Brilliant violet fluorophores: a new class of ultrabright fluorescent compounds for immunofluorescence experiments

The Nobel Prize in Chemistry was awarded in 2000 for the discovery of conductive organic polymers, which have subsequently been adapted for applications in ultrasensitive biological detection. Here, we report the first use of this new class of fluorescent probes in a diverse range of cytometric and imaging applications. We demonstrate that these "Brilliant Violet" reporters are dramatically brighter than other UV-violet excitable dyes, and are of similar utility to phycoerythrin (PE) and allophycocyanin (APC). They are thus ideally suited for cytometric assays requiring high sensitivity, such as MHC-multimer staining or detection of intracellular antigens. Furthermore, these reporters are sensitive and spectrally distinct options for fluorescence imaging, two-photon microscopy and imaging cytometry. These ultra-bright materials provide the first new high-sensitivity fluorescence probes in over 25 years and will have a dramatic impact on the design and implementation of multicolor panels for high-sensitivity immunofluorescence assays.     

Simulations of Long-Term Community Dynamics in Coral Reefs - How Perturbations Shape Trajectories

Tropical coral reefs feature extraordinary biodiversity and high productivity rates in oligotrophic waters. Due to increasing frequencies of perturbations – anthropogenic and natural – many reefs are under threat. Such perturbations often have devastating effects on these unique ecosystems and especially if they occur simultaneously and amplify each other''s impact, they might trigger a phase shift and create irreversible conditions.We developed a generic, spatially explicit, individual-based model in which competition drives the dynamics of a virtual benthic reef community – comprised of scleractinian corals and algae – under different environmental settings. Higher system properties, like population dynamics or community composition arise through self-organization as emergent properties. The model was parameterized for a typical coral reef site at Zanzibar, Tanzania and features coral bleaching and physical disturbance regimes as major sources of perturbations. Our results show that various types and modes (intensities and frequencies) of perturbations create diverse outcomes and that the switch from high diversity to single species dominance can be evoked by small changes in a key parameter.Here we extend the understanding of coral reef resilience and the identification of key processes, drivers and respective thresholds, responsible for changes in local situations. One future goal is to provide a tool which may aid decision making processes in management of coral reefs.     

LINT, a novel dL(3)mbt-containing complex, represses malignant brain tumour signature genes

Mutations in the l(3)mbt tumour suppressor result in overproliferation of Drosophila larval brains. Recently, the derepression of different gene classes in l(3)mbt mutants was shown to be causal for transformation. However, the molecular mechanisms of dL(3)mbt-mediated gene repression are not understood. Here, we identify LINT, the major dL(3)mbt complex of Drosophila. LINT has three core subunits-dL(3)mbt, dCoREST, and dLint-1-and is expressed in cell lines, embryos, and larval brain. Using genome-wide ChIP-Seq analysis, we show that dLint-1 binds close to the TSS of tumour-relevant target genes. Depletion of the LINT core subunits results in derepression of these genes. By contrast, histone deacetylase, histone methylase, and histone demethylase activities are not required to maintain repression. Our results support a direct role of LINT in the repression of brain tumour-relevant target genes by restricting promoter access.     

Type III restriction endonuclease EcoP15I is a heterotrimeric complex containing one Res subunit with several DNA-binding regions and ATPase activity

For efficient DNA cleavage, the Type III restriction endonuclease EcoP15I communicates with two inversely oriented recognition sites in an ATP-dependent process. EcoP15I consists of methylation (Mod) and restriction (Res) subunits forming a multifunctional enzyme complex able to methylate or to cleave DNA. In this study, we determined by different analytical methods that EcoP15I contains a single Res subunit in a Mod(2)Res stoichiometry. The Res subunit comprises a translocase (Tr) domain carrying functional motifs of superfamily 2 helicases and an endonuclease domain with a PD..D/EXK motif. We show that the isolated Tr domain retains ATP-hydrolyzing activity and binds single- and double-stranded DNA in a sequence-independent manner. To localize the regions of DNA binding, we screened peptide arrays representing the entire Res sequence for their ability to interact with DNA. We discovered four DNA-binding regions in the Tr domain and two DNA-binding regions in the endonuclease domain. Modelling of the Tr domain shows that these multiple DNA-binding regions are located on the surface, free to interact with DNA. Interestingly, the positions of the DNA-binding regions are conserved among other Type III restriction endonucleases.     

Synthesis of O-serogroup specific positive controls and real-time PCR standards for nine clinically relevant non-O157 STECs

Non-O157 Shiga toxin producing Escherichia coli (STEC) are gaining recognition as human pathogens, but no standardized method exists to identify them. Sequence analysis revealed that STEC can be classified on the base of variable O antigen regions into different O serotypes. Polymerase chain reaction is a powerful technique for thorough screening and complex diagnosis for these pathogens, but requires a positive control to verify qualitative and/or quantitative DNA-fragment amplification. Due to the pathogenic nature of STEC, controls are not readily available and cell culturing of STEC reference strains requires biosafety conditions of level 2 or higher. In order to bypass this limitation, controls of stacked O-type specific DNA-fragments coding for primer recognition sites were designed to screen for nine STEC serotypes frequently associated with human infection. The synthetic controls were amplified by PCR, cloned into a plasmid vector and transferred into bacteria host cells. Plasmids amplified by bacterial expression were purified, serially diluted and tested as standards for real-time PCR using SYBR Green and TaqMan assays. Utility of synthetic DNA controls was demonstrated in conventional and real-time PCR assays and validated with DNA from natural STEC strains.     

Ruthenium Oxide Nanosheets for Enhanced Oxygen Evolution Catalysis in Acidic Medium

The fabrication of highly active and robust hexagonal ruthenium oxide nanosheets for the electrocatalytic oxygen evolution reaction (OER) in an acidic environment is reported. The ruthenate nanosheets exhibit the best OER activity of all solution‐processed acid medium electrocatalysts reported to date, reaching 10 mA cm^?2 at an overpotential of only ≈255 mV. The nanosheets also demonstrate robustness under harsh oxidizing conditions. Theoretical calculations give insights into the OER mechanism and reveal that the edges are the origin of the high OER activity of the nanosheets. Moreover, the post OER analyses indicate, apart from coarsening, no observable change in the morphology of the nanosheets or oxidation states of ruthenium during the electrocatalytic process. Therefore, the present investigation suggests that ruthenate nanosheets are a promising acid medium OER catalyst with application potential in proton exchange membrane electrolyzers and beyond.