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Peter Ghazal

Publications and source records attributed to Peter Ghazal.

At least 19 recordsLinked to original sources
PubMed2006

Improved silicon nitride surfaces for next-generation microarrays.

This work reports how the use of a standard integrated circuit (IC) fabrication process can improve the potential of silicon nitride layers as substrates for microarray technology. It has been shown that chemical mechanical polishing (CMP) substantially improves the fluorescent intensity of positive control gene and test gene microarray spots on both low-pressure chemical vapor deposition (LPCVD) and plasma-enhanced chemical vapor deposition (PECVD) silicon nitride films, while maintaining a low fluorescent background. This results in the improved discrimination of low expressing genes. The results for the PECVD silicon nitride, which has been previously reported as unsuitable for microarray spotting, are particularly significant for future devices that hope to incorporate microelectronic control and analysis circuitry, due to the film's use as a final passivating layer.

Fluorescence↗
PubMed2006

Cell interaction microarray for blood phenotyping.

Microarrays promise great advances in areas of diagnostic testing where there is a need to perform multiple assays in parallel. In the short term, protein microarrays have a greater potential to impact diagnostics than DNA arrays due to their potential for direct sample measurements. Here, we report an antibody microarray technique for selectively recognizing glycan and peptide motifs on the surface of red blood cells. We present results demonstrating the optimization and efficacy of the microarray approach as a highly sensitive and specific microscale multiplex assay for blood typing. We also show that our microarray can be used to screen red blood cell surface antigens using whole blood in a label-free detection mode. Finally, our results indicate this method has potential for broader applications in biochip medicine.

Antigen-Antibody Reactions↗
PubMed2006

Gene expression profiling of mid to late secretory phase endometrial biopsies from women with menstrual complaint.

OBJECTIVE: The purpose of this study was to test whether a quantitative high-throughput molecular screen can be used to probe human endometrium and initiate the development of molecular diagnostic tools with potential for identification of therapeutic targets in women with menstrual complaints. STUDY DESIGN: Endometrium was collected from 10 patients with complaint of heavy bleeding, classified into mid or late secretory phase of the menstrual cycle by histologic dating and serum progesterone concentration. Total RNA was extracted and gene activity assessed using high-density oligonucleotide arrays. RESULTS: Statistical testing identified 83 'signature' genes whose expression levels differentiated the mid and late secretory phases of the menstrual cycle. CONCLUSION: The results show that the endometrium, a complex heterogeneous tissue, is amenable to high-throughput molecular analyses and this work provides further support for the future application of molecular profiling to clinical diagnosis.

Adult↗
PubMed2006

Locus-specific gene expression pattern suggests a unique propagation strategy for a giant algal virus.

Emiliania huxleyi virus strain 86 is the largest algal virus sequenced to date and is unique among the Phycodnaviridae since its genome is predicted to contain six RNA polymerase subunit genes. We have used a virus microarray to profile the temporal transcription strategy of this unusual virus during infection. There are two distinct transcription phases to the infection process. The primary phase is dominated by a group of coding sequences (CDSs) expressed by 1 h postinfection that are localized to a subregion of the genome. The CDS of the primary group have no database homologues, and each is associated with a unique promoter element. The remainder of the CDSs are expressed in a secondary phase between 2 and 4 hours postinfection. Compartmentalized transcription of the two distinctive phases is discussed. We hypothesize that immediately after infection the nucleic acid of the virus targets the host nucleus, where primary-phase genes are transcribed by host RNA polymerase which recognizes the viral promoter. Secondary-phase transcription may then be conducted in the cytoplasm.

Cell Nucleus↗
PubMed2006

Regulation of the transcription and replication cycle of human cytomegalovirus is insensitive to genetic elimination of the cognate NF-kappaB binding sites in the enhancer.

The role of NF-kappaB in regulating human cytomegalovirus (HCMV) replication and gene transcription remains controversial. Multiple, functional NF-kappaB response elements exist in the major immediate-early promoter (MIEP) enhancer of HCMV, suggesting a possible requirement for this transcription factor in lytic viral replication. Here we demonstrate by generating and analyzing HCMVs with alterations in the MIEP-enhancer that, although this region is essential for HCMV growth, none of the four NF-kappaB response elements contained within the enhancer are required for MIE gene expression or HCMV replication in multiple cell types. These data reveal the robustness of the regulatory network controlling the MIEP enhancer.

Base Sequence↗
PubMed2005

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

BACKGROUND: Macrophages 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. DESCRIPTION: The 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. CONCLUSION: GPX-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.

Animals↗
PubMed2005

Artificial intelligence and robotics in high throughput post-genomics.

