Vβ3 receptor, retaining good binding affinity, in the same nanomolar range as the free ligands. The antiproliferative activity of the conjugates was evaluated in three cell lines possessing different levels of αVβ3 integrin expression: human glioblastoma U87 (αVβ3+), human lung carcinoma A549 (αVβ3−) and breast adenocarcinoma MDA-MB-468 (αVβ3−). In the U87, in the MDA-MB-468, and partly in the A549 cancer cell lines, the cyclo[DKP-isoDGR]-α-amanitin conjugates bearing the lysosomally cleavable Val-Ala linker were found to be slightly more potent than α-amanitin. Apparently, for all these α-amanitin conjugates there is no correlation between the cytotoxicity and the expression of αVβ3 integrin. To determine whether the increased cytotoxicity of the cyclo[DKP-isoDGR]-α-amanitin conjugates is governed by an integrin-mediated binding and internalization process, competition experiments were carried out in which the conjugates were tested with U87  and MDA-MB-468  cells in the presence of excess cilengitide, with the aim of blocking integrins on the cell surface. Using the MDA-MB-468 cell line, a fivefold increase of the IC50 was observed for the conjugates in the presence of excess cilengitide, which is known to strongly bind not only αVβ3, but also αVβ5, αVβ6, and α5β1. These data indicate that in this case the cyclo[DKP-isoDGR]-α-amanitin conjugates are possibly internalized by a process mediated by integrins different from αVβ3 .RGD-α-amanitin and isoDGR-α-amanitin conjugates were synthesized by joining integrin ligands to α-amanitin via various linkers and spacers. The conjugates were evaluated for their ability to inhibit biotinylated vitronectin binding to the purified α Amanita Phalloides (death cap mushroom), see α-Amanitin is a bicyclic octapeptide toxin belonging to the amatoxin family, found in 50 values from 2.5 × 10−10 to 2.0 × 10−12 M. Promising results were also observed in mice bearing BxPc-3 pancreatic xenograft tumors, with complete tumor regression in 90% of the cases after two injections of the α-amanitin-anti-EpCAM ADC at a dose of 100 μg/kg with respect to α-amanitin. In these two examples, the internalization of the monoclonal antibody and subsequent release of the toxin leads to the enhancement of α-amanitin activity on the targeted cells.This strong toxicity in the presence of endocytosis mediators allowing cell permeation, aroused interest towards the use of α-amanitin as a payload for targeted cancer therapy. In 1981, Davis and Preston reported the synthesis of the antibody–drug conjugate (ADC) α-amanitin-anti-Thy 1.2 IgG, which was 47-fold more toxic than the unconjugated α-amanitin in the murine T lymphoma S49.1 cell line . The cytN-propargylasparagine of an amanitin analog to a cycloRGD integrin ligand (cyclo[RGDfK]) using a copper-catalyzed azide–alkyne cycloaddition -Val-Ala-PTX 6). Their tumor targeting ability was assessed in vitro in antiproliferative assays comparing an αVβ3 positive with an αVβ3 negative cell line -Val-Ala-PTX conjugate 5 (TI = 2.4) -α-amanitin and three cyclo[DKP-isoDGR]-α-amanitin conjugates. In these conjugates, the integrin ligands are bound to α-amanitin via a 6’-ether with two different linkers: an “uncleavable” six carbon aliphatic chain (7–11 were synthesized as described in 2NCH2-cyclo[DKP-RGD] (2) [2NCH2-cyclo[DKP-isoDGR] (4) [Cyclo[DKP-RGD]-α-amanitin and cyclo[DKP-isoDGR]-α-amanitin conjugates RGD] (2) ,29 and HDGR] (4)  to α-ama7–11 were evaluated for their ability to inhibit biotinylated vitronectin binding to the purified αVβ3 receptor. The calculated half-maximal inhibitory concentrations (IC50) are listed in 7–11 at different concentrations (10−12 to 10−5 M) in the presence of biotinylated vitronectin (1 µg mL−1) and measuring bound vitronectin.Conjugates 7 and 9 and cyclo[DKP-isoDGR]-α-amanitin conjugates 8, 10 and 11 retain good binding affinity for αVβ3 integrin, in the same range as the free ligands  were selected as αVβ3 positive, while A549 cells (human lung carcinoma) and MDA-MB-468 (breast adenocarcinoma) -uncleavable-α-amanitin (7), cyclo[DKP-isoDGR]-uncleavable-α-amanitin (8) and cyclo[DKP-RGD]-Val-Ala-α-amanitin (9) proved less potent than α-amanitin in all cell lines  -α-amanitin conjugate 10 proved slightly more potent than α-amanitin, whereas conjugate 11 showed enhanced potency compared to the free drug only on U87 and MDA-MB-468 cells.In this paper, two cyclo[DKP-RGD]-α-amanitin and three cyclo[DKP-isoDGR]-α-amanitin conjugates were prepared in good yields following a straightforward synthetic route. Conjugates Vβ3 integrin. To determine whether the slightly increased cytotoxicity of cyclo[DKP-isoDGR]-α-amanitin conjugates 10 and 11 is governed by an integrin-mediated binding and internalization process, competition experiments were carried out in which conjugates 10 and 11 were tested with U87  and MDA-MB-468  cells in the presence of 50-fold excess of cilengitide, with the aim of blocking integrins on the cell surface. Using the U87 cell line, a modest increase of the conjugate 11 IC50 was observed in the presence of cilengitide. Employing the MDA-MB-468 cell line, a more pronounced increase of IC50 was observed for both conjugates 10 and 11 in the presence of cilengitide. Therefore, it appears that blocking integrins with excess cilengitide, which is known to strongly bind not only αVβ3, but also αVβ5, αVβ6, and α5β1, results in an increase (up to 5.5 times) of the IC50 of conjugates 10 and 11 with the MDA-MB-468 cell line. These data suggest that cyclo[DKP-isoDGR]-α-amanitin conjugates 10 and 11 are possibly internalized by a process mediated by integrins different from αVβ3 , though the exact nature of this involvement is not clearly defined  (2) and H2NCH2-cyclo[DKP-isoDGR] (4) were prepared according to the literature [12a and 12b were prepared according to references [Functionalized ligands Hterature –29. 6’-Fferences –44.File 1Experimental details, characterization data and copies of spectra.


Hydrogen-bonded ladders typically encountered in ammonium carboxylate salts did not form in the presence of a pyridine acceptor group. RS)-1-phenylethan-1-aminium isonicotinate, C8H12N+·C6H4N1O2−, (I), (RS)-1-phenylethan-1-aminium flurbiprofenate [or 2-(3-fluoro-4-phenylphenyl)propanoate], C8H12N+·C15H12FO2−, (II), (RS)-1-phenylethan-1-aminium 2-chloro-4-nitrobenzoate, C8H12N+·C7H3ClNO4−, (III), (RS)-1-phenylethan-1-aminium 4-iodobenzoate, C8H12N+·C7H4IO2−, (IV), (S)-1-cyclohexylethan-1-aminium 2-chloro-4-nitrobenzoate, C8H18N+·C7H3ClNO4−, (V), 2-(cyclohex-1-en-1-yl)ethan-1-aminium 4-bromobenzoate, C8H16N+·C7H4BrO2−, (VI), and (S)-1-cyclohexylethan-1-aminium 4-bromobenzoate, C8H18N+·C7H4BrO2−, (VII). Salts (II) to (VII) feature three N+—H⋯O− hydrogen bonds, which form one-dimensional hydrogen-bonded ladders. Salts (II), (III), (IV), (V) and (VII) have a type II ladder system despite the presence of halogen bonding and other intermolecular interactions, whereas (VI) has a type III ladder system. Salt (I) has a unique hydrogen-bonded system of ladders, featuring both N+—H⋯O− and N+—H⋯N hydrogen bonds owing to the presence of the pyridine functional group. The presence of an additional hydrogen-bond acceptor on the carboxylate cation disrupts the formation of the ubiquitous type II and III ladder found predominately in ammonium carboxylate salts. Halogen bonding, however, has no influence on their formation.The crystal structures of seven ammonium carboxylate salts are reported, namely ( Thus, primarily non-covalent bonding is used to achieve the organization of molecules and ions in the solid state in order to produce materials with desired properties. Examples of such materials include organic field-effect transistors, hole collectors in organic photovoltaic cells ·(R–COO−), where R often contains a phenylethyl group generating chiral molecules : (RS)-1-phenylethan-1-aminium isonicotinate, (I)RS)-1-phenylethan-1-aminium flurbiprofenate, (II)RS)-1-phenylethan-1-aminium 2-chloro-4-nitro-benzoate, (III)RS)-1-phenylethan-1-aminium 4-iodobenzoate, (IV)S)-1-cyclohexylethan-1-aminium 2-chloro-4-nitro-benzoate, (V)S)-1-cyclohexylethan-1-aminium 4-bromobenzoate, (VII)This work will focus on the effects of hydrogen bonding. In particular, we have investigated the effects thereof of changing both the structure and stereochemistry of the constituents on the robust ionic supramolecular heterosynthons generated by ammonium carboxylate salts  (see Table 1a. The second involves the O1 atom of the isonicotinate anion in the asymmetric unit and is designated b. The third involves the pyridine ring nitrogen of a third isonicotinate anion (ii) and is designated c. The b and c hydrogen bonds form a ring structure involving two of each kind of bond, consisting of two molecules of both 1-phenylethan-1-aminium and isonicotinate . The graph set of this pattern is 88(30) ring is formed using all three hydrogen bonds involving four of both 1-phenylethan-1-aminium and isonicotinate ions. Overall, this forms a 2-D sheet as shown in Fig. 2b. As neither of the two expected type II or type III ladders are formed it seems that the additional hydrogen-bond acceptor in the form of the nitrogen atom of the pyridine ring disrupts their formation.Salt (I)II ladder system where each of the O1 atoms behaves as a bifurcated hydrogen-bond acceptor, linking the rings . This pattern has translational symmetry through a twofold screw axis along the crystallographic b axis which is inherent in the space group P21/n. As no short contacts such as halogen bonding or π-halogen interactions are observed, the fluorine atom does not disrupt the formation of the expected hydrogen-bonding patterns. However a peculiarity exists. As the cation was present as a racemate, traditionally type III ladders are expected to dominate as reported by Lemmerer and co-workers gs Fig. 3a. This II ladder is formed by the above-mentioned hydrogen bonds, as shown in Fig. 3b. The anions in adjacent rings  are connected via C—O⋯Cl halogen bonds . However, this interaction does not perturb the ladder supramolecular synthons to a significant enough degree to prevent their formation. Once again, both enantiomers of the 1-phenylethan-1-aminium were present and thus type III ladders were expected to form.In salt (III)II ladder system is observed (Table 4c). This is possible as, due to its size, iodine is very polarizable and thus the delocalized electrons in the aromatic system can create a permanent dipole in the iodine atom in the solid state. The distance of 3.740 (3) Å is similar to other molecules containing iodine interacting non-covalently with aromatic systems reported in the literature d Table 4. An inted Table 4c. This S)-1-cyclohexylethylammonium cation and one 2-chloro-4-nitro-benzoate anion, both on general positions. A type II ladder is formed as shown in Fig. 3d. No hydrogen bonding to the nitro group takes place e Table 5, which iIII ladder is observed (Table 6e). As in salt (V)In salt (VI)d Table 6, unique d Table 6e. As inS)-1-cyclohexylethylammonium cation and one 4-bromobenzoate anion, both on general positions. A type II ladder is formed (Table 7f). This is expected as the cation is enantiomerically pure d Table 7f. This II and III patterns as seen in (II)–(VII)In summary, introducing a pyridine functional group instead of a plain benzene in (I)via the slow evaporation of methanol or ethanol solutions under ambient conditions. All solutions contained a 1:1 ratio of amine and acid. Detailed masses and volumes are as follows. For (I)RS)-α-methylbenzylamine  and isonicotinic acid  in methanol (5 mL); for (II)RS)-α-methylbenzylamine  and flurbiprofen  in ethanol (8 mL); for (III)RS)-α-methylbenzylamine  and 2-chloro-4-nitro-benzoic acid  in ethanol (5 mL); for (IV)RS)-α-methylbenzylamine  and 4-iodobenzoic acid  in ethanol (5 mL); for (V)S)-1-cyclohexylethylamine  and 2-chloro-4-nitro-benzoic acid ; for (VI)S)-1-cyclohexylethylamine  and 4-bromobenzoic acid .All chemicals were purchased from commercial sources (Sigma Aldrich) and used as received without further purification. Crystals were grown 2), 0.98 Å (methylene CH3) and 0.95 Å (Ar—H)] and refined as riding with Uiso(H) = 1.2Ueq(C) or Uiso(H) = 1.5Ueq(C). The N-bound H atoms were located in difference-Fourier maps and their coordinates and isotropic displacement parameters allowed to refine freely for (I)–(VI). For (VII)Uiso(H) = 1.5Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 8For the disorder of the atom C4 in the cyclohexene ring of (VI)et al.  I, II, III, IV, V, VI, VII, shelx. DOI: 10.1107/S2056989017017856/xi2001Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989017017856/xi2001IIsup3.hklStructure factors: contains datablock(s) II. DOI: 10.1107/S2056989017017856/xi2001IIIsup4.hklStructure factors: contains datablock(s) III. DOI: 10.1107/S2056989017017856/xi2001IVsup5.hklStructure factors: contains datablock(s) IV. DOI: 10.1107/S2056989017017856/xi2001Vsup6.hklStructure factors: contains datablock(s) V. DOI: 10.1107/S2056989017017856/xi2001VIsup7.hklStructure factors: contains datablock(s) VI. DOI: 10.1107/S2056989017017856/xi2001VIIsup8.hklStructure factors: contains datablock(s) VII. DOI: Click here for additional data file.10.1107/S2056989017017856/xi2001Isup9.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989017017856/xi2001IIsup10.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989017017856/xi2001IIIsup11.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989017017856/xi2001IVsup12.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989017017856/xi2001Vsup13.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989017017856/xi2001VIsup14.cmlSupporting information file. DOI: 1811019, 1811018, 1811017, 1811016, 1811015, 1811014, 1811013CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Furthermore, 4a and 4b can induce pro-death autophagy and apoptosis simultaneously. Our study indicates that ester modification is a simple and feasible strategy to enhance the anticancer potency of Ir(III) complexes.Organometallic iridium complexes are potent anticancer candidates which act through different mechanisms from cisplatin-based chemotherapy regimens. Here, ten phosphorescent cyclometalated iridium(III) complexes containing 2,2′-bipyridine-4,4′-dicarboxylic acid and its diester derivatives as ligands are designed and synthesized. The modification by ester group, which can be hydrolysed by esterase, facilitates the adjustment of drug-like properties. The quantum yields and emission lifetimes are influenced by variation of the ester substituents on the Ir(III) complexes. The cytotoxicity of these Ir(III) complexes is correlated with the length of their ester groups. Among them, 4a and 4b are found to be highly active against a panel of cancer cells screened, including cisplatin-resistant cancer cells. Mechanism studies  Many non-platinum metal complexes, such as copper, ruthenium and osmium complexes, show promising anti-proliferative activities1Phosphorescent cyclometalated Ir(III) complexes are regarded as excellent probes for biological imaging and sensing, due to their outstanding photophysical properties, including relatively high quantum yields, long emission lifetimes, large Stokes shifts, two-photon absorption and high photobleaching resistance89111213141516141516As for drug optimization, it is important to improve ADMET 1921222dcbpy) and its diester derivatives as ligands are designed and synthesized. Their anti-proliferative activities are evaluated on several cancer cell lines. The in vitro hydrolysis of the ester bonds, as well as anticancer mechanisms including subcellular localization, impact on mitochondrial integrity, elevation of reactive oxygen species (ROS), depletion of cellular ATP production, cell cycle arrest and induction of autophagy and apoptosis, are investigated in detail.Herein, ten phosphorescent cyclometalated Ir(III) complexes containing 2,2′-bipyridine-4,4′-dicarboxylic acid (H1a‒5a) and 2-pyridine , are utilized to tune the photophysical properties of the complexes. The ligands were prepared by reacting H2dcbpy with methanol, ethanol, n-butanol or i-butanol in SOCl2 according to reported procedures1a and 1b were synthesized by refluxing the corresponding Ir(III) chloro-bridged precursors and H2dcbpy (0.4 mmol) in CH2Cl2/CH3OH with excessive Na2CO32Cl2/CH3OH, followed by anion exchange with NH4PF6 solution  complexes are shown in solution . All theOH1H NMR ‒S11 and fraction . The cry2Cl2 and CH3CN were investigated (1MLCT and 3MLCT). Upon excitation at 405 nm, 1a−5a and 1b−5b exhibit red to yellow phosphorescent emissions. Quantum yields of Ir(III) complexes range from 0.001 to 0.464, and phosphorescent lifetimes fall between 4.4 and 700.8 ns in different solvents at room temperature. Higher quantum yields and longer lifetimes are observed in organic solvents than in aqueous phase. The environmentally sensitive emission properties are commonly observed for cyclometalated Ir(III) complexes, which are considered to be advantageous for bioimaging applications1b−5b show blue shifts compared with 1a−5a, due to the electron-withdrawing F atoms on dfppy  complexes in phosphate buffered saline (PBS), CHstigated . All comstigated , which con dfppy . The ene1a (m/z 745) or 1b (m/z 817) remain unchanged. Other complexes with ester substituents show peaks assigned to intact complexes as well as peaks corresponding to hydrolytic products. The results indicate that complexes 2a‒5a and 2b‒5b can undergo hydrolysis in the presence of esterase.Ester-modified compounds have been used as prodrugs widely. They can be hydrolysed by a variety of esterases selectively or non-selectively, to release active pharmacophoresPo/w values, was determined by the shake-flask method according to a literature procedurePo/w values of these Ir(III) complexes fall between −0.81 and 2.59  show much higher cytotoxicity than the other complexes. Especially, 4a (IC50 = 1.7 μM) and 4b (IC50 = 2.3 μM) are approximately 13.9−fold and 10.3−fold more potent than cisplatin (IC50 = 23.7 μM) against A549 cells, respectively. 4a and 4b are also active in A549R cells, which may indicate that they can overcome cisplatin resistance. The results indicate that ester modification can modulate lipophilicity and anti-proliferative activity of these Ir(III) complexes. For complexes 1a and 1b, converting the carboxyl acid groups into ester groups can facilitate cellular uptake and improve their anti-proliferative effects. As carboxyl groups are usually deprotonated at physiological pH, the phenomena may be partially attributed to the alteration of molecular charge from neutral/negative to positive under physiological conditions.The cytotoxicity was evaluated in A549, A549R (cisplatin-resistant A549), HeLa , HepG2 (human hepatocellular liver carcinoma) and MCF-7 (human breast adenocarcinoma) cells. Cisplatin was also tested as a positive control. Generally, the anti-proliferative efficacy of Ir(III) complexes is in the following order: efficacy . 1a and 4a and 4b were chosen for further mechanism studies because of their relatively high anticancer potency. By virtue of their intrinsic phosphorescence, the cellular uptake behaviours of Ir(III) complexes can be investigated semi-quantitatively by confocal microscopy. Moreover, subcellular localization analysis can provide further hints for the investigation of anticancer mechanisms4a (5 μM) and 4b (5 μM) can penetrate into A549 cells rapidly in 30 min and mainly retain within cytoplasm (® Deep Red FM (MTDR), a commercial mitochondrial fluorescent probe, was co-incubated with Ir(III) complexes to further determine subcellular localization (4a or 4b with MTDR.ytoplasm . Mito-trlization 15. High 4a and 4b enter cells by non-endocytic energy dependent active transport. Our findings are consistent with the mechanisms reported for Ir(III) complexes with structural similarity15The cellular uptake mechanism was investigated by incubating cells at low temperature or pro-treating cells with carbonyl cyanide 3-chlorophenylhydrazone  or chloroquine 4a and 4b on mitochondrial integrity. Mitochondrial membrane potential (MMP) was detected by 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide (JC-1) staining. In normal mitochondria, JC-1 aggregates and shows red fluorescence. If mitochondrial membranes are depolarized, JC-1 exists as green fluorescent monomers4a or 4b. The percentage of cells with MMP loss increase from 16.1 ± 0.4% to 90.3 ± 1.0% and 88.4 ± 1.1% for 4a and 4b respectively, after 6 h treatment at a concentration of 4 μM  complexes on ATP production (4a or 4b. After 12 μM Ir(III) treatments for 6 h, the ATP level decreases to 30.0 ± 0.6% and 13.3 ± 1.5% in 4a− and 4b−treated cells, respectively. These results indicate that 4a and 4b can impair mitochondrial energy metabolism in A549 cells effectively.Mitochondria are the energy factory of cells, and they are vital for ATP productionoduction . A dose-4a and 4b can target to mitochondria and cause mitochondrial depolarization, their impact on intracellular ROS level was examined by 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) staining and analysed by flow cytometry as well as confocal microscopy. The non-fluorescent H2DCFDA can be oxidized to highly bright 2′,7′-dichlorofluorescein (DCF) by cellular ROS4a− and 4b−treated cells. Compared with the vehicle-treated control, 9.7− and 10.2−fold increase in intensity of DCF is observed in A549 cells treated with 4a  and 4b , respectively  was used to scavenge intracellular ROS4a, 4b and cisplatin in A459 cells  and 4b , respectively. These results indicate that as a consequence of mitochondrial damage, 4a and 4b induce ROS-mediated cell death.In order to investigate the role of ROS in Ir(III)-induced cell death, the antioxidant 59 cells . Compare4a or 4b at different concentrations for 24 h. Cells were stained with propidium iodide (PI) and analysed by flow cytometry. The result reveals that 4a and 4b can elicit cell cycle arrest at G0/G1 stage at lower concentrations (2 and 4 μM), which is accompanied by a decrease in the percentage of S phase cells  and 4b (4 μM) for 24 h show various multilayer structures containing cytoplasmic materials at various stages of degradation, which is typical features of autophagy (4a (8 μM) and 4b (8 μM) at a higher dose. The results indicate that 4a and 4b can induce autophagy and apoptosis simultaneously.Based on morphological criteria, classical cell death pathways include Type I (apoptosis), Type II (autophagic cell death), and Type III (necrosis)utophagy 44. Howev4a (4 μM) and 4b (4 μM) for 24 h. The well-known autophagy inducer rapamycin was used as a positive control for autophagy induction4a (4 μM) and 4b (4 μM) at 24 h show a punctate pattern of GFP-LC3 fluorescence (GFP-LC3 dots), which is similar to the phenomenon observed in cells treated with rapamycin (1 μM) (4a and 4b (Autophagy is characterized by the conversion of microtubule-associated protein 1 light chain 3 (LC3) from the cytoplasmic LC3-I to its membrane-bound form LC3-IIn (1 μM) . Westerna and 4b .4a and 4b was also detected by acridine orange (AO). AO emits green fluorescence in nuclear and cytoplasm. While in acidic compartments, it emits red fluorescence4a and 4b at a concentration of 4 μM for 24 h (4a and 4b.The formation of acidic vesicular organelles (AVOs), is another morphological characteristic of autophagy47for 24 h . The incfor 24 h . These r4a and 4b4a  and 4b , respectively. In contrast, pre-treatment of cells with 3-MA can enhance cell death induced by cisplatin, which is consistent with literature reportsAutophagy is a complicated biological process, which can play either pro-survival or pro-death roles in anticancer therapy4a and 4b. Apoptosis is featured by a range of defined morphological events  and biological events 5253Based on previous results obtained by TEM, we further characterized apoptosis induced by 4a and 4b was examined by 2′-(4-ethoxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5′-bi-1H-benzimidazole trihydrochloride (Hoechst 33342) staining. Vehicle-treated cells show normal morphology and homogeneous nuclear staining pattern . At a concentration of 8 μM, the percentage of apoptotic cells increases from 8.1% to 75.3% and 60.2% for 4a and 4b, respectively. The apoptosis-inducing capability of 4a and 4b is much higher than that of cisplatin.Annexin V-FITC/PI double staining can differentiate early apoptotic (Annexin V-FITC positive and PI negative), necrotic and late apoptotic (Annexin V-FITC positive and PI positive), and viable (Annexin V-FITC negative and PI negative) cells. Treatment of A549 cells with c phases . Apoptos® 3/7 assay was used to investigate caspase-3/7 activation induced by 4a and 4b  and 4b (by 1.5−fold) at a concentration of 8 μM. Moreover, pre-treatment of A549 cells with the pan-caspase inhibitor, z-Val-Ala-Asp-fmk (z-VAD-fmk), can impair the efficacy of cell death induced by Ir(III) complexes  complexes. Synergistic effects may be achieved by conjugating Ir(III) complexes with other drugs by ester linkage, which is underway in our laboratory.In this work, ten phosphorescent cyclometalated Ir(III) complexes containing H2dcbpy and NH4PF6 were purchased from Alfa Aesar. Cisplatin, rapamycin, DMSO, PLE, PI, NAC, MTT, H2DCFDA, JC-1, 3-MA, CCCP, CQ, AO and Annexin V-FITC apoptosis detection kit were purchased from Sigma-Aldrich. MTDR was purchased from Invitrogen. Lipofectamine® 3000 was purchased from Thermo Fisher Scientific. Hoechst 33342 and z-VAD-fmk were purchased from Beyotime. Caspase-Glo® 3/7 assay kit and CellTiter-Glo® luminescence cell viability assay kit were purchased from Promega.All solvents were of analytical grade (>95% purity). All buffer components were of biological grade and used without further purification. Iridium chloride hydrate, ppy, dfppy, H1 , L2 , L3  and L4  were synthesized according to reported procedures2Cl]2 and [Ir(dfppy)2Cl]2 were prepared according to literature methods57Ligands Lm/z values represent the major peaks in the isotopic distribution. 1H NMR spectra were recorded on a Bruker Avance 400 spectrometer (Germany). Shifts were referenced relative to the internal solvent signals. UV-vis spectra were recorded on a Varian Cary 300 spectriphotometer (USA). Emission spectra were recorded on an FLS 920 combined fluorescence lifetime and steady state spectrometer at 298 K (Japan). ICP-MS of Ir(III) complexes was recorded by X Series 2 ICP-MS . UV-vis absorbance and fluorescence/luminescence emission intensity were recorded by an Infinite M200 Pro microplate reader . TEM images were visualised by JEM 100 CX, and photographed by the Eversmart Jazz program . Confocal microscopy images were obtained by a LSM 710 confocal laser scanning fluorescence microscopy . Flow cytometry analyses were recorded by a BD FACSCaliburTM flow cytometer .Microanalysis  was conducted using a VarioEL CHNS analyser. ESI-MS spectra were recorded on a Thermo Finnigan LCQ DECA XP spectrometer. The quoted 2Cl2/CH3CN , followed by anion exchange with saturated NH4PF6 solution and purification by silica flash column chromatography eluting with CH2Cl2/CH3OH .As shown in Ir(ppy)2(Hdcbpy) (1a), [Ir(ppy)2(L2)](PF6) (3a) and Ir(dfppy)2(Hdcbpy) (1b) were synthesized by literature methods251H NMR 2SO): δ 9.34 , 8.27 , 8.13 , 8.08 , 7.95 , 7.66 , 7.16−7.09 , 7.04 , 6.92 , 6.17 , 3.98 . ESI-MS (m/z): [M−PF6]+ calcd for C36H28IrN4O4, 773.2; found, 773.2. Elemental analysis: calcd (%) for C36H28IrN4O4PF6: C, 47.11; H, 3.07; N, 6.10, found: C, 46.96; H, 2.94; N, 6.09.Red solid, yield: 73.9% (270.5 mg). 1H NMR 2SO): δ 9.27 , 8.28 , 8.11 , 7.95 , 7.68 , 7.12 , 7.04 , 6.93 , 6.16 , 4.39 , 1.80−1.66 , 1.52–1.37 , 0.93 . ESI-MS (m/z): [M−PF6]+ calcd for C42H40IrN4O4, 857.3; found, 857.3. Elemental analysis: calcd (%) for C42H40IrN4O4PF6: C, 50.35; H, 4.02; N, 5.59; found: C, 50.46; H, 3.90; N, 5.49.Red solid, yield: 74.4% (297.3 mg). 1H NMR 2SO): δ 9.24 , 8.28 , 8.18 , 8.10 , 7.95 , 7.69 , 7.19−7.09 , 7.08−7.01 , 6.93 , 6.18 , 4.25−4.12 , 2.17−1.96 , 0.99 . ESI-MS (m/z): [M−PF6]+ calcd for C42H40IrN4O4, 857.3; found, 857.3. Elemental analysis: calcd (%) for C42H40IrN4O4PF6: C, 50.35; H, 4.02; N, 5.59; found: C, 50.16; H, 3.93; N, 5.62.Red solid, yield: 74.8% (298.9 mg). 1H NMR 2SO): δ 9.40 ,8.30 , 8.17–8.10 , 8.05 , 7.74 , 7.24–7.18 , 7.02 , 5.59 , 3.99 . ESI-MS (m/z): [M−PF6]+ calcd for C36H24IrN4O4F4, 845.1; found, 845.1. Elemental analysis: calcd (%) for C36H24IrN4O4PF10: C, 43.69; H, 2.44; N, 5.66; found: C, 43.82; H, 2.49; N, 5.59.Yellow solid, yield: 73.5% (290.0 mg). 1H NMR 2SO): δ 9.36 , 8.30 , 8.17–8.10 , 8.05 , 7.75 , 7.25–7.18 , 7.02 , 5.60 , 4.45 , 1.37 . ESI-MS (m/z): [M−PF6]+ calcd for C38H28IrN4O4F4, 873.2; found, 873.2. Elemental analysis: calcd (%) for C38H28IrN4O4PF10: C, 48.25; H, 3.41; N, 5.92; found: C, 47.95; H, 3.36; N, 5.89.Yellow solid, yield: 77.9% (316.9 mg). 1H NMR 2SO): δ 9.30 , 8.31 , 8.20–8.10 , 8.05 , 7.72 , 7.27–7.15 , 7.06–6.90 , 5.61 , 4.41 , 1.81–1.68 , 1.51–1.38 , 0.95 . ESI-MS (m/z): [M−PF6]+ calcd for C42H36IrN4O4F4, 929.3; found, 929.3. Elemental analysis: calcd (%) for C42H36IrN4O4PF10: C, 46.97; H, 3.38; N, 5.22; found: C, 46.99; H, 3.37; N, 5.22.Yellow solid, yield: 74.6% (320.0 mg). 1H NMR 2SO): δ 9.30 , 8.30 , 8.19–8.12 , 8.05 , 7.76 , 7.27–7.18 , 7.03 , 5.60 , 4.24–4.12 , 2.06 , 1.00 . ESI-MS (m/z): [M−PF6]+ calcd for C42H36IrN4O4F4, 929.3; found, 929.3. Elemental analysis: calcd (%) for C42H36IrN4O4PF10: C, 46.97; H, 3.38; N, 5.22.; found: C, 46.92; H, 3.37; N, 5.24.Yellow solid, yield: 75.8% (325.3 mg). 2Cl2 solution. X-ray diffraction measurements were performed on a Bruker Smart 1000 CCD diffractometer (Germany) with Mo Kα radiation (λ = 0.71073 Å) at 298(2) K. Crystal structures of 2a (CCDC deposition no. 1469086) and 2b (CCDC deposition no. 1469087) were solved by direct methods with the program SHELXLRed crystals of 2a and yellow crystals of 2b were obtained by the diffusion of diethyl ether to the CHIr(III) complexes (20 μM) were dissolved in freshly prepared Tris-HCl  buffer. Each sample contained 100 μL Ir(III) solution and 0.6 units PLE. After the samples were incubated at 298 K for 2 h, 400 μL acetone (253 K) was added into each sample to quench the enzymatic hydrolysis. Then the samples were centrifuged at 15000 × g for 10 min. The supernatant was collected and analyzed by ESI-MS.Po/w) was determined by the flask-shaking method and analyzed by ICP-MS. Equal amounts of PBS and n-octanol were shaken in the oscillator for 24 h. The mixture was separated to obtain oil and water phases. The mixtures were centrifuged at 3000 × g for 5 min. Equal volumes of n-octanol and PBS were added to the supernatant. The mixtures were shaken for another 24 h, and then centrifuged at 3000 × g for 5 min. The oil and water phases were separated carefully and dried under vacuum. The dried sample was dissolved by HNO3  and diluted to a final volume of 10 mL with Milli-Q water containing 10 ppb In. The Ir(III) concentration (Co or Cw) was measured by ICP-MS using In as the internal reference. The Po/w value corresponds to Co/Cw.The lipophilicity . Cells were cultured in DMEM  or RPMI 1640  medium, containing 10% FBS , 100 μg/mL streptomycin and 100 U/mL penicillin (Gibco BRL). Cells were cultured in a humidified incubator, which provided an atmosphere of 95% air and 5% CO3  at room temperature for 24 h. The solution was diluted to a final volume of 10 mL with Milli-Q water containing 10 ppb In. The iridium content was measured by ICP-MS using In as the internal reference.A549 cells were cultured in 60 mm dishes and incubated for 24 h. The medium was removed and replaced with medium-DMSO  containing Ir(III) complexes (20 μM). After 1 h incubation, cells were trypsined and collected in PBS. After counted by hemocytometer, cells were digested with HNOThe cytotoxicity of the tested complexes towards different cell lines was determined by MTT assay. Cells were cultured in 96-well plates for 24 h. Cells were incubated with a series of concentrations of complexes for another 44 h. 20 μL MTT solution (5 mg/mL) was added into each well and incubated for another 4 h. Then culture media was removed and 150 μL DMSO was added into each well. The plate was shaken for 10 min. The absorbance at 595 nm was measured by a microplate reader.4a (5 μM) and 4b (5 μM) for 15 min at 310 K. MTDR (100 nM) was added and the cells were incubated for another 15 min. Cells were washed with PBS for three times and visualized immediately by confocal microscopy. The excitation wavelength of Ir(III) complexes was 405 nm. The excitation wavelength of MTDR was 543 nm. Emission was collected at 600‒700 nm (4a), 550‒650 nm (4b) and 645‒685 nm (MTDR).A549 cells were seeded in 35 mm dishes for 24 h and incubated with 4a (5 μM) or 4b (5 μM) for 30 min at 310 K or 277 K. For the inhibition studies, cells were pre-incubated with CCCP (10 μM) or CQ (50 μM) for 1 h, and then incubated with 4a (5 μM) or 4b (5 μM) for 30 min at 310 K. Cell images were captured by confocal microscopy. The excitation wavelength of Ir(III) complexes was 405 nm. Emission was collected at 600‒700 nm (4a) and 550‒650 nm (4b).A549 cells were seeded in 35 mm dishes for 24 h and incubated with 4a and 4b at the indicated concentrations for 6 h. Then cells were collected and stained with JC-1 (5 μg/mL) for 15 min at 310 K. Cells were washed twice with PBS, and analysed by flow cytometry immediately. JC-1 was excited at 488 nm and monitored simultaneously at 515‒545 nm (Green) and 575‒605 nm (Red) by flow cytometry. Data were analysed using FlowJo 7.6 software . 10, 000 events were acquired for each sample.A549 cells were seeded in 60 mm dishes and incubated with ® luminescence cell viability assay kit according to the manufacturer’s protocol. A549 cells were cultured in an opaque-walled 96-well plate and treated with 4a and 4b at the indicated concentrations for 6 h. 100 μL CellTiter-Glo® reagent was added into each well containing 100 μL medium. The plate was incubated at room temperature for 30 min. Luminescence intensity was measured by a microplate reader.ATP production was measured using the CellTiter-Glo4a and 4b at the indicated concentrations for 6 h. Then cells were collected and incubated with 10 μM H2DCFDA in serum-free DMEM at 310 K for 15 min. Cells were washed twice with serum-free DMEM, and measured by confocal microscopy immediately. The excitation wavelength of DCF was 488 nm. Emission was collected at 510‒540 nm.A549 cells were seeded in 60 mm dishes and incubated for 24 h, after which the cells were treated with 4a and 4b at the indicated concentrations for 6 h. Cells were collected and stained by 10 μM H2DCFDA in serum-free DMEM at 310 K for 15 min. Cells were analysed by flow cytometry immediately. The excitation wavelength of DCF was 488 nm. Emission was collected at 510‒540 nm. Data of flow cytometry were analysed by FlowJo 7.6 software.A549 cells were seeded in 60 mm dishes and incubated for 24 h. Then cells were treated with 4a and 4b for 24 h. Cell viability was measured by MTT assay.A549 cells were cultured in 96-well plates and grown to confluence. Cells were pre-treated with NAC (10 μM) for 1 h, and then incubated with various concentrations of 4a and 4b at the indicated concentrations for 24 h. After incubation, the cells were collected and fixed with 70% ethanol. After an overnight storage at 253 K in 70% ethanol, cells were washed twice with PBS and suspended with 500 μL of staining solution containing PI (10 μg/mL) and DNAse-free RNase (100 μg/mL), and then analysed by flow cytometry. Data of flow cytometry were analysed by ModFit LT 2.0 software .A549 cells were seeded in 60 mm dishes and incubated with 4a (5 μM) and 4b (5 μM) at 310 K for 24 h. Cells were collected and fixed overnight at 277 K in phosphate buffer (pH 7.4) containing 2.5% glutaraldehyde. Cells were then treated with osmium tetroxide, and stained with uranyl acetate and lead citrate, and visualized under transmission electron microscopy . Images were photographed using the Eversmart Jazz program (Scitex).A549 cells were treated with ® 3000 . After 24 h, cells were treated with tested complexes for 24 h and observed by the confocal microscopy. Emission was collected at 500–540 nm upon excitation at 488 nm.A549 cells were transfected with the GFP-LC3 vector using Lipofectamine4a or 4b for 24 h. Cells were collected and washed twice with ice-cold PBS, and lysed on ice in radioimmunoprecipitation assay  containing proteases inhibitors . Equal amounts of proteins were added and separated on SDS-polyacrylamide gel electrophoresis. Proteins on the gel were transferred onto a polyvinylidenedifluoride membrane. The membrane was blocked in 20 mM Tris, 150 mM NaCl, and 0.05% Tween 20  containing 5% non-fat dried milk at pH 8.0, and