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Background and Rationale Some CF patients are compound heterozygous or homozygous for nonsense mutations in the CFTR gene. Mutant CFTR gene coding for transcripts with premature termination codons (PTCs) is responsible for truncated CFTR protein and for a severe form of the disease. In a precision medicine framework the “REPAIRv2” (RNA Editing for Programmable A to I Replacement v2) tool, developed in the laboratory of Dr. Feng Zhang (USA), seems a good alternative to restore the full-length CFTR protein by editing its mRNA containing PTCs. This new approach is based on the possibility of targeting a deaminase enzyme (huADAR2) to a specific Adenosine, to be edited to Inosine (G analogue), on the mutant RNA by a specific guide RNA (gRNA), complementary to the target regions, and a Cas protein. Hypothesis and objectives We applied the new CRISPR/dCas13b based molecular tool of RNA editing (REPAIRv2) to correct the premature stop codon UGA, changing to UGG, in the H2bGFPopal and CFTRW1282X mRNAs with the purpose of recovering the full-length proteins.Essential Methods We designed and cloned the gRNAs needed to target the REPAIRv2 system to the Adenine to be modified. By site-directed mutagenesis we introduced a premature stop codon, W1282X, in the CFTR cDNA. Human HeLa cells expressing the H2BGFPopal mRNA, FRT cells expressing CFTRW1282X and IB3.1 airway epithelial human cells (CFTRΔ508/W12382X) were co-transfected with the plasmids coding for the recombinant protein dCAS13b/ADAR2DD, and for the gRNAs. Fluorescence microscopy was used to analyse the editing results. Results Direct fluorescence microscopy and immunofluorescence analyses detecting the corrected proteins (H2BGFP and CFTR, respectively) suggest that the REPAIRv2 system was able, in different cell lines, to edit the H2BGFPopal and the CFTRW1282X mRNA. However, the rate of editing does not seem high. Indeed, when RNA was purified from transfected cell, retro-transcribed and amplified base correction was not detectable by standard DNA sequencing and western blot. Conclusions Collectively, our results indicate that the REPAIRv2 tool is able to edit the UGA premature stop codon present in the HeLa-H2BGFPopal cells and in engineered FRTW1282X cells harbouring the UGA PTC in the CFTR mRNA. Furthermore, the REPAIRv2 tool worked in the IB3.1 cells suggesting its ability to edit endogenous UGA premature stop codon. Anyway, enhance the delivery of the plasmids as well increase/ stabilize the target mRNA to be edited, seem necessary to improve the efficiency of REPAIRv2. References 1. Cox DBT, Gootenberg JS, Abudayyeh OO, Franklin B, Kellner MJ, Joung J, Zhang F.- RNA editing with CRISPR-Cas13. Science. 2017 Nov 24; 358 (6366):1019-1027) 2. Lentini L, Melfi R, Di Leonardo A, Spinello A, Barone G, Pace A, Palumbo Piccionello A, Pibiri I. Toward a rationale for the PTC124 (Ataluren) promoted readthrough of premature stop codons: a computational approach and GFP-reporter cell-based assay. Mol Pharm. 2014 Mar 3;11(3):653-64. Acknowledgment FFC#5/2018 funded by FFC and supported by Delegazione FFC di Palermo

Lo studio di idrogel supramolecolari da utilizzare in ambito biomedico è in continua espansione. Questo è dovuto al fatto che, così come gli idrogel polimerici, mostrano eccellenti proprietà meccaniche, ma possono anche essere sottoposti reversibilmente a rigonfiamento o a transizioni gel-sol in risposta a stimoli esterni (temperatura, pH, enzimi, agenti redox).1Ad esempio, la buona risposta degli idrogel supramolecolari agli stimoli fisiologici, insieme alla loro elevata biodegradabilità e biocompatibilità, ne ha permesso l’utilizzo per la diagnosi tumorale. Solitamente, questi materiali sono facilmente assimilati dalle cellule e hanno la funzione di carrier per le molecole attive nel bioimaging. 1Alla luce di queste premesse e della spiccata attività antimicrobica precedentemente riscontrata su idrogel formati da sali di imidazolio,2 si è pensato di sintetizzare nuovi sali organici di imidazolio fluorescenti, che potessero assolvere alla duplice funzione di gelator e di agente per il bioimaging. Sono stati ottenuti idrogel fluorescenti che hanno mostrato interessanti proprietà chimico-fisiche (temperatura di transizione gel-sol, risposta a stimoli esterni, reologia, morfologia, indagini spettrofluorimetriche) e biologiche (attività antiproliferativa, bioimaging su cellule tumorali in vitro e rilascio controllato del gelator in soluzione fisiologica).

This preliminary report shows eight complexes of triorganotin(IV): Ph3SnOH and (CH3)3SnOH with four chelating imines on new synthesis. Of these ligands, two are salen-like (four coordination sites, two imidic, two phenoxidic) [1], one is a tetradentate pyrrole derivative [2] while the fourth, a vita- min B6 derivative, is pentadentate [3]. Ligands have been characterized by means of FT-IR, UV-Vis, Fluorescence, 1H- and 13C-NMR, LC-MS ESI triple quadrupole; complexes by means FT-IR, 1H- and 119Sn-NMR, LC-MS ESI, using the isotopic distribution pattern as a discriminant [4]. Geometry and nature of coordination complexes have been also evaluated using the 119Sn chemical shifts. Solid-state synthesis of the complexes (with a ball mill [5]) was also explored; such method reduces both solvent consumption and time – from 8-10 h under controlled atmosphere to about 1 h, with results identical to the wet synthesis. Antitumoral and antibacterial activities of the triorganotin (IV) complexes (BS01M, BS01P, BS02M, BS02P, BS03M, BS03P, BS04M, BS04P) were tested in vitro. In both analyses, the Shiff bases alone showed no biological activity. Antimicrobial activity was evaluated by Kirby-Bauer method against the Gram-negative Escherichia coli, and the two Gram-positive Kocuria rizophila and Staphylococcus aureus strains. All the ML2 complexes were active in inhibiting bacterial growth, with BS02P and BS03P showing the best antibacterial performance. Among the ML complexes, BS01M was not active, BS02M showed a weak antibacterial activity only against the Gram-positive bacteria, BS04M was mainly active against the Gram-negative E. coli and BS03M was active against all the tested strains. Antitumor activity was evaluated by MTT assay against cervical (HeLa), colon adenocarcinoma (HT- 29) and breast (MDA-MB231) cancer cell lines. Results showed that ML2 complexes are more active than ML ones, with HeLa cells more sensitive to treatments. These complexes (especially the ML2) showed promising results; their mechanism of action is under investigation. References 1. K. Tayade, S.K. Sahoo, S. Chopra, N. Singh, Inorganica Chimica Acta, 421, 538-543 (2014); https://doi.org/10.1016/j. ica.2014.05.014 2. S. Meghdadi, M. Amirnasr, K. Mereiter, Polyhedron, 30, 1651-1656 (2011); https://doi.org/10.1016/j.poly.2011.03.041 3. D. Sharma, S.K. Sahoo, S. Chaudhary, R. Kanta Bera, J.F. Callan, Analyst, 138, 3646-3650 (2013); https://doi.org/10.1039/ C3AN00199G 4. G. Lawson, R.H. Dahm, N. Ostah, E.D. Woodland, Applied Organometallic Chemistry, 10, 125-133 (1996); https://doi. org/10.1002/(SICI)1099-0739(199603)10:23.0.CO;2-1 5. G.A. Bowmaker, Chemical Communications, 49, 334-348 (2013); http://doi.org/10.1039/C2CC35694E 6. R. Di Stefano, M. Scopelliti, C. Pellerito, G. Casella, T. Fiore, G.C. Stocco, R. Vitturi, L. Ronconi, I.D. Sciacca, L. Pellerito, Journal of Inorganic Biochemistry, 98, 534-546 (2004); https://doi.org/10.1016/j.jinorgbio.2003.12.013 7. C. Pellerito, L. Nagy, L. Pellerito, A. Szorcsik, Journal of Organometallic Chemistry, 691, 1733-1747 (2006); https://doi. org/10.1016/j.jorganchem.2005.12.025

Cancer metastasis is the result of a series of deregulated biological phenomena, including alterations of cell-cell and cell-matrix interactions and of other microenvironmental conditions such as the oxygen tissue supply. Hypoxia is a well-known driver of aggressive cancer phenotypes, indeed tumors with poor prognosis have higher proportions of anoxic and hypoxic areas1. The consequences of tumour hypoxia can be local or even systemic towards distant organs, and it can evoke diversified responses: whereas low oxygen concentration in tissue environments. (pO2

Klebsiella oxytoca DSM 29614 (KO) is a strain that produces, under anaerobic conditions, bacterial exopolysaccharides (EPSs), made of four rhamnose (Rha), two glucuronic acids (GlcA) and one galactose (Gal) bound by α and β glycosidic bonds1,2, showing metal-binding properties3. In particular, KO in the presence of AgNO3 is able to synthesize silver nanoparticles (AgNPs) incorporated within the EPS (AgNPs-EPS). The AgNPs-EPS, may contain Ag+1 when KO growing in the presence of oxygen and Ag° under anaerobic conditions, giving a different biological activity4. In the present study were evaluated the cytotoxic effects of AgNPs-EPS, produced under aerobic and anaerobic conditions, on breast cancer cell line SK-BR3. Since cancer cells have an altered metabolism in the glycolytic direction (Warburg effect), and utilize glucose as an energy font even in the presence of O2, they are able to sense the presence of the sugar present in the EPS and to pick it with greater efficiency compared to normal cells. Currently, moreover, most of the silver nanoparticles are used as antimicrobial, antifungal, antioxidant and anti-infiammatory agents in biotechnology and bioengineering, in textile engineering and in water treatment, and compared to available cytotoxic chemotherapy, they could afford to developing a biocompatible and cost-effective method of treatment for cancer. The AgNPs-EPS treatments (5 μg/ml) caused a dose dependent behavior resulting in a conspicuous inhibition of cell proliferation rate, dramatic morphological changes with apoptotic features and general proteomic modulation and the most important effects were obtained by treatment with AgNPs-EPS biosynthesised aerobically, which has been demonstrated with voltammetric analysis which issues a greater presence of Ag+1. Proteomic analysis showed modulation of several proteins related to oxidative stress and apoptotic and mitochondrial pathways. Taken together, these results offer new important elements in support of the potential antitumoral activity of AgNPs-EPS.

BACKGROUND: The Human Epidermal Growth Factor Receptor 2 (HER-2), overexpressed in 25-30% of breast carcinomas (BC), is the therapeutic target for trastuzumab, a recombinant humanized monoclonal antibody. The initial response to trastuzumab is often followed by drug-insensitivity within one year. Several hypotheses have been raised to explain this event, but the mechanisms behind the responses to trastuzumab are still unclear. Aim: To study the effects of short and prolonged trastuzumab treatment on the proteomic profiles of HER-2-overexpressing SKBR-3 BC cells. MATERIALS AND METHODS: Cells were treated with trastuzumab to obtain sensitive and resistant clones. The drug effects were evaluated at the phenotypical and proteomic levels. RESULTS: In the trastuzumab-resistant cells the expression of a large amount of proteins, initially affected by treatment, reverted to levels of the untreated cells. CONCLUSION: The results obtained so far illustrate for the first time a large-scale differential protein expression between trastuzumab-treated and untreated cells, and between trastuzumab-sensitive and resistant cells. We believe that the results obtained will help to increase the knowledge of the molecular effects of trastuzumab and will be useful to better-understand the drug resistance mechanisms.

influences emanating from the tissue microenvironment, such as cell-cell and cell-matrix interactions and other local pathophysiologic conditions as hypoxia. However the hypoxic effect may be different according to the cell conditions. For example, the low concentration of tissue oxygen (pO2

A multi-centre study has been conducted, during 2005, by means of a questionnaire posted on the Italian Society of Emergency Medicine (SIMEU) web page. Our intention was to carry out an organisational and functional analysis of Italian Emergency Departments (ED) in order to pick out some macro-indicators of the activities performed. Participation was good, in that 69 ED (3,285,440 admissions to emergency services) responded to the questionnaire.The study was based on 18 questions: 3 regarding the personnel of the ED, 2 regarding organisational and functional aspects, 5 on the activity of the ED, 7 on triage and 1 on the assessment of the quality perceived by the users of the ED.The replies revealed that 91.30% of the ED were equipped with data-processing software, which, in 96.83% of cases, tracked the entire itinerary of the patient. About 48,000 patients/year used the ED: 76.72% were discharged and 18.31% were hospitalised. Observation Units were active in 81.16% of the ED examined. Triage programmes were in place in 92.75% of ED: in 75.81% of these, triage was performed throughout the entire itinerary of the patient; in 16.13% it was performed only symptom-based, and in 8.06% only on-call. Of the patients arriving at the ED, 24.19% were assigned a non-urgent triage code, 60.01% a urgent code, 14.30% a emergent code and 1.49% a life-threatening code. Waiting times were: 52.39 min for non-urgent patients, 40.26 min for urgent, 12.08 for emergent, and 1.19 for life-threatening patients.