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Cytotoxic T cell (CTL) infiltration of the tumor carries the potential to limit cancer progression, but their exclusion by the immunosuppressive tumor microenvironment hampers the efficiency of immunotherapy. Here, we show that expression of the axon guidance molecule Plexin-A4 ( Plxna4 ) in CTLs, especially in effector/memory CD8 + T cells, is induced upon T-cell activation, sustained in the circulation, but reduced when entering the tumor bed. Therefore, we deleted Plxna4 and observed that Plxna4 -deficient CTLs acquired improved homing capacity to the lymph nodes and to the tumor, as well as increased proliferation, both achieved through enhanced Rac1 activation. Mice with stromal or hematopoietic Plxna4 deletion exhibited enhanced CTL infiltration and impaired tumor growth. In a melanoma model, adoptive transfer of CTLs lacking Plxna4 prolonged survival and improved therapeutic outcome, which was even stronger when combined with anti-programmed cell death protein 1 (PD-1) treatment. PLXNA4 abundance in circulating CTLs was augmented in melanoma patients versus healthy volunteers but decreased after the first cycle of anti-PD-1, alone or in combination with anti-cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4), in those patients showing complete or partial response to the treatment. Altogether, our data suggest that Plxna4 acts as a "checkpoint," negatively regulating CTL migration and proliferation through cell-autonomous mechanisms independent of the interaction with host-derived Plxna4 ligands, semaphorins. These findings pave the way toward Plxna4-centric immunotherapies and propose Plxna4 detection in circulating CTLs as a potential way to monitor the response to immune checkpoint blockade in patients with metastatic melanoma., (©2021 The Authors; Published by the American Association for Cancer Research.)

SARS-CoV-2 infection is associated with frequent thrombotic events, at the micro and macro-vascular level, due to the perpetuation of a state of hypercoagulability. The so-called 'COVID-19 associated coagulopathy' (CAC) represents a key aspect in the genesis of organ damage from SARS-CoV-2. The main coagulative alterations described in the literature are represented by high levels of D-dimer and fibrinogen. Although CAC has some common features with disseminated intravascular coagulation and sepsis-induced coagulopathy, there are important differences between these clinical pictures and the phenotype of CAC is unique. The pathogenesis of CAC is complex and is affected by the strong interconnection between the inflammatory system and coagulation, in the phenomenon of immunothrombosis and thrombo-inflammation. Several mechanisms come into play, such as inflammatory cytokines, neutrophils, the complement system as well as an alteration of the fibrinolytic system. Finally, an altered platelet function and especially endothelial dysfunction also play a central role in the pathophysiology of CAC. Heparin has several potential effects in CAC, in fact in addition to the anticoagulant effect, it could have a direct antiviral effect and anti-inflammatory properties. The high incidence of thrombo-embolic phenomena despite the use of antithrombotic prophylaxis have led some experts to recommend the use of anticoagulant doses of heparin, but at present the optimal anticoagulant regimen remains to be determined., (Published on behalf of the European Society of Cardiology. © The Author(s) 2021.)

Purpose: The aim of this study was to evaluate mammographic findings associated with invasive lobular carcinoma in different age groups, taking into account breast composition and tumour size., Material and Methods: A total of 1023 invasive lobular carcinoma preoperative mammograms were evaluated. According to the American College of Radiology Breast Imaging Reporting and Data System, cancer mammographic findings were classified as mass, calcifications, architectural distortion, and asymmetry, and breasts were assessed as dense (C or D breast composition) or non-dense (A or B). The patient cohort was subdivided into 3 age groups (< 50, 50-69, ≥ 70 years of age). In order to make the size and age groups dichotomous variables and to perform multiple regression analysis, a cut-off of 10 mm was chosen for tumour size, and < 50-years-old and 50-69-years-old age groups were grouped together (< 70-years-old age group)., Results: Significant results of multivariate analysis were the association between mass finding and non-dense breasts and size ≥ 10 mm ( p < 0.0001), between calcifications, and dense breasts, size < 10 mm and < 70-years-old age group ( p < 0.0001), between distortion and < 70-years-old age group ( p = 0.0366), and between asymmetry and ≥ 70-years-old age group ( p = 0.0090)., Conclusions: Various mammographic findings are differently associated with age group, breast composition, and tumour size., Competing Interests: The authors report no conflict of interest., (Copyright © Polish Medical Society of Radiology 2021.)

Angiogenesis is an essential process during development. Abnormal angiogenesis also contributes to many disease conditions such as tumor and retinal diseases. Previous studies have established the Hippo signaling pathway effector Yes-associated protein (YAP) as a crucial regulator of angiogenesis. In ECs, activated YAP promotes endothelial cell proliferation, migration and sprouting. YAP activity is regulated by vascular endothelial growth factor (VEGF) and mechanical cues such as extracellular matrix (ECM) stiffness. However, it is unclear how VEGF or ECM stiffness signal to YAP, especially how dephosphorylation of YAP occurs in response to VEGF stimulus or ECM stiffening. Here, we show that protein phosphatase 2A (PP2A) is required for this process. Blocking PP2A activity abolishes VEGF or ECM stiffening mediated YAP activation. Systemic administration of a PP2A inhibitor suppresses YAP activity in blood vessels in developmental and pathological angiogenesis mouse models. Consistently, PP2A inhibitor also inhibits sprouting angiogenesis. Mechanistically, PP2A directly interacts with YAP, and this interaction requires proper cytoskeleton dynamics. These findings identify PP2A as a crucial mediator of YAP activation in ECs and hence as an important regulator of angiogenesis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Jiang, Hu, Wu, Cafarello, Di Matteo, Shen, Dong, Adler, Mazzone, Ruiz de Almodovar and Wang.)

The objective of this research was to evaluate the effect of drying temperature and innovative pre-treatments (i.e., microwave and ultrasound) on "Rocha" pear drying behavior and quality characteristics, such as color, total phenolic content and antioxidant activity. Experiments were carried out with pear slabs subjected to microwaves (2450 MHz, 539 W, 4 min, microwave oven) and ultrasounds (35 kHz, 10 min, in an ultrasonic bath) as well as control samples. The drying process was conducted in a tray dryer at three different temperatures (50, 55 and 60 °C) and a fixed air velocity of 0.75 m/s. Microwave technology resulted in a higher quality deterioration in dried pear samples compared to those of controls and ultrasound pre-treated samples. The combined application of ultrasound pre-treatment and the higher drying temperature of 60 °C was characterized by the lowest color changes (ΔE = 3.86 ± 0.23) and higher preservation of nutritional parameters (total phenolic content, TPC = 345.60 ± 8.99; and antioxidant activity, EC 50 = 8.80 ± 0.34). The drying characteristics of pear fruits were also analyzed by taking into account empirical models, with the Page model presenting the best prediction of the drying behavior. In conclusion, ultrasound application is a promising technology to obtain healthy/nutritious dried "Rocha" pear snacks as dietary sources for consumers.

