Endothelial Tie1 regulates tumor angiogenesis, vascular abnormalization and metastatic dissemination

The Angiopoietin/Tie system is one of the most important vascular-tissue specific signaling pathways, essential during embryonic vascular development and maturation, and key regulator of adult homeostasis. The Tie receptors, Tie1 and Tie2, and their secreted angiopoietin (Ang) ligands, Ang1 and Ang2, have been identified as the main factors of the system. Ang1 is primarily produced by perivascular cells and acts in a paracrine fashion as strong Tie2 agonist that mediates endothelial cell (EC) survival and maturation signals. Ang2, expressed and stored in EC, functions primarily as an antagonist of Tie2 and promotes vascular destabilization. Nevertheless, it can act as a partial agonist of Tie2 in a context-dependent manner. Tie1 and Tie2 are both expressed by EC and share high similarities in their overall domain structure. Whereas Tie2 acts as the primary signal transducing receptor, Tie1 function as a holoreceptor is not completely understood: It does not activate signaling pathways on its own but rather modulates the Tie2 kinase activity. Tie1 is highly expressed in activated endothelial cells during embryogenesis and during pathological conditions, such as tumor progression and metastasis, but it is downregulated in the adult quiescent endothelium. Thus, Tie1 global deficiency in mice leads to embryonic lethality at late gestation due to perturbed vessel integrity, while its specific deletion in the growing tumor vasculature results in decreased blood vessel density. The combination of high expression in the tumor endothelium and low expression in the adult vasculature makes Tie1 an interesting molecule and a potential therapeutic target. Angiogenesis is one of the most critical steps during tumor growth and metastasis progression. Comparative tumor and metastasis experiments in wildtype and Tie1 endothelial-deficient mice were performed to mechanistically unravel the role of the orphan receptor during individual steps of tumor progression and metastasis. Analysis of the tumor vasculature demonstrated that Tie1 endothelial deletion induces no significant changes in the early steps of tumor growth but rather affects later steps of tumor progression. Endothelial-Tie1 deletion induces progressively normalization of primary tumor blood vessels accompanied by decreased microvessel density, increased mural cell coverage, improved vessel perfusion and reduced tumor cell intravasation into the blood stream as well as extravasation at secondary sites. These effects result in almost complete inhibition of post-surgical spontaneous lung metastasis and improved overall survival of Tie1-endothelial deleted mice. Mechanistically, Tie1 targeting in the primary tumor leads to vascular normalization and stabilization by increasing the proportion of Tie2-positive endothelial cells (stalk cells) and by potentiating the Ang1/Tie2 signaling axis which is primarily essential for the maintenance of endothelial quiescence and survival. The data establish a remarkable contribution of the orphan receptor Tie1 to primary tumor angiogenesis and to individual steps of metastatic cascade. These findings contribute to the mechanism-guided validation of Tie1 as a therapeutic target to sharpen the balance between triggering vascular regression and promoting vascular normalization

Hypofractionation and Concomitant Boost in Ductal Carcinoma In Situ (DCIS): Analysis of a Prospective Case Series with Long-Term Follow-Up

We previously reported on a cohort of breast cancer patients affected with ductal carcinoma in situ (DCIS) that were treated with breast conservative surgery and hypofractionated whole-breast radiotherapy with a concomitant boost to the lumpectomy cavity. We now report on the long-term results of the oncological and toxicity outcomes, at a median follow-up of 11.2 years. We also include an analysis of the predictive factors for local recurrence (LR). Eighty-two patients with long-term observation were considered for this report. All received hypofractionated post-operative radiotherapy with a concomitant boost (45 Gy/20 fractions to the whole breast and 50 Gy/20 fractions to the lumpectomy cavity). We report on LC rates at 5 and 10 years, overall survival (OS), and breast-cancer-specific survival (BCSS), employing the Kaplan-Meier method. Cox proportional regression analysis was used to determine the role of selected clinical parameters on the risk of local recurrence, by the univariate and multivariate models. After a median follow-up of 11.2 years (range 5-15 years), 9 pts (11%) developed LR. The LR rates at 5 years and 10 years were 2.4% and 8.2%, respectively. The 5- and 10-year overall survival rates were 98.8% and 91.6%, respectively. The 5- and 10-year breast-cancer-specific survival rates were 100.0% and 99.0%. Late skin and subcutaneous toxicities were generally mild, and cosmetic results were good-excellent for most patients. For the univariate regression analysis, ER positive status (HR; 95% CI, p = 0.021), PgR positive status (HR; 95% CI, p = 0.012), and the aggregate data of positive hormonal status (HR; 95% CI, p = 0.021) were inversely correlated to LR risk. Conversely, a high tumor grade (G3) was directly correlated with the risk of LR (HR; 95% CI, p = 0.048). For the multivariate regression analysis, a high tumor grade (G3) confirmed its negative impact on LR (HR 0.40; 95% CI 0.19-0.75, p = 0.047). Our long-term data demonstrate hypofractionated whole-breast radiotherapy with a concomitant boost to be feasable, effective, and tolerable. Our experience suggests positive hormonal status to be protective with respect to LR risk. A high tumor grade is a risk factor for LR

