Raw data from the experiment

Respiration, aerobic, anaerobic</p

Hrs modifies the dominant-negative activity of Smo^−PKA12.

<p>(A) A wild-type adult wing showing interveins 1-5. (B-C) Wings from flies expressing HrsRNAi by either the wing-specific <i>MS1096</i>-Gal4 or <i>765</i>-Gal4. (D-E) Wings from flies expressing either Smo^−PKA12 alone or together with HrsRNAi by <i>C765</i>-Gal4. Arrows indicate the fusion of Vein 3 and 4 that is a partial loss of Hh phenotype. Arrowheads indicate the further fusion of Vein 3 and 4 that is caused by coexpression of HrsRNAi. (F-G) HA-Hrs was expressed either alone or together with Smo^−PKA12 by <i>C765</i>-Gal4. Arrow indicates the weaker phenotype compared to D. (H–I) Tsg101RNAi was expressed either alone or in combination with Smo^−PKA12 by <i>C765</i>-Gal4. Arrow and arrowheads indicate the enhanced fusion between Vein 3 and 4.</p

Inactivation of Hrs accumulates Smo.

<p>(A) A WT wing disc stained for Smo, Ci, and <i>ptc</i>-lacZ. Arrow indicates Smo puncta in A compartment cells where there is no Hh. White dashed line indicates the A/P boundary that is defined by Ci staining. (B) An <i>hrs</i> mutant clone marked by the lack of GFP expression was stained with the anti-Smo and anti-Rab5 antibodies. Arrows indicates the accumulated Smo and arrowheads indicate the early endosome labeled by Rab5. White dashed line marks the clone. Antibody staining outside the clone serves as control. (C) A wing disc expressing HrsRNAi by <i>act>CD2></i>Gal4 was stained with anti-Smo and anti-Hrs antibodies. The marked low levels of Hrs label the HrsRNAi expressing clone (arrow in green color panel). HrsRNAi causes Smo accumulation in puncta (arrow in red color panel). White dashed line marks the clone. Antibody staining outside the clone serves as control. (D) High magnification image from a wing disc expressing HrsRNAi by the dorsal compartment-specific <i>ap</i>-Gal4 and stained for Smo and Rab11. (E) High magnification image from a wing disc expressing HrsRNAi by <i>ap</i>-Gal4 and stained for Smo and Rab7. (F) High magnification image from a wing disc coexpressing HrsRNAi with GFP-Lamp1 by <i>ap</i>-Gal4 and stained for Smo and GFP. All wing imaginal discs shown in this study were oriented with anterior on the left and ventral on the top.</p

Direct interaction of Hrs N-terminal domains with Smo phosphorylation clusters blocks Smo phosphorylation.

<p>(A) A schematic drawing of Smo C-tail truncations and their interaction with Hrs. In the right panel, HA-Hrs was transfected in S2 cells with a series of Smo truncations. Cell extracts were immunoprecipitated with the anti-Myc antibody and subjected to a western blot with the anti-HA or anti-Myc. (B) A series of Smo internal deletion constructs was transfected in S2 cells with HA-Hrs, HrsNT1 or Hrs^ΔU. Cell extracts were immunoprecipitated with the anti-Myc antibody and subjected to a western blot with anti-HA or anti-Myc. Cell lysates were also subjected to western blot to examine the expression of Hrs constructs. Myc-Smo levels are not shown in the bottom right panels. (C) An in vitro kinase assay using the purified GST-Smo^656−678 with or without the kinase set (PKA and CK1). GST-Smo^656−678 phosphorylation was detected by western blot with the anti-SmoP antibody. The input of bacterially expressed His-HrsCT1 was detected by western blot with the anti-His antibody. (D) S2 cells were transfected with the indicated constructs followed by immunoprecipitation and western blot to examine the levels of Smo phosphorylation that was recognized by the anti-SmoP antibody. (E) An in vitro kinase assay was performed and then a GST pull-down assay was carried with the bacterially expressed His-HrsCT1 in order to examine whether phosphorylation change the interaction property of Smo. GST-Smo^656−678SD bearing phospho-mimetic mutations was also used in this experiment. The same amount of His-HrsCT1 put in the system was detected by a western blot with anti-His. (F) S2 cells were transfected with HA-Hrs and Myc-Smo or Myc-Smo^SD123 followed by OA treatment. Cell extracts were immunoprecipitated with the anti-Myc antibody and subjected to a western blot with indicated antibodies to detect the Smo-bound Hrs and the levels of Smo expression. The detection of HA with the lysates indicates the expression of Hrs.</p

Synthesis of Gold Nanoparticles from Au(I) Ions That Shuttle To Solidify: Application on the Sensor Array Design

Metal-mediated interlocking rings won the 2016 Nobel Prize in Chemistry. The metal-directed interlocking rings in macromolecular systems (e.g., proteins) may be similar to the form of Maxwell’s electromagnetic waves; the metal ions may shuttle among the rings in the special environment. To verify this hypothesis, we designed a general approach to synthesize the multicolored gold nanoparticles (GNPs) mediated by Au­(I)-directed interlocking rings in proteins. The Au­(I) ions shuttled among these interlocking rings in the strong alkaline solution. Through the rapid nucleation method, the multicolored GNPs of different morphology and sizes were synthesized in the multiple honeycombed templates. On the basis of the “three-color” principle of Thomas Young, we extracted the red, green, and blue (RGB) alterations of GNPs to fabricate a visual sensor array for protein discrimination. The fingerprints (ΔRGB) were obtained from the target proteins and fed into computer programs. The proposed sensing platform was also applied to detect lysozyme in human tears with satisfactory results. Importantly, we forecasted that lysozyme could be the effective drug for curing dacryocystitis and nasolacrimal duct obstruction diseases

Hrs UIM and N-terminal domains physically interact with Smo, but only the UIM domain is required for promoting Smo ubiquitination.

<p>(A) A schematic drawing of Hrs truncations and their ability to interact with Myc-Smo in the immunoprecipitation assay described in this Figure. (B) S2 cells were transfected with Myc-Smo and the indicated Hrs constructs followed by immunoprecipitation and western blot with the indicated antibodies. Cell lysates were also subjected to western blot to examine the expression of Hrs full-length and Hrs truncations. The bands at 25kD in the top left panel and the bands at 55kD in the top right panel indicate the IgG that served as loading control. In the last lane of top right panel, the lysate of HA-HrsNT2 was loaded in order to show the absence of the same band in the adjacent lane. (C) S2 cells were transfected with Myc-Smo and HA-Hrs constructs in combination with Flag-USP8 or USP8 RNAi to examine whether changing ubiquitination levels of Smo by USP8 could alter the interaction between Smo and Hrs. Cell extracts were immunoprecipitated by the anti-Myc antibody followed by western blot with either the anti-HA or the anti-Myc antibody to examine the amount of Smo-bound Hrs and the levels of Smo. Western blot of the cell lysates was to examine the expression of Hrs or USP8. (D) S2 cells were co-transfected with HA-HrsNT1 that contains the UIM domain and Myc-Smo or Myc-SmoK8R that bears K>R mutation in the domain binding Hrs. The immunoprecipitation assay was performed with the anti-Myc antibody and the Smo-bound Hrs was examined by the anti-HA antibody. K>R mutation did not change the interaction between Smo and HrsNT1. (E) S2 cells were transfected with the indicated constructs or Hrs dsRNA. Cell extracts were subjected to immunoprecipitation with the anti-Myc antibody followed by western blot with the anti-Myc or anti-Flag antibodies. Cell lysates were subjected to direct western blot with the anti-Flag and anti-HA antibodies to examine the protein expressed. (F) S2 cells were transfected with Myc-Smo and Hrs variants to examine the ability of different forms of Hrs in regulating Smo ubiquitination.</p

Activating Room Temperature Long Afterglow of Carbon Dots via Covalent Fixation

The achievement of long afterglow in aqueous solution and as well as with small effects of dissolved oxygen is critical for its applications, but this is still a highly difficult and challenging task. Herein, a novel strategy for facilely preparing room temperature long afterglow material is reported via covalently fixing carbon dots (CDs) onto colloidal nanosilica (nSiO₂). The as-obtained materials (named m-CDs@­nSiO₂) show not only an unexpected long afterglow emission in water dispersion (lifetime as high as 0.703 s) but also with small effects of the dissolved oxygen. Further studies revealed that the observed long afterglow of m-CDs@­nSiO₂ possesses a predominant delayed fluorescence nature and mixed with a portion of phosphorescence. Some key knowledge that can be concluded from this study are (i) covalent interaction could be employed as an option to fix and rigidify triplet emission species; (ii) covalent bonds fixation strategy could behave as a better alternative than that of the frequently used hydrogen/halogen bonds for stabilizing triplets, because this benefits in extending the occurrence of long afterglow from only solid to solution/​dispersion forms; and (iii) the containing unsaturated bonds (e.g., CC) on the surface of CDs make them to be self-protection agents from the usual quenching effects of oxygen to the triplets due to their capabilities of reaction with oxygen during the irradiation process. On the basis of the unique long afterglow features of m-CDs@­nSiO₂ in water dispersion and oxygen insensitivity, a moisture-related strategy for high-level information protection is proposed and demonstrated

Smo regulation by Tsg101.

<p>(A) A wing disc expressing Tsg101RNAi by <i>ap</i>-Gal4 was stained for Smo. GFP labels the dorsal compartment cells that express Tsg101RNAi. (B) Large magnification of a wing disc expressing Tsg101RNAi by <i>ap</i>-Gal4 and stained for Smo and Rab5. The accumulated Smo puncta do not co-localize with Rab5. (C) Large magnification of a wing disc expressing Tsg101RNAi by <i>ap</i>-Gal4 and stained for Smo and Rab7. (D) Large magnification of a wing disc expressing Tsg101RNAi by <i>ap</i>-Gal4 and stained for Smo and Hrs. The accumulated Smo puncta by Tsg101RNAi co-localize with Rab7 and Hrs. (E) S2 cells transfected with Myc-Smo followed by RNAi of the indicated endosomal components were subjected to luciferase assay with cotransfection of <i>tub-Ci</i> and the <i>ptc-luc</i> constructs. GFP RNAi served as control RNAi. RNAi efficiency was confirmed by western blot with anti-GFP or anti-Flag antibody for tagged Shi, Tsg101, and Hrs (not shown).</p

Hrs interacts with Smo and promotes Smo ubiquitination.

<p>(A) S2 cells were transfected with Myc-Smo alone or in combination with HA-Hrs or Hrs dsRNA. Cell extracts were subjected for immunoprecipitation with the anti-Myc antibody followed by western blots with anti-Ub, anti-HA, and anti-Myc to detect the ubiquitination of Smo, Smo-bound Hrs, and the levels of Myc-Smo, respectively. To normalize the levels of Smo, 50 mM MG132 and 15mM NH4Cl was used to block Smo degradation, and samples were normalized for loading. All figures in this study showing the levels of Myc-Smo were normalized by the same way. The efficiency of Hrs RNAi was determined by western blot with anti-Hrs antibody. GFP served as a transfection and loading control. (B) S2 cells were transfected with Myc-Smo alone or together with HA-Hrs, and treated with HhN-conditioned medium or control medium followed by immunoprecipitation and western blot with the indicated antibodies. Of note, the interaction between Smo and Hrs was down-regulated by Hh treatment. (C) S2 cells were transfected with Myc-Smo and treated with HhN-conditioned medium or control medium. Cell extracts were immunoprecipitated with the anti-Myc antibody and blotted with the indicated antibodies. Hh treatment reduced the interaction between Smo and endogenous Hrs.</p

High Performance Liquid Metal Battery with Environmentally Friendly Antimony–Tin Positive Electrode

For the first time, Sb–Sn alloys are reported as environmentally friendly positive electrodes for high performance liquid metal batteries (LMBs). Meanwhile, the dominant role of Sb in setting the potential and the inert “solvent” role of Sn in lowering the melting point and decreasing the cell cost are clarified on the basis of electrochemical titration and ex situ analysis. The Li||Sb–Sn LMB exhibits superior rate performance (only 13% capacity loss from 100 mA cm^–2 to 1 A cm^–2 of current densities), low materials cost (73 $ kW h^–1), and high energy density (200.4 W h kg^–1) at reduced operating temperature. Most notably, after 3500 h of operation (more than 430 full charge–discharge cycles), a discharge capacity of 20.6 Ah is maintained with a capacity retention of 96.7%, corresponding to a fade rate of 0.0078% per cycle, which potentially meets the metrics of large-scale energy storage without environmental concerns