Development of an in-vivo high throughput assay to monitor biofilm development of the pathogenic microorganism Pseudomonas aeruginosa, in C. elegans.

Bacterial biofilms are clusters of bacteria attached to host tissues and abiotic surfaces, such as medical implants, imbedded in a self-generated matrix; this is characterised by extracellular polysaccharides, proteins and eDNA, providing nutrients and protection to the bacteria, which become resistant to both the host’s immune system and antimicrobials, therefore becoming extremely difficult to treat and eradicate. About 80% of recurring chronic microbial diseases in humans are caused by bacterial biofilms, recognition of which has led to an increased attention on identifying new treatments. Particularly, biofilms are difficult to detect in clinical settings, due to lack of biofilm-specific biomarkers. Therefore, further studies are essential to identify markers unique to bacterial biofilms. In this study, we mainly focused on Quorum Sensing (QS) signalling, a well-known system of communication found in many microbial species, involved in cell density regulation for biofilm formation. Using the biofilm- former Pseudomonas aeruginosa (P. aeruginosa), we targeted QS-related genes, to build a biofilm-specific reporter, to use for in-vivo studies in the nematode Caenorhabditis elegans (C. elegans). Particularly, we aimed to create a high-throughput C. elegans in-vivo biofilm assay for industrial research and to provide a cost and time-effective protocol for it. C. elegans is extremely advantageous to address many research questions, particularly due to its transparency, short generation time and low maintenance costs; moreover, it shares more than 80% of human genes and it is colonised by pathogenic bacteria, such as P. aeruginosa. Our results show that P. aeruginosa transgenic fluorescent reporters can be employed in a C. elegans biofilm infection model, which can provide a non-invasive host’s health readout and visualisation of bacterial tissue colonisation; detection of fluorescence signal by biofilm-competent bacteria, as opposed to lack of signal by mutant biofilm-incompetent bacteria, also suggested that C. elegans could be used to monitor biofilm progression in the animal. Further studies will be needed to complement this research, to prove evidence of biofilm detection and to address standardisation of this assay for a possible industrial use; overall, this was a first step which holds promise in the attempt to bridge the gap between in-vivo clinical diagnostics and in-vitro biofilm research

Transcriptomic and genomic studies classify NKL54 as a histone deacetylase inhibitor with indirect influence on MEF2-dependent transcription

In leiomyosarcoma class IIa HDACs (histone deacetylases) bind MEF2 and convert these transcription factors into repressors to sustain proliferation. Disruption of this complex with small molecules should antagonize cancer growth. NKL54, a PAOA (pimeloylanilide o-aminoanilide) derivative, binds a hydrophobic groove of MEF2, which is used as a docking site by class IIa HDACs. However, NKL54 could also act as HDAC inhibitor (HDACI). Therefore, it is unclear which activity is predominant. Here, we show that NKL54 and similar derivatives are unable to release MEF2 from binding to class IIa HDACs. Comparative transcriptomic analysis classifies these molecules as HDACIs strongly related to SAHA/vorinostat. Low expressed genes are upregulated by HDACIs, while abundant genes are repressed. This transcriptional resetting correlates with a reorganization of H3K27 acetylation around the transcription start site (TSS). Among the upregulated genes there are several BH3-only family members, thus explaining the induction of apoptosis. Moreover, NKL54 triggers the upregulation of MEF2 and the downregulation of class IIa HDACs. NKL54 also increases the binding of MEF2D to promoters of genes that are upregulated after treatment. In summary, although NKL54 cannot outcompete MEF2 from binding to class IIa HDACs, it supports MEF2-dependent transcription through several actions, including potentiation of chromatin binding

Commercial Immunoglobulin Products Contain Neutralizing Antibodies Against Severe Acute Respiratory Syndrome Coronavirus 2 Spike Protein

BACKGROUND: Patients with antibody deficiency respond poorly to COVID-19 vaccination and are at risk of severe or prolonged infection. They are given long-term immunoglobulin replacement therapy (IRT) prepared from healthy donor plasma to confer passive immunity against infection. Following widespread COVID-19 vaccination alongside natural exposure, we hypothesised that immunoglobulin preparations will now contain neutralising SARS-CoV-2 spike antibodies which confer protection against COVID-19 disease and may help to treat chronic infection. METHODS: We evaluated anti-SARS-CoV-2 spike antibody in a cohort of patients before and after immunoglobulin infusion. Neutralising capacity of patient samples and immunoglobulin products was assessed using in vitro pseudo-virus and live-virus neutralisation assays, the latter investigating multiple batches against current circulating omicron variants. We describe the clinical course of nine patients started on IRT during treatment of COVID-19. RESULTS: In 35 individuals with antibody deficiency established on IRT, median anti-spike antibody titre increased from 2123 to 10600 U/ml post-infusion, with corresponding increase in pseudo-virus neutralisation titres to levels comparable to healthy donors. Testing immunoglobulin products directly in the live-virus assay confirmed neutralisation, including of BQ1.1 and XBB variants, but with variation between immunoglobulin products and batches.Initiation of IRT alongside Remdesivir in patients with antibody deficiency and prolonged COVID-19 infection (median 189 days, maximum over 900 days with an ancestral viral strain) resulted in clearance of SARS-CoV-2 virus at a median of 20 days. CONCLUSIONS: Immunoglobulin preparations now contain neutralising anti-SARS-CoV-2 antibodies which are transmitted to patients and help to treat COVID-19 in individuals with failure of humoral immunity

Neutralising immunity to omicron sublineages BQ.1.1, XBB, and XBB.1.5 in healthy adults is boosted by bivalent BA.1-containing mRNA vaccination and previous Omicron infection

The global COVID-19 landscape is increasingly complex; emerging new variants rapidly cause waves of infection in people with variably induced immunity. Most individuals now have so-called hybrid immunity from both infection and vaccination. However, sequential infecting variants, induction of immunity, and subsequent waning are interlinked, and immune protection against new variants is unclear

