Supplementary Figures S1-S2 and Supplementary Tables S2-S4.

BACKGROUND:The Ts1Cje mouse model of Down syndrome (DS) has partial triplication of mouse chromosome 16 (MMU16), which is partially homologous to human chromosome 21. These mice develop various neuropathological features identified in DS individuals. We analysed the effect of partial triplication of the MMU16 segment on global gene expression in the cerebral cortex, cerebellum and hippocampus of Ts1Cje mice at 4 time-points: postnatal day (P)1, P15, P30 and P84.RESULTS:Gene expression profiling identified a total of 317 differentially expressed genes (DEGs), selected from various spatiotemporal comparisons, between Ts1Cje and disomic mice. A total of 201 DEGs were identified from the cerebellum, 129 from the hippocampus and 40 from the cerebral cortex. Of these, only 18 DEGs were identified as common to all three brain regions and 15 were located in the triplicated segment. We validated 8 selected DEGs from the cerebral cortex (Brwd1, Donson, Erdr1, Ifnar1, Itgb8, Itsn1, Mrps6 and Tmem50b), 18 DEGs from the cerebellum (Atp5o, Brwd1, Donson, Dopey2, Erdr1, Hmgn1, Ifnar1, Ifnar2, Ifngr2, Itgb8, Itsn1, Mrps6, Paxbp1, Son, Stat1, Tbata, Tmem50b and Wrb) and 11 DEGs from the hippocampus (Atp5o, Brwd1, Cbr1, Donson, Erdr1, Itgb8, Itsn1, Morc3, Son, Tmem50b and Wrb). Functional clustering analysis of the 317 DEGs identified interferon-related signal transduction as the most significantly dysregulated pathway in Ts1Cje postnatal brain development. RT-qPCR and western blotting analysis showed both Ifnar1 and Stat1 were over-expressed in P84 Ts1Cje cerebral cortex and cerebellum as compared to wild type littermates.CONCLUSIONS:These findings suggest over-expression of interferon receptor may lead to over-stimulation of Jak-Stat signaling pathway which may contribute to the neuropathology in Ts1Cje or DS brain. The role of interferon mediated activation or inhibition of signal transduction including Jak-Stat signaling pathway has been well characterized in various biological processes and disease models including DS but information pertaining to the role of this pathway in the development and function of the Ts1Cje or DS brain remains scarce and warrants further investigation.

The Ts1Cje mouse model of Down syndrome (DS) has partial trisomy of mouse chromosome 16 (MMU16), which is syntenic to human chromosome 21 (HSA21). It develops various neuropathological features demonstrated by DS patients such as reduced cerebellar volume [1] and altered hippocampus-dependent learning and memory [2,3]. To understand the global gene expression effect of the partially triplicated MMU16 segment on mouse brain development, we performed the spatiotemporal transcriptome analysis of Ts1Cje and disomic control cerebral cortex, cerebellum and hippocampus harvested at four developmental time-points: postnatal day (P)1, P15, P30 and P84. Here, we provide a detailed description of the experimental and analysis procedures of the microarray dataset, which has been deposited in the Gene Expression Omnibus (GSE49050) database.

AbstractBackground: The Kimba mouse carries a human vascular endothelial growth factor transgene causing retinal neovascularisation similar to that seen in diabetic retinopathy. Here, we examine the relationship between differential gene expression induced by vascular endothelial growth factor overexpression and the architectural changes that occur in the retinae of these mice.Methods: Retinal gene expression changes in juvenile and adult Kimba mice were assayed by microarray and compared with age‐matched wild‐type littermates. Transcription of selected genes was validated by quantitative real‐time polymerase chain reaction. Protein translation was determined using immunohistochemistry and enzyme‐linked immunosorbent assay.Results: Semaphorin 3C was upregulated, and nuclear receptor subfamily 2, group 3, member 3 (Nr2e3) was downregulated in juvenile Kimba mice. Betacellulin and endothelin 2 were upregulated in adults. Semaphorin 3C colocalized with glial fibrillary acidic protein in Müller cells of Kimba retinae at greater signal intensities than in wild type. Endothelin 2 colocalised to Müller cell end feet and extended into the outer limiting membrane. Endothelin receptor type B staining was most pronounced in the inner nuclear layer, the region containing Müller cell somata.Conclusions: An early spike in vascular endothelial growth factor induced significant long‐term retinal neovascularisation associated with changes to the retinal ganglion, photoreceptor and Müller cells. Overexpression of vascular endothelial growth factor led to dysregulation of photoreceptor metabolism through differential expression of Nr2e3, endothelin 2, betacellulin and semaphorin 3C. Alterations in the expression of these genes may therefore play key roles in the pathological mechanisms that result from retinal neovascularisation.

