Test beam performance of a CBC3-based mini-module for the Phase-2 CMS Outer Tracker before and after neutron irradiation

The Large Hadron Collider (LHC) at CERN will undergo major upgrades to increase the instantaneous luminosity up to 5–7.5×10$^{34}$ cm$^{-2}$s$^{-1}$. This High Luminosity upgrade of the LHC (HL-LHC) will deliver a total of 3000–4000 fb-1 of proton-proton collisions at a center-of-mass energy of 13–14 TeV. To cope with these challenging environmental conditions, the strip tracker of the CMS experiment will be upgraded using modules with two closely-spaced silicon sensors to provide information to include tracking in the Level-1 trigger selection. This paper describes the performance, in a test beam experiment, of the first prototype module based on the final version of the CMS Binary Chip front-end ASIC before and after the module was irradiated with neutrons. Results demonstrate that the prototype module satisfies the requirements, providing efficient tracking information, after being irradiated with a total fluence comparable to the one expected through the lifetime of the experiment

Retinoic acid-dependent activation of the polycystic kidney disease-1 (PKD1) promoter

The retinoic acids all-trans retinoic acid (AT-RA) and 9-cis retinoic acid (9C-RA) and the retinoic acid receptors RAR and RXR significantly induce transcriptional activity from a 200-bp PKD1 proximal promoter in transfected mammalian cells. This PKD1 promoter region contains Ets, p53, and GC box motifs, but lacks a canonical RAR/RXR motif. Mutagenesis of the Ets sites did not affect RA induction. In contrast, GC box mutations completely blocked stimulation by AT-RA and by RXRβ or RARβ. Mithramycin A, which prevents Sp1 binding, significantly reduced basal promoter activity and suppressed upregulation by AT-RA and RXR. The 200-bp proximal promoter could not be induced by AT-RA in Drosophila SL2 cells, which lack Sp1, but could be activated in these cells transfected with exogenous Sp1. Small interfering RNA knockdown of Sp1 in mammalian cells completely blocked RXRβ upregulation of the promoter. These data indicate that induction of the PKD1 promoter by retinoic acid is mediated through Sp1 elements. RT-PCR showed that AT-RA treatment of HEK293T cells increased the levels of endogenous PKD1 RNA, and chromatin immunoprecipitation showed the presence of both RXR and Sp1 at the PKD1 proximal promoter. These results suggest that retinoids and their receptors may play a role in PKD1 gene regulation

Renal activation of extracellular signal-regulated kinase in rats with autosomal-dominant polycystic kidney disease

Renal activation of extracellular signal-regulated kinase in rats with autosomal-dominant polycystic kidney disease.BackgroundAbnormal proliferation of renal tubule epithelial cells is a central factor in the biogenesis and sustained expansion of cysts in autosomal-dominant polycystic kidney disease (ADPKD). Recent evidence from in vitro studies of human cyst wall epithelial cells has implicated a role for the mitogen-activated protein (MAP) kinase pathway in this aberrant proliferation. To determine the extent to which this signaling pathway is involved in cyst pathogenesis in vivo, we measured the expression of select components of the MAP kinase cascade in Han:SPRD rats with ADPKD at an early stage of the disease.MethodsKidneys of 8-week-old normal Han:SPRD rats (+/+) or rats heterozygous (Cy/+) for ADPKD were examined by Western blot analysis and immunohistochemistry to determine the expression of extracellular-regulated kinase (ERK), phosphorylated ERK (P-ERK), Raf-1 (MAPKKK), phosphorylated Raf-1 (P-Raf-1), B-Raf, Rap-1 and phosphorylated protein kinase A (P-PKA).ResultsP-ERK was expressed to a greater extent in Cy/+ kidneys (3.74 ± 1.07 fold) than in normal kidneys, whereas ERK abundance was not different. P-Raf-1 levels were higher in Cy/+ than in +/+ kidneys (1.53 ± 0.08 fold) consistent with upstream stimulation of receptor tyrosine kinase. B-Raf and Raf-1 abundances were greater in Cy/+ than in +/+ (1.74 ± 0.25 and 1.27 ± 0.08 fold, respectively). In Cy/+, immunohistochemistry showed increased P-ERK and B-Raf expression in the abnormal mural epithelial cells within cysts. These findings, together with the detection of P-PKA and the small G protein, Rap-1, in cyst epithelial cells, implicate a potential role for cyclic adenosine monophosphate (AMP) in the activation of ERK in ADPKD cells.ConclusionsWe conclude that the MAP kinase pathway is activated to the level of ERK in the abnormal mural epithelial cells lining cysts in animals with a dominantly inherited type of polycystic kidney disease. We suggest that cAMP, acting through PKA, Rap-1 and B-Raf, may contribute to the activation of ERK in a way that complements receptor tyrosine kinase-mediated agonists in the promotion of cyst enlargement

A mutation affecting polycystin-1 mediated heterotrimeric G-protein signaling causes PKD

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the growth of renal cysts that ultimately destroy kidney function. Mutations in the PKD1 and PKD2 genes cause ADPKD. Their protein products, polycystin-1 (PC1) and polycystin-2 (PC2) have been proposed to form a calcium-permeable receptor-channel complex; however the mechanisms by which they function are almost completely unknown. Most mutations in PKD1 are truncating loss-of-function mutations or affect protein biogenesis, trafficking or stability and reveal very little about the intrinsic biochemical properties or cellular functions of PC1. An ADPKD patient mutation (L4132Δ or ΔL), resulting in a single amino acid deletion in a putative G-protein binding region of the PC1 C-terminal cytosolic tail, was found to significantly decrease PC1-stimulated, G-protein-dependent signaling in transient transfection assays. Pkd1ΔL/ΔL mice were embryo-lethal suggesting that ΔL is a functionally null mutation. Kidney-specific Pkd1ΔL/cond mice were born but developed severe, postnatal cystic disease. PC1ΔL protein expression levels and maturation were comparable to those of wild type PC1, and PC1ΔL protein showed cell surface localization. Expression of PC1ΔL and PC2 complexes in transfected CHO cells failed to support PC2 channel activity, suggesting that the role of PC1 is to activate G-protein signaling to regulate the PC1/PC2 calcium channel

Acceleration of polycystic kidney disease progression in cpk mice carrying a deletion in the homeodomain protein Cux1

Polycystic kidney diseases (PKD) are inherited as autosomal dominant (ADPKD) or autosomal recessive (ARPKD) traits and are characterized by progressive enlargement of renal cysts. Aberrant cell proliferation is a key feature in the progression of PKD. Cux1 is a homeobox gene that is related to Drosophila cut and is the murine homolog of human CDP (CCAAT Displacement Protein). Cux1 represses the cyclin kinase inhibitors p21 and p27, and transgenic mice ectopically expressing Cux1 develop renal hyperplasia. However, Cux1 transgenic mice do not develop PKD. Here, we show that a 246 amino acid deletion in Cux1 accelerates PKD progression in cpk mice. Cystic kidneys isolated from 10-day-old cpk/Cux1 double mutant mice were significantly larger than kidneys from 10-day-old cpk mice. Moreover, renal function was significantly reduced in the Cux1 mutant cpk mice, compared with cpk mice. The mutant Cux1 protein was ectopically expressed in cyst-lining cells, where expression corresponded to increased cell proliferation and apoptosis, and a decrease in expression of the cyclin kinase inhibitors p27 and p21. While the mutant Cux1 protein altered PKD progression, kidneys from mice carrying the mutant Cux1 protein alone were phenotypically normal, suggesting the Cux1 mutation modifies PKD progression in cpk mice. During cell cycle progression, Cux1 is proteolytically processed by a nuclear isoform of the cysteine protease cathepsin-L. Analysis of the deleted sequences reveals that a cathepsin-L processing site in Cux1 is deleted. Moreover, nuclear cathepsin-L is significantly reduced in both human ADPKD cells and in Pkd1 null kidneys, corresponding to increased levels of Cux1 protein in the cystic cells and kidneys. These results suggest a mechanism in which reduced Cux1 processing by cathepsin-L results in the accumulation of Cux1, downregulation of p21/p27, and increased cell proliferation in PKD