The Eosin Y-sensitized g-C3N4/PtNi/GO-0.5% composite photocatalyst yields about 1.54 and 1178 times higher hydrogen evolution rate than the Eosin Y-sensitized g-C3N4/Pt/GO-0.5% and g-C3N4/Ni/GO-0.5% samples, respectively. Mechanism of enhanced performance for the g-C3N4/PtNi/GO composite was also investigated by different characterization, such as photoluminescence, transient photocurrent response, and TEM. These results indicated that enhanced charge separation efficiency and more reactive sites are responsible for the improved hydrogen evolution performance due to the positive synergetic effect between Pt and Ni. This study suggests that PtNi alloy can be used as an economic and effective cocatalyst for hydrogen evolution reaction.An economic and effective Pt-based alloy cocatalyst has attracted considerable attention due to their excellent catalytic activity and reducing Pt usage. In this study, PtNi alloy cocatalyst was successfully decorated on the g-C Sustainable and large-scale hydrogen evolution from water using solar energy is considered to be one of the promising method toward solving the energy crisis and environmental pollution , 2. To a2ZnSnS4 showed higher H2 production efficiency than the pure Pt loading Cu2ZnSnS4 [3Co bimetallic cocatalyst decorated CdS were prepared by Hu et al. and exhibited enhanced hydrogen evolution performance [x1 − CdxS were also evaluated in previous studies [2ZnSnS4 or high toxicity of Cd hinders their practical application on a large scale. Carbon nitride (g-C3N4) has attracted attention due to its low cost [2 evolution rate of 960 μmol g−1 h−1 was obtained over the PtCo/g-C3N4 photocatalyst under λ > 400 nm irradiation [x/g-C3N4 hybird photocatalyst was also studied by Bi et al., and a H2 evolution rate of 8456 μmol g−1 h−1 was achieved under full spectral irradiation [3N4 photocatalyst is still a little bit low due to the wide band gap of 2.7 eV and bad electron transfer ability. Eosin Y-sensitized g-C3N4 can harvest a wide range of visible light [3N4 and GO can promote the electron transfer capability in g-C3N4 and thus improve the electron-hole pair’s separation to improve the photocatalytic performance for hydrogen evolution [3N4/Pt/GO composite photocatalyst for hydrogen evolution [3N4/GO composite photocatalyst loaded with Pt-based alloy cocatalyst is useful.Yu et al. reported that PtCo or PtNi alloy cocatalyst-modified Cuu2ZnSnS4 . Pt3Co bformance . PtCo an studies , 15. Howlow cost . Han et adiation . PtNix/gadiation . Howeverle light , 20. Grale light –25. Combvolution –31. Latevolution . The exp3N4/PtNi/GO composite photocatalyst was prepared for hydrogen evolution from water. The highest hydrogen evolution rate of 5.89 mmol g−1 h−1 is obtained over the Eosin Y-sensitized g-C3N4/PtNi/GO photocatalyst, which is much higher than the Eosin Y-sensitized g-C3N4/Pt/GO and g-C3N4/Ni/GO composite samples. To the best our knowledge, there is no previous report that the Eosin Y-sensitized g-C3N4/PtNi/GO composite is employed for hydrogen production from water. The optimal molar ratio of Pt/Ni and amount of PtNi cocatalyst were screened out in detail. In addition, mechanism of enhanced photocatalystic performance for the g-C3N4/PtNi/GO composite was also investigated through different characterization methods.Here, the Eosin Y-sensitized g-C3N4 powders were synthesized as described in previous study [3N4 powders were collected for further using.g-Cus study . In a ty3 (5 g) were putted into a beaker. Then, 230 mL concentrated sulfuric acid were added, and the process must be as slow as possible. The above reaction was proceeded with stirring under ice-water bath. Next, 10 g KMnO4 was added into the mixture solution and reacted for 3 h. The temperature of solution was raised to 35 °C and was maintained for 4 h. Then, 460-mL distilled water was poured into the above solution and heated up to about 98 °C for 3 h. After reaction, a certain amount of H2O2 (30%) and concentrated hydrochloric acid were added under stirring with the purpose of removing excess KMnO4 and SO42−. Finally, the GO sample was obtained by freeze-drying for 24 h.GO was prepared using the modified Hummers’ method . Nature 3N4/PtNi/GO-X : In a typical, 133 mg of g-C3N4 were dispersed into 50 mL anhydrous ethanol. Excess NaBH4 reductant was added into the mixture solution under stirring. Then, a certain volume of NiCl2·6H2O solution (0.1 mol/L) and H2PtCl6 solution (1.0 mmol/L) were dropwise added into the above solution. In order to investigate the added procedure of NiCl2·6H2O and H2PtCl6 solutions, three methods including simultaneous loading of Pt and Ni, loading of Pt and then Ni or in reverse were chose. Then, the suspension solution was stirred for 5 h to achieve a uniform dispersion for PtNi cocatalyst in the g-C3N4. After that, the g-C3N4/PtNi-X samples were collected by centrifugation to wash away excess NaBH4 for several times. Afterwards, 67 mg GO and the g-C3N4/PtNi-X sample were dispersed into 100-mL distilled water simultaneously. The suspension solution was ultrasonicated at 500 W for 10 h. After that, a series of g-C3N4/PtNi/GO-X composite photocatalysts were obtained by centrifuged and then dried at 60 °C in a vacuum oven for one night. Other bimetallic PtNi alloy cocatalysts with different Pt/Ni molar ratio  were also prepared as same as the aforementioned method, which named as Pt9Ni1, Pt3Ni1, Pt1Ni3, and Pt1Ni9, respectively, specially, the 1:1 M ratio of Pt/Ni name as PtNi.Synthesis of g-C3N4/Ni/GO-0.5% and g-C3N4/Pt/GO-0.5% samples (0.5% represents the weight ratio of Ni or Pt to the g-C3N4/GO composite): The g-C3N4/Ni/GO-0.5% and g-C3N4/Pt/GO-0.5% samples were prepared with the same preparation procedure of g-C3N4/PtNi/GO-X samples except for adding different volume of NiCl2·6H2O solution or H2PtCl6 solution. The 2:1 weight ratio of g-C3N4 to GO is chosen in all g-C3N4/Ni/GO, g-C3N4/Pt/GO, and g-C3N4/PtxNiy/GO composite samples according to our previous study [Synthesis of g-Cus study .2SO4 solution was used as electrolyte.The XRD patterns were obtained using an X-ray diffraction diffractometer  with Cu-Kα radiation. TEM images of the samples were recorded through the transmission electron microscopy . Surface chemical states of the photocatalysts were measured by X-ray photo-electron spectroscopy  with monochromatic Al Ka X-rays (1486.6 eV). The photoluminescence (PL) spectra were measured on a JY HORIBA FluoroLog-3 spectrometer, and the excited wavelength of 460 nm was chosen. The curves of photocurrent response were carried out in an electrochemical workstation  using a conventional standard three-electrode cell under visible light irradiation (λ > 420 nm). 0.1 mol/L Na2O containing 20 vol% (v/v) triethanolamine . A 300-W xenon lamp  coupled with a UV cut-off filter (> 420 nm) was used as the light source. The amounts of hydrogen were measured through a gas chromatography .Photocatalytic experiments were conducted in a Pyrex cell with a top flat window at 6 °C. Typically, 50 mg photocatalyst powder and 50 mg Eosin Y dye were added into 100 mL H3N4/Ni/GO-0.5%, g-C3N4/Pt/GO-0.5%, and g-C3N4/PtNi/GO-0.5% samples are shown in Fig. 3N4, which arises from the in-plane structural packing motif [3N4, which corresponds to the interlayer stacking of conjugated aromatic system [3N4 and the nanosheet structure of GO. In Fig. 3N4. Compared with pure Ni and Pt, the size of the PtNi alloy cocatalyst particles is reduced and the dispersion of PtNi alloy cocatalyst is improved . The Pt 4f7/2 and 4f5/2 peaks are located at 70.97 and 74.28 eV for the g-C3N4/Pt/GO-0.5% sample, respectively, which represent the signal of Pt0 [3N4/PtNi/GO-0.5% sample, the orbital binding energies of Pt 4f shift about 0.42 eV to high binding energy compared with pure Pt. The obvious peak shift suggests that Pt electron is slight loss, which indicates that the PtNi alloy cocatalyst is formed in the g-C3N4/PtNi/GO-0.5% sample. As shown in Fig. 3/2 and Ni 2p1/2 for the g-C3N4/Ni/GO-2% sample, respectively, which are the characterization signal of Ni0 [3N4/Ni/GO-2%, the binding energies of Ni 2p shifted to low binding energy for the g-C3N4/PtNi/GO-2% sample. The result suggests that a change in the surrounding environment of Ni atoms occurs, which further confirms that the PtNi alloy cocatalyst is successfully synthesized [3N4/PtNi/GO-0.5% sample is 9:11 through XPS measurement. Based on above analysis, it can be concluded that the g-C3N4/PtNi/GO composite with PtNi alloy as cocatalyst was obtained by combing a facile liquid-phase sonochemical way with a chemical reduction method.In order to investigate the surface chemical element and valence states of g-Cectively . In Fig.l of Pt0 , 42. Forl of Ni0 . Comparethesized . The exa2 evolution rate of series of g-C3N4/PtNi/GO-0.5% samples loaded with different type of cocatalyst. Simultaneous loading of Pt and Ni names as g-C3N4/PtNi/GO-0.5%; Loading of Pt and then Ni names as g-C3N4/Pt-Ni/GO-0.5%; Loading of Ni and then Pt names as g-C3N4/Ni-Pt/GO-0.5%. For Eosin Y-sensitized g-C3N4/Ni/GO-0.5% sample with pure Ni as cocatalyst, the H2 evolution rate is very low and only reach to 0.005 mmol g−1 h−1. After replacing Ni with Pt as cocatalyst, a significant increase of H2 evolution rate is observed, which sharply increases to 3.82 mmol g−1 h−1 for the Eosin Y-sensitized g-C3N4/Pt/GO-0.5% sample. The result suggests that loading efficient Pt cocatalyst is necessary to achieve excellent performance for H2 generation. When employed PtNi alloy as cocatalyst, the Eosin Y-sensitized g-C3N4/PtNi/GO-0.5% composite shows the highest H2 evolution rate of 5.89 mmol g−1 h−1, which is about 1.54 and 1178 times higher than the Eosin Y-sensitized g-C3N4/Pt/GO-0.5% and g-C3N4/Ni/GO-0.5% samples, respectively. The enhanced performance can be attributed to the positive synergistic effect between Pt and Ni. Compared with pure Pt cocatalyst, PtNi alloy cocatalyst accelerates the accumulation of photongenerated electrons, which will provide more electrons for hydrogen evolution [2 evolution sites and enhance the electrons transfer, respectively. When loaded Pt and then Ni or in reverse, an obvious reduction of H2 evolution activity is observed. In fact, loading Pt and then Ni or in reverse does not form PtNi alloy cocatalyst [2 generation rate is strongly relied on employing efficient PtNi alloy cocatalyst.Figure volution . In addicatalyst . The res2 evolution. Therefore, the H2 production rate of Eosin Y-sensitized g-C3N4/PtxNiy/GO-0.5% samples loaded with different Pt/Ni molar ratio were investigated and the results are shown in Fig. 2 production rate is increased gradually with increasing Ni/Pt molar ratio. When the molar ratio of Ni/Pt is 1:1, the highest hydrogen production rate of 5.89 mmol g−1 h−1 is obtained. Further increasing the amount of Ni leads to a drop in the H2 evolution activity. The degradation of H2 generation performance may come from the reducing number of Pt active sites for H2 evolution. Pt active sites have stronger adsorption of hydrogen ions than Ni [2 evolution preferentially takes places on Pt instead of Ni. Figure 2 production rate of Eosin Y-sensitized g-C3N4/PtNi/GO