Background: Down syndrome (DS) individuals suffer mental retardation with further cognitive decline and early onset Alzheimer's disease.Methodology/Principal Findings: To understand how trisomy 21 causes these neurological abnormalities we investigated changes in gene expression networks combined with a systematic cell lineage analysis of adult neurogenesis using the Ts1Cje mouse model of DS. We demonstrated down regulation of a number of key genes involved in proliferation and cell cycle progression including Mcm7, Brca2, Prim1, Cenpo and Aurka in trisomic neurospheres. We found that trisomy did not affect the number of adult neural stem cells but resulted in reduced numbers of neural progenitors and neuroblasts. Analysis of differentiating adult Ts1Cje neural progenitors showed a severe reduction in numbers of neurons produced with a tendency for less elaborate neurites, whilst the numbers of astrocytes was increased.Conclusions/Significance: We have shown that trisomy affects a number of elements of adult neurogenesis likely to result in a progressive pathogenesis and consequently providing the potential for the development of therapies to slow progression of, or even ameliorate the neuronal deficits suffered by DS individuals.

Abstract Protein isoforms produced by alternative splicing (AS) of many genes have been implicated in several aspects of cancer genesis and progression. These observations motivated a genome-wide assessment of AS in breast cancer. We accomplished this by measuring exon level expression in 31 breast cancer and nonmalignant immortalized cell lines representing luminal, basal, and claudin-low breast cancer subtypes using Affymetrix Human Junction Arrays. We analyzed these data using a computational pipeline specifically designed to detect AS with a low false-positive rate. This identified 181 splice events representing 156 genes as candidates for AS. Reverse transcription-PCR validation of a subset of predicted AS events confirmed 90%. Approximately half of the AS events were associated with basal, luminal, or claudin-low breast cancer subtypes. Exons involved in claudin-low subtype–specific AS were significantly associated with the presence of evolutionarily conserved binding motifs for the tissue-specific Fox2 splicing factor. Small interfering RNA knockdown of Fox2 confirmed the involvement of this splicing factor in subtype-specific AS. The subtype-specific AS detected in this study likely reflects the splicing pattern in the breast cancer progenitor cells in which the tumor arose and suggests the utility of assays for Fox-mediated AS in cancer subtype definition and early detection. These data also suggest the possibility of reducing the toxicity of protein-targeted breast cancer treatments by targeting protein isoforms that are not present in limiting normal tissues. Mol Cancer Res; 8(7); 961–74. ©2010 AACR.

Previous diffusion tensor imaging (DTI) studies indicated microstructural disruption of white matter in alcohol dependence. To investigate the microstructure of primary neurocircuitry involved in alcohol use disorders, the present study used Tract-Based Spatial Statistics (TBSS) of DTI measures as well as probabilistic tractography. Eleven recovering alcoholics in their first week of abstinence from alcohol were compared with 10 light-drinking controls; diffusion measures were correlated with measures of neurocognition and drinking severity. Regions characterized by low fractional anisotropy and high mean diffusivity included cortico-striatal fibers and those in frontal white matter and limbic pathways. Greater diffusion abnormalities in sections of commissural fibers (inter-hemispheric connections) were associated with greater drinking severity, and lower fractional anisotropy measures in frontal and limbic fiber tracts correlated with lower visuospatial memory performance. These study findings provide direct evidence of compromised integrity of the motivational brain circuitry in alcohol use disorders. These abnormalities in fiber connections could be partially responsible for deficiencies in executive functions, behavioral regulation, and impulse control commonly described in alcohol dependence.

Background The RUNX1 transcription factor gene is frequently mutated in sporadic myeloid and lymphoid leukemia through translocation, point mutation or amplification. It is also responsible for a familial platelet disorder with predisposition to acute myeloid leukemia (FPD-AML). The disruption of the largely unknown biological pathways controlled by RUNX1 is likely to be responsible for the development of leukemia. We have used multiple microarray platforms and bioinformatic techniques to help identify these biological pathways to aid in the understanding of why RUNX1 mutations lead to leukemia. Results Here we report genes regulated either directly or indirectly by RUNX1 based on the study of gene expression profiles generated from 3 different human and mouse platforms. The platforms used were global gene expression profiling of: 1) cell lines with RUNX1 mutations from FPD-AML patients, 2) over-expression of RUNX1 and CBFβ, and 3) Runx1 knockout mouse embryos using either cDNA or Affymetrix microarrays. We observe that our datasets (lists of differentially expressed genes) significantly correlate with published microarray data from sporadic AML patients with mutations in either RUNX1 or its cofactor, CBFβ . A number of biological processes were identified among the differentially expressed genes and functional assays suggest that heterozygous RUNX1 point mutations in patients with FPD-AML impair cell proliferation, microtubule dynamics and possibly genetic stability. In addition, analysis of the regulatory regions of the differentially expressed genes has for the first time systematically identified numerous potential novel RUNX1 target genes. Conclusion This work is the first large-scale study attempting to identify the genetic networks regulated by RUNX1, a master regulator in the development of the hematopoietic system and leukemia. The biological pathways and target genes controlled by RUNX1 will have considerable importance in disease progression in both familial and sporadic leukemia as well as therapeutic implications.

