The status of the energy calibration, polarization and monochromatization of the FCC-ee

The Future Circular electron-positron Collider, FCC- ee, is designed for unprecedented precision for particle physics experiments from the Z-pole up to above the top-pair-threshold, corresponding to a beam energy range from 45.6 to 182.5 GeV. Performing collisions at various particle-physics resonances requires precise knowledge of the centre-of-mass energy (ECM) and collision boosts at all four interaction points. Measurement of the ECM by resonant depolarization of transversely polarized pilot bunches in combination with a 3D polarimeter, aims to achieve a systematic uncertainty of 4 and 100 keV for the Z-pole and W-pair-threshold energies respectively. The ECM itself depends on the RF-cavity locations, beamstrahlung, longitudinal impedance, the Earth’s tides, opposite sign dispersion and possible collision offsets. Application of monochromatization schemes are envisaged at certain beam energies to reduce the energy spread. The latest results of studies of the energy calibration, polarization and monochromatization are reported here

SARS-CoV-2 susceptibility and COVID-19 disease severity are associated with genetic variants affecting gene expression in a variety of tissues

Variability in SARS-CoV-2 susceptibility and COVID-19 disease severity between individuals is partly due to genetic factors. Here, we identify 4 genomic loci with suggestive associations for SARS-CoV-2 susceptibility and 19 for COVID-19 disease severity. Four of these 23 loci likely have an ethnicity-specific component. Genome-wide association study (GWAS) signals in 11 loci colocalize with expression quantitative trait loci (eQTLs) associated with the expression of 20 genes in 62 tissues/cell types (range: 1:43 tissues/gene), including lung, brain, heart, muscle, and skin as well as the digestive system and immune system. We perform genetic fine mapping to compute 99% credible SNP sets, which identify 10 GWAS loci that have eight or fewer SNPs in the credible set, including three loci with one single likely causal SNP. Our study suggests that the diverse symptoms and disease severity of COVID-19 observed between individuals is associated with variants across the genome, affecting gene expression levels in a wide variety of tissue types

Critical assessment of protein intrinsic disorder prediction

Abstract: Intrinsically disordered proteins, defying the traditional protein structure–function paradigm, are a challenge to study experimentally. Because a large part of our knowledge rests on computational predictions, it is crucial that their accuracy is high. The Critical Assessment of protein Intrinsic Disorder prediction (CAID) experiment was established as a community-based blind test to determine the state of the art in prediction of intrinsically disordered regions and the subset of residues involved in binding. A total of 43 methods were evaluated on a dataset of 646 proteins from DisProt. The best methods use deep learning techniques and notably outperform physicochemical methods. The top disorder predictor has Fmax = 0.483 on the full dataset and Fmax = 0.792 following filtering out of bona fide structured regions. Disordered binding regions remain hard to predict, with Fmax = 0.231. Interestingly, computing times among methods can vary by up to four orders of magnitude

Proline dehydrogenase regulation regulation by the P53 family and the regulatiory circuit with HIF-1.

Proline differs from the other amino acids because its \ue1-nitrogen is contained within a pyrrolidine ring. Therefore, it cannot be metabolized by the general transaminases and decarboxylases acting on other amino acids. Proline dehydrogenase (PRODH) is a stress-inducible, key enzyme in proline metabolism, catalyzing its conversion into \uc41-pyrroline-5-carboxylate, a crucial compound interconnecting proline metabolism with glutamate and \ue1- ketoglutarate (\ue1-KG) synthesis and with the Tricarboxylic Acids (TCA) and Urea cycles. Consequently, PRODH can influence various cellular pathways, including glutamatergic transmission, glutathione levels as well as the activity of a number of enzymes using \ue1-KG as a substrate. Proline can also be regarded as an emergency substrate, as abundant stores are released during degradation of intracellular or extracellular matrix proteins (especially collagens). PRODH is localized in the inner membrane of mitochondria and after reduction of the FAD cofactor bound to form the holoenzyme, it can directly transfer electrons to cytochrome C to generate ATP or it can oxidize O2 to generate reactive oxygen species (ROS). Thus when cells are under stress, PRODH has been proposed to act either as a survival factor, favouring maintenance of \u201csurvival energy levels\u201d, or as a cell death effector, inducing ROS-dependent apoptosis. Alterations in PRODH protein levels and catalytic activity have been implicated in diseases such as hyperprolinemia, DiGeorge syndrome, schizophrenia and cancer. For cancer in particular, several lines of evidence suggest a central role of PRODH as a mitochondrial tumor suppressor: 1) expression of PRODH is reduced in diverse colorectal and renal cancer cells as compared to normal counterparts; 2) restoration of PRODH expression in human hypo-expressing colon cancer cell lines suppresses their ability to form tumours when injected into SCID mice; 3) PRODH expression is regulated transcriptionally and posttranscriptionally by several cellular sensors of cell health and homeostasis, whose functions are deregulated during carcinogenesis, including p53, PPAR\uf067 and mTOR (mammalian target of rapamycin). However, the exact mechanisms by which these proteins control PRODH function have been only partially elucidated. Understanding transcriptional and post-transcriptional regulation of a gene and its product is clue to understanding its function. In the first two years of my PhD work we identified and characterized the p53 Response Elements (REs) in the PRODH gene, responsible for p53 binding and transactivation of this target. We confirmed p53-dependent induction of endogenous PRODH in response to genotoxic damage in cell lines of different histological origin and we established that overexpression of p73 \uf062 or p63 \uf062 is sufficient to induce PRODH expression in p53-null cells. The p53 family-dependent transcriptional activation of PRODH was linked to specific intronic response elements (REs), among those predicted by bioinformatics tools and experimentally validated by a yeast-based transactivation assay upon modulated expression of p53, p63 and p73 and by p53 occupancy measurements in HCT116 human cells by ChIP. Based on the following pieces of evidence i) it has been proposed that during nutrient stress extracellular matrix (ECM) proteins may be degraded to provide substrates for energy production (ecophagy), ii) an abundant protein in ECM is collagen, that is very rich in proline and hydroxyproline, iii) the key enzyme in hydroxyproline metabolism is hydroxyproline dehydrogenase, homologous to PRODH, whose gene (PRODH2) was also shown, although less convincingly, to be a p53 target, we decided to characterize the p53 REs present in this gene as well. We demonstrated that the PRODH2 gene was not responsive to p63 nor p73 and was at best a weak p53 target, based on minimal levels of PRODH2 transcript induction by genotoxic stress observed only in one of four p53 wildtype cell lines tested. Consistently, all predicted p53 REs in PRODH2 were poor matches to the p53 RE consensus and showed limited responsiveness, only to p53, in the functional assay. Taken together, our results highlight that PRODH but not PRODH2 expression is likely under control of the entire p53 family members, supporting a deeper link between p53 proteins and metabolic pathways, as PRODH functions in modulating the balance of proline and glutamate levels and of their derivative alpha-keto-glutarate in the metabolism under normal and pathological (tumor) conditions. Another important transcription factor that we considered for a possible role in regulation of PRODH, is the Hypoxia Inducible Factor 1 (HIF-1), whose function influences cellular metabolism and is altered during the tumourigenic process. HIF proteins are composed of two subunits, \uf061 and \uf062, both constitutively expressed in cells. However, the \ue1 subunits are rapidly degraded by the proteasome at normal oxygen concentrations found in tissues. Key to HIF-\uf031\uf061 degradation is its oxygen-dependent hydroxylation at specific residues (prolines 402 and 564) by Prolyl Hydroxylases (PHD), that target the protein for ubiquitylation and proteasomal degradation in presence of molecular oxygen, \uf061-KG and vitamin C. During hypoxia, HIF-\uf031\uf061 subunits become stabilized, which enables them to form heterodimers with HIF-\uf031\uf062, that activate numerous cell survival pathways. HIF-1 has been shown to control the expression of more than a hundred genes, either by direct transcriptional activation of protein coding genes and microRNAs (miRs), or by interacting or interfering with other transcription factors. HIF-1 activation results in profound alterations in tumour cell behaviour, which include triggering the angiogenic switch, shifting glucose metabolism towards glycolysis, promoting epithelial-to-mesenchymal transition and acquisition of an invasive phenotype, as well as increasing chemo- and radio-resistance. For this reason, tumour cells often maintain HIF-1 overexpression after they return to a normoxic environment. We tested the hypothesis that an increase in PRODH activity, by increasing \uf061-KG, would provide substrate for the hydroxylation reaction catalyzed by PHDs, thus leading to a decrease in HIF-1 \uf061 levels. indeed, ectopic expression of PRODH led to down-regulation of HIF-\uf031\uf061 and VEGF protein levels in the U87glioblastoma cell line. This finding confirmed what was already reported to occur for colon cancer cell lines. In addition to a role of PRODH in regulating HIF-1 stability in normoxia, we hypothesized that a regulatory circuit between PRODH and HIF-1 could exist. PRODH was found to be downregulated 2-fold in a transcriptomics analysis of genes regulated following induction of focal brain ischemia in rat. On the other hand, however, very recently PRODH was shown to be induced by hypoxia and this induction was AMPK-dependent and HIF-independent. Therefore, the question was still open for investigation. Our expectations are unbiased, because PRODH possesses the ability to promote either cell survival, in conditions in which energy levels are low, by producing ATP or inducing ROS dependent autophagy, or ROS induced apoptotic cell death. Of course a different outcome depending on the cell lines tested as well as on other types of stress acting on the cells concomitantly with the hypoxic stress may be expected. In a first attempt to verify if PRODH transcript levels were modified by hypoxia, we exposed cancer cell lines of different histological origin (HCT116, colon; MCF7, breast; U87MG, glia; SHSY-5Y, neural crest) to 1% hypoxia, anoxia or to treatment with CoCl2, a PHDs inhibitor, and compared the levels of expression with those obtained in the same untreated cell lines, by using real time RTqPCR. All cell lines showed a marked decrease in PRODH transcript, in particular after treatment with CoCl2, and a reduction also in protein levels, although of minor entity compared to transcript decrease. Preliminary results obtained during my PhD work confirm that PRODH-HIF-1 regulatory circuit does indeed exist, and lays the foundations for further investigations, to clarify the relationship between these two proteins to increase knowledge about PRODH regulation and its possible downregulation during the tumourigenic process

The multidimensional mechanisms of long noncoding RNA function

Abstract A major shift in our understanding of genome regulation has emerged recently. It is now apparent that the majority of cellular transcripts do not code for proteins, and many of them are long noncoding RNAs (lncRNAs). Increasingly, studies suggest that lncRNAs regulate gene expression through diverse mechanisms. We review emerging mechanistic views of lncRNAs in gene regulation in the cell nucleus. We discuss the functional interactions that lncRNAs establish with other molecules as well as the relationship between lncRNA transcription and function. While some of these mechanisms are specific to lncRNAs, others might be shared with other types of genes

Identification of a membrane-less compartment regulating invadosome function and motility

Abstract Depletion of liprin-α1, ERC1 or LL5 scaffolds inhibits extracellular matrix degradation by invasive cells. These proteins co-accumulate near invadosomes in NIH-Src cells, identifying a novel invadosome–associated compartment distinct from the core and adhesion ring of invadosomes. Depletion of either protein perturbs the organization of invadosomes without influencing the recruitment of MT1-MMP metalloprotease. Liprin-α1 is not required for de novo formation of invadosomes after their disassembly by microtubules and Src inhibitors, while its depletion inhibits invadosome motility, thus affecting matrix degradation. Fluorescence recovery after photobleaching shows that the invadosome–associated compartment is dynamic, while correlative light immunoelectron microscopy identifies bona fide membrane–free invadosome–associated regions enriched in liprin-α1, which is virtually excluded from the invadosome core. The results indicate that liprin-α1, LL5 and ERC1 define a novel dynamic membrane-less compartment that regulates matrix degradation by affecting invadosome motility

Evolution of p53 transactivation specificity through the lens of a yeast-based functional assay.

Co-evolution of transcription factors (TFs) with their respective cis-regulatory network enhances functional diversity in the course of evolution. We present a new approach to investigate transactivation capacity of sequence-specific TFs in evolutionary studies. Saccharomyces cerevisiae was used as an in vivo test tube and p53 proteins derived from human and five commonly used animal models were chosen as proof of concept. p53 is a highly conserved master regulator of environmental stress responses. Previous reports indicated conserved p53 DNA binding specificity in vitro, even for evolutionary distant species. We used isogenic yeast strains where p53-dependent transactivation was measured towards chromosomally integrated p53 response elements (REs). Ten REs were chosen to sample a wide range of DNA binding affinity and transactivation capacity for human p53 and proteins were expressed at two levels using an inducible expression system. We showed that the assay is amenable to study thermo-sensitivity of frog p53, and that chimeric constructs containing an ectopic transactivation domain could be rapidly developed to enhance the activity of proteins, such as fruit fly p53, that are poorly effective in engaging the yeast transcriptional machinery. Changes in the profile of relative transactivation towards the ten REs were measured for each p53 protein and compared to the profile obtained with human p53. These results, which are largely independent from relative p53 protein levels, revealed widespread evolutionary divergence of p53 transactivation specificity, even between human and mouse p53. Fruit fly and human p53 exhibited the largest discrimination among REs while zebrafish p53 was the least selective