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.     

Off-Pathway-Sensitive Protein-Splicing Screening Based on a Toxin/Antitoxin System

Protein-splicing domains are frequently used engineering tools that find application in the in vivo and in vitro ligation of protein domains. Directed evolution is among the most promising technologies used to advance this technology. However, the available screening systems for protein-splicing activity are associated with bottlenecks such as the selection of pseudo-positive clones arising from off-pathway reaction products or fragment complementation. Herein, we report a stringent screening method for protein-splicing activity in cis and trans, that exclusively selects productively splicing domains. By fusing splicing domains to an intrinsically disordered region of the antidote from the Escherichia coli CcdA/CcdB type II toxin/antitoxin system, we linked protein splicing to cell survival. The screen allows selecting novel cis- and trans-splicing inteins catalyzing productive highly efficient protein splicing, for example, from directed-evolution approaches or the natural intein sequence space.     

Correction to: Reliable benchmarking: requirements and solutions

International Journal on Software Tools for Technology Transfer - The article Reliable benchmarking: requirements and solutions.     

TOOLympics I: Competition on software testing

Research competitions and challenges are a driving force in transferring theoretical results into working software tools that demonstrate the state of the art in the respective field of research. Regular comparative evaluations provide guidance to practitioners that have to select new technology and tools for their development process. In order to support competitions and challenges with an appropriate publication venue, a new theme of issues in the International Journal on Software Tools for Technology Transfer was created. This issue is the inaugural issue of the newly introduced theme on “Competitions and Challenges” (CoCha). Test-Comp, the International Competition on Software Testing, is an example of a tool competition, where the research teams submit tools for test-generation, and the competition evaluates the tools and assigns scores according to achieved coverage. Test-Comp 2019 was part of the TOOLympics event, which took place as part of the 25-year celebration of the conference TACAS. Thus, it is most natural to start the new STTT-CoCha theme with a special issue that describes the results and participating systems of Test-Comp 2019. There will be a second issue on TOOLympics with contributions from other competitions.

     

Temperaturabhängiges Anzieh- und Löseverhalten von Schraubenverbindungen mit Zinklamellenüberzügen

Forschung im Ingenieurwesen - In diesem Beitrag werden die Ergebnisse von Warmlöseversuchen nach VDA 235-203/VW 01131 mit denen von Anzieh- und Löseversuchen an einem...     

Model based prognosis of contaminant leaching for reuse of demolition waste in construction projects

In this study, groundwater contamination from recycled demolitian waste in road constructions is assessed using predictions of leachate concentrations. Numerical transport simulations are performed for three scenarios (a parking lot, a noise protection barrier, and road), and using a number of characteristic subsoils of Germany, to estimate the breakthrough of different contaminant classes at the groundwater table. Conservative tracer breakthrough times (BTT) primarily depend on subsoil hydraulic properties, for organic pollutants KOC and subsoil OC are the controlling parameters. Significant concentration reductions from dispersion only occur when source concentrations decrease prior to contaminant breakthrough. If source concentrations remain high for long periods relative to peak BTT, concentration breakthrough is undamped. Accounting for biodegradation reduces breakthrough concentrations significantly. For the “noise protection barrier” and “road” scenarios, capillary barrier effects cause the seepage water to partially bypass the recycling material. Accounting for this bypass flow and spatial averaging across the constructions reduces concentrations by about 30–40?%.     

Novel Furin Inhibitors with Potent Anti‐infectious Activity

New peptidomimetic furin inhibitors with unnatural amino acid residues in the P3 position were synthesized. The most potent compound 4‐guanidinomethyl‐phenylacteyl‐Arg‐Tle‐Arg‐4‐amidinobenzylamide (MI‐1148) inhibits furin with a K_i value of 5.5 pM . The derivatives also strongly inhibit PC1/3, whereas PC2 is less affected. Selected inhibitors were tested in cell culture for antibacterial and antiviral activity against infectious agents known to be dependent on furin activity. A significant protective effect against anthrax and diphtheria toxin was observed in the presence of the furin inhibitors. Furthermore, the spread of the highly pathogenic H5N1 and H7N1 avian influenza viruses and propagation of canine distemper virus was strongly inhibited. Inhibitor MI‐1148 was crystallized in complex with human furin. Its N‐terminal guanidinomethyl group in the para position of the P5 phenyl ring occupies the same position as that found previously for a structurally related inhibitor containing this substitution in the meta position, thereby maintaining all of the important P5 interactions. Our results confirm that the inhibition of furin is a promising strategy for a short‐term treatment of acute infectious diseases.     

Studies on the potential risk of liquid metal assisted cracking (LMAC) in normal‐temperature and high‐temperature hot‐dip galvanizing of high strength bolts of dimensions greater M24

Liquid metal assisted cracking (LMAC) mainly occurs due to an unfavorable interaction of three factors: a susceptible material condition, presence of a liquid metal and sufficient tensile stress. Hot‐dip galvanizing of high‐strength bolts induces high thermal loads in bolts made of tempered steel in the presence of a zinc melt and thus, provides the boundary conditions for the above mentioned critical factors to interact. The focus of this study is on investigating thermally‐induced stresses in large diameter bolts and their impact on the formation of liquid metal assisted cracking (LMAC). In order to calculate the thermal loads in hot‐dip galvanizing, simulations were carried out regarding the thermo‐mechanical behavior of bolts during the hot‐dip galvanizing process. The simulations illustrate that cracks are most likely to occur in the first thread turn. This prediction is confirmed by experimental observations.     

Erprobungsmethodik für zyklisch überelastisch beanspruchte Bauteile unter Torsion – Bauschinger‐Kenngrößen für 54SiCr6

Power transmission contains several torsion‐stressed parts. Especially shafts and springs are numerous considered. Helical springs as a special type of springs are mainly stressed by torsion load. During operation the spring's load is basically elastic. In some cases, like overload or machine disaster, the load exceeds the spring's yield stress. For a single event the deformations can be calculated easily by using flow charts. However in other cases the spring will be stressed several times with alternating plastic load. Therefore the calculation will not be as easy as in the single load case. Especially the hardening and softening mechanisms have major influence on material behavior. For high strength steel this material behavior is not fully investigated right now. Present article shows a way how to investigate several important Bauschinger parameters based on plastic torsion load for the high strength steel 54SiCr6. Finally it can be shown that 54SiCr6 is influenced by the Bauschinger effect. On the other hand the needed material characteristic can be investigated by the shown methodology. Based on the identified data, analytical and numerical calculation of alternating plastic torsion load can be done.     

Time-Resolved Structural Kinetics of an Organic Mixed Ionic–Electronic Conductor

The structure and packing of organic mixed ionic–electronic conductors have an especially significant effect on transport properties. In operating devices, this structure is not fixed but is responsive to changes in electrochemical potential, ion intercalation, and solvent swelling. Toward this end, the steady-state and transient structure of the model organic mixed conductor, poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS), is characterized using multimodal time-resolved operando techniques. Steady-state operando X-ray scattering reveals a doping-induced lamellar expansion of 1.6 Å followed by 0.4 Å relaxation at high doping levels. Time-resolved operando X-ray scattering reveals asymmetric rates of lamellar structural change during doping and dedoping that do not directly depend on potential or charging transients. Time-resolved spectroscopy establishes a link between structural transients and the complex kinetics of electronic charge carrier subpopulations, in particular the polaron–bipolaron equilibrium. These findings provide insight into the factors limiting the response time of organic mixed-conductor-based devices, and present the first real-time observation of the structural changes during doping and dedoping of a conjugated polymer system via X-ray scattering.