High production cost of bioplastics polyhydroxyalkanoates (PHA) is a major obstacle to replace traditional petro-based plastics. To address the challenges, strategies towards upstream metabolic engineering and downstream fermentation optimizations have been continuously pursued. Given that the feedstocks especially carbon sources account up to a large portion of the production cost, it is of great importance to explore low cost substrates to manufacture PHA economically.Escherichia coli was metabolically engineered to synthesize poly-3-hydroxybutyrate (P3HB), poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P3HB4HB), and poly (PHBV) using acetate as a main carbon source. Overexpression of phosphotransacetylase/acetate kinase pathway was shown to be an effective strategy for improving acetate assimilation and biopolymer production. The recombinant strain overexpressing phosphotransacetylase/acetate kinase and P3HB synthesis operon produced 1.27 g/L P3HB when grown on minimal medium supplemented with 10 g/L yeast extract and 5 g/L acetate in shake flask cultures. Further introduction succinate semialdehyde dehydrogenase, 4-hydroxybutyrate dehydrogenase, and CoA transferase lead to the accumulation of P3HB4HB, reaching a titer of 1.71 g/L with a 4-hydroxybutyrate monomer content of 5.79 mol%. When 1 g/L of α-ketoglutarate or citrate was added to the medium, P3HB4HB titer increased to 1.99 and 2.15 g/L, respectively. To achieve PHBV synthesis, acetate and propionate were simultaneously supplied and propionyl-CoA transferase was overexpressed to provide 3-hydroxyvalerate precursor. The resulting strain produced 0.33 g/L PHBV with a 3-hydroxyvalerate monomer content of 6.58 mol%. Further overexpression of propionate permease improved PHBV titer and 3-hydroxyvalerate monomer content to 1.09 g/L and 10.37 mol%, respectively.The application of acetate as carbon source for microbial fermentation could reduce the consumption of food and agro-based renewable bioresources for biorefineries. Our proposed metabolic engineering strategies illustrate the feasibility for producing polyhydroxyalkanoates using acetate as a main carbon source. Overall, as an abundant and renewable resource, acetate would be developed into a cost-effective feedstock to achieve low cost production of chemicals, materials, and biofuels.The online version of this article (10.1186/s12934-018-0949-0) contains supplementary material, which is available to authorized users. Food and agro-based renewable bioresources, such as xylose , glucoseEscherichia coli [Cryptococcus curvatus [Clostridium sp. [Halomonas boliviensis [Moorella thermoacetica from carbon dioxide [Acetate is the second simplest carboxylic acid, and many microorganisms can utilize acetate as alternative carbon source for cell growth. The acetate assimilation species include hia coli , Cryptoccurvatus , Clostridium sp. , Halomoniviensis , and so  dioxide , 17. In  dioxide , 19. In C. curvatus [Yarrowia lipolytica [E. coli fermentation, recombinant E. coli strains were constructed to use acetate for the production of free fatty acids [Oleaginous yeasts including curvatus  and Yarrpolytica  have beety acids  and succty acids . TherefoE. coli were engineered to produce poly-3-hydroxybutyrate (P3HB), poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P3HB4HB), and poly (PHBV) using acetate as a main carbon source  medium at 37 °C and 200 rpm. LB medium contained 5 g/L yeast extract, 10 g/L Bacto tryptone, and 10 g/L NaCl. To maintain plasmid stability, 100 μg/mL ampicillin and/or 50 μg/mL kanamycin was added to the media when required.For construction of recombinants and plasmid amplification, ® High-Fidelity DNA Polymerase from New England Biolabs  was employed for PCR reactions. Plasmid isolation and DNA purification kits were purchased from Biomed . Restriction enzymes and DNA modifying enzymes were purchased from New England Biolabs.Strains and plasmids used in this study are listed in Table pta-ackA encoding phosphotransacetylase/acetate kinase and acs encoding acetyl-CoA synthetase were amplified from E. coli genomic DNA using primers pa1F/pa1R and acsF/acsR, respectively. The PCR products were purified and digested with HindIII/XbaI and inserted into the corresponding restriction sites of pEn to construct pEn-pta-ackA and pEn-acs, respectively. Moreover, Ppdc promoter and gene coding sequence were removed from pEn-derived plasmids and subcloned into kanamycin resistance carrying broad-host-range cloning vector pBBR1MCS-2 to generate pMCS-pta-ackA and pMCS-acs, respectively. Primers pa2F/pa2R were designed to amply pta-ackA from pEn-pta-ackA. The PCR product was digested with NheI/XhoI and cloned into the sucD-4hbD expression vector pMCSH5 cut with the same enzymes to form pMCSH5-pta-ackA. Primers prpPF/prpPR were designed to amply prpP encoding propionate permease from Ralstonia eutropha H16 genomic DNA. The PCR product was digested with BamHI/HindIII and cloned into pEn to generate pEn-prpP. Subsequently, Ppdc-prpP was excised from pEn-prpP using SpeI/XhoI and cloned into pMCS-pta-ackA digested with NheI/XhoI to generate pMCS-pta-ackA-prpP.The To prepare the seed cultures, 10 μL of glycerol stock was inoculated into 20 mL of LB medium and cultivated at 37 °C and 200 rpm for 12 h. For biopolymer producing shake flask studies, 4% (v/v) of the seed culture was used to inoculate into 500 mL shake flasks containing 50 mL of medium and cultured at 37 °C and 200 rpm for 48 h.phaCAB expression plasmid pBHR68, were co-transformed into E. coli JM109 to generate P3HB producing strains. Three different kinds of fermentation media were employed for P3HB production: LB, terrific broth (TB), and minimal medium (MM). TB medium contained 24 g/L yeast extract, 12 g/L Bacto tryptone, 2.31 g/L KH2PO4, and 12.54 g/L K2HPO4. MM medium comprised 2 g/L NH4Cl, 5.0 g/L (NH4)2SO4, 6.0 g/L KH2PO4, 8.4 g/L MOPS, 0.5 g/L NaCl, 0.24 g/L MgSO4, 0.002 g/L Na2MoO4 and 1 mL/L trace element solution. The trace elements solution contained (g/L): FeCl2·4H2O 3.6, CaCl2·2H2O 5.0, MnCl2·2H2O 1.3, CuCl2·2H2O 0.38, CoCl2·6H2O 0.5, ZnCl2 0.94, H3BO3 0.0311, Na2EDTA·2H2O 0.4, and thiamine-HCl 1.01. When necessary, MM medium was supplemented with a certain amount of yeast extract.The plasmids including pBBR1MCS-2, pMCS-pta-ackA, and pMCS-acs, each in combination with phaCAB-orfZ expression plasmid p68orfZ, were co-transformed into E. coli JM109SG to generate P3HB4HB producing strains. The resulting recombinants were cultivated in MM medium supplemented with 10 g/L yeast extract and 5 g/L acetate for P3HB4HB accumulation. When necessary, α-ketoglutarate or citrate was added as a second carbon source at a concentration of 1 g/L.Plasmids pMCSH5 and pMCSH5-pta-ackA, each together with pct-phaCAB expression plasmid p68-pct-CAB, were co-transformed into E. coli JM109 to generate PHBV producing strains. The resulting recombinants were cultivated in MM medium supplemented with 10 g/L yeast extract, 5 g/L acetate, and 1.5 g/L propionate for PHBV production.Plasmids pBBR1MCS-2, pMCS-pta-ackA, and pMCS-pta-ackA-prpP, each together with g for 10 min. The cell pellets were washed twice with distilled water and then lyophilized for 12 h for cell dry weight (CDW) measurement. Lyophilized cells were subjected to methanolysis at 100 °C for 4 h in the presence of 3% (v/v) H2SO4. Intracellular polymer content (wt%) and composition was analyzed using gas chromatography (GC) (Hewlett-Packard 6890) equipped with a capillary column HP-5  and a flame-ionization detector. PHBV and γ-butyrolactone purchased from Sigma-Aldrich  were used as analysis standards. Residual cell weight (rCDW) is defined as non-PHA cell mass. rCDW in each sample was determined by substracting the PHA concentration measured by GC analysis from CDW.Bacterial cells were harvested by centrifugation at 8000® HPX-87H, BioRad) and a refractive index detector . A mobile phase of 7 mM H2SO4 at a 0.6 mL/min flow rate was used.For acetate detection, the supernatant of culture broth was filtered through a 0.2-μm syringe filter and stored chilled for high-performance liquid chromatography (HPLC) analysis equipped with an ion exchange column  consumed all the acetate and yielded 1.49 g/L CDW with 0.22 g/L P3HB accumulation, which indicated that P3HB could be produced from acetate, yet the P3HB titer was much lower than that obtained from glucose [To investigate the possibility of P3HB production using acetate as a main carbon source in  glucose . TherefoE. coli, there are two potential routes for the generation of acetyl-CoA from acetate: phosphotransacetylase/acetate kinase (pta-ackA pathway) and AMP-forming acetyl-CoA synthetase (acs pathway)  consumed 5 g/L acetate and produced 0.66 g/L P3HB. The P3HB production titer of was roughly four times of that produced by control strain JM109 (pBHR68 + pBBR1MCS-2). By contrast, the overexpression of acs gene has little effect on improving P3HB accumulation  Fig. . These tynthesis , 33. It  pathway , 29. TheE. coli JM109 (pBHR68 + pMCS-pta-ackA) and control strain JM109 (pBHR68 + pBBR1MCS-2) were cultivated in shake flasks with LB, TB, and MM medium to evaluate the effects of medium composition on cell growth and P3HB accumulation  reached 1.03 g/L, and P3HB concentration was only 0.02 g/L. The CDW and P3HB accumulation increased gradually with the increase of yeast extract concentration, although the intracellular P3HB content did not change obviously. The addition of 10 g/L yeast extract yielded 3.02 g/L CDW, containing 1.27 g/L P3HB, which was the highest P3HB production titer obtained in this study. The production yield was 0.25 g P3HB/g acetate, which was 35% of the maximal theoretical yield. It has been shown that yeast extract contributes a large amount of amino acids for biomass growth, thus the addition of complex nitrogen sources such as yeast extract to defined medium saved NADPH for amino acids synthesis and increased intracellular NADPH level as well as NADPH/NADP ratio [E. coli [Next, different concentration  of yeast extract were added to MM medium to study its potential for