then incubated overnight at 277 K with the primary antibodies specific to β-actin and LC3 proteins. The membrane was incubated with corresponding horseradish peroxidase-conjugated secondary antibody after washing three times. The immunoreactivity was recorded by FluorChem M .A549 cells were seeded into 60 mm dishes and incubated for 24 h, and then exposed to complexes 4a or 4b for 24 h, after which the cells were stained with AO at a final concentration of 1 μg/mL for 15 min at 310 K. Cells were washed twice by PBS, and analysed by confocal microscopy immediately. The excitation wavelength of AO was 488 nm. Emission was collected at 500–550 nm (Green) and 670–720 nm (Red).A549 cells were seeded in 60 mm dishes and incubated for 24 h. Then the cells were treated with 4a or 4b for 24 h. Cells were stained with AO (1 μg/mL) for 15 min at 310 K and washed twice by PBS before collected. Cells were analysed by flow cytometry immediately. The excitation wavelength of AO was 488 nm. Emission was collected at 670‒710 nm. Data were analysed by FlowJo 7.6 software.A549 cells were seeded in 60 mm dishes and incubated for 24 h, then cells were treated with Cells were cultured in a 96-well plate and grown to confluence. Cells were pre-treated with or without 3-MA (5 mM) for 1 h, then incubated with the complexes at the indicated concentrations for 24 h. Cell viability was measured by MTT assay.4a or 4b at indicated concentrations for 24 h at 310 K, and then washed with ice-cold PBS and fixed with 4% paraformalclehyde for 30 min on ice. Cells were washed with ice-cold PBS twice. Then the cells were incubated with Hoechst 33342 (5 μL/mL in PBS) at room temperature in the dark for 10 min. The cells were washed twice with ice-cold PBS and visualized by confocal microscopy. The excitation wavelength of Hoechst 33342 was 405. Emission was collected at 440‒480 nm.A549 cells were seeded in 35 mm dishes for 24 h and incubated with 4a and 4b at indicated concentrations at 310 K for 24 h. Cells were collected and suspended with binding buffer, then stained with Annexin V-FITC and PI for 10 min on ice in the dark. Cells were analysed by flow cytometry immediately. Data were analysed by FlowJo 7.6 software. The excitation wavelength of both Annexin V-FITC and PI was 488 nm. Emission was collected at 505–545 nm (Annexin V-TITC) and 595–635 (PI) separately.The assay was performed according to the manufacturer’s instructions. A549 cells were seeded in 60 mm dishes and incubated for 24 h. Then the cells were incubated with ® Assay Kit according to the manufacturer’s protocol. A549 cells were cultured in opaque-walled 96-well plates and treated with 4a and 4b at the indicated concentrations for 6 h. 100 μL of Caspase-Glo® reagent was added into each well containing 100 μL culture media. The mixture was incubated at room temperature for 30 min. The luminescence was measured by a microplate reader.Caspase-3/7 activity was measured using Caspase-Glo4a and 4b at the indicated concentrations for 24 h. Cell viability was measured by MTT assay.Cells were cultured in 96-well plates and grown to confluence. Cells were pre-treated with z-VAD-fmk (25 μM) for 1 h, and then incubated with All biological experiments were performed at least twice with triplicates in each experiment. Representative results are presented as the means ± standard deviations.How to cite this article: Wang, F.-X. et al. Ester-Modified Cyclometalated Iridium(III) Complexes as Mitochondria-Targeting Anticancer Agents. Sci. Rep.6, 38954; doi: 10.1038/srep38954 (2016).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


We discovered that IL-18 acts synergistically with IL-10 to amplify the production of Mφ-derived mediators like osteopontin (OPN) and thrombin, yielding thrombin-cleaved form of OPN generation, which acts through integrins α4/α9, thereby augmenting M2 polarization of Mφ with characteristics of increasing surface CD163 expression in association with morphological alteration. Furthermore, the results of visualizing temporal behavior and morphological alteration of Mφs during angiogenesis demonstrated that M2-like Mφs induced excessive angiogenesis through the direct cell–cell interaction with endothelial cells, possibly mediated by CD163.M2 macrophage (Mφ) promotes pathologic angiogenesis through a release of pro-angiogenic mediators or the direct cell–cell interaction with endothelium in the micromilieu of several chronic inflammatory diseases, including rheumatoid arthritis and cancer, where interleukin (IL)-18 also contributes to excessive angiogenesis. However, the detailed mechanism remains unclear. The aim of this study is to investigate the mechanism by which M2 Mφs in the micromilieu containing IL-18 induce excessive angiogenesis in the  Macrophages (Mφs) play essential roles in tissue homeostasis and immunity , and exiExcessive angiogenesis is a key pathological hallmark of numerous chronic inflammatory diseases such as RA and cancer , 9. In tThe aim of this study is to investigate the mechanism by which Mφ induces excessive angiogenesis in micromilieu containing IL-18 in addition to visualize the temporal behavior and morphological alteration in Mφ during angiogenesis. As a result, we discovered here that IL-18 acts synergistically with IL-10 to amplify the production of OPN and thrombin as soluble mediators derived from Mφ, yielding Thr-OPN generation, which in turn, augments M2 polarization of Mφ with characteristics of increasing CD163 expression in association with morphological alteration. Furthermore, CD163 may be responsible for mediating the direct cell–cell interaction between these Mφs and endothelial cells, ultimately resulting in the excessive angiogenesis , and the mouse endothelial cell line, b.End5 cells , were cultured in Dulbecco’s modified Eagle’s medium supplemented with 2 mM 4 cells in 24-well plates followed by treatment with recombinant mouse TNF-α , IL-10 , IL-18 , IL-13 , and IL-33  either alone or in combination. In some experiments, hirudin, a thrombin inhibitor , or neutralizing antibodies (Abs) either against OPN , integrin α4 , integrin α9 , vascular endothelial growth factor (VEGF) , or M-CSF , or their isotype-matched control Abs were added concomitantly with the above cytokines. After incubating for 24 h at 37°C under 5% CO2, cells were harvested and rinsed with FACS wash buffer consisting of phosphate-buffered saline (PBS) supplemented with 2.5% normal horse serum, 0.1% sodium azide, and 10 mM HEPES followed by centrifugation . Cells were then stained with anti-mouse Abs against phycoerythrin (PE)-conjugated CD 54 , allophycocyanin (APC)-conjugated CD86 , fluorescein isothiocyanate (FITC)-conjugated CD68 , FITC-conjugated CD163 , FITC-conjugated CD206 , APC-conjugated M-CSF1 receptor (M-CSF1R) , Alexa Fluor 647-conjugated IL-18Rβ , FITC-conjugated integrin α4 , or PE-conjugated integrin α9  for 30 min at 4°C. After rinsing with FACS wash buffer followed by centrifugation , 300 µL of PBS was added to the residue followed by staining with propidium iodide (PI)  to exclude PI-positive dead cells. Thereafter, cells were analyzed with a CantoII FACS (BD Biosciences). Data were processed using BD FACSDiva software (BD Biosciences) to determine the mean fluorescence intensity (MFI) of each surface protein.RAW264.7 cells were seeded at 5.0 × 104 cells in 24-well plates, after which incubated with either TNF-α (5 ng/mL) or IL-10 (10 ng/mL) in the presence or absence of IL-18 (100 ng/mL) for 24 h at 37°C under 5% CO2. The cells were then rinsed with PBS to remove cytokines and supplemented with fresh medium, followed by incubation for 0, 24, or 48 h at 37°C under 5% CO2. Subsequent procedures were conducted as described above.In the experiment to check the duration of Mφ polarization status after cessation of cytokine stimuli, RAW264.7 cells were seeded at 5.0 × 104 cells in 24-well plates and stimulated by either TNF-α (5 ng/mL) or IL-10 (10 ng/mL) alone or each combined with different concentrations of IL-18 from 0.1 to 200 ng/mL. After incubating for 24 h at 37°C under 5% CO2, cells were harvested and rinsed twice with FACS wash buffer followed by centrifugation . Thereafter, the supernatant was removed, followed by addition of PBS. The remaining cells were stained with PI (2 µg/mL) and were analyzed with a CantoII FACS (BD Biosciences). Data were processed using BD FACSDiva software (BD Biosciences) to determine the percentage of PI-positive dead cells in each Mφ subset. Note that the concentrations of IL-18 used in this study are appropriate for in vitro experiments as described previously (RAW264.7 cells were seeded at 5.0 × 10eviously –33.4 cells in 24-well plates followed by treatment with IL-10 (10 ng/mL) and IL-18 (100 ng/mL) either alone or in combination for 24 h at 37°C under 5% CO2. Then, cells were washed with PBS for two times repeat and incubated with dextran-FITC  for 1 h at 37°C under 5% CO2. Thereafter, cells were washed with PBS and harvested followed by centrifugation , after which 300 µL of PBS was added to the residue and cells were stained with PI (2 µg/mL) to exclude PI-positive dead cells and subsequently analyzed with a CantoII FACS (BD Biosciences). Data were processed using BD FACSDiva software (BD Biosciences) to determine the MFI of FITC-dextran.RAW264.7 cells were seeded at 5.0 × 104 cells/100 μL) were gently seeded on top of the gel. Subsequently, polarized Mφs , VEGF , or M-CSF , or respective isotype-matched control Abs to the medium. In another experiment to assess the effect of thrombin antagonist on the tube formation induced by VEGF, b.End5 cells were incubated with media containing different doses of recombinant mouse VEGF  in the presence or absence of hirudin (1 µg/mL) or anti-VEGF Ab  on Matrigel in the same condition described above. After incubation, tube-like structures were visualized with 8 µg/mL of calcein acetoxymethylester (AM)  and photomicrographs were taken at 5–8 µm intervals for z-axis obtained at magnification 100× with a confocal laser C2 microscope (Nikon). The areas and total lengths of the tubes were calculated with NIS-Elements Ar software (Nikon) and evaluated as the degree of tube formation according to previous reports  were incubated on Matrigel for 16 h at 37°C under 5% CO2 in the presence of either TNF-α (5 ng/mL) or IL-10 (10 ng/mL) alone or each combined with IL-18 (0.1–200 ng/mL) instead of Mφs.To examine cell number-dependent changes in the degree of tube formation, b.End5 cells were seeded at 1.0–4.0 × 104 cells/well of b.End5 cells was gently seeded on top of the gel in the lower chamber. Thereafter, polarized RAW264.7 cells . The areas and total lengths of the tubes were calculated with NIS-Elements Ar software (Nikon) and evaluated as the degree of tube formation.To further determine the influence of soluble mediators derived from each Mφ subset on the angiogenesis, we constructed an indirect contact coculture model with transwell plates in the Matrigel tube formation assay. Twenty four-well transwell plates were filled with 200 µL Matrigel and allowed to solidify at 37°C for 30 min. Then, a total of 8.0 × 10ells see  were rin4 cells in 24-well plates were treated with IL-10 (10 ng/mL) in combination with IL-18 (100 ng/mL) for 24 h at 37°C under 5% CO2. The subsequent procedure was performed in a manner similar to the Matrigel Tube Formation Assay except for the cell numbers of PKH67-labeled b.End5 cells (4.0 × 104/100 μL) and PKH26-labeled Mφ (IL-10 + IL-18) (1.0 × 104/100 μL) when cocultured on Matrigel.To distinguish between endothelial cells and Mφs in the Matrigel tube formation assay, b.End5 cells were fluorescently labeled with PKH67  and RAW264.7 cells with PKH26  according to the manufacturer’s protocol. After that, PKH26-labeled RAW264.7 cells seeded at 5.0 × 104 cells and allowed to adhere to 24-well plates for 4 h at 37°C under 5% CO2. Then, 1.0 × 104 cells of Mφ (–), Mφ (IL-10), Mφ (IL-18), or Mφ (IL-10 + IL-18) were overlaid onto fluorescent b.End5 cells and cocultured for 16 h at 37°C under 5% CO2. Subsequently, photomicrographs were taken at 2–5 µm intervals for z-axis obtained at magnification 100× with a confocal laser C2 microscope (Nikon). The area of PKH67-positive region was calculated with NIS-Elements Ar software (Nikon) and evaluated as the number of surviving b.End5 cells.PKH67-labeled b.End5 cells were seeded at 4.0 × 105 cells) were gently seeded on top of the gel, followed by cocultured with PKH26 (red)-labeled Mφ (–), Mφ (IL-10), Mφ (IL-18), or Mφ (IL-10 + IL-18) (1.5 × 105 cells) on Matrigel. After that, 35-mm dish was placed on the stage of an All-in-One fluorescence microscope BZ-X710 (Keyence) equipped with an environmental chamber, which provided 37°C, humidity and 5% CO2 conditions. Time-lapse live-cell imaging of Matrigel tube formation assay was obtained at 1–3 µm intervals for the z-axis every 10 min over 16 h with a set of green and red emissions at original magnification 200×. Obtained images were processed and analyzed with BZ-X710 software (Keyence).35-mm dish was filled with 800 µL of Matrigel and allowed to solidify at 37°C for 30 min. Subsequently, PKH67 (green)-labeled b.End5 cells (6.0 × 104 cells in 24-well plates were incubated in the medium containing IL-10 (10 ng/mL) and IL-18 (100 ng/mL) alone or their combination for 24 h at 37°C under 5% CO2 condition. Thereafter, 35-mm dish was filled with 800 µL of Matrigel and allowed to solidify at 37°C for 30 min. Then, b.End5 cells (6.0 × 105 cells) were gently seeded on top of the gel. Subsequently, Mφ (–), Mφ (IL-10), Mφ (IL-18), or Mφ (IL-10 + IL-18) were rinsed with PBS followed by coculture of b.End5 cells with each subset of Mφs (1.5 × 105 cells) on Matrigel for 4 h at 37°C under 5% CO2 condition. For SEM analysis, 35-mm dishes were rinsed with 0.1 M PBS pH 7.4 at 4°C for 60 min and were fixed with 2.5% (v/v) glutaraldehyde in 0.1 M PBS pH 7.4 at 4°C for overnight. After rinsing with 0.1 M PBS pH 7.4 at 4°C for 60 min, cells were post-fixed with 1% (w/v) osmium tetroxide or at 4°C for 2 h followed by washing with 0.1 M PBS pH 7.4 at 4°C for 60 min, dehydration in an ascending ethanol series . The samples were coated with platinum-palladium freeze-dried three times with t-butyl alcohol for 20 min each and observed using a SEM SU3500 (Hitachi High-Technologies) at 15 kV.RAW264.7 cells seeded at 5 × 105 cells in six-well plates) with either IL-10 (10 ng/mL) or IL-18 (100 ng/mL) alone or their combination for 24 h at 37°C under 5% CO2, total RNA was extracted using Trizol (Thermo Fisher Scientific). The quality and quantity of total RNA were measured with a NanoDrop One spectrophotometer (Thermo Fisher Scientific) and 100–1,000 ng of total RNA was used as a template. Real-time quantitative RT-PCR amplifications were performed in 96-well optical reaction plates using an Applied Biosystems 7900HT Fast Real Time PCR System (Thermo Fisher Scientific) with a One Step SYBR Prime Script PLUS RT-PCR kit  and the gene-specific primers  at 42°C for 5 min, thereafter 95°C for 10 s followed by 40 cycles of 95°C for 5 s and 60°C for 30 s, finally 95°C for 15 s. The amounts of target mRNA, Spp1 (the gene coding OPN), Prothrombin, Mmps-2, -3, -7, and -9 were normalized to the level of glyceraldehyde-3-phosphate dehydrogenase (Gapdh) mRNA amplified from the same sample, and then to untreated control mRNA. Data were analyzed with the calibration curve method using 7900HT Fast system SDS software version 2.4 (Thermo Fisher Scientific). The gene-specific primer sequences are given in Table After treatment of RAW264.7 cells  and IL-18 (1–100 ng/mL), or concomitantly with hirudin (1 µg/mL) for 24 h at 37°C under 5% CO2. Then, cells were harvested and washed with PBS, and subsequently lysed in radio-immunoprecipitation assay (RIPA) buffer containing protease inhibitors for 30 min on ice. The supernatant of the resulting suspension was obtained after centrifugation  and collected as the total cell lysate. The total protein concentration was quantified using a Bradford protein assay kit (Bio-Rad Laboratories).RAW264.7 cells seeded at 2.0 × 10Total lysates of RAW264.7 cells were diluted with an equal volume of 2× Laemmli buffer containing 0.5 M Tris–HCl (pH 6.8), 10% sodium dodecyl sulfate (SDS), 12% β-mercaptoethanol, 20% glycerol, and 0.1% bromophenol blue, then heated for 5 min at 97°C. 10 µg of total protein were loaded and separated by SDS-polyacrylamide gel electrophoresis followed by transfer onto a nitrocellulose membrane (Bio-Rad Laboratories). The membrane was incubated in blocking buffer containing 5% non-fat dry milk  or 10% bovine serum albumin  in PBS-T (PBS pH 7.6 with 0.1% Tween-20) for 60 min at room temperature. The membrane was then probed with primary anti-mouse Abs against M-CSF , OPN , OPN N-Half [34E3 which reacts specifically with the N-terminal fragment of OPN cleaved by thrombin ], thrombin , VEGF , VEGF receptor (VEGFR) 1 , and VEGFR2  as well as actin  or GAPDH  used as internal controls in respective blocking buffer at 4°C overnight. Blots were then washed with PBS-T and incubated with horseradish peroxidase-conjugated secondary Abs against goat IgG , mouse IgG , rabbit IgG , or rat IgG   in respective blocking buffer for 60 min at room temperature. After washing with PBS-T, the immune-complexes were visualized using Pierce Western Blotting Substrate . The signal intensity of the immune reactive bands was detected with Amersham Imager 600 . The relative band intensities were quantified using Image Quant TL software . Values for M-CSF, OPN, OPN N-Half, thrombin, and VEGF relative to GAPDH, or values for VEGFR1 and VEGFR2 relative to actin were normalized to untreated cells.5 cells in six-well plates were stimulated with IL-10 (10 ng/mL) in the presence or absence of IL-18 (100 ng/mL) for 24 h at 37°C under 5% CO2. Thereafter, cells were harvested and placed in a clean test tube, and centrifuged . The supernatant was then removed, and the cells washed with PBS followed by centrifugation . After removing the supernatant, the cells were washed with PBS and further centrifuged  followed by elimination of the supernatant. The remaining cells were lysed in RIPA buffer containing protease inhibitors for 30 min on ice. Subsequently, the cell suspension was centrifuged , and the supernatant was transferred into a clean test tube as total cell lysate. The total protein concentration of each sample was quantified by the Bradford method (RAW264.7 cells seeded at 2.0 × 105 cell/dish to 35-mm glass bottom dish (Matsunami Glass) for 2 h, cells were incubated with IL-10 (10 ng/mL) in the presence or absence of IL-18 (100 ng/mL) for 24 h at 37°C under 5% CO2. Thereafter, cells were washed with PBS and fixed with 4% formaldehyde (PFA) at room temperature for 30 min followed by washing with PBS-T. Subsequently, cells were incubated in blocking buffer containing 1% BSA, 0.3 M glycine in PBS-T at room temperature for 1 h to permeabilize cell membrane and to block non-specific protein–protein interactions. After that, they were incubated with primary Abs directed against OPN , thrombin , integrin α4 , integrin α9 , or CD163  in blocking buffer at 4°C overnight. After rinses in PBS-T, cells were incubated with Alexa Fluor 488-conjugated secondary Ab against rabbit IgG , Alexa Fluor 488-conjugated secondary Ab against mouse IgG , or Alexa Fluor 594-conjugated secondary Ab against goat IgG  at room temperature for 1 h. Then, cells were washed with PBS-T followed by counterstaining with 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI) , thereafter photomicrographs were taken at 0.6–0.8 µm intervals for z-axis at original magnification 400× with a confocal laser C2 microscope (Nikon). The two- or three-dimensional images were reconstructed from obtained pictures using NIS-Elements Ar software (Nikon).After adhering RAW264.7 cells seeded at 1 × 10P < 0.05 was considered as significant.Data are presented as means ± SEM. Statistical analysis was performed using Prizm version 3 software (GraphPad Software). Statistical significance was assessed using a one-way analysis of variance followed by Dunnett’s or Tukey’s test for multiple comparisons. A value of According to the recent proposal of nomenclature for Mφ subsets linked to the stimulation scenarios , Mφs treTreatment of RAW264.7, the mouse Mφ-like cell line, with TNF-α or IL-10 increased Mφ M1 or M2 markers expression, respectively, in comparison with Mφ (–), resulting in a shift of population in CD86-positive/CD163-negative region by TNF-α or opposite shift by IL-10 Figures . These rInterleukin-18 alone influenced none of M1/M2 marker expression at concentrations from 0.1 to 200 ng/mL Figure , 27. WhiSimilar to the approach of previous studies , 35, we via direct cell–cell interaction from 3 to 8 h, driving a rapid induction of tubulogenesis, whereas Mφs (–) and Mφs (IL-18) hardly move or communicate with endothelial cells. Thereafter, numerous Mφs (IL-10 + IL-18) apparently gathered around the leading edge of the growing vascular network and/or branching points of vasculature where they interacted with endothelium, allowing vascular tube to get thicker and thicker. The acceleration of tubulogenesis was almost completed until 12 h and reached a plateau phase toward the end of observation period -labeled b.End5 cell was in contact with each other from far and wide. Subsequently, vascular segments consists of b.End5 cells connected to each other through the help of PKH26 (red)-labeled Mφs (IL-10 + IL-18) and Mφs (IL-10) mediated Because Mφs have been demonstrated to associate tightly with capillaries in the progression of angiogenesis , 54, we Spp1, the gene coding OPN . Given that IL-13 and IL-33 exhibited considerably different characters from IL-18, further studies are required to understand the influence of these cytokines on the polarization status and angiogenic capacity of Mφ.Our results identified IL-18 as a selective amplifier for an increase in CD163 level exhibited by IL-10, that is, the aug-mentation of M2 polarization Figures . By contn Figure , orientevia direct cell–cell interaction, eventually growing vascular network. In the in vivo studies, tissue resident Mφ also accelerates vascular development by bridging the neighboring tip cells and by mediating anastomosis of adjacent capillary tubes  and Mφ (IL-10 + IL-18) were similar to those in Mφ (–), while Mmp-2/9 mRNA levels were greatly increased in Mφ (TNF-α) relative to Mφ (–) , suggesting there is no involvement of MMPs in the actions of IL-10 and IL-18. Together, VEGF and MMPs detected at higher levels in the RA and tumor foci may be derived from endothelial cells or fibroblasts and all that other than Mφs among components of the micromilieu.On the other hand, OPN is cleaved not only by thrombin but also by matrix metalloproteinase (MMP)-2/3/7/9 , among wOther importance is that M-CSF has been classified as an M2 stimulus and produces some growth factors, such as VEGF . Intriguvia CD163.In conclusion, we demonstrated that IL-18 with IL-10 synergistically amplifies the expression of OPN and thrombin as soluble mediators derived from Mφ, yielding Thr-OPN generation which acts through α4/α9 integrins, ultimately augment M2 polarization of Mφ with characteristics of increasing CD163 expression. These Mφs strongly induced angiogenesis by modulating the direct contact with endothelium, highlighting a potential role of CD163 in the excessive angiogenesis probably through the direct cell–cell interaction between M2-like Mφs and endothelial cells (Figure Conceptualization: HT; methodology: TK, SH, HW, SM, and HT; formal analysis: TN and AN; investigation: TK and YY; resources: SH, AK, SN, TY, and MN; writing-original draft: TK; writing, review, and editing: TK, MN, and HT; visualization: TK, YY, and TN; supervision: SN, SM, TY, MN, and HT; funding acquisition: TK, AK, AN, MN, and HT.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Pgc-1α and its various isoforms may play a role in determining skeletal muscle mitochondrial adaptations in response to diet. 8 wks of dietary supplementation with the flavonoid quercetin (Q) or red onion extract (ROE) in a high fat diet (HFD) ameliorates HFD-induced obesity and insulin resistance in C57BL/J mice while upregulating Pgc-1α and increasing skeletal muscle mitochondrial number and function. Here, mice were fed a low fat (LF), high fat (HF), high fat plus quercetin (HF + Q), or high fat plus red onion extract (HF + RO) diet for 9 wks and skeletal muscle Pgc-1α isoform expression and DNA methylation were determined. Quantification of various Pgc-1α isoforms, including isoforms Pgc-1α-a, Pgc-1α-b, Pgc-1α-c, Pgc-1α4, total NT-Pgc-1α, and FL-Pgc-1α, showed that only total NT-Pgc-1α expression was increased in LF, HF + Q, and HF + RO compared to HF. Furthermore, Q supplementation decreased Pgc-1α-a expression compared to LF and HF, and ROE decreased Pgc-1α-a expression compared to LF. FL-Pgc-1α was decreased in HF + Q and HF + RO compared to LF and HF. HF exhibited hypermethylation at the −260 nucleotide (nt) in the Pgc-1α promoter. Q and ROE prevented HFD-induced hypermethylation. −260 nt methylation levels were associated with NT-Pgc-1α expression only. Pgc-1α isoform expression may be epigenetically regulated by Q and ROE through DNA methylation. Environsistance –11. Enerpression , 4–6, 12pression , whereaspression , 13. Trativation ; howeverl inputs , 16. Exel muscle , 16. Pgc-1α regulated posttranslationally and transcriptionally, but also several Pgc-1α isoforms with novel and specific activities have been identified [ Pgc-1α isoforms are also regulated by environmental stimuli, including cold exposure and exercise training, and by the obese state [ Pgc-1α are known to exist, arising from a combination of distinct promoter start sites and alternative splicing [ Pgc-1α-b, Pgc-1α-c, Pgc-1α4, the n-truncated splice variant NT-Pgc-1α, and the full length variant FL-Pgc-1α [ Pgc-1α-b, Pgc-1α-c, Pgc-1α4, total NT-Pgc-1α, and FL-Pgc-1α [ Pgc-1α-a, Pgc-1α-b, Pgc-1α-c, total NT-Pgc-1α, and FL-Pgc-1α are all known to play a role in exercise-induced mitochondrial adaptations in skeletal muscle [ Pgc-1α4 plays a role in promoting muscle hypertrophy [ NT-Pgc-1α , derived from either the proximal or distal promoters, have been characterized. All NT-Pgc-1α isoforms have been shown to be induced during either high intensity (NT-Pgc-1α-a) or low intensity (NT-Pgc-1α-b and NT-Pgc-1α-c) exercise, contributing to upregulation of total NT-Pgc-1α that occurs at all intensities of exercise [ NT-Pgc-1α isoforms and total NT-Pgc-1α are upregulated in skeletal muscle in response to AICAR treatment, a pharmacological activator of AMPK known to increase mitochondrial biogenesis and fatty acid oxidation in skeletal muscle [ Pgc-1α-a, Pgc-1α-b, Pgc-1α-c, Pgc-1α4, total NT-Pgc-1α, and FL-Pgc-1α. Expression patterns of these various splice variants may have the ability to prevent disease, as overexpression of total NT-Pgc-1α is also known to attenuate HFD-induced obesity [ FL-Pgc-1α and total NT-Pgc-1α expression in skeletal muscle in relation to cardiovascular disease during overweight and obesity [ Pgc-1α isoform expression may be epigenetically regulated during disease and in response to environmental stimuli, including dietary inputs.Not only isentified –19. The se state , 17–22. splicing . The mosL-Pgc-1α , 23, 24.ertrophy . More re obesity . Interes obesity  in addit obesity . Thus, P Pgc-1α.Quercetin (Q) is a bioflavanoid that protects against mitochondrial dysfunction and attenuates HFD-induced obesity and insulin resistance when supplemented in a HFD at low concentrations , 27–32.  Pgc-1α DNA methylation in skeletal muscle in association with HFD-induced obesity and insulin resistance and elaborate on how these epigenetic effects associate with splice variant expression, we used diet-induced obese C57BL/6J mice as a model system. We hypothesized that the mitochondrial gene expression patterns previously observed and published [ Pgc-1α isoforms known to play a role in skeletal muscle mitochondrial adaptations in response to environmental stimuli [To investigate the role of dietary fat, purified dietary Q, and ROE in determiningublished  would be stimuli , 21, 25.N = 10/group): LF ; high fat ; HF + Q  with 17 mg/kg quercetin aglycone ; or HF + RO (Research Diets D08072306). Mice were fed respective diets for 9 wks, during which time 48 h food consumption and body weight and composition via nuclear magnetic resonance  were assessed weekly. Mice were euthanized after 9 wks of feeding and quadriceps muscle was extracted and snap frozen in liquid nitrogen for further analyses. All experiments were reviewed and approved by the Pennington Biomedical Research Center Institutional Animal Care and Use Committee.The protocols for animal and diets have been previously published . BrieflyTo provide a more homogenous sample, frozen quadriceps muscle was ground under liquid nitrogen using a mortar and pestle. Ground muscle was then used for the extraction of genomic DNA with a DNeasy Blood and Tissue Kit (Qiagen 69581) via the manufacturer's protocol. The quantity and quality of the gDNA extracted were determined by spectrophotometry using a NanoDrop . Approximately 200 ng of gDNA was subjected to bisulfite conversion using the EpiTect Bisulfite Kit (Qiagen 59104) via the manufacturer's protocol. Pgc-1α gene. Human primer sequences that amplify the region of the Pgc-1α promoter surrounding the −260 nucleotide site have been previously published [Bisulfite sequencing was performed on the Qiagen PyroMark Q24 platform (Qiagen 9001514). Bisulfite converted gDNA was PCR amplified using the EpiTect MSP kit (Qiagen) and biotinylated primers targeting theTotal RNA was extracted from quadriceps muscle tissue using Tri-Reagent  followed by further purification with a RNeasy mini kit , as previously described . The qua Pgc-1α isoforms [qRT-PCR was performed using previously published primer pairs that targeted variousisoforms , 18, 21. P value < 0.05 was used to determine significance.The data were analyzed with GraphPad Prism 5.0 statistical analysis software. Results are expressed as means ± standard error. Food consumption and body weight composition parameters were analyzed by repeated measures ANOVA. All other measurements were analyzed by one-way ANOVA. A Tukey test was used post hoc as necessary. A Pgc-1α expression in skeletal muscle [ Pgc-1α in association with previously observed beneficial mitochondrial adaptations. Contrary to our previous findings, in the present study we found that, after 9 wks of supplementation, neither Q nor ROE altered total Pgc-1α expression despite the previously published improvements in skeletal muscle mitochondrial number in these animals , or proximal (exon 1a) promoters and also due to alternative splicing of exon 1b to produce exon 1b′, whereas NT-Pgc-1α and FL-Pgc-1α both contain the full exonic regions from 1b, 1b/, 1a, and 2  between HF and HF + Q or HF + RO.DNA methylation of the obesity , 33, 34.ptations , 4, 14. d number , 31, 32. Pgc-1α expression and mitochondrial adaptations occur in a dose- and time-dependent manner [ Pgc-1α at 8 wks of supplementation [ Pgc-1α was measured after 9 wks of supplementation. Despite the lack of increase in total Pgc-1α, we continued to observe Q- and ROE-induced increases in energy expenditure and mitochondrial number [ Pgc-1α upregulation at 8 wks. However, mitochondrial turnover occurs rapidly, ranging from ~1 to 17 days in a tissue-specific manner, and this turnover rate can be increased by dietary interventions, such as caloric restriction [ Pgc-1α expression, the continuous impetus of the HFD would lead to similar skeletal muscle physiologies in HF, HF + Q, and HF + RO groups; yet at 9 wks, we still observed increased mitochondrial number [ Pgc-1α methylation in HF + Q and HF + RO. Thus, it appears that although total Pgc-1α remains unchanged, other mechanisms may compensate for this loss of Pgc-1α upregulation to perpetuate beneficial mitochondrial adaptations in HF + Q and HF + RO.Importantly, the effects of dietary Q supplementation on skeletal musclet manner , 31. Ourentation  and, in l number , which mtriction , 36. Thul number  and decr Pgc-1α has several known isoforms that result from alternative splicing and alternate promoter start sites [ Pgc-1α splice variant expression. Indeed, we show here that Q and ROE decreased expression of FL-Pgc-1α and isoform a, without changing isoforms b, c, or 4. Additionally, Q but not ROE decreased Pgc-1α isoform B in comparison to LF but HF. Here, none of the treatments had an effect on Pgc-1α isoforms 4 and c. The only isoform shown to decrease after 9 wks of HFD feeding was NT-Pgc-1α; and, both Q and ROE were able to prevent this HFD-induced decrease. These results are consistent with changes in DNA methylation status at the −260 nt, with HF increasing in methylation but HF + Q and HF + RO showing similar levels of methylation as LF. Thus at 9 wks of feeding, it is possible that the beneficial effects of Q and ROE on mitochondrial number and function are mediated via upregulation of NT-Pgc-1α and that isoform expression is dependent on epigenetic regulation of Pgc-1α. Indeed, NT-Pgc-1α has known complementary and overlapping functions to FL-Pgc-1α and may compensate for loss of FL-Pgc-1α or other isoforms [ Pgc-1α isoform expression is epigenetically regulated by histone methylation in response to exercise, with expression of Pgc-1α-b and of Pgc-1α-c but not of Pgc-1α-a being upregulated in conjunction with increased histone methylation in the promoter of exon 1b compared to exon 1a [Interestingly, DNA methylation has been shown to drive mRNA splicing andrt sites , 21, 26.rt sites , 21, 25.isoforms , 25; and exon 1a . Pgc-1α expression and skeletal muscle mitochondrial adaptations which is located within the proximal promoter upstream of exon 1a. Given that Q and ROE decreased Pgc-1α isoforms Pgc-1α-a and Pgc-1α-b without changing Pgc-1α-c, and Pgc-1α-a includes exon 1a, Pgc-1α-b includes exon 1b, and Pgc-1α-c includes exon 1b′, it appears that −260 nt methylation does not play a role in regulating 5′ splicing or transcription initiation within Pgc-1α proximal versus alternative, distal promoters. This is consistent with the recent publication by Lochmann et al. 2015, who similarly showed that Pgc-1α methylation was not related to Pgc-1α isoform a, b, or c expression [ Pgc-1α expression was also not associated with DNA methylation at the −260 nt. However, it is unclear in previous publications what isoforms of Pgc-1α were measured and correlated with −260 nt DNA methylation. It is possible that previous reports use primers that measure isoforms which are differentially spliced at the 3′ prime end; as in the present study, although we did not observe associations between −260 nt methylation and 5′ splicing or specific promoter initiation, we did see alterations in isoform expression based on splicing at the 3′ region in association with DNA methylation levels at the −260 nt. Notably, FL-Pgc-1α decreased in HF + Q and HF + RO and total NT-Pgc-1α increased. Although alternative splicing that produces these isoforms occurs at exons 6 and 7 and the intronic region between these exons [ Pgc-1α promoter is hypermethylated [Here, we measured the percentage of −260 nt DNA methylation, a methylation site known to regulatepression . Interesse exons  and DNA  Pgc-1α −260 nt methylation induced by HFD feeding, upregulating total NT-Pgc-1α splice variant expression in skeletal muscle. Thus, the perpetuating beneficial effects of Q and ROE on body weight, body composition, energy expenditure, insulin sensitivity, and skeletal muscle mitochondrial number and function in mice after 9 wks of supplementation [ NT-Pgc-1α. There is a further need to study the functions and epigenetic regulation of total NT-Pgc-1α and other isoforms of Pgc-1α to determine their specific roles in regulating skeletal muscle adaptations and contribute to a lean, insulin sensitive state.In conclusion, Q and ROE show similar effects in preventingentation  may be m Pgc-1α hypermethylation at the −260 nt in the Pgc-1α proximal promoter in conjunction with alterations in Pgc-1α splice variant expression in skeletal muscle. Q and ROE similarly prevent HFD-induced hypermethylation of Pgc-1α and increase NT-Pgc-1α expression in skeletal muscle. Q- and ROE-induced upregulation of NT-Pgc-1α may occur through an epigenetic mechanism and may be sufficient to increase skeletal muscle mitochondrial number and complete beta oxidation of fatty acids, contributing to attenuation of obesity and insulin resistance.HFD causes


The essential part  of the molecule lies on a mirror plane, which bisects the t-butyl groups.The title compound was obtained from the condensation reaction of 3,5-di- 21H26N4O5, lies on a mirror plane, which bisects the t-butyl groups. The conformation of the C=N bond of this Schiff base compound is E, and there is an intramolecular N—H⋯O hydrogen bond present, forming an S(6) ring motif. In the crystal, molecules are linked via O—H⋯O hydrogen bonds, forming zigzag chains propagating along the a-axis direction. There are no other significant intermolecular contacts present.The essential part  of the molecule of the title compound, C They can protect polymers by increasing both their process stability and their long-term stability against oxidative degradation  ring motif -2-hydrazine (II)  and 2,4-dinitrophenylhydrazine  in methanol was refluxed for 3 h in the presence of a catalytic amount of glacial acetic acid. After cooling, the red-coloured precipitate was washed with hot methanol several times, and then dried, giving a red-coloured shiny crystalline compound in high yield 170 g (96%). Yellow block-like crystals of the title compound (m.p. 372–373 K) were obtained by slow evaporation of a solution in dichloromethane and ethanol (5:1 v/v).A mixture of 3,5-di-Uiso(H) = 1.5Ueq(O). The NH and C-bound H atoms were included in calculated positions and allowed to ride on the parent atoms: N—H = 0.86 Å, C—H = 0.93–0.96 Å with Uiso(H) = 1.5Ueq(C-methyl) and 1.2Ueq for other H atoms.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989016020107/su5340sup1.cifCrystal structure: contains datablock(s) I, Global. DOI: 10.1107/S2056989016020107/su5340Isup2.hklStructure factors: contains datablock(s) I. DOI: 1523249CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Vγ9Vδ2-T cells are considered as potent effector cells for tumor immunotherapy through directly killing tumor cells and indirectly regulating other innate and adaptive immune cells to establish antitumoral immunity. The antitumoral activity of Vγ9Vδ2-T cells is governed by a complicated set of activating and inhibitory cell receptors. In addition, cytokine milieu in tumor microenvironment can also induce the pro-tumoral activities and functional plasticity of Vγ9Vδ2-T cells. Here, we review the anti- versus pro-tumoral activities of Vγ9Vδ2-T cells and discuss the mechanisms underlying the recognition, activation, differentiation and regulation of Vγ9Vδ2-T cells in tumor immunosurveillance. The comprehensive understanding of the dual face of Vγ9Vδ2-T cells in tumor immunology may improve the therapeutic efficacy and clinical outcomes of Vγ9Vδ2-T cell-based tumor immunotherapy. Listeria monocytogenes, Cytomegalovirus, and certain hematological malignancies (Human γδ-T cells can be classified into two main subsets depending on the expression of T cell receptor (TCR) δ chain . Vδ1 γδ-gnancies , 3. In cgnancies , 4–10.The Vγ9Vδ2-T cell is an important component of immune effector cells that contribute to tumor immunosurveillance against many types of tumors, such as lymphoma, myeloma , 12, hepThe antitumor activity of Vγ9Vδ2-T cells is influenced by their functional plasticity that is driven by environmental factors . SimilarUpon phosphoantigen stimulation, Vγ9Vδ2-T cells preferentially differentiate into Th1-like cells with profound IFN-γ and TNF-α responses , 29. Th1+ T-cells, cell-to-cell contact is necessary for the B cell helper activity of the Tfh-like Vγ9Vδ2-T cells.Vγ9Vδ2-T cells with Tfh-like functions can be induced by IL-21 and phosphoantigens stimulation , 35. TheIL-17-producing γδ-T cells have been extensively discussed in the murine model . Recent Upon stimulation of IPP in the presence of IL-15 and TGF-β, the Vγ9Vδ2-T cells can be induced into transcription factor forkhead box P3 (Foxp3)-expressing Treg-like γδ-T cells with regulatory/immunosuppressive function . When coTherefore, Vγ9Vδ2-T cells can be induced into different functional subsets depending on the cytokine milieu in the tumor microenvironment.The antitumoral activity of Vγ9Vδ2-T cells has been well studied , 46. Thevia inhibition of the farnesyl pyrophosphate synthase enzyme in the isoprenoid pathway, which leads to IPP accumulation Self ligands: in addition to non-peptide ligands, Vγ9Vδ2-T cells can also recognize some molecules of cellular origin, which could be capable of indicating cellular stress or malignant transformation , 62. Sev(2)Herpes simplex , are involved in the effective triggering of antitumoral responses of Vγ9Vδ2-T cells  Figure . TogetheAnother NKR implicated in tumor recognition by Vγ9Vδ2-T cells is the DNAX accessory molecule-1 (DNAM-1) , 76. NecSimilar to NK cells, Vγ9Vδ2-T cells also express high levels of CD16 (FcγR III) upon phosphoantigen stimulation , and thuInteraction of TCR and/or NKG2D with their respective ligands can stimulate the activation of Vγ9Vδ2-T cells. Once activated, Vγ9Vδ2-T cells secrete IFN-γ and TNF-α, and increase the release of antitumor effector molecules, such as perforin and granzymes. The DNAM-1 signaling pathway can positively regulate the cytotoxic activity and IFN-γ secretion of Vγ9Vδ2-T cells against a broad range of tumors.via the binding of CD16 to antibodies, such as rituximab, trastuzumab, atumumab, and alemtuzumab, coated on the certain tumor cells , CCL4, C-X-C motif chemokine 10 (CXCL10), and CXCL13, to recruit αβ-T cells, B cells, NK cells, and macrophages/DCs to the tumor site , 31, 87.pression , 90, 91.pression , 93. Actpression . FurtherNK cells . AlthougNK cells –99, somechanisms  , L-selectin, and CD44v6, are also involved in the migration of Vγ9Vδ2-T cells to the tumor site   det det1.via phosphorylation of Zap70, Pi3K, LAT, ERK 1/2, and p38 MAPK  superfamilies. Ribot et al. suggested that CD28, which is an important member of the Ig superfamily, is constitutively expressed on lymphoid Vγ9Vδ2-T cells. Co-stimulation with CD28 promotes the survival and proliferation of Vγ9Vδ2-T cells by enhancing IL-2 production, whereas blocking antibodies target its B7 ligands (CD80 and CD86) can inhibit cell survival and proliferation . CD27 is-T cells , 114. Up-T cells . A high -T cells , 116.In addition to TCR and NKR, other surface receptors, such as adhesion molecules, are also important for regulating Vγ9Vδ2-T cell function. To date, several adhesion molecules, including LFA-1/intercellular adhesion molecule-1 (ICAM-1) , CD2/LFAHigh expressions of LFA-1 and its ligand, ICAM-1, have been detected on the surface of Vγ9Vδ2-T cells and most of the tumor cell lines, respectively. LFA-1/ICAM-1 adhesive interaction is necessary but not sufficient for IFN-γ production and the cytotoxicity of Vγ9Vδ2-T cells . The intMost importantly, the interactions between LFA-1/ICAM-1 and CD2/LFA-3 can stabilize the immunological synapses after Vγ9Vδ2-TCR/phosphoantigen triggers the formation of immunological synapses , 121.The roles of TLRs in regulating the activation of Vγ9Vδ2-T cells are not completely understood. After ligation of TLR2, TLR3, and TLR5, the Vγ9Vδ2-T cells can produce IFN-γ, TNF-α, granulocyte-macrophage colony-stimulating factor, CCL3, and CCL5 . The tri+ Vγ9Vδ2-T cells  and B- and T-lymphocyte attenuator (BTLA), act in a similar manner in Vγ9Vδ2-T cells. PD-1 and BTLA can negatively regulate Vγ9Vδ2-T cell responses. Vγ9Vδ2-T cells do not normally express detectable PD-1; however, once activated, the expression of PD-1 is upregulated in Vγ9Vδ2-T cells . Iwasakivia CD86/CTLA-4 and PD-L1/PD-1 interactions between activated Vγ9Vδ2-T cells and their responder cells , are also found to activate and expand Vγ9Vδ2-T cells by mimicking the effects of aminobisphosphonates . Upon stvia the introduction of αβ TCRs (Some recent studies have focused on the development of novel protocols to expand Vγ9Vδ2-T cells using immobilized antigens , agonist αβ TCRs , and chi αβ TCRs . Further αβ TCRs .Recent advances in Vγ9Vδ2-T cell research have paved a way for developing innovative therapeutic strategies for tumor immunotherapy. As discussed in this review, Vγ9Vδ2-T cells can recognize tumor cells through TCR and other cell surface receptors, and their antitumoral activity is strictly regulated by activating and inhibitory receptors and their ligands. In addition, stimuli/cytokine milieu in tumor microenvironment can also induce pro-tumoral activity and functional plasticity of Vγ9Vδ2-T cells. Further study on the dual face of Vγ9Vδ2-T cells in tumor immunology could optimize current therapeutic protocols and improve the therapeutic efficacy and clinical outcomes of Vγ9Vδ2-T cell-based tumor immunotherapy.ZX and WT wrote the manuscript. WT revised the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The compound crystallizes in the orthorhombic space group Pca21 and is a racemic twin [BASF = 0.402 (9)]. The geometry around the HgII atom is distorted tetrahedral, with the HgII atom coordinated to two N atoms, one Br atom, and a fourth coordination site is occupied by a mixed halide (Br/Cl). For the two ligands in the asymmetric unit, there is disorder with one of the two tert-butyl groups and benzimidazole moieties showing twofold disorder, with occupancy factors of 0.57 (2):0.43 (2) for the tert-butyl group and 0.73 (3):0.27 (3) for the benzimidazole group. In addition, there is threefold disorder for two of the four n-pentyl groups, with occupancy factors of 0.669 (4):0.177 (4):0.154 (4) and 0.662 (4):0.224 (4):0.154 (4), respectively. The molecules form a one-dimensional helical polymer propagating in the b-axis direction. The helices are held together by intra-strand C—H⋯Br and C—H⋯Cl interactions. Each strand is further linked by inter-strand C—H⋯Br and C—H⋯Cl interactions. In addition, there are weak C—H⋯N inter-strand interactions which further stabilize the structural arrangement.The mercury(II) complex of 2,2′-(5- H-benzimidazol-2-yl)benzene ligand have been well explored. This ligand is an ideal candidate for metalation due to the presence of two N atoms and one C atom which bind tightly with metal atoms . A structurally related complex has been presented recently phenyl]mercury (3) from mercury(II) chloride (C34H41N4HgCl), 1, and tetra-n-butylammonium bromide (Bu4N+Cl−) in dry acetone, the mercury complex of 2,2′-bis(1-pentyl-1H-benzimidazole) [HgBr1.52Cl0.48(C34H42N4)], 2, was isolated unexpectedly ]. The geometry around the HgII atom is distorted tetrahedral with the HgII atom coordinated to two N atoms, one Br atom, and a fourth coordination site is occupied by a mixed halide (Br/Cl). There are two such sites in the asymmetric unit and in one site, the Br/Cl occupancy is 0.599 (9):0.401 (9) and in the other site the ratio is 0.431 (9):0.569 (9), giving an overall composition (L)2HgBr1.52Cl0.48, where L is 2,2′-bis(1-pentyl-1H-benzimidazole). Unlike the case with the recently published structure , even though in the present ligand one position is open for the formation of an Hg—C bond, the basic arrangement is the same as in the previous structure.The title compound crystallizes in the orthorhombic space group tert-butyl groups and benzimidazole moieties showing twofold disorder, with occupancy factors of 0.57 (2):0.43 (2) for the tert-butyl group and 0.73 (3):0.27 (3) for the benzimidazole group. In addition, there is threefold disorder for two of the four n-pentyl groups, with occupancy factors of 0.669 (4):0.177 (4):0.154 (4) and 0.662 (4):0.224 (4):0.154 (4), respectively, as shown in Fig. 2et al., 2017II atoms, with bond angles ranging from 97.2 (2) to 124.1 (7)°, values that are more distorted than those found in the analogous complex [100.6 (1)–126.35 (7)°] due to the disorder in the anions.For the two ligands in the asymmetric unit, there is considerable disorder, with one of the two et al., 20173 group). The four sets of values are −115.6 (11)/−166.7 (11)/76.2 (19)/67.5 (19), 102.2 (12)/−61.5 (15)/−171.9 (13)/173.0 (16)°, 113.4 (10)/162.5 (9)/−174.7 (10)/−179.5 (11) and −104.2 (15)/−172.4 (13)/−78.2 (15)/177.1 (17)°. From these values it can be seen that the first two pentyl groups have adopted a slightly curled-up conformation, while the last two are in a fully extended all-trans conformation  and 88.0 (2)° Table 1, while ton Fig. 3.b direction  and N3A/N4A/C23A/C24A/C29A⋯C2B–C7B  in dry acetone was added tetra-n-butylammonium bromide  at room temperature. The reaction mixture was refluxed for 3 h under an inert atmosphere of N2 and then filtered through Whatman filter paper. The residue contained shiny mercury particles which could be witnessed with the naked eye and the solvent was evaporated from the filtrate and washed with petroleum ether and then with diethyl ether. The compound was dried under vacuo. Colorless plate-shaped single crystals were obtained from DMSO at 291 K . 1H NMR : δ 7.96–7.93 , 7.74 , 7.70 , 7.33–7.26 , 4.35 , 1.72 , 1.42 , 1.15–0.93 , 0.71 . Analysis calculated for C34H42Br1.52Cl0.48HgN4: C 48.33, H 5.01, N 6.63%; found: C 49.73, H 5.14, N 6.84%.To a solution of isoU(H) = 1.5Ueq(C) for methyl H atoms and 1.2Ueq(C) otherwise. For the two ligands in the asymmetric unit, there is considerable disorder with one of the two tert-butyl groups and benzimidazole moieties showing twofold disorder, with occupancy factors of 0.57 (2):0.43 (2) for the tert-butyl group and 0.73 (3):0.27 (3) for the benzimidazole group. The displacement parameters of the disordered tert-butyl group were restrained to be similar (SIMU command). One C atom (C22C) from the minor component was additionally restrained to be close to isotropic (ISOR 0.005 command). The two components of the benzimidazole moiety were restrained to be planar using the FLAT command and their metrical and displacement parameters were restrained using the SIMU and SADI commands in SHELXL2016 :0.177 (4):0.154 (4) and 0.662 (4):0.224 (4):0.154 (4), respectively. For one pentyl group, some atoms (C30A–C32A and C30C–C32C) of two of the three components were refined as overlapping (using EXYZ commands). For the other disordered pentyl group, all atoms of all three components adopt independent conformations. One of the occupancy rates  is shared between the two disordered sites due to mutually exclusive overlap of atoms.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989017002183/zl2695sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989017002183/zl2695Isup2.hklStructure factors: contains datablock(s) I. DOI: 1531940CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Iris kashmiriana, using a direct method of isolation, avoiding the use of chromatographic techniques. The compound was isolated by commercially viable procedure. The extraction of powdered drug (500 g) was done with petroleum ether (60–80) using a Soxhlet apparatus (24 h run). The petroleum ether extract (gums and resins 2.13 g) was obtained and the marc (400 g) was subjected to extraction with 95% methanol using a Soxhlet apparatus (24 h run). The methanolic extract (5 g) was subjected to successive fractionation with toluene, chloroform and ethyl acetate and n- butanol. On the basis of phytochemical analysis, the glycoside was present in n- butanol fraction. The n-butanol fraction (1.5 g) was taken in dried methanol, passed through activated animal charcoal and subjected to acid hydrolysis. The isoflavone (250 mg), was obtained after the usual process of separation. The purity of the compound was checked by analyzing TLC (Thin Layer chromatography) and melting point. Further, the chemical method was used to characterize the compound by shift reagents using UV spectroscopy. The quantitative estimation of isoflavone was done using RP-HPLC and was found to be 98.9% pure.The study was undertaken to develop a simplified procedure for the isolation of bioactive isoflavone from •The “previously undescribed” isoflavone was isolated by modifying approach of solvent/solvent extraction, fractionation and acid hydrolysis.•1HNMR, 13CNMR, Mass spectrometry.The spectroscopic characterization was equaly done by IR, •98.9% purity was achieved using RP-HPLC with simple solvent (Methanol and Water 55: 45). Iris kashmiriana Baker were dried, chopped, and powdered (500 g). The extraction of powdered drug was done with petroleum ether (60–80) using a Soxhlet apparatus (24 h run). The petroleum ether extract (gums and resins 2.13 g) was obtained and the marc (400 g) was subjected to extraction with 95% methanol using a Soxhlet apparatus (24 h run). The methanolic extract (5 g) was subjected to successive fractionation with toluene, chloroform and ethyl acetate and n- butanol n- butanol fraction. The n-butanol fraction (1.5 g) was taken in dried methanol (80%), passed through activated animal charcoal. The acid hydrolysis was carried out in the presence of 5% HCl (the reaction mixture was refluxed for 2–3 h at 45–50 °C and kept for night) 1HNMR, 13CNMR, Mass spectrometry and 2D NMR. Furthermore, the compound was also characterized by chemical methods by shift reagents (AlCl3 and CH3COONa) using UV spectrophotometer n-butanol, acetone and water in the ratio 4:1:5, where it was clearly observed that the sugar attached was glucose, with Rf value 0.68.Solvent-solvent extraction technique was adopted for the isolation of novel compound. In continuing with this technique the rhizomes of •This method was simple, modified, reproducible, efficient, eco-friendly and cost effective. Thus, the present work avoided the use of hyphenated chromatographic techniques such as Column chromatography & Preparative TLC (Thin Layer Chromatography).•The study deals with the isolation of “previously undescribed” molecule, together with the identification of sugar moiety from the glycoside.Why this method was adopted in the present work?10 mg test sample was dissolved in 100 mL of methanol to prepare 100 ppm solution; further dilution was made to prepare 10 ppm solution.Iris kashmiriana Baker. Agilent 1200 series HPLC binary pump system was performed on a C18 column with water: methanol (45:55), as mobile phase. Detection was performed by the DAD detector at 338 nm. This technique has been applied, for the quantitative estimation of new isoflavone. The retention time of the isoflavone was 11.2 min and no other peaks were observed. Injection volume was taken 10 μL. The purity of the compound was found to be 98.9% with single peak, which indicates the compound isolated was substantially pure and single; moreover the present study appears to be first of its kind of approach  method has been used for the quantitative estimation approach .Fig. 1Ch•Quantitative estimation of isoflavone was done by RP-HPLC using mobile phase  and the method was developed without using costly solvents such as acetonitrile. The single peak was observer after 11.2 min with significant purity (98.9%).What is the significance adopted in the present work?Ultraviolet and visible spectroscopy was one of the basic techniques routinely used for flavonoid/isoflavanoid analysis due to the existence of two characteristic UV/Vis bands in flavonoids, band I in the 300–550 nm range, arising from the B ring, and band II in the 240–285 nm range, arising from the A ring, with some variations.3+ complexation by OH groups, were also used to study flavonoid/isoflavanoid structure.Shift reagents, such as aluminium chrolide and sodium acetate, lead to shifts in the maximum wavelength of these bands due to acetate-induced deprotonation of OH groups or Al3 was added, a complex was formed between carbonyl group, hydroxyl group (at C-5) and AlCl3, resulting in the shift of λmax from 341 nm to 377.6 nm and 238.4 nm to 264.8 nm, respectively. In continuation with the procedure, few drops of HCl was added in the same mixture, there was no change in the shift of λmax except the optical density. This clearly indicates the vicinity of hydroxyl groups at C5- OH and carbonyl  scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 480 0 480 0 0 40 0 40 -480 0 -480 0 0 -40z M0 280 l0 -40 480 0 480 0 0 40 0 40 -480 0 -480 0 0 -40z"/></g></svg>O at C4) in the isoflavone moiety (The test compound (10 mg) was dissolved in 100 mL methanol and λmax was determined (341 and 238.4 nm). When few drops of AlCle moiety , Table 2The compound (10 mg) was dissolved in 100 mL methanol and λmax was determined.Since, no shift in λmax has been observed in UV region  after the addition of sodium acetate, thereby it clearly indicated, that the absence of free hydroxyl groups at C-3′,C-5′ and C-4′ positions.Why this method was adopted in the present work?This technique is inexpensive, reproducible and less time-consuming to study the flavonoids/isoflavonoid structure, though the other methods of characterization are equally important. Nowadays these techniques are not routinely used but still continue to be applied in some cases, in particular to HPLC eluates – a hyphenated LC-UV-MS has been developed using post-column UV shift reagents for the flavonoid/isoflavonoid analysis of crude extracts.14H12O6. The elemental analysis found (%) C;64.00,H;4.03,O;31.97 and calculated; C;63.21,H;3.40, O;30.83. Mass spectrometry analysis by TOF,MS, ES+. (m/z) = 301.26.The structure was further assigned by IR,1H NMR, C13 NMR, Mass spectrometry, together with U.V. RP- HPLC and chemical methods The aglycone was isolated, purified and recrystallized with methanol, the melting points was found to be 225–227 °C (uncorrected) and further analyzed for C•−1): 1518 (ArCC)], 1659 (CO), 3080 (ArC-H), 1372 (C-O), 2825(C-H Str), 3368 (Ar —OH).IR: 3.89 , 6.46  6.85 , 7.37 , 8.15,•13C- NMR assignment.The structure was further confirmed by 13C- NMR : C-4, 180.50, C-4′ 157.39, C-5 153.22 C-7 153.30, C-2 153.31, C-6 131.22, C-8a 121.92, C-8 132.47, C-3′,5′, 115.01, C-2′,6′ 129.91, C-1′,104.89, C-3 121.14, C-4a 93.71,CH3-59.80•Mass spectrometry+ 2.33e.3 m/z (rel. int): 301[M]+ (45.94), 294[M-6H]+2(13.12),285[M-CH3] + (36.79),269[M-CH3-O] + (12.05),253[M-CH3-O-O] + (63.63),237 [M-CH3-O-O-O] + (10.32), 208 [M-CH3-O-O-CO2] + (7.01),201 [M-C6H4CO ring B] + (6.63), Ring B = m/z 100. 187[M-CH3-O-O-O-C4H2] + (61.82),178[M-C6H4CO—CC—] + (28.26),162[M-C6H4CO—CC-O] + (34.94),146[M- C6H4CO—CC-O-O-] + (22.05), 136[M- C6H4CO—CC-O-C2H2] + (7.05), 117[M-CH3-3O-ring A] (4.91). Ring A = m/z116. 107[M- C6H4CO—CC-O-C2H2-CHO](59.13). 85[117-2O](100)TOF MS ESSpectroscopic characterizationPongamia pinnata, Lactone and their analogues are isolated from Inula resemosa (Pushkarmool).The RP-HPLC method developed can also be extrapolated for the standardization of herbal formulation containing isoflavone.With the help of above methodology, our team isolated more than 10 new molecules from different plant species. For example, karanjin and their new analogues are isolated from seed oils of


The Cu—N distances range from 2.050 (2) to 2.300 (2) Å. This complex cation and the two trifluoroacetate anions are connected by weak N—H⋯O and N—H⋯F hydrogen bonds, forming a three-dimensional framework. In both anions, the F atoms are disordered over two positions; in one the site-occupancy factors are 0.55 and 0.45, in the other the values are 0.69 and 0.31.In the title complex, [Cu(H DOI: 10.1107/S1600536808001438/wn2235Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


The tethered cyclopentadienyl (Cp) rings are tilted by 9.39 (18)° and assume an eclipsed conformation. The 1-oxopropane-1,3-diyl bridge has a pseudo-envelope conformation with the C=O group deviating by as much as 22.5 (2)° from coplanarity with its attached Cp ring.The crystal structure of [3]ferrocenophan-1-one, [Fe(C DOI: 10.1107/S1600536808022927/dn2366Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


II–HoIII complex ]diphenolato-1κ4               O               1,O               1′,O               6,O               6′:2κ4               O               1,N,N′,O               1′)-μ-nitrato-1:2κ2               O:O′-dinitrato-1κ4               O,O′-holmium(III)zinc(II)), [HoZn(C18H18N2O4)(NO3)3], with the hexadentate Schiff base compartmental ligand N,N′-bisethylenediamine (H2               L), the Ho and Zn atoms are triply bridged by two phenolate O atoms of the Schiff base ligand and one nitrate ion. The five-coordinate Zn atom is in a square-pyramidal geometry with the donor centers of two imine N atoms, two phenolate O atoms and one of the bridging nitrate O atoms. The HoIII center has a ninefold coordination environment of O atoms, involving the phenolate O atoms, two methoxy O atoms, two O atoms from two nitrate ions and one from the bridging nitrate ion. Weak intermolecular C—H⋯O interactions generate a two-dimensional double-layer structure.In the title heteronuclear Zn DOI: 10.1107/S1600536808013743/at2567Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials: