Molecular and cellular characterization of Space flight effects on endothelial cell function: preparatory work of the SFEF project

Exposure to microgravity during space flights (SF) of variable lenght induces a suffering of the endothelium (the diffuse organ made up of cells that line all blood vessels) mostly responsible of health problems reported by astronauts and animals returning from Space. Of interest to prenosological medicine, the effects of microgravity on astronauts are striking similar to the consequences of sedentary life, senescence and degenerative diseases on Earth, although SF effects are accelerated and reversible. Microgravity therefore represents a significant novel model to better undertstand common pathologies. A comprehensive cell and molecular biology study is necessary to explain pathophysiological findings after SF in terms of variations in genome biology. This project will study the effects of microgravity on endothelial cells (ECs) cultured on the International Space Station (ISS) through analysis of 1) cellular transcriptome and 2) methylome; 3) DNA damage and cell senescence, 4) miRNome. This project has been selected by the European Space Agency (ESA) and the Italian Space Agency and is presently in preparation: we are developing the biological and engineering conditions (with the contribution of Kayser Italia Srl) to get suitable samples after culturing, fixing and storing ECs in Space. We will culture on the ISS the human microvascular EC line HMEC-1 (CDC, Atlanta, GA USA). At this preparatory phase, we have tested different conditions for implementation of the experiment in Space, focusing on 1) designing the protocol for cell culturing, fixing and storage inside electronically controlled bioreactors; 2) testing several pre-flight incubation protocols to simulate different mission scenarios; 3) evaluating the suitability of fixed samples for subsequent experimental procedures. We expect the results will contribute to the creation of prevention and rehabilitation protocols for astronauts and for the general public suffering from inflammatory, degenerative and cardiovascular pathologies

L’impatto della diagnosi di sterilità e dei trattamenti di PMA sull’uomo: una rassegna della letteratura

Questo contributo prende in esame i vissuti e l adattamento dell uomo a seguito della diagnosi di sterilità, nonché il suo approccio ai percorsi di PMA e le principali differenze con l esperienza femminile. In realtà, il principale focus di interesse della maggior parte degli studi risulta essere la componente femminile della coppia mentre è ormai noto come la genitorialità risulti essenziale anche per l identità maschile e la mancata transizione ad essa possa determinare nell uomo gravi ricadute in termini di bassa autostima, alterazione dell immagine corporea, percezione della propria mascolinità. Il lavoro è orientato ad identificare quali componenti psicologiche giocano un ruolo nell eziologia e nell adattamento alla sterilità, nonché gli aspetti legati alla necessaria attività di counseling e psicoterapia della coppia sterile o di supporto nel caso della scelta di intraprendere un percorso di procreazione medicalmente assistita (PMA)

Quality of Life and Sexuality among People with Mental and Physical Disabilities in the Italian Context

Aim: The article analyses sexuality and quality of life among people with mental and physical disabilities in the Italian context. It is aimed at contributing to a social debate over the sexual assistant for disabled people, as this professional is not still present in Italy. The aim of this paper is therefore to reflect on the consequences of the negative attitudes towards sexual life in disabled people, as well as for their families and community. In the collective unconscious, people with physical or mental disabilities are considered unable to live a healthy sexuality. This representation contributes to the spread of false attributions and prejudices. These characteristics may also be found inside the familiar context and its parental, educational style that appears so overprotective of the disabled child, in an attempt to relieve him of any social comparisons. Conclusion: The article was aimed at encouraging a social and political debate towards the introduction of a sexual assistant in Italy where, this professional still not has an institutional recognition. Disabled people’s sexual needs and desires cannot longer be ignored. A long pathway should be undertaken to promote their well-being, towards a dynamic process of change aimed at improving the quality of life of disabled people, their family and community as well

Development of a new screening system for the identification of RNF43-related genes and characterisation of other PA-RING family members.

The E3 ubiquitin ligase RNF43 (RING finger protein 43) is an important negative modulator of the WNT signalling pathway that acts at the plasma membrane by targeting Frizzled and its co-receptor LRP for degradation. In the small intestine, this prevents uncontrolled expansion of the stem cell compartment and so it is essential to the maintenance of normal tissue homeostasis. However, despite its crucial role in fine-tuning the WNT pathway and its role as a tumour suppressor, it is unclear whether RNF43 has further binding partners and what their functional relevance is to the modulation of WNT signalling. Here, I describe the development of a new screening strategy which combines CRISPR/Cas9 technology with 3D-intestinal organoid culture for the identification of novel molecular interactors of RNF43. Overall, this study and the technology developed provide a tool to enable the detailed description of the mechanism of action of RNF43, which is important not only in order to increase our understanding of WNT pathway regulation but also to gain potential new insights into RNF43 paralogs, by analogy. The investigation of paralogs is crucial as RNF43 belongs to a newly identified family of E3 ubiquitin ligases, named the PA-RING family, whose members are still poorly characterised. The majority of PA-RING family members have not been linked to any signalling pathway, most of their targets are still unknown and in many cases their in vivo function has not been addressed. In this context, my work has specifically focused on the investigation of the potential involvement of additional PA-RING family members in WNT pathway modulation and also on target identification for selected members. The results summarised in this dissertation show that no other PA-RING family member plays a prominent role in WNT pathway modulation aside from Rnf43 and its homologue Znrf3, however, different classes of adhesion molecules are likely to be regulated by certain of these E3 ligases. In conclusion, my work has contributed to unravelling previously unexplored aspects of this protein family, with particular regard to RNF43 and its mechanism of action. Thanks to this original approach, it was possible to identify potential new players involved either in membrane clearance of Frizzled or in RNF43 maturation. In particular, my thesis focuses on the characterisation of the role of DAAM in RNF43-mediated Frizzled internalisation

Norovirus Replication in Human Intestinal Epithelial Cells Is Restricted by the Interferon-Induced JAK/STAT Signaling Pathway and RNA Polymerase II-Mediated Transcriptional Responses.

Human noroviruses (HuNoV) are a leading cause of viral gastroenteritis worldwide and a significant cause of morbidity and mortality in all age groups. The recent finding that HuNoV can be propagated in B cells and mucosa-derived intestinal epithelial organoids (IEOs) has transformed our ability to dissect the life cycle of noroviruses. Using transcriptome sequencing (RNA-Seq) of HuNoV-infected intestinal epithelial cells (IECs), we have found that replication of HuNoV in IECs results in interferon (IFN)-induced transcriptional responses and that HuNoV replication in IECs is sensitive to IFN. This contrasts with previous studies that suggested that the innate immune response may play no role in the restriction of HuNoV replication in immortalized cells. We demonstrated that inhibition of Janus kinase 1 (JAK1)/JAK2 enhanced HuNoV replication in IECs. Surprisingly, targeted inhibition of cellular RNA polymerase II-mediated transcription was not detrimental to HuNoV replication but instead enhanced replication to a greater degree than blocking of JAK signaling directly. Furthermore, we demonstrated for the first time that IECs generated from genetically modified intestinal organoids, engineered to be deficient in the interferon response, were more permissive to HuNoV infection. Taking the results together, our work revealed that IFN-induced transcriptional responses restrict HuNoV replication in IECs and demonstrated that inhibition of these responses mediated by modifications of the culture conditions can greatly enhance the robustness of the norovirus culture system.IMPORTANCE Noroviruses are a major cause of gastroenteritis worldwide, and yet the challenges associated with their growth in culture have greatly hampered the development of therapeutic approaches and have limited our understanding of the cellular pathways that control infection. Here, we show that human intestinal epithelial cells, which represent the first point of entry of human noroviruses into the host, limit virus replication by induction of innate responses. Furthermore, we show that modulating the ability of intestinal epithelial cells to induce transcriptional responses to HuNoV infection can significantly enhance human norovirus replication in culture. Collectively, our findings provide new insights into the biological pathways that control norovirus infection but also identify mechanisms that enhance the robustness of norovirus culture

Vasculitis Diagnosed on Fluorine-18 Labelled-2-Deoxy-2-Fluoro-D-Glucose Uptake in A Patient With Fever of Unknown Origin and A History of Non Hodgkin’s Lymphoma

There are increasing data demonstrating the role of 18F-flourodeoxyglucose positron emission tomography with computerized tomography fusion (18F-FDG PET/CT) in the diagnosis of large vessel vasculitis, including Takayasu arteritis and giant cell arteritis. We report a case of large vessel vasculitis detected on 18F-FDG PET/CT; a 32-year-old woman with history of Non Hodgkin Lymphoma, admitted with fever of unknown origin (FUO) of 2-months duration and asthenia. To exclude FUO of malignancy, in the suspect of NHL relapse, 18F-FDG PET/CT imaging was performed. The images demonstrated significant 18F-FDG uptake in aortic arch and no signs of NHL relapse. This case report supports the role of 18F-FDG PET/CT as a useful and noninvasive tool in diagnostic evaluation of patient with FUO, both by excluding a malignant etiology and providing information about other possible causes such as inflammation, including vasculitis. 18F-FDG PET/CT is very useful in the early diagnosis of active inflammation including vasculitis and provides timely information for appropriate therapy

Vasculitis Diagnosed on Fluorine-18 Labelled-2-Deoxy-2-Fluoro-D-Glucose Uptake in A Patient With Fever of Unknown Origin and A History of Non Hodgkin’s Lymphoma

There are increasing data demonstrating the role of 18F-flourodeoxyglucose positron emission tomography with computerized tomography fusion (18F-FDG PET/CT) in the diagnosis of large vessel vasculitis, including Takayasu arteritis and giant cell arteritis. We report a case of large vessel vasculitis detected on 18F-FDG PET/CT; a 32-year-old woman with history of Non Hodgkin Lymphoma, admitted with fever of unknown origin (FUO) of 2-months duration and asthenia. To exclude FUO of malignancy, in the suspect of NHL relapse, 18F-FDG PET/CT imaging was performed. The images demonstrated significant 18F-FDG uptake in aortic arch and no signs of NHL relapse. This case report supports the role of 18F-FDG PET/CT as a useful and noninvasive tool in diagnostic evaluation of patient with FUO, both by excluding a malignant etiology and providing information about other possible causes such as inflammation, including vasculitis. 18F-FDG PET/CT is very useful in the early diagnosis of active inflammation including vasculitis and provides timely information for appropriate therapy

A phospho-switch controls RNF43-mediated degradation of Wnt receptors to suppress tumorigenesis.

Funder: Japan Foundation for Applied Enzymology; doi: https://doi.org/10.13039/100008695Funder: Pancreas Research Foundation of Japan Collaborative Research Project Program of the Medical Institute of Bioregulation, Kyushu University, Japan Joint Research Program of the Institute for Molecular and Cellular Regulation, Gunma University, Japan Grant for Joint Research Project of the Research Institute for Microbial Diseases Osaka UniversityFunder: European Research Council (ERC (639050) and the Interpark Bio-Convergence Center Grant Program.Frequent mutation of the tumour suppressor RNF43 is observed in many cancers, particularly colon malignancies. RNF43, an E3 ubiquitin ligase, negatively regulates Wnt signalling by inducing degradation of the Wnt receptor Frizzled. In this study, we discover that RNF43 activity requires phosphorylation at a triplet of conserved serines. This phospho-regulation of RNF43 is required for zebrafish development and growth of mouse intestinal organoids. Cancer-associated mutations that abrogate RNF43 phosphorylation cooperate with active Ras to promote tumorigenesis by abolishing the inhibitory function of RNF43 in Wnt signalling while maintaining its inhibitory function in p53 signalling. Our data suggest that RNF43 mutations cooperate with KRAS mutations to promote multi-step tumorigenesis via the Wnt-Ras-p53 axis in human colon cancers. Lastly, phosphomimetic substitutions of the serine trio restored the tumour suppressive activity of extracellular oncogenic mutants. Therefore, harnessing phospho-regulation of RNF43 might be a potential therapeutic strategy for tumours with RNF43 mutations

One-step generation of conditional and reversible gene knockouts

Loss-of-function studies are key for investigating gene function, and CRISPR technology has made genome editing widely accessible in model organisms and cells. However, conditional gene inactivation in diploid cells is still difficult to achieve. Here, we present CRISPR-FLIP, a strategy that provides an efficient, rapid and scalable method for biallelic conditional gene knockouts in diploid or aneuploid cells, such as pluripotent stem cells, 3D organoids and cell lines, by co-delivery of CRISPR-Cas9 and a universal conditional intronic cassette.A.A.-R. and K.T. are supported by the Medical Research Council, A.M. is supported by Wntsapp, Marie Curie ITN. J.F. and J.C.R.S. are supported by the Wellcome Trust. W.C.S. received core grant support from the Wellcome Trust to the Wellcome Trust Sanger Institute. B.-K.K. and R.C.M. are supported by a Sir Henry Dale Fellowship from the Wellcome Trust and the Royal Society (101241/Z/13/Z) and receive a core support grant from the Wellcome Trust and MRC to the WT–MRC Cambridge Stem Cell Institute

Proceedings of the Fifth Italian Conference on Computational Linguistics CLiC-it 2018

On behalf of the Program Committee, a very warm welcome to the Fifth Italian Conference on Computational Linguistics (CLiC-­‐it 2018). This edition of the conference is held in Torino. The conference is locally organised by the University of Torino and hosted into its prestigious main lecture hall “Cavallerizza Reale”. The CLiC-­‐it conference series is an initiative of the Italian Association for Computational Linguistics (AILC) which, after five years of activity, has clearly established itself as the premier national forum for research and development in the fields of Computational Linguistics and Natural Language Processing, where leading researchers and practitioners from academia and industry meet to share their research results, experiences, and challenges