Lunar phase-dependency of <i>Siganus guttatus Period4</i> (<i>SgPer4</i>) and <i>Period2</i> (<i>SgPer2</i>) mRNA expression in the brain.

<p>(A) Experimental design using a tank cover for nocturnal moonlight interruption. See legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109119#pone-0109119-g003" target="_blank">Figure 3A</a>. (B, C) The diencephalon (n = 4) was collected at noon from the new moon to full moon phase. <i>SgPer4</i> (panel B) and <i>SgPer2</i> (panel C) mRNA levels were calculated as values relative to those of the virtual reference control gene and were defined as the average of the threshold cycles (Ct) for <i>SgEF1α</i>, <i>SgPGK and Sgβ-actin.</i> Error bars represent ± SD. Lunar phases are indicated by schematic moon images. The p values are indicated on each graph, two-way ANOVA.</p

Immunohistochemical localization of <i>Siganus guttatus</i> Cryptochrome3 (SgCRY3) in the diencephalon.

<p>(A) Drawing of the lateral view of the brain of <i>Siganus guttatus</i>. Lettered dotted lines indicate the levels of the transverse sections shown in panels C–J. Ce, cerebellum; Di, diencephalon; OpN, optic nerve; OT, optic tectum; Te, telencephalon. (B) Drawing of the transverse sections at the level of panel A. (C, D) αSgCRY3CT staining without a competitive peptide. (E, F) control sections (mouse IgG was used instead of αSgCRY3CT). (G, H) αSgCRY3CT staining in the presence of 100 µM CT17 epitope peptide. (I, J) αSgCRY3CT staining in the presence of 100 µM CT17S epitope-shuffled peptide. In the preabsorption experiment (panels G–J), CT17 or CT17S peptide (100 µM) had been incubated for 16 h at 4°C with αSgCRY3CT before the primary antibody reaction. Panels D, F, H, and J are magnified view of panels C, E, G, and I, respectively. Wash solution; PBS containing 0.25% (panels C–F) or 0.05% (panels G–J) of Triton X-100. Blocking solution; Wash solution containing 1.5% horse normal serum. Each tissue was sampled either March 23, 2012 (new moon) or June 27, 2014 (new moon).</p

Multiple sequence alignment of the deduced amino acids of <i>Siganus guttatus</i> Cryptochrome1 (SgCRY1) and Cryptochrome3 (SgCRY3), competitive ELISA and immunoblot analysis.

<p>(A) Multiple sequence alignment of the deduced amino acids of SgCRY1/3. The line above the alignment indicates the antigenic region that was conjugated to KLH or BSA and the conjugate was used as an antigen. CT17 and CT17S are epitope and epitope-shuffled peptides, respectively. (B) Competitive ELISA showing the antigen-specificity. ELISA microplate wells were coated with GST-SgCRY3CT antigen, blocked with 1% skim milk, reacted with αSgCRY3CT that had been mixed with CT17 epitope or CT17S epitope-shuffled peptide at the indicated concentration in advance. Then, the unreacted antibody was washed out and the remaining antibody was detected through use of an HRP-labeled secondary antibody. (C, D) Immunoblot analyses for validating the specificity of αSgCRY3CT to antigenic agent. GST-SgCRY3CT (0.1 µg), GST (0.1 µg), BSA-SgCRY3CT (0.15 µg), and BSA (0.15 µg) proteins were subjected to 10% polyacrylamide SDS-PAGE. In the preabsorption experiment (panel D, lanes 3–6), CT17 or CT17S peptide (100 µM) had been incubated for 1 h at 37°C with αSgCRY3CT before the primary antibody reaction.</p

Lunar phase-dependency of <i>Siganus guttatus Cryptochrome3</i> (<i>SgCry3</i>) mRNA expression in the brain.

<p>(A) Experimental design using a tank cover for nocturnal moonlight interruption. Four groups of fish were contained in tanks maintained under natural (MM, natural and natural condition) conditions or constant darkness from 30 minutes after sunset to midnight (DM, dark and natural condition) or constant darkness from midnight to 30 minutes before sunrise (MD, natural and dark condition) or constant darkness from 30 minutes after sunset to 30 minutes before sunrise (DD, dark and dark condition) from May 21 (new moon) to June 4 (full moon). Illustration shows nocturnal light conditions in tanks and the time of moonlight irradiation from May 21 to June 4, 2012. Lunar phases are indicated by schematic moon images. (B–E) Lunar changes in <i>SgCry3</i> mRNA levels in the brain. The diencephalon and optic tectum (n = 4) were collected at noon from the new moon to full moon phase. <i>SgCry3</i> mRNA levels were calculated as values relative to those of the virtual reference control gene and were defined as the average of the threshold cycles (Ct) for <i>SgEF1α</i>, <i>SgPGK</i> and <i>Sgβ-actin.</i> Error bars represent ± SD. The p values are indicated on each graph, two-way ANOVA.</p

Primers for quantitative RT-PCR.

<p>Primers for quantitative RT-PCR.</p

Absorbance changes of <i>C</i>. <i>rhodomelas</i> eyeball extract before and after NH₂OH addition and subsequent light irradiation.

<p>(A) Order Scorpaeniformes, Liparidae, snailfish. This snailfish is rosy and flexible like gelatin. They inhabit hydrothermal vent areas at depths of approximately 1500 m. The body length and eyeball diameter of the fish shown above were 127 mm and 7 mm, respectively. (B) Absorption spectra of the detergent extract from a <i>C</i>. <i>rhodomelas</i> eyeball with buffer P containing 1% CHAPS (curve "eyeball extract" in panel B). One molar NH₂OH was added to a final concentration of 100 mM (curves "NH₂OH 5 min" and "NH₂OH 10 min"). The sample was then sequentially irradiated with orange light using an O564 filter (8 mW/cm², λ_T50% = 564 nm, λ_T0.1% = 540 nm) for a total of 2 min (curves "O564 0.5 min", "O564 1 min" and "O564 2 min") and yellow light using a Y522 filter (24 mW/cm², λ_T50% = 522 nm, λ_T0.1% = 501 nm) for a total of 1 min (curve "Y522 1 min"). Inset shows difference absorbance spectra before and after the addition of NH₂OH. (C) Difference absorbance spectra before the first irradiation and after each irradiation are shown.</p

Amino acid residues at nine sites in rhodopsins of deep-sea fishes.

<p>The amino acid sites and λ_max values except for <i>S</i>. <i>altivelis</i> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135888#pone.0135888.ref029" target="_blank">29</a>] and <i>C</i>. <i>rhodomelas</i> (present study) were obtained from Hunt <i>et al</i>. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135888#pone.0135888.ref014" target="_blank">14</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135888#pone.0135888.ref016" target="_blank">16</a>].</p><p>Amino acid residues at nine sites in rhodopsins of deep-sea fishes.</p

Spectroscopic and immunoblot analyses of recombinant CrRh expressed in HEK293EBNA cells.

<p>(A) Spectral changes of recombinant CrRh caused by light irradiation. Proteins were solubilized with buffer P-10 (50 mM HEPES, 10 mM NaCl, 1 mM DTT, 4 μg/mL Aprotinin and 4 μg/mL Leupeptin) containing 1% CHAPS and 0.2 OD 11-<i>cis</i> retinal. One molar NH₂OH was added to the final concentration of 50 mM. Then the sample was sequentially irradiated with green light (501 μW/cm², λ_max = 520 nm) for totally 1 min (7.5 sec, 15 sec, 30 sec, and 60 sec). Difference absorbance spectra between those before the first irradiation and after each irradiation are shown (7.5 sec, 15 sec, 30 sec, and 60 sec). (B) Comparison between difference absorption spectrum of eyeball extract and that of recombinant CrRh. The difference absorption spectra of eyeball extract and recombinant CrRh were derived from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135888#pone.0135888.g001" target="_blank">Fig 1C</a> (O564 2min) and Fig 3A (60 sec), respectively. The spectrum of recombinant CrRh were best fitted with visual pigment template of λ_max = 480 nm, which was calculated by the method described in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135888#pone.0135888.ref036" target="_blank">36</a>]. (C) Western blot analysis of recombinant CrRh. Lanes 1 and 3, purified chicken rhodopsin (cRh; [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135888#pone.0135888.ref027" target="_blank">27</a>]). Lanes 2 and 4, recombinant CrRh proteins expressed in HEK293EBNA. Anti-rhodopsin antibody cRh-C was used. Arrowhead shows a signal considered as a monomeric form of CrRh.</p

Sequence analysis and immunoblot detection of CrRh.

<p>(A) Amino acid sequences of CrRh and the other opsins. Alignment of CrRh and representative members of vertebrate rhodopsins are shown. Amino acid sequences except for CrRh (present study) were obtained from the NCBI Entrez Protein database. <i>S</i>. <i>altivelis</i> is the deep-sea rockfish lonspine thornyhead [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135888#pone.0135888.ref029" target="_blank">29</a>]. Amino acids sequences were aligned using CLUSTAL W software. Blue-colored boxes indicate conserved regions in all members. Asterisks indicate amino acid positions with functionally important residues. Arrowheads indicate positions for spectral tuning sites based on previous study [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135888#pone.0135888.ref016" target="_blank">16</a>]. Black lines show transmembrane regions. cRh-N epitope is the epitope of polyclonal antibody (cRh-N) which recognizes N-terminal region of chicken rhodopsin (Met₁-Tyr₂₉, [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135888#pone.0135888.ref025" target="_blank">25</a>]). cRh-C epitope is the epitope of polyclonal antibody (cRh-C) which recognizes C-terminal region of chicken rhodopsin (Lys₂₉₆-Ala₃₅₁). Bold letters in the epitope regions denote amino acids identical to those of chicken rhodopsin. (B) A phylogenetic tree of opsin family proteins constructed by Neighbor-Joining method. Amino acid sequences except for <i>A</i>. <i>cornuta</i> rhodopsin [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135888#pone.0135888.ref016" target="_blank">16</a>], <i>C</i>. <i>sloani</i> rhodopsin [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135888#pone.0135888.ref016" target="_blank">16</a>], and CrRh (present study) were obtained from the NCBI Entrez Protein database. They were analyzed in the conserved region of the Rh family proteins using CLUSTAL W and NJ plot software (version 2.3). Bootstrap probabilities (p) are represented by closed circles on the nodes (p > 95%) or values near the nodes. Accession numbers for sequences obtained from the NCBI Entrez Protein database are M17718 (<i>Drosophila</i> Rh3), L11863 (Goldfish Rh), NM_131084 (Zebrafish Rh), DQ490124 (<i>Sebastolobus altivelis</i> Rh), NM_001078631 (<i>Takifugu rubripes</i> Rh), NM_001097334 (<i>Xenopus tropicalis</i> Rh), NM_001087048 (<i>Xenopus laevis</i> Rh), NM_001030606 (Chicken Rh), NM_001014890 (Bovine Rh), L11866 (Goldfish green cone), M92038.1 (Chicken green cone), L11864 (Goldfish blue cone), NM_205517 (Chicken blue cone), NM_205438 (Chicken violet cone), NM_205409 (Chicken pinopsin), L11867 (Goldfish red cone), and NM_205440 (Chicken red cone). (C) Western blot analysis and CBB stain of <i>C</i>. <i>rhodomelas</i> ocular proteins. For western blotting (lanes 1–8), eyeball extract corresponding to a presumed 0.01 retina (containing 0.0012 ODml of CrRh) of <i>C</i>. <i>rhodomelas</i> or purified chicken rhodopsin (chicken Rh, 0.5 μg, [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135888#pone.0135888.ref027" target="_blank">27</a>]) was loaded in each lane. For CBB stain (lanes 9–11), the larger amount of the extract (0.03 retina, 0.0036 ODml of CrRh) and purified chicken rhodopsin (3 μg) was loaded. Anti-rhodopsin antibodies used were: cRh-N, anti-N-terminal region of chicken rhodopsin (Met₁-Tyr₂₉, [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135888#pone.0135888.ref025" target="_blank">25</a>]); cRh-C, anti-C-terminal region of chicken rhodopsin (Lys₂₉₆-Ala₃₅₁); Toad Rh AS, anti-toad rhodopsin antiserum [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135888#pone.0135888.ref024" target="_blank">24</a>]. Concentration and dilution of the antibodies were: cRh-N (1000-fold dilution), cRh-C (2.9 ng/ml) and Toad Rh-AS (1000-fold dilution). Control anti-mouse IgG antibody was used at 2.9 ng/ml. Arrowheads indicate signals considered as monomeric form of CrRh.</p

N‑Dopant Site Formulation for White-Light-Emitting Carbon Dots with Tunable Chromaticity

Multicolor emissions from carbon dots (CDs) are vital for light-emitting diodes (LEDs), particularly for direct, white-light emission (WLE), which enables a replacement of rare earth (RE)-doped phosphors. However, the difficulty of synthesizing single-component WLE CDs with full-spectrum emission severely hinders further investigation of their emission mechanisms and practical applications. Here, we demonstrate rational design and synthesis of chromatically tunable CDs with cyan-, orange-, and white-light emission, precisely tunable along the blackbody Planckian locus by controlling the ratio of different nitrogen dopant sites. We adopted 15N solid-state nuclear magnetic resonance (NMR) spectroscopy and X-ray photoelectron spectroscopy (XPS) to identify and quantify N-dopants in different sites and environments, and we explain their influence on emission properties of these CDs. This study provides guiding principles to achieve spectrally tuned emissions, enabling us to design WLE from CDs. This study also clarifies which chemical reagents and their proportions should be used for solvothermal synthesis to realize well-defined WLE from single-component CDs and adjustable, correlated color temperature (CCT) from 6500 to 3500 K. We also demonstrate a soft lighting device by adopting an optical haze film composed of cellulose fibers with excellent light-tailoring characteristics. The proposed methodology for synthesizing WLE CDs by engineering the N-doping sites will boost the development of lighting devices with readily available materials toward realization of low-cost, environmentally friendly WLEDs, solar cells, UV-blockers, counterfeit inks, and display applications