Rapid production of interferon‐γ (IFN‐γ) in response to malaria by the innate immune system may determine resistance to infection, or inflammatory disease. However, conflicting reports exist regarding the identity of IFN‐γ‐producing cells that rapidly respond to Plasmodium falciparum. To clarify this area, we undertook detailed phenotyping of IFN‐γ‐producing cells across a panel of naive human donors following 24‐h exposure to live schizont‐infected red blood cells (iRBC). Here, we show that NK cells comprise only a small proportion of IFN‐γ‐responding cells and that IFN‐γ production is unaffected by NK cell depletion. Instead, γδ‐T cells represent the predominant source of innate IFN‐γ, with the majority of responding γδ‐T cells expressing NK receptors. Malaria‐responsive γδ‐T cells more frequently expressed NKG2A compared to non‐responding γδ‐T cells, while non‐responding γδ‐T cells more frequently expressed CD158a/KIR2DL1. Unlike long‐term γδ‐T cell responses to iRBC, αβ‐T cell help was not required for innate γδ‐T cell responses. Diversity was observed among donors in total IFN‐γ output. This was positively associated with CD94 expression on IFN‐γ+ NK‐like γδ‐T cells. Applied to longitudinal cohort studies in endemic regions, similar comparative phenotyping should allow assessment of the contribution of diverse cell populations and regulatory receptors to risk of infection and disease.

Background: The apicomplexan parasite Plasmodium falciparum causes the most severe form of malaria in humans. After invasion into erythrocytes, asexual parasite stages drastically alter their host cell and export remodeling and virulence proteins. Previously, we have reported identification and functional analysis of a short motif necessary for export of proteins out of the parasite and into the red blood cell.Results: We have developed software for the prediction of exported proteins in the genus Plasmodium, and identified exported proteins conserved between malaria parasites infecting rodents and the two major causes of human malaria, P. falciparum and P. vivax. This conserved 'exportome' is confined to a few subtelomeric chromosomal regions in P. falciparum and the synteny of these and surrounding regions is conserved in P. vivax. We have identified a novel gene family PHIST (for Plasmodium helical interspersed subtelomeric family) that shares a unique domain with 72 paralogs in P. falciparum and 39 in P. vivax; however, there is only one member in each of the three species studied from the P. berghei lineage.Conclusion: These data suggest radiation of genes encoding remodeling and virulence factors from a small number of loci in a common Plasmodium ancestor, and imply a closer phylogenetic relationship between the P. vivax and P. falciparum lineages than previously believed. The presence of a conserved 'exportome' in the genus Plasmodium has important implications for our understanding of both common mechanisms and species-specific differences in host-parasite interactions, and may be crucial in developing novel antimalarial drugs to this infectious disease.

Leukemia inhibitory factor (LIF) is required to maintain pluripotency and permit self-renewal of murine embryonic stem (ES) cells. LIF binds to a receptor complex of LIFR-beta and gp130 and signals via the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway, with signalling attenuated by suppressor of cytokine signalling (SOCS) proteins. Recent in vivo studies have highlighted the role of SOCS-3 in the negative regulation of signalling via gp130. To determine the role of SOCS-3 in ES cell biology, SOCS-3-null ES cell lines were generated. When cultured in LIF levels that sustain self-renewal of wild-type cells, SOCS-3-null ES cell lines exhibited less self-renewal and greater differentiation into primitive endoderm. The absence of SOCS-3 enhanced JAK-STAT and extracellular signal-related kinase 1/2 (ERK-1/2)-mitogen-activated protein kinase (MAPK) signal transduction via gp130, with higher levels of phosphorylated STAT-1, STAT-3, SH-2 domain-containing cytoplasmic protein tyrosine phosphatase 2 (SHP-2), and ERK-1/2 in steady state and in response to LIF stimulation. Attenuation of ERK signalling by the addition of MAPK/ERK kinase (MEK) inhibitors to SOCS-3-null ES cell cultures rescued the differentiation phenotype, but did not restore proliferation to wild-type levels. In summary, SOCS-3 plays a crucial role in the regulation of the LIF signalling pathway in murine ES cells. Its absence perturbs the balance between activation of the JAK-STAT and SHP-2-ERK-1/2-MAPK pathways, resulting in less self-renewal and a greater potential for differentiation into the primitive endoderm lineage.

