Photocaged Hoechst Enables Subnuclear Visualization and Cell Selective Staining of DNA in vivo

Selective targeting of DNA by means of fluorescent labeling has become a mainstay in the life sciences. While genetic engineering serves as a powerful technique and allows the visualization of nucleic acid by using DNA-targeting fluorescent fusion proteins in a cell-type- and subcellular-specific manner, it relies on the introduction of foreign genes. On the other hand, DNA-binding small fluorescent molecules can be used without genetic engineering, but they are not spatially restricted. Herein, we report a photocaged version of the DNA dye Hoechst33342 (pcHoechst), which can be uncaged by using UV to blue light for the selective staining of chromosomal DNA in subnuclear regions of live cells. Expanding its application to a vertebrate model organism, we demonstrate uncaging in epithelial cells and short-term cell tracking in vivo in zebrafish. We envision pcHoechst as a valuable tool for targeting and interrogating DNA with precise spatiotemporal resolution in living cells and wild-type organisms.     

The potential limitations on its basin decision-making processes of granting self-defence rights to Father Rhine

ABSTRACT

Recent grants of legal rights to rivers would seem to infuse traditional anthropocentric river governance with greater eco-centrism. Through a thought experiment, we scrutinize this proposition for the Rhine basin. We consider the governance implications of granting (procedural/material) rights to the river and elaborate on their implications for the three highly institutionalized regimes of the Rhine River of water quality, flooding and transport. Since we find that a shift to more eco-centrism has already occurred and since the right granted to the river would not be absolute, we deem radical transformations unlikely.     

Holzbrücken – Erkenntnisse zum Feuchteverhalten in den Tragquerschnitten aus Langzeitmessungen

Timber bridges – Long term monitoring results of moisture diffusion in the load carrying cross‐sections Timber road bridges have been built worldwide for centuries. The high performance of wood is approved through many constructions. However, there are still doubts about using wood by the planning engineer, which reduce the number of realized projects. The moisture induced stresses in cross‐sections according to the varied ambient climate are mainly investigated under laboratory conditions. The results show that the moisture content changes differently over the cross‐section and leads to moisture induced stresses. The discussion about methods for the assessment of the moisture induced stresses in timber bridge cross‐ections is continuing. Results observed in the long term monitoring of seven timber bridges provide first guidelines for practitioners. For all constructions, the moisture content was measured regarding the ambient micro climate. As result, the influences according to the ambient micro climate are not insignificant. Positive is that the directly measured moisture content is less compared to the equilibrium moisture content calculated from the data of a close by meteorological station. The analysis of the moisture gradient over the cross‐section allows the definition of an active or passive zone.     

Borcalein: a Carborane-Based Analogue of Baicalein with 12-Lipoxygenase-Independent Toxicity

12-Lipoxygenase is crucial for tumour angiogenesis. 5,6,7-Trihydroxy-2-phenyl-4H-1-benzopyran-4-one (baicalein) is a suitable inhibitor for this enzyme but is rapidly metabolised in vivo. Thus, an improvement of the metabolic stability is necessary to enhance the therapeutic efficiency. An emerging approach to enhance metabolic stability of carbon-based pharmaceuticals is the use of metabolically stable, non-toxic boron clusters, such as dicarba-closo-dodecaborane(12)s (carboranes) as phenyl mimetics. Therefore, the unsubstituted phenyl ring of baicalein was replaced by meta-carborane, resulting in borcalein, the carborane analogue of baicalein. This substitution resulted in a decreased inhibitory activity toward 12-lipoxygenase, but led to increased toxicity in melanoma (A375, B16, B16F10) and colon cancer cell lines (SW480, HCT116, CT26CL25) with decreased tumour selectivity in comparison to baicalein. Surprisingly, borcalein displays a different mechanism of cytotoxicity with increased intracellular production of reactive oxygen species (ROS), reactive nitrogen species (RNS) and nitric oxide (NO).     

Detailed Structure–Function Correlations of Bacillus subtilis Acetolactate Synthase

Isobutanol is deemed to be a next‐generation biofuel and a renewable platform chemical. 1 Non‐natural biosynthetic pathways for isobutanol production have been implemented in cell‐based and in vitro systems with Bacillus subtilis acetolactate synthase (AlsS) as key biocatalyst. 2 – 6 AlsS catalyzes the condensation of two pyruvate molecules to acetolactate with thiamine diphosphate and Mg²⁺ as cofactors. AlsS also catalyzes the conversion of 2‐ketoisovalerate into isobutyraldehyde, the immediate precursor of isobutanol. Our phylogenetic analysis suggests that the ALS enzyme family forms a distinct subgroup of ThDP‐dependent enzymes. To unravel catalytically relevant structure‐function relationships, we solved the AlsS crystal structure at 2.3 Å in the presence of ThDP, Mg²⁺ and in a transition state with a 2‐lactyl moiety bound to ThDP. We supplemented our structural data by point mutations in the active site to identify catalytically important residues.     

Better decision support through exploratory discrimination-aware data mining: foundations and empirical evidence

Decision makers in banking, insurance or employment mitigate many of their risks by telling “good” individuals and “bad” individuals apart. Laws codify societal understandings of which factors are legitimate grounds for differential treatment (and when and in which contexts)—or are considered unfair discrimination, including gender, ethnicity or age. Discrimination-aware data mining (DADM) implements the hope that information technology supporting the decision process can also keep it free from unjust grounds. However, constraining data mining to exclude a fixed enumeration of potentially discriminatory features is insufficient. We argue for complementing it with exploratory DADM, where discriminatory patterns are discovered and flagged rather than suppressed. This article discusses the relative merits of constraint-oriented and exploratory DADM from a conceptual viewpoint. In addition, we consider the case of loan applications to empirically assess the fitness of both discrimination-aware data mining approaches for two of their typical usage scenarios: prevention and detection. Using Mechanical Turk, 215 US-based participants were randomly placed in the roles of a bank clerk (discrimination prevention) or a citizen / policy advisor (detection). They were tasked to recommend or predict the approval or denial of a loan, across three experimental conditions: discrimination-unaware data mining, exploratory, and constraint-oriented DADM (eDADM resp. cDADM). The discrimination-aware tool support in the eDADM and cDADM treatments led to significantly higher proportions of correct decisions, which were also motivated more accurately. There is significant evidence that the relative advantage of discrimination-aware techniques depends on their intended usage. For users focussed on making and motivating their decisions in non-discriminatory ways, cDADM resulted in more accurate and less discriminatory results than eDADM. For users focussed on monitoring for preventing discriminatory decisions and motivating these conclusions, eDADM yielded more accurate results than cDADM.