[Show abstract][Hide abstract] ABSTRACT: Neonatal sepsis Whole blood Gene expression profiling Microarray Neonatal infection remains a primary cause of infant morbidity and mortality worldwide and yet our understand-ing of how human neonates respond to infection remains incomplete. Changes in host gene expression in response to infection may occur in any part of the body, with the continuous interaction between blood and tissues allowing blood cells to act as biosensors for the changes. In this study we have used whole blood tran-scriptome profiling to systematically identify signatures and the pathway biology underlying the pathogenesis of neonatal infection. Blood samples were collected from neonates at the first clinical signs of suspected sepsis alongside age matched healthy control subjects. Here we report a detailed description of the study design, includ-ing clinical data collected, experimental methods used and data analysis workflows and which correspond with data in Gene Expression Omnibus (GEO) data sets (GSE25504). Our data set has allowed identification of a patient invariant 52-gene classifier that predicts bacterial infection with high accuracy and lays the foundation for advancing diagnostic, prognostic and therapeutic strategies for neonatal sepsis. Specifications Organism/cell line/tissue Homo sapiens/whole blood Sex Male and female (continued) Specifications Sequencer or array type Illumina HT-12V3.0 Whole Human Genome microarray, CodeLink 55K Whole Human Genome microarray, Affymetrix U219 Whole Human Genome microarray and Affymetrix HG U133 Plus 2.0 Whole Human Genome microarray Data format Raw data (Tab delimited text files of background subtracted signals and .CEL files) Experimental factors Blood culture or cerebrospinal fluid positive bacterial sepsis vs. healthy control whole blood samples and culture negative suspected infected samples Experimental features A case–control gene expression profiling study of whole blood taken from neonates at the first clinical sign of sepsis and control healthy neonates. Study includes training and replication sets for blood culture positive samples and clinical evaluation set of blood culture negative sepsis cases. Results compared blood culture or cerebrospinal fluid positive septic neonates, blood culture negative septic neonates and healthy control neonates. Prior power calculations were based on Healthy Edinburgh neonates using the CodeLink platform and Gambian infants (9 months of age) were used for further refinement of power calculations using Illumina HT-12 platform. article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).
[Show abstract][Hide abstract] ABSTRACT: Understanding how human neonates respond to infection remains incomplete. Here, a system-level investigation of neonatal systemic responses to infection shows a surprisingly strong but unbalanced homeostatic immune response; developing an elevated set-point of myeloid regulatory signalling and sugar-lipid metabolism with concomitant inhibition of lymphoid responses. Innate immune-negative feedback opposes innate immune activation while suppression of T-cell co-stimulation is coincident with selective upregulation of CD85 co-inhibitory pathways. By deriving modules of co-expressed RNAs, we identify a limited set of networks associated with bacterial infection that exhibit high levels of inter-patient variability. Whereas, by integrating immune and metabolic pathways, we infer a patient-invariant 52-gene-classifier that predicts bacterial infection with high accuracy using a new independent patient population. This is further shown to have predictive value in identifying infection in suspected cases with blood culture-negative tests. Our results lay the foundation for future translation of host pathways in advancing diagnostic, prognostic and therapeutic strategies for neonatal sepsis.
[Show abstract][Hide abstract] ABSTRACT: Bootstrapping is a popular and computationally demanding resampling method
used for measuring the accuracy of sample estimates and assisting with
statistical inference. R is a freely available language and environment for
statistical computing popular with biostatisticians for genomic data analyses.
A survey of such R users highlighted its implementation of bootstrapping as a
prime candidate for parallelization to overcome computational bottlenecks. The
Simple Parallel R Interface (SPRINT) is a package that allows R users to
exploit high performance computing in multi-core desktops and supercomputers
without expert knowledge of such systems. This paper describes the
parallelization of bootstrapping for inclusion in the SPRINT R package.
Depending on the complexity of the bootstrap statistic and the number of
resamples, this implementation has close to optimal speed up on up to 16 nodes
of a supercomputer and close to 100 on 512 nodes. This performance in a
multi-node setting compares favourably with an existing parallelization option
in the native R implementation of bootstrapping.