Microarray-based gene expression analysis demonstrated that laser photocoagulation (LPC) of mouse eyes had a long-term effect on the expression of genes functionally related to tissue repair, cell migration, proliferation, ion, protein and nucleic acid metabolism, cell signaling, and angiogenesis. Six structural genes, including five crystallins (Cryaa, Cryba1, Crybb2, Crygc, Crygs) and keratin 1-12 (Krt1-12), the anti-angiogenic factor thrombospondin 1 (Tsp1), the retina- and brain-specific putative transcription factor tubby-like protein 1 (Tulp1), and transketolase (Tkt), a key enzyme in the pentose-phosphate pathway, were all shown to be up-regulated by real-time PCR and/or Western blotting. Immunohistochemistry localized five of these proteins to the laser lesions and surrounding tissue within the retina and pigmented epithelium. This is the first study demonstrating long-term changes in the expression of these genes associated with LPC. Therefore, it suggests that modulated gene expression might contribute to the long-term inhibitory effect of LPC. In addition, these genes present novel targets for gene-based therapies aimed at treating microangiopathies, especially diabetic retinopathy, a disease currently only treatable with LPC.

Central to the pathology of malaria disease are the repeated cycles of parasite invasion and destruction of human erythrocytes. In Plasmodium falciparum, the most virulent species causing malaria, erythrocyte invasion involves several specific receptor-ligand interactions that direct the pathway used to invade the host cell, with parasites varying in their dependency on these different pathways. Gene disruption of a key invasion ligand in the 3D7 parasite strain, the P. falciparum reticulocyte binding-like homolog 2b (PfRh2b), resulted in the parasite invading via a novel pathway. Here, we show results that suggest the molecular basis for this novel pathway is not due to a molecular switch but is instead mediated by the redeployment of machinery already present in the parent parasite but masked by the dominant role of PfRh2b. This would suggest that interactions directing invasion are organized hierarchically, where silencing of dominant invasion ligands reveal underlying alternative pathways. This provides wild parasites with the ability to adapt to immune-mediated selection or polymorphism in erythrocyte receptors and has implications for the use of invasion-related molecules in candidate vaccines.

Chronic microbial infections are associated with fibrotic and inflammatory reactions known as granulomas showing similarities to wound-healing and tissue repair processes. We have previously mapped three leishmaniasis susceptibility loci, designated lmr1, -2, and -3, which exert their effect independently of T cell immune responses. Here, we show that the wound repair response is critically important for the rapid cure in murine cutaneous leishmaniasis caused by Leishmania major. Mice congenic for leishmaniasis resistance loci, which cured their lesions more rapidly than their susceptible parents, also expressed differentially genes involved in tissue repair, laid down more ordered collagen fibers, and healed punch biopsy wounds more rapidly. Fibroblast monolayers from these mice repaired in vitro wounds faster, and this process was accelerated by supernatants from infected macrophages. Because these effects are independent of T cell-mediated immunity, we conclude that the rate of wound healing is likely to be an important component of innate immunity involved in resistance to cutaneous leishmaniasis.

The primary pathophysiological events contributing to fatal malaria are the cerebral syndrome, anemia, and lactic acidosis. The molecular basis of each event has been unclear. In the present study, microarray analysis of murine transcriptional responses during the development of severe disease revealed temporal, organ-specific, and pathway-specific patterns. More than 400 genes in the brain and 600 genes in the spleen displayed transcriptional changes. Dominant patterns revealed strongly suppressed erythropoiesis, starting early during infection, and highly up-regulated transcription of genes that control host glycolysis, including lactate dehydrogenase. The latter presents a mechanism that may contribute to metabolic acidosis. No evidence for hypoxia-mediated regulation of these events was observed. Interferon-regulated gene transcripts dominated the inflammatory response to cytokines. These results demonstrate previously unknown transcriptional changes in the host that may underlie the development of malarial syndromes, such as anemia and metabolic dysregulation, and increase the utility of murine models in investigation of basic malarial pathogenesis.