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c-MET/hepatocyte growth factor (HGF) system deregulation is a well-known feature of malignancy in several solid tumors, and for this reason this system and its pathway have been considered as potential targets for therapeutic purposes. In previous manuscripts we reported c-MET/HGF expression and the role in testicular germ cell tumors (TGCTs) derived cell lines. We demonstrated the key role of c-Src and phosphatidylinositol 3-kinase (PI3K)/AKT adaptors in the HGF-dependent malignant behavior of the embryonal carcinoma cell line NT2D1, finding that the inhibition of these onco-adaptor proteins abrogates HGF triggered responses such as proliferation, migration, and invasion. Expanding on these previous studies, herein we investigated the role of mitogen-activated protein kinase (MAPK)/extracellular signal regulated kinase (ERK) pathways in the HGF-dependent and HGF-independent NT2D1 cells biological responses. To inhibit MAPK/ERK pathways we chose a pharmacological approach, by using U0126 inhibitor, and we analyzed cell proliferation, collective migration, and chemotaxis. The administration of U0126 together with HGF reverts the HGF-dependent activation of cell proliferation but, surprisingly, does not exert the same effect on NT2D1 cell migration. In addition, we found that the use of U0126 alone significantly promotes the acquisition of NT2D1 «migrating phenotype», while collective migration of NT2D1 cells was stimulated. Notably, the inhibition of ERK activation in the absence of HGF stimulation resulted in the activation of the AKT-mediated pathway, and this let us speculate that the paradoxical effects obtained by using U0126, which are the increase of collective migration and the acquisition of partial epithelium–mesenchyme transition (pEMT), are the result of compensatory pathways activation. These data highlight how the specific response to pathway inhibitors, should be investigated in depth before setting up therapy.

Introduction: To assess the diagnostic accuracy of CESM and 3T MRI compared to full-field digital mammography (FFDM), plus US, in the evaluation of advanced breast lesions. Materials and Methods: Consenting women with suspicious findings underwent FFDM, US, CESM and 3T MRI. Breast lesions were histologically assessed, with histology being the gold standard. Two experienced breast radiologists, blinded to cancer status, read the images. Diagnostic accuracy of (1) CESM as an adjunct to FFDM and US, and (2) 3T MRI as an adjunct to CESM compared to FFDM and US, was assessed. Measures of accuracy were sensitivity (Se), specificity (Sp), positive predictive value (PPV) and negative predictive value (NPV). Results: There were 118 patients included along with 142 histologically characterized lesions. K agreement values were 0.69, 0.68, 0.63 and 0.56 for concordance between the gold standard and FFDM, FFDM + US, CESM and MRI, respectively (p < 0.001, for all). K concordance for CESM was 0.81 with FFDM + US and 0.73 with MRI (p value < 0.001 for all). Conclusions: CESM may represent a valuable alternative and/or an integrating technique to MRI in the evaluation of breast cancer patients.

The aim of the DeepLook project, funded by INFN (Italy), is to implement a deep learning architecture for Computed Aided Detection (CAD), based on neural networks developed with deep learning methods, for the automatic detection and classification of breast lesions in DBT images. A preliminary step (started 2 years ago and still ongoing) was the creation of a dataset of annotated images. This dataset includes images acquired with different clinical DBT units and different acquisition geometries, on several hundred patients, containing a variety of possible breast lesions and normal cases of absence of lesions. This will make the diagnostic capacity of the CAD system particularly extensive in various clinical situations and on a significant sample of patients, so allowing the network to diagnose various types of lesions (at the level of the single tomosynthesis slices) and capable of operate on commercial DBT systems, also available from different vendors, as found in breast diagnosis departments. The developed CAD and first result of the indication of the slice containing the suspected mass will be presented.

Human Space exploration has created new challenges and new opportunities for science. Reaching beyond the Earth’s surface has raised the issue of the importance of gravity for the development and the physiology of biological systems, while giving scientists the tools to study the mechanisms of response and adaptation to the microgravity environment. As life has evolved under the constant influence of gravity, gravity affects biological systems at a very fundamental level. Owing to limited access to spaceflight platforms, scientists rely heavily on on-ground facilities that reproduce, to a different extent, microgravity or its effects. However, the technical constraints of counterbalancing the gravitational force on Earth add complexity to data interpretation. In-flight experiments are also not without their challenges, including additional stressors, such as cosmic radiation and lack of convection. It is thus extremely important in Space biology to design experiments in a way that maximizes the scientific return and takes into consideration all the variables of the chosen setup, both on-ground or on orbit. This review provides a critical analysis of current ground-based and spaceflight facilities. In particular, the focus was given to experimental design to offer the reader the tools to select the appropriate setup and to appropriately interpret the results.

