AATF/Che-1-An RNA Binding Protein at the Nexus of DNA Damage Response and Ribosome Biogenesis

The DNA damage response (DDR) is a complex signaling network that is activated upon genotoxic stress. It determines cellular fate by either activating cell cycle arrest or initiating apoptosis and thereby ensures genomic stability. The Apoptosis Antagonizing Transcription Factor (AATF/Che-1), an RNA polymerase II-interacting transcription factor and known downstream target of major DDR kinases, affects DDR signaling by inhibiting p53-mediated transcription of pro-apoptotic genes and promoting cell cycle arrest through various pathways instead. Specifically, AATF was shown to inhibit p53 expression at the transcriptional level and repress its pro-apoptotic activity by direct binding to p53 protein and transactivation of anti-apoptotic genes. Solid and hematological tumors of various organs exploit this function by overexpressing AATF. Both copy number gains and high expression levels of AATF were associated with worse prognosis or relapse of malignant tumors. Recently, a number of studies have enabled insights into the molecular mechanisms by which AATF affects both DDR and proliferation. AATF was found to directly localize to sites of DNA damage upon laser ablation and interact with DNA repair proteins. In addition, depletion of AATF resulted in increased DNA damage and decrease of both proliferative activity and genotoxic tolerance. Interestingly, considering the role of ribosomal stress in the regulation of p53, more recent work established AATF as ribosomal RNA binding protein and enabled insights into its role as an important factor for rRNA processing and ribosome biogenesis. This Mini Review summarizes recent findings on AATF and its important role in the DDR, malignancy, and ribosome biogenesis

MRE11 inhibition highlights a replication stress-dependent vulnerability of MYCN-driven tumors

MRE11 is a component of the MRE11/RAD50/NBS1 (MRN) complex, whose activity is essential to control faithful DNA replication and to prevent accumulation of deleterious DNA double-strand breaks. In humans, hypomorphic mutations in these genes lead to DNA damage response (DDR)-defective and cancer-prone syndromes. Moreover, MRN complex dysfunction dramatically affects the nervous system, where MRE11 is required to restrain MYCN-dependent replication stress, during the rapid expansion of progenitor cells. MYCN activation, often due to genetic amplification, represents the driving oncogenic event for a number of human tumors, conferring bad prognosis and predicting very poor responses even to the most aggressive therapeutic protocols. This is prototypically exemplified by neuroblastoma, where MYCN amplification occurs in about 25% of the cases. Intriguingly, MRE11 is highly expressed and predicts bad prognosis in MYCN-amplified neuroblastoma. Due to the lack of direct means to target MYCN, we explored the possibility to trigger intolerable levels of replication stress-dependent DNA damage, by inhibiting MRE11 in MYCN-amplified preclinical models. Indeed, either MRE11 knockdown or its pharmacological inhibitor mirin induce accumulation of replication stress and DNA damage biomarkers in MYCN-amplified cells. The consequent DDR recruits p53 and promotes a p53-dependent cell death, as indicated by p53 loss- and gain-of-function experiments. Encapsulation of mirin in nanoparticles allowed its use on MYCN-amplified neuroblastoma xenografts in vivo, which resulted in a sharp impairment of tumor growth, associated with DDR activation, p53 accumulation, and cell death. Therefore, we propose that MRE11 inhibition might be an effective strategy to treat MYCN-amplified and p53 wild-type neuroblastoma, and suggest that targeting replication stress with appropriate tools should be further exploited to tackle MYCN-driven tumors

Clinical Multigene Panel Sequencing Identifies Distinct Mutational Association Patterns in Metastatic Colorectal Cancer

Extensive molecular characterization of human colorectal cancer (CRC) via Next Generation Sequencing (NGS) indicated that genetic or epigenetic dysregulation of a relevant, but limited, number of molecular pathways typically occurs in this tumor. The molecular picture of the disease is significantly complicated by the frequent occurrence of individually rare genetic aberrations, which expand tumor heterogeneity. Inter- and intratumor molecular heterogeneity is very likely responsible for the remarkable individual variability in the response to conventional and target-driven first-line therapies, in metastatic CRC (mCRC) patients, whose median overall survival remains unsatisfactory. Implementation of an extensive molecular characterization of mCRC in the clinical routine does not yet appear feasible on a large scale, while multigene panel sequencing of most commonly mutated oncogene/oncosuppressor hotspots is more easily achievable. Here, we report that clinical multigene panel sequencing performed for anti-EGFR therapy predictive purposes in 639 formalin-fixed paraffin-embedded (FFPE) mCRC specimens revealed previously unknown pairwise mutation associations and a high proportion of cases carrying actionable gene mutations. Most importantly, a simple principal component analysis directed the delineation of a new molecular stratification of mCRC patients in eight groups characterized by non-random, specific mutational association patterns (MAPs), aggregating samples with similar biology. These data were validated on a The Cancer Genome Atlas (TCGA) CRC dataset. The proposed stratification may provide great opportunities to direct more informed therapeutic decisions in the majority of mCRC cases

In Vitro Evaluation of Enterococcus faecalis Adhesion on Various Endodontic Medicaments

E. faecalis in endodontic infection represents a biofilm type of disease, which explains the bacteria’s resistance to various antimicrobial compounds and the subsequent failure after endodontic treatment. The purpose of this study was to compare antimicrobial activities and bacteria kinetic adhesion in vitro for three endodontic medicaments with a clinical isolate of E. faecalis. We devised a shake culture which contained the following intracanalar preparations: CPD, Endoidrox (EIX), PulpCanalSealer (PCS); these were immersed in a liquid culture medium inoculated with the microorganism. The shake system velocity was able to prevent non-specific bacteria adhesion and simulated the salivary flow. Specimens were collected daily (from both the medium and medicaments) for 10 days; the viable cells were counted by plate count, while the adhesion index AI° [E. faecalis fg DNA] /mm2 was evaluated in the pastes after DNA extraction, by quantitative real time PCR for the 16S rRNA gene. A partial growth inhibition, during the first 24 hours, was observed in the liquid medium and on the medicaments for EIX and subsequently for CPD (six logs). EIX showed the lowest adhesion coefficient (5*102 [fg DNA]/mm2) for nine days and was similar to the control. PCS showed no antimicrobial/antibiofilm properties. This showed that “calcium oxide” base compounds could be active against biofilm progression and at least in the short term (2-4 days) on E. faecalis cells growing in planktonic cultures

Advances in Computational Social Science and Social Simulation

Aquesta conferència és la celebració conjunta de la "10th Artificial Economics Conference AE", la "10th Conference of the European Social Simulation Association ESSA" i la "1st Simulating the Past to Understand Human History SPUHH".Conferència organitzada pel Laboratory for Socio­-Historical Dynamics Simulation (LSDS-­UAB) de la Universitat Autònoma de Barcelona.Readers will find results of recent research on computational social science and social simulation economics, management, sociology,and history written by leading experts in the field. SOCIAL SIMULATION (former ESSA) conferences constitute annual events which serve as an international platform for the exchange of ideas and discussion of cutting edge research in the field of social simulations, both from the theoretical as well as applied perspective, and the 2014 edition benefits from the cross-fertilization of three different research communities into one single event. The volume consists of 122 articles, corresponding to most of the contributions to the conferences, in three different formats: short abstracts (presentation of work-in-progress research), posters (presentation of models and results), and full papers (presentation of social simulation research including results and discussion). The compilation is completed with indexing lists to help finding articles by title, author and thematic content. We are convinced that this book will serve interested readers as a useful compendium which presents in a nutshell the most recent advances at the frontiers of computational social sciences and social simulation researc

An emerging connection between Nbs1 and Sonic Hedgehog (SHH) pathway is essential for cerebellar development and carcinogenesis

Genome integrity needs to be preserved for the propagation of genetic information. Inactivation of proteins involved in DNA damage responses (DDR) are often associated with cancer and/or developmental disorders of the nervous system, which appear particularly vulnerable to DNA distress. The Nijmegen Breakage syndrome (NBS), due to NBS1 gene mutations, is characterized by microcephaly, facial dysmorphisms and cancer predisposition. The neurological features of NBS patients have been modeled in the Nbs1CNS-Δ mouse, a Nbs1 CNS-restricted knock-out which shows microcephaly, severe ataxia and dramatically impaired cerebellar development. Strikingly, a very similar phenotype is also observed in mice with SHH/MYCN conditional KO, suggesting that Nbs1 and SHH/MYCN might be functionally linked. Prompted by this hypothesis, we generated new mouse models with CNS-restricted inactivation of Nbs1 in a transgenic SmoA1 cancer-prone context and new GCPs cultures for in vitro studies. The new SmoA1/Nbs1CNS-Δ mouse model showed that the absence of Nbs1 completely blocks the SmoA1-dependent tumor phenotype and causes severe cerebellar defects during postnatal development. This suggests an epistatic role of Nbs1 on the SHH pathway. The dramatic and degenerated phenotype of this model did not allow an appropriate analysis of the molecular interactions between Nbs1 and the SHH pathway. Therefore, we generated a new Nbs1GCP-Δ mouse model with the specific deletion of Nbs1 in cerebellar granule progenitors (GCPs). This model showed that the absence of Nbs1 caused defects in cerebellar development and this was associated with downregulation of the SHH pathway both in vivo and ex vivo. This was also confirmed in an inducible cell autonomous context. Given that the primary cilium is an essential structure for SHH signaling and given the emergent link between DDR proteins, centrosomes and ciliogenesis, we examined whether Nbs1 might be required for primary cilia formation and regulation, which in turn could affect SHH signaling. Accordingly, we provided strong evidence that loss of Nbs1 determines severe defects in ciliogenesis. Moreover, we showed that either the ciliary phenotype or the associated downregulation of the SHH pathway are rescued by loss of p53. In conclusion, our data support a novel function for Nbs1 in cilia-dependent SHH signaling and raise the possibility that the Nbs1-p53 axis, in addition to their conventional role in DNA damage, may regulate ciliogenesis both in physiological and pathological conditions in the cerebellum

Role of cell surface glycoconjugates in E. faecalis virulence and pathogenesis

With an increasing number of bacterial antibiotic resistances, studying bacterial pathogenicity determinants has become a focus of microbiological research. E. faecalis is one of the leading causes of nosocomial infections, posing a serious threat to hospitalized patients, due to an always growing isolation of strains resistant to even last resort antibiotics, e. g. vancomycin. In this study we examined enterococcal surface components, focusing on their role in a row of important features of pathogenicity, like biofilm formation, the tremendous importance of which in the clinical setting had been underestimated for decades. Using E. faecalis deletion mutants we were able to show that modifications of enterococcal cell surface glycoconjugates like teichoic acids, leading to alterations of biophysical properties, play an important role in bacterial adaptation to and interaction with the environment. Teichoic acids are central components of the gram positive surface. Both biofilm formation and bacteria-host interactions proved to be strongly dependent on teichoic acid structure: we showed a significant impairment of these enterococcal features in both a mutant deficient in teichoic acid alanylation and a mutant with defects in glycolipid synthesis, which is necessary for correct anchoring of the lipoteichoic acid to the cell membrane. This study will help understanding the dynamics of E. faecalis pathogenicity and contribute to the search for targets of novel antibacterial therapeutics and vaccines.Con il sempre maggiore incremento nella frequenza di isolamento di ceppi batterici multiresistenti, lo studio dei fattori di virulenza e/o dei meccanismi di patogenicità ha acquisito crescente importanza nell’ambito della ricerca microbiologica. Enterococcus faecalis è una delle principali cause di infezioni nosocomiali, e l’aumento di ceppi resistenti agli antibiotici d’elezione, quali la vancomicina, pone serie difficoltà nel trattamento di pazienti ospedalizzati. Lo scopo di questo studio si è incentrato sulla caratterizzazione di alcune componenti di superficie degli enterococci coinvolte in una serie di processi, tra cui la formazione di biofilm, che negli ultimi decenni ha acquisito sempre maggiore importanza quale fattore favorente lo sviluppo di infezioni. Manipolando geneticamente ceppi di E. faecalis, siamo stati in grado di dimostrare come alterazioni nella struttura di molecole glicosilate di superficie, tra cui gli acidi teicoici, portino a drastiche perturbazioni delle caratteristiche biofisiche del rivestimento batterico le quali, a loro volta, rivestono un ruolo di primaria importanza sia nell’adattamento del batterio all’ambiente, sia nelle diverse interazioni con superfici “animate” o “inanimate”. Gli acidi teicoici sono una delle principali componenti della superficie dei batteri gram positivi. Sia la formazione di biofilm, che le interazioni batterio-ospite si sono dimostrate fortemente dipendenti dalla struttura di queste complesse molecole di superficie: nel corso di questo studio siamo stati in grado di dimostrare che, sia un ceppo mutato nella via di modificazione degli acidi teicoici (tramite il legame di residui di alanina), che un mutante difettivo nella sintesi di glicolipidi – responsabili dell’ancoraggio dell’acido lipoteicoico alla membrana cellulare – sono difettivi in numerosi aspetti coinvolti nel meccanismo patogenetico tra cui la formazione di biofilm, l’adesione alle cellule eucariotiche, e la resistenza ai peptidi cationici antimicrobici. I dati ottenuti serviranno a migliorare la nostra comprensione dei meccanismi patogenici messi in atto da E.faecalis e potranno contribuire agli studi volti a valutare possibili bersagli per nuove strategie terapeutiche e vaccinali

Multiplex PCR Assay for Direct Identification of Group B Streptococcal Alpha-Protein-Like Protein Genes

We developed a group B streptococcus multiplex PCR assay which allows, by direct analysis of the amplicon size, determination of the surface protein antigen genes of alpha-C protein, epsilon protein, Rib, Alp2, Alp3, and Alp4. The multiplex PCR assay offers a rapid and simple method of subtyping Streptococcus agalactiae based on surface protein genes

Sistema stabile di coltura in vitro di cellule precursori granulari cerebellari (GCP), metodo stabile per la coltura in vitro di dette cellule e usi di detto sistema o metodo per la coltura in vitro

La presente invenzione si riferisce ad un sistema di coltura stabile in vitro di cellule precursori dei granuli cerebellari (GCP). Il suddetto sistema di coltura mantiene nei GCP un’elevata espressione della via di Sonic Hedgehog e un illimitato potere proliferativo garantito dagli alti livelli delle proteine staminali Sox2 e Nestin. Per tali motivi il sopra citato sistema di coltura può rappresentare un valido modello in vitro per lo studio della fisiopatologia dei granuli cerebellari, per lo studio di malattie cerebellari conseguenti a danno o a neurodegenerazione, e potenzialmente per il loro trattamento mediante approcci di terapia genica