N-acetylase is essential for the biosynthesis of mature GPI anchors and has been genetically validated in the bloodstream form of Trypanosoma brucei, which causes African sleeping sickness. We screened a focused library of zinc-binding fragments and identified salicylic hydroxamic acid as a GlcNAc-PI de-N-acetylase inhibitor with high ligand efficiency. This is the first small molecule inhibitor reported for the trypanosome GPI pathway. Investigating the structure activity relationship revealed that hydroxamic acid and 2-OH are essential for potency, and that substitution is tolerated at the 4- and 5-positions.The zinc-metalloenzyme GlcNAc-PI de- Current treatments are expensive, toxic, and difficult to administer, leaving an urgent need for improved therapeutic agents. The parasite is able to evade the host immune response by virtue of a dense surface coat composed of 5 × 106 variant surface glycoprotein (VSG) dimers that prevent lysis by the innate immune response. The coat also undergoes antigenic variation to evade the adaptive immune response.T. brucei and mammalian systems, highlighting differences in both the order of assembly and substrate specificity that can be exploited to produce species-specific inhibitors. The GlcNAc-PI de-N-acetylase is a zinc metalloenzyme that catalyses the second step in biosynthesis of GPI -d-myo-inositol-1-octadecyl phosphate (GlcN-IPC18) has been described previously.N-acetyl derivate (GlcNAc-IPC18) was prepared by treatment with acetic anhydride, and the concentration of stock solutions determined by measurement of the inositol content by selected ion-monitoring GC–MS.Trypanosome brucei (variant MITat1.4) was isolated and membranes (cell-free system) prepared as described previously and stored at −80 °C.T. brucei de-N-acetylase (GST-TbGPI12) was expressed and purified as described previously and stored at −80 °C.The synthesis of 2.2TbGPI12 (10 μg per assay) or trypanosome cell-free system  in incorporation buffer  was added to wells containing 500 pmol GlcNAc-IPC18 with or without the inhibitor and incubated at 37 °C for 1 h. The reactions were quenched by the addition of 200 μL of 5% propan-1-ol, 5 mM NH4OAc, and the glycolipids were bound to C18 resin (50 mg Isolute cartridge), washed three times with 200 μL 5% propan-1-ol, 5 mM NH4OAc and eluted with 100 μL 40% propan-1-ol, 5 mM NH4OAc. Enriched glycolipids were analysed by liquid chromatography coupled to an electrospray tandem mass spectrometer (LC–MS/MS). Samples (40 μL) were injected on to a 10 × 1 mm C18 column  and eluted using a binary gradient of 5–80% propan-1-ol in 5 mM NH4OAc (Dionex Ultimate 3000). The gradient consisted of 2 min 0% B, 2–4 min 0–100% B, 4–8 min 100%, 8–9 min 100–0% B, 9–10 min 0% B where buffer A consisted of 5% propan-1-ol, 5 mM NH4OAc and buffer B 80% propan-1-ol, 5 mM NH4OAc. The glycolipids were analysed on an electrospray triple quadrapole mass spectrometer (Micromass Quattro Ultima) in multiple reaction monitoring mode. The turnover of the substrate GlcNAc-IPC18 (m/z 715 > 223) to GlcN-IPC18 (m/z 672 > 223) was used to calculate the percentage of substrate conversion to product in a given sample.50 values were calculated using a four-parameter fit of eight-point potency curves derived from three independent experiments, and are quoted with standard deviation.Inhibition assays were performed in 100 μL final volume, with 1% v/v DMSO with or without inhibitor. Recombinant GST-2.33H]-mannose were analysed using high-performance liquid chromatography and fluorography as described previously.Trypanosome cell-free system assays, where the formation of GPI precursors is monitored by following the incorporation of : 254.1023. Found: 254.1027 (−1.4 ppm).5-Aminosalicylic acid 2.4.2O-Benzylhydroxylamine hydrochloride  was suspended in CHCl3 (85 mL) and cooled to 0 °C, and then TEA  was added. A solution of compound 9  in THF (140 mL), DMAP  and DCC  was added, and the reaction was stirred for 16 h. The solvent was removed and the solids were resuspended in EtOAc, filtered, and then washed with HCl , water (50 mL), ammonia  and filtered through cotton wool. The solvent was removed and the crude was dried on to silica then purified by column chromatography  to afford the product  as a pale yellow powder, mp 102–103 °C. 1H NMR  δ 11.33 , 9.42 , 7.63 , 7.44–7.41 , 7.39–7.34 , 7.13 , 6.90 , 6.45 , 4.97 , 1.49 ; 13C NMR  δ 157.1, 153.3, 134.9, 129.6, 129.3, 128.9, 128.7, 128.7, 126.1, 118.8, 116.0, 112.1, 80.8, 78.6, 28.3; LRMS (ES+), m/z 359.2 [M+H+]; HRMS, calcd mass for C19H23N2O5+ [M+H+]: 359.1601. Found: 359.1591 (2.8 ppm).2.4.310  was dissolved in wet TFA (5.3 mL), and the mixture was stirred for 1 h. The TFA was removed in vacuo; the residue was suspended in Et2O and filtered to afford the product  as a tan powder, mp 169–170 °C. 1H NMR  δ 11.48 , 9.96 , 7.64 , 7.47 , 7.42–7.36 , 7.31 , 7.03 , 4.96 ; 13C NMR  δ 163.4, 155.6, 135.7, 128.8, 128.29, 128.27, 126.9, 124.5, 122.7, 117.87, 115.5, 113.2, 77.0; LRMS (ES+), m/z 259.1 [M+H+]; HRMS, calcd mass for C14H15N2O3+ [M+H+]: 259.1077. Found: 259.1069 (3.3 ppm).Compound 2.4.411 , DMAP (cat.) and the acyl chloride (0.592 mmol) were dissolved in THF (2 mL) and DCE (0.5 mL). Pyridine  was added, and the reaction stirred for 24 h. The mixture was diluted with DCM (10 mL) and water (10 mL) then stirred vigorously. The mixture was passed through a phase separator, and then the solvent was removed. The crude was recrystallized from MeOH/Et2O. Characterisation of compounds 12–19 is reported in the Compound 2.4.512–19) and AcOH (2 equiv) in MeOH/THF, MeOH or THF, was passed through a H2 flow reactor  using a 20% Pd(OH)2 catalyst cartridge. The solvent was removed and the product was recrystallized from THF/MeOH, or washed with THF or MeOH. Characterisation of compounds 20–27 is reported in the A 0.05 M solution of benzyl-protected amide -d-myo-inositol-1-octadecyl phosphate (GlcNAc-IPC18) to d-glucosamine-α-(1-6)-d-myo-inositol-1-octadecyl phosphate (GlcNAc-IPC18). The replacement of the diacylglycerol moiety of GlcNAc-PI with a simple C18 alkyl chain in GlcNAc-IPC18 has no effect on the substrate recognition, and enables the use of a unique mass-transition to follow the reaction.1 −1 per non-hydrogen atom, which is over one third of the theoretical maximum affinity for organic compounds of −1.5 kcal mol−1 per non-hydrogen atom.N-hydroxyamide)-2-deoxy-β-d-glucopyranosyl]-cyclohexanediol 2-(n-octadecylphosphate) has an IC50 of 19 ± 0.5 μM and a molecular weight of 666.4,−1 per non-hydrogen atom.To discover more drug-like scaffolds for zinc-binding de-action.1 C had an T. brucei de-N-acetylase in a cell-based functional complementation assay,1  has previously been identified as an inhibitor of trypanosome alternative oxidase (TAO), and is toxic in vivo to the clinically relevant bloodstream form T. brucei.The result of the fragment screen compared favourably to our screening of 16,037 lead-like compounds from the Dundee Drug Discovery Unit’s compound collection3.21 to act as an inhibitor of the T. brucei GPI pathway was confirmed using the trypanosome cell-free system  was prevented by incubation with >300 μM of 1 , suggesting that the 5′-amide substitution was tolerated but contributed little to binding.To further explore the available chemical space, a small array of analogues of compound hlorides . After drides (1 . The twoectively , compara1 is essential for activity and substitution at the 4- and 5- position is tolerated (1 with the natural substrate GlcNAc-PI could suggest that 1 is mimicking the GlcNAc moiety, with the 2-OH of 1 mimicking the 3′-OH that is essential for substrate recognition. In the proposed reaction mechanism for the de-N-acetylase, the catalytic base D43 assists the nucleophilic attack of a zinc-bound water molecule on the activated acetamidocarbonyl, and the subsequent protonation of the tetrahedral intermediate promotes loss of acetic acid. We propose that 1 could inhibit the enzyme through the hydroxamic acid binding to the catalytic zinc, and replacing both the acetamidocarbonyl and the activated water (The structure–activity relationship derived from the data presented here suggest that the 2-OH group of olerated . Comparied water .3.41 as an inhibitor of T. brucei GlcNAc-PI de-N-acetylase with high ligand efficiency, representing the first non-substrate analogue inhibitor of the trypanosome GPI pathway. Although 1 shows modest potency with an IC50 of 63 ± 4 μM, it has impressive ligand efficiency of 0.57 kcal mol−1 per non-hydrogen atom, and thus may be a useful starting point to develop more potent inhibitors.We have identified salicylic hydroxamic acid


L) with silver nitrate (AgNO3) produced a new dinuclear silver(I) coordination complex, [Ag2(C12H8N4S)2(NO3)2]·4H2O, which crystallizes with two Ag atoms bridged by two L ligands. The Ag atom is surrounded by four N atoms of L and by one O from the nitrate anion defining a distorted square pyramid. The atoms comprising the dication are nearly coplanar, with an r.m.s. deviation of 0.1997 Å. Molecules are linked by C—H⋯O and O—H⋯O hydrogen bonds through nitrate anions and water molecules, forming a two-dimensional porous network. The overall structure involves stacking of Ag complex layers along the b axis. The cohesion in the three-dimensional architecture is ensured by O⋯Ag interactions.The self-assembly of an angular 2,5-bis(pyridin-2-yl)-1,3,4-thiadiazole ligand ( DOI: Click here for additional data file.10.1107/S1600536813014578/tk5229Isup2.hklStructure factors: contains datablock(s) I. DOI:  crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:


Eosinophilia is often linked to allergic gastrointestinal disorders linked to food allergy. EPIT using Viaskin® device has been described as a therapeutic method in food allergy. We developed a model of mice sensitized to peanut, exhibiting EoE and AE after exclusive feeding with peanut protein extracts (PPE). This study was conducted in order to evaluate the efficacy of EPIT.After oral sensitization with PPE and cholera toxin, 30 BALB/c mice were treated weekly during 8 weeks by PPE skin applications (EPIT), 20 mice were not treated (Sham) and 10 mice constituted the control group (C). Mice were then exclusively fed with PPE. Specific IgE, IgG1 and IgG2a were monitored during immunotherapy. Esophageal and jejunal samples were taken for histological analyses.sIgE increased after oral sensitization, respectively 0.207±0.03 and 0.214±0.04 μg/ml, in EPIT and Sham, with undetectable values in C. Following EPIT, sIgE decreased and sIgG2a increased, respectively 0.139±0.01 vs 0.166±0.01 μg/ml  and 14.96±0.60 vs 4.73±1.75 μg/ml (p<0.05). Esophageal eosinophilic infiltration (measured in 6 high power fields) was higher in Sham, 136±32, than in EPIT, 50±12 (p<0.05) and C, 7±3 cells/mm2 (p<0.01). Esophagus mucosa thickness was increased in Sham compared to EPIT and C (p<0.001). Sham group exhibited higher mRNA levels of cytokines than EPIT: eotaxin (p<0.05), IL-5 (p<0.05), IL-13 (p<0.05). The mRNA levels of these cytokines in EPIT were similar to C. The expression of Foxp3 mRNA increased significantky after EPIT compared with Sham and C (p<0.05). The jejunal villus/crypt ratio was lower in Sham than in EPIT and C, respectively 1.6±0.1 vs 2.3±0.2 (p<0.01) and 2.4±0.1 (p<0.001). Eosinophilic infiltration in jejunum was increased in Sham compared to EPIT (p<0.01) and C (p<0.001).EPIT is effective in preventing EoE and AE induced by oral challenge in mice sensitized to peanut.


