A conditioning model of delusion.

"Delusions" are beliefs that are false and persistent. It is suggested here that these characteristics can emerge from interplays between two fundamental learning processes: (1) the allocation of attentional resources among stimuli; and (2) the effects of feedback on learning. The former of these has been operationalized in the learned irrelevance and latent inhibition paradigms; the latter in studies of the effects of persistence-training. Normally, the attentional process functions to constrain persistence-training effects so that only valid associations acquire persistence. But when persistence-training is less influenced in this way, its mechanisms can interact with a noisy environment to gradually insulate maladaptive associations from disconfirming feedback. When unchecked, these dynamics likely lead to a systematic distortion of beliefs that can become increasingly persistent regardless of their validity. Delusions are therefore predicted to tend to arise whenever the balance of (1) is weakened in favour of (2), whether by experimental manipulation, trait-related factors, cultural causes or evolutionary history. Existing evidence is consistent with the model and further implications are discussed.

Rapid proteasomal elimination of 3-hydroxy-3-methylglutaryl-CoA reductase by interferon-γ in primary macrophages requires endogenous 25-hydroxycholesterol synthesis.

Interferons (IFNs) play a central role in immunity and emerging evidence suggests that IFN-signalling coordinately regulates sterol biosynthesis in macrophages, via Sterol Regulatory Element-Binding Protein (SREBP) dependent and independent pathways. However, the precise mechanisms and kinetic steps by which IFN controls sterol biosynthesis are as yet not fully understood. Here, we elucidate the molecular circuitry governing how IFN controls the first regulated step in the mevalonate-sterol pathway, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), through the synthesis of 25-Hydroxycholesterol (25-HC) from cholesterol by the IFN-inducible Cholesterol-25-Hydroxylase (CH25H). We show for the first 30-min of IFN stimulation of macrophages the rate of de novo synthesis of the Ch25h transcript is markedly increased but by 120-min becomes transcriptionally curtailed, coincident with induction of the Activating Transcription Factor 3 (ATF3) repressor. We demonstrate ATF3 induction by Toll-like receptors is strictly dependent on IFN-signalling. While the SREBP-pathway dependent rates of de novo transcription of Hmgcr are relatively unchanged in the first 90-min of IFN treatment, we find HMGCR enzyme levels undergo a rapid proteasomal-mediated degradation, defining a previously unappreciated SREBP-independent mechanism for IFN-action. These events precede a sustained marked reduction in Hmgcr RNA levels involving SREBP-dependent mechanisms. We demonstrate that HMGCR proteasomal-degradation by IFN strictly requires the synthesis of endogenous 25-HC and functionally couples HMGCR to CH25H to coordinately suppress sterol biosynthesis. In conclusion, we quantitatively delineate proteomic and transcriptional levels of IFN-mediated control of HMGCR, the primary enzymatic step of the mevalonate-sterol biosynthesis pathway, providing a foundational framework for mathematically modelling the therapeutic outcome of immune-metabolic pathways.

Strategic use of conformational bias and structure based design to identify potent JAK3 inhibitors with improved selectivity against the JAK family and the kinome.

Using a structure based design approach we have identified a series of indazole substituted pyrrolopyrazines, which are potent inhibitors of JAK3. Intramolecular electronic repulsion was used as a strategy to induce a strong conformational bias within the ligand. Compounds bearing this conformation participated in a favorable hydrophobic interaction with a cysteine residue in the JAK3 binding pocket, which imparted high selectivity versus the kinome and improved selectivity within the JAK family.

3-Amido pyrrolopyrazine JAK kinase inhibitors: development of a JAK3 vs JAK1 selective inhibitor and evaluation in cellular and in vivo models.

The Janus kinases (JAKs) are involved in multiple signaling networks relevant to inflammatory diseases, and inhibition of one or more members of this class may modulate disease activity or progression. We optimized a new inhibitor scaffold, 3-amido-5-cyclopropylpyrrolopyrazines, to a potent example with reasonable kinome selectivity, including selectivity for JAK3 versus JAK1, and good biopharmaceutical properties. Evaluation of this analogue in cellular and in vivo models confirmed functional selectivity for modulation of a JAK3/JAK1-dependent IL-2 stimulated pathway over a JAK1/JAK2/Tyk2-dependent IL-6 stimulated pathway.

Construction of a large scale integrated map of macrophage pathogen recognition and effector systems.

BACKGROUND: In an effort to better understand the molecular networks that underpin macrophage activation we have been assembling a map of relevant pathways. Manual curation of the published literature was carried out in order to define the components of these pathways and the interactions between them. This information has been assembled into a large integrated directional network and represented graphically using the modified Edinburgh Pathway Notation (mEPN) scheme. RESULTS: The diagram includes detailed views of the toll-like receptor (TLR) pathways, other pathogen recognition systems, NF-kappa-B, apoptosis, interferon signalling, MAP-kinase cascades, MHC antigen presentation and proteasome assembly, as well as selected views of the transcriptional networks they regulate. The integrated pathway includes a total of 496 unique proteins, the complexes formed between them and the processes in which they are involved. This produces a network of 2,170 nodes connected by 2,553 edges. CONCLUSIONS: The pathway diagram is a navigable visual aid for displaying a consensus view of the pathway information available for these systems. It is also a valuable resource for computational modelling and aid in the interpretation of functional genomics data. We envisage that this work will be of value to those interested in macrophage biology and also contribute to the ongoing Systems Biology community effort to develop a standard notation scheme for the graphical representation of biological pathways.