enhancing cell growth and P3HB accumulation  using MM medium supplemented with 10 g/L yeast extract and different concentration  of acetate to evaluate the effect of acetate concentration on P3HB accumulation. As shown in Additional file We next cultivated co-4-hydroxybutyrate) (P3HB4HB) exhibits favorable biodegradability and a wide range of physical properties ranging from highly crystalline plastic to elastic rubber, thus it is considered to be one of the most promising PHA materials. The synthesis of P3HB4HB copolymer from structural unrelated carbon sources such as glucose in E. coli has been reported [E. coli that can produce P3HB4HB from acetate as carbon source.Poly when grown in mineral medium supplemented with 10 g/L yeast extract and 5 g/L acetate  yielded 2.96 g/L CDW, containing 58.01 wt% P(3HB-co-5.79 mol% 4HB) . Similarly, the addition of 1 g/L citrate as joint carbon source resulted in 3.62 g/L CDW, containing 59.48 wt% P(3HB-co-3.43 mol% 4HB). The addition of α-ketoglutarate and citrate as additional carbon sources increased the cell growth and P3HB4HB production, but had no obvious effect on improving 4HB monomer content.Previously, the addition of TCA cycle intermediates including α-ketoglutarate and citrate were found to be effective for increasing metabolic flux into 4HB precursor when glucose was employed as carbon source . TherefoE. coli metabolizes acetate as a sole carbon and energy source, the genes involved in acetate uptake, glyoxylate cycle, TCA cycle, and gluconeogenesis were all up-regulated [When egulated . The addco-3-hydroxyvalerate) (PHBV) is more flexible and tougher than P3HB homopolymer and has been commercially produced for many years [Megasphaera elsdenii propionyl-CoA transferase and P3HB synthesis operon phaCAB, was co-transformed with pMCS-pta-ackA into E. coli JM109. The resulting recombinant reached 2.64 g/L CDW, containing 12.52 wt% P(3HB-co-6.58 mol% 3HV)  under the same culture condition. PHBV production titer was increased from 0.15 to 0.33 g/L with pta-ackA overexpression, further demonstrating that pta-ackA overexpression was an effective strategy for improving acetate-dependent biopolymer synthesis.Poly(3-hydroxybutyrate-ny years . The norny years . The conE. coli harboring phaCAB operon and propionate permease (or propionyl-CoA synthase) failed to incorporate 3HV into the biopolymer in M9 medium containing 2 g/L yeast extract, 1 g/L sodium propionate, and 20 g/L sodium acetate [Previously, recombinant  acetate . HoweverprpP gene encoding propionate permease of R. eutropha H16 was overexpressed to study its effect on PHBV synthesis. E. coli JM109 (p68-pct-CAB + pMCS-pta-ackA-prpP) resulted in 3.15 g/L CDW, containing 34.77 wt% P(3HB-co-10.37 mol% 3HV)  increased gradually with the increase of corn steep liquor concentration, yet the P3HB production exhibit a trend of first increasing then decreasing. The addition of 4 g/L and 6 g/L of corn steep liquor to MM medium containing 5 g/L acetate yielded 0.91 and 0.96 g/L P3HB, respectively. Although 10 g/L corn steep liquor addition led to highest CDW among the different corn steep liquor concentration condition, the P3HB concentration was only 0.56 g/L. Therefore, the favorable corn steep liquor concentration for P3HB production was 4–6 g/L under our culture condition. These results indicated that P3HB can be synthesized when corn steep liquor is employed as a nitrogen source, albeit with somewhat lower efficiency in comparison with yeast extract. The use of corn steep liquor for PHA production from acetate therefore merits further research.Shake flask cultivations using various concentration  of corn steep liquor instead of yeast extract were performed Fig. . The CDWE. coli JM109 (pBHR68 + pMCS-pta-ackA) showed the highest P3HB accumulation of 0.04 g/L among all strains studied when cultivated in MM medium supplemented with 6 g/L CSL. The P3HB titer obtained without acetate was much lower than that achieved by acetate supplementation. These results further demonstrated that acetate was served as the main carbon source to support effective P3HB synthesis.In addition, we performed shake flask studies without acetate addition to evaluate the contribution of CSL to P3HB production. As shown in Additional file E. coli was engineered to synthesize P3HB, P3HB4HB, and PHBV using acetate as a main carbon source. The production of biopolymers was significantly improved by the overexpression of phosphotransacetylase and acetate kinase. In shake flask cultures, the engineered E. coli produced 1.27 g/L of P3HB and 1.71 g/L P(3HB-co-5.79 mol% 4HB), respectively, with minimal medium supplemented 10 g/L yeast extract and 5 g/L acetate. When 1 g/L citrate was added as assistant carbon source, P3HB4HB production titer was increased to 2.15 g/L. The overexpression of propionyl-CoA transferase and propionate permease lead to a production titer of 1.09 g/L P(3HB-co-10.37 mol% 3HV) when 5 g/L acetate and 1.5 g/L propionate were simultaneously supplied. To our knowledge, this is the first study reporting the efficient production of P3HB, P3HB4HB, and PHBV using acetate as a main carbon source by engineered E. coli.In this study, Additional file 1: Table S1. Oligonucleotides used in this study. Table S2. P3HB production by E. coli strains cultivated in MM medium supplemented with CSL. Figure S1. Effect of acetate concentration on cell growth and P3HB production.


In each structure, the anthracene ring system and pendant ring system are almost perpendicular to each other [dihedral angles = 75.57 (7) and 70.26 (10)°]. In the extended structures, weak N—H⋯O, C—H⋯O and C—H⋯π interactions influence the centrosymmetric crystal packing. E)-1-(anthracen-9-yl)-3-(3H-indol-2-yl)prop-2-en-1-one, C25H17NO, and (E)-1-(anthracen-9-yl)-3-[4-(dimethylamino)naphthalen-1-yl]prop-2-en-1-one, C29H23NO, are reported. In each case the anthracene ring system and pendant ring system are almost perpendicular to each other . In the extended structures, weak N—H⋯O, C—H⋯O and C—H⋯π interactions influence the centrosymmetric crystal packing. Density functional theory calculations were carried out using a 6–311 G++ basis set and the calculated structures are in good agreement with the crystal structures. The compounds were also characterized by UV–Vis absorption spectroscopy and the smallest (HOMO–LUMO) energy gaps of 2.89 and 2.54 eV indicate the enhanced non-linear responses (intermolecular charge transfers) of these systems.The crystal structures of ( Each compound is made up of an anthracene ring system with the substituents indoline and N,N-dimethylnaphthalen-1-amine for (I)b. Selected calculated s-trans configuration with respect to the C15=O1 and C16=C17 bonds (Table S1). Both compounds (I)et al., 2018abcdThe enone moiety (O1/C15–C17) in (I)et al., 2008Additionally, the enone moiety for (I)via pairwise N—H⋯O hydrogen bonds (Table 122R(14) loops . These dimers are further linked into infinite sheets stacked along the bc plane. The weak C10—H10⋯Cg1 and C22—H22⋯Cg2 interactions also help to establish the packing. Overall, these links generate a three-dimensional supramolecular network.The crystal packing of (I)s Table 1, formingps Fig. 2a. Thesea-axis direction . A weak π–π interaction  with a centroid–centroid distance of 3.9432 (16) Å between C22–C27 rings is also observed. Together these interactions generate a two-dimensional supramolecular network propagating in the ab plane.In (II)on Fig. 2b. A weaet al. ]. The HOMO–LUMO energy gap represents the lowest energy for intermolecular charge transfer (ICT) where the EHOMO and ELUMO energies of the studied molecules were calculated using the B3LYP/6-311G++ basis set. The calculated energy gaps  are 3.16 eV in (I)For background to FMO analysis, which provides insight into electronic as well as optical properties of organic compounds, see: Ebenezar  al. 2013. In thiset al., 2016et al., 2015The experimental UV–vis spectrum Fig. 3 showed aet al., 2016et al. (1975E)-1-(anthracen-9-yl)-3-[4-(propan-2-yl)phenyl]prop-2-en-1-one was described by Girisha et al. (2016E)-1-(anthracen-9-yl)-3-(2-chloro-6-fluorophenyl)prop-2-en-1-one was reported by Abdullah et al. -1,3-bis(anthracen-9-yl)prop-2-en-1-one. Others related compounds include 1-(anthracen-9-yl)-2-methylprop-2-en-1-one -1- al. 2016, while  was dissolved in methanol (20 ml) over about 10–15 min. Then, indoline-2-carbaldehyde (0.5 mmol) and 4-(dimethylamino)-1-naphthaldehyde (0.5 mmol) for compound (I)Uiso(H) = 1.2 or 1.5Ueq. A rotating group model was applied to the methyl groups.Crystal data collection and structure refinement details are summarized in Table 310.1107/S2056989018013087/hb7770sup1.cifCrystal structure: contains datablock(s) I, II, global. DOI: 10.1107/S2056989018013087/hb7770Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989018013087/hb7770IIsup3.hklStructure factors: contains datablock(s) II. DOI: Click here for additional data file.10.1107/S2056989018013087/hb7770Isup4.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989018013087/hb7770IIsup5.cmlSupporting information file. DOI: 10.1107/S2056989018013087/hb7770sup6.pdfSupplementary figures and table. DOI: 1829208, 1824549CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Scientific Reports 10.1038/s41598-018-26457-7, published online 30 May 2018Correction to: This Article contains an error in the Y axis of Figure 4a, where ‘0.2’ and ‘0.4’ should read ‘0.5’ and ‘1.0’ respectively. The correct Figure 4 appears below as Figure


Cacospongia sp. (CMB-03404), obtained during deep sea commercial fishing activities off the southern coast of Australia, yielded an unprecedented family of sesterterpene α-methyl-γ-hydroxybutenolides, cacolides A–L (1–12), together with biosynthetically related norsesterterpene carboxylic acids, cacolic acids A–C (13–15). Structures were assigned on the basis of detailed spectroscopic analysis with comparisons to known natural products and biosynthetic considerations. In addition to revealing new chemical diversity, this study provided a valuable platform for comparing and contrasting the capabilities of the traditional dereplication technologies of HPLC-DAD, HPLC-MS and NMR, with those of the emerging HPLC-MS/MS approach known as global natural products social molecular networking (GNPS), as applied to marine sponge sesterterpene tetronic acids.Chemical analysis of a marine sponge,  Ircinia, Psammocinia and Sarcotragus c 0.60, MeOH); NMR  see m/z 423.2511 [M + Na]+ .Cacolide A (2). light yellow oil; [α]c 0.74, MeOH); NMR  see m/z 455.2411 [M + Na]+ .Cacolide B (3). light yellow oil; [α]c 0.79, MeOH); NMR  see m/z 455.2398 [M + Na]+ .Cacolide C (4). light yellow oil; [α]c 0.06, MeOH); NMR  see m/z 469.2559 [M + Na]+ .Cacolide D (5). light yellow oil; [α]c 0.62, MeOH); NMR  see m/z 483.2725 [M + Na]+ .Cacolide E (6). light yellow oil; [α]c 0.08, MeOH); NMR  see m/z 473.2521 [M + Na]+ .Cacolide F (7). light yellow oil; [α]c 0.05, MeOH); NMR  see m/z 501.2817 [M + Na]+ .Cacolide G (8). light yellow oil; [α]c 0.24, MeOH); NMR  see m/z 496.2678 [M + Na]+ .Cacolide H (9). light yellow oil; [α]25D − 8.1 ; NMR  see m/z 496.2682 [M + Na]+ .Cacolide I (10). light yellow oil; [α]c 0.11, MeOH); NMR  see m/z 510.2827 [M + Na]+ .Cacolide J (11). light yellow oil; [α]c 0.05, MeOH); NMR  see m/z 514.2762 [M + Na]+ .Cacolide K (12). light yellow oil; [α]c 0.15, MeOH); NMR  see m/z 514.2789 [M + Na]+ .Cacolide L (13). light yellow oil; [α]c 0.13, MeOH); NMR  see m/z 455.2397 [M + Na]+ .Cacolic acid A (14). light yellow oil; [α]c 0.07, MeOH); NMR  see m/z 373.1995 [M + Na]+ .Cacolic acid B (15). light yellow oil; [α]c 0.04, MeOH); NMR  see m/z 373.1993 [M + Na]+ .Cacolic acid C (l-glutamine (2 mM), penicillin (100 unit/mL) and streptomycin (100 μg/mL), in a humidified 37 °C incubator supplied with 5% CO2. Briefly, cells were harvested with trypsin and dispensed into 96-well microtiter assay plates at 2000–5000 cells/well after which they were incubated for 18 h at 37 °C with 5% CO2 . Test compounds were dissolved in 20% DMSO in PBS (v/v) and aliquots (10 μL) applied to cells over a series of final concentrations ranging from 10 nM to 30 μM. After 48 h incubation at 37 °C with 5% CO2 an aliquot (20 μL) of -2,5-diphenyltetrazolium bromide (MTT) in phosphate buffered saline (PBS) (5 mg/mL) was added to each well  and microtiter plates were incubated for a further 4 h at 37 °C with 5% CO2. After final incubation, the medium was aspirated and precipitated formazan crystals dissolved in DMSO (100 μL/well). The absorbance of each well was measured at 580 nm with a PowerWave XS Microplate Reader from Bio-Tek Instruments Inc. . IC50 values were calculated using Prism 7.0 , as the concentration of analyte required for 50% inhibition of cancer cell growth (compared to negative controls). Negative control comprised of 1% aqueous DMSO, while positive control was doxorubicin. All experiments were performed in duplicate.Adherent human colorectal (SW620) and lung (NCI-H460) carcinoma cells were cultured in Roswell Park Memorial Institute (RPMI) medium 1640. All cells were cultured as adherent mono-layers in flasks supplemented with 10% foetal bovine serum, 4–105 CFU/mL. The compounds to be tested were dissolved in DMSO and diluted with H2O to return 600 µM stock solutions (20% DMSO). The stock solutions were serially diluted to give concentration range of 600 µM to 0.2 µM in 20% DMSO/H2O. An aliquot (10 µL) of each dilution was transferred to a 96-well microtiter plate and freshly prepared microbial broth (190 µL) was added to each well. The plates were incubated at 37 °C for 24 h and the optical density of each well was measured spectrophotometrically at 600 nm using POLARstar Omega plate . Each test compound was screened against the Gram-negative bacterium Escherichia coli (ATCC 11775) and the Gram-positive bacterium Bacillus subtilis (ATCC 6051). Rifampicin was used as a positive control (40 µg/mL in 10% DMSO). The IC50 value was calculated as the concentration of the compound or antibiotic required for 50% inhibition of the bacterial cells using Prism 7.0 from GraphPad Software Inc. .The bacterium to be tested was streaked onto a tryptic soy agar plate and incubated at 37 °C for 24 h. One colony was then transferred to fresh tryptic soy broth (15 mL) and the cell density adjusted to 104–105 CFU/mL. The compounds to be tested were dissolved in DMSO and diluted with H2O to return 600 µM stock solutions (20% DMSO). The stock solutions were serially diluted to give concentration range of 600 µM to 0.2 µM in 20% DMSO/H2O. An aliquot (10 µL) of each dilution was transferred to a 96-well microtiter plate and freshly prepared microbial broth (190 µL) was added to each well. The plates were incubated at 37 °C for 24 h and the optical density of each well was measured spectrophotometrically at 600 nm using POLARstar Omega plate . Each test compound was screened against Candida albicans (ATCC 10231). Nystatin was used as a positive control (40 µg/mL in 10% DMSO). The IC50 value was calculated as the concentration of the compound or antibiotic required for 50% inhibition of the bacterial cells using Prism 7.0 from GraphPad Software Inc. .The fungus to be tested was streaked onto a sabouraud agar plate and incubated at 37 °C for 24 h. One colony was then transferred to fresh sabouraud broth (15 mL) and the cell density adjusted to 10n-BuOH solubles from; (A) Cacospongia sp. (CMB-03404); (B) Ircinia sp. (CMB-01064) known to produce -variabilin (16), -variabilin (17), -8-hydroxyvariabilin (18), ircinialactam A (20), 8-hydroxyircinialactam B (21) and 8-hydroxyircinialactam A (22) [C) Ircinia sp. (CMB-03363) known to produce 16–20 [D) Ircinia spp. CMB-01058, CMB-01693 and CMB-02014 that have not been subjected to detailed chemical analysis; (E) Psammocinia sp. (CMB-03231) known to produce ent-ircinialactam C [F) Psammocinia sp. (CMB-01018) known to produce 16–17 and 20, as well as ircinialactams C and G-I, ircinialactone A, irciniafuran A, -variabilin and -variabilin [G) Psammocinia sp. (CMB-03344) known to produce (–)-ircinianin, (–)-ircinianin sulphate, (–)-oxoircinianin, (–)-ircinianin lactam A [H) Psammocinia spp. CMB-01757 and CMB-02026 that have not been subjected to detailed chemical analysis; (I) Sarcotragus spp. CMB-01788, CMB-01848, CMB-02707, CMB-02717 and CMB-03390 that have not been subjected to detailed chemical analysis; and (J) authentic standards of -variabilin (16) and ircinialactam A (20). To acquire this data, aliquots (1 μL of 100 μg/mL in 100 μL MeOH) of test solutions were analysed on an Agilent 6545 Q-TOF LC/MS equipped with an Agilent 1290 infinity II UPLC system, utilising an Agilent SB-C8 1.8 μm, 2.1 × 50 mm column, with a 0.5 mL/min, 4.5 min gradient elution from 90% H2O/CH3CN to CH3CN, followed by isocratic elution with CH3CN for 1 min, with a constant isocratic 0.1% formic acid modifier. UPLC-QTOF-(–)MS/MS data acquired for all samples at a fixed collision energy of 40 eV, were converted from Agilent MassHunter data files (.d) to mzXML file format and transferred to the GNPS server (gnps.ucsd.edu). Molecular networking was performed using the GNPS data analysis workflow using the spectral clustering algorithm and a cosine score of 0.7 and a minimum of 6 matched peaks. The resulting spectral networks were imported into Cytoscape version 3.5.1 (cytoscape.org) [m/z and edge thickness corresponded to cosine scores. Separate GNPS cluster maps were generated for (I) all ×5 Ircinia spp.  Cacospongia sp. (CMB-03404) compared against data for Ircinia sp. (CMB-01064) and Ircinia sp. (CMB-03363) MS/MS data acquired on the . CMB-0104 known t. CMB-0104 known t. CMB-0104 known tape.org) , where nnia spp. , (II) alnia spp.  and  . In each


Pro-inflammatory cytokine interleukin-1 beta (IL-1β) is a key mediator of inflammation and stress in the central nervous system (CNS), and is highly expressed in the developing brain. In this study, we investigated the possible role of IL-1β in neuronal differentiation of cortical neural precursor cells (NPCs). We showed that stimulation with IL-1β increased expression levels of neurotrophin-3 (NT3) and neurogenin 1 (Ngn1) and promoted neurite outgrowth. We also found that IL-1β increased mRNA and protein levels of Wnt5a. Knockdown of Wnt5a by transfection with Wnt5a siRNA inhibited IL-1β-induced neuronal differentiation. Moreover, IL-1β-induced Wnt5a expression was regulated by nuclear factor kappa B (NF-κB) activation, which is involved in IL-1β-mediated neuronal differentiation. To examine the role of Wnt5a in neuronal differentiation of NPCs, we exogenously added Wnt5a. We found that exogenous Wnt5a promotes neuronal differentiation, and activates the RhoA/Rho-associated kinase (ROCK)/c-jun N-terminal kinase (JNK) pathway. In addition, Wnt5a-induced neuronal differentiation was blocked by RhoA siRNA, as well as by a specific Rho-kinase inhibitor (Y27632) or a SAPK/JNK inhibitor (SP600125). Furthermore, treatment with RhoA siRNA, Y27632, or SP600125 suppressed the IL-1β-induced neuronal differentiation. Therefore, these results suggest that the sequential Wnt5a/RhoA/ROCK/JNK pathway is involved in IL-1β-induced neuronal differentiation of NPCs.The online version of this article (10.1186/s13041-018-0383-6) contains supplementary material, which is available to authorized users. Interleukin-1 beta (IL-1β) is a pro-inflammatory cytokine, which is produced as an immune response to both injury and infection . IL-1β i2+ pathway, and the Wnt/planar cell polarity (PCP) pathway [Wnt signaling plays an important role in embryogenesis and in the late stages of development, by regulating cell survival, growth, and, differentiation via various signaling pathways , 10. Wnt pathway , 12. Sev pathway . A recen pathway . Wnt5a p pathway  and acts pathway . Wnt5a i pathway , and sil pathway . Further pathway . AlthougRho-family GTPases play an important role in regulating intracellular cytoskeletal and signaling pathways that facilitate axonal morphological changes , 22. TheIn the present study, we first investigated the role of IL-1β in neuronal differentiation of NPCs and found that IL-1β activated noncanonical Wnt5a signaling (Wnt5a/RhoA/ROCK/JNK pathway). We also demonstrated that IL-1β-induced noncanonical Wnt5a signaling is required for neuronal differentiation, especially for neurite outgrowth, suggesting that IL-1β can promote the neuronal differentiation of NPCs.Coon’s modified Ham’s F-12 medium and human insulin were purchased from Sigma Chemical Co . Penicillin/streptomycin solution, neurobasal medium, and B27 were purchased from Gibco , and bFGF, recombinant rat IL-1β, and recombinant Wnt5a were purchased from R&D Systems . The following antibodies were purchased: anti NT3 (#SC-547) and anti Ngn1 (#SC-19231) from Santa Cruz Biotechnology ; anti p-JNK (#9251S), anti JNK (#9252S), anti p-NF-κB (#3031S) and anti NF-κB (#6956S) from Cell Signaling Technology ; anti Wnt5a (#MABD136) from Merck ; anti β-tubulin type III (TUJ1) (#801202) from BioLegend ; anti calnexin (#ADI-SPA-860-F) from Enzo Life Sciences . Fluorescein-(DTAF)-conjugated streptavidin (#016–010-084) was purchased from Jackson Immuno Research . Y27632 and SP600125 were purchased from Abcam .2+/Mg2+-free Hank’s balanced salt solution (Gibco), seeded at 2 × 105 cells in 10-cm culture dishes , which were precoated with 15 μg/ml poly-L-ornithine (Sigma) and 1 μg/ml fibronectin . The cells were then cultured for 5–6 days in serum-free N2 medium supplemented with 20 ng/ml bFGF (R&D Systems Inc.). Cell clusters generated by precursor cell proliferation were dissociated in Hank’s balanced salt solution and plated at 2 × 104 cells per well on coated 24-well plates, 2 × 105 cells per well on coated 6-well plates, and 8 × 105 cells per dish on coated 6-cm culture dishes. All experiments were performed using passage 1 (P1) neural precursor cells.Embryonic brain cortices from E14 rat embryos were mechanically triturated in CaRhoA , Wnt5a , Control  or NF-κB p65 siRNA  were introduced into cells in knockdown experiments using a Nucleofector™ kit .cDNA was prepared using the total mRNA extracted from cells with TRIzol® reagent (Thermo Fisher Scientific); 2 μg samples of RNA were reverse-transcribed using random hexamer mixed primers. The cDNA thus formed was amplified by PCR using the following primers:Wnt3a (5′-TGCAAATGCCACGGACTATC-3′ and 5′-AGACTCTCGGTGTTTCTCTACC-3′),Wnt5a (5′-TGCCACTTGTATCAGGACCA-3′ and 5′-GGCTCATGGCATTTACCACT-3′),Wnt5b (5′-CAAGCTGGAACTGACCAACA-3′ and 5′-AAAGCAACACCAGTGGAACC-3′),Wnt7a (5′-CACAATTCCGAGAGCTAGGC-3′ and 5′-TAGCCTGAGGGGCTGTCTTA-3′),Wnt7b (5′-GCTATCAGAAGCCGATGGAG-3′ and 5′-ACGTGTTGCACTTGACGAAG-3′),Nt3 (5′-GGCACACACACAGGAAGTGTC-3′ and 5′-CTGGACGTCAGGCACGGCCTGT-3′),Ngn1 (5′-ATGCCTGCCCCTTTGGAGAC-3′ and 5′-TGCATACGGTTGCGCTCGC-3′),p65 NF-κB (5′-CTAGGAGGACTCGGGCTCTT-3′ and 5′-AGGAGCTCCACAGGACAGAA-3′),Gapdh (5′-CTCGTCTCATAGACAAGATG-3′ and 5′-AGACTCCACGACATACTCAGCAC-3′).Wnt3a, Wnt5a, Wnt5b, Wnt7a, Wnt7b, Nt3, Ngn1, p65 NF-κB, and Gapdh were as follows: denaturation at 94 °C for 30 s, annealing at 58 °C for 1 min, and extension at 72 °C for 1 min. 30 cycles were used for amplification of all cDNAs. The PCR products were analyzed on a 1.5% agarose gel. For real-time PCR, 5 μl of RT reaction product was amplified in duplicates at a final volume of 20 μl iQ™ SYBR®Green Super mix. Thermocycling conditions were 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The primer sequences for real-time PCR were same as those used for RT-PCR, and all gene expression values were normalized to those of Gapdh.The PCR conditions for amplification of 3VO4. Protein samples (20–30 μg) were loaded on 10% SDS–polyacrylamide gels, electrophoresed, and transferred to nitrocellulose membranes  after electrophoresis. After blocking with 5% non-fat dried milk for 1 h, membranes were incubated with primary antibodies followed by incubation with HRP-conjugated secondary antibody (Anti-mouse IgG (#7076) and Anti-rabbit IgG (#7074)) (1:2000) , and specific bands were detected by ECL (Amersham Pharmacia Biotech).Cells were lysed in 20 mM Tris–HCl (pH 7.5) containing 150 mM NaCl, 1 mM Ethylenediaminetetraacetic acid (EDTA), 1 mM Ethylene glycol-bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 2.5 mM sodium pyrophosphate, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1 mM Na2+ Lysis/Wash Buffer (MLB). Lysates were collected by centrifugation at 14,000×g for 5 min at 4 °C. Equal amounts of protein from cell lysates were incubated for 45 min at 4 °C with 20 μg of Rhotekin-RBD protein agarose beads. Pellets were washed with MLB and subjected to western blotting using the anti-RhoA antibody.RhoA activity in cells was measured using Rho Activation assay kit , according to the manufacturer’s instructions. Briefly, cells were washed with ice-cold PBS, and lysed with MgCells were washed with PBS and fixed with 4% paraformaldehyde in PBS, followed by three washes with PBS at room temperature. They were then permeabilized with 0.1% Triton X-100 in PBS for 10 min, followed by three washes with PBS, and then blocked with 10% normal goat serum in PBS containing 0.5% Tween 20 for 1 h at room temperature. Next, the cells were incubated with the mouse monoclonal anti-β-tubulin type III (TUJ1) antibody (1:2000 dilution) primary antibody at 4 °C. Cells were then stained with streptavidin-conjugated secondary antibody (1:400) for 1 h before mounting with Vectashield  containing 4, 6-diamidino-2-phenylindole (DAPI). Immunoreactive cells were detected using a TCS SP5 confocal imaging system  at magnification between 40× and 60 ×.w/v) picric acid/PBS containing 4% (w/v) paraformaldehyde, and incubated overnight at 4 °C with β-tubulin type III (TUJ1) antibody 1:2000. After incubation with Cy3-conjugated secondary antibody , cells were mounted on slides with Vectashield. TUJ1-positive cells were photographed using the TCS SP5 confocal imaging system (Leica Microsystems) and morphological characteristics were quantitated using Image J software (NIH-http//rsb.Info.nih.gov/ij/). The length of primary neurite was defined as the distance from the soma to the tip of the longest branch. For each graph, data on neurite length were generated from randomly selected areas of at least five independent cultures from three independent experiments and more than 100 cells were counted for each condition in each experiment.Cells were cultured on coverslips coated with fibronectin in 24 well plates, fixed with 0.1% (t test and were considered significant at p < 0.05.Cells were counted in randomly chosen (fractionator) microscope fields. Data are expressed as means ± SDs of three independent experiments. Differences were analyzed using the Student’s We first established the distribution of IL-1β in neuronal differentiation of NPCs. Nerve growth factors, such as NT3, NT4/5, and BDNF, together with the basic helix-loop-helix transcription factors neuro-D and neurogenin-1 (Ngn1) are closely associated with neuronal differentiation and can be used as markers of this process. Treatment with IL-1β increased the expression levels of NT3 and Ngn1 in a time-dependent manner (0–6 h) Fig.  and b anThe rat Wnt5a promoter contains both conserved and putative nuclear factor kappa B (NF-κB) binding sites  and its We tested whether exogenous Wnt5a could enhance neuronal differentiation. As shown in Fig. The JNK , also known as stress-activated protein kinase, has extensive implications in understanding important biological processes, such as cell growth, differentiation, tissue development and regeneration , 34. PreWe next examined whether the Wnt5a-mediated RhoA/ROCK/JNK pathway is involved in IL-1β-induced neuronal differentiation. We showed that IL-1β-induced RhoA activity was significantly decreased due to knockdown of Wnt5a using siRNA Fig.  and protIL-1β is known to be a key modulator of stress and inflammation in the CNS. Neuroinflammation is implicated in the pathophysiology of many psychiatric and neurodegenerative disorders where cognitive dysfunction and reductions in neurogenesis are evident, including Alzheimer’s disease (AD), Parkinson’s disease (PD), and depression . IL-1β hRecently, Wnt5a, identified as an axon guidance cue, was shown to activate a non-canonical pathway essential for cortical axonal morphogenesis. In this study, we found that the Wnt5a/RhoA/ROCK/JNK pathway is required for IL-1β-mediated neurite outgrowth of NPCs. The effect of Wnt5a signaling on axon outgrowth of cortical neurons is consistent with a recent study showing that Wnt5a increases axon length and promotes axon differentiation of dissociated hippocampal neurons . In addiAxon outgrowth and navigation during synapse formation are regulated by extracellular signals. The cytoskeletal mechanisms of axonal branching are regulated by Rho GTPases . Recent Here, we demonstrate a novel function of IL-1β in neuronal differentiation of NPCs. The findings of the present study are summarized in Fig. Additional file 1:Figure S1. NPCs were pretreated with 5 μM Y27632 for 1 h and stimulated with Wnt5a (20 ng/ml) for 6 h. mRNA levels of Nt3 and Ngn1 were analyzed by RT-PCR (A) and real time-RT-PCR (B). n = 3. Data are mean ± SD; Student’s t test. * p < 0.05 compared with Wnt5a-treated cells. (C) Cells were pretreated with 5 μM Y27632 for 1 h and stimulated with Wnt5a (20 ng/ml) for 2 days. Western blotting was performed using an anti-NT3, anti-Ngn1, or anti-calnexin antibodies to detect the respective protein bands. (D and E) Cells were pretreated with 5 μM Y27632 for 1 h and stimulated with Wnt5a (20 ng/ml) for 3 days. They were stained with anti-Tuj1. Scale bar, 20 μm. (E) Neurite lengths were measured in randomly selected fields using three independent experiments. n = 3 per group. Data are mean ± SD; Student’s t test. ** p < 0.01 compared with Wnt5a-treated cells. (PPTX 419 kb)


Scientific Reports 10.1038/s41598-018-30349-1, published online 08 August 2018Correction to: The Article contains an error in the legend of Table 1.†P < 0.05 (Friedman test) between pre-dose and week 4; ‡P < 0.05 between pre-dose and week 8. ns, not significant.”“Data are the mean ± standard deviation. should read:†P < 0.05 (Wilcoxon signed-rank test) between pre-dose and week 4; ‡P < 0.05 between pre-dose and week 8. ns, not significant.”“Data are the mean ± standard error of mean.


Z)-3-(2-chloro-9H-thioxanthen-9-yl)-N,N-dimethylpropan-1-amine], gives a 1:2 acid salt (II) containing a very short O—H⋯O hydrogen bond. Multiple hydrogen bonds link the ions in (I) into a complex chain of rings and those in (II) link the ions into a sheet.Co-crystallization of 3,5-dinitrobenzoic acid with trihexyphenidyl [or 1-cyclohexyl-1-phenyl-3-(piperidin-1-yl)propan-1-ol], gives a 1:1 salt (I) but chlorprothixene −, (II), the anion of which contains a very short O—H⋯O hydrogen bond, with dimensions O—H = 1.04 (3) Å, H⋯O = 1.41 (3) Å, O⋯O = 2.4197 (15) Å and O—H⋯O = 161 (3)°. In the cation of (I), the cyclohexyl and piperidyl rings both adopt chair conformations, whereas in the cation of (II), the central heterocyclic ring adopts a boat conformation, so that the dihedral angle between the two aryl rings is 41.56 (4)°. A combination of O—H⋯O, N—H⋯O and C—H⋯O hydrogen bonds links the ions of (I) into a complex chain of rings, and these chains are linked into sheets by π–π stacking interactions between inversion-related pairs of anions. In compound (II), a different combination of O—H⋯O, N—H⋯O and C—H⋯O hydrogen bonds links the ions into sheets. Comparisons are made with some related structures.Co-crystallization of racemic 1-cyclohexyl-1-phenyl-3-(piperidin-1-yl)propan-1-ol (trihexyphenidyl) with 3,5-dinitrobenzoic acid gives a simple 1:1 salt, namely 1-(3-cyclohexyl-3-hydroxy-3-phenylpropyl)piperidin-1-ium 3,5-dinitrobenzoate, C H-thioxanthen-9-yl)-N,N-dimethylpropan-1-amine, chlorprothixene, can both be used in the treatment of psychotic depression -chlorprothixene with 3,5-dintrobenzoic acid yielded a simple 1:1 salt in the case of trihexyphenidyl propan-1-ol, trihexyphenidyl, and 3--3-(2-chloro-9H-thioxanthen-9-yl)-N,N-dimethylpropan-1-amine and 3,5-dinitrobenzoic acid gave an acid salt (II)et al., 1997Co-crystallization from a methanol solution containing equimolar quantities of racemic trihexyphenidine and 3,5-dinitrobenzoic acid gave a simple 1:1 salt (I)et al., 1990et al., 19952 hydrogen bond and a two-centre C—H⋯O hydrogen bond nd Fig. 2.A, C14B, C18A and C18B are coplanar with the atoms C19 and S10, which form the bow and stern of the boat  are Q = 0.5721 (12) Å, θ = 86.71 (13)° and φ = 0.53 (14)°: for an idealized boat form the puckering angles are θ = 90.0° and φ = 60k°, where k represents an integer at Fig. 3, displacIn the anion of (I)2 system, again linking inversion-related ion pairs to form a complex motif in which a central n, n) and the fourteen-membered rings are centred at , where n represents an integer in each case, so forming a chain of rings running parallel to the [011] direction  and . These rings are strictly parallel, with an interplanar spacing of 3.4413 (6) Å: the ring-centroid separation is 3.5231 (10) Å, corresponding to a ring-centroid offset of 0.755 (2) Å on Fig. 4. Chains  Å Fig. 5, and thiet al., 1998abet al., 2000In the structure of compound (II)on Fig. 6, while ton Fig. 7. The comS(6) motif, but there are no significant direction-specific interactions between the molecules -N,N-dimethylpropan-1-amine can exist in (E) and (Z) isomers, and the structures of both forms have been reported et al., 1975et al., 2016et al., 2001et al., 2001et al., 2005et al., 20022]−, which lies across a centre of inversion in a mixed salt containing both sodium and methyltriethylammonium cations, as well as tris(thiobenzohydroximato)chromium(III) anions and water molecules  Å (Barrow v/v) and N,N-dimethylformamide.Samples of racemic trihexyphenidine and (Z)-chlorprothixene were gifts from RL Fine Chem Pvt. Ltd., Bengaluru, India. For the synthesis of compound (I)3), 0.99 Å (CH2) or 1.00 Å  and with Uiso(H) = kUeq(C), where k = 1.5 for the methyl groups, which were permitted to rotate but not to tilt, and 1.2 for all other H atoms bonded to C atoms. For atom H312 in the short O—H⋯O hydrogen bond, the atomic coordinates and the Uiso(H) value were all refined; for the remaining H atoms bonded to N or O atoms, the atomic coordinates were refined with Uiso(H) = 1.2Ueq(N) or 1.5Ueq(O). The resulting N—H and O—H distances are given in Tables 1−3, was located near the bond C17—H17, at distances from these two atoms of 1.40 and 0.62 Å, but no plausible chemical interpretation of this seems possible.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989019001385/zl2748sup1.cifCrystal structure: contains datablock(s) global, I, II. DOI: 10.1107/S2056989019001385/zl2748Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989019001385/zl2748IIsup3.hklStructure factors: contains datablock(s) II. DOI: 1883983, 1883984CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Three 1-[methyl]-4-piperazines adopt very similar molecular conformations but, while the molecules of the 3-fluorobenzoyl are linked by hydrogen bonds into a three-dimensional structure, there are no hydrogen bonds in either of the 2,6-difluorobenzoyl and 2,4-dichlorobenzoyl analogues. 19H19FN2O3 (I), 1-[methyl]-4-piperazine, C19H18F2N2O3 (II), and 1-[methyl]-4-piperazine, C19H19Cl2N2O3 (III), the piperazine rings adopt a chair conformation with the methyl substituent occupying an equatorial site: the five-membered rings are all slightly folded across the O⋯O line leading to envelope conformations. The dihedral angle between the planar amidic fragment and the haloaryl ring is 62.97 (5)° in (I) but 77.72 (12)° and 75.50 (5)° in (II) and (III), respectively. Despite their similarity in constitution and conformation, the supramolecular interactions in (I)–(III) differ: in (I), a combination of C—H⋯O and C—H⋯π(arene) hydrogen bonds links the molecules into a three-dimensional framework structure, but there are no hydrogen bonds of any sort in either (II) or (III), although the structure of (III) contains a short Cl⋯Cl contact between inversion-related pairs of molecules.In each of the compounds 1-[methyl]-4-(3-fluorobenzoyl)piperazine, C The piperazine rings all adopt chair conformations with the substituent at atom N1 in an equatorial site, while the atoms of the amide fragment  are coplanar. The only significant conformational difference between the molecules in (I)–(III) lies in the dihedral angle between the amide unit and the adjacent aryl ring (C41–C46), 62.97 (5)° in (I)In each of (I)–(III), the five-membered ring is slightly non-planar: while the atoms O11, C7et al., 1998abet al., 20001 screw axis along  to form a C(5)  into a second chain running parallel to [010] and, together, these two interactions generate a sheet lying parallel to (001)  and the symmetry codes are (i) x, −y, z) and (ii) . Hence, the two molecules providing the donor atoms here are related by inversion across . In this structure, the atoms of type O11 in the molecules at  and  are separated by a distance of only 2.7888 (18) Å. At the same time, the atoms C12 and H12 at  are distant from O11 at  by 2.66 and 3.008 (2) Å, respectively, with an associated C—H⋯O angle of 101°; the H⋯O distance is too long and the C—H⋯O angle is too small for this contact to be regarded as a hydrogen bond, but the short O⋯O distance here is perhaps associated with this ‘failed’ hydrogen bond involving atom C12.Despite their similar molecular constitutions and conformations, compounds (I)–(III) all exhibit different types of direction-specific intermolecular interactions. In the crystal structure of compound (I)1) Fig. 4. The secon Fig. 5, and chaon Fig. 5, with thD–-H⋯A angle is only 123° so that this cannot be regarded as structurally significant  and , the Cl⋯Cli distance is 3.3963 (7) Å with an associated C—Cl⋯Cli angle of 137.68 (5)° . For C—Cl⋯Cl angles of 90 and 180°, values of 1.78 and 1.58 Å have been suggested et al., 2013et al., 2019N-(4-chlorophenyl)-4-(pyrimidin-2-yl)piperazine-1-carboxamide crystallizes with Z′ = 2 in space group P21/c, and the molecules are linked by two independent N—H⋯O hydrogen bonds to form chains of C(4) in the original report –(III) with that observed in some related compounds. In 1-[methyl]-4-(pyrimidin-2-yl)piperazine (piribedil), the molecules are linked into sheets by three independent C—H⋯π hydrogen bonds  and the resulting mixtures were then stirred at 273 K for 20 min. A solution of 1-[methyl]piperazine  in N,N-dimethylformamide (5 ml) was then added to each mixture and stirring was continued overnight at ambient temperature. When the reactions were complete as confirmed using thin-layer chromatography, an excess of water was added to each of the mixtures, which were then exhaustively extracted using ethyl acetate. Each of the organic fractions was then washed successively with aqueous hydrochloric acid (1 mol dm−3), then with a saturated aqueous solution of sodium hydrogencarbonate, and finally with brine. The organic fractions were then dried over anhydrous sodium sulfate and concentrated under reduced pressure. Slow evaporation of these solutions, at ambient temperature and in the presence of air, gave crystals of compounds (I)–(III) suitable for single-crystal X-ray diffraction: m.p. (I)1-[methyl]piperazine was purchased from Sigma–Aldrich and used as received. For the synthesis of compounds (I)–(III), 1-(3-dimethylaminopropyl)-3-ethylcarbodimide , 1-hydroxybenzotriazole  and triethylamine  were added to solutions of the appropriately substituted benzoic acid [3-fluorobenzoic acid for (I)2) and with Uiso(H) = 1.2Ueq(C). For compound (I)x parameter  − (I−)]/[(I+) + (I−)] , is likewise indeterminate.