The shift of post-genomics towards a systems approach has offered an ever-increasing role for artificial intelligence (AI) and robotics. Many disciplines (e.g. engineering, robotics, computer science) bear on the problem of automating the different stages involved in post-genomic research with a view to developing quality assured high-dimensional data. We review some of the latest contributions of AI and robotics to this end and note the limitations arising from the current independent, exploratory way in which specific solutions are being presented for specific problems without regard to how these could be eventually integrated into one comprehensible integrated intelligent system.

Artificial Intelligence↗
PubMed2005

Complete genome sequence and lytic phase transcription profile of a Coccolithovirus.

The genus Coccolithovirus is a recently discovered group of viruses that infect the globally important marine calcifying microalga Emiliania huxleyi. Among the 472 predicted genes of the 407,339-base pair genome are a variety of unexpected genes, most notably those involved in biosynthesis of ceramide, a sphingolipid known to induce apoptosis. Uniquely for algal viruses, it also contains six RNA polymerase subunits and a novel promoter, suggesting this virus encodes its own transcription machinery. Microarray transcriptomic analysis reveals that 65% of the predicted virus-encoded genes are expressed during lytic infection of E. huxleyi.

Apoptosis↗
PubMed2005

Biochip sensors for the rapid and sensitive detection of viral disease.

Recent advances in DNA and protein microarray methodology and the emerging technology of cell-based sensors have massively increased the speed and sensitivity with which we can detect viral infections. The advantages of the multi-parameter microarray technologies could be combined with the speed and sensitivity of cell-based systems to give 'cell-omic' sensors.

Biosensing Techniques↗
PubMed2005

Transcriptomal analysis of varicella-zoster virus infection using long oligonucleotide-based microarrays.

Varicella-zoster virus (VZV) is a human herpes virus that causes varicella as a primary infection and herpes zoster following reactivation of the virus from a latent state in trigeminal and spinal ganglia. In order to study the global pattern of VZV gene transcription, VZV microarrays using 75-base oligomers to 71 VZV open reading frames (ORFs) were designed and validated. The long-oligonucleotide approach maximizes the stringency of detection and polarity of gene expression. To optimize sensitivity, microarrays were hybridized to target RNA and the extent of hybridization measured using resonance light scattering. Microarray data were normalized to a subset of invariant ranked host-encoded positive-control genes and the data subjected to robust formal statistical analysis. The programme of viral gene expression was determined for VZV (Dumas strain)-infected MeWo cells and SVG cells (an immortalized human astrocyte cell line) 72 h post-infection. Marked quantitative and qualitative differences in the viral transcriptome were observed between the two different cell types using the Dumas laboratory-adapted strain. Oligonucleotide-based VZV arrays have considerable promise as a valuable tool in the analysis of viral gene transcription during both lytic and latent infections, and the observed heterogeneity in the global pattern of viral gene transcription may also have diagnostic potential.

Animals↗
PubMed2005

Elimination of ie1 significantly attenuates murine cytomegalovirus virulence but does not alter replicative capacity in cell culture.

The major immediate-early (MIE) genes of cytomegaloviruses (CMV) are broadly thought to be decisive regulators of lytic replication and reactivation from latency. To directly assess the role of the MIE protein IE1 during the infection of murine CMV (MCMV), we constructed an MCMV with exon 4 of the ie1 gene deleted. We found that, independent of the multiplicity of infection, the resulting recombinant virus, MCMVdie1, which fails to express the IE1 protein, was fully competent for early gene expression and replicated in different cultured cell types with identical kinetics to those of parental or revertant virus. Immunofluorescence microscopy studies revealed that MCMVdie1 was greatly impaired in its capacity to disrupt promyelocytic leukemia bodies in NIH 3T3 cells early after infection, a process that has been proposed to increase viral transcription efficiency. We examined MCMVdie1 in the murine model using both immunocompetent BALB/c and severe combined immunodeficient (SCID) mice. When MCMVdie1 was inoculated into these two types of mice, significantly lower viral titers were detected in infected organs than in those of the wild-type virus-infected animals. Moreover, the ie1-deficient MCMV exhibited a markedly reduced virulence. While all animals infected with 5 x 10(4) PFU of parental virus died by 30 days postinfection, SCID mice infected with a similar dose of MCMVdie1 did not succumb before 60 days postinfection. The in vivo defective growth phenotype of MCMVdie1 was abrogated upon rescue of ie1. These results demonstrate the significance of the ie1 gene for promoting an acute MCMV infection and virulence yet indicate that MCMV is able to grow in vivo, although impaired, in the absence of the ie1 gene.

Animals↗
PubMed2005

Design of a herpes simplex virus type 2 long oligonucleotide-based microarray: global analysis of HSV-2 transcript abundance during productive infection.

The design and construction of a long (75-mer) oligonucleotide-based DNA microarray for herpes simplex virus type 2 transcripts is described. This array is utilized to generate an analysis of HSV-2 transcript abundance as a function of conditions of infection of human cells, and global patterns of HSV-2 transcript abundance are compared with those for HSV-1. General similarities in patterns along with notable differences in specific details are noted. These results reveal a marked conservation in the program of gene activity between phenotypically diverged strains.

Chromosome Mapping↗
PubMed2005

Practical Approaches to the Development of Biomedical Informatics: the INFOBIOMED Network of Excellence.

Biomedical Informatics (BMI) is the emerging discipline that aims to facilitate integration of Bioinformatics and Medical Informatics for the purpose of accelerating discovery and the generation of novel diagnostic and therapeutic modalities. Building on the success of the European Commission-funded BIOINFOMED Study, an INFOBIOMED Network of Excellence has been constituted with the main objective of setting a structure for a collaborative approach at a European level. Initially formed by fifteen European organizations, the main objective of the INFOBIOMED network is therefore to enable the reinforcement of European BMI at the forefront of these emergent interdisciplinary fields. The paper describes the structure of the network, the integration approaches regarding databases and four pilot applications.

Biomedical Research↗
PubMed2004

PEDRo: a database for storing, searching and disseminating experimental proteomics data.

BACKGROUND: Proteomics is rapidly evolving into a high-throughput technology, in which substantial and systematic studies are conducted on samples from a wide range of physiological, developmental, or pathological conditions. Reference maps from 2D gels are widely circulated. However, there is, as yet, no formally accepted standard representation to support the sharing of proteomics data, and little systematic dissemination of comprehensive proteomic data sets. RESULTS: This paper describes the design, implementation and use of a Proteome Experimental Data Repository (PEDRo), which makes comprehensive proteomics data sets available for browsing, searching and downloading. It is also serves to extend the debate on the level of detail at which proteomics data should be captured, the sorts of facilities that should be provided by proteome data management systems, and the techniques by which such facilities can be made available. CONCLUSIONS: The PEDRo database provides access to a collection of comprehensive descriptions of experimental data sets in proteomics. Not only are these data sets interesting in and of themselves, they also provide a useful early validation of the PEDRo data model, which has served as a starting point for the ongoing standardisation activity through the Proteome Standards Initiative of the Human Proteome Organisation.

Animals↗
PubMed2004

Neutrality of the canonical NF-kappaB-dependent pathway for human and murine cytomegalovirus transcription and replication in vitro.

Cytomegalovirus (CMV) is known to rapidly induce activation of nuclear factor kappaB (NF-kappaB) after infection of fibroblast and macrophage cells. NF-kappaB response elements are present in the enhancer region of the CMV major immediate-early promoter (MIEP), and activity of the MIEP is strongly upregulated by NF-kappaB in transient-transfection assays. Here we investigate whether the NF-kappaB-dependent pathway is required for initiating or potentiating human and murine CMV replication in vitro. We show that expression of a dominant negative mutant of the inhibitor of NF-kappaB-alpha (IkappaBalphaM) does not alter the replication kinetics of human or mouse CMV in cultured cells. In addition, mouse embryo fibroblasts genetically deficient for p65/RelA actually showed elevated levels of MCMV replication. Mutation of all NF-kappaB response elements within the enhancer of the MIEP in a recombinant mouse CMV containing the human MIEP (hMCMV-ES), which we have previously shown to replicate in murine fibroblasts with kinetics equivalent to that of wild-type mouse CMV, did not negatively affect replication in fibroblasts. Taken together, these data show that, for CMV replication in cultured fibroblasts activation of the canonical NF-kappaB pathway and binding of NF-kappaB to the MIEP are dispensable, and in the case of p65 may even interfere, thus uncovering a previously unrecognized level of complexity in the host regulatory network governing MIE gene expression in the context of a viral infection.

Animals↗
PubMed2003

Screening for mammalian neural genes via fluorescence-activated cell sorter purification of neural precursors from Sox1-gfp knock-in mice.

The transcription factor Sox1 is the earliest and most specific known marker for mammalian neural progenitors. During fetal development, Sox1 is expressed by proliferating progenitor cells throughout the central nervous system and in no tissue but the lens. We generated a reporter mouse line in which egfp is inserted into the Sox1 locus. Sox1GFP animals faithfully recapitulate the expression of the endogenous gene. We have used the GFP reporter to purify neuroepithelial cells by fluorescence-activated cell sorting from embryonic day 10.5 embryos. RNAs prepared from Sox1GFP+ and Sox1GFP- embryo cells were then used to perform a pilot screen of subtracted cDNAs prepared from differentiating embryonic stem cells and arrayed on a glass chip. Fifteen unique differentially expressed genes were identified, all previously associated with fetal or adult neural tissue. Whole mount in situ hybridization against two genes of previously unknown embryonic expression, Lrrn1 and Musashi2, confirmed the selectivity of this screen for early neuroectodermal markers.

Animals↗