Teaching and learning computational drug design: student investigations of 3D quantitative structure–activity relationships through web applications

The increasing use of information technology in the discovery of new molecular entities encourages the use of modern molecular-modeling tools to help teach important concepts of drug design to chemistry and pharmacy undergraduate students. In particular, statistical models such as quantitative structure activity relationships (QSAR)—often as its 3-D QSAR variant—are commonly used in the development and optimization of a leading compound. We describe how these drug discovery methods can be taught and learned by means of free and open-source web applications, specifically the online platform www.3d-qsar.com. This new suite of web application has been integrated into a drug design teaching course, one that provides both theoretical and practical perspectives. We include the teaching protocol by which pharmaceutical biotechnology master students at Pharmacy Faculty of Sapienza Rome University are introduced to drug design. Starting with a choice among recent articles describing the potencies of a series of molecules tested against a biological target, each student is expected to build a 3-D QSAR ligand-based model from their chosen publication, proceeding as follows: creating the initial data set (Py-MolEdit); generating the global minimum conformations (Py-ConfSearch); proposing a promising mutual alignment (Py-Align); and, finally, building and optimizing a robust 3-D QSAR models (Py-CoMFA). These student activities also help validate these new molecular modeling tools, especially for their usability by inexperienced hands. To more fully demonstrate the effectiveness of this protocol and its tools, we include the work performed by four of these students (four of the co-authors), detailing the satisfactory 3-D QSAR models they obtained. Such scientifically complete experiences by undergraduates, made possible by the efficiency of the 3-D QSAR methodology, provide exposure to computational tools in the same spirit as traditional laboratory exercises. With the obsolescence of the classic Comparative Molecular Field Analysis Sybyl host, the 3dqsar web portal offers one of the few available means of performing this well-established 3-D QSAR method

Anticancer Drugs: Recent Strategies to Improve Stability Profile, Pharmacokinetic and Pharmacodynamic Properties

In past decades, anticancer research has led to remarkable results despite many of the approved drugs still being characterized by high systemic toxicity mainly due to the lack of tumor selectivity and present pharmacokinetic drawbacks, including low water solubility, that negatively affect the drug circulation time and bioavailability. The stability studies, performed in mild conditions during their development or under stressing exposure to high temperature, hydrolytic medium or light source, have demonstrated the sensitivity of anticancer drugs to many parameters. For this reason, the formation of degradation products is assessed both in pharmaceutical formulations and in the environment as hospital waste. To date, numerous formulations have been developed for achieving tissue-specific drug targeting and reducing toxic side effects, as well as for improving drug stability. The development of prodrugs represents a promising strategy in targeted cancer therapy for improving the selectivity, efficacy and stability of active compounds. Recent studies show that the incorporation of anticancer drugs into vesicular systems, such as polymeric micelles or cyclodextrins, or the use of nanocarriers containing chemotherapeutics that conjugate to monoclonal antibodies can improve solubility, pharmacokinetics, cellular absorption and stability. In this study, we summarize the latest advances in knowledge regarding the development of effective highly stable anticancer drugs formulated as stable prodrugs or entrapped in nanosystems

Multivariate Curve Resolution Methodology Applied to the ATR-FTIR Data for Adulteration Assessment of Virgin Coconut Oil

Virgin coconut oil (VCO) is a functional food with important health benefits. Its economic interest encourages fraudsters to deliberately adulterate VCO with cheap and low-quality vegetable oils for financial gain, causing health and safety problems for consumers. In this context, there is an urgent need for rapid, accurate, and precise analytical techniques to detect VCO adulteration. In this study, the use of Fourier transform infrared (FTIR) spectroscopy combined with multivariate curve resolution–alternating least squares (MCR-ALS) methodology was evaluated to verify the purity or adulteration of VCO with reference to low-cost commercial oils such as sunflower (SO), maize (MO) and peanut (PO) oils. A two-step analytical procedure was developed, where an initial control chart approach was designed to assess the purity of oil samples using the MCR-ALS score values calculated on a data set of pure and adulterated oils. The pre-treatment of the spectral data by derivatization with the Savitzky–Golay algorithm allowed to obtain the classification limits able to distinguish the pure samples with 100% of correct classifications in the external validation. In the next step, three calibration models were developed using MCR-ALS with correlation constraints for analysis of adulterated coconut oil samples in order to assess the blend composition. Different data pre-treatment strategies were tested to best extract the information contained in the sample fingerprints. The best results were achieved by derivative and standard normal variate procedures obtaining RMSEP and RE% values in the ranges of 1.79–2.66 and 6.48–8.35%, respectively. The models were optimized using a genetic algorithm (GA) to select the most important variables and the final models in the external validations gave satisfactory results in quantifying adulterants, with absolute errors and RMSEP of less than 4.6% and 1.470, respectively

Bcl-2 family inhibitors sensitize human cancer models to therapy

Abstract BH3 mimetics, targeting the Bcl-2 family anti-apoptotic proteins, represent a promising therapeutic opportunity in cancers. ABT-199, the first specific Bcl-2 inhibitor, was approved by FDA for the treatment of several hematological malignancies. We have recently discovered IS21, a novel pan BH3 mimetic with preclinical antitumor activity in several tumor types. Here, we evaluated the efficacy of IS21 and other BH3 mimetics, both as single agents and combined with the currently used antineoplastic agents in T-cell acute lymphoblastic leukemia, ovarian cancer, and melanoma. IS21 was found to be active in T-cell acute lymphoblastic leukemia, melanoma, lung, pancreatic, and ovarian cancer cell lines. Bcl-xL and Mcl-1 protein levels predicted IS21 sensitivity in melanoma and ovarian cancer, respectively. Exploring IS21 mechanism of action, we found that IS21 activity depends on the presence of BAX and BAK proteins: complexes between Bcl-2 and Bcl-xL proteins and their main binding partners were reduced after IS21 treatment. In combination experiments, BH3 mimetics sensitized leukemia cells to chemotherapy, ovarian cancer cells and melanoma models to PARP and MAPK inhibitors, respectively. We showed that this enhancing effect was related to the potentiation of the apoptotic pathway, both in hematologic and solid tumors. In conclusion, our data suggest the use of inhibitors of anti-apoptotic proteins as a therapeutic strategy to enhance the efficacy of anticancer treatment

Prognostic impact of diabetes and prediabetes on survival outcomes in patients with chronic heart failure: A post-hoc analysis of the GISSI-HF (Gruppo Italiano per lo Studio della Sopravvivenza nella Insufficienza Cardiaca-Heart Failure) trial

826BACKGROUND: The independent prognostic impact of diabetes mellitus (DM) and prediabetes mellitus (pre-DM) on survival outcomes in patients with chronic heart failure has been investigated in observational registries and randomized, clinical trials, but the results have been often inconclusive or conflicting. We examined the independent prognostic impact of DM and pre-DM on survival outcomes in the GISSI-HF (Gruppo Italiano per lo Studio della Sopravvivenza nella Insufficienza Cardiaca-Heart Failure) trial. METHODS AND RESULTS: We assessed the risk of all-cause death and the composite of all-cause death or cardiovascular hospitalization over a median follow-up period of 3.9 years among the 6935 chronic heart failure participants of the GISSI-HF trial, who were stratified by presence of DM (n=2852), pre-DM (n=2013), and non-DM (n=2070) at baseline. Compared with non-DM patients, those with DM had remarkably higher incidence rates of all-cause death (34.5% versus 24.6%) and the composite end point (63.6% versus 54.7%). Conversely, both event rates were similar between non-DM patients and those with pre-DM. Cox regression analysis showed that DM, but not pre-DM, was associated with an increased risk of all-cause death (adjusted hazard ratio, 1.43; 95% CI, 1.28-1.60) and of the composite end point (adjusted hazard ratio, 1.23; 95% CI, 1.13-1.32), independently of established risk factors. In the DM subgroup, higher hemoglobin A1c was also independently associated with increased risk of both study outcomes (all-cause death: adjusted hazard ratio, 1.21; 95% CI, 1.02-1.43; and composite end point: adjusted hazard ratio, 1.14; 95% CI, 1.01-1.29, respectively). CONCLUSIONS: Presence of DM was independently associated with poor long-term survival outcomes in patients with chronic heart failure.openopenDauriz, Marco; Targher, Giovanni; Temporelli, Pier Luigi; Lucci, Donata; Gonzini, Lucio; Nicolosi, Gian Luigi; Marchioli, Roberto; Tognoni, Gianni; Latini, Roberto; Cosmi, Franco; Tavazzi, Luigi; Maggioni, Aldo Pietro*; Moccetti, T.; Rossi, M.G.; Pasotti, E.; Vaghi, F.; Roncarolo, P.; Zunino, M.T.; Matta, F.; Actis Perinetto, E.; Gaita, F.; Azzaro, G.; Zanetta, M.; Paino, A.M.; Parravicini, U.; Vegis, D.; Conte, R.; Ferraro, P.; De Bernardi, A.; Morelloni, S.; Fagnani, M.; Greco Lucchina, P.; Montagna, L.; Bellone, E.; Sappè, D.; Ferraro, F.; Delucchi, M.; Reynaud, S.G.; Dore, M.; La Brocca, A.; Massobrio, N.; Bo, L.; Trinchero, R.; Imazio, M.; Brocchi, G.; Nejrotti, A.; Rissone, L.; Gabasio, S.; Zocchi, C.; Randazzo, S.; Crenna, A.; Giannuzzi, P.; Bonanomi, E.; Mezzani, A.; De Marchi, M.; Begliuomini, G.; Gianonatti, C.A.; Gavazzi, A.; Grosu, A.; Dei Cas, L.; Nodari, S.; Garyfallidis, P.; Bertoletti, A.; Bonifazi, C.; Arisi, S.; Mascaro, F.; Fraccarollo, M.; Dell'Orto, S.; Sfolcini, M.; Bortolini, F.; Raccagni, D.; Turelli, A.; Santarone, M.; Miglierina, E.; Sormani, L.; Jemoli, R.; Tettamanti, F.; Pirelli, S.; Bianchi, C.; Verde, S.; Mariani, M.; Ziacchi, V.; Ferrazza, A.; Russo, A.; Bortolotti, M.; Pasini, G.F.; Volpi, A.; Jones, K.N.; Cuzzucrea, D.; Gullace, G.; Carbone, C.; Granata, A.; De Servi, S.; Del Rosso, G.; Inserra, C.; Renaldini, E.; Zappa, C.; Moretti, M.; Zanini, R.; Ferrari, M.; Moroni, E.; Cei, A.; Lissi, C.; Dovico, E.; Fiorentini, C.; Palermo, P.; Brusoni, B.; Negrini, M.; Heyman, J.; Danzi, G.B.; Finzi, A.; Frigerio, M.; Turazza, F.; Beretta, L.; Sachero, A.; Casazza, F.; Squadroni, L.; Lombardi, F.; Marano, L.; Margonato, A.; Fragasso, G.; Febo, O.C.; Aiolfi, E.; Olmetti, F.; Grieco, A.; Antonazzo, V.; Specchia, G.; Mortara, A.; Robustelli, F.; Songini, M.G.; Schweiger, C.; Frisinghelli, A.; Palvarini, M.; Campana, C.; Scelsi, L.; Ajmone Marsan, N.; Cobelli, F.; Gualco, A.; Opasich, C.; De Feo, S.; Mazzucco, R.; Iannone, M.A.; Diaco, T.; Zaniboni, D.; Milanesi, G.; Nassiacos, D.; Meloni, S.; Giani, P.; Nicoli, T.; Malinverni, C.; Gusmini, A.; Pozzoni, L.; Bisiani, G.; Margaroli, P.; Schizzarotto, A.; Daverio, A.; Occhi, G.; Partesana, N.; Bandini, P.; Rosella, M.G.; Giustiniani, S.; Cucchi, G.; Pedretti, R.; Raimondo, R.; Vaninetti, R.; Fedele, A.; Ghezzi, I.; Rezzonico, E.; Salerno Uriarte, J.A.; Morandi, F.; Salvucci, F.; Valenti, C.; Graziano, G.; Romanò, M.; Cimminiello, C.; Mangone, I.; Lombardo, M.; Quorso, P.; Marinoni, G.; Breghi, M.; Erckert, M.; Dienstl, A.; Mirante Marini, G.; Stefenelli, C.; Cioffi, G.; Buczkowska, E.; Bonanome, A.; Bazzanini, F.; Parissenti, L.; Serafini, C.; Catania, G.; Tarantini, L.; Rigatelli, G.; Boni, S.; Pasini, A.; Masini, E.; Zampiero, A.A.; Zanchetta, M.; Franceschetto, L.; Delise, P.; Marcon, C.; Sacchetta, A.; Borgese, L.; Artusi, L.; Casolino, P.; Corbara, F.; Banzato, A.; Barbiero, M.; Aldegheri, M.P.; Bazzucco, R.; Crivellenti, G.; Raviele, A.; Zanella, C.; Pascotto, P.; Sarto, P.; Milan, S.; Barbieri, E.; Girardi, P.; Dalla Villa, W.; Dalle Mule, J.; Di Sipio, M.L.; Cazzin, R.; Milan, D.; Zonzin, P.; Carraro, M.; Rossi, R.; Carbonieri, E.; Rossi, I.; Stritoni, P.; Meneghetti, P.; Risica, G.; Tenderini, P.L.; Vassanelli, C.; Zanolla, L.; Perini, G.; Brighetti, G.; Chiozza, R.; Giuliano, G.; Baldin, M.G.; Gortan, R.; Cesanelli, R.; Nicolosi, G.L.; Piazza, R.; Mos, L.; Vriz, O.; Pavan, D.; Pascottini, G.; Alberti, E.; Werren, M.; Solinas, L.; Sinagra, G.; Longaro, F.; Fioretti, P.; Albanese, M.C.; Miani, D.; Gianrossi, R.; Pende, A.; Rubartelli, P.; Magaia, O.; Domenicucci, S.; Caruso, D.; Faraguti, A.S.; Magliani, L.; Miccoli, F.; Guglielmino, G.; Bertoli, D.; Cantarelli, A.; Orlandi, S.; Vallebona, A.; Pozzati, A.; Brega, G.; Pancaldi, L.G.; Vandelli, R.; Urbinati, S.; Poci, M.G.; Zoli, M.; Costa, G.M.; Guiducci, U.; Zobbi, G.; Tartagni, F.; Tisselli, A.; Gentili, A.; Pieri, P.; Cagnetta, E.; Bendinelli, S.; Barbieri, A.; Conti, R.; Ferrari, R.; Merlini, F.; Fucili, A.; Moruzzi, P.; Buia, E.; Galvani, M.; Ferrini, D.; Baggioni, G.; Yiannacopulu, P.; Canè, G.; Bonfiglioli, A.; Zandomeneghi, R.; Brugioni, L.; Giannini, A.; Di Ruvo, R.; Giuliani, M.; Rusconi, L.; Del Corso, P.; Piovaccari, G.; Bologna, F.; Venturi, P.; Melandri, F.; Bagni, E.; Bolognese, L.; Perticucci, R.; Zuppiroli, A.; Nannini, M.; Consoli, N.; Petrone, P.; Pipitò, C.; Colombi, L.; Bernardi, D.; Mariani, P.R.; Testa, R.; Mazzinghi, F.; Cosmi, F.; Cosmi, D.; Zipoli, A.; Cecchi, A.; Castelli, G.; Ciaccheri, M.; Mori, F.; Pieri, F.; Valoti, P.; Chiarantini, D.; Santoro, G.M.; Minneci, C.; Marchi, F.; Milli, M.; Zambaldi, G.; Zipoli, A.; Brandinelli Geri, A.A.; Cipriani, M.; Alessandri, M.; Severi, S.; Stefanelli, S.; Comella, A.; Poddighe, R.; Digiorgio, A.; Carluccio, M.; Berti, S.; Rizza, A.; Bonatti, V.; Molendi, V.; Brancato, A.; D'Aprile, N.; Giappichini, G.; Del Vecchio, S.; Mantini, G.; De Tommasi, F.; Meucci, G.; Cordoni, M.; Bechi, S.; Barsotti, L.; Baldini, P.; Romei, M.; Scopelliti, G.; Lauri, G.; Pestelli, F.; Furiozzi, F.; Cocchieri, M.; Severini, D.; Patriarchi, F.; Chiocchi, P.; Buccolieri, M.; Martinelli, S.; Wee, A.; Angelici, F.; Bernardinangeli, M.; Proietti, G.; Biscottini, B.; Panciarola, R.; Marinacci, L.; Perna, G.P.; Gabrielli, D.; Moraca, A.; Moretti, L.; Partemi, L.; Gregori, G.; Amici, R.; Patteri, G.; Capone, P.; Savini, E.; Morgagni, G.L.; Paccaloni, L.; Pezzuoli, F.; Carincola, S.; Papi, S.; De