A bright FIT-PNA hybridization probe for the hybridization state specific analysis of a C → U RNA edit via FRET in a binary system

Oligonucleotide probes that show enhanced fluorescence upon nucleic acid hybridization enable the detection and visualization of specific mRNA molecules, in vitro and in cellulo. A challenging problem is the analysis of single nucleotide alterations that occur, for example, when cellular mRNA is subject to C → U editing. Given the length required for uniqueness of the targeted segment, the commonly used probes do not provide the level of sequence specificity needed to discriminate single base mismatched hybridization. Herein we introduce a binary probe system based on fluorescence resonance energy transfer (FRET) that distinguishes three possible states i.e. (i) absence of target, (ii) presence of edited (matched) and (iii) unedited (single base mismatched) target. To address the shortcomings of read-out via FRET, we designed donor probes that avoid bleed through into the acceptor channel and nevertheless provide a high intensity of FRET signaling. We show the combined use of thiazole orange (TO) and an oxazolopyridine analogue (JO), linked as base surrogates in modified PNA FIT-probes that serve as FRET donor for a second, near-infrared (NIR)-labeled strand. In absence of target, donor emission is low and FRET cannot occur in lieu of the lacking co-alignment of probes. Hybridization of the TO/JO-PNA FIT-probe with the (unedited RNA) target leads to high brightness of emission at 540 nm. Co-alignment of the NIR-acceptor strand ensues from recognition of edited RNA inducing emission at 690 nm. We show imaging of mRNA in fixed and live cells and discuss the homogeneous detection and intracellular imaging of a single nucleotide mRNA edit used by nature to post-transcriptionally modify the function of the Glycine Receptor (GlyR)

Life Cycle of Multi Technology Machine Tools – Modularization and Integral Design

AbstractFor reasons of high flexibility but still maximum productivity, machine tools integrating various production technologies have recently received particular attention. Combining and integrating multiple manufacturing techniques into one single system in early stages of the product emergence process is challenging. To keep the effort for implementation to a minimum, an initiation already in the concept phase is being actively pursued. Design guidelines are currently investigated based on the examination of different technology combinations.This approach focuses on systematic conceptual design for such hybrid machine technologies. Product architectures are used to describe the modularity and create a specific delimitation for standardization. Reference product architectures for Multi Technology Machine Tools (MTMT) carry high potential for saving expenses in product development. The main emphasis is on technology and system integration. A technological similarity assessment of the single processes involved forms the basis of this approach to assure potential for synergies. Monetary aspects in early stages of product development are considered. Based on the analysis a generic system model is connected with general product architectures for MTMT.The method introduced is validated by a Multi-Technology Machining Centre with two simultaneously usable workspaces integrating a milling spindle and two laser processing units. The research undertaken is part of the Cluster of Excellence “Integrative Production Technology for High-Wage Countries” and has been funded by German Research Foundation (DFG)

Determination of the color temperature in laser-produced shocks

Experimental results on the determination of the color temperature in shock waves produced with lasers are presented. The method is based on imaging the target rear side in two different spectral windows and on using phased zone plates to produce high-quality shocks. The shock velocity is also measured, allowing, with the use of the equation of state, the real shock temperature to be deduced and compared with the measured color temperature

Inhibitors of Helicobacter pylori Protease HtrA Found by ‘Virtual Ligand’ Screening Combat Bacterial Invasion of Epithelia

Background: The human pathogen Helicobacter pylori (H. pylori) is a main cause for gastric inflammation and cancer. Increasing bacterial resistance against antibiotics demands for innovative strategies for therapeutic intervention. Methodology/Principal Findings: We present a method for structure-based virtual screening that is based on the comprehensive prediction of ligand binding sites on a protein model and automated construction of a ligand-receptor interaction map. Pharmacophoric features of the map are clustered and transformed in a correlation vector (‘virtual ligand’) for rapid virtual screening of compound databases. This computer-based technique was validated for 18 different targets of pharmaceutical interest in a retrospective screening experiment. Prospective screening for inhibitory agents was performed for the protease HtrA from the human pathogen H. pylori using a homology model of the target protein. Among 22 tested compounds six block E-cadherin cleavage by HtrA in vitro and result in reduced scattering and wound healing of gastric epithelial cells, thereby preventing bacterial infiltration of the epithelium. Conclusions/Significance: This study demonstrates that receptor-based virtual screening with a permissive (‘fuzzy’) pharmacophore model can help identify small bioactive agents for combating bacterial infection

The effect of small-scale topography on patterns of endemism within islands

Topography influences evolutionary and ecological processes by isolating populations and by enhancing habitat diversity. While the effects of large-scale topography on patterns of species richness and endemism are increasingly well documented, the direct effect of local topography on endemism is less understood. This study compares different aspects of topographic isolation, namely the isolating effect of deep barrancos (ravines) and the effect of increasing isolation with elevation in influencing patterns of plant endemism within a topographically diverse oceanic island (La Palma, Canary Islands, Spain). We collected plant presence–absence data from 75 plots in 8 barrancos on the northern coast of La Palma, spanning an elevation gradient from 95 to 674m a.s.l. Using mixed-effects models, we assessed the effect of barranco depth and elevation on the percentage of single-island endemics, multi-island endemics and archipelago endemics. We found that percent endemism was not significantly correlated with barranco depth, and correlated negatively with elevation within barrancos (rather than the expected positive relationship). The topographic barriers associated with the deep island barrancos thus appear insufficient to drive speciation through isolation in oceanic island plants. The decrease in endemism with elevation contradicts findings by previous broader-scale studies and it may reflect local influences, such as high habitat heterogeneity at low elevations