[Show abstract][Hide abstract] ABSTRACT: Herein, we report the draft genome sequence of Pantoea sp. ED-NGS-1003, cultivated from a blood sample taken from a neonatal sepsis patient at the Royal Infirmary, Edinburgh, Scotland, United Kingdom.
[Show abstract][Hide abstract] ABSTRACT: Herein, we report the draft genome sequence of Staphylococcus aureus ED-NGS-1006, cultivated from a blood sample taken from a neonatal sepsis patient at the Royal Infirmary in Edinburgh, Scotland, United Kingdom.
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] ABSTRACT: Herein, we report the draft genome sequence for isolate ED-NGS-1015 of Serratia marcescens, cultivated from a blood sample obtained from a neonatal sepsis patient at the Royal Infirmary in Edinburgh, Scotland, United Kingdom.
[Show abstract][Hide abstract] ABSTRACT: Vaccinia virus (VACV) is a large, cytoplasmic, double-stranded DNA virus that requires complex interactions with host proteins in order to replicate. To explore these interactions a functional high throughput small interfering RNA (siRNA) screen targeting 6719 druggable cellular genes was undertaken to identify host factors (HF) influencing the replication and spread of an eGFP-tagged VACV. The experimental design incorporated a low multiplicity of infection, thereby enhancing detection of cellular proteins involved in cell-to-cell spread of VACV. The screen revealed 153 pro- and 149 anti-viral HFs that strongly influenced VACV replication. These HFs were investigated further by comparisons with transcriptional profiling data sets and HFs identified in RNAi screens of other viruses. In addition, functional and pathway analysis of the entire screen was carried out to highlight cellular mechanisms involved in VACV replication. This revealed, as anticipated, that many pro-viral HFs are involved in translation of mRNA and, unexpectedly, suggested that a range of proteins involved in cellular transcriptional processes and several DNA repair pathways possess anti-viral activity. Multiple components of the AMPK complex were found to act as pro-viral HFs, while several septins, a group of highly conserved GTP binding proteins with a role in sequestering intracellular bacteria, were identified as strong anti-viral VACV HFs. This screen has identified novel and previously unexplored roles for cellular factors in poxvirus replication. This advancement in our understanding of the VACV life cycle provides a reliable knowledge base for the improvement of poxvirus-based vaccine vectors and development of anti-viral theraputics.
PLoS ONE 01/2014; 9(6):e98431. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Herein, we report the draft genome sequence of Enterococcus faecalis ED-NGS-1009, cultivated from a blood sample taken from a neonatal sepsis patient at the Royal Infirmary in Edinburgh, Scotland, United Kingdom.
[Show abstract][Hide abstract] ABSTRACT: Herein, we report the draft genome sequence of Staphylococcus warneri ED-NGS-1001, cultivated from a blood sample taken from a preterm neonate blood sepsis patient at the Royal Infirmary, Edinburgh, Scotland, United Kingdom.
[Show abstract][Hide abstract] ABSTRACT: An MRSA assay requiring neither labeling nor amplification of target DNA has been developed. Sequence specific binding of fragments of bacterial genomic DNA is detected at femtomolar concentrations using electrochemical impedance spectroscopy (EIS). This has been achieved using systematic optimisation of probe chemistry (PNA self-assembled monolayer film on gold electrode), electrode film structure (the size and nature of the chemical spacer) and DNA fragmentation, as these are found to play an important role in assay performance. These sensitivity improvements allow the elimination of the PCR step and DNA labeling and facilitate the development of a simple and rapid point of care test for MRSA. Assay performance is then evaluated and specific direct detection of the MRSA diagnostic mecA gene from genomic DNA, extracted directly from bacteria without further treatment is demonstrated for bacteria spiked into saline (10(6) cells per mL) on gold macrodisc electrodes and into human wound fluid (10(4) cells per mL) on screen printed gold electrodes. The latter detection level is particularly relevant to clinical requirements and point of care testing where the general threshold for considering a wound to be infected is 10(5) cells per mL. By eliminating the PCR step typically employed in nucleic acid assays, using screen printed electrodes and achieving sequence specific discrimination under ambient conditions, the test is extremely simple to design and engineer. In combination with a time to result of a few minutes this means the assay is well placed for use in point of care testing.
[Show abstract][Hide abstract] ABSTRACT: Uterine NK cells (uNK) play a role in the regulation of placentation, but their functions in nonpregnant endometrium are not understood. We have previously reported suppression of endometrial bleeding and alteration of spiral artery morphology in women exposed to asoprisnil, a progesterone receptor modulator. We now compare global endometrial gene expression in asoprisnil-treated versus control women, and we demonstrate a statistically significant reduction of genes in the IL-15 pathway, known to play a key role in uNK development and function. Suppression of IL-15 by asoprisnil was also observed at mRNA level (p < 0.05), and immunostaining for NK cell marker CD56 revealed a striking reduction of uNK in asoprisnil-treated endometrium (p < 0.001). IL-15 levels in normal endometrium are progesterone-responsive. Progesterone receptor (PR) positive stromal cells transcribe both IL-15 and IL-15RA. Thus, the response of stromal cells to progesterone will be to increase IL-15 trans-presentation to uNK, supporting their expansion and differentiation. In asoprisnil-treated endometrium, there is a marked downregulation of stromal PR expression and virtual absence of uNK. These novel findings indicate that the IL-15 pathway provides a missing link in the complex interplay among endometrial stromal cells, uNK, and spiral arteries affecting physiologic and pathologic endometrial bleeding.
The Journal of Immunology 08/2013; · 5.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Herpes simplex virus type 1 (HSV-1) is a neurotropic virus causing vesicular oral or genital skin lesions, meningitis and other diseases particularly harmful in immunocompromised individuals. To comprehensively investigate the complex interaction between HSV-1 and its host we combined two genome-scale screens for host factors (HFs) involved in virus replication. A yeast two-hybrid screen for protein interactions and a RNA interference (RNAi) screen with a druggable genome small interfering RNA (siRNA) library confirmed existing and identified novel HFs which functionally influence HSV-1 infection. Bioinformatic analyses found the 358 HFs were enriched for several pathways and multi-protein complexes. Of particular interest was the identification of Med23 as a strongly anti-viral component of the largely pro-viral Mediator complex, which links specific transcription factors to RNA polymerase II. The anti-viral effect of Med23 on HSV-1 replication was confirmed in gain-of-function gene overexpression experiments, and this inhibitory effect was specific to HSV-1, as a range of other viruses including Vaccinia virus and Semliki Forest virus were unaffected by Med23 depletion. We found Med23 significantly upregulated expression of the type III interferon family (IFN-λ) at the mRNA and protein level by directly interacting with the transcription factor IRF7. The synergistic effect of Med23 and IRF7 on IFN-λ induction suggests this is the major transcription factor for IFN-λ expression. Genotypic analysis of patients suffering recurrent orofacial HSV-1 outbreaks, previously shown to be deficient in IFN-λ secretion, found a significant correlation with a single nucleotide polymorphism in the IFN-λ3 (IL28b) promoter strongly linked to Hepatitis C disease and treatment outcome. This paper describes a link between Med23 and IFN-λ, provides evidence for the crucial role of IFN-λ in HSV-1 immune control, and highlights the power of integrative genome-scale approaches to identify HFs critical for disease progression and outcome.
[Show abstract][Hide abstract] ABSTRACT: Interferons (IFN) play a pivotal role in innate immunity, orchestrating a cell-intrinsic anti-pathogenic state and stimulating adaptive immune responses. The complex interplay between the primary response to IFNs and its modulation by positive and negative feedback loops is incompletely understood. Here, we implement the combination of high-resolution gene-expression profiling of nascent RNA with translational inhibition of secondary feedback by cycloheximide. Unexpectedly, this approach revealed a prominent role of negative feedback mechanisms during the immediate (≤60 min) IFNα response. In contrast, a more complex picture involving both negative and positive feedback loops was observed on IFNγ treatment. IFNγ-induced repression of genes associated with regulation of gene expression, cellular development, apoptosis and cell growth resulted from cycloheximide-resistant primary IFNγ signalling. In silico promoter analysis revealed significant overrepresentation of SP1/SP3-binding sites and/or GC-rich stretches. Although signal transducer and activator of transcription 1 (STAT1)-binding sites were not overrepresented, repression was lost in absence of STAT1. Interestingly, basal expression of the majority of these IFNγ-repressed genes was dependent on STAT1 in IFN-naïve fibroblasts. Finally, IFNγ-mediated repression was also found to be evident in primary murine macrophages. IFN-repressed genes include negative regulators of innate and stress response, and their decrease may thus aid the establishment of a signalling perceptive milieu.
