O-methylglucose–containing lipopolysaccharides (mGLPs) proposed to modulate bacterial fatty acid metabolism. Recently, it has been shown that Mycobacterium tuberculosis mGLP specifically induces a specific subset of protective γ9δ2 T cells. Mild base treatment, which removes all the base-labile groups, reduces the specific activity of mGLP required for induction of these T cells, suggesting that acylation of the saccharide moieties is required for γ9δ2 T-cell activation. On the basis of this premise, we used analytical LC/MS and NMR methods to identify and locate the acyl functions on the mGLP saccharides. We found that mGLP is heterogeneous with respect to acyl functions and contains acetyl, isobutyryl, succinyl, and octanoyl groups and that all acylations in mGLP, except for succinyl and octanoyl residues, reside on the glucosyl residues immediately following the terminal 3-O-methylglucose. Our analyses also indicated that the octanoyl residue resides at position 2 of an internal glucose toward the reducing end. LC/MS analysis of the residual product obtained by digesting the mGLP with pancreatic α-amylase revealed that the product is an oligosaccharide terminated by α-(1→4)–linked 6-O-methyl-d-glucosyl residues. This oligosaccharide retained none of the acyl groups, except for the octanoyl group, and was unable to induce protective γ9δ2 T cells. This observation confirmed that mGLP induces γ9δ2 T cells and indicated that the acylated glucosyl residues at the nonreducing terminus of mGLP are required for this activity.Mycobacteria synthesize intracellular, 6- Mycobacterium spp. produces many exotic lipids and glycolipids that have demanded exploration into their biological functions. Many of the glycans among these glycolipids are naturally methylated (O-methyl mannopolysaccharide and 6-O-methylglucose–containing lipopolysaccharides (mGLPs)2–Rv3032 and MSMEG_5084 knockout mutants of Mycobacterium tuberculosis H37Rv and Mycobacterium smegmatis, known to be impaired in mGLP synthesis, displayed WT fatty acid contents , M. tuberculosis, Mycobacterium leprae, and Mycobacterium xenopi  (1H NMR. A clear doublet at δ 4.89 ppm (3J = 3.76 Hz) was attributed to the anomeric proton of α-Glcp-(1→2)-glyceric acid. The two overlapping doublets at δ 4.77 (3J = 7.9 Hz) and δ 4.78 ppm (3J = 8.2 Hz) confirmed the presence of two β-Glcp-(1→3)-Glcp–linked residues. The presence of one α-Glcp-(1→6)-α-Glcp linkage was evident at δ 5.05 ppm (3J = 3.84 Hz). The overlapping cluster of peaks between δ 5.25 and δ 5.38 integrated to 16 protons and was assigned to α-Glcp-(1→4)-α-glycosyl backbone  and a set of two overlapping double doublets at δ 4.07  were identified (2J) coupling between the geminal diastereotopic methylene protons of the glyceric acid residue  column and examined by NMR A 4). Th. ThA) . Thus, more detailed biochemical analyses of mGLP were pursued to further identify the nature and modification driving the biological activity.This mGP was not able to activate γO-Me-Glc, Glc, and a 3-O-Me-Glc in a ratio of 11:8:1 . Surprisingly, our mGLP preparation did not contain the 2-N-acetyl-2,6-dideoxy-β-glucopyranose as in M. bovis BCG . The resonances for the acyl substitutions were all evident between δ 0.5 and 2.6 ppm, except in this experiment succinyl resonances could not be unambiguously identified due to overlap issues. The chemical shifts at δ 2.37 , δ 1.52 , δ 1.26 , and δ 0.83 ppm  provided the evidence for the presence of the octanoyl residue. The chemical shift at δ 2.55 ppm (>CH-) and a set of two overlapping doublets at δ 1.05  and δ 1.06 ppm  were attributed to an isobutyryl residue. In addition, three differentially located acetyl groups were at δ 1.99, 2.00, and 2.03 ppm . When the spectrum was integrated with reference to eight protons at δ 1.26 ppm , the anomeric region accounted for ∼20 protons, suggesting that all the mGLP isoforms contained the octanoyl residue. However, the integral value (∼2.7) exceeds well over two protons at δ 2.37 ppm , indicating possible overlap from other acyl residues. The relative integral values (the nonoverlapping β-protons (2H) of the octanoyl group at δ 1.52 ppm was the reference integral) revealed an approximate acyl variation ratio (with reference to the octanoyl residue) as octanoyl:isobutyryl:acetyl:succinyl of 1:0.5:0.67:0.35 in the mixture of differentially acylated mGLPs.Native mGLP was analyzed first by troscopy B. The re13C resonances at δ 35.0 (Cα), δ 25.8 (Cβ), δ 29.5 (Cγ), δ 32.2 , δ 22.5 (Cζ), and δ 14.0 ppm (Me) for the octanoyl chain. The methylene carbons identified in the HSQC experiment (β) and δ 30.0 ppm (Cα) were correlated with the 1H spin system in the TOCSY experiment . As the respective methylene proton's chemical shifts were at δ 2.55 (Hα) and δ 2.37 ppm (Hβ), we assigned this methylene system to a possible succinyl residue, which was also confirmed by MS. The HSQC experiment also revealed that the methine (>CH-) proton of the isobutyryl residue at δ 2.55 ppm  overlaps with the methylene proton peak of a succinyl residue. A clear spin system correlation was observed in the TOCSY experiment  and a set of two overlapping doublets at δ 1.05  and δ 1.06 ppm . In the HSQC experiment, the 13C for the acetyls clustered around δ 20.0 ppm. As for the glyceric acid residue, the HSQC experiment showed distinct methylene carbon at δ 62.2 ppm (Cβ) with the corresponding protons at δ 4.15 and δ 4.00 ppm, respectively. The carbon centered at δ 79.8 ppm (δ 4.17 ppm (Hα)) was attributed to Cα of the glyceric acid. The different chemical shifts for the diastereotopic protons (Hβ)  for the glyceric acid has been reported earlier . The protons at δ 3.92–3.60 ppm with corresponding 13C chemical shift at δ 60.0–61.0 ppm were attributed to the H-6/C-6 of α-d-Glcp unit/s linked through (1→4)-glycosidic bonds -glycosidic bonds, were assigned to δ 3.71–3.38 ppm with the corresponding 13C chemical shift at δ 70.0–71.0 ppm -Glcp-(1→2)-glyceric acid unit  with a 13C chemical shift at δ 69.5 ppm. We assigned this to the H-2 of a Glcp that is likely to be acylated. The only other possibility, i.e. H-3 (because H-4 is perhaps glycosyl-linked), would have resulted in a more downfield shift -Glcp) and δ 4.90 ppm (α-(1→6)-Glcp)), indicating that the octanoyl chain is located on the C-2 position of α-Glcp at the reducing end. Furthermore, the α-protons of the octanoyl residue showed specific NOE correlations with protons at δ 2.55 ppm (possibly Hα of a succinyl residue (correlation with isobutyryl was ruled out because the pattern would have been different)) and acetylated methylene (-CH2-) protons at δ 4.20 ppm in addition to anomeric protons at δ 5.02–5.12 ppm (α-(1→4)-Glcp). This suggested that the octanoyl residue is possibly attached to the C-2 of a glucosyl residue with a succinyl substitution close by as in 6-substituted β(1→3)-Glcp  or the anomeric protons (δ 4.6–5.7 ppm) . This su ppm or t→3)-Glcp , 29 and m/z) 1033, identified as a contaminant and [M − 2H]2− at m/z 1918.2, and triply charged dominant species at m/z 1278.5 [M − 3H]3−, which agreed with the calculated molecular weight of mGLP based on published structural studies  mode using collision-induced dissociation . Collision energy was optimized to be 80 and 100 eV. A charge-reduced mass fragmentation pattern was observed whereby singly charged [M − H]− product ions were obtained (Identification of the site of acyl modification in mGLP was sought using tandem MS of native mGLP. The triply charged ions at obtained .m/z 263.1 (C1)− fragment and the corresponding m/z 245.1 (B1)− fragment account for a 3-O-Me-glucosyl residue substituted with one isobutyryl residue. The m/z 467.1 (C2)− fragment  is lost proved particularly informative. The m/z 1273.4 (D8-1)−, 1097.3 (D7-1)−, 921.30 (D6-1)−, 745.2 (D5-1)−, and 569.1 (D4-1)− fragments all contain three nonmethylated glucosyl units with four, three, two, and zero 6-O-Me-glucosyl residues. The m/z 569.1 (D4-1)− fragment corresponded to three nonmethylated glucosyl residues with two acetyl groups. Given the fact that the second Glc has an acetyl unit, the additional acetyl must be either on the third or fourth Glc unit. The ions at m/z 379.1 (D5-3)− and 555.1 (D5-2)− show that the second acetyl is on the fourth glucosyl residue. The ions at m/z 365.1 (D3-1)− and 717.2 (D6-2)− are consistent with this assignment. These fragments  account for a sequence of octaglucoside of the nonreducing end of mGLP of α-d-Glcp(3Me)(6isobutyryl)-(1→4)-α-d-Glcp(6Ac)-(1→4)-α-d-Glcp-(1→4)-α-d-Glcp(6Ac)-(1→4)-α-d-Glcp(6Me)-(1→4)-α-d-Glcp(6Me)-(1→4)-α-d-Glcp(6Me)-(1→4)-α-d-Glcp(6Me).The nonreducing end ment see  has the cing end . The douZ fragments (Z and X ions are numbered from the reducing end of the molecule) from m/z 2607.9 (Z12)− down to m/z 1199.4 in 176 Da shows the presence of eight additional unbranched 6-O-methylglucosyl units toward the reducing end from the nonreducing octaglucoside just described. If the next 6-O-methylglucoside was also unbranched, an ion at m/z 1023.3 should be present; however, this ion is missing. Instead the Z3− ion is present 861.3, showing that the reducing end 6-O-methylglucosyl unit (the fourth main-chain glucosyl from the reducing end) is branched with a single glucosyl unit. These leave a reducing end with four non-O-methylated glucosyl residues, one octanoyl, and one glyceric acid residue. The ions at of m/z 449.1 (D17-16)− and 347.1  contain the octanoyl group but not the glyceric acid group and thus show that the octanoyl group cannot be attached to the glyceric acid. The HSQC and NOESY NMR experiments showed that the octanoyl group is attached to the 2-position of a glucosyl unit −. The double cleave ion at m/z 1051 −, which contains the octanoyl group along with the branched 6-O-methylglucosyl residue, both β-glucosyl residues, a linear glucosyl residue, and the O-3, O-4, O-5, and O-6 oxygens of the glucosyl residue at the reducing end (but not O-2), rules out the possibility of the octanoyl group being attached to the first glucosyl residue. This conclusion is confirmed by the ion at m/z 147 −, which contains O-2 of the first glucosyl residue but no octanoyl group. Therefore the entire mGLP has the following sequence: α-d-Glcp(3Me)(6isobutyryl)-(1→4)-α-d-Glcp(6Ac)-(1→4)-α-d-Glcp-(1→4)-α-d-Glcp(6Ac)-(1→4)-α-d-Glcp(6Me)-(1→4)-α-d-Glcp(6Me)-(1→4)-α-d-Glcp(6Me)-(1→4)-α-d-Glcp(6Me)-(1→4)-α-d-Glcp(6Me)-(1→4)-α-d-Glcp(6Me)-(1→4)-α-d-Glcp(6Me)-(1→4)-α-d-Glcp(6Me)-(1→4)-α-d-Glcp(6Me)-(1→4)-α-d-Glcp(6Me)-(1→4)-α-d-Glcp(6Me)-(1→4)-α-d-Glcp(6Me)-(1→4)-α-d-Glcp(6Me)[β-d-Glcp-(1→3)]-(1→4)-α-d-Glcp(6Me)-(1→4)-α-d-Glcp[β-d-Glcp-(1→3)](2octanoyl)-(1→6)-α-d-Glcp-(1→2)-glyceric acid).The reducing end syl unit , furtherm/z 1489.06 [M − 2H]2− . This mass was attributed to the sequence α-d-Glcp(6Me)-(1→4)-α-d-Glcp(6Me)-(1→4)-α-d-Glcp(6Me)-(1→4)-α-d-Glcp(6Me)-(1→4)-α-d-Glcp(6Me)-(1→4)-α-d-Glcp(6Me)-(1→4)-α-d-Glcp(6Me)-(1→4)-α-d-Glcp(6Me)-(1→4)-α-d-Glcp(6Me)-(1→4)-α-d-Glcp(6Me)-(1→4)-α-d-Glcp(6Me)-(1→4)-α-d-Glcp(6Me)[β-d-Glcp-(1→3)]-(1→4)-α-d-Glcp(6Me)-(1→4)-α-d-Glcp[β-d-Glcp-(1→3)](2octanoyl)-(1→6)-α-d-Glcp-(1→2)-glyceric