OTULIN overexpression or knockdown affects NF-κB responses to LUBAC, TNFα, and poly(I:C) and sensitizes cells to TNFα-induced cell death. We show that OTULIN binds LUBAC and that overexpression of OTULIN prevents TNFα-induced NEMO association with ubiquitinated RIPK1. Our data suggest that OTULIN regulates Met1-polyUb signaling.The linear ubiquitin (Ub) chain assembly complex (LUBAC) is an E3 ligase that specifically assembles Met1-linked  Ub chains that regulate nuclear factor κB (NF-κB) signaling. Deubiquitinases (DUBs) are key regulators of Ub signaling, but a dedicated DUB for Met1 linkages has not been identified. Here, we reveal a previously unannotated human DUB, OTULIN , which is exquisitely specific for Met1 linkages. Crystal structures of the OTULIN catalytic domain in complex with diubiquitin reveal Met1-specific Ub-binding sites and a mechanism of substrate-assisted catalysis in which the proximal Ub activates the catalytic triad of the protease. Mutation of Ub Glu16 inhibits OTULIN activity by reducing   •FAM105B/OTULIN is a deubiquitinase exclusively hydrolyzing Met1-linked polyUb•The structure of an OTULIN-diUb complex reveals substrate-assisted catalysis•OTULIN binds LUBAC and regulates the abundance of Met1-linked chains in cells•OTULIN opposes LUBAC function in cytokine signaling OTULIN is the DUB that counteracts the recently identified linear ubiquitin chain assembly complex (LUBAC) signaling pathway and acts as a regulator of cytokine responses. Ubiquitination is an important posttranslational modification that regulates diverse processes, including protein degradation, intracellular trafficking, transcription, kinase activation, and the DNA damage response . This vaMet1-linked polyUb (Met1-polyUb) is the source of the cellular Ub pool, given that Ub is translated as a polyprotein  and postMuch less is known about DUBs that regulate Met1-polyUb chains, and a specific DUB for Met1-linkages has not been identified. Of the roughly 80 active DUBs in the human genome, many show weak or no activity toward Met1-linked chains . A potenOvarian tumor (OTU) domain DUBs regulate important cell-signaling pathways. A20 regulates NF-κB signaling , OTUD5 , and OTUHere, we identify a previously unannotated human DUB, FAM105B/OTULIN, which is specific for Met1-linked Ub chains. Structural studies reveal that this specificity is due to Met1-specific Ub-binding sites and a mechanism of substrate-assisted catalysis where a residue in a Met1-linked chain directly participates in the organization of the catalytic triad of the enzyme. Overexpression and knockdown analysis of OTULIN suggest that the protein binds LUBAC and regulates LUBAC-mediated processes in cells.76-Met1 linkage sequence between Ub moieties, given that mutant tetraUb with Ser76-Met1 linkages was not hydrolyzed  is sufficient for linkage specificity G, and OTZ score 8.7)  B. Intereore 8.7) C; e.g., ore 8.7)   A–S2C. Inore 8.7) C, and Cyore 8.7) . There wore 8.7) E and S2Fore 8.7) D. Consisore 8.7) E. Both m2 and involves the Ile44 patch that interacts with a helical arm (aa 254–264) conserved in all OTU domains  74-fold (because of a 3.5-fold lower KM for the mutant) in comparison to Met1-diUb . A polyclonal antiserum detected OTULIN in human embryonic kidney (HEK) 293ET and other cell lines  pulldown of NEMO precipitated HOIP, HOIL1, SHARPIN, and OTULIN, suggesting that these proteins might form a complex D and S5FMet1-linked polyUb is known to affect complex assembly at the TNF-RSC . IP of eprox bound to the NEMO UBAN domain with reduced affinity  against OTULIN resulted in an increase in LUBAC-induced NF-κB activation in HEK 293ET and U2OS cells and also in T-REx 293 cells stably expressing a doxycycline-inducible OTULIN-targeting microRNA (miRNA) A–S6C. LUStable OTULIN knockdown affected cytokine responses and led to enhanced NF-κB activation after poly(I:C) and TNFα E, and, eOTULIN knockdown cell lines were deregulated in their NF-κB response, but we could not observe a strong effect on canonical NF-κB signaling. Jurkat cells express higher amounts of OTULIN C, and OTHere, we identify FAM105B/OTULIN as a human OTU DUB specific for Met1-linked Ub chains. OTULIN is a missing piece in Met1-Ub chain biology, for which no linkage-specific DUB has been described to date. Our data suggest that OTULIN is an antagonist of LUBAC in vitro and in cells. We reveal the molecular basis for the remarkable Met1 linkage specificity by structural and biophysical studies and give insights into potential roles of OTULIN as a LUBAC interactor and antagonist in cells. Consistent with the biochemical findings, overexpression of OTULIN removes Met1-polyUb and dampens LUBAC-mediated NF-κB signaling, whereas knockdown of OTULIN leads to increased Met1-polyUb, IKK activation, and NF-κB signaling after TNFα stimulation. However, both overexpression and knockdown of OTULIN sensitized cells to TNFα-induced cell death, suggesting more complex roles for Met1 linkages in balancing signaling pathways downstream of the TNFα receptor.Linkage specificity in DUBs is not well understood, and the structure of apo OTULIN did not explain its Met1 specificity, because of its structural similarity to the Lys48-specific OTUB1. The key insight into linkage specificity for these two enzymes came from the complex structures of the enzyme with two Ub molecules bound across the active site.A high-affinity Ub-binding site in OTULIN allows it to select Met1-linked chains with 100-fold preference over Lys63 linkages and disallows the binding of Lys48-linked and presumably other chains. This mechanism is shared by many DUBs; e.g., TRABID  and OTUBUb-mediated substrate-assisted catalysis was recently shown to be crucial for the assembly of Lys11-linked polyUb, where Ub Glu34 complements the active site of the E2 Ub-conjugating enzyme UBE2S . The facIn transfection experiments, we demonstrated that OTULIN could counteract cellular LUBAC responses, suggesting that OTULIN helps to balance the amount of Met1-polyUb in cells. Furthermore, the mechanism of substrate-assisted catalysis suggests that OTULIN may not target individual proteins but, instead, target Met1-linked Ub chains, regardless of where they are attached. Indeed, we identified three proteins that change in their ubiquitination status when OTULIN levels are modulated. NEMO and RIPK1 are among the few reported targets of LUBAC .We provide evidence that OTULIN directly interacts with LUBAC. The observation that depletion of OTULIN leads to the modification of HOIP with Met1-polyUb suggests that HOIP, like many other E3 ligases, undergoes autoubiquitination and that OTULIN has the ability to prevent this. This most likely explains why the overexpression of OTULIN chain-binding mutants lead to increased Met1-polyUb in cells A. MoreovRegarding the functional requirement for Met1-linked chains in NF-κB responses, key questions remain to be answered. First, in contrast to Lys63-linked chains, for which roles in NF-κB signaling have long been verified, for example, by elegant replacement strategies , Met1-licpdm mice lacking SHARPIN , expressed in E. coli from pOPIN-F vector, and purified by anion exchange and gel filtration.2, 30 mM CaCl2, 10% (w/v) PEG 4k, and 20% glycerol (pH 6.5). The structure of apo OTULIN was determined by single anomalous dispersion, and subsequent structures were determined by molecular replacement.Apo, SeMet-substituted, and mutant OTULIN were crystalized from 100 mM MES and imidazole, 30 mM MgClQualitative deubiquitination assays were performed as previously described .Binding studies were performed as in FAM105B were generated according to the manufacturer’s protocol (Invitrogen). Knockdown analysis was performed in miRNA cell lines or with eight different siRNA sequences, as listed in the OTULIN and A20 were expressed from pOPIN-F or pcDNA4/TO/MRGS6H, HOIP, SHARPIN and HOIL-1L from pcDNA3.1, and GST-tagged NEMO were expressed from pEBG vectors. Stable T-REx 293 cell lines overexpressing pcDNA4/TO/MRGS6H-OTULIN or a pDEST30-EmGFP construct with an miRNA targeting the 3′ untranslated region of Renilla (Promega) luciferase plasmids by immunofluorescence with anti-p65 staining or by quantitative PCR, as described in the NF-κB activity was assessed by luciferase assays, for which cells were cotransfected with M3P sin rev κB firefly and pRL-TK Extended Experimental Proceduresp-values < 0.01 were included in the next iteration cycle. The significance of novel OTU assignments was further assessed by HMM-HMM comparison using the HHSEARCH program , Sigma and Eurogentech.The pRL-TK Renilla (Invitrogen) and the M3P sin rev κB firefly luciferase (coding for 4 NF-κB binding sites in the firefly luciferase promoter) plasmids were a kind gift of Felix Randow (MRC LMB), pcDNA3-HOIP1-/HOIL1-/Sharpin-V5-His vectors were kindly provided by Henning Walczak . pcDNA4/TO/N-MRGS6H  was a kind gift of Gerrit J.K. Praefcke (University of Cologne). pEBG-NEMO FL was a kind gift from Sally Swift (ICR London).The coding sequence for full-length Fam105B/OTULIN was obtained following PCR amplification of a Human Adult Normal Brain cDNA library (Invitrogen) using KOD HotStart DNA polymerase (Novagen) and the following primers: OTULIN fwd 5’- AAGTTCTGTTTCAGGGCCCGATGAGTCGGGGGACTATGCCCC and OTULIN rev 5’-ATGGTCTAGAAAGCTTTATAGACTGGTCTCCTCACACA CTCTG. The PCR product was cloned into pOPINF and pOPIN-GFP  using InE. coli strain Rosetta2 (DE3) pLacI. Cells were grown at 30°C in 2xTY containing 50 μg/ml ampicillin, 34 μg/ml chloramphenicol to an OD600 of 0.7. The culture was cooled to 20°C prior to induction with 150-400 μM IPTG and harvested 20 hr postinduction.OTULIN was expressed in 2+ resin (GE Life Sciences). The His6-tag was removed by overnight incubation with 3C Protease. For biochemical studies, OTULIN was subjected to size exclusion chromatography (SEC)  in buffer A . For crystallographic analysis, OTULINcat was dialyzed into 20 mM Tris, 5 mM DTT (pH 8.5) and subject to anion exchange chromatography , prior to SEC in buffer A. Purified proteins were concentrated and flash frozen.Cells were resuspended and lysed by sonication in lysis buffer , protease inhibitor cocktail (Roche)). OTULIN was purified by immobilized metal-affinity chromatography using NiQualitative deubiquitination assays were performed as previously described . BrieflyUb thioester was generated following Ub-intein cleavage as described previously . HaloalkFor monitoring Ub-probe reactivity, vOTU was used as a positive control, which displays strong reactivity toward different Ub-probes . Enzymes2, 30 mM CaCl2, 10% (w/v) PEG 4k, 20% (v/v) glycerol, pH 6.5). For SeMet-substituted crystals, native crystals were used to seed hanging drops containing SeMet-substituted OTULINcat. For Met1-diUb complex crystals, OTULINcat C129A was mixed with Met1-diUb in a 1:1.2 molar ratio, and set up at a concentration of 13 mg/ml. Crystals grew from 100 mM Bis-Tris, 2 M (NH4)2SO4, pH 6.5. Prior to data collection WT, D336A and SeMet-OTULINcat crystals were harvested and vitrified in liquid nitrogen. OTULINcat C129A-Met1-diUb crystals were soaked in 2.5 M Li2SO4 prior to vitrification.Initial hits of all crystals were obtained by sitting-drop vapor diffusion method. Native (15 mg/ml), selenomethionine (SeMet)-substituted (0.1 mg/ml) and D336A (12 mg/ml) OTULINcat crystals were grown from drops containing an equal volume of protein and reservoir . Data collection and refinement statistics can be found in Diffraction data were collected at the European Synchrotron Radiation Facility (ESRF), beam lines ID14-4 and ID23-1, and at Diamond Light Source, beam line I04-1. Diffraction images were processed and integrated using iMOSFLM  and scalUb constructs (mono- and Met1-diUb) were expressed and purified as described . In the Ub-AMC (Boston Biochem) was diluted in reaction buffer . For each reaction 10 μl of diluted substrate in a black 384-well low volume plate (Corning) was mixed with 10 μl of either: 10 pM UCH-L3  or 500 nAnalytical SEC analysis was performed on an AKTA Micro system (GE Life Sciences) using a Superdex 75 PC 3.2/30 column equilibrated in SEC buffer . Inactive OTULIN 80-352 C129A (OTULINcat C129A) was mixed with Met1-diUb variants in a 1:1.3 molar ratio (33.5 μM: 43 μM) and incubated at room temperature for 30 min. 125 μg of complex was loaded onto the column. Fractions containing protein were mixed with SDS loading buffer prior to SDS-PAGE analysis.