Nucleic Acids Research 07/2013; · 8.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Electrodeposition of platinum metal from a chloride-based platinum electroplating bath at 35 °C onto high quality titanium nitride electrodes produced by standard microfabrication techniques has been demonstrated and characterised using electrochemical methods and scanning electron microscopy. When using a relatively simple two potential step electroplating procedure, it is possible to produce good quality deposits which are both adherent and metallic in appearance, without the need for electrode pre-treatment. Cyclic voltammetric analysis of these electrodeposited Pt/TiN films in dilute sulfuric acid yields voltammograms with features characteristic of bulk polycrystalline platinum electrodes. Voltammetry in potassium ferricyanide yields reversible voltammograms, indicating the good electrical conductivity of and connectivity between the deposited Pt and the TiN substrate and hence the absence of any significant resistive surface titanium dioxide or oxynitride film. This process is compatible with the production of platinum array electrodes, which can be integrated with silicon based microelectronic circuitry to produce array based sensors and biosensors.
[Show abstract][Hide abstract] ABSTRACT: Cholesterol biosynthesis serves as a central metabolic hub for numerous biological processes in health and disease. A detailed, integrative single-view description of how the cholesterol pathway is structured and how it interacts with other pathway systems is lacking in the existing literature. Here we provide a systematic review of the existing literature and present a detailed pathway diagram that describes the cholesterol biosynthesis pathway (the mevalonate, the Kandutch-Russell and the Bloch pathway) and shunt pathway that leads to 24(S),25-epoxycholesterol synthesis. The diagram has been produced using the Systems Biology Graphical Notation (SBGN) and is available in the SBGN-ML format, a human readable and machine semantically parsable open community file format.
[Show abstract][Hide abstract] ABSTRACT: There is much that we do not understand about the immune mechanisms whereby vaccines exert their specific and non-specific effects. Most studies take the reductionist approach of examining vaccine responses at the humoral or cellular level. Whole human transcriptional profiling is becoming more accessible, and provides a picture of the entire immune response to vaccination in a single 'snapshot'. The potential uses of such information are enormous, and the data mining tools are becoming more sophisticated to handle the complex data generated. We now face the exciting prospect of gaining in depth knowledge as to exactly what vaccines do to the immune system as a whole, and identifying molecular signatures and biomarkers that can predict immediate and long term outcomes of vaccination. The challenge now is to carry out the studies and generate the much needed data.
[Show abstract][Hide abstract] ABSTRACT: The mechanisms that enable viruses to harness cellular machinery for their own survival are primarily studied in cell lines cultured in two-dimensional (2-D) environments. However, there are increasing reports of biological differences between cells cultured in 2-D versus three-dimensional (3-D) environments. Here we report differences in host-virus interactions based on differences in culture environment. Using ultrashort pulse microscopy (UPM), a form of two-photon microscopy that utilizes sub-10-fs pulses to efficiently excite fluorophores, we have shown that de novo development of extra-chromosomal virus replication compartments (VRCs) upon murine cytomegalovirus (mCMV) infection is markedly enhanced when host cells are cultured in 3-D collagen gels versus 2-D monolayers. In addition, time-lapse imaging revealed that mCMV-induced VRCs have the capacity to grow by coalescence. This work supports the future potential of 3-D culture as a useful bridge between traditional monolayer cultures and animal models to study host-virus interactions in a more physiologically relevant environment for the development of effective anti-viral therapeutics. These advances will require broader adoption of modalities, such as UPM, to image deep within scattering tissues.
Journal of Biomedical Optics 03/2013; 18(3):31111. · 2.75 Impact Factor