acid). This indicated that 6-O-Me-Glc units are responsible for enzyme resistance. Notably, the succinyl residue, along with all the acyls but octanoyl, were lost during the digestion. The product E1 was purified from the digest mixture and tested for its ability to activate γ9δ2 T cells. Compared with native mGLP, a ∼90% loss in γ9δ2 T-cell expansion ability was associated with E1 (A).Exhaustive digestion of native mGLP with porcine α-amylase yielded one enzyme major limit product, E1 , which w with E1 A.M. bovis strains, are the only vaccines known to induce protective immunity even when given to infants at birth. Despite these potent stimulatory capacities of M. tuberculosis and BCG, a third of the world is latently infected with M. tuberculosis, and ∼1.5 million people die annually from TB disease complications. Recent TB vaccine development efforts have focused almost exclusively on the induction of αβ T cells specific for immunodominant peptide epitopes, perhaps only enhancing the evolutionary advantage for the pathogen. It certainly remains possible that induction of the right combination of αβ T-cell antigen specificity and effector phenotypes will result in more successful TB vaccines. However, the current goals also involve development of novel vaccine strategies and targets in tandem with further characterization of nonconventional T cells  T cells provide a natural bridge between innate and adaptive immunity, rapidly and potently respond to pathogen infection in mucosal tissues, and are prominently induced by both TB infection and BCG vaccination. Therefore, these cells may serve as potent targets for TB immunotherapy. Recent work has demonstrated important TB-protective effects of γ9δ2 T cells against intracellular replication of mycobacteria in both nonhuman primates and human systems  sequences. Unlike αβ T cells, γδ T cells do not require antigen processing prior to recognition of certain bacterial, lipid, and tumor antigens. Recognition of these T cells by mGLP is a novel finding, and the mechanism is unclear. mGLP is not a major component of M. tuberculosis; in fact, we obtained ∼200 mg of purified mGLP from 2500 g of biomass (0.01%). Despite the presence of a number of glycosyl residues, the molecule is somewhat hydrophobic and acylated, making it behave like a lipid rather than a glycan. The octanoyl group in mGLP has been suggested to have a specific role in stabilizing the polysaccharide in helical conformation, providing it with further discriminatory capability when binding fatty-CoAs, and anchoring mGLP intermediates to the cytoplasmic membrane during the elongation steps . Following this, the product obtained after mild alkali treatment  could only weakly expand T cells, indicating that in some fashion the acyl groups contributed to the biological interplay of mGLP with γ9δ2 T cells. Next, we treated mGLP with porcine α-amylase. The major acidic product isolated after size-exclusion chromatography was analyzed and yielded one major doubly charged product with m/z 1489.06 [M − 2H]2−, confirming it to be an oligomer comprising 11 methyl-Glc, five Glc, one glyceric acid, and one octanoyl residue  whether the five small acyl groups in mGLP  influence T-cell recognition, (b) how are these distributed throughout the molecule, and (c) what is the degree of heterogeneity. Due to the complexity and heterogeneity in the molecule, we used one dominant LC-purified homogeneous isoform of molecular weight 3795 , for a detailed MS/MS experiment by sensitive ultrahigh-resolution QTOF MS/MS. A detailed fragmentation analysis indicated the two acetates to be on the 6-position of the nonreducing-end Glcp(s), and NMR and MS analyses suggest that, in the M. tuberculosis mGLP, the octanoyl group is at the C-2 position of the second Glcp from the reducing end. To summarize, from the structure/function relationship studies, it is now possible to build upon this knowledge such that synthetic intermediates can be generated in large amounts for in vivo application.For current analyses, we relied on 2D NMR and ESI-tandem MS. The questions we sought to answer were: (Sephadex G-50 (fine) was obtained from Sigma-Aldrich. All reagents for biochemical separations were obtained from Acros Organics (silica gel 60) and Sigma-Aldrich (chloroform and methanol).M. tuberculosis H37Rv) were harvested and washed twice with sterile H2O. The pellet was lyophilized and extracted with chloroform:methanol:H2O  twice at room temperature. The organic phase was dried under N2 and stored at −20 °C. Total lipid extracts were fractionated over silica gel 60  using increasing amounts of methanol in chloroform. The 100% methanol eluent was found to be enriched with mGLP  was dissolved in water (0.5 ml), applied onto the size-exclusion column , and eluted with water. The flow rate was maintained at 0.55 ml/min. Fractions  were collected. A quick α-naphthol charring thin-layer chromatography (TLC) assay was performed to identify the carbohydrate-enriched  fractions. Every three consecutive fractions were then pooled, and the monosaccharide composition was determined after derivatization using GC/MS. The analysis revealed that the 62.5–70-ml fraction had the enriched mGLP (without detectable impurities by 1H NMR). The overall purification and characterization strategy is presented as a flow sheet in Fig. S6.100% CHn NaOH (1 ml) and allowed to remain at 55 °C for 2 h. The solution was then neutralized with acetic acid, dried, and applied to a Bio-Gel P-2 column (0.5 × 50 cm) in water for desalting. The deacylated product (mGP) was checked by 1H NMR to ensure completion of deacylation.G-50–purified mGLP (1.0 mg) was dissolved in 0.2 mGP (1.0 mg) and mGLP (3.0 mg) in phosphate-buffered saline  were treated simultaneously with porcine pancreatic α-amylase  for 24 and 72 h, respectively, at 37 °C. TLC on silica gel 60 plates (Merck) with a solvent system composed of chloroform:methanol:water  was used to monitor enzyme activity , visualized by spraying with α-naphthol–sulfuric acid solution followed by charring at 120 °C. Whereas mGP was digested fully, only partial change was observed with mGLP. Enzyme was deactivated, and the digestion mixture was desalted on a Bio-Gel P-2 column (0.5 × 30 cm) followed by a G-50 column. The α-naphthol–positive fractions were used for downstream analyses.m TFA, converted to alditol acetates, and analyzed using GC/MS performed as described previously (Aliquots of G-50 column eluents were hydrolyzed with 2 eviously .1H NMR was recorded in D2O on a 400-MHz Innova (Varian), and 2D NMR  was recorded on a 500-MHz Innova (Varian) instrument at 25 °C. All chemical shifts are based on the reference to the HOD peak at δ 4.64 ppm. The default Varian parameters were used for recording spectra.All PRESAT 18, 2.1 × 150 mm, 3.5-μm particle size) held at 40 °C with a consistent 0.32 ml/min flow rate. Injections were 2 μl, and all solvents were LC/MS grade (Fisher Optima). Starting conditions, 90% solvent A (H2O with 10 mm ammonium acetate), 10% solvent B (acetonitrile with 10 mm ammonium acetate), were held for 5 min, then increased to 70% B over 10 min in a linear gradient followed by an increase to 100% B over 1 min, and then held for 4 min as a wash step. MS instrument parameter settings were as follows: gas temperature, 310 °C; vaporizer temperature, 200 °C; gas flow, 10 ml/min; nebulizer pressure, 45 p.s.i. gauge; and charging voltage, 2000 V. MS source parameter settings were set as follows: capillary voltage, 2500 V; fragmentor, 40; and skimmer1, 60.LC/MS was performed on an Agilent 1260 Infinity series HPLC in line with a 6224 time of flight (TOF) MS equipped with a multimode ESI/atmospheric pressure chemical ionization source operated in negative ESI mode. Gradient separation of a 0.4 mg/ml solution of mGLP in water was performed over an HPLC column (Waters X-Bridge C18 1.7-μm column (2.1 × 50 mm) at 40 °C. Mobile phase components were 10 mm ammonium acetate in water (A) and acetonitrile (B). The flow rate was 0.4 ml/min. The proportion of acetonitrile was increased from 10 to 70% in 3 min and then to 100% in 3.4 min and held at 100% for 1.4 min. The post-time was 2 min, and the injection volume was 3 μl. Internal instrument mass-scale calibration was performed in enhanced quadratic mode during chromatographic dead time by infusing Agilent ESI-L low concentration tuning mix.Structural elucidation of the mGLP was carried out by ultraperformance LC (UPLC) on a Waters Acquity UPLC H-Class system coupled to a Bruker MaXis Plus QTOF MS instrument. Separation was performed in gradient mode with a Waters Acquity UPLC BEH Cm/z range of 110–4000 at 1 Hz was performed in full-MS scan mode for the first 2 min, during which the internal calibrant was introduced into the LC flow. This was followed by a 1-min multiple reaction monitoring scan mode alternating collision-induced dissociation energies of 6 and 40 eV on m/z 1264.1 [M − 3H]3− parent ion with m/z width of 0 and 6, respectively. The final 3.8 min were in full-MS scan mode. Source settings for all time segments were as follows: capillary voltage, 2400 V; end-plate offset, 500 V; nebulizer gas pressure, 3 bar; drying gas flow, 10 liters/min; and drying temperature, 300 °C. Instrument controls were performed via the Bruker HyStar v4.1 software package. Data were processed using Bruker Compass 2.0 Data Analysis 4.4 software.Data acquisition in negative electrospray ion mode with an 9δ2 T cells, isolated PBMCs (1 × 106) were cultured with novel antigen fractions or controls (medium-rested and 20 μg/ml M. tuberculosis whole-cell lysate). On day 7, the PBMCs were harvested, counted, stained with anti-γδTCR (clone 11F2), anti-αβTCR (clone B3), and anti-CD3 peridinin chlorophyll protein (PerCP) (clone SK7). Absolute numbers of γ9δ2 T cells were computed by multiplying the flow cytometric percentages by the numbers of viable cells present after expansion. Expansion indices were calculated as the -fold expansion of the absolute number of γ9δ2 T cells after stimulation with treated lysates compared with the absolute number of γ9δ2 T cells after rest in medium.The assay was performed as described previously . BrieflyP. D., M. X., and C. R. formal analysis; P. D., M. M., C. M. B., K. M. D., D. H., and D. C. validation; P. D., M. M., M. X., D. C. H., K. D., T. S., and K. M. D. methodology; P. D., M. M., C. M. B., K. D., K. M. D., and D. H. writing-review and editing; M. M., C. R., and D. C. supervision; C. M. B., D. C. H., K. D., C. R., and T. S. resources; K. D. software; C. R. and D. C. visualization; K. M. D., D. H., and D. C. funding acquisition; K. M. D., D. H., and D. C. project administration; D. H. investigation; D. C. conceptualization; D. C. writing-original draft; M. X. γδ T-cell analyses.