λex = 485 nm and λem = 520 nm. Fluorescence polarization values were fitted to a one-site binding model using Graphpad Prism 5 to derive binding constants (KD).To measure binding affinities of OTULIN C129A variants to Met1-diUb variants, 10 μl of 100 nM FlAsH-tagged Met1-diUb was aliquoted into a 384 -well low volume plate (Corning). Serial dilutions in FlAsH-buffer (20 mM Tris (pH 7.4), 100 mM NaCl, 5 mM β-mercaptoethanol, 0.1 mg/ml bovine serum albumin) were prepared of OTULIN C129A variants and 10 μl of this was added to FlAsH-tagged Met1-diUb containing wells. Fluorescence polarization was recorded on a PheraStar plate reader (BMG Labtech) using an optics module with λex = 485 nm and λem = 520 nm. Met1-diUb variants were serially diluted into FlAsH-buffer and contained a fixed concentration of 300 nM FlAsH-tagged Met1-diUb, to detect changes in fluorescence anisotropy. To each well, 10 μl of substrate was mixed with 10 μl OTULIN variants in FlAsH-buffer. The change of fluorescence anisotropy was recorded over a period of 5 min. The observed fluorescence polarization values were converted to percentage of substrate cleavage by comparing to baseline values from intact substrate and FlAsH-tagged monoUb that were acquired for each experiment. All measurements were corrected by subtracting changes in fluorescent anisotropy for FlAsH-tagged Met1-diUb alone. Triplicate recordings were made for each substrate concentration. Kinetic analysis was performed in Graphpad Prism 5. Initial rates of the enzymatic reaction were calculated and plotted against substrate concentration at a fixed OTULIN concentration, allowing determination of Michaelis-Menten parameters ) and the lysate was incubated with Glutathione Sepharose 4B (GE Life Sciences). Bound protein was cleaved following overnight incubation PreScission protease. Cleaved protein was subjected to SEC  in 200 mM NaCl, 10 mM DTT, 25 mM Tris pH 8.5.The RBR and C-terminal region of HOIP   was clon2, 0.6 mM DTT). A sample was taken before addition of 10 mM ATP (t = 0 min). The reaction was incubated at 37°C for 60 min before another sample was removed. Samples were analyzed by SDS-PAGE and stained using SilverStain Plus kit (BioRad).The ligation assay was performed by mixing 100 nM E1, 2.2 μM UBE2L3, 2.5 μM HOIP 699-1072 and 0.25 mg/ml Ub or Ub E16A in reaction buffer  were grown at similar conditions in DMEM + GlutaMAX-I (GIBCO), 10% (v/v) Tet-approved FBS (Clontech), 100 U/ml penicillin, 100 μg/ml streptomycin, 5 μg/mL blasticidin (InvivoGen).HEK 293ET, HeLa, U2OS, HCT116 and MCF7 cells were cultured in DMEM + GlutaMAX-I (GIBCO), RKO and Jurkat cells in RPMI (GIBCO) both supplemented with 10% (v/v) FBS , 100 U/ml penicillin,100 μg/mL streptomycin at 37°C and 5% COEndogenous OTULIN was immunoprecipitated from HEK 293ET cells using OTULIN-specific antibodies coupled to Protein A sepharose (Roche) via Dimethylpimelimidate (Sigma). Cells were lysed in PBS, 0.1% (v/v) Triton X-100, COMPLETE protease inhibitor cocktail (Roche), 2 mM NEM and lysates were incubated overnight with Protein A-coupled OTULIN antibodies. Beads were washed five times with PBS, 0.01% (v/v) Triton X-100, COMPLETE protease inhibitor cocktail (Roche), 2 mM NEM and eluted in PBS/0.5% SDS pH 9.0 or by boiling in 2x SDS sample buffer.2, 1 mM EGTA, 0.5% (v/v) Triton X-100) supplemented with 5 mM N-Ethylmaleimide , COMPLETE protease inhibitor cocktail (Roche) and PhosSTOP (Roche) for 30 min on ice. Lysates were cleared by centrifugation and were incubated at 4°C overnight with anti-iKK γ-coupled beads (Santa Cruz Biotechnology). Beads were washed four times in 500 μl ice-cold IP buffer and bound material eluted with 0.2 M glycine, pH 2.5.HeLa cells were transfected and treated as indicated. Cells were lysed in IP buffer  NP-40, 2 mM EDTA) supplemented with 1 mM DTT, 5 mM NEM, COMPLETE protease inhibitor cocktail and PhosSTOP. Lysates were divided in two and 50 μg TUBE1 or 100 μg M1-SUB was added. Lysates were cleared, mixed with Glutathione Sepharose 4B beads  and incubated at 4°C for a minimum of 2 hr with rotation. Beads were washed four times in 500 μl ice-cold PBS Tween-20 (0.1% (v/v)). Bound material was eluted with 1xSDS sample buffer.Ubiquitin conjugates from cell lysates were pulled down in U2OS FlpIn-T-REx cells using affinity reagents. For isolation of Met1-Ub chains, recombinant protein containing one copy of the UBAN domain from human NEMO (residues 257-346) fused to Glutatione-S-transferase (GST) was used (M1-SUB). TUBE1, consisting of four UBA domains in tandem fused to GST, was used to purify all ubiquitin chains. One confluent 10 cm dish per condition was lysed in 600 μl Lysis buffer (20 mM Na7 HEK 293ET cells were transiently transfected with pOPIN-GFP-OTULIN WT or C129A for 24 hr using GeneJuice (Novagen). Cells were lysed in 200 μl GFP lysis buffer  NP40, COMPLETE protease inhibitor cocktail (Roche)) for 30 min on ice. Diluted lysates were incubated with 120 μl equilibrated Magnetic GFP-Trap beads (Chromotek) for 16 hr at 4°C and washed following the manufacturer’s protocol. Beads were then washed twice in DUB dilution buffer  and specificity assays were performed as described above using approximately 500 nM GFP-OTULIN bound to GFP-Trap beads.2 × 10T-REx293 cells were stably transfected with pcDNA/TO/N-MRGS6H-OTULIN using Gene Juice (Novagen). Selection was carried out with 150 μg/ml zeocin (InvivoGen) and individual colonies were sub-cloned, expanded and screened for OTULIN expression. OTULIN expression was induced with 1 μg/ml doxycycline for 24 hr.For stable cell lines inducibly expressing nontargeting or OTULIN targeting miRNA, the respective sequences were cloned into the mammalian pT-REx-DEST30 plasmid following the BLOCK-IT Pol II miR RNAi Expression Vector Kit with EmGFP protocol (Invitrogen). The following sequences were used: OTULIN miRNA top strand 5′-TGCTGTGCTGTTGAATCCAGACCCAAGTTTTGGCCACTGACTGACTTGGGTCTATTCAACAGCA-3′, OTULIN miRNA bottom strand 5′-CCTGTGCTGTTGAATAGACCCAAGTCAGTCAGTGGCCAAAACTTGGGTCTGGATTCAACAGCAC-3′. A nontargeting control miRNA sequence was used according to the manufacturer’s protocol. After stable transfection, selection was carried out with 500 μg/ml G418 (PAA) and individual clones were screened for GFP expression and OTULIN downregulation. OTULIN knock down was induced by 1 μg/ml doxycycline for 60-72 hr.For transient knockdown studies, 20 nM siRNA duplexes against OTULIN or HOIP were transfected into HEK 293ET or U2OS cells using INTERFERin siRNA transfection reagent (Polyplus transfection) or Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s instructions and analyzed 48 hr posttransfection. For RNA interference in Jurkat T cells, 100 nM siRNA was transfected with Atufect transfection reagent (0.5–1 mg/ml) (Silence Therapeutics) and analyzed after 72 hr.Human Fam105B/OTULIN ON-TARGETplus SMARTpool  contained the following siRNAs: GCUUAACUGUCUCGGGAAA, GGGCAUCAGAACCGAGAUU, UAGCAAAGGCAGGGCGCAA, CUUUAGUAGUAACGGGUUU. HOIP1/RNF31 ON-TARGETplus SMARTpool contained the following siRNAs: GCAGAAUACUCAUCCAAGA,CCUAGAACCUGAUCUUGCA, GGCGUGGUGUCAAGUUUAA, GCCGAGAUGUGCUGCGAUU. The ON-TARGETplus Nontargeting siRNA #1 (Dharmacon/Thermo Scientific) or sequence GGGAUACCUAGACGUUCUA served as a nontargeting control. Single siRNAs  had the following sequences: #1: GACUGAAAUUUGAUGGGAA, #2: CAAAUGAGGCGGAGGAAUA, #3: ACAGAUAGCUUGUGAUGAA, #4: GCAUCAGAACCGAGAUUAA. Sequence #4 partly overlaps with one of the SP sequences.Cells were transiently transfected with the M3P sin rev κB firefly and pRL-TK Renilla (Promega) luciferase plasmids, and with plasmids encoding indicated proteins . Cells were lysed in Passive Lysis Buffer (Promega) 15 or 24 hr posttransfection and luminescence was measured using a microplate reader (Berthold Detection Systems). For knockdown studies, cells were transfected with siRNAs (see above) 48 hr before lysis. After 24 hr, luciferase plasmids were transfected either alone or together with LUBAC; luciferase activity was assessed as described above. TNFα or poly(I:C) stimulation was carried out using the indicated concentrations for 6 hr or 20 hr, respectively.HeLa cells were grown on Glass CultureSlides (BD Biosciences) and transfected with pcDNA4/TO/N-MRGS6H-OTULIN WT, C129A, W96A or L259E 24 hr prior to stimulation with 20 ng/ml TNFα for 30 min. Stimulation was stopped by adding ice-cold PBS. Cells were fixed in PBS, 4% paraformaldehyde (w/v), permeabilized in PBS, 0.3% (w/v) Saponin and blocked with PBS, 0.3% (w/v) Saponin, 4% (w/v) BSA. Primary and Alexa labeled secondary antibodies (Invitrogen) were applied in blocking buffer. Slides were embedded in Mounting Medium with DAPI (VECTOR laboratories) and data were obtained using a LSM 710 on the inverse Axio Observer AX10 microscope and the ZEN2009 software (Zeiss).GST-NEMO WT or K285/309R (KR) was transfected with or without LUBAC into Doxycycline induced control cells or OTULIN WT overexpressing cells. 24 hr later, cells were lysed in PBS, 0.1% (v/v) Triton X-100, COMPLETE protease inhibitor cocktail (Roche), 2 mM NEM and 2.5 mg of total protein for each sample was incubated with 50 μl GSH-coupled sepharose beads 4B  overnight (o/n). Beads were washed five times with PBS, 0.01% Triton X-100 (v/v), COMPLETE protease inhibitor cocktail (Roche), 2 mM NEM and proteins were eluted by boiling in 2x SDS sample buffer. Samples and inputs were subjected to SDS-PAGE and western blotting, and detection was carried out using indicated antibodies.Stable cell lines downregulating or overexpressing OTULIN and their respective control cell lines were induced for expression with 1 μg/ml doxycycline for 72 hr (miRNA) or 24 hr (overexpression), respectively. Finally, cells were stimulated with 10 ng/ml TNFα for the indicated times. Signaling was stopped by adding ice-cold PBS. Cellular mRNA was isolated using the RNeasy Mini Kit, QIAshredder and the RNase-Free DNase Set . 1 μg mRNA was then reverse transcribed  and cDNA quantification was measured by real-time PCR using the QuantiFast SYBR Green PCR Kit (QIAGEN) in a RotorGene 6000 (Corbett Research). Data were analyzed with the RotorGene 6000 software and visualized in Excel (Microsoft). All samples were normalized to their respective GAPDH levels.2, 0.5 mM EDTA, 0.1 EGTA, 1% (v/v) Nonidet P-40, 0.5 M NaF, 1 M DTT, 1 M β-glycerophosphate, 200 mM Na vanadate, and 50x COMPLETE protease inhibitor (Roche)) according to standard protocols. Electrophoretic mobility shift assays (EMSA) were performed by using a 32P-dATP–labeled, double-stranded NF-κB oligonucleotide probe (5′-CAGGGCTGGGGATTCCCCATCTCCACAGG-3′). The samples were separated on native polyacrylamide gels prior to autoradiography.For electrophoretic mobility shift assay (EMSA), cells were lysed in whole cell lysis buffer  glycerol, 1 mM MgClStable inducible cell lines overexpressing or downregulating OTULIN and their respective control cell lines were induced for expression with 1 μg/ml doxycycline. After 24 hr (overexpression) or 72 hr (miRNA downregulation), on day 0, cells were stimulated or not with 50 ng/ml TNFα for 24 hr in doxycycline containing media. On day 1, for samples of day 2-4, media was exchanged to doxycycline containing media without TNFα. Thereby, detached cells were collected and re-plated into their respective wells. For all samples, attached and detached cells were collected after the indicated days and counted using a Vi-Cell XR cell viability analyzer (Beckman Coulter). Cells were also stained with crystal violet to visualize the cell counting results. The staining procedure included fixing in PBS, 4% (v/v) paraformaldehyde for 5 min and staining in 0.05% Crystal Violet (Sigma) for 30 min.To analyze TNFα-induced NF-κB signaling, stable cell lines downregulating or overexpressing OTULIN and their respective control cell lines were induced for expression with 1 μg/ml doxycycline for 72 hr (miRNA) and 24 hr (overexpression), respectively. Finally, cells were stimulated with 10 (miRNA) or 100 ng/ml TNFα (overexpression) for the indicated times. Addition of ice-cold PBS stopped signaling. Cells were lysed in ice-cold lysis buffer , protease inhibitor cocktail (Roche), 2 mM NEM, PhosphoSTOP (Roche)) for 30 min on ice. Lysate protein concentration was measured by Bradford assay, and equal protein amounts were subjected to SDS-PAGE and western blotting. Analysis was carried out with the indicated antibodies.TNF-RSC was purified from the indicated cell lines after stimulation with 100 ng/ml Flag-TNFα  for the indicated times. Ice-cold PBS was added to the plate to stop stimulation. Following lysis, TNF-RSC was purified by incubation with Flag M2 agarose beads (Sigma). TNFR was purified from the unstimulated sample by adding 1 μg of TNFα during lysis. The purified TNF-RSC was analyzed by western blotting for linear ubiquitination, HOIP and TNFR1.2, 20 μM ATP, 20 mM β-glycerophosphate, 50 μM Na vanadate, 1 mM DTT) in the presence of GST-IκBα (1-53) for 25 min at 37°C. After boiling in loading buffer, the kinase reactions were separated on SDS-PAGE and analyzed by autoradiography.For Kinase assays, Jurkat T cells were lysed in 900 μl co-IP buffer  NP-40, 10% (v/v) glycerol, 1 mM DTT, 10 mM sodium fluoride, 8 mM β-glycerophosphate, 300 μM sodium vanadate and protease inhibitor cocktail). After immunoprecipitation with NEMO antibody , pellets were washed and incubated in kinase assay buffer (20 mM HEPES pH 7.5, 10 mM MgCl


II atom in the title compound, [CuCl2(C16H15N3O)]·H2O, is N,N′,O-chelated by the neutral Schiff base ligand and exists in a square-pyramidal geometry. It is displaced by 0.316 (1) Å out of the square plane (r.m.s. deviation = 0.015 Å) in the direction of the apical Cl atom. The apical Cl atoms of adjacent complex units are hydrogen-bond acceptors to two water molecules, the interaction generating a centrosymmetric dimer through a cyclic R42(8) association.The Cu DOI: 10.1107/S1600536812035398/zs2227Isup2.hklStructure factors: contains datablock(s) I. DOI:  crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:


A two-dimensional polymeric framework with  topology can be observed in the ac plane, the nodes being provided by BaII ions and the connectors being N and O atoms belonging to pyrazine-2-carboxylate ligands and O atoms of bridging water molecules. Non-coordinating pyrazine-2-carboxylate ions are located between the polymeric layers in the crystal and are interconnected through extensive O—H⋯N,O hydrogen bonding. In the layered title coordination polymer, {[Ba II and SrII complexes with pyrazine-2-carboxylate as ligand, see: Ptasiewicz-Bak et al. 2(H2O)7](C5H3N2O2)2                         = 0.046                           wR(F                           2) = 0.121                           S = 1.02                           4734 reflections406 parameters1 restraintH-atom parameters constrainedmax = 1.84 e Å−3                        Δρmin = −2.62 e Å−3                        ΔρAbsolute structure: Flack 1983, 2096 FrFlack parameter: 0.04 (4)                                 SMART  used to solve structure: SHELXS97 (Sheldrick, 2008SHELXL97 (Sheldrick, 2008SHELXTL (Sheldrick, 2008SHELXTL.Data collection: 10.1107/S1600536811003023/bh2331sup1.cif            Crystal structure: contains datablocks global, I. DOI: 10.1107/S1600536811003023/bh2331Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


We evaluated the effect of mometasone furoate/formoterol (MF/F) combination therapy on SABA use with data from 3 phase III studies.An important asthma therapy goal is to limit short-acting βAll studies enrolled subjects (≥12y) with persistent asthma not well controlled on inhaled corticosteroids (ICS): study 1 ; study 2 ; study 3 . All studies included a 2-3wk MF BID run-in: P04073 (100µg), P04334(200µg), P04431(400µg). Percentage of SABA-free days/nights was a predefined secondary endpoint in all studies.P≤.003), and numerically increased for MF/F-100/10µg vs MF-100µg (14%). In study 2, change in SABA-free days/nights was significantly increased for MF/F-200/10µg (21%) vs MF-200µg (15%), F-10µg (11%), and placebo . In study 3, change in SABA-free days/nights was significantly increased for MF/F-400/10µg and MF/F-200/10µg groups (25% increase for each) vs the MF-400µg group .In study 1, mean change in the percentage of SABA-free days/ nights from baseline period (day -7 to 1) to overall treatment period was significantly increased for MF/F-100/10µg (18%) vs F-10µg (10%) and placebo (0% MF/F treatment resulted in significant improvement in the frequency of SABA-free days/nights vs individual MF and/or F monotherapies in subjects with persistent asthma not well controlled on ICS therapy.


II ion in the title complex, [PdI2(C14H10N4)], is four-coordinated in a slightly distorted square-planar environment by the two pyridine N atoms of the chelating 2,3-di-2-pyridylpyrazine (dpp) ligand and two iodide anions. In the crystal, the pyridine rings are considerably inclined to the least-squares plane of the PdI2N2 unit [maximum deviation = 0.090 (2) Å], making dihedral angles of 65.0 (2) and 66.6 (2)°. The pyrazine ring is perpendicular to the unit plane, with a dihedral angle of 89.9 (2)°. The complex molecules are connected by C—H⋯I hydrogen bonds, forming a helical chain along the b axis.The Pd DOI: 10.1107/S1600536811044023/is2795Isup2.hkl            Structure factors: contains datablock(s) I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


The CoII ion is six-coordinated in a slightly distorted octahedral environment resulting from two carboxylate O atoms of two 2-carboxymethylsulfanyl nicotinate (2-CMSN2−) anions, one water molecule and three N atoms of three 4,4′-bipyridine ligands, with one 4,4′-bipyridine ligand situated on a centre of inversion. Two neighboring CoII ions are linked by two anions, giving a dinuclear [Co2(2-CMSN)2] subunit with a Co⋯Co separation of 6.8600 (3) Å. The dinuclear subunits are joined by bridging 4,4′-bipyridine linkers, generating a three-dimensional network structure. Disordered water molecules are situated in the free space of this network. O—H⋯O hydrogen bonding within and between the subunits enhances the stability of the structure.The title complex, [Co DOI: 10.1107/S1600536813015262/wm2744Isup2.hklStructure factors: contains datablock(s) I. DOI:  crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:


It belongs to a group of compounds characterized by the general formula BaM2+2Si2O7, with M2+ = Be, Mg, Fe, Mn, Zn, Co, or Cu, among which the Be-, Fe-, and Cu-members have been found in nature. The crystal structure of clinobarylite has been re-examined in this study based on single-crystal X-ray diffraction data collected from a natural sample from the type locality . The structure of clinobarylite can be considered as a framework of BeO4 and SiO4 tetrahedra, with one of the O atoms coordinated to two Be and one Si, one coordinated to two Si, and two O atoms coordinated to one Si and one Be atom. The BeO4 tetrahedra share corners, forming chains parallel to the c axis, which are interlinked by the Si2O7 units oriented parallel to the a axis. The Ba2+ cations (site symmetry m..) are in the framework channels and are coordinated by eleven O atoms in form of an irregular polyhedron. The Si—Obr  bond length, the Si—Onbr (non-bridging O atoms) bond lengths, and the Si—O—Si angle within the Si2O7 unit are in marked contrast to the corresponding values determined in the previous study [Krivovichev et al. (2004N. Jb. Miner. Mh. pp. 373–384].Clinobarylite, ideally BaBe al. 2004. N. Jb.  Åb = 4.9175 (2) Åc = 4.6746 (2) ÅV = 267.78 (2) Å3Z = 2Kα radiationMo −1μ = 7.85 mmT = 293 K0.05 × 0.05 × 0.04 mmBruker APEXII CCD area-detector diffractometerSADABS; Sheldrick, 2005Tmin = 0.695, Tmax = 0.744Absorption correction: multi-scan (3929 measured reflections965 independent reflectionsI > 2σ(I)947 reflections with Rint = 0.020R[F2 > 2σ(F2)] = 0.011wR(F2) = 0.026S = 1.08965 reflections60 parameters1 restraintmax = 0.47 e Å−3Δρmin = −0.42 e Å−3ΔρAbsolute structure: Flack 1983, 399 FriFlack parameter: 0.502 (12)APEX2  used to solve structure: SHELXS97  I, New_Global_Publ_Block. DOI: 10.1107/S1600536812040457/wm2678Isup2.hklStructure factors: contains datablock(s) I. DOI:  crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:


The structure contains a four-coordinate Cd2+ ion in a distorted tetrahedral geometry, which lies on a crystallographic twofold rotation axis. The Cd2+ ion is bonded to two N atoms from two pyridine ligands and two carboxylate O atoms from two 5-amino-2,4,6-triiodoisophthalate anions. The Cd—O distances are 2.429 (5) and 2.305 (5) Å and the Cd—N distance is 2.236 (8) Å. The two carboxylate groups of individual 5-amino-2,4,6-triiodoisophthalate anions act as a bridge to the Cd2+ atoms. leading to a chain structure along the c axis.The hydrothermal reaction of cadmium(II) nitrate with 5-amino-2,4,6-triiodoisophthalic acid and pyridine in DMF solution leads to the formation of the title compound, [Cd(C DOI: 10.1107/S1600536810039498/si2296Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


The 9,10-dioxo-9,10-dihydroanthracene-1,4,5,8-tetracarboxylate anion is centrosymmetric with the centroid of the quinone ring located about an inversion center; the 4,4′-bipyridine ligand is also centrosymmetric with the mid-point of the C—C bond linking two pyridine rings located about another invertion center. The 9,10-dioxo-9,10-dihydroanthracene-1,4,5,8-tetracarboxylate anion and bypiridine ligand bridge the NiII cations, forming a polymeric chain along the b axis. π–π stacking is observed between pyridine and benzene rings [centroid–centroid distance = 3.705 (2) Å]. All of the coordinating water molecules are involved in O—H⋯O hydrogen bonding.In the crystal of the title polymeric complex, [Ni DOI: 10.1107/S1600536812027493/xu5560Isup2.hklStructure factors: contains datablock(s) I. DOI:  crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:


PSEN mutations alter APP processing in a manner that increases the relative abundance of longer Aβ42 peptides to that of Aβ40 peptides. The mechanisms by which Aβ40 and Aβ42 peptides are produced in a ratio of ten to one by wild type presenilin (PS) and by which Aβ42 is overproduced by FAD-linked PS variants are not completely understood. We generated chimeras of the amyloid precursor protein C-terminal fragment (C99) and PS to address this issue. We found a chimeric protein where C99 is fused to the PS1 N-terminus undergoes in cis processing to produce Aβ and that a fusion protein harboring FAD-linked PS1 mutations overproduced Aβ42. To change the molecular interactions within the C99-PS1 fusion protein, we made sequential deletions of the junction between C99 and PS1. We found differential effects of deletion in C99-PS1 on Aβ40 and 42 production. Deletion of the junction between APP CTF and PS1 in the fusion protein decreased Aβ40, while it did not decrease Aβ42 production in the presence or absence of FAD-linked PS1 mutation. These results are consistent with the idea that the APP/PS interaction is differentially regulated during Aβ40 and 42 production.Beta amyloid peptides (Aβ) play a key role in the pathogenesis of Alzheimer disease (AD). Presenilins (PS) function as the catalytic subunits of γ-secretase, the enzyme that releases Aβ from ectodomain cleaved amyloid precursor protein (APP) by intramembrane proteolysis. Familial Alzheimer disease (FAD)-linked  Alzheimer disease (AD) is a neurodegenerative disease characterized by the presence of senile plaques, of which beta amyloid peptide (Aβ) is the primary component In the present study, we examined the mechanism by which Aβ40 and Aβ42 are produced. To address this issue, we generated a series of constructs encoding chimeric proteins where APP CTF is fused to the N-terminus of PS, taking advantage of previous findings that deletion of the N-terminal domain of PS does not affect γ-secretase activity 5′-ACCCAAGCTTCACAGCTAGCGCACTCGGTG-3′), which is complementary to a sequence encoding upstream of the signal sequence at the N-terminus of C99 and includes a NheI site, and oligonucleotide C99-PS1-1r (5′-GTGGTTGTCCTCAGACTGCTCAAAGAACTTG-3′), which encodes the C-terminus of C99 without the last three amino acids of C99 and stop codon and the N-terminus of PS1 without the first 16 amino acids. The first PCR-2 was performed with PS1 cDNA as a template and the primers, oligonucleotide C99-PS1-2f (5′-AAGTTCTTTGAGCAGTCTGAGGACAACCAC-3′), which is complementary to oligonucleotide C99-PS1-1r, and oligonucleotide C99-PS1-2r (5′-GGACCTTTCCAGTGAATGGAAATCATTCCC-3′), which includes a PflmI site of PS1 cDNA. The second PCR was performed with the primers, C99-PS1-1f and C99-PS1-2r, and a mixture of the PCR products of the first PCR-1 and first PCR-2 as templates. C99-PS1D385A, C99-PS1G266S, C99-PS1R278I and C99-PS1L435H were generated using a Quick-change site directed mutagenesis kit . C99-PS2 was generated by a PCR-based method as C99-PS1. Primers for the first PCR-1 were: C99-PS2-1f, 5′-ACCCAAGCTTCACAGCTAGCGCACTCGGTG, and C99-PS2-1r, 5′-CATTAGGGACGTCCGCTGCTCAAAGAACTTG. Primers for the first PCR-2 were: C99-PS2-2f, 5′-CAAGTTCTTTGAGCAGCGGACGTCCCTAATG, and C99-PS2-2r, 5′-GCTCTAGAGTAAAACTATACAACTGCATCC. The sequence of each mutant cDNA was verified by sequencing. Details of primer sequences and PCR conditions for all cloning work are available on request. A series of deletion constructs of C99-PS1 and C99-PS1R278I were also generated using PCR-based methods and a Quick-change site directed mutagenesis kit . F-NEXT C-PS1  t-butyl ester (DAPT), was purchased from Calbiochem. Stable human embryonic kidney (HEK) 293 pools . PS1 loop antibody is a monoclonal antibody that recognizes amino acid residues 263–378 . 6E10, an anti-Aβ antibody, was purchased from Sigma. For F-NEXTΔC-PS1, PS1 N-terminal antibodies, used in SDS-PAGE was carried out on Tris/Tricine gel . Samples were mixed with 2×SDS sample buffer (Invitrogen) and incubated at 37°C for 30 min immediately prior to electrophoresis. The samples were transferred onto a polyvinylidene difluoride (PVDF) membrane . Western blotting was performed using the antibodies described above. Enhanced ChemiLuminescence (ECL) was used as the detection system.Analysis of secreted Aβ40 and 42(43) in the medium of PS dKO MEF expressing C99-PS1 chimeras was performed using a human-specific Aβ ELISA . According to the manufacturer's information, the sensitivity of human specific Aβ40 and 42 ELISA is 0.12 and 0.08 pM, respectively. DAPT treatment clearly decreased Aβ 40 and 42 levels from the basal levels in cells expressing the fusion proteins , E. Moret-test. Comparisons of the means among three or more groups were performed by analysis of variance (ANOVA). Values of p less than 0.05 were considered significant.All values are expressed as mean ± SEM. For comparisons of the means between two groups, the data were statistically analyzed by Student's 2-terminal fragment (NTF) and an ∼16–20 kDa COOH-terminal fragment (CTF) th amino acid, also reacted with these ∼50 kDa and ∼35 kDa species, indicating that the C99-PS1 fusion protein undergoes endoproteolysis as observed in wild-type PS1 protein  along with an empty vector, C99-PS1, C99-PS1D385A or C99-PS1G266S plasmid. Co-transfection of C99-PS1 and APPswe produced Aβ40 and Aβ42 , while PTo test whether C99-PS1 harboring FAD-linked PS1 mutations can overproduce Aβ42(43), we introduced PS1R278I, in addition to L435H, which was found to effectively overproduce Aβ42(43), using a random mutagenesis screen of PS1 To change the molecular interaction between APP and PS1, we made sequential deletions of the hydrophilic region between the transmembrane domain of C99 and transmembrane I domain of PS1 in the chimeric proteins. We characterized Aβ40/42 production in PS dKO MEF transiently expressing deletion mutants of C99-PS1 WT and R278I . Aβ40 prNext, to confirm that Aβ42 production by the C99-PS1R278I fusion protein was strictly γ-secretase dependent, and not due to non-specific degradation, we treated dKO MEF expressing C99-PS1R278IΔ38/Δ59 with a selective γ-secretase inhibitor, DAPT. Aβ42 production was inhibited by DAPT in dKO MEF expressing C99-PS1R278IΔ38/Δ59 , suggestThe mechanisms by which Aβ40 and Aβ42 peptides are produced in a ratio of ten to one by wild type PS and Aβ42 is overproduced by FAD-linked PS variants are not clearly elucidated . To undeBased on our findings, we suggest that the chimeric proteins characterized in this study mimic the native interaction between APP CTF and presenilin, and would be useful tools for further exploration. First, we showed that fusion of APP CTF to the N-terminus of PS1 produced Aβ by intrinsic processing upon reconstituion of γ-secretase activity in PS dKO cells. Moreover, a γ-secretase inhibitor, DAPT, inhibited Aβ production, suggesting that fusion of APP CTF to the N-terminus of PS1 produced Aβ in a γ-secretase-dependent manner. We also found that Nβ is indeed produced in K293 cells stably expressing F-NEXTΔC-PS1, the fusion protein of F-NEXTΔC, a mouse Notch1 derivative that lacks the majority of its extracellular and intracellular domains In summary, we generated fusion proteins of APP CTF to the N-terminus of PS and observed differential regulation of the APP/PS interaction during Aβ40 and 42 production. Because the crystal structures of other intramembrane proteases have been reported Text S1(DOCX)Click here for additional data file.Figure S1Comparison of expression level of C99-PS1 in COS cells and PS(−/−) cells. A higher level of protein expression was observed in COS cells than in PS(−/−) cells. Note that no reactivity with 6E10 antibody was observed in cells that expressed C99-PS1 fusion protein in PS(−/−) cells, with even high exposure.(TIF)Click here for additional data file.Figure S2Stability of C99-PS1 fusion protein in COS cells. The stability of the fusion protein was investigated using cycloheximide (30 µg/ml). Note that that stability of C99-PS1 was comparable to that of PS1. We observed that the immunoreactivity of the fusion protein was not very different between PS1 NT and 6E10 in COS cells. However, this result does not exclude that the fusion protein is being cleaved efficiently, because it is highly overexpressed in COS cells.(TIF)Click here for additional data file.Figure S3Generation of F-NEXTΔC-PS1 and detection of Nβ. The fusion protein of Notch1, another type I membrane protein, to PS1 was generated. F-NEXTΔC-PS1/K293-clone #15 expressed a high level of full-length F-NEXT-ΔC-PS1, and replacement of endogenous PS1 NTF and PS2 by full-length F-NEXT-ΔC-PS1 was observed in this cell line, but less replacement was observed in clone #4 with low expression of full-length F-NEXT-ΔC-PS1 (left panel). IP-Mass experiment revealed that Nβ was secreted in F-NEXTΔC-PS1/K293-clone #15. Thus, we confirmed that the fusion protein of flag-tagged NotchΔE fused to PS1 could be cleaved, resulting in Nβ secretion (right panel), suggesting that substrate fused to PS1 could be cleaved.(TIF)Click here for additional data file.Figure S4Validation of Aβ ELISA.Panel A: Standard curve for Aβ40 obtained using synthetic Aβ40 peptides. Panel B: Standard curve for Aβ42 obtained using synthetic Aβ42 peptides. Panel C: Comparison of secreted Aβ from PS dKO cells expressing C99-PS1 using human/rat Aβ ELISA (left panel) and human-specific Aβ ELISA (right panel). * p<0.05. Panel D: Human Aβ40 level in medium of PS dKO cells transiently transfected with vector or C99-PS1 treated with 0, 0.4, 1.1, 3.3, and 10 µM DAPT analyzed by human-specific Aβ40 ELISA. * p<0.05. Note that DAPT treatment decreased Aβ40 secreted from PS dKO cells expressing the fusion protein. Panel E: Analysis of Aβ42 level by Aβ ELISA in the medium of PS dKO cells transfected with C99 -PS1R278I Δ37/Δ59 treated with or without γ-inhibitor, DAPT. * p<0.05. Note that DAPT treatment decreased Aβ42 secreted from PS (−/−) cells expressing the fusion protein. Panel F: Analysis of Aβ level by Aβ ELISA in the medium of PS dKO cells transfected with C99/PS1Δ37/Δ65. Note that the deletion mutants of C99/PS1Δ37/Δ65 showed decreased production of Aβ42, but not Aβ40 compared to C99-PS1. * p<0.05, ** p<0.01. Panel G: Analysis of Aβ level by Aβ ELISA in the medium of PS dKO cells transfected with C99/PS1Δ37/Δ71 and Δ37/Δ72. Note that the deletion mutants of C99/PS1Δ37/Δ71 and Δ37/Δ72, which have defects in the transmembrane I domain of PS, showed disrupted production of both Aβ40 and 42 compared to C99-PS1. * p<0.05, ** p<0.01.(TIF)Click here for additional data file.Figure S5Replacement of endogenous PS1 by fusion protein. Stable human 293 pools were generated by transfecting cells with vector, or C99-PS1wt, C99-PS1D385A, or C99-PS1G266S. Note that endogenous PS1 CTF was replaced by C99-PS1D385A, which is the full-length dominant negative form.(TIF)Click here for additional data file.


The carboxylate ion and the N-heterocycle both function as bridges to link adjacent CdII atoms to result in the formation of a layer structure parallel to (010). The mid-point of the central C—C bond of the 4,4′-bipyridine ligand is located on an inversion center. In the crystal, the carboxylate ion is disordered over a twofold rotation axis in respect of its methyl group and the cyclopentane ring.In the title polymeric compound, [Cd(C R,3S)-1,2,2-trimethylcyclopentane-1,3-dicarboxylic acid, see: Adhya et al. (C10H8N2)] = 0.054                           wR(F                           2) = 0.171                           S = 1.12                           2364 reflections150 parameters51 restraintsH-atom parameters constrainedmax = 1.03 e Å−3                        Δρmin = −1.27 e Å−3                        Δρ                                 RAPID-AUTO  global, I. DOI: 10.1107/S1600536811042656/xu5349Isup2.hkl            Structure factors: contains datablock(s) I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


There are two non-coordinated molecules of acetonitrile in the formula unit. A center of inversion is present between the two metal atoms, and, consequently, there is no distinction between MnIII and MnIV metal centers. In the Mn2O2 core, the Mn—O distances are 1.817 (3) and 1.821 (3) Å. The cyclam ligand is in the cis configuration. The chloride counter-anion resides on a center of symmetry, whereas the tetraphenylborate counter-anion is in a general position. The cyclam ligand is hydrogen bonded to the acetonitrile as well as to the chloride anion. One of the phenyl rings of the anion and the acetonitrile solvent molecule are each disordered over two sets of sites.The title compound, [Mn MnO] al. 1990; Lu et a al. 2001. For str al. 1989; Levaton al. 1989. For cyc al. 1965.       2O2(C10H24N4)2](C24H20B)2Cl·2C2H3N = 0.061                           wR(F                           2) = 0.173                           S = 1.03                           6092 reflections433 parameters258 restraintsH-atom parameters constrainedmax = 0.54 e Å−3                        Δρmin = −0.81 e Å−3                        Δρ                                 APEX2  used to solve structure: SHELXS97  global, I. DOI: 10.1107/S1600536811019829/hg5039Isup2.hkl            Structure factors: contains datablock(s) I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


The three C O groups are pseudo-octahedrally disposed with C—Mo—C angles ranging from 80.7 (1) to 87.4 (1)°. The two uncoordinated 2,6-diisopropylphenyl-substituted benzene rings form dihedral angles of 75.96 (8) and 78.01 (9)° with the mean plane of the guanidine group. The coordinated benzene ring is in a slight sofa conformation with the N-substituted C atom and the bonded N atom dispaced by 0.090 (3) and 0.458 (4) Å, respectively, from the mean plane of the remaining ring atoms. In the crystal, despite there being two N—H donor groups, no conventional hydrogen bonds are present. This may be because of the steric effects of the bulky diisopropylphenyl groups.In the title compound, [Mo(C DOI: 10.1107/S1600536811021441/lh5261Isup2.hkl            Structure factors: contains datablock(s) I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


ApoE4 is the most important genetic risk factor for AD. Previous results by Cramer et al.  showed t [et al. .® , and HP-b-CD/ Tween to dissolve Bexarotene for administration in mice and dogs respectively. For acute experiments we administered a single 100 mg/kg/p.o. dose of Bexarotene  to wild type male Swiss CD1 mice and measured endogenous levels of soluble Aβx-37, Aβx-38, Aβx-40 and Aβx-42 in brain at different time points. In Beagle dogs we administered 25 and 100 mg/kg/p.o. Bexarotene and measured Aβx-37, Aβx-38, Aβx-40 and Aβx-42 levels in CSF. For chronic experiments we administered 100 mg/kg/day/p.o. Bexarotene for 19 days to 10-months-old male hAPP/PS1 mice.We used captisolIn contrast to the published data, we found that acute and chronic treatment with Bexarotene had no significant effects on Aβ levels in the brain of wild type and AD model mice and in Beagle dogs, despite high penetration of the drug into the brain. Although behavioral alterations were observed in AD model mice, adverse effects of the treatment confounded these observations.et al. [Drug formulation and pharmacokinetics might explain our contradictory observations with Cramer et al.  at least


Heterosis was found for pod number and yield. Data on general and specific combining ability (gca and sca) indicated additive and nonadditive gene actions. The gca: sca ratios were much less than unity suggesting predominant role of nonadditive gene effects. Cultivars “Binachinabadam-2” and “Dacca-1” and mutant M6/25/64-82 had the highest, second highest, and third highest pod number, as well as gca values, respectively. These two cultivars and another mutant M6/15/70-19 also had the highest, second highest, and third highest pod yield, as well as gca values, respectively. Therefore, “Dacca-1”, “Binachinabadam-2”, M6/25/64-82, and M6/15/70-19 could be used as source of salinity tolerance. Cross combinations showing high sca effects arising from parents with high and low gca values for any trait indicate the influence of nonadditive genes on their expression. Parents of these crosses can be used for biparental mating or reciprocal recurrent selection for developing high yielding varieties. Crosses with high sca effects having both parents with good gca effects could be exploited by pedigree breeding to get transgressive segregants.A study was performed using 6 × 6 F Salinity is one of the most serious environmental factors limiting crop productivity worldwide . This stArachis hypogaea L.) requires only 350 mm water to complete its life cycle [Cultivation of high water demanding crop is not possible during the dry period (November to May) in the saline areas of Bangladesh because lack of suitable irrigation water. Groundnut  of interest, it is imperative to assess the mode of its inheritance, especially with respect to being additive or nonadditive. Diallel analysis , 9 helps1 diallel population without reciprocal was used in this study. The parents of this population were discriminated as being salt tolerant/sensitive based on pod number and yield under salt stress [6/15/70-19, “Binachinabadam-1”, M6/25/64-82, and “Binachinabadam-2” and 2 sensitive cultivars: “Dacca-1” and “Zhingabadam” were crossed following the method of Kumar and Patel [6/15/70-19, and M6/25/64-82 was Mut-6. Mutant-6 was derived by irradiating the seeds of “Dacca-1”, a local land race; thus, “Dacca-1” is the common parent of “Binachinabadam-1”, “Binachinabadam-2”, M6/15/70-19, and M6/25/64-82.A 6 × 6 Ft stress . Four sand Patel  with somThe experiment was conducted in the glasshouse of Bangladesh Institute of Nuclear Agriculture (BINA), Mymensingh, with temperature and relative humidity ranges of 25–35°C and 70–90%, respectively. The study was set up as a completely randomized design with four replications, under 8 dS/m salinity level, imposed during flowering till harvest stages as flowering stage is the most sensitive stage to salinity stress . Five pr2SO4, 10% NaHCO3, CaCl2, and MgCl2 together with 5% MgSO4 by weight to simulate saline ground water of saline areas of Bangladesh [ V1 = volume of soil mixture in a pot, S1 = desired salinity - initial salinity of the soil, V2 = volume of water at 70–80% plant available water (PAW), S2 = salinity of stock solution.Saline water was synthesized by using a mixture of different salts: 50% NaCl, 15% NaV1) was determined using the following formula:V2) was determined by dividing the weight of water by its density (0.98 g/cc).Volume of soil mixture ,   k = 1,…, b (b = number of blocks/replications),  and  l = 1,…, c (c = number of observation in each plot).The mathematical model for the combining ability analysis in model 1 was as follows:Yij is the mean of i × jth genotype over k and l; m is the population mean; gi is the gca effect of ith parent; gj is the gca effect of jth parent. sij is the sca effect and 1/bc∑∑eijkl is the mean error effect.igi = 0 and ∑jsij + sii = 0 (for each i).Restrictions imposed are ∑g is the sum of squares due to gca, SSs  is the sum of squares due to sca,  Yi is the array total of the ith parent,  Yii  is the mean value of the ith parent,  Y.. is the grand total of the 1/(2n(n − 1)) crosses and parental values,  and  Yij is the progeny mean values in the diallel table.The sum of squares (SS) were calculated as follows:gi) = (n − 1)δ2e/n(n + 2),Var(sij) = 2(n − 1)δ2e/(n + 1)(n + 2).Var was calculated as the square root of the variance.1 half diallel population at 8 dS/m salinity stress are presented in P ≤ 0.01) differences between genotypes for plant height, branch number, pod number, and pod yield and not significant difference for branch number. The parents showed such significant differences for plant height and pod yield but just significant and not significant differences for pod and branch number. Like genotype, the F1s showed highly significant differences for all the traits, but the term P versus F1 appeared highly significant for pod number and yield but not significant for all the other traits.Mean squares (ANOVA) for pod yield and some related traits of a 6 × 6 F6/15/70-19 had the shortest height showing significant difference with all others except “Binachinabadam-2”. Branch number did not show any significant difference amongst the parents. Pod number was the highest in “Binachinabadam-2”, and it differed significantly with that of all the remainders. In this experiment, only “Zhingabadam” failed to produce any pod. However, 3 of the 6 mutants/varieties did not have any mature pod, and thus they have no pod yield. Of the remaining three, “Binachinabadam-2” had significantly the highest pod yield followed by “Dacca-1”.Means of pod yield and some related traits of the parents at 8 dS/m salinity stress, imposed during flowering till harvesting stages, are presented in Analysis of variance data for combining ability is presented in 6/15/70-19, had the lowest gca for plant height. The remaining genotypes had not significant negative gca values for plant height. The two genotypes with the highest pod number and yield (6/25/64-82 for pod number.Estimates of gca and sca effects of the tested genotypes are presented in Tables nd yield  also hadnd yield . In cont6/25/64-82, “Zhingabadam” × M6/15/70-19, and “Dacca-1” × “Binachinabadam-2” crosses (6/15/70-19, and M6/25/64-82 × “Binachinabadam-2”. The parents of these crosses had highly significant versus just significant positive, significant positive versus not significant negative, significant positive versus not significant negative, highly significant positive versus negative, and not significant negative versus not significant negative gca values, respectively.For plant height, the cross “Binachinabadam-1” × “Binachinabadam-2” appeared as the best specific combination followed by “Dacca-1” × M crosses . These o6/15/70-19 being the best followed by “Dacca-1” × “Binachinabadam-2”, M6/15/70-19 × “Binachinabadam-1”, “Zhingabadam” × “Binachinabadam-1”, “Zhingabadam” × M6/25/64-82, “Zhingabadam” × “Binachinabadam-2”, M6/25/64-82 × “Binachinabadam-2”, and “Zhingabadam” × M6/15/70-19 (6/15/70-19 × “Binachinabadam-2”, “Dacca-1” × M6/25/64-82, “Binachinabadam-1” × “Binachinabadam-2”, M6/15/70-19 × M6/25/64-82, and “Dacca-1” × “Binachinabadam-1” in decreasing order. These crosses were obtained from crossing between parents with highly significant negative versus highly significant positive, highly significant negative versus highly significant positive, highly significant positive versus not significant positive, highly significant negative versus not significant positive, and highly significant positive versus highly significant negative gca values, respectively.For pod number, 8 cross combinations showed highly significant positive sca with “Dacca-1” × M15/70-19 . These c6/15/70-19, “Dacca-1” × “Binachinabadam-2”, “Zhingabadam” × M6/15/70-19, “Zhingabadam” × “Binachinabadam-1”, “Zhingabadam” × M6/25/64-82, M6/15/70-19 × “Binachinabadam-1”, and M6/25/64-82 × “Binachinabadam-2”.However, for pod yield, out of 15 cross combinations, 7 had shown heterotic performances. They were “Dacca-1” × M6/15/70-19 appeared tolerant in this study as these could produce pod yield (6/15/70-19 were found to be tolerant and the remainders were found to be sensitive. This may result in screening study [Tolerance to salinity is highly influenced by the environmental factors . The varod yield  and alsong study .A detailed genetic study is a prerequisite in determining the mode of inheritance of traits which ultimately helps adopt better planning and execution in a plant breeding improvement programme. This study was to assess this with respect to pod yield and some related traits in groundnut exposed to salt stress. The term parent versus offspring appeared significant for pod number and yield only but not significant difference for plant height and branch number . This suσ2g/σ2s ratio. The closer the ratio to unity is, the greater would be the magnitude of additive genetic effects. The ratios computed in this study were much less than unity (The significant general and specific combining ability data  conform an unity  suggesti6/25/64-82 with the highest, second highest, and third highest pod number, respectively, had also highest, second highest, and third highest gca values for pod number (6/15/70-19 had highest, second highest, and third highest gca values (6/25/64-82, and M6/15/70-19 could be used as major source of salinity tolerance. The high sca effects for pod number were obtained by crossing parents with high × low, high × high, low × low, and low × high gca values (Two cultivars, “Binachinabadam-2”, and “Dacca-1” and mutant Md number . For poda values . Obviousa values . In cont


All non-H atoms lie on a crystallographic mirror plane apart from the oxide ligands, which are related by mirror symmetry. The pyridinium N atom acts as a hydrogen-bond donor to the solvent water molecule, which is in turn a hydrogen-bond donor to the both oxide ligands. These hydrogen-bonding interactions give rise to a three-dimensional network motif.The hydrazone anion in the title compound, [V(C The rea Pal 2009.       11H12N3O2)O2]·H2O = 0.039                           wR(F                           2) = 0.121                           S = 1.11                           1610 reflections125 parameters2 restraintsH atoms treated by a mixture of independent and constrained refinementmax = 0.75 e Å−3                        Δρmin = −0.72 e Å−3                        Δρ                                 APEX2  used to solve structure: SHELXS97 (Sheldrick, 2008SHELXL97 (Sheldrick, 2008X-SEED (Barbour, 2001publCIF (Westrip, 2010Data collection: 10.1107/S1600536810028886/nk2049sup1.cif            Crystal structure: contains datablocks global, I. DOI: 10.1107/S1600536810028886/nk2049Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


A μ-oxide ligand bridges two similar MoCl(di-t-Bu-bipy)O2 units, having the terminal oxide ligands mutually cis, and the chloride and μ-oxide trans to each other. In the binuclear complex, the coordination geometries of the metal atoms can be described as highly distorted octahedra. Individual complexes co-crystallize with a partially occupied water molecule of crystallization , with the crystal packing being mediated by the need to effectively fill the available space. A number of weak C—H⋯O and C—H⋯Cl interactions are present.The title hydrate, [Mo For stu al. 2011; Fernand al. 2009; Nunes e al. 2003; Pereira al. 2007.       2Cl2O5(C18H24N2)2]·0.2H2O = 0.040                           wR(F                           2) = 0.086                           S = 1.02                           10578 reflections463 parameters18 restraintsH-atom parameters constrainedmax = 0.96 e Å−3                        Δρmin = −0.67 e Å−3                        Δρ                                 APEX2  used to solve structure: SHELXTL  global, I. DOI: 10.1107/S1600536811046952/tk5013Isup2.hkl            Structure factors: contains datablock(s) I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


The present study was conducted to understand how ATP is released from DRG neurons. In an outside-out patch-clamp configuration from acutely dissociated rat DRG neurons, single-channel currents, sensitive to the P2X receptor inhibitor PPADS, were evoked by approaching the patch-electrode tip close to a neuron, indicating that ATP is released from DRG neurons, to activate P2X receptor. NA increased the frequency of the single-channel events, but such NA effect was not found for DRG neurons transfected with the siRNA to silence the cystic fibrosis transmembrane conductance regulator (CFTR) gene. In the immunocytochemical study using acutely dissociated rat DRG cells, CFTR was expressed in neurons alone, but not satellite cells, fibroblasts, or Schwann cells. It is concluded from these results that CFTR mediates NA-induced ATP efflux from DRG neurons as an ATP channel.In our earlier study, noradrenaline (NA) stimulated ATP release from dorsal root ganglion (DRG) neurons as mediated via β ATP receptors, that include ionotropic P2X receptors and metabotropic P2Y receptors, serve as a pain mediator in the DRG -9. A gro1 receptors that include α1a, α1b and α1d subtypes, α2 receptors that include α2a, α2b, α2c and α2d subtypes, and β receptors that include β1, β2, β3 and β4 subtypes. Peripheral nerve injury causes sympathetic nerve sprouting into the DRG  containing 6,7-dinitroquinoxaline-2,3-dione (DNQX)(20 μM) and DL-2-amino-5-phosphonopentanoic acid (DL-AP5)(100 μM) at a flow rate of 2 ml/min at 21-22°C. The patch electrode-filling solution was as follows: (in mM) 135 Cs-gluconate, 5 CsCl, 2 MgCl2, 5 ethyleneglycol-bis-(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid, 5 HEPES, 5 MgATP, and 1 Na2GTP, pH 7.2. Currents were analyzed using a pClamp software . NA was bath-applied with an air-pressure injector .Outside-out patches were made from acutely dissociated DRG neurons. Single-channel currents were monitored with an Axopatch-200A amplifier  in a recording chamber continuously superfused with an extracellular solution , incubated at 70°C for 1 min to break the cell membrane. Cell lysate was incubated in the presence of DNase I at 37°C for 30 min to remove genomic DNAs and at 70°C for 10 min to inactivate DNase I. Then, the sample was incubated in a DTT solution containing SuperScript III reverse transcriptase  at 55°C for 40 min followed by at 70°C for 15 min. The gene expression was amplified by double PCRs. Initially, PCR was carried out in the reaction solution containing 10× PCR buffer, dNTPs, oligonucleotide, dimethyl sulfoxide , and 5 units of Taq polymerase  with a PCR Thermal Cycler Dice  programmed as follows: first step, 98°C for 4 min, the ensuring 40 cycles, 98°C for 1 s, 65°C for 15 s, and 72°C for 30 s. Subsequently, real-time PCR was performed using the first PCR products with a SYBR Green realtime PCR Master Mix  and the Applied Biosystems 7900 real-time PCR detection system  programmed as follows: first step, 98°C for 4 min; the ensuring 40 cycles, 98°C for 1 s, 65°C for 15 s, and 72°C for 30 s. The expression level of rat CFTR mRNA was normalized by that of GAPDH mRNA. Primers used for RT-PCR were as follows: 5'-AATATCCTTAGCCCCTCGGA-3' and 5'-TGGTGGAAACAATGGCACTA-3' for CFTR (accession number M89906) and 5'-CCTTCCGTGTTCCTACCCCCAAT-3' and 5'-CCTCTCTCTTGCTCTCAGTATCCTTGCT-3' for GAPDH (accession number BC059110).Acutely dissociated DRG cells were fixed with 4% (w/v) paraformaldehyde, permeabilized with 0.3% (v/v) Triton X-100, and blocked with 10% (v/v) goat serum in phosphate buffered saline (PBS) at room temperature. Cells were reacted with a mouse monoclonal antibody for MAP2 (1:500), GFAP (1:400), S-100 (1:500) or fibronectin (1:500) and a rabbit polyclonal antibody for CFTR (1:200)(Chemicon) overnight at 4°C followed by a goat anti-rabbit IgG conjugated with Alexa 488  and a goat anti-mouse IgG conjugated with Alexa 633 (Molecular Probes) for 60 min at room temperature. Fluorescence-labeled cells were visualized with a confocal scanning laser microscope .The authors declare that they have no competing interests.All authors read and approved the final manuscript. TN. conducted the majority of the experiments and data and wrote the manuscript. TK contributed to all the experiments and data analysis.


In “Genetic Variation among Temporally and Geographically Distinct West Nile Virus Isolates, United States, 2001, 2002”, by C. Todd Davis, et al., errors occurred in Tables 1 and 2. The correct title for Table 1 is “Nucleotide mutations in sequences of the prM gene of 22 West Nile virus isolates obtained during 2001 and 2002 compared to WN-NY99.” In Table 1, line 6, Nueces Co., Tx-1, under “prM 491” should read “G (Arg)”; line 19, Galveston Co, TX-2, under “prM 679” should read “A (Thr).” The correct title for Table 2 is “Nucleotide mutations in sequences of the E gene of 22 West Nile virus isolates obtained during 2001 and 2001 compared to WNNY99.”http://wwwnc.cdc.gov/eid/article/9/11/03-0301_article.htm.In Table 2, line 3, Harris Co., TX, under “Envelope 2,392,” should read “A (Thr)”; line 8, Nueces Co., TX-2, under “Envelope 1,118,” should read “U ”; line 12, Randall Co., TX, under “1,192” should read “C (Asn)”; line 24, Galveston Co., TX-3, under “Envelope 1,192” should read “G (Ala).” Corrected versions of Tables 1 and 2 are available at: We regret any confusion these errors may have caused.


One MgII ion is octahedrally coordinated by six carboxylate O atoms. The other two cations are each octahedrally coordinated by four carboxylate O atoms and two O atoms donated by two DMF molecules: in one, the DMF molecules are cis and in the other they are trans. The three MgII octahedra form clusters, which are bridged by the BTC trianions, generating a three-dimensional structure.The asymmetric unit of the polymeric title compound, [Mg II complexes with benzene-1,3,5- tricarboxylate ligands, see: Davies et al. 2(C3H7NO)4] = 0.041wR(F2) = 0.144S = 1.004986 reflections486 parametersH-atom parameters constrainedmax = 0.48 e Å−3Δρmin = −0.38 e Å−3ΔρKM-4 Software  I, global. DOI: 10.1107/S1600536812023240/hb6785Isup2.hklStructure factors: contains datablock(s) I. DOI:  crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:


The btppy ligand acts as a N,S-bidentate ligand, forming a six-membered ring, which has an envelope conformation. The S—Ru—N bite angle is 86.76 (9)°, and the dihedral angle between the pyridine and benzene rings in btppy is 39.8 (2)°. The unit cell contains two pairs of racemic diastereomers with  and  configurations, in which the tert-butyl group on the coordinated S atom is distant from the η6-benzene ligand.In the title compound, [RuCl(C N,S-bidentate ligands in asymmetric allylic substitution reactions, see: Mellah et al. (2007tert-butylthio)phenyl)pyridine, see: Clavier et al. (C15H17NS)]PF6                         = 0.037                           wR(F                           2) = 0.078                           S = 1.03                           4474 reflections291 parameters2 restraintsH-atom parameters constrainedmax = 0.52 e Å−3                        Δρmin = −0.39 e Å−3                        ΔρAbsolute structure: Flack 1983, 1824 FrFlack parameter: 0.03 (3)                                 CrystalClear (Rigaku, 1999CrystalClear; data reduction: CrystalStructure (Rigaku/MSC, 2006SIR97 (Altomare et al., 1999SHELXL97 (Sheldrick, 2008ORTEP-3 for Windows (Farrugia, 1997CrystalStructure.Data collection: 10.1107/S1600536810049810/fj2364sup1.cif            Crystal structure: contains datablocks global, I. DOI: 10.1107/S1600536810049810/fj2364Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


II ion in the title mononuclear compound, [Co(C11H7N6)2(H2O)2]·2CH3OH, is located on an inversion center and is six-coordinated in a distorted octahedral geometry defined by four N atoms from two deprotonated 5-(pyrazin-2-yl-κN)-3-(pyridin-4-yl)-1H-1,2,4-triazol-1-ide (ppt) ligands and two water molecules. In the crystal, the complex molecules and lattice methanol molecules are linked via O—H⋯N and O—H⋯O hydrogen bonds, generating a two-dimensional supramolecular network parallel to (001). π–π interactions between the triazole and pyrazine rings and between the pyridine rings are present .The Co DOI: 10.1107/S160053681201495X/hy2533Isup2.hklStructure factors: contains datablock(s) I. DOI:  crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:


Biodegradable chitosan of different sizes were used to encapsulate antitumor drug tamoxifen (Tam) and its metabolites 4-hydroxytamoxifen (4-Hydroxytam) and endoxifen (Endox). The interactions of tamoxifen and its metabolites with chitosan 15, 100 and 200 KD were investigated in aqueous solution, using FTIR, fluorescence spectroscopic methods and molecular modeling. The structural analysis showed that tamoxifen and its metabolites bind chitosan via both hydrophilic and hydrophobic contacts with overall binding constants of Ktam-ch-15  = 8.7 (±0.5)×103 M−1, Ktam-ch-100  = 5.9 (±0.4)×105 M−1, Ktam-ch-200  = 2.4 (±0.4)×105 M−1 and Khydroxytam-ch-15  = 2.6(±0.3)×104 M−1, Khydroxytam – ch-100  = 5.2 (±0.7)×106 M−1 and Khydroxytam-ch-200  = 5.1 (±0.5)×105 M−1, Kendox-ch-15  = 4.1 (±0.4)×103 M−1, Kendox-ch-100  = 1.2 (±0.3)×106 M−1 and Kendox-ch-200  = 4.7 (±0.5)×105 M−1 with the number of drug molecules bound per chitosan (n) 2.8 to 0.5. The order of binding is ch-100>200>15 KD with stronger complexes formed with 4-hydroxytamoxifen than tamoxifen and endoxifen. The molecular modeling showed the participation of polymer charged NH2 residues with drug OH and NH2 groups in the drug-polymer adducts. The free binding energies of −3.46 kcal/mol for tamoxifen, −3.54 kcal/mol for 4-hydroxytamoxifen and −3.47 kcal/mol for endoxifen were estimated for these drug-polymer complexes. The results show chitosan 100 KD is stronger carrier for drug delivery than chitosan-15 and chitosan-200 KD.Synthetic and natural polymers are often used as drug delivery systems  Tamoxifen and 4-hydroxytamoxifen were from Sigma Chemical Company. The synthesis of endoxifen was conducted as reported An appropriate amount of chitosan was dissolved in acetate solution (pH 5.5–6.5). The Drug solutions (1 mM) tamoxifen and its metabolites in ethanol/water (25/75%) were prepared and then diluted by serial dilution in acetate buffer.−1 with a nominal resolution of 2 cm−1 and 100 scans. The difference spectra [(chitosan solution + drug solution) − (chitosan solution)] were generated using free chitosan band around 902 cm−1, as standard. This band is related to chitosan ring stretching Infrared spectra were recorded on a FTIR spectrometer , equipped with deuterated triglycine sulphate (DTGS) detector and KBr beam splitter, using AgBr windows. Solution of drug was added dropwise to the chitosan solution with constant stirring to ensure the formation of homogeneous solution and to reach the target drug concentrations of 15, 30 and 60 µM with a final chitosan concentration of 60 µM. Spectra were collected after 2 h incubation of chitosan with drug solution at room temperature, using hydrated films. Interferograms were accumulated over the spectral range 4000–600 cmex  = 270–290 nm and λem from 300 to 450 nm. The intensity of the band at 375 nm from tamoxifen and its metabolites Fluorimetric experiments were carried out on a Perkin-Elmer LS55 Spectrometer. Stock solution of drug (30 µΜ ) in acetate (pH 5.5–6.5) was also prepared at 24±1°C. Various solutions of chitosan (1 to 200 µM) were prepared from the above stock solutions by successive dilutions at 24±1°C. Samples containing 0.06 ml of the above drug solution and various polymer solutions were mixed to obtain final chitosan concentrations ranging from 1 to 200 µΜ with constant drug content (30 µΜ). The fluorescence spectra were recorded at λn) substantive binding sites for drug (Q) on polymer (B), the quenching reaction can be shown as follows:On the assumption that there are (AK), can be calculated as:Q] and [B] are the drug and polymer concentration, respectively, [nBQ] is the concentration of non fluorescent fluorophore-drug complex and [B0] gives total polymer concentration:The binding constant  can be calculated by modified Stern-Volmer equation:F0 is the initial fluorescence intensity and F is the fluorescence intensities in the presence of quenching agent (or interacting molecule). K is the Stern-Volmer quenching constant, [Q] is the molar concentration of quencher and f is the fraction of accessible fluorophore to a polar quencher, which indicates the fractional fluorescence contribution of the total emission for an interaction with a hydrophobic quencher K will be calculated from 0/FF =  K [Q] +1.The accessible fluorophore fraction (http://www.arguslab.com). The chitosan structure was obtained from literature report The docking studies were carried out with ArgusLab 4.0.1 software  and amide II band at 1540–1526 cm−1 (C–N stretching coupled with N-H bending modes) −1 were compared with those of the drug-polymer adducts in 2 groups, as well as the presence of hydrophobic contacts in drug-chitosan complexes.The drug-chitosan interactions were characterized by infrared spectroscopy and its derivative methods. The shifting and intensity variations of the chitosan amide I band at 1637–1632 cm−1 and amide II at 1540–1526 cm−1, in the difference spectra of the drug-polymer complexes (−1 (4-hydroxytamox-200) and 1656–1632 (endox-ch-15), 1656–1632 (endox-ch-100) and 1654–1624 (endox-ch-200) in the spectra of drug-chitosan complexes , a minor increase in the intensity was observed for the chitosan amide I at 1637–1632 cm−1 (4-hydroxytamox-200) and 1653–1633 (endox-ch-15), 1654–1632 (endox-ch-100), 1630–1600 cm−1 (endox-ch-200) in the spectra of drug-chitosan complexes (As drug concentration increased (30 to 60 µM), a major increase in the intensity of the polymer amide I and amide II vibrations as well as other frequencies was observed with positive features at 1633–1625 (tamox-ch-15), 1656–1632 (tamox-ch-100), 1653–1633 (tamox-ch-200) and 1655-1632 (4-hydroxtamox-ch-15), 1657–1632 (4-hydroxytamox-ch-100) and 1633–1600 cm−1 showed major shifting of polymer OH, NH and CH stretching modes (−1 (free ch-200) showed major shifting and intensity changes, in the spectra of tamoxifen and its metabolite complexes (−1 (free ch-200) exhibit major shifting upon drug complexation (2 group in drug-polymer complexes (hydrophilic contacts). However, the shifting of the polymer symmetric and antisymmetric CH stretching vibrations observed for 2920, 2851 (free ch-15), 2919, 2853 (free ch-100) and 2919, 2851 (free ch-200) in the spectra of tamoxifen, 4-hydroxytamoxifen and endoxifen-polymer complexes is related to the hydrophobic contacts in the drug-chitosan complexes F vs 1/[chitosan] is shown in K value given here averages four and six-replicate run for drug-polymer systems. Each run involves several different concentrations of chitosan (Ktam-ch-15  = 8.7 (±0.5)×103 M−1, Ktam-ch-100  = 5.9 (±0.4)×105 M−1, Ktam-ch-200  = 2.4 (±0.4)×105 M−1 and Khydroxytam-ch-15  = 2.6 (±0.3)×104 M−1, Khydroxytam – ch-100  = 5.2 (±0.7)×106 M−1 and Khydroxytam-ch-200  = 5.1 (±0.5)×105 M−1, Kendox-ch-15  = 4.1 (±0.4)×103 M−1, Kendox-ch-100  =  1.2 (±0.3)×106 M−1 and Kendox-ch-200  = 4.7 (±0.5)×105 M−1 . Therefore, ch-100 forms more stable complexes than the ch-15 and ch-200  is calculated from log [(F0 -F)/F]  =  logKS + n log [chitosan] for the static quenching n values from the slope of the straight line plot showed 0.5 to 2.8 drug molecules that are bound per chitosan molecule (The number of drug molecules bound per polymer (molecule . The res0/FF against Q to estimate the quenching constant (KQ) and the results are shown in 0/FF versus Q is a straight line for drug-chitosan adducts indicating that the quenching is mainly static in these drug-polymer complexes  are 3.2×1016 M−1/s for tamox-ch-15, 2.6× M−1/s for tamox-ch-100, 4.0×1016M−1/s for tamox-ch-200 and 8.5×1016M−1/s for 4-hydroxytamox-ch-15, 3.0×1016 M−1/s for 4-hydroxytamox-ch-100, 2.1×1017M−1/s for 4-hydroxytamox-ch-200 and 2.7×1016 M−1/s for endox-ch-15, 2.4×1016 M−1/s for endox-ch-100 and, 7.9×1016M−1/s for endox-ch-200 (10M−1/s), the static quenching is dominant in these drug-polymer complexes In order to verify the presence of static or dynamic quenching in drug-chitosan complexes we have plotted omplexes . The quex-ch-200 . Since tThe spectroscopic data were combined with docking experiments in which tamoxifen, 4-hydroxytamoxifen and endoxifen molecules were docked to chitosan to determine the preferred binding sites on the chitosan. The models of the docking for drug are shown in via different binding modes. Major hydrophobic and hydrophilic interactions via chitosan charged NH2 groups are observed in these drug-polymer complexes. The order of drug-chitosan binding is ch-100>ch-200>ch-15. Stronger complexes formed for 4-hydroxytamoxifen due to more hydrophilic and hydrophobic characters. Chitosan-100 KD is a stronger carrier for tamoxifen and its metabolites than chitosan 15 and chitosan 100 KD for drug delivery in vitro.The spectroscopic and docking results presented here show that tamoxifen and its metabolites bind chitosan


The asymmetric unit consists of two ZnII atoms, two tetrahydrofuran ligands, one 2,3,5,6-tetrafluorobenzene-1,4-dicarboxylate ligand and two half 2,3,5,6-tetrafluorobenzene-1,4-dicarboxylate ligands, which are completed by inversion symmetry. One ZnII atom has a distorted trigonal–bipyramidal coordination geometry, while the other has a distorted octahedral geometry. Two independent tetrahydrofuran ligands are each disordered over two sets of sites with occupancy ratios of 0.48 (4):0.52 (4) and 0.469 (17):0.531 (17).The title compound, [Zn DOI: 10.1107/S1600536813031887/is5307Isup2.hklStructure factors: contains datablock(s) I. DOI:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The involvement of these large molecules in the crystal structure of [Rb(18-crown-6)][Rb([2.2.2]-cryptand)]Rb2Sn9·5NH3 leads to the formation of a new structural motif, namely one-dimensionally extended double strands running parallel to [100] and built by Sn9               4− cages and Rb+ cations. The double strands are shielded by 18-crown-6 and [2.2.2]-cryptand. The cations are additionally coordinated by ammonia molecules. One of the four independent Rb+ cations is disordered over two sets of sites in a 0.74 (2):0.26 (2) ratio.The crystal structure of the title compound, poly[[rubidium] [[rubidium]di-μ-rubidium-μ-nonastannide] pentaammonia], {[Rb(C DOI: 10.1107/S1600536811013997/hp2005Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


H-benzotriazol-1-yl)methyl]-1H-1,2,4-triazole-κN               4}(sulfato-κO)copper(II) sesquihydrate, [Cu(SO4)(C9H8N6)(H2O)4]·1.5H2O, is composed of one copper atom, one 1-[(2H-benzotriazol-1-yl)methyl]-1-H-1,2,4-triazole (bmt) ligand, one sulfate ligand, four coordinated water molecules and one and a half uncoordinated water molecules. The CuII atom is six-coordinated by one N atom from a bmt ligand and five O atoms from the monodentate sulfate ligand and four water molecules in a distorted octahedral geometry. In the crystal, adjacent molecules are linked through O—H⋯O and O—H⋯N hydrogen bonds involving the sulfate anion and the coordinated and uncoordinated water molecules into a three-dimensional network.The title complex, tetraaqua{1-[(1 DOI: 10.1107/S1600536811013511/bv2183Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


Cigarette smoking is associated with a decreased incidence of Parkinson disease (PD) through unknown mechanisms. Interestingly, a decrease in the numbers of α4β2 nicotinic acetylcholine receptors (α4β2-nAChRs) in PD patients suggests an α4β2-nAChR-mediated cholinergic deficit in PD. Although oligomeric forms of α-synuclein have been recognized to be toxic and involved in the pathogenesis of PD, their direct effects on nAChR-mediated cholinergic signaling remains undefined. Here, we report for the first time that oligomeric α-synuclein selectively inhibits human α4β2-nAChR-mediated currents in a dose-dependent, non-competitive and use-independent manner. We show that pre-loading cells with guanyl-5′-yl thiophosphate fails to prevent this inhibition, suggesting that the α-synuclein-induced inhibition of α4β2-nAChR function is not mediated by nAChR internalization. By using a pharmacological approach and cultures expressing transfected human nAChRs, we have shown a clear effect of oligomeric α-synuclein on α4β2-nAChRs, but not on α4β4- or α7-nAChRs, suggesting nAChR subunit selectivity of oligomeric α-synuclein-induced inhibition. In addition, by combining the size exclusion chromatography and atomic force microscopy (AFM) analyses, we find that only large (>4 nm) oligomeric α-synuclein aggregates  exhibit the inhibitory effect on human α4β2-nAChRs. Collectively, we have provided direct evidence that α4β2-nAChR is a sensitive target to mediate oligomeric α-synuclein-induced modulation of cholinergic signaling, and our data imply that therapeutic strategies targeted toward α4β2-nAChRs may have potential for developing new treatments for PD. Parkinson disease (PD) is one of the most common neurodegenerative disorders affecting more than half a million people in the United States, with annual costs estimated at 10 billion dollars It has been reported that cigarette smoking is associated with a lower incidence of PD, attributed to a neuroprotective effect of nicotine through the activation of nicotinic acetylcholine receptors (nAChRs) Considering the significant loss of nAChRs in PD brain with α-synuclein over expression, the neurotoxicity of α-synuclein to SNc dopaminergic neurons, the extensive distribution of cholinergic innervations and their receptors in SNc dopaminergic neurons, and the neuroprotective effects provided by nAChR activation 4, 6 mm dextrose, pH 7.05, in an electroporation cuvette were mixed with α7, α4+β2, or α4+β4 subunit cDNA constructs. Samples were subjected to electroporation (Bio-Rad Gene Pulsar model 1652076) at 960 microfarads and 200 volts. After electroporation, cells were suspended to 5 ml in complete medium Human α4, α7, β2, and β4 subunits were subcloned into pcDNA3.1-zeocin and pcDNA3.1-hygromycin vectors, and transfected using established techniques Conventional whole cell current recording, coupled with techniques for fast application and removal of drugs , was applied in this study as previously described α-Synuclein was prepared and purified as previously described 3N4 AFM tips   at scan rates of 2–3 Hz and at scan resolution of 512 samples per line. Images were subjected to 2nd order polynomial flattening as needed to reduce the effects of image bowing and tilt. AFM images were analyzed with the Scanning Probe Imaging Processor (SPIP) software  to generate height distribution histograms for each sample.The lyophilized α-synuclein stock was dissolved in buffer  to a concentration of 70 µM. Oligomeric aggregates of α-synuclein were obtained by incubating at 37 °C for 7–10 days without shaking. Fibrils were obtained upon longer incubation up to 35 days. Atomic force microscopy (AFM) was used to determine the morphologies of the synthetically prepared α-synuclein aggregates. Topographic AFM images were obtained in air at room temperature using a Tapping Mode AFM with a Nanoscope IIIa controller . Images were acquired using oxide sharpened Sik), peak current (pI), and steady-state current (sI), by fitting to the mono- (or double-) exponential expression I = [(pI−sI) e−kt]+sI (or I = [(pI−iI) e−k1t]+[(iI−sI) e−k2t]+sI, where iI is the intermediate level of current and k1 and k2 are rate constants from the two separate decay processes). The statistical significance of the comparison between two groups of matched data sets was assessed as p<0.05 using two-tailed Student’s t-test. All values are expressed as mean ± SEM. For statistical analysis of data from multiple groups of data, one-way or multivariate ANOVA followed by appropriate test were applied. All experiments were performed at room temperature (23±1°C). Dose-response profiles were fit to the Hill equation and analyzed using Prizm 3.0.nAChR acute desensitization (the decline in inward current amplitude over the course of agonist application) was analyzed for decay half-time  and steady-state whole cell currents . Taken together, these data suggest that oligomeric α-synuclein aggregates acutely inhibit hα4β2-nAChR function.Morphologically distinct oligomeric and fibrillar forms of α-synuclein were generated by incubating monomeric α-synuclein for different lengths of time and aggregate morphologies were analyzed by AFM . InitialTo further characterize the specific aggregate morphologies of α-synuclein involved in altering hα4β2-nAChR function, we used size-exclusion chromatography to separate a 7-day pre-aggregated α-synuclein sample into different aggregated species. The 7-day aggregated sample contains high concentrations of different oligomeric α-synuclein species. We obtained three distinct aggregated forms of α-synuclein particles from 7-day oligomeric aggregates and determined the height distribution of these particles by AFM as previously described α4β2-nAChR-mediated whole-cell currents , respectively. These findings suggest that the inhibition of hα4β2-nAChR-mediated whole-cell currents by oligomeric α-synuclein is mediated by large size oligomeric α-synuclein.We compared the effects of the morphologically distinct oligomeric α-synuclein aggregates on hα4β2-nAChR-mediated whole-cell currents. While the large oligomeric α-synuclein aggregates significantly inhibited hcurrents , the smacurrents . Statist50 values and Hill coefficients  was used as an agonist to activate hα4β2-nAChR expressed in SH-EP1 cells. α-Synuclein inhibited nicotine-induced whole-cell currents in a concentration-dependent manner ; n = 6. p<0.01, multi-variate ANOVA); of hα7-nAChR-mediated current to 93.1±7.9% ; and of hα4β4-nAChR-mediated current to 96.5±3.8% , respectively. These results indicate that the hα4β2-nAChRs are more sensitive to α-synuclein than hα7- and hα4β4-nAChRs.Since large oligomeric α-synuclein inhibited hα4β2-nAChR currents, we studied if there was a differential effect of this α-synuclein aggregate species on different nAChR subtypes. We compared effects of the large oligomeric α-synuclein on hα4β2-, hα7- and hα4β4-nAChRs heterologously expressed in an SH-EP1 cell line. The hα4β2- and hα4β4-nAChRs were activated using 3 µM nicotine, while hα7-nAChR was activated using 3 mM choline. The results indicate that large oligomeric α-synuclein  p<0.01, t-test) and by non-repetitive challenges of nicotine, the peak current was reduced to 67.5±6.9% . No statistical significance in the nicotine-induced currents was observed between the two protocols described above  via a mechanism of open channel block, or 2) via induction of hα4β2-nAChR internalization. To test whether α-synuclein-induced inhibition operated through the open channel block mechanism, we repeatedly applied 3 µM nicotine in the continuous presence of 10 nM large oligomeric α-synuclein. Repeated application of nicotine  in the presence of 10 nM α-synuclein for 10 min led to a reduction of nAChR response a, while ed above . The absed above , A and BIn the present study, we show that pathologically-relevant levels  Misfolding and aggregation of α-synuclein has been implicated in the pathogenesis of numerous neurodegenerative diseases, particularly in PD These results clearly demonstrate that oligomeric α-synuclein selectively and partially inhibits hα4β2-nAChR function. The finding of non-competitive antagonism of hα4β2-nAChRs by oligomeric α-synuclein suggests that the large oligomeric α-synuclein species acts as a non-competitive antagonist of hα4β2-nAChRs under our experimental conditions. Our results also demonstrate that oligomeric α-synuclein, at concentrations from 1 nM to 1 µM, failed to directly induce whole-cell current responses from cells expressing hα4β2- or hα7-nAChRs or from untransfected cells (data not shown). These results indicate that α-synuclein in our hands does not have properties of a nAChR agonist.Additionally, our data show no use-dependence of large oligomeric α-synuclein-induced inhibition on hα4β2-nAChR function, suggesting that the inhibitory effects are not mediated by open channel block, although the persistence in functional block after washout of fluid phase α-synuclein suggests a “lingering” effect of the aggregates. We found that pretreatment with large oligomeric α-synuclein is necessary to induce convincing inhibition of hα4β2-nAChR-mediated whole-cell currents. However, the nature of this inhibition is still unknown. One potential explanation is that there is an aggregated α-synuclein-driven long-lasting closed conformation of nAChRs. This idea is supported by the present observation that long exposure times (10 min) to large oligomeric α-synuclein aggregates leads to persistent loss of hα4β2-nAChR function, and any loss of nAChR function does not appear to be mediated via α-synuclein-induced hα4β2-nAChR internalization. This conclusion is based on the observation that GDP-β-S (600 µM) fails to prevent the loss of hα4β2-nAChR function induced by α-synuclein pre-incubation. On the other hand, the inhibition of hα4β2-nAChR function by α-synuclein is partial, non-competitive, and reversible, which is similar to amyloid-induced inhibition on hα4β2-nAChR In addition, it is also interesting that α-synuclein selectively inhibits hα4β2-nAChR subtype rather than hα7- or hα4β4-nAChR subtypes. Although it has been reported that α7 nAChRs, shown to be unaffected in the present report by α-synuclein, are upregulated in PD Neuronal nicotinic receptors that bind radiolabeled nicotine with the highest affinity contain α4 subunits (α4*-nAChR) in vivo, which cannot be mimicked by acute, in vitro experiments. Our perspective on a primary role for hα4β2-nAChRs in low concentration effects of α-synuclein complements other findings that the modulation of nAChR function by α-synuclein could be pathologically relevant Although a partial inhibitory effect of α-synuclein on hα4β2-nAChR function was observed at pathophysiology relevant concentrations, further investigation is needed to determine whether such an effect will be large enough to be clinically relevant. It is also noteworthy that α7-nAChR binding sites are increased in PD brain tissues, suggesting that α7-nAChR might be affected by α-synuclein during PD pathogenesis. However, under our conditions, direct acute exposure of α-synuclein fails to affect hα7-nAChR function. One possible interpretation is that there may be other confounders during chronic α-synuclein accumulation to affect α7-nAChR expression Collectively, our findings demonstrate for the first time that pathologically-relevant concentrations of aggregated oligomeric α-synuclein directly inhibit neuronal human α4β2-nAChR function. We find that large oligomeric α-synuclein aggregates (>4 nm), but not monomeric or fibrillar α-synuclein, selectively inhibit hα4β2-nAChR function starting at 10 nM . Specifically, we show that predominantly larger oligomeric α-synuclein aggregates (>4 nm) but not smaller species (<4 nm) potently inhibit hα4β2-nAChRs mediated whole-cell currents. Furthermore, we elucidate pharmacological mechanisms of α-synuclein–induced inhibition, which includes dose-dependent, non-competitive, non-use-dependent manners, and this inhibition is not mediated through nAChR internalization. Finally, we demonstrate that the functional inhibition by α-synuclein exhibits nAChR subunit selectivity, occurring more profoundly in α4β2-nAChRs than in other nAChR subtypes such as α7- or α4β4-nAChRs. Our findings, along with previous reports on the roles of α6β2*-nAChRs in PD pathogenesis Figure S1p<0.01), while in the presence of Aβ1-42, α-synuclein failed to further reduce this current response (p>0.05 between Aβ1-42 and α-synuclein treated group), indicated as no significance (NS) in the figure. These results suggest that both oligomeric molecules of Aβ1-42 and α-synuclein likely bind to a common negative allosteric site to reduce human α4β2-nAChR function.Effects of pretreatment of oligomeric amyloid (Aβ1-42) on α-synuclein-induced inhibition of human α4β2-nAChRs heterologously expressed in SH-EP1 cell line. We found that after 10 min pre-treatment with 1 nM oligomeric Aβ1-42, 3 µM nicotine (around EC50 concentration)-induced inward current was reduced ((DOC)Click here for additional data file.


In this mixed-ligand metal-organic compound, Zn2+ ions are coordinated by N atoms from 2-MeImH molecules and (2-MeIm)− ions, as well as by O atoms from (BTC)3− ions. This results in two different distorted tetrahedra, viz. ZnN3O and ZnN2O2. These tetrahedra are interconnected via (BTC)3− ions and N:N′-bridging (2-MeIm)− ions, thus forming a layered structure in the bc plane. Hydrogen bonds between the O atoms of carboxylate ions and NH groups of 2-MeImH ligands link the layers into a three-dimensional structure.Hydrothermal reaction involving zinc nitrate hexahydrate, trisodium benzene-1,3,5-tricarboxylate (Na DOI: 10.1107/S1600536811015844/bt5531Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


The NiII ion is coordinated in a slightly distorted octahedral coordination enviroment by four O atoms from two bis-chelating 4-acety-3-methyl-1-phenyl-1H-pyrazol-5-olate ligands in the equatorial plane and two O atoms from two N,N-dimethylformamide ligands in the axial sites. In the crystal structure, weak intermolecular π–π stacking interactions with centroid–centroid distances of 3.7467 (13) Å link molecules into chains extending alongthe b axis.The title complex, [Ni(C DOI: 10.1107/S1600536810026231/lh5075Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


The two chelated 6-(pyridin-2-yl)-1,3,5-triazine-2,4-diamine ligands form a dihedral angle 74.75 (5)°. The mononuclear molecules are alternatively linked into layers parallel to the ac plane via N—H⋯N hydrogen bonds. Adjacent layers are connected into a three-dimensional supramolecular framework by futher N—H⋯N hydrogen-bonding interactions.In the title complex, [Mn(N DOI: 10.1107/S160053681201046X/pv2519Isup2.hklStructure factors: contains datablock(s) I. DOI:  crystallographic information; 3D view; checkCIF reportAdditional supplementary materials: