Substrate specificity of the Escherichia coli RuvC protein. Resolution of three- and four-stranded recombination intermediates

The specificity of the Escherichia coli RuvC Holliday junction resolvase has been investigated in vitro. RuvC protein cleaves synthetic DNA substrates that model three- or four-stranded recombination intermediates but fails to act upon Y junctions, G/A mismatches, heterologous loop structures, or two-stranded branched junctions. RuvC therefore differs from endonuclease VII of bacteriophage T4 which exhibits broad range specificity. Using related three- and four-stranded synthetic DNA junctions, we show that RuvC cleaves both junctions at the same DNA sequence and requires a region of homology at the junction point. The action of RuvC on three- and four-stranded recombination intermediates made by RecA was also investigated. We found that RuvC fails to resolve three-stranded intermediates in the presence of RecA, although four-stranded intermediates are resolved under the same conditions. However, both three- and four-stranded intermediates are substrates for the nuclease after removal of RecA. We interpret these differences in terms of the contiguity of the RecA nucleoprotein filament which may, under certain conditions, limit access to the Holliday junction resolvase.     

Publication outcome of research funding by the Danish Heart Foundation 1988-1990

The aim of the survey was to analyse the investment by the Danish Heart Foundation in the cardiovascular research field in the period 1988-1990 and the ensuing research results. One hundred and thirty-nine researchers were allocated a total DDK 24.1 million. Eighty percent of the researchers have concluded their research work and published 362 scientific papers in 131 journals. The total journal impact factor obtained among 270 scientific papers with known journal impact factor was 642. The median journal impact factor was 1.580. Thirty-five percent of the papers were published in journals with journal impact factor greater than three. The productivity, defined as total journal impact factor obtained divided by an estimate of the total amount (DKK 200 million) of economic support received by the researcher from all sources, was estimated to 3.2 Journal Impact Factor/DKK million. A panel of international experts reviewed the outcome of funding by the Danish Heart Foundation, and concluded that the number of publications and their impact factor was adequate in relation to the economic input.     

Environmental antigen-induced IL-13 responses are elevated among subjects with allergic rhinitis, are independent of IL-4, and are inhibited by endogenous IFN-gamma synthesis

Human immediate hypersensitivity diseases represent the most common example of chronic excessive Th2-like activation in developed nations. While IL-13 shares many functional properties with IL-4, the intensity and regulation of environmental Ag-stimulated IL-13 synthesis by allergic vs nonallergic individuals remain ill defined. Here, we examine the intensity of polyclonally and Ag-stimulated IL-13 production by PBMC of 20 subjects with seasonal allergic rhinitis and 20 healthy controls. Polyclonally driven IL-13 responses did not differ significantly (Mann-Whitney U test, p = 0.68). In contrast, the median CD4-dependent IL-13 response among atopics was markedly stronger than nonatopics in Ag-stimulated primary culture (p = 0.0031) and exhibited a strong correlation with IL-5 (r = 0.76, p = 0.0009), but not IL-4 (r = 0.14, p > 0.05), responses. IL-13 production was unaffected by blocking endogenous IL-4 or IL-5 activity or by addition of rIL-4 or rIL-5. In contrast, it was inhibited by low levels of rIFN-gamma and strongly enhanced upon addition of neutralizing anti-IFN-gamma mAb. Collectively, the data are consistent with a negative regulatory role for endogenous IFN-gamma synthesis in controlling the intensity of systemic IL-13 responses evoked in both atopic and nonatopic populations following exposure to common Ags. They also suggest that the elevated levels of IL-4 and IL-5 characteristic of type 2-dominated responses in vivo are without detectable impact on the maintenance of recall Ag-stimulated IL-13 production.     

The Chick Chorioallantoic Membrane Model: A New In Vivo Tool to Evaluate Breast Cancer Stem Cell Activity

The high plasticity of cancer stem-like cells (CSCs) allows them to differentiate and proliferate, specifically when xenotransplanted subcutaneously into immunocompromised mice. CSCs are highly tumorigenic, even when inoculated in small numbers. Thus, in vivo limiting dilution assays (LDA) in mice are the current gold standard method to evaluate CSC enrichment and activity. The chick embryo chorioallantoic membrane (CAM) is a low cost, naturally immune-incompetent and reproducible model widely used to evaluate the spontaneous growth of human tumor cells. Here, we established a CAM-LDA assay able to rapidly reproduce tumor specificities—in particular, the ability of the small population of CSCs to form tumors. We used a panel of organotropic metastatic breast cancer cells, which show an enrichment in a stem cell gene signature, enhanced CD44⁺/CD24^−/low cell surface expression and increased mammosphere-forming efficiency (MFE). The size of CAM-xenografted tumors correlate with the number of inoculated cancer cells, following mice xenograft growth pattern. CAM and mice tumors are histologically comparable, displaying both breast CSC markers CD44 and CD49f. Therefore, we propose a new tool for studying CSC prevalence and function—the chick CAM-LDA—a model with easy handling, accessibility, rapid growth and the absence of ethical and regulatory constraints.     

Structural Investigation and Molecular Modeling Studies of Strobilurin-Based Fungicides Active against the Rice Blast Pathogen Pyricularia oryzae

The increasing emergence of fungicide-resistant pathogens requires urgent solutions for crop disease management. Here, we describe a structural investigation of new fungicides obtained by combining strobilurin and succinate dehydrogenase inhibitor pharmacophores. We identified compounds endowed with very good activity against wild-type Pyricularia oryzae, combined in some cases with promising activity against strobilurin-resistant strains. The first three-dimensional model of P. oryzae cytochrome bc1 complex containing azoxystrobin as a ligand was developed. The model was validated with a set of commercially available strobilurins, and it well explains both the resistance mechanism to strobilurins mediated by the mutation G143A and the activity of metyltetraprole against strobilurin-resistant strains. The obtained results shed light on the key recognition determinants of strobilurin-like derivatives in the cytochrome bc1 active site and will guide the further rational design of new fungicides able to overcome resistance caused by G143A mutation in the rice blast pathogen.     

Direct-Ink-Writing of Electroactive Polymers for Sensing and Energy Storage Applications

Considering the high levels of materials used in the fields of electronics and energy storage systems, it is increasingly necessary to take into consideration environmental impact. Thus, it is important to develop devices based on environmentally friendlier materials and/or processes, such as additive manufacturing techniques. In this work, poly(vinylidene fluoride) (PVDF) and poly(vinylidene fluoride-co-hexafluoropropylene) (PVDF-HFP) are prepared by direct-ink-writing (DIW) by varying solvent evaporation temperature and fill density percentage. Different morphologies for both polymers are obtained, including dense films and porous membranes, as well as different electroactive β-phase content, thermal and mechanical properties. The dielectric constant and piezoelectric d₃₃ coefficient for dense films reaches up to 16 at 1 kHz and 4 pC N⁻¹, respectively for PVDF-HFP with a fill density of 80 and a solvent evaporation temperature of 50 °C. Porous structures are developed for battery separator membranes in lithium-ion batteries, with a highest ionic conductivity value of 3.8 mS cm⁻¹ for the PVDF-HFP sample prepared with a fill density of 100 and a solvent evaporation temperature of 25 °C, the sample showing an excellent cycling performance. It is demonstrated that electroactive films and membranes can be prepared by direct-ink writing suitable for sensors/actuators and energy storage systems.     

The Building Blocks of a PaaS

Traditional cloud computing providers enable developers to program and deploy applications in the cloud by using platform as a service (PaaS) providers. Despite the benefits of such an approach, this service model usually comes with a high level of the lock in effect into the service provider. The lock in effect occurs when a software developer needs to change its cloud provider. When this happens, it usually means a major application rewrite, specific for the new PaaS. This paper details the initial results of a project whose goal is to build a PaaS where vendor lock in does not occur. With this PaaS, developers wishing to deploy their applications into the cloud may continue to program using their usual development environments. There are no necessary changes required to make the application PaaS compatible. As a proof of concept, we developed an open source PaaS management application as a regular web application and then deployed it on the cloud.     

Cellular analysis by open-source software for affordable cytometry

Image cytometry is an important technique in affordable healthcare and cellular research. Some efforts toward establishing a personal, low-cost cytometer have been described in the literature. However, a self-assembled fluorescence microscope requires software for cytometric analysis. There are some open-source image-based software analysis applications available. However, for a quantitative analysis of images, software that can generate data comparable to those of previously evaluated cytometric analyses programs is required. Hence, the aim of this study is to compare results of a commercially available image cytometry program to data obtained using the open-source software CellProfiler (CP). Leukocytes and fluorescent bead images obtained using a Laser Scanning Cytometer were analyzed by CP and the results compared with those of conventional cytometric analyses' programs. Algorithms were developed enabling the analysis of leukocytes and beads by CP. CP provided similar results to those obtained by the cytometer software. Hallmark parameters, including cell count and fluorescence intensity, revealed a high correlation in the analysis of both programs. Therefore, CP is appropriate for cellular analysis on a self-assembled microscope, thereby enabling affordable cytometry.     

One-Way Functions Using Algorithmic and Classical Information Theories

We prove several results relating injective one-way functions, time-bounded conditional Kolmogorov complexity, and time-bounded conditional entropy. First we establish a connection between injective, strong and weak one-way functions and the expected value of the polynomial time-bounded Kolmogorov complexity, denoted here by?E(K ^t (x|f(x))). These results are in both directions. More precisely, conditions on?E(K ^t (x|f(x))) that imply that?f is a weak one-way function, and properties of?E(K ^t (x|f(x))) that are implied by the fact that?f is a strong one-way function. In particular, we prove a separation result: based on the concept of time-bounded Kolmogorov complexity, we find an interval in which every function?f is a necessarily weak but not a strong one-way function. Then we propose an individual approach to injective one-way functions based on Kolmogorov complexity, defining Kolmogorov one-way functions and prove some relationships between the new proposal and the classical definition of one-way functions, showing that a Kolmogorov one-way function is also a deterministic one-way function. A relationship between Kolmogorov one-way functions and the conjecture of polynomial time symmetry of information is also proved. Finally, we relate?E(K ^t (x|f(x))) and two forms of time-bounded entropy, the unpredictable entropy?H ^unp, in which ??one-wayness?? of a function can be easily expressed, and the Yao⁺ entropy, a measure based on compression/decompression schema in which only the decompressor is restricted to be time-bounded.     

Experience with model-based performance, reliability, and adaptability assessment of a complex industrial architecture

In this paper, we report on our experience with the application of validated models to assess performance, reliability, and adaptability of a complex mission critical system that is being developed to dynamically monitor and control the position of an oil-drilling platform. We present real-time modeling results that show that all tasks are schedulable. We performed stochastic analysis of the distribution of task execution time as a function of the number of system interfaces. We report on the variability of task execution times for the expected system configurations. In addition, we have executed a system library for an important task inside the performance model simulator. We report on the measured algorithm convergence as a function of the number of vessel thrusters. We have also studied the system architecture adaptability by comparing the documented system architecture and the implemented source code. We report on the adaptability findings and the recommendations we were able to provide to the system’s architect. Finally, we have developed models of hardware and software reliability. We report on hardware and software reliability results based on the evaluation of the system architecture.