Chitosan-based edible coatings represent an eco-friendly and biologically safe preservative tool to reduce qualitative decay of fresh and ready-to-eat fruits during post-harvest life due to their lack of toxicity, biodegradability, film-forming properties, and antimicrobial actions. Chitosan-based coatings modulate or control oxidative stress maintaining in different manner the appropriate balance of reactive oxygen species (ROS) in fruit cells, by the interplay of pathways and enzymes involved in ROS production and the scavenging mechanisms which essentially constitute the basic ROS cycle. This review is carried out with the aim to provide comprehensive and updated over-view of the state of the art related to the effects of chitosan-based edible coatings on anti-oxidant systems, enzymatic and non-enzymatic, evaluating the induced oxidative damages during storage in whole and ready-to-eat fruits. All these aspects are broadly reviewed in this review, with particular emphasis on the literature published during the last five years.

Background and Aims: Ulcerative colitis [UC] is associated with excessive neutrophil infiltration and collateral tissue damage, but the link is not yet completely understood. Since c-MET receptor tyrosine kinase [MET] is required for neutrophil chemoattraction and cytotoxicity in response to its ligand hepatocyte growth factor [HGF], we aimed to identify the function of HGF-MET signalling in neutrophils in UC patients and in mice during intestinal inflammation., Methods: Serum and colonic biopsies from healthy controls and UC patients with active [Mayo endoscopic subscore 2-3] and inactive [Mayo endoscopic subscore 0-1] disease were collected to assess the level of serum and colonic HGF. Disease progression and immune cell infiltration were assessed during dextran sodium sulphate [DSS] colitis in wild-type and MRP8-Cre MET-LoxP mice., Results: Increased mucosal HGF expression was detected in patients with active UC, and in mice during the inflammatory phase of DSS colitis. Similarly, serum HGF was significantly increased in active UC patients and positively correlated with C-reactive protein and blood neutrophil counts. Flow cytometric analysis also demonstrated an upregulation of colonic MET+ neutrophils during DSS colitis. Genetic ablation of MET in neutrophils reduced the severity of DSS-induced colitis. Concomitantly, there was a decreased number of TH17 cells, which could be due to a decreased production of IL-1β by MET-deficient neutrophils., Conclusions: These data highlight the central role of neutrophilic HGF-MET signalling in exacerbating damage during intestinal inflammation. Hence, selective blockade of this pathway in neutrophils could be considered as a novel therapeutic approach in UC., (© The Author(s) 2020. Published by Oxford University Press on behalf of European Crohn’s and Colitis Organisation. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Muscle regeneration is sustained by infiltrating macrophages and the consequent activation of satellite cells 1-4 . Macrophages and satellite cells communicate in different ways 1-5 , but their metabolic interplay has not been investigated. Here we show, in a mouse model, that muscle injuries and ageing are characterized by intra-tissue restrictions of glutamine. Low levels of glutamine endow macrophages with the metabolic ability to secrete glutamine via enhanced glutamine synthetase (GS) activity, at the expense of glutamine oxidation mediated by glutamate dehydrogenase 1 (GLUD1). Glud1-knockout macrophages display constitutively high GS activity, which prevents glutamine shortages. The uptake of macrophage-derived glutamine by satellite cells through the glutamine transporter SLC1A5 activates mTOR and promotes the proliferation and differentiation of satellite cells. Consequently, macrophage-specific deletion or pharmacological inhibition of GLUD1 improves muscle regeneration and functional recovery in response to acute injury, ischaemia or ageing. Conversely, SLC1A5 blockade in satellite cells or GS inactivation in macrophages negatively affects satellite cell functions and muscle regeneration. These results highlight the metabolic crosstalk between satellite cells and macrophages, in which macrophage-derived glutamine sustains the functions of satellite cells. Thus, the targeting of GLUD1 may offer therapeutic opportunities for the regeneration of injured or aged muscles.

The aim of this research was to study the evolution of carotenoid compounds, antioxidant β-ctivity, volatiles and sensory quality in two mango cultivars dried at 50, 60 and 70 °C. Total carotenoids in fresh samples were about 12 and 6 mg/100 g (dry basis) in Keitt and Osteen samples, respectively. β-carotene was the main carotenoid, representing about 50% of total carotenoids. In both cultivars, carotenoids were more susceptible to drying at 60 °C. Total phenols and metal reduction activity were higher in Osteen than in Keitt, which had higher values in radical scavenging capacity. The antioxidant activities were best preserved with drying temperatures at 50 °C in Keitt and 60 °C in Osteen fruits. Fresh Osteen mango fruits had a volatile compound content of about 37.1, while Keitt of about 5.2 mg/kg (dry basis). All the compounds with odorous impact were significantly reduced after drying. As regards organoleptic characteristics through sensory analysis, Keitt dried mangoes were quite similar to the fresh product, compared to Osteen.

The arrival of the Longobards in Northern Italy in 568 CE marked a period of renewed political stability in the Peninsula after the collapse of the Western Roman Empire. The trajectory of the spread of Longobards in Italy across the Alps and into the South is known from many literary sources. However, their mobility and residence patterns at a population level remain to be fully understood. Here we present a multi-isotopic analysis ( 87 Sr/ 86 Sr and 18 O/ 16 O) of 39 humans and 14 animals buried at the Longobard necropolis of Povegliano Veronese (VR, Italy; 6th-8th century CE), to address mode and tempo of the spread of this population in the Peninsula. The geographical location of Povegliano Veronese plays a key role: the site lies along the Via Postumia, which was one of the main ancient Roman roads of Northern Italy, representing an important route in post-classical Italy. The integration of isotopic data with the archaeological evidence allowed us to determine the presence of individuals from at least three different regions of origin, building a diachronic map of the dynamics of mobility of this group in northern Italy.

