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Multiple variants of SARS-CoV-2, since the end of 2020 have emerged in many geographical areas and are currently under surveillance worldwide highlighting the continuing need for genomic monitoring to detect variants previously not yet identified.In this study, we used whole-genome sequencing (WGS) and phylogenetic analysis to investigate A.27 lineage SARS-CoV-2 from Sardinia, Italy.The Italian A.27 lineage genomes from Sardinia appeared related in a clade with genomes from France. Among the key mutations identified in the spike protein, the N501Y and the L452R deserve attention as considered likely vaccine escape mutations. Additional mutations were also here reported.A combination of features could explain our data such as SARS-CoV-2 genetic variability, viral dynamics, the human genetic diversity of Sardinian populations, the island context probably subjected to different selective pressures. Molecular and genomic investigation is essential to promptly identify variants with specific mutations with potential impact on public health and vaccine formulation.

OBJECTIVE: Diffuse intrinsic pontine glioma (DIPG) is a rare childhood brain tumour with poor prognosis. Radiotherapy (RT) remains the only palliative intervention. In this scenario, it is essential to investigate tumor and patient microinvironment. Microbiota plays a critical role in human health and a functional link between the central nervous system and gut microbiota has been reported: the microbiota-gut-brain axis. With these premises, we have investigated the gut microbiota in DIPG. METHODS: A cohort of 18 patients was enrolled and we collected stool specimens at diagnosis (pre-RT) and after radiotherapy. Microbiota content was analysed through 16S rRNA sequencing on IONS5xl; base calling and demultiplexing were performed by Torrent Suite (ThermoFisher). Association to progression free survival (PFS) and overall survival (OS) were assessed by cox-regression univariate analyses and differential abundance (DA) analyses identified differences following RT. RESULTS: The Firmicutes/Bacteroidetes (F/B) ratio in pre-RT specimens has a median of 0.757 (range= 0.243-1.19). Having PFS as endpoint and at family classification level, an increased risk of disease progression was disclosed for Flavobacteriaceae and Bacillales with HR of 1.57 (p=0.00913) and 1.57 (p=0.215), respectively. The anaerobic bacteria Synergistaceae is found related to a decreased risk of progression with HR 0.662 (p=0.00718). These findings were also confirmed with OS as endpoint. DA analyses pointed out a number of families belonging to Firmicutes and Bacteroidetes phyla highly differentiated (log2|fold-change|>2) between pre- and post-RT. CONCLUSIONS: This study represents the first report of the microbiota-gut-brain axis role in DIPG patients. The F/B ratio, a parameter of normal intestinal homeostasis, show the presence of inflammatory patterns and some family members are associated to increased disease progression. These preliminary findings needs further validations. However, 16S microbiota has the potential ability to stratify patients and probiotic administration could restore a normal F/B ratio.

The value of product lifecycle management systems (PLMS) is more and more recognised by companies and its use current has enormously increased. It is mainly used during the product design when different roles collaborate for sharing models, take review decisions, and approve or reject preliminary results. Often, companies have a general 'picture' about the processes involving PLMS (who performs an activity, when it is performed, what is done) but this knowledge can be reinforced, improved and modified using process mining. Here the knowledge is extracted from the event logs, and model-aware analytics are generated to evaluate the modelled, known and executed process. The business rules filter the logs and verify the impact on the process mining metrics to minimise the divergences between modelled and actual processes and improve the resulting quality metrics. The results help business users to identify lines of investigation for deviations from expected behaviour and propose improvement measures.

(a) Situation faced: Like any enterprise, cultural and creative organizations have to manage their business processes, which are influenced by the internal and external environments. The corresponding dynamics and complexity require design, coordination, and awareness. However, governmental agencies often underestimate the economic potential of creative and cultural industries (CCIs), reducing them to leisure activities. These conditions have often prevented these industries from obtaining the funding and incentive they need to improve processes and envision organizational innovation. (b) Action taken: Our work has the objective of designing a process reference framework for the CCI sector. We began with an analysis of the traditional cross-industry process-classification framework provided by the APQC and then selected four organizations of the Puglia Creativa cluster association as case studies. We conducted a set of individual and group interviews with each, from which we learned about their processes and their similarities and differences. We documented our findings systematically in a new process-classification framework, which we kept largely consistent with the original structure proposed by the APQC but adapted to address the peculiarities of organizations in the CCI sector. (c) Results achieved: The comparison between the four case studies revealed that, even though the four organizations belonged to different areas of the CCI, their processes were largely the same. With the support of the managers of the four companies, we specified a process architecture by formalizing 12 groups of standard processes and their process owners. The processes were all implemented in the four organizations, regardless of the type of organization and the events they organize. Thus, our result is a first version of a new process-classification framework, inspired by the APQC, that provides an integrated view of CCI organizations’ internal and external processes. (d) Lessons learned: Our work reveals that CCI organizations do not have particularly organized processes. Activities are applied as a routine, and workers lack knowledge about techniques for standardizing and optimizing processes. The benefits of developing the process-classification framework for these organizations include the newly created process awareness of the four companies and their process owners, the Puglia Creativa Cluster association’s improved clarity regarding the similarities of its members’ processes, and the recognition of BPM’ potential in CCIs.