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989019000458/zl2747sup1.cifCrystal structure: contains datablock(s) global, I, II, III. DOI: 10.1107/S2056989019000458/zl2747Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989019000458/zl2747IIsup3.hklStructure factors: contains datablock(s) II. DOI: 10.1107/S2056989019000458/zl2747IIIsup4.hklStructure factors: contains datablock(s) III. DOI: Click here for additional data file.10.1107/S2056989019000458/zl2747Isup5.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989019000458/zl2747IIsup6.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989019000458/zl2747IIIsup7.cmlSupporting information file. DOI: 1889708, 1889709, 1889710CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Type I and type II natural killer T (NKT) cells are restricted to the lipid antigen-presenting molecule CD1d. While we have an understanding of the antigen reactivity and function of type I NKT cells, our knowledge of type II NKT cells in health and disease remains unclear. Here we describe a population of type II NKT cells that recognise and respond to the microbial antigen, α-glucuronosyl-diacylglycerol (α-GlcADAG) presented by CD1d, but not the prototypical type I NKT cell agonist, α-galactosylceramide. Surprisingly, the crystal structure of a type II NKT TCR-CD1d-α-GlcADAG complex reveals a CD1d F’-pocket-docking mode that contrasts sharply with the previously determined A’-roof positioning of a sulfatide-reactive type II NKT TCR. Our data also suggest that diverse type II NKT TCRs directed against distinct microbial or mammalian lipid antigens adopt multiple recognition strategies on CD1d, thereby maximising the potential for type II NKT cells to detect different lipid antigens. Natural killer T (NKT) cells include type I that express semi-invariant T cell receptor (TCR), and type II that cover a broader repertoire. Here the authors describe the crystal structure of a type II NKT TCR complexed with CD1d/antigen to propose that type II NKT TCRs may adapt multiple CD1d docking modes to maximise antigen recognition efficacy. There are two broad classes of NKT cells, defined as type I and type II NKT cells. The most extensively studied are the type I NKT cells, which express an invariant TCR α-chain ) (reviewed in ref. 1). Upon activation, type I NKT cells rapidly produce an array of cytokines and are implicated in a broad range of diseases through their ability to modulate Ag-presenting cells and effector T, B and natural killer (NK) lymphocytes (reviewed in ref. 1). Type II NKT cells are also CD1d restricted, but they are distinguished from type I NKT cells because they neither express the invariant TCR α-chain that characterises type I NKT cells nor do they recognise α-GalCer (reviewed in refs. 2). The specificity and function of type II NKT cells is poorly understood although some evidence suggests that they play an immunosuppressive role that may oppose the function of type I NKT cells4 (and reviewed in refs. 2). Given that type II NKT cells appear to be more abundant in humans than their type I NKT cell counterparts7 (and reviewed in ref. 8), it is important to understand the molecular mechanisms underpinning their antigen reactivity and specificity.Natural killer T (NKT) cells recognise lipid-based antigens (Ag) presented by the major histocompatibility class I (MHC I)-like molecule CD1d . Conversely, a limited understanding of the Ags recognised by type II NKT cells has hampered the study of these cells, although some studies have identified Ags recognised by some type II NKT cell lines, including sulfatide, β-glucosylceramide (β-GlcCer), phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE)20. Structural studies have determined how a type II NKT TCR can bind to sulfatide or lysosulfatide presented by mouse CD1d22, and revealed a docking mode that was distinct to the type I NKT TCR–CD1d–α-GalCer interaction (reviewed in ref. 23). Namely, type I NKT TCRs bind with a parallel docking mode over the F′-pocket of CD1d, with the TCR α-chain dominating the interaction with the galactose headgroup, while the TCR β-chain binds solely to CD1d (reviewed in ref. 23). In contrast, the sulfatide-reactive type II NKT TCR docked orthogonally over the A′-pocket of CD1d with only the CDR3β loop interacting with the sulfatide headgroup22. These studies suggests that the structural and molecular basis for CD1d–Ag recognition between type I and type II NKT cells is fundamentally distinct (reviewed in refs. 24). However, it remains to be determined whether this represents a general dichotomy that distinguishes the mode of Ag recognition by type I and type II NKT cells. We previously identified an atypical population of CD1d-restricted NKT cells that utilise a Vα10Jα50 TCR α-chain that recognise α-GalCer and α-GlcCer. Some of these atypical NKT cells were activated by a mycobacterial Ag, α-glucuronosyl-diacylglycerol (α-GlcADAG)25 presented by CD1d.The availability of α-GalCer and CD1d–α-GalCer tetramers+ NKT TCR–CD1d–α-GlcADAG complex revealed a parallel docking mode over the F′-pocket of CD1d, which contrasted the type II NKT TCR–CD1d–sulfatide ternary complexes22. Extensive CD1d mutational analysis against a panel of diverse self and microbial-antigen-reactive type II NKT TCRs reveal that type II NKT cell TCRs can adopt differing docking strategies to engage specific CD1d–Ag complexes. Thus, this study defines how a microbial-Ag is recognised by a type II NKT TCR, and demonstrates that type II NKT TCRs can bind with diverse docking modes to CD1d–Ag. This highlights the diverse lipid-based Ag recognition by type II NKT TCRs.Here, using α-GlcADAG-loaded CD1d tetramers, we characterise a population of type II NKT cells that are selectively reactive to the α-GlcADAG Ag presented by CD1d and do not recognise α-GalCer. Using a panel of synthetic α-GlcADAG analogues, we demonstrate the key moieties of this molecule that facilitate type II NKT TCR recognition. Structural analysis of a type II Vβ8.225. To specifically identify α-GlcADAG-reactive NKT cells, NKT cells were first enriched by depletion of immature (CD24+) thymocytes25, and CD1d–α-GlcADAG tetramers were used to stain mouse thymocytes from wildtype (wt), Jα18−/− and CD1d−/− mice in a BALB/c background   Fig.  althoughD69 Fig. . Collect+ cells that were sorted as single cells from both wt and Jα18−/− BALB/c thymi, following tetramer-associated magnetic enrichment (TAME) based on gates depicted in Fig. + CD1d–α-GlcADAG tetramer− type I NKT cells from wt mice were also sorted as controls. Single cell TCR α- and TCR β-chain paired analysis was performed using multiplex PCR, as previously described26  of the CD1d–α-GlcADAG tetramer+ sorted cells expressed Vα10Jα50 TCR α-chain rearrangements, similar to the Vα10+ NKT cells present in Jα18−/− mice that we previously described25. Interestingly, four CD1d–α-GlcADAG tetramer+ clones from wt BALB/c mice expressed a TCR α-chain in which the Jα50 gene was rearranged with Vα15, Vα4, Vα11, or Vα4.4, instead of the Vα10 gene. These TCR α-chains displayed little or no homology in their CDR1α and CDR2α regions, yet possessed highly similar CDR3α regions suggesting that the Jα50-encoded region confers CD1d–α-GlcADAG recognition in the context of different Vα gene usage. This may be due to the conservation of the CDR3α residues Ser109, Ser110 and Phe113 in each of these TCRs, three residues that were involved in the recognition of CD1d–α-GlcCer complexes by Vα10Jα50+ NKT TCRs25. The different CDR1α and CDR2α loops may also facilitate CD1d binding in different ways. Indeed, in a previous study we demonstrated that a Vα10Jα50+ NKT TCR utilised residues within the CDR1 and CDR2 loops to establish contact with CD1d, whilst conserved CDR3α residues contacted both CD1d and the antigen25. Interestingly two unique TCR α-chain sequences that did not express Vα14, Jα18 or Jα50 gene segments  were also identified amongst wt thymocytes sorted with CD1d–α-GlcADAG tetramers (Supplementary Table Vβ8 (24 out of 25 paired TCR sequences), consistent with predominant Vβ8 gene usage within type I NKT cells9, Vα10Jα50+ NKT cells25, and type II NKT cells28. Some TCR sequences with identical TCRα and TCRβ nucleotide homology were identified in independently sorted cells  were observed amongst Jα18−/− thymocytes sorted with CD1d–α-GlcADAG tetramers  and a Vα17Jα2-Vβ8.2 TCR (clone A17B8.2) from Jα18−/− thymus ; and an atypical NKT TCR 25.A selection of TCR sequences from CD1d–α-GlcADAG tetramer+ and Vα10Jα50-Vβ8.3+ atypical NKT clones (A10B8.2 and A10B8.3) were labelled by CD1d–α-GlcADAG tetramers, but not by CD1d–sulfatide or CD1d tetramers carrying endogenous lipid Ags (CD1d-endo) tetramers, although CD1d–α-GalCer tetramers stained with greater intensity   Fig. . Thus, t+ cell line showed clear staining with sulfatide-loaded CD1d tetramer whereas the other CD1d tetramers did not stain these cells at higher levels than CD1d–endo  was clearly labelled by CD1d–α-GlcADAG tetramer (~20× higher than CD1-endo tetramer MFI). In contrast, CD1d–α-GalCer tetramer or CD1d–sulfatide tetramers failed to provide staining of this cell line above CD1d–endo tetramers, highlighting that α-GlcADAG is indeed the preferred ligand for the A11B8.2 TCR , and interestingly, sulfatide-loaded and α-GalCer-loaded CD1d tetramers displayed a lower ability to stain this line when compared to CD1d-endo tetramers , suggesting that they can act as weaker lipid-antigens for this clone  of ~40 μM), whereas this TCR displayed stronger binding to α-GalCer-loaded CD1d, with a Kd of 11.4 μM, but was not reactive to CD1d-endo (Kd > 150 μM) . Binding of this TCR to CD1d–α-GalCer could also be detected, although with a much lower affinity (Kd = 66.5 μM) . The A10B8.2 atypical NKT TCR bound weakly to CD1d–α-GlcADAG (with a dissociation constant (μM) Fig. , consistμM) Fig. . NotablyR-tuberculostearic acid (R-TBSA) (C19:0) at sn-1 and C16:0 at sn-2 that is expressed by Mycobacterium smegmatis25  originally from Streptococcus pneumoniae35; α-galactosyldiacylglycerol  originally from Borrelia burgdorferi36; α-GlcCer (C20:2)38; the β-linked Ags sulfatide C24:115 and GD3 C26:039.The fine specificity of CD1d–α-GlcADAG-reactive TCRs was examined using CD1d tetramers loaded with a range of synthetic α-GlcADAG variants Fig. . These i+ cell line and XV19 type II NKT TCR+ cell lines showed the expected patterns of reactivity, with strong binding to CD1d tetramers loaded with α-GalCer for the former, and sulfatide for the latter  was clearly bound by the TCR at levels greater than CD1d-endo . Moderate staining of this line was achieved by the oleic-acid (C18:1/C16:0) followed by the S-TBSA variant (S-C19:0/C16:0), and to a much lower degree by the ‘isooleoyl’ regioisomer α-GlcADAG (C16:0/C18:1)  Fig. . FurtherAGs Fig. , which d22. Instead, and reminiscent of the classical type I and atypical NKT TCR–CD1d–Ag ternary complexes  and Vα10 (910 Å2) NKT TCRs. Here, the A11B8.2 TCR α- and β-chains contributed to 57% and 43% of the BSA, respectively. The CDR3α (24% BSA) and CDR3β (24% BSA) loops contributed the most to the TCR A11B8.2–CD1d–Ag interface and to a lesser extent, the CDR1α (13.5% BSA), CDR2α (9% BSA), and CDR2β (9% BSA) loops were also involved in the interaction with CD1d–α-GlcADAG   contacts with Asp153 Fig. , while T53) Fig. . The aro53) Fig. . Accordi53) Fig. . This CDoop Fig. 31. Colle45 and CD1d–α-GlcDAG-s2 (PDB code: 3T1F)35 crystal structures  and R-TBSA (sn1) chains were positioned within the A′-pocket and F′-pocket, respectively. However, the palmitoyl tail adopted the unusual counter-clockwise orientation that has been previously observed in the type I NKT TCR–CD1d–α-Gal–GSL (PDB code: 3O8X)res Fig. . While tres Fig. 23,24,46–D1d Fig. . Here, tD1d Fig. . Here, Lgs Figs.  and 3a. To more broadly investigate the docking mode of diverse type II NKT TCRs with CD1d, we generated soluble CD1d molecules with 18 single residue mutations across the length of the Ag-binding cleft and tested their ability to stimulate a panel of NKT cell lines Fig. . These m31. Further, and consistent with the crystal structure, mutational analyses indicated that Met87, Glu83, Lys86 and Val149 were critical for CD1d–Ag recognition by the A11B8.2 TCR  was impacted by the CD1d mutations: Arg79Ala, Glu83Ala, Lys86Ala, Met87Ala, Leu145Ala and Val149Ala. These residues are localised around the F′-pocket and mediate contacts with the type I NKT cell TCRs  . This raised the question of whether other type II NKT TCRs would adopt this A′-roof-binding mode with CD1d-Ag. We show that type II NKT TCRs can also bind over the F′-pocket of CD1d. Indeed, the structure of the type II A11B8.2 TCR–CD1d–α-GlcADAG complex revealed that the interactions between the two Tyr residues within the CDR2β loop with a defined region over the F′-pocket of CD1d were similar to that observed for mouse Vβ8+ NKT TCRs53. However, analysis of the binding footprint of a range of type II NKT TCRs with diverse TCR α- and β-chains, indicates that some type II NKT TCRs can bind in multiple ways to CD1d. This likely reflects the high TCR diversity of type II NKT cells and can engender differing reactivities to various CD1d-restricted Ags, including non-lipid molecules54 or even peptides and lipo-peptides55. There are some parallels with that in MR1-mediated immunity, whereby diversity in the MR1-restricted T cell repertoire can also lead to differing reactivities towards metabolite-based ligands and differing docking modes atop MR156.While several Ag-specific type II NKT cell populations have been identified7 (and reviewed in ref. 8), and considering the unique functional roles that these cells appear to play in the immune system (reviewed in ref. 2) these studies are important if we are to understand the full scope of Ags recognised by type II NKT cells and to harness the therapeutic potential of this arm of the immune system.In summary, we identify a new, microbial Ag-specific, population of type II NKT cells. Our findings also suggests that a universal potent Ag for type II NKT cells is unlikely to exist. Rather, our understanding of type II NKT cells will be built upon studies of individual Ag-specific subsets, such as those identified here. Nonetheless, given that type II NKT cells may be more abundant than type I NKT cells in humans−/−, BALB/c CD1d−/− were bred and maintained according to ethics approval from The University of Melbourne Animal Ethics Committee, under specific pathogen-free conditions at the Biological Research Facility at the Department of Microbiology and Immunology, The University of Melbourne, Australia. BALB/c wt colonies were sourced from either the same facilities, or the Animal Resources Centre . CD1d−/−  mice were sourced from the Peter MacCallum Cancer Centre  and backcrossed to BALB/c for at least 10 generations. Jα18−/− mice were originally provided by M. Taniguchi  and backcrossed to BALB/c for at least 10 generations.BALB/c wt, BALB/c Jα18g for 10 min, with no brake, at room temperature. Cells at the interphase were recovered, centrifuged at 350 × g for 4 min at 4 °C and washed with 2% FBS/PBS, or 10% FBS/RPMI for cell culture. All cells were quantified in a Neubauer counting chamber (Merck), with dead cell exclusion by trypan blue solution 0.4% (Sigma).Thymocytes were isolated by grinding thymi through a 50 μM mesh or cell strainer in the presence of 2% FBS PBS. For complement-mediated enrichment of mature thymocytes, the cell suspension was then incubated with anti-CD24 (I) (clone J11d—in house production) on ice for 30 min. Rabbit complement  and 70 μg/mL deoxyribonuclease I  were then added and allowed to incubate for another 30 min at 37 °C. Thymocytes were washed with 10% FBS/RPMI, then overlayed onto Histopaque-1083 (Sigma), and centrifuged at 1000 × All antibodies were acquired from BD Biosciences unless otherwise stated. Fluorochrome-conjugated mouse-specific antibodies include: anti-CD3-allophycocyanin (APC) or fluorescein isothiocyanate (FITC) (clone 145-2C11), anti-TCRβ APC/FITC (clone H57-597), anti-Vβ8.1/8.2 FITC/PE (clone MR5.2), anti-CD4 Brilliant Violet (BV)570/Pacific Blue/Alexa Fluor (AF)700/BV605 (clone RM4-5), anti-CD8α APC-Cy7 (clone 53-6.7), anti-CD11c FITC/PE-Cy5 (clone HL3), anti-CD11b FITC/PECy5 (clone M1/70), anti-B220 APC-Cy7/FITC/PE-Cy5 (clone 30-F11), anti-CD69 PE/APC (clone H1.2F3), and anti-CD44 FITC/AF700 (clone IM7). 7-amino-actinomycin D (7AAD) viability dye (Sigma) was included in all flow cytometry-staining panels for dead cell exclusion. Streptavidin (SAV)-PE, SAV-APC, and SAV-BV421 were purchased from BD Biosciences.59. In some assays, mouse CD1d was produced in mammalian HEK-293S.N acetylglucosaminyltransferase-I−(GnTI−) (maintained in house > 10 years) cells by cotransfection with pHLsec vectors encoding truncated mouse CD1d ectodomain with a C-terminal biotinylation motif and His6-tag (amino acid sequence at the C-terminus: GSGLNDIFEAQKIEWHEHHHHHH) and β2-microglobulin, using polyethylenimine essentially as described60. CD1d was purified from culture supernatant by immobilised metal-affinity chromatography and size-exclusion chromatography, followed by enzymatic biotinylation using biotin ligase (produced in-house), and further purification by size-exclusion chromatography, followed by storage at −80 °C.Mouse CD1d tetramers were produced in house in High Five insect cells similar to that previously describedCell suspensions were first incubated for 10 min on ice with Fc-receptor block , prior to antibody staining. For co-tetramer staining, cells were labelled with the lower affinity CD1d–lipid tetramer first, washed twice and the second CD1d–lipid tetramer  added together with surface antibodies. For TAME, anti-PE magnetic beads (Miltenyl Biotech) were added to CD1d–α-GlcADAG-labelled cells and the magnetically labelled fraction was isolated using LS columns (Miltenyl Biotech), according to manufacturer’s instructions. Cells were analysed on a BD LSRFortessa™ or BD LSR II™, or alternatively, singles cells were sorted on a BD FACSAria III™ (Becton Dickinson) directly into 96-well plates. Samples were analysed using FlowJo software (BD).Sphingomonas spp. GSL-1 (α-GlcACer C14:0) and GSL-1′  were provided by Prof. Paul Savage . Streptococcus pneumoniae α-GlcDAG (C16:0/C18:1) and Borrelia burgdorferi α-GalDAG (C17:1/C16:0) were provided by Dr. Petr Illarionov . Glycolipids were prepared in either tyloxapol-based detergent  pH 8, or Tween 20-based detergent . All glycolipids were stored at −20 °C. Glycolipids were sonicated for ~30 min prior to each use.α-GalCer (C26:0) was purchased from Alexis Biochemicals. GD3 (C26:0) was purchased from Matreya LLC. Sulfatide (C24:1) was purchased from Avanti Polar Lipids. α-GlcADAG analogues were produced in house. α-GalCer (C20:2), α-GlcCer (C20:2), were supplied by Prof. Gurdyal Besra . α-GalCer (C24:1 ‘PBS-44’) and + αβTCR+ cells was generated by the addition of 2 μl per well of buffer containing SuperScript VILO (Invitrogen) and 0.1% Triton X-100 (Sigma) and incubated according to manufacturer’s instructions. cDNA was amplified by two rounds of multiplex nested PCR with a panel of Vα-specific and Vβ primers (Supplementary Table 61 and GoTaq Master Mix (Promega). PCR products were separated on a 1.5% agarose gel, purified and sequenced .cDNA from individually sorted CD1d tetramercis-acting hydrolase element P2A-linked gene system (Addgene plasmid # 52107). The generation of TCR-transduced mouse NKT cell lines was performed similarly to what has been described for the generation of retrogenic mice62. Briefly, HEK293T cells (maintained in house > 10 years) were transfected using FuGENE-6 transfection reagent (Promega) with pMIGII expression vector containing both TCR α- and TCR β-chain-verified sequences using vector-specific primers  and used to transduce mouse BW5147.TCR α−β− thymoma cells (termed BW58—maintained in house > 10 years). The pMIG II, expression and packaging vectors were provided by Dr. Dario Vignali , and the CD3 expression vector was provided by Prof. Stephen Turner . BW58 cell lines were cultured in complete Dulbecco’s modified Eagle medium  supplemented with 10% FBS (v/v), 15 mM HEPES (Gibco), 1 mM sodium pyruvate (Gibco), 0.1 mM non-essential amino acids (Gibco), 50 μM β-mercaptoethanol (Sigma), 100 U/mL penicillin (Invitrogen), 100 μg/mL streptomycin (Invitrogen) and 2 mM l-glutamine (Invitrogen), at 37 °C, 5% CO2.T cell lines expressing αβ TCR were generated by cloning full-length genes encoding α- and β-chains into a pMSCV-IRES-GFP II (pMIG II) vector containing a For plate bound activation assays, lipid-loaded CD1d was coated in 96-well flat-bottom plates (10 μg/ml) for 3 h at 37 °C. BW58 cell lines were co-cultured with plate bound complex for 16 h in cDMEM. Cells were labelled with CD69 mAb to asses CD69 upregulation , and/or culture supernatants were collected for IL-2 cytokine analysis using cytometric bead array (CBA) flex set for mice (BD Biosciences), according to the manufacturer’s instructions.Escherichia coli BL-21 (DE3)pLysS and solubilised in 8 M urea, 0.5 mM ethylenediaminetetraacetic acid (EDTA) pH 8.0, 1 mM dithiothreitol (DTT) and 20 mM Tris–HCl pH 8.0. Soluble TCR α- or β-chain IBPs were injected into a refold buffer . Refolding occurred overnight with gentle stirring at 4 °C. Samples were dialysed for 4 h into 100 mM Urea, 10 mM Tris–HCl pH 8.0 followed by two consecutive dialysis into 10 mM Tris–HCl at pH 8.0 (first for 4 h and second overnight)50. Refolded TCRs were purified by diethylaminoethanol (DEAE) sepharose anion exchange, followed by Superdex-75 16/60 gel-filtration  and anion exchange Mono Q 10/100 GL . The molecular weight of soluble refolded TCRs was validated by liquid chromatography (LC) electrospray ionisation time of flight (ESI-TOF) mass spectrometry (MS) .Sequence-verified constructs encoding TCR α- or β-chains in pET30 or pET28 .SPR experiments were performed on a BIAcore 3000 instrument at 25 °C in HBS buffer . 1% BSA was included in the buffer to prevent any non-specific binding. On average, 3000 response units (RUs) of biotinylated-CD1d were coupled onto a SAV (SAV)-coated sensor chip. Biotin was passed over to block-free SAV sites. Serial dilutions of soluble TCRs were passed over as analytes, and experiments were referenced against an empty channel . Data were analysed using Scrubber Pro (BioLogic Software) and BIAevaluation Version 3.1 (Biacore AB). The steady-state dissociation rate  were used as separate search ensembles while for the A11B8.2–CD1d–α-GlcADAG structure, the structures of mouse CD1d minus the antigen (pdb code: 3HE6) and the previously determined A11B8.2 structure were used. The initial experimental phases showed an unbiased density for the headgroup of α-GlcADAG. The programmes BUSTER67 and COOT68 were used for refinement and model building, respectively. Subsequent refinement cycles and model building resulted in final Rwork/Rfree values of 18.1%/21.9% (For A11B8.2) and 18.7%/24.6% (for A11B8.2–CD1d–α-GlcADAG complex). The quality of both structures was validated at the Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB) Validation and Deposition Services. All presentations of molecular graphics were created with the programme PyMOL69.The A11B8.2 TCR (8–10 mg/mL) and A11B8.2–CD1d–α-GlcADAG ternary complex (5–7.5 mg/mL) were crystallised in 10–20% PEG 3350/4% Tascimate, pH 4 and 15–25% PEG 1500/10% succinate phosphate glycine, pH 6.8, respectively, using the hanging drop vapour diffusion method. The crystals were flash frozen and data were collected at the MX1 and MX2 beamlines (Wavelength 0.9537 Å and 100 K) of the Australian Synchrotron, respectively. The data for the A11B8.2 monomer and ternary complex were processed with the XDS softwareFurther information on research design is available in the Supplementary InformationPeer Review FileReporting SummarySource Data


A characteristic feature of the coordination framework is the presence of [–(μ-Cl)CuCl–]n helical chains interlinked by the btr ligands.The self-assembly of Cu 2+–chloride coordination polymer with the 4,4′-bi-1,2,4-triazole ligand (btr), [Cu4Cl8(C4H6N6)3]n, crystallizes in the non-centrosymmetric orthorhombic space group Fdd2. The two independent Cu2+ cations adopt distorted square-pyramidal geometries with {Cl2N2+Cl} coordination polyhedra. The metal atoms are bridged by μ-Cl anions forming left- and right-handed helical chains of sequence [–(μ-Cl)CuCl–]n along the c-axis direction. In the perpendicular directions, the btr ligands act in μ- and μ3– coordination modes in a 2:3 ratio. The μ-btr bridges connect neighboring helices of the same handedness, whereas the μ3-btr ligands link the helices of opposite handedness, leading to a racemic three-dimensional framework. The structure is consolidated by weak C—H⋯Cl and C—H⋯N interactions.The title Cu Four nitrogen donor sites in the btr molecule provide the possibility of different bridging modes Cl2 was isolated from an aqueous solution 2(btr)]·H2O was isolated in the presence of aqueous HCl  atoms, four chloride anions and one and a half crystallographically independent btr molecules. One btr ligand occupies a general position, while a half of btr sits on a special position .The title compound crystallizes from aqueous solution in the orthorhombic system, non-centrosymmetric space group F2N2+Cl} coordination with two triazole N atoms and two chloride anions in the plane  occupies an apical site. The copper polyhedra are linked together through the μ2-bridging Cl1 and Cl3 anions to form left- and right-handed [Cu1–Cl1–Cu2–Cl3]n helices running along the c-axis direction , with the pitch being equal to the lattice parameter c. The btr ligands adopt μ- and μ3- coordination modes in a 2:3 ratio. It is interesting to note that the μ-bridge btr molecules connect two neighboring helices of the same handedness (ΔΔ or ΛΛ). Then, each helix is connected to the other two of opposite handedness through μ3-bridging btr molecules, thus forming a three-dimensional framework structure  and 78.1 (2)° for μ-and μ3-btr, respectively.The first copper ion, Cu1, adopts a distorted square-pyramidal {Cl al. 1984 introducon Fig. 2. The helre Fig. 3. The btrv atom of the neighboring triazole fragment. There is a bifurcated contact between one C1—H1 fragment and Cl2 (major component) and Cl1iii (minor component). Two other hydrogen-bonding interactions are found between the C4—H4 and C6—H6 fragments and atoms Cl3iv and Cl2vi, respectively.In the crystal, compound (I)In conclusion, the study demonstrates that a combination of a neutral btr molecule and a chloride anion, as complementary donor units, has promising potential in the development and design of metal–organic frameworks.et al., 2016II–oxalate complex [Mn2(btr)(C2O4)2(H2O)2]·2H2O  was prepared in a yield of 60% by the literature transamination reaction between 4-amino-1,2,4-triazole and 2·2H2O  and NH4Cl  in 2 ml of water was added to a solution of btr  in water (0.5 ml). A drop of 0.10 M HCl aqueous solution was then added. The resulting green solution was left standing for several days to form green prismatic crystals. The product was filtered, washed with water and dried in air (yield 47%). Analysis calculated for C12H12Cl8Cu4N18 (I)−1): 608s, 668w, 856m, 896w, 950w, 1022s, 1044s, 1076m, 1102m, 1212w, 1308m, 1338w, 1354w, 1400w, 1498m, 1536m, 3088s, 3112s, 3120s.A solution of CuClThe thermal stability of (I)Uiso(H) = 1.2Ueq(CH).Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989019005516/hb7819sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989019005516/hb7819Isup2.hklStructure factors: contains datablock(s) I. DOI: 1911618CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information: