Correct composition of dephased behavioural models

This research is supported by EPSRC grant EP/M014290/1.Scenarios of execution are commonly used to specify partial behaviour and interactions between different objects and components in a system. To avoid overall inconsistency in specifications, various automated methods have emerged in the literature to compose (behavioural) models. In recent work, we have shown how the theorem prover Isabelle can be combined with the constraint solver Z3 to efficiently detect inconsistencies in two or more behavioural models and, in their absence, generate the composition. Here, we extend our approach further and show how to generate the correct composition (as a set of valid traces) of dephased models. This work has been inspired by a problem from a medical domain where different care pathways (for chronic conditions) may be applied to the same patient with different starting points.Postprin

A unifying perspective on protocol mediation: interoperability in the Future Internet

Given the highly dynamic and extremely heterogeneous software systems composing the Future Internet, automatically achieving interoperability between software components —without modifying them— is more than simply desirable, it is quickly becoming a necessity. Although much work has been carried out on interoperability, existing solutions have not fully succeeded in keeping pace with the increasing complexity and heterogeneity of modern software, and meeting the demands of runtime support. On the one hand, solutions at the application layer target higher automation and loose coupling through the synthesis of intermediary entities, mediators, to compensate for the differences between the interfaces of components and coordinate their behaviours, while assuming the use of the same middleware solution. On the other hand, solutions to interoperability across heterogeneous middleware technologies do not reconcile the differences between components at the application layer. In this paper we propose a unified approach for achieving interoperability between heterogeneous software components with compatible functionalities across the application and middleware layers. First, we provide a solution to automatically generate cross-layer parsers and composers that abstract network messages into a uniform representation independent of the middleware used. Second, these generated parsers and composers are integrated within a mediation framework to support the deployment of the mediators synthesised at the application layer. More specifically, the generated parser analyses the network messages received from one component and transforms them into a representation that can be understood by the application-level mediator. Then, the application-level mediator performs the necessary data conversion and behavioural coordination. Finally, the composer transforms the representation produced by the application-level mediator into network messages that can be sent to the other component. The resulting unified mediation framework reconciles the differences between software components from the application down to the middleware layers. We validate our approach through a case study in the area of conference management

Correction to: Recurrent pregnancy loss is associated to leaky gut: a novel pathogenic model of endometrium inflammation?

Background: Recurrent pregnancy loss (RPL) occurs in 3–5% in about 30% of cases no cause can be found. Women with RPL show higher prevalence of undiagnosed gut disorders. Furthermore, in endometrial tissues of RPL women, higher expression of pro-inflammatory cytokines and Nalp-3 inflammasome has been observed. Aim of this study was to investigate whether an abnormal gut permeability might occur in RPL women and allow passage into systemic circulation of pro-inflammatory molecules able to induce endometrial inflammation. Methods: 70 women with idiopathic RPL and 30 healthy women were recruited at the Recurrent Pregnancy Loss Outpatient Unit of the Gemelli Hospital of Rome from March 2013 to February 2017. Enrolled women underwent 51Cr-ethylene-diamine-tetraacetic acid absorption test to evaluate intestinal permeability. Sera obtained from enrolled women were analysed for lipopolysaccharide (LPS) by ELISA. Anxiety and depression state were evaluated by administering STAI-Y and Zung-SDS tests, respectively. Of all recruited individuals, 35 women with idiopathic RPL and 20 healthy controls accepted to undergo diagnostic hysteroscopy and endometrial biopsy. Endometrial lysates were investigated for inflammasome Nalp-3 by Western blot analysis, and caspase-1, IL-1β and IL-18 by ELISA, respectively. Results: Higher prevalence of abnormal intestinal permeability (P < 0.0001), increased circulating levels of LPS (P < 0.05), anxiety (P < 0.05) and depression (P < 0.05) were observed in RLP women compared to controls. Endometrial expression of Nalp-3, caspase-1 and IL-1β was significantly increased in RPL group (P < 0.0001; P < 0.05 and P < 0.001, respectively). IL-18 endometrial levels were not found to be higher in RPL cases. Statistically significant association between higher intestinal permeability and abnormally increased expression of endometrial Nalp-3, was observed in RPL (P < 0.01). Furthermore, higher LPS serum levels, a bacterial-derived activator of Nalp-3 complex, was shown to be statistically associated to abnormal endometrial expression of Nalp-3 inflammasome (P < 0.01) in RPL women. Conclusions: In women with RLP, leaky gut might occur and allow passage into circulation of immune triggers, potentially able to elicit endometrial innate immune response and, thus, to contribute to miscarriage pathogenesis. Diagnosis and treatment of intestinal disorders underlying leaky gut might improve endometrial environment and pregnancy outcome

Capacitative calcium influx and proliferation of human osteoblastic-like MG-63 cells

Adult bone tissue is continuously being remodelled and bone mass is maintained by a balance between osteoclastic bone resorption and osteoblastic bone formation. Alteration of osteoblastic cell proliferation may account in part for lack of balance between these two processes in bone loss of osteoporosis. There is calcium (Ca2+) control in numerous cellular functions; however, involvement of capacitative Ca2+ entry (CCE) in proliferation of bone cells is less well investigated. OBJECTIVES: The study described here was aimed to investigate roles of CCE in the proliferation of osteoblast-like MG-63 cells. MATERIALS AND METHODS: Pharmacological characterizations of CCE were undertaken in parallel, with evaluation of the expression of transient receptor potential canonical (TRPC) channels and of cell proliferation. RESULTS: Intracellular Ca2+ store depletion by thapsigargin induced CCE in MG-63 cells; this was characterized by a rapid transient increase of intracellular Ca2+ followed by significant CCE, induced by conditions that stimulated cell proliferation, namely serum and platelet-derived growth factor. Inhibitors of store-operated Ca2+ channels (2-APB and SKF-96365) prevented CCE, while voltage-dependent Ca2+ channel blockers had no effect. Expression of various TRPC channels was shown in the cells, some having been shown to be responsible for CCE. Voltage-dependent Ca2+ channel blockers had no effect on osteoblast proliferation while thapsigargin, 2-APB and SKF-96395, inhibited it. Cell cycle analysis showed that 2-APB and SKF-96395 lengthen the S and G2/M phases, which would account for the reduction in cell proliferation. CONCLUSIONS: Our results indicate that CCE, likely attributed to the activation of TRPCs, might be the main route for Ca2+ influx involved in osteoblast proliferation

Hepatocyte Growth Factor Increases Osteopontin Expression in Human Osteoblasts through PI3K, Akt, c-Src, and AP-1 Signaling Pathway

BACKGROUND: Hepatocyte growth factor (HGF) has been demonstrated to stimulate osteoblast proliferation and participated bone remodeling. Osteopontin (OPN) is a secreted phosphoglycoprotein that belongs to the SIBLING family and is present during bone mineralization. However, the effects of HGF on OPN expression in human osteoblasts are large unknown. METHODOLOGY/PRINCIPAL FINDINGS: Here we found that HGF induced OPN expression in human osteoblasts dose-dependently. HGF-mediated OPN production was attenuated by c-Met inhibitor and siRNA. Pretreatment of osteoblasts with PI3K inhibitor (Ly294002), Akt inhibitor, c-Src inhibitor (PP2), or AP-1 inhibitor (curcumin) blocked the potentiating action of HGF. Stimulation of osteoblasts with HGF enhanced PI3K, Akt, and c-Src activation. In addition, incubation of cells with HGF also increased c-Jun phosphorylation, AP-1-luciferase activity, and c-Jun binding to the AP-1 element on the OPN promoter. HGF-mediated AP-1-luciferase activity and c-Jun binding to the AP-1 element was reduced by c-Met inhibitor, Ly294002, Akt inhibitor, and PP2. CONCLUSIONS/SIGNIFICANCE: Our results suggest that the interaction between HGF and c-Met increases OPN expression in human osteoblasts via the PI3K, Akt, c-Src, c-Jun, and AP-1 signaling pathway

The Relevance of Marine Chemical Ecology to Plankton and Ecosystem Function: An Emerging Field

Marine chemical ecology comprises the study of the production and interaction of bioactive molecules affecting organism behavior and function. Here we focus on bioactive compounds and interactions associated with phytoplankton, particularly bloom-forming diatoms, prymnesiophytes and dinoflagellates. Planktonic bioactive metabolites are structurally and functionally diverse and some may have multiple simultaneous functions including roles in chemical defense (antipredator, allelopathic and antibacterial compounds), and/or cell-to-cell signaling (e.g., polyunsaturated aldehydes (PUAs) of diatoms). Among inducible chemical defenses in response to grazing, there is high species-specific variability in the effects on grazers, ranging from severe physical incapacitation and/or death to no apparent physiological response, depending on predator susceptibility and detoxification capability. Most bioactive compounds are present in very low concentrations, in both the producing organism and the surrounding aqueous medium. Furthermore, bioactivity may be subject to synergistic interactions with other natural and anthropogenic environmental toxicants. Most, if not all phycotoxins are classic secondary metabolites, but many other bioactive metabolites are simple molecules derived from primary metabolism (e.g., PUAs in diatoms, dimethylsulfoniopropionate (DMSP) in prymnesiophytes). Producing cells do not seem to suffer physiological impact due to their synthesis. Functional genome sequence data and gene expression analysis will provide insights into regulatory and metabolic pathways in producer organisms, as well as identification of mechanisms of action in target organisms. Understanding chemical ecological responses to environmental triggers and chemically-mediated species interactions will help define crucial chemical and molecular processes that help maintain biodiversity and ecosystem functionality

Editing of the urease gene by CRISPR-Cas in the diatom Thalassiosira pseudonana

Background: CRISPR-Cas is a recent and powerful addition to the molecular toolbox which allows programmable genome editing. It has been used to modify genes in a wide variety of organisms, but only two alga to date. Here we present a methodology to edit the genome of Thalassiosira pseudonana, a model centric diatom with both ecological significance and high biotechnological potential, using CRISPR-Cas. Results: A single construct was assembled using Golden Gate cloning. Two sgRNAs were used to introduce a precise 37 nt deletion early in the coding region of the urease gene. A high percentage of bi-allelic mutations (≤61.5%) were observed in clones with the CRISPR-Cas construct. Growth of bi-allelic mutants in urea led to a significant reduction in growth rate and cell size compared to growth in nitrate. Conclusions: CRISPR-Cas can precisely and efficiently edit the genome of T. pseudonana. The use of Golden Gate cloning to assemble CRISPR-Cas constructs gives additional flexibility to the CRISPR-Cas method and facilitates modifications to target alternative genes or species

Diatom-derived oxylipins induce cell death in sea urchin embryos activating caspase-8 and caspase 3/7

Diatoms are an important class of unicellular algae that produce bioactive secondary metabolites withcytotoxic activity collectively termed oxylipins, including polyunsaturated aldehydes (PUAs), hydroxy-acids (HEPEs), oxo-acids and epoxyalcohols. Previous results showed that at higher concentrations, thePUA decadienal induced apoptosis on copepods and sea urchin embryos via caspase-3 activation; atlower concentrations decadienal affected the expression levels of the caspase-8 gene in embryos of thesea urchin Paracentrotus lividus. In the present work, we studied the effects of other common oxylip-ins produced by diatoms: two PUAs (heptadienal and octadienal) and four hydroxyacids (5-, 9- 11- and15-HEPE) on P. lividus cell death and caspase activities. Our results showed that (i) at higher concentra-tions PUAs and HEPEs induced apoptosis in sea urchin embryos, detected by microscopic observationand through the activation of caspase-3/7 and caspase-8 measured by luminescent assays; (ii) at lowconcentrations, PUAs and HEPEs affected the expression levels of caspase-8 and caspase-3/7 (isolated forthe first time here in P. lividus) genes, detected by Real Time qPCR. These findings have interesting impli-cations from the ecological point of view, given the importance of diatom blooms in nutrient-rich aquaticenvironments

Native human adipose stromal cells: localization, morphology and phenotype

International audienceObjectives:Beside having roles in energy homeostasis and endocrine modulation, adipose tissue (AT) is now considered a promising source of mesenchymal stromal cells (adipose-derived stromal cells or ASCs) for regenerative medicine. Despite numerous studies on cultured ASCs, native human ASCs are rarely investigated. Indeed, the phenotype of ASCs in their native state, their localization within AT and comparison with bone marrow-derived mesenchymal stromal cells (BM-MSCs) has been poorly investigated.Design:To address these issues, the stroma vascular fraction (SVF) of human AT was extracted and native cell subtypes were isolated by immunoselection to study their clonogenic potential in culture. Immunohistology on samples of human AT in combination with reconstruction of confocal sections were performed in order to localize ASCs.Results:Compared with BM-MNCs, all native ASCs were found in the CD34(+) cell fraction of the AT-SVF. Native ASCs expressed classical mesenchymal markers described for BM-MSCs. Interestingly, CD34 expression decreased during ASC cell culture and was negatively correlated with cell proliferation rate. Immunohistological analysis revealed that native ASCs exhibited specific morphological features with protrusions. They were found scattered in AT stroma and did not express in vivo pericytic markers such as NG2, CD140b or alpha-smooth muscle actin, which appeared during the culture process. Finally, ASCs spontaneous commitment to adipocytic lineage was enhanced in AT from obese humans.Conclusions:The use of complementary methodological approaches to study native human ASCs revealed their immunophenotype, their specific morphology, their location within AT and their stemness. Furthermore, our data strongly suggest that human ASCs participate in adipogenesis during AT development.International Journal of Obesity advance online publication, 25 January 2011; doi:10.1038/ijo.2010.269