Furthermore, HIF-1α levels increase upon treatment of APL cells with all-trans retinoic acid (ATRA). As a consequence, inhibiting HIF-1α in APL mouse models delays leukemia progression and exquisitely synergizes with ATRA to eliminate leukemia-initiating cells (LICs).Acute promyelocytic leukemia (APL) is epitomized by the chromosomal translocation t and the resulting oncogenic fusion protein PML-RARα. Although acting primarily as a transcriptional repressor, PML-RARα can also exert functions of transcriptional co-activation. Here, we find that PML-RARα stimulates transcription driven by HIF factors, which are critical regulators of adaptive responses to hypoxia and stem cell maintenance. Consistently, HIF-related gene signatures are upregulated in leukemic promyelocytes from APL patients compared to normal promyelocytes. Through  Overexpressed stable forms of HIF-1α and HIF-2α as well as endogenous HIF-1α were co-immunoprecipitated with PML-RARα and with PML  due to receptor internalization in culture  increased upon serial re-plating . Unexpectedly, stable silencing of HIF-2α also impaired NB4 CFU-L re-plating efficacy and slightly prolonged mice survival, although not affecting cell migration . This occurred despite the low expression of HIF-2α in NB4 cells, which was also observed in APL patients  specifically targeting HIF-1α, while EZN-3088 is a control LNA-ON -inducible promoter  and EZN-2968 (LNA-ON for HIF-1α)  supplemented with protease inhibitor cocktail (Roche); for luciferase assays, cells were lyzed in passive lysis buffer (Promega); for co-immunoprecipitation experiments, cells were lyzed after mild cross-linking with 0.4% formaldehyde (7 min at RT) in CO-IP buffer  supplemented with protease and phosphatase inhibitors (Pierce) and then briefly sonicated to extract nuclear proteins. Total lysates were immunoprecipitated with an antibody against PML .When indicated, NB4 cells (shCTRL or shHIFs) were treated for 24 h with 100 μM CoClCell extracts and immunoprecipitates were resolved by SDS–PAGE 7.5–10% and transferred to a PVDF membrane (Biorad). Non-specific binding was blocked in 5% non-fat milk for 1 h at RT and blotted with the following antibodies: rabbit polyclonal HIF-1α (Cayman), rabbit polyclonal PML (Santa Cruz) and rabbit polyclonal PML (Novus). Mouse β-actin (Sigma) was used as internal loading control.in vitro-translated biotin-labeled PML-RARα was mixed with 5 μg of His or His-HIF-1α, GST or GST-HIF-2α in a final volume of 1 mL of binding buffer  and incubated O/N at 4°C followed by the addition of 40 μl of HIS-Select® Nichel Affinity Gel (Sigma) or glutathione–Sepharose 4B beads  and a further incubation at 4°C for 2 h. After washing, beads were analyzed by SDS–PAGE. His and GST proteins were kindly provided by V. M. Neguembor.Histidine-tagged human HIF-1α full-length protein (His-HIF-1α) was purchased from Abcam. GST-tagged HIF-2α full-length protein (GST-HIF-2α) was purchased from Abnova. Biotin-labeled PML-RARα protein was generated in reticulocyte lysates using the TNT T7 coupled transcription/translation system (Promega). 30 μL of The following antibodies were used: Streptavidin-HRP (Sigma), mouse monoclonal GST (Sigma) and mouse monoclonal HIF-1α (BD Biosciences).2. Renilla-expressing plasmid was co-transfected for transfection normalization. Dual-Luciferase® Reporter Assay System (Promega) was used to measure firefly and Renilla luciferase activities and their ratio calculated in a GloMax® luminometer (Promega).Cells were plated in 24-well plates (Costar) and transfected by Lipofectamine 2000 (Invitrogen) with a reporter plasmid expressing the luciferase gene under the control of a hypoxia responsive element (HRE)-containing promoter (kind gift of C. Simon). Plasmids containing PML, RARα, PML-RARα, NPM-RARα, PLZF-RARα and AML1-ETO or plasmids containing stable forms of HIF-1α and HIF-2α, or wild-type HIF-1α for experiments in MEFs (kind gifts of C. Simon) were transfected where indicated. HEK-293 cells were treated for 8 h with 300 μM CoClRNA was isolated with the RNeasy mini kit (Qiagen). cDNA was obtained by retro-transcription of 1 μg total RNA using Advantage RT for PCR kit (Clontech) and analyzed by real-time PCR in 7900 Fast Real-Time PCR System (Applied Biosystem).All probes for TaqMan assays were purchased from Applied Biosystem. 18S was used as internal control. The relative expression of different cDNAs was calculated using the et al, For PML-RARα DNA real-time PCR, DNA was extracted using QIAamp DNA Micro kit (Qiagen) and quantified as previously described (Nasr 3 cells were resuspended in Human Methylcellulose Base Media (R&D) and plated in 35-mm culture dishes. Cells were allowed to grow for 7–10 days and then colonies were pooled, counted, and 5 × 103 cells were re-seeded for re-plating experiments. When indicated Zn (100 μM) was added to the methylcellulose medium. In pre-treatment experiments  24 h after transfection with EZN-3088 and EZN-2968, and colonies were counted 7–10 days after plating.For human cell lines, 5 × 10ents Fig B, NB4 ce6 cells were seeded in the upper chamber of a transwell 6.5-mm diameter and 5-μM pore (Costar). Cells in the lower chamber were counted by flow cytometer as number of cells acquired per minute (LSR II Beckton Dickinson) 16 h after seeding for NB4 cells and 2.5 h after seeding for U937-PR9 and U937-MT cells. For migration experiments of BM leukemic cells 1.25 × 105 mouse leukemia cells were seeded 24 h after electroporation and migration was assessed after 4 h.For human cell lines, 1 × 10Rag2−/−γc−/− immunocompromised mice were maintained in pathogen-free animal facility and treated in accordance with European Union guidelines. All animal protocols were approved by the Institutional Animal Care and Use Commitee (IACUC).6 NB4 cells (shCTRL or shHIF-1α) and euthanized after 16 h. BM samples were stained with anti-human CD13 antibody. For survival experiments, mice were challenged i.v. with 7 × 105 NB4 cells (shCTRL or shHIFs) and sacrificed when terminally sick.For homing experiments, mice were injected intravenously (i.v.) with 5 × 10et al, 6) were inoculated i.v. into lethally irradiated syngeneic mice. Mice were monitored periodically for clinical signs of disease and leukocytosis. When terminally sick, animals were sacrificed and 1 × 106 leukemic BM cells were inoculated into syngeneic recipients.Transduction of Lin- BM cells from 129Sv mice with a modified retroviral vector (pBABE backbone with ΔNGFR as a reporter) expressing PML-RARα was performed as previously described (Minucci ex vivo with EZN-2968 and EZN-3088 in an Amaxa™ 4D-Nucleofector™ System (Lonza) and transplanted into syngeneic mice 24 h after transfection. 1 × 106 live cells per recipient mouse were transplanted for the experiments in Fig 5 cells transfected with EZN-2968 or EZN-3088 were transplanted in 4 mice/cohort, and mice were treated with ATRA for 4 consecutive days at day 21 after leukemia challenge, when control animals showed leukemia involvement in BM. Second transplantation was performed with 1 × 106 cells. ATRA was administered by subcutaneous implantation of 2 release pellets (5 mg each).Mouse APL cells were transfected t-tests were used to validate the significance of the data analyzed and considered statistically significant when P < 0.05. For survival experiments, Kaplan–Meier curves were analyzed with the Mantel–Cox test.Two-sided et al, et al, et al, n = 25) and normal promyelocytes (n = 3). The experimental platform was Affymetrix GeneChip Human Genome HG-U133A for all the samples. Raw data (retrieved as * CEL files) were pre-processed using the GCRMA library  and APL (n = 14) samples from the publicly available dataset GSE12662 , containing data acquired by means of the Affymetrix HG-U133 Plus 2.0 Array.To compare the expression levels of HIF-1α and HIF-2α (EPAS1), we retrieved CD34et al, ad hoc list of direct HIF-1α targets. A t-statistic metric was applied to establish the gene ranking, and significance was provided both in terms of nominal P-value and as FDR Q-value to take into account the adjustment for multiple hypothesis testing.Hypoxia related signatures were retrieved from the collection of curated gene sets (c2) of the Molecular Signatures Database (MSigDB). A gene set enrichment analysis (GSEA) (Subramanian Hypoxia-inducible transcription factors (HIF) have long been involved in regulating tumor progression in a variety of solid tumors. More recently, emerging literature is beginning to implicate HIF factors in leukemogenesis. In acute myeloid leukemia, HIF-1α has been found overexpressed in a sub-group of cancer cells with leukemia propagating capacity and was shown to regulate leukemia stem cell maintenance. However, a specific functional interaction of HIF-1α with oncogenic fusion proteins causative of specific leukemia sub-types has not yet been investigated.We have studied the role of HIF-1α in acute promyelocytic leukemia (APL), a sub-type of acute myeloid leukemia characterized by expression of the fusion protein PML-RARα. We found that although acting predominantly as a transcriptional repressor, PML-RARα functionally cooperates with HIF-1α and activates the expression of a number of HIF-target genes, which then regulate leukemia progression at multiple levels. As a consequence, inhibition of HIF-1α blunts leukemia progression, and also exquisitely cooperates with retinoic acid in eradicating leukemia-initiating cells.The impact of our study is twofold: on one hand, we provide molecular insights into the complex function of the PML-RARα oncoprotein, by defining a new important function of PML-RARα as a HIF-1α-transcriptional co-activator. On the other hand, our work places HIF factors as important regulators of the pathogenesis and response to therapy of acute promyelocytic leukemia, and prompt further investigation into the role of HIF-1α factors in other types of leukemia.


