Assessment of p.Phe508del-CFTR functional restoration in pediatric primary cystic fibrosis airway epithelial cells

© 2018 Sutanto et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Background Mutations in the cystic fibrosis transmembrane regulator (CFTR) gene can reduce function of the CFTR ion channel activity and impair cellular chloride secretion. The gold standard method to assess CFTR function of ion transport using the Ussing chamber requires a high number of airway epithelial cells grown at air-liquid interface, limiting the application of this method for high throughput screening of potential therapeutic compounds in primary airway epithelial cells (pAECs) featuring less common CFTR mutations. This study assessed an alternative approach, using a small scale halide assay that can be adapted for a personalized high throughput setting to analyze CFTR function of pAEC. Methods Pediatric pAECs derived from children with CF (pAEC CF ) were established and expanded as monolayer cultures, before seeding into 96-well plates for the halide assay. Cells were then transduced with an adenoviral construct containing yellow fluorescent protein (eYFP) reporter gene, alone or in combination with either wild-type CFTR (WT-CFTR) or p.Phe508-del CFTR. Four days post transduction, cells were stimulated with forskolin and genistein, and assessed for quenching of the eYFP signal following injection of iodide solution into the assay media. Results Data showed that pAEC CF can express eYFP at high efficiency following transduction with the eYFP construct. The halide assay was able to discriminate functional restoration of CFTR in pAEC CF treated with either WT-CFTR construct or the positive controls syntaxin 8 and B-cell receptor-associated protein 31 shRNAs. Significance The current study demonstrates that the halide assay can be adapted for pediatric pAEC CF to evaluate restoration of CFTR function. With the ongoing development of small molecules to modulate the folding and/or activity of various mutated CFTR proteins, this halide assay presents a small-scale personalized screening platform that could assess therapeutic potential of molecules across a broad range of CFTR mutations

Searches for the Z gamma decay mode of the Higgs boson and for new high-mass resonances in pp collisions at root s=13 TeV with the ATLAS detector

This article presents searches for the Zγ decay of the Higgs boson and for narrow high-mass resonances decaying to Zγ, exploiting Z boson decays to pairs of electrons or muons. The data analysis uses 36.1 fb−1 of pp collisions at s√=13s=13 recorded by the ATLAS detector at the CERN Large Hadron Collider. The data are found to be consistent with the expected Standard Model background. The observed (expected — assuming Standard Model pp → H → Zγ production and decay) upper limit on the production cross section times the branching ratio for pp → H → Zγ is 6.6. (5.2) times the Standard Model prediction at the 95% confidence level for a Higgs boson mass of 125.09 GeV. In addition, upper limits are set on the production cross section times the branching ratio as a function of the mass of a narrow resonance between 250 GeV and 2.4 TeV, assuming spin-0 resonances produced via gluon-gluon fusion, and spin-2 resonances produced via gluon-gluon or quark-antiquark initial states. For high-mass spin-0 resonances, the observed (expected) limits vary between 88 fb (61 fb) and 2.8 fb (2.7 fb) for the mass range from 250 GeV to 2.4 TeV at the 95% confidence level

Identification and rejection of pile-up jets at high pseudorapidity with the ATLAS detector

The rejection of forward jets originating from additional proton–proton interactions (pile-up) is crucial for a variety of physics analyses at the LHC, including Standard Model measurements and searches for physics beyond the Standard Model. The identification of such jets is challenging due to the lack of track and vertex information in the pseudorapidity range |η| > 2.5. This paper presents a novel strategy for forward pile-up jet tagging that exploits jet shapes and topological jet correlations in pile-up interactions. Measurements of the per-jet tagging efficiency are presented using a data set of 3.2 fb−1 of proton–proton collisions at a centre-of-mass energy of 13 TeV collected with the ATLAS detector. The fraction of pile-up jets rejected in the range 2.5 < |η| < 4.5 is estimated in simulated events with an average of 22 interactions per bunch-crossing. It increases with jet transverse momentum and, for jets with transverse momentum between 20 and 50 GeV, it ranges between 49% and 67% with an efficiency of 85% for selecting hard-scatter jets. A case study is performed in Higgs boson production via the vector-boson fusion process, showing that these techniques mitigate the background growth due to additional proton–proton interactions, thus enhancing the reach for such signatures

Optimized approach for the identification of highly efficient correctors of nonsense mutations in human diseases

About 10% of patients with a genetic disease carry a nonsense mutation causing their pathology. A strategy for correcting nonsense mutations is premature termination codon (PTC) readthrough, i.e. incorporation of an amino acid at the PTC position during translation. PTC-readthrough-activating molecules appear as promising therapeutic tools for these patients. Unfortunately, the molecules shown to induce PTC readthrough show low efficacy, probably because the mRNAs carrying a nonsense mutation are scarce, as they are also substrates of the quality control mechanism called nonsense-mediated mRNA decay (NMD). The screening systems previously developed to identify readthrough-promoting molecules used cDNA constructs encoding mRNAs immune to NMD. As the molecules identified were not selected for the ability to correct nonsense mutations on NMD-prone PTC-mRNAs, they could be unsuitable for the context of nonsense-mutation-linked human pathologies. Here, a screening system based on an NMD-prone mRNA is described. It should be suitable for identifying molecules capable of efficiently rescuing the expression of human genes harboring a nonsense mutation. This system should favor the discovery of candidate drugs for treating genetic diseases caused by nonsense mutations. One hit selected with this screening system is presented and validated on cells from three cystic fibrosis patients

Searches for the Z gamma decay mode of the Higgs boson and for new high-mass resonances in pp collisions at root s=13 TeV with the ATLAS detector

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