However, maximal therapeutic benefit from 131I-MIBG is likely to be obtained by its combination with chemotherapy. We previously reported enhanced antitumour efficacy of 131I-MIBG by inhibition of the poly(ADP-ribose) polymerase-1 (PARP-1) DNA repair pathway using the phenanthridinone derivative PJ34. Recently developed alternative PARP-1 inhibitors have greater target specificity and are expected to be associated with reduced toxicity to normal tissue. Therefore, our purpose was to determine whether the more specific PARP-1 inhibitors rucaparib and olaparib enhanced the efficacy of X-radiation or 131I-MIBG.The radiopharmaceutical Radiosensitisation of SK-N-BE(2c) neuroblastoma cells or noradrenaline transporter gene-transfected glioma cells (UVW/NAT) was investigated using clonogenic assay. Propidium iodide staining and flow cytometry was used to analyse cell cycle progression. DNA damage was quantified by the phosphorylation of H2AX (γH2AX).131I-MIBG activity concentration required to achieve 50 % cell kill by approximately 50 %. Rucaparib and olaparib were equally effective inhibitors of PARP-1 activity. X-radiation-induced DNA damage was significantly increased 2 h after irradiation by combination with PARP-1 inhibitors . Moreover, combination treatment (i) prevented the restitution of DNA, exemplified by the persistence of 3-fold greater DNA damage after 24 h, compared to untreated controls (p < 0.01) and (ii) induced greater G2/M arrest (p < 0.05) than either single agent alone.By combining PARP-1 inhibition with radiation treatment, it was possible to reduce the X-radiation dose or 131I-MIBG treatment. It is likely that the mechanism of radiosensitisation entails the accumulation of unrepaired radiation-induced DNA damage. Our findings suggest that the administration of PARP-1 inhibitors and 131I-MIBG to high risk neuroblastoma patients may be beneficial.Rucaparib and olaparib sensitise cancer cells to X-radiation or  Despite an incidence rate of 6 % of all childhood cancers , neurobl131I-MIBG) exploits this characteristic of neuroblastoma cells. The radiopharmaceutical 131I-MIBG is a structural analogue of noradrenaline, facilitating its selective accumulation by neuroblastoma tumour cells. 131I-MIBG has demonstrated efficacy as a single agent [131I-MIBG has yet to be defined [131I-MIBG in combination with intensive induction chemotherapy in high-risk neuroblastoma patients.Ninety percent of neuroblastoma tumours express the noradrenaline transporter (NAT) , allowinle agent , 7. Howe defined , and inc defined –11. Inde+) [BRCA-1 and BRCA-2 [PARP-1 [Poly(ADP-ribose) polymerases (PARPs) mediate the post-translational modification of target proteins following hydrolysis of the PARP substrate, nicotinamide adenine dinucleotide (NAD+) , 13. The+) , 15. Upo+) . PARP-1 d BRCA-2 , 18, twod BRCA-2 . Inhibit [PARP-1 , prompte131I-MIBG and topotecan [BRCA-deficient ovarian cancer. Both rucaparib and olaparib have shown promise in phase II/III clinical trials, both as monotherapies in BRCA-mutated breast cancer [PARP-1 inhibitors have shown great promise when used in combination with treatments that cause substantial DNA damage, including ionising radiation –23, DNA opotecan . Howeveropotecan , 28. Innopotecan , 16, 29.opotecan  and olapt cancer  and prost cancer , and in t cancer –36.131I-MIBG. We also examined the effect of combination treatment on cell cycle progression and the persistence of DNA damage.Therefore, PARP-1 inhibition is a promising approach not only to the targeting of BRCA-deficient cancers which are deficient in DNA repair capacity, but also to the enhancement of the efficacy of DNA damaging chemo- and radiotherapies. Indeed, increased PARP-1 expression has previously been associated with greater neuroblastoma cell genomic instability, higher neuroblastoma stage and poor overall survival , suggestRucaparib and olaparib were purchased from Selleckchem  and were reconstituted using phosphate buffered saline (PBS) and dimethyl sulfoxide (DMSO), respectively. Drugs were then diluted in culture medium, maximum DMSO concentration was 0.2 % (v/v). Unless otherwise stated, all other cell culture reagents were purchased from Life Technologies  and all chemicals were purchased from Sigma-Aldrich .2 in a humidified incubator, and were passaged every 3-4 days. Cell lines were cultured in this study for less than 6 months after resuscitation.Human neuroblastoma SK-N-BE(2c) cells were purchased from the American Type Culture Collection. SK-N-BE(2c) cells were maintained in high glucose Dulbecco’s Modified Eagle Medium (DMEM) containing 15 % (v/v) foetal calf serum, 2 mM L-glutamine and 1 % (v/v) non-essential amino acids. Human glioblastoma UVW cells  were tra5 cells in 25 cm2 flasks. Cells in the exponential growth phase were treated with fresh culture medium containing rucaparib or olaparib and were simultaneously irradiated using an Xstrahl RS225 X-Ray irradiator  at a dose rate of 0.93 Gy/min. After 24 h incubation at 37 °C, cells were seeded in 21.5 cm2 petri dishes at a density of 500 (SK-N-BE(2c)) or 250 (UVW/NAT) cells per dish in triplicate. After 8 days (UVW/NAT) or 14 days (SK-N-BE(2c)), colonies containing ≥50 cells were fixed with 50 % (v/v) methanol in PBS and stained with crystal violet. Stained colonies were counted and expressed as a fraction of the untreated, unirradiated control. Radiation survival curves were fitted assuming a linear-quadratic relationship between survival and radiation dose using GraphPad Prism 5.01 . The data were used to calculate the dose required to sterilise 50 % of clonogens (IC50), as well as the dose-enhancement factor at IC50 (DEF50).Monolayers were cultured at a density of 105 (SK-N-BE(2c)) or 0.5x105 (UVW/NAT) cells on to glass coverslips in 6-well plates. After 48 h, fresh medium was added containing rucaparib or olaparib, before incubating for 1.5 h at 37 °C. PARP-1 activity was stimulated by treatment with 20 mM hydrogen peroxide for 20 min at room temperature in the dark. PBS or DMSO treatment of 0.09 % (v/v) in medium constituted negative controls. Cells were fixed with ice cold methanol/acetone (1:1) on ice for 15 min, before blocking with 2 % (w/v) bovine serum albumin (BSA) in PBS for 30 min at room temperature. Fixed cells were incubated for 1 h at room temperature with a 1:200 dilution of mouse anti-PADPR monoclonal antibody  in antibody buffer  BSA in distilled water). Bound anti-PADPR primary antibody was visualised after 1 h incubation at room temperature using goat anti-mouse Alexa Fluor 488-conjugated secondary antibody , at a dilution of 1:500 in antibody buffer. Cells were fixed by treatment with 4 % (w/v) paraformaldehyde for 30 min at room temperature in the dark, before mounting on to microscope slides using Vectashield mounting medium containing DAPI nuclear stain . Fluorescence was visualised by means of a Zeiss Axio Observer LSM 780 confocal microscope, using identical laser power and gain settings for all images.Cells were seeded at a density of 1x10131I-MIBG was prepared using a solid phase system wherein the precursor of 131I-MIBG was attached to an insoluble polymer via the tin-aryl bond [131I-MIBG for 2 h, by which time 131I-MIBG uptake was maximal [No-carrier-added (n.c.a.) ryl bond , 41. Theryl bond . Cells w maximal .5 (SK-N-BE(2c)) or 4x105 (UVW/NAT) cells into 75 cm2 flasks. After 48 h, fresh medium was added containing rucaparib or olaparib and cells were simultaneously irradiated before incubating for 1.5 h at 37 °C. Cells were trypsinised and washed with PBS, before fixing with 70 % (v/v) ethanol in water at -20 °C. Ethanol was removed by washing with PBS. Cells were permeabilised by treatment with 0.05 % (v/v) Triton X-100 in PBS containing a 1:50 dilution of rabbit anti-phospho-Histone H2AX(Ser139)-Alexa Fluor 647-conjugated monoclonal antibody. After 40 min incubation at room temperature, excess antibody was removed by washing with PBST buffer (0.1 % (v/v) Tween 20 in PBS). Finally, cell pellets were resuspended in PBS containing propidium iodide (10 μg/ml) and RNase A (200 μg/ml), before analysis using a BD FACSVerse flow cytometer . FACS data were quantified using FlowJo 7.6.5 software. For cell cycle analysis, cells were treated separately, and were incubated with propidium iodide and RNase A only as detailed above.Cells were seeded at a density of 7x10Cells were seeded as for PARP-1 activity assay. Fresh medium was added containing rucaparib or olaparib, and cells were simultaneously irradiated, before incubating for 1.5 h at 37 °C. After treatment, cells were fixed with 4 % (w/v) paraformaldehyde for 30 min at room temperature before blocking with 2 % (w/v) BSA (in PBS) for 30 min at room temperature. Fixed cells were then incubated overnight at 4 °C with a 1:50 dilution of rabbit anti-phospho-Histone H2AX(Ser139)-Alexa Fluor 647-conjugated monoclonal antibody , followed by overnight incubation with a 1:250 dilution of mouse anti-β-tubulin  in antibody buffer  BSA in distilled water). Bound anti-β-tubulin primary antibody was visualised after 1 h incubation at room temperature using goat anti-mouse Alexa Fluor 488-conjugated secondary antibody , at a dilution of 1:500 in antibody buffer. Cells were mounted and fluorescence visualised as for PARP-1 activity assay.t test, or the one-way ANOVA followed by post-hoc testing using Bonferroni correction for multiple comparisons. A probability (p) value < 0.05 was considered statistically significant and < 0.01 highly significant.All statistical analyses were performed using GraphPad Prism version 5.01 . The number of experimental repeats is provided in figure legends. Data are presented as means ± standard error of the mean (SEM). Statistical significance was determined by either the unpaired Student’s two-tailed p < 0.01). Neither cell line survived 24 h treatment with 50 μM rucaparib. In contrast, 30 μM olaparib, though highly toxic to UVW/NAT cells (p < 0.01), induced modest kill of SK-N-BE(2c) clonogens .Neither rucaparib nor olaparib was cytotoxic at 1 μM. Minor yet significant clonogenic cell kill was induced by both drugs at 10 μM. Rucaparib at 30 μM was significantly toxic to SK-N-BE(2c) and UVW/NAT cells  at 20 mM – labelled no drug on Fig. p < 0.01) and 9.4-fold (p < 0.01) increase in PARP-1 activity compared to untreated SK-N-BE(2c) .Incubation with 10 μM rucaparib or olaparib  Fig.  and UVW/131I-MIBG. In combination treatments, PARP-1 inhibitors were administered at non-cytotoxic (1 μM) or cytotoxic (10 μM and 30 μM) concentrations, all of which inhibited PARP-1 activity in both cell lines.To investigate the radiosensitising potential of rucaparib and olaparib in SK-N-BE(2c) and UVW/NAT cells, clonogenic survival was assessed following drug treatment in simultaneous combination with external beam X-irradiation or treatment with the NAT-targeting radiopharmaceutical 50). In the absence of PARP-1 inhibition, the IC50 value corresponding to X-radiation treatment alone of SK-N-BE(2c) cells was 3.57 ± 0.15 Gy  or 2.52 ± 0.22 Gy by treatment with 1, 10 or 30 μM rucaparib, respectively. Exposure to 1, 10 or 30 μM olaparib reduced IC50 values to 3.42 ± 0.60, 3.22 ± 0.24 and 2.21 ± 0.18 Gy (p < 0.001) respectively, in SK-N-BE(2c) cells. Likewise in UVW/NAT cells, IC50 values observed after exposure to X-radiation alone, or in the presence of 1, 10 or 30 μM rucaparib were 4.44 ± 0.21, 3.50 ± 0.53, 2.42 ± 0.17 (p < 0.01) and 3.44 ± 0.28 Gy, respectively  and 2.09 ± 0.47 Gy (p < 0.01), respectively. These results suggest a plateau at 10 μM rucaparib or olaparib, with respect to clonogenic kill.Rucaparib and olaparib sensitised SK-N-BE(2c) cells Fig.  and UVW/ Gy Fig. . This vaely Fig. . Similar50) was calculated as the radiation dose required to achieve 50 % kill in the absence of drug divided by the radiation dose required to kill cells in the presence of drug. Therefore, a DEF50 greater than 1 is indicative of radiosensitisation. Both PARP-1 inhibitors radiosensitised SK-N-BE(2c) and UVW/NAT cells. In the case of SK-N-BE(2c) cells, the DEF50 values were 2.01 and 1.22 following 10 μM rucaparib and olaparib treatment, respectively  or 0.63 ± 0.17 MBq/ml after treatment with 10 μM rucaparib or olaparib, respectively  in the presence of rucaparib or olaparib, respectively. DEF50 values were calculated as 2.36 and 1.17 in SK-N-BE(2c) cells treated with 10 μM rucaparib or olaparib respectively  and UVW/NAT cells to treatment with IBG Fig. . In SK-Nely Fig. . Similarely Fig. . In UVW/The interaction between radiation treatment and PARP-1 inhibitors was further determined using X-irradiation doses (3 Gy) and activity concentrations (1 MBq/ml) that were responsible for 50 % kill of SK-N-BE(2c) clonogens. Combination treatment included rucaparib or olaparib at 10 μM. The expected clonogenic cell surviving fraction, if the two treatments had an additive effect, was calculated as the product of the surviving fractions resulting from single agent treatments. This is designated as “combination expected” in Fig. p < 0.05) or olaparib  with X-irradiation in SK-N-BE(2c) cells was less than that of the expected combination, indicating supra-additivity. Similarly, in UVW/NAT cells, the surviving fraction of the observed combination of rucaparib (0.25 ± 0.02) or olaparib (0.14 ± 0.02) with X-radiation was less than that of the expected combination   cells Fig. . The sur05) Fig. .131I-MIBG  cells at 0 h, to 35 ± 1 % following 3 Gy irradiation  and 32 ± 3 % (p < 0.05), 12 h and 24 h after 3 Gy irradiation, respectively  cells, from 24 ± 3 % in unirradiated controls, to 34 ± 3 % or 33 ± 3 % after administration of 10 μM rucaparib or olaparib, respectively  and 51 ± 5 % (p < 0.001) following rucaparib or olaparib combination treatment, respectively, in SK-N-BE(2c) cells. Rucaparib and olaparib, both as single agents and as components of combination therapy, produced a similar level of G2/M arrest.Rucaparib and olaparib single agent treatments significantly increased the G2/M population of UVW/NAT cells from 22 ± 1 % in unirradiated controls, to 32 ± 2 % (p < 0.01) or 30 ± 3 % (p < 0.05), respectively. Combination treatment significantly increased the G2/M population from 22 ± 1 % in unirradiated controls, to 55 ± 1 % (p < 0.001) and 61 ± 2 % (p < 0.001) following rucaparib or olaparib combination treatment, respectively. Representative histograms obtained using SK-N-BE(2c) cells are shown in Fig. Similar treatment-induced, cell cycle redistribution was observed in UVW/NAT cells Fig. . Single The generation of γH2AX foci at the site of DNA double strand breaks follows phosphorylation of the H2AX histone variant protein at serine residue 139 . γH2AX fp < 0.01) and from 2 to 21 % (p < 0.01) γH2AX positivity relative to total cellular DNA for SK-N-BE(2c) and UVW/NAT cells, respectively. Twenty four hours after irradiation, these values decreased to 14 % (SK-N-BE(2c)) and 6 % (UVW/NAT), indicating DNA repair. Combination treatment, consisting of rucaparib or olaparib with X-irradiation, resulted in greater DNA damage compared with irradiation alone. Combined X-irradiation with rucaparib caused an increase in γH2AX positivity to 45 % (p < 0.001) in SK-N-BE(2c) cells 2 h after treatment and, compared with irradiation alone, the damage was more sustained as indicated by 23 % γH2AX positivity (p < 0.05) 24 h after treatment. Similarly, combination treatment of cells with X-irradiation and olaparib caused 37 % DNA damage (p < 0.01) in SK-N-BE(2c) cells 2 h after treatment, which remained unrepaired  24 h after treatment. Therefore, combining rucaparib or olaparib with X-irradiation produced a significantly greater amount of DNA damage, compared with X-irradiation alone (p < 0.05).X-radiation treatment significantly increased DNA damage 2 h after irradiation Fig. , c and dp < 0.001) and 12 % (p < 0.01) 2 h and 24 h after treatment, respectively. Likewise, olaparib in combination with X-irradiation resulted in the greatest DNA damage 2 h after treatment  compared with single agent treatments, which remained unrepaired 24 h after treatment . Furthermore, combination treatment produced significantly greater initial DNA damage compared with X-irradiation alone (p < 0.05). These results indicate that PARP-1 inhibition prevented the restitution of radiation-induced DNA damage.Similar inhibition of DNA damage repair was observed in UVW/NAT cells Fig. . Combini131I-MIBG is effective in the clinical management of high-risk neuroblastoma. However, recent studies indicate that maximal benefit will be achieved through its administration in combination with radiosensitising drugs [131I-MIBG. Our results indicate that the mechanism of radiosensitisation entails prolonged DNA damage and accumulation of cells in G2/M phase of the cell cycle. PARP-1 inhibitors rucaparib and olaparib were comparable with respect to their potentiation of the lethality of X-irradiation or 131I-MIBG. Accordingly, both PARP-1 inhibitors may be considered of benefit to high-risk neuroblastoma patients undergoing targeted radiotherapy.Patients with high-risk neuroblastoma have an overall survival rate of 40 % despite multi-modal treatment . Therefong drugs –49. In tBRCA-mutated breast cancer [131I-MIBG. The current study focused on rucaparib and olaparib, the first PARP-1 inhibitors to enter clinical trial [Since the discovery of the synthetic lethality of PARP-1 inhibition in cells deficient in homologous recombination (HR) , 18, thet cancer , ovariant cancer  and prost cancer . Howevert cancer –36 and rt cancer . Here, wal trial –33, 36 a131I-MIBG. Simultaneous treatment with 10 μM rucaparib or olaparib effectively halved the external beam X-radiation dose or the 131I-MIBG activity concentration required to kill 50 % of clonogens (IC50) derived from human neuroblastoma SK-N-BE(2c) cells, and human glioma UVW cells genetically engineered to express the noradrenaline transporter (NAT). Rucaparib or olaparib displayed similar radiosensitising potency. Furthermore, combination treatment produced greater than additive cell kill, indicating the potential for enhanced therapeutic benefit.Although the radiosensitising capacity of PARP-1 inhibitors has previously been demonstrated in vitro , 51–55, 2/M phase of the cell cycle, which would also include a small proportion of cells in late S phase, and has been reported by others [131I-MIBG maximised the efficacy of this 3-way combination [The present study demonstrated that rucaparib, olaparib and X-irradiation monotherapies significantly increased the proportion of cells in the Gy others . This isy others , due to bination . Notablybination , suggestbination .The toxicity of PARP-1 inhibition is hypothesised to involve the accumulation of single strand breaks in irradiated cells, which are subsequently converted to double strand breaks upon collision with the advancing replication fork , 59. DouThe anti-tumour effect of PARP-1 inhibitors is not only due to inhibition of PARP-1 catalytic activity. PARP-1 inhibitor toxicity has also been attributed to ‘PARP trapping’, whereby PARP-1 is confined at the site of DNA damage, thus preventing DNA repair, replication and transcription, culminating in cell death . Indeed,MYCN amplification [MYCN amplification occurs in 25 % of all primary neuroblastomas and is used for neuroblastoma risk stratification [131I-MIBG. Abnormalities in the non-homologous end joining repair pathway, such as increased PARP-1 and DNA Ligase protein expression, have been implicated in neuroblastoma cell survival and pathogenicity [PARP-1 inhibitors are increasingly being considered for the treatment of neuroblastoma. Rucaparib has been shown to improve the efficacy of the alkylating agent temozolomide in neuroblastoma pre-clinical models . The altfication . MYCN amgenicity . Indeed,genicity , suggest131I-MIBG activity concentration required to achieve 50 % cell kill. X-radiation-induced DNA damage was significantly increased 2 h after irradiation by combination with PARP-1 inhibitors. Moreover, combination treatment (i) prevented the restitution of DNA and (ii) induced greater G2/M cell cycle arrest than single agent modalities. Finally, rucaparib and olaparib were shown to be equipotent inhibitors of PARP-1 activity and displayed analogous levels of radiosensitisation in neuroblastoma models. Our findings suggest that the administration of PARP-1 inhibition and 131I-MIBG to high-risk neuroblastoma patients may be beneficial.We have demonstrated that the third generation PARP-1 inhibitors rucaparib and olaparib sensitised tumour cells to radiation treatment. This was manifest as a 50 % reduction in the X-radiation dose or


The aim of this study was to address these questions in mice.Secretoglobin (SCGB) 3A2, a cytokine-like secretory protein of small molecular weight, is predominantly expressed in airway epithelial cells. While SCGB3A2 is known to have anti-inflammatory, growth factor, and anti-fibrotic activities, whether SCGB3A2 has any other roles, particularly in lung homeostasis and disease has not been demonstrated Scgb3a2-transgenic mouse lungs were carried out. Scgb3a2-transgenic and wild-type mice were subjected to bleomycin-induced pulmonary fibrosis model, and their lungs and bronchoalveolar lavage fluids were collected at various time points during 9 weeks post-bleomycin treatment for further analysis.A transgenic mouse line that expresses SCGB3A2 in the lung using the human surfactant protein-C promoter was established. Detailed histological, immunohistochemical, physiological, and molecular characterization of the Scgb3a2-transgenic mouse lungs expressed approximately five-fold higher levels of SCGB3A2 protein in comparison to wild-type mice as determined by western blotting of lung tissues. Immunohistochemistry showed that expression was localized to alveolar type II cells in addition to airway epithelial cells, thus accurately reflecting the site of surfactant protein-C expression. Scgb3a2-transgenic mice showed normal lung development and histology, and no overt gross phenotypes. However, when subjected to a bleomycin-induced pulmonary fibrosis model, they initially exhibited exacerbated fibrosis at 3 weeks post-bleomycin administration that was more rapidly resolved by 6 weeks as compared with wild-type mice, as determined by lung histology, Masson Trichrome staining and hydroxyproline content, inflammatory cell numbers, expression of collagen genes, and proinflammatory cytokine levels. The decrease of fibrosis coincided with the increased expression of SCGB3A2 in Scgb3a2-transgenic lungs.Adult These results demonstrate that SCGB3A2 is an anti-fibrotic agent, and suggest a possible therapeutic use of recombinant SCGB3A2 in the treatment of pulmonary fibrosis.The online version of this article (doi:10.1186/s12890-015-0065-4) contains supplementary material, which is available to authorized users. SCGB3A2erm pups . Recentlerm pups .2 (PLA2) inhibitory activity, which was thought to be at least partially responsible for the anti-inflammatory and immunomodulatory activity of SCGB1A1 [in vitro [Scgb1a1-null mice in vivo [SCGB1A1 is the most well-characterized SCGB protein, exhibiting anti-inflammatory, anti-fibrotic, and immunomodulatory functions –9. SCGB1 SCGB1A1 , 11. SCGin vitro , and the in vivo , 14.Scgb3a2 mRNA levels were reduced in the lungs of fungal-induced allergic inflammation model mice, which was almost restored by dexamethasone treatment [Scgb3a2 mRNA were inversely correlated with the increased levels of proinflammatory cytokines, IL-5 and IL-9 in bronchoalveolar lavage fluid (BALF) [Scgb3a2-null mice when subjected to OVA-inflammation model, showed exacerbated airway inflammation [SCGB3A2 also exhibits anti-inflammatory and anti-fibrotic activities , 16; thed (BALF) , 18. Whed (BALF) . Lastly,ammation . On the ammation , 21. TakScgb3a2-transgenic mouse was established that over-expresses SCGB3A2 in a lung-specific fashion under the control of the human surfactant protein C (SP-C) gene promoter. Detailed characterization demonstrated that the lungs of Scgb3a2-transgenic mice were histologically and functionally normal as compared to wild-type. When subjected to the BLM-induced pulmonary fibrosis model, however, they exhibited increased fibrosis at 3 weeks post-BLM administration, which was more quickly resolved by 6 weeks as compared to wild-type mice. These results demonstrate that SCGB3A2 has anti-fibrotic activity and suggest a potential use of SCGB3A2 as a therapeutic agent in treating lung fibrosis.To understand the role of SCGB3A2 in lung homeostasis and diseases, an Scgb3a2 cDNA that covers the entire protein coding sequence (50–427) was inserted into the SPC3.7-SV40-pUC18 plasmid. The resultant SPC3.7-SCGB3A2-SV40-pUC18 was double-digested with restriction enzymes, Nde I and Not I. The linearized SPC3.7-SCGB3A2-SV40 fragment was purified before microinjection into pronuclei of C57BL/6NCr mouse eggs. Production of Scgb3a2-transgenic mouse lines was confirmed by Southern blotting of genomic DNAs isolated from clipped mouse-tails.An expression plasmid with the human SP-C gene promoter (3.7 kb) cloned into pUC18 vector with SV40 small T intron and poly A (0.4 kb) (SPC3.7-SV40-pUC18), was provided from Dr. Jeffrey Whitsett  . The mouScgb3a2-transgenic mice was electrophoresed on 1 % agarose gel containing 0.22 M formaldehyde and transferred onto nitrocellulose membrane . Filters were hybridized with SCGB3A2 probe obtained from Eco RI digestion of the SCGB3A2/pCR2.1 construct. Hybridization was performed in Perfect Hybridization solution  at 68 °C overnight. The membrane was washed twice with 2 x SSC containing 0.1 % SDS at 68 °C for 30 min, followed