Cancer stem cell biology is tightly connected to the regulation of the pro-inflammatory cytokine network. The concept of cancer stem cells “inflammatory addiction” leads to envisage the potential role of anti-inflammatory molecules as new anti-cancer targets. Here we report on the relationship between nuclear receptors activity and the modulation of the pro-inflammatory phenotype in breast cancer stem cells.Breast cancer stem cells were expanded as mammospheres from normal and tumor human breast tissues and from tumorigenic (MCF7) and non tumorigenic (MCF10) human breast cell lines. Mammospheres were exposed to the supernatant of breast tumor and normal mammary gland tissue fibroblasts.In mammospheres exposed to the breast tumor fibroblasts supernatant, autocrine tumor necrosis factor-α signalling engenders the functional interplay between peroxisome proliferator activated receptor-α and hypoxia inducible factor-1α (PPARα/HIF1α). The two proteins promote mammospheres formation and enhance each other expression via miRNA130b/miRNA17-5p-dependent mechanism which is antagonized by PPARγ. Further, the PPARα/HIF1α interplay regulates the expression of the pro-inflammatory cytokine interleukin-6, the hypoxia survival factor carbonic anhydrase IX and the plasma lipid carrier apolipoprotein E.Our data demonstrate the importance of exploring the role of nuclear receptors (PPARα/PPARγ) in the regulation of pro-inflammatory pathways, with the aim to thwart breast cancer stem cells functioning. Jagged1) and apolipoprotein E (ApoE) in breast CSCs We recently reported the increase of NF-κB activity in breast tumor MS (T-MS), compared to their normal counterparts (N-MS) p<0.005, in MCF10-MS; +167%, p<0.005, in MCF7-MS; +63%, p<0.05, in N-MS; +76%, p<0.05 in T-MS, p<0.05, in MCF7-MS, p<0.005, in MCF10-MS; +297%, p<0.005, in MCF7-MS; +253%, p<0.05, in N-MS; +266%, p<0.05 in T-MS, p<0.05, in MCF10-MS; +76%, p<0.01, in MCF7-MS, p<0.05, in MCF10-MS; +85%, p<0.01, in MCF7-MS, p<0.005, in MCF10-MS; +122%, p<0.005, in MCF7-MS, p<0.005 and −56%, p<0.005 respectively, p<0.01, p<0.05, in MCF10-MS, +62%, p<0.01 in MCF7-MS, p<0.01, in MCF10-MS; +552%, p<0.005, in MCF7-MS, p<0.01, −43%, p<0.01, respectively, p<0.005, −36%, p<0.05, respectively, We recently reported that PPARα promotes tumor MS formation and the expression of the MS growth factor Jagged1 p<0.01, p<0.05, in MCF10-MS vs MCF10, and +82%, p<0.01, in MCF7-MS vs MCF7) and PPARγ  and PPARβ  expression (p<0.05) and in T-MS , Interleukin 8 , SLUG  and TNFα  in the breast cancer estrogen receptor-α (ERα) negative cell line MDA-MB-231  expression. This microRNA was chosen basing on two considerations: i. its capability to increase HIF1α expression via the down-regulation of the HIF1α mRNA translation inhibitory protein DDX6 Further insight into the PPARα/HIF1α interplay was obtained via the assessment of microRNA17-5p (miR17-5p), whose binding consensus is present at PPARα mRNA 3′UTR. In respect to this issue, we were allowed to observe that miR17-5p expression was decreased by TNFα administration in MCF7-MS . Furtherp<0.01) and down-regulated by HIF1α KD , as well as the PPARα agonist WY triggered  CAIXLuc activity in MCF7-MS (p<0.05) also in TAF  KD of ApoE mRNA  display increased TNFR1 compared to their normal/non tumorigenic counterparts (N-MS and MCF10-MS). Breast CSCs also exhibit an increased growth response to the TAF supernatant. Though TAF secrete low amounts of TNFα, we demonstrate that TAF elicit TNFα expression in CSCs by secreting TGFβ, thus setting up an autocrine TNFα loop that enhances MS growth. TNFR1 signalling is a major inducer of inflammatory response via NF-κB activation We then pinpoint that the core of such TAF-promoted pathway involves the PPARα/HIF1α interplay. In particular, the two proteins induce each other expression and trigger T-MS growth to a higher extent than their normal counterpart. In this regard, we show the involvement of miR130b. Over-expression of this microRNA has been previously reported in liver cancer and pluripotent stem cells in vitroWe identified two acknowledged regulators of breast CSCs, namely IL6 and SLUG as targets of the PPARα/HIF1α interplay. The former has been characterized as crucial mediator of breast CSCs growth capacity The latter target controlled by the PPARα/HIF1α interplay is ApoE, a major component of circulating lipoproteins In conclusion, we show that the PPARα/HIF1α interplay, triggered in breast CSCs by the tumor associated fibroblast secreted TGFβ, engenders the expression of two acknowledged breast CSCs regulatory genes (IL6 and SLUG), as well as up-regulates two less characterized regulators of breast CSCs, namely CAIX and ApoE . This moRecombinant human IL6, TGFβ, TNFα, and anti-human TNFα inhibitory antibody were purchased from Sigma  and dissolved in phosphate buffered saline (1 mg/mL). The PPARγ agonist PGZ  and the PPARα agonist WY (Sigma) were dissolved in dimethylsulfoxide (0.1 M).PPARα (pPPARα) and empty (pV) plasmids were obtained from Institute Pasteur . HIF1 plasmid was obtained from Eric Huang . PPARα and ApoE specific double-strand RNA oligonucleotides (siRNA) and appropriate scramble siRNA (SCR) were purchased from Origene . CAIX and HIF1 siRNA and appropriate control SCR siRNAs were purchased from Invitrogen . Pre-miRNA17-5p, miRNA130b, antago-miRNA17-5p and miRNA130b were purchased from Life Technologies .2) was generated in an Vivo2300 hypoxic workstation . MCF7-MS and MCF10-MS were generated by plating 2500 cells into 3-cm2 low-attachment wells  in mammary epithelial growth medium (MEGM), supplemented with B27, 10 ng/ml epidermal growth factor (EGF), 10 ng/ml basic fibroblast growth factor (bFGF), 10 µg/ml insulin, 10−6 M hydrocortisone . Primary MS formation usually occurs after 48 to 72 h. To examine the effects of chemicals on MS formation and MS gene expression, MCF7-MS and MCF10-MS were exposed to each molecule and assessed after 24 h to 72 h. MS with an apparent diameters ≥50 µm were scored and photographed using a inverted microscope . To examine the impact of each specific expression vector, siRNA or pre/antago-miR on MS formation and MS gene expression, adherent MCF7 or MCF10 (105 cells in a 3-cm2 well) were transfected with 1 µg/well of each siRNA or pre/antago-miR using Lipofectamine 2000 . After 6 h of incubation, cells were re-suspended, seeded in 24-well ultra-low attachment plates at a density of 2500 cells per well and assessed after 48 h or 72 h. The Effect of each specific treatment was determined by at least n = 3 independent experiments. MDA-MB231 cells were grown in RPMI 1640 medium supplemented with 10% FBS, penicillin-streptomycin and glutamine.MCF7 were grown in RPMI 1640 medium supplemented with 10% FBS, penicillin-streptomycin and glutamine. MCF10 2-well low attachment plates , filled with 3 ml MEGM, supplemented with B27, 10 ng/ml EGF, 10 ng/ml bFGF, 10 µg/ml insulin, 10−6 M hydrocortisone . Primary MS started forming after 4–6 days and were processed at day 14. Self renewal of MS was tested by assessing the capacity of primary MS to generate secondary MS after trypsin disaggregation. Transfection in primary T-MS was performed by mixing 1 µg of each expression vector or siRNA with in vitro JET-PEI reagent . The procedure was approved by the local ethical committee of Center for Applied Biomedical Research, St. Orsola-Malpighi University Hospital  (Prot n.75/2011) and by the patients’ written informed consent.Twenty-two fresh surgical specimens, obtained from patients with ductal breast carcinoma who underwent quadrantectomy or mastectomy, were collected for the study . Normal 5×g to remove debris and conserved at −80°C. For the experimental setting supernatants were diluted in MS medium (MEGM) at the final concentration of 10%, and already formed MS were exposed for 24–72 h.Fibroblasts were collected by centrifuging the Collagenase/Hyaluronidase digested tissue lysates used for MS generation at 500×g for 5 min (see above). The fibroblasts containing pellet was re-suspended and cultured in DMEM medium with 20% fetal bovine serum , penicillin-streptomycin and glutamine (Sigma), in 6-well plates. When the fibroblasts reached confluence, medium was discarded and replaced with fresh medium, containing DMEM+FBS 0.5% (1.5 mL/well), for 24 h. Supernatants were then collected, centrifuged for 5 min at 10− , where DCt values of the sample and calibrator were determined by subtracting the Ct value of the endogenous control gene from the Ct value of each target gene. RT-PCR analysis was performed using the Master RT plus PCR system kit according to the instruction of the supplier (Life Technologies). Actin was used as an internal control. RT-PCR was performed for 31 cycles  for each primer, except for CAIX that was performed for 34 cycles. Primer sequence and PCR parameters are reported in Total RNA was extracted from cultured cells, MS and fibroblasts using TRIzol  reagent following the customer’s instructions. Real-time Reverse Transcription quantitative PCR (qPCR) analysis was performed by TaqMan approach in a Gene Amp 7000 Sequence Detection System , as previously described SLUGLuc, containing the −800/+10 bp SLUG promoter sequence in the pGL3 basal vector, was kindly provided by Dr. Togo Ikuta . ERαLuc plasmid, which contains 3 copies of estrogen response element (ERE), was kindly provided by Dr. Rakesh Kumar . CAIXLuc, containing the −179/+34 bp promoter sequence of CAIX, was provided by J Pastorek . Hypoxia responding element (HRE-Luc), containing 3 copies of HIF1 consensus was kindly provided by Dr. Giovanni Melillo . IL6Luc, containing the −2161 to −41 bp IL6 promoter sequence, was kindly provided by Dr. WL Farrar . PPRELuc, containing 7 copies of PPARs consensus, was kindly provided by Professor Ronald Evans . NF-κBLuc was previously described Cell lysates were prepared, run, and blotted as previous described 5 cells per well and collected in serum-free medium for 24 h. The harvested medium was centrifuged at 500×g for 5 min (4°C) to remove floating cells and the supernatants were collected and assayed following the customer’s instructions.Determination of TNFα and TGFβ level in TAF and NAF supernatant were evaluated by ELISA . Briefly, cells were seeded in a 6-well plate at the density of 3×10p<0.05. mRNA 3′-UTR were analyzed for miRNA binding site by the on-line software Targetscan (www.Targetscan.com).Statistical significance was assessed by ANOVA followed by Bonferroni’s multiple comparison test or two-tail Student’s t-test, as appropriate, using PRISM 5.1 . The level for accepted statistical significance is Figure S1TNFα and the TAF supernatant induce Jagged1 expression in MS. Jagged1 mRNA qPCR analysis in MCF10/MCF7-MS and in N−/T-MS (samples 14–15) exposed to (A) TNFα . (B) WB analysis of PPARα protein level in TAF supernatant -exposed MCF7-MS. (C) Jagged1 mRNA qPCR analysis in MCF10/MCF7-MS and in N−/T-MS (samples 14–15) exposed to NAF and TAF supernatant . Data are expressed as mean ±S.D., n = 3, *p<0.05, #p<0.01, §p<0.005, ANOVA test.(TIF)Click here for additional data file.Figure S2Hypoxia and the TAF supernatant induce HIF1α activity and expression in MS. (A) HIF1α mRNA RT-PCR and WB analysis in TAF supernatant -exposed normoxic/hypoxic MCF7 and MCF10. HIF1α mRNA RT-PCR analysis (B) and HRELuc activity (C) in normoxic and hypoxic MCF10-MS and MCF7-MS. (D) PPARα mRNA qPCR analysis in SCR/siPPARα (72 h)-transfected normoxic and hypoxic MCF7-MS. (E) HIF1α protein WB analysis in SCR/siPPARα (72 h)-transfected and WY -exposed MCF10-MS. Data are expressed as mean ±S.D., n = 3, *p<0.05, #p<0.01, §p<0.005, ANOVA test. n.s.: not significant.(TIF)Click here for additional data file.Figure S3The PPARα/HIF1 interplay regulates SLUG and IL6 in MS. SLUG (A) and IL6 (B) qPCR mRNA analysis in pV/pPPARα transfected MCF7-MS and MCF10-MS (48 h). SLUG (C) and IL6 (D) qPCR analysis in HIF1 (48 h) or SCR/siHIF1 (72 h)-transfected MCF7-MS. Data are expressed as mean ±S.D., n = 3, *p<0.05, #p<0.01, ANOVA test.(TIF)Click here for additional data file.Figure S4Nuclear receptors expression are regulated by HIF1 in MS. (A) PPARγ WB analysis in HIF (24 h)-transfected MCF7-MS. (B) PPARα and PPARγ WB analysis in MCF10, MCF10-MS, MCF7 and MCF7-MS cells. (C) PPARβ, RXRα, RXRβ and RXRγ mRNA RT-PCR analysis in MCF10 and MCF10-MS cells. (D) HIF1α, PPARβ, RXRα, RXRβ and RXRγ mRNA RT-PCR analysis in HIF1 (24 h) and SCR/siHIF1 (72 h)-transfected MCF7-MS. Data are expressed as mean ±S.D., n = 3, *p<0.05, #p<0.01, §p<0.005, ANOVA test. n.s.: not significant. n.d.: not detected.(TIF)Click here for additional data file.Figure S5Effects of PPARγ agonist (PGZ) on CSCs pro-inflammatory pathways and on MS formation. (A) HIF1α mRNA RT-PCR analysis, (B) number of MS and (C) qPCR analysis of IL6, Notch3, Jagged1 mRNA levels in PGZ -exposed hypoxic MCF7-MS and T-MS (samples 18–20). Data are expressed as mean ±S.D., n = 3 *p<0.05, #p<0.01, §p<0.005, ANOVA test. n.s.(TIF)Click here for additional data file.Figure S6Effects of PGZ on CSCs pro-inflammatory pathways in MDA-MB-231 cells. IL6, IL8, SLUG and TNFα, mRNA RT-PCR analysis in PGZ -exposed MDA-MB-231 breast cancer cells. Data are expressed as mean ±S.D., n = 3, *p<0.05, #p<0.01, ANOVA test.(TIF)Click here for additional data file.Figure S7CAIX expression is regulated by PPARα/HIF1 interplay in MS. (A) CAIX mRNA qPCR analysis in HIF1 vector (24 h) or SCR/siHIF1 (72 h)-transfected MCF7-MS. (B) WB analysis of CAIX protein expression in hypoxia exposed MCF10, MCF10-MS, MCF7 and MCF7-MS (C), and in pV/pPPARα (24 h)-transfected MCF7-MS (D). CAIXLuc assay SCR/siPPARα (72 h)-transfected and WY -exposed hypoxic MCF7-MS. Data are expressed as mean ±S.D., n = 3, *p<0.05, #p<0.01, ANOVA test.(TIF)Click here for additional data file.Figure S8Effects of PGZ on CAIX expression in TAF. CAIX mRNA RT-PCR analysis in PGZ (20 µM)-exposed hypoxic TAF . Data are expressed as mean ±S.D., *p<0.05, ANOVA test.(TIF)Click here for additional data file.Figure S9ApoE expression is regulated by PPARα in MS. (A) WB analysis of ApoE protein expression in pV/pPPARα (24 h)-transfected MCF7-MS. (B) ApoE mRNA qPCR analysis in WY -exposed MCF10-MS and MCF7-MS. (C) WB analysis of ApoE protein expression in HIF1 vector (48 h)-transfected MCF7-MS. (D) ApoE mRNA qPCR analysis in SCR/siApoE (48 h)-transfected MCF10-MS and MCF7-MS. (E) WB analysis of ApoE protein expression in SCR/siApoE (48 h)-transfected MCF7-MS. Data are expressed as mean ±S.D., n = 3, *p<0.05, #p<0.01, §p<0.005, ANOVA test.(TIF)Click here for additional data file.Figure S10Effects of PGZ on ApoE expression in MCF10-MS. (A) ApoE mRNA qPCR analysis and (B) WB analysis of ApoE protein in PGZ -exposed MCF10-MS. Data are expressed as mean ±S.D., n = 3, n.s.: not significant.(TIF)Click here for additional data file.Table S1Clinical–pathological parameters of 22 breast carcinomas used for T-MS, N-MS and fibroblasts isolation. List of samples used with clinical and pathological parameters. Abbreviations: pT, tumor size; pN, nodal involvement; G, grade; NG, nuclear grade; ER, estrogen receptor; HER-2, ERbB2 kinase receptor; EGFR, epidermal growth factor receptor.(DOC)Click here for additional data file.Table S2List of probes used in qPCR analysis.(DOC)Click here for additional data file.Table S3List of primers used in RT-PCR analysis.(DOC)Click here for additional data file.


The dihedral angles between the central benzene ring and the imidazole rings are 4.93 (11) and 46.08 (12)°. The 1,3-bis(imidazol-1-yl)-5-methylbenzene ligand is bis-monodentate, linking symmetry-related CuII atoms into sheets in the bc plane. These sheets are further bridged into a three-dimensional supramolecular structure by O—H⋯O and C—H⋯O hydrogen bonds.In the title complex, {[Cu(NO DOI: 10.1107/S1600536812023628/lr2064Isup2.hklStructure factors: contains datablock(s) I. DOI:  crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:


The TbIII atom in the cation is coordinated by eight O atoms from two nitrate and four hexamethylphosphate ligands in a distorted square-antiprismatic geometry. Together with the two nitrate ligands, the cation is univalent, which leads to the anionic chain having a [WS4Ag] repeat unit. The polymeric anionic chain has a distorted linear configuration with W—Ag—W and Ag—W—Ag angles of 161.49 (2) and 153.743 (13) °, respectively. The title complex is isotypic with the Y, Yb, Eu, Nd, La, Dy, Sm and Lu analogues.In the title compound, {[Tb(NO For the al. 2007; Zhang 2(C6H18N3OP)4][AgWS4] = 0.050wR(F2) = 0.095S = 1.099675 reflections532 parametersH-atom parameters constrainedmax = 0.91 e Å−3Δρmin = −1.10 e Å−3ΔρCrystalClear  used to solve structure: SHELXTL  I, global. DOI: 10.1107/S1600536812023823/rz2763Isup2.hklStructure factors: contains datablock(s) I. DOI:  crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:


The iridium–butane-1,4-diyl ring has an envelope conformation. This ring is disordered because alternately the second or the third C atom of the butane-1,4-diyl fragment function as an envelope flap atom (the occupancy ratio is 1:1). In the crystal, molecules are organized into densely packed columns extending along [101]. Coherence between the molecules is essentially based on van der Waals interactions.In the mononuclear title iridium(III) complex, [Ir(C Me2] complexes, see: Conejero et al. (C15H22BN6)(C3H9P)] = 0.017wR(F2) = 0.040S = 1.087359 reflections298 parameters25 restraintsH-atom parameters constrainedmax = 0.86 e Å−3Δρmin = −0.32 e Å−3ΔρSMART  used to solve structure: SHELXS97  I, global. DOI: Click here for additional data file.10.1107/S1600536813008040/gk2565Isup2.hklStructure factors: contains datablock(s) I. DOI:  crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:


The two RhI atoms each have a distorted square-planar coordination environment, defined by a bidentate cod ligand, a bromide anion and one C atom of the bridging bidentate bis-N-heterocyclcic carbene (NHC) ligand. The average Rh—CNHC distance is 2.038 (7) Å, suggesting that the bond has a major σ contribution with very little back donation. The distances between the cod ligands and the RhI atoms vary between 2.104 (4) and 2.210 (4) Å.The title compound, [Rh N-heterocyclic carbenes (NHC) as replacements for phosphines in the area of organometallic catalysis and Rh–NHC-based complexes, see: Herrmann et al. 2(C17H32N4)] = 0.033wR(F2) = 0.078S = 1.066186 reflections402 parameters8 restraintsH atoms treated by a mixture of independent and constrained refinementmax = 0.95 e Å−3Δρmin = −0.51 e Å−3ΔρCOLLECT  I, New_Global_Publ_Block. DOI: 10.1107/S1600536814001135/im2446Isup2.hklStructure factors: contains datablock(s) I. DOI: CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


M1-gangliosidosis, GM2-gangliosidosis, and sialidosis. Glycan fingerprints were registered, and the patterns of accumulated oligosaccharides were found to reflect the specific blockages of the catabolic pathway. Our analytical approach allowed structural analysis of the excreted oligosaccharides and revealed several previously unpublished oligosaccharides. In conclusion, using online coupling of HPAEC with mass spectrometric detection, our study provides characteristic urinary oligosaccharide fingerprints with diagnostic potential for lysosomal storage disorders.Many lysosomal storage diseases are characterized by an increased urinary excretion of glycoconjugates and oligosaccharides that are characteristic for the underlying enzymatic defect. Here, we have used capillary high-performance anion-exchange chromatography (HPAEC) hyphenated to mass spectrometry to analyze free oligosaccharides from urine samples of patients suffering from the lysosomal storage disorders fucosidosis, α-mannosidosis, GThe online version of this article (doi:10.1007/s00216-012-5968-9) contains supplementary material, which is available to authorized users. M1-gangliosidosis, GM2-gangliosidosis, and sialidosis are autosomal recessive lysosomal storage diseases (LSD). These LSDs are the result of defects of one or more enzymes or cofactors involved in the catabolism of glycoconjugates that takes place in the lysosome. Fucosidosis is caused by a deficient lysosomal α-l-fucosidase (EC 3.2.1.51) and results in secretion of fucosyl-oligosaccharides [d-mannosidase (EC 3.2.1.24) causes α-mannosidosis and excessive urinary excretion of oligomannosidic glycans [M1-gangliosidosis is a neurosomatic disease due to the deficient activity of β-galactosidase (EC 3.2.1.23) [M1-gangliosides, glycoconjugates with β-galactose at the non-reducing end are increased in patients’ urine.Fucosidosis, α-mannosidosis, Gidase EC .2.1.51 a glycans . The uri glycans . GM1-gan.2.1.23) , 9. In aM2-gangliosidosis is a group of three disorders (1) Tay-Sachs disease, (2) Sandhoff disease, and (3) AB variant. For all variants of GM2-gangliosidosis, the major neural storage compound is ganglioside GM2 [N-glycan catabolism results in accumulation of oligosaccharides carrying a single N-acetylglucosamine residue at the non-reducing end in tissues and urine of Sandhoff disease patients [Gside GM2 –12. Onlyside GM2 . Blockagpatients –16. The Biochemical screening of these LSDs is usually performed using thin-layer chromatography (TLC) –19, sincM1-gangliosidosis [M1-gangliosidosis, GM2-gangliosidosis, and sialidosis patients. The results provided in glycan fingerprints that are found to be characteristic for the individual diseases and reflect the specific enzymatic defects.Hyphenation of liquid chromatography with mass spectrometry allows the detailed characterization of oligosaccharides . We haveosidosis  and galaosidosis  patientsw/w), sodium acetate, sulfuric acid, and sodium chloride were obtained from J.T. Baker . Acetonitrile was from Biosolve . All solutions were prepared using water from a Milli-Q synthesis system from Millipore BV . Details on urine samples are given in Table Analytical-reagent-grade sodium hydroxide  isocratic sodium hydroxide column wash; 88% A + 12% B (−14 to 0 min) isocratic equilibration of the column; a linear sodium acetate gradient (0–55 min) to 25.5% A + 12% B + 62.5% C was used for the separation. For separating oligosaccharides of α-mannosidosis and GM1-gangliosidosis, the following ternary gradient was used—76% A + 24% B (−20 to −14 min) isocratic sodium hydroxide column wash; 88% A + 12% B (−14 to 0 min) isocratic equilibration of the column; linear sodium hydroxide gradient (0 to 9.1 min) to 60% A + 40% B; 60% A + 40% B (9.1 to 12.5 min) isocratic; linear gradient (12.5 to 21.6 min) to 85.2% A + 12% B + 2.8% C; linear sodium acetate gradient (21.6 to 104 min) to 60.5% A + 12% B + 27.5% C. Samples were injected at time 0.0 min.The capillary chromatographic system consists of a modified Dionex BioLC system from Thermo Fisher Scientific  comprising a microbore GP40 gradient pump, a Famos micro-autosampler with a full PEEK (polyether ether ketone) injector equipped with a 1 μL loop and an ED40 electrochemical detector. BioLC control, data acquisition from the ED40 detector, and signal integration was supported by Dionex Chromeleon software (Themo Fisher Scientific). This modified system has been described in detail before . A protoE1 = 0.1 V , E2 = –2.0 V (t2 = 0.41–0.42 s), E3 = 0.6 V (t3 = 0.43 s), E4 = –0.1 V (t4 = 0.44–0.50 s) versus an Ag/AgCl reference electrode [The ED40 detector applies the following waveform to the electrochemical cell—lectrode . A 1 mm −1 [−1. The mixture was directed to the electrospray ionization interface of the Esquire 3000 used in the positive mode. The MS was operated at the following conditions: dry temperature 325 °C, nebulizer 103 kPa, dry gas 7 l min−1, capillary voltage −3,500 V, target mass m/z 850, scan speed 13,000 m/z/ s in MS mode, scan range 150–2,000 m/z, ICC target 50,000 with maximum accumulation time 50 ms. For tandem MS, automatic selection of three precursors was applied with absolute intensity threshold 10,000 and 5% relative intensity threshold (relative to the base peak intensity), using fragmentation settings of 1.40 V with smart fragmentation amplification of 30%–100% and a fragmentation time of 40 ms.Coupled to the chromatographic system was an Esquire 3000 ion trap mass spectrometer from Bruker Daltonics , equipped with an electrospray ionization source. To convert the HPAEC eluate into an ESI compatible solution, an in-line prototype desalter (Thermo Fisher Scientific) was used which was continuously regenerated with 12.5 mM sulfuric acid at a flow rate of 0.8 mL min−1 . A modif−1 lactose solution with 60 mM NaOH as eluent. The resulting MS chromatogram should pass the following criteria: The retention time has to be 7.5 min ± 15%; in the total ion current chromatogram, the baseline level intensity should be ≤4.5 e6 with the noise intensity ≤7.5 e5; for the extracted ion chromatogram (m/z 365 ± 0.5), the peak height intensity ≥4.5 e6 with a peak width at half height of ≤55 s.To check the correct functioning of the complete instrumental setup, every sequence started with analyzing a 50 nmol mLMS as well as MS/MS spectra were manually interpreted using DataAnalysis . The extracted ion chromatograms (EIC) were used in order to determine the peak area of oligosaccharides present in the MS spectra. Signals of all detected charge states and isomers corresponding to the same compound were added up. Peak areas were normalized to the sum of all glycan peak areas of one sample.M1-gangliosidosis, GM2-gangliosidosis, and sialidosis . Cross-ring cleavages at m/z 229.0 , 259.0 , and 289.0  suggest a 1–6 linkage for the fucose residue [Eight fucosylated oligosaccharides were detected in the urine sample of a fucosidosis patient, and the EIC of four of these fucosyl oligosaccharides are shown in Fig.  residue –43. Fromer m/z 37.1. Cross residue , 37, 44.N-acetylglucosaminidase cleavage products of mannose-rich oligosaccharides of composition Hex2-9HexNAc1 were detected [3HexNAc1 isomer is shown in Fig. 0,2A3 and 2,4A3 ions are typical for a 4-substituted HexNAc at the reducing end [0,3A2 (m/z 275.2) is indicative for a 6-substituted hexose [2Y2α ion  reveals the composition of the 6-antenna [2HexNAc1. The whole set of oligomannosidic structures was detected (Hex2–9HexNAc1) showing decreasing signals with increasing size.Three urine samples of three different α-mannosidosis patients from two different families were analyzed. In all three samples, 17 endo-β-detected , 45  is shown in Fig. 0,2A5 and 2,4A5 are typical for a 4-substituted reducing end HexNAc [4Y2α  reveals the composition of the 6-antenna [3HexNAc2 has been reported earlier [Extracted ion chromatograms of the disease-related glycans found in the urine of a G repeats , 34, 35.d HexNAc –43. The Ac3 m/z 7.1 is sho earlier .Fig. 5SeM2-gangliosidosis patients revealed 11 GM2-gangliosidosis-related glycan isomers . The EIC corresponding to Hex2HexNAc2 (m/z 771.5) shows the separation of these isobaric structures  and 2,4A4 (m/z 610) indicative for a 4-substituted N-acetylhexosamine [0,2A3 (m/z 508), 0,3A3 (m/z 478), and 0,4A3 (m/z 448) observed for the adjacent hexose are typical for a 6-substitution. Based on the observed MS/MS data and literature data [M2-gangliosidosis urinary tetrasaccharide GlcNAc(β1-2)Man(α1-6)Man(β1-4)GlcNAc. A lack of A3 cross-ring fragments, which is typical for a 3-substituted sugar, indicates that glycan B is the isomer GlcNAc(β1-2)Man(α1-3)Man(β1-4)GlcNAc. Hence, the linkage-specific fragmentation allowed the assignment of the observed glycans to two urinary glycans related to GM2-gangliosidosis [3HexNAc3 registered in monosodidated (m/z 1136.5) as well as disodiated (m/z 580.0) form. Figure 2HexNAc2 (both samples 7.6%), Hex2HexNAc3 (1.3% and 1.2%), Hex3HexNAc3 (8.0% and 8.2%), and Hex3HexNAc4 (14.8% and 15.5%) was observed.The analysis of the urine samples of two Grs Table  with theliosidos , 16. Hexxosamine –43. The ure data , glycan osidosis . Moreove3HexNAc2 (6.5%), HexHexNAc (5.4%), Hex3HexNAc2Neu5Ac (18.7%), and Hex5HexNAc3Neu5Ac2 (8.7%) were detected Gal(β1-4)GlcNAc and being related to sialidosis [O-sulfated oligosaccharides with terminal sialic acid residues were detected in the urine sample .One of the three isomers of Hexr et al. , 60 whilr et al. , 50, 61.alidosis , 62, 63.O-sulfated-glycan moieties to be detected. In contrast to other liquid chromatography methods relying on reducing end labeling for detection and/or separation [M1-ganglioside urine sample. Moreover, the setup used in this study enables the separation of isomeric glycans. Based on the efficient fragment ion analysis using an ion trap instrument, informative fragment spectra of sodium adducts can be obtained with minute amounts of material, allowing insights into defects of glycoconjugates degradation, and investigation of metabolic and catabolic pathways.All together, this publication shows the value of capillary HPAEC-IPAD-MS for analyzing oligosaccharides in clinical urine samples without the need for derivatization. This prototype analytical system features femtomol sensitivity for both pulsed amperometric detection and mass spectrometric detection  allowingparation , 65, HPAAlthough the used instrumentation is a prototype, similar analyses can be performed using commercially available narrow bore ion chromatographs , with prLC-MS in general and the here described method in particular are analytically powerful. In the current research paper, we demonstrated that the HPAEC-MS technology in combination with MS/MS information on structural isomers is suitable for determining characteristic glycan fingerprints in lysosomal storage diseases which may have diagnostic potential.ESM 1(PDF 662 kb)Below is the link to the electronic supplementary material.


T, was isolated from the swine intestinal tract on mucin-based media. Cells were curved rods (0.8–1.2×3.5–5.0 µm), stained Gram-negative and were non-motile with no evidence of spores. Strain CL-84T produced acetate, propionate, formate and butyrate as the end products of metabolism when grown on serine. Optimum growth occurred at 39 °C and pH 6.5. The major cellular fatty acids were iso-C15 : 0, iso-C15 : 0 3-OH, iso-C17 : 0 and C16 : 0, distinguishing strain CL-84T from closely related species. The DNA G+C content of strain CL-84T was 55.1 mol%. 16S rRNA gene sequence analysis showed that strain CL-84T shared 90–95 % similarity with characterized genera within the phylum Synergistetes, family Synergistaceae. Phylogenetic analysis showed that strain CL-84T was related to, but distinct from, Cloacibacillus evryensis. Based on these findings, we propose that strain CL-84T represents a novel species of the genus Cloacibacillus. We further propose the name Cloacibacillus porcorum sp. nov. be designated for this species. The type strain is CL-84T ( = DSM 25858T = CCUG 62631T). An emended description of the genus Cloacibacillus is provided.A novel anaerobic, mesophilic, amino-acid-fermenting bacterium, designated strain CL-84 Synergistetes have been identified in a diverse range of anaerobic environments, including anaerobic digesters  in broth, containing a basal medium, described below, and 1 % (w/v) hog gastric mucin (HGM) (Sigma-Aldrich), were used to enhance the growth of mucolytic bacteria before inoculation on solid media. Mucin-degrading bacteria were isolated on solid basal medium supplemented with 1 % (w/v) HGM after incubation at 39 °C for 5 days. Pure cultures were obtained after isolates were streaked for isolation three times. All cultures were inoculated and incubated (39 °C) in a Coy anaerobic chamber inflated with an atmosphere of N2 (85 %), CO2 (5 %) and H2 (10 %). The basal medium contained (per litre) 0.45 g CaCl2, 0.45 g MgSO4, 2.25 g KH2PO4, 2.25 g K2HPO4, 4.5 g NaCl, 4.5 g (NH4)2SO4, 0.05 % cysteine, 0.05 g haemin, 0.0001 % resazurin and 1.6 % Noble agar.Strain CL-84T grew optimally on brain heart infusion broth with 0.05 % cysteine and 0.0001 % resazurin, and supplemented with 20 mM arginine and histidine (BHIAH). This medium was used to maintain cultures. After 3 days growth at 39 °C on BHIAH medium, strain CL-84T reached a terminal OD620 of 1.2, representing 1.5×109 c.f.u. ml−1. The calculated doubling time was 8 h. Cells of strain CL-84T cultured in BHIAH broth had a curved-rod shape, were non-motile  from 4-day-old cultures, stained with uranyl acetate and lead citrate, and examined with a Tecnai 12 G2 Biotwin microscope (FEI). Micrographs revealed a cell outer envelope structure consistent with Gram-negative cells with a thin peptidoglycan layer surrounded by an outer membrane  web tools (http://rdp.cme.msu.edu/) (T within the phylum Synergistetes. The closest matches were to Cloacibacillus evryensis 158T (95 % sequence similarity) and S. jonesii 78-1T (90 %). C. evryensis was isolated from a municipal anaerobic waste digester  by the addition of mucin to the media , maximum-parsimony with dnapars v3.6 (1000 bootstraps) and maximum-likelihood with RAxML . All three analyses produced trees with the same topology, and therefore only the neighbour-joining tree is presented  , within the genus Cloacibacillus.Neighbour-joining, maximum-parsimony and maximum-likelihood phylogenetic analyses were performed based on the alignment of the 16S rRNA gene sequence from strain CL-84resented . Strain T was sequenced using a 454 titanium pyrosequencing platform (454 Life Sciences). The gyrase B gene sequence of strain CL-84T (GenBank accession no. JX443487) was obtained from the genome sequence and compared with that of C. evryensis, obtained from the finished genome sequence (IMG/GEBA database: 6858). The gyrase B nucleotide sequence from strain CL-84T showed only 90 % similarity to C. evryensis, which supports the conclusion that these are different species. Sequence analysis revealed that strain CL-84T has a DNA G+C content of 55.1 mol%.As part of a larger survey of swine intestinal microbes, the genome of strain CL-84O-glycosidic links , but grew faster on BHIAH. Growth was observed on a limited number of amino acids and whole mucin and mucin components. Growth on whole mucin and mucin O-linked glycans was also tested and observed with C. evryensis . Growth results for strain CL-84T are summarized in 620 values.Mucins are glycoproteins with carbohydrate side chains connected to a protein backbone by ic links  and are T contained butyryl-CoA : acetate CoA-transferase (E.C. 2.8.3.8) activity [specific activity = 9.88±0.45 µmol min−1 (mg protein)−1] as determined using cells cultured on basal medium with serine. The assay for transferase activity was performed on French press extracts of strain CL-84T according to  The ability of strain CL-84T to produce butyrate when grown on serine may reflect its role in the gut, as butyrate has been shown to stimulate mucin synthesis in human colonic cell lines and animal mucosa , esterase lipase (C8) and leucine arylamidase activities. Strain CL-84T tested negative for catalase.Additional biochemical characterizations were performed using Rapid ID 32 A and API ZYM test strips (bioMérieux). Strain CL-84T was pH 4–8, with optimal growth (reaching an OD620 of >1.0) at pH 6–7. Growth between 20 and 45 °C was observed. No viable cells remained after heating cultures to 80 °C for 30 min. Growth of CL-84T was inhibited by NaCl concentrations above 1.4 % (w/v), with optimal growth at 0.6–0.8 % (w/v) NaCl. Strain CL-84T cells were aerotolerant and still viable after 24 h of exposure to oxygen. Growth of CL-84T was not observed on BHIAH plates, exposed to 1.0 % oxygen, after 1 week of incubation.Heat, pH and oxygen tolerance were tested in BHIAH medium. The pH range for growth of strain CL-84T, C. evryensis 158T, was obtained from the DSM culture collection (accession no.19522). Both strain CL-84T and C. evryensis 158T were grown in 10 ml BHIAH medium to an OD620 of 1.0, and cell pellets were used for cellular fatty acid analysis. The cellular fatty acid composition was determined by Microbial ID Inc. with the GC-based MIDI Sherlock Microbial Identification System. The major cellular fatty acids of strain CL-84T were iso-C15 : 0 (27.1 %), iso-C15 : 0 3-OH (15.0 %), iso-C17 : 0 (11.7 %) and C16 : 0 (9.7 %). The profile for strain CL-84T differed from that of C. evryensis 158T in both the fatty acid types and the proportions of each component. The top three fatty acids for strain CL-84T were iso-C15 : 0, iso-C15 : 0 3-OH and iso-C17 : 0 , while the top three for C. evryensis 158T were iso-C15 : 0, iso-C15 : 0 3-OH and C17 : 1ω11c  (The closest related type strain to CL-84ctively) .T was susceptible to tylosin, lincomycin, chlortetracycline, penicillin, florphenicol, ceftiofur and carbadox (MIC <4 µg µl−1) and resistant to vancomycin and sulfathiazole (MIC 512 µg µl–1 for each). Other Synergistetes spp. have also been shown to be resistant to vancomycin (Antibiotic minimum inhibitory concentration (MIC) assays were modified from Cloacibacillus follows that of 15 : 0 and iso-C15 : 0. Differential characteristics between strain CL-84T and C. evryensis 158T and S. jonesii 78-1T are given in The description of the genus Cloacibacillus porcorum .15 : 0, iso-C15 : 0 3-OH, iso-C17 : 0 and C16 : 0. Cells are resistant to vancomycin and sulfathiazole but are susceptible to tylosin, lincomycin, chlortetracycline, penicillin, florphenicol, ceftiofur and carbadox. Optimal growth occurs at 39 °C and pH 6.5.Cells are obligately anaerobic, non-motile and curved-rod shaped. Cells ferment amino acids, and can use mucin and mucin components as a sole carbon source. Fermentation products are acetate, propionate and formate but only butyrate is produced from serine. This bacterium is an intestinal commensal of swine. Cells have a Gram-negative cell-wall structure and range in size from 0.8 to 1.2 µm wide and 3.5 to 5.0 µm long. Strong growth is obtained in BHI medium, and growth is enhanced by the addition of histidine and arginine, but not glucose. After 7 days incubation, colonies are 1 mm in diameter, circular, shiny, brown and semi-translucent. The main cellular fatty acids are iso-CT ( = DSM 25858T = CCUG 62631T), was isolated from the mucosal lining of a pig caecum in Ames, Iowa, USA. The DNA G+C content of the type strain is 55.1 mol%.The type strain, CL-84


H-pyrazole-5-carboxamide, BHX) to block Wnt signaling, and determined the absolute configuration of its precursor . We then evaluated the inhibitory effect of BHX in vitro and in vivo.Wnt/β-catenin signaling is a highly conserved pathway in organism evolution and is important in many biological processes. Overactivation of Wnt/β-catenin signaling is closely related to tumor development and progression. To identify potent small molecules that can fight aberrant Wnt/β-catenin-mediated cancer, we synthesized a novel pyrazoline derivative (N-(4-hydroxybenzyl)-1,3,4-triphenyl-4,5-dihydro-150 of 5.43 ± 1.99, 6.95 ± 0.24, and 7.62 ± 1.31 μM, respectively. BHX significantly induced apoptosis and G1 phase arrest in A549 and MGC803 cells. The β-catenin protein level was markedly reduced in A549 and MGC803 cells under BHX treatment. The inhibitory effect of BHX in vivo was investigated using a mouse xenograft model. A549 xenograft growth was suppressed by 50.96% in nude mice treated continuously with 100 mg/kg BHX for 21 d. Weight remained almost unchanged, which indicates the low toxicity of the compound.Cell proliferation was assessed in three human cancer cell lines  in the presence and absence of BHX using MTS assays. BHX effectively inhibited A549, HT29, and MGC803 cell proliferation with ICOur data suggest that BHX is a new drug candidate for cancer treatment because of its potent effect on the Wnt/β-catenin pathway and low toxicity. The Wnt signaling pathway is a network of proteins that are important in embryogenesis and cancer-4. Wnt lUnder pathological conditions, β-catenin escapes degradation and cells retain unregulated activation of canonical Wnt signaling caused by mutations in APC, axin, or β-catenin. ActivatH-pyrazole-5-carboxamid (BHX), which acts as a Wnt/β-catenin signaling inhibitor. The derivative was biologically evaluated in vitro using TOPflash reporter assay , human colon cancer cell line (HT29), and human gastric cancer cell line (MGC803) cell proliferation in a dose-dependent manner, and their IC50 values were 5.43 ± 1.99, 6.95 ± 0.4, and 7.62 ± 1.31 μM, respectively . The early apoptosis rate of the BHX-treated A549 cells (35.5%) was significantly higher than that of the vehicle-treated cells (1.23%). Similarly, the early apoptosis rate of the BHX-treated MGC803 cells (37.2%) was also significantly higher than that of the vehicle-treated cells (1.61%). However, BHX-induced late apoptosis was not observed in A549 and MGC803 cells  by subcutaneous injection. When the tumor volumes were approximately 100 mm3 to 150 mm3, the mice were randomized to receive intraperitoneal injections of vehicle or BHX at 25, 50, and 100 mg/kg for 21 consecutive days. Treatment with 100 mg/kg BHX resulted in a significant reduction in tumor volume , which indicates that BHX-associated toxicity was minimal . Several3, a single crystal for compound 3 was obtained and the absolute configuration was determined by X-ray structural analysis.In this study, we synthesized a novel small molecule BHX. To confirm the regiochemistry of the cycloaddition for Δ2-pyrazoline H-pyrazole-5-carboxylate, was obtained)/5079 were unique and observed. Empirical absorption corrections were performed using SADABS. The structures were solved by direct methods (SHELXS-97) and refined by full-matrix least squares on F2 with weight scheme w = 1/[σ2(Fo2) + (0.2000P)2 + 0.0000P] (where P = (Fo2 + 2Fc2)/3) using the SHELXL-97 program suite. All of the non-hydrogen atoms were refined anisotropically, whereas all of the hydrogen atoms were added theoretically. Crystallographic data are summarized in Table Single crystals with suitable dimensions of 0.38 mm × 0.32 mm × 0.15 mm were mounted on glass fibers. Data collection was performed on Brucker SMART Apex CCD diffractometer by ω scan technique using graphite-monochromated Mo-K2O, and then dried to obtain compound 1 as a white solid . 1H-NMR  δ: 7.65 , 7.36 , 7.27 , 7.11 , and 6.87 .Benzaldehyde  was slowly added to a mixture of phenylhydrazine  and glacial acetic acid (3.1 L). The resulting mixture was stirred at room temperature for 1 h and filtered. The precipitate was washed with acetic acid and H1  was dissolved in methanol (71.8 mL) under dim light. The resulting solution was stirred under reflux overnight. After cooling to room temperature, the reaction mixture was poured into a mixture of ethyl acetate and hexane  and filtered. The filtrate was concentrated to yield the crude product as a brown oil, which was then purified by column chromatography  to produce compound 2  as a white powder. 1H-NMR δ: 7.65 , 7.72 , 7.14 , 6.88 , 4.79 , 4.68 , 4.22 , and 1.21 .A mixture of ethyl cinnamate , chloramine-T trihydrate , and compound 2O  was added to a mixture of compound 2 , tetrahydrofuran (THF) (18.0 mL), MeOH (6.0 mL), and H2O (6.0 mL). The resulting solution was stirred for 1 h under room temperature. The reaction mixture was concentrated, dissolved in ethyl acetate (40.0 mL), and then respectively washed with 5% aqueous HCl (3 × 20 mL) and brine (3 × 20 mL). The resulting residue was dried over Na2SO4 and concentrated to produce compound 3  as a yellow solid. 1H-NMR δ: 13.32 , 7.70 , 7.28 , 7.11, 6.83, 5.11 , and 4.68 .LiOH · H3  in THF (140.0 mL). The resulting solution was stirred overnight under room temperature, and then concentrated to yield the crude product. The pure product  was obtained by column chromatography  as a pale yellow solid. 1H-NMR δ: 9.30 , 8.83 , 7.67 , 7.28 , 7.07 , 6.82 , 6.70 , 4.93 , 4.71 , and 4.21 .N-(3-Dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride , 1-hydroxybenzotriazole , and 4-(aminomethyl)phenol were added to a mixture of compound For 3--5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt (MTS) cell proliferation assays, A549, MGC803, and HT29 cells were seeded in 96-well culture plates at a density of 500 cells to 1,000 cells per well. After 24 h, BHX or dimethyl sulfoxide (DMSO) was added to the cells at concentrations between 1.25 and 40 μmol/L. Cell proliferation was determined after 72 husing CellTiter 96 AQueous One Solution (Promega). Absorbance was recorded at 490 nm using a microplate reader.Cells were seeded at approximately 50% confluence in six-well cell culture plates. BHX (5 μmol/L) or DMSO was added after 24 h, and the cells were collected after 72 h of incubation. Cell apoptosis was measured by Annexin V-FITC/propidium iodide (PI) staining. Cells were considered early apoptotic if Annexin V was positive and PI was negative, and late apoptotic if both Annexin V and PI were positive. For cell cycle analysis, cells were rinsed in PBS, fixed in 95% ethanol, pelleted by centrifugation, and resuspended in RNase A at 50 μg/mL. After incubation at 37°C for 1 h, 50 g/mL PI was added. DNA content was determined by flow cytometry, and the proportion of cells in each cell cycle phase was determined using ModFit software.3VO4, and 1 mmol/L phenylmethylsulfonyl fluoride. Lysates were then spun at 12,000 rpm for 10 min to remove insoluble materials. The protein concentration was measured by BCA protein assay (Pierce). Total proteins were fractionated using SDS–PAGE and transferred to a polyvinylidenefluoride membrane; blotted with antibodies against β-catenin, E-cadherin, and β-actin; and detected by enhanced chemiluminescence (Amersham Biosciences).Whole cell extracts were lysed in lysis buffer with the following components: 240 mmol/L Tris (pH 6.8), 2% sodium dodecyl sulfate (SDS), 0.5% glycerol, 5 mmol/L EDTA, 1 mg/L aprotinin, 1 mg/L leupeptin, 1 mg/L peptatin A, 10 mmol/L β-glycerol phosphate, 1 mmol/L Na7 A549 cells. Tumor sizes were assessed using the two largest perpendicular axes. Tumor volume was calculated using the formula V = (a × b2)/2, where a is length and b is width. When tumor volumes reached 100 mm3 to 150 mm3, the mice were randomized to drug-treated or vehicle groups (six mice per group). BHX or vehicle control was administered by daily intraperitoneal injection at dose levels of 25, 50, and 100 mg/kg body weight for 21 consecutive days. Tumor growth was monitored every 3 d using a Vernier caliper. Tumor-bearing mice were assessed for weight loss every 3 d. Tumors were removed from mice 21 d after vehicle or BHX treatment. Inhibition rates (antitumor effects) are expressed as T/C% (treatment groups versus control) by dividing the tumor volumes from treatment groups with the control group and then multiplying the quotient by 100. All animal experiments were conducted in accordance with the Animal Care Committee guidelines of Tianjin Medical University Cancer Institute and Hospital.Female BALB/c mice, weighing 17 g to 23 g, were implanted subcutaneously with 1 × 10t-test or Wilcoxon’s test. P values < 0.05 were considered statistically significant.All values are expressed as mean ± SD. Crude data were analyzed using SPSS 16.0 statistical software. The statistical significance of differential findings between experimental groups and control was determined by Student’s The authors declare that they have no potential competing interest with respect to the research, authorship and/or publication of this article.ZZ, JW, JS, YC, GY and WC acquired and analyzed data as; performed the statistical analysis; drafted the manuscript. ZY and YD conceived and designed the study; revised and approved the manuscript. All of the authors read and approved the final manuscript.3 per well were seeded into 24-well plates before transfection. TOPflash or FOPflash plasmids were co-transfected with PRL-TK plasmid . Luciferase activity was measured by Dual-Glo Luciferase Assay System after cells were cultured for 18 h. The ratio between firefly luciferase activity (TOPflash/FOPflash) and renilla activity  was used for TCF/LEF transcription activity. Data represent mean ± SD from three independent experiments. Numerous small molecules were screened for their ability to inhibit Wnt pathway downstream transcriptional activity using the TOP/FOP reporter assay. BHX was observed to reduce β-catenin/TCF-dependent transcriptional activity by 67%. 1: DMSO; 9: BHX.BHX dramatically downregulates β-catenin/TCF-dependent transcriptional activity. To measure Wnt signaling, cells at a density of 5 × 10Click here for fileEffect of BHX on cell growth of MCF-10A or A549. MCF-10A and A549 were plated in triplicate. After 24 h, the cells were treated with DMSO or 3 μmol/L BHX. Viable cell numbers were counted daily for 4 d by trypan blue exclusion. Results are presented as percentage of control. Data are presented as mean ± SD from three independent experiments.Click here for file


In order to search for new structural modification strategies on fluoroquinolones, we have designed and synthesized a series of fluoroquinolone derivatives by linking various hydrazine compounds to the C-3 carboxyl group of levofloxacin and assessed their anticancer activities. Several novel levofloxacin derivatives displayed potent cytotoxicity against the tested cancer cell lines in vitro. In the present study, we investigated the effect of 1-Cyclopropyl-6-fluoro-4-oxo-7- piperazin-1, 4-dihydro- quinoline- 3-carboxylic acid benzo  dioxol-5- ylmethylene- hydrazide (QNT11) on the apoptosis of human hepatocarcinoma cells in vitro.The inhibition effects of QNT11 on cell proliferation were examined by MTT assay. Cell apoptosis was determined by TUNEL and DNA agarose gel electrophoresis method. The topoisomerase ΙΙ activity was measured by agarose gel electrophoresis using Plasmid pBR322 DNA as the substrate. Cell cycle progression was analyzed using flow cytometry in conjunction with ethanol fixation and propidium iodide staining. Mitochondrial membrane potential (△ψm) was measured by high content screening image system. The caspase-9, caspase-8, caspase-3, Bcl-2, Bax, CDK1, Cyclin B1and cytochrome c protein expressions were detected by Western blot analysis.50 values of 2.21 μM, 2.38 μM, 3.17 μM and 2.79 μM, respectively. In contrast, QNT11 had weak cytotoxicity against mouse bone marrow mesenchymal stem cells (BMSCs) with IC50 value of 7.46 μM. Treatment of Hep3B cells with different concentrations of QNT11 increased the percentage of the apoptosis cells significantly, and agarose gel electrophoresis revealed the ladder DNA bands typical of apoptotic cells, with a decrease in the mitochondrial membrane potential. Compared to the control group, QNT11 could influence the DNA topoisomerase IIactivity and inhibit the religation of DNA strands, thus keeping the DNA in fragments. There was a significant increase of cytochrome c in the cytosol after 24 h of treatment with QNT11 and a decrease in the mitochondrial compartment. Observed changes in cell cycle distribution by QNT11 treated might be caused by insufficient preparation for G2/M transition. In addition, QNT11 increased the protein expression of Bax, caspase-9, caspase-8, caspase-3, as well as the cleaved activated forms of caspase-9, caspase-8 and caspase-3 significantly, whereas the expression of Bcl-2 decreased.QNT11 showed selective cytotoxicity against Hep3B, SMMC-7721, MCF-7 and HCT-8 cell lines with ICOur results showed that QNT11 as a fluoroquinolone derivative exerted potent and selectively anticancer activity through the mechanism of eukaryotic topoisomerase II poisoning. The growth inhibition was in large part mediated via apoptosis-associated mitochondrial dysfunction and regulation of Bcl-2 signaling pathways. Streptomyces). For this reason, antibacterial fluoroquinolones have been shown to have cytotoxic activity against cancer cells  × 100. The IC50 was the concentration that caused a 50% inhibition of cel1 proliferation.The cells were seeded at a density of 1 × l04 cells/ml) were seeded in growth medium on the cover glass slides of 6-well plates for 24 h incubation. They were then treated with the indicated concentrations of QNT11 for 24 h. Control wells consisted of cells incubated with medium only. After that, cells were examined for apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay , performed according to the manufacturer’s instructions. Cells were visualized and photographed using a fluorescent microscope . At least five randomly chosen areas in every slide were used. Percent apoptosis was determined by counting the number of apoptotic cells and dividing by the total number of cells in the areas.Hep3B cells  according to the manufacturer’s instructions. The DNA sample was incubated at 37°C for 30 min and electrophoresed on a 1% agarose gel containing 1 mg/ml ethidium bromide at 40 V/cm. Finally, the apoptotic DNA fragments were visualized under a UV transilluminator and photographed.6/ml) were washed twice with ice-cold PBS and then were re-suspended gently in 500 μl of ice-cold PBS. Thereafter, ice-cold 70% ethanol (4 ml) was added in a dropwise manner and cells were stored at 4°C for 12 h. After 12 h, cells were pelleted by centrifugation for 5 min. The supernatant was removed and cells were re-suspended in 500 μl of PBS containing propidium iodide (50 μg/ml) and incubated in dark conditions at 37°C before analysis by FACS .Hep3B cells  under increasing concentrations of QNT11. In this experiment, etoposide , a known topoisomeraseIIpoison [4 mg/L). Samples were subjected to electrophoresis in 1% agarose gels. DNA was then stained with 1 mg/ml ethidium bromide and photographed under a UV transilluminator.DNA topoisomeraseIIactivity was determined by the supercoiled pBR322 DNA relaxation assay . The expIIpoison , was useHep3B cells were treated with media containing different concentrations of QNT11 for 24 h and were then incubated with 0.5 mg/ml of the fluorescence probe JC-1  at 37°C for 20 min. The cells were washed twice thoroughly with buffer and incubated with 5 μg/ml. Hoechst 33258 for 10 min in the dark. After additional washing twice, the mitochondrial membrane potential (△ψm) was measured by high content screening (HCS) image system. .c or β-actin  overnight at 4°C. After washing and reaction with horseradish peroxidase conjugated anti-mouse IgG , or anti- rabbit IgG  secondary antibodies for 1 h, the membranes were washed with TBST buffer three times and the proteins on the membrane were detected using an enhanced chemiluminescene substrate .After treatments with different concentrations QNT11 for 24 h, Hep3B cells were lysed with ice-cold RIPA lysis buffer. Protein concentrations were determined using the Bradford method. After adjustment to a similar level of total protein concentration, samples were separated by 12% SDS–PAGE under reducing conditions and then transferred onto polyvinglidene fluoride (PVDF) membranes (Millipore). The membranes were blocked with 5% non-fat milk in TBST buffer  for 1 h at room temperature prior to incubation with specific antibodies to caspase-9, caspase-8, caspase-3, Bcl-2, Bax, CDK1, CyclinB1, Cytochrome c release from mitochondria was evaluated by western blot analysis of cytosolic protein samples. Cytosolic and mitochondrial protein fractions were prepared using the cell mitochondria isolation kit .Cytochrome t-test for paired samples. P < 0.05 was regarded as significant, and P < 0.01 as highly significant.Data are presented as the mean ± standard deviation (SD) for the indicated number of independent experiments. Statistical significance was calculated using the 50 value for 12,24, 48 and 72 h treatment was 1.92 ± 0.19 μM (r2 = 0.9601), 2.21 ± 0.20 μM (r2 = 0.9679), 2.55 ± 0.25 μM (r2 = 0.9561) and 2.70 ± 0.22 μM (r2 = 0.9802) respectively. For SMMC-7721 cells, the IC50 value for 12, 24, 48 and 72 h treatment obtained was 2.16 ± 0.28 μM (r2 = 0.9109), 2.38 ± 0.22 μM (r2 = 0.9408), 2.68 ± 0.25 μM (r2 = 0.9427) and 3.06 ± 0.19 μM (r2 = 0.9264), respectively. For MCF-7 cells, the IC50 was 2.93 ± 0.23 μM (r2 = 0.9640), 3.17 ± 0.26 μM (r2 = 0.9212), 3.11 ± 0.32 μM (r2 = 0.9490) and 3.68 ± 0.25 μM (r2 = 0.9624) for 12, 24, 48 and 72 h treatment respectively. For HCT-8 cells, the IC50 value for12, 24, 48 and 72 h treatment obtained was 2.12 ± 0.22 μM (r2 = 0.9260), 2.79 ± 0.22 μM (r2 = 0.9547), 2.86 ± 0.25 μM (r2 = 0.9139) and 3.46 ± 0.32 μM (r2 = 0.9174), respectively. In contrast, QNT11 had weak cytotoxicity against BMSCs. The IC50 value for 12, 24, 48 and 72 h treatment was 8.13 ± 0.55 μM (r2 = 0.9574), 7.46 ± 0.50 μM (r2 = 0.9034), 7.90 ± 0.55 μM (r2 = 0.9414) and 12.05 ± 0.69 μM (r2 = 0.8993), respectively.The cytotoxicity of QNT11 against cells was assessed using MTT cell viability assay. The cells were treated with various concentrations of QNT11 for 12,24, 48 and 72 h, resulting in a significant decrease in cell viability in a dose- and time-dependent manner , 10.99 ± 0.39 μM (r2 = 0.8016), 20.80 ± 0.58 μM (r2 = 0.7122) and 13.03 ± 0.46 μM (r2 = 0.8362), respectively.Levofloxacin had weak cytotoxicity against Hep3B cells. The ICAfter treatment with various concentrations of QNT11 for 24 h, TUNEL assay was used to confirm the apoptotic effect of QNT11. 24 h incubation with QNT11 increased the percentage of apoptotic Hep3B cells in a concentration-dependent manner  was detected with the fluorescent probe JC-1, which exists predominantly in monomeric form in cells with depolarized mitochondria and displays green fluorescence at 490 nm. On the other hand, JC-1 primarily forms aggregates in cells with polarized mitochondria and shows reddish-orange fluorescence. The emission intensity ratio of the 545 nm and 595 nm peaks was used as a measure of the mitochondrial depolarization; a higher ratio indicated more depolarization. Hep3B cells with QNT11 treatment for 24 h exhibited green JC-1 fluorescence in a dose-dependent manner, which is consistent with a loss of △ψm  2, 5-diphenyltetrazolium bromide; TUNEL: Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling; DMSO: Dimethysulfoxide; Bcl-2: B-cell lymphoma 2; Bax: Bcl-2–associated X protein; CDK: Cyclin-dependent kinases.The authors declare that they have no competing interests.GQH, YHK and BL designed research; JPS, ZYS and SML performed the experiments and data analysis; JBD and CSHF contributed new reagents and analytic tools; JPS, ZYS and BL wrote the paper. All authors read and approved the final manuscript.


The CuII ion is five-coordinated in a distorted square-pyramidal manner by four N atoms from two benzene-1,2-diamine ligands at the base and one O atom from a monodentate sulfate anion at the apex of the coordination polyhedron. N—H⋯O hydrogen bonding between the amino functions and the sulfate groups leads to the formation of layers parallel to (001). C—H⋯O hydrogen bonding interactions between the layers consolidate the three-dimensional set-up. There are voids in the structure filled with lattice water molecules that are disordered over three sites in a 0.430 (6):0.270 (6):0.300 (6) ratio.The title complex, [Cu(SO For gra al. 1995; Etter e al. 1990.4)(C6H8N2)2]·H2O = 0.034wR(F2) = 0.097S = 1.083555 reflections212 parameters1 restraintH-atom parameters constrainedmax = 1.29 e Å−3Δρmin = −0.64 e Å−3ΔρCOLLECT  I, global. DOI: Click here for additional data file.10.1107/S1600536812041967/wm2682Isup2.hklStructure factors: contains datablock(s) I. DOI:  crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:


III atom in the title complex, [Mn(C18H18N2O4)(CHO2)(H2O)]·2H2O, is O,N,N′,O′-chelated by the deprotonated Schiff base; the four chelating atoms form an approximate square, with the O atoms of the water molecule and the formate ion in axial positions above and below the square plane. Two metal-bearing molecules are linked by an O—Hwater⋯O hydrogen bond about a center of inversion, generating a hydrogen-bonded dinuclear species; adjacent dinuclear units are linked through the lattice water molecules, forming a three-dimensional network.The Mn III compounds with the same Schiff base, see: Bermejo et al. (CHO2)(H2O)]·2H2O = 0.067                           wR(F                           2) = 0.192                           S = 1.12                           4600 reflections280 parametersH-atom parameters constrainedmax = 1.67 e Å−3                        Δρmin = −0.96 e Å−3                        Δρ                                 APEX2  used to solve structure: SHELXS97 (Sheldrick, 2008SHELXL97 (Sheldrick, 2008X-SEED (Barbour, 2001publCIF (Westrip, 2010Data collection: 10.1107/S1600536811017648/bt5546sup1.cif            Crystal structure: contains datablocks global, I. DOI: 10.1107/S1600536811017648/bt5546Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


Amyloid-β peptide ending at 42nd residue (Aβ42) is believed as a pathogenic peptide for Alzheimer disease. Although γ-secretase is a responsible protease to generate Aβ through a processive cleavage, the proteolytic mechanism of γ-secretase at molecular level is poorly understood.We found that the transmembrane domain (TMD) 1 of presenilin (PS) 1, a catalytic subunit for the γ-secretase, as a key modulatory domain for Aβ42 production. Aβ42-lowering and -raising γ-secretase modulators (GSMs) directly targeted TMD1 of PS1 and affected its structure. A point mutation in TMD1 caused an aberrant secretion of longer Aβ species including Aβ45 that are the precursor of Aβ42. We further found that the helical surface of TMD1 is involved in the binding of Aβ45/48 and that the binding was altered by GSMs as well as TMD1 mutation.Binding between PS1 TMD1 and longer Aβ is critical for Aβ42 production. Psen  1, Psen2 or APP genes cause an increase in Aβ42 generation. Thus, Aβ42 is considered as the most pathogenic species causative for AD[Several lines of evidence suggest that the accumulation of amyloid-β peptide (Aβ), a major component of senile plaques, is a common pathological feature in Alzheimer disease (AD). Aβ is ge for AD.γ-Secretase is an intramembrane-cleaving protease complex composed of four membrane spanning proteins: PS, Nicastrin, Aph-1 and Pen-2,5. ExtenRecently, small compounds that selectively regulate Aβ42 production without affecting ϵ-cleavage emerged, which are termed γ-secretase modulators (GSMs). We havePsen1-/-/Psen2-/- double knockout mice (DKO)[bona fide molecular target of fenofibrate, in the context of modulation of intramembrane cleavage, are PS1 and SPP, i.e., the enzyme moieties.Fen-B is a derce (DKO) with or ce (DKO) were subce (DKO), was alsTo narrow down the fenofibrate binding site within PS1 NTF, we employed the limited digestion approach by inserting a thrombin substrate sequence into PS1. γ-SecreThis finding prompted us to hypothesize that TMD1 is potentially involved in the regulation of the processivity of γ-secretase to generate Aβ42. To address this issue, we screened single cysteine (single-Cys) mutants of TMD1 in cysteine-less PS1 used in SCAM. While cysteine-less PS1 increased Aβ4243)/Aβ40 ratio compared to that of wild-type PS1-expressing cells, some single-Cys mutants showed further augmentation in Aβ42(43)/Aβ40 ratio  replaced with a TMD of an unrelated membrane protein, CLAC-P[in vitro[1-110, as previously reported by Annaert et al[1-110, suggesting that TMD1 of PS1 directly binds to longer Aβ peptides, and that the cytoplasmic domain of APP is not involved in this binding. Unexpectedly, GST-TM1mt1-110 was capable of interacting with C99-FLAG as well as with peptides belonging to the Aβ40 production line . However, the binding of Aβ species of the Aβ42 production line  was significantly reduced by swapping the TMD1 sequence  in vitro-23. Morein vitro. These r, CLAC-P , indicating that TMD1 is involved in the regulation of C-terminal length of Aβ. Finally, we found that TMD1 contains a binding site for the longer Aβ species, and GSMs affect Aβ42 production by changing the affinity between TMD1 and longer Aβ. Our results suggest that TMD1 functions as a substrate binding domain during the processive cleavage by γ-secretase.GSM-1, GSM-1-BpB, NS-1017, GSM-1-amide-BpB, Fen-B and DAPT were synthesized as described,36. L-68TM strategy (Stratagene). To produce recombinant proteins, cDNAs encoding PS1 were cloned into pGEX-6P-1 vector [cDNAs encoding PS1 and APP carrying Swedish mutation (APPNL) were inserted into pMXs-puro. cDNAs elthcare). Maintenlthcare),39,44,45Preparation of samples for photoaffinity labeling experiments was perfE. Coli (BL21 DE3) (Novagen) and purified by two step procedures using glutathione sepharose and mono Q columns  as manufacturer’s instruction. All recombinant proteins were finally diluted with recombinant protein preparation buffer (10 mM HEPES (pH 7.4), 150 mM NaCl, 0.25% CHAPSO). C99-FLAG was purified from Sf9 cells infected with recombinant baculovirus encoding C99-FLAG and diluted at 1 μg/ml in recombinant protein preparation buffer. To perform binding assay, 0.5 μg of GST-fusion recombinant proteins were mixed at 1 μg of C99-FLAG, 1  or 10 (for Aβ40 and Aβ42) nM of synthetic Aβ in 1 ml of recombinant protein preparation buffer, and incubated at 4 degree overnight. After addition of glutathione sepharose, samples were then washed with the buffer and precipitates were eluted by boiling in sample buffer. For binding assay using native PS1 protein, PS1 or P88L mutant PS1 was expressed in DKO cells and solubilized in 10 mM HEPES buffer containing 1% CHAPSO. After addition of 2 ng of Aβ48 peptide, the solubilized fraction was incubated with anti-PS1 antibody G1Nr5 at 4 degree overnight. PS1-Aβ48 complex was then immunoprecipitated using Protein G sepharose 4 Fast Flow . Subsequently eluates  were analyzed by immunoblotting. We loaded 0.75 and 20% of samples as “input” and “bound”, respectively, in immunoblot of all pull down assay unless the amount of loaded proteins was otherwise indicated.GST-fusion recombinant proteins were expressed in Psen1/Psen2 double knockout mouse immortalized fibroblasts; GSM: γ-secretase modulator; NTF: Amino-terminal fragment; mt: Mutant; PS: Presenilin; SCAM: Substituted cysteine accessibility method; TMD: Transmembrane domain.AD: Alzheimer disease; CTF: Carboxyl-terminal fragment; DKO: The authors declare that they have no competing interests.YO and TT designed the research. YO, AT, SO and TT performed biochemical experiments. NS, TH, SY and TF synthesized the compounds. YO, TT and TI wrote the paper. All authors read and approved the final manuscript.


Together with full-length Aβ1–42 and Aβ1–40, N-truncated AβpE3-42 and Aβ4–42 are major variants in AD brain. Although Aβ4–42 has been known for a much longer time, there is a lack of studies addressing the question whether AβpE3-42 or Aβ4–42 may precede the other in Alzheimer’s disease pathology.The amyloid hypothesis in Alzheimer disease (AD) considers amyloid β peptide (Aβ) deposition causative in triggering down-stream events like neurofibrillary tangles, cell loss, vascular damage and memory decline. In the past years N-truncated Aβ peptides especially N-truncated pyroglutamate Aβ4-x preceded AβpE3-x intraneuronal accumulation in a transgenic mouse model for AD prior to plaque formation. The novel Aβ4-x immunoreactive antibody NT4X-167 detected high molecular weight aggregates derived from N-truncated Aβ species. While NT4X-167 significantly rescued Aβ4–42 toxicity in vitro no beneficial effect was observed against Aβ1–42 or AβpE3-42 toxicity. Phenylalanine at position four of Aβ was imperative for antibody binding, because its replacement with alanine or proline completely prevented binding. Although amyloid plaques were observed using NT4X-167 in 5XFAD transgenic mice, it barely reacted with plaques in the brain of sporadic AD patients and familial cases with the Arctic, Swedish and the presenilin-1 PS1Δ9 mutation. A consistent staining was observed in blood vessels in all AD cases with cerebral amyloid angiopathy. There was no cross-reactivity with other aggregates typical for other common neurodegenerative diseases showing that NT4X-167 staining is specific for AD.Using different Aβ antibodies specific for the different N-termini of N-truncated Aβ, we discovered that Aβ4-x precedes AβpE3-x in the well accepted 5XFAD AD mouse model underlining the significance of N-truncated species in AD pathology. NT4X-167 therefore is the first antibody reacting with Aβ4-x and represents a novel tool in Alzheimer research.Aβ Several hypotheses have been proposed and competed in trying to explain the underlying cause of Alzheimer’s disease (AD). The dominant hypothesis, since 1991, is the amyloid hypothesis that implicates amyloid-β (Aβ) deposits as the cause of this common neurodegenerative disorder. Extracellular deposits of Aβ protein and the intracellular accumulation of phosphorylated tau protein are the basis of the neuropathological characterization of AD . ContempThe “Amyloid Hypothesis” was proposed, therefore, based on the previously mentioned discovery ,7. SinceMemory loss in AD is the most prominent clinical manifestation of the disease. To that end, Haass and Selkoe  have rec1 starting with aspartate as the first amino acid, several N-truncated and modified Aβ species have been characterized [in vitro and in vivo analysis of amyloid deposits in AD [x-40 to Aβx-42) and by N-terminal truncation [4-42 is a relatively abundant species in AD, aged controls and vascular dementia patients.In addition to Aβcterized -16. In fts in AD ,17,18. Tuncation -21. Amonuncation  reportedpE3-x is present in equivalent or larger amounts than full-length Aβ in senile plaques. The suggestion that AβpE3-x precedes the deposition of unmodified Aβ (Aβ1-x) was also proposed by the authors based on their analysis of brain tissue from Down syndrome cases [pE3 and other modified Aβ species accumulate unhindered [pE3-x peptides is due to the formation of the lactam ring and loss of two negative charges and one positive charge [pE3-x peptides exhibited enhanced β-sheet formation and aggregation propensity in aqueous and hydrophobic media. They proposed that a reduction of the level of unfavorable charge repulsion between strands, brought on by the loss of the three charged groups, facilitates and stabilizes β-sheet formation. Using immunoprecipitation in combination with mass spectrometry, Portelius and colleagues [1-40, Aβ1-42, pyroglutamate AβpE3-42 and Aβ4-42 can be detected in the hippocampus and cortex of AD patients. Interestingly, it has been demonstrated that N-terminal deletions enhance Aβ aggregation when comparing Aβ4-42 with Aβ1-42[Mori and colleagues discovered that approximately 15-20% of Aβ peptides carried a pyroglutamate residue at their N-terminus . This igme cases . Saido ehindered . The agge charge . The stae charge  reportedlleagues  showed tth Aβ1-42.The weak correlation between the severity of dementia and the density and localization of amyloid plaques in the brain of AD patients is one of the major flaws in the amyloid hypothesis. Even before the primary signs of plaque deposition, memory impairment and pathological changes already appear in many AD mouse models . Soluble4–42 is highly abundant in AD brains and was the first N-truncated peptide discovered [4–42 rapidly forms aggregates and possesses a high aggregation propensity [In vitro and in vivo exposure indicated that Aβ4-42 is as toxic as AβpE3-42 and Aβ1–42. In addition, we have generated transgenic mice expressing Aβ4-42 (Tg4-42 transgenic line) that developed a massive CA1 pyramidal neuron loss in the hippocampus [4–42 alone correlated with age-dependent spatial reference memory deficits [Although Aβscovered  its possopensity . In vitrpocampus . Interesdeficits . In the 4–40 NT4X-167; DSM ACC3162) was generated by immunizing three Balb/c mice with unconjugated Aβ4–40. After preparation of the lymph nodes they were fused with the myeloma cell line P3-X63-Ag8 for generation of the hybridoma cells. The hybridoma supernatants of mixed clones were screened by ELISA and immunohistochemistry and subcloned. The idea behind the generation of novel oligomeric antibodies was that in solution Aβ4–40 peptides are forming stable aggregates that can be used as an epitope for antibodies that specifically bind at the N-terminus of Aβ4–40. Therefore Aβ4–40 was used for immunizing mice and positive clones screened in four steps. After fusion, the hybridoma cells were screened by an enzyme-linked immune-absorbent assay (ELISA) for antibody production that (1) bind Aβ4–10 and (2) Aβ4–40, but (3) not Aβ36–40. Positive antibody clones were further screened by immunohistochemical staining of human brain sections. (4) The last step of the screening procedure was that they should not preferentially bind to amyloid plaques thereby identifying NT4X-167.The novel oligomeric Aβ specific antibody NT4X-167 , transferred to 0.45 μm nitrocellulose membranes  and detected using the primary antibodies IC16 (1 μg/ml), 1–57 (1 μg/ml) and NT4X-167 (1 μg/ml). Blots were developed using Luminata Crescendo Western HRP Substrate (Millipore) and exposed with the ODYSSEY Fc (LI-COR).For Western blotting under native conditions 4-16% SERVAGel N native gels (Serva) were used under blue native conditions. Running and transfer buffers were applied according to the manufacturer instructions. Nitrocellulose membranes  were detected using the primary antibodies IC16  (generous gift by Sascha Weggen ), 1–57  were prepared, sonicated for 5 min in water bath , quickly frozen in liquid nitrogen and stored at −80°C.1–16, 2–17, 3–18, 4–19, 5–20, 6–21, 7–22, 8–23, 9–24 and 10–25) were used. The peptides were coated in a 96 well plate overnight and reacted with NT4X-167 as primary antibody followed by incubation with horse radish peroxidase conjugated secondary antibodies (Dianova).100 ng 16 amino acid long Aβ peptides . Briefly, cells were washed twice with PBS and incubated protected from light for 30 min at room temperature in the presence of 2 μM calcein-AM solution prepared in PBS. Cells were then washed twice with PBS and incubated for 15 min at room temperature in PBS containing 1% Triton X-100 (v/v). The level of calcein fluorescence was monitored by fluorescence emission at 530 nm after exciting at 485 nm, using a Fluostar microplate reader .5XFAD mice express the 695 amino acids isoform of the human amyloid precursor protein (APP695) carrying the Swedish/London/Florida mutations under the control of the murine Thy1-promoter . In addiHuman brain samples were obtained from the Netherlands Brain Bank (NBB), the Institute of Neurology, Medical University of Vienna, Austria, Department of Pathology, University of Helsinki, Finland and Department of Pathology, University of Uppsala, Sweden were approved by the local Ethical Committees.2O2 in PBS to block endogenous peroxidases and antigen retrieval was achieved by boiling sections in 0.01 M citrate buffer pH 6.0, followed by 3 min incubation in 88% formic acid. Primary antibodies were incubated overnight, followed by incubation with biotinylated secondary rabbit-anti-mouse antibodies (DAKO) before staining was visualized using the ABC method with Vectastain kit (Vector Laboratories) and diaminobenzidine as chromogen. Primary antibodies used were IC16 , 1–57  and NT4X-167 .Human and mouse tissue samples were processed as described previously . In brieDifferences between groups were tested with one-way analysis of variance (ANOVA) followed by Bonferroni multiple comparison. All data are given as means ± standard error of the mean (SEM). All statistics were calculated using GraphPad Prism version 5.04 for Windows  and SPSS statistics version 17.0 .pE3-X and Aβ4-X variants, but not with Aβ1-X. In addition to monomers and dimers, trimers and tetramers of AβpE3-42 and Aβ4–42 were recognized. AβpE3-40 and Aβ4–40 produced primarily monomers and dimers. Antibody 1–57 stained only AβpE3-40 and AβpE3-42, but no other bands as previously demonstrated [1–40 and Aβ1–42, but not any of the N-truncated peptides.Freshly dissolved Aβ peptides were subjected to SDS-PAGE to dissect the binding specificity of the three tested antibodies Figure . Under dnstrated . IC16 repE3-40 and AβpE3-42. NT4X-167 reacted with all four N-truncated Aβ peptides AβpE3-40, AβpE3-42, Aβ4–40 and Aβ4–42. The different Aβ variants produced distinct bands corresponding to approximately: Aβ1–40 (20 and 30 kDa), Aβ1–42 , AβpE3-40 , AβpE3-42 . Interestingly, Aβ4–40 and Aβ4–42 elicited only one band at approximately 50 kDa. The distinct band sizes are only an approximation based on the migration of the protein ladder under native conditions.Under native conditions Figure , freshly2–4 with the highest signal for N-truncated Aβ4-x starting with phenylalanine at position four, as compared to IC16, which preferentially bound to positions 1–3 of Aβ. Mutational analysis of Aβ4–19 replacing phenylalanine with alanine (Aβ4A-19) or proline (Aβ4P-19) completely inhibited binding of NT4X-167 antibody. Therefore, phenylalanine at position four of Aβ is the essential amino acid required for NT4X-167 antibody binding.Pepscan assays Figure  were per4–42 and the staining was visualized using a Western blot under reducing conditions , 1–57 (against AβpE3-x), and IC16 (against Aβ1-x), hemizygous and homozygous 5XFAD mice were studied using cortical sections, as this is the brain area with known abundant intraneuronal Aβ in this model [1-x accumulation was observed in young , and muPS1Δ9; ,44 or SwPS1Δ9;  APP mutaIn order to study a potential cross-reactivity with other disease-typical aggregates, brain tissue sections were stained with disease-specific markers and compared with NT4X-167 reactivity. NT4X-167 did not cross-react with other aggregated deposits of non-AD neurodegenerative disorders. The following pathological structures showed no immunoreactivity Table : (i) PhoThe amyloid-β hypothesis has been the most influential hypothesis in coining the molecular pathology of AD . Accordi1–40/1–42 as the major culprit in AD pathology.Soluble oligomers are low molecular weight non-fibrillar structures, which are stable in aqueous solution and remain soluble even after high speed centrifugation. Aβ oligomers develop preferentially within neuronal processes and synapses rather than in the extracellular space ,28. At hOf note, full-length Aβ peptides are physiological molecules produced throughout the life of a human being. The generation of N-truncated Aβ peptides has been suggested to increase toxicity . Pike et4-40, Aβ4-42, Aβ1-42 and AβpE3-42 were unstructured in the monomeric state [pE3-42, Aβ1-42 and Aβ4-42. In addition, monomeric Aβ4-42 and AβpE3-42 were rapidly converted to soluble aggregated species. Both N-truncated variants exhibited similar biochemical properties, which opens the discussion which one of them might be more important in AD pathology [We have recently extended these observations and showed that soluble aggregates have specific features responsible for their neurotoxicity . Aβ4-40,ic state . Howeverathology .4-x and AβpE3-x. In combination with two other antibodies exclusively reacting with Aβ1-x (IC16) or AβpE3-x (1–57), we were able to show that Aβ4-x preceded AβpE3-x accumulation in the brain of 5XFAD transgenic mice. More importantly, Aβ4-x was detected together with Aβ1-x in the intraneuronal compartment of cortical neurons prone to degenerate in 5XFAD mice at 12 months of age [x-42 has also been described by Mori et al. [pE3-x, no intraneuronal staining was reported [pE3-x in 6 week-old 5XFAD mice in the present study.In the present report we endeavored to address this question. We succeeded to develop an antibody differentiating between full-length Aβ and the two other major N-truncated variants, Aβs of age ,40. Earls of age ,41,54-57i et al.  studyingreported  corroborin vitro toxicity assay, we were able to demonstrate that NT4X-167 is particularly protecting against Aβ4–42 and that the binding to AβpE3-42 has no therapeutic consequence. The mechanism(s) of the diverging biological effects are not clear. The Western blot analysis might not accurately reflect the difference in affinity of NT4X-167 between Aβ4–42 and AβpE3-42. The data from the in vitro toxicity assay provides evidence that NT4X-167 preferentially binds Aβ4–42. On the other side it could also be that NT4X-167 does not efficiently bind to some toxic aggregate(s) of AβpE3-42 as it did not significantly detect the aggregate at 50 kDa as compared to 1–57 antibody under native conditions. We have previously shown that passive immunization of 5XFAD mice with 9D5, a monoclonal antibody specifically detecting low molecular weight AβpE3-x aggregates, significantly reduced overall Aβ plaque load and AβpE3-x levels, and normalized behavioral deficits [Using an deficits .PS1∆9. These data are corroborated by a previous work by Kuo et al. [x-40 is a major component. The Aβ plaques in PS1Δ9 AD cases are characterized by cotton wool morphology composed by Aβx-42 aggregates. The lack of cotton wool plaque staining using NT4X-167 further strengthens the possibility that it may prefer binding to Aβ4-40 as compared to Aβ4-42 aggregates.While amyloid plaques were observed using NT4X-167 in 5XFAD transgenic mice, it barely reacted with plaques in the brain of sporadic AD patients and familial cases with the Arctic, Swedish and the presenilin-1 mutation o et al. . They ano et al. . Human Ao et al. . Moreoveo et al. . NT4X-16pE3-40 and Aβ4–40 generated monomers and dimers, while AβpE3-42 and Aβ4–42 in addition produced trimers and tetramers as previously shown [1–42 and AβpE3-42 appeared as aggregates of different sizes with higher molecular weight aggregates. In contrast Aβ4–40 and Aβ4–42 ran as a single band at approx. 50 kDa.Selkoe and others reported that toxic Aβ oligomers are primarily dimers and trimers of Aβ ,61,62. Hly shown ,59. Usinx-40 to Aβx-42) enhances its aggregation properties. Faster aggregation leads to earlier Aβ deposition, which is believed to promote its toxicity [1-43 was discovered as a novel toxic peptide in AD [1), several N-truncated and modified Aβ species have also been described [4–42 being one of them is particularly interesting as its discovery dates back to 1985 by Masters et al. [4-42 is a relatively abundant species in AD, aged controls and vascular dementia patients. Using immunoprecipitation in combination with mass spectrometry, Portelius and colleagues [1-40, Aβ1-42, AβpE3-42 and Aβ4-42 can be detected in the hippocampus and cortex of AD patients. Moreover, it has been demonstrated that N-terminal deletions enhance Aβ aggregation comparing Aβ4-42 with Aβ1-42[1-42 and AβpE3-42 exhibited similar effects on neuronal cytotoxicity in primary cortical neurons and on memory impairment after intracerebroventricular injection in wildtype mice. AβpE3-42 is now an established factor contributing to AD pathology [x-42 are likely to be promising, by inducing the production of specific antibodies against pathological Aβ products that are, in addition, involved in the early and basic mechanisms of amyloidosis in the human brain.In fact, analysis of amyloid deposits in AD brains revealed various N- and C-terminal variants ,17,18. Ttoxicity ,21,66. Rde in AD ,68. Besiescribed -16,69. As et al. . Lewis es et al.  reportedlleagues  showed tth Aβ1-42 showed tathology  and may athology . Sergeanathology . They co4–42 is elucidated by the generation of transgenic mice (Tg4-42) expressing Aβ4–42[The importance of position four of Aβ is corroborated by Haupt et al. , who obsng Aβ4–42. The Tg42-x[1-x producing N-terminal truncated Aβ3-x. AβpE3-x formation is catalyzed by glutaminyl cyclase [3-x and Aβ4-x is unknown.At present, the enzymes responsible for N-terminal truncation are not well studied. Aminopeptidase A contributes to the N-terminal truncation of Aβ peptide producing Aβ2-x. Saido e2-x suggeste cyclase -82. Whic4-x. Phenylalanine at position four of Aβ was imperative for NT4X-167 binding. In vitro toxicity experiments demonstrated that Aβ4–42 induced neuron death was significantly rescued by NT4X-167 treatment. No rescue effect was observed for Aβ1–42 or AβpE3-42 toxicity. NT4X-167 detected only a minor fraction of plaques in brain from sporadic and familial AD patients and 5XFAD transgenic mice. It preferentially reacted with intraneuronal Aβ in young 5XFAD mice. The finding that Aβ4-x precedes AβpE3-x in the well accepted 5XFAD AD mouse model further underlines the significance of Aβ4-x. NT4X-167 did not cross-react with aggregates typical for other major neurodegenerative disorders implicating that the recognized aggregates are specific for AD. Taking all observations together, NT4X-167 represents a novel tool for AD research and therapy.The present report describes the binding properties of the novel antibody NT4X-167, which recognizes the N-terminus of N-truncated Aβ. NT4X-167 bound most efficiently to AβA patent application for NT4X-167 was filed by the University Medicine of Goettingen and TAB.TAB is the PI of this study, conceived and designed the experiments and contributed to the interpretation of findings and writing of manuscript. GA and NS performed experiments and drafted the manuscript along with TAB. YB, BCR, TP, and OW performed experiments and contributed to revising the manuscript. AP, AVA, LL, MI, GK collected samples and characterized human disease samples used in the current study. All authors read and approved the final manuscript.4-42 for sensitivity testing of NT4X-167 using freshly dissolved peptides. NT4X-167 detects monomers and dimers of Aβ4-42 up to 0.03 μg peptide.SDS-PAGE Western blot analysis of AβClick here for file