The Tgif2 gene contains a retained intron within the coding sequence

BACKGROUND: TGIF and TGIF2 are homeodomain proteins, which act as TGFβ specific Smad transcriptional corepressors. TGIF recruits general repressors including mSin3 and CtBP. The related TGIF2 protein functions in a similar manner, but does not bind CtBP. In addition to repressing TGFβ activated gene expression, TGIF and TGIF2 repress gene expression by binding directly to DNA. TGIF and TGIF2 share two major blocks of similarity, encompassing the homeodomain, and a conserved carboxyl terminal repression domain. Here we characterize two splice variants of the Tgif2 gene from mouse and demonstrate that the Tgif2 gene contains a retained intron. RESULTS: By PCR from mouse cDNA, we identified two alternate splice forms of the Tgif2 gene. One splice variant encodes the full length 237 amino acid Tgif2, whereas the shorter form results in the removal of 39 codons from the centre of the coding region. The generation of this alternate splice form occurs with the mouse RNA, but not the human, and both splice forms are present in all mouse tissues analyzed. Human and mouse Tgif2 coding sequences contain a retained intron, which in mouse Tgif2 is removed by splicing from around 25–50% of RNAs, as assessed by RT-PCR. This splicing event is dependent on sequences within the mouse Tgif2 coding sequence. Both splice forms of mouse Tgif2 encode proteins which are active transcriptional repressors, and can repress both TGFβ dependent and independent transcription. In addition, we show that human and mouse Tgif2 interact with the transcriptional corepressor mSin3. CONCLUSION: These data demonstrate that the Tgif2 gene contains a retained intron, within the second coding exon. This retained intron is not removed from the human mRNA at a detectable level, but is spliced out in a significant proportion of mouse RNAs. This alternate splicing is dependent entirely on sequences within the mouse Tgif2 coding sequence, suggesting the presence of an exonic splicing enhancer. Both splice forms of mouse Tgif2 produce proteins which are functional transcriptional repressors

Loss of Tgif Function Causes Holoprosencephaly by Disrupting the Shh Signaling Pathway

Holoprosencephaly (HPE) is a severe human genetic disease affecting craniofacial development, with an incidence of up to 1/250 human conceptions and 1.3 per 10,000 live births. Mutations in the Sonic Hedgehog (SHH) gene result in HPE in humans and mice, and the Shh pathway is targeted by other mutations that cause HPE. However, at least 12 loci are associated with HPE in humans, suggesting that defects in other pathways contribute to this disease. Although the TGIF1 (TG-interacting factor) gene maps to the HPE4 locus, and heterozygous loss of function TGIF1 mutations are associated with HPE, mouse models have not yet explained how loss of Tgif1 causes HPE. Using a conditional Tgif1 allele, we show that mouse embryos lacking both Tgif1 and the related Tgif2 have HPE-like phenotypes reminiscent of Shh null embryos. Eye and nasal field separation is defective, and forebrain patterning is disrupted in embryos lacking both Tgifs. Early anterior patterning is relatively normal, but expression of Shh is reduced in the forebrain, and Gli3 expression is up-regulated throughout the neural tube. Gli3 acts primarily as an antagonist of Shh function, and the introduction of a heterozygous Gli3 mutation into embryos lacking both Tgif genes partially rescues Shh signaling, nasal field separation, and HPE. Tgif1 and Tgif2 are transcriptional repressors that limit Transforming Growth Factor β/Nodal signaling, and we show that reducing Nodal signaling in embryos lacking both Tgifs reduces the severity of HPE and partially restores the output of Shh signaling. Together, these results support a model in which Tgif function limits Nodal signaling to maintain the appropriate output of the Shh pathway in the forebrain. These data show for the first time that Tgif1 mutation in mouse contributes to HPE pathogenesis and provide evidence that this is due to disruption of the Shh pathway

Risk of venous thromboembolism in people admitted to hospital with selected immune-mediated diseases: record-linkage study

BACKGROUND: Venous thromboembolism (VTE) is a common complication during and after a hospital admission. Although it is mainly considered a complication of surgery, it often occurs in people who have not undergone surgery, with recent evidence suggesting that immune-mediated diseases may play a role in VTE risk. We, therefore, decided to study the risk of deep vein thrombosis (DVT) and pulmonary embolism (PE) in people admitted to hospital with a range of immune-mediated diseases. METHODS: We analysed databases of linked statistical records of hospital admissions and death certificates for the Oxford Record Linkage Study area (ORLS1:1968 to 1998 and ORLS2:1999 to 2008) and the whole of England (1999 to 2008). Rate ratios for VTE were determined, comparing immune-mediated disease cohorts with comparison cohorts. RESULTS: Significantly elevated risks of VTE were found, in all three populations studied, in people with a hospital record of admission for autoimmune haemolytic anaemia, chronic active hepatitis, dermatomyositis/polymyositis, type 1 diabetes mellitus, multiple sclerosis, myasthenia gravis, myxoedema, pemphigus/pemphigoid, polyarteritis nodosa, psoriasis, rheumatoid arthritis, Sjogren's syndrome, and systemic lupus erythematosus. Rate ratios were considerably higher for some of these diseases than others: for example, for systemic lupus erythematosus the rate ratios were 3.61 (2.36 to 5.31) in the ORLS1 population, 4.60 (3.19 to 6.43) in ORLS2 and 3.71 (3.43 to 4.02) in the England dataset. CONCLUSIONS: People admitted to hospital with immune-mediated diseases may be at an increased risk of subsequent VTE. Our findings need independent confirmation or refutation; but, if confirmed, there may be a role for thromboprophylaxis in some patients with these diseases

The deposition of metal nanoparticles on carbon surfaces: the role of specific functional groups

The enormous complexity of a typical heterogeneous catalyst makes understanding the development and properties of any active nanoparticles present extremely challenging. In the case of carbon based catalysts that difficulty is compounded by the variability of the carbon powders used. We have previously developed a strategy that addresses these problems by mimicking the catalyst preparation conditions very closely but using highly ordered pyrolytic graphite crystals (HOPG) as a model surface. This enables us to examine the effects of specific functional groups on nanoparticle formation. We report here an extension of our work characterising functional groups on the HOPG surface, using XPS and AFM to explore the deposition of gold from aqueous solution onto HOPG surfaces treated in a variety of ways to alter the surface functionality. The structure and oxidation state of the resulting nanoparticles depend critically on the nature of the functional groups present and offers some insight into the development of catalysts based on these materials. Hydroxyls are identified as key functional species, reducing gold ions to their metallic state whilst being oxidised themselves to carbonyls. Carbonyls meanwhile promote the nucleation of Au3+, creating a network of islands at the HOPG surface. The results have relevance not only to catalysts using activated carbons but also the new generation of materials based on graphene and carbon nanotubes

A Role for Non-Covalent SUMO Interaction Motifs in Pc2/CBX4 E3 Activity

Background: Modification of proteins by the small ubiquitin like modifier (SUMO) is an essential process in mammalian cells. SUMO is covalently attached to lysines in target proteins via an enzymatic cascade which consists of E1 and E2, SUMO activating and conjugating enzymes. There is also a variable requirement for non-enzymatic E3 adapter like proteins, which can increase the efficiency and specificity of the sumoylation process. In addition to covalent attachment of SUMO to target proteins, specific non-covalent SUMO interaction motifs (SIMs) that are generally short hydrophobic peptide motifs have been identified. Methodology/Principal Findings: Intriguingly, consensus SIMs are present in most SUMO E3s, including the polycomb protein, Pc2/Cbx4. However, a role for SIMs in SUMO E3 activity remains to be shown. We show that Pc2 contains two functional SIMs, both of which contribute to full E3 activity in mammalian cells, and are also required for sumoylation of Pc2 itself. Pc2 forms distinct sub-nuclear foci, termed polycomb bodies, and can recruit partner proteins, such as the corepressor CtBP. We demonstrate that mutation of the SIMs in Pc2 prevents Pc2-dependent CtBP sumoylation, and decreases enrichment of SUMO1 and SUMO2 at polycomb foci. Furthermore, mutational analysis of both SUMO1 and SUMO2 reveals that the SIM-interacting residues of both SUMO isoforms are required for Pc2-mediated sumoylation and localization to polycomb foci

Experimental study of air bubbles in a simulated cardiopulmonary bypass system with flow constriction

An experimental study is performed to examine the breaking of an air bubble in the flow passage of a simulated cardiopulmonary bypass system by means of a flow constriction. The purpose of the study is to discover a geometry of the flow constriction which is efficient in breaking air bubbles while providing the least resistance to the flow of blood, i.e. to develop a new device for the oxygenation of the blood in extracorporeal circulation.Both plasma and water are used in the study. The use of plasma is to simulate the principal transport properties of the human blood and enable direct visualization of bubbles. Water is used for comparison with plasma to determine the influence of fluid properties on the breaking of bubbles. Several different shapes of flow constriction are tested. It is observed that as a result of rapid changes in the liquid pressure and bubble shape, an air bubble breaks into many bubbles at downstream from the flow constriction. The results are quantatively expressed by the number of baby bubbles vs. the flow rate.It is disclosed that the flask-shape constriction is efficient in breaking air bubbles while providing ideal passage for the flow of blood. The number of baby bubbles is found to increase with an increase in the fluid viscosity.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/32740/1/0000109.pd

Premature Senescence and Increased TGFβ Signaling in the Absence of Tgif1

Transforming growth factor β (TGFβ) signaling regulates cell cycle progression in several cell types, primarily by inducing a G1 cell cycle arrest. Tgif1 is a transcriptional corepressor that limits TGFβ responsive gene expression. Here we demonstrate that primary mouse embryo fibroblasts (MEFs) lacking Tgif1 proliferate slowly, accumulate increased levels of DNA damage, and senesce prematurely. We also provide evidence that the effects of loss of Tgif1 on proliferation and senescence are not limited to primary cells. The increased DNA damage in Tgif1 null MEFs can be partially reversed by culturing cells at physiological oxygen levels, and growth in normoxic conditions also partially rescues the proliferation defect, suggesting that in the absence of Tgif1 primary MEFs are less able to cope with elevated levels of oxidative stress. Additionally, we show that Tgif1 null MEFs are more sensitive to TGFβ-mediated growth inhibition, and that treatment with a TGFβ receptor kinase inhibitor increases proliferation of Tgif1 null MEFs. Conversely, persistent treatment of wild type cells with low levels of TGFβ slows proliferation and induces senescence, suggesting that TGFβ signaling also contributes to cellular senescence. We suggest that in the absence of Tgif1, a persistent increase in TGFβ responsive transcription and a reduced ability to deal with hyperoxic stress result in premature senescence in primary MEFs

The effect of Ku on telomere replication time is mediated by telomere length but is independent of histone tail acetylation

Peer reviewedPublisher PD

Upregulation of the cell-cycle regulator RGC-32 in Epstein-Barr virus-immortalized cells

Epstein-Barr virus (EBV) is implicated in the pathogenesis of multiple human tumours of lymphoid and epithelial origin. The virus infects and immortalizes B cells establishing a persistent latent infection characterized by varying patterns of EBV latent gene expression (latency 0, I, II and III). The CDK1 activator, Response Gene to Complement-32 (RGC-32, C13ORF15), is overexpressed in colon, breast and ovarian cancer tissues and we have detected selective high-level RGC-32 protein expression in EBV-immortalized latency III cells. Significantly, we show that overexpression of RGC-32 in B cells is sufficient to disrupt G2 cell-cycle arrest consistent with activation of CDK1, implicating RGC-32 in the EBV transformation process. Surprisingly, RGC-32 mRNA is expressed at high levels in latency I Burkitt's lymphoma (BL) cells and in some EBV-negative BL cell-lines, although RGC-32 protein expression is not detectable. We show that RGC-32 mRNA expression is elevated in latency I cells due to transcriptional activation by high levels of the differentially expressed RUNX1c transcription factor. We found that proteosomal degradation or blocked cytoplasmic export of the RGC-32 message were not responsible for the lack of RGC-32 protein expression in latency I cells. Significantly, analysis of the ribosomal association of the RGC-32 mRNA in latency I and latency III cells revealed that RGC-32 transcripts were associated with multiple ribosomes in both cell-types implicating post-initiation translational repression mechanisms in the block to RGC-32 protein production in latency I cells. In summary, our results are the first to demonstrate RGC-32 protein upregulation in cells transformed by a human tumour virus and to identify post-initiation translational mechanisms as an expression control point for this key cell-cycle regulator

LHCb Upgraded RICH 1 Engineering Design Review Report

During the Long Shutdown 2 of the LHC, the LHCb collaboration will replace the upstream Ring Imaging Cherenkov detector (RICH 1). The magnetic shield of the current RICH 1 will be modified, new spherical and plane mirrors will be installed and a new gas enclosure will be manufactured. New photon detectors (multianode photomultiplier tubes) will be used and these, together with their readout electronics, require a new mechanical support system. This document describes the new optical arrangement of RICH 1, its engineering design, installation and alignment. A summary of the project schedule and institute responsibilities is provided