2017526(金)

In the absence of a high-resolution structure

Our ELDOR experiments on YF1 H22P with inverted signal response revealed a drastically altered LOV photosensor dimer interface. The N-terminal A’α helices that in the original YF1 are embraced by the two LOV photosensors (Fig. 1A) and that play important roles in signal propagation and modulation19, 20 are displaced and possibly unstructured in H22P. As unequivocally demonstrated by ELDOR distance measurements on photo-induced NSQ radicals of the FMN chromophores, the two LOV photosensors in H22P are much closer in distance than in the original YF1. The observed distance between the two NSQ perfectly agrees with a flush packing of the LOV domains against another via their β sheets. Strikingly, this altered quaternary structure largely corresponds to the arrangement in an earlier structure of the isolated BsYtvA LOV domain that entirely lacked the A’α helices. Given its completely altered dimer interface, it is perplexing that the H22P variant still transduces LED Filament Bulbsignals, and even more so, in inverted manner.

In the absence of a high-resolution structure of the H22P variant, we modelled LED Filament Bulb-induced structural transitions on the basis of the N-terminally truncated BsYtvA LOV structure. Due to this approximation, it is challenging to extract reliable quantitative data, and the results should be considered qualitative in nature. Nonetheless, structural modelling constrained by the ELDOR data implied that the two LOV photosensors undergo a light-induced rotation and concomitant displacement of the Jα attachment sites that resemble the molecular response to light in YF1, even though the overall structure of the H22P variant is quite different from that of YF1. However, in marked difference to YF1, in the H22P variant LED Filament Bulbabsorption led to an approach of the Jα anchor sites rather than a separation, consistent with the inverted signal response. Despite different initial structure and conformational transitions of YF1 and the H22P variant, similar forces are exerted on the Jα coiled coil and give rise to a common mode of signal propagation, albeit with inverted signal polarity. These findings exemplify the remarkable malleability and robustness of signal receptors which arguably promote rapid adaption to novel stimuli and rewiring of signalling pathways during evolution. Notably, the convergence of signal mechanisms appears to be a recurring theme in sensor histidine kinases19, 36, 37: For different sensor modules signal-induced responses as diverse as pivot, piston, rotation and association reactions have been identified, yet the regulation of histidine kinase activity could well follow a unifying mechanism38.
Conclusion



2017526(金)

To determine interatomic distances

To determine interatomic distances between the labelled positions in YF1, we first recorded ELDOR traces on dark-adapted samples. The time evolution signal of all variants showed clear modulations indicative of two spatially close, interacting spin species (Suppl. Fig. S2). Following background correction (Suppl. Fig. S3), distance probability distributions p(r) were determined by Tikhonov regularisation (Fig. 2A). For positions Q44C, E55C and N84C situated in the upper half of the LOV photosensor dimer (cf. Fig. 1), the p(r) distributions showed single dominant, fairly narrow distance peaks centred at 2.8 nm (Q44C), 5.0 nm (E55C) and 6.1 nm (N84C), respectively (Fig. 2A, blue lines). The two remaining positions within the upper half of the LOV photosensor dimer featured broader distance distributions, centred at around 2.7 nm for P87C and with several distance contributions between 2.0 nm and 5.0 nm for D115C. Positions within the lower half of the LOV photosensor dimer also showed narrow distance distributions centred at 4.7 nm for D71C, 6.2 nm for D76C, 2.7 nm for Q93C and 3.6 nm for M101C. Of the positions within the coiled-coil linker, Q130C alone showed good signal-to-noise ratio (SNR) but a rather broad probability distribution of distances between 1.5 nm and 4.2 nm. By contrast, both A134C and V144C had very low SNR and were heavily affected by spurious distance contributions arising from proton and deuteron artefacts (Fig. 2A, shaded grey). Nonetheless, dominant distances of 3.7 nm (Q130C), 3.8 nm (A134C) and 3.7 nm (V144C) could be identified.
Figure 2
Figure 2

ELDOR-based structural model of light-induced transitions in YF1. (A) Distance distributions of dark-adapted (blue) and LED Candle Lights-adapted (red) states derived from ELDOR experiments (Suppl. Figs S2 and S3). Areas shaded gray indicate artefacts arising from proton and deuteron modulations. Labels positioned near the A’α helices (Q44C, E55C) and in the linker (Q130C, A134C and V144C) showed no change upon illumination, others showed shifts to larger distances. (B) Transition from dark-adapted (blue) to light-adapted (yellow) state as modelled by ENM including the P87C constraint. Predominant structural changes are marked by green arrows, and the attachment sites for the Jα linker are indicated by red spheres.
Full size image



2017525(木)

Atoms emit and absorb light

 There are many sources of light. The most common CATV splitter sources are thermal: a body at a given temperature emits a characteristic spectrum of black-body radiation. A simple thermal source is sunlight, the radiation emitted by the chromosphere of the Sun at around 6,000 kelvins (5,730 degrees Celsius; 10,340 degrees Fahrenheit) peaks in the visible region of the electromagnetic spectrum when plotted in wavelength units[17] and roughly 44% of sunlight energy that reaches the ground is visible.[18] Another example is incandescent Security alarm cable bulbs, which emit only around 10% of their energy as visible light and the remainder as infrared. A common thermal light source in history is the glowing solid particles in flames, but these also emit most of their radiation in the infrared, and only a fraction in the visible spectrum.

  The peak of the blackbody spectrum is in the deep infrared, at about 10 micrometre wavelength, for relatively cool objects like human beings. As the temperature increases, the peak shifts to shorter wavelengths, producing first a red glow, then a white one, and finally a blue-white colour as the peak moves out of the visible part of the spectrum and into the ultraviolet. These colours can be seen when metal is heated to "red hot" or "white hot". Blue-white thermal emission is not often seen, except in stars (the commonly seen pure-blue colour in a gas flame or a welder's torch is in fact due to molecular emission, notably by CH radicals (emitting a wavelength band around 425 nm, and is not seen in stars or pure thermal radiation).

  Atoms emit and absorb light at characteristic energies. This produces "emission lines" in the spectrum of each atom. Emission can be spontaneous, as in CATV splitter-emitting diodes, gas discharge lamps (such as neon lamps and neon signs, mercury-vapor lamps, etc.), and flames (light from the hot gas itself—so, for example, sodium in a gas flame emits characteristic yellow light). Emission can also be stimulated, as in a laser or a microwave maser.



2017525(木)

Light is electromagnetic radiation

Visible light" redirects here. For Cable connector that cannot be seen with human eye, see Electromagnetic radiation. For other uses, see CATV splitter (disambiguation) and Visible light (disambiguation).

  A triangular prism dispersing a beam of white light. The longer wavelengths (red) and the shorter wavelengths (blue) get separated.

  Light is electromagnetic radiation within a certain portion of the electromagnetic spectrum. The word usually refers to visible Cable connector, which is visible to the human eye and is responsible for the sense of sight.[1] Visible light is usually defined as having wavelengths in the range of 400–700 nanometres (nm), or 4.00 × 10?7 to 7.00 × 10?7 m, between the infrared (with longer wavelengths) and the ultraviolet (with shorter wavelengths).[2][3] This wavelength means a frequency range of roughly 430–750 terahertz (THz).

   Generally, EM radiation, or EMR (the designation "radiation" excludes static electric and magnetic and near fields), is classified by wavelength into radio, microwave, infrared, the visible region that we perceive as light, ultraviolet, X-rays and gamma rays.



2017524(水)

NP dilution with cell growth

NP dilution with cell growth was monitored over 6 days by ICP-MS by the quantification of intracellular levels of Zn. NB4 and THP-1 cells (0.5 × 106 cells per ml) were plated in six-well plates and incubated in serum-free RPMI-1640 with 20?μg?ml?1 of RA+NPs. After 4?h incubation, NPs that were not internalized by the cells were washed three times with PBS and the cells were left to grow at 0.2 × 106 cells per ml in complete medium for additional 4?h, 3 days and 6 days, maintaining always an exponential growth. After each incubation, cells were counted, collected by centrifugation and resuspended in nitric acid (1?ml, 69% (v/v)) for ICP analysis. The concentration of Zn was normalized per cell. The estimation of NPs was done based on Zn quantification in 20?μg of NPs. In some experiments, cells were transfected with RA+NPs labelled with TRITC, and their fluorescence monitored by flow cytometry overtime, to evaluate NPs distribution within the cells.
[3H]RA internalization studies

[11, 12-3H(N)]-Retinoic acid, 50.4?Ci?mmol?1, was purchased from Perkin Elmer. [3H]RA solution for cell culture assays was prepared on the day of experiments by dissolving [3H]RA in DMSO with unlabelled RA in a 1:1,000 ratio to a final concentration of 10?μM of RA. [3H]RA solution in DMSO for the preparation of NPs was prepared on the day of experiments using a 1:4,000 ratio of labelled to unlabelled RA. Experiments were initiated by the adding the [3H]RA solution (1?μM and 10?μM; representing less than 1% in volume of the total cell culture medium) or [3H]RA-NP suspension (1?μg?ml?1 and 10?μg?ml?1) to cultures (60,000 cells per condition, 24-well plate, 1?ml) of NB4 or U937 cells. In case of soluble RA, cells (NB4 or U937; 60,000 cells per condition, 24-well plate) were cultured with medium containing [3H]RA (1?μM and 10?μM; 1?ml of medium) for 24 or 72?h, washed with PBS (two times), collected, lysed with lysis buffer (100?ml) and kept on ice until scintillation counting procedure. In case of RA-containing NPs, cells (same conditions as for soluble RA) were cultured with [3H]RA-NPs (1?μg?ml?1 and 10?μg?ml?1) for 4?h, washed with PBS and cultured for additional 20 or 68?h in the respective culture medium. Cells were then collected to eppendorfs, washed with PBS, centrifuged (1,500?r.p.m., 5?min), lysed with lysis buffer (see above) and kept on ice until scintillation CATV splitter counting procedure. The lysed samples (100?ml) were mixed with liquid scintillation fluid (1?ml; Packard Ultima Gold) and the scintillations counted in a TriCarb 2900 TR Scintillation analyser (Perkin Elmer).
NB4 differentiation assay



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