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Cyclin-dependent kinase 4 and 6 inhibitors (CDK4/6i) have been approved in combination with endocrine therapy (ET) to treat estrogen receptor-positive (ER+) metastatic breast cancer (BC). However, drug resistance represents the leading cause of breast cancer patients mortality. This study aimed to identify novel resistance mechanisms to ER antagonists in combination with CDK4/6 inhibitors. We generated two ER+ BC cell lines, T47D and MCF7, resistant to the combination of the ER antagonist fulvestrant and CDK4/6i abemaciclib, named T47D-FAR and MCF7-FAR. Transcriptomic analysis revealed common up-regulation of genes involved in MAPK and epithelial to mesenchymal transition (EMT) pathways in FAR cells, sustaining their hyper-invasive phenotype and increased anchorage-independent growth, compared to sensitive cells. FAR cells showed higher p21-activated kinase 1 (Pak1) expression and phosphorylation levels than parental cells. PAK1 knockdown by siRNAs hampered cell proliferation, reduced anchorage-independent growth and invasive properties of T47D-FAR and MCF7-FAR, re-sensitizing them to fulvestrant and abemaciclib. Conversely, over-expression of PAK1 in MCF7 and T47D cells increased tumor spheroids' growth and invasion and reduced sensitivity to fulvestrant and abemaciclib, confirming its role in inducing drug resistance. Finally, treatment with Pak1 inhibitors, PF-3758309 (PF309) and NVS-PAK1-1, restored cell sensitivity to fulvestrant and abemaciclib of MCF7-FAR and T47D-FAR cells, both in vitro and in vivo. In conclusion, our data suggested a pivotal role for Pak1 in resistance to ET and CDK4/6i in ER+ breast cancers. These data might promote the rationale for the development of novel Pak1 inhibitors for treatment of patients with ER+ BC progressing on ET plus CDK4/6i., (© 2023. The Author(s).)

Adequate complementary feeding practices are important for short- and long-term child health. In industrialized countries, the formulation of several commercial baby foods (CBFs) and an increase in their consumption has been noticed., Aim: To update and analyze the nutritional composition of CBFs available in the Italian market., Methods: Data collection carried out in two steps (July 2018-January 2019) and updated in May-September 2021. The information on CBFs was taken from the websites of the major CBF producers available in Italy. The collected information were: Suggested initial and final age of consumption; Ingredients; Energy value; Macronutrients (protein, lipids, and carbohydrates); Fiber; Micronutrients (sodium, iron, and calcium); Presence of salt and added sugars, flavorings, and other additives., Results: Time-space for which CBFs are recommended starts too early and ends too late; protein content is adequate and even too high in some food; Amount of fats and their quality must be improved, keeping the intake of saturated fats low; Sugar content is too high in too many CBFs and salt is unnecessarily present in some of them. Finally, the texture of too many products is purée, and its use is recommended for too long, hindering the development of infants' chewing abilities.

Data assimilation (DA) is a powerful tool to optimally combine uncertain model simulations and observations. Among DA techniques, the particle filter (PF) has gained attention for its capacity to deal with nonlinear systems and for its relaxation of the Gaussian assumption. However, the PF may suffer from degeneracy and sample impoverishment. In this study, we propose an innovative approach, based on a tempered particle filter (TPF), aiming at mitigating PFs issues, thus extending over time the assimilation benefits. Probabilistic flood maps derived from synthetic aperture radar data are assimilated into a flood forecasting model through an iterative process including a particle mutation in order to keep diversity within the ensemble. Results show an improvement of the model forecasts accuracy, with respect to the Open Loop: on average the root mean square error (RMSE) of water levels decrease by 80% at the assimilation time and by 60% 2 days after the assimilation. A comparison with the Sequential Importance Sampling (SIS) is carried out showing that although SIS performances are generally comparable to the TPF ones at the assimilation time, they tend to decrease more quickly. For instance, on average TPF-based RMSE are 20% lower compared to the SIS-based ones 2 days after the assimilation. The application of the TPF determines higher critical success index values compared to the SIS. On average the increase in performances lasts for almost 3 days after the assimilation. Our study provides evidence that the application of the variant of the TPF enables more persistent benefits compared to the SIS., (© 2022. The Authors.)

The complementary feeding (CF) period that takes place between 6 and 24 months of age is of key importance for nutritional and developmental reasons during the transition from exclusively feeding on milk to family meals. In 2021, a multidisciplinary panel of experts from four Italian scientific pediatric societies elaborated a consensus document on CF, focusing in particular on healthy term infants. The aim was to provide healthcare providers with useful guidelines for clinical practice. Complementary feeding is also the time window when iron deficiency (ID) and iron deficiency anemia (IDA) are most prevalent. Thus, it is appropriate to address the problem of iron deficiency through nutritional interventions. Adequate iron intake during the first two years is critical since rapid growth in that period increases iron requirements per kilogram more than at any other developmental stage. Complementary foods should be introduced at around six months of age, taking into account infant iron status.

In an attempt to prepare sustainable epoxy thermosets, this study introduces for the first time the idea to use antagonist structures (aromatic/aliphatic) or functionalities (acid/amine) as hardeners to produce reprocessable resins based on epoxidized camelina oil (ECMO). Two kinds of mixtures were tested: one combines aromatic/aliphatic dicarboxylic acids: 2,2'-dithiodibenzoic acid (DTBA) and 3,3'-dithiodipropionic acid (DTDA); another is the combination of two aromatic structures with acid/amine functionality: DTBA and 4-aminophenyl disulfide (4-AFD). DSC and FT-IR analyses were used as methods to analyze the curing reaction of ECMO with the hardeners. It was found that the thermosets obtained with the dual crosslinked mechanism needed reduced curing temperatures and reprocessing protocols compared to the individual crosslinked thermosets. Thanks to the contribution of disulfide bonds in the network topology, the obtained thermosets showed recycling ability. The final thermomechanical properties of the virgin and mechanical reprocessed materials were analyzed by DMA and TGA. The obtained thermosets range from elastomeric to rigid materials. As an example, the ECMO/DTBA 70 4-AFD 30 virgin or reprocessed thermosets have tan δ values reaching 82-83 °C. The study also investigates the chemical recycling and the solvent resistance of these vitrimer-like materials.

Background: In 2019, a multidisciplinary panel of experts from eight Italian scientific paediatric societies developed a consensus document for the use of inhaled corticosteroids in the management and prevention of the most common paediatric airways disorders. The aim is to provide healthcare providers with a multidisciplinary document including indications useful in the clinical practice. The consensus document was intended to be addressed to paediatricians who work in the Paediatric Divisions, the Primary Care Services and the Emergency Departments, as well as to Residents or PhD students, paediatric nurses and specialists or consultants in paediatric pulmonology, allergy, infectious diseases, and ear, nose, and throat medicine., Methods: Clinical questions identifying Population, Intervention(s), Comparison and Outcome(s) were addressed by methodologists and a general agreement on the topics and the strength of the recommendations (according to the GRADE system) was obtained following the Delphi method. The literature selection included secondary sources such as evidence-based guidelines and systematic reviews and was integrated with primary studies subsequently published., Results: The expert panel provided a number of recommendations on the use of inhaled corticosteroids in preschool wheezing, bronchial asthma, allergic and non-allergic rhinitis, acute and chronic rhinosinusitis, adenoid hypertrophy, laryngitis and laryngospasm., Conclusions: We provided a multidisciplinary update on the current recommendations for the management and prevention of the most common paediatric airways disorders requiring inhaled corticosteroids, in order to share useful indications, identify gaps in knowledge and drive future research.

Given the pressure on healthcare authorities to assess whether hospital capacity allows properly responding to outbreaks such as COVID-19, there is a need for simple, data-driven methods that may provide accurate forecasts of hospital bed demand. This study applies growth models to forecast the demand for Intensive Care Unit admissions in Italy during COVID-19. We show that, with only some mild assumptions on the functional form and using short time-series, the model fits past data well and can accurately forecast demand fourteen days ahead (the mean absolute percentage error (MAPE) of the cumulative fourteen days forecasts is 7.64). The model is then applied to derive regional-level forecasts by adopting hierarchical methods that ensure the consistency between national and regional level forecasts. Predictions are compared with current hospital capacity in the different Italian regions, with the aim to evaluate the adequacy of the expansion in the number of beds implemented during the COVID-19 crisis., Competing Interests: The authors have declared that no competing interests exist.

The design of polymers from renewable resources with recycling potential comes from economic and environmental problems. This work focused on the impact of disulphide bonds in the dicarboxylic acids reactions with three epoxidized vegetable oils (EVOs). For the first time, the comparison between aromatic vs. aliphatic dicarboxylic acids, containing or not S-S bonds with EVOs was discussed and evaluated by dynamic scanning calorimetry. The obtained thermosets showed reprocessability, by the dual dynamic exchange mechanism. The virgin and reprocessed materials were characterized and the thermomechanical properties were compared. The thermosets derived from EVOs with high epoxy content combined with aromatic diacids containing disulphide bridges showed high glass transition values (~111 °C), high crosslink densities and good solvent stability.

Beyond the need to find a non-toxic alternative to DiGlycidyl Ether of Bisphenol-A (DGEBA), the serious subject of non-epichlorohydrin epoxy resins production remains a crucial challenge that must be solved for the next epoxy resin generations. In this context, this study focuses on the valorization of vegetable oils (VOs) into thermoset materials by using (i) epoxidation of the VOs through the "double bonds to epoxy" synthetic route and (ii) synthesis of crosslinked homopolymers by UV or hardener-free thermal curing processes. A thorough identification, selection and physico-chemical characterization of non-edible or non-valuated natural vegetable oils were performed. Selected VOs, characterized by a large range of double bond contents, were then chemically modified into epoxides thanks to an optimized, robust and sustainable method based on the use of acetic acid, hydrogen peroxide and Amberlite® IR-120 at 55 °C in toluene or cyclopentyl methyl ether (CMPE) as a non-hazardous and green alternative solvent. The developed environmentally friendly epoxidation process allows reaching almost complete double bond conversion with an epoxy selectivity above 94% for the 12 studied VOs. Finally, obtained epoxidized vegetable oils (EVOs), characterized by an epoxy index from 2.77 to 6.77 m eq. g -1 were cured using either UV or hardener-free thermal curing. Both methods enable the synthesis of 100% biobased EVO thermoset materials whose thermomechanical performances were proved to linearly increase with the EVOs' epoxy content. This paper highlights that tunable thermomechanical performances ( T from -19 to 50 °C and α from -34 to 36 °C) of EVO based thermoset materials can be reached by well selecting the starting VO raw materials.T g from -34 to 36 °C) of EVO based thermoset materials can be reached by well selecting the starting VO raw materials., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

The end-of-life of thermoset materials is a real issue that confronts our society, and the strategy of introducing dynamic reversible bonds can be a sustainable solution to overcome this problem. This study shows an efficient way to produce biobased and recyclable thermosets, for a circular use. To reduce the production costs linked to energy and duration, an improved curing process is proposed by combining aromatic and aliphatic diacid hardeners containing dynamic S-S bonds. The work demonstrates the increased reactivity of epoxidized vegetable oil reacted with the two diacids. The structural evolutions during the exchange reactions that allow the recyclability were followed by Fourier transformed-infrared and nuclear magnetic resonance spectroscopies, high-performance liquid chromatography, and mass spectroscopy. The curing process was studied by differential scanning calorimetry and kinetic study.

The preparation of thermosets based on epoxidized vegetable oils (EVOs) involved a peculiar attention in recent years; however, most of them cannot be recycled once cross-linked. In the present work, epoxy thermosetting resins like-vitrimers with dynamic disulfide covalent bonds were prepared by copolymerizing twelve EVOs with 2,2'-dithiodibenzoic acid, as hardener. Here, we show for the first time the reprocessability, repairability, and recyclability properties of EVOs thermosets. The 3R abilities were evaluated in correlation with the EVO epoxy contents, which influence the final thermo-mechanical properties of the recycled material. The virgin versus recycled materials' comparison was studied by FT-IR, DSC, TGA, and DMA, also comparing their swelling ability and high gel content. The study investigates, in addition, the excellent shape memory properties of the reprocessed EVOs/disulfide materials.

This work reports for the first time the copolymerization studies of 11 newly synthesized epoxidized vegetable oils (EVOs) that reacted with a disulfide-based aromatic dicarboxylic acid (DCA) to produce thermoset materials with recyclability properties. These new EVOs' reactivity and properties were compared with those of the two commercial references: epoxidized linseed oil (ELO) and epoxidized soybean oil (ESO). The structure-reactivity correlation is proposed by differential scanning calorimetry (DSC) analysis, corroborating the epoxy content of EVO monomers, the initiator effect, the copolymerization reaction enthalpy, and the temperature range. The thermomechanical properties of the obtained thermosets were evaluated and discussed in correlation with the structure and reactivity of monomers by dynamic mechanical analysis (DMA), tensile testing, and thermogravimetric analysis (TGA). It has been found that the higher the EVO functionality, the higher is the reactivity, cross-linking density, and final performances, with tan δ values ranging from 34 to 111 °C. This study investigates the chemical recycling and the solvent resistance of these vitrimer-like materials that have a high bio-based carbon content, from 58 to 79%, with potential application in coating or composite materials in the automotive sector.

Thermal degradation processes of poly(ether sulfone) random copolymers having different molar amount of diphenolic acid (DPA) units were studied by direct-pyrolysis/mass spectrometry, stepwise pyrolysis-gas chromatography/mass spectrometry and thermogravimetric techniques. Results highlighted that thermal degradation processes occur in the temperature range from 370 to 650 °C, yielding a char residue of 32-35 wt%, which decreases as the mol% of DPA units rises. The pyrolysis/mass spectra data allowed us to identify the thermal decomposition products and to deduce the possible thermal degradation mechanisms. Thermal degradation data suggest that the decarboxylation process of the pendant acid moiety mainly occurs in the initial step of the pyrolysis of the copolymers studied. Successively, the scission of the generated isobutyl groups occurs in the temperature range between 420 and 480 °C. Known processes involving the main chain random scission of the diphenyl sulfone and diphenyl ether groups were also observed.

Background: Allopurinol can induce severe cutaneous adverse reactions (SCARs), including Drug Reaction with Eosinophilia and Systemic Symptoms (DRESS) syndrome, Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN)., Aims and Methods: We analyzed the Individual Case Safety Reports (ICSRs) sent from January 2001 until April 2019 to the Campania regional Center of Pharmacovigilance (Southern Italy) that reported allopurinol as suspected, with a focus on those reporting at least one serious cutaneous adverse drug reaction (ADR). This study was aimed to describe the main characteristics of all ADRs associated with allopurinol, analyze the proportion of serious cutaneous ADRs of total ICSRs related to allopurinol and to compare the main features (age, sex, seriousness and outcome) of ICSRs that reported serious cutaneous ADRs with those that did not., Results: The Campania regional Center of Pharmacovigilance received 108 ICSRs that reported allopurinol as suspected. ADRs occurred more frequently in the elderly (median age: 71 years) and female patients (53.7%). Fifty-seven percent of all ADRs were classified as serious and 58% had a favorable outcome. Fifty-six ICSRs reported at least one serious cutaneous ADR; among these ICSRs, 37 cases of SCARs were found [DRESS syndrome (n = 3; 5.4%), SJS (n = 8; 14.3%) and TEN (n = 26; 46.4%)]. Serious cutaneous ADRs commonly occurred in the elderly (median age: 73 years) and female patients (62.5%). They frequently required hospitalization (75%) and had an unfavorable outcome (46%). No statistically significant differences were found between ICSRs that reported serious cutaneous ADRs and ICSRs that did not report serious cutaneous ADRs except for the seriousness degree "Hospitalization or its prolongation" and the outcome degrees "Unfavorable" and "Favorable"., Conclusion: This study found that 52% (56/108) of all ICSRs having allopurinol as a suspected drug were serious cutaneous ADRs. Serious cutaneous ADRs associated with allopurinol frequently required hospitalization or prolonged hospitalization, and almost half had an unfavorable outcome.

Objectives : Human papillomavirus (HPV) vaccines have proved to be effective in preventing cervical carcinoma. Although their safety profile resembled those of any other vaccine, few clinical studies showed that HPV vaccines might also induce severe adverse events. Methods : The authors aimed to investigate the safety profile of HPV vaccines, by analyzing the individual case safety reports (ICSRs) of a suspected adverse event following immunization (AEFI) concerning HPV vaccines that were sent to the Italian Pharmacovigilance Spontaneous Reporting System (RNF) in the Campania Region from January 2007 to September 2018. Results : During the study period, 82 ICSRs, covering 181 AEFIs, related to HPV vaccines were sent to the RNF in the Campania Region. The mean age of patients who experienced an AEFI after HPV vaccinations was 13 ± 4.5 years. The majority of ICSRs reported AEFIs that were considered as not serious (82%) and that had a favorable outcome (93%). Conclusion : The overall results of the study demonstrated that, except for a few cases, AEFIs related to HPV vaccines reflect those already reported in the summary of product characteristics. The authors did not identify any new safety issues or serious, rare or unexpected AEFIs that were medically confirmed to be related to HPV vaccines.

Purpose: Kisspeptin signaling, via its receptors GPR54, could be an essential players in the inhibition of mesothelioma progression, invasion and metastasis formation. The loss of KiSS1 by tumor cells has been associated with a metastatic phenotype but the mechanistic insights of this process are still unknown., Experimental Design: The blockade of the metastatic process at early stage is a hot topic in cancer research. We studied the role of KiSS1 on proliferation, invasiveness, migration abilities of mesothelioma cell lines focusing on the effect on epithelial-to-mesenchymal transition (EMT)., Results: Treatment with the KiSS1 peptide or with a synthesis peptide with longer half-life, the FTM080, significantly inhibited cell proliferation, migration and invasion of mesothelioma cell lines; the same treatment reduced the activity of MMP-2 and MMP-9 determining consequently a marked reduction in the invasiveness of primary tumors and metastases. Thespecificexpression of EMT markers, as E-caderin, Vimentin, Slug and Snail, suggested the inhibition of EMT after treatment with KiSS1 as well as the preservation of epithelial components., Conclusion: Our results support anti-proliferative effect of KiSS1 in cancer cells and suggest that targeting the KiSS1/GPR54 system may represent a novel therapeutic approach for mesothelioma., Competing Interests: CONFLICTS OF INTEREST All coauthors have no conflicts of interest to declare for the following manuscript.

Background: Safety climate is considered beneficial to the improvement of hospital safety outcomes. Nevertheless, the relations between two of its key constituents, namely those stemming from leader-subordinate relations and coworker support for safety, are still to be fully ascertained., Purpose: This article uses the theoretical lens of Social Exchange Theory to study the joint impact of leader-member exchange in the safety sphere and coworker support for safety on safety-related behavior at the hospital ward level. Social exchange constructs are further related to the existence of a shame-/blame-free environment, seen as a potential antecedent of safety behavior., Research Design: A cross-sectional study including 166 inpatients in hospital wards belonging to 10 public hospitals in Italy was undertaken to test the hypotheses developed., Methodology: Hypothesized relations have been analyzed through a fully mediated multilevel structural equation model. This methodology allows studying behavior at the individual level, while keeping into account the heterogeneity among hospital specialties., Findings: Results suggest that the linkage between leader support for safety and individual safety behavior is mediated by coworker support on safety issues and by the creation of a shame-free environment., Practical Implications: These findings call for the creation of a safety climate in which managerial efforts should be directed not only to the provision of new safety resources and the enforcement of safety rules but also to the encouragement of teamwork and freedom to report errors as ways to foster the capacity of the staff to communicate, share, and learn from each other.

Background: Several evidences suggest a marked angiogenic dependency in triple-negative breast cancer (TNBC) tumorigenesis and a potential sensitivity to anti-angiogenic agents. Herein, the putative role of Hedgehog (Hh) pathway in regulating TNBC-dependent angiogenesis was investigated., Methods: Expression and regulation of the Hh pathway transcription factor glioma-associated oncogene homolog1 protein (GLI1) were studied on the endothelial compartment and on TNBC-initiated angiogenesis. To evaluate the translational relevance of our findings, the combination of paclitaxel with the Smo inhibitor NVP-LDE225 was tested in TNBC xenografted mice., Results: Tissue microarray analysis on 200 TNBC patients showed GLI1 overexpression paired with vascular endothelial growth factor receptor 2 (VEGFR2) expression. In vitro, Hh pathway promotes TNBC progression in an autocrine manner, regulating the VEGF/VEGFR2 loop on cancer cell surface, and in a paracrine manner, orchestrating tumour vascularisation. These effects were counteracted by Smo pharmacological inhibition. In TNBC xenografted mice, scheduling NVP-LDE225 rather than bevacizumab provided a better sustained inhibition of TNBC cells proliferation and endothelial cells organisation., Conclusions: This study identifies the Hh pathway as one of the main regulators of tumour angiogenesis in TNBC, thus suggesting Hh inhibition as a potential new anti-angiogenic therapeutic option to be clinically investigated in GLI1 overexpressing TNBC patients.

Background: Data on chronic pancreatitis prevalence are scanty and usually limited to hospital-based studies., Aim: Investigating chronic pancreatitis prevalence in primary care., Methods: Participating primary care physicians reported the prevalence of chronic pancreatitis among their registered patients, environmental factors and disease characteristics. The data were centrally reviewed and chronic pancreatitis cases defined according to M-ANNHEIM criteria for diagnosis and severity and TIGAR-O classification for etiology., Results: Twenty-three primary care physicians participated in the study. According to their judgment, 51 of 36.401 patients had chronic pancreatitis. After reviewing each patient data, 11 turned out to have definite, 5 probable, 19 borderline and 16 uncertain disease. Prevalence was 30.2/100.000 for definite cases and 44.0/100.000 for definite plus probable cases. Of the 16 patients with definite/probable diagnosis, 8 were male, with mean age of 55.6 (±16.7). Four patients had alcoholic etiology, 5 post-acute/recurrent pancreatitis, 6 were deemed to be idiopathic. Four had pancreatic exocrine insufficiency, 10 were receiving pancreatic enzymes, and six had pain. Most patients had initial stage and non-severe disease., Conclusions: This is the first study investigating the prevalence of chronic pancreatitis in primary care. Results suggest that the prevalence in this context is higher than in hospital-based studies, with specific features, possibly representing an earlier disease stage., (Copyright © 2016 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.)

Inhibition of the mechanistic target of rapamycin (mTOR) is a promising treatment strategy for several cancer types. Rapamycin derivatives such as everolimus are allosteric mTOR inhibitors acting through interaction with the intracellular immunophilin FKBP12, a prolyl isomerase with different cellular functions. Although mTOR inhibitors have significantly improved survival of different cancer patients, resistance and lack of predictive factors of response remain unsolved issues. To elucidate the mechanisms of resistance to everolimus, we evaluated Met activation in everolimus-sensitive/resistant human cancer cells, in vitro and in vivo. Biochemical and computational analyses were performed. Everolimus-resistant cells were xenografted into mice (10/group) and studied for their response to everolimus and Met inhibitors. The statistical significance of the in vitro results was evaluated by Student's t test.Everolimus reduced Met phosphorylation in everolimus-sensitive cells. This event was mediated by the formation of a Met-FKBP12 complex, which in turn is disrupted by everolimus. Aberrant Met activation in everolimus-resistant cells and overexpression of wild-type/mutant Met caused everolimus resistance. Pharmacological inhibition and RNA silencing of Met are effective in condition of everolimus resistance (P<0.01). In mice xenografted with everolimus-resistant cells, the combination of everolimus with the Met inhibitor PHA665752 reduced tumor growth and induced a statistically significant survival advantage (combination vs control P=0.0005).FKBP12 binding is required for full Met activation and everolimus can inhibit Met. Persistent Met activation might sustain everolimus resistance. These results identify a novel everolimus mechanism of action and suggest the development of clinical strategies based on Met inhibitors in everolimus-resistant cancers., Competing Interests: No potential conflicts of interest were disclosed.

PEGylation of biomolecules is a major approach to increase blood stream half-life, stability and solubility of biotherapeutics and to reduce their immunogenicity, aggregation potential and unspecific interactions with other proteins and tissues. Antibodies have generally long half-lives due to high molecular mass and stability toward proteases, however their size lowers to some extent their potential because of a reduced ability to penetrate tissues, especially those of tumor origin. Fab or otherwise engineered smaller fragments are an alternative but are less stable and are much less well retained in circulation. We have here investigated the effects of various PEGylations on the binding properties and in vivo half-life of Fab fragments derived from the enzymatic splitting of Trastuzumab. We find that PEGylation increases the half-life of the molecules but also strongly affects the ability to recognize the target antigen in a way that is dependent on the extent and position of the chemical modification. Data thus support the concept that polyethylene glycol (PEG) conjugation on Trastuzumab Fabs increases half-life but reduces their affinity and this is a fine balance, which must be carefully considered for the design of strategies based on the use of antibody fragments.

Purpose: Metformin, widely used as antidiabetic drug, showed antitumoral effects expecially in combination with chemotherapy. Our group recently has demonstrated that metformin and gefitinib are synergistic in LKB1-wild-type NSCLC cells. In these models, metformin as single agent induced an activation and phosphorylation of mitogen-activated-protein-kinase (MAPK) through an increased C-RAF/B-RAF heterodimerization., Experimental Design: Since single agent metformin enhances proliferating signals through the RAS/RAF/MAPK pathway, and several MEK inhibitors (MEK-I) demonstrated clinical efficacy in combination with other agents in NSCLC, we tested the effects of metformin plus MEK-I (selumetinib or pimasertib) on proliferation, invasiveness, migration abilities in vitro and in vivo in LKB1 positive NSCLC models harboring KRAS wild type and mutated gene., Results: The combination of metformin with MEK-I showed a strong anti-proliferative and proapoptotic effect in Calu-3, H1299, H358 and H1975 human NSCLC cell lines, independently from the KRAS mutational status. The combination reduced the metastatic behaviour of NSCLC cells, via a downregulation of GLI1 trascritional activity, thus affecting the transition from an epithelial to a mesenchymal phenotype. Metformin and MEK-Is combinations also decreased the production and activity of MMP-2 and MMP-9 by reducing the NF-jB (p65) binding to MMP-2 and MMP-9 promoters., Conclusions: Metformin potentiates the antitumor activity of MEK-Is in human LKB1-wild-type NSCLC cell lines, independently from the KRAS mutational status, through GLI1 downregulation and by reducing the NF-jB (p65)-mediated transcription of MMP-2 and MMP-9.

Resistance to the EGFR tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib, often related to Ras or secondary EGFR mutations, is a relevant clinical issue in Non-Small Cell Lung Cancer (NSCLC). Although Src TK has been involved in such resistance, clinical development of its inhibitors has been so far limited. To better define the molecular targets of the Src TKIs saracatinib, dasatinib and bosutinib, we used a variety of in vitro/in vivo studies. Kinase assays supported by docking analysis demonstrated that all the compounds directly inhibit EGFR TK variants. However, in live cells only saracatinib efficiently reduced EGFR activation, while dasatinib was the most effective agent in inhibiting Src TK. Consistently, a pronounced anti-proliferative effect was achieved with saracatinib, in EGFR mutant cells, or with dasatinib, in wt EGFR/Ras mutant cells, poorly dependent on EGFR and erlotinib-resistant. We then identified the most effective drug combinations to overcome resistance to EGFR inhibitors, both in vitro and in nude mice: in T790M EGFR erlotinib-resistant cells, saracatinib with the anti-EGFR mAb cetuximab; in Ras mutant erlotinib-resistant models, dasatinib with the MEK inhibitor selumetinib. Src inhibitors may act with different mechanisms in NSCLCs, depending on EGFR/Ras mutational profile, and may be integrated with EGFR or MEK inhibitors for different cohorts of NSCLCs.

Background: Multiple lines of evidence support that the Hedgehog (Hh) signalling has a role in the maintenance and progression of different human cancers. Therefore, inhibition of the Hh pathway represents a valid anticancer therapeutic approach for renal cell carcinoma (RCC) patients. NVP-LDE225 is a Smoothened (Smo) antagonist that induces dose-related inhibition of Hh and Smo-dependent tumour growth., Methods: We assayed the effects of NVP-LDE225 alone or in combination with everolimus or sunitinib on the growth and invasion of human RCC models both in vitro and in vivo. To this aim, we used a panel of human RCC models, comprising cells with acquired resistance to sunitinib - a multiple tyrosine kinase inhibitor approved as a first-line treatment for RCC., Results: NVP-LDE225 cooperated with either everolimus or sunitinib to inhibit proliferation, migration, and invasion of RCC cells even in sunitinib-resistant (SuR) cells. Some major transducers involved in tumour cell motility, including paxillin, were also efficiently inhibited by the combination therapy, as demonstrated by western blot and confocal microscopy assays. Moreover, these combined treatments inhibited tumour growth and increased animal survival in nude mice xenografted with SuR RCC cells. Finally, lung micrometastasis formation was reduced when mice were treated with NVP-LDE225 plus everolimus or sunitinib, as evidenced by artificial metastatic assays., Conclusions: Hedgehog inhibition by NVP-LDE225 plus sunitinib or everolimus bolsters antitumour activity by interfering with tumour growth and metastatic spread, even in SuR cells. Thus, this new evidence puts forward a new promising therapeutic approach for RCC patients.

Background: Cetuximab is the only targeted agent approved for the treatment of head and neck squamous cell carcinomas (HNSCC), but low response rates and disease progression are frequently reported. As the phosphoinositide 3-kinase (PI3K) and the mammalian target of rapamycin (mTOR) pathways have an important role in the pathogenesis of HNSCC, we investigated their involvement in cetuximab resistance., Methods: Different human squamous cancer cell lines sensitive or resistant to cetuximab were tested for the dual PI3K/mTOR inhibitor PF-05212384 (PKI-587), alone and in combination, both in vitro and in vivo., Results: Treatment with PKI-587 enhances sensitivity to cetuximab in vitro, even in the condition of epidermal growth factor receptor (EGFR) resistance. The combination of the two drugs inhibits cells survival, impairs the activation of signalling pathways and induces apoptosis. Interestingly, although significant inhibition of proliferation is observed in all cell lines treated with PKI-587 in combination with cetuximab, activation of apoptosis is evident in sensitive but not in resistant cell lines, in which autophagy is pre-eminent. In nude mice xenografted with resistant Kyse30 cells, the combined treatment significantly reduces tumour growth and prolongs mice survival., Conclusions: Phosphoinositide 3-kinase/mammalian target of rapamycin inhibition has an important role in the rescue of cetuximab resistance. Different mechanisms of cell death are induced by combined treatment depending on basal anti-EGFR responsiveness.

Background and Objective: In the last 20 years, research efforts have been focused on the use of non-invasive ventilation (NIV) as a mean of avoiding tracheostomy in patients affected by neuromuscular diseases (NMD). Nocturnal NIV has been a particular focus as sleep is a risk factor for respiratory failure in NMD patients. The objective of our study was to evaluate the efficacy of nocturnal NIV in improving the respiratory function of NMD patients evaluated by polysomnography (PSG) and arterial blood gas (ABG) analysis parameters., Methods: Ten children affected by NMD underwent PSG and ABG analysis evaluation at the onset of their respiratory failure and during nocturnal NIV therapy., Results: We found a statistically significant improvement of the lowest oxygen desaturation (nadir SaO2 ), apnoea-hypopnoea index (AHI) and oxygen desaturation index (ODI) after NIV treatment in all patients. Mean SaO2 also improved, although this result was not statistical significant, while the percentage of episodes of desaturation with a SaO2 <90% and <80% decreased with a statistical significance (P < 0.0001). After NIV, only one patient showed an episode of desaturation lasting more than 5 min (10.6 min length), and we also found an improvement of daytime blood gas parameters with a normalization of these indexes., Conclusions: NIV was effective in improving respiratory parameters at night in patients affected by respiratory muscular weakness, as evaluated by PSG and ABG analysis., (© 2013 The Authors. Respirology © 2013 Asian Pacific Society of Respirology.)

Background: The human DKC1 gene is causative of X-linked dyskeratosis congenita (X-DC), a syndrome characterized by mucocutaneous features, bone marrow failure, tumor susceptibility, perturbation of stem cell function, and premature aging. DKC1 is thought to produce a single protein, named dyskerin, which shows strict nucleolar localization and participates in at least two distinct nuclear functional complexes: the H/ACA small nucleolar ribonucleoproteic complex involved in RNA pseudouridylation and the active telomerase complex., Methods: By bioinformatics and molecular analyses we identified a DKC1 splice variant able to encode a truncated form of dyskerin, confirmed its active expression in diverse human tissues by RT-PCR, and showed by immunoblotting and immunocytochemistry experiments that it actually encodes a novel protein. Stably transfected clones over-expressing the new isoform were analyzed for growth, morphology and adhesion properties., Results: Our results show that DKC1 encodes a new alternatively spliced mRNA able to direct the synthesis of a variant dyskerin with unexpected cytoplasmic localization. Intriguingly, when over-expressed in HeLa cells, the new isoform promotes cell to cell and cell to substratum adhesion, increases the cell proliferation rate and leads to cytokeratin hyper-expression., Conclusions and General Significance: Our results highlight a novel degree of complexity and regulation of the human DKC1 gene and reveal that it can play a further, unpredicted role in cell adhesion. The identification of a dyskerin cytoplasmic variant reinforces the view that other mechanisms, in addition to telomere instability, can significantly contribute to the pathogenesis of the X-DC, and suggests that DKC1 nucleolar and cytoplasmic functions might cumulatively account for the plethora of manifestations displayed by this syndrome., (2011 Elsevier B.V. All rights reserved.)

Different approaches are being developed to improve the differentiation of Leishmania genus using biochemical and molecular methods. In this study, 11 independent polymorphic microsatellites were used for the typing of strains of L. infantum isolated in Sicily. Polymerase chain reaction was employed to amplify the microsatellites contained in 12 DNA regions selected from among more investigated loci. A total of 51 isolates of L. infantum from dogs were tested by using the same locus panel. The products were successively analysed using an automatic sequence detector (ABI PRISM 3130 AB), to discover relevant microsatellite polymorphisms. It was possible to discriminate between MON-1 and non-MON-1 groups. Moreover, the method permitted to distinguish various genotypes of L. infantum isolates within each zymodema. Model- and distance-based analyses of the data set showed comparable results. The frequency of heterozygosity in the alleles analysed varied extremely between the different groups of isolates. As the method exhibits a high level of discrimination, it is suitable for characterization of closely related strains in epidemiological studies.

Patient satisfaction has become an important indicator of process quality inside hospitals. Even so, the improvement of patient satisfaction cannot simply follow from the implementation of new incentives schemes and organisational arrangements; it also depends on hospitals' cultures and climates. This paper studies the impact of alternative models of organisational climate in hospital wards on patient satisfaction. Data gathered from seven public hospitals in Italy are used to explore this relationship. The theoretical approach adopted is the Competing Value Framework which classifies organisations according to their inward or outward focus and according to the importance assigned to control vs. flexibility. Results show that both a model stressing openness, change and innovation and a model emphasising cohesion and workers' morale are positively related to patient satisfaction, while a model based on managerial control is negatively associated with patient satisfaction.

We describe an unusual case of a 48-year-old white woman diagnosed with positive antihistidyl-trna synthetase antibody polymyositis (PM) shortly after suffering from life-threatening acute respiratory distress syndrome (ARDS). In view of the fact that evaluation for infectious or noninfectious etiologies of ARDS was unrevealing, we reasoned that systemic inflammation in early subclinical PM may have precipitated alveolar-capillary membrane injury. One year after onset, PM has been maintained in remission with oral prednisone and azathioprine. This is the first report of a patient in whom ARDS was the presenting feature of anti-Jo-1 positive PM.

Aim: The aim of this study was to evaluate the sperm abnormalities in young infertile patients with hepatitis B (HBV) or C (HCV) virus infection and to evaluate the additional negative influence of varicocele on sperm parameters in these patients., Methods: Part I. Forty-two infertile patients in Child-Pugh classification A with HBV (n=23) or HCV (n=19) infection, underwent sperm analysis and quantitative detection of HBV-DNA or HCV-RNA in blood serum. Sperm parameters were compared to those of a group of 30 patients with primary infertility due to causes different from liver diseases and/or varicocele). Part II. Twenty-one infertile patients with varicocele associated to HBV (n=11) or HCV (n=10) infection were also enrolled and underwent semen analysis: a group of 39 patients without liver disease, but with varicocele alone served as matched-control group., Results: Part I. HBV patients (with a median HBV-DNA load of 6x10(5) copies/mL, range 1x10(5)-10x10(6)) showed median sperm parameters (sperm density, total number, forward motility and morphology, viability) significantly worse than those found in patients with HCV (with a median HCV-RNA load of 2.3x10(6) copies/mL, range (2x10(5)-12x10(6)). Sperm parameters showed no significant correlation with the duration of infertility neither with the duration of viral infection. Sperm morphology only, exhibited a trend (P=0.06) of negative correlationship (r=-0.59) with the viral HBV-DNA load, whereas the other sperm parameters studied showed no correlation with the viral load. Part II. The group of infertile patients with HBV and varicocele showed median values of all sperm parameter evaluated significantly worse than those found in infertile patients with varicocele alone, or with HCV infection plus varicocele., Conclusions: Patients with HBV infection show worse sperm parameters compared with HCV patients. The additional presence of varicocele further impairs sperm output in HBV patients.

Milk test-day records of 5728 lactations of Italian Simmental cows were analyzed with multivariate factor analysis in order to extract 2 common factors, whose scores were used as quantitative measures of 2 main features of lactation curve shape-i.e., the increasing rate of yield in the first part of lactation and the rate of decline of milk yield after the lactation peak. The 2 indices, objectively derived from the correlation matrix of original test-day records, showed a high discriminant power in separating lactation curves with different shapes. The weak correlation between the 2 factors (0.11), together with the high correlation of factors and the total 305-d yield (about 0.70), suggests that an increase in lactation yield could be achieved by acting only on one of the 2 factors related to lactation-curve shape, with the other kept constant at a medium or low value. The suitability of the 2 factors as descriptors of lactation patterns has been confirmed by the relationships found between factor scores and the main environmental effects known to affect the shape of the lactation curve, such as parity and season of calving.

Background: In patients with essential arterial hypertension (EAH) the left heart ventricular hypertrophy (LVH) causes structural and functional alterations of the coronary vessels that can alter the coronary blood flow reserve. The aim of this study is to evaluate in hypertensive patients with or without LVH versus normotensive subjects, the blood flow and the coronary vasodilatation capability and the coronary blood flow reserve in basal conditions and during dipiridamole i.v. infusion., Methods: Eighty patients have been selected by ECG, echo color Doppler, transesophageal echocardiography: 50 were hypertensive patients with and without LVH, from mild to moderate to severe and 30 were normotensive subjects. The enrolled patients underwent a first transesophageal echocardiography, before and during infusion of 0.86 mg/kg of dipiridamole in growing doses, 0.56 in four minutes followed after three minutes by 0.30 mg/kg. The observation lasted 18 months, and no patients left the study., Results: The coronary resistances in hypertensive patients were significintally reduced during dipiridamole infusion, maintaining their level higher compared to the normal controls. The reduced coronary vasodilatation capability in hypertensive subjects could be due to an increase of the basal vessel tone and/or a reduced compliance of the coronary resistances. The coronary blood flow reserve is significantly reduced in all hypertensive studied, included those without LVH. It is suggested that this is secondary to increase of the coronary blood flow and tone., Conclusions: In conclusion essential arterial hypertension is the cause of early anatomical and functional coronary alterations leading hypertensive subjects to risk for coronary events before LVH.

Background: The objective of this study is to evaluate the oxidative stress status in patients affected by Alzheimer's disease, considering the role played by the free radicals in the progression and determination of this disease., Methods: We have studied 13 patients aged between 65 and 84 years and diagnosed with Alzheimer's disease on the basis of Brain CT scan or MRI results, PTEA (uditive mapped slow potential waves), EEG analysis, evaluation questionnaire (MMSE, ADAS), free radicals levels., Results: This study proved the presence of an oxidative stress status in all patients studied, showing an interaction between the disease and oxidative status. A reduction of the free radicals levels after therapy with determinable anti radicals has also been observed., Conclusions: The existence between Alzheimer's disease and oxidative stress has already been demonstrated. This study is a contribution to this orientation. Further studies are suggested on the preventive effects, but particularly to demonstrate if the use of antioxidants may be able to decrease or stop the evolution of this disease.

In the various sectors of animal science there has been little exploration of the theoretical mathematical aspects of data analysis and modelling. The dominant statistical methods used for the analysis of experimental data are rarely valuable for developing a deeper understanding of the problem. In addition they do not take account of the evolution over time of those variables of major interest to be studied. Only recently have more sophisticated methods of mathematical modelling begun to be used. Nonetheless attention tends to be focused exclusively on empirical models. Mathematical models with greater explanatory power, in particular those which use differential equations, are as yet little used. This work develops a mathematical approach to a problem that is of great interest in animal science: the development over time of milk production in economically important ruminant species.

The effects of three lipid peroxidation end-products, 4-hydroxynonenal (HNE), 2-nonenal (NE) and nonanal, on phosphoinositide-specific phospholipase C (PL-C) activity were studied in HL-60 cells. Enzymatic activity was determined by measuring the amounts of inositol-P3 (Ins-P3) produced by the cells incubated at 37 degrees C in the presence of the various compounds. HNE was shown to activate PL-C at concentrations of between 10(-8) and 10(-6) M; 10(-9) and 10(-8) M of NE also strongly stimulated PL-C. In contrast, nonanal failed to modify enzymatic activity. The concentrations of HNE and NE active on PL-C showed good correspondence with those that have been reported to be chemotactic towards rat neutrophils. The pretreatment of cells with 1 microM pertussis toxin completely prevented the increase of Ins-P3 production induced by HNE and NE. Maximal PL-C stimulation was produced by 10 nM NE; the degree of inositol-P3 production induced by the simultaneous addition of an equimolar dose of HNE was not significantly different from the activity value induced by NE alone, suggesting a possible competition between the two compounds. The data indicate that both HNE and NE share a common mechanism of action which, as with other better-known chemoattractants, involves PL-C activation through a G regulatory protein.

Histiocytic Necrotising Lymphadenitis by Kikuchi-Fujimoto (NHL) is a rare disease of unknown etiopathogenesis, characterized by cervical lymphadenomegaly, fever and asthenia. It has a good prognosis with a complete functional recovery of the affected lymph nodes. In 1998 a 28 year-old patient (A. G.) was admitted in the Department of Internal Medicine, Garibaldi Hospital, Catania for fever, asthenia and cervical lymphadenopathy. Hemato-chemical tests performed during hospitalization showed only relative leukopenia and a significant increase of ESR. An initial cervical lymph node biopsy made the diagnosis of Hodgkin's lymphoma. However, the favourable clinical course and the normalization of the previous altered hematochemical tests, induced to perform a second lymph node biopsy which led to diagnosis of NHL. The patient was given antibiotics and anti-inflammatory drugs for ten days with a complete healing which persisted at a twelve-month follow-up. The conclusion is drawn that NHL of unknown etiopathogenesis and with a good prognosis is more frequent than what revealed by the data in the literature because of its insidious and aspecific clinical presentation.

Myelodysplastic syndromes (MDS) are diseases typical of the adult age, characterized by a clonal alteration of the totipotent staminal cell which causes an inefficient hemopoiesis, reduction of bone marrow cell number, increased bone marrow cell destruction, dysplasia of at least two of the three hemopoietic cell lines and by the tendency to evolve towards acute myeloid leukemia (AML). In patients with MSD, particularly in the advanced primary form, single or multiple chromosomal abnormalities can be found frequently, which may show up and/or modify themselves in any moment of the disease (multistep pathogenetic hypothesis) and whose severity influences significantly the prognosis of MSD patients. In November 1998, a 22 year old female patient (C. C.) was admitted to the Department of Internal medicine of the Garibaldi Hospital, Catania for anemia of unknown origin. The normalities of the hematochemical tests and of other instrumental examinations, induces to perform an osteo-medullary biopsy which revealed the presence of a typical MSD, refractory anemia with excess of blasts type. This disease in its primary form is rare in youth and has a high tendency to evolve in AML. The good clinical conditions of the patient, the absence of chromosomal abnormalities, the normal levels of HbF, the short time interval of the initial diagnosis induced to proceed to bone marrow transplantation, that, as shown by the data reported in the international literature, may give her a good quoad vitam prognosis.

Background: Recent findings have indicated the association between APC-resistance and cerebrovascular disease. These reports prompted us to investigate whether resistance to APC could be found in patients suffering from stroke., Methods: Therefore, we studied APC-resistance in 50 young adults (< or =45 yrs) with a history of ischemic stroke. Eleven out of fifty cerebrovascular subjects showed APC-resistance, while 2 had PC deficiency and 3 PS deficiency. No deficiencies in the anticoagulant protein AT III and in fibrinolytic proteins were found. The family history demonstrated a distribution of APC-resistance compatible with dominant autosomal inheritance. The plasma concentration of prothrombin fragment 1+2 (F1+2), which is a marker of hypercoagulable states, was also measured in patients and family members of resistant subjects (n = 38)., Results: DNA analysis showed factor V R506Q mutation (Leiden mutation) in 11 patients and their relatives with poor response to activated protein C detected by APTT tests. Of 11 investigated subjects with APC-resistance, 9 were heterozygotes and 2, with the lowest APC-ratio values, were homozygotes for factor V mutation. Among 38 relatives, 22 showed a poor response to APC and according to the APC-ratio values, 18 were heterozygotes and 4 homozygotes for FV Leiden mutation. The mutation, in heterozygous form, was also found in 2% of our normal population (n = 100). The plasma concentration of F1+2 was significantly higher both in 11 individuals carrying the FV:Q506 mutation and in 39 patients without APC-resistance compared to that found in the control group. However, the patients with FV:Q506 mutation showed the highest values in F1+2. In the studied family members F1+2 plasma levels were within normal values., Conclusions: Our findings indicate a possible involvement of APC-resistance in the pathogenesis of cerebral thrombosis in young adults and agree with the hypothesis that individuals with APC-resistance have an imbalance between pro-and anti-coagulant forces leading to increased thrombin generation and a hypercoagulable state.

To gain further insights into the immunopharmacological mode of action of the immunosuppressant antibiotic deoxyspergualin (DSP), its effects were evaluated in murine lethal endo- and exotoxemia. These are two cytokine-mediated macrophage and T cell dependent immunoinflammatory conditions that can be induced in D-Galactosamine (D-Gal) presensitized mice by the injections with either LPS or SEB, respectively. The results show that prophylactic treatment with DSP (2.5 or 5 mg/kg bd.wt. 48, 24 and 2 h prior to challenge) neither improved the rate of survival, nor influenced the massive increase in the blood levels of tumor necrosis factor-alpha which followed the challenge with LPS or SEB. In sharp contrast, these clinical and seroimmunological events were both markedly counteracted by prophylactic treatment with sodium fusidate, another immunosuppressive agent used as control.