade. The precise mechanisms by which viruses avert an inflammatory cascade are incompletely understood. Nuclear factor (NF)-κB is a central regulator of the inflammatory signaling cascade that is controlled by inhibitor of NF-κB (IκB) proteins and the IκB kinase (IKK) complex. In this study we show that murine cytomegalovirus inhibits the inflammatory cascade by blocking Toll-like receptor (TLR) and IL-1 receptor-dependent NF-κB activation. Inhibition occurs through an interaction of the viral M45 protein with the NF-κB essential modulator (NEMO), the regulatory subunit of the IKK complex. M45 induces proteasome-independent degradation of NEMO by targeting NEMO to autophagosomes for subsequent degradation in lysosomes. We propose that the selective and irreversible degradation of a central regulatory protein by autophagy represents a new viral strategy to dampen the inflammatory response.

STAT2 deficiency and susceptibility to viral illness in humans

Severe infectious disease in children may be a manifestation of primary immunodeficiency. These genetic disorders represent important experiments of nature with the capacity to elucidate nonredundant mechanisms of human immunity. We hypothesized that a primary defect of innate antiviral immunity was responsible for unusually severe viral illness in two siblings; the proband developed disseminated vaccine strain measles following routine immunization, whereas an infant brother died after a 2-d febrile illness from an unknown viral infection. Patient fibroblasts were indeed abnormally permissive for viral replication in vitro, associated with profound failure of type I IFN signaling and absence of STAT2 protein. Sequencing of genomic DNA and RNA revealed a homozygous mutation in intron 4 of STAT2 that prevented correct splicing in patient cells. Subsequently, other family members were identified with the same genetic lesion. Despite documented infection by known viral pathogens, some of which have been more severe than normal, surviving STAT2-deficient individuals have remained generally healthy, with no obvious defects in their adaptive immunity or developmental abnormalities. These findings imply that type I IFN signaling [through interferon-stimulated gene factor 3 (ISGF3)] is surprisingly not essential for host defense against the majority of common childhood viral infections.

Reversible Inhibition of Murine Cytomegalovirus Replication by Gamma Interferon (IFN-γ) in Primary Macrophages Involves a Primed Type I IFN-Signaling Subnetwork for Full Establishment of an Immediate-Early Antiviral State

ABSTRACT Activated macrophages play a central role in controlling inflammatory responses to infection and are tightly regulated to rapidly mount responses to infectious challenge. Type I interferon (alpha/beta interferon [IFN-α/β]) and type II interferon (IFN-γ) play a crucial role in activating macrophages and subsequently restricting viral infections. Both types of IFNs signal through related but distinct signaling pathways, inducing a vast number of interferon-stimulated genes that are overlapping but distinguishable. The exact mechanism by which IFNs, particularly IFN-γ, inhibit DNA viruses such as cytomegalovirus (CMV) is still not fully understood. Here, we investigate the antiviral state developed in macrophages upon reversible inhibition of murine CMV by IFN-γ. On the basis of molecular profiling of the reversible inhibition, we identify a significant contribution of a restricted type I IFN subnetwork linked with IFN-γ activation. Genetic knockout of the type I-signaling pathway, in the context of IFN-γ stimulation, revealed an essential requirement for a primed type I-signaling process in developing a full refractory state in macrophages. A minimal transient induction of IFN-β upon macrophage activation with IFN-γ is also detectable. In dose and kinetic viral replication inhibition experiments with IFN-γ, the establishment of an antiviral effect is demonstrated to occur within the first hours of infection. We show that the inhibitory mechanisms at these very early times involve a blockade of the viral major immediate-early promoter activity. Altogether our results show that a primed type I IFN subnetwork contributes to an immediate-early antiviral state induced by type II IFN activation of macrophages, with a potential further amplification loop contributed by transient induction of IFN-β.

GPX-Macrophage Expression Atlas: A database for expression profiles of macrophages challenged with a variety of pro-inflammatory, anti-inflammatory, benign and pathogen insults

BackgroundMacrophages play an integral role in the host immune system, bridging innate and adaptive immunity. As such, they are finely attuned to extracellular and intracellular stimuli and respond by rapidly initiating multiple signalling cascades with diverse effector functions. The macrophage cell is therefore an experimentally and clinically amenable biological system for the mapping of biological pathways. The goal of the macrophage expression atlas is to systematically investigate the pathway biology and interaction network of macrophages challenged with a variety of insults, in particular via infection and activation with key inflammatory mediators. As an important first step towards this we present a single searchable database resource containing high-throughput macrophage gene expression studies.DescriptionThe GPX Macrophage Expression Atlas (GPX-MEA) is an online resource for gene expression based studies of a range of macrophage cell types following treatment with pathogens and immune modulators. GPX-MEA follows the MIAME standard and includes an objective quality score with each experiment. It places special emphasis on rigorously capturing the experimental design and enables the searching of expression data from different microarray experiments. Studies may be queried on the basis of experimental parameters, sample information and quality assessment score. The ability to compare the expression values of individual genes across multiple experiments is provided. In addition, the database offers access to experimental annotation and analysis files and includes experiments and raw data previously unavailable to the research community.ConclusionGPX-MEA is the first example of a quality scored gene expression database focussed on a macrophage cellular system that allows efficient identification of transcriptional patterns. The resource will provide novel insights into the phenotypic response of macrophages to a variety of benign, inflammatory, and pathogen insults. GPX-MEA is available through the GPX website at http://www.gti.ed.ac.uk/GPX.

Molecular recognition with DNA nanoswitches: effects of single base mutations on structure.

This paper investigates the properties of a simple DNA-based nanodevice capable of detecting single base mutations in unlabeled nucleic acid target sequences. Detection is achieved by a two-stage process combining first complementary-base hybridization of a target and then a conformational change as molecular recognition criteria. A probe molecule is constructed from a single DNA strand designed to adopt a partial cruciform structure with a pair of exposed (unhybridized) strands. Upon target binding, a switchable cruciform construct (similar to a Holliday junction) is formed which can adopt open and closed junction conformations. Switching between these forms occurs by junction folding in the presence of divalent ions. It has been shown from the steady-state fluorescence of judiciously labeled constructs that there are differences between the fluorescence resonance energy transfer (FRET) efficiencies of closed forms, dependent on the target sequence near the branch point, where the arms of the cruciform cross. This difference in FRET efficiency is attributed to structural variations between these folded junctions with their different branch point sequences arising from the single base mutations. This provides a robust means for the discrimination of single nucleotide mismatches in a specific region of the target. In this paper, these structural differences are analyzed by fitting observed time-resolved donor fluorescence decay data to a Gaussian distribution of donor-acceptor separations. This shows the closest mean separation (approximately 40 A) for the perfectly matched case, whereas larger separations (up to 50 A) are found for the single point mutations. These differences therefore indicate a structural basis for the observed FRET differences in the closed configuration which underpins the operation of these devices as biosensors capable of resolving single base mutations.

Quantitative assessment of human whole blood RNA as a potential biomarker for infectious disease

Infection remains a significant cause of morbidity and mortality especially in newborn infants. Analytical methods for diagnosing infection are severely limited in terms of sensitivity and specificity and require relatively large samples. It is proposed that stringent regulation of the human transcriptome affords a new molecular diagnostic approach based on measuring a highly specific systemic inflammatory response to infection, detectable at the RNA level. This proposition raises a number of as yet poorly characterised technical and biological variation issues that urgently need to be addressed. Here we report a quantitative assessment of methodological approaches for processing and extraction of RNA from small samples of infant whole blood and applying analysis of variation from biochip measurements. On the basis of testing and selection from a battery of assays we show that sufficient high quality RNA for analysis using multiplex array technology can be obtained from small neonatal samples. These findings formed the basis of implementing a set of robust clinical and experimental standard operating procedures for whole blood RNA samples from 58 infants. Modelling and analysis of variation between samples revealed significant sources of variation from the point of sample collection to processing and signal generation. These experiments further permitted power calculations to be run indicating the tractability and requirements of using changes in RNA expression profiles to detect different states between patient groups. Overall the results of our investigation provide an essential first step toward facilitating an alternative way for diagnosing infection from very small neonatal blood samples, providing methods and requirements for future chip-based studies.

Immune oxysterols: Role in mycobacterial infection and inflammation

Infection remains an important cause of morbidity and mortality. Natural defenses to infection are mediated by intrinsic/innate and adaptive immune responses. While our understanding is considerable it is incomplete and emerging areas of research such as those related to the immune-metabolic axis are only beginning to be appreciated. There is increasing evidence showing a connection between immune signalling and the regulation of sterol and fatty acid metabolism. In particular, metabolic intermediates of cholesterol biosynthesis and its oxidized metabolites (oxysterols) have been shown to regulate adaptive immunity and inflammation and for innate immune signalling to regulate the dynamics of cholesterol synthesis and homeostasis. The side-chain oxidized oxysterols, 25-hydroxycholesterol (25HC) and vitamin D metabolites (vitamin D3 and vitamin D2), are now known to impart physiologically profound effects on immune responses. Macrophages play a frontline role in this process connecting immunity, infection and lipid biology, and collaterally are a central target for infection by a wide range of pathogens including viruses and bacteria, especially intracellular bacteria such as mycobacteria. Clinical manifestations of disease severity in the infected host are likely to pay tribute to perturbations of the metabolic-immune phenomena found in lymphocytes and myeloid cells. Historically and consistent with this notion, vitamin D based oxysterols have had a long association with promoting clinical improvements to patients infected with Mycobacterium tuberculosis. Hence understanding the role of early metabolic mediators of inflammatory responses to infection in particular oxysterols, will aid in the development of urgently needed host directed therapeutic and diagnostic design innovation to combat adverse infection outcomes and antibiotic resistance.

Electrochemical control of a DNA Holliday Junction nanoswitch by Mg2+ ions.

The molecular conformation of a synthetic branched, 4-way DNA Holliday junction (HJ) was electrochemically switched between the open and closed (stacked) conformers. Switching was achieved by electrochemically induced quantitative release of Mg(2+) ions from the oxidised poly(N-methylpyrrole) film (PPy), which contained polyacrylate as an immobile counter anion and Mg(2+) ions as charge compensating mobile cations. This increase in the Mg(2+) concentration screened the electrostatic repulsion between the widely separated arms in the open HJ configuration, inducing switching to the closed conformation. Upon electrochemical reduction of PPy, entrapment of Mg(2+) ions back into the PPy film induced the reverse HJ switching from the closed to open state. The conformational transition was monitored using fluorescence resonance energy transfer (FRET) between donor and acceptor dyes each located at the terminus of one of the arms. The demonstrated electrochemical control of the conformation of the used probe-target HJ complex, previously reported as a highly sequence specific nanodevice for detecting of unlabelled target [Buck, A.H., Campbell, C.J., Dickinson, P., Mountford, C.P., Stoquert, H.C., Terry, J.G., Evans, S.A.G., Keane, L., Su, T.J., Mount, A.R., Walton, A.J., Beattie, J.S., Crain, J., Ghazal, P., 2007. Anal. Chem., 79, 4724-4728], allows the development of electronically addressable DNA nanodevices and label-free gene detection assays.

A DNA nanoswitch incorporating the fluorescent base analogue 2-aminopurine detects single nucleotide mismatches in unlabelled targets.

DNA nanoswitches can be designed to detect unlabelled nucleic acid targets and have been shown to discriminate between targets which differ in the identity of only one base. This paper demonstrates that the fluorescent base analogue 2-aminopurine (AP) can be used to discriminate between nanoswitches with and without targets and to discriminate between matched and mismatched targets. In particular, we have used both steady-state and time-resolved fluorescence spectroscopy to determine differences in AP environment at the branchpoint of nanoswitches assembled using complementary targets and targets which incorporate single base mismatches.

Draft Genome Sequence of a Streptococcus agalactiae Strain Isolated from a Preterm Neonate Blood Sepsis Patient at the Royal Infirmary, Edinburgh, Scotland

ABSTRACT Herein, we report the draft genome sequence of Streptococcus agalactiae ED-NGS-1000, cultivated from a blood sample taken from a preterm neonate blood sepsis patient at the Royal Infirmary, Edinburgh, Scotland, United Kingdom.