Covalent PARylation of DNA base excision repair proteins regulates DNA demethylation.

The intracellular ATP-ribosyltransferases PARP1 and PARP2, contribute to DNA base excision repair (BER) and DNA demethylation and have been implicated in epigenetic programming in early mammalian development. Recently, proteomic analyses identified BER proteins to be covalently poly-ADP-ribosylated by PARPs. The role of this posttranslational modification in the BER process is unknown. Here, we show that PARP1 senses AP-sites and SSBs generated during TET-TDG mediated active DNA demethylation and covalently attaches PAR to each BER protein engaged. Covalent PARylation dissociates BER proteins from DNA, which accelerates the completion of the repair process. Consistently, inhibition of PARylation in mESC resulted both in reduced locus-specific TET-TDG-targeted DNA demethylation, and in reduced general repair of random DNA damage. Our findings establish a critical function of covalent protein PARylation in coordinating molecular processes associated with dynamic DNA methylation.

Influence of Periodic Non-Uniformities of Well-Structured Sapphire Surface by LIPSS on the Alignment of Nematic Liquid Crystal.

In this study, we report on the alignment properties of nematic liquid crystals on various transparent structured sapphire layers formed by laser-induced periodic surface structures (LIPSS). One-dimensional LIPSS (1D-LSFL) are generated by infrared femtosecond laser pulses along parallel lines covering an area of 5 × 5 mm2, with a line spacing that is varied between 7 and 17 µm. These periodic structures, employed as alignment layers, have a spatial periodicity of about 980 nm, a modulation depth of about 100 nm, and exhibit a high quality due to being characterized by a high degree of homogeneity and parallelism of the structured features. It is found that such alignment layers of the sapphire surface lead to a decreasing azimuthal anchoring energy, when the width of the unstructured gap is increased. Modifying the sapphire surface by an ITO-coating with further deposition of a polyimide film increases the azimuthal anchoring energy by a factor of about four up to Wφ ~ 4.25 × 10-6 J/m2, when the minimum width of the unstructured gap is 7 µm. Comprehensive measurements and comparisons of the azimuthal anchoring energy as well as the pretilt angle for the 1D-LSFL, unstructured gaps, and entire areas depending on the width of unstructured gaps are presented and discussed.

Ultrashort pulsed laser backside ablation of fused silica.

We report on the fabrication of rectangular microchannels with vertical sidewalls in fused silica by laser backside ablation. A 515 nm femtosecond laser is focused by an objective with a NA of 0.5 through the sample on the glass/air interface, allowing processing from the backside into the bulk material. Experimental investigations reveal a logarithmically increasing depth of the channels with an increasing number of scans, while keeping the focal position fixed. A certain number of scans has to be applied to generate rectangular shaped channels while their depth can be controlled by the applied fluence from 2.64 µm to 13.46 µm and a corresponding ablation roughness Ra between 0.20 µm and 0.33 µm. The channel width can be set directly via the number of parallel ablated lines demonstrated in a range from 10 µm to 50 µm. By adjusting the focal position after each scan the channel depth can be extended to 49.77 µm while maintaining a rectangular channel geometry. Finally, concentric rings are ablated to demonstrate the flexibility of the direct writing process.

Enhanced ablation efficiency using GHz bursts in micromachining fused silica.

We report on micromilling cavities into fused silica by a 1030 nm femtosecond laser using 2.17 GHz bursts. The milled cavities show an increased depth per layer for a higher number of pulses per burst while the ablation efficiency is also increased. The maximum ablation efficiency for the optimum fluence achieved in our experiments is 3.05mm3/min/W for a burst number of 10, which is 7.4 times higher than for the non-burst condition (0.41mm3/min/W). Furthermore, the ablation threshold for each sub-pulse is significantly reduced from 0.64J/cm2 for the non-burst condition to 0.15J/cm2 for 10 bursts. Beside the ablation efficiency, the surface roughness is also increased with the increasing burst number, while two ablation behaviors can be distinguished, namely, a gentle ablation regime for lower burst numbers and a coarse ablation regime, dominated by breaking out the surface rather than ablating it.

Inducible TDG knockout models to study epigenetic regulation.

Mechanistic and functional studies by gene disruption or editing approaches often suffer from confounding effects like compensatory cellular adaptations generated by clonal selection. These issues become particularly relevant when studying factors directly involved in genetic or epigenetic maintenance. To provide a genetic tool for functional and mechanistic investigation of DNA-repair mediated active DNA demethylation, we generated experimental models in mice and murine embryonic stem cells (ESCs) based on a minigene of the thymine-DNA glycosylase (TDG). The loxP-flanked miniTdg is rapidly and reliably excised in mice and ESCs by tamoxifen-induced Cre activation, depleting TDG to undetectable levels within 24 hours. We describe the functionality of the engineered miniTdg in mouse and ESCs (TDGiKO ESCs) and validate the pluripotency and differentiation potential of TDGiKO ESCs as well as the phenotype of induced TDG depletion. The controlled and rapid depletion of TDG allows for a precise manipulation at any point in time of multistep experimental procedures as presented here for neuronal differentiation in vitro. Thus, we provide a tested and well-controlled genetic tool for the functional and mechanistic investigation of TDG in active DNA (de)methylation and/or DNA repair with minimal interference from adaptive effects and clonal selection.

Fabrication and evaluation of negative axicons for ultrashort pulsed laser applications.

We report on the fabrication and evaluation of a sharp tip negative axicon paving the way for applications in high-power ultrashort pulsed laser systems. The negative axicon is manufactured by applying a two-step all laser-based process chain consisting of ultrashort pulsed laser ablation and CO2 laser polishing finishing the component in less than 5 minutes. The finalized negative axicon reveals a surface roughness of 18 nm, fulfilling optical quality. Two measurement setups, including the ultrashort pulsed laser itself, are used to evaluate the formation of Bessel beams in detail. By applying a focusing lens behind the negative axicon, well-developed Bessel beams are generated while their lengths depend on the distance between the negative axicon and the lens. Furthermore, the diameter of the Bessel beams increase strongly with the propagation distance. By adding a second focusing lens, Bessel beams are generated at its focal position, being almost invariant of its position. Hence, the typical Bessel beam intensity distribution is observed over an entire moving range of this second lens of 300 mm. While these Bessel beams show superior quality in terms of sharp peaks with homogeneous concentric rings, only minor deviations in intensity and diameter are observed over the moving range.

Laser-fabricated axicons challenging the conventional optics in glass processing applications.

Laser-based fabrication can be an alternative technology to mechanical grinding and polishing processes. However, the performance of these elements in real applications still needs to be validated. In this paper, we demonstrate that the subtractive fabrication technology is able to produce high-quality axicons from fused silica, which can be efficiently used for glass processing. We comprehensively investigate axicons, fabricated by ultrashort pulsed laser ablation with subsequent CO2 laser polishing, and compare their performance with commercially available axicons. We show that laser-fabricated axicons are comparable in quality with a precision commercial axicon. Furthermore, we demonstrate the intra-volume glass modification and dicing, utilising mJ-level laser pulses. We show that the tilting operation of the laser-fabricated axicons results in the formation of directional transverse cracks, which significantly enhance the 1 mm-thick glass dicing process.

Fabrication of a high-quality axicon by femtosecond laser ablation and CO2 laser polishing for quasi-Bessel beam generation.

We report on the fabrication of an axicon by applying a two-step manufacturing process including a 1030 nm femtosecond and a 10.6 µm CO2 laser. First, the pre-defined axicon geometry is generated by high-precision femtosecond layer-by-layer ablation. In order to meet high surface quality requirements, inevitable stipulated for optical use, the surface of the thus structured axicon is smoothened by a subsequent CO2 laser polishing process. The finalized axicon fulfills optical quality as the surface roughness Ra is significantly reduced from 0.56 µm to 34 nm. For the evaluation of the optical quality, the axicon is placed in a measurement setup including the femtosecond laser. Comparison between the calculated Bessel beam for an ideal axicon and the quasi-Bessel beam generated and measured by the fabricated axicon reveals excellent agreement, verifying our precise manufacturing method.

Impact of the NSAID diclofenac on survival, development, behaviour and health of embryonic and juvenile stages of brown trout, Salmo trutta f. fario.

The NSAID diclofenac is controversially discussed with respect to its environmental relevance. Since further information is need to assess whether diclofenac should be included as substance of priority in the EU water framework directive, we investigated the impact of this analgesic on the embryonic development of brown trout (Salmo trutta f. fario) from fertilized egg until the end of sac-fry stage and studied effects in juvenile fish six months post hatch. Embryos were exposed to five test concentrations (0.1, 0.5, 1, 10, 100µg/L) over 127days at 7°C. None of the treatments affected mortality, hatching, development or heart rate. Six months old juveniles exposed to five concentrations (0.1, 1, 10, 100, 200µg/L) over 25days at 7°C, however, showed increased mortality, reaching significance at 100µg/L. Furthermore, a significantly higher proportion of juvenile animals bore injuries at concentrations higher 10µg/L. Neither the levels of the stress protein Hsp70, nor the amount of lipid peroxides was affected by any of the treatments. Histological analyses of gill, liver and kidney revealed visible tissue reactions in fish from all experimental groups. Histological responses in livers of diclofenac-exposed fish outstripped the status of laboratory control fish, particularly when exposed to the two highest concentrations. Chemical analyses of fish muscle tissue revealed concentration-dependent uptake of DCF into the animal, but no relevant bioconcentration. Our study supports earlier findings indicating a lower sensitivity of trout early life stages compared to older individuals, suggesting that studies for risk assessment of diclofenac should predominantly focus on later life stages. Furthermore, fish mortality was found to increase with rising diclofenac concentrations, and the lowest observed effect concentration of 10µg/L on the organismic level emphasises the classification of diclofenac as a micropollutant that requires close attention.

Health effects of metoprolol in epibenthic and endobenthic invertebrates-A basis to validate future in vitro biotests for effect-based biomonitoring.

The aim of this study was to determine the effect data for metoprolol as a model substance for beta-blockers in aquatic invertebrates. The results will be used as a basis for the validation of future mode of action-based in vitro test systems targeting this class of pharmaceuticals. Effects of metoprolol were investigated in two autochthonous species with high relevance in stream ecology: the amphipod Gammarus fossarum and the oligochaete Lumbriculus variegatus. Mortality in G. fossarum was not observed in acute toxicity testing (48 h), and a significant increase of mortality at 45 mg/L was found when amphipods were exposed chronically (40 days). The most sensitive population-relevant endpoints were the juvenile-adult ratio and number of egg-bearing females with NOEC/LOEC-values of 5/15 mg/L. No proteotoxic effects were identified in G. fossarum. The sediment toxicity test with L. variegatus according to the OECD Guideline 225 with an exposure time of 28 days resulted in EC10-values of 92.5 and 126.1 mg/kgdw for the endpoints reproduction and biomass, respectively. In L. variegatus the response kinetics of Hsp70 showed no significant difference between the treatments. A tendency for rising lipid peroxide concentrations was found between 0.03 and 10 mg/kgdw, which were significant between the treatments, but not to the control.

The 2015 Annual Meeting of SETAC German Language Branch in Zurich (7-10 September, 2015): Ecotoxicology and environmental chemistry-from research to application.

This report provides a brief review of the 20th annual meeting of the German Language Branch of the Society of Environmental Toxicology and Chemistry (SETAC GLB) held from September 7th to 10th 2015 at ETH (Swiss Technical University) in Zurich, Switzerland. The event was chaired by Inge Werner, Director of the Swiss Centre for Applied Ecotoxicology (Ecotox Centre) Eawag-EPFL, and organized by a team from Ecotox Centre, Eawag, Federal Office of the Environment, Federal Office of Agriculture, and Mesocosm GmbH (Germany). Over 200 delegates from academia, public agencies and private industry of Germany, Switzerland and Austria attended and discussed the current state of science and its application presented in 75 talks and 83 posters. In addition, three invited keynote speakers provided new insights into scientific knowledge 'brokering', and-as it was the International Year of Soil-the important role of healthy soil ecosystems. Awards were presented to young scientists for best oral and poster presentations, and for best 2014 master and doctoral theses. Program and abstracts of the meeting (mostly in German) are provided as Additional file 1.

In permanent atrial fibrillation, PDE3 reduces force responses to 5-HT, but PDE3 and PDE4 do not cause the blunting of atrial arrhythmias.

BACKGROUND AND PURPOSE: 5-HT increases force and L-type Ca(2) (+) current (ICa,L ) and causes arrhythmias through 5-HT4 receptors in human atrium. In permanent atrial fibrillation (peAF), atrial force responses to 5-HT are blunted, arrhythmias abolished but ICa,L responses only moderately attenuated. We investigated whether, in peAF, this could be due to an increased function of PDE3 and/or PDE4, using the inhibitors cilostamide (300 nM) and rolipram (1 µM) respectively. EXPERIMENTAL APPROACH: Contractile force, arrhythmic contractions and ICa,L were assessed in right atrial trabeculae and myocytes, obtained from patients with sinus rhythm (SR), paroxysmal atrial fibrillation (pAF) and peAF. KEY RESULTS: Maximum force responses to 5-HT were reduced to 15% in peAF, but not in pAF. Cilostamide, but not rolipram, increased both the blunted force responses to 5-HT in peAF and the inotropic potency of 5-HT fourfold to sevenfold in trabeculae of patients with SR, pAF and peAF. Lusitropic responses to 5-HT were not decreased in peAF. Responses of ICa,L to 5-HT did not differ and were unaffected by cilostamide or rolipram in myocytes from patients with SR or peAF. Concurrent cilostamide and rolipram increased 5-HT's propensity to elicit arrhythmias in trabeculae from patients with SR, but not with peAF. CONCLUSIONS AND IMPLICATIONS: PDE3, but not PDE4, reduced inotropic responses to 5-HT in peAF, independently of lusitropy and ICa,L , but PDE3 activity was the same as that in patients with SR and pAF. Atrial remodelling in peAF abolished the facilitation of 5-HT to induce arrhythmias by inhibition of PDE3 plus PDE4.

Metallothionein and Hsp70 trade-off against one another in Daphnia magna cross-tolerance to cadmium and heat stress.

The association between the insensitivity of adapted ecotypes of invertebrates to environmental stress, such as heavy metal pollution, and overall low Hsp levels characterizing these organisms has been attracting attention in various studies. The present study seeks to induce and examine this phenomenon in Daphnia magna by multigenerational acclimation to cadmium in a controlled laboratory setting. In this experiment, interclonal variation was examined: two clones of D. magna that have previously been characterized to diverge regarding their cadmium resistance and levels of the stress protein Hsp70, were continuously exposed to a sublethal concentration of Cd over four generations to study the effects of acclimation on Hsp70, metallothionein (MT), reproduction and cross-tolerance to heat stress. The two clones differed in all the measured parameters in a characteristic way, clone T displaying Cd and heat resistance, lower Hsp70 levels and offspring numbers on the one hand and higher MT expression on the other hand, clone S the opposite for all these parameters. We observed only slight acclimation-induced changes in constitutive Hsp70 levels and reproductive output. The differences in MT expression between clones as well as between acclimated organisms and controls give evidence for MT accounting for the higher Cd tolerance of clone T. Overall high Hsp70 levels of clone S did not confer cross tolerance to heat stress, contrary to common expectations. Our results suggest a trade-off between the efforts to limit the proteotoxic symptoms of Cd toxicity by Hsp70 induction and those to sequester and detoxify Cd by means of MT.

Fish embryo tests with Danio rerio as a tool to evaluate surface water and sediment quality in rivers influenced by wastewater treatment plants using different treatment technologies.

In order to evaluate surface water and the sediment quality of rivers connected to wastewater treatment plants (WWTPs) with different treatment technologies, fish embryo tests (FET) with Danio rerio were conducted using native water and sediment samples collected upstream and downstream of four WWTPs in Southern Germany. Two of these WWTPs are connected to the Schussen River, a tributary of Lake Constance, and use a sand filter with final water purification by flocculation. The two others are located on the rivers Schmiecha and Eyach in the area of the Swabian Alb and were equipped with a powdered activated carbon stage 20 years ago, which was originally aimed at reducing the release of stains from the textile industry. Several endpoints of embryo toxicity including mortality, malformations, reduced hatching rate, and heart rate were investigated at defined time points of embryonic development. Higher embryotoxic potentials were found in water and sediments collected downstream of the WWTPs equipped with sand filtration than in the sample obtained downstream of both WWTPs upgraded with a powdered activated carbon stage.