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 consequences of an increase in heavy goods vehicles for passenger car drivers' mental workload and behaviour: a simulator study

The effects of an increase in Heavy Goods Vehicles (HGVs) on merging behaviour and on mental workload of motorists during filtering in and out of traffic were studied. Participants drove in a driving simulator in a total of 12 conditions; twice in each of two weather conditions and in three traffic conditions. The weather conditions were clear weather and foggy weather. The traffic conditions were without HGVs (i.e. only private cars), the current mix of HGVs and private cars, and a condition with a 70% increase of HGVs leading to an HGV column in the slow lane. The focus of the study was on assessing effects on behaviour and mental workload during filtering into traffic, and during exiting from the motorway. During the experiment driving performance was registered, behaviour was observed, self reports were collected, and the participant's heart rate was recorded. The results showed that directly after filtering into traffic the variation in driving speed increased and the minimum time headway decreased with an increase in the proportion of HGVs. Joining motorway traffic was considered to involve greater effort and risk in the condition with a column of HGVs. The effects of the conditions on heart rate are less clear, although the moment when the participants joined the traffic is clearly visible. The effects of weather conditions were limited, drivers adapting their driving behaviour in adverse weather by reducing speed. To exit the motorway is not a difficult manoeuvre. For that reason the lane change from the left hand to the right hand lane that preceded the exit was analysed. Although increased mental effort was reported and the lane change was visible in the heart rate record, no critical changes as a result of increase in proportion of HGVs were found for this manoeuvre. However, in the condition with a column of HGVs, the exit that had to be taken was most frequently missed as HGVs obstructed the view of the exit signs. It is concluded that an increase in HGVs will make merging into traffic more mentally demanding and will decrease safety margins.     

A method for evaluating personal dosemeters in workplace with neutron fields

Passive detectors, as albedo or track-etch, still dominate the field of neutron personal dosimetry, mainly due to their low-cost, high-reliability and elevated throughput. However, the recent appearance in the market of electronic personal dosemeters for neutrons presents a new option for personal dosimetry. In addition to passive detectors, electronic personal dosemeters necessitate correction factors, concerning their energy and angular response dependencies. This paper reports on the results of a method to evaluate personal dosemeters for workplace where neutrons are present. The approach here uses few instruments and does not necessitate a large mathematical workload. Qualitative information on the neutron energy spectrum is acquired using a simple spectrometer (Nprobe), reference values for H*(10) are derived from measurements with ambient detectors (Studsvik, Berthold and Harwell) and angular information is measured using personal dosemeters (electronic and bubbles dosemeters) disposed in different orientations on a slab phantom.     

Identification of a Streptococcus pneumoniae gene locus encoding proteins of an ABC phosphate transporter and a two-component regulatory system

The Escherichia coli Pst system belongs to the family of ABC transporters. It is part of a phosphate (PHO) regulon which is regulated by extracellular phosphate. Under conditions of phosphate limitation, the response regulator PhoB is phosphorylated by the histidine kinase PhoR and binds to promoters that share a consensus PHO box. Under conditions of phosphate excess, PhoR, Pst, and PhoU downregulate the PHO regulon. Screening of a library of pneumococcal mutants with defects in exported proteins revealed a putative two-component regulatory system, PnpR-PnpS, and a downstream ABC transporter, similar to the Pst system in E. coli including a gene encoding a PhoU protein. Similar to E. coli, mutagenesis of the ATP-binding cassette gene, pstB, resulted in decreased uptake of phosphate. The effects of the loss of the pneumococcal Pst system extended to decreased transformation and lysis. Withdrawal of phosphate led to transformation deficiency in the parent strain R6x but not to penicillin tolerance, suggesting that reduced bacterial death was independent of phosphate. None of these phenotypes was observed in the pneumococcal loss-of-function mutant phoU. By using a lacZ reporter construct, it was demonstrated that expression of the two-component regulatory system PnpR-PnpS was not influenced by different concentrations of phosphate. These results suggest a more complex role of the Pst system in pneumococcal physiology than in that of E. coli.     

Advanced light microscopy core facilities: Balancing service,science and career

Core Facilities (CF) for advanced light microscopy (ALM) have become indispensable support units for research in the life sciences. Their organizational structure and technical characteristics are quite diverse, although the tasks they pursue and the services they offer are similar. Therefore, throughout Europe, scientists from ALM‐CFs are forming networks to promote interactions and discuss best practice models. Here, we present recommendations for ALM‐CF operations elaborated by the workgroups of the German network of ALM‐CFs, German Bio‐Imaging (GerBI). We address technical aspects of CF planning and instrument maintainance, give advice on the organization and management of an ALM‐CF, propose a scheme for the training of CF users, and provide an overview of current resources for image processing and analysis. Further, we elaborate on the new challenges and opportunities for professional development and careers created by CFs. While some information specifically refers to the German academic system, most of the content of this article is of general interest for CFs in the life sciences. Microsc. Res. Tech. 79:463–479, 2016. © 2016 THE AUTHORS MICROSCOPY RESEARCH AND TECHNIQUE PUBLISHED BY WILEY PERIODICALS, INC.     

从灰到绿的基础设施——城市可持续设计与水资源综合管理

水基础设施是非常基本的,它可以解决很多重要的问题。现在,大多数的基础设施都是看不见的,人们一般只是使用这些设施,觉得从水龙头当中会流出无穷无尽的水。但是对于规划人员,景观设计师来说不是这样,从工程师的角度来说,这往往是灰色的角度。灰色概念从未来的角度来说,已经不太能适应城市未来的设计了,新的方式是绿色的,是非常好的。灰色的设计是在工业化时期,现代化开始的初期做的,     

Coexistence of CD14-dependent and independent pathways for stimulation of human monocytes by gram-positive bacteria

The cell wall is a key inflammatory agent of gram-positive bacteria. Possible receptors mediating cell wall-induced inflammation include CD14 and platelet-activating factor (PAF) receptor. To delineate the conditions under which these various receptors might be used, human monocytic THP-1 cells and heparinized whole human blood were stimulated with lipopolysaccharide (LPS), intact Streptococcus pneumoniae bacteria, or purified pneumococcal cell wall. THP-1 culture supernatant or cell-free plasma was analyzed for the presence of tumor necrosis factor, interleukin-1beta (IL-1beta), and IL-6. For the cultured monocytes, anti-CD14 inhibited induction of the inflammatory cytokines by the cell wall and LPS but not by intact pneumococcal bacteria. Despite the difference in CD-14 usage, the intracellular pathways induced by the three agents demonstrated similarities, as revealed in the presence of specific signal transduction inhibitors such as cholera toxin, pertussis toxin, and genistein. Cytokine production in whole human blood indicated that anti-CD14 failed to block responses to cell wall and intact pneumococci, whereas while LPS-induced responses were inhibited. PAF receptor antagonist had no effect under any conditions in both assays. These results indicate that although cell walls bind to both CD14 and PAF receptor, only CD14 appears to engender a cytokine response under restricted conditions. Furthermore, host cell responses to intact pneumococci are consistently independent of CD14 and PAF receptor.     

Microcirculatory Function during Endotoxemia—A Functional Citrulline-Arginine-NO Pathway and NOS3 Complex Is Essential to Maintain the Microcirculation

Competition for the amino acid arginine by endothelial nitric-oxide synthase (NOS3) and (pro-)inflammatory NO-synthase (NOS2) during endotoxemia appears essential in the derangement of the microcirculatory flow. This study investigated the role of NOS2 and NOS3 combined with/without citrulline supplementation on the NO-production and microcirculation during endotoxemia. Wildtype (C57BL6/N background; control; n = 36), Nos2-deficient, (n = 40), Nos3-deficient (n = 39) and Nos2/Nos3-deficient mice (n = 42) received a continuous intravenous LPS infusion alone (200 μg total, 18 h) or combined with L-citrulline (37.5 mg, last 6 h). The intestinal microcirculatory flow was measured by side-stream dark field (SDF)-imaging. The jejunal intracellular NO production was quantified by in vivo NO-spin trapping combined with electron spin-resonance (ESR) spectrometry. Amino-acid concentrations were measured by high-performance liquid chromatography (HPLC). LPS infusion decreased plasma arginine concentration in control and Nos3^−/− compared to Nos2^−/− mice. Jejunal NO production and the microcirculation were significantly decreased in control and Nos2^−/− mice after LPS infusion. No beneficial effects of L-citrulline supplementation on microcirculatory flow were found in Nos3^−/− or Nos2^−/−/Nos3^−/− mice. This study confirms that L-citrulline supplementation enhances de novo arginine synthesis and NO production in mice during endotoxemia with a functional NOS3-enzyme (control and Nos2^−/− mice), as this beneficial effect was absent in Nos3^−/− or Nos2^−/−/Nos3^−/− mice.