Their structures were elucidated on the basis of extensive spectroscopic analysis. In addition, the structure of aristolanhydride, recently isolated from the same species, was corrected by reanalysis of the published NMR data.Four hitherto unknown aristolane-type sesquiterpenes, including one novel 8,9-secoaristolane, namely secoaristolenedioic acid (The online version of this article (10.1007/s13659-019-0200-7) contains supplementary material, which is available to authorized users. Nardostachys chinensis is a perennial herb belonging to the genus Nardostachys , and mainly distributed in the Himalayan mountain areas  objective to assemble a large-scale natural product library valuable in the discovery of new drug leads from nature, the phytochemical investigation on the roots and rhizomes of N. chinensis resulted in the isolation of four new aristolane-type sesquiterpenoids, including one novel 8,9-secoaristolane, namely secoaristolenedioic acid (1), two aristolone derivatives, namely 1α,2β-dihydroxyaristolone (2), 9-epidebilon (3), and one rare aristolane-chalcone hybrid, namely 3′-hydroxynardoaristolone A (4) , and th10α-diol , 7-epi-5,11-diol , and nar,11-diol ). Herein1 was obtained as a white amorphous powder, and its molecular formula was determined to be C15H22O4 based on HRESIMS (pos.): m/z 289.1410 [M + Na]+  with five degrees of unsaturation. The IR spectrum showed characteristic absorption bands for carbonyl functionalities (1687 cm−1). The 1H NMR spectrum ], one secondary methyl [δH 0.95 ], two mutually coupled methine protons [δH 1.65  and 1.92 ], and one olefinic proton [δH 6.96 ]. The 13C and DEPT spectra (Table δC 135.1), and five quaternary carbons . The cross-peaks of H-1/H-2, H-2/H-3, H-3/H-4, and H-6/H-7 in the 1H-1H COSY spectrum disclosed the following connectivities: C-1–C-2–C-3–C-4 and C-6–C-7. A gem-dimethylcyclopropane unit was deduced from the HMBC correlations  to C-6 (δC 45.3), C-7 (δC 30.8), C-11 (δC 29.1), and C-13 (δC 32.4), from H3-13 (δH 1.32) to C-6, C-7, C-11, and C-12 (δC 16.9). Together with the HMBCs from H3-14 (δH 1.81) to C-4 (δC 37.0), C-5 (δC 40.8), and C-6, and from H3-15 (δH 0.95) to C-3 (δC 25.8), C-4, and C-5, an aristolane-type sesquiterpene skeleton was speculated for compound 1 + ion peak at m/z 289.1410, suggesting a molecular formula of C15H22O4 , while for aristolanhydride, a negative mode HRESIMS gave an [M − H]− ion peak at m/z 265.1441, but Chen and co-workers mistakenly believed that it was an [M + OH]− ion peak, and then led to a wrong dehydrated molecular formula C15H20O3. Furthermore, HPLC and TLC analysis were performed on an Agilent 1200 series HPLC system and silica gel F254 TLC plates, respectively, and a broad chromatogram peak and a long trailing spot (chloroform/methanol = 10/1) were observed, which also hinted the existence of carboxyl group not an anhydride. Above all, the structure of aristolanhydride should be revised as secoaristolenedioic acid (1)  with five degrees of unsaturation. The IR spectrum showed characteristic absorption bands for hydroxy group (3445 cm−1) and conjugated carbonyl functionalities (1642 cm−1). The 1H NMR spectrum ], one secondary methyl [δH 1.08 ], two mutually coupled methine protons [δH 1.56  and 1.74 ], and one olefinic protons [δH 5.87 ]. The 13C NMR and DEPT spectra , and four quaternary carbons (including one keto carbonyl at δC 199.7 and one olefinic at δC 168.3). The above-mentioned NMR data revealed its structural features related to those of aristolone +  with five degrees of unsaturation. The IR spectrum revealed the absorptions for hydroxy (3432 cm−1) and conjugated carbonyl groups (1646 cm−1). The NMR spectra ], one secondary methyl [δH 1.06 ], two mutually coupled up-field methine protons [δH 0.67  and 0.93 ], and one olefinic proton [δH 6.12 ] in the 1H NMR spectrum; a total of 15 carbon resonances classified as four methyls, two methylenes, five methines (including one olefinic at δC 120.8 and one oxygenated at δC 67.2), and four quaternary carbons (including one keto carbonyl at δC 199.2 and one olefinic at δC 174.7) in the 13C and DEPT spectra. The above NMR spectroscopic features were highly similar to those of debilon [9α-hydroxy-1(10)-aristolen-2-one], with the obvious difference being the chemical shift of the oxymethine + . In the NMR spectra ], one secondary methyl [δH 1.08 ], two mutually coupled methine doublets [δH 1.49  and 2.51 ], together with 15 carbon resonances  were recognized as an aristolane structural unit. Moreover, a trans-α,β-unsaturated ketone group , a chelated hydroxy [δH 13.99 ] and one aromatic proton [δH 6.05 ] due to a pentasubstituted phenyl ring, as well as one typical ABX system  were observed, which could be recognized as a chalcone structural unit. The above-mentioned NMR data were very close to those of nardoaristolone A, an unprecedented aristolane-chalcone hybrid recently isolated from the same species  and a methoxy group [δH 3.94 ] located at C-3′ and C-4′, respectively, were deduced from the HMBC correlations of the hydroxy signal (δH 5.85) to C-3′ and the methoxy signal (δH 3.94) to C-4′, together with the ROESY correlations of H-2′↔OH-3′ and H-5′↔OCH3-4′. Hence, the structure of 4 was established and named as 3′-hydroxynardoaristolone A. To our knowledge, only the two aristolane-chalcone hybrids (nardoaristolone A and 3′-hydroxynardoaristolone A) were reported until now.Compound  species , and wer18 column . Silica gel , MCI gel CHP-20P , Chromatorex C18  and Sephadex LH-20  were used for column chromatography. Fractions were monitored and analyzed using TLC, in combination with Agilent 1200 series HPLC system equipped by an Extend-C18 column .Optical rotations were measured on a Jasco P-1020 automatic digital polarimeter. UV data were obtained from HPLC online analysis. IR spectra (KBr) were obtained on a Bruker Tensor-27 infrared spectrophotometer. NMR spectra were carried out on a Bruker Avance III 600 or Bruker DRX-500 spectrometer with deuterated solvent signals used as internal standards. ESIMS and HRESIMS were measured using an Agilent G6230 time-of-flight mass spectrometer. MPLC was performed on a Büchi apparatus equipped with Büchi fraction collector C-660, Büchi pump module C-605 and manager C-615. Preparative HPLC separation was performed using an Agilent 1260 series HPLC system equipped with a Zorbax SB-CNardostachys chinensis were purchased from Luosiwan Chinese herbal medicine market in Kunming, People’s Republic of China, in August 2015 and identified by Mr. Yu Chen of Kunming Institute of Botany, Chinese Academy of Sciences. A voucher specimen (No. BBP0656026NC) was deposited at BioBioPha Co., Ltd.The roots and rhizomes of N. chinensis (12.0 kg) were extracted with 95% aqueous EtOH (3 × 18 L) at room temperature to obtain a crude extract (900 g) after evaporation of the solvent. The crude extract was fractionated by silica gel column chromatography successively eluted with a gradient of increasing acetone in petroleum ether (PE)  and then methanol to afford fractions A–H, respectively. Fraction E was separated by Sephadex LH-20 , MPLC , silica gel , and prep-HPLC  to yield compound 3 (70 mg). Fraction F was separated by silica gel , Sephadex LH-20 (MeOH), and prep-HPLC  to yield compounds 1 (47 mg) and 4 (31 mg). Fraction G was further isolated sequentially using Sephadex LH-20 (MeOH), prep-MPLC , and prep-HPLC  to afford compound 2 (14 mg). The retention times (tR) of 1–4 on an analytical HPLC Thermo Hypersil BDS C18 column  were 8.51, 7.25, 8.68, and 10.37 min, respectively.The air-dried and powdered roots and rhizomes of α]D25 −104.0 ; UV (MeOH) λmax: 215 nm; IR (KBr) νmax: 3443, 3428, 2921, 1687, 1676, 1638, 1446, 1417, 1384, 1294 cm−1; 1H and 13C NMR data: see Table m/z 289 [M + Na]+; HRESIMS (pos.): m/z 289.1410 [M + Na]+ .White amorphous powder; [α]D25 −245.3 ; UV (MeOH) λmax: 235 nm; IR (KBr) νmax: 3445, 2966, 2922, 1642, 1449, 1383, 1054 cm−1; 1H and 13C NMR data: see Table m/z 273 [M + Na]+; HRESIMS (pos.): m/z 273.1462 [M + Na]+ .White amorphous powder; [α]25 D +14.0 ; UV (MeOH) λmax: 242 nm; IR (KBr) νmax: 3432, 2967, 2942, 2882, 1710, 1646, 1455, 1383, 1193, 1053 cm−1; 1H and 13C NMR data: see Table m/z 257 [M + Na]+; HRESIMS (pos.): m/z 257.1511 [M + Na]+ .Colorless oil; [α]D25 +223.7 ; UV (MeOH) λmax: 262, 315 (sh), 379 nm; IR (KBr) νmax: 3443, 2960, 2930, 1632, 1589, 1560, 1509, 1441, 1270, 1223, 1030 cm−1; 1H and 13C NMR data: see Table m/z 569 [M + Na]+; HRESIMS (pos.): m/z 569.2147 [M + Na]+ .Orange amorphous powder; [Supplementary material 1 (DOC 1350 kb)Below is the link to the electronic supplementary material.


Size and composition of γδ T cell populations change dramatically with tissue location, during development, and in disease. Given the functional differentiation of γδ T cell subsets, such shifts might alter the impact of γδ T cells on the immune system. To test this concept, and to determine if γδ T cells can affect other immune cells prior to an immune response, we examined non-immunized mice derived from strains with different genetically induced deficiencies in γδ T cells, for secondary changes in their immune system. We previously saw extensive changes in pre-immune antibodies and B cell populations. Here, we report effects on αβ T cells. Similarly to the B cells, αβ T cells evidently experience the influence of γδ T cells at late stages of their pre-immune differentiation, as single-positive heat stable antigen-low thymocytes. Changes in these and in mature αβ T cells were most prominent with memory-phenotype cells, including both CD8+ and CD4+ populations. As previously observed with B cells, most of the effects on αβ T cells were dependent on IL-4. Unexpectedly, IL-4 seemed to be produced mainly by αβ T cells in the non-immunized mice, albeit strongly regulated by γδ T cells. Similarly to our findings with B cells, changes of αβ T cells were less pronounced in mice lacking all γδ T cells than in mice lacking only some, suggesting that the composition of the γδ T cell population determines the nature of the γδ-influence on the other pre-immune lymphocytes. TheWe initially focused our investigation on the secondary changes in B cells, and confirmed that they were the result of interactions with the altered γδ T cell populations , 23. TheIl4tm1Nnt/J) were obtained from the Jackson Laboratory and were a gift from Dr. P. Marrack at National Jewish Health. IL-4-/- mice were crossed with B6.TCR-Vγ4/6KO mice, double KO mice selected from the F2 generation and bred as a new homozygous line (B6.TCR-Vγ4/6KO/IL-4KO mice). All mice were cared for at National Jewish Health (NJH) following guidelines for normal and immune-deficient animals, and euthanized by CO2 inhalation following current guidelines of the American Veterinary Medical Association (AVMA). All efforts were made to minimize suffering. The experiments were conducted under a protocol approved by the National Jewish Health Institutional Animal Care and Use Committee (Protocol number AS2484).C57BL6J mice and γδ T cell-deficient mice of the same genetic background (B6.TCRδKO mice) were originally obtained from the Jackson Laboratory  and bred at National Jewish Health . TCR-Vγ4-/-/Vγ6-/- mice , which wAlthough the KO mice were backcrossed >10x, this approach cannot completely rule out an influence of genes closely linked to the locus of interest. Therefore, we sought to complement the genetic evidence with alternative ways of changing the same γδ T cell populations, using antibody treatment for γδ T cell-inactivation and adoptive cell-transfer for γδ T cell-reconstitution. Such experiments in our preceding studies confirmed that the altered γδ T cells mediate the changes in antibodies and B cells seen in the γδ-gene mutated mice , 23, 25.5/well) were stained in 96-well plates  for the cell surface markers shown in the figures, using the specific mAbs listed below. Lymphocytes were gated based on their forward (FSC) and side scatter (SC), and aggregates (doublets and other conjugates) were excluded by using forward scatter area (FSC-A) and forward scatter height (FSC-H). Samples were analyzed using LSR II and LSR Fortessa flow cytometers (Becton Dickinson). At least 1x105 events were acquired per gated region, and the data were analyzed using FlowJo 10.5.2 software (TreeStar).Cells obtained from single cell suspensions ; anti CD93 ; anti CD23 ; anti IgM ; anti Fas ; anti IL-4Rα ; anti CD3 ; anti CD4 ; anti CD8β.2 ; anti Fas-L ; anti TCR-β ; anti CD24 ; anti CD8α ; anti CD122 ; anti CD44 ; anti CD49d ; anti CXCR3 ; anti T-bet ; anti Eomes .in vivo, we i.v.-injected affinity-purified mAbs specific for TCR-β (clone H57.597.2), TCR-δ (clone GL3), TCR-Vγ1 (clone 2.11) and TCR-Vγ4 (clone UC3) at 200 μg/mouse as previously described in detail [in vivo capture of IL-4.In order to selectively inactivate T cells and subsets of T cells n detail . This trn detail  but effin detail . The antIn vivo production of IL-4 was measured using the Mouse IL-4 In Vivo Capture Assay Set (BD Pharmingen), following the method of Finkelman and Morris [in vivo capture period of 8.5 hrs to demonstrate the difference in IL-4 production between C57BL/6 (wt) and B6.TCR-Vγ4/6KO mice, we extended the in vivo capture period here to 24 hrs. This modification increased the IL-4 signal roughly 2x to ~ 400 pg IL-4/ml serum but abolished the difference between the mouse strains, presumably because the injected capture antibody (10 μg/mouse) becomes limiting.d Morris . Unlike d Morris , where wWe used the nomenclature for murine TCR-Vγ genes introduced by Heilig and Tonegawa .Each experiment was carried out at least 3 times independently. Data are presented as means +/- SD. The One Way Nonparametric ANOVA  test was used for group comparisons. All data generated with γδ T cell-deficient mice were compared to the data generated with wt mice. A p value <0.05 was considered statistically significant.γδ T cells encompass several subpopulations with different functional capabilities . For exaWe compared C57BL/6 mice (wt) with background-matched mice deficient in all γδ T cells (B6.TCRδKO) or in only some γδ T cells, lacking Vγ1+ cells (B6.TCR-Vγ1KO) or lacking Vγ4+ and Vγ6+ cells (B6.TCR-Vγ4/6KO). In young adult mice , the absence of all γδ T cells had little if any effect on splenic lymphocyte numbers , or splein vivo cytokine capture assay [in vitro activation, and on a per cell basis, γδ T cells produced more IL-4 than αβ T cells. But γδ T cells in blood and spleen are less frequent than αβ T cells, and we did not resolve the relative contributions of the two T cell-types to overall IL-4 serum levels. To address this question, we again used the in vivo cytokine capture assay, but this time in combination with in vivo T cell inactivation by antibody treatment [in vivo cytokine capture assay. To achieve maximum sensitivity, we used a longer in vivo capture period (24 hrs), achieving roughly 2x greater accumulation of captured IL-4 by comparison with our earlier study [We previously investigated IL-4-production in non-immunized B6.TCR-Vγ4/6KO mice . An in vre assay , employere assay . Their sreatment . Firstlyreatment . All of er study . HoweverWe previously showed that marginal zone and follicular B cells are deficient in B6.TCR-Vγ4/6KO mice, and found that removing IL-4 (B6.TCR-Vγ4/6KO/IL-4KO) restored the numbers of these B cells . ConsistIn all strains, individual mice varied in their lymphocyte numbers. In part, this might have been due to residual genetic heterogeneity in the backcrossed mice see , or age-Mice harbor substantial numbers of memory-phenotype (MP) CD8+ αβ T cells, even if they have not been exposed to foreign antigens. These cells are distinguished by their high level of expression of CD122 and CD44 . HomeostHaving found lymphopenia and higher-than wt IL-4 serum levels and production by T cells in certain γδ T cell-deficient mouse strains , 23 and gating . RepreseAIMP CD8+ αβ T cells that arise as a result of lymphopenia-induced peripheral homeostatic proliferation , “virtuaWhereas CD8+ MP αβ T cells in non-immunized mice have been examined extensively , 37, 41,In contrast to the spleen, there was no indication of lymphopenia in the thymi of γδ T cell-deficient mouse strains . NumbersIn stark contrast to the increased MP CD8+ mature thymic lymphocytes, MP CD4+ mature thymic lymphocytes were much decreased in B6.TCR-Vγ4/6KO mice when compared to wt mice , a diffeWe had previously seen large differences of mature and immature transitional B cell populations in the spleens of non-immunized γδ T cell-deficient mice (same strains as in the current study) but not of immature B cells represented by Hardy fractions  in bone Expression of the death receptor Fas can serve as a marker of activated/memory B cells . We prevThe possibility that γδ T cells affect the immune system prior to its responses has hardly been explored. To address this question, we compared immune cells in a set of genetic background-matched γδ T cell-deficient mouse strains. Between these mouse strains, without immunization, we found differences in overall leucocyte numbers, including granulocytes and B cells , 23. We Several shared features connect the current study of αβ T cells in non-immunized mice to the previous work on B cells , 23. FirThe parallels between changes of B cells and αβ T cells in the γδ T cell-deficient mice suggest that similar mechanisms are at work. This is informative because unlike B cells, αβ T cells can express TCR-γ and, sometimes, –δ genes, and thus potentially could be intrinsically affected by the mutations of the γδ T cell-deficient mouse strains examined here. However, with B cells, non-genetic manipulations of γδ T cells supported the notion of cellular interactions between γδ T cells and B cells as cause for the changes in B cells and antibodies , 23, 25.Consistently, the data imply that the γδ-influence on B cells and αβ T cells is mainly mediated by IL-4. IL-4 directly supports T and B cell-development, during the immune responses and before , 55. EarLastly, our data indicate that γδ T cells affect CD4+ and CD8+ MP αβ T cells differently. The expansion of CD8+ MP αβ T cells in B6.TCR-Vγ4/6KO mice suggests that γδ T cells deficient in these mice normally repress CD8+ MP αβ T cells, in thymus and spleen. The repression could be direct or via a second, otherwise supportive cell-type, e.g. Vγ1+ γδ T cells. The expanded CD8+ αβ T cells in B6.TCR-Vγ4/6KO mice closely resemble antigen-inexperienced memory-phenotype (AIMP) CD8+ αβ T cells previously described by others , 37. In S1 FigAdult mice were treated with i.v. injected anti TCR mAbs or saline alone, and analyzed by flow cytometry as detailed in the Methods.(PDF)Click here for additional data file.S2 FigAdult mice were treated with i.v. injected anti TCR mAbs or saline alone, and analyzed by flow cytometry as detailed in the Methods.(PDF)Click here for additional data file.S3 FigAdult mice were treated with i.v. injected anti TCR mAbs or saline alone, and analyzed by flow cytometry as detailed in the Methods.(PDF)Click here for additional data file.S4 FigFemale and male mice ages 8–12 wks were included in the comparisons shown (same mice as in (PDF)Click here for additional data file.S5 FigA) Comparison of live body weight (grams) of female C57BL/6 (B6), B6.TCRδKO, B6.TCR-Vγ1KO, B6.TCR-Vγ4/6KO, and B6.TCR-Vγ4/6KO/IL-4KO mice, at 4, 8 and 12 wks of age.B) Same comparison as in A but with male mice.n equal or greater 5 mice/group, except for B6.TCR-Vγ1KO mice  and C57BL/6 mice .(PDF)Click here for additional data file.S6 FigComparison of age-matched female and male mice for splenic lymphocyte numbers, including C57BL/6 (B6), B6.TCRδKO, B6.TCR-Vγ1KO, B6.TCR-Vγ4/6KO, and B6.TCR-Vγ4/6KO/IL-4KO mice. Female and male mice ages 8–12 wks were included in the comparison shown in (PDF)Click here for additional data file.S7 FigComparison of total numbers of TCR-β+ cells in the spleen of C57BL/6 (B6), B6.TCRδKO, B6.TCR-Vγ1KO, B6.TCR-Vγ4/6KO, and B6.TCR-Vγ4/6KO/IL-4KO mice. Female and male mice ages 8–12 wks were included in the comparison shown in (PDF)Click here for additional data file.S8 Fig(PDF)Click here for additional data file.S9 FigSplenocytes were stained and analyzed by flow cytometry as described in the Methods.(PDF)Click here for additional data file.S10 FigUnlike B6.TCR-Vγ4/6KO mice, no substantial changes in the frequencies of memory-phenotype αβ T cells were found in the spleens of B6.TCRδKO and B6.TCR-Vγ1KO mice.(PDF)Click here for additional data file.S11 Fig(PDF)Click here for additional data file.S12 FigMale and female mice, ages 8–12 wks were included (same mice as in (PDF)Click here for additional data file.


Breast cancer is the second common malignant cancer among females worldwide. Accumulating studies have indicated that deregulation of miRNA expression in breast cancer will contribute to tumorigenesis and form different cancer subtypes. However, the reported studies on miR-29b-3p-regulated breast cancer are limited so far. Herein, we investigated the role and mechanism of miR-29b-3p in the triple negative breast cancer cell line MDA-MB-231.The relative miR-29b-3p expression in different breast cancer cell lines were determined by qRT-PCR. CCK8 and colony formation assay were used to determine the influence of miR-29b-3p on cell proliferation. Migration assay and invasion assay were performed for cell migration and invasion respectively. To study the cell integrity immunofluorescence was performed. TUNEL assay, flow cytometry assay, hoechst staining and western blot were conducted to determine the influence of miR-29b-3p inhibitor on cell apoptosis. TRAF3 was found to be the target gene of miR-29b-3p using bioinformatics predictions. Dual-luciferase assay was performed to determine the relative luciferase activity in NC, miR-29b-3p mimic, miR-29b-3p inhibitor with TRAF3 3′-UTR wt or TRAF3 3′-UTR mt reporter plasmids. The proteins expression of NF-κB signaling pathway in MDA-MB-231 after transfection with NC, miR-29b-3p mimic, miR-29b-3p inhibitor were determined by western blot.The miR-29b-3p expression was significantly increased in MDA-MB-231 compare with MCF-10A. miR-29b-3p inhibitor reduced the cell viability of MDA-MB-231 and inhibited cell migration and invasion. Cell cytoskeleton integrity destroyed after miR-29b-3p inhibitor treatment. Furthermore, we identified the mechanism and found miR-29b-3p targets the TRAF3 and activates NF-κB signaling pathway.From the above studies, our results indicated that miR-29b-3p acts as a promoter for the development of MDA-MB-231. Breast cancer is considered to be one of the most common malignant cancer among females worldwide. According to the American Cancer Society, breast cancer accounts for 30% of all new cancer cases and is a major cause of cancer-related mortality in females –3. In ChmicroRNA is a kind of small, 19–22 nucleotides in length, non-coding RNA which play a crucial role in the regulation of gene expression by binding to the 3′-untranslated region (UTR) of target mRNA –12. RecemiR-29b, one of miR-29 family members, has been reported to serve as a promoter among various kinds of disease. Zhang et al.  investigIn this study, we investigated the role of miR-29b-3p expression in triple negative breast cancer cell. Furthermore, potential targets of miR-29b-3p was identified by bioinformatics analysis and dual-luciferase reporter assay. Our study revealed that inhibition of miR-29b-3p drastically reduces cell proliferation, migration, invasion in TNBC. Moreover, inhibiting miR-29b-3p also induces apoptosis in TNBC through PARP cleavage. However, mechanism involved in the inhibition of miR-29b-3p and pathogenesis and metastasis yet to be studied.2 atmosphere. miR-29b-3p mimics and miR-29b-3p inhibitor, negative control, negative control inhibitor were purchased from GenePharma . To conduct cell transfection, cells were firstly cultured to about 50–60% confluence. Then, Lipofectamine 2000  was used and transfected according to the manufacturer’s manual.Human breast cancer cell lines MDA-MB-231, MCF-7, MDA-MB-453 and human epithelial cell line MCF-10A were purchased from Procell . MDA-MB-231, MCF-7, MDA-MB-453 were cultured in complete Dulbecco’s modified Eagle’s medium  which supplemented with 10% fetal bovine serum , 1% penicillin streptomycin . MCF-10A was cultured in human epithelial cell complete medium  which containing DMEM/F12, horse serum, growth additive and P&S. All cell lines were cultured at 37 °C in 5% CO® Kit  and Applied Biosystems 7500  real time device under the standard thermal condition: 95 °C for 30 s, 40 cycles of 95 °C for 10 s and 60 °C for 30 s, followed by 95 °C for 10 s, 65 °C for 60 s and 97 °C for 1 s. All results were normalized to U6. Using the − 2△△Ct method to identify the miRNA relative expression levels. Primer sequences: U6, forward primer: 5′-CTCGCTTCGGCAGCACA-3′; Reverse primer: 5′-AACGCTTCACGAATTTGCGT-3′; miR-29b-3p, forward primer: 5′-TAGCACCATTTGAAATCAGTGTT-3′, Reverse primer: mRQ 3′ Primer . Three independent experiments were performed for each reaction in triplicate.Total RNAs were isolated from cells after 24 h transfection according to the manufacturer’s protocol , cDNA synthesis and miRNA expression were quantified using Mir-X™ miRNA qRT-PCR SYBRDoxorubicin hydrochloride was purchased from Macklin . For Doxorubicin treatment, we prepared the Doxorubicin stocking solution at 1 mM concentration in ultrapure water. Then, different concentration of Doxorubicin  were dissolved in medium and treated for 24 h, 48 h and 72 h and CCK8 assay was performed, and IC50 of DOX was used for treatment