by exposure to a phosphoimager screen . Data processing was carried out using ImageQuant TL 2005 software .Total RNA (3 μg) isolated from adult lungs of wild type and Lung from wild type and transgenic mice were frozen and crushed in 50 mM Tris-HCl, pH 8.0, 5 mM EDTA, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride (PMSF) with protein inhibitor cocktail . Protein concentrations were determined by Bradford assay  with bovine serum albumin (BSA) as standard, and samples were mixed with equal volume of 2 x SDS sample buffer . Ten microgram of sample was applied in each well of 20 % polyacrylamide gel and was run with running buffer of 50 mM Tris, 384 mM glycine, 2 % SDS. After electrophoresis, protein was transferred to Polyvinylidene fluoride (PVDF) membrane using a tank transfer system  with blotting buffer  and electric field of 30 V for 6 h. To visualize SCGB3A2 band, PVDF membrane was treated as follows; 1 h blocking with PBST  +5 % BSA, 3 h incubation with 0.2 μg/ml polyclonal rabbit anti-SCGB3A2 IgG in PBST + 5 % BSA, PBST wash 3 times, incubation with 0.1 μl/ml horseradish peroxidase (HRP)-linked anti-Rabbit IgG F(ab’) fragment  in PBST + 5 % BSA, and PBST wash 3 times before ECL plus  reaction. Polyclonal rabbit anti-SCGB3A2 antibody was produced as previously described [Scgb3a2-transgenic pregnant females at various embryonic days (E). Noon of the day on which a vaginal plug was found was considered as E 0.5. Branching degree of ex vivo cultured embryonic lungs were counted after 3 days of culture as previously described [All animal studies were carried out after approval by the National Cancer Institute Animal Care and Use Committee. Mouse embryonic lungs were collected from wild-type and escribed . Breathiescribed  accordinescribed ; 0, no bescribed . The colSCGB3A2 protein levels were determined by ELISA as previously described . BrieflyLungs were fixed in 4 % paraformaldehyde for one day at 4 °C, dehydrated, and embedded in paraffin. Lung tissues were sectioned at 4 μm and stained with hematoxyline and eosin (H&E). For immunohistochemistry, sections were at first rinsed with 0.05 % Triton-X 100 in PBS, and non-specific binding sites were blocked using 10 % normal goat serum in PBS containing 0.05 % Tween 20. Epitope retrieval was carried out using autoclave . After cooling to room temperature, the sections were incubated overnight at 4 °C with rabbit polyclonal anti-mouse SCGB3A2 [For double immunofluorescence labeling of adult lungs using two primary antibodies from the same species, the sections were first incubated with anti-SCGB3A2 antibody (1:1000) at 4 °C overnight, followed by labeled goat anti-rabbit IgG  as the secondary antibody for 1 h at room temperature. The sections were then incubated with 5 % rabbit serum for 1 h at room temperature, followed by incubation with unconjugated Fab Fragment goat anti-rabbit IgG for 1 h at room temperature. The sections were finally incubated with the secondary primary antibody, rabbit anti-pro-SP-C antibody (1:500), followed by labeled goat anti-rabbit IgG . Cell nuclei were identified by counterstaining with 4,6-diamino-2-phenylindolyl-dihydrochloride . Fluorescence images were obtained and processed using a Zeiss 780 laser-scanning confocal microscope. Matching confocal planes were analyzed in all co-localization studies.Severity of fibrosis was quantified from H&E stained entire lungs using the Ashcroft scoring system . The deg2O, using 100 μl H2O for every 10 mg of tissue. To 100 μl of tissue homogenate, 200 μl concentrated HCl (6 N) was added in a pressure-tight, teflon capped vial, and the mixture was hydrolyzed at 120 °C for 3 h, followed by filtration through a 45 μm syringe filter . Ten μl of hydrolyzed sample was transferred to a 96-well plate and was evaporated to dryness under vacuum, to which 100 μl Chloramine T reagent was added per well. After incubation at room temperature for 5 min, 100 μl p-dimethylaminobenzaldehyde reagent was added to each well and further incubated for 90 min at 60 °C. Absorbance was measured at 560 nm in a microplate reader .Hydroxyproline content was measured using hydroxyproline assay kit from Biovision  according to the manufacture’s instruction with slight modification. In brief, whole lungs were homogenized in dHScgb3a2 transgenic mice (approximately 8-weeks old) using RNeasy Mini Kit . The sample RNAs were labeled with Cy5, while pooled RNAs containing the same amount of RNA from each sample were labeled with Cy3 and used as a reference. Labeling was carried out using CyDye™ Post-Labeling Reactive Dye Pack  according to the manufacturer’s instructions. The purified Dye-coupled RNA samples were hybridized to an Agilent Whole Mouse Genome 4X44K oligo microarray kit , and were incubated for 17 h at 65 °C. The slides were washed, dried, and scanned using Agilent G26000 microarray scanner. The data were processed and analyzed by Genespring GX 11.0.2 software package (Agilent Technologies). All effective genes of microarray analysis were submitted to the Gene Expression Omnibus (GEO: ID # GSE47931).Lung total cellular RNAs were individually isolated from four mice each of no-treatment wild-type and Sftpa (forward) 5′- ACT CCC ATT GTT TGC AGA ATC -3′, (reverse) 5′- AAG GGA GAG CCT GGA GAA AG -3′; Sftpb (forward) 5′- ACA GCC AGC ACA CCC TTG -3′, (reverse) 5′- TTC TCT GAG CAA CAG CTC CC -3′; Sftpc (forward) 5′- ATG AGA AGG CGT TTG AGG TG -3′, (reverse) 5′- AGC AAA GAG GTC CTG ATG GA -3′; Sftpd (forward) 5′- GAG AGC CCC ATA GGT CCT G -3′, (reverse) 5′- GTA GCC CAA CAG AGA ATG GC -3′; Aqp1 (forward) 5′- TGC AGA GTG CCA ATG ATC TC -3′, (reverse) 5′- GGC ATC ACC TCC TCC CTA GT −3; Col1a1 (forward) 5′-TAG GCC ATT GTG TAT GCA GC-3′, (reverse) 5′- ACA TGT TCA GCT TTG TGG ACC-3′; Col3a1 (forward) 5′-TAG GAC TGA CCA AGG TGG CT-3′, (reverse) 5′- GGA ACC TGG TTT CTT CTC ACC-3′; Col4a1 (forward) 5′-CAC ATT TTC CAC AGC CAG AG-3′, (reverse) 5′- GTC TGG CTT CTG CTG CTC TT-3′; Col5a2 (forward) 5′-CAT GGA GAA GGT TTC CAA ATG-3′, (reverse) 5′- AAA GCC CAG GAA CAA GAG AA-3′; Col12a1 (forward) 5′-TGA GGT CTG GGT AAA GGC AA-3′, (reverse) 5′- GTA TGA GGT CAC CGT CCA GG-3′; Acta2 (forward) 5′-GTT CAG TGG TGC CTC TGT CA-3′, (reverse) 5′-ACT GGG ACG ACA TGG AAA AG-3′; Ctgf (forward) 5′-GCT TGG CGA TTT TAG GTG TC-3′, (reverse) 5′-CAG ACT GGA GAA GCA GAG CC-3′; Mmp2 (forward) 5′-GGG GTC CAT TTT CTT CTT CA-3′, (reverse) 5′-CCA GCA AGT AGA TGC TGC CT-3′; Mmp12 (forward) 5′-TTT GGA TTA TTG GAA TGC TGC-3′, (reverse) 5′-ATG AGG CAG AAA CGT GGA CT-3′; β-actin (forward) 5′-ATG GAG GGG AAT ACA GCC C-3′, (reverse) 5′-TTC TTT GCA GCT CCT TCG TT-3′; and 18S (forward) 5′- CGC GGT TCT ATT TTG TTG GT-3′, (reverse) 5′- AGT CGG CAT CGT TTA TGG TC-3′.Total RNAs isolated using TRIzol (Lifetechnologies) and digested with DNase I were reverse-transcribed by Superscript II reverse transcriptase (Life Technologies). Quantitative RT-PCR (qRT-PCR) was performed with ABI Prism 7900 Sequence Detection System  using SYBR Green master mixture. The ΔΔ Ct method was used using β-actin or 18S as normalization control. PCR condition used was 50 °C, 2 min and 95 °C, 10 min followed by 95 °C, 15 s and 60 °C, 40 s for 40 cycles with the following primers: t-test. P < 0.05 was considered significant.All animal studies were carried out at least twice, each using at least 5–6 mice per group. Statistical analysis was carried out between wild-type and transgenic mice of various ages using a student’s Scgb3a2 cDNA coding sequence, under control of the human SP-C gene promoter at the 5′ end, and the SV40 small T intron and poly A addition site at the 3′ end [Scgb3a2 as determined by Northern blotting  fluids at various ages from embryonic day (E) 14.5 to 12 or 24-weeks old , followed by fibrosis phase (≥7 days) . Many coScgb3a2-transgenic mice were histologically and functionally normal. When these mice were subjected to the BLM-induced pulmonary fibrosis model, a slightly exaggerated fibrosis was initially noted at 3 weeks post-BLM, however the fibrosis more rapidly resolved in Scgb3a2-transgenic mice as compared to wild-type by 6 weeks post-BLM. These results demonstrate the possible therapeutic use of SCGB3A2 in treatment of lung fibrosis.A transgenic mouse over-expressing SCGB3A2 in lung-specific fashion under the promoter of human SP-C gene was established. The lungs of the


The exobidentate hqda and 4-bpmp ligands form [Co(hqda)(4-bpmp)(H2O)2]n coordination polymer layers parallel to (110) that are anchored into the full crystal structure by O—H⋯O hydrogen bonding between aqua ligands and ligated hqda O atoms. Disordered water molecules of crystallization occupy incipient channels along [100]. However, these could not modeled reliably and so they were treated with SQUEEZE in PLATON ; the crystal data take the presence of these molecules into account. The crystal under investigation was twinned by non-merohedry, the twin fraction of the components being 53.3% and 46.7%. Only data from the major twin component were used in the refinement.In the title compound, {[Co(CSpek 2009. Acta Cr For the al. 2013.10H8O6)(C16H20N4)(H2O)2]·5H2O = 0.093wR(F2) = 0.228S = 1.102786 reflections179 parametersH-atom parameters constrainedmax = 0.71 e Å−3Δρmin = −0.89 e Å−3ΔρAPEX2  I, pub. DOI: 10.1107/S1600536814008745/tk5308Isup2.hklStructure factors: contains datablock(s) I. DOI: 997809CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Phellodendron amurense Ruprecht) and rhizoma atractylodis (Atractylodes lancea D.C). However, its effect on the anti-inflammatory activity in human dermal fibroblasts (HDFs) and the mechanism underlying this effect are unknown.Various health benefits have been attributed to Er-Miao-San (EMS), a traditional Chinese herbal formulation that contains equal amounts of cortex phellodendri (MMP-1 expression in HDFs. Our data show that EMS inhibited TNF-α-induced MMP-1 expression in a concentration-dependent manner. Interestingly, EMS maintained IκB content without inhibiting the phosphorylation of MAPKs, which are well-established upstream kinases of NF-κB. Moreover, EMS reduced the level of nuclear p65 protein in HDFs. Luciferase assay revealed that EMS inhibits the transcriptional activity of NF-κB by stabilizing IκB. Our results show that EMS exerts its anti-inflammatory effect by inhibiting NF-κB-regulated genes such as IL-1β and IL-8. Moreover, EMS effectively inhibited TNF-α-induced expression of MMP-1 via the NF-κB pathway.This study investigated the effects of EMS on TNF-α-induced Taken together, our data suggest that EMS could potentially be used as an anti-inflammatory and anti-aging treatment. Skin aging can be divided into two types, namely intrinsic aging, which is caused by the natural consequences of physical change, and extrinsic aging, which is caused by exposure to environmental factors such as ultraviolet (UV) rays and pollutions . OxidatiWrinkle formation is the most representative characteristic of skin aging, and is closely related to a reduction in skin elasticity and degeneration of the extracellular matrix (ECM) . The ECMinterleukin (IL)-1β and IL-8 [MMP-1 in dermal fibroblasts [MMP-1 expression in HDFs and inhibits skin photoaging [MMP-1, which exerts degradation of dermal type I collagen [NF-κB is one of transcription factors and forms homo-and heterodimer complexes with Rel family proteins such as RelA (p65), RelB, cRel, p50, and p52 . The traand IL-8 , 12. Accroblasts , 14. Intotoaging . Furthercollagen .Cortex Phellodendri (CP) and Rhizoma Atractylodis (RA). The major component of CP, berberine, promotes the apoptosis of cancer cells by regulating caspase-3 [Er-Miao-San (EMS) is a compound commonly found in traditional Chinese medicine (TCM) that consists of equal amounts of aspase-3 . In 3 T3aspase-3 . Moreoveaspase-3 . RA extraspase-3 , 15-lipoaspase-3 , and thraspase-3 , as wellaspase-3  and IL-2aspase-3 . Studiesaspase-3 , and thaaspase-3 , 27. HowMMP1 expression and suppresses collagen synthesis in HDFs [MMP1 expression [MMP-1 expression through suppressing NF-κB nuclear localization in HDFs. Also, we observed that EMS-mediated NF-κB inhibition was not dependent on MAPK signaling pathways in HDFs.TNF-alpha (TNF-α) is one of the major inflammatory cytokines . It was  in HDFs . After Tpression , 13. Bespression , as evidpression . Here, wMMP-1 expression. Therefore, we set the experimental conditions as followed; the treatment concentrations is 0 – 1000 μg/ml, and the treatment time is 24 h. To examine the cytotoxicity of EMS, CP, and RA, HDFs were treated with various concentrations (0–1000 μg/ml) of the reagents for 24 h, and the WST-1 assay was performed to evaluate cell viability. As shown in Figure We first investigated whether the treatments of EMS, CP and RA on human skin dermal fibroblasts will induce cytotoxic or non-cytotoxic properties, and what are the non-cytotoxic concentration ranges of EMS, CP and RA on human skin dermal fibroblasts before further experiments that investigate the EMS-induced protection effect on TNF-α-induced MMP1 expression could be regulated by treating EMS in HDFs. HDFs were seeded and pretreated with 250 and 500 μg/ml EMS for 3 h, and then exposed to TNF-α for 4 h. After TNF-α stimulation, cells were gathered and the expression level of MMP1 was investigated using RT-PCR with its specific primers. As shown in Figure MMP1 by 6.75 ± 0.81 fold compared with non-treated control cells. However, 250 and 500 μg/ml EMS treatments before TNF-α stimulation significantly inhibited the expression level of MMP1 by 4.26 ± 0.37 and 2.26 ± 0.21 fold, respectively, as compared with non-treated control cells . The each dried sample (20 g) was added to 200 ml sterilized distilled water (DW) to produce a 10% solution. For EMS, equal amounts (10 g) of the dried CP and RA were added to 200 ml sterilized DW to be 10% solution. Those solutions were subsequently extracted for 24 h at 60°C and then filtered. After the extraction and filtration, the extracts were subjected to vacuum evaporating and freeze-drying. Finally, we obtained 3.043 g of EMS, 2.548 g of CP, and 2.997 g of RA, respectively. These powders were dissolved into the tissue cell culture medium and used for experiments.2 incubator. TNF-α was purchased from Sigma-Aldrich . Cells (1 × 106 or 3 × 105) were seeded in 60 pi or 6-well cell culture dishes and incubated for overnight. After incubation, the cells were treated with 10 ng/ml TNF-α for 4 h in a serum-free media. All experiments handling human cells were carried out in line with the Tenets of the Declaration of Helsinki.HDFs were obtained from Lonza  and cultured in Dulbecco's Modified Eagle Medium  supplemented with 10% fetal bovine serum, 100 U/ml of penicillin, and 100 μg/ml of streptomycin. The cells were maintained at 37°C in a 5% COMMP-1 were 5′-TCTGACGTTGATCCCAGAGAGCAG-3′ and 5′-CAGGGTGACACCAGTGACTGCAC-3′, respectively. The forward and reverse primers for human β-actin were 5′-GGATTCCTATGTGGGCGACGA-3′ and 5′-CGCTCGGTGAGGATCTTCATG-3′, respectively. The forward and reverse primers for human IL-1β were 5′-ACAGATGAAGTGCTCCTTCCA-3′ and 5′-GTCGGAGATTCGTAGCTGGAT-3′, respectively. Also the forward and reverse primers for human IL-8 were 5′-ATGACTTCCAAGCTGGCCGTGGCT-3′ and 5′- TCTCAGCCCTCTTCAAAAACTTCTC-3′, respectively. PCR was performed using the HOT FIREPol EvaGreen PCR Mix Plus  with Line gene K software . The CT-value for MMP-1, IL-1β and IL-8 were normalized to β-actin. The 2-ΔΔCt method was used to calculate relative expression level of MMP-1, IL-1β and IL-8. Data were presented as mean ± S.D. .cDNAs were synthesized from total RNA using M-MLV reverse transcriptase  according to the manufacturer’s protocol. The forward and reverse primers for human Cell viability was assessed using the WST-1 assay according to the manufacturer’s instructions . The results are represented graphically as the measured cell viability ratio normalized to the control.Cellular proteins was extracted by lysis buffer  and EDTA-free protease and phosphatase inhibitor cocktail . Equal amounts of protein samples were separated by 10% sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and then transferred onto nitrocellulose membrane . After blocking with 5% skim milk for 1 h at room temperature, the membranes were incubated first with primary antibody at 4°C overnight and subsequently with peroxidase-conjugated secondary antibody at room temperature for 1 h. The protein bands were detected using enhanced chemiluminescence reagents. Primary antibody specific for MMP1 NF-κB-p65, IκB and Lamin B were purchased from Santa Cruz Biotechnology . Anti-β-actin antibody was purchased from Sigma-Aldrich . Anti-p38, p38, pERK, ERK, pJNK and JNK were purchased from Cell Signaling Technology .2, 10 mM KCl, 0.5 mM DTT, and 0.05% NP-40) and then incubated for 20 min on ice. The cells were centrifuged for 5 min at 3,000 × g at 4°C. Then, 25 μl of buffer B  was added, and the samples were mixed prior to centrifugation for 20 min at 12,000 × g at 4°C. The supernatant (nuclear protein extract) was collected and stored at -80°C until use.Cells were gently resuspended in 500 μl of buffer A  and incubating for 30 min on ice. After centrifugation for 30 min at 12,000 × g at 4°C, the supernatant was collected. The cell lysate was treated with luciferin (Promega) and its luminescence was measured using a Veritas Luminometer . Results shown are the averages of three independent experiments.NF-κB-luciferase reporter stable NIH-3 T3 cell line, which was stably transfected with the NF-κB-luciferase reporter vector , was purchased from Affymatrix-Panomics. pNF-κB-luc was designed to measure the transcriptional activity of NF-κB. Six copies of NF-κB binding sequences (5′-GGGAATTTCCGGGAATTTCCGGGAATTTCCGGGAATTTCCGGGAATTTCCGGGAATTTCC-3′) were subcloned into the upstream region of luciferase cDNA. The NIH-3 T3/NF-κB-luc cell line was obtained by co-transfection of pNF-κB-luc (Affymatrix-Panomics) and pHyg into NIH-3 T3 cells, followed by hygromycin selection. To test NF-κB activity, approximately 1 × 10t-test analysis. P < 0.05 was considered significant.A result of three observations per group was subjected to a statistical analysis. Data are presented as mean ± the standard deviation (S.D.). Statistical analysis was performed using two-tailed Student’s


All three compounds are isostructural to the previously reported La analogue. The asymmetric unit of [Ln(NO3)3(μ2-bpydo)2] contains one lanthanide cation, two bpydo ligands, and three nitrate anions. Both bpydo ligands act as end-to-end μ2-bridges and display nearly ideal cis and gauche conformations, respectively. The bpydo ligands link the ten-coordinate LnIII cations, forming interdigitating 44 grid-like layers extending parallel to (-101), where interdigitation of layers is promoted by C—H⋯O interactions between nitrate anions and bpydo ligands. The interdigitated layers are linked to sets of neighboring layers via further C—H⋯O and π–π interactions.Three isostructural coordination networks of Ce, Pr, and Nd nitrate with 4,4′-bipyridine  N,N′-dioxide ligands such as 4,4′-bipyridine N,N′-dioxide (bpydo) in the synthesis of lanthanide compounds comprising coordination networks has been of recent interest , [Ln(NO3)3(μ2-bpydo)2]  contains one lanthanide cation, two end-to-end bridging μ2-bpydo ligands, and three chelating nitrate anions. All atoms in the asymmetric unit lie on general positions  Fig. 3. Each laid Fig. 4.et al., 2005et al., 2012Ln—X bond lengths and bridged Ln⋯Ln distances provides evidence of the lanthanide contraction –(III) shows the expected gradual decrease in the Ln—O bond lengths from Ce (I)Ln⋯Ln distances within the layers is also consistent with the radius contraction from Ce to Nd  are observed between neighboring bpydo ligands within the coordination sphere of the LnIII cation  between the ligands of one layer and nitrate anions of the other layer  and π–π interactions between Cg1 and the inversion-related Cg1x link each set of interdigitated layers to symmetry-equivalent sets of layers above and below it , linear chains are observed (NO3)3] 3]·CH2Cl2} with Ln = Eu 3] is a two-dimensional network composed of sheets with 44 topology and is isostructural to the Ce, Pr, and Nd structures reported herein 3(NO3)6]·2CH3OH}, {[Tb(bpydo)1.5(NO3)3]·CH3OH·0.8H2O}, and {[Tb(bpydo)1.5(NO3)3]·0.4CCl4·0.8CH3OH}, two-dimensional networks composed of sheets with 4.82 topology are formed 3]·0.5H2O}, a twofold interpenetrating three-dimensional network is formed 3·6H2O (0.113 mmol) was placed in the bottom of a test tube and covered with CH2Cl2 (5 ml). 4,4′-Bipyridine-N,N′-dioxide·H2O  was dissolved in methanol (8 ml), and this solution was layered over the CH2Cl2 solution. The two solutions were allowed to slowly mix. Over a period of several weeks the Ln(NO3)3·6H2O dissolved, and red block-like crystals of [Ce(μ2-bpydo)2(NO3)3], yellow block-like crystals of [Pr(μ2-bpydo)2(NO3)3], and yellow block-like crystals of [Nd(μ2-bpydo)2(NO3)3] were formed.4,4′-bipyridine  al. 1963. All othUiso(H) = 1.2Ueq(C). Crystal data, data collection and structure refinement details are summarized in Table 6All aromatic H atoms were positioned geometrically and refined using a riding model with C—H = 0.95 Å and with 10.1107/S205698901502318X/wm5242sup1.cifCrystal structure: contains datablock(s) global, I, II, III. DOI: 10.1107/S205698901502318X/wm5242Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S205698901502318X/wm5242IIsup3.hklStructure factors: contains datablock(s) II. DOI: 10.1107/S205698901502318X/wm5242IIIsup4.hklStructure factors: contains datablock(s) III. DOI: 1440109, 1440108, 1440107CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The crystal packing features an unusual C—H⋯π interaction involving the triple bond of the propyne group which combines with C—H⋯O hydrogen bonds to stack the molecules along the  12H20NO2, was synthesized from 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (hydroxy-TEMPO) and propargyl bromide. The six-membered ring adopts a flattened chair conformation and carries a propynyloxy substituent in an equatorial orientation at the 4-position. The N—O bond length of the piperidin-1-oxyl unit is 1.289 (3) Å. In the crystal, C—H⋯O hydrogen bonds combine with unusual C—H⋯π interactions involving the alkyne unit as acceptor to generate a three-dimensional network.The title compound, C An interesting feature of these latter contacts is the support provided by C5—H5B⋯Cg interactions (Cg is the mid-point of the C8—C9 bond) involving the alkyne unit , C9—H9⋯O1 hydrogen bonds link molecules into b Table 1. Additio b Fig. 2. In an ait Fig. 3. Such cois Fig. 4.et al. , and investigates the magnetism and orientation dependent motion of the encapsulated radical. In the second, the molecule is included in the cavities of two porous frameworks derived from guanidinium cations and two organodisulfonate anions; the magnetic behaviour of the radical guest is investigated. Aryloyloxy-TEMPO derivatives are more abundant with 19 entries in the CSD  are as previously described (Gheorghe Uiso(H) = 1.2Ueq(C) for methylene H atoms, C—H = 1.00 Å and Uiso(H) = 1.2Ueq(C) for methine H atoms, C—H = 0.98 Å and Uiso(H) = 1.5Ueq(C) for methyl H atoms, and C—H = 0.95 Å and Uiso(H) = 1.2Ueq(C) for the terminal alkyne H atom. Anisiotropic refinement of the non-H atoms was constrained using the ISOR command in SHELXL to prevent atoms becoming non-positive definite. 10 reflections with Fo >> Fc were omitted from the final refinement cycles.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S1600536814017991/hb7267sup1.cifCrystal structure: contains datablock(s) global, 1. DOI: 10.1107/S1600536814017991/hb72671sup2.hklStructure factors: contains datablock(s) 1. DOI: 1017949CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


In the crystal of the orthorhombic polymorph of compound (I), the molecules are linked into chains by O—H⋯π interactions. In compound (II), carboxylic acid inversion dimers are observed; the dimers are linked into chains by C—H⋯O hydrogen bonds. 14H12O, (I), and C15H11BrO2, (II), were prepared and characterized as part of our studies of potential new photo-acid generators. In (I), which crystallizes in the orthorhombic space group Pca21, compared to P21/n for the previously known monoclinic polymorph  chains as part of the herringbone packing motif. The asymmetric unit of (II) contains two molecules with similar conformations (weighted r.m.s. overlay fit = 0.183 Å). In the crystal of (II), both molecules form carboxylate inversion dimers linked by pairs of O—H⋯O hydrogen bonds, generating R22(8) loops in each case. The dimers are linked by pairs of C—H⋯O hydrogen bonds to form [010] chains.The title compounds, Crtin 2013. Org. Le Treatment with base then solubilizes and removes the degraded polymer film in exposed areas, thereby creating a positive resist image  might occur via a stabilized carbocation intermediate 4. In the absence of an oxidant, the cyclized dihydro-phenanthrene compound 6 will equilibrate back to cis-stilbene 2. Stilbenes can also undergo 2π + 2π photochemical cycloadditions  of (I)As part of these studies, the syntheses and crystal structures of the title substituted stilbenes, (I)ar—Car—O—H (ar = aromatic) torsion angle is 172°. The molecule is close to planar and the dihedral angle between the aromatic rings is 4.35 (6)°. The bond lengths of the central unit  are consistent with data from previous studies of similar compounds There are two molecules in the asymmetric unit of (II)O) generates [001] zigzag chains, as seen in Fig. 3O) also forms [001] chains. There are also some possible very weak C—H⋯π interactions. The packing can be described as herringbone when viewed down [100] 0] Fig. 4. The mon0] Fig. 4 also fea0] Fig. 4.A and B) form carboxylic acid inversion dimers linked by pairs of O—H⋯O hydrogen bonds (Table 2A + A) and (B + B) dimers are in turn linked by pairs of C—H⋯O hydrogen bonds to generate [010] chains s Table 2, which gs Figs. 6 and 7 ▸.E-2-hydroxy stilbenes with different substituents including (E)-1,2-bis(2-hydroxyphenyl)ethene et al., 2011A survey of the Cambridge Structural Database  eluted the title compound  as a white solid (m.p. 418–419 K), which was recrystallized from hexane/diethyl ether solution to yield colourless slabs of (I)m/z 196.0886 (M+) C14H12O requires 196.0883. UV λmax(CHCl3)/nm 230 (log ∊ 4.30), 288 (4.39) and 315 (4.40). IR (νmax/cm−1) 3528s, 3019w, 2923w, 2852w, 1585s, 1498s, 1454s, 1332s, 1249s, 1195s, 1088s, 974vs, 845s, 752vs, 724vs, 691vs, 507vs. 1H NMR  δ 5.07 , 6.79 , 6.95 , 7.14 , 7.25 , 7.35 , 7.52 . 13C NMR  δ 116.1, 121.3, 123.1, 124.8, 126.7, 127.3, 127.7, 128.8, 130.3, 137.7 and 153.1 (one resonance is missing).Salicylaldehyde  and benzyltriphenylphosphonium bromide  in dry dimethylformamide (DMF) (30 ml) were treated with sodium methoxide powder  and refluxed for 4 h  eluted (II)m/z 300.9866 (M + H) C15H10O2Br requires 300.9870.2-Bromobenzaldehyde  and methyl phenylacetate  in dry DMF (30 ml) were treated with sodium methoxide powder  and refluxed for 4 h. The reaction mixture was then cooled, acidified with dilute aqueous HCl and extracted into CHO in (I)Uiso(H) = 1.2Ueq(O). The other H atoms were placed geometrically  and refined as riding atoms with Uiso(H) = 1.2Ueq. The O-bound H atoms in (II)Uiso(H) = 1.2Ueq(O). The C-bound H atoms were placed geometrically (C—H = 0.95 Å) and refined as riding atoms with Uiso(H) = 1.2Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989016002942/su5278sup1.cifCrystal structure: contains datablock(s) I, II, global. DOI: Click here for additional data file.10.1107/S2056989016002942/su5278Isup2.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989016002942/su5278IIsup3.cmlSupporting information file. DOI: 1454393, 1454392CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Although injection drug use drives antiretroviral drug resistance, the prevalence of protease inhibitor (PI) resistance among Kenyan IDUs remains undetermined. We, therefore, explored PI resistance mutations and their association with viral load and CD4+ T cell counts in HIV-1 infected IDUs  and non-drug users  naive for PI treatment from coastal Kenya.P < 0.05).Only IDUs harboured major PI resistance mutations consisting of L90M, M46I and D30 N among 3 (6.4 %) ART-experienced and 1 (3.1 %) -naive individuals. Minor PI mutations including A71T, G48E, G48R, I13V, K20I, K20R, L10I, L10V, L33F, L63P, T74S, V11I, and V32L were detected among the ART-experienced (36.2 vs. 46.9 %) and -naive (43.8 vs. 66.7 %) IDUs and non-drug users, respectively. All the four IDUs possessing major mutations had high viral load while three presented with CD4+ T cell counts of <500 cells/ml. Among the ART-naive non-drug users, CD4+ T cell counts were significantly lower in carriers of minor mutations compared to non-carriers (Transmitted drug resistance may occur in IDUs underscoring the need for genotyping resistance before initiating PI treatment. Despite up-scaling of antiretroviral drugs for treatment and prevention, HIV-1 remains an important cause of infectious disease burden in the world. In the year 2013, an estimated 35 million people globally were infected with HIV of whom 24.7 million reside in Sub-Saharan Africa . As suchThe current standard regimen for first-line HIV treatment in Kenya consists of two nucleoside reverse transcriptase inhibitors (NRTI) mainly tenofovir disoproxil fumarate (TDF) or azidovudine (AZT), lamuvudine (3TC) and one non-nucleoside reverse transcriptase inhibitor (NNRTI) efevirenz (EFV) or nevirapine (NVP) . ProteasSeveral studies across Kenya show widespread resistance to NRTIs and non-NNRTIs in ART-naive and -experienced adults and children, breastfeeding infants and IDUs –31. MoreThis cross-sectional study was conducted in HIV-1 infected IDUs and non-drug using controls at Bomu Hospital, a social enterprise facility in Mombasa City, coastal Kenya. Injection drug users were recruited via respondent-driven, snowball and makeshift-outreach sampling methods. In the context of the current study, IDUs comprised of individuals exhibiting injection needle-stick scars and reporting a history of injecting any illicit drug classified by UNODC at least once in the previous month . Those y® 903 cards  for preparing dry blood spots (DBS) that were stored at −20 °C until used for pro-viral DNA extraction. For enumerating CD4+ T cell counts, 5.0 μl of blood were stained with CD3/CD4/CD45 monoclonal antibody reagent (BD Tritest) and analysed on BD FACSCalibur™ according to the manufacturer’s protocols. HIV-1 viral load was determined in 0.2 ml plasma samples using the Abbott Molecular m2000sp sample preparation and m2000rt real-time amplification and detection systems  according to the manufacturer’s methods.In brief, about, 3.0 ml venous blood was collected from study participants in ethylenediaminetetraacetic acid vacutainer tubes  and used for enumerating CD4+ T cells, and plasma harvesting for HIV-1 viral load quantification. A drop of whole blood was applied on Whatman-FTA2, 1 U/µL Taq polymerase (KemTaq®), 200 µM of each dNTP , and 0.5 µM of each first or second round primers. Amplification consisted of initial denaturation at 94 °C for 4 min, followed by 40 cycles of 94 °C for 15 s, 50 °C for 30 s, and 72 °C for 1 min, with a final extension of 72 °C for 5 min. PCR products were electrophoresed in 1 % ethidium bromide-stained agarose gel, and visualized under ultraviolet light. PCR product sizes of approximately 330 bp were diagnostic for HIV-1 pro-viral DNA.HIV-1 pro-viral DNA was extracted from DBS using QIAamp™ DNA blood mini kit  according to the manufacturer’s recommendations. HIV-1 pro-viral DNA encoding protease gene was amplified by nested PCR with primers; Nyupol_7 (5′-GGGAATTTTCTTCAGAGCAG-3′) HXB2 nucleotide position 2125–2144, and Nyupol_8 (5′-TCTTCTGTCAATGGCCATTGT-3′) HXB2 nucleotide position 2635–2615 in the first round. Nyupol_9 (5′-TCCTTAACTTCCCTCAAATCACT-3′) HXB2 nucleotide position 2241–2264 and Nyupol_10 (5′-CTGGCACGGTTTCAATAG GACT-3′) HXB2 nucleotide position 2577–2556 were used in the second round. First and second round PCR was conducted in a 25 µl volume containing 5 µL DNA, 1× PCR buffer, 1.5 mM MgCl®, Foster City, USA) on an ABI PRISM® 3100 Genetic Analyzer  as per the manufacturer’s protocols. Primer pair Nyupol_9 and Nyupol_10 was used for forward and reverse sequencing reactions, respectively. Forward and reverse chromatograms were visually inspected and edited for base mis-calls using MEGA version 6.0 [http://www.DnaBaser.com). Protease inhibitor drug resistance mutations were identified and interpreted using Stanford web-based HIV drug resistance database [PCR amplicons were purified with QIAquick™ PCR purification kit  according to the manufacturer’s protocol. Direct sequencing was performed using BigDye™ sequencing chemistry . Age and CD4+ T cell counts and viral load were compared across study groups using Kruskal–Wallis test and Dunn’s post hoc correction for multiple comparisons. Differences in distribution of gender were compared across the study groups using Chi-square test. Rates of resistance mutations in each study group were summarised as numbers and proportions. Statistical significance was set at P < 0.05.Data analysis was conducted using IBMThis study was conducted following ethical approval obtained from Kenyatta University Ethical Review Committee, approval number PKU019/116 of 2012 in accordance with Helsinki declaration . WrittenP = 0.531). Age was significantly different across the groups (P = 0.009) with ART-naive IDUs  having younger individuals relative to ART-naive non-drug users . In addition, 20 (42.5 %), 24 (51.1 %) and 3 (6.4 %) ART-experienced IDUs while 3 (9.3 %), 7 (21.9 %) and 22 (68.8 %) ART-experienced non-drug users were on antiretroviral therapy for <1, 1–3, and >3 years, respectively. The CD4+ T cell counts were similar across the study groups (P = 0.379). The HIV-1 viral load was significantly different across the study groups (P = 0.004) with ART-naive  and -experienced  IDUs presenting with lower levels compared to ART-naive non-drug users .Baseline characteristics of the study participants are presented in Table Major and minor HIV-1 protease drug resistance mutations detected in the study participants are shown in Table In non-drug users, 15 (46.9 %) ART-experienced individuals had minor mutations consisting of 5 (15.6 %) K20R, 2 (6.3 %) L10V, 2 (6.3 %) L33F, 1 (3.1 %) I13V + L63P, 1 (3.1 %) L10V + K20R, 3 (9.4 %) L10V + T74S and 1 (3.1 %) L33F + A71T. In addition, 14 (66.7 %) ART-naive non-drug users harboured minor mutations comprising of 1 (7.1 %) K20I, 8 (38.1 %) K20R, 2 (9.5 %) L10I, 1 (7.1 %) L101 + K20R, and 2 (9.5 %) L10V + V11I.P > 0.05), but among ART-naive non-drug users, CD4+ T cell counts were significantly lower in carriers of minor mutations compared to non-carriers .Viral load and CD4+ T cell counts of individuals with major and minor mutations are presented in Tables Effective use of protease inhibitors in HIV-1 treatment and prevention is largely impeded by the increasing development and spread of acquired and transmitted resistance . AntiretIn the current study, major PI mutations were detected only in IDUs with the antiretroviral treatment-experienced  individuals having higher rates than -naive (D30N) individuals. Most importantly, major mutations, D30N and M46I co-existed with minor mutations, G48E, K20I and T74S, suggesting that these mutations mediate evolution of major drug resistance . Our finOverall, higher rates of minor mutations were found in IDUs relative to non-drug users, suggesting heterogeneity of HIV-1 strains in IDUs. The detection of A71T, G48E/R, I13V, K20I/R, L10I/V, L33F, L63P, T74S, V11I, and V32L minor mutations in this study is, in part, consistent with previous studies that reported K20R, L10I/V, L33F/I, and L63P minor mutations among HIV-positive women attending antenatal clinics in a large HIV treatment study in western Kenya . HoweverThe high viral load among individuals carrying major PI mutations is consistent with phenotypic and clinical studies showing absence or low virologic response in patients having these major mutations . It is ienv [While a prospective design would have been important in examining the emergence of PI resistance in treatment-naive and -experienced IDUs, this cross-sectional study provides the first baseline evidence of occurrence of PI resistance in IDUs from Kenya. Although specific antiretroviral regimens used by the ART-experienced individuals were not evaluated in this study, poor adherence to ART by injection drug users, generate sub-optimal antiretroviral drug levels that exert selective pressure on the viral genome leading to resistance . Since ienv  that conProtease inhibitor transmitted drug resistance is likely to occur among Kenyan IDUs. Therefore, performing HIV-1 drug resistance analyses prior to initiating PI treatment in HIV patients from high-risk population groups is critical in achieving better treatment outcomes and prolonging the effectiveness of these drugs in the country.The 125 sequences described in this study were submitted to the GenBank under accession numbers: KP791995, KP791996, KP791997, KP791998, KP791999, KP792000, KP792001, KP792002, KP792003, KP792004, KP792005, KP792006, KP792007, KP792008, KP792009, KP792010, KP792011, KP792012, KP792013, KP792014, KP792015, KP792016, KP792017, KP792018, KP792019, KP792020, KP792021, KP792022, KP792023, KP792024, KP792025, KP792026, KP792027, KP792028, KP792029, KP792030, KP792031, KP792032, KP792033, KP792034, KP792035, KP792036, KP792037, KP792038, KP792039, KP792040, KP792041, KP792042, KP792043, KP792044, KP792045, KP792046, KP792047, KP792048, KP792049, KP792050, KP792051, KP792052, KP792053, KP792054, KP792055, KP792056, KP792057, KP792058, KP792059, KP792060, KP792061, KP792062, KP792063, KP792064, KP792065, KP792066, KP792067, KP792068, KP792069, KP792070, KP792071, KP792072, KP792073, KP792074, KP792075, KP792076, KP792077, KP792078, KP792079, KP792080, KP792081, KP792082, KP792083, KP792084, KP792085, KP792086, KP792087, KP792088, KP792089, KP792090, KP792091, KP792092, KP792093, KP792094, KP792095, KP792096, KP792097, KP792098, KP792099, KP792100, KP792101, KP792102, KP792103, KP792104, KP792105, KP792106, KP792107, KP792108, KP792109, KP792110, KP792111, KP792112, KP792113, KP792114, KP792115, KP792116, KP792117, KP792118, KP792119, KT033003, KT033004, KT033005, KT033006, KT033007, KT033008, and KT033009.


As with other imidazole derivatives, metronidazole is also susceptible to protonation. However, there are few reports of the structures of metronidazolium derivatives. In the title compound, (C6H10N3O3)[AuCl4] [systematic name: 1-(2-hydroxyethyl)-2-methyl-5-nitro-1H-imidazol-3-ium tetrachloridoaurate(III)], the asymmetric unit consists of a metronidazolium cation, [H(MET)]+, and a tetrachloridoaurate(III) anion, [AuCl4]−, in which the AuIII ion is in a slightly distorted square-planar coordination environment. In the cation, the nitro group is essentially coplanar with the imidazole ring, as indicated by an O N—C=C torsion angle of −0.2 (4)°, while the hydroxyethyl group is in a coiled conformation, with an O(H)—C—C—N torsion angle of 62.3 (3)°. In the crystal, the anion and cation are linked by an intermolecular O—H⋯Cl hydrogen bond. In addition, the N—H group of the metronidazolium ion serves as a hydrogen-bond donor to the O atom of the hydroxyethyl group of a symmetry-related molecule, leading to the formation of chains along [010].Metronidazole (MET) [systematic names: 1-(2-hydroxyethyl)-2-methyl-5-nitro-1 H-imidazole and 2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethanol, is a medication that has been used for the treatment of parasitic infections, such as trichomoniasis, amoebiasis and giardiasis, and is also effective against anaerobic bacteria , marketed as flagyl, and also known by the systematic names 1-(2-hydroxyethyl)-2-methyl-5-nitro-14] consists of a metronidazolium cation, [H(MET)]+, hydrogen-bonded to a square-planar tetrachloridoaurate(III) anion, [AuCl4]−, by an O—H⋯Cl hydrogen bond as illustrated in Fig. 14]−, with an O(H)⋯Cl distance of 3.365 Å ][AuCl4]− ion , while the N—H group is the hydrogen-bond donor for metronidazolium (i.e. N—H⋯O).In the crystal, the N—H group of the metronidazolium ion serves as a hydrogen-bond donor to the oxygen atom of the hydroxyethyl group of a symmetry-related molecule, forming a chain along [010] in which each O—H group is O—H⋯Cl hydrogen bonded to a [AuClUiso(H) = 1.2Ueq(C) or 1.5Ueq(Cmethyl). H atoms bonded to N and O atoms were refined independently with isotropic displacement parameters.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989015010798/lh5766sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989015010798/lh5766Isup3.hklStructure factors: contains datablock(s) I. DOI: 1404845CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The asymmetric unit contains one TbIII ion, one formate anion (CHO2−) and half of an oxalate anion (C2O42−), the latter being completed by application of inversion symmetry. The TbIII ion is nine-coordinated in a distorted tricapped trigonal–prismatic manner by two chelating carboxylate groups from two C2O42− ligands, two carboxylate oxygen atoms from another two C2O42− ligands and three oxygen atoms from three CHO2− ligands, with the Tb—O bond lengths and the O—Tb—O bond angles ranging from 2.4165 (19) to 2.478 (3) Å and 64.53 (6) to 144.49 (4)°, respectively. The CHO2− and C2O42− anions adopt μ3-bridging and μ4-chelating-bridging coordination modes, respectively, linking adjacent TbIII ions into a three-dimensional 12-connected fcu topology with point symbol (324.436.56). The title compound exhibits thermal stability up to 623 K, and also displays strong green photoluminescence in the solid state at room temperature.The title compound, poly[( The thermal stability and luminescent properties of compound (I)Herein, we report the synthesis and structure of a terbium(III) coordination polymer containing formate and oxalate mixed ligands, [Tber Fig. 1 by two cer Fig. 1, which i3-bridging coordination mode connecting three TbIII ions, forming a two-dimensional (2-D) layer in the ac plane. In the 2-D terbium-formate monolayer, the Tb1⋯Tb1 separations along the formate ligands in syn–anti and anti–anti O1,O2-bridging coordination modes  terbium–oxalate open framework  Å with the Tb1—O1—Tb1 and Tb1—O4—Tb1 bond angles being 103.00 (9) and 102.79 (6)°, respectively. On the other hand, the channels in the 3-D open framework have an approximate rhombic shape with a Tb1⋯Tb1 separation of 6.2670 (2) Å, and are cross-linked parallel to the c axis by bridging formate ligands as shown in Fig. 4TOPOS  by linking each adjacent layer of TbIII atoms via formate and oxalate ligands.As shown in Fig. 2 Å Fig. 3. The oxark Fig. 3. The Tb1−1. This spectrum indicates the presence of the carboxylate groups of the ligands by appearance of the strong absorption bands at 1630 and 1315 cm−1 for the asymmetric (νasymCOO−) and the symmetric (νsymCOO−) carboxylate vibrations, respectively f–f transitions of TbIII ions  transitions, respectively. The most intense transition is 5D4 → 7F5, which implies the emitted light is green. The emission lifetime of (I)The photoluminescence properties of (I)viii hydrogen bonds giving S(7) graph-set motifs , oxalic acid , Na2CO3 , and a mixture (1:1 v/v) of N,N′-dimethylformamide (DMF) and water (6 ml) was sealed in a 23 ml Teflon-lined stainless steel vessel and heated under autogenous pressure at 463 K for two days. After the reactor was cooled to room temperature, colorless block-shaped crystals were filtered off and dried in air. Yield: 0.118 g (63% based on the TbIII source). Analysis (%) calculated for C3HO6Tb (291.96): C, 12.34; H, 0.35%. Found: C, 12.40; H, 0.33%. IR : 2823 (w), 2491 (w), 1630 (s), 1440 (w), 1315 (s), 1022 (m), 914 (w), 795 (s), 611 (w), 492 (s), 408 (w).All reagents were of analytical grade and were used as obtained from commercial sources without further purification. Synthesis of (I)Uiso(H) = 1.2Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S205698901502397X/zs2356sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S205698901502397X/zs2356Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S205698901502397X/zs2356Isup3.cdxSupporting information file. DOI: Click here for additional data file.10.1107/S205698901502397X/zs2356Isup4.docxSupporting information file. DOI: 1436132CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The peroxisome proliferator-activated receptor (PPAR) family plays central roles in brown adipose tissue (BAT) adipogenesis and contributes to body temperature maintenance. The transcriptional activity of PPAR family has been shown to be tightly controlled by cellular signal networks. β-arrestins function as major secondary messengers of G protein-coupled receptors (GPCR) signaling by functional interactions with diverse proteins. Here, we report that β-arrestin-1 knock-out mice show enhanced cold tolerance. We found that β-arrestin-1 directly interacts with PPARα and PPARγ through a LXXXLXXXL motif, while D371 in PPARα and L311/N312/D380 in PPARγ are required for their interactions with β-arrestin-1. Further mechanistic studies showed that β-arrestin-1 promotes PPARα- but represses PPARγ-mediated transcriptional activities, providing potential regulatory pathway for BAT function. Brown adipose tissue (BAT) produces heat through the process of non-shivering thermogenesis, which contributes to the maintenance of body temperature in response to cold challenge134The PPAR family plays central roles in brown fat adipogenesis and body temperature control. As a major activator of mitochondrial and peroxisomal fatty acid oxidation78911141516β-arrestins mediate desensitization and endocytosis of G protein-coupled receptors (GPCR) and have been considered as GPCR signal terminators21Arrb1, which were flanked by two loxP sites. The frt-Neo-frt cassette was used as a positive selection marker to get the embryonic stem cells, which were then injected into the C57BL/6 blastocysts. The resulting embryos were transferred to pseudopregnant mice to generate the chimeric mice. We mated male founders and female CreTg/Tg mice to obtain Arrb1Flox/+;CreTg/+ mice, which were then crossed with C57BL/6 mice to remove the Cre gene and back-crossed 10 times to the C57BL/6 background. All mice were genotyped by PCR analysis of genomic DNA. The resulting Arrb1-KO mice showed no detectable β-arrestin-1 left (β-arrestin-1 knock-out (Arrb1-KO) mice were generated by deletion of exons 2 and 3 in n-1 left .Ucp1, Cidea, Pgc1α, Pgc1β, Cox7a, and Cpt1β. In BAT, the expression of Ucp1, Cidea, and Pgc1β was considerable increased in Arrb1-KO mice after cold exposure compared with the wild-type mice , when nonshivering thermogenesis is not required, there were no differences in core body temperature between the wild-type and Arrb1-KO mice. However, when we exposed the mice to an ambient temperature of 5 °C, the body temperatures of Arrb1-KO mice were about 1 °C higher compared with that of the wild-type mice after 2 hours or 4 hours of cold exposure, suggesting an enhanced thermogenic capacity after cold challenge in Arrb1-KO mice . We furtype mice . In subctermates . In epidype mice . Taken tE. coli. In an in vitro pulldown assay, we observed that β-arrestin-1 bound to his-tagged PPARα-LBD and PPARγ-LBD  and affinities (KD) were presented in 2 values were above 0.97, and the χ2 values were below 0.90 for all fits. Additionally, steady-state analysis was demonstrated in on and koff values was used. The single steady-state equilibrium dissociation constants KD and the Rmax which represents the saturating binding level were presented in We then used biolayer interferometry to conduct a kinetic analysis of the interactions between β-arrestin-1 and PPARα/γ-LBD. Purified 50 μg/ml PPARα-LBD and PPARγ-LBD were biotinylated and immobilized on streptavidin biosensors (SAs). The SAs were then incubated in wells containing different concentrations of purified β-arrestin-1 0.5–32 μM). Representative data for association/dissociation phases of the curves were demonstrated in  μM. Repr50 = 0.88 μM) or PPARγ-LBD (IC50 = 0.85 μM). These results suggest that β-arrestin-1 directly interacts with PPARα-LBD and PPARγ-LBD. Furthermore, β-arrestin-1 and RXRα-LBD compete for the interaction with PPARα-LBD or PPARγ-LBD.We further examined the interactions of PPARα/γ-LBD with 9-cis-retinoic acid receptor α-ligand binding domain (RXRα-LBD) in the absence or presence of β-arrestin-1. We found that β-arrestin-1 showed no detectable interaction with RXRα-LBD . As showin vitro pulldown assay. We found that compared with the wild-type β-arrestin-1, βarr1M showed reduced interactions with both his-tagged PPARα-LBD and PPARγ-LBD  or PPARγ-LBD (KD of βarr1M vs. βarr1: 1.7 μM vs. 1.4 μM) compared to the wild-type β-arrestin-1. These results indicate that the residues Leu100 and Leu104 in LXXXLXXXL motif of β-arrestin-1 are required for the interactions with PPARα/γ-LBD.We also evaluated the kinetic properties of interactions between PPARα/γ-LBD and βarr1M. As shown in D of β-arrestin-1 peptide with PPARα/γ-LBD were significant higher compared with β-arrestin-1 (on and koff) and affinities (KD) are presented in To further test whether direct interactions between the LXXXLXXXL motif in β-arrestin-1 and PPARα/γ-LBD exist, we performed the biolayer interferometry assay using immobilized PPARα/γ-LBD and synthetic β-arrestin-1 peptides harboring the LQERLIKKL sequence. As shown in restin-1  and 2. Wrestin-1 . We alsoTo further test whether the interactions of β-arrestin-1 with PPARα and PPARγ mediate the transcriptional activities of PPARs, we performed the reporter assay by measuring the luciferase activity under the control of a PPAR response element (PPRE). As shown in 15N-labeled PPARα-LBD or PPARγ-LBD was titrated with β-arrestin-1 peptide up to a molar ratio of 1:10. Representative overlays of the 2D -HSQC spectra in the absence or presence of β-arrestin-1 peptide were shown in We further examined by nuclear magnetic resonance (NMR) spectroscopy the specific residues in PPARα and PPARγ that are critical for binding with β-arrestin-1. We carried out an NMR chemical shift perturbation assay in which a sample of To confirm the potential binding sites in PPARα and PPARγ with β-arrestin-1, we generated the following four mutants: PPARα-LBD L302G/N303G (PPARα M1), PPARα-LBD D371A (PPARα M2), PPARγ-LBD L311G/N312G (PPARγ M1), and PPARγ-LBD D380A (PPARγ M2). Then we performed the biolayer interferometry assay. We found that PPARα M2, but not M1 showed a reduced interaction with β-arrestin-1 . In addiD was obtained. Likewise, compared with the wild-type PPARγ, PPARγ M1 and M2 showed slightly increased equilibrium dissociation constants KD of interactions with β-arrestin-1 but reduced maximum responses   . Consist0.77 nm) . All theFunction of brown adipose tissue contributes to energy expenditure and adaptive thermogenesisin vivo expression of β-arrestin-1 repressed adipogenesis in WAT and diet-induced obesity21Ucp1 and Cidea in BAT of the Arrb1-KO mice. Interestingly, compared with the wild-type littermates, a brown-like phenotype in iWAT was observed in Arrb1-KO mice, which was associated with increased expression of Ucp1. However, the functional bi-direction mediation of PPARs activity by β-arrestin-1 need to be confirmed in vivo, which is now under investigation.β-arrestins function mainly by binding to diverse partners and play critical roles in regulating various signaling pathways. Studies have shown that dysfunctions of β-arrestins contribute to various disease progressions31Studies were carried out with C57BL/6 mice obtained from Shanghai Laboratory Animal Center, Chinese Academy of Sciences. All mice were maintained in pathogen-free conditions. Animal care and use were in accordance with the guidelines of the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. All animal experimental procedures were approved and overseen by the Animal Care and Use Committee of the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences.15NH4Cl was from Cambridge Isotope Laboratories. Inc. Rosiglitazone, GW7647, and n-Dodecyl β-D-maltoside (DDM) were from Sigma.Mouse anti-β-arrestin-1 polyclonal antibody was from Abmart. Rabbit anti-β-arrestin-1 (A1CT) antibody was a gift from Dr. Robert J. Lefkowitz. Rabbit anti-PPARα polyclonal antibody was purchased from Abcam. Rabbit anti-PPARγ polyclonal antibody was from Santa Cruz Biotechnology. Thrombin protease was obtained from GE Healthcare. Twelve-week-old wild-type and Arrb1-KO mice were housed individually and exposed to 5 °C for 6 hours with free access to water. Core body temperatures were measured with a rectal microprobe thermometer  before and at indicated intervals during cold exposure. After 6 hours of cold exposure, mice were sacrificed and tissues were harvested.Endogenous immunoprecipitation experiments were performed as describedrattus β-arrestin-1 was expressed and purified as reported previouslyhomo wild-type PPARα (amino acids 196–468) and PPARγ (amino acids 204–477) and their mutants were cloned into a modified pET28a vector with an N-terminal His6 tag. His6-tagged homo RXRα-LBD (amino acids 196–435) was cloned into a pET15b vector. Homo PAPRα-LBD, PPARγ-LBD, and RXRα-LBD were expressed in Escherichia coli BL21 (DE3)-CodonPlus cells. Protein purifications were performed as described2533Recombinant His-tagged PPARα/γ-LBD were mixed with β-arrestin-1 in binding buffer A  at 25 °C for 30 min. The protein mixtures were then incubated with pre-equilibrated Ni-NTA resins (QIAGEN) in binding buffer B (buffer A with 40 mM imidazole) at 4 °C for 90 min. After washing with binding buffer B, proteins were eluted and analyzed by Western blot.Bio-layer interferometry (BLI) is an optical analytical technique for measuring kinetics of interactions in real-time. The biosensor tip surface immobilized with a ligand is incubated with an analyte in solution, resulting in an increase in optical thickness at the biosensor tip and a wavelength shift, which is a direct measure of the change in thickness.on, koff and KD) and Rmax parameters were done using Octet software version 7.0 (ForteBio). The goodness of fit for the binding data was assessed by evaluation of the χ2 and R2 values generated from all the fitting analysis. All the experiments were repeated at least twice.Biolayer interferometry analysis of β-arrestin-1 or RXRα-LBD binding to PPARα-LBD and PPARγ-LBD were studied using Octet Red 96 (ForteBio). 50 μg/ml biotinylated PAPRα-LBD and PPARγ-LBD were immobilized on Streptavidin biosensor  and the typical immobilization levels were above 3 nm. Ligands-loaded SAs were then incubated with different concentrations of β-arrestin-1 or RXRα-LBD in the kinetics buffer. Global fitting of the binding curves generated a best fit with the 2:1 HL model and the kinetic association and dissociation constants were calculated. The systematic baseline drifts were corrected by subtracting the shifts recorded from sensors loaded with ligands but incubated with no analytes. All binding experiments were performed in solid-black 96-well plates containing 200 μl of solution in each well at 25 °C with an agitation speed of 1000 rpm. Curve fitting, steady state analysis, and calculation of kinetic parameters .2O. 15N-labeled PPARα-LBD and PPARγ-LBD (0.5 mM) were titrated with β-arrestin-1 peptide up to a molar ratio of 1:10. 2D [1H-15N]-HSQC spectra were recorded after each addition at 25 °C on Agilent DD2 800-MHz spectrometer equipped with a triple resonance cryo genic probe. Data were processed by NMRPipe and analyzed with NMRView.The NMR samples were prepared in a buffer containing 20 mM potassium phosphate, pH 7.4, 100 mM KCl, 2 mM DTT and 10% v/v Dt test was used to compare two groups. One-way ANOVA was used to compare three or more groups. Multiple comparisons were analyzed by two-way ANOVA followed by Bonferroni post hoc tests. Differences with P value of 0.05 or less were considered statistically significant.The quantitative results are presented as mean ± S.E.M. Unpaired two-tailed Student’s How to cite this article: Wang, C. et al. β-arrestin-1 contributes to brown fat function and directly interacts with PPARα and PPARγ. Sci. Rep.6, 26999; doi: 10.1038/srep26999 (2016).


E conformation around the imine C=N bond. Each molecule has an intramolecular N—H⋯N hydrogen bond, which generates an S(5) ring. Intermolecular N—H⋯O hydrogen bonds in the semicarbazone link the molecules into layers parallel to the bc plane, while weak intermolecular N—H⋯S hydrogen bonds in the thiosemicarbazone link the molecules into chains propagating in [110].Crotonaldehyde semicarbazone and crotonaldehyde thiosemicarbazone show the same  E)-2-[(E)-but-2-en-1-ylidene]hydrazinecarboxamide}, C5H9N3O, (I), and crotonaldehyde thiosemicarbazone {systematic name: (E)-2-[(E)-but-2-en-1-yldene]hydrazinecarbothioamide}, C5H9N3S, (II), show the same E conformation around the imine C=N bond. Compounds (I) and (II) were obtained by the condensation of crotonaldehyde with semicarbazide hydrochloride and thiosemicarbazide, respectively. Each molecule has an intramolecular N—H⋯N hydrogen bond, which generates an S(5) ring. In (I), the crotonaldehyde fragment is twisted by 2.59 (5)° from the semicarbazide mean plane, while in (II) the corresponding angle (with the thiosemicarbazide mean plane) is 9.12 (5)°. The crystal packing is different in the two compounds: in (I) intermolecular N—H⋯O hydrogen bonds link the molecules into layers parallel to the bc plane, while weak intermolecular N—H⋯S hydrogen bonds in (II) link the molecules into chains propagating in [110].Crotonaldehyde semicarbazone {systematic name: ( The semicarbazone and thiosemicarbazone fragments in the compounds show an E conformation around the imine C=N bond. The molecules  Tables 1 and 2 ▸,22(8)R ring motifs . The resulting dimers are connected through N—H⋯O hydrogen bonds, forming layers parallel to bc plane. In the crystal of (II)b).As a result of the presence of potential hydrogen-donor sites in molecules (I)fs Fig. 2a. The r1H and 13C{1H} NMR spectra were recorded on a Bruker Bio spin 400 spectrometer. Microanalysis was performed using EURO EA. Powder X-ray diffraction data were collected with Stoe Transmission diffractometer (Stadi P).All reactions and manipulations were carried out in air with reagent grade solvents. The IR spectra were recorded on a Jasco FT–IR 300E instrument. 5N3O·HCl; 0.5 g, 4.5 mmol) and sodium acetate  in 10 ml water was agitated well and crotonaldehyde  was added slowly with stirring. On completion of the addition, the reaction mixture was agitated for 24 h at room temperature. The solid product which formed was separated by filtration and washed with water and finally recrystallized from absolute ethanol to produce the product (I)For the synthesis of (I)−1): 3456, 3281, 3192 (NH2), (1668–1638) (C=O); 1H NMR  δ p.p.m. 1.76 , 6.43–5.46 , 7.39 .13C NMR  δ p.p.m. 18.52 (CH3), 130.01 (–HC=CH–), 137.62 (–HC=CH–), 145.64 (N=C), 160.19 (C=O). Analysis calculated for (I)IR  in 5 ml water and the mixture was stirred at room temperature for 24 h. The product was separated by filtration and recrystallized from absolute ethanol to produce the product (II)For the synthesis of (II)−1): 3323, 3244, 3164 (NH2), 1650(C=S). 1H NMR  δ p.p.m. 1.90 , 6.07–6.27 , 6.49 , 7.10  7.60 , 10.10 . 13C NMR  18.73 (CH3), 127.70 (–HC=CH–), 140.58 (–HC=CH–), 146.21 (N=C), 177.95 (C=S). Analysis calculated for (II)IR  selected with an incident beam curved-crystal germanium Ge(111) monochromator with a linear position-sensitive detector (PSD). The patterns were scanned over the angular range 5.0–80.0° (2θ). For pattern indexing, the extraction of the peak positions was carried out with the program WinPLOTR  = 50.5, F(20) = 71.9 ] for (I)M(20) = 61.8, F(20) = 96.0 ] for (II)1/cP2 in the monoclinic system for (I)PCrystal data, data collection and structure refinement details are summarized in Table 3et al., 1988FULLPROF Z = 2 for (II)EXPO2014 d is the sample thickness). These μ·d values were determined experimentally.The whole powder diffraction patterns from 5 to 80° (2θ) for the two compounds (I)3—CH bond was clearly stretched (close to 1.6 Å), therefore a single soft restraint was carried out to obtain a normal value (1.49 Å). The final refinement cycles were performed using isotropic atomic displacement parameters for the C, N and O atoms, an anisotropic atomic displacement parameter for S atom in (II)Before the final refinement, all H atoms were introduced in geometrically calculated positions. The coordinates of these H atoms were refined with strict restraints on bond lengths and angles until a suitable geometry was obtained, after that they were fixed in the final stage of the refinement. No soft restraints were imposed for (I)10.1107/S2056989015000663/cv5481sup1.cifCrystal structure: contains datablock(s) CROTON-CZ_Publ, I, II. DOI: 10.1107/S2056989015000663/cv5481Isup2.rtvRietveld powder data: contains datablock(s) I. DOI: 10.1107/S2056989015000663/cv5481IIsup3.rtvRietveld powder data: contains datablock(s) II. DOI: 1043290, 1043289CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


In the present study, the phenolic compounds were prepared using ultrasonic-aid extraction from sugar beet molasses (SBM).Gallic acid (GA), cyanidin-3-O-glucoside chloride (CGC) and epicatechin (EP) were produced after column chromatography from the extraction, and further detected using NMR, QTOF-MS and ESI-MS/MS.The three compounds exhibited strong antioxidant activities including DPPH radical scavenging activities, ABTS radical scavenging activities and ORAC values. GA showed the strongest antioxidant activity. Antitumor activities significantly increased in a dose-dependent manner. In particular, the CGC had growth inhibitory activities of 94.86, 87.27 and 67.13 % against the human colon (CACO-2), hepatocellular (HepG2) and breast (MCF-7) carcinoma cell lines, respectively, at the highest concentration of 400 μg/mL of the extracts. These results suggest that the three compounds are key chemical compositions valuable for preparing functional foods in the food industry.The results suggested that SBM is a natural source of antioxidant and antitumor agents for preparing functional foods. Sugar beet is an important sugar crop cultivated for sugar production , 2. SugaPhenolic contents have been demonstrated to have a variety of bioactivities such as anti-aging, anti-fatigue, anti-hypoxia, immunological, anti-radiation, anti-inflammatory, anti-proliferative and hypoglycemic effects –14. MeanThe primary objective of the present study is to separate and purify the phenolic compounds in SBM, identify the structure of prepared phenolic compounds, and study the bioactivity of the prepared phenolic compounds in SBM.SBM was provided by Xinjiang Green Xiang Sugar Industry Co., Ltd . 2,2-diphenyl-1-picryhydrazyl (DPPH), 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), ascorbic acid (Vc), fluorescein, 2,2′-azobis (2-methylpropionamidine) dihydrochloride (AAPH), trolox  and 3--2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich Chemical Co. . Dulbecco’s modified eagle medium (DMEM), penicillin-streptomycin were purchased from Gibco Co. . Fetal bovine serum (FBS) was purchased from Zhejiang Tianhang Biological Technology Co. . Methanol (HPLC-grade) was purchased from Merck Co. . Ultra-pure water was prepared by a Milli-Q system .n-butanol fraction and aqueous fraction. After drying in vacuo, five fractions were obtained. These fractions included the petroleum ether fraction , chloroform fraction , ethyl acetate fraction , n-butanol fraction , and water fraction . In the biological activity screening tests, the ethyl acetate fraction showed stronger antioxidant and antitumor activities than other four fractions. Therefore, the ethyl acetate fraction was chosen for further purification.The SBM (2.0 kg) was extracted by 30 mL 70 % (w/v) ethanol using a RK102H ultrasonic . Extraction conditions were ultrasonic power, 450 W, HCl concentrations 1.6 mol/L, temperature 40 °C and time 60 min. The extract was concentrated at 45 °C in vacuum using a rotary evaporator  to obtain the total extraction fraction (634.02 g). Then, the total extracted fraction was suspended in distilled water. The resulting solution was successively partitioned with different solvents which yielded petroleum ether fraction, chloroform fraction, ethyl acetate fraction, compound 1 (1293 mg), compound 2 (241 mg) and compound 3 (486 mg). The semi-preparative high performance liquid chromatography (HPLC) system consisted of a C18 column , a Waters 600 pump and a Waters 2998 Diode Array Detector (DAD)  analyzed at 35 °C. The mobile phase was 1 % acetic acid aqueous solution (A) and methanol (B) with a gradient program of 0–10 min, linear gradient 5–10 % B; 10–70 min, linear gradient 10–20 % B; 70–90 min, 20 % B isocratic; 90–130 min, linear gradient 20–40 % B; 130–140 min, linear gradient 40–100 % B and 140–180 min 100 % B isocratic elution at a flow rate of 15 mL/min. T 2 mL of samples were injected. Absorption wavelengths were set at 280 and 360 nm. The extraction and separation procedure of SBMP is shown in Fig. The ethyl acetate fraction (70.0 g) was loaded onto a column 3.5 cm × 100 cm) of macroporous resin D101, and the column was stepwise eluted with water, 30, 50, 70, and 95 % ethanol at a flow rate of 10 mL/min to yield five sub-fractions. After HPLC analyses, 30 % ethanol sub-fraction was further purified using a Sephadex LH-20 column chromatography and eluted with methanol, followed by a semi-preparative HPLC eluting with methanol/water, to yield 00 cm of 18 column , a Waters 600 pump and a Waters 2998 DAD , column temperature was set at 35 °C. The mobile phase was 1 % acetic acid aqueous solution (A) and methanol (B) with a gradient program of 0–5 min, linear gradient 5–10 % B; 5–35 min, linear gradient 10–20 % B; 35–45 min, 20 % B isocratic; 45–65 min, linear gradient 20–40 % B; 65–70 min, linear gradient 40–100 % B and 70–90 min 100 % B isocratic elution at a flow rate of 1 mL/min. The injection volume is 20 μL, and the detection wavelengths were set at 280 and 360 nm.Sample purity was analyzed using an HPLC-DAD system, which consisted of a C1H NMR and 13C NMR spectrometry. The ESI-MS was carried out in a LCQ-Fleet mass spectrometer , with an electrospray ionization source using a negative mode (m/z 50–800). 1H NMR spectra and 13C NMR spectra were recorded on a Bruker Avance DMX-500 spectrometer , operating at 500 and 125 MHz for 1H and 13C, respectively, using D2O or deuterated dimethyl sulfoxide (DMSO-d6). In D2O, tetramethylsilane (TMS) was used as the internal standard. In DMSO-d6, the residual solvent was used as the internal standard. Chemical shifts were expressed in δ (ppm) downfield from TMS as an internal standard, and coupling constants were reported in hertz.The purified compounds from SBM were identified by electrospray ionization-mass spectrometry (ESI-MS), −5 mol/L) and the mixture was shaken vigorously and incubated in dark at 30 °C for 30 min. Then, the absorbance was measured at 517 nm. Deionized water and ascorbic acid (VC) were used as the blank and positive control, respectively. The ability to scavenge DPPH was calculated by the eq. 0 is the absorbance of water instead of sample solution, Abs1 is the absorbance of the sample solution, and Abs2 is the absorbance of methanol instead of DPPH solution.The scavenging activity on DPPH free radical (DPPH) was measured according to the reported method with some modifications . 0.5 ml Ac is the absorbance of control without sample, As is the absorbance of the sample.The ABTS radical scavenging activity was assessed according to the recent reports with some modification , 19. BriThe oxygen radical absorbance capacity (ORAC) assay was carried out according to the modified method of Ou et al. . Briefly2 at 37 °C.Human colon (CACO-2), hepatocellular (HepG2) and breast (MCF-7) carcinoma cell lines were provided by the Medical College of Sun Yat-Sen University . The cells were cultured in DMEM containing 10 % FBS, 100 μg/mL streptomycin and 100 U/mL penicillin in a humidified incubator with 5 % CO4 cells/mL), and incubated at 37 °C in an atmosphere of 5 % CO2. After anchoring to the wells, additional medium (100 μL) containing different concentrations of test samples were added to each well. The cells were then incubated at 37 °C for 48 h in an atmosphere containing 5 % CO2. Then, 20 μL of MTT solution (5 mg/mL) was added to each well, and the incubation was continued for an additional 4 h. Then the supernatant was removed and 150 μL of DMSO was added to each well. The plate was shaken for 10 min to dissolve formazan crystals. The absorbance of the above DMSO solution was measured at 570 nm by a microplate reader. The buffer and 5-fluorouracil were used as the blank and positive control respectively. The inhibitory rate was calculated using the eq. A is the cancer cell growth inhibitory rate; Ac is the absorbance of the control; As is the absorbance of sample; and Ab is the absorbance of the blank.The antitumor activities of samples were evaluated using the MTT assay with some modifications . Brieflyp < 0.05 by analysis of variance (ANOVA) followed by Duncan’s least significant test.Data were analyzed using SPSS  and presented as mean ± SD with triplicates. Significance was determined at Three compounds isolated and obtained from SBM were identified as gallic acid (GA), cyanidin-3-O-glucoside chloride (CGC) and epicatechin (EP). Their spectroscopic data were listed below.7H6O5, white powder, purity, 99.2 %. QTOF-MS m/z: 177.0158 [M + Na-H2O]+, ESI-MS/MS m/z: 177, 153, 127. 1H-NMR  δ H: 7.12 . 13C-NMR  δ C: 121.2 (C-1), 109.4 (C-2), 145.3 (C-3), 138.6 (C-4), 145.3 (C-5), 109.4 (C-6), 168.5 (COOH) +, ESI-MS/MS m/z:450, 287. 1H-NMR  δ H: 8.92 , 6.88 , 6.91 , 8.10 , 7.02 , 8.26 , 5.28 , 3.13 , 3.38 , 3.31 , 3.48 , 3.82 , 3.71  +, ESI-MS/MS m/z: 291, 273, 165, 139. 1H-NMR  δ H: 7.03 , 6.82 , 6.77 , 5.99 , 5.89 , 4.86 , 4.11 , 2.85 , 2.72 . 13C-NMR δ C: 28.83 (C-4), 68.33 (C-3), 82.70 (C-2), 95.41 (C-8), 96.13 (C-6), 100.62 (C-10), 115.23 (C-5′), 115.70 (C-2′), 120.06 (C-6′), 132.14 (C-1′), 145.64 (C-3′), 145.72 (C-4′), 156.89 (C-9), 157.22 (C-5), 157.73 (C-7) [Epicatechin (Compound 3): C73 (C-7) , 25.The DPPH radical scavenging activities of extractions, GA, CGC and EP were shown in Fig. The ABTS radical scavenging activities of each fraction, GA, CGC and EP were shown in Fig. n-butanol fraction > water fraction > chloroform fraction > petroleum ether fraction. The ORAC values of three compounds are shown in Fig. The oxygen radical absorbance capacities of samples were shown in Fig. According to the results of DPPH, ABTS radical scavenging and ORAC assays, the ethyl acetate fraction showed the strongest antioxidant activity among five extraction fractions. The three compounds isolated from the ethyl acetate fraction could be explored as natural antioxidants as they showed strong antioxidant activities. GA showed the strongest antioxidant activity, followed by Vc which is greater than those of the CGC while EP showed the least antioxidant activity.The three compounds, GA, CGC and EP, obtained in our study are three typical phenolic compounds in plants which have been reported to possess strong antioxidant activity , 27. AccThe antitumor activities of five fractions against cancer cells are shown in Fig. As shown in Fig. Early studies showed that GA, CGC and EP had anticancer activity. GA has been shown to have antitumor activity in many cancer cells without damaging normal cells –36. WangThree compounds, Gallic acid (GA), cyanidin-3-O-glucoside chloride (CGC) and epicatechin (EP) and the main compounds in SBM with high antioxidant and antitumor activities. GA possessed the strongest antioxidant activity. CGC showed strong antitumor activities against human colon (CACO-2), hepatocellular (HepG2) and breast carcinoma cells. The antitumor activity was higher than positive control (5-fluorouracil). Further studies will be needed to investigate other chemical compounds and bioactivity of SBM. These results suggest that SBM is a potential source of antioxidant and antitumor agents for preparing functional foods.