Crescentini, S.; Gerardi, P.; Midi, P.; Gallenzi, E.; Pajes, G.; Mancone, C.; Di Spirito, V.; Di Gennaro, M.; Calcagno, S.; Toscano, S.; Antonicoli, S.; Carta, F.; Giorgi, G.; Comito, F.; Daniele, E.; Ciarla, O.; Gelfo, P.G.; Acquaviva, A.; Testa, D.; Testa, G.; Pagliaro, F.A.; Russo, F.; Vetta, F.; Marchese, I.; Di Sciascio, G.; D'Ambrosio, A.; Leggio, F.; Del Sindaco, D.; Lacchè, A.; Avallone, A.; Risa, M.P.; Azzolini, P.; Baldo, E.; Giovannini, E.; Pulignano, G.; Tondo, C.; Picchio, E.; Biffani, E.; Tanzi, P.; Pozzar, F.; Farnetti, F.; Azzarito, M.; Santini, M.; Varveri, A.; Ferraiuolo, G.; Valtorta, C.; Gaspardone, A.; Barbato, G.; Ceci, V.; Aspromonte, N.; Bellocci, F.; Colizzi, C.; Fedele, F.; Perez, F.I.; Galati, A.; Rossetti, A.; Mainella, A.; Ciuffetta, D.; Matteucci, C.; Busi, G.; De Angelis, A.; Farina, G.; Granatelli, A.; Leone, F.; Frasca, F.; Pajes, G.; Di Giovambattista, R.; Castellani, G.; Massaro, G.; Mastrogiuseppe, G.; Vacri, A.; De Sanctis, F.; Cioli, M.; Di Luzio, S.; Napoletano, C.; Piccioni, L.L.; De Simone, G.; Ottaviano, A.; Mazza, V.; Spedaliere, C.; Staniscia, D.; Calgione, E.; De Marco, G.; Chiacchio, T.; Di Napoli, T.; Romanzi, S.; Salvatore, G.; Golino, P.; Palermo, A.; Mascia, F.; Vetrano, A.; Vinciguerra, A.; Caliendo, L.; Longobardi, R.; De Caro, G.; Di Nola, R.; Piemonte, F.; Prinzi, D.; De Rosa, P.; De Rosa, V.; Riello, F.; Capuano, V.; Vecchio, G.; Landi, M.; Amato, S.; Garofalo, M.; Caruso, D.; D'Avino, M.; Sensale, P.; Maiolica, O.; Santoro, R.; Caso, P.; Miceli, D.; Maurea, N.; Bianchi, U.; Crispo, C.; Chiariello, M.; Perrone Filardi, P.; Russo, L.; Capuano, N.; Ungaro, G.; Vergara, G.; Scafuro, F.; D'Angelo, G.; Campaniello, C.; Bottiglieri, P.; Volpe, A.; Battista, R.; De Risi, L.; Cardillo, G.; Sibilio, G.; Marino, A.P.; Silvestri, F.; Predotti, P.; Iervoglini, A.; Stefanelli, S.; De Matteis, C.; Sarnicola, P.; Matarazzo, M.M.; Baldi, S.; Iuliano, V.; Astarita, C.; Cuccaro, P.; Liguori, A.; Liguori, G.; Gregorio, G.; Petraglia, L.; Antonelli, G.; Amodio, G.; De Luca, I.; Traversa, D.; Franchini, G.; Lenti, M.L.; Cavallari, D.; D'Agostino, C.; Scalera, G.; Altamura, C.M.; Russo, M.; Mascolo, A.R.; Pettinati, G.; Ciricugno, S.A.; Scrutinio, D.; Passantino, A.; Mastrangelo, D.; Di Masi, A.; De Carne, R.; Cannone, M.; Dibiase, F.; Pensato, M.; Loliva, F.; Trapani, F.; Panettieri, I.; Leone, L.; Di Biase, M.; Carrone, M.; Gallone, V.; Cocco, F.; Costantini, M.; Tritto, C.; Cavalieri, F.; Stella, L.; Magliari, F.; Callerame, M.; De Giorgi, A.; Pellegrino, L.; Correra, M.; Portulano, V.; Nisi, G.L.; Grassi, G.; Cristallo, E.; De Laura, D.; Salerno, C.; Fanelli, R.; Villella, M.; Pede, S.; Renna, A.; De Lorenzi, E.; Urso, L.; Lenti, V.; Peluso, A.; Baldi, N.; Polimeni, G.; Galati, A.; Palma, P.; Lauletta, R.; Tagliamonte, E.; Cirillo, T.; Silvestri, B.; Centonze, G.; D'Alessandro, B.; Truncellito, L.; Mecca, D.; Petruzzi, M.A.; Coviello, R.O.M.; Lopizzo, A.; Chiaffitelli, M.; Barbuzzi, S.; Gubelli, S.; Germinario, G.; Cosentino, N.; Mingrone, A.; Vico, R.; Borrello, G.; Mazza, M.L.; Cimino, R.; Galasso, D.; Cassadonte, F.; Talarico, U.; Perticone, F.; Cassano, S.; Catapano, F.; Calemme, S.; Feraco, E.; Cloro, C.; Misuraca, G.; Caporale, R.; Vigna, L.; Spagnuolo, V.; De Rosa, F.; Spadafora, G.; Zampaglione, G.; Russo, R.; Schipani, F.A.; Ferragina, A.F.; Stranieri, D.; Musca, G.; Carpino, C.; Bencardino, P.; Raimondo, F.; Musacchio, D.; Pulitanò, G.; Ruggeri, A.; Provenzano, A.; Salituri, S.; Musolino, M.; Calandruccio, S.; Marrari, A.; Tripodi, E.; Scali, R.; Anastasio, L.; Arone, A.; Aragona, P.; Donnangelo, L.; Comito, M.G.A.; Bilotta, F.; Vaccaro, I.; Rametta, R.; Ventura, V.; Bonvegna, A.; Alì, A.; Cinnirella, C.; Raineri, M.; Pompeo, F.; Cascio Ingurgio, N.; Carini, V.; Coco, R.; Giunta, G.; Leonardi, G.; Randazzo, V.; Di Blasi, V.; Tamburino, C.; Russo, G.; Mangiameli, S.; Cardillo, R.; Castelli, D.; Inserra, V.; Arena, A.; Gulizia, M.M.; Raciti, S.; Rapisarda, G.; Romano, R.; Prestifilippo, P.; Braschi, G.B.; Ledda, G.; Terrazzino, R.; De Caro, M.; Scilabra, G.; Graffagnino, B.; Grassi, R.; Di Tano, G.; Scimone, G.F.; Vasquez, L.; Coppolino, C.; Casale, A.; Castelli, M.; D'Urso, G.; D'Antonio, E.; Lo Presti, L.; Badalamenti, E.; Conti, P.; Sanfilippo, N.; Cirrincione, V.; Cinà, M.T.; Cusimano, G.; Taormina, A.; Giuliano, P.; Bajardi, A.; Mandalà, V.; Canonico, A.; Geraci, G.; Sabella, F.P.; Enia, F.; Floresta, A.M.; Lo Cascio, I.; Gumina, D.; Cavallaro, A.; Piccione, G.; Ferrante, R.; Blandino, M.; Iudicello, M.S.; Mossuti, E.; Romano, G.; Lombardo, L.; Monastra, P.; Di Vincenzo, D.; Porcu, M.; Orrù, P.; Muscas, F.; Giardina, G.; Corda, M.; Locci, G.; Podda, A.; Ledda, M.; Siddi, P.; Lai, C.; Pili, G.; Mercuro, G.; Mureddu, G.; Ganau, A.; Meloni, G.; Poddighe, G.; Sanna, G.; Barlera, Simona; Franzosi, Maria Grazia; Porcu, Maurizio; Yusuf, Salim; Camerini, Fulvio; Cohn, Jay N.; Decarli, Adriano; Pitt, Bertram; Sleight, Peter; Poole-Wilson, Philip A.; Geraci, Enrico; Scherillo, Marino; Fabbri, Gianna; Bartolomei, Barbara; Bertoli, Daniele; Cobelli, Franco; Fresco, Claudio; Ledda, Antonietta; Levantesi, Giacomo; Opasich, Cristina; Rusconi, Franco; Sinagra, Gianfranco; Turazza, Fabio; Volpi, Alberto; Ceseri, Martina; Alongi, Gianluca; Atzori, Antonio; Bambi, Filippo; Bastarolo, Desiree; Bianchini, Francesca; Cangioli, Iacopo; Canu, Vittoriana; Caporusso, Concetta; Cenni, Gabriele; Cintelli, Laura; Cocchio, Michele; Confente, Alessia; Fenicia, Eva; Friso, Giorgio; Gianfriddo, Marco; Grilli, Gianluca; Lazzaro, Beatrice; Lonardo, Giuseppe; Luise, Alessia; Nota, Rachele; Orlando, Mariaelena; Petrolo, Rosaria; Pierattini, Chiara; Pierota, Valeria; Provenzani, Alessandro; Quartuccio, Velia; Ragno, Anna; Serio, Chiara; Spolaor, Alvise; Tafi, Arianna; Tellaroli, Elisa; Ghio, Stefano; Ghizzardi, Elisa; Masson, Serge; Crociati, Lella; La Rovere, Maria Teresa; Corrà, Ugo; Di Giulio, Paola; Finzi, Andrea; Gorini, Marco; Milani, Valentina; Orsini, Giampietro; Bianchini, Elisa; Cabiddu, Silvia; Cangioli, Ilaria; Cipressa, Laura; Cipressa, Maria Lucia; Di Bitetto, Giuseppina; Ferri, Barbara; Galbiati, Luisa; Lorimer, Andrea; Pera, Carla; Priami, Paola; Vasamì, AntonellaDauriz, Marco; Targher, Giovanni; Temporelli, Pier Luigi; Lucci, Donata; Gonzini, Lucio; Nicolosi, Gian Luigi; Marchioli, Roberto; Tognoni, Gianni; Latini, Roberto; Cosmi, Franco; Tavazzi, Luigi; Maggioni, Aldo Pietro; Moccetti, T.; Rossi, M. G.; Pasotti, E.; Vaghi, F.; Roncarolo, P.; Zunino, M. T.; Matta, F.; Actis Perinetto, E.; Gaita, F.; Azzaro, G.; Zanetta, M.; Paino, A. M.; Parravicini, U.; Vegis, D.; Conte, R.; Ferraro, P.; De Bernardi, A.; Morelloni, S.; Fagnani, M.; Greco Lucchina, P.; Montagna, L.; Bellone, E.; Sappè, D.; Ferraro, F.; Delucchi, M.; Reynaud, S. G.; Dore, M.; La Brocca, A.; Massobrio, N.; Bo, L.; Trinchero, R.; Imazio, M.; Brocchi, G.; Nejrotti, A.; Rissone, L.; Gabasio, S.; Zocchi, C.; Randazzo, S.; Crenna, A.; Giannuzzi, P.; Bonanomi, E.; Mezzani, A.; De Marchi, M.; Begliuomini, G.; Gianonatti, C. A.; Gavazzi, A.; Grosu, A.; Dei Cas, L.; Nodari, S.; Garyfallidis, P.; Bertoletti, A.; Bonifazi, C.; Arisi, S.; Mascaro, F.; Fraccarollo, M.; Dell'Orto, S.; Sfolcini, M.; Bortolini, F.; Raccagni, D.; Turelli, A.; Santarone, M.; Miglierina, E.; Sormani, L.; Jemoli, R.; Tettamanti, F.; Pirelli, S.; Bianchi, C.; Verde, S.; Mariani, M.; Ziacchi, V.; Ferrazza, A.; Russo, A.; Bortolotti, M.; Pasini, G. F.; Volpi, A.; Jones, K. N.; Cuzzucrea, D.; Gullace, G.; Carbone, C.; Granata, A.; De Servi, S.; Del Rosso, G.; Inserra, C.; Renaldini, E.; Zappa, C.; Moretti, M.; Zanini, R.; Ferrari, M.; Moroni, E.; Cei, A.; Lissi, C.; Dovico, E.; Fiorentini, C.; Palermo, P.; Brusoni, B.; Negrini, M.; Heyman, J.; Danzi, G. B.; Finzi, A.; Frigerio, M.; Turazza, F.; Beretta, L.; Sachero, A.; Casazza, F.; Squadroni, L.; Lombardi, F.; Marano, L.; Margonato, A.; Fragasso, G.; Febo, O. C.; Aiolfi, E.; Olmetti, F.; Grieco, A.; Antonazzo, V.; Specchia, G.; Mortara, A.; Robustelli, F.; Songini, M. G.; Schweiger, C.; Frisinghelli, A.; Palvarini, M.; Campana, C.; Scelsi, L.; Ajmone Marsan, N.; Cobelli, F.; Gualco, A.; Opasich, C.; De Feo, S.; Mazzucco, R.; Iannone, M. A.; Diaco, T.; Zaniboni, D.; Milanesi, G.; Nassiacos, D.; Meloni, S.; Giani, P.; Nicoli, T.; Malinverni, C.; Gusmini, A.; Pozzoni, L.; Bisiani, G.; Margaroli, P.; Schizzarotto, A.; Daverio, A.; Occhi, G.; Partesana, N.; Bandini, P.; Rosella, M. G.; Giustiniani, S.; Cucchi, G.; Pedretti, R.; Raimondo, R.; Vaninetti, R.; Fedele, A.; Ghezzi, I.; Rezzonico, E.; Salerno Uriarte, J. A.; Morandi, F.; Salvucci, F.; Valenti, C.; Graziano, G.; Romanò, M.; Cimminiello, C.; Mangone, I.; Lombardo, M.; Quorso, P.; Marinoni, G.; Breghi, M.; Erckert, M.; Dienstl, A.; Mirante Marini, G.; Stefenelli, C.; Cioffi, G.; Buczkowska, E.; Bonanome, A.; Bazzanini, F.; Parissenti, L.; Serafini, C.; Catania, G.; Tarantini, L.; Rigatelli, G.; Boni, S.; Pasini, A.; Masini, E.; Zampiero, A. A.; Zanchetta, M.; Franceschetto, L.; Delise, P.; Marcon, C.; Sacchetta, A.; Borgese, L.; Artusi, L.; Casolino, P.; Corbara, F.; Banzato, A.; Barbiero, M.; Aldegheri, M. P.; Bazzucco, R.; Crivellenti, G.; Raviele, A.; Zanella, C.; Pascotto, P.; Sarto, P.; Milan, S.; Barbieri, E.; Girardi, P.; Dalla Villa, W.; Dalle Mule, J.; Di Sipio, M. L.; Cazzin, R.; Milan, D.; Zonzin, P.; Carraro, M.; Rossi, R.; Carbonieri, E.; Rossi, I.; Stritoni, P.; Meneghetti, P.; Risica, G.; Tenderini, P. L.; Vassanelli, C.; Zanolla, L.; Perini, G.; Brighetti, G.; Chiozza, R.; Giuliano, G.; Baldin, M. G.; Gortan, R.; Cesanelli, R.; Nicolosi, G. L.; Piazza, R.; Mos, L.; Vriz, O.; Pavan, D.; Pascottini, G.; Alberti, E.; Werren, M.; Solinas, L.; Sinagra, G.; Longaro, F.; Fioretti, P.; Albanese, M. C.; Miani, D.; Gianrossi, R.; Pende, A.; Rubartelli, P.; Magaia, O.; Domenicucci, S.; Caruso, D.; Faraguti, A. S.; Magliani, L.; Miccoli, F.; Guglielmino, G.; Bertoli, D.; Cantarelli, A.; Orlandi, S.; Vallebona, A.; Pozzati, A.; Brega, G.; Pancaldi, L. G.; Vandelli, R.; Urbinati, S.; Poci, M. G.; Zoli, M.; Costa, G. M.; Guiducci, U.; Zobbi, G.; Tartagni, F.; Tisselli, A.; Gentili, A.; Pieri, P.; Cagnetta, E.; Bendinelli, S.; Barbieri, A.; Conti, R.; Ferrari, R.; Merlini, F.; Fucili, A.; Moruzzi, P.; Buia, E.; Galvani, M.; Ferrini, D.; Baggioni, G.; Yiannacopulu, P.; Canè, G.; Bonfiglioli, A.; Zandomeneghi, R.; Brugioni, L.; Giannini, A.; Di Ruvo, R.; Giuliani, M.; Rusconi, L.; Del Corso, P.; Piovaccari, G.; Bologna, F.; Venturi, P.; Melandri, F.; Bagni, E.; Bolognese, L.; Perticucci, R.; Zuppiroli, A.; Nannini, M.; Consoli, N.; Petrone, P.; Pipitò, C.; Colombi, L.; Bernardi, D.; Mariani, P. R.; Testa, R.; Mazzinghi, F.; Cosmi, F.; Cosmi, D.; Zipoli, A.; Cecchi, A.; Castelli, G.; Ciaccheri, M.; Mori, F.; Pieri, F.; Valoti, P.; Chiarantini, D.; Santoro, G. M.; Minneci, C.; Marchi, F.; Milli, M.; Zambaldi, G.; Zipoli, A.; Brandinelli