Fifty years of the CERN Proton Synchrotron : Volume 2

This report sums up in two volumes the first 50 years of operation of the CERN Proton Synchrotron. After an introduction on the genesis of the machine, and a description of its magnet and powering systems, the first volume focuses on some of the many innovations in accelerator physics and instrumentation that it has pioneered, such as transition crossing, RF gymnastics, extractions, phase space tomography, or transverse emittance measurement by wire scanners. The second volume describes the other machines in the PS complex: the proton linear accelerators, the PS Booster, the LEP pre-injector, the heavy-ion linac and accumulator, and the antiproton rings.Comment: 58 pages, published as CERN Yellow Report https://cds.cern.ch/record/1597087?ln=e

Case report: Myocarditis in congenital STAT1 gain-of function

Autosomal dominant Signal transducer and activator of transcription 1 (STAT1) gain-of-function (GOF) mutations result in an inborn error of immunity characterized by chronic mucocutaneous candidiasis, recurrent viral and bacterial infections, and diverse autoimmune manifestations. Current treatment consists of chronic antifungal therapy, antibiotics for concomitant infections, and immunosuppressive therapy in case of autoimmune diseases. More recently, treatment with Janus kinases 1 and 2 (JAK1/2) inhibitors have shown promising yet variable results. We describe a STAT1 GOF patient with an incidental finding of elevated cardiac troponins, leading to a diagnosis of a longstanding, slowly progressive idiopathic myocarditis, attributed to STAT1 GOF. Treatment with a JAK-inhibitor (baricitinib) mitigated cardiac inflammation on MRI but was unable to alter fibrosis, possibly due to the diagnostic and therapeutic delay, which finally led to fatal arrhythmia. Our case illustrates that myocarditis could be part of the heterogeneous disease spectrum of STAT1 GOF. Given the insidious presentation in our case, a low threshold for cardiac evaluation in STAT1 GOF patients seems warranted

Redirecting gamma-retroviral vectors for safer gene therapy A study on p12 fusion peptides to redirect retroviral vector integration preference

In the last twenty-five years gene therapy has been used successfully to cure life-threatening monogenic inherited disorders, like ADA-SCID, X-SCID, WAS syndrome, X-CGD and β-thalassaemia. Retroviral vectors are frequently used to deliver the therapeutic genes into patients, because of their stable integration. In this thesis I will focus on gammaretroviral-based vectors, based on the Murine Leukemia Virus (MLV) platform. Gammaretroviral vectors were the first vectors used in gene therapy applications and are among the best studied and characterized. Despite successful outcome in a wide variety of clinical trials, adverse events in a subset of patients (20%) resulted in the malignant proliferation of blood cells and leukemia, due to insertional mutagenesis. Indeed, MLV-based viral vectors preferentially integrate in active promoters, enhancers, DNase hypersensitive sites (DHS) and transcription start sites (TSS). Viral vector integration in these specific regions can occasionally lead to the deregulation of the transcription of genes nearby the integration site; for example when the integration takes place near proto-oncogenes (as LMO2) it can lead to oncogenesis. Retroviral integration is not a random process, but is coordinated by cellular host factors that are co-opted by the invading viral PIC. The integration pattern of MLV and its derived viral vectors is determined by BET (bromo and ET-SEED domain containing family) proteins, which are cellular cofactors that dock to active enhancers and promoters in the chromatin and that can also bind the MLV integrase (IN). By binding to active enhancer and promoter regions, BET proteins tether the MLV viral vector integration to these regions. In 2013 it was proven that a single mutation in MLV IN (W390A) hampers the interaction between IN and BET proteins, generating a BET-independent integrase (Bin). Bin-MLV vectors have a more random and safer integration profile (from 20% to 10% integrations near TSS) compared to WT MLV vectors. In addition to integrase, the viral particle depends on p12 for efficient integration in the invaded host cell. Since MLV and derived vectors can’t pass the nuclear membrane, they require the p12 protein to tether the PIC to chromatin during mitosis, when the nuclear membrane is degraded. In this thesis I evaluated the role of the viral p12 protein on the integration sites preference of the viral vector. Previous studies showed that mutation of p12 (PM14 mutant) hampers the ability of the p12 protein to tether the PIC to the mitotic chromatin, resulting in a dead MLV virus. Introduction of different alternative chromatin binding peptides in the C-terminal domain of the p12 protein rescued this lethal PM14 mutant. In this thesis I studied the role of the p12 chromatin interaction in integration sites choice. I generated several p12-fusions and combined these with either a wild-type IN or a Bin-IN protein. Assuming that the MLV IN and BET protein interaction plays a dominant role in determining the MLV integration profile, combination of the Bin-IN together with p12-peptide fusions, might lead to an additional shift of the integration of the MLV vector to more safe regions. Briefly, I generated Bin-MLV vectors and WT vectors with the p12-PM14 protein complemented with various chromatin binding peptides. We chose to target chromatin in general with peptides that bind H2A-H2B nucleosome core histones (peptides derived from Kaposi Sarcoma-associated Herpes Virus and Prototype Foamy Virus) or the genomic DNA backbone (peptides derived from Human Papilloma Virus 8), as well as specific chromatin modifications, such as methylated histone side-chains (like Me3 of K36 at histone H3 that is recognized by a domain of LEDGF/p75). MLV vectors were produced by using a GFP transfer plasmid, a VSV-G envelope plasmid and a packaging plasmid encoding the p12-peptide fusions. WT-IN MLV and W390A-IN MLV vectors were taken as control. Both WT-IN and W390A-IN MLV vectors showed comparable transduction efficiencies, whereas the lethal PM14 p12 mutant showed no transduction, in line with previous studies. Genomic DNA of transduced SupT1 cells was also used to perform qPCR experiments to evaluate the number of integrated copies compared with the WT vector, and was used to prepare a library for Illumina Next Generation Sequencing (NGS). The protocol for library preparation was optimized and samples were sent for sequencing. The data obtained in this thesis will help to make one more step towards the engineering of safer retroviral vectors for gene therapy

First observations of intensity-dependent effects for transversely split beams during multiturn extraction studies at the CERN Proton Synchrotron

During the commissioning of the CERN Proton Synchrotron multiturn extraction, tests with different beam intensities were performed in order to probe the behavior of resonance crossing in the presence of possible space charge effects. The initial beam intensity before transverse splitting was varied and the properties of the five beamlets obtained by crossing the fourth-order horizontal resonance were studied. A clear dependence of the beamlets’ parameters on the total beam intensity was found, which is the first direct observation of intensity-dependent effects for such a peculiar beam type. The experimental results are presented and discussed in detail in this paper

Transcriptional Profiling of STAT1 Gain-of-Function Reveals Common and Mutation-Specific Fingerprints

STAT1 gain-of-function (GOF) is a primary immunodeficiency typically characterized by chronic mucocutaneous candidiasis (CMC), recurrent respiratory infections, and autoimmunity. Less commonly, also immunodysregulation polyendocrinopathy enteropathy X-linked (IPEX)-like syndromes with CMC, and combined immunodeficiency without CMC have been described. Recently, our group and others have shown that different mutation-specific mechanisms underlie STAT1 GOF in vitro, including faster nuclear accumulation (R274W), and reduced mobility (R321, N574I) to near immobility in the nucleus (T419R) upon IFNγ stimulation. In this work, we evaluated the transcriptomic fingerprint of the aforementioned STAT1 GOF mutants (R274W, R321S, T419R, and N574I) relative to STAT1 wild-type upon IFNγ stimulation in an otherwise isogenic cell model. The majority of genes up-regulated in wild-type STAT1 cells were significantly more up-regulated in cells expressing GOF mutants, except for T419R. In addition to the common interferon regulated genes (IRG), STAT1 GOF mutants up-regulated an additional set of genes, that were in part shared with other GOF mutants or mutation-specific. Overall, R274W and R321S transcriptomes clustered with STAT1 WT, while T419R and N574I had a more distinct fingerprint. We observed reduced frequency of canonical IFNγ activation site (GAS) sequences in promoters of genes up-regulated by all the STAT1 GOF mutants, suggesting loss of DNA binding specificity for the canonical GAS consensus. Interestingly, the T419R mutation, expected to directly increase the affinity for DNA, showed the most pronounced effects on the transcriptome. T419R STAT1 dysregulated more non-IRG than the other GOF mutants and fewer GAS or degenerate GAS promotor sequences could be found in the promoter regions of these genes. In conclusion, our work confirms hyperactivation of common sets of IFNγ-induced genes in STAT1 GOF with additional dysregulation of mutation-specific genes, in line with the earlier observed mutation-specific mechanisms. Binding to more degenerate GAS sequences is proposed as a mechanism toward transcriptional dysregulation in R274W, R321S, and N574I. For T419R, an increased interaction with the DNA is suggested to result in a broader and less GAS-specific response. Our work indicates that multiple routes leading to STAT1 GOF are associated with common and private transcriptomic fingerprints, which may contribute to the phenotypic variation observed in vivo.</jats:p

CERN leads the way with novel beam extraction

A team of researchers at CERN has developed a new, more-efficient technique, known as multiturn extraction, for extracting beam from a circular particle accelerator

Electron-Cloud Studies for Transversely Split Beams

Recently, resonance crossing has been proposed as a means of manipulating the transverse beam distribution. This technique has application, among other topics, to injection and extraction schemes. Moreover, the transversely split beams might also be used as a mitigation measure of electron-cloud effects. The results of detailed numerical simulations are discussed in this paper, possibly opening new options for scrubbing of beam pipes in circular accelerators

Intensity effects in the formation of stable islands in phase space during the multi-turn extraction process at the CERN PS

The CERN PS utilises a Multi-Turn Extraction (MTE) scheme to stretch the beam pulse length to optimise the filling process of the SPS. MTE is a novel technique to split a beam in transverse phase space into nonlinear stable islands. The recent experimental results indicate that the positions of the islands depend on the total beam intensity. Particle simulations have been performed to understand the detailed mechanism of the intensity dependence. The analysis carried out so far suggests space charge effects through image charges and image currents on the vacuum chamber and the magnets iron cores dominate the observed behaviour. In this talk, the latest analysis with realistic modelling of the beam environment is discussed and it is shown how this further improves the understanding of intensity effects in MTE