Readthrough Approach Using NV Translational Readthrough-Inducing Drugs (TRIDs): A Study of the Possible Off-Target Effects on Natural Termination Codons (NTCs) on TP53 and Housekeeping Gene Expression

Nonsense mutations cause several genetic diseases such as cystic fibrosis, Duchenne muscular dystrophy, β-thalassemia, and Shwachman–Diamond syndrome. These mutations induce the formation of a premature termination codon (PTC) inside the mRNA sequence, resulting in the synthesis of truncated polypeptides. Nonsense suppression therapy mediated by translational readthrough-inducing drugs (TRIDs) is a promising approach to correct these genetic defects. TRIDs generate a ribosome miscoding of the PTC named “translational readthrough” and restore the synthesis of full-length and potentially functional proteins. The new oxadiazole-core TRIDs NV848, NV914, and NV930 (NV) showed translational readthrough activity in nonsense-related in vitro systems. In this work, the possible off-target effect of NV molecules on natural termination codons (NTCs) was investigated. Two different in vitro approaches were used to assess if the NV molecule treatment induces NTC readthrough: (1) a study of the translational-induced p53 molecular weight and functionality; (2) the evaluation of two housekeeping proteins’ (Cys-C and β2M) molecular weights. Our results showed that the treatment with NV848, NV914, or NV930 did not induce any translation alterations in both experimental systems. The data suggested that NV molecules have a specific action for the PTCs and an undetectable effect on the NTCs

Heart-Kidney Biomarkers in Patients Undergoing Cardiac Stress Testing

We examined association of inducible myocardial perfusion defects with cardiorenal biomarkers, and of diminished left ventricular ejection fraction (LVEF) with kidney injury marker plasma neutrophil gelatinase-associated lipocalin (NGAL). Patients undergoing nuclear myocardial perfusion stress imaging were divided into 2 groups. Biomarkers were analyzed pre- and poststress testing. Compared to the patients in the low ischemia group (n = 16), the patients in the high ischemia group (n = 18) demonstrated a significantly greater rise in cardiac biomarkers plasma BNP, NT-proBNP and cTnI. Subjects were also categorized based on pre- or poststress test detectable plasma NGAL. With stress, the group with no detectable NGAL had a segmental defect score 4.2 compared to 8.2 (P = .06) in the detectable NGAL group, and 0.9 vs. 3.8 (P = .03) at rest. BNP rose with stress to a greater degree in patients with detectable NGAL (10.2 vs. 3.5 pg/mL, P = .03). LVEF at rest and with stress was significantly lower in the detectable NGAL group; 55.8 versus 65.0 (P = .03) and 55.1 vs. 63.8 (P = .04), respectively. Myocardial perfusion defects associate with biomarkers of cardiac stress, and detectable plasma NGAL with significantly lower LVEF, suggesting a specific heart-kidney link

Inhibition of LPAR6 overcomes sorafenib resistance by switching glycolysis into oxidative phosphorylation in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most threatening tumours in the world today. Pharmacological treatments for HCC mainly rely on protein kinase inhibitors, such as sorafenib and regorafenib. Even so, these approaches exhibit side effects and acquired drug resistance, which is an obstacle to HCC treatment. We have previously shown that selective lysophosphatidic acid receptor 6 (LPAR6) chemical antagonists inhibit HCC growth. Here, we investigated whether LPAR6 mediates resistance to sorafenib by affecting energy metabolism in HCC. To uncover the role of LPAR6 in drug resistance and cancer energy metabolism, we used a gain-of-function and loss-of-function approach in 2D tissue and 3D spheroids. LPAR6 was ectopically expressed in HLE cells (HLE-LPAR6) and knocked down in HepG2 (HepG2 LPAR6-shRNA). Measurements of oxygen consumption and lactate and pyruvate production were performed to assess the energy metabolism response of HCC cells to sorafenib treatment. We found that LPAR6 mediates the resistance of HCC cells to sorafenib by promoting lactic acid fermentation at the expense of oxidative phosphorylation (OXPHOS) and that the selective LPAR6 antagonist 9-xanthenyl acetate (XAA) can effectively overcome this resistance. Our study shows for the first time that an LPAR6-mediated metabolic mechanism supports sorafenib resistance in HCC and proposes a pharmacological approach to overcome it

Novel lysophosphatidic acid receptor 6 antagonists inhibit hepatocellular carcinoma growth through affecting mitochondrial function

Hepatocellular carcinoma (HCC) is one of the most prevalent cancers worldwide and the commonest liver cancer. It is expected to become the third leading cause of cancer-related deaths in Western countries by 2030. Effective pharmacological approaches for HCC are still unavailable, and the currently approved systemic treatments are unsatisfactory in terms of therapeutic results, showing many side effects. Thus, searching for new effective and nontoxic molecules for HCC treatment is of paramount importance. We previously demonstrated that lysophosphatidic acid (LPA) is an important contributor to the pathogenesis of HCC and that lysophosphatidic acid receptor 6 (LPAR6) actively supports HCC tumorigenicity. Here, we screened for novel LPAR6 antagonists and found that two compounds, 4-methylene-2-octyl-5-oxotetra-hydrofuran-3-carboxylic acid (C75) and 9-xanthenylacetic acid (XAA), efficiently inhibit HCC growth, both in vitro and in vitro, without displaying toxic effects at the effective doses.We further investigated the mechanisms of action of C75 and XAA and found that these compounds determine a G1-phase cell cycle arrest, without inducing apoptosis at the effective doses. Moreover, we discovered that bothmolecules act on mitochondrial homeostasis, by increasing mitochondrial biogenesis and reducing mitochondrial membrane potential. Overall, our results show two newly identified LPAR6 antagonists with a concrete potential to be translated into effective and side effect–free molecules for HCC therapy

The Role of prEoperative ultraSonography on patenCy of native arteriovenoUs accEss: RESCUE Study (in press)

The selection of an appropriate vascular access in patients with chronic kidney disease (CKD) requiring renal replacement therapy depends on various factors including vessels anatomy, variable cardiovascular risk, and overall life expectancy (1,2). The native arteriovenous fistula (AVF) often represents the preferred vascular access for CKD patients undergoing chronic hemodialysis (HD) treatment as it portends a lower incidence of complications, a longer duration, and a higher patient survival rate as compared to central venous catheters and prosthetic arteriovenous fistula (2,3). Doppler Ultrasound (DUS) is a valuable tool for the pre-operative vascular mapping and for selecting the most suitable vessels for AVF creation. Additionally, it enables early diagnosis and timely treatment of complications during AVF surveillance (4). By utilizing DUS, the number of patients eligible for AVF creation and the survival rate of AVF may improve. In the pre-operative assessment of the vessels, physical examination alone is insufficient in 25-50% of cases, whereas DUS allows a more accurate evaluation of several parameters (5). However, the literature on the use of DUS in AVF, from planning to maintenance, presents conflicting opinions and remains controversial. The 2007 European Best Practice Guidelines (EBPG) (3) suggest a clinical and ultrasound evaluation for AVF creation in all candidates. In fact, the use of ultrasound in the pre-operative phase increased the success rate of AVF, while a fistula created without ultrasound assessment is associated with three times higher risk of failure. Accordingly, in a randomised study, the primary failure rate was 25% in individuals undergoing pre-operative assessment by physical examination alone vs. 6% in those also examined by ultrasounds (6). Accordingly, in a recent study, Malovrh et al. confirmed that integrating DUS in pre-operative mapping allows an optimal planning of AVF surgery (7). On the other hand, more recently, the 2019 KDOQI guidelines (2) indicate a mandatory implementation of DUS only in patients at high risk of AVF failure, with a previous history of central venous stenosis or when the physical examination provides limited information. However, this recommendation was based on a low level of evidence. Although the Working Group recognised the strong potential of ultrasound in vascular mapping, to date, the available data were considered insufficient to recommend a wider use. In line with these observations, a recent systematic review concluded that pre-operative imaging may not increase the success rate of AVF (8) Currently, there is no common agreement on the predictive factors for success or failure of AVF. Although the relationship of vessel diameter with AVF outcome has been analysed in several studies, the ideal minimum arterial and venous diameter remain undefined. Furthermore, additional factors such as distensibility and blood flow may impart complementary information on the quality of vessels (5). With this background in mind, we planned an observational, longitudinal, multicenter cohort study, involving various Italian Nephrology and Dialysis Units. The primary aim will be to identify ultrasound parameters that may predict successful AVF maturation in patients with end stage kidney disease (ESKD) undergoing elective AVF creation. As secondary objectives we will evaluate the effectiveness of DUS in increasing the number of ESKD patients eligible for AVF. Primary and secondary endpoints will include the rate of early primary failure; as well as AVF complications such as stenosis, thrombosis, infection, and reintervention on vascular access, respectively. We will enrol patients aged 18 years or older with CKD stage 5 ND or already undergoing chronic haemodialysis by central venous catheter. Exclusion criteria will be a previous AVF creation on the same arm and a reduced life expectancy (< 6 months). On the basis of previous evidence (6,9), we estimated to need at least 113 patients recruited to answer the opening research question, taking into account a predicted percentage of 8% for AVF failure in patients undergoing preoperative evaluation with DUS, with a 95% level of confidence and a margin of error of 5%. Patients will undergo six visits by a nephrologist with acknowledged expertise in ultrasound assessment of vascular access. The first visit will take place at least one day before AVF creation, while the following will be performed within the first 24 hours from surgery and at day 7, 30, 60, and 6 months after AVF creation. During the first visit, demographic, anthropometric and clinical data will be recorded, including underlying nephropathy, comorbidities, therapy, age, sex, ethnicity weight, height, body mass index, blood pressure, heart rate and routine laboratory tests including serum urea, serum creatinine, serum sodium, serum potassium, serum calcium, serum phosphorus, parathormone, 25-OH vitamin D, serum haemoglobin, hematocrit, platelets, serum leukocytes, neutrophils, lymphocytes, monocytes, serum albumin, serum total proteins, ferritin, PCR, PT, aPTT, fasting glycaemia, fasting insulin, homocysteine, total cholesterol, HDL cholesterol, LDL cholesterol, triglycerides, serum uric acid. Clinical characteristics, vascular access complications and dialysis parameters will be updated during the following visits. In addition, self-report questionnaires, such as Injection Phobia Scale-Anxiety (IPS-ANX), Multidimensional Scale of Perceived Social Support (MSPSS), Generalized Anxiety Disorder scale-7 (GAD7), Patient Activation Measure 13 (PAM13), Edmonton Symptom Assessment System revised (ESASr), Patient Health Questionnaire-9 (PHQ9), Patient Health Questionnaire-15 (PHQ15) will be filled out during the first, fourth and last visit. At each visit, a vessel ultrasound evaluation of the upper limb will be performed in B-Mode for morphological study and in Doppler Mode for flowmetric study. Prior to AVF creation, two blinded operators will independently evaluate its feasibility based on physical examination or DUS results. The following ultrasound parameters will be measured: vessel diameter, venous depth, venous distensibility, arterial wall thickness, arterial flow rate, radial artery resistance index before and after reactive hyperaemia test. An Allen test will also be performed, and the presence and severity of vascular calcifications will be assessed. After AVF creation, the ultrasound evaluation will include arterial, venous, and anastomosis diameter of AVF, venous depth and AVF flow rate. Statistical analysis will be carried out with SPSS 21.0. Univariate and multivariate analyses by logistic regression models will be performed to determine possible associations of the variables considered with the primary and secondary outcomes. A p-value of less than 0.05 will be considered statistically significant. This study will address a conflicting issue regarding the use of ultrasonography in creating native arteriovenous access and provide further evidence on predictors of AVF failure

An observational, prospective, open-label, multicentre evaluation of aliskiren in treated, uncontrolled patients: a real-life, long-term, follow-up, clinical practice in Italy.

Introduction: The addition of the direct renin inhibitor aliskiren is demonstrated to improve blood pressure (BP) control rate and reduce progression of organ damage in treated hypertensive patients in clinical trials with a relatively short follow-up period. Aim: The objective of this study was to assess the effectiveness, safety and tolerability of aliskiren as an addon antihypertensive therapy in high-risk, treated, hypertensive patients, who were not controlled with concomitant treatment with at least two antihypertensive drugs under 'real-life' conditions, during a planned observation and treatment period of at least 12 months in Italy. Methods: Clinical data were derived from medical databases of treated, uncontrolled, hypertensive patients followed by specialized physicians operating in different clinical settings (hospital divisions or outpatient clinics) in Italy. Aliskiren was added to stable antihypertensive treatment, including at least two drug classes (independently of class or dosage) and unable to achieve BP control. Follow-up visits for measuring clinic BP levels and collecting data on drug safety and tolerability were planned at time intervals of 1, 6 and 12 months. At each predefined follow-up visit, aliskiren could be up-titrated from 150 to 300 mg daily if BP control was not achieved. Results: From May 2009 to June 2011, a total of 1186 treated, uncontrolled, hypertensive patients (46.3% female, aged 65.2 ± 11.7 years, mean duration of hypertension 13.2 ± 9.3 years, mean clinic BP levels 156.5 ± 15.9/90.3 ± 9.5mmHg) were enrolled. Systolic and diastolic BP levels were 141.1/82.4, 134.9/79.8 and 133.6/78.9 mmHg at 1-, 6- and 12-month follow-up visits, respectively (p < 0.0001 vs baseline for all comparisons). These effects were consistent in all predefined subgroups, including those with left ventricular hypertrophy, renal disease, diabetes mellitus, coronary artery disease or cerebrovascular disease. Reduced levels of microalbuminuria were also reported, without affecting other renal and electrolyte parameters. Overall, compliance to study medication was high (93.0%), with a very low proportion of patients experiencing adverse events leading to drug discontinuation (3.6%). Conclusions: In this observational, prospective, open-label, multicentre study, we reported the 12-month clinical effectiveness, safety and tolerability of adding aliskiren to treated, uncontrolled, hypertensive patients in a 'real-life' setting in Italy. This strategy leads to a significantly improved BP control rate and low incidence of drug-related side effects or discontinuations. © 2012 Springer International Publishing AG. All rights reserved

2020 Dataset on local gambling regulations in Italy

The dataset provides the complete enumeration of gambling policies implemented by Italian Municipalities between 2003 and 2021. The dataset comprises information on all municipalities existing in 2017 and following years (thus considering also merging). The following variables are available: Municipality ISTAT Code (ID), Municipality Name (Name) Province, Region, Researcher, and a series of variables identifying the number and the type of rulings adopted ('regolamento', 'ordinanza' and 'delibera'). The rulings are distinguished between identified and downloaded or only identified (because the document is no longer available). Additional variables describe municipal activism with other administrative acts (Anti-gambling Manifesto, events, projects or tax reductions). Overall, the dataset comprises 8031 units

Adipose stem cell niche reprograms the colorectal cancer stem cell metastatic machinery

Obesity is a strong risk factor for cancer progression, posing obesity-related cancer as one of the leading causes of death. Nevertheless, the molecular mechanisms that endow cancer cells with metastatic properties in patients affected by obesity remain unexplored. Here, we show that IL-6 and HGF, secreted by tumor neighboring visceral adipose stromal cells (V-ASCs), expand the metastatic colorectal (CR) cancer cell compartment (CD44v6 +), which in turn secretes neurotrophins such as NGF and NT-3, and recruits adipose stem cells within tumor mass. Visceral adipose-derived factors promote vasculogenesis and the onset of metastatic dissemination by activation of STAT3, which inhibits miR-200a and enhances ZEB2 expression, effectively reprogramming CRC cells into a highly metastatic phenotype. Notably, obesity-associated tumor microenvironment provokes a transition in the transcriptomic expression profile of cells derived from the epithelial consensus molecular subtype (CMS2) CRC patients towards a mesenchymal subtype (CMS4). STAT3 pathway inhibition reduces ZEB2 expression and abrogates the metastatic growth sustained by adipose-released proteins. Together, our data suggest that targeting adipose factors in colorectal cancer patients with obesity may represent a therapeutic strategy for preventing metastatic disease

2020 Dataset on local gambling regulations in Italy

The dataset provides the complete enumeration of gambling policies implemented by Italian Municipalities between 2003 and 2021. The dataset comprises information on all municipalities existing in 2017 and following years (thus considering also merging). The following variables are available: Municipality ISTAT Code (ID), Municipality Name (Name) Province, Region, Researcher, and a series of variables identifying the number and the type of rulings adopted (“regolamento”, “ordinanza” and “delibera”). The rulings are distinguished between identified and downloaded or only identified (because the document is no longer available). Additional variables describe municipal activism with other administrative acts (Anti-gambling Manifesto, events, projects or tax reductions). Overall, the dataset comprises 8031 units