Cycloaddition reaction between a menthone-based chiral nitrone and 2,5-dihydrofuran under microwave activation affords a enantiopure cycloadduct in good yields and with high stereoselectivity 17H28N2O3, the isoxazolidine ring adopts an envelope conformation with the O atom deviating from the mean plane of the other four ring atoms by 0.617 (1) Å. In the crystal, molecules are linked via weak C—H⋯O hydrogen bonds, forming chains which extend along the b-axis direction.In the title compound, C The present work reports the synthesis and the X-ray crystallographic study of this substituted isoxazolidine, the title compound, C17H28N2O3, (I)The 1,3-dipolar cycloaddition of nitrones to alkenes has been applied to produce substituted isoxazolidines via non-classical weak C5—H52⋯O13i hydrogen bonds, forming zigzag chains, which extend along the b-axis direction ,6(S),9(R)-6-isopropyl-4,9-dimethyl-3-oxo-1,4-diazaspiro[4.5]dec-1-ene-1-oxide] (1 eq.) in anhydrous toluene (4 ml) was introduced. The vial was flushed with argon and 2,5-dihydrofuran  was added. The vial was sealed with a septum cap and was irradiated with microwaves (temperature: 373 K)  Fig. 3. TLC monUiso(H) (in the range 1.2–1.5 times Ueq of the parent atom). These were subsequently refined with riding constraints , C7(S), C8(S), C9(S), C14(S), C20(R) assignments for the six arbitrarily named chiral centres in the molecule. The inverted structure gave a similarly high Flack factor .Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989016010641/zs2363sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S2056989016010641/zs2363Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989016010641/zs2363Isup3.cmlSupporting information file. DOI: 1489217CCDC reference: crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Bacillus licheniformis generated six novel epothilone A glycoside analouges including epothilone A 7-O-β-D-glucoside, epothilone A 7-O-β-D-galactoside, epothilone A 3,7-O-β-D-digalactoside, epothilone A 7-O-β-D-2-deoxyglucoside, epothilone A 7-O-β-L-rhamnoside, and epothilone A 7-O-β-L-fucoside. Epothilone A 7-O-β-D-glucoside was structurally elucidated by ultra-high performance liquid chromatography-photo diode array (UPLC-PDA) conjugated with high resolution quantitative time-of-flight-electrospray ionization mass spectroscopy (HR-QTOF ESI-MS/MS) supported by one-and two-dimensional nuclear magnetic resonance studies whereas other epothilone A glycosides were characterized by UPLC-PDA and HR-QTOF ESI-MS/MS analyses. The time dependent conversion study of epothilone A to epothilone A 7-O-β-D-glucoside found to be maximum (~26%) between 3 h to 5 h incubation.Epothilones are extremely cytotoxic chemotherapeutic agents with epoxide, thiazole, and ketone groups that share equipotent kinetic similarity with taxol. The in vitro glycosylation catalyzed by uridine diphosphate glucosyltransferase (YjiC) from  Sorangium cellulosum . Epothig et al. ). Differt et al. ). SimilaEothilones have been recognized as chemotherapeutic agents against different tumor cell lines including those affected by taxanes with manageable toxicity profiles demonstrated in both preclinical and clinical trials . Enzymahttp://www.cazy.org/). Carbohydrate moieties are usually attached to molecules via the O-glycosidic bond but some are linked through C– or N-glycosidic linkages . The first approach is catalyzed by enzymes called UDP-glycosyltransferases, which belong to the GT1 family proteins in the CAZy classification (d Mendez ). The nuBacillus licheniformis DSM 13. YjiC conjugates different sugar units in plant natural products with low molecular weight, including chalcone , flavony et al. ) and poty et al. ). In thiEpothilone A was available in the laboratory . UDP-D-glucose and UDP-D-galactose were purchased from Sigma-Aldrich Chemical Co. . TDP-D-2-deoxyglucose, TDP-L-rhamnose, and GDP-L-fucose were obtained from GeneChem . High performance liquid chromatography (HPLC) grade methanol and water were purchased from Mallinckrodt Baker . The remaining chemicals were high-grade products purchased from commercially available sources.E. coli BL21 (DE3)  expression host for protein production. E. coli BL21 (DE3) harboring the pET28 (a)-YjiC strain was grown in Luria- Bertani (LB) liquid medium. The culture was incubated at 37°C with kanamycin (50 μg/mL) supplementation when required.A previously constructed pET28 (a)-YjiC  plasmidE. coli BL21(DE3) harboring pET28 (a)-YjiC was cultured overnight in a shaking incubator in 37°C and 150 rpm. 500 μL of seed culture was transferred to fresh 50 mL LB medium supplemented with 50 μg/mL kanamycin. Once the optical density at 600 nm reached 0.6, the culture was induced with a 0.8 mM final concentration of isopropyl-β-D-thiogalactopyranoside and incubated for 20 h at 20°C in shaking incubator at 150 rpm. The cell pellets were harvested via centrifugation at 3,000 rpm for 15 min and washed (vortex followed by centrifugation) with buffer (50 mM Tris–HCl and 10% glycerol of pH 7.4) two times and re-suspended in 1 mL of the same buffer. The cells were sonicated, and the clear lysate was collected by high speed centrifugation at 12,000 rpm for 30 min at 4°C. The clear lysate thus obtained was packed into a His-TALON metal nickel affinity resin  for 30 min at 4°C. The resin-bound protein was eluted step by step by gravity flow with increasing concentrations of imidazole . Fractions containing purified protein were analyzed by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and further concentrated using Amicon Ultra-15 filters . The purified and concentrated protein was stored in buffer containing 50 mM Tris–HCl, pH 7.4, and 10% glycerol at −20°C.A single transformant of 2 6H2O, 2 mM substrate (dissolved in Dimethyl sulfoxide (DMSO)) and 4 mM NDP-sugars individually with 30 μg/mL of appropriately diluted enzyme, and the remaining volume was Milli-Q distilled water. The mixture was incubated at 37°C for 3 h and quenched by adding 300 μL chilled methanol (HPLC grade). The reaction mixture without enzyme was kept as a control. The reaction mixtures were centrifuged at 12,000 rpm for 20 min to remove protein precipitates. Thus, the mixture was finally analyzed by UPLC-PDA and HR-QTOF ESI-MS analyses.The glycosylation reaction was carried out under two conditions using purified protein (YjiC) with the epothilone A and UDP-D-glucose, UDP-D-galactose, TDP-D-2-deoxyglucose, TDP-L-rhamnose, and GDP-L-fucose as sugar donors. A lab-scale reaction was conducted using a 100 μL reaction volume and maintaining a final concentration of 50 mM Tris–HCl buffer at pH 7.4, 10 mM MgCl2 and incubated for 18 h at 37°C. The reaction was stopped by adding a triple volume of chilled methanol. The reaction mixture was continuously mixed by vortex followed by centrifugation at 12,000 rpm for 30 min at 4°C to remove the denatured protein. The supernatant was concentrated by evaporation and lyophilization.The preparative-scale reaction was carried out in a 5 mL volume with purified YjiC (300 μg/mL), 8 mM UDP-D-glucose (~49 mg), 5 mM substrate , 50 mM Tris–HCl (pH 7.4) buffer, and 10 mM MgCl18, 1.7 μm) connected to a PDA (UPLC LG 500 nm) at a UV absorbance of 249 nm. The binary mobile phases were composed of solvent A (HPLC grade water) and solvent B . Total flow rate was maintained at 0.4 μL/min for the 10 min program. Flow of B (MeOH) was 0% to 5 min and increased to 100% until 8 min followed by constant flow of 0% at 8–10 min and then flow was stopped at 10 min. The HR-QTOF ESI-MS analysis was performed in positive ion mode on an ACQUITY  column coupled with SYNAPT G2-S (Water Corp).The reaction mixture was analyzed by reverse-phase UPLC-PDA with a C18 column  connected to a UV detector at a UV absorbance of 249 nm using a 46 min binary program with flow of solvent B (100% MeOH) and A (HPLC grade water). Flow of B was initially maintained at 20% and increased to 75% until 25 min, 90% remained (25–35 min) and then flow was decreased to 50% at 40 min followed by 20% at 45 min and finally stopped at 46 min with a flow rate of 10 ml/min.The dried preparative scale reaction product was finally purified by preparative HPLC  with a Cd4, and subjected to 900 MHz Bruker, BioSpin nuclear magnetic resonance (NMR) analysis including one-dimensional 1H NMR, 13C NMR, and two-dimensional NMR-correlation Spectroscopy (COSY), rotating-frame nuclear Overhauser effect spectroscopy (ROESY), heteronuclear single quantum coherence (HSQC), and heteronuclear multiple bond connectivity (HMBC) when appropriate.The product was dried, lyophilized, dissolved in methanol-E. coli BL21 (DE3) soluble fraction as an N-terminal hexahistidine-tagged fusion protein. The crude protein was purified using nickel affinity chromatography. SDS-PAGE analysis of the protein produced a distinct band corresponding to hexahistidine-tagged YjiC as in our previous report  was heterologously expressed in y et al. ). The putR) of 3.51 min  and two-dimensional NMR  analyses were carried out to elucidate the structure of epothilone A glucoside. The 1H NMR study of the purified reaction product showed the presence of an anomeric proton at the chemical shift δ = 4.49 ppm , representing an anomeric proton with a beta (β) configuration of the sugar moiety, whereas other spectra were observed in the sugar region between δ = (3.0–4.0) ppm  was in a close relationship with δ = 3.2 ppm, which was annotated to H-6 of epothilone by comparing our data with 1H NMR of previously identified epothilone A and pentoside of epothilone as epothiloneoside A by Zhao et al. [Aspergillus niger , H-6 (δ = 3.29), H-2' (δ = 3.41), and H-7 (δ = 3.83) providing a clue regarding the attachment of a sugar in the C-7 hydroxyl group of epothilone A  distinctly. Similarly, the HMBC analysis , H-7 (δ = 3.83), and H-6 (δ = 3.2), confirming the glucosylation position at the C-7 hydroxyl group. The NMR data were further supported by HR-QTOF ESI-MS/MS analysis in which the fragment of glucose conjugated at C-7 hydroxyl position was observed, but we could not able to find the fragment of epothilone A conjugated at C-3 hydroxyl position  strain culture, and the glycosylated compound was detected as attachment of sugar in C-3 hydroxyl position with a molar mass of 625.29. The compound was determined to be 3-O-α-D-ribofuranosyl epothilone A  which wO-β-D-glucoside and to find out the stability of thus produced glucoside derivative in the reaction mixture. Samples were taken out at different times  and were subjected to UPLC-PDA at an absorbance of 249 nm. Under the 8 h reaction mixture analyses, the maximum glucoside conversion was achieved between 3 h to 5 h of incubation (25.55–25.56%) at 37°C, whereas product formation declined after 5 h , epothilone A 3,7-O-β-D-digalactoside      was also produced using UDP-D-galactose as the sugar donor . Drugs Microtubules play a crucial role in cellular processes such as cell motility, intracellular trafficking, cell division, and cell maintenance. Thus, current compounds targeting microtubules are being developed as cancer chemotherapeutic agents . The miO-β-D-glucoside. This concludes the C-7 hydroxyl position of epothilone A is regiospecific for the glycosylation because as we can see the C-3 hydroxyl position is much hindered by the presence of two keto-groups in parallel position.Taxens are considered to be first-line option for metastatic breast cancer, but their utility was compromised because of resistivity . Thus, Engineering the sugar moiety in natural products has always generated novel therapeutic drugs and enhanced their parental properties based on structural, functional, and cell recognition . Thus, The authors declare that they have no competing interests.1-dimensional 1H-NMR and 13C- NMR of epothilone A standard. 13C-NMR and 2-dimensional NMR analyses of epothilone A 7-O-beta-D-glucoside. HR-QTOF ESI-MS/MS analysis of diverse Epothilone A glycosides.Click here for file


Saccharomyces cerevisiae JHY605 was used as a host strain, where the production of ethanol and glycerol was largely eliminated by deleting five alcohol dehydrogenase genes  and two glycerol 3-phosphate dehydrogenase genes (GPD1 and GPD2). To improve acetoin production, acetoin biosynthetic genes from Bacillus subtilis encoding α-acetolactate synthase (AlsS) and α-acetolactate decarboxylase (AlsD) were overexpressed, and BDH1 encoding butanediol dehydrogenase, which converts acetoin to 2,3-butanediol, was deleted. Furthermore, by NAD+ regeneration through overexpression of water-forming NADH oxidase (NoxE) from Lactococcus lactis, the cofactor imbalance generated during the acetoin production from glucose was successfully relieved. As a result, in fed-batch fermentation, the engineered strain JHY617-SDN produced 100.1 g/L acetoin with a yield of 0.44 g/g glucose.Acetoin is widely used in food and cosmetic industry as taste and fragrance enhancer. For acetoin production in this study,  Acetoin, also known as 3-hydroxy-2-butanone or acetylmethylcarbinol, is widely used in food industry as a flavor enhancer, giving a buttery taste23156Bacillus subtilis, Bacillus amyloliquefaciens, Enterobacter cloacae, Serratia marcescens, and Paenibacillus polymyxa, can produce acetoin from pyruvate via α-acetolactate by two enzymatic steps catalyzed by α-acetolactate synthase and α-acetolactate decarboxylase1B. subtilis (JNA-UD-6), isolated after mutagenesis using UV irradiation with diethyl sulfate, showed a 24.3% increase in acetoin production and a 39.8% decrease in 2,3-butanediol production compared with the parental strain in batch fermentation, and produced 53.9 g/L acetoin after 144 h fermentation in fed-batch fermentation+, was overexpressed to reduce NADH-dependent 2,3-butanediol production. In S. marcescens H32, introduction of NADH oxidase from Lactobacillus brevis decreased 2,3-butanediol titer by 48% and increased acetoin titer by 33%B. subtilis and E. cloacae, resulting in 56.7 g/L and 55.2 g/L acetoin production, respectively11Many microorganisms, such as Saccharomyces cerevisiae, which is classified as generally recognized as safe (GRAS) microorganism, has been considered as a key cell factory platform for producing valuable chemicals because of its tolerance and robustness toward industrial conditions. Acetoin accumulation has been reported in commercial wine yeast engineered for glycerol overproductionGPD1 and deleting ALD6  produced 26.9 g/L glycerol with 9.5 g/L acetoin from 200 g/L glucose. In addition, several efforts have been made to engineer S. cerevisiae to produce 2,3-butanediol, a neighboring metabolite of acetoin, leading to a significant improvement in both titer and yield of 2,3-butanediol production1415S. cerevisiae has not yet been reported.S. cerevisiae strain for efficient production of 2,3-butanediol by introducing heterologous acetoin biosynthetic pathway from B. subtilis, overexpressing 2,3-butanediol dehydrogenase, and eliminating major byproduct pathways involved in ethanol and glycerol productionLactococcus lactis. In this study, for efficient production of acetoin, we additionally disrupted 2,3-butanediol dehydrogenase in adh1-5Δgpd1Δgpd2Δ strain and adopted above strategies comprised of introducing heterologous acetoin pathway and redox rebalancing.In our previous study, we developed S. cerevisiae, α-acetolactate synthase  and α-acetolactate decarboxylase  genes from B. subtilis were introduced into JHY605 strain (adh1-5Δgpd1Δgpd2Δ), which lacks five alcohol dehydrogenases (Adh1 to Adh5) and two glycerol 3-phosphate dehydrogenases (Gpd1 and Gpd2)  . The ferS. cerevisiae. By introducing this competing pathway, ethanol production yield was reduced to 0.02 g/g glucose in JHY605-SD compared with that in JHY605-C (0.12 g/g glucose).Whereas, strain JHY605 harboring p413-SD plasmid (JHY605-SD) showed an increase in glucose consumption rate and produced up to 5.9 g/L acetoin . FurtherS. cerevisiae, NAD+ regeneration for glycolysis is mainly achieved by producing ethanol and glycerol , 2,3-butanediol production from acetoin was significantly reduced to 0.2 g/L, which then contributed to the increase in acetoin production accordingly. As a result, up to 15.4 g/L acetoin was produced after 72 h fermentation in SC-His medium containing 50 g/L glucose, with a yield of 0.30 g/g glucose  showed a significant improvement in glucose consumption rate, thereby taking less time (~48 h) to completely ferment 50 g/L glucose than it was taken for JHY617-SD (~72 h)  . Moreove (~72 h) . To conf by NoxE .600 of 9.5. In fed-batch fermentation, up to 100.1 g/L acetoin was produced with a yield of 0.44 g/g glucose after 55 h cultivation, reaching 90% of maximum theoretical yield  as a template, using the primer pair of d_BDH1 F (5′-GATTTGCTCACGCTACTTTG-3′) and d_BDH1 R (5′-GCCATGCTTTGTTTTAGACG-3′). The resulting PCR product was transformed into JHY605 strain and transformants were selected on YPD plate  supplemented with 200 μg/mL G418 sulfate All strains used in this study are described in Yeast cells were cultured in YPD medium or in synthetic complete medium lacking histidine (SC-His) .alsS-expression cassette  flanked by MauBI and and NotI sites was obtained by PCR from p413_PTDH3-alsS-TCYC1 using the primers, Univ F2 (5′-GACTCGCGCGCGGGAACAAAAGCTGGAGCTC-3′) and Univ R (5′-GACTACGCGTGCGGCCGCTAATGGCGCGCCATAGGGCGAATTGGGTACC-3′), and cloned into AscI and NotI sites of p413-D plasmidnoxE-expression cassette (PFBA1-noxE-TFBA1) was additionally cloned into p413-SD as previously describedPlasmids used in this study are described in 600 of 0.3 in 10 mL of SC-His medium containing 50 g/L glucose in a 100 mL flask, and then cultivated at 30 °C with shaking at 170 rpm.For flask fermentation, yeast cells harboring appropriate plasmids were pre-cultured in 5 mL of SC-His medium containing 20 g/L glucose in a 50 mL flask, inoculated to OD600 of 9.5. The feeding solution (800 g/L glucose) was intermittently added to the culture medium when the glucose concentration was lower than 20 g/L.Fed-batch fermentation was performed in 500 mL YPD medium containing 100 g/L glucose using a 1 L bench-top fermenter FMT-DS  at 30 °C with agitation speed of 500 rpm and air flow rate of 1.0 vvm. The pH of the culture medium was maintained at 5.5 by using 4 N NaOH. Strain JHY617-SDN was pre-cultured in SC-His medium containing 20 g/L glucose and inoculated into the fermenter with initial OD600). To analyze profile of metabolites, 1 mL of culture supernatants were collected and filtered through a 0.22 μm syringe filter. The concentrations of glucose, glycerol, acetoin, 2,3-butanediol, and ethanol were determined by high performance liquid chromatography (HPLC) using UltiMate 3000 HPLC system (Thermo fishers scientific) equipped with a BioRad Aminex HPX-87H column  at 60 °C with 5 mM H2SO4 at a flow rate of 0.6 mL/min and refractive index (RI) detector at 35 °C. The intracellular concentrations of NADH and NAD+ were measured using EnzyChrom™ NAD/NADH Assay Kit . Strains JHY617-SD and JHY617-SDN were harvested at different time points of fermentation and washed with cold phosphate-buffered saline  solution. Cells of OD600 of 1.0 were pelleted and analyzed according to the manufacturer’s instructions.Cell growth was determined by measuring the optical density at 600 nm .


The down-regulation of PPARγ has gained increasing attention in neuroinflammation of Alzheimer’s disease (AD). Thus, the present study focused on exploring the influence of seipin depletion on β-amyloid (Aβ)-induced neuroinflammation and Aβ neurotoxicity.A characteristic phenotype of congenital generalized lipodystrophy 2 (CGL2) that is caused by loss-of-function of seipin gene is mental retardation. Seipin is highly expressed in hippocampal pyramidal cells and astrocytes. Neuronal knockout of seipin in mice (seipin-KO mice were treated with a single intracerebroventricular (i.c.v.) injection of Aβ25–35 (1.2 nmol/mouse) or Aβ1–42 (0.1 nmol/mouse), generally a non-neurotoxic dose in wild-type (WT) mice. Spatial cognitive behaviors were assessed by Morris water maze and Y-maze tests, and hippocampal CA1 pyramidal cells and inflammatory responses were examined.Adult male 25–35/1–42 injection in the seipin-KO mice caused approximately 30–35 % death of pyramidal cells and production of Hoechst-positive cells with the impairment of spatial memory. In comparison with the WT mice, the number of astrocytes and microglia in the seipin-KO mice had no significant difference, whereas the levels of IL-6 and TNF-α were slightly increased. Similarly, the Aβ25–35/1–42 injection in the seipin-KO mice rather than the WT mice could stimulate the activation of astrocytes or microglia and further elevated the levels of IL-6 and TNF-α. Treatment of the seipin-KO mice with the PPARγ agonist rosiglitazone (rosi) could prevent Aβ25–35/1–42-induced neuroinflammation and neurotoxicity, which was blocked by the PPARγ antagonist GW9962. In the seipin-KO mice, the level of glycogen synthase kinase-3β (GSK3β) phosphorylation at Tyr216 was elevated, while at Ser9, it was reduced compared to the WT mice, which were corrected by the rosi treatment but were unaffected by the Aβ25–35 injection.The Aβ25–35/1–42-induced neuroinflammation to cause the death of neuronal cells and cognitive deficits.Seipin deficiency in astrocytes increases GSK3β activity and levels of IL-6 and TNF-α through reducing PPARγ, which can facilitate AβThe online version of this article (doi:10.1186/s12974-016-0598-3) contains supplementary material, which is available to authorized users. Congenital generalized lipodystrophy (CGL) is an autosomal recessive disorder that is characterized by a near-total loss of adipose tissue . Genome-seipin-KO mice) reduces the expression of the hippocampal PPARγ [Seipin, an exclusive endoplasmic reticulum-residing N-glycosylated protein, can affect the generation of peroxisome proliferator-activated receptor gamma (PPARγ) . The leval PPARγ , 10, 11.al PPARγ . PPARγ aal PPARγ  and redual PPARγ . PPARγ dal PPARγ  and multal PPARγ . The leval PPARγ . The actal PPARγ . The actal PPARγ . The PPAal PPARγ . The PPAal PPARγ . The hipseipin-KO mice [seipin-KO mice showed an increase in the astrocytic differentiation of progenitor cells in the hippocampal dentate gyrus, which can be corrected by the activation of PPARγ [The beneficial effects of PPARγ ligands potentially involve the suppression of signal transducer and activator of transcription (STAT) . Li et a-KO mice . The STA-KO mice . Adult sof PPARγ . The STAof PPARγ . GSK3 isof PPARγ , 26.25–35) is the shortest fragment of Aβ that is processed in vivo by brain proteases [1–40 [seipin-KO mice with a single i.c.v. injection of Aβ25–35 at a non-neurotoxic dose in control mice [25–35 neurotoxicity  and Aβ25–35-induced neuroinflammation . To test the involvement of reduced PPARγ by seipin deficiency, we investigated the effects of the PPARγ agonist and the PPARγ antagonist on Aβ25–35-induced neuroinflammation and Aβ25–35 neurotoxicity in the seipin-KO mice. The results of the present study indicate that seipin deficiency in astrocytes facilitates the Aβ25–35-induced neuroinflammation through reducing PPARγ to elevate GSK3β activity, which causes the death of pyramidal cells leading to the spatial cognitive deficits.Among the Aβ fragments, a peptide bearing 11 amino acids (25–35)  and 5′-TCAACAGAACAGACGCT-3′ (reverse primer for loxP); and 5′-GCGGTCTGGCAGTAAAAACTATC-3′ (forward primer for nestin-Cre) and 5′-GTGAAACAGCATTGCTGTCACTT-3′ (reverse primer for nestin-Cre). All animals received a standard laboratory diet before and after all procedures. In this study, 16-week-old male seipin-KO mice (n = 88) and wild-type (WT) mice (n = 56) were used at the beginning of all experiments. All mice were randomly assigned to one of the following six experimental groups: WT mice (n = 24), Aβ25–35-treated WT mice , Aβ1–42-treated WT mice , seipin-KO mice (n = 32), Aβ25–35-treated seipin-KO mice , and Aβ1–42-treated seipin-KO mice . Each experiment was performed by two experimenters who were blinded to the experimental groups. The behavioral tests (n = 16 mice for each group) and histological observations (n = 8 mice for each group) or Western blot analyses (n = 8 mice for each group) were sequentially performed in the same cohorts.All animal handling procedures followed the guidelines for Laboratory Animal Research of Nanjing Medical University. The use of animals was approved by the Institutional Animal Care and Use Committee of Nanjing Medical University. The mice were maintained in constant environmental conditions . The neuronal lsewhere . The gen25–35  was aggregated by incubation in distilled water (1 mg/ml) at 37 °C for 4 days and diluted to the final concentration with saline immediately before the experiment [25–35 can be observed by light microscopic observation [1–42  was dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol  and then flash-freezed in liquid nitrogen and lyophilized to completely remove the solvent [1–42 was dissolved in 100 mM NaOH at a concentration of 6 mg/ml, aliquoted in 50 μl volumes [25–35 (1.2 nmol/mouse at 2 μl) or the Aβ1–42  was injected into the ventricles  using a stepper-motorized micro-syringe at a rate of 0.3 μl/min [The Aβperiment . After tervation . The Aβ1 solvent . Lyophil volumes . The mic volumes  and plac3 μl/min . The injThe PPARγ agonist rosiglitazone  and the PPARγ antagonist GW9962  were dissolved in dimethyl sulfoxide (DMSO) and then diluted in 0.9 % saline to a final concentration of 0.5 % DMSO. The oral administration (p.o.) of rosi was given daily at a dose of 5 mg/kg . The GW925–35 injection were captured via video recording  and analyzed using TopScan Lite 2.0 . Morris water maze task: The water maze task was consecutively performed to examine the spatial memory [Y-maze task: Spatial working memory performance was assessed by recording spontaneous alternation behavior in a Y-maze [The behavioral performances on days 5–14 after Aβl memory . A pool a Y-maze . A Y-mazThe mice were anesthetized with chloral hydrate  and perfused with 4 % paraformaldehyde. The brains were removed and immersed in 4 % paraformaldehyde at 4 °C overnight.Toluidine blue staining: The brains were processed for paraffin embedding, and coronal sections (5 μm) were cut. The pyramidal cells in the hippocampal CA1 region were identified using a conventional light microscope  with a 60× objective. The healthy pyramidal neurons exhibited round cell bodies with plainly stained nuclei. Stereological counting CA1 pyramidal cells: The brains were transferred into 30 % sucrose. After gradient dehydration, the coronal sections of the hippocampus (40 μm) were cut with a freezing microtome  and stained with toluidine blue. Every fourth section was obtained for consecutive cell quantification analyses. Stereological cell counting was performed by a stereological system, consisting of a light microscope with a CCD camera (Olympus DP70), a motorized specimen stage for automatic sampling , and a computer running Microbrightfield Stereo Investigator software  [VT, USA) . All couVT, USA) . The bouHoechst staining: Coronal paraffin sections (5 μm) were incubated in Hoechst 33342  for 2 min. Quantitative analyses of Hoechst-positive cells: Hoechst-positive (Hoechst+) cells in hippocampal CA1 region were observed by a conventional light microscope  with 60× objective and counted. The density of Hoechst+ cells was expressed as the number of cells per millimeter length along the pyramidal cells layer.GFAP and Iba1 staining: The hippocampal sections (40 μm) were cut using a cryostat. Every fourth section was obtained with a randomly chosen starting section. The free-floating sections were blocked with 3 % normal goat serum for 60 min. Glial fibrillary acidic protein (GFAP) and ionized calcium-binding adapter molecule 1 (Iba1) immuno-staining were performed using the following primary antibodies and incubation at 4 °C overnight: mouse anti-GFAP antibody  and goat polyclonal anti-Iba1 antibody . Then, the sections were incubated in biotin-labeled goat anti-mouse IgG antibody  or biotin-labeled rabbit anti-goat IgG antibody  for 2 h at room temperature. Immuno-reactivity was visualized based on the avidin-biotin-horseradish peroxidase complex .Counting GFAP- and Iba1-positive cells in the hippocampal CA1: The GFAP-positive (GFAP+) cells and Iba1-positive (Iba1+) cells in the hippocampal CA1 radiatum layer were counted using a conventional light microscope (Olympus DP70). The radiatum layer was outlined at ×5 magnification. Systematic random sampling was achieved with a uniform sampling grid superimposed randomly over the region. A counting frame was placed in each square of the grid, and the cells were counted within that frame at ×40 magnification. A cell was counted if the nucleus was stained and was (a) in focus, (b) within the counting frame or crossed the green counting frame inclusion line, and (c) within the optical disector. Total estimated cell number per section = number of counted cells × (1/ssf) × (1/asf) × (1/hsf), where ssf is the section sampling fraction, asf is the area sampling fraction, and hsf is the height sampling fraction [+ cells and Iba1+ cells are expressed as the mean numbers per mm3, which were normalized to the control value obtained from WT mice [fraction . The den WT mice .Seipin/GFAP or Iba1 double immuno-staining: The coronal sections (5 μm in thickness) were placed in gelatine-coated slides, blocked with 3 % normal goat serum, and then incubated in rabbit anti-seipin antibody  at 4 °C overnight, which was revealed using CY3-labeled anti-rabbit IgG antibody  or FITC-labeled anti-rabbit antibody  and mouse monoclonal anti-GFAP antibody  or goat polyclonal anti-Iba1 antibody , which was revealed using FITC-labeled anti-mouse antibody  or CY3-labeled anti-goat IgG antibody . The immune-positive cells/fibers were observed by a confocal laser-scanning microscope .The mice were decapitated under deep anesthesia with chloral hydrate. The hippocampus was quickly removed and homogenized in a lysis buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 10 mM NaF, 1 mM sodium orthovanadate, 1 % Triton X-100, 0.5 % sodium deoxycholate, 1 mM phenyl-methylsulfonyl fluoride, and a protease inhibitor cocktail . The protein concentration was determined with a BCA Protein Assay Kit . The total proteins (20 μg) were separated by SDS-poly-acrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride membrane. The membranes were incubated with 5 % nonfat dried milk in Tris-buffered saline containing 0.1 % Tween 20 for 60 min at room temperature and were then incubated with a mouse monoclonal anti-phospho-GSK3β (Tyr216) antibody , a rabbit monoclonal anti-phospho-GSK3β (Ser9) antibody, and an anti-phospho-STAT3 antibody ; a rabbit polyclonal anti-IL-6 antibody , a mouse monoclonal anti-TNF-α antibody and a PPARγ antibody ; or a mouse monoclonal anti-β-actin antibody  and a rabbit monoclonal anti-GAPDH antibody  at 4 °C overnight. The membranes were then incubated with HRP-labeled secondary antibodies . The blots were stripped by incubation in a stripping buffer  for 5 min, incubated with rabbit polyclonal antibodies for GSK3β or STAT3  and developed using an ECL Detection Kit (Millipore). The Western blot bands were scanned and analyzed with an image analysis software package .t tests or ANOVAs with or without repeated measures, followed by Bonferroni post hoc analysis for multiple comparisons. The results of the Morris water maze test were analyzed using repeated-measures ANOVA. The statistical analyses were performed using Stata 7 software . Differences were considered statistically significant at P < 0.05.The data were retrieved and processed with the Microcal Origin 8.0 software. The group data are expressed as the means ± standard errors, and significance was tested with Student’s 25–35. The mean latency required to find the visible platform on days 1–2 of training and the subsequent escape latencies to reach the hidden platform on days 3–7 of training are illustrated in Fig. F = 0.179, P = 0.674), Aβ25-35 injection  = 0.054, P = 0.818), or the genotype × Aβ25–35 interaction  = 3.159, P = 0.081). Subsequently, repeated-measures ANOVA revealed that the escape latency to reach the hidden platform progressively decreased with training days in all groups  = 71.948, P < 0.001), and this effect was influenced by genotype  = 190.133, P < 0.001), Aβ25–35  = 39.457, P < 0.001), and the genotype × Aβ25–35 interaction  = 36.701, P < 0.001). The seipin-KO mice required longer times to reach the hidden platform on days 5–7 of training than did the WT mice (P < 0.01). Furthermore, the Aβ25–35-injected (1.2 nmol/mouse) seipin-KO mice exhibited a significantly increased escape latency to the hidden platform  compared with the seipin-KO mice, but this effect was not observed in the Aβ25–35-injected WT mice  relative to the non-injected WT mice. However, the mean swimming speeds during the visible platform and hidden platform tests exhibited by Aβ25–35-mice , seipin-KO mice , and Aβ25–35-KO mice  were not significantly different from those of the WT mice (0.26 ± 0.03 m/s).Spatial memory was examined using place learning in a Morris water maze task from day 5 after the injection (i.c.v.) of AβF = 74.061, P < 0.001) and Aβ25–35  = 11.813, P = 0.001) were that the seipin-KO mice spent less swimming time in the target quadrant than the WT mice (P < 0.01). Furthermore, there was a significant interaction of genotype × Aβ25–35  = 13.716, P < 0.001). Notably, the swimming time in the target quadrant of the Aβ25–35-KO mice was shorter than that of the seipin-KO mice (P < 0.01), whereas the Aβ25–35-WT mice exhibited no significant difference compared with the WT mice (P > 0.05).A probe test was performed 24 h after the hidden platform test to measure the strength of the memory trace. The swimming times spent in the target quadrant, opposite quadrant, and right and left adjacent quadrants are showed in Fig. F = 18.472, P < 0.001; Fig. F = 0.165, P = 0.686; Fig. seipin-KO mice was reduced compared to that of the WT mice (P < 0.05). Additionally, there was a significant interaction of genotype × Aβ25–35 for the alternation ratio  = 7.214, P = 0.009). Notably, Aβ25–35-KO mice exhibited a significant reduction in the alternation ratio compared with the seipin-KO mice (P < 0.05).Spatial working memory performance was assessed with a Y-maze task. There was a main effect of genotype on the alternation ratio  = 12.778, P < 0.001; Fig. 25–35  = 0.037, P = 0.848) or genotype × Aβ25–35 interaction  = 0.374, P = 0.546). Compared with the WT mice, the level of hippocampal PPARγ was remarkably reduced in the seipin-KO mice (P < 0.05) but was not significantly different compared with that in the Aβ25–35-KO mice (P > 0.05).The level of hippocampal PPARγ exhibited a main effect of genotype  for consecutive 17 days corrected the prolongation of escape latencies to reach the hidden platform  and the decrease in the swimming time of target quadrant  and increase the swimming time in the target quadrant  and alternation ratio in the Y-maze .Consistent with the report by Zhou et al. , the treion Fig. . In compF = 1.572, P = 0.220; Fig. 25–35  = 2.091, P = 0.159) on the number of pyramidal cells in the hippocampal CA1 region; however, there was a significant genotype × Aβ25–35 interaction  = 4.294, P = 0.048). Stereological counts of the surviving pyramidal cells revealed an approximately 35 % loss of pyramidal cells in the Aβ25–35-KO mice (P < 0.01) and no loss in the Aβ25–35-mice (P > 0.05). As illustrated in Fig. +) cells were observed in the Aβ25–35-KO mice  but not in the seipin-KO mice  or the Aβ25–35-mice . The rosi treatment in the Aβ25–35-KO mice could attenuate the deaths of pyramidal cells (P < 0.05) and the number of Hoechst+ cells (P < 0.01), which were sensitive to the PPARγ antagonist GW9962 . The administration of GW9962 alone in the seipin-KO mice did not affect the survival of pyramidal cells (P > 0.05). Similarly, the Aβ1–42 injection in the seipin-KO mice rather than the WT mice caused approximately 30 % death of pyramidal cells .There was no main effect of genotype . The numbers of Iba1+ cells  and GFAP+ activated astrocytes  were significantly increased in the Aβ25–35-KO mice compared with the seipin-KO mice, which were reduced by the rosi treatment . The inhibitory effects of rosi on the activation of Iba1+ cells  and GFAP+ activated astrocytes  in the Aβ25–35-KO mice could be blocked by the PPARγ antagonist GW9962. By contrast, Aβ25–35 mice did not show the changes in the numbers of Iba1+ cells  and GFAP+ cells  compared to the WT mice. Additionally, the Aβ1–42 injection could increase the numbers of Iba1+ cells  in the seipin-KO mice but not in the WT mice, which was sensitive to the rosi treatment .Sequentially, we observed that GFAPhenotype . NotablyF = 29.253, P < 0.001; Fig. F = 65.073, P < 0.001; Fig. 25–35 interaction, because the levels of TNF-α (P < 0.01) and IL-6 (P < 0.01) were higher in the Aβ25–35-KO mice than those in the seipin-KO mice. However, the levels of TNF-α (P > 0.05) and IL-6 (P > 0.05) exhibited no differences between the WT mice and Aβ25–35 mice. Furthermore, the increased TNF-α and IL-6 levels in the seipin-KO mice  and the Aβ25–35-KO mice  were attenuated by the rosi treatment.Further experiment was designed to examine the influence of seipin deficiency on Aβ-induced inflammatory reactions using Western blot. Notably, there were a main effect of genotype for the levels of hippocampal TNF-α  and the reduction of Ser9 phospho-GSK3β . Additionally, the rosi treatment of the WT mice increased Ser9 phospho-GSK3β  but had no effect on Tyr216 phospho-GSK3β .GSK3β has been reported to modulate pro-inflammatory cytokines . GSK3β aseipin-KO mice was elevated compared with that in the WT mice  but was not affected by the Aβ25–35 injection . Additionally, the rosi treatment increased the level of phospho-STAT3 in the WT mice .The activation of GSK3β is required to activate tyrosine phosphorylation of STAT3 in astrocytes and microglia . Similar25–35/1–42-induced neuroinflammation to aggravate the Aβ25–35/1–42 neurotoxicity. This conclusion was deduced mainly from the following observations: (1) seipin was highly expressed in hippocampal pyramidal cells and astrocytes; (2) the seipin deficiency did not alter the number of neuronal cells, astrocytes, and microglia, but the levels of IL-6/TNF-α were higher in the seipin-KO mice than those in WT mice; (3) the injection (i.c.v.) of Aβ25–35 (1.2 nmol/mouse) or Aβ1–42 (0.1 nmol/mouse) could stimulate the activation of astrocytes and microglia and further increased the expression of IL-6/TNF-α in seipin-KO mice, but not in the WT mice; (4) the injection (i.c.v.) of Aβ25–35 or Aβ1–42, at the non-neurotoxic doses in WT mice, caused approximately 30–35 % death of pyramidal cells with a significant increase of apoptotic cells in hippocampal CA1 of the seipin-KO mice leading to the impairment of spatial memory.The present study provides, for the first time, in vivo evidence that the seipin deficiency in astrocytes can facilitate the Aβ1–42 can increase the production of IL-6 and TNF-α, which is inhibited by the activation of PPARγ [25–35 stimulates microglial activation in the brains of C57BL/6J mice [25–35 (1.2 nmol/mouse) or Aβ1–42 (0.1 nmol/mouse) was insufficient to stimulate the activation of astrocytes and microglia or to increase the production of TNF-α or IL-6 in WT mice. However, this injection (i.c.v.) of Aβ25–35 or Aβ1–42 in the seipin-KO mice caused the activation of astrocytes and microglia with an obvious increase in the level of TNF-α or IL-6. Interestingly, in particular, the activation of PPARγ in the seipin-KO mice was able to prevent the Aβ25–35/1–42-induced neuroinflammation. Similarly, PPARγ-deficient heterozygous mice develop an exacerbated experimental allergic encephalomyelitis (EAE) in comparison with the wild-type littermates [seipin-KO mice did not differ in comparison with the WT mice, the levels of hippocampal TNF-α and IL-6 in the seipin-KO mice were higher than those in the WT mice. In particular, the elevation of TNF-α or IL-6 in the seipin-KO mice was reduced by the PPARγ agonist. Thus, it may be possible that the reduction of PPARγ leads to the overproduction of pro-inflammatory cytokines in the seipin-KO mice, which facilitates the Aβ25–35/1–42-induced neuroinflammation. Another indirect but important supporting data is that the increased TNF-α and IL-6 in the seipin-KO mice could be corrected by the activation of PPARγ. Therefore, the findings give an indication that the seipin deficiency in astrocytes is able to facilitate the Aβ-induced neuroinflammation probably through reducing PPARγ to elevate the levels of TNF-α and IL-6.The treatment of astrocytes and microglia with Aβof PPARγ . The inj/6J mice . The injtermates . The exatermates . Astrocytermates . We obseseipin-KO mice [seipin-KO mice was enhanced as indicated by the elevation of Tyr216 phospho-GSK3β and the reduction of Ser9 phospho-GSK3β. The activation of PPARγ regulates negatively the expressions of GSK3β [seipin-KO mice. Strangely enough, the administration of rosi to the seipin-KO mice could suppress the increase of GSK3β activity but further enhanced the phosphorylation of STAT3. Therefore, it is indicated that seipin deficiency in astrocytes through reducing PPARγ enhances the GSK3β activity rather than the STAT3 signaling to increase the production of TNF-α and IL-6. On the other hand, GSK3β regulates interferon-γ (IFN-γ) signaling and is involved in IFN-γ-induced inflammation [seipin-KO mice through synergistically facilitating IFN-γ-induced STAT1 activation increases the production of TNF-α [GSK3β has been identified as a strong promoter of pro-inflammatory cytokines, including IL-6 and TNF-α , 26. The-KO mice . STAT3 a-KO mice . Notablyof GSK3β , but theammation . Thus, fof TNF-α .25–35/1–42 neurotoxicity as indicated by a massive death of pyramidal cells in the seipin-KO mice treated with the Aβ25–35 (1.2 nmol/mouse) or Aβ1–42 (0.1 nmol/mouse), generally non-neurotoxic doses in the WT mice or control mice [seipin-KO mice. The decline of PPARγ in the seipin-KO mice [25–35/1–42-induced death of pyramidal cells in the seipin-KO mice. Activated microglia and reactive astrocytes can produce cytokines, reactive oxygen species, and other neurotoxic substances to cause neuronal apoptosis and other series of pathologic events. The progressive neuroinflammation and neuronal apoptosis in AD is considered to be a consequence of the Aβ-neurotoxic properties [25–35/1–42-induced neuroinflammation in the seipin-KO mice causes the death of neuronal cells. Zhao et al. [SOD1, CAT, and GST expression in seipin-KO mice did not differ from WT mice (data not shown). Additionally, Ito et al. reported that mutations (N88S/S90L) of the seipin gene can cause the formation of cytoplasmic inclusions and enhance ubiquitination, which leads to endoplasmic reticulum (ER) stress [seipin-KO mice are not increased [A principal finding in this study is that seipin deficiency in hippocampal neuronal cells enhances the Aβrol mice . The lev-KO mice  or the n-KO mice  did not operties , 53. Ouro et al.  reported) stress . Howeverncreased .seipin-KO mice with reduction of PPARγ, we observed that seipin deficiency elevated the activity of GSK3β to enhance the production of TNF-α and IL-6, which in turn triggered and strengthened the Aβ25–35-induced inflammatory responses leading to the death of neuronal cells and the spatial cognitive deficits. This is particularly true of neuroinflammation, which contributes to a broad range of neurodegenerative diseases [Using the diseases . AlthougHippocampal GSK3β activity and IL-6/TNF-α were slightly increased in seipin-KO mice.PPARγ agonist inhibits increased GSK3β activity and IL-6/TNF-α in KO mice.25–35 causes death of pyramidal cells in KO mice, not in WT mice.Low-dose Aβ25–35 activates microglia and astrocytes to increases IL-6/TNF-α in KO mice.Aβ25–35-neuroinflammation and -neurotoxicity in KO mice.PPARγ agonist prevents Aβ+, ionized calcium-binding adaptor molecule 1-positive; IL-6, interleukin-6; PA, phosphatidic acid; PPARγ, peroxisome proliferator-activated receptor gamma; seipin-KO mice, seipin knockout mice; STAT, signal transducer and activator of transcription; TNF-α, tumor necrosis factor-αAD, Alzheimer’s disease; AGPAT2, 1-acylglycerol-3-phosphate-O-acyl transferase 2; Aβ, β-amyloid; BSCL2, Berardinelli-Seip congenital lipodystrophy 2; CGL, congenital generalized lipodystrophy; GFAP, glial fibrillary acidic protein; GSK3, glycogen synthase kinase-3; i.c.v., intracerebroventricular; Iba1


The later adopts an envelope conformation with the central methylene C atom as the flap. The benzyl and and p-tolyl rings are inclined to one another by 56.18 (15)°, and to the pyridine ring by 81.87 (14) and 47.60 (11)°, respectively. In the crystal, molecules are linked by pairs of N—H⋯Nnitrile hydrogen bonds, forming inversion dimers with an R22(12) ring motif. The dimers are linked by C—H⋯π and π–π interactions [centroid–centroid distance = 3.7211 (12) Å], forming a three-dimensional framework.The title compound, C They arH-cyclopenta[b]pyridine-3-carbonitrile -2-oxoethoxy]-6,7-dihydro-5p-toluenesulfonic acid (p-TSA) (1 mmol) was added. The reaction mixture was heated under reflux for 2–3 h. The reaction progress was monitored by thin layer chromatography. After completion of the reaction, the mixture was poured into crushed ice and extracted with ethyl acetate. The excess solvent was removed under vacuum and the residue was subjected to column chromatography using a petroleum ether/ethyl acetate mixture (97:3 v/v) as eluent to obtain the pure product. The product was recrystallized from ethyl acetate, affording colourless crystals of the title compound .A mixture of cyclopentanone (1 mmol) 1, 4-methylbenzaldehyde (1 mmol), malononitrile (1 mmol) and benzylamine were taken in ethanol (10 mL) to which iso(H) = 1.5Ueq(C) for methyl H atoms and = 1.2Ueq for other H atoms.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989015000572/su5061sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S2056989015000572/su5061Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989015000572/su5061Isup3.cmlSupporting information file. DOI: 1042906CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


A unique ‘wagging' mode involving H− motion perpendicular to the Ni(μ-H)57Fe plane was studied using 57Fe-specific nuclear resonance vibrational spectroscopy and density functional theory (DFT) calculations. On Ni(μ-D)57Fe deuteride substitution, this wagging causes a characteristic perturbation of Fe–CO/CN bands. Spectra have been interpreted by comparison with Ni(μ-H/D)57Fe enzyme mimics [(dppe)Ni(μ-pdt)(μ-H/D)57Fe(CO)3]+ and DFT calculations, which collectively indicate a low-spin Ni(II)(μ-H)Fe(II) core for Ni-R, with H− binding Ni more tightly than Fe. The present methodology is also relevant to characterizing Fe–H moieties in other important natural and synthetic catalysts.The metabolism of many anaerobes relies on [NiFe]-hydrogenases, whose characterization when bound to substrates has proven non-trivial. Presented here is direct evidence for a hydride bridge in the active site of the  Understanding the catalytic mechanism of redox-active hydrogenases is a key to efficient hydrogen production and consumption. Here, the authors use nuclear resonance vibrational spectroscopy to study [NiFe]-hydrogenase, and observe a bridging hydride structure in an EPR silent intermediate. In this regard, bidirectional hydrogenase enzymes, which catalyse both production and consumption of H2  resonance (EPR)-silent Ni-SIa, EPR-active Ni-C and another EPR-silent species known as Ni-R1617The redox-active hydrogenases are classified as either [FeFe]- or [NiFe]-hydrogenase, according to the metals present at their active sites. While both types possess high catalytic activity1213II)Fe(II) forms of Ni-R are characterized by their pH-dependent Fourier transform infrared (FT-IR) signaturesμ-H)Fe)192021μ-H)Fe)23η2-H2))25μ-H)Fe core with a protonated terminal cysteinate− is bound more strongly to Fe or Ni (ref. II) is high31Up to three isoelectronic Ni or empirical force field calculations, assisting in confident spectral assignments.The Ni-R state represents a special challenge for spectroscopy in that it is EPR-silent, photolabile (and thus difficult to study with Raman spectroscopy) and expected to feature an active site M–H moiety (being notoriously difficult to observe using infrared methods)34353657Fe-labelled enzyme active sites, avoiding interference from the thousands of protein modes present in a typical infrared or Raman spectrum. NRVS has enabled the observation of Fe–CN and Fe–CO bending and stretching modes for the active sites in [NiFe]-hydrogenases39−1, while Fe–S cluster modes only make significant contributions below 440 cm−1 (refs trans-H/D-57Fe-(CO) compound shed light on coupling of Fe–H/D and Fe–CO bending modesNRVS is uniquely suited to detailed investigations of m−1 refs , 42, 43.The present study discloses the first spectroscopic evidence for the bridging hydride in the Ni-R active site and the unprecedented NRVS observation of a Fe–H stretching mode in a synthetic Ni–H–Fe system. A combined experimental/theoretical analysis of both Ni-R and its synthetic mimics is presented here, an approach that we anticipate to be of broad utility for the characterization of (bio)inorganic Fe-hydride catalysts.II)(μ-thiolate)2(μ-H)Fe(II) core proposed for Ni-R is reproduced by the diamagnetic H2-evolving catalyst [(dppe)Ni(μ-pdt)(μ-H)Fe(CO)3]+ ethane=1,2-Ph2PCH2CH2P Ph2, pdt2−=1,3-propanedithiolate=−SCH2CH2CH2S−)44μ-pdt)(μ-H/D)57Fe(CO)3]+ in both hydride [1′H]+ and deuteride [1′D]+ forms for 57Fe NRVS analysis (see 13CO-labelled species [1′′H/D]+ were prepared as well.The Ni(ysis see . Complem1′H/D]+ are presented in −1) Fe–S modes, there are strong bands in the 440–630 cm−1 region assigned to νFe–CO stretches and δFe–CO bends 57Fe(CO)3 (1′)δFe–CO NRVS triplets for [1′H]+  and 1′  being virtually coincident Fe plane, in what is a unique ‘wagging' mode. While the Ni–H–Fe wag is relatively isolated in [1′H]+ (H motion accounts for ∼80% of the kinetic energy), the corresponding Ni-D-Fe motion in [1′D]+ is predicted to be heavily mixed with Fe–CO modes Fe(II) state of [NiFe]-hydrogenase, for which νFe–CO was observed at 559 cm−1 Fe core in Ni-R. Deuteration of Ni-R is expected to red-shift this mode into the 420–620 cm−1 Fe–CO/CN region, in agreement with the changes observed and calculated for [1′D]+. Analogous to the model complex, the Ni-D-Fe wag is strongly mixed with Fe–CO/CN modes, which, in the case of Ni-R, makes its unique assignment very difficult.NRVS analysis of [NiFe]-hydrogenase in H758 cm−1 , making 2 substrate or its heterolysis products (see −1 region of interest39η2-H2)NiFe, I or NiFe(η2-H2), II) and another where a bridging hydride is present (Ni(μ-H)Fe, III or HNi(μ-H)Fe, IV). In addition, variants of III, in which a terminal Cys ligand is protonated ((Cys546)SHNi(μ-H)Fe, V and (Cys81)SHNi(μ-H)Fe, VI), were also studied. Taking into account the Ni(II)Fe(II) Ni-R active site16II) remains low-spin, each model may exist in electronic singlet (S=0) or triplet (S=1) Ni(II) states, both of which were evaluated computationally. The DFT-calculated 57Fe PVDOS for selected models were compared with the NRVS data for Ni-R over the range 400–750 cm−1 (VS and VIS (II)) match experimental data remarkably well, with the number and positions of absorption bands being in accordance. Relative intensities of the calculated peaks are also in good agreement with our measurements.To interpret NRVS measurements in terms of suitable structural candidates for Ni-R, we performed DFT calculations on a series of active site models featuring different binding modes of the Hucts see . Limitin750 cm−1 . DFT-calVS  are derived from Fe–CO vibrations. The feature calculated at 543 cm−1 has significant νFe–CO character, while that at 613 cm−1 is assigned to a δFe–CO mode. Likewise, the 588 cm−1 band can be assigned to an H–Fe–CO bend in which H, Fe and C remain nearly collinear. The above normal modes are highly mixed, which make the assignment of individual fragments complicated.According to the normal mode analysis of model VS and VIS are predicted to exhibit a weak Ni–H–Fe out-of-plane wagging band  structure, we note that the observed error is still well within normal limits54555657−1 feature in the Ni-R NRVS spectrum as the Ni–H–Fe wag mode.Both bridging hydride models 1′H/D]+ feature characteristic Fe–H/D stretches whose energies  are comparable to those for bridging hydrides in other structures, including another recently reported Ni(μ-thiolate)2(μ-H)Fe species for which infrared spectroscopy revealed a νFe–H band at 1,687 cm−1  models (Cys81)SHNi(μ-H)Fe (VIS) and (Cys546)SHNi(μ-H)Fe (VS) reproduce the data exceptionally well, in particular with respect to the Ni–H–Fe wag in each H/D isotopologue. The model VS is also supported by the recent high-resolution crystallographic analysis of Ni-R1 (ref. II). DFT also indicated asymmetric binding of H− to the Ni(II)Fe(II) core, with the ligand more strongly bound to Ni than to Fe Ni(μ-pdt)57Fe(CO)3 (1′) via the intermediates 57Fe2I4(2-propanol)4, (dppe)Ni(μ-pdt)57FeI2 and [(dppe)Ni(μ-pdt)(μ-I)57Fe(CO)3]BF4 ([1′I]BF4) (ref. 1′ underwent facile exchange with 13CO (1 atm), allowing access to isotopologue (dppe)Ni(μ-pdt)57Fe(13CO)3 (1″). The two Ni(I)Fe(I) derivatives 1′ and 1″were then subjected to protonation and deuteronation , affording the Ni(II)Fe(II) salts [1′H]BF4, [1′D]BF4, [1′H]BF4 and [1″D]BF4 as crystalline solids. The isotopic purity of the hydrides and deuterides was confirmed using multinuclear  NMR spectroscopy, infrared spectroscopy and ESI mass spectrometry. NRVS analysis was conducted on a solid sample of each of the four model complexes (vide infra).Metallic F4) ref. . SpeciesD. vulgaris was isolated and purified as described earlier2 (1.2 bar) for 8 h. In the case of the sample in D2O, the buffer was replaced by 100 mM MES (pD=5.0) in D2O and the mixture placed under D2 (1.3 bar) for 8 h. Samples were transferred to an anaerobic chamber and loaded into NRVS cells. FT-IR spectra were recorded on a Bruker IFS66v/S FT-IR spectrometer with a 2 cm−1 spectral resolution at 293 K ([NiFe]-hydrogenase expressed in at 293 K .9 photons s−1) and BL19LXU (∼6 × 109 photons s−1) using 14.4 keV radiation at 0.8 meV resolution. The spectral maximum counts/second (cts s−1) at BL19 is ∼2.6–3 times of that at BL09. To compare the data from different beamlines, we rescale the BL19 counting time on the basis of its max cts s−1 versus the max cts s−1 at BL09 and create BL09 equivalent seconds, for example, the 10 (s) per point (s/pt) at BL19 is corresponding to 26 or 30 equivalent s/pt at BL09. Delayed nuclear and Fe K fluorescence  were recorded with a 2 × 2 APD  array in either beamline, and raw NRVS data were converted to single-phonon 57Fe PVDOS using the PHOENIX softwareThe NRVS data were collected according to a published procedure−1 is 0.6–0.8 at the Fe–CO peak (609 cm−1) and 0.10–0.12 at X–Fe–H peak (675 cm−1), while the measured dark background cts are ∼0.03 cts s−1. To improve weak features, we use 1–3 s/pt from −240 to 400 cm−1, 5–10 s/pt for the Fe–CN and Fe–CO regions, and 10–30 s/pt for the candidate X–Fe–H bending region (at 620–770 cm−1).The average cts s1′H/D]+ were extracted from the X-ray structure of [1H]BF4·3THF ɛ=4). Segmented all-electron relativistically contracted basis sets with corresponding auxiliary basis sets were used : remaining atoms). Vibrational frequencies and normal mode compositions were utilized to simulate NRVS data .DFT calculations on active-site cluster models were performed using ORCA 3.0. The initial geometry was prepared from the crystal structure of reduced [NiFe]-hydrogenase from How to cite this article: Ogata, H. et al. Hydride bridge in [NiFe]-hydrogenase observed by nuclear resonance vibrational spectroscopy. Nat. Commun. 6:7890 doi: 10.1038/ncomms8890 (2015).Supplementary Figures 1-31, Supplementary Tables 1-4, Supplementary Note 1, Supplementary Discussion, Supplementary Methods and Supplementary ReferencesModel complex [1'H]+ (H isotopomer), Fe-H stretching mode, v = 1479 cm-1Model complex [1'H]+ (H isotopomer), Ni-H stretching mode, v = 1022 cm-1Model complex [1'H]+ (H isotopomer), Ni-H-Fe wagging mode, v = 774 cm-1Enzyme cluster model V (H isotopomer), Singlet state, v = 411.00 cm-1Enzyme cluster model V (H isotopomer), Singlet state, v = 439.93 cm-1Enzyme cluster model V (H isotopomer), Singlet state, v = 460.91 cm-1Enzyme cluster model V (H isotopomer), Singlet state, v = 504.68 cm-1Enzyme cluster model V (H isotopomer), Singlet state, v = 543.53 cm-1Enzyme cluster model V (H isotopomer), Singlet state, v = 588.69 cm-1Enzyme cluster model V (H isotopomer), Singlet state, v = 613.20 cm-1Enzyme cluster model V (H isotopomer), Singlet state, v = 726.92 cm-1Enzyme cluster model V (D isotopomer), Singlet state, v = 408.20 cm-1Enzyme cluster model V (D isotopomer), Singlet state, v = 432.27 cm-1Enzyme cluster model V (D isotopomer), Singlet state, v = 438.72 cm-1Enzyme cluster model V (D isotopomer), Singlet state, v = 458.01 cm-1Enzyme cluster model V (D isotopomer), Singlet state, v = 504.14 cm-1Enzyme cluster model V (D isotopomer), Singlet state, v = 522.60 cm-1Enzyme cluster model V (D isotopomer), Singlet state, v = 544.16 cm-1Enzyme cluster model V (D isotopomer), Singlet state, v = 600.64 cm-1Enzyme cluster model V (D isotopomer), Singlet state, v = 613.26 cm-1Enzyme cluster model VI (H isotopomer), Singlet state, v = 409.49 cm-1Enzyme cluster model VI (H isotopomer), Singlet state, v = 439.68 cm-1Enzyme cluster model VI (H isotopomer), Singlet state, v = 460.61 cm-1Enzyme cluster model VI (H isotopomer), Singlet state, v = 502.78 cm-1Enzyme cluster model VI (H isotopomer), Singlet state, v = 545.14 cm-1Enzyme cluster model VI (H isotopomer), Singlet state, v = 588.10 cm-1Enzyme cluster model VI (H isotopomer), Singlet state, v = 614.86 cm-1Enzyme cluster model VI (H isotopomer), Singlet state, v = 692.53 cm-1Enzyme cluster model VI (D isotopomer), Singlet state, v = 406.20 cm-1Enzyme cluster model VI (D isotopomer), Singlet state, v = 427.43 cm-1Enzyme cluster model VI (D isotopomer), Singlet state, v = 440.82 cm-1Enzyme cluster model VI (D isotopomer), Singlet state, v = 457.11 cm-1Enzyme cluster model VI (D isotopomer), Singlet state, v = 500.43 cm-1Enzyme cluster model VI (D isotopomer), Singlet state, v = 508.69 cm-1Enzyme cluster model VI (D isotopomer), Singlet state, v = 545.50 cm-1Enzyme cluster model VI (D isotopomer), Singlet state, v = 597.65 cm-1Enzyme cluster model VI (D isotopomer), Singlet state, v = 613.77 cm-1


E. In the crystal, centrosymmetric aggregates are formed via {⋯HNCS}2 synthons which are linked into supramolecular layers by C—H⋯O interactions.Two almost planar residues in the title thione are orientated perpendicularly [dihedral angle = 82.72 (5)°]; the conformation about the imine bond is  16H16N2O2S2, comprises two almost planar residues, i.e. the phenyl ring and the remaining 14 non-H atoms (r.m.s. deviation = 0.0410 Å). These are orientated perpendicularly, forming a dihedral angle of 82.72 (5)°. An intramolecular hydroxy-O—H⋯N(imine) hydrogen bond, leading to an S(6) loop, is noted. An analysis of the geometric parameters is consistent with the molecule existing as the thione tautomer, and the conformation about the C=N bond is E. The thione S and imine H atoms lie to the same side of the molecule, facilitating the formation of intermolecular N—H⋯S hydrogen bonds leading to eight-membered {⋯HNCS}2 synthons in the crystal. These aggregates are connected by phenyl-C—H⋯O(hydroxy) interactions into a supramolecular layer in the bc plane; these stack with no directional interactions between them. An analysis of the Hirshfeld surface confirms the nature of the intermolecular interactions.The title dithiocarbazate ester, C The molecule exists in the thione tautomeric form. Consistent with this assignment, the thione C1=S2 bond length, i.e. 1.670 (2) Å, is considerably shorter than the thiol C1—S1 and, especially, C2—S1 bonds of 1.749 (2) and 1.817 (2) Å, respectively. The conformation about the C=N bond is E, and the amine-N—H atom is flanked on either side by the thione-S and imine-H atoms.The title compound, Fig. 12 synthons through the agency of thioamide-N—H⋯S(thione) hydrogen bonds, Table 1bc-plane, Fig. 2a. The layers stack along the a axis with no directional interactions between them, Fig. 2b.The most prominent feature of the packing of the title compound is the formation of centrosymmetric, eight-membered {⋯HNCS}Crystal Explorer 3.1  distance is greater than van der Waals separation, confirming the absence of π–π stacking interactions. This is also evident from the small segments delineated by blue outlines in the Hirshfeld surface mapped over curvedness, Fig. 7The overall two-dimensional fingerprint (FP) plot, Fig. 6et al., 2014i.e. 0.97, for H⋯H contacts is in accord with expectation , based on Hirshfeld surface analysis, gives further description of intermolecular interactions operating in a crystal ° in V and 63.01 (8)° in VII. By contrast, a somewhat twisted conformation is observed for each of the two independent molecules in VI. There are three examples, II–IV, of derivatives with a hydroxy group in the 2-position of the ring connected to the imine-C atom (Madanhire S-benzyldithiocarbazate (SBDTC)  was dissolved in hot absolute ethanol (100 cm3) and added to an equimolar amount of 2-hydroxy-3-methoxybenzaldehyde  in absolute ethanol (20 cm3). The mixture was heated with continuous stirring for about 30 min and was then allowed to stand overnight. The light-yellow crystals that formed were filtered and washed with absolute ethanol at room temperature. Yield: 70%. M.p.: 446–447 K. Analysis calculated for C16H16N2O2S2: C, 57.81; H, 4.85; N, 8.43. Found: C, 58.21; H, 4.87; N, 8.28%. FT–IR : 3089, ν(N—H); 1598, ν(C=N); 1030, ν(N—N); 720, ν(C=S). 13C NMR (DMSO-d6) δ (p.p.m.): 196.09 (C=S), 148.59 (C=N); 147.45–114.43 (aromatic-C), 56.43 (CH3), 38.10 (CH2). m/z calculated for C16H16N2O2S2 332.44, found 332.Following procedures adapted from the literature  set to 1.2Ueq(C). The oxygen- and nitrogen-bound H-atoms were located in a difference Fourier map but were refined with distance restraints of O—H = 0.84±0.01 Å and N—H = 0.88±0.01 Å, and with Uiso(H) set to 1.5Ueq(O) and 1.2Ueq(N).Crystal data, data collection and structure refinement details are summarized in Table 510.1107/S2056989016004291/hb7572sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989016004291/hb7572Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989016004291/hb7572Isup3.cmlSupporting information file. DOI: 1465209CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Pex11p family proteins are key players in peroxisomal fission, but their molecular mechanisms remains mostly unknown. In the present study, overexpression of Pex11pβ caused substantial vesiculation of peroxisomes in mammalian cells. This vesicle formation was dependent on dynamin-like protein 1 (DLP1) and mitochondrial fission factor (Mff), as knockdown of these proteins diminished peroxisomal fission after Pex11pβ overexpression. The fission-deficient peroxisomes exhibited an elongated morphology, and peroxisomal marker proteins, such as Pex14p or matrix proteins harboring peroxisomal targeting signal 1, were discernible in a segmented staining pattern, like beads on a string. Endogenous Pex11pβ was also distributed a striped pattern, but which was not coincide with Pex14p and PTS1 matrix proteins. Altered morphology of the lipid membrane was observed when recombinant Pex11p proteins were introduced into proteo-liposomes. Constriction of proteo-liposomes was observed under confocal microscopy and electron microscopy, and the reconstituted Pex11pβ protein localized to the membrane constriction site. Introducing point mutations into the N-terminal amphiphathic helix of Pex11pβ strongly reduced peroxisomal fission, and decreased the oligomer formation. These results suggest that Pex11p contributes to the morphogenesis of the peroxisomal membrane, which is required for subsequent fission by DLP1. At an early stage of peroxisomal fission, peroxisomes were extended, and Pex11pβ-enriched membrane subregions were generated . Overexpression of Pex11pβ caused extremely extension of peroxisomes in the fission-deficient cells .Pex11pβ induces peroxisomal fission when overexpressed in mammalian cells. To assess the effect of Pex11pβ on peroxisomal morphology, we overexpressed untagged Pex11pβ in HeLa cells . For theenerated . These senerated . This veDLP1 was knocked down in HeLa cells stably expressing EGFP tagged with peroxisomal targeting signal 1 sequence (Ser-Lys-Leu) at the C-terminus (EGFP-SKL), and the cells were immunostained with the anti-Pex11pβ antibody  . To determine the morphological effect, reconstituted liposomes were observed under confocal microscopy . In the N-lauroylsarcosine-treated control condition, liposomes were small and uniformly dispersed  . Therefoispersed , while Pispersed . Pex11pβispersed .in vitro . The average of the hydrophobic moment of this region was 0.342 in Pex11pα, 0.359 in Pex11pβ, and 0.501 in Pex11pγ . To confirm the effect, the point-mutated Myc-Pex11pβ-φ and Myc-Pex11pβ-P were overexpressed in HeLa cells, and the cells were subjected to immunostaining using antibodies to Myc and Pex14p . The effect of Pex11pα and Pex11pγ on peroxisomal fission was also examined. Both Pex11p isomers had weak effect on the fission. The fission properties of the series of Pex11p proteins were also tested in cells in which DLP1 had been knocked down. The wild-type Pex11pβ and Pex11pγ were incorporated into peroxisomes, forming Myc-Pex11p-enriched regions and enhancing peroxisomal elongation overall, while the Pex11pβ mutants and Pex11pα did not result in additional elongation of peroxisomes . Then, as the wild-type Pex11pβ  dramatically increased when Pex11pβ was reconstituted into proteo-liposomes. An increase was similarly observed in proteo-liposomes reconstituted with Pex11pα and Pex11pγ. Pex14p and the mutant forms of Pex11pβ had no effect on the percentage of large liposomal clusters.Our group earlier showed that the Pex11pβ N-terminal cytosolic region is required for its ability to enhance peroxisomal fission , and anoin vitro . This mein vitro . Using md Pex14p . While t Pex11pβ , the mutiposomes . Reconstiposomes . The sizEndogenous Pex11pβ was accumulated to specific membrane subregions in fission-deficient peroxisomes see . Next, wNext, a photobleaching assay was used to assess whether the clustered liposomes formed by reconstituting proteo-liposomes with Pex11pβ are aggregates of independent liposomes or continuous liposomes. A small fraction of clustered liposomes was repeatedly photobleached, and images were taken every 30 rounds of photobleaching . In the To observe the morphology of the liposomes in detail, negative staining electron microscopy was used. Control treatment of liposomes had no effect on their morphology, while reconstitution of liposomes with Pex11pβ caused clustering of small vesicles, as observed under confocal microscopy . When Pein vivo, the morphology and number of peroxisomes in HeLa cells that had been transfected with siRNAs targeting PEX11α, PEX11β, or PEX11γ were compared to those of control and DLP1 siRNA-treated cells. Treatment of HeLa cells with siRNA targeting PEX11α or PEX11γ for 96 h resulted in significant decreases in PEX11α and PEX11γ mRNA, respectively . This reflects the tendency of peroxisome fission to occur at the Pex11pβ-enriched regions, suggesting that these points are cleavage sites for DLP1 during the process of fission.We therefore set out to characterize the localization of Pex11pβ in mammalian cells. Both endogenous and exogenous Pex11pβ were enriched within subregions of the peroxisome membrane . Pex11pβin vivo and in vitro . In our current and earlier studies, oligomer formation of Pex11pβ was required for its activity  , corresponding to the oligomer shown in a glycerol gradient with purified protein , suggesting that the proper oligomerization of Pex11pβ in a manner dependent on its N-terminal amphiphathic region is essential for tubulation on lipid membranes.Recently, another group has demonstrated that a synthetic peptide comprising the N-terminal amphipathic helix of yeast Pex11p caused liposomes to form tubule-like structures . This acin vitro ,4. Howev. S2B–E) . N-termi. S2B–E) . Consist. S2B–E) –7. BAR a. S2B–E) . The cry. S2B–E) . Anti-pa protein . How thiin vivo experiments, the activity of Pex11pβ and Pex11pγ was similar, and Pex11pα showed less activity. The former two isoforms induced peroxisomal fission when overexpressed . While Pex11pα showed little activity, peroxisomal abundance was decreased in cells in which PEX11α was knocked down because of the formation of large peroxisome aggregates , 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), L-α-phosphatidylinositol from bovine liver (PI), L-α-phosphatidylethanolamine-N-(lissamine-rhodamine B sulfonyl) (rhodamine-PE), 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (PDPC), and 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphoethanolamine (PDPE) were purchased from Avanti Polar Lipids . Bovine brain L-α-phosphatidyl-L-serine (PS) was from Sigma . All lipids were stored in chloroform at −20°C.1,2-dioleoyl-The antibodies used were rabbit antisera to Pex14p , PMP70 , AOx Ts, ADAPS , and Pex2. DNA transfection of HeLa cells was done with Lipofectamine (Life Technologies) according to the manufacturer's protocols. Knockdown experiments were performed using Stealth siRNA duplexes (Life Technologies). The siRNA target sequences were as follows: human Pex11pγ #1, 5′-AAGAGUCGCAAGAUGGUCCUGCAGU-3′; human Pex11pγ #2, 5′-UUGCUUAGUGUAGACAAACAUGGCC-3′; and human Mff, 5′-UUAUCACACUAGCAUUUGGAACUCC-3′. Stealth RNAi Negative Control (Life Technologies) was used as a control. The siRNAs for PEX11α, PEX11β, and DLP1 were described previously  supplemented with 10% FCS under 5% COeviously . HeLa ceMBP-EGFP as a template, KpnI-BamHI fragment was amplified and ligated into pCold1 and the EcoRI-SalI fragment of the plasmid encoding Pex11pα was subsequently inserted into the plasmid. Introduction of point mutations in the N-terminal amphipathic helix of Pex11pβ was performed by site-directed mutagenesis using specific primers: Pex11pβ-P sense, 5′-TGAGCCTTGGAAGAAAGCTTCCACGC CTGGGTAACTCAGCACCTGCCCTTGAGTCAGCCAAAAG-3′; Pex11pβ-P antisense, 5′-CTTTT GGCTGACTCAAGGGCAGGTGCTGAGTTACCCAGGCGTGGAAGCTTTCTTC CAAGGCTCA-3′; Pex11pβ-φ sense, 5′-ACCTGAGCCTTGGAAGAAAGGAACTACGCGAGGGTAACTCAGAAGATGCCCTTG AGTCAGCCAA-3′; Pex11pβ-φ antisense, 5′-TTGGCTGACTCAAGGGCATCTTCTGAGTTAC CCTCGCGTAGTTCCTTTCTTCCAAGGCTCAGGT-3′. Pex11pβ-φ contains L58E, L61E, and A65E mutations, and Pex11pβ-P has point mutations to proline residues at L59 and D66. Pex14p was prepared as a GST-fusion as described previously (cDNA encoding human Pex11pβ was amplified by reverse-transcription PCR using mRNA from HeLa cells as the template. The PCR product was cloned into pcDNAZeo3.1 (Life Technologies) to generate the untagged Pex11pβ expression plasmid for HeLa cells. For the construction of His-tagged Pex11pα, the EcoRI-SalI fragment of Pex11pα was ligated into the corresponding sites of pCold1 . For His-Pex11pβ and His-Pex11pγ, the BamHI-EcoRI fragments were respectively amplified and inserted into pCold1. EGFP-fused Pex11pβ and Pex11pγ were generated by the insertion of the KpnI-BamHI fragment of plasmid encoding MBP and the BamHI-BglII fragment of plasmid encoding EGFP into the KpnI-BamHI site of the pCold1-Pex11pβ and pCold1-Pex11pγ, respectively. To construct EGFP-Pex11pα, using eviously .DLP1 or MFF. Cells were fixed with 4% paraformaldehyde for 15 min at room temperature. Peroxisomes were visualized by indirect immunofluorescence staining with the indicated antibodies. Primary antibodies were detected with goat anti-mouse and anti-rabbit IgG conjugated to Alexa Fluor 488 or Alexa Fluor 568 (Molecular Probes/Life Technologies). Cells were observed under confocal laser microscopy  with the Plan Apochromat 100 ×/1.3 NA objective lens. Line scanning was performed using the Plot profile tool in ImageJ software . For the analysis of number, size, and area of peroxisomes, images were taken of at least 30 cells by confocal microscopy. Each image was converted to a threshold image, and the number of peroxisomes was measured using the Analyze Particle command in ImageJ.For the observation of peroxisomal elongation by Pex11pβ, untagged Pex11pβ was expressed in HeLa cells. Cells were fixed at 6 h and 24 h after transfection. For the detection of peroxisomal fission induced by Pex11pα, Pex11pβ, Pex11pγ, and mutant forms of Pex11pβ, cells were transfected with the corresponding plasmids for 24 h. To verify the effect of these fission factors on Pex11pβ-dependent peroxisomal proliferation, Pex11p-expression plasmids were transfected into HeLa cells pre-treated for 48 h with siRNA for http://mobyle.pasteur.fr/cgi-bin/portal.py?form = hmoment). The helical wheel representation of the Pex11p amphipathic region was prepared using the Heliquest server (http://heliquest.ipmc.cnrs.fr).Multiple sequence alignment was prepared as described previously . The hydLiposomes were prepared essentially as described previously . The lipE. coli BL21(DE3) was transformed with pCold-Pex11p and protein expression was induced at 15°C for 24 h with 0.1 mM isopropyl β-D-thiogalactoside (IPTG). Cells were harvested and lysed in sarcosine buffer 50 mM Tris-HCl, pH 7.4, 0.5 M NaCl, 2 M Urea, 50 mM imidazole, 0.5% sodium N-lauroylsarcosine. After 15-min incubation on ice, the lysate was ultracentrifuged at 42,000 rpm for 30 min at 4°C using a Hitachi TLA100.3 rotor . The supernatant was incubated with Ni-NTA beads  at 4°C for 1 h, then the beads were collected and washed with wash buffer 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.5% sodium N-lauroylsarcosine. Protein was eluted by washing buffer containing 0.3 M imidazole. The elution buffer was used for protein reconstitution in the control of the experiment. For purification of MBP-EGFP-Pex11p proteins, cells were lysed in buffer containing n-dodecyl-β-D-maltoside , and protein was affinity-purified by amylose resin  according to the manufacturer's instructions. We named MBP-EGFP-Pex11p protein as “EGFP-Pex11p” and used as such, since the MBP-tag was not readily cleaved off by PreScission protease .Purified protein (up to 1 µg) or the same volume of the elution buffer was mixed with 50 µl of liposomes, and the mixture was frozen at −80°C for at least 5 min. Then, the mixture was thawed and diluted in 3 ml of Hepes-KOH buffer to form proteo-liposomes. Liposomes were collected by ultracentrifugation at 100,000 rpm for 1 h at 4°C, using a Hitachi TLA100.3 rotor. For the separation of protein precipitate after ultracentrifugation, the liposomal pellet was mixed with an equal volume of 60% sucrose (∼100 µl). One ml of 20% sucrose was added to the liposomes, and 100 µl of Hepes-KOH buffer was loaded on the top of the sucrose gradient. Then, the sample was centrifuged at 100,000 rpm for 1 h at 4°C using a Hitachi TLA100.3 rotor. Three serial fractions were harvested from the top of the tube by pipetting.Precipitated proteo-liposomes were mounted on glass slides by mixing with PermaFluor , and covered with glass coverslips. Samples were observed under confocal laser-scanning microscopy with a Plan Apochromat 100×/1.3 NA objective lens. Photobleaching was performed in a 1-µm circle placed at the edge of a cluster of liposomes. Images were taken before and after 30 rounds of photobleaching, and the total fluorescence intensity of the liposomal clusters was analyzed. In the control experiment, an adjacent region to the liposome clusters was photobleached and subjected to analysis.For negative staining electron microscopy, proteo-liposomes were adsorbed onto a formvar-coated 300-mesh copper grid  that had been glow-discharged just prior to use. The grid was negatively stained with 2% uranyl acetate and observed under a transmission electron microscope at 200 kV . To detect the localization of Pex11pβ, proteo-liposomes were stained with antibodies to Pex11pβ for 1 h at room temperature (RT), and labeled for 1 h with a goat anti-rabbit IgG secondary antibody conjugated to 10 nm gold particles . The liposomes were then collected by ultracentrifugation and subjected to negative staining electron microscopy. For ultra-thin sectioning, liposomes were pelleted and fixed with 3% glutaraldehyde, and further processed for embedding in Epon and staining, as described previously .Total RNA was extracted from HeLa cells using TRIzol reagent (Life Technologies), and cDNA was obtained by reverse transcription (RT),  according to the manufacturer's instructions. RT-PCR was performed using a set of specific primers: Pex11pγ sense, 5′-TGATGACCTGGCCATGTTTGTCTA-3′, and antisense, 5′-AAGTGACAGCGCCTCCGACTGCAT-3′. Primer sets for Pex11pα and actin have been described previously .


N-Methyl-N-phenylcarbamoyl)(N-methyl-N-phenylamino)sulfide and the corresponding disulfane are stable derivatives of (chlorocarbonyl)sulfenyl chloride and (chlorocarbonyl)disulfanyl chloride, respectively.( N-methyl-N-phenylamino)(N-methyl-N-phenylcarbamoyl)sulfide, C15H16N2OS, (I), and (N-methyl-N-phenylamino)(N-methyl-N-phenylcarbamoyl)disulfane, C15H16N2OS2, (II), are stable derivatives of (chlorocarbonyl)sulfenyl chloride and (chlorocarbonyl)disulfanyl chloride, respectively. The torsion angle about the S—S bond in (II) is −92.62 (6)°, which is close to the theoretical value of 90°. In the crystal of (II), non-classical intermolecular C—H⋯O hydrogen bonds form centrosymmetric cyclic dimers [graph set R22(10)], while inter-dimer C—H⋯S interactions generate chains extending along the b axis.The title compounds, ( Not surprisingly, many geometric parameters of (III) and (IV) superimpose onto the corresponding portions of (I)A search for similar structures in the Cambridge Structural Database  Fig. 3 (Barany M solution of (chlorocarbonyl)sulfenyl chloride in CHCl3 to an equal volume of a 2 M solution of N-methylaniline in CHCl3 at 273 K, followed by stirring for 30 min at 298 K  and brine (once), drying (MgSO4), filtering, and concentrating in vacuo gave the product as an oil , and recrystallization from hot hexanes (30 mL g−1) gave a white solid , m.p. 338–340 K, which was stable for several decades when stored under ambient conditions. 1H NMR : δ 7.43–7.48 , 7.37 , 7.23–7.29 , 7.12 , 6.86 , 3.41 , 3.31 . X-ray quality crystals were obtained by dissolving (I)3 (200 µL) and then adding hexane (2 mL), followed by slow evaporation of the solvent at 298 K over two days.The title compound (I)2Cl2 (15 mL) was added over 10 min to a stirred solution of N-methylaniline  in CH2Cl2 (11 mL) at 273 K. The homogeneous reaction mixture was allowed to warm to 298 K, stirred an additional 30 min, and standard extractive workup [compare to procedure above for (I)] gave the product as a brown oil . The crude product was purified by flash column chromatography, eluting with hexane–ethyl acetate (8:1), to provide a yellow oil (1.37 g), which after storing under hexanes at 253 K overnight produced the title product as an off-white solid , m.p. 326–327 K : δ 7.36–7.41 , 7.2–7.3 , 6.9–7.0 , 3.40 , 3.37 . X-ray quality crystals were prepared by dissolving (II)2Cl2 (100 µL) and then adding heptane (200 µL), followed by slow evaporation of the solvent at 278 K over 11 days.To prepare compound (II)UisoH = 1.2UeqC(aromatic) or 1.5UeqC(methyl). With (I)et al., 2013Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989015018289/zs2342sup1.cifCrystal structure: contains datablock(s) I, II, 1. DOI: 10.1107/S2056989015018289/zs2342Isup4.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989015018289/zs2342IIsup5.hklStructure factors: contains datablock(s) II. DOI: Click here for additional data file.10.1107/S2056989015018289/zs2342Isup4.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989015018289/zs2342IIsup5.cmlSupporting information file. DOI: 1428652, 1428651CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


S,3R,8S,9R,10S)-2,2-dichloro-3,7,7,10-tetramethyl-9,10-epoxytricyclododecane with a concentrated solution of hydrobromic acid. It is built up from three fused rings: a cycloheptane ring, a cyclohexyl ring bearing alkene and hydroxy substituents, and a cyclopropane ring bearing two chlorine atoms.The title compound was synthesized by treating -2,2-dichloro-3,7,7,10-tetramethyl-9,10-epoxytricyclododecane with a concentrated solution of hydrobromic acid. It is built up from three fused rings: a cycloheptane ring, a cyclohexyl ring bearing alkene and hydroxy substituents, and a cyclopropane ring bearing two chlorine atoms. The asymmetric unit contains two molecules linked by an O—H⋯O hydrogen bond. In the crystal, further O—H⋯O hydrogen bonds build up an R44(8) cyclic tetramer. One of the molecules presents disorder that affects the seven-membered ring. In both molecules, the six-membered rings display a chair conformation, whereas the seven-membered rings display conformations intermediate between boat and twist-boat for the non-disordered molecule and either a chair or boat and twist-boat for the disordered molecule owing to the disorder. The absolute configuration for both molecules is 1S,3R,8R,9R and was deduced from the chemical pathway and further confirmed by the X-ray structural analysis.The title compound, C Cedrus atlantica) is a bicyclic hydrocarbon sesquiterpene called β-himachalene -2,2-dichloro-10-methylene-3,7,7-trimethyltricyclododecan-9-ol by treating -2,2-dichloro-3,7,7,10-tetramethyl-9,10-epoxytricyclododecane with a concentrated solution of hydrobromic acid.The main constituent (50%) of the essential oil of the Atlas cedar :0.498 (8). In both molecules, the six-membered ring displays a chair conformation with puckering parameters θ = 169.3° and φ2 = 119.6° for molecule A and θ = 172.1° and φ2 = 110.0° for molecule B. The seven-membered cycloheptane ring in molecule A displays a conformation intermediate between boat and twist boat with puckering parameters q2 = 1.138 (4) Å and q3 = 0.037 (5) Å , q3 = 0.739 (6) Å] or boat and twist-boat   atom. The disorder does not affect the absolute configuration of the two molecules  and .In molecule 7C Fig. 2, resultiR44(8) cyclic tetramer .A search of the Cambridge Structural Database -2,2- dichloro-3,7,7,10-tetramethyl-9,10-epoxytricyclododecane in 25 mL of dichloromethane. The mixture was cooled to 273.15 K in an ice bath prior to dropwise addition of 8 mL of concentrated hydrobromic acid. The mixture was stirred for 30 min. TLC control showed that the reaction was complete. The reaction mixture was extracted with dichloromethane (3 × 30mL) and the organic layer was washed first with water and then with a saturated solution of NaHCO3, dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude product was purified by chromatography on silica gel (230–400 mesh) with hexane/ethyl acetate (97:3) as eluent to give the title compound in 64% yield. X-ray quality crystals were obtained by slow evaporation from a petroleum ether solution.To a 100 mL flask was added  of (1Uiso(H) = 1.2Ueq or 1.5Ueq.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989016011166/pk2583sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989016011166/pk2583Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989016011166/pk2583Isup3.cmlSupporting information file. DOI: 1491732CCDC reference: crystallographic information; 3D view; checkCIF reportAdditional supporting information: