Exploiting the Gal4/UAS System as Plant Orthogonal Molecular Toolbox to Control Reporter Expression in Arabidopsis Protoplasts

The ability of protein domains to fold independently from the rest of the polypeptide is the principle governing the generation of fusion proteins with customized functions. A clear example is the split transcription factor system based on the yeast GAL4 protein and its cognate UAS enhancer. The rare occurrence of the UAS element in the transcriptionally sensitive regions of the Arabidopsis genome makes this transcription factor an ideal orthogonal platform to control reporter induction. Moreover, heterodimeric transcriptional complexes can be generated by exploiting posttranslational modifications hampering or promoting the interaction between GAL4-fused transcriptional partners, whenever this leads to the reconstitution of a fully functional GAL4 factor. The assembly of multiple engineered proteins into a synthetic transcriptional complex requires preliminary testing, before its components can be stably introduced into the plant genome. Mesophyll protoplast transformation represents a fast and reliable technique to test and optimize synthetic regulatory modules. Remarkable properties are the possibility to transform different combinations of plasmids (co-transformation) and the physiological resemblance of these isolated cells with the original tissue. Here we describe an extensive protocol to produce and exploit Arabidopsis mesophyll protoplasts to investigate the transcriptional output of GAL4/UAS-based complexes that are sensitive to posttranslational protein modifications

Hypoxia selects bortezomib-resistant stem cells of chronic myeloid leukemia.

We previously demonstrated that severe hypoxia inhibits growth of Chronic Myeloid Leukemia (CML) cells and selects stem cells where BCR/Abl(protein) is suppressed, although mRNA is not, so that hypoxia-selected stem cells, while remaining leukemic, are independent of BCR/Abl signaling and thereby refractory to Imatinib-mesylate. The main target of this study was to address the effects of the proteasome inhibitor Bortezomib (BZ) on the maintenance of stem or progenitor cells in hypoxic primary cultures (LC1), by determining the capacity of LC1 cells to repopulate normoxic secondary cultures (LC2) and the kinetics of this repopulation. Unselected K562 cells from day-2 hypoxic LC1 repopulated LC2 with rapid, progenitor-type kinetics; this repopulation was suppressed by BZ addition to LC1 at time 0, but completely resistant to day-1 BZ, indicating that progenitors require some time to adapt to stand hypoxia. K562 cells selected in hypoxic day-7 LC1 repopulated LC2 with stem-type kinetics, which was largely resistant to BZ added at either time 0 or day 1, indicating that hypoxia-selectable stem cells are BZ-resistant per se, i.e. before their selection. Furthermore, these cells were completely resistant to day-6 BZ, i.e. after selection. On the other hand, hypoxia-selected stem cells from CD34-positive cells of blast-crisis CML patients appeared completely resistant to either time-0 or day-1 BZ. To exploit in vitro the capacity of CML cells to adapt to hypoxia enabled to detect a subset of BZ-resistant leukemia stem cells, a finding of particular relevance in light of the fact that our experimental system mimics the physiologically hypoxic environment of bone marrow niches where leukemia stem cells most likely home and sustain minimal residual disease in vivo. This suggests the use of BZ as an enhanced strategy to control CML. in particular to prevent relapse of disease, to be considered with caution and to need further deepening

Classification studies on Etruscan archaeological copper-based alloy findings from the Necropolis of ‘Pratino’ in Tuscania

In this communication, an extensive study and its results are presented about the composition of some Etruscan copper alloy findings. The objects came from Tomb 20 of the “Pratino” necropolis in Tuscania, near Viterbo (Italy), dating back to the late Hellenistic period. The excavation and study of the site was granted to the Lorenzo de' Medici Italian International Institute (Ld’M) in 2005. Since 2011, the activity is directed by the Center for Ancient Mediterranean and Near Eastern Studies (CAMNES). The archaeological materials were classified according to their composition, determined by a portable Energy Dispersive X-Ray Fluorescence (ED-XRF) instrument

Towards a middle-range theory of mental health and well-being effects of employment transitions: Findings from a qualitative study on unemployment during the 2009-2010 economic recession.

This article builds upon previous theoretical work on job loss as a status passage to help explain how people's experiences of involuntary unemployment affected their mental well-being during the 2009-2010 economic recession. It proposes a middle-range theory that interprets employment transitions as status passages and suggests that their health and well-being effects depend on the personal and social meanings that people give to them, which are called properties of the transitions. The analyses, which used a thematic approach, are based on the findings of a qualitative study undertaken in Bradford (North England) consisting of 73 people interviewed in 16 focus groups. The study found that the participants experienced their job losses as divestment passages characterised by three main properties: experiences of reduced agency, disruption of role-based identities, for example, personal identity crises, and experiences of 'spoiled identities', for example, experiences of stigma. The proposed middle-range theory allows us to federate these findings together in a coherent framework which makes a contribution to illuminating not just the intra-personal consequences of unemployment, that is, its impact on subjective well-being and common mental health problems, but also its inter-personal consequences, that is, the hidden and often overlooked social processes that affect unemployed people's social well-being. This article discusses how the study findings and the proposed middle-range theory can help to address the theoretical weaknesses and often contradictory empirical findings from studies that use alternative frameworks, for example, deprivation models and 'incentive theory' of unemployment

Cardiocirculatory intraoperative assessment during single-shot caudal anaesthesia in children: comparison between levobupivacaine and ropivacaine

BACKGROUND: Caudal block with levobupivacaine or ropivacaine is the most commonly used regional anaesthesia in children. METHODS: The aim of study was to compare the cardiocirculatory profile induced in two matched groups of young patients, submitted to caudal anaesthesia with levobupivacaine or ropivacaine for an elective subumbilical surgery. Sixty children were enrolled: thirty received levopubivacaine 0.25% and thirty ropivacaine 0.2%. Intraoperative heart rate (HR), systolic blood pressure (SBP), diastolic blood pressure (DBP) were monitored at following times: Ta0 (after anaesthesia induction), Tal (after caudal anaesthesia), Ta2 (five minutes later), Ta3 (ten minutes later), Ts1 (at surgical incision), Ts2, Ts3, Ts4, Ts5 (every 10 minutes during surgery), Taw (at the awakening). RESULTS: In both groups the cardiocirculatory trend remained within normal ranges at all times considered, demonstrating the safety of the method with both drugs. Both groups showed a similar trend at the different monitoring times: low decrease in HR, SBP and DBP after caudal block, slight increase in parameters after skin incision, slight decrease during surgery, increase at awakening. Regarding SBP and DBP, the levobupivacaine group children generally showed higher levels compared to the ropivacaine group, especially for DBP. CONCLUSIONS: Paediatric caudal anaesthesia is an effective method with an very infrequent complication rate. Possible hypotheses for differing haemodynamic behaviour could include a stronger vasoconstriction reflex of innervated areas during caudal anaesthesia with levobupivacaine and a lower levobupivacaine induced block of the sympathetic fibers, related to different pharmacokinetic profile of low concentrations of the local anaesthetics used in paediatric epidural space

Biofuels from perennial energy crops on buffer strips: A win-win strategy

The objective of this work was to assess the environmental performances of advanced biofuels produced from perennial energy crops (miscanthus and willow) grown in bioenergy buffer strips (BBS) and compare them with the environmental performances of alternative systems providing the same function, i.e. private mobility. The growing evidence of potentially negative environmental impacts of bioenergy pathways calls for renewed efforts in identifying win-win bioenergy pathways, thus capable of mitigating climate change without worsening other environmental impacts. An holistic approach encompassing all the relevant areas of environmental concern is thus fundamental to highlight environmental trade-offs. Therefore, in this study we follow an attributional Life Cycle Assessment approach, but our analysis includes detailed modelling of biogenic carbon pools, nutrients cycles, infrastructures’ impacts as well as the expansion of the system boundaries to include the fuel use. We find that the fragmented and linear configuration of the buffer strips does not affect significantly the GHG emissions of lignocellulosic ethanol for BBS compared to growing the crops in open field. Additionally, we find that ethanol from perennials grown in BBS has the potential to reduce several other environmental impacts associated to private mobility. Firstly, the cultivation of miscanthus and willow in BBS enables both the removal of nutrients from the environment and the removal of carbon from the atmosphere, through the creation of an additional terrestrial sink. Secondly, when compared to the use of fossil gasoline, bioethanol from BBS crops generates lower impacts on all other areas of environmental concern, such as resources depletion or air pollution. We also find that cars fuelled with bioethanol form buffer strips perform even better than electric vehicles in all the impact categories analysed except for acidification and particulate matter emissions, where battery electric vehicles running on renewables perform slightly better. We conclude that bioethanol from perennial crops grown in BBS is a good example of nature-based solution, able to reduce GHG emissions without shifting the environmental burden on other areas of environmental concern

Alterations in Mucin Expression in Ovarian Mucinous Tumors: Immunohistochemical Analysis of MUC2, MUC5AC, MUC6, and CD10 Expression

The aim of this study was to evaluate the immunohistochemical expression of MUC2, MUC5AC, MUC6, and CD10 in ovarian mucinous adenoma (MA), mucinous borderline tumor (MB), and mucinous adenocarcinoma (MC), and to analyze the relationship between prognosis and these expressions. The expression of MUC2, MUC5AC, MUC6, and CD10 was evaluated by immunohistochemical analysis in 29 cases of MA, 29 cases of MB, and 26 cases of MC and scored based on the percentage of positive cells. Moreover, the ovarian mucinous tumors were classified into 4 phenotypes based on the staining patterns: intestinal, gastrointestinal, gastric, and unclassified patterns. The gastrointestinal pattern and the expression of MUC2 and CD10 increased from MA to MC. Conversely, the gastric pattern and MUC5AC expression decreased from MA to MC. Low MUC2 expression in MC was correlated with a better long-term survival rate. MUC2 expression in MC may be a useful predictor of the clinical outcome. The expression patterns of MUC2, MUC5AC, MUC6, and CD10 indicated that intestinal metaplasia may arise from the gastric-like epithelium in MA and that a close association exists between carcinogenesis and intestinal metaplasia in major ovarian mucinous tumors

Exploring new visions for a sustainable bioeconomy

The Bioeconomy is both an enabler and an end for the European Green Deal transformation: achieving the EGD transformation entails transforming the very meaning of sustainable bioeconomy. Among the deepest and most effective leverage points to transform a system are the worldviews driving our behaviours: they yield an enormous power to influence the framings which determine the solution space we explore. Transforming the bioeconomy, thus, requiresreflecting on the stories we tell about ourselves, our place in nature, and our relationship with others. Scholars have highlighted how narratives surrounding the EU Bioeconomy have predominantly embraced a “Green Growth” perspective, centred around economic growth, technological innovation, and anthropocentric values, largely ignoring the social and justice dimensions, as well as not questioning the role, relations, and responsibilities of humans in the web of life. These dominant framings are increasingly contested, though, because they have failed to produce the social and ecological outcomes desired. This report introduces perspectives which have been under-represented in the Bioeconomy discourse and integrates them into an alternative vision for a “green, just and sufficient bioeconomy”. This vision places environmental sustainability and social equity at its core, regardless of economic growth; has an inclusive and participatory perspective; care, respect, and reciprocity for and with other humans and non-humans are core values; technology is important to deliver on the green and just objectives, but ethical considerations for new technologies are openly debated

THE SPACE TELESCOPE NINA: RESULTS OF A BEAM TEST CALIBRATION

Abstract In June 1998 the telescope NINA will be launched in space on board of the Russian satellite Resource-01 n.4. The main scientific objective of the mission is the study of the anomalous, galactic and solar components of the cosmic rays in the energy interval 10–200 MeV/n. The core of the instrument is a silicon detector whose performances have been tested with a particle beam at the GSI Laboratory in Germany in 1997; we report here on the results obtained during the beam calibration

Six-transmembrane epithelial antigen of the prostate and enhancer of zeste homolog 2 as immunotherapeutic targets for lung cancer

<p>Abstract</p> <p>Background</p> <p>T-cell based immunotherapy for lung cancer (LC) could be a promising and novel therapeutic approach. Six-transmembrane epithelial antigen of the prostate (STEAP) and the polycomb group protein enhancer of zeste homolog 2 (EZH2) are highly expressed in LC and since the expression of molecules in normal tissue is significantly lower as compared to tumor cells, these proteins are considered as potential tumor-associated antigens (TAAs) for developing T-cell based immunotherapy.</p> <p>Methods</p> <p>We assessed the capacity of predicted CD4 T-cell epitopes from STEAP and EZH2 to induce anti-tumor immune responses to LC cell lines.</p> <p>Results</p> <p>Out of several predicted epitopes, two synthetic peptides, STEAP_281-296and EZH2_95-109, were effective in inducing CD4 T-cell responses that were restricted by HLA-DR1, DR15, or DR53 molecules, indicating that the peptides function as promiscuous T-cell epitopes. Moreover, STEAP_281-296and EZH2_95-109-reactive T-cells could directly recognize STEAP or EZH2 expressing LC cells in an HLA-DR restricted manner. In addition, some STEAP-reactive T-cells responded to STEAP+ tumor cell lysates presented by autologous dendric cells. Most significantly, both of these peptides were capable of stimulating <it>in vitro </it>T-cell responses in patients with LC.</p> <p>Conclusions</p> <p>Peptides STEAP_281-296and EZH2_95-109function as strong CD4 T-cell epitopes that can elicit effective anti-tumor T-cell responses against STEAP or EZH2 expressing LC. These observations may facilitate the translation of T-cell based immunotherapy into the clinic for the treatment of LC.</p