Implementation of a Commercial Deep Learning-Based Auto Segmentation Software in Radiotherapy: Evaluation of Effectiveness and Impact on Workflow

Proper delineation of both target volumes and organs at risk is a crucial step in the radiation therapy workflow. This process is normally carried out manually by medical doctors, hence demanding timewise. To improve efficiency, auto-contouring methods have been proposed. We assessed a specific commercial software to investigate its impact on the radiotherapy workflow on four specific disease sites: head and neck, prostate, breast, and rectum. For the present study, we used a commercial deep learning-based auto-segmentation software, namely Limbus Contour (LC), Version 1.5.0 (Limbus AI Inc., Regina, SK, Canada). The software uses deep convolutional neural network models based on a U-net architecture, specific for each structure. Manual and automatic segmentation were compared on disease-specific organs at risk. Contouring time, geometrical performance (volume variation, Dice Similarity Coefficient-DSC, and center of mass shift), and dosimetric impact (DVH differences) were evaluated. With respect to time savings, the maximum advantage was seen in the setting of head and neck cancer with a 65%-time reduction. The average DSC was 0.72. The best agreement was found for lungs. Good results were highlighted for bladder, heart, and femoral heads. The most relevant dosimetric difference was in the rectal cancer case, where the mean volume covered by the 45 Gy isodose was 10.4 cm(3) for manual contouring and 289.4 cm(3) for automatic segmentation. Automatic contouring was able to significantly reduce the time required in the procedure, simplifying the workflow, and reducing interobserver variability. Its implementation was able to improve the radiation therapy workflow in our department

Modeling of autosomal-dominant retinitis pigmentosa in Caenorhabditis elegans uncovers a nexus between global impaired functioning of certain splicing factors and cell type-specific apoptosis

Retinitis pigmentosa (RP) is a rare genetic disease that causes gradual blindness through retinal degeneration. Intriguingly, seven of the 24 genes identified as responsible for the autosomal-dominant form (adRP) are ubiquitous spliceosome components whose impairment causes disease only in the retina. The fact that these proteins are essential in all organisms hampers genetic, genomic, and physiological studies, but we addressed these difficulties by using RNAi in Caenorhabditis elegans. Our study of worm phenotypes produced by RNAi of splicing-related adRP (s-adRP) genes functionally distinguishes between components of U4 and U5 snRNP complexes, because knockdown of U5 proteins produces a stronger phenotype. RNA-seq analyses of worms where s-adRP genes were partially inactivated by RNAi, revealed mild intron retention in developing animals but not in adults, suggesting a positive correlation between intron retention and transcriptional activity. interestingly, RNAi of s-adRP genes produces an increase in the expression of atl-1 (homolog of human ATR), which is normally activated in response to replicative stress and certain DNA-damaging agents. The up-regulation of atl-1 correlates with the ectopic expression of the pro-apoptotic gene egl-1 and apoptosis in hypodermal cells, which produce the cuticle, but not in other cell types. Our model in C. elegans resembles s-adRP in two aspects: The phenotype caused by global knockdown of s-adRP genes is cell type-specific and associated with high transcriptional activity. Finally, along with a reduced production of mature transcripts, we propose a model in which the retina-specific cell death in s-adRP patients can be induced through genomic instability

Directors' remuneration: A comparison of Italian and UK non-financial listed firms' disclosure

Directors' remuneration is a key issue for both academics and policymakers. It has caused enormous controversy in recent years. This study uses a comprehensive index to analyse the disclosure of directors' remuneration in Italian and UK listed firms. It finds that the level of voluntary disclosure is significantly associated with firm-specific incentives, such as the demand for information from investors and the need for legitimacy. It finds that the level of voluntary disclosure is significantly higher in the UK than in Italy and that firm-specific incentives to disclose voluntary information differ according to the institutional setting in which a firm operates. In the UK, firm-specific incentives mostly come from the demand for information, estimated with the level of ownership diffusion, and the need for legitimacy generated by poor market performance and shareholders' dissent. In Italy, firm-specific incentives seem to be represented by the need for legitimacy generated by media coverage. This study also provides evidence that, in both countries, the information disclosed in corporate documents does not allow readers to obtain a comprehensive picture of directors' remuneration. Bonuses are poorly disclosed even though they are a key element of directors' remuneration. This finding is clearly important for policymakers at European and national level

The rapid spread of SARS-COV-2 Omicron variant in Italy reflected early through wastewater surveillance

The SARS-CoV-2 Omicron variant emerged in South Africa in November 2021, and has later been identified worldwide, raising serious concerns. A real-time RT-PCR assay was designed for the rapid screening of the Omicron variant, targeting characteristic mutations of the spike gene. The assay was used to test 737 sewage samples collected throughout Italy (19/21 Regions) between 11 November and 25 December 2021, with the aim of assessing the spread of the Omicron variant in the country. Positive samples were also tested with a real-time RT-PCR developed by the European Commission, Joint Research Centre (JRC), and through nested RT-PCR followed by Sanger sequencing. Overall, 115 samples tested positive for Omicron SARS-CoV-2 variant. The first occurrence was detected on 7 December, in Veneto, North Italy. Later on, the variant spread extremely fast in three weeks, with prevalence of positive wastewater samples rising from 1.0% (1/104 samples) in the week 5-11 December, to 17.5% (25/143 samples) in the week 12-18, to 65.9% (89/135 samples) in the week 19-25, in line with the increase in cases of infection with the Omicron variant observed during December in Italy. Similarly, the number of Regions/Autonomous Provinces in which the variant was detected increased from one in the first week, to 11 in the second, and to 17 in the last one. The presence of the Omicron variant was confirmed by the JRC real-time RT-PCR in 79.1% (91/115) of the positive samples, and by Sanger sequencing in 66% (64/97) of PCR amplicons. In conclusion, we designed an RT-qPCR assay capable to detect the Omicron variant, which can be successfully used for the purpose of wastewater-based epidemiology. We also described the history of the introduction and diffusion of the Omicron variant in the Italian population and territory, confirming the effectiveness of sewage monitoring as a powerful surveillance tool

A Pathogenic Mechanism in Huntington's Disease Involves Small CAG-Repeated RNAs with Neurotoxic Activity

Huntington's disease (HD) is an autosomal dominantly inherited disorder caused by the expansion of CAG repeats in the Huntingtin (HTT) gene. The abnormally extended polyglutamine in the HTT protein encoded by the CAG repeats has toxic effects. Here, we provide evidence to support that the mutant HTT CAG repeats interfere with cell viability at the RNA level. In human neuronal cells, expanded HTT exon-1 mRNA with CAG repeat lengths above the threshold for complete penetrance (40 or greater) induced cell death and increased levels of small CAG-repeated RNAs (sCAGs), of ≈21 nucleotides in a Dicer-dependent manner. The severity of the toxic effect of HTT mRNA and sCAG generation correlated with CAG expansion length. Small RNAs obtained from cells expressing mutant HTT and from HD human brains significantly decreased neuronal viability, in an Ago2-dependent mechanism. In both cases, the use of anti-miRs specific for sCAGs efficiently blocked the toxic effect, supporting a key role of sCAGs in HTT-mediated toxicity. Luciferase-reporter assays showed that expanded HTT silences the expression of CTG-containing genes that are down-regulated in HD. These results suggest a possible link between HD and sCAG expression with an aberrant activation of the siRNA/miRNA gene silencing machinery, which may trigger a detrimental response. The identification of the specific cellular processes affected by sCAGs may provide insights into the pathogenic mechanisms underlying HD, offering opportunities to develop new therapeutic approaches