An image cytometric technique is a concise method to detect adenoviruses and host cell proteins and to monitor the infection and cellular responses induced

BackgroundGenetically modified adenoviruses (Ad) with preferential replications in tumor cells have been examined for a possible clinical applicability as an anti-cancer agent. A simple method to detect viral and cellular proteins is valuable to monitor the viral infections and to predict the Ad-mediated cytotoxicity.MethodsWe used type 5 Ad in which the expression of E1A gene was activated by 5′-regulatory sequences of genes that were augmented in the expression in human tumors. The Ad were further modified to have the fiber-knob region replaced with that derived from type 35 Ad. We infected human mesothelioma cells with the fiber-replaced Ad, and sequentially examined cytotoxic processes together with an expression level of the viral E1A, hexon, and cellular cleaved caspase-3 with image cytometric and Western blot analyses.ResultsThe replication-competent Ad produced cytotoxicity on mesothelioma cells. The infected cells expressed E1A and hexon 24 h after the infection and then showed cleavage of caspase-3, all of which were detected with image cytometry and Western blot analysis. Image cytometry furthermore demonstrated that increased Ad doses did not enhance an expression level of E1A and hexon in an individual cell and that caspase-3-cleaved cells were found more frequently in hexon-positive cells than in E1A-positive cells. Image cytometry thus detected these molecular changes in a sensitive manner and at a single cell level. We also showed that an image cytometric technique detected expression changes of other host cell proteins, cyclin-E and phosphorylated histone H3 at a single cell level.ConclusionsImage cytometry is a concise procedure to detect expression changes of Ad and host cell proteins at a single cell level, and is useful to analyze molecular events after the infection

CA-ARBAC: privacy preserving using context-aware role-based access control on Android permission system

Existing mobile platforms are based on manual way of granting and revoking permissions to applications. Once the user grants a given permission to an application, the application can use it without limit, unless the user manually revokes the permission. This has become the reason for many privacy problems because of the fact that a permission that is harmless at some occasion may be very dangerous at another condition. One of the promising solutions for this problem is context-aware access control at permission level that allows dynamic granting and denying of permissions based on some predefined context. However, dealing with policy configuration at permission level becomes very complex for the user as the number of policies to configure will become very large. For instance, if there are A applications, P permissions, and C contexts, the user may have to deal with A × P × C number of policy configurations. Therefore, we propose a context-aware role-based access control model that can provide dynamic permission granting and revoking while keeping the number of policies as small as possible. Although our model can be used for all mobile platforms, we use Android platform to demonstrate our system. In our model, Android applications are assigned roles where roles contain a set of permissions and contexts are associated with permissions. Permissions are activated and deactivated for the containing role based on the associated contexts. Our approach is unique in that our system associates contexts with permissions as opposed to existing similar works that associate contexts with roles. As a proof of concept, we have developed a prototype application called context-aware Android role-based access control. We have also performed various tests using our application, and the result shows that our model is working as desired

A Possible Anticancer Agent, Type III Interferon, Activates Cell Death Pathways and Produces Antitumor Effects

Recently identified interleukin-28 and -29 belong to a novel type III interferon (IFN) family, which could have distinct biological properties from type I and II IFNs. Type I IFNs, IFN-α/β, have been clinically applied for treating a certain kind of malignancies for over 30 years, but a wide range of the adverse effects hampered the further clinical applications. Type III IFNs, IFN-λs, have similar signaling pathways as IFN-α/β and inhibits proliferation of tumor cells through cell cycle arrest or apoptosis. Restricted patterns of type III IFN receptor expression in contrast to ubiquitously expressed IFN-α/β receptors suggest that type III IFNs have limited cytotoxicity to normal cells and can be a possible anticancer agent. In this paper, we summarize the current knowledge on the IFN-λs-mediated tumor cell death and discuss the functional difference between type I and III IFNs

Midkine is a potential novel marker for malignant mesothelioma with different prognostic and diagnostic values from mesothelin

BACKGROUND: We evaluated possible diagnostic and prognostic values of serum midkine in malignant pleural mesothelioma in comparison with those of serum mesothelin, a well-established diagnostic biomarker. METHODS: Serum mesothelin and midkine levels were determined with an enzyme-linked immunosorbent assay. We examined specimens from 95 Turkish cases with malignant pleural mesothelioma, 56 metastatic cancers to pleura, 27 other types of benign pleural diseases and 20 benign asbestos pleurisy. The cut-off values were 1.5 nmol/L for mesothelin and 421 pg/mL for midkine. RESULTS: Sensitivity and specificity of mesothelin were 51.6 and 71.4%, 51.6 and 85.2%, and 51.6 and 85% for differentiating mesothelioma from metastatic cancers to pleura, other benign pleural diseases and benign asbestos pleurisy, respectively. Sensitivity and specificity of midkine were 61.1 and 41.1%, 61.1 and 48.1%, and 61.1 and 75% to distinguish mesothelioma from metastatic cancers to pleura, other benign pleural diseases and benign asbestos pleurisy, respectively. Combination of both biomarkers did not improve the differential diagnostic efficacy. Mesothelin levels were elevated in the epitheloid type and in the advanced cases, but were not related to the prognosis. In contrast, elevated baseline levels of midkine were independently associated with a poor prognosis of mesothelioma patients after adjusting for the stage, the histological subtypes and treatment schedules (HR = 1.84; 95% CI: 1.09-3.09) (p = 0.022). CONCLUSIONS: Serum mesothelin showed moderate sensitivity and high specificity to differentiate malignant pleural mesothelioma from metastatic malignancy to pleura and from benign pleural diseases. In contrast, midkine was a useful marker for predicting prognosis of mesothelioma patients

An intrapleural administration of zoledronic acid for inoperable malignant mesothelioma patients: a phase I clinical study protocol

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Metformin produces growth inhibitory effects in combination with nutlin-3a on malignant mesothelioma through a cross-talk between mTOR and p53 pathways

BackgroundMesothelioma is resistant to conventional treatments and is often defective in p53 pathways. We then examined anti-tumor effects of metformin, an agent for type 2 diabetes, and combinatory effects of metformin and nutlin-3a, an inhibitor for ubiquitin-mediated p53 degradation, on human mesothelioma.MethodsWe examined the effects with a colorimetric assay and cell cycle analyses, and investigated molecular events in cells treated with metformin and/or nutlin-3a with Western blot analyses. An involvement of p53 was tested with siRNA for p53.ResultsMetformin suppressed cell growth of 9 kinds of mesothelioma including immortalized cells of mesothelium origin irrespective of the p53 functional status, whereas susceptibility to nutlin-3a was partly dependent on the p53 genotype. We investigated combinatory effects of metformin and nutlin-3a on, nutlin-3a sensitive MSTO-211H and NCI-H28 cells and insensitive EHMES-10 cells, all of which had the wild-type p53 gene. Knockdown of p53 expression with the siRNA demonstrated that susceptibility of MSTO-211H and NCI-H28 cells to nutlin-3a was p53-dependent, whereas that of EHMES-10 cells was not. Nevertheless, all the cells treated with both agents produced additive or synergistic growth inhibitory effects. Cell cycle analyses also showed that the combination increased sub-G1 fractions greater than metformin or nutlin-3a alone in MSTO-211H and EHMES-10 cells. Western blot analyses showed that metformin inhibited downstream pathways of the mammalian target of rapamycin (mTOR) but did not activate the p53 pathways, whereas nutlin-3a phosphorylated p53 and suppressed mTOR pathways. Cleaved caspase-3 and conversion of LC3A/B were also detected but it was dependent on cells and treatments. The combination of both agents in MSTO-211H cells rather suppressed the p53 pathways that were activated by nutrin-3a treatments, whereas the combination rather augmented the p53 actions in NCI-H28 and EHMES-10 cells.ConclusionThese data collectively indicated a possible interactions between mTOR and p53 pathways, and the combinatory effects were attributable to differential mechanisms induced by a cross-talk between the pathways

Combination of a third generation bisphosphonate and replication-competent adenoviruses augments the cytotoxicity on mesothelioma

BackgroundApproximately 80 % of mesothelioma specimens have the wild-type p53 gene, whereas they contain homozygous deletions in the INK4A/ARF locus that encodes p14 ARF and the 16 INK4A genes. Consequently, the majority of mesothelioma is defective of the p53 pathways. We examined whether zoledronic acid (ZOL), a third generation bisphosphonate, and adenoviruses with a deletion of the E1B-55kD gene (Ad-delE1B55), which augments p53 levels in the infected tumors, could produce combinatory anti-tumor effects on human mesothelioma cells bearing the wild-type p53 gene.MethodsCytotoxicity of ZOL and Ad-delE1B55 was assessed with a WST assay. Cell cycle changes were tested with flow cytometry. Expression levels of relevant molecules were examined with western blot analysis to investigate a possible mechanism of cytotoxicity. Furthermore, the expressions of Ad receptors on target cells and infectivity were estimated with flow cytometry. Viral replication was assayed with the tissue culture infection dose method.ResultsA combinatory use of ZOL and Ad-delE1B55 suppressed cell growth and increased sub-G1 or S-phase populations compared with a single agent, depending on cells tested. The combinatory treatment up-regulated p53 levels and subsequently enhanced the cleavage of caspase-3, 8, 9 and poly (ADP-ribose) polymerase, but expression of molecules involved in autophagy pathways were inconsistent. ZOL-treated cells also increased Ad infectivity with a dose-dependent manner and augmented Ad replication although the expression levels of integrin molecules, one of the Ad receptors, were down-regulated.ConclusionsThese findings indicated that ZOL and Ad-delE1B55 achieved combinatory anti-tumor effects through augmented apoptotic pathways or increased viral replication

Cytotoxicity of replication-competent adenoviruses powered by an exogenous regulatory region is not linearly correlated with the viral infectivity/gene expression or with the E1A-activating ability but is associated with the p53 genotypes

BackgroundReplication-competent adenoviruses (Ad) produced cytotoxic effects on infected tumors and have been examined for the clinical applicability. A biomarkers to predict the cytotoxicity is valuable in a clinical setting.MethodsWe constructed type 5 Ad (Ad5) of which the expression of E1A gene was activated by a 5′ regulatory sequences of survivin, midkine or cyclooxygenase-2, which were highly expressed in human tumors. We also produced the same replication-competent Ad of which the fiber-knob region was replaced by that of Ad35 (AdF35). The cytotoxicity was examined by a colorimetric assay with human tumor cell lines, 4 kinds of pancreatic, 9 esophageal carcinoma and 5 mesothelioma. Ad infectivity and Ad-mediated gene expression were examined with replication-incompetent Ad5 and AdF35 which expressed the green fluorescence protein gene. Expression of cellular receptors for Ad5 and AdF35 was also examined with flow cytometry. A transcriptional activity of the regulatory sequences was investigated with a luciferase assay in the tumor cells. We then investigated a possible correlation between Ad-mediated cytotoxicity and the infectivity/gene expression, the transcriptional activity or the p53 genotype.ResultsWe found that the cytotoxicity was greater with AdF35 than with Ad5 vectors, but was not correlated with the Ad infectivity/gene expression irrespective of the fiber-knob region or the E1A-activating transcriptional activity. In contrast, replication-competent Ad produced greater cytotoxicity in p53 mutated than in wild-type esophageal carcinoma cells, suggesting a possible association between the cytotoxicity and the p53 genotype.ConclusionsSensitivity to Ad-mediated cytotoxic activity was linked with the p53 genotype but was not lineally correlated with the infectivity/gene expression or the E1A expression

Heat-resistant DNA tile arrays constructed by template-directed photoligation through 5-carboxyvinyl-2′-deoxyuridine

Template-directed DNA photoligation has been applied to a method to construct heat-resistant two-dimensional (2D) DNA arrays that can work as scaffolds in bottom-up assembly of functional biomolecules and nano-electronic components. DNA double-crossover AB-staggered (DXAB) tiles were covalently connected by enzyme-free template-directed photoligation, which enables a specific ligation reaction in an extremely tight space and under buffer conditions where no enzymes work efficiently. DNA nanostructures created by self-assembly of the DXAB tiles before and after photoligation have been visualized by high-resolution, tapping mode atomic force microscopy in buffer. The improvement of the heat tolerance of 2D DNA arrays was confirmed by heating and visualizing the DNA nanostructures. The heat-resistant DNA arrays may expand the potential of DNA as functional materials in biotechnology and nanotechnology