Understanding molecular mechanisms responsible for bone cells unbalance in microgravity would allow the development of better countermeasures for astronauts, and eventually advancing terrestrial osteoporosis treatments. We conduct a unique investigation by using a controlled 3D in vitro cell model to mimic the bone microenvironment in microgravity aboard the SpaceX Dragon cargo ferry to the ISS. Osteoblasts (OBs), osteoclasts (OCs), and endothelial cells (ECs), seeded on Skelite discs, were cultured w/ or w/o rec-Irisin and exposed to 14 days of microgravity in the eOSTEO hardware. Gene expression analysis was assessed, and results were compared to ground controls treated within identical payloads. Our results show that the microgravity-induced downregulation of mRNA levels of genes encoding for OB key transcription factors (Atf4 -75%, P < .01; RunX2 -87%, P < .001, Osterix -95%, P < .05 vs ground) and proteins (Collagen I -84%, P < .05; Osteoprotegerin -94%, P < .05) were prevented by irisin. Despite it was not effective in preventing Trap and Cathepsin K mRNA increase, irisin induced a 2.8-fold increase of Osteoprotegerin (P < .05) that might act for reducing osteoclastogenesis in microgravity. Our results provide evidence that irisin supports OB differentiation and activity in microgravity and it might represent a countermeasure to prevent bone loss in astronauts.

The direct impact of microgravity exposure on male germ cells, as well as on their malignant counterparts, has not been largely studied. In previous works, we reported our findings on a cell line derived from a human seminoma lesion (TCam-2 cell line) showing that acute exposure to simulated microgravity altered microtubule orientation, induced autophagy, and modified cell metabolism stimulating ROS production. Moreover, we demonstrated that the antioxidant administration prevented both TCam-2 microgravity-induced microtubule disorientation and autophagy induction. Herein, expanding previous investigations, we report that simulated microgravity exposure for 24 h induced the appearance, at an ultrastructural level, of cell-to-cell junctional contacts that were not detectable in cells grown at 1 g. In line with this result, pan-cadherin immunofluorescence analyzed by confocal microscopy, revealed the clustering of this marker at the plasma membrane level on microgravity exposed TCam-2 cells. The upregulation of cadherin was confirmed by Western blot analyses. Furthermore, we demonstrated that the microgravity-induced ROS increase was responsible for the distribution of cadherin nearby the plasma membrane, together with beta-catenin since the administration of antioxidants prevented this microgravity-dependent phenomenon. These results shed new light on the microgravity-induced modifications of the cell adhesive behavior and highlight the role of ROS as microgravity activated signal molecules.

Background Cancer and coagulation activation are tightly related. The extent to which factors related to both these pathologic conditions concur to patient prognosis intensely animates the inherent research areas. The study herein presented aimed to the development of a tool for the assessment and stratification of risk of death and disease recurrence in early breast cancer. Methods Between 2008 and 2010, two hundreds thirty-five (N: 235) patients diagnosed with stage I–IIA breast cancer were included. Data on patient demographics and clinic-pathologic features were collected in course of face-to-face interviews or actively retrieved from clinical charts. Plasma levels of plasminogen activator inhibitor type 1 (PAI-1), fragment 1 + 2 (F1 + 2), thrombin antithrombin complex (TAT), factor VIII (FVIII), and D-dimer (DD) were measured at breast cancer diagnosis and prior to any therapeutic procedure, including breast surgery. The risk of death was computed in terms of overall survival (OS), which was the primary outcome. For a subset of patients (N = 62), disease free survival (DFS) was also assessed as a measure of risk of disease recurrence. Results Median follow up was 95 months (range 6–112 months). Mean age at diagnosis was 60.3 ± 13.4 years. Cancer cases were more commonly intraductal carcinomas (N: 204; 86.8%), pT1 (131; 55.7%), pN0 (141; 60%) and G2 (126; 53.6%). Elevated levels of PAI-1 (113; 48.1%) represented the most frequent coagulation abnormality, followed by higher levels of F1 + 2 (97; 41.3%), DD (63; 27.0%), TAT (34; 40%), and FVIII (29; 12.3%). In univariate models of OS, age, pT, DD, FVIII were prognostically relevant. In multivariate models of OS, age (p = 0.043), pT (p = 0.001), levels of DD (p = 0.029) and FVIII (p = 0.087) were confirmed. In the smaller subgroup of 62 patients, lymph node involvement, percent expression of estrogen receptors and levels of FVIII impacted DFS significantly. Conclusions We developed a risk assessment tool for OS including patient- and cancer-related features along with biomarkers of coagulation activation in a cohort of early BC patients. Further studies are warranted to validate our prognostic model in the early setting and eventually extend its application to risk evaluation in the advanced setting for breast and other cancers. Electronic supplementary material The online version of this article (10.1186/s12967-018-1511-x) contains supplementary material, which is available to authorized users.

Evidence on everolimus in breast cancer has placed hyperglycemia among the most common high grade adverse events. Anthropometrics and biomarkers of glucose metabolism were investigated in a observational study of 102 postmenopausal, HR + HER2- metastatic breast cancer patients treated with everolimus-exemestane in first and subsequent lines. Best overall response (BR) and clinical benefit rate (CBR) were assessed across subgroups defined upon fasting glucose (FG) and body mass index (BMI). Survival was estimated by Kaplan-Meier method and log-rank test. Survival predictors were tested in Cox models. Median follow up was 12.4 months (1.0–41.0). The overall cohort showed increasing levels of FG and decreasing BMI (p

This study analyzed discourses influencing the meaning of Sustainable Forest Management in Catalonia (Spain) and the effects of the European Natura 2000 policy on these discourses. It focused on the Montseny area and on the Administration and Practitioner stakeholder groups. Based on literature review and interviews, we found six discourses influencing the meanings of Sustainable Forest Management. Two of these discourses underwent changes due to the effects of Natura 2000 implementation while the others did not, showing the substantial role played by local elements and actors in shaping the discursive framework of Sustainable Forest Management. Based on empirical findings, the study provided conclusions valid at European level: (1) implementing Natura 2000 does not mean revolutionizing local nature conservation systems, but rather adapting them to European requirements; (2) in order to increase local policy impact, the implementation of European environmental policies needs to be backed up by economic compensation of local actors.

In 2015, the European Union’s Birds and Habitats Directives underwent an evaluation in which selected national stakeholders provided their perspectives on the directives’ implementation. Analyzing the views of different stakeholder groups from eight European member states, this study found that these views could be synthesized into three more general perspectives. The first perspective focuses on problems, indicating that these are caused by legislative drawbacks in the directives. The second perspective holds that problems are generated by improper implementation by member states. The third perspective commends the benefits of the directives in face of the existing implementation problems. Interest groups and to a minor extent governmental bodies espoused the first perspective, and environmental non-governmental organizations especially favored the third. The struggle between these three perspectives reflects ongoing debates regarding positive and negative aspects of the directives and possibilities for improving their implementation. We conclude that the relevance and impact of conservation policies should never be seen as self-evident. In order to reach the conservation goals envisaged, continuous efforts are needed to enforce and maintain environmental legislation.

A seashell dump from the site of San Vincenzo (Stromboli, Aeolian islands, Italy) was studied using an interdisciplinary approach to identify the type of molluscs and the formation process of the deposit, with the aim of reconstructing the most important cultural and social aspects of the meal. A large village occupied Stromboli during the Early and Middle Bronze Age. The village, at least during its later phases, had a modular topographical structure made up of rectangular enclosures with imposing stone elements at the corners. A large pit filled almost exclusively with marine shells was found near one of these stones and by an enclosure wall at the southernmost edge of the village. A sample of the deposit where the shells are embedded in their originally sandy matrix was extracted, investigated by means of CT scanning and reconstructed in 3D, confirming deposition after a single discard event. Radiometric, stratigraphic, typological and archaeometric data indicate that the meal was consumed contemporaneously with the beginning of interactions with the Mycenaeans, and discharged in a specific location possibly marking a major topographical transformation. The quantity of molluscs consumed was reconstructed experimentally by comparing the ancient record with the corresponding quantity of meat provided by contemporary animals collected as a specimen from the rocky shores of the island. Shell deposits of the Mediterranean Bronze Age have been rarely studied in such detail and this case study may reveal forms of ancient communal consumption of such food at the beginning of the Middle Bronze Age, and therefore important feasting contexts in which ancient communities materially interacted and exchanged information.

A hypercoagulable state may either underlie or frankly accompany cancer disease at its onset or emerge in course of cancer development. Whichever the case, hypercoagulation may severely limit administration of cancer therapies, impose integrative supporting treatments and finally have an impact on prognosis. Within a flourishing research pipeline, a recent study of stage I-IIA breast cancer patients has allowed the development of a prognostic model including biomarkers of coagulation activation, which efficiently stratified prognosis of patients in the study cohort. We are now validating our risk assessment tool in an independent cohort of 108 patients with locally advanced breast cancer with indication to neo-adjuvant therapy followed by breast surgery. Within this study population, we will use our tool for risk assessment and stratification in reference to 1. pathologic complete response rate at definitive surgery, intended as our primary endpoint, and 2. rate of thromboembolic events, intended as our secondary endpoint. Patients’ screening and enrollment procedures are currently in place. The trial will be shortly enriched by experimental tasks centered on next-generation sequencing techniques for identifying additional molecular targets of treatments which may integrate current standards of therapy in high-risk patients.

EXPLORA Siamo soli nell’universo? Qual è l’origine della vita sulla Terra? Potremo un giorno realizzare insediamenti umani al di fuori del nostro pianeta utilizzando le piante quale fonte di energia e vita? Il viaggio inizia ...... e ci porta nello spazio profondo, dove solo sonde sofisticate possono arrivare. Ma saliremo anche a bordo della stazione spaziale internazionale dove astronauti, provenienti da ogni parte del mondo, soggiornano ininterrottamente da più di 15 anni, alternando vari equipaggi. Esploriamo per comprendere l’universo che ci circonda, la capacità della vita di adattarsi a condizioni estreme e ricercarla altrove. Abbiamo esplorato pianeti rocciosi, pianeti giganti e messo in luce il grande interesse esobiologico di mondi quali Europa, Encelado e Ganimede che nascondono, con ogni probabilità, un oceano di acqua al di sotto della loro superficie. Come Marte, di cui, nel 2018, è stata accertata, da un radar italiano a bordo della sonda Mars Express, la presenza di acqua allo stato liquido nel sottosuolo. Una grandissima scoperta scientifica che fa onore al nostro paese. Siamo atterrati sulla Luna, Venere, Marte, Titano e persino su una cometa, la Churyumov-Gerasimenko dove abbiamo trovato il più semplice degli amminoacidi (la glicina), ma anche il fosforo (che si trova negli acidi nucleici). L’uomo, nel frattempo, ha costruito una casa/laboratorio, che viaggia veloce , orbitando 400Km sopra alla Terra. Questa sofisticata e complessa struttura potrà fornire indicazioni e risposte utili sulla nostra concreta possibilità di vivere su altri pianeti. Perché è lì che un giorno vorremmo arrivare. E così, i viaggi robotici interplanetari e le missioni umane vanno a braccetto. Gli uni esplorando i pianeti e, le altre, la possibilità che l’uomo possa vivere in luoghi diversi e lontani scrutati e studiati dalle sonde che lo hanno preceduto. Ad oggi Marte è considerata la meta più probabile, ma sono diverse le destinazioni potenzialmente interessanti nel nostro sistema solare. Questo piccolo, grande viaggio è quello che faremo insieme nelle pagine che seguono...... R. Battiston

In this explorative study, we compare national policies affecting production and use of forest-based bioenergy in five European countries: Finland, Germany, Norway, Slovenia and Spain. Our methodological approach combines horizontal and vertical policy interaction with three policy layers: policy objectives, policy instruments and thematic elements in implementation. Great variations are found in national bioenergy policies, with complex interlinkages revealed to national renewable energy and forest policies (horizontal dimension) and to EU policies (vertical dimension). Horizontally, the national bioenergy objectives are closely linked to forest policy objectives and, in Finland, also to renewable energy objectives. Policy instruments promoting bioenergy demand interact to various degrees with both renewable energy and forest policies, while supply side instruments, as well as implementation aspects, interact most closely with forest policies. Vertically, highest degree of interactions with EU polices are in this study revealed for policy objectives and instruments, less so for implementation. It is concluded that consideration of policy layers are useful for increasing the understanding of the complexity in horizontal and vertical policy interactions, and thus an important basis for understanding how to minimize conflicts and enhancing synergies among multiple forest objectives. We argue that the chosen analytical framework can enhance the understanding of complex interlinkages between bioenergy and broader policy and market developments. Identifying these complex interactions can contribute to facilitate policy developments promoting and regulating future production and use of forest-based bioenergy, while taking other forest objectives into consideration.

In environmental governance, the European Union draws on norms of effectiveness, decentralisation, and participation to ensure that its policies and regulations are considered legitimate. This article analyses how the construction of legitimacy in European nature policy has changed over time. Although the norms of participation and decentralisation are increasingly evoked to address the needs of stakeholders and member states in the implementation and financing of Natura 2000, the norm of effectiveness continues to dominate the construction of legitimacy. Effectiveness first acquired its meaning in the context of a science-based approach to Natura 2000 to emphasise the importance of achieving its conservation objectives. More recently, it has become increasingly re-articulated as cost-effectiveness, which reflects a growing influence of neoliberal discourse. The article concludes by discussing the implications of the findings for the legitimacy of European environmental governance.

International audience; Natura 2000, which is the core pillar of the European Union's biodiversity conservation policy, is an ambitious and complex venture that requires funding to be successful. A major challenge is said to be a lack of available funding, and a low uptake of allocated funds is also reported. However, in in-depth analysis has still not been produced to assess the approaches to funding, the reasons for these approaches and their impact regarding the achievement of the aims of Natura 2000. Thus, with this article, we intend to fill this gap. To accomplish this, a case study analysis was carried out in six selected EU Member States: Austria, France, Germany, the Netherlands, Spain, and the UK In our study, we perceived different approaches which we sum up to two main types of approaches that were present in the Member States to different degrees. The first type was to find the funding necessary for the required activities, and the second was to delay the implementation of Natura 2000. The major reasons for the different approaches were related to domestic political power realities. The funding approaches impacted onto the attractiveness of EU co-financing instruments, and the sustainability of the schemes. Alternative approaches were either absent or declining in importance. The economic benefits were not perceived on the ground. We conclude that neither a "one size fits all" approach to funding Natura 2000 will work nor will a universal claim for "more money". Therefore, a successful funding strategy ultimately necessitates effective interventions at institutional levels, the business environment and the local level.

The establishment of Natura 2000, the European Union’s network of protected areas, has been a challenging process and has caused a variety of conflicts. These conflicts are related to contradictory stakeholder interests and perceptions, as well as to procedural issues and feelings of exclusion, especially by concerned local land user groups. To prevent further conflict, local participation has been stressed as an important tool to increase the inclusiveness of Natura 2000 and its acceptance among land users. In this paper, we present an analysis of participation practices related to the Natura 2000 implementation processes in six EU member states. Based on material collected from semi-structured interviews and document analysis, we describe the organisational settings of the participatory processes, focusing, among other things, on the type of participants involved, the level and intensity of their involvement, and the goal of participation. In addition, we also describe the local context in which the participation processes have been embedded. Finally, we assess the outcomes of the participatory processes in terms of their impact on forest and nature conservation management practices. Our results show that local participation practices were shaped not just by the Natura 2000 policy, but also by the history of the area, including, for example, earlier conflicts among the local actors. We also show that although the participation process leads to a greater acceptance of the Natura 2000 policy, this does not relate to significant changes in management practices among local actors. These findings, however, do not suggest that participation is irrelevant. Rather, we conclude that participation involves context-dependent, localised learning processes that can only be understood by taking the historical socio-economic and institutional context in which they are situated into account.

The study of how mechanical forces may influence cell behavior via cytoskeleton remodeling is a relevant challenge of nowadays that may allow us to define the relationship between mechanics and biochemistry and to address the larger problem of biological complexity. An increasing amount of literature data reported that microgravity condition alters cell architecture as a consequence of cytoskeleton structure modifications. Herein, we are reporting the morphological, cytoskeletal, and behavioral modifications due to the exposition of a seminoma cell line (TCam-2) to simulated microgravity. Even if no differences in cell proliferation and apoptosis were observed after 24 hours of exposure to simulated microgravity, scanning electron microscopy (SEM) analysis revealed that the change of gravity vector significantly affects TCam-2 cell surface morphological appearance. Consistent with this observation, we found that microtubule orientation is altered by microgravity. Moreover, the confocal analysis of actin microfilaments revealed an increase in the cell width induced by the low gravitational force. Microtubules and microfilaments have been related to autophagy modulation and, interestingly, we found a significant autophagic induction in TCam-2 cells exposed to simulated microgravity. This observation is of relevant interest because it shows, for the first time, TCam-2 cell autophagy as a biological response induced by a mechanical stimulus instead of a biochemical one.

Summary The glial cell line–derived neurotrophic factor (GDNF) has multiple functions that promote cell survival, proliferation and migration in different cell types. The experimental over-expression of GDNF in mouse testis leads to infertility and promotes seminomatous germ cell tumours in older animals, which suggests that deregulation of the GDNF pathway may be implicated in germ cell carcinogenesis. GDNF activates downstream pathways upon binding to its specific co-receptor GDNF family receptor-a 1 (GFRA1). This complex then interacts with Ret and other co-receptors to activate several intracellular signalling cascades. To explore the involvement of the GDNF pathway in the onset and progression of testicular germ cell tumours, we analysed GFRA1 and Ret expression patterns in seminoma samples. We demonstrated, via immunohistochemistry, that GFRA1, but not Ret, is over-expressed in in situ carcinoma (CIS) and in intratubular and invasive seminoma cells compared with normal human germ cells. Functional analysis of the GDNF biological activity was performed on TCam-2 seminoma cell line. Reverse transcription-PCR (RT-PCR) and immunohistochemical analyses demonstrate that TCam-2 cells express both GFRA1 and Ret mRNA, but only GFRA1 was detected at the protein level. In TCam-2 cells, although GDNF is not mitogenic, it is able to induce migration, as demonstrated by a Boyden chamber assay, possibly through the Src and MEK pathways. Moreover, GDNF promotes invasive behaviour, an effect dependent on pericellular protease activity, possibly through the activity of matrix metalloproteinases. GFRA1 over-expression in CIS and seminoma cells, along with the functional analyses in TCam-2 cells, suggests an involvement of the GDNF pathway in the progression of testicular germ cell cancer.

We have studied the effects of HGF on BTB dynamics in adult rats. We demonstrate that, at stages VII–VIII of the epithelium wave when germ cells traverse the BTB, HGF reduces the levels of occludin and influences its distribution pattern and assembling. Moreover, we report that, at stages VII–VIII, HGF significantly increases the amount of active TGF-β and the amount of uPA present in the tubules. For the first time we report that, in the same stages, HGF reduces the amount of actin present in the BTB region, in which occludin levels are highest, and modifies the morphology of the actin cytoskeleton network. At the level of maximal intensity of occludin fluorescence, we report that HGF also modifies the colocalization of occludin and actin. Lastly, we demonstrate that HGF is maximally expressed at stages VII–VIII, whereas its levels fall in the subsequent stages.

The implementation of Natura 2000, Ecological Network of protected areas established in the European territory under Council Directive 92/43/EEC, has encountered serious problems in many European States. This is proven by conspicuous juridical interventions initiated by the European Union against Member States failing to comply with the Directive’s requirements; by the aversion of stakeholders involved in the use of protected areas and by the criticisms that environmental non-governmental organizations expressed about the governmental approaches towards the network’s realization. This paper presents a critical reflection on the problems in the realization of Natura 2000 Network, by analysing and comparing the experiences of two Member States: the Netherlands and Italy. Despite the differences in national natural heritages and nature conservation policy traditions, the two Natura 2000 implementation processes present interesting similarities. These allowed the authors to identify theoretical and practical obstacles that are making the network’s implementation problematic and to reflect on the most important challenges to the realization of Natura 2000 Network in the two countries, as well as in other Member States that experienced similar implementation problems.

Mouse embryonic stem (ES) cells were stimulated to differentiate either as adherent monolayer cultures in DMEM/F12 supplemented with N2/B27, or as floating embryoid bodies (EBs) exposed to 1 microM retinoic acid (RA) for 4 days, starting from 4 DIV, and subsequently re-plated in DMEM/F12 medium. Adherent monolayer cultures of ES cells expressed mGlu5 receptors throughout the entire differentiation period. Selective pharmacological blockade of mGlu5 receptors with methyl-6-(phenylethynyl)-pyridine (MPEP) (1 microM, added once a day) accelerated the appearance of the neuronal marker, beta-tubulin. In addition, treatment with MPEP increased the number of cells expressing glutamate decarboxylase-65/67 (GAD(65/67)), a marker of GABAergic neurons. In floating EBs, mGlu5 receptors are progressively replaced by mGlu4 receptors. The orthosteric mGlu4/6/7/8 receptor agonist, L-2-amino-4-phosphonobutanoate (L-AP4), or the selective mGlu4 receptor enhancer, PHCCC,--both combined with RA at concentrations of 30 microM--increased the expression of both beta-tubulin and GAD(65/67), inducing the appearance of fully differentiated neurons that released GABA in response to membrane depolarization. We conclude that mGlu receptor subtypes regulate neuronal differentiation of ES cells in a context-dependent manner, and that subtype-selective ligands of these receptors might be used for the optimization of in vitro protocols aimed at producing GABAergic neurons from ES cells.

Testicular vasculogenesis is one of the key processes regulating male gonad morphogenesis. The knowledge of the molecular cues underlining this phenomenon is one of today's most challenging issues and could represent a major contribution toward a better understanding of the onset of testicular morphogenetic disorders. R-spondin 1 has been clearly established as a candidate for mammalian ovary determination. Conversely, very little information is available on the expression and role of R-spondin 1 during testicular morphogenesis. This study aims to clarify the distribution pattern of R-spondin 1 and other partners of its machinery during the entire period of testicular morphogenesis and to indicate the role of this system in testicular development. Our whole mount immunofluorescence results clearly demonstrate that R-spondin 1 is always detectable in the testicular coelomic partition, where testicular vasculature is organized, while Dickkopf-1 is never detectable in this area. Moreover, organ culture experiments of embryonic male UGRs demonstrated that Dickkopf-1 acted as an inhibitor of testis vasculature formation. Consistent with this observation, real-time PCR analyses demonstrated that DKK1 is able to slightly but significantly decrease the expression level of the endothelial marker Pecam1. The latter experiments allowed us to observe that DKK1 administration also perturbs the expression level of the Pdgf-b chain, which is consistent with some authors' observations relating this factor with prenatal testicular patterning and angiogenesis. Interestingly, the DKK1 induced inhibition of testicular angiogenesis was rescued by the co-administration of R-spondin 1. In addition, R-spondin 1 alone was sufficient to enhance, in culture, testicular angiogenesis.

The study explored the sub-national implementation of the Pan-European Criteria and Indicators (C&I) for Sustainable Forest Management in Italy, based on the statistical analysis of answers to a questionnaire provided by representatives of the Italian sub-national forest administrations. The questionnaire explored the implementation of C&I in five applications and through the analytical lenses of ‘suitability of C&I to sub-national contexts’ and ‘adaptability of administrations to C&I’. The analysis of suitability and adaptability made it possible to identify and compare challenges in, and successful experiences with, the implementation of C&I, which were then used to formulate recommendations aimed at encouraging implementation and promoting academic and policy debate. The study reveals that C&I were implemented to a moderate extent. This level of implementation was determined most of all by the suitability of C&I to the Italian sub-national contexts, and less by the adaptability of administratio...

European forests are a resource that is targeted by several EU environmental and land use policies as forests can be of critical importance to mitigate climate change. At the same time, they are central to the EU's biodiversity policy, and particular the Natura 2000 network of protected areas. Yet, the interlinkage between climate change and biodiversity policy is complex and discursively contested. In this paper, we assess how the debate on climate change adaptation affects forest conservation and management under Natura 2000. Drawing on the concept of argumentative discourse analysis, we present evidence from 213 qualitative interviews with policy stakeholders and practitioners that were conducted at both the European policy level and the local country level in 6 EU member states. Our results demonstrate that the nexus between climate change adaptation and forest conservation policy is conceptualised differently by different stakeholders and practioners at different levels. Three major discourses can be made out (pragmatic discourse, dynamics discourse, threat discourse), which are characterised by a set of partially overlapping story lines. These discourses are employed by four discourse coalitions (environmental, forest users’, expert, and grass root coalition). As a general rule, debates at the European level are more polarised and politicised, while the local debates on climate change and Natura 2000 remain rather vague and are less polarised. This seems to indicate that the link between climate change adaptation and forest conservation is mostly an issue for an abstract high-level policy debate. At this level, climate change is used to influence well-known policies, and to legitimise distinct interests that were already present before the climate change debate has emerged.

This paper analyses Natura 2000 as a shifting configuration of different approaches to nature conservation and discusses the consequences of these shifts for the roles of the stakeholders affected by this policy. Natura 2000 started with a technocratic approach that privileged conservation experts and marginalised socio-economic stakeholders. Over time, this approach has been complemented with participatory and economic approaches that offered scope for the inclusion of land users and business actors. However, the analysis also shows that the selective inclusion of economic values and stakeholders in the Natura 2000 framework risks marginalising other important socio-environmental actors.

Scientific studies reveal significant consequences of climate change for nature, from ecosystems to individual species. Such studies are important factors in policy decisions on forest conservation and management in Europe. However, while research has shown that climate change research start to impact on European conservation policies like Natura 2000, climate change information has yet to translate into management practices. This article contributes to the on-going debates about science–society relations and knowledge utilization by exploring and analysing the interface between scientific knowledge and forest management practice. We focus specifically on climate change debates in conservation policy and on how managers of forest areas in Europe perceive and use climate change ecology. Our findings show that forest managers do not necessarily deny the potential importance of climate change for their management practices, at least in the future, but have reservations about the current usefulness of available knowledge for their own areas and circumstances. This suggests that the science–management interface is not as politicized as current policy debates about climate change and that the use of climate change ecology is situated in practice. We conclude the article by discussing what forms of knowledge may enable responsible and future oriented management in practice focusing specifically on the role of reflexive experimentation and monitoring.

This volume presents the fruits of research that began in the 1980s concerning a class of pottery that has assumed increasing importance in Italian late prehistory, namely pottery of Mycenaean type or style, usually decorated, dating from the 17th to 11th century BC, and found throughout peninsular Italy, Sicily and Sardinia. Its significance lies in the way this pottery reflects Italy's growing connections with the outside world at this time, mainly with the Aegean but also further afield to the east. Establishing that much of this pottery was made within Italy has led to its labelling 'Italo-Mycenaean'. Following the book's introduction, there is a gazetteer of sites where this and related pottery has been found in Italy. The next chapter provides a comparative chronology between the Aegean and Italy. There is then the presentation of the pottery itself, its characterisation by style and with science-based analysis to determine its origins and technological attributes; the results of experimental reconstruction are included. The impact of external influences on the indigenous cultures within Italy and Italy's role in the so-called Late Bronze Age 'International Age' in the Mediterranean are among the main issues considered in the last chapter.

Breast cancer remains a leading cause of mortality among women. In metastasis, cascade migration of cancer cells and invasion of extracellular matrix (ECM) represent critical steps. Urokinase-type plasminogen activator (uPA), as well as metalloproteinases MMP-2 and MMP-9, strongly contribute to ECM remodelling, thus becoming associated with tumour migration and invasion. In addition, the high expression of cytoskeletal (CSK) proteins, as fascin, has been correlated with clinically aggressive metastatic tumours, and CSK proteins are thought to affect the migration of cancer cells. Consumption of fruits and vegetables, characterized by high procyanidin content, has been associated to a reduced mortality for breast cancer. Therefore, we investigated the biological effect of grape seed extract (GSE) on the highly metastatic MDA-MB231 breast cancer cell line, focusing on studying GSE ability in inhibiting two main metastatic processes, i.e., cell migration and invasion. After MDA-MB231 breast cancer cells stimulated with GSE migration and invasion were evaluated by means of trans-well assays and uPA as well as MMPs activity was detected by gelatin zymography. Fascin, β-catenin and nuclear factor-κB (NF-κB) expression were determined using western blot technique. β-Catenin localization was observed by confocal microscopy. We observed that high concentrations of GSE inhibited cell proliferation and apoptosis. Conversely, low GSE concentration decreased cell migration and invasion, likely by hampering β-catenin expression and localization, fascin and NF-κB expression, as well as by decreasing the activity of uPA, MMP-2 and MMP-9. These results make GSE a powerful candidate for developing preventive agents against cancer metastasis.

Seminoma is one of the most common Testicular Germ Cell Tumours that originates during embryonic development due to an alteration of the local niche that in turn results in a delayed or blocked differentiation of Primordial Germ Cells. The block of differentiation is actually a common way to develop cancer disease as postulated by the "embryonic rest theory of cancer". In agreement with this theory different studies have demonstrated that embryonic cues display the capacity of reprogramming aggressive cancer cells towards a less aggressive phenotype. Herein we investigate the ability of a culture medium added with 10% egg albumen (EW, Egg White) to modulate seminoma cell phenotype and behaviour, by ensuring a proper set of morphogenetic signals. We chose to use the TCam-2 seminoma cell line that has been established as the only available cell line, obtained from a primary testicular seminoma. EW is able to: 1) modify TCam-2 cell spreading rate and cell-substrate adhesion without affecting proliferation and survival indexes; 2) modulate TCam-2 actin distribution pattern increasing cortical localization of actin filaments; 3) increase TCam-2 cell-cell junction capability; 4) decrease both chemo-sensitive and collective TCam-2 migratory behaviour. According to these observations morphometric fractal analysis revealed the ability of EW to increase Circularity and Solidity parameters and, consequently, to decrease Fractal dimension. Prompted by these observations we hypothesize that EW treatment could rescue, at least in part, the neoplastic-metastatic behaviour of seminoma cells.

The hepatocyte growth factor (HGF) is a pleiotropic cytokine and a well-known regulator of mouse embryonic organogenesis. In previous papers, we have shown the expression pattern of HGF and its receptor, C-MET, during the different stages of testis prenatal development. We demonstrated that C-MET is expressed in fetal Leydig cells (FLCs) and that HGF stimulates testosterone secretion in organ culture of late fetal testes. In the present study, we analyzed the proliferation rate, apoptotic index, and differentiation of FLCs in testicular organ culture of 17.5 days postcoitum (17.5 dpc) embryos to clarify the physiological role of HGF in late testis organogenesis. Based on our data, we conclude the following: 1) HGF acts as an antiapoptotic factor that is able to reduce the number of apoptotic FLCs and testicular caspase-3 active fragment; 2) HGF does not affect FLC proliferation; 3) HGF significantly increases expression of insulin-like 3 (INSL3), a marker of Leydig cell terminal differentiation, without affecting 3beta-hydroxysteroid dehydrogenase (3betaHSD) expression; 4) HGF significantly decreases the expression of nestin, a marker of Leydig cell progenitors; and 5) HGF significantly increases the number of fully developed FLCs. Taken together, these observations demonstrate that HGF is able to act in vitro as a survival and differentiation factor in FLC population.

The hepatocyte growth factor (HGF) is a pleiotropic cytokine and a well-known regulator of mouse embryonic organogenesis. In previous papers, we have shown the expression pattern of HGF and its receptor, C-MET, during the different stages of testis prenatal development. We demonstrated that C-MET is expressed in fetal Leydig cells (FLCs) and that HGF stimulates testosterone secretion in organ culture of late fetal testes. In the present study, we analyzed the proliferation rate, apoptotic index, and differentiation of FLCs in testicular organ culture of 17.5 days postcoitum (17.5 dpc) embryos to clarify the physiological role of HGF in late testis organogenesis. Based on our data, we conclude the following: 1) HGF acts as an antiapoptotic factor that is able to reduce the number of apoptotic FLCs and testicular caspase-3 active fragment; 2) HGF does not affect FLC proliferation; 3) HGF significantly increases expression of insulin-like 3 (INSL3), a marker of Leydig cell terminal differentiation, without affecting 3beta-hydroxysteroid dehydrogenase (3betaHSD) expression; 4) HGF significantly decreases the expression of nestin, a marker of Leydig cell progenitors; and 5) HGF significantly increases the number of fully developed FLCs. Taken together, these observations demonstrate that HGF is able to act in vitro as a survival and differentiation factor in FLC population.

Thirty-two patients with recurrent (skin) or metastatic (skin, node, or both) melanoma have been treated with a hyperthermia-cisplatin regimen. The hyperthermic treatment was carried out for 60 minutes at 43 degrees C with the MHS-SMA and the Sapic SVO3 ALENIA devices once a week. When the tumor temperature reached 42 degrees C, cisplatin was administered at a dosage of 50 mg/m2 given by intravenous bolus infusion. The treatment was repeated four times and the tumor response evaluated 4 weeks after the last treatment. Significant systemic or local toxicity was not seen. In terms of results, there were 9 patients with complete responses (28.1%), 13 with partial responses (40.6%), 8 with no change (25.0%), and two with disease progression (6.3%). The objective response rate was 68.7%. The response duration for those with complete responses ranged from 4 to 49 months (median 20 months). The median time to progression for patients with partial responses and those with no change was 6 and 5 months, respectively, with ranges of 1-7 and 1-10 months, respectively. The 4-year actuarial survival rates were 47.6% and 20.3% for the complete and incomplete responders, respectively. These results can be considered satisfactory, taking into account that most patients were pretreated with radiotherapy, chemotherapy or both, confirming the therapeutic potential of the hyperthermia and cisplatin regimen.

609 Background: The aims of the study were 1) to assess the accuracy of a new intra-operative molecular diagnostic tool named OSNA, based on the measurement of cytokeratin 19 (CK19) mRNA, in the detection of axillary sentinel lymph node (SLN) metastases in patients with breast carcinoma 2) to determine the concordance of OSNA analysis with multilevel haematoxylin and eosin (H&E) and immunohistochemical (IHC) examination. Methods: A prospective series of 247 consecutive SLNs from 187 breast cancer patients was evaluated. The OSNA assay (Sysmex, Kobe - Japan) follows a short sample preparation step and subsequent rapid amplification of CK19 mRNA based on reverse transcription loop-mediated isothermal amplification. Each SLN was immediately divided into four slices. Two alternate slices were used for the intra-operative OSNA assay. The remaining two slices were investigated by six-level histopathology with 100 μm skip ribbons using routine H&E and CK19 IHC staining. The results of histology and OSNA were then compared. Results: Pts characteristics: infiltrating ductal/infiltrating lobular/mucinous/medullar/DCIS: 130/10/1/1/25. OSNA and histo-pathological methods identified 54 SLNs positive and 185 negative cases (2/3 contained isolated tumour cells). We found 8 discordant cases, 2 OSNA negative with micrometastasis found by IHC/H&E analysis, 5 OSNA positive for micrometastases but IHC/H&E negative and 1 case macrometastatic at OSNA, while negative at IHC/H&E analysis. The overall concordance of OSNA with histopathology was 96.7% with a specificity of 96.8% and sensitivity of 96.4%. These discordances could be due to sampling bias such that a micrometastasis was confined to the slices used for OSNA or the slices used for histology. Conclusions: The current study suggests that the performance provided by OSNA assay is comparable to intensive histopathological work-up even when using only half a lymph node. The method could be applied as a rapid and reliable intra-operative diagnostic tool thus preventing breast cancer patients from a diagnostic delay or second surgery due to a postoperatively diagnosed positive SLN. No significant financial relationships to disclose.