Defective heart development in hypomorphic Lsd1 mice

Lysine-specific demethylase 1 (Lsd1/Aof2/Kdm1a), the first enzyme with specific lysine demethylase activity to be described, demethylates several target proteins, including histones, DNMT1 and p53. Lsd1 can act as either a transcriptional activator or repressor, depending on the histone lysine it demethylates; its specificity for either H3K4 or H3K9 appears to be determined by its binding partners. We have previously demonstrated that a complete loss of this protein results in early embryonic lethality. However, it was also noted that no adult mice homozygous for the floxed allele survived, even though this allele should be indistinguishable from wild-type Lsd1 after splicing removes the LoxP sites. Homozygous pups die perinatally, with most animals showing major heart development defects. The Aof22lox allele contains two point mutations; the resulting protein shows reduced interaction with known binding partners and decreased enzymatic activity. The expression of a very limited subset of genes is altered in the hearts. This includes an increase in CK2beta expression, the regulatory subunit of the CK2 kinase, which results in E-cadherin hyperphosphorylation. These results identify a previously unknown role for Lsd1 in heart development, through the control of E-cadherin phosphorylation

Test of short model and prototype of the HL-LHC D2 orbit corrector based on CCT technology

In the frame of the high-luminosity upgrade project for the large hadron collider, new twin aperture beam orbit corrector magnets will be installed near the recombination dipole (D2). These magnets are 2.2 m long canted cosine theta NbTi dipoles, with two independently powered apertures oriented such that their field vectors are perpendicular to each other and to the direction of the beams. A 0.5 m model magnet in single and double aperture configuration and a full-length double aperture prototype were built and tested at CERN. In this paper, the performance of these magnets at 1.9 K in terms of training behavior, quench detection and protection, and other tests is discussed. In addition, the thermal response of the magnet to a hypothetical beam discharge is simulated and analyzed

Test of the First Full-Length Prototype of the HL-LHC D2 Orbit Corrector Based on Canted Cosine Theta Technology

In the context of CERN's high-luminosity upgrade project (HL-LHC) for the Large Hadron Collider (LHC), a new double aperture beam orbit corrector magnets will be installed near the recombination dipole (D2). These 2.2 m long NbTi dipoles are built with the canted cosine theta (CCT) technique. The two independently powered apertures are oriented such that their field vectors are perpendicular to each other and to the direction of the beams. A full-length double aperture prototype was built and tested at CERN in the SM18 test facility. Here we present the results of powering tests at 1.9 and 4.5 K: training of each aperture, magnetic field quality and cross-talk effects, quench detection system effectiveness, quench protection performance and quench-back with several energy extraction systems

Immunohistochemistry of the hypomorphic hearts.

<p>(A) Staining with an antibody specific for the phosphorylation of E-cadherin is strongly increased in the <i>Aof2</i>^2lox/2lox hearts compared to the control. The arrows indicate the regions (grey for heart wall, black for septum) from which the higher magnification images (on right) originate. (B) β-catenin localization is altered, with more of the protein present at the plasma membrane in <i>Aof2^2lox/2lox</i> hearts. (C) Lsd1 staining shows slightly decreased signals in <i>Aof2^2lox/2lox</i> hearts. (D) Staining with a non-specific IgG control antibody confirms the specificity of the staining. To minimize background, no counterstain was used. All photomicrographs constitute representative fields; magnification factor is provided above or beside the photographs.</p

The 2lox Lsd1 variant shows decreased binding to known interactors.

<p>(A) Immunoblots of FLAG-based coimmunoprecipitations of NIH 3T3 cells transfected with the Lsd1 variants demonstrate that the 2lox protein exhibits greatly reduced complex formation with known binding partners CoREST and HDAC1. For the two single point mutants, the M448V mutation has a more profound effect on CoREST binding. *: immunoglobulin heavy chain. (B) The relative coimmunoprecipitation of the Lsd1 binding partner CoREST was quantitated using ImageJ software. Data represent the mean +/− SD for two independent experiments. (C) Purification of endogenous Lsd1 complexes from MEF lines confirmed a decrease in Lsd1-CoREST interaction in cells expressing the mutant Lsd1. All westerns are representative of results from experiments performed in triplicate. (D) The decrease in the endogenous interaction of Lsd1 and CoREST by the 2lox variant was quantitated, using ImageJ software. Data represent the mean +/− SD for three independent experiments. **: p<0.001; *: p<0.005, compared to binding by wild-type Lsd1.</p

Statistical analysis of microarray data (significant probes only).

<p>Legend: ID, Affymetrix probe ID; logFC, log value of the Fold change in expression in the 2lox/2lox hearts; AveExpr, average expression in wild-type hearts;</p><p>P.value, unadjusted P-value; adj.P.Val, adjusted P-value.</p

Lsd1 expression in the developing heart.

<p>Wild-type embryos at the indicated development stages (E8.5 to E13.5, and E18.5) were dissected and processed for immunohistochemistry. H&E staining was performed, along with staining with anti-Lsd1 to visual the Lsd1 expression pattern during heart development. Note that in all panels, the black bar represents 100 µm.</p