Geri, A. A.; Cipriani, M.; Alessandri, M.; Severi, S.; Stefanelli, S.; Comella, A.; Poddighe, R.; Digiorgio, A.; Carluccio, M.; Berti, S.; Rizza, A.; Bonatti, V.; Molendi, V.; Brancato, A.; D'Aprile, N.; Giappichini, G.; Del Vecchio, S.; Mantini, G.; De Tommasi, F.; Meucci, G.; Cordoni, M.; Bechi, S.; Barsotti, L.; Baldini, P.; Romei, M.; Scopelliti, G.; Lauri, G.; Pestelli, F.; Furiozzi, F.; Cocchieri, M.; Severini, D.; Patriarchi, F.; Chiocchi, P.; Buccolieri, M.; Martinelli, S.; Wee, A.; Angelici, F.; Bernardinangeli, M.; Proietti, G.; Biscottini, B.; Panciarola, R.; Marinacci, L.; Perna, G. P.; Gabrielli, D.; Moraca, A.; Moretti, L.; Partemi, L.; Gregori, G.; Amici, R.; Patteri, G.; Capone, P.; Savini, E.; Morgagni, G. L.; Paccaloni, L.; Pezzuoli, F.; Carincola, S.; Papi, S.; De Crescentini, S.; Gerardi, P.; Midi, P.; Gallenzi, E.; Pajes, G.; Mancone, C.; Di Spirito, V.; Di Gennaro, M.; Calcagno, S.; Toscano, S.; Antonicoli, S.; Carta, F.; Giorgi, G.; Comito, F.; Daniele, E.; Ciarla, O.; Gelfo, P. G.; Acquaviva, A.; Testa, D.; Testa, G.; Pagliaro, F. A.; Russo, F.; Vetta, F.; Marchese, I.; Di Sciascio, G.; D'Ambrosio, A.; Leggio, F.; Del Sindaco, D.; Lacchè, A.; Avallone, A.; Risa, M. P.; Azzolini, P.; Baldo, E.; Giovannini, E.; Pulignano, G.; Tondo, C.; Picchio, E.; Biffani, E.; Tanzi, P.; Pozzar, F.; Farnetti, F.; Azzarito, M.; Santini, M.; Varveri, A.; Ferraiuolo, G.; Valtorta, C.; Gaspardone, A.; Barbato, G.; Ceci, V.; Aspromonte, N.; Bellocci, F.; Colizzi, C.; Fedele, F.; Perez, F. I.; Galati, A.; Rossetti, A.; Mainella, A.; Ciuffetta, D.; Matteucci, C.; Busi, G.; De Angelis, A.; Farina, G.; Granatelli, A.; Leone, F.; Frasca, F.; Pajes, G.; Di Giovambattista, R.; Castellani, G.; Massaro, G.; Mastrogiuseppe, G.; Vacri, A.; De Sanctis, F.; Cioli, M.; Di Luzio, S.; Napoletano, C.; Piccioni, L. L.; De Simone, G.; Ottaviano, A.; Mazza, V.; Spedaliere, C.; Staniscia, D.; Calgione, E.; De Marco, G.; Chiacchio, T.; Di Napoli, T.; Romanzi, S.; Salvatore, G.; Golino, P.; Palermo, A.; Mascia, F.; Vetrano, A.; Vinciguerra, A.; Caliendo, L.; Longobardi, R.; De Caro, G.; Di Nola, R.; Piemonte, F.; Prinzi, D.; De Rosa, P.; De Rosa, V.; Riello, F.; Capuano, V.; Vecchio, G.; Landi, M.; Amato, S.; Garofalo, M.; Caruso, D.; D'Avino, M.; Sensale, P.; Maiolica, O.; Santoro, R.; Caso, P.; Miceli, D.; Maurea, N.; Bianchi, U.; Crispo, C.; Chiariello, M.; Perrone Filardi, P.; Russo, L.; Capuano, N.; Ungaro, G.; Vergara, G.; Scafuro, F.; D'Angelo, G.; Campaniello, C.; Bottiglieri, P.; Volpe, A.; Battista, R.; De Risi, L.; Cardillo, G.; Sibilio, G.; Marino, A. P.; Silvestri, F.; Predotti, P.; Iervoglini, A.; Stefanelli, S.; De Matteis, C.; Sarnicola, P.; Matarazzo, M. M.; Baldi, S.; Iuliano, V.; Astarita, C.; Cuccaro, P.; Liguori, A.; Liguori, G.; Gregorio, G.; Petraglia, L.; Antonelli, G.; Amodio, G.; De Luca, I.; Traversa, D.; Franchini, G.; Lenti, M. L.; Cavallari, D.; D'Agostino, C.; Scalera, G.; Altamura, C. M.; Russo, M.; Mascolo, A. R.; Pettinati, G.; Ciricugno, S. A.; Scrutinio, D.; Passantino, A.; Mastrangelo, D.; Di Masi, A.; De Carne, R.; Cannone, M.; Dibiase, F.; Pensato, M.; Loliva, F.; Trapani, F.; Panettieri, I.; Leone, L.; Di Biase, M.; Carrone, M.; Gallone, V.; Cocco, F.; Costantini, M.; Tritto, C.; Cavalieri, F.; Stella, L.; Magliari, F.; Callerame, M.; De Giorgi, A.; Pellegrino, L.; Correra, M.; Portulano, V.; Nisi, G. L.; Grassi, G.; Cristallo, E.; De Laura, D.; Salerno, C.; Fanelli, R.; Villella, M.; Pede, S.; Renna, A.; De Lorenzi, E.; Urso, L.; Lenti, V.; Peluso, A.; Baldi, N.; Polimeni, G.; Galati, A.; Palma, P.; Lauletta, R.; Tagliamonte, E.; Cirillo, T.; Silvestri, B.; Centonze, G.; D