We established a semi-high-throughput in vivo screening platform using hyperactive piggyBac (hyPB) transposons (designated as PB-miR) to identify microRNAs (miRs) that inhibit hepatocellular carcinoma (HCC) development in vivo, following miR overexpression in hepatocytes. PB-miRs encoding six different miRs from the miR-17-92 cluster and nine miRs from outside this cluster were transfected into mouse livers that were chemically induced to develop HCC. In this slow-onset HCC model, miR-20a significantly inhibited HCC. Next, we developed a more aggressive HCC model by overexpression of oncogenic Harvey rat sarcoma viral oncogene homolog (HRAS G12V ) and c-MYC oncogenes that accelerated HCC development after only 6 weeks. The tumor suppressor effect of miR-20a could be demonstrated even in this rapid-onset HRAS G12V /c-MYC HCC model, consistent with significantly prolonged survival and decreased HCC tumor burden. Comprehensive RNA expression profiling of 95 selected genes typically associated with HCC development revealed differentially expressed genes and functional pathways that were associated with miR-20a-mediated HCC suppression. To our knowledge, this is the first study establishing a direct causal relationship between miR-20a overexpression and liver cancer inhibition in vivo. Moreover, these results demonstrate that hepatocyte-specific hyPB transposons are an efficient platform to screen and identify miRs that affect overall survival and HCC tumor regression., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Drying characteristics of persimmon, cv. "Rojo Brillante", slabs were experimentally determined in a hot air convective drier at drying temperatures of 45, 50, 55, 60, and 65 °C at a fixed air velocity of 2.3 m/s. It was observed that the drying temperature affected the drying time, shrinkage, and colour. Four empirical mathematical models namely, Enderson and Pabis, Page, Logarithmic, and Two term, were evaluated in order to deeply understand the drying process (moisture ratio). The Page model described the best representation of the experimental drying data at all investigated temperatures (45, 50, 55, 60, 65 °C). According to the evaluation of the shrinkage models, the Quadratic model provided the best representation of the volumetric shrinkage of persimmons as a function of moisture content. Overall, higher drying temperature (65 °C) improved the colour retention of dried persimmon slabs., Competing Interests: The authors declare no conflict of interest.

Among mammary tumor-infiltrating immune cells, the highest expression of podoplanin (PDPN) is found in a subset of tumor-associated macrophages (TAMs). We hereby demonstrate that PDPN is involved in the attachment of this TAM subset to lymphatic endothelial cells (LECs). Mechanistically, the binding of PDPN to LEC-derived galectin 8 (GAL8) in a glycosylation-dependent manner promotes the activation of pro-migratory integrin β1. When proximal to lymphatics, PDPN-expressing macrophages (PoEMs) stimulate local matrix remodeling and promote vessel growth and lymphoinvasion. Anti-integrin β1 blockade, macrophage-specific Pdpn knockout, or GAL8 inhibition impairs TAM adhesion to LECs, restraining lymphangiogenesis and reducing lymphatic cancer spread. In breast cancer patients, association of PoEMs with tumor lymphatic vessels correlates with incidences of lymph node and distant organ metastasis., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The reduction of alcohol content in wines has two main objectives, the former is decreasing the wines' strength and the latter is producing new low alcohol beverages. To accomplish the latter, in this study, we focused on the dealcoholization of a white wine (cv Falanghina , 12.5 vol%) obtained from an ancient Italian grape variety that has recently aroused a renewed interest. It was dealcoholized at various alcohol content levels ranging from 9.8 to 0.3 vol% through the osmotic distillation process, and the main quality parameters of the obtained dealcoholized samples were evaluated. No significant differences ( p  < 0.05) in total phenols, flavonoids, organic acids and total acidity were observed among the wine samples at different alcohol content levels. On the contrary, the volatile compounds content decreased with increasing alcohol removal. Specifically, almost 50% of higher alcohols with acids and lactones were preserved in dealcoholized wine at 9.8 vol% alcohol content, but this percentage reduced to 30% in the sample at 6.8 vol%, and was even lower in the dealcoholized wine with lower alcohol content. It was argued that the transport of volatile compounds through the membrane, beside the membrane selectivity, is highly correlated with the Henry constant (R 2  > 0.8021 for 9.8 vol% of dealcoholized wine). Moreover, results of the sensory evaluation indicated a significant change in terms of acidity, odour, sweetness and body taste in dealcoholized wine (0.3 vol%), giving an overall perceived imbalance and unacceptable taste with respect to the original wine. Therefore, in order to balance acid sensation and enhance body and aftertaste, an attempt was made to formulate an alcohol-free wine-based beverage with enhanced odour and sweetness, by adding some floral wine flavours, up to the amount present in the original wine.

In mammalian cells, aberrant iNOS induction may have detrimental consequences, and seems to be involved in the proliferation and progression of different tumors, such as malignant gliomas. Therefore, selective inhibition of iNOS could represent a feasible therapeutic strategy to treat these conditions. In this context, we have previously disclosed new acetamidines able to inhibit iNOS with a very high selectivity profile over eNOS or nNOS. Here we report the synthesis of a new series of compounds structurally related to the leading scaffold of N-[(3-aminomethyl)benzyl] acetamidine (1400 W), together with their in vitro activity and selectivity. Compound 39 emerged as the most promising molecule of this series, and it was ex vivo evaluated on isolated and perfused resistance arteries, confirming a high selectivity toward iNOS inhibition. Moreover, C6 rat glioma cell lines biological response to 39 was investigated, and preliminary MTT assay showed a significant decrease in cell metabolic activity of C6 rat glioma cells. Finally, results of a docking study shed light on the binding mode of 39 into NOS catalytic site., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Due to their interesting ferroelectric, conductive and dielectric properties, in recent years, perovskite-structured materials have begun to attract increasing interest in the biosensing field. In this study, a strontium titanate perovskite layer (SrTiO₃) has been synthesized on a platinum electrode and exploited for the development of an impedimetric label-free immunosensor for Escherichia coli O157:H7 detection. The electrochemical characterization of the perovskite-modified electrode during the construction of the immunosensor, as well as after the interaction with different E. coli O157:H7 concentrations, showed a reproducible decrease of the total capacitance of the system that was used for the analytical characterization of the immunosensor. Under optimized conditions, the capacitive immunosensor showed a linear relationship from to 1 to 7 log cfu/mL with a low detection limit of 1 log cfu/mL. Moreover, the atomic force microscopy (AFM) technique underlined the increase in roughness of the SrTiO₃-modified electrode surface after antibody immobilization, as well as the effective presence of cells with the typical size of E. coli ., Competing Interests: The authors declare no conflict of interest.

Oxygen-dependent HIF1α hydroxylation and degradation are strictly controlled by PHD2. In hypoxia, HIF1α partly escapes degradation because of low oxygen availability. Here, we show that PHD2 is phosphorylated on serine 125 (S125) by the mechanistic target of rapamycin (mTOR) downstream kinase P70S6K and that this phosphorylation increases its ability to degrade HIF1α. mTOR blockade in hypoxia by REDD1 restrains P70S6K and unleashes PP2A phosphatase activity. Through its regulatory subunit B55α, PP2A directly dephosphorylates PHD2 on S125, resulting in a further reduction of PHD2 activity that ultimately boosts HIF1α accumulation. These events promote autophagy-mediated cell survival in colorectal cancer (CRC) cells. B55α knockdown blocks neoplastic growth of CRC cells in vitro and in vivo in a PHD2-dependent manner. In patients, CRC tissue expresses higher levels of REDD1, B55α, and HIF1α but has lower phospho-S125 PHD2 compared with a healthy colon. Our data disclose a mechanism of PHD2 regulation that involves the mTOR and PP2A pathways and controls tumor growth., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Objective: To correlate imaging parameters from baseline MRI diffusion-weighted imaging (DWI) and fludeoxyglucose (FDG) positron emission tomography (PET)-CT with synchronous and metachronous metastases in mucinous carcinoma (MC) and non-mucinous carcinoma (NMC) rectal cancer., Methods: 111 patients with extraperitoneal locally advanced rectal cancer, who underwent pelvic MRI, DWI and FDG PET-CT, were stratified into MC (n = 23) and NMC (n = 88). We correlated adverse morphologic features on MRI [mT4, mesorectal fascia involvement, extramural venous invasion (mEMVI), mN2] and quantitative imaging parameters [minimum apparent diffusion coefficient (ADC min ), maximum standardized uptake value, total lesion glycolysis, metabolic tumour volume, T 2 weighted and DWI tumour volumes] with the presence of metastatic disease. All patients underwent pre-operative chemoradiation therapy (CRT); 100/111 patients underwent surgery after CRT and were classified as pathological complete response (PCR) and no PCR [tumour regression grade (TRG)1 vs TRG2-5] and as ypN0 and ypN1-2. Median follow-up time was 48 months. Metastases were confirmed on FDG PET-CT and contrast-enhanced multidetector CT., Results: The percentage of mucin measured by MRI correlates with that quantified by histology. On multivariate analysis, the synchronous metastases were correlated with mEMVI [odds ratio (OR) = 21.48, p < 0.01] and low ADC min (OR = 0.04, p = 0.038) in NMC. The difference of metachronous recurrence between the MC group (10-90% mucin) and NMC group was significant (p < 0.01) (OR = 21.67, 95% confidence interval 3.8-120.5). Metachronous metastases were correlated with ypN2 (OR = 8.24, p = 0.01) in MC and in NMC. In NMC, mEMVI correlated with no PCR (p = 0.018) and ypN2 (p < 0.01)., Conclusion: mEMVI could identify patients with NMC, who are at high risk of synchronous metastases. The MC group is at a high risk of developing metachronous metastases. Advances in knowledge: Patients at high risk of metastases are more likely to benefit from more aggressive neoadjuvant therapy.

In this work the development and optimization of an impedimetric label free immunosensor for the detection of Ochratoxin A (OTA) is reported. Two antibody immobilization methods (oriented and not oriented) were compared highlighting a lower limit of detection (5pg/ml) for the not oriented immobilization but a closer linear range in contrast to oriented anti-OTA immunosensors which showed linearity in the range of 0.01-5ng/mL OTA. The analysis of the Atomic Force Microscopy (AFM) images showed two different nanostructures indicating that the use of oriented immobilization created a more ordered and highly dense antibody surface. Finally the oriented immunosensor was used to quantify OTA in spiked cocoa bean samples and the results were compared with those registered with competitive ELISA kit. The immunosensor was sensitive to OTA lower than 2μg/kg that represents the lower acceptable limit of OTA established by European legislation for the common food products., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Background: Our objective was to investigate the relationship between asymmetric increase in breast vascularity (AIBV) and pathologic profiles of breast cancer. We also addressed the prognostic performance of AIBV and of vascular maps reduction after neoadjuvant chemotherapy (NAC) in predicting pathologic complete response (pCR) at surgery and outcome at follow-up., Materials and Methods: Two hundred nineteen patients with unilateral locally advanced breast cancer (LABC) underwent magnetic resonance imaging before and after NAC. Axial, sagittal, and coronal maximum intensity projections were obtained in a subjective comparative evaluation. Asymmetrical versus symmetrical breast vascularity was defined through number of vessels, diameter, and signal intensity. Kaplan-Meier methodology was employed for late survival (31.4 ± 18 months follow-up)., Results: AIBV ipsilateral to LABC occurred in 62.5% (P < .001). AIBV was significantly associated with invasive ductal carcinoma, G3, triple-negative, HER2+, and hybrid phenotypes (P < .001). pCR was more frequent among patients with AIBV (24%) (P = .001). After NAC, the vascular map was significantly reduced, particularly in patients with pCR (P < .001). At follow-up, the recurrence rate was 22% (6.1% mortality). AIBV after NAC was associated with worse late survival (P = .036). A trend towards worse late survival existed among patients with AIBV before NAC. We did not observe statistically different survival according to the variation of vascularity after NAC., Conclusion: LABC with ipsilateral AIBV before NAC is associated with more aggressive pathologic profiles. Nonetheless, it is more sensitive to NAC and shows a higher frequency of pCR. The persistence of AIBV after NAC entails a worse late prognosis and should prompt more aggressive therapeutic strategies., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Hypoxic tumor-associated macrophages (TAMs) acquire angiogenic and immunosuppressive properties. Yet it remains unknown if metabolic changes influence these functions. Here, we argue that hypoxic TAMs strongly upregulate the expression of REDD1, a negative regulator of mTOR. REDD1-mediated mTOR inhibition hinders glycolysis in TAMs and curtails their excessive angiogenic response, with consequent formation of abnormal blood vessels. Accordingly, REDD1 deficiency in TAMs leads to the formation of smoothly aligned, pericyte-covered, functional vessels, which prevents vessel leakiness, hypoxia, and metastases. Mechanistically, highly glycolytic REDD1-deficient TAMs outcompete endothelial cells for glucose usage that thwarts vascular hyperactivation and promotes the formation of quiescent vascular junctions. Tuning down glycolysis in REDD1 knockout TAMs re-establishes abnormal angiogenesis and metastases. On this basis, we prove that the anti-tumor effect of mTOR inhibitors is partly countered by the deleterious outcome of these drugs on TAMs. Our data provide a functional link between TAM metabolism and tumor angiogenesis., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Nitric oxide (NO) is an essential endogenous mediator with a physiological role in the central nervous system as neurotransmitter and neuromodulator. A growing number of studies have demonstrated that abnormal nitrergic signaling is a crucial event in the development of neurodegeneration. In particular, the uncontrolled production of NO by neuronal nitric oxide synthase (nNOS) is observed in several neurodegenerative diseases. Moreover, it is well recognized that specific isoforms of human carbonic anhydrase (hCA) physiologically modulate crucial pathways of signal processing and that low expression of CA affects cognition, leading to mental retardation, Alzheimer's disease, and aging-related cognitive impairments. In light of this, dual agents that are able to target both NOS (inhibition) and CA (activation) could be useful drug candidates for the treatment of Alzheimer's disease, aging, and other neurodegenerative diseases. In the present work, we show the design, synthesis, and in vitro biological evaluation of new nitrogen-based heterocyclic compounds. Among the tested molecules, 2-amino-3-(4-hydroxyphenyl)-N-(1H-indazol-5-yl)propanamide hydrochloride (10 b) was revealed to be a potent dual agent, able to act as a selective nNOS inhibitor and activator of the hCA I isoform., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The most frequently used treatment for hormone receptor positive breast cancer in post-menopausal women are aromatase inhibitors. In order to develop new aromatase inhibitors, we designed and synthesized new imidazolylmethylpiperidine sulfonamides using the structure of the previously identified aromatase inhibitor SYN 20028567 as starting lead. By this approach, three new aromatase inhibitors with IC50 values that are similar to that of letrozole and SYN 20028567 were identified., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Objective: Cancer immunology is a growing field of research whose aim is to develop innovative therapies and diagnostic tests. Starting from the hypothesis that immune cells promptly respond to harmful stimuli, we used peripheral blood monocytes in order to characterise a distinct gene expression profile and to evaluate its potential as a candidate diagnostic biomarker in patients with colorectal cancer (CRC), a still unmet clinical need., Design: We performed a case-control study including 360 peripheral blood monocyte samples from four European oncological centres and defined a gene expression profile specific to CRC. The robustness of the genetic profile and disease specificity were assessed in an independent setting., Results: This screen returned 43 putative diagnostic markers, which we refined and validated in the confirmative multicentric analysis to 23 genes with outstanding diagnostic accuracy (area under the curve (AUC)=0.99 (0.99 to 1.00), Se=100.0% (100.0% to 100.0%), Sp=92.9% (78.6% to 100.0%) in multiple-gene receiver operating characteristic analysis). The diagnostic accuracy was robustly maintained in prospectively collected independent samples (AUC=0.95 (0.85 to 1.00), Se=92.6% (81.5% to 100.0%), Sp=92.3% (76.9% to 100.0%). This monocyte signature was expressed at early disease onset, remained robust over the course of disease progression, and was specific for the monocytic fraction of mononuclear cells. The gene modulation was induced specifically by soluble factors derived from transformed colon epithelium in comparison to normal colon or other cancer histotypes. Moreover, expression changes were plastic and reversible, as they were abrogated upon withdrawal of these tumour-released factors. Consistently, the modified set of genes reverted to normal expression upon curative treatment and was specific for CRC., Conclusions: Our study is the first to demonstrate monocyte plasticity in response to tumour-released soluble factors. The identified distinct signature in tumour-educated monocytes might be used as a candidate biomarker in CRC diagnosis and harbours the potential for disease follow-up and therapeutic monitoring., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)

Nitric Oxide Synthase (NOS) inhibitors could play a powerful role in inflammatory and neurodegenerative diseases. In this work, novel acetamidine derivatives of NOS were synthesized and the inhibitor activity was evalued. To screen the activity and selectivity, the l-citrulline residue, after the enzymatic NOS assay, was derivatized with o-phthaldialdehyde/N-acetyl cysteine (OPA/NAC) and then evaluated by RP-HPLC method with fluorescence detection. All compounds did not affect the activity of endothelial and neuronal isoforms, while nine of them possessed a percentage of iNOS activity at 10μM lower than 50%, and were selected for IC50 evaluation. Among them, a compound emerged as a very potent (IC50 of 53nM) and selective iNOS inhibitor., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Duchenne muscular dystrophy (DMD) is a genetic neuromuscular disorder caused by the absence of dystrophin. We developed a novel gene therapy approach based on the use of the piggyBac (PB) transposon system to deliver the coding DNA sequence (CDS) of either full-length human dystrophin (DYS: 11.1 kb) or truncated microdystrophins (MD1: 3.6 kb; MD2: 4 kb). PB transposons encoding microdystrophins were transfected in C2C12 myoblasts, yielding 65±2% MD1 and 66±2% MD2 expression in differentiated multinucleated myotubes. A hyperactive PB (hyPB) transposase was then deployed to enable transposition of the large-size PB transposon (17 kb) encoding the full-length DYS and green fluorescence protein (GFP). Stable GFP expression attaining 78±3% could be achieved in the C2C12 myoblasts that had undergone transposition. Western blot analysis demonstrated expression of the full-length human DYS protein in myotubes. Subsequently, dystrophic mesoangioblasts from a Golden Retriever muscular dystrophy dog were transfected with the large-size PB transposon resulting in 50±5% GFP-expressing cells after stable transposition. This was consistent with correction of the differentiated dystrophic mesoangioblasts following expression of full-length human DYS. These results pave the way toward a novel non-viral gene therapy approach for DMD using PB transposons underscoring their potential to deliver large therapeutic genes., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: We evaluated the diagnostic performance of the baseline diffusion weighted imaging (DWI) and the apparent diffusion coefficient (ADC) in the prediction of a complete pathologic response (pCR) to neoadjuvant chemotherapy (NAC) in patients with breast cancer stratified according to the tumor phenotype., Patients and Methods: We retrospectively studied 225 patients with stage II, III, and IV breast cancer who had undergone contrast-enhanced magnetic resonance imaging (MRI) and DWI before and after NAC, followed by breast surgery., Results: The tumor phenotypes were luminal (n = 143; 63.6%), triple-negative (TN) (n = 37; 16.4%), human epidermal growth factor receptor 2 (HER2)-enriched (n = 17; 7.6%), and hybrid (hormone receptor-positive/HER2(+); n = 28; 12.4%). After NAC, a pCR was observed in 39 patients (17.3%). No statistically significant difference was observed in the mean ADC value between a pCR and no pCR in the general population (1.132 ± 0.191 × 10(-3) mm(2)/s vs. 1.092 ± 0.189 × 10(-3) mm(2)/s, respectively; P = .23). The optimal ADC cutoff value in the general population was 0.975 × 10(-3) mm(2)/s (receiver operating characteristic [ROC] area under the curve [AUC], 0.587 for the prediction of a pCR). After splitting the population into subgroups according to tumor phenotype, we observed a significant or nearly significant difference in the mean ADC value among the responders versus the nonresponders in the TN (P = .06) and HER2(+) subgroups (P = .05). No meaningful difference was seen in the luminal and hybrid subgroups (P = .59 and P = .53, respectively). In contrast, in the TN and HER2(+) subgroups (cutoff value, 0.995 × 10(-3) mm(2)/s and 0.971 × 10(-3) mm(2)/s, respectively), we observed adequate ROC AUCs (0.766 and 0.813, respectively)., Conclusion: The pretreatment ADC value is not capable of predicting the pCR in the overall population of patients with locally advanced breast cancer. Nonetheless, an ameliorated diagnostic performance was observed in specific phenotype subgroups (ie, TN and HER2(+) tumors)., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Mutations or amplification of the MET proto-oncogene are involved in the pathogenesis of several tumours, which rely on the constitutive engagement of this pathway for their growth and survival. However, MET is expressed not only by cancer cells but also by tumour-associated stromal cells, although its precise role in this compartment is not well characterized. Here we show that MET is required for neutrophil chemoattraction and cytotoxicity in response to its ligand hepatocyte growth factor (HGF). Met deletion in mouse neutrophils enhances tumour growth and metastasis. This phenotype correlates with reduced neutrophil infiltration to both the primary tumour and metastatic sites. Similarly, Met is necessary for neutrophil transudation during colitis, skin rash or peritonitis. Mechanistically, Met is induced by tumour-derived tumour necrosis factor (TNF)-α or other inflammatory stimuli in both mouse and human neutrophils. This induction is instrumental for neutrophil transmigration across an activated endothelium and for inducible nitric oxide synthase production upon HGF stimulation. Consequently, HGF/MET-dependent nitric oxide release by neutrophils promotes cancer cell killing, which abates tumour growth and metastasis. After systemic administration of a MET kinase inhibitor, we prove that the therapeutic benefit of MET targeting in cancer cells is partly countered by the pro-tumoural effect arising from MET blockade in neutrophils. Our work identifies an unprecedented role of MET in neutrophils, suggests a potential 'Achilles' heel' of MET-targeted therapies in cancer, and supports the rationale for evaluating anti-MET drugs in certain inflammatory diseases.

N-[(3-Aminomethyl)benzyl]acetamidine derivatives were synthesized and in vitro evaluated as inhibitors of the inducible isoform of nitric oxide synthase (iNOS). Because of the high potency of action and the excellent selectivity over the endothelial nitric oxide synthase (eNOS), compound 10 was ex vivo evaluated on isolated and perfused resistance arteries. The results confirm that compound 10 selectively inhibits the iNOS, without affecting the endothelial isoform. The outcome of the docking studies showed that the hydrophobic interaction is the driving force of the binding process, especially for iNOS, where the binding pocket is characterized by a significant lipophilic region.

The robustness and safety of liver-directed gene therapy can be substantially improved by enhancing expression of the therapeutic transgene in the liver. To achieve this, we developed a new approach of rational in silico vector design. This approach relies on a genome-wide bio-informatics strategy to identify cis-acting regulatory modules (CRMs) containing evolutionary conserved clusters of transcription factor binding site motifs that determine high tissue-specific gene expression. Incorporation of these CRMs into adeno-associated viral (AAV) and non-viral vectors enhanced gene expression in mice liver 10 to 100-fold, depending on the promoter used. Furthermore, these CRMs resulted in robust and sustained liver-specific expression of coagulation factor IX (FIX), validating their immediate therapeutic and translational relevance. Subsequent translational studies indicated that therapeutic FIX expression levels could be attained reaching 20-35% of normal levels after AAV-based liver-directed gene therapy in cynomolgus macaques. This study underscores the potential of rational vector design using computational approaches to improve their robustness and therefore allows for the use of lower and thus safer vector doses for gene therapy, while maximizing therapeutic efficacy.

The development of robust nonviral vectors could facilitate clinical gene therapy applications and may overcome some of the immune complications of viral vectors. Nevertheless, most nonviral gene deliver approaches typically yield only transient and/or low gene expression. To address these caveats, we have explored piggyBac transposons to correct hemophilia B by liver-directed factor IX (FIX) gene therapy in hemophilic mice. To achieve this, we combined the use of: (i) a hyperactive codon-optimized piggyBac transposase, (ii) a computationally enhanced liver-specific promoter, (iii) a hyperfunctional codon-optimized FIX transgene (FIX R338L Padua), and (iv) a modification of the transposon terminal repeats. This combination strategy resulted in a robust 400-fold improvement in vector performance in hepatocytes, yielding stable supraphysiologic human FIX activity (>1 year). Liver-specific expression resulted in the induction of FIX-specific immune tolerance. Remarkably, only very low transposon/transposase doses were required to cure the bleeding diathesis. Similarly, PB transposons could be used to express supraphysiologic factor VIII levels using low transposon/transposase doses. PB transposition did not induce tumors in a sensitive hepatocellular carcinoma-prone mouse model. These results underscore the potency and relative safety of the latest generation PB transposons, which constitutes a versatile platform for stable and robust secretion of therapeutic proteins.

Aim: The estimation of response to neoadjuvant chemotherapy (NAC) is useful in the surgical decision in breast cancer. We addressed the diagnostic reliability of conventional MRI, of diffusion weighted imaging (DWI) and of a merged criterion coupling morphological MRI and DWI. Diagnostic performance was analysed separately in different tumor subtypes, including HER2+ (human epidermal growth factor receptor 2)/HR+ (hormone receptor) (hybrid phenotype)., Materials and Methods: Two-hundred and twenty-five patients underwent MRI before and after NAC. The response to treatment was defined according to the RECIST classification and the evaluation of DWI with apparent diffusion coefficient (ADC). The complete pathological response - pCR was assessed (Mandard classification)., Results: Tumor phenotypes were Luminal (63.6%), Triple Negative (16.4%), HER2+ (7.6%) or Hybrid (12.4%). After NAC, pCR was observed in 17.3% of cases. Average ADC was statistically higher after NAC (p<0.001) among patients showing pCR vs. those who had not pCR. The RECIST classification showed adequate performance in predicting the pCR in Triple Negative (area under the receiver operating characteristic curve, ROC AUC=0.9) and in the HER2+ subgroup (AUC=0.826). Lower performance was found in the Luminal and Hybrid subgroups (AUC 0.693 and 0.611, respectively), where the ADC criterion yielded an improved performance (AUC=0.787 and 0.722). The coupling of morphological and DWI criteria yielded maximally improved performance in the Luminal and Hybrid subgroups (AUC=0.797 and 0.761)., Conclusion: The diagnostic reliability of MRI in predicting the pCR to NAC depends on the tumor phenotype, particularly in the Luminal and Hybrid subgroups. In these cases, the coupling of morphological MRI evaluation and DWI assessment may facilitate the diagnosis., (Copyright © 2014. Published by Elsevier Ireland Ltd.)

In winemaking gluconic acid is an important marker for quantitative evaluation of grape infection by Botrytis cinerea. A screen-printed amperometric bienzymatic sensor for the determination of gluconic acid based on gluconate kinase (GK) and 6-phospho-D-gluconate dehydrogenase (6PGDH) coimmobilized onto polyaniline/poly (2-acrylamido-2-methyl-1-propanesulfonic acid; PANI-PAAMPSA) is reported in this study. The conductive polymer electrodeposed on the working electrode surface allowed the detection of NADH at low potential (0.1 V) with a linear range from 4 × 10(-3) to 1 mM (R2 = 0.99) and a sensitivity of 419.44 nA∙mM(-1). The bienzymatic sensor has been optimized with regard to GK/6PGDH enzymatic unit ratio and ATP/NADP+ molar ratio which resulted equal to 0.33 and 1.2, respectively. Under these conditions a sensitivity of 255.2 nA∙mM(-1), a limit of detection of 5 μM and a Relative Standard Deviation (RSD) of 4.2% (n = 5) have been observed. Finally, the biosensor has been applied for gluconic acid measurements in must grape samples and the matrix effect has been taken into consideration. The results have been compared with those obtained on the same samples with a commercial kit based on a spectrophotometric enzyme assay and were in good agreement, showing the capability of the bienzymatic PANI-PAAMPSA biosensor for gluconic acid measurements and thus for the evaluation of Botrytis cinerea infection in grapes.

Nitric oxide synthase (NOS) inhibitors are potential drug candidates due to the critical role of an excessive production of nitric oxide in a range of diseases. At present, the radiometric detection of L-[(3)H]-citrulline produced from L-[(3)H]-arginine during the enzymatic reaction is one of the most accepted methods to assess the in vitro activity of NOS inhibitors. Here we report a fast, easy, and cheap reversed-phase high-performance liquid chromatography method with fluorescence detection, based on the precolumn derivatization of L-citrulline with o-phthaldialdehyde/N-acetyl cysteine, for the in vitro screening of NOS inhibitors. To evaluate enzyme inhibition by the developed method, N-[3-(aminomethyl)benzyl]acetamidine, a potent and selective inhibitor of inducible NOS, was used as a test compound. The half maximal inhibitory concentration obtained was comparable to that derived by the well-established radiometric assay., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In an effort to develop safe and efficacious compounds for the treatment of metabolic disorders, new compounds based on a combination of clofibric acid, the active metabolite of clofibrate, and trans-stilbene, chalcone, and other lipophilic groups were synthesized. They were evaluated for PPARα transactivation activity; all branched derivatives showed an increase of the transcriptional activity of receptor compared to the linear ones. Noteworthy, stilbene and benzophenone branched derivatives activated the PPARα better than clofibric acid.

Carotenoids are natural compounds whose nutritional importance comes from the provitamin A activity of some of them and their protection against several serious human disorders. The degradation of carotenoids was investigated during apricot drying by microwave and convective hot-air at 60 and 70 °C. Seven carotenoids were identified: antheraxanthin, lutein, zeaxanthin, β-cryptoxanthin, 13-cis-β-carotene, all-trans-β-carotene and 9-cis-β-carotene; among these, all-trans-β-carotene was found to be about 50 % of total carotenoids. First-order kinetic models were found to better describe all-trans-β-carotene reduction during drying, with a degradation rate constant (k1) that increased two folds when temperatures increased by 10 °C, in both methods. No differences were found in k1 between apricots dried by hot air at 70 °C (k1 = 0.0340 h(-1)) and by microwave at 60 °C. The evolution of total carotenoids (117.1 mg/kg on dry basis) during drying highlighted a wider decrease (about 50%) when microwave heating was employed, for both set temperatures. Antheraxantin was found to be the carotenoid most susceptible to heat, disappearing at 6 h during both trials with microwave as well as during convective hot-air at 70 °C. For this reason, antheraxanthin could be a useful marker for the evaluation of thermal damage due to the drying process. Also the degree of isomerization of all-trans-β-carotene could be a useful marker for the evaluation of the drying process.

Osmotic distillation technique was used for the total dealcoholization of a red wine (Aglianico grape variety) up to 0.19 vol.%. The dealcoholization process was performed in subsequent cycles which gave rise wine samples at different alcoholic degrees. The effect of processing on the main chemical and physical properties of Aglianico wine was evaluated. Among wine samples, no significant differences (p<0.05) of oenological parameters such as pH, total acidity were found. Similarly, the total phenolic, flavonoids and tartaric esters content and the composition of organic acids did not show significant differences (p<0.05) during the process. On the contrary, colour intensity and tonality of wine samples changed significantly when the alcohol reduction was over the 6.5 vol.%. Finally, the total dealcoholized wine showed properties similar to Aglianico wine except for the volatile compounds, which decreased over 98%. Hence, flavour enrichment may be required to produce a pleasurable and delicious non alcoholic beverage from wine., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The identification of novel PPAR ligands represents an attractive research to fully understand the complex biological pathways regulated by these receptors. Selective PPAR modulators, inverse agonists and antagonists of three PPAR isoforms could help to clarify biological effects on lipid and glucose homeostasis. Here we describe the identification of a group of N-(methylsulfonyl)amides, derived from PPARα agonist carboxylic acids. Transactivation and FRET assay confirmed an antagonist behaviour on PPARα for some of these compounds, with submicromolar IC(50). A preliminary analysis on selectivity α/γ revealed different profiles of inhibition or activation., (Copyright © 2012. Published by Elsevier Masson SAS.)

Gene therapy may provide a cure for hemophilia and overcome the limitations of protein replacement therapy. Increasing the potency of gene transfer vectors may allow improvement of their therapeutic index, as lower doses can be administered to achieve therapeutic benefit, reducing toxicity of in vivo administration. Here we generated codon-usage optimized and hyperfunctional factor IX (FIX) transgenes carrying an R338L amino acid substitution (FIX Padua), previously associated with clotting hyperactivity and thrombophilia. We delivered these transgenes to hemophilia B mice by hepatocyte-targeted integration-competent and -defective lentiviral vectors. The hyperfunctional FIX transgenes increased FIX activity reconstituted in the plasma without detectable adverse effects, allowing correction of the disease phenotype at lower vector doses and resulting in improved hemostasis in vivo. The combined effect of codon optimization with the hyperactivating FIX-R338L mutation resulted in a robust 15-fold gain in potency and therefore provides a promising strategy to improve the efficacy, feasibility, and safety of hemophilia gene therapy.

Introduction: The continuous improvement of gene transfer technologies has broad implications for stem cell biology, gene discovery, and gene therapy. Although viral vectors are efficient gene delivery vehicles, their safety, immunogenicity and manufacturing challenges hamper clinical progress. In contrast, non-viral gene delivery systems are less immunogenic and easier to manufacture., Areas Covered: In this review, we explore the emerging potential of transposons in gene and cell therapy. The safety, efficiency, and biology of novel hyperactive Sleeping Beauty (SB) and piggyBac (PB) transposon systems will be highlighted for ex vivo gene therapy in clinically relevant adult stem/progenitor cells, particularly hematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs), myoblasts, and induced pluripotent stem (iPS) cells. Moreover, efforts toward in vivo transposon-based gene therapy will be discussed., Expert Opinion: The latest generation SB and PB transposons currently represent some of the most attractive systems for stable non-viral genetic modification of primary cells, particularly adult stem cells. This paves the way toward the use of transposons as a non-viral gene therapy approach to correct hereditary disorders including those that affect the hematopoietic system. The development of targeted integration into "safe harbor" genetic loci may further improve their safety profile.

The PiggyBac (PB) transposon system was originally derived from the cabbage looper moth Trichoplusia ni and represents one of the most promising transposon systems to date. Engineering of the PB transposase enzyme (PBase) and its cognate transposon DNA elements resulted in a substantial increase in transposition activities. Consequently, this has greatly enhanced the versatility of the PB toolbox. It is now widely used for stable gene delivery into a broad variety of cell types from different species, including mammalian cells. This opened up new perspectives for potential therapeutic applications in the fields of gene therapy and regenerative medicine. In particular, we have recently demonstrated that PB transposons could be used to stably deliver genes into human CD34(+) hematopoietic stem cells (HSCs) resulting in sustained transgene expression in its differentiated progeny. The PB transposon system is particularly attractive for the generation of induced pluripotent stem cells (iPS). Typically, this can be accomplished by stable gene transfer of genes encoding one or more reprogramming factors (i.e., c-MYC, KLF-4, OCT-4, and/or SOX-2). We have generated a PB-based nonviral reprogramming toolbox that contains different combinations of these reprogramming genes. The main advantage of using this PB toolbox for iPS generation is that the reprogramming cassette can be excised by de novo transposase expression, without leaving any molecular trace in the target cell genome. This "traceless excision" paradigm obviates potential risks associated with inadvertent re-expression of reprogramming factors in the iPS progeny. These various applications in gene therapy, stem cell engineering, and regenerative medicine underscore the emerging versatility of the PB toolbox.

Factors contained in physiological microenvironments in tissues where mast cells differentiate and reside may influence mast cell responsiveness and modify antigen-dependent activation. A possible direct or indirect role of mast cell responses in diabetes mellitus prompted us to study the impact of insulin treatment on antigen triggered signaling pathways downstream of FcepsilonRI in bone marrow-derived mouse mast cells (BMMCs). We found that insulin alone stimulates tyrosine phosphorylation of tyrosine kinases Lyn, Syk, Fyn, the adapter protein Gab2 (Grb2-associated binding protein 2), Akt and activates ERK, JNK and p38 kinase. Effect of insulin on FcepsilonRI signaling pathways was marked by enhanced phosphorylation of Lyn, Fyn, Gab2 and Akt. Furthermore, BMMC stimulated with antigen in the presence of insulin responded with enhanced protein kinase theta (PKCtheta) activity and increased JNK phosphorylation when compared to BMMC triggered with antigen alone. Functional studies reveal enhanced degranulation and altered cytoskeletal rearrangement when BMMCs were treated simultaneously with insulin and antigen. Our results suggest that insulin tunes antigen-mediated responses of mast cells., ((c) 2009 Elsevier Ltd. All rights reserved.)

The province of Siena, Italy, enacted a new garbage plan (NGP) with the objective of increasing separate waste collection (SWC), shutting down six landfills and increasing incineration. The aim of the paper is to evaluate costs and benefits of the NGP. The hypothesis is that willingness to pay (WTP) should reflect the value to the community of having better environmental quality, according to the Contingent Valuation literature. The paper reports the results of a contingent valuation (CV). The sample was divided into two subsets: firms and households. Through the information gathered via a detailed questionnaire, parametric and non-parametric estimates were elaborated to analyse the WTP of the population for the benefits flowing from increased SWC, increased incineration and shutting down of landfills. These values were expressed as a share of the tax actually paid. Although a small subset of firms and households valued increasing incineration less positively, requesting compensation, on the whole interviewees (with large differences between firms and households) had a net positive WTP for the provisions included in NGP. Parametric estimation procedures enabled us to analyse the economic as well as social and demographic factors affecting these results. These elements are useful for computing a value for the waste charge that also reflects external effects. Finally, we estimated household income elasticity of WTP for the increase in SWC and found less than one: environmental quality is not a luxury good.

Aim: Despite strong evidence of benefit, few stroke units exist in New Zealand. In this paper, we describe the process and outcome for the country's first, comprehensive Acute Stroke Unit (ASU), established at Middlemore Hospital in 2001., Methods: The evaluation comprised: (a) two independent 'before and after' audits of medical records of a random selection of patients (2 x n=100) identified from Diagnostic Related Group (DRG) discharge codes for stroke in 1999 (12 months) and 2001-02 (9 months); (b) a review of all DRG stroke outcome data and internal cost analyses for the study periods; and (c) a 'time-in-motion' study of nursing care requirements., Results: The DRG data showed an increase in separations (538 vs 613); stable re-admissions (8% vs 7%); and declines in average length of stay (6.1 vs 5.4 days), deaths (14.0% vs 8.8%), and referrals for rehabilitation (127 vs 67); while the audit indicated shorter times from admission to brain imaging, and swallow and allied health assessments, for stroke f rom 1999 to 2001-02. A 1:4 nurse:patient ratio seems to provide an optimum level of care for costs., Conclusions: The introduction of the ASU has been associated with improvements in several key indicators of quality of care for patients with stroke.

The aim of this study was to examine if the growth factor, transforming growth factor beta 1 (TGF beta 1), could prevent induction of nitric oxide synthase and cytokine-mediated inhibitory effects in the insulin-containing, clonal beta cell line RINm5F. Treatment of RINm5F cells for 24 h with interleukin-1 beta (IL-1 beta) (100 pM) induced expression of nitric oxide synthase and inhibited glyceraldehyde-stimulated insulin secretion. Combinations of IL-1 beta (100 pM), tumour necrosis factor-alpha (100 pM) and interferon-gamma (100 pM) reduced RINm5F cell viability (determined by the 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium (MTT) reduction assay) and de novo protein synthesis, as measured by incorporation of radiolabelled amino acids into perchloric acid-precipitable protein. Pretreatment of RINm5F cells with TGF beta 1 (10 pM) for 18 or 24 h, prior to the addition of either IL-1 beta or combined cytokines, prevented cytokine-induced inhibition of insulin secretion, protein synthesis and the loss of cell viability. TGF beta 1 pretreatment inhibited cytokine-induced expression and activity of nitric oxide synthase in RINm5F cells as determined by Western blotting and by cytosolic conversion of radiolabelled arginine into labelled citrulline and nitric oxide. Chemically generated superoxide also induced expression of nitric oxide synthase possibly due to direct activation of the nuclear transcription factor NF kappa B, an effect prevented by both an antioxidant and TGF beta 1 pretreatment. In conclusion, the mechanism of action of TGF beta 1 in blocking cytokine inhibitory effects was by preventing induction of nitric oxide synthase.

We have studied inhibition of glucose-stimulated insulin secretion in islets of Langerhans isolated from adult Sprague-Dawley rats and treated with different alkylating agents. Streptozotocin (STZ), N-methyl-N-nitrosourea (MNU), and N-ethyl-N-nitrosourea (ENU) all released nitric oxide, as demonstrated by an increase in medium nitrite and cellular cyclic GMP. Methyl methanesulphonate (MMS) and ethyl methanesulphonate (EMS), which do not possess a nitroso group, did not show evidence of nitric oxide release. All five compounds, however, decreased glucose-stimulated insulin release, suggesting that nitric oxide release was not necessary for the inhibition of secretion. Lack of involvement of nitric oxide was further suggested by the failure of oxyhaemoglobin to reverse STZ and MNU inhibition of insulin secretion. Since ENU was at least as effective as MNU in inhibiting insulin secretion, it appears that alkylation of DNA at the O6 position of guanine may not be involved in this process.