Calreticulin (CALR) is a Ca2+-binding protein that shuttles among cellular compartments with proteins bound to its N/P domains. The knowledge that activation of the human erythropoietin receptor induces Ca2+ fluxes prompted us to investigate the role of CALR in human erythropoiesis. As shown by Western blot analysis, erythroblasts generated in vitro from normal sources and JAK2V617F polycythemia vera (PV) patients expressed robust levels of CALR. However, Ca2+ regulated CALR conformation only in normal cells. Normal erythroblasts expressed mostly the N-terminal domain of CALR (N-CALR) on their cell surface (as shown by flow cytometry) and C-terminal domain (C-CALR) in their cytoplasm (as shown by confocal microscopy) and expression of both epitopes decreased with maturation. In the proerythroblast (proEry) cytoplasm, C-CALR was associated with the glucocorticoid receptor (GR), which initiated the stress response. In these cells, Ca2+ deprivation and inhibition of nuclear export increased GR nuclear localization while decreasing cytoplasmic detection of C-CALR and C-CALR/GR association and proliferation in response to the GR agonist dexamethasone (Dex). C-CALR/GR association and Dex responsiveness were instead increased by Ca2+ and erythropoietin. In contrast, JAK2V617F proErys expressed normal cell-surface levels of N-CALR but barely detectable cytoplasmic levels of C-CALR. These cells contained GR mainly in the nucleus and were Dex unresponsive. Ruxolitinib rescued cytoplasmic detection of C-CALR, C-CALR/GR association, and Dex responsiveness in JAK2V617F proErys and its effects were antagonized by nuclear export and Ca2+ flux inhibitors. These results indicates that Ca2+-induced conformational changes of CALR regulate nuclear export of GR in normal erythroblasts and that JAK2V617F deregulates this function in PV.

Cultural and Creative industry (CCI) is a wide sector with different specializations and application areas. It refers to the creation and distribution of services or product of a cultural or artistic nature. The study investigates the lean thinking methodology application to the management of the production processes in the cultural and creative industries (CCIs) sector for realizing live performance. Applying a lean methodology to this processes costs and production times can be reduced and the overall performance increase. To address this need, a theoretical processes’ framework is proposed for process re-engineering and better allocation of human and physical resources. This result is reached by the interaction between researchers with many years of experience in business process and the expertise of four main cultural operators of the Apulia region.

Planning and executing missions in terms of trajectory generation are challenging problems in the operational phase of unmanned aerial vehicles (UAVs) lifecycle. The growing adoption of UAVs in several civil applications requires the definition of precise procedures and tools to safely manage UAV missions that may involve flight over populated areas. The paper aims at providing a contribution toward the definition of a reliable environment, called FLIP (flight planner) for route planning and risk evaluation in the framework of mini- and micro-UAV missions over populated areas. The environment represents a decision support system (DSS) for UAV operators and other decision makers, like airports authorities and aviation agencies. A new ICT tool integrating an innovative procedure for evaluating the risk related to the use of UAV over populated areas is proposed.

In manufacturing companies, computer-aided design (CAD)/computer-aided manufacturing (CAM) feature-based approaches have been developed for faster numerical control (NC) programming. They allow to automatically generate toolpath, recognizing both standard and custom machining features, and defining for each of them the best or preferred machining process based on predefined rules. The definition of Feature Based Manufacturing (FBM) rules requires advanced competences and skills, furthermore, the standardization required by these instruments is too rigorous for real machining practices. It is therefore necessary to extend the Product Lifecycle Management (PLM) environment in order to be able to make explicit and manage manufacturing rules based on industrial best practice. The paper addresses these problems presenting a possible solution to optimize FBM information management and integration within the product lifecycle. A data model extension, covering new items such as &ldquo, manufacturing rules&rdquo, and &ldquo, tool setting preferences&rdquo, and a new methodology for rules management and deployment are proposed.

The Italian cultural heritage is the richest in the world and its attrac-tiveness is still far from a complete exploitation. The technological innovation is an opportunity to enhance the valorisation process and cultural institutions are willing to exploit this potential in order to attract even more visitors. New com-munication paradigms are needed to satisfy the emerging need for cultural knowledge and experience of citizens and tourists. This paper introduces an in-novative technological and methodological framework aimed at facilitating the collaborative creation and sharing of cultural narrative experiences. The potential benefits and implication related to the framework development are described. The framework starts from the research and systematization of cultural heritage contents, which are then digitalized and virtually reconstructed in order to create interactive and immersive experiences.

In manufacturing companies, technologists use CAD/CAM tools for NC programming. Feature based approaches enable to faster programming, but require advanced competences and a standardization too rigorous for real machining practices. It is necessary a data and manufacturing rules management environment, in which knowledge engineer can define the rules based on industrial best practice and CAM Experts can customize them for production requirements. A possible solution is to extend the FBM software module with an easy-to-use system that simplifies feature based rules management and deployment. © 2018 Faculty of Mechanical Engineering and Naval Architecture. All rights reserved.

Since 2001 the Istituto Superiore di Sanità established a quality assurance programme for molecular genetic testing that covers four pathologies: Cystic Fibrosis (CF), Beta Thalassemia (BT), Fragile X Syndrome (FX), and Familial Adenomatous Polyposis Coli (APC). Since 2009 this activity is an institutional activity and participation is open to both public and private laboratories. Seven rounds have been performed until now and the eighth is in progress. Laboratories receive 4 DNA samples with mock clinical indications. They analyze the samples using their routine procedures. A panel of assessors review the raw data and the reports; all data are managed through a web utility. In 2010 the number of participants was 43, 17, 15, 5 for CF, BT, FX, APC schemes respectively. Genotyping results were correct in 96%, 98.5%, 100%, and 100% of CF, BT, FX, and APC samples, respectively. Interpretation was correct in 74%, 91%, 88%, and 60% of CF, BT, FX, and APC reports, respectively; however in most of them it was not complete but a referral to genetic counseling was given. Reports were satisfactory in more than 60% of samples in all schemes. This work presents the 2010 results in detail comparing our data with those from other European schemes.

Most business processes are today rooted into an informa-tion system recording operational events in log files. Process Mining algorithms exploit this information to discover and qualify differences between observed and modelled process. However, the output of these algorithms are not clearly con-nected with business properties. Our work faces these lim-itations by proposing an approach for calibrating Process Mining results based on the Business Rules adopted by an organisation. The general idea relates on applying Process Mining algorithms on subsequent refinements of the event log, flltering process executions based on Business Rules. This way we are able to associate these results with specific characterisations of the process, as entailed by the corre-sponding Business Rules. This approach is confronted to a real world scenario using data provided by an Italian man-ufacturing company.

In our previous studies, the bisindolic alkaloid voacamine (VOA), isolated from the plant Peschiera fuchsiaefolia, proved to exert a chemosensitizing effect on cultured multidrug resistant (MDR) osteosarcoma cells exposed to doxorubicin (DOX). In particular, VOA was capable of inhibiting P-glycoprotein action in a competitive way, thus explaining the enhancement of the cytotoxic effect induced by DOX on MDR cells. Afterwards, preliminary observations suggested that such an enhancement did not involve the apoptotic process but was due instead to the induction of autophagic cell death. The results of the present investigation demonstrate that the plant alkaloid VOA is an autophagy inducer able to exert apoptosis-independent cytotoxic effect on both wild-type and MDR tumor cells. In fact, under treatment condition causing about 50 percent of cell death, no evidence of apoptosis could be revealed by microscopical observations, Annexin V-FITC labeling and analysis of PARP cleavage, whereas the same cells underwent apoptosis when treated with apoptosis inducers, such as doxorubicin and staurosporine. Conversely, VOA-induced autophagy was clearly evidentiated by electron microscopy observations, monodansylcadaverine staining, LC3 expression, and conversion. These results were confirmed by the analysis of the modulating effects of the pretreatment with autophagy inhibitors prior to VOA administration. In addition, transfection of osteosarcoma cells with siRNA against ATG genes reduced VOA cytotoxicity. In conclusion, considering the very debated dual role of autophagy in cancer cells (protective or lethal, pro- or anti- apoptotic) our findings seem to demonstrate, at least in vitro, that a natural product able to induce autophagy can be effective against drug resistant tumors, either used alone or in association with conventional chemotherapeutics.

Elevated fetal hemoglobin (HbF) ameliorates the clinical severity of hemoglobinopathies such as β-thalassemia and sickle cell anemia. Currently, the only curative approach for individuals under chronic transfusion/chelation support therapy is allogeneic stem cell transplantation. However, recent analyses of heritable variations in HbF levels have provided a new therapeutic target for HbF reactivation: the transcriptional repressor BCL11A. Erythroid-specific BCL11A abrogation is now actively being sought as a therapeutic avenue, but the specific impact of such disruption in humans remains to be determined. Although single nucleotide polymorphisms in BCL11A erythroid regulatory elements have been reported, coding mutations are scarcer. It is thus of great interest that patients have recently been described with microdeletions encompassing BCL11A. These patients display neurodevelopmental abnormalities, but whether they show increased HbF has not been reported. We have examined the hematological phenotype, HbF levels, and erythroid BCL11A expression in 3 such patients. Haploinsufficiency of BCL11A induces only partial developmental g-globin silencing. Of greater interest is that a patient with a downstream deletion exhibits reduced BCL11A expression and increased HbF. Novel erythroid-specific regulatory elements in this region may be required for normal erythroid BCL11A expression, whereas loss of separate elements in the developing brain may explain the neurological phenotype.

Splenomegaly is a major manifestation of primary myelofibrosis (PMF) contrib- uting to clinical symptoms and hematologic abnormalities. The spleen from PMF patients contains increased numbers of hematopoietic stem cells (HSC) and megakar- yocytes. These megakaryocytes express high levels of P-selectin (P-sel) that, by trig- gering neutrophil emperipolesis, may cause TGF-β release and disease progression. This hypothesis was tested by deleting the P-sel gene in the myelofibrosis mouse model carrying the hypomorphic Gata1low mutation that induces megakaryocyte abnormalities that recapitulate those observed in PMF. P-selnullGata1low mice survived splenectomy and lived three months longer than P-selWTGata1low littermates and did not express fibrosis and osteosclerosis in the marrow or splenomegaly. Furthermore, deletion of P-sel disrupted megakaryocyte/neutrophil interactions in spleen, reduced TGF-β content and corrected the HSC distribution that in Gata1low mice, as in PMF patients, is abnormally expanded in spleen. Conversely, pharmacological inhibition of TGF-β reduced P-sel expression in megakaryocytes and corrected HSC distribution. Spleens, but not marrow, of Gata1low mice contained numerous cKITpos activated fibro- cytes, probably of dendritic cell origin, whose membrane protrusions interacted with megakaryocytes establishing niches hosting immature cKITpos hematopoietic cells. These activated fibrocytes were not detected in spleens from P-selnullGata1low or TGF-β-inhibited Gata1low littermates and were observed in spleen, but not in marrow, from PMF patients. Therefore in Gata1low mice, and possibly in PMF, abnormal P-sel expres- sion in megakaryocytes may mediate the pathological cell interactions that increase TGF-β content in MK and favor establishment of a microenvironment that supports myelofibrosis-related HSC in spleen., Italian Journal of Anatomy and Embryology, Vol. 120, No. 1 (Supplement) 2015

Expansion of erythroblasts from human peripheral blood mononuclear cells is 4- to 15-fold more efficient than that of CD34(+) cells purified from peripheral blood mononuclear cells. In addition, purified CD34(+) and CD34(-) populations from blood do not reconstitute this erythroid yield, suggesting a role for feeder cells present in blood mononuclear cells that increase hematopoietic output. Immunodepleting peripheral blood mononuclear cells for CD14(+) cells reduced hematopoietic stem and progenitor cell expansion. Conversely, the yield was increased upon co-culture of CD34(+) cells with CD14(+) cells (full contact or transwell assays) or CD34(+) cells re-constituted in conditioned medium from CD14(+) cells. In particular, CD14(++)CD16(+) intermediate monocytes/macrophages enhanced erythroblast outgrowth from CD34(+) cells. No effect of CD14(+) cells on erythroblasts themselves was observed. However, 2 days of co-culturing CD34(+) and CD14(+) cells increased CD34(+) cell numbers and colony-forming units 5-fold. Proliferation assays suggested that CD14(+) cells sustain CD34(+) cell survival but not proliferation. These data identify previously unrecognized erythroid and non-erythroid CD34(-) and CD34(+) populations in blood that contribute to the erythroid yield. A flow cytometry panel containing CD34/CD36 can be used to follow specific stages during CD34(+) differentiation to erythroblasts. We have shown modulation of hematopoietic stem and progenitor cell survival by CD14(+) cells present in peripheral blood mononuclear cells which can also be found near specific hematopoietic niches in the bone marrow.

Background: Resistance to antitumor agents is a major cause of treatment failure in patients with cancer. Some mechanisms of tumor resistance to cytotoxic drugs may involve increased acidification of extracellular compartments. We investigated whether proton pump inhibitors (PPIs), currently used in the anti-acid treatment of peptic disease, could inhibit the acidification of the tumor microenvironment and increase the sensitivity of tumor cells to cytotoxic agents. Methods: We pretreated cell lines derived from human melanomas, adenocarcinomas, and lymphomas with the PPIs omeprazole, esomeprazole, or pantoprazole and tested their response to cytotoxic drugs in cell death assays. We also evaluated extracellular and intracellular pH and vacuolar-H + -ATPase (V-H + -ATPase) expression, distribution, and activity in PPI-pretreated cells by using western blot analyses, immunocytochemistry, laser scanning confocal analysis, and bioluminescence assays. Finally, we evaluated human melanoma growth and cisplatin sensitivity with or without omeprazole pretreatment in xenografted SCID/SCID mice. Results: PPI pretreatment sensitized tumor cell lines to the effects of cisplatin, 5-fluorouracil, and vinblastine, with an IC 50 value reduction up to 2 logs. PPI pretreatment was associated with the inhibition of V-H + -ATPase activity and increases in both extracellular pH and the pH of lysosomal organelles. PPI pretreatment induced a marked increase in the cytoplasmic retention of the cytotoxic drugs, with clear targeting to the nucleus in the case of doxorubicin. In in vivo experiments, oral pretreatment with omeprazole was able to induce sensitivity of human solid tumors to cisplatin. Conclusion: Our results open new possibilities for the treatment of drug-resistant tumors through combination strategies based on the use of well-tolerated pH modulators such as PPIs.

Particulate matter (PM), a component of urban air pollution that derives primarily from the combustion of fossil fuels, is responsible for a number of health effects in humans. Recent studies have demonstrated that the fine particles (PM(2.5)) present in high numbers in PM samples can be more harmful than larger particles, since they are more efficiently retained in the peripheral lung. In the present study, we have investigated the biological effects of PM(2.5) on human lung epithelial cell line A549. Morphological analysis performed by immunofluorescence and electron microscopy showed that fine particles interact with the cell surface, where they induce evident alterations and, subsequently, are internalized in the cytoplasm. Cytoskeletal components, in particular microfilaments and microtubules, cause modifications upon challenge with PM(2.5). Of interest, an early cell response to the fine particulate is an increase of reactive oxygen species content, which can account for the observed cytoskeletal changes and the production of proinflammatory cytokines in A549 cells. In particular, exposure to PM(2.5) promoted a dose- and time-dependent release of TNF-alpha and IL-6 in the cell medium.

The search for innovative therapeutic approaches based on the use of new substances is gaining more interest in clinical oncology. In this in vitro study the potential anti-tumoral activity of tea tree oil, distilled from Melaleuca alternifolia, was analyzed against human melanoma M14 WT cells and their drug-resistant counterparts, M14 adriamicin-resistant cells. Both sensitive and resistant cells were grown in the presence of tea tree oil at concentrations ranging from 0.005 to 0.03%. Both the complex oil (tea tree oil) and its main active component terpinen-4-ol were able to induce caspase-dependent apoptosis of melanoma cells and this effect was more evident in the resistant variant cell population. Freeze-fracturing and scanning electron microscopy analyses suggested that the effect of the crude oil and of the terpinen-4-ol was mediated by their interaction with plasma membrane and subsequent reorganization of membrane lipids. In conclusion, tea tree oil and terpinen-4-ol are able to impair the growth of human M14 melanoma cells and appear to be more effective on their resistant variants, which express high levels of P-glycoprotein in the plasma membrane, overcoming resistance to caspase-dependent apoptosis exerted by P-glycoprotein-positive tumor cells.

Salivary gland tumours are rare tumours characterized by histopathologic complexity and a wide variety of morphologic features. Studies on genetic changes in different histological subtypes of salivary gland tumours are important to better understand molecular pathogenetic mechanisms and to identify diagnostic and prognostic markers. Data are even more scanty dealing with unusual subtypes of these tumours. The aim of the present study was to analyse two high grade transformation adenoid cystic carcinomas (hgACC) and one hybrid tumour in order to identify, by mutational and microsatellite analysis, genetic alterations in TP53, CDKN2A/ARF, RAS, BRAF, PTEN, MAPK2 and EGFR genes. The two hgACCs showed snps missense in RAS genes and alterations with allelic instability in CDKN2A/ARF; moreover, a double mutation in TP53 was detected in one case. The hybrid tumour showed alterations in CDKN2A/ARF gene and snps missense in NRAS genes. Our data suggest that CDKN2A/ARF pathway might be involved in pathogenesis of the salivary gland tumours analysed. Further molecular analyses of these very rare tumours are necessary to better understand the role of other genetic alterations detected in our study.

Myelofibrosis (MF) is characterized by hyperactivation of thrombopoietin (TPO) signaling, which induces a RPS14 deficiency that de-regulates GATA1 in megakaryocytes by hampering its mRNA translation. As mice carrying the hypomorphic Gata1low mutation, which reduces the levels of Gata1 mRNA in megakaryocytes, develop MF, we investigated whether the TPO axis is hyperactive in this model. Gata1low mice contained two times more Tpo mRNA in liver and TPO in plasma than wild-type littermates. Furthermore, Gata1low LSKs expressed levels of Mpl mRNA (five times greater than normal) and protein (two times lower than normal) similar to those expressed by LSKs from TPO-treated wild-type mice. Gata1low marrow and spleen contained more JAK2/STAT5 than wild-type tissues, an indication that these organs were reach of TPO-responsive cells. Moreover, treatment of Gata1low mice with the JAK inhibitor ruxolitinib reduced their splenomegaly. Also in Gata1low mice activation of the TPO/MPL axis was associated with a RSP14 deficiency and a discordant microarray ribosome signature (reduced RPS24, RPS26 and SBDS expression). Finally, electron microscopy revealed that Gata1low megakaryocytes contained poorly developed endoplasmic reticulum with rare polysomes. In summary, Gata1low mice are a bona fide model of MF, which recapitulates the hyperactivation of the TPO/MPL/JAK2 axis observed in megakaryocytes from myelofibrotic patients.

Our study showed the detection of S. suis in DNA from tissue belonging to 4 patients out of 8 with salivary gland tumors. To our knowledge there are no prevalence data of S. suis in the region where the patients were recruited; however, over the past few years the number of reported S. suis infections in humans has increased significantly (5) and it is worthwhile to report this finding to accumulate significant data about possible risk factors for these rare tumors.

Recently, the tendency of companies to adopt a PLM (product lifecycle management) strategy is increasing and asks for guidelines and reference frameworks to which screen the own organizational situation and drive improvements. Several scholars and managers have argued about PLM using a business and a technological view. Based on the need of Finmeccanica corporate to provide its companies with a reference guidelines on PLM addressing organizational and technological elements, the paper systematically analyzes models and frameworks available in literature and web-sites. The aim is to provide a set of structured key components useful to guide an exploitation of a corporate associates PLM. Models and frameworks have been organized in three categories (Academic Models, Commercial Models and Enterprise Architecture Frameworks) and fully analyzed. A four-sections PLM model has been specified supported by the evidences coming from literature. The paper provides insights for companies working in complex environment and for theoreticians.

Steady state erythropoiesis is regulated by interaction of EPO with its receptor, EPO-R which activates Ca2+signaling and terminal maturation [Miller, Blood 1989;73:1188]. Under stress, GR switches EPO-R signaling to a proliferation mode allowing erythroblast (Ery) amplification [Zhang, Nature 2013;499:92]. However, for recovery from anemia to occur, a mechanism, still to be identified, must counteract GR and switch EPO-R signaling back to a maturation mode. CALR chaperones other proteins to their active sites[Michalak, Biochem J 1999;344: 281]. By importing GR from the nucleus, CALR resets the stress response of murine fibroblasts. This function is regulated by the exposure of its C-terminal domain (C-CALR) sustained by Ca2+ [Holaska, Mol Cell Biol 2002;22:6286]. Whether CALR regulates nuclear import of GR in human Ery and how this regulation is modulated by Ca2+and EPO is unknown. Studies were performed to fill this gap. By WB, human Ery expanded in vitro from adult blood expressed robust levels of CALR, detectable with N-CALR- and C-CALR antibodies, and of GR. By flow cytometry, N-CALR and C-CALR were detectable on the surface of cells from the proerythroblast (proEry) to polychromatic stage. Expression of N-CALR, the epitope most exposed on the surface, was up-regulated by EPO. By confocal microscopy, both N-CALR and C-CALR were detected in non-permeabilized Ery but only C-CALR was detectable in cells permeabilized to reveal their cytoplasmic content. Intra-cytoplasmic C-CALR staining was robust in proEry, barely detectable in orthochromatic cells and enhanced by adding Ca2+ to the permeabilization buffer. Double staining of C-CALR with GR or LaminB1 (an inner nuclear membrane protein) indicated that C-CALR/GR were associated in the perinuclear area of the cytoplasm but that C-CALR remained distinct from Lamin B1, suggesting that C-CALR/GR co-localize at the cytoplasm/nuclear boundary. These results suggest that C-CALR mediates nuclear export of GR and that this function is regulated by Ca2+. This hypothesis was tested first by determining content and localization of GRα and C-CALR in nuclear and cytoplasmic fractions of Ery treated with Dex, EPO and SCF by WB. Although none of these stimulations altered the total content of these proteins, some of them affected their compartmentalization: Dex rapidly (15') induced GR S211 phosphorylation and increased the levels of GRα in nuclear fractions. These effects were antagonized by addition of the GR inhibitor RU486. The nuclear levels of GRα were modestly increased also by SCF but strongly decreased by EPO. By contrast, regardless of the stimuli, C-CALR was detected mostly in cytoplasmic fractions. The effects of these stimulations were also assessed by confocal microscopy. Exposure to Dex and EPO increased by 15' but decreased by 4h the nuclear localization of GR and significantly increased the levels of C-CALR and of C-CALR/GR co-localization detected in the cytoplasm by 4h, suggesting that C-CALR drives nuclear export of GR. In agreement with this hypothesis, multi-regression analyses comparing single C-CALR and total and cytoplasmic GR signals as independent parameters against merged signals as dependent parameter in 410 individual proEry indicated that the levels of C-CALR/GR co-localization directly correlated with the cytoplasmic levels of both C-CALR and GR but that the greatest p-values were observed for C-CALR (p=0.0000 vs 0.045). Moreover, treatment with the nuclear export inhibitor Leptomycin greatly decreased C-CALR/GR co-localization, increased nuclear localization of GR in proEry, both in untreated and EPO-treated cells, and reduced Ery proliferation in response to Dex down to levels sustained by Dex with RU486. To assess the role of Ca2+ in the nuclear export activity of C-CALR confocal microscopy and proliferation studies on Ery exposed or not to the Ca2+ chelator BAPTA in combination with Ionomycin (Io, a raiser of intracellular Ca2+) or Thapsigargin (TG, a reducer of Ca2+in endoplasmic reticulum) were performed. These treatments greatly decreased C-CALR and C-CALR/GR co-localization in the cytoplasm, increased GR signals in nuclei and reduced the proliferation of Ery in response to Dex. These results suggest that conformation changes of C-CALR induced by Ca2+signaling downstream to EPO-R sustain nuclear export of GR, resetting the stress-response of normal proEry allowing them to undergo terminal maturation. Disclosures No relevant conflicts of interest to declare.

Product lifecycle management (PLM) has become more important in companies providing technologies and methodologies to manage data, information, and knowledge along the whole product lifecycle. In recent years, several authors have argued about PLM using a managerial or a technological view. The paper analyses these studies and integrates different author's points of view using focus groups, blogs, and face-to-face meetings in a university community of practice. Three sets of features (i.e., managerial, technological, and collaborative ones) have been used to review the existing definitions shared between academic and industrial ones and to propose an extended PLM definition describing its key concepts. The paper is a useful reference for managers and academics who want to have a clear and critical understanding of PLM using a unique source to collect lines of evidence on several PLM definitions, features, and concepts.

Elevation of cAMP inhibits the proliferation and expression of transformed phenotype in several cell types, including breast cancer cells. Leptin has been shown to act as a mitogen/survival factor in many types of cancer cells. In the present work, we have studied the impact of cAMP elevation on leptin-induced proliferation of breast cancer cells. Here we report that treatment of estrogen receptor negative human breast cancer cell line MDA-MB-231 with leptin or cAMP elevating agents has positive and negative effects on cell proliferation, respectively. Surprisingly, we find that leptin strongly potentiates the anti-proliferative action of cAMP elevating agents, by concurring to cell cycle arrest at G1 phase and inducing apoptosis. Pretreatment with the PKA inhibitor KT-5720 completely prevented the anti-proliferative effects induced by the combination between leptin and cAMP elevating agents. The above anti-proliferative effects were paralleled by the decrease of cyclin D1 and A and by the increase of inhibitor p27kip1 cell cycle regulating protein levels. In these conditions we found also a strong decrease of anti-apopotic Bcl2 protein levels. Altogether, our data extend the evidence of adenylate cyclase/cAMP/PKA as a growth suppressor system and of leptin as a growth promoting factor in breast cancer cells. Remarkably, our results suggest that when cAMP levels are increased, leptin drives cells towards apoptosis, and that targeting both cAMP levels and leptin signalling might represent a simple novel way for therapeutic intervention in breast cancer.

A correlation between regulation of cell proliferation and polyamine metabolism is described. The latter can enter protein synthesis through the modification of eukaryotic initiation factor 5A (eIF5A) and the formation of the peculiar amino acid hypusine. Specific inhibitors of hypusine formation induce apoptosis that can be potentiated by the combination with cytokines such as interferonalpha (IFNalpha) that itself decreases hypusine synthesis. We have also demonstrated that the concomitant treatment of cancer cells with IFNalpha and the protein synthesis inhibitor fusion protein TGFalpha/Pseudomonas Aeruginosa toxin synergize in inducing cancer cell growth inhibition. Another way used by polyamines to induce apoptosis is the generation of intracellular oxidative stress through the interaction with bovine serum amine oxidase (BSAO). This enzyme used simultaneously to spermine induces apoptosis, necrosis, inhibition of cell proliferation and inhibition of DNA and protein synthesis in several cell types. The enzymatic oxidation products of polyamine, H2O2 and aldehyde(s) cause these effects. We have recently found that the cytotoxicity of anti-cancer agents, either etoposide or docetaxel, in cancer cells is potentiated in the presence of BSAO/Spermine. In conclusion, polyamine metabolites could be useful in the design of new therapeutic strategies.

Hyperthermia is currently receiving widespread attention when associated with other therapeutic modalities, such as irradiation or chemotherapy, in the treatment of cancer. The occurrence of resistance to cytotoxic pharmacological agents in tumor cells, associated with several phenotypic alterations, is one of the major obstacles to successful anticancer chemotherapy. We investigated a new strategy to overcome multidrug resistance (MDR) cancer cells, using bovine serum amine oxidase (BSAO), which forms toxic products from spermine (H2O2 and aldehydes). The cytotoxicity of the products was evaluated in drug-sensitive (LoVo WT) and multidrug-resistant (LoVo DX) colon adenocarcinoma cells at 37 and 42 degrees C, using a clonogenic cell survival assay. Cytotoxicity was considerably enhanced at 42 degrees C. Both toxic species contributed to the thermal enhancement of cytotoxicity induced by BSAO and spermine. Cytotoxicity was eliminated in the presence of catalase and aldehyde dehydrogenase (ALDH). An interesting finding was that BSAO and spermine at

In our previous studies, voacamine, a bisindolic alkaloid extracted from Peschiera fuchsiaefolia, was examined for its possible capability of enhancing the cytotoxic effect of doxorubicin (DOX) on multidrug resistant (MDR) human osteosarcoma cells (U-2 OS-R). Voacamine induced in resistant cells a significant increase of drug retention and intranuclear location which became comparable to those observed in the parental sensitive counterparts (U-2 OS-WT). In the present study, the cell survival analysis and the electron microscopic observations confirmed the evident cytotoxicity of DOX on MDR cells after pre-treatment with the plant extract. Moreover, an increase of the reactivity of P-glycoprotein (P-gp) with the monoclonal antibody UIC2, which recognizes an epitope of the drug transporter in its functional conformation, was revealed, demonstrating that voacamine is a substrate of P-gp, thus acting as a competitive antagonist of the cytotoxic agent. Moreover, to investigate if the enhancement of the cytotoxic effect induced by voacamine could be due to an apoptotic process, we carried out the analysis of cell morphology after Hoechst staining and the quantification of apoptosis by Annexin V-FITC assay. These evaluations showed a very low rate of apoptosis in U-2 OS-R cells treated with voacamine and DOX given in association. In addition, the combined treatment induced ultrastructural modifications suggestive of autophagic cell death. In particular, transmission electron microscopy observations revealed the presence of numerous lysosomes and the formation of a large number of autophagosomes containing residual digested material. In conclusion, these findings seem to indicate that voacamine is capable of enhancing the cytotoxic effect of DOX on MDR cells by favouring a lethal autophagic process.

In situ formation of cytotoxic metabolites by an enzyme-catalyzed reaction is a recent approach in cancer chemotherapy. We demonstrate that multidrug resistant human melanoma cells (M14 ADR) are more sensitive than the corresponding wild type cells (M14 WT) to hydrogen peroxide and aldehydes, the products of bovine serum amine oxidase (BSAO)-catalyzed oxidation of spermine. Hydrogen peroxide was mainly responsible for the loss of cell viability. With about 20%, the aldehydes formed from spermine contribute also to cytotoxicity. Elevation of temperature from 37 degrees C to 42 degrees C decreased survival of both cell lines by about one log unit. Pre-treatment with N1,N4-bis(2,3-butadienyl)-1,4-butanediamine (MDL 72527), a lysosomotropic compound, sensitized cells to toxic spermine metabolites. MDL 72527 (at 300 microM) produced in M14 cells numerous cytoplasmic vacuoles which, however, disappeared by 24 h, even in the presence of the drug. Mitochondrial damage, as observed by transmission electron microscopy, correlated better with the cytotoxic effects of the treatment than vacuole formation. Since the release of lysosomal enzymes causes oxidative stress and apoptosis, we suggest that the lysosomotropic effect of MDL 72527 is the major reason for its sensitizing effect.

Mixed cationic liposomes composed by different ratios of dimyristoyl-sn-glycero-phosphatidylcoline (DMPC) and a cationic gemini surfactant have been studied by various physicochemical tools as vehicles for m-tetrahydroxyphenylchlorin (m-THPC), a photosensitizer used in photodynamic therapy. Entrapment and location of m-THPC within the lipid double layer have been evaluated by different techniques and the new formulations have been tested on a stabilized cell line from a human colon tumor, COLO206. A correlation between the physicochemical features of formulations and their efficiency as photosensitizers vector was found.