Evo-Devo and the evolution of cancer: a hypothesis for metamorphic therapies for the cancers of prolactin-influenced tumourigenesis: with special reference to glioblastoma multiforme (GBM).

Recalling the remarkable developmental similarities between cancer cells and embryonic tissues, this paper argues that, by the process of retrodifferentiation and heterochronization, stem cells that have become neoplastic could be said to have undergone "cellular heterochrony." It theorizes, therefore, that hormones are the major factor in the non-random regulation of cellular heterochrony in tumourigenesis. Two recent articles confirm that there is low thyroxine and high prolactin in glioblastomas. Thyroxine metamorphoses vertebrates' tissues so as to mature the tissues, e.g., in amphibian metamorphosis. In 1896, thyroxine (horse thyroid extract) was the first successful hormonal product to be used against a fulminating breast cancer. Recent work confirms the important role of prolactin in the induction and progression of mammary, prostate and colorectal tumours. Although the pituitary is the main source of prolactin in vertebrates, there is also placental production of prolactin, and paracrine production of prolactin by tumours themselves. Since tumours produce their own prolactin, shutting down the pituitary source has not proven wholly successful. Research to find prolactin receptor antagonists is ongoing. Therefore, prolactin inhibitors (dopamine agonists), prolactin receptor antagonists, plus thyroxine comprise a plausible metamorphic therapy for shrinking solid tumour mass. By contrast with "differentiation" therapies currently sought by stem cell oncologists, this paper advocates "metamorphic" therapies, to introduce hormonal oncological knowledge of how to modulate signalling pathways that are aberrant in the stem cells that give rise to tumours. Despite subtle differences in these signalling translation pathways and cascades, strategies exist that will allow these evolved populations, going back to their stem precursors, to "metamorphose" or perhaps apoptotically cease proliferation.

Heart energy signature spectrogram for cardiovascular diagnosis.

A new method and application is proposed to characterize intensity and pitch of human heart sounds and murmurs. Using recorded heart sounds from the library of one of the authors, a visual map of heart sound energy was established. Both normal and abnormal heart sound recordings were studied. Representation is based on Wigner-Ville joint time-frequency transformations. The proposed methodology separates acoustic contributions of cardiac events simultaneously in pitch, time and energy. The resolution accuracy is superior to any other existing spectrogram method. The characteristic energy signature of the innocent heart murmur in a child with the S3 sound is presented. It allows clear detection of S1, S2 and S3 sounds, S2 split, systolic murmur, and intensity of these components. The original signal, heart sound power change with time, time-averaged frequency, energy density spectra and instantaneous variations of power and frequency/pitch with time, are presented. These data allow full quantitative characterization of heart sounds and murmurs. High accuracy in both time and pitch resolution is demonstrated. Resulting visual images have self-referencing quality, whereby individual features and their changes become immediately obvious.

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.

Innate immune response gene expression profiles of N9 microglia are pathogen-type specific.

Glial cells, particularly microglia, are thought to play a pivotal role in initiating and guiding innate immune responses to CNS infections and in perpetuating inflammation and pathology in CNS diseases such as multiple sclerosis and Alzheimer's disease. We describe here the development and use of a new microarray designed to specifically profile transcript expression of innate immunity genes. Microarray analysis validated by quantitative PCR demonstrated an extensive range of pattern recognition receptor gene expression in resting N9 microglia, including Toll-like receptors, scavenger receptors and lectins. Stimulation with LPS or infection with virus modulated pattern recognition receptor, cytokine, chemokine and other innate immune transcripts in a distinct and stimulus-specific manner. This study demonstrates that a single glial cell phenotype has an innate capability to detect infection, determine its form and generate specific responses.

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.

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.

Expression profiling reveals novel innate and inflammatory responses in the jejunal epithelial compartment during infection with Trichinella spiralis.

Infection with intestinal nematodes induces profound pathological changes to the gut that are associated with eventual parasite expulsion. We have applied expression profiling as an initial screening process with oligonucleotide microarrays (Affymetrix MG-U74AV2 gene chips) and time course kinetics to investigate gene transcription triggered by the intraepithelial nematode Trichinella spiralis in jejunal epithelium from BALB/c mice. Of the 4,114 genes detected, 2,617 were present in all uninfected and T. spiralis-infected replicates, 8% of which were notably upregulated, whereas 12% were downregulated at the time of worm expulsion (day 14 postinfection). Upregulation of goblet cell mucin gene transcripts intestinal mucin gene 3 (MUC3), calcium chloride channel 5 (CLCA5), and goblet cell gene 4 (GOB4) is consistent with enhanced production and alteration of mucus, whereas a 60- to 70-fold upregulation of transcripts for mast cell proteases 1 and 2 (MCPT-1 and -2) is consistent with intraepithelial mucosal mast cell recruitment. Importantly, there was novel expression of sialyltransferase 4C (SIAT4C), small proline-rich protein 2A (SPRR2A), and resistin-like molecule beta (RELMbeta) on day 14 postinfection. In contrast, DNase I and regenerating protein 3 (REG3) transcripts were substantially downregulated. Time course analyses revealed early (within 48 h of infection) induction of Siat4c, Sprr2A, and Relmbeta and later (within 120 h) induction of Mcpt-1 and -2. The findings demonstrate early innate responses and later inflammatory changes within the epithelium. The early epithelial responses may be associated both with repair (Sprr2A) and with the development of innate immunity (Siat4c and Relmbeta).

Identification of a region of the virus genome involved in murine gammaherpesvirus 68-induced splenic pathology.

Infection with the murine gammaherpesvirus MHV-68 has profound effects on splenic and mediastinal lymph node pathology in mice which lack the interferon-gamma receptor (IFN-gamma R(-/-)). In these mice MHV-68 infection causes fibrosis and loss of lymphocytes in the spleen and the mediastinal lymph node as well as interstitial pulmonary fibrosis and fibrotic changes in the liver. The changes are associated with transient elevated latent virus loads in the spleen. Four independent virus mutants with insertions and/or deletions in the left end of the genome fail to induce the pathological changes and establish latency at normal levels in the spleen. The data indicate that the pathology does not correlate with any of the known genes encoded within this region of the genome, genes M1-M4 and the eight vtRNAs. Northern analysis of mRNAs transcribed by wild-type and mutant viruses shows that at least two uncharacterized transcripts are encoded within this region. These transcripts are absent in the mutant viruses and are candidates for the virus genes responsible for the aberrant pathology in IFN-gamma R(-/-) mice.

Triple-target microarray experiments: a novel experimental strategy.

BACKGROUND: High-throughput, parallel gene expression analysis by means of microarray technology has become a widely used technique in recent years. There are currently two main dye-labelling strategies for microarray studies based on custom-spotted cDNA or oligonucleotides arrays: (I) Dye-labelling of a single target sample with a particular dye, followed by subsequent hybridisation to a single microarray slide, (II) Dye-labelling of two different target samples with two different dyes, followed by subsequent co-hybridisation to a single microarray slide. The two dyes most frequently used for either method are Cy3 and Cy5. We propose and evaluate a novel experiment set-up utilising three differently labelled targets co-hybridised to one microarray slide. In addition to Cy3 and Cy5, this incorporates Alexa 594 as a third dye-label. We evaluate this approach in line with current data processing and analysis techniques for microarrays, and run separate analyses on Alexa 594 used in single-target, dual-target and the intended triple-target experiment set-ups (a total of 18 microarray slides). We follow this by pointing out practical applications and suitable analysis methods, and conclude that triple-target microarray experiments can add value to microarray research by reducing material costs for arrays and related processes, and by increasing the number of options for pragmatic experiment design. RESULTS: The addition of Alexa 594 as a dye-label for an additional--third--target sample works within the framework of more commonplace Cy5/Cy3 labelled target sample combinations. Standard normalisation methods are still applicable, and the resulting data can be expected to allow identification of expression differences in a biological experiment, given sufficient levels of biological replication (as is necessary for most microarray experiments). CONCLUSION: The use of three dye-labelled target samples can be a valuable addition to the standard repertoire of microarray experiment designs. The method enables direct comparison between two experimental populations as well as measuring these two populations in relation to a third reference sample, allowing comparisons within the slide and across slides. These benefits are only offset by the added level of consideration required in the experimental design and data processing of a triple-target study design. Common methods for data processing and analysis are still applicable, but there is scope for the development of custom models for triple-target data. In summary, we do not consider the triple-target approach to be a new standard, but a valuable addition to the existing microarray study toolkit.

Expression profiling of the developing testis in wild-type and Dazl knockout mice.

Genetic understanding of male-factor infertility requires knowledge of gene expression patterns associated with normal germ cell differentiation. The mouse is one of the best models of mammalian fertility due to its well-characterized genetics and the existence of many infertile mutants both naturally occurring and experimentally induced. We used cDNA microarrays firstly to investigate normal gene expression in the wild-type (wt) testis and secondly to gain a better insight into the effect of the disruption of the Dazl gene on spermatogenesis. We constructed a cDNA microarray from a subtracted and normalized adult testis library and focused on six developmental time-points during the initial synchronous wave of spermatogenesis. The results suggest that in the wild-type testis, 89.5% of genes on our chip change expression dramatically during the time-course. To identify patterns in the gene-expression data, a k-means clustering algorithm and principal component analysis were used. In the Dazl knockout testes, the majority of genes remain at baseline levels of expression, because absence of Dazl has a severe effect on cell-types present in the testis. Although in the prepubescent Dazl-null mice the final point reached in germ cell development is the leptotene-zygotene stage, the microarray results suggest that lack of Dazl expression has a detectable effect on the mRNA complement of germ cells as early as day 5 when only type A spermatogonia are present. Mol. Reprod. Dev. 67: 26-54, 2004.