to identify the miRNA relative expression.Cell proliferation of MDA-MB-231 was detected using Cell Counting Kit-8 (CCK8) . Briefly, 8000 cells per well were seeded in 96-well plate with 100 μL complete medium and cultured for 24 h. After Doxorubicin or transfection treatment, 100 μL medium containing 10 μL CCK8 was added to the wells and incubated for 4 h later absorbance was measured using EnSpire Multimode Plate Reader  at 450 nm. Each group in triplicate.2 incubator. After colony formation, 4% paraformaldehyde (PFA)  was used to fix the cells for 20 min and stained with 1% crystal violate  for 15 min at room temperature. The number of colony- forming units were calculated in three random wells.Single cell suspension was obtained by trypsinization after transfection for 24 h. 500 cells were seeded in 6-well plate and cultured it for 2 weeks with complete medium in 5% CO5 MDA-MB-231 cells were seeded in 24-well and culture overnight. After 80–90% confluence transient transfection was performed for 24 h, later 1 μL sterile tip was used to make a straight wound. Scratch photo was taken at 0 h, 6 h and 12 h by microscope . Distance migration was measured by calculating the area of the wound using Image J software and represented in the form of bar graph.To study the migration ability of MDA-MB-231 migration assay were conducted. Briefly, 5 × 104 cells were harvested and seeded in the 8 μm pore size upper Transwell chamber  coated with Matrigel , which cultured in basic medium. The optimal coating concentration ratio was DMEM serum-free medium: Matrigel = 8:1 (V/V), and 2 h incubation at 37 °C. 900 μL complete medium containing 5% FBS was added into the lower chamber as a chemoattractant. After 24 h incubation cells were fixed with 4% paraformaldehyde and stained with 1% crystal violate. Migratory cells in lower chamber were counted from each well and photographed at 20× . The experiment was repeated three times.To detect the invasive ability of MDA-MB-231 invasion assay was performed. After 24 h of transient transfection, 2.5 × 105 MDA-MB-231 cells were seeded on cover slips in 24 well plate. After 36 h of NC, miR-29b-3p mimics and miR-29b-3p inhibitor treated cells were fixed with 4% paraformaldehyde for 15 min at room temperature and then washed with PBS twice. Later, the cells were washed with a quenching solution (0.1% glycine in PBS) twice, permeated with 0.1% Triton X-100 for 10 min and blocked using a blocking solution (10% FBS in PBS) for 1 h at room temperature. Later the cells were incubated for overnight with primary antibody against α-Tubulin (anti α-Tubulin (11H10), 1:100, 2125, Cell Signaling Technology, USA) followed by secondary antibody Alexa Fluor 594 Conjugated goat anti-rabbit IgG . Nuclei were stained with 4,6-diamidino-2 phenylindole DAPI and the cover slip was mounted using anti-fluorescent quencher . Immunofluorescence image were captured under LSM 710 Laser scanning confocal microscope . Digital images were optimized for image resolution , brightness, and contrast using Adobe Photoshop 7.0 . No alteration was made in image such as adding or removal of image details.To study the cytoskeleton reorganization in breast cancer cell line MDA-MB 231 immunofluorescence technique was performed. 1 × 105 MBA-MB-231 and MCF-7 cells were seeded on the cover slip in 24 well plate and transfected with NC, miR-29b-3p mimics and miR-29b-3p inhibitor for 36 h. The cells were fixed for 20 min with 4% PFA then washed with PBS, incubated with 0.3% Triton-X 100 for 5 min. Later cells were washed and 100 μL Hoechst 33258  was added to cells and incubated for 30 min in dark. The cover slip was mounted using anti-fluorescent quencher . Photographed under the LSM 710 Laser scanning confocal microscope at 40× .To study the nuclear morphology of the MDA-MB-231 and MCF-7, Hoechst staining was performed. 1 × 105 MDA-MB-231 cells were seeded in 24-well plate and transfected with NC, miR-29b-3p mimics and miR-29b-3p inhibitor for 36 h. The cells were fixed with 4% paraformaldehyde for 30 min then wash with PBS. Later 0.3% Triton-X 100 in PBS was added and incubated for 5 min. One-step TUNEL apoptosis detection kit  was used to estimate the apoptosis efficiency according the manufacture’s instruction. In brief, cells were incubated with 50 μL TUNEL reaction buffer in a 37 °C humidified chamber for 1 h, nucleus was counterstained with DAPI for 1 min at room temperature in dark later mounted with anti-fade mounting medium. Stained apoptotic cells were visualized at 20× by LSM 710 Laser scanning confocal microscope .1 × 106 MDA-MB-231 cells were seeded in 6 well plate after 24 h the cells were transfected with NC, miR-29b-3p mimics and miR-29b-3p inhibitor. The cells were trypsinized and single cells suspension was added with 5 μL Annexin V-FITC followed by 5 μL PI solution in dark and incubated for 15 min. Soon 400 μL Annexin V Binding Solution was added and analyzed by BD Accuri C6 Plus (BD) flow cytometer. Necrotic, early and late apoptosis cells were identified by BD Accuri C6 Plus software.Annexin V/propidium iodide staining for apoptosis was conducted using Annexin V-FITC Detection kit  according to the manufacturer’s instructions. Briefly, 1 × 10MDA-MB-231 cells were seeded into 24-well plates and after 24 h incubation the confluence reaches to 60–70%. Psi-CHECK2/TRAF3-3′-UTR wt and Psi-CHECK2/TRAF3-3′-UTR mutant reporter plasmids were constructed in advanced. According to the manufacturer’s instruction, MDA-MB-231 cells were transiently co-transfected with miR-29b-3p mimics or miR-29b-3p inhibitor together with 0.1 μg psi-CHECK2/TRAF3-3′-UTR wt or psi-CHECK2/TRAF3-3′-UTR mutant reporter plasmids using Lipofectamine 2000. After 48 h, Dual-luciferase Reporter Assay System  was used to detect firefly and Renilla luciferase activities and recorded using GloMax 96 Microplate Luminometer .6 cells/each well in 6-well plates) was transfected with NC, miR-29b-3p mimics and miR-29b-3p inhibitor for 48 h. Cell lysates were collected using 1 mL RIPA  with 10 μL PMSF. Protein concentration of cell lysates were measured with a BCA Protein Assay Kit . Western blot was carried out by separating 40 µg of protein by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electro-transferred onto a polyvinylidene difluoride (PVDF) membrane . The membranes were blocked with 5% non-fat milk and washed with TBST buffer and then incubated with primary antibody Cleaved PARP (Asp214) (D64E10), NF-κB p65 (D14E12), phospho-NF-κB p65 (ser536), IκB-α (44D4), phospho-IκB-α (14D4),  at a dilution of 1:1000 in Primary Antibody Dilution Buffer  at 4 °C overnight. Next, membrane treated with primary antibody were washed 3 times with TBST and incubated with an anti-rabbit HRP-linked antibody at a 1:5000 dilutions for 1 h at room temperature, β-Actin was used as an internal reference. The signals were visualized by Immobilon Western Chemiluminescent HRP Substrate  with the Amersham Imager 600 imagers .Breast cancer cell line MDA-MB-231 . Therefore, based on the TargetScan predictions, it showed that TRAF3 is targeted by miR-29b-3p. Dual-luciferase reporter assay showed that significant decrease in relative luciferase activity was noted when psi-CHECK2/TRAF3-3′-UTR was co-transfected with the miR-29b-3p mimics compare with NC group, it indicated that miR-29b-3p mimics bind to the 3′UTR of the TRAF3, thereby inhibiting the luciferase activity. However, the relative luciferase activity was increased when psi-CHECK2/TRAF3 3′-UTR was co-transfected with the miR-29b-3p inhibitor compare with NC group. It indicated that miR-29b-3p inhibitor inhibits the endogenous miR-29b-3p binding to the TRAF3 3′-UTR, resulting in stronger relative luciferase activity than the NC group. But in miR-29b-3p mimics group, the relative luciferase activity was decreased compare with NC group. Moreover, no significant difference among groups when psi-CHECK2/TRAF3 3′-UTR mutant co-transfected with NC, miR-29b-3p mimics and miR-29b-3p inhibitor. The results showed that miR-29b-3p binds to TRAF3 and miR-29b-3p inhibitor inhibited miR-29b-3p. Therefore, TRAF3 is one target gene of miR-29b-3p. TRAF3, one of the six members of this family of proteins in humans and mice, has been reported to interact with other members of the TNFR family [Next, we predicated and validated the possible target mRNAs of miR-29b-3p. Previous studies have shown that miR-29b-3p targets many genes and exerts different biological functions, such as MCMBP, PI15, COL3A1 (TargetScan v7.2; R family , 47 and R family . DiffereR family  and was R family , 51. ActR family , 53. DurR family . In accoTaken together, we identified miR-29b-3p as an important regulator in TNBC which enhances cell proliferation, migration and invasion. Moreover, inhibition of miR-29b-3p induces cell apoptosis by suppressing NF-κB signaling pathway in MDA-MB-231. Altogether our results showed that miR-29b-3p has a key role in MDA-MB-231.Our findings indicated that miR-29b-3p was highly expressed in MDA-MB-231 cells. Inhibition of miR-29b-3p could suppress the cancer cells proliferation and induced the apoptosis. In addition, it influenced the formation of microtubule and suppressed the process of cell migration and invasion. Moreover, miR-29b-3p targets to the 3′-UTR of TRAF3 and activates the NF-κB signaling pathway. Therefore, miR-29b-3p could be an underlying biomarker and it can be developed as novel therapeutic target against the TNBC.


E configuration about the C=C bond and the carbonyl group is syn with respect to the C=C bond. In the crystal, the molecules are connected into a tape structure by C—H⋯O and C—H⋯π interactions.The molecule has an  17H14ClFO3, consists of a 4-chloro-3-fluorophenyl ring and a 3,4-dimethoxyphenyl ring linked via a prop-2-en-1-one spacer. The molecule has an E configuration about the C=C bond and the carbonyl group is syn with respect to the C=C bond. The F and H atoms at the meta positions of the 4-chloro-3-fluorophenyl ring are disordered over two orientations, with an occupancy ratio of 0.785 (3):0.215 (3). In the crystal, molecules are linked via pairs of C—H⋯O interactions with an R22(14) ring motif, forming inversion dimers. The dimers are linked into a tape structure running along [10The molecular structure of the title compound, C Chalcones attract significant attention because of their availability of high optical non-linearities arising from the delocalization of π-conjugated electron clouds throughout the chalcone system, which provides a large charge-transfer axis with appropriate substituents on the terminal aromatic rings. π-conjugated systems have been studied extensively for their optoelectronic properties  Å for C16 and 0.209 (2) Å for C17. The 4-chloro-3-fluorophenyl fragment is disordered over two orientations around the C9—C10 bond axis, with an occupancy ratio of 0.785 (3):0.215 (3).The title compound Fig. 1 is compovia pairs of C—H⋯O interactions , O⋯C/C⋯O (1.7%), Cl⋯C/C⋯Cl (1.1%), F⋯F (0.9%), Cl⋯F/F⋯Cl (0.7%) and F⋯O/O⋯F (0.2%).The Hirshfeld surface and two-dimensional fingerprint plots of the title compound were calculated using rm Fig. 4, the whiet al., 2016E)-1,3-diphenylprop-2-en-1-one as the main skeleton revealed 3314 hits. Six structures containing the (E)-1,3-diphenylprop-2-en-1-one framework with different substituents that are similar to the title compound were found, viz. 3-(3-chlorophenyl)-1-prop-2-en-1-one -3-(2-bromophenyl)-1-prop-2-en-1-one -1--3-prop-2-en-1-one -1-(3-bromophenyl)-3-prop-2-en-1-one ° for VIDVEM, 17.91 (17)° for BIBWOB, 9.3 (2) and 19.4 (2)°  for LAPREB, 5.46 (2)° for KUZFOB, 26.59 (9)° for LAQWUX and 47.81 (6) and 50.18 (5)°  for MEGQOF. In the crystals of VIDVEM and BIBWOB, molecules are linked by C—H⋯O hydrogen bonds, forming dimers with et al., 2013abThe title compound was synthesized as per the procedure reported earlier (Kumar Uiso(H) = 1.2 or 1.5Ueq(C). The 4-chloro-3-fluorophenyl fragment was found to be disordered in a difference-Fourier map, and the F and H atoms at the meta positions of the benzene ring were treated as disordered over two sites with an occupancy ratio of 0.785 (3):0.215 (3).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989019007783/is5515sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989019007783/is5515Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989019007783/is5515Isup3.cmlSupporting information file. DOI: 1919577CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


E,4E)-6-hexa-2,4-dien-1-ylidene}pyrimidine-2,4,6-trione or TMI, C25H29N3O3, and 1,3-diethyl-2-sulfanylidene-5-[2-ethylidene]dihydropyrimidine-4,6-dione or DTB, C21H25N3O2S, are described. These compounds contain the same indole derivative donor group and differ in their acceptor groups  and the length of the π-bridge.The synthesis and crystal structures of 1,3-diethyl-5-{-6-hexa-2,4-dien-1-ylidene}pyrimidine-2,4,6-trione or TMI, C25H29N3O3, and 1,3-diethyl-2-sulfanylidene-5-[2-ethylidene]dihydropyrimidine-4,6-dione or DTB, C21H25N3O2S, have been crystallized and studied. These compounds contain the same indole derivative donor group and differ in their acceptor groups  and the length of the π-bridge. In both materials, molecules are packed in a herringbone manner with differences in the twist and fold angles. In both structures, the molecules are connected by weak C—H⋯O and/or C—H⋯S bonds.Two compounds, 1,3-diethyl-5-{(2 The double and single bonds in the TMI π-bridge vary in length from 1.372 (2) to 1.410 (2) Å, the difference between the single and double bonds getting smaller closer to the acceptor, indicating a higher degree of conjugation in this region. The dihedral angles between donor group and the bridge and between the bridge and the acceptor group are 9.10 (12) and 7.44 (12)°, respectively. All three fragments of the TMI structure are slightly distorted from a planar configuration, as shown by the r.m.s. deviations of 0.022 and 0.039 Å, respectively, for atoms in the donor and acceptor groups.Both title compounds have the same donor 2,3-dihydro-1,3,3-trimethyl-1MI Fig. 1a and 1,TB Fig. 1b. The det al., 2019Comparing DTB to TMI, it is observed that DTB possesses a more planar and rigid structure, consistent with previously reported results for studies of push–pull chromophores with different π-bridge lengths , C12—C13 1.386 (1), C13—C14 1.404 (1) Å], the central fragment can be presented as C8—C12=C13—C14, which indicates that the contribution of the zwitterionic form in the molecular structure of DTB. It should be mentioned that measurements of the first molecular hyperpolarizability, β, have positive values for dyes with hexamethine bridges, such as TMI, while dyes with a dimethine bridge have negative β values (Ortiz a) and a twist angle of 54.90 (7)° and fold angle of 78.96 (3)° in DTB . In both compounds, molecules are hold together via three hydrogen bonds, of the C—H⋯O type in TMI and of the C—H⋯O and C—H⋯S types in DTB ° and fold angle of 57.08 (1)° in TMI Fig. 2a and a TB Fig. 2b. In bo Tables 1.P21/c. According to the bond order alternation pattern in these structures (see the supporting information), we suggest that they have the potential to be used as non-linear optical materials, but for this application they should be either be embedded in a polymer matrix or recrystallized under different conditions to attain an acentric packing mode.For push–pull molecules be applied in the form of non-linear crystalline materials, they should exhibit a non-centrosymmetric type of packing. TMI and DTB both crystallize in the centrosymmetric space group et al., 2016et al., 2007et al., 2015et al., 1994et al., 2013et al., 1994et al., 1976et al., 1976et al., 1999et al., 2017et al., 1994et al., 2015et al., 2005ortho position of the acceptor ring, and a cyano group in the meta position instead of an ethyl group ethylidene]dihydropyrimidine-4,6-dione (DTB):in vacuo to give 1,3-diethyl-2-sulfanylidene-5-[2-ethylidene]dihydropyrimidine-4,6-dione as transparent red crystals . 1H NMR 8.69 , 7.70 , 7.40 , 7.26 , 7.12 , 4.55 , 4.54 , 3.59 , 1.73 , 1.27 , 1.26  ppm.2-acetaldehyde  and diethylthiobarbituric acid  were dissolved in about 35 mL of absolute ethanol with stirring and sonication. After stirring for 1 h at room temperature, the product was precipitated by adding distilled water. The mixture was then filtered and the residue redissolved in EtOH and precipitated again. The precipitant was washed with hexane and dried Single crystals of both DTB and TMI were grown by vapour diffusion using chloroform as the solvent and cyclohexane as the antisolvent. Crystallization took place over a three week period to give DTB crystals of suitable size and quality.Uiso(H) = 1.2Ueq(C) or 1.5Ueq(C-methyl).Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989019011071/yk2125sup1.cifCrystal structure: contains datablock(s) TMI, DTB. DOI: 10.1107/S2056989019011071/yk2125TMIsup2.hklStructure factors: contains datablock(s) TMI. DOI: 10.1107/S2056989019011071/yk2125DTBsup3.hklStructure factors: contains datablock(s) DTB. DOI: Click here for additional data file.10.1107/S2056989019011071/yk2125TMIsup4.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989019011071/yk2125DTBsup5.cmlSupporting information file. DOI: 1946233, 1946232CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


O-β-d-arabinopyranosyl-(2′′→1′′′)-O-β-d-arabinopyrnosyl-2′′′-O-3′′′′, 7′′′′-dimethylnonan-1′′′′-oate (1), 5-hydroxy-7, 4′-dimethoxyflavone-5-O-α-d-arabinopyranosyl-(2"→1′′′)-O-α-d-arabinopyranosyl-2′′′-O-3′′′′, 7′′′′-dimethylnonan-1′′′′-oate (2), 5-hydroxy-7, 4′-dimethoxyflavone-5-O-β-d-arabinofuranosyl-(2"→1′′′)-O-β-d-arabinopyranosyl-2′′′-O-lanost-5-ene (3) and 4′,4′′-diferuloxy feruloyl-O-α-d-arabinopyranosyl-(2a→1b)-O-α-d-arabinopyranosyl-(2b→1c)-O-α-d-arabinopyranosyl-(2c→1d)-O-α-d-arabinopyranosyl-(2d→1e)-O-α-d-arabinopyranosyl-2e-3′′′, 7′′′-dimethylnonan-1′′′-oate (4), along with three known compounds (5–7) were isolated from Oryza sativa leaves and straw. The structures of new and known compounds were elucidated by 1D (1H and 13C NMR) and 2D NMR spectral methods, viz: COSY, HMBC, and HSQC aided by mass techniques and IR spectroscopy. The cytotoxicity of these constituents was assessed by using (RAW 264.7) mouse macrophage cell line, and allelopathic effects of compounds (1–7) on the germination and seedling growth characteristics such as seedling length and root length of barnyardgrass (Echinochloa oryzicola) were evaluated. Significant inhibitory activity was exhibited by compounds comprising flavone derivatives such as (1–3) on all of seed germination characteristics. The allelopathic effect of flavone derivatives were more pronounced on seedling length and root length than the germination characteristics. The higher concentration of flavone derivatives showed stronger inhibitory effects, whereas the lower concentrations showed stimulatory effects in some cases.Four new constituents, as 5, 7-dihydroxy-4′-methoxyflavonol-3- Oryza sativa L.) is the major staple food in Asia and generally exists as two types, white hulled and colored hulled. The most common type (85%) is white-hulled rice. The germination of rice is of great agricultural importance and has long been known to be influenced by compounds present in the seed coat (hull) D24 –39.1 ; IR (KBr) νmax 3450, 3358, 3271, 2939, 2836, 1721, 1685, 1602, 1514, 1428, 1367, 1226, 1164, 1121, 1025, 833, 752 cm−1; for 1H and 13C NMR spectroscopic data, see m/z (rel. int.): 719 [M + H]+ (C36H47O15) (1.3), 299 (10.3), 287 (37.1), 155 (97.2); HRFTMS m/z 719.2915 .-d-Arabinopyranosyl-(2"→1′′′)-O-α-d-Arabinopyranosyl-2′′′-3′′′′, 7′′′′-Dimethylnonan-1′′′′-Oate5-Hydroxy-7, 4′-Dimethoxyflavone-5-O-α (2): Yellow powder; mp. 220–225 °C; Rf 0.49 (chloroform: methanol:9:1); [α]D24 −69.1 ; IR (KBr) νmax 3451, 3369, 3283, 2938, 2832, 1720, 1607, 1513, 1456, 1424, 1346, 1260, 1174, 1118, 1024, 836 cm−1; for 1H and 13C NMR spectroscopic data, see m/z (rel. int.): 717 [M + H]+ (C37H49O15) (2.4), 429 (5.1), 419 (5.3), 297 (6.8), 287 (4.7), 155 (100); HRFTMS m/z 717.3123 .-d-Arabinofuranosyl-(2"→1′′′)-O-β-d-Arabinopyranosyl-2′′′-O-Lanost-5-Ene5-Hydroxy-7, 4′-Dimethoxyflavone-5-O-β (3): Semi-solid; Rf 0.69 (chloroform:methanol:9:1); [α]D24 –59.1 ; IR (KBr) νmax 3450, 3355, 3281, 2937, 2841, 1690, 1635, 1525, 1428, 1367, 1228, 1164, 1120, 1026, 835 cm−1; for 1H and 13C NMR spectroscopic data, see m/z (rel. int.): 972 [M + H]+ (C57H81O13) (2.2), 675 (4.1), 430 (8.3), 427 (20.5), 297 (15.7); HRFTMS m/z 973.5677 .-d-Arabinopyranosyl-(2a→1b)-O-α-d-Arabinopyranosyl-(2b→1c)-O-α-d-Arabinopyranosyl-(2c→1d)-O-α-d-Arabinopyranosyl-(2d→1e)-O-α-d-Arabinopyranosyl-2e-3′′′, 7′′′-Dimethylnonan-1′′′-Oate4′, 4′′-Diferuloxy Feruloyl-O-α (4): Gummy; Rf 0.79 (chloroform:methanol:9:1); [α]D24 −79.1 ; IR (KBr) νmax 3515, 3420, 3356, 3237, 2916, 2848, 1722, 1653, 1602, 1513, 1480, 1425, 1336, 1264, 1223, 1116, 1070, 1022, 718 cm−1; ESIMS m/z (rel. int.): 1361 [M + H]+ (C65H85O31) (1.3), 1205 (1.6), 1189 (1.8), 544 (13.8), 528 (4.7), 368 (5.2), 352 (5.5), 303 (9.1), 289 (9.7), 192 (6.4), 176 (4.9), 155 (19.3); HRFTMS m/z 1361.5076 .1−4 (10 mg each) were heated at 70−80 °C with diluted 2 M hydrochloric acid (2.5 mL) in 70% aqueous ethanol (3 mL) for 30 min. The reaction mixture was dried under vacuum, neutralized with aqueous sodium bicarbonate, and extracted with chloroform (3 × 5 mL) to separate the flavonoid moiety. The chloroform extract was washed with water (3 × 5 mL), dried over anhydrous sodium sulfate, and evaporated to produce aglycone moieties. The mother liquor, after separation of the flavonoid, was treated with dilute 2 M hydrochloric acid to liberate the other aglycone moiety and was re-extracted with chloroform (3 × 5 mL) to obtain the compound. The presence of sugars in the aqueous solution was determined by co-TLC.Compounds O. sativa was determined by the method of Gyamfi et al. [50 value (µM) is the concentration at which scavenging activity is 50%.The DPPH radical scavenging activity of the seven of chemical constituents isolated from rice plant (straw and leaves) of i et al. . Initial2 throughout the study and routinely grown in 25 cm2 culture flasks and trypsinized to harvest after attaining confluence. Meanwhile, some differences were considered regarding trypsinization procedure for RAW 264.7 semi adherent cells versus two other adherent cells. Following centrifugation (1300 g for 7 min), cells were resuspended in the culture medium and used for the following study.Raw 264.7 cells were purchased from American Type Culture Collection  and grown in Dulbecco′s modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. All the cells were maintained at 37 °C in a humidified atmosphere of 5% CO5 cells per well and then incubated at 37 °C in 5% CO2 to allow cell attachment. The medium was removed and replaced with fresh medium containing various concentrations  of seven of chemical constituents. After treatment for 24 h, 100 μL MTT (1 mg/mL) was added to each well and the plate was further incubated. Four hours later, all remaining supernatant was removed and 100 μL of DMSO was added to each well to dissolve the resulting formazan crystals. Finally, absorbance was read at 570 nm using a multilabel plate reader  and the cell viability percentage was calculated using the equation: (mean OD of treated cells/mean OD of control cells) × 100.To evaluate the cytotoxicity of the seven isolated chemicals on RAW 264.7 cell, viability tests were applied using the MTT colorimetric assay. Briefly, all cell lines were seeded in 96-well plates at a density of 2 × 10E. oryzicola) found in rice paddies of Korea were collected during 1997 and 1998. The seeds were dried (water content 12.04% at fresh weight basis) and stored at 5 °C in the dark. Before the start of experiment, the seeds were soaked with distilled water for one day to remove the germination inhibition compound in the seed coat , and their germination was determined, which was >80% in all cases. The germination test was carried out in sterile petri dishes (15 cm) lined with Whatman No. 2 filter paper . Seeds of maize were sterilized with NaOCl 10% for 2–3 min. Thereafter, the seeds were thoroughly rinsed three times with sterile water. Aqueous extracts of different concentrations (10 mL) were pipetted to the filter paper placed in petri dishes and distilled water was used as control treatment. The petri dishes were set in the laboratory at room temperature ranging from 28–30 °C. The experiment extended over a period of 7 d to allow the last seed’s germination. A seed was considered as germinated when a radical emerged. Germination was recorded on daily basis. The results were determined by counting the number of germinated seeds and measuring the lengths of both root and shoot. At the end of the last day of germination, germination characteristics and seedling growth including fresh and dry weight were recorded. Final germination percentage (FGP) was calculated by using FGP = Ng/Nt × 100, where Ng is total number of germinated seeds and Nt is total number of evaluated seeds. The germination index (GI) of the seeds were estimated as follow GI = (13 × N1) + (12 × N2) + ... + (1 × N13). N1, N2 and … are the number of germinated seeds at first day, second, and other days and numbers 9, 10 and ... are respectively the weights imposed on the number of seeds germinated at first, second, and other days. Also, the coefficient of velocity of germination (CVG) was calculated by using CVG = 100 × ΣNi/ΣNiTi formula, where Ni is number of germinated seeds per day and Ti is number of days from the start of the experiment. Germination speed (Rs) was estimated based on the Magour method and by using the following equation, Rs = ΣSi/Di 20, where Si is the number of germinated seeds in it h day and Di is the day number. The calculation of mean of germination time (MGT) was done using the following equations: MGT = ΣNiTi/ΣNi = 100/CVG [Seeds of barnyardgrass . Critical differences (CDs) were calculated at O. sativa, along with their biological activities, including cytotoxicity and allelopathic activities, on weed germination. To the best of our knowledge, there has been no prior report on the phytochemistry of O. sativa straw and leaves.We have reported the isolation of four new and three known compounds from


Prop⋯OImdzln (Prop = prop-2-yn-1-yl and Imdzln = imidazolidine) hydrogen bonds link the molecules into infinite chains along the b-axis direction. Two weak C—HPhen⋯π interactions are also observed.The title molecule consists of an imidazolidine unit linked to two phenyl rings and two prop-2-yn-1-yl moieties. The imidazolidine ring is oriented at dihedral angles of 79.10 (5) and 82.61 (5)° with respect to the phenyl rings, while the dihedral angle between the two phenyl rings is 62.06 (5)°. In the crystal, C—H 21H16N2O2, consists of an imidazolidine unit linked to two phenyl rings and two prop-2-yn-1-yl moieties. The imidazolidine ring is oriented at dihedral angles of 79.10 (5) and 82.61 (5)° with respect to the phenyl rings, while the dihedral angle between the two phenyl rings is 62.06 (5)°. In the crystal, intermolecular C—HProp⋯OImdzln (Prop = prop-2-yn-1-yl and Imdzln = imidazolidine) hydrogen bonds link the molecules into infinite chains along the b-axis direction. Two weak C—HPhen⋯π interactions are also observed. The Hirshfeld surface analysis of the crystal structure indicates that the most important contributions for the crystal packing are from H⋯H (43.3%), H⋯C/C⋯H (37.8%) and H⋯O/O⋯H (18.0%) interactions. Hydrogen bonding and van der Waals interactions are the dominant interactions in the crystal packing. Computational chemistry indicates that the C—HProp⋯OImdzln hydrogen-bond energy in the crystal is −40.7 kJ mol−1. Density functional theory (DFT) optimized structures at the B3LYP/6–311G level are compared with the experimentally determined molecular structure in the solid state. The HOMO–LUMO behaviour was elucidated to determine the energy gap.The title compound, C Atoms O1, O2, C16 and C19 are at distances of 0.0271 (12), −0.1040 (12), 0.1657 (19) and −0.0336 (19) Å from the mean plane of the imidazolidine ring, A. The orientation of the prop-2-yn-1-yl moieties with respect to the imidazolidine unit can be described by the C3—N1—C16—C17 and C3—N2—C19—C20 torsion angles of −115.3 (2) and 76.6 (2)°, respectively.The title molecule consists of an imidazolidine unit linked to two phenyl rings and two prop-2-yn-1-yl moieties Fig. 1. The plaProp⋯OImdzln (Prop = prop-2-yn-1-yl and Imdzln = imidazolidine) hydrogen bonds  analysis , Fig. 6d, has a pair of spikes with the tips at de + di = 2.24 Å.The overall two-dimensional fingerprint plot, Fig. 6dnorm plotted onto the surface are shown for the H⋯H, H⋯C/C⋯H and H⋯O/O⋯H interactions in Fig. 7a–c.The Hirshfeld surface representations with the function et al., 2015The Hirshfeld surface analysis confirms the importance of H-atom contacts in establishing the packing. The large number of H⋯H, H⋯C/C⋯H and H ⋯ O/O⋯H interactions suggest that van der Waals interactions and hydrogen bonding play the major roles in the crystal packing  is the sum of electrostatic (Eele), polarization (Epol), dispersion (Edis) and exchange-repulsion (Erep) energies  was calculated to be −15.3 (Eele), −3.2 (Epol), −52.2 (Edis), 37.6 (Erep) and −40.7 (Etot) for the C—HProp⋯NImdzln interaction.The intermolecular interaction energies Table 3 were calvia density functional theory (DFT) calculations using the standard B3LYP functional and 6–311G basis set , hardness (η), potential (μ), electrophilicity (ω) and softness (σ) are recorded in Table 3σ is to evaluate both the reactivity and stability of a compound. The electron transition from the HOMO to the LUMO energy level is shown in Fig. 8E = ELUMO - EHOMO] of the molecule is about 5.8874 eV, and the frontier molecular orbital energies, EHOMO and ELUMO are −6.6964 and −0.8090 eV, respectively.The optimized structure of the title compound in the gas phase was generated theoretically n-pentyl-5,5-diphenylimidazolidine-2,4-dione  at room temperature. The reaction was monitored using TLC. After removal of the inorganic salt by filtration, the solution was evaporated under reduced pressure. The residue was separated by chromatography on a column of silica gel with ethyl acetate–hexane (v:v 3:7) as eluent. The isolated solid was crystallized from ethanol solution to afford colourless crystals (yield: 82%).The appropriate bromide propargil  was added to a solution of 5,5 diphenylhydantoin  in DMF (50 ml), potassium carbonate  and tetra-et al., 2013bCrystal data, data collection and structure refinement details are summarized in Table 510.1107/S2056989019007801/lh5908sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989019007801/lh5908Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989019007801/lh5908Isup3.cdxSupporting information file. DOI: Click here for additional data file.10.1107/S2056989019007801/lh5908Isup4.cmlSupporting information file. DOI: 1919743CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


To harness the synthetic power of modular polyketide synthases (PKSs), many aspects of their biochemistry must be elucidated. A robust platform to study these megadalton assembly lines has not yet been described. Here, we in vitro reconstitute the venemycin PKS, a short assembly line that generates an aromatic product. Incubating its polypeptides, VemG and VemH, with 3,5-dihydroxybenzoic acid, ATP, malonate, coenzyme A, and the malonyl-CoA ligase MatB, venemycin production can be monitored by HPLC and NMR. Multi-milligram quantities of venemycin are isolable from dialysis-based reactors without chromatography, and the enzymes can be recycled. Assembly line engineering is performed using pikromycin modules, with synthases designed using the updated module boundaries outperforming those using the traditional module boundaries by over an order of magnitude. Using combinations of VemG, VemH, and their engineered derivatives, as well as the alternate starter unit 3-hydroxybenzoic acid, a combinatorial library of six polyketide products is readily accessed. A robust platform to study modular polyketide synthases (PKSs) in vitro is still unavailable. Here, the authors report the reconstitution of the venemycin PKS, engineer hybrid venemycin/pikromycin PKSs, and obtain much improved yields through employing the updated module boundaries. While synthetic chemists and material scientists alike yearn to reprogram these carbon–carbon bond forming, stereocenter-setting machines to produce new medicines and polymers, they can be challenging to work with, as they are comprised of several, difficult-to-purify polypeptides encoded by large, GC-rich genes. The only reconstituted PKS assembly line that has been reported is the erythromycin synthase; however, its first polypeptide was expressed in three pieces, and the product of the assembly line was only detectable by mass spectrometry3.Modular polyketide synthases (PKSs) are among the most miniature assembly lines known, yet they produce some of the most important human medicines, such as the antibacterial erythromycin and the anticancer agent epothilone6. As one of the shortest polyketide assembly lines (some contain >30 modules) and as the producer of an easily detectable product, it provides a tractable platform and a foothold to learn more about these molecule factories. Since few successes in engineering assembly lines with the traditional boundaries have been reported, our lab is particularly interested in testing a recent hypothesis concerning the boundaries of modules: bioinformatic investigation of related aminopolyol-producing synthases revealed that the set of domains that genetically co-migrates during the evolution of polyketide assembly lines differs from the traditional definition of a module and suggested that the downstream end of a module should include the KS traditionally considered to be part of the subsequent module7. We show that synthases engineered using the updated boundaries are over an order of magnitude more active than those engineered using the traditional boundaries11.We in vitro reconstitute the three-module venemycin synthase  Fig. 4–6. As oStreptomyces venezuelae ATCC 10712 genomic DNA into expression vectors such that the histidine tags used in the purification are positioned at the assembly line termini, distant from the docking domain interface formed by the C- and N-terminal ends of VemG and VemH, respectively  domains of the synthase are phosphopantetheinylated by the promiscuous enzyme Sfp (engineered into the Escherichia coli K207-3 expression host) and are thus functionalized to relay the growing chain in a hand-over-hand manner through the assembly line14. Extender units are supplied by a malonyl-CoA regeneration system comprised of Streptomyces coelicolor MatB, malonate, CoA, and ATP15. Through optimizing the reaction conditions  and the concentrations of the enzymes  and substrates  100 µL reactions went to completion in 2 h (turnover rate = 37 min−1)  over 16 h. The polyketide product can be rapidly isolated without chromatography and characterized , and the enzymes can be recycled in subsequent reactors with a half-life of 2 days.Scaled-up reactions were performed in 45 mL reactors, in which a dialysis bag maintains a high enzyme concentration and reaction progress can be monitored by HPLC or NMR analysis of the surrounding solution Fig. . In a tyS. venezuelae ATCC 15439) and the second and third modules from the venemycin assembly line was designed  method18. Purified PikAI/VemG and VemH were entered into a reactor in which both malonyl- and methylmalonyl-CoA are regenerated by MatB, as the pikromycin loading module makes a propionyl starter unit through the decarboxylation of a methylmalonyl group obtained from methylmalonyl-CoA. As MatB prefers malonate to methylmalonate, 1.6 mM malonate and 8.4 mM methylmalonate were supplied19. The anticipated pyrone (2) was produced quantitatively (11 min−1). A hybrid synthase comprised of the first two modules of the venemycin assembly line and the last module of the pikromycin assembly line (VemG and VemH/PikAIV) produced the anticipated methyl-substituted venemycin (3) quantitatively (15 min−1). Combining PikAI/VemG and VemH/PikAIV also yielded the anticipated methyl-substituted pyrone (4), albeit at a slower rate (0.93 min−1).Using the updated module definition, a hybrid synthase comprised of the first module from the pikromycin assembly line . The synthase composed of both PikAI/VemG and VemH/PikAIV was also less active than the synthase constructed with updated module boundaries (27-fold).21. However, the medicinal chemistry involved in exploring variations of such stereochemically rich compounds can be prohibitively challenging. By collaborating with the assembly lines that naturally biosynthesize these molecules, combinatorial libraries of polyketide drug leads may be accessable. As proof of this concept, a combinatorial library of six compounds was biocatalytically generated using modules from the venemycin and pikromycin assembly lines and the alternative starter unit 3-hydroxybenzoic acid  assembly lines  through bioinformatics7.The lower activities of synthases engineered using the traditional module boundaries may be due to weaker interactions between ACPs and downstream KSs that did not evolutionarily co-migrate. Previous studies have shown that maintaining ACPs with the KSs naturally C-terminal to them in an engineered assembly line can be beneficialtrans-AT assembly lines is localized on a LPTYPFxxxxxW motif C-terminal to the KS flanking subdomain24 , dehydratase (DH), enoylreductase (ER)] or smaller versions that contain fewer or none of these processing enzymes. Having essentially one module type may confer an evolutionary advantage to cis-AT assembly lines in that productive recombination events can occur in more locations, as observed in variants of the rapamycin synthase in which junctions occurred internal to KS and AT domains25. Even when recombination in a cis-AT assembly line separates an ACP from the enzymes of its module the recombined synthase may still be functional, as ACPs from different cis-AT modules are quite homogenous, especially in comparison to ACPs from different trans-AT module types. Our group chose a recombination site N-terminal to the flanking subdomain connecting the KS and AT domains so that its structural integrity would not be jeopardized26.The junction between modules in n24 Fig.  and is e28. We generated an all-atom model of the venemycin synthase to aid in these structure/function studies  assembly lines to produce diverse, stereocomplex products. The described platform should significantly aid efforts to construct larger assembly lines that synthesize bioactive molecules and, ultimately, new medicines.Many facets of assembly line biochemistry can be studied with the described platform, such as enzyme specificities, processivity versus iteration, ACP docking, unstructured linkers, and docking domains. The structural biology of assembly line enzymes also merits deeper investigationd-thiogalactopyranoside (IPTG) is from Carbosynth. Ni-NTA affinity resin and tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl) are from Thermo Fisher Scientific. MonoQ 5/50, Superdex 200 Increase 10/300, Sephacryl S-300 HR, HiLoad 16/600 Superdex 200 pg, and Superdex 200 Increase 16/600 GL are from GE Healthcare. Magnesium chloride is from MACRON. Tris(hydroxymethyl)aminomethane is from MP Biomedicals. Malonic acid and methylmalonic acid are from Oakwood Chemical. 3,5-Hydroxybenzoic acid, magnesium sulfate, sodium succinate, 4-hydroxy-6-methyl-2-pyrone, adenosine triphosphate (ATP), imidazole, and glycine are from Sigma-Aldrich. Coenzyme A (CoA) is from Oriental Yeast Co., Ltd. 3-Hydroxybenzoic acid is from Alfa Aesar. Amicon Ultracentrifugal protein concentrators filters are from Millipore. Ethyl acetate (EtOAc), acetonitrile, methanol, chloroform, and hexanes are from Fisher Scientific. D2O, CDCl3, and DMSO-d6 are from Cambridge Isotope Laboratories. SiliaFlash Irregular Silica Gels are from SiliCycle.The KAPA HiFi DNA polymerase is from KAPA Biosystems. All restriction enzymes are from New England Biolabs. All primers were custom ordered from Sigma-Aldrich. Luria-Bertani (LB) Miller Broth, sodium phosphate, potassium phosphate, sodium chloride, glycerol, and HEPES are from Fisher Scientific. Kanamycin sulfate, dithiothreitol (DTT), and sodium citrate dihydrate are from VWR. Carbenicillin is from Amersham. Isopropyl-β-1H and 13C NMR spectra were collected on a Bruker AVIII HD 400 (400 MHz) supported by Grant Willson and Benjamin Keitz (CHE-1626211) and Bruker AVIII HD 500 (500 MHz) supported by Jonathan Sessler (NIH 1 S10 OD021508-01).FPLC chromatography was performed using a Biologic DualFlow instrument (Bio-Rad). HPLC analysis was carried out on a Waters 1525 HPLC system. High-resolution mass spectral analysis was conducted on a 6230 TOF LC/MS (Agilent Technologies). E. coli DH5α was used for plasmid construction. BL21(DE3)star pLysS cells were used for MatB expression. E.coli K207-3 cells were used for the expression of assembly line enzymes13.Streptomyces venezuelae ATCC 10712. The resulting vemG and vemH amplicons were SLiCE assembled into NdeI/XhoI sites of pET28b-His10  star (pLysS) cells. The E. coli transformed with the expression plasmid were grown on LB medium containing 50 mg mL−1 kanamycin to OD600 = 0.6 at 37 °C, shaking at 225 rpm. Cultures were induced through the addition of 0.5 mM IPTG and then incubated for 18 h at 15 °C. Cells were harvested by centrifugation (4000 × g for 20 min), resuspended in lysis buffer , sonicated, and the cell debris was removed by centrifugation . The supernatant was applied to a Ni-NTA column (2 × 4 cm) and washed with 5 column volumes of lysis buffer containing 15 mM imidazole. The His-tagged protein was eluted with 2 column volumes of lysis buffer containing 250 mM imidazole. His-tagged MatB was concentrated using an Amicon Ultra centrifugal filter (Merck), and the buffer was exchanged for storage buffer . His-tagged VemG was further purified using a Superdex 200 Increase 10/300 GL column equilibrated with size exclusion buffer . His-tagged PikAI/VemG chimeric proteins were further purified using a HiLoad Superdex 16/600 200 pg column  equilibrated with size exclusion buffer. His-tagged VemH and VemH/PikAIV chimeric proteins were applied to a MonoQ 5/50 column equilibrated with 95% Buffer A  and 5% Buffer B . The proteins were eluted with a linear gradient of 5–50% Buffer B. Final purification was performed with a Superdex 200 Increase 10/300 GL column equilibrated with the size exclusion buffer. Protein concentrations were estimated using the ε280 values calculated by ExPASY ProtParam (https://web.expasy.org/protparam/): ε280 (His10 + VemG) = 156,760 M−1 cm−1, ε280(VemH + His6) = 125,820 M−1 cm−1, ε280(VemH/PikAIV(updated) + His6) = 150,800 M−1 cm−1, ε280 + His6) = 152,290 M−1 cm−1, ε280(His6 + PikAI/VemG(updated)) = 292,740 M−1 cm−1, ε280) = 292,740 M−1 cm−1, and ε280(His6 + MatB) = 33,920 M−1 cm−1. Protein quality was assessed by SDS-PAGE analysis and size-exclusion chromatography , 1 mM CoA, 10 µM MatB, 8 µM VemG, and 8 µM VemH at pH 7.5. After preincubating the reaction mixture at 25 °C for 5 min, the reaction was initiated through the addition of VemG and VemH and allowed to proceed for 1–10 min. The reaction was quenched by the rapid addition of 70% v/v perchloric acid (5 µL) and then neutralized by the addition of 1 M sodium bicarbonate (25 µL). Insoluble material was removed by centrifugation at 15,000 × g for 5 min. The supernatant (10 µL) was subjected to HPLC analysis on a Waters 1525 HPLC system equipped with a Microsorb-MV 300–5 C18 column (4.6 × 250 mm). The HPLC conditions were as follows: flow rate 1 mL min−1; solvent A (water with 0.1% v/v formic acid) and solvent B (acetonitrile). After column equilibration with 5% B, 5–100% B over 15 min, and then 100% B for 3 min. To determine turnover numbers, six independent experiments were set up per synthase (three from one synthase prep and three from a separate prep).Assays were performed in a final volume of 100 μL containing 400 mM potassium phosphate buffer, 5 mM TCEP, 9 mM MgClKd) to extract the turnover number and apparent Kd.y = initial velocity, c = concentration of VemH , x = concentration of VemG, K = apparent Kd, and t = turnover number.Initial velocities (µM product/min/µM VemH) were measured with 0.2 µM VemH and varying concentrations of VemG 0.1–8.0 µM). The resulting curve was fit using GraphPad Prism 8.1 and the following quadratic Eq. –8.0 µM. 1). IUPAC name: 6--4-hydroxy-2H-pyran-2-one. Formula: C11H8O5. The reactor was set up with a reaction solution of 45 mL comprised of 400 mM potassium phosphate, 5 mM TCEP, 9 mM MgCl2, 9 mM ATP, 0.75 mM 3,5-dihydroxybenzoic acid, 10 mM malonate, and 1 mM CoA, at pH 7.5. The reaction was initiated by adding a 100-kDa MWCO dialysis bag containing Ni-NTA column purified VemG (65 µM), VemH (100 µM), and MatB (10 µM) in 2 mL. After 20 h the reaction was saturated with ammonium sulfate, extracted with EtOAc (3 × 50 mL), dried with MgSO4, filtered, and concentrated in vacuo. Column chromatography (SiliaFlash F60) with EtOAc/hexanes (2/1) → EtOAc as eluent. TLC : Rf = 0.35; 1H NMR : δ 11.77 , 9.57 , 6.62 , 6.47 , 6.30 , 5.31  (D2O suppressed). 1H NMR : δ 10.06 , 6.76 , 6.36 , 6.30 , 5.01 . 13C NMR : δ 171.2 (C2), 163.6 (C4), 160.6 (C6), 159.3 , 133.2 (C7), 105.6 (C10), 103.9 , 98.6 (C5), 89.9 (C3).Venemycin (2). IUPAC name: 6-ethyl-4-hydroxy-2H-pyran-2-one. Formula: C7H8O3. The reactor was set up with a reaction solution of 45 mL comprised of 400 mM potassium phosphate, 5 mM TCEP, 12 mM MgCl2, 12 mM ATP, 1.6 mM malonate, and 8.4 mM methylmalonate at pH 7.5. The reaction was initiated by adding a 100 kDa MWCO dialysis bag containing Ni-NTA column purified PikAI/VemG (60 µM), VemH (100 µM), and MatB (10 μM) in 2 mL. After 2 d the reaction was saturated with ammonium sulfate, extracted with EtOAc (3 × 50 mL), dried with MgSO4, filtered, and concentrated in vacuo. Column chromatography (SiliaFlash F60) with EtOAc/hexanes (1/1) → (2/1) with 1% formic acid as eluent. TLC : Rf = 0.53; 1H NMR : δ 5.96 , 5.56 , 2.53 , 1.22 . 1H NMR : δ 5.96 , 5.20 , 2.50 , 1.17 . 13C NMR (CDCl3): δ 172.3 (C2), 168.5 (C4), 167.8 (C6), 100.3 (C5), 89.8 (C3), 26.9 (C7), 10.8 (C8).Pyrone (3). IUPAC name: 6--4-hydroxy-3-methyl-2H-pyran-2-one. Formula: C12H10O5. The reactor was set up with a reaction solution of 45 mL comprised of 400 mM potassium phosphate, 5 mM TCEP, 9 mM MgCl2, 9 mM ATP, 0.75 mM 3,5-dihydroxybenzoic acid, 1.6 mM malonate and 8.4 mM methylmalonate at pH 7.5. The reaction was initiated by adding a 100 kDa MWCO dialysis bag containing Ni-NTA column purified VemG (71 µM), VemH/PikAIV (100 µM), and MatB (10 µM) in 2 mL. After 2 d the reaction was saturated with ammonium sulfate, extracted with EtOAc (3 × 50 mL), dried with MgSO4, filtered, and concentrated in vacuo. Column chromatography (SiliaFlash F60) with EtOAc/hexanes (1/1) → (2/1) with 1% formic acid as eluent. TLC  Rf = 0.45; 1H NMR : δ 9.55 , 6.55 , 6.44 , 6.26 , 1.76 . 1H NMR : δ 10.05 , 6.76 , 6.35 , 1.67 . 13C NMR : δ 164.8 (C2), 159.4 , 157.0 (C6), 133.5 (C7), 105.2 (C10), 103.5 , 98.5 (C5), 98.3 (C3), 9.2 (CH3).Methylvenemycin (4). IUPAC name: 6-ethyl-4-hydroxy-3-methyl-2H-pyran-2-one. Formula: C8H10O3. The reactor was set up with a reaction solution of 45 mL comprised of 400 mM potassium phosphate, 5 mM TCEP, 12 mM MgCl2, 12 mM ATP, 1.6 mM malonate, and 8.4 mM methylmalonate at pH 7.5. The reaction was initiated by adding a 100-kDa MWCO dialysis bag containing Ni-NTA column purified PikAI/VemG (124 µM), VemH/PikAIV (208 µM), and MatB (10 µM) in 2 mL. After 5 d the reaction was saturated with ammonium sulfate, extracted with EtOAc (3 × 50 mL), dried with MgSO4, filtered, and concentrated in vacuo. Less than one milligram of 4 was obtained. Column chromatography (SiliaFlash F60) with EtOAc/hexanes (1/1) → (2/1) with 1% formic acid as eluent. 1H NMR : δ 5.71 , 2.50 , 1.91 , 1.25 .Methylpyrone (5). IUPAC name: 4-hydroxy-6-(3-hydroxyphenyl)-2H-pyran-2-one. Formula: C11H8O4. The reactor was set up with a reaction solution of 45 mL comprised of 400 mM potassium phosphate, 5 mM TCEP, 9 mM MgCl2, 9 mM ATP, 0.75 mM 3-hydroxybenzoic acid, and 10 mM malonate at pH 7.5. The reaction was initiated by adding a 100 kDa MWCO dialysis bag containing Ni-NTA column purified VemG (65 µM), VemH (100 µM), and MatB (10 µM) in 2 mL. After 1 d the reaction was saturated with ammonium sulfate, extracted with EtOAc (3 × 50 mL), dried with MgSO4, filtered, and concentrated in vacuo. Column chromatography (SiliaFlash F60) with EtOAc/hexanes (1/1) → (2/1) as eluent. TLC : Rf = 0.50; 1H NMR : δ 9.76 , 7.29 , 7.26 , 7.18 , 6.88 , 6.55 , 5.22 . 1H NMR : δ 7.32–7.26 , 7.19 , 6.89 , 6.51 , 4.99 . 13C-NMR : δ 171.8 (C2), 164.2 (C4), 160.4 (C6), 158.1 (C9), 132.8 (C7), 130.8 (C11), 118.5 (C10), 116.8 (C12), 112.4 (C8), 99.3 (C5), 89.8 (C3).Deshydroxyvenemycin (6). IUPAC name: 4-hydroxy-6-(3-hydroxyphenyl)-3-methyl-2H-pyran-2-one. Formula: C12H10O4. The reactor was set up with a reaction solution of 45 mL comprised of 400 mM potassium phosphate, 5 mM TCEP, 9 mM MgCl2, 9 mM ATP, 0.75 mM 3-hydroxybenzoic acid, 1.4 mM malonate, and 8.4 mM methylmalonate at pH 7.5. The reaction was initiated by adding a 100-kDa MWCO dialysis bag containing Ni-NTA column purified VemG (69 µM), VemH/PikAIV (100 µM), and MatB (10 µM) in 2 mL. After 2 d the reaction was saturated with ammonium sulfate, extracted with EtOAc (3 × 50 mL), dried with MgSO4, filtered, and concentrated in vacuo. Column chromatography (SiliaFlash F60) with EtOAc/hexanes (1/2) → (1/1) as eluent. TLC : Rf = 0.60; 1H NMR : δ 9.80 , 7.31 , 7.18 , 7.14 , 6.88 , 6.63 , 1.83 . 1H NMR : δ 7.26–7.22 , 7.16 , 6.86–6.80 , 6.41 , 1.68 . 13C-NMR : δ 165.5 (C2), 164.7 (C4), 158.3 (C6), 157.0 (C9), 132.9 (C7), 130.7 (C11), 118.1 (C10), 116.2 (C12), 112.0 (C8), 98.8 (C3), 98.3 (C5), 9.1 (CH3).Deshydroxymethylvenemycin (29. The A domains were threaded through a dimeric long-chain fatty acid CoA ligase (PDB 3G7S). KR0 was threaded through the monomeric ketoreductase from the second module of the erythromycin assembly line (PDB 2FR0). KS and AT domains were threaded through the dimeric KS and monomeric AT domains from the third and fourth modules of the erythromycin assembly line (PDB 6C9U). ACP domains were threaded through the monomeric ACP domain from the eighth module of the mycolactone assembly line . The TE domain was threaded through the dimeric TE domain from the tautomycetin assembly line (PDB 3LCR). The docking domains were threaded through the dimeric docking domains connecting the second and third polypeptides of the erythromycin assembly line (PDBs 1PZR and 1PZQ). Domains were relatively oriented using the program Pymol , and flexible loop residues were built with the program Coot30.Several solved structures were combined to generate the homodimeric synthase comprised of the 232-kDa VemG and the 140-kDa VemG (Supplementary Fig. Further information on research design is available in the Supplementary InformationReporting SummaryDescription of Additional Supplementary FilesSupplementary Data 1