Methodological framework for an integrated multi-scale vulnerability and resilience assessment

The deliverable illustrates the methodological framework to assess vulnerability and resilience across different temporal and spatial scales, acknowledging the different domains where the latter may manifest, and in particular in the natural and the built environment, allocating a large importance to the so called “critical infrastructures”, in social and economic systems. A set of four matrices has been developed to identify what aspects should be looked at before the impact, that is to say what shows the potential ability or inability to cope with an extreme; at the impact, addressing in particular the capacity (or incapacity) to sustain various types of stresses (in the form of acceleration, pressure, heat…); in the time immediately after the impact, as the ability (or inability) to suffer losses and still continue functioning; and in the longer term of recovery, as the capacity to find a new state of equilibrium in which the fragilities manifested during and after the impact are addressed. Developing the framework, a particular attention has been paid to the relationships among systems within the same matrix and among matrices, across spatial and temporal scales. A set of matrices has been developed for different natural hazards, including in particular landslides and floods, trying to include as much as possible what past cases, the international literature and prior experience of involved partners have indicated as relevant parameters and factors to look at. In this regard, the project builds on the state of the art, embedding what has been learned until now in terms of response capacity to a variety of stresses and in the meantime identifying gaps to be addressed by future research

Identification and Molecular Characterization of a Novel Large-Scale Variant (Exons 4_18 Loss) in the LDLR Gene as a Cause of Familial Hypercholesterolaemia in an Italian Family

Abstract: Next-generation sequencing (NGS) is nowadays commonly used for clinical purposes, and represents an efficient approach for the molecular diagnosis of familial hypercholesterolemia (FH). Although the dominant form of the disease is mostly due to the low-density lipoprotein receptor (LDLR) small-scale pathogenic variants, the copy number variations (CNVs) represent the underlying molecular defects in approximately 10% of FH cases. Here, we reported a novel large deletion in the LDLR gene involving exons 4–18, identified by the bioinformatic analysis of NGS data in an Italian family. A long PCR strategy was employed for the breakpoint region analysis where an insertion of six nucleotides (TTCACT) was found. Two Alu sequences, identified within intron 3 and exon 18, could underlie the identified rearrangement by a nonallelic homologous recombination (NAHR) mechanism. NGS proved to be an effective tool suitable for the identification of CNVs, together with small-scale alterations in the FH-related genes. For this purpose, the use and implementation of this cost-effective, efficient molecular approach meets the clinical need for personalized diagnosis in FH cases

A Novel GCK Large Genomic Rearrangement in a Patient with MODY-2 Detected by Clinical Exome Sequencing

Maturity-onset diabetes of the young (MODY) is a rare form of non-autoimmune diabetes with an autosomal dominant inheritance. To date, 14 genes have been reported as genetic basis of MODY. GCK gene, encoding the glucokinase enzyme, was the first MODY gene to be identified. GCK heterozygous inactivating variants cause the GCK-MODY or MODY2 subtype. However, partial or whole gene deletions have been rarely identified, showing it to be a rare cause of GCK-MODY. We reported the molecular evaluation of a Ukrainian patient with clinical diagnosis of MODY2. We performed the Next generation sequencing of the clinical exome using the Clinical Exome Solution® kit (SOPHiA Genetics), followed by the design of a 14 genes virtual panel related to the suggestive diagnosis of MODY. Bioinformatics analysis was performed using the SOPHiA DDM platform (SOPHiA Genetics). The SALSA MLPA kit for MODY (MRC-Holland) was used for relative quantification of GCK exons. From the molecular evaluation, no pathogenic sequence variants were detected in the investigated genes. Copy Number Variation analysis was able to identify a large deletion involving the last three exons of the GCK gene. This result was confirmed by MLPA. To the best of our knowledge, the identified rearrangement has never been reported in the literature

Sex-Based Dimorphism of Anticancer Immune Response and Molecular Mechanisms of Immune Evasion

PURPOSE: We previously demonstrated that sex influences response to immune checkpoint inhibitors. In this article, we investigate sex-based differences in the molecular mechanisms of anticancer immune response and immune evasion in patients with NSCLC. EXPERIMENTAL DESIGN: We analyzed (i) transcriptome data of 2,575 early-stage NSCLCs from seven different datasets; (ii) 327 tumor samples extensively characterized at the molecular level from the TRACERx lung study; (iii) two independent cohorts of 329 and 391 patients, respectively, with advanced NSCLC treated with anti–PD-1/anti–PD-L1 drugs. RESULTS: As compared with men, the tumor microenvironment (TME) of women was significantly enriched for a number of innate and adaptive immune cell types, including specific T-cell subpopulations. NSCLCs of men and women exploited different mechanisms of immune evasion. The TME of females was characterized by significantly greater T-cell dysfunction status, higher expression of inhibitory immune checkpoint molecules, and higher abundance of immune-suppressive cells, including cancer-associated fibroblasts, MDSCs, and regulatory T cells. In contrast, the TME of males was significantly enriched for a T-cell–excluded phenotype. We reported data supporting impaired neoantigens presentation to immune system in tumors of men, as molecular mechanism explaining the findings observed. Finally, in line with our results, we showed significant sex-based differences in the association between TMB and outcome of patients with advanced NSCLC treated with anti–PD-1/PD-L1 drugs. CONCLUSIONS: We demonstrated meaningful sex-based differences of anticancer immune response and immune evasion mechanisms, that may be exploited to improve immunotherapy efficacy for both women and men. TRANSLATIONAL RELEVANCE: It is well known that sex (i.e., the biological differences between men and women) and gender (i.e., behavioral differences associated with being male or female) are variables that affect immune responses to both foreign and selfantigens. Such sex- and gender-based dimorphism of immune system function, in turn reflects complex interactions between genes, hormones, the environment, and commensal microbiome composition. In our previous works, we showed that patients' sex is significantly associated with effectiveness of immune checkpoint inhibitors (ICIs) in patients with several solid tumors, including NSCLC. Here, we identified meaningful differences in molecular mechanisms that drive anticancer immune response as well as in immune evasion mechanisms exploited by NSCLCs arising in men and women. Importantly, we showed that all the findings reported, were not related to other variables potentially associated with sex such as patients' age, stage of disease, tumor histotype, and smoking status. The findings reported in this our work explain our previous clinical observations and can open this area to different immunotherapy strategies in males and females with NSCLC to further improve prognosis of both

PAT-ChIP coupled with laser microdissection allows the study of chromatin in selected cell populations from paraffin-embedded patient samples

Background: The recent introduction of pathology tissue-chromatin immunoprecipitation (PAT-ChIP), a technique allowing chromatin immunoprecipitation from formalin-fixed and paraffin-embedded (FFPE) tissues, has expanded the application potential of epigenetic studies in tissue samples. However, FFPE tissue section analysis is strongly limited by tissue heterogeneity, which hinders linking the observed epigenetic events to the corresponding cellular population. Thus, ideally, to take full advantage of PAT-ChIP approaches, procedures able to increase the purity and homogeneity of cell populations from FFPE tissues are required. Results: In this study, we tested the use of both core needle biopsies (CNBs) and laser microdissection (LMD), evaluating the compatibility of these methods with the PAT-ChIP procedure. Modifications of the original protocols were introduced in order to increase reproducibility and reduce experimental time. We first demonstrated that chromatin can be prepared and effectively immunoprecipitated starting from 0.6-mm-diameter CNBs. Subsequently, in order to assess the applicability of PAT-ChIP to LMD samples, we tested the effects of hematoxylin or eosin staining on chromatin extraction and immunoprecipitation, as well as the reproducibility of our technique when using particularly low quantities of starting material. Finally, we carried out the PAT-ChIP using chromatin extracted from either normal tissue or neoplastic lesions, the latter obtained by LMD from FFPE lung sections derived from mutant K-rasv12transgenic mice or from human adeno- or squamous lung carcinoma samples. Well characterized histone post-translational modifications (HPTMs), such as H3K4me3, H3K27me3, H3K27Ac, and H3K9me3, were specifically immunoselected, as well as the CTCF transcription factor and RNA polymerase II (Pol II). Conclusions: Epigenetic profiling can be performed on enriched cell populations obtained from FFPE tissue sections. The improved PAT-ChIP protocol will be used for the discovery and/or validation of novel epigenetic biomarkers in FFPE human samples

Dual inhibition of CDK12 and CDK13 uncovers actionable vulnerabilities in patient-derived ovarian cancer organoids

Background: High grade serous ovarian cancer (HGSOC) is highly lethal, partly due to chemotherapy resistance and limited availability of targeted approaches. Cyclin dependent kinases 12 and 13 (CDK12/13) are promising therapeutic targets in human cancers, including HGSOC. Nevertheless, the effects of their inhibition in HGSOC and the potential synergy with other drugs are poorly known. Methods: We analyzed the effects of the CDK12/13 inhibitor THZ531 in HGSOC cells and patient-derived organoids (PDOs). RNA sequencing and quantitative PCR analyses were performed to identify the genome-wide effects of short-term CDK12/13 inhibition on the transcriptome of HGSOC cells. Viability assays with HGSOC cells and PDOs were performed to assess the efficacy of THZ531 as single agent or in combination with clinically relevant drugs. Results: The CDK12 and CDK13 genes are deregulated in HGSOC and their concomitant up-regulation with the oncogene MYC predicts poor prognosis. HGSOC cells and PDOs display high sensitivity to CDK12/13 inhibition, which synergizes with drugs in clinical use for HGSOC. Transcriptome analyses revealed cancer-relevant genes whose expression is repressed by dual CDK12/13 inhibition through impaired splicing. Combined treatment with THZ531 and inhibitors of pathways regulated by these cancer relevant genes (EGFR, RPTOR, ATRIP) exerted synergic effects on HGSOC PDO viability. Conclusions: CDK12 and CDK13 represent valuable therapeutic targets for HGSOC. We uncovered a wide spectrum of CDK12/13 targets as potential therapeutic vulnerabilities for HGSOC. Moreover, our study indicates that CDK12/13 inhibition enhances the efficacy of approved drugs that are already in use for HGSOC or other human cancers

Status and challenges for the concept design development of the EU DEMO Plant Electrical System

The EU DEMO Plant Electrical System (PES) main scopes are to supply all the plant electrical loads and to deliver to the Power Transmission Grid (PTG) the net electrical power generated. The studies on the PES during the Pre-Concept Design (PCD) Phase were mainly addressed to understand the possible issues, related to the special features both of the power generated, with respect to a power plant of the same size, and of the power to be supplied to the electrical loads. For this purpose, the approach was to start the design of the different PES components adopting technologies already utilized in fusion experiments and in Nuclear Power Plants (NPP) to verify their applicability and identify possible limits when scaled to the DEMO size and applied to the specific pulsed operating conditions. This work is not completed, however several issues have been already identified related to the pulsed operation of the turbine generator, the large amount of recirculation power, the very high peaks of active power required for the plasma formation and control, the huge reactive power demand, if thyristor converter technology was adopted to supply the superconducting coils, etc.. The paper gives an overview on the features and scope of the PES and its subsystems, on the main achievements during the Pre-Concept Design (PCD) Phase, on the challenges for the development of the conceptual design in the next framework program and on the plan to face them

Biocompatible cellulose nanocrystal-based Trojan horse enables targeted delivery of nano-Au radiosensitizers to triple negative breast cancer cells

: A hybrid cellulose-based programmable nanoplatform for applications in precision radiation oncology is described. Here, sugar heads work as tumor targeting moieties and steer the precise delivery of radiosensitizers, i.e. gold nanoparticles (AuNPs) into triple negative breast cancer (TNBC) cells. This "Trojan horse" approach promotes a specific and massive accumulation of radiosensitizers in TNBC cells, thus avoiding the fast turnover of small-sized AuNPs and the need for high doses of AuNPs for treatment. Application of X-rays resulted in a significant increase of the therapeutic effect while delivering the same dose, showing the possibility to use roughly half dose of X-rays to obtain the same radiotoxicity effect. These data suggest that this hybrid nanoplatform acts as a promising tool for applications in enhancing cancer radiotherapy effects with lower doses of X-rays