samples loaded with different amounts of PtNi alloy cocatalyst. When the weight content of PtNi alloy cocatalyst is 0.5%, a hydrogen evolution rate of 2.45 mmol g−1 h−1 is obtained for the g-C3N4/PtNi/GO-0.25% sample. The hydrogen evolution rate increases from 2.45 mmol g−1 h−1 to the highest value of 5.89 mmol g−1 h−1 after the amount of PtNi alloy cocatalyst up to 0.5%. In further increasing the amount of PtNi alloy cocatalyst, the hydrogen evolution performance shows a slight decrease. Excess PtNi alloy cocatalyst can hinder the light absorption of Eosin Y and g-C3N4 and thus degrade the photocatalytic performance. The stability of hydrogen generation for the g-C3N4/PtNi/GO-0.5% sample was also measured and the result is shown in Fig. 3N4/PtNi/GO-0.5% sample shows a slight decrease, which indicates that the g-C3N4/PtNi/GO-0.5% composite sample has relatively stability for hydrogen evolution. A strong covalent bond between the carbon and nitride in C3N4 and the weak degradation of Eosin Y are the two main reasons for the hydrogen evolution stability of g-C3N4/PtNi/GO-0.5% sample [The composition of PtNi alloy cocatalyst has an important effect on the catalytic activity for H than Ni . Therefo% sample , 45.Fig.3N4/PtNi/GO sample with PtNi alloy as cocatalyst, two possible reasons of light absorption and charge separation efficiency were evaluated. Figure 3N4/Ni/GO-0.5%, g-C3N4/Pt/GO-0.5%, and g-C3N4/PtNi/GO-0.5% samples. The three different samples exhibit an obvious absorption after about 450 nm, which is from the metal cocatalyst [3N4/Ni/GO-0.5% sample with pure Ni as cocatalyst shows the strongest absorption after about 450 nm. However, the H2 evolution rate for the g-C3N4/Ni/GO-0.5% sample is the lowest. The result suggests that the improved H2 evolution performance is not coming from the enhanced light absorption.In order investigate mechanism for the improved photocatalystic performance of g-Ccatalyst . The g-C3N4/Ni/GO-0.5%, g-C3N4/Pt/GO-0.5%, and g-C3N4/PtNi/GO-0.5% samples. Only the Eosin Y solution without photocatalyst exhibits an extensive emission peak at about 540 nm because of Eosin Y’s conjugate xanthene structure and strong recombination capacity of photogenerated electron-hole pairs in excited Eosin Y. Obvious fluorescence quenching is observed after adding different type of photocatalysts into the Eosin Y solution. The fluorescence quenching suggests that the electrons transfer to photocatalysts from excited Eosin Y and then migrate to cocatalyst for proton reduction. In addition, there is a small blue shift (ca. 1.3 nm) of PL quenching spectra for the three different evaluated composite photocatalysts, which can be attributed to the noncovalent π-π stacking interaction among Eosin Y, g-C3N4 and GO [3N4/Pt/GO-0.5% sample shows a moderated intensity, which is lower than the g-C3N4/Ni/GO-0.5% sample. Importantly, the g-C3N4/PtNi/GO-0.5% sample exhibits the lowest fluorescence intensity, which implies that the PtNi alloy cocatalyst is the most efficient cocatalyst for improving the charge separation efficiency than pure Pt or Ni. The result is consistent with the H2 evolution activity , which further confirms employing PtNi alloy cocatalyst is essential to improve charge separation efficiency. Based on above results, the improved H2 evolution activity for the Eosin Y-sensitized g-C3N4/PtNi/GO-0.5% composite is attributed to the enhanced charge separation efficiency.Charge separation efficiency can be characterized by the photoluminescence (PL) quenching spectra , 47. In 4 and GO . The PL 2 evolution process for the Eosin Y-sensitized g-C3N4/PtNi/GO composite sample (Fig. 3N4 and/or GO and then to the PtNi alloy cocatalyst for protons reduction. Meanwhile, the photoexcited electrons in the CB of g-C3N4 also flow to the PtNi alloy cocatalyst for H2 evolution reaction. At the same time, the photogenerated holes or oxidized Eosin Y dyes directly oxide the TEOA sacrificial agent.According to above results and mechanism analysis, we propose a schematic diagram to understand the Hple Fig. . Under v3N4/PtNi/GO composite was synthesized by combing a facile liquid-phase sonochemical way with a chemical reduction method. The Eosin Y-sensitized g-C3N4/PtNi/GO-0.5% composite shows the highest hydrogen evolution rate of 5.89 mmol g−1 h−1, which is about 1.54 and 1178 times higher than the Eosin Y-sensitized g-C3N4/Pt/GO-0.5% and g-C3N4/Ni/GO-0.5% samples, respectively. The enhanced photocatalytic activity can be ascribed to the positive synergetic effect between Pt and Ni as well as more reactive sites, which leads to efficient photoexcited electron-hole pairs separation. This study demonstrates that PtNi alloy can be employed as an economic and efficient cocatalyst for photocatalytic hydrogen evolution.Ternary g-C


Skint1, a butyrophilin-like (Btnl) gene expressed specifically by thymic epithelial cells and suprabasal keratinocytes. However, the generality of this mechanism has remained opaque, since neither Skint1 nor DETCs are evolutionarily conserved. Here, Btnl1 expressed by murine enterocytes is shown to shape the local TCR-Vγ7+ γδ compartment. Uninfluenced by microbial or food antigens, this activity evokes the developmental selection of TCRαβ+ repertoires. Indeed, Btnl1 and Btnl6 jointly induce TCR-dependent responses specifically in intestinal Vγ7+ cells. Likewise, human gut epithelial cells express BTNL3 and BTNL8 that jointly induce selective TCR-dependent responses of human colonic Vγ4+ cells. Hence, a conserved mechanism emerges whereby epithelia use organ-specific BTNL/Btnl genes to shape local T cell compartments.Many body surfaces harbor organ-specific γδ T cell compartments that contribute to tissue integrity. Thus, murine dendritic epidermal T cells (DETCs) uniquely expressing T cell receptor (TCR)-Vγ5 chains protect from cutaneous carcinogens. The DETC repertoire is shaped by   •Btnl1) shapes the local γδ T cell compartmentGut epithelial butyrophilin-like 1 (•Btnl genes select cognate γδ cells in other sitesOther organ-specific epithelial •Btnl heteromers can specifically activate γδ T cells with cognate T cell receptors•BTNL genes reveal a conserved biology of epithelial T cell regulationHuman  Epithelial cells provide signals that instruct the development and function of their local γδ T cell compartments so that these immune cells can support the non-immune functions of the different barrier tissues. Likewise, differentiated epithelia provide physical and chemical barriers to pathogens and toxins    limit in (DETCs) . Moreoveed roles .+ IELs are largely unaffected by major histocompatibility complex (MHC) antigens that shape αβ T cell repertoires. Likewise, whereas some γδ TCRs bind CD1 and MR1, genetic studies have largely excluded these MHC-related molecules from the selection of murine γδ cell repertoires  genes, of which mice have two and humans six. Butyrophilin genes derive their name (“butter-loving”) from the founding member, Btn1a1/BTN1A1, that encodes a milk fat micelle-associated protein , although it is not known whether this is mediated by direct TCR-BTN3A1 binding , TIGIT (an inhibitory co-receptor), and the TCR (detected with anti-CD3 antibodies) and low levels of RNA for Rorgc and Sox13, two transcription factors contributing to γδ T cell differentiation (− IELs (mostly Vγ1 or Vγ4) did not show this phenotype, and whereas both Vγ7+ and Vγ7− IEL subsets were mostly CD45RBhi, CD44+, and CCR9+, Vγ7+ IELs were distinct in being Lag3+, Thy1−, CD69+, CD5−, and CD8αα+  from putative Vγ7+ IEL progenitors   B, with telection .hi Vγ7+ and CD122lo Vγ7+ IELs were purified from the same day 14–17 mice on four independent occasions and assessed by RNA sequencing (RNA-seq)  or downregulated  in CD122hi versus CD122lo Vγ7+ cells were likewise regulated by Skint1 selection of DETC progenitors  C. ConsisRNA-seq) C. Furthegenitors F and S1Chi Vγ7+ cells were enriched in cell-cycle genes, consistent with which ∼100% of Vγ7+ IELs at day 21–24 were Ki67+ , compared to <40% of Vγ7− cells (p < 0.0001) (+ IELs at day 28 phenocopied rapidly dividing thymocytes in that ∼10% incorporated ethynyldeoxyuridine (EdU) (a labeled nucleotide) during a 3-hr pulse, compared to only 4% of Vγ7− IELs  G. Likewiγ7− IELs D.hi, Thy1−, TIGIT+, Lag3+, CD8αα+, CD5−, CD24−, TCRhi Vγ7+ cells that by weeks 3–4 dominate the γδ IEL compartment. After week 5, the fraction of cycling (Ki67+) Vγ7+ IELs at steady state declined to levels comparable to Vγ7− IELs , the compartment was again dominated by CD122hi Vγ7+ IELs. Moreover, ∼25% of Vγ7+ IELs in NU/NU and in euthymic mice reacted with antibody GL2 that detects Vδ4 (TRDV2-2 encoded) chains. Consistent with this, TRDV2-2 sequences accounted for ∼25% of TCRδ chain RNAs expressed by purified Vγ7+ IELs  mice  mice  that folia) mice F.+ and Vγ7GL2+ IEL compartments comparable to conventionally housed counterparts  of IEL maturation in weanling mice, microbial and/or food antigens were logical candidates. However, C57Bl/6 mice bred into and maintained in a germ-free environment and/or on elemental, protein-antigen-free diet displayed Vγ7terparts B. The Vγree mice B. Thus, Btnl1, Btnl4, and Btnl6, that are closely related to Skint1 , and a strain with an internally deleted Btnl1 locus generated by clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 in mouse eggs (Btnl RNAs (In seeking that element(s), we focused on three genes, o Skint1 . Btnl4 wabilized C. Expresabilized G. Btnl1 ntestine H, where e thymus I. These use eggs J. The sttnl RNAs I–S2K.Btnl1−/− strains each displayed major, highly selective losses of Vγ7+ IELs, assessed by flow cytometry or confocal microscopy  mice, but this was set against a background of dramatically reduced γδ IEL numbers  and comparable representation and phenotypes of Vγ7+ thymocytes from day 4 to week 8  transfer to irradiated 8- to 10-week, γδ T cell-deficient TCRδ−/− mice -inducible promoter  regulated by the ubiquitously expressed Rosa26 promoter. As reported, adding low-dose Dox to drinking water had little overt impact on the gut over a several-week period (Btnl1 de novo in Btnl1−/− bitransgenic (BiTg) mice that inherited both rtTA and the inducible Btnl1 transgene. Conversely, BiTg mice administered sugar water and Dox-treated single-transgenic (SiTg) Btnl1−/− mice (that inherited only the Btnl1 transgene) served as controls. Btnl1 induction in mice of appropriate genotypes was validated by qPCR .When several W11 Rβ+ IELs D–S5F. ThBtnl1 induction to mature enterocytes by generating Btnl1−/− mice transgenic for rtTA expressed from the villin promoter. Within 1–2 weeks of Dox-treatment of several W11 BiTg Btnl1−/− mice, most Vγ7+ IELs had become Lag3hi, Thy1−, and CD122hi, of which the majority were also Ki67+  was greatly enhanced by co-transfection with Btnl4 or Btnl6 , Btnl6 (L6), Btnl1 plus Btnl6 (L1+6), or empty vector (EV) were co-cultured with freshly explanted IELs that were then assayed for CD25 (IL-2Rα chain) upregulation, which is among the most robust readouts of TCR stimulation for systemic T cells  (MODE-K cells stably transduced with  T cells . Whereas− cells) A. Signif− cells)   B.gfp mice in which GFP is upregulated by nuclear factor of activated T cells (NFAT) activation downstream of TCR signaling , CCL4, and granulocyte macrophage colony-stimulating factor (GM-CSF) among 36 cytokines tested H. These +Vδ2+ cells that dominate the peripheral blood  , Vγ4 was− cells . Indeed,1+ cells A.Btnl2-proximal amplicon on mouse chromosome 17 that encodes Btnl1, Btnl4, and Btnl6 , BTNL8 (L8), BTNL3 and BTNL8 (L3+8), or EV  cells showed marked TCR downregulation when co-cultured with L3+8 cells , or EV F. For th2− cells D. Althou2− cells E, and CD2− cells G.− cells responded to L3+8  were biased toward Vγ3  might have a broad role in γδ cell regulation by Btnl/BTNL genes ; Btnl1 can directly appose IEL at steady state Mbp) and e.s. cells for Btnl4−/− (Btnl4tm1(KOMP)Mbp) mice were obtained from the international mouse phenotyping consortium (IMPC) (project IDs: CSD67994 and CSD81524). Btnl1indel/indel mice were generated using CrisprCas Technology. Briefly: Two independent short guide RNAs, targeting the intronic region: between exon1 and 2 and between exon 5 and 6 were identified using the online tool: http://crispr.mit.edu/.Wild-type (WT) C57Bl/6 mice were obtained from Charles River and Harlan. 3 independently derived embryonic stem (e.s.) cells for Intron1/2: CCAGCTCCAAGATCCCCCTTGGG Intron5/6: TCCATAGCACCTTATCCGGTTGGThe sg RNAs & PAM sequences were cloned into the g-RNA basic vector, translated in vitro, purified and co-injected with Cas9 into day 1 zygotes and transferred into pseudopregnant foster mice.Btnl-knockout lines were generated and maintained at The Francis Crick Institute’s Biological resource facilities. B6.Cg-Foxn1 < Nu > /J (NU/NU) mice were obtained from the Jackson Laboratory. Nur77.gfp mice were kindly provided by K. Hogquist  mice were kept in plastic isolators  with autoclaved food, bedding and water. Sterility of animals was checked bi-weekly by culturing faeces in thioglycollate medium under aerobic and anaerobic conditions for at least ten days. All handling procedures for GF mice were conducted in a laminar flow hood under sterile conditions. All experiments were conducted according to the German animal protection law.Food antigen-free (FAF) mice were raised on an amino acid-containing diet for up to five generations. Pellets of FAF diet  contained all essential vitamins, minerals, trace elements, fat, dextrin, sucrose and free amino acids equimolar to the protein content of normal rodent chow .Btnl1-Tg mice were generated by injection of Btnl1−/− blastocysts with a linearized cassette containing a TRE/CMV-promoter upstream of the Btnl1-ORF. The TRE/CMV cassette has been previously described  or local (Villin) induction of Btnl1 transgene expression by doxycycline administered to drinking water . Animal experiments were undertaken in full compliance with UK Home Office regulations and under a project license to A.H. (80/2480).Doxycycline (Dox)-inducible escribed . R26-rtTescribed  or Villiescribed  mice werAntibodies for mouse: CD3 APC Cy7 (17A2); CD3 PerCPCy5.5 (145-2C11); TCRβ Brilliant Violet 421 (H57-597); TCRβ APC (H57-597); CD122 PE (TMβ1); CD122 Brilliant Violet 421 (TMβ1); CD122 APC (TMβ1); TIGIT PE (GIGD7); CD45RB APC Cy7 (C363-16A); Thy1.2 Brilliant Violet 510 (53-2.1); Lag3 PerCP-efluor 710 (C9B7W); CD5 PE (53-7.3); CD24 FITC (M1/69); CD24 PECy7 (M1/69); CD8α PECy7 (53-6.7); CD8α PECy7 (53-6.7); TCR Vδ4 FITC (GL-2); TCR Vδ4 PE (GL-2); CD8β PerCpCy5.5 (YTS156.7.7); CD25 PerCpCy5.5 (PC61); CD69 PECy7 (H1.2F3); CCR9 PECy7 (CW-1.2); CD44 PECy7 (IM7); TCRVγ7 (F2.67) was provided by Pablo Pereira ; TCRVγ1 APC (2.11); TCRVγ4 APC (UC3-10A6); TCRδ BV421 (GL3); Ki67 FITC (B56/MOPC-21); CD45 Qdot 605 (30-F11); CD5 Brilliant Violet 510 (53-7.3); TCRδ PeCy7 (GL3); CD161/NK1.1 Brilliant Violet 650 (PK136); CD4 Brilliant Violet 786 (GK1.5); CD8α AlexaFluor 700 (53-6.7); CD25 APC (PC61); GITR PE (DTA-1); CD44 FITC (IM7); CD62L PerCP-Cy5.5 (MEL-14); KLRG1 BV421 (2F1); CD11c BV786 (HL3); CD11b BV510 (M1/70); F4/80 PerCPCy5.5 (BM8); Ly6G APC (1A8); Ly6C AlexaFluor 700 (AL-21); CD103 PE (M290); CD317 Brilliant Violet 650 (927); MHCII/IA/IE FITC (2G9); CD86 Pe-Cy7 (GL1); CD3 Brilliant Violet 421 (145-2C11); CD19 Brilliant Violet 421 (1D3); CD161/NK1.1 (lin) Brilliant Violet 421 (PK136); IgG1 PE (A85-1); B220 (CD45R) AlexaFluor 700 (RA3-6B2); IgM Brilliant Violet 786 (R6-60.2); IgD PerCPCy5.5 (11-26c.2a); GL-7 AlexaFluor 647 (GL7); CD95 PECy7 (Jo2); CD138 Brilliant Violet 650 (281-2); CD21/35 FITC (7G6); CD23 Brilliant Violet 421 (B-ly6). Antibodies for human: CD25 Brilliant Violet 421 (BC96); CD25 PE (BC96); CD3 Brilliant Violet 510 (OKT3); CD3 BUV (UCHT 1); EpCAM eFlour 660 (1B7); Streptavidin APC-Cy7; Streptavidin Brilliant Violet 421; TCRγδ PeCy7 (IMMU510); Vγ9 PC5 (IMMU360); Vγ9 PE (B3); Vδ1 APC (REA173); Vδ2 PerCP (B6); Vγ2/3/4 biotin (23D12), Vγ3/5 biotin (56.3) and Vγ8 biotin (R4.5.1) were provided by D. Kabelitz and D. Wesch (University of Kiel). Other antibodies: DYKDDDDK-PE (Flag); DYKDDDDK-APC (Flag); HA-DyLight 650; 6x-Histidine-PE.Flow cytometry was performed using the following antibodies, coupled to the indicated fluorochromes (see key resources table). Commercial antibodies were purchased from Biolegend, eBioscience, BD-Bioscience, Thermo Fisher Scientific or Miltenyi (see key resource Information). Viability dyes (near IR or Blue) were from Invitrogen. Anti TCRVγ7 (F2.67) was purified from hybridoma supernatant using the mouse TCS purification system (Abcam) and conjugated to biotin or AF647 (see key resources table).Ki-67 staining was performed on cells fixed and permeabilised using the Foxp3 staining buffer set (eBioscience). BrdU (Sigma-Aldrich) and EdU incorporation was assessed 3h post-intraperitoneal injection (50mg/kg) by immunohistochemistry or by flow cytometry , respectively.Anti-TCRVγ7  was purified from hybridoma supernatant using the mouse TCS purification system (abcam-ab128749). Purified anti-TCRVγ7 was conjugated to biotin  or to AF647 . Anti-human Vγ2/3/4 , Vγ5/3  and Vγ8  were provided by D. Kabelitz & D.Wesch .Flow cytometry data analysis was performed on FlowJo (Version 9.9).R) was constructed by replacing the PuromycinR/mIR cassette from the pAPM vector , BTNL8S (GenBank:NM_024850), and BTNL8 (GenBank:NM_001040462) were cloned from Caco-2 cells by conventional RT-PCR, using the following primers (See BTNL3 For 5′-GAATATCCATGGCTTTTGTGC-3′BTNL3 Rev 5′-GTCTTCTCTGTCTCATCCCC-3′BTNL8 For 5′-CCATTCACAGAACACATCCATG-3′BTNL8S Rev 5′-TATGGGTTACAGTTTTCAGATCAG-3′BTNL8 Rev 5′-GTGGGATGTGATTCATCCTAC-3′cDNAs were (sub-)cloned into pCSIGPW or variant vectors see . Btnl1, escribed . BTNL3  using the following primers See :Vγ2/3/4 BTNL3 and BTNL8 was checked by conventional RT-PCR using the primers indicated above. BTN3A1, BTN3A2, EPCAM and GAPDH were used as control genes  (env) . Btnl/BTNL expression was checked 48h post-transfection. Lentiviral particles were produced in HEK293T cells by co-transfection of pCSIGPW or pCSIGHW either empty or containing gag/pol) , and pHIl) (env) . TransduSamples were stored in RNAlater (Ambion) or directly frozen in RLT buffer prior to RNA purification (QIAGEN RNeasy kit). cDNA was generated using Superscript-II (Invitrogen) and analyzed using Sybr-green assay (Invitrogen) using a ViiA7 Real-time PCR machine (Applied Biosystems) See .Btnl1 For: 5′-TGACCAGGAGAAATCGAAGG-3′Btnl1 Rev: 5′-CACCGAGCAGGACCAATAGT-3′Btnl4 For: 5′-CATTCTCCTCAGAGACCCACACTA-3′Btnl4 Rev: 5′-GAGAGGCCTGAGGGAAGAA-3′Bntl6 For: 5′-GCACCTCTCTGGTGAAGGAG-3′Btnl6 Rev: 5′-ACCGTCTTCTGGACCTTTGA-3′β-Actin For: 5′-CAGCTTCTTTGCAGCTCCTT-3′β-Actin Rev: 5′-CACGATGGAGGGGAATACAG-3′Sox-13 For: 5′-CTCCAGGCCTTCCCAGAC-3′Sox-13 Rev: 5′-CATGGACTTCCAGCGAGAAC-3′Rorγc For: 5′-GGTGACCAGCTACCAGAGGA-3′Rorγc Rev: 5′-CCACATACTGAATGGCCTCA-3′Tbp For: 5′-GGGGAGCTGTGATGTGAAGT-3′Tbp Rev: 5′-CCAGGAAATAATTCTGGCTCA-3′CycloFor: 5′-CAAATGCTGGACCAAACACAA-3′Cyclo Rev: 5′-CCATCCAGCCATTCAGTCTTG-3′Btnl1 For: 5′-ACTGGCTTCCTCAGAGTCAT-3′Btnl1 Rev: 5′-CAGTAGTGAATGGCCCCTGA-3′Btnl4 For: 5′-GACCAACGCTTCCCTACCTC-3′Btnl4 Rev: 5′-GCCTTGGGTCCAACAAGACA-3′Btnl1-Tg-Ex3-For: 5′-GGTTTTCTGTGAAGGGACCA-3′Btnl1-Tg-Ex4-Rev: 5′-GGTCTGCAACTCAGAGGAGG-3′Southern blots were performed with probes generated using a Dig-Probe labeling kit; blots were hybridized in DIG-Easy-hyb buffer overnight, and developed using the DIG-Luminescence Detection Kit (Sigma-Aldrich). For probe sequences see Supplementary Information. DIG labeled probes for Southern blotting were generated using the following primers See :Btnl1 FoBtnl1, GenBank:NM_001111094.1 (576-1723); Btnl4, GenBank:NM030746.1 (560-968); Btnl6, GenBank:NM_030747.1 (245-1552) (See RNAscope was performed on paraffin embedded sections using probes and kits obtained from Advanced Cell Diagnostics using the RNAscope 2.0 HD Reagent Kit-BROWN. Reference sequences are as follows: 552) See .IEL were isolated from mouse small intestine as previously described . Small iwww.immunophenotyping.org). In brief, Spleen and MLN were digested with collagenase (1mg/ml)/DNAse (0.1 mg/ml) in 2% FCS PBS (+ Ca/Mg) for 20 minutes at 37°C and filtered through 30μm cell strainers. Cells were plated on 96 well V-bottom plates, washed in PBS and stained with Zombie Near-IR (Biolegend) for live/dead discrimination. Antibody stains were performed at 4°C for 20mins. Full details regarding phenotyping panels are included in Comprehensive immunophenotyping of Btnl1-/- mice was performed using a platform developed by the Wellcome Trust Infection and Immunity Immunophenotyping (3i) consortium (5 MODE-K were seeded in 48-well plates 24h prior to the addition of 105 unsorted or (where indicated) positively FACS-sorted (CD45+Vγ7+) IEL and incubated for 16-18h in 10% CO2 unless indicated otherwise. For transwell assays, 2x105 MODE-K cells were seeded onto 24-well transwell plates (3 μm pore size - Corning) 24h prior to the addition of 3x105 IEL, either in direct contact (below), sequestered from (above), or split 50:50 with MODE-K cells (above and below the transwell).Cells were co-cultured in RPMI 1640 supplemented with 10% FCS, Pen/Strep, 2.5% HEPES, 1% Glutamine, 1% non-essential amino acids, 1% sodium pyruvate, 0.2% β-mercapto-ethanol (GIBCO) and cytokines including IL-2 (10U/ml), IL-15 (10 ng/ml) (Immunotools), IL-3 (100U/ml), IL-4 (200U/ml) (R&D). 102 prior to analysis.96-well U bottom plates were coated overnight with 10 μg/ml LEAF-Purified anti-mouse CD3-ε or Hamster IgG Isotype control (Biolegend) at 4°C and washed once with PBS 1x before seeding IEL. 100,000 IEL were seeded per well. Cells were incubated at 37°C for 16-18h in 10% COProximal small intestine (SI) samples were fixed in Zamboni’s fixative, blocked with normal goat serum and stained with antibodies against TCRβ, TCRδ, TCRVδ4 (encoded by TRDV2-2) (GL2), CD3 and Vγ7. Z-Sections were acquired on a confocal-LSM-710 microscope (Zeiss) and processed and analyzed using Imaris Software (Bitplane Scientific Solutions).6 donor bone marrow cells and analyzed 4-12 weeks later.10-12 week old recipient mice were irradiated with 950Rads 24h, injected (IV) with 5-10x10−/− and TCRδ−/−Btnl1−/− recipients. Analysis was performed 2-3 weeks later.IEL harvested from 4 week-old WT mice were column-purified using CD45 microbeads (MACS Miltenyi biotec) and IV-injected into 6 week-old TCRδ+CD122hi and Vγ7+CD122lo IEL were sorted from from pooled D14-17 pups directly into RLT buffer. RNA was prepared using the RNA-Micro-plus kit (QIAGEN). RNA libraries were generated using the KAPA Stranded RNA-seq Kit with RiboErase (HMR) (KAPA BIOSYSTEMS). Paired-end sequencing on HiSeq 2500 (illumina) using rapid run chemistry (read length: 100bp).Vγ7Endoscopic biopsies were obtained from the ascending colon of adult donors undergoing routine diagnostic colonoscopy after informed consent and in compliance with local ethical approval (REC number 07/H0803/237). Excess resected skin discarded at the time of cutaneous or reconstructive surgery was obtained from adult donors after informed consent and in compliance with local ethical approval (REC number 06/Q0704/18). This study was conducted adhering to the principles of the Declaration of Helsinki.Primary gut lymphocytes were obtained using an adaptation of the method of Kupper and Clarke  Figure . 9mm x 9PBMC were isolated by Ficoll gradient from blood obtained from the blood donation service.+ cells were sorted by flow cytometry directly into RLT lysis buffer. RNA and cDNA were prepared as described above.Colonic samples were incubated with 5 mM 1,4-dithiothreitol (Sigma), followed by enzymatic digestion with 1.5mg/ml collagenase VIII (Sigma) and 0.05 mg/mL DNase I (Sigma). EpCAM5 HEK293T cells, transduced with either empty vector (EV), BTNL3, BTNL8 or BTNL3+8 and 2x105 freshly harvested primary human lymphocytes were co-cultured in 96-well plates with complete medium (see Supplementary Information) without supplementary cytokine and incubated at 37°C at 5% CO2 for 16hrs .5x10+ IEL was performed using the Amp2Seq Platform (iRepertoire).Human TCRG Vγ gene: Amplification and sequencing of TCRγ CDR3 was performed using the immunoSEQ Platform (Adaptive Biotechnologies).Mouse TRDV gene: Amplification and sequencing of TCRδ CDR3 from RNA purified from sorted Vγ7ns > 0.05).Unless stated otherwise, bar/spider charts display mean ± SD and p values were derived from unpaired two tailed t tests, assuming equivalent SD  with a 40x oil objective . 3D image analysis on z-stacks was carried out using Imaris (Bitplane). The surfaces tool was used to identify CD3hi and CD122lo Vγ7+ IEL groups using DESeq2   with Bowg DESeq2  was idenGSE85422.The accession number for the RNA sequencing data reported in this paper is GEO: Conceptualization, A.H.; Methodology, A.H., A.J., L.D., R.D.M.B., P.V., R.J.D., O.N., U.S., and R.H.; Resources, D.G., P.M.I., P.P., and U.S.; Investigation, R.D.M.B., N.A.R., R.J.D., P.V., A.J., O.N., L.D., S.C., R.H., M.L.I., A.L., B.S.-D., P.E., and U.S.; Validation, R.J.D., L.D., and A.J.; Data Curation, R.D.M.B., B.S.-D., P.E., P.V., and A.H.; Writing – Original Draft, A.H.; Writing – Review & Editing, A.H., U.S., R.D.M.B., N.A.R., R.J.D., P.V., A.J., L.D., P.P., and S.C.; Visualization, R.D.M.B., N.A.R., R.J.D., A.J., R.H., B.S-D., P.V., and A.L.; Supervision, A.H.; Funding Acquisition, U.S. and A.H.


UBE2L3 polymorphisms.Caspase-1 activation by inflammasome signaling scaffolds initiates inflammation and antimicrobial responses. Caspase-1 proteolytically converts newly induced pro-interleukin 1 beta (IL-1β) into its mature form and directs its secretion, triggering pyroptosis and release of non-substrate alarmins such as interleukin 1 alpha (IL-1α) and HMGB1. While some caspase-1 substrates involved in these events are known, the identities and roles of non-proteolytic targets remain unknown. Here, we use unbiased proteomics to show that the UBE2L3 ubiquitin conjugase is an indirect target of caspase-1. Caspase-1, but not caspase-4, controls pyroptosis- and ubiquitin-independent proteasomal degradation of UBE2L3 upon canonical and non-canonical inflammasome activation by sterile danger signals and bacterial infection. Mechanistically, UBE2L3 acts post-translationally to promote K48-ubiquitylation and turnover of pro-IL-1β and dampen mature-IL-1β production. UBE2L3 depletion increases pro-IL-1β levels and mature-IL-1β secretion by inflammasomes. These findings regarding UBE2L3 as a molecular rheostat have implications for IL-1-driven pathology in hereditary fever syndromes and in autoinflammatory conditions associated with   •Caspase-1 inflammasomes induce loss of UBE2L3 in macrophages and dendritic cells•UBE2L3 loss is proteasome-dependent, ubiquitin- and pyroptosis-independent•UBE2L3 participates in K48 ubiquitylation and proteasomal turnover of pro-IL-1β•UBE2L3 modulates levels of pro-IL-1β available for processing by caspase-1 Eldridge et al. show that the UBE2L3 ubiquitin E2 conjugating enzyme is targeted for proteasomal degradation by inflammasomes and is therefore an indirect target of human and mouse caspase-1. They also find that UBE2L3 is a post-translational regulator of pro-IL-1β protein levels and thus regulates mature IL-1β production. Multi-molecular signaling scaffolds, called inflammasomes, control the cysteine protease caspase-1 . Caspase+ efflux, double-stranded DNA (dsDNA), and bacterial flagellin that activate of NLRP3, AIM2, and NLRC4, respectively  cause hereditary monogenic fever syndromes driven by excessive IL-1β or IL-18 , and HMGB1 release  left untreated or treated with nigericin to induce Kreatment A and S1Areatment B. Prolonreatment B. In ordreatment . Glycinereatment B. The en release C. Import release C. These  release B. UBE2L3 release D. Extracepletion D. Thus, epletion A–1D.Xenopus). We therefore asked whether hUBE2L3 was depleted by caspase-1. Indeed, PMA-differentiated THP-1 macrophage-like cells treated with LPS plus nigericin lost UBE2L3, and extracellular K+ prevented both caspase-1 activation and UBE2L3 loss , a bacterial pathogen that can strongly activate NLRP3 via its listeriolysin toxin, AIM2, by release of genomic DNA, and weakly activate the NLRC4 pathway because of downregulation of flagellin expression (Lm infection resulted in robust caspase-1-dependent UBE2L3 depletion in mouse (Lm (LmΔhlyA), which does not escape from vacuoles, does not stimulate caspase-1 activation or UBE2L3 depletion  resulted in f UBE2L3 B. AIM2-dits loss C. Based pression , should in mouse C and humin mouse D cells. epletion D. Thus, epletion E and S2FSalmonella enterica Typhimurium (STm) that selectively activates NLRC4 when grown under conditions that induce high expression of the Salmonella Pathogenicity Island-1 (SPI-1) type 3 secretion system (T3SS)  bacteria . We testm (T3SS) . While m protein G; the STe UBE2L3 G. Importll lysis H. Nlrc4− protein D. Remark/− cells D. This c/− cells D. Asc−/−p10 form D, but thp10 form . TherefoE. coli LPS along with cholera toxin B (CTB) that delivers LPS into the cytosol. UBE2L3 depleted in wild-type iBMDMs treated with CTB+LPS, but its levels remained unchanged in Casp4−/− and Casp1/4−/− cells  and Il18r1−/− macrophages  depletion of UBE2D3 (UBCH5C) and UBE2N (UBC13) in mouse embryonic fibroblasts . We therrophages A. This r/− cells C. This etivators  or in retivators C. Taken Atg7 to block autophagy and used MG132 or epoxomicin to inhibit distinct protease activities of the proteasome. Atg7 silencing did not prevent loss of UBE2L3, ruling out a role for autophagy  in Gsdmd−/− iBMDMs and assessed their depletion by caspase-1. Remarkably, both wild-type and UBE2L318R proteins were degraded similarly upon caspase-1 activation , THP1YFP-UBE2L3 cells showed diminished LPS-induced pro-IL-1β . Interestingly, UBE2L3 silencing led to ∼6-fold higher levels of LPS-induced pro-IL-1β protein as compared to control cells . In contrast, pathogenic STm and Lm stimulated robust caspase-1 activity and UBE2L3 depletion. Thus, during weak or absent caspase-1 activation, sustained UBE2L3 expression promoted pro-IL-1β turnover  was also proteasome-dependent. Interestingly, ubiquitin-independent degradation has been reported for several common proteins such as IκBε  in ignaling . NF-κB rignaling . SNP genignaling . Our resignaling , UBE2L3 ignaling , and embIL-1β is an endogenous pyrogen and has potent inflammatory properties . TherefoSTm ATCC 14028s and ΔprgH  were grown to induce high SPI-1 expression. Briefly, standing cultures of STm were grown overnight at 37°C in LB containing 300 mM NaCl, re-inoculated into fresh LB-NaCl (1:60 dilution), and incubated in a shaker until OD600 ∼0.9–1.2. Bacteria were washed two times in DMEM before use. High SPI-1 expressing STm were tested to only activate NLRC4 inflammasomes in murine iBMDMs. Lm 10403s and ΔhlyA  were grown overnight in BHI medium in a shaker at 37°C and washed three times in DMEM before use. Infections were synchronized by centrifuging bacteria on macrophages at 750 × g for 10 min. Gentamycin (100 μg/mL) was added 30 min (STm) or 60 min (Lm) post-infection, and cells were incubated further for 2–4 hr. Treatments for immunoblotting were performed in OptiMEM plus sodium pyruvate, followed by precipitation with 4 vol of acetone for 16–20 hr at −20°C. Cell lysates were prepared in 2× Laemmli loading buffer supplemented with complete protease inhibitor and phosphatase inhibitor tablets (Thermo Fisher Scientific), 1 mM PMSF, 10 μM MG132, 10 μM PR619, 10 mM EDTA, and 5% 2-mercaptoethanol. Proteins were separated by SDS-PAGE using Tris-Tricine (for caspase-1 p10) or Tris-Glycine buffer systems and transferred to PVDF membranes (Bio-Rad Laboratories). Additional details are provided in the iBMDMs were grown in DMEM plus penicillin and streptomycin (PS), 10% heat-inactivated fetal bovine serum (HI-FBS), and 20% L929-spent medium. THP1 cells were maintained in RPMI plus PS, HEPES, sodium pyruvate, and 10% HI-FBS, and differentiated with 100 ng/mL phorbol 12-myristate 13-acetate (PMA) for 3 days. Cells were primed with ultrapure O111:B4 LPS  or PAM3CSK4 , followed by treatment with ATP (5 mM) or nigericin (20 μM) for 60 min or times as indicated. Cells were transfected for 5 hr with poly(dA:dT) (5 μg/mL) or LPS (5 μg/mL) using Lipofectamine 2000. CTB (20 μg/mL) was used along with 5–10 μg/mL LPS for 5 hr. MG132 (20 μM) was added 5 min after ATP or nigericin, or at times as indicated. Recombinant, cell permeable C3 Exoenzyme was used to activate Pyrin and a mixture of anthrax lethal factor and activated protective antigen to activate NLRP1. All experiments were repeated at least twice. Immunoblot quantification used images acquired on a Chemidoc MP (Bio-Rad), analyzed using Image Lab software (Bio-Rad Laboratories). For ELISA, qRT-PCR, LDH release, and PI uptake assays, two to three technical replicates were used to estimate mean from one experiment. Means from two or more independent experiments were analyzed by two-tailed Student’s t test or two-way ANOVA and the Benjamini-Hochberg (BH) false-discovery rate was used to account for multiple comparisons . Means tConceptualization, A.R.S.; Investigation, M.J.G.E., J.S.-G., P.J.G., G.F.H., and A.R.S.; Validation, M.J.G.E., J.S.-G., and P.J.G.; Visualization, M.J.G.E., J.S.-G., and A.R.S.; Writing – Original Draft, A.R.S.; Writing – Review & Editing, M.J.G.E. and A.R.S.; Supervision, A.R.S.; Funding Acquisition, A.R.S.


The structure of title co-crystal consists of a 2,3,5,6-tetrakis(pyridin-2-yl)pyrazine coformer and hydrogen peroxide solvent molecules in a ratio of 1:4.75. 24H16N6·4.75H2O2, consists of a 2,3,5,6-tetrakis(pyridin-2-yl)pyrazine coformer and hydrogen peroxide solvent molecules in an overall ratio of 1:4.75. Three of the six H2O2 molecules modelled in the structure were found to be cross-orientationally disordered over two positions with occupancy ratios 0.846 (9):0.154 (9), 0.75 (2):0.25 (2), and 0.891 (9):0.109 (9). In the crystal, all of the peroxide molecules are linked into hydrogen-bonded chains that propagate parallel to the a axis. These chains are further linked by O—H⋯N hydrogen bonds to the pyridine groups of the main molecule.The structure of the title co-crystal, C Nowadays, some peroxosolvates find widespread use as environmentally friendly decontaminating and bleaching compounds pyrazine coformer and six crystallographically independent peroxide molecules Fig. 1, namely ns Fig. 2. This tyet al., 1992In the organic molecule, all four pyridin-2-yl substituents are significantly inclined with respect to the central pyrazine ring Fig. 3, such thIn the peroxide molecules, the O—O distances range between 1.44 (4) and 1.485 (5) Å. The mean value of 1.465 Å is close to those previously observed in the accurately determined structures of crystalline hydrogen peroxide  mode Fig. 4. This coa-axis A1 motif n⋯Hal−⋯(H2O2)k⋯  by Schumb  al. 1955.2O2 molecules were found to be cross-orientationally disordered over two positions with occupancy ratios 0.846 (9):0.154 (9), 0.75 (2):0.25 (2), and 0.891 (9):0.109 (9), and were refined with restrained O—O distances.Crystal data, data collection and structure refinement details are summarized in Table 2i/H61i  was found to be partially occupied. Simultaneous refinement of occupancy and thermal parameters for atom O61 was not stable and resulted in oscillating occupancies between 0.46 and 0.53 for consecutive cycles of refinement. It was therefore fixed at 0.5 for the final refinement.The centrosymmetric peroxide molecule modelled as H61/O61/O61Uiso(H) = 1.2Ueq(C). Peroxide hydrogen atoms were placed on the lines connecting hydrogen-bonded atoms at a distance of 0.80 Å from the corresponding O atoms. They were refined as riding atoms with relative isotropic displacement parameters Uiso(H) = 1.5Ueq(O).Aromatic H atoms were placed in calculated positions with C—H = 0.95 Å and refined as riding atoms with relative isotropic displacement parameters 10.1107/S2056989017015328/pk2607sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989017015328/pk2607Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989017015328/pk2607Isup3.cmlSupporting information file. DOI: 1581165CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


However, the molecular mechanisms underlying altered RyR-mediated intracellular Ca2+ release in AD remain to be fully elucidated. We report here that RyR2 undergoes post-translational modifications  in SH-SY5Y neuroblastoma cells expressing the β-amyloid precursor protein (βAPP) harboring the familial double Swedish mutations (APPswe). RyR2 macromolecular complex remodeling, characterized by depletion of the regulatory protein calstabin2, resulted in increased cytosolic Ca2+ levels and mitochondrial oxidative stress. We also report a functional interplay between amyloid β (Aβ), β-adrenergic signaling, and altered Ca2+ signaling via leaky RyR2 channels. Thus, post-translational modifications of RyR occur downstream of Aβ through a β2-adrenergic signaling cascade that activates PKA. RyR2 remodeling in turn enhances βAPP processing. Importantly, pharmacological stabilization of the binding of calstabin2 to RyR2 channels, which prevents Ca2+ leakage, or blocking the β2-adrenergic signaling cascade reduced βAPP processing and the production of Aβ in APPswe-expressing SH-SY5Y cells. We conclude that targeting RyR-mediated Ca2+ leakage may be a therapeutic approach to treat AD.Alteration of ryanodine receptor (RyR)-mediated calcium (Ca Alzheimer disease (AD)2+ homeostasis deregulation in AD pathological process  (––2+ release likely contributes to ER Ca2+ deregulation in AD (Data are now converging to suggest an important contribution of endoplasmic reticulum (ER) Ca process , 9. Thise (IP3R) –12, and  (IP3R) ––16. Impo2+ leak in AD are still not fully understood. RyR macromolecular complexes include four RyR protomers (565 kDa each) and several regulatory proteins including the RyR channel-stabilizing subunit calstabin. Recent studies have revealed that RyR-mediated ER Ca2+ depletion is linked to post-translational modifications  of the RyR macromolecular complexes resulting in calstabin depletion and “leaky RyR channels”  have been implicated in the development of AD both in humans and in AD animal models –23. Howe–in vitro AD study model. We used human SH-SY5Y neuroblastoma cells stably expressing pcDNA3.1 (control) or human βAPP harboring the double Swedish mutations (APPswe: APPKM670/671NL) constructs. We previously reported that SH-SY5Y cells expressing APPswe yield increased levels of APP C-terminal fragments (CTFs) fragments (C99 and C83) and of Aβ peptides . RyR2 macromolecular complexes from APPswe-expressing cells also exhibited reduced levels of serine/threonine protein phosphatase 1 (PP1) and spinophilin  (26–A–D) but significantly prevented dissociation of calstabin2 from RyR2 channels (A and E). S107 did not restore PP1 and spinophilin levels in RyR2 complexes . PKA phosphorylation of RyR2 occurs downstream of β2-AR activation  individually caused a ∼2-fold and, in combination, caused an ∼4-fold decrease in the binding affinity of calstabin2 to neuronal RyR2 (–A–E) without affecting PP1 and spinophilin levels on RyR2 complex .RyR2 was immunoprecipitated and immunoblotted for protein kinase A (PKA) phosphorylation (at residue Ser-2808), oxidation ), nitrosylation (anti-Cys NO), and levels of the channel stabilizing subunit calstabin2 (FKBP12.6) in the RyR2 macromolecular complex. Neuronal RyR2 from control SH-SY5Y cells had no biochemical remodeling of the RyR2 macromolecular complex, whereas APPswe-expressing cells exhibited RyR2 PKA phosphorylation, oxidation, nitrosylation, and calstabin2 depletion , A–E. Ry, and G) , 25. We , and G) , 26–28. nd G) 26–, A–D butchannels , A and Etivation , 25. In nal RyR2 . Becausenal RyR2 –23, we eal RyR2 –, A–E witA and B), the PKA inhibitor (H-89) (C and D), or the β1-AR antagonist CGP 20712A (E and F). We show that complete blockade of RyR2 phosphorylation and calstabin2 dissociation from RyR2 was obtained with ICI at 10 nm, 0.1 μm, and 1 μm (A and B). Accordingly, we also observed a dose-dependent blockade of RyR2 post-translational remodeling in APPswe cells with H-89 with a maximal effect at 1 μm and 5 μm (C and D). Importantly, treatment of APPswe cells with the β1-AR antagonist CGP 20712A up to 1 μm did not modify either RyR2 phosphorylation or calstabin2 binding to RyR2 (E and F). Although H-89 does not behave as a fully specific inhibitor of PKA (A–F).We further performed several tests to ascertain that RyR2 post-translational remodeling occurs through β2-AR in our study model. Indeed, SH-SY5Y cells express both β1- and β2-AR but not β3-AR . APPswe r (H-89) , C and DP 20712A , E and Fand 1 μm , A and Band 5 μm , C and D to RyR2 , E and Fr of PKA , it suppr of PKA , A–F.I and Ref. m, 30–60 min) resulted in RyR2 PKA phosphorylation, oxidation, nitrosylation, and depletion of calstabin2 from the RyR2 macromolecular complex (A–E). However, in contrast to SH-SY5Y-APPswe cells, the amounts of PP1 and spinophilin in the RyR2 complex were unaffected by acute treatment with Aβ . Pretreatment with S107 reduced Aβ-induced calstabin2 depletion from the RyR2 macromolecular complex (A and E). We also show that treatment of SH-SY5Y control cells with increasing concentrations of the β-AR agonist isoproterenol induces RyR2 phosphorylation associated with a dissociation of calstabin2 from RyR2 (G and H). ICI inhibited Aβ-induced RyR2 remodeling, thus preventing RyR2 PKA phosphorylation, oxidation, nitrosylation, and calstabin2 depletion (A–E). Neither S107 nor ICI affected PP1 and spinophilin levels in the RyR2 complex. Acute Aβ exposure increased cAMP levels (14.2 ± 0.2 versus 8.0 ± 0.3 pmol/mg), which was inhibited by ICI (7.9 ± 0.1 pmol/mg) , suggesting that Aβ acts upstream of β2-AR signaling. These data and those of A and H) and to β2-AR activation (A–H). Because our Aβ preparation contains monomeric and low molecular weight oligomeric forms of Aβ, we cannot attribute the observed effects to a specific Aβ species. We could detect only a moderate elevation of cAMP levels in APPswe model , but data were obtained at only one time point. Thus, other experiments will be necessary to unravel time-dependent modulation of cAMP production in the APPswe model.We then sought to determine the potential influence of Aβ or other APP metabolites on the RyR2 macromolecular complex remodeling. We used an Aβ preparation containing soluble Aβ oligomers (considered as the most toxic forms of Aβ)  or γ-secretase  inhibitors and investigate the potential implication of other βAPP metabolites in RyR2 remodeling.We also used a pharmacological approach to modulate APP metabolism by using β-secretase . As expected, β- and γ-secretase inhibitors fully block intracellular and extracellular Aβ production (L–O). Interestingly, blocking β-secretase or γ-secretase reduces RyR2 PKA phosphorylation, oxidation, nitrosylation, and calstabin2 depletion in APPswe-expressing cells (P–T).Although inhibition of β-secretase reduces C99 fragment production and enhances the level of C83 fragment, γ-secretase inhibition enhances the levels of both C99 and C83 fragments , J and Koduction , L–O. Inng cells , P–T.2+ due to increased Ca2+ release from the ER via IP3R and RyR and enhanced Ca2+ entry via voltage-dependent and voltage-independent plasma membrane Ca2+ channels . Accordingly, exogenous Aβ treatment of SH-SY5Y cells for 12 h  also results in elevated basal [Ca2+]cyt (C and D). As previously reported (2+]cyt (E and F). Interestingly, Aβ-induced Ca2+ elevation was prevented by S107, ICI, or ryanodine pretreatment (ΔF/F0 = 1.95 ± 0.16 (n = 57), 0.63 ± 0.08 (n = 31), 0.28 ± 0.08 (n = 44), 0.63 ± 0.09 (n = 23) in Aβ, Aβ+S107-, Aβ+ICI-, and Aβ+ryanodine-treated cells, respectively) (E and F). Our finding is in accordance with data showing that preincubation with inhibitory ryanodine virtually eliminated the generation of Ca2+ signals elicited by oligomeric Aβ in primary hippocampal neurons  , E and F neurons .2+]cyt. This may indicate that, in addition to leaky RyR2, there are other components that contribute to the Aβ-induced rise in [Ca2+]cyt. Possibilities include: 1) intracellular Ca2+ release via the IP3Rs and 2) Ca2+ entry through the plasma membrane as previously reported .S107, ICI, and ryanodine did not completely reverse the rise in [Careported . Accordi2+]cyt in APPswe cells untreated or treated with S107 or ICI (A and B). We show that S107 or blocking β2-adrenergic signaling by ICI significantly reduce basal [Ca2+]cyt in APPswe-expressing cells (A and B).We also analyzed basal cyt . All over, these data demonstrate that cytosolic Ca2+ increase in APPswe cells and in Aβ-treated cells is largely contributed by RyR-mediated Ca2+ release.It is known that IPand RyRs . Accordi the RyR . We shown APPswe , G–J conn by ICI , G0-J, bresponse , G–J. SiCa2+]cyt  (supplemi.e. APPswe cells) and exogenous application of Aβ (i.e. treatment of wild-type SH-SY5Y cells with Aβ for 12 h) resulted in increased mitochondrial reactive oxygen species (ROS) production (K and L). Interestingly, mitochondrial ROS production stimulated by chronic Aβ production in SH-SY5Y-APPswe cells was partially inhibited by both ICI and S107 (M). We also show that acute application of Aβ enhanced MitoSOX fluorescence until reaching a steady state plateau (N). Bar graphs show the mean plateau value of ΔF/F ± S.E. obtained in each experimental condition and reveal a significant decrease of MitoSOX fluorescence intensity in Aβ+ICI cells versus Aβ alone and Aβ+S107 (O). S107 pretreatment reduced Aβ-mediated ROS production , but this reduction was not statistically significant. These data reveal that Aβ may contribute to mitochondrial ROS elevation that is significantly reversed by ICI and to a lesser extent with S107 (N and O). These data may indicate that mitochondrial ROS elevation in APPswe likely results from both β-adrenergic and RyR-mediated Ca2+ leak. In Aβ-treated cells, mitochondrial ROS elevation likely occurs in a β-adrenergic-dependent manner and is only partially due to RyR-mediated ER Ca2+ leak.Next we found that both endogenous production  . As the control, we used dantrolene to block RyR-mediated Ca2+ release . C99 undergoes cleavage by γ-secretase to yield Aβ40 and Aβ42 peptides and AICD (F and K) and of total intracellular Aβ (F and J). Interestingly, the total amount of secreted Aβ42, but not of Aβ40, was also significantly reduced by S107 treatment in SH-5Y5Y cells expressing APPswe . Thus, S107 treatment reduces γ-secretase-mediated AICD production in an APPswe-expressing cells membrane preparation, similar to γ-secretase inhibition (A and B).Next, we determined whether S107-mediated inhibition of RyR2 leak could affect βAPP processing and Aβ production in SH-SY5Y cells expressing APPswe. Interestingly, the levels of C83 and C99 were reduced by S107 treatment . We show that S107 did not alter βAPP localization in early endosome (EEA1) , ER (SERCA2b) , recycling endosomes (CD71) , late endosomal and lysosomal membranes (CD63) , and the trans-Golgi network (TGN46) .We also evaluated whether S107 affects βAPP trafficking . This was performed using immunofluorescence and confocal imaging with antibodies recognizing specific cellular compartments involved in βAPP trafficking. The cellular localization of βAPP was assessed using Pearson's correlation and Mander's Overlap coefficients , H–I. WeA and B), whereas the blockade of β1-AR with CGP did not (C and D). The effect of ICI and S107 on C99 production was not additive (E and F). In accordance with ICI data, inhibition of PKA by H-89 reduced [Ca2+]cyt , and C99 production (G and H). Thus, RyR2 channel remodeling occurring downstream of β2-adrenergic signaling contributes to regulation of βAPP processing.As with S107 treatment, blockade of β2-AR with ICI reduced the production of the C99 fragment in APPswe-expressing SH-SY5Y cells , A and B did not , C and Dadditive , E and Foduction , G and H2+ release in an in vitro AD study model. Enhanced neuronal RyR2-mediated ER Ca2+ leak is linked to pathophysiological post-translational modifications in the macromolecular RyR complex  is decreased in the AD brain , 41. We in vitro and in vivo, and to oxidative stress , norepinephrine, and noradrenergic receptors in AD as the LC widespread projections terminate in areas important for learning and memory such as the hippocampus and the cortex. Neuronal cell death in the LC and other brain stem nuclei is a well defined characteristic of AD pathology . It has 2+ leak .Targeting of β2-adrenergic signaling to reverse/prevent AD pathology remains a controversial therapeutic track. Genetic knockdown or pharmacologic blockade of β2-AR elicits beneficial effects on taupathies  as well 2+]cyt was observed by different laboratories in various AD models  were cultured following the manufacturer's instructions. SH-SY5Y cells stably expressing empty pcDNA3.1 vector (Control) or pcDNA3.1 bearing APPswe cDNA were generated as already described  and mainm final concentration and vehicle  was used as control  (m) for the indicated times ; H-89 2HCl (Sigma) , a PKA inhibitor; ICI 118,551, hydrochloride ((±)-1--3-[(1-methylethyl) amino]-2-butanol hydrochloride) (Sigma) , a highly selective β2-adrenoreceptor antagonist; CGP, 20712A, ((±)-2-hydroxy-5-[2-[[2-hydroxy-3-[4-[1-methyl-4-(trifluoromethyl)-1H-imidazol-2-yl]phenoxy]propyl] amino]ethoxy]-benzamide methanesulfonate salt), a highly selective and potent β1-adrenoreceptor antagonist (Sigma) ; isoproterenol hydrochloride ((−)-isoprenaline hydrochloride, (−)-N-isopropyl-l-noradrenaline hydrochloride, (R)-3,4-dihydroxy-α-(isopropylaminomethyl)benzyl alcohol hydrochloride), a β-adrenergic agonist (Sigma) . ICI, CGP, and H-89 were applied for 12 h. Isoproterenol was applied for 1 h.Cells were treated overnight for 12 h with β- or γ-secretase inhibitors. γ-Secretase inhibitor ELND006 was used at a 5 μ control , 34. β-S control , synthesivative) , 26, 28 LDN-expressing CHO cells  were obtained by stable transfection of pcDNA4 empty vector (Control) and hAPP695 cDNA harboring London mutation (APPLDN: APPV642I) and subcloned in pcDNA4 vector. Cells were maintained in DMEM containing 10% FBS, sodium hypoxanthine-thymidine supplement, and 300 μm proline . Secretions were centrifuged (1000 × g for 10 min) and then concentrated into Amicon Ultra-15 3000 filters (4000 × g for 30 min). One-milliliter aliquots of Aβ preparation concentrates were stored at −80 °C until use. Aβ preparations were controlled on Tris-Tricine gels as already described  using human C-terminal Aβ antibodies.Detection of Aβ40 and Aβ42 peptides secreted in cell media were measured by an ELISA kit  overnight at 4 °C. The RyR2-specific antibody is an affinity-purified polyclonal rabbit antibody custom-made by Yenzym Antibodies  using the peptide CKPEFNNHKDYAQEK corresponding to amino acids 1367–1380 of mouse RyR2 with a cysteine residue added to the N terminus. The immune complexes were incubated with protein A-Sepharose beads (Sigma) at 4 °C for 1 h, and the beads were washed three times with radioimmune precipitation assay buffer. The immunoprecipitates were size-fractionated on SDS-PAGE gels  and transferred onto nitrocellulose membranes for 2 h at 200 mA. Immunoblots were developed using the following primary antibodies: anti-RyR2 , anti-phospho-RyR-Ser(P)-2808 ). All immunoblots were developed with the Odyssey system  using IR-labeled anti-mouse and anti-rabbit IgG  secondary antibodies.RyR2 was immunoprecipitated from cell lysates with an RyR2-specific antibody (2 μg) in 0.5 ml of a modified radioimmune precipitation assay buffer . Cells were then incubated at 4 °C overnight with primary antibodies diluted in BSA (3%). After 3 washes, coverslips were incubated with secondary antibodies ) at room temperature for 1 h. Immunofluorescence images were acquired on a Leica SP5 confocal microscope using excitation filters 488 and 594 nm. Images were analyzed using ImageJ software. Images were background-corrected, and the colocalization of red and green staining was determined using JACoP plugging. Pearson's correlation coefficient and Mander's Overlap Coefficient were used to evaluate the extent of colocalization as described .2+-ATPase of the endoplasmic reticulum; EEA1 antibody  targeting early endosome antigen 1; CD63 antibody  targeting antigens of lysosome membrane and late endosome membrane; CD71 antibody  targeting transferrin receptor: TGN46 antibody .We used βAPP C-terminal antibody (Sigma) recognizing 676–695 residues of βAPP. Other antibodies recognizing the following proteins were: SERCA2b antibody  targeting SR Cam MitoSOX red prepared in a KRB  supplemented with 1 mm CaCl2 (KRB/CaCl2) at 37 °C for 30 min. After a brief wash, Z-stack images were acquired on a SP5 confocal microscope (Leica). Dye intensity was quantified on Z-stack projection of images after thresholding using Leica SP5 confocal microscope and ImageJ software. Atpenin A5, a specific potent inhibitor of mitochondrial respiratory chain complex II, was used as the positive control (data not shown) (versus respective controls in each experiment). Kinetic measurements of MitoSOX are presented as ΔF/F to measure Aβ-mediated MitoSOX elevations correcting for differences in basal fluorescence or probe loading.We used MitoSOX (Invitrogen) Red fluorogenic dye to detect superoxide in the mitochondria of living cells . Cells gt shown) . We presm Fluo-4, AM (cytosolic Ca2+ probe), prepared in a KRB/CaCl2 at 37 °C for 30 min. After a brief washout, Z-stack images were acquired on a SP5 confocal microscope (Leica). Cellular loading of dyes was quantified on maximal projection of Z-stack images after thresholding using ImageJ software.Cells grown on 24-mm coverslips were loaded with 5 μ2+ according to the formula,dK for Fluo4 is 345 nm, F is the background-corrected fluorescence intensity recorded from cells during the experiment, Fmin is the Ca2+-free indicator fluorescence (determined in the ionophore (ionomycin 0.5 μm)/40 mm MnCl2 solution), and Fmax is the Ca2+-saturated indicator fluorescence (determined in the ionophore (ionomycin 0.5 μm)/1 mm Ca2+ rich solution)  .E, F, H, I, and J) changes were normalized and presented as ΔF/F0 to measure Aβ- or carbachol-mediated Ca2+ elevations correcting for differences in basal fluorescence or probe loading  following the manufacturer's instruction.m Tris, pH 10.8, 25 mm betaine and diluted in 2× Tris-Tricine loading buffer . Proteins were resolved by 16.5% Tris-Tricine SDS-PAGE and transferred onto PVDF membranes. Membranes were boiled in PBS and incubated overnight with specific antibodies.To detect CTFs (C99 and C83) and Aβ peptide, protein extracts (40 μg) were incubated with 70% formic acid (Sigma) and SpeedVac-evaporated for 40 min. The pellets were dissolved in 1 Aβ, β-CTF (C99), and full-length βAPP were detected using 6E10 antibody , which recognizes 1–16 residues of Aβ. α- and β-APP CTFs  were detected using the APP C-terminal antibody (Sigma) recognizing 676–695 residues of βAPP. Full-length βAPP was also detected using APP  antibody . β-Actin (Sigma) was used as loading control.In vitro γ-secretase activity was assessed as described  and dimethylammonio]-2-hydroxy-1-propanesulfonate 1% (v/v) supplemented with protease inhibitor mixture. All steps were performed at 4 °C. Solubilized membranes were diluted once with sodium citrate buffer (150 mm pH 6.4) and with reaction buffer . The resulting reaction mixtures were then either incubated with constant agitation for 16 h at 37 °C or stored at 4 °C (negative controls). Samples were then supplemented with 2× Tris-Tricine loading buffer, boiled for 5 min, and subjected to 16.5% Tris-Tricine SDS-PAGE.escribed , 96. 20 t test. Minimum statistically significant differences were established at p < 0.05. Non-statistically significant differences are not shown in the graphs.Data are expressed as the mean ± S.E. Sample size for each experiment is stated in the figure captions. Statistical analyses were performed using one-way or two-way ANOVA and Dunnett's, Bonferroni's, or Tukey's post-tests or 2+ experiments. M. C., A. L., X. L., and R. Z. analyzed ROS production measurements. A. R. M., M. C., F. C., and A. L. analyzed the data and wrote and edited the manuscript.A. R. M., A. L., and M. C. independently conceived the study. R. B., F. C., M. C., A. R. M., A. L., X. L., and S. R. designed the experiments. S. R. and V. S. conducted all the biochemistry assays of RyR2. R. B., M. C., and C. M. conducted the biochemistry assays of APP processing, immunofluorescence, and γ-secretase activity experiments. R. B., M. C., A. L., and X. L. conducted the Ca


Prnp, has been reported to be a receptor of β-amyloid. Their interaction is mandatory for neurotoxic effects of β-amyloid oligomers. In this study, we aimed to explore whether the cellular prion protein participates in the spreading of α-synuclein. Results demonstrate that Prnp expression is not mandatory for α-synuclein spreading. However, although the pathological spreading of α-synuclein can take place in the absence of Prnp, α-synuclein expanded faster in PrPC-overexpressing mice. In addition, α-synuclein binds strongly on PrPC-expressing cells, suggesting a role in modulating the effect of α-synuclein fibrils.The cellular prion protein, encoded by the gene The online version of this article (doi:10.1007/s12035-017-0451-4) contains supplementary material, which is available to authorized users. C), a glycosylphosphatidylinositol (GPI)-anchored protein, participates in several neural functions )). More relevantly, p-α-synuclein aggregates (after mouse or human fibril treatment) were detected in identified axons and in the cytoplasm of cultured neurons in A as well in B  antibody, no labelling was observed in the absence of α-synuclein fibril cultures. However, after α-synuclein fibril treatment in A Fig. d, immunon B Fig. b–g. In a9S/81A ann B Fig. . After mn B Fig.  43, 46], 46). MoC has been described as a receptor for β-amyloid [C in the intercellular β-amyloid transport in microfluidic devices remains elusive, its expression seems to be needed to trigger β-amyloid-mediated effects in treated neurons , 22.22% (Prnp+/+) and 16.66% (Tga20) of the cultures showed no α-synuclein transport in our microfluidic devices. In fact, these cultures displayed very few axons crossing between reservoirs as determined by Fluo4-AM staining ) ).As indicated above, PrP-amyloid , 49. AltC overexpression enhanced α-synuclein interaction with cells  and processed for α-synuclein immunolabelling  or mock (pcDNA) plasmids and then incubated 24 h later with mouse α-synuclein fibrils; their cellular binding was analysed using western blot and immunocytochemistry for PrPC and α-synuclein  identified cells from three different experiments demonstrated that 91.08% of the analysed cells with relevant α-synuclein labelling were also positive for PrPC  ΔF35/pcDNA = 2.15 ± 0.38; ΔCC/pcDNA = 0.92 ± 0.27; ΔHR/pcDNA = 1.11 ± 0.21; PrPC/pcDNA = 1.14 ± 0.09; mean ± SEM)  of the ΔCC-labelled cells were α-synuclein-positive )  ) Fig. h–k and PC. Thus, Prnp expression is not mandatory for α-synuclein transport in the mouse brain, although increased levels of α-synuclein transport can be seen in wild-type and overexpressing mice. In fact, it has been reported that the absence of PrPC does not modify the appearance or temporal evolution of p-α-synuclein deposits in transgenic mice overexpressing human α-synuclein driven by a platelet-derived growth factor-β promoter [Prnp dosage do not alter α-synuclein expression in adult mice (not shown). Although data using additional models of synucleinopathies (e.g. A53T mice) have not been published, the present results are in line with these observations [Prnp-overexpressing mice which suggest that, as also reported for β-amyloid, PrPC might participate in the cellular binding of α-synuclein and its expansion.In this study, we determined that α-synuclein fibrils can be transported to different brain regions of wild-type mice after injection in the postcommissural striatum. Our results reinforce the notion that the spreading of p-α-synuclein pathology does not occur by diffusion or non-specific transport . The trapromoter . Furtherrvations . HoweverC is well characterized [Prnp constructs, that their overexpression can enhance the binding of α-synuclein in HEK293 cells with low endogenous capability of α-synuclein binding  or α-synuclein fibrils with plasma membrane has been well described [Prnp genotypes (GEO database ref. GSE16223). However, and since a functional interaction between LAG3 with PrPC cannot be ruled out, we hypothesise that PrPC as well as other proteins (e.g. GRP78) may cooperate with LAG3 in neuronal α-synuclein transport. This is also relevant if considered that the absence of LAG3 does not fully impair α-synuclein protofibril transport in vitro and in vivo [The interaction of several amyloids to PrPcterized , 15–17. see also  for detasee also –61. In fescribed –60, 62, escribed  or GRP78escribed ). To thiescribed  reviewedescribed  points tescribed . mRNA le in vivo .C has been reported as a receptor for Aβ (see above). The present study indicates that PrPC might contribute to enhanced binding of α-synuclein fibrils to the plasma membrane, in line with other studies, reporting an increased level of binding between Aβ and PrPC during ageing in several mouse models of Alzheimer’s disease [C domains have been revealed as putative pharmacological targets for Alzheimer’s disease [C as a putative target to modulate α-synuclein expansion.Although not described for other fibrillar peptides, PrP disease . In addi disease , 53 in c disease –67, our Supplementary Fig. 1.a-b Electron microscopy photomicrograph illustrating recombinant mouse α-synuclein fibers in aggregated stage (a) and after sonication procedure (b). c Western blot of sonicated α-synuclein fibers. Note the appearance of the ≈ 17 kDa band as well as the ≈ 35 kDa band typical of non-monomeric α-synuclein. Similar band pattern was observed in the case of human α-synuclein fibrils d-e Low power photomicrographs illustrating p-α-synuclein staining in sham-operated (d) and mouse α-synuclein-operated (e) Prnp+/+ mice. p129S/81A p-α-synuclein labelling can be seen in parietal and cingular neocortex, hippocampus, mamillothalamic tract, fornix, globus pallidus, caudate putamen and white matter. However, after injection, p-α-synuclein deposits as LBL or LNL aggregates (arrows in e) can clearly be seen over the background using the p129S/81A antibody. f-g High power photomicrographs illustrating p-α-synuclein LBL after mouse fibril injection in neocortex (f) or amygdala (g). Abbreviations: A, amygdala; CP, caudate putamen; EC, entorhinal cortex; GP, globus pallidus; H, hippocampus; T, thalamus. Scale bar: d = 500 μm pertains to e; f = 25 μm pertains to g. (JPEG 8544 kb)Supplementary Fig. 2.a-b Examples of Fluo4-AM labelling (FITC optics) of microfluidic devices after 7 DIV. Note the difference in the number of labelled axons between (a) and (b) in the microchannels. c Example of western blotting of α-synuclein in a device without relevant interconnection reservoirs (a case). Two different exposures (10 seg and 4 min) are shown in the panel. Note the presence of a very pale band of α-synuclein in the B cell extract (asterisk) only detectable after 4 min of exposure. In these cultures, no endogenous α-synuclein labelling was observed, demonstrating detected α-synuclein in B derived from interneuronal transport of exogenous α-synuclein protofibrils as also demonstrated in α-synuclein Analysis of mouse α-synuclein transport using microfluidic devices. ein Fig. . Anti-β-Supplementary Fig. 3.a 2D representation of the two PDMS devices used in the present study. b-g Primary cortical cultures of Prnp+/+ were maintained in the devices for 5-7 days. Then human recombinant α-synuclein was added to A reservoir (asterisk) , neuronal presence of p-α-synuclein in A was analysed (c), and their transport to B reservoir was analysed with p-α-synuclein immunocytochemistry (d-g). b-c Examples of double-labelled neurons (TUJ1/p-α-synuclein) in A reservoir (indicated with camera icon) showing p-α-synuclein labelling as LBL (arrows in b and c). d-g Examples of p-α-synuclein-labelled neurons and axons  in B reservoir (indicated with camera icon). Scale bar: b and f = 40 μm pertains to c-e and g, respectively. (JPEG 8502 kb)Analysis of human α-synuclein fibril transport using microfluidic devices. Supplementary Fig. 4.Overexposed (15 min) uncropped films showing the absence of α–synuclein in cultured media of B in contrast to A reservoir, indicating the absence of fluidic flux between reservoirs illustrated in Fig. Supplementary Fig. 5.a Western blot illustrating the overexpression of the different PrPC constructs  in HEK293 cells. The cellular distribution of the constructs in the plasma membrane can be seen in [b Histogram illustrating the activation of caspase 3 by the different PrPC constructs (including ΔCD and ΔCR constructs). Data represent the mean ± S.E.M. of three different experiments. *** P < 0.01, ANOVA Bonferroni post hoc test. (JPEG 1479 kb) seen in  b Histog


They are nonplanar structures whose crystal arrangement is governed by Csp2—H⋯A  hydrogen bonding. The inherent complexity of the supramolecular arrangements of compounds (I) (Z′ = 2) and (II) (Z′ = 4) into tapes, helices and sheets is the result of the additional participation of π–n–π*  is self-paired, probably due to the effect of the steric demand of the methyl group on the crystal packing. Theoretical ab initio calculations confirmed that the presence of the arene ring at the benzimidazole 2-position increases the rotational barrier of the nitrobenzene ring and also supports the electrostatic nature of the orthogonal ONO⋯Csp2 and Npy⋯NO2 interactions.A detailed structural analysis of the benzimidazole nitroarenes 1-(4-nitrophenyl)-1 IR spectra was obtained with a 3100 FT–IR Excalibur Series spectrophotometer.The uncorrected melting points were measured in open-ended capillary tubes in an Electrothermal apparatus IA 9100. GAUSSIAN09 package  level of theory were performed without any symmetry restraints using the 2CO3  in dimethyl sulfoxide (13 ml) at 393 K for 20 h, as reported for 2-(4-bromophenyl)-1-(4-nitrophenyl)-1H-benzimidazole , 8.20 , 7.92 , 7.41 , 7.62 , 7.77 . 13C NMR: δ 146.7 (C-13), 144.6 (C-9), 141.9 (C-10), 141.8 (C-2), 133.0 (C-8), 126.1 , 124.8 (C-6), 124.0 (C5), 123.9 , 121.4 (C-4), 110.5 (C-7). IR : 1595, 1507 (C=C Ar), 1347 (NO2), 848, 754 (C—H out of plane).Compound (I)H-1,3-benzimidazole  and 1-fluoro-4-nitrobenzene  in a basic medium of K2CO3 , dimethylformamide (2 ml) and CuCl (11 mg) as catalyst, as a yellow solid in 72% yield (m.p. 421–423 K). Crystals suitable for X-ray diffraction were obtained from a hexane/ethyl acetate solution. 1H NMR: δ 8.34 , 7.88 , 7.48 , 7.34 . 13C NMR: δ 152.4 (C2), 147.2 (C9), 143.4 (C13), 142.8 (C10), 136.4 (C8), 130.2 (C19), 129.7 (C16), 129.5 , 128.9 (C17), 128.1 , 125.5 , 124.3 (C6), 124.0 (C5), 120.6 (C4), 110.1 (C7). IR : 1590, 1526 (C=C Ar), 1350 (NO2), 778, 746, 694 (C—H out of plane).Compound (II)m-tolyl-1H-1,3-benzimidazole  as a yellow solid in 20% yield after silica-gel chromatography (m.p. 447.7–449.0 K). Crystals suitable for X-ray diffraction were obtained from an hexane/ethyl acetate solution. 1H NMR: δ 8.38 , 7.90 , 7.54 , 7.50 , 7.34 , 2.34 . 13C NMR: δ 152.7 (C2), 147.2 (C9), 143.4 (C10), 142.8 (C13), 139.0 (C20), 136.3 (C8), 131.1 (C18), 130.5 (C17), 129.3 (C16), 128.6 (C19), 128.1 , 126.7 (C21), 125.1 , 124.2 (C6), 124.0 (C5), 120.6 (C4), 110.1 (C7), 21.6 (Me). IR : 1678 (C=N), 1590, 1515 (C=C Ar), 1347, 1303 (NO2), 791, 756, 745, 695 (C—H out of plane).Compound (III)Uiso(H) = 1.5Ueq(C) for methyl H atoms or 1.2Ueq(C) otherwise.Crystal data, data collection and structure refinement details are summarized in Table 1i.e. A (atoms N1/C2/N3/C4–C15/N13/O13A/O13B) and B (N21/C22/N23/C24–C35/N33/O33A/O33B), appear in the asymmetric unit of compound (I)C2/c. Molecules (IA) and (IB) are related by a second-order pseudo-helicoidal axis. The nitrobenzene ring (denoted N-nitroBz) is twisted from the mean benzimidazole (Bzm) plane by 35.71 (9) and 40.11 (7)° in molecules (IA) and (IB), respectively  [C14—C13—N13—O13B = −2.2 (4)°] and twisted in (IB) [C34—C33—N33—O33B = −22.4 (4)°]. However, the C—NO2 bond lengths are equal in both molecules and are also in the expected range  molecules form centrosymmetric dimers, i.e.A2, through C12—H12⋯O13Ai interactions, describing a twisted et al., 1995a). Furthermore, a meso helix is developed along the [030] direction through C7—H7⋯Cg2v T-shaped interactions linking the A2 dimers , and Cg2⋯Cg7v, between the aromatic ring (Cg2 is the centroid of the C4—C9 ring and Cg7 is the centroid of the C30–C35 ring) of the Bzm moiety of molecule (IA) and the nitrobenzene ring of molecule (IB). The intercentroid Cg2⋯Cg7v distance [3.6123 (17) Å] is very close to the interplanar distance [3.4361 (12) Å], in agreement with a face-to-face interaction  between phenylalanine and nitrobenzene  Å and N33⋯O33B⋯C2 = 93.7 (2)°  . This interaction has been described in 3,3′-dinitro-2,2′-bipyridine N-oxides, with distances in the range 2.762 (4)–2.789 (3) Å -3-(4-nitrophenyl)-2-(1-phenyl-1H-benzimidazol-2-yl)acrylonitrile  (atoms N1/C2/N3/C4–C15/N13/O13A/O13B), (IIB) (N21/C22/N23/C24–C35/N33/O33A/O33B), (IIC) (N41/C42/N43/C44–C55/N53/O53A/O53B) and (IID) (N61/C62/N63/C64–C75/N73/O73A/O73B). Molecules (IIA) and (IIC), as well as (IIB) and (IID), are related by a local pseudocentre of inversion located at the fractional coordinates  and , respectively, in the asymmetric unit. This condition has frequently been observed in PZ′ structures  and (IIB) have similar angles, as have molecules (IIC) and (IID), judged by the mean values of the angles between the planes. The N-nitroBz ring in compound (II)et al., 2014et al., 1992Compound (II)it Fig. 3, namely A) and (IIB) are linked through N⋯NO2 interactions (N13⋯N23 and N33⋯N3), with the participation of the pyridine-like N atom as the donor and the N atom of the nitro group as the acceptor, forming b axis. Chains of (IIA) and (IIB) molecules are linked through C6—H6⋯O13Ai and C26—H26⋯O33Biv soft hydrogen bonds to develop a sheet within the ab plane. C7—H7⋯O33Bii and C27—H27⋯O13Av soft hydrogen bonds are responsible for linking two (IIA)/(IIB) planes along the c-axis direction, i.e. (A2B2)n . These C—H⋯O interactions are of the bifurcated type with respect to the acceptor O atoms, i.e. H6⋯O13A⋯H27 and H7⋯O33B⋯H26. The geometrical parameters and symmetry codes of the hydrogen-bonding and N⋯NO2 interactions are listed in Tables 4Molecules (II)n Fig. 4a. TheseC) and (IID) develop a (DC2D)n ribbon within the (10b and 4c), also through N⋯NO2 and C—H⋯O interactions . The (A2B2)n double sheets and (DC2D)n ribbons are interleaved to develop the three-dimensional structure along the c-axis direction through C—H⋯X (X = N and O) interactions , with the participation of the pyridine-like N and nitro O atoms as acceptors.Molecules  interaction plays a crucial role in the molecular self-assembly and crystal packing of compound (II)et al., 1986et al., 20052 interactions in (II)et al., 1992Remarkably, the intermolecular N2⋯Csp2 and Npy⋯NO2 interactions present in (I)n–π* type, since the donor atom approaches in a perpendicular manner to the plane that includes the acceptor. N⋯NO2 interactions have been envisaged as an entry to supramolecular cages without using metals to make orthogonal corners Pa). Both N-nitroBz and C2-MeBz rings are twisted from the mean Bzm plane, the angles between the planes being 67.74 (4) (Bzm and N-nitroBz), 28.21 (5) (Bzm and C2-MeBz) and 64.77 (5)° (N-nitroBz and C2-MeBz), i.e. more twisted than in compound (II)2 group is almost in the plane of the N-nitroBz ring [C14—C13—N13—O13B = 6.9 (2)°].Compound (III)it Fig. 5a. Both i interactions in the form of an b). Infinite tapes propagating along the a-axis direction are developed by C12—H12⋯N3ii soft hydrogen bonds, forming an c). Finally, the two- and three-dimensional structures are arranged through C14—H14⋯Cg2iii and N13—O13A⋯Cg3iv dispersive interactions  . The geometrical parameters and symmetry codes of the hydrogen bonding for compound (III)Molecules of (III)ng Fig. 5b. Infinif Fig. 5c. Final2] Fig. 5d. The gAb initio theoretical density functional theory (DFT) calculations at the B3LYP/6-31G level of theory were performed to support the experimental findings. The calculated geometric parameters are in agreement with the experimental ones. In general, the differences between the geometrical parameters in the experimental and optimized geometries are in most cases 0.01 Å (bond lengths) and 0.5° (bond angles), but large differences are observed for torsion angles that might be attributed to the gas-phase calculations without considering the crystal-packing forces. Additionally, the greater differences in favour of the N-nitroBz ring might be attributed to the presence of the nitro group, which is involved in intermolecular interactions.sp2—H hydrogens bear a significant positive charge, particularly H12 and H14, which are both in ortho positions with respect to the nitro group. The calculated MKS charges are listed in Table 7n–π* donor–acceptor interactions ONO⋯Csp2 and Npy⋯NO2 observed in (I)py) of the heterocycle bears the most negative charge, followed by the O atoms of the nitro group. The C2-Ph substitution in compound (II)The nitro group retrieves electronic density from the benzene ring, so the CA) in the crystal lattice, where the N-nitroBz ring is twisted from the mean Bz plane, with a C8—N1—C10—C11 torsion angle of −39.43  versus −32.47° . The maximum energy values of 3.27 or 2.10 kcal mol−1 were found when the N-nitroBz ring is coplanar or perpendicular to the benzimidazole heterocycle, respectively. Because of symmetry reasons and the small cost in energy, the calculated rotamer with a C8—N1—C10—C11 torsion angle of 39.43° is equally probable . The rotational barriers of compounds (I)–(III) are listed in Table S1 in the supporting information.The theoretical energy profiles of compounds (I)–(III) were also calculated to estimate the energy involved in the interconversion between the N-nitroBz and C2-Ph rotamers. The experimental and theoretical torsion-angle values are listed in Table 8A)–(IID) found in the asymmetric unit . The N1—C2—C16—C17 torsion angle was fixed at 33.73° to calculate the rotational barrier of the N-nitroBz ring. A maximum peak of energy 8.95 kcal mol−1 was found when the N-nitroBz ring is in the same plane as the Bz heterocycle and of only 1.60 kcal mol−1 when perpendicular . Therefore, the rotational barrier of the C2-Ph ring is just 40% of the energy required to rotate the N-nitroBz ring.In the case of compound (II)ar Fig. 6. Thus, tvide supra). However, the energy profile and the maximum peak of energy 9.20 kcal mol−1 were found to be very similar to those exhibited by compound (II)supporting information).The calculated C8—N1—C10—C11 torsion angle of 58.72° contrasts with the experimental value of 72.23 (19)° for compound (III)i.e. an electron-withdrawing nitro group at one end and an electron-donating amino group at the other end. However, the structures lack strong hydrogen-bond donors. Thus, the crystal networks are developed by dispersive soft interactions, with the participation of the nitro group, namely Csp2—H⋯ONO, n–π* –(III) are nonplanar molecules having two strong hydrogen-bonding acceptors groups, ns Fig. 7.Z′-structure where equi-energetic conformers co-exist in the crystal network. Calculations also supported the fact that orthogonal ONO⋯Csp2 and Npy⋯Theoretical calculations confirmed that the presence of the C2-Bz ring increases the rotational barrier of the N-nitroBz ring, thus fewer conformers are expected. However, compound (II)10.1107/S2053229618003406/uk3146sup1.cifCrystal structure: contains datablock(s) I, II, III, global. DOI: 10.1107/S2053229618003406/uk3146Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2053229618003406/uk3146IIsup3.hklStructure factors: contains datablock(s) II. DOI: 10.1107/S2053229618003406/uk3146IIIsup4.hklStructure factors: contains datablock(s) III. DOI: 10.1107/S2053229618003406/uk3146sup5.pdfTheoretical rotation profiles and calculated rotation barriers. DOI: 1826170, 1565535, 1565536CCDC references:


S(6). In addition, molecules are stacked along the [100] through C=O⋯Cg interactions, as suggested by the Hirshfeld surface, which also indicates that the most important contributions for the crystal structure cohesion are O⋯H (28.5%) and H⋯H (26.7%) interactions. An in silico evaluation of the title compound with the dihydrofolate reductase enzyme was performed and N—H⋯O and Cg⋯Cg interactions were found.The molecule of 5-nitroisatin-3-phenylhydrazone deviates slightly from a planar geometry. In the crystal, molecules are linked by hydrogen bonding into a two-dimensional polymer along (120), forming rings of graph-set motifs  14H10N4O3, whose molecular structure deviates slightly from a planar geometry . An intramolecular N—H⋯O hydrogen bond is present, forming a ring of graph-set motif S(6). In the crystal, molecules are linked by N—H⋯O and C—H⋯O hydrogen-bonding interactions into a two-dimensional network along (120), and rings of graph-set motif R22(8), R22(26) and R44(32) are observed. Additionally, a Hirshfeld surface analysis suggests that the molecules are stacked along [100] through C=O⋯Cg interactions and indicates that the most important contributions for the crystal structure are O⋯H (28.5%) and H⋯H (26.7%) interactions. An in silico evaluation of the title compound with the DHFR enzyme (dihydrofolate reductase) was performed. The isatin–hydrazone derivative and the active site of the selected enzyme show N—H⋯O(ASP29), N—H⋯O(ILE96) and Cg⋯Cg(PHE33) interactions.The reaction between 5-nitroisatin and phenylhydrazine in acidic ethanol yields the title compound, C As sugg0] Fig. 3. The sep0] Fig. 3.et al., 2012de (y axis) and di (x axis) values are the closest external and internal distances (in Å) from given points on the Hirshfeld surface : H⋯C = 17.7, H⋯N = 8.9, C⋯O = 8.2, C⋯C = 5.5 and C⋯N = 3.3. The Hirshfeld surface graphical representation with transparency and labelled atoms Figs. 4 and 5 ▸ −1, gradient normal = 0.763, HOMO = −8.96 eV, LUMO =-1.66 eV and energy gap = 7.30 eV. The target prediction for 5-nitroisatin-3-phenylhydrazone was calculated with the SwissTargetPrediction webserver based on the bioisosteric similarity to the isatin entity  (1.928 Å), N4—H5⋯O(ILE96) (1.925 Å) and Cg⋯Cg(PHE33) (3.567 Å)  was carried out. Initially, the semi-empirical equilibrium energy of the small molecule was obtained using the PM6 Hamiltonian, but the experimental bond lengths were conserved. The calculated parameters were: heat of formation = 149.41 kJ molÅ) Fig. 7.et al., 2016E)-5-nitro-3-(2-phenylhydrazinylidene)-1H-indol-2(3H)-one, which may be compared with that of the title compound. The molecular structure deviates slightly from the ideal planar geometry and the C⋯C contacts between the planes are observed. The molecules are linked by N—H⋯O and C—H⋯Cl interactions into a two-dimensional hydrogen-bonded polymer, a quite similar structure to the title compound. The in silico evaluation of 5-chloroisatin-phenylhydrazone, a molecule with similar crystal packing to the title compound, with and the DNA topoisomerase IIα enzyme was performed and the global free energy of −26.59 kJ mol−1 was found. The evaluation agrees with the literature data for molecular docking and cytotoxic activity of hydrazone derivatives against breast cancer cells  on slow evaporation of the solvent.All starting materials are commercially available and were used without further purification. The synthesis of the title compound was adapted from a procedure reported previously (Fonseca Uiso(H) = 1.2Ueq, and with C—H = 0.95 Å and N—H = 0.88 Å.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989016020375/rz5203sup1.cifCrystal structure: contains datablock(s) I, publication_text. DOI: 10.1107/S2056989016020375/rz5203Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989016020375/rz5203sup3.pdfSwissTargetPrediction Report for 5-nitroisatin-3-phenylhydrazone. DOI: Click here for additional data file.10.1107/S2056989016020375/rz5203Isup4.cmlSupporting information file. DOI: 1524161CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information: